CN114990185A - Preparation method of oligopeptide capable of improving defecation function of pet dogs and cats - Google Patents
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Abstract
The invention discloses a preparation method of oligopeptide for improving defecation function of pet dogs and cats, relates to the field of preparation of active peptides, and particularly relates to a method for preparing wheat oligopeptide containing homocysteine and oxides thereof by carrying out prehydrolysis on wheat gluten, oxidizing and breaking protein disulfide bonds by using amylase, glucose oxidase and ultrasonic cavitation and continuing proper enzymolysis. The invention overcomes the defect that gluten powder is easy to adhere to form a cluster when being stirred in water, effectively opens a disulfide bond with firm protein, has low hydrolysis degree of the obtained wheat oligopeptide and high cysteine content, can effectively improve the gastrointestinal condition of pet dogs and cats, and reduces the problems of non-pathological diarrhea, soft stool and the like caused by the change of feeding areas or daily ration of the pet dogs and cats.
Description
Technical Field
The invention relates to the field of preparation of active peptides, in particular to a preparation method of oligopeptide capable of improving defecation function of pet dogs and cats.
Background
Wheat gluten contains 75-85% protein and 5-7% starch. The wheat protein mainly comprises gliadin and glutenin and is rich in cysteine, wherein the content of the cysteine accounts for about 2.0-2.5 percent of the total amino acid content and is higher than that of common plant protein, and the sulfydryl of the cysteine exists in the form of intramolecular and intermolecular disulfide bonds, so the problems of poor solubility, low bioavailability and the like are caused.
With the intensive research on protein nutrition, researchers find that the protein is absorbed by animals not only in the form of free amino acid but also in the form of oligopeptide, and the latter has the advantages of high absorption speed, difficulty in saturation, capability of promoting the absorption of amino acid and the like, so that more and more proteins are hydrolyzed into polypeptide for utilization. The wheat gluten powder is enzymolyzed by protease, so that the solubility of the wheat gluten powder can be increased, the wheat peptide with various biological activities of reducing blood pressure, resisting oxidation, improving immunity and the like can be obtained, the polypeptide containing cysteine also has the functions of broad-spectrum antibiosis, metal ion chelation and the like, and can also play an important physiological role in vivo as a cysteine sustained-release precursor substance: (1) directly removing sulfydryl and amino to generate pyruvic acid which participates in glycometabolism; (2) sulfydryl is oxidized into sulfinyl, then amino and sulfinyl are removed, and finally pyruvic acid and sulfurous acid are generated, wherein the former can participate in glycometabolism, and the latter can be changed into sulfuric acid after oxidation; (3) after sulfydryl is oxidized into sulfinyl, hypotaurine is formed through decarboxylation, the hypotaurine is a good antioxidant and can be oxidized into taurine, the taurine is an active substance for regulating normal physiological activities of an organism, has the functions of diminishing inflammation, easing pain, maintaining osmotic pressure balance of the organism, maintaining normal vision and the like, and is also a component of bile acid, and the formed bile acid salt is beneficial to promoting the digestive absorption of lipid; (4) cysteine is also a raw material for synthesizing glutamic acid, and further synthesizing glutathione, so that the cysteine can help dogs and cats to keep normal immune system functions, and has the functions of antioxidation and detoxification.
The more the number of disulfide bonds of the protein is, the greater the stability of protein molecules against the influence of external factors is, the protease hardly contacts the enzymolysis sites protected by the disulfide bonds, and the resistance to digestion by the protease is relatively strong, so that the wheat gluten is enzymolyzed by the conventional method at a lower hydrolysis degree (less than or equal to 8%), the cysteine content of the oligopeptide is low (less than or equal to 0.25%), and the cysteine content in the oligopeptide can be increased (more than or equal to 1%) at a higher hydrolysis degree (more than or equal to 15%), but the excessive hydrolysis can cause the loss of the activity of the polypeptide and the high content of free amino acids in hydrolysate (more than or equal to 25%). The enrichment and characterization of wheat gluten protein wheat peptide containing cysteine [ J ] the food and fermentation industry, 2020,46(19):64-69.) utilizes mercaptopropyl resin Sepharose 6B to enrich wheat peptide containing cysteine, and the sulfhydryl content is increased from original 195 mu mol/g protein to 994.95 mu mol/g protein, but the method has complex process and high cost, and the process of directly obtaining wheat oligopeptide with high cysteine content under the condition of lower hydrolysis degree has not been reported at present.
