CN112391305B - Streptococcus salivarius F286 and uses thereof - Google Patents

Streptococcus salivarius F286 and uses thereof Download PDF

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CN112391305B
CN112391305B CN201910754930.9A CN201910754930A CN112391305B CN 112391305 B CN112391305 B CN 112391305B CN 201910754930 A CN201910754930 A CN 201910754930A CN 112391305 B CN112391305 B CN 112391305B
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streptococcus salivarius
breast milk
streptococcus
mice
cells
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CN112391305A (en
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申剑
李娜
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Shanghai Jiaotong University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • 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
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/21—Streptococcus, lactococcus
    • A23V2400/245—Salivarius
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus

Abstract

The invention provides streptococcus salivarius F286 with a preservation number of CGMCC No.17579. In the invention, streptococcus salivarius F286 is collected by an inventor, is subjected to gavage on human breast milk and is fed to 8-week-old sterile mice growing in a sterile bag, and each mouse is subjected to secondary gavage on every other day by using the same breast milk. After the completion of the gavage, the mice were further kept in a sterile bag for 8 weeks, and the mice were subjected to bacterial isolation using the feces of the mice at the 8 th week. The streptococcus salivarius F286 can be used as probiotic preparation, food additive, or effective component of medicine, especially for infantile breast milk, breast milk substitute, infant formula, dairy product or related product lacking streptococcus.

Description

Streptococcus salivarius F286 and uses thereof
Technical Field
The invention relates to a bioengineering bacterium, in particular to streptococcus salivarius F286 and application thereof.
Background
Human breast milk not only provides nutrition for the newborn but also human symbiotic bacteria, and studies have shown that milk of healthy mothers contains 10 2 –10 5 CFU/ml bacteria. The breast milk is provided to the infants fed with pure breast milk on a daily basis of 10 ml per day, calculated on the average per day of 800 ml breast milk fed to them 4 –10 8 And (4) bacterial cells. Bacteria in breast milk colonize the newborn intestinal tract immediately after birth, are the "pioneer flora" of the newborn intestinal tract, and play a key role in the development of the newborn immune system.
Existing research has focused mainly on isolating bifidobacteria (bifidobacteria) and lactobacilli (lactobacilli) from breast milk and investigating the immunomodulatory effects of these traditional probiotics. However, not all breast milk contains viable bifidobacteria and lactobacilli which can be isolated in culture, and studies of the flora structure of breast milk in larger, larger populations have shown that the bacterium which is most abundant in breast milk and which is detectable in breast milk of different people is Streptococcus (Streptococcus). Jost et al collected fresh milk from 7 mothers, only bifidobacteria were isolated from 2 of them, lactobacilli from 1 mother, but streptococci from all 7 mothers; moreover, the literature indicates that, among the bacteria isolated from human breast milk, bifidobacteria account for only 1.7% and streptococci for 17.9%. In addition, streptococcus is also the predominant bacterium in the gut of newborns within 2 weeks after birth. These results suggest that breast milk streptococcus is a human commensal bacterium that most mothers transmit to their offspring through milk and has important probiotic effects. Therefore, there is a need to provide probiotic formulations of streptococcus in human breast milk.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present invention aims to provide a streptococcus salivarius F286 and its use for solving the problem of streptococcus deficiencies in some breast milk, breast milk substitutes, infant formulas, dairy products or related products in the prior art.
In order to achieve the above objects and other related objects, the invention provides streptococcus salivarius F286 with a preservation number of CGMCC No.17579.
In the invention, streptococcus salivarius F286 is obtained by the inventor through human breast milk and is perfused into 8-week-old sterile mice growing in a sterile bag, and the same breast milk is used for carrying out secondary perfusion on each mouse every other day. After the completion of the gavage, the mice were further kept in a sterile bag for 8 weeks, and the mice were subjected to bacterial isolation using the feces of the mice at the 8 th week.
