Effect of Lactiplantibacillus plantarum X22-2 on Biogenic Amine Formation and Quality of Fermented Lamb Sausage during Storage

Abstract

In this study, the safety of fermented lamb sausage was examined. The aim was to investigate the effect of Lactiplantibacillus plantarum X22-2 (LP X22-2) on the quality of, and biogenic amine (BA) formation in, fermented lamb sausages during fermentation, maturation, and storage. The results showed that LP X-22 was effective in increasing the number of lactic acid bacteria (LAB) and in significantly inhibiting the formation of putrescine, histamine, cadaverine, and tyramine in fermented lamb sausage (p < 0.05). The total volatile basic nitrogen (TVB-N) content, peroxide value (POV) of fat, pH, water activity (AW), and viscosity were lower in the LF group compared to other groups (commercial starter group—CF, and natural fermentation group—NF) (p < 0.05). Furthermore, sensory evaluation and texture profile analysis (TPA) indicated that LP X-22 significantly increased the a* value, chewiness, and hardness of the sausages (p < 0.05). Therefore, LP X-22 is recommended as a natural and safe protective culture for preserving fermented lamb sausage and maintaining the color of the sausages while improving their sensory quality and inhibiting the accumulation of BAs.

1. Introduction

Fermented lamb sausage is a highly nutritious meat product characterized by unique flavor, high protein content, good texture, and excellent storage stability due to microbial fermentation [1]. Inevitably, the growth of spoilage bacteria during the fermentation process leads to deterioration in the flavor, color, freshness, and quality of fermented sausages, and even results in frequent food safety problems. Therefore, it is crucial to improve the eating quality and safety of fermented lamb sausages during storage. Common metrics, such as biogenic amine content, total volatile basic nitrogen (TVB-N) content, colony counts, and pH, are commonly used to assess product freshness and safety [2,3].

Biogenic amines, such as histamine, putrescine, phenylethylamine, tyramine, tryptamine, spermine, and cadaverine, are mainly produced by microbial amino acid decarboxylases acting on amino acid decarboxylation [4,5,6]. An overdose of biogenic amines can trigger serious allergic reactions, such as changes in blood pressure, respiratory problems, palpitations, headaches, nausea, and other serious life-threatening reactions [7]. Both Gram-negative and Gram-positive bacteria present in fermented sausages are able to produce biogenic amines. Depending on the degree and rate of acidification in fermented sausages, Micrococcus, Staphylococcus, and Enterobacteriaceae with decarboxylase activity are inhibited, and the content of biogenic amines is reduced [8,9]. Enterobacteriaceae were reduced in the presence of lactic acid fermenters compared to the natural fermentation group. Researchers have found high levels of histidine, lysine, histidine, and ornithine decarboxylase activity in Enterobacteriaceae from fermented sausages [5]. Low pH is the most important factor in inhibiting the growth of amine decarboxylase-positive microorganisms. High storage stability is a characteristic feature of high-acid fermented sausages with pH values below 5.4. Strains containing biogenic amine-degrading enzymes in fermented meat products inhibit or degrade the synthesis of BAs by oxidizing biogenic amines to aldehyde [10,11]. Methods for reducing biogenic amine levels include the inoculation of strains, control of microbial increase, and improvement in fermentation conditions [12]. Elevated temperatures may allow microorganisms with aminoacyl decarboxylases to produce biogenic amines through amino acid catabolism from protein hydrolysis in foods with high protein content [13]. In a study by Martuscelli et al., staphylococci with amine oxidase activity, isolated from Italian sausages, showed high histamine levels, but inoculation with Lactiplantibacillus plantarum (LP) significantly reduced putrescine and cadaverine. LP as a fermenting agent for fermented sausages shortens the sausage fermentation cycle, inhibits the production of harmful substances in the human gut, and significantly improves nutrient absorption and safety.

Currently, one of the most commonly used methods for detecting the presence of BAs in food is high-performance liquid chromatography (HPLC) [14]. Researchers used high-performance liquid chromatography to analyze data from 120 fermented sausages sold in Turkey and noted that the presence of Lactobacillus, which has strong oxidative enzymatic properties, affected the amount of BA in sausages during fermentation [15]. However, there are few reports on strains screened from traditional meat products to control the increase in biogenic amines. In this study, Lactiplantibacillus plantarum (LP X22-2) with a good fermentation capacity and a better ability to reduce BAs, was screened from traditional fermented meat products as a natural preservative. LP X22-2 was added to fermented lamb sausages to analyze the effects on lamb sausage quality and BAs during product maturing and storage [16]. The results of this analysis provide a new strategy for improving the safety and physical properties of fermented lamb sausage and improving the production process of fermented meat products.

2. Materials and Methods

2.1. Materials

Fermented lamb sausages were divided into three groups: natural fermentation group (non-starter culture added, NF group); commercial starter group (commercial starter culture added, CF group, viable count: ≥10⁷ CFU/g); LF group (LP X22-2 strain was added, LF group, viable count: ≥10⁷ CFU/g).

