Compound bacteria, meat fermentation composition and meat fermentation method

Abstract

The invention relates to a compound bacterium, a meat fermentation composition and a meat fermentation method, the compound bacterium is used for meat fermentation, and the compound bacterium comprises at least one of the following three bacteria: lactobacillus plantarum XC-1-3 preserved in the China General Microbiological Culture Collection Center (CGMCC) with the preservation number of CGMCC No.36530, lactobacillus plantarum XC-1-3 preserved in the China General Microbiological Culture Collection Center (CGMCC), lactobacillus plantarum XC-1-3 preserved in the China General Microbiological Culture Collection Center (CGMCC), and lactobacillus plantarum XC-1-3 preserved in the China General Microbiological Culture Collection Center (CGMCC). The lactobacillus plantarum YC-1-4 is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number of the lactobacillus plantarum YC-1-4 is CGMCC No.36529, the lactobacillus plantarum MXC-4 is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number of the lactobacillus plantarum MXC-4 is CGMCC No.36528, and the lactobacillus plantarum MXC-4 is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number of the lactobacillus plantarum MXC-4 is CGMCC No.36528. In the synergistic fermentation, the compound bacteria can effectively improve the flavor and quality of the fermented meat, the vinasse liquid can promote the high-value utilization of vinasse byproducts, and the synergistic effect of the compound bacteria and the vinasse liquid realizes the dual improvement of the flavor and the shelf life of the fermented meat product.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, and in particular to a compound microbial and meat fermentation composition and a meat fermentation method. Background Technology

[0002] Fermented meat products are a type of processed meat food that is widely popular. Traditional fermentation processing often relies on a single fermenting agent or exogenous flavoring additives, as exemplified by Chinese invention patent application number CN202411005866.1 (publication number CN118853476A). Traditional preparation methods make it difficult to simultaneously achieve significant flavor enhancement and extended quality stability in fermented meat products.

[0003] Furthermore, brewing byproducts such as distiller's grains are rich in free amino acids, organic acids, and various flavor precursors, and carry complex microbial communities, making them potential natural flavor and functional ingredients. However, their direct application in meat processing faces technical bottlenecks such as microbial safety risks, unstable flavor components, and poor process controllability, and has not yet been fully utilized in industrialization.

[0004] Furthermore, lactic acid bacteria, as the core microorganisms in food fermentation, can reduce the risk of harmful microorganisms and nitrosamines by producing organic acids and antibacterial substances, and can also improve flavor and texture. However, when lactic acid bacteria are used directly in meat products, their insufficient environmental adaptability and limited metabolic spectrum often result in insufficient flavor complexity and limited shelf life extension. Summary of the Invention

[0005] The first technical problem to be solved by the present invention is to provide a compound bacteria that can be used for meat fermentation and can improve the flavor and quality of fermented meat, in contrast to the prior art.

[0006] The second technical problem to be solved by the present invention is to provide a meat fermentation composition having the above-mentioned complex bacteria, in contrast to the prior art.

[0007] The third technical problem to be solved by the present invention is to provide a meat fermentation method that is different from the prior art.

[0008] The technical solution adopted by the present invention to solve the above-mentioned technical problems is: a compound bacteria, characterized in that it is used for meat fermentation and includes at least one of the following three bacteria:

[0009] Lactiplantibacillus plantarum XC-1-3 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 36530;

[0010] Lactiplantibacillus plantarum YC-1-4 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36529.

[0011] Lactiplantibacillus plantarum MXC-4 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36528.

[0012] Furthermore, the compound bacteria include *Lactobacillus plantarum* XC-1-3 and *Lactobacillus plantarum* YC-1-4.

[0013] Furthermore, the compound bacteria consists of *Lactobacillus plantarum* XC-1-3 and *Lactobacillus plantarum* YC-1-4, and the bacterial suspensions of the two are compounded at the same concentration and in equal volume.

[0014] The technical solution adopted to further solve the second technical problem mentioned above is: a meat fermentation composition, characterized in that it includes the compound bacteria as described above.

[0015] Furthermore, the meat fermentation composition also includes lees.

[0016] The technical solution adopted to further solve the third technical problem mentioned above is: a meat fermentation method, characterized in that it uses the meat fermentation composition described above.

[0017] Furthermore, the meat fermentation method is characterized by including the following steps:

[0018] S1: Take the lees, add purified water, mix well and heat to boiling to remove bacteria and off-flavors. After cooling to room temperature, divide into sterilized fermentation containers for later use.

[0019] S2: Add the activated compound bacteria to the lees extract prepared in step S1 and mix thoroughly;

[0020] S3: The inoculated lees extract is left to ferment statically to activate the metabolic activity of the complex bacteria and form a fermentation broth;

[0021] S4: After removing impurities from the raw meat, clean it thoroughly, remove visible connective tissue, fat and other non-muscle tissue, then cut it into pieces according to the predetermined specifications and freeze it at low temperature for later use.

