WO2021217803A1 - 一种减少肠道气体产生的组合物 - Google Patents

一种减少肠道气体产生的组合物 Download PDF

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WO2021217803A1
WO2021217803A1 PCT/CN2020/095967 CN2020095967W WO2021217803A1 WO 2021217803 A1 WO2021217803 A1 WO 2021217803A1 CN 2020095967 W CN2020095967 W CN 2020095967W WO 2021217803 A1 WO2021217803 A1 WO 2021217803A1
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lactose
bifidobacterium
infants
tetraose
nutritional composition
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PCT/CN2020/095967
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French (fr)
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刘斐童
王园园
莱恩乔纳森
王欣
胡瑞标
陈桔淳
杨珂
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合生元（广州）健康产品有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • 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
    • A23L33/10—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
    • 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
    • A61K31/7016—Disaccharides, e.g. lactose, lactulose
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/10—Laxatives
    • 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
    • A23V2400/11—Lactobacillus
    • A23V2400/147—Helveticus
    • 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
    • A23V2400/513—Adolescentes
    • 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
    • A23V2400/517—Bifidum
    • 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
    • A23V2400/519—Breve
    • 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
    • A23V2400/521—Catenulatum
    • 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
    • A23V2400/529—Infantis
    • 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
    • A23V2400/533—Longum
    • 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
    • A23V2400/535—Pseudocatenulatum
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a composition, in particular, to a nutritional composition or nutritional additive for inhibiting and reducing the production of gas in the intestinal tract of infants and young children.
  • Necrotizing enterocolitis is a serious disease threatening the health of newborns, especially premature infants. Its main characteristics are acute onset, severe illness, and high mortality. Clinically, the main symptoms are vomiting, diarrhea, abdominal distension and blood in the stool, and intestinal perforation is often associated. The cause of NEC is not fully understood, and the morbidity and mortality of premature infants and very low birth weight infants are relatively high. It is generally believed that immature intestinal development (gastrointestinal absorption function or maturity of host defense mechanism, gastrointestinal osmotic pressure, intestinal microecology, etc.) is a risk factor for NEC. Diet and feeding patterns (such as milk composition, increase The rate of milk production), infection, and hypoxia are also considered to be the causes of NEC.
  • the microorganisms in the intestine can produce unabsorbed carbohydrates and non-digestible carbohydrates in yeast milk and milk powder, produce ammonia, methane and carbon dioxide and other gases, and cause abdominal distension and gas in the intestinal cavity. Increased intestinal pressure can reduce intestinal mucosal blood flow, secondary to intestinal ischemia and mucosal damage.
  • Clostridium perfringens also known as Clostridium weichii, is a conditional pathogen that can cause antibiotic-related diarrhea and food poisoning. Clostridium perfringens is divided into five toxin subtypes, each of which can produce alpha toxin, which is one of its most important toxins. The relationship between Perfringens and NEC has always attracted much attention. Existing clinical observations and experimental studies on NEC have found that Perfringens can affect the development of NEC and the severity of the disease.
  • the colonization of Cp in the intestines is related to the food and feeding methods of infants and young children.
  • the number of Perfringens perfringens in the intestine of breastfed infants is lower than that of milk powder/mixed-fed infants.
  • the alpha-toxin protein of Perfringens can be identified and detected in the strains isolated from the intestinal tract of children with NEC.
  • the purpose of the present invention is to provide a method for effectively inhibiting the growth of Clostridium perfringens in the gastrointestinal tract of infants and effectively reducing the production of gas in the intestinal tract of infants.
  • the combination is used to prepare a nutritional composition or preparation, and the nutritional composition is used for:
  • the nutritional composition includes:
  • a human milk oligosaccharide comprising: 2'-fucosyllactose (2'-FL), 3'-fucosyllactose (3'-FL), lactose- Disaline algae tetraose (DFL/LDFT), lactose-N-algae pentose I (LNFP-I), lactose-N-algae pentose II (LNFP-II), lactose-N-algae Pentose III (LNFP-III), lactose-N-tetraose (LNT), lactoyl-N-neotetraose (LNnT), 3'-sialyllactose (3SL), 6'-sialyllactose (6SL), saliva Lactosyl-N-tetraose (LST) and/or Lactosyl-N-tetraose (DS).
  • LNFP-I lac
  • a probiotic microorganism including bifidobacteria.
  • the infant refers to a population of 0-24 months of age, preferably a population of 0-12 months of age, and more preferably of a population of 0-6 months of age. crowd.
  • the infants include infants with normal intestinal function.
  • the infants include infants with abdominal distension, bloating, abdominal cramps, or gastrointestinal discomfort.
  • the intestinal tract refers to the digestive tract of the human body from the pylorus of the stomach to the anus.
  • the preparation can also be used for:
  • the Clostridium perfringens includes: Clostridium perfringens, Enterococcus faecium, or a combination thereof.
  • the overgrowth refers to a concentration higher than 3-30% of the normal value, preferably 3-15% higher than the normal value, and more preferably 3-10% higher than the normal value.
  • the nutritional composition further includes: fucosylated oligosaccharides, fructooligosaccharides, galactooligosaccharides, lactose, or a combination thereof.
  • the fucosylated oligosaccharide is selected from the group consisting of 2'-fucosylgalactose, 2'-fucosyllactose, 3'-fucosyllactose, Difucosyllactose, lactose-N-fucopentose I, lactose-N-fucopentose II, lactose-N-fucopentose III, lactose-N-fucopentose V, lactose-N -Fucose, lactose-N-difucohexose I, fucosyllactose-N-hexose, fucosyllactose-N-new hexose I, fucosyllactose-N-new Hexose II, difucosyl lactose-N-hexose I, difucosyl lactose-N-n
  • the HMO includes: 2'-fucosyllactose (2'-FL), lactose, or a combination thereof.
  • the HMO is 2'-fucosyllactose (2'-FL).
  • the probiotic microorganisms also include bacteria selected from the group consisting of Bifidobacterium species (Bifidobacterium spp.), Lactobacillus species (Lactobacillus spp.), Streptococcus species (Streptococcus spp.) ), Enterococcus species (Enterococcus spp.), and Saccharomyces species (Saccharomyces spp.), or a combination thereof.
  • the Bifidobacterium species is selected from the group consisting of Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium longum.
  • Bifidobacterium Bifidobacterium bifidum
  • Bifidobacterium adolescentis Bifidobacterium adolescentis
  • Bifidobacterium pseudocatenulatum Bifidobacterium pseudocatenulatum
  • Bifidobacterium catenulatum Bifidobacterium Catenulatum
  • the bifidobacterium is Bifidobacterium longum (Bifidobacterium longum), Bifidobacterium breve (Bifidobacterium breve), Bifidobacterium infantis (Bifidobacterium infantis), and Bifidobacterium (Bifidobacterium bifidum), Or a combination.
