Fermentation inoculant for preparing dried milk cake with special flavor and storage resistance and application of fermentation inoculant

Abstract

The invention discloses a fermentation inoculant for preparing dried milk cakes with special flavor and storage resistance and application of the fermentation inoculant, belongs to the technical field of microorganisms, and provides the fermentation inoculant for preparing the dried milk cakes with special flavor and storage resistance, the fermentation inoculant comprises lactobacillus pentosus YN-04 and lactobacillus plantarum YN-05, the lactobacillus pentosus YN-04 and the lactobacillus plantarum YN-05 are preserved in the China Center for Type Culture Collection on March 13, 2025, and the preservation number is CGMCC NO. The preservation address is Wuhan University, Wuhan, China, and the preservation numbers are respectively CCTCC (China Center for Type Culture Collection) NO: M2025469 and CCTCC NO: M2025470. The zymophyte agent can synthesize enzymes capable of promoting special flavor and facilitating storage, the enzymes synthesized by the two bacteria are complementary in variety, and the zymophyte agent has excellent fermentation characteristics.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a fermentation agent for preparing milk tofu with special flavor and good storage resistance and application thereof. Background Art

[0002] Probiotics play a crucial role in maintaining human health. Their primary function, after ingestion, is to improve the structure of the intestinal flora, promote the proliferation of beneficial bacteria, inhibit the growth of harmful bacteria, and enhance the body's specific and nonspecific immunity, thereby helping to combat various diseases. In recent years, probiotics have demonstrated broad application prospects in a variety of fields, including food processing, healthcare, and animal husbandry. Probiotic preparations have broad application value in agriculture, food, and medicine. Traditional bacterial preparations generally suffer from low survival rates (<50% survival rate when stored at room temperature) and limited functionality.

[0003] Lactobacillus pentosus ( Lactiplantibacillus pentosus ) is a lactic acid bacterium with special metabolic characteristics. It can use a variety of carbon sources for fermentation and produce organic acids, antibacterial substances and other beneficial metabolites. Studies have shown that Lactobacillus pentosus not only has an inhibitory effect on harmful intestinal bacteria, but also promotes the absorption of nutrients. Therefore, it has important application value in functional foods and feed additives. Lactiplantibacillus plantarum Lactobacillus plantarum is a typical member of the Lactobacillus genus and is widely found in fermented foods. This strain possesses strong tolerance, allowing it to survive in the gastrointestinal tract and exert probiotic effects. Furthermore, Lactobacillus plantarum can secrete antioxidants and degrade anti-nutritional factors, thereby improving host health.

[0004] As resident beneficial microorganisms in the gastrointestinal tract, Lactobacillus pentosus and Lactobacillus plantarum play a key role in the stability and health of the intestinal microecological environment. Numerous clinical studies, animal experiments, and in vitro cell experiments have demonstrated that these two bacteria not only lower the intestinal pH by producing organic acids such as lactic acid and acetic acid, effectively inhibiting the growth of harmful bacteria such as Escherichia coli and Salmonella, and creating a suitable living environment for beneficial bacteria, but also stimulate the intestinal immune system, enhance the intestinal mucosal barrier function, and promote the repair and renewal of intestinal epithelial cells, thereby maintaining normal physiological metabolism and digestive and absorptive functions of the intestine. Furthermore, they have also shown positive effects in improving the symptoms of lactose intolerance and assisting in regulating blood lipid and blood sugar levels.

[0005] Despite significant progress in probiotic research, including a deeper understanding of the basic properties and some functions of Lactobacillus pentosus and Lactobacillus plantarum, research on specific strains remains limited. While Lactobacillus pentosus and Lactobacillus plantarum, as recognized safe microorganisms, are already used in fermented foods, their synergistic effects and mechanisms remain largely unexplored. Summary of the Invention

[0006] To solve the above technical problems, the present invention proposes a fermentation agent for preparing milk tofu with a special flavor and good storage resistance and its application. The fermentation agent includes Lactobacillus pentosus YN-04 and Lactobacillus plantarum YN-05, which can synthesize enzymes that promote special flavor and are beneficial for storage. The types of enzymes that can be synthesized by the two are complementary, have excellent fermentation characteristics, and show potential application value in the fermentation industry.

[0007] To achieve the above object, the present invention provides a fermentation agent for preparing milk tofu with special flavor and good storage resistance, wherein the fermentation agent comprises Lactobacillus pentosus ( Lactiplantibacillus pentosus )YN-04 and Lactobacillus plantarum ( Lactiplantibacillus plantarum )YN-05; The Lactobacillus pentosus YN-04 was deposited in the China Center for Type Culture Collection on March 13, 2025, at Wuhan University, Wuhan, China, with the deposit number: CCTCC NO: M2025469; The plant lactobacillus YN-05 was deposited in the China Center for Type Culture Collection on March 13, 2025, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being: CCTCC NO: M2025470.

[0008] Preferably, the mass ratio of Lactobacillus pentosus YN-04 to Lactobacillus plantarum YN-05 in the fermentation agent is 1:1; the effective viable cell count of Lactobacillus pentosus YN-04 in the fermentation agent is ≥10 9 CFU / mL, the effective viable count of Lactobacillus plantarum YN-05 in the fermentation agent is ≥10 8 CFU / mL.

[0009] The present invention also provides application of the fermentation bacteria agent in preparing fermentation products with special flavor.

[0010] Preferably, the fermented product is milk tofu.

[0011] The present invention also provides the use of the fermentation agent in preparing a fermentation product that is storable.

[0012] Preferably, the fermented product is milk tofu.

[0013] The present invention also provides a method for preparing milk tofu using the fermentation agent, comprising the following steps: sterilizing raw milk, inoculating the fermentation agent into the sterilized raw milk, fermenting until the pH value is 4.6, heating to drain whey, kneading into a mass, cutting, and compression molding to obtain milk tofu.

[0014] Preferably, the raw milk is sterilized by placing the raw milk in an 80° C. water bath, stirring for 15 minutes, and then cooling.

[0015] Preferably, the inoculation amount of the fermentation bacteria is calculated based on 3% of the volume fraction of the sterilized raw milk; and the fermentation temperature is 25°C.

[0016] Preferably, the whey is heated and discharged in a water bath at 65°C.

