CN112870233B - Composition containing bifidobacterium lactis and breast milk oligosaccharide and application thereof - Google Patents

Abstract

The invention mainly relates to a composition containing bifidobacterium lactis and breast milk oligosaccharide and application thereof, wherein the composition is a nutritional composition containing bifidobacterium lactis (Bifidobacterium lactis) and breast milk oligosaccharide (Human Milk Oligosaccharides). The composition of the invention can effectively improve the immunity of the gastrointestinal tract, and can be added into various health-care foods.

Description

Composition containing Bifidobacterium lactis and human milk oligosaccharide and application thereof

Technical Field

The present invention mainly relates to a composition comprising bifidobacterium lactis and human milk oligosaccharides and its application, in particular to a nutritional composition comprising bifidobacterium lactis and human milk oligosaccharides (HMOs), which can be added to various health foods.

Background Art

In the last millennium, medical literature has documented that infants who are not breastfed have higher rates of illness and mortality. Breast milk not only provides the necessary nutrition for infants, but the active ingredients in breast milk also provide protection for the development of the infant's intestines and the improvement of immunity. Compared with infants fed with formula milk, infants fed with breast milk have a higher relative abundance of beneficial bacteria in their intestinal flora, especially bifidobacteria and lactobacilli.

Breast milk, through the transmission of flora, combined with active ingredients such as human milk oligosaccharides and cytokines in breast milk, establishes a healthy intestinal flora for newborns. Infants ingest 10 ⁷ -10 ⁸ bacteria, including lactic acid bacteria and bifidobacteria, through breast milk every day. These bacteria are directly transmitted to infants through breast milk, and some can colonize in the infant's intestines, promoting the establishment of intestinal flora in early life. The establishment of infant intestinal flora has short-term and even lifelong effects on the development of the intestine, as well as health and the immune system.

Human Milk Oligosaccharides (HMOs) are the third most abundant substance in breast milk after lactose and fat. Its total content varies in various stages of lactation, ranging from about 12-14 g/L in mature milk to about 20-24 g/L in colostrum. The structure of each human milk oligosaccharide has a lactose at the reducing end, most of which use polylactosamine as the main structural chain and contain fucose, sialic acid or both at the chain end. The presence and content of HMOs vary from person to person and are related to the Lewis secretory composition of the lactating mother. Since the raw material of infant formula is usually cow's milk, which usually contains no or very little of this type of oligosaccharide, HMOs have become a gap that infant formula must cross if it wants to get closer to the composition of breast milk.

In the 1990s, 2-fucosyllactose (2’-FL), an HMO found in most breast milk, was found to be effective in reducing the toxicity of stable toxins in Escherichia coli; in 2003, this oligosaccharide was reported to inhibit the attachment and infection of Campylobacter jejuni. Subsequently, three major functions of human milk oligosaccharides were gradually reported and discovered: (1) inhibiting the attachment and infection of specific pathogens; (2) acting as a prebiotic to promote the growth of bacteria in the intestinal symbiotic system; and (3) directly reducing the inflammatory response of the mucosa to toxic stimuli. The first clinical intervention trial using 2’-FL confirmed that the addition of this specific ingredient to a low-calorie formula was not only safe but also allowed formula-fed infants to have a growth rate comparable to that of breast-fed infants. 2’-FL is also used as a nutritional supplement for adults to relieve irritable bowel syndrome or inflammatory bowel disease, or as a prebiotic to maintain intestinal flora balance.

Intestinal flora is an important component of the human intestinal microecological system and plays an important role in human health, such as providing essential nutrients, producing vitamin K, assisting the digestive process, and promoting angiogenesis and intestinal nerve function. Prebiotics and probiotics are regarded as microecological management tools to improve the health of the body, which can change, regulate and reorganize the existing intestinal flora.

At present, in the field of infant formula powder, complementary food and nutritional supplements, there is a need for solutions to relieve intestinal discomfort in infants and young children and enhance their own immunity. At the same time, in the field of children over 3 years old, adolescents and adults, there is also a need to maintain the balance of intestinal flora and regulate immunity.

Summary of the invention

One object of the present invention is to provide a nutritional composition that can improve gastrointestinal immunity.

The inventors of the present invention have found in their research that the combination of Bifidobacterium lactis and human milk oligosaccharides has a synergistic effect in improving gastrointestinal immunity.

Thus, in one aspect, the present invention provides a nutritional composition comprising Bifidobacterium lactis and human milk oligosaccharides.

According to a specific embodiment of the present invention, in the composition of the present invention, the human milk oligosaccharides include one or more of 2'-fucosyllactose, 3'-fucosyllactose, lactose-N-tetraose, 3'-sialyllactose, and 6'-sialyllactose.

2’-fucosyllactose is a trisaccharide structure formed by fucose and lactose. It is prepared by microbial fermentation and has the same structure as the oligosaccharides found in human milk.

3’-fucosyllactose is a trisaccharide structure formed by fucose and lactose, and is an isomer of 2’-fucosyllactose. It is prepared by microbial fermentation and has the same structure as the oligosaccharides found in human milk.

