Preparation method of high-yield protease lactic acid bacteria auxiliary fermentation sausage

Abstract

The invention discloses a preparation method of a high-yield protease lactic acid bacteria auxiliary fermentation sausage, and belongs to the technical field of fermentation product preparation, and the preparation method comprises the following steps: mincing meat blocks to obtain raw material meat; vegetable protein powder and water are mixed, lactobacillus acidophilus is inoculated for fermentation, and fermented plant whey is obtained after fermentation is finished; uniformly mixing the pretreated raw material meat with table salt, sugar, a leavening agent, seasonings and fermented plant whey; according to the method, a fermentation agent is added into a casing for fermentation, the uniformly mixed materials are poured into the casing for fermentation to obtain the fermented sausage, the fermentation agent comprises lactic acid bacteria, the classification name of a strain is pediococcus pentosaceus IMAUYR3-1, the strain is preserved in the China Center for Type Culture Collection on April 29, 2025, and the preservation number is CCTCC NO: M 2025938. The pediococcus pentosaceus IMAUYR3-1 disclosed by the invention has good salt resistance, bile salt resistance, nitrite resistance and acid resistance, meets the requirements of meat product leavening agents, has good antibacterial activity, and can promote macromolecular muscle protein to be hydrolyzed into small molecular substances.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and more particularly to a method for preparing a sausage assisted by fermentation of high-protease lactic acid bacteria. Background Art

[0002] During the fermentation of meat products, muscle protein (primarily sarcoplasmic and myofibrillar proteins) is degraded by the combined action of endogenous muscle enzymes and microbial proteases. Microbial enzymes can degrade large protein molecules into small peptides and free amino acids (AA). Many AA are flavor compounds or their precursors, generating amines and organic acids through decarboxylation and deamination, which in turn form volatile flavor compounds in the product. Lactic acid bacteria (LAB) also play a crucial role in sausage flavor formation by releasing and degrading free AA, regulating the composition of non-volatile metabolites. The application of LAB with high protease activity in fermented sausages not only improves the quality characteristics of fermented meat products but also increases the concentration of free AA and short peptides, positively impacting the maturation of fermented meat products. Therefore, screening LAB that can hydrolyze large muscle proteins into small molecules and applying them to fermented sausages is of great significance for sausage processing. Summary of the Invention

[0003] Another object of the present invention is to provide a method for preparing a high-yield protease lactic acid bacteria-assisted fermented sausage. Pediococcus pentosaceus IMAUYR3-1 has good salt resistance, bile salt resistance, nitrite resistance and acid resistance, which meets the requirements of meat product starter culture. It also has good antibacterial properties and can promote the hydrolysis of large-molecule muscle proteins into small-molecule substances.

[0004] In order to achieve these objects and other advantages of the present invention, a method for preparing a high-yield protease-producing lactic acid bacteria-assisted fermented sausage is provided, comprising the following steps: Grind the meat chunks to obtain raw meat; The plant protein powder and water are mixed in a weight ratio of 1:5-10, and Lactobacillus acidophilus is inoculated and fermented at a temperature of 35°C-38°C for 12-24 hours. After the fermentation is completed, liquid whey is obtained by centrifugation. The liquid whey is heated at 80°C for 10 minutes, cooled, and then the pH of the liquid whey is adjusted to 5.8-6.0 with 0.1 mol / L phosphate buffer to obtain fermented plant whey. The amount of Lactobacillus acidophilus added is 0.05-0.15% of the total weight of the plant protein powder and water; The pretreated raw meat is mixed evenly with salt, sugar, a fermentation agent, seasonings and fermented plant whey, wherein the amount of the fermentation agent added is 0.1-0.3% of the total weight of the pretreated raw meat, and the amount of the fermented plant whey added is 10-16% of the total weight of the pretreated raw meat; Stirring the evenly mixed materials into casings for fermentation to obtain fermented sausages; Among them, the fermentation agent includes lactic acid bacteria, and the classification name of the strain is Pediococcus pentosaceus Pediococcus pentosaceus IMAUYR3-1 was deposited in the China Center for Type Culture Collection (deposit address: Wuhan University, Wuhan, China) on April 29, 2025, with the deposit number CCTCC NO: M 2025938.

[0005] Preferably, the fermentation conditions are: temperature 22-28°C, relative humidity 92-98%, and time 1-3 days; The drying conditions are as follows: the temperature of the first stage is 10-20°C, the relative humidity is 71-80%, and the time is 2-4 days; the temperature of the second stage is 7-13°C, the relative humidity is 60-70%, and the time is 5-7 days.

[0006] Preferably, at the 24th hour of fermentation, the sausage is immersed in the permeate for 60-80 minutes. After the soaking, the surface is wiped dry and the fermentation is continued. The permeate comprises flavor protease, fermented plant whey, sodium lactate, lysozyme, and the remainder is water. The mass concentration of the fermented plant whey in the permeate is 10-20%, the concentration of sodium lactate is 0.1-0.3 mol / L, the amount of lysozyme added accounts for 0.02-0.05% of the total weight of the permeate, and the weight ratio of flavor protease to lysozyme is 1:0.15-0.25.

[0007] Preferably, the permeate further comprises an enzyme composite carrier, which is composed of liposome-encapsulated lysozyme and flavor protease, and the weight ratio of liposome to permeate is 1:8-12; The preparation method of liposomes is as follows: Lecithin and cholesterol were dissolved in chloroform at a weight ratio of 3-5:1 and rotary evaporated to form a lipid film; Mixing lysozyme, flavor protease, and fermented plant whey in a weight ratio of 1:1:3-5 to obtain an enzyme mixture; The enzyme mixture was added to the lipid film, hydrated under nitrogen for 30 minutes, and then ultrasonically disrupted to form polydisperse crude liposomes at an ultrasonic power of 300 W for 5 minutes. The crude liposomes were passed through a polycarbonate membrane extruder at 35-40°C for 5-7 cycles, using filter membranes with pore sizes of 1 μm, 400 nm, and 200 nm in sequence to obtain an enzyme-containing composite carrier.

[0008] Preferably, the preparation of the enzyme mixture comprises the following steps: Preheating the fermented plant whey to 35-38° C., adding 0.06-0.08% by weight of L-cysteine ​​hydrochloride, stirring and dissolving, to obtain a solution; Add flavor protease to the dissolving solution, activate it under nitrogen protection at 25-28°C for 15 minutes to obtain a mixed solution; Dissolve lysozyme in citric acid buffer at 2-5°C and pH 5.0-5.5 to obtain a lysozyme solution at a concentration of 8-12 mg / mL; The lysozyme solution was added dropwise to the mixed solution at a flow rate of 0.5 mL / min. The system temperature was maintained at 20-22°C during the addition process. Stirring was continued for 5-20 minutes after the addition was completed. After stirring, the solution was stored at 2-4°C for later use.

[0009] Preferably, the mixing method of the pretreated raw meat and the fermented plant whey is: First, pre-mix the starter culture with 25-35% fermented plant whey at 12-15°C for 5-10 minutes to obtain an activated fermentation liquid; Mixing the pre-treated raw meat, salt, sugar, seasonings and the remaining fermented plant whey at 0-4°C and stirring until the mixture becomes viscous to obtain minced meat; Add the activated fermentation liquid to the minced meat at 18-20°C, and mix for 3-5 minutes at a vacuum degree of -0.04~-0.06MPa and a rotation speed of 20-30r / min.

[0010] Preferably, the activated fermentation liquid is mixed with the minced meat by: The activated fermentation liquid was pre-cooled to 4°C, formed into droplets using a droplet generator, and sprayed into a -25°C cold air channel at a wind speed of 2-3 m / s to freeze and form fermentation agent microspheres; At a speed of 25-28 r / min, the starter microspheres were added to the minced meat in three batches: the first batch, 40% of the total weight of the starter microspheres, was mixed for 1 minute, the second batch, 30% of the total weight of the starter microspheres, was mixed for 1 minute, and the third batch, 30% of the total weight of the microspheres, was mixed for 1-3 minutes; Before adding each batch of starter microspheres, atomized liquid of fermented plant whey saturated with CO2 is sprayed on the surface of the minced meat, with a droplet size of 20-50 μm and a spraying amount of 0.5-1.0% of the weight of the minced meat.

[0011] The present invention has at least the following beneficial effects: First, Pediococcus pentosaceus IMAUYR3-1, a strain with a strong muscle protein degradation ability, was identified from a specialty fermented food source in Inner Mongolia. This strain exhibits excellent salt, bile, nitrite, and acid tolerance, meeting the requirements of a meat starter culture and exhibiting excellent antibacterial properties. Pediococcus pentosaceus IMAUYR3-1 can hydrolyze large muscle proteins into smaller molecules, with the strain showing the greatest efficacy in degrading myofibrillar proteins.

[0012] Second, the use of Pediococcus pentosaceus IMAUYR3-1 as a starter culture in fermented sausages significantly reduced the Aw value, pH, TVB-N content, and TBARS content, while effectively improving the sausage's color, firmness, elasticity, and cohesion. Pediococcus pentosaceus IMAUYR3-1 also altered the moisture distribution of the fermented sausages, reducing the amount of immobile water. It also effectively strengthened the internal gel network structure of the fermented sausages, improving their water retention. Pediococcus pentosaceus IMAUYR3-1 significantly increased the NPN, PI, and AN contents of the fermented sausages, demonstrating its potent muscle protein degradation. A total of 18 alcohols, 17 esters, 6 aldehydes, 17 terpenes, 5 acids, 3 ketones, 5 ketones, and 4 other substances were detected in the three groups of fermented sausages.

[0013] Fourth, the present invention ferments plant whey with Lactobacillus acidophilus and then adds it to the filling. The soluble components in the plant whey act as hydrophilic colloids in the minced meat, increase the viscosity of the system, and form a gel network, thereby improving the texture evaluation of the fermented sausage. Through pre-activation of the starter, construction of a low-temperature minced meat matrix, and medium-temperature vacuum mixing, the effects of the plant whey and Pediococcus pentosaceus IMAUYR3-1 can be maximized to form a highly water-holding, dense and uniform protein gel network, thereby improving the hardness, elasticity, and cohesion of the fermented sausage. The synergistic effect of the microspheroidized starter, batch embedding, and carbon dioxide makes the protein network more uniform and controllable, and penetrates layer by layer into the deep layer of the minced meat, thereby improving the texture of the sausage.