[0017] Compared with the prior art, the present invention has the following advantages and technical effects: The present invention provides a fermentation agent for preparing milk tofu with special flavor and good storage resistance, comprising Lactobacillus pentosus YN-04 and Lactobacillus plantarum YN-05. Lactobacillus pentosus YN-04 and Lactobacillus plantarum YN-05 have good gastrointestinal fluid tolerance and bile salt tolerance, and also have excellent fermentation characteristics; Lactobacillus pentosus YN-04 and Lactobacillus plantarum YN-05 have strong protein decomposition and amino acid synthesis capabilities, and after preparing milk tofu, proline, creatine, L-threonine, glycine, pyruvate, L-valine, L-isoleucine, and L-isoleucine are significantly formed. The two strains are complementary in terms of the types of synthetase: no acetate kinase, phosphate acetyltransferase and alcohol dehydrogenase were found in Lactobacillus plantarum YN-05, but it has abundant aminopeptidases and amino acid synthetases, which can produce a variety of amino acids. Lactobacillus pentosus YN-04 has the above-mentioned related enzymes, and Lactobacillus plantarum YN-05 contains L-lactate dehydrogenase and malate / lactate dehydrogenase. The complementarity of the types of synthetase between the two can enhance the special flavor of the prepared milk tofu and facilitate its storage. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 This is the phylogenetic tree diagram of Lactobacillus pentosus YN-04; Figure 2 is the cholesterol standard curve; Figure 3 The following are the base composition distribution diagrams of second-generation sequencing reads and the length distribution diagrams of third-generation sequencing reads, among which A is the base composition distribution diagram of second-generation sequencing reads of Lactobacillus plantarum YN-05, B is the base composition distribution diagram of second-generation sequencing reads of Lactobacillus pentosus YN-04, C is the length distribution diagram of third-generation sequencing reads of Lactobacillus plantarum YN-05, and D is the length distribution diagram of third-generation sequencing reads of Lactobacillus pentosus YN-04; Figure 4 is the gene length distribution diagram, where A is Lactobacillus plantarum YN-04 and B is Lactobacillus pentosus YN-05; Figure 5 This is a COG functional annotation map, where A represents Lactobacillus plantarum YN-05 and B represents Lactobacillus pentosus YN-04; Figure 6 GO functional annotation diagram, where A is Lactobacillus plantarum YN-05 and B is Lactobacillus pentosus YN-04; Figure 7 This is the KEGG functional annotation map of Lactobacillus plantarum YN-05; Figure 8 : The radar chart of the response values ​​of the electronic nose and electronic tongue for fermented milk tofu, wherein a is the radar chart of the response value of the electronic nose, b is the radar chart of the response value of the electronic tongue, A in the figure represents milk tofu fermented by Lactobacillus pentosus YN-04, B in the figure represents milk tofu fermented by Lactobacillus plantarum YN-05, C in the figure represents milk tofu fermented by a fermentation agent composed of Lactobacillus plantarum YN-04 and Lactobacillus plantarum YN-05 of the present invention, and D in the figure represents commercially available milk tofu; Figure 9 This is a PCA analysis of fermented milk tofu metabolites, wherein a is a comparison of A and D, b is a comparison of B and D, and c is a comparison of C and D. In the figure, A represents milk tofu fermented by Lactobacillus pentosus YN-04, B represents milk tofu fermented by Lactobacillus plantarum YN-05, C represents milk tofu fermented by a fermentation agent composed of Lactobacillus plantarum YN-04 and Lactobacillus plantarum YN-05 of the present invention, and D represents commercially available milk tofu; Figure 10OPLS-DA analysis of fermented milk tofu metabolites, wherein a is the OPLS-DA score graph of A vs D, b is the model scatter plot of A vs D, c is the OPLS-DA score graph of B vs D, d is the model scatter plot of B vs D, e is the OPLS-DA score graph of C vs D, and f is the model scatter plot of C vs D. In the figure, A represents milk tofu fermented by Lactobacillus pentosus YN-04, B represents milk tofu fermented by Lactobacillus plantarum YN-05, C represents milk tofu fermented by a fermentation agent composed of Lactobacillus plantarum YN-04 and Lactobacillus plantarum YN-05 of the present invention, and D represents commercially available milk tofu; Figure 11 Volcano plots of differential metabolites in fermented milk tofu, where A compares milk tofu fermented by strain YN-04 with commercial milk tofu, B compares milk tofu fermented by strain YN-05 with commercial milk tofu; C compares milk tofu fermented by a fermentation agent composed of Lactobacillus pentosus YN-04 and Lactobacillus plantarum YN-05 of the present invention with a commercial milk tofu group; Figure 12 KEGG bubble diagram of differential metabolites of fermented milk tofu, wherein A is a comparison between milk tofu fermented by strain YN-04 and commercial milk tofu, B is a comparison between milk tofu fermented by strain YN-05 and commercial milk tofu; C is a comparison between milk tofu fermented by the fermentation agent composed of Lactobacillus pentosus YN-04 and Lactobacillus plantarum YN-05 of the present invention and a commercial milk tofu group; Figure 13 This is the phylogenetic tree diagram of Lactobacillus plantarum YN-05; Figure 14 It is a gene annotation statistical diagram, where A is Lactobacillus plantarum YN-05 and B is Lactobacillus pentosus YN-04; Figure 15 This is the circle map of the genome of Lactobacillus plantarum YN-05; Figure 16 These are morphological images of Lactobacillus pentosus YN-04 and Lactobacillus plantarum YN-05, wherein A is Lactobacillus pentosus YN-04, B is Lactobacillus plantarum YN-05, and the scale bar is 10 μm; Figure 17 This is the KEGG functional annotation map of Lactobacillus pentosus YN-04; Figure 18 This is the circle map of the genome of Lactobacillus pentosus YN-04; Figure 19 The pie chart shows the classification and proportion of metabolites in milk tofu samples.

[0020] Certificate of Deposit Lactobacillus pentosus YN-04, Latin name Lactiplantibacillus pentosusYN-04, this strain is deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, China, the deposit date is March 13, 2025, and the deposit number is: CCTCC NO: M 2025469.

[0021] Lactobacillus plantarum YN-05, Latin for Lactiplantibacillus plantarum YN-05, this strain is deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, China, the deposit date is March 13, 2025, and the deposit number is: CCTCC NO: M 2025470. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] The sources of materials used in the present invention are: raw milk from cows under grazing conditions comes from herders in Abaga Banner, Bai Banner and Zhenglan Banner of Xilin Gol League, Inner Mongolia; raw milk from cows under stall feeding conditions comes from pastures in Abaga Banner, Bai Banner and Zhenglan Banner of Xilin Gol League, Inner Mongolia; buffer S, buffer GA, buffer GB, buffer DV, buffer BV, buffer W1 and buffer W2 containing RNaseA are all from Illumina Company in the United States.

[0028] The MRS broth medium (solid) used in the present invention has the following formula: 10 g of peptone (animal source), 8 g of beef extract powder, 4 g of yeast extract powder, 20 g of glucose, 1 mL of Tween-80, 2 g of dipotassium hydrogen phosphate, 5 g of anhydrous sodium acetate, 2 g of triammonium citrate, 0.05 g of manganese sulfate tetrahydrate, 0.2 g of magnesium sulfate heptahydrate, 15 g of agar powder, and distilled water to a volume of 1 L. The initial pH is adjusted to 6.20, and the mixture is sterilized at 121° C. for 15 min.

[0029] The formula of the MRS broth medium (liquid) used in the present invention is: 10 g of peptone (animal source), 8 g of beef extract powder, 4 g of yeast extract powder, 20 g of glucose, 1 mL of Tween-80, 2 g of dipotassium hydrogen phosphate, 5 g of anhydrous sodium acetate, 2 g of triammonium citrate, 0.05 g of manganese sulfate tetrahydrate, 0.2 g of magnesium sulfate heptahydrate, and distilled water to 1 L. The initial pH is adjusted to 6.20 and sterilized at 121° C. for 15 min.

[0030] The present invention does not particularly limit the sources of peptone (animal source), beef extract powder, yeast extract powder, glucose, Tween-80, dipotassium hydrogen phosphate, anhydrous sodium acetate, triammonium citrate, manganese sulfate tetrahydrate, magnesium sulfate heptahydrate, and agar powder, and conventional commercially available products well known to those skilled in the art can be used.

[0031] The skim milk culture medium used in the present invention is as follows: 100.0 g / L skim milk powder is dissolved by heating, divided into portions, and sterilized by high pressure at 121° C. for 15 min.

[0032] The cholesterol (TC) culture medium used in the present invention comprises the following steps: accurately weigh 0.1 g of cholesterol in a small beaker, add 1 mL of Tween-80 and 0.1 g of sucrose ester, stir evenly, add 5.0 mL of glacial acetic acid, heat and stir until fully dissolved, ultrasonicate for 15 minutes, add to MRS culture medium, stir while adding, adjust the cholesterol concentration to 0.1 mg / mL, then add 0.2% sodium thioglycolate, adjust the pH to 6.0, and sterilize at 121°C for 15 minutes.

[0033] Example 1 1. Isolation and screening of Lactobacillus pentosus YN-04 and Lactobacillus plantarum YN-05.

