CN114958951B - Preparation method of casein phosphopeptide for improving digestion function of pet dogs and cats - Google Patents

Preparation method of casein phosphopeptide for improving digestion function of pet dogs and cats Download PDF

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CN114958951B
CN114958951B CN202210794516.2A CN202210794516A CN114958951B CN 114958951 B CN114958951 B CN 114958951B CN 202210794516 A CN202210794516 A CN 202210794516A CN 114958951 B CN114958951 B CN 114958951B
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casein
casein phosphopeptide
cats
zinc
dogs
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CN114958951A (en
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李云亮
马海乐
吕少骏
阮思煜
王智超
赵博博
徐雅宣
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Weishi Pet Nutrition Research Institute Jiangsu Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/40—Feeding-stuffs specially adapted for particular animals for carnivorous animals, e.g. cats or dogs
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
    • Y02P60/87—Re-use of by-products of food processing for fodder production

Abstract

The invention discloses a preparation method of casein phosphopeptide for improving digestion function of dogs and cats, and relates to the field of preparation of polypeptide-metal chelate. In particular to a casein phosphopeptide with high zinc content, which is prepared by firstly carrying out enzymolysis on casein under an acidic condition, then reducing and breaking protein disulfide bonds by utilizing hydrogen generated by the reaction of zinc and hydrochloric acid, and utilizing the chelation effect of zinc ions, cysteine and methionine in order to protect free sulfhydryl groups. The invention overcomes the defect of low enzymolysis efficiency caused by poor solubility of casein in a water-soluble environment, and zinc ion chelation effectively avoids reagglomeration of opened protein disulfide bonds, and the obtained casein phosphopeptide can promote appetite of dogs and cats of pets and improve digestion and absorption rates of the casein phosphopeptide to proteins, fat and the like.

