CN111134231B - Bacillus Zhangzhou and method for fermenting mulberry leaf powder by using same - Google Patents
Bacillus Zhangzhou and method for fermenting mulberry leaf powder by using same Download PDFInfo
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- CN111134231B CN111134231B CN202010066397.XA CN202010066397A CN111134231B CN 111134231 B CN111134231 B CN 111134231B CN 202010066397 A CN202010066397 A CN 202010066397A CN 111134231 B CN111134231 B CN 111134231B
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- mulberry leaf
- bacillus
- leaf powder
- zhangzhou
- fermented
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Abstract
The invention relates to bacillus Zhangzhou and a method for fermenting mulberry leaf powder by using the bacillus Zhangzhou, and belongs to the technical field of fermented feeds. The bacillus Zhangzhou JSSW-BP 44%Bacillus zhangzhouensis) JSSW-BP44 is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2019989. The bacillus Zhangzhou JSSW-BP44 can be used for liquid fermentation of feed mulberry leaf powder, the obtained fermented mulberry leaf powder contains rich protein, small peptide, amino acid, vitamin, polysaccharide and other nutrient substances, and compared with Sang Shelai, tannin, phytic acid and other anti-nutrient factors are obviously reduced, so that the absorption of livestock and aquatic animals to the nutrient substances can be improved, and the growth performance and disease resistance of the livestock and aquatic animals can be improved.
Description
Technical Field
The invention relates to bacillus Zhangzhou and a method for fermenting mulberry leaf powder by using the bacillus Zhangzhou, and belongs to the technical field of fermented feeds.
Background
The deciduous tree of Moraceae Morus can be planted and grown normally in most areas of China. Mulberry is the main product of mulberry, and has abundant protein content (15% -28%) and high amino acid content (34.7%), and also has abundant mineral substances and various bioactive components such as 1-Deoxynojirimycin (DNJ), flavone, polyphenols and the like. Wherein DNJ can regulate blood sugar level of organism, and flavone and polyphenol substances can reduce inflammatory factor level by improving oxidation resistance of organism, inhibiting lipid peroxidation, and relieving inflammatory reaction, thereby enhancing disease resistance of organism and improving lipid metabolism function. The ministry of health in China recognizes mulberry leaves as a plant resource of 'homology of medicine and food' at the beginning of the 20 th century. At present, research indicates that Sang Sheneng can be used as a vegetable protein source to be applied to compound feeds of fish such as tilapia and grass carp so as to save the application of high-value protein sources such as fish meal and bean pulp, but when a large amount of mulberry leaf powder is added, the high-content cellulose content, tannin, phytic acid and other anti-nutritional factors have an inhibition effect on the growth performance and the antioxidation function of fish bodies in use.
Chinese patent No. CN201910640354.5 discloses a strain of Bacillus zhuzhou (Bacillus zhangzhouensis) Z-XWW for degrading ammonia nitrogen and application thereof in degrading ammonia nitrogen. The invention evaluates the ammonia nitrogen degradation capability from 196 marine strains, screens out bacillus Zhangzhou (Bacillus zhangzhouensis) Z-XWW77, and is used for degrading ammonia nitrogen in ammonia-containing pollutants such as sewage, wastewater, household garbage and animal feces, wherein the treatment condition is that the temperature is 25-45 ℃ and the pH is 6-7.5.
The present invention aims at decomposing macro-molecules, such as crude fiber and protein, in mulberry leaves, which are difficult to digest into small-molecule nutrients which are easy to be absorbed by the body by means of enzyme systems produced by microorganisms through a microbial fermentation method. The anti-nutritional factors such as tannin, phytic acid and the like can be digested by microorganisms in the fermentation process, so that the palatability and the nutritional value of a fermentation product are improved, and the application range of the mulberry leaf powder in aquatic animals is further enlarged and promoted.
Disclosure of Invention
The invention aims to overcome the defects, and provides bacillus Zhangzhou and a method for fermenting mulberry leaf powder by using the bacillus Zhangzhou, and the prepared fermented mulberry leaf powder has higher nutrition substances such as protein, free amino acid, small peptide and the like than unfermented mulberry leaf powder, so that the feeding nutrition value of the mulberry leaf powder is improved.
The technical scheme of the invention is that the preparation method of the 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 slurry;
(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 in the step (4) for fermentation;
(5) And (3) drying: and (5) freeze-drying after fermentation is finished, so that a finished product of the fermented mulberry leaf powder can be obtained.
