CN117987328B

CN117987328B - Functional mulberry leaf feed and preparation method and application thereof

Info

Publication number: CN117987328B
Application number: CN202410391819.9A
Authority: CN
Inventors: 斯大勇; 李金转; 阳田恬; 张皓森; 蒋晓晗; 姜伯嵩; 卫旭彪; 张日俊
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2024-04-02
Filing date: 2024-04-02
Publication date: 2024-07-16
Anticipated expiration: 2044-04-02
Also published as: CN117987328A

Abstract

The invention relates to the technical field of biological feeds, in particular to a functional mulberry leaf feed and a preparation method and application thereof. When the fermentation strain composition provided by the invention is used for fermenting mulberry leaves, the strains are in synergistic interaction, so that the content of crude fibers in the fermentation mulberry leaves can be effectively reduced, the content of crude proteins is increased, the lactic acid content is increased, the pH is reduced, the functionality of the fermentation mulberry leaves to livestock and poultry is improved, the fermentation strain composition can be used for preparing functional mulberry leaf feeds through multi-strain synergistic fermentation, the antioxidant capacity and the immune function of the livestock and poultry can be obviously improved, the intestinal health of the livestock and poultry can be improved, the meat quality can be effectively improved, the comprehensive economic benefit of livestock and poultry cultivation is improved, and a starter resource and an effective method are provided for the application of the mulberry leaves in animal production.

Description

Functional mulberry leaf feed and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological feeds, in particular to a functional mulberry leaf feed and a preparation method and application thereof.

Background

In recent years, with the improvement of the living standard of people, the pursuit of high-quality life has been an urgent need of most people, and the demands for nutrients such as meat, eggs, milk and the like have been increasing, so that the increase of the livestock breeding amount is promoted, and the demand for feed resources is increasing, wherein the serious shortage of protein feed is one of the problems faced in the production of animal husbandry.

The mulberry belongs to Moraceae (Moraceae) Morus (Morus), is a perennial woody plant, and the mulberry leaf is the main product. Mulberry leaves have been mainly used for silkworm spinning for over 5000 years. The mulberry can adapt to various complex environments, and can grow in most temperate, tropical and subtropical areas, and the mulberry is spread all over the world. The mulberry leaves are comprehensive and balanced in nutrition components, and the content of the nutrition components such as Crude Protein (CP) (15-35%), crude fat (EE) (3.5-5.57%), vitamins (2.42-4.71% Ca, 0.23-0.97% P), amino acids and the like is higher than that of alfalfa (13.0-28% CP, 1.3-3.0% EE). In addition, the mulberry leaf contains natural active substances such as flavonoid, alkaloid, polysaccharide, polyphenol and the like, and has obvious effects of reducing blood sugar, reducing blood fat, resisting bacteria, resisting oxidation, resisting inflammation and the like. Therefore, the mulberry leaf is suitable to be used as a high-quality protein feed raw material in animal production, such as pigs, chickens, ruminants, fish and the like.

At present, the mulberry leaves are mostly utilized to directly cut fresh feed, or fresh mulberry leaves are dried (or dried) and crushed into mulberry leaf powder and then added into animal feed, and the utilization modes are simple and easy to implement, and achieve a certain effect in the aspect of animal cultivation, but a plurality of problems are gradually exposed in the cultivation process. On one hand, the mature mulberry leaves and mulberry branches have higher crude fiber content and poorer palatability; on the other hand, sang Shecu has high protein and water content, is extremely easy to spoil, is not suitable for long-term storage, has seasonality and limited utilization time, and consumes a large amount of energy when drying mulberry leaves. Researches show that the nutrient content of the mulberry leaves can be improved, the palatability is improved, the preservation time can be prolonged, and the fresh mulberry leaves can be fully utilized by utilizing different microorganisms to ferment the mulberry leaves. However, the fermented mulberry leaves prepared by the existing fermentation strain and method are mainly used as feed raw materials to replace protein feeds such as soybean meal, and the effect of improving the oxidation resistance, immunity, intestinal health, meat quality and the like of livestock and poultry is still limited. Therefore, developing the functional mulberry leaf feed improves the immunity of livestock and poultry and the oxidation resistance of the livestock and poultry while realizing the reduction substitution of protein feed, and has important theoretical value and practical significance for producing high-quality livestock and poultry products.

Disclosure of Invention

The invention provides a functional mulberry leaf feed and a preparation method and application thereof.

The fermentation strain has important influence on the composition and content of the nutritional ingredients and the functional ingredients of the fermented mulberry leaves, and the strain for preparing the functional mulberry leaf feed is screened in order to improve the functionality of the fermented mulberry leaves to livestock and poultry. According to the invention, a plurality of strains of lactic acid bacteria and bacillus are separated and screened from the chyme of livestock and poultry, the mulberry leaves are fermented by utilizing different strains and different combination modes thereof, a method for preparing the functional mulberry leaf feed through multi-bacteria collaborative fermentation is systematically researched, the optimal fermentation strain combination is screened, and the functional mulberry leaf feed prepared by fermenting the fermentation strain combination can obviously improve the oxidation resistance and the immunity functions of the livestock and poultry, and meanwhile, the intestinal health is improved, and the meat quality is improved.

Specifically, the invention provides the following technical scheme:

In a first aspect, the present invention provides a fermentation broth composition comprising Pediococcus acidilactici (Pediococcus acidilactici), pediococcus pentosaceus (Pediococcus pentosaceus), bacillus subtilis (Bacillus subtilis), and Bacillus licheniformis (Bacillus licheniformis);

The Pediococcus acidilactici, pediococcus pentosaceus, bacillus subtilis and Bacillus licheniformis are all preserved in the China general microbiological culture Collection center (CGMCC) No. 28922, CGMCC No. 28921, CGMCC No. 28923 and CGMCC No. 28924.

In the invention, the pediococcus acidilactici is pediococcus acidilactici A62, and the strain is preserved in China general microbiological culture Collection center (CGMCC) at 11 and 9 days of 2023, and is classified and named as pediococcus acidilactici Pediococcus acidilactici with a preservation number of CGMCC No. 28922, wherein the CGMCC is the No. 3 of West-line 1 in the Korean area North Star of Beijing, and the pediococcus acidilactici is the microbiological institute of China.

Pediococcus pentosaceus is Pediococcus pentosaceus A7, which is preserved in China general microbiological culture Collection center (CGMCC) at the date of 2023, 11 and 9, and has the classification name Pediococcus pentosaceus Pediococcus pentosaceus and the preservation number of CGMCC No. 28921, wherein the CGMCC is called as North Star Xiding No. 1, 3, the university of Korea, beijing, and the microorganism institute, and the mail code is 100101.

The bacillus subtilis KO1 is preserved in China general microbiological culture collection center (CGMCC) of China general microbiological culture Collection center (address: north Star Xway No. 1,3 of the Korean area of Beijing, and the institute of microorganisms, post code 100101) on day 11 and 9 of 2023, and is classified and named as bacillus subtilis Bacillus subtilis with a preservation number of CGMCC No. 28923.

The bacillus licheniformis is bacillus licheniformis DO1, and the strain is preserved in China general microbiological culture Collection center (CGMCC) of China Committee for culture Collection of microorganisms (address: north Star Xway No. 1,3 of the university of China, and postal code 100101) at the 11 th month and 9 th year of 2023, and is classified and named as bacillus licheniformis Bacillus licheniformis, and the preservation number is CGMCC No. 28924.

The Pediococcus acidilactici, pediococcus pentosaceus, bacillus subtilis and Bacillus licheniformis are obtained by separating and screening from minced food of livestock and poultry, and the combination of the Pediococcus acidilactici, the Pediococcus pentosaceus, the bacillus subtilis and the Bacillus licheniformis is used for preparing the functional mulberry leaf feed by multi-bacteria collaborative fermentation, so that the crude fiber content in the prepared functional mulberry leaf feed can be obviously reduced, the content of crude protein in the functional mulberry leaf feed is increased, the pH is reduced, the lactic acid content is increased, the antioxidant capacity and the immune function of the livestock and poultry can be obviously improved, the intestinal health of the livestock and poultry can be obviously improved, the growth performance of the livestock and poultry can be improved, the meat quality and the nutritional value can be improved, and the comprehensive economic benefit of livestock and poultry cultivation can be improved.

Preferably, in the fermentation cylinder composition, the ratio of the effective viable count of Pediococcus acidilactici, pediococcus pentosaceus, bacillus subtilis and Bacillus licheniformis is (1-3): (1-3): (3-10): (3-10).

