A mixed lactic acid bacterial strain with a deodorizing effect, a preparation method and applications thereof

Abstract

The present application relates to the field of microbial technology, and specifically discloses a mixed lactic acid bacterial strain with a deodorizing effect, a preparation method and application thereof, which comprises Lactobacillus brevis (Lb. Latilactobacillus curvatus ) and Lactobacillus plantarum (Lb. Lactiplantibacillus plantarum ). The present application separates and identifies lactic acid bacteria in hand-made goat cheese and fresh goat milk, screens lactic acid bacteria CS0002 and CS0020 with potential probiotic properties and strong fermentation performance, and compounding the lactic acid bacteria as a compound starter according to a proportion, so as to be applied to the preparation of goat dairy products. The lactic acid bacteria can obviously reduce the bad odor in goat cheese and yogurt, improve the product flavor, make the goat dairy products be accepted, loved and ingested by more people, and increase the selection willingness of consumers for the goat dairy products.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a mixed lactic acid bacteria strain with anti-odor effect, its preparation method, and its application. Background Technology

[0002] Goat milk is an excellent source of protein and minerals essential for the human body, with higher levels of calcium, potassium, phosphorus, magnesium, and vitamin A than cow's milk. Goat milk is also more easily absorbed by the body than cow's milk because its fat globules and casein molecules are smaller. However, goat milk and its products (such as cheese and yogurt) generally have a distinctive, strong goaty odor, primarily derived from the high levels of short-chain fatty acids (such as caproic acid, caprylic acid, and capric acid) in goat milk fat. This peculiar odor significantly impacts the product's taste and flavor appeal, limiting the expansion of the consumer base and the further development of the goat milk industry.

[0003] Currently, in industrial production, physical, chemical, or biological methods are commonly used to mask or remove the odor of goat milk. Physical methods, such as deodorization and vacuum processing, require large investments in equipment and consume a lot of energy, and may lead to the loss of heat-sensitive nutrients. Chemical methods, such as adding flavor masking agents, acids, or alkalis, may introduce foreign chemical substances, which does not meet modern consumers' demands for "clean labels" and natural foods.

[0004] Biological methods, particularly lactic acid bacteria fermentation, are considered a more natural, healthy, and effective means of suppressing goaty odor. Current research has attempted to improve the flavor of goat milk products using single lactic acid bacteria strains, but the effects are often limited or unstable. Single strains typically have limited functional characteristics and cannot comprehensively address the complex composition of goaty odor compounds. Therefore, we propose a mixed lactic acid bacteria strain with anti-goaty odor effects, its preparation method, and its applications. Summary of the Invention

[0005] The purpose of this invention is to provide a mixed lactic acid bacteria strain with anti-goat odor effect, a preparation method and its application, so as to solve the problem of reducing the goat odor of goat milk products and improving the product flavor mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a mixed lactic acid bacteria strain with anti-odor effect, wherein the strain is *Lactobacillus curvilinearis* (… Latilactobacillus curvatus ) and Lactobacillus plantarum ( Lactiplantibacillus plantarum It is a mixture; The curved Lactobacillus ( Latilactobacillus curvatus It is deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 20251551, and the deposit date is July 8, 2025; The plant lactobacillus ( Lactiplantibacillus plantarumIt is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M 20251552, and the deposit date is July 8, 2025.

[0007] Preferably, the *Lactobacillus curvilinearis* and *Lactobacillus plantarum* are mixed in a 1:1 ratio. Specifically, the overnight culture media of *Lactobacillus curvilinearis* and *Lactobacillus plantarum* are adjusted to the same OD600 = 1.0 before being mixed in a 1:1 (v / v) ratio.

