CN111134231A - Zhangzhou bacillus and method for fermenting mulberry leaf powder by using Zhangzhou bacillus - Google Patents
Zhangzhou bacillus and method for fermenting mulberry leaf powder by using Zhangzhou bacillus Download PDFInfo
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Abstract
The invention relates to Zhangzhou bacillus and a method for fermenting mulberry leaf powder by using the Zhangzhou bacillus, and belongs to the technical field of fermented feed. Zhangzhou bacillus JSSW-BP 44: (Bacillus zhangzhouensis) JSSW-BP44, preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2019989. The Zhangzhou bacillus JSSW-BP44 disclosed by the invention can be used for liquid fermentation of feed mulberry leaf powder, the obtained fermented mulberry leaf powder contains rich nutrients such as proteins, small peptides, amino acids, vitamins and polysaccharides, and anti-nutritional factors such as tannin and phytic acid are remarkably reduced compared with mulberry leaves, so that the absorption of nutrients by livestock, poultry and aquatic animals can be improved, and the growth performance and disease resistance of the livestock, poultry and aquatic animals are improved.
Description
Technical Field
The invention relates to Zhangzhou bacillus and a method for fermenting mulberry leaf powder by using the Zhangzhou bacillus, and belongs to the technical field of fermented feed.
Background
Deciduous trees of Moraceae (Moraceae) and Morus (Morus) can be planted and grown normally in most areas of China. Mulberry leaves are the main product of mulberry trees, and besides the mulberry leaves have abundant protein content (15% -28%) and high amino acid content (34.7%), the mulberry leaves also have abundant minerals and various bioactive components such as 1-Deoxynojirimycin (DNJ), flavone and polyphenols. Wherein DNJ can regulate blood sugar level of organism, and flavone and polyphenol can reduce inflammatory factor level and relieve inflammatory reaction by improving oxidation resistance of organism and inhibiting lipid peroxidation, thereby enhancing disease resistance of organism and improving lipid metabolism function. The Ministry of health in China considers mulberry leaves as a plant resource with homology of medicine and food at the beginning of the 20 th century. At present, researches indicate that mulberry leaves can be used as a vegetable protein source to be applied to compound feed of tilapia, grass carp and other fishes so as to save the application of high-value protein sources such as fish meal, bean pulp and the like, but when a large amount of mulberry leaf powder is added, high-content cellulose content, tannin, phytic acid and other anti-nutritional factors have an inhibiting effect on the growth performance and the anti-oxidation function of the fish body in use.
Chinese patent CN201910640354.5 discloses Zhangzhou bacillus (Bacilluszhangzhuchouensis) Z-XWW77 for degrading ammonia nitrogen and application thereof in ammonia nitrogen degradation. The invention carries out the evaluation of ammonia nitrogen degradation capability from 196 strains of ocean, screens out Zhangzhou Bacillus (Bacillus zhangzhuensis) Z-XWW77 for degrading ammonia nitrogen in ammonia-containing pollutants, such as sewage, wastewater, household garbage and animal manure, and the treatment conditions are that the temperature is 25-45 ℃ and the pH is 6-7.5.
The invention aims to decompose crude fiber, protein and other difficultly digestible macromolecules in mulberry leaves into micromolecule nutrient substances which are easily absorbed by organisms by using an enzyme system generated by microorganisms through a microbial fermentation method. Anti-nutritional factors such as tannin, phytic acid and the like can also be digested by microorganisms in the fermentation process, so that the palatability and the nutritional value of the fermentation product are improved, and the application range of the mulberry leaf powder in aquatic animals is further expanded and popularized.
Disclosure of Invention
The invention aims to overcome the defects and provide Zhangzhou bacillus and a method for fermenting mulberry leaf powder by using the Zhangzhou bacillus, and the nutrient substances such as protein, free amino acid, small peptide and the like in the prepared fermented mulberry leaf powder are obviously higher than those in the unfermented mulberry leaf powder, so that the feeding nutritional value of the mulberry leaf powder is improved.
