CN115191615B - Lactobacillus bacteriocin with intestinal microecology regulating function - Google Patents
Lactobacillus bacteriocin with intestinal microecology regulating function Download PDFInfo
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- CN115191615B CN115191615B CN202210540829.5A CN202210540829A CN115191615B CN 115191615 B CN115191615 B CN 115191615B CN 202210540829 A CN202210540829 A CN 202210540829A CN 115191615 B CN115191615 B CN 115191615B
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- 230000001681 protective effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000009711 regulatory function Effects 0.000 description 1
- 229960002477 riboflavin Drugs 0.000 description 1
- 235000019192 riboflavin Nutrition 0.000 description 1
- 239000002151 riboflavin Substances 0.000 description 1
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- 238000000926 separation method Methods 0.000 description 1
- 238000011451 sequencing strategy Methods 0.000 description 1
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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Abstract
The invention discloses a lactobacillus bacteriocin with intestinal microecology regulating function, which comprises the following components: the lactobacillus Class IIb bacteriocin plantarciin NC8. The invention also provides application of the lactobacillus Class IIb bacteriocin plant NC8 in regulating intestinal flora of human bodies, wherein the lactobacillus bacteriocin plant NC8 can increase abundance of the human intestinal pralidoxime Lei Wote bacteria_9 (prevotella_9), bifidobacterium (Bifidobacterium), shi He Aixi bacteria (Escherichia-Shigella) and Mi Suoke bacteria (Mitsuokella) on a 'genus' level.
Description
Technical Field
The invention belongs to the technical field of food biology, and particularly relates to a method for regulating intestinal flora of a human body by using bacteriocin of lactobacillus.
Background
The human gastrointestinal tract (GIT) has a complex community of 100 trillion microorganisms including bacteria, fungi, archaebacteria and virus-like particles, collectively referred to as intestinal flora. The microbial diversity in the human intestinal tract develops with maturation of feeding and eating patterns. By the age of 3-5 years, the microbiota resembles an adult, and the intestinal flora is a micro-ecosystem, complex and dense. Intestinal flora can colonize intestinal mucosa to form a natural protective barrier. Through the fermentation process, the anaerobic enterobacteria metabolize the substrate to form end products, such as organic acids and gases. This anaerobic metabolism has a positive effect on the daily energy demand of the intestine and the homeostasis in the intestine. Ideally, a human host and its complex intestinal flora live in harmony with a state that promotes physiological elasticity. However, dysbiosis can lead to challenges such as medication, infection, aging, lifestyle, surgery, and malnutrition. Therapeutic studies in recent years have shown that many intestinal diseases are treated with antibiotics. However, antibiotic resistance leads to an increase in the failure rate of infectious disease treatment, and early life exposure to antibiotics has a long-term effect on intestinal homeostasis and epithelial barrier function, and antibiotics disrupt the expression of intestinal antibacterial proteins, mucins, and fibronectin (TJPs). However, few new antibacterial agents are currently being developed, and few antibacterial agents are available that can treat gram-negative intestinal bacteria.
Lactic acid bacteria are used as main flora in intestinal tract, and can produce lactic acid, acetic acid and bacteriocin during fermentation, wherein the bacteriocin has inhibiting effect on pathogenic bacteria such as Escherichia coli, staphylococcus aureus, salmonella, etc. Some lactobacillus species can compete for intestinal niches, metabolize to produce lactic acid, bacteriocin and the like by being planted in the intestinal tract, so that invasion and planting of pathogenic bacteria are inhibited, intestinal immune function is regulated, and barrier function of the intestinal tract is improved.
Lactic acid bacteria can improve intestinal environment to promote host health, bacteriocins are important factors for the lactic acid bacteria to play a probiotic role, and potential therapeutic application of the lactic acid bacteria in human and veterinary medicines represents a relatively new field in research. The ability of lactic acid bacteria to produce bacteriocins is a common feature of many bacteria associated with a complex natural ecosystem and may decisively influence the stability of their microbial population. Bacteriocins are antimicrobial peptides produced by microorganisms that help to promote the defensive mechanisms of the microorganisms themselves. These peptides are generally classified based on their structural features into class IV, common bacteriocins are class I and class II bacteriocins, class I bacteriocins generally include posttranslationally introduced small peptides with unusual amino acids (< 5 kDa), such as lanthionine and β -methyllanthionine, class II bacteriocins are generally synthesized and processed in precursor form (< 10 kDa), and include bacteriocins consisting of two peptides (class IIb), such as PLNC8 αβ (PLNC 8). Bacteriocin Plantarcicin NC8 (PLNC 8) is a kind of dipeptide bacteriocin with broad-spectrum antibacterial property, has inhibition effect on most gram-positive bacteria, few gram-negative bacteria and partial fungi, and the total antibacterial activity of PLNC8 is realized by the complementary effect of PLNC8 alpha and beta in a molar ratio of 1:16.
