CN114287638A - Small molecule composite oligopeptide and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the field of functional foods or health-care products, and discloses a micromolecule composite oligopeptide, a preparation method and an application thereof, wherein the micromolecule composite oligopeptide comprises the following main materials and auxiliary materials in parts by weight: main materials: 25-32% of walnut oligopeptide, 16-20% of peanut oligopeptide, 10-13% of fish skin oligopeptide, 17-22% of bovine bone oligopeptide, 9-11% of oat oligopeptide and 4-6% of corn oligopeptide; auxiliary materials: 2 to 3 percent of gamma-aminobutyric acid, 1.5 to 2 percent of N-acetylneuraminic acid and 0.2 to 0.5 percent of stevioside; the walnut oligopeptide, the peanut oligopeptide, the fish skin oligopeptide, the beef bone oligopeptide, the oat oligopeptide and the corn oligopeptide are obtained by an enzymolysis method. The composite polypeptide calcium powder combines the plant-derived bioactive peptide with the animal-derived bioactive peptide, can enhance the immunity of an organism, can regulate the immune system of the organism, is beneficial to relieving physical fatigue and increasing bone density, and is suitable for the adjuvant therapy of patients with susceptibility to fatigue and osteoporosis.

Description

Small molecule composite oligopeptide and preparation method and application thereof

Technical Field

The invention belongs to the field of functional foods or health-care products, and particularly relates to a small-molecule compound peptide and a preparation method and application thereof.

Background

With the rapid development of social economy, the pace of life is accelerated continuously, the pressure from the society, life and work is increased, and people in sub-health state are more and more, so that a plurality of unstable and discordant potential factors are brought to individuals, families and society, and the social productivity and the life quality of people are seriously influenced. Among them, insomnia and the related symptoms caused by insomnia are more common and prominent in many sub-health states, and are receiving more and more attention.

Insomnia is a disease characterized by frequent failure to obtain normal sleep, and is mainly characterized by short sleep time, insufficient deep sleep, difficulty in sleeping for light people, difficulty in sleeping for sleeping without soundness, incapability of sleeping when sleeping or after waking, and insomnia for heavy people all over night. Insomnia has now become the second largest disease in neurology clinics following headache.

At present, the medicines for treating insomnia are mainly western medicines and partial traditional Chinese medicines, and the medicines have the problems of inaccurate effect, long-term administration, immediate attack after medicine withdrawal, high possibility of generating dependence, high toxic and side effects and the like. The long-term taking of the medicine is harmful to the physical health of patients and even causes other iatrogenic diseases. Therefore, the development of safe and effective novel sleep-aiding medicines, health-care products or functional foods is inevitably a development trend in the field, attention is paid to the adoption of dietary therapy for relieving or eradicating certain diseases including insomnia in recent years, and no satisfactory dietary therapy scheme or product exists on the market so far.

Therefore, the development of the sleep-aiding product with remarkable sleep-aiding effect and small side effect has important research significance and popularization value.

Disclosure of Invention

The invention aims to overcome the defect of food therapy schemes or products with obvious sleep-aiding effect in the prior art and provides a small molecular composite oligopeptide. The micromolecule composite oligopeptide provided by the invention takes compatibility of various micromolecule peptides as a main material, and is supplemented with neurotrophic factors such as gamma-aminobutyric acid, N-acetylneuraminic acid, stevioside and the like, so that the raw material is green and natural, the taste is fine and mellow, the obvious sleep-aiding effect is achieved, and the defects of unobvious efficacy and great side effect of the existing sleep-aiding product are overcome to a great extent; and the preparation is in the form of a solid preparation, so that the problems that related liquid preparations are difficult to store and carry and the like are avoided.

The invention also aims to provide a preparation method of the small molecule composite oligopeptide.

The invention also aims to provide application of the small molecule compound oligopeptide in preparing medicines, health products or functional foods or preparations for aiding sleep, restoring nutrition and improving body immunity or beautifying.

In order to make up the defects of the prior art, the invention provides a micromolecule composite oligopeptide with rich nutrition and capable of enhancing immunity, and a preparation method and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme: a micromolecule composite oligopeptide comprises the following main materials and auxiliary materials in parts by weight: main materials: 25-32% of walnut oligopeptide, 16-20% of peanut oligopeptide, 10-13% of fish skin oligopeptide, 17-22% of bovine bone oligopeptide, 9-11% of oat oligopeptide and 4-6% of corn oligopeptide; auxiliary materials: 2 to 3 percent of gamma-aminobutyric acid, 1.5 to 2 percent of N-acetylneuraminic acid and 0.2 to 0.5 percent of stevioside; the walnut oligopeptide, the peanut oligopeptide, the fish skin oligopeptide, the beef bone oligopeptide, the oat oligopeptide and the corn oligopeptide are obtained by an enzymolysis method.

