CN114568520A

CN114568520A - Infant formula powder containing bifidobacterium longum subspecies infantis and application thereof

Info

Publication number: CN114568520A
Application number: CN202011381138.2A
Authority: CN
Inventors: 杜阿如娜; 刘彪; 李威; 孔小宇; 卡洛琳·安妮卡·凡·鲁-鲍曼; 盖瑞特·施密特; 吉塞拉·阿德里安娜·怀斯; 王雯丹; 司徒文佑; 刘伟贤; 洪维鍊
Original assignee: Inner Mongolia Yili Industrial Group Co Ltd
Current assignee: Inner Mongolia Yili Industrial Group Co Ltd
Priority date: 2020-11-30
Filing date: 2020-11-30
Publication date: 2022-06-03

Abstract

The invention provides an infant formula powder containing bifidobacterium longum subspecies infants and application thereof. The invention firstly provides a maternal emulsion infant formula powder, which comprises Bifidobacterium longum subsp. The breast milk infant formula powder of the present invention may further comprise breast milk oligosaccharides. The maternal emulsion infant formula powder can effectively improve the gastrointestinal tract immunity.

Description

Infant formula powder containing bifidobacterium longum subspecies infants and application thereof

Technical Field

The invention mainly relates to infant formula powder containing Bifidobacterium longum subspecies and application thereof, in particular to maternal emulsified infant formula powder containing Bifidobacterium longum subspecies (Bifidobacterium longum subsp.

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 reconstituting the already existing intestinal flora.

At present, in the fields of infant formula powder and the like, a solution for relieving infant intestinal discomfort and improving autoimmune ability is needed.

Disclosure of Invention

The invention aims to provide infant formula powder capable of relieving intestinal discomfort of infants and improving gastrointestinal tract immunity.

The inventor of the present invention finds in research that Bifidobacterium longum subsp. infantis with the preservation number of CCTCC No. m2011122 has the effect of inhibiting adhesion of intestinal pathogenic bacteria, improving intestinal barrier integrity, inhibiting the generation of intestinal inflammatory factors, and relieving inflammatory reaction caused by the intestinal pathogenic bacteria. Furthermore, when Bifidobacterium longum subsp. infantis (Bifidobacterium longum subsp. infantis) with the preservation number of CCTCC No. M2011122 and breast milk oligosaccharide are combined for application, the composition has a synergistic effect on improving the gastrointestinal immunity.

The bifidobacterium longum subspecies infantis with the preservation number of CCTCC No. M2011122 is also named as bifidobacterium longum subspecies infantis GB-1496 in the invention. The strain is preserved in China Center for Type Culture Collection (CCTCC) with the preservation unit address: wuhan university, Wuhan, China, 430072; the preservation date is as follows: 2011, 04 month 10 days; the preservation number is: CCTCC NO: m2011122, category naming: bifidobacterium longum subsp. The bifidobacterium longum subspecies infantis CCTCC No. M2011122 also has better free radical scavenging capacity and reducing capacity, and can induce Caco-2 cells to increase the expression of antioxidant enzymes, so the bifidobacterium longum subspecies infantis GB-1496 strain also has the activity effect of resisting oxidation, and can reduce the concentration of free radicals to inhibit organ aging.

In one aspect, the invention provides a maternal emulsion infant formula powder, which comprises Bifidobacterium longum subsp.

According to a specific embodiment of the present invention, the breast milk oligosaccharide is further included in the present maternal emulsion infant formula.

According to a specific embodiment of the invention, in the maternal emulsified infant formula 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, 2 ' -FL or 2FL) is a trisaccharide structure formed by fucose and lactose, and is a representative substance of fucosyl oligosaccharide. Commercially available materials are usually prepared by microbial fermentation and have the same structure as oligosaccharides found in human milk.