At present, only Nisin (Nisin) is a bacteriocin which is allowed to be used as a food additive worldwide, and has no inhibition effect on intestinal pathogens such as gram-negative bacteria escherichia coli and the like because only gram-positive pathogens can be inhibited, so that the application of Nisin is limited. Therefore, the discovery of new bacteriocins with bacteriostasis to enteropathogenic bacteria such as salmonella, escherichia coli and the like is urgent.
The fifteenth annual meeting of the Chinese food science and technology (2018) published in plant NC8 antibacterial mode and action target research reports the following: plantarcicin NC8 is Class IIb bacteriocin and consists of two oligopeptides PLNC8 alpha and PLNC8 beta. Plantarciin NC8 is capable of inhibiting a wide variety of gram-positive and gram-negative bacteria, among which micrococcus luteus is most susceptible and has a minimum inhibitory concentration MIC of 800. Mu.M. Is sensitive to alpha-chymotrypsin, trypsin, pepsin and papain, but is insensitive to lysozyme, lipase, alpha-amylase, ribonuclease a and proteinase K. Experimental results show that Lipid II is not an action target of bacteriocin plant apicin NC8. However, the article does not show any correlation of the lactobacillin plantarciin NC8 with human intestinal flora.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a new application of the lactobacillus Class IIb bacteriocin plant NC8, namely, the regulation effect of the lactobacillus bacteriocin plant NC8 on microecology of human intestinal tracts (human intestinal tracts simulated in vitro).
In order to solve the technical problems, the invention provides a lactic acid bacteria bacteriocin with intestinal microecology regulating function: is lactobacillus Class IIb bacteriocin plantarciin NC8.
The invention also provides application of the lactobacillus Class IIb bacteriocin plantarciin NC8 in regulating intestinal flora of human bodies. That is, the lactic acid bacteria bacteriocin NC8 has a regulating effect on the micro-ecology of human intestinal tracts (human intestinal tracts simulated in vitro).
As an improvement of the application of the invention: the lactic acid bacteriocin plant NC8 increases the abundance of Proteus Lei Wote _9 (Prevolella_9), bifidobacterium (Bifidobacterium), shigella He Aixi (Escherichia-Shigella) and Mitsukelela Mi Suoke (Mitsukelela) in the human intestinal tract at the "genus" level.
As a further improvement of the application of the invention: the Lactobacillus bacteriocin NC8 can promote the growth of bifidobacteria and Lactobacillus in human intestinal tract, thereby promoting the metabolism of intestinal flora to produce short chain fatty acid.
As a further improvement of the application of the invention: aiming at different intestinal type people, the microbial flora has different regulating effects on human intestinal tracts (in-vitro simulated human intestinal tracts).
As a further improvement of the application of the invention:
1. when the test intestinal type (intestinal type of test volunteers) is ET B intestinal type:
the numbers of bifidobacteria and lactobacilli increased for NC83 and NC830 relative to F;
both NC83 and NC830 were reduced relative to group F with respect to the number of kola species;
both NC83 and NC830 were reduced relative to group F in terms of the number of blautia;
description:
bifidobacteria and lactobacillus are beneficial microorganisms in human intestinal tracts, and have important roles in inhibiting the growth of harmful bacteria of human bodies, resisting pathogenic bacteria infection, synthesizing vitamins required by human bodies, promoting the absorption of minerals by human bodies, stimulating the immune system of human bodies, improving disease resistance and the like;
it has now been found that the body weight and fat mass of humans are inversely related to the abundance of kola species, and thus the present invention can help predict the risk of obesity;
it has been found that consumption of the genus blautia in the obese pediatric microbiota exacerbates intestinal inflammation and metabolic phenotype;
2. when the test intestinal type (intestinal type of test volunteers) is ET P intestinal type:
the numbers of bifidobacteria and kola were increased for NC83 and NC830 relative to F;
both NC83 and NC830 were reduced relative to group F in terms of the number of blautia;
the group F is a natural fermentation group, the group NC83 is a fermentation group inoculated with PLNC8 at a low concentration (3. Mu.M), and the group NC830 is a fermentation group inoculated with PLNC8 at a high concentration (30. Mu.M).
