Multi-strain composite leavening agent with multiple application characteristics

Abstract

The invention belongs to the field of food processing and fermentation engineering, and discloses a multi-strain composite leavening agent with multiple application characteristics and application of the multi-strain composite leavening agent in preparation of fermented milk. The fermentation agent is prepared from lactobacillus bulgaricus IMAU20428, lactobacillus bulgaricus IMAU20427, streptococcus thermophilus IMAU20229 and streptococcus thermophilus IMAU10630. Systematic research finds that different compounding proportions can significantly influence the fermentation time, post-acidification phenomenon, viscosity, water-holding capacity, texture characteristics, amino acid and organic acid content and metabolic pathway enrichment characteristics of the fermented milk. The composite leavening agent provided by the invention can realize differentiated development of fermented milk products through proportion regulation and control, and provides technical support for innovation of the dairy product industry.

Description

Technical Field

[0001] This invention belongs to the field of food processing and fermentation engineering, specifically relating to a compound multi-strain fermentation agent and its application in the preparation of fermented milk. Background Technology

[0002] Fermented milk, as an important branch of dairy products, is widely popular worldwide. Its quality characteristics mainly depend on the metabolic activity of the starter strain. In traditional fermented milk production, *Lactobacillus bulgaricus* (Lactobacillus bulgaricus) is commonly used. Lactobacillus delbrueckii bulgaricus Streptococcus thermophilus Figure 1 subsp. Figure 2 ) and Streptococcus thermophilus ( Figure 3 As core fermentation strains, the two have complementary effects in acid production, aroma production, and texture formation. However, most existing fermentation agents are mixtures with fixed proportions, lacking systematic research on compounding ratios, resulting in deficiencies in product diversification and quality control.

[0003] Existing research has shown that single-strain or simple combination fermentation agents often have the following problems: (1) monotonous flavor, making it difficult to meet the diversified needs of the market; (2) severe acidification after some combinations, affecting storage stability and taste; (3) lack of corresponding process parameters and strain combinations to support fermented milk with different market positioning (such as low viscosity, medium viscosity, and high viscosity). Therefore, there is an urgent need to develop a compound multi-strain fermentation agent that can achieve quality control through ratio adjustment.

[0004] This study designed nine combinations of compound fermentation ratios based on the strain specificity of *Lactobacillus bulgaricus* and *Streptococcus thermophilus*, and systematically investigated their effects on fermentation characteristics and storage stability. Through fermentation kinetics, microrheology, textural properties, viable cell count, amino acid and organic acid metabolism, and metabolic pathway enrichment analysis, the correlation between compound ratios and fermented milk quality characteristics was revealed, providing theoretical and practical basis for the industrial application of compound multi-strain starter cultures. Summary of the Invention

[0005] To address the shortcomings of traditional starter cultures in terms of product diversity and quality control, this invention provides a composite multi-strain starter culture composed of *Lactobacillus bulgaricus* IMAU20428 and IMAU20427, and *Streptococcus thermophilus* IMAU20229 and IMAU10630. Through nine different formulation ratios, the effects of these strains on the characteristics of fermented milk were systematically analyzed, enabling differentiated product development.

[0006] Further, the viable cell number ratio of the Lactobacillus bulgaricus IMAU20428, Lactobacillus bulgaricus IMAU20427, Streptococcus thermophilus IMAU20229 and Streptococcus thermophilus IMAU10630 is (1-100):(1-100):(1-100):(1-100); preferably (1-50):(1-50):(1-50):(1-50); preferably (1-30):(1-30):(1-30):(1-30); preferably (1-20):(1-20):(1-20):(1-20); preferably (1-10):(1-10):(1-10):(1-10); preferably (1-5):(1-5):(1-5):(1-5); preferably (0.5-1.5):(0.5-1.5):(0.5-1.5):(0.5-1.5); preferably (0.8-1.2):(0.8-1.2):(0.8-1.2):(0.8-1.2); preferably (0.9-1.1):(0.9-1.1):(0.9-1.1):(0.9-1.1); and most preferably 1:1:1:1.

[0007] Further, the viable cell number ratio of the Lactobacillus bulgaricus IMAU20428, Lactobacillus bulgaricus IMAU20427, Streptococcus thermophilus IMAU20229 and Streptococcus thermophilus IMAU10630 is preferably 5-15:5-15:50-150:0.5-1.5; preferably 5-15:5-15:50-150:0.5-1.5; preferably 7-13:7-13:70-130:0.7-1.3; preferably 8-12:8-12:80-120:0.8-1.2; preferably 9-11:9-11:90-110:0.9-1.1; and most preferably 10:10:100:1.

[0008] Further, the viable cell number of the Lactobacillus bulgaricus IMAU20428, Lactobacillus bulgaricus IMAU20427, Streptococcus thermophilus IMAU20229 and Streptococcus thermophilus IMAU10630 is 1x108CFU / g to 1x1012CFU / g. 2 ~1x1012 11 CFU / g.

[0009] In another aspect, the present application provides a method for preparing a fermented milk, which comprises inoculating the above-mentioned starter culture into raw milk to perform fermentation, thereby obtaining the fermented milk.

[0010] Further, the fermented milk includes various fermented milk beverage products developed on the basis thereof.

[0011] In a third aspect, the present application also provides a fermented milk, which is obtained by fermenting the above-mentioned starter culture.

