CN112544787A

CN112544787A - Method for fermenting paper mulberry compound feed by bacteria and enzyme in synergy mode and compound feed thereof

Info

Publication number: CN112544787A
Application number: CN202011200251.6A
Authority: CN
Inventors: 汪以真; 苏维发; 路则庆; 姜子鹏; 郝丽红; 王成; 王凤芹
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2020-11-02
Filing date: 2020-11-02
Publication date: 2021-03-26

Abstract

The invention discloses a method for synergistically fermenting paper mulberry compound feed with bacteria and enzymes and the compound feed. (1) Preparation of fermentation raw materials; (2) Preparation of activation medium; (3) Activation of strains and preparation of fermented seed liquid, Saccharomyces cerevisiae and Lactobacillus plantarum are connected to the activation medium for activation and mixed according to the volume ratio of 2:1, and acid is added. The protease is mixed evenly to obtain the fermented seed liquid and then inoculated; (4) bagged for fermentation, put into a breathing bag and fermented for 3-5 days before use. The invention can significantly increase the crude protein, small peptides, probiotics, organic acids and flavonoids in the paper mulberry compound feed, enhance the total antioxidant capacity of the feed, effectively reduce the tannin and crude fiber content, and improve the paper mulberry compound feed in vitro for pigs. digestibility, has a good application prospect.

Description

Method for fermenting paper mulberry compound feed by bacteria and enzyme in synergy mode and compound feed thereof

Technical Field

The invention belongs to the technical field of animal feed, and particularly relates to a method for fermenting paper mulberry compound feed by bacteria and enzyme in a synergistic manner and the compound feed.

Background

In recent years, the feed industry has changed greatly due to African swine fever, and the development and transformation of the feed industry are promoted by banning of resistance policies, so that the biological feed technology is developed vigorously. The reasonable development and utilization of different kinds of feeds and unconventional feeds by adopting a biological fermentation technology and the exploration of fermentation processes aiming at different raw materials are an important development trend of future biological feeds or can effectively relieve the problem of feed resource shortage.

The paper mulberry is also called milk tree, and is an unconventional feed raw material widely applied to livestock breeding. The planting area of the national broussonetia papyrifera in 2017 is over 30 ten thousand acres, and the feed raw material catalog is included in 2018. According to different harvesting heights, the paper mulberry contains 15-24% of crude protein, 5-7% of crude fat and 12-21% of crude fiber, and is rich in more than 16 amino acids and minerals such as Ca, P, Zn, Fe, Mn, Cu and the like. In addition, the paper mulberry contains more than 70 flavonoid compounds, and has various biological activities such as antibiosis, antioxidation, anti-inflammation and the like. However, the paper mulberry serving as a woody feed raw material contains a large amount of tannin and crude fiber, has poor palatability, and seriously influences the feed intake of animals and the digestion and absorption of nutrients.

The microbial fermentation can effectively degrade anti-nutritional factors in the feed, promote digestion and absorption of nutrients, provide beneficial bacteria and metabolites thereof, regulate the stable state of intestinal flora, reduce the use of antibiotics and further improve the health of animals. Therefore, suitable probiotics are screened according to the characteristics of the raw materials and are fermented by a suitable process, and the nutritive value and the utilization efficiency of the raw materials can be effectively improved. However, reports of the process for fermenting the paper mulberry compound feed by using bacterial enzymes in a synergistic manner and related products applied to fattening pigs are quite rare at present.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a method for fermenting paper mulberry compound feed by using bacteria and enzymes and the compound feed.

A method for fermenting Broussonetia papyrifera compound feed by virtue of bacteria and enzyme cooperation comprises the following steps:

1) preparing fermentation raw materials

The fermentation raw materials comprise, by mass, 22% of soybean meal, 38% of corn, 15% of paper mulberry branches and leaves, 15% of molasses soybean hull and 10% of bran, and are crushed and mixed uniformly;

2) preparing an activation medium

The saccharomyces cerevisiae activation medium comprises 5% of soybean meal powder, 1% of yeast powder, 5% of molasses and the balance of water by mass percent, and is cooled to room temperature after being heated to 100 ℃; the lactobacillus plantarum activation medium comprises 5% of soybean meal powder, 1% of yeast powder, 2% of molasses, 1% of tomato powder, 0.1% of monopotassium phosphate and the balance of water by mass percent, is heated to 100 ℃ and then is cooled to room temperature;