As a further improvement of the application of the invention:
the lactobacillus bacteriocin plant NC8 is prepared by mixing PLNC8 alpha and PLNC8 beta according to the equimolar ratio.
The bacteriocin plant NC8 has wide bacteriostasis spectrum and good inhibition effect on enteropathogenic bacteria such as salmonella enteritidis, escherichia coli and the like; this is a known technique, and PLNC8 α and PLNC8 β can be synthesized by Shanghai Jier corporation using a solid phase synthesis method.
The invention utilizes an in-vitro intestinal canal simulation experiment to explore the effect of PLNC8 on intestinal canal flora, and comprises the following steps:
(1) eight healthy volunteers between 20-40 years of age were selected and provided with fresh faeces. Fecal suspension was measured in YCFA medium at 10% inoculation in anaerobic work station (Do Whitley Scientific), V/V.
(2) The low concentration bacteriocin PLNC8 (NC 83, 3. Mu.M) and the high concentration bacteriocin PLNC8 (NC 830, 30. Mu.M) were added to the YCFA medium to which the fecal suspension was added as experimental groups; YCFA medium with fecal suspension (group F) was added as a blank. After culturing in a constant temperature incubator (SPX-150B-Z, shanghai Boqing Co., ltd.) at 37deg.C for 24 hr, collecting fecal fermentation broth, centrifuging at 8000rpm for 5min, collecting supernatant, storing in a new EP tube, storing in a refrigerator at-20deg.C, and retaining the rest precipitate in the original EP tube for subsequent DNA extraction and flora structure analysis
(3) And (3) extracting bacterial DNA from the sediment of the fecal fermentation broth obtained by centrifugation in the step (2), and immediately sending the sediment to Beijing Baimeike biotechnology Co., ltd for further analysis after the bacterial DNA is extracted and is well stored by dry ice.
(4) After the samples in the step (3) are sent to Beijing Baimai biotechnology Co., ltd, the company uses an Illumina sequencing platform to perform high throughput sequencing on the microbial community in 3 groups (F groups (natural fermentation groups), NC83 (low concentration PLNC8 inoculated fermentation groups), NC830 (high concentration PLNC8 inoculated fermentation groups) and 8 tested volunteer fecal samples in each group, and determines the change of fecal flora and the intestinal type of the microbial community.
(5) The supernatant of the fermentation sample obtained in the step (2) was subjected to chromatography using a DB-FFAP gas chromatography column (GC plus 2010, shimadzu) and a capillary column FFAP (InterCap, 0.25 mm. Times.30 m. Times.0.25 μm) having a specification of 0.32 mm. Times.30 m. Times.0.5 μm with crotonic acid as a reference, and the content of short chain fatty acids such as propionic acid, butyric acid, acetic acid, valeric acid, isobutyric acid in the fermentation broth was measured.
(6) Combining the sequencing result of the microbial community in the fecal sample obtained in the step (4) with the result of the short-chain fatty acid content obtained in the step (5) to obtain the influence condition of the bacteriocin PLNC8 on the microbial abundance and short-chain fatty acid in the intestinal tract of the human body, thereby further determining the regulation effect of the bacteriocin PLNC8 on the intestinal tract of the human body.
In the invention, an in-vitro intestinal canal simulation experiment is carried out by using the artificially synthesized bacteriocin plant arcin NC8, and the prepared bacteriocin plant arcin NC8 has the function of improving intestinal canal flora (namely, is beneficial to the regulation of human intestinal canal flora).
The artificially synthesized bacteriocin plant NC8 is used for promoting the growth of bifidobacteria (Bifidobacterium) and Lactobacillus (Lactobacillus) in intestinal tracts, thereby promoting the metabolism of intestinal flora to produce short chain fatty acids.
The bacteriocin plant NC8 synthesized by the invention has very remarkable effect on intestinal tract regulation (intestinal flora and flora metabolites), has remarkable promotion effect on the growth of bifidobacteria (bifidobacteria) in the ET P intestinal type and the ET B intestinal type and Lactobacillus (Lactobacillus) in the ET B intestinal type, and the bifidobacteria have a certain promotion effect on inhibiting the growth of pathogenic bacteria and promoting the metabolism of intestinal flora of human bodies to generate Short Chain Fatty Acids (SCFAs) as species with the best promotion effect and remarkable difference in both intestinal types.
In conclusion, the invention utilizes the synthesized bacteriocin plant NC8 to carry out in-vitro intestinal canal simulation experiments, and discovers that the bacteriocin plant NC8 is beneficial to promoting the growth of beneficial intestinal canal bacteria and realizes the function of improving the health of the intestinal canal.