Description

Preparation method of casein phosphopeptide for improving digestion function of pet dogs and cats
Technical Field
The invention relates to the field of polypeptide-metal chelate preparation, in particular to a preparation method of high-zinc casein phosphopeptide and application thereof in pets.
Background
Zinc is one of essential trace elements for animals, and plays a role in the physiological processes of growth, development, immunity, endocrine and the like of animals. Zinc has effects of improving taste, stimulating appetite, enhancing immunity, and promoting brain development. The young dogs and cats are in the growth phase, the digestive system is not yet developed completely, and the intestinal tract cannot effectively absorb nutrients. Therefore, the young dogs and cats are easy to have symptoms such as gastrointestinal discomfort, dyspepsia and the like, which can cause low immunity, easily cause diseases and even influence normal growth and development. The polypeptide has the characteristics of small irritation, easy absorption and the like, so that zinc and the polypeptide are chelated, the absorption efficiency of the zinc can be improved, and the digestibility of dogs and cats can be improved.
Casein is a hard and compact curd, has the defects of poor solubility, difficult digestion by full enzymolysis and the like, and accounts for 80% -82% of cow milk protein, wherein αs1-casein is the main component in the cow milk protein, is highly phosphorylated protein (1 molecule contains 8 phosphate groups), and can obtain casein phosphopeptide after enzymatic hydrolysis, and has the effects of reducing blood pressure, resisting oxidation, promoting the absorption of metal ions by organisms and the like. Casein is rich in cysteine (about 3%), which has the functions of broad-spectrum antibiosis, chelating metal ions, protecting sulfhydryl protease and liver cells from damage, stimulating hematopoiesis function, increasing white blood cells and promoting repair of skin injury, promoting recovery of liver function, participating in reduction process of cells and phospholipid metabolism in liver, and is also an important constituent amino acid of proteins such as hair, etc. The sulfhydryl groups of these cysteines are mainly present in disulfide form. The more disulfide bonds of the protein are, the more compact the protein is crosslinked, the more difficult the protein is to dissolve in water, so that protease is difficult to 'attack' the disulfide bond protected enzymolysis site, and the enzymolysis efficiency of casein is low, so that the preparation of casein phosphopeptide with high cysteine content becomes a research hot spot by improving the enzymolysis efficiency. The conventional method generally adopts two steps: in the first step, disulfide bonds of the protein are reduced to free sulfhydryl groups by a reduction method (such as adding a reducing agent sodium bisulfite); and secondly, carrying out proteolysis under neutral or alkaline conditions to form oligopeptide with higher cysteine content. However, since the free thiol group of cysteine is easily re-oxidized to disulfide bond under neutral or alkaline conditions, protection of the free thiol group is required.
The zinc ion and the sulfhydryl of cysteine can coordinate to form a finger-like structure, thereby playing a role in protecting free sulfhydryl. The metal zinc-hydrochloric acid reduction method is a common method for preparing L-cysteine by reducing L-cystine, but the method utilizes zinc to simultaneously realize reduction of casein disulfide bonds, protection of free sulfhydryl groups and reduction of separation difficulty of casein phosphopeptide which is a subsequent product, and research on using the casein phosphopeptide prepared by the method for improving digestion functions of dogs and cats of pets has not been reported yet.
Disclosure of Invention
To overcome the defects of the prior method, the invention aims at providing a preparation method of casein phosphopeptide-zinc chelate and provides the application of the casein phosphopeptide-zinc chelate in pet functional foods.
The preparation method of casein phosphopeptide for improving digestion function of pet dogs and cats provided by the invention comprises the following steps:
(1) Casein enzymolysis: 10 parts of casein is added with 250 to 500 parts of water, the pH is regulated to 1.5 to 2.0, 0.02 to 0.05 part of pepsin is added, the temperature is maintained at 35 to 42 ℃, and the enzymolysis is carried out until the hydrolysis degree is 15 to 20 percent.
(2) Zinc reduces disulfide bonds: concentrated hydrochloric acid is added into casein hydrolysate, the final concentration of the hydrochloric acid is regulated to 1.5-2.5 mol/L, 2-5 parts of zinc powder is added, and the reaction is carried out for 1.5-2.5 h at 50-60 ℃.
By this setting, H is generated by the reaction of zinc powder and hydrochloric acid 2 The cysteine disulfide bond in casein and hydrolysate thereof can be reduced into free sulfhydryl, and meanwhile, the free sulfhydryl can be further chelated with zinc ions, so that the solubility of the polypeptide is reduced.
(3) Casein phosphopeptide precipitation: adding 0.1-0.2 part of methionine, slowly adding NaOH solid, stirring, regulating the pH to 5.5-6.5, cooling to 0-10 ℃, standing for at least 1h, centrifuging, removing the supernatant, taking the precipitate, washing with 95% ethanol to remove impurities, and drying to obtain casein phosphopeptide.
According to the setting, free sulfhydryl, carboxyl and amino groups on a plurality of polypeptide and amino acid side chains and zinc ion chelates can be further chelated and bridged with methionine to form molecular aggregates to further reduce the solubility of the complex, and when the pH is 5.5-6.5, the net charge amount of the complex is minimum, and the solubility is minimum, so that the solubility is further reduced by reducing the temperature, and casein phosphopeptide can be obtained by simple centrifugation.
The casein phosphopeptide obtained by the method is applied to pet foods for improving the digestion function of young pet dogs and cats.
The invention has the specific advantages that:
(1) The process is as follows: the first step is to carry out enzymolysis under an acidic condition, and after the reaction is finished, the protease can be directly subjected to enzyme deactivation treatment by adjusting the concentration of hydrochloric acid to be 1.5-2.5 mol/L, so that the process is simple; second step, zinc is reacted with hydrochloric acid to produce H 2 And zinc ions, the disulfide bond is reduced, and meanwhile, the zinc ions are not required to be added in the subsequent process; the third step of chelating simple zinc ions with oligopeptide, the precipitation is slow to form, the yield is low, more amino groups for coordination are provided by adding methionine, the chelation rate is obviously increased, and the precipitation generation speed and the precipitation yield are further improved (by more than 88 percent) by adjusting pH and reducing temperature;
(2) The effect is that: the casein phosphopeptide prepared by the method contains a high proportion of organic chelated zinc, reduced cysteine and glutamine. The organic chelated zinc has the advantages of small irritation and high absorptivity while realizing zinc supplementation, and can promote appetite and improve the digestion function of animals on proteins, fat and the like. The cysteine not only can be used for supplementing nutrition for the body, but also can be further converted into taurine to participate in metabolism of the body, has the functions of diminishing inflammation, easing pain, maintaining osmotic pressure balance of the body, maintaining normal vision, promoting digestion and absorption of lipid and the like, and in addition, the glutamine has a promoting effect on biochemical synthesis of hexosamine and glucosamine which are epithelial components of gastrointestinal mucosa, and can play a role in protecting the intestinal mucosa and promoting the intestinal absorption capacity. After the daily ration of the casein phosphopeptide prepared by the invention is taken for one week, the digestibility of the young Chinese rural dogs on crude protein and crude fat is increased by at least 21% and 22%, the digestibility of the young Chinese rural dogs on crude protein and crude fat is increased by at least 19% and 10%, and the average daily feed intake is improved by more than 10g/d, which indicates that the casein phosphopeptide prepared by the invention can promote the appetite of the pet dogs and cats and improve the digestibility of the pet dogs and the fat and the like.