The strain is determined to be streptococcus salivarius through 16S rRNA gene amplification sequence developmental tree analysis, and according to the international naming rule: the strain is named by the genus name, the species name and the strain name, the genus name, the species name and the strain name are respectively streptococcus, streptococcus salivarius strains and F286, the strain is named as streptococcus salivarius F286, the strain is sent to a common microorganism center of China Committee for microorganism preservation management in 2019 and 19 months, and the preservation number is as follows: CGMCC No.17579.
Further, the nucleotide sequence of the streptococcus salivarius F286 is shown as SEQ ID NO. 5.
The invention also provides application of the streptococcus salivarius F286 in preparing food, health-care products or medicines.
Further, the food or health product may be specific for infants, for example, it may be a probiotic preparation, an infant formula, or a liquid beverage, such as a dairy product.
Further, the streptococcus salivarius F286 can be live or killed.
In another aspect of the invention, a food or health product is provided, which comprises streptococcus salivarius F286 or a streptococcus salivarius F286 lysate.
Further, the food or health product may be specific for infants, for example, it may be a probiotic preparation, an infant formula, or a liquid beverage, such as a dairy product.
Further, the streptococcus salivarius F286 can be live or killed.
Further, the content of streptococcus salivarius F286 in the food or health product is 10 2 CFU/ml-10 10 CFU/ml。
Further, the food or health care product has the functions of promoting the development of mammals, activating the immune function of the mammals and/or improving the immunity of the mammals.
Further, the food or health care product has the function of promoting peripheral blood mononuclear cells of mammals to secrete Interleukin (IL) -12 and Interleukin (IL) -10.
According to a further aspect of the present invention there is provided a medicament comprising streptococcus salivarius F286 or a streptococcus salivarius F286 lysate as hereinbefore described.
Further, the streptococcus salivarius F286 can be live or killed.
Furthermore, the medicine has the function of promoting the peripheral blood mononuclear cells of the mammal to secrete Interleukin (IL) -12 and Interleukin (IL) -10.
Further, the medicament has the effects of promoting the development of mammals, activating the immune functions of the mammals and/or improving the immunity of the mammals.
Further, the medicament may be for use with infants and young children.
As described above, streptococcus salivarius F286 and uses thereof according to the present invention have the following beneficial effects:
the streptococcus salivarius F286 can be used as a probiotic preparation, a food additive or an effective component of a medicament, and particularly aims at the condition that streptococcus is deficient in infant breast milk, breast milk substitutes, infant formula milk, dairy products or related products. Streptococcus salivarius F286 is safe and effective. Experiments prove that the compound can effectively promote human peripheral blood mononuclear cells to secrete Interleukin (IL) -12 and Interleukin (IL) -10, and can also promote the expression of immune genes of nematodes and prolong the life of the nematodes.
The preservation information of the strains of the invention is as follows:
the strain name: streptococcus salivarius
The preservation number is as follows: CGMCC No.17579;
the preservation date is as follows: 19/4/2019;
the name of the depository: china general microbiological culture Collection center;
the preservation unit is abbreviated as: CGMCC;
the address of the depository: xilu No.1 Hospital No.3, beijing, chaoyang, north.
Drawings
FIG. 1 shows the F286 ERIC map of Streptococcus salivarius.
FIG. 2 shows the evolutionary tree of S.salivarius F286, based on the full-length sequence of the 16S rRNA gene.
FIG. 3 shows that the bacterial cells of Streptococcus salivarius F286 stimulate human Peripheral Blood Mononuclear Cells (PBMC) to secrete Interleukin (IL) -12 and Interleukin (IL) -10 in amounts, and the stimulation of the bacterial cells of Streptococcus salivarius F286 is stronger than that of the existing commercial probiotic Lactobacillus rhamnosus GG (LGG).
FIG. 4 shows that the somatic cells of Streptococcus salivarius F286 significantly prolonged the life of C.elegans (Caenorhabditis elegans), and that the ability of the somatic cells of Streptococcus salivarius F286 to prolong the life of nematodes is comparable to that of the existing commercial probiotic Lactobacillus rhamnosus GG (LGG).