Drawings
The following describes the embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 relative abundance of intestinal microbiota at portal and genus levels in different broths and different intestinal types;
in fig. 1:
A-B: ET B is composed of flora at the phylum and genus level;
C-D: ET P is composed of flora at the phylum and genus level.
In A-D:
f represents the average level of 8 replicates of the fecal-like group;
NC83 represents the average level of 8 replicates of a PLNC8 vaccinated fermentation group at a concentration of 3 μm;
NC830 represents the average level of 8 replicates of the PLNC8 vaccinated fermentation group at a concentration of 30 μm;
description: in A-D: there is a one-to-one correspondence between each small legend on the right and layering of each bar graph from top to bottom.
Specifically:
FIG. 1 shows the relative abundance of "phylum", "genus" flora after fermentation in an in vitro intestinal model, and results obtained for the exemplary three methods of the present embodiments. When the population abundance of eight volunteers tested was analyzed at the "genus" level, four of them were found to be intestinal type 1 and the remaining four subjects were intestinal type 2. Wherein intestinal form 1, which may be denoted ET B, is the best indicator group; intestinal form 2, which may also be denoted ETP, is driven by Prevotella, whose abundance is generally inversely proportional to the abundance of bacteroides sp. The ET B enteric form is mainly involved in the synthesis of biotin, riboflavin, pantothenic acid and ascorbic acid, while the ET P enteric form is mainly involved in the synthesis of thiamine and folic acid. The composition of the colonies at the system level and the genus level for group F and group PLNC8 are shown in FIG. 1 (A-B) for group ET B and FIG. 1 (C-D) for group ET P. It is evident that the dominant flora of ET B is bacterioides, whereas the dominant flora of ET P is Prevotella. At the gating level, the overall levels of bacteroides, actinomycetes and proteobacteria were increased in group B compared to group F, while the overall levels of Verrucous, firmicutes, streptococcus were decreased. At the genus level, actinomycetes (actinomycetes), bacteroides (bacterioides), bifidobacteria (bifidobacteria), megamonas (Megamonas) and shi He Shiai (Escherichia-Shigella) of ET B were significantly increased over group F (p < 0.05). In addition, PLNC8 increased the abundance of P.sup. Lei Wote _9 (Prevotella_9), bifidobacterium (Bifidobacterium), shigella He Aixi (Escherichia-Shigella) and Mi Suoke (Mitsuokella) in group B compared to group F.
FIG. 2 shows the variation of bifidobacterium, lactobacillus, blueTorilis and kola after ET B intestinal in vitro intestinal model fermentation;
in fig. 2:
a is the growth of bifidobacteria;
b is the growth condition of lactobacillus;
c is the growth of the genus kola;
d is the growth of BlueTorula;
in A-D:
f represents the average level of 8 replicates of the fecal-like group;
NC83 represents the average level of 8 replicates of a PLNC8 vaccinated fermentation group at a concentration of 3 μm;
NC830 represents the average level of 8 replicates of the PLNC8 vaccinated fermentation group at a concentration of 30 μm.
FIG. 3 shows the variation of Bifidobacterium, bluestone's bacteria and kola's bacteria after ET P intestinal in vitro intestinal model fermentation;
in fig. 3:
a is the growth of bifidobacteria;
b is the growth condition of the Bruetzia;
c is the growth of the genus kola;
in A-C:
f represents the average level of 8 replicates of the fecal-like group;
NC83 represents the average level of 8 replicates of a PLNC8 vaccinated fermentation group at a concentration of 3 μm;
NC830 represents the average level of 8 replicates of the PLNC8 vaccinated fermentation group at a concentration of 30 μm.
FIG. 4 is a PCoA analysis;
in fig. 4:
PCoA analysis of ET B intestinal volunteers;
b is PCoA analysis of ET P enteric volunteers;
in A-B:
f represents the average level of 8 replicates of the fecal-like group;
NC83 represents the average level of 8 replicates of a PLNC8 vaccinated fermentation group at a concentration of 3 μm;
NC830 represents the average level of 8 replicates of the PLNC8 vaccinated fermentation group at a concentration of 30 μm.
