**New Formulation towards Healthier Meat Products:** *Juniperus communis* **L. Essential Oil as Alternative for Sodium Nitrite in Dry Fermented Sausages**

**Vladimir Tomovi´c <sup>1</sup> , Branislav Šoji´c 1,\*, Jovo Savanovi´c 1,2, Sunˇcica Koci´c-Tanackov <sup>1</sup> , Branimir Pavli´c <sup>1</sup> , Marija Jokanovi´c <sup>1</sup> , Vesna Ðordevi´c ¯ 3 , Nenad Parunovi´c <sup>3</sup> , Aleksandra Martinovi´c <sup>4</sup> and Dragan Vujadinovi´c <sup>5</sup>**


Received: 8 July 2020; Accepted: 3 August 2020; Published: 6 August 2020

**Abstract:** The effect of *Juniperus communis* L. essential oil (JEO) addition at concentrations of 0.01, 0.05 and 0.10 µL/g on pH, instrumental parameters of color, lipid oxidation (2-Thiobarbituric acid reactive substances (TBARS)), microbial growth, texture and sensory attributes of dry fermented sausages produced with different levels of fat (15 and 25%) and sodium nitrite (0, 75 and 150 mg/kg) was assessed. Reduced level of sodium nitrite (75 mg/kg) in combination with all three concentrations of JEO (0.01–0.10 µL/g) resulted in satisfying physico-chemical (color and texture) properties and improved oxidative stability (TBARS < 0.3 mg MDA/kg) of dry fermented sausages produced with 25% of fat. However, sausages produced with 0.10 µL/g of JEO had untypical flavor. No foodborne pathogens (*Escherichia coli*, *Listeria monocytogenes*, *Salmonella* spp. and sulfite-reducing clostridia) were detected in any sample throughout the storage period (225 days). The results of this study revealed significant antioxidative activity of JEO and consequently its high potential as effective partial replacement for sodium nitrite in dry fermented sausages.

**Keywords:** *Juniperus communis* L.; essential oil; sodium nitrite; dry fermented sausage

#### **1. Introduction**

Fermented sausages have been manufactured in many countries worldwide. Currently, customers are becoming progressively aware of these meat products for their unique sensory characteristics and important health benefits [1]. Dry fermented sausages are produced using fresh or frozen meat (70–80%) and back fat (20–30%), salt, starter cultures, spices and food additives [2,3]. Owing to the relatively high level of fat and distinctive processing technology (e.g., using diverse raw materials, absence of thermal treatment), fermented sausages are highly susceptible to quality deterioration, including lipid oxidation and bacterial growth [2,3].

Lipid oxidation is one of the chief non-microbial factors in quality deterioration in meat and meat-derived products [4]. It is well known that meat products become very susceptible to oxidative

deterioration due to high levels of unsaturated lipids (e.g., polyunsaturated fatty acids, phospholipids and cholesterol), a variety of oxidizing agents in the muscle tissue, the presence of metal catalysts, heme pigments, etc. Lipids (triacyl-glycerides, phospholipids and sterols) are largely spread in both the intra- and extracellular space of muscle tissue. Oxidation of lipids is a three-step radical chain reaction which involves: initiation, propagation and termination with the free radical's formation [5]. It should be highlighted that lipid oxidation leads to loss of nutritional quality, reduced shelf life, intensified toxicity and reduction of the market value of meat and meat-derived products [4].

Spoilage (*Acinetobacter*, *Lactobacillus* spp., *Pseudomonas*, *Proteus* spp., *Enterobacter*, *Leuconostoc* spp., *Moraxella*, etc., yeasts and molds) and pathogenic (e.g., *Salmonella* spp., *Campylobacter jejuni*, *Listeria monocytogenes*, *Escherichia coli* O157:H7, *Clostridium* spp.) microorganisms can diminish the quality of meat and meat products and consequently induce numerous foodborne contaminations [6]. The growth of spoilage microorganisms causes the degradation of lipids and proteins present in meat and meat products and affects the development of unpleasant quality characteristics (e.g., discoloration, slime and gas production, off-odors and off-flavors). On the other hand, pathogenic bacteria are primarily responsible for foodborne diseases and food poisoning of meat and meat-derived products. Furthermore, in past decades, foodborne diseases have been marked as essential factors of growing public health and economic problems all around the world.

Therefore, lipid oxidation and microbiological deterioration of meat and meat products can be marked as major limitations in the modern meat industry [6].

The use of synthetic additives is one of the main approaches for preventing microbial growth and oxidative reactions in meat products [7]. Nitrites (sodium and potassium nitrite) are well known food additives and curing agents in meat processing [8]. They are officially registered as preservatives by European Union directives [9]. During the process of curing, nitrites are applied in order to improve the product's shelf life because they efficiently suppress the growth of many harmful microorganisms and impart significant antioxidant potential to meat products [10,11]. Besides the strong preservative effect, the use of nitrites contributes to the of development of the typical reddish-pink color and flavor of cured meat products [11,12]. However, these preservatives were recently marked as unhealthy to humans because they promote the formation of carcinogenic N-nitroso-compounds [13,14].

Hence, consumers are increasingly demanding fresh, natural, and negligibly processed products with lower content of artificial additives [12,15]. Essential oils are defined as volatile oils with peculiar scents isolated from aromatic and medicinal plants by hydro-distillation or by cold pressing from citrus fruit peel. It is well known that essential oils obtained from different aromatic and medicinal plants possess a significant antioxidant and antimicrobial potential and therefore they are progressively used as natural additives in the modern food industries [16,17]. They represent the complex mixture of terpenoid compounds which can be present in different parts of herbs, particularly in their waxy channels, glands and trichomes. From a chemical point of view, essential oils are usually multipart mixtures of different organic compounds (e.g., terpenoids), aldehydes, ketones, esters, acids and alcohols, where the main constituents commonly constitute up to 85% of the essential oils, while minor compounds and trace elements constitute up to 15% [18]. Predominantly, essential oils are attracting attention as natural food additives (antioxidants and/or antimicrobials), as they are "generally recognized as safe" (GRAS) and have a wide customer acceptance [19]. Hence, several authors have investigated the application of essential oils as natural additives in dry fermented sausages [20–22], as well as potential replacements for nitrites in processing of cooked [12,16,23] and dry cured meat products [24].

*Juniperus communis* L. is an evergreen coniferous plant widespread throughout Europe, North America and North Asia [25]. The berries obtained from the medicinal herb *Juniperus communis* L. are conventionally well known as a strong immune system booster and powerful detoxifier [26]. *Juniperus communis* L. is most frequently used in natural remedies for respiratory infections, sore throat, arthritis, muscle aches and fatigue. It has been found that plant stems have also been used in order to prevent both short- and long-term illnesses. *Juniperus communis* L. essential oil has been assessed and

established for its in vitro antiradical and antioxidant activities which are mostly dependent on its chemical shape [25,26].

Due to its strong antioxidant, antibacterial, antifungal, and anti-inflammatory properties, *Juniperus communis* L. and its essential oil are widely used in food processing, and in the pharmaceutical and cosmetic industries. Terpenoids (e.g., α-pinene, limonene and myrcene) determine the strong and distinctive aroma of juniper essential oil [27].

Recently, the application of *Juniperus communis* L. essential oil as natural additive was investigated in several studies [28–30]. Selim et al. [29] found that *Juniperus communis* L. essential oil added at concentrations of 0.1, 0.5, and 1% possesses a weak inhibitory effect towards *Enterococci* and *Escherichia coli* O157:H7 that were inoculated in ground beef meat, stored at a temperature of 7 ◦C for 14 days. However, in an earlier study, Schelz et al. [28] determined the strong antimicrobial potential of *Juniperus communis* L. essential oil against *Saccharomyces cerevisiae*. In our previous research, we found that *Juniperus communis* L. essential oil efficiently suppressed lipid oxidation and microbial growth and enhanced the color of cooked pork sausages [30].

A literature review has exposed only a few published research papers that discuss the application of essential oil as natural additive in dry fermented sausage processing. There is also a lack of data regarding the application of essential oils as sodium nitrite replacements in this type of dry cured meat product. Regarding its strong antioxidant and antimicrobial potential, we hypothesized that *Juniperus communis* L. essential oil could be used as an alternative for sodium nitrite in meat processing. Thus, the aim of this study was to assess the effect of *Juniperus communis* L. essential oil as an alternative for sodium nitrite in dry fermented sausages. For these purposes, several physicochemical (pH, color and texture), microbiological (total plate count, lactic acid bacteria) and sensory (color, odor and flavor) parameters of dry fermented sausages were determined.

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

#### *2.1. Juniperus communis L. Essential Oil*

#### GC-MS Profile of Terpenoid Compounds

*Juniperus communis* L. essential oil (JEO) was purchased from the manufacturer Herba doo (Belgrade, Serbia). JEO was kept in dark glass bottles at 4 ◦C prior to the experiments.

For identification of volatile terpenoids from JEO, GC-MS analysis was used according to the method described by Pavli´c et al. [31]. Agilent GC890N system coupled to mass spectrometer Agilent MS 5759, with HP-5MS column (0.25 mm inner diameter and 0.25 µm film thickness, 30 m length), was applied for the characterization of terpenoid profile. Flow rate of helium was 2 mL/min. JEO was dissolved in dichloromethane (approx. 1 mg/mL) and 5 µL of solution was injected in the device with split ratio 30:1. Temperature conditions were: injector temperature 250 ◦C, detector temperature 300 ◦C, initial 60 ◦C with linear increase of 4 ◦C/min up to 150 ◦C. The NIST 05 and Wiley 7n data base were used for compound identification. Retention equations, which describe dependence of peak area on different concentration (*R* <sup>2</sup> > 0.99), were obtained using standard compounds dissolved in dichloromethane at different concentrations (1–500 µg/mL). Results were expressed as relative percentage (%).

#### *2.2. Samples*

Dry fermented sausages were created with two levels (15 and 25%) of pork back fat (FC). In both obtained batters, sodium nitrite (NC) was added at three concentrations (0, 75 and 150 mg/kg). Next, each batter was divided into four parts, and into each part the corresponding concentrations of JEO (0.00, 0.01, 0.05 and 0.10 µL/g) were added. The total number of batches (B) was: FC (2) × NC (3) × JEO (4) = 24 (Figure 1). Samples were collected at different storage periods (SD) involving three randomly

selected dry fermented sausages from each batch at the end of drying (0) and after 75, 150 and 225 days of storage. The total number of samples was: B (24) × SD (4) × 3 = 288. randomly selected dry fermented sausages from each batch at the end of drying (0) and after 75, 150 and 225 days of storage. The total number of samples was: B (24) × SD (4) × 3 = 288.

**Figure 1.** Photograph of the inside surfaces of the sausages at the end of storage. **Figure 1.** Photograph of the inside surfaces of the sausages at the end of storage.

#### *2.3. Preparation of Dry Fermented Pork Sausages*

*2.3. Preparation of Dry Fermented Pork Sausages*  Dry fermented sausages were produced in a local industrial plant (A.D. Dim-Dim, Laktaši, Bosnia and Herzegovina). Batters were produced using lean pork shoulder and pork back fat in the ratio 75:25 and 85:15%. The amounts of other ingredients were calculated in relation to raw material weight, and were as follows: NaCl (2.50%), gluconic delta-lactone (0.70%), spice mix (0.50%), dextrose (0.10%), sodium nitrite (0, 75 and 150 mg/kg) and JEO (0.00, 0.01, 0.05 and 0.10 µL/g.) The meat and back fat were minced using a cutter (Krämer & Grebe, Germany), and then the other ingredients were Dry fermented sausages were produced in a local industrial plant (A.D. Dim-Dim, Laktaši, Bosnia and Herzegovina). Batters were produced using lean pork shoulder and pork back fat in the ratio 75:25 and 85:15%. The amounts of other ingredients were calculated in relation to raw material weight, and were as follows: NaCl (2.50%), gluconic delta-lactone (0.70%), spice mix (0.50%), dextrose (0.10%), sodium nitrite (0, 75 and 150 mg/kg) and JEO (0.00, 0.01, 0.05 and 0.10 µL/g.) The meat and back fat were minced using a cutter (Krämer & Grebe, Germany), and then the other ingredients were added and mixed with them until the required temperature (1 ◦C) was achieved. The sausages

added and mixed with them until the required temperature (1 °C) was achieved. The sausages were

were stuffed in 37 mm diameter collagen casings and were placed in a climate chamber (Frigovent, Serbia) for 21 days. The processes of fermentation, smoking, drying and ripening were performed at a temperature of 14–16 ◦C and a relative humidity of 80–95%. Produced sausages were vacuum packed (Multivac C500, Wolfertschwenden, Germany) and stored at 15 ± 1 ◦C for 225 days. The proximate chemical compositions of the sausages produced with 15 and 25% of back fat at the end of drying process are presented in Table S1 (Supplementary material).

#### *2.4. Physico-Chemical Analysis*

The proximate chemical composition (moisture, protein, fat and ash) was determined according to International Organization for Standardization (ISO) procedures [32–35].

The pH was evaluated using a digital pH meter Testo 205 (Testo AG, USA). Before measurement it was calibrated using standard buffers (pH = 4.00 ± 0.05 and pH = 7.00 ± 0.01 at 20 ± 2 ◦C). pH values were determined for three samples, from each group of dry fermented sausages, in duplicate.

Color (CIE-*LAB* values: *L\**—lightness; *a\**—redness; *b\**—yellowness) of each sample of the dry fermented sausages was measured on fresh cross cut immediately after slicing. The *L\**, *a\** and *b\** color coordinates were determined using a MINOLTA Chroma Meter CR-400 (Minolta Co., Ltd., Osaka, Japan) using D-65 lighting, a 2◦ standard observer angle and an 8-mm aperture in the measuring head [16]. Prior to measurement it was calibrated using a Minolta calibration plate (No. 11333090; Y = 92.9, x = 0.3159; y = 0.3322). Color was measured for three samples (2 cm thick) from each group of dry fermented sausages in triplicate.

The TPA (Texture profile analysis) test was conducted at room temperature using TA.XT2 Texture Analyzer (Texture Technologies Corp., Scarsdale, NY/Stable MicroSystems, Godalming, UK) equipped with a standard ∅ 75 mm cylindrical plate. TPA parameters hardness (g), springiness, cohesiveness, and chewiness (g) were determined as described by Ikoni´c et al. [36]. The cylindrical shape samples (2.54 cm in diameter, 2 cm thick) were taken from the central part of the sausage, and were analyzed in two cycle compressions to 50% of their original thickness at a constant test speed of 1 mm/s. Peak force during the first compression cycle was marked as hardness. The rate at which a deformed sample goes back to its undeformed condition after the deforming force is removed was defined as springiness. The ratio of the area under the second and first curve was defined as cohesiveness. Lastly, by multiplying hardness, cohesiveness and springiness, chewiness was obtained. TPA was performed for three samples from each group of dry fermented sausages in duplicate.