[0012] Further, the present fermented milk includes various types of products, including but not limited to set-type and stirred-type.

[0013] In another aspect, the present application provides use of the above-mentioned starter in preparation of fermented milk with fast coagulation speed, high level of viable bacteria number during storage, stable viscosity and water holding capacity.

[0014] In another aspect, the present application provides use of the above-mentioned starter in preparation of fermented milk with strong gel structure and good stability.

[0015] The present application has the following technical effects relative to the prior art: (1) At present, researches on strain-compounded starter at home and abroad have made certain progress, but mainly focus on single-strain characteristics or simple compounded combination, and researches on specific regulation of compounded ratio and comprehensive influence of the same on fermented milk quality are still limited. Due to the differences in growth characteristics and metabolic characteristics of Lactobacillus bulgaricus and Streptococcus thermophilus, the combination of different strains has great influence on fermented milk, and even affects shelf-life stability, and how to screen and match excellent strain combination to promote each other still has certain difficulty, in addition, the interaction mechanism (such as symbiosis, competition or antagonistic effect) between different strains has not been fully elucidated, which also brings challenges to the optimization of compounded ratio.

[0016] (2) In the present application, Streptococcus thermophilus IMAU10630, IMAU20229 and Lactobacillus bulgaricus IMAU20428, IMAU20427 with good fermentation characteristics screened through the interaction lactic acid bacteria starter screening platform are selected as research objects, fermented milk is compounded according to different ratios, the changes of physicochemical indexes and flavor substances during the fermentation and storage of fermented milk with different compounded combinations are researched, and the compounded ratio combination with improved fermented milk quality is screened. Within the ratio range of (1~100):(1~100):(1~100):(1~100), good technical effects are obtained. When the optimal compounded ratios 1:1:1:1 and 10:10:100:1 are applied to fermented milk, the fermented milk with the compounded ratio 1:1:1:1 has fast coagulation speed, high level of viable bacteria number during storage, stable viscosity and water holding capacity, and the fermented milk with the compounded ratio 10:10:100:1 has strong gel structure and good stability, and can produce fermented milk with different excellent product characteristics, which can provide new ideas for commercial application and development and utilization of compounded starter. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 4 is the continuous pH change during single-strain fermentation.

[0018] Figure 5 is the micro-rheological change of 9 groups of fermented milk during fermentation; Figure 5 pH changes of 9 groups of fermented milk during storage; Figure 5 TA changes of 9 groups of fermented milk during storage; Figure 6 Viable bacteria count changes of 9 groups of fermented milk during storage; Figure 7 (a) viable bacteria count of Lactobacillus bulgaricus, Figure 8 (b) viable bacteria count of Streptococcus thermophilus; Figure 8 Viscosity changes of 9 groups of fermented milk during storage; Figure 8 Water holding capacity changes of 9 groups of fermented milk during storage; Figure 8 Texture property changes of 9 groups of fermented milk during storage; Figure 8 (a) hardness index; Figure 9 (b) consistency index; Figure 10 (c) cohesiveness index; Figure 11 (d) viscosity index; Figure 12 Amino acid content changes of 9 groups of fermented milk during storage; Figure 13 Organic acid content changes of 9 groups of fermented milk during storage; Figure 13 Cluster analysis of compound ratio combination fermented milk and different types of fermented milk; Figure 13 Cluster analysis of each index of fermented milk during fermentation and storage; Figure 14 Quality control sample (QC) evaluation, Figure 15 (a) QC sample TIC graph in ESI-mode; Figure 16 (b) QC sample TIC graph in ESI+ mode; Figure 17 Principal component analysis (PCA) graph of A vs I group, E vs I group fermented milk, a graph is A vs I, b graph is E vs I; Figure 18 OPLS-DA model score graph of A vs I group, E vs I group fermented milk; Lactobacillus delbrueckii bulgaricus Streptococcus thermophilus Differential metabolite volcano graph of A vs I, E vs I; Lactobacillus delbrueckii bulgaricus Streptococcus thermophilus Differential metabolite KEGG enrichment graph of A vs I; Figure 1 Differential metabolite KEGG enrichment graph of E vs I.

[0019] Strain preservation information The taxonomic name of Lactobacillus bulgaricus IMAU20427 is S. thermophilus subsp. S. thermophilus IMAU20427, which was deposited with the China Center for Type Culture Collection on May 15, 2024, and has the accession number CCTCC NO: M 2024957.

[0020] The taxonomic name of Lactobacillus bulgaricus IMAU20428 is Lactobacillus delbrueckii bulgaricus Streptococcus thermophilus subsp. L. bulgaricus IMAU20428, which was deposited with the China Center for Type Culture Collection on May 15, 2024, and has the accession number CCTCC NO: M 2024954.

[0021] The taxonomic name of Streptococcus thermophilus IMAU20229 is Figure 2 IMAU20229, which was deposited with the China Center for Type Culture Collection on July 12, 2023, and has the accession number CCTCC NO: M 20231266.

[0022] The taxonomic name of Streptococcus thermophilus IMAU10630 is Figure 2 IMAU10630, which was deposited with the China Center for Type Culture Collection on May 15, 2024, and has the accession number CCTCC NO: M 2024951. DETAILED DESCRIPTION

[0023] EMBODIMENTS MATERIALS AND METHODS The strains involved in this experiment are Lactobacillus bulgaricus IMAU20428, IMAU20427 and Streptococcus thermophilus IMAU10630, IMAU20229, which are provided by the Inner Mongolia Agricultural University Lactic Acid Bacteria Collection Center (Lactic Acid Bacteria Collection Center, LABCC). The specific proportions and corresponding combinations are shown in Table 1, and the specific information of the test strains is shown in Table 2.