[0014] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the morphology of the strain under an optical microscope (1000 times magnification) according to one of the technical solutions of the present invention; Figure 2 This is the growth curve of Pediococcus pentosaceus IMAUYR3-1, one of the technical solutions of the present invention; Figure 3 This is one of the technical solutions of the present invention, the salt tolerance and nitrite tolerance of Pediococcus pentosaceus IMAUYR3-1, wherein the left figure shows salt tolerance and the right figure shows nitrite tolerance; Figure 4 This is a graph showing the temperature tolerance and free amino acid nitrogen changes of Pediococcus pentosaceus IMAUYR3-1, one of the technical solutions of the present invention. The left graph shows the temperature tolerance, and the right graph shows the changes in free amino acid nitrogen with increasing fermentation days. Figure 5This is an electrophoresis analysis diagram of Pediococcus pentosaceus IMAUYR3-1, one of the technical solutions of the present invention; Figure 6 This is a graph showing the pH change and volatile basic nitrogen change of fermented sausages according to one of the technical solutions of the present invention, wherein the left graph shows the pH change and the right graph shows the volatile basic nitrogen; Figure 7 This is the NPN content of fermented sausage according to one of the technical solutions of the present invention; Figure 8 This is one of the technical solutions of the present invention, which is the distribution of water in different forms during the processing and storage of fermented sausages; Figure 9 This is a scanning electron microscope image of the fermented sausage structure according to one of the technical solutions of the present invention. DETAILED DESCRIPTION

[0016] The present invention is further described in detail below with reference to the embodiments so that those skilled in the art can implement the invention with reference to the description.

[0017] <Example 1> Screening and Characterization Analysis of Lactic Acid Bacteria with High Protease Production 1. Initial screening of lactic acid bacteria with high protease production Extraction of myofibrillar protein: 10 g of mutton was thoroughly ground and then added to 0.03 mol / L phosphate buffer (pH 6.5) at a mass-to-volume ratio (w / v) of 1:10. The mixture was homogenized with a magnetic stirrer for 4 min at 4°C and centrifuged at 10,000 rpm for 20 min at the same temperature. The supernatant was discarded. 0.03 mol / L phosphate buffer was then added at a mass-to-volume ratio (w / v) of 1:10. The mixture was homogenized with a magnetic stirrer for 4 min at 4°C and centrifuged at 10,000 rpm for 20 min at the same temperature. This procedure was repeated three times. Then, 0.1 mol / L phosphate buffer (pH 6.5) (containing 0.7 mol / L KI and 0.02% NaN3) was added at a mass-to-volume ratio (w / v) of 1:4. The mixture was homogenized with a magnetic stirrer for 4 min at 4°C and centrifuged at 10,000 rpm for 20 min at the same temperature. The supernatant was dialyzed overnight at 4°C to obtain myofibrillar protein.

[0018] Myofibrillar proteins were extracted from mutton and perforated in a myofibrillar protein culture medium using a 6 mm diameter cork punch. 60 μL of activated bacterial culture was injected into the wells and incubated for 48 hours. After incubation, the agar layer was removed from the culture dish and stained with Coomassie Brilliant Blue and decolorized. The diameter of the clearing zone was observed and measured. The presence and size of the clearing zone determined whether the strain had the ability to hydrolyze muscle proteins. The absence of a clearing zone indicated that the strain did not hydrolyze muscle proteins.

[0019] 52 strains isolated from specialty fermented foods in Inner Mongolia were purified by streak culture. Strains with poor growth and unstable genetic performance were screened out. The hydrolysis zone diameters of myofibrillar protein were measured. Based on the measurement results, these 6 strains were selected for further rescreening. The strain numbers and hydrolysis zone diameters (mm) were as follows: ALS-2 (12.23±0.31 c )、R1-2-4(11.38±0.31 d )、YR3-1(16.62±0.27 a )、YR2-3-2(10.22±0.25 e ), BP-4 (15.82±0.59 b ), where different lowercase letters indicate significant differences ( P <0.05), YR3-1 is Pediococcus pentosaceus IMAUYR3-1.

[0020] 2. Rescreening of lactic acid bacteria with high protease production Drawing of standard curve: draw the tyrosine standard curve according to the method of GB / T23527-2009. The linear regression equation of the tyrosine standard curve is y=0.0044x+0.0969, and the linear correlation coefficient R 2 =0.9992, indicating a good linear relationship; The 6 strains obtained from the initial screening were subjected to protease activity assay, and the tyrosine content was obtained according to the standard curve.

[0021] Protease activity was calculated according to the formula: enzyme activity / (μg·mL −1 )=(K×OD×n×4) / 10, where K represents the reciprocal of the slope of the standard curve; 4 means 1 mL of the reaction solution was taken out for measurement from 4 mL (i.e., 4 times); n represents the dilution factor of the enzyme solution; and 10 means the reaction lasted 10 min.

[0022] The results showed that the protease activity of Pediococcus pentosaceus IMAUYR3-1 was (23.13±0.40) U / mL, significantly higher than that of the other strains (P<0.05). Therefore, strain YR3-1 was rescreened for the next step of the experiment.

[0023] 3. Morphological observation and physiological and biochemical tests of the re-screened lactic acid bacteria strains The isolated and purified Pediococcus pentosaceus IMAUYR3-1 was inoculated on MRS plates and cultured for 1 to 2 days. The colonies of Pediococcus pentosaceus IMAUYR3-1 on the culture medium were round in shape, with most colonies having a convex center, a diameter of 1 mm to 2 mm, neat edges, white color, opaque color, and no mucus production.

[0024] After Gram staining, the bacterial morphology was observed. The morphology of the strain under an optical microscope (1000 times) was as follows: Figure 1 As shown, Pediococcus pentosaceus IMAUYR3-1 is spherical in shape, Gram-positive in staining, and has no spores.

[0025] The activated 3rd generation strain was tested for glucose gas production, glucose fermentation, hydrogen sulfide production and bioamine production. Glucose gas production assay: The activated third-generation strain was inoculated into a glucose gas production biochemical tube and cultured at 37°C for 24 hours. The small inverted tube was observed to see if there were bubbles. No bubbles were found. Glucose fermentation test: The activated third-generation strain was inoculated into a glucose fermentation biochemical tube and cultured at 37°C for 24 hours. The color of the culture medium was observed to see if it changed from blue to yellow. The result was yellow. Hydrogen sulfide production test: The activated third-generation strain was inoculated into a hydrogen sulfide biochemical tube solid culture medium and cultured at 37°C for 24 hours. The culture medium was observed to see if there was any black precipitate. No black precipitate was found. Bioamine production test: The activated third-generation strain was inoculated on four biogenic amine solid culture media (arginine, histidine, lysine, and tyrosine), and an uninoculated sterile biogenic amine solid culture medium was used as a blank control. The culture was cultured at 37°C for 2-3 days, and the culture medium was observed to see if it changed color (from yellow to purple). If the result was yellow, the Pediococcus pentosaceus IMAUYR3-1 met the requirements for the starter strain.

[0026] Based on the comprehensive screening and physiological and biochemical test results, Pediococcus pentosaceus IMAUYR3-1 was selected for identification.

[0027] 4. Molecular biological identification and growth ability determination of Pediococcus pentosaceus IMAUYR3-1 16S rDNA sequence analysis: Genomic DNA extraction procedure: Take 1.5 mL of bacterial culture and centrifuge at 4500 rpm for 5 minutes to obtain a bacterial pellet. Add 567 μL of TE buffer to the bacterial pellet and repeatedly pipette to resuspend the pellet. Add 3 μL of proteinase K (20 mg / mL) and incubate at 37°C for 1 hour to obtain a bacterial lysate. Add 100 μL of CTAB and 100 μL of 0.7 mol / L NaCl to the bacterial lysate, mix thoroughly, and incubate at 65°C for 10 minutes to obtain a crude extract. Add 700 μL of phenol / chloroform / isoamyl alcohol to the crude extract, mix thoroughly by inversion, and centrifuge at 12000 rpm for 5 minutes. Collect the supernatant. Add 700 μL of chloroform / isoamyl alcohol to the supernatant, mix thoroughly by inversion, and centrifuge again at 12000 rpm for 5 minutes. Collect the supernatant. 500 μL of isoamyl alcohol was added to the supernatant and centrifuged at 12000 rpm for 5 minutes to obtain a DNA precipitate. The DNA precipitate was washed with 1 mL of 70% ethanol, dried naturally, and then dissolved to obtain total DNA. The sequencing was subsequently completed by Hohhot Skyray Medical Laboratory Co., Ltd. The results showed that Pediococcus pentosaceus IMAUYR3-1 and Pediococcus pentosaceus ( Pediococcus pentosaceus ) was 99%, so it was determined that Pediococcus pentosaceus IMAUYR3-1 was Pediococcus pentosaceus.

[0028] 5. Growth curve and acid production capacity analysis of Pediococcus pentosaceus IMAUYR3-1 Growth curve drawing: The activated third generation of Pediococcus pentosaceus IMAUYR3-1 was inoculated into MRS liquid medium and cultured in a 37°C incubator for 24 hours. The OD was measured every 2 hours. 600nm Value and pH value; Growth curve of Pediococcus pentosaceus IMAUYR3-1 (OD 600nm , viable bacterial count and acid production capacity) such as Figure 2 As shown ( Figure 2 The middle left picture shows the number of viable cells and OD 600nm , the right picture shows the acid production capacity, Figure 2 YR3-1 in the table represents Pediococcus pentosaceus IMAUYR3-1. The strain entered the logarithmic phase at 4 hours and gradually reached the stationary phase between 12 and 14 hours. The pH of the strain rapidly decreased between 0 and 5 hours, falling below 4.5 after 6 hours of culture. This indicates that Pediococcus pentosaceus IMAUYR3-1 has good acid production capacity. Therefore, Pediococcus pentosaceus IMAUYR3-1 meets the fermentation and acid production requirements for use as a starter culture.

[0029] 6. Tolerance test of Pediococcus pentosaceus IMAUYR3-1 The tests used Lactobacillus plantarum XAR-10 (Lactobacillus plantarum XAR-10 was from the free bacteria library and was an existing known strain) as the positive control group, and Pediococcus pentosaceus IMAUYR3-1 as the experimental group; 6.1 Salt tolerance The detection method is as follows: inoculate the activated bacteria of three generations into MRS liquid culture medium with NaCl mass fraction of 3.0%, 6.0%, 7.5%, 9.0% and 12.0%, culture at 37℃ for 24 h, and measure the absorbance value (OD) at a wavelength of 600 nm. 600nm value), the result is as follows Figure 3 (Left figure, in the figure, YR3-1 represents Pediococcus pentosaceus IMAUYR3-1, XAR-10 represents Lactobacillus plantarum XAR-10). Figure 3 It can be seen that when the mass fraction of NaCl is 6%, both Lactobacillus plantarum XAR-10 and Pediococcus pentosaceus IMAUYR3-1 grow well, and Pediococcus pentosaceus IMAUYR3-1 has the strongest tolerance. With the increase of salt concentration and the increase of osmotic pressure of fermentation liquid, the growth ability of the strain is gradually inhibited ( P <0.05). When the NaCl concentration reached 12%, Lactobacillus plantarum XAR-10 and Pediococcus pentosaceus IMAUYR3-1 almost stopped growing.