  • the bifidobacterium is of human origin.
  • the probiotic microorganisms include Lactobacillus helveticus R52 (Lactobacillus helveticus R52), Bifidobacterium infantis R33 (Bifidobacterium infantis R33), Bifidobacterium bifidum (Bifidobacterium bifidum R71), Bifidobacterium breve ( Bifidobacterium breve M-16V), or a combination thereof.
  • the total amount of the HMO is 10-200 g/kg, preferably 60-170 g/kg, more preferably 80-150 g/kg.
  • the total amount of the HMO is 1%-20% by weight, preferably 6%-17% by weight, more preferably 8%-15% by weight.
  • the concentration of the probiotic microorganisms is 1 ⁇ 10 6 -1 ⁇ 10 14 CFU/g, preferably 1 ⁇ 10 8 -1 ⁇ 10 10 CFU/g , More preferably 1 ⁇ 10 9 -1 ⁇ 10 10 CFU/g.
  • a nutritional composition which includes:
  • a human milk oligosaccharide comprising: 2'-fucosyllactose (2'-FL), 3'-fucosyllactose (3'-FL), lactose- Disaline algae tetraose (DFL/LDFT), lactose-N-algae pentose I (LNFP-I), lactose-N-algae pentose II (LNFP-II), lactose-N-algae Pentose III (LNFP-III), lactose-N-tetraose (LNT), lactoyl-N-neotetraose (LNnT), 3'-sialyllactose (3SL), 6'-sialyllactose (6SL), saliva Lactosyl-N-tetraose (LST) and/or Lactosyl-N-tetraose (DS).
  • LNFP-I lac
  • a probiotic microorganism including bifidobacteria.
  • a method for preparing the nutritional composition according to the second aspect of the present invention comprising the steps of: mixing the following components (i) and (ii) to obtain The nutritional composition according to the second aspect of the present invention;
  • a human milk oligosaccharide comprising: 2'-fucosyllactose (2'-FL), 3'-fucosyllactose (3'-FL), lactose- Disaline algae tetraose (DFL/LDFT), lactose-N-algae pentose I (LNFP-I), lactose-N-algae pentose II (LNFP-II), lactose-N-algae Pentose III (LNFP-III), lactose-N-tetraose (LNT), lactoyl-N-neotetraose (LNnT), 3'-sialyllactose (3SL), 6'-sialyllactose (6SL), saliva Lactosyl-N-tetraose (LST) and/or Lactosyl-N-tetraose (DS).
  • LNFP-I lac
  • a probiotic microorganism including bifidobacteria.
  • a method for inhibiting the overgrowth of Clostridium perfringens in the intestine of infants and young children is provided, and the nutritional composition according to the second aspect of the present invention is administered to a subject in need.
  • the method of administration includes oral, injection, infusion, or a combination thereof.
  • the subject includes infants and children aged 0-24 months, preferably infants and children aged 0-12 months, and more preferably children aged 0-6 months. Infants and young children.
  • the infants include infants with normal intestinal function.
  • the infants include infants who have abdominal distension, bloating, abdominal cramps, or gastrointestinal discomfort.
  • Figure 1 shows the comparison of the gas production results of breast-fed infants and milk-fed infants with different substrates for intestinal flora.
  • Figure 2 shows the effect of different substrates on gas production in the intestinal tract of bloated infants.
  • Figure 3 shows the negative correlation between the content of bifidobacteria in the intestines of infants and gas production in the intestines
  • Figure 4 shows the determination of aerobic bacteria in the intestinal tract of infants by metagenomic sequencing technology.
  • the present invention has been completed on this basis.
  • the terms "nutritional composition” and “combination of the present invention” are used interchangeably, and refer to the second aspect of the present invention including a breast milk oligosaccharide and a probiotic microorganism that can be used to improve infants Nutritional composition for intestinal function.
  • infant refers to a child aged 0-24 months, preferably 0-12 months.
  • infants and young children are in the stage of rapid growth and development, and their bodies and functions are constantly developing and improving. Diet not only greatly affects the growth and development of healthy infants and young children, but also has a positive preventive and intervention effect on various pathological or sub-health symptoms that appear during their growth and development.
  • breast milk is not only a child’s food, it has an impact on the baby’s life.
  • the World Health Organization recommends that babies should receive exclusive breastfeeding during the first six months of life to achieve optimal growth and development.
  • the breastfeeding rate is only 38% globally. This makes it a technological trend to develop infant nutrition with food ingredients close to breast milk or nutritional supplements with functions close to breast milk.
  • the intestinal flora plays an important role early in life. It is generally believed that the gastrointestinal tract of a baby is sterile before birth, and colonization begins within 48 hours after birth. The first bacteria in the neonatal gastrointestinal tract come from the mother's birth canal, environment and breast milk. The gastrointestinal micro-ecology of infants born by caesarean section is established earlier in natural delivery. The initial gastrointestinal microecology of breastfed infants and artificially fed infants is different. Breast-fed full-term infants are dominated by bifidobacteria in the micro-ecology, and the diversity of intestinal flora in artificially-fed infants has increased, accompanied by a significant increase in the number of Bacteroides.
  • Clostridium is also a normal type of dominant bacteria in the intestinal flora. It is very important to the human body. It mainly includes two types of beneficial Clostridium and harmful Clostridium. Beneficial Clostridium participates in many physiological functions such as metabolism, immunity, and regulation of microecological balance. Harmful Clostridium is mostly conditional pathogenic bacteria, which can cause a variety of basic intestinal and other related diseases, such as osteomyelitis and bacteremia.
  • HMO is a type of complex mixed oligosaccharides present in human milk, and is the third largest solid component in breast milk. At present, more than 200 different HMO structures have been identified. Among them, 2'-fucosyllactose (2'-FL) and similar HMO structures linked by ⁇ -1,2 fucose are the most abundant in HMO .
  • HMO plays a very important role in the early growth and development of infants, such as 1) regulating the intestinal flora, especially promoting the proliferation of beneficial bacteria (such as bifidobacteria); 2) by enhancing the symbiont of non-pathogenic bacteria Its competitive advantage can indirectly inhibit the growth of pathogenic bacteria, and it can also directly act as an anti-adhesive antibacterial agent to reduce microbial infections; 3) directly and indirectly regulate the immune system and improve immunity; 4) promote brain development, etc.
