CN117337992A - Probiotic prebiotic composition and application thereof - Google Patents
Probiotic prebiotic composition and application thereof Download PDFInfo
- Publication number
- CN117337992A CN117337992A CN202311240013.1A CN202311240013A CN117337992A CN 117337992 A CN117337992 A CN 117337992A CN 202311240013 A CN202311240013 A CN 202311240013A CN 117337992 A CN117337992 A CN 117337992A
- Authority
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- composition
- bifidobacterium
- cfu
- product
- salmonella
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- Granted
Links
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Classifications
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- 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
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- 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
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/385—Concentrates of non-alcoholic beverages
- A23L2/39—Dry compositions
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- 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
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- 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
- A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
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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
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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
- A61P1/12—Antidiarrhoeals
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- 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
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- 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
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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
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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- 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 relates to the technical field of foods, in particular to a probiotic prebiotic composition and application thereof, wherein the composition comprises breast milk oligosaccharide, bifidobacterium bifidum and bifidobacterium longum subspecies of infants, and has the advantages of improving the resistance of gastrointestinal tract to pathogenic bacteria infection and reducing salmonella colonization.
Description
Technical Field
The invention relates to the technical field of foods, in particular to a probiotic prebiotic composition and application thereof.
Background
Infant diarrhea is a gastrointestinal dysfunction in infant stage, and is mainly characterized by diarrhea and vomiting, and a part of newborns and infants can be affected by intense and excessive angina, and pathogenic factors are divided into three aspects, namely physique, infection and digestive dysfunction. Clinically, the number of times of defecation is increased, defecation and water electrolyte disturbance are mainly manifested, if the treatment is not carried out in time, serious water electrolyte disturbance occurs, and the life of infants can be endangered.
At present, the treatment medicines mainly comprise antibacterial and anti-inflammatory medicines, antidiarrheal medicines, traditional Chinese medicines, hormone medicines and the like, but the effects are poor, the hormone medicines can treat the symptoms without treating the root causes, and obvious side effects can be generated; the traditional Chinese medicine has more impurities, is not suitable for infants to take, and cannot take effect rapidly.
Diarrhea mainly occurs because pathogenic bacteria (including salmonella) cause unbalance of intestinal flora after infection, thereby causing dysfunction of intestinal absorption and water retention. However, probiotics are active microorganisms which play a role by improving the balance of intestinal microbial flora of a host, and have important physiological effects of improving the structure of the intestinal microbial flora, inhibiting pathogenic bacteria, eliminating cancerogenic factors, improving the immunity of the organism, reducing cholesterol and the like.
By taking probiotics, especially probiotics which can produce bacteriocin and antibacterial substances, intestinal harmful bacteria can be well inhibited. The balance of beneficial bacteria in the intestinal tract is regulated, so that the intestinal immune system is improved, and the immune system capacity is improved, so that diarrhea symptoms are effectively relieved.
Chinese patent application CN112244302a discloses a probiotic drop for improving infantile diarrhea and a preparation method thereof, comprising the following raw materials: bifidobacterium animalis, bifidobacterium lactis, bifidobacterium breve, lactobacillus rhamnosus, lactobacillus reuteri, lactobacillus acidophilus, lactobacillus fermentum, galacto-oligosaccharides, xylo-oligosaccharides, resistant dextrins, alginic acid, natural vitamin a, natural vitamin D, natural vitamin E, unsaturated fatty acids, vegetable oils, and phospholipid oils. However, the drop has a complicated formulation, and the diarrhea improving effect is still to be further improved.
Chinese patent application CN104206539A discloses a probiotic infant milk powder, which comprises 10-40 parts of lactose, 10-40 parts of desalted whey powder, 5-25 parts of skim milk powder, 0-12 parts of concentrated whey protein powder, 1-4.5 parts of composite prebiotics, 0.5-2.5 parts of mineral premix, 0.3-1.8 parts of arachidonic acid superfine powder, 0.04-0.4 parts of composite probiotic freeze-dried powder and the like; the composite probiotics freeze-dried powder comprises lactobacillus reuteri with a strain number of DSM17938 and bifidobacterium animalis with a strain number of Bb-12, and the composite probiotics comprises at least one of fructo-oligosaccharide, galacto-oligosaccharide, polydextrose and fructo-oligosaccharide.
