CN116769655A - Streptococcus salivarius thermophilus JIAN+ and application thereof - Google Patents
Streptococcus salivarius thermophilus JIAN+ and application thereof Download PDFInfo
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- CN116769655A CN116769655A CN202310706813.1A CN202310706813A CN116769655A CN 116769655 A CN116769655 A CN 116769655A CN 202310706813 A CN202310706813 A CN 202310706813A CN 116769655 A CN116769655 A CN 116769655A
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- streptococcus salivarius
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Abstract
The application relates to the technical field of microorganisms, and provides streptococcus salivarius subspecies thermophilus JIAN+ and application thereof. The preservation number of the streptococcus salivarius thermophilus JIAN+ is CGMCC No.27130. The streptococcus salivarius thermophilus JIAN+ can effectively inhibit alpha-amylase and alpha-glucose glycanase, and the composition of the streptococcus salivarius thermophilus JIAN+ and white kidney beans has synergistic effects on inhibiting the activity of the alpha-amylase and the alpha-glucose glycanase; meanwhile, the catalyst has good scavenging ability to free radicals DPPH and OH and has oxidation reduction resistance; has good antibacterial effect on escherichia coli; the strain has good gastric juice pancreatic juice tolerance, is a natural strain and high in safety, and can provide a new probiotic source for development of functional products such as weight-losing products, blood sugar-reducing products and antioxidant products.
Description
Technical Field
The application relates to the technical field of microorganisms, in particular to streptococcus salivarius subspecies jian+ and application thereof.
Background
With the improvement of social and economic levels and human living levels, obese people gradually increase, and the obesity not only affects the image of the people, but also brings negative effects to life and physical health. The development of obesity is a result of a number of factors, including excessive carbohydrate intake, intestinal flora, chronic inflammation, and hormones, among others, which can lead to obesity.
Diabetes is a chronic disease which is widely popular worldwide, various vascular syndromes caused by diabetes seriously affect human health, after carbohydrate is eaten, substances such as starch and the like are degraded into disaccharides or oligosaccharides by alpha-amylase, and then glucose is further generated through enzymolysis under the action of intestinal mucosa alpha-glucosidase and finally absorbed, so that the alpha-amylase and the alpha-glucosidase are inhibited, the absorption of the carbohydrate can be effectively inhibited, the generation of obesity is reduced, the blood sugar content is reduced, and diabetes is prevented.
Free radicals are a class of atoms or groups with unpaired electrons that are extremely reactive chemically. The free radicals are from in vivo and in vitro, such as cell activity, aerobic respiration, kitchen fume, smoking, automobile exhaust, and inhalation of environmental pollutants such as industrial waste gas, and a large amount of free radicals can be generated in cosmetic use. The free radical is beneficial to human body in a certain range, but beyond a certain amount, the uncontrolled free radical can bring various harm to the organism, such as attack on biological macromolecules in the organism, such as proteins, lipids, nucleic acids and the like, and deprive electrons in the organism, thereby causing different health problems, such as cerebral apoplexy, senile dementia, arteriosclerosis, cancers, diabetes and the like. In addition, more and more researches show that the aging of organisms is closely related to the oxidation process, and the decomposition of free radicals or oxidants on cells and tissues is an important factor for triggering the aging, so that the free radicals in the bodies are timely removed, and the eating of antioxidant substances is an important method for preventing and reducing the diseases and delaying the aging.
Currently, the clinical blood glucose-lowering drugs of inhibitors for inhibiting alpha-glucosidase mainly include acarbose, voglibose, miglitol and the like, but the drug treatment can produce certain side effects on organisms and is not suitable for long-term use as daily diet. The most effective method for combating free radicals is to administer anions, i.e. antioxidant substances which are effective in scavenging free radicals.
Therefore, how to develop a safe natural probiotic which can inhibit alpha-amylase and alpha-glucosidase and can effectively remove free radicals, and provide a new probiotic source for the development of food and health care products with the functions of daily weight loss, diabetes prevention, excessive free radical removal and the like for human beings, and the technical problem which is solved by the person skilled in the art.
Disclosure of Invention
In order to solve the defects of the prior art mentioned in the background art, the application provides a streptococcus salivarius thermophilus jian+, streptococcus salivarius thermophilus jian+ (Streptococcus salivarius subsp. Thermophilus jian+) which is preserved in the China general microbiological culture Collection center of China general microbiological culture Collection center for 4 months and 17 days in 2023, wherein the preservation address is North Chen Silu No. 1, 3 in the Korean region of Beijing, and the preservation number is CGMCC No.27130.
The present application provides a composition comprising the streptococcus salivarius subspecies jian+ as described above.
In one embodiment, the composition comprises one of a microbial preparation, a food, a health product, and a medicament.
In one embodiment, the composition comprises the Streptococcus salivarius subspecies thermophilus JIAN + 6 CFU/mL or ≡1X10. 6 CFU/g. In one embodiment, the composition comprises the Streptococcus salivarius subspecies thermophilus JIAN + 8 CFU/mL or ≡1X10. 8 CFU/g。
In one embodiment, the composition comprises one or more combinations of a streptococcus salivarius jian+ non-inactivated bacteria, a streptococcus salivarius jian+ inactivated bacteria, a metabolite of a streptococcus salivarius jian+ strain, a streptococcus salivarius jian+ lyophilized strain.
The application also provides a ferment obtained by fermentation of the streptococcus salivarius subspecies thermophilus jian+ as described above.
The application also provides the use of streptococcus salivarius subspecies jian+ and/or a fermentation thereof as described above for the preparation of a functional product.
In one embodiment, the functional product comprises a food, a health product, or a pharmaceutical product.
