CN116064272A

CN116064272A - Streptococcus thermophilus with serotonin level improving function and application thereof

Info

Publication number: CN116064272A
Application number: CN202210831191.0A
Authority: CN
Inventors: 赵建新; 刘小鸣; 于朋; 杨波; 陈海琴; 崔树茂; 陈卫
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2022-07-14
Filing date: 2022-07-14
Publication date: 2023-05-05

Abstract

The invention discloses streptococcus thermophilus with the effect of improving serotonin level and application thereof, belonging to the technical field of dairy processing and fermentation. The invention screens a streptococcus thermophilus GDMCC No.60810 from yak triala in Qinghai-Xiding region, the strain is a food-grade safe strain, and can obviously improve the serotonin level in animal serum, and the up-regulation multiple reaches 6.7 times. In addition, the streptococcus thermophilus GDMCC No.60810 has good tolerance to the gastrointestinal tract and can increase the abundance of bifidobacteria in the intestinal tract. Therefore, the streptococcus thermophilus GDMCC No.60810 can be used for preparing various foods and microbial preparations, and can also be used for preparing medicines for treating symptoms related to serotonin change, such as depression, anxiety symptoms, PTSD symptoms, parkinsonism and the like.

Description

Streptococcus thermophilus with serotonin level improving function and application thereof

Technical Field

The invention relates to streptococcus thermophilus with serotonin level improving function and application thereof, belonging to the technical field of dairy processing and fermentation.

Background

Serotonin is an important monoamine neurotransmitter of the brain, and more than 90% of serotonin is produced by converting tryptophan from colonic enterochromaffin cells. Recent studies have shown that the effects of serotonin on the body may be affected by intestinal microorganisms. Some microorganisms in the gut have the ability to produce serotonin, and at the same time, the microbiota and its metabolites (e.g. butyric acid) can regulate the body's serotonin level by affecting tryptophan hydroxylase activity or tryptophan metabolic pathways. In turn, serotonin and its reuptake inhibitors can modulate intestinal microbial composition and function. Serotonin, an important signal molecule connecting intestinal microorganisms and the brain, plays an important role in regulating and controlling nervous system, gastrointestinal functions, organism lipid metabolism, immune system and the like. Studies show that serotonin is related to the functions of regulating emotion, energy, memory and the like, and the lack of serotonin is closely related to the occurrence of depression.

Intestinal flora is involved in the regulation of central and peripheral nervous functions. Studies have shown that bioactive nutrients and intestinal flora can alter DNA methylation and histone characteristics through a variety of mechanisms, and that microorganisms in the human intestinal tract may play an important role in regulating various elements of the brain axis. Currently, certain bifidobacteria and lactobacilli are able to regulate neurological functions: a strain of Lactobacillus rhamnosus which increases the level of the neurotransmitter 5-hydroxytryptamine in the brain is disclosed in patent CN11454024A, a strain of Lactobacillus reuteri which increases the level of the neurotransmitter 5-hydroxytryptamine in the peripheral blood of rats is disclosed in patent CN107523526A, a strain of Bifidobacterium breve which increases the level of 5-hydroxytryptamine in serum of mice with Alzheimer's disease is disclosed in patent CN113215034A, and a strain of Lactobacillus lactis which increases the level of central 5-hydroxytryptamine is disclosed in patent CN 113512514A.

Streptococcus thermophilus is one of the important fermentation strains developed in the dairy industry, and researches on the streptococcus thermophilus are mostly focused on fermentation characteristics, such as the influence of the streptococcus thermophilus on the acidogenesis, the flavor formation and the texture characteristics of the fermented milk, and the streptococcus thermophilus serving as a common food fermentation strain has important significance if the streptococcus thermophilus can be further mined to play a role in host metabolism regulation after being ingested.

