A strain of Neurospora crassa and its application in preparing protein feed by fermenting honeysuckle residue

Abstract

The present invention belongs to the field of feed, and specifically relates to a strain of Neurospora crassa and its application in fermenting honeysuckle residues to prepare protein feed. The present invention provides a strain of Neurospora crassa isolated from honeysuckle residues, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Committee, with a deposit number of CGMCC No.40085, a deposit date of March 1, 2022, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The use of the strain to ferment and prepare Chinese medicine residue feed can effectively reduce the content of crude fiber and improve the fermentation effect of honeysuckle residues.

Description

Technical Field

[0001] The invention belongs to the field of feed, and particularly relates to a strain of Neurospora crassa and application of the strain in preparing protein feed by fermenting honeysuckle residues. Background Art

[0002] Abuse or irrational use of antibiotics will lead to an increase in drug-resistant bacteria and drug-resistant genes, and will also cause antibiotic residues in the environment and the spread of bacterial resistance. Currently, the use of antibiotics in feed has been completely banned. Commonly used "antibiotic alternatives" added to feed include traditional Chinese medicine and probiotics. The effect of traditional Chinese medicine is the most recognized, followed by probiotics. Fermenting feed with probiotics can not only deeply process feed raw materials and improve the nutritional value of the feed itself, but also help develop local feed and utilize crop by-products, reduce feed costs, and improve production efficiency.

[0003] Honeysuckle has antibacterial, antiviral and immune-enhancing effects. Currently, honeysuckle residues are mainly disposed of by landfill or incineration, which not only wastes resources but also pollutes the environment. Studies have found that honeysuckle residue extract can improve the quality of fish, indicating that honeysuckle residues still contain a large amount of active ingredients.

[0004] The Chinese invention patent application with application publication number CN107897523A discloses a fermented feed of traditional Chinese medicine residues for livestock and poultry, and records the preparation of feed by fermenting traditional Chinese medicine residues containing honeysuckle residues with fermentation liquid. After research, it was found that conventional bacterial agents have limited adaptability in fermenting honeysuckle residues, and the fermentation effect on honeysuckle residues needs to be improved. Summary of the invention

[0005] The invention aims to provide a strain of Neurospora crassa, so as to improve the fermentation effect on honeysuckle residues.

[0006] The second object of the present invention is to provide the use of the above Neurospora crassa in fermenting honeysuckle residues to prepare protein feed.

[0007] In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0008] Neurospora crassa, which is Neurospora crassa JBH-BCZ1, with the deposit number being CGMCC No.40085.

[0009] The present invention provides a strain of Neurospora crassa isolated from honeysuckle residues, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Committee, with a deposit number of CGMCC No. 40085, a deposit date of March 1, 2022, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The use of the strain to ferment and prepare Chinese medicine residue feed can effectively reduce the crude fiber content and improve the fermentation effect of honeysuckle residues.

[0010] Application of Neurospora crassa in fermenting honeysuckle residue to prepare protein feed.

[0011] Using this strain to ferment honeysuckle residue protein feed can further improve the fermentation effect of honeysuckle residue, achieve the superposition of the effects of probiotics and traditional Chinese medicine, save resources and protect the environment.

[0012] Preferably, the preparation of the protein feed comprises solid-state fermentation of a solid fermentation medium containing the honeysuckle residues with a fermentation seed liquid, wherein the fermentation seed liquid contains the Neurospora crassa.

[0013] More preferably, the fermentation seed liquid further contains Bacillus subtilis and Bacillus coagulans. More preferably, Neurospora crassa, Bacillus subtilis and Bacillus coagulans are inoculated onto the solid fermentation medium in the form of bacterial liquid, and the number of viable bacteria of the bacterial liquid of Neurospora crassa, Bacillus subtilis and Bacillus coagulans is (1-2)×10 9 cfu / mL, (7-8)×10 8 cfu / mL, (6-7)×10 8 cfu / mL, and the inoculation amount is 2-5%, 1-4%, 0.1-2%, respectively. The inoculation amount of Neurospora crassa, Bacillus subtilis, and Bacillus coagulans is more preferably 2-4%, 1-2%, 0.7-2%. The inoculation amount "%" refers to the volume of the bacterial solution inoculated per 100g of solid fermentation medium (mL).

