Bifidobacterium longum subsp. longum IMAU12449 and its application

Abstract

The present invention discloses a Bifidobacterium longum subsp. longum IMAU12449 and its application, belonging to the technical field of microbiology. It provides a Bifidobacterium longum subsp. longum IMAU12449, which was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on July 8, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is: CGMCC NO. 31225. It provides the application of Bifidobacterium longum subsp. longum IMAU12449 in the preparation of fermented milk and / or soy milk. The present invention uses Bifidobacterium longum subsp. longum IMAU12449 as a potential probiotic to prepare milk and soy milk, and the resulting fermented milk has a stable viable count of bacteria, a moderate acidity, and no whey separation.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to Bifidobacterium longum subsp. longum IMAU12449 and its application. Background Art

[0002] At present, most traditional fermented milks are fermented using a commercial starter composed of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus.

[0003] Bifidobacterium is a genus name of bacteria. The genus Bifidobacterium is a genus of Gram-positive, non-motile, rod-shaped cells, sometimes bifurcated at one end, and strictly anaerobic bacteria, which are widely present in habitats such as the digestive tract, vagina and oral cavity of humans and animals. Bacteria of the genus Bifidobacterium are one of the important members of the intestinal flora of humans and animals. Some strains of Bifidobacterium can be used as probiotics in food, medicine and feed. As an important probiotic, Bifidobacterium has been widely used in the fields of medicine, food science, etc. The addition of probiotic Bifidobacterium can change the quality of fermented milk, and some fermented milks containing probiotic Bifidobacterium components also have probiotic effects on the body.

[0004] However, at present, the resources of Bifidobacterium from the intestine are relatively scarce, and there are few Bifidobacterium with excellent probiotic characteristics. Moreover, the research on the fermentation of soy milk is also relatively less. In summary, screening a strain of Bifidobacterium with potential probiotic ability and adding it as a probiotic to fermented milk and soy milk is of great significance for the development of intestinal-source probiotic fermented milk and soy milk. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides Bifidobacterium longum subsp. longum IMAU12449 and its application. The present invention uses Bifidobacterium longum subsp. longum IMAU12449 as a potential probiotic, and performs compound fermentation with Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus to prepare milk and soy milk. The process is simple, and the viable count of the fermented milk obtained is stable, the acidity is moderate, and there is no whey separation.

[0006] To achieve the above object, the present invention provides a Bifidobacterium longum subsp. longum IMAU12449, which was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on July 8, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is: CGMCC NO. 31225.

[0007] The present invention also provides the application of the Bifidobacterium longum subsp. longum IMAU12449 in the preparation of fermented milk.

[0008] The present invention also provides the application of the Bifidobacterium longum subsp. longum IMAU12449 in the preparation of fermented soy milk.

[0009] The present invention also provides a method for preparing fermented milk, which is prepared by using the Bifidobacterium longum subsp. longum IMAU12449 and includes the following steps:

[0010] (1) Preheat the milk, then mix it with sucrose, homogenize it, pasteurize it, and quickly cool it to obtain pretreated milk.

[0011] (2) Inoculate the Bifidobacterium longum subsp. longum IMAU12449 and a starter culture into the pretreated milk obtained in step (1), and ferment until the pH reaches 4.5 - 4.6 to obtain fermented milk.

[0012] Preferably, in step (1), the preheating treatment preheats the milk to 55 - 65°C; the dosage of sucrose in step (1) is 6 - 7 wt%; the pressure of homogenization in step (1) is 15 - 25 MPa; the temperature of pasteurization in step (1) is 95°C, and the time of pasteurization is 3 min.

[0013] Preferably, the inoculation amount of the Bifidobacterium longum subsp. longum IMAU12449 in step (2) is 5×10 6 CFU / mL; the starter culture in step (2) is composed of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus, and the inoculation amount of the starter culture is 0.003 wt%; the fermentation in step (2) is anaerobic culture, the fermentation temperature is 37°C, and the fermentation time is 6 - 7 h.

