阅读说明：本技术 增强免疫复合蛋白肽酶解液的制备方法、增强免疫复合蛋白肽饮品及其制备方法 (Preparation method of immune-enhancing compound protein peptidase hydrolyzed liquid, immune-enhancing compound protein peptide beverage and preparation method thereof ) 是由 许团辉 陈璇 李媛媛 郑晓辉 吴奕武 陈蝶玲 于 2020-06-26 设计创作，主要内容包括：一种增强免疫复合蛋白肽酶解液的制备方法,包括下述步骤：(1)配备原料；(2)植物蛋白酶解液的制备,其中酶解用碱性蛋白酶、风味蛋白酶和木瓜蛋白酶分两步进行；(3)动物蛋白酶解液的制备,其中酶解用碱性蛋白酶、胰蛋白酶和中性蛋白酶分两步进行；(4)离心；(5)脱色和过滤,得到增强免疫复合蛋白肽酶解液。本发明还提供含有增强免疫复合蛋白肽酶解液的增强免疫复合蛋白肽饮品,以及这种增强免疫复合蛋白肽饮品的一种制备方法。本发明能够使每种蛋白都能够充分得到酶解,并保留活性肽,使所得的复合蛋白肽容易被快速吸收,且具有快速补充营养、提高免疫力的作用；增强免疫复合蛋白肽饮品具有显著的增强免疫效果,且较为可口。(A preparation method of an immune-enhancing compound protein peptidase hydrolysate comprises the following steps: (1) preparing raw materials; (2) preparing a vegetable protein enzymolysis liquid, wherein the enzymolysis is carried out by two steps of alkaline protease, flavourzyme and papain; (3) preparing animal protein enzymolysis liquid, wherein the enzymolysis is carried out by two steps of alkaline protease, trypsin and neutral protease; (4) centrifuging; (5) decolorizing and filtering to obtain the immune-enhancing compound protein peptidase hydrolyzed solution. The invention also provides an immune-enhancing composite protein peptide beverage containing the immune-enhancing composite protein peptidase hydrolyzed solution and a preparation method of the immune-enhancing composite protein peptide beverage. The invention can lead each protein to be fully enzymolyzed and retain active peptide, thus leading the obtained compound protein peptide to be easily and rapidly absorbed and having the functions of rapidly supplementing nutrition and improving immunity; the immune-enhancing compound protein peptide beverage has a remarkable immune enhancing effect and is delicious.)

1. A preparation method of an immune-enhancing compound protein peptidase hydrolysate is characterized by comprising the following steps:

(1) preparing the following raw materials in dry weight: 5-18% of spirulina, 3-15% of soybean protein isolate, 2-10% of mung bean protein isolate, 2-12% of wheat protein, 18-32% of cod skin, 24-36% of yak bone and 5-9% of whey protein;

(2) preparation of plant protein enzymolysis liquid

(2-1) Spirulina pretreatment

Pulverizing Spirulina to obtain Spirulina powder; then mixing the spirulina powder with purified water according to the proportion of 1: 4-1: 8, mixing, uniformly stirring and swelling; then, treating the swollen spirulina powder by using a high-pressure homogenizer to obtain pretreated feed liquid;

(2-2) proteolysis of plant

Adding the pretreated feed liquid obtained in the step (2-1) into an enzyme reaction kettle, adding soybean protein isolate, mung bean protein isolate and wheat protein, adding purified water to prepare feed liquid with the dry matter content of 8-22%, and adjusting the pH value of the feed liquid to 9.2-9.5;

then carrying out enzymolysis by two steps of alkaline protease, flavourzyme and papain, wherein the dosage of the alkaline protease is 1-3% of the dry weight of the feed liquid, the dosage of the flavourzyme is 0.5-2% of the dry weight of the feed liquid, and the dosage of the papain is 1-3% of the dry weight of the feed liquid;

firstly adding alkaline protease for enzymolysis for 2-4 hours at 47-58 ℃, and then heating the enzymolysis liquid to 80-90 ℃ to inactivate the enzyme for 20-30 minutes; cooling the enzymolysis liquid to 48-64 ℃, adding flavourzyme and papain for enzymolysis for 3-6 hours, and then heating the enzymolysis liquid to 80-90 ℃ to inactivate enzyme for 20-30 minutes to obtain plant protease enzymolysis liquid;

(3) preparation of animal protein enzymolysis liquid

(3-1) pretreatment of cod skin

Cleaning the cod skin with water, adding water, soaking for 5-10 minutes, adding a sodium bicarbonate solution, and soaking for 6-9 hours; then cleaning with water, and adding hydrogen peroxide for decoloring for 3-6 hours; cleaning with water, adding purified water with weight 3-5 times of the dry weight of the cod skin, and mincing cod skin with a mincing machine to obtain cod skin slurry;

(3-2) Yak bone pretreatment

Washing yak bones with NaCl solution with the weight percentage concentration of 0.2-0.5%, and then washing with flowing clear water until no blood color exists; then drying the yak bones, and crushing the dried yak bones into yak bone powder by a crusher; adding yak bone powder into EDTA solution with weight percentage concentration of 0.5-1%, soaking for 24-36 hr, filtering out EDTA solution, and washing with clear water for 1-3 times;

(3-3) cooking

Mixing the cod skin slurry obtained in the step (3-1) and the yak bone powder obtained in the step (3-2), adding into an autoclave, and cooking at the temperature of 110-;

