CN113826764B - Preparation method of soybean small peptide chelated iron - Google Patents
Preparation method of soybean small peptide chelated iron Download PDFInfo
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- CN113826764B CN113826764B CN202111121409.5A CN202111121409A CN113826764B CN 113826764 B CN113826764 B CN 113826764B CN 202111121409 A CN202111121409 A CN 202111121409A CN 113826764 B CN113826764 B CN 113826764B
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Classifications
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- 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
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/105—Aliphatic or alicyclic compounds
-
- 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
- A23K20/163—Sugars; Polysaccharides
-
- 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
- A23K20/20—Inorganic substances, e.g. oligoelements
- A23K20/30—Oligoelements
-
- 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
- A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
-
- 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
- A23K50/60—Feeding-stuffs specially adapted for particular animals for weanlings
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Polymers & Plastics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Food Science & Technology (AREA)
- Birds (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Inorganic Chemistry (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Fodder In General (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention belongs to the technical field of preparation of soybean small peptide chelated iron, and particularly relates to a preparation method of soybean small peptide chelated iron. According to the preparation method of the soybean small peptide protein peptide iron chelate, the iron element is chelated with the soybean small peptide, and the soybean small peptide chelated iron prepared by the steps of pretreatment, enzymolysis reaction, chelation reaction, spray drying and the like of soybean meal has good stability, has double functions of metal ions and small peptide, has high small peptide content, has the peptide content of less than 1000Da and more than 75%, has the peptide content of less than 500Da and more than 50%, is easy to be absorbed and utilized by organisms, can meet the requirement of the soybean small peptide on the iron element when being applied to weaned pigs, reduces the emission of iron, and simultaneously has the growth performance and the in-vivo iron content of the suckling pigs.
Description
Technical Field
The invention belongs to the technical field of preparation of soybean small peptide chelated iron, and particularly relates to a preparation method of soybean small peptide chelated iron.
Background
Iron is a component of various compounds required for the oxidative energy supply process of substances in the body, is an important component of cytochromes, haemoglobin, myoglobin and enzymes, such as xanthine oxidase, cytochrome oxidase, peroxidase and the like, and participates in important biochemical reactions and substance metabolism in the body, and plays an important role in the transportation of oxygen in the body and various oxidation systems. Iron deficiency of livestock and poultry often manifests as anemia, diarrhea, reduced feed conversion rate, reduced immunity, and persistent influence on the immune system, resulting in lower body weight and length development than normal index. The young livestock and poultry are in the rapid growth period, need a large amount of iron elements to maintain the normal growth and development of organisms, and are easy to cause symptoms such as anemia, slow growth and development, reduced resistance of the organisms to diseases and the like due to iron deficiency. The currently used iron supplement for the suckling pigs is the dextran iron, which can prevent the anemia of the suckling pigs, but because the iron supplement is an injection, the injection part has more residues, is orally toxic, has higher requirements on the suckling pigs, cannot pass through placenta barriers of the sow and the like, and therefore, the use of the iron supplement for the suckling pigs is limited.
In practical application, a large amount of soybean protein resources such as soybean meal and the like are directly used as feed, so that the digestion and absorption of protein by young and aquatic organisms with simple and short digestive tracts, which have not yet been sound in digestive function, are very difficult, and small peptide substances generated by degradation can be absorbed and utilized by organisms. Depending on the molecular weight, less than 500Da is small peptide, 500 to 1000Da is oligopeptide, and 1000 to 10000 Da is polypeptide. The amino acid absorption of oligopeptides is slightly slower than that of small peptides. As the amino acid content increases, the rate of absorption of the small peptide decreases significantly, and it is believed that the small peptide is absorbed intact. The peptide content of the soybean small peptide chelated iron is affected by different protease enzymolysis or other hydrolysis modes, CN 109868302A is hydrolyzed by protease, and the proportion of small molecular peptide with the relative molecular weight of 180-1000Da in the finished product accounts for more than 70 percent of the total protein, and the proportion below 180Da is 11.8 percent. Zhang Monan the molecular weight of soybean polypeptide prepared by enzymatic hydrolysis of soybean with trypsin is mainly distributed between 2-10 kDa. Li Wenjun et al have the problems of poor product stability, low content of effective polypeptide and the like because the molecular weight of soybean polypeptide prepared by hydrolyzing soybean with lactobacillus bulgaricus is mostly between 1000Da and 2500 Da.
