CN112870233A - Composition containing bifidobacterium lactis and breast milk oligosaccharide and application thereof - Google Patents
Composition containing bifidobacterium lactis and breast milk oligosaccharide and application thereof Download PDFInfo
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- CN112870233A CN112870233A CN202011370298.7A CN202011370298A CN112870233A CN 112870233 A CN112870233 A CN 112870233A CN 202011370298 A CN202011370298 A CN 202011370298A CN 112870233 A CN112870233 A CN 112870233A
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- China
- Prior art keywords
- bifidobacterium lactis
- composition
- breast milk
- cfu
- fucosyllactose
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Abstract
The invention mainly relates to a composition containing Bifidobacterium lactis and breast Milk oligosaccharide and application thereof, and the composition is a nutritional composition containing Bifidobacterium lactis and breast Milk oligosaccharide (Human Milk Oligosaccharides). The composition of the present invention can effectively improve the gastrointestinal tract immunity, and the composition can be added into various health foods and health foods.
Description
Technical Field
The invention mainly relates to a composition containing Bifidobacterium lactis and breast Milk Oligosaccharides and application thereof, in particular to a nutritional composition containing Bifidobacterium lactis and breast Milk Oligosaccharides (HMOs), and the composition can be added into various health foods and health foods.
Background
Over the last thousand years, medical literature has documented a high rate of morbidity and mortality in infants who are not breastfed. The breast milk not only provides the required nutrition for the infant, but also provides the guarantee for the intestinal development and the immunity improvement of the infant by the active ingredients in the breast milk. Breast-fed infants have a higher relative abundance of beneficial bacteria, particularly bifidobacteria and lactic bacteria, in the gut flora compared to formula-fed infants.
The breast milk is transferred by flora, and active ingredients such as breast milk oligosaccharide and cytokine in the breast milk are added to establish healthy intestinal flora for the newborn. The infant intakes 10 via breast milk every day7-108Individual bacteria, including lactic acid bacteria and bifidobacteria. The bacteria are directly transmitted to the infant through breast milk, and part of the bacteria can be planted in the intestinal tract of the infant, so that the establishment of the intestinal flora in the early life is promoted. The establishment of the infant's intestinal flora has short-term, even lifelong effects on the development of its intestinal tract, as well as on the health and immune system.
Breast Milk Oligosaccharides (HMOs) belong to the third most abundant substances in breast Milk, except lactose and fat. The total content varies at various stages of lactation, and is about 12-14g/L in mature milk and about 20-24g/L in colostrum. Each breast milk oligosaccharide has a lactose at the reducing end, mostly with poly lactosamine as the structural backbone, and fucose, sialic acid, or both at the chain end. HMOs are present in individual differences in content and are associated with the lewis secretory component of the nursing mother. Since the raw material of infant formula is usually cow's milk, which usually contains no or very little such oligosaccharides, HMOs constitute a gap that infant formula is expected to approach the breast milk.
In the last 90 s of the century, HMO, 2-fucosyllactose (2' -FL), contained in most breast milk, was found to be effective in reducing the toxicity of stable toxins in escherichia coli; by 2003, the oligosaccharides were reported to inhibit the attachment and infection of jejunum flexuosum. Subsequently, three major functions of breast milk oligosaccharides were gradually reported and discovered: (1) inhibiting attachment and infection of specific pathogens; (2) as a prebiotic, the growth of bacteria in the intestinal tract symbiotic system is promoted; (3) directly slow down the inflammatory reaction of mucosa under toxic stimulation. The first clinical intervention trial with 2' -FL demonstrated that the addition of this specific ingredient to a low calorie formula was not only safe but also allowed formula-fed infants to grow at a rate comparable to breast-fed infants. 2' -FL is also used as a nutritional supplement for adults, to alleviate irritable bowel syndrome or inflammatory bowel disease, or as a prebiotic to maintain intestinal flora balance.
The intestinal flora is an important component of a human intestinal microecosystem and has important effects on human health, such as supplying essential nutrients, generating vitamin K, assisting a digestion process and promoting angiogenesis and intestinal nerve. Prebiotics and probiotics are considered as micro-ecological management tools for improving the health of the body, altering, regulating and recombining the already existing intestinal flora.
Currently, in the field of infant formula powder, supplementary food and nutritional supplements, solutions for alleviating infant intestinal discomfort and improving autoimmune ability are needed. Meanwhile, in the fields of children, teenagers and adults over 3 years old, the balance of intestinal flora needs to be maintained, and the immunity is regulated.
Disclosure of Invention
It is an object of the present invention to provide a nutritional composition that enhances the immune competence of the gastrointestinal tract.
The inventor of the present invention finds that the combination of Bifidobacterium lactis (Bifidobacterium lactis) and breast milk oligosaccharides has a synergistic effect on improving the gastrointestinal tract immunity.
Thus, in one aspect, the present invention provides a nutritional composition comprising Bifidobacterium lactis (Bifidobacterium lactis) and breast milk oligosaccharides.
According to a particular embodiment of the invention, in the composition of the invention, the breast milk oligosaccharides comprise one or more of 2 '-fucosyllactose, 3' -fucosyllactose, lacto-N-tetraose, 3 '-sialyllactose, 6' -sialyllactose.
2 '-fucosyllactose (2' -fucosyllactose), a trisaccharide structure formed by fucose and lactose, is prepared by microbial fermentation and has the same structure as oligosaccharides found in human milk.
