WO2022100758A1 - 一种含乳双歧杆菌与母乳低聚糖的组合物及其应用 - Google Patents

一种含乳双歧杆菌与母乳低聚糖的组合物及其应用 Download PDF

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WO2022100758A1
WO2022100758A1 PCT/CN2021/130978 CN2021130978W WO2022100758A1 WO 2022100758 A1 WO2022100758 A1 WO 2022100758A1 CN 2021130978 W CN2021130978 W CN 2021130978W WO 2022100758 A1 WO2022100758 A1 WO 2022100758A1
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cfu
composition
staphylococcus aureus
food
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French (fr)
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怀斯吉塞拉·阿德里安娜
凡·鲁-鲍曼卡洛琳·安妮卡
施密特盖瑞特
王雯丹
司徒文佑
谷方婕
刘彪
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内蒙古伊利实业集团股份有限公司
内蒙古乳业技术研究院有限责任公司
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Priority to US18/253,171 priority Critical patent/US20240000868A1/en
Priority to AU2021377524A priority patent/AU2021377524A1/en
Priority to EP21891278.0A priority patent/EP4245309A1/en
Publication of WO2022100758A1 publication Critical patent/WO2022100758A1/zh

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/20—Dietetic milk products not covered by groups A23C9/12 - A23C9/18
    • A23C9/203—Dietetic milk products not covered by groups A23C9/12 - A23C9/18 containing bifidus-active substances, e.g. lactulose; containing oligosaccharides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/51—Bifidobacterium
    • A23V2400/531—Lactis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K2035/11—Medicinal preparations comprising living procariotic cells
    • A61K2035/115—Probiotics

Definitions

  • the present invention mainly relates to a kind of probiotics and prebiotics composition which can improve the organism's resistance to Staphylococcus aureus infection and its application, specifically refers to a kind of probiotics and prebiotics composition comprising Bifidobacterium lactis (Bifidobacterium lactis) HN019 and breast milk oligosaccharides
  • Bifidobacterium lactis Bifidobacterium lactis
  • breast milk oligosaccharides The composition of (Human Milk Oligosaccharides, HMOs for short) can effectively improve the organism's resistance to Staphylococcus aureus infection, and the composition can be added to various health food and health food.
  • HMOs Human Milk Oligosaccharides
  • lactose and fat are the third most abundant substances in breast milk except lactose and fat. Its total content varies in various stages of lactation, about 12 to 14 g/L in mature milk, and about 20 to 24 g/L in colostrum.
  • the structure of each breast milk oligosaccharide has a lactose at the reducing end, most of which have polylactosamine as the structural backbone, and contain fucose, sialic acid or both at the chain end.
  • Breast milk oligosaccharides are mainly composed of three categories: (1) Fucosyl oligosaccharides, represented by 2'-fucosyl oligosaccharides and 3'-fucosyl oligosaccharides; (2) Sialyl oligosaccharides, represented by 3'-sialyllactose and 6'-sialyllactose; (3) Core sugar chain structure without fucosyl or sialic acid groups The oligosaccharides formed are represented by lactose-N-tetrasaccharide and lactose-N-neotetrasaccharide.
  • HMOs have become a gap that infant formula powder must bridge to be closer to the composition of breast milk.
  • the first clinical intervention trial using 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 rates comparable to breast-fed infants.
  • 2'-FL is also used as a nutritional supplement in adults to relieve irritable bowel syndrome or inflammatory bowel disease, or as a prebiotic to maintain the balance of intestinal flora.
  • Intestinal flora is an important constituent of the human intestinal micro-ecosystem 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 effects.
  • Both prebiotics and probiotics are regarded as micro-ecological management tools to improve the body's health, which can modify, regulate and reorganize the existing intestinal flora.
  • Caenorhabditis elegans (C.elegans), as a model organism, has good application prospects in preclinical research and evaluation. It has a short life cycle (21 days), is reproducible and reproducible, is simple to operate, transparent and easy to cultivate. Its genome has been fully sequenced, and a quarter of its genes are homologous to the human genome. Gene-mutated nematode organisms produced by editing the nematode's genes can be used as an experimental tool for genetic analysis. Nematodes are not currently considered an animal in European legislation. It is widely used as in vitro assays such as transcriptomics, proteomics, metabolomics, etc. As a model organism, it is often used as the first step in the evaluation of raw materials. Before the design of functional raw materials, in vitro enzyme or cell experiments, mouse models and clinical experiments, nematodes are often used as a high-throughput method to screen test raw materials. some of the characteristics.
  • Staphylococcus aureus is a Gram-positive bacterium belonging to the genus Staphylococcus and is a common food-borne pathogenic microorganism. It exists widely in the natural environment, and under appropriate conditions, it can produce enterotoxin and can cause food poisoning, causing many foodborne microbial food poisoning events caused by Staphylococcus aureus. After the human body is infected by Staphylococcus aureus, common symptoms of food poisoning such as nausea, vomiting, dizziness, etc. may occur, and symptoms such as enteritis, pneumonia, skin infection, wound infection and ulceration, and meningitis may occur.
  • One object of the present invention is to provide a composition that can improve the resistance of an organism to Staphylococcus aureus infection.
  • the inventor of the present case found in research that the combination of Bifidobacterium lactis and breast milk oligosaccharides has a synergistic effect on improving the organism's resistance to Staphylococcus aureus infection.
  • the present invention provides a composition comprising Bifidobacterium lactis and breast milk oligosaccharides.
  • the Bifidobacterium lactis comprises Bifidobacterium lactis HN019 strain.
  • Bifidobacterium lactis HN019 strain a commercial strain, can be provided by DuPont Danisco.
  • the breast milk oligosaccharides include 2'-fucosyllactose, 3'-fucosyllactose, lactose-N-tetrasaccharide, 3'-fucosyllactose One or more of sialyllactose, 6'-sialyllactose.
  • 2'-fucosyllactose 2'-FL
  • fucose and lactose 2'-fucosyllactose
  • 2'-FL a trisaccharide structure formed by fucose and lactose
  • Commercially available substances are usually prepared by microbial fermentation and have the same structure as oligosaccharides found in human milk.
  • 3-fucosyllactose is a trisaccharide structure formed by fucose and lactose, and is an isomer of each other with 2'-fucosyllactose. It is a representative substance of fucosyl oligosaccharides. This substance is prepared by microbial fermentation and has the same structure as oligosaccharides found in human milk.
  • Lactose-N-tetraose is a hexasaccharide structure formed by lactose and tetrasaccharide. This substance is prepared by microbial fermentation. It is based on the core sugar chain and does not contain fucose. It is a representative substance of oligosaccharides based on sialyl or sialic acid groups and has the same structure as the oligosaccharides found in human milk.
  • 3'-sialyllactose 3'-SL
  • 3'-SL 3'-sialyllactose
  • This substance is prepared by microbial fermentation and has the same structure as oligosaccharides found in human milk.
  • 6'-sialyllactose (6'-SL), a trisaccharide structure formed by sialic acid and lactose, is an isomer with 3'-sialyllactose, and is a sialic acid group Representative substances of oligosaccharides. This substance is prepared by microbial fermentation and has the same structure as oligosaccharides found in human milk.
  • the content of 2'-fucosyllactose in the breast milk oligosaccharide is 0%-73%, preferably 0%-63%, and further It is preferably 0%-58%, more preferably 2%-55%, such as 3%-8%, 25%-35% or 51%-56%, more specifically 5%, 30% or 53% %.
  • each breast milk oligosaccharide is 2'-fucosyllactose, 3'-fucosyllactose, lactose-N-tetrasaccharide, The sum of the total weight of 3'-sialyllactose and 6'-sialyllactose is 100%.