Lipid oxidation of dry fermented sausages was assessed using the 2-Thiobarbituric acid reactive substances (TBARS) test according to the method of Botsoglou et al. [37], with some modifications. The final step of the extraction procedure was carried out with total volume (10 mL) of TCA (trichloroacetic acid) in ultrasonic bath XUB 12 (Grant Instruments, Cambridge, UK). Spectrophotometer Jenway 6300 (Jenway, Felsted, UK) was used for absorbance measurement at 532 nm. The results of the TBARS test were expressed as milligrams of malondialdehyde per kilogram of sample (mg MDA/kg). TBARS was determined on three samples from each group of dry fermented sausages in duplicate.

#### *2.5. Microbiological Analysis*

Microbiological analyses were performed on three samples from each group of dry fermented sausages in duplicate. Samples (20 g) were homogenized in 180 mL 1 g/L buffered peptone water (Merk, Darmstadt) for 10 min at 200 rpm (Unimax 1010, Heidolph, Germany) and the serial of decimal dilutions were prepared (up to 7–10). From each dilution 1 mL was placed in a sterile Petri plate and poured with appropriate media depending on the type of tested microorganisms. The following microorganisms were determined: total plate count (TPC), lactic acid bacteria (LAB), *Escherichia coli*, *Salmonella* spp., *Listeria monocytogenes* and sulfite-reducing clostridia count [38–43]. TPC was enumerated in Plate Count Agar (PCA) (Merk, Darmstadt, Germany) and incubated at 30 ◦C for 72 h; LAB was enumerated in de Man, Rogosa and Sharpe (MRS) Agar (Merk, Darmstadt, Germany) and incubated at 30 ◦C for 72 h; *Escherichia coli* was determined on Tryptone Bile Glucuronic Agar (TBX

agar) (Merk, Darmstadt, Germany) after an incubation at 44 ◦C for 24 h; *Salmonella* spp. was determined on Xylose Lysine Deoxycholate (XLD) agar (Merk, Darmstadt, Germany) after an incubation at 37 ◦C for 24 h; *Listeria monocytogenes* was determined on Listeria agar acc. Ottaviani and Agosti (ALOA) (Merk, Darmstadt, Germany) after an incubation at 37 ◦C for 24 h; sulfite-reducing clostridia count was determined on Tryptone Sulfite Cycloserine (TSC) Agar (Merk, Darmstadt, Germany) after an incubation at 37 ◦C for 24–48 h under anaerobic conditions. After incubation, microscopic observation of cell morphology and biochemical tests were used for typical and atypical grown colonies identification. Results were expressed as a log number of colony forming units per gram (log CFU/g).

#### *2.6. Sensory Analyses*

Sensory analysis was carried out by a trained panel consisting of ten members, aged 25 to 50 years, per two sessions. All panelists work at the Faculty of Technology Novi Sad, Serbia, and have wide expertise in the sensory evaluation of foods. Panelists were trained according to methods described in ISO 8586 [44], in a sensory laboratory equipped according to ISO 8589 [45]. Evaluation of sensory attributes (color, odor and flavor) was performed using the difference-from-control test [46]. Prior to analyses, sausages were equilibrated to room temperature for about 15 min. and marked with a three-digit sample number. The sausages were sliced into 2 mm thick pieces and placed on a white porcelain plate. Consumers were firstly questioned to evaluate the control sample (without JEO and with the corresponding contents of fat and nitrite) and afterward to determine how different the coded samples were from the control one. The difference was rated on a scale from 0 to 6, where 0 = no difference; 1 = very slight difference; 2 = slight/moderate difference; 3 = moderate difference; 4 = moderate/large difference; 5 = large difference; and 6 = very large difference.

#### *2.7. Statistical Analysis*

The statistical program STATISTICA 13.0 (TIBCO Software Inc., Palo Alto, CA, USA) was used for data analyses. The main effects (fat content, nitrite content, JEO content and storage day) were compared. All data were expressed as mean value with their standard deviation (Stdev). The two-way, three-way and four-way interactions between these effects were also tested. Differences among treatment means were compared according to *t*-test and Duncan's multiple range test (*p* < 0.05).

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

#### *3.1. Chemical Profile of JEO*

Chemical profile of JEO was determined by GC-MS and results are presented in Table 1.

It can be observed that β-myrcene (14.12%) was the predominant compound in JEO, obtained using the conventional technique of hydro distillation. Other compounds detected in JEO with relative percentage higher than 1% were: (1) monoterpene hydrocarbons: sabinene (9.51%), β-pinene (5.39%), α-terpinene (1.95%), *p*-cymene (3.92%), d,l-limonene (8.36%), γ-terpinene (3.38%) and α-terpinolene (2.80%); (2) oxygenated monoterpenes: 4-terpineol (6.88%); (3) sesquiterpene hydrocarbons: α-cubebene (1.22%), α-copaene (1.39%), β-elemene (3.38%), caryophyllene (3.94%), α-humulene (3.26%), germacrene D (3.81%), ledene (1.40%), α-muurolene (1.30%), α-amorphene (5.43%) and germacrene B (3.74%), while all other compounds were present in content less than 1%. Results suggested a majority of terpenoids with hydrocarbons, while a lower amount could be accounted for by the oxygenated monoterpenes and sesquiterpenes. Present results were in accordance with similar studies since it was reported that monoterpene hydrocarbons [α-pinene (31.1%), β-myrcene (16.3%), sabinene (7.5%), limonene (6.2%) and β-pinene (3.7%)] were the major compounds identified in commercial JEO [47]. Similar results were also reported by Radoukova et al. [48] and Zheljazkov et al. [49]. It should be highlighted that variations in chemical profile of JEO could be related to genetic properties, geographical origin and climate conditions. Besides conventional technique of hydro distillation, Orav et al. [50] and Markovi´c et al. [51] investigated the possibility of using a novel technique of extraction for JEO

recovery. Orav et al. [50] reported that the JEO obtained using supercritical fluid extraction (SFE) with carbon-dioxide had a lower content of monoterpenes and a higher content of sesquiterpenes compared to JEO obtained using conventional hydro distillation. On the contrary, Markovi´c et al. [51] determined a similar chemical profile for JEOs obtained using conventional hydro distillation and novel microwave-assisted hydro distillation. Therefore, it could be assumed that supercritical fluid extraction will cause co-extraction of other lipophilic compounds which could further alter the bioactivity of these extracts. Besides that, juniper variety, geographical origin, climate and post-harvest processing could significantly affect JEO yield and chemical profile of terpenoids. Furthermore, the possibility of utilization of other juniper materials, such as the needles (leaves) during hydro distillation cannot be excluded [47].


**Table 1.** Chemical profile of JEO determined by GC-MS.


**Table 1.** *Cont.*

<sup>1</sup> Not identified.

#### *3.2. pH and Instrumental Parameters of Color of Dry Fermented Sausages*

The pH values of dry fermented sausages are presented in Table 2.


**Table 2.** pH, instrumental parameters of color and 2-Thiobarbituric acid reactive substances (TBARS) values of dry fermented sausages.

FC—fat content; NC—nitrite content; JC— *Juniperus communis* L. essential oil (JEO) content; SD—storage day; Means <sup>±</sup> Stdev with different letters (a–d) in the same column are significantly different (*<sup>p</sup>* <sup>&</sup>lt; 0.05).

The fat content and storage time had a significant (*p* < 0.05) effect on the pH values. The samples produced with 15% of fat had a higher pH value. Regarding storage time, it can be observed that pH values inconsistently increased throughout storage, probably as the result of formation of amino-compounds during the proteolysis in fermented sausages [52,53]. The two-way (SD × JC), three-way (FC × NC × SD, FC × SD × JC) and four-way (FC × NC × SD × JC) interactions had a significant (*p* < 0.05–0.001) effect on the pH values (Table S2—Supplementary material). Values of pH ranged from 5.11 (FC = 25%, NC = 0 mg/kg, SD = 0, JC = 0.05 µL/g) to 5.63 (FC = 15%, NC = 150 mg/kg, SD = 75, JC = 0.10 µL/g). Similar results were observed by Kur´cubi´c et al. [52] and Ozaki et al. [54] in fermented meat products.

Color is one of the key quality parameters for meat and meat products [16]. The instrumental parameters of color (*L\**, *a\** and *b\**) are displayed in Table 2. The contents of fat, nitrite and JEO, as well as storage time, had a significant (*p* < 0.05) effect on *L\** values. As expected, the samples produced with 15% of fat had lower *L\** values. Moreover, storage time had the effect of decreasing *L\** values, according with the findings of Pateiro et al. [55]. Finally, the addition of JEO decreased the *L\** value, probably as the result of interactions among bioactive compounds of JEO (phenolics, terpenes) and myoglobin [30]. The two-way (FC × SD) and four-way (FC × NC × SD × JC) interactions were also significant (*p* < 0.05) for *L\** values (Table S2). The *L\** values ranged across a wide interval from 43.92 (FC = 15%, NC = 150 mg/kg, SD = 225, JC = 0.10 µL/g) to 56.64 (FC = 25%, NC = 0 mg/kg, SD = 0, JC = 0.00 µL/g).

Fat content and storage time had a significant (*p* < 0.05) effect on the *a\** values. As expected, the samples produced with 15% of fat had higher *a\** values. Concerning storage time, the increasing of *a\** values after the 150th day of storage can be noticed. This is in accordance with the findings of Pateiro et al. [55]. The increase of *a\** values could be related to the growth of the *Staphylococcus* species [56]. Faustman and Cassens [56] reported that enzymes (NADH-cytochrome b5 reductase systems, metmyoglobin reductase and nitrate reductase) of *S. carnosus* or *S. xylosus* can alter metmyoglobin to form red myoglobin derivatives and enhance the color of meat products. Two-way (FC × NC) and three-way interactions (FC × NC × SD and FC × SD × JC) suggested a significant (*p* < 0.05–0.01) effect of using both sodium nitrite (150 mg/kg) and JEO (0.10 µL/g) for enhancing the redness of low-fat (15%) dry fermented sausages (Table S2). The lowest (9.38) and the highest (15.95) *a\** values were determined in the samples: FC = 25%, NC = 0 mg/kg, JC = 0.00 µL/g, SD = 0; FC= 15%, NC = 150 mg/kg, JC = 0.10 µL/g SD = 225. No significant (*p* > 0.05) four factor interaction was detected for the *a\** value.

Fat content and storage time had a significant (*p* < 0.05) effect on the *b\** values. Surprisingly, the samples produced with 15 fat had higher *b\** values. After the 150th day of storage, the increase in *b\** value can also be noticed. Similar findings were observed by Rubio et al. [57] for comparable meat products. Two-way (FC × SD, FC × JC, NC × JC, SD × JC), three-way (FC × NC × SD, NC × SD × JC) and four-way (FC × NC × SD × JC) interactions were significant (*p* < 0.05–0.001) for *b\** values (Table S2). The lowest (5.88) and the highest (10.17) *b\** value was detected in the samples: FC = 25%, NC = 0 mg/kg, SD = 75, JC = 0.01 µL/g; FC = 15%, NC = 150 mg/kg, SD = 225, JC = 0.01 µL/g.

#### *3.3. TBARS Values of Dry Fermented Sausages*

Lipid oxidation is one the most important parameters of quality for meat and meat products [30]. TBARS values of dry fermented sausages are presented in Table 2. The contents of nitrite and JEO and storage time had a significant (*p* < 0.05) effect on TBARS values. The inclusion of sodium nitrite (75 and 150 mg/kg) decreased TBARS values. This was probably the result of the antioxidant activity of sodium nitrite [11]. Furthermore, Honikel [11] reported that antioxidant activity of nitrites is associated with the ability of NO to fix and stabilize heme iron (Fe) of meat myoglobin, making it unavailable to catalyze reactions of oxidation. Also, Karwowska et al. [58] reported that the reduction of nitrites, from 150 to 50 mg/kg, increased TBARS values in cooked meat products. Moreover, samples produced with the addition of JEO (0.05 and 0.10 µL/g) had lower TBARS values compared to samples produced

without JEO. This is the consequence of the strong antioxidant potential of JEO. Höferl et al. [25] reported that juniper berry oil significantly prevented the formation of lipid peroxidation by-products caused by TBA. Certain compounds, such as α-terpinene, γ-terpinene and α-terpinolene exhibit strong antioxidant activity in prevention of lipid oxidation which could be compared with α-tocopherol [59]. On the other hand, certain compounds from JEO (pinene, sabinene and limonene) have a rather weak effect. Similar findings of the antioxidant effects of JEO in meat products were observed in our previous study [30]. As expected, storage time had a significant (*p* < 0.05) effect on increasing TBARS values, as the result of lipid oxidation [2]. The two-way interactions (FC × SD and NC × SD) were significant (*p* < 0.05) for TBARS values. Moreover, three-way (FC × NC × SD and FC × SD × JC) and four-way interactions had a significant (*p* < 0.05–0.001) effect on TBARS values (Table S2). The highest TBARS value (0.398 mg MDA/kg) was observed in the sample: FC = 15%, NC = 0 mg/kg, SD = 225, JC = 0.05 µL/g. At the same time, TBARS values in the samples were: FC = 25%, NC = 75 mg/kg, SD = 225, JC = 0.01 µL/g and FC = 25%, NC = 75 mg/kg, SD = 225, JC = 0.05 µL/g amounted 0.117 and 0.110 mg MDA/kg, respectively. According to Melton [60], the TBARS value of 0.3 mg MDA/kg is marked as the threshold for rancidity of meat products. The obtained results suggested that interaction between sodium nitrite (75 mg/kg) and JEO (0.01 and 0.05 µL/g) efficiently reduced the lipid oxidation in high-fat (25%) dry fermented sausages. Regarding the strong lipo-solubility of terpenoid compounds (e.g., β-myrcene, sabinene, β-pinene, limonene) JEO possessed a higher antioxidant potential in dry fermented sausages produced with higher fat content (25%).

#### *3.4. Microbiological Analysis of Dry Fermented Sausages*

Total plate count (TPC) and lactic acid bacteria (LAB) of dry fermented sausages are presented in Table 3.


#### **Table 3.** Microbiological quality of dry fermented sausages.

TPC—total plate count; LAB—lactic acid bacteria; FC—fat content; NC—nitrite content; JC—JEO content; SD—storage day; Means <sup>±</sup> Stdev with different letters (a–d) in the same column are significantly different (*<sup>p</sup>* <sup>&</sup>lt; 0.05).

