Method for Preparing Feed by Bacteria-enzyme Synergistic Fermentation

Abstract

The disclosure discloses a method for preparing feed by bacteria-enzyme synergistic fermentation, belonging to the technical field of fermentation engineering. According to the disclosure, Lactobacillus plantarum JUN-DY-6, protease and cellulase are used as a starter, and Camellia seed meal or rapeseed meal is used as a substrate. The bacteria-enzyme fermentation product has higher yield of organic acids and flavor substances and better palatability, and can be used for preparing feed additives. The L. plantarum of the disclosure can inhibit growth of harmful bacteria such as Escherichia coli, S. aureus and Salmonella in the intestinal tract of poultry and livestock, and is good for health of the intestinal tract. The method increases the added value of Camellia seed meal and rapeseed meal, and is conductive to reuse of waste.

Claims

1. A method for preparing feed, comprising using Lactobacillus plantarum (L. plantarum) JUN-DY-6 and enzymes to co-treat a raw material; wherein the raw material is rapeseed meal or Camellia seed meal; the enzymes comprise protease and cellulase; a moisture content of the raw material is 30-50%, and a content of the cellulase is 300-400 U/g substrate; the protease is neutral protease or alkaline protease; a content of the alkaline protease is 800-1200 U/g substrate; and a content of the neutral protease is 1350-1500 U/g substrate.

2. The method according to claim 1, wherein the collection number of the L. plantarum JUN-DY-6 is CCTCC NO: M 2017138.

3. The method according to claim 2, wherein the L. plantarum JUN-DY-6, the alkaline protease and the cellulase are added to an environment containing Camellia seed meal and then fermentation is carried out; wherein the moisture content is 30-50%, the protease is the alkaline protease, the content of the alkaline protease is 800-1500 U/g substrate, and an inoculum size of the L. plantarum JUN-DY-6 is 1-5%.

4. The method according to claim 3, wherein the fermentation is fermentation at 35° C.-37° C. for at least 20 h.

5. The method according to claim 4, wherein a cell concentration of the L. plantarum JUN-DY-6 is ≥10.sup.6 CFU/g substrate.

6. The method according to claim 1, wherein the L. plantarum JUN-DY-6, the cellulase and the neutral protease are added to an environment containing rapeseed meal and then fermentation is carried out; and the protease is the neutral protease, and an amount of the neutral protease used is 800-1500 U/g rapeseed meal.

7. The method according to claim 6, wherein the fermentation is fermentation at 35° C.-37° C. for 40-60 h.

8. The method according to claim 1, wherein contents of organic acids in the feed are increased.

9. The method according to claim 1, wherein contents of organic acids in the feed are increased, and a content of glucosinolates is decreased.

10. The method according to claim 9, wherein the organic acids comprise one or more of lactic acid, citric acid and malic acid.

11. A starter, containing L. plantarum JUN-DY-6, protease and cellulase; wherein an enzyme activity unit ratio of the protease to the cellulase is (3-4):(8-15).

12. The starter according to claim 11, comprising the L. plantarum JUN-DY-6, alkaline protease and the cellulase; wherein an enzyme activity unit ratio of the cellulase to the alkaline protease in the starter is (3-4):(8-15); and a cell concentration of the L. plantarum JUN-DY-6 is ≥10.sup.7CFU/g or ≥10.sup.7CFU/mL.

13. The starter according to claim 11, comprising the L. plantarum JUN-DY-6, neutral protease and the cellulase; wherein an enzyme activity unit ratio of the cellulase to the neutral protease in the starter is (3-4):(12-15); and a cell concentration of the L. plantarum JUN-DY-6 is ≥10.sup.7CFU/g or ≥10.sup.7CFU/mL.

Description

BRIEF DESCRIPTION OF FIGURES

[0041] FIG. 1 shows morphology of L. plantarum JUN-DY-6 in a plating medium.

[0042] FIG. 2 shows a growth curve of L. plantarum JUN-DY-6 in an MRS medium.

[0043] FIG. 3 shows inhibition zones of L. plantarum DY1-DY6 in a zone of inhibition test.

[0044] FIG. 4 is a graph showing contents of main organic acids in a fermentation supernatant after bacteria-enzyme synergistic fermentation of Camellia seed meal.

[0045] FIG. 5 is a graph showing changes of flavor substances before and after bacteria-enzyme synergistic fermentation of Camellia seed meal.

DETAILED DESCRIPTION

[0046] (I) Method for determining diameter of inhibition zone of strain

[0047] Preparation of indicator bacterial suspension: three indicators, namely E. coli, Salmonella and S. aureus, are inoculated in an LB liquid medium, and cultured at 37° C. for 24 h.

