CN115997937B - Nutritional composition containing milk fat globule membrane, application and food - Google Patents

Abstract

The invention provides a nutritional composition containing milk fat globule membrane, application and food, wherein the nutritional composition comprises a milk fat globule membrane component (MFGM) and an alkaline protein component, the content ratio of the alkaline protein to the MFGM is more than 1:100 by mass, and the nutritional composition has a synergistic gain promotion effect on bone health (skeleton development, bone density improvement and bone metabolism regulation).

Description

Nutritional composition containing milk fat globule membrane, application and food

Technical Field

The present invention relates generally to the field of food products. In particular, the present invention relates to a nutritional composition for promoting bone health (bone development, improving bone density, regulating bone metabolism), a nutritional/health food comprising the nutritional composition, and the use of the nutritional composition. More particularly, the present invention relates to a nutritional composition comprising basic protein and Milk Fat Globule Membrane (MFGM), a food product comprising the nutritional composition, and the use of the nutritional composition for non-therapeutic purposes for improving the height and growth of infants and children, in particular for promoting bone development in children, increasing bone density.

Background

Osteoporosis is a systemic skeletal disease characterized by a decrease in bone strength, which depends on bone density and bone mass, and an increased risk of fracture. The difference in bone density in the middle-aged and elderly people is closely related to the peak bone mass achieved in young age and the rate of bone loss in middle-aged and elderly people.

Normal total bone mass peaks after epiphyseal closure of the long bones, this peak bone mass being called Peak Bone Mass (PBM). It is counted that every 10% increase in peak bone mass, the risk of developing osteoporotic fracture will be reduced by 50% in the future, or the age of onset of osteoporosis will be delayed for 13 years, it being seen that peak bone mass is critical for maintaining bone strength, reducing the risk of developing osteoporosis. Half of the peak bone mass in individuals accumulates during childhood and adolescence, and over 1/4 of the peak bone mass rapidly accumulates during adolescence, up to 18 years, and has reached 90% of the peak bone mass in individuals.

Therefore, the childhood and adolescence are key periods of peak bone mass accumulation, and if the rapid bone mass accumulation period can be fully utilized, the body is promoted to reach higher peak bone mass, and the method has positive significance for preventing osteoporosis in middle-aged and elderly people. It is important to recognize skeletal development characteristics of children and teenagers and to explore factors influencing the development of the children and teenagers so as to promote the body to obtain ideal peak bone mass.

The growth and development of bones in children and adolescents are rapid, bone construction is mainly performed, bone transformation is accelerated, and bone formation is dominant, so that bone mass steadily increases. The weight of the bones is 70-95g when the human body is born, and the average total weight of the bones reaches 2400g when the female reaches 3300g when the female reaches the end of puberty. Bone growth includes linear growth and bone mass accumulation, with childhood being a major linear growth, with an average rate of growth of about 5-6cm per year. Adolescence, especially adolescence, is dominated by the accumulation of bone mass, with about 40% -60% of adult bone mass being achieved in adolescence, by the age of 18, 90% of peak bone mass has been accumulated.

Therefore, the normal growth and development of the bones in the childhood and adolescence stage has important significance for obtaining ideal peak bone mass and reducing diseases such as osteoporosis and the like in the future. However, there is room for further improvement in the current research on the state of skeletal growth and development and its influencing factors in the childhood and adolescence stage. The growth and development of the bones of children and teenagers are complicated by factors such as heredity, endocrine, nutrition, movement, diseases, medicines and the like, the normal growth and development of the bones at the stage are concerned, the interference of adverse factors is reduced, the endocrine regulation and control are actively improved, the nutrition and movement are enhanced, the positive balance of calcium is promoted, the deficiency of vitamin D is corrected, and the method has profound significance for the bone health in the future.

The nutrition plays an important role in bone growth and development, and the nutrition elements such as protein, fat, calcium, vitamin D and the like are worth paying attention.

In addition, the 'milk' is a complex suspension, and the fat is one of the most main components of the milk, and the ratio of the fat to the milk reaches 3% -5%. Milk fat is present in milk in the form of small droplets, and is spherical, and is therefore called milk fat globules. The diameter of the milk fat globules is about 0.2-15 μm, and the surface is covered with a thin film of 10-20 nm called milk fat globule membrane (Milk fat globule membranes, MFGM). The emulsion can ensure the stability of fat globules in milk to a great extent, and protect the fat globules from aggregation and decomposition. The content of MFGM in cow's milk is indicated in reference 1 to be about 3.6g/L, and the protein and lipid contents are about 22.3% and 71.8%, respectively.

In recent years, milk fat globule membrane proteins are widely applied to the ingredient list of infant formula milk powder, and the special nutritional value and functional characteristics of the milk fat globule membrane proteins are increasingly concerned by the food industry, in particular the infant formula milk powder industry. It has an important role in breast milk, and can promote brain development of infants and improve cognitive ability, improve metabolism, reduce gastrointestinal tract infection and improve immunity (cited document 2).

In addition, colostrum alkaline protein (CBP) is a small molecule active peptide prepared from bovine colostrum by the processes of sterilization, degreasing, centrifugal separation, casein removal, alpha-lactalbumin, beta-lactoglobulin, microfiltration, ultrafiltration, freeze drying and the like, and researches show that the small molecule active peptide can improve the utilization rate of Ca ions, promote osteoblast proliferation, inhibit osteoclast proliferation and stimulate the secretion of growth factors. More and more businesses begin to add CBP to the product and thus the colostrum alkaline protein is either a new resource food.