The technical scheme of the invention is that a preparation method of fermented mulberry leaf powder comprises the following steps:
(1) crushing mulberry leaves: drying fresh mulberry leaves to constant weight, crushing and sieving to obtain mulberry leaf powder;
(2) preparing mulberry leaf pulp: adding water into the mulberry leaf powder obtained in the step (1) and uniformly stirring to obtain mulberry leaf pulp;
(3) and (3) sterilization: adjusting the pH value of the mulberry leaf pulp obtained in the step (2), and sterilizing;
(4) fermentation: inoculating a fermentation strain into the sterilized mulberry leaf pulp obtained in the step (4) for fermentation;
(5) and (3) drying: and (4) after fermentation, carrying out freeze drying to obtain a finished product of the fermented mulberry leaf powder.
Further, in the step (1), drying at 60-65 ℃ to constant weight, crushing mulberry leaves for 2-10min at the rotating speed of 20000r/min-24000r/min, and sieving by a 60-mesh sieve to obtain the mulberry leaf powder.
Further, the fresh mulberry leaves are fresh mulberry leaves harvested 4 months per year.
Further, the drying is carried out until the constant weight is achieved, and the water content is 2.0% -4.0%.
Further, 940-950mL of water is added to every 50-60g of the sieved mulberry leaf powder in the step (2), and the mixture is fully and uniformly stirred to prepare mulberry leaf pulp.
Further, in the step (3), the pH of the mulberry leaf pulp is adjusted to 7-8 by using a sodium hydroxide solution with a mass concentration of 20-30% and a hydrochloric acid solution, and then the mulberry leaf pulp is autoclaved at 121 ℃ for 30 min.
Further, fermenting the sterilized mulberry leaf pulp by using Zhangzhou bacillus JSSW-BP44 in the step (4); the content of viable bacteria of 1-2mL is 10 per 50mL of mulberry leaf pulp7CFU/mL Zhangzhou bacillus suspension, wherein the fermentation conditions comprise a material-water ratio of 5.0-6.0%, a liquid filling amount of a fermentation bottle of 10.0-12.0%, the fermentation time of 48-72h and the fermentation temperature of 33-35 ℃.
Further, the microbial strain supposed in the step (4) is Zhangzhou Bacillus (Bacillus zhangzhuangzhouensis) JSSW-BP44 which is classified and named as Zhangzhou Bacillus (Bacillus zhangzhuensis), and the preservation number is CCTCC NO: m2019989, the preservation date is 12 months and 2 days in 2019, and the culture is preserved in China center for type culture Collection with the addresses as follows: wuhan university in Wuhan, China.
Zhangzhou Bacillus JSSW-BP44(Bacillus zhangzhuensis) JSSW-BP44 has the biochemical characteristics of generating alkaline phosphatase, esterase, lipoid esterase, lipase, valine arylamine enzyme, chymotrypsin, naphthol-AS-BI-phosphate and β -glucosidase, has cellulase activity and can utilize glycerol, L-arabinose, ribose, D-xylose, galactose, glucose, fructose, mannose, mannitol, arbutin, esculin, saligenin, cellobiose, melibiose, sucrose, trehalose and D-tagatose.
Further, the temperature of freeze drying in the step (5) is-40 to-60 ℃, the vacuum degree is 30-40Pa, and the drying time is 24-36 h.
The content of crude protein in the fermented mulberry leaf powder prepared by the invention is 18-20%, the content of free amino acid is 22-25mg/g, and the content of small peptide is 220 mg/g.
The application of the fermented mulberry leaf powder is to apply the fermented mulberry leaf powder as one of raw materials in the preparation of aquatic feeds.
The invention has the beneficial effects that: according to the invention, a strain of Zhangzhou bacillus (Bacillus zhangzhuhuangyensis) JSSW-BP44 is obtained through screening, and is used for producing feed mulberry leaf powder, the obtained fermented mulberry leaf powder has aromatic smell, a certain food calling effect and good palatability, the contents of crude protein, small peptide and free amino acid in the fermented mulberry leaf powder are respectively increased by 18.6% -22%, 59.2% -70% and 226.2-8.4% compared with the contents evaluated before fermentation, and the content of nutrient components is remarkably higher than that of unfermented mulberry leaf powder, so that the absorption of nutrient substances by livestock, poultry and aquatic animals can be improved, and the growth performance and disease resistance of the livestock, poultry and aquatic animals are improved.
The fermented mulberry leaf powder is added into the aquatic animal feed, so that the fermented mulberry leaf powder can partially replace vegetable proteins such as bean pulp and the like, the weight gain rate, the specific growth rate and the protein efficiency of the carassius auratus gibelio are obviously improved, the feed coefficient is obviously reduced, the feed investment cost is reduced, and the culture economic benefit is improved.
