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 breast milk oligosaccharides, and the streptococcus can grow by taking breast milk oligosaccharides as a carbon source, the utilization rate of 2'-fucosyllactose, 6'-sialyllactose and lacto-N-tetraose reaches more than 97.26%, and the streptococcus is helpful to promoting the growth of short bifidobacterium which cannot utilize 6'-SL.

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 humans has been proven to be safe and probiotic, and 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 proven 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 the adaptability of themselves or other communities. 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.

[0005] Therefore, screening streptococcus strains that can decompose and utilize human milk oligosaccharides is of great significance for regulating the intestinal microecology and immune response of infants, and promoting the development and application of infant microecological preparations. SUMMARY

[0006] The present application aims to solve the problems in the prior art, and provides a streptococcus capable of decomposing and utilizing human milk oligosaccharides.

[0007] The present application provides a Streptococcus sp. CCFM1330, which is preserved in Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC NO: 63649, and the preservation date is July 14, 2023.

[0008] The Streptococcus sp. CCFM1330 is derived from breast milk of a healthy female, and the strain is identified by the steps of genomic DNA extraction, 16S rDNA specific primer PCR amplification, amplification product purification, DNA sequencing, and sequence alignment. The results show that the strain is Streptococcus sp., named Streptococcus sp. CCFM1330, and stored in Jiangnan University Food Biotechnology Culture Collection Center.

[0009] The colony of the Streptococcus sp. CCFM1330 on M17 solid culture medium is protruding, smooth, round, milky white, translucent, and has a diameter of 0.45 mm.

[0010] The present application provides a microbial preparation containing the Streptococcus sp. CCFM1330.

[0011] In one embodiment, the viable bacterial count of Streptococcus sp. in the microbial preparation is not less than 7×10 8 CFU / mL or 7×10 8 CFU / g.

[0012] The present application provides a food containing the Streptococcus sp. CCFM1330. The Streptococcus sp. CCFM1330 has the ability to decompose and utilize 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. Therefore, the food can provide a theoretical basis and feasibility for the development of probiotic products utilizing breast milk oligosaccharides.

[0013] In one embodiment, the viable bacterial count of Streptococcus sp. in the food is not less than 7×10 8 CFU / mL or 7×10 8 CFU / g.

[0014] In one embodiment, the food is a freeze-dried powder.

[0015] In an embodiment, the preparation method of the freeze-dried powder is: inoculating the Streptococcus CCFM1330 into a culture medium for culture to obtain a seed liquid; inoculating the seed liquid into a culture medium for culture to obtain a culture liquid; centrifuging the culture liquid to collect bacterial slurry; resuspending the bacterial slurry after washing with normal saline to obtain a resuspension liquid; adding a freeze-drying protective agent to the resuspension liquid to obtain a mixed liquid; vacuum freeze-drying the mixed liquid to obtain the freeze-dried powder.

[0016] In an embodiment, the seed liquid is inoculated into the culture medium for culture at an inoculation amount of 2-4% (v / v).

[0017] In an embodiment, the components of the freeze-drying protective agent include skimmed milk powder, trehalose, sucrose and water.

[0018] 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.

[0019] 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.

[0020] In an embodiment, the freeze-drying protective agent is added to the resuspension liquid in an amount of 2-4 times the total weight of the bacterial slurry.

[0021] In an embodiment, the seed culture medium is MRS solid culture medium, and the fermentation culture medium is MRS liquid culture medium.

[0022] In an embodiment, the MRS liquid culture medium is MRS liquid culture medium added with cysteine hydrochloride.

[0023] In an embodiment, the cysteine hydrochloride is added in an amount of 0.04-0.1% by mass fraction.

[0024] In an embodiment, the inoculation amount of the seed liquid into the MRS liquid culture medium for culture is 2-4%, and the culture conditions are: anaerobic culture at 34-38℃ for 24-36 h, centrifugation at 7000-12000 rpm for 20-30 min, and resuspension after washing with normal saline for 3-4 times.

[0025] The application also provides the use of the Streptococcus CCFM1330 in decomposing human milk oligosaccharides.

[0026] 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).

[0027] The application also provides the use of the streptococcus CCFM1330 in promoting the growth of Bifidobacterium breve.

[0028] In one embodiment, the use is to promote the growth and / or increase the number of Bifidobacterium breve in an environment containing human milk oligosaccharides.

