Composite multi-strain leavening agent for Maillard browning fermented milk

Abstract

The invention belongs to the technical field of microorganisms, and discloses a composite multi-strain leavening agent for Maillard browning fermented milk. According to the invention, two groups of bacterial strain combinations with optimal fermentation performance are screened out, wherein the two groups of bacterial strain combinations are as follows: a group T fermentation agent (Lactobacillus bulgaricus) IMAU95052 + Lactobacillus bulgaricus (Lactobacillus bulgaricus) IMAU40073 + Streptococcus thermophilus IMAU80806 + Streptococcus thermophilus IMAU10630 and a group D fermentation agent (Lactobacillus bulgaricus (Lactobacillus bulgaricus) IMAU-1, and the group D fermentation agent is Lactobacillus bulgaricus (Lactobacillus bulgaricus) IMAU-1. The strain has excellent performance in multiple fermentation characteristics and storage stability indexes, and has the potential of being developed into a special leavening agent for brown fermented milk.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a compound multi-strain fermentation agent for Maillard browning fermented milk. Background Technology

[0002] In recent years, brown fermented milk products have gradually gained popularity in the market due to their unique caramel flavor and appealing color. However, while the Maillard reaction imparts a unique flavor and color to brown fermented milk, it also brings a series of quality problems. Among them, the most prominent is the decline in the texture and other qualities of the fermented milk, which seriously affects the taste and stability of the product and limits the further development of the brown fermented milk industry.

[0003] Maillard browning increases the free amino nitrogen content in milk, shortens fermentation time, and accelerates the formation of milk gel during fermentation, exhibiting high viscoelasticity, low solid-liquid equilibrium value, and fluid gel structure characteristics. Meanwhile, Turbiscan stability analysis shows that the stability of browned fermented milk is worse than that of ordinary fermented milk, and microstructural comparison reveals that the structure of browned fermented milk is looser than that of ordinary fermented milk. During storage, the extracellular polysaccharide content, various textural parameters, and viscosity of Maillard browned fermented milk are generally lower than those of ordinary fermented milk, while the average pH value of browned fermented milk is significantly higher, exhibiting weak post-acidification characteristics. In summary, Maillard browning imparts unique flavor characteristics to fermented milk, shortens fermentation time, and provides weak post-acidification; however, it also affects storage stability.

[0004] Currently, there are relatively few solutions on the market for addressing the quality issues of brown fermented milk, and traditional starter cultures cannot effectively adapt to the complex changes brought about by the Maillard reaction. Developing a dedicated starter culture for brown fermented milk is urgently needed. This study aims to construct a starter culture suitable for brown fermented milk by screening lactic acid bacteria strains with specific metabolic characteristics, thereby mitigating its negative impact on the texture and other quality aspects of fermented milk.

[0005] Furthermore, developing starter cultures suitable for brown fermented milk has significant commercial value and plays a vital role in driving industrial development. For businesses, high-quality starter cultures can improve product quality, enhance market competitiveness, meet consumer demand for high-quality brown fermented milk, thereby expanding market share and increasing economic benefits. From an industry development perspective, the successful development of specialized starter cultures helps standardize the production process of brown fermented milk, promotes the healthy and sustainable development of the entire brown fermented milk industry, and provides new ideas and technical support for innovative development in the food industry. Simultaneously, it also provides reference and guidance for research on other foods with quality problems caused by the Maillard reaction.

[0006] Meanwhile, existing research on fermented milk starter cultures primarily employs a combination of a single Lactobacillus bulgaricus strain and a single Streptococcus thermophilus strain. However, this combination has several drawbacks: First, it results in a monotonous flavor, homogeneous metabolites, and a lack of differentiation in fermented milk products, making it difficult to meet diverse consumer taste demands. Second, the strains have limited functionality, making it difficult to simultaneously possess desirable traits such as rapid acid production, good viscosity production, and phage resistance. This makes them susceptible to fermentation failure and deterioration in texture due to phage contamination, leading to low cost-effectiveness in factory applications. Third, the interspecies interaction is disrupted by phages, prolonging fermentation time and reducing the number of lactic acid bacteria in the final product, thus affecting quality and stability. Summary of the Invention To address the aforementioned issues, including the decline in texture and quality of brown fermented milk and the limited variety of existing starter cultures, this study screened two strain combinations with optimal fermentation performance: Group T starter culture (Lactobacillus bulgaricus (…)). Lactobacillus delbrueckii subsp. bulgaricus IMAU95052+ Lactobacillus bulgaricus ( Lactobacillus delbrueckii subsp. bulgaricus IMAU40073+ thermophilic streptococci ( Streptococcus thermophilus IMAU80806+ Streptococcus thermophilus ( Streptococcus thermophilus IMAU10630 and Group D starter culture (Lactobacillus bulgaricus ( Lactobacillus delbrueckii subsp. bulgaricus IMAU95052+ Lactobacillus bulgaricus ( Lactobacillus delbrueckii subsp. bulgaricus IMAU40073+ thermophilic streptococci ( Streptococcus thermophilus IMAU80806+ Streptococcus thermophilus ( Streptococcus thermophilus IMAU20326 exhibits excellent performance in multiple fermentation characteristics and storage stability indicators, demonstrating the potential to be developed into a dedicated starter culture for brown fermented milk. Specifically, the T-group starter culture produces more volatile flavor compounds during storage, and the resulting fermented milk has a high content of extracellular polysaccharides, good stability, and relatively minimal negative impact from the Maillard reaction on its various quality indicators. The D-group starter culture produces brown fermented milk with a higher total amount of volatile flavor compounds, and some Maillard reaction products indicate a strong ability of its lactic acid bacteria to utilize substrates to produce flavor compounds. Therefore, the T-group starter culture can be applied to the development of starter cultures for brown fermented milk products that emphasize flavor preservation and product stability, especially suitable for long-shelf-life products, maintaining good flavor and stable texture over extended storage periods. The D-group starter culture, on the other hand, is more suitable for brown fermented milk products that emphasize unique flavor and maximize flavor release in a short time, such as short-shelf-life or freshly prepared brown fermented milk, fully leveraging its advantage of producing a large amount of flavor compounds in the early stages.

[0007] On one hand, the present invention provides a fermentation agent comprising *Lactobacillus bulgaricus* IMAU95052, *Lactobacillus bulgaricus* IMAU40073, *Streptococcus thermophilus* IMAU80806, and *Streptococcus thermophilus* IMAU10630. On the other hand, the present invention provides a fermentation agent comprising *Lactobacillus bulgaricus* IMAU95052, *Lactobacillus bulgaricus* IMAU40073, *Streptococcus thermophilus* IMAU80806, and *Streptococcus thermophilus* IMAU20326.

[0008] Further, the live count ratio of Lactobacillus bulgaricus IMAU95052, Lactobacillus bulgaricus IMAU40073, Streptococcus thermophilus IMAU80806, and Streptococcus thermophilus IMAU10630 is (0.1~10):(0.1~10):(10~1000):(10~1000); preferably (0.2~5):(0.2~5):(20~500):(20~500); preferably (0.3~3):(0.3~3):(50~300):(50~300); preferably (0.5~2):(0.5~2):(50~200):(50~200); preferably (0.8~1.5):(0.8~1.5):(80~150):(80~150); most preferably 1:1:100:100.

[0009] Furthermore, the viable count of IMAU95052, Lactobacillus bulgaricus IMAU40073, Streptococcus thermophilus IMAU80806, and / or Streptococcus thermophilus IMAU10630 is 1×10⁻⁶. 2 -1×10 10 ; 1×10 3 ; 1×10 4 ; 1×10 5 ; 1×10 6 ; 1×10 7 ; 1×10 8 ; 1×10 9 CFU / g.

[0010] Further, the live bacteria ratio of Lactobacillus bulgaricus IMAU95052, Lactobacillus bulgaricus IMAU40073, Streptococcus thermophilus IMAU80806, and Streptococcus thermophilus IMAU20326 is (0.1~10):(0.1~10):(10~1000):(10~1000); preferably (0.2~5):(0.2~5):(20~500):(20~500); preferably (0.3~3):(0.3~3):(50~300):(50~300); preferably (0.5~2):(0.5~2):(50~200):(50~200); preferably (0.8~1.5):(0.8~1.5):(80~150):(80~150); most preferably 1:1:100:100.

[0011] Furthermore, the viable count of *Lactobacillus bulgaricus* IMAU95052, *Lactobacillus bulgaricus* IMAU40073, *Streptococcus thermophilus* IMAU80806, and / or *Streptococcus thermophilus* IMAU20326 is 1 × 10⁻⁶. 2 -1×10 10 ; 1×10 3 ; 1×10 4 ; 1×10 5 ; 1×10 6 ; 1×10 7 ; 1×10 8 ; 1×10 9 CFU / g.

[0012] On the other hand, the present invention provides a method for preparing brown fermented milk, the method comprising inoculating the above-mentioned starter culture into raw milk for fermentation to obtain brown fermented milk.

[0013] Furthermore, the brown fermented milk category includes brown fermented milk beverage products developed based on this.