Specifically:
FIG. 4 shows Beta diversity-PCoA analysis of intestinal flora in different intestinal forms to demonstrate intestinal flora distribution. Intestinal microorganisms and their metabolites regulate human health. They are bridges between diet and host, with metabolic functions not possessed by the human body. To clarify the effect of F and NC8 on intestinal microbiologic diversity, PCoA was used to rank the eigenvalues and eigenvectors, and the eigenvalues of the first few bits were selected. Fig. 4A and 4B show PCoA scores for ET B and ET P, respectively. On the generic level, there was a clear separation between the metabolites of group F and NC8 (fig. 4). There was a significant difference between group F and group NC8, but no significant difference between group NC83 and group NC 830. However, ET P in groups F, NC83 and NC830 were significantly different (P < 0.05), indicating that they differ in intestinal microbiota diversity.
FIG. 5 shows the in vitro fermentation produced SCFAs content;
in fig. 5:
A-E are the contents of total acid, propionic acid, butyric acid, acetic acid and isovaleric acid produced by in vitro fermentation of the ET B intestinal volunteers; F-J are the contents of total acids, propionic acid, butyric acid, acetic acid, isovaleric acid produced by the in vitro fermentation of ET P intestinal volunteers. Fermentation time is 24 hours, the horizontal coordinates are different culture mediums, and the vertical coordinates are concentrations;
in A to J:
f represents the average level of 8 replicates of the fecal-like group;
NC83 represents the average level of 8 replicates of a PLNC8 vaccinated fermentation group at a concentration of 3 μm;
NC830 represents the average level of 8 replicates of the PLNC8 vaccinated fermentation group at a concentration of 30 μm.
Specifically:
FIG. 5 is the effect of PLNC8 on the metabolic production of short chain fatty acids by the intestinal mimicking system flora in vitro. SCFAs are the main fermentation products of intestinal anaerobic bacteria metabolism, including acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, etc., and have positive effects on the human body. As shown in FIG. 5, after 24 hours of fermentation, the SCFAs content did not accumulate with the increase of fermentation time. There was no significant difference in SCFAs for ET B (FIGS. 5A-E) and ET P (FIGS. 5F-J). For ET B, NC83 group had a higher isovaleric acid content than group F and NC 830. And the acetic acid content of NC830 was higher than that of F and NC 83. While in ET P there was no significant difference in SCFAs levels in any two of the three groups.
Detailed Description
The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:
the lactobacillus bacteriocin NC8 of the invention is prepared from PLNC8 alpha and PLNC8 beta according to the following ratio of 1:1 in a molar ratio.
1. Setting an experiment system:
calcium chloride solution: accurately weighing 1.25g of calcium chloride powder, and fixing the volume of ultrapure water to 1L.
Magnesium sulfate solution: accurately weighing 0.09g of magnesium sulfate powder, and fixing the volume of ultrapure water to 1L.
Resazurin solution: accurately weighing 1.25g of resazurin powder, and fixing the volume of ultrapure water to 1L.
Heme solution: accurately weighing 0.50g of heme powder, dissolving in 1mL of 1mol/L NaOH, fixing the volume of ultrapure water to 100mL, and preserving at 4 ℃ in a dark place for later use.
YCFA medium: accurately weighing 10g of peptone, 2.5g of yeast extract, 1g of L-cysteine, 2mL of heme solution, 9g of NaCl, 125 mu L of calcium chloride solution and KH 2 PO 4 0.45g，K 2 HPO 4 0.45g of magnesium sulfate solution 500 mu L is dissolved in 1L of ultrapure water, after the complete dissolution, 1mL of resazurin solution is added, the mixture is boiled until the culture medium turns from red to yellow, the surface of the culture medium is kept anaerobic immediately by nitrogen blowing, the culture medium is packaged into penicillin bottles (5 mL/bottle) by peristaltic pumps, the penicillin bottles are sealed by a gland, and the penicillin bottles are used after autoclaving (121 ℃ for 15 min).
PLNC8 sample group: taking bacteriocin PLNC8 (lactobacillus bacteriocin NC 8) powder, and stabilizing the average value of the minimum bacteriostasis concentration of the bacteriocin PLNC8 to intestinal flora such as escherichia coli, salmonella enteritidis and the like at 3 mu M through the experimental result in the earlier stage of a laboratory. Therefore, the concentration of the solid powder was diluted to 3. Mu.M as a low concentration test group and 30. Mu.M as a high concentration test group.
Fecal bacterial suspension: fecal samples were provided by 8 volunteers (4 men and 4 women), all enrolled in the food microbiology technology key laboratory in Zhejiang province, subjects aged 20-30 years, subjects had no antibiotics, prebiotics, and no gastrointestinal disease within three months prior to sampling, and their fresh feces were taken 2g in a sampler. Fresh feces (0.80 g/person) was weighed from the feces sampler and placed into a 10mL centrifuge tube, then sterilized 0.1mM PBS (ph=6.8) was added to 8mL, vortex shaking was thoroughly mixed, large particles (pore size of the filter screen 80 mesh) were filtered out with a filter screen, and each volunteer was prepared with a single portion of bacterial suspension for use.
