Application of regulator in regulation of prokaryote methylation level
By regulating the methylation level of prokaryotic DNA, the problem of dairy fermentation failure caused by phage contamination is solved, the bacteria's resistance and survival rate to phages is improved, and a broad-spectrum anti-phage solution is provided.
Patent Information
- Application Number
- CN202310842637.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-07-10
- Publication Date
- 2025-01-10
AI Technical Summary
The prior art is difficult to effectively combat the problem of dairy fermentation failure caused by bacteriophage contamination, especially the lack of solutions for broad-spectrum bacteria.
Regulate the methylation level of prokaryotic DNA through regulators (such as inhibitors and activators of DNA methyltransferase), change the properties of bacteria, improve resistance and survival rate to bacteriophages, and apply it to bacterial culture and fermentation. .
The effective resistance to phages and enhanced bacterial survival rate are achieved, the quality of fermentation products is improved, and a broad-spectrum anti-phage solution is provided.
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Abstract
Description
Technical Field
[0001] The present application relates to the field of microorganisms. Specifically, it relates to the use of a regulator in regulating the methylation level of prokaryotic nucleic acids, and the use of a regulator in preparing a prokaryotic culture medium or food. The present application also relates to a method for regulating the methylation level of prokaryotic nucleic acids, and a method for changing bacterial properties (e.g., improving bacterial resistance to bacteriophages, enhancing bacterial survival rates, and changing bacterial fermentation products). Background Art
[0002] Phages often cause fermentation failures in dairy plants, resulting in huge economic losses. About one-third of the 500 million tons of milk produced each year worldwide is converted into fermented products. Of this, two-thirds of processed milk is fermented by Lactococcus and Candida albicans, and the rest is fermented by Thermomyces lactis and Streptococcus thermophilus. It is estimated that 0.1% to 10% of all milk fermentations are negatively affected by virulent phages. Phage contamination can slow down or even stop the fermentation process of milk. During the fermentation process, the consequences of phage contamination include: changes in product quality (such as taste, flavor and texture), as well as the formation of microbial flora. Phage contamination can also lead to the formation of microbial flora with negative effects during the fermentation process due to delayed lactic acid production. In the most serious cases of phage contamination, the contaminated fermented milk must be discarded.
[0003] Currently, the dairy industry has developed many methods to reduce the impact of phages on its production systems. These methods include: a) using multiple strains to ferment milk to limit the impact of single phage contamination; b) carefully controlling hygiene and using cleaning agents that are effective against phages; c) developing phage-resistant strains through internal screening and selection processes; d) using genetic modification to design strains with multiple phage resistance; e) establishing a set of strains with different phage susceptibilities that can be regularly interchanged to produce products with the same characteristics while minimizing the chance of phage damage. Each of these methods has certain advantages, but none of them provides a solution for all phages. In the case of probiotics, each strain has its own unique health benefits, and the methods cannot be universally used for any probiotic strain.
[0004] Therefore, it is particularly important to provide a technical solution for fighting bacteriophages that can be applied to a wide spectrum of bacteria. Summary of the invention
[0005] The applicant of the present application has experimentally confirmed that the same regulator has significant differences in the ability to regulate DNA methylation levels in eukaryotes and prokaryotes. Further, the applicant has experimentally obtained a regulator that can effectively regulate the DNA methylation level of prokaryotes, and by exposing Lactobacillus acidophilus to different regulators, 385 differentially expressed genes have been obtained. Applying these regulators to the cultivation and / or fermentation of bacteria will help change the properties of bacteria, particularly change the fermentation products of bacteria, improve the ability of bacteria to resist bacteriophages, improve the survival rate of bacteria after freeze-drying, etc. Therefore, these regulators have strong application potential in the cultivation and / or fermentation of bacteria, and in the preparation of food containing bacteria.
[0006] Therefore, in a first aspect, the present application provides a use of a regulator in regulating the methylation level of prokaryotic nucleic acid, wherein the regulator is selected from an inhibitor of DNA methyltransferase (DNMT), an activator of DNMT, or any combination thereof.
[0007] In certain embodiments, the nucleic acid is DNA and / or RNA.
[0008] In certain embodiments, the methylation level is selected from: 5 C modification level, m 6 A modification level, m 7 G modification level, m 3 C modification level, or any combination thereof.
[0009] In certain embodiments, the modulator is used to modulate the mRNA expression of prokaryotic DNA. 5 C modification level, m 6 A modification level, m 3 C modification level, and / or m 7 G modification level.
[0010] In certain embodiments, the prokaryotic organism is selected from bacteria (eg, probiotics), actinomycetes, rickettsia, chlamydia, mycoplasma, or any combination thereof.
[0011] In certain embodiments, the bacteria is selected from the genus Lactobacillus, Bifidobacterium, Bacillus, Propionibacterium, Streptococcus, Lactococcus, Pediococcus, Enterococcus, Staphylococcus, or any combination thereof.
[0012] In certain embodiments, the bacteria is selected from Lactobacillus acidophilus, Escherichia coli, Bifidobacterum lactis, Lactobacillus infantis, Lactobacillus paracasei, or any combination thereof.
[0013] In certain embodiments, the modulator is selected from caffeic acid, epigallocatechin gallate (EGCG), benzoic acid, genistein, anacardic acid, sodium butyrate, diallyl disulfide, DL-Sulphoraphane, resveratrol, L-methionine, or any combination thereof.
[0014] In certain embodiments, the DNMT inhibitor is sodium butyrate.
[0015] In certain embodiments, the DNMT activator is selected from genistein, diallyl disulfide, DL-Sulphoraphane, epigallocatechin gallate (EGCG), or any combination thereof.
[0016] In certain embodiments, the CAS number of caffeic acid is 331-39-5. In certain embodiments, the CAS number of epigallocatechin gallate is 989-51-5. In certain embodiments, the CAS number of benzoic acid is 65-85-0. In certain embodiments, the CAS number of genistein is 446-72-0. In certain embodiments, the CAS number of anacardic acid is 16611-84-0. In certain embodiments, the CAS number of sodium butyrate is 156-54-7. In certain embodiments, the CAS number of diallyl disulfide is 2179-57-9. In certain embodiments, the CAS number of sulforaphane is 4478-93-7. In certain embodiments, the CAS number of resveratrol is 501-36-0. In certain embodiments, the CAS number of L-methionine is 63-68-3.