[0014] Preferably, the solid fermentation medium is prepared by mixing solid raw materials with liquid medium, wherein the solid raw materials are prepared by the following raw materials in parts by weight: 300-500 parts of honeysuckle residues, 200-300 parts of rice straw, 100-200 parts of peat, 50-100 parts of bran, and 200 parts of water; the liquid medium provides a nitrogen source, a phosphorus source, and magnesium sulfate. The rice straw is crushed and passed through a 40-80 mesh sieve to obtain rice straw powder.

[0015] Further preferably, the composition of the liquid culture medium is: peptone 0.1-1 g / 100 mL, KH2PO4 0.1-0.5 g / 100 mL, MgSO4 0.05-0.2 g / 100 mL, NH4Cl 0.1-0.5 g / 100 mL, and the solvent is water. The more preferred composition of the liquid culture medium is: peptone 0.3-0.6 g / 100 mL, KH2PO4 0.1-0.3 g / 100 mL, MgSO4 0.05-0.1 g / 100 mL, NH4Cl 0.2-0.4 g / 100 mL, and the solvent is water.

[0016] More preferably, the weight ratio of the solid raw material to the liquid culture medium is (1-5): 1. More preferably, the weight ratio of the solid raw material to the liquid culture medium is (2-3):1.

[0017] Preferably, the solid-state fermentation is carried out at a temperature of 25° C. to 35° C., at a humidity of 45% to 75%, for 5 to 15 days.

[0018] Preferably, after the solid-state fermentation is completed, the fermentation product is dried at 60-80° C. to obtain a protein feed with a moisture content of less than 10%. The moisture content is more preferably 5-8%. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a colony image of Neurospora crassa JBH-BCZ1 screened in the present invention;

[0020] Figure 2 This is a mycelium image of Neurospora crassa JBH-BCZ1 screened and obtained in the embodiment of the present invention;

[0021] Figure 3 This is a phylogenetic tree diagram of Neurospora crassa JBH-BCZ1 screened in the examples of the present invention. DETAILED DESCRIPTION

[0022] The present invention is based on a strain of Neurospora crassa isolated from honeysuckle residues to prepare functional protein feed with honeysuckle residues. The fermented seed liquid based on the strain can improve the fermentation effect of honeysuckle residues, and finally obtain a protein feed with high crude protein content, low crude fiber content and pH value, wherein the mass percentage of crude protein is 48-53%, the mass percentage of crude fiber is less than 10%, and it also contains 0.018-0.029% vitamin B 12 .

[0023] In addition, the nutritional conditions of Neurospora crassa are simple, the growth is rapid, the fermentation cycle is short, and the actual production process is easy to control, which can produce vitamin B 12(can promote the growth and development of poultry, especially young poultry and livestock, improve the utilization rate of feed protein, and treat emaciation of cattle and sheep caused by cobalt deficiency) and high-activity cellulase, and can also lower the pH value to avoid mold and odor during product storage; Bacillus subtilis is the most commonly used probiotic, which can produce amylase, lipase and protease, etc.; Bacillus coagulans is a type of lactic acid bacteria that can form spores, which can maintain the balance of animal intestinal microecology, stimulate immunity, and improve animal digestive function and health level.

[0024] The fermented honeysuckle residue can improve the palatability of feed, promote animal feed intake, and increase the digestibility of nutrients; the combined effects of the effective ingredients of traditional Chinese medicine and probiotics in the functional protein feed are conducive to the growth of beneficial bacteria in the animal intestines, inhibit the proliferation of harmful bacteria, and help animals establish a complete and balanced intestinal microbial system. It can also improve the animal's body's defense function, enhance immunity, reduce stress response, improve the absorption capacity of trace elements, and better prevent diseases. Therefore, this product can replace antibiotics and meet the requirements of the era of antibiotic prohibition.

[0025] The preparation process of Neurospora crassa liquid (fermentation liquid), Bacillus subtilis liquid (fermentation liquid), and Bacillus coagulans (fermentation liquid) involved in the fermentation seed liquid is described as follows:

[0026] The preparation method of Neurospora crassa fermentation liquid is as follows: inoculating Neurospora crassa into a fermentation medium with a liquid volume of 30%, and fermenting at 28°C and 150 r / min for 36 to 48 hours; the formula of the fermentation medium (g / 100 mL) is: 3 sucrose, 1.5 cottonseed powder, 0.1 KH2PO4, 0.2 MgSO4·7H2O, and pH 6.8.