(3-4) proteolysis of animal protein

Adding the mixed feed liquid obtained in the step (3-3) and the lactalbumin into an enzyme reaction kettle, then adding purified water to prepare feed liquid with the dry matter content of 15-24%, and adjusting the pH value of the feed liquid to 9.2-9.5;

then carrying out enzymolysis by two steps of alkaline protease, trypsin and neutral protease, wherein the dosage of the alkaline protease is 1-3% of the dry weight of the feed liquid, the dosage of the trypsin is 0.5-2% of the dry weight of the feed liquid, and the dosage of the neutral protease is 1-3% of the dry weight of the feed liquid;

firstly adding alkaline protease for enzymolysis for 2-4 hours at 47-58 ℃, and then heating the enzymolysis liquid to 80-90 ℃ to inactivate the enzyme for 20-30 minutes; cooling the enzymolysis liquid to 42-55 ℃, adding trypsin and neutral protease for enzymolysis for 3-6 hours, and then heating the enzymolysis liquid to 80-90 ℃ to inactivate the enzyme for 20-30 minutes to obtain animal protease enzymolysis liquid;

(4) centrifugation

Mixing the plant protein enzymolysis liquid and the animal protein enzymolysis liquid, and centrifuging by using a centrifugal machine to obtain a supernatant;

(5) decolorization and filtration

Pumping the supernatant into a decoloring tank, and decoloring; filtering after decoloring treatment to obtain the immune-enhancing compound protein peptidase hydrolysate.

2. The method for preparing the enhanced immune complex protein peptidase hydrolyzed solution according to claim 1, wherein: in the step (2-1), stirring at the speed of 30-50 r/min, and swelling for 1-2 hours at the temperature of 45-65 ℃; the pressure of the high-pressure homogenizer is 40-60MPa, and the treatment lasts 20-30 minutes.

3. The method for preparing the enhanced immune complex protein peptidase hydrolyzed solution according to claim 1, wherein: in the step (2-2), the activity unit of the alkaline protease is 20-50 ten thousand units, the activity unit of the papain is 50-80 ten thousand units, and the activity unit of the flavourzyme is 5-10 ten thousand units.

4. The method for preparing the enhanced immune complex protein peptidase hydrolyzed solution according to claim 1, wherein: the sodium bicarbonate solution and the hydrogen peroxide used in the step (3-1) are both food grade; the weight percentage concentration of the sodium bicarbonate solution is 2-5%; the concentration of the hydrogen peroxide is 3-6% by weight.

5. The method for preparing the enhanced immune complex protein peptidase hydrolyzed solution according to claim 1, wherein: the EDTA used in the step (3-2) is food grade; the power of the pulverizer is 30-40 kW; stirring at the speed of 100 revolutions per minute in the soaking process; the EDTA solution was replaced every 12 hours.

6. The method for preparing the enhanced immune complex protein peptidase hydrolyzed solution according to claim 1, wherein: in the step (3-3), an autoclave with the power of 14-21.5kW is adopted for cooking; the pressure in the autoclave is 0.1-0.15MPa during cooking;

in the step (3-4), the activity unit of the alkaline protease is 20-50 ten thousand units, the activity unit of the trypsin is 5-20 ten thousand units, and the activity unit of the neutral protease is 10-30 ten thousand units;

in the step (4), the centrifuge adopts a disc centrifuge, and the centrifuge is carried out at the rotation speed of 4000-;

in the step (5), activated carbon and kieselguhr are added into the decoloring tank, wherein the dosage of the activated carbon is 1-3% of the supernatant, and the dosage of the kieselguhr is 1-3% of the supernatant; stirring at 30-50 rpm, and decolorizing at 50-60 deg.C for 40-60 min.

7. The method for preparing the enhanced immune complex protein peptidase hydrolyzed solution according to claim 1, wherein the filtering in the step (5) comprises the steps of:

(5-1) filtering the destaining solution by using a plate frame to obtain a composite protein peptide solution;

(5-2) adding the composite protein peptide solution into a stainless steel storage tank, and pretreating by using tubular ceramic microfiltration equipment;

(5-3) ultrafiltration: performing ultrafiltration on the composite protein peptide solution pretreated in the step (5-2) by using ultrafiltration equipment to obtain a small molecular composite protein peptide solution;

(5-4) nanofiltration: and (4) carrying out nanofiltration on the small molecular compound protein peptide solution obtained in the step (5-3) to obtain the immune-enhancing compound protein peptidase hydrolysate.

8. The method for preparing the enhanced immune complex protein peptidase hydrolyzed solution according to claim 1, wherein:

in the step (5-1), the filter cloth used for plate-frame filtration is made of nylon material, and the aperture of the filter cloth is 1-10 microns;

in the step (5-2), the aperture of the ceramic membrane filter element used by the tubular ceramic microfiltration equipment is 0.1-0.2 micron;

in the step (5-3), the membrane filter element used by the ultrafiltration device allows substances with molecular weight lower than 5000-10000Da to pass through;

in the step (5-4), a membrane filtering filter core used by the nanofiltration equipment allows substances with molecular weight lower than 150-300Da to pass through, continuous three-stage nanofiltration is carried out, the trapped liquid after the first-stage nanofiltration is feed liquid of the second-stage nanofiltration, and the trapped liquid of the second-stage nanofiltration is feed liquid of the third-stage nanofiltration; the primary nanofiltration pressure is 0.35MPa, the secondary nanofiltration pressure is 0.4MPa, and the tertiary nanofiltration pressure is 0.5 MPa.

9. The immune-enhancing compound protein peptide beverage is characterized by comprising the following components in parts by weight: 6-18% of the immune-enhancing composite protein peptidase-hydrolyzed liquid prepared according to any one of claims 1-8, 2-8% of erythritol, 2-8% of honey, 0.1-5% of medlar extract, 0.1-2% of vitamin C, 0.2-4% of spina date seed extract, 0.02-1% of ginseng extract, 0.01-3% of yeast extract, 0-5% of pineapple fruit powder, 0-3% of passion fruit powder, 0-2% of strawberry fruit powder, 0-6% of blackberry concentrated fruit juice, 0-3% of grapefruit concentrated fruit juice, 0-0.9% of malic acid, 0-1% of citric acid, 0-0.6% of pectin, 0-0.2% of xanthan gum, 0-0.4% of sodium carboxymethylcellulose, 0.005-0.025% of sucralose and the balance of purified water.

