WO2016141812A1 - 核苷酸组合物及其在食品中的应用 - Google Patents

核苷酸组合物及其在食品中的应用 Download PDF

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Publication number
WO2016141812A1
WO2016141812A1 PCT/CN2016/074813 CN2016074813W WO2016141812A1 WO 2016141812 A1 WO2016141812 A1 WO 2016141812A1 CN 2016074813 W CN2016074813 W CN 2016074813W WO 2016141812 A1 WO2016141812 A1 WO 2016141812A1
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Prior art keywords
cmp
ump
gmp
composition
nucleotide
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PCT/CN2016/074813
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English (en)
French (fr)
Inventor
方芳
李婷
赵子夫
齐炳理
司徒文佑
李威
刘彪
赵红霞
陈青山
Original Assignee
内蒙古伊利实业集团股份有限公司
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Priority to MYPI2017703243A priority Critical patent/MY186145A/en
Priority to ES16761039T priority patent/ES2805825T3/es
Priority to SG11201707213PA priority patent/SG11201707213PA/en
Priority to NZ735399A priority patent/NZ735399B2/en
Priority to DK16761039.3T priority patent/DK3266310T3/da
Priority to US15/555,867 priority patent/US20180049460A1/en
Priority to CN201680013841.3A priority patent/CN107529809B/zh
Priority to AU2016228670A priority patent/AU2016228670B2/en
Priority to EP16761039.3A priority patent/EP3266310B1/en
Publication of WO2016141812A1 publication Critical patent/WO2016141812A1/zh

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    • 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
    • A23C9/1526—Amino acids; Peptides; Protein hydrolysates; Nucleic acids; Derivatives thereof
    • 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
    • A23L33/13—Nucleic acids or derivatives thereof
    • 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
    • A23C9/16—Agglomerating or granulating milk powder; Making instant milk powder; Products obtained thereby
    • 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

Definitions

  • the present invention relates to a nucleotide composition for use as a food additive. Still further, the invention also relates to a food product comprising the nucleotide composition and the use of the nucleotide composition in the preparation of a food product.
  • nucleotides in organisms, such as nucleotides, which can enhance the body's immune system and the function of the gastrointestinal system.
  • milk-based formulas are an important food supplement source for infants and young children. Because nucleotides and their derivatives are low in cow's milk, exogenous nucleotides are added to milk-based formulas. Among them, it has important significance for the growth and development of the body, especially infants and young children (such as gastrointestinal development) and immunity.
  • the inventors conducted extensive experiments to adjust the nucleotides in the nucleic acid composition.
  • the composition and ratio of the components are maximized to maximize the effect of the composition as a nucleotide supplement.
  • the inventors have surprisingly found that, with respect to the products used in the prior art, by the specific composition and ratio of the nucleotide composition of the present invention, it is possible to provide more Good results.
  • the invention relates to a nucleotide composition for use as a food additive.
  • the nucleotide composition of the invention consists essentially of CMP, AMP, UMP, GMP, and IMP or consists of CMP, AMP, UMP, GMP, and IMP, wherein the ratio of components is by weight. : CMP: 58 to 70%, AMP: 7.5 to 12.5%, UMP: 12 to 16.5%, GMP: 10 to 13%, and IMP: 0 to 2.5%, provided that the sum of the various components is 100%.
  • the nucleotide composition of the invention consists essentially of CMP, AMP, UMP, GMP and IMP or consists of CMP, AMP, UMP, GMP and IMP, wherein the proportion of each component by weight It is: CMP: 60 to 65%, AMP: 8 to 12%, UMP: 14 to 16%, GMP: 11 to 12%, and IMP: 0 to 2%, provided that the sum of the various components is 100%.
  • the nucleotide composition of the invention consists essentially of CMP, AMP, UMP and GMP or consists of CMP, AMP, UMP and GMP, wherein the ratio of components is by weight: CMP: 58 to 72%, AMP: 6 to 14%, UMP: 10 to 18%, and GMP: 8 to 14%, provided that the sum of the various components is 100%.
  • the nucleotide composition of the invention consists essentially of CMP, AMP, UMP and GMP or consists of CMP, AMP, UMP and GMP, wherein the ratio of components is by weight: CMP: 60 to 70%, AMP: 8 to 12%, UMP: 12 to 16%, and GMP: 10 to 12%, provided that the sum of the various components is 100%.
  • the nucleotide composition of the invention consists essentially of CMP, AMP, UMP and GMP or consists of CMP, AMP, UMP and GMP, wherein the ratio of components is by weight: CMP: 60 to 65%, AMP: 10 to 12%, UMP: 14 to 16%, and GMP: 11 to 12%, provided that the sum of the various components is 100%.
  • the nucleotide composition of the invention consists essentially of CMP, AMP, UMP and GMP or consists of CMP, AMP, UMP and GMP, wherein the ratio of components is by weight: CMP: 65 to 70%, AMP: 8 to 10%, UMP: 12 to 14%, and GMP: 10 to 11%, provided that the sum of the various components is 100%.
  • the nucleotide composition of the invention consists essentially of CMP, AMP, UMP and GMP or consists of CMP, AMP, UMP and GMP, wherein the ratio of components is by weight: CMP: 60%, AMP: 12%, UMP: 16%, and GMP: 12%.
  • the nucleotide composition of the invention consists essentially of CMP, AMP, UMP and GMP or consists of CMP, AMP, UMP and GMP, wherein the ratio of components is by weight: CMP: 65%, AMP: 10%, UMP: 14%, and GMP: 11%.
  • the nucleotide composition of the invention consists essentially of CMP, AMP, UMP and GMP or consists of CMP, AMP, UMP and GMP, wherein the ratio of components is by weight: CMP: 70%, AMP: 8%, UMP: 12%, and GMP: 10%.
  • the food product is an infant food, and more preferably, the food product is in the form of a dairy product, such as a milk powder or a liquid dairy product, such as for infants and toddlers. Milk powder or liquid dairy products.
  • the invention relates to a food product comprising a nucleotide composition of the invention.
  • the food product is an infant food.
  • the food product is in the form of a dairy product, such as in the form of a milk powder or a liquid dairy product, such as a milk powder or liquid dairy product for use by an infant.
  • the invention also relates to a method of preparing a food product, the method comprising adding a nucleotide composition of the invention to a raw material of the food product.
  • the food product is an infant food.
  • the food product is in the form of a dairy product, such as in the form of a milk powder or a liquid dairy product, such as a milk powder or liquid dairy product for use by an infant.
  • the invention also relates to the use of a nucleotide composition of the invention in the preparation of a food product.
  • the food product is an infant food.
  • the food product is in the form of a dairy product, such as in the form of a milk powder or a liquid dairy product, such as a milk powder or liquid dairy product for use by an infant.
  • the food product is used to provide an immunostimulatory effect.
  • the food product is used to promote growth and development (e.g., gastrointestinal development) and to promote repair after intestinal damage (e.g., to promote repair of cells, particularly intestinal epithelial cells).
  • the food product is used to promote the growth of intestinal beneficial microorganisms.
  • the present invention is also directed to enhancing immunity of an individual, promoting growth and development (such as gastrointestinal development), and promoting repair after intestinal damage (for example, promoting repair of cells, particularly intestinal epithelial cells, such as by oxidation) A method of causing damage) and/or promoting growth of an intestinal beneficial microorganism of an individual and/or any combination thereof, the method comprising administering to the individual a nucleotide composition or food of the invention.
  • the individual is a human, preferably a human infant.
  • Fig. 1 is a graph showing the results of cell survival experiments.
  • Fig. 2 is a graph showing the results of the SOD activity experiment.
  • Fig. 3 is a graph showing the results of an LDH activity experiment.
  • Fig. 4 is a graph showing the results of an MDA content experiment.
  • Fig. 5 is a graph showing the results of a cell proliferation experiment.
  • nucleotide refers to a compound consisting of a purine or pyrimidine base, ribose or deoxyribose, and a phosphate group.
  • nucleotides can be classified into ribonucleotides and deoxynucleotides depending on the sugar.
  • nucleotides can be divided into adenine nucleotides, guanine nucleotides, cytosine nucleotides, uracil nucleotides, thymidine nucleotides, and hypoxanthine nucleotides. Wait.
  • NMP nucleoside diphosphate
  • NTP nucleoside triphosphate
  • nucleotide as used herein also encompasses cytosine (C), uracil (U), adenine (A), and guanine (G) which are present in various forms in the nucleotide compositions of the present invention.
  • And/or hypoxanthine (I) such as ribonucleosides, ribonucleotides, RNA phosphates, and any other form of derivative or precursor, as long as it is capable of being transformed or metabolized to the corresponding nucleotide in vivo or in vitro. form.
  • the nucleotides used are mainly CMP (cytosine nucleotide), UMP (uracil nucleotide), AMP (adenine nucleotide), GMP (guanine nucleotide), IMP (hypoxanthine nucleotide) and the like.
  • CMP cytosine nucleotide
  • UMP uracil nucleotide
  • AMP adenine nucleotide
  • GMP guanine nucleotide
  • IMP hypoxanthine nucleotide
  • 5'-mixed nucleotides currently on the market contain 5'-adenylate (AMP), 5'-cytosine (CMP), 5'-guanylic acid (GMP), 5'-uridine. Acid (UMP) and 5'-inosinic acid (IMP).
  • such 5'-mixed nucleotides may, for example, be present in two types when used in combination: one is that 5'-adenylate and 5'-cytidine are present in the form of a free acid, and
  • the other three nucleotides exist in the form of a sodium salt, abbreviated as the trisodium diacid type; the other is in the form of a sodium salt, referred to as pentasodium type.
  • nucleotide as used herein also encompasses the form of its salt, such as an alkali metal or alkaline earth metal salt such as a sodium salt, a potassium salt or a calcium salt, etc., such as a monosodium salt or a disodium salt, etc., such as CMPNa. 2 , AMPNa 2 , UMPNa 2 , GMPNa 2 , IMPNa 2 and CMPK 2 , AMPK 2 , UMPK 2 , GMPK, IMPK 2 and the like.
  • an alkali metal or alkaline earth metal salt such as a sodium salt, a potassium salt or a calcium salt, etc.
  • a monosodium salt or a disodium salt etc.
  • CMPNa. 2 AMPNa 2 , UMPNa 2 , GMPNa 2 , IMPNa 2 and CMPK 2 , AMPK 2 , UMPK 2 , GMPK, IMPK 2 and the
  • each component of the nucleotide compositions of the present invention may each optionally and independently present in various salt forms, including but not limited to the above mentioned “sodium trisodium phosphate" Or the individual components and salts thereof may be present in any combination, for example, only GMP is present in the form of a salt, only CMP is present in the form of a salt, and the like. Alternatively, each component of the nucleotide composition of the present invention may be present in the form of a salt, including but not limited to the "penta-sodium form" mentioned above.
  • nucleotide as used herein also encompasses solvates (such as hydrates) in various forms thereof.
  • the ratio of these forms in the composition should be calculated after conversion to their corresponding nucleotide molecules. For example, when CMP is present in its disodium salt form (CMPNa 2 ), it should be converted to CMP to calculate the corresponding weight ratio.
  • CMPNa 2 disodium salt form
  • the nucleotide composition of the invention consists essentially of or consists of CMP, AMP, UMP, GMP, and IMP. In another embodiment, the nucleotide composition of the invention consists essentially of or consists of CMP, AMP, UMP and GMP.
  • CMP comprises from 58 to 72%, preferably from 60 to 70%, such as from 60 to 65% and from 65 to 70% by weight of the nucleotide composition of the invention. It should be understood that these ranges cover all of the point values such as, but not limited to, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70 %, 71%, etc.; and various sub-ranges composed of these point values, such as, but not limited to, 60 to 63%, 60 to 68%, 63 to 70%, and the like. In a particular embodiment, CMP comprises 60%, 65% or 70% by weight of the nucleotide composition of the invention.
  • AMP comprises from 6 to 14%, preferably from 8 to 12%, such as from 8 to 10% and from 10 to 12% by weight of the nucleotide composition of the invention. It should be understood that these ranges encompass all of the point values, such as, but not limited to, 7%, 8%, 9%, 10%, 11%, 12%, 13%, etc.; and various sub-ranges consisting of these point values, For example, it is not limited to 8 to 9%, 8 to 11%, 9 to 12%, and the like. In a particular embodiment, the AMP comprises 8%, 10% or 12% by weight of the nucleotide composition of the invention.
  • UMP is in the nucleotide composition of the invention by weight 10 to 18%, preferably 12 to 16%, for example 12 to 14% and 14 to 16%. It should be understood that these ranges encompass all of the point values, such as but not limited to 11%, 12%, 13%, 14%, 15%, 16%, 17%, etc.; and various sub-ranges consisting of these point values, For example, it is not limited to 12 to 15%, 14 to 15%, and the like. In a particular embodiment, UMP comprises 12%, 14% or 16% by weight of the nucleotide composition of the invention.
  • GMP comprises from 8 to 14%, preferably from 10 to 12%, such as from 10 to 11% and from 11 to 12% by weight of the nucleotide composition of the invention. It should be understood that these ranges encompass all of the point values, such as, but not limited to, 9%, 10%, 11%, 12%, 13%, etc.; and various sub-ranges consisting of these point values, such as, but not limited to, 8 11%, 10 to 14%, etc. In a particular embodiment, GMP comprises 10%, 11% or 12% by weight of the nucleotide composition of the invention.
  • the IMP comprises from 0 to 2.5%, preferably from 0 to 2%, more preferably 0% by weight of the nucleotide composition of the invention (ie, the nucleotide composition of the invention is not With IMP). It should be understood that these ranges encompass all of the point values therein, such as, but not limited to, 1%, 2%, etc.; and various sub-ranges consisting of these point values, such as, but not limited to, 0 to 1%, 1-2%, and the like.
  • the above-listed ranges and specific values for the various components may be arbitrarily combined and selected with each other as long as such a combination satisfies the condition that the sum of the selected components is 100%.
  • the content of CMP when the content of CMP is 60 to 70%, such as 60 to 65% or 65 to 70%, the content of AMP may be 8 to 12%, such as 8 to 10% or 10 ⁇ 12%.
  • the content of UMP when the content of CMP and AMP is in any of the above ranges, the content of UMP may be 12 to 16%, such as 12 to 14% or 14 to 16%.
  • the content of GMP may be 10 to 12%, such as 10 to 11% or 11 to 12%.
  • the content of IMP may be 0 to 2.5%, such as 0 to 2% or 0% (ie, the nucleotide composition of the present invention is not With IMP).
