Streptococcus and application thereof

Abstract

The application discloses a streptococcus and application thereof, and belongs to the technical field of microorganisms. The application screens a streptococcus capable of decomposing and utilizing human milk oligosaccharides, and the streptococcus can grow by taking human milk oligosaccharides as a carbon source; the utilization rate of 2'-fucosyllactose, 6'-sialyllactose and lacto-N-tetraose reaches more than 95.98%; and the streptococcus is helpful for promoting the growth of short bifidobacterium which cannot utilize human milk oligosaccharides.

Description

TECHNICAL FIELD

[0001] The present application relates to a strain of streptococcus and its application, and belongs to the technical field of microorganisms. BACKGROUND

[0002] At present, people pay more and more attention to physical health, and the demand for functional foods is also increasing day by day. Among them, probiotic foods and health products have developed rapidly. Streptococcus is one of the symbiotic bacteria of human body, and is an important part of the intestinal flora of infants. Streptococcus isolated from healthy human body has been proved to be safe and have probiotic properties, so it has been widely recognized. Streptococcus thermophilus, Streptococcus salivarius and Lactococcus can promote the health of the host body through their own physiological activity.

[0003] Human milk oligosaccharides (HMO) are a class of water-soluble compounds that cannot be digested and absorbed by the human body, mainly composed of five basic monosaccharides, galactose (Gal), glucose (Glc), N-acetylglucosamine (GlcNAc), sialic acid (Sia) and fucose (Fuc), which are combined in various glycosidic bonds. The content of human milk oligosaccharides in breast milk is high, second only to lactose and lipids. Human milk oligosaccharides have been proved to be beneficial to the healthy development of newborns. Human milk oligosaccharides can regulate the immune response of intestinal tissue cells in infants, and systematically affect the immune system response, playing an anti-inflammatory, anti-viral and antibacterial role.

[0004] The utilization of human milk oligosaccharides by intestinal microorganisms is an important way for them to exert physiological activity. Human milk oligosaccharides are the main carbon source for intestinal microorganisms of breast-fed infants, and intestinal microorganisms metabolize human milk oligosaccharides in different ways, taking lactic acid and SCFAs as metabolic products, and promoting the growth of different bacteria. These bacteria use different sugar utilization systems to utilize diet and host-derived glycan, share digested oligosaccharides, carbohydrate-active enzymes and fermentable intermediates to maintain intestinal microbial symbiosis and improve their own or other community's adaptability. In addition, the metabolic products produced by the utilization of HMO increase the acidity of the intestinal environment, which can inhibit the growth of pathogenic bacteria and protect infants from harmful intestinal infections. Therefore, strains with the ability to utilize human milk oligosaccharides can regulate the structure and composition of the intestinal flora of infants, and regulate the immune system response of the infant body. Screening of streptococcus strains that can decompose and utilize human milk oligosaccharides can be used to regulate the intestinal microecology and immune response of infants, promote the development and application of infant microecological preparations, and has important significance. SUMMARY

[0005] The present application aims to solve the problems in the prior art, and provides a streptococcus capable of decomposing and utilizing human milk oligosaccharides.

[0006] This invention provides a strain of Streptococcus sp. CCFM1328, which is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 63647, deposit date of July 14, 2023, and address of Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] The Streptococcus CCFM1328 strain was derived from the breast milk of a healthy woman. The strain was identified as a species through steps such as genomic DNA extraction, PCR amplification with 16S rDNA specific primers, purification of amplification products, DNA sequencing, and sequence alignment. The results showed that the strain belonged to the genus Streptococcus and was named Streptococcus CCFM1328. It was preserved at the Food Biotechnology Culture Collection Center of Jiangnan University.

[0008] The colony protrusions of Streptococcus CCFM1328 on M17 solid medium are smooth, round, milky white, translucent, and 0.45 mm in diameter.

[0009] The present invention provides a microbial preparation containing the Streptococcus CCFM1328.

[0010] In one embodiment, the viable count of Streptococcus in the microbial preparation is not less than 4 × 10⁻⁶. 8 CFU / mL or 4×10 8 CFU / g.

[0011] This invention proposes a food product containing the aforementioned Streptococcus CCFM1328. The Streptococcus CCFM1328 possesses the ability to decompose and utilize human milk oligosaccharides (HMIs), and can grow using HMIs as a carbon source, particularly using HMIs as the sole carbon source. Therefore, this food product provides a theoretical basis and feasibility for the development of probiotic products utilizing HMIs.