In an embodiment, the functional product comprises at least one of the following functions:
(1) Inhibiting alpha-amylase enzyme activity;
(2) Inhibiting alpha-glucohydrolase activity;
(3) Scavenging free radical DPPH;
(4) Removing OH free radicals;
(5) Has reducing ability.
(6) Has remarkable inhibiting effect on Escherichia coli.
In one embodiment, the functional products include antioxidant products, hypoglycemic products, and weight-loss functional products.
The application also provides the use of a streptococcus salivarius subspecies thermophilus jian+ as described above or a composition as described above in the preparation of a fermented food product.
The present application also provides a white kidney bean complex comprising white kidney bean powder, the complex comprising the streptococcus salivarius subspecies jian+ and/or a ferment thereof as described above. Wherein the white kidney bean complex includes at least one of the following functions: the complex of the streptococcus salivarius thermophilus JIAN+ and the white kidney beans has a synergistic effect on inhibiting the activity of alpha-amylase, and the complex of the streptococcus salivarius thermophilus JIAN+ and the white kidney beans has a synergistic effect on inhibiting the activity of alpha-glucose glycase.
Based on the above, compared with the prior art, the streptococcus salivarius subspecies jian+ provided by the application has the following beneficial effects:
the streptococcus salivarius subspecies thermophilus JIAN+ provided by the application can effectively inhibit alpha-amylase and alpha-glucohydrolase, and the composition of the streptococcus salivarius subspecies thermophilus JIAN+ and white kidney beans has a synergistic effect on inhibiting the activity of the alpha-amylase and the alpha-glucohydrolase; meanwhile, the catalyst has good scavenging ability to free radicals DPPH and OH and has oxidation reduction resistance; has good antibacterial effect on escherichia coli; the strain has good gastric juice pancreatic juice tolerance, is a natural strain and high in safety, and can provide a new probiotic source for development of functional products such as weight-losing products, blood sugar-reducing products and antioxidant products.
Additional features and advantages of the application will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the application. The objectives and other advantages of the application will be realized and attained by the structure particularly pointed out in the written description and claims hereof as well as the appended drawings.
Drawings
For a clearer description of embodiments of the application or of the solutions of the prior art, the drawings that are needed in the description of the embodiments or of the prior art will be briefly described, it being obvious that the drawings in the description below are some embodiments of the application, and that other drawings can be obtained from them without inventive effort for a person skilled in the art; the positional relationships described in the drawings in the following description are based on the orientation of the elements shown in the drawings unless otherwise specified.
FIG. 1 is a colony morphology of Streptococcus salivarius subspecies thermophilus JIAN+.
FIG. 2 is a scanning electron microscope image of Streptococcus salivarius subspecies thermophilus JIAN+.
FIG. 3 is an agarose gel electrophoresis of 16S r DNA fragment of S.salivarius JIAN+.
FIG. 4 is a phylogenetic tree of the 16S rDNA gene of the Streptococcus salivarius subspecies thermophilus JIAN+.
Fig. 5 is a graph of the zone of inhibition of streptococcus salivarius jian+ against escherichia coli.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions of the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application, and it is apparent that the described embodiments are some embodiments of the present application, but not all embodiments of the present application; the technical features designed in the different embodiments of the application described below can be combined with each other as long as they do not conflict with each other; all other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
In the description of the present application, it should be noted that all terms used in the present application (including technical terms and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs and are not to be construed as limiting the present application; it will be further understood that terms used herein should be interpreted as having a meaning that is consistent with their meaning in the context of this specification and the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
The application also provides the following embodiments:
the streptococcus salivarius thermophilus JIAN+ (Streptococcus salivarius subsp. Thermophilus JIAN+) is obtained from a fresh milk sample through natural fermentation in a laboratory.
The operation example of extracting the bacteria from fresh milk is as follows:
EXAMPLE 1 screening separation of bacteria
Taking fresh milk sample, culturing at 37deg.C for 3-7 days, selecting natural fermented yogurt sample with yogurt flavor, and diluting with dilution factor of 10 -3 、10 -4 、10 -5 0.1mL of the strain is coated in MC culture medium for 36-72 hours at the constant temperature of 37 ℃, colony morphology is observed, red single colonies are selected, after 3 times of purification, inhibition effect analysis of alpha-amylase and alpha-glucosidase is carried out, and the strain with excellent comprehensive inhibition rate is selected for preservation and named as JIAN+.
Example 2 identification of species
2.1 morphological observation of bacteria
The colony morphology of JIAN+ is shown in FIG. 1, the cell morphology is shown in FIG. 2, and the main morphology characteristics of JIAN+ are as follows: the bacterial colony is round, red, smooth and opaque on MC culture medium, and the ellipse of bacterial body is nearly round, the width of bacterial body is 0.6-1.0 μm, and the length is 0.8-1.3 μm.
2.2 analysis of carbon Source utilization by bacteria
Using HBI lactobacillus biochemical identification strips (standard GB4789.35, purchased from the seaborne organism), one biochemical identification strip was taken and a portion was selected from the jian+ single colony and inoculated into the following medium: esculin, cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin, lactose, 1% sodium hippurate. Marking after inoculation, covering a cover, placing at 37+ -1deg.C for culturing for 24-48h, observing and recording culture result; wherein, after the culture, 0.2ml of ninhydrin solution is slowly added along the tube wall without shaking, and the result is interpreted after being placed in a water bath at 36+/-1 ℃ for 10min, and the judging result is shown in table 1.
From the results in table 1, it can be seen that: the available carbon sources of JIAN+ are mainly cellobiose, maltose, salicin, sucrose, lactose, etc.