[0008] A method for preparing a mixed lactic acid bacteria strain with anti-odor effect includes the following steps: S1. Take samples of handmade goat cheese and fresh goat milk, aseptically homogenize and serially dilute them, then spread them on MRS solid medium. After incubation, single colonies are picked for purification. Gram staining and catalase test are used to screen out suspected lactic acid bacteria, and the samples are stored at -20℃. S2. Inoculate the strains selected in step S1 into seed culture medium and culture them to the logarithmic phase. Extract the total DNA from them using a bacterial genomic DNA extraction kit. S3. PCR amplification and identification of the 16S rDNA fragment of lactic acid bacteria; S31. PCR amplification was performed using primers 27F and 1492r; The reaction system includes template, primers, 2×Taq PCR Mix and dd H2O; S32. The PCR product was detected by agarose gel electrophoresis and observed and photographed in a gel imaging system. At the same time, it was detected whether there was a fluorescent band of about 1500 bp. S33. Analyze and sequence the amplified products that show a positive PCR result; S34. Classify the species and genera based on 16S rDNA sequence homology greater than 99%, and then perform 16S rDNA sequence analysis on the lactic acid bacteria strains to be tested and related type strains.

[0009] Preferably, in step S1, the handmade goat cheese sample is ground in a mortar under aseptic conditions.

[0010] Preferably, in step S1, suspected lactic acid bacteria that are Gram-positive and catalase-negative are stored in a 50% sterile glycerol solution at -20°C for later use. During preservation, the ratio of sterile glycerol solution to bacterial solution is 1:1, v / v.

[0011] Preferably, in step S31, the primers include an upstream primer and a downstream primer, wherein the upstream primer is 27F (5'-AGAGTTTGATCMTGGTCAG-3') and the downstream primer is 1492r (5'-TACGGYTACCTTGTTACGACTT-3').

[0012] The PCR reaction program consisted of 94℃ pre-denaturation for 5 min, 94℃ denaturation for 1 min, 53℃ annealing for 1 min, and 72℃ extension for 2 min, with 30 cycles.

[0013] Preferably, in step S32, bacterial strains with bands around 1500 bp are screened, and their amplification products are subjected to base sequence detection.

[0014] Application of a mixed lactic acid bacteria strain with anti-goat odor effect in the preparation of goat cheese.

[0015] Application of a mixed lactic acid bacteria strain with anti-goat odor properties in the production of goat fermented milk.

[0016] This invention has at least the following beneficial effects: This invention isolates and identifies lactic acid bacteria in handmade goat cheese and fresh goat milk, and selects CS0002 and CS0020, which have potential probiotic properties and strong fermentation performance. These lactic acid bacteria are then compounded in a certain proportion as a compound fermentation agent and applied to the preparation of goat dairy products. This can significantly reduce the unpleasant goaty smell in goat cheese and yogurt and improve the flavor of the products.

[0017] This invention, while inhibiting the goaty smell through bacterial strains, can generate flavor substances through normal fermentation, giving goat cheese and yogurt a milder acidity and a richer flavor. It avoids the problem of monotonous flavor or off-flavors that may be caused by traditional methods of inhibiting goaty smell, and achieves the dual effect of removing goaty smell and enhancing aroma.

[0018] The strains selected in this invention are all isolated from natural goat milk products, accurately identified by 16S rDNA sequencing, and deposited at the China Center for Type Culture Collection (CCTCC). The strains have clear identities, high genetic stability, and are safe and traceable for industrial applications.

[0019] The strain of this invention undergoes a multi-step screening process to precisely eliminate contaminating bacteria, ensuring that the final strain has high purity and accurate identification. At the same time, the preservation method of 50% glycerol at -20℃ can effectively maintain the activity of the strain, making it convenient for long-term storage and subsequent production use, and reducing the cost of strain preservation.

[0020] By adding this patented strain, ordinary goat dairy products can be upgraded into low-goat-flavored specialty products, forming a differentiated competitive advantage and helping companies increase product premium. At the same time, lactic acid bacteria are food-grade microorganisms, which meet consumers' demand for natural and healthy foods, further enhancing the health attributes of the products and improving market competitiveness. Attached Figure Description

[0021] Figure 1 These are images of the morphology of two lactic acid bacteria strains observed under a microscope. Figure 2 This is a diagram showing the changes in the microstructure of cheese during its ripening process. Figure 3 This is a graph showing the changes in free amino acids during cheese ripening. Figure 4 A graph showing the percentage of volatile compounds during cheese ripening; Figure 5 Sensory score graph for cheese during ripening. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example 1

[0023] Isolation and purification of lactic acid bacteria Handmade goat cheese from Dali, Yunnan and unpasteurized fresh goat milk from Yantai, Shandong were collected and sealed in aseptic bags or bottles. They were then transported at low temperature in an insulated box with ice packs, and the dilution and coating of the samples were completed within 24 hours.

