**E**ff**ect of Fermentation, Drying and Roasting on Biogenic Amines and Other Biocompounds in Colombian Criollo Cocoa Beans and Shells**

## **Johannes Delgado-Ospina 1,2,\*, Carla Daniela Di Mattia 1, Antonello Paparella 1, Dino Mastrocola 1, Maria Martuscelli 1,\* and Clemencia Chaves-Lopez <sup>1</sup>**


Received: 21 March 2020; Accepted: 14 April 2020; Published: 21 April 2020

**Abstract:** The composition of microbiota and the content and pattern of bioactive compounds (biogenic amines, polyphenols, anthocyanins and flavanols), as well as pH, color, antioxidant and reducing properties were investigated in fermented Criollo cocoa beans and shells. The analyses were conducted after fermentation and drying (T1) and after two thermal roasting processes (T2, 120 ◦C for 22 min; T3, 135 ◦C for 15 min). The fermentation and drying practices affected the microbiota of beans and shells, explaining the great variability of biogenic amines (BAs) content. Enterobacteriaceae were counted in a few samples with average values of 10<sup>3</sup> colony forming units per gram (CFU g−1), mainly in the shell, while *Lactobacillus* spp. was observed in almost all the samples, with the highest count in the shell with average values of 10<sup>4</sup> CFU g<sup>−</sup>1. After T1, the total BAs content was found to be in a range of 4.9÷127.1 mg kg−<sup>1</sup> DFW; what was remarkable was the presence of cadaverine and histamine, which have not been reported previously in fermented cocoa beans. The total BAs content increased 60% after thermal treatment *T2*, and of 21% after processing at *T3*, with a strong correlation (*p* < 0.05) for histamine (ß = 0.75) and weakly correlated for spermidine (ß = 0.58), spermine (ß = 0.50), cadaverine (ß = 0.47) and serotonine (ß = 0.40). The roasting treatment of *T3* caused serotonin degradation (average decrease of 93%) with respect to unroasted samples. However, BAs were detected in a non-alarming concentration (e.g., histamine: n.d <sup>÷</sup> 59.8 mg kg−<sup>1</sup> DFW; tyramine: n.d. <sup>÷</sup> 26.5 mg kg−<sup>1</sup> DFW). Change in BAs level was evaluated by principal component analysis. PC1 and PC2 explained 84.9% and 4.5% of data variance, respectively. Antioxidant and reducing properties, polyphenol content and BAs negatively influenced PC1 with both polyphenols and BA increasing during roasting, whereas PC1 was positively influenced by anthocyanins, catechin and epicatechin.

**Keywords:** biogenic amines; polyphenols; histamine; microbiota; roasting

#### **1. Introduction**

In the last years, an increase in global cocoa production has been observed with a market demand of high-quality cocoa products [1]. Colombian cocoa was declared by the International Cocoa Organization as "fine" and "flavour" due to the agro-ecological characteristics of the areas in which it is cultivated and the adequate fermentation and drying processes that are carried out. In particular, Criollo is a variety of Colombia and other Latin American countries, known for its high quality.

Cocoa is produced from cocoa beans that undergo several processes such as fermentation, drying, roasting, dutching, conching, and tempering. In the first stages, cocoa pods (fruits) are picked from the

trees (*Theobroma cacao*), collected in piles and immediately opened or left to stand for a few days (pod storage) to obtain positive effects on the quality of the final products. After harvesting, beans together with mucilage are removed from the pod, fermented, dried, and roasted [2].

Fermentation is essential for the degradation of mucilage thanks to the production of ethanol, which kills cocoa bean cotyledons, and to the production of different organic acids and important volatile compounds that diffuse into the interior of the beans and react with substances responsible for the flavour of final products during the subsequent roasting process. In addition, fermentation influences some functional properties such as antiradical activity and reduces the power of cocoa beans [3,4]. However, biogenic amines (BAs) can be formed during this step, with detrimental effects on cocoa quality and human health. The occurrence of BAs in food originates from decarboxylation of free amino acids, amination and transamination of ketones and aldehydes or during thermal processes. In fermented products, the concentration of BAs is the result of a balance between formation and degradation reactions in which several microorganisms are involved. In fact, cocoa microbiota may present strains with decarboxylase activity [5–8] and amino-oxidase activity [9].

A decrease of bioactive compounds, such as BAs and polyphenolic compounds, occurs in different steps of the cocoa beans processing, affecting their final content and functional properties in cocoa derivatives [10–12]. During roasting, physical and chemical changes occur in the beans, such as differences in colour, removal of undesirable volatile compounds, formation of desirable aroma and flavour, reduction of water content (up to 2%), and formation of a brittle structure, as well as changes in flavanols, proanthocyanidins and antioxidant activity [13,14]. In addition, peculiar cocoa volatile compounds are generated by Maillard reactions and their release is favoured by modifications of the matrix structure [15]. In spite of this, during roasting critical changes may also take place such as the formation of water-insoluble melanoidins, the degradation of catechin-containing compounds [16], the reduction of polyphenol content and antioxidant activity [17], and an increase of the biogenic amines content [12]. If some Maillard Reaction products, such as melanoidins, are required for the development of the peculiar cocoa sensory characteristics and brown colour, some furanic compounds are supposed to have negative effects on human health, as they can show cytotoxicity at high concentration and are "possibly carcinogenic to humans" [18]. Furthermore, since the presence of cocoa shell in cocoa beans derivatives adversely affects the final product quality [19], beans should be peeled before or after roasting [20,21].

The present research was aimed to study the effect of fermentation, drying and roasting on the microbiological, physical and chemical characteristics of Colombian Criollo cocoa (bean and shell), with a particular focus on the content of bioactive compounds such as BAs and polyphenols.

#### **2. Materials and Methods**

#### *2.1. Origin of the Samples*

Criollo cocoa bean samples were collected in spring 2018 directly from 18 farms (identified with a numerical code) located in three Departments of Colombia, with different environmental conditions and different fermentation and drying systems (Table 1). Thirteen samples were from Valle del Cauca, located in the western part of the country, between 3◦05 and 5◦01 N latitude, 75◦42 and 77◦33 W longitude; four samples from Cauca, located in the southwest of the country on the Andean and Pacific regions, between 0◦58 54" N and 3◦19 04" N latitude, 75◦47 36" W and 77◦57 05" W longitude; one sample from Nariño, located in the west of the country (1◦16 0.01" N latitude and 77◦22 0.12" W longitude) and, despite low altitude, affected by cold winds from the south of the continent.



*Foods* **2020**, *9*, 520

#### *2.2. Samples Preparation and Defatting*

The samples were collected at the final stage of fermentation and drying (step T1). Moreover, dried cocoa beans were divided into two batches and treated in a convection oven (Memmert UN110, Büchenbach, Germany) in two different commercial roasting conditions: T2, 120 ◦C for 22 min; T3, 135 ◦C for 15 min.

After cooling, the shell was removed manually from the cocoa beans. After the removal of the external skin and grinding (IKA M20, Staufen, Germany), cocoa samples were defatted by three cycles of hexane washing (8 g of cocoa sample in 50 mL of hexane), following the method described by Di Mattia et al. [4]. Four grams of sample were weighed and 25 mL of hexane added, then the mixture was vortexed for 1 min and centrifuged (2325× *g* for 10 min), each time discharging the supernatant. To completely remove the hexane from the sample, the lipid-free solids were air-dried at room temperature. The fat-free samples were then used for the extraction of the phenolic fraction and other chemical determinations.

#### *2.3. Moisture and pH Determination*

The pH of defatted cocoa nibs was measured by diluting in distilled water (1:1) by using an electrode probe connected to a pHmeter (FE20, Mettler Toledo, Columbus, OH, USA).

Moisture content was determined according to the official procedure adopted by the Association of Official Analytical Chemists (AOAC) [22]. In particular, 1 g of sample was dried in a forced-air drying oven at 105 ◦C up to a constant weight.

#### *2.4. Microbiological Analyses*

Microbiological analyses were performed according to Chaves et al. [23]. From samples of dried cocoa beans, the beans (from here they are beans without shell) and the shells were obtained by manual separation. Twenty grams of cocoa nibs and separate shells were homogenized in a Stomacher Lab-blender (Thomas Scientific, Swedesboro, NJ, USA) in 90 mL phosphate buffer solution (PBS, Biolife, Milan, Italy) sterile solution, pH 7.4. Decimal dilutions of the suspension were prepared in PBS, plated and incubated as follows: Enterobacteriaceae were counted and isolated in Violet Red Bile Glucose Agar (Oxoid, Basingstoke, UK) at 37 ◦C in anaerobiosis for 48 h; mesophilic aerobic bacteria in Plate Count Agar (PCA) at 30 ◦C for 48 h; thermophilic aerobic bacteria in PCA and incubated at 45 ◦C for 48 h; lactobacilli in De Man Rogose and Sharp (MRS) Broth (Oxoid, Basingstoke, UK) at 37 ◦C in anaerobiosis for 72 h; lactic streptococci in M17 agar (Oxoid, Basingstoke, UK) at 37 ◦C in anaerobiosis for 72 h; yeasts in Yeast Extract-Peptone-Dextrose (YPD) agar medium and Walerstein Laboratory (WL) medium agar (Biolife, Milan, Italy) at 25 ◦C for 48 h; moulds in DG18 Agar (Oxoid, Basingstoke, UK) and Czapec-Agar (Biolife, Milan, Italy) added with 150 ppm chloramphenicol (Sigma-Aldrich Italy, Milan, IT) for 5 days.

#### *2.5. Biogenic Amines Determination*

Defatted samples were subjected to BAs extraction, detection, identification and quantification by high-performance liquid chromatography (HPLC) using an Agilent 1200 Series (Agilent Technologies, Milano, Italy), optimizing the method described by Chaves-Lopez et al. [23]. Shortly after, 1.0 g of sample was added of 5.0 mL of 0.1 N HCl and stirred in vortex (1 min) and ultrasound (20 min). It was centrifuged (Hettich Zentrifugen, Tuttlingen, Germany) at relative centrifugal force of 2325× *g* for 10 min and the supernatant recovered. Then, 150 μL of saturated NaHCO3 was added to 0.5 mL of the supernatant, adjusting the pH to 11.5 with 0.1 N NaOH. For derivatization, 2.0 mL of dansyl chloride/acetone (10 mg mL<sup>−</sup>1) was added and incubated at 40 ◦C for 1 h under agitation (195 stokes) (Dubnoff Bath-BSD/D, International PBI, Milano, Italy). To remove excess of dansyl chloride, 200 μL of 30% ammonia was added, allowed to stand for 30 min at room temperature, and diluted with 1950 μL of acetonitrile.

In a Spherisorb S30ODS Waters C18-2 column (3 μm, 150 mm × 4.6 mm ID), 10 μl of sample were injected with gradient elution, acetonitrile (solvent A) and water (solvent B) as follows: 0–1 min 35% B isocratic; 1–5 min, 35%–20% B linear; 5–6 min, 20%–10% linear B; 6–15 min, 10% B isocratic; 15–18 min, 35% linear B; 18–20 min, 35% B isocratic. Identification and quantification of cadaverine (CAD), dopamine (DOP), ethylamine (ETH), histamine (HIS), 2-phenylethylamine (PHE), putrescine (PUT), serotonin (SER), spermidine (SPD), spermine (SPM), and tyramine (TYR) was performed by comparing retention times and calibration curves of pure standards. The results were reported as mg of BA kg−<sup>1</sup> of defatted dry weight (of DFW).

#### *2.6. Colour Analysis*

Colour analysis of the cocoa samples was carried out by a Minolta Bench-top Colorimeter CR-5 (Konica Minolta, Tokyo, Japan) CM-500 spectrophotometer. Before analysis, two calibrations were carried out, one with black standard and the other one with white standard. For each measurement, a single layer of grounded cocoa beans was spread on a Petri dish. The analysis was repeated three times on each sample.

The instrument gave the results in terms of CIE L\* a\* b\* parameters (CIELAB is a colour space specified by the International Commission on Illumination, French Commission Internationale de l'éclairage, CIE), where L\* indicates the lightness within the range from 0 (black) to 100 (white); a\* ranges from −60 (green) to +60 (red); b\* ranges from −60 (blue) to +60 (yellow). a\* and b\* indicate colour direction and from these values we obtained the Hue angle (h◦), calculated as h◦ = arctan(b\*/a\*)

#### *2.7. Anthocyanin Determination*

The total anthocyanins content was determined by the method described by do Carmo Brito et al. [11]. In brief, 1.0 mL of 95% ethanol and 1.5 N hydrochloric acid solution (85:15 *v*/*v*) were added to 0.1 g of defatted cocoa sample, then stirred in vortex for two min and allowed to stand overnight. The sample was centrifuged at 10,000× *g*, and the supernatant was suitably diluted to measure its absorbance at 535 nm by a spectrometer (Eppendorf Biospectrometer kinetic, Hamburg, Germany. The results were reported as mg of anthocyanins g−<sup>1</sup> of sample.

#### *2.8. Extraction of the Phenolic Fraction*

The defatted samples were further ground with mortar and pestle to reduce the powder size and to allow better contact of the extracting solvent with the sample. The sample extraction was carried out according to Di Mattia et al. [4] with some modifications. One gram of defatted sample was added to 5 mL of 70:29.5:0.5 acetone/water/acetic acid; the mixture was vortexed for 1 min, then sonicated in an ultrasonic bath (Labsonic LBS 1, Falc, Treviglio, Bergamo, Italy) at 20 ◦C for 10 min and finally centrifuged (2325× *g* for 10 min). The surnatant liquid was recovered and filtrated through cellulose filters. The extracted polyphenols were then stored in the freezer at −32 ◦C until analyses. This extract was used for the evaluation of total polyphenol content, radical scavenging activity and ferric reducing properties. For flavanols analysis, samples were extracted, kept at −80 ◦C and analysed on the same day or at the latest a few days after extraction.

#### 2.8.1. Total Polyphenols Content (TPC)

The total polyphenol content (TPC) was determined according to a procedure modified from Di Mattia et al. [10]. To a volume of 0.1 mL of diluted defatted sample extract, water was added up to a volume of 5 mL, and 500 μL of Folin–Ciocalteu reagent was added. After 3 min, 1.5 mL of a 25% (*w*/*v*) Na2CO3 solution was added and then deionized water up to 10 mL of the final volume. Solutions were maintained at room temperature under dark conditions for 60 min and the total polyphenols content was determined at 765 nm using a spectrophotometer (Lambda Bio 20 Perkin Elmer, Waltham, MA, USA) Gallic acid standard (Fluka, Buchs, Switzerland) solutions were used for calibration. Results were expressed as milligrams of gallic acid equivalents (GAE) per gram of defatted and dry weight.

#### 2.8.2. Flavanols Identification and Quantification

HPLC (high-performance liquid chromatography) was used for separation, quantitative determination and identification of flavonoids. The chromatographic analyses were performed on a 1200 Agilent Series HPLC (Agilent Technologies, Milano, Italy) equipped with a quaternary pump, a degasser, a column thermostat, an autosampler injection system and a diode array detector (DAD). The system was controlled by Agilent ChemStation for Windows (Agilent Technologies). Flavanols determination was carried out according to Ioannone et al. [13]. The sample (20 μL) was injected into a C18 reversed-phase column. Separation of phenolic compounds was carried out at a flow rate of 1 mL/min with a non-linear gradient from A (1% acetic acid solution) to B (ACN). Gradient elution was as follows: from 6% to 18% B from 0 to 40 min, from 18% to 100% B from 40 to 45 min, from 100% to 6% B from 45 to 50 min, isocratic from 50 to 53 min. The DAD acquisition range was set from 200 to 400 nm. The calibration curves were made with epicatechin and catechin, and the results were expressed as mg per gram of defatted and dry weight.

#### 2.8.3. ABTS (2,2 -azino-bis(3-ethylbenzthiazoline-6-sulfuric acid)) Assay

The radical scavenging activity was measured by ABTS (2,2 -azino-bis (3-ethylbenzthiazoline-6-sulfuric acid)) radical cation decoloration assay, as described by Re et al. [24]. The ABTS radical stock solution was prepared by dissolving ABTS in water to a 7 mM concentration and by making this solution react with 2.45 mM of potassium persulfate. The mixture was then left in the dark at room temperature for 12–16 h before use. The ABTS+• stock solution was diluted in water to an Abs of 0.70 ± 0.02 for the analysis, and the reaction was started by the addition of 30 μL of cocoa extract to 2.97 mL of ABTS+• radical solution. The bleaching rate of ABTS+• in the presence of the sample was monitored at 25 ◦C at 734 nm using a spectrophotometer (Lambda Bio 20, Perkin Elmer, Boston, MA, USA) and decoloration after 5 min was used as the measure of antioxidant activity. Radical scavenging activity was expressed as Trolox Equivalents Antioxidant Capacity (TEAC-μmol of Trolox equivalents per g of defatted and dry weight), calculated by the ratio between the correlation coefficient of the dose–response curve of the sample and the correlation coefficient of the dose–response curve of Trolox, the standard compound.