A preparation method of small molecule composite oligopeptide comprises the following steps:

(1) respectively pretreating walnut meal, peanut meal, fish skin, beef bone, oat meal and corn meal, and then carrying out enzymolysis, separation and purification to respectively obtain walnut oligopeptide, peanut oligopeptide, fish skin oligopeptide, beef bone oligopeptide, oat oligopeptide and corn oligopeptide;

(2) mechanically mixing the obtained walnut oligopeptide, peanut oligopeptide, fish skin oligopeptide, cow bone oligopeptide, oat oligopeptide, corn oligopeptide, citric acid, malic acid and stevioside, spray drying to obtain powder, sieving the powder with a 50-100 mesh sieve, and packaging.

Furthermore, the oat oligopeptide and the corn oligopeptide are prepared by the following method: mixing oat meal, corn meal and water according to a weight ratio of 1: (6-10), mixing, adding alkaline protease and trypsin for enzymolysis, keeping the enzymolysis temperature at 45-65 ℃, keeping the pH value of the enzymolysis solution at 7-9, keeping the temperature for 2-3.5h, filtering to remove impurities in the enzymolysis solution, carrying out secondary enzymolysis on the filtrate, keeping the enzymolysis conditions the same as the first time, adding acid into the secondary enzymolysis solution to adjust the pH value to 4-6, and carrying out centrifugal separation to obtain a supernatant; and (3) decoloring and deodorizing the supernatant through macroporous resin at the speed of 2-3mL/min, and spray-drying to obtain powder of oat oligopeptide and corn oligopeptide.

Further, the fish skin oligopeptide is obtained by adopting the following method: cleaning and drying marine fish skin, soaking in 0.05mol/L sodium hydroxide solution, soaking at normal temperature for 2-4h, separating marine fish skin, and cleaning with distilled water; putting the processed marine fish skin into distilled water which is 8-10 times of the weight of the marine fish skin, adding 1-1.5% of neutral protease for enzymolysis for 4-8h to obtain collagen solution, wherein the enzymolysis temperature is 25-45 ℃, and the enzymolysis pressure is 0.01-0.03 MPa; adding 1.5-2.5% of activated carbon into the collagen solution obtained by enzymolysis for adsorption, controlling the pressure at 0.03-0.05MPa at normal temperature, separating and purifying the collagen solution after 30-50min, adding beta-cyclodextrin into the collagen solution for coating, and then carrying out spray drying at 40-70 ℃ to obtain the fish skin oligopeptide.

Further, the walnut oligopeptide and the peanut oligopeptide are obtained by the following method: drying walnut meal and peanut meal in vacuum, then crushing, adding water, adjusting the pH value to 5-6, heating to 50-60 ℃, adding compound protease and flavourzyme for enzymolysis, wherein the mass ratio of the compound protease to the flavourzyme is 1: 1, carrying out enzymolysis for 3h, adjusting the pH once every half hour in the enzymolysis process to maintain the pH value at 5-6, inactivating enzyme after the enzymolysis is finished, filtering to obtain a clear solution, concentrating the filtrate, and then carrying out spray drying to obtain the walnut oligopeptide and the peanut oligopeptide.

Further, the bovine bone oligopeptide is obtained by adopting the following method: cleaning the ox bone pieces crushed into 2-3 cm after impurity removal, putting the bone pieces into a cooking pot, adding water for cooking, carrying out oil-water separation on cooking liquor, cooling the obtained water phase to 50-55 ℃ to obtain a crude collagen solution, adding a high-efficiency compound enzyme preparation with the dosage of 0.05-0.15% of the weight of the raw materials, uniformly mixing, and carrying out enzymolysis for 0.5-2 h under natural conditions without adjusting the pH value and controlling the temperature to obtain a collagen peptide solution; filtering with an inorganic nanofiltration ceramic membrane with the molecular weight cutoff of 800Da, and performing high-temperature and high-pressure treatment on the obtained high-purity collagen peptide solution to obtain a treatment solution; the conditions of the high-temperature high-pressure treatment comprise that the temperature is 110-130 ℃, the pressure is 0.1-0.3 MPa, and the time is 5-20 min; and (3) carrying out reduced pressure heating concentration on the treatment solution, and carrying out spray drying on the concentrated solution to obtain the bovine bone oligopeptide.