The diarrhea is a symptom which often occurs in pet dogs and cats, and not only can affect the normal physiological activities of the pet dogs and cats, but also can cause great damage to the health status of the pet, and further can seriously affect the interaction between the pet and human beings. The causes of the soft diarrhea of pet dogs and cats can be classified into pathological and non-pathological. The pathological diarrhea is usually caused by the fact that pets are infected with certain pathogenic bacteria to cause inflammation of intestinal tracts or normal microbial population change of the intestinal tracts, so that the absorption of nutrient substances such as excrement moisture and the like by the intestinal tracts is influenced, and the condition of diarrhea is caused; the non-pathological diarrhea is 70-80% caused by stress state of pet dogs and cats caused by food change or sudden change of living environment, influences the digestive tract condition or microenvironment such as intestinal flora, and deteriorates the absorption capacity of nutrient substances, thereby causing the content of nutrient substances such as feces moisture to be increased. At present, a plurality of products aiming at the pathological diarrhea of pet dogs and cats exist in the market, but no report is found in a research for relieving or improving the diarrhea of pet dogs and cats caused by stress by using wheat oligopeptide.
Disclosure of Invention
In order to overcome the defects of the existing methods, the invention aims to provide a method for preparing wheat oligopeptide containing homocysteine and oxide thereof, and provides an application method of the wheat oligopeptide in pet functional food.
The invention provides a preparation method of oligopeptide for improving defecation function of pet dogs and cats, which comprises the following steps:
(1) pre-hydrolysis of wheat gluten
Adding 10 parts of wheat gluten into 125-200 parts of water, adding 0.05-0.1 part of neutral protease, maintaining the pH at 5.5-7.0 and the temperature at 50-60 ℃, and carrying out enzymolysis for a period of time until the hydrolysis degree is 3% -5%.
Through the setting, the molecular weight of wheat gluten is reduced through the prehydrolysis of protease, a suspension with lower adhesiveness can be formed, the defect that the wheat gluten is easy to adsorb and bond into lumps when being stirred in water can be overcome, and the subsequent reaction is facilitated.
(2) Cysteine oxidation of wheat gluten
Adjusting the pH value of prehydrolyzed wheat gluten powder to 5.5-7.0, adding 0.005-0.01 part of amylase (saccharifying enzyme) and 0.005-0.01 part of glucose oxidase, pumping air at a constant temperature of 40-50 ℃, applying divergent ultrasonic treatment (ultrasonic power density is 10-50W/L and ultrasonic frequency is 40-60 kHz), and stirring for 15-20 min.
By the setting, starch in wheat gluten is hydrolyzed into glucose, and glucose oxidase is utilized to catalyze the reaction of the glucose and oxygen dissolved in enzymolysis liquid to generate gluconic acid-delta-lactone and H 2 O 2 Instantaneous high temperature assist H by cavitation of ultrasonic waves 2 O 2 Free sulfydryl and disulfide bond of cysteine are oxidized, so that a local compact structure of the protein is opened, and subsequent proteolysis is facilitated.
(3) Enzymatic hydrolysis of oxidized wheat gluten
And (2) adding no protease or 0.02-0.05 part of neutral protease into the pretreated wheat gluten, maintaining the pH value at 5.5-7.0 and the temperature at 50-60 ℃, continuing enzymolysis for a period of time until the hydrolysis degree is 10-12%, boiling for 15-20 min after the reaction is finished, inactivating the enzyme, centrifuging, taking the supernatant, and performing spray drying to obtain the wheat oligopeptide.
The neutral protease can tolerate H 2 O 2 Oxidation and small enzyme activity loss, so that the neutral protease can be continuously reacted without being supplemented.
2. The wheat oligopeptide obtained by the method is applied to pet foods for improving the sub-health conditions of non-pathological diarrhea, soft stool and the like of pet dogs and cats.
