Streptococcus salivarius thermophilic subspecies JIAN+ and its application

Abstract

This invention relates to the field of microbial technology, providing *Streptococcus salivarius* subsp. *thermophilus* JIAN+ and its applications. The preservation number of this *Streptococcus salivarius* subsp. *thermophilus* JIAN+ is CGMCC No. 27130. This *Streptococcus salivarius* subsp. *thermophilus* JIAN+ can effectively inhibit α-amylase and α-glucosidase, and its combination with white kidney bean has a synergistic effect on inhibiting α-amylase activity and α-glucosidase. Simultaneously, it has good scavenging ability against free radicals DPPH and OH, exhibiting antioxidant and reducing capabilities; it also has good antibacterial activity against *Escherichia coli*; it also has good tolerance to gastric and pancreatic juices, and as a natural strain, it has strong safety, providing a new probiotic source for the development of functional products such as weight loss products, blood sugar lowering products, and antioxidant products.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to Streptococcus salivarius subsp. JIAN+ and its applications. Background Technology

[0002] With the improvement of socioeconomic levels and living standards, the obese population is gradually increasing. Obesity not only affects one's appearance but also has negative impacts on life and health. Obesity is the result of multiple factors, including excessive carbohydrate intake, gut microbiota, chronic inflammation, and hormones.

[0003] Diabetes is a prevalent chronic disease worldwide. Various vascular syndromes caused by diabetes seriously affect human health. After carbohydrates are ingested, substances such as starch are degraded into disaccharides or oligosaccharides by α-amylase. These are further enzymatically broken down into glucose by α-glucosidase in the intestinal mucosa and eventually absorbed. Therefore, inhibiting α-amylase and α-glucosidase can effectively inhibit the absorption of carbohydrates, not only reducing obesity but also lowering blood sugar levels and preventing diabetes.

[0004] Free radicals are atoms or groups with unpaired electrons and are highly reactive chemically. Free radicals can originate from both inside and outside the body, such as from cellular activity, aerobic respiration, inhalation of environmental pollutants like kitchen fumes, smoking, vehicle exhaust, and industrial waste gases, as well as from the use of cosmetics. Within a certain range, free radicals can be beneficial to the human body, but when they exceed a certain level, uncontrolled free radicals can cause various harms, such as attacking biomolecules like proteins, lipids, and nucleic acids, stealing electrons and leading to various health problems such as stroke, Alzheimer's disease, arteriosclerosis, cancer, and diabetes. Furthermore, increasing research shows that aging is closely related to oxidation processes; the breakdown of cells and tissues by free radicals or oxidants is a significant factor in aging. Therefore, timely removal of free radicals from the body and the consumption of antioxidants are important methods for preventing and reducing the aforementioned diseases and delaying aging.

[0005] Currently, the main hypoglycemic drugs used clinically to inhibit α-glucosidase include acarbose, voglibose, and miglitol. However, drug treatment can produce certain side effects and is not suitable for long-term use as part of daily diet. The most effective way to combat free radicals is to provide negative ions, that is, to provide antioxidants that can effectively scavenge free radicals.

[0006] Therefore, how to develop a safe and natural probiotic that can inhibit α-amylase and α-glucosidase, has reducing ability, and can effectively scavenge free radicals, to provide a new source of probiotics for the development of products for daily weight loss, diabetes prevention, and scavenging of excess free radicals, is a technical problem that those skilled in the art are committed to solving. Summary of the Invention

[0007] To address the shortcomings of the prior art mentioned in the background section, this invention provides a *Streptococcus salivarius* subsp. *thermophilus* JIAN+, *Streptococcus salivarius* subsp. *thermophilus* JIAN+ ( Streptococcus salivarius subsp.thermophilus JIAN+ was deposited on April 17, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.27130.

[0008] The present invention provides a composition containing the thermophilic subsp. saliva of Streptococcus as described above.

[0009] In one embodiment, the composition includes one of a microbial preparation or a pharmaceutical.

[0010] In one embodiment, the number of *Streptococcus salivarius* subsp. *thermophilus* JIAN+ in the composition is ≥1×10⁻⁶. 6 CFU / mL or ≥1×10 6 CFU / g. In one embodiment, the number of *Streptococcus salivarius* subsp. *thermophilus* JIAN+ in the composition is ≥1×10⁻⁶. 8 CFU / mL or ≥1×10 8 CFU / g.

[0011] In one embodiment, the composition comprises one or more combinations of uninactivated Streptococcus thermophilus subsp. JIAN+, inactivated Streptococcus thermophilus subsp. JIAN+, metabolites of Streptococcus thermophilus subsp. JIAN+ strains, and freeze-dried Streptococcus thermophilus subsp. JIAN+ strains.

[0012] The present invention also provides a fermentation broth, which is obtained by fermentation of Streptococcus salivarius subsp. JIAN+ as described above.

[0013] This invention also provides the application of *Streptococcus salivarius* subsp. *thermophilus* JIAN+ as described above in the preparation of functional products;

[0014] The functional product includes at least one of the following functions:

[0015] (1) Inhibits α-amylase activity;

[0016] (2) Inhibits α-glucosidase activity;

[0017] (3) Scavenging free radicals DPPH;

[0018] (4) Scavenging OH free radicals;

[0019] (5) It has an inhibitory effect on Escherichia coli.