[0029] In one embodiment, the human milk oligosaccharides include one or more of 2'-fucosyllactose, 6'-sialyllactose, and lacto-N-tetraose.

[0030] In one embodiment, the use is to co-culture the streptococcus CCFM1330 with Bifidobacterium breve.

[0031] Advantages:

[0032] (1) The streptococcus CCFM1330 provided by the 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 CCFM1330 provided by the application has certain advantages over traditional food, and the strain can be used to prepare probiotic preparations, etc., and has a broad market prospect.

[0033] (2) The streptococcus CCFM1330 provided by the 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, 6'-sialyllactose, and lacto-N-tetraose.

[0034] (3) The streptococcus CCFM1330 provided by the 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 99.96%, and the utilization rate of LNT is 97.26%.

[0035] (4) The streptococcus CCFM1330 provided by the 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.

[0036] (5) The end product of the streptococcus CCFM1330 provided by the application decomposing 2'-FL is acetic acid and lactic acid, and the ratio of the two is 1:1.67; the end product of decomposing 6'-SL is acetic acid and lactic acid, and the ratio of the two is 1:1.23; and the product of decomposing LNT is acetic acid and lactic acid, and the ratio of the two is 1:0.77.

[0037] (6) The Streptococcus CCFM1330 provided by the application can feed the Bifidobacterium breve SH-SJ-MZM1 which cannot utilize 6'-SL when the Streptococcus CCFM1330 grows in the 6'-SL-M17 medium.

[0038] Biological material preservation

[0039] A Streptococcus sp. CCFM1330, taxonomically named Streptococcus sp., has been preserved in the Guangdong Microbial Culture Collection Center on July 14, 2023, with a preservation number of GDMCC NO: 63649 and a preservation address of No. 59 Building, 5th Floor, 100 Middle Liangma Street, Guangzhou. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 ANI and dDDH values of Streptococcus CCFM1330 and its closely related strains.

[0041] Figure 2 Growth curve of Streptococcus CCFM1330 when using human milk oligosaccharides as the sole carbon source.

[0042] Figure 3 pH curve of Streptococcus CCFM1330 when using human milk oligosaccharides as the sole carbon source.

[0043] Figure 4 Residual amount of human milk oligosaccharides of Streptococcus CCFM1330 when using human milk oligosaccharides as the sole carbon source.

[0044] Figure 5 Intermediate product of Streptococcus CCFM1330 when using human milk oligosaccharides as the sole carbon source.

[0045] Figure 6 End product of Streptococcus CCFM1330 when using human milk oligosaccharides as the sole carbon source.

[0046] Figure 7 Streptococcus CCFM1330 feeding Bifidobacterium breve SH-SJ-MZM1 using human milk oligosaccharides.

[0047] Figure 8 Bacterial number of Streptococcus CCFM1330 and Bifidobacterium breve in pure culture and co-culture on 6'-SL.

[0048] Figures 1-8 In the table, “*”, “**”, “***”, and “****” all represent significant differences from the no-sugar group, and the more stars, the greater the significant difference. DETAILED DESCRIPTION

[0049] The application will be further described below in combination with specific embodiments and the accompanying drawings.

[0050] Streptococcus CCFM1330 used in the following examples was deposited in Guangdong Microbial Culture Collection Center on July 14, 2023, with the accession number GDMCC NO: 63649. Bifidobacterium breve SH-SJ-MZM1 is a strain screened from breast milk.

[0051] The culture media involved in the following examples are as follows:

[0052] M17 liquid medium: tryptone 5 g / L, soybean peptone 5 g / L, beef extract 5 g / L, yeast extract powder 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.

[0053] M17 solid medium: tryptone 5 g / L, soybean peptone 5 g / L, beef extract 5 g / L, yeast extract powder 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 / fructose oligosaccharide) 5 g / L, agar powder 20 g / L.

[0054] MRS liquid medium: MRS liquid medium: tryptone 10 g / L, beef extract 10 g / L, yeast powder 5 g / L, glucose 20 g / L, anhydrous sodium acetate 2 g / L, magnesium sulfate heptahydrate 0.5 g / L, manganese sulfate monohydrate 0.25 g / L, diammonium hydrogen citrate 2 g / L, dipotassium hydrogen phosphate trihydrate 2.6 g / L, Tween 80 1 mL / L, cysteine hydrochloride 0.5 g / L.