The contents of fat and nitrite did not exhibit a significant (*p* > 0.05) effect on TPC and LAB. In the case of JEO, the addition of this essential oil (≥0.05 µL/g) had a tendency to reduce the TPC (for 0.14 log cfu/g), but differences among the samples were not significant (*p* > 0.05). Hence, further optimization with a higher concentration is necessary. Moreover, in our previous study [30] we found that JEO addition (≥0.10 µL/g) efficiently reduced TPC in cooked pork sausages. On the contrary, Selim et al. [29] showed that JEO had no effect on the reduction of microbial growth in fresh beef meat. The antimicrobial potential of essential oil depends of its chemical shape. Generally, monoterpenes from the JEO (α-pinene, β-pinene, sabinene, γ-terpinene, β-myrcene, and limonene) are not efficient antimicrobials when applied singly [61]. However, a mixture of these compounds with the presence of other JEO constituents present as a minor content could result in additive or synergistic antimicrobial effects [62]. As mentioned, the utilization of a novel extraction technique (e.g., SFE) could be a good solution in order to improve the chemical profile of JEO [50]. Orav et al. [50] found that JEO obtained using SFE contained less monoterpenes (5.1%) and more sesquiterpenes and oxygenated sesquiterpenes (69.8%) with a higher antimicrobial potential. As expected, storage time significantly (*p* < 0.05) affected TPC and LAB. TPC decreased during the first 75 days of storage, then increased until the 150th day of storage and again decreased until the end of storage. This trend could be related to the reduction of LAB during storage, especially after the 150th day of storage. As mentioned, the population of LAB decreased throughout storage, probably as the consequences of low storage temperature (15 ◦C) and the exhaustion of sugar [63]. No significant (*p* > 0.05) two, three or four factor interactions were detected for both TPC and LAB (Table S2). It can also be noticed that foodborne pathogens (*Escherichia coli*, *Listeria monocytogenes*, *Salmonella* spp. and sulfite-reducing clostridia) were not detected in any sample throughout the storage. The obtained results suggested that all treatments provided a satisfactory microbiological quality according to EU regulation [64].

#### *3.5. Texture Analysis of Dry Fermented Sausages*

Results of instrumental determination of texture characteristics are shown in Table 4.

As expected, fat content significantly changed (*p* < 0.05) the texture parameters (hardness, springiness, cohesiveness and chewiness). Samples with lower fat content showed a higher value of hardness and chewiness. An increase in hardness and chewiness as fat content decreases in dry fermented sausages was also reported by other authors [65,66], probably as a result of a more pronounced moisture loss in sausages with higher proportions of lean meat [66]. During the storage, hardness and chewiness values showed significant (*p* < 0.05) increase until the 150th day of storage, followed by decrease till day 225. Springiness value showed constant increase, while cohesiveness showed constant decrease until the 150th day of storage. Rubio et al. [67] reported increase of hardness, springiness, cohesiveness and chewiness of dry fermented sausage over the whole storage period (till 210 days), while Severini et al. [68] reported decrease in firmness, due to proteolysis. In the case of nitrites, it can be observed that the addition of sodium nitrite significantly (*p* < 0.05) affected the cohesiveness of dry fermented sausages. A similar finding was observed by Dong et al. [69] in cooked pork sausages. Moreover, Villaverdre et al. [70] found that the sodium nitrite addition at the levels of 75 and 150 mg/kg increased the hardness of fermented sausages. This could be related to the ability of sodium nitrite to promote protein oxidation and Schiff base formation [70]. Regarding JEO, it has been noticed that JEO addition had no impact on the texture parameters (hardness, cohesiveness and chewiness) of dry fermented sausages. Similarly, Viuda-Martos et al. [71] reported that rosemary essential oil has no effect on texture parameters of cooked sausages. Two-way (FC × SD) and four-way interactions were also significant (*p* < 0.05–0.001) for all texture parameters (Table S2). The main texture parameters (hardness and chewiness) ranged in interval from 3539 (FC = 25%, NC = 75 mg/kg, SD = 0, JC = 0.00 µL/g) to 10,990 g (FC = 15%, NC = 0 mg/kg, SD = 150, JC = 0.05 µL/g) and from 926 (FC = 25%, NC = 0 mg/kg, SD = 225, JC = 0.05 µL/g) to 2759 g (FC = 15%, NC = 150 mg/kg, SD = 225, JC = 0.05 µL/g), respectively. Similar results for hardness of different dry fermented sausages were observed by Triki et al. [65] and Rubio et al. [67].


**Table 4.** Texture parameters of dry fermented sausages.

FC—fat content; NC—nitrite content; JC—JEO content; SD—storage day; Means <sup>±</sup> Stdev with different letters (a–c) in the same column are significantly different (*p* < 0.05).

#### *3.6. Sensory Analysis of Dry Fermented Sausages*

Results of sensory analysis are shown in Table 5.

The fat content, JEO content and storage time had a significant (*p* < 0.05) effect on the sensory attribute of color. Two-way (FC × NC, FC × SD, NC × SD, FC × JC, SD × JC), three-way (FC × NC × SD, FC × NC × JC, FC × SD × JC, NC × SD × JC) and four-way interactions were also significant (*p* < 0.05–0.001) for this sensory attribute (Table S2). Moreover, nitrite content, JEO content and storage had a significant (*p* < 0.05) effect on sensory attribute of odor. Furthermore, two-way (FC × NC, NC × JC, SD × JC) and three-way (FC × NC × JC, NC × SD × JC) interactions had a significant (*p* < 0.05–40.001) effect on odor (Table S2). It should also be noticed that the numerical data for the sensory attributes of color and odor did not exceed the values of 2.0 (slight differences, less than 1.67 (color) and 1.78 (odor)), in any samples. Hence, the obtained results suggested that the fluctuations of fat, sodium nitrite and JEO, as well as storage time had no negative impact on these sensory attributes. Nitrite content, JEO addition and storage time had a significant (*p* < 0.05) effect on sensory attribute of flavor. Two-way (FC × JC, NC × JC, SD × JC), three-way (FC × NC × JC, FC × SD × JC, NC × SD × JC) and four-way interactions had also a significant (*p* < 0.05–0.001) effect on the flavor (Table S2). The highest differences (>3, higher than moderate) of typical flavor were observed in the samples: FC = 25%, NC = 150 mg/kg, SD = 225, JC = 0.10 µL/g; FC = 15%, NC = 150 mg/kg, SD = 225, JC = 0.10 µL/g. Regarding JEO content of 0.05 µL/g, the highest difference (1.33) was observed in samples: FC = 15%, NC = 150 mg/kg, SD = 225, JC = 0.05 µL/g; FC = 15%, NC = 75 mg/kg, SD = 150, JC = 0.05 µL/g). This difference could be the result of interaction among the sodium nitrite and terpenoid-compounds of JEO. In our previous study we also determined that a high percentage of JEO had a significant effect on the strong aroma of cooked pork sausages [30]. Using novel extraction techniques (e.g., supercritical fluid extraction) at optimum conditions results in extracts which possess a strong antioxidant and

antimicrobial potential, as well as mild flavor, which enables their application at lower concentration in meat processing [17,23].


**Table 5.** Sensory parameters of dry fermented sausages.

FC—at content; NC—nitrite content; JC—JEO content; SD—storage day; Means <sup>±</sup> Stdev with different letters (a–d) in the same column are significantly different (*p* < 0.05).

#### **4. Conclusions**

Monoterpene hydrocarbon β-myrcene (14.12%) was the most abundant compound identified in JEO. The sausages produced with a lower fat content were significantly darker and redder (*p* < 0.05). Moreover, the values of hardness and chewiness were significantly (*p* < 0.05) higher in the samples produced with a lower fat content. The variations in the contents of nitrite and JEO had no negative impact on the color and texture parameters of dry fermented sausages. No foodborne pathogens were detected in any samples. The highest concentration of JEO (0.10 µL/g) had negative impact on flavor. The addition of JEO (0.01 and 0.05 µL/g) combined with reduced concentration of sodium nitrite (75 mg/kg) efficiently retarded the lipid oxidation of high-fat (25%) dry fermented sausages during 225 days of storage. Hence, JEO with evident antioxidant potential could be used as a partial replacement for sodium nitrite in fermented sausages processing. In order to enhance the antimicrobial potential of JEO, the usage of novel extraction technique (e.g., SFE) could be an effective solution. Further investigations are needed to analyze the synergistic effects of different natural extracts, isolated from various plant sources, on improving the quality and shelf-life of meat products.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2304-8158/9/8/1066/s1. Table S1. Proximate chemical composition of dry ferment sausages; Table S2. The effect of two-way, three-way and four-way interactions among processing parameters on the quality of dry fermented sausages expressed as *p*-value.

**Author Contributions:** Conceptualization, V.T.; methodology, V.T. and B.Š.; software, V.T.; formal analysis, B.Š., J.S., S.K.-T., B.P. and M.J.; investigation, V.T. and B.Š.; resources, J.S., N.P., D.V.; writing—original draft preparation, B.Š. and B.P.; writing—review and editing, V.T., V.Ð., N.P. and A.M.; supervision, D.V.; project administration, V.T. and A.M.; All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Ministry of Education, Science and Technological Development, Republic of Serbia, under Grant 451-03-68/2020-14/200134. Also, this research has been done in liaison with the activities defined by the grant for the establishment and implementation of the research-innovation-scientific program "Centre of Excellence (CoE) for digitalization of microbial food safety risk assessment and quality parameters for accurate food authenticity certification (FoodHub)", financed by the Ministry of Science of Montenegro, under the Grant No. 01-3660/2.

**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* **Phosphate Elimination in Emulsified Meat Products: Impact of Protein-Based Ingredients on Quality Characteristics**

**Olivier Goemaere, Seline Glorieux, Marlies Govaert , Liselot Steen and Ilse Fraeye \***

Research Group for Technology and Quality of Animal Products, Department of Microbial and Molecular Systems, Leuven Food Science and Nutrition Research Centre (LFoRCe), KU Leuven Ghent Technology Campus, Gebroeders De Smetstraat 1, B-9000 Gent, Belgium; olivier.goemaere@kuleuven.be (O.G.); seline.glorieux@tradelio.eu (S.G.); marliesgovaert@hotmail.com (M.G.); Liselot.Steen@solina-group.eu (L.S.) **\*** Correspondence: ilse.fraeye@kuleuven.be; Tel.: +32-9-331-66-17

**Abstract:** The addition of phosphates to meat products improves the emulsifying and gelling properties of meat proteins, in turn enhancing overall product quality. The current market trend towards additive-free products and the health issues related to phosphate challenge the industry to develop phosphate-free meat products. The aim of this study was to evaluate the potential of seven proteinbased ingredients (pea, blood plasma, gelatin, soy, whey, egg, and potato) to remediate quality losses of emulsified meat products (cooked sausages) upon phosphate elimination. First, the intrinsic gelling and emulsifying characteristics of the proteins were assessed. Next, the proteins were added to phosphate-free sausages, of which quality characteristics during production (viscoelastic behavior and emulsion stability) and of the final products (texture, cooking loss, and pH) were screened. Blood plasma and soy were superior in phosphate-free cooked sausages, as no significant differences in hardness, cooking yield, or stability were found compared to phosphate-containing sausages. Egg and pea also improved the previously mentioned quality characteristics of phosphate-free sausages, although to a lesser extent. These insights could not entirely be explained based on the intrinsic gelling and emulsifying capacity of the respective proteins. This indicated the importance of a well-defined standardized meat matrix to determine the potential of alternative proteins in meat products.

**Keywords:** phosphate elimination; emulsified meat products; proteins; standardized meat matrix

## **1. Introduction**

Food phosphates exist in different types (mono-, di-, tri-, and polyphosphates) and are often used in the meat industry due to their impact on pH, chelation, ionic strength, and antibacterial activity. They fulfill several functional properties in meat products such as a good buffering capacity (monophosphates) and the ability to dissociate the actomyosin complex of meat (diphosphates) and activate the meat proteins by chelating the protein-bound Mg2+ and Ca2+, leading to increased solubilization of the meat proteins and depolymerization of the thick and thin filaments (tri- and polyphosphates) [1,2]. Due to these effects, meat proteins can maximally exert their emulsifying and gelling properties, which are very important with regard to water holding capacity (WHC) and fat emulsification. In addition, most di- or polyphosphates contribute to an increase in pH or ionic strength, respectively. Both effects result in increased electrostatic repulsion and consequently more space to bind water and fat between the meat proteins, which again contributes to increased water and fat stabilization. The different phosphate types (or blends) in meat products can be added to a maximum amount of 0.5% (expressed as P2O5) according to European legislation [3]. However, in a former study of Ritz, et al. [4], an association was found between a high intake of phosphate additives and cardiovascular morbidity and mortality. This health issue was already recognized for chronic kidney disease patients, but questions arose with regard to the general population. The EFSA Panel

**Citation:** Goemaere, O.; Glorieux, S.; Govaert, M.; Steen, L.; Fraeye, I. Phosphate Elimination in Emulsified Meat Products: Impact of Protein-Based Ingredients on Quality Characteristics. *Foods* **2021**, *10*, 882. https://doi.org/10.3390/foods10040882

Academic Editor: Gonzalo Delgado-Pando

Received: 18 March 2021 Accepted: 12 April 2021 Published: 17 April 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 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 (https:// creativecommons.org/licenses/by/ 4.0/).

on Food Additives and Flavorings further investigated the matter and provided a scientific opinion re-evaluating the safety of phosphates as food additives in 2019. They considered phosphates to be of low acute oral toxicity, and there was no concern with respect to genotoxicity and carcinogenicity. Furthermore, the Panel considered an acceptable daily intake (ADI) of 40 mg/kg body weight per day. However, this ADI does not apply to humans with a reduction in renal function. Ten percent of the general population might have chronic kidney disease with reduced renal function and they may not tolerate the proposed ADI [5].

In a recent study, it was shown that the current amount of P2O<sup>5</sup> added to emulsified meat products (cooked sausages) can be strongly reduced with minimal loss in product quality [6]. Nevertheless, the market trend towards additive-free products [2,7] and the negative effect of phosphates on human health for certain population groups justify attempts to develop phosphate-free emulsified meat products. Unfortunately, phosphate elimination results in decreased meat protein functionality, which causes quality defects such as compromised water and fat stabilization [6]. Therefore, alternative ingredients or even innovative technologies are needed to compensate for this functionality loss. These include pH improving ingredients, starches, hydrocolloids, or the use of high-pressure technology [2,8–11]. Additionally, proteins, from both animal and vegetable sources, can act as enhancers to compensate for the loss of functionalized meat proteins due to phosphate elimination in meat products. This is mainly related to their gelling and emulsification properties. They have already been proven useful to boost the quality characteristics of meat products related to water and fat binding properties, gel network formation, texture, and/or sensorial properties. In this respect, they have been successfully deployed as fat-replacers, processing aids of low-cost meat products, and substitutes for meat proteins [12–20].

However, only a limited number of studies aimed to investigate the opportunities of reduction/replacement of phosphate in meat products [11], especially with regard to the use of alternative proteins. Hurtado, et al. [21] concluded that porcine blood plasma was a useful functional ingredient to replace phosphate and caseinate in frankfurters. Pereira, et al. [22] stated that the addition of collagen fibers improved cooking yield and hardness in phosphate-free sausages. Enhancement of water holding capacity, sensorial attributes, color, and microbial stability could also be achieved by replacing phosphates with a purified beef collagen powder in injected beef strip loins [23]. Furthermore, Muguruma, et al. [24] stated that the addition of biopolymers containing soybean and milk proteins may permit a reduction in phosphate content without a loss of the texture of chicken sausages.