[0020] In one embodiment, the functional products include antioxidant products, blood sugar lowering products, and weight loss products.

[0021] The present invention also provides a white kidney bean complex containing white kidney bean powder, wherein the complex contains *Streptococcus thermophilus* subsp. *salivarius* JIAN+ as described above. The white kidney bean complex includes at least one of the following functions: the complex of *Streptococcus thermophilus* subsp. *salivarius* JIAN+ and white kidney bean has a synergistic effect on inhibiting α-amylase activity; the complex of *Streptococcus thermophilus* subsp. *salivarius* JIAN+ and white kidney bean has a synergistic effect on inhibiting α-glucosidase activity.

[0022] Based on the above, compared with the prior art, the *Streptococcus salivarius* subsp. *thermophilus* JIAN+ provided by the present invention has the following beneficial effects:

[0023] The *Streptococcus thermophilus* subsp. *salivarius* JIAN+ provided by this invention can effectively inhibit α-amylase and α-glucosidase, and its combination with white kidney bean has a synergistic effect on inhibiting α-amylase activity and α-glucosidase. Simultaneously, it has good scavenging ability against free radicals DPPH and OH, exhibiting antioxidant and reducing capabilities; it also has good antibacterial activity against *Escherichia coli*; it has good tolerance to gastric and pancreatic juices; and as a natural strain, it has strong safety. It can provide a new probiotic source for the development of functional products such as weight loss products, blood sugar lowering products, and antioxidant products.

[0024] Other features and beneficial effects of the invention will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other beneficial effects of the invention can be realized and obtained by means of the structures particularly pointed out in the description, claims and drawings. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Unless otherwise specified, the positional relationships shown in the drawings in the following description are based on the direction in which the components are drawn in the figure.

[0026] Figure 1 This is a colony morphology diagram of Streptococcus salivarius subsp. JIAN+.

[0027] Figure 2 Scanning electron microscope image of Streptococcus salivarius subsp. JIAN+.

[0028] Figure 3 Agarose gel electrophoresis image of the amplified 16S rDNA target fragment of Streptococcus salivarius subsp. JIAN+.

[0029] Figure 4 Phylogenetic tree diagram of the 16S rDNA gene of Streptococcus salivarius subspecies JIAN+.

[0030] Figure 5 The inhibition zone diagram of *Streptococcus thermophilus* subsp. JIAN+ inhibiting *Escherichia coli*. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. The technical features designed in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0032] In the description of this invention, it should be noted that all terms used in this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and should not be construed as limiting the invention; it should be further understood that the terms used in this invention should be understood to have the same meaning as those in the context of this specification and in the relevant field, and should not be understood in an idealized or overly formal sense, except as expressly defined in this invention.

[0033] The present invention also provides the following embodiments:

[0034] This invention relates to *Streptococcus salivarius* subsp. *thermophilus* JIAN+ ( Streptococcus salivarius subsp.thermophilus JIAN+ It is obtained from fresh milk samples through natural fermentation in the laboratory.

[0035] The following is an example of the operation of extracting this bacterium from fresh milk according to the present invention:

[0036] Example 1: Screening and Isolation of Bacteria

[0037] Take fresh milk samples and incubate them at 37℃ for 3-7 days. Select naturally fermented yogurt samples that have coagulated naturally and have a yogurt aroma, and dilute them by 10. -3 , 10 -4 , 10 -5 0.1 mL of the culture medium was spread on MC medium and kept at 37℃ for 36-72 h. The colony morphology was observed. Red single colonies were selected and purified three times. The inhibitory effects of α-amylase and α-glucosidase were analyzed. The strain with the best overall inhibition rate was selected, preserved and named JIAN+.

[0038] Example 2: Identification of bacterial strains

[0039] 2.1 Morphological observation of bacteria

[0040] JIAN+ colony morphology as follows Figure 1 As shown, the bacterial cell morphology is as follows Figure 2 As shown, the main morphological characteristics of JIAN+ are as follows: the colonies are round, red, smooth, and opaque on MC medium, with oval to nearly round cells, 0.6-1.0 μm wide and 0.8-1.3 μm long.

[0041] 2.2 Analysis of carbon source utilization by bacteria

[0042] HBI lactic acid bacteria biochemical identification strips (GB4789.35 standard, purchased from Haibo Biotechnology) were used. One strip was taken, and a portion was picked from a single colony of JIAN+ and inoculated into the following culture medium: esculin, cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin, lactose, and 1% sodium hippurate. After inoculation, the strips were labeled, capped, and incubated at 37±1℃ for 24-48 hours. The results were observed and recorded. For the 1% sodium hippurate, after incubation, 0.2 ml of ninhydrin solution was slowly added along the wall of the test tube without shaking. The tube was then incubated in a 36℃±1℃ water bath for 10 minutes before interpreting the results. The interpretation results are shown in Table 1.

[0043] According to the results in Table 1, the main carbon sources available to JIAN+ are cellobiose, maltose, salicin, sucrose, and lactose.

