Active peptide feed additive prepared through cooperation of low-intensity magnetic field and microbial fermentation and application of active peptide feed additive

Abstract

The invention belongs to the crossing field of animal nutrition and bioengineering, and particularly relates to an active peptide feed additive prepared through cooperation of a low-intensity magnetic field and microbial fermentation and application of the active peptide feed additive. The feed additive is used for improving the digestibility, antioxidant activity and palatability of the feed. The active peptide feed additive is prepared through cooperation of a low-intensity magnetic field and microbial fermentation, and the feed additive is goose hemoglobin peptide powder.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of animal nutrition and bioengineering, and specifically relates to a weak magnetic field synergistic microbial fermentation method for preparing active peptide feed additives and application thereof. The prepared small molecule active peptides can be widely used as feed additives in animal feed such as livestock and poultry, aquatic products, etc., for improving the digestibility, antioxidant activity and palatability of feed. BACKGROUND

[0002] In modern livestock and poultry breeding, the quality of feed directly affects the growth performance, health level and feed conversion rate of animals. Traditional feed is mostly based on raw materials such as soybean meal, fish meal and grains. Although it can provide basic energy and protein, it has the disadvantages of poor palatability, high anti-nutritional factors and weak functional activity. Especially during the stress period of weaning and group transfer, animals are prone to have problems such as reduced feed intake and intestinal inflammation, which affects production efficiency.

[0003] With the implementation of the "antibiotic ban", finding green substitutes with natural anti-inflammatory, immune regulation and appetite induction functions has become a research hotspot in the feed industry. Active polypeptides, especially small molecule peptides produced by hydrolysis or fermentation of animal and plant proteins, have attracted widespread attention due to their easy absorption and strong function. Among them, some polypeptides have the effect of inducing flavor enhancement and promoting feeding, such as glutamyl peptides and aromatic amino acid peptides. Some other small peptides show good anti-inflammatory, antioxidant and immune regulation functions, such as peptides rich in histidine, arginine and hydroxyproline, which can significantly reduce the expression of pro-inflammatory factors and alleviate animal stress response.

[0004] Currently, the main methods for preparing polypeptides include chemical synthesis, enzymatic hydrolysis and microbial fermentation. Among them, microbial fermentation is more suitable for functional application of feed due to its greenness, safety and suitability for industrial scaling. However, existing technologies mainly focus on the development of single-function polypeptides (such as taste peptides or anti-inflammatory peptides), and lack strategies for designing and preparing polypeptide compositions with synergistic optimization of flavor and function. At the same time, there is no systematic research and application report on how to promote the release of small peptides through fermentation process and enrich effective peptides with dual functions.

[0005] In recent years, it has been found that low-intensity magnetic field stimulation (such as static magnetic field and alternating magnetic field) can be used as a new type of green physical intervention method to promote the growth of various microorganisms, enhance the activity of enzyme systems and regulate metabolic pathways, which can effectively stimulate microorganisms to produce secondary metabolites and thus improve the quality and functionality of fermentation products. It has been preliminarily applied in the fields of food fermentation, microbial synthesis of amino acids and polysaccharides. However, the research on the synergistic use of magnetic field technology and microbial fermentation for the preparation of functional peptides in animal feed is still in its infancy, and there is no mature process or industrialization scheme.

[0006] Therefore, it is urgent to develop a green and efficient magnetic field-microorganism synergistic fermentation technology path for preparing a feed functional peptide product with clear biological activity and excellent palatability, so as to break through the constraints of the existing technology and meet the urgent needs of modern breeding for high-performance feed additives. SUMMARY

[0007] The present application aims to overcome the problems of low functional peptide yield, insufficient biological activity and poor flavor in the process of preparing feed functional peptides by microbial fermentation in the prior art, and provides a preparation method based on weak magnetic field synergistic microbial fermentation for efficiently producing multifunctional small molecule active peptide feed additives with antioxidant activity, enhanced palatability and improved digestibility.

[0008] To achieve the above-mentioned purpose, the present application provides the following technical solutions: The active peptide feed additive prepared by the weak magnetic field synergistic microbial fermentation is goose hemoglobin peptide powder.