[0012] In one embodiment, the viable count of Streptococcus in the food is not less than 2 × 10⁻⁶. 9 CFU / mL or 2×10 9 CFU / g.

[0013] In one embodiment, the food is freeze-dried powder.

[0014] In one embodiment, the freeze-dried powder is prepared by: inoculating the Streptococcus CCFM1328 into a culture medium for cultivation to obtain a seed culture; inoculating the seed culture into a culture medium for cultivation to obtain a culture solution; centrifuging the culture solution and collecting the bacterial sludge; washing the bacterial sludge with physiological saline and resuspending it to obtain a resuspension; adding a freeze-drying protectant to the resuspension to obtain a mixture; and freeze-drying the mixture under vacuum to obtain freeze-dried powder.

[0015] In an embodiment, the seed liquid is inoculated into the culture medium at an inoculation amount of 2-4% (v / v) for culture.

[0016] In an embodiment, the components of the freeze-drying protective agent include skimmed milk powder, trehalose, sucrose and water.

[0017] In an embodiment, the components of the freeze-drying protective agent are 80-120 g / L skimmed milk powder, 80-140 g / L trehalose, 140-180 g / L sucrose and water.

[0018] In an embodiment, the components of the freeze-drying protective agent include 100 g / L skimmed milk powder, 100 g / L trehalose, 160 g / L sucrose and water.

[0019] In an embodiment, the freeze-drying protective agent is added in the resuspension liquid at an amount of 2-4 times the total weight of the bacterial slurry.

[0020] In an embodiment, the seed culture medium is MRS solid medium, and the fermentation culture medium is MRS liquid medium.

[0021] In an embodiment, the MRS liquid medium is MRS liquid medium added with cysteine hydrochloride.

[0022] In an embodiment, the cysteine hydrochloride is added at a mass fraction of 0.04-0.1%.

[0023] In an embodiment, the seed liquid is inoculated into the MRS liquid medium at an inoculation amount of 2-4% for culture, and the culture conditions are as follows: anaerobic culture at 34-38°C for 24-36 h, centrifugation at 7000-12000 rpm for 20-30 min, collection of the bacterial slurry, washing with normal saline for 3-4 times and resuspension.

[0024] The application also provides use of the streptococcus CCFM1328 in decomposition of human milk oligosaccharides.

[0025] In an embodiment, the human milk oligosaccharides include one or more of 2'-fucosyllactose (2'-FL), 6'-sialyllactose (6'-SL) and lacto-N-tetraose (LNT).

[0026] The application also provides use of the streptococcus CCFM1328 in promoting growth of Bifidobacterium breve.

[0027] In an embodiment, the use is to promote growth and / or increase in number of Bifidobacterium breve in an environment containing human milk oligosaccharides.

[0028] In an embodiment, the human milk oligosaccharides include one or more of 2'-fucosyllactose, 6'-sialyllactose, lacto-N-tetraose.

[0029] In an embodiment, the application is to co-culture the Streptococcus CCFM1328 with Bifidobacterium breve.

[0030] Beneficial effects:

[0031] (1) The Streptococcus CCFM1328 provided by the present application is isolated from breast milk, and the strain has no toxic side effects on the human body. Therefore, the food prepared by using the Streptococcus CCFM1328 provided by the present application has certain advantages compared with traditional food, and the strain can be used for making probiotic preparations and the like, and has a broad market prospect.

[0032] (2) The Streptococcus CCFM1328 provided by the present application has the ability to decompose breast milk oligosaccharides, and can grow with breast milk oligosaccharides as a carbon source, especially can grow with human milk oligosaccharides as the only carbon source. The breast milk oligosaccharides include one or more of 2'-fucosyllactose (2'-FL), 6'-sialyllactose (6'-SL) and lacto-N-tetraose (LNT).

[0033] (3) The Streptococcus CCFM1328 provided by the present application has a high utilization rate of breast milk oligosaccharides, and can use it to maintain its own growth and metabolism. The utilization rate of 2'-FL is 99.78%, the utilization rate of 6'-SL is 97.65%, and the utilization rate of LNT is 95.98%.

[0034] (4) The Streptococcus CCFM1328 provided by the present application can decompose 2'-FL to produce intermediate products lactose and fucose; can decompose 6'-SL to produce intermediate products lactose and sialic acid; and can decompose LNT to produce intermediate product lactose.

