Preparation method of high-protease lactic acid bacteria assisted fermented sausage

Abstract

The application discloses a preparation method of high-protease lactic acid bacteria auxiliary fermented sausages, and belongs to the technical field of fermented product preparation, and comprises the following steps: grinding meat blocks to obtain raw material meat; mixing plant protein powder with water, inoculating lactobacillus acidophilus for fermentation, and obtaining fermented plant whey after the fermentation; uniformly mixing the pretreated raw material meat with salt, sugar, leavening agent, flavoring agent and fermented plant whey; filling the uniformly mixed material into a casing for fermentation to obtain fermented sausages, wherein the leavening agent comprises lactic acid bacteria, and the classification name of the strain is Pediococcus pentosaceus Pediococcuspentosaceus IMAUYR3-1 was preserved in China Center for Type Culture Collection on April 29, 2025, and the preservation number is CCTCC NO: M 2025938. The Pediococcus pentosaceus IMAUYR3-1 of the application has good salt tolerance, bile salt tolerance, nitrite tolerance and acid tolerance, meets the requirements of meat product leavening agents, has good antibacterial property, and can also promote hydrolysis of large molecular muscle proteins into small molecular substances.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbiological technology. More particularly, the present application relates to a preparation method of high protease-producing lactic acid bacteria assisted fermented sausages. BACKGROUND

[0002] In the fermentation process of meat products, the degradation of muscle proteins (mainly sarcoplasmic proteins and myofibrillar proteins) is the result of the combined action of endogenous muscle enzymes and microbial proteases. Microbial enzymes can degrade large molecular proteins into small peptides and free amino acids (AA), and many AA are flavor substances or their precursors, which generate amines and organic acids through decarboxylation and deamination, and then form volatile flavor substances in the product. At the same time, lactic acid bacteria (LAB) play an important role in the formation of sausage flavor by releasing and degrading free AA, regulating the composition of non-volatile metabolites, and improving the quality characteristics of fermented meat products. The application of lactic acid bacteria with high protease activity in fermented sausages not only improves the quality characteristics of fermented meat products, but also promotes the increase of free AA and short peptide concentration, which has a positive effect on the maturation of fermented meat products. Therefore, it is of great significance to screen lactic acid bacteria that can hydrolyze large molecular muscle proteins into small molecular substances and apply them to fermented sausages. SUMMARY

[0003] It is another object of the present application to provide a preparation method of high protease-producing lactic acid bacteria assisted fermented sausages. Pediococcus pentosaceus IMAUYR3-1 has good salt tolerance, bile salt tolerance, nitrite tolerance and acid tolerance, meets the requirements of meat product fermentation agents, has good antibacterial properties, and can also promote the hydrolysis of large molecular muscle proteins into small molecular substances.

[0004] In order to achieve these objects and other advantages in accordance with the present application, a preparation method of high protease-producing lactic acid bacteria assisted fermented sausages is provided, comprising the following steps:

[0005] The meat pieces are ground to obtain raw meat;

[0006] The plant protein powder is mixed with water at a weight ratio of 1:5-10, inoculated with Lactobacillus acidophilus for fermentation at a temperature of 35-38°C for 12-24 hours, and then centrifuged to obtain liquid whey. The liquid whey is heated at 80°C for 10 minutes, cooled, and then adjusted to a pH of 5.8-6.0 using 0.1 mol / L phosphate buffer to obtain fermented plant whey. The addition amount of Lactobacillus acidophilus is 0.05-0.15% of the total weight of the plant protein powder and water;

[0007] Mix the pretreated raw meat with salt, sugar, leavening agent, seasoning and fermented plant whey, the leavening agent is added in an amount of 0.1-0.3% of the total weight of the pretreated raw meat, and the fermented plant whey is added in an amount of 10-16% of the total weight of the pretreated raw meat;

[0008] Fill the mixed material into a casing for fermentation to obtain the fermented sausage;

[0009] The leavening agent comprises lactic acid bacteria, and the classification name of the strain is Pediococcus pentosaceus Pediococcus pentosaceus IMAUYR3-1, preserved in the China Center for Type Culture Collection (CCTCC) on April 29, 2025 (address: Wuhan University, Wuhan, China), with the preservation number CCTCC NO:M 2025938.

[0010] Preferably, the fermentation conditions are as follows: temperature 22-28℃, relative humidity 92-98%, and time 1-3d;

[0011] The drying conditions are as follows: first stage temperature 10-20℃, relative humidity 71-80%, time 2-4d; second stage temperature 7-13℃, relative humidity 60-70%, time 5-7d.

[0012] Preferably, at the 24th hour of fermentation, the sausage is immersed in a permeation liquid for 60-80 minutes, and then the surface is wiped dry after soaking, and the fermentation is continued, wherein the permeation liquid comprises flavor protease, fermented plant whey, sodium lactate, lysozyme, and the rest water, the mass concentration of the fermented plant whey in the permeation liquid is 10-20%, the concentration of the sodium lactate is 0.1-0.3mol / L, the added amount of the lysozyme accounts for 0.02-0.05% of the total weight of the permeation liquid, and the weight ratio of the flavor protease to the lysozyme is 1:0.15-0.25.

[0013] Preferably, the permeation liquid further comprises an enzyme complex carrier, which is composed of liposomes embedding lysozyme and flavor protease, and the weight ratio of the liposomes to the permeation liquid is 1:8-12.

[0014] The preparation method of the liposomes is as follows:

[0015] Dissolve lecithin and cholesterol in chloroform at a weight ratio of 3-5:1 to form a lipid film by rotary evaporation;

[0016] Mix lysozyme, flavor protease and fermented plant whey at a weight ratio of 1:1:3-5 to obtain an enzyme mixture;

[0017] Add the enzyme mixture into the lipid film, hydrate for 30 minutes under nitrogen protection, and then ultrasonically break to form coarse liposomes, the ultrasonic power is 300W, and the time is 5 minutes.

[0018] The crude liposome is circulated and extruded 5-7 times through polycarbonate membrane extruder at a temperature of 35-40°C, and 1 μm, 400 nm and 200 nm pore size filters are used in sequence to obtain the enzyme-containing composite carrier.

[0019] Preferably, the preparation of the enzyme mixture comprises the following steps:

[0020] The fermented whey is preheated to 35-38°C, and 0.06-0.08% L-cysteine hydrochloride by weight of the fermented whey is added and stirred to dissolve, to obtain a dissolved solution;

[0021] The flavour protease is added to the dissolved solution, and activated at 25-28°C for 15 minutes under nitrogen protection to obtain a mixed solution;

[0022] The lysozyme is dissolved in a citric acid buffer at 2-5°C and pH 5.0-5.5, and the concentration of the lysozyme is 8-12 mg / mL, to obtain a lysozyme solution;

[0023] The lysozyme solution is added dropwise to the mixed solution at a flow rate of 0.5 mL / min, and the temperature of the system is maintained at 20-22°C during the dropwise addition. After the dropwise addition is completed, stirring is continued for 5-20 minutes, and then the mixture is stored at 2-4°C for use.

[0024] Preferably, the mixing method of the pretreated raw meat and the fermented whey is as follows:

[0025] The starter culture is first pre-mixed with 25-35% of the fermented whey at 12-15°C for 5-10 minutes to obtain an activated fermentation solution;

[0026] The pretreated raw meat, salt, sugar, flavouring and the remaining fermented whey are mixed at 0-4°C until a viscous state is obtained, to obtain a meat paste;

[0027] The activated fermentation solution is added to the meat paste at 18-20°C, and mixed at a vacuum degree of -0.04~-0.06 MPa and a rotation speed of 20-30 r / min for 3-5 minutes.

[0028] Preferably, the mixing method when the activated fermentation solution is added to the meat paste is as follows:

[0029] The activated fermentation solution is pre-cooled to 4°C, and droplets are formed using a droplet generator and sprayed into a cold air channel at -25°C at a wind speed of 2-3 m / s, to freeze and form starter culture microspheres;

[0030] The starter culture microspheres are added to the meat paste in three batches at a rotation speed of 25-28 r / min: the first batch is 40% of the total weight of the starter culture microspheres, mixed for 1 minute, the second batch is 30% of the total weight of the starter culture microspheres, mixed for 1 minute, and the third batch is 30% of the total weight of the microspheres, mixed for 1-3 minutes;

[0031] Wherein, before each batch of leavening agent microspheres is added, the surface of the meat paste is sprayed with a CO2 saturated fermented plant whey atomized liquid, the spray droplet particle size is 20-50 microns, and the spraying amount is 0.5-1.0% of the weight of the meat paste.

[0032] The present application at least includes the following beneficial effects:

[0033] First, Pediococcus pentosaceus IMAUYR3-1 with strong muscle protein degradation ability is screened from Inner Mongolia characteristic fermented food, the bacteria has good salt tolerance, bile salt tolerance, nitrite tolerance and acid tolerance, meets the requirements of meat product leavening agent, and has good antibacterial property. Pediococcus pentosaceus IMAUYR3-1 can promote the hydrolysis of large molecular muscle protein into small molecular substances, and the degradation effect of Pediococcus pentosaceus IMAUYR3-1 on myofibrillar protein is optimal.