[0024] Take 10 g of handmade goat cheese, ground under aseptic conditions in a mortar, and place it into a sterile homogenizing bag containing 90 mL of sterile physiological saline. Homogenize for 5 min using a sterile homogenizer. Then, take 1 mL of the homogenate and dilute it 10-fold in 0.9% sterile physiological saline. -3 10 -4 and 10 -5Diluted solutions were prepared by spreading 100 μL onto MRS solid medium (Hangzhou Best Biotechnology Co., Ltd.) using a plating method with three replicates for each gradient. The plates were then incubated overnight at approximately 37°C in a biochemical incubator for 48 hours. Once colonies were formed, single colonies of different morphologies and colors were repeatedly streaked onto plates. Microscopic examination confirmed pure colonies. Gram staining and catalase tests were performed. Suspected lactic acid bacteria showing Gram-positive staining but catalase-negative results were preserved in 50% sterile glycerol solution at -20°C for later use (sterile glycerol solution: bacterial culture = 1:1, v / v).

[0025] like Figure 1 As shown, both purified strains stained purple, indicating they were Gram-positive bacteria, catalase-negative, and their cells exhibited a rod-shaped morphology. Example 2

[0026] Molecular biological identification of lactic acid bacteria (1) Extraction of lactic acid bacteria DNA: The strains screened from the samples provided in Example 1 were inoculated into seed culture medium and cultured to the logarithmic growth phase. Total DNA was extracted according to the instructions of the bacterial genomic DNA extraction kit. The bacterial genomic DNA extraction kit was obtained from Shanghai Sangon Biotech Co., Ltd.

[0027] (2) PCR amplification of the 16S rDNA fragment of lactic acid bacteria: PCR amplification primers: upstream primer 27F (5'-AGAGTTTGATCMTGGTCAG-3'), downstream primer 1492r (5'-TACGGYTACCTTGTTACGACTT-3').

[0028] 25 μL PCR reaction system: 1 μL template, 1 μL each of forward and reverse primers (bacterial 16S rDNA universal primers 27F and 1492r, Shanghai Sangon Biotech Co., Ltd.). 12.5 μL 2×Taq PCR Mix, made up to 25 μL with dd H2O.

[0029] PCR reaction program: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 1 min, 53℃ annealing for 1 min, 72℃ extension for 2 min, 30 cycles.

[0030] PCR products were detected by electrophoresis on a 1% agarose gel and observed and photographed using a gel imaging system. The presence of a fluorescent band of approximately 1500 bp was also checked. Results showed that the bands of both strains were clear without significant tailing, and both bands were around 1500 bp. Therefore, their amplification products could be sequenced.

[0031] The amplified products with positive PCR results were sent to Sangon Biotech (Shanghai) Co., Ltd. for analysis and sequencing. Homology analysis and comparison of the PCR product sequencing results were performed on the NCBI website, and the 16S rDNA sequence information of relevant bacterial species was obtained from the database. Species classification was based on a 16S rDNA sequence homology greater than or equal to 99%, and then the 16S rDNA sequence analysis was performed on the lactic acid bacteria strains to be tested. The identification results are shown in Table 1, where CS0002 is *Lactobacillus curvilinearis* (…). Latilactobacillus curvatus CS0020 is *Lactobacillus plantarum* ( Lactiplantibacillus plantarum ).