  • the expression "consisting essentially of” as used herein means that, in addition to the defined nucleotide component, the nucleotide composition of the present invention may optionally comprise a food or physiologically acceptable carrier, if desired.
  • Excipients or adjuvants such as preservatives, antioxidants, binders, thickeners, diluents and the like, as well as various impurities which may be present.
  • These carriers, excipients or adjuvants and impurities are inert to the active ingredient (nucleotide component) in the composition, and the presence or amount thereof does not interfere with or significantly interfere with the function of the active ingredient.
  • nucleotide composition of the present invention may or may not contain the above-mentioned carrier, excipient or adjuvant.
  • carrier, excipient or adjuvant means that the nucleotide composition of the present invention may or may not contain the above-mentioned carrier, excipient or adjuvant.
  • food as used herein has the meaning commonly understood by one of ordinary skill in the art, for example, it refers to materials that are available for human consumption or consumption, including processed foods, semi-finished and unprocessed foods, and the like.
  • infant food generally refers to a food intended for infants other than breast milk, which usually adds various ingredients such as nucleotides, fatty acids, vitamins, carbohydrates, vegetable oils for the growth and development of infants and young children.
  • a trace element or the like for example, a nucleotide composition of the present invention.
  • examples of such foods are dairy products (such as milk powder and liquid dairy products), purees, rice flour, and the like.
  • the infant food is in the form of a dairy product, such as a milk powder or a liquid dairy product, such as an infant formula or a liquid dairy product to which the nucleotide composition of the invention is added.
  • infant and toddler generally refers to a human individual from 0 to 3 years of age.
  • the use of the nucleotide composition of the present invention is not limited to this age group.
  • the nucleotide composition of the present invention or a food product comprising the nucleotide composition can be administered to a human subject of a greater age, such as 4 years old, 5 years old, 6 years old, 7 years old, 8 years old, 9 Years old, 10 years old or older.
  • the present invention also provides a method of preparing a food product comprising adding a nucleotide composition of the present invention to the food product.
  • the food product is an infant food.
  • the food product is in the form of a dairy product, such as a milk powder or a liquid dairy product, such as a milk powder or liquid dairy product for use in infants and young children.
  • liquid dairy product and “liquid milk” or “liquid milk” have the same meaning and are used interchangeably and are intended to mean a dairy product in liquid form, which contains various types for use by the human body. Nutrients and energy, such as those shown below.
  • the nucleotide composition of the present invention is used in the preparation of the milk powder of the present invention in an amount of from 0.2 to 0.58 parts by weight based on 1000 parts by weight of the milk powder.
  • the milk powder of the present invention comprises: 120 to 160 parts by weight of skim milk powder and 240 to 280 of lactose based on 1000 parts by weight of the milk powder.
  • whey protein powder 180-210 parts by weight, whey protein powder (WPC 34%) 90-120 parts by weight, sunflower oil 155-180 parts by weight, corn oil 35-55 parts by weight, soybean oil 40-60 weight A portion, 0.35 to 0.5 parts by weight of the nucleotide composition of the present invention, 1 to 2.5 parts by weight of soybean phospholipid, 0.1 to 0.15 parts by weight of bifidobacteria, 4 to 5 parts by weight of oligofructose powder, and 10 to 5 parts by weight of oligogalactose pulp. 12 parts by weight, a nutritionally acceptable amount of vitamins and a nutritionally acceptable amount of trace element supplement.
  • the main preparation process of the milk powder of the present invention comprises: compounding, preheating, homogenizing, concentrated sterilization, spray drying, dry mixing, and then obtaining a final product, wherein the nucleotide composition of the present invention is It is added to the spray-dried milk powder together with DHA, ARA and Bifidobacteria, and then mixed.
  • the liquid dairy product of the present invention comprises the following components (per 100 g of liquid dairy product): 1.75 g to 4.26 g of protein other than lactoferrin, 1.75 g to 4.97 g of fat, 250 kJ to 355 kJ of energy, vitamins A 42.5 ⁇ g ⁇ 191.7 ⁇ g RE, vitamin D 0.625 ⁇ g ⁇ 2.6625 ⁇ g, vitamin E ⁇ 0.375mg ⁇ -TE, vitamin K1 ⁇ 2.5 ⁇ g, vitamin B1 ⁇ 27.5 ⁇ g, vitamin B2 ⁇ 27.5 ⁇ g, vitamin B6 ⁇ 27.5 ⁇ g, vitamin B12 ⁇ 0.1 ⁇ g, niacin (or nicotinamide) ⁇ 275 ⁇ g, folic acid ⁇ 2.5 ⁇ g, pantothenic acid ⁇ 175 ⁇ g, vitamin C ⁇ 4.5mg, biotin ⁇ 1 ⁇ g, sodium ⁇ 71mg, potassium 45mg ⁇ 244.95mg, copper 17.5 ⁇ g ⁇ 124.25 ⁇ g , magnesium ⁇ 3.5mg, iron 0.625
  • the fat used is provided in one or more of anhydrous butter, soybean oil, corn oil, sunflower seeds in any ratio and combination.
  • some food-acceptable food additives such as an emulsion stabilizer.
  • the food additive and amount preferably used are, for example but not limited to, carrageenan 0.005 wt% to 0.05 wt%, glyceryl monostearate 0.01 wt% to 1 wt%, guar gum 0.01 wt% to 0.1.
  • carrageenan 0.005 wt% to 0.05 wt%
  • glyceryl monostearate 0.01 wt% to 1 wt%
  • guar gum 0.01 wt% to 0.1.
  • the invention also relates to the use of the nucleotide composition of the invention in the preparation of a food product.
  • the food product is an infant food.
  • the food product is in the form of a dairy product, such as a milk powder or a liquid dairy product, such as a milk powder or liquid dairy product for use in infants and young children.
  • the nucleotide composition of the invention and the food product comprising the composition provide an immunostimulatory effect upon consumption.
  • the "immunostimulation" effect described herein refers to the effect of improving the immune function of an individual, for example, in improving lymphocyte transformation function, enhancing macrophage phagocytosis, enhancing NK cell activity, and improving immune response and antibody production, such as Those effects provided by the Examples section of the specification.
  • the individual is a human, preferably a human infant.
  • the nucleotide composition of the present invention and the food containing the composition can promote the growth and development of the individual (e.g., gastrointestinal development) and promote the repair after intestinal damage. For example, it promotes the growth and maturation of cells in the intestinal tract (such as the small intestine), repairs damage to the intestinal tract (such as the small intestine), protects the cells of the intestinal tract (such as the small intestine) from free radical attacks, and reduces inflammation of the intestinal tract (such as the small intestine). Occurs.
  • the nucleotide composition of the present invention can promote the growth of intestinal epithelial cells, promote the proliferation of intestinal epithelial cells, and have a protective effect on intestinal epithelial cells and hepatocytes.
  • damage may, for example, be oxidative damage, such as damage caused by reactive oxygen species.
  • the individual is a human, preferably a human infant.
  • the nucleotide composition of the present invention and the food containing the composition can promote the growth of the intestinal beneficial microorganisms (such as bifidobacteria and lactobacilli) of the individual after consumption, for example, in favor of the growth of bifidobacteria, For example, increase the content of bifidobacteria and lactobacilli in the intestines and feces (these flora can inhibit the growth of anaerobic pathogens and Escherichia coli), reduce the proportion of harmful bacteria in the intestines, and thus benefit the health of the individual.
  • the intestinal beneficial microorganisms such as bifidobacteria and lactobacilli
  • the composition is added in the intestinal environment or in an in vitro environment similar to the intestinal environment, and the number of probiotics and lactic acid bacteria in the environment, especially the proliferation of bifidobacteria, is found.
  • the nucleotide composition of the invention exhibits a significant growth promoting effect on intestinal beneficial microorganisms.
  • the individual is a human, preferably a human infant.
  • composition and composition of the test samples used in the examples of the present application are as follows (by weight):
  • nucleotide composition of the present application (see also claim 5):
  • Comparative sample 1 CMP: 33.7%, AMP: 20.3%, UMP: 23.1%, GMP: 7.6%, and IMP: 15.3%
  • the main reagents used in this example are: RPMI-1640 medium (Gibco); fetal bovine serum (Gibco); concanavalin A (ConA) (Sigma); lymphocyte separation solution, whole blood and tissue dilution, cells Washing liquid (Tianjin Yuyang Biological Products Technology Co., Ltd.); 5% chicken red cell suspension (laboratory preparation); MTT kit, Hank's solution (Biyuntian Biotechnology Research Institute); Giemsa dyeing solution (Zhuhaibei) Suo Biotechnology Co., Ltd.; EDTA anticoagulant tube (BD); 96-well cell culture plate (Corning); Baicheng brand bovine colostrum capsule (Shanghai Fuzheng Biotechnology Co., Ltd.); nucleotide sample (Nanjing Tongkai Zhao) industry).
  • CO 2 incubator Sanyo MCO-18AIC (UV)); ultra clean bench (AIRTECH); inverted microscope (OLYMPUS); Hitachi horizontal centrifuge (himac-CT6EL); microplate reader (BIO-RAD Model 680); OLYMPUS microscope (BX51); DNP constant temperature incubator (Shanghai Jinghong Experimental Equipment Co., Ltd.).
  • mice After adaptively feeding KM mice for 7 days, they were randomly divided into 8 groups, 20 in each group, namely, blank control group, positive control group, high, medium and low dose groups of sample 1, and high of comparative sample 1. Medium and low dose groups. Each group of animals was intragastrically dissolved with sterile water of each sample (see below), and the blank control group was intragastrically administered with the same volume of sterile water. Once a day, feed or gavage for 30 days.
  • the dose in the high dose group was 67.37 mg ⁇ kg -1 ⁇ d -1
  • the dose in the middle dose group was 41.82 mg ⁇ kg -1 ⁇ d -1
  • the dose in the low dose group was 23.23 mg ⁇ kg -1 . d -1
  • the dose of the positive control group was 150.17 mg ⁇ kg -1 ⁇ d -1 .
  • Positive control group bovine colostrum capsules.
  • Sample 1 is a nucleotide composition of the present invention.
  • Comparative Sample 1 is a nucleotide composition prepared according to the nucleotide components and ratios used in a commercially available nucleotide-added infant food.
  • Lymphocyte transformation experiments On the 38th day of the experiment, 10 mice were randomly selected, 2mL EDTA anticoagulant tube was used, eyeballs were removed, and lymphocytes were aseptically isolated by Ficoll density gradient centrifugation. Take 1 mL of fresh anticoagulant, mix it with whole blood and tissue dilution 1:1, then carefully add to the surface of an equal volume of lymphocyte separation. The cells were centrifuged at 1500 rpm for 15 minutes in a horizontal centrifuge, and a ring-shaped milky white lymphocyte layer was collected, which was washed twice with a cell washing solution.
  • mice were randomly selected, and each mouse was intraperitoneally injected with 5% chicken red blood cell suspension 1 mL. After 30 minutes, the mice were sacrificed by cervical dislocation. Inject 1 mL of normal saline into the abdominal cavity, gently rub the abdomen of the mouse for 1 minute, cut the skin of the abdominal wall, open on the muscle layer, and pipette into the abdominal cavity to absorb 1 mL of the peritoneal fluid, and drop it on a clean glass slide. The slides were placed in an enamel box with wet gauze and then incubated in a 37 ° C incubator for 30 minutes.
  • the SI values of the high, medium and low dose groups of sample 1 were significantly higher than the blank control group (p ⁇ 0.05), and were dose-dependent; the SI value of the high dose group of the comparative sample 1 was significantly higher than that of the sample 1
  • nucleotide composition of the present invention can significantly enhance the lymphocyte proliferation function of mice, and the effect is superior to the comparative sample.
  • the percentages of phagocytosis and phagocytic index of the high, medium and low dose groups of sample 1 were significantly higher than those of the blank control group (p ⁇ 0.05); the percentage of phagocytosis and phagocytic index of the high dose group of the comparative sample 1 were significantly higher than that of the control group.
  • the blank control group (p ⁇ 0.05), the percentage of phagocytosis and the phagocytic index of the middle and low dose groups of the comparison sample 1 were not significantly different from the blank control group (p>0.05); the percentage of phagocytosis and the phagocytic index of the low dose group of sample 1 were significant. Higher than the middle dose group of Comparative Sample 1 (p ⁇ 0.05).
  • nucleotide composition of the present invention can significantly increase the phagocytosis function of mouse peritoneal macrophages, and the effect is superior to the comparative sample.
  • the nucleotide composition of the present invention can significantly improve lymphocyte transformation function, enhance the phagocytosis function of peritoneal macrophages, and has an immune function, and the immunomodulatory effect is significantly higher than that of the comparative sample.
  • Freezer high speed centrifuge (SIGMA 3-30K), oscillator (Vortex4digital), sample gun (Eppendorf, Germany), whole blood cell analyzer (Ultra U-2900PLUS), carbon dioxide incubator (Thermo311), microplate reader (Biotek H4), spectrophotometer (Shanghai Precision Instrument Science Instrument Co., Ltd. 722s), ultra-clean workbench (Shangyu Star Instrument Equipment Co., Ltd. SW-CJ-2D), autoclave (Japan Sanyo MLS-3781-PC 75L) , low temperature refrigerator (Hangzhou Aipu Instrument Equipment Co., Ltd.
  • Cyclophosphamide (Jiangsu Hengrui Pharmaceutical Co., Ltd.), dipotassium edetate (Beijing Reagent Factory), RpMI1640 cell culture solution (Shanghai Yuanlong Biotechnology Co., Ltd.), calf serum, 2-mercaptoethanol, penicillin , Streptomycin, ConA solution (Beijing Mengyimei Biotechnology Co., Ltd.), sterile Hank's solution, BrdUp labeling solution (Beijing Mengyimei Biotechnology Co., Ltd.), dinitrofluorobenzene (Beijing Qingshengda Chemical Technology) Ltd.), strontium sulfide, sheep red blood cells (SRBC), guinea pig serum, Indian ink (Shanghai Yuanmu Biotechnology Co., Ltd.), YAC-1 cells (Shanghai Enzyme Research Biotechnology Co., Ltd.), Tris-HCL buffer (Beijing Dream) Yimei Biotechnology Co.,
  • a total of 770 animals were divided into 5 large immunization groups for feeding.
  • Each of the 154 large immunized groups was randomly divided into 11 groups (5 nucleotide samples in high and low dose groups, 10 groups in total, 1 group as control group) , 14 in each group.