[0030] 6.2 Nitrite tolerance The detection method is as follows: inoculate the activated bacteria of the third generation into MRS liquid culture medium with NaNO2 concentrations of 30 mg / L, 60 mg / L, 90 mg / L, 120 mg / L, and 150 mg / L, culture for 24 h, and measure the absorbance value (OD) at a wavelength of 600 nm. 600nm value), the result is as follows Figure 3 (right picture). Figure 3 (Right figure) It can be seen that when the nitrite concentration is 150 mg / L, the OD of the two strains 600nm The values ​​are all greater than 1, which means they can still grow. The OD values ​​of Pediococcus pentosaceus IMAUYR3-1 and the control group Lactobacillus plantarum XAR-10 are 600nm The values ​​were 1.45±0.01 and 1.44±0.01, respectively, indicating good growth ability. At the same time, with the increase of NaNO2 concentration, the growth ability of the strain showed a significant downward trend ( P <0.05).

[0031] 6.3 Bile Salt Tolerance The detection method is as follows: 0.2 mL of activated three-generation bacterial solution was inoculated into 20 mL of MRS liquid medium containing 0.03%, 0.30% and 0.50% bile salt respectively, and cultured at 37°C for 24 h. The absorbance of the culture medium was measured at a wavelength of 600 nm (OD 600nm The tolerance of the strain to different concentrations of bile salts was calculated. The calculation results of bile salt tolerance are shown in Table 1: Bile salt tolerance = OD value of bile salt-added medium / OD value of blank medium × 100% Table 1 Bile salt tolerance test results of Pediococcus pentosaceus IMAUYR3-1 It can be seen from Table 1 (in the table, YR3-1 represents Pediococcus pentosaceus IMAUYR3-1, and XAR-10 represents Lactobacillus plantarum XAR-10) that when the bile salt content is 0.30%, the bile salt tolerance of Pediococcus pentosaceus IMAUYR3-1 is 14.37%, and Pediococcus pentosaceus IMAUYR3-1 shows strong bile salt tolerance, that is, Pediococcus pentosaceus IMAUYR3-1 has better bile salt tolerance than Lactobacillus plantarum XAR-10.

[0032] 6.4 pH tolerance The detection method is as follows: inoculate 4.0% of the inoculum into MRS liquid medium, culture at different pH (3.5, 4.5, 5.5, 6.5, 7.5) for 24 h, and measure the absorbance value (OD) of the strain growth at each pH at a wavelength of 600 nm. 600nm The results showed that both strains grew best at a pH of 6.5. At pH 6.5, the absorbance of Pediococcus pentosaceus IMAUYR3-1 was 1.6767, while that of Lactobacillus plantarum XAR-10 was 1.6893. Therefore, Pediococcus pentosaceus IMAUYR3-1 grows best under slightly acidic conditions.

[0033] 6.5 Temperature tolerance The detection method is as follows: inoculate 4.0% of the strain into MRS liquid medium, culture at different temperatures (4°C, 20°C, 30°C, 37°C, 42°C) for 24 h, and measure the absorbance value (OD) of the strain growth at each temperature at a wavelength of 600 nm. 600nm value), the result is as follows Figure 4 (Left picture). Figure 4 (Left) It can be seen that at 4℃, the growth ability of the two strains is significantly lower than that at other temperatures ( P<0.05), and the growth ability showed a trend of first increasing and then decreasing with the increase of temperature. This may be because the metabolic rate of Pediococcus IMAUYR3-1 slowed down at 4℃, which affected the growth and reproduction of the strain. At 37℃, the OD 600nm The value was 1.68±0.01, second only to the control Lactobacillus plantarum XAR-10.

[0034] 7. Antibacterial analysis Lactobacillus plantarum XAR-10 was used as the positive control group and Pediococcus pentosaceus IMAUYR3-1 was used as the antibacterial activity test group. The antibacterial ability was determined by the Oxford cup diffusion method: the indicator bacteria Escherichia coli ( Escherichia coli ATCC25922) and Staphylococcus aureus ( Staphylococcus aureus ATCC 6538) was evenly spread on nutrient agar medium, a sterile Oxford cup was placed in the culture dish, 200 μL of the strain supernatant was added, and the mixture was diffused at 4°C for 6 hours and then cultured at 37°C for 48 hours. The diameter of the inhibition zone was measured with a vernier caliper. The experimental results showed that the diameter of the inhibition zone of Pediococcus pentosaceus IMAUYR3-1: Staphylococcus aureus was 14.44±0.33 a The diameter of the inhibition zone of Escherichia coli was 19.52±0.42 a ; XAR-10: The diameter of the inhibition zone of Staphylococcus aureus was 14.07±0.71 ab The diameter of the inhibition zone of Escherichia coli was 15.66±0.23 b ; The experimental results show that the metabolites of the two strains have a certain inhibitory effect on two common pathogens, and the inhibitory effect of the strain Pediococcus pentosaceus IMAUYR3-1 on pathogenic bacteria is better than that of Lactobacillus plantarum XAR-10.

[0035] 8. Protein hydrolysis capacity analysis 8.1 Cell wall protease activity Lactobacillus plantarum XAR-10 was used as the positive control group, and Pediococcus pentosaceus IMAUYR3-1 was used as the cell wall protease activity test group. The specific method was as follows: the strain was expanded and cultured at 37℃ for 20 h until the logarithmic growth phase, and then the strain was taken out and centrifuged at 4500 r / min for 20 min at 4℃. The strain was washed with 50 mmol / L Tris-HCl (containing 30 mmol / L CaCl). 2+ The cells were washed three times with a buffer solution containing 50 mmol / L EDTA-Na2 and 50 mmol / L Tris-HCl and incubated at 37°C for 3 h. The supernatant was then used to measure the cell wall protease activity (enzyme activity unit is U·mL). -1), the results showed that the enzyme activity of Pediococcus pentosaceus IMAUYR3-1 was 12.62±0.36 a The enzyme activity of Lactobacillus plantarum XAR-10 was 11.32±0.37 b .

[0036] 8.2 Free amino acid nitrogen The myofibrillar protein fermentation system was established by inoculating a third-generation activated strain of Pediococcus pentosaceus IMAUYR3-1 into a myofibrillar protein liquid medium and cultivating it at 37°C. An uninoculated strain served as a blank control. The free amino acid nitrogen content in the myofibrillar protein fermentation broths at different fermentation days (0, 1, 2, 3, and 4) was determined using an o-phthalaldehyde derivatization colorimetric method. The absorbance was measured at a wavelength of 340 nm using a spectrophotometer. A standard curve was constructed using phenylalanine as a standard. The linear regression equation for the phenylalanine standard curve was y = 0.491x - 0.0011, with a linear correlation coefficient of 0. R 2 =0.9993, indicating a good linear relationship. The free amino acid nitrogen content was calculated according to the standard curve regression equation, such as Figure 4 (right picture). Figure 4 It can be seen that in the fermentation simulation system with different fermentation times, the content of free amino acid nitrogen in the Pediococcus pentosaceus IMAUYR3-1 group was significantly higher than that in the blank control group ( P <0.05), which proved that Pediococcus pentosaceus IMAUYR3-1 had good proteolytic ability.

[0037] 8.3 Myofibrillar Protein SDS-PAGE Electrophoresis Sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed as follows: 3 generations of activated Pediococcus pentosaceus IMAUYR3-1 were inoculated into myofibrillar protein liquid culture medium and cultured at 37°C for 48 hours to form the myofibrillar protein fermentation system. An uninoculated strain served as the blank control group. The myofibrillar protein fermentation broth from the 48-hour culture was added with 5× protein loading buffer at a volume ratio of 4:1. The sample was boiled for 10 minutes. The marker and sample were loaded in 10 μL volumes for protein electrophoresis analysis. Figure 5 As shown; Depend on Figure 5As shown, the brightness of each protein band in the experimental group was significantly reduced compared to the blank control, indicating that the strain promoted the degradation of myofibrillar proteins. The actin band (Band 1, 45 kDa) in the treated group was lighter than that in the blank group, indicating that IMAUYR3-1 has a strong ability to degrade muscle proteins. The brightness of the tropomyosin (Band 2, 40 kDa) and troponin (Band 3, 37 kDa) bands was significantly reduced, and these bands in the IMAUYR3-1 group were almost absent. These results indicate that IMAUYR3-1 has a strong ability to degrade myofibrillar proteins. The myosin light chain (Band 5, 12 kDa) band was more prominent in the experimental group compared to the blank control group, indicating that IMAUYR3-1 hydrolyzes large protein fragments into smaller ones. Therefore, IMAUYR3-1 can effectively degrade muscle proteins.

[0038] <Example 2> Whole genome sequencing of Pediococcus pentosaceus IMAUYR3-1 1. Genomic DNA extraction and sequencing Genomic DNA was extracted using an optimized SDS extraction method. Bacteria collected by centrifugation during the logarithmic phase were ground in liquid nitrogen and lysed using lysis buffer. Appropriate amounts of proteinase K and mercaptoethanol were added, and the lysis process was gently inverted to mix thoroughly. After lysis, the supernatant was cooled to room temperature and centrifuged. Two extractions were performed using chloroform / isoamyl alcohol (24:1). DNA was precipitated with isopropanol, gently inverted to mix thoroughly, and then centrifuged. The waste solution was discarded, and the precipitate was washed twice with 75% ethanol. Purification was performed using OMEGA purification columns and Ampure XP beads. Quality control was performed using Nanodrop and QbUIT, and electrophoresis.

[0039] 2. Genomic library construction and sequence determination DNA libraries from strains that passed quality inspection were sequenced using the Illumina Novaseq 6000 high-throughput sequencing platform. Sequencing was commissioned to Hangzhou Lianchuan Biotechnology Co., Ltd.