FIG. 5 shows that the streptococcus salivarius F286 somatic cells significantly improve the expression level of nematode (Caenorhabditis elegans) immunity genes, and the capability of the streptococcus salivarius F286 somatic cells in improving the nematode immunity gene expression is equivalent to that of the existing commercial probiotic Lactobacillus rhamnosus GG (LGG).
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, sambrook et al: a LABORATORY MANUAL, second edition, cold Spring Harbor LABORATORY Press,1989and Third edition,2001; ausubel et al, current PROTOCOLS IN MOLECULAR BIOLOGY, john Wiley & Sons, new York,1987and periodic updates; the series METHODS IN ENZYMOLOGY, academic Press, san Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, third edition, academic Press, san Diego,1998; (iii) METHODS IN ENZYMOLOGY, vol.304, chromatin (P.M.Wassarman and A.P.Wolffe, eds.), academic Press, san Diego,1999; and METHODS IN MOLECULAR BIOLOGY, vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, totowa,1999, etc.
Materials and sources thereof:
sterile mice: shanghai Slek's laboratory animal center, C57BL/6J mice
Wilkins-Chalgren (WCH) medium: qingdao, haibop; the goods number is: HB0261
M17 medium: qingdao, haibop; the goods number is: HB0391
The NGM culture medium is prepared by the following reagents:
NaCl,Sigma S7653-1KG
Peptone，Sigma P6713-500G
Agar，Sigma 05038-500G
CaCl 2 .2H 2 O,Sigma C3881-500G
MgSO 4 .7H 2 O，Sigma M1880-500G
KH 2 PO 4 ,Sigma P0662-500G
K 2 HPO 4 .3H 2 O，Sigma,P9666-500G
Cholesterol，Sigma C8667-1G
the NGM culture medium configuration method comprises the following steps: 3g of NaCl, 2.5g of Peptone and 20g of Agar are weighed, 975mL of single distilled water is added for dissolution, the mixture is sterilized by high pressure steam at 121 ℃ for 20min, and the mixture is cooled to about 65 ℃. Under sterile conditions, the following sterile solutions were added: 1mL 1M CaCl 2 ，1mL 1M MgSO 4 25mL of 1M potassium phosphate buffer (pH 6.0), and 1mL of 5mg/mL of Cholesterol. Mixing, subpackaging with sterile pipette into 6 cm culture dish (15 mL each), standing at room temperature for solidification to obtain NGM agar plate
Nematodes: american nematode Collection CGC (Camphorhabditis Genetics Centre, university of Minnesota, minneapolis)
Coli OP50: american nematode Collection CGC (Camphorhabditis Genetics Centre, university of Minnesota, minneapolis)
Example 1 isolation of Streptococcus salivarius F286
100 microliters of freshly collected human breast milk was gavaged into 8-week-old sterile mice grown in sterile bags, and each mouse was gavaged twice every other day with the same breast milk. Mice were kept on sterile bags for 8 weeks after the gavage was completed. Bacterial isolation was performed using mouse feces from week 8.
1mL of sterile 0.01mol/L phosphate buffer (PBS, 0.1% by weight of L-Cysteine) was immediately added to the mouse feces, and the mixture was sufficiently shaken to make the feces sample uniformly turbid. Making the fecal suspension into 10 -2 ，10 -3 ，10 -4 ，10 -5 ，10 -6 ，10 -7 ，10 -8 ，10 -9 Dilutions of each gradient were plated at 100. Mu.L each on Wilkins-Chalgren (WCH) and M17 plates of two solid media (1.2% agar), and three plates were repeated for each dilution gradient. All plates were placed upside down in an anaerobic incubator and incubated at 37 ℃. After 48 hours of culture, the plate with moderate colony number and more separable single colonies is selected. According to different coloniesMorphology and size, colonies were randomly picked on each plate of each medium, transferred to the corresponding new WCH and M17 solid medium plates, respectively, and numbered. Each single colony was streaked three times. And (3) picking the purified single colony into a liquid M17 culture medium, carrying out enrichment culture for 24 hours, and storing and subsequently identifying the obtained culture.
