A fermented mucus lactobacillus YYS-K2 and its application

Abstract

The present invention relates to the field of microbial technology, and provides a fermented mucus lactobacillus YYS-K2 and an application thereof. The deposit number of the fermented mucus lactobacillus YYS-K2 is CGMCC No.27129. The fermented mucus lactobacillus YYS-K2 can produce ethanol dehydrogenase (ADH), has good ethanol tolerance and degradation ability, can grow in a high-concentration ethanol culture medium, and at the same time, the bacteria has a significant inhibitory effect on α-amylase inhibition, has significant OH and DPPH free radical scavenging ability, and has good reducing ability. The bacteria can provide a new probiotic source for the development of functional products such as hangover products, antioxidant products and weight loss products (α-amylase inhibition functional products), and has important application value.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a fermented mucus lactobacillus YYS-K2 and an application thereof. Background Art

[0002] Alcohol has been an indispensable food in human life since ancient times. Excessive drinking will metabolize in the body to produce free radicals and other substances. Long-term drinking is extremely harmful to the liver and can easily cause alcoholic liver, chronic ethanol poisoning, beer belly, obesity, etc. At the same time, alcohol will enter the blood-brain barrier and cause brain dysfunction.

[0003] However, as an important food for the development of human civilization, wine plays an irreplaceable role in social etiquette, relieving emotional stress, and invigorating the spirit. The pleasure that drinking brings to humans is immeasurable. Therefore, how to let humans experience the pleasure of drinking while minimizing the harm to the body is a very meaningful and important task.

[0004] Free radicals are atoms or groups with unpaired electrons that are extremely active in chemical reactions. In addition to being produced in the body by excessive drinking, they are also commonly found in environmental pollutants such as aerobic respiration, cell activity, house fumes, smoking, automobile exhaust, and industrial waste gas. Excessive free radicals and oxidants can cause various harms to the body, including attacking cells and decomposing tissues, and ultimately causing different health problems such as cerebral stroke, Alzheimer's disease, arteriosclerosis, cancer, diabetes, and oxidative aging.

[0005] In addition, drinking can easily lead to sub-health problems such as beer belly and obesity. Inhibiting α-amylase can reduce the body's absorption of carbohydrates during drinking, and can play a certain inhibitory role in the obesity problem caused by it.

[0006] Probiotics are a type of microorganisms that are beneficial to human health. Studies have shown that probiotics can regulate the body's balance in a variety of ways. Some probiotics can use ethanol as a carbon source to metabolize ethanol during their growth, while others can produce alcohol dehydrogenase to degrade ethanol. However, at present, in this field, there is still a gap in the research of strains that have the functions of sobering up, anti-oxidation, and inhibiting α-amylase.

[0007] How to develop a probiotic that can significantly degrade ethanol (detoxify alcohol), scavenge free radicals, and relieve obesity caused by drinking is the problem and difficulty that technicians in this field are committed to solving. Summary of the invention

[0008] In order to solve the deficiencies of the prior art mentioned in the above background technology, the present invention provides a fermented mucus lactobacillus YYS-K2. Fermented mucus lactobacillus YYS-K2 (Limosilactobacillus fermentum YYS-K2) was deposited in the General Microbiological Center of China National Committee for the Preservation of Microorganisms on April 17, 2023, with a preservation address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and a preservation number of CGMCC No. 27129.

[0009] The present invention also provides a composition, which contains the fermented Lactobacillus mucilaginosus YYS-K2 as described above.

[0010] In one embodiment, the composition includes one of a microbial preparation, a food, a health product, and a medicine.

[0011] In one embodiment, in the composition, the amount of the fermented Lactobacillus mucilaginosus YYS-K2 is ≥ 1×10 6 CFU / mL or ≥1×10 6 In one embodiment, in the composition, the number of the fermented Lactobacillus mucilaginosus YYS-K2 is ≥ 1×10 8 CFU / mL or ≥1×10 8 CFU / g.

[0012] In one embodiment, the composition comprises one or more combinations of non-inactivated fermented Lactobacillus mucoides YYS-K2, inactivated fermented Lactobacillus mucoides YYS-K2, metabolites of fermented Lactobacillus mucoides YYS-K2 strains, and freeze-dried fermented Lactobacillus mucoides YYS-K2 strains.

[0013] The present invention also provides a fermented product, which is obtained by fermenting the fermented Lactobacillus mucilaginosus YYS-K2 as described above.

[0014] The present invention also provides the use of the fermented Lactobacillus mucilaginosus YYS-K2 and / or its fermentation product in preparing functional products.

[0015] In one embodiment, the functional product includes food, health care products or medicines.

[0016] In one embodiment, the functional product includes at least one of the following functions:

[0017] (1) Degradation of ethanol;

[0018] (2) Producing ADH enzyme;

[0019] (3) scavenging the free radical DPPH;

[0020] (4) scavenging OH free radicals;

[0021] (5) Having the ability to restore;

[0022] (6) Inhibit α-amylase activity.

[0023] In one embodiment, the functional products include alcohol sobering products, antioxidant products and α-amylase inhibiting products.

[0024] The present invention also provides use of the fermented mucus lactobacillus YYS-K2 or the composition described above in preparing fermented food.

[0025] In one embodiment, fermented Lactobacillus mucilaginosus YYS-K2 is used as a probiotic, and fruits and Chinese herbs are fermented to prepare fermented food.

[0026] Based on the above, compared with the prior art, the fermented mucus Lactobacillus YYS-K2 provided by the present invention has the following beneficial effects:

[0027] The fermented mucus lactobacillus YYS-K2 provided by the invention can produce ethanol dehydrogenase (ADH), has good ethanol tolerance and degradation ability, can grow in a high-concentration ethanol culture medium, and at the same time, the bacteria has a significant inhibitory effect on alpha-amylase inhibition, has significant OH and DPPH free radical scavenging ability, and has good reducing ability. The bacteria can provide a new probiotic source for the development of functional products such as alcohol sobering products, antioxidant products and weight loss products (products with alpha-amylase inhibition function), and has important application value.