Chinese patent application CN110432494a discloses a probiotic composition for preventing and treating infant allergic eczema and/or diarrhea, a preparation method and application thereof. The probiotic composition is prepared from the following components in parts by weight: 0.2-1 part of lactobacillus rhamnosus HN 001; 0.4-1.5 parts of bifidobacterium lactis HN 019; 45-55 parts of resistant dextrin; 35-45 parts of galacto-oligosaccharide; 5-12 parts of fructo-oligosaccharide.
However, the effects of improving immunity and adjusting intestinal flora of the product are still to be further improved.
It is therefore highly desirable to develop a probiotic prebiotic composition that addresses the above-described technical problems.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a probiotic prebiotic composition with good effects of improving immunity, improving diarrhea and regulating intestinal flora and capable of reducing salmonella colonisation and application thereof.
The invention is realized by the following technical scheme:
a probiotic prebiotic composition comprising breast milk oligosaccharide, bifidobacterium bifidum and bifidobacterium longum subspecies infancy.
Preferably, the composition consists of breast milk oligosaccharides, bifidobacterium bifidum and bifidobacterium longum subspecies infantis.
Preferably, the breast milk oligosaccharide is 2' -fucosyllactose.
More preferably, the bifidobacterium bifidum is bifidobacterium bifidum R0071.
More preferably, the bifidobacterium longum subspecies infancy is bifidobacterium subspecies infancy R0033.
More preferably, the concentration of said 2' -fucosyllactose in said composition is between 10mg and 50g/100g.
Preferably, the concentration of bifidobacterium bifidum in the composition is 1×10 6 -1×10 14 CFU/100g, more preferably 10 8 -10 12 CFU/100g。
Preferably, the concentration of the bifidobacterium longum subspecies infantis in the composition is 1 x 10 6 -1×10 14 CFU/100g, more preferably 10 8 -10 12 CFU/100g。
More preferably, the viable count ratio of bifidobacterium bifidum to bifidobacterium longum subspecies infancy is 1:0.1 to 10, preferably 1:0.5 to 5, more preferably 1:1.
More preferably, the mass ratio of the 2' -fucosyllactose to the total viable count of the bifidobacterium bifidum and bifidobacterium longum subspecies infantis is 1mg:10 4 -10 12 CFU, preferably 1mg:10 6 -10 10 CFU, further preferably 1mg:10 6 -10 8 CFU。
In another embodiment of the invention, the use of the above composition for the preparation of an immunity enhancing product is also disclosed.
Preferably, the enhancing immunity is specifically an increase in the resistance of the gastrointestinal tract to pathogenic infection.
More preferably, the pathogenic bacteria is salmonella.
Preferably, the product is a food or a pharmaceutical.
Preferably, the product comprises a nutritional supplement, a solid beverage, a lozenge, an oral cleanser or a probiotic micro-foam tablet.
Preferably, the immunity enhancing is specifically an immunity enhancing of a secretory infant and/or child.
Preferably, the enhancing immunity is specifically preventing and/or ameliorating diarrhea.
In another embodiment of the invention, the use of the above composition for the preparation of a product for regulating intestinal flora is also disclosed.
Preferably, the modulation of intestinal flora is in particular a reduction of salmonella colonization.
Preferably, the product is a food or a pharmaceutical.
More preferably, the product comprises a nutritional supplement, a solid beverage, a lozenge, an oral cleanser, or a probiotic micro-foam tablet.
In another embodiment of the invention, the use of the above composition for the preparation of a product for the treatment of diarrhea is also disclosed.
Preferably, the product is a food or a pharmaceutical.
Preferably, the product comprises a nutritional supplement, a solid beverage, a lozenge, an oral cleanser or a probiotic micro-foam tablet.