  • a human milk oligosaccharide comprising: 2'-fucosyllactose (2'-FL), 3'-fucosyllactose (3'-FL), lactose- Disaline algae tetraose (DFL/LDFT), lactose-N-algae pentose I (LNFP-I), lactose-N-algae pentose II (LNFP-II), lactose-N-algae Pentose III (LNFP-III), lactose-N-tetraose (LNT), lactoyl-N-neotetraose (LNnT), 3'-sialyllactose (3SL), 6'-sialyllactose (6SL), saliva Lactosyl-N-tetraose (LST) and/or Lactosyl-N-tetraose (DS).
  • LNFP-I lac
  • a probiotic microorganism including bifidobacteria.
  • the HMO is 2'-fucosyllactose (2'-FL).
  • the bifidobacteria of the present invention are of human origin.
  • the Bifidobacterium is Bifidobacterium longum (Bifidobacterium longum), Bifidobacterium breve (Bifidobacterium breve), Bifidobacterium infantis (Bifidobacterium Infantis), Bifidobacterium (Bifidobacterium bifidum) ), or a combination thereof.
  • the probiotic microorganisms include Lactobacillus helveticus R52 (Lactobacillus helveticus R52), Bifidobacterium infantis R33 (Bifidobacterium infantis R33), Bifidobacterium bifidum (Bifidobacterium bifidum R71), Bifidobacterium breve ( Bifidobacterium breve M-16V), or a combination thereof.
  • the total amount of the HMO is 10-200 g/kg, preferably 60-170 g/kg, more preferably 80-150 g/kg.
  • the total amount of the HMO is 1%-20% by weight, preferably 6%-17% by weight, more preferably 8%-15% by weight Parts by weight.
  • the concentration of the probiotic microorganisms is 1 ⁇ 10 6 -1 ⁇ 10 14 CFU/g, preferably 1 ⁇ 10 8 -1 ⁇ 10 10 CFU/g, and more preferably 1 ⁇ 10 8 -1 ⁇ 10 10 CFU/g. ⁇ 10 9 -1 ⁇ 10 10 CFU/g.
  • the nutritional composition of the present invention can be used to: (a) reduce the production of intestinal gas in infants and young children; (b) inhibit the overgrowth of Clostridium perfringens in the intestines of infants and young children; (c) balance the intestinal flora of infants and young children; (d) ) Improve infantile constipation; and/or (e) Improve infantile intestinal metabolism.
  • the present invention screens out an HMO prebiotic through an in vitro fermentation model, which can significantly reduce gas production in the intestinal tract of infants and young children.
  • the present invention selects a kind of HMO prebiotics combined with human-derived bifidobacteria through an in vitro fermentation model to have a more excellent effect of reducing gas production in the intestinal tract of infants and young children.
  • the present invention verifies through high-throughput sequencing that the HMO and human-derived bifidobacteria formula can better improve the intestinal flora of infants with abdominal distension and colic.
  • Example 1 Fermentation of the intestinal flora of healthy infants and young children to screen HMO with low gas production
  • the infant feeding method is breastfeeding (the number of times of formula feeding ⁇ 2 times per day) or formula feeding (the number of breastfeeding times per day ⁇ 2 times).
  • Baby stool collection Open the baby's diaper, collect a stool sample from the diaper, and put it into a sampling tube.
  • the sampling tube is placed in a sealed bag, stored in a low temperature, and sent to the laboratory within 48 hours after collection. After the sample was collected, it was divided into two parts, and one part was measured for 16s rRNA in feces to determine the structure of the flora. The other part was prepared with anaerobic PBS to prepare a fecal suspension, and then immediately inoculated into the in vitro fermentation system after filtration. Put the fresh feces into PBS buffer, shake and mix well, and filter to prepare a 10% fecal suspension.
  • the operating unit of the system consists of a batch fermentation tank and a fecal gas detector.
  • the fermenter contains carbon-free basal medium (YCFA).
  • the basic fermentation medium does not contain carbon source, only fructooligosaccharide (FOS), galactooligosaccharide (GOS), lactose (lactose) and 2'-fucosylgalactose (2'-FL), 2'- Fucosylgalactose + Bifidobacterium breve (M16-V) as the sole carbon source and raw material were added to the culture medium.
  • Basal medium (YCFA) without carbohydrates was used as a control.
  • the 0-hour fermentation sample flora structure was used as the baseline of the experiment to compare the changes of the flora after fermentation.
  • the gas production of breast-fed infant feces after fermentation is significantly lower than that of milk-fed infants, which proves that the difference between the nutritional composition of breast milk and the nutritional composition of formula milk powder has a significant impact on the intestinal flora and fecal fermentation metabolites.
  • oligosaccharides in breast milk that are more suitable for infants and young children. They are the main fermented carbohydrates of the intestinal flora of breast-fed infants. They are also an important difference in nutrients for different feeding methods. Therefore, 2'-FL (fucosyl half Lactose), as the highest content component of breast milk oligosaccharides (HMO), is an important factor in controlling gas production in the intestinal tract.
  • Example 2 Using HMO to Intervene Fermentation of Intestinal Flora in Infants with Abdominal Distension
  • Abdominal bloating is mostly caused by diet. Infant bloating caused by improper diet is based on the clinical diagnosis of the pediatrician, confirmed by physical examination and/or abdominal imaging examination (B-ultrasound or standing abdominal plain film) and other indicators, and Exclude occasional abdominal distension in infants caused by the mother’s diet.
  • Sample collection method Open the baby's diaper, collect a stool sample from the diaper, and put it into a sampling tube.
  • the sampling tube is placed in a sealed bag, stored in a low temperature, and sent to the laboratory within 48 hours after collection. After the sample was collected, it was divided into two parts, and one part was measured for 16s rRNA in feces to determine the structure of the flora.
  • the other part was prepared with anaerobic PBS to prepare a fecal suspension, and then immediately inoculated into the in vitro fermentation system after filtration. Put the fresh feces into PBS buffer, shake and mix well, and filter to prepare a 10% fecal suspension.
  • the operating unit of the system consists of a batch fermentation tank and a fecal gas detector.
  • the fermentation tank contains carbon-free basal medium (YCFA).
  • the basic fermentation medium does not contain carbon source, only 0.8% fructooligosaccharide (FOS), galactooligosaccharide (GOS), lactose (lactose), 2'-fucosylgalactose (2'-FL) and 2'-Fucosylgalactose + Bifidobacterium breve (M16-V) as the sole carbon source and raw materials were added to the medium separately.
  • Basal medium (YCFA) without carbohydrates was used as a control.
  • the fecal suspension was inoculated in the fermentor and fermented for 24 hours at 37°C to monitor the gas production and gas composition of the feces in real time; the microbial structure was determined by 16s rRNA sequencing and metagenomic methods to determine the microbial composition and quantity.
  • the 0-hour fermentation sample flora structure was used as the baseline of the experiment to compare the changes of the flora after fermentation.