10. A preparation method of an immune-enhancing composite protein peptide beverage is characterized by comprising the following steps:

(1') preparing the following raw materials by weight: 6-18% of the immune-enhancing composite protein peptidase-hydrolyzed liquid prepared according to any one of claims 1-8, 2-8% of erythritol, 2-8% of honey, 0.1-5% of medlar extract, 0.1-2% of vitamin C, 0.2-4% of spina date seed extract, 0.02-1% of ginseng extract, 0.01-3% of yeast extract, 0-5% of pineapple fruit powder, 0-3% of passion fruit powder, 0-2% of strawberry fruit powder, 0-6% of blackberry concentrated fruit juice, 0-3% of grapefruit concentrated fruit juice, 0-0.9% of malic acid, 0-1% of citric acid, 0-0.6% of pectin, 0-0.2% of xanthan gum, 0-0.4% of sodium carboxymethylcellulose, 0.005-0.025% of sucralose and the balance of purified water;

(2 ') mixing the various raw materials prepared in the step (1'), uniformly stirring, and then filling the mixture into brown glass bottles by using a filling machine to obtain bottled immune-enhancing composite protein peptide beverage;

(3') sterilizing the bottled immune-enhancing complex protein peptide beverage at the temperature of 110-121 ℃ for 15-25 minutes.

Technical Field

The invention relates to the technical field of protein peptides and protein peptide drinks, in particular to a preparation method of an immune-enhancing compound protein peptidase hydrolysate, an immune-enhancing compound protein peptide drink containing the immune-enhancing compound protein peptidase hydrolysate and a preparation method of the immune-enhancing compound protein peptide drink.

Background

The immune active peptide mainly refers to a peptide segment which has a regulating effect on the immune system of a human body. The immune active peptide can enhance the immunity of organisms to a certain extent, enhance the phagocytosis capability of macrophages, the proliferation of lymphocytes and other physiological functions, has the advantages of strong stability, small molecular weight, high biological activity, weak immunogenicity and the like, can be prepared into various immune preparations and immune health-care nutriments, and has wide application prospect. The amino acid sequence with biological activity hidden in the food-borne protein molecule from plants or animals can be released after being subjected to enzymolysis by proper protease, thereby having certain immunologic function and showing the effects of enhancing body defense, preventing diseases, promoting health, regulating circadian rhythm and the like. The animal and plant source proteins of which the immune protein peptide is obtained at present mainly comprise spirulina protein, chlorella protein, fish skin protein, wheat protein, mushroom protein, sea cucumber protein, mung bean protein, soybean protein, whey protein and the like.

Spirulina (Spirulina) belongs to Cyanophyta, Oscillatoriaceae, and is a kind of spiral prokaryotic algae composed of single cell or multiple cells, with protein content as high as 60-70%, and simultaneously contains abundant VA, VB and minerals. A large number of researches show that the spirulina has the effects of supplementing high-quality protein, vitamins and minerals, relieving fatigue, promoting the differentiation and proliferation of hematopoietic progenitor cells and promoting protein synthesis; has effects in scavenging free radicals, resisting oxidation and inflammation, enhancing immunity, and promoting immune system function; has effects in protecting nervous tissue and DNA from damage; has anti-tumor effect, and can inhibit growth of tumor cells and induce apoptosis of tumor cells; can improve the radiation resistance of the body. The effects of the above aspects are generally accepted and widely used in health food and medicine development. At present, spirulina powder, tablets and capsules are mainly used in the market, and due to the reasons that spirulina is poor in solubility, strong in fishy smell and poor in protein stability, and is easy to precipitate and denature, the loss of biological activity is further caused, and meanwhile, the spirulina is not easy to digest and absorb by human bodies due to large protein molecular weight, so that the further development of the spirulina in the fields of health food and medicines is limited.

The collagen peptide is a transparent light yellow collagen extract and consists of two to twenty amino acids, and the functions of the collagen peptide expressed by different molecular weights and amino acid arrangement sequences of the collagen peptide are different. Collagen is an important component of the human body and is an important raw material for repairing damaged tissues. The collagen peptide has high hydroxyproline, hydroxylysine and proline contents, is easy to absorb, can provide nutrition for body tissues such as skin, cartilage, ligament, bone and the like, maintains health, and is a good nutritional health-care product. The concept of collagen supplementation is deep into the heart of Chinese people for thousands of years, people usually supplement collagen through pig trotters, fish gelatin, donkey-hide gelatin and the like, but because the collagen has a compact structure, the digestion and absorption utilization rate is low, and the effect is poor. The collagen peptide has good solubility, water solubility, thickening property, high biological tissue safety and good thermal stability, is suitable for being added into various liquid fruit juice, milk beverage or instant powder for production, and is widely applied to the aspects of biomedicine, material engineering and the like by virtue of good bioactivity, excellent histocompatibility, low immunogenicity, biodegradability, high physical and mechanical properties and the like. The main manifestations reported in the current research are: protein nutrition effect; the immunity of the organism is improved; inhibition of platelet aggregation; protecting gastric mucosa, and preventing and treating ulcer; relieving the pain of joints, bones and muscles; the antioxidant effect and the effect of eliminating free radicals; lowering blood pressure; anti-tumor; promoting the regeneration and metabolism of skin collagen, and delaying skin aging; activating cell function; promoting mineral absorption; controlling body weight and other physiological functions.