Disclosure of Invention

The inventor screens a streptococcus thermophilus from yak triton in Qinghai Xining region, the strain can obviously improve the host serotonin level, and can increase the abundance of bifidobacterium in feces, so that theoretical guidance is provided for the research of the diversity of the streptococcus thermophilus strain and the application of the streptococcus thermophilus strain, and meanwhile, the fermentation fungus library resources in China can be enriched, and the streptococcus thermophilus strain has important significance for industry development.

A first object of the present invention is to provide a Streptococcus thermophilus (Streptococcus thermophilus) which has been selected from Yak Trela collected in Qinghai-Xiding region of China and which has been deposited at the microorganism culture Collection of Cantonese, 10 months and 16 days of 2019, under the accession number GDMCC No.60810, at accession number Guangzhou, first, china, no. 100, university, no. 59 building 5.

In one embodiment, the streptococcus thermophilus GDMCC No.60810 strain has the following properties:

(1) Strains can significantly up-regulate serotonin levels in host serum;

(2) The strain can obviously promote the abundance of bifidobacteria in intestinal tracts;

(3) The strain has good gastrointestinal tract tolerance and in vivo proliferation capacity.

A second object of the present invention is to provide a method for culturing said Streptococcus thermophilus GDMCC No.60810, inoculating said Streptococcus thermophilus into M17 medium and culturing at 35-40 ℃.

A third object of the present invention is to provide a composition comprising said Streptococcus thermophilus GDMCC No.60810.

In one embodiment, the composition comprises one or more of a live strain of Streptococcus thermophilus GDMCC No.60810, a dried strain of Streptococcus thermophilus GDMCC No.60810, a metabolite of Streptococcus thermophilus GDMCC No.60810, an inactivated Streptococcus thermophilus GDMCC No.60810, a fermentation broth of Streptococcus thermophilus GDMCC No.60810, a lysate of Streptococcus thermophilus GDMCC No.60810, or an extract of Streptococcus thermophilus GDMCC No.60810.

In one embodiment, the composition has a live Streptococcus thermophilus bacterial count of not less than 5X 10 ⁹ CFU/mL or 5X 10 ⁹ CFU/g。

It is a fourth object of the present invention to provide a product containing said composition.

In one embodiment, the product includes, but is not limited to, a microbial agent, a food or a pharmaceutical product.

In one embodiment, the product is used to increase serotonin levels and/or to increase the abundance of bifidobacteria in the gut.

Preferably, the product is for increasing serotonin levels in serum.

More preferably, the product can raise serotonin levels in serum to alleviate or modulate symptoms of depression, anxiety, PTSD and parkinson's disease.

In one embodiment, the pharmaceutical product comprises the streptococcus thermophilus GDMCC No.60810, a pharmaceutical carrier and/or a pharmaceutical excipient.

In one embodiment, the pharmaceutical carrier comprises microcapsules, microspheres, nanoparticles, and/or liposomes.

In one embodiment, the pharmaceutical excipients comprise excipients and/or additives.

In one embodiment, the excipient comprises a binder, filler, disintegrant, and/or lubricant.

In one embodiment, the additive comprises a solubilizing agent, a co-solvent, a latent solvent, and/or a preservative.

In one embodiment, the pharmaceutical product is in the form of powder, granule, capsule, tablet, pill or oral liquid.

In one embodiment, the product has a viable count of Streptococcus thermophilus GDMCC No.60810 of no less than 5×10 ⁹ CFU/mL or 5X 10 ⁹ CFU/g。

In one embodiment, the food product is a health food product; or the food is dairy product, bean product or fruit and vegetable product produced by using a starter containing the streptococcus thermophilus GDMCC No. 60810; or the food is a beverage or snack containing the streptococcus thermophilus GDMCC No.60810.

In one embodiment, the fermented food is produced by fermentation using the streptococcus thermophilus GDMCC No.60810, and comprises solid food, liquid food and semi-solid food.

In one embodiment, the fermented food product comprises a dairy product, a soy product, or a fruit and vegetable product; the dairy product comprises milk, yoghurt, sour cream or cheese; the bean product comprises soybean milk and sour soybean milk; the fruit and vegetable product comprises yellow peach, hawthorn, cucumber, carrot, beet, celery or cabbage product.