[0034] Second, Pediococcus pentosaceus IMAUYR3-1 as a leavening agent applied in fermented sausages can significantly reduce the Aw value, pH value, TVB-N content and TBARS content, while effectively improving the color, hardness, elasticity and cohesiveness of the sausages; Pediococcus pentosaceus IMAUYR3-1 changes the water distribution state of fermented sausages, reduces the content of non-flowing water; effectively enhances the internal gel network structure of fermented sausages, and improves the water holding capacity of sausages. Pediococcus pentosaceus IMAUYR3-1 significantly improves the NPN content, PI and AN content of fermented sausages, proving that the strain has strong degradation effect on muscle protein. 3 groups of fermented sausages are detected to contain 18 kinds of alcohols, 17 kinds of esters, 6 kinds of aldehydes, 17 kinds of terpenes, 5 kinds of acids, 3 kinds of ketones, 5 kinds of ketones and 4 kinds of other substances.

[0035] Fourth, the plant whey is fermented by Lactobacillus acidophilus in the present application before being added to the stuffing, the soluble components in the plant whey play the role of hydrophilic colloid in the meat paste, increase the viscosity of the system and can form a gel grid, thereby improving the texture evaluation of fermented sausages; through pre-activation of the leavening agent, construction of low-temperature meat paste matrix and vacuum mixing at medium temperature, the effects of plant whey and Pediococcus pentosaceus IMAUYR3-1 can be maximized, forming a protein gel network with high water holding capacity, compactness and uniformity, realizing the improvement of the hardness, elasticity and cohesiveness of fermented sausages; the microspherized leavening agent, batch embedding and the synergistic effect of carbon dioxide make the protein network more uniform and controllable, penetrate into the deep layer of the meat paste layer by layer, and realize the improvement of the texture of sausages.

[0036] Other advantages, objects, and features of the present application will be apparent from the following specification, and will be appreciated by those skilled in the art. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1Strain morphology under optical microscope (1 000 times) of one of the technical solutions of the present application;

[0038] Figure 2 Growth curve of Pediococcus pentosaceus IMAUYR3-1, one of the technical solutions of the present application;

[0039] Figure 3 Salt tolerance and nitrite tolerance of Pediococcus pentosaceus IMAUYR3-1, one of the technical solutions of the present application, wherein the left graph is salt tolerance and the right graph is nitrite tolerance;

[0040] Figure 4 Temperature tolerance and free amino acid nitrogen change graph of Pediococcus pentosaceus IMAUYR3-1, one of the technical solutions of the present application, wherein the left graph is temperature tolerance and the right graph is the change of free amino acid nitrogen with the increase of fermentation days;

[0041] Figure 5 Electrophoresis analysis graph of Pediococcus pentosaceus IMAUYR3-1, one of the technical solutions of the present application;

[0042] Figure 6 pH change and volatile basic nitrogen change graph of fermented sausage, one of the technical solutions of the present application, wherein the left graph is pH change and the right graph is volatile basic nitrogen;

[0043] Figure 7 NPN content of fermented sausage, one of the technical solutions of the present application;

[0044] Figure 8 Different forms of water distribution in the process of processing and storage of fermented sausage, one of the technical solutions of the present application;

[0045] Figure 9 Scanning electron microscope graph of fermented sausage, one of the technical solutions of the present application. DETAILED DESCRIPTION

[0046] The present application will be further described in detail below with reference to examples, so that those skilled in the art can implement the present application according to the description.

[0047] <Example 1>

[0048] Screening and property analysis of lactic acid bacteria with high protease production

[0049] 1. Preliminary screening of lactic acid bacteria with high protease production

[0050] Extraction of myofibrillar protein: Take 10 g of mutton, fully pulverize and add 0.03 mol / L, pH 6.5 phosphate buffer solution at a mass-volume ratio (w / v) of 1:10, homogenize at 4°C with a magnetic stirrer for 4 min, centrifuge at 10000 r / min at the same temperature for 20 min, discard the supernatant, and then add 0.03 mol / L phosphate buffer solution at a mass-volume ratio (w / v) of 1:10, homogenize at 4°C with a magnetic stirrer for 4 min, centrifuge at 10000 r / min at the same temperature for 20 min. Repeat the above operation 3 times, then add 0.1 mol / L, pH 6.5 (containing 0.7 mol / L KI and 0.02% NaN3) phosphate buffer solution at a mass-volume ratio (w / v) of 1:4, homogenize at 4°C with a magnetic stirrer for 4 min, centrifuge at 10000 r / min at the same temperature for 20 min, and take the supernatant to dialyze overnight at 4°C. The myofibrillar protein is obtained.

[0051] Myofibrillar protein was extracted from mutton, and a punch with a diameter of 6 mm was used to punch holes in the myofibrillar protein medium. 60 μL of activated bacterial solution was injected into the holes, and cultured for 48 h. After culture, the agar layer was taken out of the culture dish, and subjected to Coomassie brilliant blue staining and decolorization. The presence and size of the transparent circle were observed and measured to determine whether the strain had the ability to hydrolyze muscle protein. If there was no transparent circle, it indicated that the strain did not have the ability to hydrolyze muscle protein.

[0052] The 52 strains isolated from the characteristic fermented foods in Inner Mongolia were purified by plate streaking culture, and strains with poor growth and unstable genetic properties were screened out. The diameters of the hydrolysis circles of the strains on myofibrillar protein were measured, and the 6 strains were further selected according to the measurement results. The strain numbers and hydrolysis circle diameters (mm) were 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 ), respectively. Different lowercase letters represent significant differences P <0.05), and YR3-1 is Pediococcus pentosaceus IMAU YR3-1.

[0053] 2. Further screening of lactic acid bacteria with high protease production

[0054] Preparation of standard curve: The tyrosine standard curve was prepared according to the method of GB / T 23527-2009, and the linear regression equation of the tyrosine standard curve was y=0.0044x+0.0969, and the linear correlation coefficient R2 =0.9992, indicating a good linear relationship;

[0055] The 6 strains obtained by preliminary screening were subjected to protease activity determination, and the tyrosine content was obtained according to the standard curve.

[0056] The protease activity was calculated according to the formula: enzyme activity / (μg·mL −1 )=(K×OD×n×4) / 10, wherein K represents the reciprocal of the slope of the standard curve; 4 represents 1 mL of the reaction solution taken out of 4 mL (i.e. 4 times); n represents the dilution multiple of the enzyme solution; and 10 represents 10 min of reaction.

[0057] The results show that the protease activity of P. pentosus IMAUYR3-1 is (23.13±0.40) U / mL, which is significantly higher than that of other strains (P<0.05). Therefore, the strain YR3-1 is rescreened for further tests.

[0058] 3. Morphological observation and physiological and biochemical tests of the rescreened lactic acid bacterial strain

[0059] P. pentosus IMAUYR3-1 obtained by separation and purification was inoculated on MRS plates and cultured for 1-2 days. The colony shape of P. pentosus IMAUYR3-1 on the culture medium was round, most of the colony centers were convex, the diameter was generally 1-2 mm, the edge was neat, the color was white, the colony was opaque, and none of the colonies produced mucus.

[0060] After Gram staining, the bacterial morphology was observed under an optical microscope (1000 times), and the morphology of P. pentosus IMAUYR3-1 was as shown in Figure 1 The morphology of P. pentosus IMAUYR3-1 was spherical, and the Gram staining was positive without spores.

[0061] The activated 3rd generation strain was subjected to glucose gas production, glucose fermentation, hydrogen sulfide production, and biogenic amine production tests;

[0062] Glucose gas production test: the activated 3rd generation strain was inoculated in a glucose gas production biochemical tube and cultured at 37℃ for 24 h, and whether there was gas bubble in the small inverted tube was observed. The result was no gas bubble;

[0063] Glucose fermentation test: the activated 3rd generation strain was inoculated in a glucose fermentation biochemical tube and cultured at 37℃ for 24 h, and whether the color of the culture medium changed from blue to yellow was observed. The result was yellow;

[0064] Hydrogen sulfide production test: the activated 3rd generation strain was inoculated in a hydrogen sulfide biochemical tube solid culture medium and cultured at 37℃ for 24 h, and whether black precipitate was generated in the culture medium was observed. The result was no black precipitate;

[0065] Biogenic amine production experiment: The activated third generation strain was inoculated on four kinds of biogenic amine solid medium (arginine, histidine, lysine, tyrosine), and un-inoculated sterile biogenic amine solid medium was used as a blank control. The culture was incubated at 37°C for 2-3 days, and the color change of the medium (from yellow to purple) was observed. The result was yellow, which met the requirements of the fermenting strain for P. pentosaceus IMAUYR3-1.

[0066] Based on the comprehensive screening and physiological and biochemical test results, P. pentosaceus IMAUYR3-1 was selected for identification.