  • the content of 3'-fucosyllactose in the breast milk oligosaccharide is 0%-61%, preferably 11%-51%, and further It is preferably 16%-46%, more preferably 20%-44%, such as 18%-23%, 25%-35% or 40%-45%, more specifically 21%, 30% or 41% %.
  • the content of lactose-N-tetrasaccharide in the breast milk oligosaccharide is 0%-52%, preferably 6%-42%, more preferably 11-37%, more preferably 13%-35%, such as 13%-18%, 22%-27% or 30%-35%, more specifically 16%, 25% or 32%.
  • the content of 3'-sialyllactose in the breast milk oligosaccharide is 0%-42%, preferably 0%-32%, more preferably 0%-27%, more preferably 2%-25%, such as 2%-7%, 10%-15% or 20%-25%, more specifically 5%, 12% or 22% .
  • the content of 6'-sialyllactose in the breast milk oligosaccharide is 0%-25%, preferably 0%-15%, more preferably It is 0%-10%, more preferably 0%-8%, for example, it can be 3%-8%, and more specifically, it can be 3% or 5%.
  • the breast milk oligosaccharide is composed of 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL) ), lactose-N-tetrasaccharide (LNT), at least two, at least three or at least one of 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL) A combination of four components.
  • the breast milk oligosaccharide comprises a weight ratio of (0%-73%):(0%-61%):(0%-52%):( 0%-42%): (0%-25%) 2'-fucosyllactose, 3'-fucosyllactose, lactose-N-tetrasaccharide, 3'-sialyllactose, 6' - at least three or four of the sialyllactoses.
  • the breast milk oligosaccharide comprises a weight ratio (0%-63%):(11%-51%):(6%-42%):( 0%-32%): (0%-15%) 2'-fucosyllactose, 3'-fucosyllactose, lactose-N-tetrasaccharide, 3'-sialyllactose, 6' - at least three or four of the sialyllactoses.
  • the breast milk oligosaccharide comprises a weight ratio (0%-58%):(16%-46%):(11%-37%):( 0%-27%): (0%-10%) 2'-fucosyllactose, 3'-fucosyllactose, lactose-N-tetrasaccharide, 3'-sialyllactose, 6' - at least three or four of the sialyllactoses.
  • the breast milk oligosaccharide comprises a weight ratio (0%-55%):(20%-44%):(13%-35%):( 2%-25%): (0%-8%) 2'-fucosyllactose, 3'-fucosyllactose, lactose-N-tetrasaccharide, 3'-sialyllactose, 6' - Sialyl lactose.
  • the breast milk oligosaccharide comprises a weight ratio of (0-53%):(21-41%):(16-32%):(5-22%) %):(0-5%), preferably (5-53%):(21-41%):(16-32%):(5-22%):(0-5%), more preferably (5 ⁇ 53%):(21 ⁇ 41%):(16 ⁇ 32%):(5 ⁇ 22%):(3 ⁇ 5%) 2'-fucosyllactose, 3'-fucose Syllactose, lactose-N-tetrasaccharide, 3'-sialyllactose, 6'-sialyllactose.
  • the breast milk oligosaccharide comprises a weight ratio (30%-53%):(21%-30%):(16%-25%):( 5%-12%): (3%-5%) of 2'-fucosyllactose, 3'-fucosyllactose, lactose-N-tetrasaccharide, 3'-sialyllactose, 6' - Sialyl lactose.
  • the breast milk oligosaccharide comprises a weight ratio (33%-73%):(1%-41%):(0%-36%):( 0%-25%):(0%-25%), preferably (43%-63%):(11%-31%):(6%-26%):(0%-15%):( 0%-15%), more preferably (48%-58%):(16%-26%):(11%-21%):(0%-10%):(0%-10%), Most preferred are (51%-56%):(18%-23%):(13%-18%):(2%-7%):(3%-8%) 2'-fucosyl Lactose, 3'-fucosyllactose, lactose-N-tetrasaccharide, 3'-sialyllactose, 6'-sialyllactose.
  • the ratio of Bifidobacterium lactis to breast milk oligosaccharides is 1 ⁇ 10 3 CFU to 1 ⁇ 10 12 CFU: 0.1 g to 10 g, preferably 1 ⁇ 10 6 CFU ⁇ 1 ⁇ 10 10 CFU: 1g ⁇ 10g.
  • the ratio of Bifidobacterium lactis to breast milk oligosaccharides is 1 ⁇ 10 8 CFU: 0.08g ⁇ 0.3g.
  • the ratio of the Bifidobacterium lactis to the breast milk oligosaccharide is based on the amount of the two in the same composition.
  • the present invention also provides the application of the composition in the preparation of food or medicine which can effectively improve the organism's resistance to Staphylococcus aureus infection, and can improve the organism's innate immunity and/or anti-aging effect .
  • the organism in the application of the composition of the present invention, includes animals or humans.
  • the improving the organism's resistance to Staphylococcus aureus infection includes: improving the individual's ability to prevent Staphylococcus aureus infection, and reducing the ability of the individual to prevent Staphylococcus aureus infection. and/or relieve various symptoms caused by Staphylococcus aureus infection such as food poisoning (including nausea, vomiting, dizziness, etc.), enteritis, pneumonia, skin infection, wound ulceration or meningitis.
  • the composition of the present invention can be used to prepare various health foods, health care products, medicines, and the like.
  • the 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 added to infant food (including infant formula powder, complementary food, nutritional supplements), It also has broad application prospects in nutritional supplements or foods (such as milk powder) for children over 3 years old, adolescents and adults.
  • the medicine may be an oral preparation or an external preparation, such as a smeared ointment and the like.
  • the present invention also provides a method for improving the resistance of an organism to Staphylococcus aureus infection, improving the innate immunity and/or anti-aging of the organism, the method comprising: administering an effective amount of the present invention to the organism the described composition.
  • the improving the organism's resistance to Staphylococcus aureus infection includes: improving the individual's ability to prevent Staphylococcus aureus infection; reducing the ability of the individual to infect the Staphylococcus aureus; and/or relieving the individual caused by Staphylococcus aureus infection Food poisoning, enteritis, pneumonia, skin infection, wound ulceration and/or meningitis.
  • the composition is administered to the organism in the form of a food product, the food product being a liquid beverage, a solid beverage, an oral liquid, a dairy product, a tablet or a capsule, such as a milk powder, in particular Infant formula powder.
  • the application amount of Bifidobacterium lactis in food is 1 ⁇ 10 3 CFU/day to 1 ⁇ 10 12 CFU/day, more preferably 1 ⁇ 10 6 CFU/day to 1 ⁇ 10 11 CFU/day.
  • the application amount of breast milk oligosaccharides in food is 0.015g/day-15g/day, more preferably 0.1g/day-12g/day, more preferably 1.5g/day-10g/day.
  • the present invention also provides a food comprising the composition of the present invention.
  • the application amount of the Bifidobacterium lactis in the food is 1 ⁇ 10 3 CFU/day ⁇ 1 ⁇ 10 12 CFU/day, preferably 1 ⁇ 10 6 CFU/day ⁇ 1 ⁇ 10 11 CFU/day. In some specific embodiments of the present invention, the application amount of Bifidobacterium lactis in the food is 1 ⁇ 10 8 CFU/day.
  • the application amount of the breast milk oligosaccharide in the food is 0.015g/day ⁇ 15g/day, more preferably 0.1g/day ⁇ 12g/day, more preferably 1.5g/day day ⁇ 10g/day.