[0014] Thirdly, the present invention also provides a brown fermented milk, which is obtained by fermentation using the above-mentioned fermenting agent.

[0015] Furthermore, the brown fermented milk category includes various types of products, including but not limited to set-type and stirred-type.

[0016] On the other hand, the present invention provides the application of the above-mentioned fermenting agent in the preparation of brown fermented milk.

[0017] Compared with the prior art, the present invention has the following technical advantages: (1) The Maillard reaction that occurs during prolonged heating of milk significantly affects the properties of fermented milk. Brown fermented milk takes longer to form a gel but has greater strength, and its pH and TA values ​​are significantly higher and lower than those of ordinary fermented milk, respectively, during storage.p <0.05%, which is presumably related to lactose consumption and buffer production during the Maillard reaction; in addition, affected by the Maillard reaction, the viscosity, water-holding capacity, viable cell count, and extracellular polysaccharide content of browned fermented milk were all significantly reduced ( p <0.05). The T-group starter culture selected in this invention significantly reduced the negative impact of the Maillard reaction on the textural properties and storage stability of fermented milk. Differences existed in the volatile flavor compounds and metabolites produced by different starter culture combinations. The HD-group fermented milk was the richest in volatile flavor compounds, and its Maillard reaction products were significantly higher than those of the HT-group, indicating that the D-group starter culture strains had a stronger ability to utilize substrates to produce flavor compounds. Fermented milk prepared using the T-group consistently produced more volatile flavor compounds at the end of storage. Metabolomics analysis of differential metabolites and related metabolic pathways revealed that the BT-group was enriched with more metabolites related to glycolysis pathways associated with extracellular polysaccharide synthesis than the BD-group. This may explain why the extracellular polysaccharide content of fermented milk prepared with the T-group starter culture was significantly higher than that prepared with the D-group starter culture during storage. During storage, the viable cell count of the HT-group fermented milk was generally higher than that of the HD-group, a phenomenon that can be explained by the significant upregulation of α-ketoglutarate and acetoacetic acid. In the HT and HD groups, α-ketoglutarate and acetoacetic acid were significantly upregulated in the degradation pathways of valine, leucine, and isoleucine, as well as in the histidine metabolic pathway.

[0018] (2) Due to the specificity of the strains, compared with a single combination of Lactobacillus bulgaricus and Streptococcus thermophilus, the combination of strains has a richer metabolic network and functional complementarity. Different strains can synergistically produce a variety of extracellular polysaccharides, esters, and aldehydes and ketones, making the fermented milk aroma more three-dimensional and the taste smoother. At the same time, the multi-strain system occupies different ecological niches through quorum sensing and secretes broad-spectrum antimicrobial peptides, which can significantly inhibit phage proliferation and reduce the risk of fermentation failure. In addition, the complementary proteolytic enzyme system and acid and oxygen resistance among the strains can shorten the fermentation cycle, increase the number of viable bacteria, and endow the product with potential probiotic functions such as regulating intestinal flora and enhancing immunity, providing a new path for developing highly stable and differentiated fermented dairy products. Attached Figure Description

[0019] Figure 1 The microrheological changes during the fermentation of four groups of fermented milk are shown; Note: HT is brown fermented milk fermented with starter culture T; HD is brown fermented milk fermented with starter culture D; BT is ordinary fermented milk fermented with starter culture T; BD is ordinary fermented milk fermented with starter culture D. Figure 1 a is the elasticity factor. Figure 1 b is the solid-liquid equilibrium value. Figure 1 c is the viscosity factor. Figure 1 d is the flow factor.

[0020] Figure 2The pH values ​​of the four groups of fermented milk during storage ( Figure 2 a) and TA ( Figure 2 The change in b); Figure 3 The changes in viscosity of the four groups of fermented milk during storage; Figure 4 The changes in water-holding capacity of the four groups of fermented milk during storage; Figure 5 The changes in viable bacterial counts during storage of four groups of fermented milk; Figure 5 (a) represents the viable count of Streptococcus thermophilus. Figure 5 (b) is the number of viable Lactobacillus bulgaricus bacteria; Figure 6 The changes in EPS content during the storage of four groups of fermented milk; Figure 7 Backscattered light during the fermentation process of fermented milk; Figure 7 (a) is the HT backscattered light. Figure 7 (b) is the BD backscattered light. Figure 7 (c) represents the BT backscattered light. Figure 7 (d) represents HD backscattered light; Figure 8 The stability index of the fermentation process of fermented milk; Figure 9 The change in organic acid content of fermented milk before and after fermentation; Figure 9 (a) shows the change in malic acid content. Figure 9 (b) shows the change in itaconic acid content. Figure 9 (c) shows the change in lactic acid content. Figure 9 (d) shows the change in vanillic acid content. Figure 9 (e) shows the change in salicylic acid content. Figure 9 (f) shows the change in tartaric acid content. Figure 9 (g) Changes in adipic acid content Figure 9 (h) represents the change in 4-OH phenyllactic acid content. Figure 9 (i) shows the change in gibberellin content. Figure 9 (j) represents the change in acrylic acid content. Figure 9 (k) represents the change in oxalic acid content. Figure 9 (l) represents the change in acetic acid content; Figure 10 The change in amino acid content of fermented milk before and after fermentation; Figure 10 (a) shows the change in glutamine content. Figure 10 (b) shows the change in arginine content. Figure 10 (c) shows the change in asparagine content. Figure 10 (d) shows the change in aspartic acid content. Figure 10 (e) shows the change in cystine content. Figure 10(f) shows the change in DL-2 amino adipic acid content. Figure 10 (g) Changes in glutamic acid content Figure 10 (h) represents the change in glycine content. Figure 10 (i) represents the change in histidine content. Figure 10 (j) represents the change in isoleucine content. Figure 10 (k) represents the change in lysine content. Figure 10 (l) represents the change in methionine content. Figure 10 (m) represents the change in phenylalanine content. Figure 10 (n) represents the change in serine content. Figure 10 (o) represents the change in threonine content. Figure 10 (p) represents the change in tryptophan content. Figure 10 (q) represents the change in tyrosine content. Figure 10 (r) represents the change in valine content; Figure 11 Continuous pH determination for fermentation of single strains of Streptococcus thermophilus IMAU80806, Streptococcus thermophilus IMAU10630, Lactobacillus bulgaricus IMAU40073, and Lactobacillus bulgaricus IMAU95052; Figure 12 The images are scanning electron microscope (SEM) images of fermented milk; a) BT group (ordinary fermented milk fermented with starter culture T); b) HT group (brown fermented milk fermented with starter culture T); c) BD group (ordinary fermented milk fermented with starter culture D); d) HD group (brown fermented milk fermented with starter culture D).

[0021] Strain Preservation Information The classification name of Lactobacillus bulgaricus IMAU95052 is... Lactobacillus delbrueckii subsp. bu lgaricus IMAU95052 was deposited at the China Center for Type Culture Collection on May 22, 2024, with accession number CCTCC NO:M 2024955.

[0022] The classification name of Lactobacillus bulgaricus IMAU40073 is... Lactobacillus delbrueckii subsp. bu lgaricus IMAU40073 was deposited at the China Center for Type Culture Collection on May 22, 2024, with accession number CCTCC NO:M 2024958.

[0023] The classification name of Lactobacillus bulgaricus IMAU20427 is... Lactobacillus delbrueckii subsp. bu lgaricusIMAU20427 was deposited at the China Center for Type Culture Collection on May 22, 2024, with accession number CCTCC NO:M 2024957.

[0024] The classification and naming of Streptococcus thermophilus IMAU80806 Streptococcus thermophilus IMAU80806 was deposited at the China Center for Type Culture Collection on May 22, 2024, with accession number CCTCC NO:M 2024959.

[0025] The classification and naming of Streptococcus thermophilus IMAU10630 Streptococcus thermophilus IMAU10630 was deposited at the China Center for Type Culture Collection on May 22, 2024, with accession number CCTCC NO:M 2024951.

[0026] The classification and naming of Streptococcus thermophilus IMAU20326 Streptococcus thermophilus IMAU20326 was deposited at the China Center for Type Culture Collection on May 22, 2024, with accession number CCTCC NO:M 2024953. Detailed Implementation

[0027] Example Materials and Methods The strain information of the lactic acid bacteria strains used in this experiment from the Inner Mongolia Agricultural University Lactic Acid Bacteria Strain Resource Bank is shown in Table 1.

[0028] Table 1 Information on tested strains Experimental methods Strain activation The bacterial strains preserved in the ampoules of the bacterial bank were revitalized using the following procedure: Ampoules were placed in a sterile operating table, and bacterial cells were streaked onto the surface of a solid culture medium using an inoculation loop to activate the strain. After colony formation, a single colony was transferred to 10 mL of liquid culture medium and cultured under suitable conditions. To ensure bacterial purity, samples were taken for microscopic examination after each subculture. The third generation of bacterial culture was obtained after two consecutive generations. The culture was then centrifuged (4000 r / min, 10 min, 4℃), the culture medium was discarded, and the cells were washed with PBS buffer (Na₂HPO₄-1.15 g / L, KH₂PO₄-0.2 g / L, NaCl-8 g / L) and stored at 4℃ for later use.