[0009] Further optimization of the technical solution, the preparation method of the feed additive comprises the following steps: Raw material pretreatment: select goose hemoglobin as raw material, configure fermentation medium, configure 1L of fermentation medium according to goose hemoglobin powder: water = 1:10 (w / v); Strain activation: inoculate Bacillus subtilis into a shake flask medium with an inoculation amount of 1%, which is used for the activation process of the strain, and the culture conditions are 37℃, 180-220 rpm / min, and the culture time is 10-12h; after continuous activation of the strain for three generations, centrifuge at 4℃, 8000 rpm / min for 15min, collect the bacterial precipitate, and adjust the concentration of the bacterial liquid to 107 CFU / mL with sterile physiological saline before inoculation; Substrate fermentation: add the activated Bacillus subtilis culture solution to the fermentation medium at a proportion of 8.2%, place it in a constant temperature of 35-37℃, 180-220 rpm / min, and culture for 24h-48h; after the fermentation is completed, place the fermentation liquid in 95℃ for 30min to inactivate the enzyme, cool it, and then centrifuge at 4℃, 8000-12000 rpm / min for 15-30min, collect the supernatant, concentrate the collected supernatant by a rotary evaporator, and then freeze-dry the concentrated liquid to obtain goose hemoglobin crude peptide; Small molecule peptide enrichment: enrich small molecule peptides from goose hemoglobin crude peptide by ultrafiltration <3 kDa, and obtain goose hemoglobin peptide powder by concentration and freeze-drying.

[0010] The further optimization of the technical scheme is that a magnetic field is applied to assist the fermentation in the substrate fermentation process, and the magnetic field assisted fermentation is that the fermentation liquid is treated with a weak magnetic field of 1.0-5.0 mT for 2-6 h after inoculation, and then the fermentation process is continued at 37 DEG C until the fermentation is completed.

[0011] The application further provides an active peptide feed prepared by the method.

[0012] The further optimization of the technical scheme is that the feed comprises corn, soybean meal, wheat bran, rapeseed meal, goose hemoglobin peptide powder, oil, yeast culture, calcium hydrogen phosphate, glucose, salt, lysine, acidifying agent, compound enzyme preparation and premix.

[0013] The further optimization of the technical scheme is that the feed comprises corn 56%, soybean meal 18%, wheat bran 7%, rapeseed meal 4.5%, goose hemoglobin peptide powder 5%, oil 3.4%, yeast culture 1%, calcium hydrogen phosphate 1.3%, glucose 1%, salt 0.3%, lysine 0.3%, acidifying agent 0.6%, compound enzyme preparation 0.3% and premix 1.3% in terms of mass percentage.

[0014] The further optimization of the technical scheme is that the preparation method of the feed is as follows: the yeast culture, calcium hydrogen phosphate, glucose, salt, lysine, acidifying agent, compound enzyme preparation and premix are premixed for 2-3 min to obtain mixture A; then the corn, soybean meal, wheat bran and rapeseed meal are crushed and sieved by a pulverizer, the particle size is controlled to be 1.5 mm, and after the sieving is completed, the mixture is mixed at 75-80 DEG C for 2-3 min, and then the goose hemoglobin peptide powder is added after cooling to room temperature, and the mixture is mixed for 1-2 min to obtain mixture B; and A and B are mixed for 3-5 min to obtain mixture C, i.e. the active peptide feed.

[0015] The application further provides an application of the active peptide feed additive prepared by the method in feed preparation.

[0016] Compared with the prior art, the technical scheme has the following beneficial effects: 1. The application provides a preparation method of goose hemoglobin peptide powder, and the method promotes the release of small molecule active peptides compared with a traditional fermentation method.

[0017] 2. The taste characteristics and antioxidant activity of the goose hemoglobin peptide powder are determined.

[0018] 3. The compounding use of the goose hemoglobin peptide powder not only improves the resource utilization rate of by-products, but also is suitable for the fields of animal feed and functional cosmetics. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The evaluation results (A), soluble peptide content determination (B), and DPPH free radical scavenging rate (C) of the electronic tongue of different treatment groups are shown in the schematic diagram; Figure 2 The schematic diagram of the influence of different treatment groups on the growth performance of finishing pigs is shown in the schematic diagram; Figure 3 The schematic diagram of the influence of different treatment groups on the feed to gain ratio (F / G) during the feeding process of finishing pigs is shown in the schematic diagram. DETAILED DESCRIPTION

[0020] In order to explain the technical content, structural features, purposes and effects of the technical scheme in detail, the following will be described in detail in combination with specific embodiments and the accompanying drawings.