[0067] 4. Molecular biological identification and growth ability determination of P. pentosaceus IMAUYR3-1

[0068] 16S rDNA sequence analysis: Genomic DNA extraction process: 1.5 mL of bacterial culture was centrifuged at 4500 rpm for 5 minutes to obtain bacterial precipitate. 567 μL of TE buffer was added to the bacterial precipitate, and the precipitate was resuspended by repeated blowing. 3 μL of proteinase K (20 mg / mL) was added, and the bacterial lysate was obtained by incubating at 37°C for 1 hour. 100 μL of CTAB and 100 μL of 0.7 mol / L NaCl were added to the bacterial lysate, mixed, and incubated at 65°C for 10 minutes to obtain the crude extract. 700 μL of phenol / chloroform / isoamyl alcohol was added to the crude extract, mixed by inverting, and then centrifuged at 12000 rpm for 5 minutes to obtain the supernatant. 700 μL of chloroform / isoamyl alcohol was added to the supernatant, mixed by inverting, and then centrifuged at 12000 rpm for 5 minutes to obtain the supernatant. 500 μL of isoamyl alcohol was added to the supernatant, and centrifuged at 12000 rpm for 5 minutes to obtain the DNA precipitate. The DNA precipitate was washed with 1 mL of 70% ethanol, naturally dried, and then resuspended to obtain the total DNA. Sequencing was completed by Hohhot Skyray Medical Test Co., Ltd., and the results showed that the sequence similarity of P. pentosaceus IMAUYR3-1 and P. pentosaceus was 99%. Therefore, P. pentosaceus IMAUYR3-1 was determined to be P. pentosaceus. Pediococcus pentosaceus ) of 99%, and therefore P. pentosaceus IMAUYR3-1 was determined to be P. pentosaceus.

[0069] 5. Growth curve and acid production ability analysis of P. pentosaceus IMAUYR3-1

[0070] Growth curve: The activated third generation P. pentosaceus IMAUYR3-1 was inoculated into MRS liquid medium and cultured in a 37°C incubator for 24 hours. The OD 600nm value and pH value were measured every 2 hours.

[0071] The growth curve (OD 600nm , viable cell count, and acid production ability) of P. pentosaceus IMAUYR3-1 is shown in the following table: Figure 2 ​Figure 2 The left graph is the viable cell count and OD 600nm , and the right graph is the acid production capacity, Figure 2 YR3-1 represents Pediococcus pentosaceus IMAU YR3-1. Pediococcus pentosaceus IMAU YR3-1 entered the logarithmic phase at 4 h, and the growth gradually reached the stationary phase at 12-14 h. The pH of Pediococcus pentosaceus IMAU YR3-1 rapidly decreased at 0-5 h, and the pH value decreased to below 4.5 at 6 h. Pediococcus pentosaceus IMAU YR3-1 has good acid production capacity. Therefore, Pediococcus pentosaceus IMAU YR3-1 meets the fermentation and acid production conditions as a starter.

[0072] 6. Pediococcus pentosaceus IMAU YR3-1 tolerance test

[0073] The detection was performed with Lactobacillus plantarum XAR-10 (Lactobacillus plantarum XAR-10 from the Free Bacterial Bank, an existing known strain) as a positive control group and Pediococcus pentosaceus IMAU YR3-1 as an experimental group.

[0074] 6.1 Salt tolerance

[0075] The detection method was as follows: inoculate the activated 3rd generation bacterial solution into MRS liquid medium with NaCl mass fraction of 3.0%, 6.0%, 7.5%, 9.0%, and 12.0%, respectively, and cultivate at 37°C for 24 h. The absorbance value (OD 600nm value) was measured at a wavelength of 600 nm. The results are shown in Figure 3 (the left graph, wherein YR3-1 represents Pediococcus pentosaceus IMAU YR3-1, and XAR-10 represents Lactobacillus plantarum XAR-10). It can be seen that Figure 3 when the mass fraction of NaCl is 6%, both Lactobacillus plantarum XAR-10 and Pediococcus pentosaceus IMAU YR3-1 grow well, and Pediococcus pentosaceus IMAU YR3-1 has the strongest tolerance. With the increase of salt concentration and the increase of osmotic pressure of the fermentation broth, the growth ability of the strain is gradually inhibited (P<0.05). When the NaCl concentration reaches 12%, Lactobacillus plantarum XAR-10 and Pediococcus pentosaceus IMAU YR3-1 hardly grow any more. P

[0076] 6.2 Nitrite tolerance

[0077] The detection method was as follows: inoculate the activated 3rd generation bacterial solution into MRS liquid medium with NaNO2 mass concentration of 30 mg / L, 60 mg / L, 90 mg / L, 120 mg / L, and 150 mg / L, respectively, and cultivate for 24 h. The absorbance value (OD 600nm value) was measured at a wavelength of 600 nm. The results are shown in Figure 3 ​(Right) shown. By Figure 3 (Right) can be seen that when the nitrite concentration is 150 mg / L, the OD 600nm values of the two strains are >1, and they can still grow, among which the OD 600nm values of P. pentosus IMAUYR3-1 and the control group P. acidilactici XAR-10 are 1.45 ± 0.01 and 1.44 ± 0.01, respectively, and the growth ability is good. At the same time, with the increase of the mass concentration of NaNO2, the growth ability of the strains shows a significant downward trend (P < 0.05). P

[0078] 6.3 Cholate salt tolerance

[0079] The detection method is as follows: 0.2 mL of activated third-generation bacterial solution is inoculated into 20 mL of MRS liquid medium containing cholate salt at 0.03%, 0.30% and 0.50%, respectively, and cultured at 37°C for 24 h. Then, the absorbance value (OD 600nm value) of the culture solution is measured at a wavelength of 600 nm. The tolerance of the strains to different concentrations of cholate salt is calculated, and the calculation results of cholate salt tolerance are shown in Table 1:

[0080] Cholate salt tolerance = OD value of cholate salt culture medium / OD value of blank culture medium x 100%

[0081] Table 1 Test results of cholate salt tolerance of P. pentosus IMAUYR3-1

[0082]

[0083] As can be seen from Table 1 (in the table, YR3-1 represents P. pentosus IMAUYR3-1, and XAR-10 represents P. acidilactici XAR-10), when the cholate salt content is 0.30%, the cholate salt tolerance of P. pentosus IMAUYR3-1 is 14.37%, and P. pentosus IMAUYR3-1 shows strong cholate salt tolerance, i.e., the cholate salt tolerance of P. pentosus IMAUYR3-1 is better than that of P. acidilactici XAR-10.

[0084] 6.4 pH tolerance

[0085] The detection method is as follows: 4.0% of the inoculum is inoculated into MRS liquid medium, and cultured at different pH (3.5, 4.5, 5.5, 6.5, 7.5) for 24 h. Then, the absorbance value (OD 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.

[0086] 6.5 Temperature tolerance

[0087] 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.

[0088] 7. Antibacterial analysis

[0089] 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 ;

[0090] The experimental results show that the metabolic products of the two strains have certain inhibitory effect on the two common pathogenic bacteria, and the inhibitory effect of P. pentosus IMAUYR3-1 on pathogenic bacteria is better than that of L. plantarum XAR-10.

[0091] 8. Analysis of proteolytic ability

[0092] 8.1 Cell wall protease activity

[0093] L. plantarum XAR-10 was used as a positive control group, and P. pentosus IMAUYR3-1 was used as a cell wall protease activity test group. The specific method was as follows: the strain was cultured at 37°C for 20 h to the logarithmic growth phase, centrifuged at 4500 r / min for 20 min at 4°C, washed with 50 mmol / L Tris-HCL (containing 30 mmol / L Ca 2+ , pH 7.8) buffer solution three times, and the bacterial cells were collected. The bacterial cells were suspended in a solution containing 50 mmol / L EDTA-Na2 and 50 mmol / L Tris-HCL, and incubated at 37°C for 3 h, then centrifuged, and the supernatant was collected to determine the cell wall protease activity (the enzyme activity unit was U·mL -1 ). The results were as follows: the enzyme activity of P. pentosus IMAUYR3-1 was 12.62±0.36 a , and the enzyme activity of L. plantarum XAR-10 was 11.32±0.37 b .

[0094] 8.2 Free amino acid nitrogen

[0095] P. pentosus IMAUYR3-1 of the third generation was inoculated in myofibrillar protein liquid medium and cultured at 37°C, which was a myofibrillar protein fermentation system. The un-inoculated strain was used as a blank control group. The content of free amino acid nitrogen in the myofibrillar protein fermentation broth at different fermentation times (0 d, 1 d, 2 d, 3 d, and 4 d) was determined by an o-phthalaldehyde derivatization colorimetric method. The absorbance was measured at 340 nm by a spectrophotometer, and at the same time, phenylalanine was used as a standard, and a standard curve was drawn. The linear regression equation of the phenylalanine standard curve was y=0.491x-0.0011, and the linear correlation coefficient R 2 =0.9993, indicating a good linear relationship. The content of free amino acid nitrogen was calculated according to the standard curve regression equation, as shown in Figure 4 . It can be seen from Figure 4 that in the fermentation simulation system at different fermentation times, the content of free amino acid nitrogen in the P. pentosus IMAUYR3-1 group was significantly higher than that in the blank control group P (<0.05), which proved that P. pentosus IMAUYR3-1 had good proteolytic ability.

[0096] 8.3 Myofibrillar protein SDS-PAGE electrophoresis

[0097] Sodium dodecyl sulfate polyacrylamide gel electrophoresis, method: activated 3 generation of Pediococcus pentosaceus IMAUYR3-1 was inoculated in myofibrillar protein liquid medium, cultured at 37℃ for 48 h, which was myofibrillar protein fermentation system, and the un-inoculated strain was blank control group. The myofibrillar protein fermentation broth cultured for 48 h was added with 5x protein loading buffer at a volume ratio of 4:1, and the sample was boiled for 10 min. The marker and sample loading amount were both 10 μL, and protein electrophoresis analysis was performed, as shown in Figure 5 .