Yak bone is rich in collagen, polysaccharide, enzyme, bone matrix, chondroitin sulfate, calcium, phosphorus, sodium, magnesium, iron, etc., wherein 20-30% of the yak bone is collagen. The yak bone collagen peptide can promote osteoblast proliferation, has interactive regulation and control effects on multiple metabolic pathways such as bile secretion, protein digestion and absorption, mineral substance absorption amino acid metabolism and lipid metabolism, cellular immunity, nervous system, carbon metabolism and endocrine system, can intervene multiple signal pathways simultaneously, and has effects on prevention and improvement of osteoporosis through multiple links, multiple targets and multiple dimensions. Because the collagen contains a large amount of hydroxyproline, the action of the hydroxyproline and the vitamin C can maintain the activity of hydroxyproline hydroxylase and lysyl oxidase, and the collagen has the capacity of eliminating free radicals and the antioxidant capacity.

The soybean peptide is a mixed oligopeptide usually obtained by taking soybean protein or soybean meal as a basic raw material and carrying out chemical treatment, biological treatment or enzymolysis treatment, usually consists of 3-6 amino acids, is rich in 22 amino acids, contains 9 essential amino acids for human bodies, and has rich and balanced essential amino acid content. The soybean peptide has the health functions of resisting fatigue, quickly supplementing and balancing human nutrients, improving immunity, enhancing vitality, promoting fat metabolism, enhancing physical strength and the like, and is known as super amino acid. The soybean peptide can quickly supplement energy, remarkably delay the catabolism of protein in muscles during exercise and improve the exercise capacity. The soybean peptide contains a substance capable of promoting phagocytosis of bacteria by mononuclear leukocytes, can stimulate phagocytosis of macrophages, thereby improving immunity of the organism and being easy to absorb, and is particularly suitable for people with poor digestion and absorption of protein.

The mung bean belongs to leguminous plants, not only can be eaten, but also has a certain medicinal effect, and the nutritional ingredients in the mung bean comprise protein, starch, fat, carbohydrate, various mineral substances such as calcium, phosphorus, iron and the like and vitamins. The protein content of the mung bean is 19.5-33.1%, and the mung bean has known pharmacological effects of clearing away heat and toxic materials, reducing blood fat, reducing cholesterol, resisting bacteria, resisting allergy, resisting oxidation, resisting tumor, inducing diuresis to reduce edema, regulating immunity and the like, and has good medicinal and edible values. The mung bean protein peptide has the activity of improving the immunity of a human body, and researches show that the mung bean protein peptide can improve the number of antibody cells generated by the spleen, the half hemolysis value and the lymphocyte proliferation capacity of mice, and can promote the phagocytic capacity of macrophages. In recent years, researches show that the mung bean protein peptide not only contains immune active peptide, but also contains active peptides such as antioxidant active peptide, ACE inhibitory active peptide and the like.

Wheat protein, commonly known as buerger gluten flour, also known as active gluten flour, is a natural protein extracted from wheat flour, and the main components of wheat protein are gliadin and glutenin, both of which are proteins rich in glutamic acid and proline. The wheat polypeptide obtained by the enzymolysis of the wheat protein has good emulsifying function, foaming function, water absorbing function, thickening function and adhesion function. The peptide substance with opioid activity in the pepsin hydrolysate of wheat gluten, the activity of which can be reversed by naloxone, is called exogenous opioid active peptide, and the opioid active peptides have the traditional analgesic effect and also have important effects on improving cardiovascular function, respiratory function, digestive function, immunity, pituitary hormone secretion, sleep, memory and the like. The wheat gluten protein contains exogenous opioid active peptide, and can significantly promote the growth of immune organs, enhance the phagocytic capacity of abdominal macrophages, improve the transformation of blood lymphocytes, promote the secretion of intestinal tracts and strengthen mucosal immunity. Gluten protein is rich in glutamine peptide which is the key for maintaining muscle metabolism and structure, is a precursor for synthesizing lymphocyte pyrimidine and purine nucleotide, is an indispensable nitrogen source for synthesizing nucleic acid, and is an important regulating factor for protein metabolism. Glutamine peptide is an important respiratory fuel for intestinal mucosal cells and fast proliferating cells of immune cells, can enhance the immune response of organisms, maintains the barrier function of intestinal tracts, and has important significance for preventing translocation of bacteria and toxins and promoting recovery of intestinal functions.

Whey protein is a generic term for the various protein components that remain in the supernatant after separation of the casein precipitate. Whey protein is mainly composed of beta-lactoglobulin (55-60%), alpha-lactalbumin (15-20%), bovine serum albumin (11%), immunoglobulin (9%), and other components such as lactoferrin, lipoproteins, bioactive factors and enzymes (lactoperoxidase) which are present in small amounts. The whey protein peptide has extremely high nutritive value and physiological health care function, plays an important role in promoting the growth and development of organisms, preventing and treating diseases and the like, and can be applied to daily life of people as functional food or food additive or even medicine. The active peptide types currently found in whey protein are mainly opioid peptides, antihypertensive peptides, antioxidant peptides, antibacterial peptides and immunomodulatory peptides. Exogenous opioid peptides stimulate the secretion of insulin and gut somatostatin, regulate animal behavior, promote the absorption of moisture and electrolytes by the gut, regulate gut motility, stimulate food intake, inhibit respiration, and modulate sleep patterns. The polypeptide generated by the degradation of the lactalbumin plays an important role in the immune regulation of organisms, can stimulate the proliferation of mouse spleen cells, and the enzymolysis product can obviously increase the secretion of gamma-interferon.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a preparation method of the enhanced immune composite protein peptidase hydrolysate, each protein can be sufficiently enzymolyzed by adopting the preparation method of the enhanced immune composite protein peptidase hydrolysate, and active peptide is reserved, so that the obtained composite protein peptide is easily and quickly absorbed, and the preparation method has the functions of quickly supplementing nutrition and improving immunity; on the basis, the immune-enhancing composite protein peptide beverage containing the immune-enhancing composite protein peptidase hydrolyzed solution and the preparation method thereof are provided, and the immune-enhancing composite protein peptide beverage has a remarkable immune-enhancing effect and is delicious. The technical scheme is as follows:

a preparation method of an immune-enhancing compound protein peptidase hydrolysate is characterized by comprising the following steps:

(1) preparing the following raw materials in dry weight: 5-18% of spirulina, 3-15% of soybean protein isolate, 2-10% of mung bean protein isolate, 2-12% of wheat protein, 18-32% of cod skin, 24-36% of yak bone and 5-9% of whey protein;

(2) preparation of plant protein enzymolysis liquid

(2-1) Spirulina pretreatment

Pulverizing Spirulina to obtain Spirulina powder; then mixing the spirulina powder with purified water according to the proportion of 1: 4-1: 8, and swelling (preferably stirring at 30-50 rpm; swelling at 45-65 deg.C for 1-2 hours); treating the swollen spirulina powder with a high-pressure homogenizer (preferably at 40-60MPa for 20-30 min) to obtain pretreated material liquid;

(2-2) proteolysis of plant

Adding the pretreated feed liquid obtained in the step (2-1) into an enzyme reaction kettle, adding soybean protein isolate, mung bean protein isolate and wheat protein, adding purified water to prepare feed liquid with the dry matter content of 8-22%, and adjusting the pH value of the feed liquid to 9.2-9.5;

then carrying out enzymolysis by two steps of alkaline protease, flavourzyme and papain, wherein the dosage of the alkaline protease is 1-3% of the dry weight of the feed liquid, the dosage of the flavourzyme is 0.5-2% of the dry weight of the feed liquid, and the dosage of the papain is 1-3% of the dry weight of the feed liquid;

firstly adding alkaline protease for enzymolysis for 2-4 hours at 47-58 ℃, and then heating the enzymolysis liquid to 80-90 ℃ to inactivate the enzyme for 20-30 minutes; cooling the enzymolysis liquid to 48-64 ℃, adding flavourzyme and papain for enzymolysis for 3-6 hours, and then heating the enzymolysis liquid to 80-90 ℃ to inactivate enzyme for 20-30 minutes to obtain plant protease enzymolysis liquid;

(3) preparation of animal protein enzymolysis liquid

(3-1) pretreatment of cod skin

Cleaning the cod skin with water, adding water, soaking for 5-10 minutes, adding a sodium bicarbonate solution, and soaking for 6-9 hours; then cleaning with water, and adding hydrogen peroxide for decoloring for 3-6 hours; cleaning with water, adding purified water with weight 3-5 times of the dry weight of the cod skin, and mincing cod skin with a mincing machine to obtain cod skin slurry;

(3-2) Yak bone pretreatment

Washing yak bones with NaCl solution with the weight percentage concentration of 0.2-0.5%, and then washing with flowing clear water until no blood color exists; then drying the yak bones, and crushing the dried yak bones into yak bone powder by a crusher (preferably crushing the yak bone powder to pass through a 20-mesh sieve); adding yak bone powder into EDTA solution with weight percentage concentration of 0.5-1%, soaking for 24-36 hr, filtering out EDTA solution, and washing with clear water for 1-3 times;

(3-3) cooking

Mixing the cod skin slurry obtained in the step (3-1) and the yak bone powder obtained in the step (3-2), adding into an autoclave, and cooking at the temperature of 110-;

(3-4) proteolysis of animal protein

Adding the mixed feed liquid obtained in the step (3-3) and the lactalbumin into an enzyme reaction kettle, then adding purified water to prepare feed liquid with the dry matter content of 15-24%, and adjusting the pH value of the feed liquid to 9.2-9.5;

then carrying out enzymolysis by two steps of alkaline protease, trypsin and neutral protease, wherein the dosage of the alkaline protease is 1-3% of the dry weight of the feed liquid, the dosage of the trypsin is 0.5-2% of the dry weight of the feed liquid, and the dosage of the neutral protease is 1-3% of the dry weight of the feed liquid;

firstly adding alkaline protease for enzymolysis for 2-4 hours at 47-58 ℃, and then heating the enzymolysis liquid to 80-90 ℃ to inactivate the enzyme for 20-30 minutes; cooling the enzymolysis liquid to 42-55 ℃, adding trypsin and neutral protease for enzymolysis for 3-6 hours, and then heating the enzymolysis liquid to 80-90 ℃ to inactivate the enzyme for 20-30 minutes to obtain animal protease enzymolysis liquid;

(4) centrifugation

Mixing the plant protein enzymolysis liquid and the animal protein enzymolysis liquid, and centrifuging by using a centrifugal machine to obtain a supernatant;

(5) decolorization and filtration

Pumping the supernatant into a decoloring tank, and decoloring; filtering after decoloring treatment to obtain the immune-enhancing compound protein peptidase hydrolysate.

In the preferable step (2-1), the spirulina powder and the purified water are added into a stainless steel tank together, and the stainless steel tank is provided with a stirring system and a circulating water system; the stirring system is used for stirring the materials in the stainless steel tank; the circulating water system keeps the temperature inside the stainless steel tank at 45-65 ℃ for swelling.

In the step (2-2), the pH of the feed solution is usually adjusted with sodium hydroxide.

Preferably, in the step (2-2), the activity unit of the alkaline protease is 20-50 ten thousand units, the activity unit of the papain is 50-80 ten thousand units, and the activity unit of the flavourzyme is 5-10 ten thousand units.