3 ' -fucosyllactose (3-fucosyllactose, 3 ' -FL or 3FL) is a trisaccharide structure formed by fucose and lactose, and is an isomer of 2 ' -fucosyllactose. Is a representative substance of fucosyl oligosaccharide. The substance is prepared by microbial fermentation, and has the same structure as oligosaccharide found in human milk.

lacto-N-tetraose (LNT), which is a hexasaccharide structure formed by lactose and tetraose, is a representative substance of oligosaccharides having a core sugar chain as a basic structure and containing no fucosyl or sialyl group. The substance is prepared by microbial fermentation, and has the same structure as oligosaccharide found in human milk.

3 ' -sialyllactose (3 ' -sialyllactose, 3 ' -SL or 3SL) is a trisaccharide structure formed by sialic acid and lactose, and is a representative substance of sialyl oligosaccharides. The substance is prepared by microbial fermentation, and has the same structure as oligosaccharide found in human milk.

6 ' -sialyllactose (6 ' -sialyllactose, 6 ' -SL or 6SL), which is a trisaccharide structure formed by sialic acid and lactose, is a representative of sialic acid-based oligosaccharides. The substance is prepared by microbial fermentation, and has the same structure as oligosaccharide found in human milk.

According to a specific embodiment of the present invention, in the breast milk oligosaccharide of the present invention, the breast milk oligosaccharide comprises 2 '-fucosyllactose, 3' -fucosyllactose, lacto-N-tetraose, 3 '-sialyllactose, 6' -sialyllactose in a weight ratio of (0-18): (1-8): (1-6): (1-4): 0-12).

According to a specific embodiment of the present invention, the breast milk oligosaccharides in the present breast milk formula comprise, in weight percent (0-55%) (3-44.4%) (3-33.3%) (3-22.2%) (0-36%) 2 '-fucosyllactose, 3' -fucosyllactose, lacto-N-tetraose, 3 '-sialyllactose, 6' -sialyllactose. The weight percentage is based on the total amount of the breast milk oligosaccharide as 100%.

According to some preferred embodiments of the present invention, the breast milk oligosaccharide is present in the maternal emulsion infant formula of the present invention in an amount of 0.1 to 6.7%, preferably 0.3 to 5%, more preferably 0.7 to 3.5%. In the present invention, the contents or ratios are weight contents or ratios unless otherwise noted.

According to some preferred embodiments of the present invention, the content of bifidobacterium longum subspecies infants with the preservation number of CCTCC No. m2011122 in the breast-milk infant formula powder of the present invention is 1 × 10³CFU/g～1×10¹²CFU/g, preferably 1X 10⁶CFU/g～1×10¹⁰CFU/g。

According to some preferred embodiments of the invention, the maternal emulsified infant formula powder of the invention has a total protein content of 10-20 g/100g, and whey protein accounts for 50-70% of the total protein; the fat content is 15-30 g/100 g; the carbohydrate content is 50-70 g/100 g. Preferably, the linoleic acid content in the maternal emulsified infant formula powder is 2700-4500 mg/100g, and the alpha-linolenic acid content is 270-450 mg/100 g.

According to a particular embodiment of the invention, the present marmem infant formula may comprise, in addition to the above-mentioned components, conventional components of infant formula, for example a built nutrient package.

According to the specific embodiment of the invention, in the maternal emulsified infant formula powder of the invention, the protein and fat indexes can be further subdivided according to the development stage of the infant. Specifically, for infants between 0 and 6 months: the total protein content of the milk powder is 10-15 g/100g, the proportion of whey protein to the total protein is 60-70%, the fat content is 23-30 g/100g, the linoleic acid content is 2700-4500 mg/100g, the alpha-linolenic acid content is 270-450 mg/100g, and the carbohydrate content is 52-56 g/100 g. For older infants between 6-12 months: the milk powder contains 10-20 g/100g of total protein, 50-70% of whey protein, 20-30 g/100g of fat, 2700-4500 mg/100g of linoleic acid, 270-450 mg/100g of alpha-linolenic acid and 52-56 g/100g of carbohydrate. Aiming at the infants of 12-36 months: the milk powder contains 12-20 g/100g of total protein, 50-70% of whey protein, 20-30 g/100g of fat, 2700-4500 mg/100g of linoleic acid, 270-450 mg/100g of alpha-linolenic acid and 52-56 g/100g of carbohydrate.

