CN113826764A - Preparation method of soybean small peptide chelated iron - Google Patents
Preparation method of soybean small peptide chelated iron Download PDFInfo
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- CN113826764A CN113826764A CN202111121409.5A CN202111121409A CN113826764A CN 113826764 A CN113826764 A CN 113826764A CN 202111121409 A CN202111121409 A CN 202111121409A CN 113826764 A CN113826764 A CN 113826764A
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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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
- A23K20/163—Sugars; Polysaccharides
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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
- A23K20/20—Inorganic substances, e.g. oligoelements
- A23K20/30—Oligoelements
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- 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
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- 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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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. The invention provides a preparation method of soybean small peptide protein peptide iron chelate, which chelates iron element with soybean small peptide, and the soybean meal is pretreated, enzymolyzed, chelated, spray dried, the prepared soybean small peptide chelated iron has good stability, has double functions of metal ion and small peptide, and has high small peptide content, peptide content with molecular weight below 1000Da is more than 75%, peptide content below 500Da is more than 50%, the soybean small peptide chelated iron is easy to be absorbed and utilized biologically, and can meet the requirement of weaned suckling pigs on iron element when being used as feed additive, reduce the discharge amount of iron, and simultaneously increase 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 oxidation and energy supply processes of substances in vivo, is an important component of cytochrome, hemoglobin, myoglobin and enzymes, such as xanthine oxidase, cytochrome oxidase, peroxidase and the like, participates in important biochemical reactions and substance metabolism in the body, and plays an important role in oxygen transportation and various oxidation systems in the body. Iron deficiency of livestock and poultry often shows as anemia, diarrhea, reduced feed conversion rate and reduced immunity, and has lasting influence on immune system, so that the weight and body length development is lower than normal indexes. When young livestock and poultry are in a rapid growth period, a large amount of iron is needed to maintain normal growth and development of the organism, and symptoms such as anemia, growth and development retardation, reduction of the resistance of the organism to diseases and the like are easily caused by iron deficiency. Currently, the more used iron supplement for the piglets is iron dextran and can prevent the anemia of the piglets, but the iron supplement is an injection, has more residues on injection parts, is toxic by oral administration, has higher requirements on the used piglets, cannot pass the placenta barrier of the sows and the like, and is limited in use.
In practical application, a large amount of soybean protein resources such as soybean meal and the like are directly used as feeds, the digestion and absorption of proteins by young animals with incomplete digestion functions and aquatic organisms with simple and short digestive tracts are very difficult, and small peptide substances generated by degradation can be absorbed and utilized by organisms. According to the molecular weight, less than 500Da is small peptide, 500 to 1000Da is oligopeptide, and 1000 to 10000 Dalton is polypeptide. Oligopeptides absorb amino acids slightly more slowly than small peptides. The rate of absorption of small peptides decreases significantly with increasing amino acid content, and it is believed that small peptides are absorbed intact. Different protease enzymolysis or other hydrolysis modes influence the peptide content of the soybean small peptide chelated iron, CN 109868302A utilizes protease hydrolysis, the proportion of the small molecule peptide with the relative molecular weight of 180-1000Da in the finished product accounts for more than 70% of the total protein, and the proportion below 180Da is 11.8%. The molecular weight of the soybean polypeptide prepared by hydrolyzing soybeans with trypsin is mainly distributed between 2 and 10 kDa. The molecular weight of the soybean polypeptide prepared by hydrolyzing soybeans by utilizing lactobacillus bulgaricus by Li Wen Jun and the like is mostly between 1000Da and 2500 Da, and the problems of poor product stability, low content of effective polypeptide and the like exist.
Because small peptide substances have independent absorption ways and have the characteristics of good chemical stability, higher biological value, high solubility, easy digestion and absorption, interference resistance and the like, the preparation of low molecular weight peptides by utilizing enzymolysis has important significance. In recent years, with the intensive research on iron chelate, amino acid iron chelate is mainly used at present, and peptide iron chelate contains essential iron ions required for growth and organic components for promoting iron ion absorption, has special biological activity, and is considered to be a more ideal feed and food additive at present. However, in the current feed production, the content of trace elements in 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 the trace elements in the feed exceeds the standard. However, the excessive addition of the inorganic salt can cause high residue of trace elements in animal livers or tissues, the inorganic salt is easy to cause oxidative stress damage to organisms in the absorption process and influence the production performance of animals, the absorption and utilization rate of the animals on the non-polar iron element in the feed is low, the trace element resource waste is often caused in the actual production, and the animals excrete in large quantities, so that the environment is easily polluted.