[0014] The present invention also provides a method for preparing fermented soy milk, which is prepared by using the Bifidobacterium longum subsp. longum IMAU12449 and includes the following steps:

[0015] S1. Preheat the soy milk, then mix it with sucrose and distilled water, homogenize it, pasteurize it, and quickly cool it to obtain pretreated soy milk.

[0016] S2. Inoculate the Bifidobacterium longum subsp. longum IMAU12449 and the starter culture into the pre-treated soy milk described in S1, and ferment until the pH reaches 4.5 - 4.6 to obtain fermented soy milk.

[0017] Preferably, in S1, the preheating treatment preheats the soy milk to 55 - 65°C; the dosage of sucrose in S1 is 7 - 9 wt%; the dosage of distilled water in S1 is 26 - 28 wt%; the pressure of homogenization in S1 is 15 - 25 MPa; the temperature of pasteurization in S1 is 95°C, and the time of pasteurization is 3 min.

[0018] Preferably, the inoculation amount of the Bifidobacterium longum subsp. longum IMAU12449 in S2 is 5×10 6 CFU / mL; the starter culture in S2 consists of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus, and the inoculation amount of the starter culture is 0.003 wt%; the fermentation in S2 is anaerobic culture, the fermentation temperature is 37°C, and the fermentation time is 6 - 7 h.

[0019] The present invention also provides the fermented soy milk prepared by the method for preparing fermented soy milk described above.

[0020] Compared with the prior art, the present invention has the following advantages and technical effects:

[0021] The present invention uses Bifidobacterium longum subsp. longum IMAU12449 as a potential probiotic, and performs compound fermentation with Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus to prepare cow milk and soy milk. The process is simple, and the obtained fermented milk has stable viable bacteria count, moderate acidity, and no whey separation. After storage at 4°C for 21 days, the viable bacteria count in the prepared fermented cow milk and fermented soy milk is still above 10 7 CFU / mL. Therefore, the present invention broadens the application prospect and industrialization resources of Bifidobacterium longum subsp. longum in fermented dairy products, discloses the function of Bifidobacterium longum subsp. longum IMAU12449 in improving whey separation and texture during the shelf life of fermented milk, as well as its excellent survival ability during the shelf life. The present invention obtains fermented milk with stable texture, providing a new candidate strain for the development of intestinal source probiotic fermented milk. Description of the Drawings

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0023] Figure 1 Colony and morphology diagrams of Bifidobacterium longum subsp. longum IMAU12449. Among them, A is the colony diagram and B is the morphology diagram;

[0024] Figure 2 Graph for determining the optimum growth temperature of Bifidobacterium longum subsp. longum IMAU12449;

[0025] Figure 3 Growth curve diagram of Bifidobacterium longum subsp. longum IMAU12449 in MRS + L-cysteine hydrochloride medium. Among them, A shows the changes in viable cell count and pH, and B shows the changes in OD value;

[0026] Figure 4 Graph for determining the antibacterial ability of Bifidobacterium longum subsp. longum IMAU12449;

[0027] Figure 5 Graph showing the changes in pH and titratable acidity during the storage of the fermented milk prepared in Example 2. Among them, A is the pH change graph. N: IMAU12449 represents the co-fermentation of milk by the starter culture and Bifidobacterium longum subsp. longum IMAU12449, N: control group represents the fermentation of milk by the starter culture, D: IMAU12449 represents the co-fermentation of soy milk by the starter culture and Bifidobacterium longum subsp. longum IMAU12449, D: control group represents the fermentation of soy milk by the starter culture; B is the titratable acidity change graph. N: IMAU12449 represents the co-fermentation of milk by the starter culture and Bifidobacterium longum subsp. longum IMAU12449, N: control group represents the fermentation of milk by the starter culture, D: IMAU12449 represents the co-fermentation of soy milk by the starter culture and Bifidobacterium longum subsp. longum IMAU12449, D: control group represents the fermentation of soy milk by the starter culture;