[0034] 10g of fresh milk sample was collected from each of the PXN and HXN groups (P = grazing; H = confinement; XN = raw milk) under different feeding conditions and added to 90mL of normal saline. The mixture was shaken for 30 minutes. The samples were serially diluted and plated on a plate to isolate lactic acid bacteria. After incubation at 37°C for 48 hours, colony morphology was observed. Single colonies with distinct characteristics were picked and streaked three times onto MRS broth (solid) plates for purification. Gram staining and catalase assays were performed. The 109 selected and purified lactic acid bacteria strains were stored in a 30% glycerol solution and stored at -80°C until further use.

[0035] 2. Extraction of strain DNA.

[0036] First, prepare 1.0×10 9 CFU / mL of bacterial culture is then centrifuged to remove the supernatant. The precipitate is then suspended in buffer S containing RNase A. Lysozyme stock solution is then added, mixed thoroughly, and allowed to stand for 5 minutes. EDTA is then added, mixed, and then placed in an ice bath for 5 minutes. Buffer GA is then added, vortexed, and incubated in a 65°C water bath for 10 minutes. Buffer GB and pre-chilled buffer DV are then added, mixed, and centrifuged. The upper phase is discarded, and the precipitate and lower phase are retained. These are then washed with buffer DV and the upper phase is discarded. The filtrate is then centrifuged using a filter, and buffer BV is added, mixed thoroughly, and centrifuged. Finally, the precipitate is washed with buffers W1 and W2, followed by deionized water to elute the DNA. The extracted DNA is then centrifuged.

[0037] 3. PCR amplification of the strain genome.

[0038] Table 1 PCR amplification reaction system ;

[0039] For the PCR amplification reaction system, add the components listed in Table 1 to a 0.2 mL centrifuge tube. Gently flick the tube to mix thoroughly, centrifuge briefly to collect droplets from the tube wall and transfer to the bottom. Perform the PCR reaction in a PCR amplifier using the following parameters: initial denaturation (95°C, 5 min), denaturation (95°C, 30 s), annealing (58°C, 30 s), extension (72°C, 90 s), and final extension (72°C, 7 min), for 35 cycles.

[0040] After the reaction, 3 μL of PCR product was subjected to 1% agarose gel electrophoresis to confirm the PCR amplification fragment. PCR products were then recovered using AxyPrep DNA gel electrophoresis. Purified PCR products from each bacterial species were sequenced on an ABI 3730-XL sequencer. Sequences of model strains with high homology were downloaded and phylogenetic relationships were constructed using the neighbor-joining (NJ) method using MEGA 7.0 software. Lactic acid strains were then counted and identified.

[0041] like Figure 1 As shown, this is the phylogenetic tree diagram of Lactobacillus pentosus YN-04, as Figure 13 Shown is the phylogenetic tree of Lactobacillus plantarum YN-05.

[0042] The nucleotide sequences of 16srRNA of Lactobacillus pentosus YN-04 and Lactobacillus plantarum YN-05 are shown in SEQ ID NO. 1 and SEQ ID NO. 2.

[0043] 4. Screening of strains.

[0044] The isolated and purified lactic acid bacteria strains were cultured in MRS broth (liquid) for preliminary screening of target strains. The main goal of the screening was to find strains with rapid acid production, good coagulation, and a rich flavor.

[0045] (1) Determination of the coagulation characteristics and acid production ability of the strain: A. Inoculate the isolated and purified strain into a 100 g / L skim milk culture medium (sterilized) at a 3% (v / v) inoculation ratio. Then culture at 25°C for 48 hours and observe the time required for coagulation and its state.

[0046] B. Using the national food safety standard GB5009.239-2016, the acidity and pH of the curd during the curdling process were measured, and the acid production rate was calculated using the following formula: △T=°T / t; Among them, △T represents the average acid production rate, °T represents the fermentation acidity, and t represents the coagulation time.

[0047] (2) Determination of the heat coagulation property of the strain: The purified strain was inoculated at a 3% (v / v) inoculum into a sterile skim milk culture medium containing 100 g / L of culture medium and cultured at 25°C for 48 hours. After culture, the curd samples were heated in a 65°C water bath for 5 minutes, until the temperature reached approximately 85°C. The curdling properties and flavor profile developed during heating were then observed and recorded. This approach allowed the removal of strains with poor curdling characteristics and unsatisfactory flavor.

[0048] (3) Determination of the growth ability of the strain in skim milk: The target strain obtained after the initial screening was added to 100 g / L sterilized skim milk according to the addition amount of A, and fermented at a constant temperature of 25°C. Samples were taken at 0, 6, 12, 16, 18, 20, and 24 hours, and the acidity and pH values ​​were measured according to step B.

[0049] 5. Colony morphological characteristics.

[0050] The Lactobacillus pentosus YN-04 and Lactobacillus plantarum YN-05 of the present invention form milky white opaque colonies with a diameter of 0.5 to 1 mm, a round and flat or micro-papillary shape, and a flat edge on an MRS broth medium (solid).

[0051] In a clean bench, use an inoculation loop to pick up the purified bacterial sludge of Lactobacillus pentosus YN-04 and Lactobacillus plantarum YN-05 for smearing. After drying, perform Gram staining. Observe and record the morphology and staining characteristics of the strains under a microscope. Figure 16 China A and Figure 16 As shown in B, the strain is a Gram-positive bacterium observed under a microscope. The bacteria are rod-shaped and mostly appear alone, in pairs, in chains, or in clusters. The colony diameter and colony edges on MRS broth medium (solid) are neat, creamy white, and opaque.

[0052] The Lactobacillus pentosus YN-04 and Lactobacillus plantarum YN-05 of the present invention have the following biological characteristics: the bacteria are rod-shaped under a microscope, and the arrangement state is also single, paired, chain or clustered, but not forming a clear chain structure, without flagella or cilia, and will not move.

[0053] Example 2 Probiotic properties of Lactobacillus pentosus YN-04 and Lactobacillus plantarum YN-05.

[0054] 1. Determination of acid and bile salt resistance: After the strain was activated for two generations, the strain was inoculated at a 3% (v / v) addition rate into MRS broth medium with a pH of 2.5 and an MRS broth medium with a 3% ox bile salt concentration. The culture was constant temperature at 37°C, and the viable bacteria were counted at 0 and 3 hours to determine the survival rate of the strain. The formula for calculating the survival rate of the strain is: Survival rate (%) = N1 / N0 × 100%; Wherein, N1 represents the number of viable bacteria measured at 3 h, CFU / mL; N0 represents the number of viable bacteria measured at 0 h, CFU / mL.

[0055] The acid and bile salt resistance results of Lactobacillus pentosus YN-04 and Lactobacillus plantarum YN-05 are shown in Table 2 below.

[0056] Table 2 Acid and bile salt resistance of Lactobacillus pentosus YN-04 and Lactobacillus plantarum YN-05 ;

[0057] 2. Determination of the antioxidant capacity of the strain: The MRS activated bacterial solution was centrifuged at 5500 r / min for 15 min, washed with PBS and resuspended twice to prepare bacterial suspension (OD 600 value is 1.0) for backup.

[0058] (1) DPPH free radical scavenging ability: A 0.4 mmol / L DPPH solution was prepared using anhydrous ethanol. 2 mL of DPPH solution was mixed with the bacterial suspension and allowed to react at room temperature in the dark for 30 minutes. Subsequently, the mixture was centrifuged at 6000 rpm for 10 minutes. The supernatant was obtained after centrifugation and its absorbance was measured at 517 nm in triplicate. Finally, the DPPH free radical scavenging rate was calculated using the following formula: DPPH free radical scavenging rate (%) = [1-(A b -A c ) / A a ]×100%; Among them, A b is the OD value after the bacterial suspension is mixed with the DPPH solution; A c is the OD value after the bacterial suspension is mixed with anhydrous ethanol; A a It is the OD value after mixing pure water and DPPH solution.