Lactose-N-tetraose, produced by microbial fermentation, has the same structure as the oligosaccharides found in human milk.

3’-sialyllactose is a trisaccharide structure formed by sialic acid and lactose. It is prepared by microbial fermentation and has the same structure as the oligosaccharides found in human milk.

6’-sialyllactose is a trisaccharide structure formed by sialic acid and lactose, and is an isomer of 3’-sialyllactose. It is prepared by microbial fermentation and has the same structure as the oligosaccharides found in human milk.

According to a specific embodiment of the present invention, in the composition of the present invention, the human milk oligosaccharides include 2'-fucosyllactose, 3'-fucosyllactose, lactose-N-tetraose, 3'-sialyllactose, and 6'-sialyllactose in a weight ratio of (0-10):(4-8):(3-6):(1-4):(0-1).

According to a specific embodiment of the present invention, in the composition of the present invention, the human milk oligosaccharide comprises 2'-fucosyllactose, 3'-fucosyllactose, lactose-N-tetraose, 3'-sialyllactose, and 6'-sialyllactose in weight percentages (0-53%): (21%-44%): (16%-32%): (5%-22%): (0-5%). The weight percentages are based on the total amount of human milk oligosaccharides as 100%.

According to a specific embodiment of the present invention, in the composition of the present invention, the Bifidobacterium lactis includes the Bifidobacterium lactis HN019 strain and/or the Bifidobacterium lactis with a preservation number of CGMCC No.15650.

Bifidobacterium lactis HN019 strain is a commercial strain and can be provided by DuPont Danisco.

The Bifidobacterium lactis with a deposit number of CGMCC No.15650 is also named Bifidobacterium lactis BL-99. The strain has gastric acid resistance, and the survival rate of live bacteria is more than 62% when treated in gastric acid solution with a pH of 2.5 for 30 minutes, and the survival rate of live bacteria is more than 61% when treated for 2 hours. The Bifidobacterium lactis BL-99 provided by the present invention also has intestinal fluid resistance, and the survival rate of live bacteria is more than 70% when treated in small intestinal fluid with a pH of 6.8 for 2 hours. Mouse experiments show that the strain has no oral acute toxicity, no antibiotic tolerance, and can be safely used in food processing. The strain has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration (CGMCC) (Address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences) on April 26, 2018, and is classified and named: Bifidobacterium lactis; the deposit number is CGMCC No.15650.

According to a specific embodiment of the present invention, in the composition of the present invention, the ratio of Bifidobacterium lactis to human milk oligosaccharide is 1×10 ³ CFU to 1×10 ¹² CFU: 5 mg, preferably 1×10 ⁶ CFU to 1×10 ¹⁰ CFU: 5 mg.

According to a specific embodiment of the present invention, in the composition of the present invention, the amount of Bifidobacterium lactis used in the nutritional composition is 1×10 ³ CFU to 1×10 ¹² CFU/day, preferably 1×10 ⁶ CFU to 1×10 ¹¹ CFU/day.

According to a specific embodiment of the present invention, in the composition of the present invention, the amount of human milk oligosaccharides used in the nutritional composition is 1 mg to 15 g/day.

On the other hand, the present invention also provides the use of the composition in preparing health food or medicine having the effect of improving gastrointestinal immunity.

According to a specific embodiment of the present invention, in the application of the composition of the present invention, the improvement of gastrointestinal immunity includes resisting the invasion of pathogenic bacteria in the intestinal system, maintaining the intestinal barrier function, and/or reducing the release of inflammatory factors IL-8 and/or IP-10 by intestinal cells.

According to a specific embodiment of the present invention, the nutritional composition of the present invention can be used to prepare various health foods, etc. Specifically, the health food can be a liquid beverage, a solid beverage, an oral liquid, a dairy product, a tablet or a capsule, etc., for example, it can be used to add to infant health food (including infant formula powder, complementary food, nutritional supplements), and nutritional supplements or health foods for children over 3 years old, adolescents and adults, and has broad application prospects. Preferably, the amount of the Bifidobacterium lactis HN019 added to the health food is 1×10 ³ CFU~1×10 ¹² CFU/day, and more preferably 1×10 ⁶ CFU~1×10 ¹¹ CFU/day. Preferably, the amount of the Bifidobacterium lactis BL-99 added to the health food is 1×10 ³ CFU~1×10 ¹² CFU/day, and more preferably 1×10 ⁶ CFU~1×10 ¹¹ CFU/day. The amount of human milk oligosaccharides added to health food can be 1mg-15g/day.

The health food or medicine comprising the nutritional composition of the present invention has the function of improving the gastrointestinal immunity due to the nutritional composition.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the effect of human milk oligosaccharide mixture on the survival rate of the tested pathogenic bacteria EPEC.

FIG. 2 shows the effects of Bifidobacterium lactis HN-019 and BL-99 strains on the survival rate of the tested pathogenic bacteria EPEC.