Example 2 identification of Streptococcus salivarius F286
(1) Extracting genome DNA: after 24 hours of anaerobic liquid culture, 3mL of the bacterial solution was collected, 9000g of the solution was centrifuged for 5 minutes to collect the cells, 475. Mu.L of TE buffer (10 mM Tris-HCl,1mM EDTA, pH 8.0) and 25. Mu.L of lysozyme (lysozyme, 50 mg/mL) were added, shaking and incubation were carried out at 37 ℃ for 1 hour, 5. Mu.L (20. Mu.g/mL) of proteinase K and 50. Mu.L (20%) of SDS were added, shaking and mixing were carried out, and then incubation was carried out at 55 ℃ for 30 minutes. Adding equal volume of about 550 μ L phenol chloroform isoamyl alcohol (volume ratio 25. The supernatant was added with two volumes (about 800. Mu.L) of absolute ethanol and 80. Mu.L (3M) of sodium acetate, which had been placed at-20 ℃ in advance, and left to stand in a refrigerator at-20 ℃ for 2 hours to precipitate DNA, and centrifuged at 14000rpm for 15min to collect DNA. After drying in vacuo at low temperature, the mixture was dissolved in 50. Mu.l of TE buffer (Tris-HCl, pH8, 10 mM). Mu.l RNase (20 mg/mL) was added thereto, and the mixture was gently mixed and incubated at 37 ℃ for 30min to digest RNA. DNA concentration was quantified using a microplate reader SpectraMax M5 (Molecular Devices, san Francisco, USA) in combination with PicoGreen fluorescent dye (Thermo Fisher Scientific, sunnyvale, USA).
(2) ERIC-PCR (Enterobacterial reliable interactive consensus sequence-PCR) fingerprinting: the upstream of an amplification primer of the ERIC-PCR is ERIC1 (SEQ ID NO.1: 5'-atgtaagctcctggggattcac-3'), the downstream is ERIC2 ((SEQ ID NO.2: 5'-aagtaagtgactggggtgagcg-3') 25 mu L PCR amplification system contains 20ng of bacterial genome DNA, the concentration of each of four deoxynucleotides (dNTPs) is 200mM,2.5U of TaKaRa rTaq DNA polymerase (Takara, dalian, china), 1 XPCR buffer (Mg 2+ free), 2mM MgCl 2 Primers were 10pM each. The PCR procedure was: pre-denaturation at 95 ℃ for 7min; denaturation at 95 ℃ for 30s, annealing at 52 ℃ for 1min, extension at 65 ℃ for 8min, and circulation for 30 times; finally, extension is carried out for 16min at 65 ℃. Get 400ng ERIC-PCR product was subjected to 1.5% (w/v) agarose electrophoresis using the UVI gel imaging System (Tanon 3500, tanon science)&Technology co., ltd., china) was photographed to obtain a fingerprint (fig. 1).
The ERIC map is a representation of a specific genome of the strain and can be used as a characteristic map of a streptococcus salivarius F286 strain.
(3) PCR amplification, cloning, sequencing and evolutionary position analysis of 16S rRNA gene full-length sequence of streptococcus salivarius F286 CGMCC No.17579 strain:
primers used for PCR amplification of the full-length sequence of the 16S rRNA gene of the strain were 27f (SEQ ID NO.3: 5'-agagtttgatcctggctcag-3') and 1492r (SEQ ID NO.4: 5'-cggcttaccttgttacgactt-3'). The 25. Mu.L system included 0.75U rTaq DNA polymerase (Takara, dalian, china), 1 XPCR buffer (Mg 2+ free), 2mM MgCl 2 10pmol of each primer, 200. Mu.M of each of the four deoxynucleotides, and 10ng of bacterial genomic DNA as a template. The amplification procedure was as follows: pre-denaturation at 95 ℃ for 7min; denaturation at 94 deg.C for 30s, annealing at 52 deg.C for 1min, and extension at 65 deg.C for 8min, and repeating the above steps for 25 times; finally, extension was carried out at 65 ℃ for 16min.
