Bifidobacterium longum subsp. Longum IMAU12449 and application thereof

Abstract

The invention discloses bifidobacterium longum subsp. Longum IMAU12449 and application thereof, belongs to the technical field of microorganisms, and provides the bifidobacterium longum subsp. Longum IMAU12449, the bifidobacterium longum subsp. Longum IMAU12449 is preserved in the China General Microbiological Culture Collection Center on July 8, 2024, the preservation address is No. 3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO. The preservation number is CGMCC (China General Microbiological Culture Collection Center) The invention provides application of the bifidobacterium longum subsp. Longum IMAU12449 in preparation of fermented cow milk and / or soybean milk. The bifidobacterium longum subsp. Longum IMAU12449 is used as a potential probiotic to prepare cow milk and soybean milk, and the prepared fermented milk is stable in viable count, moderate in acidity and free of whey precipitation.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to Bifidobacterium longum subspecies longum IMAU12449 and an application thereof. Background Art

[0002] Currently, most traditional fermented milk starter cultures are fermented using a commercial starter culture consisting of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus.

[0003] Bifidobacterium is the name of a genus of bacteria. Bifidobacterium is a genus of Gram-positive, non-motile, rod-shaped cells, sometimes forked at one end, strictly anaerobic bacteria, widely found in the digestive tract, vagina and oral cavity of humans and animals. Bacteria of the genus Bifidobacterium are an important member 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 and food science. The addition of probiotic bifidobacteria can change the quality of fermented milk, and some fermented milk containing probiotic bifidobacteria ingredients also have a probiotic effect on the body.

[0004] However, at present, the resources of bifidobacteria from the intestine are relatively scarce, and there are few bifidobacteria with excellent probiotic properties. In addition, there are relatively few studies on soy milk fermentation. In summary, screening a strain of bifidobacteria with potential probiotic ability and adding it as a probiotic to fermented cow's milk and soy milk is of great significance for the development of intestinal probiotic fermented cow's milk and soy milk. Summary of the invention

[0005] In order to solve the above technical problems, the present invention proposes a Bifidobacterium longum subsp. longum IMAU12449 and an application thereof. The present invention utilizes Bifidobacterium longum subsp. longum IMAU12449 as a potential probiotic, and performs compound fermentation with Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus to prepare cow's milk and soy milk. The process is simple, and the prepared fermented milk has a stable number of viable bacteria, moderate acidity, and no whey precipitation.

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

[0007] The present invention also provides the use of the Bifidobacterium longum subspecies longum IMAU12449 in preparing fermented milk.

[0008] The present invention also provides the use of the Bifidobacterium longum subspecies longum IMAU12449 in preparing fermented soy milk.

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

[0010] (1) The milk is preheated, then mixed with sucrose, homogenized, pasteurized, and rapidly cooled to obtain pretreated milk;

[0011] (2) inoculating the Bifidobacterium longum subspecies longum IMAU12449 and the basic starter into the pretreated milk of step (1), and fermenting until the pH value is 4.5-4.6 to obtain fermented milk.

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

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

[0014] The present invention also provides a method for preparing fermented soymilk, which is prepared by using the Bifidobacterium longum subspecies longum IMAU12449, and comprises the following steps:

[0015] S1, preheating the soy milk, then mixing it with sucrose and distilled water, homogenizing it, pasteurizing it, and rapidly cooling it to obtain pretreated soy milk;

[0016] S2, inoculating the Bifidobacterium longum subspecies longum IMAU12449 and the basic starter into the pretreated soymilk described in S1, and fermenting until the pH value is 4.5-4.6 to obtain fermented soymilk.

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

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

[0019] The invention also provides the method for preparing fermented soymilk and the fermented soymilk obtained.