In a second aspect, the present invention provides a mulberry leaf feed starter comprising the above-described combination of fermentation broths.

In the mulberry leaf feed starter, the Pediococcus acidilactici, pediococcus pentosaceus, the bacillus subtilis and the bacillus licheniformis in the fermentation strain composition can be respectively and independently packaged in liquid or solid dosage forms such as bacterial liquid or dry powder, or the bacterial liquid or dry powder of at least two strains can be mixed and packaged.

Preferably, in the starter, the ratio of the effective viable count of Pediococcus acidilactici, pediococcus pentosaceus, bacillus subtilis and Bacillus licheniformis is (1-3): (1-3): (3-10): (3-10).

Besides the fermentation strain composition, the ferment can also contain carriers (such as bran, rice hull powder, secondary powder, corncob powder, corn gluten meal and the like) or auxiliary materials allowed in the field of microbial preparations. The fermentation agent may be a liquid formulation (e.g., bacterial suspension, etc.) or a solid formulation (e.g., dry powder).

In a third aspect, the present invention provides a method for preparing the mulberry leaf feed starter as described above, comprising the step of culturing the Pediococcus acidilactici, pediococcus pentosaceus, bacillus subtilis and Bacillus licheniformis.

The culturing of Pediococcus acidilactici, pediococcus pentosaceus, bacillus subtilis and Bacillus licheniformis can be performed by using culture media and methods of Pediococcus acidilactici, pediococcus pentosaceus, bacillus subtilis and Bacillus licheniformis which are commonly used in the art. The above-mentioned respective strains are preferably cultured separately.

In some embodiments of the invention, the culture medium used for the cultivation of Pediococcus acidilactici and Pediococcus pentosaceus comprises the following components: 4 to 6 percent of molasses, 1 to 3 percent of bean pulp, 0.08 to 0.15 percent of ammonium sulfate, 0.08 to 0.15 percent of sodium acetate, 0.4 to 0.6 percent of dipotassium hydrogen phosphate, 0.01 to 0.03 percent of anhydrous magnesium sulfate, 0.004 to 0.006 percent of manganese sulfate, 0.08 to 0.15 percent of tween-80 (v/v) and pH of 6.3 to 6.8.

In some embodiments of the invention, the culture medium used for the culture of bacillus subtilis and bacillus licheniformis comprises the following components: glucose 0.4-0.6%, soybean meal 2-4%, corn flour 2-4%, yeast extract powder 0.3-0.5%, manganese sulfate 0.02-0.04%, and pH 6.8-7.5.

The temperature of the cultivation of Pediococcus acidilactici, pediococcus pentosaceus, bacillus subtilis and Bacillus licheniformis is preferably 35-37 ℃.

In a fourth aspect, the invention provides the use of the above-described composition of fermentation broth or the above-described mulberry leaf feed starter in the preparation of fermented mulberry leaf or functional mulberry leaf feed.

Applications described above include: the mulberry leaves are fermented by adopting the fermentation strain composition or the mulberry leaf feed starter.

In a fifth aspect, the present invention provides a method for preparing a functional mulberry leaf feed, the method comprising: fermenting the mulberry leaves by using pediococcus acidilactici, pediococcus pentosaceus, bacillus subtilis and bacillus licheniformis;

Preferably, in the fermentation, the ratio of the number of viable bacteria inoculated by Pediococcus acidilactici, pediococcus pentosaceus, bacillus subtilis and Bacillus licheniformis is (1-3): (1-3): (3-10): (3-10).

Preferably, pediococcus acidilactici, pediococcus pentosaceus, bacillus subtilis and Bacillus licheniformis are respectively activated, cultured in a liquid culture medium until the logarithmic phase to obtain bacterial liquid, and then mixed according to the ratio of the number of the active bacteria and inoculated.

In some embodiments of the invention, the ratio of the number of viable bacteria inoculated from Pediococcus acidilactici, pediococcus pentosaceus, bacillus subtilis, and Bacillus licheniformis is 1:1:3:3, 3:1:3:3, 1:3:3, 1:1:10:3, or 1:1:3:10. Preferably 1:1:3:3.

Preferably, the total inoculation amount of the pediococcus acidilactici, the pediococcus pentosaceus, the bacillus subtilis and the bacillus licheniformis is 2% -8% (v/w).

Preferably, the fermentation is anaerobic fermentation.

Preferably, the fermentation temperature is 23-42 ℃. More preferably 30-37 ℃.

Preferably, the fermentation time is 7-30 days.

Preferably, the fermented bottom material is further added with fermentation auxiliary materials, and the fermentation auxiliary materials are one or more selected from bran, corn flour and molasses.

In some embodiments of the invention, the fermentation adjunct is bran. Preferably, the addition amount of the bran is 5-15% of the mulberry leaf by mass ratio.

In some embodiments of the invention, the method comprises: uniformly mixing mulberry leaves with 5% -15% (w/w) bran, inoculating fermentation strain mixed liquor consisting of Pediococcus acidilactici, pediococcus pentosaceus, bacillus subtilis and bacillus licheniformis according to an inoculum size of 2% -8% (v/w), fully uniformly mixing, and performing anaerobic fermentation at 23-42 ℃ for 7-30 days. Preferably, the mulberry leaves are cut to be within 3 cm or pulped mulberry leaves.

In a sixth aspect, the present invention provides a functional mulberry leaf feed prepared by the preparation method described above.

According to the invention, through experimental verification, the economic benefit of cultivation can be remarkably improved by adding the functional mulberry leaf feed into daily ration; the addition of 4-12% of the functional mulberry leaf feed can obviously improve the apparent digestibility of crude fiber; the pH _24h, the flesh color, the marbling, the intramuscular fat content, the protein content and the essential amino acid content of the muscle are obviously improved, and the meat quality is integrally improved; the daily ration is added with 4-8% of functional mulberry leaf feed, so that the total saturated fatty acid and monounsaturated fatty acid content of muscles can be obviously reduced, and the polyunsaturated fatty acid and functional fatty acid content and the PUFA/SFA ratio can be obviously improved; the functional mulberry leaf feed can also obviously improve the oxidation resistance and the immunity by adding the functional mulberry leaf feed into daily ration; and simultaneously improve intestinal morphology, intestinal fatty acid content and intestinal flora.

In a seventh aspect, the present invention provides any one of the following uses of the functional mulberry leaf feed described above:

(1) The application in improving the comprehensive economic benefit of livestock and poultry cultivation;

(2) The application in improving the digestion and utilization rate of the livestock and poultry to the nutrients;

(3) The application in improving the quality of livestock and poultry meat;

(4) The application of the composition in improving the content of fatty acid and/or amino acid in livestock and poultry muscles;

(5) The application in improving the oxidation resistance of livestock and poultry;

(6) The application in improving the immune function of livestock and poultry;

(7) The application of the composition in improving the intestinal health of livestock and poultry and/or increasing the content of short chain fatty acid in the intestinal tract of livestock and poultry;

(8) The application in improving the intestinal flora of livestock and poultry.

The invention also provides application of the functional mulberry leaf feed in preparing a product with any one or more functions of (1) - (8).

In the above (1), the comprehensive economic benefit specifically includes gross profit and the like.

In the above (2), the digestion and utilization ratio of the nutrient specifically includes the apparent digestion ratio of the nutrient and the like.

In the above (3), the meat quality specifically includes muscle pH, flesh color, marbling, muscle crude protein content, intramuscular fat content, muscle tenderness, moisture content, muscle shear force, etc.

In (4) above, improving muscle fatty acid content includes reducing total saturated fatty acid and monounsaturated fatty acid content, increasing polyunsaturated fatty acid and functional fatty acid content, and/or increasing PUFA/SFA ratio. Improving the amino acid content of muscle includes increasing the content of essential amino acids, etc.

In the above (7), improving intestinal health includes improving intestinal morphology and the like.

In the above (8), improving the intestinal flora of livestock and poultry comprises improving the diversity of the intestinal flora, increasing the abundance of beneficial bacteria and/or reducing the abundance of harmful bacteria.

In the invention, the livestock and poultry comprise pigs, cattle, sheep, horses, donkeys, chickens, ducks, geese and the like.

In some embodiments of the invention, the livestock and poultry are pigs.

The beneficial effects of the invention at least comprise: when the fermented strain composition is used for fermenting mulberry leaves, the strains cooperate, so that the content of crude fibers in the fermented mulberry leaves can be effectively reduced, the content of crude proteins is increased, the content of lactic acid is increased, the pH is reduced, the prepared functional mulberry leaf feed can remarkably improve the oxidation resistance and the immune function of livestock and poultry, improve the intestinal health of livestock and poultry, simultaneously can effectively improve the meat quality, improve the comprehensive economic benefit of livestock and poultry cultivation, and provide starter resources and an effective method for the application of mulberry leaves in animal production.