In one embodiment, the preparation method of the starter comprises inoculating the streptococcus thermophilus GDMCC No.60810 into a culture medium according to an inoculum size accounting for 2-4% of the total mass of the culture medium, and culturing for 18h at 37 ℃ to obtain a culture solution; centrifuging the culture solution to obtain thalli; washing thalli with normal saline for 3 times, and then re-suspending the thalli with a freeze-drying protective agent to obtain re-suspension; lyophilizing the heavy suspension by vacuum freezing to obtain the starter.

In one embodiment, the lyoprotectant is added to the suspension before lyophilization, wherein the mass ratio of lyoprotectant to thallus is 2:1.

In one embodiment, the lyoprotectant is skimmed milk powder at a concentration of 130 g/L.

The invention has the beneficial effects that:

1. the streptococcus thermophilus GDMCC No.60810 can obviously improve the serotonin level in serum, and the up-regulation rate is up to 6.7 times from 0.47mg/L to 3.6mg/L, so that the streptococcus thermophilus GDMCC No.60810 plays a role in regulating serotonin-change-related symptoms such as depression, anxiety symptoms, PTSD symptoms, parkinsonism and the like;

2. the streptococcus thermophilus GDMCC No.60810 can remarkably promote the abundance of bifidobacteria in intestinal tracts, good gastrointestinal tract tolerance and in vivo proliferation capacity;

3. the streptococcus thermophilus GDMCC No.60810 is a safe strain which can be used for food, has no other chemical substances, is safe and healthy, can be used for preparing various foods, medicines and microbial preparations, and improves the flavor of the product.

Preservation of biological materials

The streptococcus thermophilus provided by the invention is named Streptococcus thermophilus in taxonomy, is preserved in the microorganism strain collection of Guangdong province at 10 months 16 in 2019, and has a preservation number of GDMCC No.60810 and a preservation address of building 5 of Guangzhou Xian Zhou 100 university, guangzhou City.

Drawings

FIG. 1 is a photograph of a colony of Streptococcus thermophilus GDMCC No.60810 on M17 solid medium;

FIG. 2 is a photograph of a gram-stained strain of Streptococcus thermophilus GDMCC No.60810 under a 1000-fold microscope;

FIG. 3 shows the increase in serotonin change in mice with Streptococcus thermophilus GDMCC No. 60810;

FIG. 4 is a graph showing the promotion of bifidobacteria in mouse faeces by Streptococcus thermophilus GDMCC No. 60810;

FIG. 5 is a graph showing the evaluation of gastrointestinal tract tolerance and proliferation in vivo of Streptococcus thermophilus GDMCC No.60810.

FIG. 6 shows the growth of Streptococcus thermophilus GDMCC No.60810 in skim milk fermentation;

FIG. 7 shows the acidogenesis of Streptococcus thermophilus GDMCC No.60810 in the fermentation of skim milk;

FIG. 8 shows the volatile content of Streptococcus thermophilus GDMCC No.60810 after fermentation of skim milk.

Detailed Description

Definition of the definition

As used herein, the following belongs to the following definitions.

"serotonin" or 5-hydroxytryptamine (abbreviated "5 HT") is commonly known as a neurotransmitter in the CNS, and it is involved in emotional processing (influencing emotion) and cognition.

The term "strain" as used herein refers to a microorganism of a particular species having common characteristics. The terms "strain" and "cell" are used interchangeably herein unless indicated to the contrary.

The term "plate" as used herein refers to a plate culture medium, which is the most commonly used form of solid medium used to obtain pure culture of microorganisms, and which is a solid plane of the medium formed by cooling solidified solid medium in a sterile petri dish, often referred to simply as a culture plate, or plate.

The term "medium" as used herein refers to a medium comprising the chemical elements necessary for the growth of the microorganism together with at least one carbon source and one nitrogen source.