[0040] 3. Genome sequence assembly and quality control analysis The raw data quality control, genome assembly, and genome structure analysis were entrusted to Hangzhou Lianchuan Biotechnology Co., Ltd.; 4. Genome functional analysis The whole genome of Pediococcus pentosaceus IMAUYR3-1 was analyzed by orthologous clustering, gene ontology, Kyoto Encyclopedia of Genes and Genomes, and carbohydrate-active enzyme function on the Lianchuan BioCloud platform; The genome of Pediococcus pentosaceus IMAUYR3-1 consists of a 1,785,490-bp circular chromosome and a plasmid. The circular chromosome has a G+C content of 37.23%. A total of 1,879 coding genes, 1,779 predicted coding sequences (CDS), one genomic island, four prophages, and two potential CRISPR sequences were identified within the genome. The genome also contains 55 tRNA genes, 15 rRNA genes (five each for 23S rRNA, 5S rRNA, and 16S rRNA), and one tmRNA gene.

[0041] 4.1 COG functional annotation Functional category, a total of 1778 genes of Pediococcus pentosaceus IMAUYR3-1 were annotated into 23 functional categories, accounting for 94.62% of the total number of genes. The number of coding genes annotated into different categories is as follows: C category (energy production and conversion) 63, D category (cell cycle regulation, cell division, chromosome distribution) 31, E category (amino acid transport and metabolism) 128, F category (nucleotide transport and metabolism) 97, G category (carbohydrate transport and metabolism) 150, H category (coenzyme transport and metabolism) 76, I category (lipid transport and metabolism) 75, J category (translation, ribosome structure and biogenesis) 190, K category (transcription) 158, L category (replication, recombination and repair) 104, M category (cell wall / membrane / envelope biosynthesis) 10 ) 107, N category (cell motility) 14, O category (post-translational modification, protein turnover, molecular chaperone) 56, P category (inorganic ion transport and metabolism) 82, Q category (secondary metabolite biosynthesis, transport and catabolism) 22, R (general function prediction only) 172, S category (unknown function) 78, T category (signal transduction mechanism) 78, U category (intracellular transport, secretion and vesicle transport) 19, V category (defense mechanism) 43, W category (extracellular structure) 4, X category (mobile genetic elements: phage, transposon) 30, Z category (cytoskeleton) 1.

[0042] 4.2 GO functional annotation The GO database functional annotation information of Pediococcus pentosaceus IMAUYR3-1 selected the top 20 most annotated GOslim secondary categories under each category for mapping. 1320 genes were annotated to biological processes, 955 genes were annotated to cellular components, and 2168 genes were annotated to molecular functions. In the secondary functional classification of biological processes, the largest number of coding genes were annotated to translation (59) and phosphorylation (57). The rest were transmembrane transport (13), cell morphology regulation (17), DNA template transcription regulation (20), protein hydrolysis (32), phosphoenolpyruvate-dependent sugar phosphotransferase system (32), peptidoglycan biosynthesis process (15), negative regulation of DNA template transcription (16), cell wall organization (18), cell division (19), carbohydrate metabolism process (15), DNA repair (16), 17 genes were annotated to DNA recombination, 12 genes were annotated to DNA integration, biological processes of inosinic acid synthesis, and cell cycle, 11 genes were annotated to DNA replication, glycolysis, and fatty acid biosynthesis, and 11 genes were annotated to cytoplasmic membrane (218 genes) in the second-level functional categories of cellular components, followed by cytosol (189) and cytoplasm (171), 8 genes were annotated to small ribosomal subunits, 15 genes were annotated to ribosomes, 4 genes were annotated to ribonucleoside diphosphate reductase complexes, 14 genes were annotated to ribonucleoprotein complexes, and 11 genes were annotated to proton transport ATP. 5 genes in the catalytic core F(1) of the synthase complex, 7 genes in the protein-DNA complex, 5 genes in the periplasmic space defined by the outer membrane, 162 genes in the membrane, 10 genes in the extracellular region, 10 genes in the cytoplasmic small ribosomal subunit, 20 genes in the cytoplasmic large ribosomal subunit, 6 genes in the chromosome, 4 genes in the cell envelope Sec protein transport complex, 4 genes in the cell division site, 6 genes in the bacterial microcompartment, 8 genes in the DNA-directed RNA polymerase complex, and 11 genes in the ATP-binding cassette (ABC) transporter complex. Among the secondary functional categories included in the molecular function, the number of genes encoding ATP binding (203) was the largest, followed by DNA binding (130), zinc ion binding (8), transmembrane transporter activity (31), transferase activity (36), transcription cis-regulatory region binding (18), transfer RNA binding (23), ribosomal structural components (53), and ribosomal RNA 31 binding, 28 oxidoreductase activities, 22 nucleic acid binding, 86 metal ion binding, 46 magnesium ion binding, 23 kinase activities, 35 hydrolase activities, 15 acyltransferase activities and transfer of non-amino acyl groups, 34 RNA binding, 21 guanosine triphosphate (GTP) binding, 60 DNA binding transcription factor activities, and 56 ATP hydrolysis activities.

[0043] 4.3 KEGG functional annotation KEGG database functional annotation information for Pediococcus pentosaceus IMAUYR3-1 includes: Cellular Processes (45 prokaryotic cell populations), Cell Motility (7), Cell Growth and Death (10); Environmental Information Processing (34 signal transduction, 93 membrane transport); Genetic Information Processing (79 translation, 6 transcription, 45 replication and repair, and 32 folding, sorting, and degradation); Biological Systems (3 immune system, 3 environmental adaptation); Metabolism (16 xenobiotic biodegradation and metabolism, 81 nucleotide metabolism, 13 terpenoid and polyketide metabolism, 31 other amino acid metabolism, 76 cofactor and vitamin metabolism, 49 lipid metabolism, 64 glycan biosynthesis and metabolism, 408 global and overview maps, 45 energy metabolism, 157 carbohydrate metabolism, 22 other secondary metabolite biosynthesis, and 70 amino acid metabolism). The results indicate that this strain has a robust metabolism and strong growth and reproduction capabilities.

[0044] 4.4 Carbohydrate enzyme annotation The CAZy database annotation of Pediococcus pentosaceus IMAUYR3-1 identified 42 carbohydrate-active enzymes, including 20 glycosyltransferases, 17 glycoside hydrolases, 4 carbohydrate esterases, and 1 auxiliary oxidoreductase. Among the predicted glycosyl hydrolase families, protein family GT51 was found in Pediococcus pentosaceus IMAUYR3-1, which indirectly participates in proteolysis regulation and enhances protease activity, demonstrating that Pediococcus pentosaceus IMAUYR3-1 possesses good proteolytic capacity in fermented meat products.

[0045] 4.5 Protein degradation genes Genes related to protein degradation in Pediococcus pentosaceus IMAUYR3-1: signal peptidase II (lspA), ATP-dependent proteases (clpA, ClpE, clpX, clpQ, clpC, hslU), proline peptidase (pepD), DNA-binding transcription repressor (LexA), oligopeptidase (pepF), oligopeptidase (pepB), glutamyl aminopeptidase (pepA), dipeptidase (pepDB), transpeptidase A (srtA), aminopeptidase (ampS, pepS, ampT), carboxyl-terminal processing protease (ctpA), penicillin-binding protein (pbp2A), tripeptidyl aminopeptidase (pepT), methionyl aminopeptidase (map), regulator of sigma E protease (rseP), DNA repair protein (radC), cell division protease (ftsH, hflB), matrix metalloproteinase (MMP24), puromycin-sensitive aminopeptidase (NPEPPS), heat shock protein (htpX), L -proline amidohydrolase (laaA), X-Pro dipeptidase (pepX), non-heme chloroperoxidase (cpo), putative serine protease (pepD); Genome-wide functional gene annotation of Pediococcus pentosaceus IMAUYR3-1 based on the COG, GO, and KEGG databases revealed that IMAUYR3-1 possesses functional gene segments involved in proteolysis, a process involved in protein catabolism. Genes encoding oligopeptidases (including dipeptidases and tripeptidases) include pepF, pepB, pepDA, pepDB, pepX, and pepT, which cleave peptide bonds in proteins, hydrolyzing them into smaller peptides and / or amino acids. The gene encoding glutamine aminopeptidase, pepA, specifically hydrolyzes peptide bonds containing glutamyl groups, releasing free glutamate. Glutamate contributes significantly to the flavor of fermented meats, enhancing their umami flavor.

[0046] <Example 3> Preparation of fermented sausages Mutton was used as the raw material: the raw meat (lamb hind leg meat and sheep tail fat were minced and mixed, wherein the amount ratio of lamb hind leg meat and sheep tail fat was 8:2), and the following materials were added according to the mass ratio with the raw meat: salt 20 g / kg, glucose 5 g / kg, sucrose 5 g / kg, dried ginger powder 2 g / kg, pepper powder 2 g / kg, pepper powder 1 g / kg, sodium nitrite 0.1 g / kg, white wine 25 mL / kg, ascorbic acid 0.5 g / kg, corn starch 10 g / kg, whey protein powder 5 g / kg, the number of viable bacteria in the starter was 1×10 7 CFU / g; The sausage preparation was divided into three groups according to different lactic acid bacteria starter cultures: natural fermentation group (CK, negative control), Lactobacillus plantarum XAR-10 group (XAR-10 group, positive control), and Pediococcus pentosaceus IMAUYR3-1 group (YR3-1 group); Fermented sausage production and parameters: Mince the raw meat, add spices and a starter culture (mix directly), marinate at 4°C for 12 hours, and then fill the meat filling into a 25mm diameter collagen casing. Ferment in a constant temperature and humidity chamber (25°C, 95% relative humidity) for 1-2 days. Fermentation is completed when the pH is <5, determined by pH testing (the fermented sausages of this invention are all fermented within 1 day). Drying: The first stage is 2-4 days (temperature 15°C, relative humidity 75%), and the second stage is 5-7 days (temperature 10°C, relative humidity 65%). Finally, store at 4°C.

[0047] <Example 4> A method for preparing high-protease lactic acid bacteria-assisted fermentation sausage comprises the following steps: Grinding the meat to obtain raw meat (the lamb hind leg meat and lamb tail fat are minced and mixed, wherein the lamb hind leg meat and lamb tail fat are used in a ratio of 8:2); The plant protein powder (pea protein powder) and water are mixed in a weight ratio of 1:7, and Lactobacillus acidophilus is inoculated and fermented at a temperature of 35°C-38°C for 18 hours. After the fermentation is completed, liquid whey is obtained by centrifugation. The liquid whey is heated at 80°C for 10 minutes, cooled, and then the pH of the liquid whey is adjusted to 5.8-6.0 with 0.1 mol / L phosphate buffer to obtain fermented plant whey. The amount of Lactobacillus acidophilus added is 0.05-0.15% of the total weight of the plant protein powder and water; The pretreated raw meat is uniformly mixed with salt (20 g / kg), sugar (5 g / kg glucose), a starter culture, seasonings (5 g / kg sucrose, 2 g / kg dried ginger powder, 2 g / kg pepper powder, 1 g / kg Sichuan pepper powder, 0.1 g / kg sodium nitrite, 25 mL / kg white wine, 0.5 g / kg ascorbic acid, 10 g / kg corn starch, 5 g / kg whey protein powder) and fermented plant whey, wherein the amount of the starter culture added is 0.1-0.3% of the total weight of the pretreated raw meat, and the amount of the fermented plant whey added is 10-16% of the total weight of the pretreated raw meat; Stirring the evenly mixed materials into casings for fermentation to obtain fermented sausages; Among them, the fermentation agent includes lactic acid bacteria, and the classification name of the strain is Pediococcus pentosaceus Pediococcus pentosaceusIMAUYR3-1 was deposited in the China Center for Type Culture Collection (deposit address: Wuhan University, Wuhan, China) on April 29, 2025, with the deposit number CCTCC NO: M 2025938. The viable cell count in the starter culture was 1 × 10 7 CFU / g.

