Novel vegetable protein raw material as well as preparation method and application thereof

Abstract

The invention discloses a novel vegetable protein raw material as well as a preparation method and application thereof, and belongs to the technical field of feeds. The preparation method of the novel plant protein raw material comprises the following steps: fermenting agricultural wastes and an enzyme preparation so as to reduce the content of mycotoxin in the agricultural wastes and obtain fermented protein; wherein zymoprotein with the amino acid sequence as shown in SEQ ID NO.1 serves as an active ingredient of the enzyme preparation, the mycotoxin comprises aflatoxin B1, vomitoxin and zearalenone, and the agricultural waste is selected from corn steep liquor and mushroom residues. According to the scheme, the content of mycotoxin in the corn steep liquor and the mushroom residues can be effectively reduced, then the fermented protein meeting the feed hygienic standard can be prepared, and a safe, economical and feasible scheme is provided for soybean meal reduction substitution.

Description

Technical Field

[0001] The invention belongs to the technical field of feed, and in particular relates to a novel plant protein raw material and a preparation method thereof and application thereof in preparing livestock and poultry feed. Background Art

[0002] Mycotoxin contamination of feed has always been a major problem that plagues the development of the feed industry. Mycotoxins are a large class of secondary metabolites produced by fungi and molds, which pose serious risks to human and animal health. Mycotoxin contamination can endanger the health of humans and animals and can cause damage to the liver, intestines, kidneys and immune systems of humans and animals. It is worth noting that humans are exposed to mycotoxins not only through eating plant-based foods contaminated with mycotoxins, but also through eating animal products containing mycotoxins and their metabolites, such as animal tissues, milk and eggs. The presence of mycotoxins can seriously affect the global economy and international trade. According to estimates by the Food and Agriculture Organization of the United Nations, 25% of the world's grains are contaminated with mycotoxins each year, and an average of 2% is wasted because they cannot be eaten. High-quality new protein sources are another major issue affecting the development of the feed industry. Limited by the "soybean meal" feed formula system, my country's soybean meal consumption is extremely high, and the dependence on soybean imports remains above 80%. The price of soybean meal has repeatedly broken historical highs, and soybean meal reduction and substitution technology is imperative. Although there are many options for new protein raw materials, the existence of mycotoxin contamination makes it difficult for feed companies to select materials, and the current shortage of protein raw materials has not been significantly improved.

[0003] In feed, the common and most harmful mycotoxins are aflatoxin (AF), deoxynivalenol (DON) and zearalenone (ZEN). Aflatoxin is a bifuranoid toxin produced by molds such as Aspergillus flavus and Aspergillus parasiticus. There are more than 20 derivatives of AF, which are named B1, B2, G1, G2, M1, M2, GM, P1, Q1, and toxic alcohol according to their structure, properties and luminescence color. Among them, AFB1 is the most toxic, mainly targeting the liver, and has strong carcinogenic, mutagenic and teratogenic properties. Deoxynivalenol is a metabolite produced by Fusarium. Its chemical structure is stable, heat-resistant, pressure-resistant, and does not decompose in weak acid. Only part of its toxicity can be destroyed after adding alkali and high-pressure treatment. Zearalenone is mainly an estrogen analogue produced by Fusarium. It has reproductive toxicity and cytotoxicity, and can also damage the liver and kidney systems.

[0004] Studies have shown that many agricultural wastes are expected to provide protein sources for feed, such as corn slurry and mushroom residue. Corn slurry is one of the by-products of the wet process of producing corn starch. my country produces a large amount of corn slurry every year. After the corn is soaked, most of the toxins are retained in the soaking water and exist in the corn slurry when it is concentrated. Corn slurry contains a large amount of soluble protein, L-lactic acid, soluble sugars, various minerals and vitamins, and is an ideal feed raw material. However, the mycotoxin content in corn slurry is seriously exceeded, especially aflatoxin B1 and vomitoxin. Mushroom residue is an agricultural waste after the harvest of various edible fungi such as shiitake mushrooms and oyster mushrooms, which are produced with cottonseed hulls, sawdust, straw, corn cobs, sugarcane bagasse and various crop straws as the main raw materials. There are also a large number of mycotoxins in mushroom residue. In view of the impact of mycotoxins, although corn slurry and mushroom residue have been used in the feed industry, there are still prominent problems such as limited dosage and high safety risks. Summary of the invention

[0005] In view of the problem that corn slurry and mushroom residue are difficult to be effectively used in the feed industry, the present invention aims to develop a high-quality, new type of plant protein raw material based on corn slurry and mushroom residue, providing a safe and economically feasible solution for reducing and replacing soybean meal.