FIG3 shows the effect of the combination of Bifidobacterium lactis HN019 and a human milk oligosaccharide mixture on the survival rate of the tested pathogenic bacteria EPEC.

FIG. 4 shows the effect of the combination of Bifidobacterium lactis BL-99 and a human milk oligosaccharide mixture on the survival rate of the tested pathogenic bacteria EPEC.

Figure 5A shows the results of the experiment on the adhesion of the pathogenic bacteria EPEC to intestinal cells Caco-2 by the combination of Bifidobacterium lactis HN019 (10 ⁸ ) and human milk oligosaccharide mixture A; Figure 5B shows the effect of the combination of different concentrations of Bifidobacterium lactis HN019 and different ratios of human milk oligosaccharide mixture on the intestinal adhesion of the tested pathogenic bacteria EPEC.

Figure 6A shows the results of the experiment on the adhesion of the pathogenic bacteria EPEC to intestinal cells Caco-2 by the combination of Bifidobacterium lactis BL-99 (10 ⁸ ) and human milk oligosaccharide mixture B; Figure 6B shows the effect of the combination of different concentrations of Bifidobacterium lactis BL-99 and different ratios of human milk oligosaccharide mixture on the intestinal adhesion of the tested pathogenic bacteria EPEC.

Figure 7A shows the effect of the combination of Bifidobacterium lactis HN019 (10 ⁸ ) and human milk oligosaccharide mixture A on the intestinal barrier; Figure 7B shows the effect of the combination of 10 ⁸ of Bifidobacterium lactis HN019 and different proportions of human milk oligosaccharide mixtures on the transmembrane resistance TEER under ETEC invasion conditions; Figure 7C shows the effect of the combination of 10 ⁶ of Bifidobacterium lactis HN019 and human milk oligosaccharide composition A on the transmembrane resistance TEER under pathogenic bacteria ETEC invasion conditions.

FIG8 shows the effect of the combination of 10 ⁸ of Bifidobacterium lactis BL-99 and different ratios of human milk oligosaccharide mixture on the transmembrane resistance TEER under ETEC invasion conditions.

9A and 9B show the effect of the combination of Bifidobacterium lactis HN019 and a human milk oligosaccharide mixture on the secretion of inflammatory factors IL-8 and IP-10 by intestinal cells when Escherichia coli was not added for co-culture.

10A and 10B show the effect of the combination of Bifidobacterium lactis HN019 and a human milk oligosaccharide mixture on the secretion of inflammatory factors IL-8 and IP-10 by intestinal cells when Escherichia coli was added for co-culture.

Patented procedure for preservation of microorganisms:

Bifidobacterium lactis BL-99 of the present invention:

Deposit date: April 26, 2018;

Depository: China General Microbiology Center (CGMCC);

Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

Deposit number: CGMCC No.15650;

Classification name: Bifidobacterium lactis.

DETAILED DESCRIPTION

In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below in conjunction with specific examples and drawings. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. Unless otherwise specifically defined, all technical and scientific terms used herein have the same meaning as those commonly understood by ordinary technicians in the relevant fields.

The inventors of this case have proved through specific experiments that the probiotic-prebiotic composition of the present invention has a synergistic effect in regulating gastrointestinal immunity.

The experimental methods and test substances used in each embodiment and control are as follows:

1. Experimental preparation steps

1.1 Culture medium preparation and storage and preparation of the tested prebiotics

The culture medium containing prebiotics (see Table 1 for prebiotic types) and probiotics was freshly prepared in a sterile environment on the day of each experiment and preheated to 37°C. The tested prebiotics/HMOs were stored in a dry, dark environment at room temperature. The concentration used in the experiment was 5 g/L.

Table 1

serial number name abbreviation Chinese name 1 2'-fucosyllactose 2'-FL 2'-fucosyllactose 2 3'-fucosyllactose 3'-FL 3'-fucosyllactose 3 3'-sialyllactose 3'-SL 3'-Sialyl lactose 4 6'-sialyllactose 6’-SL 6'-Sialyl lactose 5 Lacto-N-tetraose LNT Lactose-N-tetraose

1.2 Probiotic culture, growth curve drawing and activity identification

Probiotic powders were delivered to the laboratory in a freeze-dried state before the experiments. Probiotics were inoculated on MRS-agar plates, individual strain colonies were taken for 16S rDNA identification, and used to prepare glycerol stocks (stored at -80°C). Before each experiment, probiotics were removed from the glycerol stock and inoculated on MRS plates at 37°C, harvested in a static state, and washed with MEM medium before the experiment. Probiotic activity and cell count were measured using a fluorescence-activated cell sorting system before the probiotics were tested. The concentration of probiotics used in the experiments was 1×10 ⁸ CFU/mL. Bifidobacterium lactis HN019 or BL-99 was cultured under anaerobic conditions at 37°C, and growth curves were drawn before the experiments. The activity of the probiotic strains was tested by flow cytometry upon receipt of the probiotic raw materials and before the start of all experiments. 16S sequencing was used to determine the strain identity. Probiotic activity and cell count were measured using a fluorescence-activated cell sorting system before the probiotics were tested in each specific experiment.