The PCR product was purified according to the instructions of the Gel Extraction Kit 200 (Omega, USA). The purified PCR product was ligated with the Vector pGEM-T Easy Vector (Promega, madison, USA) according to the ligation kit instructions, and the ligation product was transformed into host E.coli DH 5. Alpha. Competent cells (Transgen, beijing, china). Then, the cells were spread on LB-ampicillin (100. Mu.g/mL) plates to which IPTG and X-Gal were added in advance at a predetermined concentration, cultured at 37 ℃ for 12 hours, and white spot positive clones were randomly selected and sequenced (Life Technologies, shanghai, china). The full-length sequence of the 16S rRNA gene of the streptococcus salivarius F286 strain is shown in SEQ ID NO. 5: g a c g a a c g c t g g c g g c g t g c c t a a t a c a t g c a a g t a g a t c g c t g a a g a g a g g a g c t t g c t c t t c t t g g a t g a g t t g c g a a c g g g t g a g t a a c g c g t a g g t a a c c t g c c t t g t a g c g g g g g a t a a c t a t t g g a a a c g a t a g c t a a t a c c g c a t a a c a a t g g a t g a c a c a t g t c a t t t a t t t g a a a g g g g c a a t t g c t c c a c t a c a a g a t g g a c c t g c g t t g t a t t a g c t a g t a g g t g a g g t a a c g g c t c a c c t a g g c g a c g at a c a t a g c c g a c c t g a g a g g g t g a t c g g c c a c a c t g g g a c t g a g a c a c g g c c c a g a c t c c t a c g g g a g g c a g c a g t a g g g a a t c t t c g g c a a t g g g g g c a a c c c t g a c c g a g c a a c g c c g c g t g a g t g a a g a a g g t t t t c g g a t c g t a a a g c t c t g t t g t a a g t c a a g a a c g a g t g t g a g a g t g g a a a g t t c a c a c t g t g a c g g t a g c t t a c c a g a a a g g g a c g g c t a a c t a c g t g c c a g c a g c c g c g g t a a t a c g t a g g t c c c g a g c g t t g t c c g g a t t t a t t g g g c g t a a a g c g a g c g c a g g c g g t t t g a t a a g t c t g a a g t t a a a g g c t g t g g c t c a a c c a t a g t t c g c t t t g g a a a c t g t c a a a c t t g a g t g c a g a a g g g g a g a g t g g a a t t c c a t gt g t a g c g g t g a a a t g c g t a g a t a t a t g g a g g a a c a c c g g t g g c g a a a g c g g c t c t c t g g t c t g t a a c t g a c g c t g a g g c t c g a a a g c g t g g g g a g c g a a c a g g a t t a g a t a c c c t g g t a g t c c a c g c c g t a a a c g a t g a g t g c t a g g t g t t g g a t c c t t t c c g g g a t t c a g t g c c g c a g c t a a c g c a t t a a g c a c t c c g c c t g g g g a g t a c g a c c g c a a g g t t g a a a c t c a a a g g a a t t g a c g g g g g c c c g c a c a a g c g g t g g a g c a t g t g g t tt a a t t c g a a g c a a c g c g a a g a a c c t t a c c a g g t c t t g a c a t c c c g a t g c t a t t t c t a g a g a t a g a a a g t t a c t t c g g t a c a t c g g t g a c a g g t g g t g c a t g g t t g t c g t c a g c t c g t g t c g t g a g a t g t t g g g t t a a g t c c c g c a a c g a g c g c a a c c c c t a t t g t t a g t t g c c a t c a t t c a g t t g g g c a c t c t a g c g a g a c t g c c g g t a a t a a a c c g g a g g a a g g t g g g g a t g a c g t c a a a t c a t c a t g c c c c t t a t g a c