Therefore, a novel iron supplement with high bioavailability and small addition amount is needed, and the iron supplement can enhance the oxidation stress resistance and immunity of piglets and reduce the emission of iron elements in excrement.
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 double functions of metal ions and small peptides, the content of the small peptides is high, the content of the peptides with the molecular weight of below 1000Da accounts for more than 75%, the content of the peptides with the molecular weight of below 500Da accounts for more than 50%, the soybean small peptide chelated iron is easy to be absorbed and utilized by organisms, the chelation rate of iron elements and the soybean small peptides is high, and the effect is excellent when the soybean small peptide chelated iron is applied to feed additives.
The technical scheme of the invention is as follows:
a preparation method of soybean small peptide chelated iron comprises the following steps:
s1, preprocessing: adding water into the soybean meal, uniformly stirring to prepare a protein solution with the concentration of 10-12%, then adding an enzymolysis promoter with the mass of 1.3-2.1% of the soybean meal, and carrying out homogenization treatment for 2-3 times under the condition of 15-30 Mpa to obtain a homogenized mixed soybean meal protein solution;
s2, enzymolysis reaction: adjusting the pH value of the mixed soybean meal protein liquid obtained in the step S1 to 8-9, adding compound protease, carrying out enzymolysis for 3-5 h at 40-50 ℃, heating to 80-95 ℃, inactivating enzyme for 10-30 min, centrifuging for 20min at 4000rpm/min, collecting supernatant, and carrying out 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 the mixture 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, adjusting the pH value to 5-6, starting timing when the temperature is increased to 45-65 ℃, and reacting for 0.4-1.5 h;
s4, spray drying: after the reaction is finished, the soybean small peptide chelated iron is obtained by spray drying.
Further, the protein content of the soybean meal in the step S1 is 30-50%.
Further, the enzymolysis accelerant in the step S1 is polyvinyl alcohol and reduced glutathione in a weight ratio of (5-8): 3, and (3).
Further, the compound protease in the step S2 is alkaline protease, neutral protease and trypsin, and the enzyme activity ratio is 3:2:1, and the total enzyme activity of the compound protease is 4000-6000U/g of soybean meal.
Further, the antioxidant in step S3 is one or a combination of two or more of sodium D-erythorbate, vitamin C, astragalus polysaccharides and sodium sulfite.
Furthermore, the antioxidant in the step S3 is composed of D-sodium erythorbate, astragalus polysaccharide and sodium sulfite according to a weight ratio of 7-11: 2-5: 1-4.
Furthermore, the antioxidant in the step S3 is composed of sodium D-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 10-20% ferrous sulfate solution.
Further, the mass ratio of the ferrous iron in the inorganic ferrous salt solution in the step S3 to the soybean oligopeptide powder is 1: 3-5.
Further, in the step S4, the temperature of the air inlet is 180-220 ℃ and the temperature of the air outlet is 90-100 ℃.
The iron element plays an important role in animal organisms, iron deficiency of livestock and poultry is a normal state, and a dextran iron needle usually selected for supplementing iron to suckling pigs brings a series of adverse effects on the animal organisms. High intake can lead to high excretion, and metal elements remained in the livestock and poultry breeding excrement not only can influence the soil environment, but also can influence the health of people and livestock through the biological amplification effect of the crop absorption through a food chain. According to the invention, the soybean small peptide and the iron element are chelated, and in the pretreatment process, an enzymolysis accelerant with a certain proportion is added into the soybean meal protein liquid, and then the homogenization treatment is carried out, so that the viscosity of the protein solution can be reduced, the soybean meal protein can be dispersed in water more uniformly and stably, meanwhile, the structure of the soybean protein can be destroyed, the disulfide bond is broken, the peptide bond is unfolded, the soybean protein can be hydrolyzed more efficiently by compound protease in the subsequent enzymolysis process, the hydrolysis efficiency is improved, meanwhile, the soybean protein peptide with smaller molecular weight can be obtained, the obtained soybean small peptide has more excellent biological activity, and simultaneously, the soybean small peptide has higher metal chelating activity, the chelating reaction with the iron element can be favorably carried out, the content of chelated iron is improved, and the iron supplement effect of the product is better exerted. Then, hydrolyzing the soybean meal protein liquid by adopting compound protease consisting of alkaline protease, neutral protease and trypsin according to the enzyme activity ratio of 3:2:1, and further improving the enzymolysis effect by controlling the conditions of enzymolysis time, enzymolysis pH, temperature and the like, wherein 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 chelation reaction, an antioxidant which is composed of D-sodium erythorbate, astragalus polysaccharide and sodium sulfite according to a certain mass ratio is added, and a mode of adding the antioxidant in two times is adopted, so that active groups on a soybean small peptide molecular chain in the chelation reaction process can be effectively maintained, the physiological activity of low-molecular-weight soybean protein peptide is favorably maintained, the chelation reaction is promoted to be carried out, the chelation rate of the obtained soybean small peptide chelated iron is high, the soybean small peptide chelated iron is applied to the feed industry, the absorption of animals to feed products is more favorably realized, the disease resistance and the immunity of the animals can be improved, and the emission of iron elements in piglet excrement can be reduced.
