CN116491655A - Application of probiotic prebiotic composition in preparation of food for improving intestinal probiotic colonization - Google Patents
Application of probiotic prebiotic composition in preparation of food for improving intestinal probiotic colonization Download PDFInfo
- Publication number
- CN116491655A CN116491655A CN202310440525.6A CN202310440525A CN116491655A CN 116491655 A CN116491655 A CN 116491655A CN 202310440525 A CN202310440525 A CN 202310440525A CN 116491655 A CN116491655 A CN 116491655A
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- Prior art keywords
- bifidobacterium
- probiotic
- colonization
- lactobacillus
- strains
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- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
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Classifications
-
- 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
- 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
- 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
- A23C9/156—Flavoured milk preparations ; Addition of fruits, vegetables, sugars, sugar alcohols or sweeteners
-
- 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
- A23C9/16—Agglomerating or granulating milk powder; Making instant milk powder; Products obtained thereby
-
- 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
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
-
- 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
- A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
- A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7016—Disaccharides, e.g. lactose, lactulose
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- 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
- A61K35/745—Bifidobacteria
-
- 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
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- 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
-
- 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
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- 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
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- 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
- C12R2001/225—Lactobacillus
-
- 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
- C12R2001/225—Lactobacillus
- C12R2001/23—Lactobacillus acidophilus
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses application of a probiotic prebiotic composition in preparation of medicines or foods for improving colonization of intestinal probiotics, wherein the intestinal probiotics are selected from lactobacillus and/or bifidobacteria. The probiotic prebiotic composition can promote the colonization of most of breast milk oligosaccharide non-metabolizing strains through the combination of bifidobacterium bifidum R0071 and 2 '-fucosyllactose, has no inhibition effect on the colonization of the breast milk oligosaccharide metabolizing strain R0033, and further shows that the combination of bifidobacterium bifidum R0071 and 2' -fucosyllactose can promote the colonization of the breast milk oligosaccharide non-metabolizing strains by promoting the growth of the breast milk oligosaccharide non-metabolizing strains.
Description
Technical Field
The invention belongs to the technical field of medical foods, and particularly relates to application of a probiotic prebiotic composition in preparation of medicines or foods for improving intestinal probiotic field planting.
Background
A probiotic is a living microorganism and helps the host (human or other organism) to be healthy when ingested in an appropriate amount. At present, china adopts an approval form for infant edible strains, and 14 strains are included in a list. Although these strains have been demonstrated to have some adhesion to intestinal cells in vitro experiments, most strains still have short colonization time in the clinic, resulting in poor clinical results (Candela et al 2005; capurso 2019;Gopal et al.2001;Greene and Klaenhammer 1994;Jungersen et al.2014;Olivares et al.2006;Toscano et al.2014;Xiao et al.2021). Probiotic colonization is not simply to adhere to epithelial cells, but after adhesion probiotics can be subject to harsh environmental challenges of the gastrointestinal tract, such as acidic environments, antibiotics, pathogenic bacteria, salt tolerance, washout of fluids, etc. Although most commercial probiotic strains have been subjected to most environmental tolerance evaluations, under the environmental pressure of antibiotics, we cannot use drug resistant strains, and therefore it is necessary to develop a safe way to improve the environmental tolerance of probiotics and their colonization.
Biofilm (Biofilm) is a new strategy which has been attracting attention by a great number of students, and it means that bacteria adhere to a certain interface and self-secrete Extracellular matrix, mainly Extracellular Polysaccharide (EPS), protein, extracellular DNA (eDNA), etc., forms a closed space, and the inside bacteria adhere to each other to form a form of co-existence of bacterial population. Numerous studies have shown that biofilms have physical stability, surface inductivity, gradient and diversity of internal environmental profiles, and the like, which can make bacteria (e.g., probiotics) within the biofilm lower than adverse environmental conditions, such as antibiotics (Salas-Jara et al 2016). Meanwhile, studies have shown that knockout strains of genes related to biofilm formation (such as luxS, rfbP, etc.) have significantly reduced colonization in vivo, which has shown that the biofilm formation capacity of probiotics is one of the key factors for their colonization (Xiao et al 2021). However, most commercial strains do not have the capability of biological film measurement, and a few strains do relevant evaluation, but the environment simulated by experiments adopts common carbon source (glucose) which does not accord with the environmental conditions of intestinal tracts, so that the carbon source simulated by prebiotics is more reasonable.