Fermented sausage consists mainly of 20% lamb tail oil and 80% lean meat from the hind legs and belly. By mass of the minced meat, 2.5% salt, 0.5% sucrose, 0.5% dextrose, 0.5% ascorbic acid, 0.5% cumin, and 0.5% black pepper were added to it. Immediately after that, sodium nitrite and sodium nitrate were added at 70 mg/kg and 100 mg/kg, and finally, together with a leavening agent, they were mixed in well and the entire mixture was loaded into a collagen enteric coating. The starter culture was obtained as follows: LP X22-2 strain was obtained from Meat Laboratory (College of Food Science and Engineering, Inner Mongolia Agricultural University, Inner Mongolia, China). LP X22-2, which does not produce biogenic amines and is inhibitory to amine-producing bacteria, was identified [16]; Co-Hansen: commercial starter culture (Staphylococcus xylosus, Pediococcus pentosaceus pentose flake cocci).

2.2. Sausage Sample Preparation

Three different batches of sausages were repeated on different days with different raw materials. There were 12 sausages per treatment group for a total of 108 samples. Fermented sausages were made from a combination of fresh lamb tail fat and hind leg meat. The sinews of the hind leg meat were removed and the lamb tail fat was diced. Then, lamb meat and fat were mixed with auxiliary ingredients and cured overnight at a low temperature (4 °C, 12 h). Fermented lamb sausages with a diameter of 35 mm were prepared using a starter mix. After inoculation, the sausages were placed in an incubator at constant temperature and humidity (conditions: 24–25 °C, 95–98% relative humidity, 3 days). Directly after fermentation, drying and post-ripening were carried out (conditions: 14–15 °C, 72–90% relative humidity, 11 days), and after drying, the sausages were vacuum-packed and stored at 4 °C.

Samples were collected after 11 d, 21 d, 41 d, and 61 d of storage (4 °C, vacuum), respectively. Samples were collected randomly and three samples were taken from each group for the determination of quality indicators, microbiological indicators, and biogenic amine content of sausages.

2.3. Microbiological Index Determination

Prior to testing, the casings were removed from the sausages under aseptic conditions in order to collect sausage samples from the inside of the sausage bars. A sausage sample weighing 25 g was placed in a sterile homogenization cup containing 225 mL of normal saline and homogenized for 2 min at 8000 rpm. The solution was diluted 1:10. After the plates were thoroughly cleaned and placed in an incubator for 48 h (30 °C, aerobic conditions), the diluted sample was transferred to the culture medium. The total viable count was determined using a Plate Count Agar (PCA) (HuanKai, Guangdong, China) medium, and the number of lactic acid bacteria (LAB) was determined using DeMan, Rogosa, and Sharpe (MRS) (HuanKai, Guangdong, China) media.

2.4. Determination of Physical and Chemical Indicators

2.4.1. pH Value

A 10 g sample was homogenized in 90 mL of normal saline using a homogenizer (Silverson L5M-A, Chesham, UK). The sample was measured after calibrating the PB-10 digital pH meter (PB-10, Zhicheng Analytical Instrument Manufacturing Co., Ltd., Shanghai, China) at 20 °C. The standard buffer solution of the pH meter was pH 4.00, pH 6.88, and pH 9.23, respectively.

2.4.2. Water Activity (Aw)

The samples were crushed and the Aw values were measured using the HD-3A Intelligent Water Activity Meter (Huake Instrument Co., Ltd., Wuxi, China).

2.4.3. Quality Characteristics

Texture Profile Analysis (TPA) was performed on sausage samples using a texture analyzer (TMS-PRO, Food Technology Corporation, Atlanta, GA, USA) equipped with a P100 probe. Measures of hardness, tenderness, gumminess, and cohesiveness were determined by taking a portion near the center of the sausage sample and cutting it into a cube with side lengths of 1 cm. It was placed on the carrier stage, aligned with the center of the probe. Immediately after that, the test speed of the probe was set to 120 mm/min, and the compression ratio was set to 50% to set the texture profile of the sample, the measurement was cycled twice, and then the whole process was carried out at room temperature.

2.4.4. Color

The sausage color was measured after slicing the sausage, and each sample was recorded three times to calculate the average value. The results were expressed in a* (redness value), b* (yellowness value), and L* (brightness value) systems using a TCP2 (Aoyike Optoelectronic Instrument Co., Ltd., Tongzhou, Beijing) fully automatic colorimeter. The chromometer was calibrated with a standardized white tile, at a 2 °C observer angle, 50 mm aperture size, and the illuminant D65. The e value is used as the main parameter for judging the color of sausages. e-value represents the value of reddishness of the sample under the influence of brightness and yellowness [17]. The higher the e-value, the higher the redness value of the sausage. The Formula (1) for calculating this parameter is expressed as follows:

$$e=\frac{a*}{b*}+\frac{a*}{L*}$$

(1)

2.4.5. Thiobarbituric Acid Values (TBA)

The samples weighed 10 g, so 50 mL of 75% trichloroacetic acid (including 1% EDTA) was added, and then the product was shaken for 30 min and filtered with double-layer filter paper. Then, 5 mL of 0.02 M TBA solution was added to the 5 mL supernatant, warmed in a 90 °C water bath for 40 min, then cooled for 1 h. The cooled sample was centrifuged for 5 min (16,000 rpm), 5 mL of chloroform was added to the supernatant and shaken well, then left to stand for stratification, before the supernatant was removed, and the optical density (OD) value was measured at 532 nm and 600 nm.