[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 carries out compound fermentation with Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus to prepare cow milk and soybean milk. The process is simple, and the number of viable bacteria in the fermented milk is stable, the acidity is moderate, and no whey precipitation occurs. After storage at 4°C for 21 days, the number of viable bacteria in the fermented cow milk and fermented soybean milk is still 10 7 CFU / mL or more. Therefore, the present invention broadens the application prospects and industrializable resources of Bifidobacterium longum subspecies long in fermented dairy products, discloses the function of Bifidobacterium longum subspecies longum IMAU12449 in improving the whey precipitation and texture of fermented milk during the shelf life, and its excellent survival ability during the shelf life. The present invention obtains fermented milk with stable texture, and provides a new candidate strain for the development of intestinal probiotic fermented milk. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 : The colony and morphology of Bifidobacterium longum subspecies longum IMAU12449, wherein A is the colony image and B is the morphology image;

[0024] Figure 2 This is a graph showing the optimum growth temperature for Bifidobacterium longum subsp. longum IMAU12449;

[0025] Figure 3 This is a growth curve of Bifidobacterium longum subspecies longum IMAU12449 in MRS+L-cysteine ​​hydrochloride medium, where A is the viable count and pH change, and B is the OD value change;

[0026] Figure 4 This is a graph showing the antibacterial ability of Bifidobacterium longum subspecies longum IMAU12449;

[0027] Figure 5 The graphs of pH and titrated acidity changes during storage of the fermented milk prepared in Example 2, wherein A is a graph of pH changes, N: IMAU12449 represents milk fermented with the basic fermentation agent and Bifidobacterium longum subspecies IMAU12449, N: the control group represents milk fermented with the basic fermentation agent, D: IMAU12449 represents soy milk fermented with the basic fermentation agent and Bifidobacterium longum subspecies IMAU12449, and D: the control group represents soy milk fermented with the basic fermentation agent; B is a graph of titrated acidity changes, N: IMAU12449 represents milk fermented with the basic fermentation agent and Bifidobacterium longum subspecies IMAU12449, N: the control group represents milk fermented with the basic fermentation agent, D: IMAU12449 represents soy milk fermented with the basic fermentation agent and Bifidobacterium longum subspecies IMAU12449, and D: the control group represents soy milk fermented with the basic fermentation agent;

[0028] Figure 6 This is a graph showing the change in the number of live bacteria during storage of the fermented milk prepared in Example 2, N: IMAU12449 represents the basic starter and Bifidobacterium longum subspecies IMAU12449 co-fermented cow milk, N: the control group represents the basic starter fermented cow milk, D: IMAU12449 represents the basic starter and Bifidobacterium longum subspecies IMAU12449 co-fermented soy milk, D: the control group represents the basic starter fermented soy milk;

[0029] Figure 7 This is a graph showing the viscosity change of the fermented milk prepared in Example 2 during storage, N: IMAU12449 represents the basic starter and Bifidobacterium longum subspecies IMAU12449 co-fermented cow milk, N: the control group represents the basic starter fermented cow milk, D: IMAU12449 represents the basic starter and Bifidobacterium longum subspecies IMAU12449 co-fermented soy milk, D: the control group represents the basic starter fermented soy milk;

[0030] Figure 8 This is a graph showing the change in water holding capacity of the fermented milk prepared in Example 2 during storage, N: IMAU12449 represents the basic starter and Bifidobacterium longum subspecies IMAU12449 co-fermented cow milk, N: the control group represents the basic starter fermented cow milk, D: IMAU12449 represents the basic starter and Bifidobacterium longum subspecies IMAU12449 co-fermented soy milk, D: the control group represents the basic starter fermented soy milk;