[0032] Table 1. Results of strain identification

[0033] Example 3 Example 3 is a determination of the low pH tolerance of the two strains from Example 2. The specific implementation method is as follows: Preparation of bacterial suspension: Lactic acid bacteria strains were statically cultured in a medium at 37℃ for 24 h. After three generations of activation, the culture was centrifuged at 7000 rpm for 10 min. The supernatant was discarded, and the suspension was washed three times with phosphate-buffered saline (PBS) (pH 7.4). PBS was then added and the suspension was shaken thoroughly to prepare a suspension. The bacterial concentration was adjusted to 1.0 × 10⁻⁶ cells / mL. 8 CFU / mL (OD600 nm=1.0).

[0034] Take 10 mL of bacterial suspension and adjust the pH to 2.0. Incubate the suspension at 37°C for 3 h. Take samples at 0 h and 3 h, dilute them, and spread them onto MRS solid medium. Count the viable bacteria using the plating method. Perform three replicates for each group and take the average value. The calculation formula is as follows:

[0035] Where: N th The number of viable bacteria in the culture medium after 3 h of incubation at pH 2, expressed in CFU / mL. N 0h The number of viable bacteria in the culture medium after 0 h of culture at pH 2, expressed in CFU / mL. The results showed that strains CS0002 and CS0020 had a tolerance rate of over 35% when cultured for 3 hours under high acid conditions at pH 2. Example 4

[0036] Example 3 is a determination of the tolerance of the two strains from Example 2 to simulated gastrointestinal fluid. The specific implementation method is as follows: (1) Tolerance of the strain to artificial gastric fluid: Take 1 mL of the bacterial suspension prepared in Example 3 and mix it with 9 mL of simulated artificial gastric fluid at pH 3.0. Incubate at 37°C. Take samples at 0 h and 3 h, dilute them serially and spread them on MRS solid medium. Use the plate method to count the number of viable bacteria. Perform 3 parallels for each group and take the average value. The survival rate is calculated using the same formula as in Example 3.

[0037] (2) Tolerance of the strain to artificial intestinal fluid: 1 mL of bacterial culture that had been subjected to simulated gastric fluid was mixed with 9 mL of artificial intestinal fluid at pH 8.0 and incubated at 37°C. Samples were taken at 0 h and 3 h, serially diluted and spread onto MRS solid medium. The number of viable bacteria was counted using the plate method. Three replicates were performed for each group and the average value was taken. The survival rate was calculated using the same formula as in Example 3.

[0038] After being absorbed by the body, probiotics begin to exert their effects in the stomach. These probiotics can only be effective if they colonize the lower intestine. Results showed that the tolerance rates of each strain to intestinal fluid were stronger than those to gastric fluid. CS0002 showed tolerance rates of (71.64±3.11)% to artificial gastric fluid and (83.55±7.7)% to artificial intestinal fluid, while CS0020 showed tolerance rates of (71.64±3.11)% to artificial gastric fluid and (83.55±7.7)% to artificial intestinal fluid. Example 5

[0039] Example 5 illustrates the application of two lactic acid bacteria strains in the preparation of goat fermented milk. The lactic acid bacteria suspension described in Example 3 was inoculated at 4% (v / v) into fresh goat milk that had been pasteurized and homogenized. The mixture was incubated at 37°C for 12 h. The curd time, curd acidity, post-curd acidity (curd acidity after 1 day of refrigeration), viscosity, and sensory scores (see Table 2 for sensory scoring) were measured. At least three replicates were performed for each group, and the average value was taken. Acidity was calculated using Gilnell degrees (°T). The specific method followed GB 5009.239-2016, "Determination of Acidity in Foods." Viscosity was measured using a viscometer.

[0040] Among them, strain CS0004, isolated and purified from goat cheese, is used. Lactococcus lactis ), CS0007 ( Leuconostoc mesenteroides As a control, CS0004 was identified as being similar to Lactococcus lactis (…). Lactococcus lactis The homology was 99%, and CS0007 was similar to Leuconostoc mesenteroides (Enteromonas vaginalis). Leuconostoc mesenteroides The homology reached 100%.