[0044] Table 1. JIAN+ sugar compounds and major biochemical reactions

[0045]

[0046] Note: "+" indicates that more than 90% of the strains are positive, and "-" indicates that more than 90% of the strains are negative.

[0047] 2.3 Molecular biological identification of JIAN+

[0048] ① Extraction of bacterial genomic DNA: Bacterial genomic DNA was extracted using the TIANGEN bacterial genomic DNA extraction kit. The specific extraction steps were described in the kit's instructions.

[0049] ② PCR amplification of the 16S rDNA sequence: The primers used to amplify the 16S rDNA gene sequence are:

[0050] ② PCR amplification of 16S rDNA sequence: The primers used to amplify the 16S rDNA gene sequence were F9-27: 5'-GAGTTT GAT CCT GGC TCA G-3'; R1525-1542: 5'-AGA AAG GAG GTG ATC CAG CC-3'; PCR reaction system: 12.5 μL of 2*Mix, 1 μL each of primers and DNA, and 9.5 μL of ddH2O.

[0051] PCR amplification program: 93℃ pre-denaturation for 4 min. Then denature at 94℃ for 30 s, 55℃ (16S rDNA), 72℃ extension for 90 s, for a total of 30 cycles. Finally, extend at 72℃ for 10 min and store at 4℃.

[0052] ③ PCR product detection and sequencing analysis: 5 μL of PCR product was separated and examined by gel electrophoresis in 1.0% agarose gel containing EB. The amplified 16S rDNA target fragment was approximately 1500 bp in length (see agarose gel electrophoresis image of 16S rDNA target fragment amplification). Figure 3 ).

[0053] ④ Phylogenetic analysis: Blast alignment analysis was performed on each 16S rRNA sequence in NCBI data to obtain the sequence and... Streptococcus thermophilus (After the name change) Streptococcus salivarius subsp.thermophilus The sequence homology of all sequences was greater than 99%, and the developmental tree was constructed using the Neighbor-joining method in MEGA 4.1 (results are shown in [link to results]). Figure 4 The gene sequence is as follows:

[0054]

[0055] Based on morphological observation, biochemical identification, and homology analysis in the DNA phylogenetic tree, JIAN+ bacteria were identified as *Streptococcus salivarius* subspecies *thermophilus*. Streptococcus salivarius subsp.thermophilus ) bacterial strains.

[0056] The performance characterization of *Streptococcus salivarius* subsp. *thermophilus* JIAN+ provided by this invention is as follows:

[0057] Example 3: Analysis of the inhibitory effect of *Streptococcus salivarius* subsp. *thermophilus* JIAN+ and its complex with white kidney bean on α-amylase.

[0058] (1) Three test solutions were prepared as follows:

[0059] ① JIAN+ bacterial suspension: Specifically, the fermentation broth of *Streptococcus salivarius* subsp. *thermophilus* JIAN+ cultured for 48 h was centrifuged at 4500 r / min and 4℃ for 10 min. The supernatant was discarded, and the precipitate was washed twice with 0.85% physiological saline and resuspended. The OD of the bacterial suspension was adjusted to 1.5 and 1.0 (×10⁻⁶). The total bacterial count determined by flow cytometry was 4.34 and 2.36 (×10⁻⁶). 8 (cfu / mL) to obtain bacterial suspension samples for testing;

[0060] ② Prepare the white kidney bean test solution: Add white kidney bean powder to PBS buffer (pH=6.8, 0.1mol / L) to prepare a 1mg / mL white kidney bean solution, which is then used as the white kidney bean test solution;

[0061] ③ White kidney bean and JIAN+ composition: White kidney bean powder was added to bacterial suspensions with different OD values ​​in ① to prepare a white kidney bean (1mg / mL) + JIAN+ bacterial suspension (OD values ​​of 1.5 and 1.0 respectively) composition.

[0062] α-Amylase inhibition test:

[0063] Add 100 μL of PBS solution containing 0.6 mg / mL α-amylase to the microplate, add 100 μL of the sample solution to be tested and react at 37°C for 30 minutes, add 100 μL of starch solution to the system and react at 37°C for another 15 minutes, finally add 5 μL of dilute iodine solution and measure the OD value at 660 nm on the microplate reader.

[0064] The formula for calculating the α-amylase inhibition rate is as follows:

[0065] α-Amylase inhibition rate / % = [(BC) - (BA)] / (BC) × 100%,

[0066] Where: A is the experimental group, B is the blank group without α-amylase, and C is the control group without test samples.

[0067] (2) The test results are shown in Table 2 below. It can be seen that:

[0068] JIAN+ bacterial suspension has a good inhibitory effect on α-amylase, at OD 600 The inhibition rates of α-amylase were 67.04% and 49.28% when the concentrations were 1.5 and 1.0, respectively; under the conditions of this experiment, no inhibitory effect of 1 mg / mL white kidney bean solution on α-amylase was detected.

[0069] In the combination of white kidney bean + bacterial suspension 1, the inhibition rate of α-amylase was detected to be 75.96%, which was significantly different from the bacterial suspension 1 treatment without white kidney bean at the P=0.05 level, indicating that JIAN+ and white kidney bean can achieve a synergistic effect in inhibiting α-amylase.