The invention has the following specific advantages:
(1) the wheat gluten powder of the invention is prehydrolyzed by protease to overcome the defect that the wheat gluten powder is easy to adhere and agglomerate when stirred in water, and the subsequent H is treated 2 O 2 The disulfide bonds of oxidized cysteines open the robust disulfide structure of proteins and it is highly advantageous to prepare homocysteine (and its derivatives) wheat oligopeptides at low degrees of hydrolysis. Compared with the traditional process, the product prepared by the method has high cysteine content>1.1%) low degree of hydrolysis of the oligopeptide<12%) and less free amino acids (<10 percent), no excessive enzymolysis, low bitter taste, good palatability, and the gluconic acid-delta-lactone contained in the product can also play the roles of a stabilizer, an acidulant, a preservative and a preservative;
(2) the wheat oligopeptide rich in cysteine can be used for supplementing nutrition for a body, can be further converted into taurine to participate in body metabolism, has the effects of diminishing inflammation, easing pain, maintaining the osmotic pressure balance of an organism, maintaining the normal visual function, promoting the digestion and absorption of lipid and the like, has high proportion of glutamine (about 20 percent), has the effect of promoting the biochemical synthesis of hexosamine and glucosamine which are epithelial components of gastrointestinal mucosa, and can play roles in protecting the intestinal mucosa and promoting the absorption capacity of the intestinal tract.
(3) Animal experiments prove that the wheat oligopeptide prepared by the invention can effectively improve the gastrointestinal condition of pet dogs and cats, reduce the problems of non-pathological diarrhea, soft feces and the like of the pet dogs and cats caused by the change of feeding areas or daily rations, increase the defecation normal rate of the dogs from 30% to over 90%, increase the defecation normal rate of the cats from 20% to over 70%, enhance the adaptability of the pet dogs and cats to new environments and new diets, and provide convenience for pet owners to feed the dogs and cats.
Detailed Description
The terms used in the present invention have generally the meanings that are commonly understood by those of ordinary skill in the art, unless otherwise specified. The present invention is described in further detail below with reference to specific examples and with reference to the data. It will be understood that these examples are for the purpose of illustrating the invention and are not intended to limit the scope of the invention in any way.
In the following examples, various procedures and methods not described in detail are conventional methods well known in the art. The source, trade name and composition of the reagents used are indicated at the first appearance and the same reagents used thereafter are the same as indicated for the first time unless otherwise specified.
Method for determining free amino acids: weighing a sample containing 10-20 mg of protein (about 50-100 mg, accurate to 0.1mg, and the sample amount is matched with the dilution multiple, so that the final concentration of each amino acid is 50-200 nmol/mL, or the mass concentration of the total amino acid is 0.1-0.3 mg/mL) in a 20mL anaerobic tube. 10mL of 6mol/L HCl (500 mL of high-grade pure hydrochloric acid (36% -38%) was added to the solution, and 1g of phenol was added thereto to make the volume 1000 mL. Putting the hydrolysis tube into a refrigerant, freezing for 3-5 min, connecting to an exhaust tube of a vacuum pump, vacuumizing (close to 0Pa), and then filling high-purity nitrogen; vacuumizing and filling nitrogen again, repeating the steps for three times, and sealing or screwing down the screw cap in the nitrogen filling state. And (3) putting the sealed hydrolysis tube into a constant-temperature oven at 110 +/-1 ℃, hydrolyzing for 22h, taking out and cooling. After hydrolysis, cooling, mixing, opening the tube, filtering (filter paper) and fixing the volume. Taking a proper amount (about 0.2 mL) of filtrate, putting the filtrate into a 1.5mL centrifuge tube, putting the centrifuge tube in a concentrator, vacuumizing the centrifuge tube at the temperature lower than 60 ℃, evaporating the filtrate to dryness, adding a little water if necessary, and repeatedly evaporating the filtrate to dryness for 1-2 times. Adding the sample liquid and testing on the machine.