[0006] The technical solution of the present invention is as follows: The first aspect of the present invention provides a method for preparing a novel plant protein raw material, specifically: fermenting agricultural waste with an enzyme preparation to reduce the mycotoxin content in the agricultural waste and obtain fermented protein; wherein the enzyme preparation has an enzyme protein with an amino acid sequence as shown in SEQ ID NO.1 as an active ingredient.

[0007] In the method of the present invention, the enzyme protein whose amino acid sequence is shown in SEQ ID NO.1 is a uridine diphosphate glucuronyl transferase, which can be prepared and produced on a large scale by existing technology. The enzyme protein screened and provided by the present invention has the ability to degrade mycotoxins, especially has good degradation ability for three common mycotoxins, aflatoxin B1, vomitoxin and zearalenone.

[0008] Preferably, in the above method, the fermentation reaction temperature is 20-80°C and the reaction pH is 5-8. The UDP-glucuronyl transferase provided by the present invention has high stability and excellent heat resistance and acid and alkali resistance, thereby providing a guarantee for better degradation of mycotoxins in different agricultural wastes in a wider temperature and pH range.

[0009] Preferably, in the above method, the mass ratio of agricultural waste to enzyme protein is 1:(0.01-0.03), and the fermentation reaction time is 10-75 h.

[0010] In the method of the present invention, agricultural waste includes but is not limited to corn steep liquor, mushroom residue, corn germ meal, mash, etc. In some embodiments of the present invention, agricultural waste consists of corn steep liquor and mushroom residue, and the mass ratio of the two is preferably (1-2):(2-3); experimental data show that after corn steep liquor and mushroom residue are fermented with the above-mentioned UDP-glucuronyl transferase, the obtained fermented protein not only has a significantly reduced mycotoxin content to meet the feed hygiene standards, but also is rich in nutrition and can replace soybean meal for livestock and poultry feed.

[0011] The second aspect of the present invention seeks to protect the novel plant protein raw material prepared according to the above method of the present invention.

[0012] The third aspect of the present invention provides the use of the novel plant protein raw material in preparing livestock and poultry feed.

[0013] In the above livestock and poultry feed, the soybean meal can be directly partially or completely replaced by the new plant protein raw material; preferably, in the above livestock and poultry feed, the addition amount of the new plant protein raw material is 10-25 wt%.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The scheme of the present invention can effectively degrade mycotoxins in agricultural waste (such as corn steep liquor and mushroom residue), especially has a high degradation rate for aflatoxin B1, vomitoxin and zearalenone, and provides a new way to effectively solve the restrictions on the application of mycotoxins to agricultural waste.

[0015] The enzyme preparation used in the scheme of the present invention forms a complex by combining with mycotoxins, hydrolyzing or decomposing toxin groups after oxidation, destroying the molecular structure of the toxin, and converting the mycotoxins into non-toxic metabolites, thereby ensuring the safety of the fermented protein. Moreover, in view of the high temperature resistance and acid and alkali resistance of the enzyme preparation used in the present invention, the scheme of the present invention has a wide range of applications and can be used for the deep treatment of mycotoxins in different plant raw materials. In addition, the enzyme protein used in the scheme of the present invention itself is harmless to livestock and poultry. Test data show that the enzyme protein can even promote the absorption of nutrients by livestock and poultry and improve feed utilization.

[0016] The novel plant protein raw material prepared by using corn steep liquor and mushroom residue as raw materials can effectively replace soybean meal in livestock and poultry feed, and provides a new solution for replacing soybean meal in feed. DETAILED DESCRIPTION

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. The term "comprises" and any variations thereof in the specification and claims of the present invention are intended to cover non-exclusive inclusions.