[0022] S5: Rinse the thawed meat in running water to thoroughly remove blood, impurities and surface mucus, improving the sensory quality of the meat and the uniformity of subsequent marinating;

[0023] S6: Add water to a pot, enough to cover the meat, add appropriate seasonings, bring to a boil and simmer over low heat to remove any off-flavors, then remove and let cool naturally to room temperature.

[0024] S7: Place the cooled meat pieces into the fermentation liquid prepared in step S3, ensuring that the meat pieces are completely submerged in the fermentation liquid for low-temperature fermentation;

[0025] S8: After fermentation is complete, remove the meat pieces, divide them into quantitative portions, add a small amount of the fermentation liquid prepared in step S3, vacuum seal, and store at low temperature.

[0026] Preferably, in step S2, the amount of compound bacterial suspension added is 6% of the volume of the distiller's grains liquid, and the concentration of the compound bacterial suspension is 1×10⁻⁶. 8 CFU / mL.

[0027] Preferably, the preparation conditions of the fermentation broth in step S3 are: static fermentation for 24 hours at a temperature of 30°C and a humidity of 65%.

[0028] The conditions for the low-temperature fermentation in step S7 are: fermentation for 24 hours at a temperature of 15°C and a humidity of 65%.

[0029] Preferably, the mass ratio of the fermentation liquid added in step S8 to the meat chunks is 1:6.

[0030] Compared with the prior art, the advantages of the present invention are as follows: The *Lactobacillus plantarum* XC-1-3, YC-1-4, and MXC-4 obtained by screening in this invention are all deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date for all three is November 7, 2025. Specifically, the accession number for *Lactobacillus plantarum* XC-1-3 is CGMCC No. 36530, for *Lactobacillus plantarum* YC-1-4 it is CGMCC No. 36529, and for *Lactobacillus plantarum* MXC-4 it is CGMCC No. 36528.

[0031] This invention utilizes a synergistic fermentation method combining "fermentation liquor + compound bacteria" to improve the flavor and quality of fermented meat. The combined action of the fermentation liquor and lactic acid bacteria effectively improves the color characteristics of the fermented meat, giving it a more vibrant appearance. The combination of *Lactobacillus plantarum* XC-1-3 and *Lactobacillus plantarum* YC-1-4 mitigates the negative textural effects of fermentation liquor treatment, enhancing product texture, effectively inhibiting lipid oxidation, significantly reducing TBARS values, delaying oxidative deterioration, ensuring the quality stability of fermented meat products, and extending their shelf life. Furthermore, synergistic fermentation enhances the release and accumulation of amino acids in the meat and exhibits advantages in the production of flavor compounds such as alcohols, aldehydes, ketones, and esters, thus enriching the flavor profile of the fermented meat with more complex and diverse characteristics.

[0032] In summary, the compound bacteria in the synergistic fermentation of this invention can effectively improve the flavor and quality of fermented meat, while the lees can promote the high-value utilization of by-products of distiller's grains. The two work together to achieve a dual improvement in the flavor and shelf life of fermented meat products. Attached Figure Description

[0033] Figure 1 The above are electrophoretic analysis images of *Lactobacillus plantarum* XC-1-3, YC-1-4, and MXC-4 in the embodiments of the present invention.

[0034] Figure 2 The growth curves of *Lactobacillus plantarum* XC-1-3, YC-1-4, and MXC-4 in the embodiments of the present invention are shown.

[0035] Figure 3 The acid production curves of *Lactobacillus plantarum* XC-1-3, YC-1-4, and MXC-4 in the embodiments of the present invention are shown.

[0036] Figure 4 This invention relates to a test of the antagonistic ability of *Lactobacillus plantarum* strains XC-1-3+YC-1-4, XC-1-3+MXC-4, and YC-1-4+MXC-4 in the embodiments of the present invention.

[0037] Figure 5 The biofilm yields of *Lactobacillus plantarum* XC-1-3, YC-1-4, and MXC-4 and various mixed bacterial combinations in the embodiments of the present invention are shown, where X is XC-1-3, Y is YC-1-4, and M is MXC-4 (the same below).

[0038] Figure 6 The AI-2 signal molecule yields of *Lactobacillus plantarum* XC-1-3, YC-1-4, and MXC-4 and various mixed bacterial combinations in the embodiments of the present invention;

[0039] Figure 7The diagram shows the color change of fermented meat in the embodiments of the present invention. In the diagram, XC-YWL is the XC-1-3 + fermented liquid group, YC-YWL is the YC-1-4 + fermented liquid group, MXC-YWL is the MXC-4 + fermented liquid group, MIX-YWL is the mixed bacteria + fermented liquid group, YWL is the fermented liquid group, and CK is the blank group (the same below).