Although some research has been conducted on milk fat globule membranes in terms of exploration and application, there is still a wide exploration space for their development or for their compounding with other nutritional components.

Citation literature:

citation 1: FONG B Y, NORRIS C S, MAGIBBON AK H.protein and lipid composition of bovine milk-fat-global membrane.International Dairy Journal,2007,17 (4): 275-288.

Citation 2: "research progress of milk fat globule membrane", liu Tingting et al, milk science and technology, 2019, vol 42, no.3.

Disclosure of Invention

Problems to be solved by the invention

As described above, although some studies have been made on the properties and effects of milk fat globule membranes, there is still a need to expand the application thereof, mainly focusing on the promotion of immunity, brain development, advocating health, and the like.

Surprisingly, when the animal experiment is used for evaluating the influence of the nutritional composition consisting of the colostrum basic protein and the milk fat globule membrane on bone development, the combination of the two can synergistically promote bone development and increase bone density through further research. These findings have led to an unexpected expansion of the range of potential applications of both components, whether as pharmaceuticals or in the form of foods or nutritional or dietary supplements, suitable for improving skeletal development.

Solution for solving the problem

Specifically, the invention is implemented by the following technical scheme:

[1] the present invention first provides a nutritional composition, wherein the nutritional composition comprises a milk fat globule membrane component (MFGM) and an alkaline protein component; wherein the content ratio of the alkaline protein to the MFGM is more than 1:100 by mass.

[2] The nutritional composition according to [1], wherein the alkaline protein is milk-derived alkaline protein, preferably the alkaline protein is milk alkaline protein, preferably colostrum alkaline protein fraction (CBP).

[3] The nutritional composition of [1] or [2], wherein the MFGM is derived from animal milk; the content ratio of the CBP to the MFGM is more than 1:80 by mass.

[4] The nutritional composition according to any one of [1] to [3], wherein the content ratio of CBP to MFGM is 1:60 to 1:10 by mass.

[5] Further, the present invention also provides a food which is a nutritional food or a health food, wherein the food comprises or uses the nutritional composition according to any one of the above [1] to [4] as a raw material.

[6] The food according to [5], wherein the content of the MFGM is 0.05 to 10% by mass and the content of the CBP is 0.01 to 1% by mass based on the total mass of the food.

[7] The food according to [5] or [6], wherein the food is a powdery infusible food or a liquid beverage.

[8] The food according to any one of [5] to [7], wherein the food comprises: infant food, child food or adult food.

[9] The food according to any one of [5] to [8], wherein the food comprises: infant formula, infant complementary food, child formula, pregnant and lying-in women's milk powder, middle aged and elderly milk powder or nutritional or dietary supplements.

[10] The present invention also provides the use of the nutritional composition according to any one of the above [1] to [4] or the food according to any one of the above [5] to [9] for promoting bone health.

ADVANTAGEOUS EFFECTS OF INVENTION

The invention evaluates the effect of basic protein, especially milk basic protein, especially colostrum basic protein of cow milk and milk fat globule membrane composition on bone development through experiments. The nutritional composition is prepared by scientifically matching the (colostrum) basic protein and the milk fat globule membrane components, and experiments prove that the nutritional composition can remarkably promote bone development, improve bone density and regulate bone metabolism. Specifically, the effects of the present invention are as follows:

the first object of the present invention is to provide a combination comprising an alkaline protein, particularly a milk-derived alkaline protein, particularly (bovine) colostrum alkaline protein and milk fat globules, which can exert an effect of preventing or promoting bone development and improving bone density by compounding in a specific ratio.

It is a further object of the present invention to provide a method for preventing or promoting bone development in children comprising administering to a subject in need thereof a combination of colostrum basic protein and milk fat globules, optionally in combination with or embedded in a food or nutritional or dietary supplement, containing basic protein, in particular a combination of milk-derived basic protein, in particular (cow's milk) colostrum basic protein and milk fat globules, to obtain the above-mentioned effects.

Drawings

Fig. 1: schematic of the release of milk fat globules and composition of MFGM

Fig. 2: process flow for separating MFGM from cow milk

Detailed Description

The following describes embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the configurations described below, and various modifications are possible within the scope of the invention as claimed, and embodiments and examples obtained by appropriately combining the technical means disclosed in the different embodiments and examples are also included in the technical scope of the present invention.

< definition >

In the present specification, the numerical range indicated by the term "numerical value a to numerical value B" means a range including the end point numerical value A, B.

In the present specification, a numerical range indicated by "above" or "below" is a numerical range including the present number.

In the present specification, the meaning of "can" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

In this specification, the use of "optional" or "optional" means that certain substances, components, steps of performing, conditions of applying, etc. may or may not be used.

In the present specification, unless otherwise specified, "normal temperature" as used herein refers generally to a temperature of 25 ℃.

In the present specification, unit names used are international standard unit names, and "%" used represent weight or mass% unless otherwise specified.

In the present specification, "parts" are used in parts by weight.

Reference throughout this specification to "some specific/preferred embodiments," "other specific/preferred embodiments," "an embodiment," and so forth, means that a particular element (e.g., feature, structure, property, and/or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the elements may be combined in any suitable manner in the various embodiments.

In the present specification, the term "infant" means a person of 0 to less than 6 months of age.

The term "older infant" refers to a person from 6 to less than 12 months of age.

The term "young child" refers to a person of 12 to 36 months of age.

The term "infant" refers to a person 0-36 months of age.

The term "child" refers to a person of 3-18 years of age.

The term "adult" refers to a person over 18 years of age.

The term "middle aged" refers to persons 41-less than 65 years of age.