The alkaline protease is a serine type endoprotease, can hydrolyze peptide chains of protein molecules to generate polypeptide or amino acid, is stable at a pH value of 5-10, has high specificity to a substrate, can only hydrolyze the peptide chains of the protein, has specificity to the carboxyl side of an opening point, and has strong capability of decomposing the protein. The flavor protease is exonuclease, more small molecular peptides can be obtained by combining with endonuclease, meanwhile, hydrophobic amino acid of the plant protein is exposed in the enzymolysis process, a large amount of bitter taste can be generated, and the flavor protease can reduce the bitter taste and improve the mouthfeel. Papain is extracted from papaya latex and roughly prepared, mainly contains papain, chymopapain and lysozyme, and also contains a small amount of proteases such as cysteine protease, cellulase, glutamine and the like, and is thiol protease, can hydrolyze carboxyl terminals of arginine and lysine in protein and polypeptide, and can preferentially hydrolyze peptide bonds of amino acids with 2 carboxyl groups or aromatic L amino acids at the N terminal of the peptide bonds; the protein has the characteristics of high temperature resistance, strong activity, good stability, strong protein hydrolysis capacity and the like, and is insensitive to PH change, metal ions and detergents; the protein hydrolysate has the activity of protease and esterase, has wide specificity, has strong enzymolysis capacity on animal and plant proteins, polypeptides, esters, amides and the like, also has synthesis capacity, can be used for synthesizing protein substances from protein hydrolysates, and can be used for improving the nutritional value or functional property of the animal and plant proteins. The invention adopts a plurality of enzymes to hydrolyze the plant protein step by step, combines the incision enzyme and the excision enzyme, enables the protein to be hydrolyzed fully, and keeps the active peptide. Through years of laboratory research and production practice, the applicant can fully hydrolyze each plant protein and retain active peptides by selecting proper types of enzymes and proportion, addition sequence and enzymolysis process conditions thereof. The small molecular peptide obtained by the spirulina through biological enzymolysis not only greatly improves the solubility and improves the taste, but also obtains active peptides such as high-activity immunoregulation peptide, antioxidant peptide, anti-tumor peptide, antihypertensive peptide, hypoglycemic peptide and the like, and the small molecular active peptide can be directly and quickly absorbed, thereby improving the digestion, absorption and utilization rate.

Generally, the sodium bicarbonate solution and the hydrogen peroxide used in the step (3-1) are both food grade. In the preferable step (3-1), the concentration of the sodium bicarbonate solution is 2-5% by weight. In the preferable step (3-1), the concentration of the hydrogen peroxide is 3-6% by weight.

In the step (3-2), the bone meal is soaked in EDTA solution to decalcify the bone meal. Generally, the EDTA (i.e., ethylenediaminetetraacetic acid) used in step (3-2) is food grade. Preferably, in the step (3-2), the power of the pulverizer is 30-40 kW. Preferably, in the step (3-2), the stirring is carried out at a speed of 100 rpm during the soaking. Preferably, in step (3-2), the EDTA solution is replaced every 12 hours.

In the step (3-3), an autoclave with the power of 14-21.5kW can be adopted for cooking. In the step (3-3), the pressure in the autoclave during the cooking is usually 0.1 to 0.15 MPa. Generally, in the step (3-3), after the cod skin slurry and the yak bone powder are mixed, a proper amount of purified water is added and then the mixture is steamed, wherein the amount of the purified water is 3-8 times of the dry weight of the yak bone powder.

In the step (3-4), the pH of the feed solution is usually adjusted with sodium hydroxide.

Preferably, in the step (3-4), the activity unit of the alkaline protease is 20-50 ten thousand units, the activity unit of the trypsin is 5-20 ten thousand units, and the activity unit of the neutral protease is 10-30 ten thousand units.

The alkaline protease is a serine type endoprotease, has good stability, high specificity to a substrate, specificity to the carboxyl side of an opening point, strong capability of decomposing protein for animal protein, capability of hydrolyzing the animal protein into smaller protein and peptide and contribution to further hydrolysis. The neutral protease is bacterial protease, is endoprotease, has the characteristics of strong hydrolysis capability, high speed, little bitter taste of hydrolysate and the like, can decompose peptide bonds in a protein peptide chain, and belongs to metalloenzyme and Ca simultaneously²⁺Is indispensable, Mg²⁺、Zn²⁺、Mn²⁺The animal protein selected by the invention contains high content of calcium and a plurality of beneficial minerals, and neutral protease is not inhibited by the minerals and is beneficial to playing the role. Trypsin is an endopeptidase, a serine protease, which cleaves the carboxyl side of lysine and arginine residues in polypeptide chainsThe protease is the most specific protease. The invention hydrolyzes animal protein by adopting a plurality of enzymes step by step, combines incision enzyme and excision enzyme, enables the protein to be fully hydrolyzed and retains active peptide. The invention hydrolyzes animal protein by adopting a plurality of enzymes step by step, combines incision enzyme and excision enzyme, enables the protein to be fully hydrolyzed and retains active peptide. Through years of laboratory research and production practice, the applicant can fully hydrolyze each animal protein and retain active peptides by selecting proper types of enzymes and proportion, addition sequence and enzymolysis process conditions thereof.

Preferably, in the step (4), the centrifuge adopts a disk centrifuge, and the centrifugation is carried out at the rotation speed of 4000-. The power of the disc centrifuge is 4-15 kW.

In the preferable step (5), activated carbon and kieselguhr are added into the decoloring tank, wherein the dosage of the activated carbon is 1-3% of the supernatant, and the dosage of the kieselguhr is 1-3% (preferably 2%) of the supernatant; stirring at 30-50 rpm, and decolorizing at 50-60 deg.C for 40-60 min. More preferably, the activated carbon used in step (5) is plant activated carbon with a particle size of 300-.

In a preferred embodiment, the filtration in step (5) comprises the following steps:

(5-1) filtering the destaining solution by using a plate frame to obtain a composite protein peptide solution;

(5-2) adding the composite protein peptide solution into a stainless steel storage tank, and pretreating by using tubular ceramic microfiltration equipment;

(5-3) ultrafiltration: performing ultrafiltration on the composite protein peptide solution pretreated in the step (5-2) by using ultrafiltration equipment to obtain a small molecular composite protein peptide solution;

(5-4) nanofiltration: and (4) carrying out nanofiltration on the small molecular compound protein peptide solution obtained in the step (5-3) to obtain the immune-enhancing compound protein peptidase hydrolysate.

