Composition containing Bifidobacterium breve i1088 and human milk oligosaccharide and its application

Abstract

The present invention discloses a composition containing Bifidobacterium breve i1088 and human milk oligosaccharides and its application, belonging to the technical field of microbial engineering. The Bifidobacterium breve i1088 disclosed in the present invention is deposited in the China Center for Type Culture Collection of the Institute of Microbiology, Chinese Academy of Sciences. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is May 8, 2023, and the deposit number is CGMCC NO. 27281. The Latin name is Bifidobacterium breve i1088. The Bifidobacterium breve i1088 has an adhesion effect on intestinal epithelial cells, protects the integrity of the intestinal epithelial cell layer, and can be applied to the preparation of fermented milk and probiotics. The strain survival rate in the prepared probiotic powder is high.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial engineering, and relates to a composition containing Bifidobacterium breve, specifically a composition containing Bifidobacterium breve i1088 and human milk oligosaccharides and its application. Background Art

[0002] Bifidobacterium breve is a bacterium of the genus Bifidobacterium, which is Gram-positive and is a dominant flora naturally present in the human colon. It plays an important role in regulating the balance of intestinal flora and promoting the normal development of the intestine. In order to effectively exert its probiotic function in the human gastrointestinal tract, Bifidobacterium breve must have good intestinal adhesion and tolerance to the gastrointestinal tract.

[0003] Human milk oligosaccharides are important bioactive components in human milk and are also one of the important factors affecting the composition of the infant intestinal flora. They can exert a prebiotic effect in the infant's intestine, guide the stability of the infant intestinal flora, establish a healthier intestinal barrier function, and promote the development of the mucosal immune system, but their colonization ability in the intestine is weak.

[0004] Therefore, there is an urgent need for a composition with stronger adhesion ability to intestinal epithelial cells. Summary of the Invention

[0005] To solve the above-mentioned deficiencies in the prior art, one object of the present invention is to provide a composition containing Bifidobacterium breve i1088 and human milk oligosaccharides to achieve the effect of improving the adhesion of intestinal epithelial cells; another object of the present invention is to provide the application of the above composition, and by compounding human milk oligosaccharides in Bifidobacterium breve i1088, to achieve the effect of preparing a probiotic powder for protecting the intestinal barrier.

[0006] To achieve the above object, the technical solutions adopted by the present invention are as follows.

[0007] A composition containing Bifidobacterium breve i1088 and human milk oligosaccharides, wherein the Bifidobacterium breve i1088 is preserved in the China Center for Type Culture Collection of the Institute of Microbiology, Chinese Academy of Sciences. The preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The preservation date is May 8, 2023, and the preservation number is CGMCC NO. 27281, and the Latin name is Bifidobacterium breve i1088.

[0008] As a limitation of the present invention, the full length of the 16S rDNA sequence of Bifidobacterium breve i1088 is 1403 bp, and the specific sequence is as shown in SEQ ID NO.1, which is:

[0009] ACACATGCAGTCGAACGGGATCCATCAAGCTTGCTTGGTGGTGAGAGTGGCGAA

[0010] CGGGTGAGTAATGCGTGACCGACCTGCCCCATGCACCGGAATAGCTCCTGGAAACGG

[0011] GTGGTAATGCCGGATGCTCCATCACACTGCATGGTGTGTTGGGAAAGCCTTTGCGGCA

[0012] TGGGATGGGGTCGCGTCCTATCAGCTTGATGGCGGGGTAACGGCCCACCATGGCTTCG

[0013] ACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACATTGGGACTGAGATACGGCCCAGA

[0014] CTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGC

[0015] GACGCCGCGTGAGGGATGGAGGCCTTCGGGTTGTAAACCTCTTTTGTTAGGGAGCAA

[0016] GGCATTTTGTGTTGAGTGTACCTTTCGAATAAGCACCGGCTAACTACGTGCCAGCAGC

[0017] CGCGGTAATACGTAGGGTGCAAGCGTTATCCGGAATTATTGGGCGTAAAGGGCTCGT

[0018] AGGCGGTTCGTCGCGTCCGGTGTGAAAGTCCATCGCTTAACGGTGGATCCGCGCCGGG

[0019] TACGGGCGGGCTTGAGTGCGGTAGGGGAGACTGGAATTCCCGGTGTAACGGTGGAAT

[0020] GTGTAGATATCGGGAAGAACACCAATGGCGAAGGCAGGTCTCTGGGCCGTTACTGAC

[0021] GCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCC

[0022] GTAAACGGTGGATGCTGGATGTGGGGCCCGTTCCACGGGTTCCGTGTCGGAGCTAAC

[0023] GCGTTAAGCATCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGAAATTGA

[0024] CGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACC

[0025] TTACCTGGGCTTGACATGTTCCCGACGACCCCAGAGATGGGGTTTCCCTTCGGGGCGG

[0026] GTTCACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCC

[0027] CGCAACGAGCGCAACCCTCGCCCCGTGTTGCCAGCGGATTGTGCCGGGAACTCACGG

[0028] GGGACCGCCGGGGTTAACTCGGAGGAAGGTGGGGATGACGTCAGATCATCATGCCCC

[0029] TTACGTCCAGGGCTTCACGCATGCTACAATGGCCGGTACAACGGGATGCGACAGCGC

[0030] GAGCTGGAGCGGATCCCTGAAAACCGGTCTCAGTTCGGATCGCAGTCTGCAACTCGA

[0031] CTGCGTGAAGGCGGAGTCGCTAGTAATCGCGAATCAGCAACGTCGCGGTGAATGCGT

[0032] TCCCGGGCCTTGTACACACCGCCCGTCAAGTCATGAAAGTGGGCAGCACCCGAAGCC

[0033] GGTGGCCTAACCCCTTGCGGGAGGGAGCCGTC

[0034] The full length of the tuf gene sequence of Bifidobacterium breve i1088 is 979 bp, and the specific sequence is shown in SEQ ID NO.2 as follows:

[0035] TCAACATCGCCCACATCGAGTACCAGACCGAGAAGCGTCACTACGCTCACGTCGACTGCCCGGGCCACGCCGACTTCGTGAAGAACATGATCACCGGTGCTGCCCAGATGGATGGCGCCATCCTCGTTGTGGCCGCCACCGACGGCCCGATGGCTCAGACCCGCGAGCACGTGCTGCTCGCTCGTCAGGTGGGCGTCCCGAAGATCCTTGTCGCCCTGAACAAGTGCGACATGGTTGAGGATGAGGAGCTCATCGAGCTCGTCGAAGAAGAGGTCCGCGACCTCCTCGACGAGAACGGCTTCGACCGCGACTGCCCGGTCATCCACACCTCCGCTTACGGTGCTCTGCACGACGACGCTCCGGACCACGAGAAGTGGGTCCAGTCCGTTAAGGACCTCATGGACGCCGTCGACGACTACATCCCGACCCCGGTCCACGACCTCGACAAGCCGTTCCTGATGCCGATCGAGGACGTCTTCACCATCTCCGGCCGTGGTACCGTTGTCACCGGTCGTGTTGAGCGTGGCCAGCTGGCCGTCAACACTCCGGTCGAGATCGTTGGTATCCGTCCGACCCAGACCACCACCGTCACCTCCATCGAGACCTTCCACAAGACCATGGACGCCTGCGAGGCTGGCGACAACACCGGTCTGCTGCTGCGTGGTCTTGGCCGTGAAGATGTCGAGCGTGGCCAGGTTGTGGCCAAGCCGGGCTCCGTCACCCCGCACACCAAGTTCGAGGGCGAAGTCTACGTGCTGACCAAGGACGAGGGTGGCCGTCACTCGCCGTTCTTCTCCAACTACCGTCCGCAGTTCTACTTCCGTACCACCGACGTCACCGGCGTCATCGAGCTGCCGGAAAGGCGTCGAGATGGTTCAGCCGGGCGACCACGCTACCTTCACCGTTGAGCTGATTCAGCCCATCGCTATGGAGGAAGGCCTGACCTTCGCTGTGCGTGAAGTGGCCCCACCGTTGGCTC

[0036] As the second limitation of the present invention, the method for isolating and identifying Bifidobacterium breve i1088 is as follows:

[0037] (1) Source of infant feces

[0038] Collect fresh feces from breastfed newborns aged 3 to 6 months. The collection and transportation processes are carried out in an anaerobic environment. Put the collected fresh feces into a feces box and seal it immediately, and then put it into a disposable plastic self-sealing bag containing an anaerobic gas-generating bag and seal it well.

[0039] (2) Isolation and purification of strains

[0040] a. Weigh 0.5 - 1 g of fresh feces and mix and dissolve it with a feces diluent to obtain a feces solution;

[0041] b. Take 200 μl of the feces solution at a dilution of 10 -2 and spread it evenly on Plate I of TOS medium, and culture it under anaerobic conditions at 37°C for 48 - 72 h;

[0042] c. Pick single colonies of Bifidobacterium on Plate I of the above TOS medium, inoculate them into a new Plate II of TOS medium using the plate streaking isolation method, culture them under anaerobic conditions at 37°C for 48 - 72 h, then pick single colonies on this Plate II of TOS medium and continue to streak and inoculate them onto a new sterile solid MRS medium plate. Continuously culture until the colony morphology in the medium is consistent, and after observing that the cell morphology of the strain is single under a microscope, the single strain to be verified is obtained;

[0043] d. Pick the above single strain to be verified into a liquid MRS medium supplemented with lithium mupirocin and cysteine hydrochloride, and culture it at 37°C for 48 h to obtain the final pure culture strain;

[0044] The whole process from sampling to obtaining the final pure culture strain should be completed within 12 hours.

[0045] (3) Conduct strain characteristic identification and molecular identification on the above pure culture strain.

[0046] As the third limitation of the present invention, the human milk oligosaccharides include at least two of 2'-fucosyllactose, 3'-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, and lacto-N-neotetraose.

[0047] As the fourth limitation of the present invention, the human milk oligosaccharides are 2'-fucosyllactose and lacto-N-neotetraose.

[0048] As the fifth limitation of the present invention, in the composition, the concentration of Bifidobacterium breve i1088 is 3.3×10 9 ~7.7×109 cfu / g.

[0049] As the sixth limitation of the present invention, in the composition, the concentration of human milk oligosaccharide is 2 g / 100 g to 2.9 g / 100 g.

[0050] The present invention also provides an application of a composition containing Bifidobacterium breve i1088 and human milk oligosaccharide, and the composition containing Bifidobacterium breve i1088 and human milk oligosaccharide is used for preparing probiotic powder.