[0040] Figure 8 This is a graph showing the textural changes of fermented meat in an embodiment of the present invention;

[0041] Figure 9 This is a PCA principal component analysis diagram of fermented meat using an electronic nose in an embodiment of the present invention.

[0042] Figure 10 This is a graph showing the change in thiobarbituric acid content in fermented meat in an embodiment of the present invention.

[0043] Figure 11 This is a schematic diagram showing the free amino acid content of fermented meat in an embodiment of the present invention;

[0044] Figure 12 A schematic diagram illustrating the DPPH free radical scavenging ability of fermented meat in this embodiment of the invention. Detailed Implementation

[0045] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0046] The main culture media used in each embodiment are as follows:

[0047] MRS liquid culture medium: peptone 10.0 g / L, beef extract 8.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1 g / L, pH 5.7 ± 0.2.

[0048] MRS solid medium: peptone 10.0 g / L, beef extract 8.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, agar 14.0 g / L, Tween 80 1.0 g / L, pH 6.5 ± 0.2.

[0049] Example 1: Screening of bacterial strains

[0050] Weigh 5g of each of the fermented products (pickled vegetables, pickled fish, and pickled meat) (purchased from retail markets in Keqiao District, Shaoxing, Zhejiang Province) and place them in a sterile homogenizing bag. Add 45mL of sterile physiological saline, mix in a sterile beater for 2min, and perform 10-fold serial dilutions. Select an appropriate dilution, take 100uL of the diluted solution and spread it on an MRS agar plate containing 0.2% bromocresol purple, and incubate at 37℃ for 48h.

[0051] Yellow single colonies were picked from MRS agar plates, purified by streak plating twice, and then expanded in MRS broth. Eight strains of lactic acid bacteria were preserved in 50% glycerol tubes and stored at -80°C for later use. Safety screening of the eight purified strains was performed, including testing for amino acid decarboxylase negativity, glucose fermentation gas production negativity, litmus milk positivity, and hydrogen sulfide production negativity. The results are shown in Table 1. Five strains meeting the requirements were finally obtained and named: XC-1-3, YC-1-4, MXC-4, FJY-N-1, and C8.

[0052] Through screening for tolerance to 6% ethanol, 6% salt, and 6% nitrite, and combined with a comprehensive evaluation of safety and tolerance, three suitable lactic acid bacteria strains, XC-1-3, YC-1-4, and MXC-4, were obtained from Shaoxing pickled vegetables and salted vegetables, respectively.

[0053] Table 1. Characterization of safety and tolerance performance of lactic acid bacteria

[0054]

[0055] Example 2: Molecular biological identification and growth capacity determination of lactic acid bacteria

[0056] (1) 16S rDNA sequence analysis: After the XC-1-3, YC-1-4 and MXC-4 strains stored at -80℃ were activated in MRS broth for two generations, DNA was extracted using the FastPure Bacteria DNA Isolation Mini Kit and sequenced by Hangzhou Youkang Biotechnology Co., Ltd.

[0057] The obtained rRNA sequences were compared with the National Center for Biotechnology Information (NCBI) database. 16S rDNA identification results showed that strains XC-1-3, YC-1-4, and MXC-4 had a 99% similarity to *Lactiplantibacillus plantarum* in the NCBI database, confirming that the three lactic acid bacteria belong to *Lactiplantibacillus plantarum*. Therefore, they were renamed *Lactiplantibacillus plantarum* XC-1-3, *Lactiplantibacillus plantarum* YC-1-4, and *Lactiplantibacillus plantarum* MXC-4, respectively. The 16S rDNA sequences of the screened *Lactiplantibacillus plantarum* XC-1-3, YC-1-4, and MXC-4 were amplified and sequenced. The electrophoretic bands of each *Lactiplantibacillus plantarum* are shown below. Figure 1 As shown.

[0058] (2) Growth curves and acid production capacity analysis of Lactobacillus plantarum XC-1-3, YC-1-4 and MXC-4

[0059] Growth curve plotting: Two generations of activated *Lactobacillus plantarum* XC-1-3, YC-1-4, and MXC-4 were centrifuged at 8000 r / min for 3 min at 4℃. The cells were collected, washed three times with sterile physiological saline, and the viable cell count was determined. Based on the viable cell count, the concentrations of *Lactobacillus plantarum* XC-1-3, YC-1-4, and MXC-4 were adjusted to 1×10⁻⁶ using sterile physiological saline. 8 CFU / mL, 1 mL was inoculated into 100 mL of fresh LMR syrup liquid medium and incubated at 37°C for 24 h. OD was measured every 2 h. 600 nm Value and pH value.