[0098] As can be seen from Figure 5 , compared with the blank control group, the brightness of each protein band in the test group was significantly weakened, indicating that the strain promoted the degradation of myofibrillar protein. The actin band (Band1, 45 k Da) of Pediococcus pentosaceus IMAUYR3-1 added in the test group was shallower than that of the blank group, indicating that Pediococcus pentosaceus IMAUYR3-1 had good ability to degrade muscle protein. The brightness of tropomyosin (Band2, 40 k Da) and troponin (Band3, 37 k Da) bands was significantly weakened, and the band of Pediococcus pentosaceus IMAUYR3-1 group almost disappeared. The results showed that Pediococcus pentosaceus IMAUYR3-1 had strong degradation ability to myofibrillar protein. Compared with the blank control group, the myosin light chain (Band5, 12 k Da) band in the test group was more obvious, indicating that Pediococcus pentosaceus IMAUYR3-1 hydrolyzed the large molecular fragments in the protein into small molecular fragments. Therefore, Pediococcus pentosaceus IMAUYR3-1 can effectively degrade muscle protein.

[0099] <Example 2>

[0100] Whole genome sequencing of Pediococcus pentosaceus IMAUYR3-1

[0101] 1. Genomic DNA extraction and sequencing

[0102] The optimized SDS extraction method was used to extract genomic DNA. The bacteria collected by centrifugation in the logarithmic phase were ground in liquid nitrogen, and the lysis solution was used for lysis. Appropriate amount of proteinase K and mercaptoethanol were added, and the lysis process was gently inverted and mixed. After cooling to room temperature after lysis, centrifugation was performed, and the supernatant was added with chloroform / isoamyl alcohol (24:1) for extraction. Extraction was performed twice, and DNA was precipitated with isopropanol. After gentle inversion and mixing, centrifugation was performed, the waste liquid was discarded, and the precipitate was washed with 75% ethanol twice. Purification was performed using a purification column (OMEGA), and purification was performed using Ampure XP beads after purification. Nanodrop and Qbuit were used for quality inspection, and electrophoresis was used for quality inspection.

[0103] 2. Genome library construction and sequence determination

[0104] The DNA library of the strain qualified by quality inspection was sequenced by Illumina Novaseq 6000 high-throughput sequencing platform. The sequencing was completed by Hangzhou Lianche Biological Technology Co., Ltd.

[0105] 3. Genome sequence assembly and quality control analysis

[0106] The original data quality control, genome assembly and genome structure analysis were commissioned by Hangzhou Lianche Biological Technology Co., Ltd.

[0107] 4. Genome function analysis

[0108] The direct homologous clusters, gene ontology, Kyoto Encyclopedia of Genes and Genomes, and carbohydrate-active enzyme function analysis of the whole genome of P. pentosus IMAUYR3-1 were performed on the Lianche biological cloud platform.

[0109] The genome of P. pentosus IMAUYR3-1 contains a circular chromosome of 1785490 bp and a plasmid. The G+C content of the circular chromosome is 37.23%, and 1879 coding genes, 1779 predicted coding sequences (CDS), 1 gene island, 4 prophages, 2 potential CRISPR sequences, etc. are identified in the whole genome of P. pentosus IMAUYR3-1. Meanwhile, the genome contains 55 tRNA genes, 15 rRNA genes (5 23S rRNA, 5 5S rRNA, and 5 16S rRNA) and 1 tmRNA gene.

[0110] 4.1 COG function annotation

[0111] The functional categories, Pediococcus pentosaceus IMAUYR3-1 has 1778 genes annotated into 23 functional categories, accounting for 94.62% of the total number of genes. The number of genes annotated into different categories is as follows: 63 genes in category C (energy production and conversion), 31 genes in category D (cell cycle control, cell division, chromosome partitioning), 128 genes in category E (amino acid transport and metabolism), 97 genes in category F (nucleotide transport and metabolism), 150 genes in category G (carbohydrate transport and metabolism), 76 genes in category H (coenzyme transport and metabolism), 75 genes in category I (lipid transport and metabolism), 190 genes in category J (translation, ribosomal structure and biogenesis), 158 genes in category K (transcription), 104 genes in category L (replication, recombination and repair), 107 genes in category M (cell wall / membrane / envelope biogenesis), 14 genes in category N (cell motility), 56 genes in category O (posttranslational modification, protein turnover, chaperones), 82 genes in category P (inorganic ion transport and metabolism), 22 genes in category Q (secondary metabolites biosynthesis, transport and catabolism), 172 genes in category R (general function prediction only), 78 genes in category S (function unknown), 78 genes in category T (signal transduction mechanisms), 19 genes in category U (intracellular transport, secretion, and vesicular transport), 43 genes in category V (defense mechanisms), 4 genes in category W (cellular processes and signaling), 30 genes in category X (mobile elements: prophages and transposons), and 1 gene in category Z (cellular processes and signaling).

[0112] 4.2 GO functional annotation

[0113] The GO database function annotation information of P. pentosaceus IMAUYR3-1 was selected to draw the secondary classification of the top 20 annotations of GOslim under each classification. 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 number of genes encoding was the largest in translation (59) and phosphorylation (57). The rest, transmembrane transport 13, cell morphology regulation 17, DNA template transcription regulation 20, protein hydrolysis 32, phosphoenolpyruvate-dependent sugar phosphate transferase 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, DNA recombination 17, DNA integration, biosynthesis of guanylic acid, cell cycle, all 12, DNA replication, glycolysis process, fatty acid biosynthesis process, all 11; In the secondary functional categories contained in the cellular components, the number of genes encoding was the largest in the cytoplasmic membrane (218), followed by the cytosol (189) and the cytoplasm (171), small ribosomal subunit 8, ribosome 15, ribonucleoside-diphosphate reductase complex 4, ribonucleoprotein complex 14, proton transport ATP synthase complex catalytic core F(1) 5, protein-DNA complex 7, outer membrane bounded periplasmic space 5, membrane 162, extracellular region 10, cytoplasmic small ribosomal subunit 10, cytoplasmic large ribosomal subunit 20, chromosome 6, cell envelope Sec protein transport complex 4, cell division site 4, bacterial microcompartments 6, DNA-directed RNA polymerase complex 8, ATP-binding cassette (ABC) transporter complex 11; In the secondary functional categories contained in the molecular functions, the number of genes encoding was the largest in ATP binding (203), followed by DNA binding (130), zinc ion binding 8, transmembrane transporter activity 31, transferase activity 36, transcriptional cis-regulatory region binding 18, transport RNA binding 23, ribosomal structural constituent 53, ribosomal RNA binding 31, oxidoreductase activity 28, nucleic acid binding 22, metal ion binding 86, magnesium ion binding 46, kinase activity 23, hydrolase activity 35, acyltransferase activity and transfer of non-amino acyl groups 15, RNA binding 34, guanosine triphosphate (GTP) binding 21, DNA binding transcription factor activity 60, ATP hydrolysis activity 56.

[0114] 4.3 KEGG function annotation

[0115] KEGG database functional annotation information of P. pentosus IMAUYR3-1, cell process: 45 prokaryotic cell groups, 7 cell motility, 10 cell growth and death, environmental information processing: 34 signal transduction, 93 membrane transport, genetic information processing: 79 translation, 6 transcription, 45 replication and repair, 32 folding, sorting and degradation, biological system: 3 immune system, 3 environmental adaptation, metabolism: 16 xenobiotic biodegradation and metabolism, 81 nucleotide metabolism, 13 terpenes and polyketones metabolism, 31 other amino acid metabolism, 76 cofactor and vitamin metabolism, 49 lipid metabolism, 64 polysaccharide biosynthesis and metabolism, 408 global and overview map, 45 energy metabolism, 157 carbohydrate metabolism, 22 other secondary metabolite biosynthesis, 70 amino acid metabolism. The results showed that the strain had a strong metabolism and a strong growth and reproduction ability.

[0116] 4.4 Carbohydrate enzyme annotation

[0117] P. pentosus IMAUYR3-1 was annotated in CAZy database, a total of 42 carbohydrate active enzymes were annotated, including 20 glycosyltransferases, 17 glycoside hydrolases, 4 carbohydrate esterases, and 1 auxiliary oxidoreductase. Among the predicted glycosyl hydrolase families, it was found that P. pentosus IMAUYR3-1 contained protein family GT51, which was indirectly involved in protein hydrolysis regulation and could improve the protease activity of the strain, reflecting that P. pentosus IMAUYR3-1 had good proteolytic ability in fermented meat products.