TABLE 1 JIAN+ sugar water Compounds and Main Biochemical reaction conditions
Note that: "+" indicates that 90% or more of the strains were positive, and "-" indicates that 90% or more of the strains were negative.
2.3 molecular biological characterization of JIAN +
(1) Extraction of bacterial genome DNA: the bacterial genome DNA extraction kit of TIANGEN company is adopted for extraction, and specific extraction steps refer to the instruction book of the kit.
(2) PCR amplification of 16S rDNA sequence: the 16S rDNA gene sequence is amplified by using the following primers:
(2) PCR amplification of 16S rDNA sequence: the 16SrDNA gene sequence is amplified by using the primers F9-27: 5'-GAGTTT GAT CCT GGC TCA G-3'; r1525-1542: 5'-AGA AAG GAG GTG ATC CAG CC-3'; PCR reaction system: 2 x 12.5 μl, primer and DNA 1 μl each, ddH 2 09.5μL。
PCR amplification procedure: pre-denatured at 93 ℃ for 4min. Then denatured at 94℃for 30s,55℃for 16SrDNA, extended at 72℃for 90s for 30 cycles, and finally fully extended at 72℃for 10min, stored at 4 ℃.
(3) PCR product detection and sequencing analysis: and 5uL of PCR product is taken and subjected to gel electrophoresis separation detection in agarose of 1.0% added with EB, and the target fragment of 16SrDNA with about 1500bp length is obtained by amplification (the agarose electrophoresis diagram of the amplification of the target fragment of 16SrDNA is shown in figure 3).
(4) Phylogenetic analysis: blast alignment analysis was performed on each 16S rRNA sequence in NCBI data to obtain sequences with sequence homology of greater than 99% to Streptococcus thermophilus (Streptococcus salivarius subsp. Thermophilus after renaming), and development tree construction analysis was performed by Neighbor-joining method in MEGA 4.1 (see fig. 4 for results). The gene sequence is as follows:
GCAAGCGGGGTGCTATACATGCAGTCGAACGCGTTGGCCCAATTGATTGACGGTGCTTGCACCTGATTGATTTTGGTCGCCAACGAGTGGCGGACGGGTGAGTAACACGTAGGTAACCTGCCCAGAAGCGGGGGACAACATTTGGAAACAGATGCTAATACCGCATAACAACGTTGTTCGCATGAACAACGCTTAAAAGATGGCTTCTCGCTATCACTTCTGGATGGACCTGCGGTGCATTAGCTTGTTGGTGGGGTAACGGCCTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACAATGGGACTGAGACACGGCCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAACACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTAAAGAAGAACAC
GTATGAGAGTAACTGTTCATACGTTGACGGTATTTAACCAGAAAGTCACGGCTAACTACGTGCCA
GCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGAGAGTGCAGG
CGGTTTTCTAAGTCTGATGTGAAAGCCTTCGGCTTAACCGGAGAAGTGCATCGGAAACTGGATAA
CTTGAGTGCAGAAGAGGGTAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGA
ACACCAGTGGCGAAGGCGGCTACCTGGTCTGCAACTGACGCTGAGACTCGAAAGCATGGGTAGCG
AACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCC
GCCCTTCAGTGCCGGAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAA
CTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGA
AGAACCTTACCAGGTCTTGACATCTTGCGCCAACCCTAGAGATAGGGCGTTTCCTTCGGGAACGC
AATGACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACG
AGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGTTGGGCACTCTAGTGAGACTGCCGGTGACAA
ACCGGAGGAAGGTGGGGACGACGTCAGATCATCATGCCCCTTATGACCTGGGCTACACACGTGCT
ACAATGGACGGTACAACGAGTCGCGAACTCGCGAGGGCAAGCAAATCTCTTAAAACCGTTCTCAG
TTCGGACTGCAGGCTGCAACTCGCCTGCACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATG
CCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACC
CAAAGTCGGTGGGGTAACCTTTTAGGAGCCAGCCGCCTAAGGTGGGACAGATGATTAGGGTGAAG
TCGTAACAAGGTAGCGAGGGCCC。
combining morphological observation, biochemical identification and homology analysis in DNA phylogenetic tree of the JIAN+ bacteria, the JIAN+ bacteria are determined to be streptococcus salivarius subsp thermophilus (Streptococcus salivarius subsp. Thermophilus) species.
The performance of the streptococcus salivarius subspecies thermophilus JIAN+ provided by the application is characterized as follows:
example 3 analysis of the inhibition of alpha-amylase by Streptococcus salivarius subspecies thermophilus JIAN+, its Complex with white kidney beans
(1) Three liquids to be measured are prepared, respectively:
(1) jian+ bacterial suspension: specifically, after culturing for 48h, the streptococcus salivarius thermophilus subspecies JIAN+ fermentation broth 4500r/min and centrifuging at 4deg.C for 10min, pouring out supernatant, collecting precipitate, cleaning with 0.85% physiological saline for 2 times, and re-suspending, adjusting bacterial suspension density OD to 1.5 and 1.0 (total bacterial count measured by flow cytometry is 4.34 and 2.36 (×10) 8 cfu/mL), obtaining a sample to be tested of the bacterial suspension;
(2) white kidney bean test solution: adding white kidney bean powder into PBS buffer solution (pH=6.8, 0.1 mol/L) to prepare white kidney bean solution of 1mg/mL, and preparing white kidney bean solution to be detected;
(3) white kidney beans with jian+ composition: white kidney bean powder is added into bacterial suspensions with different OD values in the step (1) to prepare white kidney bean (1 mg/mL) +JIAN+ bacterial suspension (OD is 1.5 and 1.0 respectively).