Drawings

In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the growth curves of 11 candidate strains in example 1 of the present invention, wherein A is the growth curve of 7 lactic acid bacteria and B is the growth curve of 4 Bacillus.

FIG. 2 is a pH change curve of 11 candidate strains in inventive example 1, wherein A is 7 strain lactic acid bacteria pH change curve and B is 4 strain Bacillus pH change curve.

FIG. 3 is a diagram showing the antibacterial ability of Pediococcus acidilactici (CGMCC No. 28922), pediococcus pentosaceus (CGMCC No. 28921) and Lactobacillus Agilis (LA) oxford in example 1 of the present invention, wherein SA6538 represents Staphylococcus aureus ATCC6538, SE10467 represents Salmonella enteritidis CICC10467, and K88 represents Escherichia coli K88.

FIG. 4 shows the enzyme productivity of 11 candidate strains in example 1, wherein A is the cellulase production capacity of 11 strains, and B is the protease production capacity of 11 strains.

FIG. 5 shows stress resistance of 11 strains in example 1, wherein A is acid resistance of 7 strains of lactic acid bacteria and B is acid resistance of 4 strains of Bacillus.

FIG. 6 shows stress resistance of 11 strains in example 1, wherein A is 7 strains of lactic acid bacteria and B is 4 strains of Bacillus.

FIG. 7 shows stress resistance of 11 strains in example 1, wherein A is bile salt tolerance of 7 strains of lactic acid bacteria and B is bile salt tolerance of 4 strains of Bacillus.

FIG. 8 shows the tolerance of 11 strains to artificial gastrointestinal fluids in example 1 of the present invention.

FIG. 9 shows the effect of different single strain fermentations on crude protein (A) and crude fiber (B) of functional mulberry leaf feed in example 2 of the present invention.

FIG. 10 shows the effect of fermentation of different single strains on pH (A) and lactic acid content (B) of functional mulberry leaf feed in example 2 of the present invention.

FIG. 11 shows the effect of different single strain fermentations on acetic acid content (A) and lactic acid bacteria number (B) of functional mulberry leaf feed in example 2 of the present invention.

FIG. 12 shows the effect of the combined fermentation of different strains on the crude protein, crude fiber, pH, lactic acid content, acetic acid content and lactic acid bacteria amount of the functional mulberry leaf feed in example 3 of the present invention.

FIG. 13 shows the effect of different inoculum size fermentations on crude protein, crude fiber, pH, lactic acid content, acetic acid content and lactic acid bacteria count of functional mulberry leaf feed in example 3 of the present invention.

FIG. 14 shows the effect of fermentation at different inoculation ratios on crude protein, crude fiber, pH, lactic acid content, acetic acid content and lactic acid bacteria amount of functional mulberry leaf feed in example 3 of the present invention.

FIG. 15 shows the effect of fermentation with different types of auxiliary materials on the crude protein, crude fiber, pH, lactic acid content, acetic acid content and lactic acid bacteria amount of the functional mulberry leaf feed in example 3 of the present invention.

FIG. 16 shows the effect of different amounts of bran added in fermentation on the crude protein, crude fiber, pH, lactic acid content, acetic acid content and lactic acid bacteria amount of the functional mulberry leaf feed in example 3 of the present invention.

FIG. 17 shows the effect of fermentation on crude protein, crude fiber, pH, lactic acid content, acetic acid content and lactic acid bacteria amount of functional mulberry leaf feed under different temperature conditions in example 3 of the present invention.

FIG. 18 shows the effect of different fermentation times on the crude protein, crude fiber, pH, lactic acid content, acetic acid content and lactic acid bacteria amount of the functional mulberry leaf feed in example 3 of the present invention.

FIG. 19 shows the effect of the functional mulberry leaf feed on the antioxidant capacity of swine muscle in example 7 of the present invention.

FIG. 20 shows the effect of the functional mulberry leaf feed of example 7 of the present invention on the antioxidant capacity of pig serum.

FIGS. 21 and 22 show the effect of the functional mulberry leaf feed of example 7 of the present invention on the immune performance of pigs.

FIG. 23 shows the effect of the functional mulberry leaf feed of example 8 of the present invention on short chain fatty acid content in the cecum of pigs.

In the above drawings, CGMCC28922 is Pediococcus acidilactici CGMCC No. 28922, CGMCC28921 is Pediococcus pentosaceus CGMCC No. 28921, CGMCC28923 is Bacillus subtilis CGMCC No. 28923, CGMCC28924 is Bacillus licheniformis CGMCC No. 28924, LS is Lactobacillus salivarius, LG is Lactobacillus gasseri, LA is Acinetobacter, LR4 is Lactobacillus reuteri, LR9 is Lactobacillus reuteri, BS is Bacillus subtilis, and BC is Bacillus coagulans.

Detailed Description

Firstly researching biological characteristics of candidate strains, preparing functional mulberry leaf feed by utilizing single strain fermentation, selecting strains with better fermentation quality from the strains according to fermentation results, carrying out combination screening on the strains obtained by screening, and determining optimal fermentation strain combination; and then taking the optimal fermentation strain combination as a starter, determining the optimal fermentation condition through fermentation condition optimization, and obtaining a method for preparing the functional mulberry leaf feed by multi-strain synergistic fermentation and the functional mulberry leaf feed prepared by the method, and being practically applied to the pig breeding process.

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

EXAMPLE 1 analysis of biological Properties of candidate strains

1. Material

1.1 Candidate strains

Pediococcus acidilactici (CGMCC No. 28922), pediococcus pentosaceus (CGMCC No. 28921), lactobacillus Gasseri (LG), lactobacillus Salivarius (LS), lactobacillus Agilus (LA), lactobacillus reuteri (LR 4), lactobacillus reuteri (LR 9), bacillus subtilis (CGMCC No. 28923), bacillus licheniformis (CGMCC No. 28924), bacillus Coagulans (BC) and Bacillus Subtilis (BS) are all screened and stored in the earlier stage of the laboratory of the inventor.

1.2 Indicator strain

Coli (EHEC O157: H7, K88, K99), salmonella (CVCC 519, CICC 10467, ATCC 14028), staphylococcus aureus (ATCC 43300, ATCC 6538, ATCC 25923) and Listeria monocytogenes (ATCC 19115) were all kept in the laboratory of the inventors and were purchased commercially.

1.3 Main culture medium

MRS medium: soybean peptone 10 g, yeast extract 5g, beef extract 10 g, triammonium citrate 2g, sodium acetate 5g, glucose 20g, dipotassium hydrogen phosphate 2g, tween 80 1.0 mL, salt solution 5.0 mL, adding deionized water to dissolve to 1000 mL, sterilizing 15-20 g (solid culture medium) of agar, and sterilizing 20min at 121 ℃.

Salt solution: magnesium sulfate heptahydrate 11.5 g, manganese sulfate monohydrate 2.8 g, and deionized water are added to dissolve the mixture to a volume of 100 mL.

LB medium: tryptone 10 g, yeast extract 5g, sodium chloride 10 g, deionized water to 1000 mL, agar 15-20 g (solid medium) and sterilizing at 121 ℃ 20min.

BPY medium: beef extract 5g, tryptone 10g, yeast extract 5g, sodium chloride 5g and glucose 5g, adding deionized water to dissolve to volume of 1000 mL, agar 15-20 g (solid medium), and sterilizing at 121deg.C 20 min.

2. Method of

2.1 Strain activation

And streaking the candidate strain stored at the temperature of minus 80 ℃ on a solid culture medium plate, culturing for 24-48 hours at the temperature of 37 ℃, picking a single bacterial drop into a liquid culture medium, culturing for 12-24 hours at the temperature of 37 ℃ and 180 rpm, and activating for two generations for later use.

2.2 Growth curve

Inoculating the activated strain into 100 mL corresponding liquid culture medium according to 2% inoculum size, culturing 7 strains of lactobacillus for 48 h at 37 ℃ under 180 rpm, culturing 4 strains of bacillus for 36 h, sampling every 2h, measuring bacterial liquid OD ₆₀₀, and drawing a growth curve by taking time as an abscissa and OD ₆₀₀ as an ordinate.

2.3 PH change curve

Inoculating the activated strain into a corresponding liquid culture medium of 100 mL at an inoculum size of 2%, culturing 7 strains of lactic acid bacteria for 48 h at 37 ℃ and 180: 180 rpm, culturing 4 strains of bacillus for 36 h, sampling every 2: 2h, measuring the pH of the bacterial liquid, and drawing a pH change curve by taking time as an abscissa and pH as an ordinate.