  • the food or medicine comprising the composition of the present invention can improve the organism's resistance to Staphylococcus aureus infection due to the composition.
  • Figure 1 shows the effect of 10 mg/mL of breast milk oligosaccharide composition A in combination with Bifidobacterium lactis HN019 on the survival of C. elegans against S. aureus infection.
  • the intervening substances probiotics and/or HMO added in the infection stage for each group were the same as in the culture stage.
  • Figure 2 shows the effect of 30 mg/mL of breast milk oligosaccharide composition A in combination with Bifidobacterium lactis HN019 on the survival of C. elegans against S. aureus infection.
  • the intervening substances probiotics and/or HMO added in the infection stage for each group were the same as in the culture stage.
  • Figure 3 shows the effect of the composition of breast milk oligosaccharide composition A and Bifidobacterium lactis HN019 strain on the survival rate of Caenorhabditis elegans on the third day of infection with Staphylococcus aureus.
  • the intervening substances probiotics and/or HMO added in the infection stage for each group were the same as in the culture stage.
  • Figure 4 is a graph showing the effect of the composition of human milk oligosaccharide composition A and Bifidobacterium lactis HN019 strain on the survival rate of C. elegans on the fifth day of infection with Staphylococcus aureus.
  • the intervening substances probiotics and/or HMO added in the infection stage for each group were the same as in the culture stage.
  • Figure 5 is a graph showing the results of the gentamicin disc diffusion inhibition zone experiment.
  • Figure 6 is a graph showing the results of Bifidobacterium lactis HN019 disc diffusion inhibition zone experiment.
  • Figure 7 shows the different effects of co-cultivation of HN019 (1 x 108 CFU) with HMO Composition A ( 10 mg/mL) on enhancing nematode survival before and/or during S. aureus infection.
  • Figure 8 shows the effect of HMO composition A (8 mg/mL) and Bifidobacterium lactis HN019 (1 x 10 8 CFU) alone and after forming the composition on nematode survival under S. aureus infection.
  • HMO composition G (8 mg/mL) and Bifidobacterium lactis HN019 (1 ⁇ 10 8 CFU) alone and after forming the composition on the survival rate of nematodes in the case of S. aureus infection.
  • Figure 10 shows the effect of HMO composition H (8 mg/mL) and Bifidobacterium lactis HN019 (1 x 10 8 CFU) alone and after forming the composition on nematode survival under S. aureus infection.
  • Example 1 Effects of a combination of human milk oligosaccharides and Bifidobacterium lactis HN019 composition on the survival rate of C. elegans infection with Staphylococcus aureus
  • composition A of five HMOs the composition ratios are: 53% 2FL, 21% 3FL, 16% LNT, 5% 3SL, 5% 6SL.
  • Human milk oligosaccharide solutions were prepared in distilled water and cultured in petri dishes containing different final concentrations (1, 10, 30 mg/mL, respectively) of nematode growth medium.
  • the tested probiotics were grown in MRS medium supplemented with 1% cysteine and incubated overnight at 37°C in an anaerobic environment. Cells were harvested and rinsed with physiological saline solution, and plated at a final concentration of 1 x 108 CFU in plates containing nematode growth medium adjusted for bacterial concentration.
  • Age-matched nematodes were obtained and cultured in petri dishes containing nematode agar medium (NGM medium, nematode medium containing Escherichia coli OP50 as food), supplemented with different doses of HMO combinations (8 mg/mL, 10 mg /mL, 30 mg/mL) co-cultured with a probiotic (1 x 10 8 CFU) composition (total volume of liquid added to each nematode petri dish was 10 mL.
  • NVM medium nematode medium containing Escherichia coli OP50 as food
  • HMO combinations 8 mg/mL, 10 mg /mL, 30 mg/mL
  • a probiotic (1 x 10 8 CFU) composition total volume of liquid added to each nematode petri dish was 10 mL.
  • each dish plate For the HMO test group at a concentration of 10 mg/mL, each The final amount of HMO in each dish was 100 mg; for the HMO test groups with concentrations of 8 mg/mL and 30 mg/mL, respectively, the final amount of HMO in each dish was 80 mg and 300 mg, respectively. Therefore, in each dish plate , the proportion of probiotics and HMO added is: 1 ⁇ 10 8 CFU: 80mg-300mg). After the nematodes became adults, they were transferred to petri dishes inoculated with Staphylococcus aureus ATCC25923 at 10 8 -10 9 CFU/mL to simulate infection with Staphylococcus aureus.
  • the intervening substances (probiotics and/or HMO) added in the infection stage of each group were the same as those in the culture stage. Two controls were used, conditions without pathogenic bacteria (nematode petri dishes containing E. coli OP50) and those with pathogenic bacteria S. aureus infection without any intervention (dishes containing S. aureus only) .
  • a group in which an intervening substance was added in the culture phase and no intervening substance in the infection phase and a group in which the intervening substance was not added in the cultivation phase but added in the infection phase were set.
  • Discs were made with sterile filter paper and infused with the tested HMO substances (10 mg/mL, 30 mg/mL) or probiotics (1 ⁇ 10 8 CFU), and the discs were dried overnight under sterile conditions.
  • Staphylococcus aureus ATCC25923 (1.0 ⁇ 10 6 CFU/mL) was evenly spread on the surface of the NGM agar plate, and then the disc was placed on the surface of the inoculated agar plate petri dish.
  • Gentamicin 200 ⁇ g/mL
  • All dishes were incubated at 37°C for 18 hours. Whether the test substance can directly inhibit bacterial growth or has antibacterial activity is confirmed by observing whether a transparent annular ring is formed around the disc.
  • Figures 3 and 4 show a comparison of the effects of HMO Composition A and probiotic-formed compositions on C. elegans survival on day three and day five of infection. On the fifth day of infection, the composition formed by prebiotics and HN019 was more effective than other test groups.
  • the experiments of the present invention show that the C. elegans survival rate of the group in which the combined intervention of HMO and HN019 was added in the culture stage and no intervention was added in the infection stage was significantly higher than that in the culture stage and the infection stage without any intervention. group of things.
  • each breast milk oligosaccharide monomer could not directly inhibit the growth of Staphylococcus aureus. It can be seen that the protective effect of each breast milk oligosaccharide and probiotic HN019 composition reflected in this study on nematodes infected by Staphylococcus aureus is not due to the direct sterilization of the substances.
  • Example 2 Experiments on improving innate immunity and anti-aging effects of different compositions of breast milk oligosaccharide combination and Bifidobacterium lactis HN019 composition
  • Test composition efficacy verification experiment 1 (to investigate the preventive effect of the test substance in "improving the organism's resistance to Staphylococcus aureus infection, improving the organism's innate immunity and/or anti-aging"): Age-consistent results were obtained. Nematodes were cultured in petri dishes containing nematode agar medium supplemented with a proportion of HMO composition A (10 mg/mL) and HN019 probiotic (1 x 10 8 CFU) composition for co-cultivation until the nematodes reached adulthood . Thereafter, the nematodes were transferred to infected plates to receive infection with the pathogenic bacteria Staphylococcus aureus ATCC25923 without the addition of the HMO and probiotic composition.
  • Efficacy verification experiment 2 of the test composition (to investigate the therapeutic effect of the test substance in "improving the organism's resistance to Staphylococcus aureus infection, improving the organism's innate immunity and/or anti-aging"): obtained age-consistent results. nematodes and grow them in petri dishes containing nematode agar medium. When the nematodes became adults, the nematodes were transferred to plates, which were supplemented with a certain ratio of HMO Composition A (10 mg/mL) and HN019 probiotics (1 x 10 8 CFU), and received the pathogenic bacteria Vitis aureus Infection with coccus ATCC25923.