[0028] Figure 6 Graph showing the changes in viable cell count during the storage of the fermented milk prepared in Example 2. N: IMAU12449 represents the co-fermentation of milk by the starter culture and Bifidobacterium longum subsp. longum IMAU12449, N: control group represents the fermentation of milk by the starter culture, D: IMAU12449 represents the co-fermentation of soy milk by the starter culture and Bifidobacterium longum subsp. longum IMAU12449, D: control group represents the fermentation of soy milk by the starter culture;

[0029] Figure 7 Graph showing the changes in viscosity during the storage of the fermented milk prepared in Example 2. N: IMAU12449 represents the co-fermentation of milk by the starter culture and Bifidobacterium longum subsp. longum IMAU12449, N: control group represents the fermentation of milk by the starter culture, D: IMAU12449 represents the co-fermentation of soy milk by the starter culture and Bifidobacterium longum subsp. longum IMAU12449, D: control group represents the fermentation of soy milk by the starter culture;

[0030] Figure 8 Figure showing the change in water holding capacity during storage of the fermented milk prepared in Example 2. N: IMAU12449 represents the co-fermentation of cow milk by the starter culture and Bifidobacterium longum subsp. IMAU12449. N: Control group represents the fermentation of cow milk by the starter culture. D: IMAU12449 represents the co-fermentation of soy milk by the starter culture and Bifidobacterium longum subsp. IMAU12449. D: Control group represents the fermentation of soy milk by the starter culture.

[0031] Figure 9 Figure showing the sensory score during storage of the fermented milk prepared in Example 2. NI1 represents the storage of cow milk co-fermented by the starter culture and Bifidobacterium longum subsp. IMAU12449 for 1 day. NI7 represents the storage of cow milk co-fermented by the starter culture and Bifidobacterium longum subsp. IMAU12449 for 7 days. NI14 represents the storage of cow milk co-fermented by the starter culture and Bifidobacterium longum subsp. IMAU12449 for 14 days. NI21 represents the storage of cow milk co-fermented by the starter culture and Bifidobacterium longum subsp. IMAU12449 for 21 days. NC1 represents the storage of cow milk fermented by the starter culture for 1 day. NC7 represents the storage of cow milk fermented by the starter culture for 7 days. NC14 represents the storage of cow milk fermented by the starter culture for 14 days. NC21 represents the storage of cow milk fermented by the starter culture for 21 days. DI1 represents the storage of soy milk co-fermented by the starter culture and Bifidobacterium longum subsp. IMAU12449 for 1 day. DI7 represents the storage of soy milk co-fermented by the starter culture and Bifidobacterium longum subsp. IMAU12449 for 7 days. DI14 represents the storage of soy milk co-fermented by the starter culture and Bifidobacterium longum subsp. IMAU12449 for 14 days. DI21 represents the storage of soy milk co-fermented by the starter culture and Bifidobacterium longum subsp. IMAU12449 for 21 days. DC1 represents the storage of soy milk fermented by the starter culture for 1 day. DC7 represents the storage of soy milk fermented by the starter culture for 7 days. DC14 represents the storage of soy milk fermented by the starter culture for 14 days. DC21 represents the storage of soy milk fermented by the starter culture for 21 days. Detailed implementation manners

[0032] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0033] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0035] Without departing from the scope or spirit of the present invention, various improvements and variations can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are merely exemplary.

[0036] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0037] Sources of materials used in the present invention: The starter culture (composed of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus) was purchased from Chr. Hansen (Denmark); The DNA extraction kit was purchased from (KG203-02, Beijing, China).