1.2.1 Bifidobacterium lactis BL-99

The Bifidobacterium lactis BL-99 of the present invention is isolated from the intestinal tract of infants. The strain has been deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration Committee (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences) on April 26, 2018, and the classification name is Bifidobacterium lactis; the deposit number is CGMCC No.15650.

1.2.1.1 Taxonomic characteristics of Bifidobacterium lactis BL-99

Physical and chemical test results:

1.2.1.2 Tolerance of Bifidobacterium lactis BL-99 to artificial gastric and intestinal juices

Bifidobacterium is a genus of bacteria that is generally not acid-resistant. In this example, the artificial gastric juice and intestinal juice tolerance of the Bifidobacterium lactis BL-99 of the present invention was tested. At the same time, the Bifidobacterium lactis BL-99, which is currently recognized in the field as having excellent acid resistance and can survive through the gastrointestinal tract, was used as the For comparison.

Test method: After culturing Bifidobacterium lactis BL-99 strain in MRS liquid medium at 37°C for 16 hours, centrifuge at 4°C and 2500 rpm for 10 minutes to collect the bacteria.

The strains to be tested were cultured in artificial gastric juice and artificial intestinal juice, and viable bacteria were counted and analyzed after treatment at 37°C for 0, 30 minutes, and 2 hours, and the acid resistance and intestinal fluid resistance of the strains were evaluated by the survival rate. Survival rate = (number of viable bacteria after treatment/number of viable bacteria at time 0) × 100%.

The survival rate test results of the strains in artificial gastric acid (pH2.5) are shown in Table 2. When BB-12 is treated in artificial gastric acid (pH2.5) for 30 minutes, the survival rate of live bacteria is 7.04%, and the survival rate of live bacteria is only 1.64% after being treated for 2 hours; while the survival rate of live bacteria of the Bifidobacterium lactis BL-99 of the present invention is 62.60% when it is treated in artificial gastric acid (pH2.5) for 30 minutes, and the survival rate of live bacteria is 61.83% after being treated for 2 hours. It shows that the Bifidobacterium lactis BL-99 of the present 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 rate of strains in artificial gastric acid (pH 2.5)

The results of the survival rate test of the strains in artificial intestinal fluid (pH 6.8) are shown in Table 3. The data show that the survival rate of BB-12 in artificial intestinal fluid (pH 6.8) for 2 hours is only 28.95%, while the survival rate of the Bifidobacterium lactis BL-99 in the present invention is 70.23% in artificial intestinal fluid (pH 6.8) for 2 hours. This shows that the Bifidobacterium lactis BL-99 in the present invention has excellent intestinal fluid resistance and can survive and colonize in the intestine.

Table 3 Survival rate of strains in artificial intestinal fluid (pH 6.8)

1.2.1.3 Virulence experiment and safety test of Bifidobacterium lactis BL-99

The lactobacillus BL-99 of the present invention is inoculated in a BBL liquid culture medium, and anaerobically cultured at 36±1°C for 48±2 hours, and the number of viable bacteria of the lactobacillus BL-99 in the culture medium is counted to be 3.7×10 ⁸ cfu/mL, and the original liquid and the 5-fold concentrated liquid of the culture are orally gavaged to the test mice at 20.0mL/kg BW for 3 consecutive days, and observed for 7 days. The test sets a control group of the original liquid of the culture medium and the 5-fold concentrated liquid. The test results show that the BBL culture original liquid and the 5-fold concentrated liquid group of the lactobacillus BL-99 have no statistically significant effect on the weight growth of mice compared with the respective control groups (p＞0.05), and no toxic reaction or death of the test mice is observed.

The antibiotic sensitivity of Bifidobacterium lactis BL-99 was evaluated using the method of SN/T 1944-2007 "Determination of bacterial resistance in animals and their products". The evaluation results showed that Bifidobacterium lactis BL-99 was sensitive to Ampicillin, Penicillin G, Erythromycin, Chloramphenicol, Clindamycin, Vancomycin and Tetracycline. It meets the requirements of the European Food Safety Authority for the evaluation of edible bacteria resistance. Bifidobacterium lactis BL-99 does not contain exogenous antibiotic resistance genes and is safe for consumption.

1.2.2 Bifidobacterium lactis HN019

The HN019 strain was provided by DuPont Danisco.

1.3 Preparation of different combinations of prebiotics and probiotics

The concentration of the tested prebiotics in the combination was 5 g/L; the number of the tested probiotics was 1×10 ⁸ CFU/mL.

First round of testing: Five combinations of prebiotics: 2’-FL: 3-FL: LNT: 3-SL: 6-SL = 10: 4: 3: 1: 1

Second round of testing: The proportions and percentages of prebiotics in the five combinations are shown in Tables 4 and 5 below.