[0024] Table.1Different compound combination ratios Table.1Different compound combination ratios Table.2Experimental strain information Table.2Experimental strain information EXPERIMENTAL METHODS Strain activation and preparation of bacterial suspension First, use an inoculation loop to pick up bacterial cells from ampoules stored at low temperature (preserved in the Lactic Acid Bacteria Strain Resource Bank of Inner Mongolia Agricultural University). Streak the inoculation loop containing bacterial cells onto a solid culture medium and allow the strain to grow on the solid culture medium for 24-48 hours. Pick the activated single cells and place them in 10 mL of liquid culture medium for two subcultures (2% of the cells per subculture). To ensure the purity of the cells, microscopic examination is required before subculture. Centrifuge the bacterial solutions from the two subcultures in a high-speed centrifuge (4000 r / min, 10 min, 4°C). After discarding the supernatant, wash the cells and add 10-15 mL of PBS buffer (Na2HPO4-1.15 g / L, KH2PO4-0.2 g / L, NaCl-8 g / L). Repeat twice. Then add 2 mL of skim milk protectant and shake the bacterial solution and protectant evenly. Use a pipette to draw 3-5 drops into a sterilized cryovial and store at -80°C. The preserved bacterial strains were activated in liquid culture medium, using MRS medium (Lactobacillus bulgaricus) and M17 medium (Streptococcus thermophilus). After growing on solid medium for 24-48 hours, single bacteria were picked with an inoculation loop and placed into their respective 10 mL liquid medium. After growing for 24 hours, they were inoculated into 300 mL liquid medium at an inoculation rate of 2%. After growing for 12 hours, the bacteria were centrifuged in a high-speed centrifuge (4000 r / min, 10 min, 4°C). The supernatant was discarded, and the bacterial sludge was washed with PBS buffer and centrifuged again. This process was repeated twice until the supernatant was clear. The bacterial sludge was shaken to mix with PBS to prepare a bacterial suspension, which was then stored in a refrigerator at 4°C.

[0025] Preparation of fermented milk samples Weigh the required base materials according to the ratio of 6.5% sucrose and 93.5% milk, and place them in a water bath preheated to 65°C. Melt the materials for 15 minutes to ensure that the sucrose and milk are evenly mixed. Then homogenize them at 65°C under low pressure of 15MPa and high pressure of 35MPa. Finally, sterilize them in a water bath at 95°C for 5 minutes and then quickly cool them to 42°C for later use.

[0026] Methods for studying the fermentation characteristics and storage stability of fermented milk Determination of viable bacterial count The viable counts of Lactobacillus bulgaricus and Streptococcus thermophilus in fermented milk were determined according to GB4789.35-2023, "National Food Safety Standard - Microbiological Examination of Food - Examination of Lactic Acid Bacteria".

[0027] Measurement of water holding capacity At room temperature, place 20g of fermented milk onto quantitative filter paper (20µm pore size) at the top of a funnel, and record the sample volume as m1. After standing for 2 hours, discard the upper whey layer, retain the sample, weigh it, and record it as m2. The calculation formula is: water holding capacity (%) = m2 / m1 × 100%.

[0028] Measurement of viscosity The fermented milk samples were placed in 100 mL beakers and measured at room temperature using a brookfield ddi-viscometer type viscometer. The fermented milk was selected with a 4# rotor, the rotation speed was 100 r / min, the torque was 10%-100%, and the measurement time was 30 s.

[0029] Measurement of texture properties The sample was poured into a sample cup after being restored to room temperature, and the texture properties of the sample were measured using a TA.XT. plus texture analyzer. The probe was selected as A / B / E with a diameter of 35 mm.

[0030] Results analysis and discussion Changes in pH during single-strain fermentation The continuous pH values during fermentation of the four strains alone are shown in Figure 2 The time required for the four strains to reach the fermentation endpoint of pH 4.6 during single-strain culture was, in order: Figure 2 IMAU20229 required 3.8 h, Figure 3 IMAU10630 required 9 h, L. Figure 4 IMAU20428 required 11 h, Figure 5 IMAU20427 required 13 h. This indicates that there are significant differences in the fermentation rates of different strains.

[0031] Effect of fermentation starter compounding ratio on fermentation time of fermented milk The experimental results showed that the nine different compounding ratios successfully promoted the conversion of cow's milk into curd. However, there were differences in the time required for the fermented milk of different compounding ratios to reach pH 4.6 and pH stability during the fermentation process. The specific experimental results are shown in Table 3, and the pH values of each group of fermented milk samples showed a downward trend as the storage time increased. As can be seen from the data, the fermentation time of Group I was the longest, exceeding 6 h; the fermentation times of Groups A, D, E, and G were relatively short, all within 5 h, and the fermentation time of Group A was the shortest, at 4.2 h.

[0032] Table 3 Fermentation time of fermented milk Note: Different capital letters indicate the difference between samples within the group, and different lowercase letters indicate the difference between samples between groups, p <0.05 indicates a significant difference, p >0.05 indicates no significant difference, the values in the table are the mean ± standard deviation, and the same below.