[0017] In certain embodiments, the modulator is food safe.
[0018] In certain embodiments, the information of the above-mentioned modulator is as follows:
[0019]
[0020]
[0021] In certain embodiments, the modulator is capable of increasing the m 5 C modification level. In certain embodiments, the modulator is selected from DL-Sulphoraphane, Genistein, Diallyl Disulfide, Anacardic Acid, or any combination thereof.
[0022] In certain embodiments, the modulator is capable of reducing the m 5 C modification level. In certain embodiments, the modulator is selected from Sodium Butyrate, Caffeic Acid, Benzoic Acid, or any combination thereof.
[0023] In certain embodiments, the modulator is capable of increasing the m 6 A modification level. In certain embodiments, the modulator is selected from DL-Sulphoraphane, epigallocatechin gallate (EGCG), genistein, anacardic acid, or any combination thereof.
[0024] In certain embodiments, the modulator is capable of reducing the m 6 A modification level. In certain embodiments, the modulator is selected from sodium butyrate, caffeic acid, or any combination thereof.
[0025] On the other hand, the present application provides a prokaryotic culture medium, which contains a regulator capable of regulating the methylation level of prokaryotic nucleic acid, wherein the regulator is selected from an inhibitor of DNA methyltransferase (DNMT), an activator of DNMT, or any combination thereof.
[0026] In certain embodiments, the prokaryotic organism is selected from bacteria (eg, probiotics), actinomycetes, rickettsia, chlamydia, mycoplasma, or any combination thereof.
[0027] In certain embodiments, the bacteria is selected from the genus Lactobacillus, Bifidobacterium, Bacillus, Propionibacterium, Streptococcus, Lactococcus, Pediococcus, Enterococcus, Staphylococcus, or any combination thereof.
[0028] In certain embodiments, the bacteria is selected from Lactobacillus acidophilus, Escherichia coli, Bifidobacterum lactis, Lactobacillus infantis, Lactobacillus paracasei, or any combination thereof.
[0029] In certain embodiments, the culture medium is in solid form, liquid form, or semi-solid form.
[0030] In certain embodiments, the culture medium further comprises: ingredients for providing nutrients, ingredients for fermentation, prebiotics, growth factors, water, or any combination thereof.
[0031] In certain embodiments, the nucleic acid is as previously defined.
[0032] In certain embodiments, the methylation is as previously defined.
[0033] In certain embodiments, the modulator is as previously defined.
[0034] On the other hand, the present application provides a use of a regulator in preparing a prokaryotic culture medium; wherein the regulator is selected from an inhibitor of DNA methyltransferase (DNMT), an activator of DNMT, or any combination thereof.
[0035] In certain embodiments, the culture medium is used to regulate the growth, reproduction and / or fermentation of a prokaryotic organism.
[0036] In certain embodiments, the prokaryotic organism is selected from bacteria (eg, probiotics), actinomycetes, rickettsia, chlamydia, mycoplasma, or any combination thereof.
[0037] In certain embodiments, the bacteria is selected from the genus Lactobacillus, Bifidobacterium, Bacillus, Propionibacterium, Streptococcus, Lactococcus, Pediococcus, Enterococcus, Staphylococcus, or any combination thereof.
[0038] In certain embodiments, the bacteria is selected from Lactobacillus acidophilus, Escherichia coli, Bifidobacterum lactis, Lactobacillus infantis, Lactobacillus paracasei, or any combination thereof.
[0039] In certain embodiments, the methylation is as previously defined.
[0040] In certain embodiments, the modulator is as previously defined.
[0041] On the other hand, the present application provides a use of a regulator in preparing food, wherein the regulator is selected from an inhibitor of DNA methyltransferase (DNMT), an activator of DNMT, or any combination thereof.
[0042] In certain embodiments, the food product comprises bacteria (eg, probiotics).
[0043] In certain embodiments, the modulator is used to modulate the growth, proliferation and / or fermentation of probiotics contained in the food product.
[0044] In certain embodiments, the modulator is as previously defined.
[0045] In certain embodiments, the food product is in solid form, liquid form, or semi-solid form.
[0046] In certain embodiments, the food product is a dairy product.
[0047] In certain embodiments, the food is selected from milk, yogurt, milk powder, lactic acid beverage, fermented milk beverage, popsicle, ice cream, or any combination thereof.
[0048] On the other hand, the present application provides a method for regulating the methylation level of prokaryotic nucleic acid, the method comprising contacting the prokaryotic organism with an effective amount of a regulator; wherein the regulator is selected from an inhibitor of DNA methyltransferase (DNMT), an activator of DNMT, or any combination thereof.
[0049] In certain embodiments, an effective amount of a modulator is contacted with the prokaryote during culturing (eg, subculturing) of the prokaryote.
[0050] In certain embodiments, an effective amount of a modulator is contacted with the prokaryote during activation of the prokaryote.
[0051] In certain embodiments, an effective amount of a modulator is contacted with the prokaryote during fermentation of the prokaryote.
[0052] In certain embodiments, the nucleic acid is DNA and / or RNA.
[0053] In certain embodiments, the methylation level is selected from: 5 C modification level, m 6 A modification level, m 7 G modification level, m 3 C modification level, or any combination thereof.
[0054] In certain embodiments, the modulator is used to modulate the mRNA expression of prokaryotic DNA. 5 C modification level, m 6 A modification level, m 3 C modification level, and / or m 7 G modification level.
[0055] In certain embodiments, the prokaryotic organism is selected from bacteria (eg, probiotics), actinomycetes, rickettsia, chlamydia, mycoplasma, or any combination thereof.
[0056] In certain embodiments, the bacteria is selected from the genus Lactobacillus, Bifidobacterium, Bacillus, Propionibacterium, Streptococcus, Lactococcus, Pediococcus, Enterococcus, Staphylococcus, or any combination thereof.
[0057] In certain embodiments, the bacteria is selected from Lactobacillus acidophilus, Escherichia coli, Bifidobacterum lactis, Lactobacillus infantis, Lactobacillus paracasei, or any combination thereof.