[0028] Other features and beneficial effects of the present invention will be described in the following description, and partly become apparent from the description, or understood by practicing the present invention. The purpose and other beneficial effects of the present invention can be realized and obtained by the structures particularly pointed out in the description, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work. The positional relationships described in the drawings in the following description are based on the directions in which the components are drawn in the diagrams, unless otherwise specified.

[0030] Figure 1 This is the colony morphology of fermented Lactobacillus mucilaginosus YYS-K2.

[0031] Figure 2 This is a scanning electron micrograph of fermented Lactobacillus mucilaginosus YYS-K2.

[0032] Figure 3 Agarose electrophoresis diagram of 16S r DNA target fragment amplification of fermenting Lactobacillus mucilaginosus YYS-K2.

[0033] Figure 4 This is the phylogenetic tree of the 16S rDNA gene of fermentative Lactobacillus mucilaginosus YYS-K2. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments; the technical features designed in different implementation modes of the present invention described below can be combined with each other as long as they do not conflict with each other; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0035] In the description of the present invention, it should be noted that all terms used in the present invention (including technical terms and scientific terms) have the same meanings as those generally understood by ordinary technicians in the field to which the present invention belongs, and cannot be understood as limiting the present invention; it should be further understood that the terms used in the present invention should be understood to have the same meanings as these terms in the context of this specification and in the relevant fields, and should not be understood in an idealized or overly formal sense, unless explicitly defined in the present invention.

[0036] The present invention also provides the following operation examples and embodiments:

[0037] The fermented mucus lactobacillus YYS-K2 (Limosilactobacillus fermentum YYS-K2) of the invention is obtained from milk fans through laboratory breeding.

[0038] The present invention is to extract the bacteria from milk fan operation example is as follows:

[0039] Example 1 Screening and Isolation of Bacteria

[0040] Take about 1g of the milk fan sample and place it in an MRS liquid medium with an ethanol concentration of 8% (v / v), and culture it at 37°C for 1-3 days until the medium becomes turbid to obtain the first generation culture solution. Place the first generation culture solution in an MRS liquid medium with an ethanol concentration of 12% (v / v), and culture it at 37°C for 1-3 days until the medium becomes turbid to obtain the culture medium sample of the ethanol-resistant strain. Dilute the ethanol-resistant fermentation medium sample to 10 -3 , 10 -4 , 10-5 0.1 mL was applied to a solution containing 12% ethanol and 2% CaCO 3 The culture medium was incubated at 37°C for 48-72 hours. Several single colonies of suspected lactobacilli that could produce large lysis circles were selected and purified three times. The activity of alcohol dehydrogenase (ADH) was tested and the strain with the highest ADH enzyme activity was selected and preserved and named YYS-K2.

[0041] Example 2 Identification of strains

[0042] 2.1 Morphological observation of YYS-K2 bacteria

[0043] The colony morphology of YYS-K2 is as follows Figure 1 As shown, the bacterial morphology is Figure 2 The main morphological characteristics of YYS-K2 are as follows: the colonies are round, milky white, smooth and opaque on MRS medium; the bacteria are rod-shaped, 0.6-0.9μm wide and 1-2.5μm long.

[0044] 2.2 Analysis of carbon source utilization of YYS-K2 bacteria

[0045] HBI lactic acid bacteria biochemical identification strips (GB4789.35 standard, purchased from Haibo Biotechnology) were used. Specifically, a biochemical identification strip was taken out, and some colonies of purified YYS-K2 were picked and inoculated into the following culture media: esculin, cellobiose, maltose, mannitol, maltose, salicin, sorbitol, sucrose, raffinose, inulin, lactose, and 1% sodium hippurate. After inoculation, the strips were marked, covered, and placed at 36±1℃ for incubation. After the incubation, they were placed on a record card for observation.

[0046] Among them, 1% sodium hippurate also needs to be added slowly with 0.2 mL of ninhydrin solution along the wall of the test tube after the culture is completed. Do not shake it and place it in a water bath at 36℃±1℃ for 10 minutes before judging the results. The judgment results are detailed in Table 1.

[0047] Table 1 YYS-K2 glycosylated compounds and main biochemical reactions

[0048]

[0049] Note: “+” indicates a positive test, and “-” indicates a negative test.

[0050] Conclusion: According to the results in Table 1, YYS-K2 can utilize sugars such as maltose, sucrose, raffinose and lactose, but cannot utilize esculin, cellobiose, mannitol, salicin, sorbitol, inulin and sodium hippurate.

[0051] 2.3 Molecular biological identification of YYS-K2

[0052] ① Extraction of YYS-K2 bacterial genomic DNA: The bacterial genomic DNA extraction kit of TIANGEN Company was used for extraction. The specific extraction steps refer to the instruction manual of the kit.

[0053] ② PCR amplification of 16S rDNA sequence: Amplify the 16S rDNA gene sequence, the primers used are:

[0054] ② PCR amplification of 16S rDNA sequence: Amplify the 16S rDNA gene sequence, the primers used are F 9-27: 5'-GAGTTT GAT CCT GGC TCA G-3'; R 1525-1542: 5'-AGA AAG GAG GTG ATC CAG CC-3'; PCR reaction system: 2×Mix 12.5μL, primers and DNA 1μL each, ddH 2 09.5μL.

[0055] PCR amplification program: 93°C pre-denaturation for 4 min, then 94°C denaturation for 30 s, 55°C (16S rDNA), 72°C extension for 90 s, a total of 30 cycles, and finally 72°C full extension for 10 min, and stored at 4°C.