Alpha-amylase inhibition test:
100uL of PBS solution containing 0.6mg/mL alpha-amylase is dripped into the ELISA plate, 100uL of sample solution to be detected is added to react for 30 minutes at 37 ℃, 100uL of starch solution is dripped into the system to react for 15 minutes at 37 ℃, 5uL of dilute iodine solution is dripped finally, and OD value is measured at 660nm on an ELISA instrument;
wherein, the calculation formula of the alpha-amylase inhibition rate is as follows:
alphase:Sub>A-amylase inhibition rate/% = [ (B-C) - (B-A) ]/(B-C) ×100%,
wherein: a is the experimental group, B is the blank group without alpha-amylase sample, and C is the control group without test sample.
(2) The test results are shown in table 2 below, and it can be seen that:
the JIAN+ bacterial suspension has good inhibition effect on alpha-amylase and has good inhibition effect on OD 600 The inhibition rates for alpha-amylase at values of 1.5 and 1.0 were 67.04% and 49.28, respectively; under the experimental conditions, the inhibition effect of 1mg/mL white kidney bean solution on alpha-amylase is not detected;
in the composition of white kidney bean + bacterial suspension 1, the inhibition rate of alpha-amylase was detected to be 75.96%, and compared with the bacterial suspension 1 treatment without white kidney bean, there was a significant difference in the p=0.05 level, indicating that the JIAN + and white kidney bean combination can achieve a synergistic effect on alpha-amylase inhibition.
TABLE 2 analysis of inhibition of alpha-amylase by JIAN+ and white kidney bean compositions
| Treatment group | Alpha-amylase inhibition/% | Treatment group | Alpha-amylase inhibition/% |
| Physiological saline | - | White kidney bean (1 mg/mL) | - |
| Bacterial suspension 1 | 67.04±5.00b | Bacterial suspension 1+white kidney beans | 75.96±3.46a |
| Bacterial suspension 2 | 49.28±2.33c | Bacterial suspension 2+ white kidney beans | 50.04±4.35c |
Note that: the bacterial suspension 1 is obtained by centrifuging JIAN+ thallus and adjusting OD with physiological saline 600 =1.5, bacterial suspension 2 was OD 600 =1.0; the concentration of white kidney beans was 1mg/mL. The different letters indicated a significant difference (P.ltoreq.0.05) between treatments, and "-" indicated no inhibition.
Example 4 analysis of the inhibition of alpha-glucosidase by Streptococcus salivarius subspecies thermophilus JIAN+, its mixture with white kidney beans
(1) Three liquids to be measured are prepared, respectively:
(1) jian+ fermentation supernatant: centrifuging the streptococcus salivarius thermophilus subspecies JIAN+ fermentation liquor 4500r/min after 48h culture at 4 ℃ for 10min, taking supernatant, adjusting the supernatant to 3 concentrations respectively, namely fermentation supernatant 1 as a stock solution, fermentation supernatant 2 (supernatant: PBS (v/v) =1:1) and fermentation supernatant 3 (supernatant: PBS (v/v) =1:2), and taking a non-inoculated MC culture medium as a blank control group to obtain a JIAN+ fermentation supernatant to-be-tested liquid;
(2) white kidney bean test solution: adding white kidney bean powder into PBS buffer solution (pH=6.8, 0.1 mol/L) to prepare white kidney bean solution of 1mg/mL, and preparing white kidney bean solution to be detected;
(3) white kidney bean + JIAN + composition: white kidney beans (1 mg/mL) +JIAN+ fermentation supernatant compositions were prepared by adding white kidney beans to the fermentation supernatants of different concentrations in (1).
Determination of the inhibition effect of alpha-glucosidase: 50uL of PBS buffer solution (0.1 mol/L, pH 6.8) is added dropwise into a microplate, 50uL of alpha-glucosidase solution (1.5 u/mL, PBS buffer solution is used as solvent) is added, 50uL of bacteria solution to be detected is added for reaction for 10 minutes at 37 ℃,50 uL of PNPG solution (1 mg/mL) is added, and after reaction for 30 minutes at 37 ℃,80 uL of Na is added 2 CO 3 After the reaction, the OD value of the stop solution is measured at 405nm by an enzyme-labeled instrument.
(2) The results of the α -glucosidase inhibition are shown in table 3, which shows that:
the inhibition rates of the fermentation supernatant liquid and the 1-time and 2-time dilution liquid of the JIAN+ fermentation supernatant liquid to the alpha-glucosidase are 58.13 percent, 41.3 percent and 24.57 percent respectively;
the corresponding inhibition rates of the fermentation supernatant and the white kidney bean composition on the alpha-glucosidase are 62.73%, 51.00% and 37.83%, which are obviously higher than those of the non-combined group, so that the JIAN+ fermentation supernatant and the diluent have good alpha-glucosidase inhibition effect on the alpha-glucosidase, and the composition can be compounded with the white kidney bean to form the synergistic effect of the composition on the alpha-glucosidase inhibition effect.
TABLE 3 analysis of inhibition of alpha-glucosidase by JIAN+ and white kidney bean compositions
Note that: "-" means no inhibition
EXAMPLE 5 analysis of the ability of Streptococcus salivarius thermophilus JIAN+ to scavenge 2, 2-Biphenyl-1-picrylhydrazyl (DPPH) and OH free radicals
(1) Preparation of JIAN+ suspension method As in example 3 above, the density OD of the suspension was adjusted 600nm About 1.0, obtaining a sample to be tested of the JIAN+ bacterial suspension;
cell disruption supernatant preparation: centrifuging the cultured bacterial liquid for 10min at 4500r/min and 4 ℃, pouring out the supernatant, taking the precipitate, washing with physiological saline with the mass fraction of 0.85% for 2 times, then resuspending, adjusting the density OD of bacterial suspension to be about 1.0, crushing cells in an ultrasonic crusher, setting the power to be 40%, stopping 5s for 5s, and centrifuging for 20min at 10000r/min after crushing, and taking the supernatant to obtain the supernatant to be measured liquid of the cell crushed material.