  • nematodes After the nematodes had been cultured for several days, their survival was counted daily. If the worm does not respond to the platinum wire, it can be considered dead. In addition, two groups of controls were included, one without the addition of pathogenic bacteria (common culture plate plus Escherichia coli OP50 as food), and the other group as the infection control group (only S. aureus was added to the plate). Two independent assays were performed for each condition. Statistical comparative analysis of survival curves was performed, and log rank survival significance analysis was performed using the GraphPad Prism 9 statistical software package.
  • the survival curve shows that from the first day to the fifth day after culture, whether the test substance is added to the co-culture before infection or during infection, the survival rate of nematodes in the case of Staphylococcus aureus infection has a good improvement. and protection.
  • the nematodes only added the test substance during the infection of S. aureus, and the test substance was present from the beginning to the end, both of which had similar effects on improving the survival rate and protecting the S. aureus, and were similar to adding the test substance only before infection.
  • the case where the test substance is added in the infection stage and the case where the test substance is present in the whole process has a higher protective effect on the survival rate of nematodes.
  • HMO composition H (8mg/mL) and Bifidobacterium lactis HN019 alone and after forming the composition on the survival rate of nematodes under the condition of Staphylococcus aureus infection
  • Example 3 Infant formula powder with added breast milk oligosaccharide combination and Bifidobacterium lactis HN019 composition
  • This embodiment provides an infant formula milk powder aged 0-6 months, the total protein content of the formula milk powder is 11.1g/100g powder, the fat content is 28.3g/100g powder, the carbohydrate content is 52.9g/100g powder, and the breast milk content is 52.9g/100g powder.
  • the oligosaccharide composition content is 2.5g/100g powder, and the Bifidobacterium lactis is 4 ⁇ 10 8 CFU/100g.

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Abstract

一种含乳双歧杆菌与母乳低聚糖的组合物及其应用，所述组合物是一种包含乳双歧杆菌(Bifidobacterium lactis)与母乳低聚糖(Human Milk Oligosaccharides，简称HMOs)的组合物。该组合物可有效提升生物体对金黄色葡萄球菌感染的抵御能力、可提高生物体先天免疫和/或抗衰老，可添加在各种健康食品以及保健食品中。

Description

一种含乳双歧杆菌与母乳低聚糖的组合物及其应用 技术领域
本发明主要是关于一种可提升生物体对金黄色葡萄球菌感染抵御能力的益生菌益生元组合物及其应用，具体是指一种包含乳双歧杆菌(Bifidobacterium lactis)HN019与母乳低聚糖(Human Milk Oligosaccharides，简称HMOs)的组合物，可有效提升生物体对金黄色葡萄球菌感染的抵御能力，该组合物可添加在各种健康食品以及保健食品中。
背景技术
在上个千年，已有医学文献记载，未经母乳喂养的婴儿有较高的疾病率和死亡率。母乳不仅为婴儿提供所需营养，母乳中的活性成分更为婴儿的肠道发育和免疫力完善提供保障。母乳喂养的婴儿与配方奶喂养的婴儿相比，肠道菌群中有益菌的相对丰度更高，特别是双歧杆菌和乳酸菌。
母乳通过菌群的传递，加上母乳低聚糖和母乳中的细胞因子等活性成分，为新生儿建立起健康的肠道菌群。婴儿每天通过母乳摄入10 7～10 8个细菌，包括乳酸菌和双歧杆菌。这些菌通过母乳直接传递给婴儿，部分可在婴儿肠道定植，促进生命早期肠道菌群的建立。婴儿肠道菌群的建立，对其肠道的发育，以及对健康和免疫***有着短期，甚至终生的影响。
母乳低聚糖(Human Milk Oligosaccharides，简称HMOs)属于母乳中除乳糖和脂肪外，含量第三丰富的物质。其总含量在泌乳期的各个阶段有变化，在成熟乳中大约是12～14g/L，而初乳中大约是20～24g/L。每一种母乳低聚糖的结构在还原端都有一个乳糖，大部分以聚乳糖胺作为结构主链，并在链端含有岩藻糖、唾液酸或二者均有。母乳低聚糖主要由三大类组成：(1)岩藻糖基类低聚糖，以2’-岩藻糖基低聚糖和3’-岩藻糖基低聚糖为代表性物质；(2)唾液酸基类低聚糖，以3’-唾液酸基乳糖和6’-唾液酸基乳糖为代表性物质；(3)不含岩藻糖基或唾液酸基的核心糖链结构形成的低聚糖，以乳糖-N-四糖和乳糖-N-新四糖为代表性物质。