[0060] Lactobacillus plantarum XC-1-3, YC-1-4 and MXC-4 in OD 600 nm The number of live bacteria is as follows Figure 2 As shown, the acid-producing capacity is as follows Figure 3 As shown, *Lactobacillus plantarum* XC-1-3, YC-1-4, and MXC-4 enter the logarithmic growth phase at 4 hours and gradually reach the stationary phase at 10–12 hours. The pH of *Lactobacillus plantarum* XC-1-3, YC-1-4, and MXC-4 rapidly decreases from 0 to 8 hours, dropping below 4.5 after 8 hours of culture. All three *Lactobacillus plantarum* XC-1-3, YC-1-4, and MXC-4 exhibit good acid-producing capabilities.

[0061] (3) Test of antagonistic ability among Lactobacillus plantarum strains XC-1-3, YC-1-4 and MXC-4

[0062] Two generations of activated *Lactobacillus plantarum* XC-1-3, YC-1-4, and MXC-4 were centrifuged at 8000 r / min for 3 min at 4 °C, and the bacterial cells were collected. After washing three times with sterile physiological saline, the viable cell count was determined. Based on the viable cell count, the concentrations of *Lactobacillus plantarum* XC-1-3, YC-1-4, and MXC-4 were adjusted to 1 × 10⁻⁶ using sterile physiological saline. 8 CFU / mL.

[0063] Strands of *Lactobacillus plantarum* XC-1-3+YC-1-4, XC-1-3+MXC-4, and YC-1-4+MXC-4 were inoculated onto solid MRS medium along two perpendicular lines. The inoculated medium was then incubated at 37°C for 48 hours. Colony growth at the junction of the two streaks was observed: no colony growth at the junction indicated antagonism between the two strains (positive); colony growth at the junction indicated no antagonism between the two strains (negative).

[0064] The results are as follows Figure 4 As shown, no obvious inhibition zones were observed at the interface between strains XC-1-3 and YC-1-4, XC-1-3 and MXC-4, and YC-1-4 and MXC-4, indicating that no significant antagonistic effect was observed among the three strains. Under co-culture conditions, the growth of each strain did not inhibit the others, and they were able to coexist stably in the same environment, and may further exert their respective advantages through synergistic effects.

[0065] (4) Preparation of Lactobacillus plantarum XC-1-3, YC-1-4 and MXC-4 and mixed starter culture

[0066] Lactobacillus plantarum XC-1-3, YC-1-4, and MXC-4 were activated twice in MRS broth medium, and the cells were collected after centrifugation at 8000 r / min for 3 min at 4℃. The cells were washed three times with sterile physiological saline, and the viable cell count was determined. Based on the viable cell count, the concentration of Lactobacillus plantarum XC-1-3, YC-1-4, and MXC-4 was adjusted to 1×10⁻⁶ using sterile physiological saline. 8 CFU / mL, for later use. The adjusted bacterial strains were mixed and compounded at a volume ratio of 1:1 according to the following combinations: *Lactobacillus plantarum* XC-1-3+YC-1-4, XC-1-3+MXC-4, and YC-1-4+MXC-4. The total bacterial count of the compound groups was maintained at 1×10⁻⁶. 8 CFU / mL, for later use.

[0067] (5) Determination of biofilm production and AI-2 signal molecule production of Lactobacillus plantarum XC-1-3, YC-1-4 and MXC-4 and mixed fermentation agent

[0068] Biofilm formation capacity was determined using a 96-well plate crystal violet staining method. The specific procedure was as follows: A 100-fold diluted bacterial suspension was added at 200 μL / well to modified MRS medium in a 96-well plate. After static incubation at 37°C for 24 h to form a biofilm, the medium and airborne bacteria were carefully aspirated from the wells. The plates were then gently washed three times with 250 μL of sterile PBS to remove any unattached bacteria. Subsequently, 200 μL of 90% methanol was added for fixation for 30 min, the fixative was discarded, and the plates were allowed to air dry. 100 μL of 0.1% crystal violet was added to each well for staining for 15 min, followed by washing three times with PBS to remove free dye and drying. Finally, 200 μL of 33% glacial acetic acid was added to dissolve the stain at 37°C for 20 min, and the absorbance was measured at 595 nm.

[0069] like Figure 5 As shown, under single-strain culture conditions, the biofilm contents of XC-1-3, YC-1-4, and MXC-4 were 1.814, 1.571, and 1.811, respectively; under mixed-culture conditions, the biofilm contents of XC-1-3+YC-1-4, XC-1-3+MXC-4, and YC-1-4+MXC-4 were 2.363, 2.077, and 2.051, respectively. This indicates that the biofilm contents in the mixed-culture groups were all higher than those in the corresponding single-strain groups, suggesting that different strains can mutually promote biofilm formation under co-culture conditions, thereby enhancing the overall stability and environmental adaptability of the biofilm.