Further, in the step (1), the mulberry leaves are dried to constant weight at the temperature of 60-65 ℃, crushed for 2-10min at the rotating speed of 20000-24000 r/min, and sieved by a 60-mesh sieve, so as to obtain the mulberry leaf powder.
Further, the fresh mulberry leaves are fresh mulberry leaves harvested every 4 months of the year.
Further, the drying to constant weight specifically contains 2.0% -4.0% of water.
Further, 940-950mL of water is added into every 50-60g of the sieved mulberry leaf powder in the step (2), and the mulberry leaf powder is fully and uniformly stirred to prepare the mulberry leaf slurry.
Further, in the step (3), the pH of the mulberry leaf slurry is adjusted to 7-8 by adopting a sodium hydroxide solution and a hydrochloric acid solution with the mass concentration of 20-30%, and then the mulberry leaf slurry is autoclaved for 30min at the temperature of 121 ℃.
Further, in the step (4), the sterilized mulberry leaf pulp is fermented by adopting bacillus zhuzhou JSSW-BP 44; 1-2mL of living bacteria with the content of 10 are inoculated into each 50mL of mulberry leaf pulp 7 The fermentation condition of the bacillus Zhangzhou suspension with CFU/mL is that the material-water ratio is 5.0% -6.0%, the liquid filling amount of a fermentation bottle is 10.0% -12.0%, the fermentation time is 48-72h, and the fermentation temperature is 33-35 ℃.
Further, the microorganism strain in the step (4) is bacillus zhuzhou (Bacillus zhangzhouensis) JSSW-BP44, and is classified and named as bacillus zhuzhou (bacillus), and the preservation number is CCTCC NO:2019989, the date of preservation is 2019, 12 months and 2 days, and the Chinese typical culture collection is provided with the following addresses: university of martial arts in chinese.
Bacillus Zhangzhou JSSW-BP44 (Bacillus zhangzhouensis) JSSW-BP44 has the biochemical characteristics of being capable of producing alkaline phosphatase, esterase, lipoid esterase, lipase, valine arylamine enzyme, chymotrypsin, naphthol-AS-BI-phosphate and beta-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 freeze-drying temperature in the step (5) is-40 to-60 ℃, the vacuum degree is 30-40Pa, and the drying time is 24-36h.
The crude protein content in the fermented mulberry leaf powder prepared by the invention is 18-20%, the free amino acid content is 22-25mg/g, and the small peptide content is 205-220mg/g.
The application of the fermented mulberry leaf powder is that the fermented mulberry leaf powder is used as one of raw materials to be applied to the preparation of aquatic feed.
The invention has the beneficial effects that: according to the invention, a strain of bacillus Zhangzhou (Bacillus zhangzhouensis) 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 feeding attraction effect and good palatability, and the content of crude protein, small peptide and free amino acid in the fermented mulberry leaf powder is increased by 18.6% -22%, 59.2% -70% and 26.2% -28.4% respectively compared with the content of the crude protein, small peptide and free amino acid in the fermented mulberry leaf powder evaluated before fermentation, and the content of nutritional ingredients is obviously higher than that of the unfermented mulberry leaf powder, so that the absorption of livestock and poultry aquatic animals to nutritional substances can be improved, and the growth performance and disease resistance of the livestock and poultry aquatic animals can be improved.
The fermented mulberry leaf powder is added into the aquatic animal feed, so that plant proteins such as bean pulp can be partially replaced, the weight gain rate, specific growth rate and protein efficiency of the carassius auratus gibelio are obviously improved, the feed coefficient is obviously reduced, the feed input cost is reduced, and the cultivation economic benefit is improved.
The mulberry leaves and other leaf raw materials have wide sources and low price, the preparation process of the bacillus Zhangzhou JSSW-BP44 fermented mulberry leaves is simple, the fermented mulberry leaves are used as an effective plant protein in feed, and a new direction is provided for effective utilization of agricultural leaf resources.
Preservation of biological material samples: bacillus Zhangzhou JSSW-BP44 (Bacillus zhangzhouensis) JSSW-BP44 is classified and named as Bacillus zhangzhou (Bacillus) with a preservation number of CCTCC NO:2019989, the date of preservation is 2019, 12 months and 2 days, and the Chinese typical culture collection is provided with the following addresses: university of martial arts in chinese.
Drawings
FIG. 1A photograph of the morphology of mulberry leaf powder before fermentation in example 4.
FIG. 2A photograph of the morphology of mulberry leaf powder after fermentation in example 4.