[0029] Preparation of fermented milk samples This study employed two different processing methods to prepare fermented dairy products. For the preparation of ordinary fermented milk, the milk was first precisely weighed according to a ratio of 93.5% pure milk to 6.5% sucrose. The milk was preheated to 65°C, and then sucrose was added under water bath conditions with continuous stirring for 15 minutes until completely dissolved. The dissolved mixture was homogenized under high pressure at 20 MPa, followed by pasteurization at 95°C for 5 minutes, and then rapidly cooled to 42-45°C. Fermentation was then carried out under aseptic conditions with the inoculum amount shown in Table 4. For the preparation of brown fermented milk, pure milk was first heated in a 96-98°C water bath for 3 hours to induce browning. Subsequent homogenization, sterilization, and cooling steps were the same as for ordinary fermented milk. Both types of fermented milk were fermented at 42°C until the pH dropped to 4.6, then immediately cooled in an ice-water bath, and demulsification and packaging were completed under aseptic conditions. The prepared samples were stored at 4°C and sampled and tested according to the experimental design time points.

[0030] Table 4. Inoculum amounts for different experimental combinations Note: LB1: IMAU95052; LB2: XI82-2-1; LB3: MGC17-4; ST1: IMAU80806; ST2: IMAU10630; ST3: IMAU20326.

[0031] Methods for studying the fermentation characteristics and storage stability of fermented milk Measurements included pH, titratable acidity (TA), viscosity, water-holding capacity, viable cell count, microrheological properties, textural properties, extracellular polysaccharide (EPS) content, Turbiscan stability analysis during fermentation, and microstructure observation of fermented milk.

[0032] Determination of organic acids and amino acids in fermented milk Sample pretreatment and sample testing.

[0033] Determination of volatile flavor compounds in fermented milk Sample extraction process and chromatographic and mass spectrometric acquisition conditions.

[0034] Determination of non-targeted metabolites in fermented milk Extraction and instrumental analysis of metabolites.

[0035] Data Analysis In the above experimental results, the microrheological results were analyzed using the Rheolaser Master optical microrheological method, and the microrheological data were processed using the Smart software installed in the instrument. For all fermented milk indicators, three or more parallel sets of data were obtained. After summarizing using Office Excel, one-way ANOVA was performed using IBM SPSS Statistics 27 (significance level set to 0.05), and plotting was done using Origin Pro 2021. The raw data obtained after determining the metabolites in the fermented milk were converted to mzXML format using the Proteo Wizard tutorial and then identified as metabolites through the HMDB database. Differential metabolites were screened based on the t-test p-value < 0.05 and the chi-square value criterion of Variable Importance in the Projection (VIP) > 1. Metabolic pathway enrichment analysis was then performed using the KEGG PATHWAY database, and finally, visualization analysis was performed using R language.

[0036] Results Analysis and Discussion Screening of 2LB-2ST combination Based on the fermentation characteristics of previous strains and the prediction of multi-strain interactions using artificial intelligence, three strains of Lactobacillus bulgaricus and three strains of Streptococcus thermophilus were screened, as shown in Table 5. Table 5. Strain combination groups and screening Note: LB1: IMAU95052; LB2: XI82-2-1; LB3: MGC17-4; ST1: IMAU80806; ST2: IMAU10630; ST3: IMAU20326.

[0037] Based on the above determination of the viscosity and water-holding capacity of fermented milk, combination 1 and combination 2 were selected as two excellent starter cultures. Combination 1 was named group T starter culture and combination 2 group D starter culture, and the same applies below.

[0038] Methods for studying the fermentation characteristics and storage stability of fermented milk Microrheological properties analysis during fermentation During fermentation, the decrease in pH leads to protein denaturation and aggregation, and rearrangement of casein micelles, resulting in a gelled network. An optical microrheometer can detect the rheological properties of fermented milk samples by observing particle motion trajectories at a microscopic level during fermentation. Specifically, it reflects the material structure of the fermented milk sample during fermentation by examining four parameters: elasticity factor, solid-liquid equilibrium value, viscosity factor, and flow factor. The results are as follows: Figure 1 As shown.

[0039] like Figure 1As shown in Figure a, in the early stage of fermentation, the fermented milk did not form a gel structure, and the EI values ​​of the four groups of samples remained stable. As fermentation time progressed, the EI values ​​of the four groups of fermented milk increased significantly. The EI value trends of the ordinary fermentation groups (BD, BT) were nearly identical, indicating that the gel structure formation time of the two types of fermented milk tended to be consistent, both forming a gel structure around 4 hours. The browning fermentation groups (HD, HT) showed significant differences in the time point of gel structure formation, with the HT group forming a gel structure significantly earlier than the HD group. The HT group began to form a gel structure around 5 hours and reached a stable state around 6 hours, while the HD group formed a gel structure around 6 hours and reached a stable state around 8 hours, tending to be at the same level. Overall, the browning fermentation group formed a gel later than the ordinary fermentation group, and the gel strength formed was greater than that of the ordinary fermented milk. Studies by Yang Shujuan et al. found that within a certain range, fermented milk samples with higher protein content had lower corresponding EI values. Most Maillard intermediates formed in dairy products are bound to milk proteins; therefore, it is speculated that the high-temperature process of the Maillard reaction affects protein structure, thereby altering the EI value of microrheology.

[0040] Solid-liquid equilibrium (SLB) directly reflects the degree to which a product is more solid or liquid over time, and can be expressed as a function of SLB over time. When SLB is between 0 and 0.5, the product exhibits an elastic modulus, leaning more towards solid properties; while when SLB is between 0.5 and 1, the product exhibits a viscous modulus, leaning more towards liquid properties. Because micellar-bound whey protein aggregates modify the micelle surface, as the isoelectric point of casein approaches, the repulsive forces between casein particles decrease, hydrophobic interactions increase, and rapid aggregation leads to extensive rearrangement within the protein network. The figure shows the changes in SLB during fermentation. In the early stages of fermentation, the SLB of the fermented milk sample fluctuates, possibly due to changes in microbial activity, substrate utilization, or other factors. As fermentation progresses, lactic acid bacteria continuously produce acid, and the SLB value no longer fluctuates but slowly increases, with its elastic modulus leaning towards solid. At this point, casein aggregates, the viscosity of the fermented milk system increases, and the sample reaches its gel point. The groups reaching the gel point fastest, from fastest to slowest, were BT, BD, HT, and HD, stabilizing at 4 hours, 4 hours, 5 hours, and 7 hours, respectively. When the SLB (solubilization volume) approached equilibrium, the SLB values ​​of the fermented milk in groups BT, BD, and HD were all greater than 0.5, while the lowest SLB value was in group HT at 0.49, indicating that the fermented milk in group HT tended to be solid. Furthermore, the order of reaching the curd point shown in the SLB changes was consistent with that presented by EI (Extracorporeal Intake).

[0041] Viscosity factor (MVI), as a function of time, reflects changes in the viscosity of fermented milk. During fermentation, the MVI changes as follows: Figure 1 As shown in c, in the early stage of fermentation, the MVI values ​​of each group were around 10. -6 -10-4 The viscosity fluctuated during this period because the gel network structure had not yet formed, resulting in an initial stagnant phase with low viscosity. The viscosity factor of the ordinary fermented milk sample increased significantly after 4 hours and reached an inflection point first. The HT of the brown fermented milk then reached an inflection point at 5 hours, and finally the HD of the brown fermented milk reached an inflection point at 6 hours. The MVI values ​​of ordinary and brown fermented milk tended to be at the same level, with the brown fermented milk showing a higher value than the ordinary fermented milk, a phenomenon consistent with the EI value plot above. This indicates that, under the microstructure, the viscosity of brown fermented milk is higher than that of ordinary fermented milk from the same source.

[0042] Changes in flow factor reflect the velocity of microscopic particles in a fermented milk sample. A high flow factor (approximately 10 Hz) indicates that the sample exhibits liquid properties, while a low flow factor (approximately 10 Hz) indicates a liquid state. -2 A FI value (Hz) indicates that the sample exhibits solid-state properties. A higher FI value indicates faster particle velocity, and therefore greater sample fluidity. For example... Figure 1 As shown in d, in the early stage of fermentation, the flow factor (FI) value of ordinary fermented milk is much higher than that of browned fermented milk. The FI values ​​of both groups fluctuate, indicating they are liquids. The flow factors of ordinary fermented milk in groups BD and BT drop sharply around 4 hours, reaching an inflection point around 5 hours, and then tend to stabilize. The flow factors of groups HT and HD start to drop sharply at 5 hours and 7 hours, respectively, and tend to stabilize at 7 hours and 8 hours, respectively. The flow factor of group HT is the lowest, indicating that it moves slowly, has weak fluidity, and high viscosity, which corresponds to the MVI mentioned above.