[0056] ③PCR product detection and sequencing analysis: Take 5ul of PCR product and separate and test it by gel electrophoresis in 1.0% agarose gel added with EB. The amplified 16SrDNA target fragment is about 1500bp long (see the agarose gel electrophoresis diagram of 16S rDNA target fragment amplification for details). Figure 3 );

[0057] ④ Phylogenetic analysis: Blast alignment analysis was performed on each 16S rRNA sequence in the NCBI data, and the obtained sequences had a homology of more than 99% with the sequence of Limosilactobacillus fermentum. The neighbor-joining method in MEGA 4 was used to construct a phylogenetic tree (the results are shown in Figure 4 ). The gene sequence is as follows:

[0058] GCAAGCGGGGTGCTATACATGCAGTCGAACGCGTTGGCCCAATTGATTGACGGTGCTTGCACCTGATTGATTTTGGTCGCCAACGAGTGGCGGACGGGTGAGTAACACGTAGGTAACCTGCCCAGAAGCGGGGGACAACATTTGGAAACAGATGCTAATACCGCATAACAACGTTGTTCGCATGAACAACGCTTAAAAGATGGCTTCTCGCTATCACTTCTGGATGGACCTGCGGTGCATTAGCTTGTTGGTGGGGTAACGGCCTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACAATGGGACTGAGACACGGCCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAACACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTAAAGAAGAACACGTATGAGAGTAACTGTTCATACGTTGACGGTATTTAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGAGAGTGCAGGCGGTTTTCTAAGTCTGATGTGAAAGCCTTCGGCTTAACCGGAGAAGTGCATCGGAAACTGGATAACTTGAGTGCAGAAGAGGGTAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTACCTGGTCTGCAACTGACGCTGAGACTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGGAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAA

[0059] CTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGA

[0060] AGAACCTTACCAGGTCTTGACATCTTGCGCCAACCTAGAGATAGGGCGTTTCCTTCGGGAACGC

[0061] AATGACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACG

[0062] AGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGTTGGGCACTCTAGTGAGACTGCCGTGACAA

[0063] ACCGGAGGAAGGTGGGGACGACGTCAGATCATCATGCCCCTTATGACCTGGGCTACACACGTGCT

[0064] ACAATGGACGGTACAACGAGTCGCGAACTCGCGAGGGCAAGCAAATCTCTTAAAACCGTTCTCAG

[0065] TTCGGACTGCAGGCTGCAACTCGCCTGCACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATG

[0066] CCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACC

[0067] CAAAGTCGGTGGGGTAACCTTTTAGGAGCCAGCCGCCTAAGGTGGGACAGATGATTAGGGTGAAG

[0068] TCGTAACAAGGTAGCGAGGGCCC.

[0069] Conclusion: Combining the morphological observation of YYS-K2, biochemical identification of HBI lactic acid bacteria, and homology analysis in the DNA phylogenetic tree, it was determined that YYS-K2 was a strain of Limosilactobacillus fermentum.

[0070] The performance characteristics of the fermented mucus lactobacillus YYS-K2 provided by the present invention are as follows:

[0071] Example 3 Growth of fermented Lactobacillus mucilaginosus YYS-K2 on culture media with different ethanol concentrations

[0072] (1) The fermented Lactobacillus mucilaginosus YYS-K2 mother liquor was inoculated at 2% into the following MRS medium with different ethanol concentrations and cultured anaerobically at 37°C. The fermented liquid was taken at different fermentation times (6, 12, 24, and 36 h) and the bacterial OD was measured. 600nm Values, uninoculated MRS medium with the same ethanol concentration was used as blank control, each treatment was repeated three times, bacterial growth;

[0073] Among them, the OD of the fermented Lactobacillus mucilaginosus YYS-K2 mother solution in MRS medium with different ethanol concentrations 600nm The △OD value is obtained by deducting the blank effect. 600nm The calculation formula is:

[0074] △OD 600nm =OD 样品 -OD 空白 ;

[0075] Among them, OD 空白 It represents the OD value measured by MRS medium with the same ethanol concentration but without bacteria.

[0076] (2) By measuring the growth of bacteria in different ethanol concentrations (see Table 2 for details), it was found that:

[0077] YYS-K2 can grow well in MRS medium with ethanol concentration ≤12%, and the △OD value of the bacteria is greater than 1.0 after 36h of fermentation; in MRS medium with ethanol concentration of 18-24(%), after 6-24 days of adaptation, some bacteria can grow significantly, and at 36h of fermentation, the △OD value of the bacteria is greater than 1.0. 600nm The value is greater than 0.5, indicating that strain YYS-K2 can grow in a high concentration of 24% ethanol stress environment, which provides a good basis for its further degradation of ethanol in a high concentration ethanol environment.

[0078] Table 2 Growth status of YYS-K2 in medium containing different ethanol concentrations (△OD 600nm value)

[0079]

[0080] Note: Different letters ag in the table indicate significant differences between samples.

[0081] Example 4 Test on the tolerance of fermented Lactobacillus mucilaginosus YYS-K2 to high concentration of ethanol

[0082] (1) Based on the characteristic that strain YYS-K2 can grow in an environment with an ethanol concentration of 24%, this study analyzes its tolerance in an ethanol environment with a concentration greater than 24%. The specific test method is as follows:

[0083] YYS-K2 cultured for 24 hours was centrifuged and diluted to the original volume to prepare a bacterial suspension. 1 mL of the bacterial suspension was added to physiological saline containing different concentrations of ethanol to prepare a bacterial suspension containing YYS-K2 with a final concentration of 25-45% ethanol. The suspension was allowed to stand at 37°C for 2 hours. 1 mL of the treated sample was added to 0.1 mL of PI diluent (PI refers to propidium iodide stain) and stained at 37°C for 10 minutes. The total number of bacteria P1 and the number of dead bacteria P2 were determined by flow cytometry, where the bacterial survival rate % = (P1-P2) / P1.