[0021] The application provides a preparation method of an active peptide feed additive prepared based on a weak magnetic field and microbial fermentation, comprising the following steps: Raw material pretreatment: selecting goose hemoglobin as the raw material, configuring the fermentation medium, and configuring 1L of the fermentation medium according to goose hemoglobin powder:water = 1:10 (w / v).

[0022] Strain activation: the Bacillus subtilis is inoculated into the shake flask medium, the inoculation amount is 1%, which is used for the activation process of the strain, the culture conditions are 37℃, 180-220 rpm / min, and the culture time is 10-12h. After three generations of continuous activation of the strain, the bacterial body precipitate is collected under the conditions of 4℃ and 8000 rpm / min for 15min. Before inoculation, the concentration of the bacterial liquid is adjusted to 10 7 CFU / mL using sterile normal saline.

[0023] Substrate fermentation: the activated Bacillus subtilis culture solution is added into the fermentation medium at a proportion of 8.2%, and is placed in a constant temperature of 37℃, 180-220 rpm / min for 24h-48h. After the fermentation is completed, the fermentation liquid is placed in 95℃ for 30min to inactivate the enzyme, and after cooling, the supernatant is collected under the conditions of 4℃, 8000-12000 rpm / min for 15-30min. The collected supernatant is concentrated by a rotary evaporator. Then the concentrated liquid is freeze-dried, and the goose hemoglobin crude peptide can be obtained.

[0024] Magnetic field assisted fermentation: after inoculation, the fermentation liquid is treated with different intensities of weak magnetic field (1.0~5.0 mT) for 2-6h, and then the fermentation process is continued at 37℃ until the fermentation is completed.

[0025] Small molecule peptide enrichment: small molecule peptides are enriched by ultrafiltration (<3 kDa), and goose hemoglobin peptide powder can be obtained by concentration and freeze-drying.

[0026] Determination of peptide content: The casein was diluted with distilled water to obtain a series of standard solution with concentration of 0, 10, 20, 30, 40, 50 mg / mL. The standard solution was mixed with biuret reagent at a ratio of 2:3, and then allowed to stand for 30 min. After centrifugation at 2000 r / min at 4°C for 10 min, the absorbance of the supernatant was measured at 540 nm. The sample solution was mixed with 10% TCA solution at a ratio of 1:1 and allowed to stand for 10 min. Then, the mixture was centrifuged at 4000 r / min for 15 min, and the supernatant was mixed with biuret reagent at a ratio of 2:3, allowed to stand for 30 min, and then centrifuged and measured for absorbance under the same conditions.

[0027] Polypeptide content (mg / g) = CV / m Note: C represents the polypeptide concentration, with units of mg / mL; V represents the fermentation liquid volume, with units of mL; and m represents the goose hemoglobin powder mass, with units of g.

[0028] Preparation of the basic feed: corn 61%, soybean meal 18%, wheat bran 7%, rapeseed meal 4.5%, oil 3.4%, yeast culture 1%, calcium hydrogen phosphate 1.3%, glucose 1%, salt 0.3%, lysine 0.3%, acidifier 0.6%, complex enzyme preparation 0.3%, and premix 1.3%, in terms of mass percentage.

[0029] Preparation of the experimental feed: corn 56%, soybean meal 18%, wheat bran 7%, rapeseed meal 4.5%, goose hemoglobin peptide powder 5%, oil 3.4%, yeast culture 1%, calcium hydrogen phosphate 1.3%, glucose 1%, salt 0.3%, lysine 0.3%, acidifier 0.6%, complex enzyme preparation 0.3%, and premix 1.3%, in terms of mass percentage.