#### 2.8.4. Ferric Reducing Antioxidant Power (FRAP)

The reducing activity of the samples was determined according to the method described by Benzie and Strain [25] with some modifications. One hundred microliters of suitably diluted sample extract were added to 2900 μL of the FRAP reagent obtained by mixing acetate buffer (300 mM, pH 3.6), TPTZ (2,4,6-tripyridyl-s-triazine), 10 mM solubilized in HCl 40 mM and FeCl3 20 mM in the ratio 10:1:1. The absorbance change was followed at 593 nm for 6 min. A calibration plot based on FeSO4·7H2O was used, and results were expressed as mmols of Fe2<sup>+</sup> per gram of defatted and dry weight.

#### *2.9. Statistical Analyses*

All determinations were done in triplicate, except where differently indicated. Means and relative standard deviations were calculated. Analysis of variance (ANOVA) was performed to test the significance of the effects of the factor variables (processing steps); differences among means were separated by the least significant differences (LSD) test. Statistical analysis of data was performed using XLSTAT software version 2019.1 for Microsoft Excel (Addinsoft, New York, NY, USA). All results were considered statistically significant at *p* < 0.05.

The multivariate descriptive analysis was used to understand the presence of the main descriptors related to the BAs content of cocoa. The principal components analysis (PCA) started with the analysis of a matrix (18 × 55) that consisted of 18 samples of Criollo cocoa. The analyses were performed in triplicate. The 55 conformations of the values of the evaluated variables were gathered by the following tests: roasting temperature (raw, 120 ◦C for 22 min and 135 ◦C for 15 min), pH, content of total polyphenols (TPC), anthocyanins, antioxidant activity (FRAP and TEAC), flavonols (catechin and epicatechin), levels of the main microorganisms groups in cocoa beans and shell, ethylamine (ETH), dopamine (DOP), 2-phenylethylamine (PHE), putrescine (PUT), cadaverine (CAD), serotonin (SER), histamine (HIS), tyramine (TYR), spermidine (SPD), and spermine (SPM).

#### **3. Results**

#### *3.1. Characterization of Fermented and Dried Cocoa Beans*

Several indicators are used to measure the quality of cocoa beans. These include, in addition to microbiota, composition, colour and acidity of the beans [26].

#### 3.1.1. Microbiota

Some researchers found that variations in the content of BAs in cocoa are mainly affected by fermentation, which is directly correlated to the type and quantity of microbial populations [11]. Figure 1a,b shows the distribution of microorganism groups enumerated both in cocoa beans and cocoa shells of investigated samples. A large variability was observed, confirming that postharvest practices carried out in the different Colombian farms affected microbiota, which in turn can explain the great diversity of decarboxylation products, such as BAs content at the T1 step. The microbial load of the shell was determined because many of the metabolites produced in the shell during fermentation and drying can migrate to the beans, causing a pH decrease due to the accumulation of organic acids. The microbiological analyses showed the presence of enterobacteria, total aerobic mesophiles, total aerobic thermophiles, acetic bacteria, spore-forming bacteria, lactobacilli, lactococci, fungi, and yeasts that are mainly involved in fermentation and drying. Variations in microbial counts and species were observed in the different samples, likely due to different fermentation and drying practices (pod ripeness, postharvest pod storage, variations in pulp/bean ratio, fermentation method, batch size, frequency of bean mixing or turning, and fermentation time), as well as due to some characteristics of the environment where the cultivation takes place (farm, weather conditions, pod diseases) [27,28].

A great difference was also observed between the microbial community found in the shell and inside the beans. As expected, the shell contained a greater number of microorganisms because sugars and other rapidly degradable nutrients are concentrated here, while a smaller number of microbial populations could adapt to the conditions of the beans. According to Lima et al. [29], average levels of total aerobic microorganisms and aerobic total spores are reduced in the beans, while Enterobacteriaceae and fungi were not detected.

Generally, the production of BAs is attributed to certain species of Enterobacteriaceae, mainly *Clostridium* spp., *Lactobacillus* spp., *Streptococcus* spp., *Micrococcus* spp., and *Pseudomonas* spp. [30]. Two of these groups were found in cocoa samples; Enterobacteriaceae were counted in a few samples with average values of 103 CFU g−1, mainly in the shell and probably due to contamination during outdoor drying, while *Lactobacillus* spp. was observed in almost all the samples, with the highest count in the shell with average values of 104 CFU g<sup>−</sup>1.

#### 3.1.2. pH, Moisture and Colour

The characteristics of Colombian Criollo cocoa samples at the end of fermentation and drying (T1) are shown in Table 2. The great variability found in the samples depends on several factors, namely fermentation and drying, as well as some intrinsic characteristics of the farming system. The organic acids produced by lactic and acetic bacteria during fermentation diffuse within the beans and cause a pH decrease; low pH values are considered an index of appropriate fermentation while pH values above 5.5 may indicate an inadequate or incomplete fermentation [31]. The pH of the samples ranged between 4.43 and 6.17 (C.V. 10%). With the exception of sample 18, it can be stated that the samples coming from Valle de Cauca were generally characterized by lower pH values compared to Cauca samples.

(**b**)

**Figure 1.** Levels of the main microorganisms groups in beans (**a**) and shells (**b**) of Colombian Criollo cocoa samples (after fermentation and drying, step T1).


**Table 2.** Chemico-physical and colour parameters (L\*, a\*, b\* and h◦) for fermented and dried samples (T1). The data are expressed as mean ± standard deviation.

The moisture in bean samples ranged between 1.2% (sample 6) and 6.2% (sample 14), with differences depending on process conditions (solar or artificial dryers) and processing time. However, for all the samples, moisture content was below 12% which is considered the threshold value for optimal beans storage, corresponding to inhibition of both enzymatic reactions and fungal growth that can produce undesired metabolites during storage, such as mycotoxins.

The lightness (L\*) of the 18 samples had a mean value of 40.8 (±4.03), ranging from 48.92, observed in sample 2, to 32.96 in sample 18. For redness values (a\*), a mean of 7.78 (±1.94) was detected with 10.04 as the maximum value (in sample 13) and 3.64 as the minimum (in sample 2). For yellowness (b\*), we observed the highest value in sample 13 (11.11 ± 2.07) and the lowest value in sample 6 (6.70 ± 0.54). Finally, for hue angle (h◦), a mean value of 55.12 ± 8.26 was determined with a range from 68.77 (sample 2) to 36.49 (sample 6). Other authors reported L\* values quite different from those obtained in the present study, while results for parameters of a\* and b\* were similar [32]. The values obtained for h◦ were similar to those reported by Sacchetti et al. [14].

#### *3.2. Biogenic Amines Profile*

The BAs profile of the Criollo cocoa beans under investigation is described in Table 3. In unroasted samples (T1), the total BAs amount was found to be 57.5 (±37.5) mg kg−<sup>1</sup> DFW, with a minimum value of 4.9 mg kg−<sup>1</sup> DFW (in sample 16, from Nariño region) and a maximum of 127.1 mg kg−<sup>1</sup> DFW (in sample 4, from Valle de Cauca). As far as the BAs pattern is concerned, the most represented BAs in unroasted beans (T1) were CAD, SER, HIS, SPD, and SPM (Table 3); DOP was also detected in unroasted sample 15 (from Cauca).

The Pearson correlation coefficient between total BAs content and each single BA was calculated. A strong correlation was only found with HIS (ß = 0.75); tot BAs correlated weakly with SPD (ß = 0.58), SPM (ß = 0.50), CAD (ß = 0.47), and SER (ß = 0.40), while no significant correlation was found with other amines.





**Table 3.** *Cont.*

*Foods* **2020**, *9*, 520 spermine (SPM); nd, not detectable.

To the best of our knowledge, there are no studies reporting the occurrence of CAD and HIS in raw cocoa beans, although there are few studies where BAs are identified in cocoa. Some authors [12] found tyramine, 2-phenylethylamine, tryptamine, serotonin, and dopamine in different varieties of raw cocoa beans; other authors [11] also found spermidine and spermine in Brazilian samples during fermentation.

Most of the analysed samples presented similar profiles of BAs that might be explained by the fact that they belong to the same variety. However, variations in their concentration were found and can be explained by the difference between cultivars, different growth, fermentation and drying conditions, as well as the microbiota of beans and shell (see Figure 1a,b).

Polyamines can also occur naturally due to the large proliferation of cells that occur in the early stages of growth or germination caused by physiological changes in tissues [33,34]. In fact, being that the cocoa bean is a seed and germination does not start if the optimal fermentation conditions are not present, a consequence of this could be that secondary metabolites such as the aliphatic amines (PUT, CAD, SPM and SPD) could be accumulated in cells. To our best knowledge, very few studies have been published on the relation between the physiological conditions and the BAs content in cocoa seeds, thus these aspects should be thoroughly investigated.

Although the development of microorganisms with amino acid decarboxylases activity occurs in environments with optimal pH between 4.0 and 5.5, no correlation was observed between BAs content and low pH.

No direct relationship was found between the content of polyphenols and the content of BAs in cocoa beans (see below). However, it is possible to hypothesize that the presence of metabolites as polyamines may influence antioxidant activity in cocoa samples or exhibit pro-oxidant properties [35].

#### *3.3. E*ff*ect of Roasting on the BAs Content*

A significant effect of both the roasting processes on total BAs content was found in all the samples; in particular, the beans treated at T2 (120 ◦C for 22 min) showed an increase of 60% with respect to the raw beans samples (T1), whilst the roasting process T3 (135 ◦C, 15 min) caused an increase of 21% compared to T1 samples.

In our experiments, we observed a large variability in the behaviour of each BA in the samples (Table 3); after the high temperature treatment, we determined the presence of TYR, 2-PHE, ETH, and PUT that were not detected in unroasted beans (T1). On the other hand, the roasting treatment increased the concentration of DOP and SPM with the increase of the temperature, while CAD and SPD levels decreased dramatically.

Several factors could affect the final accumulation of BAs. In particular, some authors have reported that Strecker degradation is responsible for the formation of BAs during the thermal decarboxylation of amino acids in the presence of α-dicarbonyl compounds formed during the Maillard reaction [12,36] or lipid peroxidation [37].

After treatment at 120 ◦C (T2), total BAs concentration correlated significantly with SPM (ß = 0.77), SPD (ß = 0.67) and PUT (ß = 0.60), while at 135 ◦C (T3) there was a strong correlation between tot BAS and SPD (ß = 0.85), HIS (ß = 0.81), and PUT, CAD and SPM (ß > 0.70).

Some authors suggested that serotonin could be formed as a result of the transformation of its precursors (tryptophan and 5-hydroxytryptophan) at very high temperatures [38]. In this study, we detected an increase in the concentration of serotonin only in three samples after T2 treatment, while this monoamine neurotransmitter in most of the samples decreased considerably after roasting at 135 ◦C (T3) with respect to unroasted samples (T1); a similar behaviour was observed for histamine in 50% of investigated samples. These results are in contrast with other authors who demonstrated the histamine thermostability during cooking processes [39,40].

It was also observed that the histamine level increased in foods after frying and grilling [41]. However, other authors elucidated the mechanism by which certain cooking ingredients and common organic acids destroy histamine [42], so it could be very interesting to deepen this aspect by considering

the occurrence of bio-compounds that develop following the roasting process of cocoa and their possible role in the control of the BAs levels in food.

#### *3.4. Anthocyanins, Total Polyphenols and Flavanols Content*

The results on the content of anthocyanins, total polyphenols and flavanols of the eighteen cocoa samples at different process steps (T1, T2 and T3) are reported in Table 4.

After fermentation and drying treatment (T1), the anthocyanin concentration was between 0.17 and 3.36 mg g−<sup>1</sup> DFW, with an average value of 1.02 mg g−<sup>1</sup> DFW; these pigments disappeared during fermentation [11], reaching low values on the sixth day of fermentation, and they are a good parameter to determine the progress or status of the fermentation. The contents found are similar to other cocoa varieties from Colombia [43], but inferior to those found in other studies conducted on Ghana cocoa varieties [44]. In unroasted samples (T1), the average content of total polyphenols was 45.50 mg GAE g−<sup>1</sup> DFW, values that are similar to other cocoa varieties planted in Colombia [43,45], with the only exception being sample 4 which presented higher contents (over 80 mg GAE g−<sup>1</sup> DFW). These are more similar to the values found in other studies carried out on varieties planted in Ghana, as well as in other varieties planted in Colombia [44,46]. It is important to point out that these results may have been affected by the fact that each single phenol shows a different response to the Folin-Ciocalteau reagent [14].

According to Carrillo et al. [45], the cocoa-producing region can have a significant effect on the total polyphenol content, as a proportional relationship was found between polyphenols content and altitude of plant crops. Their results suggest that plants grown at lower altitude accumulate more polyphenols compared to plants grown at higher altitude. In the present study, the TPC determined for sample 16 (from geographical area at 30 m.a.s.l) was lower than other cocoa samples so it seems that the theory proposed by Carillo et al. is not confirmed by our data, although this aspect would be worth investigating with a large number of samples.

Roasting did not cause a statistically significant decrease in anthocyanin content in samples from all the three regions, with the following exception: a decrease of 50%–60% was observed in roasted cocoa beans in the sample of the Narino region (sample 16) due to its highest values at the end of fermentation. The decrease in anthocyanin content is in accordance with data observed by other authors [12,43] for different roasting temperatures. Regarding the TPC, a not statistically significant increase was found from 45.50 mg GAE g−<sup>1</sup> DFW for T1 to 55.26 mg GAE g−<sup>1</sup> DFW (+21%) for T2 and 62.01 mg GAE g−<sup>1</sup> DFW (+14%) for T3. However, an increase in TPC values after the roasting process is consistent with the data reported by Ioannone et al. [13]; these authors suggested that an increase in TPC is dependent on temperature and exposure time, as a series of condensation and polymerization reactions occur with the formation of complex molecules such as pro-anthocyanidins from lower molecular weight compounds such as phenols and anthocyanins. Additionally, through Maillard reactions, melanoidins can be formed from reducing sugars and free amino acids; as a consequence, melanoidins can have reducing properties that affect the response to the Folin-Ciocalteu reagent, thus causing an overestimation of the TPC values [14].

The occurrence of flavanols before and after roasting was also investigated in Criollo cocoa samples and the results are reported in Table 3. Moreover, Table S1 shows the epicatechin to catechin ratio (epi/cat) for both unroasted and roasted samples. Catechin was found in all unroasted samples (ranging from n.d. to 4.43 ± 0.13) with the exception of the samples 1, 5 and 9. Epicatechin was detected in all the samples with a maximum value of 5.7 <sup>±</sup> 0.17 mg g−<sup>1</sup> (in sample 12) and a minimum of 0.45 <sup>±</sup> 0.01 mg g−<sup>1</sup> (in sample 11). Similar catechin contents were found by Loureiro et al. in dried cocoa beans from Latin America [47].



\*\**p* < 0.01; \*\*\* *p* < 0.001; n.s. not significant.

#### *Foods* **2020**, *9*, 520

The epi/cat ratio is a widely used index as it may be associated with the degree of cocoa processing [48,49] (Table S1). Generally, with the increase of temperature the epi/cat ratio tends to decrease due to isomerization reactions and the faster degradation of epicatechin with respect to catechin [50]. The major flavanol present in unroasted samples was (−)-epicatechin. According to Hurst et al. [51], the high temperatures may induce the epimerization of this flavanol to (–)-catechin, and (+)-catechin to (+)-epicatechin. This behaviour was noticed in many samples, even though in other cases the opposite was observed. Moreover, in many cases the ratio could not be calculated since either catechin or epicatechin was not detected. Finally, it can be said that both roasting conditions had a similar effect on flavanols.

#### *3.5. Trolox Equivalent Antioxidant Capacity (TEAC) and the Ferric Reducing Antioxidant Power (FRAP) Assays*

Table 5 shows the effect of the different roasting treatments on the radical scavenging activity (TEAC) and the reducing activity (FRAP) assays with respect to unroasted cocoa bean samples. The Pearson correlation coefficient was calculated: a strong correlation was found between TPC content and TEAC (ß = 0.88, *p* < 0.05) and between TPC content and FRAP (ß = 0.92, *p* < 0.05).