[0084] (2) The above method was used to test the bacterial survival rate to characterize the tolerance of the bacteria in ethanol. The tolerance test results are shown in Table 3;

[0085] Conclusion: The test results show that after the bacteria YYS-K2 was placed in a physiological saline stress environment with an ethanol concentration of 25-40% for 2 hours, the survival rate of the bacteria was between 26.23-12.21(%). In a physiological saline environment with an ethanol concentration of up to 45%, the survival rate of the bacteria can still be maintained at around 8.32%, indicating that the bacteria YYS-K2 has good ethanol tolerance.

[0086] Table 3 Tolerance of YYS-K2 in high concentration ethanol stress environment

[0087]

[0088] Example 5 Degradation rate of ethanol by fermentation of Lactobacillus mucilaginosus YYS-K2

[0089] (1) Analyze the degradation rate of bacteria YYS-K2 in different ethanol concentrations. The specific degradation rate test method is as follows:

[0090] The YYS-K2 fermentation liquid cultured for 24 hours was inoculated into 200 mL of MRS medium containing different concentrations of ethanol at an inoculum size of 5%. The uninoculated MRS medium with the same ethanol concentration was used as the control group. The culture was allowed to ferment at 37°C for 24 hours to obtain the samples to be tested.

[0091] The ethanol content of the sample to be measured is determined according to the national standard GB / T 15038-2006, specifically:

[0092] Take 100mL of each sample to be tested in a 500mL distillation bottle containing a small amount of glass beads, add 100mL of pure water, use a full glass distiller to distill the sample and collect the distillate to about 100mL, stabilize the distillate in a 20℃ water bath for 30min, use an alcohol meter to test the alcohol content and temperature of the distillate, and calculate the actual alcohol content of each treatment through the alcohol meter temperature concentration conversion table;

[0093] The calculation formula of ethanol degradation rate is:

[0094] Ethanol degradation rate / %=(control ethanol content-fermentation ethanol content)×100% / control ethanol content.

[0095] The control ethanol content refers to the ethanol content of the sample to be tested in the uninoculated control group.

[0096] The ethanol content after fermentation is: the ethanol content of the sample to be tested is obtained by inoculating the strain YYS-K2 at an inoculation rate of 5% into 200 mL of MRS medium containing different concentrations of ethanol and fermenting.

[0097] (2) The degradation rate of ethanol by YYS-K2 obtained by testing is shown in Table 4 below. According to the data, it can be seen that:

[0098] When the ethanol content was 4%, 8% and 16% (v / v), the degradation rates of ethanol by YYS-K2 were 74.28%, 52.90% and 29.21%, respectively, indicating that YYS-K2 had a good degradation effect on ethanol.

[0099] Table 4 Analysis of the degradation of ethanol by YYS-K2

[0100] Ethanol content / % 4 8 16 Ethanol degradation rate % 74.28±1.54 52.90±0.97 29.21±3.97

[0101] Example 6 Ability of fermenting Lactobacillus mucilaginosus YYS-K2 to produce ethanol dehydrogenase

[0102] (1) The specific test method for the ability of strain YYS-K2 to produce ethanol dehydrogenase is:

[0103] YYS-K2 was fermented in MRS liquid medium for 24 h, 50 mL of fermentation broth was taken, centrifuged at 5500 rpm / min for 10 min, and the supernatant was taken for testing;

[0104] At the same time, take another 5 mL of the supernatant stock solution, add it to 5 mL of artificial gastric juice with a pH of 2.5 and mix well. Quickly take 1 mL of the mixed solution as the 0 h control group for the determination of ADH enzyme. The remaining mixed solution is placed in a 37 ° C water bath, and 1 mL is sampled at 1 h and 2 h respectively to obtain the gastric juice environment treated supernatant for the determination of ADH enzyme. Each treatment is repeated three times.

[0105] Among them, ADH enzyme detection was performed using an alcohol dehydrogenase (ADH) activity detection kit (Solarbio LifeSciences). The method was referred to the kit instructions. The specific operation is as follows:

[0106] Blank control tube: add 20ul ddH 2 0, 8ul reagent 2, 152ul reagent 1, 20ul reagent 3, add to the same microplate and mix quickly, measure the absorbance at 340nm, record the absorbance at 15s and 75s, respectively, record as A1 and A2, Δ blank tube = A1-A2 (only 1-2 blank tubes are made);

[0107] Determination tube: add 20ul sample supernatant, 8ul reagent 2, 152ul reagent 1, and 20ul reagent 3 to a 96-well microplate (UV plate) in sequence, mix quickly after adding to the same microplate, and measure the absorbance at 340nm. Record the absorbance at 15s and 75s, which are recorded as A3 and A4 respectively;

[0108] Among them, Δ blank tube = A3-A4; supernatant ADH (U / mL) = 2.68 × (ΔA assay tube - ΔA blank tube). ADH (U / mL) after gastric juice treatment = 2.68 × (ΔA assay tube - ΔA blank tube) × supernatant dilution factor (the dilution factor in this experiment is 2).

[0109] (2) The ADH enzyme test results of the YYS-K2 fermentation supernatant and the supernatant gastric juice treatment liquid are shown in Table 5. The test results show that: the ADH enzyme produced per 1 mL of YYS-K2 fermentation supernatant per 1 min is 0.121 U / mL. After being treated in a gastric juice environment with a pH value of 2.5 for 1 h and 2 h, the enzyme activity was not significantly different from that at 0 h (P≤0.05), indicating that YYS-K2 not only produces a higher amount of ADH enzyme, but also has good gastric juice tolerance.