The small peptide substance has the characteristics of single absorption path, good chemical stability, higher biological potency, high solubility, easy digestion and absorption, interference resistance and the like, so that the preparation of the low molecular weight peptide by utilizing enzymolysis has important significance. With the intensive research of chelate iron in recent years, amino acid chelate iron is mainly adopted at present, and peptide chelate iron contains necessary iron ions required for growth and organic components for promoting iron ion absorption, has special biological activity and is considered to be a relatively ideal feed and food additive at present. However, in the current feed production, the content of trace elements in the feed raw materials is generally not considered, and the trace elements are directly added according to 1-3 times of the recommended amount in the feeding standard, so that the content of trace elements in the feed exceeds the standard. However, excessive addition can cause high residue of trace elements in animal livers or tissues, inorganic salts not only easily cause oxidative stress damage of organisms in the absorption process and influence animal production performance, but also have low absorption and utilization rate of electrodeless iron elements in feeds, so that trace element resource waste is often caused in actual production, and the animals excrete in large quantities, so that the environment is easily polluted.
Therefore, a novel iron supplement with higher bioavailability and small addition amount is needed, and the problems of improving the oxidation stress resistance and immunity of the suckling pigs, reducing the emission of iron in the feces and the like are solved.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a preparation method of soybean small peptide chelated iron. The soybean small peptide chelated iron prepared by the preparation method provided by the invention has the dual functions of metal ions and small peptides, the small peptide content is high, the peptide content with the molecular weight below 1000Da is more than 75%, the peptide content below 500Da is more than 50%, the absorption and utilization of organisms are easy, the chelation rate of iron element and soybean small peptide is high, and the effect is excellent when the soybean small peptide chelated iron is applied to a feed additive.
The technical scheme of the invention is as follows:
a preparation method of soybean small peptide chelated iron comprises the following steps:
S1, pretreatment: adding water into soybean meal, uniformly stirring to prepare protein liquid with the concentration of 10-12%, adding an enzymolysis promoter with the mass of 1.3-2.1% into the soybean meal, and homogenizing for 2-3 times under the condition of 15-30 Mpa to obtain homogenized mixed soybean meal protein liquid;
S2, enzymolysis reaction: regulating the pH of the mixed soybean meal protein liquid obtained in the step S1 to 8-9, adding compound protease, carrying out enzymolysis for 3-5 hours at 40-50 ℃, heating to 80-95 ℃, inactivating enzyme for 10-30 minutes, centrifuging for 20 minutes at 4000rpm/min, collecting supernatant, and freeze-drying to obtain soybean small peptide powder;
S3, chelation reaction: adding water into the soybean small peptide powder obtained in the step S2, uniformly stirring to prepare soybean small peptide solution with the concentration of 10-20%, then adding antioxidant with the mass of 0.5-1% of the soybean small peptide powder and inorganic ferrous salt solution with the concentration of 10-20%, uniformly stirring, then adding antioxidant with the mass of 0.2-0.5% of the soybean small peptide powder, uniformly mixing, regulating the pH value to 5-6, heating to 45-65 ℃, starting timing, and reacting for 0.4-1.5 h;
s4, spray drying: and after the reaction is finished, spray drying to obtain the soybean small peptide chelated iron.
Further, the protein content of the soybean meal in the step S1 is 30-50%.
Further, the enzymolysis promoter in the step S1 is polyvinyl alcohol and reduced glutathione according to the weight ratio of (5-8): 3.
Further, the composite protease in the step S2 is composed of alkaline protease, neutral protease and trypsin according to an enzyme activity ratio of 3:2:1, and the total enzyme activity of the composite protease is 4000-6000U/g soybean meal.
Further, the antioxidant in the step S3 is one or a combination of more than two of sodium D-erythorbate, vitamin C, astragalus polysaccharide and sodium sulfite.