According to a specific embodiment of the present invention, in the maternal emulsified infant formula of the present invention, the raw materials for providing total protein comprise: one or more of raw milk, whole milk powder, skimmed milk powder, whey protein powder, and desalted whey powder. Based on 1000 parts by weight of the infant formula powder, the infant formula powder comprises the following raw materials: 800-3000 parts of raw milk, 0-300 parts of whole milk powder, 0-120 parts of whey protein powder, 0-300 parts of skimmed milk powder and/or 0-300 parts of desalted whey powder.

According to a specific embodiment of the present invention, in the emulsion infant formula powder of the present invention, the raw material for providing fat comprises: one or more of milk fat-containing raw materials, mixed vegetable oil and OPO structure fat. The mixed vegetable oil comprises 15-50 parts of corn oil, 40-70 parts of soybean oil, 0-100 parts of sunflower seed oil and/or 0-115 parts of OPO structural grease.

According to a specific embodiment of the present invention, in the maternal emulsion infant formula of the present invention, the carbohydrate providing raw material comprises: 50-450 parts of lactose and/or the protein raw material containing lactose.

According to the inventionAccording to a specific embodiment, the raw materials of the maternal emulsion infant formula powder also comprise appropriate compound nutrients containing vitamins and minerals. The compound nutrient mainly comprises: compounding vitamins: taurine, vitamin A, vitamin D, vitamin E and vitamin K₁Vitamin B₁Vitamin B₂Vitamin B₆Vitamin B₁₂Nicotinic acid, folic acid, pantothenic acid, vitamin C, choline and biotin; compounding minerals: sodium, potassium, calcium, phosphorus, copper, iron, zinc, manganese, iodine, selenium, magnesium, potassium and derivatives thereof. The source, content and usage amount of the vitamins and minerals should meet the national standards and relevant regulations of infant formula food.

On the other hand, the invention also provides a method for preparing the breast-emulsified infant formula powder, which adopts a wet method or a dry method production process to mix bifidobacterium longum subspecies infants, breast milk oligosaccharide and other raw materials in the formula to prepare the breast-emulsified infant formula powder. The preparation process mainly comprises the following steps: preparing materials, homogenizing, concentrating, sterilizing, spray drying, and dry mixing to obtain the final product.

In the present invention, bifidobacterium longum subspecies infantis may be mixed together at the time of compounding (in inactivated form in the infant formula), preferably added during post-mixing (in viable form in the infant formula).

In the invention, the breast milk oligosaccharide can be mixed together during the compounding process, and can also be added in the post-mixing process.

On the other hand, the invention also provides application of the infant formula powder in preparing food with the effect of improving gastrointestinal immunocompetence. Specifically, the improvement of the gastrointestinal tract immunity comprises: improving intestinal bacterial infection resistance, resisting pathogenic bacteria invasion in intestinal system, maintaining intestinal shielding function, preventing diarrhea caused by pathogenic bacteria, and/or reducing intestinal cell release inflammatory factor IP-10.

In conclusion, the bifidobacterium longum subspecies of infants is found to have the functions of reducing the adhesion capability of pathogenic bacteria to intestinal epithelial cells, improving the intestinal barrier function and activating the intestinal immunity, and has the functions of resisting infection, regulating the intestinal health and the immunity and wide application prospect when being applied to infant formula powder.

Drawings

FIG. 1 shows the effect of various monomers and compositions of breast milk oligosaccharides on the survival of EPEC, a pathogen tested.

FIG. 2 shows the results of the adhesion experiments of probiotic bacteria and their compositions with various proportions of breast milk oligosaccharides on the action of pathogenic bacteria EPEC on intestinal cells Caco-2.

Fig. 3 shows the effect of probiotics and their compositions with various proportions of breast milk oligosaccharides on intestinal barrier disruption by escherichia coli.

FIG. 4 shows the effect of probiotics and their combination with breast milk oligosaccharides on the secretion of inflammatory factor IP-10 by intestinal cells when cocultured with the addition of E.coli.