**Table 5.** Results of Trolox Equivalent Antioxidant Capacity (TEAC) and the Ferric Reducing Antioxidant Power (FRAP) assays on the Criollo cocoa samples after fermentation and drying (T1) and after roasting (T2, 120 ◦C for 22 min; T3, 135 ◦C for 15 min). The data are expressed as mean of triplicate analysis.


Legend: data followed by different superscript letters, in the same line, are significantly different (LSD test, *p* < 0.05); asterisks indicate significance at \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001; n.s. not significant.

Regarding the reducing capacity as evaluated by FRAP assay (Table 5), a mean value of 395 μmol Fe<sup>2</sup>+/g was determined in unroasted samples, which is in agreement with other authors [4,52].

The trend of values of FRAP was similar to those obtained in the ABTS assay. Moreover, in roasted samples results of TEAC and FRAP were comparable with values found by Ioannone et al. [13]. Generally, the antioxidant activity was higher in roasted samples compared with unroasted ones, with the exception of some cases (samples 3, 4, 7, 11, 14 and 18).

The improvement in the antioxidant and reducing properties after the roasting process may be related to the formation of reducing molecules, not quantified in the present work, as well as to the occurrence of condensation reactions among polyphenols, as evidenced by the results reported in Table 4.

#### *3.6. Principal Component Analysis*

A principal component analysis (PCA) was performed to highlight how factors or variables can influence the changes in the BA level in raw cocoa beans after roasting. Figure 2 shows the distribution of the variables analysed in the two first principal components which represent 90.1% of data variance. Usually, the two first principal components are sufficient to explain the maximum variation in all data [31]. PC1 and PC2 explained 84.9% and 5.2% of date variance related to the BA content of cacao. In order to better describe the data set, the following results and information were included: microbial counts, polyphenols (TPC), anthocyanins, antioxidant activity (FRAP and TEAC), flavonols (catechin and epicatechin), BA content at different processing conditions (T1, T2, and T3) and origin of the samples.

**Figure 2.** Principal component analysis related to the content of biogenic amines in Colombian criollo cocoa samples. Legend: empty square, T1 (raw cocoa beans); gray triangle, T2 (120 ◦C for 22 min)**;** empty circle, T3 (135 ◦C for 15 min).

Concerning PC1, antioxidant activity (FRAP and TEAC) (−1.23 to −1.01), polyphenol content (TPC), and BA (−0.72 to −0.56) showed a negative influence on this component, while FRAP, TEAC, and TPC showed a significant increase in concentration in the same way as BA during roasting conditions. On the other side, anthocyanins, catechin, and epicatechin (0.43 to 0.55) showed a positive influence on this component. The anthocyanin content is a good parameter to determine if fermentation is carried out properly since they decrease as the fermentation progresses; therefore, the correlation found between a high anthocyanin content in raw cocoa (T1) and a high BA content may be related to a non-ideal fermentation process in which, for different reasons, the enzymatic activity of the grains remained active, generating metabolic intermediates such as BAs.

As for the individual BAs, a positive influence was found for DOP (0.51) under initial conditions (T1) after treatment at 120 ◦C (T2) for PUT, CAD, and SPD (0.49 to 0.54), and at 135 ◦C (T3) for CAD, SPD, SER, HIS, and SPM (0.44 to 0.56), while the other BAs showed no influence on this component.

The variables pH (0.1), region (0.41), shell microbiota (−0.26), and bean microbiota (−0.12) showed a weak correlation with each other. The pH is important to select the type of microorganisms that can grow and therefore quantity, and on the type of BAs they can generate [53]; in the present study, the values found for pH were not low enough to inhibit Enterobacteriaceae, which is one of the main groups that can produce BAs [31]. Moreover, pH values were in the optimal range that can favour BAs accumulation. The synthesis of polyamines, such as spermine and spermidine, occurs in response to high pH environments; these BAs act as inhibitors of carbonic anhydrase enzymes that catalyse the interconversion of carbon dioxide and water into bicarbonate and protons and vice versa [54].

According to Lima et al. [29], average levels of microorganisms are lower in the beans compared to those found in the shell due to the lower availability of nutrients, which can cause the activation of metabolic pathways in some groups of microorganisms that can lead to the accumulation of decarboxylation products such as BAs; however, no influence was observed in this component. Regarding the origin, the difference between cultivars, different growth, and postharvest conditions may be related to the presence of these BAs, but no influence in this component was established between the different sites where the samples were taken.

Concerning PC2, this component was mainly influenced by roasting. On the positive axis, the characteristics of the beans without heat treatment (T1) were located predominantly, differing from the samples T2 and T3 that were located on the negative axis of the component. Although a statistically significant difference was found in the content of BAs at T1, T2 and T3 in most of the samples, in the PC2 component only a small correlation was evident among them.

#### **4. Conclusions**

The present study aimed to evaluate the accumulation of bioactive compounds in eighteen Criollo cocoa beans samples from Colombia, with a special focus on biogenic amines and polyphenols, after fermentation and drying and after two different roasting processes commonly used in cocoa factories.

The samples showed a similar BAs profile, with a variability in their concentration as a consequence of both cocoa beans and shell microbiota, as well as differences among cultivars, growth conditions and fermentation and drying treatments. High temperature seems to correlate with the occurrence of TYR, PHE, ETH and PUT; moreover, the roasting process significantly increased the concentration of DOP and SPM, whilst CAD and SPD levels generally decreased. The total phenolic content was positively affected by the roasting processes; even without a statistically significant difference a remarkable improvement in the antioxidant and reducing properties were observed, showing an enhancement of their functionality.

No direct relationship was found between the content of polyphenols and the content of BAs in cocoa beans, even if it can be speculated that polyamines could have a role by influencing the antioxidant activity or exhibiting pro-oxidant properties in cocoa beans. Therefore, the correlation found between a high anthocyanin content and a high BAs content in unroasted cocoa samples (T1) could be attributable to a non-ideal fermentation process. One important result that it is worth pointing out is that the quantities of BAs found in the unroasted cocoa beans were not alarming, especially with regard to HIS and TYR, the amines of toxicological interest.

Moreover, low BAs amounts were also found in roasted samples, which is of crucial importance considering that such values that were calculated for defatted samples will be further processed and used as ingredients in complex formulations.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2304-8158/9/4/520/s1, Table S1: The ratio of epicatechin to catechin (epi/cat) in cocoa bean samples, fermentation and drying (T1) and after roasting (T2 and T3).

**Author Contributions:** Conceptualization, C.C.-L., M.M., C.D.D.M. and J.D.O.; methodology, M.M., C.D.D.M. and C.C.-L.; software, M.M. and J.D.-O.; formal analysis, J.D.-O., C.D.D.M., M.M.; investigation, J.D.-O., C.D.D.M., and M.M.; resources, J.D.-O., C.D.D.M. and M.M.; data curation, M.M.; writing—original draft preparation, J.D.-O., M.M.; writing—review and editing, M.M. and C.D.D.M.; visualization, M.M. and C.C.-L.; supervision, D.M., A.P.; project administration, C.C.-L.; funding acquisition, J.D.-O. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by "Colciencias, Patrimonio Autónomo Fondo Nacional de Financiamiento para la Ciencia, la Tecnología y la Innovación Francisco José de Caldas" (C. 808-2018. Agreement 240-2019. Number 123280864259).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **E**ff**ects of Soaking and Fermentation Time on Biogenic Amines Content of** *Maesil* **(***Prunus Mume***) Extract**

## **So Hee Yoon 1, Eunmi Koh 2, Bogyoung Choi <sup>2</sup> and BoKyung Moon 1,\***


Received: 30 October 2019; Accepted: 15 November 2019; Published: 19 November 2019

**Abstract:** *Maesil* extract, a fruit-juice concentrate derived from *Prunus mume* prepared by fermenting with sugar, is widely used with increasing popularity in Korea. Biogenic amines in *maesil* extract were extracted with 0.4 M perchloric acid, derivatized with dansyl chloride, and detected using high-performance liquid chromatography. Among 18 home-made *maesil* extracts collected from different regions, total biogenic amine content varied from 2.53 to 241.73 mg/L. To elucidate the effects of soaking and fermentation time on biogenic amine content in *maesil* extract, *maesil* was soaked in brown sugar for 90 days and the liquid obtained was further fermented for 180 days at 15 and 25 ◦C, respectively. The main biogenic amines extracted were putrescine and spermidine and the total biogenic amine content was higher at 25 ◦C than at 15 ◦C. Soaking at 15 and 25 ◦C increased the total biogenic amines content from 14.14 to 34.98 mg/L and 37.33 to 69.05 mg/L, respectively, whereas a 180 day fermentation decreased the content from 31.66 to 13.59 mg/L and 116.82 to 57.05 mg/L, respectively. Biogenic amine content was correlated with total amino acid content (particularly, arginine content). Based on these results, we have considered that biogenic amine synthesis can be reduced during *maesil* extract production by controlling temperature and fermentation time.

**Keywords:** biogenic amine; *maesil*; amino acids; soaking; fermentation; temperature

#### **1. Introduction**

*Maesil* (*Prunus mume*) known as Japanese *Ume* has been used not only as a food but also as a medicine on account of its various functionalities [1–3]. As the seed of *maesil* has a toxic substance called amygdalin [4], *maesil* has been processed into various products such as alcoholic beverage, juice, pickle or extract rather than eaten raw [5]. *Maesil* extract is a fruit-juice concentrate produced by the fermentation of *maesil* and sugar. Recently, it has been increasingly used as a seasoning to impart sweetness and a unique flavor to foods [5–8]. Traditionally, *maesil* extract is soaked for a long period (90 days) at room temperature and fermented naturally under different conditions in individual households. Therefore, uncontrolled fermentation can lead to the formation of biogenic amines, which are produced by molds and bacteria.

As biogenic amines are mainly produced by the microbial decarboxylation of free amino acids, they are easily found in fermented foods [9,10]. These biogenic amines have been reported to be abundant and they have been found in a wide range of food products, including fish products, soy sauce, *Chunjang* (traditional fermented soybean paste in Korea and China), and agricultural products [11–15]. As a high intake of biogenic amines can cause various detrimental effects such as migraine and gastrointestinal problems, their ingestion needs to be restricted [16,17]. Indeed, their content is currently regulated in certain food products. For example, the histamine content in fish products is regulated by the US Food and Drug Administration (FDA, 50 mg/kg) and the European

Union (100 mg/kg) in fish products [18]. The formation of biogenic amines is influenced by microbial flora and their growth as well as the fermentation conditions used in the production of fermented foods [19,20]. To date, however, studies on the changes in biogenic amines during fruit fermentation have mainly focused on wine [9]. Moreover, little research has been conducted on the fermentation of biogenic amines during the fermentation of other fruits.

Therefore, in this study, we tried to monitor the biogenic amine content of *maesil* extracts and determine the effect of fermentation conditions on the changes in biogenic amines in *maesil* extracts during fermentation. For this purpose, we (i) determined the content of biogenic amines content in 18 home-made *maesil* extracts collected from different households in Korea and (ii) monitored the content of biogenic amines during the fermentation of *maesil* extracts at two different temperatures, 15 and 25 ◦C, over a period of 9 months.

#### **2. Materials and Methods**

#### *2.1. Chemicals*

Biogenic amine standards (histamine dihydrochloride (HIS), tryptamine hydrochloride (TRP), 2-phenylethylamine (2-PHE), putrescine dihydrochloride (PUT), cadaverine dihydrochloride (CAD), tyramine hydrochloride (TYR), spermidine trihydrochloride (SPD), and spermine tetrahydrochloride (SPM)) and dansyl chloride were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Perchloric acid, 25% ammonium hydroxide solution, sodium hydroxide sodium hydrogen carbonate, and diethyl ether were acquired from Daejung Chemical Co. (Siheung, Korea). Acetone and acetonitrile (High-performance liquid chromatography (HPLC) grade) were purchased from Tedia Co. (Fairfield, OH, USA). Compound mixtures of amino acids, borate buffer, *o*-phthalaldehyde (OPA) and 9-fluorenylmethoxycarbonyl chloride (FMOC-Cl) were obtained from Agilent Technologies (Andover, MA, USA).

#### *2.2. Preparation of Food Samples*

During the period from 2010 to 2014, we collected samples 18 *maesil* extracts from different households in Korea for analysis of biogenic amines content. We also prepared our own *maesil* extract, following the method of Choi and Koh (2016) [5], and the process of preparation is shown in Figure 1. *Maesil* fruits obtained from a local market were washed with pure water, and drained at room temperature (23 ± 1 ◦C). To the 400 g of *maesil*, we added 400 g of brown sugar and the mixture was then placed in 1 L clear plastic jars, which were maintained in incubators set at 15 ◦C and 25 ◦C, respectively. After the *maesil* fruits were taken out from the jar after 90 days of soaking, the obtained liquid (490 mL at 15 ◦C and 476 mL at 25 ◦C) was further fermented for the next 180 days in the same jar. Biogenic amines were analyzed at 30, 45, 75, and 90 days of soaking period and 30, 60, 120, 150, and 180 days of fermentation.

**Figure 1.** The fermentation process used for producing *maesil* extract.888.

#### *2.3. pH Measurement*

To measure the pH, *maesil* extract (10 g) was mixed with 10 mL deionized water for 3 min and then filtered through Whatman paper No.2 filter paper (Advantec, Tokyo, Japan). The pH was measured using a pH meter (Beckman Coulter, FL, USA) following the method of Shukla et al. [21].

#### *2.4. Amino Acids Analysis*

Amino acids in the *maesil* extract were analyzed using an HPLC system (Dionex Ultimate 3000, Thermo Fisher Scientific, Waltham, MA, USA), equipped with a 1260 Infinity fluorescence detector (Agilent Technologies, Waldbronn, Germany), following the method described by Jajic et al. (2013) [22] with slight modifications. The samples were derivatized with OPA and FMOC via a programmed autosampler. After derivatization, samples (0.5 μL) were injected into an Inno-C18 column (4.6 × 50 mm, 5 μm, Youngin Biochrom, Korea) at 40 ◦C. The fluorescence was detected at excitation and emission wavelengths of 340 and 450 mm, respectively for OPA, and at 266 and 305 nm, respectively, for FMOC. Primary and secondary amino acids were analyzed based on the OPA and FMOC derivatives, respectively. The mobile phase solvent A was 40 mM sodium phosphate (pH 7), and solvent B was a 10:45:45 (*v*/*v*) mixture of distilled water, acetonitrile, and methanol. The gradient program was run at a flow rate of 1.0 mL/min as follows: 5% B for 3 min; followed by elution with 5% to 55% B in 24 min; 55% to 90% B in 25 min; maintained 90% of B for next 6 min; and 90% to 5% B for 3.5 min, maintained for 0.5 min.

#### *2.5. Biogenic Amine Analysis*

#### 2.5.1. Extraction of Biogenic Amines

Biogenic amines were extracted from the *maesil* extract using the method of Shukla et al. (2014) [21] with slight modifications. Briefly, 10 mL of 0.4 M perchloric acid solution was mixed with 5 g *maesil* extract, homogenized for 3 min, and then centrifuged at 3000× *g* for 10 min at 4 ◦C. The residue was re-extracted with 0.4 M perchloric acid solution (10 mL). After the supernatants were combined and 0.4 M perchloric acid solution was added to adjust the final volume to 50 mL. After filtering through Whatman filter paper No.1 (11 μm, Adventec, Tokyo, Japan), 1 mL of the extract was used for derivatization with dansyl chloride.

#### 2.5.2. Derivatization of Biogenic Amines

Biogenic amines were derivatized following the methods described by Shukla et al. (2010) [23] and Frias et al. (2007) [24]. An extract sample (1 mL) or standard solution mixture (1 mL) was mixed with 200 μL 2 M sodium hydroxide; next, 300 μL of sodium hydrogen carbonate solution was added to saturate the solution. To the mixture, 1 mL of a dansyl chloride solution (10 mg/mL in acetone) was added and kept for 45 min at 40 ◦C. To stop the reaction, 100 μL of 25% ammonium hydroxide was added to the mixture and reacted for 30 min at 25 ◦C. Then, the derivatized biogenic amines were extracted twice with 1 mL of diethyl ether. Subsequent to drying in a nitrogen stream, the extract was redissolved in acetonitrile (1 mL) and filtered through a 0.22 μm polyvinylidene fluoride (PVDF) filter (Millipore Co., Bedford, MA, USA) for injection into the HPLC system.