[0059] (2) OH scavenging ability: Prepare a 9mmol / L FeSO4 solution, a 9mmol / L salicylic acid-ethanol solution, and an 8.8mmol / L H2O2 solution. Add 1mL of each of the three solutions to 1mL of the bacterial suspension, let it react at room temperature for 20 minutes, and measure the absorbance at 510nm. Measure the absorbance in triplicate at each set of three. Calculate the hydroxyl radical scavenging rate using the following formula: Hydroxyl radical scavenging rate (%) = [1-(B b -B a ) / B c ]×100%; Among them, B b is the OD value of bacterial suspension and mixed solution; B a is the OD value of pure water instead of H2O2 solution; B c is the OD value of pure water and mixed solution.

[0060] (3) Cholesterol capacity determination: The cholesterol-degrading ability of the target strain was determined using a phosphorus-sulfur-iron colorimetric method. The phosphorus-sulfur-iron reagent was prepared as follows: 2.50 g of FeCl₃·6H₂O was accurately weighed and dissolved in 100 mL of phosphoric acid to produce an FeCl₃ solution. Next, 88 mL of this solution was added with concentrated sulfuric acid and the volume was brought to 100 mL to prepare the phosphorus-sulfur-iron colorimetric reagent.

[0061] Cholesterol standard curve drawing: establish a cholesterol standard curve (such as Figure 2 As shown), the regression equation is y=0.4629x+0.0141, R 2 =0.9948.

[0062] A 0.1 mg / mL cholesterol culture medium was prepared, and the cholesterol degradation rate of lactic acid bacteria was determined using the phosphorus-sulfur-iron colorimetric method. Activated strains VN-04 and YN-05 were inoculated into the cholesterol culture medium at a concentration of 3%. A blank control group consisted of uninoculated culture medium. After incubation at 37°C for 24 hours, the culture medium was mixed with anhydrous ethanol and centrifuged. The supernatant was added with a phosphorus-sulfur-iron colorimetric reagent. After cooling to room temperature, the absorbance was measured at 550 nm. Three replicates were used for each group.

[0063] The cholesterol degradation rate is calculated as follows: Cholesterol degradation rate (%) = (CA) / C × 100%; Wherein, C represents the cholesterol concentration of the blank control group (mg / mL); A represents the cholesterol concentration in the sample (mg / mL).

[0064] The antioxidant capacity of Lactobacillus pentosus YN-04 and Lactobacillus plantarum YN-05 includes DPPH free radical scavenging rate, hydroxyl free radical scavenging capacity and cholesterol degradation rate, as shown in Table 3 below.

[0065] Table 3 DPPH free radical scavenging rate, hydroxyl free radical scavenging ability and cholesterol degradation rate of the strains ;

[0066] 3. Determination of antibacterial ability: The double-layer agar diffusion method was used to select Escherichia coli, Staphylococcus aureus, and Salmonella typhimurium as control bacteria. The Oxford cup method was then used to measure the antibacterial activity of the two target strains. First, a sterilized culture dish was poured into 10 mL of nutrient agar. After cooling slightly, three Oxford cups were placed. Then, 1 mL of indicator bacteria solution (concentration of 10 7 After mixing the sample (100 mL of nutrient agar) with 10 mL of nutrient agar and allowing it to solidify, remove the Oxford cup and add 0.2 mL of lactic acid bacteria fermentation broth to the well. Finally, incubate at 37°C for 24 hours, and measure the diameter of the inhibition zone.

[0067] The antibacterial abilities of Lactobacillus pentosus YN-04 and Lactobacillus plantarum YN-05 against Escherichia coli, Staphylococcus aureus and Salmonella typhimurium are shown in Table 4 below.

[0068] Table 4 Antibacterial ability of the strains against Escherichia coli, Staphylococcus aureus and Salmonella typhimurium ;

[0069] Example 3 1. Whole genome sequencing of the strain.

[0070] Purification and detection of strain DNA: The sequencing strain was cultured at 37°C for 24 h, 1 mL of bacteria was collected, stored on dry ice, and sent to Meiji Biotechnology for whole genome sequencing.

[0071] Illumina library construction and Denovo sequencing of strains: Whole-genome sequencing was performed using the Illumina HiSeq and PacBio sequencing platforms. The extracted DNA was first fragmented to generate a DNA fragment of approximately 10 kb. Sequencing data reads were finally obtained through a series of steps, including end-filling, 3' A-tailing, ligation of SMRT sequencing adapters at both ends, library preparation, paired-end sequencing, and data analysis.

[0072] Data quality control and sequence assembly: The raw sequencing data was sheared to obtain high-quality data. Low-quality reads, reads with a high N-content, and reads that were too short were removed. Unicycler software and the PacBio database were used for data assembly. Finally, the assembly results were optimized and corrected using Illumina data to obtain the final data.

[0073] Gene prediction and annotation: Coding genes were predicted using Glimmer software, plasmid genes were predicted using GeneMarkS (4.3), tRNAs, rRNAs, and sRNAs were predicted and annotated using tRNAscan-SE (2.0.12), Infernal (1.1.4), and the Rfam database, respectively. Interspersed repeats and tandem repeats were predicted using Repeatmasker and Tandem Repeats Finder, respectively. Genomic islands, prophages, and CRISPR-Cas sequences were predicted using IslandViewer (1.2), Phage Finder, and Minced (0.2.0).

[0074] Protein function annotation of CDSs was performed using tools such as BLAST, Diamond, and HMMER in conjunction with databases such as NR, GO, COG, and KEGG. Secondary metabolite gene clusters were identified and analyzed using antiSMASH. Furthermore, Circos was used to create genomic circular maps of YN-04 and YN-05, providing bioinformatics support for subsequent research.

[0075] 2. Basic characteristics of the whole genome of Lactobacillus pentosus YN-04 and Lactobacillus plantarum YN-05.

[0076] After DNA extraction and purification, sequencing libraries were constructed for Lactobacillus pentosus YN-04 and Lactobacillus plantarum YN-05. Subsequently, their complete genomes were sequenced using a combination of second-generation Illumina sequencing technology and third-generation PacBio sequencing technology.

[0077] After filtering, the raw data from the third-generation sequencing of Lactobacillus plantarum YN-05 and Lactobacillus pentosus YN-04 were 222.39 Mb and 109.79 Mb, respectively, with a total of 26,038 and 13,332 reads. The longest read lengths were 38,504 bp and 19,495 bp, respectively, with average read lengths of 8,540.99 bp and 8,234.85 bp, respectively. The Q30 values ​​were 97.28% and 95.62%, respectively, and the Q20 values ​​were 99.19% and 98.64%, respectively. According to the Illumina requirements of Q20 > 80% and Q30 > 70% (Q20: the percentage of bases with a base call accuracy greater than 99%; Q30: the percentage of bases with a base call accuracy greater than 99.9%), confirming the accuracy of the raw data from the sequencing of Lactobacillus plantarum YN-04 and Lactobacillus plantarum YN-05.

[0078] Illumina sequencing technology generates massive amounts of read data. This data is so massive that it's impossible to fully characterize the quality of each read. Therefore, statistical analysis is necessary to comprehensively assess the quality of both library construction and sequencing from a macroscopic perspective, including base composition distribution maps and base quality distribution statistics.

[0079] like Figure 3 China A and Figure 3Figure B shows the base composition distribution of quality control pre-sequence reads from next-generation sequencing of two strains, Lactobacillus plantarum YN-05 and Lactobacillus pentosus YN-04. The horizontal axis represents the base position of the reads, arranged from the 5' to the 3' end, while the vertical axis reflects the percentage of A, C, G, T, and N bases at each sequencing position. Different bases are represented by different colors. At the beginning of the sequence, the ratio of A, C, G, and T bases fluctuates due to the influence of the sequencing primer adapter; however, the base ratio tends to stabilize as the sequence continues. The figure shows that the low proportion of N bases indicates that there are fewer unknown bases in the sample, indicating high sequencing data quality and minimal impact of systematic AT bias. These results indicate that the sequencing data after quality control has good accuracy, stable base composition, and effective control of bias during the sequencing process.