[0048] Fermentation is carried out in a constant temperature and humidity chamber for 1-2 days (temperature of 25°C, relative humidity of 95%). Fermentation is completed when the pH is less than 5. The fermentation completion time is specifically determined according to pH detection (the fermented sausages of the present invention are all fermented within 1 day). Drying: the first stage is 2-4 days (temperature of 15°C, relative humidity of 75%), the second stage is 5-7 days (temperature of 10°C, relative humidity of 65%), and finally stored at 4°C.

[0049] At the 24th hour of fermentation, the sausage is immersed in the permeate for 60-80 minutes. After the soaking, the surface is wiped dry and the fermentation is continued. The permeate includes flavor protease, fermented plant whey, sodium lactate, lysozyme, and the remaining water. The mass concentration of fermented plant whey in the permeate is 5%, the concentration of sodium lactate is 0.2 mol / L, the amount of lysozyme added accounts for 0.05% of the total weight of the permeate, and the weight ratio of flavor protease to lysozyme is 1:0.2.

[0050] The permeate also contains an enzyme composite carrier, which is composed of liposome-encapsulated lysozyme and flavor protease, and the weight ratio of liposome to permeate is 1:10; The preparation method of liposomes is as follows: Phosphatidylcholine and cholesterol were dissolved in chloroform at a weight ratio of 4:1 and rotary evaporated to form a lipid film; Mixing lysozyme, flavor protease, and fermented plant whey in a weight ratio of 1:1:4 to obtain an enzyme mixture; The enzyme mixture was added to the lipid film, hydrated under nitrogen for 30 minutes, and then ultrasonically disrupted to form polydisperse crude liposomes at an ultrasonic power of 300 W for 5 minutes. The crude liposomes were passed through a polycarbonate membrane extruder at 35-40°C for 5-7 cycles, using filter membranes with pore sizes of 1 μm, 400 nm, and 200 nm in sequence to obtain an enzyme-containing composite carrier.

[0051] The preparation of the enzyme mixture comprises the following steps: Preheat the fermented plant whey to 35-38° C., add 0.07% by weight of L-cysteine ​​hydrochloride, and stir to dissolve to obtain a solution; Add flavor protease to the dissolving solution, activate it under nitrogen protection at 25-28°C for 15 minutes to obtain a mixed solution; Dissolve lysozyme in citric acid buffer at 2-5°C and pH 5.0-5.2 to obtain a lysozyme solution at a concentration of 10 mg / mL; The lysozyme solution was added dropwise to the mixed solution at a flow rate of 0.5 mL / min. The system temperature was maintained at 20-22°C during the addition process. Stirring was continued for 14 minutes after the addition was completed. After stirring, the solution was stored at 2-4°C for later use.

[0052] The mixing method of pretreated raw meat and fermented plant whey is: First, pre-mix the starter culture with 25-35% fermented plant whey at 12-15°C for 10 minutes to obtain an activated fermentation liquid; Mixing the pre-treated raw meat, salt, sugar, seasonings and the remaining fermented plant whey at 0-4°C and stirring until the mixture becomes viscous to obtain minced meat; Add the activated fermentation liquid to the minced meat at 18-20°C, and mix for 3-5 minutes at a vacuum degree of -0.04~-0.06 MPa and a rotation speed of 20-30 r / min.

[0053] The mixing method when adding activated fermentation liquid to minced meat is as follows: The activated fermentation liquid was pre-cooled to 4°C, formed into droplets using a droplet generator, and sprayed into a -25°C cold air channel at a wind speed of 2-3 m / s to freeze and form fermentation agent microspheres; At a speed of 25 r / min, the starter microspheres were added to the minced meat in three batches: the first batch contained 40% of the starter microspheres by weight and mixed for 1 min, the second batch contained 30% of the starter microspheres by weight and mixed for 1 min, and the third batch contained 30% of the microspheres by weight and mixed for 1-3 min. Before adding each batch of starter microspheres, atomized liquid of fermented plant whey saturated with CO2 was sprayed on the surface of the minced meat. The droplet size was 40 μm and the spraying amount accounted for 1.0% of the weight of the minced meat.

[0054] Comparative Example 1: Fermented sausages were prepared using the method of Example 4, except that the plant protein powder was not fermented after being mixed with water. That is, the plant protein powder was mixed with water to obtain a protein liquid. The fermented plant whey used in subsequent steps was all protein liquid, and the remaining steps were the same.

[0055] Comparative Example 2: Fermented sausages were prepared using the method of Example 4, except that no permeate soaking treatment was used at the 24th hour of fermentation, that is, the uniformly mixed material was poured into the casing for fermentation. The fermentation conditions were: temperature 25°C, relative humidity 95%, and time 1-3 days; drying conditions were: temperature 15°C, relative humidity 75%, and time 2-4 days in the first stage; temperature 10°C, relative humidity 65%, and time 5-7 days in the second stage. Fermented sausages were obtained upon completion of fermentation.

[0056] Comparative Example 3: Fermented sausages were prepared using the method of Example 4, except that the enzyme composite carrier was not included in the permeate.

[0057] Comparative Example 4: Fermented sausage was prepared using the method of Example 4, except that the enzyme mixture was prepared by mixing lysozyme, flavor protease, and fermented plant whey in a weight ratio of 1:1:4 and dissolving the mixture in a citric acid buffer solution at pH 5.0-5.2 to obtain the enzyme mixture.

[0058] Comparative Example 5: Fermented sausages were prepared using the method of Example 4, except that the pretreated raw meat and fermented plant whey were directly mixed.

[0059] Comparative Example 6: Fermented sausages were prepared using the method of Example 4, except that the activated fermentation liquid was directly added to the minced meat for mixing.

[0060] <Representation> 1. Determination of physical and chemical indicators of fermented sausages 2.1 pH determination pH value determination: refer to the method of GB5009.237-2016 "National Food Safety Standard - Determination of pH Value of Food"; The pH value changes of fermented sausage during processing and storage are caused by Figure 6 (Left) As shown, the pH value changes of the three groups of sausages in Example 3 were basically similar. At the end of fermentation (1 day), the pH value of the sausages in the XAR-10 group and the YR3-1 group was significantly lower than that of the fermented sausages in the CK group ( P <0.05). Subsequently (days 4-60), the pH values ​​of the sausages in the XAR-10 and YR3-1 groups showed an overall upward trend. This was mainly due to the microbial decomposition of proteins to produce some free amino acids and peptides, which led to the accumulation of some nitrogen-based compounds and alkaline substances such as ammonia, which increased the pH value of the fermented sausages.

[0061] 2.2 Changes in water activity Determination of water activity (Aw): Refer to the method in GB5009.3-2016 "National Food Safety Standard - Determination of Water in Food" and use the HD-3A intelligent water activity meter to measure the change of Aw; The changes in water activity (Aw) values ​​of fermented sausages during processing and storage are shown in Table 2; Table 2 shows the changes in water activity Comparative analysis of the CK, XAR-10, and YR3-1 groups revealed that water activity first decreased and then increased with processing and storage time. On day 0 and day 1 of fermentation, the Aw values ​​of the CK, XAR-10, and YR3-1 fermented sausages showed little change. This is due to the relatively high humidity in the first stage of fermentation, resulting in a relatively small change trend. Reducing water activity is crucial to ensuring the shelf life and safety of fermented sausages. As fermentation progressed, the Aw values ​​of all three groups decreased significantly ( P <0.05), which may be due to the evaporation of water during the fermentation process. At the finished product stage (9 days), the Aw values ​​of the fermented sausage samples of the XAR-10 and YR3-1 groups were significantly lower than those of the CK group ( P <0.05). After 60 days of storage, the Aw values ​​of the three groups of fermented sausages increased slowly, which may be due to moisture absorption during storage, but the Aw values ​​were still below 0.88, which effectively inhibited the growth of spoilage bacteria; By comparing Example 4, Comparative Example 1, Comparative Example 2, and Comparative Example 3, it can be seen that the development trends of Example 4, Comparative Example 1, Comparative Example 2, and Comparative Example 3 are all consistent with those of the YR3-1 group, but Example 4 is the best, and on the 60th day, Example 4 is significantly better than the other experimental groups (YR3-1 group, XAR-10 group), indicating that the preparation method of the fermented sausage of the present invention can effectively reduce the influence of moisture absorption during storage.

[0062] 2.3 Color difference changes Inoculation fermentation has a certain effect on the color difference of fermented sausages. The changes in color difference during processing and storage of the fermented sausages of Example 3 (CK group, YR3-1 group, XAR-10 group, Example 4, Comparative Examples 1-5) were tested. The results are shown in Tables 3-5.

[0063] Table 3 is the fermented sausage value Table 4 is the fermented sausage value Table 5 is the fermented sausage value From Tables 3, 4 and 5, we can see that sausage and The values ​​showed a trend of decreasing first and then increasing. At the finished product stage (9 days), the fermented sausages of XAR-10 group and YR3-1 group and The values ​​were significantly lower than those in the CK group ( P <0.05). The sausages prepared in all experimental groups The value slowly increased and then stabilized. At the finished product stage (9 days), the The values ​​were significantly higher than those at each processing stage, indicating that the addition of lactic acid bacteria and the preparation method of the fermented sausage of the present invention were helpful for the production of redness of the fermented sausage. and This may be because a small amount of fat seeps out of the sausage during storage, and moisture absorption occurs, which increases the moisture content and increases the brightness and yellowness of the surface of the fermented sausage.

[0064] The results showed that adding Pediococcus pentosaceus IMAUYR3-1 and changing the preparation method of fermented sausages can effectively improve the color of fermented sausages.