[0110] Table 5 Ethanol dehydrogenase production capacity of strain YYS-K2

[0111]

[0112] Example 7 Test of the OH and DPPH free radical scavenging ability and reducing power of fermented Lactobacillus mucilaginosus YYS-K2

[0113] (1) The specific test methods for OH and DPPH free radical scavenging and reducing power are as follows:

[0114] YYS-K2 bacterial suspension sample preparation: The fermented Lactobacillus mucosa YYS-K2 fermented liquid after 24 hours of culture was centrifuged at 4500r / min and 4°C for 10min, the supernatant was discarded, and the precipitate was washed twice with 0.85% saline and resuspended to adjust the bacterial suspension density OD 600nm is about 1.0 (the number of bacteria is 3.0×10 8 cfu / mL) to obtain the bacterial suspension sample to be tested;

[0115] 1) Determination of OH free radical scavenging ability:

[0116] 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 solution to be tested, then add 1 mL of FeSO 4 solution (2.5 mmol / L) and 0.5 mL of H 2 O 2 Solution (20 mmol / L) was placed in a 37°C water bath for 1 h, and its OD value was detected at 536 nm.

[0117] The calculation formula of OH radical scavenging rate is:

[0118] OH free radical scavenging rate / % = (A 实验 -A 对照 ) / (A 空白 -A 对照 )×100%;

[0119] Among them, A 对照 A is the absorbance value of the sample replaced by an equal volume of PBS solution; 空白 Replace HO with an equal volume of PBS 2 O 2 Solution absorbance, A 实验 is the absorbance value measured for the bacterial solution sample to be tested.

[0120] 2) 2,2-Biphenyl-1-picrylhydrazyl (DPPH) free radical scavenging ability experiment:

[0121] Take 2 mL of the above bacterial suspension samples, add them into 2 mL of anhydrous ethanol solution containing 0.2 mmol / L DPPH, mix well, react at room temperature in the dark for 30 min, centrifuge at 8000 r / min for 10 min, and measure the OD value at 517 nm.

[0122] The calculation formula of DPPH free radical scavenging rate is as follows:

[0123] DPPH free radical scavenging rate (%) = [1-(A 1 -A 2 ) / A 0]×100%;

[0124] Among them A 1 is the absorbance value of the experimental group, A 0 A is the absorbance value of the sample group replaced by an equal volume of anhydrous ethanol. 2 It is the absorbance value of DPPH anhydrous ethanol solution replaced by an equal volume of anhydrous ethanol.

[0125] 3) Determination of reducing power:

[0126] Take the YYS-K2 bacterial suspension sample to be tested, mix 0.5mL of 1% potassium ferricyanide, 0.5mL of PBS buffer (pH6.6) and 0.5mL of sample solution, and place in a 50°C water bath for 20min, then quickly cool the mixed liquid; then add 0.5mL of 10% trichloroacetic acid, centrifuge at 4000r / min for 10min, take 1mL of the sample to be tested, add 1mL of ultrapure water and 1mL of 0.1% ferric chloride solution, shake and mix evenly, place at room temperature for 10min, and measure the OD value at 700nm.

[0127] The calculation formula of reduction capacity is as follows:

[0128] Reducing ability / % = (A 1 -A 0 ) / A 1 ×100%;

[0129] Among them, A 1 is the absorbance value of the sample group; A 0 The absorbance value of the sample is replaced by PBS buffer.

[0130] (2) The test results of the above OH radical scavenging ability, DPPH radical scavenging rate and reduction rate are shown in Table 6. According to the test results, it can be seen that:

[0131] The OH free radical scavenging rate of YYS-K2 bacterial suspension was 47.46%, indicating that the fermented mucus lactobacillus YYS-K2 had significant OH free radical scavenging ability.

[0132] The scavenging rate of YYS-K2 bacterial suspension on DPPH free radicals was 16.90%, indicating that the fermented mucus lactobacillus YYS-K2 had a certain scavenging ability on DPPH free radicals.

[0133] The reduction rate of YYS-K2 bacterial suspension was 15.46%, indicating that the fermented mucus lactobacillus YYS-K2 had a certain reduction ability.

[0134] Table 6 Free radical scavenging ability of fermented mucus lactobacillus YYS-K2

[0135] sample OH free radical scavenging rate / % DPPH free radical scavenging rate / % Reduction power / % YYS-K2 bacterial suspension 47.46±3.37 16.90±2.98 15.46±1.35

[0136] Example 8: Inhibition of α-amylase by fermentation of Lactobacillus mucilaginosus YYS-K2

[0137] (1) The test method for the inhibition of α-amylase is:

[0138] YYS-K2 bacterial suspension was prepared by centrifuging the fermented Lactobacillus mucosa YYS-K2 at 4000 r / min and 4°C for 10 min after culturing for 24 h, discarding the supernatant and washing the precipitate twice with 0.85% saline and resuspending the precipitate to adjust the bacterial suspension density OD to 1.5 and 1.0 (the number of bacteria was 6.7 and 3.0 × 10 8 cfu / mL) to obtain the bacterial suspension sample to be tested.

[0139] α-Amylase inhibition test: add 100uL of 0.6mg / mL α-amylase PBS solution to the ELISA plate, add 100uL of the sample solution to react at 37℃ for 30 minutes, add 100uL of starch solution to the system, and react at 37℃ for 15 minutes. Finally, add 5uL of dilute iodine solution and measure the OD value at 660nm on the ELISA reader;

[0140] The calculation formula of α-amylase inhibition rate is:

[0141] α-amylase inhibition rate / % = [(BC)-(BA)] / (BC)×100%;

[0142] A is the experimental group, B is the blank group without α-amylase sample, and C is the control group without test sample.

[0143] (2) The test results are shown in Table 7. It can be seen that: 600nm When the values ​​were 1.5 and 1.0, the inhibition rates of α-amylase were 68.54% and 41.82%, respectively, indicating that the YYS-K2 bacterial suspension had a good inhibitory effect on α-amylase.