[0035] (5) The end product of the Streptococcus CCFM1328 provided by the present application decomposing 2'-FL is acetic acid and lactic acid, and the ratio of the two is 1:2.19; the end product of decomposing 6'-SL is acetic acid and lactic acid, and the ratio of the two is 1:0.99; and the product of decomposing LNT is acetic acid and lactic acid, and the ratio of the two is 1:0.89.

[0036] (6) The Streptococcus CCFM1328 provided by the present application can feed the Bifidobacterium breve SH-SJ-MZM1 which cannot utilize 6'-SL when growing in a 6'-SL-M17 medium.

[0037] Biological material preservation

[0038] A strain of Streptococcus sp. CCFM1328, taxonomically named Streptococcus sp., has been deposited with the Guangdong Microbial Culture Collection Center on July 14, 2023, and has the accession number GDMCC NO: 63647, and the deposit address is No. 59 Building, 5th Floor, 100, Martyrs' Avenue, Guangzhou. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 ANI and dDDH values of Streptococcus sp. CCFM1328 and its close relative strains.

[0040] Figure 2 Growth curve of Streptococcus sp. CCFM1328 when using human milk oligosaccharides as the sole carbon source.

[0041] Figure 3 pH curve of Streptococcus sp. CCFM1328 when using human milk oligosaccharides as the sole carbon source.

[0042] Figure 4 Residual amount of human milk oligosaccharides of Streptococcus sp. CCFM1328 when using human milk oligosaccharides as the sole carbon source.

[0043] Figure 5 Intermediate product of Streptococcus sp. CCFM1328 when using human milk oligosaccharides as the sole carbon source.

[0044] Figure 6 End product of Streptococcus sp. CCFM1328 when using human milk oligosaccharides as the sole carbon source.

[0045] Figure 7 Streptococcus sp. CCFM1328 feeding Bifidobacterium breve SH-SJ-MZM1 with human milk oligosaccharides.

[0046] Figure 8 Bacterial number of Streptococcus sp. CCFM1328 and Bifidobacterium breve in pure culture and co-culture on 6'-SL.

[0047] Figures 1-8 In the present application, “*”, “**”, “***”, “****” all represent significant difference from the sugar-free group, and the more stars, the greater the significant difference. DETAILED DESCRIPTION

[0048] The present application will be further described below in conjunction with specific examples and drawings.

[0049] Streptococcus sp. CCFM1328 used in the following examples has been deposited with the Guangdong Microbial Culture Collection Center on July 14, 2023, and has the accession number GDMCC NO: 63647. Bifidobacterium breve SH-SJ-MZM1 was screened from human milk.

[0050] The culture media involved in the following examples are as follows:

[0051] M17 liquid medium: Tryptone 5 g / L, soybean peptone 5 g / L, beef extract 5 g / L, yeast extract 2.5 g / L, magnesium sulfate heptahydrate 0.25 g / L, ascorbic acid 0.5 g / L, sodium β-glycerophosphate pentahydrate 19 g / L, carbon source (2'-FL / 6'-SL / LNT / glucose) 5 g / L.

[0052] M17 solid medium: Tryptone 5 g / L, soybean peptone 5 g / L, beef extract 5 g / L, yeast extract 2.5 g / L, magnesium sulfate heptahydrate 0.25 g / L, ascorbic acid 0.5 g / L, sodium β-glycerophosphate pentahydrate 19 g / L, carbon source (2'-FL / 6'-SL / LNT / glucose / oligofructose) 5 g / L, agar powder 20 g / L.

[0053] MRS liquid medium: Tryptone 10 g / L, beef extract 10 g / L, yeast powder 5 g / L, glucose 20 g / L, sodium acetate anhydrous 2 g / L, magnesium sulfate heptahydrate 0.5 g / L, manganese sulfate monohydrate 0.25 g / L, diammonium hydrogen citrate 2 g / L, dipotassium phosphate trihydrate 2.6 g / L, Tween 80 1 mL / L, cysteine hydrochloride 0.5 g / L.

[0054] MRS solid medium: Tryptone 10 g / L, beef extract 10 g / L, yeast powder 5 g / L, glucose 20 g / L, sodium acetate anhydrous 2 g / L, magnesium sulfate heptahydrate 0.5 g / L, manganese sulfate monohydrate 0.25 g / L, diammonium hydrogen citrate 2 g / L, dipotassium phosphate trihydrate 2.6 g / L, Tween 80 1 mL / L, cysteine hydrochloride 0.5 g / L, agar powder 20 g / L. When isolating bacteria, 15 mL / L of 0.5% bromocresol purple solution was added to the solid medium as an indicator.