#### 2.5.3. HPLC Analysis of Biogenic Amines

Biogenic amines were analyzed using an HPLC system consisting of an Alliance 2695 separations module (Waters, Milford, MA, USA) and Ultra violet (UV)/Visible detector 2487 (Waters, Milford, MA, USA) with a Capcell Pak C18 column (4.6 × 250 mm i.d., 5 μm; Shiseido, Kyoto, Japan), thermostated at 30 ◦C, and detected at 210 nm [24–26]. The injection volume was 20 μL and the mobile phase consisted of solvent A (water) and B (acetonitrile) run at a flow rate of 0.8 mL/min with the following gradient

elution program for 35 min: 65:35 (A:B, *v*/*v*), followed by 45% B for 5 min, elution with 45% to 65% B in 10.05 min, 65% to 80% B in 17.05 min, 80% to 90% B up to 26.25 min, and 90% to 35% B in 35 min.

#### *2.6. Method Validation*

The HPLC method for biogenic amines analysis was validated for linearity, limits of detection (LOD) and limits of quantification (LOQ), accuracy, and precision [22]. The linearity was evaluated using five concentrations (0.5, 1, 2, 5, and 10 mg/L) of each the biogenic amine standards (PUT, CAD, HIS, TRP, 2-PHE, TYR, SPD, and SPM) by constructing a calibration curve. The LOD and LOQ values were calculated using the following equations: LOD = 3.3 × (standard deviation (SD)/slope of calibration curve) and LOQ = 10 × (SD/slope of calibration curve). The accuracy of the method was verified by triplicate analysis of spiked samples at two different levels (5 and 10 mg/L) and expressed as % recovery. The recoveries were calculated by contrasting the peak area of measured concentration with the peak area of the spiked concentrations. To evaluate the precision, repeatability, inter-day, and intra-day were performed and expressed as the percentage relative standard deviation (RSD) of the peak area measurements. Repeatability was estimated by analysis of six consecutively injected samples. The inter-day precision was determined at two different levels, 5 and 10 mg/L, and the analyses were performed over a period of three consecutive days. The intra-day precision was determined by spiking five blank samples at concentrations levels of 5 and 10 mg/mL and the evaluation was based on the results obtained using the method operating over a single day under the same conditions.

#### *2.7. Statistical Analysis*

Quantitative data are expressed as the means ± SD of at least three measurements. Statistical analysis was performed using a one-way analysis of variance (ANOVA) and Duncan's multiple range test by SAS software, version 8.0 for Windows (SAS Institute, Cary, NC, USA). The probability value of *p* < 0.05 was considered statistically significant.

#### **3. Results and Discussion**

#### *3.1. Method Validation*

The results obtained from the different method validations are presented in Table 1. Standard curves for biogenic amines were constructed from triplicate analyses of five concentrations in the range 0.5–10 mg/L. With the exception of spermine (correlation coefficient (R2) > 0.998), the linearity of the calibration curves for each biogenic amine was >0.999. The precision, expressed as %RSD, of inter-day variation was between 0.17% and 5.20%, and the RSD values for intra-day variation were between 0.07% and 6.46%. The LOD and LOQ of the biogenic amines ranged from 0.01 to 0.20 mg/L and 0.02 to 0.61 mg/L, respectively. The accuracy of the method with regard to recovery was between 89.4% and 110.8%.


**Table 1.** Summary results relating to validation of the high-performance liquid chromatography (HPLC) method used for biogenic amines.

LOD: Limits of detection; LOQ: Limits of quantification; RSD: Relative standard deviation HIS: Histamine; TRP: Tryptamine; PUT: Putrescine; 2-PHE: 2-phenylethylamine; CAD: Cadaverine; TYR: Tyramine; SPM: Spermine; SPD: Spermidine.

#### *3.2. Content of Biogenic Amines in Home-Made Maesil Extract*

Among the 18 home-made maesil extracts analyzed, the total content of biogenic amines ranged from 2.5 to 241.7 mg/L, the major individual biogenic amines were putrescine (not detectable (ND)-80.82 mg/L) and spermidine (ND-219.20 mg/L), followed by tryptamine (Table 2). Putrescine, histamine, tyramine, cadaverine, 2-phenylethylamine, spermidine, spermine, agmatine, and tryptamine are the main biogenic amines in wine [27]. Among these amines, putrescine has been reported to be generated from the raw material or by microbial decarboxylation [28]. In the case of wine, putrescine content has been found to be influenced by geographical region and grape variety [29]. Histamine and spermine detected in wine [27,29,30] are known to have toxicity or play a role in enhancing toxicity [11,31]. However, we were unable to detect either of these two amines in the 18 maesil extracts examined in the present study. These results imply that the amount and composition of biogenic amines may differ widely among different fruit-derived products and that these differences could be attributed to differences in manufacturing practice and fruit material.


**Table 2.**Biogenic amines content (mg/L) in 18 home-made*maesil*extracts prepared in individual households.

The content of biogenic amines is known to be affected by fermentation conditions, including temperature, microorganisms, and the synthetic pathways of the biogenic amine formation [32–35]. In wine, cadaverine, histamine, putrescine, and tyramine are mainly detected, the content of which can vary depending on fermentation factors, storage, microbial decarboxylase activity, and vinification [27,30,36]. Marcobal et al. (2006) have reported that the content of biogenic amines in wine ranged from ND to 54.02 mg/L [31]. Garai et al. (2006) found that the main biogenic amine in commercial apple ciders was putrescine and that the total biogenic amine content ranged from ND to 23.26 mg/L [12]. In comparison, the results of this study indicate that the biogenic amine content in home-made *maesil* extracts is considerably higher than that reported in wine or apple ciders [12,27,30], thereby emphasizing the necessity to control biogenic amines productions during the fermentation of *maesil* extract.

#### *3.3. Content of Biogenic Amines During Soaking and Fermentation*

During the 90 day soaking of *maesil* examined in the present study, we found that the total biogenic amines content increased from 14.1 to 35.0 mg/L and 37.3 to 69.1 mg/L at 15 and 25 ◦C, respectively, indicating that the content was higher at the latter temperature throughout the soaking period (Figure 1a). Previous studies have reported that biogenic amines are generated via the catalytic activity of decarboxylase enzymes produced during the growth of microorganisms such as lactic acid bacteria [37], and thus, the increase in biogenic amines during the soaking period might be caused by microbial decarboxylase activity. At both incubation temperatures we assessed, the predominant biogenic amines detected in *maesil* extract were putrescine and spermidine (Figure 2b,c), and the latter comprised approximately 80% of the total biogenic amines.

**Figure 2.** The content (mg/L) of total biogenic amines (**a**), spermidine (**b**), and putrescine (**c**) during soaking at 25 ◦C ( ) and 15 ◦C ( ).

After removing the *maesil* fruit from the sample jars at the end of the soaking period, the residual liquid was subsequently fermented for 180 days, during which, the content of biogenic amines decreased from 31.7 to 13.6 mg/L and 116.8 to 57.1 mg/L at 15 and 25 ◦C, respectively (Table 3). Generally, the extracts fermented at 25 ◦C exhibited biogenic amines content that was twice as high as that obtained at 15 ◦C. Moreover, at the end of the fermentation period, the total biogenic amines content at 15 ◦C was 23.8% of that at 25 ◦C. In addition to putrescine and spermidine, tryptamine was also detected at 0.33 mg/L when *maesil* was fermented at the higher temperature for 30 days. In this regard, Chong et al. (2011) have reported that temperature was the most important factor affecting biogenic amines formation [38], and Pinho et al. (2001) reported a higher increase in biogenic amines at a storage temperature of 21 ◦C than at 4 ◦C [35]. In addition, Kim et al. (2002) found that 25 ◦C was the optimum temperature for histamine production in fish muscles [39]. *Maesil* extract is typically produced by natural fermentation without controlling the temperature or starter culture. Moreover, it is sometimes consumed immediately after a 90 day soaking without subsequent fermentation. On the basis of the results obtained in this study, we recommend that, to yield a product with lower levels of biogenic amines, *maesil* extract should be fermented at a relatively low temperature and for a long period of time.

#### *3.4. E*ff*ects of Processing Factors on Biogenic Amines Formation*

The pathways implicated in the synthesis of biogenic amines can vary depending on the temperature, sugar content, precursors, and microorganisms involved in the fermentation of various food items [16,32,40]. Generally, putrescine is derived from the decarboxylation of arginine and ornithine or is already present in raw materials [28,32], whereas, spermidine is produced from arginine and ornithine or is converted from putrescine by spermidine synthase [16,31]. Poveda (2019) and Bardocz (1995) reported that most putrescine is either converted to spermidine or spermine, or is catabolized to succinate and other amino acids via succinate [28,31].

In the present study, to determine the effects of the factors influencing biogenic amines formation, we performed Pearson's correlation. Among these factors, we detected no significant correlation between biogenic amines content and pH, which had a narrow range (pH 2.9–3.3) during the fermentation.

Arena et al. (2008) reported a negative correlation between biogenic amines and sugar concentration and found that; the additions of glucose and fructose at 5 and 20 g/L reduced biogenic amines production by 82%–93% and 61%–99%, respectively [40]. Cid et al. (2008) reported that lower glucose concentration is associated with a high activity of ornithine-decarboxylase produced by *Lactobacillus* [41]. In our study, we found that, although there was a negative correlation between sugar content and biogenic amines content (Figure 3a), the relationship was not significant, which could be attributable to the narrow range of sugar content (61 to 81 ◦Brix) during fermentation.



**Table3.**Biogenicaminescontent(mg/L)duringfermentationatdifferenttemperatures.

161

**Figure 3.** Correlations between sugar content and biogenic amine content (mg/L) (**a**); pH and biogenic amine content (mg/L) (**b**); the content (mg/L) of total amino acid and total biogenic amines (**c**); and the content (mg/L) of biogenic amines and arginine (**d**).

The total biogenic amines content showed a positive correlation with the total amounts of amino acids (R = 0.6581, *p* < 0.05), which could be explained by the fact that amino acids are precursors of biogenic amines [42]. We also detected a strong positive correlation between the amounts of putrescine and spermidine (R = 0.9277, *p* < 0.01; data not shown), consistent with the findings of Bardocz (1995) [31] and Nuriez et al. (2016) [16], which indicates that putrescine is a precursor of spermidine. However, apart from a positive correlation between arginine and total biogenic amines content (R = 0.6910, *p* < 0.05), we detected no correlation between individual biogenic amines and their respective precursor amino acid, which is consistent with the findings reported by Soufleros et al. (1998) [43]. Gezginc et al. (2013) reported that arginine serves as a precursor of putrescine, which can, in turn, be converted to spermidine [32]. Furthermore, it has been found that, in plants and some microorganisms, there are alternative pathways in which putrescine is generated from arginine via agmatine [33]. These results indicate that the fermentation of *maesil* extract at low temperature could reduce the production of biogenic amines. In addition, biogenic amine formation in *maesil* extract could be affected by the origin of *maesil*, the number of amino acids as well as the content of biogenic amine precursors.

#### **4. Conclusions**

The present study was conducted to evaluate the changes in biogenic amines formation and the relationship between biogenic amines and amino acids in *maesil* extract during the fermentation of this product. Although the consumption of *maesil* extract is currently increasing, there has, to date, been a lack of studies on the changes that biogenic amines undergo during *maesil* extract fermentation. The results of this study showed that the biogenic amines content in *maesil* extract is affected by both the inherent amino acids content and fermentation temperature and time. Moreover, the content of some biogenic amines may also be affected by the presence of other biogenic amines. We found that

#### *Foods* **2019**, *8*, 592

both amino acids and biogenic amines content was lower during fermentation at 15 ◦C than at 25 ◦C and decreased with increasing fermentation time. Accordingly, these observations indicate that employing protracted low-temperature fermentations could be an effective approach for reducing the production of biogenic amines in *maesil* extract. In further research, it will be necessary to study the types of microorganisms and formation on biogenic amines in *maesil* extract.

**Author Contributions:** Conceptualization, S.H.Y. and B.C.; methodology, S.H.Y. and B.C.; validation, S.H.Y., B.C., and E.K.; formal analysis, S.H.Y., B.C., and E.K.; investigation, S.H.Y. and B.C.; resources, B.M.; data curation, S.H.Y. and B.C.; writing—original draft preparation, S.H.Y. and B.C.; writing—review and editing, B.M.; supervision, B.M.; project administration, B.M.

**Funding:** This research received no external funding.

**Acknowledgments:** This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## **Casing Contribution to Proteolytic Changes and Biogenic Amines Content in the Production of an Artisanal Naturally Fermented Dry Sausage**

#### **Annalisa Serio, Jessica Laika, Francesca Maggio, Giampiero Sacchetti, Flavio D'Alessandro, Chiara Rossi, Maria Martuscelli, Clemencia Chaves-López \* and Antonello Paparella**

Faculty of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, via Balzarini 1, 64100 Teramo, Italy; aserio@unite.it (A.S.); jessica.laika@hotmail.com (J.L.); fmaggio@unite.it (F.M.); gsacchetti@unite.it (G.S.); flavio\_dale@live.it (F.D.); crossi@unite.it (C.R.); mmartuscelli@unite.it (M.M.); apaparella@unite.it (A.P.)

**\*** Correspondence: cchaveslopez@unite.it

Received: 26 August 2020; Accepted: 8 September 2020; Published: 13 September 2020

**Abstract:** The effect of two kinds of casings on the production and characteristics of a dry fermented sausage was investigated. In detail, an Italian product, naturally fermented at low temperatures and normally wrapped in beef casing instead of the most diffused hog one, was selected. Two different productions (one traditionally in beef casing (MCB) and another in hog casing (MCH)) were investigated over time to determine the differences particularly regarding proteolytic changes during fermentation and ripening. First of all, the product in hog casing required a longer ripening time, up to 120 days, instead of 45–50 days, because of the lower drying rate, while the microbial dynamics were not significantly modified. Conversely, the proteolysis showed a different evolution, being more pronounced, together with the biogenic amines content up to 341 mg/Kg instead of 265 mg/Kg for the traditional products. The latter products were instead characterized by higher quantities of total free amino acids, 3-methyl butanoic acid, 3-Methyl-1-butanal, and 2-Methylpropanal, enriching the final taste and aroma. The traditional product MCB also showed lower hardness and chewiness than MCH. The results highlight how the choice of casing has a relevant impact on the development of the final characteristics of fermented sausages.

**Keywords:** proteolysis; dry fermented sausage; casing; biogenic amines; volatile compounds; texture; low temperature

#### **1. Introduction**

The characteristics of dry fermented sausages depend on many factors, including the ingredients, the recipe, the microbiota composition, and the different production steps. These factors, combined together, determine the variety of products widespread in different European countries [1]. Many fermented meats are produced in Italy; among them, a very particular one is produced during autumn and winter in the Abruzzo Region (Central Italy), and is called "*Mortadella di Campotosto*". It is made of lean pork meat mixed with salt, pepper, and nitrite and it contains a bar of lard, previously cured with salt and spices for about two weeks. It shows a characteristic sub-ovoid shape and length about 15–20 cm, with a diameter of 8–10 cm. Among the typical characteristics, this product is handmade and wrapped (instead of being stuffed) into a natural beef casing; afterwards, it is fermented at low temperature without any starter addition. Traditionally, the product is exposed to dry cold northern winds that provide the ideal conditions for product ripening, which lasts about 40–50 days, with temperatures varying from −1 ◦C to about 10 ◦C.

As for this product, the traditional casing is made of beef middles, we decided to evaluate the effect of both the traditional casing and a more usual hog casing on the characteristics of the sausage.

In fact, although "*Mortadella di Campotosto*" sausage manufacturing does not have any particular specifications about the natural casing to use, most of the producers use beef casing, but some may use hog casing because of its larger availability.

As regards fermented sausages, besides containing the meat batter, the casing exerts mechanical protection and guarantees permeability, which is the basis of the exchange of water and oxygen necessary for adequate and homogeneous drying and ripening [2]. Many features affect the quality of natural casings, such as the portion of the intestinal tract used, the manipulation of the product [3] and the mechanical and physical characteristics such as casing elasticity, permeability to water and gases, diameter and its uniformity, adhesion, and resistance to temperature variations [2]. In addition, casing can be a relevant source of enzymes, while casing microbiota is important overall when starter cultures are not added for fermentation [4].

In light of these considerations, we hypothesized that different casings could affect ripening by modifying sausage microbiota, water diffusion, and proteolysis dynamics, with a potentially significant impact on the product quality. Therefore, in this research, we aimed to deepen the knowledge on the influence of the type of casing on the characteristics of "*Mortadella di Campotosto*"-like sausages and to highlight the effect of casing on specific quality traits of the product. For these reasons, the specific objectives were: to follow the evolution of the principal microbial groups during the production process; to evaluate the protein hydrolysis of the fermented meats soon after stuffing and during ripening; to evaluate the texture and flavor of the finished product; and to follow the production of biogenic amines during the process.