[0048] Oxford cup assay: Plates having a diameter of about 90 mm are taken, 15-20 mL of heated and melted nutrient agar is respectively poured into the plates and is made uniformly spread in the plates, and the plates are placed on a horizontal table to solidify the nutrient agar as a bottom layer. An appropriate amount of semisolid nutrient agar medium (with an agar content of 1%) is heated and melted, and cooled to 48-50° C. 0.1-0.2 mL of indicator bacterial suspension is added to every 50-100 mL of the medium. 5 mL of the indicator bacterial suspension is added to each plate, and is made uniformly spread on the bottom layer to serve as a bacterial layer.4-5 Oxford cups are uniformly placed in each plate at equal intervals for later use. 200 μL of Lactic acid bacteria supernatant is respectively dripped into the Oxford cups in each double-layer plate, and cultured at 37° C. for 10-13 h. Then, the diameter of each inhibition zone is measured to make an evaluation.

[0049] (II) Method for Determining Contents of Organic Acids

[0050] The contents of organic acids in a fermentation supernatant were determined by an ultraviolet process. The concentration of the organic acid standard is 1 g/L, the temperature of the organic acid column (Aninex Hpx-87H ion exchange column) is 30° C., the mobile phase is a 5 mmol/L H.sub.2SO.sub.4 solution, the flow rate is 0.6 mL/min, the injection volume is 20 μL, and the standard and the sample are made to run for 14 min. Spectra are output and analyzed. The peak time and peak area of the sample are compared with those of the standard, and the contents of various organic acids in the sample are calculated.

[0051] (III) Method for Determining Contents of Flavor Substances

[0052] 2 g of fermented meal is accurately weighed and put in a 20 mL headspace bottle. Headspace conditions: The equilibrium temperature is 120° C., the transmission line temperature is 120° C., the sample loop temperature is 120° C., the pressurization time is 0.5 min, the equilibrium time is 30 min, the cycle time is 50 min, the sample loop filling time is 0.5 min, the sample loop equilibrium time is 0.5 min, and the injection time is 1 min.

Example 1: Screening of Strains

[0053] Strains were Gram-positive strains with good bacteriostatic effect separated and screened from a Camellia seed meal sample by a plate-dilution separation method. The separation and screening method was as follows:

[0054] 1. Dilution of mixed strains: The Camellia seed meal sample was weighed, 1 g of the Camellia seed meal was put into an MRS medium and cultured at 37° C. for 24 hours to obtain a bacterial suspension with a cell concentration on the order of magnitude of 1×10.sup.7 CFU/mL, and the bacterial suspension was subjected to gradient dilution.

[0055] 2. Preparation of MRS medium: 10.0 g of peptone, 8.0 g of beef extract, 4.0 g of yeast powder, 20.0 g of glucose, 2.0 g of dipotassium hydrogen phosphate, 2.0 g of triammonium citrate, 5.0 g of sodium acetate, 0.58 g of magnesium sulfate heptahydrate, 0.25 g of manganese sulfate tetrahydrate, 1 mL of Tween 80 and 1 L of distilled water were sterilized at 115° C. for 20 minutes.

[0056] 3. Primary screening of strains: 100 μL of bacterial suspension subjected to gradient dilution in step 1 was spread on an MRS solid medium plate with bromocresol purple for primary screening, and cultured at 37° C. for 24 hours. Strains with high growth speed, large colonies and large yellow circle were selected (referring to FIG. 1). After several times of primary screening, 6 Lactic acid bacteria strains were obtained, and numbered DY1-DY6 (referring to FIG. 2 for the growth curve of DY6).

[0057] 4. Secondary screening of strains: The 6 strains DY1-DY6 obtained by primary screening were inoculated into a liquid medium for secondary screening (10.0 g of peptone, 8.0 g of beef extract, 4.0 g of yeast powder, 20.0 g of glucose, 2.0 g of dipotassium hydrogen phosphate, 2.0 g of triammonium citrate, 5.0 g of sodium acetate, 0.58 g of magnesium sulfate heptahydrate, 0.25 g of manganese sulfate tetrahydrate, 1 mL of Tween 80 and 1 L of distilled water, pH 6.5), and cultured at 37° C. at 200 rpm for 24 h. The bacteriostatic effect of DY1-DY6 was determined.

[0058] The results showed that DY6 had better inhibitory effect on E. coli, Salmonella and S. aureus than the other 5 strains (referring to Table 1 and FIG. 3). The strain DY6 was finally screened out as the strain for fermenting Camellia seed meal.

TABLE-US-00001 TABLE 1 Bacteriostatic effect of L. plantarum Strain number E. coli (mm) Salmonella (mm) S. aureus (mm) DY1 11.61 10.20 11.45 DY2 12.31 12.84 13.35 DY3 10.46 10.75 11.40 DY4 11.42 10.88 11.26 DY5 11.24 10.86 12.14 DY6 13.27 12.38 13.67

[0059] Identification of strain: The obtained strain DY6 was spread on an MRS solid medium, a single colony was picked and amplified using universal primers 1492R and 27F, and the amplification product was delivered to Sangon Biotech (Shanghai) Co., Ltd., and subjected to 16S rRNA sequencing. The sequencing result was compared for homology by Nucleotide BLAST in NCBI. The comparison result showed that the strain has 99% similarity to the 16sRNA of the related type strain (Lactobacillus plantarum WCFS 1, No. 1108) in Genbank, and the strain was determined to be L. plantarum, named Lactobacillus plantarum JUN-DY-6.