3 ' -fucosyllactose (3 ' -fucosyllactose) is a trisaccharide structure formed by fucose and lactose, and is an isomer of 2 ' -fucosyllactose. The substance is prepared by microbial fermentation, and has the same structure as oligosaccharide found in human milk.
lacto-N-tetraose (lacto-N-tetraose), a hexasaccharide structure formed from lactose and tetrasaccharide, is produced by microbial fermentation and has the same structure as oligosaccharides found in human milk.
3 '-sialyllactose (3' -sialyllactose), a trisaccharide structure formed by sialic acid and lactose, is prepared by microbial fermentation and has the same structure as oligosaccharides found in human milk.
6 ' -sialyllactose (6 ' -sialyllactose) is a trisaccharide structure formed by sialic acid and lactose, and is an isomer of 3 ' -sialyllactose. The substance is prepared by microbial fermentation, and has the same structure as oligosaccharide found in human milk.
According to a particular embodiment of the invention, the breast milk oligosaccharides comprise 2 '-fucosyllactose, 3' -fucosyllactose, lacto-N-tetraose, 3 '-sialyllactose, 6' -sialyllactose in a weight ratio of (0-10): (4-8): (3-6): (1-4): 0-1).
According to a particular embodiment of the invention, the composition of the invention comprises the human milk oligosaccharides 2 '-fucosyllactose, 3' -fucosyllactose, lacto-N-tetraose, 3 '-sialyllactose, 6' -sialyllactose in a weight percentage (0-53%) (21% -44%) (16% -32%) (5% -22%) (0-5%). The weight percentage is based on the total amount of the breast milk oligosaccharide as 100%.
According to a particular embodiment of the invention, in the composition of the invention, the Bifidobacterium lactis comprises the Bifidobacterium lactis including the Bifidobacterium lactis (Bifidobacterium lactis) HN019 strain and/or Bifidobacterium lactis (Bifidobacterium lactis) with the accession number CGMCC No. 15650.
Bifidobacterium lactis (Bifidobacterium lactis) HN019 is a commercial strain available from Dupont Danisco.
The bifidobacterium lactis with the preservation number of CGMCC No.15650 is also named as bifidobacterium lactis BL-99. The strain has gastric acid resistance, and the survival rate of viable bacteria is more than 62% when the strain is treated in gastric acid liquid with pH of 2.5 for 30min and more than 61% when the strain is treated for 2 hours. The bifidobacterium lactis BL-99 provided by the invention also has intestinal juice resistance, and the survival rate of viable bacteria is more than 70% after being treated in small intestinal juice with pH of 6.8 for 2 hours. Mouse experiments show that the strain has no oral acute toxicity, no antibiotic tolerance and safety and can be used for food processing. The strain has been preserved in China general microbiological culture Collection center (CGMCC) 26.04.2018 (address: No. 3 Xilu-Beijing institute of microbiology, institute of China academy of sciences, North Cheng-Yang district, Beijing city), and is named after classification: bifidobacterium lactis (Bifidobacterium lactis); the preservation number is CGMCC No. 15650.
According to a particular embodiment of the invention, the ratio of bifidobacterium lactis to breast milk oligosaccharides in the composition of the invention is 1 × 103CFU～1×1012CFU: 5mg, preferably 1X 106CFU～1×1010CFU：5mg。
According to a particular embodiment of the invention, the bifidobacterium lactis is present in the nutritional composition in an amount of 1 x 103CFU～1×1012CFU/day, preferably 1X 106CFU～1×1011CFU/day.
According to a particular embodiment of the invention, the breast milk oligosaccharides are applied in the nutritional composition in an amount of 1mg to 15 g/day.
In another aspect, the invention also provides the application of the composition in preparing food or medicine with the effect of improving gastrointestinal tract immunity.
According to a particular embodiment of the invention, the use of the composition according to the invention for increasing the immunological competence of the gastrointestinal tract comprises combating pathogenic bacteria invasion in the intestinal system, maintaining the barrier function of the intestinal tract, and/or reducing the release of inflammatory factors IL-8 and/or IP-10 by the intestinal cells.
According to the bookAccording to the specific embodiment of the invention, the nutritional composition can be used for preparing various health foods, health foods and the like. In particular, the food can be liquid beverage, solid beverage, oral liquid, dairy products, tablets or capsules, and the like, and can be added into infant food (including infant formula powder, complementary food and nutritional supplements) and nutritional supplements or foods for children, teenagers and adults over 3 years old, so that the food has wide application prospect. Preferably, the addition amount of the bifidobacterium lactis HN019 in the food is 1 x 103CFU～1×1012CFU/day, more preferably 1X 106CFU～1×1011CFU/day. Preferably, the addition amount of the bifidobacterium lactis BL-99 in the food is 1 x 103CFU～1×1012CFU/day, more preferably 1X 106CFU～1×1011CFU/day. The addition amount of breast milk oligosaccharide in food is 1mg-15 g/day.
The food or medicine comprising the nutritional composition of the present invention has a function of improving the immunocompetence of the gastrointestinal tract due to the inclusion of the nutritional composition.
Drawings
FIG. 1 shows the effect of a mixture of breast milk oligosaccharides on the survival of the tested pathogenic bacteria EPEC.
FIG. 2 shows the effect of Bifidobacterium lactis HN-019 and BL-99 strains on the survival rate of the tested pathogenic bacteria EPEC.
Figure 3 shows the effect of bifidobacterium lactis HN019 in combination with a mixture of breast milk oligosaccharides on the survival rate of the tested pathogen EPEC.