At present, the number of probiotics which are approved to be used is large, but the prebiotic metabolic capacities of different strains are different, the biggest difference is the metabolic capacity of breast milk oligosaccharide (human milk oligosaccharides, HMO), only a few probiotics strains can be metabolized, and most probiotics strains cannot be utilized, so that most commercial probiotics strains cannot play the biggest role of HMO when being combined with HMO. In order to promote the health efficacy of HMO and the colonization amount of most of HMO probiotics which cannot be metabolized, the invention develops a probiotic-prebiotic combination HMO probiotics to be applied to the colonization of probiotics in the gastrointestinal tract by exploring the interaction of the probiotics under different prebiotics.
The difficulty of the invention is that there are various interactions, such as synergism, antagonism, symbiosis, competition, etc. between strains, so that multi-strain probiotic products often have the potential risk of antagonism between strains. Several studies have shown in the past that combinations of certain strains do not exert a synergistic effect on certain functions. Mcfarland found by meta analysis of clinical studies that the combined use of lactobacillus rhamnosus GG with other strains (bifidobacterium animalis subspecies lactis Bb-12 or HN019, or bifidobacterium longum subspecies longum Bb 536) was less effective in preventing necrotic colitis than single use. In contrast, the combination of Lactobacillus rhamnosus GG animals with Bifidobacterium animalis subspecies Bb-12 significantly increased the eradication rate of helicobacter pylori compared to the single strain. This suggests that the same probiotic combination may not always maintain a synergistic effect in different functional directions and may even act as an antagonistic effect. In addition, the interaction relationship between strains also changes with changes in environmental conditions. At present, the research on the interaction relation of probiotics is still very limited, and few researches use the same strain to explore the difference in efficacy between the two. Therefore, the invention screens the combination of different probiotics and probiotics, and develops the application of the probiotic prebiotics combination HMObiotics to synergistically enhance the colonization of probiotics in the gastrointestinal tract.
Disclosure of Invention
In order to make up the defects of the prior art, the invention aims to provide an application of a probiotic prebiotic composition HMObiotics in improving intestinal probiotic field planting. In order to achieve the purpose of the invention, the following technical scheme is adopted:
the invention provides an application of a probiotic prebiotic composition in improving intestinal tract probiotic colonization, which is characterized in that the probiotic in the probiotic prebiotic composition is bifidobacterium bifidum R0071, the prebiotic in the probiotic prebiotic composition is 2' -fucosyllactose, and the intestinal tract probiotic is lactobacillus and/or bifidobacterium.
Use according to claim 1, said lactobacillus or bifidobacterium being selected from at least one of bifidobacterium animalis subspecies lactis or bifidobacterium animalis Bb-12 (Bifidobacterium animalis subsp.lacts or Bifidobacterium animalis Bb-12), bifidobacterium longum subspecies infantis or bifidobacterium infantis R0033 (Bifidobacterium longum subsp.infantis or Bifidobacterium infantis R0033), bifidobacterium animalis subspecies lactis or bifidobacterium lactis HN019 and Bi-07 (Bifidobacterium animalis subsp.lactis or Lactobacillus lactis HN019, bifidobacterium animalis subsp.lactis or Lactobacillus lactis Bi-07), bifidobacterium breve M16V (Bifidobacterium breve M V), lactobacillus helveticus R0052 (Lactobacillus helveticus R0052), lactobacillus rhamnosus LGG or GG (Lactobacillus rhamnosus LGG or GG, lactobacillus rhamnosus HN 001), bifidobacterium subspecies longum Bb536 (Bifidobacterium longum subsp.longum Bb 536), lactobacillus acidophilus NCFM (Lactobacillus acidophilus NCFM).
In a preferred embodiment of the invention, the concentration of the probiotic is 1X 10 6 -1×10 14 CFU/100g, preferably 1X 10 8 -1×10 14 CFU/100g。
In a preferred embodiment of the invention, the food product is a formula or a nutritional composition.