Cysteine determination method: putting a sample containing 7.5-25 mg of protein into a 20mL anaerobic tube (without cutting and adhering to the wall); placing in ice water at 0 deg.C, 2mL of pre-cooled performic acid solution (30% H) 2 O 2 And 88% formic acid as 1: 9(V/V), placing for 1h at room temperature, placing in ice water at 0 ℃ for cooling for 30min, preparing before use, and wetting the sample completely when adding liquid, but not shaking; in a refrigerator at 2 deg.CPlacing for 15h or placing in 55 deg.C water bath for 15 min; the excessive performic acid in the solution can be kept stand for 30min in ice bath by adding 168mg of sodium metabisulfite; 7.5mL of HCl (6.8mol/L) was added; and (3) putting the mixture into a constant-temperature oven at 110 +/-1 ℃ for hydrolysis for 22-24 h. And after the hydrolysis is finished, cooling, uniformly mixing, transferring the hydrolysate to a 50mL volumetric flask by using ultrapure water, adding a proper amount of sodium hydroxide solution to adjust the pH value to 2.2, metering the volume, shaking and uniformly mixing, filtering a proper amount of sample by using a 0.22-micrometer filter membrane, and then using the filtered sample for measurement on a machine.
Glutamine determination method: refer to methods of Jun Guanjun et al (Zheng Zhou food academy of academic, 1999,20(4): 65-67).
Neutral protease (unit enzyme activity is 10 ten thousand U/g), amylase (saccharifying enzyme, unit enzyme activity is 10 ten thousand U/g) and glucose oxidase (unit enzyme activity is 1 ten thousand U/g) used in the method are purchased from Penbo bioengineering company Limited in Nanning.
Comparative example 1
Adding 10 parts of wheat gluten into 200 parts of water, adding 0.05 part of neutral protease, maintaining the pH at 6.5 and the temperature at 50 ℃, carrying out enzymolysis for a period of time until the hydrolysis degree is 8%, boiling for 15min after the reaction is finished to inactivate the enzyme, centrifuging to obtain supernatant, and carrying out spray drying to obtain the wheat oligopeptide, wherein the content of the obtained polypeptide cysteine is 0.25%, the content of free amino acid is 15%, and the content of glutamine is 18%.
Comparative example 2
Adding 10 parts of wheat gluten into 200 parts of water, adding 0.05 part of neutral protease, maintaining the pH at 6.5 and the temperature at 50 ℃, carrying out enzymolysis for a period of time until the hydrolysis degree is 18%, boiling for 15min after the reaction is finished to inactivate the enzyme, centrifuging to obtain supernatant, and carrying out spray drying to obtain the wheat oligopeptide, wherein the content of the obtained polypeptide cysteine is 0.86%, the content of free amino acid is 28.0%, and the content of glutamine is 20.2%.
Example 1
Adding 10 parts of wheat gluten into 200 parts of water, adding 0.05 part of neutral protease, maintaining the pH at 6.5 and the temperature at 50 ℃, and carrying out enzymolysis for a period of time until the hydrolysis degree is 3%.
The pH of the prehydrolyzed wheat gluten was adjusted to 5.5, 0.005 part amylase (saccharifying enzyme), 0.01 part glucose oxidase were added, constant temperature 50 deg.C, air was pumped in, divergent ultrasonic treatment (10W/L, 60kHz) was applied and stirring was carried out for 20 min.
Adding 0.05 part of neutral protease into the pretreated wheat gluten, maintaining the pH at 6.5 and the temperature at 50 ℃, performing enzymolysis for a period of time until the hydrolysis degree is 10%, boiling for 15min after the reaction is finished, inactivating the enzyme, centrifuging, taking the supernatant, and performing spray drying to obtain the wheat oligopeptide. The obtained wheat oligopeptide has cysteine content of 1.13%, free amino acid content of 9.2%, and glutamine content of 18.2%.
Example 2
Adding 10 parts of wheat gluten into 125 parts of water, adding 0.1 part of neutral protease, maintaining the pH at 7.0 and the temperature at 60 ℃, and performing enzymolysis for a period of time until the hydrolysis degree is 5%.