[0043] In summary, there are certain differences in the rheological properties between ordinary fermented milk and brown fermented milk. The microstructures of the two groups of ordinary fermented milk are similar, while the HT group in brown fermented milk shows an earlier appearance of gel structure, tends to be solid, and has higher viscosity. Analysis of acidity changes during storage The acidity changes of fermented milk during storage have a significant impact on its flavor and determine its shelf life. The storage pH of commercially available fermented milk is typically between 3.9 and 4.2. Acidity gives fermented milk its unique flavor, distinguishing it from regular milk. Acidity is an important indicator of the strength of the acidity and the overall condition of the fermented milk. Excessive acidity makes it unsuitable for consumption and often leads to a decline in quality later in storage; conversely, insufficient acidity results in a lack of flavor and inadequate fermentation.

[0044] During storage, the lactic acid bacteria in fermented milk continue to grow and metabolize at a slow rate under low temperatures, producing lactic acid and its metabolites. The acidity value gradually decreases with prolonged storage; the lower the pH, the higher the TA value. The pH and TA changes of the four groups of fermented milk during storage are shown above. Figure 2 As shown.

[0045] like Figure 2a. The pH level decreased uniformly over 21 days, and the pH level of all fermented milk groups decreased significantly during the storage period. p <0.05), the pH of brown fermented milk was significantly higher than that of the corresponding ordinary fermented milk, exhibiting a characteristic of weak post-acidification. Among them, the pH of the HD group after browning was on average 3%-4% higher than that of the BD group. The pH of the HT group after browning was on average about 1% higher than that of the control group BT group. The pH of the HD group was significantly higher than that of the HT group during storage.

[0046] like Figure 2 b, The level of TA indicates the amount of 0.1 mol / L NaOH standard solution consumed per unit sample. (From...) Figure 4 b. The TA values ​​of the four fermented milk groups showed a linear upward trend from 1 to 21 days, with all TA values ​​below 100 °T at day 21. The rate of change was uniform, without any sudden spikes. The BT and HT groups remained essentially parallel, while the difference between the HD and BD groups was larger than that between the HT and BT groups. Figure 2 The pH phenomenon is consistent with that of a.

[0047] In summary, assuming no changes in starter culture, fermentation conditions, etc., there were significant differences in pH and TA before and after browning of the same fermented milk. The pH of brown fermented milk was significantly higher than that of (…). p <0.05) The TA value of ordinary fermented milk stored at the same time was significantly lower than that of ( p <0.05) This may be due to the large-scale consumption of lactose as a reactant in the Maillard reaction, reducing the substrate for lactic acid bacteria fermentation and leading to a decrease in lactic acid production and a less significant pH drop. Another possible reason is the production of buffering substances in the Maillard reaction. Arginine is the main amino acid produced in the Maillard reaction, and its production rate is highest at pH=8. Arginine is absorbed by cells within the biofilm, easily metabolized, and improves pH homeostasis by altering the microbial ecology, making it a significant contributor to pH restoration and neutralization. Therefore, it is speculated that arginine in brown fermented milk neutralizes pH by altering the microbial ecology, resulting in a generally higher pH in browned fermented milk compared to ordinary fermented milk. For the two starter cultures studied in this experiment, the difference in pH and TA in group D fermented milk was greater after browning, and browned fermented milk generally exhibited a weak post-acidification characteristic.

[0048] Analysis of viscosity changes during storage Viscosity is an important indicator for evaluating the quality of fermented milk, and it plays a significant role in the evaluation of taste. The viscosity changes of the four groups of fermented milk during the storage period are as follows: Figure 3 As shown.

[0049] Depend on Figure 3It can be seen that the viscosity of group BD was significantly higher than that of brown fermented milk HD made with the same starter culture during the storage period. The highest viscosity values ​​for both groups occurred on the first day, at 2379 mPa·s and 1174 mPa·s, respectively, and decreased by 102%, 59%, 90%, and 92% during the storage period, respectively. Groups BT and HT showed better viscosity characteristics during the storage period, and were significantly higher than those of groups BD and BT during the same period. p <0.05), the viscosity of ordinary fermented milk in both groups was significantly higher than that of brown fermented milk at 1, 7, and 14 days. p <0.05), with no significant difference at 21 days, decreasing by 22%, 23%, 18%, and 0%, respectively. The highest viscosity was observed on day 1, at 3964 mPa·s and 3244 mPa·s, respectively. In conclusion, the Maillard reaction reduced the viscosity of fermented milk, a trend that was more pronounced between the BT and HT groups, with viscosity differences within 25%.

[0050] Analysis of changes in water-holding capacity during storage period Changes in water-holding capacity during storage can be used to assess the stability of the fermented milk's tissue state and characterize the degree of whey separation. The changes in the four groups of samples during storage are as follows: Figure 4 As shown.

[0051] As shown in the figure, the water-holding capacity of the samples fluctuated within the range of 60%-65%, and the overall trend, similar to the viscosity changes, showed that the water-holding capacity of brown fermented milk made with the same starter culture was lower than that of ordinary fermented milk. The water-holding capacity remained stable or fluctuated from day 1 to 14, decreased after day 14, and was lowest at day 21. This indicates that whey separation occurred significantly in the last 7 days of storage, leading to a substantial decrease in water-holding capacity. The water-holding capacity of the HT group decreased by 6%, 5%, 4%, and 5% respectively at the four time points compared to ordinary fermented milk (BT) made with the same starter culture, while the water-holding capacity of the HD group decreased by 4%, 8%, 7%, and 7% respectively at the four time points compared to ordinary fermented milk (BD) made with the same starter culture. Based on the above analysis, it was found that the Maillard reaction significantly reduces the water-holding capacity of fermented milk. p <0.05), which may be related to its viscosity. The water-holding capacity of fermented milk in group D decreased more significantly after the Maillard reaction, while that of fermented milk in group T decreased more gradually.

[0052] Analysis of changes in viable bacterial count during storage period The number of viable bacteria in fermented milk is one of the important indicators for measuring product quality and functionality. It reflects the growth of lactic acid bacteria during the storage of fermented milk, and the number of lactic acid bacteria determines the flavor, texture, and other characteristics of fermented milk. Figure 5As shown in Figure a, the viable count of Streptococcus thermophilus in brown fermented milk was significantly lower than that in ordinary fermented milk produced with the same starter culture at most time points. This may be because the Maillard reaction produces certain inhibitory substances that affect the growth of Streptococcus thermophilus. In the BT group, the viable count of Streptococcus thermophilus was significantly higher than other groups at 7 days of storage, reaching its maximum value during storage. The BD and HD groups reached their maximum viable count at 14 days. The activity of starter culture D was relatively gradual, with the peak viable count occurring later than in the BT group. In the HT group, the viable count of Streptococcus thermophilus showed an increasing trend during the 21-day storage period, reaching its highest value at 21 days. This indicates that the activity of starter culture T in brown yogurt was relatively slow, but gradually increased in the later stages.

[0053] like Figure 5 The viable count of *Streptococcus thermophilus* in brown fermented milk using starter cultures a and D was significantly lower than that in regular fermented milk during storage. The viable count of *Streptococcus thermophilus* in brown yogurt using starter cultures T was significantly lower than that in regular fermented milk from day 1 to 14, but higher at day 21. The viable count in brown fermented milk was significantly lower than in regular yogurt from day 1 to 14, but higher at day 21. This indicates that the activity of starter culture T in brown yogurt was initially low, but gradually recovered and even exceeded that of regular yogurt later. This may be because the strains in starter culture T have a stronger ability to adapt to the environment, especially in the later stages of brown yogurt production, overcoming the effects of initial inhibitory factors. The activity of starter culture D in both regular and brown yogurt was relatively gradual, with the viable count peaking at day 14. This indicates that the activity of starter culture D in brown yogurt was inhibited or that environmental conditions were unfavorable for its growth, suggesting that the strains in starter culture D have poor environmental adaptability to brown yogurt.

[0054] like Figure 5 b. The viable count of *Lactobacillus bulgaricus* in brown fermented milk was significantly lower than that in ordinary fermented milk produced with the same starter culture during storage. All four groups of samples showed a slow increase over 1-7 days, peaking at 7 days, followed by a decline, reaching its lowest point at 21 days. Fermented milk produced with starter culture group T showed a significantly higher viable count than group D during storage. The decline rate in ordinary fermented milk was slow over 14-21 days, while the decline in brown fermented milk was rapid over the same period. At 21 days, the viable counts of BT, BD, HT, and HD were 3.82 × 10⁻⁶. 6 CFU / g, 3.4×10 6 CFU / g, 1.55×10 6 CFU / g, 5×10 5 The CFU / g level may be due to the Maillard reaction occurring during the heating process of the brown fermented milk, producing compounds such as 5-hydroxymethylfurfural. 5-hydroxymethylfurfural inhibits the growth and survival of bacilli and streptococci, especially during the later stages of storage when the metabolic products accumulate, making the inhibitory effect more pronounced.

[0055] Studies have found that the number of viable *Streptococcus thermophilus* bacteria in brown fermented milk was lower than that in regular fermented milk for most of the time, and the number of viable *Lactobacillus bulgaricus* bacteria in brown fermented milk was significantly lower than that in regular fermented milk during storage. It is speculated that the inhibitory substances produced by the Maillard reaction affected their growth, or that the Maillard reaction consumed some of the substrate in the raw milk, thus affecting their growth. The T starter culture showed slow activity in brown yogurt initially, gradually increasing in later stages. This starter culture exhibited stronger adaptability to the brown yogurt environment in the later stages.