Table 4

Proportion 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 F 12.5 5.75 4.25 1.5 1

Table 5

1.4 Caco-2 cell culture

Human colon tumor cell line (Caco-2) was purchased from DSMZ (Braunschweig, Germany) and cultured in the presence of 5% CO ₂ at 37°C with humidity. Caco-2 cells from passage 40 to 44 were used for the experiments. MEM medium was supplemented with 20% (vol/vol) fetal bovine serum (FBS), 1% non-essential amino acids, 1% Glutamax, 1% sodium pyruvate, with or without 1% penicillin-streptomycin solution, and 50 μg/mL gentamicin (all purchased from Invitrogen, Breda, The Netherlands). Cells were grown in T75 culture flasks to 80% confluence and harvested by trypsinization.

1.5 Cultivation of pathogenic bacteria ETEC and EPEC

ETEC cell line H10407 (ATCC 35401) was cultured in BHI-B medium (Merck, New York, USA). After overnight culture at 37°C under anaerobic conditions, pathogenic bacteria were cultured again to reach mid-log phase before infection. Cells were collected by centrifugation, washed and resuspended in PBS before the experiment.

EPEC serotype O119 strain was purchased from DSMZ under freeze-dried conditions (DSM8699). The strain was cultured in BHI-B medium (Merck, New York, USA). After overnight culture at 37°C under anaerobic conditions, the pathogenic bacteria were cultured again to reach mid-log phase before infection. The cells were collected by centrifugation, rinsed and resuspended in PBS before the experiment.

1.6 Data Analysis

If possible, three replicates (sometimes six replicates) were used for statistical analysis of each individual test. Anti-adhesion data were log10 transformed. One-way ANOVA was used for statistical analysis of log10-transformed anti-adhesion data and epidermal signaling data. Dunnett’s posthoc test was used to identify statistical differences with negative control (Neg. control) or under conditions of stimulation with E. coli. In the transmembrane resistance TEER test, the statistical differences between the negative control and the test group at each time point were analyzed using two-way ANOVA and Dunnett’s posthoc test. In the test of inflammatory factors IL-8 and IP-10, one-way ANOVA statistical analysis was performed, and Dunnett’s posthoc test was used for significance analysis. Significant differences are indicated by asterisks: * represents p<0.05, ** represents p<0.01, *** represents p<0.001, and **** represents p<0.0001. P<0.05 is considered to be significantly different. The groups marked with asterisks are statistically significantly different from the groups without asterisks, and the degree of difference varies depending on the number of asterisks. Because Dunnett’s posthoc test uses ANOVAMSResidual as a pooled estimate of differences and uses a modified significance value to adjust for the number of comparisons, the same result may be significant in one graph but not in another.

2. Specific experimental steps

2.1 Anti-adhesion test

Caco-2 cells were cultured in 24-well plates. On the day of the experiment, Caco-2 cells were washed with pre-warmed PBS. Test substances were added to Caco-2 cells in triplicate. Cells were incubated with test substances for 1 hour. Pathogenic bacteria Escherichia coli were then added at a multiplicity of infection (MOI) of 50:1 (final concentration of 10 ⁷ CFU/mL) and incubated with the test substances for 1 hour at 37°C. As a negative control, Caco-2 cells were incubated with pathogens alone in the culture medium. 1 mM zinc oxide (ZnO) was used as a positive control as it has been reported to reduce pathogen adsorption. After incubation, Caco-2 cells were washed and lysed, and pathogens were then plated on agar. After overnight incubation on agar plates at 37°C, the 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 with the anti-adhesion test, E. coli (final concentration of) 10 ⁷ CFU/mL was added to 1 mL of the five tested combinations and co-cultured at 37°C for 1 hour to measure activity. After incubation, E. coli were collected from each sample by centrifugation, resuspended in PBS, and plated on agar plates. After incubation on agar plates at 37°C overnight, the 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

An ideal intestinal epithelial barrier function is a prerequisite for protecting the host from invading pathogens and/or toxins derived from pathogens. In this study, barrier integrity in vitro was assessed by measuring the transepidermal electrical resistance (TEER) of the intestinal cell layer. Food components may have a protective function against the decline in intestinal barrier function after infection (mitigating the decline in TEER after infection). To investigate the effects of prebiotics and probiotics on infection, TEER changes over time were measured before and after infection with Escherichia coli.

Caco-2 cells were seeded into Transwell polycarbonate cell culture inserts with an average pore size of 0.4 μm and an area of 0.33 cm ² until fully differentiated (±1000Ω). Transepithelial electrical resistance (TEER) was measured using an EVOM2 epidermal voltmeter purchased from World Precision Instruments to measure barrier integrity.

On the day of the test, cells were washed and incubated for 1 hour at 37°C in medium without antibiotics and serum but containing the test substances. E. coli were then added to the test substances (MOI 200:1) and incubated for 6 hours. TEER was measured before the start of the experiment (t = -1), after 1 hour of exposure to the test substances and before the addition of the pathogen (t = 0), and at 1 hour, 2 hours, 3 hours, 4 hours and 6 hours after the pathogen contact. The TEER values of the individual conditions after the contact with the pathogen were related to their respective TEER values at t = 0 and expressed as ΔTEER (Ω.Cm2). Negative controls (only E. coli added) and positive controls not exposed to the pathogen or the test substances were also included in the experimental groups. All conditions were measured in triplicate, and some controls were measured in 6 replicates.