In summary, alternative proteins have been proven to function as functional ingredients in different meat systems on account of their gelling and emulsifying capacities. In contrast, the more specific ability to act as a phosphate replacer, in order to manage the loss of functionalized meat proteins, has only been studied for a limited number of proteins. Furthermore, standardized comparison between proteins remains difficult, because these surveys were conducted on different meat matrices (difference in meat product class, composition of recipes, processing conditions) and different analyzing techniques were applied, making it impossible to identify the most promising protein. Xiong [25] stated that a valid comparison between proteins is only possible if the screening is made under identical processing and storage conditions. Therefore, the aim of this study was to evaluate the potential of seven protein-based ingredients, from both animal and vegetable sources, to counter the loss of quality due to phosphate elimination in emulsified meat products (cooked sausages). In the first stage, the intrinsic protein characteristics (gelation and emulsification potential), related to improving the quality of meat products, are studied. In the second stage, the ability of the proteins to enhance the properties of phosphate-free cooked sausages (viscoelastic and textural properties, emulsion stability, cooking loss, and pH) is evaluated. This research is of important industrial relevance, since phosphate elimination in emulsified meat products will decrease potential health concerns and is a promising step towards clean-label products.

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

#### *2.1. Determination of the Intrinsic Characteristics of Selected Proteins*

The proteins discussed in this study are egg white (Pulviver), pea (Nutralys), potato (KMC), soy concentrate (Pulviver), blood plasma (Veos), gelatin (Rousselot), and whey (Caldic) protein. Proteins were selected based on their industrial relevance. In addition, a balanced distribution between animal and vegetable proteins was envisioned. In order to learn more about their intrinsic properties related to enhancing meat product quality, their gelation and emulsification potential were studied in a watery environment. It is well described that the salt level and acidity of a medium have an important impact on protein characteristics [12,26,27]. In order to create an aqueous medium that reflects the composition of emulsified meat products, proteins were suspended in a 0.05 M Na phosphate buffer (pH = 6) containing 3.5% NaCl and stirred (800 rpm) for 105 min at room temperature before analysis. The applied protein concentration was dependent on the screening technique used, as described below. The concentration of salt corresponds to its quantity in the cooked sausage model (see Section 2.2) expressed in the water phase, and the pH value is in the range of common meat products.

#### 2.1.1. Gelation Potential

The gelation properties of the proteins were determined through rheological measurements using an AR2000ex stress-controlled rheometer (TA instruments, New Castle, DE, USA) equipped with a 40-mm parallel plate system. A crosshatched upper plate and a lower plate were used to prevent slippage of the sample. The gap was set at 500 µm. The AR2000ex was supplemented with an efficient Peltier temperature control system and an upper heated plate (TA Instruments) to control the sample temperatures accurately. Temperature sweeps were conducted to investigate structural changes of the protein suspensions (4.5% protein) during a heating and cooling process, representative of the manufacturing process of emulsified meat products. The following profile was applied: (1) a heating step from 20 to 76 ◦C at a constant heating rate of 2 ◦C/min; (2) an isothermal heating step at 76 ◦C for 3 min; (3) a cooling step from 76 to 20 ◦C at a constant cooling rate of 2 ◦C/min. Oscillation measurements during the entire process were performed at a fixed frequency of 1 Hz and a strain of 0.025, a value found to be within the linear viscoelastic region based on preliminary experiments. The storage modulus (G') and phase angle (δ, with δ of 90◦ representing a fully viscous material and δ of 0◦ representing a fully elastic material) at the end of the isothermal heating step and cooling step (G'<sup>76</sup> ◦C,suspension, δ<sup>76</sup> ◦C,suspension and G'end,suspension, δend,suspension, respectively) were derived from the temperature sweep profiles using the software (Rheology Advantage Data Analysis, v. 5.7.0, TA Instruments). All G'-values are expressed logarithmically. Protein suspensions were made in duplicate for each protein, and determination of the gelling potential was performed in duplicate per suspension.

#### 2.1.2. Emulsification

The emulsifying properties of the proteins were screened according to the procedure described by Steen, et al. [28], which was based upon the turbidimetric method of Pearce and Kinsella [29]. Emulsions were prepared by mixing 2.0 mL sunflower oil and 8.0 mL protein suspensions (0.15% protein) for 1 min at a speed of 12,000 rpm and room temperature using an Ultra-Turrax homogenizer (model T25, IKA-Werke GmbH, Staufen, Germany). Immediately and 10 min after emulsion formation, 50 µL of the emulsion was taken and diluted with 5 mL of 0.1% sodium dodecyl sulfate (SDS) solution. Absorbance values were measured at 500 nm (A500) and used to calculate the emulsifying activity index (EAI, m2/g) according to the equations below.

$$\text{EAI} \left(\text{m}^2/\text{g}\right) = \frac{2\text{x}T\text{x}F}{\text{g}\text{ }\text{C}}\tag{1}$$

$$T = \frac{2.303 \text{xA500}}{L} \tag{2}$$

where A<sup>500</sup> represents the absorbance at 500 nm, *L* the light path length (*L* = 0.01 m), *ϕ* the volume fraction (v/v) of the dispersed phase (*ϕ* = 0.20), *C* the protein concentration (*C* = 1500 g/m<sup>3</sup> ) before emulsification, *T* the turbidity, and *F* the dilution factor (*F* = 100). The emulsion activity index immediately after emulsion formation is represented by the abbreviation EAI0. Emulsion stability (ES) was the percentage of emulsion turbidity remaining after 10 min. Emulsions were made in duplicate for each protein, and determination of the emulsifying properties was performed in duplicate for each emulsion.

#### *2.2. Manufacturing of Cooked Sausage*

Cooked sausages were prepared in the pilot plant of the research group "Technology and Quality of Animal Products" (KU Leuven Technology Campus Gent, Belgium). Raw materials (pork shoulder and pork backfat) were obtained from a local industrial meat supplier (De Lausnay Rene bvba, Destelbergen, Belgium), chopped, homogenized to generate one batch, vacuum-packed, and stored at –18 ◦C until preparation. Cooked sausages contained pork shoulder (35/100 g), pork backfat (35/100 g), and ice (30/100 g), together with nitrite curing salt (1.5/100 g), sugar (0.5/100 g), white pepper (0.2/100 g), foil (0.05/100 g), ascorbic acid (0.05/100 g), glutamate (0.05/100 g), coriander (0.025/100 g), and cardamom (0.025 g/100 g). All non-meat ingredients were purchased from Solina Group (Eke-Nazareth, Belgium). Ingredients were calculated relative to the total mass of meat raw materials (pork shoulder and pork backfat) and ice. First, a standardized reference treatment was prepared, containing 0.32/100 g tetrasodium pyrophosphate (TSPP) (Solina Group, Eke-Nazareth, Belgium), which is equal to 0.171% P2O5, a standard amount used in the meat industry for emulsified meat products. The reference containing phosphate will be referred to as M+TSPP. Secondly, TSPP was eliminated and standardized phosphate-free cooked sausages were prepared. These cooked sausages will be referred to as M-TSPP. Finally, the seven above-described protein-based ingredients were added to the phosphate-free treatment. All proteins were added in a mass fraction of 2/100 g, a commonly used dosage [17]. Proteins were calculated relative to the total mass of meat raw materials (pork shoulder and pork backfat) and ice. These phosphate-free treatments containing protein-based ingredients will be referred to as M-TSPP+"corresponding protein source". During manufacturing of the M+TSPP preparations, the raw lean meat was first pre-chopped together with ice, salt, and TSPP in a bowl cutter for 7 min and 30 s (Stephan cutter UM12, Hameln, Germany), corresponding to a final temperature of 5 ◦C. Next, the pork backfat was added to the meat batter together with the remaining food ingredients. The total mass was ground under vacuum for 4 min and 30 s to obtain a homogenous batter. The temperature did not exceed 14 ◦C during processing to avoid protein denaturation and fat coalescence. Phosphate-free sausages were prepared the same way, but without the addition of TSPP. When phosphate-free sausages containing proteinbased ingredients were prepared, these proteins were added during the first grinding step of the manufacturing process instead of TSPP. Part of the batter, prepared in duplicate per treatment, was immediately analyzed for dynamic viscoelastic properties (Section 2.3) and emulsion stability (Section 2.4). In order to standardize the cooking process, the remainder of the batter was filled into cans of standardized dimensions (diameter 7 cm, height 5 cm, mass ± 250 g, Crown Verpakking België NV, Hoboken, Belgium), cooked at 76 ◦C (core temperature 72 ◦C) for 90 min in a cooking chamber (Rational Climaplus Combi CPC 61, Claes Machines, Paal, Belgium) and finally cooled to 4 ◦C. The resulting meat products, cooked in cans, served as a model product for cooked sausage and will be referred to as "cooked sausages". Each treatment, including the reference products with or without TSPP, was manufactured in duplicate. One week after the manufacturing process, three sausages per replicate were analyzed for cooking loss (Section 2.5), pH (Section 2.6), and textural properties (Section 2.7). The number of measuring points is described in the respective analyses below.

#### *2.3. Dynamic Viscoelastic Properties*

The dynamic viscoelastic properties of the batters were analyzed using the same equipment as described in Section 2.1.1. The gap was set at 1000 µm for both rheological procedures described below (stress sweep and temperature sweeps).

Stress sweeps were conducted at a temperature of 13 ◦C, between 0.1 and 1000 Pa, and at a fixed frequency of 1 Hz to determine the linear viscoelastic region (LVR). Hereby, parameters G', G" (storage and loss modulus respectively), and δ were directly obtained from the software. The complex modulus (G\*), representing the materials' overall rigidity or resistance to deformation, was calculated by the following formula,

$$\mathbf{G}\* = \sqrt{\mathbf{G}'^2 + \mathbf{G}''^2} \tag{3}$$

The LVR represents the stress range within which G\* (and thus G',G") is independent of the imposed stress amplitude and is determined according to Glorieux, Goemaere, Steen and Fraeye [6]. LVR is determined in duplo per replicate of each treatment and referred to as LVRbatter. Furthermore, the corresponding G\*batter, expressed logarithmically, and δbatter within the LVR are reported.

Temperature sweeps were conducted to investigate the impact of phosphate elimination and use of alternative proteins on the structure formation of meat batters during a heating and cooling procedure, representative of the manufacturing process of cooked sausages. Similar profiles and conditions were applied as described in Section 2.1.1., except for the initial (before heating) and final (end of cooling) temperatures, which were both set at 13 ◦C in accordance with the final temperature of the raw batter at the end of the cutter process. The parameters G' and δ at the end of the isothermal heating step and cooling step (G'76◦C,batter, δ76◦C, batter and G'end,batter δend,batter, respectively) were derived from the temperature sweep profiles using the software. G'-values are expressed logarithmically. All rheological parameters (G'76◦C,batter, δ76◦C,batter, G'end,batter, δend,batter) were determined in duplo per replicate of each treatment.

#### *2.4. Emulsion Stability*

Emulsion stability of the meat batter was determined immediately after the grinding process, according to Glorieux, Goemaere, Steen and Fraeye [6] with slight modifications. Summarized, emulsion stability is expressed as drip loss upon heating (30 min, 70 ◦C) and centrifugation at 4230× *g* (6000 rpm in a rotor Cat. No. 1620 A, Hettich, Germany) at 25 ◦C for 3 min, of a pre-weighed amount of raw batter. The percentage of total expressible fluid (TEF) was expressed as follows:

$$\text{TEF} \left( \% \right) = \frac{drip \text{ loss meat battery}}{\text{initial weight meat battery}} \times 100 \tag{4}$$

Furthermore, the relative amount of water, next to the fat in the drip, was determined. Therefore drip loss after centrifugation was weighed before and after drying in an oven (Typ U 40, Memmert, Germany) for 24 h. The relative amount of water in the drip loss was expressed as follows:

$$\text{Relative amount of H}\_2\text{O in dirp}\_{\prime} \text{ (\%)} = \frac{drip \text{ before drying} - drip \text{ after drying}}{drip \text{ before drying}} \times 100 \text{ (5)}$$

TEF and Relative amount of H2O in drip were determined six times per replicate of each treatment.

#### *2.5. Cooking Loss*

Cooking loss (CL) of the cooked sausages of each treatment was measured according to Glorieux, Goemaere, Steen and Fraeye [6]. CL was calculated as follows:

$$\text{CL } (\%) = \frac{drip \, loss \, usage\,age}{initial \, weight \, usage\,age} \times 100\tag{6}$$

Measurements were determined in triplicate per replicate of each treatment.

#### *2.6. pH Measurement*

The pH of the cooked sausages was measured three times on three different sausages (nine measurements) per replicate of each treatment, according to the methods described in Glorieux, Goemaere, Steen and Fraeye [6].

#### *2.7. Texture*

The hardness of the cooked sausages was analyzed using a Lloyd Texture Analyzer (Model LF plus, Lloyd Instruments, Bognor Regis, UK) and expressed as the maximum force (N) to penetrate the sample, as described in Glorieux, Goemaere, Steen and Fraeye [6]. Per replicate of each treatment, hardness was measured three times on three different sausages (nine measurements).

#### *2.8. Statistical Analysis*

Results are expressed as mean ± standard deviation. All results were evaluated by one-way ANOVA. A Tukey's post hoc test was performed with a significance level of *p* < 0.05 to identify significant differences. Statistical analysis was performed using the software IBM SPSS Statistics 25 (IBM, Armonk, NY, USA).

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

#### *3.1. Intrinsic Characteristics of Selected Proteins*

Screening of the intrinsic characteristics of functional ingredients is often executed in watery media. It is a rather quick and easy method to evaluate ingredient functionality that requests no specific and often expensive process equipment to imitate industrial food products. Moreover, it can provide a broad view of the application potential of the ingredients in several food products. Food proteins are mainly applied in meat products in relation to their gelling and emulsifying properties, enabling them to improve overall meat product quality. Results regarding these intrinsic characteristics are described below in Sections 3.1.1 and 3.1.2.