[0055] The detection methods involved in the following examples are as follows:

[0056] (1) Determination method of growth curve and pH value:

[0057] The overnight cultured Streptococcus CCFM1328 culture solution was inoculated into M17 liquid medium with 2'-FL, 6'-SL or LNT as the sole carbon source at an inoculation amount of 1%, and cultured in a 37°C constant temperature incubator for 24 h. Every 2 h, the OD 600 of the bacterial solution was determined by visible spectrophotometer, and the pH value of the bacterial solution was determined by pH meter, with M17 liquid medium with glucose and oligofructose (GOS) as the carbon source as the positive control, and M17 medium without sugar as the negative control. Each sample was tested in triplicate.

[0058] (2) Determination method of residual amount of human milk oligosaccharides and intermediate product generation amount:

[0059] The fermentation broth of Streptococcus sp. CCFM1328 at the mid and late fermentation stage was precisely taken, the protein was removed by Savage method, MiliQ water was added for dilution by 5 times, mixed, centrifuged at 8000g at 4℃ for 5min, and after centrifugation, the filtrate was filtered through a sterile microporous filter membrane of 0.22μm. The samples after gradient dilution were detected for the contents of 2’-FL, 6’-SL, LNT, GOS, fucose, sialic acid, lactose, galactose and glucose in the fermentation broth by high performance liquid chromatography-mass spectrometry (HPLC-MS). The instrument parameter settings were as follows: a BEH Amide column was used, mass spectrometry was used as the detector, the mobile phases A and B were 25mM ammonium acetate and acetonitrile respectively, the flow rate was 0.3mL / min, and the column temperature was maintained at 50℃. Three biological parallel experiments were performed for each sample.

[0060] (3) Determination method of metabolic end product generation amount of human milk oligosaccharides:

[0061] The fermentation broth of Streptococcus sp. CCFM1328 at the late fermentation stage was precisely taken, the protein was removed by Savage method, MiliQ water was added for dilution by 5 times, mixed, centrifuged at 8000g at 4℃ for 5min, and after centrifugation, the filtrate was filtered through a sterile microporous filter membrane of 0.22μm. The samples after gradient dilution were detected for the contents of acetic acid, lactic acid and 1,2-propanediol in the fermentation broth by high performance liquid chromatography (HPLC). The instrument parameter settings were as follows: an Aminex HPX-87H column (300mm×7.8mm, Bio Rad) was used, a refractive index detector (RID) was used as the detector, 5mM H2SO4 was used as the mobile phase, the flow rate was 0.5mL / min, and the column temperature was maintained at 50℃. Three biological parallel experiments were performed for each sample.

[0062] (4) Cross-feeding detection method with human milk oligosaccharides as carbon source:

[0063] Streptococcus sp. CCFM1328 was cultured in M17 medium with 5g / L 6’-SL as carbon source for 10h. The fermentation broth was taken and filtered through a sterile microporous filter membrane of 0.22μm to obtain sterile fermentation supernatant, which was mixed with fresh sugar-free MRS medium (without additional sugar carbon source) at 1:1, and Bifidobacterium breve SH-SJ-MZM1 was inoculated into the mixed medium mixed with fermentation broth at an inoculation amount of 1%, and incubated at 37℃ for 48h, and whether the bacterial solution was turbid was observed.

[0064] Example 1: Screening and identification of Streptococcus sp. CCFM1328

[0065] 1. Sample collection

[0066] The breast milk samples were collected in Wuxi, Jiangsu Province, and were placed in sample tubes containing 30% glycerol and stored in a heat preservation box with ice bags. After being brought back to the laboratory, the samples were quickly placed in a -80°C refrigerator for separation and screening.

[0067] 2. Isolation and purification of the strain

[0068] (1) Dilution and coating: 0.5 mL of the breast milk sample stored in 30% glycerol was added to a 7 mL centrifuge tube containing 4.5 mL of physiological saline under sterile conditions to obtain a 10 -1 dilution solution. The above dilution step was repeated to obtain 10 -2 and 10 -3 dilution solutions, respectively.

[0069] (2) Coating culture: 100 μL of the above 10 -1 , 10 -2 , and 10 -3 gradient dilution solutions were respectively taken and uniformly coated on M17 solid medium with 2'-FL as the sole carbon source and bromocresol purple as the indicator, and cultured at 37°C. During the culture, the surrounding yellow colonies were observed and picked after 48 h.