  • Example 3 Correlation analysis of 2'-FL with intestinal flora and gas production
  • the stool sample was extracted with a stool extraction kit to extract the sample DNA, and the DNA band size and integrity were detected by 1% agarose gel electrophoresis, and the genome concentration, A260/A280 and other values were determined.
  • the DNA of the above-mentioned extracted sample was sequenced using the Ilumina Hiseq platform, and a paired-end sequencing (Paired-End) method was used to construct a small fragment library for sequencing. Each DNA sample was sequenced twice in parallel, and differentiated bacteria with smaller deviations from the parallel sequencing were selected to compare the abundance values, and the numerical differences were recorded at the family level.
  • 2’-FL promotes the growth of beneficial bifidobacteria, reduces the number of Clostridium perfringens, and affects the amount of gas produced in the intestinal tract
  • Typical clinical manifestations of children with NEC include abdominal distension, vomiting, and blood in the stool.
  • Treatment measures include fasting, antibiotics, supportive therapy, and surgery.
  • For infants and young children with abdominal distension in addition to the use of drugs or physical therapy to reduce gastrointestinal pressure, improve intestinal microcirculation and promote intestinal peristalsis to discharge intestinal gas, the most important and commonly used preventive and intervention method is through dietary intervention, such as providing access Breast milk nutritious food or provide useful probiotics to prevent or treat abdominal distension and related gastrointestinal diseases.

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Abstract

一种组合的用途，所述组合用于制备一营养组合物或制剂，所述营养组合物用于：（a）减少婴幼儿肠道气体的产生；和/或（b）抑制婴幼儿肠道产气梭菌的过度生长；其中，所述营养组合物中包括：（i）一种母乳低聚糖（HMO）；和（ii）一种益生菌微生物，所述益生菌微生物包括双歧杆菌。显著降低婴幼儿肠道产气、较优的改善腹胀以及腹绞痛婴儿的肠道菌群。

Description

一种减少肠道气体产生的组合物 技术领域
本发明涉及一种组合物，具体地，涉及一种用于抑制和减少婴幼儿肠道气体产生的营养组合物或营养添加剂。
背景技术
新生儿坏死性小肠结肠炎(neonatal necrotizing enterocolitis,NEC)是一种威胁新生儿特别是早产儿健康的严重疾病，其主要特点是发病急、病情重、死亡率高。临床上以呕吐、腹泻、腹胀和便血为主要症状，还常合并肠穿孔。NEC的病因还未完全清楚，早产儿和极低出生体重儿发病率和死亡率较高。一般认为肠道发育不成熟(胃肠吸收功能或宿主防御机制的成熟度、胃肠道渗透压、肠道微生态等)是NEC发生的危险因子，饮食和喂养模式(如奶的成分、增加奶量的速度)、感染、缺氧因素也被认为是发生NEC的诱因。
肠管内的微生物可发酵母乳和奶粉中未被吸收的碳水化合物和非消化性碳水化合物，产生氨气、甲烷和二氧化碳等气体，而出现腹胀和肠腔积气。肠腔压力增高可减少肠粘膜血流，继发肠缺血和粘膜损伤。
产气荚膜梭菌(Clostridium perfringens，Cp)，又称魏氏梭菌(Clostridum weichii)，属于条件致病菌，可引起抗生素相关性腹泻和食物中毒等。产气荚膜梭菌分为五种毒素亚型，每种亚型均可产生α毒素，是其最重要的毒素之一。产气荚膜杆菌与NEC的关系一直备受关注，现有对NEC的临床观察和实验研究，均发现产气荚膜杆菌可影响NEC的发展和疾病严重级别。
Cp在肠道的定殖与婴幼儿的食物及喂养方式有关，母乳喂养儿肠道内的产气荚膜杆菌数量较奶粉/混合喂养儿更低。在NEC患儿的肠道分离的菌株中可鉴定检测到产气荚膜杆菌α毒素蛋白。
在一项针对早产儿的研究中，建议以肠道气体产生量作为诊断产气荚膜杆菌Cp感染的指标。
因此，本领域迫切需要开发一种有效抑制婴儿胃肠道产气荚膜梭菌生长，并且有效减少婴儿肠道气体产生的方法。
发明内容