(1.1) analysis of radical scavenging ability of 2, 2-biphenyl-1-picrylhydrazyl (DPPH):
respectively taking 2mL of the sample to be detected of the bacterial suspension and 2mL of the sample to be detected of the supernatant of the cell disruption, adding the sample to be detected into 2mL of an absolute ethyl alcohol solution containing 0.2mmoL/L DPPH, uniformly mixing, carrying out light-proof reaction at room temperature for 30min, centrifuging at 4500r/min for 10min, and measuring an OD value at 517 nm;
wherein, the calculation formula of DPPH free radical clearance is:
DPPH radical scavenging rate/% = [1- (a) 1 -A 2 )/A 0 ]×100％；
Wherein A is 1 For the absorbance of the experimental group, A 0 For the absorbance of the sample group replaced by the equal volume of absolute ethyl alcohol, A 2 The absorbance value of the DPPH absolute ethyl alcohol solution is replaced by the equal volume absolute ethyl alcohol.
(1.2) determination of OH free radical scavenging ability:
1mL of 1, 10-phenanthroline solution (2.5 mmol/L), 1mL of PBS (pH 7.4) and 0.5mL of bacteria solution to be tested are uniformly mixed, and then 1mL of FeSO is added 4 Solution (2.5 mmol/L) and 0.5mL of H 2 O 2 The solution (20 mmol/L) was incubated at 37℃for 1h in a water bath and the OD at 536nm was measured.
Wherein, the calculation formula of the OH free radical clearance is:
OH radical scavenging rate/% = (a Experiment -A Control )/(A Blank space -A Control )×100％；
Wherein A is Control Absorbance values for the samples were replaced with equal volumes of PBS solution; a is that Blank space Equal volume PBS instead of H 2 O 2 Absorbance of the solution.
(2) The test results of DPPH radical scavenging rate and OH radical scavenging rate are shown in Table 4, and it can be seen that:
the clearance rate of the JIAN+ bacterial suspension to DPPH free radicals is up to 84.24%, and the clearance rate of the cell disruption supernatant to DPPH free radicals is 29.56%, which shows that the streptococcus salivarius thermophilus JIAN+ has good DPPH free radical clearance capability.
The JIAN+ bacterial suspension had a removal rate of 38.56% for OH free radicals, indicating that the Streptococcus salivarius thermophilus JIAN+ has a significant removal capacity for OH free radicals.
TABLE 4 ability of Streptococcus salivarius thermophilus JIAN+ to scavenge free radicals
| Sample of | JIAN+ fermentation broth | JIAN+ cell lysate supernatant |
| DPPH radical scavenging/% | 84.24±0.03 | 29.56±6.07 |
| OH radical scavenging/% | 38.56±2.33 | 3.51±0.02 |
Example 6 determination of the reducing Capacity of Streptococcus salivarius subspecies thermophilus JIAN +
The reducing ability (antioxidant ability) means that electrons are given out to achieve the effect of scavenging free radicals by the reduction of the reducing ability, and the stronger the reducing ability is, the stronger the antioxidant ability is, and the stronger the ability to resist damage of free radicals and oxidants is.
Taking a bacterial suspension sample to be detected and a cell disruption supernatant sample to be detected in the embodiment 5, mixing 0.5mL of 1% potassium ferricyanide, 0.5mL of PBS buffer solution (pH 6.6) and 0.5mL of sample solution, and cooling the mixed solution at an extremely high speed after 20min in a water bath at 50 ℃; then 0.5mL of 10% trichloroacetic acid is added, the mixture is centrifuged for 10min at 4000r/min, 1mL of ultrapure water and 1mL of 0.1% ferric trichloride solution are added into the mixture, the mixture is mixed evenly by shaking, and after the mixture is placed at normal temperature for 10min, the OD value at 700nm is measured.
The calculation formula of the reducing ability is:
reduction capacity/% = (a 1 -A 0 )/A 1 ×100％；
Wherein A is 1 Absorbance values for the sample set; a is that 0 Absorbance values for PBS buffer instead of samples.
(2) The reduction rate test results are shown in Table 5, and it can be seen that:
the reduction rate of the JIAN+ bacterial suspension is 32.9%, and the reduction rate of the supernatant of the cell disruption product is 46.30%, which shows that the JIAN+ bacterial suspension of streptococcus salivarius subspecies thermophilus and the cell content have obvious reduction capacity or antioxidation capacity.
TABLE 5 determination of reducing Capacity of Streptococcus salivarius thermophilus JIAN +
| Sample of | JIAN+ bacterial suspension | JIAN+ cell lysate supernatant |
| Reducing power/% | 32.90±1.58 | 46.30±10.03 |
Example 7 analysis of the tolerability of Streptococcus salivarius subspecies thermophilus JIAN+ in simulated Artificial gastric fluid Environment
Taking bacteria JIAN+ fermented for 24-48h, centrifuging for 10min at 4500r/min to collect thallus, adding physiological saline (0.85%) with the same volume, and mixing; compounding artificial gastric juice (125mM NaCl,7mM KCl,45mM NaHCO) 3 And 3g/L pepsin), adjusting the pH value to 2.0, 2.5 and 3.0, filtering by a 0.22 mu M microporous filter membrane for later use, taking 1mL of treated bacterial liquid, placing the bacterial liquid in 9mL of artificial gastric juice with different pH values, culturing at a constant temperature of 37 ℃, sampling at the time of culturing 1, 2 and 3 hours, diluting 0.9mL each time, adding 0.1mL of PI diluent, dyeing for 10min at 37 ℃, detecting the total particle number P1 and the death particle number P2 of bacteria by a flow cytometer, and calculating the bacterial survival rate according to the formula:
bacterial jian+ viability/% = (P1-P2)/p1×100%.