Figure 4 shows the effect of bifidobacterium lactis BL-99 in combination with a mixture of breast milk oligosaccharides on the survival of the tested pathogenic bacteria EPEC.
FIG. 5A shows Bifidobacterium lactis HN019 (10)8) The adhesion experiment result of the combination of the mixture and the breast milk oligosaccharide mixture A on the pathogenic bacteria EPEC acting on the intestinal tract cells Caco-2; figure 5B shows the effect of different concentrations of bifidobacterium lactis HN019 in combination with different proportions of a mixture of breast milk oligosaccharides on the intestinal adhesion of the test pathogen EPEC.
FIG. 6A shows Bifidobacterium lactis BL-99 (10)8) Combination with Breast milk oligosaccharide mixture BThe adhesion experiment result of the pathogen EPEC acting on the intestinal tract cells Caco-2; fig. 6B shows the effect of different concentrations of bifidobacterium lactis BL-99 in combination with different proportions of the mixture of breast milk oligosaccharides on the intestinal adhesion of the test pathogen EPEC.
FIG. 7A shows Bifidobacterium lactis HN019 (10)8) Effect on intestinal barrier in combination with breast milk oligosaccharide mixture a; FIG. 7B shows Bifidobacterium lactis 108The effect of the combination of HN019 with different proportions of breast milk oligosaccharide mixtures on transmembrane resistance TEER under ETEC challenge conditions; FIG. 7C shows 106The effect of the combination of bifidobacterium lactis HN019 and the breast milk oligosaccharide composition a on transmembrane resistance TEER under pathogenic bacterium ETEC attack conditions.
FIG. 8 shows 108The effect of the combination of bifidobacterium lactis BL-99 with different proportions of a mixture of breast milk oligosaccharides on the transmembrane resistance TEER under ETEC challenge conditions.
FIGS. 9A and 9B show the effect of Bifidobacterium lactis HN019 in combination with a mixture of breast milk oligosaccharides on secretion of inflammatory factors IL-8 and IP-10 by intestinal cells in the absence of E.coli coculture.
FIGS. 10A and 10B show the effect of Bifidobacterium lactis HN019 in combination with a mixture of breast milk oligosaccharides on the secretion of inflammatory factors IL-8 and IP-10 by intestinal cells when cocultured with E.coli.
Microbial preservation of the patent procedure:
bifidobacterium lactis BL-99 of the present invention:
the preservation date is as follows: 26/04/2018;
the preservation unit: china general microbiological culture Collection center (CGMCC);
the address of the depository: xilu No.1 Hospital No. 3, the institute of microbiology, China academy of sciences, Beijing, Chaoyang
The preservation number is: CGMCC No. 15650;
and (3) classification and naming: bifidobacterium lactis (Bifidobacterium lactis).
Detailed Description
For a more clear understanding of the technical features, objects and advantages of the present invention, reference is now made to the following detailed description of the embodiments of the present invention taken in conjunction with the accompanying drawings, which are included to illustrate and not to limit the scope of the present invention. Unless specifically defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the relevant art.
The inventor proves that the probiotic composition has a synergistic effect in the aspect of regulating the gastrointestinal tract immunity through specific experiments.
The experimental methods and test substance cases used in the examples and the control are as follows:
1. preparation of the experiment
1.1 media preparation and storage and preparation of prebiotics tested
The medium containing prebiotics (prebiotic species see table 1) and probiotics was freshly prepared in a sterile environment on the day of each experiment and pre-warmed to 37 ℃. The prebiotics/HMOs tested were stored in a dry, dark room temperature environment. The concentration used in the experiment was 5 g/L.
TABLE 1
| Numbering | Name(s) | Abbreviations | Name of Chinese |
| 1 | 2’-fucosyllactose | 2’-FL | 2' - |
| 2 | 3’-fucosyllactose | 3’-FL | 3' - |
| 3 | 3’-sialyllactose | 3’-SL | 3' - |
| 4 | 6’-sialyllactose | 6’-SL | 6' -sialyllactose |
| 5 | Lacto-N-tetraose | LNT | lacto-N-tetraose |
1.2 probiotic culture, growth curve plotting and activity identification
The probiotic powder was sent to the laboratory in a freeze-dried state prior to the experiment. Probiotics were inoculated onto MRS-agar plates and individual populations of strains were taken for 16S rDNA identification and used to prepare glycerol stocks (stored at-80 ℃). Prior to each experiment, the probiotic was removed from the glycerol stock and inoculated on MRS plates at 37 ℃, harvested at rest, and rinsed with MEM medium prior to the experiment. Probiotic activity and cell number were measured with a fluorescence activated cell sorting system prior to probiotic testing. The probiotic concentration used in the experiment was 1X 108CFU/mL. Culturing Bifidobacterium lactis HN019 or BL-99 at 37 deg.C under anaerobic condition, and drawing raw material before experimentLong curve. The activity of the probiotic strains was tested with a flow cytometer before the probiotic raw material was received and all experiments were started. 16S sequencing was used to determine strain identity. Before testing of each experiment of the probiotics, the activity and the cell number of the probiotics are measured by a fluorescence activated cell sorting system.
1.2.1 Bifidobacterium lactis BL-99
The bifidobacterium lactis BL-99 is separated from the intestinal tract of the infant. The strain has been preserved in China general microbiological culture Collection center (CGMCC) 26.04.2018 (address: No. 3 Xilu-Beijing institute of microbiology, institute of China academy of sciences, North Cheng-Yang district, Beijing city), and is named after classification: bifidobacterium lactis (Bifidobacterium lactis); the preservation number is CGMCC No. 15650.