[0065] 2.4 Texture changes Texture measurement: The hardness, elasticity, cohesion and chewiness of the fermented sausages prepared in Example 3 (three groups), Example 4, Comparative Example 1, Comparative Example 2, Comparative Example 5 and Comparative Example 6 were tested using a TA.XT PlusC texture analyzer. The test results are shown in Tables 6, 7 and 8. Different lowercase letters indicate significant differences between different processing times in the same group ( P <0.05), different capital letters indicate significant differences among different groups with the same processing time ( P <0.05); Table 6 Changes in hardness of fermented sausage during processing and storage (hardness g) Table 7 Changes in elasticity during processing and storage of fermented sausages (elasticity / mm) Table 8 Changes in cohesion during processing and storage of fermented sausages Comparative analysis of the texture data of the control group, XAR-10 group, and YR3-1 group in Tables 6, 7, and 8 shows that at the end of fermentation (1 day), the elasticity, hardness, and cohesion of the fermented sausage samples in the YR3-1 group were significantly higher than those in the XAR-10 group and the CK group. During the storage period (30 days and 60 days), the hardness, elasticity, and cohesion of the YR3-1 group were slightly higher than those in the XAR-10 group and the CK group. This indicates that the use of Pediococcus pentosaceus IMAUYR3-1 can effectively improve the texture characteristics of sausages. Comparative analysis of Example 4, Comparative Example 1, Comparative Example 2, Comparative Example 5, and Comparative Example 6 in Tables 10, 11, and 12 shows that the data of Example 4 is better than that of Comparative Example 2, which is better than that of Comparative Example 1, which is better than that of Comparative Example 5, and which is better than that of Comparative Example 6. This indicates that when plant whey is fermented with Lactobacillus acidophilus and then added to the filling, the soluble components in the plant whey act as hydrophilic colloids in the meat emulsion, increasing the viscosity of the system and forming a gel network, thereby improving the texture evaluation of the fermented sausage. The pre-activation of the starter, the construction of a low-temperature meat emulsion matrix, and the medium-temperature vacuum mixing can maximize the effects of the plant whey and Pediococcus pentosaceus IMAUYR3-1, forming a highly water-holding, dense, and uniform protein gel network, thereby improving the hardness, elasticity, and cohesion of the fermented sausage. The synergistic effects of the microspheroidized starter, batch embedding, and carbon dioxide enable the formation of a uniform and controllable protein network, which gradually penetrates deep into the meat emulsion, allowing the storage of salt-soluble proteins for dissolution, thereby improving the texture of the sausage.

[0066] 2.5 Changes in Volatile Basic Nitrogen Determination of Total Volatile Base Nitrogen (TVB-N): Determination was carried out according to the semi-micro nitrogen determination method in GB5009.228-2016 “Determination of Volatile Basic Nitrogen in Foods”. The results are as follows: Figure 6 As shown in the figure (right), TVB-N content showed an overall upward trend during the processing of the three sausage groups. At the end of drying (day 4), the TVB-N content in the CK group was significantly higher than in the XAR-10 and YR3-1 groups. This is because the lactic acid bacteria fermentation of the sausages lowered the pH, inhibited the growth of stray bacteria, prevented the production of volatile nitrogen compounds, and improved the quality of the sausages. During storage (days 30-60), the rate of increase in TVB-N content in the CK group slowed, but remained significantly higher than in the XAR-10 and YR3-1 groups. This demonstrates that Pediococcus pentosaceus IMAUYR3-1 inhibits the production of alkaline nitrogenous substances, thereby effectively improving the quality of fermented sausages.

[0067] 2.6 Changes in Thiobarbituric Acid Determination of Thiobarbituric Acid Reactive Substances (TBARS): The results of Example 3, Example 4, and Comparative Examples 1-3 are shown in Table 9. Table 9 shows the thiobarbituric acid value (mg / 100g) Comparative analysis of the CK, XAR-10, and YR3-1 groups revealed that TBARS values ​​of the three fermented sausages showed a significant upward trend over time during processing and storage. At the finished product stage (9 days), TBARS values ​​of the XAR-10 and YR3-1 groups were significantly lower than those of the CK group. This may be because the addition of lactic acid bacteria reduced the water activity of the fermented sausages, thereby mitigating lipid oxidative rancidity. At 60 days of storage, TBARS values ​​of the YR3-1 group were significantly lower than those of the XAR-10 and CK groups, indicating that Pediococcus pentosaceus IMAUYR3-1 has a strong ability to inhibit lipid oxidative rancidity in fermented sausages.

[0068] A comparative analysis of Example 4, Comparative Example 1, Comparative Example 2, and Comparative Example 3 showed that the development trends of Example 4, Comparative Example 1, Comparative Example 2, and Comparative Example 3 were consistent with those of the YR3-1 group, but the data of Example 4 was better than that of the YR3-1 group, indicating that the preparation method of the fermented sausage of the present invention can effectively reduce the content of thiobarbituric acid.

[0069] 3. Determination of microbial indicators of fermented sausages Under sterile conditions, 25 g of fermented sausage was weighed from each group, chopped, and placed in 225 mL of normal saline. After vortexing for 5 minutes, the mixture was serially diluted and poured onto plates. The mixture was then incubated at 37°C for 48 hours. The total colony count was determined using PCA solid medium, and the lactic acid bacteria count was determined using MRS solid medium. The lactic acid bacteria count and total colony count during the processing and storage of the fermented sausages are shown in Table 10. Table 10 Changes in microbial counts during processing and storage of fermented sausages As shown in Table 10, the change trends of the number of lactic acid bacteria and the total number of colonies in the control group and the experimental group were basically the same, both showing a trend of first increasing and then decreasing. On day 0, due to the addition of fermentation agents in the XAR-10 and YR3-1 groups, the number of lactic acid bacteria was 10 7 cfu / g, significantly higher than that of the CK group without adding fermentation agent ( P <0.05). At the end of fermentation (1 day), the number of lactic acid bacteria reached a maximum of 10 8 cfu / g, the total colony count and lactic acid bacteria count of the fermented sausage samples in the starter groups (XAR-10 group and YR3-1 group) were significantly higher than those in the natural fermentation CK group ( P <0.05). Subsequently, due to the decrease in processing temperature and Aw, the number of lactic acid bacteria and total colony count showed a downward trend. During the storage period (30 days to 60 days), the number of lactic acid bacteria in each group of fermented sausages showed a slow downward trend.

[0070] 4. Changes in moisture distribution of fermented sausages Each group of samples was processed into small cubes of 1cm×1cm×1cm. Samples of similar size and weight were selected and wrapped with plastic wrap to reduce water loss. They were then placed at the bottom of the nuclear magnetic resonance tube, and the moisture distribution of different groups of fermented sausage samples was detected using low-field nuclear magnetic resonance technology. The transverse relaxation time T2 of the sample was determined using the CPMG sequence, and the measurement was repeated 3 times for each treatment group sample. The main parameters are: nuclear magnetic tube cavity temperature 30°C, sampling frequency 200 KHz, main frequency 20MHz, number of echoes 15000, frequency offset 796383.67 Hz, 90-degree pulse 7.00μs, 180-degree pulse width 13.52μs, cumulative number 8, and analog gain 18.0dB. The scanned curve was inverted to obtain the T2 distribution of the sample, and the moisture distribution of different forms during the processing and storage of fermented sausages was shown. Figure 8 As shown, Figure 8 A is the CK group; B is the XAR-10 group; C is the YR3-1 group; During sausage processing, the transverse relaxation time T2 can reflect the degree of freedom of water. The larger the T2, the smaller the water binding force and the higher the degree of freedom. The curve mainly shows four water distribution states, namely: strongly bound water T2, 20 (0.1~1ms), weakly bound water T 21 (relaxation time 1 to 10 ms), non-flowing water T 22 (relaxation time 10-100 ms) and free water T 23 (relaxation time 100~1000 ms), A 20 、A 21 、A 22 and A 23 is the relaxation peak area corresponding to different forms of water. During processing and storage, the T 20 、A 20 There was no significant change within or between groups. This may be because the water content in this part is extremely stable and is not affected by changes in protein structure. Therefore, whether or not lactic acid bacteria starter is added has no effect on this part of water during sausage processing and storage.

[0071] Depend on Figure 8 As shown in A, B, and C, at the end of fermentation (1 day), the water distribution in the three groups of fermented sausages was mainly composed of non-mobile water T 22 The difference among the groups was significant ( P <0.05), followed by bound water T 21 and Free Water T 23 The proportion is relatively small, and the A content of fermented sausages in the starter groups (YR3-1 group and XAR-10 group) is 22 Significantly lower than the naturally fermented CK group ( P <0.05), among which YR3-1 group T22 The degree of migration to the left is more obvious. This is because under acidic conditions, muscle protein is destroyed, water is lost, and it is difficult for the water to flow to migrate to the left as a whole. The three groups of sausage samples T 22 、A 22 All of them decreased significantly with the increase of processing and storage time ( P <0.05), T 23 、A 23 Both increased significantly with the increase of processing and storage time ( P <0.05), indicating that both the distribution and proportion of free water increased, leading to a decrease in water retention. However, after 60 days of storage, the increase in the naturally fermented CK group was greater than that in the starter cultures (YR3-1 and XAR-10 groups). This suggests that the addition of the Pediococcus pentosaceus IMAUYR3-1 starter culture can reduce the proportion of free water in fermented sausages during storage, thereby slowing the decline in water retention.

[0072] 5. Observation of the microstructure of fermented sausage Scanning electron microscopy was used to compare and observe each group of fermented sausage samples. The samples were cut into small rectangular blocks with a side length of 5 mm and a thickness of 2 mm, and then vacuum-freeze-dried in a freeze dryer before being taken out. The processed samples were adhered to double-sided adhesive tape with a conductive carbon film and observed and photographed using a scanning electron microscope. The microstructure of the samples of the three groups of fermented sausages at the finished stage (9 days) is shown in Figure 2. Figure 9 As shown, A is the CK group; B is the XAR-10 group; C is the YR3-1 group; Depend on Figure 9 Compared with the naturally fermented CK group, the sausages from the starter cultures (YR3-1 and XAR-10 groups) had a more compact structure and smaller pores. The YR3-1 group had a smaller and more uniform pore structure, suggesting that the addition of Pediococcus pentosaceus IMAUYR3-1 refined the sausage surface, strengthened its internal gel network, and made the structure more compact, further improving water retention.