HMOs的存在与含量存在个体差异，并与哺乳母亲的路易斯分泌型组成有关。由于婴幼儿配方粉的原料通常是牛乳，而牛乳中通常不含或含有很少这类低聚糖物质， HMOs便成为了婴幼儿配方粉想要更加接近母乳成分所必须跨越的一道鸿沟。
上世纪90年代，作为在大部分母乳中均含有的HMO，2-岩藻糖基乳糖(2’-FL)被发现可有效地减轻大肠杆菌中稳定毒素的毒性；到了2003年，该低聚糖被报道可抑制弯曲空肠杆菌的附着和感染。随后，母乳低聚糖的三大主要功能被逐步报道和发现：(1)抑制特定病菌的附着和感染；(2)作为益生元，促进肠道共生***里面细菌的生长；(3)直接减缓粘膜在有毒刺激下的炎症反应。使用了2’-FL的首个临床干预试验证实了在低卡路里配方中加入这个特定成分不仅安全还可以让配方奶喂养的婴儿生长速率与母乳喂养的婴儿具有可比性。2’-FL也被用作成年人的营养补充剂，缓解肠道应激性综合症或炎症性肠病，或被用作益生元维持肠道菌群平衡。
肠道菌群是人体肠道微生态***的重要组成物质，对人类健康有重要作用，如可提供必需营养素，生成维生素K，辅助消化过程与促进血管生成和肠道神经作用。益生元和益生菌均被视为改善机体健康的微生态管理工具，可改变、调节和重组已经存在的肠道菌群。
目前在婴幼儿配方粉、辅食及营养补充剂领域，需要有缓解婴幼儿肠道不适及提升自身抵御致病菌如金黄色葡萄球菌感染能力的解决方案。同时，在3岁以上儿童、青少年及成人领域，也需要缓解肠道不适及提升自身抵御致病菌如金黄色葡萄球菌感染能力的解决方案。
秀丽隐杆线虫(C.elegans)作为一种模型生物，在临床前研究与评估中有较好的应用前景。它具有较短的生命周期(21天)，可复制和再生性较强，操作简便、透明且易于培养。它的基因组已全部被测序，并且有四分之一的基因与人类基因组是同源的。对线虫的基因进行编辑从而产生的基因突变的线虫生物体，可用作基因分析的实验手段。目前线虫在欧洲的立法中并不被认为是一种动物。它被广泛用作活体外的测定，比如转录组学、蛋白组学、代谢组学等。作为模式生物，它也常常被作为原料评估的第一个步骤，在功效原料的设计、体外酶或细胞实验、小鼠模型和临床实验前，往往用线虫作为高通量的手段来筛选测试原料的一些特性。
金黄色葡萄球菌(Staphylococcus aureus,S.aureus)是一种革兰氏阳性菌，属于葡萄球菌属，是常见的食源性致病微生物。它广泛存在于自然环境中，在适当条件下，能产生肠毒素并能引起食物中毒，引发过多起由金黄色葡萄球菌导致的食源性微生物食物中毒事件。人体在受到金黄色葡萄球菌感染后，可能会出现恶心、呕吐、头晕等 常见的食物中毒症状，并可能出现肠炎、肺炎、皮肤感染、伤口感染溃烂、脑膜炎等症状。
发明内容
本发明的一个目的在于提供一种可提升生物体对金黄色葡萄球菌感染抵御能力的组合物。
本案发明人在研究中发现，乳双歧杆菌(Bifidobacterium lactis)和母乳低聚糖组合，对于提高生物体对金黄色葡萄球菌感染抵御能力具有协同作用。
从而，一方面，本发明提供了一种组合物，其包括乳双歧杆菌(Bifidobacterium lactis)及母乳低聚糖。
根据本发明的具体实施方案，本发明的组合物中，所述乳双歧杆菌包括乳双歧杆菌(Bifidobacterium lactis)HN019菌株。
乳双歧杆菌(Bifidobacterium lactis)HN019菌株，为商业化菌株，可由杜邦丹尼斯克公司提供。
根据本发明的具体实施方案，本发明的组合物中，所述母乳低聚糖包括2’-岩藻糖基乳糖、3’-岩藻糖基乳糖、乳糖-N-四糖、3’-唾液酸基乳糖、6’-唾液酸基乳糖中的一种或多种。
2’-岩藻糖基乳糖(2’-fucosyllactose，2’-FL)，为岩藻糖与乳糖形成的三糖结构，是岩藻糖基类低聚糖的代表性物质。市售该物质通常为经微生物发酵法制备，与人乳中发现的寡糖具有相同结构。
3-岩藻糖基乳糖(3-fucosyllactose，3-FL)，为岩藻糖与乳糖形成的三糖结构，与2’-岩藻糖基乳糖互为同分异构体。是岩藻糖基类低聚糖的代表性物质。该物质经微生物发酵法制备，与人乳中发现的寡糖具有相同结构。
乳糖-N-四糖(lacto-N-tetraose，LNT)，为乳糖与四糖形成的六糖结构，该物质经微生物发酵法制备，是以核心糖链为基础结构，且不含岩藻糖基或唾液酸基的低聚糖的代表性物质，且与人乳中发现的寡糖具有相同结构。
3’-唾液酸基乳糖(3’-sialyllactose，3’-SL)，为唾液酸与乳糖形成的三糖结构，与6’-唾液酸基乳糖互为同分异构体，是唾液酸基类低聚糖的代表性物质。该物质经微生物发酵法制备，与人乳中发现的寡糖具有相同结构。
6’-唾液酸基乳糖(6’-sialyllactose，6’-SL)，为唾液酸与乳糖形成的三糖结构，与3’-唾液酸基乳糖互为同分异构体，是唾液酸基类低聚糖的代表性物质。该物质经微生物发酵法制备，与人乳中发现的寡糖具有相同结构。
根据本发明的一些具体实施方案，本发明的组合物中，2’-岩藻糖基乳糖在所述母乳低聚糖中的含量为0％-73％，优选为0％-63％，进一步优选为0％-58％，更进一步优选为2％-55％，例如可以为3％-8％、25％-35％或51％-56％，更具体可以为5％、30％或53％。本发明中，除特别注明外，各母乳低聚糖的含量是以母乳低聚糖中的2’-岩藻糖基乳糖、3’-岩藻糖基乳糖、乳糖-N-四糖、3’-唾液酸基乳糖、6’-唾液酸基乳糖的总重量之和为100％计。
根据本发明的一些具体实施方案，本发明的组合物中，3’-岩藻糖基乳糖在所述母乳低聚糖中的含量为0％-61％，优选为11％-51％，进一步优选为16％-46％，更进一步优选为20％-44％，例如可以为18％-23％、25％-35％或40％-45％，更具体可以为21％、30％或41％。
根据本发明的一些具体实施方案，本发明的组合物中，乳糖-N-四糖在所述母乳低聚糖中的含量为0％-52％，优选为6％-42％，进一步优选为11-37％，更进一步优选为13％-35％，例如可以为13％-18％、22％-27％或30％-35％，更具体可以为16％、25％或32％。
根据本发明的一些具体实施方案，本发明的组合物中，3’-唾液酸基乳糖在所述母乳低聚糖中的含量为0％-42％，优选为0％-32％，进一步优选为0％-27％，更进一步优选为2％-25％，例如可以为2％-7％、10％-15％或20％-25％，更具体可以为5％、12％或22％。
根据本发明的一些具体实施方案，本发明的组合物中，6’-唾液酸基乳糖在所述母乳低聚糖中的含量为0％-25％，优选为0％-15％，进一步优选为0％-10％，更进一步优选为0％-8％，例如可以为3％-8％，更具体可以为3％或5％。
根据本发明的一些具体实施方案，本发明的组合物中，所述母乳低聚糖是由2’-岩藻糖基乳糖(2’-FL)、3-岩藻糖基乳糖(3-FL)、乳糖-N-四糖(LNT)、3’-唾液酸基乳糖(3’-SL)和6’-唾液酸基乳糖(6’-SL)中的至少二种、至少三种或至少四种组成的组合。
根据本发明的一些具体实施方案，本发明的组合物中，所述母乳低聚糖包括重量 比例(0％-73％):(0％-61％):(0％-52％):(0％-42％):(0％-25％)的2’-岩藻糖基乳糖、3’-岩藻糖基乳糖、乳糖-N-四糖、3’-唾液酸基乳糖、6’-唾液酸基乳糖中的至少三种或四种。