2.4 Bacteriostatic ability

2.4.1 Indicating strain activation

And (3) selecting the indication strains by using an inoculating loop, respectively scribing on an LB solid culture medium, culturing for 24-48 hours at 37 ℃ in an inverted mode, selecting single bacterial colonies, inoculating the single bacterial colonies into an LB liquid culture medium, culturing for 5-12 hours at 37 ℃ at 180 rpm, and adjusting the concentration of the bacterial suspension of the indication strains to be 1 multiplied by 10 ⁷ CFU/mL.

2.4.2 Preparation of candidate Strain supernatant

Inoculating seed solution of 11 candidate strains into a corresponding liquid culture medium according to an inoculum size of 2% (v/v), culturing at 37 ℃ and 180 rpm for 12-24 hours, collecting bacterial liquid, centrifuging at 4 ℃ and 8000 rpm for 20 and min to obtain bacterial supernatant, filtering by a 0.45 mu m filter membrane, and preserving at 4 ℃ for later use.

2.4.3 Oxford cup method for measuring bacteriostatic ability of candidate strain

Pouring about 15 mL nutrient agar culture medium into a culture dish, horizontally placing, standing until the nutrient agar culture medium is solidified, inoculating 100 mu L of indicator bacteria suspension, uniformly coating, vertically placing an oxford cup on the surface of the culture medium, adding 200 mu L of candidate strain supernatant into the oxford cup, taking a blank culture medium as a reference, setting 3 repetitions each group, standing at room temperature for 2h, culturing at 37 ℃ for 24 h, measuring the diameter of a bacteriostasis ring, and judging the bacteriostasis performance of the strain according to the diameter of the bacteriostasis ring.

2.5 Enzyme production ability

2.5.1 Measurement of cellulase Activity

Cellulase activity was measured by the 3, 5-dinitrosalicylic acid (DNS) chromogenic method with reference to NY/T912-2020.

2.5.2 Determination of protease Activity

Protease activity was determined with reference to GB/T23527.1-2023 using Fu Lin Fenfa.

2.6 Stress resistance

2.6.1 Acid tolerance test

The pH of the liquid culture medium was adjusted to 2.5, 3.5, 4.5, 5.5, 6.5 and 7.0 by using 1 mol/L HCl, the activated candidate strain fermentation broth was inoculated into the liquid culture medium of the corresponding strain with different pH at an inoculum size of 2%, 24 h was cultured at 37℃and 180 rpm, and the OD ₆₀₀ of the bacterial broth was measured, with the blank medium as a control.

2.6.2 Temperature tolerance test

The activated candidate strain was inoculated into the corresponding strain liquid medium at an inoculum size of 2%, and cultured at 30, 37, 40, 45 ℃ and 180 rpm for 24h, respectively, and the OD ₆₀₀ of the bacterial liquid was measured.

2.6.3 Bile salt tolerance test

The activated candidate strain was inoculated in 2% inoculum size into corresponding liquid culture medium with bile salt concentration of 0.1%, 0.3%, 0.5% and 1%, respectively, and cultured at 37℃and 180 rpm for 24 h, and OD ₆₀₀ of the bacterial liquid was measured by using the blank culture medium as a control.

2.6.4 Simulated gastrointestinal tolerability test

Preparing artificial gastric juice: adjusting the pH value of the sterile physiological saline solution to 2.0 by using hydrochloric acid with the volume fraction of 9.5% -10.5%, adding 2.5 g/L pepsin, fully dissolving, and filtering and sterilizing by using a sterile filter membrane with the volume fraction of 0.22 mu m.

Preparing artificial intestinal juice: dissolving 3.4 g potassium dihydrogen phosphate in 250 mL sterile physiological saline solution, adjusting pH to 6.8 with 0.4% sodium hydroxide solution, adding 1g trypsin into each 100 mL liquid, mixing, and filtering with 0.22 μm sterile filter membrane for sterilization.

Mixing 500 μl of bacterial liquid of the activated candidate strain with 4.5 mL artificial gastric juice in 5mL shake tube, culturing at 37deg.C and 180 rpm for 4h, and counting viable bacteria of each strain; then, 500 mu L of mixed gastric juice cultured by 4h is inoculated into 4.5 mL artificial intestinal juice, 4h is cultured under the conditions of 37 ℃ and 180 rpm, viable count is carried out on each strain, and the survival rate is calculated according to the following formula:

survival rate (%) = (number of viable bacteria of gastric juice or intestinal juice culture 4 h/number of viable bacteria of 0 h) ×100%.

3. Results

3.1 Strain growth curve

As can be seen from FIG. 1, 7 strains of lactobacillus enter the logarithmic phase after culturing 2h, and strains CGMCC No. 28922, CGMCC No. 28921, LS and LR4 enter the stationary phase after culturing 12 h; strain LA and LR9 are cultured for 15 h and then enter a stable period; the strain LG has a longer growth period, and the logarithmic phase is 2-18 h. The 4 bacillus strains enter the logarithmic phase after 2h of the bacillus strains are cultivated, and the bacterial strain BS enters the stationary phase after 15 h of the bacillus strains are cultivated; the strain CGMCC No. 28923, CGMCC No. 28924 and BC is cultured to 18 and h and then enters a stable period.

3.2 Acid producing characteristics of the strain

As can be seen from FIG. 2, the strains CGMCC No. 28922, CGMCC No. 28921, LG and LA have fast pH falling within 2-8 hours, the strain LS has fast pH falling within 2-6 hours, the strains LR4 and LR9 have fast pH falling within 2-15 hours, and the strains LR4 and LR9 have slow pH falling within 15-48 hours. The pH of the 4 strains of bacillus was minimized at 4h and remained on an upward trend after 4: 4 h.

3.3 Bacterial strain bacteriostatic ability

As can be seen from FIG. 3 and Table 1, the bacterial strains CGMCC No. 28922, CGMCC No. 28921 and LA have a bacteriostasis ring diameter of more than 12 mm for all indicator bacteria and have broad-spectrum bacteriostasis effects for Escherichia coli, salmonella, staphylococcus aureus and listeria; the diameters of the inhibition zones of other lactic acid bacteria on all indicator bacteria are larger than 7 mm, and the bacteria have certain inhibition capacity; the diameter of the bacteriostasis circle of the bacillus BC to individual pathogenic bacteria is larger than 7 mm, the bacillus BC has a certain bacteriostasis effect, and other bacillus has no bacteriostasis effect.

TABLE 1 diameter of zone of inhibition (mm) for candidate strains

Note that: the diameter of the inhibition zone "-" indicates < 7mm (no bacteriostatic activity); "+" indicates 8-12 mm; "++" means 12-16 mm; "+". ++'s representation of 16-20 mm; "+". ++ + "means >20 mm; the oxford cup had a diameter of 7 mm.

3.4 Enzyme production ability of the strain

As can be seen from fig. 4, the cellulase production capacity of the strains BS and BC is significantly higher than that of other strains (P < 0.05), while the cellulase production capacity of the strain CGMCC No. 28923 is significantly higher than that of CGMCC No. 28924 and 7 lactic acid bacteria (P < 0.05). The capacity of the strain CGMCC No. 28923 for producing protease is obviously higher than that of other strains (excluding CGMCC No. 28924) (P < 0.05), the capacity of the strain CGMCC No. 28924 for producing protease is obviously higher than that of the strains CGMCC No. 28922, LS, LA and LR4 (P < 0.05), and no obvious difference exists among other strains (P < 0.05).

3.5 Stress resistance of strains

3.5.1 Acid tolerance

As shown in FIG. 5, when the pH of the strains CGMCC No. 28922, CGMCC No. 28921 and LA is less than 3.5, the growth is inhibited and basically no growth occurs, and the other 4 strains have stronger acid tolerance and can grow; in the pH range of 3.5-7.0, the growth capacity of 7 strains of lactic acid bacteria is gradually enhanced along with the increase of the pH. When the pH is less than 3.5, the growth of 4 strains of bacillus is inhibited, when the pH of the strain CGMCC No. 28924 is less than 4.5, the growth is still inhibited, and the growth condition of 4 strains of bacillus is better within the pH range of 5.5-7.0.

3.5.2 Temperature tolerance

As can be seen from FIG. 6, when the temperature is higher than 37 ℃, the OD ₆₀₀ of 7 strains of lactic acid bacteria gradually decreases, and the growth is inhibited; when the temperature is higher than 30 ℃, the bacterial strains CGMCC No. 28923, CGMCC No. 28924 and the OD ₆₀₀ of the BS are in a descending trend, and the bacterial strain BC can grow well in the temperature range of 30-45 ℃.