In the process of TBA value determination, the OD value was measured and the TBA value was calculated using the appropriate Formula (2).

$$\mathrm{TBA} (\mathrm{mg}/100 \mathrm{g})=\frac{(\mathrm{O}\mathrm{D}532-\mathrm{O}\mathrm{D}600)}{155\times \left(1/10\right)}\times 72.6\times 100$$

(2)

2.4.6. Total Volatile Basic Nitrogen (TVB-N)

The content of TVB-N in fermented sausages was determined according to the Chinese national standard (GB 5009.228-2016 Determination of volatile salty nitrogen in food). For this purpose, 10 g of sausage was ground and stirred before being put into a distillation tube, to which 75 mL of water was added, and the sausage was fully shaken and macerated for 30 min so that the sausage and water were completely mixed. Then, 1 g of magnesium chloride powder was added into the distillation tube and immediately connected to an automatic Kjeldahl nitrogen analyzer (FOSS, Copenhagen, Denmark). Thereafter, the automatic Kjeldahl nitrogen analyzer was operated according to the instrument instructions. Finally, the obtained distillate was titrated, and the content of TVB-N was calculated according to the results based on the appropriate formula.

$$\mathrm{X} (\mathrm{m}\mathrm{g}/100 \mathrm{g})=\frac{\left(\mathrm{V}1-\mathrm{V}2\right)\times \mathrm{c}\times 14}{\mathrm{m}}\times 100$$

(3)

X—the content of volatile salt nitrogen in the sample. The unit is milligrams per hundred grams (mg/100 g); V1—the volume of hydrochloric acid standard titration solution consumed by the sample. The unit is milliliters (mL); V2—the volume of hydrochloric acid titration solution consumed by the reagent blank. The unit is milliliters (mL); c—the concentration of the standard hydrochloric acid titration solution. The unit is moles per liter (mol/L); m—the mass of the sample. The unit is grams.

2.4.7. Peroxide Value (POV)

The peroxide value of the fermented sausage was determined according to the Chinese national standard (GB 5009.227-2016 Determination of peroxide value in foodstuffs). A mixture of trichloromethane and glacial acetic acid, a saturated solution of potassium iodide, a 1% starch indicator, a standard solution of sodium thiosulfate, and a pretreatment of petroleum ether were prepared according to the standard. After 10 g of fermented sausage was crumbled and added with 3 times the sample volume of petroleum ether, it was mixed thoroughly and left to stand for 12 h, then filtered through a funnel containing anhydrous sodium sulfate. The resulting filtrate was rotary evaporated at 40 degrees Celsius to remove the petroleum ether, and then the sample was obtained.

Thereafter, 2.5 g of the sample was weighed and placed into a 30 mL mixture of chloroform and glacial acetic acid to dissolve the sample completely. Then, 1.00 mL of saturated potassium iodide solution was added. The solution was shaken well for 0.5 min and allowed to stand in the dark for 3 min. Titration was immediately performed with sodium thiosulfate standard solution (0.002 mol/L standard solution) by adding 100 mL of water and shaking well. When the titration color turned light yellow, 1 mL of the starch indicator was added. The titration continued and was shaken until the blue color of the solution disappeared. At the same time, a blank test was performed. The volume V0 of the 0.01 mol/L sodium thiosulfate solution used in the blank test did not exceed 0.1 mL.

$$\mathrm{X} (\mathrm{g}/100 \mathrm{g})=\frac{\left(\mathrm{V}-\mathrm{V}0\right)\times \mathrm{c}\times 0.1269}{\mathrm{m}}\times 100$$

(4)

X—peroxide value, in grams per hundred grams (g/100 g); V—the volume of sodium thiosulfate standard solution consumed by the sample, in mL; V0—the volume of sodium thiosulfate standard solution consumed in blank test, unit: mL; c—concentration of sodium thiosulfate standard solution, unit: mol/L; 0.1269—the mass of iodine equivalent to 1.00 mL sodium thiosulfate standard titration solution [c (Na₂S₂O₃) = 1.000 mol/L]; m: Sample mass, in grams (g); 100: conversion factor.

