A strain of Streptococcus salivarius thermophilus subspecies against Helicobacter pylori and its postbiotics and application

Abstract

The present invention discloses a thermophilic subspecies of Streptococcus salivarius against Helicobacter pylori and its postbiotics and applications, belonging to the field of microbial fermentation technology. The present invention screens and obtains a thermophilic subspecies of Streptococcus salivarius, obtains the bacterial body and its postbiotics by culture, and forms a complex with papaya extract, which exhibits a synergistic effect, specifically manifested in that Helicobacter pylori is significantly inhibited and eventually effectively eliminated, and the RUT test results in some mice are negative, reducing the incidence of gastric diseases. The present invention is not only of practical value for the comprehensive utilization of thermophilic subspecies of Streptococcus salivarius and its postbiotics, but also of important value for the body's resistance to Helicobacter pylori.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, and in particular to a thermophilic subspecies of Streptococcus salivarius resistant to Helicobacter pylori and its postbiotics and applications. Background Art

[0002] Helicobacter pylori ( Helicobacter pylori ) is a gram-negative bacterium of the genus Helicobacter in the family Helicobacteraceae. It is a spiral-shaped, microaerophilic bacillus. Helicobacter pylori disease includes gastritis, peptic ulcer, lymphoproliferative gastric lymphoma, etc. caused by Helicobacter pylori infection. In worse cases, Helicobacter pylori disease can lead to gastric cancer.

[0003] Streptococcus salivarius subsp. thermophilus ( Streptococcus salivarius subsp. thermophilus ) is a homolactic fermentation, facultative anaerobic Gram-positive bacterium. As a recognized safe strain in the genus Streptococcus, it is widely used in the dairy industry due to its acid and aroma production characteristics. S. salivarius thermophilus subsp. is mainly used in fermented dairy products to improve lactose intolerance, regulate immunity, and improve constipation and diarrhea.

[0004] pawpaw( Pseudocydonia sinensis (Dum.Cours.) CKSchneid.), is the only species of the genus Chaenomeles in the Rosaceae family. Chaenomeles are shrubs or small trees native to China, distributed in most provinces and regions of East China, Central China, and South China. Chaenomeles are used as both medicine and food. The fruit tastes astringent and can be eaten after being boiled or candied. It is also the source of the Chinese medicinal material "Quanqiu", which has the effects of dispelling rheumatism, relieving cough and reducing phlegm.

[0005] Although people in all walks of life have a certain understanding of the efficacy of thermophilic Streptococcus salivarius and papaya, there has been no report on the combined use of the above two substances for a long time, and the efficacy of adding thermophilic Streptococcus salivarius and its postbiotics against Helicobacter pylori is also unknown. Summary of the invention

[0006] In view of the above-mentioned prior art, the purpose of the present invention is to provide a strain of Streptococcus salivarius thermophilic subspecies that is resistant to Helicobacter pylori and its postbiotics and applications.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] In a first aspect of the present invention, a strain of Streptococcus salivarius thermophilic subspecies resistant to Helicobacter pylori is provided. The Streptococcus salivarius thermophilic subspecies is Streptococcus salivarius thermophilic subspecies WZA-ST57, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20241481.

[0009] The second aspect of the present invention provides the use of the thermophilic subspecies of Streptococcus salivarius in the preparation of an anti-Helicobacter pylori product, wherein the number of viable bacteria of the thermophilic subspecies of Streptococcus salivarius is greater than 1×10 8 cfu / mL.

[0010] The third aspect of the present invention provides a postbiotic of Streptococcus salivarius thermophilus subsp., wherein the postbiotic of Streptococcus salivarius thermophilus subsp. is prepared by the following method:

[0011] The thermophilic subspecies of Streptococcus salivarius is activated and inoculated into a fermentation medium for fermentation at a fermentation temperature of 20-50°C for a fermentation time of 5-50 hours; the fermented product is centrifuged and the supernatant is freeze-dried to obtain the postbiotics of the thermophilic subspecies of Streptococcus salivarius.