The term "elderly" or "elderly" refers to people over 65 years of age.

Unless defined otherwise, other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The invention provides a nutritional composition which comprises milk fat globule membrane component (MFGM) and alkaline protein, especially milk-derived alkaline protein, especially (cow's milk) colostrum alkaline protein Component (CBP) in specific proportion, and can promote skeleton development of infants. Specifically, the present invention is based on the following findings:

the prior art and products do not mention the effect of the co-formulation of the colostrum basic protein and the milk fat globule composition on promoting bone development, improving bone density, in particular the proportions are not referred to in the combined formulation. According to the invention, through a large number of experiments, the formula combination and proportion are optimized, and the food composition for promoting skeletal development is prepared according to the characteristics of infant and children skeletal development, so that the requirements of the market on the functional composition are met, as infant diet is more and more emphasized and breast milk research is continued to be deep, new functional compositions gradually appear, and the breast milk process of infant formula milk powder is promoted. The two functional components of the basic protein and the milk fat globule membrane exist in breast milk, researches show that the basic protein and the milk fat globule membrane have a certain effect on promoting brain development, and the basic protein and the milk fat globule membrane have unexpectedly found that compared with a common composition, the basic protein and the milk fat globule membrane can synergistically promote the bone development of an organism, improve the bone density and regulate bone metabolism.

Milk fat globule membrane

Milk is a complex suspension, and fat is one of the most important components of milk. Milk fat is present in milk in the form of small droplets, and is spherical, and is therefore called Milk Fat Globules (MFG). The diameter of the milk fat globules is about 0.2-15 μm, and the surface is covered with a thin film of 10-20 nm called milk fat globule membrane (Milk fat globule membranes, MFGM).

MFGM is generally considered to have a three-layer membrane structure, and is a "natural" emulsifier that can prevent coagulation and aggregation of fat globules in milk and protect fat from the action of enzymes.

Further, MFGM is a protein-lipid complex from a compositional point of view.

Prior studies have shown that in milk fat globule membranes, milk fat globule membrane proteins are distributed in an asymmetric manner in the milk fat globule membrane in an amount of about 25% to 70% of the total MFGM. The positions of milk fat globule membrane proteins can be classified into: integrins, peripherins, proteins loosely bound to the membrane.

For MFGM proteins, which are of a wide variety, the most predominant MFGM proteins are: milk philin (BTN, glycoprotein), mucin 1 (MUC 1, glycoprotein), xanthine oxidoreductase/dehydrogenase (XO/XDH, glycoprotein), milk collectin (MFG-E8 or PAS 6/7), mucin 15 (PAS iii, glycoprotein), CD36 (or PAS iv, glycoprotein), adipose differentiation-related proteins (ADPH, non-glycoprotein) and fatty acid binding proteins (FABP, non-glycoprotein), and the like.

For MFGM lipids, the lipid component of MFGM consists mainly of polar lipids (e.g., phospholipids) that also contain some neutral lipids (e.g., triglycerides), which in turn are divided into phospholipids and sphingolipids. The polar lipids of MFGM are often used as emulsifiers. It was found that the polar lipids in MFGM mainly include lecithin (PC), phosphatidylethanolamine (phosphatidyl ethanolamines, PE), sphingomyelin (SM), phosphatidylinositol (PI) and Phosphatidylserine (PS), with the PC being the highest and the PS and PI being relatively low.

Further, the source of the milk fat globule membrane of the present invention is not particularly limited in principle and may generally be obtained by extraction from animal milk or products thereof, which in some preferred embodiments may be cow milk, sheep milk, camel milk, horse milk or dairy products based thereon (e.g. cheese) or the like, more preferably may be extracted from cow milk, including cow colostrum or cow regular milk.

The method for extracting MFGM from the animal milk or the product thereof is not particularly limited, and for example, the MFGM may be isolated by an acidification precipitation-centrifugation-isoelectric enrichment-drying method, or by a conventional membrane filtration method. For a typical method of separating MFGM from cow's milk, reference is made to the process flow in fig. 2.

In addition, the MFGM may be obtained from commercial products, and the commercial sources of the MFGM according to the present invention includeMFGM-10、/>PL-20、/>Lipid-rich MFGM fraction or buttermilk concentrate BPC50, BPC60, G600, PC700, < >>WPC 7500MEGM ENRICHED WPC, etc.

Alkaline proteins

The alkaline protein of the invention, in some specific embodiments, may be a milk-derived alkaline protein, particularly a milk alkaline protein.

In some preferred embodiments of the present invention, the basic protein is selected from colostrum basic protein (Colostrum basic protein), also called CBP for short, which is only four parts per million extracted from bovine colostrum, and is small molecule active peptide prepared from bovine colostrum by the processes of sterilization, degreasing, centrifugal separation, casein removal, alpha-lactalbumin, beta-lactoglobulin, microfiltration, ultrafiltration, freeze drying, etc., and has high protein content, smaller molecular weight, and only 30KD.

It is generally believed that colostrum basic protein can act directly on bone cells, promoting bone metabolism. Can coordinate the activities of osteoblast and osteoclast, maintain the dynamic balance of osteoblast and osteoclast, promote bone growth and repair bone mass. In addition, the composition has a good preventive effect on osteoporosis.

Further, CBP may be obtained by commercial products, and commercial sources of CBP of the present invention include CBP023502092150 from bioNatin BV, netherlands.

Nutritional composition

Although the prior researches prove that the CBP has the effect of promoting the bone growth, through the verification of various animal experiments, the invention discovers that when the CBP is compounded with the MFGM which is not considered to have obvious effect of promoting the bone growth in a specific proportion, the synergistic gain effect can be obtained in the aspects of promoting the bone growth and bone health.