Microbial deposits for patent procedures:

GB-1496 strain of the invention

The preservation date is as follows: 2011 10/04/month

The preservation unit is as follows: china Center for Type Culture Collection (CCTCC)

The address of the depository: wuhan university, Wuhan, China 430072

The preservation number is: CCTCC NO: m2011122

And (3) classification and naming: bifidobacterium longum subsp

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.

Example 1

The embodiment provides 0-6-month-old infant formula powder containing breast milk oligosaccharides and bifidobacterium longum subsp.

1000 parts of raw milk, 350 parts of lactose, 120 parts of mixed vegetable oil, 110 parts of OPO structural grease, 125 parts of desalted whey powder, 50 parts of skim milk powder, 70 parts of whey protein powder, 2 parts of breast milk oligosaccharide (2' -FL), 2 parts of bifidobacterium longum subspecies infantis CCTCC NO: m20111220.18 parts, compound vitamin 2.5 parts, and compound mineral substance 8 parts.

The infant formula powder of the embodiment is prepared by adopting a dry production process, and mainly comprises the following steps: preparing materials, preheating, homogenizing, concentrating, sterilizing, spray drying, dry mixing, and adding HMO and Bifidobacterium longum subspecies of infant to obtain the final product.

Through detection, the content of each component in the milk powder meets the requirement.

Example 2

The embodiment provides a 6-12 month old older infant formula powder containing breast milk oligosaccharides and bifidobacterium longum subsp.

1500 parts of raw milk, 220 parts of desalted whey powder, 170 parts of lactose, 170 parts of skim milk powder, 70 parts of mixed vegetable oil, 70 parts of OPO structural fat, 40 parts of whey protein powder, 4 parts of breast milk oligosaccharides (2' -FL, 3-FL, LNT and 3-SL mixture), 4 parts of bifidobacterium longum subspecies of infants CCTCC NO: m20111220.18 parts, 3 parts of compound vitamin and 6 parts of compound mineral.

Example 3

The embodiment provides a 12-36 month-old infant formula powder containing breast milk oligosaccharides and bifidobacterium longum subsp.

1500 parts of raw milk, 250 parts of skim milk powder, 180 parts of lactose, 150 parts of desalted whey powder, 70 parts of mixed vegetable oil, 65 parts of OPO structural grease, 35 parts of whey protein powder, 8 parts of breast milk oligosaccharides (2' -FL, 3-FL, LNT, 3-SL, 6-SL mixture), 8 parts of bifidobacterium longum subspecies of infants CCTCC NO: m20111220.18 parts, 3 parts of compound vitamin and 6 parts of compound mineral.

Bifidobacterium longum subspecies infantis CCTCC NO: m2011122 and breast milk oligosaccharide efficacy experiment

The experimental methods and test article cases are as follows:

1. preparation of the experiment

1.1 media preparation and storage and preparation of prebiotics tested

The medium containing the prebiotics 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, whether monomer or composition, was 5 g/L.

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, probiotics were removed from glycerol stocks and inoculated on MRS plates at 37 ℃, harvested at rest, rinsed with DPBS, and resuspended in 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, and in particular cases, 1X 10 was selected⁶CFU/mL。

The bifidobacterium longum subspecies infantis GB-1496 is derived from human breast milk, is preserved in China Center for Type Culture Collection (CCTCC), and has the preservation unit address: wuhan university, Wuhan, China, 430072; the preservation date is as follows: 2011, 04 month 10 days; the preservation number is: CCTCC NO: m2011122, category naming: bifidobacterium longum subsp.

The taxonomical characteristics of the strain were confirmed based on the results of 16S rDNA sequence analysis and analysis by the API bacterial identification system. The morphological and general characteristics of Bifidobacterium longum subspecies infantis GB-1496 are detailed in Table 1.

TABLE 1

Bifidobacterium longum subspecies infantis GB-1496 strain is preserved at-80 ℃ in MRS medium containing 20% glycerol. Before use, the mixture was activated twice (24 hours) at 37 ℃ with MRS broth (DIFCO) containing 0.05% L-cysteine.