Compared with the prior art, the soybean small peptide chelated iron provided by the invention has the following advantages:
(1) the method adopts the soybean meal as the protein source, has low cost, easily obtained raw materials and wide sources of various amino acids, the chelated iron prepared by the method can be produced in an industrial scale, the reaction product is easy to separate and purify, the operation is simple, the cost is lower, the physical and chemical properties of the product are stable, and the soybean small peptide chelated iron prepared by the method has high purity and good crystal structure, thereby being beneficial to accurately controlling the use amount.
(2) The soybean small peptide chelated iron prepared by the preparation method has small molecular weight, the molecular weight is more than 75% below 1000Da, the molecular weight is more than 50% below 500Da, the molecular weight is more than 14% below 180Da, 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, the absorption and utilization rate of a target animal to iron is increased, on one hand, the nutritional functionality and the biological potency of the iron element are improved, the absorption and utilization of organisms are facilitated, on the other hand, the content of the iron element in excrement is reduced, and therefore, the environmental pollution is reduced.
(3) The soybean small peptide chelated iron prepared by the preparation method has good palatability, comprehensive nutrition and easy digestion, ensures that piglets can be smoothly weaned, can alleviate the strong stress on the piglets caused by weaning, and reduces the wound caused by converting pig milk into solid feed.
Drawings
FIG. 1 is an infrared spectrum of soybean small peptide chelated iron prepared by the embodiment 2 of the invention;
FIG. 2 is a UV spectrum of soybean small peptide chelated iron prepared by the embodiment 2 of the invention;
FIG. 3 structural analysis diagram of soybean small peptide chelated iron.
Detailed Description
The present invention is further illustrated by the following description of specific embodiments, which are not intended to limit the invention, and various modifications and improvements can be made by those skilled in the art based on the basic idea of the invention, but the invention is within the protection scope of the invention.
The soybean meal is purchased from Shandong Guocheng chemical science and technology Limited; astragalus polysaccharides CAS 89250-26-0, available from Nanjing Dow Biotech, Inc.; reduced glutathione, Shanghai Michelin Biochemical technology Limited, with a product number of L810392-100 g; polyvinyl alcohol 1799, CAS:9002-89-5, available from Shanghai Violet-reagent, Inc. (Clara, Inc.); alkaline protease, neutral protease, trypsin were purchased from Shanghai-derived leaf Biotech, Inc.
In addition, other reagents used in the invention are 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, preprocessing: weighing 1000kg of soybean meal, adding 10000kg of water to prepare 10% protein solution, adding 1.3% enzymolysis accelerator by mass of the soybean meal, and homogenizing for 2 times under 15Mpa to obtain homogenized mixed soybean meal protein solution;
s2, enzymolysis reaction: adjusting the pH value of the mixed soybean meal protein liquid obtained in the step S1 to 8 by using 2mol/L NaOH and HCl solution, then adding compound protease, carrying out enzymolysis for 3h at 40 ℃, heating to 80 ℃, inactivating the enzyme for 10min, centrifuging for 20min at 4000rpm/min, collecting supernatant, and carrying out 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, uniformly stirring to prepare a soybean small peptide solution with the concentration of 10%, then adding an antioxidant with the mass of 0.5% of the soybean small peptide powder and a ferrous sulfate solution with the concentration of 10%, uniformly stirring, then adding an antioxidant with the mass of 0.2% of the soybean small peptide powder, uniformly mixing, adjusting the pH value to 5 by using sodium hydroxide with the concentration of 20%, heating to 45 ℃, starting timing, and reacting for 0.4 h;
the enzymolysis accelerant in the step S1 is polyvinyl alcohol 1799 and reduced glutathione according to the weight ratio of 5: 3, and (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 iron in the ferrous sulfate solution in the step S3 to the soybean small peptide powder is 1: 5.