1.2.1.1 taxonomic characterization of Bifidobacterium lactis BL-99
The results of the physical and chemical tests are as follows:
1.2.1.2 tolerance of Bifidobacterium lactis BL-99 to artificial gastric juice and intestinal juice
Bifidobacteria are genera that are generally not acid-fast. In this example, the tolerance of bifidobacterium lactis BL-99 of the present invention to artificial gastric juice and intestinal juice was tested, and bifidobacterium lactis which had been recognized in the art as having excellent acid resistance and survived through the gastrointestinal tract
For comparison.
The test method comprises the following steps: culturing Bifidobacterium lactis BL-99 strain in MRS liquid culture medium at 37 deg.C for 16 hr, centrifuging at 4 deg.C and 2500rpm for 10min, and collecting thallus.
Respectively culturing the strains to be tested in artificial gastric juice and artificial small intestine juice, processing at 37 ℃ for 0, 30min and 2h, and then performing viable count analysis to evaluate the acid resistance and intestinal juice resistance of the strains according to the survival rate. Survival rate (viable cell count after treatment/viable cell count at time 0) × 100%.
The survival rate detection result of the bacterial strain in artificial gastric acid (pH2.5) is shown in Table 2, the survival rate of the viable bacteria is 7.04% when BB-12 is treated in the artificial gastric acid (pH2.5) for 30min, and the survival rate of the viable bacteria is only 1.64% after 2 hours of treatment; the survival rate of the live bacteria of the bifidobacterium lactis BL-99 is 62.60 percent when the bifidobacterium lactis BL-99 is treated in artificial gastric acid (pH2.5) for 30min, and the survival rate of the live bacteria is 61.83 percent when the bifidobacterium lactis BL-99 is treated for 2 hours. The bifidobacterium lactis BL-99 disclosed by the invention has excellent gastric acid resistance and can smoothly pass through the stomach to reach the intestinal tract to play a probiotic role.
TABLE 2 survival of the strains in artificial gastric acid (pH2.5)
The survival rate of the strain in the artificial small intestine solution (pH6.8) is determined and shown in Table 3. The data show that the viable bacteria survival rate of BB-12 in artificial small intestine solution (pH6.8) for 2 hours is only 28.95%; the viable bacteria survival rate of the bifidobacterium lactis BL-99 is 70.23 percent when the bifidobacterium lactis BL-99 is treated in artificial gastric acid (pH2.5) for 2 hours. The bifidobacterium lactis BL-99 disclosed by the invention has excellent intestinal juice resistance and can survive and colonize in intestinal tracts.
TABLE 3 survival rate of the strains in artificial intestinal juice (pH6.8)
1.2.1.3 toxicity test and safety detection of Bifidobacterium lactis BL-99
Inoculating the bifidobacterium lactis BL-99 of the invention into BBL liquid culture medium, anaerobically culturing for 48 plus or minus 2 hours at 36 plus or minus 1 ℃, and counting in the culture solutionThe viable count of the bifidobacterium lactis BL-99 is 3.7 multiplied by 108cfu/mL, stock solutions and 5-fold concentrates of the cultures were continuously gavaged to 20.0mL/kg BW for 3 days and observed for 7 days. The experiment was performed with a control group of 5-fold concentrated solution and a stock solution of the medium. The test result shows that: the BBL culture stock solution and 5-fold concentrated solution of Bifidobacterium lactis BL-99 had no statistical effect on the weight gain of mice (p > 0.05) compared with the respective control group, and no toxic reaction or death of the tested mice was observed.
The antibiotic sensitivity of the bifidobacterium lactis BL-99 is evaluated by adopting an SN/T1944-2007 method of determination of bacterial resistance in animals and products thereof. The evaluation results show that the bifidobacterium lactis BL-99 is sensitive to Ampicillin Ampicillin, penicillin G Penicillin G, Erythromycin Erythromycin, Chloramphenicol Chloramphenicol, Clindamycin Clindamycin, Vancomycin Vancomycin, Tetracycline and the like. Meets the requirements of European Food Safety Authority (European Food Safety Authority) on the evaluation specification of the resistance of the edible bacteria. The bifidobacterium lactis BL-99 does not contain exogenous antibiotic resistance genes and is safe to eat.
1.2.2 Bifidobacterium lactis HN019
The HN019 strain was supplied by Dupont Danisco.
1.3 formulation of different combinations of prebiotics and probiotics
The concentration of prebiotics tested in the combination was 5 g/L; the number of probiotics tested is 1 × 108CFU/mL。
The first round of testing: five combined prebiotics 2' -FL:3-FL: LNT:3-SL:6-SL ═ 10:4:3:1
And (3) testing in a second round: the proportions and percentages of the five combined prebiotics are shown in tables 4 and 5 below.
TABLE 4
| Ratio of | 2’-FL | 3-FL | LNT | 3-SL | 6- |
| Composition A | |||||
| 10 | 4 | 3 | 1 | 1 | |
| |
10 | 4 | 3 | 2 | 0 |
| Composition C | 9 | 6 | 3 | 2 | 0 |
| |
0 | 8 | 6 | 4 | 0 |
| Composition F | 12.5 | 5.75 | 4.25 | 1.5 | 1 |
TABLE 5
1.4Caco-2 cell culture
Human colon tumor cell line (Caco-2) was obtained from DSMZ (Bilelix, Germany) and cultured in the presence of 5% CO2Culturing at 37 deg.C under certain humidity. Caco-2 cells from passage 40-44 were used for the experiments. To MEM medium was added 20% (v/v) Fetal Bovine Serum (FBS), 1% nonessential amino acids, 1% Glutamax, 1% sodium pyruvate, with or without 1% penicillin-streptomycin solution, and 50. mu.g/mL gentamicin (all available from Invitrogen corporation, Dutch Brada). Cells were grown to 80% abundance in T75 flasks and harvested by trypsinization.