[0070] (3) Primary purification culture: the dilution and coating plates with the number of colonies in the range of 30-300 were taken, and 10 single colonies with white or milky white color, smooth surface, and neat edges of different sizes were randomly selected from each sample and streaked on M17 solid medium containing glucose and cultured at 37°C for 48 h to obtain single colonies, which were named CCFM1328.

[0071] (4) Secondary purification culture: the single colonies on the streaking plates of step (3) were inoculated in M17 liquid medium containing glucose and cultured at 37°C for 20 h to obtain secondary purification culture solution.

[0072] 3. Strain preservation and identification

[0073] (1) Strain preservation

[0074] The above secondary purification culture solution was mixed, 1 mL of the bacterial solution was taken into 2 mL of clean strain preservation tubes in parallel for 5 times. After centrifugation at 6000 rpm for 3 min, the supernatant was discarded, and the bacterial body was resuspended with 0.9% physiological saline. The above operation was repeated for 3 times. Four of them were centrifuged again and the supernatant was discarded, and 1 mL of 30% glycerol was added for resuspension. After 30 min of standing, they were stored in a -80°C refrigerator. One was used for strain identification.

[0075] (2) Amplification and homology analysis of 16S rDNA gene sequence

[0076] The bacterial liquid used in step (1) for strain identification was used as a template for 16S rDNA PCR. The procedure of 16S rDNA PCR was as follows: first step: 94°C, 5 min; second step: 94°C, 30 s; third step: 55°C, 30 s, fourth step: 72°C, 2 min; fifth step: 72°C, 10 min; wherein, the second to fourth steps were repeated for 30 cycles. The system configuration was as follows: forward primer 27F (5'-AGAGTTTGATCCTGGCCTCA-3') 0.25 μL, reverse primer 1492R (GGTTACCTTGTTACGACTT) 0.25 μL, Taq enzyme Mixture 12.5 μL, template 1 μL, double distilled water 11 μL. After the PCR product was confirmed by nucleic acid electrophoresis analysis, it was sent to Suzhou Jinvivoscience Biotechnology Co., Ltd. for sequencing; the sequencing return sequence was compared with NCBI (https: / / www.ncbi.nlm.nih.gov / ) and EzTaxon database (https: / / www.ezbiocloud.net / ) and the species and genus were confirmed.

[0077] (3) Genomic analysis of new species

[0078] The purified CCFM1328 strain was added to the MRS culture medium at an inoculation amount of 2% and cultured to the third generation, and the bacterial slurry was collected and sent to Shanghai Meiji Biomedicine Technology Co., Ltd. for sketch sequencing. The nucleotide sequence of 16S rDNA is shown as SEQ ID NO. 1, and the online analysis platform ANI Calculator (https: / / www.ezbiocloud.net / ) and Genome-to-Genome Distance Calculator (https: / / ggdc.dsmz.de / home.php) were used to analyze the average nucleotide similarity (Average Nucleotide Identity, ANI) and DNA-DNA hybridization (DNA-DNA hybridization, dDDH) of the strain CCFM1328 and its close strains.

[0079] The results of gene sequence alignment showed that the 16S rDNA gene sequence similarity of strain CCFM1328 with Streptococcus infantis, Streptococcus oralis, Streptococcus mitis, Streptococcus pseudopneumoniae, Streptococcus toyakuensis, Streptococcus pneumoniae was 99.30%, 98.66%, 98.59%, 98.38%, 98.38%, 98.10%, respectively. Strain CCFM1328 was preliminarily identified as Streptococcus. It can be seen from Figure 1 that the ANI values of Streptococcus CCFM1328 and its close source strains were 94.91%, 82.33%, 82.34%, 82.40%, 82.53%, 81.87%, respectively, and the dDDH values were 59.10%, 26.00%, 26.20%, 26.30%, 26.40%, 26.00%, respectively, which were much lower than the threshold of bacterial species level identification (ANI>95%, dDDH>70%), so strain CCFM1328 was a potential new species of Streptococcus.

[0080] Example 2: Preparation of Streptococcus CCFM1328 fermentation broth

[0081] The preparation method of Streptococcus CCFM1328 fermentation broth is as follows:

[0082] (1) Dip the bacterial liquid of Streptococcus CCFM1328 from the glycerol tube on the M17 solid culture medium containing glucose, and incubate at 37°C for 48h to obtain single colonies; pick the single colonies and inoculate in M17 liquid medium containing glucose, and incubate at 37°C for 48h in anaerobic environment for activation culture, repeat this operation for 3 times to obtain the activated bacterial liquid.