The source of leaf raw materials such as folium mori and the like is wide, the price is low, the preparation process of the Zhangzhou bacillus JSSW-BP44 fermented folium mori is simple, the fermented folium mori is used as an effective plant protein to be applied to feed, and a new direction is provided for the effective utilization of agricultural leaf resources.
Biological material sample preservation: zhangzhou Bacillus JSSW-BP44(Bacillus zhangzhzhouensis) JSSW-BP44, classified and named as Zhangzhou Bacillus (Bacillus zhangzhzhouensis), and the preservation number is CCTCC NO: m2019989, the preservation date is 12 months and 2 days in 2019, and the culture is preserved in China center for type culture Collection with the addresses as follows: wuhan university in Wuhan, China.
Drawings
FIG. 1 photograph of mulberry leaf powder before fermentation in example 4.
FIG. 2 photograph of mulberry leaf powder after fermentation in example 4.
Detailed Description
The technical solution of the present invention will be described in detail by specific examples.
The experimental methods in the following examples are all conventional methods unless otherwise specified; the experimental materials used, unless otherwise specified, were purchased from conventional biochemical manufacturers.
Example 1 screening and identification of Bacillus Zhangzhou (Bacillus zhangzhouensis) JSSW-BP44
(1) Screening: collecting a sample in a pond of the black carp in the Wuxi goose lake, weighing 10g of pond sediment in a 250mL triangular flask filled with 90mL of sterile water and a small amount of glass beads, oscillating for 30min, and standing. Taking supernatant, inoculating into cellulose culture medium and protease culture medium, culturing at 37 deg.C for 3-5 days, and separating strain containing cellulase and protease. Sucking 1.0mL of each sample, heating at 80 ℃ for 10min, sucking 0.1mL of each sample on a basal medium by a pipette, coating, and culturing at 37 ℃. Selecting single bacteria, repeatedly streaking and purifying for 3 times, randomly selecting and purifying colonies after the bacteria grow out, dibbling a cellulase culture medium and a protein culture medium, verifying the cellulase activity and the protease activity of the strains, performing a mulberry leaf powder fermentation performance test, selecting strains which have strong cellulase and protease activity, can well grow in the mulberry leaf powder fermentation culture medium, and have high crude protein content, small peptides and free amino acids after fermentation of the mulberry leaf powder, and numbering as JSSW-BP 44.
The basic culture medium consists of (g/L): 5g of glucose, 1g of peptone, 5g of beef extract, 5g of NaCl and pH 7.0.
The cellulose culture medium consists of (g/L): k2HPO40.50，MgSO4·7H2O0.25, CMC-Na1.88, Congo red 0.20, agar 16.00, gelatin 2.00, pH7.0.
The protein culture medium consists of (g/L): 5% of milk powder and 1.5% of agar powder.
The mulberry leaf powder fermentation medium comprises the following components in g/L: 50-100 parts of mulberry leaf powder, and is prepared by distilled water with constant volume and pH of 7.0-8.0.
(2) Strain identification
a. Morphological characteristics: the strain JSSW-BP44 grows on the surface of nutrient agar, and the colony features circular, jagged edge, milky white, smooth surface, opacity and no luster.
b. Biochemical characteristics of the strain JSSW-BP44 are capable of producing alkaline phosphatase, esterase, lipoid esterase, lipase, valine arylamine, chymotrypsin, naphthol-AS-BI-phosphate, β -glucosidase, and can utilize glycerol, L-arabinose, ribose, D-xylose, galactose, glucose, fructose, mannose, mannitol, arbutin, esculin, saligenin, cellobiose, melibiose, sucrose, trehalose, D-tagatose.
(3)16S rRNA sequence analysis and construction of phylogenetic trees
The 16S rRNA gene sequence of the strain JSSW-BP44, the length of the 16S rRNA gene is 1450BP, which is shown as SEQ ID NO. 1; performing Blast analysis on the gene sequence and a nucleic acid sequence known from GenBank, selecting a sequence with higher homology, performing sequence comparison on Cluster X software, and constructing a phylogenetic tree by using MEGA4.1 software after the comparison is finished.