And (5-1) mainly filtering the decolorant added in the decoloration process, wherein the obtained composite protein peptide solution is light yellow to orange red and is bright. Further, in the step (5-1), the filter cloth used for plate-frame filtration is made of nylon material, and the aperture of the filter cloth is 1-10 microns.

And (5-2) pretreating by using a tubular ceramic microfiltration device, aiming at removing macromolecular substances such as macromolecular protein, pigment molecules and the like in the composite protein peptide solution and residual decolorizing agent, and preparing for ultrafiltration. Further, in the step (5-2), the aperture of the ceramic membrane filter element used by the tubular ceramic microfiltration equipment is 0.1-0.2 micron.

Preferably, in step (5-3), the membrane filter cartridge used in the ultrafiltration apparatus allows substances having a molecular weight of less than 5000-.

In the preferable step (5-4), a membrane filtering filter core used by the nanofiltration equipment allows substances with the molecular weight lower than 150-300Da to pass through, continuous three-stage nanofiltration is carried out, the trapped liquid after the first-stage nanofiltration is the feed liquid of the second-stage nanofiltration, and the trapped liquid of the second-stage nanofiltration is the feed liquid of the third-stage nanofiltration; the primary nanofiltration pressure is 0.35MPa, the secondary nanofiltration pressure is 0.4MPa, and the tertiary nanofiltration pressure is 0.5 MPa. And filtering by a nanofiltration membrane to remove univalent cations such as sodium and the like through continuous three-stage nanofiltration, wherein the trapped liquid after the first-stage nanofiltration is feed liquid of the second-stage nanofiltration, the trapped liquid of the second-stage nanofiltration is feed liquid of the third-stage nanofiltration, and the trapped liquid of the third-stage nanofiltration is the required enhanced immune complex protein peptidase hydrolyzed liquid. The three-stage nanofiltration greatly improves the salt ion removal efficiency and the concentration efficiency, avoids the conditions of activity reduction, denaturation, deterioration and the like caused by long-term treatment of the protein peptide, can be efficiently concentrated at the same time, enables the content of the composite protein peptide to reach more than 25-30 percent, well protects the activity of the composite protein peptide, greatly reduces the utilization of energy, and has the cost only one tenth of the falling film concentration cost.

The invention carries out pre-treatment on a composite protein raw material, selects proper enzyme to carry out enzymolysis in two steps under proper conditions, obtains a crude enzymolysis liquid of the composite protein peptide through centrifugation, decoloration and plate-and-frame filtration, removes macromolecular impurities such as macromolecular protein, active carbon, pigment molecules and the like through the pre-treatment of filtration of a ceramic filter core, removes larger peptide segments through ultrafiltration membrane filtration of 5000 plus 10000Da so that the content of small molecular peptide less than 3000Da is more than 90 percent, and removes mineral substances through nanofiltration membrane filtration, wherein the removal rate reaches more than 80 percent, so that the obtained composite protein peptide can be directly absorbed, is fast to absorb and easy to dissolve, and simultaneously retains active substances such as active polysaccharide, vitamins and the like in the composite protein.

The immune-enhancing compound protein peptide beverage is characterized by comprising the following components in parts by weight: 6-18% of immunity-enhancing compound protein peptidase hydrolysate, 2-8% of erythritol, 2-8% of honey, 0.1-5% of medlar extract, 0.1-2% of vitamin C, 0.2-4% of spina date seed extract, 0.02-1% of ginseng extract, 0.01-3% of yeast extract, 0-5% of pineapple fruit powder, 0-3% of passion fruit powder, 0-2% of strawberry fruit powder, 0-6% of blackberry concentrated juice, 0-3% of grapefruit concentrated juice, 0-0.9% of malic acid, 0-1% of citric acid, 0-0.6% of pectin, 0-0.2% of xanthan gum, 0-0.4% of sodium carboxymethylcellulose, 0.005-0.025% of sucralose and the balance of purified water.

The erythritol, the honey, the vitamin C (namely VC), the malic acid, the citric acid, the pectin, the xanthan gum, the sodium carboxymethylcellulose and the sucralose are all food-grade products, the wolfberry extract is 10-30 times of the wolfberry extract, the spina date seed extract is 10-30 times of the spina date seed extract, the ginseng extract is 10-30 times of the ginseng extract, the yeast extract is a yeast wall-broken extract, the pineapple fruit powder is pineapple juice spray-dried powder, the passion fruit powder is passion fruit juice spray-dried powder, the strawberry is strawberry juice spray-dried powder, the blackberry concentrated juice is blackberry juice concentrated solution, and the grapefruit concentrated juice is grapefruit juice concentrated solution.

The invention also provides a preparation method of the immune-enhancing composite protein peptide beverage, which is characterized by comprising the following steps:

(1') preparing the following raw materials by weight: 6-18% of immunity-enhancing compound protein peptidase hydrolysate, 2-8% of erythritol, 2-8% of honey, 0.1-5% of medlar extract, 0.1-2% of vitamin C, 0.2-4% of spina date seed extract, 0.02-1% of ginseng extract, 0.01-3% of yeast extract, 0-5% of pineapple fruit powder, 0-3% of passion fruit powder, 0-2% of strawberry fruit powder, 0-6% of blackberry concentrated juice, 0-3% of grapefruit concentrated juice, 0-0.9% of malic acid, 0-1% of citric acid, 0-0.6% of pectin, 0-0.2% of xanthan gum, 0-0.4% of sodium carboxymethylcellulose, 0.005-0.025% of sucralose and the balance of purified water, wherein the protein peptide content is calculated by weight;

(2 ') mixing the various raw materials prepared in the step (1'), uniformly stirring, and then filling the mixture into brown glass bottles by using a filling machine to obtain bottled immune-enhancing composite protein peptide beverage;

(3') sterilizing the bottled immune-enhancing complex protein peptide beverage at the temperature of 110-121 ℃ for 15-25 minutes.