[0080] like Figure 3 Middle C and Figure 3 Figure D shows the length distribution of clean reads from third-generation sequencing of Lactobacillus plantarum YN-05 and Lactobacillus pentosus YN-04, respectively. The horizontal axis represents the length of sequencing reads, and the vertical axis represents the number of reads of varying lengths. The black curve reflects the change in read count as read length increases; the green curve shows the change in the total accumulated base count as read length increases, although the green area is not meaningful. As can be seen from the figure, the third-generation sequencing data for strains YN-04 and YN-05 have long reads and no ambiguous bases. Therefore, by splicing and assembling the third-generation sequencing data and combining it with the second-generation data, the complete genome sequence was ultimately obtained.

[0081] In summary, the genome sequencing and assembly results of Lactobacillus pentosus YN-04 and Lactobacillus plantarum YN-05 were ideal, and the sequencing depth met the expected requirements. Further bioinformatics analysis can be carried out next.

[0082] The basic information of the genomes of the two strains of Lactobacillus pentosus YN-04 and Lactobacillus plantarum YN-05 is shown in Table 5 below.

[0083] Table 5 Basic genomic characteristics of Lactobacillus pentosus YN-04 and Lactobacillus plantarum YN-05 ;

[0084] As shown in Table 5, a total of 2558 genes were identified in the Lactobacillus plantarum YN-05 strain, of which 67 tRNA sequences and 19 rRNA sequences were successfully predicted. A total of 3156 genes were identified in the Lactobacillus pentosus YN-04 strain, including 68 tRNA sequences and 16 rRNA sequences. Figure 4The YN-04 gene length distribution diagram shown in A and Figure 4 The gene length distribution of YN-05 (B) shows that the majority of genes are between 200 and 1000 bp in length, but a significant proportion of long genes (greater than 1000 bp) also exist. In strain YN-04, 984 genes are longer than 1000 bp, accounting for 31.18% of all coding genes. In strain YN-05, 737 genes are longer than 1000 bp, accounting for 31.18% to 28.81% of all coding genes. These results indicate that while most genes are of moderate length, some relatively long genes exist in both strains, demonstrating the diversity and complexity of their genomic structures.

[0085] 3. tRNA gene prediction.

[0086] Lactobacillus pentosus YN-04 and Lactobacillus plantarum YN-05 contain 68 and 67 tRNAs, respectively. Table 6 below is a statistical table of tRNA prediction results.

[0087] Table 6 Statistics of tRNA prediction results ;

[0088] Tandem Repeats Finder software was used to predict tandem repeat sequences in the two strains. The results showed that the genomes of YN-05 and YN-04 contained 36 and 71 repeat sequences, totaling 9,420 and 24,404 bp, respectively, accounting for 0.44% and 0.88% of the genome, respectively. These data provide key clues for analyzing the genomic structure and DNA characteristics of the two strains and are of great significance for understanding gene evolution, expression regulation, and disease resistance.

[0089] Interspersed repetitive sequences (transposable elements) include DNA transposons and retrotransposons (such as LTRs, LINEs, and SINEs). Using Repeatmasker software, the genomes of the two strains were predicted to contain four DNA transposons in YN-05 and two in YN-04, respectively, and three retrotransposons: SINEs (11 and 10), LINEs (11 and 9), and LTRs (0 and 2).

[0090] Example 4 1. COG functional annotation.

[0091] like Figure 5 China A and Figure 5As shown in Figure B, strains YN-05 and YN-04, respectively, have 2129 and 2748 fully annotated genes. Functionally, the largest number of genes belongs to metabolism (including classes C, E, F, G, H, I, P, and Q), with 855 for YN-05 and 1094 for YN-04. This is followed by cellular process and signaling-related genes (506 and 695, respectively), followed by information storage and processing genes (including classes D, M, N, O, T, U, V, and W), with 487 and 616, respectively. Finally, genes of unclear function (class S) are present, with 281 and 343, respectively.

[0092] The functions with the largest number of annotated functions in strain YN-05 are: translation, ribosome structure and biogenesis (J, 202), amino acid transport and metabolism (E, 183), carbohydrate transport and metabolism (G, 178), transcription (K, 171), general function prediction (R, 166), and cell wall / cell membrane / extracellular membrane biosynthesis (M, 143); the functions with the largest number of annotated functions in strain YN-04 are: transcription (K, 262), carbohydrate transport and metabolism (G, 244), amino acid transport and metabolism (E, 255), translation, ribosome structure and biogenesis (J, 222), general function prediction (R, 219), and cell wall / cell membrane / extracellular membrane biosynthesis (M, 165).

[0093] 2. GO functional annotation.

[0094] Comparison of the GO annotation database revealed that strains YN-05 and YN-04 had 926 and 1147 genes associated with cell composition (CC), 1490 and 1908 genes associated with molecular function (MF), and 952 and 1164 genes associated with biological process (BP), respectively. In these three GO categories, strains YN-05 and YN-04 had 1811 and 2314 genes annotated, respectively, representing 70.8% and 73.32% of their coding genes.

[0095] like Figure 6 Medium A (YN-05) and Figure 6As shown in Figure B (YN-04), a comparison of the GO database reveals that strain YN-05 has the most common cellular component annotations for cell membrane, cytoplasm, plasma membrane, and ribosome. These genes may have transmembrane transport capabilities. Molecular function annotations, on the other hand, are more common for ATP binding, DNA binding, and hydrolase activity. Biological process annotations, on the other hand, are more common for phosphorylation and translation. In strain YN-04, cellular component annotations are more common for membrane components and cytoplasm. Molecular function annotations, on the other hand, are more common for attachment activity, reflecting the crucial role of biomolecular interactions in biological reactions, with attachment being a prominent example. Specifically, the ATP binding mechanism is a typical example. ATP-binding genes specifically attach to ATP molecules, acting on their phosphate groups and catalyzing ATP hydrolysis, thereby releasing energy for other biological processes. This process is not only a core component of energy conversion but also fundamental to the smooth functioning of numerous metabolic activities within the cell. Phosphorylation and proteolysis are more common in biological processes.

[0096] 3. KEGG functional annotation.

[0097] The KEGG database provides functional annotations for the whole genomes of Lactobacillus pentosus YN-04 and Lactobacillus plantarum YN-05. KEGG is a professional database that compares and analyzes bacterial genes and metabolic pathways at the molecular level. It can explore the molecular level of bacteria and study the relationship between functional genes and biological molecules in various biological pathways. The KEGG database divides the biological pathways of microorganisms into six categories, including metabolism, genetic information processing, environmental information processing, cellular processes, organism systems, and human diseases. Figure 7 (YN-05) and Figure 17 As shown in Figure 5 (YN-04), 1980 and 2281 functional genes were annotated for Lactobacillus pentosus YN-04 and Lactobacillus plantarum YN-05, respectively, through KEGG, which were mainly concentrated in metabolism, especially carbohydrate (PXN 179; HXN 196), amino acid and nucleotide metabolism (PXN 147; HXN 135), indicating that these two strains have strong metabolic capabilities.

[0098] 4. CAZy functional annotation.

[0099] The Carbohydrate Active Enzyme (CAZy) database, used to annotate carbohydrate-related enzymes, is divided into six major categories: glycoside hydrolases, glycosyltransferases, polysaccharide lyases, carbohydrate esterases, carbohydrate-binding modules, and auxiliary oxidoreductases. According to the CAZy database, 73 and 96 carbohydrate-active enzyme genes were annotated for Lactobacillus pentosus YN-04 and Lactobacillus plantarum YN-05, respectively, belonging to four (YN-05) and five (YN-04) protein families. YN-05 and YN-04 contain two and 11 auxiliary oxidoreductase genes, 13 and 18 carbohydrate esterase genes, 37 and 39 glycoside hydrolase genes, 21 and 27 glycosyltransferase genes, respectively. YN-04 also contains one carbohydrate-binding module gene.