In particular, the nutritional composition of the invention comprises milk fat globule Membrane Fraction (MFGM) and an alkaline protein, in particular a colostrum alkaline protein fraction (CBP), and the content ratio of alkaline protein, in particular CBP to MFGM, is above 1:80 by mass, in some specific embodiments below 1:1, preferably the content ratio of alkaline protein, in particular CBP to MFGM, is between 1:70 and 1:5, more preferably between 1:60 and 1:10, even more preferably between 1:50 and 1:20. Also, the content ratio of CBP to MFGM that may be cited may also be 1:77, 1:75, 1:73, 1:70, 1:67, 1:65, 1:63, 1:57, 1:55, 1:53, 1:47, 1:45, 1:43, 1:40, 1:37, 1:35, 1:33, 1:30, 1:27, 1:25, 1:23, 1:17, 1:15, 1:13, 1:11, 1:9, 1:7, etc.

Further, as a further object of the present invention, the present invention provides the use of the above-mentioned nutritional composition comprising a specific basic protein, in particular the content ratio of CBP to MFGM, for promoting bone health in humans. The promotion of human skeletal health according to the present invention includes one or more aspects of promoting human skeletal development, improving/increasing bone density, and regulating bone metabolism. The invention also provides the use of the above-described nutritional composition for the preparation of a composition for promoting bone health in humans, especially infants and children. Also, in some specific embodiments, the nutritional compositions described above may be used or applied for a non-therapeutic purpose or effect.

Nutritional/health food

Further, the present invention also provides a food product comprising the above-described nutritional composition as a raw material, which may be a nutritional or health-care type food product. The nutritional/health food has the effect of promoting bone health of human body by adding or using the nutritional composition. And the above-mentioned nutritional/health food of the present invention also includes any nutritional/health food obtained by subjecting a food precursor obtained by adding the above-mentioned nutritional composition as a raw material to a further food processing process.

In addition, the above-mentioned nutritional/health food may contain other ingredients, which may be added or used according to any desired, in addition to the defined nutritional composition.

A milk-containing component or a protein component, the milk-containing component including milk including fresh milk derived from raw fresh cow (sheep) milk, powdered milk, whey protein, cheese, or the like; the protein component may be derived from vegetable proteins, such as soy protein, peanut protein, and the like.

Plant or plant extract components including fructus fici, fructus Punicae Granati, fructus Actinidiae chinensis, fructus Citri Tangerinae, fructus Citri Junoris, fructus Ananadis Comosi, strawberry, fructus Mali Pumilae, rubber, fructus Vitis Viniferae, fructus Pyri, fructus Pruni Pseudocerasi, fructus Myrtilli, blackcurrant, cranberry, raspberry, fructus melo, fructus Phyllanthi and fructus Mori, or their extracts; fruit and vegetable substances such as oceanic, cucumber, tomato, cauliflower, carrot, spinach, cabbage mustard, head cabbage, garlic, basil, and herba Cymbopogonis Citrari, or extracts thereof; grains such as rice (indica rice, japonica rice, glutinous rice), wheat (wheat, barley, oat, rye), corn, sorghum, millet, yellow rice, buckwheat, soybean, broad bean, pea, mung bean, red bean, kidney bean, etc., or their extracts; nut substances such as walnut, pistachio, cashew, hazelnut, almond, pine nut, peanut, melon seed, chestnut, hawaii nut, gingko and the like or extracts thereof; coffee or an extract thereof.

Animal components including meat product components of cattle, sheep, fish or poultry.

A fat component, which may include at least one of saturated fatty acids, polyunsaturated fatty acids, monounsaturated fatty acids, OPO structured fat, DHA, EPA, ARA, phospholipids, more specifically safflower seed oil, walnut oil, peanut oil, corn oil, soybean oil, argan, olive oil, tea oil, mayonnaise oil, olive oil, coconut oil, perilla oil, deep sea fish oil, cocoa butter, palm oil, tallow, butter, lard, medium chain triglycerides or lecithins, etc.

Functional additive components including vitamins (one or more of vitamin A, beta-carotene, vitamin D3, vitamin E, vitamin K1, vitamin B2, vitamin B6, vitamin B12, vitamin C, pantothenic acid, folic acid, nicotinic acid, choline, inositol, biotin), starch, modified starch, amino acids (L-lysine-L-glutamic acid, L-arginine, L-tryptophan, L-glutamine, taurine, L-valine, L-isoleucine or L-leucine, etc.), chinese medicine or Chinese medicine extract, dietary fiber (inulin, konjak powder, galactooligosaccharide, fructooligosaccharide, isomaltooligosaccharide, soybean polysaccharide, cyclodextrin, resistant dextrin or soybean fiber, etc.).

The trace element supplement may include metal ion salts of organic acids, such as one or more of calcium citrate, calcium L-lactate, calcium hydrogen phosphate, potassium gluconate, sodium citrate, ferrous gluconate, potassium iodide, zinc gluconate, sodium selenite, copper gluconate, chromium sulfate, manganese gluconate, and magnesium gluconate.

Any food acceptable auxiliary material including, but not limited to, solvents, antioxidants, antibacterial agents, thickeners, diluents, co-solvents, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweeteners, flavoring agents, food colors, and the like.

When the nutritional/health food contains other milk or protein components, it is preferable that the ratio of total basic proteins, especially total CBP and total MFGM, contained in the nutritional/health food is such that the ratio of total basic proteins, especially total CBP and total MFGM, satisfies the ratio of the total basic proteins to total MFGM contained in the nutritional composition of the present invention.