c t g g g c t a c a c a c g t g c t a c a a t g g t t g g t a c a a c g a g t t g c g a g t c g g t g a c g g c a a g c t a a t c t c t t a a a g c c a a t c t c a g t t c g g a t t g t a g g c t g c a a c t c g c c t a c a t g a a g t c g g a a t c g c t a g t a a t c g c g g a t c a g c a c g c c g c g g t g a a t a c g t t c c c g g g c c t t g t a c a c a c c g c c c g t c a c a c c a c g a g a g t t t g t a a c a c c c g a a g t c g g t g a g g t a a c c t t t t g g a g c c a g c c g c c t a a g g t g g g a t a g a t g a t t g g g g t g
BLAST (http:// www.ncbi.nlm.ni h. Gov/BLAST) alignment of the obtained 16S rRNA gene sequences in the Genbank database was performed, and the bacteria having the most similar sequences to the known bacteria in the database were: streptococcus salivaria strain NCTC8618 has a similarity of 99.93%. A phylogenetic tree (Neighbor-joiningphylogenetic tree) was constructed using MEGA 5 software (Molecular evolution Genetics Analysis package), showing that the F286 strain is S.salivarius (FIG. 2).
Example 3 Heat-killed bacterial cells of Streptococcus salivarius F286 promote secretion of Interleukin 12 (IL-12) and Interleukin 10 (IL-10) by human Peripheral Blood Mononuclear Cells (PBMCs)
Streptococcus salivarius F286 and lactobacillus rhamnosus LGG were cultured in M17 (china, qingdao, haibo) and MRS (china, qingdao, haibo) liquid medium, respectively, for 8 hours, reaching the plateau phase. The culture was centrifuged at 5,000g for 10min, and cells of Streptococcus salivarius F286 and Lactobacillus rhamnosus LGG were collected. After resuspending the bacterial cells with PBS, the cells were centrifuged at 5,000g for 10min, and washed 2 times to remove the bacterial culture medium. The cell concentration of the cells was adjusted to 10 with PBS 8 CFU/ml and 10 9 CFU/ml, water bath at 65 deg.C for 20min, killing thallus cells by heat, and freezing to-80 deg.C for use.
In 24-well plates, 2X 10 inoculations per well 6 After the addition of 20. Mu.l of 1X 10 cells (PBMC) to individual Peripheral Blood Mononuclear Cells (PBMC) 8 CFU/ml or 1X 10 9 CFU/ml heat-killed Streptococcus salivarius F286 thallus and Lactobacillus rhamnosus LGG thallus with final volume of 1ml, constructed cells and bacteriaRatios of 1:1 and 1:10 in a co-incubation system. In the negative control group, no bacterial cells were added, and 20. Mu.l of PBS was added. The cell culture medium is 1640 cell culture medium containing 10% fetal bovine serum and 1% streptomycin and ampicillin. The mixture was incubated at 37 ℃ in a 5% carbon dioxide incubator for 24 hours. The cell culture supernatants were collected and tested for interleukin 10 and interleukin 12 concentrations using an ELISA kit.
As shown in FIG. 3, the bacterial cells of Streptococcus salivarius F286 stimulated more Interleukin (IL) -12 and Interleukin (IL) -10 secretion from human Peripheral Blood Mononuclear Cells (PBMC) (FIG. 3), while the bacterial cells of Lactobacillus rhamnosus LGG stimulated only a small amount of Interleukin (IL) -12 and Interleukin (IL) -10 secretion from human Peripheral Blood Mononuclear Cells (PBMC).