[0118] 4.5 Protein degradation gene

[0119] P. pentosaceus IMAUYR3-1 protein degradation related genes: signal peptidase II (lspA), ATP-dependent proteases (clpA, ClpE, clpX, clpQ, clpC, hslU), proline peptidase (pepD), DNA-binding transcriptional repressor (LexA), oligopeptidase (pepF), oligopeptidase (pepB), glutamyl amino peptidase (pepA), dipeptidase (pepDB), transpeptidase A (srtA), aminopeptidase (ampS, pepS, ampT), carboxy-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);

[0120] Based on the information obtained from the COG, GO and KEGG databases for the whole genome functional gene annotation of P. pentosaceus IMAUYR3-1, it was found that P. pentosaceus IMAUYR3-1 has functional gene fragments involved in protein catabolic process proteolysis, wherein the genes encoding oligopeptidases (including dipeptidases and tripeptidases) include pepF, pepB, pepDA, pepDB, pepX and pepT, which are genes that hydrolyze proteins into smaller polypeptides and / or amino acids by cleaving the peptide bonds of proteins. The gene encoding glutamine aminopeptidase is pepA, and this enzyme can specifically hydrolyze glutamyl-containing peptide bonds to release free glutamic acid, which plays a great role in improving the flavor of fermented meat products during meat fermentation, and has the effect of enhancing umami taste.

[0121] <Example 3>

[0122] Preparation of fermented sausages

[0123] Sheep meat as raw material: raw meat (goat hind leg meat, goat tail fat, after being ground and mixed, wherein the amount of goat hind leg meat and goat tail fat is 8:2), the following materials are 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, baijiu 25 mL / kg, ascorbic acid 0.5 g / kg, corn starch 10 g / kg, whey protein powder 5 g / kg, and the number of viable bacteria in the starter culture is 1 x 107 CFU / g;

[0124] The preparation of the fermented sausages was divided into 3 groups according to different lactic acid bacteria starters: a natural fermentation group (CK, negative control), a Pediococcus pentosaceus XAR-10 group (XAR-10 group, positive control), and a Pediococcus pentosaceus IMAUYR3-1 group (YR3-1 group);

[0125] Preparation and parameters of the fermented sausages: the raw meat was ground and mixed with spices and a starter (directly mixed), and then was placed at 4℃ for 12h of curing. The meat stuffing was filled into collagen casings with a diameter of 25mm. The fermentation was carried out in a constant temperature and humidity box for 1-2d (temperature 25℃, relative humidity 95%), and the fermentation was completed when the pH was less than 5. The fermentation completion time was determined according to the pH detection (the fermented sausages of the present application were all completed in 1d). Drying: the first stage was 2-4d (temperature 15℃, relative humidity 75%), the second stage was 5-7d (temperature 10℃, relative humidity 65%), and finally the sausages were stored at 4℃.

[0126] <Embodiment 4>

[0127] A preparation method of a high-proteinase-yielding lactic acid bacteria assisted fermented sausage, comprising the following steps:

[0128] The meat pieces were ground to obtain raw meat (goat hind leg meat and goat tail fat were ground and mixed, and the amount ratio of the goat hind leg meat to the goat tail fat was 8:2);

[0129] The plant protein powder (pea protein powder) was mixed with water at a weight ratio of 1:7, and then Lactobacillus acidophilus was inoculated at a temperature of 35-38℃ for 18h of fermentation. After the fermentation was completed, the liquid whey was obtained by centrifugal separation. The liquid whey was heated at 80℃ for 10min, and then was cooled and adjusted to a pH of 5.8-6.0 by using 0.1 mol / L phosphate buffer. The fermented plant whey was obtained, and the addition amount of the Lactobacillus acidophilus was 0.05-0.15% of the total weight of the plant protein powder and water.

[0130] The pretreated raw meat was uniformly mixed with salt (20 g / kg of salt), sugar (5 g / kg of glucose), a starter, flavorings (5 g / kg of sucrose, 2 g / kg of dry ginger powder, 2 g / kg of pepper powder, 1 g / kg of pepper powder, 0.1 g / kg of sodium nitrite, 25 mL / kg of baijiu, 0.5 g / kg of ascorbic acid, 10 g / kg of corn starch, and 5 g / kg of whey protein powder), and the fermented plant whey. The addition amount of the starter was 0.1-0.3% of the total weight of the pretreated raw meat, and the addition amount of the fermented plant whey was 10-16% of the total weight of the pretreated raw meat.

[0131] The uniformly mixed material was filled into casings for fermentation to obtain the fermented sausage.

[0132] The fermenting agent comprises lactic acid bacteria, and the classification name of the strain is Pediococcus pentosaceus Pediococcus pentosaceus IMAUYR3-1, which was preserved in the China Center for Type Culture Collection (CCTCC) on April 29, 2025, and the preservation number is CCTCC NO: M 2025938, and the viable bacterial count in the fermenting agent is 1 x 10 7 CFU / g.

[0133] Fermentation is carried out in a constant-temperature and constant-humidity box for 1-2 days (temperature 25 DEG C, relative humidity 95%), and the fermentation is completed when the pH is less than 5, and the fermentation completion time is determined according to the pH detection (the fermented sausages of the present application are all completed in 1 day), drying: first stage 2-4 days (temperature 15 DEG C, relative humidity 75%), second stage 5-7 days (temperature 10 DEG C, relative humidity 65%), and finally stored at 4 DEG C.

[0134] In the 24th hour of fermentation, the sausage is immersed in a permeation liquid for 60-80 minutes, and the surface is wiped dry after soaking, and the fermentation is continued, wherein the permeation liquid comprises flavor protease, fermented plant whey, sodium lactate, lysozyme, and the rest water, the mass concentration of the fermented plant whey in the permeation liquid is 5%, the concentration of the sodium lactate is 0.2 mol / L, the addition amount of the lysozyme accounts for 0.05% of the total weight of the permeation liquid, and the weight ratio of the flavor protease to the lysozyme is 1:0.2.

[0135] The permeation liquid further comprises an enzyme composite carrier, which is composed of liposomes embedding lysozyme and flavor protease, and the weight ratio of the liposomes to the permeation liquid is 1:10.

[0136] The preparation method of the liposomes is as follows:

[0137] Egg phosphatidylcholine and cholesterol are dissolved in chloroform at a weight ratio of 4:1 to form a lipid film by rotary evaporation;

[0138] Lysozyme, flavor protease and fermented plant whey are mixed at a weight ratio of 1:1:4 to obtain an enzyme mixture;

[0139] The enzyme mixture is added to the lipid film, and is hydrated for 30 minutes under nitrogen protection, and then is ultrasonically broken to form coarse liposomes, the ultrasonic power is 300 W, and the time is 5 minutes;

[0140] The coarse liposomes are extruded through a polycarbonate membrane extruder at a temperature of 35-40 DEG C for 5-7 times, and 1 μm, 400 nm and 200 nm pore size filters are used in sequence to obtain the enzyme composite carrier.

[0141] The preparation of the enzyme mixture comprises the following steps:

[0142] The fermented whey is preheated to 35-38℃, 0.07% L-cysteine hydrochloride by weight of the fermented whey is added, stirred and dissolved to obtain a dissolved solution;

[0143] Flavourzyme is added to the dissolved solution, activated for 15 minutes under nitrogen protection at 25-28℃ to obtain a mixed solution;

[0144] Lysozyme is dissolved in a citric acid buffer at 2-5℃ and pH 5.0-5.2, and the concentration of lysozyme is 10 mg / mL to obtain a lysozyme solution;

[0145] The lysozyme solution is added to the mixed solution at a flow rate of 0.5 mL / min, the system temperature is maintained at 20-22℃ during the addition process, and the stirring is continued for 14 minutes after the addition is completed. After stirring, it is stored at 2-4℃ for use.

[0146] The mixing method of the pretreated raw meat and the fermented whey is as follows:

[0147] First, the starter culture is pre-mixed with 25-35% of the fermented whey at 12-15℃ for 10 minutes to obtain an activated fermentation liquid;

[0148] The pretreated raw meat, salt, sugar, flavorings and the remaining fermented whey are mixed at 0-4℃ to obtain a meat paste;

[0149] The activated fermentation liquid is added to the meat paste at 18-20℃, and the mixing is carried out at a vacuum degree of -0.04~-0.06 MPa and a rotation speed of 20-30 r / min for 3-5 minutes.

[0150] The mixing method of the activated fermentation liquid added to the meat paste is as follows:

[0151] The activated fermentation liquid is pre-cooled to 4℃, liquid droplets are formed using a liquid droplet generator, and are sprayed into a cold air channel at -25℃ with a wind speed of 2-3 m / s to freeze and form starter culture microspheres;

[0152] The starter culture microspheres are added to the meat paste in three batches at a rotation speed of 25 r / min: the first batch is 40% of the total weight of starter culture microspheres, mixed for 1 minute, the second batch is 30% of the total weight of starter culture microspheres, mixed for 1 minute, and the third batch is 30% of the total weight of microspheres, mixed for 1-3 minutes;

[0153] Before each batch of starter culture microspheres is added, the surface of the meat paste is sprayed with CO2-saturated fermented whey atomized liquid with a droplet size of 40 μm, and the spraying amount is 1.0% by weight of the meat paste.

[0154] Comparative Example 1: Fermented sausages were prepared by the method of Example 4, except that the plant protein powder was mixed with water without fermentation, i.e. the plant protein powder was mixed with water to obtain a protein liquid, and the fermented plant whey used in the subsequent steps was all protein liquid, and the remaining steps were the same.