[0060] The Lactobacillus plantarum JUN-DY-6 has been disclosed in the patent application CN107446852A, and has been collected by China Center for Type Culture Collection on Jun. 5, 2017, and the collection number is CCTCC NO: M 2017138.

Example 2: Fermentation of Camellia Seed Meal with L. plantarum

[0061] In order to explore the nutrient composition and fermentation technique having optimal bacteriostatic activity after bacteria-enzyme synergistic fermentation, on the basis of an MRS medium, orthogonal testing was designed to study the effect of the following four components on the fermentation of the Camellia seed meal: water, cellulase, alkaline protease, and L. plantarum JUN-DY-6 bacterial suspension with a cell concentration on the order of magnitude of 1×10.sup.7 CFU/mL. The factor levels were shown in Table 2.

TABLE-US-00002 TABLE 2 Screening factors and levels of bacteria-enzyme synergistic fermentation components Water (mass Cellulase Alkaline Factor fraction %) (U/g) protease (U/g) JUN-DY-6 (%) Level 30 200  800 3 40 300 1200 4 50 400 1500 5

TABLE-US-00003 TABLE 3 Results of orthogonal experiments Factor Alkaline Diameter of Experiment Cellulase protease JUN-DY-6 inhibition number Water (%) (U/g) (%) zone (mm) Experiment 1 30 200 800 3 14.40 Experiment 2 30 300 1200 4 16.32 Experiment 3 30 400 1500 5 15.86 Experiment 4 40 300 1200 5 16.09 Experiment 5 40 400 1500 3 15.85 Experiment 6 40 400 800 4 16.55 Experiment 7 50 200 1500 4 16.04 Experiment 8 50 300 800 5 16.62 Experiment 9 50 400 1200 3 16.19

[0062] Through the analysis of the orthogonal experiments (referring to Table 3), preferred technological conditions for bacteria-enzyme synergistic fermentation of Camellia seed meal were as follows: the moisture content was 50%, the content of the cellulase was 300 U/g substrate, the content of the alkaline protease was 800 U/g substrate, and the inoculum size of the JUN-DY-6 was 5%.

[0063] A 96-well plate method was used to determine the inhibitory effect of a fermentation supernatant of Camellia seed meal on E. coli.

[0064] 50 μL of E. coli bacterial suspension with a cell concentration of 10.sup.8 CFU/mL was added to a 96-well plate with 150 μL of filter-sterilized fermentation supernatant (the substrate Camellia seed meal, in which the moisture content was 50% (m/m), the content of cellulase was 300 U/g substrate, the content of alkaline protease was 800 U/g substrate and the inoculum size of JUN-DY-6 was 5% (v/m), was fermented in an MRS fermentation medium at 35° C.-37° C. for 24 h, 2 g of the obtained solid fermentation product was dissolved in 10 mL of sterile water, the mixture was mixed thoroughly and uniformly by vortex for 10 minutes, dispensed in 1.5 mL sterile centrifuge tubes, centrifuged at 12000 rpm for 5 min, and filtered through a sterile filter membrane with a pore size of 0.22 μm on an ultraclean bench to remove solid particles), and cultured at 37° C. for 24 h. Then, the OD.sub.600 value was determined using a microplate reader. The bacterial suspension inoculated with E. coli and sterile water were used as the control group. Fermented meal (fermented Camellia seed meal) with smaller OD.sub.600 than control group was screened out.

TABLE-US-00004 TABLE 4 96-Well plate test data (OD.sub.600) Technique Control group Fermentation group Parallel 1 0.412 0.269 Parallel 2 0.384 0.258 Parallel 3 0.391 0.275 Control 0.17 ± 0.7 Background 0.182 0.195

[0065] Note: To parallels 1, 2 and 3 in the control group, 50 μL of E. coli bacterial suspension and 150 μL of supernatant of Camellia seed meal not subjected to bacteria-enzyme synergistic fermentation were added; to parallels 1, 2 and 3 in the fermentation group, 50 μL of E. coli bacterial suspension and 150 μL of supernatant of bacteria-enzyme synergistic fermentation were added; and the control was 50 μL of E. coli bacterial suspension and 150 μL of sterile water.

[0066] The bacteriostasis rate is calculated as follows:

[00001]$\mathrm{Bacteriostasis}\mathrm{rate}=\left(1-\frac{O{D}_{600}\mathrm{value}\mathrm{of}\mathrm{fermentation}\mathrm{group}-\mathrm{background}}{O{D}_{600}\mathrm{value}\mathrm{of}\mathrm{control}\mathrm{group}}\right)\times 100\%$

[0067] The results (shown in Table 4) showed that the bacteriostasis rate of the control group was −11%, and the bacteriostasis rate of the fermentation group was 62%. The negative bacteriostasis rate in the control group indicated that E. coli continued to grow using unfermented Camellia seed meal as the growth medium.