The pH of the prehydrolyzed wheat gluten was adjusted to 6.5, 0.01 part amylase (saccharifying enzyme), 0.01 part glucose oxidase were added, constant temperature 40 deg.C, air was pumped in, divergent ultrasonic treatment (50W/L, 40kHz) was applied and stirring was carried out for 15 min.
Adding protease into pretreated wheat gluten, maintaining pH at 7.0 and temperature at 60 deg.C, performing enzymolysis for a period of time until the hydrolysis degree is 12%, boiling for 20min to inactivate enzyme after reaction, centrifuging to obtain supernatant, and spray drying to obtain wheat oligopeptide. The obtained wheat oligopeptide has cysteine content of 1.78%, free amino acid content of 5.3%, and glutamine content of 21.5%.
Example 3
Adding 10 parts of wheat gluten into 200 parts of water, adding 0.075 part of neutral protease, maintaining the pH at 5.5 and the temperature at 55 ℃, and carrying out enzymolysis for a period of time until the hydrolysis degree is 4%.
The pH of the prehydrolyzed wheat gluten was adjusted to 6.5, 0.0075 parts amylase (saccharifying enzyme), 0.0075 parts glucose oxidase were added, constant temperature 45 deg.C, air was pumped in, divergent ultrasonic treatment (30W/L, 50kHz) was applied and stirring was carried out for 15 min.
And (3) adding no protease into the pretreated wheat gluten powder, maintaining the pH value at 5.5 and the temperature at 55 ℃, performing enzymolysis for a period of time until the hydrolysis degree is 10%, boiling for 20min after the reaction is finished, inactivating the enzyme, centrifuging, taking the supernatant, and performing spray drying to obtain the wheat oligopeptide. The obtained wheat oligopeptide has cysteine content of 1.23%, free amino acid content of 8.71%, and glutamine content of 19.6%.
Example 4
Adding 10 parts of wheat gluten into 125 parts of water, adding 0.08 part of neutral protease, maintaining the pH at 6.5 and the temperature at 60 ℃, and performing enzymolysis for a period of time until the hydrolysis degree is 4%.
The pH of the prehydrolyzed wheat gluten was adjusted to 7.0, 0.005 part amylase (saccharifying enzyme), 0.005 part glucose oxidase were added, constant temperature 50 deg.C, air was pumped in, divergent ultrasonic treatment (30W/L, 50kHz) was applied and stirring was carried out for 15 min.
Adding 0.02 part of neutral protease into the pretreated wheat gluten, maintaining the pH at 6.5 and the temperature at 60 ℃, performing enzymolysis for a period of time until the hydrolysis degree is 12%, boiling for 20min after the reaction is finished, inactivating the enzyme, centrifuging, taking the supernatant, and performing spray drying to obtain the wheat oligopeptide. The obtained wheat oligopeptide has cysteine content of 1.81%, free amino acid content of 4.9%, and glutamine content of 24.1%.
The beneficial effects of the present invention are illustrated by the following animal experimental examples.
1. Experimental test method
Selecting 60 healthy cats with the age of 1-3 years and the weight of 1.5-4 kg in a certain breeding base; selecting 60 healthy dogs aged 1-6 years and weighing 5-10 kg. Dividing dogs and cats into 10 groups at random, 2 groups as cat comparison groups, and 4 groups as cat experiment groups; group 2 was used as canine control group and group 4 was used as canine experimental group. The 120 pet dogs and cats are transported to a new breeding base (new brand daily ration is changed) which is 25 km away, the dog and cat experimental groups 1, 2, 3 and 4 freely eat the new brand daily ration added with 2% of the oligopeptide in the examples 1, 2, 3 and 4 respectively, the dog and cat comparison groups 1 and 2 freely eat the new brand daily ration added with 2% of the oligopeptide in the comparative examples 1 and 2 respectively, after 2 days continuously, all dog and cat excreta are observed, collected and stored on the 3 rd day, dried in an oven at 105 ℃ until the constant weight, the moisture content is measured, and the results are shown in tables 3 and 4.
And scoring the defecation condition of the pet according to the following table, wherein the total score of the defecation condition is the defecation frequency score plus the defecation form score.