Further, the content of the basic protein, particularly CBP or MFGM, in the nutritional/health food is not particularly limited in principle, as long as it complies with the specifications of laws and regulations. In some specific embodiments of the present invention, the MFGM is contained in an amount of 0.5 to 10% by mass, preferably 1 to 8% by mass, more preferably 1 to 5% by mass, and particularly preferably 1 to 4% by mass, based on the total mass of the nutritional/health food, in the composition in view of having a desired effect of promoting bone health, in satisfying various regulations (national or regional regulations) concerning use safety in the food field; the content of the basic protein, particularly CBP, is 0.01 to 1% by mass, preferably 0.02 to 1% by mass, more preferably 0.02 to 0.5% by mass, and particularly preferably 0.05 to 0.5% by mass.

Meanwhile, for the edible composition of the present invention, other additives for improving skin condition, such as N-acetylneuraminic acid, nicotinamide, sodium hyaluronate, etc., may be further included.

Further, the specific form of the nutritional/health food of the present invention is not particularly limited, and may be solid (powder, cake, etc.), semi-solid (soft, pasty, thick, etc.), or liquid.

In some specific embodiments, the nutritional or health food may be a powdered infusible food (solid beverage, milk powder, instant coffee, cereal powder, nut powder, lotus root starch, etc.), a liquid beverage (carbonated beverage, juice beverage, functional beverage, tea beverage, milk beverage, alcoholic beverage, etc.).

Typically, the above-mentioned food product comprises: infant formula, infant complementary food, children formula, pregnant and lying-in women formula, middle-aged and elderly milk powder or nutrition or dietary supplements, etc.

Examples

The technical scheme of the invention will be further described through specific experimental examples:

1. materials and methods

1.1 major reagents and instruments

The main reagents and instruments used in this experiment are shown in Table 1-1.

TABLE 1-1 Main reagents and instruments

Name of the name	Production unit
		Colostrum basic protein	BioNatIn BV Co., netherlands
MFGM	Hilmar Co., ltd
		N-terminal pro peptide kit for type I procollagen	Beijing Chenglin biotechnology Co., ltd
Bone alkaline phosphatase kit	Beijing Chenglin biotechnology Co., ltd
		Collagen cross-linking C-terminal peptide kit	Beijing Chenglin biotechnology Co., ltd
Kit for osteocalcin	Beijing Chenglin biotechnology Co., ltd
		Dual-energy X-ray bone densimeter	Le Jie Hologic Co., ltd. In America
VD-1320 type clean workbench	BEIJING DONGLIAN HAR INSTRUMENT MANUFACTURING Co.,Ltd.
		HPG-9245 oven	Beijing Donglian Harbin instruments manufacturing Co.Ltd
Full-series Eppendorf pipettor	Eppendorf, germany
		GL-21M high-speed refrigerated centrifuge	R.B. CARR ENGINEERING, Inc.
PL2002 type and AL104 type electronic balance	Shanghai Meite Teler Tolyduo instruments Co Ltd
		Vortex oscillator	Shanghai Qingpu Huxi Instrument Factory
Model 680 type enzyme mark instrument	Beckman Co Ltd

1.2 laboratory animals

Young (3-4 weeks old) male C57 mice with stable health status, SPF grade, were scheduled for purchase, about 170. Feeding environment: the room temperature is 20-25 ℃, the illumination is changed in darkness every 12 hours, the relative humidity is about 50%, the feed, the drinking water and the like are uniformly matched, the indoor environment is kept well ventilated, the indoor environment is cleaned every day, and the good sanitary environment in the cage is maintained; animal welfare: in the whole treatment process of the experiment, reasonable means are carried out on the mice, which accords with the relevant regulations of the ethical committee of experimental animals, and the mice are specifically carried out according to IAC and other relevant standard operation regulations (SOP); the method for killing comprises the following steps: and (3) carrying out anesthesia on the mice after the experiment is finished and the mice are removed, carrying out abdominal artery bloodletting and sacrifice, then storing the cadavers in a special refrigerator container, and finally carrying out harmless unified treatment.

1.3 Experimental grouping

170 male C57 mice of 3-4 weeks of age were randomly divided into 17 groups of 10, the specific grouping information being shown in Table 1-1. The formal experiment was started 7 days after the adaptive feeding. The blank control group is fed with standard feed, the low-calcium control group and the experimental group are fed with low-calcium feed, the experimental group is filled with gastric test samples (total gastric lavage 39 times) for 11 weeks every other day, the blank control group and the low-calcium control group are filled with normal saline (total gastric lavage 39 times) for 11 weeks every other day, and after the gastric lavage is finished, the experimental samples are collected. As shown in tables 1-2.

TABLE 1-2 animal experiment grouping information table

1.4 index detection

1.4.1 body weight

All mice were weighed and recorded before death, average body weights were calculated, and differences between groups were analyzed.

1.4.2 determination of femoral bone Density

And separating the femur on the right side of the mouse, removing the attached soft tissues, and measuring the midpoint density of the femur by using a dual-energy X-ray bone densitometer, wherein the midpoint measuring point of the femur is the midpoint of the long axis of the femur.

1.4.3 determination of femur dry weight and Length

The right femur was baked to constant weight in a 105 ℃ oven and the bone dry weight was weighed with an analytical balance. The total length of the femur after constant weight was measured with a vernier caliper.