[0070] Table 2. Analysis of the inhibitory effects of JIAN+ and its combination with white kidney bean on α-amylase.

[0071]

[0072] Note: Bacterial suspension 1 is JIAN+ bacterial cells centrifuged and then adjusted for OD with physiological saline. 600 =1.5, bacterial suspension 2 is OD 600 =1.0; the concentration of white kidney bean was 1 mg / mL. Different letters indicate significant differences between treatments (P≤0.05), and "-" indicates no inhibitory effect.

[0073] Example 4: Analysis of the inhibitory effect of Streptococcus salivarius subsp. JIAN+ and its mixture with white kidney bean on α-glucosidase.

[0074] (1) Three test solutions were prepared as follows:

[0075] ①JIAN+ fermentation supernatant: Specifically, the fermentation broth of Streptococcus salivarius subsp. thermophilus JIAN+ cultured for 48 h was centrifuged at 4500 r / min and 4℃ for 10 min. The supernatant was collected and adjusted to three concentrations: fermentation supernatant 1 as the original solution, fermentation supernatant 2 (supernatant: PBS (v / v) = 1:1), and fermentation supernatant 3 (supernatant: PBS (v / v) = 1:2). Uninoculated MC medium was used as a blank control group to obtain the JIAN+ fermentation supernatant test solution.

[0076] ②Prepare the white kidney bean test solution: Add white kidney bean powder to PBS buffer (pH=6.8, 0.1mol / L) to prepare a 1mg / mL white kidney bean solution, which is then used to prepare the white kidney bean test solution;

[0077] ③ White kidney bean + JIAN + composition: White kidney beans were added to fermentation supernatants of different concentrations in ① to prepare a white kidney bean (1mg / mL) + JIAN + fermentation supernatant composition.

[0078] Assay for α-glucosidase inhibition: 50 μL of PBS buffer (0.1 mol / L, pH 6.8) was added to a microplate, followed by 50 μL of α-glucosidase solution (1.5 u / mL, using PBS buffer as solvent). 50 μL of the bacterial culture to be tested was added and reacted at 37℃ for 10 minutes. Then, 50 μL of PNPG solution (1 mg / mL) was added, and the reaction was continued at 37℃ for 30 minutes. Finally, 80 μL of Na2CO3 stop solution was added. After the reaction was complete, the OD value was measured at 405 nm using a microplate reader.

[0079] (2) The results of α-glucosidase inhibition rate are shown in Table 3, which show that:

[0080] The JIAN+ fermentation supernatant stock solution, and the 1-fold and 2-fold dilutions of the fermentation supernatant, showed inhibition rates of 58.13%, 41.3%, and 24.57% against α-glucosidase, respectively.

[0081] The corresponding fermentation supernatant and white kidney bean combination showed inhibition rates of 62.73%, 51.00%, and 37.83% on α-glucosidase, respectively, all significantly higher than the uncombined group. This indicates that both JIAN+ fermentation supernatant and dilution have good inhibitory effects on α-glucosidase, and can be combined with white kidney beans to exert a synergistic effect on the inhibitory effect on α-glucosidase.

[0082] Table 3. Analysis of the inhibitory effects of JIAN+ and its combination with white kidney bean on α-glucosidase.

[0083]

[0084] Note: "-" indicates no inhibitory effect.

[0085] Example 5: Analysis of the scavenging ability of *Streptococcus salivarius* subsp. *JIAN* against 2,2-biphenyl-1-picrylhydrazine (DPPH) and OH free radicals.

[0086] (1) The method for preparing JIAN+ bacterial suspension is the same as in Example 3 above, except that the bacterial suspension density OD is adjusted. 600nm The value was around 1.0, and a JIAN+ bacterial suspension was obtained as the test sample.

[0087] Preparation of cell lysate supernatant: Centrifuge the cultured bacterial suspension at 4500 r / min and 4℃ for 10 min, discard the supernatant, wash the precipitate twice with 0.85% physiological saline, and resuspend it. Adjust the density (OD) of the bacterial suspension to approximately 1.0. Then, disrupt the cells in an ultrasonic disruptor with a power setting of 40%, working for 5 seconds and stopping for 5 seconds, for a total effective working time of 450 seconds. After disruption, centrifuge at 10000 r / min for 20 min and collect the supernatant to obtain the cell lysate supernatant for testing.

[0088] (1.1) Analysis of the free radical scavenging ability of 2,2-biphenyl-1-picrylhydrazyl (DPPH):

[0089] Take 2 mL of the bacterial suspension and cell lysate supernatant samples respectively, add 2 mL of 0.2 mmol / L DPPH anhydrous ethanol solution, mix well, react at room temperature in the dark for 30 min, centrifuge at 4500 r / min for 10 min, and measure the OD value at 517 nm.

[0090] The formula for calculating the DPPH free radical scavenging rate is as follows:

[0091] DPPH free radical scavenging rate / % = [1 - (A1 - A2) / A0] × 100%;

[0092] Where A1 is the absorbance of the experimental group, A0 is the absorbance of the sample group with an equal volume of anhydrous ethanol, and A2 is the absorbance of the DPPH anhydrous ethanol solution with an equal volume of anhydrous ethanol.