[0058] In certain embodiments, the modulator is selected from caffeic acid, epigallocatechin gallate (EGCG), benzoic acid, genistein, anacardic acid, sodium butyrate, diallyl disulfide, DL-Sulphoraphane, resveratrol, L-methionine, or any combination thereof.
[0059] In certain embodiments, the DNMT inhibitor is sodium butyrate.
[0060] In certain embodiments, the DNMT activator is selected from genistein, diallyl disulfide, DL-Sulphoraphane, epigallocatechin gallate (EGCG), or any combination thereof.
[0061] In certain embodiments, the modulator is food safe.
[0062] In certain embodiments, the modulator is capable of increasing the m 5 C modification level. In certain embodiments, the modulator is selected from DL-Sulphoraphane, Genistein, Diallyl Disulfide, Anacardic Acid, or any combination thereof.
[0063] In certain embodiments, the modulator is capable of reducing the m 5 C modification level. In certain embodiments, the modulator is selected from Sodium Butyrate, Caffeic Acid, Benzoic Acid, or any combination thereof.
[0064] In certain embodiments, the modulator is capable of increasing the m 6 A modification level. In certain embodiments, the modulator is selected from DL-Sulphoraphane, epigallocatechin gallate (EGCG), genistein, anacardic acid, or any combination thereof.
[0065] In certain embodiments, the modulator is capable of reducing the m 6 A modification level. In certain embodiments, the modulator is selected from sodium butyrate, caffeic acid, or any combination thereof.
[0066] In certain embodiments, the effective amount of caffeic acid is 500 mM or less, for example, 2.5 μM to 50 μM, 50 μM to 500 μM, 500 μM to 2.5 mM, 2.5 mM to 5 mM, 5 mM to 500 mM.
[0067] In certain embodiments, the effective amount of epigallocatechin gallate (EGCG) is 500 mM or less, for example, 100 μM to 500 μM, 500 μM to 1 mM, 1 mM to 2.5 mM, 2.5 mM to 5 mM, 5 mM to 500 mM.
[0068] In certain embodiments, the effective amount of the benzoic acid is 500 mM or less, for example, 15 μM to 50 μM, 50 μM to 500 μM, 500 μM to 2.5 mM, 2.5 mM to 5 mM, 5 mM to 500 mM.
[0069] In certain embodiments, the effective amount of genistein is 500 mM or less, for example, 5 μM to 50 μM, 50 μM to 500 μM, 500 μM to 2.5 mM, 2.5 mM to 5 mM, 5 mM to 500 mM.
[0070] In certain embodiments, the effective amount of anacardic acid is 500 mM or less, for example, 300 μM to 500 μM, 500 μM to 1 mM, 1 mM to 2.5 mM, 2.5 mM to 5 mM, 5 mM to 500 mM.
[0071] In certain embodiments, the effective amount of sodium butyrate is 500 mM or less, for example, 40 μM to 100 μM, 100 μM to 500 μM, 500 μM to 2.5 mM, 2.5 mM to 5 mM, 5 mM to 500 mM.
[0072] In certain embodiments, the effective amount of diallyl disulfide is 500 mM or less, for example, 40 μM to 100 μM, 100 μM to 500 μM, 500 μM to 2.5 mM, 2.5 mM to 5 mM, 5 mM to 500 mM.
[0073] In certain embodiments, the effective amount of DL-Sulphoraphane is 500 mM or less, for example, 40 μM to 100 μM, 100 μM to 500 μM, 500 μM to 2.5 mM, 2.5 mM to 5 mM, 5 mM to 500 mM.
[0074] In certain embodiments, the effective amount of resveratrol is 500 mM or less, for example, 40 μM to 100 μM, 100 μM to 500 μM, 500 μM to 2.5 mM, 2.5 mM to 5 mM, 5 mM to 500 mM;
[0075] In certain embodiments, the effective amount of L-methionine is 500 mM or less, for example, 40 μM to 100 μM, 100 μM to 500 μM, 500 μM to 2.5 mM, 2.5 mM to 5 mM, 5 mM to 500 mM.
[0076] On the other hand, the present application provides a method for preparing a culture medium for a prokaryote, the method comprising adding an effective amount of a regulator to the culture medium of the prokaryote; wherein the regulator is selected from an inhibitor of DNA methyltransferase (DNMT), an activator of DNMT, or any combination thereof.
[0077] In certain embodiments, the culture medium is in solid form, liquid form, or semi-solid form.
[0078] In certain embodiments, the culture medium further comprises: ingredients for providing nutrients, ingredients for fermentation, prebiotics, growth factors, water, or any combination thereof.
[0079] In certain embodiments, the prokaryotic organism is selected from bacteria (eg, probiotics), actinomycetes, rickettsia, chlamydia, mycoplasma, or any combination thereof.
[0080] In certain embodiments, the bacteria is selected from the genus Lactobacillus, Bifidobacterium, Bacillus, Propionibacterium, Streptococcus, Lactococcus, Pediococcus, Enterococcus, Staphylococcus, or any combination thereof.
[0081] In certain embodiments, the bacteria is selected from Lactobacillus acidophilus, Escherichia coli, Bifidobacterum lactis, Lactobacillus infantis, Lactobacillus paracasei, or any combination thereof.
[0082] In certain embodiments, the modulator is as previously defined.
[0083] On the other hand, the present application provides a method for fermenting a product containing a prokaryote, the method comprising contacting an effective amount of a regulator with the prokaryote; wherein the regulator is selected from an inhibitor of DNA methyltransferase (DNMT), an activator of DNMT, or any combination thereof.
[0084] In certain embodiments, an effective amount of a modulator is contacted with the prokaryote during culturing (eg, subculturing) of the prokaryote.
[0085] In certain embodiments, an effective amount of a modulator is contacted with the prokaryote during activation of the prokaryote.
[0086] In certain embodiments, an effective amount of a modulator is contacted with the prokaryote during fermentation of the prokaryote.
[0087] In certain embodiments, the prokaryotic organism is selected from bacteria (eg, probiotics), actinomycetes, rickettsia, chlamydia, mycoplasma, or any combination thereof.
[0088] In certain embodiments, the bacteria is selected from the genus Lactobacillus, Bifidobacterium, Bacillus, Propionibacterium, Streptococcus, Lactococcus, Pediococcus, Enterococcus, Staphylococcus, or any combination thereof.