Example 3: Changes of Contents of Organic Acids Before and After Bacteria-Enzyme Synergistic Fermentation

[0068] Acidulants can lower the pH of feed, lower the pH in the stomach and increases the activity of digestive enzymes. Acidulants are inferior to organic acids in building healthy intestinal flora of poultry and livestock. For the disease resistance of poultry and livestock, excessive acidulants are often added to feed, which affects the palatability of the feed and increases the cost.

[0069] In this example, the fermentation product lactic acid obtained after bacteria-enzyme synergistic fermentation of Camellia seed meal was used instead of the acidulants to well make up for the deficiency of the acidulants in the ability of building healthy intestinal flora.

[0070] The contents of organic acids in the fermentation supernatant obtained in Example 2 were determined. The results showed that the contents of lactic acid, citric acid and malic acid were significantly increased (referring to Table 5 and FIG. 4). The content of lactic acid was increased by 6.3 times after the fermentation.

TABLE-US-00005 TABLE 5 Changes of contents of organic acids before and after fermentation Type of Content in control Content in organic acid group (%) fermentation group (%) Lactic acid 0.34 2.13 Citric acid 0.19 0.33 Malic acid 0.08 0.22

Example 4: Aromatic Substances for Improving Palatability of Feed by Fermentation

[0071] Feed flavors are also known as feed attractants and appetite stimulants, and their action principle is closely related to the taste, smell, respiratory system, digestive system and other functions of animals. The feed flavors can improve the palatability of feed.

[0072] The contents of flavor substances in the fermentation supernatant obtained in Example 2 were analyzed by gas chromatography. The detection results showed that among the main flavor substances in the fermentation supernatant obtained in Example 2, the contents of acetyl methyl carbinol, ethyl caprylate, 1-octen-3-ol, octanoic acid, ethyl caprate and ethyl laurate are relatively high (referring to Table 6 and FIG. 5). Flavor substances changed significantly after bacteria-enzyme synergistic fermentation with L. plantarum JUN-DY-6, including acetyl methyl carbinol, isovalaric acid, 2,3-butanedione, ethyl laurate, nonanoic acid and the like. Acetyl methyl carbinol, often used as a pharmaceutical intermediate and a food flavoring, is mainly used for preparing cream, milk, yogurt and strawberry type flavors, has a strong creamy, fatty and buttery fragrance, and has a pleasant milk fragrance after being highly diluted. After the fermentation, the content of acetyl methyl carbinol was increased by 61%. Isovalaric acid has a pungent rancid smell, and has a sweet fruity aroma after being highly diluted. The isovalaric acid is often used in baked foods and meat products and mostly used for production of flavors. After the fermentation, the content of isovalaric acid was increased by 13 times. After the bacteria-enzyme synergistic fermentation, different contents of 2,3-butanedione, ethyl laurate and nonanoic acid were detected. These are common materials for making essences and flavors. Benzoic acid can be used as a bacteriostatic agent. In addition, 3% of benzaldehyde was detected in the fermentation product. Although the content was not high, there was a slight pungent odor. After the fermentation, the content of benzaldehyde was reduced to 1.5%. Methylcyclopentane is irritating to eyes, skin, mucosae and upper respiratory tract. After the fermentation, no methylcyclopentane was detected.

TABLE-US-00006 TABLE 6 Changes of flavor substances before and after fermentation Main flavor substance Control group (%) Fermentation group (%) Methylcyclopentane 3.51 — Ethyl alcohol 1.4 1.01 2,4-Dimethyldecane 6.44 2.04 2,2,4,4-Tetramethylpropane 9.13 — 2-Hexanol 1.16 — 1-Butanol-3-methyl 1.09 1.05 Acetyl methyl carbinol 10.43 16.81 n-Hexanol 1.01 — Nonyl aldehyde 2.92 — Ethyl caprylate 5.07 3.42 1-Octen-3-ol 1.79 — 1-Heptanol 1.59 — Furan-3-carboxaldehyde 1.2 — Acetic acid 2.48 9.93 Benzaldehyde 3 1.29 Cyclopropane-pentyl 1.79 1.2 Ethyl caprate 1.21 1.22 Isovaleric acid 1.04 13.46 Dimethoxyphenol 3.42 1.72 2,3-Butanedione — 3.63 Butane-2,3-diol — 3.35 Ethyl laurate — 1.47 Benzoic acid — 1.14 Nonanoic acid — 1.51

Example 5 Preparation of Starter

[0073] Preparation of L. plantarum JUN-DY-6 bacterial suspension: L. plantarum JUN-DY-6 was inoculated in an MRS medium and cultured at 37° C. at 200 r.Math.min.sup.−1 for 24 h to obtain the L. plantarum bacterial suspension. Optionally, an appropriate amount of protective agent was added to the bacterial suspension, and the mixture was freeze-dried to prepare bacterial powder.