Further, the antioxidant in the step S3 is composed of D-sodium erythorbate, astragalus polysaccharide and sodium sulfite according to the weight ratio of 7-11:2-5:1-4.
Further, the antioxidant in the step S3 is composed of D-sodium erythorbate, astragalus polysaccharide and sodium sulfite according to a weight ratio of 10:3:2.
Further, the inorganic ferrous salt solution in the step S3 is a ferrous sulfate solution with the concentration of 10-20%.
Further, the mass ratio of the ferrous iron in the inorganic ferrous salt solution in the step S3 to the soybean small peptide powder is 1:3-5.
Further, in the step S4, the temperature of the spray drying air inlet is 180-220 ℃, and the temperature of the air outlet is 90-100 ℃.
The iron element plays an important role on animal bodies, iron deficiency of livestock and poultry is normal, and a dextran iron needle which is commonly selected for supplementing iron for suckling pigs can bring adverse effects to the animal bodies. The high intake can lead to high excretion, and the metal elements remained in the feces of livestock and poultry cultivation can not only affect the soil environment, but also affect the health of people and livestock through the biological amplification effect of food chain by crop absorption. In the invention, soybean small peptide and iron element are adopted for chelation, and in the pretreatment process, a certain proportion of enzymolysis promoter is added into soybean meal protein liquid, and then homogenization treatment is carried out, so that the viscosity of protein solution can be reduced, soybean meal protein can be more uniformly and stably dispersed in water, meanwhile, the structure of soybean protein can be destroyed, disulfide bonds are broken, peptide bonds are unfolded, the soybean protein can be more efficiently hydrolyzed by compound protease in the subsequent enzymolysis process, the hydrolysis efficiency is improved, the soybean protein peptide with smaller molecular weight can be obtained, the obtained soybean small peptide has more superior bioactivity, and meanwhile, the metal chelation activity is higher, the chelation reaction with iron element is facilitated, the content of chelated iron is improved, and the iron supplementing effect of the product is better exerted. Then, the soybean meal protein liquid is hydrolyzed by adopting alkaline protease, neutral protease and trypsin according to the enzyme activity ratio of 3:2:1, and the enzymolysis effect is further improved by controlling the conditions of enzymolysis time, enzymolysis pH, temperature and the like, the molecular weight of the prepared soybean small peptide is mainly concentrated within 1000Da, and the proportion of collagen peptide with the molecular weight within 1000Da reaches more than 75%.
Furthermore, during the chelation reaction, the antioxidant comprising D-sodium erythorbate, astragalus polysaccharide and sodium sulfite according to a certain mass ratio is added, and an active group on a soybean small peptide molecular chain in the chelation reaction process can be effectively maintained by adopting a mode of adding the antioxidant for two times, so that the physiological activity of the soybean protein peptide with low molecular weight is beneficial to maintaining, the chelation reaction is promoted, the chelation rate of the obtained soybean small peptide chelated iron is high, and the chelation rate of the soybean small peptide chelated iron is more beneficial to the absorption of animals to feed products when the chelation reaction is applied to the feed industry, and the disease resistance and the immunity of animals can be improved, and meanwhile, the emission of iron element in piglet manure can be reduced.
Compared with the prior art, the soybean small peptide chelated iron provided by the invention has the following advantages:
(1) The protein source adopted by the invention is soybean meal, the cost is low, the raw materials are easy to obtain, the source is rich in various amino acids, the chelate iron prepared by the method can be produced in industrial scale, the reaction product is easy to separate and purify, the operation is simple, the cost is low, the physicochemical property of the product is stable, and the soybean small peptide chelate iron prepared by the method has high purity and good crystal form structure, thereby being beneficial to accurately controlling the use amount.