Further, the high-efficiency compound enzyme preparation consists of 30-40 wt% of compound protease, 25-30 wt% of flavourzyme, 25-30 wt% of alkaline protease, 7-8 wt% of animal proteolytic enzyme and 2-3 wt% of enzyme activator, wherein the enzyme activator contains a reducing agent and divalent metal ions; the reducing agent is dithiothreitol and/or beta-mercaptoethanol; the molar ratio of the reducing agent to the divalent metal ions in the enzyme activator is 1 (0.5-2); the divalent metal ion is a mixture of Mg2+, Zn2+ and Ca2 +.

The small molecular compound oligopeptide is applied to the preparation of sleep-aiding, nutrition-repairing, organism immunity-improving or cosmetic medicines, health-care products or functional foods or preparations.

The causes of sleep disorders include various factors, such as sleep mood, physical and mental fatigue, synthesis and secretion deficiency of sleep factors, and the like. From the several aspects, the obtained multi-angle sleep-assisting product has a better sleep-assisting effect and a wider application range.

On one hand, the oat oligopeptide and the corn oligopeptide can promote the repair of damaged skeletal muscle cells, maintain the integrity of the skeletal muscle cells, reduce the generation of oxygen free radicals of myocardial mitochondria, maintain the normal structure and function of the myocardial mitochondria, protect the heart and improve the oxygen resistance; on the other hand, oat oligopeptide and corn oligopeptide which are rich in homoglutamine can effectively regulate nerves. Thereby promoting sleep by reducing physical and mental fatigue.

Tryptophan in the walnut oligopeptide and the peanut oligopeptide can promote the secretion of serotonin and melatonin, and glutamic acid can promote the synthesis of inhibitory neurotransmitters such as serotonin and gamma-aminobutyric acid in brain, so that the active effect of improving sleep is exerted.

The rich tryptophan in fish skin oligopeptide and ox bone oligopeptide is the main raw material for synthesizing 5-hydroxytryptamine by brain, and can improve the mood before sleep, inhibit nerve activity and shorten the sleep latency.

Repeated researches show that the specific plant small molecular peptide: walnut, peanut and oat, corn, and certain animal small molecule peptides: the fish skin oligopeptide and the bovine bone oligopeptide are compounded, so that nerve cells can be well nourished, and the repairing and the improvement of brain cell metabolism are participated; the gamma-aminobutyric acid has the functions of calming, helping sleep and resisting anxiety; n-acetylneuraminic acid has the functions of participating in maintaining the structural and functional integrity of brain nerves, promoting the differentiation, development, regeneration and synaptic transmission of nerve cells and the like.

Compared with the prior art, the invention has the following beneficial effects: (1) the specific micromolecule composite oligopeptide peptide combination is used as the main material, so that the comprehensive sleep-aiding effect can be cooperatively exerted from the aspects of improving sleep emotion, reducing physical and mental fatigue, increasing synthesis and secretion of sleep factors and the like, and the phenomenon of poor sleep-aiding effect caused by single component is reduced; meanwhile, functional neurotrophic factors are used as auxiliary materials, and the main materials and the auxiliary materials are compounded to obtain the small molecular compound oligopeptide which has green and natural raw materials, fine and mellow taste and obvious sleep-aiding effect, can greatly shorten the sleep latency time and prolong the sleep effective period time, and overcomes the defects of unobvious efficacy and great side effect of the existing sleep-aiding products to a great extent; the preparation is in a solid preparation form, so that the problems that related liquid preparations are difficult to store and carry and the like are avoided;

(2) the preparation method provided by the invention has simple process, different protein peptides are prepared by an enzymolysis process, the preparation process is simple, green and environment-friendly, the foreign proteins and various fishy and foreign flavors in the protein peptides can be fully removed, and the preparation method is easy to popularize and produce in a large scale; the micromolecule composite oligopeptide is easy to redissolve and reduce when meeting water, and is beneficial to digestion and absorption; easy to store and carry, suitable for the current fast-paced life style and has higher social and economic benefits.

Detailed Description

In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.

Example 1

The micromolecule composite oligopeptide comprises the following main materials and auxiliary materials in parts by weight: main materials: 25% of walnut oligopeptide, 16% of peanut oligopeptide, 13% of fish skin oligopeptide, 22% of beef bone oligopeptide, 11% of oat oligopeptide and 6% of corn oligopeptide; auxiliary materials: 2% of gamma-aminobutyric acid, 1.5% of N-acetylneuraminic acid and 0.2% of stevioside.