[0012] Furthermore, the fermentation medium is composed of: 10-30 parts of lactose, 100-200 parts of enzymatic defatted milk, 10-30 parts of soy oligopeptides, 10-30 parts of whey protein powder, 1-10 parts of histidine, 1-10 parts of isoleucine, 0.1-10 parts of tyrosine, 0.1-10 parts of cysteine, 0.1-10 parts of glutamic acid, 0.001-1 parts of niacin, 0.1-1 parts of ascorbic acid, 0.01-1 parts of magnesium chloride, 0.001-1 parts of calcium pantothenate, 0.001-1 parts of thiamine hydrochloride, 0.1-1 parts of calcium chloride, and 800-1200 parts of distilled water.

[0013] The fourth aspect of the present invention provides the use of the postbiotics of the thermophilic subspecies of Streptococcus salivarius in the preparation of an anti-Helicobacter pylori product, wherein the anti-Helicobacter pylori product is prepared by mixing the postbiotics of the thermophilic subspecies of Streptococcus salivarius and papaya extract in a mass ratio of (0.5-5):(0.5-5).

[0014] Furthermore, the papaya extract is prepared by the following method:

[0015] Take fresh ripe papaya, wash and remove the seeds, cut into pieces and dry in a 60℃ oven, crush to 400 mesh and sieve to obtain papaya powder; extract the papaya powder in 75% v / v ethanol at room temperature with a mass volume ratio of 1g:3ml, each extraction time is 9h, and a total of 4 extractions; combine the extracts obtained each time, concentrate under reduced pressure at 60℃, and then dry in a 75℃ oven to obtain papaya extract.

[0016] Beneficial effects of the present invention:

[0017] (1) The present invention obtains the thermophilic subspecies of Streptococcus salivarius through screening. The bacterium has the characteristics of acid resistance, can resist Helicobacter pylori, and reduce the incidence of gastric diseases.

[0018] (2) The present invention ferments the thermophilic subspecies of Streptococcus salivarius, freeze-dries the supernatant to obtain the postbiotics of the thermophilic subspecies of Streptococcus salivarius, and forms a complex with the postbiotics and papaya extract, namely, the thermophilic subspecies of Streptococcus salivarius postbiotic complex.

[0019] (3) The present invention uses the postbiotic complex of thermophilic subspecies of Streptococcus salivarius for experiments. It can be seen from the experimental results that in the in vitro antibacterial detection experiment, the antibacterial rate of Helicobacter pylori was 56.8%; in the animal experiment, among the 24 positive experimental animals, after 4 weeks of administration, 14 turned negative in the RUT test, and the eradication rate of Helicobacter pylori was 58.3%. This result not only has a significant advantage over the control group, but also has a significant improvement compared with the postbiotic group, papaya group, and probiotic group used alone, reflecting a synergistic effect. Specifically, Helicobacter pylori was significantly inhibited and eventually effectively eliminated. The RUT test results in some mice were negative, reducing the incidence of gastric diseases. The present invention not only has practical value for the comprehensive utilization of thermophilic subspecies of Streptococcus salivarius and its postbiotics, but also has important value for the body's resistance to Helicobacter pylori. DETAILED DESCRIPTION

[0020] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0021] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0022] The experimental materials used in the examples of the present invention that are not specifically described are all conventional experimental materials in the art and can be purchased through commercial channels. The Helicobacter pylori used is Helicobacter pylori ATCC A43504, purchased from Shanghai Beino Biotechnology Co., Ltd. The Kunming mice used were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., and animal welfare was guaranteed throughout the experiment.

[0023] The deposit information of Streptococcus salivarius thermophilus subspecies used in the present invention is as follows:

[0024] Chinese name: Streptococcus salivarius thermophilus subspecies WZA-ST57;

[0025] Latin name: Streptococcus salivarius subsp. thermophilus WZA-ST57;

[0026] Deposit number: CCTCC NO:M 20241481;

[0027] Deposit date: July 4, 2024;

[0028] Depository: China Center for Type Culture Collection (CCTCC);

[0029] Deposit: Wuhan University, Wuhan, China.