(2) The soybean small peptide chelated iron prepared by the preparation method disclosed by the invention has the advantages that the molecular weight is small, the molecular weight is less than 1000Da and accounts for more than 75%, the molecular weight is less than 500Da and accounts for more than 50%, the molecular weight is less than 180Da and accounts for more than 14%, meanwhile, the iron content is more than 11%, the chelation rate is more than 80%, the iron element is chelated with the soybean small peptide, so that the absorption and utilization rate of the iron element by target animals is increased, the nutrition functionality and biological valence of the iron element are improved, the absorption and utilization of organisms are facilitated, and the iron element content in excrement is reduced, so that the environmental pollution is reduced.
(3) The soybean small peptide chelated iron prepared by the preparation method provided by the invention has the advantages of good palatability, comprehensive nutrition and easy digestion, ensures that piglets are weaned smoothly, can alleviate strong stress on the piglets caused by weaning, and reduces the wounds caused by converting pig milk into solid feed.
Drawings
FIG. 1 is an infrared spectrum of the soybean small peptide chelated iron prepared in example 2 of the invention;
FIG. 2 is an ultraviolet spectrum of the soybean small peptide chelated iron prepared in example 2 of the invention;
FIG. 3 is a diagram showing the structural analysis of soybean small peptide chelated iron.
Detailed Description
The invention is further illustrated by the following description of specific embodiments, which are not intended to be limiting, and various modifications or improvements can be made by those skilled in the art in light of the basic idea of the invention, but are within the scope of the invention as long as they do not depart from the basic idea of the invention.
The soybean meal is purchased from Shandong Gucheng chemical technology Co., ltd; astragalus polysaccharides CAS 89250-26-0, available from Nanjing doffer Biotechnology Co., ltd; reduced glutathione, shanghai Miclin Biochemical technology Co., ltd., product number is L810392-100g; polyvinyl alcohol 1799, CAS:9002-89-5, available from Shanghai purple-reagent plant (Klamal); alkaline protease, neutral protease, trypsin were purchased from Shanghai Seiyaka Biotechnology Co.
In addition, other reagents used in the invention are all common reagents and can be purchased from conventional reagent production and sale companies.
Example 1 preparation method of Soybean small peptide chelated iron
The preparation method of the soybean small peptide chelated iron comprises the following steps:
S1, pretreatment: weighing 1000kg of soybean meal, adding 10000kg of water to prepare a protein solution with the concentration of 10%, adding an enzymolysis promoter with the mass of 1.3% of the soybean meal, and homogenizing for 2 times under the condition of 15Mpa to obtain a homogenized mixed soybean meal protein solution;
S2, enzymolysis reaction: regulating the pH of the mixed soybean meal protein solution obtained in the step S1 to 8 by using 2mol/L NaOH and HCl solution, then adding compound protease, carrying out enzymolysis for 3 hours at 40 ℃, heating to 80 ℃, inactivating enzyme for 10 minutes, centrifuging for 20 minutes under the condition of 4000rpm/min, collecting supernatant, and freeze-drying to obtain soybean small peptide powder;
S3, chelation reaction: weighing 900kg of the soybean small peptide powder obtained in the step S2, adding 9000kg of water, stirring uniformly to prepare soybean small peptide solution with the concentration of 10%, adding antioxidant with the mass of 0.5% of the soybean small peptide powder and ferrous sulfate solution with the concentration of 10%, stirring uniformly, adding antioxidant with the mass of 0.2% of the soybean small peptide powder, regulating the pH value to 5 by using sodium hydroxide with the concentration of 20% after mixing uniformly, heating to 45 ℃, starting timing, and reacting for 0.4h;
the enzymolysis promoter in the step S1 is polyvinyl alcohol 1799 and reduced glutathione with the weight ratio of 5: 3.
The compound protease in the step S2 is composed of alkaline protease, neutral protease and trypsin according to the enzyme activity ratio of 3:2:1, and the total enzyme activity of the compound protease is 4000U/g soybean meal.
The antioxidant in the step S3 is composed of D-sodium erythorbate, astragalus polysaccharide and sodium sulfite according to the weight ratio of 7:2:4.
The mass ratio of the ferrous in the ferrous sulfate solution to the soybean small peptide powder in the step S3 is 1:5.
And in the step S4, the temperature of the spray drying air inlet is 180 ℃ and the temperature of the spray drying air outlet is 90 ℃.