1.5 cultivation of pathogenic bacteria ETEC and EPEC
ETEC cell line H10407(ATCC35401) was cultured in BHI-B medium (Merck, N.Y.). After overnight incubation at 37 ℃ under anaerobic conditions, the pathogen was re-incubated prior to infection to reach mid-log phase. Cells were harvested by centrifugation, washed and resuspended in PBS prior to the experiment.
Strain EPEC serotype O119 was purchased from DSMZ under freeze-dried conditions (DSM 8699). The strains were cultured in BHI-B medium (Merck, N.Y. USA). After overnight incubation at 37 ℃ under anaerobic conditions, the pathogen was re-incubated prior to infection to reach mid-log phase. Cells were harvested by centrifugation, washed and resuspended in PBS prior to the experiment.
1.6 data analysis
If possible, three replicates (sometimes six replicates) were used to perform the statistical analysis of each individual test. Anti-adhesion data were transformed with log 10. Statistical analysis was performed using one-way ANOVA for anti-adhesion data and epidermal signal transmission data after log10 transformation. The Dunnett's posthoc test was used to identify statistical differences from negative controls (neg. control) or with e.coli stimulation conditions. Statistical differences between the negative control and the test groups at each time point in the transmembrane resistance TEER test were analyzed using the two-way ANOVA and Dunnett's posthoc test. One-way ANOVA statistical analysis was performed in the testing of the inflammatory factors IL-8 and IP-10, and significance analysis was performed using the Dunnett's posthoc test. Significant differences are indicated by asterisks: represents p <0.05, represents p <0.01, represents p <0.001, represents p < 0.0001. P <0.05 was considered to be significantly different. The groups marked with asterisks have significant statistical differences with the groups not marked with asterisks, and the difference degree is different according to the number of asterisks. Since Dunnett's posthoc test used ANOVA MSResidual as a pooled assessment of differences and modified significance values to adjust the number of comparisons, the same results may be significant in one graph and not in the other.
2. Specific experimental procedures
2.1 anti-adhesion test
Caco-2 cells were cultured in 24-well plates. On the day of the assay, Caco-2 cells were washed with pre-warmed PBS. The test substances were added to Caco-2 cells in triplicate. Cells were cultured with the test substance for 1 hour. Pathogenic E.coli was then added, at a multiple of infection (MOI) 50: 1 addition (final concentration 10)7CFU/mL), and the test substance were incubated at 37 ℃ for 1 hour. As a negative control, Caco-2 cells were cultured in medium with pathogenic bacteria only. 1mM Zinc oxide (because it is reported to reduce pathogen adsorptionZnO) was used as a positive control. After incubation, the Caco-2 cells were washed and lysed, and then the pathogen was seeded on agar. After overnight incubation on agar plates at 37 ℃, CFU colonies of bacteria were counted to measure pathogen adsorption. The number of growing E.coli colonies was counted and recorded as CFU/mL. In parallel to the anti-adhesion test, Escherichia coli (final concentration of) 107CFU/mL was added to 1mL of the five combinations tested and co-incubated at 37 ℃ for 1 hour to measure activity. After incubation, E.coli was harvested from each sample by centrifugation, resuspended in PBS, and plated on agar plates. After overnight incubation on agar plates at 37 ℃, CFU colonies of bacteria were counted to measure pathogen adsorption. The number of growing E.coli colonies was counted and recorded as CFU/mL.
2.2 intestinal Barrier integrity test
The ideal small intestinal epithelial barrier function is a prerequisite to protect the host from pathogenic invasion and/or pathogenic toxins. In this study, barrier integrity in vitro was demonstrated by measuring the transepidermal electrical resistance (TEER) of the intestinal cell layer. Food ingredients may have the function of protecting the intestinal barrier function from decreasing after infection (reducing the decrease in TEER after infection). To study the effect of prebiotics and probiotics on infection, the TEER was measured before and after infection with e.
Caco-2 cells were seeded into Transwell polycarbonate cell culture inserts with an average pore size of 0.4um and an area of 0.33cm2Until complete differentiation (± 1000 Ω). Trans-epithelial electrical resistance (TEER) was measured with an EVOM2 epidermal voltmeter purchased from a world precision instrument to measure barrier integrity.
On the day of testing, cells were washed and cultured for 1 hour at 37 ℃ in medium without antibiotics and serum, but containing the test substance. Immediately thereafter, E.coli was added to the test substance (the infection magnification MOI was 200:1) and cultured for 6 hours. TEER was measured 1 hour, 2 hours, 3 hours, 4 hours and 6 hours after exposure of the test substance and before addition of the pathogen before the start of the experiment (t ═ 1), and 1 hour, 2 hours, 3 hours, 4 hours and 6 hours after pathogen exposure, respectively. The TEER values under the individual conditions after exposure to a pathogen correlate with their TEER values at t ═ 0 and are expressed as Δ TEER (Ω. Cm 2). Negative controls (addition of E.coli only) and positive controls not exposed to pathogenic bacteria or test substances were also included in the experimental group. All conditions were assayed in triplicate and some controls were assayed in 6 replicates.