1.3 formulation of different combinations of prebiotics and probiotics

The tested concentration of the prebiotics or the compositions thereof is 5 g/L; the number of the tested probiotics is 1 multiplied by CFU/mL, and under special conditions, 1 multiplied by 10 is selected⁶CFU/mL。

The proportion and the percentage of the five prebiotics in the combination are shown in the table 2 and the table 3.

TABLE 2

Ratio of	2’-FL	3-FL	LNT	3-SL	6-SL
						Composition A
	10	4	3	1	1
						Composition B	10	4	3	2	0
Composition C	9	6	3	2	0
						Composition D	0	8	6	4	0
Composition E	18	1	1	1	12
						Composition F	12.5	5.75	4.25	1.5	1

TABLE 3

Percent by weight%	2’-FL	3-FL	LNT	3-SL	6-SL
						Composition A	53	21	16	5	5
Composition B	53	21	16	10	0
						Composition C	45	30	15	10	0
Composition D	0	44.4	33.3	22.2	0
						Composition E	55	3	3	3	36
Composition F	50	23	17	6	4

1.4 Caco-2 cell culture

A human colon tumor cell line (Caco-2) was obtained from the German Collection of microorganisms DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) in the presence of 5% CO₂Culturing at 37 deg.C under certain humidity. Caco-2 cells at passages 40-44 were used for the experiments. MEM medium was added at 20% (by volume)Volume) of Fetal Bovine Serum (FBS), 1% of 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, Blanda, Netherlands). Cells were grown to 80% abundance in T75 flasks and harvested by trypsinization.

1.5 cultivation of pathogenic bacteria ETEC and EPEC

Two pathogens were used in this study, enterotoxigenic E.coli ETEC H10407 and the enteropathogenic E.coli EPEC serotype O119. These two bacteria can be used to mimic small bowel infection in vitro. Both of these are common pathogens causing infantile diarrhea and traveler's diarrhea, especially in developing areas with poor hygiene. ETEC H10407 is a well-defined model strain commonly used in vitro experiments and has been widely used in other studies evaluating probiotics and prebiotics for pathogen adsorption and inflammatory signaling. This strain is also used in animal testing for the development of vaccines. EPEC serotype O119 is isolated from infant faeces and prebiotics have been shown to reduce its adsorption.

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 assay for inflammatory factor IP-10, and significance analysis was performed using 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 porous response analysis and modified significance values were used to adjust the number of comparisons, the same results may be significant in one graph and not in another.

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 substance was added to Caco-2 cells in triplicate. The cells were incubated 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 (ZnO) was used as a positive control, as it was reported to reduce pathogen adsorption. 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 test combination species and co-incubated at 37 ℃ for 1 hour to measure activity. After incubation, E.coli was collected by centrifugation from each sample, resuspended in PBS and culturedThe agar plates were inoculated. 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. All conditions were performed in triplicate.

2.2 intestinal Barrier integrity test

The ideal small intestinal epithelial barrier function is a prerequisite for protection of the host from pathogenic invasion and/or toxins of pathogenic origin. 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 0.4 μm 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 IP-10 in the supernatant. 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 IP-10, while anti-inflammatory prebiotics and probiotics may decrease the production of 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 the 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 cocktail of culture media 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 ℃. IP-10 was 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 bacteria adsorption is associated with pathogenic bacteria activity, pathogenic bacteria activity was also tested after co-culturing prebiotics with probiotics and cells. As shown in FIG. 1, similar to other prebiotics tested, the survival rate of E.coli EPEC 0119 bacteria was not significantly affected by the addition of the test substance, each of the breast milk oligosaccharides 2 ' -FL, 3 ' -FL, LNT, 3 ' -SL, etc. or combinations thereof. Therefore, the survival rate of the Escherichia coli EPEC is not influenced by breast milk oligosaccharide.