(2) The bacterial viability results of bacterial jian+ are shown in table 6, from the data:
the survival rate of the JIAN+ in the artificial simulated gastric fluid environment with the pH of 2.5 and 3.0 is between 53.23 and 67.69 (%), and the survival rate remained for 3 hours in the gastric fluid environment is still more than 50%, which indicates that the JIAN+ has good gastric fluid tolerance.
TABLE 6 survival rate/%of Streptococcus salivarius thermophilus JIAN+ in an artificial simulated gastric fluid environment
Example 8 analysis of the tolerability of Streptococcus salivarius subspecies thermophilus JIAN+ in simulated Artificial pancreatic juice Environment
Taking bacteria JIAN+ fermented for 24-48h, centrifuging for 5min at 8000r/min to collect thalli, adding physiological saline (0.85%) with the same volume, and mixing well for standby; preparing protein pancreatic juice (0.1% pancreatic juice w/v,0.15% oxgall), respectively adjusting pH values to 7.5 and 8.0, filtering with a 0.22 mu M microporous filter membrane for later use, taking 1mL of treated bacterial liquid to 9mL of protein pancreatic juice with different pH values, placing the treated bacterial liquid in constant temperature culture at 37 ℃, sampling at 3 and 6 hours, diluting 0.9mL each time, adding 0.1mL of PI (PI refers to propidium iodide dye liquor), dyeing at 37 ℃ for 10 minutes, detecting total bacterial particle number P1 and death particle number P2 by a flow cytometer, and calculating bacterial survival rate;
the bacterial viability was calculated as:
bacterial jian+ viability/% = (P1-P2)/p1×100%.
(2) The bacterial viability results of bacterial jian+ are shown in table 7, from the data:
after the JIAN+ is treated for 6 hours in an artificial simulated pancreatic juice environment with the pH value of 7.5, the survival rate is as high as 99.66 percent, and after the JIAN+ is treated for 3-6 hours in a pancreatic juice environment with the pH value of 8.0, the survival rate is stabilized between 98.11 and 99.32 percent, which shows that the JIAN+ has good pancreatic juice tolerance.
TABLE 7 survival rate/%of Streptococcus salivarius thermophilus JIAN+ in an artificially simulated pancreatic juice environment
Example 9 inhibitory Effect of Streptococcus salivarius subspecies thermophilus JIAN+ on E.coli
The oxford cup (diameter 6 mm) is placed in LB culture medium coated with escherichia coli (E.coli) ATCC35150, 200 μl of JIAN+ fermentation liquor is taken and cultured at a constant temperature of 37 ℃ for 16 hours in the oxford cup, and the antibacterial effect of the JIAN+ fermentation liquor on the escherichia coli is measured, so that the antibacterial diameter is 18.7+/-1.5 mm (shown in FIG. 5 in particular), and the JIAN+ has good antibacterial effect on the escherichia coli.
EXAMPLE 10 preparation of probiotic agent by Streptococcus salivarius subspecies thermophilus JIAN +
Inoculating Streptococcus salivarius thermophilus JIAN+ into a culture medium such as MC culture medium, culturing at 30-42deg.C for more than 15 hr, centrifuging to collect bacterial cells, and suspending in physiological saline or PBS buffer solution, to obtain liquid bacterial preparation containing Streptococcus salivarius thermophilus JIAN+. Alternatively, the streptococcus salivarius subspecies jian+ cells are resuspended in a cytoprotective agent and carrier, and lyophilized to obtain a solid powder formulation containing streptococcus salivarius subspecies jian+.
Alternatively, the streptococcus salivarius subspecies jian+ may be used as a raw material component in an antioxidant-type product, a hypoglycemic product, a slimming product (e.g., food, health product, pharmaceutical product), and the streptococcus salivarius subspecies jian+ may be present in the product in the form of a liquid or solid formulation; further, the streptococcus salivarius jian+ as a raw material component can be used together with antioxidant food, hypoglycemic and slimming food substances commonly used in foods or health foods to prepare foods, health foods or medicines.
EXAMPLE 11 preparation of fermented food from Streptococcus salivarius subspecies thermophilus JIAN +
Preparing fermentation mother liquor of streptococcus salivarius thermophilus subspecies JIAN+, inoculating various saccharides serving as auxiliary materials into the fermentation mother liquor of streptococcus salivarius subspecies JIAN+ under a certain temperature condition (30-42 ℃) for fermentation for a certain time, preparing a fermentation product, carrying out inactivation or non-inactivation treatment, diluting the stock liquor or different proportions, and adding common beverage auxiliary materials to prepare the fermented food.