[0089] In certain embodiments, the bacteria is selected from Lactobacillus acidophilus, Escherichia coli, Bifidobacterum lactis, Lactobacillus infantis, Lactobacillus paracasei, or any combination thereof.
[0090] In certain embodiments, the modulator is as previously defined.
[0091] On the other hand, the present application provides a method for preparing food containing prokaryotes, the method comprising contacting an effective amount of a regulator with the prokaryote; wherein the regulator is selected from an inhibitor of DNA methyltransferase (DNMT), an activator of DNMT, or any combination thereof.
[0092] In certain embodiments, an effective amount of a modulator is contacted with the prokaryote during culturing (eg, subculturing) of the prokaryote.
[0093] In certain embodiments, an effective amount of a modulator is contacted with the prokaryote during activation of the prokaryote.
[0094] In certain embodiments, an effective amount of a modulator is contacted with the prokaryote during fermentation of the prokaryote.
[0095] In certain embodiments, the food product is in solid form, liquid form, or semi-solid form.
[0096] In certain embodiments, the food product is a dairy product.
[0097] In certain embodiments, the food is selected from milk, yogurt, milk powder, lactic acid beverage, fermented milk beverage, popsicle, ice cream, or any combination thereof.
[0098] In certain embodiments, the prokaryotic organism is selected from bacteria (eg, probiotics), actinomycetes, rickettsia, chlamydia, mycoplasma, or any combination thereof.
[0099] In certain embodiments, the bacteria is selected from the genus Lactobacillus, Bifidobacterium, Bacillus, Propionibacterium, Streptococcus, Lactococcus, Pediococcus, Enterococcus, Staphylococcus, or any combination thereof.
[0100] In certain embodiments, the bacteria is selected from Lactobacillus acidophilus, Escherichia coli, Bifidobacterum lactis, Lactobacillus infantis, Lactobacillus paracasei, or any combination thereof.
[0101] In certain embodiments, the modulator is as previously defined.
[0102] On the other hand, the present application provides a method for changing bacterial performance (for example, improving bacterial resistance to bacteriophages, enhancing bacterial survival, and changing bacterial fermentation products), the method comprising contacting an effective amount of a regulator with the prokaryote; wherein the regulator is selected from an inhibitor of DNA methyltransferase (DNMT), an activator of DNMT, or any combination thereof.
[0103] In certain embodiments, the bacteria are probiotics.
[0104] In certain embodiments, the bacteria is selected from the genus Lactobacillus, Bifidobacterium, Bacillus, Propionibacterium, Streptococcus, Lactococcus, Pediococcus, Enterococcus, Staphylococcus, or any combination thereof.
[0105] In certain embodiments, the bacteria is selected from Lactobacillus acidophilus, Escherichia coli, Bifidobacterum lactis, Lactobacillus infantis, Lactobacillus paracasei, or any combination thereof.
[0106] In certain embodiments, the modulator is as previously defined.
[0107] Definition of terms
[0108] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics and recombinant DNA procedures used herein are conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.
[0109] As used herein, the term "and / or" used in phrases such as "A and / or B" is intended to include "A and B", "A or B" as well as "A" and "B".
[0110] As used herein, the term "epigenetic" refers to the phenomenon that the DNA sequence does not change, but the expression of the gene undergoes a heritable change. DNA epigenetic modification refers to the heritable chromatin modification in cells in addition to genetic information. It is widely present in the physiological and biochemical reactions of living organisms, such as restriction and modification systems (RM systems), DNA replication and repair, transcription, translation, etc. The forms of epigenetic inheritance include: DNA modification, histone modification, non-coding RNA regulation, chromatin remodeling, and nucleosome positioning.
[0111] As used herein, the term "DNA methylation" is a form of DNA modification that can change genetic expression without changing the DNA sequence. DNA methylation refers to the covalent bonding of a methyl group to the 5th carbon position of cytosine in genomic CpG dinucleotides under the action of DNA methyltransferase. A large number of studies have shown that DNA methylation can cause changes in chromatin structure, DNA conformation, DNA stability, and the way DNA interacts with proteins, thereby controlling gene expression.
[0112] As used herein, the term "regulating the methylation level of prokaryotic nucleic acid" refers to changing the methylation level of prokaryotic nucleic acid, or changing the degree of methylation of prokaryotic nucleic acid. The regulation includes increasing and decreasing the methylation level of prokaryotic nucleic acid. Similarly, the term "regulating the methylation level of prokaryotic DNA" refers to changing the methylation level of prokaryotic nucleic acid, or changing the degree of methylation of prokaryotic nucleic acid. 5 "C modification level" refers to the change (including increase and decrease) of the m 5 C modification level.
[0113] As used herein, the term "DNMT" is a term known in the art, and the full name of DNMT is DNA methyltransferase. DNMT refers to an enzyme that catalyzes the transfer of methyl groups to DNA. Non-limiting examples of DNA methyltransferases include DNMT, DNMT3A, DNMT3B, DNMT3C and DNMT3L.
[0114] As used herein, the term "effective amount" refers to an amount of a compound or modulator sufficient to achieve a purpose. For example, an amount of a compound or modulator sufficient to modulate the methylation level of a prokaryotic organism.
[0115] As used herein, the term "food safe" means that the substance can be added or present in food in any form without causing safety problems to the food. In certain embodiments, the substance can be present in food with other ingredients as an additive, or added to food during food preparation.
[0116] Advantageous Effects of the Invention
[0117] The present application experimentally confirmed that the same regulator has significant differences in its ability to regulate DNA methylation levels in eukaryotes and prokaryotes. Furthermore, the present application experimentally obtained a regulator that can effectively regulate the DNA methylation level of prokaryotes, and obtained 385 differentially expressed genes by exposing Lactobacillus acidophilus to different regulators.
[0118] Applying these regulators to bacterial culture and / or fermentation will help change the properties of bacteria, especially change the fermentation products of bacteria, improve the ability of bacteria to resist bacteriophages, improve the survival rate of bacteria after freeze-drying, etc. Therefore, these regulators have strong application potential in bacterial culture and / or fermentation, as well as in the culture and / or fermentation of foods containing bacteria.
[0119] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS
[0120] Figure 1 The effect of different concentrations of caffeic acid on the growth of Lactobacillus acidophilus.