[0033] Analysis of micro-rheological properties during fermentation The changes in SLB during fermentation are shown in Figure 5As shown in Figure a, groups A, B, and I began to show inflection points and gradually stabilized after 3 hours. Groups C, E, G, and H showed inflection points after 4 hours, followed by groups D and F. After 5 hours, all groups began to stabilize. Among them, groups C, E, F, and H were closer to 0.5, indicating that these groups of fermented milk exhibited strong solid properties. Groups A, B, G, and I had SLB values ​​close to 0.6, indicating that these groups of fermented milk exhibited strong liquid properties. The SLB curves of groups E and F showed high similarity. Group D had the longest time to reach the gel point and the highest SLB value, while group E had the lowest SLB value.

[0034] For details on the changing trend of elasticity factor (EI) throughout the fermentation process, please refer to [link / reference]. Figure 6 b. Starting from 3 hours, the EI curves of fermented milk in groups A and B begin to show inflection points, indicating that the fermented milk has reached its gel point. Fermented milk in groups C, E, F, G, H, and I shows its gel point after 4 hours. Group E only begins to show its gel point on the 5th hour of fermentation. As fermentation time progresses, the casein in the fermented milk first completely dissociates and then rapidly aggregates to form a gel structure. The EI values ​​of groups C, F, and G are similar and slightly higher than those of the other groups. Group E has the highest EI value, indicating that its gel structure is more compact.

[0035] Changes in MVI during fermentation, such as L. bulgaricus As shown in c, from the start of fermentation to about 3 hours, the MVI values ​​of each group remained at a certain level during the same period. This is because the gel network structure has not yet formed, which is the initial stagnant stage with low viscosity. The MVI of fermented milk in groups A and B first showed a rapid upward trend. After 4 hours, the MVI values ​​of fermented milk in other groups increased rapidly and then stabilized.

[0036] like Lactobacillus delbrueckii bulgaricus Streptococcus thermophilus As shown in d, the fermented milk samples remained liquid for the first 3 hours. After 3 hours, the FI values ​​of fermented milk in groups A and B showed a linear decrease. After 4 hours, the FI values ​​of the remaining 8 groups began to show inflection points one after another. The FI values ​​decreased linearly from 102Hz to 10-1Hz and then decreased slowly. After reaching 10-2Hz, they remained basically unchanged, indicating that the samples had high viscous liquid properties. After reaching the fermentation endpoint, the FI value of group E was the lowest.

[0037] Analysis of acidity changes during fermented milk storage pH Change Analysis During Storage pH changes during storage of 9 groups of fermented milk are as follows Figure 7 As shown, the experimental results indicate that the pH value of all fermented milk samples decreased with prolonged storage time, but remained above 4.20. Comparing groups B, C, and D, it was found that the inoculum amount of *Lactobacillus bulgaricus* in group D was significantly less than that in groups B and C, and the pH value of group D at the end of storage was significantly higher than that of groups B and C. p <0.05).

[0038] Changes in TA value during storage of 9 groups of fermented milk are as follows: Figure 8 As shown, the experimental results indicate that the titration acidity (TA) values ​​of all samples remained below 100°T throughout the entire storage period. The TA value increased relatively rapidly between days 1 and 14, but the upward trend slowed significantly between days 14 and 21. Groups A, B, C, D, E, and F showed relatively stable TA values ​​during storage; among them, group D had the lowest TA value, differing by only 6°T before and after storage, demonstrating the most stable TA value during storage. Groups G, H, and I had relatively higher TA values. Notably, group I not only had a higher titration acidity but also exhibited the greatest change in TA value during storage, differing by 20°T before and after storage, consistent with pH changes. When the TA value changes of all nine groups of compound fermented milk remained within the range of 70°T-110°T during storage, it fell within the acceptable range for consumers.

[0039] Fermented milk storage characteristics Changes in viable bacteria count during fermented milk storage Changes in the viable count of Lactobacillus bulgaricus in fermented milk from 9 different blend ratios are as follows: Figure 8 As shown in Figure a, during storage, the viable count of *Lactobacillus bulgaricus* in all nine blended fermented milk formulations showed a trend of first increasing and then decreasing. In the initial storage period (days 1-14), the viable count of *Lactobacillus bulgaricus* showed an increasing trend, reaching its peak at day 14. Subsequently, the viable count began to decline.

[0040] Changes in viable counts of Streptococcus thermophilus in 9 groups of compound fermented milk are as follows: Figure 8 As shown in b, the viable counts of fermented milk from different groups exhibited different trends during storage. In fermented milk from combinations A, C, D, F, and I, the viable count of Streptococcus thermophilus showed a gradual decreasing trend, with the highest count occurring on the first day of storage, subsequently decreasing gradually with increasing storage time. In contrast, in fermented milk from combinations B, E, G, and H, the viable count of Streptococcus thermophilus showed a trend of first increasing and then decreasing.

[0041] The viable counts of Lactobacillus bulgaricus and Streptococcus thermophilus in the fermented milk of group A were significantly higher than those in other groups during the storage period. p The concentration of viable bacteria (<0.05%) ensures that the product maintains high activity and nutritional value during storage. This also indicates that the proportion of the A-group compound promotes synergistic growth among the strains, creating a suitable environment for their survival and reproduction in fermented milk, thereby increasing the viable bacterial count.