本发明的目的是提供一种有效抑制婴儿胃肠道产气荚膜梭菌生长，并且有效减少婴儿肠道气体产生的方法。
在本发明的第一方面，提供了一种组合的用途，其特征在于，所述组合用于制备一营养组合物或制剂，所述营养组合物用于：
(a)减少婴幼儿肠道气体的产生；和/或
(b)抑制婴幼儿肠道产气梭菌的过度生长；
其中，所述营养组合物中包括：
(i)一种母乳低聚糖(HMO)，所述HMO包括：2’-岩藻糖基乳糖(2’-FL)、3’-岩藻糖基乳糖(3’-FL)、乳糖-二盐藻四糖(DFL/LDFT)、乳糖-N-盐藻基戊糖I(LNFP-I)、乳糖-N-盐藻基戊糖II(LNFP-II)、乳糖-N-盐藻基戊糖III(LNFP-III)、乳糖-N-四糖(LNT)、乳酰-N-新四糖(LNnT)、3’-唾液乳糖(3SL)、6’-唾液乳糖(6SL)、唾液酸基乳糖基-N-四糖(LST)和/或二唾液酸基乳糖基-N-四糖(DS-LNT)；和
(ii)一种益生菌微生物，所述益生菌微生物包括双歧杆菌。
在另一优选例中，所述婴幼儿是指月龄为0-24个月的人群，优选地为月龄为0-12个月的人群，更优选为月龄为0-6个月的人群。
在另一优选例中，所述婴幼儿包括肠道功能正常的婴幼儿。
在另一优选例中，所述婴幼儿包括具有腹胀、气涨、腹绞痛、或胃肠道不适的婴幼儿。
在另一优选例中，所述肠道是指人体从胃幽门至肛门的消化管。
在另一优选例中，所述制剂还可用于：
(c)平衡婴幼儿肠道菌群；
(d)改善婴幼儿便秘；和/或
(e)改善婴幼儿肠道代谢。
在另一优选例中，所述产气梭菌包括：产气荚膜梭菌、屎肠球菌，或其组合。
在另一优选例中，所述过度生长是指浓度高于正常值的3-30％，较佳地高于正常值的3-15％，更佳地高于正常值的3-10％。
在另一优选例中，所述营养组合物还包括：岩藻糖基化低聚糖、低聚果糖、低聚半乳糖、乳糖，或其组合。
在另一优选例中，所述岩藻糖基化低聚糖选自下组：2’-岩藻糖基半乳糖、2’- 岩藻糖基乳糖、3’-岩藻糖基乳糖、二岩藻糖基乳糖、乳糖-N-岩藻戊糖I、乳糖-N-岩藻戊糖II、乳糖-N-岩藻戊糖III、乳糖-N-岩藻戊糖V、乳糖-N-岩藻己糖、乳糖-N-二岩藻己糖I、岩藻糖基乳糖-N-己糖、岩藻糖基乳糖-N-新己糖I、岩藻糖基乳糖-N-新己糖II、二岩藻糖基乳糖-N-己糖I、二岩藻糖基乳糖-N-新己糖I、二岩藻糖基乳糖-N-新己糖II、岩藻糖基-对-乳糖-N-己糖，或其组合。
在另一优选例中，所述HMO包括：2’-岩藻糖基乳糖(2’-FL)、乳糖，或其组合。
在另一优选例中，所述HMO为2’-岩藻糖基乳糖(2’-FL)。
在另一优选例中，所述益生菌微生物还包括选自下组的细菌：双歧杆菌属物种(Bifidobacterium spp.)、乳酸杆菌属物种(Lactobacillus spp.)、链球菌属物种(Streptococcus spp.)、肠球菌属物种(Enterococcus spp.)、和酵母属物种(Saccharomyces spp.)，或其组合。
在另一优选例中，所述双歧杆菌属物种选自下组：长双歧杆菌(Bifidobacterium longum)、短双歧杆菌(Bifidobacterium breve)、婴儿双歧杆菌(Bifidobacterium infantis)、两歧双歧杆菌(Bifidobacterium bifidum)、青春双歧杆菌(Bifidobacterium adolescentis)、假链状双歧杆菌(Bifidobacterium pseudocatenulatum)、链状双歧杆菌(Bifidobacterium Catenulatum)，或其组合。
在另一优选例中，所述双歧杆菌为长双歧杆菌(Bifidobacterium longum)、短双歧杆菌(Bifidobacterium breve)、婴儿双歧杆菌(Bifidobacterium infantis)、两歧双歧杆菌(Bifidobacterium bifidum)，或其组合。
在另一优选例中，所述双歧杆菌是人源的。
在另一优选例中，所述益生菌微生物包括瑞士乳杆菌R52(Lactobacillus helveticus R52)、婴儿双歧杆菌R33(Bifidobacterium infantis R33)、两歧双歧杆菌(Bifidobacterium bifidum R71)、短双歧杆菌(Bifidobacterium breve M-16V)，或其组合。
在另一优选例中，所述营养组合物中，所述HMO的总量为10-200g/kg，较佳地为60-170g/kg，更佳地为80-150g/kg。
在另一优选例中，所述营养组合物中，以干重计，所述HMO的总量为1％-20％重量份，较佳地为6％-17％重量份，更佳地为8％-15％重量份。
在另一优选例中，所述营养组合物中，所述益生菌微生物的浓度为1×10 6-1×10 14CFU/g，较佳地1×10 8-1×10 10CFU/g，更佳地1×10 9-1×10 10CFU/g。
在本发明的第二方面，提供了一种营养组合物，所述营养组合物中包括：
(i)一种母乳低聚糖(HMO)，所述HMO包括：2’-岩藻糖基乳糖(2’-FL)、3’-岩藻糖基乳糖(3’-FL)、乳糖-二盐藻四糖(DFL/LDFT)、乳糖-N-盐藻基戊糖I(LNFP-I)、乳糖-N-盐藻基戊糖II(LNFP-II)、乳糖-N-盐藻基戊糖III(LNFP-III)、乳糖-N-四糖(LNT)、乳酰-N-新四糖(LNnT)、3’-唾液乳糖(3SL)、6’-唾液乳糖(6SL)、唾液酸基乳糖基-N-四糖(LST)和/或二唾液酸基乳糖基-N-四糖(DS-LNT)；和
(ii)一种益生菌微生物，所述益生菌微生物包括双歧杆菌。
在本发明的第三方面，提供了一种制备如本发明第二方面所述的营养组合物的方法，包括步骤：将以下组分(i)和组分(ii)进行混合，从而得到如本发明第二方面所述的营养组合物；
(i)一种母乳低聚糖(HMO)，所述HMO包括：2’-岩藻糖基乳糖(2’-FL)、3’-岩藻糖基乳糖(3’-FL)、乳糖-二盐藻四糖(DFL/LDFT)、乳糖-N-盐藻基戊糖I(LNFP-I)、乳糖-N-盐藻基戊糖II(LNFP-II)、乳糖-N-盐藻基戊糖III(LNFP-III)、乳糖-N-四糖(LNT)、乳酰-N-新四糖(LNnT)、3’-唾液乳糖(3SL)、6’-唾液乳糖(6SL)、唾液酸基乳糖基-N-四糖(LST)和/或二唾液酸基乳糖基-N-四糖(DS-LNT)；和
(ii)一种益生菌微生物，所述益生菌微生物包括双歧杆菌。