[0030] Effect of adding goose hemoglobin peptide powder in the diet on the growth performance of fattening pigs: Two groups were set up, namely the basic feed group and the experimental feed group. Thirty healthy weaned fattening pigs were randomly divided into two groups, and the experiment lasted for 4 weeks. The body weight of each pig was measured at 0 d, 14 d, and 28 d in the evening, and the initial body weight and the body weight after the experiment were recorded. In addition, the pigs were fed at a fixed time every day, and the weight and loss of the feed were recorded. The average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (F / G) were calculated.

[0031] ADG (g / d) = total gain (g) / {number of pigs × feeding days} ADFI (g / d) = total feed intake (g) / {number of pigs × feeding days} F / G = total feed intake (g) / total gain (g) Results analysis: As shown in Table 1, after the goose hemoglobin was treated by magnetic field, the content of free amino acids changed significantly compared with the single fermentation group (without magnetic field). The content of most amino acids, especially the umami amino acids (Asp and Glu) and sweet amino acids (Thr, Gly, Ala) increased significantly. The results showed that the magnetic field helped to promote the fermentation process, which may be due to the weak magnetic field promoting the growth and metabolic activity of bacterial cells. And existing studies have found that low-intensity magnetic field treatment can assist the fermentation and enzymolysis process, which may increase the reaction rate between the substrate and the enzyme, and improve the kinetic properties of the enzyme.

[0032] Table 1 Changes of free amino acids before and after preparation of goose hemoglobin peptide

[0033] Figure 1 A shows the electronic tongue evaluation results of small molecule peptides after ultrafiltration. The results show that the umami of the magnetic field treatment group is significantly improved compared with the single fermentation group, and the bitterness decreases. In addition, although the salty taste is negative, the salty taste also improves after the magnetic field treatment. The above results show that the magnetic field treatment may promote the release of flavor molecules. Figure 1 B The determination results of soluble peptide content further show the promotion effect of magnetic field on the release of small molecule peptides.

[0034] In addition, the oxidation of feed can easily lead to changes in nutritional ingredients, increased production costs and other problems. Therefore, the antioxidant activity of goose hemoglobin peptide powder was determined, and the results showed that the peptide powder obtained after fermentation had high DPPH free radical scavenging rate, and the antioxidant activity increased significantly with the addition of magnetic field. These results further show the nutritional value and palatability of goose hemoglobin peptide.

[0035] To explore the effect of fermented hemoglobin peptide powder on the growth performance of fattening pigs, the average daily gain (ADG) and average daily feed intake (ADFI) of the basic diet group and the treatment group with fermented hemoglobin peptide powder added to the basic diet were compared. The results are shown in Figure 2 As shown in Table 2, compared with the basic diet group, the addition of fermented hemoglobin peptide powder significantly improved the ADG and ADFI of fattening pigs p <0.05). Among them, the ADG of the treatment group was significantly higher than that of the control group, indicating that it had obvious advantages in promoting the weight gain of pigs; at the same time, the ADFI of the treatment group also increased significantly, indicating that the additive had good palatability and could effectively stimulate feeding. Figure 3 The results showed that the addition of goose hemoglobin peptide powder reduced the F / G value. Therefore, the comprehensive results showed that the addition of fermented hemoglobin peptide powder during the fattening stage could significantly improve the feeding behavior and growth efficiency of fattening pigs.

[0036] It is to be noted that, in the present text, terms such as first and second, and the like, merely serve to identify a difference between one entity or action and another entity or action, and do not necessarily require or imply that there is any such actual relationship or order between these entities or actions. Moreover, the terms "comprising", "including", or any other variant thereof are intended to cover a non-exclusive inclusion, such that processes, methods, articles, or apparatuses that comprise a list of elements are not required to comprise only those elements, but can include other elements not expressly listed or inherent to such processes, methods, articles, or apparatuses. Without further limitation, an element preceded by "comprises a" or "comprises" does not, without more limitations, preclude the existence of further elements of the process, method, article, or apparatus that includes the element. Furthermore, in the present text, "greater than", "less than", "exceed", and the like are understood to exclude the number itself; "and above", "and below", "and within", and the like are understood to include the number itself.

[0037] Although the above-mentioned embodiments have been described, those skilled in the art can make further changes and modifications to these embodiments once they know the basic inventive concept, so the above description is only for the embodiments of the present application, and does not limit the patent protection scope of the present application, and any equivalent structure or equivalent process transformation using the content of the present application specification and drawings, or direct or indirect application in other related technical fields, are also included in the patent protection scope of the present application.