#### **2. Materials and Methods**

#### *2.1. Samples Production*

"*Mortadella di Campotosto*"-like sausages were manufactured in autumn–winter in Macelleria "L'Olmo", sited in Scanno (AQ), Italy. The sausages were produced according to the traditional formulation and procedure: pork lean meat (ham, shoulder, and loin) and bacon were first minced, then mixed with salt (20 g/Kg), black pepper (1.0 g/Kg), and a mixture of sucrose, glucose, ascorbic acid (E300), and potassium nitrite (E252) in a total quantity of 4.0 g/Kg. After 12 h at 4 ◦C, the meat mix was then minced again, and about 420 g was taken for each sausage. A stripe of back lard was previously cured at 4 ◦C with salt, spices (pepper, oregano, rosemary, pimento, juniper, and cilantro), and a mixture of sucrose, glucose, ascorbic acid, and potassium nitrite, for about 14 days, after which it was cut into portions of about 3 × 3 × 10 cm that were inserted into the batter. Then, the product was shaped in its typical oval form. Successively, the meat balls were hand-wrapped with the natural casing, previously washed with water and vinegar, then air-dried for about 10 h. Then, the products were left in air for 3–4 h, to favor the sealing of the casing edges; afterward, they were linked with a cotton string of medium caliber and tied in pairs. Thirty-one samples for each batch were used. The size of the samples at T0 was 17 ± 2 cm in length × 9 ± 2 cm in width and the weight was 452 ± 18 g. At the end of ripening, the size was about 10 ± 1 cm in length × 6 ± 1 cm in width and about 20 ± 1.5 in diameter and a final weight of about 270 ± 10 g.

Two different batches were produced: batch A, named MCB, wrapped with beef casing (beef middles)**,** and batch B, named MCH, wrapped with hog casing (hog bung). All samples were transferred to a fermentation room, in which the temperature varied from −2 and 5 ◦C for 5 days. The samples were then moved to a drying–ripening room, where they stayed up to 120 days. In the room, natural ventilation was favored, with variable relative humidity (depending on the external weather conditions) and temperatures below 12 ◦C.

#### *2.2. Microbiological Analysis*

Microbiological analyses were carried out on the batter section, excluding the lard, after casing removal. Ten grams of the sausages were homogenized with 90 mL of sterile 0.1% (*w*/*v*) peptone water for 2 min, using sterile plastic pouches in a Stomacher Lab-Blender 400 (PBI International Milan, Italy). Serial 10-fold dilutions were prepared in sterile peptone water solution and inoculated in duplicate in appropriate culture media. The following microbial groups were determined: aerobic mesophilic bacteria on Plate Count Agar (PCA) at 30 ◦C for 48 h; mesophilic lactobacilli (LAB) and cocci on MRS agar and M17, respectively, at 30 ◦C for 48–72 h under anaerobiosis; presumptive enterococci on Slanetz and Bartley agar (S&B) at 37 ◦C for 48 h; total enterobacteria on Violet Red Bile Glucose Agar (VRBGA) at 37 ◦C for 24 h; micrococci and staphylococci on Mannitol Salt Agar (MSA) at 30 ◦C for 72 h; yeasts on Yeast extract-Peptone-Dextrose agar (YPD), added with 150 ppm of chloramphenicol, at 30 ◦C for 72 h.

Mold development on the casing was evaluated by sampling 10 cm2 of casing and determining mold growth on YPD added with 150 ppm of chloramphenicol. All the culture media were from Oxoid SpA (Rodano, Italy).

#### *2.3. Physical Analyses*

Measurement of water activity (aw) was performed by the Aqualab instrument CX/2 (Series 3, Decagon Devices, Inc., Pullman, WA, USA). Samples (10 g) were randomly obtained from the sausage (batter section). Moisture (g water/100 g sample) was measured by drying a 3 g sample at 100 ◦C to constant weight [5], and the pH values were obtained using a MP 220 pH meter (Mettler-Toledo, Columbus, OH, USA).

The weight loss of "*Mortadella di Campotosto*"-like samples during drying was gravimetrically determined and calculated as shown in the following Equation (1):

$$\text{Weight loss (\%)} = \text{[(m0 -- mt)/m0]} \times 100\tag{1}$$

where m0 is the weight of sausage obtained after filling and mt is the weight of the sausages after a specific processing time (0, 6, 11, 18, 30, 45, and 120 days).

The measurements of moisture were performed by air oven drying [6], while for the NaCl content, the method of Volhard (ISO 1841-2: 1996) was used [7].

#### *2.4. Chemical Determinations*

Total nitrogen content (TN, % *w*/*w*) was determined by the Kjeldahl method, while proteins were obtained by multiplying TN × 6.25 [8]. Non-protein nitrogen content (NPN, % *w*/*w*) was measured by the precipitation of proteins with trichloroacetic acid, followed by determination of the nitrogen in the extract by the Kjeldahl method. Proteolysis Index (PI, %) was calculated as the ratio between NPN and TN (PI % <sup>=</sup> <sup>100</sup> <sup>×</sup> NPN <sup>×</sup> TN<sup>−</sup>1), as previously reported [9].

To evaluate the intensity of the primary proteolytic changes during the process, sarcoplasmic and myofibrillar proteins were extracted [10]. The protein concentrations were determined using Bradford reagent (Sigma-Aldrich, Milan, Italy) and bovine serum albumin (BSA, Sigma-Aldrich) as standard reference, according to Bradford (1976) [11]. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze proteins [12] by Mini Protean III electrophoresis equipment (Bio-Rad, Segrate, Italy), as previously described [10]. The GS-800™ Calibrated Densitometer (Bio-Rad, Segrate, Italy) was used to quantify the relative abundance of each protein band.

Total amino acids were extracted and measured on 2 g of each sample, using the Cadmium-ninhydrin method [13]. For the extraction of the free amino acids, the method proposed by Berardo and colleagues [14] was followed and concentrations were determined by Reverse-phase high performance liquid chromatography (RP-HPLC), as previously reported [15], by using the Waters AccQ Tag method (Millipore Co-Operative, Milford, MA, USA). Amino acids were converted to stable fluorescent derivatives by reaction with AccQ·Fluor reagent (6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate). RP-HPLC was performed using a Waters liquid chromatography system consisting of a Waters™ 626 pump, Waters™ 600 S controller, and Waters™ 717 S autosampler (Millipore Co-operative, Milford, MA, USA), by means of a Nova-Pak™ C18 column (4 μm, 3.9 × 4.6 mm), heated to 37 ◦C in a Shimadzu model

CTO-10AC column oven. Elution was performed in a gradient of solvent A (Waters AccQ·Tag eluent A), solvent B (acetonitrile: Aldrich Chemical Co., Milan, Italy), and solvent C (20% methanol in Milli-Q water), prepared as follows: initial eluent 100% A; 99% A and 1% B at 0.5 min; 95% A and 5% B at 18 min; 91% A and 9% B at 19 min; 83% A and 17% B at 29.5 min; 60% B and 40% C at 33 min and held under these conditions for 20 min before returning to 100% A. The concentration of A was maintained at 100% up to 65 min, after which the gradient was changed to 60% B and 40% C for 35 min, before returning to the starting conditions. The single amino acids were identified by comparing their retention times with calibration standards. Peak areas were processed using Millennium 32 software v.4.0 (Waters, Milford, MA, USA).

#### *2.5. Determination of Volatile Compounds*

Volatile compounds were determined by solid phase micro-extraction coupled with gas chromatography mass spectrometry (SPME/GC-MS) [16] on 5 g of MCB or MCH at the selected sampling times. Volatile peaks identification was carried out by computer matching of mass spectral data with those of the compounds contained in the Agilent Hewlett-Packard NIST 98 and Wiley v. 6 mass spectral database. The volatile compounds content was expressed as relative percentage area.

#### *2.6. Determination of Biogenic Amines*

The following eight biogenic amines were detected, identified, and quantified: tryptamine (TRP), β-phenylethylamine (β-PHE), putrescine (PUT), cadaverine (CAD), histamine (HIS), tyramine (TYR), spermidine (SPD), and spermine (SPM).

The procedure of amines extraction and derivatization was carried out as described by Martuscelli et al. [17]: an aliquot of 2 g was homogenized (in Stomacher Lab blender 400, International PBI, Milan, Italy) with 10 mL of 5% trichloroacetic acid (TCA) and centrifuged (Hettich Zentrifugen, Tuttlingen, Germany) at a relative centrifugal force of 2325× *g* for 10 min; the supernatant was recovered and the extraction was performed with 5% TCA acid. The two acid extracts were mixed and made up to 50 mL with 5% TCA acid; the final acid extract was filtered through Whatman 54 paper (Carlo Erba, Milan, Italy). For derivatization of the samples, an aliquot of each acid extract (0.5 mL) was mixed with 150 μL of a saturated NaHCO3 solution and the pH was adjusted to 11.5 with about 150 μL NaOH 1.0 M. Dansyl chloride (Fluka, Milan, Italy) solution (2 mL of 10 mg/mL dansyl chloride/acetone) was added to the alkaline amine extract. Derivatized extracts were transferred to an incubator and kept for 60 min at 40 ◦C under agitation (195 stokes) (Dubnoff Bath-BSD/D, International PBI, Milano, Italy). The residual dansyl chloride was removed by adding 200 mL of 300 g/L ammonia solution (Carlo Erba). After 30 min at 20 ± 1 ◦C and protected from light, each sample was brought up to 5 mL with acetonitrile (Carlo Erba) and filtered through a 0.22 μm PTFE filter (Alltech, Sedriano, Italy).

Biogenic amines were determined, after extraction and derivatization, by high-performance liquid chromatography (HPLC) using an Agilent 1200 Series (Agilent Technologies, Milano, Italy). In a Spherisorb S30ODS Waters C18-2 column (3 μm, 150 mm × 4.6 mm ID), 10 μL of sample was injected with gradient elution, acetonitrile (solvent A), and water (solvent B) as follows: 0–1 min 35% B isocratic; 1–5 min, 35%–20% B linear; 5–6 min, 20%–10% linear B; 6–15 min, 10% B isocratic; 15–18 min, 35% linear B; 18–20 min, 35% B isocratic. Identification of the biogenic amines (BAs) was based on their retention times and BAs content was reported as mg/kg of product.

#### *2.7. Texture Analysis*

Textural properties were evaluated at room temperature (22 ± 2 ◦C) using an Instron Universal Testing Machine (mod. 5422, Instron LTD, Wycombe, UK) equipped with a 500 N load cell.

Slices (1 cm thick) were transversally cut from the central part of the sausage. Cubic samples (1 × 1 × 1 cm) were cut from the inner part of the slices placed between the lard and the casing. Samples were compressed by a plunger with a plane circular surface (35 mm diameter) using a crosshead speed of 0.5 mm/s. Two different tests were carried out for the textural characterization:


Each test was carried out on 10 samples of each batch (MCB and MCH). Since, in both tests, the experimental conditions in the first compression stage were the same, 20 samples were used for hardness calculation.

#### *2.8. Experimental Design and Statistical Analysis*

Two batches of sausages characterized by different casings were analyzed over time. In detail, three sausages per batch were randomly taken and analyzed at each ripening time (0, 6, 11, 18, 30, and 45 days). Time zero was considered as the time in which the batter was just wrapped in the casings, thus samples of batter (which was the same for the two batches) were taken just before being wrapped. All the data were subjected to two-way analysis of variance (ANOVA) to test the significance of individual (casing, ripening time) and interactive (casing × ripening time) effect. The model used for the two-way ANOVA is presented in Equation (2):

$$Y\_{\rm ijk} = \mu + \infty\_i + \beta\_{\rm j} + \gamma\_{\rm ij} + \varepsilon\_{\rm ijk} \tag{2}$$

where μ is the intercept; α the casing factor; β the time factor; γ the interaction; ε the error; i and j are the level of the first and second factor; k the number of within group replicates. The significance of the effects was tested by Fisher's F value and the associated *p* value.

Since the MCH batch was still not ripened after 45 days, an additional sampling was carried out only for this batch at 120 days. As the definitive experimental design was incomplete, data were further analyzed by the two-way nested ANOVA and the model used is presented in Equation (3):

$$Y\_{\rm ijk} = \mu + \alpha\_i + \beta\_{\rm j(i)} + \varepsilon\_{\rm ijk} \tag{3}$$

where β is the time factor nested with the casing factor. Post hoc mean comparison was carried out on the time nested with casing effect using the Tukey's HSD test.

The mold load and textural properties of the fully ripened sausages (MCB at 45 days and MCH at 120 days) were eventually compared among them using the Student's *t*-test for independent samples in order to test significant differences between the two groups of samples.

Statistical analyses were performed by using Statistica v. 6.1 (Statsoft Europe, Hamburg, Germany).

#### **3. Results and Discussion**

#### *3.1. E*ff*ect of the Di*ff*erent Casings on Microbial Growth*

Differently from other Mediterranean dry fermented sausages, in which the fermentation time is 1–2 days at 18–24 ◦C or 1 week at relatively low temperatures (10–12 ◦C), the fermentation of "*Mortadella di Campotosto*" sausages is carried out at very low temperatures, below 4 ◦C. These conditions, together with the absence of starters, cause a slow growth of lactic acid bacteria and therefore, an extension of the fermentation time.

Table 1 depicts the behavior of the different microbial groups during time. As evidenced, during the fermentation phase (up to 11 days) and the first week of ripening (day 18), no statistically significant differences were noticed in the growth dynamics of all microbial groups between MCB and MCH products. As expected, Enterobacteriaceae were not detected after 6 days of production, probably as a consequence of the progressive pH reduction. With the extension of the ripening time (from 30 days), statistically significant differences were observed in yeast and CNS counts that were lower in MCB. In detail, the yeast count increased in MCH samples at 30 days, probably because of a succession of different species. It has to be underlined that at 30 days, the aw values of MCB and MCH samples were significantly different and the higher MCH aw allowed a greater microbial growth with more abundant cells loads. After that, the number of cells progressively decreased during the ripening time.

In addition, in this study, we observed that the type of casing used can affect the colonization of molds, which reached values of 5.60 Log CFU/cm<sup>2</sup> in MCB and < 2.0 Log CFU/cm<sup>2</sup> in MCH at the end of the process.

#### *3.2. E*ff*ect of the Di*ff*erent Casings on Physicochemical Parameters*

In Table 1, the changes in pH values during fermentation and ripening of the samples in beef casing (MCB) and in hog casing (MCH) are reported. A significant (*p* < 0.05) pH decrease was detected in both cases up to day 11, which could thus be presumably considered as the end of fermentation. The pH decrease was concurrent with the increasing number of presumptive LAB that reached levels of 7.66 and 7.85 Log CFU/g for MCH and MCB, respectively. After that, pH slowly increased, due to the typical phenomena of ripening, starting with proteolysis in both batches, but with different rates throughout ripening. The end of ripening (45 days for MCB and 120 days for MCH, respectively) was first evaluated by professional manufacturers, who tested product hardness, as perceived by digital pressure, and flavor sniffing, and then, confirmed by textural analysis before final sampling. At the end of ripening, the pH reached levels of about 5.67 for MCB and of 5.90 for MCH, respectively.

The type of casing exerted a significant effect on the drying rate, as highlighted by moisture (Figure 1) and aw (Table 1) data; no water losses were observed during fermentation but, during ripening, the moisture dramatically differed between the two batches. After 45 days of ripening (the end of the ripening for MCB products), MCB batches reached moisture (31.2%) and aw (0.841) values significantly lower than MCH samples, in which the values of moisture and aw were 53.7% and 0.923, respectively. These differences can be attributed to the physical characteristics of the two types of casing, such as the degree of casing permeability, which influences the level of exchange between the filling and the external environment. In fact, hog bung casings had greater thickness (about 3-fold) than MCB, leading to a lower water vapor transmission rate and higher aw [4]. The degree of casing permeability to water, gas, and light affects water loss, fat hydrolysis, fat oxidation, as well as pH and aw [2,4].

As regards NaCl content, given as g 100/g total solids, slightly significant differences were observed at the end of the ripening time in both types of sausages, which showed values of 4.77 ± 0.01% and 4.97 ± 0.16% for MCB and MCH, respectively.



*Foods* **2020**, *9*, 1286

**Figure 1.** Evolution of the relative humidity of "*Mortadella di Campotosto*"-like sausages produced with beef (MCB) and hog (MCH) casings over time.

#### *3.3. E*ff*ect on Proteolysis*

Protein hydrolysis was evaluated by gel electrophoresis, as well as by measuring the content of total free amino acids, volatiles, and amines that greatly influence the texture, flavor, and safety of dry fermented sausages [19]. The hydrolysis of sarcoplasmic and myofibrillar proteins, determined via SDS-PAGE analysis, was influenced by the ripening time and the type of casing (Figure 2).