[0027] Furthermore, before fermentation culture, Neurospora crassa is first activated on a slant and cultured on a seed basis, and then the seed solution is inoculated into a fermentation medium at an inoculation amount of 2-15% (preferably 10%) by volume concentration; the slant activation method is as follows: inoculating Neurospora crassa into a slant culture medium, culturing at 20-37° C. for 12-72 hours (preferably culturing at 28° C. for 48 hours) to obtain slant bacteria; the slant culture medium (potato glucose agar culture medium) has a formula (g / 100 mL): 18-22 potatoes, 2-2.5 glucose, 1.5-2 agar, the solvent is deionized water, and the pH is natural. The seed expansion culture method comprises: inoculating the slant bacteria into the seed culture medium, and culturing at 25°C and 150 r / min for 24 to 48 hours to obtain the seed solution; the seed culture medium formula (g / 100 mL) is 2.5 sucrose, 0.3 yeast powder, 1 peptone, 0.1 K2HPO4, 0.05 MgSO4·7H2O, and pH 6.8.

[0028] The preparation method of Bacillus subtilis fermentation liquid is as follows: inoculating Bacillus subtilis seeds into a fermentation medium with a liquid volume of 10%, culturing at 37°C and 150r / min until OD600 is 0.6-2.5, and obtaining a fermentation liquid; the fermentation medium (g / 100mL) is: 0.2-5 maltose, 0.2-5 peptone, and the solvent is deionized water, and the pH is 5.0-9.0. Preferably, the fermentation medium (g / 100mL) is composed of: 0.2 maltose, 0.2 peptone, and the solvent is distilled water, and the initial pH is 8.0.

[0029] Furthermore, before inoculation, the Bacillus subtilis is first activated by slant and cultured for seed expansion, and then the seed liquid is inoculated into the fermentation medium at an inoculum amount of 2-15% (preferably 10%) by volume concentration. The slant activation method is as follows: inoculating the Bacillus subtilis into the slant culture medium, culturing at 20-37° C. for 12-72 hours (preferably culturing at 37° C. for 24 hours) to obtain slant bacteria; the composition (g / 100 mL) of the slant culture medium is 0.5-2 glucose, 0.5-2 peptone, 0.1-2 beef extract, 0.5-2 sodium chloride, 2 agar, and pH 6.0-7.5; preferably, the composition (g / 100 mL) of the slant culture medium is 0.5 glucose, 1 peptone, 0.1 beef extract, 0.5 NaCl, 2 agar, and the solvent is distilled water, and the pH is 7.3-7.4. The seed expansion culture method comprises: inoculating the slant bacteria into a seed culture medium, culturing at 37°C and 200 r / min for 12 hours, and obtaining a seed solution; the composition of the seed culture medium (g / 100 mL) is: 0.5-2 glucose, 0.5-2 peptone, 0.5-2 beef extract, 0.5-2 sodium chloride, and pH 6.0-7.5, and the preferred final concentration composition formula of the seed culture medium (g / 100 mL) is: 0.5 glucose, 1 peptone, 1 beef extract, 0.5 NaCl, and pH 7.3-7.4.

[0030] The preparation method of Bacillus coagulans fermentation broth is as follows: inoculating Bacillus coagulans into a fermentation medium, culturing at 37°C, 150 r / min, and a liquid volume of 50%, for 24 to 28 hours to obtain a fermentation broth; the fermentation medium composition (g / 100 mL) is: 2 glucose, 1 yeast extract, 2 tryptone, and pH 6.8 to 7.0.

[0031] Furthermore, before inoculation, the coagulant Bacillus is first activated on a slant and cultured for seed expansion, and then the seed solution is inoculated into the fermentation medium at an inoculation amount of 2-10% (preferably 10%) by volume concentration, the slant activation method is: inoculating the coagulant Bacillus into the slant culture medium, culturing at 37°C for 24 hours, and obtaining the slant bacteria; the slant culture medium (g / 100mL) is: 0.5 beef extract, 1 tryptone, 0.5 sodium chloride, pH 6.9-7.1. The seed expansion culture method is: inoculating the slant bacteria into the seed culture medium, culturing at 37°C, 150r / min for 12-72 hours, and obtaining the seed solution; the seed culture medium (g / 100mL) is: 1 glucose, 1 tryptone, 0.5 yeast extract, pH 6.8-7.0.