The preparation method of the small molecule composite oligopeptide comprises the following steps:

(1) mixing oat meal, corn meal and water according to a weight ratio of 1: (6-10), mixing, adding alkaline protease and trypsin for enzymolysis, keeping the enzymolysis temperature at 45-65 ℃, keeping the pH value of the enzymolysis solution at 7-9, keeping the temperature for 2-3.5h, filtering to remove impurities in the enzymolysis solution, carrying out secondary enzymolysis on the filtrate, keeping the enzymolysis conditions the same as the first time, adding acid into the secondary enzymolysis solution to adjust the pH value to 4-6, and carrying out centrifugal separation to obtain a supernatant; and (3) decoloring and deodorizing the supernatant through macroporous resin at the speed of 2-3mL/min, and spray-drying to obtain powder of oat oligopeptide and corn oligopeptide.

(2) Cleaning and drying marine fish skin, soaking in 0.05mol/L sodium hydroxide solution, soaking at normal temperature for 2-4h, separating marine fish skin, and cleaning with distilled water; putting the processed marine fish skin into distilled water which is 8-10 times of the weight of the marine fish skin, adding 1-1.5% of neutral protease for enzymolysis for 4-8h to obtain collagen solution, wherein the enzymolysis temperature is 25-45 ℃, and the enzymolysis pressure is 0.01-0.03 MPa; adding 1.5-2.5% of activated carbon into the collagen solution obtained by enzymolysis for adsorption, controlling the pressure at 0.03-0.05MPa at normal temperature, separating and purifying the collagen solution after 30-50min, adding beta-cyclodextrin into the collagen solution for coating, and then carrying out spray drying at 40-70 ℃ to obtain the fish skin oligopeptide.

(3) Drying walnut meal and peanut meal in vacuum, then crushing, adding water, adjusting the pH value to 5-6, heating to 50-60 ℃, adding compound protease and flavourzyme for enzymolysis, wherein the mass ratio of the compound protease to the flavourzyme is 1: 1, carrying out enzymolysis for 3h, adjusting the pH once every half hour in the enzymolysis process to maintain the pH value at 5-6, inactivating enzyme after the enzymolysis is finished, filtering to obtain a clear solution, concentrating the filtrate, and then carrying out spray drying to obtain the walnut oligopeptide and the peanut oligopeptide.

(4) Cleaning the ox bone pieces crushed into 2-3 cm after impurity removal, putting the bone pieces into a cooking pot, adding water for cooking, carrying out oil-water separation on cooking liquor, cooling the obtained water phase to 50-55 ℃ to obtain a crude collagen solution, adding a high-efficiency compound enzyme preparation with the dosage of 0.05-0.15% of the weight of the raw materials, uniformly mixing, and carrying out enzymolysis for 0.5-2 h under natural conditions without adjusting the pH value and controlling the temperature to obtain a collagen peptide solution; filtering with an inorganic nanofiltration ceramic membrane with the molecular weight cutoff of 800Da, and performing high-temperature and high-pressure treatment on the obtained high-purity collagen peptide solution to obtain a treatment solution; the high-efficiency compound enzyme preparation consists of 30-40 wt% of compound protease, 25-30 wt% of flavourzyme, 25-30 wt% of alkaline protease, 7-8 wt% of animal proteolytic enzyme and 2-3 wt% of enzyme activator, wherein the enzyme activator contains a reducing agent and divalent metal ions; the reducing agent is dithiothreitol and/or beta-mercaptoethanol; the molar ratio of the reducing agent to the divalent metal ions in the enzyme activator is 1 (0.5-2); the divalent metal ions are a mixture of Mg2+, Zn2+ and Ca2+, and the conditions of high-temperature and high-pressure treatment comprise that the temperature is 110-130 ℃, the pressure is 0.1-0.3 MPa, and the time is 5-20 min; and (3) carrying out reduced pressure heating concentration on the treatment solution, and carrying out spray drying on the concentrated solution to obtain the bovine bone oligopeptide.

(5) Mechanically mixing the obtained walnut oligopeptide, peanut oligopeptide, fish skin oligopeptide, cow bone oligopeptide, oat oligopeptide, corn oligopeptide, citric acid, malic acid and stevioside, spray drying to obtain powder, sieving the powder with a 50-100 mesh sieve, and packaging.

Example 2

The micromolecule composite oligopeptide comprises the following main materials and auxiliary materials in parts by weight: main materials: 32% of walnut oligopeptide, 20% of peanut oligopeptide, 10% of fish skin oligopeptide, 17% of beef bone oligopeptide, 9% of oat oligopeptide and 4% of corn oligopeptide; auxiliary materials: 2% of gamma-aminobutyric acid, 2% of N-acetylneuraminic acid and 0.5% of stevioside.

The preparation method of the small molecule composite oligopeptide in this example is the same as that in example 1.