[0031] Fig. 9 It is a sensory score graph of fermented milk during storage prepared in Example 2, NI1 represents the basic fermentation agent and Bifidobacterium longum subspecies IMAU12449 co-fermented milk for 1 day, NI7 represents the basic fermentation agent and Bifidobacterium longum subspecies IMAU12449 co-fermented milk for 7 days, NI14 represents the basic fermentation agent and Bifidobacterium longum subspecies IMAU12449 co-fermented milk for 14 days, NI21 represents the basic fermentation agent and Bifidobacterium longum subspecies IMAU12449 co-fermented milk for 21 days, NC1 represents the basic fermentation agent fermented milk for 1 day, NC7 represents the basic fermentation agent fermented milk for 7 days, NC14 represents the basic fermentation agent fermented milk for 14 days, and NC21 represents the basic fermentation agent fermented milk for 21 days. 12449 co-fermented soy milk and stored for 21 days; DI1 represents soy milk fermented by basic fermentation agent and Bifidobacterium longum subsp. IMAU12449 and stored for 1 day; DI7 represents soy milk fermented by basic fermentation agent and Bifidobacterium longum subsp. IMAU12449 and stored for 7 days; DI14 represents soy milk fermented by basic fermentation agent and Bifidobacterium longum subsp. IMAU12449 and stored for 14 days; DI21 represents soy milk fermented by basic fermentation agent and Bifidobacterium longum subsp. IMAU12449 and stored for 21 days; DC1 represents soy milk fermented by basic fermentation agent and stored for 1 day; DC7 represents soy milk fermented by basic fermentation agent and stored for 7 days; DC14 represents soy milk fermented by basic fermentation agent and stored for 14 days; DC21 represents soy milk fermented by basic fermentation agent and stored for 21 days. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] Sources of materials used in the present invention: Basic fermentation agent (consisting of Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus thermophilus) was purchased from Chr. Hansen ( Denmark); DNA extraction kit was purchased from (KG203-02, Beijing, China).

[0038] Example 1

[0039] 1. Isolation and identification of strains

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

[0041] DNA was extracted using a DNA extraction kit. The extracted DNA was amplified by PCR and then sequenced by 16S rRNA. The sequencing results were compared with the EZBioCloud database to determine the species status. The bifidobacteria isolated were screened, and finally a strain of Bifidobacterium longumsubsp.longum IMAU12449 with excellent probiotic properties was obtained.

[0042] The strain was identified physiologically and biochemically and observed morphologically. The characteristics are as follows: The strain is a Gram-positive, catalase-negative anaerobic bacterium. Figure 1 Middle A and Figure 1 As shown in B, the colonies of strain IMAU12449 on the solid medium of MRS+L-cysteine ​​hydrochloride are small, milky white, round colonies with a raised center, regular edges, and a shiny surface. Under an optical microscope, they appear to be curved rods and branches, arranged in a V shape. This strain is consistent with existing morphological studies on B. longum subsp. longum.

[0043] 2. Study on the physiological and biochemical characteristics of B. longum subsp. longum IMAU12449

[0044] 1. Optimum growth temperature

[0045] After culturing IMAU12449 at different temperatures (20℃, 30℃, 37℃, 45℃, 55℃) for 36h, there were significant differences in its growth (P<0.05). The strain grew fastest at 37℃, and the cell density showed a significant growth trend from 2h to 14h, and gradually stabilized after 14h. When cultured at 20℃ and 55℃, the cell density grew slowly, so the optimal growth temperature of the strain was determined to be 37℃ (such as Figure 2 shown).

[0046] 2. Determination of Growth Curve

[0047] The growth curve of strain IMAU12449 in MRS + L-cysteine ​​hydrochloride medium at 37°C for 36 h is shown in Figure 3 Middle A and Figure 3 As shown in B. The initial inoculum size of strain IMAU12449 was 2.30×10 7 CFU / mL, the lag phase is 0-2h, during which the number of cells grows slowly and the pH value shows a slowly decreasing trend; after 2h, it enters the logarithmic phase, the number of viable bacteria and cell density OD 600nm The value increased rapidly, the acid production increased, and the pH value dropped rapidly; the number of viable bacteria reached the maximum value of 1.25×109 CFU / mL, after 14h, the number of viable bacteria slowly decreased, and the cell density OD 600nm The value remains stable.