[0038] Example 1

[0039] I. Isolation and Identification of Strains

[0040] The fecal samples collected from Hohhot were serially diluted, and appropriate dilutions were plated on MRS medium supplemented with L-cysteine hydrochloride. The plates were inverted and cultured in an anaerobic incubator at 37 °C for 72 h. Strains with different colony morphologies were selected for streak purification. The purified strains were subjected to Gram staining and catalase tests. Strains that were Gram-positive and catalase-negative were selected for preservation.

[0041] DNA was extracted using a DNA extraction kit. After PCR amplification of the extracted DNA, 16S rRNA sequencing was performed. The sequencing results were compared using the EZBioCloud database to determine the species status. Among them, the isolated Bifidobacterium was screened, and finally a strain of Bifidobacterium longum subsp. longum IMAU12449 with excellent probiotic characteristics was obtained.

[0042] The strain was subjected to physiological and biochemical identification and morphological observation, and the characteristics were as follows: The strain belongs to anaerobic bacteria that are Gram-positive and catalase-negative. Its colony and morphological results are as Figure 1 shown in A and Figure 1 shown in B. The colonies of strain IMAU12449 on the solid medium of MRS + L-cysteine hydrochloride were small, milky white, convex in the center, regular in edge, and shiny circular colonies. Under the optical microscope, it presented curved rod-shaped and branched forms, arranged in a V shape. This strain was consistent with the existing morphological studies on B. longum subsp. longum.

[0043] II. Study on the physiological and biochemical characteristics of B. longum subsp. longum IMAU12449

[0044] 1. Optimum growth temperature

[0045] After strain IMAU12449 was cultured for 36 h under different temperature conditions (20 °C, 30 °C, 37 °C, 45 °C, 55 °C), there were significant differences in its growth conditions (P < 0.05). The strain had the fastest growth rate at 37 °C, and the cell density showed a significant increasing trend from 2 h to 14 h, and then gradually leveled off. When cultured at 20 °C and 55 °C, the cell density increased slowly. Therefore, the optimum growth temperature of the strain was determined to be 37 °C (as Figure 2 shown).

[0046] 2. Determination of the growth curve

[0047] The growth curve of strain IMAU12449 continuously cultured for 36 h at 37 °C in MRS + L-cysteine hydrochloride medium is as Figure 3 shown in A and Figure 3 shown in B. The initial inoculation amount of strain IMAU12449 was 2.30×10 7 CFU / mL, and the lag phase was 0 - 2 h. During this period, the cell number increased slowly, and the pH value showed a slow downward trend; at 2 h, it began to enter the logarithmic phase, and the viable cell count, cell density OD 600nm value increased rapidly, the acid production increased, and the pH value decreased rapidly; at 14 h, the viable cell count reached the maximum value, which was 1.25×109 CFU / mL. After 14 h, the viable cell count decreased slowly and the cell density OD 600nm value remained stable.

[0048] 3. API50 CHL test

[0049] The results of the API50 CHL test are shown in Table 1 below. Strain IMAU12449 can utilize L - arabinose, D - ribose, D - xylose, D - galactose, D - glucose, D - fructose, D - maltose, D - lactose, D - melibiose, D - sucrose, D - melezitose, D - raffinose and D - turanose.

[0050] Table 1 API50 CHL results of strain IMAU12449

[0051]

[0052]

[0053] Note: "+" indicates utilization and "-" indicates non - utilization.

[0054] III. Determination of the tolerance of B. longum subsp. longum IMAU12449 to simulated artificial gastrointestinal fluids

[0055] The strain IMAU12449 activated to the third generation was prepared into a bacterial suspension. The bacterial suspension was serially diluted for counting to obtain the viable cell count of the original bacterial solution. 1.0 mL of the bacterial suspension was added to the prepared artificial gastric juice (9.0 mL), and it was placed in a 37 °C water bath for anaerobic culture. After 3 h, the viable cells were counted. At the same time, 1.0 mL of the liquid after 3 h of culture was transferred into the prepared artificial intestinal juice (9.0 mL), and the viable cells were counted at 4 h and 8 h respectively. The calculation formulas are as follows:

[0056] Survival rate in artificial gastric juice (%) = N1 / N0 × 100%;

[0057] Survival rate in artificial intestinal juice (%) = N2 / N1 × 100%;

[0058] Where: N0: represents the viable cell count of the original bacterial solution (CFU / mL);

[0059] N1: represents the viable cell count after 3 h of culture in gastric juice (CFU / mL);

[0060] N2: represents the viable cell count after 4 h and 8 h of culture in intestinal juice (CFU / mL).