[0051] As a limitation of the present invention, the preparation method of the probiotic powder includes the following steps carried out in sequence:

[0052] S1. Preparation of raw materials

[0053] 1) Preparation of cryoprotectant

[0054] By weight percentage, the raw materials for making the effective components of the cryoprotectant are skim milk powder 12%, 2'-fucosyllactose 2% - 2.6%, lacto-N-neotetraose 1.4% - 1.8%, trehalose 3%, glycerol 1.5%, L-cysteine hydrochloride 0.4% and the balance of water;

[0055] 2) Preparation of Bifidobacterium breve i1088 bacterial sludge

[0056] Inoculate Bifidobacterium breve i1088 in a culture medium for activation and subculture, and centrifuge to collect the Bifidobacterium breve i1088 bacterial sludge;

[0057] Among them, the viable count in the Bifidobacterium breve i1088 bacterial sludge is 1×10 10 ~2×10 10 cfu / g;

[0058] S2. Mixing

[0059] Mix the cryoprotectant and the Bifidobacterium breve i1088 bacterial sludge according to a weight ratio of 1.6 - 2:1, dry and freeze-dry to obtain the probiotic powder.

[0060] Due to the adoption of the above technical solutions, compared with the prior art, the beneficial effects achieved by the present invention are:

[0061] (1) The composition containing Bifidobacterium breve i1088 and human milk oligosaccharide provided by the present invention has the ability to adhere to intestinal epithelial cells. Under the stimulation of human milk oligosaccharide, its adhesion ability is significantly improved, which can reduce the damage of intestinal epithelial cells, protect the integrity of the intestinal epithelial cell layer, and protect the intestinal barrier;

[0062] (2) Use of the composition containing Bifidobacterium breve i1088 and human milk oligosaccharide provided by the present invention for preparing probiotic powder, the operation method of the probiotic powder is simple, and the strain survival rate in the prepared probiotic powder is high;

[0063] (3) Bifidobacterium breve i1088 provided by the present invention has strong acid and bile salt tolerance and excellent ability to tolerate the reverse environment of the digestive tract. When ingested orally and entering the human body, it can tolerate the gastric environment and reach the intestine with a high survival rate. Among them, the survival rate of Bifidobacterium breve i1088 in fermented milk after standing in artificial gastric juice for 2 h is higher than that of commercial Bifidobacterium A, and the survival rate can reach 68.9% after simulated artificial gastrointestinal digestion, which is 11.1% higher than that of commercial Bifidobacterium A;

[0064] (4) When Bifidobacterium breve i1088 provided by the present invention is co-fermented with the JLB-1510 starter, it shows a faster fermentation rate, improves production efficiency, and has more advantages in industrial production;

[0065] (5) The post-acidification of the fermented milk prepared by co-fermenting Bifidobacterium breve i1088 provided by the present invention with the JLB-1510 starter is weakened, which can better ensure the product quality and reduce the occurrence of unacceptable over-acidity and various sensory quality degradation phenomena;

[0066] (6) The fermented milk added with Bifidobacterium breve i1088 of the present invention has higher stability, a more stable system, excellent water-holding capacity, is not prone to whey separation, has a good yogurt texture, and more excellent shelf-life stability;

[0067] (7) The fermented milk prepared by co-fermenting Bifidobacterium breve i1088 provided by the present invention with the JLB-1510 has a clean aroma without peculiar smell, appropriate acidity and viscosity, and good overall acceptability.

[0068] Biological preservation description:

[0069] Bifidobacterium breve i1088 was deposited in the China Center for Type Culture Collection of the Institute of Microbiology, Chinese Academy of Sciences. The deposit address is No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is May 8, 2023, and the deposit number is CGMCC NO. 27281, and the Latin name is Bifidobacterium breve i1088. Description of the drawings

[0070] The present invention will be further described in detail below with reference to the drawings and specific embodiments.

[0071] Figure 1 It is the Gram staining result diagram of Bifidobacterium breve i1088 in Example 1;

[0072] Figure 2Adhesion rate result chart of the adhesion effect of Bifidobacterium breve i1088 on intestinal epithelial cells in Example 1;

[0073] Figure 3 Result chart of the gastric juice survival rates of #2 and #3 in the acid and bile salt tolerance test of Bifidobacterium breve in the fermented milk prepared in Example 6;

[0074] Figure 4 Result chart of the digestive survival rates of #2 and #3 in the acid and bile salt tolerance test of Bifidobacterium breve in the fermented milk prepared in Example 6;

[0075] Figure 5 Result chart of the change of the kinetic stability index values of #2 and #3 over time on the 7th day in the kinetic stability experiment of the fermented milk prepared in Example 6;

[0076] Figure 6 Result chart of the change of the kinetic stability index values of #2 and #3 over time on the 14th day in the kinetic stability experiment of the fermented milk prepared in Example 6;

[0077] Figure 7 Result chart of the change of the kinetic stability index values of #2 and #3 over time on the 21st day in the kinetic stability experiment of the fermented milk prepared in Example 6. Detailed implementation manners

[0078] The present invention will be further described in detail below through specific examples. It should be understood that the preferred examples described herein are only for explaining and understanding the present invention, and are not used to limit the present invention.

[0079] The materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial channels without special instructions. The experimental methods without specific conditions in the examples usually follow conventional conditions or the conditions recommended by the manufacturers.