Preferably, the diarrhea treatment product is an infant diarrhea treatment product.
Preferably, the diarrhea is caused by salmonella.
In another embodiment of the invention, the use of the above composition for the preparation of a product for reducing salmonella colonization is also disclosed.
Preferably, the product is a food or a pharmaceutical.
Preferably, the product comprises a nutritional supplement, a solid beverage, a lozenge, an oral cleanser or a probiotic micro-foam tablet.
In another embodiment of the invention, there is also a product for enhancing immunity comprising the composition described above.
In another embodiment of the invention, it also relates to a product for regulating intestinal flora, comprising a composition as described above.
The beneficial effects of the invention are as follows:
the invention combines breast milk oligosaccharide, bifidobacterium bifidum and bifidobacterium longum subspecies for infants, has obvious synergistic effect, and has obvious effects in improving the resistance of gastrointestinal tract to pathogenic bacteria infection, treating diarrhea or regulating intestinal flora and reducing salmonella colonisation.
Furthermore, the invention has obvious synergistic effect by combining 2' -fucosyllactose, bifidobacterium bifidum R0071 and bifidobacterium longum subspecies R0033, and has obvious effects in improving the resistance of gastrointestinal tract to pathogenic bacteria infection, treating diarrhea or regulating intestinal flora and reducing salmonella colonization.
Drawings
FIG. 1 shows Salmonella SL 1344 colonization in cecal tissue of non-secretory and secretory mice.
FIG. 2 shows the results of the adhesion rate of different oligosaccharides to E.coli stdABCD strain to Caco-2 cells.
Detailed Description
The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.
Example 1
65 mother and infant volunteers were recruited from the hospital, and the criteria for the selection were healthy, term (gestational age > 37 weeks), vaginal delivery; medical science has proved to be healthy infants: asymptomatic and disease-free signs. The infant mother who participated in the experiment signed an informed protocol and ensured pure breast feeding during the experiment.
Mother breast milk samples were collected at the third month T3 (90 d±2d) by first month T1 (30 d±2d), second month T2 (60 d±2d) and third month T3 (90 d±2d) after baby delivery. Breast milk samples will be divided into secreted and non-secreted forms for analysis to detect α1, 2-fucosylated breast milk oligosaccharide content in three periods of mom's breast milk, including 2' -fucosyllactose (2 ' -fucesyl), lacto-N-fucoidan (LDFT), lacto-N-fucopentaose I (LNFP-I), lacto-lactose-N-difucose I (N-difucose I, LNDFH-I & lnnffh-I), monofucosyllacto-N-hexasaccharide I (monofucesyl-N-hexaose I, mfh-I), bifucosyl lactose-N-hexasaccharide (a). The results are shown in Table 1.
Infant faeces samples were also collected every two weeks during three months after delivery, and conventional tests were performed to detect common pathogenic bacteria causing diarrhea in infant faeces samples, such as campylobacter jejuni, escherichia coli diarrhoea, shigella, salmonella, aeromonas, rotavirus, and the like. The infant was then followed up to 2 years old, its diarrhea recorded and infant saliva samples were collected for secretion assays. Wherein infants and young children have three or more watery stools within 24 hours and are considered diarrhea, or two or more loose or watery stools exceeding the daily stool frequency of children as determined by researchers. The results are shown in Table 2.
Finally, the correlation between the content of alpha 1, 2-fucosylation breast milk oligosaccharide in breast milk at different stages and the content of bifidobacterium in the infant feces sample and the infection condition of pathogenic bacteria of the infant is found through statistics. The results are shown in tables 3 and 4. The correlation of the occurrence of diarrhea in infants after 1 year old with the genotype of the infants was also recorded during the follow-up, and the results are shown in Table 5.
TABLE 1 content of alpha 1, 2-fucosylated breast oligosaccharide in breast milk at different lactation phases
* And (3) injection: the same letter superscript for the same row of data indicates that the difference is not significant (p > 0.05), and the different letter indicates that the difference is significant (p < 0.05).