[0061] As shown in Table 2 below, the survival rate of strain IMAU12449 in gastric juice was 62.38%. As shown in Table 3 below, the survival rate after 4 hours in intestinal juice was 51.49%, and the survival rate after 8 hours was 40.30%. It has a good ability to tolerate artificial gastrointestinal fluids.

[0062] Table 2 Evaluation of the tolerance of strain IMAU12449 to simulated artificial gastric juice

[0063]

[0064] Table 3 Evaluation of the tolerance of strain IMAU12449 to simulated artificial intestinal juice

[0065]

[0066] IV. Safety evaluation of B. longum subsp. longum IMAU12449

[0067] 1. Antibiotic susceptibility test

[0068] Referring to the international standard ISO10932-2010, the growth of strain IMAU12449 in a medium containing 12 different antibiotics was determined, and its minimum inhibitory concentration value (MIC) was calculated. The minimum inhibitory concentration value of each strain was compared with the resistance standard of Bifidobacterium published by the European Food Safety Authority (EFSA). If the minimum inhibitory concentration value of the strain was higher than the resistance threshold, it was resistant; if it was less than or equal to the threshold, it was sensitive. The sensitivity results of IMAU12449 to 12 antibiotics are shown in Table 4. The results show that IMAU12449 is sensitive to streptomycin, neomycin, tetracycline, ampicillin, linezolid, ciprofloxacin, chloramphenicol and trimethoprim; it is resistant to kanamycin, clindamycin, erythromycin and rifampicin.

[0069] Table 4 Antibiotic susceptibility of strain IMAU12449

[0070] Serial number Antibiotic IMAU12449 Serial number Antibiotic IMAU12449 1 Streptomycin Sensitive 7 Linezolid Sensitive 2 Kanamycin Tolerant 8 Erythromycin Tolerant 3 Neomycin Sensitive 9 Rifampicin Tolerant 4 Clindamycin Tolerant 10 Ciprofloxacin Sensitive 5 Tetracycline Sensitive 11 Chloramphenicol Sensitive 6 Ampicillin Sensitive 12 Trimethoprim Sensitive

[0071] 2. Hemolytic activity

[0072] The evaluation of the hemolytic activity of strain IMAU12449 showed that there was no obvious change around the colonies grown after culturing on a 5% sheep blood plate at 37°C for 72 hours, indicating that the hemolytic type of IMAU12449 was γ-hemolysis, that is, it did not have hemolytic activity.

[0073] 3. Nitrate reductase activity

[0074] The nitrate reductase activity of strain IMAU12449 was determined by the color change of nitrate medium, and the results showed that the strain was negative for nitrate reductase.

[0075] V. Bacteriostatic ability of B. longum subsp. longum IMAU12449

[0076] The bacteriostatic abilities of B. longum subsp. longum IMAU12449 against Escherichia coli, Staphylococcus aureus, Bacillus cereus, Shigella flexneri and Salmonella were determined. First, Escherichia coli, Staphylococcus aureus, Bacillus cereus, Shigella flexneri and Salmonella were activated with LB medium, and the pathogenic bacteria were diluted to 10 4 -10 5 CFU / mL with sterile normal saline. Then, 100 μL was taken and spread on LB agar medium. Three sterile Oxford cups were placed in the plate, and 100 μL of the fermentation supernatant of B. longum subsp. longum IMAU12449 (prepared by centrifuging at 4000 r / min for 10 min and filtering with a 0.22 μm filter membrane) was placed in each Oxford cup. The plate was slowly placed in an incubator at 37 °C for 24 h, and then the size of the inhibition zone was observed.