[0048] 3. API50 CHL test

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

[0050] Table 1 API50 CHL results of strain IMAU12449

[0051]

[0052]

[0053] Note: “+” means use, “-” means not use.

[0054] 3. Tolerance test of B. longum subsp. longum IMAU12449 in simulated artificial gastrointestinal fluid

[0055] Prepare the bacterial suspension of the activated third-generation strain IMAU12449. Perform gradient dilutions on the bacterial suspension and count the number of live bacteria in the original bacterial solution. Add 1.0 mL of the bacterial suspension to the prepared artificial gastric fluid (9.0 mL), place it in a 37°C water bath for anaerobic culture, and count the live bacteria after 3 hours. At the same time, transfer 1.0 mL of the liquid after 3 hours of culture to the prepared artificial intestinal fluid (9.0 mL), and count the live bacteria after 4 hours and 8 hours, respectively. The calculation formula is as follows:

[0056] Artificial gastric juice survival rate (%) = N1 / N0 × 100%;

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

[0058] Where: N0: represents the number of viable bacteria in the original bacterial solution (CFU / mL);

[0059] N1: represents the number of viable bacteria cultured in gastric juice for 3 h (CFU / mL);

[0060] N2: represents the number of viable bacteria (CFU / mL) cultured in intestinal fluid for 4h and 8h.

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

[0062] Table 2 Evaluation of strain IMAU12449's resistance to simulated artificial gastric fluid

[0063]

[0064] Table 3 Evaluation of strain IMAU12449's resistance to simulated artificial intestinal fluid

[0065]

[0066] 4. Safety evaluation of B. longum subsp. longum IMAU12449

[0067] 1. Antibiotic sensitivity test

[0068] According to the international standard ISO10932-2010, the growth of strain IMAU12449 in a culture medium containing 12 different antibiotics was measured, and its minimum inhibitory concentration (MIC) was calculated. The minimum inhibitory concentration value of each strain was compared with the bifidobacterium resistance standard issued by the European Food Safety Authority (EFSA). If the minimum inhibitory concentration value of the strain is higher than the resistance threshold, it is resistant, and if it is less than or equal to the threshold, it is 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 tolerant to kanamycin, clindamycin, erythromycin and rifampicin.

[0069] Table 4 Antibiotic sensitivity of strain IMAU12449

[0070] Serial number antibiotic IMAU12449 Serial number antibiotic IMAU12449 1 Streptomycin sensitive 7 Linezolid sensitive 2 Kanamycin Tolerance 8 Erythromycin Tolerance 3 Neomycin sensitive 9 Rifampin Tolerance 4 Clindamycin Tolerance 10 Ciprofloxacin sensitive 5 tetracycline sensitive 11 Chloramphenicol sensitive 6 Ampicillin sensitive 12 Trimethoprim sensitive

[0071] 2. Hemolytic activity

[0072] The hemolytic activity of strain IMAU12449 was evaluated. The results showed that there was no obvious change around the colonies grown by IMAU12449 after culturing on 5% sheep blood plates at 37°C for 72 hours, indicating that the hemolytic type of IMAU12449 was γ-hemolytic, that is, it had no hemolytic activity.

[0073] 3. Nitrate reductase activity

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

[0075] 5. Antibacterial ability of B. longum subsp. longum IMAU12449

[0076] The antibacterial ability of B. longum subsp. longum IMAU12449 against Escherichia coli, Staphylococcus aureus, Bacillus cereus, Shigella flexneri and Salmonella was 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, and then 100 μL was spread on LB agar medium, and 3 sterile Oxford cups were placed on the plate, and 100 μL of L. longum subsp. longum IMAU12449 fermentation supernatant (prepared by centrifugation at 4000 r / min for 10 min and filtration with a 0.22 μm filter membrane) was placed in each Oxford cup. The plate was slowly placed in a 37°C incubator for 24 hours and the size of the inhibition zone was observed.