Detailed Description
The technical scheme 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 specified, are all purchased from conventional biochemical reagent manufacturers.
Example 1 screening and identification of Bacillus Zhangzhou (Bacillus zhangzhouis) JSSW-BP44
(1) Screening: the sample is collected in a tin-free goose lake black carp pond, 10g of pond sediment is weighed into a 250mL triangular flask filled with 90mL of sterile water and a small amount of glass beads, and the mixture is oscillated for 30min and kept stand. Taking supernatant, inoculating in cellulose culture medium and protease culture medium, culturing at 37deg.C for 3-5 days, and separating strain with cellulase and protease. Each sample was pipetted 1.0mL, heated at 80℃for 10min, pipette was used to aspirate 0.1mL onto basal medium, spread, and incubated at 37 ℃. Selecting single bacteria, repeatedly streaking and purifying for 3 times, randomly selecting colonies after bacterial colonies grow out, purifying, then dibbling a cellulase culture medium and a protein culture medium, verifying the cellulase activity and the protease activity of the strain, performing a mulberry leaf powder fermentation performance test, selecting a strain which has strong cellulase and protease activity and can well grow in the mulberry leaf powder fermentation culture medium and has high crude protein content, small peptide and free amino acid content after fermentation of the mulberry leaf powder, and numbering as JSSW-BP44.
The basic culture medium composition is (g/L): glucose 5g, peptone 1g, beef extract 5g,NaCl 5g,pH 7.0.
The cellulose culture medium composition is (g/L): k (K) 2 HPO 4 0.50，MgSO 4 ·7H 2 O0.25, CMC-Na1.88, congo red 0.20, agar 16.00, gelatin 2.00, pH7.0.
The composition of the protein culture medium is (g/L): milk powder 5% and agar powder 1.5%.
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 the pH value is 7.0-8.0.
(2) Identification of species
a. Morphological characteristics: the strain JSSW-BP44 grows on the surface of nutrient agar, and the colony is characterized by being round, with sawtooth-shaped edges, milky white, smooth in surface, opaque and matt.
b. Biochemical characteristics: the strain JSSW-BP44 can produce alkaline phosphatase, esterase, lipoid esterase, lipase, valine arylaminase, chymotrypsin, naphthol-AS-BI-phosphate and beta-glucosidase, and can utilize glycerol, L-arabinose, ribose, D-xylose, galactose, glucose, fructose, mannose, mannitol, arbutin, esculin, saligenin, cellobiose, melibiose, sucrose, trehalose and D-tagatose.
(3) 16S rRNA sequence analysis and phylogenetic tree construction
The 16S rRNA gene sequence of the strain JSSW-BP44, the length of the 16S rRNA gene is 1450BP, and is shown as SEQ ID NO. 1; and (3) performing Blast analysis on the sequences with high homology with the known nucleic acid sequences in GenBank, selecting the sequences with high homology, performing sequence alignment in Cluster X software, and constructing a phylogenetic tree by using MEGA4.1 software after the alignment is finished.
Sequencing result of the strain JSSW-BP44 gene sequence: the 16S rRNA gene sequence amplified by the strain is subjected to homology search at NCBI through Blast, and the 16S rRNA gene sequence of lactobacillus is searched out as a result, a molecular development tree of the strain is constructed by adopting an adjacent method, and the isolated strain belongs to the same branch (SEQ ID NO. 1) with Bacillus zhangzhouensis (accession number: JOTP 01000027) on the phylogenetic tree. The isolated strain was identified as bacillus zhangzhou by combining morphological and physiological biochemical characteristics.
EXAMPLE 2 measurement of cellulase and protease Activity of Bacillus Zhangzhou (Bacillus zhangzhouensis) JSSW-BP44
The bacillus Zhangzhou JSSW-BP44 is inoculated on a cellulose culture medium and a protein culture medium, and transparent circles appear on a flat plate to show that the strain has the capability of producing cellulase and protease, and the formula Up= (D/D) is used for preparing the strain 2 To indicate the hydrolysis capacity, D is the diameter of the transparent ring (mm), and D is the diameter of the colony (mm); the specific results are shown in Table 1.
TABLE 1 determination of Bacillus Zhangzhou enzyme production characteristics
| Enzymes | 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 that: the sheet diameter was 0.6cm.
As can be seen from the results in Table 1, the bacillus Zhangzhou JSSW-BP44 obtained by screening has strong cellulase and protease activities, and can effectively degrade macro-molecules such as crude fibers, proteins and the like in the mulberry leaves, which are difficult to digest.