[0080] Example 1 A strain of Bifidobacterium breve i1088

[0081] (1) Isolation and purification of Bifidobacterium breve i1088

[0082] (1) Source of infant feces

[0083] Collect fresh feces of newborns aged 3 to 6 months who are breastfed. The collection and transportation process is carried out in an anaerobic environment. Put the collected fresh feces into a feces box and seal it immediately, and then put it into a disposable plastic self-sealing bag containing an anaerobic gas-generating bag and seal it.

[0084] (2) Isolation and purification of the strain

[0085] a. Sample preparation

[0086] Weigh 1 g of fresh feces, add feces diluent, and fully oscillate and mix until the feces are dissolved to obtain a feces solution;

[0087] b. Sample enrichment

[0088] Dilute the feces solution with feces diluent in a 10-fold gradient, take 200 μl of the feces solution at a dilution of 10 -2 and evenly coat it on the TOS medium plate I, and culture it under anaerobic conditions at 37°C for 72 h;

[0089] c. Strain isolation

[0090] Pick the single colonies of Bifidobacterium on the above TOS medium plate I, isolate and streak inoculate them into a new TOS medium plate II, culture them under anaerobic conditions at 37°C for 48 h, then pick the single colonies on this TOS medium plate II and continue to streak inoculate them onto a new sterile solid MRS medium plate. Continuously culture three times until the colony morphology in the medium is consistent. After observing that the cell morphology of the strain is single under the microscope, the single strain to be verified is obtained and labeled as i1088;

[0091] d. Single strain expansion and preparation

[0092] Pick the above single strain i1088 to be verified into the improved liquid MRS medium and culture it at 37°C for 48 h to obtain the final pure culture strain i1088;

[0093] e. Strain preservation

[0094] Take 800 μL of the above pure culture strain i1088 and mix it with sterilized 50% glycerol, place it in a strain preservation tube, mix well and store it at -80°C. At the same time, inoculate the inclined plane of the improved MRS solid medium test tube for temporary preservation.

[0095] (2) Identification of Bifidobacterium breve i1088

[0096] (1) Strain characteristic identification:

[0097] Inoculate the pure culture strain i1088 obtained in step d onto the MRS solid medium plate, culture it under anaerobic conditions at 37°C for 72 h with inverted streaking, observe the cell morphology and conduct physical and chemical experiments such as Gram staining, oxidase test, catalase test, and carbohydrate acid production (API 50CH). The results are shown in Table 1, and the Gram staining result diagram is as Figure 1 ,

[0098] Table 1 Results of cell morphology and physical and chemical experiments of i1088

[0099]

[0100] ② Molecular identification:

[0101] Molecular identification was performed on the pure cultured strain i1088 obtained in step d: Using the genomic DNA of this pure cultured strain i1088 as a template, it was amplified with universal primers for bacterial 16S rDNA, and its 16S rDNA sequence was obtained, as shown in SEQ ID NO.1. Its tuf gene sequence was amplified, and the result was as shown in SEQ ID NO.2.

[0102] Through analysis and comparison, this pure cultured strain i1088 was identified as Bifidobacterium breve i1088. This strain has been deposited in the China Center for Type Culture Collection, Institute of Microbiology, Chinese Academy of Sciences. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is May 8, 2023, and the deposit number is CGMCC NO.27281, with the Latin name Bifidobacterium breve i1088.

[0103] Characteristic verification

[0104] This effect experiment was used to verify the ability of Bifidobacterium breve i1088 to synergistically enhance the adhesion ability of intestinal epithelial cells with human milk oligosaccharides (HMOs).

[0105] Experiment design:

[0106] The CaCo-2 cells in intestinal epithelial cells were continuously cultured and passaged until they could evenly cover the 24-well plate and the confluence reached more than 90%, and then the culture was stopped;

[0107] Bifidobacterium breve i1088 and commercial Bifidobacterium breve A were respectively cultured in MRS medium supplemented with lithium mupirocin and cysteine hydrochloride at 37°C for 20 h, activated 3 times, and subjected to primary counting, recorded as J1. 10 6 CFU / mL of Bifidobacterium breve i1088 and commercial Bifidobacterium breve A bacterial solutions were centrifuged, washed 2 times with PBS, and suspended and cultured with serum-free and antibiotic-free MEM medium for standby. Among them, the concentration of the bacterial suspension in the MEM medium was 10 6 CFU / mL.

[0108] Experiment grouping:

[0109] Bifidobacterium breve i1088 group: 300 μL of the MEM medium bacterial suspension containing Bifidobacterium breve i1088 and 50 μL of pure MEM medium were added to each well in the 24-well plate containing the above CaCo-2 cells;

[0110] Commercial Bifidobacterium breve A group: 300 μL of the MEM medium bacterial suspension containing commercial Bifidobacterium breve A and 50 μL of pure MEM medium were added to each well in the 24-well plate containing the above CaCo-2 cells;

[0111] Bifidobacterium breve i1088 + HMOs group: Add 300 μL of MEM medium bacterial suspension containing Bifidobacterium breve i1088 and 50 μL of HMOs solution to each well of the 24-well plate containing the above CaCo-2 cells;

[0112] Commercial Bifidobacterium breve A + HMOs group: Add 300 μL of MEM medium bacterial suspension containing commercial Bifidobacterium breve A and 50 μL of HMOs solution to each well of the 24-well plate containing the above CaCo-2 cells;

[0113] Blank control group: Add 350 μL of pure MEM medium to each well of the 24-well plate containing the above CaCo-2 cells.