#### 3.1.1. Gelation Potential

The gelling characteristics of proteins are one of the key reasons they are applied for meat product improvement. Figure 1 shows the gelling properties of the proteins upon heating, with the exception of gelatin. Gelatin is a cold-gelling protein that solubilizes during heating [30] and can therefore only participate in gel network formation at sufficiently low temperatures. The critical temperature below which gelling can occur is dependent on gelatin concentration, cooling rate, and maturing temperature [31]. The applied thermal processing and used gelatin (concentration, source) did not allow the expression of the cold gelling character of gelatin. For all other protein suspensions, the heating and subsequent cooling process caused in general an overall increase in G' and a decrease of δ. This suggests the formation of a gel-like structure and increased elastic behavior. G'76◦C,suspension and G'end,suspension are highest for potato and egg white protein, indicating the strongest gelling potential of all screened proteins. The irreversible heat coagulation of egg white proteins involves the formation of spherical aggregates via hydrophobic interactions, which are further stiffened through sulfhydryl–disulfide reactions to finally give rise to a gel, which explains the rather high values of G'76◦C,suspension. Furthermore, the G'-values of egg white protein still increase (G'76◦C,suspension vs. G'end,suspension) during cooling, which can

be attributed to the numerous hydrogen bonds that are formed at lower temperatures [32]. The suspension of potato protein also exerted very good gelling properties upon heating. The low denaturation temperature of patatin, one of the main potato protein fractions, may be partially responsible for this. The denaturation temperature is roughly 20 ◦C lower compared to common food proteins as ovalbumin (egg) or soy glycinin [33,34]. Figure 1 reveals no significant difference between G'76◦C,suspension of the egg white protein and G'76◦C,suspension of soy concentrate, indicating good gelation characteristics of the latter. Pea proteins are mainly composed of globulins. Pea globulins are recognized for their lower gelling ability compared to their soy counterparts. This can also be observed in Figure 1, where G'76◦C,suspension and G'end,suspension of pea proteins are significantly lower than the values of soy concentrate. The gelation of pea proteins appeared to be governed mainly by nonspecific interactions, whereas the involvement of disulfide bonds was reported for soy proteins [35]. Furthermore, high temperatures are required to induce the gelation of the pea proteins because of their high denaturation temperature (>85 ◦C) [36]. The applied thermal processing in this research was therefore not sufficient to obtain proper gelling of pea proteins. Whey protein suspensions start to form gels at concentrations higher than 80 mg whey protein/g H2O when heated above 75 ◦C [37]. The rather short heating time above 75 ◦C and applied concentration could therefore explain the somewhat low values of G'76◦C,suspension, and G'end,suspension of whey protein. Blood plasma also showed relatively low values of both G'76◦C,suspension, and G'end,suspension and is probably attributed to the same reasons as described for whey protein. Research stated that heating to 75 ◦C was a necessity to create strong gels from 4% *w*/*v* plasma protein solutions [38]. Other sources claimed that suspensions containing 4–5% blood plasma already tend to form firm and irreversible gels when temperatures over 70 ◦C are applied [39]. roughly 20 °C lower compared to common food proteins as ovalbumin (egg) or soy glycinin [33,34]. Figure 1 reveals no significant difference between G'76°C,suspension of the egg white protein and G'76°C,suspension of soy concentrate, indicating good gelation characteristics of the latter. Pea proteins are mainly composed of globulins. Pea globulins are recognized for their lower gelling ability compared to their soy counterparts. This can also be observed in Figure 1, where G'76°C,suspension and G'end,suspension of pea proteins are significantly lower than the values of soy concentrate. The gelation of pea proteins appeared to be governed mainly by nonspecific interactions, whereas the involvement of disulfide bonds was reported for soy proteins [35]. Furthermore, high temperatures are required to induce the gelation of the pea proteins because of their high denaturation temperature (>85 °C) [36]. The applied thermal processing in this research was therefore not sufficient to obtain proper gelling of pea proteins. Whey protein suspensions start to form gels at concentrations higher than 80 mg whey protein/g H2O when heated above 75 °C [37]. The rather short heating time above 75 °C and applied concentration could therefore explain the somewhat low values of G'76°C,suspension, and G'end,suspension of whey protein. Blood plasma also showed relatively low values of both G'76°C,suspension, and G'end,suspension and is probably attributed to the same reasons as described for whey protein. Research stated that heating to 75 °C was a necessity to create strong gels from 4% w/v plasma protein solutions [38]. Other sources claimed that suspensions containing 4–5% blood plasma already tend to form firm and irreversible gels when temperatures over 70 °C are applied [39]. Based upon the gathered data and literature study, potato and egg white protein show the most potential for use in meat products. Their gelling properties may lead to a better structure formation of the meat gel and as a consequence to improved water binding or texture of the sausages.

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which can be attributed to the numerous hydrogen bonds that are formed at lower tem-

upon heating. The low denaturation temperature of patatin, one of the main potato protein fractions, may be partially responsible for this. The denaturation temperature is

**Figure 1.** G'76°C,suspension and δ76°C,suspension represent the elasticity modulus and phase angle of protein suspensions at the end of the isothermal heating at 76 °C for 3 min. G'end,suspension and δend,suspension represent the elasticity modulus and phase angle of protein suspensions after further cooling from 76 to 20 °C. (\*) = no value of gelatin could be obtained. Mean values and standard deviations are presented (n = 4). Letters a–d: different letters indicate significant differences (*p* < 0.05) between **Figure 1.** G'76◦C,suspension and δ76◦C,suspension represent the elasticity modulus and phase angle of protein suspensions at the end of the isothermal heating at 76 ◦C for 3 min. G'end,suspension and δend,suspension represent the elasticity modulus and phase angle of protein suspensions after further cooling from 76 to 20 ◦C. (\*) = no value of gelatin could be obtained. Mean values and standard deviations are presented (n = 4). Letters a–d: different letters indicate significant differences (*p* < 0.05) between different proteins.

different proteins.

Based upon the gathered data and literature study, potato and egg white protein show the most potential for use in meat products. Their gelling properties may lead to a better structure formation of the meat gel and as a consequence to improved water binding or texture of the sausages.

#### 3.1.2. Emulsification upon the measurement of oil droplet particle sizes. The same research presented potato

different proteins.

*Cooked Sausage*

coalescence, creaming, and/or flocculation [28].

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In addition to their gelation potential, proteins are also of interest to the meat industry because of their ability to stabilize emulsions. The intrinsic emulsifying and emulsionstabilizing properties of the proteins can be derived from Figure 2. The EAI0 indicates the area of interface stabilized per unit weight of protein (m2/g) and is associated with the ability of the protein to coat the water–oil interface immediately after emulsion formation. ES represents the percentage of emulsion turbidity remaining after 10 min and therefore refers to the ability of an emulsion to resist changes in its properties over time, e.g., droplet

Gelatin possesses good emulsifying properties, as can be noticed by the high values of both EAI0 and ES in Figure 2. Gelatin is capable of reducing the surface tension of aqueous environments and forming the necessary identically charged film around the fat droplets of the dispersed phase. Therefore, the isoelectric point (IEP) is of great importance in the surface activity effects of the used gelatin [40]. The protein carried a net negative charge under the conditions in which this analysis was performed. Whey proteins are well-known for their ability to stabilize interfaces, explaining their great emulsifying properties, as seen in the present research [41]. Figure 2 also shows that blood plasma and egg white protein exerted excellent emulsifying properties. Research by Rodriguez Furlán*, et al.* [42] confirmed the good emulsifying properties of blood plasma. Yet, literature stated that ovalbumin, the major protein in egg white, may perform good emulsifying ability and stability under extreme acidic conditions, which is in contrast to the watery suspensions applied in this research, while under neutral and alkaline pH the stability of egg white emulsions was limited [43]. The emulsifying capacity of soy concentrate was rather limited, as indicated by the low value of EAI0 in Figure 2. The study of Amine*, et al.* [44] also indicated soy protein as a poor emulsifier for oil in water emulsions, based

3.1.2. Emulsification

In addition to their gelation potential, proteins are also of interest to the meat industry because of their ability to stabilize emulsions. The intrinsic emulsifying and emulsionstabilizing properties of the proteins can be derived from Figure 2. The EAI0 indicates the area of interface stabilized per unit weight of protein (m2/g) and is associated with the ability of the protein to coat the water–oil interface immediately after emulsion formation. ES represents the percentage of emulsion turbidity remaining after 10 min and therefore refers to the ability of an emulsion to resist changes in its properties over time, e.g., droplet coalescence, creaming, and/or flocculation [28]. protein as the better emulsifier compared to soy and pea proteins, as was the case in this study. Pea protein also exhibited poor emulsifying properties, as seen in Figure 2. Several studies concluded that pea proteins are usually inferior to traditional emulsifiers such as milk and egg proteins [45]. Results indicated the use of egg white protein, blood plasma, gelatin, or whey protein may be more beneficial in stabilizing meat emulsion regarding water and fat binding compared to the other screened proteins because of their high initial emulsion activity in combination with their good emulsion stability.

**Figure 2.** Comparison of emulsifying activity index immediately after emulsion formation (EAI0, m2/g) and emulsion stability (ES) of several protein sources. Mean values and standard deviations are presented (n = 4). Letters a–e: different letters indicate significant differences (*p* < 0.05) between **Figure 2.** Comparison of emulsifying activity index immediately after emulsion formation (EAI0, m2/g) and emulsion stability (ES) of several protein sources. Mean values and standard deviations are presented (n = 4). Letters a–e: different letters indicate significant differences (*p* < 0.05) between different proteins.

*3.2. Impact of Seven Different Protein-Based Ingredients on the Quality Characteristics of*  The results presented in the following sections deal with the impact of the selected proteins on several quality characteristics of phosphate-free sausage. Gelatin possesses good emulsifying properties, as can be noticed by the high values of both EAI0 and ES in Figure 2. Gelatin is capable of reducing the surface tension of aqueous environments and forming the necessary identically charged film around the fat droplets of the dispersed phase. Therefore, the isoelectric point (IEP) is of great importance in the surface activity effects of the used gelatin [40]. The protein carried a net negative charge under the conditions in which this analysis was performed. Whey proteins are well-known for their ability to stabilize interfaces, explaining their great emulsifying properties, as seen in the present research [41]. Figure 2 also shows that blood plasma and egg white protein exerted excellent emulsifying properties. Research by Rodriguez Furlán, et al. [42] confirmed the good emulsifying properties of blood plasma. Yet, literature stated that ovalbumin, the major protein in egg white, may perform good emulsifying ability and stability under extreme acidic conditions, which is in contrast to the watery suspensions applied in this research, while under neutral and alkaline pH the stability of egg white emulsions was limited [43]. The emulsifying capacity of soy concentrate was rather limited, as indicated by the low value of EAI0 in Figure 2. The study of Amine, et al. [44] also indicated soy protein as a poor emulsifier for oil in water emulsions, based upon the measurement of oil droplet particle sizes. The same research presented potato protein as the better emulsifier compared to soy and pea proteins, as was the case in this study. Pea protein also exhibited poor emulsifying properties, as seen in Figure 2. Several studies concluded that pea proteins are usually inferior to traditional emulsifiers such as milk and egg proteins [45].

> Results indicated the use of egg white protein, blood plasma, gelatin, or whey protein may be more beneficial in stabilizing meat emulsion regarding water and fat binding compared to the other screened proteins because of their high initial emulsion activity in combination with their good emulsion stability.

#### *3.2. Impact of Seven Different Protein-Based Ingredients on the Quality Characteristics of Cooked Sausage*

The results presented in the following sections deal with the impact of the selected proteins on several quality characteristics of phosphate-free sausage.

3.2.1. Dynamic Viscoelastic Properties of Meat Batters Influenced by Protein Source

Stress sweeps were performed to study the structure of the raw meat batter immediately after the grinding process, prior to thermal processing. Data (Table 1, Stress sweeps) indicated that the LVR, the stress range in which the structure of the sample remains intact, significantly (*p* < 0.05) increased when TSPP was eliminated (M-TSPP) compared to the model preparation containing phosphate (M+TSPP). Since TSPP has the ability to dissociate the actomyosin complex [1], the M+TSPP batter was presumably more sensitive to external deformation. This is reflected in a significantly (*p* < 0.05) lower δbatter value compared to the M-TSPP sample, the latter having more "solid-like" behavior. In parallel, the G\*batter of M-TSPP was significantly higher compared to M+TSPP, indicating that M-TSPP showed high resistance to deformation. A higher LVRbatter, lower δbatter, and higher G\*batter as a result of phosphate elimination were also seen in our previous study [6].

The addition of protein-based ingredients to phosphate-free raw sausage batters did not affect the LVRbatter or G\*batter compared to the M-TSPP preparations, with the exception of the preparation with gelatin (M-TSPP+gelatin) and egg white protein (M-TSPP+egg). The addition of gelatin to phosphate-free raw sausage batter (M-TSPP+gelatin) significantly increased the G\*batter, which can possibly be attributed to the cold gelling capacity of the protein [46]. Raw phosphate-free sausage batter containing 2% egg white protein (M-TSPP+egg) gave rise to a significantly lower LVRbatter, and, at the same time, a remarkably high G\*batter compared to M-TSPP. An explanation of this striking observation is given in Appendix A.

To study the rheological properties of the sausage batters during thermal processing, all samples were subjected to a temperature sweep as described in Section 2.3. The heat causes the myofibrillar proteins to unfold and/or dissociate, followed by association and aggregation, resulting in a gelled system in which water and fat are entrapped [47,48]. The high G'batter-values in Figure 3 confirmed the formation of gel structures. δ76◦C,batter is lower than 10◦ for all batters, indicating a strong elastic behavior of the formed network. Significant differences in δ76◦C,batter between batters have little relevance. *Foods* **2021**, *10*, x FOR PEER REVIEW 10 of 17

**Figure 3.** G'76°C,batter and δ76°C,batter represent the elasticity modulus and phase angle, respectively, of sausage batters during rheological measurements at the end of isothermal heating at 76 °C. G'end,batter and δend,batter represent the elasticity modulus and phase angle of sausage batters after further cooling from 76 to 20 °C. Mean values and standard deviations are presented (n = 2). Abbreviations used: M (model) and TSPP (tetrasodium pyrophosphate). Letters a–e: different letters indicate significant differences (*p* < 0.05) between treatments. **Figure 3.** G'76◦C,batter and δ76◦C,batter represent the elasticity modulus and phase angle, respectively, of sausage batters during rheological measurements at the end of isothermal heating at 76 ◦C. G'end,batter and δend,batter represent the elasticity modulus and phase angle of sausage batters after further cooling from 76 to 20 ◦C. Mean values and standard deviations are presented (n = 2). Abbreviations used: M (model) and TSPP (tetrasodium pyrophosphate). Letters a–e: different letters indicate significant differences (*p* < 0.05) between treatments.

gel strength at the end of heating prior to cooling. On the other hand, upon subsequent cooling, no significant differences could be observed anymore between M+TSPP and M-TSPP (G'end,batter and δend,batter). The stronger increase of G' during heating upon the addition of phosphates was possibly caused by conformation transitions, exposure of hydrophobic groups, and the formation of more disulfide bonds of the meat proteins [49]. In other words, TSPP promoted gelation, as it aids in the extraction of myofibrillar proteins that will subsequently aggregate and gel upon thermal processing [48,50–52]. However, Sun and Holley [48] also reported that it was possible that polyphosphates do not influence myofibrillar gel strength, as this is dependent on the applied protein source and prepara-

tion and gelation conditions that are used.

Comparison between M+TSPP and M-TSPP indicated that the elimination of phos-

The strong gelling properties of potato protein nullified the drop in G'76°C,batter due to phosphate elimination (M-TSPP+potato). A similar observation can be made when egg white protein (M-TSPP+egg) is used, although the total impact of phosphate elimination on G'76,batters could not be compensated, since a significant difference in G'76°C,batters between M+TSPP and M-TSPP+egg remained. Furthermore, G'end,batter and δend,batter significantly increased (*p* < 0.05) with the addition of egg white (M-TSPP+egg) and potato proteins (M-TSPP+potato) compared to M-TSPP. Potato and egg white proteins probably formed additional protein networks or improved interactions for gel formation compared to the other proteins, leading to increased structure formation. Studies on the impact of egg albumin on the thermal gelation of myofibrillar proteins are contradictory. Some authors concluded egg proteins caused disruption of the meat gel by interfering with the gelling process of the myofibrillar proteins or by the formation of mixed egg–myofibrillar protein gels, while others reported egg proteins participated in meat gel network formation [53]. Hunt*, et al.* [54] also observed a positive effect on gelation characteristics of Alaska pollock fish protein upon the addition of dried egg white protein. No significant difference in

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**Table 1.** Structural parameters of sausage batters (stress sweeps) and hardness, pH, water, and fat binding characteristics (CL, TEF, and relative amount of H2O) of cooked sausages with phosphate (M+TSPP), without phosphate (M-TSPP), and several protein-based ingredients instead of phosphate (M-TSPP+protein). Mean values and standard deviations are presented (n = 2). Different letters indicate significant differences at *p* < 0.05. Abbreviations used: M (Model), TSPP (tetrasodium pyrophosphate), LVRbatter (linear viscoelastic region of the sausage batter), G\*batter (complex modulus of the sausage batter), δbatter (phase angle of the sausage batter), TEF (total expressible fluid), and CL (cooking loss). Superscripts a–e: different letters indicate significant differences (*p* < 0.05) between different treatments.