Example 4 somatic cells of Streptococcus salivarius F286 increase expression levels of nematode immunity genes and prolong nematode longevity
(1) Somatic cells of streptococcus salivarius F286 prolong life of nematodes
Streptococcus salivarius F286 and Lactobacillus rhamnosus LGG were cultured in M17 medium (China, qingdao, haibo) and MRS (China, qingdao, haibo) medium, respectively, at 37 ℃ anaerobic workstation (DG 500, DWS, united Kingdom) for 8 hours. Escherichia coli OP50 was cultured overnight at 37 ℃ in an aerobic condition. Centrifuging the cultured bacterial liquid for 10min at 15000 Xg, removing supernatant, collecting streptococcus salivarius F286, lactobacillus rhamnosus LGG and escherichia coli OP50 thalli, centrifuging for 10min under the condition of 15000 Xg by using a sterile M9 buffer solution, and washing the bacterial thalli twice. The concentration of the washed cells was adjusted to 10 mg/100. Mu.l (wet weight/volume) with M9 buffer solution, and the mixture was mixed. 100. Mu.l of bacterial resuspension containing 10mg of cells was dropped on a 6 cm diameter NGM (mNGM) plate to which no peptone was added.
After the nematodes growing to the L3 stage on the peptone-added NGM plates and feeding on the Escherichia coli OP50, the nematodes were transferred to the mNGM plates, respectively feeding on the Streptococcus salivarius F286, lactobacillus rhamnosus LGG and Escherichia coli OP50, and cultured at 25 ℃. Streptococcus salivarius F286, lactobacillus rhamnosus LGG and Escherichia coli OP50 were individually plated on 5 parallel plates (20-25 plates) for a total of 100-125 nematodes for life testing and comparison. In the course of the experiment, the nematodes were considered dead in the absence of a slight stimulation of the platinum wire. Kaplan-Meier Survival Analysis was performed using OASIS 2 (Online Application for surveyal Analysis 2) Online software and the difference in mean life span of nematodes fed different bacterial strains was examined using log-rank.
The results are shown in fig. 4, where both somatic cells of streptococcus salivarius F286 and LGG cells of lactobacillus rhamnosus significantly prolonged the life span of the nematodes, compared to the standard food escherichia coli OP50 of nematodes; meanwhile, the two have no difference in the capability of prolonging the life span of the nematodes. The safety and the probiotics of the streptococcus salivarius F286 are illustrated.
(2) Extraction of RNA of nematode of thallus cell of streptococcus salivarius F286
After 14 days of feeding Streptococcus salivarius F286, lactobacillus rhamnosus LGG, or Escherichia coli OP50, 500 nematodes were collected, and RNAs were extracted after the nematodes were lysed using TRIZOL reagent (Invitrogen), the RNAs were purified using RNeasy Mini Kit (Qiagen), and residual DNAs were removed using DNaseI Kit (Invitrogen).
(3) Method for measuring thallus cells of streptococcus salivarius F286 by qPCR (quantitative polymerase chain reaction) method to improve expression level of nematode immune genes, and purifying nematode RNA by using SuperScript TM First-Strand Synthesis System for RT-PCR (Invitrogen) reverse transcription kit, first cDNA Strand was synthesized using oligo (dT) as primer. Quantitative detection of nematode immunity genes was performed using SYBR Green Supermix (BIO-RAD) (Roche, lightCycler 96). Using housekeeping gene act-1 as reference gene and 2 -ΔΔCt The relative expression level of the immune gene was calculated (see FIG. 5).
The results show that the somatic cells of streptococcus salivarius F286 have obviously improved the expression level of nematode immunity genes (cpr-1, cpr-5, lys-7, clec-60, clec-85, C15C8.3) compared with the standard food escherichia coli OP50 of nematodes, and the capacity of the somatic cells of streptococcus salivarius F286 to improve the expression of the nematode immunity genes is equivalent to that of lactobacillus rhamnosus LGG.
The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the methods and compositions set forth herein, as well as variations of the methods and compositions of the present invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.