M17 liquid Medium (1L): 5.0g of soybean peptone, 2.5g of casein peptone, 2.5g of yeast extract powder, 5.0g of beef extract powder, 5.0g of lactose, 0.5g of sodium ascorbate, 19.0g of beta-sodium glycerophosphate, 0.25g of magnesium sulfate and a pH value of 7.2+/-0.2. Adding distilled water to dissolve completely, sterilizing at 115 deg.C for 20min at pH of 6.8-7.2.

M17 solid medium (1L): prepared according to the M17 liquid medium, and then 18g of agar powder is added. Sterilizing at 115 deg.C for 20min.

Male C57BL/6 mice: purchased from beijing velutinin inc.

Control strain LMD9: is a commercial starter, and is purchased from China center for type culture Collection of microorganisms. LMD9 has been reported to have a certain in vivo colonisation capacity and a certain in vivo regulatory effect. Thus, LMD9 was used as a control bacterium in studying streptococcus thermophilus host regulation.

Serum metabolite detection method: 100. Mu.L of serum was carefully aspirated into a 1.5mL centrifuge tube; adding 400 mu L of methanol (pre-cooling at-20 ℃ in advance) to precipitate protein, and generating a large amount of white floccules after adding; vortex 30s, placing the sample in a refrigerator at-20 ℃ for incubation for 1h, and improving the protein precipitation rate; high-speed centrifugation at 15000rpm for 15min at 4 ℃; 400 μl of the supernatant was evaporated to dryness. 100 μl methanol was added: vortex 30s after water (4:1, v/v), redissolve; centrifuging at 15000rpm for 15min at 4deg.C, removing supernatant, and loading into sample injection vial for machine detection; QC preparation: transferring the same volume from the prepared sample to be measured to a new sample injection bottle, and uniformly mixing; and (3) Blank preparation: 1mL of methanol: and (3) uniformly mixing water (4:1, v/v) to a sample injection bottle. Metabolome analysis was performed in full scan mode using an UltiMate 3000HPLC system (Thermo Fisher Scientific, MA, USA) in tandem with a high resolution Q-exact mass spectrometer (Thermo Fisher Scientific, MA, USA). The chromatographic separation of the metabolites was carried out using a Waters Acquity UPLCHSS reverse phase T3 column (Waters, M, USA; 2.1X100 mm. Times.1.8 μm) at a column temperature of 35 ℃.

The present invention will now be described in detail with reference to the embodiments thereof as illustrated in the accompanying drawings, wherein like numerals refer to like features throughout. While specific embodiments of the invention are shown in the drawings, it should be understood that the invention may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the suitable methods, examples of materials, are set forth below for purposes of illustration only and are not intended to be limiting.

Further, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic can be included in at least one implementation of the invention.

Example 1: method for separating and identifying streptococcus thermophilus GDMCC No.60810 strain

(1) Preparation of a suitable sample dilution gradient and incubation

And (5) taking out the yak qula in Qinghai Xining region stored in a refrigerator at the temperature of-80 ℃ and thawing the yak qula on ice. Adding 0.5mL sample into 4.5mL sterile physiological saline after shaking and mixing uniformly, completing 10 times dilution once, taking out 0.5mL diluent from the diluent after shaking and mixing uniformly, adding into 4.5mL sterile physiological saline, completing 10 times dilution for the second time, and so on until dilution reaches 10 times ^-6 100 mu L of each gradient of diluent is sucked, uniformly coated on an M17 solid culture medium plate, inverted, placed in anaerobic condition at 37 ℃ for culture for 36-48 h, and observed in time.

(2) Scribing separation and purification

And (3) taking out the plate with the colonies, selecting a gradient plate with obvious single colonies, picking out the colonies with different colony forms, and carrying out secondary streaking until all the single colonies are purified.

(3) Gram staining and catalase experiments

Picking single colony on a glass slide, performing smear, drying, fixing, primary dyeing, water washing, mordant dyeing, water washing, decoloring, counterstaining, water washing, drying, microscopic examination, and recording gram dyeing results (see figure 2); and picking single colony on the glass slide, adding 3% hydrogen peroxide solution, observing whether bubbles are generated or not, and recording the catalase contact result.