[0056] Analysis of changes in extracellular polysaccharide content during storage The metabolic activities of the bacterial strain produce EPS (extracellular polymeric substances). EPS in fermented milk possesses good rheological properties and water retention capacity, thus its content contributes to the stability of the fermented milk system. A standard curve for glucose content was plotted using the glucose content and its corresponding absorbance values, and the regression equation was obtained: y = 0.0172x + 0.0995 (R²). 2 =0.9969), from which the change in EPS content in fermented milk during storage can be calculated, such as Figure 6 As shown.

[0057] The content of EPS gradually increased during storage from day 1 to day 7, reaching its highest value at day 7, with HD, HT, BD, and BT contents of 160 mg / L, 174 mg / L, 175 mg / L, and 185 mg / L, respectively. It decreased after day 7, reaching its lowest value at day 21, consistent with the aforementioned viscosity and textural characteristics. The extracellular polysaccharide (EPS) content in conventional fermented milk produced with the same starter culture was significantly higher than that in brown fermented milk, possibly due to the Maillard reaction consuming lactose in the milk, leading to reduced EPS production. The EPS content in group T starter culture was significantly higher than that in group D. p <0.05), consistent with the viscosity trend mentioned above.

[0058] Analysis of changes in texture properties during storage Table 64 shows the changes in textural properties of fermented milk during storage. Note: HT represents brown fermented milk fermented with starter culture T; HD represents brown fermented milk fermented with starter culture D; BT represents regular fermented milk fermented with starter culture T; BD represents regular fermented milk fermented with starter culture D. Different letters indicate intra-group variability in the samples. p <0.05 indicates a significant difference. p >0.05 indicates no significant difference.

[0059] Table 6 above shows the data on changes in textural properties during storage. As can be seen from the table, the indicators for the HT, BD, and BT fermented milk groups increased from day 1 to day 7, reaching their highest values ​​during storage at day 7, then decreased significantly at day 14, and fluctuated somewhat at day 21. For the HD group, the indicators showed no significant changes from day 1 to day 7, then decreased significantly after day 7, reaching their lowest values ​​at days 14 and 21. The reason for this may be that in the early stages of fermented milk storage, the activity of lactic acid bacteria is high, and continuous acid production and metabolic activity lead to protein coagulation, forming a gel structure similar to yogurt. Therefore, textural indicators (such as hardness and viscosity) gradually increase. Furthermore, lactic acid bacteria convert lactose into lactic acid, causing a decrease in pH, further promoting the coagulation of milk proteins and the formation of network structures. At day 7, the gel structure of milk proteins reaches its optimal state, and the pH and gel structure of the fermented milk are relatively stable, exhibiting the best textural performance. In the later stages of storage, due to the accumulation of lactic acid, the pH further decreases, leading to whey separation and a significant decrease in textural indicators. Prolonged storage may cause whey to separate, further reducing the firmness and other texture parameters of yogurt.

[0060] In summary, the textural properties of ordinary fermented milk stored under the same starter culture and storage conditions were significantly higher than those of brown fermented milk. While the Maillard reaction imparts a unique flavor and color to brown fermented milk, it also negatively impacts its textural properties. Possible reasons for this include: firstly, the Maillard reaction leads to the denaturation or cross-linking of milk proteins such as casein, potentially weakening their ability to form gel structures, thus reducing the firmness, viscosity, and elasticity of the yogurt; secondly, brown fermented milk consumes some lactose and protein during the Maillard reaction, resulting in a reduction in available substrates for lactic acid bacteria during fermentation, leading to lower lactic acid production and affecting gel formation. Since starter culture T produces more extracellular polysaccharides, it also affects the texture of the yogurt. Overall, starter culture T performs better than starter culture D in terms of textural stability and may be more suitable for producing yogurt for long-term storage.

[0061] Turbiscan stability analysis during fermentation In fermentation, Turbiscan, as a highly efficient stability analysis tool, has been widely used to monitor sample dispersion characteristics, particle aggregation behavior, sedimentation processes, and solvation. Based on Multiple Light Scattering (MLS) technology, this device can capture dynamic changes in samples in real time, providing comprehensive experimental data for stability assessment of fermentation broths or products. Specifically, Turbiscan technology accurately measures the particle size distribution and concentration changes of particulate matter by detecting the intensity of scattered light at different angles and positions in the sample. When the particle concentration in the sample is low, some light can still be transmitted after multiple scattering, while another portion is scattered in the opposite direction to the incident light, known as backscattered light. Using the initial state of the sample as a reference, the difference between the backscattered light and the sample is the rate of change (ΔBS); the larger the value, the worse the stability of the system. Using Turbisoft Lab 2.0 software, the stability index (Turbiscan Stability Index, TSI) can be further calculated based on the changes in backscattered light during protein particle aggregation and dynamic migration. The TSI value comprehensively reflects the dynamic changes in concentration and particle size of the sample during the measurement period. The higher the value, the worse the stability of the system.

[0062] like Figure 7 As shown, the changes in BT, BD, HT, and HD were 2.5%, 3%, 3.2%, and 4%, respectively. The stability ranking was BT > BD > HT > HD. This change is mainly attributed to the increase in average particle size, which is essentially an instability process caused by particle aggregation. Furthermore, during fermentation, such as... Figure 8 The results showed that the TSI values ​​of all four fermented milk groups increased over time, indicating that protein aggregates gradually promoted the formation of gel structures during fermentation. Based on the TSI value ranking (HD > HT > BD > BT), the relative stability of each group could be determined as BT > BD > HT > HD, a conclusion completely consistent with the trend of ΔBS. This study further validates the reliability and practicality of Turbiscan technology in fermentation broth stability analysis, providing important experimental evidence for research in related fields.

[0063] Analysis of changes in organic acid content in fermented milk To further investigate the effect of the Maillard reaction on carbon sources in fermented milk, this study focused on organic acids in fermented milk. Carbon sources are the main raw materials for the production of organic acids by microorganisms. Microorganisms convert carbon sources into organic acids through metabolic pathways such as glycolysis and the tricarboxylic acid cycle. Lactic acid is the main organic acid in fermented milk, such as... Figure 9 After fermentation, lactic acid bacteria grow in large quantities, and lactic acid levels rise significantly. p<0.05). Both *Lactobacillus bulgaricus* and *Streptococcus thermophilus* metabolize lactose to produce lactic acid, acetic acid, and carbon dioxide, promoting milk coagulation and giving fermented milk its unique texture and flavor. The malic acid content of the four groups BD-1d, HD-1d, BT-1d, and HT-1d was significantly higher than that of the MB-B and MB-H combination (…). p <0.05%), malic acid is a metabolite of the tricarboxylic acid cycle, which can improve the microbial stability of fermented products. It can also be converted into lactic acid by lactic acid bacteria, which can harmonize the flavor of yogurt and make its texture smoother. Furthermore, studies have found that vanillic acid produced by lactic acid bacteria has antifungal properties. During the fermentation process of milk under the action of lactic acid bacteria, the vanillic acid content increases significantly ( p <0.05).

[0064] Analysis of changes in amino acid content in fermented milk To further investigate the effect of the Maillard reaction on nitrogen sources in fermented milk, this study focused on amino acids in fermented milk. Amino acids are a class of organic compounds containing amino and carboxylic acid groups; they are the basic building blocks of proteins. They also play an important role as non-volatile flavor compounds in fermented foods. Figure 10 As shown, there were no significant differences in phenylalanine and tryptophan levels. Variations in valine, tyrosine, glycine, isoleucine, asparagine, aspartic acid, DL-2-aminoadipic acid, methionine, threonine, and serine were consistent across groups; MB-B < MB-H, with the HD group significantly higher than other groups. p <0.05). Keto acid derivatives such as valine, tyrosine, glycine, and isoleucine can synthesize diacetyl or other ketones during sugar metabolism, thus producing caramel and creamy aromas. Aspartic acid can effectively inhibit the formation of burnt and sour tastes and promote sweetness. The content trends of aspartic acid and asparagine are consistent: HT > BT; HD > BD. p <0.05). Studies have shown that during the Maillard reaction, flavor nucleotides such as adenine nucleotides (AMP) and inosinic acid (IMP), as well as flavor amino acids such as aspartic acid and asparagine, are gradually released as the reaction proceeds. They have a synergistic effect and work together to enhance the umami flavor of food.

[0065] like Figure 10 The content of glutamine (MB-B) is greater than that of MB-H, and BD and BT are significantly higher than those of HD and HT. Glutamine deamidation produces colloidal substances, which can participate in the Maillard reaction to produce various aromatic compounds such as furfural, furanone, Strecker aldehyde, pyrazine, and pyrrole. This indicates that a large amount of glutamine participates in the Maillard reaction to form aromatic compounds.