[0121] Figure 2 The effect of different concentrations of epigallocatechin gallate (EGCG) on the growth of Lactobacillus acidophilus.
[0122] Figure 3 The effect of different concentrations of benzoic acid on the growth of Lactobacillus acidophilus.
[0123] Figure 4 The effect of different concentrations of genistein on the growth of Lactobacillus acidophilus.
[0124] Figure 5 The effect of different concentrations of anacardic acid on the growth of Lactobacillus acidophilus.
[0125] Figure 6 The effect of different concentrations of sodium butyrate on the growth of Lactobacillus acidophilus.
[0126] Figure 7 The effect of different concentrations of diallyl disulfide on the growth of Lactobacillus acidophilus.
[0127] Figure 8 The effect of different concentrations of DL-Sulphoraphane on the growth of Lactobacillus acidophilus.
[0128] Fig. 9 The effect of different concentrations of 5-aza-2'-deoxycytidine on the growth of Lactobacillus acidophilus.
[0129] Fig.10 The effect of different concentrations of TSA on the growth of Lactobacillus acidophilus.
[0130] Fig.11 The relative quantitative analysis results of 5-mC DNA methylation levels in Lactobacillus acidophilus treated with different compounds. a-d Tukey matching results showed that there were significant differences between Lactobacillus acidophilus samples under different treatments (P<0.05).
[0131] Fig.12A circular diagram of the Lactobacillus acidophilus genome, listing the differentially expressed genes (DEGs) based on the fold-change in expression values collected by RNA sequencing.
[0132] Fig.13 Comparison of the expression levels of methylation-related genes in epigenetically treated Lactobacillus acidophilus and untreated control strains.
[0133] Fig.14 Comparison of the expression levels of transcription factor-related genes in epigenetically treated Lactobacillus acidophilus and untreated control strains.
[0134] Fig.15 Comparison of the expression levels of transposase-related genes in epigenetically treated Lactobacillus acidophilus and untreated control strains.
[0135] Fig.16 The inhibitory activity of different compounds on histone deacetylase (HDAC) in eukaryotic organisms (yeast); among them, 4-PBA is 4-phenylbutyric acid. DETAILED DESCRIPTION
[0136] The present invention will now be described with reference to the following examples which are intended to illustrate the present invention (but not to limit the present invention). Unless otherwise specified, the experiments and methods described in the examples were performed essentially according to conventional methods well known in the art and described in various references.
[0137] In addition, if the specific conditions are not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be obtained commercially. It is known to those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the present invention. All public cases and other references mentioned herein are incorporated herein by reference in their entirety.
[0138] Example 1. Effects of 8 compounds on the growth of Lactobacillus acidophilus
[0139] This example evaluates the effects of eight candidate compounds and two control chemicals on the growth of Lactobacillus acidophilus. The eight candidate compounds are caffeic acid, epigallocatechin gallate (EGCG), benzoic acid, genistein, anacardic acid, sodium butyrate, diallyl disulfide and DL-sulphoraphane. The two control chemicals were 5-aza-2'-deoxycytidine and TSA (purchased from Sigma Aldrich (Merck, St. Louis, Missouri, United States, catalog numbers: A3656 and T8552, respectively). The optimal concentration of each compound was determined by comparing the growth curves of Lactobacillus acidophilus treated with different concentrations of the compounds. At the optimal concentration, the bacteria may have the greatest epigenetic effect and the least effect on bacterial growth.
[0140] Specifically, the growth curve of Lactobacillus acidophilus was determined in a 96-well microplate with 200 μL. 600 Adjusted to 0.95, and added to a 96-well plate at a ratio of 3%, while the candidate compound (dissolved in DMSO) was added to the 96-well plate with its relative concentration (detailed information is shown in Table 1). The absorbance (600 nm) was continuously measured for 96 hours using a FLUOstar Omega microplate reader (BMG LABTECH, Ortenberg, Germany).
[0141] Table 1. Relative concentrations of compounds
[0142]
[0143] The experimental results are as follows Figure 1-10 As shown. Among them, Figure 1 The effect of different concentrations of caffeic acid on the growth of Lactobacillus acidophilus. Figure 2 The effect of different concentrations of epigallocatechin gallate (EGCG) on the growth of Lactobacillus acidophilus. Figure 3 The effect of different concentrations of benzoic acid on the growth of Lactobacillus acidophilus. Figure 4 The effect of different concentrations of genistein on the growth of Lactobacillus acidophilus. Figure 5The effect of different concentrations of anacardic acid on the growth of Lactobacillus acidophilus. Figure 6 The effect of different concentrations of sodium butyrate on the growth of Lactobacillus acidophilus. Figure 7 The effect of different concentrations of diallyl disulfide on the growth of Lactobacillus acidophilus. Figure 8 The effect of different concentrations of DL-Sulphoraphane on the growth of Lactobacillus acidophilus. Fig. 9 The effect of different concentrations of 5-aza-2'-deoxycytidine on the growth of Lactobacillus acidophilus. Fig.10 The effect of different concentrations of TSA on the growth of Lactobacillus acidophilus.
[0144] Example 2. Screening of modulators based on their ability to inhibit / activate DNMTs
[0145] This example evaluates the effects of eight candidate compounds and two control chemicals on the methylation level of Lactobacillus acidophilus. The DNA methylation level of 5-mC of Lactobacillus acidophilus was detected by two ELISA test kits (MethylFlash TM , EpiGentek, United States).
[0146] The results of the 5-mC DNA methylation level test are as follows Fig.11 As shown, the calculation method of these results is calculated by comparing with the positive DNA control in each kit according to the method described in the kit manual. The ordinate in the figure is the relative methylation level, which represents the percentage of the two methylation patterns in the total amount of Lactobacillus acidophilus DNA detected by the specific antibody. The test was repeated at least 4 times to ensure the accuracy of the data, and the results are expressed as mean + SD. The letters above each bar graph (group) indicate significant differences based on Tukey paired mean comparison results (P < 0.05), and groups with the same letters indicate that there are no significant differences between them (P > 0.05).