2.4.8. Biogenic Amines (BAs) in Fermented Sausages

Biogenic amines in fermented lamb sausage were determined using the method described by Mazzucco et al. [18]. Standards of biogenic amines (BAs, histamine, putrescine, tyramine, tryptamine, phenylethylamine, cadaverine, spermine, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) and dansyl chloride (Macklin, Shanghai, China) were used. Identification was performed using high-performance liquid chromatography (HPLC) [19]. Samples were prepared and extracted according to the method described by Eerola et al. [20]. HPLC analysis was performed on an Agilent 1260 series liquid chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a diode array detector (DAD), Chemstation software, a binary pump, a vacuum degasser, an autosampler, and a thermostatic column chamber. HPLC reference conditions were as follows: column length 250 mm; column inner diameter 4.6 mm; column packing size 5 μm; UV detection wavelength 254 nm; injection volume 20 μm; column temperature 35 °C; mobile phase A, 90% acetonitrile 10% (0.01 mol/L ammonium acetate solution containing 0.1% acetic acid); mobile phase B, 10% acetonitrile 90% (0.01 mol/L ammonium acetate solution containing 0.1% acetic acid); mobile phase B, 10% acetonitrile 90% (0.01 mol/L ammonium acetate solution containing 0.1% acetic acid); mobile phase B, 10% acetonitrile 90% (0.01 mol/L ammonium acetate solution containing 0.1% acetic acid); at a flow rate of 0.8 mL/min. Table S1 shows the gradient elution procedure. The column temperature was 40 °C, and the flow rate was 1.5 mL/min. The standard curve for biogenic amines was obtained with reference to Sun et al. [16].

$$\mathrm{X} (\mathrm{m}\mathrm{g}/\mathrm{k}\mathrm{g})=\frac{\mathrm{C}\times \mathrm{V}\times \mathrm{f}}{\mathrm{m}}$$

(5)

X—the amount of the measured component in the sample. The unit is milligrams per kilogram (mg/kg); c—the concentration of the measured component in the sample solution. The unit is microgram per liter (mg/L); V—the volume of the sample solution. The unit is milliliters (mL); f—the number of dilutions; m—the mass of the sample. The unit is grams (g).

2.5. Data Analysis

Data were analyzed using SPSS 26 (IBM, Amonk, NY, USA). R (v4.1.1) was used for data statistics. The Kruskal–Wallis test was used to compare differences between different groups at the same time, the Friedman test was used for the same group at different times, and Bonferroni Correction was used to correct the obtained p-value. Adjust p < 0.05 was considered a significant difference. All results are expressed as mean ± standard errors (S.E.). A Random Forest classification model was constructed using the R package “Random Forest” and “rfPermute” to screen the indicators that significantly contributed to the differences among the three groups. Principal component analysis (PCA) was performed on the screened indicators using the R package “factoextra”, and the differences among the three groups on PC1 and PC2 were compared using ANOVA and Tukey’s test. The R package “vegan” was used for the p-value of Adonis.

3. Results and Discussion

3.1. Analysis of the Total Viable Count, LAB Count, pH, and Water Activity

The changes in the total viable count (TVC) and the total number of lactic acid bacteria colonies, as well as the pH and Aw values of the three groups of fermented lamb sausages during storage were examined. The TVC of all three groups of fermented sausages decreased (10⁶–10⁸ cfu/g) throughout the storage process (Figure 1A). The CF group showed the most significant decrease in TVC from day 11 to day 21 (p < 0.05). However, the CF group had the highest TVC of the three groups by day 41 (p < 0.05). For the NF and CF groups, the trend of TVC was similar, with a decrease in TVC from day 11 to day 41, but an increase in TVC from day 41 to day 61. The NF group had the lowest TVC on day 11 compared to the other two groups (p < 0.05) because of its natural fermentation, without the addition of fermentation agents. For the LF group, the TVC on days 41 and 61 were significantly lower than the TVC on day 11 (p < 0.05). The trends regarding the changes in LAB count during storage were similar for all three groups (Figure 1B). The changes for each group were not significant during the storage period (p > 0.05). However, on the 41st day, the LF group had a significantly higher number of lactic acid bacteria colonies of 10⁸ CFU/g than both the NF group and the CF group (p < 0.05).

The pH values of all three groups changed similarly during storage (Figure 1C). However, the LF group had consistently lower values than the other two groups (p < 0.05). The rise in pH of the groups during storage may be due to microorganisms acting with enzymes to break down proteins and fats, thus producing some alkaline substances.

The Aw of all three groups decreased steadily during fermentation (Figure 1D). On the 11th, 21st, and 61st days of storage, the Aw values of the NF group were significantly higher than those of the other two groups (p < 0.05). All three groups decreased below 0.8 in water activity. In addition, the Aw values of the LF group were significantly lower than those of the other two groups on the 41st and 61st days compared with those recorded on the 11th day (p < 0.05). The reduction in Aw values and pH values inhibited the growth of microorganisms [21]. The AW values of the control group were higher than those of the other two groups, which could be attributed to the ability of the fermenters to accelerate the evaporation of water from the fermented sausage and reduce the moisture content in the finished product, resulting in uniform drying of the sausage as well as inhibiting the growth of microorganisms.

3.2. Analysis of Color

Color is a critical quality indicator for fermented sausages, with redness (a*) as the primary reference. Lightness (L*) and yellowness (b*) values also affect color perception. In particular, the higher a* values observed in the LF group (Figure 2B) indicate that LP X22-2 has a beneficial influence on the color of fermented sausages. This may be due to the fact that this strain promotes the synthesis of red substances, including myoglobin nitrite and hemoglobin nitrite [22]. The color assessment of fermented sausages has an important role in the determination of their overall quality. Protein denaturation can result from phenomena related to pH and temperature. At pH 6.2, proteins begin to denature, causing the filamentous lattice to contract and increasing free water and light scattering. As temperature increases [23,24,25], protein denaturation increases, and the meat becomes lighter in color. The recorded pH was below 5.6 in all three groups, indicating that most of the subproteins, among all groups, were not denatured.