[0093] (1.2) Determination of OH radical scavenging ability:

[0094] Mix 1 mL of 1,10-phenanthroline solution (2.5 mmol / L), 1 mL of PBS (pH 7.4), and 0.5 mL of the bacterial culture to be tested thoroughly. Then add 1 mL of FeSO4 solution (2.5 mmol / L) and 0.5 mL of H2O2 solution (20 mmol / L), incubate at 37°C for 1 h, and measure the OD value at 536 nm.

[0095] The formula for calculating the OH radical scavenging rate is as follows:

[0096] OH radical scavenging rate / % = (A 实验 -A 对照 ) / (A 空白 -A 对照 )×100%;

[0097] Among them, A 对照 The absorbance of the sample was replaced by an equal volume of PBS solution; A 空白 The absorbance is calculated using an equal volume of PBS instead of H2O2 solution.

[0098] (2) The test results of DPPH radical scavenging rate and OH radical scavenging rate are shown in Table 4. It can be seen that:

[0099] JIAN+ bacterial suspension showed a DPPH free radical scavenging rate of up to 84.24%, while its cell lysate supernatant showed a DPPH free radical scavenging rate of 29.56%, indicating that JIAN+, a thermophilic subspecies of Streptococcus salivarius, has a good DPPH free radical scavenging ability.

[0100] The JIAN+ bacterial suspension showed a OH free radical scavenging rate of 38.56%, indicating that *Streptococcus salivarius* subsp. *thermophilus* JIAN+ has a significant OH free radical scavenging ability.

[0101] Table 4. Free radical scavenging ability of *Streptococcus thermophilus* subsp. *salivarius* JIAN+

[0102]

[0103] Example 6: Determination of the reducing power of Streptococcus salivarius subsp. JIAN+

[0104] Reducing capacity (antioxidant capacity) refers to the ability to eliminate free radicals by donating electrons through its own reduction process. The stronger the reducing capacity, the stronger the antioxidant capacity and the stronger the ability to resist damage from free radicals and oxidants.

[0105] Take the bacterial suspension and cell lysate supernatant samples from Example 5, mix 0.5 mL of 1% potassium ferricyanide, 0.5 mL of PBS buffer (pH 6.6) and 0.5 mL of sample solution, incubate at 50°C for 20 min, and then rapidly cool the mixture. Add 0.5 mL of 10% trichloroacetic acid, centrifuge at 4000 r / min for 10 min, take 1 mL of the sample to be tested, add 1 mL of ultrapure water and 1 mL of 0.1% ferric chloride solution, shake to mix evenly, let stand at room temperature for 10 min, and then measure the OD value at 700 nm.

[0106] The formula for calculating restorative power is:

[0107] Reduction capacity / % = (A1 - A0) / A1 × 100%;

[0108] Where A1 is the absorbance value of the sample group; A0 is the absorbance value of the PBS buffer used to replace the sample.

[0109] (2) The test results of the reduction rate are shown in Table 5. It can be seen that:

[0110] The reduction rate of JIAN+ bacterial suspension was 32.9%, and the reduction rate of its cell lysate supernatant was 46.30%, indicating that both the JIAN+ bacterial suspension and cell contents of Streptococcus salivarius thermophilus have significant reducing or antioxidant capabilities.

[0111] Table 5. Determination of reducing power of Streptococcus salivarius subsp. JIAN+

[0112]

[0113] Example 7: Tolerance analysis of Streptococcus salivarius subsp. JIAN+ in a simulated gastric juice environment

[0114] Collect JIAN+ bacteria fermented for 24-48 hours, centrifuge at 4500 r / min for 10 min to collect bacterial cells, add an equal volume of physiological saline (0.85%) and mix well for later use; prepare artificial gastric fluid (125 mM NaCl, 7 mM KCl, 45 mM NaHCO3 and 3 g / L pepsin), adjust the pH to 2.0, 2.5 and 3.0, filter through a 0.22 μM microporous membrane and set aside. Take 1 mL of the treated bacterial culture and add it to 9 mL of artificial gastric fluid at different pH values, incubate at 37 ℃, and take samples at 1, 2 and 3 h of incubation. Take 0.9 mL of the sample each time, dilute it, add 0.1 mL of PI dilution solution, stain at 37 ℃ for 10 min, and detect the total number of bacterial particles P1 and the number of dead particles P2 by flow cytometry. The formula for calculating bacterial survival rate is:

[0115] Survival rate of bacteria JIAN+ / % = (P1-P2) / P1×100%.

[0116] (2) The bacterial survival rate of JIAN+ is shown in Table 6. According to the data, the following can be seen:

[0117] The survival rate of JIAN+ in artificial simulated gastric juice environments at pH 2.5 and 3.0 ranged from 53.23% to 67.69%. Even after 3 hours of retention in the gastric juice environment, the survival rate was still greater than 50%, indicating that JIAN+ has good gastric juice tolerance.

[0118] Table 6. Survival rate (%) of Streptococcus salivarius subsp. JIAN+ in simulated gastric juice environment

[0119]

[0120] Example 8: Tolerance analysis of Streptococcus salivarius subsp. JIAN+ in a simulated artificial pancreatic juice environment.