From the results of the above examples, it was found that the streptococcus salivarius subspecies thermophilus jian+ provided by the present application has the following properties and effects:
a. carbon sources such as cellobiose, maltose, salicin, sucrose, lactose, etc. can be used.
b. The survival rate of the mixture is higher than 50% when the mixture stays for 0 to 3 hours in gastric juice environment with pH value of 2.5 to 3.0, and the survival rate of the mixture stays for 0 to 6 hours in pancreatic juice environment with pH value of 7.5 and is higher than 99%.
c. Can inhibit the alpha-amylase and the alpha-glucohydrolase effectively, and the inhibition rate of the alpha-amylase is 49.27-67.04 percent; the inhibition rate of the alpha-glucosidase is 58.13-24.57 (%).
d. The complex of the streptococcus salivarius thermophilus JIAN+ and the white kidney beans has remarkable synergistic effect on the inhibition rate of alpha-amylase and alpha-gluco-glycation, the inhibition rate of the complex of the streptococcus salivarius thermophilus JIAN+ and the white kidney beans is 50.04-75.96 (%) on the alpha-amylase, and the inhibition rate of the complex of the streptococcus salivarius thermophilus and the white kidney beans is 37.83-62.73 (%) on the alpha-glucosidase, which is higher than any single component, and the complex of the streptococcus salivarius thermophilus with the white kidney beans shows remarkable synergistic effect.
e. The streptococcus salivarius jian+ bacterial suspension has good scavenging ability to DPPH and OH free radicals, and the scavenging rate is 84.23 percent and 39.56 percent respectively; meanwhile, the cell disruption supernatant has better scavenging ability to DPPH and OH; and, the trivalent iron in potassium ferricyanide can be reduced to divalent iron by the JIAN+ bacterial suspension and the cell disruption supernatant, the reduction rates are respectively 32.9% and 46.3%, and the reduction capability is good.
f. The streptococcus salivarius thermophilus JIAN+ has remarkable inhibiting effect on escherichia coli
g. The streptococcus salivarius thermophilus JIAN+ is obtained from daily fresh milk through natural fermentation, has high edible safety, can be used as a weight-losing product, a blood sugar reducing product and an antioxidant product for common foods, health-care foods and medicines, and has wide application prospect.
In summary, compared with the prior art, the streptococcus salivarius subspecies jian+ provided by the application has the following beneficial effects:
the streptococcus salivarius subspecies thermophilus JIAN+ provided by the application can effectively inhibit alpha-amylase and alpha-glucohydrolase, and the composition of the streptococcus salivarius subspecies thermophilus JIAN+ and white kidney beans has a synergistic effect on inhibiting the activity of the alpha-amylase and the alpha-glucohydrolase; meanwhile, the catalyst has good scavenging ability to free radicals DPPH and OH and has oxidation reduction resistance; has good antibacterial effect on escherichia coli; the strain also has good gastric juice pancreatic juice tolerance capability, is a natural strain and strong in safety, and can provide a new probiotic source for development of functional products such as weight-losing products, blood sugar-reducing products, antioxidant products and the like, for example:
(1) The streptococcus salivarius thermophilus JIAN+ can be used as a raw material component of the composition to prepare the composition with the functions of inhibiting the activity of alpha-amylase, inhibiting the activity of alpha-glucokinase, scavenging free radicals DPPH, scavenging OH free radicals, reducing the capability, inhibiting bacteria of escherichia coli and the like; wherein the composition includes but is not limited to microbial preparations, foods, health care products or medicaments, and the like.
Wherein the species is present in the composition in a form that includes, but is not limited to, one or more combinations of a streptococcus salivarius subspecies jian+ inactivated bacteria, a metabolite of a streptococcus salivarius subspecies jian+ strain, and a streptococcus salivarius subspecies jian+ lyophilized strain. Preferably, in the composition, the amount of the Streptococcus salivarius JIAN+ is not less than 1×10 6 CFU/mL or ≡1X10. 6 CFU/g. Further preferably, in the composition, the amount of JIAN+ of Streptococcus salivarius subspecies thermophilus is 1×10 or more 8 CFU/mL or ≡1X10. 8 CFU/g。
(2) Can be prepared by taking various plants (such as fruits, chinese herbal medicines, grains and the like) as raw materials, matching with various ingredients, inoculating streptococcus salivarius thermophilus subspecies JIAN+ for fermentation treatment, and preparing a ferment which can be applied to preparing products with the functions of resisting oxidation, reducing blood sugar and losing weight;
wherein, the fermentation raw material can be various plant fermentation raw materials which are conventionally used, and the auxiliary materials can also be the conventional auxiliary materials, including but not limited to the scheme selection. The ferment is not limited to be used for preparing fermented foods, and can also be products such as health care products, medicines and the like;
in summary, streptococcus salivarius jian+ and/or its fermentate may be applied, according to its characteristics, in functional products comprising at least one of the following functions:
(1) Significantly inhibiting the level of alpha-amylase enzyme activity;
(2) Significantly inhibiting the level of alpha-glucosidase activity;
(3) Significant scavenging of free radical DPPH levels;
(4) Significant OH radical scavenging levels;
(5) Has remarkable reducing capability.
(6) Has remarkable antibacterial effect on Escherichia coli
Wherein, the products with the functions of (1) - (6) comprise but are not limited to antioxidant products and weight-losing functional products, have the effects of resisting oxidation, reducing blood sugar, losing weight and the like, and can also be products with other effects based on the functions of (1) - (6); wherein the functional product includes, but is not limited to, food, health care products or medicines.
Because the complex of the streptococcus salivarius thermophilus JIAN+ and the white kidney beans has a synergistic effect on inhibiting the activity of alpha-amylase and inhibiting the function of alpha-glucose glycanase, the complex can be compounded with the white kidney beans for use, and the formed complex is applied to weight-losing products for inhibiting the activity of alpha-amylase and inhibiting the function of alpha-glucose glycanase.
Similarly, the complex of streptococcus salivarius jian+ and white kidney beans is not limited to functional products for losing weight, namely, the complex is not limited to the effects of reducing blood sugar, losing weight and the like, but also can be products with other effects of inhibiting the activity of alpha-amylase and inhibiting the function of alpha-gluco-glycanase; wherein the functional product includes, but is not limited to, food, health care products or medicines.
It should be noted that:
(1) Definition:
the term food as used herein is used in a broad sense to include human foods and drinks. In certain embodiments, the food product is suitable and designed for human feeding. The application can be used for preparing solid preparations such as powder, tablets and the like, and also can be dispersed in liquid to prepare liquid preparations, including but not limited to powder.