Example 3 fermentation culture of Bacillus Zhangzhou (Bacillus zhangzhouensis) JSSW-BP44
(1) Activating strains: sterile opening freeze-dried deposited strain of bacillus Zhangzhou JSSW-BP44, streaking and inoculating to a nutrient agar test tube inclined plane, culturing for 24-48h at 30-37 ℃, and then streaking and transferring to a nutrient agar eggplant bottle inclined plane, and culturing for 24-72h at 30-37 ℃; sporulation in the microscopic thallus, namely maturation; repeatedly activating for 2-3 times to obtain seed bacterial suspension;
the composition of the slant culture medium is calculated in g/L: peptone 10, beef extract 3, naCl 5, agar 15-20, distilled water to constant volume, pH 7.0-7.2;
(2) Fermentation culture: inoculating the seed bacterial suspension obtained in the step (1) into a triangular flask filled with a fermentation culture medium according to the volume ratio of 1% -10%, wherein the volume ratio of the liquid in the triangular flask is 10% -20%, and the rotation speed is 100-180rpm, and carrying out constant-temperature shaking culture for 48-72h at 30-37 ℃ to obtain fermentation liquor;
fermentation medium composition in g/L: 50-100 parts of mulberry leaf powder, and is prepared by distilled water with constant volume, and the pH value is 7.0-8.0.
Example 4 preparation method of fermented mulberry leaf powder
(1) Crushing mulberry leaves: drying fresh mulberry leaves at 65 ℃ to constant weight (water content is 3.0%), grinding and sieving with a 60-mesh sieve; the obtained mulberry leaf powder is shown in figure 1, and has yellow green color before fermentation.
(2) Preparation of mulberry leaf pulp: and adding 380mL deionized water into 20g of the sieved mulberry leaf powder, and uniformly stirring to obtain 5.0% mulberry leaf slurry.
(3) And (3) sterilization: the pH of the mulberry leaf slurry obtained in step 2) was adjusted to 7.5 with a 23% sodium hydroxide solution. After filling 50mL of mulberry leaf slurry into 500mL triangular flasks, the mulberry leaf slurry was sterilized in a high pressure steam sterilizer at 121℃for 30min.
(4) Fermentation: the sterilized mulberry leaf slurry was cooled to room temperature and 1mL (10) was inoculated into each flask 7 CFU/mL) was subjected to liquid fermentation at 34 ℃ for 55h.
(5) And (3) drying: freeze drying after fermentation, maintaining the temperature at-50deg.C and vacuum degree at 30Pa, and drying for 28 hr to obtain fermented folium Mori powder; the state of the fermented mulberry leaf powder is shown in figure 2, and the fermented mulberry leaf powder is greenish brown.
The crude protein content, small peptide content and free amino acid content of the mulberry leaf powder before fermentation and the mulberry leaf powder obtained after fermentation in example 4 were respectively measured, and specific results are shown in table 2.
TABLE 2 nutrient comparison of unprocessed mulberry leaves with fermented mulberry leaves of example 1
The results show that the crude protein, small peptide and free amino acid contents in the mulberry leaf powder of the fermented feed are increased by 22.2%, 70.0% and 26.2% respectively compared with the evaluation before fermentation, and the nutrient content is obviously higher than that of the unfermented mulberry leaf powder.
EXAMPLE 5 preparation method of fermented mulberry leaf powder
(1) Crushing mulberry leaves: fresh mulberry leaves were dried to constant weight (3.5% moisture) at 65 c and then ground and sieved through a 60 mesh sieve.
(2) Preparation of mulberry leaf pulp: and adding 330mL of deionized water into 20g of the sieved mulberry leaf powder, and uniformly stirring to obtain 5.7% mulberry leaf slurry.
(3) And (3) sterilization: the pH of the mulberry leaf slurry obtained in step 2) was adjusted to 7.8 with 25% sodium hydroxide solution. After filling 500mL triangular flask with each 60mL mulberry leaf slurry, sterilizing in a high-pressure steam sterilizing pot at 121 ℃ for 30min.
(4) Fermentation: the sterilized mulberry leaf slurry was cooled to room temperature and 2mL (10) was inoculated into each flask 7 CFU/mL) was subjected to liquid fermentation at 33 ℃ for 62h.
(5) And (3) drying: and (3) freeze-drying after fermentation, maintaining the temperature at-40 ℃ and the vacuum degree at 40Pa, and drying for 36 hours to obtain the finished product of the fermented feed mulberry leaves.