[0147] The results of 5-mC are as follows Fig.11As shown. The results showed that the methylation level of Lactobacillus acidophilus treated with diallyl disulfide (1.05%) was significantly higher than that of the untreated wild-type strain (0.74%) and the strain treated with the positive sample control 5-aza-2'-deoxycytidine (0.50%). Among them, 5-aza-2'-deoxycytidine is a recognized DNA methyltransferase (DNMT) inhibitor. Compared with the positive sample control, the 5-mC methylation level of Lactobacillus acidophilus treated with DL-Sulphoraphane (1.02%) and 500μM genistein (0.84%) was also significantly higher. It is worth noting that sodium butyrate (0.63%), as a potential DNMT inhibitor, showed almost equivalent methylation level regulation ability to the positive control 5-aza-2'-deoxycytidine, and showed the strongest DNMT inhibitory activity among all compounds. Furthermore, the regulatory abilities of DL-Sulphoraphane and diallyl disulfide were significantly different from those of sodium butyrate, both of which showed stronger DNMT activation abilities.
[0148] In general, sodium butyrate showed the most significant DNMT inhibition ability among all candidate epigenetic compounds. In contrast, genistein showed DNMT activation ability, and diallyl disulfide, DL-Sulphoraphane and EGCG also showed certain DNMT activation ability.
[0149] Example 3. Transcriptome analysis of epigenetically treated Lactobacillus acidophilus
[0150] In this example, the candidate compound is used to treat Lactobacillus acidophilus, and transcriptome sequencing analysis is performed on the treated Lactobacillus acidophilus.
[0151] Transcriptome analysis was performed by AZENTA Life Sciences (Suzhou, China) on an Illumina Novaseq platform using RNA sequencing (in 2x150bp paired-end configuration). RNA sequencing results were condensed into Fig.12In the circular diagram shown, a total of 385 differentially expressed genes are shown after Lactobacillus acidophilus is treated with different modulators (5-aza-2'-deoxycytidine, Caffeic Acid, Diallyl Disulfide, Genistein, Resveratrol, L-methionine, Sodium Butyrate). The outermost circle represents the chromosome and base pair position (kb) of Lactobacillus acidophilus; the second outer circle represents the 385 genes that are differentially expressed when the strain is treated with different compounds (compared to the wild-type control), and the names of the related genes that have been identified so far. A to G in the figure are heat maps showing the differential expression levels of related genes under different treatments of 7 epigenetically active compounds. The genomic mapping and visualization of DEGs were performed using Circos (version 0.69-8).
[0152] Figure 13-15 Figure 2 is the expression level of some specific genes related to methylation, transcription factors and transposase in Lactobacillus acidophilus treated with modulators compared with the untreated control strain.
[0153] Example 4. Differences in the effects of regulators in eukaryotes and prokaryotes
[0154] This example compares the difference in the regulatory activity of the same compound on DNA methylation levels in eukaryotes (yeast) and prokaryotes. By testing whether the candidate compound has histone deacetylase (HDAC) inhibitory activity in Saccharomyces cerevisiae, it is tested whether it has DNA methylation level regulatory activity in eukaryotes (because DNA methylation levels cannot be directly measured in yeast, the ability of the compound to regulate DNA methylation levels is reflected by measuring the HDAC inhibitory activity of the compound).
[0155] Fig.16 The results show the inhibitory activity of HDAC in Saccharomyces cerevisiae after treatment with different compounds. Compared with HeLa cells not treated with inhibitors, TSA at different concentrations showed strong HDAC inhibitory activity, indicating that it has a strong ability to regulate DNA methylation levels in eukaryotes. However, 5-aza-2'-deoxycytidine has no HDAC inhibitory activity at all, indicating that it has a very weak ability to regulate DNA methylation levels in eukaryotes.
[0156] The activity of the above two compounds in regulating DNA methylation levels in eukaryotes is completely opposite to that in prokaryotes! Fig.11As shown in the results, the regulatory activity of TSA is almost the same as that of the untreated wild type, that is, TSA has basically no ability to regulate DNA methylation levels in prokaryotes. However, the regulatory activity of 5-aza-2'-deoxycytidine is significantly different from that of the untreated wild type, that is, 5-aza-2'-deoxycytidine has a stronger ability to regulate DNA methylation levels in prokaryotes.
[0157] Therefore, the same compound has significant differences in its regulatory activity on DNA methylation levels in eukaryotes (eg, yeast) and prokaryotes.
[0158] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.
Claims
1. Use of a regulator in regulating the methylation level of prokaryotic nucleic acid, wherein: The regulator is selected from an inhibitor of DNA methyltransferase (DNMT), an activator of DNMT, or any combination thereof.
2. The use according to claim 1, wherein The nucleic acid is DNA and / or RNA; Preferably, the methylation level is selected from: 5 C modification level, m 6 A modification level, m 7 G modification level, m 3 C modification level, or any combination thereof; Preferably, the regulator is used to regulate the m 5 C modification level, m 6 A modification level, m 3 C modification level, and / or m 7 G modification level.
3. The use according to claim 1 or 2, wherein The prokaryotic organism is selected from bacteria (e.g., probiotics), actinomycetes, rickettsia, chlamydia, mycoplasma, or any combination thereof; Preferably, the bacteria is selected from the group consisting of Lactobacillus, Bifidobacterium, Bacillus, Propionibacterium, Streptococcus, Lactococcus, Pediococcus, Enterococcus, Staphylococcus, or any combination thereof; Preferably, the bacteria is selected from Lactobacillus acidophilus, Escherichia coli, Bifidobacterum lactis, Lactobacillus infantis, Lactobacillus paracasei, or any combination thereof.
4. The use according to any one of claims 1 to 3, wherein The regulator is selected from caffeic acid, epigallocatechin gallate (EGCG), benzoic acid, genistein, anacardic acid, sodium butyrate, diallyl disulfide, DL-sulphoraphane, resveratrol, L-methionine, or any combination thereof; Preferably, the DNMT inhibitor is sodium butyrate; Preferably, the DNMT activator is selected from genistein, diallyl disulfide, DL-Sulphoraphane, epigallocatechin gallate (EGCG), or any combination thereof; Preferably, the conditioning agent is food safe.