In the step S4, the temperature of the air inlet is 180 ℃ and the temperature of the 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, preprocessing: weighing 1000kg of soybean meal, adding 9091kg of water to prepare protein liquid with the concentration of 11%, then adding an enzymolysis accelerator with the mass of 1.8% of the soybean meal, and carrying out homogenization treatment for 3 times under the condition of 25Mpa to obtain homogenized mixed soybean meal protein liquid;
s2, enzymolysis reaction: adjusting the pH value of the mixed soybean meal protein liquid obtained in the step S1 to 8.5 by using 2mol/L NaOH and HCl solution, then adding compound protease, carrying out enzymolysis for 4h at 45 ℃, heating to 90 ℃, inactivating the enzyme for 20min, centrifuging for 20min at 4000rpm/min, collecting supernatant, and carrying out 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, uniformly stirring to prepare a soybean small peptide solution with the concentration of 15%, then adding an antioxidant with the mass of 0.8% of the soybean small peptide powder and a ferrous sulfate solution with the concentration of 15%, uniformly stirring, then adding an antioxidant with the mass of 0.3% of the soybean small peptide powder, uniformly mixing, adjusting the pH value to 5.5 by using sodium hydroxide with the concentration of 20%, heating to 55 ℃, starting timing, and reacting for 1.0 h;
the enzymolysis accelerant in the step S1 is polyvinyl alcohol 1799 and reduced glutathione according to the weight ratio of 7: 3, and (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.
The mass ratio of the ferrous iron in the ferrous sulfate solution in the step S3 to the soybean small peptide powder is 1: 4.
In the step S4, the temperature of the air inlet is 200 ℃ and the temperature of the 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, preprocessing: weighing 1000kg of soybean meal, adding 8333kg of water to prepare protein liquid with the concentration of 12%, then adding an enzymolysis accelerator with the mass of 2.1% of the soybean meal, and carrying out homogenization treatment for 3 times under the condition of 30Mpa to obtain homogenized mixed soybean meal protein liquid;
s2, enzymolysis reaction: adjusting the pH value of the mixed soybean meal protein liquid 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 the enzyme for 30 minutes, centrifuging for 20 minutes at 4000rpm/min, collecting supernatant, and carrying out 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, uniformly stirring 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%, uniformly stirring, then adding an antioxidant with the mass of 0.5% of the soybean small peptide powder, uniformly mixing, adjusting the pH value to 6 by using sodium hydroxide with the concentration of 20%, heating to 65 ℃, starting timing, and reacting for 1.5 h;
the enzymolysis accelerant in the step S1 is polyvinyl alcohol 1799 and reduced glutathione according to the weight ratio of 8: 3, and (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.
The mass ratio of the ferrous iron in the ferrous sulfate solution in the step S3 to the soybean small peptide powder is 1: 3.
In the step S4, the temperature of the air inlet is 220 ℃ and the temperature of the air outlet is 100 ℃.
Comparative example 1 preparation method of soybean small peptide chelated iron
Compared with example 2, comparative example 1 is different in that no enzymatic hydrolysis promoter is added in the pretreatment of step S1, and other parameters and operations are the same as those of example 2.
Comparative example 2 preparation method of soybean small peptide chelated iron
Compared with example 2, comparative example 2 is different in that polyvinyl alcohol 1799 is not added to the enzymolysis promoter in the pretreatment of step S1, and other parameters and operation are the same as those of example 2.
Comparative example 3 preparation method of soybean small peptide chelated iron
Compared with example 2, comparative example 3 is different in that reduced glutathione is not added to the enzymatic hydrolysis promoter in the pretreatment of step S1, and other parameters and operations are the same as those of example 2.
Comparative example 4 preparation method of soybean small peptide chelated iron
In comparison with example 2, comparative example 4 is different in that astragalus polysaccharide is not added to the antioxidant of step S3, and other parameters and operations are the same as those of example 2.
Comparative example 5 preparation method of soybean small peptide chelated iron
Comparative example 5 is different from example 2 in that the antioxidant in step S3 is composed of sodium D-erythorbate, 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
In comparison with example 2, comparative example 5 is different in that the chelation reaction of step S3 only adds the first antioxidant, and after adding the ferrous sulfate solution, does not add the antioxidant, and other parameters and operations are the same as those of example 2.