TABLE 2 symptoms of diarrhea in infants of breast milk type and 3 months after delivery
| Type of breast milk | Secretion type (n=48) | Non-secretory (n=17) |
| Infection and diarrhea symptoms | 1 | 4 |
| Infection is asymptomatic | 22 | 10 |
| Uninfected with | 25 | 3 |
TABLE 3 average amount of alpha 1, 2-fucosylated breast oligosaccharide in breast milk taken by diarrhea symptoms in infants three months after delivery
* And (3) injection: the same letter superscript for the same row of data indicates that the difference is not significant (p > 0.05), and the different letter indicates that the difference is significant (p < 0.05).
TABLE 4 Bifidobacterium content in stool samples for infants with different symptoms early in life
* And (3) injection: the same row of data superscripts contained the same letter indicating that the difference was not significant (p > 0.05) and the different letters indicating that the difference was significant (p < 0.05).
As can be seen from tables 1-4, most of the alpha 1, 2-fucosylated breast milk oligosaccharides in secreted breast milk were significantly higher than non-secreted breast milk three months before delivery; in the breast feeding stage, whether it is secretory or non-secretory breast feeding, infants are very low in frequency of infection and diarrhea symptoms (5/65), 49% (32/65) infants are infected but have no diarrhea symptoms, and the rest infants have no infection; in different lactation period, the average intake of alpha 1, 2-fucosylated breast milk oligosaccharides from breast milk is significantly lower in few infants with diarrhea symptoms than in asymptomatic or uninfected infants (p < 0.05), which in total might help to reduce the risk of diarrhea in infants and young children in early life (three months before delivery). In addition, the results of the stool samples of table 4 show that bifidobacteria are significantly lower in the infant gut flora with diarrhea symptoms than in asymptomatic infants, presumably helping the infant gut early in life to establish an effective barrier to reduce the risk of diarrhea symptoms caused by pathogenic infection.
Table 51 correlation of infant diarrhea Rate after the age of 48 with infant genotype
| Infant type | Secretion type (n=51) | Non-secretory (n=14) |
| Symptoms of diarrhea have occurred | 25 | 5 |
| No diarrhea symptom | 26 | 9 |
The results in table 5 show that after weaning, the diarrhea frequency of infants is higher and the diarrhea risk of secreted infants is significantly higher than that of non-secreted infants by losing the protective effect of breast milk, and that according to the results of breast feeding and correlation analysis described above, alpha 1, 2-fucosylated breast milk oligosaccharides and bifidobacteria may be the key to protect secreted infants from diarrhea, as further verified using animal experiments.
Example two
Fut2 knockout C57BL/6 mice were used as non-secretory populations, while wild type C57BL/6 mice were used as secretory populations. Pathogenic bacteria selected from Salmonella enterica subspecies enterica serogroup SL 1344 (Salmonella enterica subsp.enterica serovar Typhimurium, ST) for inducing diarrhea in mice by administering 10 6 The CFU salmonella bacteria are resuspended in 0.1mL of sterile physiological saline to prepare pathogenic bacteria suspension, then the bacteria suspension prepared in the above is inoculated by oral gavage at 21 days of age of mice, the colonization amount of salmonella in the cecum tissue of mice is observed at 1 day, 3 days and 7 days after infection, and the effect of Fut2 gene on salmonella colonization is verified. Wherein the blank control group is not infected by pathogenic bacteria, and 0.1mL of physiological saline is orally taken. The amount of colonization of pathogenic bacteria was determined by a viable count method (CFU), a cecal tissue sample was collected, the content was separated, the cecal tissue was homogenized in an appropriate amount of PBS after separation, and the homogenized solution was gradient diluted and cultured using LB agar medium containing 100 μg/mL streptomycin as a selective medium for salmonella SL 1344. Record the secretory group as Fut2 +/+ +st group, non-secretory group recorded as Fut2 -/- + ST group. Salmonella SL 1344 colonization in cecal tissue of two groups of mice is shown in FIG. 1. * P is p<0.033，***p<0.001。
As shown in fig. 1, although salmonella colonization was higher in the cecum of non-secreting mice at the initial stage of infection (day 1); however, over time, when day 7 post-infection was reached, salmonella colonization was significantly reduced in non-secreting mice (p < 0.0001), and non-secreting groups were significantly lower than secreting groups. The above results demonstrate that the intestinal tract of the secretory individuals is more susceptible to long-term colonisation by salmonella SL 1344 and has a higher risk of disease.