[0155] Comparative Example 2: Fermented sausages were prepared by the method of Example 4, except that at the 24th hour of fermentation, the material was not soaked with the permeate solution, i.e. the uniformly mixed material was filled into the casing for fermentation, and the fermentation conditions were: temperature 25℃, relative humidity 95%, time 1-3d; the drying conditions were: first stage temperature 15℃, relative humidity 75%, time 2-4d; second stage temperature 10℃, relative humidity 65%, time 5-7d, and the fermented sausage was obtained after fermentation was completed.

[0156] Comparative Example 3: Fermented sausages were prepared by the method of Example 4, except that the permeate solution did not include the enzyme complex carrier.

[0157] Comparative Example 4: Fermented sausages were prepared by the method of Example 4, except that the preparation method of the enzyme mixture was to mix lysozyme, flavor protease and fermented plant whey at a weight ratio of 1:1:4, and dissolve in a citric acid buffer solution with pH 5.0-5.2 to obtain the enzyme mixture.

[0158] Comparative Example 5: Fermented sausages were prepared by the method of Example 4, except that the pre-processed raw meat and the fermented plant whey were directly mixed.

[0159] Comparative Example 6: Fermented sausages were prepared by the method of Example 4, except that the activated fermentation liquid was directly added to the meat paste for mixing.

[0160] <Characterization>

[0161] 1. Determination of physicochemical indexes of fermented sausages

[0162] 2.1 Determination of pH value

[0163] Determination of pH value: reference to the method of GB5009.237-2016 "National Food Safety Standard-Determination of pH value of food";

[0164] Changes in pH value of fermented sausages during processing and storage Figure 6 (left), the pH values of the sausages in the XAR-10 and YR3-1 groups were significantly lower than that of the fermented sausage in the CK group at the end of fermentation (1d) P<0.05). Subsequently (4-60 days), the pH values of the fermented sausages in the XAR-10 group and the YR3-1 group showed an overall upward trend, mainly due to the decomposition of proteins by microorganisms to produce some free amino acids and peptides, resulting in the accumulation of some basic nitrogen compounds and ammonia, thereby causing the pH value of the fermented sausage to increase.

[0165] 2.2 Change in water activity

[0166] Determination of water activity (Aw): According to the method of GB5009.3-2016 "National Food Safety Standard-Determination of Water in Food", the change in Aw was determined using an HD-3A intelligent water activity measuring instrument;

[0167] The change in water activity (Aw) of the fermented sausage during processing and storage is shown in Table 2.

[0168] Table 2 Change in water activity

[0169]

[0170] Comparative analysis of the CK group, the XAR-10 group, and the YR3-1 group showed that the water activity decreased and then increased with increasing processing and storage time. At 0 d and 1 d of fermentation, the changes in the Aw values of the fermented sausages in the CK, XAR-10, and YR3-1 groups were small, which was due to the high humidity in the environment during the first stage of fermentation, resulting in a small change trend. The decrease in water activity is crucial for ensuring the shelf life and safety of fermented sausages. With the progress of fermentation, the Aw values of the fermented sausages in the three groups were significantly reduced (P < 0.05), which may be due to water evaporation during fermentation. At the finished product stage (9 d), the Aw values of the fermented sausage samples in the XAR-10 group and the YR3-1 group were significantly lower than those in the CK group (P < 0.05). At 60 d of storage, the Aw values of the fermented sausages in the three groups slowly increased, which may be due to moisture absorption during storage, but the Aw values were still lower than 0.88, effectively inhibiting the growth of spoilage bacteria. P <0.05). Subsequently (4-60 days), the pH values of the fermented sausages in the XAR-10 group and the YR3-1 group showed an overall upward trend, mainly due to the decomposition of proteins by microorganisms to produce some free amino acids and peptides, resulting in the accumulation of some basic nitrogen compounds and ammonia, thereby causing the pH value of the fermented sausage to increase. P <0.05). At 60 d of storage, the Aw values of the fermented sausages in the three groups slowly increased, which may be due to moisture absorption during storage, but the Aw values were still lower than 0.88, effectively inhibiting the growth of spoilage bacteria.

[0171] Comparative 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 Example 4 was the best, and at 60 days, Example 4 was 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 application can effectively reduce the influence of moisture absorption during storage.

[0172] 2.3 Change in color difference

[0173] 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.

[0174] Table 3 is the fermented sausage value

[0175]

[0176] Table 4 is the fermented sausage value

[0177]

[0178] Table 5 is the fermented sausage value

[0179]

[0180] 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.

[0181] 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.

[0182] 2.4 Texture changes

[0183] 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 difference between different processing time groups P <0.05);

[0184] Table 6 Change of hardness during processing and storage of fermented sausages (hardness g)

[0185]

[0186] Table 7 Change of springiness during processing and storage of fermented sausages (springiness / mm)

[0187]

[0188] Table 8 Change of cohesiveness during processing and storage of fermented sausages

[0189]

[0190] 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 (1d), the springiness, hardness and cohesiveness of the fermented sausage samples of the YR3-1 group were significantly higher than those of the XAR-10 group and the CK group; during the storage period (30 days, 60 days), the hardness value, springiness and cohesiveness of the YR3-1 group were slightly higher than those of the XAR-10 group and the CK group; this shows that the use of Pediococcus pentosaceus IMAU YR3-1 can effectively improve the texture properties of sausages;

[0191] 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, which is better than that of Comparative Example 6; this shows that after the plant whey is fermented by Lactobacillus acidophilus and then added to the stuffing, on the one hand, the soluble ingredients in the plant whey play a hydrophilic colloid role in the meat paste, increasing the viscosity of the system and forming a gel network, thereby improving the texture evaluation of fermented sausages; through pre-activation of the starter, construction of a low-temperature meat paste matrix and vacuum mixing at medium temperature, the effect of plant whey and Pediococcus pentosaceus IMAU YR3-1 can be maximized, forming a high water-holding, dense and uniform protein gel network, which realizes the improvement of the hardness, springiness and cohesiveness of fermented sausages; the microspherization of the starter, the batch embedding and the synergistic effect of carbon dioxide make the protein network form a uniform and controllable layer-by-layer penetration into the deep layer of the meat paste and the storage of salt-soluble protein dissolution, which realizes the improvement of the texture of sausages.

[0192] 2.5 Change of volatile basic nitrogen

[0193] Determination of Total Volatile Base Nitrogen (TVB-N): The determination was carried out according to the semi-micro nitrogen determination method in GB5009.228-2016 "Determination of Total Volatile Base Nitrogen in Foods", and the determination results were as shown in Table 6. Figure 6 As shown in FIG. 2 (right), it can be seen that the TVB-N content of the three groups of fermented sausages showed an overall upward trend during the processing. At the end of drying (4d), the TVB-N content of the CK group was significantly higher than that of the XAR-10 and YR3-1 groups, because the lactic acid bacteria fermentation of the fermented sausage led to a decrease in pH, inhibited the growth of stray bacteria, prevented the production of volatile nitrogen compounds, and improved the quality of the fermented sausage. During the storage stage (30-60d), the increase rate of the TVB-N content of the CK group tended to be slow, and was still significantly higher than that of the XAR-10 and YR3-1 groups. It is proved that P. pentosaceus IMAU YR3-1 has an inhibitory effect on the production of basic nitrogen-containing substances, thereby effectively improving the quality of the fermented sausage.

[0194] 2.6 Change of thiobarbituric acid

[0195] Determination of Thiobarbituric Acid Reactive Substances (TBARS): The determination results of Example 3, Example 4, Comparative Examples 1-3 are shown in Table 9;

[0196] Table 9 is the value of thiobarbituric acid (mg / 100g)

[0197]

[0198] Comparative analysis of the CK group, the XAR-10 group and the YR3-1 group showed that the TBARS value of the three groups of fermented sausages showed a significant upward trend with time during the processing and storage. At the finished product stage (9d), the TBARS value of the fermented sausage samples of the XAR-10 group and the YR3-1 group was significantly lower than that of the CK group, which may be because the addition of lactic acid bacteria reduced the water activity of the fermented sausage, thereby slowing down the degree of lipid oxidation and rancidity. At the storage stage of 60d, the TBARS value of the fermented sausage samples of the YR3-1 group was significantly lower than that of the XAR-10 group and the CK group, indicating that P. pentosaceus IMAU YR3-1 has a strong ability to inhibit the lipid oxidation and rancidity in the fermented sausage.

[0199] Comparative analysis of Example 4, Comparative Examples 1, 2 and 3 showed that the development trend of Example 4, Comparative Examples 1, 2 and 3 was consistent with that 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 application can effectively reduce the content of thiobarbituric acid.

[0200] 3. Determination of microbial indicators of fermented sausage

[0201] 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.

[0202] Table 10 Changes in microbial counts during processing and storage of fermented sausages

[0203]

[0204] 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.

[0205] 4. Changes in moisture distribution of fermented sausages

[0206] 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;

[0207] 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.

[0208] 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 T 22 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.