[0070] The activity of AI-2 quorum sensing signal molecules was detected using Vibrio harveyi BB170 strain. The experimental procedure was as follows: The activated bacterial suspension was inoculated into 100 mL of modified MRS medium at a 1% inoculum. After static incubation at 37°C for 24 hours, 10 mL of the culture was centrifuged at 6000 rpm (4°C, 5 min) to obtain the supernatant, which was then sterilized through a 0.22 μm filter membrane. Simultaneously, Vibrio harveyi BB170 was inoculated into AB medium at a 1% inoculum and cultured at 30°C with shaking at 120 rpm until the logarithmic growth phase (OD200). 600 nm (0.6-0.8). Transfer 20 μL of this culture to 100 mL of fresh AB medium and add 1 mL of *Lactobacillus plantarum* supernatant or biofilm sample, and continue culturing under the same conditions. Dynamically monitor fluorescence intensity (λex = 485 nm, λem = 538 nm) using a microplate reader. Measure every hour during the initial stage of culture (0-6 h), and every 3 hours after 6 hours. Finally, plot the fluorescence intensity versus time curve to analyze AI-2 activity.

[0071] The results are as follows Figure 6As shown, compared with single bacteria, strain combinations under co-culture conditions generally exhibited higher AI-2 signaling molecule content. In particular, some co-culture combinations showed significantly increased signaling molecule levels, indicating a synergistic effect among strains in quorum sensing. These results suggest that different strains, under co-culture conditions, can promote information transmission by enhancing AI-2 signaling molecule secretion, which contributes to synergistic effects among bacterial communities and biofilm formation.

[0072] Based on the combined results of antagonistic effect detection, biofilm formation ability, and AI-2 signaling molecule secretion level, it is evident that *Lactobacillus plantarum* XC-1-3 and YC-1-4 exhibit a strong synergistic effect under co-culture conditions. They can not only coexist stably but also significantly improve the signaling molecule level and biofilm formation. Therefore, the combination of *Lactobacillus plantarum* XC-1-3 and YC-1-4 was selected for subsequent experiments.

[0073] Example 3: Preparation of marinated chicken

[0074] The preparation steps for marinated chicken are as follows:

[0075] (S1) Take 10 parts of distiller's grains (purchased from Ningbo Laodizi Food Technology Co., Ltd.), add 30 parts of purified water, mix well, heat to a boil, and maintain boiling for 10 minutes to remove bacteria and off-flavors, to obtain distiller's grains extract. After cooling to room temperature, dispense into sterilized fermentation tanks for later use.

[0076] (S2) Grouping of lactic acid bacteria: XC-1 group (with fermentation agent Lactobacillus plantarum XC-1-3 added alone); YC-1 group (with fermentation agent Lactobacillus plantarum YC-1-4 added alone); MXC-4 group (with fermentation agent Lactobacillus plantarum MXC-4 added alone); MIX group (with compound fermentation agents Lactobacillus plantarum XC-1-3 and Lactobacillus plantarum YC-1-4 added); YWL group (with lees added alone); CK group (no fermentation agent added, no lees added). In the ultra-clean workbench, the activated Lactobacillus plantarum was added to the lees extract (obtained from step S1) at a ratio of 6% of the volume of the lees, and mixed thoroughly.

[0077] (S3) The inoculated lees extract was allowed to ferment at 30°C and 65% humidity for 24 hours to activate the metabolic activity of Lactobacillus plantarum and form a fermentation system.

[0078] (S4) Raw material pretreatment: After removing impurities, the raw chicken (purchased from Longhu Century Hualian Supermarket in Beilun District, Ningbo City, Zhejiang Province) is cleaned, and visible connective tissue, fat and other non-muscle tissues are removed. Then it is cut into pieces (3cm×3cm) according to the predetermined specifications and frozen at low temperature for later use.

[0079] (S5) Rinse the thawed chicken in running water to thoroughly remove blood, impurities and surface mucus, improving the sensory quality of the meat and the uniformity of subsequent marinating.

[0080] (S6) Add water to a pot, enough to cover the chicken, and add appropriate seasonings (sliced ​​ginger, scallion segments, cooking wine). After the water boils, simmer for 10 minutes to remove any unpleasant taste. After simmering, remove the chicken and let it cool naturally to room temperature.

[0081] (S7) Place the cooled chicken pieces into the fermented mash liquid (along with the tank) that has completed its first fermentation, ensuring that the chicken pieces are completely submerged in the fermentation system. Ferment at a low temperature of 15°C and 65% humidity for 24 hours.