And II: group fermentation of human body excrement suspension
Taking 500 mu L of the fecal suspension obtained in the step one, centrifuging at 8000rpm for 5min, taking supernatant, adding 100 mu L of the crotonic metaphosphate solution, placing into a 2mL centrifuge tube, fully mixing, and freezing in a refrigerator at-20 ℃ to be used as an analysis sample before human fecal fermentation for short-chain fatty acid analysis.
The bacterial suspension samples of 8 tested volunteers obtained in the step one were inoculated with 10% (V/V) fecal bacterial suspension of each tested volunteer in three cylinder bottles containing YCFA medium in an anaerobic workstation to prepare three duplicate fecal fermentation samples.
Three replicates of each volunteer were divided into three experimental groups F, NC, NC830, resulting in 8 samples per experimental group, i.e., 8 volunteers each provided one fecal fermentation broth sample. The three groups total 24 samples.
F represents fecal panel: 500 μl of fecal suspension;
NC83 represents the low concentration PLNC8 experimental group: 500. Mu.L of fecal suspension, 5mL of YCFA medium, PLNC8 sample at a final concentration of 3. Mu.M;
NC830 represents the high concentration PLNC8 experimental group: 500. Mu.L of fecal suspension, 5mL of YCFA medium, PLNC8 sample at 30. Mu.M final concentration;
the fermentation samples corresponding to the experimental groups are placed in a constant temperature incubator at 37 ℃ for culturing for 24 hours. And then taking out the sample, vibrating uniformly, opening the bottle cap of the cylinder bottle by using a bottle opener, sucking 500 mu L of fermentation suspension by using a pipetting gun, placing the fermentation suspension in a 2mL centrifuge tube, centrifuging at a speed of 8000rpm for 5min, taking fermentation supernatant, adding 100 mu L of crotonic metaphosphate solution into the fermentation supernatant, vibrating and mixing uniformly, storing the fermentation supernatant in a refrigerator at a temperature of 20 ℃ below zero for 24 hours, and taking the fermentation supernatant as an analysis sample after human excrement fermentation for subsequent short-chain fatty acid analysis, wherein the sediment of the residual fermentation broth is left for subsequent DNA extraction and colony structure analysis, so that 8*3 =24 analysis samples after human excrement fermentation are obtained.
Thirdly,: the extraction method of the fecal suspension fermentation sample DNA and the 16S rDNA sequencing analysis can be specifically operated by referring to the conventional extraction method of the DNA extraction kit as follows:
DNA extraction was performed on the pellet of fecal fermentation broth after centrifugation in example two using the Chejj QI Aamp Power Fecal DNA Kit kit.
The sequencing of the region V3-V4 of the 16S rDNA gene is carried out immediately after the bacterial DNA is extracted (the region V3-V4 of the 16S rDNA gene can be stored by dry ice and sent to Beijing Baimeike biotechnology Co., ltd.) and the sequencing platform is MiSeqPE250 and the sequencing primer is a universal primer. The amplification upstream primer is 338F (ACTCCTACGGGAGGCAGCA), the downstream primer is GGACTACHVGGGTWTCTAAT, the target fragment length is 480bp, and the Miseq-PE250 is adopted as a sequencing strategy. The microbiota of the fecal samples from 3 groups (natural fermentation group indicated as F, low concentration PLNC8 inoculated fermentation group indicated as NC83, high concentration PLNC8 inoculated fermentation group indicated as NC 830) were subjected to high throughput sequencing using an Illumina sequencing platform, and the data obtained was screened, optimized and spliced to analyze the specific composition of each sample (group) at different classification levels (and to examine if there were statistical differences between groups). The difference in flora structure and species associated with the difference between different samples (groups) is further measured by a variety of multivariate statistical analysis tools.