[0100] And by Figure 14 China A and Figure 14 It can be seen from Figure B that in Lactobacillus plantarum YN-05 and Lactobacillus pentosus YN-04, auxiliary oxidoreductases accounted for 2.74% and 11.76% of the total carbohydrate active enzyme genes, carbohydrate esterase genes accounted for 17.81% and 18.75% respectively, glycoside hydrolase genes accounted for 50.68% and 40.63% respectively, and glycosyltransferase genes accounted for 28.78% and 28.13% respectively, while the carbohydrate binding module genes of strain YN-04 accounted for 1.04%.

[0101] 5. Genome circle map.

[0102] like Figure 15 and Figure 18Shown are genome maps of strains YN-05 and YN-04. This clearly structured map reveals key features of the genome from the outside in. The first and fourth circles of the map highlight the coding sequences (CDS) on the plus and minus strands, respectively. Color-coded COG functional groups are carefully distinguished, allowing readers to easily identify the distribution of various functional genes. The second and third circles illustrate the distribution of CDS, tRNA, and rRNA on the plus and minus strands, providing valuable insights into the transcription and translation processes of the genome. The fifth circle focuses on GC content. Outward-facing regions indicate regions with higher GC content than the genome-wide average, with the height of the peak directly corresponding to the difference from the average. Conversely, inward-facing regions indicate regions with lower GC content than the genome-wide average, and the significance of the peak also directly reflects the degree of difference from the average. This layered analysis helps reveal base composition biases within the genome and their potential functional significance. The sixth circle displays the GC-Skew value, which, through the sharp contrast between green (skew+) and purple (skew-), visually demonstrates the imbalanced distribution of G and C bases in the genome. Skew+ indicates a higher G content than C, while skew- indicates the opposite, with a lower G content than C. This value, calculated as (GC) / (G+C), not only helps us distinguish between leading and lagging strands (generally, leading strand GC skew > 0, lagging strand GC skew < 0), but also plays a crucial role in determining the origin (minimum cumulative skew) and endpoint (maximum cumulative skew) of replication, particularly in analyzing circular genomes. The innermost circle clearly indicates the genome size.

[0103] 6. Analysis of genes related to flavor substance metabolism.

[0104] The unique flavor of fermented dairy products is formed through complex biochemical reactions between lactic acid bacteria (LAB) and enzymes. LAB metabolize lactose to produce lactic acid, which in turn changes the flavor of dairy products. Flavor formation primarily involves carbohydrate conversion, fat breakdown, and amino acid metabolism. Key enzymes in carbohydrate conversion include citrate lyase, oxaloacetate decarboxylase, and lactate dehydrogenase; key enzymes in amino acid metabolism include peptidases, transaminases, hydroxyacid dehydrogenases, and phosphotransacylases.

[0105] Lactic acid bacteria (LAB) are typical amino acid auxotrophs. Due to their limited amino acid synthesis capacity, they primarily rely on external sources for essential amino acids to sustain growth. These amino acids are primarily obtained through two pathways: hydrolysis of casein in milk and direct absorption of free amino acids from the environment. Amino acids are not only essential nutrients for LAB growth but also serve as crucial precursors for the production of flavor compounds during their metabolism. Whole-genome sequencing analysis of YN-05 and YN-04 (Tables 7 and 8) revealed that YN-05 harbors 183 genes involved in amino acid synthesis and 178 genes involved in carbohydrate metabolism. Genes associated with flavor synthesis include gene0444 (L-lactate dehydrogenase), gene1294 (L-lactate dehydrogenase), and gene0455 (β-galactosidase). YN-04, on the other hand, harbors 225 genes involved in amino acid synthesis and 244 genes involved in carbohydrate metabolism. Genes involved in flavor synthesis include gene0205 (acetate kinase), gene0297 (acetate kinase), and gene0314 (alcohol dehydrogenase).

[0106] Table 7 Genes related to flavor production and metabolism in YN-05 ; Table 7 ; Table 7 ; Table 7 ; Table 7 ; Table 7 ; Table 7 ; Table 7 ; Table 7 ;

[0107] Table 8 YN-04 flavor production and metabolism related genes ; Table 8 ; Table 8 ; Table 8 ; Table 8 ; Table 8 ; Table 8 ; Table 8 ; Table 8 ; Table 8 ; Table 8 ; Table 8 ;

[0108] While acetate kinase, phosphate acetyltransferase, and alcohol dehydrogenase were absent in YN-05, it possessed a rich array of aminopeptidases and amino acid synthetases, enabling the production of a wide range of amino acids. YN-04, on the other hand, possessed these related enzymes, while YN-05 contained L-lactate dehydrogenase and malate / lactate dehydrogenase, enzymes absent in YN-04. These enzymes complement each other in a complementary relationship. Both strains possess genes involved in carbohydrate, amino acid, and lipid metabolism. Therefore, further investigation into the flavor formation mechanisms and metabolic pathways of these two strains will help predict their flavor synthesis potential and provide theoretical support for their industrial application.

[0109] 7. Functional gene characteristics of Lactobacillus pentosus YN-04 and Lactobacillus plantarum YN-05.

[0110] Tolerance-related functional genes of Lactobacillus pentosus YN-04 and Lactobacillus plantarum YN-05 were annotated, and 9 protease-related genes (htpX, ftsH, clpC, clpE, gluP, ysxB, hslU, clpX, rseP) and 10 protease-related genes (gluP, ftsH, clpX, clpE, ysxB, clpC, clpP, rseP, dam, degP) were annotated, respectively.

[0111] Plant Bacillus pentosus YN-04 and Lactobacillus plantarum YN-05 were compared against the VFDB and VirulenceFinder databases, using sequence similarity >90% and sequence coverage >60% as screening criteria. The results showed that no potential virulence-associated genes were detected in the ProSci-246 genome. Protease-related genes were detected and annotated in the genomes of Plant Bacillus pentosus YN-04 and Lactobacillus plantarum YN-05. The specific genes are shown in Table 9 below.

[0112] Table 9 Protease-related genes in the genomes of Lactobacillus pentosus YN-04 and Lactobacillus plantarum YN-05 ;

[0113] Example 5 1. Preparation of milk tofu auxiliary fermentation agent using Lactobacillus pentosaceus YN-04 and Lactobacillus plantarum YN-05.

[0114] Raw milk → sterilization → cooling → adding fermentation agent → heating to drain whey → kneading into a dough → cutting → pressing → compression molding → milk tofu finished product.

[0115] Key Points: Lactobacillus pentosus YN-04 and Lactobacillus plantarum YN-05 were mixed in a 1:0, 0:1, and 1:1 mass ratio to form a bacterial suspension. A 3% v / v inoculum was inoculated into a sterilized raw milk sample (the milk was placed in an 80°C waterbath, stirred for 15 minutes, and then cooled). The suspension was then placed in a 25°C incubator to simulate natural fermentation conditions. Fermentation reached its endpoint when the pH reached approximately 4.6, the acidity reached 65°C, and a small amount of whey precipitated. The curd was then poured into a beaker and placed in a 65°C waterbath with continuous stirring to ensure even heating. The whey was then drained until the curds became smaller, shrunken, and elastic. The curds were then kneaded into a ball, cut, and pressed into a mold to form a milk tofu sample.