Example 3

The micromolecule composite oligopeptide comprises the following main materials and auxiliary materials in parts by weight: main materials: 25% of walnut oligopeptide, 16% of peanut oligopeptide, 10% of fish skin oligopeptide, 22% of beef bone oligopeptide, 11% of oat oligopeptide and 6% of corn oligopeptide; auxiliary materials: 3% of gamma-aminobutyric acid, 2% of N-acetylneuraminic acid and 0.3% of stevioside.

The preparation method of the small molecule composite oligopeptide in this example is the same as that in example 1.

Example 4

The micromolecule composite oligopeptide comprises the following main materials and auxiliary materials in parts by weight: main materials: 25% of walnut oligopeptide, 20% of peanut oligopeptide, 10% of fish skin oligopeptide, 22% of beef bone oligopeptide, 9% of oat oligopeptide and 6% of corn oligopeptide; auxiliary materials: 2% of gamma-aminobutyric acid, 2% of N-acetylneuraminic acid and 0.4% of stevioside.

The preparation method of the small molecule composite oligopeptide in this example is the same as that in example 1.

Example 5

The micromolecule composite oligopeptide comprises the following main materials and auxiliary materials in parts by weight: main materials: 32% of walnut oligopeptide, 16% of peanut oligopeptide, 13% of fish skin oligopeptide, 17% of beef bone oligopeptide, 11% of oat oligopeptide and 4% of corn oligopeptide; auxiliary materials: 3% of gamma-aminobutyric acid, 1.5% of N-acetylneuraminic acid and 0.5% of stevioside.

The preparation method of the small molecule composite oligopeptide in this example is the same as that in example 1.

In order to show that the small molecule compound oligopeptides prepared in the embodiments 1 to 5 have the health care effects of relieving physical fatigue and increasing bone density, the beneficial effects of relieving physical fatigue and increasing bone density of the small molecule compound oligopeptides prepared in the embodiments 1 to 5 are studied, and the specific method is as follows:

first, mouse load swimming experiment

(1) Experimental animals: 48 Kunming mice, 24 male and female mice respectively, and the individual mass is 20-25 g.

(2) An experimental instrument: swimming box (40cm is multiplied by 55cm)

(3) The experimental method comprises the following steps: the method comprises the following steps of randomly dividing 48 mice into 6 groups, dividing the mice into experiment 1-5 groups and blank control groups, wherein 8 mice in each group of the experiment 1-5 groups and 8 blank control groups are provided, half of the males and females in each group are provided with deionized water in the process of experimental study, the products in the examples 1-5 are respectively provided for the experiment 1-5 groups for gastric perfusion, each experiment group is divided into 3 groups, 8 Kunming mice in each group are perfused for 10d according to the weight of 0.3g/kg, and the anti-fatigue index of the mice is measured after the test object is tested for 30 min:

(4) weight bearing swimming test

After 8 mice in each group were gazed for 30min, the tail root was loaded with 5% by mass of lead wire, and the mice were placed in a swimming tank at a water depth of 30cm and a water temperature of 25 ℃ and the time from swimming to death of the mice was recorded.

(5) Blood lactic acid determination after each group of 8 mice was gavaged for 30min, the tail root was loaded with 5% by mass of lead wire, the mice were placed in a swimming box at a water depth of 30cm and a water temperature of 25 ℃ for 10min, blood was taken from the angular venous plexus of the eye of the mice before and after 0, 15 and 60min after swimming, 0.48ml of 1% by mass NaF solution was added to a 5ml centrifuge tube, and 20 μ l of whole blood was accurately aspirated and added to the bottom of the test tube. Washing the micropipette several times with the test tube supernatant, adding 1.5ml protein precipitant, shaking, mixing, centrifuging at 3000r/min for 10min, and collecting the supernatant for determination.

(1) Experimental results of load swimming test

TABLE 1 influence of Small Compound oligopeptide on weight bearing swimming time of mice

Group of	Drench material	Dosage (g/kg)	Animal number (only)	Swimming time (min)
					Blank control group	Deionized water	0.3	8	5.59±1.04
Experiment 1 group	Example 1	0.3	8	13.02±2.22
					Experiment 2 groups	Example 2	0.3	8	10.98±1.37
Experiment 3 groups	Example 3	0.3	8	8.98±2.44
					Experiment 4 groups	Example 4	0.3	8	11.27±2.31
Experiment 5 groups	Example 5	0.3	8	11.92±1.49

As can be seen from table 1, the swimming time of the mice in examples 1 to 5 is longer than that of the blank control group, which indicates that the anti-fatigue capability of the mice can be improved and the weight bearing time of the mice can be obviously prolonged after the mice are drenched with the small molecule compound oligopeptide disclosed by the invention, the weight of the small molecule compound oligopeptide of 0.3g/kg in example 1 can obviously prolong the weight bearing time of the mice, and the difference is significant; the small molecule compound oligopeptide prepared by the invention has obvious anti-fatigue effect, and the swimming time of the example 1 is higher than that of the examples 2-5 compared with that of other examples, thus the anti-fatigue capability of the small molecule compound oligopeptide of the example 1 is better than that of the small molecule compound oligopeptides of the examples 2-5.