In a preferred embodiment of the invention, the formula or nutritional composition is an infant formula or infant nutritional composition product.
In a preferred embodiment of the present invention, the infant milk powder or the nutritional composition has a content of 2' -fucosyllactose of 10mg/100g to 10X 10 3 mg/100g, preferably 10X 10 1 ～10×10 3 mg/100g; the content of Bifidobacterium bifidum R0071 is 1×10 6 -1×10 14 CFU/100g, preferably 1X 10 8 -1×10 14 CFU/100g。
The invention has the advantages that: the probiotic prebiotic composition can promote the colonization of most of breast milk oligosaccharide non-metabolizing strains through the combination of bifidobacterium bifidum R0071 and 2 '-fucosyllactose, has no inhibition effect on the colonization of the breast milk oligosaccharide metabolizing strain R0033, and further shows that the combination of bifidobacterium bifidum R0071 and 2' -fucosyllactose can promote the colonization of the breast milk oligosaccharide non-metabolizing strains by promoting the growth of the breast milk oligosaccharide non-metabolizing strains.
Drawings
FIG. 1 oligosaccharide metabolizing ability of different probiotic strains;
FIG. 2 effect of bifidobacterium bifidum R0071 in combination with 2' -FL on intestinal probiotic colonization.
Note that: asterisks indicate significant differences (P < 0.05) compared to the test strain single strain culture
FIG. 3 effect of bifidobacterium bifidum R0071 in combination with FOS on intestinal probiotic colonization.
Note that: asterisks indicate significant differences (P < 0.05) compared to the test strain single strain culture
FIG. 4 effect of bifidobacterium bifidum R0071 in combination with glucose on intestinal probiotic colonization.
Note that: asterisks indicate significant differences (P < 0.05) compared to the test strain single strain culture
Detailed Description
In order to further understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Unless otherwise specified, all reagents involved in the examples of the present invention are commercially available products and are commercially available.
Example 1:
1 Experimental method
1.1 preparation of Medium
cfMRS broth medium formulation (1L) containing different carbon sources: 10g of carbon source, 10g of peptone, 5g of yeast extract, 1mL of Tween 80,2g K 2 HPO 4 ，5g CH 3 COONa·3H 2 o,2g of tri-ammonium citrate, 200mg of MgSO 4 ·7H 2 O,50mg MnSO 4 ·4H 2 O,0.5g of cysteine salt, 1L of ultrapure water, and the pH was adjusted to 6.2 to 6.5. Wherein the carbon source comprises 2' -fucosyllactose (2 ' -FL), 6-sialyllactose (6 ' -SL), lactose-N-neotetraose (LNnT), fructo-oligosaccharide (FOS), galacto-oligosaccharide (GOS), glucose (Gluose).
1.2 Experimental strains and culture activation thereof
The study used 4 strains of lactobacillus and 8 strains of bifidobacteria in total, wherein the lactobacillus includes lactobacillus acidophilus NCFM, lactobacillus helveticus R0052, lactobacillus rhamnosus GG, lactobacillus rhamnosus HN019; the bifidobacterium includes Bifidobacterium longum subspecies infancy R0033, bifidobacterium longum subspecies infancy M63, bifidobacterium longum subspecies BB536, bifidobacterium animalis subspecies lactis BI-07, bifidobacterium animalis subspecies lactis HN019, bifidobacterium animalis subspecies Bb-12, bifidobacterium bifidum R0071, and Bifidobacterium breve M-16V. Table 1 below:
TABLE 1 list of probiotic strains
The probiotic strain frozen stock solution or the bacterial powder is respectively inoculated into an MRS agar culture medium by a plate marking or coating mode, and after anaerobic culture is carried out for 48 hours at 37 ℃, the MRS agar plate rich in single colony is obtained. Selecting single colony of each strain from MRS agar plate with sterile gun head or inoculating loop, inoculating into MRS broth, culturing at 37deg.C for anaerobic overnight to obtain activated probiotic bacteria liquid, and using for subsequent experiment, wherein bacteria liquid concentration passes Live/head TM The assay was performed using a kit (thermo fisher).