Detailed Description
The terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art unless otherwise indicated. The invention will be described in further detail below in connection with specific examples and with reference to the data. It should be understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
In the following examples, various processes and methods, which are not described in detail, are conventional methods well known in the art. The sources of the reagents used, the trade names and the necessary list the constituents are all indicated at the first occurrence, and the same reagents used thereafter, unless otherwise indicated, are all the same as the first indicated.
The method for measuring the protein content in the comparative example and the example refers to national standard GB/T5009.5-2016.
The method for measuring the oil content in the comparative example and the example is referred to national standard GB/T5009.6-2016.
The method for determining the chelation rate content in the comparative example and the example refers to small sages, zhang Min, he Guanhao, and the like, the enzymatic preparation of milk-derived zinc chelate and the chelation characteristic analysis [ J ]. Food and fermentation industry, 2019,45 (10): 135-141.
Pepsin used in the invention is purchased from Nanning Pang Bo biological engineering Co., ltd, and the unit enzyme activity is 3000NF U/mg.
Comparative example 1
Adding 500g of water into 10g of casein, regulating the pH to 2.0, adding 0.05g of pepsin, maintaining the temperature at 35 ℃, and carrying out enzymolysis until the hydrolysis degree is 15%.
Slowly adding NaOH solid, stirring, regulating pH to 5.5, cooling to 10deg.C, standing for at least 1 hr, centrifuging, removing supernatant, collecting precipitate, washing with 95% ethanol to remove impurities, and oven drying to obtain casein phosphopeptide with yield of 17%.
Example 1
(1) Adding 500g of water into 10g of casein, regulating the pH to 2.0, adding 0.05g of pepsin, maintaining the temperature at 35 ℃, and carrying out enzymolysis until the hydrolysis degree is 15%.
(2) Concentrated hydrochloric acid is added into casein hydrolysate, the final concentration of the hydrochloric acid is regulated to 2.5mol/L, 2g of zinc powder is added, and the reaction is carried out for 1.5h at 60 ℃.
(3) Adding 0.2g of methionine, slowly adding NaOH solid, stirring, regulating pH to 5.5, cooling to 10 ℃, standing for at least 1h, centrifuging, removing supernatant, taking precipitate, washing with 95% ethanol to remove impurities, and drying to obtain casein phosphopeptide with the yield of 37%.
Example 2
(1) Adding 250g of water into 10g of casein, regulating the pH to 1.5, adding 0.02g of pepsin, maintaining the temperature at 42 ℃, and carrying out enzymolysis until the hydrolysis degree is 20%.
(2) Concentrated hydrochloric acid is added into casein hydrolysate, the final concentration of the hydrochloric acid is regulated to 1.5mol/L, 5g of zinc powder is added, and the reaction is carried out for 2.5 hours at 50 ℃.
(3) Adding 0.1g of methionine, slowly adding NaOH solid, stirring, regulating pH to 6.5, cooling to 0 ℃, standing for at least 1h, centrifuging, removing supernatant, taking precipitate, washing with 95% ethanol to remove impurities, and drying to obtain casein phosphopeptide with a yield of 32%.
Example 3
(1) 375g of water is added into 10g of casein, the pH is regulated to 1.5, 0.04g of pepsin is added, the temperature is maintained at 37.5 ℃, and the enzymolysis is carried out until the hydrolysis degree is 18%.
(2) Concentrated hydrochloric acid is added into casein hydrolysate, the final concentration of the hydrochloric acid is regulated to 2.0mol/L, 4g of zinc powder is added, and the reaction is carried out for 2 hours at 55 ℃.
(3) Adding 0.15g of methionine, slowly adding NaOH solid, stirring, regulating pH to 6.0, cooling to 4 ℃, standing for at least 1h, centrifuging, removing supernatant, taking precipitate, washing with 95% ethanol to remove impurities, and drying to obtain casein phosphopeptide with a yield of 47%.
The beneficial effects of the present invention are described below by way of animal experiments.
The experimental method comprises the following steps: 40 young Chinese rural dogs (age 5-7 months, weight 5.0-7.5 kg) and 40 young Chinese rural cats (age 4-7 months, weight 1.0-3.0 kg) were selected and randomly divided into 4 groups of 10 kittens and 10 kittens, respectively, wherein the males: female = 5:5. the control group was fed with 0.01% of the ration of comparative example 1 (see tables 1 and 2), the dogs and cats experimental groups 1, 2 and 3 were fed with 0.01% of the ration of examples 1, 2 and 3, respectively, and fed with free feeding, and fed continuously for 7 days, and the feed intake and body weight of the dogs and cats were measured every day, and the feces were collected on day 8, so as to perform the digestibility detection of crude proteins and crude fats.
Dogs and cats have been subjected to the necessary immunization, insect repellent treatment prior to the experiment, and were not fed any drugs (e.g., antibiotics) that would alter the intestinal microbiota 15 days prior to the start of the experiment. Keeping the house clean, each dog and cat is fed daily diet regularly, and free drinking and feeding are ensured during the whole experiment.
Table 1 nutritional levels of basic ration for young chinese rural dogs
Composition of the components Content/%
Crude protein 32.0
Crude fat 13.0
Coarse fibers 5.0
Moisture content 10.0
Calcium 1.2
Total phosphorus 1.0
Lysine 1.2
Coarse ash 10.0
Water-soluble chloride (in Cl) - Meter (C) 0.4
Table 2 nutritional levels of basic ration for young chinese garden cats
Experimental results:
as shown in table 3, examples 1, 2, and 3 all effectively increased appetite in pet dogs and cats. After 7 days of feeding, the dogs of examples 1, 2, 3 added the average daily ration of at least 153.47g for the experimental groups 1, 2, 3, with significant differences compared to 142.48g for the control group. The cats of experimental groups 1, 2, 3 of examples 1, 2, 3 were added with an average daily ration of at least 54.56g, whereas the cat control group had a feed intake of 43.74g, with significant differences.
TABLE 3 growth of pet dogs and cats
Group of Average daily feed intake g/d
Dog control group 142.48
Dog experiment group 1 159.25
Dog experiment group 2 153.47
Dog experiment group 3 156.93
Cat control group 43.74
Cat experiment group 1 59.47
Cat experiment group 2 54.56
Cat experiment group 3 55.93
As shown in Table 4, examples 1, 2 and 3 all improved the digestion of dogs and cats. The dogs of experimental groups 1, 2, 3 with examples 1, 2, 3 added had a minimum of 81.68% and 84.44% crude protein digestibility and crude fat digestibility, which were improved by at least 19.52% and 10.71% compared to the dogs of the control group, 7 days after feeding. The cat experimental groups 1, 2, 3 to which examples 1, 2, 3 were added had crude protein digestibility and crude fat digestibility of at least 71.87% and 73.79%, whereas the cat control group had crude protein digestibility and crude fat digestibility of 59.34% and 60.25%, which were very different.
TABLE 4 digestibility of crude protein and crude fat by pet dogs and cats
The foregoing embodiments are merely examples of the present invention, and the scope of the present invention includes, but is not limited to, the forms and styles of the foregoing embodiments, and any suitable changes or modifications made by those skilled in the art, which are consistent with the claims of the present invention, shall fall within the scope of the present invention.