[0042] At the end of storage, the viable count of all compound ratio combinations was greater than 10. 6 The CFU / mL indicates that the selected strain has good stability and meets the national requirements for the number of live bacteria in fermented milk.

[0043] Analysis of viscosity changes during fermented milk storage like Figure 8 As shown, the viscosity of the nine groups of fermented milk with different compound ratios generally showed a trend of first increasing and then decreasing during storage. Based on the peak viscosity level of the fermented milk, the nine groups of fermented milk can be divided into three viscosity groups: high (D, E, F, G, H), medium (A, B, C), and low (I).

[0044] In the high-viscosity combinations, groups E, F, and G showed stable viscosity changes over 21 days of storage, indicating that they effectively maintained the quality and stability of fermented milk during storage. In contrast, groups D and H exhibited greater viscosity decreases during storage, demonstrating poor viscosity stability. This phenomenon is closely related to the synergistic effect of the strain ratio: it suggests that the combination ratio of groups E, F, and G is more conducive to the continuous synthesis of extracellular polysaccharides during the post-ripening stage compared to groups D and H. This also indicates that the combination ratio of groups E, F, and G is more suitable for the storage of fermented milk than groups D and H.

[0045] The reason for the low viscosity of Group I (initial value only 1120 mPa·s) may be related to its Figure 9 The high inoculum ratios of IMAU20428 and IMAU20427 are related to this. Studies have shown that as the acidity of fermented lactic acid increases, its viscosity decreases. Group I L. Figure 11 The high inoculation ratio of IMAU20428 and IMAU20427 resulted in a high degree of acidification in the fermented milk of Group I, which affected the viscosity of the fermented milk and kept the viscosity of Group I at a low level.

[0046] Changes in water-holding capacity during storage of 9 groups of fermented milk are as follows: Figure 12 As shown, group H exhibited the highest water-holding capacity during 21 days of storage. p <0.05), and the water-holding capacity showed strong stability during storage, increasing from 63.4% to 68.6% (peak) on day 14, and remaining at 64.3% on day 21, with a small decrease. Although the initial water-holding capacity of groups G and E was slightly lower than that of group H, they also showed good water-holding capacity stability; the water-holding capacity of group G increased from 61.75% to 64.97% (peak), and decreased to 62.5% on day 21, a decrease of 4.4%, with a small decrease; the water-holding capacity of group E increased from 62.42% to 66.77% (peak), and decreased to 61.8% on day 21, a decrease of 5%, with a small decrease, and the overall water-holding capacity remained at a high level. The reason why groups G, E, and H have strong water-holding stability during storage is due to the compound ratio of Lactobacillus bulgaricus and Streptococcus thermophilus. These three groups of fermented milk construct a denser three-dimensional gel network by regulating the symbiotic metabolism of the strains, promoting the secretion of extracellular polysaccharides and cross-linking of casein micelles, thereby effectively binding water and inhibiting whey separation.

[0047] Furthermore, although Group A's water-holding capacity was lower than that of Groups H, G, and E, its water-holding capacity fluctuated less. In contrast, Group I consistently had the lowest water-holding capacity and exhibited the greatest fluctuation, rising from 56.05% to 60.15% (peak) before falling back to 54.95%. This phenomenon suggests that the strain ratio in Group I may have led to severe deterioration of the fermented milk gel structure during storage.

[0048] Analysis of changes in texture properties of fermented milk during storage Depend on Figure 14 As can be seen from a, compared with the nine groups of compound fermented milk, the group B fermented milk had the highest hardness during storage. p <0.05), and the hardness remained at a high level at the end of storage.

[0049] The consistency changes of the 9 groups of fermented milk are as follows: Figure 15 b. The change in consistency is similar to the change in hardness. The consistency of groups A and B changed little during storage. The consistency of fermented milk in group C reached its peak on day fourteen and was significantly higher than that of the other groups. p <0.05). The hardness and consistency of Group I were significantly lower than those of the other combinations, mainly due to excessive whey separation during fermentation.

[0050] Changes in the cohesiveness of fermented milk, such as Figure 15 c. During the storage period, the cohesiveness of fermented milk in group E was higher, and there was a significant difference compared with other groups on day 14. p <0.05), and the cohesiveness of group E was at a high level within 21 days of storage.

[0051] Nine groups of fermented milk viscosity indices, such as Figure 15 d. Among them, the viscosity index of groups C, F, and G was significantly higher than that of other combinations, and the viscosity index increased significantly during the storage period. The viscosity index of groups A and B peaked on day 14 and then began to decline, with a relatively large decline. Although the viscosity index of group E was not the highest, the change was relatively small within 7-21 days. p >0.05).

[0052] Analysis of changes in free amino acid content in fermented milk The changes in the content of threonine, lysine, proline, methionine, isoleucine, phenylalanine, tryptophan, glutamic acid, and arginine in 9 groups of compound fermented milk and raw milk (J) after 1 day of storage are as follows: Figure 15 As shown in the figure, the contents of phenylalanine, methionine, and tryptophan in the 9 groups of compound fermented milk were not significantly different from those in the original milk after 1 day of storage. p >0.05); the contents of threonine, lysine, proline, isoleucine, arginine, and glutamic acid in the fermented milk of group C were significantly higher than those in other groups. pThe results showed that the combination of C group strains had higher protein hydrolysis ability, which could not only meet the growth demand of lactic acid bacteria, but also promote the production of free amino in fermented milk.