During the storage period, the a* values of the LF group were significantly higher than those of the NF group (p < 0.05). In addition, the LF group had higher a* values on days 21–41 than the other two groups (p < 0.05). The fermented sausages of the LF group had significantly higher a* values on days 21 and 61 than on day 41 (p < 0.05), and the L* values of the NF and LF groups were significantly higher than those of the CF group on days 21 and 61 (p < 0.05) (Figure 2A). During storage, the b* values of the LF group were higher than those of the NF group (Figure 2C), indicating that the LF group probably promoted the oxidation of lipids. The b* values of the fermented sausage were significantly higher (p < 0.05) in the LF group on days 21 and 61 as compared with those on day 41. Regarding changes in the e-value of fermented lamb sausage in the three groups (Figure 2D), the e-values of all three groups increased at the beginning and then decreased during the drying and ripening processes. The e-values reached a peak at the end of the treatment (11 days). After 41 days of storage, the e-value of the LF group showed a significant increase (p < 0.05), while those of the other two groups showed no significant change (p > 0.05).

In conclusion, color is an important aspect in the quality evaluation of fermented sausages, where the a* value is the main reference factor. The a*, b*, and L* values of fermented sausages were increased by the activity of LP X22-2, probably due to the fact that LP X22-2 was able to better nitrosate myoglobin in the meat and form a stable nitrosomyoglobin pigment with nitrites, which release nitrogen oxides, and increase the level of lipid oxidation. The potential of LP X22-2 to change the color of fermented sausages was demonstrated in these studies.

3.3. Analysis of TBARS, TVB-N, and POV

The maximum value of TBARS in the LF group was 0.478 mg/kg on the 11th day (Figure 3A), which was significantly higher than that in the CF group (p < 0.05). At the same time, the value of TBARS in the LF group was significantly higher than that in the CF group on the 41st and 61st days (p < 0.05). The TBARS value indicates the amount of malondialdehyde in the product and represents the degree of lipid secondary oxidation. The TBARS values of all three groups of fermented lamb sausages decreased during storage. The suitable moisture temperature during the processing of the fermented products could help in the decomposition of the unsaturated fatty acids and aldehydes. The reduction in TBARS values during storage might be due to the carboxyammonia reaction of aldehydes with substances such as proteins and amino acids containing amino side-chain radicals [26]. The higher the TBARS value, the higher the degree of lipid oxidation. Lipid oxidation proceeds through a free radical chain mechanism to produce a number of end products. The most widely studied of these is malondialdehyde (MDA) because of its association with off-flavors in meat products [27].

Throughout the storage of fermented lamb sausages, TVB-N increased in all groups (Figure 3B). At the end of the drying process, the TVB-N levels in the LF group were significantly lower than those in the NF and CF groups (p < 0.05). The most significant difference in TVB-N among the groups was observed on day 21, when the TVB-N levels were 49.230 mg/100 g in the NF group, 30.973 mg/100 g in the CF group, and only 19.940 mg/100 g in the LF group. On day 41, the LF group TVB-N was significantly lower than those of both the NF and CF groups (p < 0.05). A primary indicator of meat quality and sanitation, TVB-N, an alkaline nitrogenous substance produced by the enzymatic breakdown of proteins in meat products, contributes to a sour odor. Elevated levels of TVB-N have been shown to have adverse effects on human health [28]. The increase in TVB-N observed in the fermented lamb sausages suggests the production of nitrogenous substances. This production is facilitated by microorganisms and enzymes. In particular, the LF group had significantly lower TVB-N levels than the other two groups. This suggests that LP X22-2 can reduce the production of TVB-N in fermented lamb sausages and improve the safety of the products. In other words, the increase in biogenic amines was reduced through the use of Lactobacillus strains in LF group samples. The possible mechanism for this effect could be a reduction in the production of amino acids, which are the precursors of biogenic amines, due to a decrease in protein hydrolysis capacity.

POV reflects the accumulation of peroxides and is an indicator of primary lipid oxidation. The POV value of all groups increased and then decreased during storage, which is consistent with the findings of Sohn et al. [29]. During storage, the POV values of the three groups of sausages changed slightly, but did not change significantly (p > 0.05) with time. This may be due to the fact that peroxides, which can easily be further oxidized and produce other substances, are the main products of lipid oxidation. As shown in Figure 3C, the NF group reached a maximum POV value of 0.069 (g/100 g) at 41 d, while the CF and LF groups reached maximum POV values of 0.115 (g/100 g) at 21 d of storage. At 11 d, the POV values of all three groups were similar 0.070 (g/100 g). At 21–61 d, the POV values of the NF and LF groups were significantly lower than those of the CF group (p < 0.05). At 41 d, the LF group had significantly lower POV values than the CF group (p < 0.05) and similar values to the NF group (p > 0.05). POV decreased to 0.043 (g/100 g) (NF), 0.083 (g/100 g) (CF), and 0.060 (g/100 g) (LF) after 61 days of storage. These results suggest that LP X22-2 may contribute to the maintenance of the quality and safety of fermented lamb sausages by reducing the primary oxidation of lipids.