[0083] (2) The activated bacterial liquid obtained in step (1) is inoculated into M17 liquid medium containing 5g / L corresponding carbon source (2'-FL / 6'-SL / LNT / glucose / GOS) or without additional sugar carbon source at an inoculation amount of 1% (v / v), and incubated at 37°C for 24h to obtain the fermentation broth of Streptococcus CCFM1328.

[0084] Example 3: Utilization ability of Streptococcus CCFM1328 to different human milk oligosaccharides

[0085] The preparation method of the bacterial suspension is the same as step (1) in Example 1. The obtained activated bacterial suspension is inoculated into M17 liquid medium containing 5 g / L of the corresponding carbon source (2'-FL / 6'-SL / LNT / glucose) or without additional sugar carbon source at an inoculation rate of 1% (v / v). The OD of the bacterial suspension is measured every 2 hours using a visible light spectrophotometer. 600 The pH value of the bacterial solution was measured using a pH meter.

[0086] Depend on Figure 2 It was found that Streptococcus CCFM1328 grew rapidly on a medium with 5 g / L glucose, GOS, or three human milk oligosaccharides as the sole carbon source, reaching its maximum biomass (OD) within 24 hours. 600 >2.0). Among them, the growth rate is higher when glucose is the sole carbon source, and relatively slower when GOS, 2'-FL, 6'-SL or LNT are the sole carbon sources. It can be seen that Streptococcus CCFM1328 can utilize 2'-FL, 6'-SL and LNT for growth and reproduction.

[0087] Depend on Figure 3 It was found that when 5 g / L glucose, GOS, or the three human milk oligosaccharides were used as the sole carbon source, the pH value of the fermentation broth of Streptococcus CCFM1328 decreased rapidly, reaching its lowest value (pH < 5.5) within 24 hours. The pH decrease was faster when glucose was used as the sole carbon source, and relatively slower when GOS, 2'-FL, 6'-SL, or LNT were used as the sole carbon source. This indicates that Streptococcus CCFM1328 can utilize 2'-FL, 6'-SL, and LNT for fermentation and acid production.

[0088] Example 4: Utilization efficiency of human milk oligosaccharides by Streptococcus CCFM1328

[0089] The fermentation broth was prepared according to Example 2. The residual amount of human milk oligosaccharides in the fermentation broth of Streptococcus CCFM1328 fermented with different carbon sources was detected by high performance liquid chromatography-mass spectrometry (HPLC-MS).

[0090] Depend on Figure 4 It was found that after culturing Streptococcus CCFM1328 for 24 h, the levels of 2'-FL, 6'-SL, and LNT in the culture medium were significantly reduced (P<0.05). The utilization rates of 2'-FL, 6'-SL, and LNT by the strain reached 99.78%, 97.65%, and 95.98%, respectively. Therefore, Streptococcus CCFM1328 can efficiently decompose and utilize 2'-FL, 6'-SL, and LNT, with particularly high utilization efficiency for 2'-FL.

[0091] Example 5: Intermediate products of human milk oligosaccharides metabolized by Streptococcus CCFM1328

[0092] The fermentation broth was prepared according to Example 2. The contents of fucose, sialic acid and lactose in the fermentation broth of Streptococcus CCFM1328 were detected by high performance liquid chromatography-mass spectrometry (HPLC-MS).

[0093] Depend on Figure 5 It was found that after 10 h of culture, *Streptococcus* CCFM1328 produced fucose or sialic acid in the medium with 2'-FL or 6'-SL as the sole carbon source, respectively. After 24 h of culture, the fucose content further increased, while the sialic acid content decreased (<1 ppm). Furthermore, small amounts of lactose (5-25 ppm) appeared in both 2'-FL and 6'-SL media after 10 h of culture, but significantly decreased (<1 ppm) after 24 h. This indicates that the strain decomposes human milk oligosaccharides during growth, producing their respective structural monomers, including fucose, sialic acid, and lactose. Lactose and sialic acid are further decomposed and utilized by the strain to maintain growth and metabolic activities; fucose is not further decomposed, or is only partially decomposed, thus accumulating in the fermentation broth. Furthermore, no lactose production was detected in the LNT medium during the culture process. However, based on Example 3, Streptococcus CCFM1328 is known to decompose and utilize LNT, suggesting that the strain could decompose LNT to produce lactose. However, the lactose was rapidly metabolized and broken down by the strain, resulting in a low lactose concentration in the fermentation broth. In summary, Streptococcus CCFM1328 can decompose 2'-FL to produce the intermediate products fucose and lactose, decompose 6'-SL to produce sialic acid and lactose, and decompose LNT to produce lactose.