[0077] The bacteriostatic results are as Figure 4 shown. B. longum subsp. longum IMAU12449 showed different degrees of bacteriostatic ability against common pathogenic bacteria. Among them, the strain IMAU12449 had the best inhibitory effect on Bacillus cereus (24.67 ± 0.30 mm). Followed by Staphylococcus aureus and Salmonella.

[0078] Example 2

[0079] Application of B. longum subsp. longum IMAU12449 in the preparation of fermented milk

[0080] 1. Preparation of fermented milk

[0081] (1) The milk was preheated to 60 °C, then mixed with 6.5 wt% sucrose, homogenized at 20 MPa, pasteurized at 95 °C for 3 min, and quickly cooled to obtain pretreated milk;

[0082] (2) The bacteria were inoculated into the pretreated milk and anaerobically cultured and fermented at 37 °C for 6.5 h until the pH reached 4.5 to obtain fermented milk.

[0083] S1. The soy milk was preheated to 60 °C, then mixed with 8 wt% sucrose and 27 wt% distilled water, homogenized at 20 MPa, pasteurized at 95 °C for 3 min, and quickly cooled to obtain pretreated soy milk;

[0084] S2. Inoculate the bacteria into the pretreated soymilk and anaerobically culture and ferment at 37 °C for 6.5 h until the pH reaches 4.5 to obtain fermented soymilk.

[0085] The inoculum amount is shown in Table 5. Aliquot the prepared fermented milk samples into sample bottles, cool them, and store them at 4 °C. Measure various indexes of the fermented milk samples at 1 d, 7 d, 14 d, and 21 d of storage.

[0086] Table 5 Experimental groups and inoculum amounts of IMAU12449

[0087]

[0088] 2. Determination of acidity during fermentation and storage of fermented milk

[0089] As can be seen from A in Figure 5 and B in Figure 5 , at the initial stage of fermentation, there were no obvious changes in the pH values and titratable acidities of the N group and the D group. After 2 h of fermentation, the pH values of the N group and the D group decreased rapidly and the titratable acidities showed a significant upward trend. The N group reached the fermentation end point at about 6.5 h, and the D group reached the fermentation end point at about 7.5 h. At this time, in the N group, the titratable acidity of the control group was 83 °T, and the titratable acidity of the IMAU12449 group was 84 °T; in the D group, the titratable acidity of the control group was 73 °T, and the titratable acidity of the IMAU12449 group was 76 °T. There was no significant difference in the titratable acidities between the N group and the D group at the fermentation end point (P > 0.05).

[0090] At the end of storage, the titratable acidity of the N group IMAU12449 group was the highest, at 103 °T. During storage, the pH values of the four groups of fermented milk decreased continuously. At the end of storage, the pH values of the N group IMAU12449 group were the lowest, at 4.1, and the pH values of the D group IMAU12449 group were the lowest, at 4.2. The pH values of the fermented milk in the N group and the D group were within the optimal drinking range during storage.

[0091] 3. Determination of viable count during fermentation and storage of fermented milk

[0092] As the fermentation and storage time of the four groups of fermented milk extended, the viable count showed a trend of first increasing and then decreasing. The results are shown in Figure 6 . During fermentation, the viable counts of the four groups of fermented milk all showed an upward trend. The viable count reached the highest at 1 d after fermentation to after-ripening. At this time, the viable count of IMAU12449 in the N group was 3.08×10 8 CFU / mL, and the viable count of IMAU12449 in the D group was 1.60×10 8CFU / mL. After 7 days of storage, the viable cell counts of the fermented milk in the N group and the D group began to decline slowly. At 21 days, the viable cell counts of IMAU12449 in the N group and the D group were 2.70×10 7 CFU / mL and 1.42×10 7 CFU / mL respectively, both greater than the national standard of 10 6 CFU / mL. This indicates that the addition of IMAU12449 to fermented milk can exert its probiotic effect, and compared with fermented soy milk, the fermentation activity of IMAU12449 in cow's milk is more excellent.