3.4. Analysis of Biogenic Amines (BAs)

3.4.1. Biogenic Amine Standard Curve

The biogenic amine standard curve was constructed by plotting standard solution concentrations (0.1 to 5 μg/mL) on the horizontal axis and corresponding internal standard peak area ratios on the vertical axis. The resulting standard curves (Table S2) showed linear relationships between peak area ratios and seven biogenic amine concentrations, with correlation coefficients greater than 0.99. The elution procedure used in this study effectively separated the seven biogenic amine monomers within 10 to 28 min, providing accurate measurements of the biogenic amines present in the samples. These results are evidence of the utility of this method for the analysis of biogenic amines [16].

3.4.2. Biogenic Amines

Proteins in fermented sausages are broken down into amino acids through the action of enzymes. The amino acids are easily decarboxylated to form biologically active small molecules called biogenic amines (BAs). It is important to control the presence of these amines in fermented lamb sausages, as excessive intake of BAs can be toxic to humans [30]. The balance between amino acid decarboxylase and biogenic amine oxidase determines the biogenic amine content of fermented foods. The introduction of a starter culture for reducing the amine content is usually achieved in one of two ways. One is by producing metabolites and the other is by inhibiting decarboxylation reactions [31]. Oxygen depletion and high acidity during fermentation may also prevent biogenic amine oxidase from effectively controlling the amine content of fermented foods [30].

The proliferation of aminogenic microbes is inhibited by the presence of LP. It competes for nutrients (organic acids, ammonia peroxide, bacteriocins, etc.) that favor the proliferation of certain types of amine-producing bacteria [32]. The acidic environment results in the degradation and denaturation of the salt-soluble proteins present in meat, which can improve its texture and increase its absorption rate. The physiological role of BA synthesis seems to be related to the defense mechanism of bacteria against acidic environments. LP may control the oxidation of BAs in food to aldehydes, hydrogen peroxide, and ammonia through its amine oxidase activity [33]. Several studies have found that using LP as a fermenting agent for fermented sausages can rapidly lower the pH of the sausages and effectively prevent the increase in amine-producing bacteria, thereby inhibiting the production of BAs [34,35].

The levels of tryptamine in the three groups of fermented lamb sausages increased with time (Figure 4A). The tryptamine contents in the LF group were significantly lower than those in the NF group throughout the storage period (p < 0.05). This indicates that the LP X22-2 strain effectively inhibited tryptamine production. According to the research report, the national regulations for tryptamine content standards are basically in the range of 5 to 27 (mg/kg). Adding starter culture during fermentation reduced tryptamine to 5 mg/kg, indicating that using a starter culture can reduce tryptamine production in fermented sausages during storage. At the beginning of the storage period (11 days), the level of phenylethylamine was significantly higher in the NF group than in the CF and LF groups (p < 0.05). Phenylethylamine levels (Figure 4B) initially increased and then decreased in all groups during subsequent storage periods. The levels of the NF group decreased from 5.62 to 4.13 (mg/kg), those of the CF group decreased from 7.23 to 3.46 (mg/kg), and those of the LF group decreased from 4.53 to 1.59 (mg/kg). Similar to the findings of Amonlaya et al., Lactiplantibacillus inhibited the production of tryptamine and reduced its content to 30 mg/kg in the fermented sausages [36]. The content of phenylethylamine was also significantly reduced due to the starter culture. During high-temperature fermentation, phenylalanine decarboxylation leads to significant phenylethylamine accumulation [37]. However, the activity of the decarboxylase decreases during the subsequent storage periods at a lower temperature (4 °C), which reduces the accumulation of phenylethylamine and promotes its degradation by the oxidase. On the 61st day, the LF group had a significantly lower level of phenylethylamine than the NF group (p < 0.05), and no significant difference was observed between the LF group and the CF group (p > 0.05).

At the end of the drying period (11 days), the putrescine content (Figure 4C) of the NF group was significantly higher than those of the CF and LF groups (p < 0.05). All three groups showed a trend of decreasing and then increasing putrescine, with the lowest values observed at 21 days of storage. At day 61, the CF and NF groups had significantly higher putrescine levels than the LF group (p < 0.05). In a parallel comparison, the addition of LP X22-2 as a starter culture resulted in more stability during storage, with significantly lower putrescine content than the other two groups (p < 0.05). The putrescine content of the CF group was significantly lower than that of the NF group (p < 0.05). Under the same storage conditions, the cadaverine contents of the CF and LF groups were 86.34 and 92.90 (mg/kg), respectively. Both were significantly lower than that of the NF group (p < 0.05) at the end of the drying period (11 days). After 21 days, the cadaverine content increased from 152.34 to 251.01 mg/kg in the NF group, increased from 86.34 to 107.19 mg/kg in the CF group, and decreased from 92.90 to 66.78 mg/kg in the LF group. The addition of the starter culture reduced the cadaverine contents (Figure 4D) in the fermented lamb sausages (p < 0.05). The study found that the histamine content in fermented lamb sausages increased with the fermentation time and reached 29.19 mg/kg at the end of fermentation [38]. The study showed the inoculation of probiotics into fermented sausages with a histamine content of 12.78 mg/kg after 22 days of storage [39]. The sausages were found to contain less than 13 mg/kg of histamine during 11 d–61 d of storage, which is lower compared to other studies.