2.3 Inflammatory factor release test

Prebiotics and probiotics can have immunomodulatory (pro- or anti-inflammatory) effects, which can increase resistance to infection or promote intestinal health. The immunomodulatory effects of prebiotics and probiotics can be measured by measuring cytokine/chemokine production by intestinal epithelial cells in the presence or absence of pro-inflammatory stimuli. The effects 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 acute autoimmune reactions and can lead to the accumulation of neutrophils. IP-10 is important in the secondary response of immunity. It can attract monocytes and macrophages, including Th1 cells, which are important for clearing infections. Pro-inflammatory prebiotics and probiotics can increase the production of IL-8 and/or IP-10, while anti-inflammatory prebiotics and probiotics can reduce the production of IL-8 and/or IP-10.

Caco-2 cells were grown to appropriate confluence in 96-well plates. At the beginning of the experiment, the cells were washed once with medium without antibiotics. The monolayers were co-cultured with the test substances at 37°C in medium without antibiotics for 1 hour, repeated three times. E. coli were added to stimulate the cells (MOI 200:1). After 1 hour of incubation, the monolayers were co-cultured with pathogenic bacteria, washed with medium containing the test substances and 50 μg/mL gentamicin and incubated overnight. As a blank control, only medium was used without stimulation with E. coli. The medium stimulated with E. coli but without the test substances was used as a control for the response of E. coli. In addition, as a control for the response of Caco-2 cells, the cells were stimulated with a mixture of medium containing Rec TNFα (10 ng/mL) and Rec IFNγ (5 ng/mL) (both purchased from R&D systems, Abingdon, UK). The supernatant was collected after 24 hours of stimulation and stored at -20°C. IL-8 and IP-10 were assayed using the Bio-Plex kit (BioRad, CA, USA) according to the manufacturer's instructions.

3. Test on the effect of the test substance on the survival rate of pathogenic bacteria EPEC

To verify whether the reduction in pathogen adsorption is related to pathogen activity, the activity of pathogens was also detected after the prebiotics and probiotics were cultured. The results are shown in Figures 1, 2, 3, and 4. Similar to other tested probiotics or prebiotics, the addition of the test substance HN019 or HMO mixture did not significantly affect the survival rate of Escherichia coli EPEC 0119. The addition of the combination B of the two slightly reduced the survival rate of Escherichia coli. Similar to other tested probiotics or prebiotics, the addition of the test substance BL-99 or HMO mixture, or the combination of the two, did not significantly affect the survival rate of Escherichia coli EPEC 0119.

Example 1: Results of the experiment on the adhesion of HN019 and human milk oligosaccharide mixture to the intestinal tract of pathogenic bacteria EPEC

Please refer to the above paragraphs for the preparatory steps and specific experimental methods. The tested Bifidobacterium lactis HN019 had a normal growth curve.

The protective effect of probiotic and prebiotic combinations on preventing the adsorption of pathogenic bacteria to intestinal epithelial cells was studied using a common diarrhea-causing strain (EPEC O119).

As shown in Figure 5A, the human milk oligosaccharide composition A alone had no significant difference in the adhesion of pathogenic bacteria Escherichia coli O119 to intestinal cells Caco-2 compared with the negative control, while the positive control zinc oxide significantly reduced the adsorption of pathogenic bacteria to intestinal cells (p<0.0001). The probiotic HN019 (10 ⁸ ) alone had no significant difference in the adhesion of pathogenic bacteria Escherichia coli O119 to intestinal cells Caco-2 compared with the negative control. However, when the prebiotic, namely the human milk oligosaccharide composition A, was used together with the probiotics as the test substances in the treatment group, both significantly reduced the adsorption of pathogenic bacteria EPEC 0119 to intestinal cells (p<0.05).

Figure 5B shows the experimental results of the composition formed by different concentrations of Bifidobacterium lactis HN019 and different proportions of HMO mixtures in another batch of experiments for inhibiting Escherichia coli EPEC from adhering to Caco-2 cells. The experimental results (mean ± standard error, repeated three times) are shown in Table 6. Due to the large number of groups, two different experimental measurements were performed. After comparing the negative control with the test group using Dunnett's posthoc test, it was found that in experimental group 1, the mixture of HN019 and composition A at a concentration of 10 ⁶ was significantly lower than the negative control (P<0.05). In experimental group 2, HN019 at a concentration of 10 ⁸ significantly reduced the adhesion of pathogenic bacteria (P<0.0001), composition A also significantly reduced the adsorption of pathogenic bacteria (P<0.0001), and the mixture of HN019 at a concentration of 10 ⁸ and composition A also significantly reduced the adhesion of pathogenic bacteria (P<0.0001); composition B significantly reduced the adsorption of pathogenic bacteria (P<0.01), and the mixture of HN019 at a concentration of 10 ⁸ and composition B more significantly reduced the adhesion of pathogenic bacteria (P<0.0001), reflecting a synergistic effect; the mixture of HN019 at a concentration of 10 ⁸ and composition F also significantly reduced the adhesion of pathogenic bacteria (P<0.0001).