[0030] Example 1: Screening of Streptococcus salivarius thermophilus subsp.

[0031] 1. Isolation and initial screening of strains:

[0032] Collect 10 g of fermented milk sample, take 1 g of each sample and put it into a 50 mL centrifuge tube containing 9 mL of sterile saline, shake for 15 min, mix thoroughly and then dilute in gradients, pour into M17 solid culture medium containing 0.05% bromocresol purple, and culture at 42±1℃ for 36 h. Pick a single colony and determine the morphology of the colony by Gram staining; take a drop of the strain after 12 h of culture on a slide, add a drop of 3% H 2 O 2 On the droplet, observe whether bubbles are generated. The strains that do not produce gas are streaked and purified, and after the single colony morphology is stable, it is numbered and stored.

[0033] 2. Strain screening: acid resistance

[0034] The strains were treated in simulated gastric fluid at pH = 3 for 0 h, 1 h, 2 h, 3 h, and 4 h, and the survival rate was recorded; strains with higher survival rates were selected for subsequent identification.

[0035] 3. Identification of strains:

[0036] The strain was sent to Henan Industrial Microbial Engineering Technology Research Center for 16S rDNA gene sequence identification, the sequence is shown in SEQ ID NO: 1, and the identification results showed that the strain belonged to Streptococcus salivarius thermophilic subspecies ( Streptococcus salivarius subsp. thermophilus ). The strain WZA-ST57 was biologically deposited.

[0037] Example 2: Preparation of postbiotics of Streptococcus salivarius subsp. thermophilus

[0038] The preparation method is as follows:

[0039] (1) The thermophilic Streptococcus salivarius subsp. preserved in Example 1 was activated to obtain an activated bacterial solution of the thermophilic Streptococcus salivarius subsp. The activation temperature was 37° C. and the activation time was 24 h. The activation medium was a Columbia blood agar plate having the following composition: 23.0 g of special peptone, 1.0 g of soluble starch, 5.0 g of sodium chloride, 10.0 g of agar, pH=7.3, and 1.0 L of distilled water.

[0040] (2) The activated bacterial liquid of thermophilic Streptococcus salivarius obtained in step (1) is inoculated into a fermentation medium at an inoculation amount of 3wt% (g / g) of the fermentation medium to obtain a thermophilic Streptococcus salivarius fermentation liquid. The fermentation temperature is 37° C., the fermentation time is 48 hours, and the composition of the fermentation medium used is as follows: 20 g lactose, 170 g enzymatic skim milk, 19 g soy oligopeptide, 17 g whey protein powder, 1.6 g histidine, 1.3 g isoleucine, 0.9 g tyrosine, 0.12 g cysteine, 0.15 g glutamic acid, 2 mg nicotinic acid, 0.5 g ascorbic acid, 40 mg magnesium chloride, 2 mg calcium pantothenate, 4 mg thiamine hydrochloride, 0.4 g calcium chloride, 1.0 L distilled water, pH=6.5.

[0041] (3) taking the fermentation broth of Streptococcus salivarius thermophilus subsp. salivarius obtained in step (2), centrifuging it at 30° C., and freeze-drying the supernatant to obtain postbiotics of Streptococcus salivarius thermophilus subsp. salivarius.

[0042] The content of thermophilic Streptococcus salivarius subspecies in the precipitate was determined by spectrophotometry. The viable bacterial count was 5×10 8 The cfu / ml concentration was stored in a refrigerator at 4°C.