[0055] MRS solid medium: MRS liquid medium: tryptone 10 g / L, beef extract 10 g / L, yeast powder 5 g / L, glucose 20 g / L, anhydrous sodium acetate 2 g / L, magnesium sulfate heptahydrate 0.5 g / L, manganese sulfate monohydrate 0.25 g / L, diammonium hydrogen citrate 2 g / L, dipotassium hydrogen 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 is added to the solid medium as an indicator.

[0056] The detection methods involved in the following examples are as follows:

[0057] (1) Determination method of growth curve and pH value:

[0058] Streptococcus sp. CCFM1330 overnight culture 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 incubated in a 37°C constant temperature incubator for 24h. The OD of the bacterial solution was measured every 2h with a visible spectrophotometer, and the pH of the bacterial solution was measured with a pH meter. M17 liquid medium with glucose and GOS as the carbon source was used as the positive control, and M17 medium without sugar was used as the negative control. Each sample was tested in triplicate. 600 The pH of the bacterial solution was measured with a pH meter, and M17 liquid medium with glucose and GOS as the carbon source was used as the positive control, and M17 medium without sugar was used as the negative control. Each sample was tested in triplicate.

[0059] (2) Method for determining the residual amount of breast milk oligosaccharides and the amount of intermediate products generated:

[0060] The fermentation broth of Streptococcus sp. CCFM1330 at the mid and late stages of fermentation was accurately pipetted, the protein was removed by the Savage method, MiliQ water was added to dilute 5 times, mixed well, and centrifuged at 8000g at 4°C for 5min. After centrifugation, the filtrate was filtered through a sterile microporous filter membrane of 0.22μm. The gradient-diluted sample was used to detect 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 parameters were set as follows: 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°C. Each sample was tested in triplicate.

[0061] (3) Method for determining the amount of metabolic end products of breast milk oligosaccharides:

[0062] The fermentation broth of Streptococcus sp. CCFM1330 at the late stage of fermentation was accurately pipetted, the protein was removed by the Savage method, MiliQ water was added to dilute 5 times, mixed well, and centrifuged at 8000g at 4°C for 5min. After centrifugation, the filtrate was filtered through a sterile microporous filter membrane of 0.22μm. The gradient-diluted sample was used to detect the contents of acetic acid, lactic acid and 1,2-propanediol in the fermentation broth by high performance liquid chromatography (HPLC). The instrument parameters were set as follows: Aminex HPX-87H column (300mm×7.8mm, Bio Rad) was used, 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°C. Each sample was tested in triplicate.

[0063] (4) Method for detecting cross-feeding with breast milk oligosaccharides as the carbon source:

[0064] Streptococcus CCFM1330 was cultured in M17 medium with 5g / L 6'-SL as carbon source for 10h. The fermentation broth was filtered with 0.22μm sterile microporous filter to obtain sterile fermentation supernatant, mixed with fresh MRS medium without sugar (without additional sugar carbon source) at 1:1, and Bifidobacterium breve SH-SJ-MZM1 was inoculated into the mixed medium with 1% inoculation amount, and then incubated at 37℃ for 48h. Whether the broth was turbid was observed.

[0065] Example 1: Screening and identification of Streptococcus CCFM1330

[0066] 1. Sample collection

[0067] The breast milk samples were collected from 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℃ refrigerator for separation and screening.

[0068] 2. Isolation and purification of strains

[0069] (1) Dilution and coating: 0.5mL of breast milk sample stored in 30% glycerol was added to a 7mL centrifuge tube containing 4.5mL 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.

[0070] (2) Coating culture: 100μL of the above 10 -1 , 10 -2 and 10 -3 dilution solutions were respectively taken and coated on M17 solid medium with 2'-FL as the only carbon source and bromocresol purple as the indicator, and then incubated at 37℃. During the incubation, the yellowing colonies around were observed and picked after 48h.

[0071] (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, neat edge and different sizes were randomly selected from each sample and streaked on M17 solid medium containing glucose, and then incubated at 37℃ for 48h to obtain single colonies, which were named as CCFM1330.

[0072] (4) Secondary purification culture: the single colonies on the streaking plates of step (3) were inoculated in M17 liquid medium containing glucose, and then incubated at 37℃ for 20h to obtain secondary purification culture.