The results obtained are the differences in microbial composition and abundance at the "genus" level in the in vitro fermentation system, as shown in figures 2, 3. There was a significant variation in Bifidobacterium (bifidobacteria), lactobacillus (Lactobacillus), koala (phascolaarcobacterium), blauta (Blautia) between the different dose groups. As shown in fig. 2A and 2B, the numbers of bifidobacteria and lactobacillus in NC83 group and NC830 group in ET B intestinal group are increased relative to the control group, and bifidobacteria and lactobacillus are beneficial microorganisms in human intestinal tract, and have important effects in inhibiting growth of harmful bacteria in human body, resisting infection of pathogenic bacteria, synthesizing vitamins required by human body, promoting absorption of minerals by human body, stimulating human immune system, improving disease resistance and the like. The number of Couloses in FIG. 2C also decreased in both NC83 and NC830 relative to the blank, and it has now been found that the body weight and fat mass of humans are inversely related to the abundance of the genus kola, thus helping to predict obesity risk. The b.brucellosis in experimental groups NC83 and NC830 in fig. 2D were reduced relative to the control group, and it was found that the consumption of b.brucellosis in the obese pediatric microbiota aggravated intestinal inflammation and metabolic phenotype.
In FIG. 3, the numbers of bifidobacteria and kola in the NC83 and NC830 groups in the ET P intestinal type population shown in FIGS. 3A and 3C are both increased relative to the control group, while the ratio of BlueTorilis is decreased relative to the control group in FIG. 3B. As can be seen from fig. 2 and 3, PLNC8 at both concentrations increased the numbers of bifidobacteria and kola in both ET B and ET P intestinal volunteers, and inhibited the growth of the genus blautia.
Figure 4 shows Beta diversity of intestinal flora of volunteers of different intestinal types. For ET B, the Beta diversity of the intestinal microorganisms of the bacteriocin group is very similar, but very dissimilar to control group F. The Beta diversity of the intestinal flora of the ET P intestinal type three groups is not similar and also has obvious differences, which indicates that bacteriocin plantarciin NC8 has an influence on the diversity of intestinal microorganisms. In SCFAs produced in vitro (fig. 5), the three groups did not have any difference, especially in terms of total acid production. However, in ET B intestinal form, the propionic and isovaleric acid content of NC83 was slightly higher than in groups F and NC 830.
Fourth, the method comprises the following steps: detection of short chain fatty acids in fecal suspension fermentation samples by gas chromatography
The content of short chain fatty acid is a product generated by the metabolism of microorganisms in human intestinal tracts, and is an important index for measuring the growth and metabolism conditions of intestinal flora, and the short chain fatty acid comprises acetic acid, propionic acid, butyric acid, isovaleric acid, isobutyric acid and the like. Taking out the sample to be tested (analysis sample before human excrement fermentation and analysis sample after human excrement fermentation) from a refrigerator at-20 ℃, thawing at normal temperature, centrifuging (4 ℃ C., 10000rpm,3 min) after complete thawing, taking the supernatant, and filtering with a 0.22 μm filter membrane. And adding 100 mu L of filtrate of the sample into a gas phase sample bottle, tightly covering with a cover to remove bubbles, loading the sample for analysis, and preserving the rest filtrate at-20 ℃ for later use. Gas chromatography conditions:
chromatographic column: agilentFFAP30 m 0.25mm 0.25 μm;
column temperature: heating to 180deg.C at 75deg.C at 20deg.C/min for 1min, heating to 220deg.C at 50deg.C/min for 1min;
sample inlet temperature: 250 ℃;
sample injection amount: 1.0. Mu.L;
split ratio: 5:1;
carrier gas: high purity nitrogen;
the carrier gas flow rate is kept at 2.5mL/min for 6.5min, and the carrier gas flow rate is increased from 2.8mL/min to 2.8mL/min for 2min; a detector: FID;
temperature: 250 ℃;
tail blowing: 20mL/min;
hydrogen gas: 30mL/min;
air: 300mL/min.
In the ion monitoring mode of mass spectrum, the peak area on the extracted gas chromatographic ion is recorded, and a standard curve is drawn. The specific concentration of each short chain fatty acid component in each sample was then calculated using the peak area ratio and standard curve for short chain fatty acid standard.
As shown in FIG. 5, after 24 hours of fermentation of the fecal sample, the amounts of SCFAs in the two intestinal forms did not increase significantly with the increase in fermentation time. There was no significant difference in SCFAs as shown in FIGS. ET B (FIGS. 5A-E) and ET P (FIGS. 5F-J). But in the group of test volunteers of different intestinal type, it was found that for the ET B intestinal type of test volunteers, as shown in fig. 5D, the NC830 group had higher acetic acid content than the remaining two experimental groups F and NC83 groups at the acetic acid level. At the isovaleric acid level, as shown in fig. 5E, the isovaleric acid content of NC83 was higher than that of the remaining two experimental groups F and NC 830. In the ET P intestinal type subjects, there was no significant difference in SCFAs levels in any two of the three groups.