Example 2 preparation method of Soybean small peptide chelated iron
The preparation method of the soybean small peptide chelated iron comprises the following steps:
S1, pretreatment: weighing 1000kg of soybean meal, adding 9091kg of water to prepare a protein solution with the concentration of 11%, adding an enzymolysis promoter with the mass of 1.8% of the soybean meal, and homogenizing for 3 times under the condition of 25Mpa to obtain a homogenized mixed soybean meal protein solution;
S2, enzymolysis reaction: regulating the pH of the mixed soybean meal protein solution obtained in the step S1 to 8.5 by using 2mol/LNaOH and HCl solution, adding compound protease, carrying out enzymolysis for 4 hours at 45 ℃, heating to 90 ℃, inactivating enzyme for 20 minutes, centrifuging for 20 minutes under the condition of 4000rpm/min, collecting supernatant, and freeze-drying to obtain soybean small peptide powder;
S3, chelation reaction: weighing 900kg of the soybean small peptide powder obtained in the step S2, adding 6000kg of water, stirring uniformly to prepare soybean small peptide solution with the concentration of 15%, adding antioxidant with the mass of 0.8% of the soybean small peptide powder and ferrous sulfate solution with the concentration of 15%, stirring uniformly, adding antioxidant with the mass of 0.3% of the soybean small peptide powder, regulating the pH to 5.5 after uniformly mixing, heating to 55 ℃, starting timing, and reacting for 1.0h;
the enzymolysis promoter in the step S1 is polyvinyl alcohol 1799 and reduced glutathione with the weight ratio of 7: 3.
The compound protease in the step S2 is composed of alkaline protease, neutral protease and trypsin according to the enzyme activity ratio of 3:2:1, and the total enzyme activity of the compound protease is 5000U/g soybean meal.
The antioxidant in the step S3 is composed of D-sodium erythorbate, astragalus polysaccharide and sodium sulfite according to the weight ratio of 10:3:2.
And the mass ratio of the ferrous in the ferrous sulfate solution to the soybean small peptide powder in the step S3 is 1:4.
And in the step S4, the temperature of the spray drying air inlet is 200 ℃, and the temperature of the spray drying air outlet is 95 ℃.
Example 3 preparation method of Soybean small peptide chelated iron
The preparation method of the soybean small peptide chelated iron comprises the following steps:
S1, pretreatment: weighing 1000kg of soybean meal, adding 8333kg of water to prepare a protein solution with the concentration of 12%, adding an enzymolysis promoter with the mass of 2.1% of the soybean meal, and homogenizing for 3 times under the condition of 30Mpa to obtain a homogenized mixed soybean meal protein solution;
S2, enzymolysis reaction: regulating the pH of the mixed soybean meal protein solution obtained in the step S1 to 9 by using 2mol/L NaOH and HCl solution, then adding compound protease, carrying out enzymolysis for 5 hours at 50 ℃, heating to 95 ℃, inactivating enzyme for 30 minutes, centrifuging for 20 minutes under the condition of 4000rpm/min, collecting supernatant, and freeze-drying to obtain soybean small peptide powder;
S3, chelation reaction: weighing 900kg of the soybean small peptide powder obtained in the step S2, adding 4500kg of water, stirring uniformly to prepare a soybean small peptide solution with the concentration of 20%, then adding an antioxidant with the mass of 1% of the soybean small peptide powder and a ferrous sulfate solution with the concentration of 20%, stirring uniformly, then adding an antioxidant with the mass of 0.5% of the soybean small peptide powder, stirring uniformly, regulating the pH to 6 by using sodium hydroxide with the concentration of 20%, heating to 65 ℃, starting timing, and reacting for 1.5h;
the enzymolysis promoter in the step S1 is polyvinyl alcohol 1799 and reduced glutathione with a weight ratio of 8: 3.
The compound protease in the step S2 is composed of alkaline protease, neutral protease and trypsin according to the enzyme activity ratio of 3:2:1, and the total enzyme activity of the compound protease is 6000U/g soybean meal.