1.3 determination of the oligosaccharide Metabolic Capacity of the probiotics
Culture solution for each strain at night(method 1.2) dilution with cfMRS broth without carbon source and dilution with 10 6 CFU/mL was inoculated into cfMRS broth containing different oligosaccharides (2 '-FL,6' -SL, LNnT, FOS, GOS), cfMRS culture without carbon source was used as negative control, cfMRS broth containing glucose was used as positive control. After incubation in an anaerobic incubator at 37℃for 72h, OD measurements were performed at a wavelength of 595 nm.
1.4 probiotic interaction Co-culture experiments
Based on the experimental results of 1.3, 3 strains were selected for co-cultivation experiments, lactobacillus helveticus R0052, bifidobacterium longum subspecies infantis R0033, and bifidobacterium bifidum R0071, respectively. The culture solution (method 1.2) of each strain is 10 percent 6 CFU/mL was inoculated into cfMRS broth containing 2' -FL, and a total of 3 experimental groups were cultured as a single strain, and 3 experimental groups were cultured as a double strain (r0052+r0033, r0052+r0071, r0033+r0071). The cfMRS broth without carbon source was inoculated with a single strain as a negative control group. After anaerobic culture for 20 hours at 37 ℃, a proper amount of sample is taken, DNA extraction is carried out by using a bacterial DNA small extraction kit (DNB 361-03B, mabio), quality inspection is carried out on the DNA sample by using a Nanodrop 2000 (ThermoFisher), and the qualified sample is subjected to quantitative analysis on the cell number of each strain in the sample by qPCR.
quantitative analysis of qPCR adopts an external standard method, firstly, a standard curve of a threshold cycle number Cq value and DNA copy number is drawn, concentration determination is carried out on DNA samples of single strains of each probiotics through Nanodrop 2000, then double distilled water is used for 10 times dilution, and gradient copy number concentration (10 2 To 10 9 ) A qPCR amplification system (table 2 below) was configured, and the Cq value of each sample was determined using each strain-specific primer (table 4 below) in a qPCR apparatus according to a set procedure (table 3 below) to obtain a standard curve of Cq value versus DNA copy number calculated by the following formula:
for each probiotic bacterial liquid (10 5 -10 7 CFU/mL), determining the Cq value at each concentration by qPCR, obtaining the corresponding DNA copy number according to a standard curve, then calculating the ratio of the viable count of each strain to the total number of DNA copy numbers, and taking the average value of the ratios at three concentration gradients as a conversion coefficient (a). The cell number of each strain in the final sample was quantitatively analyzed by the following formula:
test strain cell number (cell number/mL) =test strain copy number (copies/. Mu.L) ×conversion coefficient a
TABLE 2 qPCR amplification System configuration
TABLE 3 qPCR amplification procedure
TABLE 4 specific forward and reverse strand primers for each species
1.5 determination of the amount of probiotic colonization
Based on the experimental results of 1.3 and 1.4, 5 strains were selected for experiments to investigate the effect of bifidobacterium bifidum R0071 on the colonization amount of probiotics, the strains involved were lactobacillus helveticus R0052, bifidobacterium longum subspecies infantis R0033, bifidobacterium bifidum R0071, bifidobacterium longum subspecies BB536, and bifidobacterium animalis subspecies lactis HN019, respectively. The experiment will be performed at three different carbon sources, including 2' -FL, FOS and glucose, and the effect of the combination of different carbon sources with bifidobacterium bifidum R0071 on the probiotic colonization. The bacterial liquid (method 1.2) was cultured overnight at 10 6 CFU/mL was inoculated into cfMRS broth containing 2' -FL, respectively. For a single strain group, each strain dilution was combined with cfMRS broth medium containing 2' -FL1, the method comprises the following steps: 1, mixing in proportion; for the two strain group, the bifidobacterium bifidum dilution was at 1:1 was mixed with the rest of the experimental strains. Then, an appropriate amount (600. Mu.L) of the mixture was added to a 48-well microplate, and the microplate was left to stand still for anaerobic culture at 37℃for 24 hours. To remove free cells (non-adherent cells), microwells were gently washed twice with appropriate PBS; the biofilm at the bottom of the well plate was then scraped off using a sterile gun head and resuspended with 600 μl PBS. Staining the resuspended bacteria liquid according to the instruction of a nucleic acid staining kit (Thermo), incubating for 15min under dark, and quantitatively determining fluorescence by using a flow cytometer, and finally characterizing the fixed planting amount by the total bacteria number. The above experiments were repeated after replacing 2' -FL with FOS or glucose to investigate the effect of different carbon sources in combination with bifidobacterium bifidum R0071 on the probiotic colonization.