Sequencing result of JSSW-BP44 gene sequence: homology search is carried out on the 16S rRNA gene sequence amplified by the strain at NCBI through Blast, the result is searched out to be the 16S rRNA gene sequence of lactobacillus, a strain molecular development tree is constructed by adopting an adjacent method, and the strain is separated from the strain on the phylogenetic tree to belong to the same branch (SEQ ID NO.1) as Zhangzhou Bacillus (accession number: JOTP 01000027). The isolated strain was identified as Zhangzhou bacillus by a combination of morphological and physiological biochemical characteristics.
Example 2 measurement of cellulase and protease Activity of Bacillus Zhangzhou (Bacillus zhangzhouensis) JSSW-BP44
Zhangzhou bacillus JSSW-BP44 is dibbled in a cellulose culture medium and a protein culture medium, and a transparent ring appears on a flat plate to show that the strain has the capability of producing cellulase and protease, and the formula Up ═ D/D2D is the diameter (mm) of the transparent ring, D is the diameter (mm) of the bacterial colony; specific results are shown in table 1.
TABLE 1 determination result of enzyme production characteristics of Zhangzhou bacillus
| Enzyme | Cellulase enzymes | Protease enzyme |
| d/mm | 6.12±0.88 | 7.02±0.46 |
| D/mm | 12.25±0.51 | 15.96±0.32 |
| Up | 4.00±0.58 | 5.15±0.49 |
Note: the diameter of the sheet was 0.6 cm.
From the results in table 1, the screened bacillus zhangzhou JSSW-BP44 has strong cellulase and protease activities, and can effectively degrade the indigestible macromolecules such as crude fiber and protein in the mulberry leaves.
Example 3 fermentation culture of Bacillus Zhangzhou (Bacillus zhangzhouensis) JSSW-BP44
(1) Activating strains: aseptically starting a freeze-dried preservation strain of Zhangzhou bacillus JSSW-BP44, streaking and inoculating the strain to a nutrient agar test tube inclined plane, culturing for 24-48h at 30-37 ℃, streaking and transferring to a nutrient agar eggplant bottle inclined plane, and culturing for 24-72h at 30-37 ℃; microscopic examination of spore formation in the thallus, namely maturity; repeatedly activating for 2-3 times to obtain seed bacteria suspension;
the composition of the slant culture medium is measured in g/L: 10 parts of peptone, 3 parts of beef extract, 5 parts of NaCl and 15-20 parts of agar, and the components are prepared by distilled water with constant volume and pH of 7.0-7.2;
(2) fermentation culture: inoculating the seed bacteria suspension obtained in the step (1) into a triangular flask filled with a fermentation culture medium according to the inoculation amount of 1-10% of the volume ratio, wherein the liquid filling amount of the triangular flask is 10-20% of the volume ratio, the rotating speed is 100-;
the fermentation medium composition is calculated by g/L: 50-100 parts of mulberry leaf powder, and is prepared by distilled water with constant volume and pH of 7.0-8.0.
Example 4 preparation method of fermented Mulberry leaf powder
(1) Crushing mulberry leaves: drying fresh folium Mori at 65 deg.C to constant weight (water content is 3.0%), grinding, and sieving with 60 mesh sieve; the obtained mulberry leaf powder is yellow green before fermentation as shown in FIG. 1.
(2) Preparing mulberry leaf pulp: 20g of the sieved mulberry leaf powder is added with 380mL of deionized water and stirred uniformly to obtain 5.0% mulberry leaf pulp.
(3) And (3) sterilization: adjusting pH of the mulberry leaf pulp obtained in the step 2) to 7.5 with 23% sodium hydroxide solution. Each 50mL of the mulberry leaf pulp is put into a 500mL triangular flask and sterilized in an autoclave at 121 ℃ for 30 min.
(4) Fermentation: cooling the sterilized mulberry leaf pulp to room temperature, and adding 1mL (10) of the sterilized mulberry leaf pulp into each triangular flask7CFU/mL) of the bacillus Zhangzhou suspension, and fermenting for 55 hours at 34 ℃.
(5) And (3) drying: after fermentation, freeze drying is carried out, the temperature is kept at minus 50 ℃, the vacuum degree is maintained at 30Pa, and drying is carried out for 28 hours, thus obtaining a finished product of the fermented mulberry leaf powder; the state of the fermented mulberry leaf powder is shown in FIG. 2, and the fermented mulberry leaf powder is green brown.
The crude protein content, the small peptide content and the free amino acid content of the mulberry leaf powder before fermentation and the mulberry leaf powder obtained after fermentation in example 4 were measured, respectively, and the specific results are shown in table 2.