The compositions include, but are not limited to, microbial preparations, foods, nutraceuticals, pharmaceuticals, and the compositions containing streptococcus salivarius subspecies jian+ can be used in other forms of products.
The presence of the streptococcus salivarius subspecies jian+ in the composition includes, but is not limited to, non-inactivated bacteria, metabolites, lyophilized strains, and the like, and it is contemplated that the streptococcus salivarius subspecies jian+ may also be present in the composition in other forms.
The functional product comprises the functions of scavenging free radicals DPPH, scavenging OH free radicals, reducing, inhibiting alpha-amylase activity, inhibiting alpha-glucokinase activity, inhibiting escherichia coli and the like, and the obvious effects of the functional product applied to the functional product are antioxidation, blood sugar reduction and weight reduction according to the functions; because the Chinese medicinal composition has the effects of scavenging free radicals DPPH, scavenging OH free radicals, reducing capacity, inhibiting alpha-amylase enzyme activity, inhibiting bacteria of escherichia coli and the like, the effects of the application products of the Chinese medicinal composition include, but are not limited to, antioxidation, blood sugar reduction and weight reduction, and other effects on human bodies can be achieved through the effects.
(2) Related prior art means or prior art terms to which the present application relates:
the "OD" value is an abbreviation of optical density (also called absorbance), and the difference between the energy of light passing through the object to be measured and the energy of light passing through the object to be measured is the energy absorbed by the object to be measured. "OD 600 "the optical density value measured when the wavelength is set to 600 nm" is a standard index for tracking the microorganism density in a liquid culture and is usually used to indicate the cell density of a cell. Among these, the "OD" value determination method is prior art, and the principle and method thereof will not be described here.
"DPPH" is free radical 2, 2-biphenyl-1-picrylhydrazyl, and "OH" is free radical hydroxyl.
The use of flow cytometry to determine the total number of bacteria P1 and the dead number of bacteria P2 is prior art, the principles and methods of which are not described here.
The method for measuring the alcohol content and the temperature of the distilled liquid by adopting an alcohol meter and calculating the actual alcohol content of each treatment by adopting an alcohol meter temperature concentration conversion table is the prior art, and the principle and the method are not further described here.
The principle and method of the prior art, which are not described here, are analyzed by using the HBI lactobacillus biochemical identification strip.
(3) The formulation of each medium used in the examples is as follows:
the MC culture medium (1L) comprises 5.0 parts of soybean peptone, 3.0 parts of beef extract powder, 3.0 parts of yeast extract powder, 20.0 parts of glucose, 20.0 parts of lactose, 10.0 parts of calcium carbonate and 6.0+/-0.1 parts of pH value (the solid culture medium is added with neutral red 0.05 and agar 20.0 parts on the basis of the culture medium), and the packaging is carried out for 20 minutes after the packaging and the high-pressure sterilization are carried out at 121 ℃.
The formula of the LB culture medium is as follows: 10.0g of tryptone, 5g,NaCl 10g,pH 7.0 g of yeast extract powder and 20.0g of agar powder; and autoclaving at 115℃for 20min.
The experimental procedures referred to in the examples of the present application are conventional in the art, and reagents or instruments referred to are commercially available from regular sources, unless explicitly indicated otherwise.
In addition, it should be understood by those skilled in the art that although many problems exist in the prior art, each embodiment or technical solution of the present application may be modified in only one or several respects, without having to solve all technical problems listed in the prior art or the background art at the same time. Those skilled in the art will understand that nothing in one claim should be taken as a limitation on that claim.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present application, and not for limiting the same; although the application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the application.
Claims (10)
1. A streptococcus salivarius subspecies thermophilus jian+, characterized by: the preservation number is CGMCC No.27130.
2. A composition characterized by: comprising the streptococcus salivarius subspecies jian+ according to claim 1.
3. The composition of claim 2, wherein: the composition comprises one of a microbial preparation, food, health care product and medicine.
4. A ferment, characterized in that: which is obtained by fermentation of the streptococcus salivarius subspecies thermophilus jian+ according to claim 1.
5. Use of streptococcus salivarius subspecies jian+ and/or a fermentation thereof for the preparation of a functional product, characterized in that: the streptococcus salivarius subspecies jian+ is the streptococcus salivarius subspecies jian+ according to claim 1.
6. The use according to claim 5, characterized in that: the functional product comprises food, health product or medicine.
7. The use according to claim 5, characterized in that: the functional product comprises at least one of the following functions:
(1) Inhibiting alpha-amylase enzyme activity;
(2) Inhibiting alpha-glucohydrolase activity;
(3) Scavenging free radical DPPH;
(4) Removing OH free radicals;
(5) The device has reducing capability;
(6) Has inhibiting effect on Escherichia coli.
8. The use according to claim 5, characterized in that: the functional products comprise antioxidant products, hypoglycemic products and weight-losing functional products.
9. Use of streptococcus salivarius subspecies jian+ as defined in claim 1 or a composition as defined in any one of claims 2 to 3 in the preparation of a fermented food product.
10. A white kidney bean complex comprising white kidney bean flour, characterized in that: the complex contains streptococcus salivarius subspecies jian+ and/or a fermentation thereof;
wherein the streptococcus salivarius subspecies jian+ is the streptococcus salivarius subspecies jian+ of claim 1.
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| CN116574634B (en) * | 2023-03-13 | 2023-11-03 | 广东悦创生物科技有限公司 | Streptococcus salivarius thermophilus subspecies JF2 and application thereof in preparation of anti-inflammatory and lipid-relieving food and drug |
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