[0032] The implementation process of the present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0033] Example 1 Screening of Neurospora crassa

[0034] The screening process of Neurospora crassa JBH-BCZ1 in this example is described as follows:

[0035] (1) Strain screening

[0036] The composition of broth agar medium (g / 100mL) is as follows: 1 part peptone, 0.3 part beef extract, 0.5 part sodium chloride, 1.5 part agar, the solvent is deionized water, and the pH value is 7.4.

[0037] The honeysuckle residues obtained from a traditional Chinese medicine production enterprise in Henan were diluted with sterile water at different times, and the dilutions were spread on PDA agar plates and cultured in a 37°C incubator overnight. The colonies with light yellow mycelium grown on the PDA agar plates were dipped and inoculated on fresh PDA agar plates by streaking and cultured for 36 hours. The above method was used for separation and purification three times to obtain a pure strain.

[0038] (2) Identification of bacterial species

[0039] 1) Morphological characteristics

[0040] The colony of strain L5 (i.e. JBH-BCZ1) is as follows: the single colony is round, and the colony size varies depending on the number of single colonies on the plate. The colonies are all white at the beginning. After 2 days, the middle of the colony is white, and a circle of light yellow hyphae is formed around it ( Figure 1 ), later on, as the aerial hyphae grow, the colonies all turn yellow, and finally orange, and different colonies connect together, and the entire plate is covered with orange aerial hyphae. Under the microscope, the hyphae are light yellow or light orange, and are distributed in a dendrite-like manner ( Figure 2 ), spores are mostly spherical or oval, with a diameter of 6 to 8 μm.

[0041] 2) Identification by molecular biology methods

[0042] 2.1. Preparation of mycelium

[0043] The purified strain was inoculated into ordinary broth culture medium and cultured at 28°C for 36 to 48 hours. The bacterial solution was centrifuged at 5000 r / min for 20 minutes, and the obtained precipitate (mycelium) was frozen and stored at -20°C for 24 hours.

[0044] 2.2 DNA purification

[0045] Thaw the frozen mycelium and shake it well, ultrasonically vibrate for 10 minutes, and then centrifuge it at 5000r / min for 10 minutes. Add benzyl chloride extraction buffer (pH=9.0) to the precipitate and shake it well, place it in a 50℃ water bath for 1-2 hours, and shake it every 10 minutes. After the mycelium is completely dissolved, add 0.1M sodium acetate solution (pH=5.2), then ice bath for 15 minutes, and centrifuge it at 5000r / min for 15 minutes after ice bath. Add isopropanol to the supernatant and mix it well, then precipitate it at room temperature for 20 minutes, and then centrifuge it at 5000r / min for 10 minutes. Wash the precipitate twice with 80% ethanol, and add 1×TE to dissolve it to obtain crude DNA. Add an equal volume of phenol / chloroform / isoamyl alcohol to the crude DNA, gently turn it upside down 30 times, and centrifuge it at 18℃ 12000r / min for 5 minutes. The supernatant is pure DNA.

[0046] 2.3 PCR amplification and electrophoresis detection

[0047] Using bacterial DNA as a template, 18S rRNA universal primers NS1 (5'-GTAGTCATATGCTTGTCTC-3') and NS2 (5'-GGCTGCTGGCACCAGACTTGC-3') were used to amplify the 18S rRNA gene fragment of the strain by PCR.

[0048] PCR amplification system 20 μL: 0.5 μL of upstream and downstream primers, 0.5 μL of extracted DNA template (10-100 ng / μL), 8.5 μL of sterile ultrapure water, 10 μL of 2×Ex Tap enzyme. PCR amplification program: (94°C, 5 min) + {(94°C, 30 s) + (55°C, 30 s) + (72°C, 1 min)} × 30 + (72°C, 7 min). The PCR product was verified by electrophoresis with 1% agarose gel and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0049] The sequence peak graph of the sequencing results was checked by BioEdit 7.2.6, and the base sequences with clear and continuous peaks and valleys were selected. The target sequence was homologously compared with the sequences in the nucleic acid sequence library GenBank in the NCBI database through the BLAST system, and the strain was determined to be Neurospora crassa. Multiple similar sequences were downloaded, and the phylogenetic tree was constructed using MEGA8.5 software. The results are shown in the figure. Figure 3 .