3.5.3 Bile salt tolerance

As is clear from FIG. 7, when the bile salt concentration was 0.3%, the growth of 7 lactic acid bacteria was best, and the growth was inhibited at both too low and too high bile salt concentrations. The growth of bacillus BC is inhibited when the concentration of bile salts is more than 0.3%, and the growth of other 3 strains of bacillus BC is inhibited when the concentration of bile salts is more than 0.5%.

3.5.4 Resistance to artificial gastrointestinal fluids

As shown in FIG. 8, after 11 strains are treated by the artificial gastrointestinal fluid, the survival rates of the strains CGMCC No. 28922, CGMCC No. 28921, CGMCC No. 28923 and CGMCC No. 28924 can reach more than 80%, and the survival rates of other strains can reach more than 70%.

The biological material preservation information of the strains obtained by screening is as follows:

Pediococcus acidilactici A62 is preserved in China general microbiological culture collection center (CGMCC) at 11 and 9 days of 2023, and is classified and named as Pediococcus acidilactici Pediococcus acidilactici with a preservation number of CGMCC No. 28922, wherein the CGMCC is address: north Star, west way 1, china academy of sciences, and a microbiological study, post code 100101).

Pediococcus pentosaceus A7 is preserved in China general microbiological culture collection center (CGMCC) at 11 and 9 of 2023, and has a classification of Pediococcus pentosaceus Pediococcus pentosaceus and a preservation number of CGMCC No. 28921, wherein the CGMCC is called by the classification of Pediococcus pentosaceus No. 3, the institute of microbiology, national academy of sciences, and the Korean area North Star, the West road No. 1, of Beijing.

The bacillus subtilis KO1 is preserved in China general microbiological culture collection center (CGMCC) at 11 and 9 days of 2023, and is classified and named as bacillus subtilis Bacillus subtilis with a preservation number of CGMCC No. 28923, wherein the CGMCC is called by the Beijing Kogyo area North Star, the Xiya institute of China academy of sciences, and the post code is called 100101.

Bacillus licheniformis DO1 is preserved in China general microbiological culture Collection center (CGMCC) at 11 and 9 days of 2023, and is classified and named as Bacillus licheniformis Bacillus licheniformis with a preservation number of CGMCC No. 28924, wherein the CGMCC is called by the Beijing Kogyo area North Star, west Liu No. 1, institute of microbiology, and mail code 100101).

Example 2 preparation of functional Mulberry leaf feed by Single fermentation

1. Method of

Streaking 7 candidate lactic acid bacteria in the embodiment 1 on an MRS solid culture medium plate, culturing for 24-48 h at 37 ℃, selecting a single colony, inoculating the single colony into an MRS liquid culture medium, culturing for 12-24 h at 37 ℃, activating for two generations to obtain an activated strain, inoculating the activated strain into a 5mL MRS culture solution, standing and culturing for 12-15 h at 37 ℃ to obtain a primary seed solution, inoculating the primary seed solution into the MRS liquid culture medium at 2 percent of inoculation amount, and culturing for 12-15 h at 37 ℃ to obtain a secondary seed solution (the number of viable bacteria is greater than 10 ⁸ CFU/mL); streaking 4 candidate bacillus strains in the embodiment 1 on a BPY solid culture medium plate, culturing for 12-24 hours at 37 ℃, picking a single bacterial colony, inoculating the single bacterial colony into a BPY liquid culture medium, culturing for 12-24 hours at 37 ℃ and 180 rpm, and activating for two generations to obtain an activated bacterial strain; inoculating the activated strain into a culture solution of 5mL BPY, culturing at 37 ℃ for 12-18 h at 180: 180 rpm (the number of viable bacteria is greater than 10 ⁸ CFU/mL), obtaining a primary seed solution, inoculating the primary seed solution into a BPY liquid culture medium at an inoculum size of 2%, and culturing at 37 ℃ for 12-18 h at 180: 180 rpm (the number of viable bacteria is greater than 10 ⁸ CFU/mL), thus obtaining a secondary seed solution.

Cutting fresh mulberry leaves to be within 3 cm percent or pulping, adding 10 percent (w/w) of bran into the mulberry leaves to obtain fermentation base materials, inoculating secondary seed liquid of each candidate strain into the fermentation base materials according to the inoculum size of 5 percent (v/w), fully mixing, filling a sample about 200 g into a feed fermentation bag (23 cm multiplied by 30 cm), sealing and fermenting for 7d, wherein the fermentation temperature is 37 ℃, and setting three groups of tests in parallel.

2. Results

The fermentation detection indexes mainly comprise: crude protein, crude fiber, pH, lactic acid content, acetic acid content, microbial count (lactic acid bacteria), and the like.

As can be seen from FIG. 9, FIG. 10 and FIG. 11, the crude protein content after fermenting folium Mori with CGMCC No. 28922, CGMCC No. 28921, LS and LG and CGMCC No. 28923 is significantly higher than that of CON group (P < 0.05). The crude fiber content of the bacillus BC and BS after fermenting the mulberry leaves is obviously lower than that of CON group, strain CGMCC No. 28921 and LS (P < 0.05). The pH of the lactobacillus CGMCC No. 28922 and CGMCC No. 28921 fermentation groups is obviously lower than that of other groups (P < 0.05). The lactic acid content of the lactobacillus CGMCC No. 28922 and CGMCC No. 28921 fermentation groups is obviously higher than that of other groups (P < 0.05), and the lactic acid content of the bacillus CGMCC No. 28924 fermentation groups is obviously higher than that of the lactobacillus LS, LR4 and bacillus BC and BS fermentation groups (P < 0.05). The acetic acid content of the lactobacillus LG, LA and LR4 fermentation groups is obviously higher than that of the CON group, the lactobacillus CGMCC No. 28922, the lactobacillus CGMCC No. 28921 and other 4 bacillus fermentation groups (P < 0.05). The number of lactic acid bacteria in all lactic acid bacteria fermented groups was significantly higher than in the CON group and the bacillus fermented group (P < 0.05). And (3) combining the results, selecting lactobacillus CGMCC No. 28922, CGMCC No. 28921, LS, LA and LR9 and bacillus CGMCC No. 28923 and CGMCC No. 28924 as subsequent fermentation strains to perform multi-bacteria collaborative fermentation.

Example 3 preparation of functional Mulberry leaf feed by Multi-bacteria synergistic fermentation

According to the result of preparing the functional mulberry leaf feed by single-bacteria fermentation, pediococcus acidilactici CGMCC No. 28922, pediococcus pentosaceus CGMCC No. 28921, lactobacillus salivarius LS, lactobacillus agili LA, lactobacillus reuteri LR9, bacillus subtilis CGMCC No. 28923 and bacillus licheniformis CGMCC No. 28924 are selected for multi-bacteria synergistic fermentation to prepare the functional mulberry leaf feed.

1. Different candidate strain combinations: the fermentation condition is that the inoculation amount is 5%, fresh mulberry leaves are cut to be within 3 cm or pulped, 10% (w/w) bran is added into the mulberry leaves to be used as a fermentation base material, the fermentation time is 7 d, the fermentation temperature is 37 ℃, 7 strains of Pediococcus acidilactici CGMCC No. 28922, pediococcus pentosaceus CGMCC No. 28921, lactobacillus salivarius LS, lactobacillus agilis LA, lactobacillus reuteri LR9, bacillus subtilis CGMCC No. 28923 and bacillus licheniformis CGMCC No. 28924 are selected for combination, and the combination of the strains comprises the following combination 1: pediococcus acidilactici, pediococcus pentosaceus, lactobacillus salivarius, lactobacillus agilis and Lactobacillus reuteri; combination 2: pediococcus acidilactici, pediococcus pentosaceus, bacillus subtilis and Bacillus licheniformis; combination 3: lactobacillus salivarius, lactobacillus agilus, lactobacillus reuteri, bacillus subtilis and bacillus licheniformis; combination 4: pediococcus acidilactici, pediococcus pentosaceus, lactobacillus salivarius, lactobacillus agilus, lactobacillus reuteri, bacillus subtilis and Bacillus licheniformis. From the results of fig. 12, the crude fiber content of the strain combination 2 treatment group was significantly lower than that of the strain combination 1 treatment group (P < 0.05), the pH was significantly lower than that of the strain combination 3 treatment group (P < 0.05), and the lactic acid content was significantly higher than that of the other treatment groups (P < 0.05). Combining the results to determine the optimal fermentation strain combination as combination 2: pediococcus acidilactici CGMCC No. 28922 +Pediococcus pentosaceus CGMCC No. 28921 +Bacillus subtilis CGMCC No. 28923 +Bacillus licheniformis CGMCC No. 28924.