TABLE 2 comparison of nutrients in raw mulberry leaves with fermented mulberry leaves of example 1
The results show that the contents of crude protein, small peptide and free amino acid in the mulberry leaf powder of the fermented feed are respectively increased by 22.2%, 70.0% and 26.2% compared with the evaluation before fermentation by implementing the example, and the content of nutrient components is obviously higher than that of the unfermented mulberry leaf powder.
Example 5 preparation of fermented Mulberry leaf powder
(1) Crushing mulberry leaves: drying fresh folium Mori at 65 deg.C to constant weight (water content of 3.5%), grinding, and sieving with 60 mesh sieve.
(2) Preparing mulberry leaf pulp: 20g of the sieved mulberry leaf powder is added with 330mL of deionized water and stirred uniformly to obtain 5.7% mulberry leaf pulp.
(3) And (3) sterilization: adjusting pH of the mulberry leaf pulp obtained in the step 2) to 7.8 with 25% sodium hydroxide solution. Each 60mL of the mulberry leaf pulp is put into a 500mL triangular flask and sterilized in an autoclave at 121 ℃ for 30 min.
(4) Fermentation: cooling the sterilized mulberry leaf pulp to room temperature, and inoculating 2mL (10 mL) of the sterilized mulberry leaf pulp into each triangular flask7CFU/mL) of the bacillus Zhangzhou suspension, and fermenting for 62 hours at 33 ℃.
(5) And (3) drying: after fermentation, freeze drying is carried out, the temperature is kept at-40 ℃, the vacuum degree is maintained at 40Pa, and drying is carried out for 36 hours, thus obtaining the finished product of the mulberry leaf fermented feed.
The crude protein content, the small peptide content and the free amino acid content of the mulberry leaf powder before fermentation and the mulberry leaf powder obtained after fermentation in example 5 were measured, respectively, and the specific results are shown in table 3.
TABLE 3 comparison of nutrients in raw mulberry leaves with fermented mulberry leaves of example 2
The results show that the contents of crude protein, small peptide and free amino acid in the mulberry leaf powder of the fermented feed are respectively increased by 18.6%, 59.2% and 28.4% compared with the evaluation before fermentation by implementing the example, and the content of nutrient components is obviously higher than that of the unfermented mulberry leaf powder.
Example 6 addition application effect of fermented mulberry leaf powder in aquatic animal feed in this example, 3 groups of compound feeds for experiments were designed, using fish meal, cottonseed meal, rapeseed meal, and soybean meal as main protein sources, and soybean oil as main fat sources, and using fermented mulberry leaf powder (protein content 18.1%) instead of 0%, 5%, and 10% soybean meal respectively, 3 groups of compound feeds for experiments were designed, and the feed formulas and nutritional levels were respectively a control group, a low-fermented mulberry leaf powder group, and a high-fermented mulberry leaf powder group, as shown in table 4. Table 4 test feed formulations used in this example
Note: fish meal, bean pulp, rapeseed meal, and cottonseed meal are provided by Tongwei corporation (China, Wuxi); choline chloride (50%), premix, vitamin C were provided by the stannless huanowei animal health products limited (china, stannless); zeolite powder, microcrystalline cellulose, monocalcium phosphate were provided by Shanghai Fei Atke technologies, Inc. (China, Shanghai).
Crushing fish meal, cottonseed meal, rapeseed meal, soybean meal and fermented mulberry leaf powder in daily ration according to a conventional method, sieving with a 40-mesh sieve, fully and uniformly mixing the fish meal, the soybean meal, the rapeseed meal, the cottonseed meal, wheat flour, microcrystalline cellulose, a premix, zeolite powder, monocalcium phosphate, choline chloride (50%), vitamin C and other raw materials by a gradual mixing method from few to many applications, adding soybean oil, stirring and uniformly mixing, preparing into granulated feed with the particle size of 2mm by using an F-26 small granulator, and airing at normal temperature for use.