(2) Results of blood lactic acid measurement

TABLE 2 Effect of small molecule complex oligopeptides on serum lactose in mice

Group of

Drench material

Dosage (g/kg)

Animal number (only)

Before swimming

After swimming (0min)

After swimming (15min)

After swimming (60min)

Blank control group

Deionized water

0.3

8

24.7±3.5

43.7±4.3

30.7±2.2

19.5±2.3

Experiment 1 group

Example 1

0.3

8

22.9±4.2

40.5±4.2

24.3±3.0

16.8±3.4

Experiment 2 groups

Example 2

0.3

8

22.5±4.8

40.9±3.4

23.3±3.2

15.7±2.5

Experiment 3 groups

Example 3

0.3

8

21.6±6.2

40.5±4.7

23.0±4.2

16.2±2.7

Experiment 4 groups

Example 4

0.3

8

23.1±3.6

38.9±4.5

22.6±2.7

15.9±3.8

Experiment 5 groups

Example 5

0.3

8

22.1±4.6

37.0±3.9

21.1±3.7

14.6±2.6

In the experiment 1-5 groups, the blood lactic acid content is respectively reduced by about 25% (e <0.051) at 0min and 15min after swimming compared with the control group, and the lactic acid content before swimming tends to be at 15min, which is shown in table 2. The blood lactic acid content is respectively reduced within 15min after exercise, which shows that the small molecular compound oligopeptide has the positive effect of promoting physical fatigue elimination, and has similar effects in medium-high and low doses. And the blood lactose of the examples 1 to 4 is slightly higher than the blood lactose of the example 5 compared with the blood lactose of the example 5, which shows that the small molecule compound oligopeptide of the example 5 has better physical fatigue elimination promotion than the small molecule compound oligopeptide of the examples 1 to 4.

Second, the experiment for the influence of bone mineral density in mice

The method is operated according to the technical specification for health food inspection and evaluation (2003 edition) issued by the Ministry of health of the people's republic of China. The anti-osteoporosis effect of the rat is mainly used for inspecting the body weight of the rat; femur length, wet weight, dry weight; femoral bone density and bone hardness;

(1) experimental animals: 6-month-old SD rats 70, SPF grade, body weight 80 + -10 g, female and male halves.

(2) An experimental instrument: XR-36 TYPE DOUBLE ENERGY X-RAY BONE DENSITY INSTRUMENT

(3) The experimental method comprises the following steps:

after the rats are fed for 3 days in an adaptive manner, the rats are randomly divided into 7 groups, each group comprises 10 rats including a blank control group, a calcium carbonate control group and an experiment 1-experiment 5 group, each group of 10 rats in the experiment 1-experiment 5 group is fed with feed with the Ca content of 0mg/kg body weight to the blank control group, and 1.0mL of deionized water is fed per 100g of body weight by intragastric administration every day; feeding calcium carbonate of 160mg/kg body weight to calcium carbonate control group; experiments 1-5 were fed with the small molecule compound oligopeptides prepared in examples 1-5, respectively, and 10 rats fed in the experiments 1-5 were fed with a test substance group (300mg/kg body weight) 10 times of the human recommended amount per group, and the administration method was gavage. After continuously feeding the rats of each group for 3 weeks, carrying out a 3d calcium metabolism experiment, measuring the calcium absorption rate, continuously feeding the rats for 13 weeks, and detecting the length, the weight change and the density content of thighbone of the rats of each group when the experiment is finished.