  • Immunization group 1 delayed type hypersensitivity
  • immunization group 2 mouse lymphocyte transformation test, NK cell activity measurement, and visceral ratio
  • immunization group 3 half of hemolysis value, antibody-producing cell number
  • immunization group 4 carbon clearance test
  • Immunization group 5 mouse peritoneal macrophage phagocytosis fluorescent microsphere test.
  • Each sample was administered by gavage for 28 days, once a day, and the volume of the gavage was 0.2 mL/10 g.
  • the control group was given distilled water by gavage. At the end of the administration, the mice were sacrificed and various immunological indexes were determined.
  • Samples 1-3 are the nucleotide compositions of the present application. Comparative Samples 2 and 3 are the other two nucleotide compositions disclosed in the prior art. Two samples of low and high doses were set for each sample, which were 120.7 mg/kg and 1207.0 mg/kg, respectively.
  • Organ/body weight ratio The mice were weighed and weighed as initial weight and final body weight after 28 days of initial administration. The mice were dislocated and sacrificed. The spleen and thymus were taken, and the fascia was removed. The surface of the organ was stained with filter paper, weighed, and the spleen/body weight ratio and thymus/body weight ratio were calculated.
  • mice After 28 days of continuous administration, the mice were sacrificed and disinfected in a 75% alcohol beaker. The spleen was aseptically placed and placed in a 3 cm x 3 cm area. In a small layer of four-layer gauze (autoclaved), add appropriate amount of sterile Hank's solution, wrap the spleen with gauze, and gently grind the spleen with an elbow to make a single cell suspension. It was washed twice with Hank's solution and centrifuged at 1000 rpm for 10 min each time.
  • MTT method ConA-induced lymphocyte transformation in mice
  • the cells were suspended in 2 mL of the complete culture solution, and the number of viable cells was counted, and the cell concentration was adjusted to 5 ⁇ 10 6 /mL.
  • the cell suspension was further divided into two wells and added to a 24-well culture plate, 1 mL per well, and 75 ⁇ L of ConA solution (corresponding to 7.5 ⁇ g/mL) was added to one well, and the other well was used as a control. Incubate for 72 h at 37 ° C with 5% CO 2 .
  • Antibody-producing cell test After 28 days of continuous administration, the sheep blood was taken and washed with physiological saline for 3 times. Each rat was immunized by intraperitoneal injection of 2% (V/V, prepared with physiological saline) and SRBC 0.2 mL. . Mice immunized 4 days after SRBC immunization were sacrificed, and spleens were taken to prepare a cell suspension of 5 ⁇ 10 6 cells/mL.
  • Serum hemolysin half hemolysis value (HC 50 ): After 28 days of continuous administration, the sheep blood was taken and washed three times with normal saline. Each mouse was intraperitoneally injected with 2% (V/V, prepared with physiological saline). The SRBC 0.2 mL was immunized. After 4 days, the eyeballs were removed and blood was taken in a 1.5 mL centrifuge tube, and placed at 4 ° C for about 1 h. The serum was analyzed by centrifugation, centrifuged at 2000 rpm for 10 min, and serum was collected. Serum was taken and diluted 100-fold with SA buffer.
  • the diluted serum was added to a 96-well plate at 100 ⁇ L per well, followed by 10% (v/v) SRBC 50 ⁇ L, 100 ⁇ L of complement (diluted 1:8 with SA solution), and incubated in a constant temperature water bath at 37 ° C for 30 min. Centrifuge at 1500 rpm for 10 min. Then, 50 ⁇ L of the supernatant was taken from each of the sample well and the blank control well, and added to another 96-well culture plate, and 150 ⁇ L of Gavinius reagent was added. At the same time, half of the hemolytic holes were set, and 10% (v/v) SRBC 12.5 ⁇ L was added, and then Wenwen's reagent was added to 200 ⁇ L. The mixture was thoroughly mixed with an oscillator, and after standing for 10 minutes, the optical density value of each well was measured at 540 nm using a fully automatic microplate reader.
  • the amount of hemolysin is expressed as half of the hemolysis value (HC 50 ) and is calculated according to the following formula:
  • Sample HC 50 (sample optical density value / optical density value at half half of SRBC hemolysis) ⁇ dilution factor
  • mice were intraperitoneally injected with 0.2 mL of 2% SRBC to activate mouse macrophages 4 days before the end of the gavage. On the day of the experiment, the mice were sacrificed by cervical dislocation. The Hank's solution containing calf serum was intraperitoneally injected with 3 mL/s of calf serum, and gently rubbed into the abdomen 20 times to fully wash out the peritoneal macrophages.
  • the abdominal wall was cut into a small mouth, 2 mL of the peritoneal washing solution was aspirated, and the mixture was filtered into a test tube with a 75 ⁇ m filter to adjust the number of macrophages to 4 to 6 ⁇ 10 5 /mL.
  • NK cell activity (Lactate dehydrogenase (LDH) assay) : Animals were administered continuously for 28 days, and the target cells YAC-1 were subcultured 24 hours before the start of the experiment, and washed twice with Hank's solution before use. The cell concentration was adjusted to 1 ⁇ 10 5 /mL (target cells) using RPMI 1640 complete medium containing 10% calf serum. The mice were sacrificed by cervical dislocation and aseptically taken from the spleen. The spleen cell suspension was prepared, washed twice with Hank's solution, centrifuged at 1000 rpm for 10 min, and resuspended in 2 mL of RPMI 1640 complete medium containing 10% calf serum.
  • LDH lactate dehydrogenase
  • Trypan blue viable cell staining count (the number of viable cells should be above 95%), adjust the cell concentration to 1 ⁇ 10 7 /mL (effector cells), so that the ratio of effector cells to target cells is 100:1.
  • 100 ⁇ L of the target cells and effector cells were added to the U-shaped 96-well culture plate, and the target cells were naturally released from the wells plus 100 ⁇ L of the target cells and the culture medium, and the target cells were maximally released and the target cells and 100% of the 1% NP40 were set.
  • Three parallel wells were incubated for 4 hours at 37 ° C in a 5% CO 2 incubator.
  • the 96-well culture plate was centrifuged at 1500 rpm for 5 min, and the supernatant was aspirated into a 100 ⁇ L flat-bottomed 96-well culture plate. At the same time, 100 ⁇ L of LDH substrate solution was added for 3 min, and then 30 ⁇ L of 1 mol/L HCL solution was added to each well to terminate the reaction. The OD value was measured at 490 nm, and the NK activity was calculated as follows:
  • Both of the experimental results in the mononuclear-macrophage function test program were positive, or the results of the two dose groups in either experiment were positive, and the mononuclear-macrophage function was positive.
  • the results of more than one dose group in the NK cell activity assay were positive, and the NK cell activity result was positive.
  • the nucleotide composition of the present invention has more immunostimulatory positive indicators from the aspect of the above (1.6) dose group, thereby exhibiting stronger And a more comprehensive immune stimulating effect.
  • sample 1 has cellular immunity, other samples do not have; 2) sample 1 has humoral immunity, other samples do not have; 3) samples 1 and 2 are positive under carbon clearance test indicators, and other samples do not; 4 Samples 2, 3 and Comparative Sample 3 had macrophage phagocytosis, and other samples did not; 5) Samples 1, 2, 3 and Comparative Sample 3 were positive for NK cell activity.
  • nucleotide composition of the present invention can effectively stimulate immune function
  • the effect is good and the effect is superior to the prior art compositions.
  • a stronger immunomodulatory effect means that the same or similar effects can be achieved with less dose, which has a significant economic cost advantage for large scale industrial production.
  • the purpose of this example was to demonstrate the effect of the nucleotide composition of the present invention in providing protection and repair against damage.
  • RPMI-1640 medium and DMEM high sugar medium were purchased from Gibco.
  • 5'-AMP A1752
  • 5'-CMP-Na 2 C1006
  • 5'-GMP-Na 2 G8377
  • 5'-UMP-Na 2 U6375
  • thiazolyl MTT
  • insulin Since Sigma.
  • Fetal bovine serum was purchased from Hangzhou Sijiqing.
  • the kits used in this example, such as LDH, SOD, and MDA test kits, were purchased from Nanjing Jiancheng Bioengineering Research Institute.
  • the trypsin, double antibody, and BCA protein assay kits were purchased from Biyuntian.
  • the remaining reagents such as dimethyl sulfoxide (DMSO) and hydrogen peroxide were domestically analyzed and purely purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd. 25 cm 2 flasks, 96-well cell culture plates, 6-well cell culture plates, 60 mm cell culture dishes, 50 mL centrifuge tubes, and 15 mL centrifuge tubes were purchased from Coming, USA. Disposable needle filters were purchased from Millipore.
  • the main experimental equipments include Heal Force Biosafety Cabinet (Hong Kong, China), Countstar Cell Counter (Inno-Alliance Biotech, USA), EVOS FL Fluorescence Microscope (Thermo Fisher), and microplate reader (Rebec, Finland).
  • Rat small intestine crypt epithelial cells (IEC-6 cells) were obtained from the Institute of Basic Cells of the Chinese Academy of Medical Sciences.
  • Rat normal liver cells (BRL 3A cells) were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences.
  • Samples 1-3 are the nucleotide compositions of the present application.
  • Comparative Samples 2 and 3 are the nucleotide compositions disclosed in the prior art (see above). Each sample was set at three concentrations of low, medium and high (62.5, 250, 1000 ⁇ mol/L, respectively).
  • Oxygen injury is one of the most typical and common body injuries (such as intestinal damage). Many diseases of humans and animals, such as digestive diseases, are known to be closely related to free radicals and reactive oxygen species, such as oxygen damage, which has been proven to be one of the causes of inflammatory bowel disease.
  • the intracellular redox reaction maintains equilibrium under normal physiological conditions, and the oxygen free radicals and antioxidant systems in the body are important factors in maintaining this balance. Once the oxygen free radicals are produced in large quantities or the antioxidant system is weakened, it will cause damage to the tissue cells.
  • SOD superoxide dismutase
  • SOD superoxide dismutase
  • lactate dehydrogenase (LDH) released into the culture supernatant by cells is a marker of cell membrane integrity or an indicator of cell necrosis.
  • intracellular oxides/peroxides can attack polyunsaturated fatty acids in biofilms, causing lipid peroxidation to form lipid peroxides such as malondialdehyde (MDA).
  • MDA malondialdehyde
  • Oxygen free radicals not only cause cell damage through the peroxidation of polyunsaturated fatty acids in biofilms, but also cause cell damage through the decomposition products of lipohydroperoxides. Therefore, the amount of MDA can usually reflect the degree of lipid peroxidation. Reflects the extent of cellular damage.
  • the concentration or activity of these substances or changes thereof and cell viability can be used to evaluate the protective effect of the nucleotide composition of the present invention. See, "Remediation of the Effect of Rhubarb Polysaccharide on Intestinal Epithelial Cell Injury and Its Mechanism", Ph.D. Thesis, Fourth Military Medical University, Liu Linna, May 1, 2005; "Baolong Longyanshen on Rats Induced by Carbon Tetrachloride” The protective effect of hepatocyte injury", Duan Xiaoqun et al., Chinese Pharmacy, Vol. 17 No. 15, 2006, 1132-1143; “Protective effect of Baoganning on hepatocytes damaged by hydrogen peroxide in rats", Zhao Jinjun et al Journal of Guangzhou University of Traditional Chinese Medicine, Vol. 19, No. 3, 2002, 211-213.
  • the protective effect of the composition of the present invention on cellular oxidative damage was investigated by treating cells with a nucleotide composition and inducing oxidative damage of cells with H 2 O 2 .
  • the test indicators were cell viability and SOD activity.
  • the test indicators are LDH activity and MDA content.
  • IEC-6 cells were cultured in RPMI-1640 medium containing 5% FBS and 2 mg/L insulin at 37 ° C in a 5% CO 2 incubator and subcultured every 3 days.
  • BRL 3A cells were cultured in DMEM high glucose medium containing 10% FBS at 37 ° C in a 5% CO 2 incubator and subcultured every 4 days.
  • the nucleotide composition was formulated into 50 mmol/L mother liquor with PBS, sterilized by filtration, and stored at -20 ° C until use.
  • cells in logarithmic growth phase were inoculated into 60 mm cell culture dishes at 7 ⁇ 10 5 / dish. After the cells were attached, the cells were cultured for 24 h with different concentrations of the nucleotide composition samples (3 replicates for each concentration). The experiment was performed with normal control without H 2 O 2 and H 2 O 2 only. Positive control. After 24 h, a certain concentration of H 2 O 2 was added to induce oxidative damage of the cells, and the culture was continued. After 2 h, the culture solution was aspirated, and the supernatant was centrifuged to detect relevant indexes. The cells were digested with trypsin, washed once with PBS, then resuspended in 700 ⁇ l of PBS, and the cells were disrupted by sonication, and all indicators were detected within 2 days.
  • the experimental results of the cell viability of BRL 3A cells are shown in Fig. 1.
  • the H 2 O 2 treatment resulted in a significant decrease in the cell viability of BRL 3A (P ⁇ 0.01).
  • Cell viability increased to varying degrees by treating cells with nucleotide composition samples, especially samples 1 and 2.
  • the statistical results showed that the cell viability of all the concentration groups of sample 1, the low-medium concentration group of sample 2, and the low concentration group of sample 3 were significantly higher than that of the H 2 O 2 control (P ⁇ 0.01 or P ⁇ 0.05).
  • the cell viability of all the concentration groups of Sample 1 and the low concentration group of Sample 2 were significantly higher than those of Comparative Samples 2 and 3 (P ⁇ 0.01 or P ⁇ 0.05).
  • the survival rate of the oxidatively damaged cells can be effectively enhanced, thereby exhibiting a protective effect against oxidative damage and further promoting repair after cell damage. Moreover, such a protective effect is superior to prior art nucleotide compositions.
  • the results of SOD activity experiments of BRL 3A cells are shown in Fig. 2.
  • the H 2 O 2 treatment caused a significant decrease in SOD activity in the cells (P ⁇ 0.01).
  • the SOD activity in the cells is increased to varying degrees.
  • the SOD activity in all the concentration groups (P ⁇ 0.05), the middle concentration group (P ⁇ 0.05), and the medium high concentration group (P ⁇ 0.01) of sample 1 were significantly higher than those of H 2 O 2 .
  • the SOD activity of the concentration group in the sample 3 was significantly higher than that of the middle-high concentration group of the comparative sample 2 (P ⁇ 0.05) and all the concentration groups of the comparative sample 3 (P ⁇ 0.05).