Example III
Past studies have shown that salmonella recognizes the a1, 2-fucosylation receptor on the surface of intestinal epithelial cells via std gene cluster for colonisation, whereas fucosylation receptor in the intestinal tract is mainly concentrated in secretory individuals, so inhibiting adhesion of std encoded flagellin is a key to reducing the risk of long-term infection of secretory individuals with salmonella. Since std encodes a flagellin with low expression level under in vitro culture conditions, stable expression by molecular cloning is required. Plasmid p4394 was constructed using a pWSK29 low copy cloning vector, and the prepared p4394 plasmid contained a dehydrated tetracycline-inducible promoter and the salmonella stdABCD operon (encoding an adhesin), and the p4393 plasmid was transformed into a Turbo competent E.coli K-12 strain (New England Biolab), and finally a monoclonal strain (E.coli stdABCD strain) was obtained on LB agar medium containing the corresponding antibiotics.
Activating the escherichia coli stdABCD strain in an LB culture medium containing the anhydrotetracycline, culturing to a logarithmic phase and inducing the expression of the flagella adhesion to obtain a bacterial suspension. Caco-2 cells were subcultured in complete medium (Gibco RPMI 1640 medium plus 10% fetal bovine serum, gibco 1% green-streptomycin, gibco 1% MEM nonessential amino acid solution) and the activated cells were cultured in 10 5 Concentration of individual/well inoculated in 24 well plate at CO 2 Cell incubator culture (37 ℃,5% CO) 2 Saturated humidity), the medium was changed every 2 days, and the cells were cultured for 15 days to completely differentiate Caco-2 cells, and the cell fusion was confirmed by a microscope.
To verify the different oligosaccharides against E.coli stdABCDEffect of plant adhesion, incubation with Caco-2 differentiated cells for 30min was performed using pre-chilled complete medium containing 0.1% of different types of oligosaccharides, followed by addition of 10 5 Cell adhesion is carried out on CFU/hole escherichia coli stdABCD strain for 1h, and the whole adhesion experiment is carried out at 4 ℃ to prevent pathogenic bacteria from infecting cells. The oligosaccharides used in the treatment group included 2' -FL, lactose-N-neotetraose (LNnT), fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), and the positive control group used 0.1% JINGDOU lectin I (Ulex europaeus agglutinin-I, UEA-I), while the blank group was normal saline. The treatment, positive control and blank groups had the same volume of solution.
Adherent bacteria were quantified by viable count by washing Caco-2 cells 5 times with PBS to remove non-adherent bacteria, followed by lysing the cells with 1% triton X-100 and resuspending adherent bacteria. The bacterial suspension diluted in 10-time gradient is cultured on LB culture medium to determine the number of adhered bacteria, and then the bacterial adhesion rate is calculated, wherein the bacterial adhesion rate=the bacterial adhesion amount of an experimental group/the bacterial adhesion amount of a blank group multiplied by 100%. The adhesion rate of different oligosaccharides to E.coli stdABCD strain to Caco-2 cells is shown in FIG. 2.
As can be seen from fig. 2, both UEA-I, which is a lectin specifically adhering to a1, 2-fucosyl chains, and 2'-FL, significantly inhibited std flagellin-mediated cell adhesion (p < 0.05), demonstrating that 2' -FL has a similar function as a receptor to capture e.coli stdABCD strain, thereby reducing the infection rate.