#### 3.3.1. Sarcoplasmic Fraction

The electrophoretic separation of sarcoplasmic proteins of MCB and MCH at different processing stages is illustrated in Figure 2a. Proteolysis took place during fermentation, as revealed by the slight changes from the first fermentation phase; the bands most susceptible to degradation were those of about 61 and 56 kDa, followed by a huge band of about 48 kDa, and the most intensive degradation was observed in MCH samples. In addition, two fragments were generated at 36 and 35 KDa, which were assumed to be glyceraldehyde-3-phosphate dehydrogenase [20], and 18 KDa in both types of samples.

In addition, during ripening, starting from day 18, a more intense hydrolysis was observed in MCH samples, as indicated by the intensity decrease in the band at 45 kDa (data at 120 days not shown), which is assumed to be creatine kinase [21]. This band completely disappeared after 45 days, while the intensity of the bands of about 74, 37, 36, 18, and 12 KDa increased overall in the MCB samples. The appearance and increase in polypeptides in the range of 14–100 kDa have been observed also by other authors [10,22].

As aw strongly affects the activities of all endogenous proteinases [23], the differences between MCB and MCH could be ascribable to the higher aw values of MCH samples (Table 1). Thus, it could be possible that the highest aw values in MCH batches could have favored the activity of cathepsins B, which are able to break down sarcoplasmic proteins [24]. However, the contribution of bacterial enzymes to protein degradation needs to be taken into account, since LAB counts at day 30 reached higher values in MCH samples (8.24 Log CFU/g) than in MCB samples (6.48 Log CFU/g). In this context, in addition to LAB microbial enzymes, also *Staphylococcus carnosus* and *Staphylococcus simulans* proteases are capable to hydrolyze sarcoplasmic proteins [25].

**Figure 2.** SDS-PAGE electrophoretic profiles of "*Mortadella di Campotosto*"-like sausages produced with beef (MCB) and hog (MCH) casings over time. Panel (**a**) sarcoplasmic fraction; panel (**b**) myofibrillar fraction. Batter: 0 days. Values on the ordinates refer to the marker and express the bands dimension in KDa.

#### 3.3.2. Myofibrillar Fraction

Myofibrillar proteins play the most critical role during meat processing, as they are responsible for the cohesive structure and firm texture of meat products [26]. As evidenced, this protein fraction was less susceptible to degradation and the hydrolysis dynamics in MCB and MCH showed very similar profiles. Recently, Berardo et al. [14] reported that actin (45 kDa) is highly broken down during fermentation; nevertheless, we did not evidence any change of this protein during fermentation in both MCB and MCH samples, in agreement with other studies [27,28]. On the contrary, the generation of polypeptides and large peptides with molecular weight from 50 to 100 kDa was more evident at day 11.

During ripening, when the endogenous enzymes are affected by the aw decrease, having particular impact on cathepsins and alanyl- and pyroglutamyl-amino-peptidases [29], an important degradation of the band of about 48 kDa was detected (day 18); afterwards, it disappeared in both batches. In the meantime, a band of about 33 KDa, probably corresponding to β-tropomyosin, appeared in MCH samples at day 18, while in MCB samples, it was detected at 30 days of ripening [30].

Differences between the two products were clear from the 30th day of the process, in which the aw values were 0.890 ± 0.008 and 0.940 ± 0.012 for MCB and MCH, respectively.

As regards Myosin Heavy Chain (220 kDa), an important degradation for at least 45% in MCH samples was detected at day 45, in contrast with MCB samples, in which it remained almost unchanged during ripening. The hydrolysis of actin (45 kDa) was less severe than that of myosin (25 kDa), and it was clear from day 30 in MCB, in which the reduction was about 15%. These results are in accordance with other authors [31], who reported a lower degradation of actin in fermented sausages with higher pH, suggesting that this might be due to the low optimum pH of cathepsin D-like muscle enzymes, playing a major role in actin hydrolysis.

Additionally, changes in tropomyosin (35 kDa) were more intense in MCB samples that presented also hydrolysis of the bands at 48 and 54 kDa, probably corresponding to desmin. Moreover, the myosin II short-chain (about 18 kDa) showed an intensity increase of approximatively 10% in MCH samples; muscle proteinases predominate in proteolysis evolution along the dry fermented sausage ripening, while those from bacteria mainly act during fermentation [32]. Nevertheless, the major proteolysis of the myofibrillar fractions in MCH (presenting aw values of 0.890 at day 30) could be attributed to particular bacteria or yeast species present in the meat or in the casing, well adapted to the particular environment of this sausage and probably dominating during fermentation and ripening. Moreover, on the MCB casing surface, the molds, reaching loads of 5.60 Log CFU/g, could have promoted the greater proteolysis [33]. In fact, during ripening, when aw decreases, the molds, and especially strains of *Aspergillus* and *Penicillium* genera, tend to dominate due to their capability to overcome xerophilic and halophilic conditions. *Penicillium chrysogenum* and *P. nalgiovense* contribute to proteolytic activities [34] and *Penicillium chrysogenum* Pg222 proteolytic enzymes show activity on the principal myofibrillar proteins, including actin, myosin, tropomyosin, and troponin [35].

#### *3.4. Total Amino Acids Content*

The generation of free amino acids (FAA) is the final outcome of proteolysis, and it contributes to the specific taste and also to the generation of volatile compounds, which provide the flavor in fermented sausages. The FAA content, expressed as mM of leucine, of both kinds of samples, was analyzed during the experimental period. As expected, the low temperatures applied in the production of the samples resulted in a limited generation of FAA, and their content was significantly different (*p* < 0.05) depending on the type of casing.

The quantification of total free amino acids (TFAA) is reported in Table 2. As evidenced, the initial batter contained about 361.25 ± 13.32 mg amino acids/100 g of dry-matter, and during the process, this concentration changed over time to a final concentration of 84.35 ± 19.18 and 235.59 ± 6.59 mg/100 g of dry-matter for MCB and MCH, respectively, at 45 and 120 days, with the major contribution of arginine (Arg) and alanine (Ala), followed by leucine (Leu) and valine (Val). The observed fluctuations in the content of each individual amino acid could be ascribed to the balance between FAA produced by protein breakdown and microbial activity. Among the bacteria, coagulase-negative staphylococci (CNS), *Lactobacillus sakei*, *Lactobacillus curvatus*, and some yeasts such as *Saccharomyces cerevisiae* have been reported to be directly involved in meat proteolysis and in free amino acids generation [15]. At the same time, many of these microorganisms use free amino acids as substrate for further metabolic reactions (deamination, dehydrogenation, and transamination), which are related to the development of aroma and flavor that characterize the final fermented sausage [36,37]. Moreover, arginine reduction in MCH samples could also be correlated to decarboxylation reactions, with the consequent production of putrescine, starting from day 45.


Val—valine; FAA—free amino acids. Means with different letters in the same column are significantly different (*<sup>p</sup>* < 0.05). Fisher's F value of casing (F(C)), time (F(t)), combined casing and time (F(C × t)), and time nested with casing (F(t(C))) factors calculated for each analytical determination. ANOVA significant differences were indicated by F values. \*\* *p* <0.01. \*\*\* *p* <0.005. n.d.—not detectable.

#### *Foods* **2020** , *9*, 1286

The major differences in FAA were observed during ripening, in which hydrophobic amino acids were accumulated. In addition, significant differences (*p* < 0.05) were observed for the greater amounts of Arg and Ala in MCB samples up to 30 days. This concentration appeared dramatically reduced at day 45, particularly for Arg in MCH samples and for Ala in MCB ones. In this respect, two hypotheses can be proposed: (1) the different environmental conditions present in MCB after 30 days could have selected microorganisms with a highly efficient arginine-converting machinery, with the aim of obtaining energy from arginine in the absence of glucose, as reported for some *Lactobacillus*, CNS and *Pseudomonas* species [38–40], and this might be reflected by the presence of *Pseudomonadaceae* at values of 5.11 ± 0.2 Log CFU/g in MCB samples at day 45; or (2) a possible oxidation of this amino acid could have happened by means of free radicals generated by lipolysis, leading to the formation of carbonyl groups [41]. In fact, amino acids such as lysine, threonine, arginine, and proline are easily attacked by these radicals.

In the case of MCH, the major FAA at the end of ripening was Ala with amounts of 123.20 mg/100 g of dry-matter.

#### *3.5. Volatile Compounds Derived from Amino Acids*

FAA are very important in fermented sausages, both for their contribution to the specific taste and for their involvement in degradation reactions that generate volatile compounds, which provide the flavor in this type of product. It is documented that the pH rise during fermentation is due to the microbial degradation of FAA by decarboxylation and deamination [42]. The transamination and decarboxylation of valine, isoleucine, and leucine, which are branched amino acids, produce the respective branched aldehydes, alcohols, and/or acids. Additionally, amino acids such as Phe, Thr, Try, Tyr, etc., are transformed into their respective aldehydes, such as phenylacetaldehyde from phenylalanine, and indole compounds from tryptophan, while the degradation of the sulfur amino acids cysteine and methionine produces sulfur volatile compounds [43]. In particular, we analyzed the accumulation dynamics of compounds derived from branched amino acids (branched aldehydes, alcohols, and carboxylic acids), such as 2-methylpropanal derived from valine (Val), 2-methylbutanal from isoleucine (Ile), 3-methylbutanal from leucine (Leu), and phenylacetaldehyde, benzaldehyde, phenylethyl alcohol, and ethyl benzoate ester that derived from phenylalanine (Phe) and the results are shown in Table 3.

In general, a greater relative abundance of branched chain aldehydes, alcohols, and acids from the catabolism of branched chain amino acids was detected in MCB samples. In particular, 3-Methyl-1-butanal was the most abundant compound in both types of samples, being more present during fermentation (up to 11 days) in MCH and during ripening in MCB samples. In this respect, other authors [44] suggested that 3-Methyl-butanoic acid and 3-Methyl-1-butanal are markers of the CNS activity in fermented meats.

On the other hand, the decrease in 3-Methyl butanol in MCB samples could be ascribed to its conversion into the corresponding 3-Methyl-butanoic acid (Table 3). MCB samples were characterized also by a major presence of 2-Methylpropanal and 2-Methylpropanoic acid, which increased over time and were not detected in MCH samples during the entire production period. On the contrary, phenyl ethyl alcohol concentration increased only in MCH batches.



2-MPA-2-Methylpropanoic acid; 3-MBA-3-Methylbutanoic acid; 3-MB-3-Methylbutanol; PEA-phenyl ethyl alcohol; 3-M1-B-3-Methyl-1-butanal; 2-MP-2-Methylpropanal. Means with different letters in the same column are significantly different (*<sup>p</sup>* < 0.05). Fisher's F value of casing (F(C)), time (F(t)), combined casing and time (F(C × t)), and time nested with casing (F(t(C))) factors calculated for each analytical determination. ANOVA significant differences were indicated by F values. \* *p* < 0.05, \*\* *p* < 0.01. \*\*\* *p* < 0.005; n.d.—not detectable.

#### *3.6. Texture Analysis*

The textural properties of "*Mortadella di Campotosto*"-like sausages were studied both by stress relaxation and TPA (Texture Profile) analysis. TPA parameters of the two types of samples at the end of ripening are shown in Table 4. At the end of ripening, corresponding to 45 days for MCB and 120 days for MCH, the two types of samples showed very similar textural profiles, except for small differences in springiness and chewiness.

**Table 4.** Results of texture profile analysis and compression–relaxation test of ripened "*Mortadella di Campotosto*"-like sausages produced with beef (MCB) and hog (MCH) casings.


Data in the same column with different letters are significantly different at a *p* < 0.05 level.

No statistically significant differences were found in hardness, despite MCB showing lower aw and moisture values and a slightly higher proteolysis index (12.92% vs. 12.13%) than MCH. In general, protein breakdown during fermented sausages ripening contributes to hardness decrease [45]. However, beside proteolysis, drying is a major factor affecting the binding and textural properties of fermented meat products. In most cases, the effect of dehydration, which increases hardness by promoting the elastic behavior, could counteract and even overcome the effect of proteolysis [28,46].

MCH samples showed a more elastic physical behavior and a consequently higher chewiness. Chewiness resulted positively affected by proteolysis in many studies, independently from positive or negative hardness changes [28,47,48]. Since in TPA analysis, chewiness is the product of hardness × cohesiveness × springiness, the higher chewiness of the MCH product observed in this study is due to its higher springiness, since no differences were found in hardness and cohesiveness. Springiness, which is a measure of elasticity [18], is depleted by moisture content, since water acts as a plasticizer and promotes viscous behavior. Despite differences in springiness, observed by measuring the recovery of the deformation after uniaxial compression, no significant differences in elastic behavior were observed when a stress–relaxation test was applied, as the force dissipated by viscous flow was identical in the two samples (Table 4).

In this section, the effect of proteolysis and dehydration were discussed since they are the main factors affecting sausage texture, but it should be considered that also lipolysis may contribute to the final texture.

#### *3.7. Biogenic Amines (BA) Content*

High quantities of proteins, associated with the proteolytic activity of endogenous enzymes and decarboxylase activity of wild microbiota, can support the accumulation of biogenic amines in fermented sausages [49,50], although the final balance depends on the equilibrium between BAs formation and degradation [51]. Figure 3 depicts the BAs content in "*Mortadella di Campotosto*"-like samples up to the end of ripening (45 days for MCB, panel a; 120 days for MCH, panel b).

In general, tryptamine, phenylethylamine, and spermine were not detected, while tyramine (TYR) and polyamines such as cadaverine (CAD), putrescine (PUT), and spermidine (SPD) were found during the entire production process, although with differences between the two products. Histamine was not detected in MCH, while it was found in MCB at low concentration (up to 17 mg/Kg, after the drying step). In addition, during fermentation (up to 11 days), TYR and SPD were the most abundant amines in MCB sausages and were detected in similar concentrations in MCB and MCH samples at up to 45 days of ripening. Tyramine production has been associated with the presence of LAB and

enterococci that usually possess high amino acid decarboxylase activity [52]. This characteristic is strain-dependent and could be expressed during drying and ripening [53].

**Figure 3.** Putrescine (put), cadaverine (cad), histamine (his), tyramine (tyr), spermidine (spd), and sum of biogenic amines (as mg/Kg) content in "*Mortadella di Campotosto*"-like sausages produced with beef (panel (**a**), MCB) and hog (panel (**b**), MCH) casings, over time.

The sum of BAs content at the end of ripening resulted significantly different between the two types of products (265 ± 6 and 341 ± 23 mg/Kg, respectively, for MCB and MCH). These BAs levels are commonly found in other types of dry sausages produced by natural fermentation. Again, the differences in BAs content could be attributed to the higher water activity in MCH, as well as to the lower NaCl concentration due to scarce water loss, optimal for microbial development and BAs accumulation, in particular of tyramine and putrescine [54]. The significant reduction in total BAs at the end of ripening was associated with PUT decrease (more than 80%) in MCH samples, and with the decline of TYR, close to 40%, in MBH samples. Biogenic amines degradation was probably due to microorganisms possessing amino-oxidase enzymes, such as particular strains of the genera

*Lactobacillus*, *Pediococcus*, *Micrococcus*, as well as *Staphylococcus carnosus* [55]. The activity of these enzymes is pH-dependent and particularly active at pH values close to neutrality [56].

In the absence of a legal limit for biogenic amines content in dry fermented sausages, European Food Safety Authority (EFSA) stated that up to 50 mg of histamine and 60 mg of tyramine can be considered safe for healthy individuals; however, these limits fall dramatically if an individual takes anti-MAO drugs or is particularly sensitive to these amines [57]. Suzzi and Gardini [53] identified a sum of 200 mg/Kg of vasoactive amines (tyramine, histamine, tryptamine, and 2-phenylethylamine), as an indicator of good manufacturing procedure for fermented sausages. Among the investigated samples, only in MCH this limit was reached 45 days after the start of the manufacturing process and was still exceeded (although reduced) at the end of ripening.

#### **4. Conclusions**

The effect of different casing on some characteristics of dry fermented sausages produced at very low temperatures was investigated. This study demonstrated that "*Mortadella di Campotosto*"-like sausages did not show an intense proteolysis during fermentation and ripening, probably because production is carried out at very low temperatures. Nevertheless, the type of casing had a strong effect on ripening time, proteolysis, and production of some volatile compounds. On the other hand, although the presence of biogenic amines is considered as unavoidable in fermented meat products, the results highlighted that in "*Mortadella di Campotosto*"-like sausages processed with the traditional beef casing, the risk associated with the presence of bioactive amines is low. Furthermore, in addition to the better texture performance, when beef casing is used for this type of sausage, the process is significantly shorter in comparison to hog casing, with important positive effects on production costs for ripening and storage. For all these considerations, and despite its lower availability on the market, beef casings, traditionally used for this kind of product, are determinant for the final characteristics of this type of product.