[0144] Table 7 Inhibitory effect of YYS-K2 on α-amylase

[0145]

[0146] Example 9 Survival analysis of fermented mucus lactobacillus YYS-K2 in artificial simulated gastric fluid environment and gastric fluid environment containing ethanol

[0147] (1) The survival rate test process is as follows:

[0148] Take the YYS-K2 strain that has been fermented for 24 hours, centrifuge at 5500r / min for 10min to collect the bacteria, add the same volume of physiological saline (0.85%) and mix well for later use;

[0149] Prepare artificial gastric juice (125 mM NaCl, 7 mM KCl, 45 mM NaHCO 3 and 3g / L pepsin), adjust the pH to 2.0, 2.5, and 3.0, filter through a 0.22μM microporous filter membrane for later use; take 1mL of the treated sample in 9mL of artificial gastric juice with a pH of 2.75, place it in a constant temperature culture at 37°C, take samples at 1, 2, 3, and 5h of treatment, take 0.9mL each time, add 0.1mL PI (PI refers to propidium iodide stain), stain at 37°C for 10min, take 0.1ml and put it in 0.9mL of ultrapure water, detect the total bacterial count P1 / % and the death count P2 / % by flow cytometry, and calculate the bacterial survival rate;

[0150] The calculation formula of bacterial survival rate is:

[0151] Bacterial survival rate / %=(P1 / %-P2 / %) / P1 / %.

[0152] (2) The survival rates of bacteria YYS-K2 in different gastric fluid environments are shown in Table 8 below. According to the data, it can be seen that:

[0153] Bacteria YYS-K2 can survive for 1 hour in a gastric juice pH environment of 2.0, with a survival rate of 91.31%; when treated in a simulated gastric juice environment with a pH value of 2.5 for 1-5 hours, the survival rate of YYS-K2 was 82.71-93.94%; when treated in a simulated gastric juice environment with a pH value of 3.0 for 1-5 hours, the survival rate of YYS-K2 was 86.78-95.41%, indicating that YYS-K2 has a strong tolerance in the artificial simulated gastric juice environment, which provides a good basis for its degradation of ethanol in the gastric juice environment.

[0154] Table 8 Survival rate of YYS-K2 in artificial simulated gastric fluid environment / %

[0155]

[0156] Example 10 Survival rate analysis of fermented mucus lactobacillus YYS-K2 in gastric juice environment containing ethanol

[0157] (2) Survival test process in gastric fluid containing ethanol:

[0158] Prepare an artificial gastric juice + ethanol environment (artificial gastric juice adjusted to pH 2.5 + different concentrations of ethanol), take 1 mL of the treated bacterial solution and put it in 9 mL of artificial gastric juice with a pH value of 2.75, place it in a constant temperature culture at 37°C, take samples at 1, 2, 3, and 4 hours of treatment, take 0.9 mL each time, add 0.1 mL of PI, dye at 37°C for 10 min, take 0.1 ml and put it in 0.9 mL of ultrapure water, and use flow cytometry to detect the total bacterial count P1 / % and the death count P2 / %, and calculate the bacterial survival rate;

[0159] The calculation formula of bacterial survival rate is:

[0160] Bacterial survival rate / %=(P1 / %-P2 / %) / P1 / %.

[0161] The survival rate data of bacteria YYS-K2 in artificial simulated gastric fluid (pH = 2.75) + ethanol environment are shown in Table 9. According to the data, it can be seen that:

[0162] When YYS-K2 was treated in gastric juice with 5% ethanol content for 1-4 hours, the survival rate of YYS-K2 was 77.73-87.01%, which was an extremely high survival rate; when treated in gastric juice environment with 10% ethanol concentration for 1 hour and 2 hours, the survival rates were 81.81% and 29.24% respectively, which was an excellent survival effect.

[0163] Table 9 Survival rate of YYS-K2 in artificial simulated gastric fluid (pH=2.5) + ethanol environment / %

[0164]

[0165] Example 11 Analysis of the viability of fermented mucus lactobacillus YYS-K2 in a simulated artificial pancreatic juice environment

[0166] (1) Take the YYS-K2 bacteria fermented for 24 hours, centrifuge at 8000r / min for 5min to collect the bacteria, add the same volume of physiological saline (0.85%) and mix well for later use; prepare protein pancreatic juice (0.1% pancreatin w / v, 0.15% ox bile), adjust the pH value to 7.5 and 8.0 respectively, filter through 0.22μM microporous filter membrane and set aside, take 1mL of the treated bacterial liquid and add it to 9mL of protein pancreatic juice with different pH values, place it in a constant temperature culture at 37℃, take samples at 3 and 6(h) of treatment, take 0.9mL each time, add 0.1ml PI diluent, stain at 37℃ for 10min, detect the total number of bacteria P1 / % and the mortality rate P2 / % by flow cytometry, and calculate the bacterial survival rate;

[0167] The calculation formula of bacterial survival rate is:

[0168] Bacterial survival rate / %=P1 / %-P2 / %.

[0169] (2) The bacterial survival rate of strain YYS-K2 is shown in Table 10. According to the data, it can be seen that:

[0170] When the bacteria YYS-K2 was treated in a pancreatic juice environment with a pH of 7.5 for 3 and 6 (h), the survival rate was greater than 99%, indicating that it was not affected by the 7.5 pancreatic juice environmental stress; the survival rate in a pancreatic juice environment with a pH of 8.0 was 51.28-58.12%, and it had good pancreatic juice tolerance.

[0171] Table 10 Survival rate of YYS-K2 in artificial pancreatic juice environment / %

[0172]

[0173] The present invention also provides the following application examples of fermented mucus lactobacillus YYS-K2:

[0174] Example 12 Preparation of probiotics by fermentation of Lactobacillus mucilaginosus YYS-K2

[0175] The fermented mucus lactobacillus YYS-K2 is inoculated into a culture medium, such as an MRS culture medium, and cultured at 0-38°C for more than 15 hours, and the bacterial cells are collected by centrifugation, and resuspended in, for example, physiological saline or PBS buffer to prepare a liquid bacterial preparation containing the fermented mucus lactobacillus YYS-K2. Optionally, the fermented mucus lactobacillus YYS-K2 bacterial cells are resuspended in a cell protectant and a carrier, and freeze-dried to obtain a solid bacterial powder preparation containing the fermented mucus lactobacillus YYS-K2.