Test example I, measurement of molecular weight distribution and content of active ingredient of soybean small peptide chelated iron peptide
1. Test materials: the soybean small peptide chelated iron prepared in the examples 1 to 3 and the comparative examples 1 to 6.
2. The test method comprises the following steps: the molecular weight of the peptide, the total content of iron elements and the chelating rate of the soybean small peptide prepared in the above examples 1 to 3 and comparative examples 1 to 6 are detected. The peptide content is measured according to the high-efficiency gel filtration chromatography in GB/T22729-2008, the measurement of the molecular weight distribution of the soybean small peptide is entrusted to the analysis and test center of the third-party Jiangnan university for detection, the infrared spectrum analysis of the small peptide chelated iron structure is entrusted to the institute of university, and the iron content is detected by the o-dinitrogen phenanthrene method.
3. Test results
The test results are shown in table 1.
TABLE 1 molecular weight distribution of soybean mini-peptide chelated iron peptide
TABLE 2 iron content and chelating rate of soybean small peptide chelate
| Group of | Iron content/%) | Chelating ratio/% |
| 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 proportion of the small-molecule peptides with the relative molecular weight of less than 1000Da of the soybean small-peptide chelated iron peptides prepared according to embodiments 1-3 of the invention in the total protein reaches more than 75%, wherein the proportion of the small-molecule peptides with the molecular weight of less than 1000Da in embodiment 2 is up to 77.86%, the iron content is more than 11%, and the chelation rate is 83.43%.
Test example two, content test of effective ingredient
1. Infrared spectrogram of chelated iron of soybean small peptide prepared in example 2 of the invention
From FIG. 1, it is presumed that the metal ion is coordinated to the peptide fragment.
2. Soybean small peptide chelated iron ultraviolet spectrogram prepared in example 2 of the invention
See figure 2.
3. Structural analysis of bean small peptide chelated iron prepared by the invention
See figure 3.
Test example three, Effect test
1. Test animals: 150 heads of the grown ternary hybrid healthy weaned piglets which are 21 +/-2 days old and grown up are selected to have the weight of 7.80 +/-0.10 kg.
2. The test method comprises the following steps:
(1) grouping tests: the 150-year-old healthy weaned piglets are divided into 5 groups according to weight, litter size and sex, each group is provided with three repetitions, each repetition is 10, and the test period is 42 days. Treating 1 to obtain blank group, and feeding basic ration; treatment 2 was a control group, fed basal diet + (soy small peptides + inorganic Fe); treatments 3-5 were test groups 1-3, and were fed with basal diet plus soy minor peptide chelated iron prepared in examples 1-3. The basic ration is formulated according to the NRC 2012 standard.
(2) Feeding management: feeding according to the pig feeding standard NYT 65-2004. Feeding experimental weaned piglets by adopting a high bed, naturally ventilating, and having good environmental sanitation conditions, wherein the experimental weaned piglets are fed 3 times (1 time for each of 7:00, 13:00 and 20: 00) a day and are freely drunk; anthelmintic and immune treatments were performed according to the pig farm protocol. The animal feeding and health conditions were observed at any time. All diarrhea pigs were not treated.
(3) Collecting samples: after the test is finished, 2 boars and 1 sow are slaughtered repeatedly, 15mL of blood is collected from the anterior vena cava of each test pig, the pig is kept still in a water bath at 37 ℃ until serum is separated out, the pig is centrifuged at 3500r/min for 10min, and the separated serum is stored in an Eppendorf tube at-80 ℃ and is used for measuring serum trace elements and biochemical indexes.
(4) Detection indexes are as follows: the method comprises the steps of measuring the number of red blood cells, the concentration of hemoglobin, the content of serum iron and the content of immunoglobulin G of piglets by using a full-automatic blood analyzer through adopting piglet marginal venous blood before and after a test respectively, recording the growth, diarrhea and death and elutriation conditions of the piglets, and detecting the content of iron in excrement. The test results are shown in table 3.