[0114] Among them, the HMOs solution contains: 1.2 mg / mL of 2'-fucosyllactose, 0.6 - 0.85 mg / mL of 3'-fucosyllactose, 0.2 mg / mL of 3'-sialyllactose, 0.4 mg / mL of 6'-sialyllactose, 0.6 mg / mL of lacto-N-neotetraose. Distilled water is used as the solvent, and it is filtered and sterilized with a 0.22 μm filter membrane. Its content is added according to the approved use of HMOs in infant formula in EU Implementing Regulation 2017 / 2470 and the EU Novel Food List and Amendments.

[0115] Put the 24-well plates of the above 5 groups into an incubator at 37 °C and 5% CO2 - 95% air for 6 hours, wash twice with PBS to remove unadhered Bifidobacterium breve, pipette and elute each well plate with Triton X-100 for 10 minutes, collect the eluate and perform plate counting, denoted as J2, and calculate the adhesion rate according to the following formula. The counting results are as Figure 2 shown.

[0116] Adhesion rate = J2 / J1 × 100%

[0117] As Figure 2 can be seen, the adhesion rate of CaCo-2 cells in the Bifidobacterium breve i1088 group is 67.19%, lower than 68.35% in the commercial Bifidobacterium breve A group. However, the adhesion rate of CaCo-2 cells in the Bifidobacterium breve i1088 group with added HMOs is 76.81%, significantly higher than 68.35% in the commercial Bifidobacterium breve A group and 70.79% in the commercial Bifidobacterium breve A + HMOs group, indicating that Bifidobacterium breve i1088 has an adhesion effect on intestinal epithelial cells, and this adhesion effect is significantly enhanced under the stimulation of the combination with HMOs. Bifidobacterium breve i1088 can be combined with HMOs as the main component of a probiotic preparation for protecting the intestinal barrier, has good adhesion to intestinal epithelial cells, can reduce the damage of intestinal epithelial cells, and protect the integrity of the intestinal epithelial cell layer.

[0118] Example 2 A composition containing Bifidobacterium breve i1088 and human milk oligosaccharides

[0119] This example is a composition containing Bifidobacterium breve i1088 and human milk oligosaccharides. The human milk oligosaccharides are 2'-fucosyllactose and lacto-N-neotetraose. In the composition, the concentration of Bifidobacterium breve i1088 is 3.3×10 9 cfu / g, and the concentration of human milk oligosaccharides is 2 g / 100 g.

[0120] Example 3 A composition containing Bifidobacterium breve i1088 and human milk oligosaccharides

[0121] This example is a composition containing Bifidobacterium breve i1088 and human milk oligosaccharides. The human milk oligosaccharides are 2'-fucosyllactose, 3'-fucosyllactose and 3'-sialyllactose. In the composition, the concentration of Bifidobacterium breve i1088 is 7.7×10 9 cfu / g, and the concentration of human milk oligosaccharides is 2.2 g / 100 g.

[0122] Example 4 A composition containing Bifidobacterium breve i1088 and human milk oligosaccharides

[0123] This example is a composition containing Bifidobacterium breve i1088 and human milk oligosaccharides. The human milk oligosaccharides are 2'-fucosyllactose, 3'-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose and lacto-N-neotetraose. In the composition, the concentration of Bifidobacterium breve i1088 is 7.7×10 9 cfu / g, and the concentration of human milk oligosaccharides is 2.9 g / 100 g.

[0124] Example 5 Application of a composition containing Bifidobacterium breve i1088 and human milk oligosaccharides

[0125] This example is about the application of a composition containing Bifidobacterium breve i1088 and human milk oligosaccharides.

[0126] Compared with frozen products and liquid bifidobacterium preparations, freeze-dried probiotic powder is easy to use and has a longer viable bacteria preservation period. This example uses vacuum freeze-drying technology to provide a freeze-drying protectant formula for human milk oligosaccharides containing 2'-fucosyllactose and lacto-N-neotetraose to prepare probiotic powder. The specific preparation method is as follows:

[0127] S1. Preparation of raw materials

[0128] 1) Preparation of protectant:

[0129] Based on weight percentage, the raw materials for making the active ingredient of the protective agent are 12% skim milk powder, 2% 2'-fucosyllactose, 1.4% lacto-N-neotetraose, 3% trehalose, 1.5% glycerol, 0.4% L-cysteine hydrochloride, and the balance water;

[0130] Among them, the human milk oligosaccharides are 2'-fucosyllactose and lacto-N-neotetraose, and their proportion in the protective agent is 3.4%.

[0131] (2) Preparation of Bifidobacterium breve i1088 bacterial sludge:

[0132] Using the two-stage fermentation method, inoculate a cryopreservation tube containing 100 g of Bifidobacterium breve i1088 with 2×10 8 cfu / g into the modified MRS liquid medium for activation. After anaerobic culture for 48 h, transfer it to a new modified MRS liquid medium, and perform scale-up culture for 48 h using the same medium. Centrifuge and collect 1 g of bacterial sludge with 2×10 10 cfu;

[0133] (3) Mixing

[0134] Mix the protective agent and Bifidobacterium breve i1088 bacterial sludge in a weight ratio of 1.8:1, put them into a vacuum freeze dryer, and freeze-dry for 24 h to obtain the probiotic powder.

[0135] Among them, the concentration of human milk oligosaccharides in the probiotic powder is 2.2 g / 100 g, and the concentration of Bifidobacterium breve i1088 is 7.1×10 9 cfu / g.