[0018] In view of the problem that products that may be used as soybean meal substitutes contain mycotoxins, the present invention provides a solution based on screening the obtained enzyme protein, specifically: fermenting agricultural waste with an enzyme preparation to reduce the mycotoxin content in the agricultural waste and obtain fermented protein; wherein the enzyme preparation has an enzyme protein with an amino acid sequence as shown in SEQ ID NO.1 as an active ingredient, and the agricultural waste includes but is not limited to corn steep liquor, mushroom residue, etc.

[0019] By optimizing the amount of enzyme protein used, fermentation reaction time, reaction pH value and reaction temperature, the degradation rate of aflatoxin B1, vomitoxin and zearalenone in mycotoxins can be maintained at 85%-96%. A large number of experiments have shown that the degradation rate of aflatoxin B1 is time-dependent and enzyme protein dose-dependent. After 2 hours of reaction, pH has a great influence on the degradation rate of aflatoxin B1, and this phenomenon gradually disappears as the reaction time increases; the degradation rate of vomitoxin is time-dependent and enzyme protein dose-dependent, and the weight is: dose>reaction time>pH value; and for zearalenone, in the presence of enzyme protein, pH value has the greatest impact on the degradation rate of zearalenone, and the degradation rate of zearalenone can reach 89.76%-98.40%, among which pH 5.5-6.5 is the best.

[0020] It is understandable that the choice of agricultural waste is another key factor affecting whether the fermented protein can successfully replace soybean meal and the amount used in feed. The experiment of the present invention shows that the fermented protein obtained by using a specific ratio of corn steep liquor and mushroom residue as raw materials is rich in nutrients, can effectively replace soybean meal, and has a positive effect on the growth of chicks.

[0021] The technical solution of the present invention will be described clearly and completely below in conjunction with specific embodiments. It should be understood that the embodiments described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.

[0022] In the following examples, the contents of aflatoxin B1, vomitoxin and zearalenone were detected by ELISA method, and the contents of various mycotoxins in the corn steep liquor (purchased from Jingliang Group) and mushroom residue (purchased from Shandong Youhong Biotechnology Co., Ltd.) used in the following examples are specifically shown in Table 1: Table 1 Original content of mycotoxins in corn steep liquor and mushroom residue

[0023] If no specific techniques or conditions are specified in the following examples, all of them are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions; if the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0024] Example 1 In this example, corn steep liquor was used as raw material, and UDP-glucuronyl transferase with an amino acid sequence as shown in SEQ ID NO.1 was used for fermentation under different conditions to obtain fermented protein. The specific process was: the enzyme protein was mixed with corn steep liquor, fermented, dried and then crushed. The test parameters and test results are shown in Table 2.

[0025] Table 2 Degradation rate of various mycotoxins in corn steep liquor

[0026] Example 2 In this example, corn steep liquor and mushroom residue were used as raw materials, and fermentation was performed using uridine diphosphate glucuronyl transferase with an amino acid sequence as shown in SEQ ID NO.1, thereby providing a new type of plant protein raw material.

[0027] The specific operation is as follows: 1 kg enzyme, 10 kg water and 100 kg mushroom residue are mixed and then left to stand for 3 hours to dry, then mixed with 100 kg corn pulp and 2 kg enzyme, reacted at room temperature for 72 hours, dried at 65°C overnight, and crushed.

[0028] Example 3 Different from Example 2, the reaction process of this example is as follows: 2.5 kg of enzyme and 100 kg of corn pulp are mixed, and then 250 kg of mushroom residue is added and mixed, and the mixture is placed at room temperature for 72 hours, dried at 65° C. overnight, and crushed.

[0029] The fermented proteins obtained in Examples 2 and 3 were tested for mycotoxins and nutritional indicators, and the results are shown in Tables 3-4.

[0030] Table 3 Mycotoxin content in fermented protein

[0031] Table 4 Test results of various nutritional indicators in fermented protein

[0032] Example 4 In this example, the fermented protein prepared in Example 3 was used to replace soybean meal to prepare chick feed. Specifically, the fermented protein was used to replace the soybean meal in the basic diet shown in Table 5 in the same proportion.