Table 6

Example 2: Results of the experiment on the adhesion of BL-99 and human milk oligosaccharide mixture to the intestinal tract of pathogenic bacteria EPEC

Please refer to the above paragraphs for the preparatory steps and specific experimental methods. The tested Bifidobacterium lactis BL-99 had a normal growth curve.

The protective effect of probiotic and prebiotic combinations on preventing the adsorption of pathogenic bacteria to intestinal epithelial cells was studied using a common diarrhea-causing strain (EPEC O119).

As shown in Figure 6A, the human milk oligosaccharide composition B alone had no significant difference in the adhesion of pathogenic bacteria Escherichia coli O119 to intestinal cells Caco-2 compared with the negative control, while the positive control zinc oxide significantly reduced the adsorption of pathogenic bacteria to intestinal cells (p<0.0001). The probiotic BL-99 (10 ⁸ ) alone had no significant difference in the adhesion of pathogenic bacteria Escherichia coli O119 to intestinal cells Caco-2 compared with the negative control. However, when the prebiotic, namely the human milk oligosaccharide composition B, was used together with the probiotics as the test substances in the treatment group, both significantly reduced the adsorption of pathogenic bacteria EPEC 0119 to intestinal cells (p<0.05).

FIG6B shows the experimental results of the compositions formed by different concentrations of Bifidobacterium lactis BL-99 and different proportions of HMO mixtures in another batch of experiments for inhibiting the adhesion of Escherichia coli EPEC O119 to Caco-2 cells. The experimental results (mean ± standard error, repeated three times) are shown in Table 7. Due to the large number of groups, two different experimental determinations were performed. After comparing the negative control with the test group using Dunnett's posthoc test, it was found that in experimental group 1, the composition of BL-99 and composition A at a concentration of 10 ⁶ was significantly lower than the negative control (P<0.05); the mixture of probiotic BL-99 and HMO composition C at a concentration of 10 ⁸ had a tendency to reduce EPEC adhesion. In experimental group 2, BL-99 at a concentration of 10 ⁸ significantly reduced the adhesion of pathogenic bacteria (P<0.01), composition A also significantly reduced the adhesion of pathogenic bacteria (P<0.01), and the mixture of BL-99 at a concentration of 10 ⁸ and composition A more significantly reduced the adhesion of pathogenic bacteria (P<0.0001), reflecting a synergistic effect; the mixture of BL-99 at a concentration of 10 ⁸ and composition B significantly reduced the adhesion of pathogenic bacteria (P<0.001), reflecting a synergistic effect; composition F also significantly reduced the adhesion of pathogenic bacteria (P<0.01), and the mixture of BL-99 at a concentration of 10 ⁸ and composition F more significantly reduced the adhesion of pathogenic bacteria (P<0.001), reflecting a synergistic effect.

Table 7

Example 3: Experimental results of the effect of the combination of HN019 and human milk oligosaccharide mixture on transmembrane electrical resistance (TEER)

Please refer to the above paragraphs for the preparatory steps and specific experimental methods. The tested Bifidobacterium lactis HN019 had a normal growth curve.

The results are shown in Figure 7A. The effects of the test substances on transmembrane resistance were tested at different time points. The human milk oligosaccharide combination A or probiotic HN019 (10 ⁸ ) alone had basically no effect on the decrease of transmembrane resistance TEER, which was close to the negative control group. The combination of probiotic HN019 and human milk oligosaccharide mixture A can, at multiple time points, to a certain extent increase the transmembrane resistance TEER value to make it closer to the blank group.

FIG7B shows the effects of probiotics HN019 (10 ⁸ ) alone and HMO compositions A, B and F in different proportions on transmembrane resistance TEER when exposed to E. coli ETEC H10407 in another batch of experiments. The experimental results (mean ± standard error, repeated three times) are shown in Table 8. At t = 1, HN019 (10 ⁸ ) + composition A significantly improved the intestinal barrier, which was significantly different from the negative control (P < 0.05). At t = 2, HN019 (10 ⁸ ) improved the intestinal barrier, which was significantly different from the negative control (P <0.01); HN019 (10 ⁸ ) + composition A also improved the intestinal barrier, which was significantly different from the negative control (P <0.01); HN019 (10 ⁸ ) + composition F improved the intestinal barrier, which was significantly different from the negative control (P < 0.05). At t=4, HN019(10 ⁸ ) improved the intestinal barrier property, which was significantly different from the negative control (P<0.05), while HN019(10 ⁸ )+composition A improved the intestinal barrier property more significantly, which was significantly different from the negative control (P<0.01), reflecting the synergistic effect; HN019(10 ⁸ )+composition F improved the intestinal barrier property, which was significantly different from the negative control (P<0.05). At t=6, HN019(10 ⁸ )+composition A improved the intestinal barrier property, which was significantly different from the negative control (P<0.01), reflecting the synergistic effect; HN019(10 ⁸ )+composition F also improved the intestinal barrier property, which was significantly different from the negative control (P<0.01), reflecting the synergistic effect. In addition, two-way ANOVA and Dunnett's posthoc test were used to perform statistical analysis on the probiotic HN019 (10 ⁸ ) alone and the combinations of different probiotics and HMOs. It was found that at t=6, HN019 (10 ⁸ ) + combination A (P<0.05) and HN019 (10 ⁸ ) + combination F (P<0.01) were significantly different from the probiotic HN019 (10 ⁸ ) alone, reflecting a synergistic effect.