[0066] like Figure 10As shown in Figure e, the cystine content in milk decreased significantly after browning treatment (MB-B > MB-H), and the cystine content in fermented milk from group T was significantly higher than that in other groups. The study indicates that the Maillard reaction heat treatment process denatures cysteine ​​and degrades cystine in milk, leading to the formation of sulfides. Dicarbonyl compounds (diacetyl and 2,3-pentanedione) coexist, producing hydrogen sulfide. This demonstrates that the Maillard reaction significantly reduces cystine content, and the lactic acid bacteria in group T produce a larger yield of cystine through metabolism, thus mitigating the negative impact of the Maillard reaction on cystine production.

[0067] like Figure 10 As shown in k, the lysine content MB-H > MB-B, but there was no significant increase; HD > HT > BT and BD ( p <0.05). Studies have shown that lysine content is significantly positively correlated with the degree of Maillard reaction. This indicates that the Maillard reaction increases lysine in milk to a certain extent, but not significantly, while the participation of lactic acid bacteria significantly increases the lysine content in fermented milk.

[0068] In summary, comparing the changes in amino acid content (MB-B, MB-H) before and after browning of raw milk, it can be concluded that the Maillard reaction reduces the content of some amino acids, but the change is not significant. Under the action of lactic acid bacteria, after the fermentation process, a large number of amino acids are produced, especially the content of amino acids in brown fermented milk is significantly higher than that in ordinary fermented milk.

[0069] Single strain continuous pH measurement The pH changes of four lactic acid bacteria strains—Streptococcus thermophilus IMAU80806, Streptococcus thermophilus IMAU10630, Lactobacillus bulgaricus IMAU40073, and Lactobacillus bulgaricus IMAU95052—over 24 hours are shown in the table below. Figure 11 .

[0070] Fermented milk microstructure Figure 12 These are scanning electron microscope (SEM) images of four groups of fermented milk stored for one day. The images clearly show no obvious difference between the BT and HT groups, while the HD group is relatively looser than the BD group. It is speculated that the high-temperature environment during the Maillard reaction caused protein denaturation in the milk, leading to the formation of relatively uneven casein micelles in the browned fermented milk system. The high extracellular polysaccharide production of the T group starter may have altered the milk's gel structure, resulting in the HT group appearing more compact than the HD group in the microstructure.

[0071] Analysis of volatile flavor compounds Sample grouping and changes in metabolites in each group The aforementioned studies on organic acids indicate that the Maillard reaction alters their content and affects the flavor of fermented milk. To further investigate the effects of the Maillard reaction and starter cultures on the flavor of fermented milk, the following study was conducted focusing on volatile flavor compounds. Table 7 below shows samples of fermented milk prepared before and after browning, using starter cultures T and D, stored for 1 day and 21 days. The table presents the differences in the number of metabolites and their up- and down-regulation data for each group. The D-group starter culture produced higher levels of volatile flavor compounds than the T-group starter culture in both regular and brown fermented milk. Notably, the volatile flavor compounds decreased in HD-21d compared to HD-1d, while HT-21d showed a significant increase compared to HT-1d. It is speculated that the lactic acid bacteria in the T-group starter culture produced more volatile flavor compounds during storage.

[0072] Table 7. Number of differentially expressed metabolites and data on up- and down-regulation in each group. Composition of total metabolite categories Ten categories of substances were detected in the 10 samples, including 318 aldehydes, ketones, and esters (33.16%), 193 hydrocarbons (20.13%), 139 alcohols and amines (14.49%), 90 heterocyclic compounds (9.38%), 72 terpenoids (7.51%), 65 benzene and its substituted derivatives (6.78%), 30 organic acids and their derivatives (3.13%), 18 nitrogen-containing compounds (1.88%), 14 ethers (1.46%), and 13 halogenated hydrocarbons (1.36%).

[0073] Key volatile flavor compounds and their rOAV values Volatile esters have a significant impact on the flavor of probiotic beverages. Their rOAV≥1 ester metabolites include ethyl 3-methylbutyrate, γ-decanolide, γ-caprolactone, γ-dodecanolactone, γ-undecanolide, γ-pentylbutyrolactone, γ-octanolide, methyl benzoate, heptyl propionate, and methyl heptanoate. Among these, methyl benzoate and ethyl 3-methylbutyrate have fruity aromas and are often used as food flavorings. In aroma addition experiments, the combination of γ-octanolide and diacetyl in binary mixtures showed the most significant effect in enhancing the flavor of milk.

[0074] Aldehydes are mainly produced by amino acid metabolism or unsaturated fatty acid oxidation. They have a low flavor threshold in fermented milk beverages and significantly affect flavor. Their rOAV≥1 ester metabolites include (2E,4E)-2,4-octadienal, (E)-2-decenal, (E)-2-octenal, (E,E)-2,6-nonadienal, (R)-3,7-dimethyl-6-octenal, nonanal, and undecenal. Among these, (E)-2-decenal exhibits a pleasant flavor, while nonanal and (E)-2-octenal have been studied extensively and exhibit sweet, fruity flavors.

[0075] Ketones are generated through the thermal decomposition of amino acids, the Maillard reaction, and the oxidative decomposition of unsaturated fatty acids. Due to their low threshold, they significantly influence the aroma of dairy products. Three ketones were detected: 3-ethyl-2-hydroxy-2-cyclopentenone, 4-hydroxy-2,5-dimethyl-3(2H)-furanone, and tetrahydro-6-methyl-2H-pyran-2-one. Tetrahydro-6-methyl-2H-pyran-2-one is a key flavor compound in fruits such as strawberries. 4-hydroxy-2,5-dimethyl-3(2H)-furanone is a Maillard reaction product, and its methyl ether, 2,5-dimethyl-4-methoxy-3(2H)-furanone, is an imported aromatic chemical considered a key flavor compound in many fruits. Due to its unique sensory properties, it is highly valued by the food industry.

[0076] Alcohols can be produced by amino acid metabolism and glucose degradation, and are closely related to processes such as lactose metabolism and methyl ketone reduction. 3-Furfural, 6-undecyl alcohol, (E)-2-octen-1-ol, (Z)-2-octen-1-ol, 6-methyl-2-heptanol, 2-octen-1-ol, (Z)-3-hexenol, benzenethiol, and (E)-3-hexenol are examples of flavor compounds. (Z)-3-hexenol and (E)-3-hexenol provide a natural and refreshing flavor profile in food, enhancing its freshness and naturalness, adding unique flavor layers, and imparting a fresh aroma to fruits such as blueberries. (Z)-3-hexenol and the aforementioned γ-decyl lactones combine in aqueous systems to promote flavor formation. 3-Furfural provides an ice cream flavor to fermented milk.

[0077] Differential metabolite category composition Compared to the BT-1d group, the HT-1d group showed an upregulation of 8 flavor compounds, including 1-phenyl-2-butanone, 2-hexanol, ethyl 2-hydroxy-4-methylvalerate, and 2,3-butanedione monooxime. 33 flavor compounds showed a downregulation, including alcohols such as (E)-3-hexenol, (Z)-3-hexenol, 2-furanethanol, and 2-decyl alcohol; ketones such as 1,3-diazabicyclo[3.2.2]non-4-one and 1,3-dimethylimidazolidine-2,4-dione; esters such as ethyl mandelate, propyl pyruvate, and cyclobutyl ethyl phthalate; and heterocyclic compounds such as 4-methyl-2H-pyran and 4-methylisothiazolium.

[0078] This study compares the fold changes in metabolite quantification information between the BT-1d and HT-1d groups. The results for the top 20 metabolites with the largest fold changes in each group are presented. Among the important flavor compounds are 2,3-butanedione monooxime, a derivative of 2,3-butanedione, also known as diacetyl, which has a typical milky and creamy aroma and is a characteristic flavor of many fermented dairy products; 2-furanethanol, a Maillard reaction compound, affects color, aroma, and flavor, exhibiting better antioxidant properties and higher inhibitory activities on xanthine oxidase, tyrosinase, and acetylcholinesterase; ethyl 3-methylbutyrate, with a fruity aroma, is commonly used as a food flavoring; and (E)-3-hexenol, commonly known as leaf alcohol, has a fresh grassy aroma and is an important component of natural aroma in food.

[0079] Compared to the BD-1d group, the HD-1d group showed an upregulation of 8 flavor compounds, including heptyl propionate, cyclobutyl ethyl phthalate, and 2-propyl-1-heptanol. Forty-two flavor compounds were downregulated, including alcohols such as (Z)-3-hexenol, (E)-3-hexenol, 2-hexanol, (E)-3-hexenol, and 2-furanethanol; ketones such as 2,3-butanedione monooxime, 3-methyl-2(5H)-furanone, and 3-(hydroxymethyl)-2-nonanone; esters such as ethyl 3-methylbutyrate and dodecyl methacrylate; and heterocyclic compounds such as 4-methyl-2H-pyran and 2,4-dimethyl-1H-pyrrole.