The femoral density measuring method comprises the following steps: measuring the bone density of the midpoint and the far end of the femur sample by an XR-36 type dual-energy X-ray bone densitometer;

(4) the experimental results are as follows:

TABLE 3 Effect of small molecule complex oligopeptides on rat body length

Group of	Drench material	Dosage (g/kg)	Initial length/cm	The length of the body/cm in the fourth week
					Blank control group	1.0ml of deionized water	0.0	14.82±0.32	18.21±0.45
Calcium carbonate control group	160mg/kg body weight calcium carbonate	0.16	14.84±0.43	19.83±0.44
					Experiment 1 group	Example 1	0.3	14.92±0.35	21.85±0.32
Experiment 2 groups	Example 2	0.3	14.82±0.26	23.12±0.38
					Experiment 3 groups	Example 3	0.3	14.84±0.41	21.98±0.35
Experiment 4 groups	Example 4	0.3	15.07±0.26	24.43±0.31
					Experiment 5 groups	Example 5	0.3	15.08±0.21	24.84±0.33

As can be seen from Table 3, there was no significant difference in initial length between the calcium carbonate control group fed with 160mg/kg body weight and the rats fed with 300mg/kg body weight in the experimental groups 1 to 5, but there was a significant difference in comparison of body length at week 4, which was significant in comparison with the calcium carbonate control group (P <0.01), wherein the body length of the animals in the group of example 5 fed with week 4 had the greatest increase in body length compared with the calcium carbonate control group at the same level. And the body length of the rat in the example 1-3 is slightly shorter than that of the rat in the example 4-5 compared with that of the rat in the example 4-5, which shows that the small molecule compound oligopeptide in the example 4-5 promotes the growth of the rat better than that of the small molecule compound oligopeptide in the example 1-3.

TABLE 4 influence of small molecule compound oligopeptide on bone density of rat femur

Group of	Drench material	Dosage (g/kg)	Middle point bone density/g.cm2	Cm2 is the distal bone density/g
					Blank control group	1.0ml of deionized water	0	0.216±0.010	0.312±0.002
Calcium carbonate control group	160mg/kg body weight calcium carbonate	0.16	0.238±0.002	0.344±0.008
					Experiment 1 group	Example 1	0.3	0.261±0.005	0.394±0.006
Experiment 2 groups	Example 2	0.3	0.275±0.003	0.471±0.014
					Experiment 3 groups	Example 3	0.3	0.252±0.007	0.387±0.021
Experiment 4 groups	Example 4	0.3	0.289±0.012	0.487±0.011
					Experiment 5 groups	Example 5	0.3	0.282±0.007	0.491±0.003

As can be seen from Table 4, the bone density at the midpoint and far ends of the left femur of the animals in the experiment 2, 4 and 5 groups is increased compared with the low-calcium control group and the calcium carbonate control group, and the difference has very significant significance (P is less than 0.01) through statistical test, which indicates that the bone density result is positive. And the left femoral midpoint and distal bone densities of examples 1 and 3 are slightly lower than the left femoral midpoint and distal bone densities of examples 2, 4 and 5, which indicates that the small molecule compound oligopeptides of examples 2, 4 and 5 promote the left femoral midpoint and distal bone densities to be better than the small molecule compound oligopeptides of examples 1 and 3.

And (4) experimental conclusion: the health-care effect of the embodiment 1 is obvious in fatigue resistance, the effect of the embodiment 5 is obvious in bone density improvement, and the health-care effects of the embodiments 2, 4 and 5 are also certain in fatigue resistance and bone density improvement.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Claims (8)

1. The micromolecule composite oligopeptide is characterized by comprising the following main materials and auxiliary materials in parts by weight: main materials: 25-32% of walnut oligopeptide, 16-20% of peanut oligopeptide, 10-13% of fish skin oligopeptide, 17-22% of bovine bone oligopeptide, 9-11% of oat oligopeptide and 4-6% of corn oligopeptide; auxiliary materials: 2 to 3 percent of gamma-aminobutyric acid, 1.5 to 2 percent of N-acetylneuraminic acid and 0.2 to 0.5 percent of stevioside; the walnut oligopeptide, the peanut oligopeptide, the fish skin oligopeptide, the beef bone oligopeptide, the oat oligopeptide and the corn oligopeptide are obtained by an enzymolysis method.

2. A preparation method of small molecule composite oligopeptide is characterized by comprising the following steps:

(1) respectively pretreating walnut meal, peanut meal, fish skin, beef bone, oat meal and corn meal, and then carrying out enzymolysis, separation and purification to respectively obtain walnut oligopeptide, peanut oligopeptide, fish skin oligopeptide, beef bone oligopeptide, oat oligopeptide and corn oligopeptide;

(2) mechanically mixing the obtained walnut oligopeptide, peanut oligopeptide, fish skin oligopeptide, cow bone oligopeptide, oat oligopeptide, corn oligopeptide, citric acid, malic acid and stevioside, spray drying to obtain powder, sieving the powder with a 50-100 mesh sieve, and packaging.