Comparison between M+TSPP and M-TSPP indicated that the elimination of phosphate had an effect on the viscoelastic behavior of the meat batter during heating (G'76◦C,batter δ76◦C,batter). Values of G'76,batters revealed that phosphate elimination significantly reduced gel strength at the end of heating prior to cooling. On the other hand, upon subsequent cooling, no significant differences could be observed anymore between M+TSPP and M-TSPP (G'end,batter and δend,batter). The stronger increase of G' during heating upon the addition of phosphates was possibly caused by conformation transitions, exposure of hydrophobic groups, and the formation of more disulfide bonds of the meat proteins [49]. In other words, TSPP promoted gelation, as it aids in the extraction of myofibrillar proteins that will subsequently aggregate and gel upon thermal processing [48,50–52]. However, Sun and Holley [48] also reported that it was possible that polyphosphates do not influence myofibrillar gel strength, as this is dependent on the applied protein source and preparation and gelation conditions that are used.

The strong gelling properties of potato protein nullified the drop in G'76◦C,batter due to phosphate elimination (M-TSPP+potato). A similar observation can be made when egg white protein (M-TSPP+egg) is used, although the total impact of phosphate elimination on G'76,batters could not be compensated, since a significant difference in G'76◦C,batters between M+TSPP and M-TSPP+egg remained. Furthermore, G'end,batter and δend,batter significantly increased (*p* < 0.05) with the addition of egg white (M-TSPP+egg) and potato proteins (M-TSPP+potato) compared to M-TSPP. Potato and egg white proteins probably formed additional protein networks or improved interactions for gel formation compared to the other proteins, leading to increased structure formation. Studies on the impact of egg albumin on the thermal gelation of myofibrillar proteins are contradictory. Some authors concluded egg proteins caused disruption of the meat gel by interfering with the gelling process of the myofibrillar proteins or by the formation of mixed egg–myofibrillar protein gels, while others reported egg proteins participated in meat gel network formation [53]. Hunt, et al. [54] also observed a positive effect on gelation characteristics of Alaska pollock fish protein upon the addition of dried egg white protein. No significant difference in G'76◦C,batters, G'end,batter, or δend,batter between M-TSPP and preparations with pea, gelatin, whey, blood plasma, or soy concentrate (M-TSPP+pea, M-TSPP+gelatin, M-TSPP+whey, M-TSPP+plasma, and M-TSPP+soy, respectively) could be observed. On the other hand, the studies of Wang, et al. [55] and Li, et al. [56] claimed an improvement of the gelling characteristics and structural strength of myofibrillar protein gels upon the addition of soy protein. Additionally, the addition of blood plasma has been shown to affect the thermal gelation of myofibrils and therefore influence the final gel strength [57,58]. Sun and Holley [48] stated that due to a lack of interaction between nonmeat and muscle proteins, it is possible that texture is negatively affected by interference with the gelation of the myofibrillar proteins. This could not be deducted from Figure 3, as the final gel strength (G'end,batter) of all phosphate-free batches with different proteins is similar or higher on average compared to M-TSPP.

The rather strong gelation potential of egg white and potato protein in the meat matrix during heating was also seen in the watery medium (Figure 1), as described in Section 3.1.1. Despite this similarity, batter parameters G'76◦C,batter and G'end,batter were significantly higher for potato protein compared to egg white protein, which was not the case for G'76◦C, suspension and G'end,suspension of the same proteins. Furthermore, blood plasma and whey protein resulted in similar values of G'76◦C,batter and G'end,batter compared to egg white protein, which was not observed in G'76◦C, suspension and G'end,suspension of the same proteins. In contrast, soy concentrate resulted in a significantly lower value of G'76◦C,batter compared to egg white protein, while this was not the case for G'76◦C,suspension. On the other hand, G'76◦C,batter was lowest for preparations with pea protein and gelatin, which was identically reflected in G'76◦C,suspension.

Evaluating these insights, it seems that the gelation potential of the different proteins, as determined in an aqueous medium during thermal processing (Section 3.1.1), was not always clearly noticeable in a meat system. This suggests the importance of a well-defined

meat matrix, imitating industrial meat products, to determine and understand the impact of ingredient functionality. A food environment is a more complex system, where ingredients and other components (i.e., salts, lipids, and proteins) may interact, thus modifying the added value to the product quality of the functional ingredient.

3.2.2. Emulsion Stability of Meat Batters and Cooking Loss of Cooked Sausages Influenced by Protein Source

Significant (*p* < 0.05) differences in emulsion stability and cooking loss (CL) were found between the different preparations (Table 1). Elimination of TSPP (M-TSPP) resulted in a significant increase of total expressible fluid (TEF) and thus lower emulsion stability, and increased CL compared to M+TSPP. These findings are in line with our former study [6]. It is known from the literature that TSPP is able to dissociate the actomyosin complex, releasing myosin, which can act as a natural emulsifier. Additionally, more myofibrillar proteins are extracted by TSPP, helping to stabilize the protein matrix in which water and fat are entrapped [1].

Preparations containing egg white protein (M-TSPP+egg), pea (M-TSPP+pea), soy concentrate (M-TSPP+soy), and blood plasma proteins (M-TSPP+plasma) significantly (*p* < 0.05) reduced TEF compared to M-TSPP and even resulted in similar percentages of TEF as the preparation containing phosphate (M+TSPP), indicating an equal stabilization of moisture and fat in the meat matrix. These proteins were thus able to compensate for the decreased emulsion stability due to phosphate elimination. On the other hand, there was no significant difference in TEF between M-TSPP and preparations with the addition of potato (M-TSPP+potato), whey proteins (M-TSPP+whey), and gelatin (M-TSPP+gelatin). Furthermore, the use of some proteins also caused a shift in composition (water vs. fat) of the drip loss. The relative amount of fat in the drip loss was significantly higher when adding blood plasma (M-TSPP+plasma), gelatin (M-TSPP+gelatin), soy concentrate (M-TSPP+soy), or whey protein (M-TSPP+whey) to phosphate-free sausages (M-TSPP). This could mean that fat stabilization in the meat matrix could be altered by using additional proteins, which could affect the final product characteristics such as texture or mouthfeel [59].

In almost all cases, CL significantly (*p* < 0.05) decreased with the addition of proteinbased ingredients compared to M-TSPP. The addition of blood plasma (M-TSPP+plasma), whey proteins (M-TSPP+whey), egg white proteins (M-TSPP+egg), and soy concentrate (M-TSPP+soy) even resulted in similar CL as the cooked sausages containing phosphate (M+TSPP). Blood plasma proteins are good emulsifiers [9] and were found to be a useful substitute for polyphosphate in frankfurters, as they did not affect the water holding capacity and cooking losses compared to frankfurters containing 0.5% sodium tripolyphosphate [21]. Research by Prabhu [60] also indicated blood plasma was suitable to improve the emulsion stability, texture, flavor, and juiciness of comminuted meat products. Additionally, the use of pea protein (M-TSPP+pea) could significantly decrease CL compared to M-TSPP, although to a lesser extent than the previously mentioned proteins. On the other hand, the addition of gelatin (M-TSPP+gelatin) or potato protein (M-TSPP+potato) did not change CL compared to M-TSPP. This was in contrast with the study by Nieto, Castillo, Xiong, Álvarez, Payne and Garrido [20] in which cooking losses were reduced when 2.5% hydrolyzed potato proteins were added to phosphate-free meat emulsions.

Comparison between the intrinsic properties of the proteins discussed in Section 3.1 and their impact on fat and water binding characteristics of cooked sausages indicated limited analogy. Stronger gelation potential of the protein samples as measured in the watery medium would suggest better water and especially fat binding in meat products. Furthermore, proteins with good emulsifying capacities are expected to be able to stabilize emulsified meat products to a greater extent and contribute to reducing cooking loss (especially fat release). While potato and egg white protein both showed very good gelation properties, only the latter could positively improve the cooking yield. In contrast, pea proteins showed both low emulsifying capacity and gelling behavior in the watery medium, while in the cooked sausages, they could reduce cooking loss and TEF. Results even surpassed those of potato protein. Blood plasma proteins, showing an average

gelation potential and good attribution to emulsion stability in the watery environment, outperformed the other proteins, with the exception of soy concentrate, regarding water and fat binding in cooked sausages. Again, these results underline the importance of a welldefined meat matrix, close to industrial meat products, to determine and fully understand the impact of ingredient functionality.

#### 3.2.3. pH of Cooked Sausages Influenced by Alternative Protein Source

Data (Table 1) showed that the elimination of TSPP (M-TSPP) resulted in significantly lower pH values compared to the model system containing TSPP (M+TSPP), which was in line with our former study [6]. The pH of TSPP (1% solution) is equal to 10.2 [1], which explains the pH difference between preparations M-TSPP and M+TSPP. Due to phosphate elimination, the pH of the meat product was decreased and was closer to the iso-electric pH of the myofibrillar proteins. This led to a reduction in their net charge and repulsion between proteins, causing a negative impact on water and fat binding, as seen in Section 3.2.2 [61]. The decrease in pH by phosphate elimination could not be compensated by the addition of protein-based ingredients, as seen in Table 1. Velemir, et al. [62] determined no significant difference in pH upon the addition of 1.5% whey or soy protein to sausages. Blood plasma, despite its higher pH, could also not remediate the lower pH of phosphate-free sausages, which was also seen in the research of Hurtado, Saguer, Toldrà, Parés and Carretero [21]. The proteins could therefore not contribute to water binding by generating a higher concentration of negative meat protein charges.

#### 3.2.4. Textural Properties of Cooked Sausages Influenced by Protein Type

The differences in hardness of the different preparations are limited (Table 1). Phosphate elimination (M-TSPP) did not significantly affect the hardness of the cooked sausages, which was in line with our former study [6]. A lower hardness might be expected when TSPP is eliminated, since TSPP dissociates the actomyosin complex, resulting in more proteins being available for emulsification and the formation of a more stable gel matrix during heating. Yet, gel strength at the end of thermal processing (see Section 3.2.1) also revealed no difference in G'end,batter between M+TSPP and M-TSPP. On the other hand, the increase in CL when phosphate is eliminated could lead to a firmer meat product. The addition of gelatin (M-TSPP+gelatin) increased hardness compared to M-TSPP, despite no significant difference in CL being measured. Therefore, it could be concluded that gelatin itself had an impact on the final hardness of the phosphate-free cooked sausage, which could probably be attributed to its cold gelling properties [46]. The addition of the other protein-based ingredients did not significantly affect hardness compared to M-TSPP, but a significant increase in hardness compared to the reference sausage containing phosphate (M+TSPP) was determined upon the addition of pea, potato, egg white protein, and again gelatin. Nieto, Castillo, Xiong, Álvarez, Payne and Garrido [20] found that the addition of 2.5% hydrolyzed potato proteins had no effect on the hardness of phosphate-free frankfurters, which was also in line with our results. Furthermore, Youssef and Barbut [14] concluded that soy protein could increase or decrease the product texture depending on the type of soy used. The impact of whey proteins on hardness is linked to their degree of denaturation, which is dependent on their production process. In general, undenatured whey protein preparations deteriorate textural properties, while partially denatured whey concentrates enhance the binding and texture of sausages and other comminuted meat products [25]. This could possibly explain the mild impact on the observed hardness of whey proteins. Fernandez, et al. [63] also found no difference in hardness when 2% of dried egg white was added to chicken meat batters. Cofrades, Guerra, Carballo, Fernández-Martín and Colmenero [19] noted an increase in the product hardness of Bologna sausages when blood plasma was applied. This observation could not be established in this research.

#### **4. Conclusions**

The elimination of phosphate had a negative impact on several quality characteristics of cooked sausages. Next to an increase in cooking loss and reduced emulsion stability, a change in gel network formation during thermal processing could be observed, although the final gel strength was not influenced. The cause of these quality losses is mainly related to the reduced functionality of the myofibrillar proteins due to phosphate elimination. This research indicated that the addition of specific proteins could remediate the negative impact of phosphate elimination. However, it is important to keep in mind that different protein sources exhibit varying potential in this respect. Hereby, it is crucial to evaluate the potential of the proteins in a well-defined standardized meat matrix. The intrinsic protein properties, gelation and emulsification, related to improving meat quality are often evaluated in aqueous media. This study showed that protein characteristics determined in this manner did not entirely reflect their capacity to enhance the characteristics of phosphate-free emulsified meat products.

In phosphate-free cooked sausages, blood plasma and soy protein overall showed the most promising results, as no significant differences in terms of product hardness, cooking yield, or emulsion stability could be found compared to standard phosphate-containing sausages. These proteins may therefore provide an added value for the meat industry to further reduce E-numbers and contribute to the healthy image of meat products. Other screened proteins, such as egg white, pea, and whey protein, also proved to be beneficial, yet the quality level of the phosphate-containing sausages could not be equaled. Potato protein and gelatin showed the least improvement to the phosphate-free cooked sausages.

Future research can be conducted on the use of combinations of different protein sources or mixtures of proteins with certain hydrocolloids to further remediate the loss of quality due to phosphate elimination in emulsified meat products.

**Author Contributions:** Conceptualization: O.G. and I.F.; formal analysis: S.G.; funding acquisition: I.F.; investigation: O.G., M.G., and L.S.; methodology: O.G. and I.F.; project administration: I.F.; resources: I.F.; supervision: I.F.; validation: M.G. and L.S.; visualization: O.G.; writing—original draft: O.G. and S.G.; writing—review and editing: M.G., L.S., and I.F. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was 80% funded by Flanders Food and the Agency for Innovation by Science and Technology (IWT), grant number IWT 130500, and 20% co-funded by the Flemish meat and ingredient producing industry.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** Participating companies in the research (co-funding partners) had the opportunity to propose ingredients to be included in the study. However, the final decision of ingredient choice was solely and independently made by the researchers. Protein-based ingredients were chosen to obtain a good mix of different animal and vegetable sources and with relevance to the broad industry. Furthermore, they had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

#### **Appendix A**

Raw phosphate-free sausage batter containing 2% egg white protein (M-TSPP+egg) gave rise to a significantly lower LVRbatter, and, at the same time, a remarkably high G\*batter compared to M-TSPP, as seen in Table 1 (main text). In order to gain insight into this striking observation, the stress sweeps as seen in Figure A1 were evaluated.