[0073] 3. Strain preservation and identification

[0074] (1) Strain preservation

[0075] Mix the above-mentioned secondary purified culture medium thoroughly. Transfer 1 mL of the bacterial culture to a clean 2 mL culture preservation tube, and repeat the process in five parallel fractions. Centrifuge at 6000 rpm for 3 min, discard the supernatant, and resuspend the bacterial cells in 0.9% physiological saline. Repeat the above operation three times. Centrifuge four fractions again, discard the supernatant, add 1 mL of 30% glycerol, resuspend, let stand for 30 minutes, and then store at -80℃. One fraction is used for bacterial identification.

[0076] (2) Amplification and homology analysis of the 16S rDNA gene sequence

[0077] Step (1) uses the bacterial culture for bacterial identification as the template for 16S rDNA PCR. The 16S rDNA PCR procedure is as follows: Step 1: 94℃, 5min; Step 2: 94℃, 30s; Step 3: 55℃, 30s; Step 4: 72℃, 2min; Step 5: 72℃, 10min; Steps 2 to 4 are repeated 30 times. The system configuration is as follows: forward primer 27F (5'-AGAGTTTGATCCTGGCCTCA-3') 0.25μL, reverse primer 1492R (GGTTAC CTTGTTACGACTT) 0.25μL, Taq enzyme Mixture 12.5μL, template 1μL, double-distilled water 11μL. After the PCR products were confirmed by nucleic acid electrophoresis analysis, they were sent to Suzhou Genewise Biotechnology Co., Ltd. for sequencing. The sequenced sequences were compared with the NCBI (https: / / www.ncbi.nlm.nih.gov / ) and EzTaxon database (https: / / www.ezbiocloud.net / ) for species confirmation.

[0078] (3) Genomic analysis of new species

[0079] The purified CCFM1330 strain was added to MRS medium at a 2% inoculum and cultured to the third generation. The mycelial sludge was collected and sent to Shanghai Meiji Biotechnology Co., Ltd. for draft sequencing. The nucleotide sequence of the 16S rDNA is shown in SEQ ID NO.1. The average nucleotide similarity (ANI) and DNA-DNA hybridization (dDDH) of strain CCFM1330 and its closely related strains were analyzed using the online analysis platforms ANI Calculator (https: / / www.ezbiocloud.net / ) and Genome-to-Genome Distance Calculator (https: / / ggdc.dsmz.de / home.php).

[0080] The results of gene sequence alignment showed that the 16S rDNA gene sequence similarity of strain CCFM1330 with Streptococcus mitis, Streptococcus oralis, Streptococcus infantis, Streptococcus pseudopneumoniae, Streptococcus pneumoniae, Streptococcus toyakuensis was 99.09%, 99.02%, 98.81%, 98.81%, 98.60%, 98.60%, respectively. Strain CCFM1330 was preliminarily identified as Streptococcus. It can be seen that the ANI value of Streptococcus CCFM1330 and its close source strain was 82.89%, 82.52%, 82.86%, 88.38%, 82.43%, 82.78%, respectively, and the dDDH value was 27.00%, 26.20%, 27.50%, 35.00%, 26.70%, 27.20%, respectively, which was far lower than the threshold value of bacterial species level identification (ANI>95%, dDDH>70%). Therefore, strain CCFM1330 is a potential new species of Streptococcus. Figure 1

[0081] Example 2: Preparation of Streptococcus CCFM1330 fermentation broth

[0082] The preparation method of Streptococcus CCFM1330 fermentation broth is as follows:

[0083] (1) The bacterial liquid of Streptococcus CCFM1330 was taken from the glycerol tube and streaked on M17 solid medium containing glucose, and incubated at 37°C for 48h to obtain single colonies; the single colonies were picked and inoculated in M17 liquid medium containing glucose, and incubated at 37°C for 48h in anaerobic environment for activation culture, and the operation was repeated for 3 times to obtain the activated bacterial liquid.

[0084] (2) The activated bacterial liquid obtained in step (1) was 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 CCFM1330.

[0085] Example 3: Utilization ability of Streptococcus CCFM1330 to different human milk oligosaccharides

[0086] ​The preparation method of the bacterial suspension is the same as step (1) in Example 2. 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.

[0087] Depend on Figure 2 It was found that Streptococcus CCFM1330 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 was higher when glucose was the sole carbon source, relatively slower when GOS, 2'-FL, or 6'-SL were the sole carbon sources, and the slowest when LNT was the sole carbon source. It can be seen that Streptococcus CCFM1330 can utilize 2'-FL, 6'-SL, and LNT for growth and reproduction.