[0074] The L. plantarum JUN-DY-6, neutral protease and cellulase were mixed to prepare the starter. An enzyme activity unit ratio of the cellulase to the neutral protease in the starter was (3-4):(12-15). A cell concentration of the L. plantarum JUN-DY-6 was ≥10.sup.7CFU/g or ≥10.sup.7 CFU/mL.

[0075] The starter also contains auxiliary materials. The auxiliary materials can be conventional auxiliary materials in the art, preferably including one or more of water, lactose, sucrose, maltodextrin, sodium glutamate, gelatin, glycerin, sorbitol, trehalose, yeast extract and β-cyclodextrin.

Example 6 Effect of Different Inoculum Size on Fermented Rapeseed Meal

[0076] Water, L. plantarum JUN-DY-6, cellulase and protease were added to rapeseed meal and then fermentation was carried out. The L. plantarum JUN-DY-6 was cultured in an MRS medium at 37° C. for 24 h to obtain a L. plantarum JUN-DY-6 bacterial suspension with a cell concentration on the order of magnitude of 1×10.sup.8 CFU/mL. The bacterial suspension was added to the rapeseed meal according to the inoculum size of 1%, 2%, 3%, 4% and 5% (v/m, mL/g substrate). The moisture content in the rapeseed meal raw material for fermentation was adjusted to 50%, the fermentation temperature was controlled at 37° C., the fermentation time was 48 h, the amount of cellulase added was 400 U/g, the protease was neutral protease, and the amount of protease added was 1500 U/g substrate. The detection results of the fermentation products were shown in Table 7.

TABLE-US-00007 TABLE 7 Effect of different inoculum size in rapeseed meal on contents of various substance Inoculum size (%) 1 2 3 4 5 Small peptides (mg .Math. g.sup.−1) 85.65 87.13 86.61 86.12 85.75 Glucosinolates (μmol .Math. g.sup.−1) 22.1 18.64 15.21 16.91 15.98 Total acids (%) 3.69 3.9 4.86 4.54 5.35 Lactic acid (g .Math. L.sup.−1) 0.91 1.16 2.01 1.83 2.16

Example 7 Effect of Different Amount of Neutral Protease Added on Fermented Rapeseed Meal

[0077] The L. plantarum JUN-DY-6 was cultured according to the method in Example 6, the inoculum size was adjusted to 3% (v/m, mL/g), and the bacterial suspension was added to the substrate such that the cell concentration reached 1×10.sup.6 CFU/g substrate. Water, the L. plantarum JUN-DY-6, cellulase and protease were added to the rapeseed meal and then fermentation was carried out. The rapeseed meal was fermented according to the neutral protease content of 1200, 1350 and 1500 U/g. The moisture content of the entire fermentation raw material was 50%, the fermentation temperature was 37° C., the content of cellulase was 400 U/g substrate, and the fermentation time was 48 h. The rest operations were the same as in Example 6. The detection results of the fermentation products were shown in Table 8.

TABLE-US-00008 TABLE 8 Effect of amount of neutral protease added in rapeseed meal on contents of various substances Enzyme activity (U/g) 1200 1350 1500 Small peptides (mg .Math. g.sup.−1) 80.84 86.23 86.19 Glucosinolates (μmol .Math. g.sup.−1) 15.98 15.49 15.55 Total acids (%) 4.85 5.27 5.04 Lactic acid (g .Math. L.sup.−1) 1.95 2.09 2.11

Example 8 Effect of Different Fermentation Time on Fermented Rapeseed Meal

[0078] The specific implementation was the same as in Example 6. The L. plantarum JUN-DY-6 was cultured according to the method in Example 6. The bacterial suspension was added to the substrate according to the inoculum size of 3% (v/m, mL/g) such that the cell concentration reached 1×10.sup.6 CFU/g substrate. The amount of neutral protease added was 1350 U/g. Water, the L. plantarum JUN-DY-6, cellulase and the protease were added to rapeseed meal and then fermentation was carried out. The fermentation time was respectively 12 h, 24 h, 36 h, 48 h and 60 h. The moisture content of the entire fermentation raw material was 50%, the fermentation temperature was 37° C., and the amount of cellulase added was 300 U/g substrate. The detection results of the fermentation products were shown in Table 9.

TABLE-US-00009 TABLE 9 Effect of different fermentation time of rapeseed meal on contents of various substances Time (h) 12 24 36 48 60 Small peptides 78.91 80.57 84.5 86.35 85.88 (mg .Math. g.sup.−1) Glucosinolates 18.32 17.41 16.34 15.48 15.1 (μmol .Math. g.sup.−1) Total acids (%) 2.83 3.42 4.8 5.35 5.45 Lactic acid (g .Math. L.sup.−1) 1.32 1.73 1.9 2.16 2.31

Example 9 Effect of Different Amount of Cellulase Added on Fermented Rapeseed Meal

[0079] The specific implementation was the same as in Example 6. The L. plantarum JUN-DY-6 was cultured according to the method in Example 6. The bacterial suspension was added to the substrate according to the inoculum size of 3% (v/m, mL/g) such that the cell concentration reached 1×10.sup.6 CFU/g substrate. The amount of neutral protease added was adjusted to 1350 U/g, and the fermentation time was 48 h. Water, the L. plantarum JUN-DY-6, cellulase and the protease were added to rapeseed meal and then fermentation was carried out. The amount of cellulase was respectively 300, 350 and 400 U/g. The fermentation temperature was 37° C., and the moisture content of the entire fermentation raw material was 50%. The detection results of the fermentation products were shown in Table 10.