2. Inoculation amount: the fermentation condition is 10% of the addition amount of bran, the fermentation time is 7 d, the fermentation temperature is 37 ℃, and the combination of strains is the optimal combination determined by the screening: pediococcus acidilactici, pediococcus pentosaceus, bacillus subtilis and Bacillus licheniformis (1:1:3:3), and fermenting at inoculum size of 2%, 4%, 5%, 6% and 8%, respectively. From the results of fig. 13, it can be seen that the pH of the 8% inoculum size treated group was significantly lower than that of the 2% and 5% inoculum size treated groups (P < 0.05), the lactic acid content of the 8% inoculum size treated group was significantly higher than that of the 2% and 5% inoculum size treated groups (P < 0.05), and the lactic acid bacteria count of the 8% inoculum size treated group was significantly higher than that of the other groups (P < 0.05), whereby the optimal inoculum size was determined to be 8%.

3. Inoculation ratio: the fermentation condition is that the addition amount of bran is 10%, the fermentation time is 7 d, the fermentation temperature is 37 ℃, the bacterial strain combination is Pediococcus acidilactici, pediococcus pentosaceus, bacillus subtilis and bacillus licheniformis, the inoculation amount is 8%, and the fermentation is carried out after inoculation according to the effective viable count ratio of Pediococcus acidilactici, pediococcus pentosaceus, bacillus subtilis and bacillus licheniformis of 1:1:3:3, 3:1:3:3, 1:3:3:1:1:10:3 and 1:1:3:10 respectively. As can be seen from the results of FIG. 14, the crude fiber content of the treated groups (Pediococcus acidilactici, pediococcus pentosaceus, bacillus subtilis and Bacillus licheniformis inoculated with viable bacteria at 3X 10 ⁹、3×10⁹、1×10¹⁰、1×10¹⁰ CFU/kg fermentation substrate) with an effective viable bacteria ratio of 1:1:3:3 was significantly lower than that of the treated groups (P < 0.05) with an inoculation ratio of 3:1:3:3 and 1:3:3, the pH was significantly lower than that of the treated groups (P < 0.05) with an inoculation ratio of 1:3:3:3, and the lactic acid content was significantly higher than that of the treated groups (P < 0.05) with an inoculation ratio of 1:3:3:3, whereby the optimal inoculation ratio of Pediococcus acidilactici, pediococcus pentosaceus, bacillus subtilis and Bacillus licheniformis was obtained with an effective viable bacteria ratio of 1:1:1:3:3.

4. Auxiliary material types: the optimized strain is utilized to combine pediococcus acidilactici, pediococcus pentosaceus, bacillus subtilis and bacillus licheniformis, the inoculation amount is 8%, the effective viable count ratio of inoculation is 1:1:3:3, the fermentation time is 7 d, the fermentation temperature is 37 ℃, and the auxiliary materials such as bran, corn meal, molasses, bran and corn meal are screened. From the results of fig. 15, it can be seen that the lactic acid content of the bran-added treatment group was significantly higher than that of the bran-added and corn flour treatment groups (P < 0.05), while the lactic acid bacteria count of the bran-added treatment group was significantly higher than that of the other groups (P < 0.05), thereby selecting bran as an adjunct.

5. Bran addition amount: and (3) screening 5%, 10% and 15% of bran addition by utilizing the optimized strain combination pediococcus acidilactici, pediococcus pentosaceus, bacillus subtilis and bacillus licheniformis with an inoculum size of 8%, an effective viable count ratio of 1:1:3:3 and a fermentation time of 7 d at a fermentation temperature of 37 ℃. From the results of fig. 16, it can be seen that the crude protein content of the 10% bran-added treatment group was significantly higher than that of the 15% bran-added treatment group (P < 0.05), and the pH of the 10% bran-added treatment group was significantly lower than that of the 15% bran-added treatment group (P < 0.05), whereby the optimum addition amount of bran was 10%.

6. Fermentation temperature: the optimized strain is utilized to combine Pediococcus acidilactici, pediococcus pentosaceus, bacillus subtilis and Bacillus licheniformis, the inoculation amount is 8%, the ratio of the effective viable bacteria to the inoculation amount is 1:1:3:3, the bran addition amount is 10%, and the fermentation temperature is respectively set at 23 ℃,37 ℃ and 42 ℃ for fermentation. From the results of fig. 17, it was found that the number of lactic acid bacteria in the 37 ℃ treatment group was significantly higher than that in the 42 ℃ treatment group (P < 0.05), whereby the optimum fermentation temperature was 37 ℃.

7. Fermentation time: the optimized strain is utilized to combine pediococcus acidilactici, pediococcus pentosaceus, bacillus subtilis and bacillus licheniformis, the inoculation amount is 8%, the ratio of the effective viable bacteria to the inoculation amount is 1:1:3:3, the bran addition amount is 10%, the fermentation temperature is 37 ℃, and the fermentation time is respectively set to 3 d, 7 d, 14 d, 30 d and 60 d. As can be seen from the results of fig. 18, the pH of the 14 d, 30 d and 60 d treatment groups is significantly lower than that of the 3 d and 7 d treatment groups (P < 0.05), the lactic acid content of the 14 d treatment group is significantly higher than that of the 3 d treatment group (P < 0.05), the lactic acid bacteria count of the 3 d and 7 d treatment groups is significantly higher than that of the other groups (P < 0.05), and the fermentation time is preferably 7-30 d by combining the indexes.

Example 4 preparation of Pediococcus acidilactici preparation, pediococcus pentosaceus preparation, bacillus subtilis preparation, bacillus licheniformis preparation and Compound microorganism preparation

1. Preparation of Pediococcus acidilactici preparation

Inoculating activated pediococcus acidilactici CGMCC No. 28922 into an MRS culture medium, culturing at 37 ℃ for 12-18 h, then inoculating 5-10% (v/v) of the pediococcus acidilactici into a fermentation culture medium, placing the fermentation culture medium into a fermentation tank, and culturing at 60-rpm and 37 ℃ for 18-24 h to obtain pediococcus acidilactici CGMCC No. 28922 fermentation liquor (liquid).

Wherein the fermentation medium formula of Pediococcus acidilactici is: molasses 5%, soybean meal 2%, ammonium sulfate 0.1%, crystalline sodium acetate 0.1%, dipotassium hydrogen phosphate 0.5%, anhydrous magnesium sulfate 0.02%, manganese sulfate 0.005%, tween-80.1% (v/v), ph=6.5±0.2.

Uniformly mixing the fermentation liquor of Pediococcus acidilactici CGMCC No. 28922 with the carrier according to the weight ratio of the fermentation liquor to the carrier of 0.5-0.8:1, and then drying and crushing at low temperature to obtain the Pediococcus acidilactici CGMCC No. 28922 dry powder. The carrier can be one or more of bran, rice hull powder, secondary powder, corncob powder and corn gluten meal, and in the embodiment, the carrier is bran.

2. Preparation of Pediococcus pentosaceus preparation

The preparation method of the pediococcus pentosaceus preparation is the same as that of the pediococcus acidilactici preparation, and the strain used is pediococcus pentosaceus CGMCC No. 28921.

3. Preparation of bacillus subtilis preparation

Inoculating the activated bacillus subtilis CGMCC No. 28923 into a BPY culture medium, culturing at a constant temperature of 37 ℃ for 16: 16 h, then inoculating into a fermentation tank according to an inoculum size of 5% -10%, and culturing at 37 ℃ for 24-36 h to obtain bacillus subtilis CGMCC No. 28923 fermentation liquor (liquid).

The formula of the bacillus subtilis fermentation medium is as follows: glucose 0.5%, soybean meal 3%, corn flour 3%, yeast extract powder 0.4%, manganese sulfate 0.03%, and ph=7.0±0.2.

Uniformly mixing the bacillus subtilis CGMCC No. 28923 fermentation liquor and a carrier according to the weight ratio of 0.5-0.8:1, drying and crushing to obtain the bacillus subtilis CGMCC No. 28923 dry powder. The carrier can be one or more of bran, rice hull powder, secondary powder, corncob powder and corn gluten meal, and in the embodiment, the carrier is bran.

4. Preparation of Bacillus licheniformis preparation

The preparation method of the bacillus licheniformis preparation is the same as that of the bacillus subtilis preparation, and the strain used is bacillus licheniformis CGMCC No. 28924.