根据本发明的一些具体实施方案，本发明的组合物中，所述母乳低聚糖包括重量比例(0％-63％):(11％-51％):(6％-42％):(0％-32％):(0％-15％)的2’-岩藻糖基乳糖、3’-岩藻糖基乳糖、乳糖-N-四糖、3’-唾液酸基乳糖、6’-唾液酸基乳糖中的至少三种或四种。
根据本发明的一些具体实施方案，本发明的组合物中，所述母乳低聚糖包括重量比例(0％-58％):(16％-46％):(11％-37％):(0％-27％):(0％-10％)的2’-岩藻糖基乳糖、3’-岩藻糖基乳糖、乳糖-N-四糖、3’-唾液酸基乳糖、6’-唾液酸基乳糖中的至少三种或四种。
根据本发明的一些具体实施方案，本发明的组合物中，所述母乳低聚糖包括重量比例(0％-55％):(20％-44％):(13％-35％):(2％-25％):(0％-8％)的2’-岩藻糖基乳糖、3’-岩藻糖基乳糖、乳糖-N-四糖、3’-唾液酸基乳糖、6’-唾液酸基乳糖。
根据本发明的一些具体实施方案，本发明的组合物中，所述母乳低聚糖包括重量比例(0～53％):(21～41％):(16～32％):(5～22％):(0～5％)，优选为(5～53％):(21～41％):(16～32％):(5～22％):(0～5％)，更优选为(5～53％):(21～41％):(16～32％):(5～22％):(3～5％)的2’-岩藻糖基乳糖、3’-岩藻糖基乳糖、乳糖-N-四糖、3’-唾液酸基乳糖、6’-唾液酸基乳糖。
根据本发明的一些具体实施方案，本发明的组合物中，所述母乳低聚糖包括重量比例(30％-53％):(21％-30％):(16％-25％):(5％-12％):(3％-5％)的2’-岩藻糖基乳糖、3’-岩藻糖基乳糖、乳糖-N-四糖、3’-唾液酸基乳糖、6’-唾液酸基乳糖。
根据本发明的一些具体实施方案，本发明的组合物中，所述母乳低聚糖包括重量比例(33％-73％):(1％-41％):(0％-36％):(0％-25％):(0％-25％)，优选为(43％-63％):(11％-31％):(6％-26％):(0％-15％):(0％-15％)，更优选为(48％-58％):(16％-26％):(11％-21％):(0％-10％):(0％-10％)，最优选为(51％-56％):(18％-23％):(13％-18％):(2％-7％):(3％-8％)的2’-岩藻糖基乳糖、3’-岩藻糖基乳糖、乳糖-N-四糖、3’-唾液酸基乳糖、6’-唾液酸基乳糖。
根据本发明的具体实施方案，本发明的组合物中，所述乳双歧杆菌与母乳低聚糖 的比例为1×10 3CFU～1×10 12CFU：0.1g～10g，优选为1×10 6CFU～1×10 10CFU：1g～10g。在本发明的一些更具体实施方案中，所述乳双歧杆菌与母乳低聚糖的比例为1×10 8CFU：0.08g～0.3g。所述乳双歧杆菌与母乳低聚糖的比例是以二者在同一组合物中的用量计。
另一方面，本发明还提供了所述组合物在制备具有可有效提升生物体对金黄色葡萄球菌感染的抵御能力、可提高生物体先天免疫和/或抗衰老功效的食品或药品中的应用。
根据本发明的具体实施方案，本发明的组合物的应用中，所述生物体包括动物或人。
根据本发明的具体实施方案，本发明的组合物的应用中，所述提升生物体对金黄色葡萄球菌感染抵御能力包括：提升个体预防金黄色葡萄球菌感染的能力，降低金黄色葡萄球菌感染个体的能力，和/或缓解金黄色葡萄球菌感染引起的各种症状例如食物中毒(包括恶心、呕吐、头晕等)、肠炎、肺炎、皮肤感染、伤口溃烂或脑膜炎等。
根据本发明的具体实施方案，本发明的组合物可用于制备各种健康食品、保健品以及药品等。具体地，所述食品可以为液体饮料、固体饮料、口服液、奶制品、片剂或胶囊等，例如可以是用于添加在婴幼儿食品(包括婴幼儿配方粉、辅食、营养补充剂)，以及3岁以上儿童、青少年和成人的营养补充剂或食品(例如奶粉)中，具有广阔的应用前景。所述药品可以是口服制剂，也可以是外用制剂，例如涂抹用药膏等。
另一方面，本发明还提供了一种提升生物体对金黄色葡萄球菌感染的抵御能力、提高生物体先天免疫和/或抗衰老的方法，所述方法包括：给予生物体有效量的本发明所述的组合物。其中，所述提升生物体对金黄色葡萄球菌感染抵御能力包括：提升个体预防金黄色葡萄球菌感染的能力；降低金黄色葡萄球菌感染个体的能力；和/或缓解金黄色葡萄球菌感染引起的个体食物中毒、肠炎、肺炎、皮肤感染、伤口溃烂和/或脑膜炎。在本发明的一些具体实施方案中，所述的组合物是以食品的形式给予生物体，所述食品为液体饮料、固体饮料、口服液、奶制品、片剂或胶囊，例如奶粉，特别是婴幼儿配方粉。优选地，乳双歧杆菌在食品中的应用量为1×10 3CFU/天～1×10 12CFU/天，更优选为1×10 6CFU/天～1×10 11CFU/天。优选地，母乳低聚糖在食品中的应用量为0.015g/天～15g/天，进一步优选为0.1g/天～12g/天，更优选为1.5g/天～10g/天。
本发明还提供了一种食品，其包括本发明所述的组合物。
根据本发明的具体实施方案，所述乳双歧杆菌在所述食品中的应用量为1×10 3CFU/天～1×10 12CFU/天，优选为1×10 6CFU/天～1×10 11CFU/天。在本发明的一些具体实施例方案中，乳双歧杆菌在所述食品中的应用量为1×10 8CFU/天。
根据本发明的具体实施方案，所述母乳低聚糖在所述食品中的应用量为0.015g/天～15g/天，进一步优选为0.1g/天～12g/天，更优选为1.5g/天～10g/天。
本发明的包括所述组合物的食品或药品，因包括所述组合物而具有可提升生物体对金黄色葡萄球菌感染抵御能力。
附图说明
图1显示10mg/mL的母乳低聚糖组合物A与乳双歧杆菌HN019的组合物对秀丽隐杆线虫受到金黄色葡萄球菌感染时存活率的影响。图中各组别在感染阶段添加的干预物(益生菌和/或HMO)情况分别与培养阶段相同。
图2显示30mg/mL的母乳低聚糖组合物A与乳双歧杆菌HN019的组合物对秀丽隐杆线虫受到金黄色葡萄球菌感染时存活率的影响。图中各组别在感染阶段添加的干预物(益生菌和/或HMO)情况分别与培养阶段相同。
图3为母乳低聚糖组合物A与乳双歧杆菌HN019菌株的组合物对秀丽隐杆线虫受到金黄色葡萄球菌感染时第三天的存活率的影响。图中各组别在感染阶段添加的干预物(益生菌和/或HMO)情况分别与培养阶段相同。
图4为母乳低聚糖组合物A与乳双歧杆菌HN019菌株的组合物对秀丽隐杆线虫受到金黄色葡萄球菌感染时第五天的存活率的影响。图中各组别在感染阶段添加的干预物(益生菌和/或HMO)情况分别与培养阶段相同。
图5为庆大霉素圆片扩散抑菌圈实验结果图。
图6为乳双歧杆菌HN019圆片扩散抑菌圈实验结果图。
图7显示HN019(1×10 8CFU)与HMO组合物A(10mg/mL)在金葡菌感染前和/或感染期间共培养对于提升线虫存活率的不同效果。
图8显示HMO组合物A(8mg/mL)与乳双歧杆菌HN019(1×10 8CFU)单独及形成组合物后对于金葡菌感染情况下线虫存活率的影响。
图9显示HMO组合物G(8mg/mL)与乳双歧杆菌HN019(1×10 8CFU)单独及 形成组合物后对于金葡菌感染情况下线虫存活率的影响。
图10显示HMO组合物H(8mg/mL)与乳双歧杆菌HN019(1×10 8CFU)单独及形成组合物后对于金葡菌感染情况下线虫存活率的影响。
图中：*代表p<0.05,**代表p<0.01,***代表p<0.001,****代表p<0.0001，NS代表没有显著性差异(not significant)。