Claims

1. A method for preparing active peptide feed additive by weak magnetic field and microbial fermentation, characterized in that, The feed additive is goose hemoglobin peptide powder.

2. The low field synergistic microbial fermentation process for producing active peptide feed additive as claimed in claim 1, wherein, The preparation method of the feed additive comprises the following steps: Raw material pretreatment: select goose hemoglobin as raw material, configure fermentation medium, according to goose hemoglobin powder: water = 1: (8-10) (w / v), configure 1L of fermentation medium; Strain activation: Bacillus subtilis was inoculated into a shake flask medium at an inoculation amount of 1% for the strain activation process, and the culture conditions were 35-37°C, 180-220 rpm / min, and a culture time of 10-12 h. After three generations of continuous activation of the strain, the bacterial body precipitate was collected by centrifugation at 4°C and 8000 rpm / min for 15 min, and the concentration of the bacterial solution was adjusted to 10 7 CFU / mL with sterile normal saline before inoculation. Substrate fermentation: add the activated bacillus subtilis culture solution to the fermentation medium according to the proportion of 8.2%, place it in a constant temperature of 37℃, 180-220 rpm / min, cultivate for 24h-48h, after the fermentation is completed, place the fermentation liquor in 95℃ for 30min to kill the enzyme, after cooling, centrifuge at 4℃, 8000-12000 rpm / min for 15-30min, collect the supernatant, concentrate the collected supernatant through a rotary evaporator, then freeze-dry the concentrated solution, and the goose hemoglobin crude peptide can be obtained; Small molecule peptide enrichment: the goose hemoglobin crude peptide is enriched by ultrafiltration <3 kDa to obtain small molecule peptides, and the goose hemoglobin peptide powder can be obtained by concentration and freeze-drying.

3. The low field synergistic microbial fermentation process for producing active peptide feed additive as claimed in claim 1, wherein, The substrate fermentation process is assisted by a magnetic field, and the magnetic field assisted fermentation is as follows: after inoculation, the fermentation liquor is treated with a weak magnetic field of 1.0-5.0 mT for 2-6h, and then the fermentation process is continued at 37℃ until the fermentation is completed.

4. A method for preparing active peptide feed by weak magnetic field synergistic microbial fermentation, characterized in that, The feed comprises corn, soybean meal, wheat bran, rapeseed meal, goose hemoglobin peptide powder, oil, yeast culture, calcium hydrogen phosphate, glucose, salt, lysine, acidifying agent, complex enzyme preparation, premix.

5. The weak magnetic field synergistic microbial fermentation preparation of active peptide feed of claim 4, characterized in that, The feed comprises corn 56%, soybean meal 18%, wheat bran 7%, rapeseed meal 4.5%, goose hemoglobin peptide powder 5%, oil 3.4%, yeast culture 1%, calcium hydrogen phosphate 1.3%, glucose 1%, salt 0.3%, lysine 0.3%, acidifying agent 0.6%, complex enzyme preparation 0.3%, and premix 1.3%, in terms of mass percentage.

6. The active peptide feed prepared by the synergistic microbial fermentation under a weak magnetic field according to claim 4, wherein the active peptide feed is prepared by the synergistic microbial fermentation under a weak magnetic field. The preparation method of the feed is as follows: the yeast culture, calcium hydrogen phosphate, glucose, salt, lysine, acidifying agent, complex enzyme preparation, and premix are premixed for 2-3min to obtain mixture A; then the corn, soybean meal, wheat bran, and rapeseed meal are crushed and sieved through a pulverizer, and the particle size is controlled to be 1.5mm; after sieving, mix them under the condition of 75-80℃ for 2-3min, cool to room temperature, add the goose hemoglobin peptide powder, mix for 1-2min to obtain mixture B, mix A and B for 3-5min to obtain mixture C, that is, the feed added with active peptide.

7. The weak magnetic field synergistic microbial fermentation preparation of active peptide feed of claim 4, characterized in that, 8. Use of the weak magnetic field synergistic microbial fermentation active peptide feed additive in the preparation of feed. ​

Images