  • the SOD activity of the high concentration group of the sample 3 was significantly higher than that of the high concentration group of the comparative sample 2 (P ⁇ 0.05) and the low high concentration group of the comparative sample 3 (P ⁇ 0.01). It can be seen that the nucleotide composition of the present invention can effectively increase the concentration of an oxidatively damaged intracellular antioxidant active substance (such as SOD), thereby exhibiting a protective effect against oxidative damage, and the protective effect is superior to that of the prior art. Glycoside composition.
  • an oxidatively damaged intracellular antioxidant active substance such as SOD
  • the results of the LDH activity test of IEC-6 cells are shown in Fig. 3.
  • treatment of cells with H 2 O 2 resulted in higher LDH content in the culture supernatant than in the normal control (P ⁇ 0.01).
  • the LDH content in the supernatant was decreased to some extent by treating the cells with the nucleotide composition, and the LDH content in the low concentration group of sample 2 and the medium concentration group of sample 3 was the most significant, which was not statistically different from the normal control.
  • the medium concentration group of sample 1 (P ⁇ 0.05), the low high concentration group of sample 2 (P ⁇ 0.01 or P ⁇ 0.05), and the low medium concentration group of sample 3 (P ⁇ The LDH content of 0.01) was significantly decreased.
  • nucleotide composition of the present invention can effectively reduce the concentration of a substance (for example, LDH) representing an unfavorable index in the supernatant of oxidatively damaged cells, thereby exhibiting a protective effect against oxidative damage, and the protective effect is superior.
  • a substance for example, LDH
  • the nucleotide composition of the present invention can effectively reduce the concentration of a substance (e.g., lipid peroxide MDA) which represents an adverse index in an oxidatively damaged cell, thereby exhibiting a protective effect against oxidative damage.
  • a substance e.g., lipid peroxide MDA
  • the nucleotide compositions of the present invention exhibit significant and consistent protective effects over all concentration ranges.
  • prior art nucleotide compositions do not exhibit a protective effect at higher concentrations (eg, moderately high concentrations). This further illustrates the advantages of the nucleotide compositions of the present invention over the prior art.
  • the survival rate of oxidatively damaged cells can be improved, the factor (e.g., SOD) activity/concentration having an oxidative protective effect in the cells can be increased, and the adverse effects can be lowered.
  • Activity/content of factors eg LDH and MDA.
  • the nucleotide composition of the present invention can promote the recovery of the equilibrium state before the injury, thereby promoting the repair after the injury.
  • the effects of the nucleotide composition of the present invention can be expressed not only in intestinal cells but also in other parts of the digestive system such as the liver, thereby exhibiting a positive effect on the entire digestive system.
  • Example 3 In which the promotion of cell proliferation by the nucleotide composition of the present invention was verified using IEC-6 cells.
  • the effect of the nucleotide composition on the proliferation of IEC-6 cells was examined using the MTT method.
  • IEC-6 cells in the logarithmic growth phase were taken, and the cells were digested with trypsin and centrifuged to prepare a cell suspension.
  • the cells were counted by a cell counter, seeded in a 96-well plate at 5000 cells/well, and cultured overnight at 37 ° C under 5% CO 2 to adhere the cells.
  • the culture medium was changed the next day, and 200 ⁇ L of a cell culture solution containing different concentrations of the nucleotide composition (each sample was set to 6 concentrations of 62.5, 250, and 1000 ⁇ mol/L) was added to each well.
  • Each sample has 6 replicate wells per concentration, and there is also a control group without nucleotide composition and a zero-free well without cells (normal medium only), and the culture solution is changed every 24 hours. .
  • 96-well plates were taken at 24 h after the nucleotide composition treatment, 10 ⁇ L of MTT was added to each well, gently shaken, and returned to the incubator for 4 h. After 4 hours, discard the supernatant, add DMSO 150 ⁇ L/well, and mix well in the dark. After the blue purple formazan crystals are dissolved, place the plate in the microplate reader and measure the OD value at 490 nm to avoid the blank of the cells. Group zero. The curve is drawn with the OD value.
  • the results are shown in Figure 5.
  • the results in this figure indicate that the proliferation of IEC-6 cells was significantly altered compared to the control after addition of the nucleotide sample.
  • the cell proliferation rate of the high concentration group of sample 1 and the concentration group of sample 3 was significantly higher than that of the normal control group (P ⁇ 0.01); the cell proliferation rate of the middle concentration group of sample 2 was also higher than that of the control group (P ⁇ 0.05).
  • the cell proliferation in the low concentration group of Comparative Sample 2 was significantly lower than that in the high concentration group of Sample 1, the low medium concentration group of Sample 2, and the low, medium, and high concentration group of Sample 3 (P ⁇ 0.01).
  • the cell proliferation of the high concentration group of sample 2 was also significantly higher than that of the low concentration group of the comparative sample 2 (P ⁇ 0.05).
  • nucleotide composition of the present application can promote cell proliferation, in particular, promote intestinal cell proliferation and growth, thereby exhibiting an effect of promoting intestinal epithelial cell growth, increasing intestinal epithelial cell proliferation, and the effect is superior to the prior art. Nucleotide composition.
  • This example demonstrates the stimulatory effect of the nucleotide compositions of the present application on the beneficial intestinal flora. Two concentrations were used for each sample (low dose: 1 g/100 ml; high dose: 2 g/100 ml), and no nucleotides were added to the control.
  • EMB Eosin blue agar
  • TSC pancreas - sulfite-cycloserine agar
  • BBL bifidobacterium agar medium
  • a total of about 0.5 g of fecal samples were taken from the anus of 5 SPF mice in a sterile table, placed in a sterile test tube, and 3 sterilized glass beads were added. After vortexing, gradient dilution was performed and diluted to 10 -2 Level, as a seed solution for subsequent experiments.