[0209] 5. Microstructure observation of fermented sausages

[0210] The samples of each group of fermented sausages were observed and compared by using a scanning electron microscope. The samples were cut into small cubes with a side length of 5 mm and a thickness of 2 mm, and then taken out after vacuum freeze-drying on a freeze dryer. The treated samples were adhered to the double-sided adhesive tape of the conductive carbon film, and observed and photographed by using a scanning electron microscope. The microstructure of the samples of the fermented sausages at the finished product stage (9 days) of the three groups is shown in FIG. 2, wherein A is the CK group; B is the XAR-10 group; and C is the YR3-1 group. Figure 9

[0211] As can be seen from FIG. 2, compared with the naturally fermented CK group, the structure of the sausage of the starter group (YR3-1 and XAR-10 groups) is more compact and has smaller voids. The pore structure of the sausage of the YR3-1 group is smaller and more uniform, which indicates that the addition of Pediococcus pentosaceus IMAU YR3-1 can make the surface structure of the sausage delicate, enhance the internal gel network structure of the sausage, and make the tissue state more compact, thereby further improving the water retention effect. Figure 9 6. Sensory evaluation of fermented sausages

[0212] Thirteen panelists were selected by a basic taste identification program and trained for one week to taste commercial sausage products to familiarize them with the characteristics of the samples to be evaluated. The test sausages were cut into slices with a thickness of about 4-5 mm for sensory evaluation. The mouth was washed with water before tasting different samples. The evaluation criteria are shown in Table 11, and the evaluation results are shown in Table 12.

[0213] Table 11. Sensory evaluation table of fermented sausages

[0214]

[0215] Table 12. Sensory evaluation results of fermented sausages

[0216]

[0217] As shown in Table 12, compared with the XAR group and the CK group, the sausage of the YR3-1 group has the best acceptability, and the taste and tissue state are better than those of the other two groups, and the scores of all indicators are higher than those of the CK group. This indicates that the use of Pediococcus pentosaceus YR3-1 for fermentation of sausages can reduce the pH value and the water activity of the fermented sausages, and can improve the hardness and elasticity of the sausages. Therefore, the sausage of the YR3-1 group has good taste and texture, and greatly improves the quality of the fermented sausages. The color of the sausage of the XAR-10 group is better than that of the other two groups, but the taste is poor. The flavor scores of the sausages of the YR3-1 and XAR-10 groups are similar.

[0218]

[0219] ​​Compared with the group of Example 4, the acceptability and mouth texture of Example 4 were better than those of YR3-1 group, and Example 4 was better than Comparative Examples 1-6. By comparing Example 4 with Comparative Example 1, it was found that the addition of fermented plant whey could effectively improve the sensory evaluation of fermented sausages and improve the overall acceptability. By comparing Example 4 with Comparative Examples 2-4, it was found that the methods of immersing sausages in a penetration solution, adding an enzyme complex carrier to the penetration solution, and preparing an enzyme mixed solution could effectively improve the sensory evaluation of sausages. By comparing Example 4 with Comparative Examples 5 and 6, it was found that by changing the mixing method of the pretreated raw meat and the fermented plant whey and changing the mixing method of the activated fermentation liquid added to the meat paste, the sensory evaluation of the fermented sausages could be effectively improved.

[0220] 7. Protein hydrolysis characteristics of fermented sausages

[0221] 7.1 Analysis of changes in non-protein nitrogen content in fermented sausages

[0222] Non-protein nitrogen content (NPN) is an index for evaluating the degree of protein hydrolysis of a fermenting agent in a fermented sausage.

[0223] 2 g of a sample and 18 mL of distilled water were added to a 50 mL centrifuge tube, homogenized for 2 min, centrifuged at 10,000 x g at 5°C for 15 min, and the supernatant was filtered twice with Whatman 1# chromatography paper. The volume (V) of the filtrate was recorded. 15 mL of the filtrate and 15 mL of 10% trichloroacetic acid were mixed and allowed to stand for 30 min, and then centrifuged under the same conditions as above. The supernatant was filtered with Whatman 4# chromatography paper, and 5 mL of the filtrate was used to determine the nitrogen content N1. The non-protein nitrogen (NPN) content N0 was calculated by 0.2V x N1, and the total nitrogen content was N.

[0224] NPN content during the processing and storage of fermented sausages Figure 7 As shown in the left graph, during the fermentation-drying-maturing process, the NPN content of the three groups of fermented sausages showed an upward trend, and tended to be stable during the storage period. At the end of fermentation (1 d), the NPN content of the XAR-10 group and the YR3-1 group was significantly higher than that of the natural fermentation CK group. This is because under the action of proteolytic enzymes, the total content of polypeptides, short peptides and free AA increases, resulting in an increase in the degree of protein degradation in sausages. During the storage stage (30-60 d), the NPN content of the three groups of fermented sausages tended to be stable, but the NPN content of the fermentation agent groups (XAR-10 group and YR3-1 group) was still significantly higher than that of the natural fermentation CK group.

[0225] 7.2 Determination of protein hydrolysis index of fermented sausages

[0226] 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;

[0227] Table 13 is the protein hydrolysis index of fermented sausage

[0228]

[0229] 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;

[0230] 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.

[0231] 7.3 Determination of amino nitrogen content in fermented sausages

[0232] The amino nitrogen (AN) content was determined by formaldehyde titration. Calculation was performed according to formula (3):

[0233] Amino nitrogen content (g / 100mL) = ((V1-V0)×C×0.014×100) / (10×V / 100) (3)

[0234] 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;

[0235] 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<The AN content of the fermented sausages of the Example 4 group (0.468 g / 100 g) was higher than that of the Comparative Example 1 group (0.457 g / 100 g). During the storage stage (30-60 d), the AN content of the fermented sausages of all groups tended to be stable, and the rate of proteolysis slowed down, which can be attributed to the inhibition of the growth and metabolism of microorganisms and the decrease in enzyme activity, further maintaining the stability of the AN content (the AN content increased in the first 9 days, and tended to be stable in the 9-60 days), but the AN content of the Example 4 group (0.659 g / 100 g) during the storage stage was higher than that of the Comparative Example 1 group (0.659 g / 100 g), the RY3-1 group (0.630 g / 100 g), the RY3-1 group was higher than that of the XAR-10 group (0.614 g / 100 g) and the CK group (0.483 g / 100 g). This indicates that the plant whey prepared by the present application can effectively increase the AN content.

[0236] 8. Measurement of volatile flavor substances of fermented sausages

[0237] Detection method: 5 g of sausage sample was weighed into a sample bottle, an extraction needle was inserted above the sample bottle, and after adsorption at 60°C for 40 min, it was taken out and then inserted into the GC inlet, and resolved at 250°C for 3 min. GC-MS conditions: TR-5 chromatographic column (30 m x 0.25 mm, 0.25 μm), carrier gas He; carrier gas flow rate 1.0 mL / min; sample and interface temperature 250°C; temperature program: initial temperature 40°C, hold for 5 min, increase to 200°C at 5°C / min, hold for 5 min, then increase to 250°C at 20°C / min, hold for 5 min, no split injection; ion source temperature 250°C; transfer line temperature 250°C; mass scan range 30-400 m / z; solvent delay 1 min. Mass spectral data were searched against Meanlib, Nistdemo and Wiley Library for qualitative analysis, and a matching degree greater than 800 was used as the identification basis. The peak area percentage of each component was calculated by area normalization.

[0238] A total of 72 types of volatile compounds were detected during fermentation, 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 changed differently over time in each group, with alcohols, esters and terpenes being the main components, and the amount of the three types of substances in the YR3-1 group and the XAR-10 group was more than that in the CK group.

[0239] The alcohol substances mainly include ethanol, heptanol, nonanol, octanol, 2,3-butanediol, 2-ethylcyclobutanol, camphor alcohol, alpha-terpineol, carveol, citronellol, 1-octene-3-ol, 3-cyclohexene-1-ol, terpinen-4-ol, isopropyl alcohol and the like. Among them, the contents of ethanol and octanol are relatively high. Ethanol can help meat products release flavor substances, and itself can be converted into esters (such as ethyl acetate) to give fruity aroma. Octanol has mushroom, earthy aroma, and is an important component of the complex flavor of fermented sausages. 1-octene-3-ol has a special mushroom smell and is an important flavor contributor in fermented meat products. From the test results, the content of octanol: the content of octanol in the YR3-1 group (9d was 4.54±0.11 a , 30d was 0.14±0.01 a , 60d was 0.13±0.04 a ), the content of octanol in the XAR-10 group (9d was 4.05±1.82 a , 30d was 0.13±0.02 a , 60d was 0.14±0.03 a ), the content of octanol in the CK group (9d was 0.13±0.02 b , 30d was 0.14±0.02 a , 60d was 0.23±0.10 a ), the content of octanol in the example 4 group (9d was 5.71±0.18 a , 30d was 0.53±0.02 b , 60d was 0.49±0.02 a ), the content of octanol in the comparative example 3 group (9d was 4.34±0.06 b , 30d was 0.39±0.01 a , 60d was 0.26±0.07 a ), the content of octanol in the comparative example 4 group (9d was 5.24±0.27 a , 30d was 0.49±0.14 a , 60d was 0.41±0.05 c ); the content of 1-octene-3-ol: the content of 1-octene-3-ol in the YR3-1 group (9d was 3.42±2.01 a , 30d was 1.23±0.96 ab , 60d was 3.25±0.51 a , the content of 1-octene-3-ol in the XAR-10 group (9d was 1.81±0.24 ab , 30d was 2.07±1.02 a, 30d was nd, 60d was nd (nd = not detected), Example 4 group 1-octen-3-ol content (9d was 4.61 ± 1.18 a , 30d was 4.05 ± 0.98 a , 60d was 4.59 ± 1.07 a ), Comparative Example 3 group 1-octen-3-ol content (9d was 3.77 ± 1.43 a , 30d was 2.61 ± 1.12 a , 60d was 2.73 ± 1.35 a ), Comparative Example 4 group 1-octen-3-ol content (9d was 3.94 ± 1.27 c , 30d was 2.87. ± 1.36 a , 60d was 3.72 ± 0.96 a );