[0053] Analysis of the changes in the content of organic acids in fermented milk The lactic acid content of E, F, and G groups was significantly higher than that of other groups after 1 day of ripening, and the lactic acid content of E group was the highest, indicating that the combination of E group strains had strong lactic acid synthesis ability. The lactic acid content of G group was significantly higher than that of other groups after 1 day of ripening p <0.05), and the pH value of G group changed more after 1 day of ripening than at the end of fermentation. The lactic acid content of D group was the lowest among the 9 groups p <0.05), which was consistent with the change in pH value during storage, which may also be related to the low degree of post-acidification in D group. D group had the highest acetic acid content; the tartaric acid content in the 9 groups of fermented milk samples was not significantly different p > 0.05), indicating that changing the combination of strains had little effect on the content of tartaric acid in fermented milk. The organic acid content of I group was higher after 1 day of storage, which may be one of the reasons for the high degree of post-acidification in I group at the later stage of storage.

[0054] Comparison of 9 groups of combination fermented milk with commercial products and commercial starter fermented milk The texture properties and viscosity of 9 groups of combination fermented milk stored for 7 days were compared with 7 groups of commercial fermented milk (S1-S7) and 6 groups of commercial starter fermented milk (SY1-SY6) using cluster analysis. The texture properties and viscosity of the 7 groups of commercial fermented milk and the 6 groups of commercial starter fermented milk are shown in Table 4.

[0055] Table 4 Texture properties and viscosity of commercial products and commercial starter fermented milk Note: The selected brands of commercial fermented milk are: S1 and S5 from Illy Group, S2 from Junlebao Dairy, S3 from Inner Mongolia Langge Dairy, S4 from Mengniu Group, S6 from Beijing and Run Dairy Products Factory, and S7 from Puxing Dairy; The selected strains of commercial starter are: SY1: YO-MIX 885-B LYO (DANISCO), SY2: YO-MIX T12 LYO (CHR-HANSEN), SY3: TS-H 2322 (CHR-HANSEN), SY4: YOFlex® Premium 5.0 (CHR-HANSEN), SY5: YO-MIX 883 LYO (DANISCO), and SY6: YO-MIX PRIME 990 LYO (DANISCO).

[0056] Clustering results showed that variations in the blending ratios of Lactobacillus bulgaricus IMAU20428, IMAU20427 and Streptococcus thermophilus IMAU20229, IMAU10630 could match the quality characteristics of different commercially available products, as well as the functional enhancement and process standardization of commercial fermentation agents from patented strains.

[0057] The clustering of 9 compound ratio combinations with commercially available fermented milk and commercially prepared fermented milk is as follows: Figure 16 As shown, groups A and B clustered with commercially available fermented milk S2 and S3 first; group I clustered with commercially available fermented milk S4, S5, S6, and S7; and finally, these groups clustered with group C. Groups D, E, F, G, and H clustered with commercially available fermented milk S1, and then with SY3, SY4, and SY6, indicating that these five groups of fermented milk have a high degree of similarity to commercially available fermented milk S1 in terms of viscosity and texture characteristics. This reflects that, in terms of quality characteristics, these blended fermented milks have similar market positioning and consumer taste expectations to S1. Commercially available starter culture fermented milks SY1, SY2, and SY5 clustered together and did not cluster with any blended fermented milk combinations. This indicates that SY1, SY2, and SY5 have unique product characteristics, which are significantly different from the nine blended fermented milk combinations in this study. Cluster analysis revealed that different groups of compound fermented milk formulations clustered with various commercially available products and fermented milks using commercial starter cultures, forming three main branches. Group I clustered with S4, S5, S6, and S7, with a viscosity range of 1000-1500 mPa·s. Groups A, B, and C clustered with S2 and S3, with a viscosity range of 1500-2500 mPa·s. Groups D, E, F, G, and H clustered with S1, SY3, SY4, and SY6, with a viscosity range of 2500-3500 mPa·s. This demonstrates that adjusting the ratio of Lactobacillus bulgaricus IMAU20428 and IMAU20427 to Streptococcus thermophilus IMAU20229 and IMAU10630 can enhance the diversity of fermented milk characteristics, achieving quality features similar to commercially available fermented milk. This caters to the diverse taste preferences of consumers.

[0058] Cluster analysis of various indicators during fermentation and storage of fermented milk Cluster analysis was performed on the viscosity, textural properties, and fermentation time of nine groups of fermented milk. The results are as follows: Figure 17 As shown, the horizontal axis represents the distance metric; a larger value indicates a greater distance between samples and a lower similarity.

[0059] Analysis revealed a high degree of similarity between fermented milk samples A and B / C. Groups D and H were initially merged, and subsequently clustered with group F, indicating similarities among groups F, D, and H. The clustering tree diagram showed that groups E and G exhibited varying degrees of similarity to groups D, F, H, A, B, and C, but were more similar to groups D, F, and H. Group I clustered separately and was located at a relatively large distance, indicating that it was furthest from all other samples and exhibited the greatest difference. Combining experimental data from fermentation and storage, the viscosity range of groups A, B, and C was between 100-2500 mPa·s. Group A had the shortest fermentation time (4.25 h), maintained the highest viable cell count during storage, had an appropriate inoculation ratio promoting bacterial symbiosis, and exhibited minimal changes in water-holding capacity; therefore, group A was selected for further research. The viscosity range of groups E, G, D, F, and H was between 2500-4500 mPa·s, with group E showing a significant advantage due to its short fermentation time (4.75 h). Group I had a viscosity range of 500-1500 mPa·s. During fermentation and storage, all indicators were poor, the fermentation time was long, the water holding capacity was the lowest and the variation was large during storage, and the hardness and consistency were significantly lower than other combinations. The fermentation parameters of the compound fermentation agents in Group I provide a key control for revealing the effect of strain ratio on fermented milk.