Example IV
The effect of different bifidobacteria species in combination with 2' -FL on pathogenic-induced inflammatory responses of intestinal epithelial cells was explored using in vitro studies. Using HT-29 cells as a model, cells were subcultured in complete medium (Gibco RPMI 1640 medium plus 10% fetal bovine serum, gibco 1% green-streptomycin, gibco 1% MEM nonessential amino acid solution) and activated cells were passaged 10 5 Concentration of individual/well inoculated in 24 well plate at CO 2 Cell incubator culture (37 ℃,5% CO) 2 Saturated humidity), the culture medium is replaced every 2 days,culturing for 21 days allowed HT-29 cells to fully differentiate and cell fusion was confirmed by microscopy.
TABLE 6 list of experimental strains
Each experimental strain (as in Table 6) was cultured using the corresponding agar medium and single colonies were obtained, wherein bifidobacteria were cultured using MRS agar medium supplemented with 0.05% cysteine, and Salmonella was cultured using BHI agar medium. Then, single colonies are picked and inoculated to MRS or BHI broth culture medium for overnight culture to activate, supernatant is removed by centrifugation to obtain thalli, the thalli are washed three times by PBS, then a proper amount of cell complete culture medium is added to re-suspend the thalli, and if two strains of probiotics are 1:1, and finally the concentration of the single strain or the mixed bacterial suspension of the two strains reaches 10 8 CFU/mL. Bacteria (probiotics or pathogenic) and cells (HT-29 cells) at 10:1 (number ratio). If 2' -FL is intervened, the cell culture solution is added at a concentration of 1% (cell culture solution concentration 10) 6 Individual cells/mL). I.e. the ratio of probiotics to 2' -FL is 10 6 CFU 1mg. HT-29 cells were pre-incubated with 2' -FL and/or probiotics for 2h prior to Salmonella infection, following specific intervention protocols:
experiment group 1: PSB buffer solution
Experiment group 2:2' -FL
Experiment group 3:2' -FL+ Bifidobacterium longum subspecies infantis R0033
Experiment group 4:2' -FL+ bifidobacterium animalis subspecies lactis Bb-12
Experimental group 5:2' -FL+ bifidobacterium bifidum R0071
Experiment group 6:2' -FL+ bifidobacterium breve M-16V
Experiment group 7:2' -FL+ Bifidobacterium pseudolongum subspecies pseudolongum ATCC 25526
Experiment group 8:2' -FL+ bifidobacterium catenulatum ATCC 27539
Experiment group 9:2' -FL+R0033+R0071
Experimental group 10:2' -FL+R0033+Bb-12
Experiment group 11:2' -FL+R0033+M-16V
Experiment group 12:2' -FL+R0033+ATCC 25526
Experiment group 13:2' -FL+R0033+ATCC 27539
Experiment group 14:2' -FL+R0071+Bb-12
Experiment group 15:2' -FL+R0071+M-16V
Experiment group 16:2' -FL+R0071+ATCC 25526
Experiment group 17:2' -FL+R0071+ATCC 27539
Experimental group 18: R0033+R0071.
After the probiotic bacteria or/and 2' -FL are used for pretreating the cells, pathogenic bacteria salmonella CICC21848 is added for treatment for 4 hours, then cell supernatant is collected, sediment is removed by centrifugation at 4 ℃, and the samples are stored at-20 ℃ until analysis and determination. The cell supernatant was subsequently assayed for inflammatory factor IL-8 protein and biologically repeated three times according to the kit operating manual. The results are shown in Table 7.