5. The use according to any one of claims 1 to 4, wherein The modulator has one or more characteristics selected from the following: (1) The regulator can increase the m 5 C modification level; Preferably, the regulator is selected from DL-Sulphoraphane, Genistein, Diallyl Disulfide, Anacardic Acid, or any combination thereof; (2) The regulator can reduce the m 5 C modification level; Preferably, the regulator is selected from sodium butyrate, caffeic acid, benzoic acid, or any combination thereof; (3) The regulator can increase the m 6 A modification level; (4) The regulator can reduce the m 6 A modification level.
6. A prokaryotic culture medium, comprising a regulator capable of regulating the methylation level of prokaryotic nucleic acid, wherein: The regulator is selected from an inhibitor of DNA methyltransferase (DNMT), an activator of DNMT, or any combination thereof.
7. The culture medium according to claim 6, wherein The prokaryotic organism is selected from bacteria (e.g., probiotics), actinomycetes, rickettsia, chlamydia, mycoplasma, or any combination thereof; Preferably, the bacteria is selected from the group consisting of Lactobacillus, Bifidobacterium, Bacillus, Propionibacterium, Streptococcus, Lactococcus, Pediococcus, Enterococcus, Staphylococcus, or any combination thereof; Preferably, the bacteria is selected from Lactobacillus acidophilus, Escherichia coli, Bifidobacterum lactis, Lactobacillus infantis, Lactobacillus paracasei, or any combination thereof.
8. The culture medium according to claim 6 or 7, wherein The culture medium is in solid form, liquid form, or semi-solid form; Preferably, the culture medium further comprises: ingredients for providing nutrition, ingredients for fermentation, prebiotics, growth factors, water, or any combination thereof; Preferably, the nucleic acid is as defined in any one of claims 1 to 5; Preferably, the methylation is as defined in any one of claims 1-5; Preferably, the modulator is as defined in any one of claims 1-5.
9. Use of a regulator in preparing a prokaryotic culture medium; wherein: The regulator is selected from a DNA methyltransferase (DNMT) inhibitor, a DNMT activator, or any combination thereof; Preferably, the culture medium is used to regulate the growth, reproduction and / or fermentation of prokaryotes.
10. The use according to claim 9, wherein The prokaryotic organism is selected from bacteria (e.g., probiotics), actinomycetes, rickettsia, chlamydia, mycoplasma, or any combination thereof; Preferably, the bacteria is selected from the group consisting of Lactobacillus, Bifidobacterium, Bacillus, Propionibacterium, Streptococcus, Lactococcus, Pediococcus, Enterococcus, Staphylococcus, or any combination thereof; Preferably, the bacteria is selected from Lactobacillus acidophilus, Escherichia coli, Bifidobacterum lactis, Lactobacillus infantis, Lactobacillus paracasei, or any combination thereof; Preferably, the methylation is as defined in any one of claims 1-5; Preferably, the modulator is as defined in any one of claims 1-5.
11. Use of a regulator in preparing food, wherein: The regulator is selected from a DNA methyltransferase (DNMT) inhibitor, a DNMT activator, or any combination thereof; Preferably, the food comprises bacteria (e.g., probiotics); Preferably, the regulator is used to regulate the growth, reproduction and / or fermentation of probiotics contained in the food; Preferably, the modulator is as defined in any one of claims 1-5.
12. The use according to claim 11, wherein The food is in solid form, liquid form, or semi-solid form; Preferably, the food is a dairy product; Preferably, the food is selected from milk, yogurt, milk powder, lactic acid beverage, fermented milk beverage, popsicle, ice cream, or any combination thereof.
13. A method for regulating the methylation level of a prokaryotic nucleic acid, the method comprising contacting an effective amount of a regulator with the prokaryotic organism; wherein: The regulator is selected from an inhibitor of DNA methyltransferase (DNMT), an activator of DNMT, or any combination thereof.
14. The method of claim 13, wherein: The nucleic acid is DNA and / or RNA; Preferably, the methylation level is selected from: 5 C modification level, m 6 A modification level, m 7 G modification level, m 3 C modification level, or any combination thereof; Preferably, the regulator is used to regulate the m 5 C modification level, m 6 A modification level, m 3 C modification level, and / or m 7 G modification level.
15. The method according to claim 13 or 14, wherein The prokaryotic organism is selected from bacteria (e.g., probiotics), actinomycetes, rickettsia, chlamydia, mycoplasma, or any combination thereof; Preferably, the bacteria is selected from the group consisting of Lactobacillus, Bifidobacterium, Bacillus, Propionibacterium, Streptococcus, Lactococcus, Pediococcus, Enterococcus, Staphylococcus, or any combination thereof; Preferably, the bacteria is selected from Lactobacillus acidophilus, Escherichia coli, Bifidobacterum lactis, Lactobacillus infantis, Lactobacillus paracasei, or any combination thereof.
16. The method according to any one of claims 13 to 15, wherein The regulator is selected from caffeic acid, epigallocatechin gallate (EGCG), benzoic acid, genistein, anacardic acid, sodium butyrate, diallyl disulfide, DL-sulphoraphane, resveratrol, L-methionine, or any combination thereof; Preferably, the DNMT inhibitor is sodium butyrate; Preferably, the DNMT activator is selected from genistein, diallyl disulfide, DL-Sulphoraphane, epigallocatechin gallate (EGCG), or any combination thereof; Preferably, the conditioning agent is food safe.
17. The method according to any one of claims 13 to 16, wherein The modulator has one or more characteristics selected from the following: (1) The regulator can increase the m 5 C modification level; Preferably, the regulator is selected from DL-Sulphoraphane, Genistein, Diallyl Disulfide, Anacardic Acid, or any combination thereof; (2) The regulator can reduce the m 5 C modification level; Preferably, the regulator is selected from sodium butyrate, caffeic acid, benzoic acid, or any combination thereof; (3) The regulator can increase the m 6 A modification level; Preferably, the regulator is selected from DL-Sulphoraphane, epigallocatechin gallate (EGCG), genistein, anacardic acid, or any combination thereof; (4) The regulator can reduce the m 6 A modification level; Preferably, the regulator is selected from sodium butyrate, caffeic acid, or any combination thereof.