[0073] 6. Sensory evaluation of fermented sausage Thirteen panelists were selected and trained for one week using a basic taste test procedure. They tasted commercial sausage products to familiarize themselves with the characteristics of the samples to be evaluated. The test sausages were sliced ​​into approximately 4-5 mm thick slices for sensory evaluation. The panelists rinsed their mouths with water before tasting the different samples. The evaluation criteria are shown in Table 11, and the evaluation results are shown in Table 12. Table 11 Sensory evaluation of fermented sausage Table 12 shows the sensory evaluation results of fermented sausages Analyzing Table 12, compared to the XAR and CK groups, the sausages in the YR3-1 group had the best acceptability, with better taste and texture than the other two groups, and all indicators scored higher than the CK group. This indicates that fermenting sausages with Pediococcus pentosaceus YR3-1 reduced the pH and water activity of the fermented sausages, increasing their firmness and elasticity. Therefore, the YR3-1 sausages had a good taste and texture, significantly improving the quality of the fermented sausages. The XAR-10 sausages had a better color than the other two groups, but their taste was poor. The flavor scores of the YR3-1 and XAR-10 sausages were similar. Compared with the Example 4 group, the acceptability and mouth feel of Example 4 are better than those of the YR3-1 group, and Example 4 is better than Comparative Examples 1-6. A comparative analysis of Example 4 and Comparative Example 1 shows that the addition of fermented plant whey can effectively improve the sensory evaluation of the fermented sausage and improve the overall acceptability. Analysis of Example 4 and Comparative Examples 2-4 shows that the sensory evaluation of the sausage can be effectively improved by immersing the sausage in the permeate and adding an enzyme composite carrier to the permeate, as well as the preparation method of the enzyme mixture. Analysis of Example 4 and Comparative Examples 5 and 6 shows that the present invention can effectively improve the sensory evaluation of the fermented sausage by changing the mixing method of the pretreated raw meat and the fermented plant whey and changing the mixing method when the activated fermentation liquid is added to the minced meat.

[0074] 7. Analysis of hydrolysis characteristics of fermented sausage protein 7.1 Analysis of changes in non-protein nitrogen content in fermented sausages Non-protein nitrogen content (NPN) is an indicator for evaluating the extent of protein hydrolysis in fermented sausages by starter cultures; Add 2 g of sample and 18 mL of distilled water to a 50 mL centrifuge tube, homogenize for 2 minutes, and centrifuge at 10,000 × g for 15 minutes at 5°C. Filter the supernatant twice through Whatman #1 chromatography paper, and record the filtrate volume (V). Mix 15 mL of the filtrate with 15 mL of 10% trichloroacetic acid, let it stand for 30 minutes, and centrifuge under the same conditions as above. Filter the supernatant through Whatman #4 chromatography paper, and take 5 mL of the filtrate to determine the nitrogen content (N1). Calculate the non-protein nitrogen (NPN) content (N0) by 0.2V × N1. The total nitrogen content is N. NPN content in fermented sausage during processing and storage Figure 7As shown in the left figure, NPN content in the three fermented sausages increased during the fermentation-drying-ripening process, then leveled off during storage. At the end of fermentation (day 1), NPN content in the sausages fermented with XAR-10 and YR3-1 was significantly higher than that in the naturally fermented CK group. This is because proteolytic enzymes increase the total content of polypeptides, short peptides, and free AA, leading to increased protein degradation in the sausages. During storage (days 30-60), NPN content in the three fermented sausages stabilized, but NPN content in the starter cultures (XAR-10 and YR3-1) remained significantly higher than that in the naturally fermented CK group.

[0075] 7.2 Determination of protein hydrolysis index of fermented sausage The proteolysis index (PI) of fermented sausage was calculated according to the formula: PI = N0 / N × 100%. The changes in PI during the processing and storage of fermented sausage are shown in Table 13; Table 13 is the protein hydrolysis index of fermented sausage Comparative analysis of the CK group, XAR-10 group, and YR3-1 group showed that the PI of the three fermented sausages increased significantly with the extension of processing time ( P <0.05). In the finished product period (9 days), the XAR-10 group and the YR3-1 group were significantly higher than the CK group ( P <0.05). This is because LAB produces proteases, which accelerate protein degradation, leading to a rapid increase in NPN and PI values. However, compared with the XAR-10 group, the PI of the fermented sausages in the YR3-1 group was higher, indicating that Pediococcus pentosaceus IMAUYR3-1 has a stronger proteolytic ability; A comparative analysis of Example 4 and Comparative Example 1 shows that the trends of Example 4 and Comparative Example 1 are consistent with those of the YR3-1 group, but both the Example 4 group and the Comparative Example 1 group are higher than the YR3-1 group, indicating that the plant whey liquid prepared by the present invention can effectively increase the content of PI.

[0076] 7.3 Determination of amino nitrogen content in fermented sausages The amino nitrogen (AN) content was determined by formaldehyde titration. Calculation was performed according to formula (3): Amino nitrogen content (g / 100mL) = ((V1-V0)×C×0.014×100) / (10×V / 100) (3) Where: V is the volume of the sample diluent, mL; C is the concentration of the NaOH standard solution, mol / L; V1 is the recorded titration volume; V0 is the blank titration volume; The results showed that the AN content of the fermented sausages continued to increase throughout the processing, which was consistent with the change trend of NPN content. During the drying period (4 days), there was no significant difference in the AN content of the fermented sausage samples between the XAR-10 group (0.449g / 100g) and the YR3-1 group (0.448g / 100g). P >0.05), but significantly higher than the CK group (0.339, P< 0.05), and Example 4 (0.468 g / 100g) was higher than Comparative Example 1 (0.457 g / 100g). During the storage period (30-60 days), the AN content of all fermented sausages stabilized, and the rate of proteolysis slowed. This was likely due to the inhibition of microbial growth and metabolism, as well as the reduction of enzyme activity, which further maintained the stability of AN content (AN content initially increased from 0 to 9 days and stabilized from 9 to 60 days). However, Example 4 (0.659 g / 100g) had a higher AN content during storage than Comparative Example 1 (0.659 g / 100g) and RY3-1 (0.630 g / 100g). The RY3-1 group, in turn, had a higher AN content than the XAR-10 group (0.614 g / 100g) and the CK group (0.483 g / 100g). This demonstrates that the plant whey liquid prepared by the present invention can effectively increase AN content.

[0077] 8. Determination of volatile flavor compounds in fermented sausages Detection Method: 5 g of sausage sample was weighed and placed into a vial. An extraction needle was inserted above the vial, and the sample was adsorbed at 60°C for 40 minutes. The sample was then removed and inserted into the GC inlet for desorption at 250°C for 3 minutes. GC-MS conditions included a TR-5 column (30 m × 0.25 mm, 0.25 μm), He carrier gas at a flow rate of 1.0 mL / min, a sample inlet and interface temperature of 250°C, a temperature program of 40°C for 5 minutes, a temperature increase of 5°C / min to 200°C for 5 minutes, and then a temperature increase of 20°C / min to 250°C for 5 minutes, with splitless injection. The ion source temperature was 250°C, the transfer line temperature was 250°C, the mass scan range was 30–400 m / z, and the solvent delay was 1 minute. Mass spectral data were identified by searching with Meanlib, Nistdemo, and Wiley Library, with a match greater than 800 considered the basis for identification. The peak area percentage of each component was calculated using the area normalization method.

[0078] A total of 72 volatile compounds were detected during the fermentation process, including 18 alcohols, 17 esters, 6 aldehydes, 17 terpenes, 5 acids, 3 ketones, 5 ketones, and 4 other substances. The amount of each volatile flavor compound detected varied over time in each group, with alcohols, esters, and terpenes being the dominant compounds. These three compounds were found in greater quantities in the fermented sausages from the YR3-1 and XAR-10 groups than in the CK group.

[0079] Alcohol substances mainly include ethanol, heptanol, nonanol, octanol, 2,3-butanediol, 2-ethylcyclobutanol, camphorol, α-terpineol, carveol, citronellol, 1-octen-3-ol, 3-cyclohexene-1-ol, terpinene-4-ol, isopropanol, etc. Among them, the content of ethanol and octanol is relatively high. Ethanol can help meat products release flavor substances, and at the same time it may be converted into esters (such as ethyl acetate) to give fruity aroma. Octanol has a mushroom and earthy aroma and is an important component of the complex flavor of fermented sausages. 1-octen-3-ol has a special mushroom smell and is an important flavor contributor in fermented meat products. It can be seen from the test results that the octanol content: the octanol content of the YR3-1 group (9d is 4.54±0.11 a , 30d is 0.14±0.01 a , 60d is 0.13±0.04 a ), the content of octanol in the XAR-10 group (9d was 4.05±1.82 a , 30d is 0.13±0.02 a , 60d is 0.14±0.03 a ), the content of octanol in the CK group (9d was 0.13±0.02 b , 30d is 0.14±0.02 a , 60d is 0.23±0.10 a ), the content of octanol in Example 4 (9d was 5.71±0.18 a , 30d is 0.53±0.02 b , 60d is 0.49±0.02 a ), the content of octanol in the comparative example 3 group (9d was 4.34±0.06 b , 30d is 0.39±0.01 a , 60d is 0.26±0.07 a ), the content of octanol in the comparative example 4 group (9d was 5.24±0.27 a , 30d is 0.49±0.14 a , 60d is 0.41±0.05 c ); 1-octen-3-ol content: YR3-1 group 1-octen-3-ol content (9d is 3.42±2.01 a, 30d was 1.23±0.96 ab , 60d is 3.25±0.51 a , the content of 1-octen-3-ol in XAR-10 group (9d was 1.81±0.24 ab , 30d is 2.07±1.02 a , 60d was nd), the content of 1-octen-3-ol in group CK (9d was 0.33±0.05, 30d was nd, 60d was nd (nd means not detected), the content of 1-octen-3-ol in group Example 4 (9d was 4.61±1.18 a , 30d was 4.05±0.98 a , 60d is 4.59±1.07 a ), the content of 1-octen-3-ol in the comparative example 3 group (9d was 3.77±1.43 a , 30d is 2.61±1.12 a , 60d is 2.73±1.35 a ), the content of 1-octen-3-ol in the comparative example 4 group (9d was 3.94±1.27 c , 30d is 2.87.±1.36 a , 60d is 3.72±0.96 a ); Esters are produced through the esterification reaction of alcohols and acids. Many ester compounds are believed to be the source of the fruity and caramel flavors in fermented sausages. Esters include ethyl 3-methylbutyrate, ethyl heptanoate, ethyl valerate, ethyl acetate, ethyl 2-hydroxypropionate, ethyl nonanoate, ethyl stearate, ethyl undecanoate, ethyl arachidate and ethyl laurate. Ethyl acetate, ethyl valerate and ethyl heptanoate are typical fruity esters that can balance the saltiness in fermented meat products. The precursor of ethyl valerate is leucine produced by the hydrolysis of muscle protein, which has a unique flavor. At the end of fermentation (1d) and the finished product period (9d), the YR3-1 group (0.20±0.03 on 1d) a , 9d is 0.33±0.05 a ) and XAR-10 group (1d was 0.19±0.04 a , 9d is 0.25±0.02 a ) The content of ethyl valerate was significantly higher than that of the natural fermentation CK group, and the Example 4 group (1 day was 0.24±0.03 a , 9d is 0.40±0.02 a , 60d is 0.62±0.06 a ) The content of ethyl valerate was higher than that of the control group 3 (1d was 0.21±0.03 a , 9d is 0.35±0.01a, 60d is 0.57±0.04a ) and control group 4 (1d was 0.22±0.01 a , 9d is 0.37±0.02 a , 60d is 0.59±0.04 a ).