[0176] Optionally, fermented Lactobacillus mucus YYS-K2 can be used as a raw material component in alcohol-resolving products, antioxidant products, and weight-loss (inhibiting α-amylase) products (such as foods and health products), and fermented Lactobacillus mucus YYS-K2 can be present in the product in the form of a liquid or solid preparation; further, fermented Lactobacillus mucus YYS-K2 can be used as a raw material component and combined with commonly used alcohol-resolving foods, antioxidant foods, and weight-loss food substances in foods or health foods to prepare foods, health foods or medicines.

[0177] Example 13 Fermentation of Lactobacillus mucilaginosus YYS-K2 to prepare fermented food

[0178] The method comprises the following steps: preparing fermented mucus lactobacillus YYS-K2 yeast solution, taking various fruits, Chinese herbal medicines, cereal raw materials and various sugars as auxiliary materials, inoculating fermented mucus lactobacillus YYS-K2, fermenting for a certain time under certain temperature conditions (30-38 DEG C) to prepare fermented products, inactivating or not inactivating the fermented products, and adding common beverage auxiliary materials to prepare fermented foods after diluting the original solution or diluting the fermented products in different proportions.

[0179] According to the results of the above examples, the fermented mucus Lactobacillus YYS-K2 provided by the present invention has the following properties and effects:

[0180] a. Carbon sources such as cellobiose, maltose, salicin, sucrose, and lactose can be used.

[0181] b. It can survive for 1 hour in a gastric juice environment with a pH of 2.0, with a survival rate as high as 91.31 (%). It can survive for at least 5 hours in an artificial simulated gastric juice environment with a pH of 2.5-3.0, with a survival rate between 82.71-95.41 (%).

[0182] c. When treated in a gastric juice environment containing 5% (v / v) ethanol for 0-4 hours, the survival rate was between 77.73-87.01 (%), and when treated in a gastric juice environment containing 10% (v / v) ethanol for 0-2 hours, the survival rate was between 29.24-81.81%.

[0183] d. After being treated in a pancreatic juice environment with a pH of 7.5 for 6 hours, the survival rate was as high as 99.81%. After being treated in a pancreatic juice environment with a pH of 8.0 for 6 hours, the survival rate was greater than 51%.

[0184] e. Positive growth can be achieved in the MRS medium stress environment with an ethanol concentration of up to 24% (v / v). After 36 hours of fermentation, the bacterial OD 600nm Values ​​greater than 0.5.

[0185] f. It can survive for 2 hours in a stress environment with an ethanol concentration of up to 25-45(%) (v / v), with a survival rate of 8.32-26.23(%).

[0186] g. ADH enzyme can be produced. When the ADH enzyme is treated in the gastric fluid environment for 0-2 hours, its enzyme activity is not affected by the gastric fluid environment.

[0187] h. It has good ethanol degradation ability, can significantly inhibit α-amylase activity, has significant OH and DPPH free radical scavenging ability, and has good reducing ability.

[0188] i. Bacteria YYS-K2 is isolated from daily food and has high food safety. It can be used as a weight loss, antioxidant, and hangover product in ordinary food, health food, and medicine, and has broad application prospects.

[0189] In summary, compared with the prior art, the fermented mucus Lactobacillus YYS-K2 provided by the present invention has the following beneficial effects:

[0190] The fermented mucus lactobacillus YYS-K2 can produce ethanol dehydrogenase (ADH), has good ethanol tolerance and degradation ability, can grow in high-concentration ethanol culture medium, and has a significant inhibitory effect on α-amylase inhibition, has significant OH and DPPH free radical scavenging ability, and has good reducing ability. The bacteria provides a new probiotic source for the development of functional products and has important application value, such as:

[0191] (1) Fermented Lactobacillus mucilaginosus YYS-K2 can be used as a raw material component of the composition to prepare a composition with the above functions; wherein the composition includes but is not limited to microbial preparations, foods, health products or medicines.

[0192] The bacterial species present in the composition include, but are not limited to, one or more combinations of non-inactivated fermented Lactobacillus mucoides YYS-K2, inactivated fermented Lactobacillus mucoides YYS-K2, metabolites of fermented Lactobacillus mucoides YYS-K2 strains, and freeze-dried strains of fermented Lactobacillus mucoides YYS-K2. Preferably, in the composition, the number of fermented Lactobacillus mucoides YYS-K2 is ≥ 1×10 6 CFU / mL or ≥1×10 6 CFU / g. Further preferably, the number of the fermented mucus lactobacillus YYS-K2 is ≥1×10 8 CFU / mL or ≥1×10 8 CFU / g.

[0193] (2) Various plants (such as fruits, Chinese herbal medicines, grains, etc.) can be used as raw materials, combined with various ingredients, inoculated with fermented Lactobacillus mucilaginosus YYS-K2 for fermentation to prepare fermented products, which can be used to prepare products with alcohol sobering, anti-oxidation or weight loss functions;

[0194] The fermentation raw materials can be various types of plant fermentation raw materials commonly used, and the auxiliary materials can also use existing conventional auxiliary materials, including but not limited to the above-mentioned scheme selection. The fermented product is not limited to being used for preparing fermented food, but can also be a health product, medicine and other products;

[0195] In summary, the fermented Lactobacillus mucilaginosus YYS-K2 and / or its fermented product can be applied to functional products including at least one of the following functions according to their characteristics:

[0196] (1) Significantly reduce ethanol levels;

[0197] (2) Producing ADH enzyme;

[0198] (3) Significant free radical scavenging DPPH level;

[0199] (4) Significant scavenging of OH free radicals;

[0200] (5) Have significant reduction capabilities;

[0201] (6) It has a significant inhibitory effect on the activity of α-amylase.