TABLE 7 influence of different intervention groups on secretion of the immune factor IL-8 by HT-29 cells
| Group of | Inflammatory factor IL-8 secretion pg/mL (mean+ -SD) |
| Experiment group 1 | 1962.3±262.1 a |
| Experiment group 2 | 1558±132.1 b |
| Experiment group 3 | 1239±38.5 c |
| Experiment group 4 | 1414.7±57.1 bc |
| Experiment group 5 | 1245±30.8 c |
| Experiment group 6 | 1303.7±59.7 bc |
| Experiment group 7 | 1414±80.6 bc |
| Experiment group 8 | 1353±31.6 bc |
| Experiment group 9 | 606.3±30.0 d |
| Experiment group 10 | 1124.3±10.3 c |
| Experiment group 11 | 1123±67.4 c |
| Experiment group 12 | 1095±86.6 c |
| Experiment group 13 | 1019±61.7 c |
| Experiment group 14 | 1014±36.2 c |
| Experiment group 15 | 1085±58.9 c |
| Experiment group 16 | 1016.7±110.6 c |
| Experiment group 17 | 1045.7±130 c |
| Experiment group 18 | 1282.3±46.8 bc |
* And (3) injection: the same column of data superscripts contained the same letter indicating that the difference was not significant (p > 0.05) and the different letters indicating that the difference was significant (p < 0.05).
As shown in Table 7, the cells in the blank (test group 1) secreted the highest amount of the pro-inflammatory factor IL-8, and Salmonella, similar to the previous study, promoted secretion of the inflammatory factor IL-8. And the combination of different probiotics and 2' -FL can reduce the secretion of the proinflammatory factor IL-8 after acting on cells. The greater reduction of IL-8 secretion in panels 3-8 compared to panel 2 suggests that 2'-FL works better when combined with a single strain than with a single 2' -FL, with panels 3 and 5 showing more prominence. In addition, the effect of inhibiting the proinflammatory factor of experiment group 9 is optimal under the combination of the two strains, which shows that the combination of 2' -FL and probiotics R0033 and R0071 can regulate inflammatory response caused by pathogenic bacteria, and the effect is better.
Experiment group 9 (2 '-FL and two strains) showed significantly improved effect of inhibiting pro-inflammatory factors compared to experiment group 3, experiment group 5 (2' -FL and single strain); meanwhile, the experimental group 9 also has more remarkable inhibition effect compared with the experimental groups 2 and 18 (two-strain combination), which shows that the combination of the probiotics R0033 and R0071 and the combination of the 2' -FL and the probiotics R0033 and R0071 have remarkable synergistic effect in enhancing the immunity.
Example five
Animal experiments were used to verify the effect of a particular bifidobacteria combination with 2' -FL on the in vivo colonization of salmonella, the experimental animals were selected from wild type mice 129X1/SvJ, nutrient intervention was started at 10 days of age of the mice, and after 21 days of age of the mice, a model of chronic enteritis was induced in the mice by infection with salmonella SL 1344, 10 8 The salmonella bacterial suspension of CFU was inoculated by oral gavage and the salmonella content in the mouse faeces was detected 7 days after infection, the nutrient intervention was continued until the end of the experiment, the specific intervention protocol was as follows:
blank group: physiological saline
Experiment group 1:2' -FL (1 g/kg body weight)
Experiment group 2: bifidobacterium longum subspecies infantis R0033
Experiment group 3: bifidobacterium bifidum R0071
Experiment group 4:2' -FL (1 g/kg body weight) +R0033
Experimental group 5:2' -FL (1 g/kg body weight) +R0071
Experiment group 6: R0033+R0071 (R0033:R0071 live bacteria number=1:1)
Experiment group 7:2' -FL (1 g/kg body weight) +R0033+R0071 (R0033: R0071 viable count=1:1).
The oligosaccharide 2' -FL used in the above experiments was dissolved in the drinking water of mice at a probiotic dose of 10 8 CFU/g, i.e. probiotic to 2' -FL usage ratio of 10 8 CFU 1mg. And ensuring that each experimental group has more than 6 experimental mice. The results are shown in Table 8.
Table 8 number of live Salmonella bacteria in feces at day 7 post-infection for each group
* And (3) injection: the same letter superscript for the same row of data indicates that the difference is not significant (p > 0.05), and the different letter indicates that the difference is significant (p < 0.05).