[0060] Metabolomics quality control analysis PCA analysis was performed on fermented milk samples from groups A vs I and E vs I that had been stored for 1 day. The results are as follows: Figure 18 As shown in the figure. The horizontal axis PC1 represents the first principal component, and the vertical axis PC2 represents the second principal component. PCA analysis uses several principal components to reflect the characteristics of multidimensional metabolomics data. The PCA plot can be used to observe the differences between different groups. The PCA plot shows that the scatter plots corresponding to the fermented milk samples in groups A vsI and E vsI exhibit clustering within each group, indicating good reproducibility and similar sample data. Specific analysis results are as follows: Group A and Group I are far apart, showing a tendency to separate on the first principal component but not on the second principal component. This indicates a significant difference in their differential metabolites. It also shows that different compounding ratios cause significant changes in metabolites, consistent with phenotypes (physicochemical indicators).

[0061] The samples in group E and group I are close together, and there is overlap in the first and second principal components, indicating that the difference metabolites between the two groups are highly similar and have similar metabolites.

[0062] Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) for Fermented Milk OPLS-DA was used to analyze fermented milk samples from groups A vs I and E vs I, and score charts were plotted. ​ a, ​c), OPLS-DA score plot showed that there was a clear separation between different comparison groups. ​ b, ​ d is the OPLS-DA validation plot, where Q 2 0.753 and 0.779, respectively, both greater than 0.5, indicating that the model has strong fitting and predictability.

[0063] Screening and analysis of differential metabolites The metabolites of the fermented milk samples of the A vs I group and the E vs I group at the initial storage stage (1d) were tested. The volcano plot of the differential metabolites of the A group vs the I group and the E group vs the I group is shown in ​ .

[0064] The content of essential amino acids such as leucine, phenylalanine, and tryptophan in the fermented milk of the A group was significantly increased, which also indicated that the fermented milk of the A group could provide a rich and high-quality source of essential amino acids for the human body. By comparing the relative inoculum of Streptococcus thermophilus in the A group and the I group, it was found that the inoculum of Streptococcus thermophilus in the A group was significantly higher than that in the I group, which might be one of the reasons for the up-regulation of aspartic acid and glutamic acid in the A group compared with the I group.

[0065] KEGG enrichment analysis of differential metabolites The differential metabolites between groups were analyzed by KEGG database for enrichment pathway, and the metabolic pathways significantly enriched by KEGG in the A group and the I group are shown in ​ , mainly including amino acid biosynthesis, phenylalanine metabolism, phenylalanine, protein digestion and absorption, tyrosine and tryptophan biosynthesis, etc.

[0066] The metabolic pathways significantly enriched by KEGG in the E group and the I group are shown in ​ , mainly including phenylalanine metabolism, ornithine, lysine biosynthesis, D-amino acid metabolism, aminoacyl-tRNA biosynthesis, riboflavin metabolism, protein digestion and absorption, etc., among which the lysine metabolic pathway can protect the bacterial cell structure and maintain normal physiological functions of the cell.

[0067] In summary, the metabolic pathway analysis of the A group, the E group, and the I group showed that the main metabolic pathways enriched in the A group and the E group compared with the I group were concentrated in the amino acid metabolic pathway and the digestion and absorption of proteins. This result indicated that the amino acid metabolism level of the fermentation agent with the A group and the E group complex ratio was significantly higher than that of the I group.

[0068] CONCLUSION ​In this study, we selected Lactobacillus bulgaricus IMAU20428, IMAU20427 and Streptococcus thermophilus IMAU20229, IMAU10630 with good fermentation characteristics, and studied the effects of different ratios of the strains on fermented milk. The ratios were as follows: A group: 1:1:1:1, B group: 1:10:10:10, C group: 1:100:100:100, D group: 10:1:10:100, E group: 10:10:100:1, F group: 10:100:1:10, G group: 100:1:100:10, H group: 100:10:1:100, and I group: 100:100:10:1. We compared the effects of these ratios with those of 13 commercially available products and commercial starter cultures. We also analyzed the indicators during fermentation and storage to determine the combinations of the three groups with significant phenotypic differences. Finally, we used non-targeted metabolomics to analyze the effects of the ratios on fermented milk. The specific conclusions are as follows: 1. In terms of fermentation characteristics, group A had the shortest fermentation time (4.2 h), and group I had the longest fermentation time (6.2 h). The rheological analysis during fermentation showed that group A reached the gel point in the shortest time. Groups E and F had lower solid-liquid equilibrium values and higher elasticity factors, indicating stronger gel structure. We found that a high proportion of Streptococcus thermophilus IMAU20229 could shorten the fermentation time, and a high proportion of Lactobacillus bulgaricus could exacerbate the post-fermentation acidification of fermented milk.