2 experimental results
2.1 oligosaccharide metabolism Capacity of different probiotic strains
As shown in FIG. 1, only R00033, M63 and R0071 in 2' -FL can grow well; only M63 and R0071 were able to grow well in 6' -SL; only R0033, M63, R0071 and M-16V were allowed to grow in LNnT; but R0071, GG and HN001 cannot grow on FOS; in addition, GG and HN001 also have limited growth in GOS. In conclusion, R0071 has strong metabolism ability on all tested breast milk oligosaccharides and is used as a representative of HMO decomposers; r0033 has metabolic capacity for part of the tested breast milk oligosaccharide metabolism, to be representative of part of HMO decomposers; r0052 has no catabolic ability on the tested breast milk oligosaccharides, and can be used as a representative of unavailable HMO, and the three can be used for carrying out subsequent interaction experiments to explore the interaction relationship among the strains.
2.2 synergistic growth between probiotics
Tables 5-7 show the total number of strains after co-cultivation of different probiotics in 2' -FL. Consistent with the results of 2.1, the total bacterial count of the R0052 single strain in the culture with 2' -FL as a carbon source did not significantly increase, which indicates that R0052 cannot metabolize 2' -FL for growth, while the total bacterial count of the R0033 and R0071 single cultures were significantly increased, which indicates that both strains can metabolize 2' -FL for growth. Furthermore, in experiments with co-cultivation of both strains, bifidobacterium bifidum R0071 was able to increase the cell numbers of bifidobacterium longum subspecies infantis R0033 and lactobacillus helveticus R0052, which results indicate that R0071 might decompose 2' -FL into fucose and lactose by releasing extracellular enzymes, thereby providing nutritional components for R0033 and R0052 and promoting growth and proliferation thereof.
TABLE 5 cell count of each strain after R0033 and R0071 were cultured alone or co-cultured in 2' -FL
Note that: the asterisks indicate significant differences (P < 0.05) compared to single strain cultures of the same line
TABLE 6 cell count of each strain after R0052 and R0071 were cultured alone or co-cultured in 2' -FL
Note that: the asterisks indicate significant differences (P < 0.05) compared to single strain cultures of the same line
TABLE 7 cell count of each strain after R0052 and R0033 were cultured alone or co-cultured in 2' -FL
Note that: the asterisks indicate significant differences (P < 0.05) compared to single strain cultures of the same line
2.3 effects of Bifidobacterium bifidum on intestinal probiotic colonization by different carbon source combinations
As shown in FIG. 2, the results demonstrate that the total number of probiotics in the mixed biofilm formed by R0071 and 9 different probiotic strains respectively is significantly increased compared with the single biofilm by taking 2'-FL as a carbon source, and the total number of probiotics comprises R0052, GG, HN001, NCFM, BB536, HN019, BI-07, bb12 and M-16V, which indicates that the combination of R0071 and 2' -FL can synergistically promote the colonization of different intestinal probiotics. The total number of probiotics in the mixed biofilm of R0071 and R0033 is equivalent to that of the biofilm of the single strain of R0033, which indicates that the combination of R0071 and 2' -FL does not influence the colonization of R0033.
As shown in FIG. 3, the result shows that the total bacterial count of probiotics in the mixed biofilm formed by R0071 and 5 different probiotic bacterial strains respectively is obviously increased compared with a single biofilm by taking FOS as a carbon source, wherein the total bacterial count comprises R0052, BB536 and GG, NCFM, BI-07; the total number of probiotics in the mixed biofilm of R0071 and 3 probiotics with different strains is obviously reduced compared with that of the biofilm with a single strain, and the mixed biofilm comprises R0033, HN019 and M-16V. This suggests that the combination of R0071 and FOS inhibits colonization of a portion of the strain.