**Author Contributions:** Conceptualization, A.P. and C.C.-L.; methodology, C.C.-L., A.S.; software, C.C.-L., G.S.; validation, A.S., C.C.-L. and A.P.; formal analysis, J.L., F.M., F.D., C.R.; investigation, J.L., F.M., F.D., C.R., M.M.; data curation, A.S., C.C.-L., A.P., M.M., G.S.; writing—original draft preparation, C.C.-L., A.S.; writing—review and editing, C.C.-L., A.S., A.P., G.S.; supervision, A.P.; project administration, A.P.; funding acquisition, A.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors gratefully thank Macelleria "L'Olmo", sited in Scanno (AQ), Italy for the helpfulness, and for having provided all the experimental samples.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Modeling Some Possible Handling Ways with Fish Raw Material in Home-Made Sushi Meal Preparation**

#### **Hana Buchtova 1,\*, Dani Dordevic 2,3, Iwona Duda 4, Alena Honzlova <sup>5</sup> and Piotr Kulawik <sup>4</sup>**


Received: 14 August 2019; Accepted: 4 October 2019; Published: 8 October 2019

**Abstract:** The aim of this work was to simulate selected ways of handling with raw fish after its purchase. The experiment was designed as three partial simulations: (a) trend in the biogenic amines formation in raw fish caused by breakage of cold chain during the transport after purchase, (b) the use of a handheld gastronomic unit as an alternative method of smoking fish with cold smoke in the household with regard to a possible increase in polycyclic aromatic hydrocarbon content, and (c) whether the cold smoked fish affects selected sensory parameters of nigiri sushi meal prepared by consumers. The material used in the research consisted of: yellowfin tuna (*Thunnus albacares*) sashimi fillets and the Atlantic salmon (*Salmo salar*) fillets with skin. The control (fresh/thawed tuna; without interrupting the cold chain) and experimental (fresh/thawed tuna; cold chain was interrupted by incubation at 35 ◦C/6 h) samples were stored at 2 ± 2 ◦C for 8 days and analyzed after 1st, 4th and 8th day of the cold storage. Histamine content was very low throughout the experiment, though one exception was found (thawed tuna without interrupting the cold chain: 272.05 <sup>±</sup> 217.83 mg·kg−1/8th day). Tuna samples contained more PAH (4.22 <sup>μ</sup>g·kg−1) than salmon samples (1.74 <sup>μ</sup>g·kg−1). Alarming increases of benzo(a)anthracene (1.84 <sup>μ</sup>g·k−1) and chrysene (1.10 <sup>μ</sup>g·kg<sup>−</sup>1) contents in smoked tuna were detected.

**Keywords:** nigiri sushi; polycyclic aromatic hydrocarbons; histamine; household smoker unit

#### **1. Introduction**

Currently, sushi meals are becoming popular worldwide [1]. Sushi meals have developed from a simple street food to sophisticated cuisine. Many studies have dealt with the health benefits and health hazards associated with the sushi cuisine [2]. In the past, high attention has been devoted to studies of microbiological [3], chemical [4,5] or parasitic [6] hazards in fishery products, like the toxicological risks of diseases after consumption of raw fish or foodstuffs that include raw fish flesh [1]. Recorded cases of acute gastric anisakiasis are a serious warning to consumers [7]. Sushi belongs to ready to eat foods and is predisposed to contamination with food pathogens, such as *Listeria monocytogenes* [8,9].

Consumer concerns about food safety might disrupt a healthy food choice [10]. Risks associated with the consumption of fish might impose barriers to consumption, though fish is considered an important component of the human diet [11].

A new look at research on sushi meal assessment, including simulation of model of real consumer behavior and culinary practices by chefs, should shift research to a higher level of knowledge. In recent years there has been an increase in collaboration between researchers and chefs in the field of gastronomy [12]. Modern trends of molecular gastronomy that works with human senses, is a fast food preparation method using portable, easy-to-use applications, developed specifically for chefs, to create new unusual flavors.

The biggest interruption in the cold chain occurs after product purchase and during its delivery to the household. Consumer behavior and the ambient temperature largely influence the shelf life and food safety [13].

In recent years, also in the Czech Republic, there has been a growing trend of self-preparation of sushi food by consumers. In our research, based on the buying habits of some consumers and their creativity approach to treat raw fish raw using a handheld smoker unit, we wanted to connect partial studies on fish handling and sushi preparation into one model experiment.

The experiment was designed in the form of three partial simulations: (a) trend in biogenic amines formation caused by severe breakage of cold chain during the transport of fish raw material after purchase to household, (b) the use of a handheld gastronomic unit as an alternative method of smoking fish with cold smoke in the household with regard to a possible increase in polycyclic aromatic hydrocarbons (PAH) content, and (c) whether the cold smoked fish affects selected sensory parameters of nigiri sushi meal prepared by consumers in their households.

#### **2. Materials and Methods**

Fresh sashimi fillets of the yellowfin tuna (*Thunnus albacares*, caught, FAO 71 area, category of fishing gear: seines) and fresh fillets with skin of the Atlantic salmon (*Salmo salar*, farmed, Norway) were bought from a retail shop (Ocean48, Brno, Czech Republic).

#### *2.1. Trend in the Biogenic Amines Formation Caused by Severe Breakage of Cold Chain during the Transport of Fish Raw Material after Purchase Place to Household*

Tuna sashimi fillet was used to a case study focused on simulating conditions sale of fresh and thawed fish and consumer behavior (compliance/interruption with/of the cold chain) and to determine how this behavior affects the formation of biogenic amines total content and its spectrum (tryptamine TRP, 2-phenylethylamine 2-PHE, putrescine PUT, cadaverine CAD, histamine HIS, tyramine TYR, spermidine SPD, spermine SPR). The experiment was carried out in four separate replicates. Tuna fillets purchased for the fifth repetition had to be excluded from the experiment because of the high histamine content at the start of the storage, which significantly exceeded the limit set out in Regulation (EC) No 2073/2005 [see in Appendix A1] at the start of storage (the possible reason for higher histamine content will be commented on in the Results and Discussion section).

The case study was based on the use of four different sashimi fillets (1, 2, 3, 4). Four types of samples A, B, C, D from each tuna fillet were prepared simultaneously. Characteristics of the samples were as follows: A: control sample, fresh tuna was cold stored at +2 ± 2 ◦C, without interrupting the cold chain after buying the fish in a store; B: experimental sample, fresh tuna, cold chain of fresh sample was interrupted before the cold storage in a laboratory by incubation of the sample (35 ◦C/6 h) to simulate the possible consumer behavior in the summer after buying the fish in a store, subsequently samples were cold stored at +2 ± 2 ◦C; C: experimental sample, thawed tuna, after buying the fish in a store the sample was experimentally frozen (−35 ◦C) and stored for two weeks in a frozen state (−18 ◦C), then the samples were thawed in the refrigerator (+2 ± 2 ◦C/12 h) and subsequently cold stored at +2 ± 2 ◦C; D: experimental sample, thawed tuna, after buying the fish in a store, the sample was experimentally frozen (−35 ◦C) and stored two weeks in a frozen state (−18 ◦C), the cold chain of thawed sample was interrupted by incubation the sample (35 ◦C/6 h) to simulate the possible consumer behavior in the summer after buying the fish in a store, the samples were subsequently cold stored at

+2 ± 2 ◦C. All samples (A, B, C, D) were stored at +2 ± 2 ◦C for 8 days, the samples were analyzed after 1st, 4th and 8th days of cold storage.

The biogenic amines analysis was performed according to the method described by [14]. The chromatographic separation was performed using a Dionex Ultimate 3000 HPLC apparatus (Thermo Scientific, Waltham, MA, USA) with a FLD 3400RS four channel fluorescent detector (Thermo Scientific) and a low pressure gradient pump with a four channel mixer. The detector settings were set to 340 nm for excitation and 540 nm for emission. The separation was performed on a Kromasil 100-5-C18 4.6 × 250 mm column (Akzo Nobel, Amsterdam, The Netherlands) and the column temperature of 30 ◦C. Flow rate was 0.8 mL/min with two mobile phases: (A) acetonitrile (Merck, Darmstadt, Germany) and (B) water (Merck). The detection limit for each biogenic amine was 0.005 mg·kg<sup>−</sup>1. The samples were analyzed in duplicate and triplicate and injections into HPLC were carried out on each duplicate (N = 4 × 2 × 3).

*2.2. The Part of the Research Consisting of Testing the Use of a Handheld Gastronomic Unit as an Alternative Method of Smoking Fish With Cold Smoke in the Household with Regard to a Possible Increase in Polycyclic Aromatic Hydrocarbons (PAH) Content and Whether the Cold Smoked Fish A*ff*ects Selected Sensory Parameters of Nigiri Sushi Meal Prepared by Consumers at Their Household*

Tuna and salmon fillets were used for preparation of nigiri sushi with not-smoked (raw, control samples) and smoked (experimental samples) samples. Smoked muscle of both fish was prepared with application of a smoker unit (Super Aladin smoker, Manihi s.r.o., Praha, Czech Republic). Cold smoke (20 ◦C; Aladin oak chips) was applied on meat surface beneath the glass hatch for 5 min. The experiment was carried out in five separate replicates. Sensory attributes (saltiness, bitterness, juiciness, consistency) of sushi meal and a question focused on examining the fact whether the conscious consumption of smoked fish in sushi can affect the consumer's confidence in the health safety of this food were monitored by a group of trained evaluators and evaluated on the basis of questionnaires.

Nigiri sushi samples with tuna and salmon meat (smoked and not smoked) were prepared in the Sensory Laboratory at the Department of Meat Hygiene and Technology (Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic). Fillets were frozen according to the Commission regulation (EC) No 853/2004 (Annex III, Section VIII, Chapter III, Part D, Point 2a [see in Appendix A2]) at −40 ◦C using quick freezing unit F.R.C. BF 031AF (Friulinox, Taiedo di Chions, Italy) to muscle core temperature of −20 ◦C and were stored in a frozen chamber with regulated temperature (−20 ± 2 ◦C) for 2 weeks. Then the samples were thawed in refrigerator (+2 ± 2 ◦C/12 h) and subsequently divided into two parts, the one was used as control (raw not smoked) samples for sushi preparation and the second one was used for cold smoking by Super Aladin smoker and for sushi preparation. The rice was cooked in rice cooker (42507 Design Reiskocher, Gastroback, Hollenstedt, Germany). Information about sushi ingredients are following: sushi rice (short grain variety, Yutaka, Italy), 8% vinegar (apple vinegar, Bzenecky Ocet, Bzenec, Czech Republic), 6% sugar (sugar crystal, producer: Korunni, Hrušovany nad Jevišovkou, Czech Republic) and 2% cooking salt with iodine (NaCl 98%, J 20-34 mg·kg<sup>−</sup>1, K+S Czech Republic a.s., Olomouc, Czech Republic), wasabi paste (Yutaka, China). The experimental design is shown in Figure 1.

Sushi samples were sensory evaluated in the laboratory equipped according to ISO 8589:2008 [see in Appendix A3]. The protocol consisted of unstructured graphical scales of 100 mm length, with one edge of the scale representing the strongest expressed attribute and the second one the weakest expressed attribute. Saltiness (2% salt addition) was evaluated by respondents' comparison with sushi samples prepared without salt (0 point) and with 3% salt content (100 points) added to rice.

Twenty panelists took part in the sensory evaluation, where they assessed selected parameters: (saltiness, bitterness, juiciness, consistency) and consumer confidence in the food safety (expressed in their own words). Ingredients' weights (g) of nigiri sushi are given in Table 1.

**Figure 1.** Experimental design of sushi production samples used in the research.


**Table 1.** Ingredients' portions (%) of nigiri sushi prepared with salmon and tuna.

The thawed raw tuna and salmon samples were used for chemical analysis (total protein, total fat and dry matter content). The same thawed not smoked and smoked tuna and salmon samples were used for determination of polycyclic aromatic hydrocarbons (PAH).

The total protein content (ISO 937:1978 [see in Appendix A4]) was determined as the amount of organically bound nitrogen (recalculating coefficient *f* = 6.25) using the analyzer Kjeltec 2300 (FOSS Tecator, Höganäs, Sweden). The total lipid content was determined quantitatively (ISO 1443:1973 [see in Appendix A5]) by extraction in solvents using Soxtec 2055 (FOSS Tecator). The dry matter was determined gravimetrically according to the Czech National Standard (ISO 1442:1997 [see in Appendix A6]) by drying the sample to a constant weight at +103 ± 2 ◦C (Binder FD 53, Tuttlingen, Germany).

PAH were determined by accredited method (no. 19 Standard operating procedure 8.15A) using HPLC/FLD in the laboratory of State Veterinary Institute Jihlava (Jihlava, Czech Republic). Each sample was analyzed in parallel. A thoroughly homogenized samples, after trituration with anhydrous sodium sulphate p.a. (Lach-Ner, sro, Tovarni 157, 27711, Neratovice, Czech Republic) and after addition of internal standard 2-methylchrysene (Dr. Ehrenstorfer GmbH, Augsburg, Germany) were extracted with diethyl ether. Extracts were filtered through glass fiber filter paper (Cat. No. 516-0867, VWR International bvba, Leuven, Belgien, the solvent was evaporated on a Büchi R-134 rotary evaporator (BÜCHI Labortechnik) AG, Flawil, Switzerland) at a maximum temperature of 30 ◦C. The residue was carefully blown off with a stream of nitrogen. The extracted fat was dissolved in chloroform (Cat. No. 20034-UT2-M2500-7 (Macron 6754), Lach-Ner, Ltd., Neratovice, Czech Republic). An aliquot of the solution was purified by gel permeation chromatography on a Gilson Aspec XL system (Gilson, Middleton, WI, USA) using a PAH prep column (500 × 8 mm) packed with gel (styrene divinylbenzene copolymer) (Watrex Praha, sro, Carolina Center, Prague, Czech Republic). Purified samples were evaporated to near dryness on a Büchi R-134 rotary evaporator at a maximum temperature of 30 ◦C

or the remaining solvent was blown off with a stream of nitrogen. The residue was dissolved in acetonitrile and used for fluorescence detection by liquid chromatography.

Chromatographic analysis was performed on Waters Alliance e2695 liquid chromatograph with 2475 fluorescence detector (Waters Corporation, Milford, MA, USA) on Waters PAH column (250 mm × 4.6 mm × 5 μm) using gradient elution with mobile phase. Gradient mobile phase consisted out of acetonitrile/redistilled water (75/25 100/0), flow rate 0.7 mL/min, injection 10 μL, column temperature 30 ◦C. Detection was performed by fluorescence detector with programmable wavelength change (excitation wavelength—265 nm for benzo(a)anthracene and chrysene, 290 nm for benzo(b)fluoranthene and benzo(a)pyrene; wavelength 380 nm for benzo(a)anthracene and chrysene and 430 nm for benzo(b)fluoaranthene and benzo(a)pyrene). PAH Calibration Mix, CRM47940 (Supelco Analytical, Bellefonte, PA, USA) was used for the calibration. The limit of quantification was 0.25–0.29 μg/kg, the repeatability of the method was 10% and yield 65–95%.

#### *2.3. Statistical Analysis*

The results of chemical composition were evaluated (mean ± s. d.) in the program Microsoft Office Excel 2007 (Microsoft Corp., Redmond, WA, USA). Statistically significant differences of biogenic amines (BA) spectrum were performed at levels of α = 0.05 (*p* < 0.05) using the UNISTAT 6.0 (Unistat® Ltd, London, UK) statistical package (multiple comparison, Tukey's HSD test).