[0050] The strain is deposited in the General Microbiology Center of China Microorganism Culture Collection, with the deposit number: CGMCC No.40085, the deposit date: March 1, 2022, the deposit unit: General Microbiology Center of China Microorganism Culture Collection, and the deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0051] Example 2 Preparation of fermentation seed liquid

[0052] This example describes in detail the preparation process of the fermented seed liquid used in the preparation of protein feed:

[0053] (1) Composition of fermentation seed liquid

[0054] The solid-state fermentation seeds used in the present invention contain fermentation broths of Neurospora crassa, Bacillus coagulans and Bacillus subtilis, and the fermentation broths of the three strains are mixed in different proportions. Neurospora crassa is separated from honeysuckle residues, Bacillus coagulans and Bacillus subtilis are strains preserved in this laboratory, and their deposit numbers are CGMCC 22951 and CGMCC No 15560, respectively. Bacillus coagulans is described in detail in the Chinese invention patent application with publication number CN113652373A, and Bacillus subtilis is described in detail in the Chinese invention patent application with publication number CN109022318A.

[0055] Neurospora crassa has simple nutritional conditions, rapid growth, short fermentation cycle, and easy-to-control production process. It can produce high-activity cellulase and vitamin B during the fermentation production process. 12 Cellulase is beneficial to the decomposition of cellulose in Chinese medicine residues, vitamin B 12It can promote the growth and development of poultry, especially young poultry and livestock, improve the utilization rate of feed protein, and treat emaciation caused by cobalt deficiency in cattle and sheep; Bacillus subtilis is a star strain among probiotics, because it can produce protease, amylase and lipase, promote animal digestion, improve feed utilization, and reduce feed-to-meat ratio. It is the earliest and most widely used probiotic feed additive; Bacillus coagulans is a new favorite among probiotics. It is a bacterium that can both form spores and produce lactic acid. The lactic acid and H2O2 it produces play an important role in maintaining the balance of animal intestinal microecology, stimulating immunity and promoting digestion. Because it can produce spores, Bacillus coagulans has unique advantages such as strong resistance to stress, resistance to gastric acid, resistance to drying, resistance to high temperature and high pressure, and easy storage.

[0056] (2) Culture medium formulation

[0057] The composition (g / 100mL) of the Neurospora crassa slant culture medium (potato dextrose agar medium) is: 20 potatoes, 2 glucose, 1.5 agar, natural pH; the composition (g / 100mL) of the seed culture medium is: 2.5 sucrose, 0.3 yeast powder, 1 peptone, 0.1 K2HPO4, 0.05 MgSO4·7H2O, pH 6.8; the composition (g / L) of the fermentation culture medium is: 3 sucrose, 1.5 cottonseed meal, 0.1 KH2PO4, 0.2 MgSO4·7H2O, pH 6.8.

[0058] The composition of the Bacillus subtilis slant culture medium (i.e., ordinary broth agar culture medium) (g / 100mL) is: 0.5 glucose, 1 peptone, 0.1 beef extract, 0.5 NaCl, 2 agar, the solvent is distilled water, and the pH value is 7.3-7.4; the composition of the seed culture medium (i.e., ordinary broth culture medium) (g / 100mL) is: 0.5 glucose, 1 peptone, 0.1 beef extract, 0.5 NaCl, the solvent is distilled water, and the pH value is 7.3-7.4; the fermentation culture medium formula (g / 100mL) is: 0.2 maltose, 0.2 peptone, and the initial pH value is 8.0.

[0059] The formula of Bacillus coagulans slant culture medium (g / 100mL) is: 0.5 beef extract, 1 tryptone, 0.5 sodium chloride, pH 7.0±0.1; the formula of seed culture medium (g / 100mL) is: 1 glucose, 1 tryptone, 0.5 yeast extract, pH 6.8-7.0; the formula of fermentation culture medium (g / 100mL) is: 2 glucose, 1 yeast extract, 2 tryptone, the solvent is deionized water, pH 6.8-7.0.

[0060] (3) Bacteria culture process

[0061] Before fermentation, Neurospora crassa was first activated on a slant and expanded with seeds. The seed solution was then inoculated into the fermentation medium at a volume concentration of 10%. The culture was then heated at 28°C and 150 r / min for 72 h. The number of viable bacteria in the resulting fermentation liquid was 1.5×10 9 cfu / mL.

[0062] Before inoculation, Bacillus subtilis was activated on a slant and expanded in seed culture. The seed solution was then inoculated into the fermentation medium at a volume concentration of 10% and cultured at 37°C and 200 r / min for 24 h. The number of viable bacteria in the resulting fermentation liquid was 7.1×10 8 cfu / mL.