[0093] 4. Determination of viscosity during fermentation and storage of fermented milk

[0094] Viscosity is one of the important indicators for evaluating the quality of fermented milk, and the factors affecting viscosity include acidity, temperature, concentration, etc. Viscosity is produced by the action of lactic acid, the acidic metabolite of bacteria, and lactate dehydrogenase on casein. During fermentation, the viscosity decreases due to two factors: the coagulation of casein and the secretion of exopolysaccharides by bacteria. As Figure 7 can be seen (the letters in the figure represent significant differences), from 0 h to 2 h of fermentation, the viscosity values of the N group and the D group increased slowly with little change, and at 4 h of fermentation, the viscosity values increased significantly (P<0.05). At the end of fermentation, the viscosity of the IMAU12449 group in the N group was 2050 mPa·s, and the viscosity of the IMAU12449 group in the D group was 2024 mPa·s. During the whole fermentation process, there was a significant difference in the viscosity values between the N group and the D group (P<0.05). During storage, the viscosity values of the four groups of fermented milk showed an upward trend from 1 day to 14 days. At 14 days of storage, the viscosity reached the maximum value, and the viscosity values of the IMAU12449 group in the N group and the IMAU12449 group in the D group were 2545 mPa·s and 2436 mPa·s respectively. During the period from 14 days to 21 days of storage, the pH values of each group of fermented milk showed a gradually decreasing trend, and the acidity also increased accordingly. During storage, the structure of proteins was damaged, the solubility gradually decreased with the extension of time, and whey separation occurred, which was the main reason for the decrease in viscosity.

[0095] 5. Determination of water-holding capacity during fermentation and storage of fermented milk

[0096] Water-holding capacity plays a key role in evaluating the quality of fermented milk. Low water-holding capacity often leads to whey separation of fermented milk during storage, which will have an adverse impact on the sensory characteristics, taste experience and color of the product. Therefore, improving the water-holding capacity of the product is of great significance for extending the shelf life of fermented milk. As Figure 8As shown (the letters in the figure indicate significant differences), during fermentation, the water-holding capacity of the four groups of fermented milk showed an increasing trend. At the end of fermentation, the water-holding capacity of group N (IMAU12449 group) was the highest, at 61.43%. During the storage period from 1 d to 14 d, the water-holding capacity of the four groups of fermented milk continued to increase and reached the maximum value at 14 d. During the storage period from 14 d to 21 d, the water-holding capacity showed a slow downward trend, which was consistent with the above results of viscosity. During the entire storage period, the water-holding capacity of group N (IMAU12449 group) was the highest. The results showed that compared with the control group, adding strain IMAU12449 could improve the water-holding capacity of fermented milk to a certain extent, thereby enhancing its storage stability.

[0097] 6. Sensory evaluation of fermented milk during storage

[0098] As Figure 9 shown, it is the sensory score chart of the fermented milk samples of group N and group D after 21 d of storage. It can be seen that as the storage time extends, the sensory score of fermented milk gradually increases during the period from 1 d to 14 d, which may be related to the increase in the viscosity and water-holding capacity of fermented milk. However, during the storage period from 14 d to 21 d, due to the continuous accumulation of acidity in the fermented milk, the water-holding capacity of the fermented milk decreased and whey separated out, so the sensory scores of the fermented milk samples in each group decreased. The sensory score of group D was lower than that of group N. Whey separated out in the later stage of storage, resulting in a decrease in the viscosity of the fermented milk and affecting the taste. At the end of storage, the score of group N (IMAU12449 group) was the highest, at 84 points. From the results of sensory scores, it can be seen that fermented cow milk has the sweet taste of raw milk and is also superior to fermented soy milk in texture and taste. Therefore, compared with fermented soy milk, fermented cow milk is more popular among the public.