[0041] Table 2 Sensory Evaluation Table for Goat Fermented Milk

[0042] Only when the acidity rises to a certain level will the proteins in goat milk form a gel network and begin to coagulate. Prolonged coagulation time may lead to the growth of harmful bacteria, affecting the quality of the yogurt. Lower acidity indicates a weaker acid-producing capacity of the strain, which is not conducive to masking the goaty odor. The results are shown in Table 3. Among them, strain CS0002 showed the greatest advantage in fermenting goat milk in terms of coagulation time, coagulation acidity, post-coagulation acidity, viscosity, and sensory scores. P <0.05), followed by CS0020 and CS0004, while CS0007 fermented goat milk with a longer curd time, lower curd acidity and post-curd acidity, and lower viscosity, which also resulted in its sensory score being lower than other strains.

[0043] Table 3 Data on fermentation of goat milk by single lactic acid bacteria strains

[0044] Note: Different letters in each column indicate significant differences in fermented goat milk from different single strains. P < 0.05). Example 6

[0045] Example 6 illustrates the application of a compound starter culture, formed by combining two lactic acid bacteria strains in a certain proportion, in the preparation of goat fermented milk. The specific implementation method is as follows: Single-strain fermented dairy products have drawbacks such as slow fermentation time and monotonous flavor. However, when multiple strains are cultured together, the interactions between the strains promote the synthesis of flavor compounds and enhance the sensory qualities of the dairy products. Based on Example 5, CS0007 was removed, and referring to previous strain compounding methods, compound starter cultures F1 (CS0020:CS0004=1:1), F2 (CS0020:CS0002=1:1), and F3 (CS0004:CS0002=1:1) were obtained. Goat fermented milk was prepared according to the method described in Example 5. Its coagulation time, coagulation acidity, post-coagulation acidity (coagulation acidity after 1 day of refrigeration), viscosity, and sensory score were measured (specific testing methods are the same as in Example 5).

[0046] Table 4 Data on fermented goat milk with compound lactic acid bacteria

[0047] Note: Different letters in each column indicate significant differences in fermentation of goat milk by different compound starter cultures. P < 0.05).

[0048] As shown in the table, the compound strain F2 has the best effect. Therefore, CS0002:CS0020 = 1:1 was selected as the goat cheese re-fermentation agent. Example 6

[0049] Example 6 illustrates the application of two lactic acid bacteria strains in the preparation of goat cheese. The preparation process for goat cheese is as follows: Fresh goat milk → homogenization and sterilization → room temperature cooling → addition of starter culture → addition of calcium chloride and rennet → curdling → cutting of curd → settling and stirring → heating and stirring, draining whey → composting → pressing → refrigeration → finished product.

[0050] The key points of goat cheese preparation are as follows: (1) Homogenization and sterilization: Fresh goat milk was homogenized three times at 20 MPa using a high-pressure homogenizer and then pasteurized at 63℃ for 30 min.

[0051] (2) Preparation of fermentation agent: The suspension prepared by activating strains CS0002 and CS0020 for three generations was mixed at a ratio of 1:1 and inoculated into sheep milk at 37°C at an inoculation rate of 3% for 4 h.

[0052] (3) Let the milk stand to form curd: When the pH of the milk drops to 5.5~6.3, add 0.02% calcium chloride and 0.01~0.03% rennet while stirring quickly and evenly, and let it stand until curd forms.

[0053] (4) Curd cutting: When the curd can be separated from the fermentation tank wall, it indicates that the curd can be cut. Use a cheese knife to cut the curd into 1 cm pieces. 3 Let the small squares on the left and right stand at room temperature for about 10 minutes.

[0054] (5) Hanging bag filtration: filter with sterile gauze and hang it in a constant temperature and humidity chamber at 10-15℃ for 4 hours. Raise the temperature to 36-44℃ and drain the whey until it stops dripping.

[0055] (6) Pressing: Place the cheese into a homemade cheese trough and press it in a constant temperature and humidity chamber at 10-15℃ for 24 hours.