Intestinal adhesion experiment result of probiotics GB-1496 and composition of probiotics GB-1496 and breast milk oligosaccharide to pathogenic bacteria EPEC

The protective effect of prebiotic and probiotic compositions to prevent pathogenic bacteria from adsorbing to small intestine epithelial cells was investigated by common diarrheagenic strains (EPEC O119). The figure shows the results of experiments on the inhibition of the adherence of the escherichia coli EPEC to Caco-2 cells of the combination of the probiotic GB-1496 with 2' -FL or HMO compositions a to F (mean ± standard error, three repeated measurements).

Because the group of each experiment is limited, the experiments are carried out in two times. See table 4 and fig. 2 for experimental results.

TABLE 4

In the first experiment (left part of FIG. 2), 5g/L of 2' -FL significantly reduced the adhesion of EPEC (P) compared to the negative control<0.01)；GB-1496(10⁸) +2 '-FL significantly reduced the adhesion of EPEC and was more significant than 2' -FL alone (P)<0.001), the synergistic effect of the probiotics and the HMO is shown; composition C alone did not reduce the adhesion of EPEC, but after the addition of probiotic bacteria, GB-149610⁸+ mix C significantly reduced the adhesion of EPEC (P)<0.01) which embodies the synergistic effect of the prebiotics and the probiotics; composition D alone significantly reduced EPEC adhesion (P) compared to the negative control<0.05), the reduction of probiotic and composition D was more pronounced after probiotic addition (P)<0.01)。

In a second experiment (right part of fig. 2), the negative control was compared to the test group, probiotic 10⁶Significantly reduced pathogen adherence (P) over the negative control<0.0001), probioticsBacterium 10⁸Significantly reduced pathogen adherence (P) over the negative control<0.0001), composition A significantly reduced adhesion of pathogenic bacteria (P) over the negative control<0.0001)，GB-1496 10⁸+ composition A also significantly reduced the adhesion of pathogenic bacteria (P)<0.0001); composition B significantly reduced the adherence of pathogenic bacteria (P)<0.01),GB-1496 10⁸+ HMO composition B also significantly reduced the adhesion of pathogenic bacteria, and to a greater extent (P)<0.0001), the synergistic effect of the prebiotics and the probiotics is embodied; HMO composition E significantly reduced adhesion of pathogenic bacteria (P)<0.001),GB-1496 10⁸+ HMO composition E also significantly reduced the adhesion of pathogenic bacteria, and to a greater extent (P)<0.0001), embodies the synergistic effect of the prebiotics and the probiotics. HMO composition F significantly reduced the adhesion of pathogenic bacteria (P)<0.0001),GB-1496 10⁸+ HMO composition F also significantly reduced the adhesion of pathogenic bacteria (P)<0.0001)。

Experimental results of the effect of probiotic GB-1496 and composition with breast milk oligosaccharide on transmembrane resistance (TEER)

The experimental results of the effect of various human milk oligosaccharides and combinations with probiotic GB-1496 on transmembrane resistance (TEER) are shown in Table 5 and FIG. 3.

TABLE 5

Fig. 3 shows the effect of probiotic GB-1496 alone with HMO composition A, B, E or F on transmembrane resistance TEER when exposed to ETEC H10407 (mean ± standard error, triplicate measurements). GB-1496 (10)⁸) With HMO composition A at t ═ 1 (P)<0.05)、t＝2(P<0.05) and t ═ 6 (P)<0.05), intestinal barrier was significantly improved compared to the negative control. GB-1496 (10)⁸) + HMO composition E significantly improved intestinal barrier compared to the negative control at time point t ═ 6 (P)<0.05)。

GB-1496(10⁸) + HMO composition a at t ═ 4 (P)<0.05) and t ═ 6 (P)<0.01) two time points, all compared to probiotic GB-1496 (10) alone⁸) The improvement is remarkable, and the synergistic effect of prebiotics and probiotics is reflected; and GB-1496 (10)⁸) + HMO composition E at t ═ 6 (P)<0.05), also compared to GB-1496 (10)⁸) The group has obvious improvement on TEER, and a synergistic effect is embodied; and GB-1496 (10)⁸) + HMO composition F at t ═ 6 (P)<0.05) time point, ratio GB-1496 (10)⁸) The group also had a significant improvement in TEER, representing a synergistic effect.