Table 8

FIG7C shows the effects of probiotics HN019 (10 ⁶ ) alone and HMO composition A on transmembrane resistance TEER when exposed to E. coli ETECH10407. The experimental results (mean ± standard error, repeated three times) are shown in Table 9.

Table 9

At t=2, HN019(10 ⁶ )+composition A was significantly improved compared with the negative control (P<0.05), reflecting a synergistic effect; at t=4, HN019(10 ⁶ )+composition A was significantly improved compared with probiotic HN019(10 ⁶ ) alone (P<0.05), reflecting a synergistic effect.

Example 4: Transmembrane electrophoresis of a combination of Bifidobacterium lactis BL-99 and a mixture of human milk oligosaccharides in different proportions Resistance test

Please refer to the above paragraphs for the preparatory steps and specific experimental methods. The tested Bifidobacterium lactis BL-99 had a normal growth curve.

FIG8 shows the effects of probiotic BL-99 (10 ⁸ ) alone and HMO compositions A, B and F in different ratios on transmembrane resistance TEER when exposed to E. coli ETEC H10407. The experimental results (mean ± standard error, repeated three times) are shown in Table 10.

Table 10

At t=6, BL-99(10 ⁸ )+composition A significantly improved the intestinal barrier property, which was significantly different from the negative control (P<0.05), reflecting the synergistic effect. At t=4, BL-99(10 ⁸ )+composition B significantly improved the intestinal barrier property, which was significantly different from the negative control (P<0.05), reflecting the synergistic effect. At t=4, BL-99(10 ⁸ )+composition A significantly improved the intestinal barrier property, which was significantly different from the negative control (P<0.05).

Example 5: Effect of HN019 combined with a mixture of human milk oligosaccharides on the secretion of inflammatory factors IL-8 and IP- 10 Impact

Please refer to the above paragraphs for the preparatory steps and specific experimental methods. The tested Bifidobacterium lactis HN019 had a normal growth curve.

As shown in Figures 9A, 9B, 10A, and 10B, when not co-cultured with pathogenic bacteria ETEC, the levels of IL-8 and IP-10 released by Bifidobacterium lactis HN019 and its combination with breast milk oligosaccharide 2'-FL were significantly lower than those of ETEC compared with pathogenic bacteria ETEC. And the composition of HN019 with HMO mixtures C and D showed a certain synergistic effect on the release of IL-8. When pathogenic bacteria ETEC and the test substance were present in the culture system at the same time, HN019 and the composition of HN019 and HMO mixture C did not affect the release of IL-8, but the composition of HN019 and HMO mixture D significantly reduced the release of IL-8, showing a synergistic effect; in addition, HN019 and its composition with HMO mixtures C or D can significantly reduce the release of IP-10. It shows that the nutritional composition of HN019+HMO mixture has a certain regulatory effect on the release of inflammatory factors.

Claims (7)

1. A nutritional composition comprises Bifidobacterium lactis (Bifidobacterium lactis) and breast milk oligosaccharide;

wherein the bifidobacterium lactis is bifidobacterium lactis (Bifidobacterium lactis) HN019 strain or bifidobacterium lactis (Bifidobacterium lactis) with the preservation number of CGMCC No. 15650;

wherein the breast milk oligosaccharide consists of (by weight) 2 '-fucosyllactose, 3' -fucosyllactose, lactose-N-tetraose, 3 '-sialyllactose and 6' -sialyllactose in percentage by weight (0-53%) (21% -44%) (16% -32%) (5% -22%) (0-5%);

The ratio of bifidobacterium lactis to breast milk oligosaccharide is 1×10 ⁶CFU~1×10¹⁰ CFU:5mg.

2. Use of the composition of claim 1 for the preparation of a health food or pharmaceutical product having the efficacy of enhancing gastrointestinal immunity;

Wherein the enhancing gastrointestinal immunity comprises: against pathogenic bacteria invasion in the intestinal system, or maintain intestinal tract shielding function, or reduce inflammatory factor IL-8 and/or IP-10 release by intestinal cells.

3. The use according to claim 2, wherein the health food is a liquid beverage.

4. The use according to claim 2, wherein the health food is a solid beverage.

5. The use according to claim 2, wherein the health food is an oral liquid.

6. The use according to claim 2, wherein the health food is a dairy product.

7. The use according to claim 2, wherein the health food is a tablet or capsule.