2.3 inflammatory factor Release assay
Prebiotics and probiotics can have immunomodulatory (promoting or anti-inflammatory) effects, can increase resistance to infection or promote gut health. The immunomodulatory effects of prebiotic probiotics can be measured by measuring cytokine/chemokine production by small intestine epithelial cells in the presence or absence of a pro-inflammatory stimulus. The effect of prebiotics and probiotics on chemokine/cytokine production can be screened by stimulating Caco-2 cells with E.coli strains and measuring the production of IL-8 and IP-10 in the supernatant. IL-8 is important for an urgent autoimmune response, which can lead to the aggregation of neutrophils. IP-10 is important in the secondary response of immunity. It attracts monocytes and macrophages, also including Th1 cells, which play an important role in the clearance of infection. Pro-inflammatory prebiotics and probiotics may increase the production of IL-8 and/or IP-10, while anti-inflammatory prebiotics and probiotics may decrease the production of IL-8 and/or IP-10.
Caco-2 cells were cultured in 96-well plates to appropriate abundance. At the beginning of the experiment, cells were washed once with medium without antibiotics. The monolayer cells were co-cultured with the test substance at 37 ℃ for 1 hour in a medium containing no antibiotic, and this was repeated three times. Coli stimulating cells (MOI 200:1) were added. After 1 hour incubation, the monolayer cells were co-cultured with the pathogens and rinsed and incubated overnight with medium containing the test substance and 50 μ g/mL gentamicin. As a Blank control (Blank), only culture medium was used without stimulation with E.coli. Culture broth stimulated with E.coli but without test substance was used as a control for E.coli response. In addition, as a control for Caco-2 cell response, cells were stimulated with a mixture of cultures containing Rec TNF α (10ng/mL) and Rec IFN γ (5ng/mL), both purchased from R & D systems of Abindion, UK. Supernatants were collected after 24 hours of stimulation and stored at-20 ℃. IL-8 and IP-10 were tested using the Bio-Plex kit (BioRad, Calif., USA) according to the manufacturer's instructions.
3. Test for influence of test substance on EPEC survival rate of pathogenic bacteria
To verify whether the reduction in pathogenic adsorption is associated with pathogenic activity, pathogenic activity was also tested after culturing prebiotics and probiotics. The results are shown in fig. 1, fig. 2, fig. 3 and fig. 4, and similar to other tested probiotics or prebiotics, the survival rate of escherichia coli EPEC0119 bacteria is not significantly affected after the tested substance HN019 or HMO mixture is added. The survival rate of E.coli was slightly reduced by adding the two compositions B. Similar to other probiotics or prebiotics tested, the survival rate of E.coli EPEC0119 bacteria was not significantly affected by the addition of the test substance BL-99 or HMO mixture, or a combination of both.
Example 1: test result of intestinal adhesion of pathogenic bacteria EPEC (enteroadhesion promoting factor) by combination of HN019 and breast milk oligosaccharide mixture
The preparation steps before the experiment and the specific experimental methods are described in the preceding paragraphs. The tested lactobacillus bifidus HN019 has a normal growth curve.
The protective effect of probiotics and prebiotic compositions to prevent pathogenic bacteria from adsorbing to small intestine epithelial cells was investigated by common diarrheagenic strains (EPEC O119).
As shown in FIG. 5A, the adhesion of the breast milk oligosaccharide composition A alone to the pathogenic bacteria Escherichia coli O119 to the intestinal tract cells Caco-2 is not significantly different from that of the negative control, while the positive control zinc oxide can significantly reduce the adsorption of the pathogenic bacteria to the intestinal tract cells (p)<0.0001). Probiotics HN019 alone (10)8) The adhesion effect of the pathogenic bacterium Escherichia coli O119 on intestinal tract cells Caco-2 is not significantly different from that of a negative control. However, when prebiotics, i.e. breast milk oligosaccharide composition A, were used as the test substance in the treatment group together with probiotics, both significantly reduced the adsorption of the pathogenic bacterium EPEC0119 to intestinal cells (p)<0.05)。
FIG. 5B shows the different concentrations of Bifidobacterium lactis HN019 in another batch of experiments withThe results of experiments with compositions of different ratios of HMO mixtures for inhibiting the adherence of the EPEC of escherichia coli to Caco-2 cells are shown in table 6 (mean ± standard error, triplicate measurements). Due to the large number of groups, the test is divided into two different experimental determinations. After comparing the negative control with the test group using the Dunnett's posthoc test, 10 were found in the experimental group 16The mixture of HN019 and composition a at concentration was significantly lower than the negative control (P)<0.05). In Experimental group 2, 108HN019 at concentration significantly reduced pathogen adherence (P)<0.0001), composition A also significantly reduced pathogenic bacterial attachment (P)<0.0001)，108The combination of HN019 with composition A at concentrations also significantly reduced pathogen adherence (P)<0.0001); composition B significantly reduced pathogen adsorption (P)<0.01)，108The combination of HN019 with composition B at concentrations more significantly reduced pathogen adherence (P)<0.0001), a synergistic effect is embodied; 108The combination of HN019 with composition F at concentrations also significantly reduced pathogen adherence (P)<0.0001)。
TABLE 6
Example 2: results of intestinal adhesion experiment of BL-99 and breast milk oligosaccharide mixture on pathogenic bacteria EPEC
The preparation steps and specific experimental methods before the experiment are described in the preceding paragraphs. The tested bifidobacterium lactis BL-99 has a normal growth curve.