[0121] Collect JIAN+ bacteria fermented for 24-48 h, centrifuge at 8000 r / min for 5 min to collect the bacterial cells, add the same volume of physiological saline (0.85%) and mix well for later use; prepare pancreatic protein solution (0.1% secretin w / v, 0.15% ox bile), adjust the pH to 7.5 and 8.0 respectively, filter through a 0.22 μM microporous membrane for later use, take 1 mL of the treated bacterial solution into 9 mL of pancreatic protein solution at different pH values, incubate at 37℃, take samples at 3 and 6 h, take 0.9 mL each time, dilute, add 0.1 mL of PI (PI refers to propidium iodide staining solution), stain at 37℃ for 10 min, and detect the total number of bacterial particles P1 and the number of dead particles P2 by flow cytometry to calculate the bacterial survival rate;

[0122] The formula for calculating bacterial survival rate is:

[0123] Survival rate of bacteria JIAN+ / % = (P1-P2) / P1×100%.

[0124] (2) The bacterial survival rate of JIAN+ is shown in Table 7. According to the data, the following can be seen:

[0125] After treatment in an artificially simulated pancreatic juice environment at pH 7.5 for 6 hours, the survival rate of JIAN+ reached 99.66%. After treatment in a pancreatic juice environment at pH 8.0 for 3-6 hours, the survival rate remained stable between 98.11% and 99.32%, indicating that JIAN+ has good pancreatic juice tolerance.

[0126] Table 7 Survival rate (%) of Streptococcus salivarius subsp. JIAN+ in an artificial simulated pancreatic juice environment

[0127]

[0128] Example 9: Inhibitory effect of *Streptococcus thermophilus* subsp. *JIAN+* on *Escherichia coli*.

[0129] Oxford cups (6 mm in diameter) were placed in LB medium coated with E. coli ATCC35150. 200 μl of JIAN+ fermentation broth was added to each Oxford cup, and the mixture was incubated at 37°C for 16 h. The antibacterial effect of the JIAN+ fermentation broth on E. coli was then determined, and the inhibition diameter was found to be 18.7 ± 1.5 mm (specifically as shown in the figure). Figure 5 As shown in the figure, JIAN+ has a good antibacterial effect on Escherichia coli.

[0130] Example 10: Preparation of probiotic agent from *Streptococcus thermophilus* subsp. *salivarius* JIAN+

[0131] *Streptococcus thermophilus* subsp. *JIAN+* is inoculated into a culture medium, such as MC medium, and cultured at 30-42°C for more than 15 hours. The bacterial cells are collected by centrifugation and resuspended in, for example, physiological saline or PBS buffer to prepare a liquid bacterial preparation containing *Streptococcus thermophilus* subsp. *JIAN+*. Optionally, *Streptococcus thermophilus* subsp. *JIAN+* bacterial cells are resuspended in a cell protectant and a carrier, and then freeze-dried to obtain a solid bacterial powder preparation containing *Streptococcus thermophilus* subsp. *JIAN+*.

[0132] Optionally, *Streptococcus thermophilus* subsp. JIAN+ can be used as a raw material component in antioxidant products, hypoglycemic products, and weight loss products (such as pharmaceuticals). *Streptococcus thermophilus* subsp. JIAN+ can be present in the products in liquid or solid dosage form.

[0133] Example 11: Preparation of fermented food from *Streptococcus salivarius* subsp. *thermophilus* JIAN+

[0134] To prepare a fermenting yeast broth of Streptococcus salivarius subsp. thermophilus JIAN+, various fruits, Chinese herbal medicines, grain raw materials, and various sugars are used as auxiliary materials. The broth is inoculated with Streptococcus salivarius subsp. thermophilus JIAN+ and fermented for a certain period of time under certain temperature conditions (30-42℃) to prepare a fermented product. The product is then inactivated or not inactivated, diluted in its original form or at different ratios, and then mixed with common beverage additives to prepare a fermented food.

[0135] Based on the results of the above embodiments, the *Streptococcus salivarius* subsp. *thermophilus* JIAN+ provided by the present invention has the following properties and effects:

[0136] It can utilize carbon sources such as cellobiose, maltose, salicin, sucrose, and lactose. Its survival rate is above 50% after 0–3 hours in gastric juice at pH 2.5–3.0, and above 99% after 0–6 hours in pancreatic juice at pH 7.5. It effectively inhibits α-amylase and α-glucosidase, with inhibition rates of 49.27–67.04% for α-amylase and 58.13–24.57% for α-glucosidase. The complex of *Streptococcus thermophilus* subsp. JIAN+ and white kidney bean shows a significant synergistic effect on the inhibition rates of α-amylase and α-glucosidase. The combination of this compound and white kidney bean shows an inhibition rate of 50.04–75.96% for α-amylase and 37.83–62.73% for α-glucosidase, higher than either component alone, demonstrating a significant synergistic effect. The suspension of *Streptococcus thermophilus* subsp. *salivarius* JIAN+ exhibits good scavenging ability against DPPH and OH free radicals, with scavenging rates of 84.23% and 39.56%, respectively. Simultaneously, its cell lysis supernatant also shows good scavenging ability against DPPH and OH. Furthermore, both the JIAN+ suspension and its cell lysis supernatant can reduce ferric iron in potassium ferricyanide to ferrous iron, with reduction rates of 32.9% and 46.3%, respectively, demonstrating good reducing ability. *Streptococcus thermophilus* subsp. *salivarius* JIAN+ has a significant inhibitory effect on *Escherichia coli*. *Streptococcus thermophilus* JIAN+ is obtained from fresh milk through natural fermentation, making it highly safe for consumption. It can be used in pharmaceuticals as a weight-loss product, a hypoglycemic agent, and an antioxidant, showing broad application prospects. In summary, compared with existing technologies, the *Streptococcus thermophilus* subsp. *salivarius* JIAN+ provided by this invention has the following beneficial effects:

[0137] The *Streptococcus thermophilus* subsp. *JIAN+* provided by this invention can effectively inhibit α-amylase and α-glucosidase, and its combination with white kidney bean has a synergistic effect on inhibiting α-amylase activity and α-glucosidase. Simultaneously, it has good scavenging ability against free radicals DPPH and OH, exhibiting antioxidant and reducing capabilities; it also has good antibacterial activity against *Escherichia coli*; it also has good tolerance to gastric and pancreatic juices, and as a natural strain, it has strong safety. It can provide a new probiotic source for the development of functional products such as weight loss products, blood sugar lowering products, and antioxidant products, for example:

[0138] (1) Streptococcus salivarius subsp. JIAN+ can be used as a raw material component of the composition to prepare a composition with the above-mentioned functions of inhibiting α-amylase activity, inhibiting α-glucosidase activity, scavenging free radical DPPH, scavenging OH free radical, having reducing ability, and inhibiting Escherichia coli; wherein, the composition includes, but is not limited to, microbial preparations, drugs, etc.

[0139] The bacterial strains present in the composition include, but are not limited to, one or more combinations of the following: non-inactivated *Streptococcus thermophilus* subsp. *salivarius*, inactivated *Streptococcus thermophilus* subsp. *salivarius*, metabolites of *Streptococcus thermophilus* subsp. *salivarius* strains, and freeze-dried *Streptococcus thermophilus* subsp. *salivarius* strains. Preferably, in the composition, the number of *Streptococcus thermophilus* subsp. *salivarius* is ≥1×10⁻⁶. 6 CFU / mL or ≥1×10 6 CFU / g. More preferably, in the composition, the number of *Streptococcus salivarius* subsp. *thermophilus* JIAN+ is ≥1×10⁻⁶. 8 CFU / mL or ≥1×10 8 CFU / g.

[0140] (2) Various plants (such as fruits, Chinese herbal medicines, grains, etc.) can be used as raw materials, combined with various ingredients, and inoculated with Streptococcus salivarius subsp. JIAN+ for fermentation treatment to prepare fermented products. These fermented products can be used to prepare products with antioxidant, hypoglycemic and weight loss functions.

[0141] The fermentation raw materials can be various commonly used plant fermentation raw materials, and the auxiliary materials can also be existing conventional auxiliary materials, including but not limited to the above-mentioned options. The fermented product is not limited to products used in the preparation of fermented foods, but can also be health products, pharmaceuticals, etc.

[0142] In summary, *Streptococcus thermophilus* subsp. JIAN+ and / or its ferments, based on their characteristics, can be used in functional products that include at least one of the following functions:

[0143] (1) Significantly inhibits α-amylase activity levels;

[0144] (2) Significantly inhibits α-glucosidase activity;

[0145] (3) Significantly reduced DPPH levels, which scavenges free radicals;

[0146] (4) Significantly high level of OH radical scavenging;

[0147] (5) It has significant reduction ability.

[0148] (6) It has a significant inhibitory effect on Escherichia coli.

[0149] Among them, products with the above-mentioned functions (1)-(6) include, but are not limited to, antioxidant products and weight loss products, which have obvious effects such as antioxidant, blood sugar reduction and weight loss, and may also be products with other obvious effects based on the functions (1)-(6); wherein, the functional products include, but are not limited to, pharmaceuticals.

[0150] Since the complex of *Streptococcus thermophilus* subsp. JIAN+ and white kidney bean has a synergistic effect in inhibiting α-amylase activity and α-glucosidase, it can be used in combination with white kidney bean. The resulting complex can be applied in weight loss products that inhibit α-amylase activity and α-glucosidase function.

[0151] Similarly, the complex of *Streptococcus thermophilus* subsp. JIAN+ and white kidney bean is not limited to functional products for weight loss, that is, it is not limited to the obvious effects of lowering blood sugar and weight loss, but can also be a product with other obvious effects based on inhibiting α-amylase activity and inhibiting α-glucosidase function; wherein, the functional products include, but are not limited to, pharmaceuticals.

[0152] It should be noted that:

[0153] (1) Definition:

[0154] The term "food" as used herein is used in a broad sense, encompassing both human food and drink. In some embodiments, the food product is suitable for and designed for human consumption. This application can be used to prepare solid dosage forms such as powders and tablets, and also to disperse in liquids to prepare liquid dosage forms, including but not limited to powders.