(4) Preservation of bacterial species

Single colony of each strain after purification is picked into 5mL of liquid M17 culture medium, placed under anaerobic condition at 37 ℃ for static culture for 20-24 h, 1mL of bacterial liquid is sucked into a bacteria-preserving tube, centrifuged at 6000rpm for 3min, supernatant is poured out, 1mL of 37% sterile glycerol solution is added, resuspended and placed at-80 ℃ for preservation.

(5) 16S rDNA sequence amplification

Sucking 1mL of bacterial liquid, centrifuging at 6000rpm for 3min, pouring out the supernatant, washing twice, centrifuging, pouring out the supernatant to obtain bacterial mud, and performing PCR amplification by taking the bacterial mud as a template, wherein the flow is as follows:

1) Amplification System 20. Mu.L:

wherein the template amount is 1 mu L, the two-way primers are 1 mu L each, and the Taq enzyme MasterMix is 10 mu L, and the ddH is the same as that of the two-way primer ₂ O was 7. Mu.L.

The primers used were 27F and 1492R,

27F：AGA GTT TGA TCC TGG CCT CA(SEQ ID No.1)，

1492R：GGT TAC CTT GTT ACG ACT T(SEQ ID No.2)。

2) Amplification conditions:

pre-denaturation: 3min at the temperature of 95 ℃,

first step denaturation: at 94 ℃ for 1min,

and a second step of annealing: the temperature of the mixture is 60 ℃ for 37s,

and a third step of extension: the temperature is 72 ℃ for 2min,

cycle times: the cycle number is 37 and the cycle time is 37,

and a fourth step of final extension: at the temperature of 72 ℃ for 5min,

and a fifth step of maintaining: 10min at 12 ℃.

(6) Agarose gel electrophoresis

Weighing 80mL of agarose, adding 80mL of 1 xTAE into a conical flask, heating for 4min by microwave intermittently until the liquid is clear and transparent, slightly cooling, adding 4 mu L of nucleic acid dye, shaking uniformly, pouring into a gel plate groove without bubbles, cooling for 40min for solidification, placing into an electrophoresis groove, exhausting bubbles, sequentially adding PCR amplification products, adding 2 mu L of PCR amplification products into each hole, running gel for 120V for 15min, taking out after finishing, placing into a gel electrophoresis imager for photographing and preserving, recording the serial number of a PCR successful sample, and placing the successful PCR products into a refrigerator at the temperature of minus 20 ℃ for preserving.

(7) Sequencing and identification

And (3) sending the sample with successful PCR to a Instrongback (Shanghai) trade company for detection, carrying out BLAST search in a National Center for Biotechnology Information (NCBI) sequence database (http:// www.ncbi.nlm.nih.gov/BLAST) according to the fed-back sequence result (the sequencing result is shown as SEQ ID NO. 1), and selecting strain information with highest matching degree for result recording. The strain provided by the invention is streptococcus thermophilus (Streptococcus thermophilus) through analysis and identification.

Example 2: streptococcus thermophilus GDMCC No.60810 for increasing serum serotonin level in mice

Healthy male C57BL/6 mice weighing 18-20g were taken as experimental animal strains, and animals were divided into 3 groups, namely a normal Control group (Control), a Streptococcus thermophilus Control strain LMD9 group and a Streptococcus thermophilus GDMCC No.60810 group. Strain group was perfused with the corresponding strain daily at a concentration of 10 per mouse ⁹ CFU/only; normal control group is irrigated with the same volume of physiological saline. All groups were free drinking and ingestion. Mice were tested twice weekly for body weight and food intake. On day 28, mice were anesthetized and quickly collected from the eyeball to take blood after 12 hours fasted after the end of gavage, and sacrificed in combination with cervical dislocation. Blood samples of the sacrificed mice were collected, and the blood samples were allowed to stand at 4℃for 1h, centrifuged at 3000rpm for 15min, and the supernatant serum was carefully collected. Serum metabolites were assayed and the serotonin content of the serum was measured as shown in FIG. 3.