[0056] (7) Packaging and refrigeration: Place the pressed cheese into an aseptic cheese packaging box and store it at 9°C. Turn it over every day. When it matures to the 5th day, vacuum pack it immediately.

[0057] Based on response surface methodology, the optimal cheese-making process is as follows: fresh goat milk → homogenization and sterilization → room temperature cooling → addition of starter culture → pH reduction to 5.68 → addition of calcium chloride → addition of rennet (0.0175%) → curdling → clot cutting → settling and stirring → heating (42.13℃), stirring, and whey removal → composting and whey removal → pressing → refrigeration → finished product. At this stage, the goat cheese yield can reach 14.38%. Example 7

[0058] Example 7 illustrates the changes in the microstructure of goat cheese during maturation. The microstructure was determined using scanning electron microscopy, as follows: The samples were fixed in 0.1 mol / L phosphate buffer (pH=7.0) containing 2.5% glutaraldehyde for at least 24 h. After fixation, they were dehydrated with ethanol solution for 15 min and then immersed in tert-butanol for 30 min before being sputter-coated with gold. After gold sputtering, the samples were observed and images were acquired under a Hitachi S-3000N scanning electron microscope at an accelerating voltage of 3.0 kV and magnifications of 300× and 1000×.

[0059] The results are as follows Figure 2 As shown, the cheese texture is tightly connected with no obvious gaps between 0-10 days. Under a 1000× microscope, it appears as cloud-like structures, formed by the disordered accumulation of casein, minerals, and fat particles. Between 20-30 days, the microstructure of the cheese changes, with cracks and cavities gradually increasing in size. This is likely due to the increasing degree of protein hydrolysis and the gradual disappearance of casein micelles and fat globules. Example 8

[0060] Example 8 describes the analysis of free amino acid content during the maturation of goat cheese. A high-speed amino acid analyzer (L-8900, Hitachi) was used to detect the free amino acid content in cheese at different maturation times. The high-speed amino acid analyzer system used lithium citrate as the mobile phase and performed UV-Vis detection at 570 nm and 440 nm. The mobile phase flow rate was 0.35 mL / min, and the derivatization reagent flow rate was 0.3 mL / min. The column temperature was set to 38°C, and the post-column reaction equipment was maintained at 130°C. The autosampler temperature was 4°C, and the injection volume for both standards and samples was 20 μL. The peak areas of the compounds were compared with those of the amino acid standards. A control group was prepared using a commercial starter culture according to the goat cheese production process described in Example 6.

[0061] The results are as follows Figure 3As shown, free amino acids have a significant impact on cheese flavor. They not only act as precursors to certain flavor compounds but can also directly impart flavors such as umami, bitterness, and sweetness to cheese. Throughout the maturation period, the content of free amino acids in both the experimental and control groups gradually increased, with the experimental group showing a higher content than the control group. Higher glutamic acid (Glu) content is more conducive to reducing bitterness in cheese. Glutamic acid (Glu) and aspartic acid (Asp) are umami amino acids, and during this period, the contents of aspartic acid (Asp) and glutamic acid (Glu) in the experimental group were higher than those in the control group. Alanine (Ala) is a sweet amino acid, and its content in both the experimental and control groups showed an increasing trend during maturation, reaching maximum values ​​of 118.51 nmol / L and 102.75 nmol / L, respectively, at 30 days. Alanine also helps reduce unpleasant tastes in food. Overall, the experimental group had higher contents of umami amino acids and total free amino acids than the control group during maturation, reflecting to some extent that the experimental group's cheese had a richer flavor than the control group. Example 7

[0062] Example 7 describes the determination of volatile compounds in goat cheese: solid-phase microextraction combined with gas chromatography-triple quadrupole mass spectrometry (GC-MS / MS) was used to identify volatile flavor compounds during cheese maturation. The GC conditions were as follows: column: HP-INNOWAX (60 m × 0.25 mm × 0.25 μm); injection port temperature: 250℃; temperature program: 40℃ for 4 min, ramp to 100℃ at 3℃ / min, hold for 1 min, ramp to 150℃ at 4℃ / min, and finally ramp to 240℃ at 10℃ / min, hold for 4 min; carrier gas: helium (99.99% purity); flow rate: 2 mL / min; injection method: splitless injection. The mass spectrometry conditions were: electron ionization energy: 70 eV; ion source temperature: 230℃; quadrupole temperature: 150℃; emission current: 35 μA; scan rate: 1.9 scans·s. -1 Quality scan range: 30-400 amu.