3) activating strain and preparing fermented seed liquid

Inoculating Saccharomyces cerevisiae powder into Saccharomyces cerevisiae activation culture medium according to 0.1% of the culture medium by mass, and introducing oxygen at 37 deg.C for culturing for 12 h; inoculating lactobacillus plantarum ZJUAF-5 powder into a lactobacillus plantarum activation culture medium according to 0.1% of the mass of the culture medium, and standing and culturing at 37 ℃ for 18 h; uniformly mixing the activated saccharomyces cerevisiae and lactobacillus plantarum according to the volume ratio of 2:1, and then adding acid protease with the enzyme activity of 10000U/g according to 0.2% of the total fermentation system mass to uniformly mix to obtain fermented seed liquid; the saccharomyces cerevisiae is purchased from China general microbiological culture Collection center, and the number of the strain catalog is as follows: 2.3973, respectively; the lactobacillus plantarum ZJUAF-5 is purchased from the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms, and the catalog number of the culture is as follows: 1.510;

4) fermentation of

Adding the fermentation seed liquid obtained in the step 3) into the fermentation raw material in the step (1) according to the proportion of 10% of the total fermentation system mass, adding warm water at 37 ℃ to enable the total water content to be 45% of the total fermentation system mass, uniformly stirring, then filling into a breathing bag, fermenting for 1-2 days in an open way through the breathing bag, and then sealing and fermenting for 2-3 days to obtain the fermentation paper mulberry compound feed.

The method comprises the steps of performing bacterial enzyme synergistic fermentation, and performing aerobic and anaerobic two-step fermentation in a breathing bag at the temperature of 37 ℃.

The bacterial enzyme synergistic fermentation broussonetia papyrifera compound feed prepared by the method.

The invention has the advantages that:

according to the invention, the contents of crude protein, small peptide, probiotics, organic acid and flavone in the broussonetia papyrifera compound feed are obviously improved by using the compatibility of saccharomyces cerevisiae, lactobacillus plantarum and acid protease and the two-step fermentation process in the breathing bag, the total oxidation resistance of the feed is enhanced, the contents of tannin and crude fiber are effectively reduced, and the in vitro digestibility of pigs in the broussonetia papyrifera compound feed is improved.

Drawings

FIG. 1 is scanning electron microscope images of Broussonetia papyrifera compound feed before and after fermentation.

FIG. 2 is protein electrophoresis chart before and after fermentation of paper mulberry compound feed.

Detailed Description

The invention is further illustrated below with reference to the figures and examples.

Example 1 preparation method of laboratory

Adding 5% of soybean meal powder, 1% of yeast powder and 5% of molasses into water according to the mass percentage of a saccharomyces cerevisiae activation medium, uniformly stirring, heating to 100 ℃, and cooling to room temperature; the lactobacillus plantarum activation medium is prepared by adding 5% of soybean meal powder, 1% of yeast powder, 2% of molasses, 1% of tomato powder and 0.1% of monopotassium phosphate into water according to mass percentage, uniformly stirring, heating to 100 ℃, and cooling to room temperature.

Inoculating Saccharomyces cerevisiae powder into Saccharomyces cerevisiae activation culture medium according to 0.1% of the culture medium by mass, and introducing oxygen at 37 deg.C for culturing for 12 h; the lactobacillus plantarum ZJUAF-5 powder is inoculated into a lactobacillus plantarum activation culture medium according to 0.1 percent of the mass of the culture medium, and is subjected to static culture at 37 ℃ for 18 hours. And uniformly mixing the activated saccharomyces cerevisiae and lactobacillus plantarum according to the volume ratio of 2:1, and then adding acid protease with the enzyme activity of 10000U/g according to 0.2% of the total fermentation system mass to uniformly mix to obtain the fermented seed liquid.

Adding 200g of substrate (22% of soybean meal, 38% of corn, 15% of branches and leaves of paper mulberry, 15% of molasses soybean hull and 10% of bran) into the prepared fermented seed liquid according to 10% of the mass of the total fermentation system, adding warm water at 37 ℃ to enable the total water content to be 45% of the mass of the total fermentation system, uniformly stirring, transferring into a 1000mL conical flask, covering with a sterile membrane, and carrying out aerobic fermentation at 37 ℃ for 24 hours. And then replacing the sample with a sealing film, fermenting for 48 hours at 37 ℃ under an anaerobic condition, taking part of wet sample, and rapidly detecting microbial metabolites and in-vitro digestibility. The samples were then dried at 65 ℃ for 12h, cooled and ground for nutritional analysis.