As can be seen from FIG. 3, streptococcus thermophilus GDMCC No.60810 caused a significant change in serotonin in normal mice serum, up-regulated 6.7-fold from 0.47mg/L to 3.6mg/L for the Control group. And at the same time is significantly greater than 0.8mg/L of the control strain LMD 9. Thus, streptococcus thermophilus GDMCC No.60810 has the function of remarkably regulating serotonin in serum.

Example 3: streptococcus thermophilus GDMCC No.60810 promotes proliferation of bifidobacteria in mouse faeces

Healthy male C57BL/6 mice weighing 18-20g were taken as experimental animal strains, and animals were divided into 3 groups, namely a normal Control group (Control), a Streptococcus thermophilus Control strain LMD9 group and a Streptococcus thermophilus GDMCC No.60810 group. The corresponding strain was perfused daily with a concentration of 10 per mouse ⁹ CFU/alone, all groups were free drinking and ingestion. Mice were tested twice weekly for body weight and food intake. On day 28, feces of each group of mice were collected, placed on ice, immediately transferred to a-80 ℃ refrigerator, DNA from the feces was extracted using a DNA rapid extraction kit according to the protocol steps, PCR amplification was performed on the V3-V4 region of the sample 16S rDNA, the amplified fragments were purified and recovered using a gel recovery kit, high throughput sequencing was performed on the Illumina miseq pe platform, and amplicon was analyzed using a QIIME2 analysis platform.

Serotonin changes in serum are to some extent regulated by intestinal microorganisms. Some microorganisms in the intestinal tract have the ability to produce serotonin, and at the same time, the microbiota and its metabolites can regulate the body's serotonin level by affecting tryptophan hydroxylase activity or tryptophan metabolic pathways. Studies have shown that bifidobacteria change is closely related to the level of serotonin in the body. As can be seen from fig. 4, the streptococcus thermophilus GDMCC No.60810 was able to significantly increase the abundance of bifidobacteria in the stool from 2.8% to 7.0% after 28 days of gastric lavage compared to the Control group, consistent with the result that the streptococcus thermophilus GDMCC No.60810 was able to significantly increase the serotonin content in the serum. Whereas the control strain LMD9 significantly reduced the abundance of bifidobacteria.

Example 4: gastrointestinal tract tolerance and in vivo proliferation assessment of streptococcus thermophilus GDMCC No.60810

(1) Inoculating Streptococcus thermophilus GDMCC No.60810 stored at-80deg.C into M17 liquid culture medium, culturing at 37deg.C for 24 hr, subculturing for 2-3 times until the bacterial concentration reaches 10 ⁸ ～10 ⁹ CFU/mL; 10. Mu.L of the bacterial liquid after activation in M17 medium was taken out and added to 0.99mL of simulated gastric fluid and simulated intestinal fluid, and the mixture was vortexed and vibrated 3 times in a vortex mixer. The bacterial liquid simulating gastric juice is added and cultured for 0h at 37 ℃ and then subjected to living bacteria counting experimental operation. The bacterial solution of the simulated intestinal juice is added and cultured for 0h and 1h at 37 ℃ for living bacteria counting experimental operation.

The simulated gastric fluid and intestinal fluid were formulated as follows:

(1) simulating gastric juice:

a fixed amount of pepsin (Sigma, p 7000) was weighed and dissolved in 2/3 of physiological saline (0.5% w/v), pH was adjusted to 3.0 with concentrated hydrochloric acid, and the solution was filtered through a 0.22 μm sterile filter membrane to a desired volume to give a final concentration of 3g/L. Simulate gastric juice to be used at present.

(2) Simulation of intestinal juice:

a fixed amount of trypsin (Sigma, p 1500), bile salts (oxoid, LP 0055) were weighed and dissolved in 2/3 of the desired volume of physiological saline (0.5% w/v), pH was adjusted to 8.0 with 0.1mol/L NaOH, and the final concentration was adjusted to 1g/L and 0.3% by filtration through a 0.22 μm sterile filter to the desired volume. The simulated intestinal juice is prepared at present.