  • a beef paste peptone liquid medium was used as a base medium, and a nucleotide sample was added thereto at 1 g/100 ml, 2 g/100 ml, respectively, and the seed liquid prepared in 2.1 was added at 1% by volume.
  • the bifidobacteria in the culture solution were subjected to anaerobic culture for 48 hours at 36 +1 ° C using a specific selective medium (BBL agar), and colony counts were carried out, and the results were expressed in Lg CFU/ml.
  • the raw materials used in the milk powder of the present invention are: skim milk powder 140 kg, lactose 260 kg, desalted whey powder 200 kg, whey protein powder (WPC 34%) 100 kg, corn oil 52 kg, soybean oil 42 kg, sunflower oil 170 Kilograms, 4.8 kg of oligofructose powder, 12 kg of oligogalactose syrup, 6.4 kg of compound nutrients, and 2 kg of soybean phospholipid.
  • the end product can be obtained by filling the evenly mixed milk powder with nitrogen.
  • An additional 0.5 kg of the nucleotide composition of the invention, 0.1 kg of Bifidobacterium, 5.2 kg of DHA and 6.7 kg of ARA were added and mixed by a dry mixer.
  • the end product can be obtained by filling the evenly mixed milk powder with nitrogen.
  • Example 9 Preparation of baby liquid milk (based on 100 g liquid milk)
  • the liquid milk of the present invention comprises the following nutrients:
  • the nucleotide composition was 2.7 mg with an energy of about 300 kJ.
  • the fat is supplied from anhydrous butter, soybean oil, corn oil, sunflower seeds in any ratio, but contains 0.5 g of linoleic acid in a total amount of 100 g.
  • the preparation method is as follows (the raw materials and processes are in line with relevant national standards):

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Abstract

一种用作食品添加物的核苷酸组合物，由58-72%CMP、6-14%AMP、10-18%UMP和8-14%GMP组成，或者由58-70%CMP、7.5-12.5%AMP、12-16.5%UMP、10-13%GMP和0-2.5%IMP组成。该组合物可用于制备食品，例如婴幼儿食品，乳制品，能实现以下效果：提供免疫刺激、促进生长发育、促进肠道损伤后的修复、促进肠道有益微生物的生长和/或它们的任意组合。

Description

核苷酸组合物及其在食品中的应用
本申请要求2015年3月6日提交的题为“核苷酸组合物及其在食品中的应用”的第201510099850.6号中国专利申请的优先权。该申请的内容整体援引加入本文。
技术领域
本发明涉及一种用作食品添加物的核苷酸组合物。更进一步地，本发明还涉及包含所述核苷酸组合物的食品及所述核苷酸组合物在制备食品中的应用。
背景技术
近年来，有许多实验已经证明了核苷酸在生物体中的重要作用，例如核苷酸可以提高机体的免疫系统和胃肠道系统的功能。同时，以牛奶为基础的代乳品是目前婴幼儿重要的食品补充来源，由于核苷酸及其衍生物在牛乳中含量很低，因此将外源核苷酸添加到以牛奶为基础的代乳品中，对机体尤其是婴幼儿的生长发育(如胃肠道发育)及免疫力增强都有重要的意义。
US 4,994,442中指出将各种核苷酸添加入婴儿配方食品中可以增强T细胞免疫应答，但是在该文献中各种核苷酸组分(CMP、AMP、UMP、GMP和IMP)是以等量添加至配方食品中。EP 1549158中公开了一种婴儿奶粉组合物，其包含3.2-15.4mg/L的CMP、1.8-11.0mg/L的UMP、1.8-8.0mg/L的GMP、0.1-2.2mg/L的IMP和2.5-13.2mg/L的AMP，但是其仅意图给早产婴儿使用，而且也没有公开任何可能实现的效果。
在现有技术的基础上，为了充分发挥核苷酸对于机体生长发育的作用，优化用作食品添加物的核苷酸的效果，发明人进行了广泛的实验，调整了核酸组合物中核苷酸组分的组成及配比，以最大化该组合物作为核苷酸补充添加物的效果。同时，发明人出人意料地发现，相对于现有技术中所用的产品，通过本发明的核苷酸组合物所具有特定组成和配比，能够提供更 好的效果。
发明内容
在第一方面，本发明涉及一种用作食品添加物的核苷酸组合物。
在一实施方案中，本发明的核苷酸组合物基本上由CMP、AMP、UMP、GMP和IMP组成或者由CMP、AMP、UMP、GMP和IMP组成，其中按重量计，各成分的比例为：CMP：58～70％、AMP：7.5～12.5％、UMP：12～16.5％、GMP：10～13％以及IMP：0～2.5％，条件是各种组分之和为100％。
在优选的实施方案中，本发明的核苷酸组合物基本上由CMP、AMP、UMP、GMP和IMP组成或者由CMP、AMP、UMP、GMP和IMP组成，其中按重量计，各成分的比例为：CMP：60～65％、AMP：8～12％、UMP：14～16％、GMP：11～12％以及IMP：0～2％，条件是各种组分之和为100％。
在另一实施方案中，本发明的核苷酸组合物基本上由CMP、AMP、UMP和GMP组成或者由CMP、AMP、UMP和GMP组成，其中按重量计，各成分的比例为：CMP：58～72％、AMP：6～14％、UMP：10～18％以及GMP：8～14％，条件是各种组分之和为100％。
在另一实施方案中，本发明的核苷酸组合物基本上由CMP、AMP、UMP和GMP组成或者由CMP、AMP、UMP和GMP组成，其中按重量计，各成分的比例为：CMP：60～70％、AMP：8～12％、UMP：12～16％以及GMP：10～12％，条件是各种组分之和为100％。
在另一实施方案中，本发明的核苷酸组合物基本上由CMP、AMP、UMP和GMP组成或者由CMP、AMP、UMP和GMP组成，其中按重量计，各成分的比例为：CMP：60～65％、AMP：10～12％、UMP：14～16％以及GMP：11～12％，条件是各种组分之和为100％。
在另一实施方案中，本发明的核苷酸组合物基本上由CMP、AMP、UMP和GMP组成或者由CMP、AMP、UMP和GMP组成，其中按重量计，各成分的比例为：CMP：65～70％、AMP：8～10％、UMP：12～14％以及GMP：10～11％，条件是各种组分之和为100％。
在优选的实施方案中，本发明的核苷酸组合物基本上由CMP、AMP、UMP和GMP组成或者由CMP、AMP、UMP和GMP组成，其中按重量计，各成分的比例为：CMP：60％、AMP：12％、UMP：16％以及GMP：12％。
在优选的实施方案中，本发明的核苷酸组合物基本上由CMP、AMP、UMP和GMP组成或者由CMP、AMP、UMP和GMP组成，其中按重量计，各成分的比例为：CMP：65％、AMP：10％、UMP：14％以及GMP：11％。
在优选的实施方案中，本发明的核苷酸组合物基本上由CMP、AMP、UMP和GMP组成或者由CMP、AMP、UMP和GMP组成，其中按重量计，各成分的比例为：CMP：70％、AMP：8％、UMP：12％以及GMP：10％。
在上述第一方面的各个实施方案中，优选地，所述食品为婴幼儿食品，更加优选地，所述食品为乳制品的形式，例如奶粉或液态乳制品的形式，如供婴幼儿使用的奶粉或液态乳制品。
在第二方面，本发明涉及一种食品，其包含本发明的核苷酸组合物。在一优选的实施方案中，所述食品为婴幼儿食品。在更加优选的实施方案中，所述食品为乳制品的形式，例如奶粉或液态乳制品的形式，如供婴幼儿使用的奶粉或液态乳制品。
在第三方面，本发明还涉及一种制备食品的方法，所述方法包括向食品的原料中添加本发明的核苷酸组合物。在一优选的实施方案中，所述食品为婴幼儿食品。在更加优选的实施方案中，所述食品为乳制品的形式，例如奶粉或液态乳制品的形式，如供婴幼儿使用的奶粉或液态乳制品。
在第四方面，本发明还涉及本发明的核苷酸组合物在制备食品中的应用。在一优选的实施方案中，所述食品为婴幼儿食品。在更加优选的实施方案中，所述食品为乳制品的形式，例如奶粉或液态乳制品的形式，如供婴幼儿使用的奶粉或液态乳制品。在一优选实施方案中，所述食品用于提供免疫刺激效果。在另一优选实施方案中，所述食品用于促进生长发育(如胃肠道发育)及促进肠道损伤后的修复(例如促进细胞特别是肠上皮细胞损伤修复)。在另一优选实施方案中，所述食品用于促进肠道有益微生物的生长。
在第五方面，本发明还涉及用于提高个体的免疫力、促进生长发育(如胃肠道发育)、促进肠道损伤后的修复(例如促进细胞特别是肠上皮细胞损伤修复，如由氧化导致的损伤)和/或促进个体的肠道有益微生物的生长和/或它们的任意组合的方法，所述方法包括向所述个体施用本发明的核苷酸组合物或食品。在优选实施方案中，所述个体为人，优选人的婴幼儿。
附图说明
图1为示出细胞存活率实验结果的图。
图2为示出SOD活性实验结果的图。
图3为示出LDH活性实验结果的图。
图4为示出MDA含量实验结果的图。
图5为示出细胞增殖实验结果的图。
具体实施方式
除非另外专门定义，本文使用的所有技术和科学术语都与相关领域普通技术人员的通常理解具有相同的含义。除非另外专门定义，本文中使用的比例(包括百分比)均按重量计。
核苷酸
本文所用的术语“核苷酸”指由嘌呤或嘧啶碱基、核糖或脱氧核糖以及磷酸基团组成的化合物。例如，根据糖的不同，核苷酸可以分为核糖核苷酸及脱氧核苷酸。例如，根据碱基的不同，核苷酸可以分为腺嘌呤核苷酸、鸟嘌呤核苷酸、胞嘧啶核苷酸、尿嘧啶核苷酸、胸腺嘧啶核苷酸及次黄嘌呤核苷酸等。当核苷酸分子中只有一个磷酸基时，其称为一磷酸核苷酸(NMP)。5′-核苷酸的磷酸基还可进一步磷酸化生成二磷酸核苷(NDP)及三磷酸核苷(NTP)。
本文所用的术语“核苷酸”还涵盖以各种形式存在于本发明的核苷酸组合物中的胞嘧啶(C)、尿嘧啶(U)、腺嘌呤(A)、鸟嘌呤(G)和/或次黄嘌呤(I)，例如核糖核苷、核糖核苷酸、RNA磷酸酯以及任何其他形式的衍生物或前体，只要其在体内或体外能够转化或代谢为相应的核苷酸形式。
例如，在食品添加领域，所用的核苷酸主要有CMP(胞嘧啶核苷酸)、UMP(尿嘧啶核苷酸)、AMP(腺嘌呤核苷酸)、GMP(鸟嘌呤核苷酸)、IMP(次黄嘌呤核苷酸)等。例如，目前市场上出售的5′-混合核苷酸含有5′-腺苷酸(AMP)、5′-胞苷酸(CMP)、5′-鸟苷酸(GMP)、5′-尿苷酸(UMP)和5′-肌苷酸(IMP)。通常，在组合使用的时候，这种5′-混合核苷酸可以例如以两种类型存在：一种是5′-腺苷酸和5′-胞苷酸是以游离酸的形式存在，而其他3种核苷酸以钠盐形式存在，简称两酸三钠型；另一种则均以钠盐的形式存在，简称五钠型。因此，本文所用的术语“核苷酸”还涵盖其盐的形式，例如碱金 属盐或碱土金属盐，如钠盐、钾盐或钙盐等，例如一钠盐或二钠盐等，如CMPNa2、AMPNa2、UMPNa2、GMPNa2、IMPNa2和CMPK2、AMPK2、UMPK2、GMPK、IMPK2等。本领域技术人员应当理解，本发明的核苷酸组合物中的各个组分可以各自任选地且各自独立地以各种盐形式存在，包括但不限于上文提及的“两酸三钠型”；或者各个组分及其盐可以以任意组合的形式存在，例如仅GMP以盐形式存在，仅CMP以盐形式存在等。可选地，本发明的核苷酸组合物中的各个组分可以均以盐的形式存在，包括但不限于上文提及的“五钠型”。
同样，本文所用的术语“核苷酸”还涵盖其各种形式的溶剂合物(如水合物)。因此，当本发明的核苷酸组合物包含核苷酸组分的诸如上文所列的其他形式时，这些形式在组合物中的配比应当换算成其对应的核苷酸分子后计算。例如，当CMP以其二钠盐形式(CMPNa2)存在时，应当换算成CMP来计算相应的重量比。
核苷酸组合物
在一实施方案中，本发明的核苷酸组合物基本上由以下成分组成或者由以下成分组成：CMP、AMP、UMP、GMP和IMP。在另一实施方案中，本发明的核苷酸组合物基本上由以下成分组成或者由以下成分组成：CMP、AMP、UMP和GMP。
在一实施方案中，按重量计，CMP在本发明的核苷酸组合物中占58～72％，优选60～70％，例如60～65％和65～70％。应当理解，这些范围涵盖其中所有的点值，例如但不限于59％、60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、71％等；以及由这些点值组成的各种亚范围，例如但不限于60～63％、60～68％、63～70％等。在具体实施方案中，按重量计，CMP在本发明的核苷酸组合物中占60％、65％或70％。
在一实施方案中，按重量计，AMP在本发明的核苷酸组合物中占6～14％，优选8～12％，例如8～10％和10～12％。应当理解，这些范围涵盖其中所有的点值，例如但不限于7％、8％、9％、10％、11％、12％、13％等；以及由这些点值组成的各种亚范围，例如但不限于8～9％、8～11％、9～12％等。在具体实施方案中，按重量计，AMP在本发明的核苷酸组合物中占8％、10％或12％。
在一实施方案中，按重量计，UMP在本发明的核苷酸组合物中占 10～18％，优选12～16％，例如12～14％和14～16％。应当理解，这些范围涵盖其中所有的点值，例如但不限于11％、12％、13％、14％、15％、16％、17％等；以及由这些点值组成的各种亚范围，例如但不限于12～15％、14～15％等。在具体实施方案中，按重量计，UMP在本发明的核苷酸组合物中占12％、14％或16％。
在一实施方案中，按重量计，GMP在本发明的核苷酸组合物中占8～14％，优选10～12％，例如10～11％和11～12％。应当理解，这些范围涵盖其中所有的点值，例如但不限于9％、10％、11％、12％、13％等；以及由这些点值组成的各种亚范围，例如但不限于8～11％、10～14％等。在具体实施方案中，按重量计，GMP在本发明的核苷酸组合物中占10％、11％或12％。