[0240] Esters are produced by esterification of alcohols and acids, and many ester compounds are considered to be the source of fruitiness and caramel flavor in fermented sausages. Ester compounds include ethyl 3-methylbutanoate, ethyl heptanoate, ethyl valerate, ethyl acetate, ethyl 2-hydroxypropanoate, ethyl nonanoate, ethyl stearate, ethyl undecanoate, ethyl arachidate, and ethyl laurate. Ethyl acetate, ethyl valerate, and ethyl heptanoate are typical fruit-flavored esters, which can balance the saltiness in fermented meat products, and ethyl valerate, a precursor of which is leucine produced by muscle protein hydrolysis, has a unique flavor. At the end of fermentation (1d) and at the product stage (9d), the ethyl valerate content of the YR3-1 group (1d was 0.20 ± 0.03 a , 9d was 0.33 ± 0.05 a ) and the XAR-10 group (1d was 0.19 ± 0.04 a , 9d was 0.25 ± 0.02 a ) was significantly higher than that of the natural fermentation CK group, and the ethyl valerate content of the Example 4 group (1d was 0.24 ± 0.03 a , 9d was 0.40 ± 0.02 a , 60d was 0.62 ± 0.06 a ) was higher than that of the Comparative Example 3 group (1d was 0.21 ± 0.03 a , 9d was 0.35 ± 0.01a, 60d was 0.57 ± 0.04 a ) and the Comparative Example 4 group (1d was 0.22 ± 0.01 a , 9d was 0.37 ± 0.02 a , 60d was 0.59 ± 0.04 a ).

[0241] The additives and spices used in the sausage can generate terpene substances, which help to form the flavor of the fermented sausage. The detection results show that the terpene substances mainly include caryophyllene, phenol, camphene, cucurbitene, isocaryophyllene, styrene, a-farnesene, 1-methyl-4-cyclohexene and D-limonene, etc. Among them, camphene has a slight citrus and herbal flavor, which can balance the greasy feeling in meat products, and cucurbitene gives the fermented meat products a smoked flavor. The contents of these two substances are relatively high. The terpene substances in the fermented sausage can enhance the layering of the sausage flavor, thereby making the sausage taste rich. For cucurbitene: the content of cucurbitene in the YR3-1 group (1d is 0.16±0.01 b , 30d is 0.11±0.02 a , 60d is 0.77±1.14 a ) is lower than that in the XAR-10 group (1d is 0.79±0.15 a , 30d is 0.85±0.01 a , 60d is 0.93±0.72 a ), but the content of cucurbitene in the YR3-1 group is significantly higher than that in the natural fermentation CK group (1d is 0.06±0.01 b , 30d is 0.10±0.01 a , 60d is 0.13±0.01 a ) at the storage time (60d), which indicates that the effect of using P. pentosaceus IMAU YR3-1 alone in the YR3-1 group on the content of cucurbitene is lower than that of P. plantarum XAR-10; the content of cucurbitene in the example 4 group (1d is 0.84±0.02 a , 30d is 0.65±0.03 b , 60d is 1.17±0.05 b ) is higher than that in the comparative example 3 group (1d is 0.80±0.01 b , 30d is 0.55±0.04 a , 60d is 0.98±0.02 b ) and the comparative example 4 group (1d is 0.82±0.03 a , 9d is 0.37±0.02 a , 60d is 1.02±0.06 b );

[0242] Aldehydes are the key to the fresh and complex flavor of fermented sausages, and aldehydes participate in the Maillard reaction, which promotes the formation of color on the surface of sausages. 3-methylhexanal, which has a grassy and nutty aroma, is due to the Strecker degradation of leucine. At the finished period (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 sausages citrus and fat aroma, which is formed by the oxidation of oleic acid or linoleic acid. Ketones usually give sausages a buttery aroma. Secondly, there are some other aromatic hydrocarbons in fermented sausages, such as toluene and ethylbenzene, but such substances are less and have less impact on the flavor of fermented sausages. For 3-methylhexanal: the content of 3-methylhexanal in the YR3-1 group at different periods (9d was 0.17±0.02 a , 30d was 0.15±0.01 b , 60d was 0.13±0.04 a , the content of 3-methylhexanal in the XAR-10 group at different periods (9d was 0.15±0.03 a , 30d was 0.14±0.02 a , 60d was 0.13±0.05 a ), the content of 3-methylhexanal in the CK group at different periods (9d was 0.06±0.02 b , 30d was 0.18±0.01 a , 60d was 0.20±0.05 a ), the content of 3-methylhexanal in Example 4 at different periods (9d was 0.26±0.03 b , 30d was 0.28±0.02 a , 60d was 0.24±0.05 a ), the content of 3-methylhexanal in Comparative Example 1 at different periods (9d was 0.19±0.01 a , 30d was 0.22±0.03 a , 60d was 0.17±0.01 a ), the content of 3-methylhexanal in Comparative Example 2 at different periods (9d was 0.21±0.03 a , 30d was 0.24±0.01 c , 60d was 0.20±0.05 a ); other ingredients were detected, which are not listed in this application;

[0243] From the above analysis, it can be seen that the addition of Pediococcus pentosaceus IMAU YR3-1 has a certain effect on the flavor components of fermented sausages, but the addition of the permeate solution during the preparation of sausages can significantly retain the content of flavor components in fermented sausages and improve the taste of fermented sausages.

[0244] While embodiments of the application have been disclosed in connection with the above specification, it will be apparent to those skilled in the art that numerous modifications can be made thereto without departing from the overall concept of the application. Accordingly, it is intended that all such modifications be included within the scope of the claims and their equivalents.

Claims

1. A method for preparing a high-protease lactic acid bacteria-assisted fermented sausage, characterized by, The method comprises the following steps: mincing the meat to obtain raw meat; mixing plant protein powder and water in a weight ratio of 1:5-10, inoculating Lactobacillus acidophilus at a temperature of 35-38°C for 12-24 hours of fermentation, centrifuging to obtain liquid whey after the fermentation is completed, heating the liquid whey at 80°C for 10 minutes, cooling, and then adjusting the pH of the liquid whey to 5.8-6.0 using 0.1 mol / L phosphate buffer to obtain fermented plant whey, and the addition amount of Lactobacillus acidophilus is 0.05-0.15% of the total weight of the plant protein powder and water; mixing the pretreated raw meat, salt, sugar, leavening agent, flavoring agent and fermented plant whey uniformly, the addition amount of the leavening agent is 0.1-0.3% of the total weight of the pretreated raw meat, and the addition amount of the fermented plant whey is 10-16% of the total weight of the pretreated raw meat; filling the uniformly mixed material into a casing for fermentation to obtain fermented sausages; The fermenting agent comprises lactic acid bacteria, and the classification name of the strain is Pediococcus pentosaceus (IMAUYR3-1) which is preserved in China Center for Type Culture Collection on April 29, 2025, and the preservation number is CCTCC NO:M2025938. Pediococcus pentosaceus ) IMAUYR3-1, preserved in China Center for Type Culture Collection on April 29, 2025, and the preservation number is CCTCC NO:M2025938.

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

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

25.

4. The method for preparing a high-protease lactic acid bacteria-assisted fermented sausage according to claim 3, characterized by, The penetration liquid further comprises an enzyme complex carrier, the carrier is composed of liposomes embedding lysozyme and flavor protease, and the weight ratio of the liposomes to the penetration liquid is 1:8-12. The preparation method of the liposomes is as follows: dissolving lecithin and cholesterol in chloroform in a weight ratio of 3-5:1 to form a lipid film by rotary evaporation; mixing lysozyme, flavor protease and fermented plant whey in a weight ratio of 1:1:3-5 to obtain an enzyme mixture; adding the enzyme mixture into the lipid film, hydrating for 30 minutes under the protection of nitrogen, and then ultrasonically crushing to form coarse liposomes, the ultrasonic power is 300 W, and the time is 5 minutes; extruding the coarse liposomes through a polycarbonate membrane extruder at a temperature of 35-40°C for 5-7 times, and sequentially using filter membranes with pore sizes of 1 μm, 400 nm and 200 nm to obtain the enzyme complex carrier.

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

6. The method for preparing a high-protease lactic acid bacteria-assisted fermented sausage according to claim 2, characterized by, The mixing method of the pretreated raw meat and the fermented plant whey is as follows: First, the starter culture is mixed with 25-35% of the fermented plant whey at 12-15 ℃ for 5-10 minutes to obtain an activated fermentation liquid; The pretreated raw meat, salt, sugar, spices, and the remaining fermented plant whey are mixed at 0-4 ℃ until a sticky state is achieved, to obtain a meat paste; The activated fermentation liquid is added to the meat paste at 18-20 ℃, and the mixture is mixed at a vacuum degree of -0.04~-0.06 MPa and a rotation speed of 20-30 r / min for 3-5 minutes.

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

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