TABLE 1 nutritional ingredient changes before and after fermentation of paper mulberry compound feed

Index%	Compound feed for unfermented broussonetia papyrifera	Fermented paper mulberry compound feed
			Dried substance	90.78	91.39
Crude protein	17.09^b	19.69^a
			Acid soluble protein	9.58^b	25.86^a
Coarse fiber	10.48^a	8.18^b
			Acid detergent fiber	15.83^a	12.56^b
Neutral detergent fiber	26.11^a	21.70^b
			Tannin	0.33^a	0.16^b
Arginine	0.80^b	0.88^a
			Leucine	1.09^b	1.16^a
Isoleucine	0.48^b	0.53^a
			Methionine	0.13^b	0.17^a
Threonine	0.52^b	0.57^a
			Lysine	0.73^b	0.77^a
Phenylalanine	0.69	0.71
			Histidine	0.34	0.36
Valine	0.66	0.73
			Cysteine	0.32	0.36
Aspartic acid	1.33	1.36
			Tyrosine	0.36	0.37
Serine	0.65^b	0.72^a
			Glycine	0.61^b	0.69^a
Alanine	0.71^b	1.03^a
			Glutamic acid	2.52^b	2.90^a
Proline	0.84^b	0.98^a
			Total amino acids	12.79^b	14.27^a

Note: the difference of the lower case letters on the shoulder marks of the same line is significant (P<0.05)。

As can be seen from Table 1, the crude protein, acid soluble protein and total amino acid of the fermented broussonetia papyrifera compound feed are respectively increased by 2.60%, 16.28% and 1.48% compared with the unfermented broussonetia papyrifera compound feed. The degradation rates of the crude fiber, the acid washing fiber, the neutral washing fiber and the tannin respectively reach 21.95 percent, 20.66 percent, 16.89 percent and 51.52 percent. In conclusion, the two-step fermentation process cooperated with the bacterial enzyme effectively reduces the antinutritional factors in the paper mulberry compound feed and improves the nutritional value of the feed.

TABLE 2 in vitro digestibility of pig before and after fermentation of paper mulberry compound feed

Index%	Compound feed for unfermented broussonetia papyrifera	Fermented paper mulberry compound feed
			Dried substance	54.38^b	56.74^a
Crude protein	71.13^b	77.61^a
			Arginine	65.47^b	82.92^a
Histidine	57.04^b	70.37^a
			Leucine	57.24^b	74.67^a
Isoleucine	54.30^b	75.24^a
			Lysine	63.95^b	76.78^a
Methionine	48.83^b	77.28^a
			Valine	38.22^b	58.95^a
Threonine	58.09^b	75.20^a
			Phenylalanine	64.70^a	48.80^b
Aspartic acid	62.83^b	79.44^a
			Glutamic acid	72.50^b	86.97^a
Tyrosine	74.32^b	80.94^a
			Serine	55.29^b	74.50^a
Glycine	49.01^b	67.60^a
			Alanine	60.74^b	81.41^a
Cysteine	29.53^b	49.06^a
			Proline	59.52^b	73.82^a
Total amino acids	60.61^b	75.73^a

As can be seen from Table 2, the results of the two-step in vitro digestibility determination method using pig pepsin-trypsin show that the in vitro digestibility of dry matter, crude protein and amino acid in the broussonetia papyrifera compound feed is significantly improved by the bacterial enzyme synergistic two-step fermentation process, so that the utilization of nutrients is improved.

TABLE 3 Broussonetia papyrifera compound feed before and after fermentation change of microorganisms and functional substances

As can be seen from Table 3, the organic acid content of the fermented broussonetia papyrifera compound feed is remarkably improved, the lactic acid content reaches 4.31 percent, the pH value of the feed is reduced to 4.51, and the viable count of the saccharomyces cerevisiae and the lactobacillus plantarum respectively reaches 4.33 multiplied by 10⁸cfu/g and 35.09X 10⁸cfu/g, and in addition, the flavone content and the total antioxidant capacity are obviously improved. In conclusion, the broussonetia papyrifera compound feed fermented by the bacterial enzyme is rich in probiotics and organic acid, the flavone content and the total antioxidant capacity are improved, and the broussonetia papyrifera compound feed canEffectively relieve the stress reaction of the fattening pigs.

As can be seen from figure 1, the paper mulberry fiber in the paper mulberry compound feed has smooth and complete surface, becomes more broken after the synergistic fermentation of bacterial enzymes, has an aggregated surface, has more pores, and is enriched with probiotics on the surface and in the pores. The change of the structure of the broussonetia papyrifera compound feed by the bacterial enzyme synergistic fermentation is prompted, and the higher digestibility after fermentation is explained from another angle.