在本发明的第四方面，提供了一种抑制婴幼儿肠道产气梭菌的过度生长的方法，向有所需要的对象施用如本发明第二方面所述的营养组合物。
在另一优选例中，所述施用的方式包括：口服、注射、输注，或其组合。
在另一优选例中，所述对象包括月龄为0-24个月的婴幼儿，优选地为月龄为0-12个月的婴幼儿，更优选为月龄为0-6个月的婴幼儿。
在另一优选例中，所述婴幼儿包括肠道功能正常的婴幼儿。
在另一优选例中，所述婴幼儿包括具有腹胀、气涨、腹绞痛，或胃肠道不适的婴幼儿。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了母乳喂养婴儿和奶粉喂养婴儿肠道菌群使用不同底物体外发酵的产气结果比较。
图2显示了不同底物对腹胀婴儿肠道产气的影响。
图3显示了婴儿肠道中的双歧杆菌含量与肠道产气的负相关性
图4显示了宏基因组测序技术确定婴儿肠道的产气菌。
具体实施方式
本发明人经过长期而深入的研究，通过大量的筛选，意外地开发出一种有效抑制婴儿胃肠道产气荚膜梭菌生长，并且有效减少婴儿肠道气体产生的营养组合物，该营养组合物中包括2’-FL和人源的双歧杆菌。实验证明，用该营养组合物能够显著抑制婴幼儿胃肠道产气荚膜梭菌过度增殖、缓解令肠道微生态不平衡导致的腹胀，并且可促进双歧杆菌的增殖，减少肠道气体产生，从而预防和/或治疗NEC。在此基础上完成了本发明。
营养组合物
如本文所用，术语“营养组合物”、“本发明的组合”可互换使用，是指本发明第二方面所述的包括一种母乳低聚糖和一种益生菌微生物的可用于改善婴儿肠道功能的营养组合物。
如本文所用，术语“婴儿”是指月龄在0-24个月，优选0-12个月的小孩。
众所周知，婴幼儿处于快速生长发育阶段，其身体及其功能都在不断发展和完善中。饮食不仅极大的影响健康婴幼儿的生长发育，也对其生长发育过程中出现的各种病理或亚健康症状有着积极的预防及干预效果。正如母乳不仅是孩子的粮食，它对婴儿的一生都是有影响的。世界卫生组织建议，婴儿应当在生命最初的六个月内接受纯母乳喂养，以实现最佳的成长和发育。然而由于现代生活方式的变化，母乳喂养率全球仅达38％，这使得开发食物成分接近母乳的婴幼儿营养品或功能接近母乳的营养强化剂成为技术趋势。
肠道菌群在生命早期发挥着重要作用。通常认为，婴儿在出生前，其胃肠道是无菌的，出生后48小时之内开始有菌定殖。新生儿胃肠道内最初的细菌来自于母亲的产道、环境以及母乳。自然分娩比剖腹产的婴幼儿胃肠道微生态 更早建立。母乳喂养儿与人工喂养儿最初的胃肠道微生态有所不同。母乳喂养的足月儿微生态以双歧杆菌为主，人工喂养儿肠道菌群多样性增加，伴随有拟杆菌数量的显著上升。
从婴儿期来看，由于环境的影响，最早建立的肠道菌群为大肠杆菌和链球菌。随后定植的为两种非常重要的有益细菌-双歧杆菌和乳酸杆菌，而且双歧杆菌数量迅速上升，成为婴幼儿肠道的优势菌群。这些肠道有益菌对于婴幼儿早期胃肠道免疫系统的发育成熟、营养功能如缓解乳糖不耐受症、加强脂类和蛋白质代谢，以及维生素的合成起重要作用。婴幼儿的肠道有益菌越早建立，越能保护婴儿免受感染性疾病、过敏和各种消化道问题。在肠道微生态建立的这一关键时期，及早给婴幼儿补充高质量的益生菌，促进健康微生态的形成，对婴幼儿期以及成人后的健康都会产生深远的正面影响。
虽然NEC的致病机制不明，但目前均认为新生儿和婴幼儿肠道早期的微生物定殖对NEC的发展有着重要的影响。婴幼儿肠道早期优势微生物除了双歧杆菌以外，梭菌也是肠道菌群中的一类正常的优势细菌，对人体非常重要，其主要包括有益的梭菌和有害的梭菌两类。有益梭菌参与代谢、免疫以及调节微生态平衡等诸多生理功能，有害梭菌多为条件致病菌，能引起多种肠道基本及其他相关疾病，如骨髓炎、菌血症等。
对于NEC研究已十分丰富，然而目前主要通过抗生素及手术治疗NEC，还未有通过食物干预来影响NEC的发展，达到预防或减轻NEC症状的方法。
母乳及奶粉是新生儿的主要食物，研究显示，母乳喂养的婴儿，其肠道中产气荚膜杆菌的数量更少，NEC的发生率更低。母乳中含有多种活性物质，其中HMOs是重要的碳水化合物组分。HMO是一类存在于人乳中的复杂混合低聚糖，是母乳中第三大固体成分。目前已鉴定出超过200中不同的HMO结构，其中2’岩藻糖基乳糖(2’-FL)以及与其类似的通过α-1,2岩藻糖连接的HMO结构，是HMO中含量最高的。研究显示，HMO对于婴儿早期生长发育起到非常重要的作用，如1)调节肠道菌群，尤其是促进有益菌(如，双歧杆菌)的增殖；2)通过提高非致病菌共生体的竞争优势来间接抑制致病菌的生长，也能直接充当抗黏附抗菌剂减少微生物感染；3)直接和间接调节免疫系统，提高免疫力；4)促进大脑发育等。
在本发明中，提供了一种营养组合物，其包括：
(i)一种母乳低聚糖(HMO)，所述HMO包括：2’-岩藻糖基乳糖(2’-FL)、3’-岩 藻糖基乳糖(3’-FL)、乳糖-二盐藻四糖(DFL/LDFT)、乳糖-N-盐藻基戊糖I(LNFP-I)、乳糖-N-盐藻基戊糖II(LNFP-II)、乳糖-N-盐藻基戊糖III(LNFP-III)、乳糖-N-四糖(LNT)、乳酰-N-新四糖(LNnT)、3’-唾液乳糖(3SL)、6’-唾液乳糖(6SL)、唾液酸基乳糖基-N-四糖(LST)和/或二唾液酸基乳糖基-N-四糖(DS-LNT)；和
(ii)一种益生菌微生物，所述益生菌微生物包括双歧杆菌。
在一个优选的实施方式中，所述HMO为2’-岩藻糖基乳糖(2’-FL)。
优选地，本发明的双歧杆菌是人源的。在一个优选的实施方式中，所述的双歧杆菌为长双歧杆菌(Bifidobacterium longum)、短双歧杆菌(Bifidobacterium breve)、婴儿双歧杆菌(Bifidobacterium infantis)、两歧双歧杆菌(Bifidobacterium bifidum)，或其组合。
在一个实施方式中，所述的益生菌微生物包括瑞士乳杆菌R52(Lactobacillus helveticus R52)、婴儿双歧杆菌R33(Bifidobacterium infantis R33)、两歧双歧杆菌(Bifidobacterium bifidum R71)、短双歧杆菌(Bifidobacterium breve M-16V)，或其组合。
在本发明的营养组合物中，所述HMO的总量为10-200g/kg，较佳地为60-170g/kg，更佳地为80-150g/kg。