[0063] Before inoculation, Bacillus coagulans was activated on a slant and expanded in seed culture. The seed solution was then inoculated into the fermentation medium at a volume concentration of 5%. The culture was maintained at 37°C and 150 rpm for 24 h. The number of viable bacteria in the resulting fermentation liquid was 6.3 × 10 8 cfu / mL.

[0064] Example 3 Application in the preparation of protein feed

[0065] This example describes the process of preparing functional protein feed using honeysuckle residues as follows:

[0066] (1) Preparation of solid fermentation medium

[0067] The solid raw material consists of 400 parts of honeysuckle residue (water content 30-40%), 250 parts of rice straw, 150 parts of peat and 75 parts of bran. The rice straw that has been crushed and passed through a 40-mesh sieve is mixed with the honeysuckle residue, peat, bran and 200 parts of water and then sterilized to obtain the solid raw material. 5g of peptone, 2g of KH2PO4, 0.8g of MgSO4 and 3g of NH4Cl are added to 1L of water, and the liquid culture medium is obtained after stirring and dissolving.

[0068] The solid raw material and the liquid culture medium are uniformly mixed in a mass ratio of 5:1 to obtain a solid fermentation medium.

[0069] (2) Solid-state fermentation

[0070] The fermentation broths of Neurospora crassa, Bacillus subtilis and Bacillus coagulans in Example 2 were mixed in a certain proportion (Table 1) and sprayed onto the solid fermentation medium. After stirring evenly, the mixture was spread in a culture container and placed in a sterile room at 28°C and 65% humidity for fermentation for 7 days. Stir once a day to promote the growth of probiotics. After the solid-state fermentation, the fermentation product was dried at 60°C to a moisture content of <10% to obtain a functional protein feed. The pH value was determined by a pH meter, and the crude protein content in the feed was determined by the Kjeldahl method; the crude fiber content in the feed was determined by near-infrared spectroscopy; the amino acid content in the feed was determined by high performance liquid chromatography; and the vitamin B in the feed was determined by reversed-phase high performance liquid chromatography. 12 , the results are shown in Table 1 below.

[0071] Table 1 Effect of starter culture composition on the nutritional components of fermented feed

[0072]

[0073]

[0074] (Note: A total of 6.6 mL of three bacterial agents were inoculated into every 100 g of solid fermentation medium. The ratios of Neurospora crassa: Bacillus subtilis: Bacillus coagulans fermentation broth were as follows: Group 1 was a blank control without adding fermentation agent, Group 2 was 0:5:2, Group 3 was 10:5:2, Group 4 was 3:2:2, and Group 5 was 5:10:1.)

[0075] From the results in Table 1, it can be seen that when the proportion of Bacillus subtilis in the starter formula is large, the crude protein content increases; when the proportion of Neurospora crassa is large, the crude fiber content decreases, and the vitamin B 12 As the content increases, the yellow color deepens; as the number of Bacillus coagulans increases, the pH value decreases, thus preventing the feed from stinking due to contamination by foreign bacteria.

[0076] Experimental Example 4 Evaluation of functional fermented feed prepared using honeysuckle residues

[0077] The feeding method is as follows: 500 piglets of similar weight (18.5±1.0kg) were selected, and a single-factor completely randomized experimental design was adopted, divided into 5 treatment groups, 10 replicates in each group, 10 pigs in each replicate, and the experimental period was 30 days. The pigs were fed with 25% of the fermented feed of the present embodiment and 75% of the commercial feed, and the control group was fed with 100% commercial feed, and the other feeding conditions were the same. The average feed intake (ADFI), average daily weight gain (ADG) and feed-to-meat ratio (F / G) of each group of pigs were calculated before and at the end of the experiment. The effect of the fermented feed of the present invention on the growth performance of fattening pigs was investigated. The diarrhea rate and the odor of feces were also investigated. The test results are shown in Table 2.

[0078] SAS (Statistical Analysis) version 7.2 was used for statistical analysis and variance analysis, and the results were considered significant when P < 0.05. Significant differences were expressed by letter marking: first, all the averages were arranged in order from large to small, and then the letter a was marked on the largest average; and the average was compared with the following averages, and all those with insignificant differences were marked with the letter a, until a certain average with a significant difference was marked with the letter b; then the average marked with b was used as the standard, and compared with the averages above that were larger than it, and all those with insignificant differences were also marked with the letter b; then the largest average marked with b was used as the standard, and compared with the following unmarked averages, and all those with insignificant differences continued to be marked with the letter b, until a certain average with a significant difference was marked with c.