[0033] Table 5 Basic diet composition

[0034] The premix in Table 5 provides per kilogram of feed: 5,000 IU of vitamin A, 9.8 mg of vitamin B1, 28.8 mg of vitamin B2, 19.6 mg of vitamin B6, 0.1 mg of vitamin B12, 1 OOOO IU of vitamin D, 75.0 mg of vitamin E, 38.8 mg of vitamin K, 4.0 mg of copper, 40.0 mg of iron, 37.6 mg of zinc, 50.0 mg of molybdenum, 0.2 mg of selenium, 0.2 mg of iodine, 196.5 mg of niacin, 4.8 mg of folic acid, and 2.5 mg of biotin.

[0035] 21-day-old chicks with similar body weight and good health were randomly selected and divided into 3 treatment groups, with 2 replicates in each treatment group and 100 chicks in each replicate. The control group was fed with a basal diet; the experimental group 1 was fed with a diet replaced by 10% fermented protein (i.e., the basal diet was supplemented with 10% fermented protein and 10% soybean meal was reduced); the experimental group 2 was fed with a diet replaced by 20% fermented protein (i.e., the basal diet was supplemented with 20% fermented protein and 20% soybean meal was reduced). The chicks were raised in fully enclosed cages, fed artificially, and had free access to food and water. The experiment lasted for 15 days.

[0036] From the beginning of the experiment to the end of the experiment, the actual growth of the chicks was observed every day, and the health of the chicks and feed consumption were recorded. The dead and sick chicks were eliminated because they did not meet the test requirements. The following two indicators were calculated according to the following formula: Average daily gain (ADG) = (chicken weight at the end of the experimental period − initial weight) / number of experimental days; Average daily feed intake (ADFI) = total feed intake of chicks during the experimental period / number of experimental days.

[0037] Specifically, the growth conditions of the chicks are shown in Table 6.

[0038] Table 6 Test results of related indicators of chick growth performance

[0039] In addition, the chicks' organs were tested after the experiment, and the results are shown in Table 7.

[0040] Table 7 Results of chick organ index test

[0041] From the experimental results of this example, it can be seen that the fermented protein provided by the present invention will not affect the growth performance and health of chicks.

[0042] In summary, the scheme of the present invention can effectively reduce the mycotoxins in feed raw materials, and at the same time provides a specific and feasible scheme for effectively replacing soybean meal in livestock and poultry feed with corn steep liquor and mushroom residue.

[0043] It should be noted that the above embodiments are only part of the embodiments of the present invention rather than all the embodiments, and are only used to illustrate the technical solution of the present invention rather than to limit it. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

Claims

1. A method for preparing a novel plant protein raw material, characterized in that: Agricultural waste is fermented with an enzyme preparation to reduce the mycotoxin content in the agricultural waste and obtain fermented protein; wherein the enzyme preparation has an enzyme protein with an amino acid sequence as shown in SEQ ID NO.1 as an active ingredient.

2. The preparation method according to claim 1, characterized in that: The mycotoxins include aflatoxin B1, vomitoxin and zearalenone.

3. The preparation method according to claim 1, characterized in that: The mass ratio of the agricultural waste to the enzyme protein is 1:(0.01-0.03).

4. The preparation method according to claim 1, characterized in that: In the fermentation, the reaction temperature is 20-80°C, and the reaction pH is 5-8.

5. The preparation method according to claim 1, characterized in that: The agricultural waste includes corn steep liquor and mushroom residue.

6. The preparation method according to claim 5, characterized in that: The agricultural waste consists of corn pulp and mushroom residue, and the mass ratio of the two is (1-2):(2-3).

7. A novel plant protein raw material prepared according to any one of claims 1 to 6.

8. Use of the novel plant protein raw material according to claim 7 in preparing livestock and poultry feed.

9. The use according to claim 8, characterized in that: In the livestock and poultry feed, soybean meal is replaced by a new type of plant protein raw material.

10. The use according to claim 8, characterized in that The addition amount of the novel plant protein raw material is 10-25 wt %.

Images