The protective effect of probiotics and prebiotic compositions to prevent pathogenic bacteria from adsorbing to small intestine epithelial cells was investigated by common diarrheagenic strains (EPEC O119).
Results see FIG. 6A, for breast milk oligosaccharide composition B alone against the pathogenic bacterium Escherichia coli O119 against the intestinal tractThe adhesion effect of the Caco-2 cells is not obviously different from that of a negative control, and the positive control zinc oxide can obviously reduce the adsorption of pathogenic bacteria to intestinal cells (p)<0.0001). Probiotics BL-99 alone (10)8) The adhesion effect of the pathogenic bacterium Escherichia coli O119 on intestinal tract cells Caco-2 is not significantly different from that of a negative control. However, when prebiotics, i.e. breast milk oligosaccharide composition B, were used as the treatment group test substance together with probiotics, both significantly reduced the adsorption of the pathogenic bacterium EPEC0119 to intestinal cells (p)<0.05)。
Fig. 6B shows the results of experiments on the inhibition of the adhesion of e.coli EPEC O119 to Caco-2 cells using compositions of different concentrations of bifidobacterium lactis BL-99 and different ratios of HMO mixtures in another batch of experiments, with the data of the results (mean ± standard error, three repeated measurements) shown in table 7. Due to the large number of groups, the test is divided into two different experimental determinations. After comparing the negative control with the test group using the Dunnett's posthoc test, 10 were found in the experimental group 16BL-99 in combination with composition A at a concentration significantly lower than the negative control (P)<0.05)；108The mixture of probiotic BL-99 with HMO composition C at concentration has a tendency to reduce the adherence of EPEC. In Experimental group 2, 108BL-99 at concentrations significantly reduced pathogen adherence (P)<0.01), composition A also significantly reduced the adhesion of pathogenic bacteria (P)<0.01) and 10)8The mixture of BL-99 with composition A at a concentration more significantly reduced the adhesion of pathogenic bacteria (P)<0.0001), a synergistic effect is embodied; 108The mixture of BL-99 with composition B at a concentration significantly reduced pathogenic bacterial adherence (P)<0.001), a synergistic effect is embodied; composition F also significantly reduced pathogen adherence (P)<0.01) and 10)8The mixture of BL-99 with composition F at a concentration more significantly reduced the adhesion of pathogenic bacteria (P)<0.001) exhibiting a synergistic effect.
TABLE 7
Example 3: experimental results on the Effect of HN019 in combination with a Breast milk oligosaccharide mixture on transmembrane resistance (TEER)
The preparation steps before the experiment and the specific experimental methods are described in the preceding paragraphs. The tested lactobacillus bifidus HN019 has a normal growth curve.
Results referring to figure 7A, the effect of test substances on transmembrane resistance was tested at different time points, breast milk oligosaccharide combination a alone or probiotic HN019 (10)8) The reduction of transmembrane resistance TEER is basically not influenced and is close to that of a negative control group, and the combination of the probiotic HN019 and the breast milk oligosaccharide mixture A can increase the transmembrane resistance TEER value to a certain extent at a plurality of time points so that the transmembrane resistance TEER value is closer to that of a blank group.
Figure 7B shows another batch of probiotic HN019 alone in the experiment (10)8) The results of the experimental data (mean ± standard error, triplicate measurements) for the effect of HMO composition A, B and F in different proportions on transmembrane resistance TEER in the case of exposure to e.coli ETEC H10407 are shown in table 8. When t is 1, HN019 (10)8) + composition A significantly improved intestinal barrier properties, significantly different from the negative control (P)<0.05). When t is 2, HN019 (10)8) The intestinal barrier property is improved, and the intestinal barrier property is obviously different from that of a negative control (P)<0.01)；HN019(108) + composition A also improved intestinal barrier, with a significant difference from the negative control (P)<0.01)；HN019(108) + composition F improved intestinal barrier, a significant difference from the negative control (P)<0.05). When t is 4, HN019 (10)8) The intestinal barrier property is improved, and the intestinal barrier property is obviously different from that of a negative control (P)<0.05), and HN019 (10)8) + composition A more significantly improved intestinal barrier properties, significantly different from the negative control (P)<0.01), a synergistic effect is embodied; HN019 (10)8) + composition F improved intestinal barrier, a significant difference from the negative control (P)<0.05). When t is 6, HN019 (10)8) + the composition A improves the intestinal barrier property and the function of yinThe sexual control is significantly different (P)<0.01), a synergistic effect is embodied; HN019 (10)8) + composition F also improved intestinal barrier, with a significant difference from the negative control (P)<0.01), a synergistic effect is embodied. Probiotic bacteria HN019 alone were also tested in a two-way ANOVA and Dunnett's posthoc test (10)8) And statistical analysis between different probiotic and HMO combinations, found HN019 (10) at t ═ 68) + composition A (P)<0.05) and HN019 (10)8) + composition F (P)<0.01) are mixed with separate probiotics HN019 (10)8) Has obvious difference and shows synergistic effect.
TABLE 8
Figure 7C shows probiotic bacteria HN019 alone (10)6) The data of the results of the experiments (mean ± standard error, triplicate measurements) with HMO composition a on the effect of transmembrane resistance TEER in the case of exposure to e.coli ETEC H10407 are shown in table 9.
TABLE 9
When t is 2, HN019 (10)6) + composition A showed a significant increase compared to the negative control (P)<0.05), a synergistic effect is embodied; when t is 4, HN019 (10)6) + composition A with probiotic bacteria HN019 alone (10)6) Compared with the prior art, has obvious improvement (P)<0.05), a synergistic effect is embodied.