[0043] Example 3: Preparation of a postbiotic complex of Streptococcus salivarius subsp. thermophilus

[0044] Papaya Extract Preparation

[0045] Take fresh ripe papaya, wash and remove the seeds, cut into pieces and dry in a 60℃ oven, crush to 400 mesh and sieve to obtain papaya powder; extract the papaya powder in 75% v / v ethanol at room temperature with a mass volume ratio of 1g:3ml, each extraction time is 9h, and the total extraction is 4 times; the extracts obtained each time are combined, concentrated under reduced pressure at 60℃, and then dried in a 75℃ oven to obtain papaya extract.

[0046] Preparation of complexes

[0047] The postbiotics of Streptococcus salivarius thermophilus subsp. prepared in Example 2 and the papaya extract prepared in Example 3 were mixed in a mass ratio of 1:1 to obtain a Streptococcus salivarius thermophilus subsp. postbiotic complex.

[0048] Test Example 1: Helicobacter pylori antibacterial detection

[0049] Take a 96-well plate and add 100 μL of Helicobacter pylori culture solution (the bacteria are in the logarithmic phase and the concentration is 1×10 6cfu / mL) and 95 μL of TSB medium, 5 μL of the postbiotics of Streptococcus salivarius thermophilus subsp. prepared in Example 2, the papaya extract prepared in Example 3, the preserved Streptococcus salivarius thermophilus subsp. prepared in Example 2 (1 ml is regarded as 1 g) and the postbiotic complex of Streptococcus salivarius thermophilus subsp. were added to the four experimental groups respectively to form a postbiotic group, a papaya group, a probiotic group and a complex group; 5 μL of DMSO solution was added to the control group; 3 replicates were set for each treatment. The 96-well plate was placed at 37°C and 150 rpm for 48 h of shaking culture. The OD 600 The value reflects the concentration of bacterial solution, and the inhibition rate (%) of each group is calculated by the following formula:

[0050] Inhibition rate (%) = (OD 600 Control group bacterial solution - OD 600 Experimental group bacterial solution) / OD 600 Control group bacterial solution × 100%.

[0051] The test results are shown in Table 1 below.

[0052] Table 1 Helicobacter pylori inhibition rate

[0053]

[0054] As shown in Table 1, Helicobacter pylori was inhibited in all experimental groups. Among them, the compound group performed the best, and the Helicobacter pylori inhibition rate in the compound group was 56.8%. It can be seen that after the compound group was treated, the growth of Helicobacter pylori was more inhibited, and the compound group treatment showed a synergistic effect, which could better exert the anti-Helicobacter pylori effect.

[0055] Test Example 2: Helicobacter pylori animal experiment

[0056] Six-week-old healthy Kunming mice were selected, half male and half female, and given a mixed antibiotic solution (ampicillin 10 mg / mL, gentamicin 1.2 mg / mL, azithromycin 10 mg / mL) by gavage, with a dose of 0.3 mL per mouse, once a day for three consecutive days. Seven days later, the mice were gavaged with 0.3 mL of freshly cultured Helicobacter pylori (1×10 9 cfu / mL), once every other day, for a total of 5 times. The mice were fasted for 24 hours before gavage and 2 hours after gavage. Two weeks after the last gavage, 2 mice were randomly selected, and the gastric antrum tissue was taken for rapid urease test (RUT) and hematoxylin-eosin staining (HE staining) to determine whether the infection was positive. Both RUT and HE staining results showed positive, indicating that the model was successfully established.

[0057] The mice with successful modeling were divided into half male and half female, randomly divided into groups of 8 mice in each group, and three experimental groups were set in parallel, and the grouping was set as follows: postbiotic group, the additive was the postbiotic of the thermophilic subspecies of Streptococcus salivarius prepared in Example 2; papaya group, the additive was the papaya extract prepared in Example 3; probiotic group, the additive was the thermophilic subspecies of Streptococcus salivarius prepared and preserved in Example 2 (1 ml was regarded as 1 g); complex group, the additive was the thermophilic subspecies of Streptococcus salivarius postbiotic complex prepared in Example 3; each additive group was gavaged according to the weight of the animal at a dose of 3 mg / kg, the unmodeled mice were used as the negative group, and the modeled mice were used as the control group, and the same amount of normal saline was given. In addition to feeding with the same normal feed, each group was gavaged once a day until the end of the experiment.