As shown in FIG. 4, the results indicate that R0071 forms a mixed biofilm with 3 different probiotic strains with significantly increased total numbers of probiotics, including R0052, R0033 and BI-07, respectively, using glucose as a carbon source, compared to a single biofilm; the total number of probiotics in the mixed biofilm of R0071 and 4 probiotics with different strains is obviously reduced compared with that of the biofilm with a single strain, including BB536, HN019, NCFM and M-16V, which shows that the combination of R0071 and glucose can inhibit the colonization of partial strains.
In conclusion, the combination effect of R0071 and FOS is poor, and the effect of promoting colonization is not ideal, probably because R0071 cannot metabolize FOS, and thus does not play a role in synergy. The combination effect of R0071 and glucose is poor, and the strains compete with each other probably because the glucose belongs to a broad-spectrum carbon source, so that the synergistic effect is not achieved. The combination effect of R0071 and 2' -FL is optimal, has no inhibition effect on the colonization of all test strains, and can promote the colonization of most (9/10) test strains.
The results combined with 2.1,2.2 and 2.3 show that probiotics are difficult to colonize in the gut of infants fed with normal breast milk, as most probiotics cannot utilize breast milk oligosaccharides. Two strains of the metabolism strain of the breast milk oligosaccharide (2' -FL) exist, and only R0071 can promote the growth of other probiotics strains (R0052) in the breast milk oligosaccharide, so that the combination of the R0071 and the breast milk oligosaccharide can synergistically promote the growth metabolism of the non-metabolism strain of the breast milk oligosaccharide. Meanwhile, the combination of R0071 and 2'-FL can promote the colonization of most of the breast milk oligosaccharide non-metabolizing strains, and has no inhibition effect on the colonization of the breast milk oligosaccharide metabolizing strain R0033, so that the combination of R0071 and 2' -FL can promote the colonization of the breast milk oligosaccharide non-metabolizing strains by promoting the growth of the breast milk oligosaccharide non-metabolizing strains.
The foregoing describes preferred embodiments of the present invention, but is not intended to limit the invention thereto. Modifications and variations to the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.
Claims (6)
1. The application of a probiotic prebiotic composition in preparing a medicament or food for improving intestinal probiotic colonization is characterized in that the probiotic in the probiotic prebiotic composition is bifidobacterium bifidum R0071, the prebiotic in the probiotic prebiotic composition is 2-fucosyllactose, and the intestinal probiotic is selected from lactobacillus and/or bifidobacterium.
2. The use according to claim 1, said lactobacillus or bifidobacterium being selected from at least one, two, three or more of bifidobacterium animalis subsp.or bifidobacterium animalis Bb-12 (Bifidobacterium animalis subsp.lacts or Bifidobacterium animalis Bb-12), bifidobacterium longum subsp.infantis or bifidobacterium infantis R0033 (Bifidobacterium longum subsp.infantis or Bifidobacteriuminfantis R0033), bifidobacterium animalis subsp.lactis or bifidobacterium lactis HN019 and Bi-07 (Bifidobacterium animalis subsp.lactis or Lactobacillus lactis HN019, bifidobacterium animalis subsp.lactis or Lactobacillus lactis Bi-07), bifidobacterium breve M16V (Bifidobacterium breve M V), lactobacillus helveticus R0052 (Lactobacillus helveticus R0052), lactobacillus rhamnosus LGG or GG (Lactobacillus rhamnosus LGG or GG, lactobacillus rhamnosus HN 001), bifidobacterium longum subsp Bb536 (Bifidobacterium longum subsp.longum Bb 536), lactobacillus acidophilus NCFM (Lactobacillus acidophilus NCFM).
3. The use according to claim 1, wherein the concentration of the probiotic is 1 x 10 6 -1×10 14 CFU/100g。
4. The use according to claim 1, wherein the food product is a formula or a nutritional composition.
5. The use according to claim 4, wherein the formula or nutritional composition is an infant formula or infant nutritional composition product.
6. The use according to claim 5, wherein the infant milk powder has a content of 2' -fucosyllactose of 10mg/100g to 10X 10 3 mg/100g; the content of Bifidobacterium bifidum R0071 is 1×10 6 -1×10 14 CFU/100g。
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