在一实施方案中，按重量计，IMP在本发明的核苷酸组合物中占0～2.5％，优选0～2％，更优选为0％(即，本发明的核苷酸组合物不含IMP)。应当理解，这些范围涵盖其中所有的点值，例如但不限于1％、2％等；以及由这些点值组成的各种亚范围，例如但不限于0～1％、1～2％等。
本领域技术人员应当理解，上述对各种组分的所列的范围和具体值可以任意地互相组合和选择，只要这样的组合满足所选的各种组分之和为100％的条件。例如，在本发明的核苷酸组合物中，当CMP的含量为60～70％如60～65％或65～70％时，AMP的含量可以为8～12％如8～10％或10～12％。同样，当CMP和AMP的含量取任何上述范围时，UMP的含量可以为12～16％如12～14％或14～16％。类似地，当CMP、AMP和UMP的含量取任何上述范围时，GMP的含量可以为10～12％如10～11％或11～12％。基于同样的原则，当CMP、AMP、UMP和GMP的含量取任何上述范围时，IMP的含量可以为0～2.5％如0～2％或0％(即，本发明的核苷酸组合物不含IMP)。本领域技术人员应当理解，上述组合的选择条件应当满足的条件是所选的各种组分之和为100％。
本文所用的表述“基本上由...组成”表示除了所限定的核苷酸组分以外，若需要，本发明的核苷酸组合物还可以任选地包含食品或生理学上接受的载体、赋形剂或辅剂，例如防腐剂、抗氧化剂、粘合剂、增稠剂、稀释剂等以及可能存在的各种杂质。这些载体、赋形剂或辅剂和杂质对于组合物中的活性成分(核苷酸组分)而言是惰性的，其存在或量并不会干扰或明显干扰所述活性成分的功能。而且，当计算本发明的核苷酸组合物中各核苷酸 组分的配比时，不考虑这些可能存在的载体、赋形剂或辅剂和杂质。另外，本领域技术人员应当理解，表述“基本上由...组成”涵盖“由...组成”的含义。
本文所用的术语“任选(地)”指可以存在或不存在。例如，所述“任选地包含食品或生理学上接受的载体、赋形剂或辅剂”指本发明的核苷酸组合物可以包含上述载体、赋形剂或辅剂，也可以不包含上述载体、赋形剂或辅剂。
食品
本文所用的术语“食品”具有本领域普通技术人员通常理解的含义，例如其表示可供人类食用或饮用的物质，包括加工食品、半成品和未加工食品等。
本文所用的术语“婴幼儿食品”通常指除了母乳以外的供婴幼儿使用的食品，其通常为了婴幼儿生长发育的需要而添加各种成分，例如核苷酸、脂肪酸、维生素、碳水化合物、植物油、微量元素等，例如本发明的核苷酸组合物。这样的食品的实例有乳制品(例如奶粉和液态乳制品)、果泥、米粉等。根据本发明的具体实施方案，所述婴幼儿食品为乳制品的形式，如奶粉或液态乳制品，例如添加了本发明的核苷酸组合物的婴幼儿奶粉或液态乳制品。
本文所用的术语“婴幼儿”通常指0～3岁的人个体。但是，本发明的核苷酸组合物的应用并不局限于这一年龄段。若需要，可以将本发明的核苷酸组合物或包含所述核苷酸组合物的食品施用于更大年龄的人个体，例如4岁、5岁、6岁、7岁、8岁、9岁、10岁或更大。
在另一方面，本发明还提供了一种食品的制备方法，其包括向所述食品中添加本发明的核苷酸组合物。在一实施方案中，所述食品为婴幼儿食品。在优选的实施方案中，所述食品为乳制品的形式，例如奶粉或液态乳制品的形式，如供婴幼儿使用的奶粉或液态乳制品。
在整个说明书中，术语“液态乳制品”和“液态奶”或“液态乳”具有相同的含义，可以互换使用，并且意图表示以液体形式存在的乳制品，其包含供人体使用的各种营养成分和能量，例如下文所示的那些成分。
根据一实施方案，本发明的核苷酸组合物在制备本发明所述的奶粉中的用量为0.2～0.58重量份(基于1000重量份的奶粉)。例如，本发明的奶粉，基于1000重量份的所述奶粉，包含：脱脂奶粉120～160重量份、乳糖240～280 重量份、脱盐乳清粉180～210重量份、乳清蛋白粉(WPC34％)90～120重量份、葵花籽油155～180重量份、玉米油35～55重量份、大豆油40～60重量份、本发明的核苷酸组合物0.35～0.5重量份、大豆磷脂1～2.5重量份、双歧杆菌0.1～0.15重量份、低聚果糖粉4～5重量份、低聚半乳糖浆10～12重量份，营养学上可接受量的维生素以及营养学上可接受量的微量元素补充剂。
在一具体实施方案中，本发明的奶粉主要的制备工艺流程包括：配料、预热、均质、浓缩杀菌、喷雾干燥、干混，然后获得最终产品，其中将本发明的核苷酸组合物和DHA、ARA及双歧杆菌一起添加至喷雾干燥后奶粉中，然后混合。
根据另一实施方案，本发明的液态乳制品包含以下成分(以每100g液态乳制品计)：乳铁蛋白以外的蛋白质1.75g～4.26g、脂肪1.75g～4.97g、能量250kJ～355kJ、维生素A 42.5μg～191.7μg RE、维生素D 0.625μg～2.6625μg、维生素E≥0.375mgα-TE、维生素K1≥2.5μg、维生素B1≥27.5μg、维生素B2≥27.5μg、维生素B6≥27.5μg、维生素B12≥0.1μg、烟酸(或烟酰胺)≥275μg、叶酸≥2.5μg、泛酸≥175μg、维生素C≥4.5mg、生物素≥1μg、钠≤71mg、钾45mg～244.95mg、铜17.5μg～124.25μg、镁≥3.5mg、铁0.625mg～1.775mg、锌0.25mg～1.065mg、钙≥42.5mg、磷≥20.75mg、碘≥3.5μg、氯≤184.6mg、乳铁蛋白5～13mg和碳水化合物，其中本发明的核苷酸的量为2.64～7.66mg，并且碳水化合物的量使得其与蛋白质和脂肪一共提供的能量为250kJ～355kJ。
所用的脂肪由无水奶油、大豆油、玉米油、葵花籽中的一种或多种以任意比例和组合提供。另外，为了提高液态奶体系的稳定性等性质，还可以向其中添加一些食品上可接受的食品添加剂，如乳化稳定剂等。在本发明中，优选使用的食品添加剂及量例如但不限于：卡拉胶0.005重量％～0.05重量％、单硬脂酸甘油酯0.01重量％～1重量％、瓜尔豆胶0.01重量％～0.1重量％、刺槐豆胶0.01重量％～0.1重量％中的一种或者任意多种的组合。
最后，本发明还涉及本发明的核苷酸组合物在制备食品中的应用。在一实施方案中，所述食品为婴幼儿食品。在优选的实施方案中，所述食品为乳制品的形式，例如奶粉或液态乳制品的形式，如供婴幼儿使用的奶粉或液态乳制品。
免疫刺激
在一优选实施方案中，本发明的核苷酸组合物和包含所述组合物的食品经食用后可以提供免疫刺激效果。本文所述的“免疫刺激”效果指具有提高个体的免疫功能的作用，例如表现在提高淋巴细胞转化功能，增强巨噬细胞吞噬功能，增强NK细胞活性，提高免疫应答和抗体生成等方面，如说明书的实施例部分所提供的那些效果。在优选的实施方案中，所述个体为人，优选人的婴幼儿。
促进生长发育及促进肠道损伤后的修复
本发明的核苷酸组合物和包含所述组合物的食品经食用后可以促进个体的生长发育(如胃肠道发育)及促进肠道损伤后的修复。例如，促进肠道(如小肠)细胞的生长发育及成熟，修复肠道(如小肠)的损伤，保护肠道(如小肠)细胞免受自由基的攻击，降低肠道(如小肠)炎症的发生等。发明人在体外实验中观察到，本发明的核苷酸组合物可促进肠上皮细胞生长，促进肠上皮细胞的增殖，对肠上皮细胞和肝细胞具有损伤保护作用。这样的损伤，例如可以是氧化损伤，例如由活性氧物质引起的损伤。在优选的实施方案中，所述个体为人，优选人的婴幼儿。
促进肠道有益微生物的生长
本发明的核苷酸组合物和包含所述组合物的食品经食用后可以促进个体的肠道有益微生物(如双歧杆菌及乳酸杆菌)的生长，例如体现在有利于双歧杆菌的生长，如提高肠道及粪便中双歧杆菌及乳酸杆菌含量(这些菌群可抑制厌酸型病原菌和大肠杆菌的繁殖)，降低肠道有害菌的比例，从而有益于个体的健康。对此，发明人研究发现，在肠道环境中或类似肠道环境的体外环境中添加该组合物，通过观察环境中益生菌和乳酸菌的增殖数量，尤其是双歧杆菌的增殖数量，发现本发明的核苷酸组合物对肠道有益微生物显示出显著的促生长作用。在优选的实施方案中，所述个体为人，优选人的婴幼儿。
实施例
在本申请的实施例中所用的测试样品的组成和配比如下(按重量计)：
本申请的核苷酸组合物(还参见权利要求5)：
样品1：CMP：60％、AMP：12％、UMP：16％和GMP：12％
样品2：CMP：65％、AMP：10％、UMP：14％和GMP：11％
样品3：CMP：70％、AMP：8％、UMP：12％和GMP：10％
现有技术的核苷酸组合物：
比较样品1：CMP：33.7％、AMP：20.3％、UMP：23.1％、GMP：7.6％和IMP：15.3％
比较样品2：CMP：60％、AMP：14.5％、UMP：18.2％和GMP：7.3％
比较样品3：CMP：42.7％、AMP：13.4％、UMP：24.2％和GMP：19.7％
实施例1：免疫刺激效果(一)
本实施例意图证实本发明的核苷酸组合物在免疫刺激方面的效果，其原理和步骤基于“保健食品检验与评价技术规范(2003年版)”第二部分“功能学检验方法”第一章的规定和要求。
1.材料与方法
1.1.主要试剂
本实施例中所用的主要试剂有：RPMI-1640培养基(Gibco)；胎牛血清(Gibco)；刀豆蛋白A(ConA)(Sigma)；淋巴细胞分离液、全血及组织稀释液、细胞洗涤液(天津市灏洋生物制品科技有限责任公司)；5％鸡红细胞悬液(实验室制备)；MTT试剂盒、Hank′s液(碧云天生物技术研究所)；Giemsa染液(珠海贝索生物技术有限公司)；EDTA抗凝管(BD)；96孔细胞培养板(Corning)；百呈牌牛初乳胶囊(上海孚正生物科技有限公司)；核苷酸样品(南京同凯兆业)。
1.2.主要仪器设备
CO2培养箱(三洋MCO-18AIC(UV))；超净工作台(AIRTECH)；倒置显微镜(OLYMPUS)；日立水平离心机(himac-CT6EL)；酶标仪(BIO-RAD Model680)；OLYMPUS显微镜(BX51)；DNP恒温培养箱(上海精宏实验设备有限公司)。
1.3.实验动物
5～6周龄雄性KM小鼠160只，由上海斯莱克实验动物有限责任公司提供。
1.4.动物分组
使KM小鼠适应性饲养7天后，将其随机分为8组，每组20只，即空白对照组，阳性对照组，样品1的高、中、低剂量组以及比较样品1的高、 中、低剂量组。向每组动物分别灌胃溶解了各样品(见下文)的无菌水，空白对照组灌胃相同体积的无菌水。每天1次，连续饲喂或灌胃30天。
高剂量组的饲喂的剂量为67.37mg·kg-1·d-1，中剂量组的剂量为41.82mg·kg-1·d-1，低剂量组的剂量为23.23mg·kg-1·d-1。阳性对照组的剂量为150.17mg·kg-1·d-1。
阳性对照组：牛初乳胶囊。
实验组：样品1为本发明的核苷酸组合物。比较样品1为根据市售的某款添加核苷酸的婴幼儿食品中所用的核苷酸组分和配比制备的核苷酸组合物。
1.5.检测指标
淋巴细胞转化实验：各组于实验第38天，随机取10只小鼠，使用2mL EDTA抗凝管，摘除眼球采血，采用Ficoll密度梯度离心法，无菌分离淋巴细胞。取1mL新鲜抗凝血，将其与全血和组织稀释液以1：1混匀，然后小心加于等体积淋巴细胞分离液的液面上。用水平离心机以1500转/分钟离心15分钟，收集环状乳白色淋巴细胞层，将其用细胞洗涤液洗涤2次。用RPMI-1640完全培养液配制为1×107个/mL，然后加入96孔细胞培养板，每孔100μL，每鼠6孔，其中3孔加入ConA(终浓度为5μg/mL)，另3孔不加作为对照。在5％CO2和37℃的CO2培养箱中培养68小时后，加入MTT(5mg/mL)溶液(10μL/孔)，继续培养4小时后，每孔加入100μL Formazan(甲臜)溶液，然后在37℃的CO2培养箱内继续孵育，直至观察到Formazan全部溶解。在酶标仪上以波长570nm测定OD值，计算刺激指数(SI)：SI＝ConA刺激管OD平均值/对照管OD平均值。
腹腔巨噬细胞吞噬实验：各组于实验第38天，随机取10只小鼠，每鼠腹腔注射5％鸡红细胞悬液1mL。30分钟后，以颈椎脱臼法处死小鼠。腹腔注入1mL生理盐水，轻揉小鼠腹部1分钟，剪开腹壁皮肤，在肌肉层上开口，用吸管伸入腹腔吸取腹腔液1mL，滴加于洁净的载玻片上。将载玻片放入垫有湿纱布的搪瓷盒中，然后放入37℃培养箱孵育30分钟。取出载玻片，漂洗去除上清液和未粘附在玻片上的细胞，然后在室温下晾干，用1：1丙酮/甲醇溶液固定后，置于Giemsa染色液中，浸染15～30分钟，然后水洗并晾干。在高倍镜下计数巨噬细胞，每张玻片计数100个，分别计算吞噬百分率和吞噬指数。
Figure PCTCN2016074813-appb-000001
Figure PCTCN2016074813-appb-000002
1.6.统计学分析
数据均以
Figure PCTCN2016074813-appb-000003
表示，采用SPSS 13.0统计软件对数据进行方差分析，两两比较采用snk法，检验水准为α＝0.05。
2.结果
2.1.样品对淋巴细胞增殖功能的影响
表1样品对小鼠淋巴细胞增殖功能的影响
Figure PCTCN2016074813-appb-000004
Figure PCTCN2016074813-appb-000005
注：*与空白对照组相比差异显著(p＜0.05)
由表1可见，样品1的高、中、低剂量组的SI值均显著高于空白对照组(p＜0.05)，并且呈剂量依赖性；比较样品1的高剂量组的SI值显著高于空白对照组(p＜0.05)，比较样品1的中、低剂量组SI值与空白对照组差异不显著(p＞0.05)；样品1的低剂量组的SI值显著高于比较样品1中剂量组(p＜0.05)。
以上结果表明本发明的核苷酸组合物可显著提高小鼠淋巴细胞增殖功能，且效果优于比较样品。
2.2.样品对巨噬细胞吞噬功能的影响
表2样品对小鼠巨噬细胞吞噬功能的影响
Figure PCTCN2016074813-appb-000006
Figure PCTCN2016074813-appb-000007
注：*与空白对照组相比差异显著(p＜0.05)
由表2可见，样品1的高、中、低剂量组的吞噬百分率及吞噬指数均显著高于空白对照组(p＜0.05)；比较样品1的高剂量组的吞噬百分率及吞噬指数显著高于空白对照组(p＜0.05)，比较样品1的中、低剂量组的吞噬百分率及吞噬指数与空白对照组差异不显著(p＞0.05)；样品1的低剂量组的吞噬百分率及吞噬指数显著高于比较样品1的中剂量组(p＜0.05)。
以上结果表明本发明的核苷酸组合物可显著提高小鼠腹腔巨噬细胞吞噬功能，且效果优于比较样品。
3.结论
从以上实验结果可知，本发明的核苷酸组合物可显著提高淋巴细胞转化功能，增强腹腔巨噬细胞吞噬功能，具有提高免疫功能的作用，且免疫调节作用显著高于比较样品。
从另一个角度而言，更强的免疫调节作用意味着以更少的剂量能够实现相同或相似的效果，这对于大规模工业生产会具有经济成本上的明显优势。
在此基础上，发明人还重复了以上实验，其中将样品1分别替换为样品2和样品3。对于样品2和样品3，同样获得了与样品1类似的结果。
实施例2：免疫刺激效果(二)
本实施例意图进一步证实本发明的核苷酸组合物在免疫刺激方面的效果，其原理和步骤基于“保健食品检验与评价技术规范(2003年版)”第二部分 “功能学检验方法”第一章的规定和要求。
1.材料与方法
1.1主要仪器