Selecting healthy carassius auratus gibelio (initial weight 15.34 +/-0.59 g) and temporarily culturing 180 fish in 9 circular culture tanks (specification psi 820 multiplied by 700mm, 300L/tank), setting 3 times of experiments, and repeating 20 fish times of experiments, and filtering circulating water by taking activated carbon and corallite as biological filter materials. The feeding is carried out for 4 times at regular time every day in the culture test period, the feeding is respectively 8:00, 10:30, 13:30 and 16:00, the feeding is carried out after apparent satiety, and the culture period is 8 weeks. Controlling the temperature during the culture period to 26 +/-1 ℃, keeping the pH value at 7.2-7.8, keeping the dissolved oxygen content at more than or equal to 5mg/L, keeping the ammonia nitrogen concentration of the culture water body at less than or equal to 0.05mg/L, and keeping the nitrite concentration at less than or equal to 0.05 mg/L.
At the end of 8 weeks of culture, all test fish in the culture bucket were weighed and counted to compare the culture effect of different test feeds (table 5). The calculation formula is as follows:
the feed coefficient is the total feed intake/(total fish body final weight-total fish body initial weight)
Weight gain ratio (%) < 100 × (fish body end weight-fish body initial weight)/fish body initial weight
Specific growth (%/d) of 100 × (end weight of Ln fish body-initial weight of Ln fish body)/days of culture
Protein efficiency (%) ═ 100 x total weight gain of fish/(total feed intake x feed protein content)
Growth data were statistically analyzed using SPSS20.0, and control and fermented mulberry leaf powder groups were analyzed for significance using independent T-tests (marked by differences in the two groups, test level P < 0.05). The growth results are shown in table 5.
Table 5 growth effect of carassius auratus gibelio of this example
| Raw materials (g/kg) | Control group | Fermented mulberry leaf powder group |
| Coefficient of feed | 2.34±0.28 | 1.71±0.11* |
| Weight gain (%) | 97.38±5.68 | 136.29±8.11* |
| Specific growth (%/d) | 1.05±0.12 | 1.43±0.15* |
| Protein efficiency (%) | 105.28±3.89 | 123.63±2.95* |
Through the application of the embodiment, the fact that 15% of folium mori powder fermented by bacillus Zhangzhou is added into the carassius auratus gibelio compound feed can remarkably promote the weight gain rate, the specific growth rate and the protein efficiency of the carassius auratus gibelio, remarkably reduce the feed coefficient, reduce the feed input cost and improve the culture economic benefit.
Sequence listing
<110> research center of freshwater fishery of Chinese aquatic science research institute
Chilo biological research Co., Ltd, Jiangsu province Su
<120> Zhangzhou bacillus and method for fermenting mulberry leaf powder by using Zhangzhou bacillus
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>1450
<212>DNA
<213> 16S rRNA of Zhangzhou bacillus JSSW-BP44 (2 Ambystoma laterale x Ambystoma jeffersonia)
<400>1
ctctgtactt cggcggctgg ctccataaag gttacctcac cgacttcggg tgttgcaaac 60
tctcgtggtg tgacgggcgg tgtgtacaag gcccgggaac gtattcaccg cggcatgctg 120
atccgcgatt actagcgatt ccagcttcac gcagtcgagt tgcagactgc gatccgaact 180
gagaacagat ttatgggatt ggctaaacct tgcggtctcg cagccctttg ttctgtccat 240
tgtagcacgt gtgtagccca ggtcataagg ggcatgatga tttgacgtca tccccacctt 300
cctccggttt gtcaccggca gtcaccttag agtgcccaac taaatgctgg caactaagat 360
caagggttgc gctcgttgcg ggacttaacc caacatctca cgacacgagc tgacgacaac 420
catgcaccac ctgtcactct gtccccgaag ggaaagcccc tatctctagg gttgtcagag 480
gatgtcaaga cctggtaagg ttcttcgcgt tgcttcgaat taaaccacat gctccaccgc 540
ttgtgcgggc ccccgtcaat tcctttgagt ttcagtcttg cgaccgtact ccccaggcgg 600
agtgcttaat gcgttagacg cagcactaag gggcggaaac cccctaacac ttagcactca 660
tcgtttacgg cgtggactac cagggtatct aatcctgttc gctccccacg ctttcgctcc 720
tcagcgtcag ttacagacca gagagtcgcc ttcgccactg gtgttcctcc acatctctac 780
gcatttcacc gctacacgtg gaattccact ctcctcttct gcactcaagt ttcccagttt 840
ccaatgaccc tccccggttg agccgggggc tttcacatca gacttaagaa accgcctgcg 900
agccctttac gcccaataat tccggacaac gcttgccacc tacgtattac cgcggctgct 960