或者，所述营养组合物中，以干重计，所述HMO的总量为1％-20％重量份，较佳地为6％-17％重量份，更佳地为8％-15％重量份。
或者，所述营养组合物中，所述益生菌微生物的浓度为1×10 6-1×10 14CFU/g，较佳地1×10 8-1×10 10CFU/g，更佳地1×10 9-1×10 10CFU/g。
本发明的营养组合物可用于：(a)减少婴幼儿肠道气体的产生；(b)抑制婴幼儿肠道产气梭菌的过度生长；(c)平衡婴幼儿肠道菌群；(d)改善婴幼儿便秘；和/或(e)改善婴幼儿肠道代谢。
本发明的主要优点包括：
1)本发明通过体外发酵模型筛选出一种HMO类的益生元可以显著降低婴幼儿肠道产气。
2)本发明通过体外发酵模型帅选出一种HMO类益生元结合人源性双歧杆菌具有更优异的降低婴幼儿肠道产气的效果。
3)本发明通过高通量测序验证了该HMO与人源性双歧杆菌的配方可较优的改善腹胀以及腹绞痛婴儿的肠道菌群。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例1：健康婴幼儿肠道菌群发酵筛选产气量低的HMO
1.1.研究方法：
研究对象：
从儿童医院收集40-60位0-6个月健康婴儿的粪便样品。其中包括30位母乳喂养和30位配方乳喂养的婴儿粪便样品。婴儿年龄分段为：≤1个月，分别10例母乳喂养和配方奶喂养婴儿；＞1个月并≤3个月，分别10例母乳喂养和配方奶喂养婴儿；＞3个月并≤6个月，分别10例母乳喂养和配方奶喂养婴儿。
入选标准：
1)健康、足月儿(胎龄≥37周)、阴道分娩；
2)经过医学上证明是健康婴儿：无症状和无疾病体征。
3)年龄为0-6个月；
4)收集粪便样品前，婴儿喂养方式为母乳喂养(每天配方奶粉喂养次数≤2次)或配方奶粉喂养(每天母乳喂养次数≤2次)。
1.2.实验方法：
婴儿粪便采集：打开婴儿的纸尿裤，从纸尿裤上收集大便样品，装入采样管中。采样管放入密封袋中，低温冷冻保存，在采集后48小时之内送至实验室。样品收集后分成两份，一份测定粪便16s rRNA测定菌群结构。另一份用厌氧的PBS配制粪便悬浮液，过滤后立刻接种到体外发酵系统中。新鲜粪便放入PBS缓冲液中，震荡混匀，过滤，制成10％粪便悬浮液。
体外发酵实验：该系统操作单元由批量发酵罐与粪便气体检测仪组成。发酵罐内含有无碳源基础培养基(YCFA)。发酵基础培养基不含碳源，仅将低聚果糖(FOS)、低聚半乳糖(GOS)，乳糖(lactose)和2’-岩藻糖基半乳糖(2’-FL),2’-岩藻糖基半乳糖+短双歧杆菌(M16-V)作为唯一碳源和原料分别加入到培养基 中。不加碳水化合物的基础培养基(YCFA)作为对照。37度条件下发酵48小时,实时监测粪便产气情况及气体组成；每隔6小时取样一次，于第48小时测量气体压力，并取样通过16s rRNA测序及宏基因组方法测定菌群构成及数量。以0小时发酵样品菌群结构作为实验的基准线用以对比发酵后菌群的变化。
1.3.实验结果：
结果如图1所示，无论是于母乳喂养婴儿还是奶粉喂养婴儿，粪便菌群发酵2’-FL的产气量都显著低于发酵乳糖、FOS和GOS的产气量，尤其是对于母乳婴儿，2’-FL和2’-FL+短双歧杆菌发酵气体量与对照培养基接近。
母乳喂养婴儿粪便在发酵后产气显著低于奶粉喂养婴儿，证明母乳营养成分的组成与配方奶粉营养成分组成的差别对肠道菌群及粪便发酵代谢产物有显著影响。母乳中存在更适于婴幼儿的低聚糖成分，是母乳喂养婴儿肠道菌群的主要发酵碳水化合物，也是不同喂养方式营养物的重要区别，因此，2’-FL(岩藻糖基半乳糖)作为母乳低聚糖(HMO)的最高含量成分，是控制肠道产气的重要因素。
实施例2：采用HMO干预腹胀婴幼儿肠道菌群发酵
2.1.研究方法：
研究对象:
挑选收集3个月以下腹胀婴儿新鲜粪便生物样本。
腹部胀气多是由饮食引起，婴儿饮食不当引起的腹胀婴儿的判断是基于儿科医生的临床诊断，通过查体和/或腹部影像学检查(B超或立位腹平片)等指标确认，并排除因母亲饮食引发的婴儿偶发性腹胀情况。
2.2.实验方法：
样品采集方法：打开婴儿的纸尿裤，从纸尿裤上收集大便样品，装入采样管中。采样管放入密封袋中，低温冷冻保存，在采集后48小时之内送至实验室。样品收集后分成两份，一份测定粪便16s rRNA测定菌群结构。另一份用厌氧的PBS配制粪便悬浮液，过滤后立刻接种到体外发酵系统中。新鲜粪便放入PBS缓冲液中，震荡混匀，过滤，制成10％粪便悬浮液。
体外发酵实验：该系统操作单元由批量发酵罐与粪便气体检测仪组成。发 酵罐内含有无碳源基础培养基(YCFA)。发酵基础培养基不含碳源，仅将0.8％的低聚果糖(FOS)、低聚半乳糖(GOS)，乳糖(lactose)、2’-岩藻糖基半乳糖(2’-FL)和2’-岩藻糖基半乳糖+短双歧杆菌(M16-V)作为唯一碳源和原料分别加入到培养基中。不加碳水化合物的基础培养基(YCFA)作为对照。在发酵罐中接种粪便悬浮液，在37度条件下发酵24小时,实时监测粪便产气情况及气体组成；菌群结构通过16s rRNA测序及宏基因组方法测定菌群构成及数量。以0小时发酵样品菌群结构作为实验的基准线用以对比发酵后菌群的变化。
2.3.实验结果：
如图2结果显示，对于腹胀婴儿的粪便菌群来说，在培养基底物中添加乳糖、低聚果糖、低聚葡萄糖会进一步提高菌群的产气量。而添加岩藻糖基半乳糖(2’-FL)的培养基与基础培养基组产气量相当，说明2’-FL不为产气菌完全利用，相对于其他碳水化合物，能够显著抑制腹胀婴儿肠道产气增加。
实施例3：2’-FL与肠道菌群以及产气量的关联分析
3.1.研究方法：
将上述实施例1和2中的样品进行菌群16s rDNA测序和宏基因组测序分析肠道菌群。
3.2.实验方法：
将粪便样品用粪便提取试剂盒抽提样品DNA，使用1％琼脂糖凝胶电泳检测DNA条带大小和完整性，并测定基因组浓度、A260/A280等数值。
将样品DNA分别稀释至10ng、1ng、0.1ng作为模板，用细菌基因组16S通用引物27F和1492R扩增所提的样品DNA，同时设置以ddH20为模板的阴性对照，以检测样品基因组提取质量。设置25μL反应体系，反应完成后，1％琼脂糖凝胶电泳检测条带。
对样品DNA进行Hind III限制性酶切，配置20μL酶切反应体系：DNA500ng，Hind III 1μL，10×Buffer 2μL，补ddH20至20μL，置于37℃反应过夜酶切。酶切完成后，将产物进行1％琼脂糖凝胶电泳观察条带差异。