Example 4: transmembrane electrode formed by combination of bifidobacterium lactis BL-99 and breast milk oligosaccharide mixture in different proportions
Resistance test
The preparation steps and specific experimental methods before the experiment are described in the preceding paragraphs. The tested bifidobacterium lactis BL-99 has a normal growth curve.
FIG. 8 shows probiotic BL-99 alone (10)8) With different proportions of HMO compositionA. The data of the results of the experiments (mean ± standard error, triplicate measurements) on the effect of the transmembrane resistance TEER in the case of exposure to e.coli ETEC H10407 are shown in table 10.
When t is 6, BL-99 (10)8) + composition A significantly improved intestinal barrier properties, significantly different from the negative control (P)<0.05), a synergistic effect is embodied. When t is 4, BL-99 (10)8) + composition B significantly improved intestinal barrier properties, significantly different from the negative control (P)<0.05), a synergistic effect is embodied. When t is 4, and BL-99 (10)8) + composition A significantly improved intestinal barrier properties, significantly different from the negative control (P)<0.05)。
Example 5: combination of HN019 and breast milk oligosaccharide mixture for secreting inflammatory factors IL-8 and IP-
10 influence of
The preparation steps before the experiment and the specific experimental methods are described in the preceding paragraphs. The tested lactobacillus bifidus HN019 has a normal growth curve.
Results referring to figures 9A, 9B, 10A, 10B, bifidobacterium lactis HN019, and its combination with the breast milk oligosaccharide 2' -FL, released both IL-8 and IP-10 factors at significantly lower levels than the pathogenic bacterium ETEC without co-cultivation with the pathogenic bacterium ETEC. And the combination of HN019 with HMO mixtures C and D exhibited some synergistic effect on IL-8 release. When pathogenic bacteria ETEC and a tested substance exist in a cultured system at the same time, the release of IL-8 is not influenced by the composition of HN019, HN019 and HMO mixture C, but the release of IL-8 is obviously reduced by the composition of HN019 and HMO mixture D, and a synergistic effect is presented; furthermore, HN019, and its combination with HMO blends C or D, significantly reduced the release of IP-10. The HN019+ HMO mixture nutritional composition has a certain regulation effect on the release of inflammatory factors.
Claims (10)
1. A nutritional composition comprises Bifidobacterium lactis (Bifidobacterium lactis) and breast milk oligosaccharides.
2. The composition according to claim 1, wherein the Bifidobacterium lactis comprises Bifidobacterium lactis (Bifidobacterium lactis) strain HN019 and/or Bifidobacterium lactis (Bifidobacterium lactis) with accession No. CGMCC No. 15650.
3. The composition of claim 1, wherein the breast milk oligosaccharides comprise one or more of 2 '-fucosyllactose, 3' -fucosyllactose, lacto-N-tetraose, 3 '-sialyllactose, 6' -sialyllactose.
4. The composition of claim 3, wherein the breast milk oligosaccharide comprises 2 '-fucosyllactose, 3' -fucosyllactose, lacto-N-tetraose, 3 '-sialyllactose, 6' -sialyllactose in a weight ratio of (0-10): (4-8): (3-6): (1-4): 0-1).
5. The composition of claim 3, wherein the human milk oligosaccharide comprises, in weight percent (0-53%) (21-44%) (16-32%) (5-22%) (0-5%) 2 '-fucosyllactose, 3' -fucosyllactose, lacto-N-tetraose, 3 '-sialyllactose, 6' -sialyllactose.
6. Composition according to claim 1 or 2, wherein the ratio of bifidobacterium lactis to breast milk oligosaccharides is 1 x 103CFU～1×1012CFU: 5mg, preferably 1X 106CFU～1×1010CFU：5mg。
7. Composition according to claim 1 or 2, wherein the bifidobacterium lactis is applied in the nutritional composition in an amount of 1 x 103CFU～1×1012CFU/day, preferably 1X 106CFU～1×1011CFU/day.
8. Use of the composition according to any one of claims 1 to 7 for the preparation of a food or pharmaceutical product having the efficacy of increasing the immunocompetence of the gastrointestinal tract.
9. The use of claim 8, wherein the enhancing gastrointestinal immune competence comprises: resist pathogenic bacteria invasion in intestinal tract system, maintain intestinal tract shielding function, and/or reduce release of inflammatory factor IL-8 and/or IP-10 from intestinal tract cells.
10. Use according to claim 8, wherein the food product is a liquid beverage, a solid beverage, an oral liquid, a dairy product, a tablet or a capsule.
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| CN113796546A (en) * | 2021-09-13 | 2021-12-17 | 合生元（广州）健康产品有限公司 | Breast milk oligosaccharide composition for improving intestinal barrier function and application thereof |
| CN116196319A (en) * | 2021-11-30 | 2023-06-02 | 内蒙古伊利实业集团股份有限公司 | Use of oligosaccharides for protecting cartilage |
| WO2023098518A1 (en) * | 2021-11-30 | 2023-06-08 | 内蒙古伊利实业集团股份有限公司 | Use of bifidobacterium lactis bl-99 in protection of cartilage |
| CN116196319B (en) * | 2021-11-30 | 2024-06-04 | 内蒙古伊利实业集团股份有限公司 | Use of oligosaccharides for protecting cartilage |
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| CN112870232A (en) | 2021-06-01 |
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