[0094] Example 6: End products of streptococcal CCFM1328 metabolizing human milk oligosaccharides

[0095] The fermentation broth was prepared according to Example 2. The contents of lactic acid and acetic acid in the fermentation broth of Streptococcus CCFM1328 fermented with different carbon sources were detected by high performance liquid chromatography.

[0096] Depend on Figure 6It can be seen that Streptococcus CCFM1328 produced acetic acid and lactic acid after 24h cultivation in the medium with 2'-FL, 6'-SL or LNT as the sole carbon source. The mass ratio of acetic acid and lactic acid in the 2'-FL medium was 1:2.19, in which the acetic acid concentration was 0.648±0.013 mg / mL and the lactic acid concentration was 1.420±0.005 mg / mL; the mass ratio of acetic acid and lactic acid in the 6'-SL medium was 1:0.99, in which the acetic acid concentration was 1.171±0.003 mg / mL and the lactic acid concentration was 1.156±0.045 mg / mL; the mass ratio of acetic acid and lactic acid in the LNT medium was 1:0.89, in which the acetic acid concentration was 1.060±0.003 mg / mL and the lactic acid concentration was 0.947±0.045 mg / mL. Therefore, Streptococcus CCFM1328 can decompose and utilize 2'-FL, 6'-SL and LNT to produce end products acetic acid and lactic acid.

[0097] Example 7: Streptococcus CCFM1328 utilizes 6'-SL to feed Bifidobacterium breve SH-SJ-MZM1

[0098] The mixed medium containing Streptococcus CCFM1328-6'-SL fermentation broth was prepared as follows:

[0099] The preparation method of Streptococcus CCFM1328 fermentation broth was the same as that of Example 2, except that the cultivation time was 10h. The fermentation broth was filtered with a sterile microporous filter membrane of 0.22 μm, and mixed with fresh sugar-free MRS medium (without additional sugar as carbon source) at a volume ratio of 1:1, which was used as the culture medium for Bifidobacterium breve SH-SJ-MZM1.

[0100] The Streptococcus-Bifidobacterium co-culture system was designed and the bacterial count was performed as follows:

[0101] Streptococcus CCFM1328 and Bifidobacterium breve SH-SJ-MZM1 were inoculated into MRS liquid medium for activation three times, and the activated two bacteria were inoculated into MRS liquid medium containing 5 g / L 6'-SL at an inoculation amount of 1%, alone or mixed at a Streptococcus:Bifidobacterium number ratio of 1:1, and placed in a 37°C anaerobic incubator for cultivation for 48h, followed by counting.

[0102] The number of bacteria after the single or co-culturing of Streptococcus CCFM1328 and Bifidobacterium breve SH-SJ-MZM1 was determined by pouring method. The specific determination method was as follows: the bacteria liquid after 48h of culture was gradiently diluted and the appropriate dilution gradient was selected. The bacteria liquid cultured alone was poured on normal MRS solid plate, the co-cultured bacteria liquid was poured on Streptococcus selective culture medium (MRS plate with 6'-SL as the only carbon source) and Bifidobacterium breve selective culture medium (MRS plate with fucose as the only carbon source) respectively, and was placed in 37℃ anaerobic incubator for 48h of culture and then counting. The design of selective culture medium was based on the utilization characteristics of carbon source of the strain. Streptococcus could utilize 6'-SL for growth and reproduction, while Bifidobacterium breve could grow in the culture medium with fucose as the only carbon source.