Sequence listing
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<120> Streptococcus salivarius F286 and uses thereof
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gacgaacgct ggcggcgtgc ctaatacatg caagtagatc gctgaagaga ggagcttgct 60
cttcttggat gagttgcgaa cgggtgagta acgcgtaggt aacctgcctt gtagcggggg 120
ataactattg gaaacgatag ctaataccgc ataacaatgg atgacacatg tcatttattt 180
gaaaggggca attgctccac tacaagatgg acctgcgttg tattagctag taggtgaggt 240
aacggctcac ctaggcgacg atacatagcc gacctgagag ggtgatcggc cacactggga 300
ctgagacacg gcccagactc ctacgggagg cagcagtagg gaatcttcgg caatgggggc 360
aaccctgacc gagcaacgcc gcgtgagtga agaaggtttt cggatcgtaa agctctgttg 420
taagtcaaga acgagtgtga gagtggaaag ttcacactgt gacggtagct taccagaaag 480
ggacggctaa ctacgtgcca gcagccgcgg taatacgtag gtcccgagcg ttgtccggat 540
ttattgggcg taaagcgagc gcaggcggtt tgataagtct gaagttaaag gctgtggctc 600
aaccatagtt cgctttggaa actgtcaaac ttgagtgcag aaggggagag tggaattcca 660
tgtgtagcgg tgaaatgcgt agatatatgg aggaacaccg gtggcgaaag cggctctctg 720
gtctgtaact gacgctgagg ctcgaaagcg tggggagcga acaggattag ataccctggt 780
agtccacgcc gtaaacgatg agtgctaggt gttggatcct ttccgggatt cagtgccgca 840
gctaacgcat taagcactcc gcctggggag tacgaccgca aggttgaaac tcaaaggaat 900
tgacgggggc ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc 960
ttaccaggtc ttgacatccc gatgctattt ctagagatag aaagttactt cggtacatcg 1020
gtgacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc 1080
aacgagcgca acccctattg ttagttgcca tcattcagtt gggcactcta gcgagactgc 1140
cggtaataaa ccggaggaag gtggggatga cgtcaaatca tcatgcccct tatgacctgg 1200
gctacacacg tgctacaatg gttggtacaa cgagttgcga gtcggtgacg gcaagctaat 1260
ctcttaaagc caatctcagt tcggattgta ggctgcaact cgcctacatg aagtcggaat 1320
cgctagtaat cgcggatcag cacgccgcgg tgaatacgtt cccgggcctt gtacacaccg 1380
cccgtcacac cacgagagtt tgtaacaccc gaagtcggtg aggtaacctt ttggagccag 1440
ccgcctaagg tgggatagat gattggggtg 1470

Claims (3)

1. The application of streptococcus salivarius F286 in preparing medicines is disclosed, wherein the preservation number of the streptococcus salivarius F286 is CGMCC No.17579, and the medicines have the effect of promoting peripheral blood mononuclear cells of mammals to secrete interleukin IL-12 and interleukin IL-10.
2. Use according to claim 1, characterized in that: the streptococcus salivarius F286 is killed.
3. Use according to claim 1, characterized in that: the content of streptococcus salivarius F286 in the medicine is 10 8 CFU/ml -10 10 CFU/ml。
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WO2001027143A1 (en) * 1999-10-12 2001-04-19 Blis Technologies Limited Lantibiotic
WO2010034844A1 (en) * 2008-09-29 2010-04-01 Actogenix N.V. Reduced colonization of microbes at the mucosa
EP3351259A1 (en) * 2017-01-18 2018-07-25 Symrise AG Probiotics for aggregation with disease-associated species in the oral cavity
CN109675017A (en) * 2019-02-28 2019-04-26 北京大学人民医院(北京大学第二临床医学院) Application of the streptococcus salivarius element in the drug of preparation prevention and/or treatment autoimmune disease

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曹杰，申剑.人母乳细菌Streptococcus salivarius F286 和S. parasanguinis F278 对HT29细胞的细胞毒性.《基因组学与应用生物学》.2019,第38卷(第7期),第3030-3036页. *

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