TABLE-US-00010 TABLE 10 Effect of different amount of cellulase added in rapeseed meal on contents of various substances Enzyme activity (U/g) 300 350 400 Small peptides (mg .Math. g.sup.−1) 85.9 86.01 85.93 Glucosinolates (μmol .Math. g.sup.−1) 15.44 15.18 15.29 Total acids (%) 4.86 4.95 5.13 Lactic acid (g .Math. L.sup.−1) 2.03 2.27 2.25

Example 10 Effect of Different Temperature on Fermented Rapeseed Meal

[0080] The specific implementation was the same as in Example 6. The L. plantarum JUN-DY-6 was cultured according to the method in Example 6. The bacterial suspension was added to the substrate according to the inoculum size of 3% (v/m, mL/g) such that the cell concentration reached 1×10.sup.6 CFU/g substrate. The amount of neutral protease added was 1350 U/g, the fermentation time was 48 h, and the amount of cellulase added was 350 U/g. Water, the L. plantarum JUN-DY-6, the cellulase and the protease were added to rapeseed meal and then fermentation was carried out. The fermentation temperature of the rapeseed meal was respectively controlled to 30° C., 35° C., 37° C. and 40° C., and the moisture content of the entire fermentation raw material was 50%. The detection results of the fermentation products were shown in Table 11.

TABLE-US-00011 TABLE 11 Effect of different fermentation temperature of rapeseed meal on contents of various substances Temperature (° C.) 30 35 37 40 Small peptides (mg .Math. g.sup.−1) 83.21 85.05 86.65 1350 Glucosinolates (μmol .Math. g.sup.−1) 17.28 16.22 15.72 350 Total acids (%) 3.93 4.27 5.23 113.21 Lactic acid (g .Math. L.sup.−1) 1.25 1.49 2.22 26.32

Example 11 Effect of Different Moisture Content on Fermented Rapeseed Meal

[0081] The specific implementation was the same as in Example 6. The L. plantarum JUN-DY-6 was cultured according to the method in Example 6. The bacterial suspension was added to the substrate according to the inoculum size of 3% (v/m, mL/g) such that the cell concentration reached 1×10.sup.6 CFU/g substrate. The amount of neutral protease added was 1350 U/g, the fermentation time was 48 h, and the amount of cellulase added was 350 U/g, and the fermentation temperature was 37° C. Water, the L. plantarum JUN-DY-6, the cellulase and the protease were added to rapeseed meal and then fermentation was carried out. The moisture content in the fermentation raw material was respectively adjusted to 10%, 30%, 50% and 60%. The detection results of the fermentation products were shown in Table 12.

TABLE-US-00012 TABLE 12 Effect of different moisture content of rapeseed meal on contents of various substances Moisture content (%) 10 30 50 60 Small peptides (mg .Math. g.sup.−1) 82.3 85.43 86.15 93.32 Glucosinolates (μmol .Math. g.sup.−1) 16.19 15.88 15.58 16.22 Total acids (%) 4.72 4.84 5.12 5.03 Lactic acid (g .Math. L.sup.−1) 1.84 1.95 2.06 2.19

Example 12 Bacteria-Enzyme Synergistic Fermentation of Rapeseed Meal

[0082] The L. plantarum JUN-DY-6 was cultured according to the method in Example 6. Water, the L. plantarum JUN-DY-6, cellulase and protease were added to rapeseed meal and then fermentation was carried out. The moisture content in the rapeseed meal was 50% (m/m), the content of cellulase was 400 U/g substrate, the protease was neutral protease, and the content of protease was 1500 U/g substrate. The bacterial suspension was added to the substrate according to the inoculum size of the L. plantarum JUN-DY-6 of 5% (v/m, mL/g) such that the cell concentration reached 1×10.sup.6 CFU/g substrate.

[0083] The rapeseed meal, into which the protease had been added and the L. plantarum had been inoculated, was fermented at 37° C. for 48 h. It was determined that after the completion of the fermentation, the content of small peptides was 85.75 mg/g, the content of glucosinolates was 15.98 μmol.Math.g.sup.−1, the content of total acids was 5.35%, and the content of lactic acid was 2.16 g.Math.L.sup.−1 in the rapeseed meal. The content of small peptides was increased by 41.59%, the content of glucosinolates was reduced by 53.27%, the content of total acids was increased by 19.58 times, and the content of lactic acid was increased by 3.24 times.