1.4.4 serum Biochemical index determination

Centrifuging the mouse blood sample at 3000r/min for 15min, and preserving at-80deg.C. After thawing the serum samples, ELISA was performed to detect bone formation markers, type I procollagen N-terminal propeptide (Procollagen type IN-terminal propeptide, PINP), bone alkaline phosphatase (bone alkaline phosphatase, BALP), bone resorption markers, type I collagen cross-linked C-terminal peptide (carboxyl-terminal telopeptide of type I collagen, CTX-I) in serum, strictly as required by the specification.

The specific method of enzyme-linked immunosorbent assay (ELISA) is as follows: ELISA kits were equilibrated at room temperature for 15-30min prior to ELISA detection.

(1) And (5) dilution and sample addition of the standard substance. The ELISA plate is provided with 10 holes of standard substance holes, 100 mu L of standard substance is added into 1 and 2 holes, 50 mu L of standard substance diluent is added into 1 and 2 holes, and then the mixture is uniformly mixed, 100 mu L of diluent is taken from 1 and 2 holes, 3 and 4 holes are added into the mixture, 50 mu L of diluent is added into the mixture, the mixture is uniformly mixed, 50 mu L of diluent is discarded, 50 mu L of diluent is taken from 1 and 2 holes, 5 and 6 holes are added into the mixture, and 50 mu L of diluent is added into the mixture for uniform mixing. Mixing, removing 50 μl of the solution, adding 5 or 6 holes to 50 μl, adding 50 μl of the diluted solution, and mixing. Mixing, adding 50 μl of 7 and 8 holes, and adding 50 μl of diluent. mu.L of the mixture was added to 9 and 10 wells, 50. Mu.L of the diluted solution was added thereto and mixed well, and 50. Mu.L of the solution was discarded.

(2) And (5) sample adding. Blank holes (without adding samples and enzyme-labeled reagents) and holes to be tested (40 mu L of diluent and 10 mu L of sample to be tested) are respectively arranged, the samples are added at the bottom of the enzyme-labeled holes, the walls of the holes are not touched as much as possible, and the holes are gently shaken and uniformly mixed.

(3) Incubation. After sealing with a sealing plate, incubating for 30min at 37 ℃.

(4) Preparing liquid. The washing liquid is concentrated by 30 times, and is diluted by 30 times with double distilled water for standby.

(5) And (5) washing. Carefully removing the sealing plate film, discarding the liquid, and spin-drying. Each well was topped up with wash solution, left to stand for 30s, and discarded, and repeated 5 times. And (5) drying.

(6) Enzyme is added. 50 mu L of enzyme-labeled reagent is added to each well, and blank wells are not added.

(7) Incubation. After sealing with a sealing plate, incubating for 30min at 37 ℃.

(8) And (5) washing. Removing the sealing plate membrane, discarding the liquid, and spin-drying. The wells were filled with wash solution, left to stand for 30s, discarded, and the wells were repeated 5 times and patted dry.

1.5 statistical analysis

Experimental data are expressed as mean ± standard deviation (mean ± SD), each experiment is repeated at least 6 times independently, excel 2018 is plotted, SPSS 16.0 software is used to statistically analyze the experimental data, and single-factor analysis of variance (Duncan's multiple range test) is used. ^* P is less than 0.05; ^** represents the ratio of P </to low-calcium control group0.01； ^*** P < 0.001 is shown compared with the low-calcium control group.

2. Experimental results

2.1 Effect of each subject on mouse body weight

To evaluate the effect of the treatment alone or in combination on bone health in mice, the body weight of each group of mice was measured as shown in Table 2-1.

Table 2-1: effects of each subject on mouse body weight

Note that: ^* p is less than 0.05; ^** p is less than 0.01 compared with a low-calcium control group; ^*** p is less than 0.001 compared with a low-calcium control group;

sample treatment results showed that the weight gain of colostrum basic protein was significant in the experimental dose range of 125mg/kg and MFGM was 4500mg/kg, and the other dose was not significant as compared to the low-calcium control group, however, the present inventors found that the weight of mice could be synergistically increased and the growth and development could be promoted when the two components were used in combination, as the feeding time was prolonged.

Experiment 7 corresponds to the combination of comparative examples 3 and 4, and the weight gain (1.99) in experiment 7 was greater than the sum (1.88) of the weight gain of comparative examples 3 and 4, respectively, by 1.99, 1.69, 0.19 for each of experiment 7 and comparative examples 3 and 4, respectively, relative to the low calcium control group.

In the same way, experimental examples 1, 4, 6 and 9 all lead to this conclusion.

Therefore, when the CBP and the MFGM are used in combination, the weight can be obviously increased, and the specific proportion of the two components has a synergistic effect, so that the weight can be synergistically increased, and the growth and development can be promoted.

2.2 Effect of each subject on the right femoral Dry weight of mice

To evaluate the effect of the test substances alone or in combination on the bones of the mice, the dry weight of the femur of the right side of each group of mice was measured as shown in tables 2-2.

Table 2-2: influence of each test on the dry weight of the right femur of the mice

Note that: ^* p is less than 0.05; ^** p is less than 0.01 compared with a low-calcium control group; ^*** p is less than 0.001 compared with a low-calcium control group;

sample treatment results showed that the amount of colostrum basic protein increased significantly with the increase of feeding time compared to the low-calcium control group, and the amount of MFGM increased significantly with the experimental dose range of 4500mg/kg, while the other dose was not significant, however, the inventors found that the two components, when used in combination, were able to synergistically increase the dry weight of the femur of the mice, promoting bone growth and development.