Besides enhancing histamine and tyramine (Figure 4E) toxicity, cadaverine and putrescine generate amyl nitrite in meat products through nitrite reaction with amine oxidase. Putrescine levels escalate with increases in ornithine and spermidine (Figure 4F) due to protein breakdown over time and increases in various bacteria. Putrescine levels are significantly reduced with the addition of starter culture. The decarboxylation of lysine in fermented sausages leads to a gradual accumulation of cadaverine. The LF group had significantly lower levels of cadaverine than the NF and CF groups. The addition of the enzymes inhibits the activity of decarboxylase and reduces the accumulation of cadaveric amines. On day 41, the rate of cadaverine increase exceeded the oxidase activity, resulting in rapid cadaverine escalation. Cadaveric amines were reduced by oxidase, and LP X22-2 efficiently reduced their levels.

At the end of the drying process (11 days), the amount of histamine in each group was NF > CF > LF group. The amount of histamine in the NF group was significantly higher than those in the other two groups (p < 0.05). After 61 days of storage, the NF group histamine content increased from 20.14 to 30.37 (mg/kg), that of the CF group from 8.75 to 12.60 (mg/kg), and that of the LF group from 4.28 to 7.9 (mg/kg). In the cardiovascular system, histamine is an important neuromodulatory substance that aids in the conduction of nerve impulses. High levels of histamine can cause severe toxic effects, with a threshold of ≥40 mg [40]. In the present study, the histamine level in the NF group was significantly higher than those in the CF and LF groups. However, similar to the results of Gençcelep [41], the addition of a starter culture was found to have strong histamine metabolizing enzyme activity, which inhibited decarboxylase activity and reduced histamine accumulation.

As for the tyramine content, there was a significant difference (p < 0.05) among the three groups of fermented sausages at the end of the drying process (11 days). After 21 days of storage, the tyramine content was the lowest among the three groups (NF: 402.93 mg/kg, CF: 279.693 mg/kg, LF: 247.753 mg/kg). The tyramine content of the group with the starter culture was significantly lower (p < 0.05) than that of the NF group. However, from 41 to 61 days of storage, the tyramine content increased significantly in all three groups (p < 0.05). The tyramine content in the CF and LF groups was always lower than that in the NF group, which means that inoculating the fermenting agent can reduce the accumulation of tyramine. The tyramine content in all three groups of fermented lamb sausages was lower than the maximum level of tyramine in food (800 mg/kg) [42]. Tyramine was the most abundant biogenic amine in fermented lamb sausages in Switzerland, with a maximum concentration of 474.43 mg/kg [43]. Studies found 273.91 mg/kg tyramine in Turkish fermented sausages and these results are similar to the present study [15]. During the storage of fermented lamb sausages, the concentration of spermidine remained relatively constant, averaging between 3.0 and 6.0 mg/kg. There was no clear pattern of changes in spermine content among the different groups. Spermine is not a microbial metabolite, but is converted from putrescine [44,45].

3.5. Principle Component Analysis (PCA)

The dataset records were summarized in a confusion matrix. This matrix ordered the records according to the true category and the predicted category judgment using the classification model. The matrix had true rows and predicted columns (Table S3). All three groups, each consisting of 36 samples, were accurately predicted out of a total of 108 samples. Random Forest was used to screen the features and to visualize the data (Figure S1). In order to filter out important features that contribute to the classification of the storage phase of the three fermented lamb sausage treatments, the Random Forest algorithm was applied. A total of 14 important features were identified. These included histamine, putrescine, cadaverine, POV, TBARS, tryptamine, phenylethylamine, chewiness, cohesiveness, tyramine, adhesion, hardness, TVB-N, and AN.

To further analyze the data and extract linear combinations of biosafety and product quality attributes important for classification, Principal Component Analysis (PCA) was performed. The first two principal components (PC1 and PC2) accounted for 36.6% and 22.2% of the variance (Figure 5). Region X, located far from the origin on the positive half-axis of PC1, was identified as high in tryptamine, histamine, cadaverine, tyramine, putrescine, phenylethylamine, spermidine, and TVB-N. The NF and CF groups were located in the positive half-axis of PC1. This suggests that LP 22-2 has a stronger inhibitory effect on these important features. The results of this study demonstrate the usefulness of Random Forest and PCA for identifying important features in classifying fermented lamb sausages.

3.6. Texture Profile Analysis (TPA)

The chewiness of fermented lamb sausages showed an increasing trend in all groups during storage (

Table 1. Differences in TPA among the three groups on different days (mean ± S.E.). Different capital letters in the same row indicate significant differences between the same group of fermented sausage at different storage times. Different lowercase letters in the same column indicate significant differences among the three groups of fermented sausage at the same storage time.