18. The method according to any one of claims 13 to 17, wherein The modulator has one or more characteristics selected from the following: (1) The effective amount of caffeic acid is 500 mM or less, for example, 2.5 μM to 50 μM, 50 μM to 500 μM, 500 μM to 2.5 mM, 2.5 mM to 5 mM, 5 mM to 500 mM; (2) the effective amount of epigallocatechin gallate (EGCG) is 500 mM or less, for example, 100 μM to 500 μM, 500 μM to 1 mM, 1 mM to 2.5 mM, 2.5 mM to 5 mM, 5 mM to 500 mM; (3) The effective amount of the benzoic acid is 500 mM or less, for example, 15 μM to 50 μM, 50 μM to 500 μM, 500 μM to 2.5 mM, 2.5 mM to 5 mM, 5 mM to 500 mM; (4) The effective amount of genistein is 500 mM or less, for example, 5 μM to 50 μM, 50 μM to 500 μM, 500 μM to 2.5 mM, 2.5 mM to 5 mM, 5 mM to 500 mM; (5) The effective amount of anacardic acid is 500 mM or less, for example, 300 μM to 500 μM, 500 μM to 1 mM, 1 mM to 2.5 mM, 2.5 mM to 5 mM, 5 mM to 500 mM; (6) The effective amount of sodium butyrate is 500 mM or less, for example, 40 μM to 100 μM, 100 μM to 500 μM, 500 μM to 2.5 mM, 2.5 mM to 5 mM, 5 mM to 500 mM; (7) The effective amount of diallyl disulfide is 500 mM or less, for example, 40 μM to 100 μM, 100 μM to 500 μM, 500 μM to 2.5 mM, 2.5 mM to 5 mM, 5 mM to 500 mM; (8) The effective amount of DL-Sulphoraphane is 500 mM or less, for example, 40 μM to 100 μM, 100 μM to 500 μM, 500 μM to 2.5 mM, 2.5 mM to 5 mM, 5 mM to 500 mM; (9) The effective amount of resveratrol is 500 mM or less, for example, 40 μM to 100 μM, 100 μM to 500 μM, 500 μM to 2.5 mM, 2.5 mM to 5 mM, 5 mM to 500 mM; (10) The effective amount of L-methionine is 500 mM or less, for example, 40 μM to 100 μM, 100 μM to 500 μM, 500 μM to 2.5 mM, 2.5 mM to 5 mM, or 5 mM to 500 mM.
19. A method for preparing a culture medium for a prokaryote, the method comprising adding an effective amount of a regulator to the culture medium for the prokaryote; wherein: The regulator is selected from an inhibitor of DNA methyltransferase (DNMT), an activator of DNMT, or any combination thereof.
20. The method of claim 19, wherein: The culture medium is in solid form, liquid form, or semi-solid form; Preferably, the culture medium further comprises: ingredients for providing nutrition, ingredients for fermentation, prebiotics, growth factors, water, or any combination thereof; Preferably, the prokaryotic organism is selected from bacteria (e.g., probiotics), actinomycetes, rickettsia, chlamydia, mycoplasma, or any combination thereof; Preferably, the bacteria is selected from the group consisting of Lactobacillus, Bifidobacterium, Bacillus, Propionibacterium, Streptococcus, Lactococcus, Pediococcus, Enterococcus, Staphylococcus, or any combination thereof; Preferably, the bacteria is selected from Lactobacillus acidophilus, Escherichia coli, Bifidobacterum lactis, Lactobacillus infantis, Lactobacillus paracasei, or any combination thereof; Preferably, the modulator is as defined in any one of claims 13-18.
21. A method for fermenting a product containing a prokaryotic organism, the method comprising contacting an effective amount of a regulator with the prokaryotic organism; wherein: The regulator is selected from an inhibitor of DNA methyltransferase (DNMT), an activator of DNMT, or any combination thereof.
22. The method of claim 21, wherein: The prokaryotic organism is selected from bacteria (e.g., probiotics), actinomycetes, rickettsia, chlamydia, mycoplasma, or any combination thereof; Preferably, the bacteria is selected from the group consisting of Lactobacillus, Bifidobacterium, Bacillus, Propionibacterium, Streptococcus, Lactococcus, Pediococcus, Enterococcus, Staphylococcus, or any combination thereof; Preferably, the bacteria is selected from Lactobacillus acidophilus, Escherichia coli, Bifidobacterum lactis, Lactobacillus infantis, Lactobacillus paracasei, or any combination thereof; Preferably, the modulator is as defined in any one of claims 13-18.
23. A method for preparing a food containing a prokaryotic organism, the method comprising contacting an effective amount of a regulator with the prokaryotic organism; wherein: The regulator is selected from an inhibitor of DNA methyltransferase (DNMT), an activator of DNMT, or any combination thereof.
24. The method of claim 23, wherein: The food is in solid form, liquid form, or semi-solid form; Preferably, the food is a dairy product; Preferably, the food is selected from milk, yogurt, milk powder, lactic acid beverage, fermented milk beverage, popsicle, ice cream, or any combination thereof; Preferably, the prokaryotic organism is selected from bacteria (e.g., probiotics), actinomycetes, rickettsia, chlamydia, mycoplasma, or any combination thereof; Preferably, the bacteria is selected from the group consisting of Lactobacillus, Bifidobacterium, Bacillus, Propionibacterium, Streptococcus, Lactococcus, Pediococcus, Enterococcus, Staphylococcus, or any combination thereof; Preferably, the bacteria is selected from Lactobacillus acidophilus, Escherichia coli, Bifidobacterum lactis, Lactobacillus infantis, Lactobacillus paracasei, or any combination thereof; Preferably, the modulator is as defined in any one of claims 13-18.
25. A method for changing bacterial performance (e.g., improving bacterial resistance to bacteriophage, enhancing bacterial survival, changing bacterial fermentation products), the method comprising contacting an effective amount of a regulator with the prokaryotic organism; wherein, The regulator is selected from an inhibitor of DNA methyltransferase (DNMT), an activator of DNMT, or any combination thereof.
26. The method of claim 25, wherein: The bacteria are probiotics; Preferably, the bacteria is selected from the group consisting of Lactobacillus, Bifidobacterium, Bacillus, Propionibacterium, Streptococcus, Lactococcus, Pediococcus, Enterococcus, Staphylococcus, or any combination thereof; Preferably, the bacteria is selected from Lactobacillus acidophilus, Escherichia coli, Bifidobacterum lactis, Lactobacillus infantis, Lactobacillus paracasei, or any combination thereof; Preferably, the modulator is as defined in any one of claims 13-18.