具体实施方式
为了对本发明的技术特征、目的和有益效果有更加清楚的理解，现对本发明的技术方案进行以下详细说明，但不能理解为对本发明的可实施范围的限定。
为了对本发明的技术特征、目的和有益效果有更加清楚的理解，现对本发明的技术方案进行以下详细说明，但不能理解为对本发明的可实施范围的限定。
实施例1：母乳低聚糖组合与乳双歧杆菌HN019组合物对秀丽隐杆线虫受到金黄色葡萄球菌感染时存活率的影响
1.实验材料与方法
1.1受试母乳低聚糖样品见表1。
表1
Figure PCTCN2021130978-appb-000001
五种HMO的组合物A中，组成比例为：53％2FL,21％3FL,16％LNT,5％3SL,5％6SL。
用蒸馏水配置母乳低聚糖溶液，并在含有不同终浓度(分别是1、10、30mg/mL)的线虫生长培养基的培养皿中进行培养。
用补充了1％半胱氨酸的MRS培养基来培养受试的益生菌，并在37℃厌氧环境 中孵育过夜。收获细胞并用生理盐水溶液冲洗，调整细菌浓度将其接种在含有线虫生长培养基的平皿中，终浓度为1×10 8CFU。
1.2线虫感染模型
获取了年龄一致的线虫并将其培养在含线虫琼脂培养基(NGM培养基，线虫培养基中含有大肠杆菌OP50为食物)的培养皿中，添加了不同剂量的HMO组合(8mg/mL，10mg/mL，30mg/mL)与益生菌(1×10 8CFU)组合物进行共培养(每个线虫培养皿中加入的液体总体积是10mL。对于浓度为10mg/mL的HMO受试组，每个培养皿中HMO的最终量是100mg；对于浓度分别为8mg/mL和30mg/mL的HMO受试组，每个培养皿中HMO的最终量分别是80mg和300mg。因此每个培养皿平板中，益生菌与HMO的添加量比例为：1×10 8CFU：80mg-300mg)。在线虫成年后，它们被转移到接种了金黄色葡萄球菌ATCC25923的培养皿中，添加量为10 8～10 9CFU/mL，来模拟受到金黄色葡萄球菌感染的情况。各组别在感染阶段添加的干预物(益生菌和/或HMO)情况分别与培养阶段相同。使用了两个对照，分别是没有致病菌的条件(线虫培养皿中含有大肠杆菌OP50)，以及有致病菌金黄色葡萄球菌感染但没有任何干预物的(只含有金黄色葡萄球菌的培养皿)。另设置培养阶段添加干预物而感染阶段没有任何干预物的组别、以及培养阶段未添加干预物而感染阶段添加干预物的组别。
将线虫培养几天后，对它们的存活率进行每日计数。如果线虫对铂丝没有反应，则可被认为是死亡。每种条件均进行了两次独立测定。
对存活曲线进行了统计学的比较分析，用GraphPad Prism 4统计学软件包进行了log rank survival显著性分析。关于对线虫存活率的影响各组间在每一天体现的差异，先用Two-way ANOVA进行分析，再用Tukey’s post hoc test进行各组间的比较，对于HMO组合物单独或益生菌单独组同益生元益生菌混合组的区别，在图上用星号标出。用One-way ANOVA与Dunnett’s post hoc test分析感染第三天或第五天各组存活率的显著性差异。
1.3圆片扩散抑菌圈实验
为确定受试物质是否本身具有抗菌效果，进行了圆片扩散的抑菌圈实验。用无菌的滤纸制成圆片，并接入受试的HMO物质(10mg/mL，30mg/mL)或益生菌(1×10 8CFU)，并将圆片在无菌条件下干燥过夜。将金黄色葡萄球菌ATCC25923(1.0×10 6 CFU/mL)均匀涂抹在NGM琼脂平板表面，随后再将圆片放置于接种的琼脂平板培养皿表面。庆大霉素(200μg/mL)作为阳性对照。所有培养皿在37摄氏度下培养18小时。通过观察在圆片周围是否形成透明的环状圈来确认受试物质是否能直接抑制细菌生长或者具有抗菌活性。
2.实验结果
乳双歧杆菌HN019与两种不同剂量的五种HMO组合物A对秀丽隐杆线虫受到金葡菌感染存活率的影响如图1和图2所示。在整个实验周期，HMO组合物在10mg/mL时，益生元益生菌组合物比HMO组合物单独或益生菌单独的效果均显著的好(P<0.0001)。而HMO组合物A在30mg/mL时，在整个实验周期，HMO与益生菌组合物并未体现优势。
如图1所示，在HMO组合物A为10mg/mL时，益生元HMO和益生菌HN019组合物在第4、5天效果显著好于益生菌HN019单独的组(p<0.05)，在第2、4、5天显著好于HMO组合物单独的组(第2天：p<0.0001；第4、5天：p<0.05)。在观察是否有协同效应时，将HMO与益生菌组合物的存活率数值与单独HMO组合或单独益生菌作比较，发现HMO与益生菌组合物存活率在第4和第5天均显著高于单独HMO组合与单独益生菌存活率数值的加和，体现了HMO与益生菌组合物的协同效应(表2)。
表2
Figure PCTCN2021130978-appb-000002
图3和图4显示了在感染第三天和第五天，HMO组合物A与益生菌形成的组合物对秀丽隐杆线虫存活率影响的比较。在感染第五天，益生元与HN019形成的组合物相比于其他受试组，效果较好。
此外，本发明的实验显示，培养阶段添加了HMO和HN019的组合干预物而感染阶段没有添加任何干预物的组别的秀丽隐杆线虫存活率，显著高于培养阶段及感染 阶段均未添加干预物的组别。
为了研究在此前测试中所出现的，HMO与益生菌组合物对于应对金黄色葡萄球菌感染起到的保护作用，是否来源于直接的杀菌或者抑菌功能，进行了受试物质和菌群一起培养并观察是否有抑菌圈形成的测试。如图5、图6所示，乳双歧杆菌HN019菌株未在接种点周围形成透明的环状抑菌圈(图6)。而阳性对照庆大霉素(gentamicin)则显示出了抑菌效果，以透明环状抑菌圈的产生作为标志(图5)。此外，各HMO单体在两个不同浓度下(10mg/mL，30mg/mL)的抑菌圈实验也有类似结果，各母乳低聚糖单体并不能直接抑制金葡菌生长。可见，本研究中体现的各母乳低聚糖和益生菌HN019组合物对于线虫受到金黄色葡萄球菌感染的保护作用并非源于物质可以直接杀菌。
实施例2：不同组成的母乳低聚糖组合与乳双歧杆菌HN019组合物的提升先天免疫和抗衰老功效实验
本实施例中，受试母乳低聚糖组合物的各组分及含量比例见表3。
表3
Figure PCTCN2021130978-appb-000003
受试组合物功效验证实验1(考察受试物在“提升生物体对金黄色葡萄球菌感染的抵御能力、提高生物体先天免疫和/或抗衰老”方面的预防效果)：获取了年龄一致的线虫并将其培养在含线虫琼脂培养基的培养皿中，添加了一定比例的HMO组合物A(10mg/mL)与HN019益生菌(1×10 8CFU)组合物进行共培养，直到线虫成年。此后，将线虫转移到感染的平板中，接受致病菌金黄色葡萄球菌ATCC25923的感染，不添加HMO与益生菌组合物。
受试组合物功效验证实验2(考察受试物在“提升生物体对金黄色葡萄球菌感染的抵御能力、提高生物体先天免疫和/或抗衰老”方面的治疗效果)：获取了年龄一致的线虫并将其培养在含线虫琼脂培养基的培养皿中。当线虫成年后，将线虫转移到平板中，平板中添加了一定比例的HMO组合物A(10mg/mL)与HN019益生菌(1×10 8 CFU)组合物，并接受致病菌金黄色葡萄球菌ATCC25923的感染。
此外，另有一组，将线虫在感染前与感染后的阶段均添加了HMO与益生菌组合物，并与上述功效验证实验1、2两种情形作为对照组比较。