As can be seen from FIG. 2, the protein with a larger molecular weight (35-55 kDa) in the fermented paper mulberry compound feed is degraded, and the content of the protein with a smaller molecular weight (15-25 kDa) is increased, which explains that the fermented paper mulberry compound feed has a higher crude protein digestion rate in vitro.

Example 2 preparation method of Broussonetia papyrifera compound feed by bacterial enzyme synergistic fermentation in production

Preparing an activation medium

Preparing fermented seed liquid

Two-step fermentation of breath bag

Adding fermented seed liquid into 40kg of fermented raw materials (according to the mass ratio of 22% of soybean meal, 38% of corn, 15% of paper mulberry branches and leaves, 15% of molasses soybean hull and 10% of bran) according to the mass ratio of 10% of the total fermented system, adding warm water at 37 ℃ to enable the total water content to be 45% of the total fermented system mass, uniformly stirring, then putting into a breathing bag, carrying out open aerobic fermentation for 2 days, and sealing and fermenting for 2 days to obtain the fermented paper mulberry compound feed product.

TABLE 4 Change before and after fermentation of Broussonetia papyrifera composite fodder

As can be seen from Table 4, compared with the broussonetia papyrifera compound feed raw material, the fermented broussonetia papyrifera compound feed product obtained by the synergistic two-step fermentation process of the respiring bag bacterial enzyme has the advantages that the crude protein is improved by 2.61%, the acid soluble protein is 2.84 times of that before fermentation, the lactic acid content is 2.81 times of that before fermentation, the degradation rate of crude fiber reaches 40.79%, and the fermented broussonetia papyrifera compound feed product is rich in probiotic live bacteria.

The embodiments in the above description can be further combined or replaced, and the embodiments are only described as preferred embodiments of the present invention, and do not limit the concept and scope of the present invention, and various changes and modifications made to the technical solution of the present invention by those skilled in the art without departing from the design concept of the present invention belong to the protection scope of the present invention. The scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. a method for bacterial enzyme synergistic fermentation of paper mulberry compound feed, is characterized in that, step is as follows:

1) Preparation of fermentation raw materials

According to the quality of the fermentation raw materials, including 22% soybean meal, 38% corn, 15% twig leaves, 15% molasses soybean hull and 10% bran, they are crushed and mixed evenly;

2) Prepare activation medium

Saccharomyces cerevisiae activation medium contains 5% soybean meal powder, 1% yeast powder, 5% molasses by mass percentage, and the balance is water, heated to 100 ° C and cooled to room temperature; Lactobacillus plantarum activation medium by mass percentage contains 5% % soybean meal powder, 1% yeast powder, 2% molasses, 1% tomato powder, 0.1% potassium dihydrogen phosphate, the balance is water, heated to 100 ℃ and cooled to room temperature;

3) Activation of strains and preparation of fermented seed liquid

Saccharomyces cerevisiae powder was added to Saccharomyces cerevisiae activation medium according to 0.1% of the quality of the medium, and cultured at 37 °C for 12 hours; In the base, stand at 37°C for 18h; mix the above activated Saccharomyces cerevisiae and Lactobacillus plantarum in a ratio of 2:1 by volume, and then add acid protease with an enzyme activity of 10000U/g according to 0.2% of the total fermentation system mass Mix well to obtain fermented seed liquid; the above-mentioned Saccharomyces cerevisiae was purchased from the General Microorganism Center of the China Microorganism Culture Collection Management Committee, the strain catalog number: 2.3973; the above-mentioned Lactobacillus plantarum ZJUAF-5 was purchased from the China Microorganism Culture Collection Management Committee Ordinary Microorganisms Center, strain catalog number: 1.510;

4) Fermentation

Add the fermented seed liquid obtained in step 3) to the fermentation raw materials in step (1) according to the proportion of 10% of the mass of the total fermentation system, and add warm water at 37°C to make the total moisture account for 45% of the mass of the total fermentation system, stir evenly, and then pack Put it into a breathing bag, ferment the bag open for 1-2 days, and then seal it for 2-3 days to obtain the fermented paper mulberry compound feed.

2 . The method according to claim 1 , wherein the bacteria and enzymes are fermented cooperatively, and in the breathing bag, aerobic and anaerobic two-step fermentation are carried out, and the temperature is 37° C. 3 .

3. A method according to claim 1 prepares the compound feed of mycoplasma-enzyme synergistic fermentation paper mulberry.