[0080] The additives and spices used in sausages will generate terpenes, which contribute to the flavor formation of fermented sausages. The test results show that terpenes mainly include caryophyllene, phenol, camphene, cucurbitacin, isomerized cinnamene, styrene, α-farnesene, 1-methyl-4-cyclohexene and D-limonene, among which camphene has a slight citrus and herbal flavor, which can balance the greasiness in meat products, and cucurbitacin gives fermented meat products a smoky flavor. The content of these two substances is relatively high. Terpenes in fermented sausages can enhance the layering of sausage flavor, thereby enriching the taste of sausages. Among them, for cucurbitacin: YR3-1 group (1d is 0.16±0.01 b , 30d is 0.11±0.02 a , 60d is 0.77±1.14 a ) was lower than that of the XAR-10 group (1d was 0.79±0.15 a , 30d is 0.85±0.01 a , 60d is 0.93±0.72 a ) cucurbitacin content, but the YR3-1 group was significantly higher than the natural fermentation CK group (1 day was 0.06±0.01) during storage (60 days). b , 30d is 0.10±0.01 a , 60d is 0.13±0.01 a ), which shows that the effect of using Pediococcus pentosaceus IMAUYR3-1 alone on the content of cucurbitacin in the YR3-1 group was lower than that of Lactobacillus plantarum XAR-10; Example 4 group (1d was 0.84±0.02 a , 30d is 0.65±0.03 b , 60d is 1.17±0.05 b ) The content of cucurbitacin was higher than that of the control group 3 (1d was 0.80±0.01 b , 30d is 0.55±0.04 a , 60d is 0.98±0.02 b ) and control group 4 (1d was 0.82±0.03 a , 9d is 0.37±0.02 a , 60d is 1.02±0.06 b ); Aldehydes are the key to the freshness and complexity of fermented sausages. At the same time, aldehydes participate in the Maillard reaction and promote the formation of the surface color of the sausage. Among them, 3-methylhexanal has a grassy and nutty aroma, which is produced by the Strecker degradation of leucine. At the finished product stage (9d), the content of 3-methylhexanal in the YR3-1 group was significantly higher than that in the CK group. Secondly, hexanal, heptanal, nonanal and decanal give the sausage a citrus and fatty aroma, which are formed by the oxidation of oleic acid or linoleic acid. Ketones usually give the sausage a creamy aroma. Secondly, there are some other aromatic hydrocarbons in the fermented sausage, such as toluene and ethylbenzene, but these substances are relatively rare and have little effect on the flavor of the fermented sausage. Among them, for 3-methylhexanal: the content of 3-methylhexanal in the YR3-1 group at different stages (9d was 0.17±0.02 a , 30d is 0.15±0.01 b , 60d is 0.13±0.04 a , the content of 3-methylhexanal in the XAR-10 group at different stages (9d was 0.15±0.03 a , 30d is 0.14±0.02 a , 60d is 0.13±0.05 a ), the content of 3-methylhexanal in the CK group at different stages (9d was 0.06±0.02 b , 30d is 0.18±0.01 a , 60d is 0.20±0.05 a ), the content of 3-methylhexanal at different stages in Example 4 (9d was 0.26±0.03 b , 30d is 0.28±0.02 a , 60d is 0.24±0.05 a ), the content of 3-methylhexanal at different periods in Comparative Example 1 (9d was 0.19±0.01 a , 30d is 0.22±0.03 a , 60d is 0.17±0.01 a ), the content of 3-methylhexanal at different periods in Comparative Example 2 (9d was 0.21±0.03 a , 30d is 0.24±0.01 c , 60d is 0.20±0.05 a ); Other ingredients have been tested and are not listed in this application; From the above analysis, it can be seen that the addition of Pediococcus pentosaceus IMAUYR3-1 has a certain effect on the flavor components of fermented sausages, but the addition of permeate during the preparation of sausages can significantly retain the content of flavor components in the fermented sausages and improve the taste of the fermented sausages.

[0081] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and embodiments shown and described herein.

Claims

1. A method for preparing a high-yield protease lactic acid bacteria-assisted fermented sausage, characterized in that: The following steps are involved: Grind the meat chunks to obtain raw meat; The plant protein powder and water are mixed in a weight ratio of 1:5-10, and Lactobacillus acidophilus is inoculated and fermented at a temperature of 35°C-38°C for 12-24 hours. After the fermentation, liquid whey is obtained by centrifugation. The liquid whey is heated at 80°C for 10 minutes, cooled, and then the pH of the liquid whey is adjusted to 5.8-6.0 with 0.1 mol / L phosphate buffer to obtain fermented plant whey. The amount of Lactobacillus acidophilus added is 0.05-0.15% of the total weight of the plant protein powder and water. The pretreated raw meat is mixed evenly with salt, sugar, a fermentation agent, seasonings and fermented plant whey, wherein the amount of the fermentation agent added is 0.1-0.3% of the total weight of the pretreated raw meat, and the amount of the fermented plant whey added is 10-16% of the total weight of the pretreated raw meat; Stirring the evenly mixed materials into casings for fermentation to obtain fermented sausages; The fermentation agent includes lactic acid bacteria, and the classification name of the strain is Pediococcus pentosaceus ( Pediococcus pentosaceus )IMAUYR3-1, deposited in the China Center for Type Culture Collection on April 29, 2025, with the deposit number CCTCC NO:M2025938.

2. The method for preparing a high-yield protease lactic acid bacteria-assisted fermented sausage according to claim 1, wherein: Fermentation conditions are: temperature 22-28°C, relative humidity 92-98%, and time 1-3 days; The drying conditions are as follows: the temperature of the first stage is 10-20°C, the relative humidity is 71-80%, and the time is 2-4 days; the temperature of the second stage is 7-13°C, the relative humidity is 60-70%, and the time is 5-7 days.

3. The method for preparing a high-yield protease lactic acid bacteria-assisted fermented sausage according to claim 2, wherein: At the 24th hour of fermentation, the sausage is immersed in the permeate for 60-80 minutes. After the soaking, the surface is wiped dry and the fermentation is continued. The permeate comprises flavor protease, fermented plant whey, sodium lactate, lysozyme, and the balance water. The mass concentration of the fermented plant whey in the permeate is 10-20%, the concentration of sodium lactate is 0.1-0.3 mol / L, the amount of lysozyme added accounts for 0.02-0.05% of the total weight of the permeate, and the weight ratio of flavor protease to lysozyme is 1:0.15-0.

25.

4. The method for preparing a high-yield protease lactic acid bacteria-assisted fermented sausage according to claim 3, wherein: The permeate also contains an enzyme composite carrier, which is composed of liposome-encapsulated lysozyme and flavor protease, and the weight ratio of liposome to permeate is 1:8-12; The preparation method of liposomes is as follows: Lecithin and cholesterol were dissolved in chloroform at a weight ratio of 3-5:1 and rotary evaporated to form a lipid film; Mixing lysozyme, flavor protease, and fermented plant whey in a weight ratio of 1:1:3-5 to obtain an enzyme mixture; The enzyme mixture was added to the lipid film, hydrated under nitrogen for 30 minutes, and then ultrasonically disrupted to form polydisperse crude liposomes at an ultrasonic power of 300 W for 5 minutes. The crude liposomes were passed through a polycarbonate membrane extruder at 35-40°C for 5-7 cycles, using filter membranes with pore sizes of 1 μm, 400 nm, and 200 nm in sequence to obtain an enzyme-containing composite carrier.

5. The method for preparing high-yield protease lactic acid bacteria-assisted fermented sausage according to claim 4, characterized in that: The preparation of the enzyme mixture comprises the following steps: Preheating the fermented plant whey to 35-38° C., adding 0.06-0.08% by weight of L-cysteine ​​hydrochloride, stirring and dissolving, to obtain a solution; Add flavor protease to the dissolving solution, activate it under nitrogen protection at 25-28°C for 15 minutes to obtain a mixed solution; Dissolve lysozyme in citric acid buffer at 2-5°C and pH 5.0-5.5 to obtain a lysozyme solution at a concentration of 8-12 mg / mL; The lysozyme solution was added dropwise to the mixed solution at a flow rate of 0.5 mL / min. The system temperature was maintained at 20-22°C during the addition process. Stirring was continued for 5-20 minutes after the addition was completed. After stirring, the solution was stored at 2-4°C for later use.

6. The method for preparing high-yield protease lactic acid bacteria-assisted fermented sausage according to claim 2, characterized in that: The mixing method of pretreated raw meat and fermented plant whey is: First, pre-mix the starter culture with 25-35% fermented plant whey at 12-15°C for 5-10 minutes to obtain an activated fermentation liquid; Mixing the pre-treated raw meat, salt, sugar, seasonings and the remaining fermented plant whey at 0-4°C and stirring until the mixture becomes viscous to obtain minced meat; Add the activated fermentation liquid to the minced meat at 18-20°C, and mix for 3-5 minutes at a vacuum degree of -0.04~-0.06MPa and a rotation speed of 20-30r / min.

7. The method for preparing high-yield protease lactic acid bacteria-assisted fermented sausage according to claim 6, characterized in that: The mixing method when adding activated fermentation liquid to minced meat is as follows: The activated fermentation liquid was pre-cooled to 4°C, formed into droplets using a droplet generator, and sprayed into a -25°C cold air channel at a wind speed of 2-3 m / s to freeze and form fermentation agent microspheres; At a speed of 25-28 r / min, the starter microspheres were added to the minced meat in three batches: the first batch, 40% of the total weight of the starter microspheres, was mixed for 1 minute, the second batch, 30% of the total weight of the starter microspheres, was mixed for 1 minute, and the third batch, 30% of the total weight of the starter microspheres, was mixed for 1-3 minutes; Before adding each batch of starter microspheres, atomized liquid of fermented plant whey saturated with CO2 is sprayed on the surface of the minced meat, with a droplet size of 20-50 μm and a spraying amount of 0.5-1.0% of the weight of the minced meat.

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