[0155] The composition includes, but is not limited to, microbial preparations and pharmaceuticals, and the composition containing *Streptococcus thermophilus* subsp. *salivarius* JIAN+ can be used in other forms of products.

[0156] The presence of *Streptococcus thermophilus* subsp. JIAN+ in the composition includes, but is not limited to, non-inactivated bacteria, inactivated bacteria, metabolites, freeze-dried strains, etc. It is anticipated that *Streptococcus thermophilus* subsp. JIAN+ may also exist in the composition in other forms.

[0157] The functional product includes the aforementioned functions of scavenging free radicals (DPPH and OH), possessing reducing ability, inhibiting α-amylase activity, inhibiting α-glucosidase activity, and inhibiting Escherichia coli. Based on these functions, its directional effects in the functional product are antioxidant, blood sugar lowering, and weight loss. Because it has the aforementioned functions of scavenging free radicals (DPPH and OH), possessing reducing ability, inhibiting α-amylase activity, and inhibiting Escherichia coli, the effects of its application in the product include, but are not limited to, antioxidant, blood sugar lowering, and weight loss. Other effects on the human body that can be achieved through the aforementioned functions are also included.

[0158] (2) The relevant prior art means or prior art terms involved in this application:

[0159] "OD" is an abbreviation for optical density, also known as absorbance. The energy difference before and after light passes through an analyte is the energy absorbed by the analyte. At a specific wavelength, there is a quantitative relationship between the concentration of the same analyte and the absorbed energy, which can be used to determine the concentration of the analyte. 600 "OD" is the optical density value measured when the wavelength is set to 600nm. It is a standard indicator for tracking the density of microorganisms in liquid cultures and is usually used to indicate the density of bacterial cells. The method for measuring the "OD" value is existing technology, and its principle and method will not be described here.

[0160] “DPPH” stands for free radical 2,2-biphenyl-1-picrylhydrazine, and “OH” stands for free radical hydroxyl.

[0161] The determination of total bacterial count P1 and dead bacterial count P2 using flow cytometry is an existing technology, and its principles and methods will not be elaborated here.

[0162] The method of using an alcohol meter to test the alcohol content and temperature of the distillate, and then calculating the actual alcohol content of each treatment using an alcohol meter temperature-concentration conversion table, is an existing technology, and its principle and method will not be elaborated here.

[0163] Carbon source utilization analysis was performed using HBI lactic acid bacteria biochemical identification strips. This is an existing technology, and its principles and methods will not be elaborated here.

[0164] (3) The formulations of the culture media used in the examples are as follows:

[0165] The MC medium (1L) formula is as follows: soybean peptone 5.0, beef extract 3.0, yeast extract 3.0, glucose 20.0, lactose 20.0, calcium carbonate 10.0, pH 6.0 ± 0.1 (Note: For solid medium, add neutral red 0.05 and agar 20.0 to this medium). After dispensing, autoclave at 121℃ for 20 min.

[0166] The LB medium formula is as follows: 10.0g tryptone, 5g yeast extract, 10g NaCl, pH 7.0, 20.0g agar powder; autoclave at 115℃ for 20 min.

[0167] Unless otherwise specified, the experimental procedures involved in the embodiments of the present invention are conventional experimental procedures in the art, and the reagents or instruments involved can be obtained from legitimate channels.

[0168] Furthermore, those skilled in the art should understand that although many problems exist in the prior art, each embodiment or technical solution of the present invention can be improved in only one or a few aspects, without necessarily solving all the technical problems listed in the prior art or the background art simultaneously. Those skilled in the art should understand that any content not mentioned in a claim should not be construed as a limitation on that claim.

[0169] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A thermophilic subspecies of Streptococcus salivarius ( Streptococcus salivarius subsp. thermophilus JIAN+, characterized by: Its accession number is CGMCC No. 27130.

2. A composition, characterized in that: Contains JIAN+, a thermophilic subspecies of Streptococcus salivarius as described in claim 1.

3. The composition according to claim 2, characterized in that: The composition includes one of microbial preparations and pharmaceuticals.

4. Fermentation broth, characterized in that: It is obtained by fermentation of *Streptococcus salivarius* subsp. JIAN+ as described in claim 1.

5. The application of *Streptococcus salivarius* subsp. *thermophilus* JIAN+ in the preparation of functional products, characterized by: The thermophilic subspecies of Streptococcus salivarius JIAN+ is the Streptococcus salivarius JIAN+ as described in claim 1; The functional product includes at least one of the following functions: (1) Inhibits α-amylase activity; (2) Inhibits α-glucosidase activity; (3) Scavenging free radicals DPPH; (4) Scavenging OH free radicals; (5) It has an inhibitory effect on Escherichia coli.

6. The application according to claim 5, characterized in that: The functional products include antioxidant products, blood sugar lowering products, and weight loss products.

7. A white kidney bean complex containing white kidney bean powder, characterized in that: The complex contains *Streptococcus salivarius* subsp. *thermophilus* JIAN+; Wherein, the thermophilic subspecies of Streptococcus salivarius JIAN+ is the thermophilic subspecies of Streptococcus salivarius JIAN+ as described in claim 1.

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