TABLE 1 Pet dog and cat defecation frequency scoring table
TABLE 2 Pet dog and cat defecation form scoring table
2. Results of the experiment
As shown in table 3, the average moisture content of the excreta of the cat comparison groups 1 and 2 is 85.6 percent and 75.8 percent, the minimum moisture content of the excreta of the cat in the experimental group is 61.1 percent, and the improvement is obvious compared with the comparison group; the average moisture content of the excrement of the dog comparison groups 1 and 2 is 79.3 percent and 74.4 percent, the minimum moisture content of the excrement of the dog experiment group is 34.7 percent, the improvement is obvious compared with the comparison group, and the improvement effect of the dog is better than that of the cat.
TABLE 3 Pet dog and cat excreta moisture content determination
As can be seen from table 4, after feeding for 3 days, the average scores of defecation of the cat comparison groups 1 and 2 are 8.7 and 8.4, and the average scores of defecation of the cat experiment groups 1, 2, 3 and 4 are 4.6, 5.2, 5.1 and 4.8 respectively, which indicates that the examples 1, 2, 3 and 4 have better effect on improving the defecation of the pet cat under stress state, wherein the experimental groups 1 and 4 are optimal and have obvious difference compared with the comparison group; the average scores of the defecation conditions of the dog comparison groups 1 and 2 are 8.5 and 8.2, and the average scores of the defecation conditions of the dog experiment groups 1, 2, 3 and 4 are 3.0, 3.4, 2.6 and 3.1 respectively, which illustrate that the example 1, 2, 3 and 4 have excellent effect on improving the defecation conditions of pet dogs in a stress state, wherein the experiment groups of dog 1 and dog 3 are optimal and have obvious difference compared with the comparison group.
TABLE 4 Pet dog and cat defecation scoring table
The above embodiments are only specific examples of the present invention, and the protection scope of the present invention includes but is not limited to the product forms and styles of the above embodiments, and any suitable changes or modifications made by those skilled in the art according to the claims of the present invention shall fall within the protection scope of the present invention.
Claims (3)
1. A preparation method of oligopeptide for improving defecation function of pet dogs and cats is characterized by comprising the following steps:
(1) pre-hydrolysis of wheat gluten
Adding 10 parts of wheat gluten into 125-200 parts of water, adding 0.05-0.1 part of neutral protease, maintaining the pH at 5.5-7.0 and the temperature at 50-60 ℃, and carrying out enzymolysis for a period of time until the hydrolysis degree is 3% -5%;
(2) cysteine oxidation of wheat gluten
Adjusting the pH value of prehydrolyzed wheat gluten to 5.5-7.0, adding 0.005-0.01 part of amylase (saccharifying enzyme) and 0.005-0.01 part of glucose oxidase, pumping air at a constant temperature of 40-50 ℃, applying divergent ultrasonic treatment and stirring for 15-20 min;
(3) enzymatic hydrolysis of oxidized wheat gluten
And (2) adding no protease or 0.02-0.05 part of neutral protease into the pretreated wheat gluten, maintaining the pH value at 5.5-7.0 and the temperature at 50-60 ℃, continuing enzymolysis for a period of time until the hydrolysis degree is 10-12%, boiling for 15-20 min after the reaction is finished, inactivating the enzyme, centrifuging, taking the supernatant, and performing spray drying to obtain the wheat oligopeptide.
2. The method for preparing oligopeptide capable of improving defecation function of pet dogs and cats according to claim 1, wherein the divergent ultrasonic treatment parameters in step (2) are ultrasonic power density of 10-50W/L and ultrasonic frequency of 40-60 kHz.
3. The use of the wheat oligopeptide obtained by the method for preparing the oligopeptide capable of improving the defecation function of the pet dogs and cats in the pet food for improving the sub-health conditions of non-pathological diarrhea, soft stool and the like of the pet dogs and cats according to the claims 1-2.
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| CN115521963A (en) * | 2022-10-28 | 2022-12-27 | 华南理工大学 | Glutamine peptide and preparation method and application thereof |
| WO2024103619A1 (en) * | 2022-11-15 | 2024-05-23 | 南京纽邦生物科技有限公司 | Preparation method for light-colored water-soluble vegetable protein |
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