3. The method for preparing a small molecule composite oligopeptide according to claim 2, wherein the oat oligopeptide and the corn oligopeptide are prepared by the following steps: mixing oat meal, corn meal and water according to a weight ratio of 1: (6-10), mixing, adding alkaline protease and trypsin for enzymolysis, keeping the enzymolysis temperature at 45-65 ℃, keeping the pH value of the enzymolysis solution at 7-9, keeping the temperature for 2-3.5h, filtering to remove impurities in the enzymolysis solution, carrying out secondary enzymolysis on the filtrate, keeping the enzymolysis conditions the same as the first time, adding acid into the secondary enzymolysis solution to adjust the pH value to 4-6, and carrying out centrifugal separation to obtain a supernatant; and (3) decoloring and deodorizing the supernatant through macroporous resin at the speed of 2-3mL/min, and spray-drying to obtain powder of oat oligopeptide and corn oligopeptide.

4. The method for preparing a small molecule composite oligopeptide according to claim 2, wherein the fish skin oligopeptide is obtained by the following method: cleaning and drying marine fish skin, soaking in 0.05mol/L sodium hydroxide solution, soaking at normal temperature for 2-4h, separating marine fish skin, and cleaning with distilled water; putting the processed marine fish skin into distilled water which is 8-10 times of the weight of the marine fish skin, adding 1-1.5% of neutral protease for enzymolysis for 4-8h to obtain collagen solution, wherein the enzymolysis temperature is 25-45 ℃, and the enzymolysis pressure is 0.01-0.03 MPa; adding 1.5-2.5% of activated carbon into the collagen solution obtained by enzymolysis for adsorption, controlling the pressure at 0.03-0.05MPa at normal temperature, separating and purifying the collagen solution after 30-50min, adding beta-cyclodextrin into the collagen solution for coating, and then carrying out spray drying at 40-70 ℃ to obtain the fish skin oligopeptide.

5. The method for preparing a small molecule compound oligopeptide according to claim 2, wherein the walnut oligopeptide and the peanut oligopeptide are obtained by the following method: drying walnut meal and peanut meal in vacuum, then crushing, adding water, adjusting the pH value to 5-6, heating to 50-60 ℃, adding compound protease and flavourzyme for enzymolysis, wherein the mass ratio of the compound protease to the flavourzyme is 1: 1, carrying out enzymolysis for 3h, adjusting the pH once every half hour in the enzymolysis process to maintain the pH value at 5-6, inactivating enzyme after the enzymolysis is finished, filtering to obtain a clear solution, concentrating the filtrate, and then carrying out spray drying to obtain the walnut oligopeptide and the peanut oligopeptide.

6. The method for preparing small molecule composite oligopeptide according to claim 2, wherein the bovine bone oligopeptide is obtained by the following method: cleaning the ox bone pieces crushed into 2-3 cm after impurity removal, putting the bone pieces into a cooking pot, adding water for cooking, carrying out oil-water separation on cooking liquor, cooling the obtained water phase to 50-55 ℃ to obtain a crude collagen solution, adding a high-efficiency compound enzyme preparation with the dosage of 0.05-0.15% of the weight of the raw materials, uniformly mixing, and carrying out enzymolysis for 0.5-2 h under natural conditions without adjusting the pH value and controlling the temperature to obtain a collagen peptide solution; filtering with an inorganic nanofiltration ceramic membrane with the molecular weight cutoff of 800Da, and performing high-temperature and high-pressure treatment on the obtained high-purity collagen peptide solution to obtain a treatment solution; the conditions of the high-temperature high-pressure treatment comprise that the temperature is 110-130 ℃, the pressure is 0.1-0.3 MPa, and the time is 5-20 min; and (3) carrying out reduced pressure heating concentration on the treatment solution, and carrying out spray drying on the concentrated solution to obtain the bovine bone oligopeptide.

7. The method for preparing small molecule compound oligopeptide according to claim 6, wherein the high efficiency compound enzyme preparation comprises 30-40 wt% of compound protease, 25-30 wt% of flavourzyme, 25-30 wt% of alkaline protease, 7-8 wt% of animal proteolytic enzyme and 2-3 wt% of enzyme activator, wherein the enzyme activator contains reducing agent and divalent metal ions; the reducing agent is dithiothreitol and/or beta-mercaptoethanol; the molar ratio of the reducing agent to the divalent metal ions in the enzyme activator is 1 (0.5-2); the divalent metal ion is a mixture of Mg2+, Zn2+ and Ca2 +.

8. Use of the small molecule compound oligopeptide of any one of claims 1 to 7 in preparation of sleep-aiding, nutritional restoration, immunity-enhancing or cosmetic drugs, health products or functional foods or preparations.