5. Preparation of composite microbial preparation

The pediococcus acidilactici preparation, the pediococcus pentosaceus preparation, the bacillus subtilis preparation and the bacillus licheniformis preparation are mixed according to different proportions to prepare a compound liquid preparation or dry powder.

Example 5 application of functional Mulberry leaf feed in maintaining pig growth Property, improving economic benefits and nutrient apparent digestibility

1. Materials and methods

The preparation method of the functional mulberry leaf feed comprises the following steps: cutting fresh mulberry leaves to be within 3 cm or pulping, and adding 10% (w/w) bran into the mulberry leaves to obtain a fermentation base material; mixing fermentation liquor of Pediococcus acidilactici CGMCC No. 28922, pediococcus pentosaceus CGMCC No. 28921, bacillus subtilis CGMCC No. 28923 and Bacillus licheniformis CGMCC No. 28924 according to an effective viable count of 1:1:3:3 to obtain a fermentation agent, inoculating the fermentation agent into the fermentation base material according to an inoculation amount of 8% (v/w) (the inoculation viable count of Pediococcus acidilactici, pediococcus pentosaceus, bacillus subtilis and Bacillus licheniformis is 3× ⁹、3×10⁹、1×10¹⁰、1×10¹⁰ CFU/kg fermentation base material respectively), fully mixing, filling into a feed fermentation bag, sealing and fermenting by a sealing machine, wherein the fermentation time is 7 d, and the fermentation temperature is 37 ℃.

96 Ternary hybrid fattening pigs (Changbai X Da Baidu Rock) with initial weight of 83 kg or so are randomly divided into 4 groups of 4 replicates, and 6 pigs are replicated. The control group (CON) was fed with basal diet, group i trial (FML 4), group ii trial (FML 8) and group iii trial (FML 12), and the basal diet was fed with diet with 4%, 8% and 12% functional mulberry leaf diet, respectively, instead of part of corn, soybean meal and bran. The test period was 35 d at 08 per day: 00 and 17: and feeding in equal amount twice at 00 times, and drinking water freely. The basic diet is prepared according to the nutrient requirement of growing-finishing pigs recommended by NY/T65-2004. The diet composition and nutrient levels are shown in table 2. Pig killing samples were taken for analysis 35 days after feeding.

The test data were subjected to ANOVA analysis using SPSS 26.0 and multiple comparisons were made using Tukey and Dunnett's methods. Drawing was performed with GraphPad 8.0 software. The test data are expressed as averages, P < 0.05 indicates significant differences, and P > 0.05 is considered no significant differences.

Table 2 test diet composition and nutrient level (dry matter basis)

Note that: ① Each kilogram of premix is provided to the ration: iron 1500.00 mg, zinc 1075.00 mg, manganese 400.00 mg, copper 75.00 mg, selenium 3.00 mg, iodine 10.00 mg, vitamin a 130 KIU, vitamin D ₃ 30 KIU, vitamin E300.00 mg, vitamin K ₃ 40.00.00 mg, vitamin B ₁ (thiamine) 25.00 mg, vitamin B ₂ (riboflavin) 75.00 mg, vitamin B ₆ 35.00.00 mg, vitamin B ₁₂ 0.25 mg, nicotinamide 475.00 mg, biotin 1.00 mg, folic acid 14.00 mg, pantothenic acid 280.00 mg, choline chloride 5000.00 mg, calcium 10%, total phosphorus 2.00%, sodium chloride 6%, lysine 3.0%, phytase 12500U, moisture 10%.

② The calculated values are the digestive energy and the measured values are the other nutritional components.

2. Results

2.1 Influence of functional mulberry leaf feed on pig growth performance

The effect of functional mulberry leaf feed on pig growth performance during the test period is shown in table 3. On days 1-20, there was no significant difference (P > 0.05) between the medium body weight, average daily gain and the feed weight ratio of FML4 group, FML8 group and CON group. There was no significant difference (P > 0.05) between the end body weights, average daily gain and the weight-to-charge ratios of FML4, FML8 and CON groups on days 21-35. During the whole test period (1-35 d), the average daily gain and the feed-weight ratio of the FML4 group, the FML8 group and the CON group are not obviously different (P is more than 0.05). Thus, the growth performance of pigs in FML4 and FML8 groups was not affected compared to CON groups.

TABLE 3 Effect of functional Mulberry leaf feed on pig growth Properties

Note that: the same row of data shoulder letters are different to indicate that the difference is significant (P < 0.05), and the same or no label indicates that the difference is not significant (P > 0.05).

2.2 Influence of functional mulberry leaf feed on pig economic benefit

The economic benefit analysis is shown in Table 4. The gross profit of the FML8 group is higher than that of the CON group, and is improved by 19.55 percent compared with the CON group; the feed cost of CON group, FML4 group, FML8 group and FML12 group is 10.17, 10.43, 9.00 and 9.78 yuan for every 1kg live pig, which shows that adding 8% of functional mulberry leaves, namely FML8 group, can obviously improve economic benefit.

TABLE 4 influence of functional Mulberry leaf feed on economic benefits of fattening pigs

2.3 Influence of functional mulberry leaf feed on apparent digestibility of pig nutrients

The effect of functional mulberry feed on apparent digestibility of pig nutrients is shown in table 5. There was no significant difference between the apparent digestibility of crude fat in FML8, FML12 and CON groups (P > 0.05), and the apparent digestibility of crude fiber in FML4, FML8 and FML12 groups was significantly higher than that in CON group (P < 0.05).

TABLE 5 Effect of functional Mulberry leaf feed on apparent digestibility of pig nutrients

Example 6 application of functional Mulberry leaf feed to improving pork quality and increasing muscle fatty acid and amino acid content

1. Materials and methods

The preparation of the functional mulberry leaf feed is the same as in example 5.

The feed, management, feeding, etc. of the test pigs were the same as in example 5.

The data analysis method was the same as in example 5.

2. Results

2.1 Influence of functional mulberry leaf feed on pork quality

The effect of functional mulberry leaf feed on pork quality is shown in table 6. Muscle pH _24h was significantly higher in FML4, FML8 and FML12 groups than in CON group (P < 0.05), flesh color scores were significantly higher in FML4 and FML8 groups than in CON group (P < 0.05), marbling scores were significantly higher in FML8 and FML12 groups than in CON group (P < 0.05), muscle moisture content was significantly higher in FML4 and FML8 groups than in CON group (P < 0.05), and muscle crude protein content was significantly higher in FML4 and FML8 groups than in CON group (P < 0.05), muscle intramuscular fat content was significantly higher in FML12 groups than in other groups (P < 0.05) as the functional mulberry leaf feed addition increased. The results show that the functional mulberry leaf feed is added into animal feed, and the added amount of the functional mulberry leaf feed is increased, and especially the FML8 group and the FML12 group can improve the pH _24h of muscles, the flesh color, the marbling, the crude protein and the intramuscular fat content, increase the tenderness of muscles and improve the meat quality.

TABLE 6 influence of functional Mulberry leaf feed on pork quality

2.2 Influence of functional mulberry leaf feed on fatty acid content of pig muscle

The effect of functional mulberry leaf feed on the fatty acid content of the pig muscle is shown in table 7. The total saturated fatty acid content of the FML12 group is significantly higher than that of the other three groups (P < 0.05), wherein SFA C14:0 of the FML4 group and the FML8 group is significantly lower than that of the CON group and the FML12 group (P < 0.05); MUFA C16:1 was significantly higher in the FML12 group than in the FML4 group (P < 0.05). The total polyunsaturated fatty acid content of the FML4 and FML8 groups was significantly higher than that of the CON and FML12 groups (P < 0.05), with the PUFAs C18:2n6c of the FML4 and FML8 groups being significantly higher than that of the FML12 groups (P < 0.05), the PUFAs C20:3n6 and C22:6n3 of the FML8 groups being significantly higher than those of the other three groups (P < 0.05), and the PUFAs C20:3n3 of the FML4 and FML8 groups being significantly higher than those of the CON and FML12 groups (P < 0.05). The PUFA/SFA ratios were significantly higher in the FML4 and FML8 groups than in the FML12 group (P < 0.05); n-3 PUFA of FML8 group was significantly higher than that of CON and FML12 groups (P < 0.05); n-6 PUFA was significantly higher in the FML4 and FML8 groups than in the CON and FML12 groups (P < 0.05). The above results indicate that FML8 group significantly reduced total saturated and monounsaturated fatty acid content, significantly increased polyunsaturated and functional fatty acid content and PUFA/SFA ratio.