As can be seen from FIG. 5A, the Streptococcus thermophilus GDMCC No.60810 strain has a higher acid and bile salt resistance in vitro than the control strain. Indicating that Streptococcus thermophilus GDMCC No.60810 has good gastrointestinal tract tolerance.

(2) Measurement of intestinal proliferation ability of Streptococcus thermophilus

Faeces were collected at

days

0, 7 and 28 according to the animal design of the experiment of example 3 and the amount of Streptococcus thermophilus in the faeces was measured. DNA in the feces was extracted according to the protocol using a DNA rapid extraction kit, amplified using primers specific for Streptococcus thermophilus, and the absolute biomass of Streptococcus thermophilus was detected using a qPCR instrument.

The primer sequences are as follows:

F：5’-TTATTTGAAAGGGGCAATTGCT-3’；

R：5’-GTGAACTTTCCACTCTCACAC-3’。

at the same time, with continued strain intervention, the proliferation potency of streptococcus thermophilus in vivo increased significantly after 7 days of gastric lavage and remained stable at days 7, 28 (fig. 5B). It is suggested that GDMCC No.60810 may have better proliferation capability in the intestinal tract.

Example 5: growth of streptococcus thermophilus GDMCC No.60810 in skimmed milk fermentation

Inoculating Streptococcus thermophilus GDMCC No.60810 stored at-80deg.C into M17 liquid culture medium, culturing at 37deg.C for 24 hr, subculturing for 2-3 times until the bacterial concentration reaches 10 ⁸ ～10 ⁹ CFU/mL; taking out the bacterial liquid activated in the M17 culture medium, inoculating the bacterial liquid into skim milk according to the volume ratio of 2 percent, and leading the bacterial amount in the system to reach 10 ⁶ ～10 ⁷ CFU/g; the inoculated samples were placed in an incubator at 42℃for fermentation, sampled every 6 hours, and the change in the bacterial load during fermentation was detected, and the results are shown in FIG. 6.

As can be seen from FIG. 6, after 6 hours of fermentation, streptococcus thermophilus GDMCC No.60810 increased by about 1.5 orders of magnitude and reached a viable count equivalent to LMD9 for 24 hours, and thus Streptococcus thermophilus GDMCC No.60810 had good growth in skim milk, which could be the slow fermenting strain of choice.

Example 6: acid production of streptococcus thermophilus GDMCC No.60810 in skimmed milk fermentation

Inoculating Streptococcus thermophilus GDMCC No.60810 stored at-80deg.C into M17 liquid culture medium, culturing at 37deg.C for 24 hr, subculturing for 2-3 times until the bacterial concentration reaches 10 ⁸ ～10 ⁹ CFU/mL; taking out the bacterial liquid activated in the M17 culture medium, inoculating the bacterial liquid into skim milk according to the volume ratio of 2 percent, and leading the bacterial amount in the system to reach 10 ⁶ ～10 ⁷ CFU/g; the inoculated samples were placed in an incubator at 42℃for fermentation, samples were taken every 6 hours, and the pH change during fermentation was measured, and the results are shown in FIG. 7. As can be seen from FIG. 7, after 12h of fermentation, streptococcus thermophilus GDMCC No.60810 reached a pH of 4.94 and at 24h the pH reached 4.53. Whereas LMD9 fermentation for 12h and 24h produced acid fast and much more, indicating that streptococcus thermophilus GDMCC No.60810 could be used as a strain for slow fermentation of fermented milk and it might slow down the post acidification process.

Example 7: streptococcus thermophilus GDMCC No.60810 in skim milkContent of volatile substances after fermentation

Fermentation was performed under the fermentation conditions of examples 5 and 6, and the fermented milk was sampled at 12 hours for volatile matter content measurement.