[0202] Among them, products with the above-mentioned functions (1)-(6) include but are not limited to alcohol sobering products, antioxidant products and weight loss (inhibition of α-amylase) functional products, and have obvious effects such as alcohol sobering, antioxidant, and weight loss; they can also be products with other obvious effects generated based on the functions of (1)-(6); wherein the functional products include but are not limited to food, health products or medicines.

[0203] It should be noted that:

[0204] (1) Definition:

[0205] The term "food" as used herein is broad and includes food and drink for humans. In certain embodiments, the food product is suitable for and designed for human consumption. The present application can be used to prepare solid preparations such as powders and tablets, and can also be dispersed in liquids to prepare liquid preparations and other preparations suitable for oral administration to humans, including but not limited to powders.

[0206] The composition includes but is not limited to microbial preparations, foods, health products, and medicines. The composition containing fermented mucus lactobacillus YYS-K2 can be used in other forms of products.

[0207] The fermented mucus lactobacillus YYS-K2 may exist in the composition in forms including but not limited to non-inactivated bacteria, inactivated bacteria, metabolites, freeze-dried strains, etc. It is expected that the fermented mucus lactobacillus YYS-K2 may also exist in the composition in other forms.

[0208] The functional product includes the above-mentioned effects of degrading ethanol, producing ADH enzyme, scavenging free radicals DPPH, scavenging OH free radicals, having reducing ability, inhibiting α-amylase enzyme activity, etc. According to the above-mentioned effects, the manifest effects of its application to the functional product are hangover, anti-oxidation and weight loss; because it has the above-mentioned effects of degrading ethanol, producing ADH enzyme, scavenging free radicals DPPH, scavenging OH free radicals, having reducing ability, inhibiting α-amylase enzyme activity, etc., the effects of its application products include but are not limited to hangover, anti-oxidation and weight loss, and other effects on the human body that can be achieved through the above-mentioned effects are all acceptable.

[0209] (2) Related prior art means or prior art terms involved in this application:

[0210] "OD" is the abbreviation of optical density, also known as absorbance. When light passes through the object, the energy difference before and after is the energy absorbed by the object. At a specific wavelength, there is a quantitative relationship between the concentration of the same object and the absorbed energy. This quantitative relationship can be used to determine the concentration of the object. x" is the optical density value measured when the wavelength is set to Xnm. It is a standard indicator for tracking the density of microorganisms in liquid cultures and is usually used to indicate bacterial cell density. Among them, the "OD" value determination method is a prior art, and its principles and methods are not repeated here.

[0211] "DPPH" is the free radical 2,2-biphenyl-1-picrylhydrazyl, and "OH" is the free radical hydroxyl.

[0212] The use of flow cytometry to determine the total bacterial count P1 and the number of dead bacteria P2 is an existing technology, and its principles and methods will not be repeated here.

[0213] The method of using an alcohol meter to test the alcohol content and temperature of the distillate and calculating the actual alcohol content of each treatment through the alcohol meter temperature concentration conversion table is a prior art, and its principle and method will not be repeated here.

[0214] The HBI lactic acid bacteria biochemical identification strips are used to analyze carbon source utilization. This is a prior art, and its principles and methods will not be described again here.

[0215] (3) The formulas of the culture media used in the examples are as follows:

[0216] MRS medium (g / L): casein peptone 10, beef extract 10, yeast extract 5, glucose 5, sodium acetate 5, K2HPO4 2, diammonium citrate 2, MgSO 4 .7H 2 O 0.2, MnSO 4 .H 2 O 0.05, Tween 80 1; pH 6.2, solid medium plus 2% agar and 2% CaCO 3 , sterilize at 121℃ for 15min.

[0217] MRS medium with X% ethanol concentration: After sterilizing the above MRS medium, add different amounts of ethanol according to the volume ratio, where X% refers to the ethanol concentration value in the MRS medium.

[0218] Unless otherwise specified, the experimental operation methods involved in the embodiments of the present invention are conventional experimental operation methods in the art, and the reagents or instruments involved can be purchased from regular channels.

[0219] In addition, those skilled in the art should understand that, although there are many problems in the prior art, each embodiment or technical solution of the present invention can be improved in only one or several aspects, without having to solve all the technical problems listed in the prior art or background technology at the same time. Those skilled in the art should understand that the content not mentioned in a claim should not be used as a limitation on the claim.

[0220] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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 fermented mucus lactobacillus ( Limosilactobacillus fermentum )YYS-K2, characterized by: Its deposit number is CGMCC No. 27129.

2. A composition, characterized in that: Contains the fermented mucus lactobacillus YYS-K2 as claimed in claim 1.

3. The composition according to claim 2, characterized in that: The composition is one of a microbial preparation, a food and a health product.

4. The composition according to claim 2, characterized in that: The composition comprises one or more combinations of non-inactivated fermented Lactobacillus mucoides YYS-K2 bacteria, inactivated fermented Lactobacillus mucoides YYS-K2 bacteria, metabolites of fermented Lactobacillus mucoides YYS-K2 strains, and freeze-dried fermented Lactobacillus mucoides YYS-K2 strains.

5. A fermented product, characterized in that: The fermented lactobacillus YYS-K2 is obtained by fermentation as claimed in claim 1.

6. Application of fermented Lactobacillus mucilaginosus YYS-K2 and / or its fermented product in the preparation of functional products, characterized in that: The fermented mucus lactobacillus YYS-K2 is the fermented mucus lactobacillus YYS-K2 as claimed in claim 1; and the functional product is a food or a health product.

7. The use according to claim 6, characterized in that: The functional product includes at least one of the following functions: (1) Degradation of ethanol; (2) Producing ADH enzyme; (3) Remove the free radical DPPH; (4) Remove OH free radicals; (5) Having the ability to restore; (6) Inhibit α-amylase activity.

8. The use according to claim 6, characterized in that: The functional products are hangover sobering products, antioxidant products and weight loss functional products.

9. Use of the fermented mucus Lactobacillus YYS-K2 according to claim 1 or the composition according to any one of claims 2 to 4 in the preparation of fermented food.

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