冷冻高速离心机(SIGMA 3-30K)、振荡器(Vortex4digital)、加样枪(德国eppendorf)、全血细胞分析仪(优利特U-2900PLUS)、二氧化碳培养箱(Thermo311)、酶标仪(Biotek H4)、分光光度计(上海精密仪器科学仪器有限公司722s)、超净工作台(上虞市星星仪器设备有限公司SW-CJ-2D)、高压灭菌器(日本三洋MLS-3781-PC 75L)、低温冰箱(杭州艾普仪器设备有限公司DW-40L058)、倒置显微镜(日本尼康Eclipse Ti-S)、游标卡尺(美国Standard Gage)、电子天平(赛多利斯BSA124S-CW)、足跖容积测量仪(济南益言科技发展有限公司YLS-7C)、溶血空斑自动图像分析仪(北京安泰永信医疗科技有限公司AT-Spot 5100)、流式细胞仪(美国BD FACSCalibur)。
1.2主要试剂
环磷酰胺(江苏恒瑞医药股份有限公司)、乙二胺四乙酸二钾(北京试剂厂)，RpMI1640细胞培养液(上海元龙生物技术有限公司)、小牛血清、2-巯基乙醇、青霉素、链霉素、ConA液(北京梦怡美生物科技有限公司)、无菌Hank’s液、BrdUp标记液(北京梦怡美生物科技有限公司)、二硝基氟苯(北京市庆盛达化工技术有限公司)、硫化钡、绵羊红细胞(SRBC)、豚鼠血清、印度墨汁(上海远慕生物科技有限公司)、YAC-1细胞(上海酶研生物科技有限公司)、Tris-HCL缓冲液(北京梦怡美生物科技有限公司)。
1.3实验动物
3-4周龄健康雄性BALB/c小鼠770只，体重11-13g，由北京华阜康生物科技有限责任公司提供。饲养于中国医学科学院协和药物研究所二级动物房：维持室温(25±2℃)，相对湿度(55±2)％，12h/12h光照，自由进食和饮水，动物4-5只每笼，适应性喂养3天后开始正式实验。
动物共770只，分为5大免疫组进行喂养，每个免疫大组154只，随机分为11组(5种核苷酸样品高低剂量各一组，共10组，1组作为对照组)，每组14只。免疫1组：迟发型变态反应；免疫2组：小鼠淋巴细胞转化试验、NK细胞活性测定、脏体比值；免疫3组：半数溶血值、抗体生成细胞数；免疫4组：碳廓清试验；免疫5组：小鼠腹腔巨噬细胞吞噬荧光微球试验。以灌胃方式给药各样品28天，每天灌胃1次，灌胃体积为0.2mL/10g。 对照组灌胃给予蒸馏水。给药结束后处死小鼠并测定各项免疫指标。
1.4测试样品
样品1-3为本申请的核苷酸组合物。比较样品2和3为现有技术中公开的另外两种核苷酸组合物。每种样品设低、高两个剂量组，分别为120.7mg/kg和1207.0mg/kg。
1.5检测项目和指标
1.5.1脏器/体重比值：小鼠初始称重和给药28天后称重分别作为初始体重和终体重。将小鼠脱臼处死，取脾脏和胸腺，去尽筋膜，用滤纸吸干脏器表面血污，称重，计算脾脏/体重比值和胸腺/体重比值。
1.5.2迟发型变态反应(足趾增厚法，DTH)：动物连续给药28天后，每鼠腹腔注射2％压积SRBC(v/v，用生理盐水配制)SRBC 0.2mL。致敏4天后，测量左后足跖部厚度，同一部位测量3次，取平均值。然后在测量部位皮下注射20％SRBC 20μL，注射后于24h测量左后足跖部厚度，同一部位测量3次取平均值，以攻击前后足趾厚度的差值(足趾肿胀度)来表示DTH的程度。
1.5.3ConA诱导的小鼠淋巴细胞转化(MTT法)：动物连续给药28天后，将小鼠处死，在75％酒精的烧杯中消毒后，无菌取脾，置于装有3cm×3cm四层纱布(高压灭菌)的小平皿中，加入适量无菌Hank’s液，用纱布将脾包住，用弯头镊轻轻将脾磨碎，制成单细胞悬液。将其用Hank’s液洗涤2次，每次以1000rpm离心10min。然后将细胞悬浮于2mL的完全培养液中，计数活细胞数，调整细胞浓度为5×106个/mL。再将细胞悬液分两孔加入24孔培养板中，每孔1mL，在其中一孔加入75μLConA液(相当于7.5μg/mL)，另一孔作为对照。5％CO2，37℃下培养72h。培养结束前4h，每孔轻轻吸去上清液0.7mL，加入0.7mL不含小牛血清的RPMI 1640培养液，同时加入MTT(5mg/mL)50μL/孔，继续培养4h。培养结束后，每孔加入1mL酸性异丙醇，吹打均匀，使紫色结晶完全溶解。然后分装到96孔培养板中，每个孔分装3孔作为平行样，用酶联免疫检测仪，以570nm波长测定光密度值。淋巴细胞的增殖能力用加ConA孔的光密度值减去不加ConA孔的光密度值表示。
1.5.4抗体生成细胞检测：动物连续给药28天后，取羊血，生理盐水洗涤3次，每只鼠经腹腔注射2％(V/V，用生理盐水配制)压积SRBC 0.2mL 进行免疫。将SRBC免疫4天后的小鼠处死，取脾，制成5×106个细胞/mL的细胞悬液。将琼脂糖加热溶解后，与等量双倍Hank’s液混合，分装小试管，每管0.5mL，再向管内加20％(V/V，用生理盐水配制)压积SRBC 50μL，脾细胞悬液200μL，迅速混匀，倾倒至已刷琼脂糖薄层的六孔板上，待琼脂凝固后，放入二氧化碳培养箱中继续孵育1h，然后加入SA缓冲液稀释的补体(1∶10)，继续孵育2h，计数溶血空斑数。
1.5.5血清溶血素半数溶血值(HC50)：动物连续给药28天后，取羊血，生理盐水洗涤3次，每只鼠经腹腔注射2％(V/V，用生理盐水配制)压积SRBC0.2mL进行免疫。4天后，摘除眼球取血于1.5mL离心管内，4℃放置约1h，使血清充分析出，2000rpm离心10min，收集血清。取血清，用SA缓冲液稀释100倍。将稀释后的血清加入96孔板，每孔100μL，再依次加入10％(v/v)SRBC 50μL，补体100μL(用SA溶液按1∶8稀释)，在37℃恒温水浴中保温30min，然后以1500rpm离心10min。然后样品孔和空白对照孔各取上清液50μL，加入另一个96孔培养板内，加文齐氏试剂150μL。同时设半数溶血孔，加入10％(v/v)SRBC 12.5μL，再加文齐氏试剂至200μL。用震荡器充分混匀，放置10min后，于540nm处用全自动酶标仪测定各孔光密度值。
溶血素的量以半数溶血值(HC50)表示，按以下公式计算：
样品HC50＝(样品光密度值/SRBC半数溶血时的光密度值)×稀释倍数
1.5.6小鼠腹腔巨噬细胞吞噬荧光微球试验：动物连续给药28天后，灌胃结束前4天给每只小鼠腹腔注射0.2mL 2％SRBC激活小鼠巨噬细胞。实验当天用颈椎脱臼法处死小鼠，腹腔注射加小牛血清的Hank′s液3mL/只，轻轻按揉腹部20次，以充分洗出腹腔巨噬细胞。然后将腹壁剪开一个小口，吸取腹腔洗液2mL，用75μm过滤器过滤至试管内，调整巨噬细胞数为4～6×105/mL。用移液枪吸取1mL腹腔洗液于6孔培养板中，加入已经预调理过的荧光微球(1×107/板)，37℃二氧化碳细胞培养箱避光孵育120分钟。孵育结束后弃上清(含未贴壁细胞和多余荧光微球)，每次使用1.0mL PBS缓冲液轻轻洗涤2次，去除上清后再加入4℃的PBS缓冲液0.3mL，用细胞刮刮下贴壁细胞，轻轻吹打均匀后经75μm过滤器过滤后上机分析。
结果按下式计算：
Figure PCTCN2016074813-appb-000008
Figure PCTCN2016074813-appb-000009
1.5.7NK细胞活性测定(乳酸脱氢酶(LDH)测定法)：动物连续给药28天，开始实验前24小时将靶细胞YAC-1进行传代培养，用前以Hank’s液洗涤2次，用含10％小牛血清的RPMI 1640完全培养液调整细胞浓度为1×105个/mL(靶细胞)。小鼠颈椎脱臼处死，无菌取脾，制成脾细胞悬液，用Hank’s液洗涤2次，以1000rpm离心10min，再用2mL含10％小牛血清的RPMI 1640完全培养基重悬，用台盼蓝活细胞染色计数(活细胞数应在95％以上)，调整细胞浓度为1×107个/mL(效应细胞)，使效应细胞和靶细胞的比率为100∶1。取靶细胞和效应细胞各100μL，加入U型96孔培养板中，靶细胞自然释放孔加靶细胞和培养液各100μL，靶细胞最大释放孔加靶细胞和1％NP40各100μL，上述均设三个平行孔，37℃，5％CO2培养箱中培养4小时。将96孔培养板以1500rpm离心5min，每孔吸取上清100μL置平底96孔培养板中，同时加入LDH基质液100μL，反应3min，然后每孔加入1mol/L的HCL溶液30μL终止反应，在酶标仪490nm处测OD值，NK活性按下式计算：
Figure PCTCN2016074813-appb-000010
1.5.8碳廓清实验：动物连续给药28天，称体重，尾静脉注射印度墨汁，注入墨汁后2、10min，取血20μl，加入2ml 0.1％碳酸钠溶液中，在600nm处测定OD值。将小鼠处死，取肝脏和脾脏，用滤纸吸干脏器表面血污，称重。
以吞噬指数表示小鼠碳廓清的能力，按照以下公式进行计算：
Figure PCTCN2016074813-appb-000011
Figure PCTCN2016074813-appb-000012
1.6判定标准：在细胞免疫、体液免疫功能、单核-巨噬细胞功能、NK细胞活性四个方面任两个方面结果阳性，可判定该样品具有增强免疫力功能作用。其中细胞免疫功能测定项目中的两个实验结果均为阳性，或任一个实验的两个剂量组结果阳性，可判定细胞免疫功能测定结果为阳性。体液免疫功能测定项目中的两个实验结果均为阳性，或任一个实验的两个剂量组结果阳性，可判定体液免疫功能测定结果为阳性。单核-巨噬细胞功能测定项目中的两个实验结果均为阳性，或任一个实验的两个剂量组结果阳性，可判定单核-巨噬细胞功能结果阳性。NK细胞活性测定实验的一个以上剂量组结果阳性，可判定NK细胞活性结果阳性。
2.结果：
经口给予小鼠各试验样品28天后，各项免疫功能检测指标如下：
体重及脏器比结果各样品组与正常组比较，均无显著差异。
所有阳性和阴性结果如表3所示。
表3
Figure PCTCN2016074813-appb-000013
从表3的结果可以看出，根据整体的四个大方面(即上文1.6节所述的四个方面)的判断标准，样品1、2、3和比较样品3判定为阳性，因此具有免疫刺激功效。相比之下，比较样品2则不判定为阳性。
在此基础上，对于样品1-3和比较样品3，从上文(1.6节)的剂量组方面而言，本发明的核苷酸组合物的免疫刺激阳性指标更多，从而表现出更强和更全面的免疫刺激功效。例如1)样品1具有细胞免疫功效，其他样品不具有；2)样品1具有体液免疫功效，其他样品不具有；3)样品1和2在碳廓清试验指标下为阳性，其他样品不具有；4)样品2、3和比较样品3具有巨噬细胞吞噬功能，其他样品不具有；5)样品1、2、3和比较样品3的NK细胞活性为阳性。
由以上结果可知，本发明的核苷酸组合物能够实现有效地刺激免疫功 能的效果，且效果优于现有技术的组合物。同样，如实施例1中所述，更强的免疫调节作用意味着以更少的剂量能够实现相同或相似的效果，这对于大规模工业生产会具有经济成本上的明显优势。
实施例3：核苷酸组合物的损伤保护和修复作用
本实施例的目的是用于证实本发明的核苷酸组合物在提供针对损伤的保护和修复方面的效果。
1.材料与方法
1.1仪器与试剂
RPMI-1640培养基和DMEM高糖培养基购自Gibco。5’-AMP(A1752)、5’-CMP-Na2(C1006)、5’-GMP-Na2(G8377)、5’-UMP-Na2(U6375)、噻唑兰(MTT)、胰岛素均购自Sigma。胎牛血清购自杭州四季青。本实施例所用的试剂盒如LDH、SOD、MDA检测试剂盒均购自南京建成生物工程研究所。胰酶、双抗、BCA蛋白测定试剂盒购自碧云天。其余试剂如二甲亚砜(DMSO)，过氧化氢等均为国产分析纯，均购自上海国药集团化学试剂有限公司。25cm2培养瓶、96孔细胞培养板、6孔细胞培养板、60mm细胞培养皿、50mL离心管、15mL离心管均购自美国Coming公司。一次性针头过滤器购自Millipore公司。主要实验设备有Heal Force生物安全柜(中国香港力康公司)，Countstar细胞计数仪(美国Inno-Alliance Biotech公司)，EVOS FL荧光型显微镜(Thermo Fisher公司)，酶标仪(芬兰雷勃)，721可见分光光度计(上海仪电分析仪器有限公司)，恒温水浴箱(上海博迅)，超声波破碎仪(宁波新芝生物科技股份有限公司)，倒置显微镜(日本尼康)，冷冻离心机(Thermo Fisher公司)。
1.2实验材料
大鼠小肠隐窝上皮细胞(IEC-6细胞)获得自自中国医学科学院基础细胞研究所。大鼠正常肝细胞(BRL 3A细胞)获得自中科院上海细胞库。
1.3测试样品
样品1-3为本申请的核苷酸组合物。比较样品2和3为现有技术中公开的核苷酸组合物(见上文)。每种样品均设低、中、高三个浓度(分别为62.5、250、1000μmol/L)。
1.4实验模型和指标
氧损伤是最典型和常见的机体损伤(如肠道损伤)之一。已知人和动物的许多疾病如消化系统疾病与自由基和活性氧物质密切相关，例如氧损伤已经证实是炎性肠病的原因之一。细胞内的氧化还原反应在正常生理状态下会保持平衡，而体内的氧自由基和抗氧化系统是维持该平衡的重要因素。一旦氧自由基大量产生或抗氧化系统功能减弱，都会引起组织细胞的损伤。SOD(超氧化物歧化酶)则属于重要的抗氧化酶，能清除过氧化物产生的O2 -，保护细胞免受损伤，对细胞内的氧化/抗氧化平衡起到重要作用。对于氧化物/过氧化物引起的损伤，细胞释放入培养上清的乳酸脱氢酶(LDH)是细胞膜完整性的标志或者说是细胞坏死的指标。另外，细胞内的氧化物/过氧化物能够攻击生物膜中的多不饱和脂肪酸，引起脂质过氧化作用，从而形成脂质过氧化物，如丙二醛(MDA)。氧自由基不但通过生物膜中多不饱和脂肪酸的过氧化引起细胞损伤，而且还能通过脂氢过氧化物的分解产物引起细胞损伤，因而MDA的量通常可以反映脂质过氧化的程度，进而反映出细胞损伤的程度。因此，这些物质的浓度或活性或者其变化以及细胞存活率可以用来评价本发明的核苷酸组合物的保护效果。参见，“大黄多糖对肠上皮细胞损伤的修复作用及其机理研究”，第四军医大学博士毕业论文，刘琳娜，2005年5月1日；“保内龙眼参多糖对四氯化碳诱导大鼠原代肝细胞损伤的保护作用”，段小群等人，中国药房，2006年第17卷第15期，1132-1143；“保肝宁对过氧化氢损伤大鼠肝细胞的保护作用”，赵进军等，广州中医药大学学报，2002年第19卷第3期，211-213。
另外，经申请人实验证实，600μmol/L的H2O2会显著降低BRL 3A细胞的存活率(P＜0.01)，因此，在BRL 3A氧化损伤中采用600μmol/L的H2O2诱导细胞氧化损伤。类似地，对于IEC-6细胞采用100μmol/L的H2O2诱导细胞氧化损伤。
基于以上情况，在这个实施例的实验中，通过用核苷酸组合物处理细胞，用H2O2诱导细胞氧化损伤，来研究本发明的组合物对于细胞氧化损伤的保护效果。对于BRL 3A细胞，测试指标有细胞存活率和SOD活性。对于IEC-6细胞，测试指标有LDH活性和MDA含量。
1.5细胞培养及样品准备
IEC-6细胞采用含5％FBS、2mg/L胰岛素的RPMI-1640培养基，于37℃、5％CO2培养箱中培养，每隔3天传代一次。BRL 3A细胞采用含10％FBS 的DMEM高糖培养基，于37℃、5％CO2培养箱中培养，每隔4天传代一次。核苷酸组合物用PBS配成50mmol/L母液，过滤除菌，在-20℃下储存备用。
1.6实验方法
对于细胞存活率，取对数生长期的细胞，以5000个/孔接种于96孔板，待细胞贴壁后采用含不同浓度核苷酸组合物样品的培养基培养24h(每个浓度均设6个复孔)，实验同时设不加H2O2的正常对照和只加H2O2的阳性对照。24h后分别加入H2O2诱导细胞氧化损伤。2h后用MTT法检测细胞存活率，细胞存活率＝实验组的OD值/正常对照的OD值×100％。
对于其他指标，取对数生长期的细胞，按照7×105个/皿接种到60mm细胞培养皿中。待细胞贴壁后采用含不同浓度核苷酸组合物样品的培养基培养24h(每个浓度均设3个重复)，实验同时设不加H2O2的正常对照和只加H2O2的阳性对照。24h后加入一定浓度的H2O2诱导细胞氧化损伤，继续培养，2h后吸取培养液，离心取上清检测相关指标。细胞用胰酶消化，离心后用PBS洗涤一次，然后重悬于700μlPBS，超声波破碎细胞，于2日内检测所有指标。