[0116] The milk tofu fermented by Lactobacillus pentosus YN-04 (1:0) was group A, the milk tofu fermented by Lactobacillus plantarum YN-05 (1:0) was group B, the milk tofu fermented by Lactobacillus pentosus YN-04 and Lactobacillus plantarum YN-05 (1:1) was group C, and the commercial milk tofu was group D. The physical and chemical indicators of the prepared milk tofu are shown in Table 10, and the sensory properties are shown in Figure 8 A and Figure 8 As shown in (b), Group C had a significantly lower moisture content (p < 0.05) than the other three groups. This low moisture content aids in the preservation and development of milk tofu and effectively inhibits the growth and reproduction of harmful microorganisms. Furthermore, Group C had significantly higher ash and protein contents (p < 0.05) than the other three groups. Ash is an important indicator of food nutritional value, and its level reflects the overall value of a food. The protein content of milk tofu plays a key role in the coagulation and syneresis processes of milk. Changes in the relative content of specific protein components can significantly affect the quality of traditional cheese. Compared to Group D, there were no significant differences in fat content among the three fermentation groups.

[0117] Table 10 Physical and chemical indicators of milk tofu prepared in different groups ;

[0118] 2. Qualitative and quantitative analysis of milk tofu metabolites based on GC-MS technology.

[0119] (1) Extraction of metabolites.

[0120] A 50 mg sample was weighed and 500 μL of pre-chilled extraction solution (methanol:chloroform, volume ratio 3:1, containing L-2-chlorophenylalanine) was added. After vortex mixing for 30 seconds, steel beads were added and the sample was milled at 40 Hz for 4 minutes. The sample was then sonicated in an ice-water bath for 5 minutes (repeated three times). The sample was centrifuged at 12,000 rpm for 15 minutes at 4°C. 100 μL of the supernatant was transferred to a 1.5 mL EP tube and dried. 40 μL of each sample was pooled to form a QC sample. Then, 40 μL of methoxyamine salt reagent was added and the sample was incubated in an 80°C oven for 30 minutes. Subsequently, 60 μL of BSTFA was added and the sample was incubated at 70°C for 1.5 hours. Finally, the sample was cooled to room temperature, 5 μL of FAMEs were added, and the sample was analyzed in random order.

[0121] (2) GC-MS detection conditions.

[0122] A SHIMADZU GC-2030 gas chromatograph coupled to a QP2020 NX mass spectrometer was used, employing a DB-5MS column (30 m × 250 μm × 0.25 μm). The sample was injected in splitless mode at a volume of 1 μL. Helium was used as the carrier gas, with a front-end inlet purge flow rate of 3 mL / min and an in-column gas flow rate of 1 mL / min. The column temperature program was as follows: initial temperature at 50°C, held for 1 min, then increased to 310°C at a rate of 8°C / min, and held at 310°C for 11.5 min. The inlet, transfer line, and ion source temperatures were 280°C, 280°C, and 200°C, respectively. The ionization voltage was -70 eV. Mass spectral data were acquired in full-scan mode over a mass range (m / z) of 50–500, at an acquisition rate of 12.5 spectra per second, and with a solvent delay time of 7.2 min.

[0123] (3) Data processing.

[0124] ChromaTOF software (V 4.3x, LECO) was used to analyze the mass spectrometry data, including peak extraction, baseline correction, deconvolution, peak integration, and peak alignment. The LECO-Fiehn Rtx5 database was used for qualitative analysis, including mass spectrum matching and retention time index matching. The retention time indices are shown in Table 11.

[0125] Table 11 Retention time index table ;

[0126] Four kinds of milk tofu samples were tested by GC-MS technology, such as Figure 19 As shown, a total of 645 metabolites from 12 categories were identified. The four main groups of milk tofu substances include organic acids and their derivatives, organic oxygen compounds, lipids and lipid molecules, and organic heterocyclic compounds, accounting for a total of 72.56%. Organic acids enhance the flavor of milk tofu and impart prebiotic properties, improving its texture and positively impacting intestinal health. During the production of milk tofu, lipids play a crucial role in its flavor and preservation. The flavor of milk tofu depends largely on the breakdown of fat and its interaction with other components. Organic heterocyclic compounds are highly recognized in the flavoring field, many of which have unique aromas and extremely low olfactory thresholds. Therefore, differences in the types and content of these compounds in milk tofu may be key factors contributing to the flavor differences.

[0127] PCA analysis of metabolites of fermented milk tofu Figure 9 Middle a, Figure 9 Zhongb and Figure 9 As shown in Figure c, the samples are in different quadrants and the samples between groups are clustered, indicating that they are significantly different. Principal component analysis can be used to distinguish milk tofu samples fermented with different bacterial strain combinations. The OPLS-DA model score diagram and verification results between the groups are shown in Figure 4. Figure 10 a is the scatter plot of OPLS-DA model scores of group A and group D, Figure 10 Middle b is the OPLS-DA permutation test dot plot of group A and group D, Figure 10 Middle c is the scatter plot of OPLS-DA model scores of group B and group D, Figure 10 d in the middle is the OPLS-DA permutation test dot plot of group B and group D, Figure 10 Figure e is the scatter plot of the OPLS-DA model scores of group C and group D. Figure 10 Figure f is the OPLS-DA permutation test dot plot of group C and group D. Figure 10 As shown in Figure a, the milk tofu fermented by Lactobacillus pentosus YN-04 represented by Group A and the commercial milk tofu samples of Group D are in different quadrants and far away from each other, indicating that there are significant differences in the metabolites of milk tofu fermented by Lactobacillus pentosus YN-04 and commercial milk tofu. Figure 10 Middle C and Figure 10 As can be seen from Figure e, there are significant differences in metabolites between the milk tofu fermented by Lactobacillus plantarum YN-05 represented by Group B and the commercial milk tofu represented by Group D. There are also significant differences in metabolites between the milk tofu fermented by the fermentation agent composed of Lactobacillus pentosus YN-04 and Lactobacillus plantarum YN-05 represented by Group C and the commercial milk tofu represented by Group D. Figure 10 Middle b, Figure 10 Medium d and Figure 10As can be seen from Figure 5, the OPLS-DA replacement points are all located on the left, and the intersection of their regression lines and the vertical axis is significantly lower than the R² and Q² values ​​of the original model, indicating that the original model is not overfitting and the results are significantly effective. This proves that the metabolites of milk tofu prepared by groups A, B, and C are significantly different from those of commercially available milk tofu.

[0128] Depend on Figure 11 As shown in Figure A, a total of 148 differential metabolites were screened between Groups A and D, mainly including organic acids and their derivatives, organic oxygen compounds, lipids and lipid-like molecules, organic nitrogen compounds, benzene and substituted derivatives, and nucleosides, nucleotides and their analogs. Among them, 38 differential metabolites such as glycolic acid, L-valine, D-glutamic acid and citric acid were upregulated, while 110 differential metabolites such as maleimide, L-alanine, lactamide and octanoic acid were downregulated.

[0129] Depend on Figure 11 As shown in Figure B, a total of 177 differential metabolites were screened between Group B and Group D, mainly divided into categories such as lipids and lipid-like molecules, organic acids and their derivatives, organic oxygen compounds, nucleosides, nucleotides and their analogs, phenylpropanoids and polyketides, and organic heterocyclic compounds. Among them, 75 differential metabolites such as L-homoserine, L-alanine, glycerol phosphate, and myristic acid were upregulated, and 102 differential metabolites such as pyruvate, lactamide, L-malic acid, and 3-aminoglutaric acid were downregulated.

[0130] Depend on Figure 11 As shown in Figure C, a total of 148 differential metabolites were screened between Groups C and D, mainly classified into lipids and lipid-like molecules, organic acids and their derivatives, organic oxygen compounds, phenylpropanoids and polyketides, and organic heterocyclic compounds. Among them, 144 differential metabolites such as L-alanine, glycine, L-homoserine, and lauric acid were upregulated, while 35 differential metabolites such as sorbitol, lactobionic acid, hippuric acid, and 4-hydroxyquinoline were downregulated.