[0082] (S8) After fermentation, take out the chicken pieces, divide them into 180g bags, add 20g of the corresponding fermentation liquid to each bag, and seal them with a vacuum packaging machine.

[0083] (S9) The sealed product is stored at 4°C. Sensory analysis, physicochemical index determination or flavor component detection can be carried out later according to the experimental purpose.

[0084] Example 4: Determination of Fermented Meat Indicators

[0085] (1) Determination of the color of fermented meat

[0086] The color difference of fermented meat was measured using a handheld colorimeter (SWG-2300, Sanenshi Technology Co., Ltd.). The results are shown in Table 2. At least three parallel tests were performed on each sample.

[0087] Table 2. Color of Braised Pork

[0088]

[0089]

[0090] As shown in Table 2, compared with the control group, the addition of fermented meat residue and lactic acid bacteria significantly reduced the brightness (L* value) of the fermented meat, while significantly increasing the redness (a* value) and yellowness (b* value). This result indicates that the combined effect of fermented meat residue and lactic acid bacteria can effectively improve the color characteristics of fermented meat, giving it a more vibrant color.

[0091] (2) Determination of the texture of fermented meat

[0092] Using a P50 probe (TA.XT Plus, SteadyMicro Systems, UK), with a deformation set to 50% and a measurement speed of 5 mm / s, two extrusion tests were performed. At least three replicates were tested for each sample group. The texture determination results are shown below. Figure 8The textural changes were compared between single-strain treatment groups (XC-1, YC-1, MXC-4), mixed-strain treatment group (MIX), group with only added residue, and untreated control group. Figure 7 and Figure 8 It is evident that the mixed-culture treatment group (MIX) significantly outperformed the group treated with only fermented liquid and the control group in terms of firmness and adhesiveness, with the mixed-culture group exhibiting the highest firmness among all groups. The XC-1 group made a significant contribution to chewiness, and the mixed-culture group combined the advantages of both. Therefore, it is clear that inoculating XC-1-3 and YC-1-4 together can mitigate the negative textural impact of fermented liquid treatment on fermented meat, thereby improving the product's textural quality.

[0093] (3) Determination of electronic nose for fermented meat

[0094] The testing method for the electronic nose of fermented meat is as follows: Transfer the samples to the measuring bottles and cool them to room temperature. Contact the electronic nose sensor with the air in the measuring bottle to generate corresponding sensor response values ​​(SS1-SS10). Set the electronic nose detection conditions as follows: carrier gas flow rate 0.2L / min, sensor cleaning time 100s, sample preparation time 5s, and sampling time 120s.

[0095] Figure 9 Principal component analysis (PCA) diagrams of fermented meat under different treatment methods obtained by electronic nose show significant differences in spatial distribution among the treatment groups: the YWL group and the CK group have high separation, indicating that the fermentation liquid treatment can significantly change the odor characteristics of meat products; the distribution of the three single-strain treatment groups is similar, indicating that their odor fingerprints are similar and their volatile substance metabolism has certain commonalities; the mixed-strain MIX group is independently distributed in the third quadrant, maintaining a large distance from the other groups, forming a unique flavor characteristic that is different from single-strain, fermentation liquid and control groups. This result may be due to the synergistic metabolic effect among mixed strains, which can endow fermented meat products with a unique odor fingerprint.

[0096] (4) Determination of thiobarbituric acid in fermented meat

[0097] Determination of thiobarbituric acid (TBA) value: Take 1.00 g of meat sample, add 4.00 mL of distilled water and homogenize, then add 4.00 mL of 10% trichloroacetic acid (TCA) solution and homogenize thoroughly. Filter and prepare the sample. Take 1.00 mL of the filtrate, add 0.25 mL of 0.06 mol·L⁻¹ TBA solution, mix well, and react in a metal bath at 80℃ for 90 min. After the reaction, cool to room temperature and centrifuge (or let stand). Take the supernatant and measure the absorbance at 532 nm. Calculate the MDA concentration C (mol·L⁻¹) according to the TEP (or MDA) standard curve, and calculate the TBARS value (mg·kg⁻¹) using the following formula. -1 )

[0098]

[0099] Where V is the final volume of the reaction system, M is the molar mass of MDA, and m is the sample mass (g). All samples were tested at least three times, and the results are expressed as mean ± standard deviation.

[0100] Depend on Figure 10 The test results show that the control group (CK) had the highest thiobarbituric acid value, significantly higher than other treatment groups. The TBARS values ​​of YWL in the single-bacterial inoculation group, mixed-bacterial MIX group, and lees treatment group were all significantly reduced, with little difference between them. Only the lees group (YWL) was slightly higher than the lactic acid bacteria treatment group.