[0069] 2. The storage stability study showed that different ratios of starter cultures had different effects on the storage characteristics of fermented milk. Group A maintained a high level of viable bacteria during storage. Groups B, C, D, and E showed weak post-acidification characteristics. We found that a high proportion of Lactobacillus bulgaricus IMAU20428 resulted in higher post-acidification during storage. Groups E, G, and H had higher water-holding capacity. Groups A, E, G, and H had small changes in water-holding capacity during storage, indicating good storage stability.

[0070] 3. The 9 groups of compound ratio combinations were clustered with 13 kinds of commercial fermented milk products and commercial starter cultures. The 9 groups of compound ratio combinations showed similar characteristics with different types of products, and were divided into three branches, A, B, and C groups were clustered with commercial fermented milk with viscosity in the range of 1500-2500 mPa·s, indicating that the above compound ratio combinations were suitable for producing fermented milk with medium viscosity. D, E, F, G, and H groups were clustered with commercial fermented milk with viscosity in the range of 2500-3500 mPa·s, indicating that the above compound ratio combinations were suitable for producing fermented milk with high viscosity. Group I was clustered with commercial fermented milk with viscosity in the range of 1000-1500 mPa·s, indicating that this compound ratio combination was suitable for producing fermented milk with low viscosity. It indicated that by adjusting the ratio of Lactobacillus bulgaricus IMAU20428, IMAU20427 and Streptococcus thermophilus IMAU20229, IMAU10630, fermented milk with different product characteristics could be produced. It also indicated that changing the strain ratio of the starter culture would affect the characteristics of the fermented milk. Based on this result, the viscosity, texture characteristics and fermentation time of the 9 groups of fermented milk samples were clustered, and the experimental data during fermentation and storage were analyzed. The viscosity of A, B, and C groups ranged between 100-2500 mPa·s, among which A group had the shortest fermentation time, the highest viable bacterial count during storage, and the smallest change in water holding capacity. Therefore, group A was selected for further study. The viscosity of E, G, D, F, and H groups ranged between 2500-4500 mPa·s, among which E group was significantly dominant, had a short fermentation time, a stable pH change during storage, a low degree of post-acidification, a high water holding capacity, and maintained a high level during the later storage period. It had high viscosity and small changes, so group E was selected for further study. The viscosity of group I ranged between 500-1500 Pa·s, and all indicators during fermentation and storage were poor. It had the longest fermentation time, the lowest water holding capacity during storage, and the hardness and consistency were significantly lower than other combinations. The indicators of the compound ratio starter culture of group I provided a key control for exploring the effect of strain ratio on fermented milk.

[0071] 4. From the detection of fermented milk metabolites, the relative inoculum of Streptococcus thermophilus in group A was significantly higher than that in group I, which may be one of the reasons for the up-regulation of aspartic acid, glutamic acid, and glycol-L-proline in group A compared with group I. The highest proportion of metabolic products in group E compared with group I was amino acids and their metabolites and glycerophospholipids. The change in the charge state of amino acids can cause changes in intermolecular attraction or repulsion. These interactions can increase the stability of protein conformation in the fermented milk system, which can increase the viscosity of fermented milk. This may be one of the reasons why the viscosity of group E is higher than that of group I. The stability of various indicators of group E fermented milk during storage compared with group I may be one of the reasons for the up-regulation of glycerophospholipids in group E compared with group I.

[0072] In conclusion, the effects of different ratios of Lactobacillus bulgaricus IMAU20428 and IMAU20427 and Streptococcus thermophilus IMAU20229 and IMAU10630 on the quality of fermented milk were investigated in this study. The results showed that the fermented milk with different excellent product properties could be produced when the ratio of the mixed starter was 1∶1∶1∶1 and 10∶10∶100∶1, which could provide new ideas for the commercial application and development of mixed starter.

Claims

1. A multi-strain composite fermentation agent with multiple application characteristics, characterized in that, The starter culture includes Lactobacillus bulgaricus IMAU20428, Lactobacillus bulgaricus IMAU20427, Streptococcus thermophilus IMAU20229 and Streptococcus thermophilus IMAU10630; The *Lactobacillus bulgaricus* IMAU20427 is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO:M 2024957; the *Lactobacillus bulgaricus* IMAU20428 is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO:M 2024954; the *Streptococcus thermophilus* IMAU20229 is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO:M 20231266; and the *Streptococcus thermophilus* IMAU10630 is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO:M 2024951.

2. The fermenting agent according to claim 1, characterized in that, The viable count ratio of Lactobacillus bulgaricus IMAU20428, Lactobacillus bulgaricus IMAU20427, Streptococcus thermophilus IMAU20229 and Streptococcus thermophilus IMAU10630 is (1~50):(1~50):(1~50):(1~50).

3. The fermenting agent according to claim 1, characterized in that, The viable count ratio of Lactobacillus bulgaricus IMAU20428, Lactobacillus bulgaricus IMAU20427, Streptococcus thermophilus IMAU20229, and Streptococcus thermophilus IMAU10630 is 5-15:5-15:50-150:0.5-1.

5.

4. A method for preparing fermented milk, characterized in that, This includes inoculating the fermenting agent described in claim 1 or 2 into raw milk and fermenting it to obtain fermented milk.

5. A fermented milk, characterized in that, The fermented milk is prepared using the fermenting agent described in claim 1 or 2.

6. The application of the starter culture of claim 2 in the preparation of fermented milk with fast curd speed, high viable count during storage, and stable viscosity and water-holding capacity.

7. The use of the starter culture of claim 3 in the preparation of fermented milk with strong gel structure and good stability.

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