The antioxidant in the step S3 is composed of D-sodium erythorbate, astragalus polysaccharide and sodium sulfite according to the weight ratio of 11:5:1.
And the mass ratio of the ferrous in the ferrous sulfate solution to the soybean small peptide powder in the step S3 is 1:3.
And in the step S4, the air inlet temperature is 220 ℃ and the air outlet temperature is 100 ℃ in spray drying.
Comparative example 1 preparation method of Soybean small peptide chelated iron
Compared with example 2, the difference of comparative example 1 is that no enzymolysis promoter is added in the pretreatment of step S1, and other parameters and operations are the same as in example 2.
Comparative example 2 preparation method of Soybean small peptide chelated iron
Compared with example 2, the difference of comparative example 2 is that the enzymolysis accelerator in the pretreatment of step S1 is not added with polyvinyl alcohol 1799, and other parameters and operations are the same as those of example 2.
Comparative example 3 preparation method of Soybean small peptide chelated iron
Compared with example 2, the difference of comparative example 3 is that reduced glutathione is not added to the enzymolysis accelerator in the pretreatment of step S1, and other parameters and operations are the same as in example 2.
Comparative example 4 preparation method of Soybean small peptide chelated iron
Compared with example 2, the difference of comparative example 4 is that astragalus polysaccharide is not added to the antioxidant of step S3, and other parameters and operations are the same as example 2.
Comparative example 5 preparation method of Soybean small peptide chelated iron
Compared with example 2, the difference of comparative example 5 is that the antioxidant in step S3 is composed of sodium D-isoascorbate, astragalus polysaccharide and sodium sulfite in a weight ratio of 1:1:1, and other parameters and operations are the same as example 2.
Comparative example 6 preparation method of Soybean small peptide chelated iron
The difference of comparative example 5 compared to example 2 is that the step S3 chelation reaction is added with only the first antioxidant, and no antioxidant is added after the ferrous sulfate solution is added, and other parameters and operations are the same as example 2.
Test example I, molecular weight distribution of soybean small peptide chelated iron peptide and content test of effective component
1. Test materials: examples 1 to 3, comparative examples 1 to 6.
2. The test method comprises the following steps: the soybean small peptides prepared in comparative examples 1 to 6 were subjected to chelation in examples 1 to 3 above, and the molecular weight of the peptides, the total content of iron elements and the chelation rate were measured. The peptide content is measured according to high-efficiency gel filtration chromatography in GB/T22729-2008, the measurement of the soybean small peptide molecular weight distribution is entrusted to the detection of a third party Jiangnan university analysis test center, and the infrared spectrum analysis of the small peptide chelated iron structure is entrusted to the Dalian institute, and the phenanthroline method is used for detecting the iron content.
3. Test results
The test results are shown in Table 1.
TABLE 1 molecular weight distribution of Soybean small peptide chelated iron peptides
TABLE 2 iron content and chelation Rate of Soybean small peptide chelated iron
| Group of | Iron content/% | Chelation rate/% |
| Example 1 | 11.71 | 82.39 |
| Example 2 | 12.13 | 83.43 |
| Example 3 | 11.42 | 82.33 |
| Comparative example 1 | 5.68 | 62.38 |
| Comparative example 2 | 7.41 | 70.26 |
| Comparative example 3 | 7.65 | 69.33 |
| Comparative example 4 | 8.99 | 74.15 |
| Comparative example 5 | 9.08 | 73.58 |
| Comparative example 6 | 7.12 | 72.65 |
As can be seen from tables 1 and 2, the ratio of the small molecular peptides with the relative molecular weight below 1000Da to the total protein of the small molecular peptides with the molecular weight below 1000Da in the soybean small peptide chelated iron peptides prepared in the embodiments 1-3 reaches more than 75%, wherein the ratio of the small molecular peptides with the molecular weight below 1000Da in the embodiment 2 reaches up to 77.86%, the iron content is more than 11%, and the chelation rate is 83.43.
Test example two, active ingredient content test
1. The soybean small peptide chelated iron infrared spectrum obtained in the embodiment 2 of the invention
From FIG. 1, it is assumed that the metal ion coordinates with the peptide fragment.