[0101] The above results demonstrate that introducing lactic acid bacteria during fermentation can effectively inhibit lipid oxidation in meat products, significantly reduce TBARS values, and delay product oxidative deterioration. In contrast, the effect of adding fermented mash alone is limited, while the application of lactic acid bacteria can stably inhibit lipid oxidation and ensure the quality stability of fermented meat products.

[0102] (5) Determination of free amino acid content in fermented meat

[0103] The free amino acid content was determined using the Suzhou Grees Biotech Amino Acid (AA) Content Reagent Kit. Different treatment methods had a significant impact on the free amino acid content of meat products, as shown in the results below. Figure 11 As shown. By Figure 11 It is evident that the mixed-culture treatment group had the highest amino acid level, significantly better than the other groups; the single-culture treatment group was second, with little difference among the single cultures; the treatment group with only added lees had a further decrease in amino acid content, while the control group was at the lowest level.

[0104] The above indicates that mixed fermentation can enhance the release and accumulation of amino acids, which is speculated to be related to the synergistic effect formed between different strains in protein degradation and amino acid metabolism. However, when there is only lees or no fermentation, the protein degradation efficiency is insufficient, resulting in a low level of amino acid accumulation. In this case, amino acids not only directly contribute to umami and taste, but can also serve as flavor precursors to further participate in Maillard reactions and metabolic transformations. Therefore, the higher amino acid level in the mixed group may provide meat products with richer and more complex flavor characteristics.

[0105] (6) Determination of DPPH free radical scavenging ability of fermented meat

[0106] The DPPH method was used, following the instructions for the DPPH free radical scavenging ability test kit (micro-method) provided by Nanjing Jiancheng Bioengineering Research Institute. Results are as follows: Figure 12As shown, all experimental groups that added fermentation broth and residue exhibited significantly higher DPPH radical scavenging rates than the control group. Although no statistically significant differences in scavenging rates were observed between the experimental groups, it can be seen that the mixed group achieved the highest DPPH radical scavenging rate, possibly reflecting the synergistic effect between different lactic acid bacteria. Lactic acid bacteria can comprehensively and effectively delay the oxidative deterioration of meat products through their antioxidant effects, thereby extending shelf life and maintaining their quality.

[0107] (7) Determination of the flavor of fermented pork

[0108] The flavor profile of fermented pork was determined using gas phase ion mobility spectrometry (GC-MS) to analyze volatile compounds. Sample preparation method:

[0109] The sample was rapidly chopped, and 2.00 g was weighed into an SPME headspace extraction vial. Volatile compounds in the static headspace were extracted for 35 min at 70 °C using an SPME extraction head, and then separated and identified by GC-MS. Chromatographic conditions: Agilent HP5MS (Agilent Technologies), capillary column (30 m × 0.25 mm × 0.25 μm); carrier gas: He, flow rate: 1 mL / min. Injector temperature: 250 °C, splitless injection, resolution time: 5 min. Temperature program: initial temperature 40 °C, hold for 3 min, increase to 80 °C at 5 °C / min, hold for 1 min, increase to 120 °C at 5 °C / min, hold for 1 min, then increase to 230 °C at 6 °C / min, hold for 8 min.

[0110] Mass spectrometry conditions: Agilent 5975MSD mass spectrometer, electron ionization (EI) ion source; electron energy 70 eV; GC to MS interface temperature 280 °C; ion source temperature 230 °C; mass scan range 10–450 anm; electron detector detection voltage 350 V.

[0111] Qualitative and quantitative identification of volatile flavor components: The NIST 20 spectral library was searched, and the retention index and similarity greater than 80% were used as identification results, which were compared with the results reported in the literature for auxiliary identification. The quantification of volatile compounds was performed using the internal standard method, with a methanol-soluble solution of 2-methylheptanone as the internal standard.

[0112] The results are shown in Table 3. This invention detected a variety of flavor-related compounds in the products of different fermentation groups, including alcohols, aldehydes, esters, and ketones. Regarding alcohols, the control group had a higher alcohol content, but it was dominated by 1-octen-3-ol, which had a simple structure. In contrast, the fermentation groups, especially MXC-4 and MIX, detected a richer variety of alcohols, such as 1-hexanol, 2-propyl-1-pentanol, phenylethanol, and linalool, making the overall alcohol composition closer to the characteristic flavor compounds of meat. Regarding aldehydes, typical lipid oxidation products such as hexanal, octanal, nonanal, and heptanal were detected in all groups, but the total aldehyde content was significantly higher in the control group, while it was lower in the fermentation groups. The MIX group maintained a moderate level of aldehyde types and content, avoiding an overly strong grassy or oily odor caused by a high content of a single aldehyde. Regarding esters, various esters, such as vinyl hexanoate and ethyl phenylacetate, were detected in the fermentation products of the added strains. These substances are generally associated with fruity and floral aromas, contributing to improved flavor appeal. The XC-1 group, in particular, exhibited higher ester content, displaying a more pronounced ester aroma characteristic. As for ketones, 2,3-octanedione, 2-heptanone, and 2-decanone were commonly detected in the fermentation groups, especially in the MIX and YWL groups. These substances are associated with milky and caramel aromas, helping to enhance the complexity of meat flavors. Furthermore, among aromatic and heterocyclic aldehydes, benzaldehyde and furfural were detected in some fermentation groups, imparting nutty and caramel aromas and adding complexity to the overall flavor.