[0058] After 4 weeks of administration, gastric antrum tissues of mice were taken for rapid urease test. If the reagent turns red within 12 hours, it is positive, otherwise it is negative. Negative means that Helicobacter pylori is completely eradicated. The test results are shown in Table 2 below.

[0059] Table 2 Animal experiments on Helicobacter pylori

[0060]

[0061] As shown in Table 2, compared with the control group, the experimental animals in each experimental group turned negative. Among them, the compound group performed the best. In the compound group, 24 positive experimental animals turned negative in the RUT test after 4 weeks of administration, and the Helicobacter pylori eradication rate was 58.3%. It can be seen that after the treatment of the compound group, Helicobacter pylori in the mice was significantly inhibited and eventually effectively eliminated. The RUT test results were negative, reducing the incidence of gastric diseases.

[0062] In summary, the complex group performed best. In the in vitro antibacterial test, the Helicobacter pylori inhibition rate was 56.8%; in the animal experiment, among the 24 positive experimental animals, after 4 weeks of administration, 14 turned negative in the RUT test, and the Helicobacter pylori eradication rate was 58.3%. This result not only has a significant advantage over the control group, but also has a significant improvement compared to the postbiotic group, papaya group, and probiotic group used alone, reflecting a synergistic effect. Specifically, Helicobacter pylori was significantly inhibited and eventually effectively eliminated. The RUT test results in some mice were negative, reducing the incidence of gastric diseases.

[0063] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of a postbiotic of Streptococcus salivarius thermophilus subspecies in the preparation of an anti-Helicobacter pylori product, characterized in that: The anti-Helicobacter pylori product is prepared by mixing postbiotics of Streptococcus salivarius thermophilus subspecies and papaya extract in a mass ratio of (0.5-5):(0.5-5); The thermophilic subspecies of Streptococcus salivarius is the thermophilic subspecies of Streptococcus salivarius ( Streptococcus salivarius subsp. thermophilus ) WZA-ST57, deposited in China Center for Type Culture Collection, with the deposit number CCTCCNO:M 20241481; The postbiotics of Streptococcus salivarius thermophilic subspecies are prepared by the following method: The thermophilic subspecies of Streptococcus salivarius is activated and inoculated into a fermentation medium for fermentation at a fermentation temperature of 20-50° C. for a fermentation time of 5-50 hours; the fermented product is centrifuged and the supernatant is freeze-dried to obtain the postbiotics of the thermophilic subspecies of Streptococcus salivarius; The papaya extract is prepared by the following method: Take fresh ripe papaya, wash and remove the seeds, cut into pieces and dry in a 60℃ oven, crush to 400 mesh and sieve to obtain papaya powder; extract the papaya powder in 75% v / v ethanol at room temperature with a mass volume ratio of 1g:3ml, each extraction time is 9h, and a total of 4 extractions; combine the extracts obtained each time, concentrate under reduced pressure at 60℃, and then dry in a 75℃ oven to obtain papaya extract.

2. The use according to claim 1, characterized in that: The composition of the fermentation medium is as follows: 10-30 parts of lactose, 100-200 parts of enzymatic defatted milk, 10-30 parts of soybean oligopeptides, 10-30 parts of whey protein powder, 1-10 parts of histidine, 1-10 parts of isoleucine, 0.1-10 parts of tyrosine, 0.1-10 parts of cysteine, 0.1-10 parts of glutamic acid, 0.001-1 parts of niacin, 0.1-1 parts of ascorbic acid, 0.01-1 parts of magnesium chloride, 0.001-1 parts of calcium pantothenate, 0.001-1 parts of thiamine hydrochloride, 0.1-1 parts of calcium chloride, and 800-1200 parts of distilled water.

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