As can be seen from table 8, experimental group 1 significantly reduced salmonella colonization in vivo, demonstrating that 2' -FL, similar to in vitro assays, was able to reduce pathogenic colonization as a receptor, and in addition, in part, could be through modulation of intestinal flora, thereby reducing the susceptibility of the body to salmonella; while experimental groups 2 and 3 (single probiotics) also significantly reduced pathogenic colonization in vivo, experimental groups 4, 5 and 6 significantly reduced pathogenic colonization, and the results indicated that the combination of 2' -FL with specific probiotics provided the body with a good intestinal barrier against pathogenic infection early in life, reduced salmonella colonization, and modulation of intestinal flora. Furthermore, the significantly reduced pathogenic bacteria content in the feces of experimental group 7 compared to experimental groups 1, 4, 5, and 6, demonstrated that the combination of probiotics R0033 and R0071, and the combination of 2' -FL and probiotics R0033 and R0071 reduced pathogenic bacteria colonisation in vivo had a synergistic effect.
The foregoing detailed description is directed to one of the possible embodiments of the present invention, which is not intended to limit the scope of the invention, but is to be accorded the full scope of all such equivalents and modifications so as not to depart from the scope of the invention.
Claims (20)
1. A probiotic prebiotic composition comprising breast milk oligosaccharide, bifidobacterium bifidum and bifidobacterium longum subspecies infancy.
2. The composition of claim 1, wherein the human milk oligosaccharide is 2' -fucosyllactose.
3. The composition according to claim 2, wherein the bifidobacterium bifidum is bifidobacterium bifidum R0071.
4. A composition according to claim 3, wherein the bifidobacterium longum subspecies infancy is bifidobacterium subspecies infancy R0033.
5. The composition according to claim 4, wherein the concentration of 2' -fucosyllactose in the composition is from 10mg to 50g/100g.
6. The composition according to any one of claims 1-5, wherein the concentration of bifidobacterium bifidum in the composition is 1 x 10 6 -1×10 14 CFU/100g, preferably 10 8 -10 12 CFU/100g; the concentration of the Bifidobacterium longum subspecies infantis in the composition is 1X 10 6 -1×10 14 CFU/100g, preferably 10 8 -10 12 CFU/100g。
7. The composition of claim 6, wherein the viable count ratio of bifidobacterium bifidum to bifidobacterium longum subspecies infancy is 1:0.1 to 10, preferably 1:0.5 to 5, further preferably 1:1; the mass ratio of the 2' -fucosyllactose to the total viable count of the bifidobacterium bifidum and bifidobacterium longum subspecies infantis is 1mg:10 4 -10 12 CFU, preferably 1mg:10 6 -10 10 CFU, further preferably 1mg:10 6 -10 8 CFU。
8. Use of a composition according to any one of claims 1 to 7 for the preparation of an immunity enhancing product.
9. The use according to claim 8, wherein the enhancing immunity is in particular increasing the resistance of the gastrointestinal tract against pathogenic infections.
10. The use according to claim 8, wherein the product is a food or a pharmaceutical.
11. The use according to claim 8, wherein the product comprises a nutritional supplement, a solid beverage, a lozenge, an oral cleanser or a probiotic micro-foam tablet.
12. The use of claim 9, wherein the pathogenic bacteria is salmonella.
13. Use according to claim 8, wherein the enhancing immunity is in particular enhancing immunity of a secretory infant and/or child.
14. Use according to claim 8, wherein the enhancing immunity is in particular preventing and/or ameliorating diarrhea.
15. Use of a composition according to any one of claims 1 to 7 for the preparation of a product for regulating intestinal flora.
16. Use according to claim 15, wherein the modulation of intestinal flora is in particular a reduction of salmonella colonization.
17. The use according to claim 15, wherein the product is a food or a pharmaceutical.
18. The use according to claim 17, wherein the product comprises a nutritional supplement, a solid beverage, a lozenge, an oral cleanser or a probiotic micro-foam tablet.
19. An immunity enhancing product comprising the composition of any one of claims 1-7.
20. A product for regulating intestinal flora, comprising a composition according to any of claims 1-7.
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