[0136] Effect experiment

[0137] To verify the survival effect of Bifidobacterium breve i1088 probiotic powder, the experimental groups are divided as follows:

[0138] Experimental group 1: 1 g of Bifidobacterium breve i1088 bacterial sludge containing 2×10 10 cfu / g + 2 g of probiotic powder containing the protective agent with HMOs;

[0139] Experimental group 2: 1 g of commercial Bifidobacterium breve A bacterial sludge containing 2×10 10 cfu / g + 2 g of probiotic powder containing the protective agent with HMOs;

[0140] Control group 1: 1 g of Bifidobacterium breve i1088 bacterial sludge containing 2×10 10 cfu / g + 2 g of probiotic powder containing the protective agent without HMOs;

[0141] Control group 2: 1 g of Bifidobacterium breve i1088 bacterial sludge containing 2×10 101 g of commercial Bifidobacterium breve A bacterial sludge with cfu / g + 2 g of probiotic powder of a protective agent without HMOs.

[0142] Among them, the preparation method of commercial Bifidobacterium breve A bacterial sludge is similar to that of Bifidobacterium breve i1088 bacterial sludge; the preparation method of the protective agent without HMOs is similar to the preparation steps of the protective agent in the above step (1), the difference is that 2'-fucosyllactose and lacto-N-neotetraose are not added.

[0143] Accurately weigh 1 g of each freeze-dried probiotic powder, perform plate counting according to the method in Example 1, and calculate the survival rate after freeze-drying. The results are shown in Table 2.

[0144] Survival rate = (number of viable bacteria in the freeze-dried powder / number of viable bacteria in the mixture of bacterial sludge and protective agent before freeze-drying) × 100%

[0145] Table 2 Survival rate of Bifidobacterium breve in probiotic powder

[0146] Group Experimental Group 1 Experimental Group 2 Control Group 1 Control Group 2 Survival Rate 81.39％±1.22％ 70.25％±1.63％ 69.81％±2.3％ 68.08％±3.1％

[0147] It can be seen from Table 2 that the protective agent added with HMOs has an obvious promoting effect on the viability of Bifidobacterium breve i1088 during freeze-drying, while it has almost no effect on Bifidobacterium breve A. Therefore, in the presence of HMOs, Bifidobacterium breve i1088 has a higher survival rate in extreme environments, which is beneficial to the production of probiotic powder.

[0148] Example 6 Application of Bifidobacterium breve i1088

[0149] This example is the application of using Bifidobacterium breve i1088 and starter JLB-1510 to prepare fermented milk. The specific preparation process of the fermented milk is as follows:

[0150] (1) Add 70 g of granulated sugar to 1 L of milk, heat to 65 °C, homogenize, and sterilize in a water bath at 90 °C for 15 min;

[0151] (2) Cool down to 42 °C, after inoculating 0.05 g of 50 g / t starter JLB-1510, inoculate 200 mL of Bifidobacterium breve i1088 bacterial liquid;

[0152] (3) Ferment statically at 42 °C until the pH of the fermented milk reaches 4.5, then stop fermentation;

[0153] (4) After cooling, stir at a speed of 850 rpm / min for 2 min;

[0154] (5) After completion of stirring, place at 4 °C overnight for after-ripening, and the fermented milk containing Bifidobacterium breve i1088 is obtained;

[0155] Among them, the Bifidobacterium breve i1088 bacterial liquid is: centrifuged after culturing in a modified MRS medium supplemented with lithium mupirocin and cysteine hydrochloride for 48 h, and the bacterial sludge is washed twice with sterile physiological saline and reserved for use, with a concentration of 2×10 7 cfu / mL of Bifidobacterium breve i1088 bacterial liquid.

[0156] Effect experiment

[0157] This effect experiment is used to verify the acid and bile salt tolerance of Bifidobacterium breve i1088, the post-acidification ability, shelf-life stability ability and sensory evaluation of the fermented milk prepared by compounding Bifidobacterium breve i1088 and JLB-1510.

[0158] Experimental design:

[0159] Bifidobacterium breve i1088 and commercial Bifidobacterium breve A are respectively centrifuged after culturing in a modified MRS medium supplemented with lithium mupirocin and cysteine hydrochloride for 48 h, and the bacterial sludge is washed twice with sterile physiological saline to obtain bacterial liquid for standby.

[0160] After testing, neither Bifidobacterium breve i1088 nor commercial Bifidobacterium breve A can coagulate milk when fermented alone. Therefore, the starter JLB-1510 is used in combination for the preparation of fermented milk.

[0161] Experimental grouping:

[0162] #1: Add 70 g of granulated sugar to 1 L of milk, add 0.05 g of starter JLB-1510 at 50 g / t, and do not add strains;

[0163] #2: Add 70 g of granulated sugar to 1 L of milk, add 0.05 g of starter JLB-1510 at 50 g / t and 200 mL of Bifidobacterium breve i1088 bacterial liquid at 2×10 7 cfu / mL;

[0164] #3: Add 70 g of granulated sugar to 1 L of milk, add 0.05 g of starter JLB-1510 at 50 g / t and 200 mL of commercial Bifidobacterium breve A bacterial liquid at 2×10 7 cfu / mL;

[0165] The three groups are respectively fermented at 42 °C, and the fermentation end point is set when the pH value reaches 4.5.