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

[0078] Example 2

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

[0080] 1. Preparation of fermented milk

[0081] (1) preheating milk to 60° C., then mixing with 6.5 wt % sucrose, homogenizing at 20 MPa, pasteurizing at 95° C. for 3 min, and rapidly cooling to obtain pretreated milk;

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

[0083] S1, preheating the soy milk to 60°C, then mixing with 8wt% sucrose and 27wt% distilled water, homogenizing at 20MPa, pasteurizing at 95°C for 3min, and rapidly cooling to obtain pretreated soy milk;

[0084] S2. Inoculate the bacteria into the pretreated soy milk, and ferment at 37° C. for 6.5 hours until the pH reaches 4.5 to obtain fermented soy milk.

[0085] The inoculum amount is shown in Table 5. The prepared fermented milk samples were dispensed into sample bottles, stored at 4°C after cooling, and various indicators of the fermented milk samples were measured at 1d, 7d, 14d, and 21d of storage.

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

[0087]

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

[0089] Depend on Figure 5 Middle A and Figure 5 As shown in Figure B, at the beginning of fermentation, the pH value and titrated acidity of group N and group D did not change significantly. After 2 hours of fermentation, the pH value of group N and group D decreased rapidly, and the titrated acidity showed a significant upward trend. Group N reached the end of fermentation at about 6.5 hours, and group D reached the end of fermentation at about 7.5 hours. At this time, in group N, the titrated acidity of the control group was 83°T, and the titrated acidity of the IMAU12449 group was 84°T; in group D, the titrated acidity of the control group was 73°T, and the titrated acidity of the IMAU12449 group was 76°T. There was no significant difference in titrated acidity between group N and group D at the end of fermentation (P>0.05).

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

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

[0092] As the fermentation and storage time of the four groups of fermented milk increased, the number of live bacteria increased first and then decreased. Figure 6 During the fermentation period, the number of live bacteria in the four groups of fermented milk showed an upward trend. The number of live bacteria reached the highest level from the end of fermentation to 1 day after ripening. At this time, the number of live bacteria in group N IMAU12449 was 3.08×10 8 CFU / mL, the viable count of IMAU12449 in group D was 1.60×10 8After 7 days of storage, the number of viable bacteria in the fermented milk of groups N and D began to slowly decrease. At 21 days, the number of viable bacteria of IMAU12449 in groups N and D was 2.70×10 7 CFU / mL and 1.42×10 7 CFU / mL, all greater than the national standard of 10 6 CFU / mL. This indicates that adding 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 better.

[0093] 4. Determination of Viscosity of Fermented Milk during Fermentation and Storage

[0094] Viscosity is one of the important indicators for evaluating the quality of fermented milk. Factors that affect viscosity include acidity, temperature, concentration, etc. Viscosity is produced by the action of lactic acid and lactate dehydrogenase, the acidic metabolites of bacteria, on casein. During the fermentation process, the viscosity decreases due to the coagulation of casein and the secretion of extracellular polysaccharides by bacteria. Figure 7 It can be seen (the letters in the figure indicate significant differences) that the viscosity values ​​of group N and group D increased slowly from 0h to 2h of fermentation, with little change, and the viscosity value increased significantly at 4h of fermentation (P<0.05). At the end of fermentation, the viscosity of group N IMAU12449 was 2050mPa·s, and the viscosity of group D IMAU12449 was 2024mPa·s. During the entire fermentation process, the viscosity value of group N was significantly different from that of group D (P<0.05). During storage, the viscosity values ​​of the four groups of fermented milk showed an upward trend from 1d to 14d, and reached the maximum value at 14d of storage. The viscosity values ​​of group N IMAU12449 and group D IMAU12449 were 2545mPa·s and 2436mPa·s, respectively. During storage from 14d to 21d, the pH value of the fermented milk in each group showed a trend of gradual decrease, and the acidity also increased accordingly. During storage, the structure of protein is destroyed, the solubility gradually decreases with time, and whey precipitates, which is the main reason for the decrease in viscosity.