ggcacgtagt tagccgtggc tttctggtta ggtaccgtca aggtgcgaag cagttactct 1020
cgcacttgtt cttccctaac aacagagctt tacgatccga aaaccttcat cactcacgcg 1080
gcgttgctcc gtcagacttt cgtccattgc ggaagattcc ctactgctgc ctcccgtagg 1140
agtctgggcc gtgtctcagt cccagtgtgg ccgatcaccc tctcaggtcg gctacgcatc 1200
gtcgccttgg tgagccatta ccccaccaac tagctaatgc gccgcgggtc catctgtaag 1260
tgacagccga aaccgtcttt catccttgaa ccatgcggtt caaggaacta tccggtatta 1320
gctccggttt cccggagtta tcccagtctt acaggcaggt tacccacgtg ttactcaccc 1380
gtccgccgct aacatccggg agcaagctcc cttctgtccg ctcgacttgc atgtatagca 1440
cgccgcccgt 1450
Claims (9)
1. A preparation method of fermented mulberry leaf powder is characterized by comprising the following steps:
(1) crushing mulberry leaves: drying fresh mulberry leaves to constant weight, crushing and sieving to obtain mulberry leaf powder;
(2) preparing mulberry leaf pulp: adding water into the mulberry leaf powder obtained in the step (1) and uniformly stirring to obtain mulberry leaf pulp;
(3) and (3) sterilization: adjusting the pH value of the mulberry leaf pulp obtained in the step (2), and sterilizing;
(4) fermentation: inoculating a fermentation strain into the sterilized mulberry leaf pulp obtained in the step (4) for fermentation;
(5) and (3) drying: and (4) after fermentation, carrying out freeze drying to obtain a finished product of the fermented mulberry leaf powder.
2. The method for preparing fermented mulberry leaf powder according to claim 1, wherein the fermentation step comprises the following steps: in the step (1), the fresh mulberry leaves are harvested in 4 months per year; drying at 60-65 deg.C to constant weight, specifically containing 2.0-4.0% water; pulverizing folium Mori at 20000-24000 r/min for 2-10min, and sieving with 60 mesh sieve to obtain folium Mori powder.
3. The method for preparing fermented mulberry leaf powder according to claim 1, wherein the fermentation step comprises the following steps: and (3) adding 940 and 950mL of water into every 50-60g of the sieved mulberry leaf powder in the step (2), and fully and uniformly stirring to prepare the mulberry leaf pulp.
4. The method for preparing fermented mulberry leaf powder according to claim 1, wherein the fermentation step comprises the following steps: and (3) adjusting the pH value of the mulberry leaf pulp to 7.0-8.0 by adopting a sodium hydroxide solution with the mass concentration of 20-30% and a hydrochloric acid solution, and then carrying out autoclaving at 121 ℃ for 30 min.
5. The method for preparing fermented mulberry leaf powder according to claim 1, wherein the fermentation step comprises the following steps: step (4) adopts Zhangzhou spore rodsFermenting the sterilized mulberry leaf pulp by using JSSW-BP 44; the content of viable bacteria of 1-2mL is 10 per 50mL of mulberry leaf pulp7CFU/mL Zhangzhou bacillus suspension, wherein the fermentation conditions comprise a material-water ratio of 5.0-6.0%, a liquid filling amount of a fermentation bottle of 10.0-12.0%, a fermentation time of 59-62h and a fermentation temperature of 33-35 ℃.
6. The method for preparing fermented mulberry leaf powder according to claim 1, wherein the fermentation step comprises the following steps: in the step (5), the temperature of freeze drying is-40 to-60 ℃, the vacuum degree is 30 to 40Pa, and the drying time is 24 to 36 hours.
7. Zhangzhou bacillus JSSW-BP 44: (Bacillus zhangzhouensis) JSSW-BP44, classified and named as Zhangzhou bacillus (Zhangzhou bacillus) (Zhangzhou bacillus)Bacillus zhangzhouensis) The preservation number is CCTCC NO: m2019989, the preservation date is 12 months and 2 days in 2019, and the culture is preserved in China center for type culture Collection with the addresses as follows: wuhan university in Wuhan, China.
8. The 16S rRNA gene sequence of the Zhangzhou bacillus JSSW-BP44 is shown as SEQ ID No. 1.
9. The application of the fermented mulberry leaf powder is characterized in that: the fermented mulberry leaf powder is used as a raw material to be applied to the preparation of aquatic feeds.
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