Example 13 Bacteria-Enzyme Synergistic Fermentation of Rapeseed Meal

[0084] The L. plantarum JUN-DY-6 was cultured according to the method in Example 6. Water, the L. plantarum JUN-DY-6, cellulase and protease were added to rapeseed meal and then fermentation was carried out. The moisture content in the rapeseed meal was 50% (m/m), the content of cellulase was 300 U/g substrate, the protease was neutral protease, and the content of protease was 1350 U/g substrate. The bacterial suspension was added to the substrate according to the inoculum size of the L. plantarum JUN-DY-6 of 3% (v/m, mL/g) such that the cell concentration reached 1×10.sup.6 CFU/g substrate.

[0085] The rapeseed meal, into which the protease had been added and the L. plantarum had been inoculated, was fermented at 37° C. for 60 h. It was determined that after the completion of the fermentation, the content of small peptides was 85.88 mg/g, the content of glucosinolates was 15.10 μmol.Math.g.sup.−1, the content of total acids was 5.45%, and the content of lactic acid was 2.31 g.Math.L.sup.−1 in the rapeseed meal. The content of small peptides was increased by 41.80%, the content of glucosinolates was reduced by 55.84%, the content of total acids was increased by 19.96 times, and the content of lactic acid was increased by 3.53 times.

Example 14 Bacteria-Enzyme Synergistic Fermentation of Rapeseed Meal

[0086] Water, the L. plantarum JUN-DY-6, cellulase and protease were added to the rapeseed meal and then fermentation was carried out. The moisture content was 50% (m/m), the content of cellulase was 350 U/g substrate, the protease was neutral protease, and the content of protease was 1350 U/g substrate. The L. plantarum JUN-DY-6 was cultured according to the method in Example 2, and the bacterial suspension was added to the substrate according to the inoculum size of 3% (v/m, mL/g) such that the cell concentration reached 1×10.sup.6 CFU/g substrate.

[0087] The rapeseed meal, into which the protease had been added and the L. plantarum had been inoculated, was fermented at 40° C. for 48 h. It was determined that after the completion of the fermentation, the content of small peptides was 113.21 mg/g, the content of glucosinolates was 26.32 μmol.Math.g.sup.−1, the content of total acids was 2.29%, and the content of lactic acid was 1.12 g.Math.L.sup.−1 in the rapeseed meal. The content of small peptides was increased by 86.94%, the content of glucosinolates was reduced by 23.04%, the content of total acids was increased by 10.26 times, and the content of lactic acid was increased by 1.20 times.

Example 15 Bacteria-Enzyme Synergistic Fermentation of Rapeseed Meal

[0088] (1) Water, L. plantarum JUN-DY-6, cellulase and protease were added to the rapeseed meal and then fermentation was carried out. The moisture content in the rapeseed meal was 50% (m/m), the content of cellulase was 400 U/g substrate, the protease was neutral protease, and the content of protease was 1500 U/g substrate. The L. plantarum JUN-DY-6 was cultured according to the method in Example 2, and the bacterial suspension was added to the substrate according to the inoculum size of 4% (v/m, mL/g) such that the cell concentration reached 1×10.sup.6 CFU/g substrate.

[0089] The rapeseed meal, into which the protease had been added and the L. plantarum had been inoculated, was fermented at 37° C. for 48 h. It was determined that after the completion of the fermentation, the content of small peptides was 86.12 mg/g, the content of glucosinolates was 16.91 μmol.Math.g.sup.−1, the content of total acids was 4.54%, and the content of lactic acid was 1.83 g.Math.L.sup.−1 in the rapeseed meal. The content of small peptides was increased by 42.17%, the content of glucosinolates was reduced by 50.56%, the content of total acids was increased by 16.46 times, and the content of lactic acid was increased by 2.59 times.

Example 16 Bacteria-Enzyme Synergistic Fermentation of Rapeseed Meal

[0090] (1) Water, L. plantarum JUN-DY-6, cellulase and protease were added to the rapeseed meal and then fermentation was carried out. The moisture content in the rapeseed meal was 30% (m/m), the content of cellulase was 350 U/g substrate, the protease was neutral protease, and the content of protease was 1500 U/g substrate. The L. plantarum JUN-DY-6 was cultured according to the method in Example 2, and the bacterial suspension was added to the substrate according to the inoculum size of 3% (v/m, mL/g) such that the cell concentration reached 1×10.sup.6 CFU/g substrate.

[0091] The rapeseed meal, into which the protease had been added and the L. plantarum had been inoculated, was fermented at 37° C. for 48 h. It was determined that after the completion of the fermentation, the content of small peptides was 85.07 mg/g, the content of glucosinolates was 17.12 μmol.Math.g.sup.−1, the content of total acids was 4.79%, and the content of lactic acid was 1.69 g.Math.L.sup.−1 in the rapeseed meal. The content of small peptides was increased by 40.47%, the content of glucosinolates was reduced by 49.94%, the content of total acids was increased by 17.42 times, and the content of lactic acid was increased by 2.31 times.