2. The soybean small peptide chelate iron violet prepared in example 2 of the present invention has an external spectrum
See fig. 2.
3. The bean small peptide chelated iron structure analysis prepared by the invention
See fig. 3.
Test example III, effect test
1. Test animals: 150 healthy weaned piglets with the weight of 7.80+/-0.10 kg are selected from Du long and large ternary hybridization of 21+/-2 days old.
2. The test method comprises the following steps:
(1) Test grouping: the 150 healthy weaned pigs are divided into 5 groups according to body weight, nest and sex, three repeats are arranged in each group, 10 repeats are arranged in each group, and the test period is 42 days. Treatment 1 is a blank group and is fed with basic ration; treatment 2 is a control group fed basal diet + (soybean small peptide + inorganic Fe); treatments 3 to 5 were test groups 1 to 3, and basic ration + soybean small peptide chelated iron prepared in examples 1 to 3 was fed. The basic ration is formulated according to NRC 2012 standard.
(2) And (3) feeding management: raising according to pig raising standard NYT 65-2004. The weaned pigs are fed by a high bed, naturally ventilated, and have good environmental sanitation conditions, and are fed 3 times a day (1 time for 7:00, 13:00 and 20:00) and are free to drink water; insect repellent and immune were treated according to pig farm protocol. And observing the feeding and health conditions of animals at any time. All diarrhea pigs were not treated.
(3) Sample collection: at the end of the test, 2 boars and 1 sow are slaughtered repeatedly, 15mL of the anterior vena cava of each test pig is sampled, the test pigs are kept stand in a water bath at 37 ℃ until serum is separated out, centrifugation is carried out for 10min at 3500r/min, and the separated serum is stored in an Eppendorf tube at-80 ℃ and used for measuring serum microelements and biochemical indexes.
(4) Detecting the index: and (3) utilizing a full-automatic blood analyzer to measure the red blood cell number, the hemoglobin concentration, the serum iron content and the immunoglobulin G content of the piglets by adopting the piglet ear vein blood before and after the test, recording the growth, diarrhea and death and panning of the piglets, and detecting the iron content in the feces. The test results are shown in Table 3.
TABLE 3 Biochemical indices of growth status and serum of piglets
| Group of | Blank group | Comparison group | Test group 1 | Test group 2 | Test group 3 |
| Initial average weight (kg) | 7.81 | 7.84 | 7.80 | 7.86 | 7.79 |
| Last average weight (kg) | 27.13 | 28.42 | 29.22 | 29.70 | 29.21 |
| Average daily feed intake ADFI (kg/d) | 0.71 | 0.75 | 0.69 | 0.68 | 0.67 |
| Average daily gain ADG (kg/d) | 0.46 | 0.49 | 0.51 | 0.52 | 0.51 |
| Ratio of weight to material (F/G) | 1.54 | 1.53 | 1.35 | 1.31 | 1.31 |
| Diarrhea Rate (%) | 3.05 | 2.68 | 2.03 | 1.92 | 1.92 |
| Rate of death panning (%) | 3.33 | 3.33 | 0 | 0 | 0 |
| Hemoglobin concentration HGB (g/L) | 90.23 | 92.87 | 97.54 | 98.78 | 97.45 |
| Serum ferritin SF (g/L) | 15.25 | 17.56 | 20.84 | 21.02 | 20.35 |
| Red blood cell number RBC (10 12/L) | 4.79 | 5.02 | 5.23 | 5.26 | 5.21 |
| Immunoglobulin IgG (g/L) | 3.42 | 3.56 | 3.81 | 3.90 | 3.80 |
| Fe content in feces (mg/kg) | 79.80 | 115.41 | 85.24 | 78.04 | 80.26 |
As can be seen from Table 1, the soybean small peptide chelated iron obtained by the preparation method of examples 1-3 of the invention can obviously improve the hemoglobin concentration of weaned pigs, obviously increase the feed intake and weight gain and reduce the death rate of diarrhea rate compared with the comparison example and the blank group. In contrast, soybean small peptide and inorganic iron are added into the basic ration respectively, and in this case, compared with the embodiments 1-3 of the present invention, the effects of hemoglobin concentration, feed intake and weight gain are all reduced, and the feed conversion ratio, diarrhea rate and death rate are also increased. Therefore, the soybean small peptide chelated iron provided by the invention has the advantages that through a special preparation process and the selection and content proportion of raw materials in a formula, the components are synergistic, the intestinal tract can be regulated, the healthy development of the intestinal tract is promoted, and the hematopoietic capacity and the immunity of piglets are improved.