TABLE 7 Effect of functional Mulberry leaf feed on the fatty acid content of pig muscle

2.3 Influence of functional mulberry leaf feed on amino acid content of pig muscle

The effect of functional mulberry leaf feed on the amino acid content of the pig muscle is shown in table 8. The total amino acid content of FML4 group was significantly higher than that of CON group and FML12 group (P < 0.05), the essential amino acid content of FML4 group and FML8 group was significantly higher than that of FML12 group (P < 0.05), and there was no significant difference from CON group (P > 0.05). The isoleucine content of FML8 group was significantly higher than that of CON and FML12 groups (P < 0.05), the histidine content of FML4 and FML8 groups was significantly higher than that of CON and FML12 groups (P < 0.05), while the aspartic acid and cystine content of FML4 and FML8 groups was significantly higher than that of FML12 groups (P < 0.05), and the tyrosine content of FML12 group was significantly higher than that of CON and FML4 groups (P < 0.05). From the results, the animal feed can be added with functional mulberry leaf feed, especially FML8 group can increase the content of essential amino acids in pig muscle.

TABLE 8 influence of functional mulberry leaf feed on the amino acid content of pig muscle

Example 7 application of functional Mulberry leaf feed in improving antioxidant capacity and immunity of pigs

1. Materials and methods

The preparation method of the functional mulberry leaf feed is the same as in example 5.

The data analysis method was the same as in example 5.

2. Results

2.1 Influence of functional mulberry leaf feed on pig serum biochemical index

The effect of the functional mulberry leaf feed on pig serum biochemical index is shown in table 9. The total protein content of the FML12 group is obviously higher than that of the FML4 group (P < 0.05), the albumin content of the FML12 group is obviously higher than that of the other three groups (P < 0.05), the urea nitrogen content of the FML4 group is obviously lower than that of the other three groups (P < 0.05), the glutamic pyruvic transaminase content of the FML4 group is obviously higher than that of the FML12 group (P < 0.05), and the indexes are not obviously different between the FML8 group and the CON group (P > 0.05).

TABLE 9 influence of functional Mulberry leaf feed on pig serum Biochemical index

2.2 Influence of functional mulberry leaf feed on oxidation resistance of pig muscle

The effect of functional mulberry leaf feed on the antioxidant capacity of pig muscle is shown in fig. 19. Glutathione peroxidase GSH-Px activity of FML4, FML8 and FML12 is significantly higher than that of CON group (P < 0.05), catalase CAT activity of FML8 group is significantly higher than that of CON group and FML12 group (P < 0.05), and total antioxidant capacity T-AOC, superoxide dismutase SOD activity and malondialdehyde MDA content of each group of muscle are not significantly different (P > 0.05). From this, it is clear that FML group 8 can enhance the antioxidant capacity of pig muscle.

2.3 Effect of functional Mulberry leaf feed on antioxidant Capacity of pig serum

The effect of functional mulberry leaf feed on the antioxidant capacity of pig serum is shown in figure 20. The total antioxidant capacity T-AOC of the FML4 group is obviously higher than that of the FML12 group (P is less than 0.05), the catalase CAT activity of the three test groups is obviously higher than that of the CON group (P is less than 0.05), and the serum superoxide dismutase SOD activity, the glutathione peroxidase GSH-Px activity and the malondialdehyde MDA content of the three test groups are not obviously different (P is more than 0.05). Therefore, the functional mulberry leaf feed can enhance the antioxidant capacity of the pig serum.

2.4 Influence of functional mulberry leaf feed on pig immunity

The effect of functional mulberry leaf feed on swine immune performance is shown in fig. 21 and 22. Serum IgG levels were significantly increased in FML8 compared to FML4 and FML12 (P < 0.05), serum IL-6 levels were significantly lower in FML12 than in CON and FML4 (P < 0.05), serum IL-8 levels were significantly lower in the test and FML4 and FML8 than in CON and FML12 (P < 0.05). Furthermore, there was no significant difference between the serum IgM, igA, IL-1 beta and TNF-alpha levels (P > 0.05) between the groups. Therefore, the pig immunity performance can be enhanced by adding the functional mulberry leaf feed.

Example 8 application of functional Mulberry leaf feed to improving intestinal morphology, intestinal short-chain fatty acid content and intestinal flora of pigs

1. Materials and methods

The data analysis method was the same as in example 5.

2. Results

2.1 Influence of functional mulberry leaf feed on intestinal morphology of small intestine of pig

The effect of the functional mulberry leaf feed on the intestinal morphology of the small intestine of pigs is shown in table 10. There was no significant difference (P > 0.05) between the duodenal, ileal villus height, crypt depth, and ileal villus to crypt ratio (VH/CD) groups, but all showed a trend of increasing villus height and decreasing crypt depth, whereas the duodenal villus to crypt ratio was significantly higher for the three test groups than for the CON group (P < 0.05). Therefore, the functional mulberry leaf feed group can be added to improve the intestinal morphology of pigs.

TABLE 10 influence of functional mulberry leaf feed on pig intestinal morphology

2.2 Influence of functional mulberry leaf feed on content of short-chain fatty acid in cecum of pigs

The effect of functional mulberry leaf feed on the short chain fatty acid content of the cecum of pigs is shown in figure 23. The acetic acid content and the total short-chain fatty acid content of the feed group added with the functional mulberry leaves are obviously higher than those of the CON group (P < 0.05), the butyric acid content of the FML4 group is obviously higher than those of the FML8 group and the FML12 group (P < 0.05), and the propionic acid content of each group is not obviously different (P > 0.05). From the results, the functional mulberry leaf feed can obviously improve the total short-chain fatty acid content of the cecum of pigs.

In addition, the effect of the functional mulberry leaf feed on the pig intestinal flora is analyzed, and the result shows that the test group can change the pig intestinal flora diversity, and the FML8 group obviously improves the relative abundance of beneficial bacteria.

In summary, the optimal conditions for preparing the functional mulberry leaf feed are that 4 strains of Pediococcus acidilactici (CGMCC No. 28922), pediococcus pentosaceus (CGMCC No. 28921), bacillus subtilis (CGMCC No. 28923) and bacillus licheniformis (CGMCC No. 28924) are selected as fermentation mixed strains to be combined according to a ratio of 1:1:3:3, the optimal inoculation amount is 8%, the bran addition amount is 10%, the fermentation temperature is 37 ℃, and the fermentation time is 7-30 d. In addition, the functional mulberry leaf prepared by the invention can improve the oxidation resistance and immunity of animals, maintain intestinal health and improve meat quality.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a functional mulberry leaf feed for pigs, which is characterized by comprising the following steps: fermenting fresh folium Mori with Pediococcus acidilactici (Pediococcus acidilactici), pediococcus pentosaceus (Pediococcus pentosaceus), bacillus subtilis (Bacillus subtilis) and Bacillus licheniformis (Bacillus licheniformis);

the Pediococcus acidilactici, pediococcus pentosaceus, bacillus subtilis and Bacillus licheniformis are all preserved in the China general microbiological culture Collection center (CGMCC) No. 28922, CGMCC No. 28921, CGMCC No. 28923 and CGMCC No. 28924;

In the fermentation, the ratio of the number of inoculated viable bacteria of Pediococcus acidilactici, pediococcus pentosaceus, bacillus subtilis and Bacillus licheniformis is (1-3): (1-3): (3-10): (3-10).

2. The method of claim 1, wherein the fermentation is anaerobic fermentation;

And/or, the temperature of the fermentation is 23-42 ℃;

and/or the fermentation time is 7-30 days.

3. The preparation method according to claim 1 or 2, characterized in that a fermentation auxiliary material is further added to the fermented base material;

The fermentation auxiliary material is one or more selected from bran, corn flour and molasses.

4. A functional mulberry leaf feed for pigs, characterized in that it is prepared by the preparation method of any one of claims 1-3.

5. Use of the functional mulberry leaf feed of claim 4 for any one of the following non-therapeutic purposes:

(1) The application in improving the digestion and utilization rate of the pig to the nutrients;

(2) Use in improving pork color, muscle pH _24h, marbling, crude protein, intramuscular fat content and/or muscle tenderness of pork;

(3) Use in improving the fatty acid and/or amino acid content of porcine muscle;

(4) The application in improving the antioxidation capability of pigs;

(5) The application in improving the immune function of pigs;

(6) The application of the polypeptide in improving the intestinal morphology of pigs and/or increasing the content of short chain fatty acids in the intestinal tracts of the pigs;

(7) The application in improving the intestinal flora of pigs.