[0079] Table 2 Effects of fermented feed on growth performance of fattening pigs

[0080] project Group 1 Group 2 Group 3 Group 4 Group 5 ADG / g <![CDATA[587±6.35 b ]]> <![CDATA[596±7.24 b ]]> <![CDATA[693±6.34 a ]]> <![CDATA[623±10.76 b ]]> <![CDATA[619±8.32 b ]]> ADFI / g <![CDATA[2134±10.65 c ]]> <![CDATA[2198±6.32 b ]]> <![CDATA[2315±9.34 a ]]> <![CDATA[2235±11.87 b ]]> <![CDATA[2212±13.21 b ]]> F / G <![CDATA[3.64±0.34 a ]]> <![CDATA[3.63±0.23 a ]]> <![CDATA[3.34±1.01 b ]]> <![CDATA[3.58±0.31 a ]]> <![CDATA[3.57±0.56 a ]]> Diarrhea rate / % <![CDATA[6.13±0.23 a ]]> <![CDATA[4.23±0.14 c ]]> <![CDATA[2.21±0.09 d ]]> <![CDATA[5.21±0.21 b ]]> <![CDATA[4.98±0.43 b ]]>

[0081] From the results in Table 2, it can be seen that after adding the feed prepared by fermentation of medicinal residues, the average feed intake and average daily weight gain (ADG) of fattening pigs increased, and the feed-to-meat ratio decreased. In addition, the fecal odor of the experimental group was significantly reduced. In particular, as the proportion of Neurospora crassa in the starter formula increased, the feed intake increased significantly.

Claims

1. A strain of Neurospora crassa, characterized in that Neurospora crassa (Neurospora crassa) JBH-BCZ1, the deposit number is: CGMCC No. 40085.

2. Application of Neurospora crassa in fermenting honeysuckle residues to prepare protein feed, characterized in that: The preparation of protein feed comprises solid-state fermentation of a solid fermentation medium containing the honeysuckle residue with a fermentation seed liquid, wherein the fermentation seed liquid contains the crassa Neurospora, and the fermentation seed liquid also contains Bacillus subtilis and Bacillus coagulans; the preservation number of the crassa Neurospora is CGMCC No. 40085; the preservation number of the Bacillus coagulans is CGMCC22951; and the preservation number of the Bacillus subtilis is CGMCC NO 15560.

3. The use according to claim 2, characterized in that Neurospora crassa, Bacillus subtilis and Bacillus coagulans were inoculated into solid fermentation medium in the form of bacterial liquid. The number of viable bacteria in the bacterial liquid of Neurospora crassa, Bacillus subtilis and Bacillus coagulans were (1-2)×10 9 cfu / mL, (7~8)×10 8 cfu / mL, (6~7)×10 8 cfu / mL, and the volume of the bacterial liquid inoculated per 100 g of solid fermentation medium was 2~5 mL, 1~4 mL, and 0.1~2 mL, respectively.

4. The use according to claim 2, characterized in that The solid fermentation culture medium is prepared by mixing solid raw materials with liquid culture medium, wherein the solid raw materials are prepared from the following raw materials in parts by weight: 300-500 parts of honeysuckle residues, 200-300 parts of rice straw, 100-200 parts of peat, 50-100 parts of bran, and 200 parts of water; the liquid culture medium provides a nitrogen source, a phosphorus source, and magnesium sulfate.

5. The use according to claim 4, characterized in that The liquid culture medium is composed of: peptone 0.1-1 g / 100 mL, KH2PO4 0.1-0.5 g / 100 mL, MgSO4 0.05-0.2 g / 100 mL, NH4Cl 0.1-0.5 g / 100 mL, and the solvent is water.

6. The use according to claim 5, characterized in that The weight ratio of the solid raw material to the liquid culture medium is (1-5):

1.

7. The use according to any one of claims 2 to 6, characterized in that: The solid-state fermentation is carried out at a temperature of 25° C. to 35° C., a humidity of 45% to 75%, and a time of 5 to 15 days.

8. The use according to claim 7, characterized in that After the solid-state fermentation is completed, the fermentation product is dried at 60-80° C. to obtain a protein feed with a moisture content of less than 10%.

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