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

[0096] Water holding capacity plays a key role in evaluating the quality of fermented milk. Low water holding capacity often leads to whey precipitation during storage, which in turn has an adverse effect on the sensory properties, 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. Figure 8As shown in the figure (the letters in the figure indicate significant differences), during the fermentation period, the water holding capacity of the four groups of fermented milk showed a continuous upward trend. When the fermentation end point was reached, the water holding capacity of the N group IMAU12449 group was the highest, at 61.43%. During the storage period of 1d to 14d, the water holding capacity of the four groups of fermented milk continued to increase and reached the maximum value at 14d. During the storage period of 14d to 21d, the water holding capacity showed a slow downward trend, which was consistent with the above viscosity results. During the entire storage period, the water holding capacity of the N group IMAU12449 group was the highest. The results showed that compared with the control group, the addition of strain IMAU12449 can 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] like Fig. 9 As shown in the figure, the sensory score of fermented milk samples in groups N and D after storage for 21 days. It can be seen that with the extension of storage time, the sensory score of fermented milk gradually increased during the period of 1d to 14d, which may be related to the increase in viscosity and water holding capacity of fermented milk. During storage for 14d to 21d, the acidity in the fermented milk continued to accumulate, resulting in a decrease in the water holding capacity of the fermented milk and whey precipitation, so the sensory scores of fermented milk samples in each group decreased. Compared with group N, group D had a lower sensory score. In the later stage of storage, whey precipitation occurred, resulting in a decrease in the viscosity of the fermented milk and affecting the taste. At the end of storage, group N IMAU12449 scored the highest with 84 points. From the sensory score results, it can be seen that fermented cow's milk has the sweet taste of raw milk, and is also better than fermented soy milk in texture and taste. Therefore, compared with fermented soy milk, fermented cow's milk is more popular with the public.

[0099] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A Bifidobacterium longum subsp. longum IMAU12449, characterized in that The Bifidobacterium longum subspecies longum IMAU12449 was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on July 8, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is: CGMCC NO.31225.

2. Use of Bifidobacterium longum subspecies longum IMAU12449 as claimed in claim 1 in preparing fermented milk.

3. Use of Bifidobacterium longum subspecies longum IMAU12449 as claimed in claim 1 in preparing fermented soymilk.

4. A method for preparing fermented milk, characterized in that: The method is prepared by using the Bifidobacterium longum subspecies longum IMAU12449 described in claim 1, comprising the following steps: (1) The milk is preheated, then mixed with sucrose, homogenized, pasteurized, and rapidly cooled to obtain pretreated milk; (2) inoculating the Bifidobacterium longum subspecies longum IMAU12449 and the basic starter into the pretreated milk of step (1), and fermenting until the pH value is 4.5-4.6 to obtain fermented milk.

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

6. The method for preparing fermented milk according to claim 4, characterized in that: The inoculum size of Bifidobacterium longum subspecies longum IMAU12449 in step (2) is 5×10 6 CFU / mL; the basic fermentation agent in step (2) is composed of Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus thermophilus, and the inoculation amount of the basic fermentation agent is 0.003wt%; the fermentation in step (2) is anaerobic culture, the fermentation temperature is 37°C, and the fermentation time is 6 to 7h.

7. A method for preparing fermented soybean milk, characterized in that: The method is prepared by using the Bifidobacterium longum subspecies longum IMAU12449 described in claim 1, comprising the following steps: S1, preheating the soy milk, then mixing it with sucrose and distilled water, homogenizing it, pasteurizing it, and rapidly cooling it to obtain pretreated soy milk; S2, inoculating the Bifidobacterium longum subspecies longum IMAU12449 and the basic starter into the pretreated soymilk described in S1, and fermenting until the pH value is 4.5-4.6 to obtain fermented soymilk.

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

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

10. The fermented soymilk prepared by the method for preparing fermented soymilk according to any one of claims 7 to 9.

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