将上述提取样品的DNA，使用Ilumina Hiseq平台测序，利用双末端测序(Paired-End)的方法，构建小片段文库测序。每种DNA样品平行测序两次，挑选两 次平行测序偏差较小的差异化细菌比较丰度数值，以科(Family)水平记录数值差异。
3.3.实验结果：
如图3所示，粪便中双歧杆菌数量越高，产气越低。
肠道菌群与产气关联分析：
如图4所示，宏基因组测序结果发现主要的产气疑似菌包括Clostridium perfringens。
2’-FL促进有益双歧杆菌生长，降低产气荚膜梭菌数量，并且影响肠道内气体的生成量
讨论
对于NEC研究已十分丰富，然而目前主要通过抗生素及手术治疗NEC，还未有通过食物干预来影响NEC的发展，达到预防或减轻NEC症状的方法。
典型的NEC患儿临床表现包括腹胀、呕吐和便血，治疗措施包括禁食、抗生素、支持疗法和外科手术等。针对婴幼儿腹胀，除了使用药物或理疗减轻胃肠内压力、改善肠道微循环促进肠道蠕动以使肠内气体排出以外，最重要及常用的预防和干预手段就是通过饮食干预，如提供接近母乳营养成分的食物或提供有用益生菌等方式来预防或治疗腹胀及其相关的胃肠道疾病。
由于NEC的急发病和高致死率，抑制肠道内产气荚膜梭菌的数量和产气，有利于帮助降低NEC的发生和发展，所以缺乏一种非医学手段来预防NEC的营养接近方案。
使用HMO和益生菌的组合，或益生菌的组合来预防和/或治疗婴幼儿胃肠道产气及其有关的胃肠道病症、腹绞痛和气涨的应用有少量已公开文献曾有报道。然而，现有文献均未涉及婴幼儿母乳化HMOs成分和双歧杆菌组合应用于抑制产气荚膜梭菌，调节肠道内双歧杆菌数量，减少婴幼儿胃肠道气体及其有关的胃肠胀气、腹胀、腹绞痛及其相关病症。
因此，本发明技术方案在本领域有着极佳的应用前景。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申 请所附权利要求书所限定的范围。

Claims (10)

  1. 一种组合的用途，其特征在于，所述组合用于制备一营养组合物或制剂，所述营养组合物用于：
    (a)减少婴幼儿肠道气体的产生；和/或
    (b)抑制婴幼儿肠道产气梭菌的过度生长；
    其中，所述营养组合物中包括：
    (i)一种母乳低聚糖(HMO)，所述HMO包括：2’-岩藻糖基乳糖(2’-FL)、3’-岩藻糖基乳糖(3’-FL)、乳糖-二盐藻四糖(DFL/LDFT)、乳糖-N-盐藻基戊糖I(LNFP-I)、乳糖-N-盐藻基戊糖II(LNFP-II)、乳糖-N-盐藻基戊糖III(LNFP-III)、乳糖-N-四糖(LNT)、乳酰-N-新四糖(LNnT)、3’-唾液乳糖(3SL)、6’-唾液乳糖(6SL)、唾液酸基乳糖基-N-四糖(LST)和/或二唾液酸基乳糖基-N-四糖(DS-LNT)；和
    (ii)一种益生菌微生物，所述益生菌微生物包括双歧杆菌。
  2. 如权利要求1所述的用途，其特征在于，所述婴幼儿是指月龄为0-24个月的人群，优选地为月龄为0-12个月的人群，更优选为月龄为0-6个月的人群。
  3. 如权利要求1所述的用途，其特征在于，所述营养组合物还包括：岩藻糖基化低聚糖、低聚果糖、低聚半乳糖、乳糖，或其组合。
  4. 如权利要求1所述的用途，其特征在于，所述HMO包括：2’-岩藻糖基乳糖(2’-FL)、乳糖，或其组合。
  5. 如权利要求1所述的用途，其特征在于，所述双歧杆菌为长双歧杆菌(Bifidobacterium longum)、短双歧杆菌(Bifidobacterium breve)、婴儿双歧杆菌(Bifidobacterium infantis)、两歧双歧杆菌(Bifidobacterium bifidum)，或其组合。
  6. 如权利要求1所述的用途，其特征在于，所述双歧杆菌是人源的。
  7. 如权利要求1所述的用途，其特征在于，所述益生菌微生物包括瑞士乳杆菌R52(Lactobacillus helveticus R52)、婴儿双歧杆菌R33(Bifidobacterium infantis R33)、两歧双歧杆菌(Bifidobacterium bifidum R71)、短双歧杆菌(Bifidobacterium breve M-16V)，或其组合。
  8. 如权利要求1所述的用途，其特征在于，所述营养组合物中，所述益生菌微生物的浓度为1×10 6-1×10 14CFU/g，较佳地1×10 8-1×10 10CFU/g，更佳地1×10 9-1×10 10CFU/g。
  9. 一种营养组合物，其特征在于，所述营养组合物中包括：
    (i)一种母乳低聚糖(HMO)，所述HMO包括：2’-岩藻糖基乳糖(2’-FL)、3’-岩藻糖基乳糖(3’-FL)、乳糖-二盐藻四糖(DFL/LDFT)、乳糖-N-盐藻基戊糖I(LNFP-I)、乳糖-N-盐藻基戊糖II(LNFP-II)、乳糖-N-盐藻基戊糖III(LNFP-III)、乳糖-N-四糖(LNT)、乳酰-N-新四糖(LNnT)、3’-唾液乳糖(3SL)、6’-唾液乳糖(6SL)、唾液酸基乳糖基-N-四糖(LST)和/或二唾液酸基乳糖基-N-四糖(DS-LNT)；和
    (ii)一种益生菌微生物，所述益生菌微生物包括双歧杆菌。
  10. 一种制备如权利要求2所述的营养组合物的方法，其特征在于，包括步骤：将以下组分(i)和组分(ii)进行混合，从而得到如权利要求2所述的营养组合物；
    (i)一种母乳低聚糖(HMO)，所述HMO包括：2’-岩藻糖基乳糖(2’-FL)、3’-岩藻糖基乳糖(3’-FL)、乳糖-二盐藻四糖(DFL/LDFT)、乳糖-N-盐藻基戊糖I(LNFP-I)、乳糖-N-盐藻基戊糖II(LNFP-II)、乳糖-N-盐藻基戊糖III(LNFP-III)、乳糖-N-四糖(LNT)、乳酰-N-新四糖(LNnT)、3’-唾液乳糖(3SL)、6’-唾液乳糖(6SL)、唾液酸基乳糖基-N-四糖(LST)和/或二唾液酸基乳糖基-N-四糖(DS-LNT)；和
    (ii)一种益生菌微生物，所述益生菌微生物包括双歧杆菌。
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CN112890200B (zh) * 2021-03-08 2021-11-12 合生元（广州）健康产品有限公司 用于促进低体重婴幼儿生长追赶的一种营养组合物
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