1.7数据分析
所有数据均以平均数±标准差
Figure PCTCN2016074813-appb-000014
表示，采用SPSS17.0软件进行统计分析，组间差异比较采用单因素方差分析，P＜0.05有统计学意义，P＜0.01有高度统计学意义。
2.结果
2.1细胞存活率实验
BRL 3A细胞的细胞存活率的实验结果示于图1中。从该图可见，H2O2处理使得BRL 3A的细胞存活率显著下降(P＜0.01)。通过用核苷酸组合物样品处理细胞，细胞存活率有不同程度升高，尤其是样品1和2。统计结果表明，样品1的所有浓度组、样品2的低中浓度组以及样品3的低浓度组的细胞存活率均显著高于H2O2对照(P＜0.01或P＜0.05)。此外，样品1的所有浓度组及样品2的低浓度组的细胞存活率均显著高于比较样品2和3的所有浓度组(P＜0.01或P＜0.05)。可见，通过用本发明的核苷酸组合物处理细胞，能够有效地提高氧化损伤的细胞的存活率，从而表现出针对氧化损伤的保护效果，进而促进细胞损伤后的修复。而且这样的保护效果优于 现有技术的核苷酸组合物。
2.2SOD活性实验
BRL 3A细胞的SOD活性实验结果示于图2中。从该图可见，H2O2处理使得细胞中的SOD活性显著下降(P＜0.01)。通过用核苷酸组合物处理细胞，细胞中的SOD活性有不同程度的升高。其中样品1的所有浓度组(P＜0.05)、样品2的中浓度组(P＜0.05)、样品3的中高浓度组(P＜0.01)的细胞中的SOD活性都显著高于H2O2对照。并且，样品3中浓度组细胞SOD活性显著高于比较样品2的中高浓度组(P＜0.05)及比较样品3的所有浓度组(P＜0.05)。样品3的高浓度组细胞SOD活性显著高于比较样品2的高浓度组(P＜0.05)及比较样品3的低高浓度组(P＜0.01)。可见，本发明的核苷酸组合物能够有效地提高氧化损伤的细胞内抗氧化活性物质(如SOD)的浓度，从而表现出针对氧化损伤的保护效果，而且保护效果优于现有技术的核苷酸组合物。
2.3.LDH活性实验
IEC-6细胞的LDH活性实验的结果示于图3。从该图可见，用H2O2处理细胞会使得培养液上清中的LDH含量高于正常对照(P＜0.01)。通过用核苷酸组合物处理细胞，上清中LDH含量有不同程度的下降，其中样品2的低浓度组和样品3的中浓度组中LDH含量下降最明显，与正常对照没有统计学差别。另外，与H2O2对照相比，样品1的中浓度组(P＜0.05)、样品2的低高浓度组(P＜0.01或P＜0.05)、样品3的低中浓度组(P＜0.01)的LDH含量均显著下降。而且，与比较样品2的高浓度相比，样品2的低浓度组(P＜0.05)以及样品3的低中浓度组(P＜0.01)的LDH含量明显下降。可见，本发明的核苷酸组合物能够有效地降低氧化损伤的细胞的上清液中代表不利指标的物质(例如LDH)的浓度，从而表现出针对氧化损伤的保护效果，而且保护效果优于现有技术的核苷酸组合物。
2.4.MDA浓度实验
IEC-6细胞的MDA浓度实验的结果示于图4中。从该图可见，用H2O2处理细胞后，细胞中MDA含量会显著升高(P＜0.01)。通过用核苷酸组合物处理细胞，样品1-3的所有浓度组的MDA含量均有不同程度的下降，且显著低于H2O2对照。相比之下，比较样品2和3的中高浓度组则相对H2O2对照无显著变化。可见，本发明的核苷酸组合物能够有效地降低氧化损伤 的细胞内代表不利指标的物质(例如脂质过氧化物MDA)的浓度，从而表现出针对氧化损伤的保护效果。特别地，本发明的核苷酸组合物在所有浓度范围下均能表现出显著和一致的保护效果。相比之下，现有技术的核苷酸组合物在浓度较高的情况下(例如中和高浓度)则无法体现出保护效果。这进一步说明本发明的核苷酸组合物相对于现有技术的优势。
3.讨论
从以上结果可见，通过用本申请的核苷酸组合物进行处理，能够提高氧化损伤的细胞的存活率，提高细胞内具有氧化保护效果的因子(例如SOD)活性/浓度，降低代表不利影响的因子的活性/含量(例如LDH和MDA)。这意味着本申请的核苷酸组合物能够提供针对细胞氧化损伤的保护效果，且效果优于现有技术中使用的核苷酸组合物。同时，通过增加具有保护效果的因子活性和降低不利因子的含量，本发明的核苷酸组合物能够促使细胞恢复损伤前的平衡状态，从而促进损伤后的修复。而且，本发明的核苷酸组合物的效果不仅可以表现在肠细胞，还可以表现在消化系统的其他部分(如肝脏)，从而表现出对整个消化系统的积极的作用。
实施例4：细胞增殖效果
本实施例中所用的仪器和试剂参见实施例3，其中使用IEC-6细胞验证本发明的核苷酸组合物对细胞增殖的促进作用。
在本实施例中，采用MTT法检测核苷酸组合物对IEC-6细胞增殖的影响。取处于对数生长期的IEC-6细胞，采用胰酶消化细胞，离心后制备成细胞悬液。采用细胞计数仪计数，按照5000个/孔接种于96孔板，置37℃，5％CO2条件下培养过夜，使细胞贴壁。次日更换培养液，各孔分别加入含有不同浓度核苷酸组合物(每个样品均设62.5、250、1000μmol/L 3个浓度)的细胞培养液200μL。每个样品的每个浓度均设6个复孔，同时还设有不加核苷酸组合物的对照组及不加细胞的调零孔(只加正常培养基)，每24h更换一次培养液。在核苷酸组合物处理后的第24h取96孔板，每孔加入MTT 10μL，轻轻震荡，放回培养箱中孵育4h。4h后弃上清液，加入DMSO150μL/孔，避光震荡混匀，待蓝紫色甲臜结晶溶解后，将培养板放入酶标仪中，于490nm波长测定OD值，以不加细胞的空白组调零。以OD值绘制曲线。
所有数据均以平均数±标准差
Figure PCTCN2016074813-appb-000015
表示，采用SPSS17.0软件进行统计分析，组间差异比较采用单因素方差分析，P＜0.05有统计学意义，P＜0.01有高度统计学意义。
结果示于图5中。该图中的结果表明：加入核苷酸样品后，IEC-6细胞的增殖与对照相比有明显变化。样品1的高浓度组、样品3的所有浓度组的细胞增殖速度明显高于正常对照(P＜0.01)；样品2的中浓度组的细胞增殖速度也高于对照组(P＜0.05)。比较样品2低浓度组的细胞增殖明显低于样品1的高浓度组、样品2的低中浓度组、样品3的低中高浓度组(P＜0.01)。样品2的高浓度组细胞增殖也明显高于比较样品2的低浓度组(P＜0.05)。可见，本申请的核苷酸组合物能够促进细胞增殖，特别是促进肠道细胞增殖和生长，从而表现出促进肠上皮细胞生长，提高肠上皮细胞增殖的效果，并且效果优于现有技术的核苷酸组合物。
实施例5：对肠道的有益菌群的效果
本实施例证实本申请的核苷酸组合物对于肠道有益菌群的刺激作用。每个样品采用2个浓度(低剂量：1g/100ml；高剂量：2g/100ml)，对照组中不加入核苷酸。
1.材料与方法
1.1.主要试剂
伊红美蓝琼脂(EMB)；胰
Figure PCTCN2016074813-appb-000016
-亚硫酸盐-环丝氨酸琼脂(TSC)；双歧杆菌琼脂培养基(BBL)；氢氧化钠；盐酸；卵黄乳液50％；D-环丝氨酸；核苷酸样品(南京同凯兆业)。
1.2.主要仪器设备
自动立体蒸汽灭菌器(LDZX-30FBS，上海申安医疗器械厂)；二氧化碳培养箱(HH.CP-TW9，上海申贤恒温设备厂)；电子天平(XS1003S，瑞士)；洁净工作台(VS-130L-U，苏州安泰空气技术有限公司)；菌落计数仪(DIGITAL S 4905000，西班牙Selecta)；密封培养盒(C-32日本三菱)。
2.研究方法
2.1.肠道菌群的获取
在无菌台中从5只SPF级小鼠肛门处取共约0.5g粪便样品，置于无菌试管中，加入灭菌的玻璃珠3颗，涡旋震荡后进行梯度稀释，稀释至10-2 水平，作为后续试验的种子液。
2.2.培养液的制备
采用牛肉膏蛋白胨液体培养基作为基础培养基，在其中分别按照1g/100ml、2g/100ml添加核苷酸样品，按照1％(体积)接入2.1中制备的种子液。
2.3.菌落计数
使用特定的选择性培养基(BBL琼脂)对培养液中的双歧杆菌在36+1℃的条件下厌氧培养48h后进行菌落计数进行测定，结果以Lg CFU/ml表示。
2.4.数据统计
使用SPSS17.0统计软件对数据进行分析，采用单因素方差分析对各组数据进行比较，以α＝0.05作为差异判定标准。结果如表4所示：
表4
Figure PCTCN2016074813-appb-000017
*代表显著性增长
统计结果表明，以双歧杆菌为例，用本申请的核苷酸组合物处理之后，双歧杆菌的生长相比对照明显提高(Lg CFU)，表明本申请的核苷酸组合物能够刺激肠道有益菌群的生长。
实施例6：婴儿奶粉制备(1000公斤，干法添加)
本发明的奶粉所用的原料有：脱脂奶粉140千克、乳糖260千克、脱盐乳清粉200千克、乳清蛋白粉(WPC34％)100千克、玉米油52千克、大豆油42千克、葵花籽油170千克、低聚果糖粉4.8千克、低聚半乳糖浆12千克、复配营养素6.4千克以及2千克的大豆磷脂。将上述原料混合均匀后，经巴氏杀菌、均质、蒸发浓缩和喷雾干燥成粉状半成品，然后添加0.38千克本发明的核苷酸组合物、0.1千克双歧杆菌、4.32千克DHA、 6.0千克ARA，并经过干混机混合。将混合均匀后的奶粉充氮包装即得终产品。
实施例7：婴儿奶粉制备(1000公斤，干法添加)
将脱脂奶粉150千克、乳糖250千克、脱盐乳清粉190千克、乳清蛋白粉(WPC34％)110千克、玉米油44千克、大豆油44千克、葵花籽油175千克、低聚果糖粉4.5千克、低聚半乳糖浆11千克、复配营养素7千克、2千克大豆磷脂混合均匀，然后进行巴氏杀菌、均质、蒸发浓缩和喷雾干燥，制成成粉状半成品。再添加0.5千克本发明的核苷酸组合物、0.1千克双歧杆菌、5.2千克DHA和6.7千克ARA，通过干混机混合。将混合均匀后的奶粉充氮包装即得终产品。
实施例8：婴儿奶粉食品制备(1000公斤，湿法添加)
将脱脂奶粉150千克、乳糖250千克、脱盐乳清粉190千克、乳清蛋白粉(WPC34％)110千克、玉米油43千克、大豆油44.7千克、葵花籽油178千克、低聚果糖粉4.5千克、低聚半乳糖浆11千克、复配营养素7千克、0.4千克本发明的核苷酸组合物和2千克的大豆磷脂混合均匀。然后进行巴氏杀菌、均质、蒸发浓缩和喷雾干燥制成粉状半成品。再添加0.1千克双歧杆菌、3.9千克DHA和5.4千克ARA，经过干混机混合，混合均匀后的奶粉充氮包装即得终产品。
实施例9：婴儿液态奶的制备(按100g液态乳计)
本发明的液态乳包含以下营养成分：
蛋白质2.2g、脂肪3.0g、乳糖9.0g、维生素A80μgRE、维生素D 1.5μg、维生素E 1mgα-TE、维生素K16μg、维生素B170μg、维生素B270μg、维生素B6200μg、维生素B120.5μg、烟酸350μg、叶酸3.5μg、泛酸200μg、维生素C 7mg、生物素2μg、钠50mg、钾70mg、铜80μg、镁30mg、铁1mg、锌0.7mg、钙80mg、磷45mg、碘20μg、氯100mg、乳铁蛋白10mg以及本发明的核苷酸组合物2.7mg，其中能量为约300kJ。
脂肪由无水奶油、大豆油、玉米油、葵花籽由以任意比例组合提供，但其中以100g总配料量计，含亚油酸0.5g。
制备方法如下(原料及工艺均符合国家相关标准)：
1、原料乳验收、检验和优选。
2、(1)用温水溶解蛋白质(包括酪蛋白、乳清蛋白、乳铁蛋白)、B族维生素和维生素C以及本发明的核苷酸组合物；
(2)用温水溶解碳水化合物类原料和卡拉胶、单硬脂酸甘油酯、瓜尔豆胶等食品添加剂；用温水溶解矿物质；并用40℃～50℃的脂肪类料液溶解其它原料；然后将上述三份溶解好的料液混合均匀；
(3)将步骤(1)得到的料液通过膜过滤除菌处理、杀菌；
(4)将步骤(2)得到的混合均匀的料液通过UHT(超高温瞬时处理)灭菌处理；
(5)将上述步骤(3)和(4)得到的料液进行无菌混合，并用水定容至总配料量；
(6)将上述混合后的料液进行无菌均质；以及
(7)无菌灌装、包装。
除非另有说明，本说明书(包括权利要求书)中使用的所有表示成分、细胞培养、处理条件等的量的数字应当理解为在所有条件下受到术语“约”的修饰。因此，除非另有相反的说明，数值参数为近似值，并且可以根据通过本发明试图获得的期望特性而变化。除非另有说明，一系列元素之前的术语“至少”应当理解为指该系列中的每个元素。本领域技术人员会认识到或者能够利用不超过常规实验确定本文所述的本发明的具体实施方案的许多等同物。所附权利要求书意图涵盖这类等同物。
本领域技术人员会清楚，可以进行本发明的许多修改和变化而不背离其精神和范围。本文所述的具体实施方案仅通过实例的方式提供，并不意味着以任何方式限制。本发明的真正范围和精神通过所附权利要求书示出，说明书和实施例仅是示例性的。

Claims (19)

  1. 一种用作食品添加物的核苷酸组合物，其中按重量计，所述组合物基本上由CMP、AMP、UMP和GMP组成，并且各组分的比例为CMP：58～72％、AMP：6～14％、UMP：10～18％以及GMP：8～14％，条件是各种组分之和为100％。
  2. 权利要求1的组合物，其中各组分的比例为CMP：60～70％、AMP：8～12％、UMP：12～16％以及GMP：10～12％，条件是各种组分之和为100％。
  3. 权利要求1的组合物，其中各组分的比例为CMP：60～65％、AMP：10～12％、UMP：14～16％以及GMP：11～12％，条件是各种组分之和为100％。
  4. 权利要求1的组合物，其中各组分的比例为CMP：65～70％、AMP：8～10％、UMP：12～14％以及GMP：10～11％，条件是各种组分之和为100％。
  5. 权利要求1-4中任一项的组合物，其中各组分的比例为
    CMP：60％、AMP：12％、UMP：16％以及GMP：12％；
    CMP：65％、AMP：10％、UMP：14％以及GMP：11％；或者
    CMP：70％、AMP：8％、UMP：12％以及GMP：10％。
  6. 一种用作食品添加物的核苷酸组合物，其中按重量计，所述组合物基本上由CMP、AMP、UMP、GMP和IMP组成，并且各组分的比例为CMP：58～70％、AMP：7.5～12.5％、UMP：12～16.5％、GMP：10～13％以及IMP：0～2.5％，条件是各种组分之和为100％。
  7. 权利要求6的组合物，其中各组分的比例为CMP：60～65％、AMP：8～12％、UMP：14～16％、GMP：11～12％以及IMP：0～2％，条件是各种组分之和为100％。
  8. 权利要求1-7中任一项的组合物，其中所述食品为婴幼儿食品。
  9. 权利要求1-8中任一项的组合物，其中所述食品为乳制品的形式。
  10. 权利要求1-9中任一项的组合物，其中所述食品为奶粉或液态乳制品的形式。
  11. 一种食品，其包含权利要求1-10中任一项的核苷酸组合物。
  12. 权利要求11的食品，其为婴幼儿食品。
  13. 权利要求11或12的食品，其中所述食品为乳制品的形式。
  14. 权利要求11-13中任一项的食品，其中所述食品为为奶粉或液态乳制品的形式。
  15. 权利要求1-10中任一项的核苷酸组合物在制备食品中的应用。
  16. 权利要求15的应用，其中所述食品为婴幼儿食品。
  17. 权利要求15或16的应用，其中所述食品为乳制品的形式。
  18. 权利要求15-17中任一项的应用，其中所述食品为奶粉或液态乳制品的形式。
  19. 权利要求15-18中任一项的应用，其中所述食品用于实现以下方面的效果：提供免疫刺激效果、促进生长发育、促进肠道损伤后的修复、促进肠道有益微生物的生长和/或它们的任意组合。
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