[0088] 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 CCFM1330 decreased rapidly, reaching its lowest value (pH < 5.5) within 24 hours. Specifically, the pH decreased most rapidly when glucose was the sole carbon source, relatively more slowly when GOS, 2'-FL, or 6'-SL were used, and the slowest decrease occurred when LNT was used as the sole carbon source. This indicates that Streptococcus CCFM1330 can utilize 2'-FL, 6'-SL, and LNT for fermentation and acid production.

[0089] Example 4: Utilization efficiency of human milk oligosaccharides by Streptococcus CCFM1330

[0090] The fermentation broth was prepared according to Example 2. The residual amount of human milk oligosaccharides in the fermentation broth of Streptococcus CCFM1330 fermented with different carbon sources was detected by high performance liquid chromatography-mass spectrometry (HPLC-MS).

[0091] Depend on Figure 4 It was found that after culturing Streptococcus CCFM1330 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%, 99.96%, and 97.26%, respectively. Therefore, Streptococcus CCFM1330 can efficiently decompose and utilize 2'-FL, 6'-SL, and LNT, with higher utilization rates for 2'-FL and 6'-SL.

[0092] Example 5: Intermediate products of human milk oligosaccharides metabolized by Streptococcus CCFM1330

[0093] The fermentation broth was prepared according to Example 2. The contents of fucose, sialic acid and lactose in the fermentation broth of Streptococcus CCFM1330 were detected by high performance liquid chromatography-mass spectrometry (HPLC-MS).

[0094] Depend on Figure 5 It was found that after culturing Streptococcus CCFM1330 for 10 h, fucose (328.55±7.04 ppm) or sialic acid (34.43±0.09 ppm) were produced in the culture medium with 2'-FL or 6'-SL as the sole carbon source, respectively; after culturing for 24 h, the contents of fucose (229.32±12.18 ppm) and sialic acid (14.12±0.97 ppm) decreased significantly. In addition, after culturing for 10 h, only a small amount of lactose (<2 ppm) was detected in the three culture media. According to Example 3, Streptococcus CCFM1330 can decompose and utilize 2'-FL, 6'-SL and LNT. Therefore, it is speculated that the strain can decompose 2'-FL, 6'-SL and LNT and produce the structural unit lactose. However, the lactose is rapidly metabolized and decomposed by the strain, resulting in a low lactose concentration in the fermentation broth. Therefore, during its growth, the strain decomposes human milk oligosaccharides, producing their respective structural monomers, including fucose, sialic acid, and lactose. Lactose and sialic acid are further broken down and utilized by the strain to maintain growth and metabolic activities; fucose is not further broken down, or is only partially broken down, thus accumulating in the fermentation broth. In summary, Streptococcus CCFM1330 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.

[0095] Example 6: End products of streptococcal CCFM1330 metabolizing human milk oligosaccharides

[0096] The fermentation broth was prepared according to Example 1, and the contents of lactic acid and acetic acid in the fermentation broth of Streptococcus CCFM1330 were detected by high performance liquid chromatography.

[0097] Depend on Figure 6It can be seen that Streptococcus CCFM1330 produces acetic acid and lactic acid after 24h culture 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 is 1:1.67, in which the acetic acid concentration is 0.632±0.008mg / mL and the lactic acid concentration is 1.054±0.012mg / mL; the mass ratio of acetic acid and lactic acid in the 6'-SL medium is 1:1.23, in which the acetic acid concentration is 1.101±0.007mg / mL and the lactic acid concentration is 1.353±0.026mg / mL; the mass ratio of acetic acid and lactic acid in the LNT medium is 1:0.77, in which the acetic acid concentration is 1.231±0.005mg / mL and the lactic acid concentration is 0.952±0.017mg / mL. Therefore, Streptococcus CCFM1330 can decompose and utilize 2'-FL, 6'-SL and LNT to produce end products acetic acid and lactic acid.

[0098] Example 7: Streptococcus CCFM1330 utilizes 6'-SL to feed Bifidobacterium breve SH-SJ-MZM1

[0099] The mixed medium containing Streptococcus CCFM1330-6'-SL fermentation broth is prepared as follows:

[0100] The preparation method of Streptococcus CCFM1330 fermentation broth is the same as that of Example 2, except that the culture time is 10h. The fermentation broth is filtered with a sterile microporous filter membrane of 0.22μm, and mixed with fresh sugar-free MRS medium (without additional sugar as carbon source) at a volume ratio of 1:1, which is used as the culture medium for Bifidobacterium breve SH-SJ-MZM1.