[0063] Identification of flavor compounds: Compounds were obtained by comparing mass spectrometry (MS) data from a database. The Kovar retention index (RI) of the compounds eluted before and after the specified time was calculated based on the retention times of a saturated alkane standard (C7-C30) measured under the same procedure. Qualitative identification was performed by matching the RI values ​​with those reported in the NIST (National Institute of Standards and Technology) spectral library. Quantitative analysis used cyclohexanone (1.11 μg / μL) as an internal standard, employing a relative internal standard quantification method. The peak areas of volatile flavor compounds were compared with the peak areas of the internal standard to quantify the content of the compounds.

[0064] A control group cheese was prepared using a commercial starter culture and the goat cheese production process described in Example 6. The control group cheeses were designated D0, D10, D20, and D30. Flavor comparisons were performed between the control and experimental group cheeses during maturation. The experimental group cheeses were designated S0, S10, S20, and S30. A graph showing the percentage of volatile compounds during cheese maturation is provided. Figure 4 The following table shows the differences in the concentration of each aroma compound between the two groups of cheese samples at different maturation stages.

[0065] Table 5. Volatile compound content during cheese maturation

[0066]

[0067] Note: S represents the experimental group cheese, D represents the control group cheese, and 0-30 represents the maturation days. Different letters in each row indicate significant differences in the same type of cheese at different maturation days. P < 0.05), ND indicates not detected.

[0068] The results of the examples show that, under the same maturation conditions, the relative peak areas (GC-MS) of 3-hydroxy-2-butanone, 2,3-butanediol, 2-heptanone, 2-nonanone, isoamyl alcohol, amyl butyrate, ethyl octanoate, ethyl decanoate, benzaldehyde, and nonanal in the volatile components of cheese in the experimental group were all higher than those in the control group, which impart milky, fruity, and nutty aromas. Conversely, the relative peak areas of butyric acid, hexanoic acid, octanoic acid, and decanoic acid, which produce a rancid or sour odor, were all lower in the experimental group than in the control group. These differences resulted in the experimental group having a total sensory evaluation score greater than or equal to that of the control group, and a lower off-odor score, thus verifying the technical effectiveness of the strain of this invention in improving the sensory quality of cheese.

[0069] Acetic acid is one of the most abundant acids during the maturation period of both cheeses, giving them a sour taste. Butyric acid and hexanoic acid have a rancid and sweaty odor. The butyric acid content in both cheeses increases during 20-30 days, with maximum values ​​reaching (491.88±2.19) μg / Kg and (749.81±0.17) μg / Kg, respectively.

[0070] Caprylic and capric acids have a typical goaty odor, which is unacceptable to most consumers. The caprylic and capric acid content in both groups of cheeses gradually increases during maturation. PAlthough the concentration of caprylic acid was less than 0.05%, the content of caprylic acid and decanoic acid in the experimental group was lower than that in the control group. During the 0-30 day period, the caprylic acid content in the experimental group was 53.86%, 13.34%, 55.87%, and 85.34% of that in the control group, respectively. During the 10-30 day period, the decanoic acid content in the experimental group was 14.58%, 79.46%, and 91.86% of that in the control group, respectively. This indicates that the compound fermentation agent F2 (CS0020:CS0002=1:1) has a reducing effect on the muttony odor compared to commercial fermentation agents.

[0071] Example 8 Example 8 is a sensory evaluation of the goat cheese prepared in Example 6. The sensory evaluation was conducted by 10 food science students who had received sensory evaluation training. The fermented goat milk samples were stored at 4°C for 24 hours and then equilibrated to room temperature. They were placed in tasting cups with random number codes. Drinking water was also provided to avoid taste interference between fermented goat milk samples. The sensory evaluation of the fermented goat milk was based on the scores. The sensory scoring criteria are as follows.