[0166] After experiments, the fermentation times of three groups were 6.2 h, 5.6 h, and 5.9 h respectively. From the fermentation times, it can be seen that compared with the fermentation using only the JLB-1510 starter and the co-fermentation of commercial Bifidobacterium breve A and the JLB-1510 starter, the co-fermentation of Bifidobacterium breve i1088 and the JLB-1510 starter showed a faster fermentation rate, which can improve production efficiency and has more advantages in industrial production.

[0167] The acid and bile salt tolerance tests, the post-acidification ability determination of fermented milk, the shelf-life stability monitoring, and the sensory evaluation were carried out on the three groups respectively.

[0168] (1) Acid and bile salt tolerance test of Bifidobacterium breve i1088

[0169] The in vitro gastric juice and intestinal juice tolerance tests were carried out on the fermented milk of #2 and #3 respectively to evaluate the acid and bile salt tolerance ability of Bifidobacterium breve i1088.

[0170] Experimental design:

[0171] Preparation of artificial gastric juice:

[0172] The solution containing 0.2 g / 100 mL of NaCl and 0.35 g / 100 mL of pepsin was adjusted to pH 3.0 with 1 mol / L HCl and then filtered and sterilized for standby.

[0173] Preparation of artificial intestinal juice:

[0174] Pancreatic juice: The solution containing 1.1 g / 100 mL of sodium bicarbonate, 0.2 g / 100 mL of NaCl, and 0.1 g / 100 mL of trypsin was adjusted to pH 8.0 and then filtered and sterilized for standby;

[0175] Bile acid solution: The solution containing 0.9 g / 100 mL of Bile Salts was adjusted to pH 8.0 and then filtered and sterilized for standby;

[0176] The above-mentioned pancreatic juice and bile acid solution were mixed at a volume ratio of 2:1 to obtain artificial intestinal juice.

[0177] Experimental method:

[0178] 1 mL of the fermented milk of #2 and #3 was taken respectively and added to 9 mL of artificial gastric juice and shaken well. The viable cell count of the mixed solution was immediately detected and recorded as the viable cell count at 0 h; it was left standing at 37 °C for 2 h, and the viable cell count of the mixed solution was detected and recorded as the viable cell count at 2 h, which was marked as the 2-h treatment solution of artificial gastric juice;

[0179] 1 mL of the 2-h treatment solution of artificial gastric juice of #2 and #3 was taken respectively and added to 9 mL of artificial intestinal juice. After mixing, it was left standing at 37 °C for 4 h, and its viable cell count was detected and recorded as the viable cell count at 6 h;

[0180] Among them, the calculation methods of the indexes of gastric juice survival rate and digestion survival rate are as follows:

[0181] Gastric juice survival rate (%) = [(number of viable bacteria at 2 h × 10) / number of viable bacteria at 0 h] × 100%

[0182] Digestion survival rate (%) = [(number of viable bacteria at 6 h × 10) / number of viable bacteria at 0 h] × 100%

[0183] The experimental results are as Figure 3 and Figure 4 shown. It can be seen from the experimental results that Bifidobacterium breve i1088 has good acid and bile salt tolerance characteristics. The survival rate of Bifidobacterium breve i1088 in #2 after standing in artificial gastric juice for 2 h is 88.5%, higher than 85.8% of commercial Bifidobacterium A in #3. After simulated artificial gastrointestinal digestion, the survival rate can reach 68.9%, an increase of 11.1% compared with the survival rate of commercial Bifidobacterium A, indicating that Bifidobacterium breve i1088 proposed by the present invention has excellent ability to tolerate the adverse environment of the digestive tract. When ingested orally and entering the human body, it can tolerate the gastric environment and reach the intestine with a relatively high survival rate.

[0184] (2) Post-acidification ability of fermented milk

[0185] The three groups of fermented milk were respectively placed at 4 °C, and the pH values and titratable acidity values of each group were measured on the 7th, 14th, and 21st days. The experimental results are shown in Table 3.

[0186] Among them, the titratable acidity was measured according to GB 5009.239—2016.

[0187] Table 3 Determination of post-acidification ability of three groups of fermented milk

[0188]

[0189] It can be seen from the above determination results of the post-acidification ability of fermented milk that on the 21st day, the pH value of #2 is greater than that of #3, and the titratable acidity value of #2 is less than that of #3, indicating that the post-acidification of the fermented milk prepared by compounding Bifidobacterium breve i1088 and JLB-1510 is weakened, which can better ensure the product quality and reduce the occurrence of unacceptable over-acidity and various sensory quality degradation phenomena.

[0190] (3) Monitoring the shelf-life stability of fermented milk

[0191] The three groups of fermented milk were respectively placed at 4 °C, and the stability of each group of fermented milk was monitored for 21 days.

[0192] ① Water-holding capacity monitoring experiment

[0193] The water-holding capacity was determined by the centrifugation method: 20.0 g of each of the three groups of fermented milk was accurately weighed and placed into centrifuge tubes respectively. The rotation speed of the centrifuge was set at 4000 r / min and the centrifugation time was 10 min. The water-holding rate was expressed as WHC, and WHC=(m1 - m0) / m2×100%, where m0 was the mass of the centrifuge tube; m1 was the total mass of the sample and the centrifuge tube after centrifugation; m2 was the mass of the sample, with the unit of g;

[0194] The monitoring results of the 21-day stability of the three groups of fermented milk are shown in Table 4,

[0195] Table 4 Monitoring of the water-holding capacity of the three groups of fermented milk for 21 days

[0196]

[0197]

[0198] From the above monitoring results of the water-holding capacity, it can be seen that on the 7th day, 14th day and 21st day, the water-holding capacity of #2 was better than that of #1 and #3, indicating that the fermented milk added with Bifidobacterium breve i1088 had higher stability than the fermented milk without added strains and the fermented milk added with commercial Bifidobacterium breve A, was less likely to undergo whey separation, and had a good texture of yogurt.