[0101] The design of Streptococcus-Bifidobacterium co-culture system and the method of bacterial counting are as follows:

[0102] Streptococcus CCFM1330 and Bifidobacterium breve SH-SJ-MZM1 are inoculated into MRS liquid medium for activation three times. The activated two bacteria are inoculated into MRS liquid medium containing 5g / L 6'-SL at an inoculation amount of 1%, alone or mixed at a ratio of 1:1 (Streptococcus:Bifidobacterium), and placed in a 37℃ anaerobic incubator for culture for 48h, followed by counting.

[0103] The number of bacteria after the single or co-culturing of Streptococcus CCFM1330 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.

[0104] From the above results, it can be seen that Streptococcus CCFM1330 could not utilize 6'-SL for growth and reproduction, and the OD Figure 7 (A: Bifidobacterium breve SH-SJ-MZM1 grew with glucose as carbon source, B: Bifidobacterium breve SH-SJ-MZM1 grew with 6'-SL as carbon source, C: Bifidobacterium breve SH-SJ-MZM1 grew with the fermentation liquid of Streptococcus CCFM1330 after utilizing 6'-SL as 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 CCFM1330, and the OD 600 of 48h of culture was 1.102, and the viable bacteria count was (7.7±0.5)×10 8 CFU / mL. Therefore, Streptococcus CCFM1330 could decompose 6'-sialyllactose by extracellular enzyme to release lactose and sialic acid, and feed Bifidobacterium breve SH-SJ-MZM1 which had no 6'-sialyllactose utilization ability.

[0105] The viable bacteria count of Streptococcus and Bifidobacterium in 6'-SL culture medium after pure culture and co-culture was shown in Table 1. Figure 8 In Table 1, the black column represented the viable bacteria count of Bifidobacterium in the two culture modes, and the gray column represented the viable bacteria count of Streptococcus in the two culture modes. The viable bacteria count 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 count after co-culture increased to 3.37×10 8 CFU / mL. The viable bacteria count of Streptococcus CCFM1330 after pure culture and co-culture was similar, about 3.5×10 8 CFU / mL. Therefore, there was cross-feeding between Streptococcus CCFM1330 and Bifidobacterium breve SH-SJ-MZM1 in 6'-SL culture medium, and the viable bacteria count of Bifidobacterium was significantly improved.

[0106] Example 8: Preparation of freeze-dried preparation of Streptococcus CCFM1330

[0107] The specific steps are as follows:

[0108] (1) Activation of the strain: the bacterial solution of Streptococcus CCFM1330 was streaked on MRS solid medium from a glycerol tube, and incubated at 37°C for 48 h to obtain single colonies; the single colonies were inoculated into MRS liquid medium and incubated at 37°C for 24 h for activation culture, and this operation was repeated 3 times to obtain the activated bacterial solution.

[0109] (2) The bacterial solution obtained in step (1) was inoculated into MRS liquid medium at an inoculation amount of 2%, and incubated at 37°C for 24 h to obtain the fermentation broth; the fermentation broth was centrifuged at 8000 rpm for 20 min to collect the bacterial slurry, which was washed with normal saline for 3 times and then reserved for use, and the viable cell count was adjusted to 1×10 11 CFU / mL.

[0110] (3) Preparation of freeze-drying protectant: 100 g / L skim milk powder, 100 g / L trehalose, 160 g / L sucrose and the rest water were mixed to obtain the freeze-drying protectant.

[0111] (4) The freeze-drying protectant prepared above was added to the bacterial slurry obtained in step (2), and the weight of the freeze-drying protectant was 3 times of the weight of the bacterial slurry, and then mixed uniformly and vacuum freeze-dried, and finally the freeze-dried preparation was vacuum packaged.

[0112] Although the present application has been disclosed with the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.

Claims

1. A strain of Streptococcus ( Streptococcus sp.) CCFM1330, characterized in that, It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 14, 2023, with accession number GDMCC NO: 63649.

2. A microbial preparation containing the Streptococcus CCFM1330 of claim 1.

3. The microbial preparation according to claim 2, characterized in that, The viable count of streptococci in the microbial preparation is not less than 7 × 10⁻⁶. 8 CFU / mL or 7×10 8 CFU / g.

4. Food containing the Streptococcus CCFM1330 as described in claim 1.

5. The application of Streptococcus CCFM1330 according to 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 CCFM1330 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 CCFM1330 with Bifidobacterium breve.

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