Claims (1)

1. The preparation method of casein phosphopeptide for improving digestion function of pet dogs and cats is characterized by comprising the following steps:
(1) Casein enzymolysis: adding 250-500 parts of water into 10 parts of casein, regulating the pH to 1.5-2.0, adding 0.02-0.05 part of pepsin, maintaining the temperature to 35-42 ℃, and carrying out enzymolysis until the hydrolysis degree is 15% -20%;
(2) Zinc reduces disulfide bonds: adding concentrated hydrochloric acid into casein hydrolysate, regulating the final concentration of the hydrochloric acid to 1.5-2.5 mol/L, adding 2-5 parts of zinc powder, and reacting for 1.5-2.5 h at 50-60 ℃;
(3) Casein phosphopeptide precipitation: adding 0.1-0.2 part of methionine, slowly adding NaOH solid, stirring, regulating the pH to 5.5-6.5, cooling to 0-10 ℃, standing for at least 1h, centrifuging, removing the supernatant, taking the precipitate, washing with 95% ethanol to remove impurities, and drying to obtain casein phosphopeptide.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5834427A (en) * 1995-03-23 1998-11-10 Sang Kee Han Casein phosphopeptide, casein containing same and process for the preparation thereof
CN103494214A (en) * 2013-09-29 2014-01-08 安徽农业大学 Casein phosphopeptide and zinc chelate compound
CN107141240A (en) * 2017-06-19 2017-09-08 广汉隆达饲料有限公司 The preparation method of feed grade hydroxy methionine chelates of zinc
CN107557418A (en) * 2016-06-30 2018-01-09 天津唐朝食品工业有限公司 CPP extracting method

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5834427A (en) * 1995-03-23 1998-11-10 Sang Kee Han Casein phosphopeptide, casein containing same and process for the preparation thereof
CN103494214A (en) * 2013-09-29 2014-01-08 安徽农业大学 Casein phosphopeptide and zinc chelate compound
CN107557418A (en) * 2016-06-30 2018-01-09 天津唐朝食品工业有限公司 CPP extracting method
CN107141240A (en) * 2017-06-19 2017-09-08 广汉隆达饲料有限公司 The preparation method of feed grade hydroxy methionine chelates of zinc

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