[0072] Table 6 Sensory Rating Table for Goat Cheese

[0073] The results are as follows Figure 5 As shown, the compound fermentation agent F2 (CS0020:CS0002=1:1) has a less pronounced muttony odor and better sensory characteristics compared to commercial fermentation agents.

[0074] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0075] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A mixed lactic acid bacteria strain with anti-odor effect, characterized in that: The strain is *Lactobacillus curvilinearis* (… Latilactobacillus curvatus ) and Lactobacillus plantarum ( Lactiplantibacillus plantarum It is a mixture; The curved Lactobacillus ( Latilactobacillus curvatus It is deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 20251551, and the deposit date is July 8, 2025; The plant lactobacillus ( Lactiplantibacillus plantarum It is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M 20251552, and the deposit date is July 8, 2025.

2. The mixed lactic acid bacteria strain with anti-odor effect according to claim 1, characterized in that: The *Lactobacillus curvilinearis* and *Lactobacillus plantarum* were mixed in a 1:1 ratio.

3. A method for preparing a mixed lactic acid bacteria strain with anti-odor effect, characterized in that, Includes the following steps: S1. Take samples of handmade goat cheese and fresh goat milk, aseptically homogenize and serially dilute them, then spread them on MRS solid medium. After incubation, single colonies are picked for purification. Gram staining and catalase test are used to screen out suspected lactic acid bacteria, and the samples are stored at -20℃. S2. Inoculate the strains selected in step S1 into seed culture medium and culture them to the logarithmic phase. Extract the total DNA from them using a bacterial genomic DNA extraction kit. S3. PCR amplification and identification of the 16S rDNA fragment of lactic acid bacteria; S31. PCR amplification was performed using primers 27F and 1492r; The reaction system includes template, primers, 2×Taq PCR Mix and dd H2O; S32. The PCR product was detected by agarose gel electrophoresis and observed and photographed in a gel imaging system. At the same time, it was detected whether there was a fluorescent band of about 1500 bp. S33. Analyze and sequence the amplified products that show a positive PCR result; S34. Classify the species and genera based on 16S rDNA sequence homology greater than 99%, and then perform 16S rDNA sequence analysis on the lactic acid bacteria strains to be tested.

4. The method for preparing the mixed lactic acid bacteria strain with anti-odor effect according to claim 3, characterized in that: In step S1, the handmade goat cheese sample is ground in a mortar under aseptic conditions.

5. The method for preparing the mixed lactic acid bacteria strain with anti-odor effect according to claim 3, characterized in that: In step S1, suspected lactic acid bacteria that are Gram-positive and catalase-negative are stored in a 50% sterile glycerol solution at -20°C for later use. During preservation, the ratio of sterile glycerol solution to bacterial solution is 1:1, v / v.

6. The method for preparing the mixed lactic acid bacteria strain with anti-odor effect according to claim 3, characterized in that: In step S31, the primers include an upstream primer and a downstream primer, the upstream primer being 27F (5'-AGAGTTTGATCMTGGTCAG-3') and the downstream primer being 1492r (5'-TACGGYTACCTTGTTACGACTT-3'). The PCR reaction program consisted of 94℃ pre-denaturation for 5 min, 94℃ denaturation for 1 min, 53℃ annealing for 1 min, and 72℃ extension for 2 min, with 30 cycles.

7. The method for preparing the mixed lactic acid bacteria strain with anti-odor effect according to claim 3, characterized in that: In step S32, bacterial strains with bands around 1500 bp are screened, and their amplification products are subjected to base sequence detection.

8. The use of a mixed lactic acid bacteria strain with anti-goat odor effect as described in any one of claims 1-3 in the preparation of goat cheese.

9. The use of a mixed lactic acid bacteria strain with anti-goat odor effect as described in any one of claims 1-3 in the production of goat fermented milk.

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