Future research can be conducted on the use of combinations of different protein sources or mixtures of proteins with certain hydrocolloids to further remediate the loss of

**Author Contributions:** conceptualization: O.G. and I.F.; formal analysis: S.G.; funding acquisition: I.F.; investigation: O.G., M.G., and L.S.; methodology: O.G. and I.F.; project administration: I.F.; resources: I.F.; supervision: I.F.; validation: M.G. and L.S.; visualization: O.G.; writing—original draft: O.G. and S.G.; writing—review and editing: M.G., L.S., and I.F. All authors have read and agreed to

**Funding:** This research was 80% funded by Flanders Food and the Agency for Innovation by Science and Technology (IWT), grant number IWT 130500, and 20% co-funded by the Flemish meat and

**Conflicts of Interest:** Participating companies in the research (co-funding partners) had the opportunity to propose ingredients to be included in the study. However, the final decision of ingredient choice was solely and independently made by the researchers. Protein-based ingredients were chosen to obtain a good mix of different animal and vegetable sources and with relevance to the broad industry. Furthermore, they had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Raw phosphate-free sausage batter containing 2% egg white protein (M-TSPP+egg)

ing observation, the stress sweeps as seen in Figure A1 were evaluated.

quality due to phosphate elimination in emulsified meat products.

the published version of the manuscript

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable. **Data Availability Statement:** Not applicable.

ingredient producing industry.

**Appendix A** 

**Figure A1.** Stress sweep curves of meat batters containing phosphate (M+TSPP, dash-dot line), without phosphate (M-TSPP, dotted line), and without phosphate-containing egg white proteins (M-TSPP+egg, full line). The complex modulus (G\*, black color) and phase angle (δ, grey color) are displayed on the primary and secondary y-axis, respectively. Confidence intervals are not in-**Figure A1.** Stress sweep curves of meat batters containing phosphate (M+TSPP, dash-dot line), without phosphate (M-TSPP, dotted line), and without phosphate-containing egg white proteins (M-TSPP+egg, full line). The complex modulus (G\*, black color) and phase angle (δ, grey color) are displayed on the primary and secondary y-axis, respectively. Confidence intervals are not included to enhance the readability of the graph (n = 2).

cluded to enhance the readability of the graph (n = 2).

According to Glorieux, Goemaere, Steen and Fraeye [6], the LVR is calculated as the stress level at which G\* deviates more than 5% from a constant G\* (plateau) value and indicates irreversible structure breakdown. However, the raw sausage batter containing egg white proteins (M-TSPP+egg white) contained two plateau regions in which G' and G" (and thus G\*) were independent of the applied stress amplitude. The first plateau was characterized by a high Gbatter\* value and ranged up to ±10 Pa, the stress value at which structure breakdown occurred. However, from around a stress value of 30 Pa, the structure stabilized again, resulting in another plateau that reached stress values of ±200 Pa until irreversible structure breakdown occurred (Figure A1). The two LVR regions could possibly be explained by the presence of two distinct protein structures. The first plateau is characterized by G\*batter higher than G\*batter of M-TSPP, which may be attributed to the presence of the egg white proteins. The second plateau is characterized by G\*batter values in the same order of magnitude as M-TSPP, and the LVR ends at a comparable stress value, presumably indicating that this part of the LVR was stabilized independently of the added protein-based ingredient.

#### **References**


## *Article* **Replacement of Sodium Tripolyphosphate with Iota Carrageenan in the Formulation of Restructured Ostrich Ham †**

**Sumari Schutte 1,2, Jeannine Marais <sup>2</sup> , Magdalena Muller <sup>2</sup> and Louwrens C. Hoffman 1,3,\***


**Abstract:** The influence of iota carrageenan (iota-CGN) as a partial replacement of sodium tripolyphosphate (STPP) was investigated on the physical (pH, yield, instrumental color, texture profile analysis), chemical (moisture, protein, total fat, ash, phosphate) and sensory (descriptive analysis, acceptance testing) quality of restructured ostrich ham (95% lean meat plus fat). Treatments consisted of five decreasing levels of STPP (0.70%, 0.53%, 0.35%, 0.18% and 0%) that were simultaneously substituted with five increasing levels of iota-CGN (0%, 0.1%, 0.2%, 0.3% and 0.4%). Cooked yield, hardness, cohesiveness, and gumminess of restructured ostrich ham increased (*p* ≤ 0.05) with decreasing levels of STPP (and increased levels of iota-CGN). No significant trend in instrumental color measurements or springiness were observed between treatments. Ostrich ham with 0.35% STPP and lower had increased ostrich meat aroma and flavor, while spicy aroma and flavor, mealiness and consumer acceptance decreased. Iota carrageenan can be substituted for STPP (up to 0.35% STPP and 0.2% iota-CGN) to produce reduced STPP ham.

**Keywords:** iota carrageenan; chemical composition; consumer acceptance; descriptive analysis; ham; ostrich; phosphate; polysaccharide; processed; restructured meat; sensory profile

## **1. Introduction**

Restructured ham is usually prepared from large pieces of meat that are molded together to resemble a whole muscle meat product after cooking. The actual binding of adjacent meat pieces relies on extraction of myofibrillar proteins by salt (NaCl), phosphate and mechanical action (massaging or tumbling). During subsequent heating, the latter proteins, of which myosin is the major protein, coagulate and act as a bonding agent holding the meat pieces together [1–5]. The binding properties of restructured ham are essential to produce a uniformly attractive product with desirable slicing characteristics. The most desirable properties of high-quality cooked ham are cohesiveness, textural firmness, and juiciness.

Polyphosphates are used extensively in restructured meat products due to their functional properties of increasing binding strength, water holding capacity and yield [4,6–10]. Polyphosphate action is ascribed to the increase in the pH and ionic strength in meat products [11,12]. Tripolyphosphates (TPP) are the most widely used of all the phosphates utilized in meat processing and are typically permitted up to 3.5% of final product weight. However, there is an increase in the demand for meat products with reduced phosphate levels [13].

**Citation:** Schutte, S.; Marais, J.; Muller, M.; Hoffman, L.C. Replacement of Sodium Tripolyphosphate with Iota Carrageenan in the Formulation of Restructured Ostrich Ham. *Foods* **2021**, *10*, 535. https://doi.org/ 10.3390/foods10030535

Academic Editors: Gonzalo Delgado-Pando, Tatiana Pintado and Andrea Garmyn

Received: 28 December 2020 Accepted: 2 March 2021 Published: 5 March 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 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 (https:// creativecommons.org/licenses/by/ 4.0/).

The presence of excessive amounts of phosphates in the diet may influence the calcium, iron, and magnesium balance in the human body, and can increase the risk of bone diseases [14–16]. Furthermore, consumers and retailers generally associate polyphosphates with cost reduction and lower quality products. Consumers also seem to associate the term 'polyphosphates' with non-food applications, viewing them as 'chemical products'. The former indicates an opportunity for the use of alternatives to phosphates in restructured cooked meat products [3,5,13,17]. Numerous non-meat functional ingredients, mainly proteins and polysaccharides, have been applied as binders, fillers, and extenders to improve the quality of restructured meat products [4,5,18,19]. These ingredients are primarily used for their water binding ability and texture modification functionality [20].

Hydrocolloids with their unique characteristics in building texture, stability and emulsification are of great interest in the low-fat processed meat area due to their ability to bind water and form gels [21]. Carrageenan (CGN), a sulphated polysaccharide extracted from seaweed, is a hydrocolloid used extensively in the food industry in a broad range of applications because of its water binding, thickening and gelling properties [22,23]. There are three major types: kappa (κ, gelling); iota (ι, gelling); and lambda-CGN (λ, non-gelling). These differ in degree and manner of sulphation, the position of the 3–6 anhydrogalactose residues, their pyranose ring conformations, and the cations associated with the sulphate groups [23]. Carrageenans, alone or combined with other ingredients, have been used extensively in restructured meat products [24–29] for their ability to form gels, retain water and to provide a desirable texture [30,31]. Various levels of ingredients in combination with CGN have been studied; for example, the use of 1.5% salt with iota-CGN improved the cooking yield, juiciness, and tenderness of restructured pork nuggets [25]. Kappa-CGN favorably affected hydration properties and thermal stability, yielding lower cooking loss, purge, and expressible moisture of beef gels [27], whilst kappa-CGN increased the sliceability and rigidity in roasted turkey breasts [24], and improved the adhesion in pork hams [26].

Ostrich meat is frequently marketed as a healthy alternative to other red meats as it has a favorable fatty acid profile and a low intramuscular fat content [32–34]. Ostrich meat has a high ultimate pH of ca. 6.0 [35] and should by implication have a high-water binding capacity and thus be able to retain high levels of moisture. Therefore, moisture-retaining agents, such as phosphates, in restructured ostrich meat products could be reduced.

To maintain the health characteristics of ostrich meat, it is suggested that an alternative ingredient, that mimics the textural, functional and flavor characteristics of phosphate, be introduced in the formulation of restructured meat products. Therefore, the aim of this study was to investigate the effect of replacement of sodium tripolyphosphate (STPP) with iota carrageenan (iota-CGN) on the physical, chemical, sensory characteristics, and consumer acceptability of restructured cooked ostrich ham.

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

#### *2.1. Ham Manufacture*

Five different ham formulations with decreased levels of STPP and increased levels of iota-CGN were produced (Table 1). Each treatment was formulated to contain a 95% Total Meat Equivalent (TME) on chemical analysis (lean meat and fat). Brine ingredients, expressed as percentage in the brine, consisted of 9% NaCl, 0.25% sodium erythorbate, 1% curing salt (NaCl + 0.6% nitrite), 20% starch (corn flour), 1% ground garlic, 1% ground ginger, STPP (3.5%, 2.63%, 1.75%, 0.88% and 0%, respectively), iota-CGN (0%, 0.5%, 1.0%, 1.5% and 2.0%, respectively), and water (64.25%, 64.62%, 65.00%, 65.37% and 65.75%, respectively). The corn flour was added to the brine and the meat after the first tumble cycle.


**Table 1.** Formulation of five ostrich ham treatments.

\* Salt (1.8%), curing salt (0.2%), sodium erythorbate (0.05%), ginger (0.2%), garlic (0.2%), starch (4%).

Ostrich (*Struthio camelus* var. *domesticus*) fan fillets (n = 40 different birds; 1–1.5 kg weight per fan fillet) were obtained from a local European Union approved abattoir, Mosstrich (Mossdustria, Mossel Bay, South Africa), with all the muscles being randomly sampled from one day's kill. The meat was vacuum packed and frozen before being transported to Stellenbosch; where it was stored at <sup>−</sup><sup>20</sup> ◦C until used. Iota-CGN (GENU® texturizer type MB-150F) from Tranarc (Tranarc Holdings Pty Ltd., Benmore, South Africa) was used. All the remaining ingredients were provided by a single provider, Deli Spices (Epping, Cape Town, South Africa).

The thawed (24 h at 4 ◦C) ostrich fan fillets (n = 10 fillets per batch) were cut into fist sized pieces (±100 g per piece) and mixed in a container. The meat structure was subsequently further disrupted by the mild shearing action of passing through a meat mincing machine without any cutting blades or plates. The latter opened the meat structure to facilitate brine penetration and protein extraction, without reducing particle size. The meat from each batch was then divided into five smaller batches—one batch per treatment. The brine mixture for each treatment was then added to the meat and the latter mixture was tumbled (Biro VTS-41) under vacuum (25 kPa) for 6 h (4 ◦C) with a cycle of 20 min tumble and 10 min rest. After tumbling, the ham mixtures were vacuum stuffed (Talsa Model T0101, Germany) into impermeable plastic casings. The above-mentioned procedures were followed four times to produce four replications per treatment. Each replicate sample weighed approx. 1.5 kg and was 30 cm in length and 12 cm in diameter. Each stuffed casing within each treatment was weighed and cooked in a water bath until a core temperature of 72 ◦C was reached (approximately 1 h). The internal temperature of the ham was measured using a thermocouple probe inserted into the center of the product. After cooking, the hams were immediately immersed in cold water containing ice for 15 min before refrigeration at 4 ◦C prior to subsequent analyses.

#### *2.2. Chemical Analyses*

Homogenized samples of the five ham treatments (of a randomly selected ham within each treatment) were analyzed in duplicate for total percentages of moisture, ash, and phosphorus (according to AOAC Official Methods 934.01, 942.05, and 960.03, respectively) [36]. The total crude protein content was determined on dried (60 ◦C for 24 h), defatted and ground (with a pestle and mortar to a fine powder) samples (0.1 mg) encapsulated in LecoTM foil sheets and analyzed using a Leco Protein Analyzer (FP-528, Leco Corporation). An EDTA calibration sample (Leco Corporation, St. Joseph, HI, USA, Part number 502–092, lot number 1038) was analyzed before and after every 10 samples, with the intention of ensuring the accuracy and recovery rate of each sample. A Nitrogen conversion factor of 6.25 was used to determine the total protein content. The total fat content was determined by extracting the fat with a 2:1 mixture of chloroform:methanol [37]. The laboratory at the Department of Animal Sciences, Stellenbosch University, is accredited by the Agricultural Laboratory Association of South Africa (AgriLASA) to perform accurate and reliable proximate analyses. For validation of accuracy and repeatability, the laboratory partakes in the monthly National Inter-laboratory Scheme where blind tests are conducted. The lean meat equivalent (LME) was calculated using a conversion factor of 30 to convert protein to lean meat and the total meat equivalent (TME) was obtained through the summation of the LME and fat.

#### *2.3. Physical Analyses*

The pH of the refrigerated (4 ◦C) cooked hams was measured with the use of a calibrated (standard buffers pH 4.0 and 7.0) portable Testo 502 pH-meter. Cooked yield, color (CIE lightness L\*, a\* and b\* color coordinates) and Texture Profile Analysis (TPA) measurements were recorded on each of the four ham replicates per treatment. Cooking yield was expressed as follows:

Cooked yield (%) = (W1 − W2) × 100 where W1 = ham weight after cooking and W2 = ham weight before cooking

The weight of the cooked product was recorded after 24 h chilling (4 ◦C), when the products were removed from the casings, touch dried with absorbent paper, and casing weight recorded, separate from product weight. Product weight losses occurred primarily during thermal processing; weight loss due to the exudate remaining in the tumbler was small (about 1%) as the tumbler surfaces had been scraped with a spatula to reclaim as much exudate as possible.

Instrumental color measurements of cooked ham were recorded on three slices obtained from each of the four ham replicates per treatment [38]. A color-guide 45◦/O◦ colorimeter (Cat no: 6805; BYK-Gardner, BYK-Instruments, Orlando, FL, USA) was used; the colorimeter was calibrated using the supplied calibration white tile according to the supplier's instruction before and between every 10 samples. Three ham slices (1.5 to 2.0 cm thick) of each treatment were allowed to "bloom" for 30 min at ambient temperature (*ca.* 20 ◦C) prior to color measurements. Four color measurements were recorded for each slice at randomly selected positions and expressed by the coordinated L\*, a\* and b\* of the CIELab colorimetric space. In the color space L\* indicates lightness and a\* and b\* are the chromaticity coordinates, where a\* is the red-green range, and b\* the yellow-blue range of the color spectrum.