The HGB concentration begins to decline when the iron reserves in the body are exhausted, and the HGB concentration is used as an index for diagnosing the iron level of the suckling pigs, so that the HGB concentration has hysteresis and is unfavorable for the prevention and treatment of the iron of the suckling pigs. SF mass concentration is positively correlated with iron reserve in vivo, and can directly reflect iron reserve in vivo, and HGB and SF mass concentration are combined, so that the method has specificity and sensitivity, is an effective index for diagnosing the nutrition status of the suckling pig iron and the anemia or not, and is an index for evaluating the iron supplementing effect of the iron supplementing agent. The embodiments 1-3 of the invention have remarkable effects on improving the serum hemoglobin and serum ferritin concentration of piglets, and can be well applied to weaned piglet feeds as a feed additive.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Claims (4)
1. The preparation method of the soybean small peptide chelated iron is characterized by comprising the following steps:
S1, pretreatment: adding water into soybean meal, uniformly stirring to prepare protein liquid with the concentration of 10-12%, adding an enzymolysis promoter with the mass of 1.3-2.1% into the soybean meal, and homogenizing for 2-3 times under the condition of 15-30 mpa to obtain homogenized mixed soybean meal protein liquid;
S2, enzymolysis reaction: regulating the pH of the mixed soybean meal protein liquid obtained in the step S1 to 8-9, adding compound protease, carrying out enzymolysis for 3-5 hours at 40-50 ℃, heating to 80-95 ℃, inactivating enzyme for 10-30 minutes, centrifuging for 20 minutes at 4000rpm/min, collecting supernatant, and freeze-drying to obtain soybean small peptide powder;
S3, chelation reaction: adding water into the soybean small peptide powder obtained in the step S2, uniformly stirring to prepare a soybean small peptide solution with the concentration of 10-20%, then adding an antioxidant with the mass of 0.5-1% of the soybean small peptide powder and an inorganic ferrous salt solution with the concentration of 10-20%, uniformly stirring, then adding an antioxidant with the mass of 0.2-0.5% of the soybean small peptide powder, uniformly mixing, regulating the pH value to 5-6, heating to 45-65 ℃, starting timing, and reacting for 0.4-1.5 h;
S4, spray drying: after the reaction is finished, spray drying is carried out to obtain soybean small peptide chelated iron; the enzymolysis promoter in the step S1 is polyvinyl alcohol and reduced glutathione according to the weight ratio of (5-8): 3, composing; the compound protease in the step S2 is composed of alkaline protease, neutral protease and trypsin according to an enzyme activity ratio of 3:2:1, and the total enzyme activity of the compound protease is 4000-6000U/g soybean meal; the antioxidant in the step S3 is composed of D-sodium erythorbate, astragalus polysaccharide and sodium sulfite according to the weight ratio of 7-11:2-5:1-4; and the inorganic ferrous salt solution in the step S3 is 10-20% ferrous sulfate solution.
2. The method for preparing soybean small peptide chelated iron according to claim 1, wherein the antioxidant in the step S3 is composed of sodium D-isoascorbate, astragalus polysaccharide and sodium sulfite in a weight ratio of 10:3:2.
3. The method for preparing soybean small peptide chelated iron according to claim 1, wherein the mass ratio of ferrous iron in the inorganic ferrous salt solution to soybean small peptide powder in the step S3 is 1:3-5.
4. The method for preparing soybean small peptide chelated iron according to claim 1, wherein in the step S4, the spray drying air inlet temperature is 180-220 ℃, and the air outlet temperature is 90-100 ℃.
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