Finally, it should also be noted that the above list is merely a few specific embodiments of the present invention. Obviously, the invention is not limited to the above embodiments, but many variations are possible. All modifications directly derived or suggested to one skilled in the art from the present disclosure should be considered as being within the scope of the present invention.
Sequence listing
<110> Zhejiang university of Industrial and commercial university
<120> a lactic acid bacteria bacteriocin having intestinal microecological regulatory function
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<213> Artificial sequence (Artificial Sequence)
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Claims (5)
1. The application of lactobacillus Class IIb bacteriocin plantarciin NC8 in preparing a composition for regulating intestinal flora of human body is characterized in that: the lactic acid bacteriocin plant NC8 increases the abundance of Proteus Lei Wote _9 (Prevolella_9), bifidobacterium (Bifidobacterium), shigella He Aixi (Escherichia-Shigella) and Mitsukelela Mi Suoke (Mitsukelela) in the human intestinal tract at the "genus" level.
2. The use according to claim 1, characterized in that: the Lactobacillus bacteriocin NC8 can promote the growth of bifidobacteria and Lactobacillus in human intestinal tract, thereby promoting the metabolism of intestinal flora to produce short chain fatty acid.
3. Use according to claim 1 or 2, characterized in that: aiming at different intestinal type people, the traditional Chinese medicine composition has different regulating effects on intestinal flora of human bodies.
4. A use according to claim 3, characterized in that:
1. when the test intestinal type is ET B intestinal type:
the numbers of bifidobacteria and lactobacilli increased for NC83 and NC830 relative to F;
both NC83 and NC830 were reduced relative to group F with respect to the number of kola species;
both NC83 and NC830 were reduced relative to group F in terms of the number of blautia;
2. when the test intestinal type is ET P intestinal type:
the numbers of bifidobacteria and kola were increased for NC83 and NC830 relative to F;
both NC83 and NC830 were reduced relative to group F in terms of the number of blautia;
the group F is a natural fermentation group, the group NC83 is a fermentation group inoculated with PLNC8 at a low concentration of 3 mu M, and the group NC830 is a fermentation group inoculated with PLNC8 at a high concentration of 30 mu M.
5. Use according to claim 1 or 2, characterized in that:
the lactobacillus bacteriocin plant NC8 is prepared by mixing PLNC8 alpha and PLNC8 beta according to the equimolar ratio.
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| WO2006063428A1 (en) * | 2004-12-17 | 2006-06-22 | Canbiocin Inc. | Lactic acid bacteria for the treatment of food |
| WO2016176729A1 (en) * | 2015-05-01 | 2016-11-10 | Rmit University | Bacteriocin polypeptides and uses thereof |
| CA3096807A1 (en) * | 2018-02-20 | 2019-08-29 | Torbjorn BENGTSSON | Plantaricin nc8.alpha..beta. markedly enhances the effects of antibiotics |
| CN110964655A (en) * | 2018-09-30 | 2020-04-07 | 内蒙古伊利实业集团股份有限公司 | Bifidobacterium lactis BL-99 capable of regulating gastrointestinal flora and application thereof |
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| IT1399793B1 (en) * | 2010-01-12 | 2013-05-03 | Giuliani Spa | PROCEDURE FOR THE PREPARATION OF A BIOMASS INCLUDING PLANTARICINA AND ITS USES IN MEDICAL FIELD. |
| EP3675639A4 (en) * | 2017-08-31 | 2021-09-01 | Syngulon SA | Methods and compositions for making bacteriocins and antimicrobial peptides |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006063428A1 (en) * | 2004-12-17 | 2006-06-22 | Canbiocin Inc. | Lactic acid bacteria for the treatment of food |
| WO2016176729A1 (en) * | 2015-05-01 | 2016-11-10 | Rmit University | Bacteriocin polypeptides and uses thereof |
| CA3096807A1 (en) * | 2018-02-20 | 2019-08-29 | Torbjorn BENGTSSON | Plantaricin nc8.alpha..beta. markedly enhances the effects of antibiotics |
| CN112004548A (en) * | 2018-02-20 | 2020-11-27 | 托尔比约恩·本特松 | The lactobacillus plantarum NC8 alpha beta obviously enhances the effect of antibiotics |
| CN110964655A (en) * | 2018-09-30 | 2020-04-07 | 内蒙古伊利实业集团股份有限公司 | Bifidobacterium lactis BL-99 capable of regulating gastrointestinal flora and application thereof |
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