[0080] Compare the fold changes in metabolite quantification information between the BT-1d and HT-1d groups. The results for the top 20 metabolites with the highest fold changes in each group comparison are shown. These metabolites are extremely similar to the differentially expressed compounds in the BT-1d vs HT-1d groups, all containing 2-furanethanol, 3-furanethanol, (E)-3-hexenol, (Z)-3-hexen-1-ol, ethyl 3-methylbutyrate, and 2,3-butanedione monooxime. This suggests that these compounds may be Maillard reaction flavor products common to brown fermented milk.

[0081] Compared to the HD-1d vs HT-1d control group, 34 flavor compounds were upregulated, while 6 flavor compounds were downregulated. The upregulated compounds included alcohols and amines such as 2-furanethanol, 3-furanethanol, (E)-3-hexenol, (Z)-3-hexenol, 2-hexanol, and bicyclo[2.2.1]heptane-2-ol; ketones such as 3-methyl-2(5H)-furanone and 4-methylcyclohex-3-en-1-one; esters such as 4-hexenyl acetate, leaf ester acetate, and dodecyl methacrylate; hydrocarbons such as 6-methyl-1-octene, (Z)-3-methyl-5-undecene, (Z)-9-methyl-2-undecene, and ethylenecyclohexane; and heterocyclic compounds such as N-benzo[1,2,5]-5-thiadiazolylacetamide and 4-methyl-2H-pyran. The heterocyclic compounds that were significantly downregulated included 4-amino-3-hydroxytetrahydrothiophene-1,1-dioxide and 2-peroxyhydro-1,4-dioxane; the ester compounds included (E,Z)-2-butenoic acid-3-hexenyl ester and ethyl mandelate; the hydrocarbon compound 1-heptadecene; the alcohol compound 2-decyl alcohol; and the benzene derivative 1,2,3,4-tetramethylbenzene.

[0082] Results of the top 20 metabolites with the largest fold differences in each group comparison are presented: 3-hexen-1-ol acetate and (E)-3-hexen-1-ol acetate (Z)- were identified as characteristic odor compounds with strong floral, fruity, and sweet aromas. 2-Furfural is one of the important flavor compounds produced by the Maillard reaction during coffee roasting, contributing significantly to the roasted and sweet aromas of coffee. (Z)-3-hexenol and (E)-3-hexenol provide a natural and refreshing flavor profile in food, enhancing its freshness and naturalness, adding unique flavor layers, and imparting a refreshing aroma to fruits such as blueberries.

[0083] The above analysis revealed that the Maillard reaction in fermented milk produces a series of unique flavor compounds, such as 2-furanol and 3-furanol. The brown fermented milk produced with Group D starter culture had higher levels of volatile flavor compounds than that produced with Group T starter culture. Furthermore, significant differences in some Maillard reaction products suggest that the lactic acid bacteria in Group D starter culture may be better able to utilize the substrate to produce flavor compounds.

[0084] Metabolomics analysis To further investigate the metabolic differences between the two groups of starter cultures in conventional and brown fermented milk, these compounds were classified into 15 categories according to the HMDB primary classification. The compounds with higher abundance included: organic acids and their derivatives: 158 (19.17%), benzene and its substituted derivatives: 119 (14.44%), heterocyclic compounds: 87 (10.56%), aldehydes, ketones, and esters: 68 (8.25%), fatty acids (FA): 67 (8.13%), amino acids and their metabolites: 63 (7.65%), others: 52 (6.31%), carbohydrates and their metabolites: 51 (6.19%), and glycerophospholipids (GP): 48. (5.83%), alcohols and amines: 33 (4.00%), nucleotides and their metabolites: 25 (3.03%), hormones and hormone-related compounds: 14 (1.70%), flavonoids: 11 (1.33%), bile acids: 6 (0.73%), glycolipids (GL): 4 (0.49%), terpenes: 4 (0.49%), coenzymes and vitamins: 3 (0.36%), phenolic acids: 3 (0.36%), tryptamine, choline, and pigments: 3 (0.36%), alkaloids: 2 (0.24%), lignans and coumarins: 2 (0.24%), steroids: 1 (0.12%).

[0085] Screening and analysis of differential metabolites Compared to the HT-1d group, the BT-1d group showed significant upregulation of 156 metabolites and significant downregulation of 167 metabolites. Screening of Grade A and Grade B compounds revealed that the BT-1d group had 38 significantly upregulated metabolites and 40 significantly downregulated metabolites compared to the HT-1d group. Upregulated metabolites included carbohydrates such as isomalttriose; heterocyclic compounds such as pyridoxal, indole-2-carboxylic acid, and taurine; organic acids and their derivatives such as 6-hydroxyhexanoic acid and chlorogenic acid; and amino acids and their metabolites such as O-acetyl-L-serine, L-hydroxyproline-L-isoleucine, glycyl-L-proline, Nα-acetyl-L-arginine, glutamine-glutamine, 5-methoxy-DL-tryptophan, phenylalanine-aspartic acid, and N-phenylacetylglycine. Benzoic acid, phenylacetic acid, N-acetylethylamine, and other benzene and their derivatives were also included. Isomaltotriose can promote the growth of beneficial intestinal bacteria such as Lactobacillus and is widely used in health products, food, and pharmaceuticals. This explains the significantly higher viable bacterial count in ordinary fermented milk compared to brown fermented milk. Benzoic acid is the most commonly used preservative in food and is generally considered safe. In this study, benzoic acid was significantly upregulated in the BT-1d group compared to the HT-1d group, indicating that benzoic acid is crucial for the quality stability of fermented milk during storage.

[0086] Compared to the HT-1d group, the BT-1d group showed significantly downregulated metabolites including N-acetyl-D-galactosamine and other carbohydrates and their metabolites; dopamine, hexaethylene glycol and other alcohols and amines; oxaloacetic acid, glyceric acid, 3-hydroxybutyric acid, 3-methyl-2-oxovaleric acid and other organic acids and their derivatives; methionine, L-tryptophan, proline-alanine, L-arginyl-L-phenylalanine, leucyl-tryptophan and other amino acids and their metabolites; and 3-indole-lactic acid, salicylic acid, methyl cinnamate and other benzene and their derivatives. Methionine has a faint sulfide odor and a slightly bitter taste. In some fermented foods, the presence of methionine and its metabolites can have a certain impact on the flavor. Its contribution to flavor formation is mainly through thermal degradation or thermal interaction with other food components, especially reducing sugars. Yuxia pointed out that methionine and its metabolites are important participants in the production of flavor compounds through the Maillard reaction.

[0087] Compared to the HT-1d group, the HD-1d group showed 152 significantly upregulated metabolites and 112 significantly downregulated metabolites. Screening of Grade A and Grade B compounds revealed that the D group showed 28 significantly upregulated metabolites and 33 significantly downregulated metabolites compared to the E group.

[0088] The upregulated compounds include four organic acids: 3-guanidinopropionic acid, 2-phospho-D-glyceric acid, and itaconic acid; six amino acid metabolites: L-arginyl-L-methionine, 5-L-glutamyl-L-alanine, and N-methyl-L-glutamic acid; five heterocyclic compounds: D-fructose and ε-caprolactam; and four benzene compounds and their derivatives: hydroquinone and mandelic acid.

[0089] The substances downregulated included six amino acids and their metabolites, such as N-acetyl-ornithine and 3-methyl-L-histidine; nine organic acids, such as α-ketoglutarate and 2-methylsuccinic acid; six benzene compounds and their derivatives, such as vanillic acid and 3-methylsalicylic acid; and some carbohydrates and heterocyclic compounds with significant differences. Notably, glycerophospholipids showed significant differences. KEGG enrichment analysis of differential metabolites The study examined the KEGG enrichment maps of differentially metabolites between the BD and BT groups, highlighting the glycolysis pathway. During extracellular polysaccharide synthesis, key enzymes such as glycosyltransferases utilize precursors like UDP-glucose produced by glycolysis to transfer glycosyl groups one by one to the synthesizing polysaccharide chain, achieving polysaccharide chain elongation and polymerization, ultimately forming extracellular polysaccharides. Four differentially metabolites were enriched in the glycolysis pathway: salicin, 3-phosphoglycerate, and dihydroxyacetone phosphate were downregulated; α-D-glucose was upregulated. Notably, 3-phosphoglycerate is a crucial metabolite in glycolysis. During glycolysis, 1,3-diphosphoglycerate, through the action of phosphoglycerate kinase, transfers a high-energy phosphate group to ADP to generate ATP, simultaneously producing 3-phosphoglycerate. As a key intermediate in the glycolysis pathway, 3-phosphoglycerate indirectly affects the production of nucleotide sugars by regulating metabolic flux, thereby influencing EPS synthesis. This explains why, as mentioned earlier, the extracellular polysaccharide content of fermented milk produced by group T starter culture during storage was significantly higher than that of fermented milk produced by group D starter culture.