The volatile matter measuring method comprises the following steps: gas chromatography conditions and temperature program: rtx-WAX capillary (30 m×0.25mm, 0.25 mm), sample inlet temperature 225 ℃, split ratio 10, carrier gas (helium) flow rate 1mL/min were selected. Setting an initial temperature of 30 ℃ by a heating program, keeping the temperature for 3min, heating to 225 ℃ at 15 ℃/min, and keeping the temperature for 5min; mass spectrometry conditions: ionization mode EI, emission energy 70eV, emission current 200 muA, detector voltage 1.4kV, ion source temperature 240 ℃, interface temperature 230 ℃, quaternary rod temperature 150 ℃ and mass scanning range m/z 30-500; and (3) carrying out substance characterization on the spectrogram obtained by GC-MS in the retrieval and standard substance comparison of NIST 2001 standard spectrum library, and quantifying by a peak area normalization method. The results are shown in FIG. 8.

As can be seen from FIG. 8, after 12 hours of fermentation of Streptococcus thermophilus GDMCC No.60810, the content of acetaldehyde and acetoin produced is more than 2 times that produced by the strain LMD9, especially acetoin, and can reach 24 mug/Kg. Can provide creamy taste to fermented milk. Thus, streptococcus thermophilus GDMCC No.60810 has excellent aroma-producing properties.

Example 8: preparation of microbial agent containing streptococcus thermophilus GDMCC No.60810

Scribing the streptococcus thermophilus GDMCC No.60810 on an M17 solid culture medium, and culturing for 48 hours at 37 ℃ to obtain single colonies; single colony is selected and inoculated in MRS liquid culture medium, and is cultured for 18 hours at 37 ℃ for activation, and the activation is carried out for two generations continuously, so as to obtain an activation solution; inoculating the activating solution into a culture medium according to the inoculum size accounting for 2-4% of the total mass of the culture medium, and culturing for 18h at 37 ℃ to obtain bacterial solution; centrifuging 8000g of bacterial liquid for 10min to obtain bacterial mud; washing the bacterial mud with physiological saline for 3 times, and re-suspending with protective agent to a concentration of 1×10 ¹⁰ CFU/mL to obtain bacterial suspension, namely the liquid bacterial agent; the bacterial suspension is pre-cultured for 60min at 37 ℃ and then freeze-dried, and the streptococcus thermophilus GDMCC No.60810 bacterial powder is obtained.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A strain of streptococcus thermophilus (Streptococcus thermophilus), which has been deposited at the cantonese province microorganism strain collection at 10/16 of 2019 under the accession number GDMCC No.60810.

2. A composition comprising streptococcus thermophilus according to claim 1.

3. The composition of claim 2, wherein the composition comprises one or more of a live strain of streptococcus thermophilus, a dried strain of streptococcus thermophilus, a metabolite of streptococcus thermophilus, an inactivated streptococcus thermophilus, a fermentation broth of streptococcus thermophilus, a lysate of streptococcus thermophilus, or an extract of streptococcus thermophilus.

4. A composition according to claim 2 or 3, wherein the number of viable streptococcus thermophilus bacteria in the composition is not less than 5 x 10 ⁹ CFU/mL or 5X 10 ⁹ CFU/g。

5. A product comprising a composition according to any one of claims 2 to 4.

6. The product of claim 5, wherein the product comprises a microbial agent, a food or a pharmaceutical product.

7. The product according to claim 5 or 6, wherein the product is for increasing serotonin levels.

8. The product according to claim 6 or 7, for use in increasing the abundance of bifidobacteria in the gut.

9. The streptococcus thermophilus product of claim 1, wherein the pharmaceutical product contains the streptococcus thermophilus of claim 1, a pharmaceutical carrier and/or a pharmaceutical excipient.

10. The method of claim 2, wherein the food product is a health product; or the food is a dairy product, a bean product or a fruit and vegetable product prepared by using the streptococcus thermophilus of claim 1 or a starter culture containing the streptococcus thermophilus of claim 1; or the food product is a beverage or snack containing the streptococcus thermophilus of claim 1.