[0199] ② Kinetic stability experiment

[0200] Using the kinetic stability index (TSI) as an index, the Turbiscan Lab stability analyzer was used for on-machine analysis to characterize the physical overall instability phenomenon under the conditions of no dilution, no disturbance and no contact of the sample.

[0201] TSI can be used to determine the physical stability of the system and is convenient for comparing the stability differences between different samples. This evaluation index refers to the average change rate of the backscattered light within the observation time of the sample. TSI is negatively correlated with the system stability. The smaller the TSI value, the more stable the system, and the larger the TSI value, the more unstable the system.

[0202] The TSI values of the fermented milk of #2 and #3 were measured respectively, and the results are as Figures 5 - 7 shown. It can be seen from Figures 5 - 7 that the TSI values of #2 on the 7th day, 14th day and 21st day were all less than those of #3, indicating that the fermented milk added with Bifidobacterium breve i1088 had a more stable system and better shelf-life stability than the fermented milk without added strains and the fermented milk added with commercial Bifidobacterium breve A.

[0203] (4) Sensory evaluation

[0204] A total of 24 professional sensory evaluation personnel were convened to conduct a comprehensive evaluation on the three groups of fermented milk after ripening for 12 h.

[0205] The sensory evaluation method is as follows: For the overall taste preference, the 9-point intensity scale method is used (9 → 1 indicates like → dislike, <5 indicates not liked), and for other sensory characteristics, the 10-point intensity scale method is used (10 → 0 indicates extremely strong → undetectable). Using this method, the overall preference, total aroma, milk aroma, fermentation aroma, viscosity in the mouth, acidity, and powdery feeling of the above three groups of fermented milks were evaluated. The results are shown in Table 5.

[0206] Table 5 Sensory evaluation results of each group of fermented milks

[0207]

[0208]

[0209] SPSS was used for the analysis of significant differences. a, ab, b indicate significant differences, P < 0.05.

[0210] The above sensory evaluation results show that when Bifidobacterium breve i1088 and JLB-1510 are co-fermented, the overall preference of sensory evaluators is high, the total aroma and milk aroma are more obvious. At the same time, the sensory degrees of acidity and powdery feeling are weaker. Therefore, the fermented milk prepared by co-fermenting Bifidobacterium breve i1088 and JLB-1510 in the present invention has good sensory and flavor characteristics.

[0211] It should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still modify the technical solutions recorded in the above embodiments or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A composition containing Bifidobacterium breve i1088 and human milk oligosaccharide, characterized in that, The Bifidobacterium breve i1088 is deposited in the China Center for Type Culture Collection of the Institute of Microbiology, Chinese Academy of Sciences. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is May 8, 2023, and the deposit number is CGMCC NO. 27281. The Latin name is Bifidobacterium breve i1088, The human milk oligosaccharides include at least two of 2'-fucosyllactose, 3'-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, and lacto-N-neotetraose.

2. The composition containing Bifidobacterium breve i1088 and human milk oligosaccharide according to claim 1, characterized in that, The human milk oligosaccharides are 2'-fucosyllactose and lacto-N-neotetraose.

3. The composition containing Bifidobacterium breve i1088 and human milk oligosaccharide according to claim 1, wherein In the composition, the concentration of Bifidobacterium breve i1088 is 3.3×10 9 ~7.7×10 9 cfu / g.

4. The composition containing Bifidobacterium breve i1088 and human milk oligosaccharide according to any one of claims 1 to 3, characterized in that, In the composition, the concentration of the human milk oligosaccharides is 2 g / 100 g to 2.9 g / 100 g.

5. Use of a composition containing Bifidobacterium breve i1088 and human milk oligosaccharide according to any one of claims 1 to 4, characterized in that, The composition containing Bifidobacterium breve i1088 and human milk oligosaccharides is used for preparing a probiotic powder.

6. Use of the composition containing Bifidobacterium breve i1088 and human milk oligosaccharide according to claim 5, characterized in that, The preparation method of the probiotic powder includes the following steps carried out in sequence: S1. Preparation of raw materials 1) Preparation of cryoprotectant By weight percentage, the raw materials for making the effective components of the cryoprotectant are 12% skim milk powder, 2% - 2.6% 2'-fucosyllactose, 1.4% - 1.8% lacto-N-neotetraose, 3% trehalose, 1.5% glycerol, 0.4% L-cysteine hydrochloride, and the balance of water; 2) Preparation of Bifidobacterium breve i1088 bacterial sludge Inoculate Bifidobacterium breve i1088 in a medium for activation and subculture, and centrifuge to collect the Bifidobacterium breve i1088 bacterial sludge; Among them, the viable count in the Bifidobacterium breve i1088 bacterial sludge is 1×10 10 ~2×10 10 cfu / g; S2. Mixing Mix the cryoprotectant and the Bifidobacterium breve i1088 bacterial sludge in a weight ratio of 1.6 - 2:1, dry and freeze-dry to obtain the probiotic powder.

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