#### **3. Results and Discussion**

Fresh fishery products are among the most perishable food commodities Their quality is influenced by a number of factors (the origin-wild/farmed, water temperature and level of environmental pollution, compliance with veterinary and hygienic standards during hunting and after capture, species and health and nutritional status/age/sex/phase of sexual cycle/size/weight of fish, way of treatment-gutting/cutting, initial microbiological contamination, keeping the cold chain) which can vary in time and therefore consequently the quality can vary significantly from batch to batch [15]. Freezing is an excellent way to extend shelf life of fish meat during long-term transport or frozen storage [16]. Offering thawed fishery products is a common way of selling fish in landlocked countries [17]. On the other hand, cold chain breakage can cause potentially serious alimentary intoxication, such as histamine content increment. Therefore, growth and activity of histamine-producing bacteria can become dangerous especially in fish which muscle tissue contain a high concentration (about 10,000 mg·kg−1) of free histidine in muscle tissue as tuna fillets. Though, salmon has a low concentration (about 100 to 200 mg·kg−1) of free histidine [18]. Especially, *Photobacterium phosphoreum* and psychrotolerant bacteria similar to *Morganella morganii* are known to be present in fresh fish tissue and form histamine at low temperatures (under 5 ◦C) [19]. Appearance freshness is often unlawfully restored by an injection or immersion of fillets in the nitrites solution to change the dark red or brown colour (visually less fresh meat) to red pigmentation. Meat after this application looks fresh but histamine levels can be high. Also, inappropriate practices of freezing with subsequent illegal treatment are often used (tuna fish originally of canning grade can be illegally sold as sushi grade of tuna). According to the regulation (EC) no 853/2004 [see in Appendix A2], fishery products shall be frozen below −18 ◦C, for canning industry; unprocessed fish initially is tolerated to be frozen in brine at −9 ◦C. Subsequent illegal treatment of tuna fillets and application of nitrites/nitrates (e.g., salt, additives or vegetable extracts containing high level of nitrites) or using of gas carbon monooxide (CO) is not authorized according to the regulation (EC) no 1333/2008 [see in Appendix A7]. Approximately 25,000 tons of tuna per year undergo this treatment [20].

Besides the fact that consumers may be deceived by the quality of fillets, their health can also be compromised. The high level of histamine can cause allergic syndrome [21], nitrites may lead to formation of nitrosamines that have carcinogenic effects [22]. As we wrote in the Materials and Methods section, fresh tuna fillets purchased for the fifth replicate of the experiment, could be hypothetically treated by any of the above illegal practices. Fresh/thawed samples were stored at +2 ± 2 ◦C; the

samples contained in the 1st day of 781/195, 4th day of 1851/1542 and 8th day of 1717/2914 mg·kg−<sup>1</sup> of histamine in meat. This fifth fillet was therefore excluded from the further experimentation. However, we have shown that even frozen tuna fillet can become a serious threat to human health due to the increment of BA.

#### *3.1. Trends in Biogenic Amines Formation Caused by Severe Breakage of the Cold Chain during the Transport to Households of Fish Raw Material after Purchase*

Significant qualitative and quantitative variability in the observed BA was found between sample groups (A, B, C, D) depending on the sampling day (Table 2) as well as between the individual sampling days within one particular group (Table 3).

Regarding the criteria for foodstuffs (Regulation (EC) No 2073/2005, Chap. I, Point 1.26 [see in Appendix A1]), histamine content was very low throughout the experiment, with one exception (C/8 day) which will be further commented (Table 2). The fresh sample of tuna (A) contained the highest (*p* < 0.05) histamine content in the samples measured after 4 days (Table 3).

The fresh samples (group B) that were exposed to 35 ◦C/6 h and subsequently cold-stored at +2 ± 2 ◦C during 8 days had almost the same histamine contents during the storage period (Table 3). Distinctly higher histamine contents were found in C samples (thawed/subsequently cold-stored at +2 <sup>±</sup> 2 ◦C), after 8th day of storage (the mean of four different batches: 272.05 <sup>±</sup> 217.83 mg·kg−1). The noticeably high value of the standard deviation (s. d.) draws attention to possible differences in quality among purchased lots of fillets; due to these differences the results of thawed fillets (C) obtained for the 8th day of sampling are presented in Table 4. Based on the partial results for each of the four lots, it can be concluded that the three lots were probably more contaminated with microorganisms capable to decarboxylation activity. Due to their activities, histamine content of three tuna lots (8 days of storage) increased to 354.74 <sup>±</sup> 185.67 mg·kg−1; PUT, CAD and TYR contents were significantly (*p* < 0.05) higher and caused five times higher total biogenic amine content (563.32 <sup>±</sup> 252.61 mg·kg<sup>−</sup>1) for these three tuna lots compared to the fourth one (Table 4).

In the fourth lot, the HIS content (23.98 <sup>±</sup> 0.93 mg·kg−1) was very low, but compared to the other three lots, the fillet contained significantly more SPD, though the BA total was low overall (108.67 <sup>±</sup> 1.67 mg·kg−1) (Table 4). In the thawed samples of group D tuna, that were experimentally exposed to temperatures of 35 ◦C/6 h and subsequently cold-stored at +2 ± 2 ◦C during 8 days, the HIS content remained virtually unchanged between 1 and 4 days, and we found only a significant reduction in the HIS content on day 8 (Table 3). Based on higher standard deviation (s. d.) for PUT, CAD, TYR, SPD (Table 2), differences in quality were observed for group D tuna batches (similar to the batches of group C tuna). Three batches were found to have significantly (*p* < 0.05) higher amounts of CAD. Consequently, the total BA content in three batches of fillets was approximately two times higher (*p* < 0.05) in comparison to the fourth fillet (Table 4).

The total BA contents of all sample groups were less than 100 mg·kg<sup>−</sup>1, except for samples from group C and D/8 days (Table 2, BA sum). Each of studied sample groups (A, B, C, D) showed a different trend depending on the storage time (Table 3). BA contents in fresh (A) and thawed samples (C) without cold chain break taken on the day 1 were very low (*p* > 0.05) (Table 2).



*Foods* **2019** , *8*, 459


S. s. \* \* \* *p* < 0.05 \* *p* < 0.05 \* \* *p* < Statistically significant differences (*<sup>p</sup>* < 0.05) for a particular biogenic amine (lower case "a", "b" in columns) and between biogenic amines (capital letters "*A"* to *"G"* in italic independingofnumberofbatchesgiven.\*valueswithoutstatisticallysignificantdifferences(S.s.).

1/D

 are

 are

 ±

0.06 ± 0.01 a*A*

 ±

0.62 ± 0.02 a*A*

 ±

9.75 ± 0.35 a*D*

 ±

7.04 ± 0.19 a*C*

 ±

2.36 ± 0.10 a*B*

 ±

25.51 ± 0.59 a*E*

 ±

27.59 ± 0.25 a*F*

 ±

1.94 ± 0.05 a*B*

 ± 74.87 ± 1.12 a

0.05

 rows)

#### *Foods* **2019** , *8*, 459

The BA content in fresh samples (A) from day 4 was significantly higher compared to day 1, followed by a significant (*p* < 0.05) decrease in BA content on the day 8 (Table 3). Thawed samples (C) contained very low levels of BA on day 1 and day 4, while BA samples were significantly (*p* < 0.05) 13 times higher on day 8. In the group of fresh (B) and thawed samples (D), where the cold chain was experimentally broken, the BA formation dynamics was more uniform, but the trend was the opposite. Overall, the BA content of Group B samples gradually decreased (significant differences were observed between days 1 and 4, 8); in contrast, in samples of group D, BA content increased gradually (significant differences were found between days 1 and 8) (Table 3).

The spectrum of BA was formed in larger quantities (above 10 mg·kg<sup>−</sup>1) mainly TYR and SPD and in isolated cases PUT (C and D/8th day), CAD (D/4th and 8th day). HIS contents were already discussed (A and D/4th day, C/8th day), same as SPR (B and D/1st day, D/4th and 8th day). TRP and 2-PHE levels were very low and oscillated between 0 and 3.0 mg·kg−<sup>1</sup> in individual samples. Statistically significant (*p* < 0.05) differences between the biogenic amine values are given for each group and each day of the storage in Table 3 (capital letters "*A*" to "*D*" in rows).

*3.2. Testing of Use of a Handheld Gastronomic Unit as an Alternative Method of Smoking Fish with Cold Smoke in the Household with Regard to a Possible Increase in Polycyclic Aromatic Hydrocarbons (PAH) Content and Whether the Cold Smoked Fish A*ff*ects Selected Sensory Parameters of Nigiri Sushi Meal Prepared by Consumers at Their Household*

The factors affecting food or meal acceptance among consumers have changed rapidly. Sushi is admired by many consumers worldwide due to its appearance and taste [23–25].

A new non-traditional or unusual treatment of meal can increase consumer interest in its taste and, maybe, in its safety. We have found no previous reports of sushi meal containing tuna or salmon smoked with cold smoke for a very short period. It is possible to predict that Super Aladin smoker will be used more frequently in the practice of molecular gastronomy or home-made sushi preparation to imitate its sensory qualities and bring it closer to that of Philadelphia rolls, which is sometimes prepared with salmon smoked with cold or hot smoke.

Appearance, touch, odor, texture and taste represent the sensory properties of foods/meals. Sensory properties are one of the main factors influencing consumers' acceptance and purchase of meals. Health consciousness (including lowering sodium content, the presence of biogenic amines or smoked products) is another factor that can have significant influence on meal acceptance. Certain sushi ingredients (such as vinegar, wasabi and sugar) provide specific sensory properties to this meal.

The sensory properties of prepared sushi samples, smoked and not smoked, are shown in Table 5. Higher values estimated by panelists for juiciness and consistency indicate worse evaluation of these sensory properties. Bigger values for bitterness are emphasizing savoury intensity. Juiciness of sushi prepared without salt was evaluated with higher values then the rest of sushi samples, though statistical significance was not observed (*p* > 0.05).

Health aspects of certain meal are getting priority over shelf life and nutritional profile [26]. Dealing with issues concerning salt consumption is also important due to the fact that salty foods belong to the group of foods toward which consumers can develop addictive tendencies [27].

Sodium intake according to World Health Organization (WHO) should not exceed 2 g per day or 5 g of natural salt (NaCl) [28]. Worldwide salt intake exceeds this limit and ranges from 9 g/day to 12 g/day, which is equivalent to 3.6 g/day to 4.8 g/day [29].

Published information about cold-smoked tuna is sparse, though cold-smoked tuna processing is similar to that of cold-smoked salmon, in which both processing and product characteristics have been extensively studied [4,18].

Warm or hot smoked seafood is accepted and consumed by consumers due to its unique taste, texture and color. Additionally, due to dehydrating, bactericidal and antioxidant properties, smoking processes increase food shelf life. The problem with smoking of foods is that during the process of smoking considerable amount of polycyclic aromatic hydrocarbons (PAH) can be formed due to incomplete wood combustion. Phenols present in wood smoke belong to desirable molecules, since they positively affect food sensory properties and shelf life, but PAH compounds are undesirable molecules [30].


**Table 5.** Sensory attributes evaluation for nigiri sushi meal with not smoked and smoked samples of tuna and salmon fillets.

Saltiness (2% salt addition) was evaluated by respondents' comparison with sushi samples prepared without salt (0 point) and with 3% salt content (100 points) added to rice.

Table 6 shows contents of polycyclic aromatic hydrocarbons (PAH) in the samples of smoked and not smoked tuna and salmon. Tuna samples contained more PAH (4.22 <sup>μ</sup>g·kg<sup>−</sup>1) than salmon samples (1.74 <sup>μ</sup>g·kg<sup>−</sup>1).


**Table 6.** The content of polycyclic aromatic hydrocarbons (PAH) in <sup>μ</sup>g·kg−1.

B(a)A—benzo[a]anthracene; CHr—chrysene; B(b)F—benzo[b]fluoranthene; B(a)P—benzo[a]pyrene; \* values under limit of detection (LOD).

Despite alarming content findings of B(a)A (1.84 <sup>μ</sup>g·kg<sup>−</sup>1) and CHr (1.10 <sup>μ</sup>g·kg<sup>−</sup>1) in smoked tuna, the content of B(a)P and sum of PAH in our samples were lower than the maximum levels written in the regulation (EC) no. 1881/2006, Annex, Section 6 (B(a)P [see in Appendix A8]: 2 <sup>μ</sup>g·kg−1, sum of PAH: 12 <sup>μ</sup>g·kg<sup>−</sup>1).

The existence of several factors affecting PAH content in smoked fish has been scientifically confirmed. PAHs are produced during combustion processes and smoke formation. The type of used matrix (wood), combustion temperature, smoke generation technique, filtration, temperature and smoke composition. Following factors are also influencing PAH formation: size, treatment and chemical composition of smoked fish. Regarding the effect on food (fish), the diffusion intensity of PAH below its surface into the muscle is relatively low. This fact that PAHs are mainly concentrated in the surface layers means that their content in the food is determined by food surface, same as total weight of the food. Surface/weight ratio is also probably responsible for higher levels of PAH in smoked tuna. Certainly, shape of smoked food (thickness and weight) are influencing PAH levels too. The content of PAHs is also associated with the fat content of food. Reference [31] also found that B(a)A content can differ significantly in dependence of seafood species.

Higher fat content in salmon samples influences higher PAH contents than in tuna samples [4,32,33]. The findings of these authors are not in agreement with the results of our chemical composition analysis

(Table 7). However, the fat content, which we determined in our samples, did correspond to published values [34]. Fluctuations in filtering capacity of the smoker could also influence PAH amounts, though the processes of sample smoking took place in the laboratory under the same conditions (time of smoke/cover of samples with glass lid).


**Table 7.** Chemical composition of sashimi tuna fillets and salmon fillets with skin (in %).

Beside the antibacterial and antioxidant properties of smoking that are connected with phenolic compounds present in wood smoke, from our results a negative impact of seafood smoking represented as an increase of PAH compounds can be also seen. The importance of PAH level control in food is important, since these compounds are carcinogenic, mutagenic and endocrine disrupting. PAH compounds (there are more than 660 identified PAH compounds) are produced in wood smoke during pyrolysis (depolymerisation) of lignin and then condensation of the lignin components in lignocelluloses at temperature above 350 ◦C [35,36]. Aside from PAH increment, smoking changes color of food due to Maillard reaction (change coloration occurs due to the reaction of carbonyl groups in smoke with amino groups present on the surface of smoked food [36]. Phenols from wood smoke enter seafood by diffusion and capillary action, changing its flavor, color and prolonging shelf life [30]. PAH compounds in canned smoked tuna were 17.67 <sup>μ</sup>g·kg−<sup>1</sup> [37].

#### **4. Conclusions**

The main finding of the research is highlighting a food safety issue that was found by the experiments with a gastronomic smoking unit due to increased amounts of polycyclic aromatic hydrocarbons. The Super Aladin smoker unit is a patented product, but the manufacturer does not comment on the PAH hazards associated with food safety in the user documentation. The producers of this smoker units should at least include in the manuals the maximum smoking time depending on the type of food and its fatness. In this way they would alert users to the possible danger, which is the adhesion of harmful PAH on smoked food. The minimum or maximum exposure time of food to smoke is not specified or restricted for specific food types. For prolonged smoking (up to 24 h!), the manufacturer recommends intermittent repeated batches of smoke under the hatch at multiple time intervals as required (optical smoke density control under the hatch). In addition to commercial hardwood chips (oak, beech, Jack Daniels), users can use other alternative matrices including aromatic oils for the development of smoke. Due to the lipophilic nature of PAH, these substances could hypothetically be added to the smoke and subsequently increase PAH level to even more harmful concentrations. Certainly, that further experiments with household gastronomic smokers, such as, Super Aladin smoker, will probably give broader and more precise picture about all possibilities and issues concerning these types of devices.

In the case of the experiment aimed at monitoring the content of biogenic amines in tuna samples, we found much more favorable results than expected. Serious interruption of thawed tuna samples cold chain after purchase did not result in an increase of biogenic amines to levels that could represent a health risk for consumers. Manufacturers inform consumers that they should store purchased fish at 0–2 ◦C and consume it within 2 days. According to the results obtained in our experiment, tuna samples could be considered suitable for consumption after 4 days in terms of BA content and even up to 8 days (except for thawed samples without cold chain interruption) after purchase. However, we cannot recommend this practice due to the possibility that purchased tuna could have higher histamine content developed before purchase. The risk of intoxication with histamine becomes more realistic with each new day of storage. Laboratory examinations of fish species associated with high histamine content in muscle should therefore be a normal part of quality controls by sellers so that they do

not have to passively rely on written statements from fish suppliers regarding histamine content in commercial or veterinary evidences of their origin.

**Author Contributions:** H.B. conceived and designed the experiments. H.B. and D.D. performed the basic chemical and sensory analyses and processed all laboratory data including statistical analysis, A.H. carried out the determination of the polycyclic aromatic hydrocarbons include software service and validation of this methodology. I.D. and P.K. carried out the determination of the biogenic amine spectrum include software service and validation of this methodology. The first draft of the manuscript was prepared by H.B. and D.D. and it was revised and substantially improved by D.D. and H.B.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Appendix A**

#### *Appendix A.1 Legislation and Procedures*


#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

MDPI St. Alban-Anlage 66 4052 Basel Switzerland Tel. +41 61 683 77 34 Fax +41 61 302 89 18 www.mdpi.com

*Foods* Editorial Office E-mail: foods@mdpi.com www.mdpi.com/journal/foods

MDPI St. Alban-Anlage 66 4052 Basel Switzerland

Tel: +41 61 683 77 34 Fax: +41 61 302 89 18