Example 17 Bacteria-Enzyme Synergistic Fermentation of Rapeseed Meal

[0092] (1) Water, L. plantarum JUN-DY-6, cellulase and protease were added to the rapeseed meal and then fermentation was carried out. The moisture content in the rapeseed meal was 60% (m/m), the content of cellulase was 300 U/g substrate, the protease was neutral protease, and the content of protease was 1350 U/g substrate. The L. plantarum JUN-DY-6 was cultured according to the method in Example 2, and the bacterial suspension was added to the substrate according to the inoculum size of 3% (v/m, mL/g) such that the cell concentration reached 1×10.sup.6 CFU/g substrate.

[0093] The rapeseed meal, into which the protease had been added and the L. plantarum had been inoculated, was fermented at 30° C. for 36 h. It was determined that after the completion of the fermentation, the content of small peptides was 80.82 mg/g, the content of glucosinolates was 17.34 μmol.Math.g.sup.−1, the content of total acids was 2.89%, and the content of lactic acid was 1.01 g.Math.L.sup.−1 in the rapeseed meal. The content of small peptides was increased by 33.45%, the content of glucosinolates was reduced by 49.3%, the content of total acids was increased by 10.12 times, and the content of lactic acid was increased by 0.98 time.

Example 18 Comparison of Components in Fermented Rapeseed Meal Prepared Under Different Conditions

[0094] (1) Changes of Contents of Total Acids and Organic Acids Before and After Fermentation

[0095] Acidulants can lower the pH of feed, lower the pH in the stomach and increases the activity of digestive enzymes. Acidulants are inferior to organic acids in building healthy intestinal flora of poultry and livestock. For the disease resistance of poultry and livestock, excessive acidulants are often added to feed, which affects the palatability of the feed and increases the cost.

[0096] In this example, the fermentation product lactic acid obtained after bacteria-enzyme synergistic fermentation of rapeseed meal was used instead of the acidulants to well make up for the deficiency of the acidulants in the ability of building healthy intestinal flora. The contents of total acids and organic acids in the fermentation products obtained in different examples were determined. It was found that the contents of total acids and organic acids were significantly increased (referring to Table 13). The content of total acids could be increased by up to 19.96 times, and the content of lactic acid could be increased by up to 3.5 times.

[0097] (2) Changes of Content of Glucosinolates Before and After Bacteria-Enzyme Synergistic Fermentation

[0098] Glucosinolates are the main antinutritional factors in rapeseed meal that limit the feedability of rapeseed meal. Microbial fermentation can reduce the content of glucosinolates in the rapeseed meal and lower the toxicity of the rapeseed meal. After determining the content of glucosinolates in the fermented rapeseed meal supernatant in different examples, it can be seen that after the bacteria-enzyme synergistic fermentation, the content of glucosinolates in the rapeseed meal was significantly reduced (Table 13) by up to 55.84%.

[0099] (3) Changes of Content of Small Peptides Before and After Bacteria-Enzyme Synergistic Fermentation

[0100] The increase in the content of small peptides is mainly due to the enzymolysis of macromolecular proteins in the rapeseed meal by the protease. Through the comparison of degradation effects of different proteases, it was determined that the content of small peptides in the fermented rapeseed meal in different examples could be increased by up to 86.94%.

TABLE-US-00013 TABLE 13 Changes of contents of various substances in rapeseed meal before and after fermentation Rapeseed meal raw Type material Example 12 Example 13 Example 14 Example 15 Example 16 Example 18 Small peptides (mg .Math. g.sup.−1) 60.56 85.75 85.88 113.21 86.12 85.07 80.82 Total acids (%) 0.26 5.35 5.45 2.93 4.54 4.79 2.89 Lactic acid (g .Math. L.sup.−1) 0.51 2.16 2.31 1.12 1.83 1.69 1.01 Citric acid (g .Math. L.sup.−1) 0.10 0.45 0.44 0.18 0.45 0.21 0.22 Malic acid (g .Math. L.sup.−1) 0.12 0.18 0.25 0.15 0.24 0.18 0.17 Glucosinolates (μol .Math. g.sup.−1) 34.20 15.98 15.10 26.32 16.91 17.12 17.34

Comparative Example 1 Preparation of Fermented Rapeseed Meal with Different L. plantarum Strains

[0101] The L. plantarum JUN-DY-6 was replaced with other L. plantarum strains preserved in the laboratory, and fermentation was carried out according to the same method as in Example 12. The contents of various substances in the fermented feed were detected. The results were shown in Table 14.

TABLE-US-00014 Glucosinolates Small peptides Total acids Strain (μmol/g) (mg/g) (%) DY1 20.25 84.31 4.00 DY2 30.93 84.10 3.63 DY3 26.03 84.85 2.12 DY4 33.14 82.81 1.82 DY5 25.79 86.44 2.24

[0102] Although the disclosure has been disclosed as above in the preferred examples, it is not intended to limit the disclosure. Any person familiar with the art can make various changes and modifications without departing from the spirit and scope of the disclosure. Therefore, the protection scope of the disclosure should be as defined in the claims.