Effect of probiotics GB-1496 and composition of probiotics GB-1496 and breast milk oligosaccharide on secretion of inflammatory factor IP-10 by intestinal cells

See table 6 and fig. 4 for experimental results.

TABLE 6

FIG. 4 shows the effect of the combination of GB-1496 and 2' -FL on the production of the inflammatory factor IP-10 by Caco-2 cells following stimulation with ETEC (mean. + -. standard error, triplicate measurements). It can be seen that the individual probiotics are in 10⁶At a concentration lower than that of the negative control, the production of the inflammatory factor IP-10 (P) can be reduced<0.01); and the individual probiotics are in 10⁸Under these conditions, and at a concentration of 5g/L for the breast milk oligosaccharide 2' -FL alone, there was no difference from the negative control, but after the probiotic had formed a composition with the HMO, GB-1496 was 10⁸Under the condition, the composition with 2' -FL can obviously reduce the generation (P) of inflammatory factor IP-10<0.001)。

Claims

1. A kind of mother emulsified infant formula powder comprises Bifidobacterium longum subsp.

2. The maternal emulsion infant formula of claim 1, further comprising breast milk oligosaccharides.

3. The maternal emulsified infant formula of claim 2, wherein the breast milk oligosaccharides comprise one or more of 2 '-fucosyllactose, 3' -fucosyllactose, lacto-N-tetraose, 3 '-sialyllactose, 6' -sialyllactose.

4. The maternal emulsion infant formula of claim 3, wherein said maternal oligosaccharides comprise 2 '-fucosyllactose, 3' -fucosyllactose, lacto-N-tetraose, 3 '-sialyllactose, 6' -sialyllactose in a weight ratio of (0-18): (1-8): (1-6): (1-4): 0-12; or,

the breast milk oligosaccharide comprises (by weight percentage) 0-55%, (3-44.4%), 3-33.3%, (3-22.2%), and (0-36%) 2 '-fucosyllactose, 3' -fucosyllactose, lacto-N-tetraose, 3 '-sialyllactose, and 6' -sialyllactose.

5. A maternal emulsified infant formula according to any one of claims 2 to 4, wherein the content of breast milk oligosaccharides in the maternal emulsified infant formula is 0.1-6.7%, preferably 0.3-5%, more preferably 0.7-3.5%.

6. The maternal emulsion infant formula powder according to claim 1 or 2, wherein the content of bifidobacterium longum subsp³CFU/g～1×10¹²CFU/g, preferably 1X 10⁶CFU/g～1×10¹⁰CFU/g。

7. The maternal emulsified infant formula powder according to claim 1 or 2, wherein the maternal emulsified infant formula powder has a total protein content of 10-20 g/100g, and the whey protein accounts for 50-70% of the total protein; the fat content is 15-30 g/100 g; the carbohydrate content is 50-70 g/100 g;

preferably, the content of linoleic acid in the maternal emulsion infant formula powder is 2700-4500 mg/100g, and the content of alpha-linolenic acid is 270-450 mg/100 g.

8. A process for preparing a maternal emulsion infant formula according to any one of claims 1 to 7, wherein a wet or dry manufacturing process is used to prepare said maternal emulsion infant formula by mixing the sub-species bifidobacterium longum infant with the accession number CCTCC No. m2011122 with the other raw materials of the formula.

9. Use of a maternal emulsified infant formula according to any one of claims 1 to 7 in the manufacture of a food product having the efficacy of enhancing the immunocompetence of the gastrointestinal tract.

10. The use of claim 9, wherein the enhancing gastrointestinal immune competence comprises: improving intestinal bacterial infection resistance, resisting pathogenic bacteria invasion in intestinal system, maintaining intestinal shielding function, preventing diarrhea caused by pathogenic bacteria, and/or reducing intestinal cell release inflammatory factor IP-10.