将线虫培养几天后，对它们的存活率进行每日计数。如果线虫对铂丝没有反应，则可被认为是死亡。此外还包含了两组对照，一组没有添加致病菌(普通培养平板加上大肠杆菌OP50作为食物)，另一组为感染对照组(平板上只添加了金葡菌)。每种条件均进行了两次独立测定。对存活曲线进行了统计学的比较分析，用GraphPad Prism 9统计学软件包进行了log rank survival显著性分析。
实验过程中其余操作参照实施例1。
实验结果：
1.受试物质与线虫在感染前和/或感染期间共培养对于实验结果的影响
如前所述，为了判断受试物质是否有预防效果，仅将线虫与HMO组合物A与HN019在金葡菌感染前进行共培养。为了判断受试物质是否有治疗效果，仅将线虫与HMO组合物A与HN019在金葡菌感染的同时进行共培养。同样，还包括了一种情况，即将受试物质与线虫在感染前和感染期间都共培养。
结果参见图7所示。存活曲线显示，在培养后第一天到第五天，不管是将受试物质在感染前还是感染期间加入共培养，对于提升线虫在金葡菌感染情况下的存活率都有较好的提升和保护作用。而线虫在金葡菌感染期间才加入受试物质，和从始至终都有受试物质的情况，二者对金葡菌存活率的提升和保护作用都相似，且与仅在感染前加入受试物质相比，在感染阶段加入受试物质的情况，以及全过程都有受试物质的情况，对于线虫存活率的保护作用更高。
2.HMO组合物A(8mg/mL)与乳双歧杆菌HN019单独及形成组合物后对于金葡菌感染情况下线虫存活率的影响
结果参见图8。可以看出，在培养后第一天到第五天，8mg/mL的HMO组合物A与单独的益生菌HN019(10 8cfu)均可显著提升线虫在金葡菌感染条件下的存活率，且当二者形成组合物之后，提升效果更显著(P<0.0001)。体现了益生菌HN019与HMO组合物A(8mg/mL)的较好的协同效应。
3.HMO组合物G(8mg/mL)与乳双歧杆菌HN019单独及形成组合物后对于金葡菌感染情况下线虫存活率的影响
结果参见图9。可以看出，在培养后第一天到第五天，8mg/mL的HMO组合物G与单独的益生菌HN019(10 8cfu)均可显著提升线虫在金葡菌感染条件下的存活率，当二者形成组合物之后，提升效果有更显著的趋势。体现了益生菌HN019与HMO组合物G(8mg/mL)的较好的组合效果。
4.HMO组合物H(8mg/mL)与乳双歧杆菌HN019单独及形成组合物后对于金葡菌感染情况下线虫存活率的影响
结果参见图10。可以看出，在培养后第一天到第五天，8mg/mL的HMO组合物H与单独的益生菌HN019(10 8cfu)均可显著提升线虫在金葡菌感染条件下的存活率，当二者形成组合物之后，提升效果有更显著的趋势。体现了益生菌HN019与HMO组合物H(8mg/mL)的较好的组合效果。
实施例3：添加的母乳低聚糖组合与乳双歧杆菌HN019组合物的婴儿配方粉
本实施例提供一种0～6月龄的婴儿配方奶粉，该配方奶粉中总蛋白质含量为11.1g/100g粉，脂肪含量为28.3g/100g粉，碳水化合物含量为52.9g/100g粉，母乳低聚糖组合物含量2.5g/100g粉，乳双歧杆菌为4×10 8CFU/100g。HMOs组合物的组成及比例如下，2’-岩藻糖基乳糖(2’-FL)：3’-岩藻糖基乳糖(3’-FL)：乳糖-N-四糖(LNT)：3’-唾液酸基乳糖(3’-SL)：6’-唾液酸基乳糖(6’-SL)＝53：21：16：5：5。

Claims (14)

  1. 一种组合物，其包括乳双歧杆菌(Bifidobacterium lactis)及母乳低聚糖。
  2. 根据权利要求1所述的组合物，其中，所述乳双歧杆菌包括乳双歧杆菌(Bifidobacterium lactis)HN019菌株。
  3. 根据权利要求1或2所述的组合物，其中，所述母乳低聚糖包括2’-岩藻糖基乳糖、3’-岩藻糖基乳糖、乳糖-N-四糖、3’-唾液酸基乳糖、6’-唾液酸基乳糖中的一种或多种；
    优选地，所述母乳低聚糖包括重量比例(0％-73％):(0％-61％):(0％-52％):(0％-42％):(0％-25％)的2’-岩藻糖基乳糖、3’-岩藻糖基乳糖、乳糖-N-四糖、3’-唾液酸基乳糖、6’-唾液酸基乳糖中的至少三种或四种；更优选地，所述母乳低聚糖包括重量比例(0～53％):(21～41％):(16～32％):(5～22％):(0～5％)的2’-岩藻糖基乳糖、3’-岩藻糖基乳糖、乳糖-N-四糖、3’-唾液酸基乳糖、6’-唾液酸基乳糖。
  4. 根据权利要求1或2所述的组合物，其中，所述乳双歧杆菌与母乳低聚糖的比例为1×10 3CFU～1×10 12CFU：0.1g～10g，优选为1×10 6CFU～1×10 10CFU：1g～10g。
  5. 权利要求1～4中任一项所述的组合物在制备具有可有效提升生物体对金黄色葡萄球菌感染的抵御能力、可提高生物体先天免疫和/或抗衰老功效的食品或药品中的应用。
  6. 根据权利要求5所述的应用，其中，所述提升生物体对金黄色葡萄球菌感染抵御能力包括：提升个体预防金黄色葡萄球菌感染的能力，降低金黄色葡萄球菌感染个体的能力，和/或缓解金黄色葡萄球菌感染引起的个体食物中毒、肠炎、肺炎、皮肤感染、伤口溃烂和/或脑膜炎。
  7. 根据权利要求5所述的应用，其中，所述食品为液体饮料、固体饮料、口服液、奶制品、片剂或胶囊，例如奶粉，特别是婴幼儿配方粉。
  8. 根据权利要求5或7所述的应用，其中，所述乳双歧杆菌在组合物中的应用量为1×10 3CFU/天～1×10 12CFU/天，优选为1×10 6CFU/天～1×10 11CFU/天。
  9. 根据权利要求5或7所述的应用，其中，所述母乳低聚糖在组合物中的应用量为0.015g/天～15g/天，进一步优选为0.1g/天～12g/天，更优选为1.5g/天～10g/天。
  10. 一种食品，其中包含有权利要求1～4中任一项所述的组合物；
    优选地，所述乳双歧杆菌在食品中的应用量为1×10 3CFU/天～1×10 12CFU/天，更 优选为1×10 6CFU/天～1×10 11CFU/天；
    优选地，所述母乳低聚糖在食品中的应用量为0.015g/天～15g/天，进一步优选为0.1g/天～12g/天，更优选为1.5g/天～10g/天。
  11. 母乳低聚糖在用于提升乳双歧杆菌HN019在提升生物体对金黄色葡萄球菌感染的抵御能力、可提高生物体先天免疫和/或抗衰老方面的用途。
  12. 一种提升生物体对金黄色葡萄球菌感染的抵御能力、提高生物体先天免疫和/或抗衰老的方法，所述方法包括：
    给予生物体有效量的权利要求1～4中任一项所述的组合物。
  13. 根据权利要求12所述的方法，其中，所述提升生物体对金黄色葡萄球菌感染抵御能力包括：
    提升个体预防金黄色葡萄球菌感染的能力；
    降低金黄色葡萄球菌感染个体的能力；和/或
    缓解金黄色葡萄球菌感染引起的个体食物中毒、肠炎、肺炎、皮肤感染、伤口溃烂和/或脑膜炎。
  14. 根据权利要求12或13所述的方法，其中，权利要求1～4中任一项所述的组合物以食品的形式给予生物体，所述食品为液体饮料、固体饮料、口服液、奶制品、片剂或胶囊，例如奶粉，特别是婴幼儿配方粉；
    优选地，乳双歧杆菌在食品中的应用量为1×10 3CFU/天～1×10 12CFU/天，更优选为1×10 6CFU/天～1×10 11CFU/天；
    优选地，母乳低聚糖在食品中的应用量为0.015g/天～15g/天，进一步优选为0.1g/天～12g/天，更优选为1.5g/天～10g/天。
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