Items	Groups	11d	21d	41d	61d
Hardness/g	NF	1721.05 ± 302.3917 Cb	2481.2 ± 96.6158 ABCb	3088.54 ± 69.9037 ABb	3432.13 ± 630.0595 Ab
	CF	2848.75 ± 423.9597 Ba	2789.89 ± 372.1003 Bab	4226.39 ± 203.0002 ABa	4537.09 ± 65.8715 Aab
	LF	2846.38 ± 221.572 Ba	4098.07 ± 398.1491 ABa	4348.78 ± 385.0237 Aba	7049.03 ± 652.2639 Aa
Elasticity	NF	0.62 ± 0.0859 ABa	0.62 ± 0.0694 Aa	0.51 ± 0.0004 Bb	0.52 ± 0.029 ABa
	CF	0.53 ± 0.0094 ABa	0.55 ± 0.0098 Aab	0.55 ± 0.0246 ABa	0.51 ± 0.0128 Ba
	LF	0.62 ± 0.4058 Aa	0.52 ± 0.0203 Ab	0.53 ± 0.0108 Aab	0.52 ± 0.0331 Aa
Cohesiveness	NF	0.49 ± 0.0797 Aab	0.48 ± 0.0091 Aa	0.47 ± 0.0075 Aab	0.48 ± 0.0311 Aab
	CF	0.52 ± 0.0389 Aa	0.48 ± 0.010 Aa	0.49 ± 0.0115 Aa	0.49 ± 0.0175 Aa
	LF	0.45 ± 0.0275 Bb	0.49 ± 0.0099 Aa	0.45 ± 0.0053 Bb	0.45 ± 0.0303 ABb
Chewiness	NF	501.84 ± 102.6476 Bb	732.96 ± 164.9974 ABb	722.81 ± 62.2123 Ab	581.97 ± 150.4956 ABb
	CF	762.72 ± 14.5363 ABa	731.49 ± 110.7989 Bb	1153.67 ± 69.9084 Aa	1159.01 ± 64.3266 Aab
	LF	782.35 ± 76.8353 Ba	992.5 ± 71.7348 ABa	980.8 ± 59.6045 ABab	2071.3 ± 105.899 Aa
Adhesion	NF	834.2 ± 222.8949 Bb	1187.16 ± 57.7083 Ba	1382.72 ± 176.5026 ABb	1827.81 ± 78.7324 Ab
	CF	1445.32 ± 184.6078 Ba	1408.52 ± 330.1148 Ba	1998.9 ± 189.3273 ABa	2189.24 ± 104.8466 Aab
	LF	1261 ± 207.7469 Bb	1902.54 ± 524.1228 ABa	2084.45 ± 219.8095 ABa	3153.98 ± 202.0457 Aa

In the same column, different letters mean that values are significantly different (p < 0.05). In the same row, different letters mean that values are significantly different (p < 0.05) differences in values between maturity and storage periods. Results are expressed as mean ± standard error (n = 24).

). The chewiness of the groups with added starter culture tended to be higher than that of the NF group at the later stages of storage. At 41 d, both the CF and LF groups had significantly higher chewiness than the NF group (p < 0.05). At 61 d, the chewiness of the LF group was significantly higher than that of the CF and NF groups (p < 0.05). The addition of starter culture had no significant effect on the viscosity or elasticity of fermented lamb sausages (p > 0.05). Furthermore, the viscosity and elasticity of dried fermented lamb sausages did not change significantly with storage time after drying (p > 0.05).

The hardness and adhesiveness of all three groups of sausages increased with storage time, and the LF group showed higher hardness and adhesiveness than the other two groups at an earlier stage of 21 d. At 41 d, the hardness and adhesiveness of the LF and CF groups were similar (p > 0.05), but significantly higher than the NF group (p < 0.05). At 61 d, the hardness and adhesion of the LF group were significantly higher than those of the other two groups (p < 0.05). The higher chewiness of the LF group was attributed to the rapid acid production by the starter culture, resulting in the meat losing water-holding capacity [46]. The viscosity and elasticity of the dried fermented lamb sausages did not change significantly with the storage time, suggesting that these properties reached stability after the drying process, when the Aw decreased to less than 0.8 in all the groups. The increase in hardness and adhesion could be attributed to the increased protein oxidation of lamb sausage by LP, resulting in changes in water-holding capacity, emulsification capacity, and protein solubility [47].

4. Conclusions

To summarize, the results of this study demonstrate the potential of LP X22-2 as a meat starter. The addition of Lactiplantibacillus plantarum X22-2 reduced pH, Aw, viscosity, TVB-N, and POV in fermented lamb sausage. It prevented primary fat oxidation. It also significantly increased the a* value, chewiness, and hardness of the sausages while improving the color and texture of the product. In addition, the strain effectively inhibited the increase in, and accumulation of, putrescine, histamine, cadaverine, and tyramine, thereby improving the safety of the fermented lamb sausage.