[0131] like Figure 12 Middle A, Figure 12 Middle B and Figure 12 As shown in C, it is a bubble diagram of the different metabolic pathways between the fermentation group and the commercial group. Figure 12 As shown in Figure A, compared with group D, group A had six significant metabolic pathways, including arginine and proline metabolism, glyoxylate and dicarboxylic acid metabolism, pyrimidine metabolism, glycerolipid metabolism, linoleic acid metabolism, and valine, leucine, and isoleucine biosynthesis (pathway p < 0.05). Figure 12 Figure B represents the metabolic pathway enrichment map of group B and group D. The two groups have six significant metabolic pathways, including valine, leucine and isoleucine biosynthesis, arginine and proline metabolism, glycerolipid metabolism, pyrimidine metabolism, glycine, serine and threonine metabolism, and linoleic acid metabolism (pathway p < 0.05). Figure 12As shown in C, compared with group D, group C had 9 significant metabolic pathways, including arginine and proline metabolism, glycine, serine and threonine metabolism, valine, leucine and isoleucine biosynthesis, pantothenic acid and coenzyme A biosynthesis, glycerolipid metabolism, aminoacyl-tRNA biosynthesis, sulfur metabolism, linoleic acid metabolism and arachidonic acid metabolism (pathway p < 0.05).

[0132] Metabolite and KEGG enrichment analysis revealed an increase in significantly enriched metabolic pathways in both groups C and D, with a significant increase in amino acid-related pathways compared to single-strain fermentation. Among the nine differential metabolic pathways, amino acid-related pathways were predominant, with common differential metabolites including proline, creatine, L-threonine, glycine, pyruvate, L-valine, L-isoleucine, and succinate. Proline and glycine, which have a slightly sweet taste, can mitigate any bitterness or other irritation that may be present in milk tofu, resulting in a more balanced and mellow flavor. Creatine, a naturally occurring compound primarily found in animal-derived foods, is also a functional dietary supplement widely used in sports nutrition and health. It has numerous important biological functions in the body, including enhancing energy metabolism, promoting muscle cell growth and repair, and exerting antioxidant effects. L-valine undergoes Strecker degradation to produce isobutyraldehyde, a volatile compound with nutty and malty notes that significantly enhances cheese flavor. L-isoleucine is degraded by Strecker to produce 2-methylbutanal, a volatile compound with nutty and caramel notes that enhances the flavor complexity of cheese.

[0133] 3. Changes in milk tofu quality during storage.

[0134] The above fermentation agents were used to prepare milk tofu as the experimental group, and traditional milk tofu (purchased from Changhong Dairy Factory, Zhenglan Banner, Xilin Gol League, Inner Mongolia) was selected as the control group. The changes in physical and chemical indicators and total bacterial counts of the two groups of milk tofu during storage (0, 15, 30, and 45 days) were investigated, as shown in Tables 12 and 13.

[0135] The above fermentation agent is used to prepare milk tofu: the raw milk is sterilized (the raw milk is placed in an 80°C water bath, stirred for 15 minutes, and then cooled), the fermentation agent is inoculated into the sterilized raw milk (the inoculation amount of the fermentation agent is calculated according to 3% of the volume fraction of the sterilized raw milk), fermented at 25°C to a pH of 4.6, heated in a water bath at 65°C to drain the whey, kneaded into a ball, cut, and compressed to obtain milk tofu.

[0136] Table 12 Changes in basic physical and chemical indicators of milk tofu during storage ;

[0137] Note: Different letters in the same column indicate significant differences (P < 0.05); Same letters in the same column indicate no significant differences (P > 0.05).

[0138] Table 13 Changes in the total bacterial count of milk tofu during storage ;

[0139] During storage, traditional milk tofu exhibited significant changes in basic physicochemical and microbiological indicators. Table 12 shows that with increased storage time, the control group's traditional milk tofu exhibited significant degradation of both fat and protein, resulting in significant quality loss. Compared to traditional milk tofu, the experimental group exhibited minimal fat degradation during the early stages of storage, but this remained stable after 15 days, indicating that the experimental group's milk tofu did not experience significant fat oxidation during storage. Furthermore, while protein degradation occurred to some extent around 30 days of storage, protein retention was significantly better than that of the control group, indicating that the starter culture is beneficial for maintaining the storage quality of milk tofu.

[0140] As shown in Table 13, in order to simulate the refrigerator storage conditions in the sales process, the storage temperature of milk tofu was set at 10°C. Under this condition, the total colony count of traditional milk tofu increased from the initial 3.42×10 4 The CFU / mL increased significantly to 9.84×10 6 CFU / mL, an increase of 2 orders of magnitude; while the total colony count in the experimental group remained at a low level, slowly increasing from the lower limit of detection (<10 CFU / mL) to 8.68×10 3 CFU / mL. It is worth noting that the total amount of microorganisms in the experimental group after 45 days of storage was still lower than the baseline value of the traditional group on the first day, which fully proves that the experimental group products have better storage stability during circulation. In a low-temperature environment of 4°C, although microbial proliferation was inhibited, the traditional milk tofu still showed a two-order-of-magnitude increase (final value 8.56×10 4 CFU / mL), while the microbial proliferation rate in the experimental group was significantly slowed, which is closely related to the antibacterial active substances formed within it. Fresh milk tofu is rich in nutrients and easily becomes a substrate for rapid microbial growth, causing rancidity. The specialized starter culture developed in this research creates an antibacterial barrier within the milk tofu system through specific metabolic pathways, effectively inhibiting the proliferation dynamics of microorganisms during storage, keeping the total colony count within a safe threshold, thereby simultaneously improving food safety and extending the shelf life.

[0141] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A fermentation agent for preparing storage-stable flavored milk tofu, characterized in that: The fermentation bacteria include Lactobacillus pentosus ( Lactiplantibacillus pentosus )YN-04 and Lactobacillus plantarum ( Lactiplantibacillus plantarum )YN-05; The Lactobacillus pentosus YN-04 was deposited in the China Center for Type Culture Collection on March 13, 2025, at Wuhan University, Wuhan, China, with the deposit number: CCTCC NO: M2025469; The plant lactobacillus YN-05 was deposited in the China Center for Type Culture Collection on March 13, 2025, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being: CCTCC NO: M2025470.

2. The fermentation agent according to claim 1, characterized in that The mass ratio of Lactobacillus pentosus YN-04 and Lactobacillus plantarum YN-05 in the fermentation agent is 1:1; the effective viable cell count of Lactobacillus pentosus YN-04 in the fermentation agent is ≥10 9 CFU / mL, the effective viable count of Lactobacillus plantarum YN-05 in the fermentation agent is ≥10 8 CFU / mL.

3. Use of the fermentation agent according to claim 1 or 2 in the preparation of flavor fermented products.

4. The application according to claim 3, characterized in that The fermented product is milk tofu.

5. Use of the fermentation agent according to claim 1 or 2 in the preparation of a fermentation product that is storable.

6. The application according to claim 5, characterized in that The fermented product is milk tofu.

7. A method for preparing milk tofu using the fermentation agent according to claim 1 or 2, characterized in that: The following steps are involved: The raw milk is sterilized, the fermentation agent is inoculated into the sterilized raw milk, fermented until the pH value is 4.6, heated to drain the whey, kneaded into a mass, cut, and compressed to obtain milk tofu.

8. The method for preparing milk tofu according to claim 7, wherein: The raw milk sterilization is as follows: the raw milk is placed in a water bath at 80° C., stirred for 15 minutes, and then cooled.

9. The method for preparing milk tofu according to claim 7, wherein: The inoculation amount of the fermentation bacteria agent is calculated based on 3% of the volume fraction of the sterilized raw milk; the fermentation temperature is 25°C.

10. The method for preparing milk tofu according to claim 7, characterized in that: The whey is heated in a water bath at 65°C.

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