[0103] From the above results, it can be seen that Streptococcus CCFM1328 could not grow and reproduce with 6'-SL as the only carbon source, and the OD Figure 7 (A: Bifidobacterium breve SH-SJ-MZM1 grew with glucose as the carbon source, B: Bifidobacterium breve SH-SJ-MZM1 grew with 6'-SL as the carbon source, C: Bifidobacterium breve SH-SJ-MZM1 grew with the fermentation liquid of Streptococcus CCFM1328 after utilizing 6'-SL as the carbon source) it can be seen that Bifidobacterium breve SH-SJ-MZM1 could not grow and reproduce with 6'-SL as the only carbon source, and the OD 600 of 48h of culture was 0.089; while it could grow and reproduce with the carbon source in the 6'-SL fermentation liquid of Streptococcus CCFM1328, and the OD 600 of 48h of culture was 0.831, and the viable bacteria number was (4.3±0.6)×10 8 CFU / mL. Therefore, Streptococcus CCFM1328 could decompose 6'-sialyllactose through extracellular enzyme to release lactose and sialic acid, and feed Bifidobacterium breve SH-SJ-MZM1 which had no 6'-sialyllactose utilization ability.

[0104] The viable bacteria number of Streptococcus and Bifidobacterium in 6'-SL culture medium after pure culture and co-culture was shown in Table 2. Figure 8 The black column represented the viable bacteria number of Bifidobacterium under different culture modes, and the gray column represented the viable bacteria number of Streptococcus under different culture modes. The viable bacteria number of Bifidobacterium breve SH-SJ-MZM1 in 6'-SL culture medium after pure culture was 2.83×10 5 CFU / ml, and the viable bacteria number after co-culture increased to about 2.2×10 8 CFU / ml. The viable bacteria number of Streptococcus CCFM1328 after pure culture and co-culture was similar, about 1.9×10 8 CFU / ml. Therefore, there was cross-feeding when Streptococcus CCFM1328 and Bifidobacterium breve SH-SJ-MZM1 were co-cultured in 6'-SL culture medium, and the viable bacteria number of Bifidobacterium was significantly improved.

[0105] Example 8: Preparation of lyophilized formulation of Streptococcus CCFM1328

[0106] The specific steps are as follows:

[0107] (1) Activation of the strain: Take the bacterial solution of Streptococcus CCFM1328 from the glycerol tube and streak it on MRS solid medium. Incubate at 37℃ for 48h to obtain a single colony. Pick a single colony and inoculate it into MRS liquid medium. Incubate at 37℃ for 24h for activation culture. Repeat this operation 3 times to obtain the activated bacterial solution.

[0108] (2) The bacterial culture obtained in step (1) was inoculated into MRS liquid medium at an inoculation rate of 2%, and cultured at 37°C for 24 hours to obtain the fermentation broth. The obtained fermentation broth was centrifuged at 8000 rpm for 20 minutes, and the bacterial sludge was collected. The bacterial sludge was washed three times with physiological saline and then used for later use. The viable cell count was adjusted to 1×10⁻⁶. 11 CFU / mL.

[0109] (3) Preparation of freeze-drying protectant: Mix 100g / L skim milk powder, 100g / L trehalose, 160g / L sucrose and the remaining water to obtain freeze-drying protectant.

[0110] (4) Add the above-prepared freeze-drying protectant to the fungal sludge obtained in step (2), wherein the weight of the freeze-drying protectant is 3 times the weight of the fungal sludge. After mixing evenly, perform vacuum freeze-drying and finally vacuum package the freeze-dried preparation.

[0111] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of streptococcus ( Streptococcus The streptococcus (sp.) CCFM1328 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 63647 and deposit date of July 14, 2023.

2. A microbial preparation containing the Streptococcus CCFM1328 of claim 1.

3. The microbial preparation according to claim 2, characterized in that, The viable count of Streptococcus CCFM1328 in the microbial preparation is not less than 4 × 10⁻⁶. 8 CFU / mL or 4×10 8 CFU / g.

4. Food containing the Streptococcus CCFM1328 as described in claim 1.

5. The application of Streptococcus CCFM1328 as described in claim 1 in the preparation of food products that decompose human milk oligosaccharides, characterized in that, The human milk oligosaccharides include 2'-fucosylated lactose, 6'-sialylated lactose, and lactyl- N - One or more of the four sugars.

6. The use of the Streptococcus CCFM1328 of claim 1 in the preparation of probiotic products that promote the growth of Bifidobacterium breve.

7. The application according to claim 6, characterized in that, The application promotes the growth and / or increase in number of Bifidobacteria brevis in an environment containing human milk oligosaccharides.

8. The application according to claim 7, characterized in that, The human milk oligosaccharides include 2'-fucosylated lactose, 6'-sialylated lactose, and lactyl- N - One or more of the four sugars.

9. The application according to any one of claims 6 to 8, characterized in that, The application involves co-culturing the Streptococcus CCFM1328 with Bifidobacterium breve.

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