Instrumental textural properties were analyzed using the Instron Universal Testing Machine (UTM, model 3344, 825 University Ave, Norwood, MA, 02062–2643, USA). Texture Profile Analysis (TPA) was performed on five cores (2.5 cm height and 2 cm diameter) per slice (two slices of each of the four replicates within the five treatments = 40 measurements per treatment). The cores were placed on the platform of the UTM. A circular plate of 2.5 cm diameter was attached to a 50 N load cell and the sample was compressed to 50% of its original height at a cross head speed of 200 mm/min twice in two cycles [39]. Hardness (N), springiness (mm), cohesiveness (ratio) and gumminess (N) were calculated for each sample [39].

#### *2.4. Sensory Evaluation*

#### 2.4.1. Descriptive Sensory Analysis

Descriptive sensory analysis (DSA) was conducted to determine the effect of STPP reduction on the sensory quality characteristics of all five treatments of ostrich ham [40]. For each treatment four replicate encased hams were produced. The encased hams (stored at 4 ◦C) were opened 2 h prior to sensory analysis, sliced into 3.5 mm thick slices and vacuum packed (Multivac C200, Bahnhofstraße 4, D-87787 Wolfertschwenden, Germany). Four slices were placed next to each other and the slices did not overlap when vacuum packed.

A panel of assessors (n = 8), with extensive experience in DSA of meat, was trained in two interactive sessions to familiarize them with the treatments and to identify the aroma, flavor and mouthfeel characteristics associated with the respective treatments. Reference standards were also used to enable the assessors to calibrate their sensory perception during

training, thereby allowing them to recognize and score all the characteristics tested in the respective treatments. The reference standards included commercial beef fillet, ostrich fan fillet and pork ham, resembling the meaty, ostrich meat and spicy aroma and flavor notes, respectively. Beef liver was used to illustrate a mealy meat texture. The questionnaire was compiled during the first training session and refined and tested during the second training session. Unstructured 100-point line scales were used to analyze the sensory characteristics. Table 2 depicts the sensory characteristics and definitions used.


**Table 2.** Definitions of sensory characteristics for descriptive sensory analysis of five ostrich ham treatments.

Sensory testing was performed in individual booths fitted with Compusense® software (Compusense, Guelph, ON, Canada) in a temperature—(20 ◦C) and light-controlled (equivalent to daylight) sensory evaluation area. A sample of each of the five treatments was served to the assessors in a randomized order in four replicate test sessions (two sessions per day). The sample size per treatment per test session was one slice, with each assessor receiving an eighth (1/ <sup>8</sup>) of a slice. Each sample was coded with a three-digit blinding code and served at a refrigeration temperature of ca. 6–10 ◦C. Assessors were provided with distilled water, dried apple pieces and water biscuits as palate cleansers.

#### 2.4.2. Acceptance Testing

Sensory acceptance testing was conducted with a hundred target consumers (79 females, 21 males) recruited among staff and students at Stellenbosch University, Stellenbosch, South Africa. The consumers tested three of the treatments (STPP levels 0.70%, 0.35% and 0.00%), without any knowledge of the formulation of the products. The sample size per treatment per consumer was an eighth (1/8) of a slice. Samples were coded with three-digit blinding codes and served in a random order to each consumer at a refrigeration temperature of ca. 6–10 ◦C. Testing was done in a temperature- (20 ◦C) and light-controlled (equivalent to daylight) sensory evaluation area. Consumer acceptance testing was tested using the traditional nine-point hedonic scale ranging from 1 (dislike extremely) to 9 (like extremely) [40].

#### *2.5. Statistical Analysis*

The experimental design consisted of five treatments and four replicates per treatment. One-way analysis of variance (ANOVA) was performed to compare treatment means in terms of chemical, physical and sensory data, using SAS version 9.1 statistical software [41]. The Shapiro–Wilk test was performed to test for non-normality [42]. In some cases, deviations from normality were the cause of one or two outliners, which were removed before the final analysis [43]. Student's t-Least Significant Difference (LSD) was calculated at a 5% significant level to compare treatment means. Pearson correlation coefficients were

also calculated to measure the strength and direction of the linear relationship between selected variables.

For the consumer data, hedonic score values of three of the treatments were subjected to one-way ANOVA. Student's t-Least Significant Difference (LSD) was calculated at a 5% significant level to compare treatment means.

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

#### *3.1. Chemical and Physical Characteristics*

The chemical composition, total meat equivalent (TME), product pH, cooking yield, textural properties, and instrumental color of the five ham treatments with decreasing levels of STPP are presented in Table 3.

**Table 3.** Means (±SD) of the chemical and physical characteristics of five ostrich hams manufactured with decreasing Sodium tripolyphosphate levels (n = 4 per treatment) \*.


\* Statistical analyses were performed on all data except for phosphorus, TME (Total Meat Equivalent: % Lean Meat Equivalent + % Total Fat) and pH, as these were measured/calculated only once per treatment; SD, Standard Deviation; LSD, Least Significant Difference (*p* = 0.05); a–d Means within the same row with different superscripts differ significantly (*<sup>p</sup>* <sup>≤</sup> 0.05), where L\* represents white (100) to black (0), a\* represents green (−ve values) to red (+ve values) and b\* represents blue (−ve values) to yellow (+ve values).

#### 3.1.1. Chemical Composition

The ham formulated with 0.18% STPP presented the highest moisture content of 74.3% that differed (*p* ≤ 0.05) from the hams formulated with 0.70%, 0.53% and 0% STPP (Table 3). As expected, since no fat was added during the manufacturing process, there were no differences (*p* > 0.05) in the lipid and protein content between the five ham treatments. In an earlier study [44], the lipid content of restructured pork shoulder was found to be in a range of 23% to 25%. This is much higher than the lipid content (2.5% to 2.9%) in the present study, which could be attributed to the low intramuscular fat content of ostrich meat [32]. The ash content decreased (*p* ≤ 0.05) with decreased levels of STPP; the ham formulated with 0.70% STPP had the highest ash content (4.0%) whilst the ham formulated with 0% STPP had the lowest (3.2%). As the spice content was kept constant, the decrease in ash content may be attributed to the decreasing STPP levels. As expected, the phosphorus content in the hams also decreased with decreasing levels of STPP. However, the phosphorus content measured in the end-product proved to be much higher than the expected calculated phosphate content. These elevated values could be due to the natural phosphorus content (0.51%) of the meat as reflected in the ham formulated with no STPP added to the brine. Since a

constant amount of phosphate was incrementally decreased in the formulation, it could be assumed that the discrepancies in the elevated phosphorus values were due to either sampling error or increased phosphorus content of a specific batch. Decreasing levels of STPP were found to have no effect on the pH of the cooked product.

#### 3.1.2. Total Meat Equivalent (TME)

In this study the TME values of the hams formulated with 0.70%, 0.53% and 0% STPP were higher than the targeted value of 95% and therefore exceeded legal requirements, whereas the TME value of the 0.18% STPP level ham was lower (93.28%) (Table 3). Once more, the reason for this variation is unknown but may be linked to the latter sample having a lower protein and higher (*p* ≤ 0.05) moisture content thus resulting in the calculated difference.

#### 3.1.3. Cooked Yield

The decrease in STPP levels with a concomitant increase in iota-CGN levels resulted in an increase (*p* ≤ 0.05) in the cooked yield of the restructured ostrich ham (Table 3). The latter can be attributed to the gelling properties and increased water binding capacity of the increased iota-CGN content [4]. During cooking, water and water-soluble components are released from myofibrils caused by the heat denaturation of the muscle proteins. Carrageenan develops a gel layer on the surface of the ham, which has a sealing effect, thereby decreasing the loss of the internal components [4]. The cooked yield levels observed in this experiment (86.0% to 94.1%) are substantially lower than that reported by Fisher and co-workers [44], who found that an ostrich ham-like product formulated with 0.3% and 1.5% phosphate produced a cooking yield of 99.21% and 99.42%, respectively. This difference could be due to different processing techniques, i.e., Fisher and co-workers [44] tumbled the meat for 20 min, whereas in this study, the meat was tumbled for 6 h.

#### 3.1.4. Instrumental Color

The lightness (L\* value) of the samples ranged between 48.1 and 51.7, redness (a\* value) between 8.3 and 9.8 and yellowness (b\* values) between 11.4 and 13.0 units (Table 3). The ham formulated with 0.35% STPP, was found to be the lightest (51.7) and least red (8.3) in color. However, the instrumental color measurements of the different ostrich ham samples revealed no pattern with relation to the decrease in STPP levels. This result is supported by a visually observed variation in the composition of each of the sample slices. Ostrich meat is known to have a darker color than other red meat types [45]. This is also evident in this study where the range of a\* values (redness) in ostrich ham (8.3 to 9.8) are much higher than that of, for example, restructured beef steaks (3.82 to 5.94) [46]. Though not measured, it was observed that storage of the chilled (<4 ◦C) ham under lighting conditions (exposure of ham to light) between manufacture and consumption (over a 2-week period) led to browning of the product (decrease in redness). Light has a pro-oxidant effect that provokes a decrease in a\* values due to oxidation and degradation of the nitroso-pigment [47,48]. This rapid oxidation warrants further investigation as does the use of higher nitrite levels to minimize this phenomenon.

#### 3.1.5. Instrumental Texture Properties

The effect of the variation of the composition within each sample slice was reflected in the results for instrumental texture as no significant pattern was observed with the incremental decrease in the STPP levels (Table 3). However, significant differences in hardness, cohesiveness and gumminess were only observed with relation to the extreme manipulation of STPP (0.70% and 0%) during this experiment. The 0.53%, 0.35% and 0.18% STPP levels did not have a significant effect on the mentioned characteristics. The observed increase in the measured textural properties may be the results of increased levels of iota-CGN that forms a firm cohesive gel structure during cooling. These findings agree with results by Ulu [49], who studied the effect of carrageenan on the cooking and textural properties of low-fat meatballs.

#### *3.2. Sensory Characteristics and Consumer Acceptance*

The effect of reduced STPP on the sensory profile of five ham treatments is shown in Table 4. A meaty aroma was found to be the highest in the ham formulated with 0.35% (30.9), followed by 0.18% and 0.70% (25.7 and 25.4, respectively) STPP. Additionally, the ham formulated with 0.35% STPP was found to have the strongest (*p* ≤ 0.05) meaty flavor, compared to the other ham treatments. All ham treatments illustrated perceptible meaty aromas and flavors, irrespective of STPP level. Ostrich meat aroma and flavor for the ham formulated with 0.18% and 0% STPP was found to be much stronger (*p* ≤ 0.05) than the other ham treatments. The assessors were not able to discriminate (*p* > 0.05) between the ham treatments formulated with 0.70%, 0.53% and 0.35% STPP in terms of ostrich meat aroma and flavor. Therefore, a STPP level in ostrich ham of 0.18% and lower, does not conceal the typical aroma and flavor of ostrich meat even though spices were included at a constant level in all five treatments. Ginger and garlic were included in the formulae to mask the typical ostrich meat aroma and flavor. The sensory assessors perceived a slight spicy aroma and flavor in all ham treatments, which was perceived at lower intensities in the ham treatments with lower STPP levels (0.18% and 0%). Mealiness was defined as the mouthfeel experienced when the meat pieces separate upon chewing. This perception is indicative of the degree of cohesion between the meat pieces of the restructured ham. It seemed that STPP levels of 0.35% and higher resulted in increased mealiness (*p* ≤ 0.05), significantly more than STPP levels 0.18% and 0.00%. Mealiness also correlated negatively (*r* > −0.9; *p* ≤ 0.05) with the instrumental textural properties, particularly with the instrumental variables, hardness, and cohesiveness [49,50]. This increased mealiness could also be attributed to the increased cooking loss (Table 3) experienced in the higher % STPP inclusion treatments. This indicates that decreasing levels of STPP (coupled with increasing levels of iota-CGN) has a negative impact on the textural quality of the product as perceived by a trained taste panel.

**Table 4.** Means (±SD) for the sensory characteristics and hedonic scores (±SE) of five ostrich hams manufactured with decreasing Sodium tripolyphosphate levels (n = 4 per treatment).


a–c Means within the same row with different superscripts differ significantly (*<sup>p</sup>* <sup>≤</sup> 0.05); SD, Standard Deviation; SE, Standard Error; LSD, Least Significant Difference (*p* = 0.05); NE, Not Evaluated. Sensory characteristics were scored on 100-point scales, whereas the 9-point hedonic scale was used to score consumer preference.

> Table 4 illustrates the degree of liking, as perceived by a group of target consumers, for three of the ostrich ham treatments. This group of consumers equally liked the ham formulated with 0.70% and 0.35% STPP (*p* > 0.05). However, the ostrich ham prepared with 0% STPP was found to be significantly (*p* ≤ 0.05) less liked (an average value of 5.4 translates to neither like nor dislike on the nine-point hedonic scale). Therefore, it can be concluded that the STPP level in ostrich ham can be successfully reduced to an acceptable level of

0.35%. These results serve as a further confirmation that further product development is necessary to produce a feasible phosphate-free ostrich ham to the consumer [51].

#### **4. Conclusions**

The results from this study indicate that the production of a reduced STPP ostrich ham is a viable option for the ostrich meat industry. Due to the variation in the composition within the replicate samples of each treatment, no significant tendency was found with decreasing levels of STPP with relation to the chemical composition and physical properties measured. However, decreasing levels of STPP showed significant increases in the cooked yield, which could be attributed to the water binding ability of the increased levels of iota-CGN. The low-fat content of ostrich ham makes it a healthy option for the consumer. Descriptive sensory analysis and consumer acceptance results revealed that the STPP level in ostrich ham could be reduced to an acceptable level of 0.35%. Further research should investigate the use of other alternatives to substitute phosphate compounds and focus on optimizing the processing technique (i.e., tumbling time) for optimum myofibrillar protein extraction to manufacture a product with optimum textural and sensory quality. Further research should also include the use of antioxidants to control color changes and shelf-life studies of the product.

**Author Contributions:** Conceptualization and investigation, L.C.H., M.M. and S.S.; supervision, L.C.H. and M.M.; writing—review and editing, L.C.H., M.M., S.S. and J.M.; funding acquisition, L.C.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research is supported by the South African Research Chairs Initiative (SARChI) and partly funded by the South African Department of Science and Technology (UID number: 84633), as administered by the National Research Foundation (NRF) of South Africa and by Stellenbosch University. Any opinions, findings and conclusions or recommendations expressed in this material are that of the author(s) and the National Research Foundation does not accept any liability in this regard.

**Institutional Review Board Statement:** Ethical review and approval were waived for this study as the meat products were processed according to South African legislation in an approved food grade facility.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in sensory and acceptance tests.

**Acknowledgments:** The inputs of Francois Mellett are appreciated.

**Conflicts of Interest:** The authors wish to declare no conflict of interest. The funders had no role in the design of the study; collection, analysis, and interpretation of data; writing of the manuscript, or in the decision to publish the research.

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