In the step (3'), the bottled immune-enhancing composite protein peptide beverage can be sterilized by a high-temperature high-pressure sterilization pot, and a finished product of the immune-enhancing composite protein peptide beverage is obtained after sterilization.

Ginseng has effects of tonifying spleen, lung channel and heart channel, invigorating primordial qi, tranquilizing, improving intelligence, invigorating qi, promoting fluid production, tonifying deficiency, strengthening body resistance, prolonging life, benefiting qi, promoting blood production, nourishing blood, invigorating heart qi, tranquilizing, and improving intelligence. Modern medicine also proves that ginseng has the effects of nourishing and building body, enhancing immunity, preventing cancer, resisting aging, treating stomach and liver diseases, diabetes and the like. The ginsenoside and the ginseng polysaccharide are active ingredients of the ginseng for regulating the immunity function, and the immunity can be obviously enhanced through animal experiments and human clinical experiments.

The medlar contains rich medlar polysaccharide, beta-carotene, vitamin E, selenium, flavonoids and the like, has good antioxidation, and can resist free radical peroxidation, relieve the damage of the free radical peroxidation, delay aging and prolong life. Fructus Lycii has effects of improving immunity, invigorating qi, replenishing essence, nourishing liver and kidney, resisting aging, quenching thirst, warming body, and resisting tumor. In addition, fructus Lycii has effects of lowering blood pressure, blood lipid and blood glucose, preventing atherosclerosis, protecting liver, inhibiting fatty liver, and promoting liver cell regeneration.

The spina date seed is sweet and sour in taste, enters heart, liver and gallbladder meridians, has the effects of nourishing heart and liver, calming heart and tranquilizing mind, arresting sweating and promoting fluid production, and is mainly used for treating dysphoria and insomnia, palpitation and dreaminess, body deficiency and hyperhidrosis, body fluid deficiency and thirst. The Shen nong Ben Cao Jing carries the actions of the wild jujube for calming the five internal organs, reducing weight and prolonging life. It is a kind of Chinese medicine for treating neurasthenia, vexation, insomnia, dreaminess, night sweat, convulsion and other diseases and has high tranquilizing effect.

In the immune-enhancing composite protein peptide beverage, the immune-enhancing composite protein peptide hydrolysate contains composite protein peptides of spirulina peptide, collagen peptide, yak bone peptide, soybean peptide, mung bean peptide, wheat peptide and whey protein peptide, is easy to absorb quickly, and has the functions of quickly supplementing nutrition and improving immunity; meanwhile, herbal essences such as a medlar extract, a spina date seed extract and a ginseng extract and antioxidant components such as VC are added, so that the immunity enhancing effect is remarkable; and pineapple fruit powder, passion fruit powder, strawberry fruit powder, blackberry concentrated juice, grapefruit concentrated juice, malic acid, citric acid and the like are added as auxiliary materials, so that the drink is sour, sweet and delicious in taste.

The invention has the following technical advantages and effects:

the immune-enhancing compound protein peptidase hydrolysate containing the spirulina peptide, the collagen peptide, the yak bone peptide, the soybean peptide, the mung bean peptide, the wheat peptide and the whey protein peptide is obtained by a proper biological enzymolysis process, is easy to absorb quickly, and has the functions of quickly supplementing nutrition and improving immunity. The protein peptide beverage in the market at present is compounded into the protein peptide beverage after directly purchasing protein peptide powder raw materials, the cost is high, the quality of various protein peptides is uniform, and the quality cannot be ensured.

The proportion of various amino acids of each protein raw material is greatly different, the protein contains different active peptides, and proper enzymes are selected and enzymolysis conditions are controlled, so that the active peptides can be prevented from being subjected to enzymolysis while the enzymolysis efficiency is ensured. Through years of laboratory research and production practice, the applicant develops an enzyme and an enzymolysis process special for enzymolysis of animal protein and plant protein on the basis of experiments of proper enzyme selection, enzyme proportion, addition sequence and enzymolysis conditions on each protein raw material, and performs enzymolysis on the plant compound protein and the animal compound protein separately, so that each protein can be hydrolyzed fully, and active peptides are reserved.

Each enzyme has a specific enzymolysis site, and the mixed enzymolysis of multiple proteins can improve the utilization rate of the proteins and obtain more active peptides. The enzymolysis is carried out by multiple enzymes step by step, the enzyme has multiple enzyme cutting sites, the incision enzyme and the exonuclease are combined and mutually supplemented, the enzymolysis can be more thorough, and meanwhile, the two-step enzymolysis is adopted, so that each enzyme can play a role under the proper enzymolysis condition, the mutual inhibition effect among the enzymes is reduced, and the enzymolysis efficiency is improved.

In the immune-enhancing composite protein peptide beverage, the immune-enhancing composite protein peptide hydrolysate contains composite protein peptides of spirulina peptide, collagen peptide, yak bone peptide, soybean peptide, mung bean peptide, wheat peptide and whey protein peptide, is easy to absorb quickly, and has the functions of quickly supplementing nutrition and improving immunity; meanwhile, herbal essences such as a medlar extract, a spina date seed extract and a ginseng extract and antioxidant components such as VC are added, so that the immunity enhancing effect is remarkable; and pineapple fruit powder, passion fruit powder, strawberry fruit powder, blackberry concentrated juice, grapefruit concentrated juice, malic acid, citric acid and the like are added as auxiliary materials, so that the drink is sour, sweet and delicious in taste.

Detailed Description