TABLE 3 piglet growth status and serum biochemical indices
| Group of | Blank group | Comparison | Test group | 1 | Test group 2 | Test group 3 |
| Weight balance (kg) | 7.81 | 7.84 | 7.80 | 7.86 | 7.79 | |
| Average weight of powder (kg) | 27.13 | 28.42 | 29.22 | 29.70 | 29.21 | |
| Average daily food 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 | |
| Material to weight ratio (F/G) | 1.54 | 1.53 | 1.35 | 1.31 | 1.31 | |
| Diarrhea Rate (%) | 3.05 | 2.68 | 2.03 | 1.92 | 1.92 | |
| Death rate (%) | 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, compared with the comparative example group and the blank group, the soybean small peptide chelated iron obtained by the preparation methods of the embodiments 1 to 3 of the invention can obviously improve the hemoglobin concentration of weaned piglets, obviously increase the feed intake and weight gain, and reduce the diarrhea rate and death and culling rate. In contrast, in the case that the soybean peptide and the inorganic iron were added to the basal diet, the effect of increasing hemoglobin concentration, feed intake, and weight gain was reduced, and the feed conversion ratio, diarrhea rate, and mortality and culling rate were increased, as compared to examples 1 to 3 of the present invention. Therefore, through the special preparation process and the selection and content proportion of the raw materials in the formula, the soybean small peptide chelated iron has synergistic effect among the components, can regulate intestinal tracts, promote the healthy development of the intestinal tracts, and improve the hematopoietic capacity of piglets and the immunity of the piglets.
The HGB concentration begins to decline when the iron reserve in the body is exhausted, and the HGB concentration is used as an index for diagnosing the iron level of the suckling pigs, has hysteresis and is not beneficial to the prevention and treatment of the suckling pigs. The mass concentration of SF is positively correlated with the mass concentration of iron in vivo, the iron storage in vivo can be directly reflected, and the combination of the mass concentration of HGB and the mass concentration of SF has specificity and sensitivity, is an effective index for diagnosing the nutritional status and anemia of the suckling pig iron, and is an effective index for evaluating the iron supplementing effect of the iron supplementing agent. The embodiments 1-3 of the invention have obvious effects on improving the concentration of hemoglobin and serum ferritin of piglet serum, and can be used as a feed additive to be well applied to weaned piglet feed.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (9)
1. A preparation method of soybean small peptide chelated iron is characterized by comprising the following steps:
s1, preprocessing: adding water into the soybean meal, uniformly stirring to prepare a protein solution with the concentration of 10-12%, then adding an enzymolysis promoter with the mass of 1.3-2.1% of the soybean meal, and carrying out homogenization treatment for 2-3 times under the condition of 15-30 Mpa to obtain a homogenized mixed soybean meal protein solution;
s2, enzymolysis reaction: adjusting the pH value of the mixed soybean meal protein liquid obtained in the step S1 to 8-9, adding compound protease, carrying out enzymolysis for 3-5 h at 40-50 ℃, heating to 80-95 ℃, inactivating enzyme for 10-30 min, centrifuging for 20min at 4000rpm/min, collecting supernatant, and carrying out 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 the mixture 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, adjusting the pH value to 5-6, starting timing when the temperature is increased to 45-65 ℃, and reacting for 0.4-1.5 h;
s4, spray drying: after the reaction is finished, the soybean small peptide chelated iron is obtained by spray drying.
2. The method for preparing soybean small peptide chelated iron as claimed in claim 1, wherein the enzymatic hydrolysis promoter in step S1 is polyvinyl alcohol and reduced glutathione in a weight ratio of (5-8): 3, and (3).
3. The method for preparing soybean small peptide chelated iron as claimed in claim 1, wherein the compound protease in 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.
4. The method for preparing soybean small peptide chelated iron as claimed in claim 1, wherein the antioxidant in step S3 is one or a combination of two or more of D-sodium erythorbate, vitamin C, astragalus polysaccharides and sodium sulfite.
5. The method for preparing soybean small peptide chelated iron as claimed in claim 4, wherein the antioxidant in step S3 is composed of sodium D-erythorbate, astragalus polysaccharides and sodium sulfite in a weight ratio of 7-11: 2-5: 1-4.
6. The method for preparing soybean small peptide chelated iron as claimed in claim 5, wherein the antioxidant in step S3 is composed of sodium D-erythorbate, Astragalus polysaccharides and sodium sulfite in weight ratio of 10:3: 2.
7. The method for preparing soybean small peptide chelated iron as claimed in claim 1, wherein the inorganic ferrous salt solution in step S3 is 10-20% ferrous sulfate solution.
8. The method for preparing soybean small peptide chelated iron as claimed in claim 1, wherein the mass ratio of ferrous iron in the inorganic ferrous salt solution in step S3 to soybean small peptide powder is 1: 3-5.
9. The method for preparing soybean small peptide chelated iron as claimed in claim 1, wherein in step S4, the temperature of the air inlet is 180-220 ℃ and the temperature of the air outlet is 90-100 ℃.
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