In particular, experimental example 7 corresponds to the combination of comparative examples 3 and 4 in terms of the components used and the amounts used, and experimental examples 7, comparative examples 3, 4 increased the dry weight of femur by 6.33, 1.83, 0.5, respectively, relative to the low-calcium control group, and the increment (6.33) of the dry weight of femur in experimental example 7 was greater than the sum of the respective increments (5.33) of comparative examples 3 and 4.

Experimental example 8 corresponds to the combination of comparative examples 3 and 5, and experimental example 8, comparative examples 3 and 5 increased the dry weight of femur by 7.00, 4.83, and 1.67, respectively, relative to the low-calcium control group, and the increment (7.00) of the dry weight of femur in experimental example 8 was greater than the sum of the respective increments (6.5) of comparative examples 3 and 5.

Similarly, experimental examples 1, 3, 4 and 5 all reached this conclusion.

Therefore, when the CBP and the MFGM are used in combination, the dry weight of the femur can be obviously increased, a synergistic effect exists between the two components in a specific proportion, the dry weight of the femur can be synergistically increased, and the growth and development of the bone can be promoted.

2.3 Effect of each subject on the length of the right femur of the mice

To evaluate the effect of the test substances alone or in combination on the bones of the mice, the length of the right femur of each group of mice was measured as shown in tables 2 to 3.

Table 2-3: influence of each test on the length of the right femur of the mice

Note that: ^* p is less than 0.05; ^** p is less than 0.01 compared with a low-calcium control group; ^*** p is less than 0.001 compared with a low-calcium control group;

sample treatment results showed that the amount of colostrum basic protein increased significantly with the increase of feeding time in the range of experimental dose of 125mg/kg and MFGM in the range of 4500mg/kg, and the other dose was not significant, compared to the low-calcium control group, however, the present inventors found that the two components were used in combination to synergistically increase the femoral length of mice and promote bone growth and development.

In particular, experimental example 7 corresponds to the combination of comparative examples 3 and 4, and experimental example 7, comparative examples 3 and 4 increased the femur length by 2.14, 1.65, and 0.05, respectively, with respect to the low-calcium control group, and the increment (2.14) of the femur length in experimental example 7 was greater than the sum (1.7) of the increments of comparative examples 3 and 4, respectively.

Similarly, experimental examples 1, 2, 3, 4, 5, 6, 8, and 9 all reached this conclusion.

Therefore, when the CBP and the MFGM are used in combination, the length of the femur can be obviously increased, a certain proportion between the two components has a synergistic effect, the length of the femur can be synergistically increased, and the growth and development of bones can be promoted.

2.4 Effect of each subject on mouse bone Density

To evaluate the effect of the test substances alone or in combination on the bone of mice, the bone density of each group of mice was measured as shown in tables 2 to 4.

Tables 2 to 4: effects of each test on bone Density in mice

Note that: ^* p is less than 0.05; ^** p is less than 0.01 compared with a low-calcium control group; ^*** p is less than 0.001 compared with a low-calcium control group;

bone mineral density is an important index for evaluating bone quality, and can reflect the degree of osteoporosis, and is also an important basis for evaluating bone loss caused by preventing bone metabolic diseases and predicting fracture risk. Sample treatment results showed that the bone density increased significantly with the increase in the amount of colostrum basic protein at 125mg/kg and MFGM at 4500mg/kg experimental dose, and not with the other doses, compared to the low-calcium control group, however, the inventors found that the bone density of mice could be increased synergistically when the two components were used in combination, promoting bone growth and development.

In particular, in terms of the components used and the amounts used, experimental example 7 corresponds to the combination of comparative examples 3 and 4, and experimental example 7, comparative examples 3 and 4 increased bone density by 0.37 and 0.25, respectively, relative to the low-calcium control group, and the increase in bone density (0.37) in experimental example 7 was greater than the sum of the respective increases (0.25) of comparative examples 3 and 4.

Experimental example 8 corresponds to the combination of comparative examples 3 and 5, and experimental example 8, comparative examples 3 and 5 increased bone density by 0.39, 0.25 and 0.03, respectively, with respect to the low-calcium control group, and the increase in bone density (0.39) in experimental example 8 was greater than the sum of the respective increases (0.28) of comparative examples 3 and 5.

Similarly, experimental examples 1, 2, 3, 4, 5, and 6 all reached this conclusion.

Therefore, when the CBP and the MFGM are used in combination, the bone density length can be obviously increased, a synergistic effect exists between the two components in a specific proportion, the bone density can be synergistically increased, and the bone growth and development can be promoted.

2.5 Effect of each test on the osteocalcin content in rat serum

To evaluate the effect of the treatment alone or in combination on bone health in mice, the serum osteocalcin content of each group of mice was measured as shown in tables 2 to 5.

Tables 2-5: effect of each subject on osteocalcin in mouse serum

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Note that: ^* p is less than 0.05; ^** p is less than 0.01 compared with a low-calcium control group; ^*** p is less than 0.001 compared with a low-calcium control group;

osteocalcin (BGP) is a non-collagen protein containing vitamin-dependent amino acids, and is mainly produced and secreted by osteoblasts, and it can maintain the normal mineralization rate of bone, inhibit abnormal hydroxyapatite crystal formation and cartilage mineralization rate, and can directly reflect the functional state of osteoblasts and the condition of bone formation. Sample treatment results showed that the amounts of colostrum basic protein 50, 125mg/kg, MFGM in the experimental dose ranges of 3000, 4500mg/kg increased significantly with the prolongation of feeding time compared to the low-calcium control group, and the other doses were not significant, however, the present inventors found that when the two components were used in combination, the content of osteocalcin in the serum of mice could be synergistically increased, promoting bone metabolism.