[0090] The study investigated the KEGG enrichment maps of differentially metabolites between the HD and HT groups. The valine, leucine, and isoleucine degradation pathways and histidine metabolic pathways showed significant performance across multiple indicators, making them metabolic pathways worthy of close attention. The valine, leucine, and isoleucine degradation pathway is an important branch of amino acid metabolism, primarily involving the catabolism of these three branched-chain amino acids (BCAAs). BCAAs initially form branched-chain α-keto acids through transamination by branched-chain aminotransferases. Specifically, leucine is converted to α-ketoisocaproic acid, isoleucine to α-ketomethylvaleric acid, and valine to α-ketoisovaleric acid. Ultimately, these BCAAS are lost as CO, while another portion can generate acetyl-CoA and succinyl-CoA, entering the tricarboxylic acid cycle (TCA cycle). The TCA cycle is a common pathway for the oxidation of nutrients for energy in organisms. While the intermediate products of branched-chain amino acid degradation are present in low concentrations in the body, they participate extensively in the healthy growth process as signaling molecules, energy substrates, and other substances. As a small molecule with biological activity, it has the effect of protecting tissues and organs such as nerves and cardiovascular system, and has a protective function in alleviating osteopenia.

[0091] Four differentially expressed metabolites in the valine, leucine, and isoleucine degradation pathway were enriched in both the HD and HT groups. Acetoacetate, isobutyryl-CoA, 2-methylcrotonyl-CoA, and 3-methyl-2-oxobutyrate were all significantly downregulated. Acetyl-CoA is a potential precursor for acetoacetate synthesis and participates in metabolism in the cytosol, mitochondria, peroxisomes, and nucleus. It is a substrate of the TCA cycle, producing ATP and precursor metabolites of amino acids, nucleotide bases, and porphyrins. Acetyl-CoA is also a structural unit of fatty acid synthesis and an end product of fatty acid degradation. Furthermore, acetyl-CoA is a substrate for protein acetylation, playing a role in the regulation of enzyme function and DNA transcription. The T group starter culture showed a stronger acetoacetate production capacity, providing energy for the growth and metabolism of the bacteria in the fermented milk. During storage, the viable cell count in the HT group fermented milk was generally higher than that in the HD group.

[0092] Four differentially expressed metabolites were found in the histidine metabolic pathway between the HD and HT groups. L-aspartic acid, L-histamine, 3-methyl-L-histidine, and α-ketoglutarate were all significantly downregulated. Aspartic acid catabolism involves a two-step reaction to produce pyruvate. First, aspartic acid is converted to oxaloacetate by aspartate transaminase, and then oxaloacetate is decarboxylated by oxaloacetate carboxylase to produce pyruvate. Pyruvate can be further converted to lactic acid, acetic acid, or acetolactate, and subsequently to diacetyl and acetoin. Both have creamy, buttery aromas, but diacetyl's aroma intensity is several tens of times greater than acetoin's. Due to the downregulation of L-aspartic acid, the production of diacetyl and acetoin in the fermented milk of the HD group may be reduced, affecting the product's flavor richness. α-Ketoglutarate is an intermediate in the tricarboxylic acid cycle and is also a keto acid produced by the deamination of glutamate. In the tricarboxylic acid cycle, α-ketoglutarate is generated by the oxidative decarboxylation of isocitrate (catalyzed by isocitrate dehydrogenase), and then used to generate succinyl-CoA (catalyzed by α-ketoglutarate dehydrogenase). During glutamine degradation, glutamine is converted to glutamate, and then to α-ketoglutarate, which can be introduced into the tricarboxylic acid cycle as a compensatory substrate. α-ketoglutarate also inhibits starvation-induced autophagy. The significant downregulation of α-ketoglutarate in the HD group may make the microorganisms in fermented milk more susceptible to stress conditions such as starvation, leading to increased autophagy and thus affecting the quality stability of fermented milk. This is also reflected in the significantly higher levels of *Lactobacillus bulgaricus* and *Streptococcus thermophilus* in the HT group during the later stages of storage, explaining the weak post-acidification characteristic of the HD group.

[0093] A study of KEGG enrichment maps of differentially metabolites between the BT and HT groups revealed that ABC transporters exhibited superior performance across multiple indicators, making them a metabolic pathway worthy of close attention. ABC transporters are a class of transmembrane proteins widely distributed in organisms, capable of actively transporting various molecules, from simple molecules (fatty acids, sugars, nucleosides, and amino acids) to complex organic compounds (lipids, oligonucleotides, polysaccharides, and proteins). In lactic acid bacteria, ABC transporters are responsible for transporting metabolites such as lactic acid, amino acids, and sugars, influencing the growth and metabolic efficiency of lactic acid bacteria. By regulating the transport of metabolites in lactic acid bacteria, ABC transporters may affect the acidity and flavor compound (such as acetic acid and propionic acid) formation in fermented milk. ABC transporters participate in the transport of flavor precursors such as amino acids and short-chain fatty acids in lactic acid bacteria, thereby affecting the flavor characteristics of fermented milk. Some ABC transporters may be involved in transporting amino acids outside the cell, where they are further metabolized into volatile flavor compounds. In probiotics, ABC transporters may participate in the transport of beneficial metabolites such as polysaccharides and short-chain fatty acids, enhancing the probiotic function of fermented milk. In many cases, ABC transporters may not directly transport lipid substrates, but they do influence lipid arrangement. For example, Khakhaina et al. identified the mechanisms by which ABC transporters regulate lipid transport and rearrangement in *S. cerevisiae* cells. This study provides evidence that ScPdr5 / ScYor1 negatively regulates Lem3 flipperase, while Pdr1 regulates permeability and sphingolipid biosynthesis, respectively. These transporters utilize energy from ATP hydrolysis to transport substrates from inside the cell to outside, or from outside the cell to inside, thus exhibiting highly efficient active transport capabilities. ABC transporters in lactic acid bacteria may participate in the transport of toxins or metabolic waste, helping cells resist external stresses (such as acid, salt, and temperature), thereby improving the stability and survival rate of strains during fermented milk production.

[0094] The study found six differentially expressed metabolites in the ABC transporter pathway between the BT and HT groups: betaine, biotin, guanosine, riboflavin, taurine, and isomaltotriose. All six metabolites were upregulated. Jean-Marc Collar stated that taurine promotes microbial growth and its metabolism plays a crucial role in shaping the mouse gut microbiota. Biotin, also known as vitamin B7, is essential for maintaining natural growth, development, and normal physiological functions in the human body. Biotin plays a vital role in various physiological functions, including maintaining metabolism, cell lysis, and nervous system health, as well as relieving muscle pain and preventing hair loss. Biotin affects intracellular transport mechanisms in bacteria, promoting the proliferation of lactic acid bacteria. Riboflavin is an important micronutrient, a precursor to coenzyme flavin mononucleotide and flavin adenine dinucleotide, and is essential for biochemical reactions in all living cells. For decades, one of the most important applications of riboflavin has been its use as a nutritional supplement for animals and humans worldwide. In summary, taurine, biotin, and isomaltotriose, when enriched in differential metabolites along the ABC transporters pathway, all promote the growth of lactic acid bacteria in fermented milk. This is consistent with the observation that the levels of Lactobacillus bulgaricus and Streptococcus thermophilus were higher in the BT group than in the HT group during storage.

Claims

1. A fermenting agent, characterized in that, The starter culture includes Lactobacillus bulgaricus IMAU95052, Lactobacillus bulgaricus IMAU40073, Streptococcus thermophilus IMAU80806 and Streptococcus thermophilus IMAU10630. The *Lactobacillus bulgaricus* IMAU95052 is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2024955; the *Lactobacillus bulgaricus* IMAU40073 is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2024958; and the *Streptococcus thermophilus* IMAU80806 is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2024959. Streptococcus thermophilus IMAU10630 is deposited at the China Center for Type Culture Collection, accession number CCTCC NO:M2024951.

2. The fermenting agent according to claim 1, characterized in that, The viable count ratio of Lactobacillus bulgaricus IMAU95052, Lactobacillus bulgaricus IMAU40073, Streptococcus thermophilus IMAU80806 and Streptococcus thermophilus IMAU10630 is (0.1~10):(0.1~10):(10~1000):(10~1000).

3. A fermenting agent, characterized in that, The fermentation agent includes Lactobacillus bulgaricus IMAU95052, Lactobacillus bulgaricus IMAU40073, Streptococcus thermophilus IMAU80806, and Streptococcus thermophilus IMAU20326; The *Lactobacillus bulgaricus* IMAU95052 is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2024955; the *Lactobacillus bulgaricus* IMAU40073 is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2024958; and the *Streptococcus thermophilus* IMAU80806 is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2024959. Streptococcus thermophilus IMAU20326 is deposited at the China Center for Type Culture Collection, accession number CCTCC NO:M2024953.

4. The fermentation agent according to claim 3, wherein the ratio of viable bacteria of Lactobacillus bulgaricus IMAU95052, Lactobacillus bulgaricus IMAU40073, Streptococcus thermophilus IMAU80806, and Streptococcus thermophilus IMAU20326 is (0.1~10):(0.1~10):(10~1000):(10~1000).

5. A method for preparing brown fermented milk, characterized in that, The preparation method includes inoculating the fermenting agent according to claim 1 or 3 into raw milk for fermentation to obtain brown fermented milk.

6. A brown fermented milk, wherein the brown fermented milk is obtained by fermentation using the fermenting agent according to claim 1 or 3.

7. The use of the starter culture according to claim 1 or 3 in the preparation of brown fermented milk.

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