[0099] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. Bifidobacterium longum subsp. longum IMAU12449, characterized in that, The Bifidobacterium longum subsp. longum IMAU12449 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on July 8, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is: CGMCC NO. 31225; the Bifidobacterium longum subsp. longum IMAU12449 is used for preparing fermented milk and fermented soy milk, improving the viscosity of fermented milk and fermented soy milk, improving the viscosity of fermented milk stored for 14 days and fermented soy milk stored for 14 days, improving the water-holding capacity of fermented milk and fermented soy milk, and improving the water-holding capacity of fermented milk during storage and fermented soy milk during storage.

2. The application of the Bifidobacterium longum subsp. longum IMAU12449 as described in claim 1 in the preparation of fermented milk.

3. The application of the Bifidobacterium longum subsp. longum IMAU12449 as described in claim 1 in the preparation of fermented soy milk.

4. A method for preparing fermented milk, characterized in that, The preparation using the Bifidobacterium longum subsp. longum IMAU12449 as described in claim 1 includes the following steps: (1) Preheat the milk, then mix it with sucrose, homogenize it, pasteurize it, and quickly cool it to obtain pretreated milk. (2) Inoculate the Bifidobacterium longum subsp. longum IMAU12449 and the starter culture into the pretreated milk in step (1), and ferment until the pH reaches 4.5 - 4.6 to obtain fermented milk.

5. The method for preparing fermented milk according to claim 4, wherein In step (1), the preheating treatment preheats the milk to 55 - 65 °C; the dosage of sucrose in step (1) is 6 - 7 wt%; the pressure of homogenization in step (1) is 15 - 25 MPa; the temperature of pasteurization in step (1) is 95 °C, and the time of pasteurization is 3 min.

6. The method for preparing fermented milk according to claim 4, wherein The inoculation amount of *Bifidobacterium longum* subsp. *longum* IMAU12449 described in step (2) is 5×10 6 CFU / mL; the starter culture described in step (2) is composed of *Lactobacillus delbrueckii* subsp. *bulgaricus* and *Streptococcus thermophilus*, and the inoculation amount of the starter culture is 0.003 wt%; the fermentation in step (2) is anaerobic culture, the temperature of the fermentation is 37 °C, and the time of the fermentation is 6-7 h.

7. A method for preparing fermented soy milk, characterized in that, The preparation using the Bifidobacterium longum subsp. longum IMAU12449 as described in claim 1 includes the following steps: S1. Preheat the soy milk, then mix it with sucrose and distilled water, homogenize it, pasteurize it, and quickly cool it to obtain pretreated soy milk. S2. Inoculate the Bifidobacterium longum subsp. longum IMAU12449 and the starter culture into the pretreated soy milk in S1, and ferment until the pH reaches 4.5 - 4.6 to obtain fermented soy milk.

8. The method for preparing fermented soybean milk according to claim 7, characterized in that, In S1, the preheating treatment preheats the soy milk to 55 - 65 °C; the dosage of sucrose in S1 is 7 - 9 wt%; the dosage of distilled water in S1 is 26 - 28 wt%; the pressure of homogenization in S1 is 15 - 25 MPa; the temperature of pasteurization in S1 is 95 °C, and the time of pasteurization is 3 min.

9. The method for preparing fermented soybean milk according to claim 7, wherein, The inoculation amount of Bifidobacterium longum subsp. longum IMAU12449 described in S2 is 5×10 6 CFU / mL; the starter culture described in S2 is composed of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus, and the inoculation amount of the starter culture is 0.003 wt%; the fermentation described in S2 is anaerobic culture, the temperature of the fermentation is 37°C, and the time of the fermentation is 6-7 h.

10. The fermented soy milk prepared by the method for preparing fermented soy milk as described in any one of claims 7 - 9.

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