In particular, experimental example 5 corresponds to a combination of comparative examples 2 and 5 in terms of components used and amounts, and experimental examples 5, comparative examples 2, 5 respectively increased BGP by 0.39, 0.12, 0.13, and the increment (0.39) of BGP in experimental example 5 was greater than the sum (0.25) of the respective increments of comparative examples 2 and 5, relative to the low-calcium control group.

This conclusion can be reached in experimental examples 1, 2, 3, 4, 6, 7, 8.

Therefore, when the two components are combined, the serum calcitonin content can be obviously increased, a synergistic effect exists between the two components in a specific proportion, bone metabolism can be synergistically improved, and bone health is promoted.

2.6 Effect of each test on the content of type I collagen cross-linked carboxy terminal peptide (ICTP) in rat serum

To evaluate the effect of the treatment alone or in combination on bone health in mice, the amount of type I collagen cross-linked carboxy terminal peptide (ICTP) in the serum of each group of mice was determined as shown in tables 2-6.

Tables 2-6: effect of each subject on type I collagen cross-linked carboxy terminal peptide (ICTP) in mouse serum

Note that: ^* p is less than 0.05; ^** p is less than 0.01 compared with a low-calcium control group; ^*** p is less than 0.001 compared with a low-calcium control group;

the type I collagen cross-linked carboxy terminal peptide (ICTP) is linked by a 3-valent cross-link and is released into the blood upon degradation of type I collagen in bone resorption and loose connective tissue, where it exists as an intact immunogenic protein, a specific indicator reflecting the high sensitivity of bone resorption. The increase in serum concentration is associated with increased osteolysis and can be used as an indicator of osteolytic bone metabolism and collagen conversion.

Sample treatment results showed that the amounts of colostrum basic protein (50, 125 mg/kg) and MFGM (3000, 4500 mg/kg) were significantly reduced in the experimental dose range of type I collagen cross-linked carboxy-terminal peptide (ICTP) and the other dose was not significant as compared with the low-calcium control group, however, the present inventors found that the amounts of type I collagen cross-linked carboxy-terminal peptide (ICTP) in the serum of mice could be synergistically increased, bone metabolism was promoted, and osteolysis was reduced when the two components were used in combination.

In particular, experimental example 5 corresponds to a combination of comparative examples 2 and 5 in terms of components used and amounts, and experimental examples 5, comparative examples 2, 5 respectively reduced type I collagen cross-linked carboxy terminal peptide (ICTP) by 15.17, 4.34, 5.5, relative to the low calcium control group, the increase (15.17) of type I procollagen amino terminal pro peptide PICP in experimental example 5 being greater than the sum (9.84) of the respective increases of comparative examples 2 and 5.

This conclusion can be reached in experimental examples 1, 2, 3, 4, 6, 7, 8.

Therefore, when the two components are combined, the content of the type I collagen cross-linked carboxyl terminal peptide (ICTP) in serum can be obviously reduced, a synergistic effect exists between the two components in a specific proportion, the activity of osteoclast can be synergistically inhibited, the synthesis of new bone can be increased, and the bone health can be promoted.

2.7 summary

From the above test data, it can be seen that when the content ratio of CBP to MFGM is controlled to be 1:100 or more, the promotion effect of the combination of CBP and MFGM on bone health can be obviously observed, except for the weight index (the reference index is lower than other indexes because the weight index is a macroscopic comprehensive index).

In a word, the invention provides a new idea for developing future functional foods in the research of bone development, especially bone density improvement and bone metabolism improvement of the colostrum basic protein and milk fat globule membrane composition. The colostrum alkaline protein and the milk fat globule membrane have wide prospects in the aspect of promoting bone development of organisms, and research shows that the colostrum alkaline protein and the milk fat globule membrane have better synergistic effects in increasing bone density, improving bone development and enhancing bone metabolism.

Industrial applicability

The nutritional compositions provided by the invention can be prepared industrially.

Claims (9)

1. A nutritional composition comprising milk fat globule membrane MFGM and colostrum basic protein CBP; wherein, the content ratio of the colostrum basic protein CBP to the milk fat globule membrane MFGM is 1:100-1:10 by mass.

2. Nutritional composition according to claim 1, wherein the milk fat globule membrane MFGM is derived from animal milk; the content ratio of the colostrum basic protein CBP to the milk fat globule membrane MFGM is 1:80-1:10 by mass.

3. Nutritional composition according to claim 1 or 2, characterized in that the content ratio of colostrum basic protein CBP to milk fat globule membrane MFGM is 1:60 to 1:10 by mass.

4. A food product which is a nutritional or health food, characterized in that the food product comprises or uses the nutritional composition according to any one of claims 1-3 as a raw material.

5. The food product according to claim 4, wherein the milk fat globule membrane MFGM is present in an amount of 0.05-10 mass% and the colostrum basic protein CBP is present in an amount of 0.01-1 mass%, based on the total mass of the food product.

6. The food product according to claim 4 or 5, characterized in that the food product is a powdered infusible food product or a liquid beverage.

7. The food product according to claim 4 or 5, characterized in that it comprises: infant food, child food or adult food.

8. The food product according to claim 4 or 5, characterized in that it comprises: infant formula, infant complementary food, children formula, pregnant and lying-in women formula, middle-aged and elderly milk powder or dietary supplements.

9. Use of the nutritional composition according to any one of claims 1 to 3 or the food according to any one of claims 4 to 8 for non-therapeutic purposes to promote bone health.