[0113] In summary, the control group was dominated by aldehydes and had a relatively simple flavor, while the single-strain fermentation group showed certain advantages in terms of alcohols or esters. The three-strain mixed fermentation (MIX group) had the most balanced composition and proportion of flavor substances such as alcohols, aldehydes, ketones and esters, showing the flavor characteristics closest to real meat.

[0114] Table 3 Flavor compounds of fermented pork belly

[0115]

[0116]

[0117]

Claims

1. A composite bacteria, characterized in that, The meat fermentation composition can be used for meat fermentation, and comprises at least one of the following three bacteria: Lactiplantibacillus plantarum XC-1-3, which is preserved in the China General Microbiological Culture Collection Center and has a preservation number of CGMCC No. 36530; Lactiplantibacillus plantarum YC-1-4, which is preserved in the China General Microbiological Culture Collection Center and has a preservation number of CGMCC No. 36529, Lactiplantibacillus plantarum MXC-4, which is preserved in the China General Microbiological Culture Collection Center and has a preservation number of CGMCC No. 36528.

2. The complex bacteria as claimed in claim 1, wherein, The meat fermentation composition comprises Lactiplantibacillus plantarum XC-1-3 and Lactiplantibacillus plantarum YC-1-4.

3. The complex bacteria as claimed in claim 2, wherein, The meat fermentation composition consists of Lactiplantibacillus plantarum XC-1-3 and Lactiplantibacillus plantarum YC-1-4, and the bacterial suspensions of the two are compounded at the same concentration and in the same volume.

4. A meat fermentation composition, characterized by, The meat fermentation composition comprises the compound bacteria according to any one of claims 1-3.

5. The meat fermentation composition of claim 4, wherein, The meat fermentation composition further comprises liquor.

6. A meat fermentation method, characterized by, The meat fermentation composition is used in the meat fermentation.

7. The meat fermentation method according to claim 6, characterized in that The meat fermentation composition comprises the following steps: S1: taking distiller's grains, adding pure water, mixing uniformly, heating and boiling to remove miscellaneous bacteria and odors, cooling to room temperature, and then being divided into sterile fermentation containers for standby; S2: adding the activated compound bacteria to the distiller's grains extract prepared in step S1 and mixing uniformly; S3: inoculating the distiller's grains extract to activate the metabolic activity of the compound bacteria and form a fermentation liquor; S4: after the raw meat is treated to remove impurities, the meat is cleaned, and visible connective tissue, fat and other non-muscle tissues are removed, and then the meat is cut into pieces according to the predetermined specification and stored in a low-temperature condition for standby; S5: the thawed meat is washed in flowing clean water to completely remove blood water, impurities and surface mucus, and improve the sensory quality of the meat pieces and the uniformity of subsequent marinating; S6: adding clean water to a pot, covering the meat with water, adding appropriate accessories, boiling the water, and then cooking on a small fire to remove odors, and then taking out the meat and naturally cooling to room temperature; S7: placing the cooled meat pieces into the fermentation liquor prepared in step S3 to ensure that the meat pieces are completely immersed in the fermentation liquor for low-temperature fermentation; S8: after the fermentation is completed, the meat pieces are taken out, quantitatively divided, and a small amount of the fermentation liquor prepared in step S3 is added, vacuum packaged, and stored in a low-temperature condition.

8. The meat fermentation method according to claim 7, characterized in that, The amount of the complex bacteria suspension added in the step S2 is 6% of the volume of the vinasse, and the concentration of the complex bacteria suspension is 1 x 10 8 CFU / mL.

9. The meat fermentation method according to claim 7, characterized in that, The preparation condition of the fermentation liquor in step S3 is that the fermentation is carried out at a temperature of 30℃ and a humidity of 65% for 24 hours. The low-temperature fermentation condition in step S7 is that the fermentation is carried out at a temperature of 15℃ and a humidity of 65% for 24 hours.

10. The meat fermentation method according to claim 7, characterized in that, The mass ratio of the added fermentation liquor to the meat pieces in step S8 is 1:6.

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