The crude protein content, small peptide content and free amino acid content of the mulberry leaf powder before fermentation and the mulberry leaf powder obtained after fermentation in example 5 were respectively measured, and specific results are shown in table 3.
TABLE 3 nutrient comparison of raw mulberry leaves with fermented mulberry leaves of example 2
The results show that the crude protein, small peptide and free amino acid contents in the mulberry leaf powder of the fermented feed are increased by 18.6%, 59.2% and 28.4% respectively compared with those evaluated before fermentation, and the nutrient content is obviously higher than that of the unfermented mulberry leaf powder.
EXAMPLE 6 additive application Effect of fermented mulberry leaf powder in aquatic animal feed
In this example, 3 groups of compound feeds for experiments were designed, wherein fish meal, cotton meal, rapeseed meal and soybean meal are used as main protein sources, soybean oil is used as main fat sources, and fermented mulberry leaf powder (protein content 18.1%) is used to replace 0%, 5% and 10% of soybean meal respectively, 3 groups of compound feeds for experiments were designed, namely a control group, a low-fermentation mulberry leaf powder group and a high-fermentation mulberry leaf powder group, and the feed formula and the nutrition level are shown in Table 4. Table 4 test feed formulation used in this example
Note that: fish meal, soybean meal, rapeseed meal, cotton meal are supplied by Tongwei stock, inc. (China, no tin); choline chloride (50%), premix, vitamin C were supplied by the tinless Hua Nuowei animal health products limited (china, tinless); zeolite powder, microcrystalline cellulose and monocalcium phosphate are supplied by Shanghai feiya technology limited (china, shanghai).
According to the conventional method, the fish meal, the cotton meal, the rapeseed meal, the soybean meal and the fermented mulberry leaf powder in the daily ration are crushed and then are sieved by a 40-mesh sieve, the raw materials of the fish meal, the soybean meal, the rapeseed meal, the cotton meal, the wheat flour, the microcrystalline cellulose, the premix, the zeolite powder, the monocalcium phosphate, the choline chloride (50%), the vitamin C and the like are fully and uniformly mixed from few to many, the soybean oil is added after the raw materials are fully and uniformly mixed by a step-by-step mixing method, and the mixture is prepared into a pellet feed with the particle size of 2mm by an F-26 small granulator after being stirred and uniformly mixed, and the pellet feed is used after being dried at normal temperature.
180 carassius auratus gibelio (initial weight 15.34+/-0.59 g) with healthy physique is selected and temporarily cultivated in 9 circular cultivation barrels (specification psi 820X 700mm, 300L/barrel), 3 repeats are arranged for each test group, 20 fish are repeated, and the circulating water is filtered by using activated carbon and coral stone as biological filter materials. The feeding was timed 4 times per day during the cultivation test, 8:00, 10:30, 13:30 and 16:00 respectively, the apparent satiety feeding was performed, and the cultivation period was 8 weeks. The temperature is controlled during the cultivation period, the temperature is 26+/-1 ℃, the pH value is kept at 7.2-7.8, the content of dissolved oxygen is more than or equal to 5mg/L, the ammonia nitrogen concentration of the cultivation water body is less than or equal to 0.05mg/L, and the nitrite concentration is less than or equal to 0.05mg/L.
At the end of 8 weeks of cultivation, all test fish in the cultivation barrels were weighed and counted and the cultivation effect of the different test feeds was compared (table 5). The calculation formula is as follows:
feed coefficient = total feed intake/(total fish tail-total initial fish weight)
Weight gain (%) =100× (fish body weight last-fish body weight initial)/fish body weight initial
Specific growth (%/d) =100× (Ln body weight-Ln body initial weight)/days of cultivation
Protein efficiency (%) =100×total weight gain of fish/(total feed intake×feed protein content)
The growth data were statistically analyzed by SPSS20.0 and the control and fermented mulberry leaf meal groups were subjected to significance analysis by independent T-test (x represents significant differences between the two groups at a test level P < 0.05). The results of the growth are shown in Table 5.
TABLE 5 Carassius gibelio growth effects of this example
| Raw material (g/kg) | Control group | Fermented mulberry leaf powder group |
| Feed coefficient | 2.34±0.28 | 1.71±0.11 * |
| Weight gain Rate (%) | 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 addition of 15% of mulberry leaf powder fermented by bacillus alzhuzhou in the compound feed for the carassius auratus gibelio can obviously promote the weight gain rate, the specific growth rate and the protein efficiency of the carassius auratus gibelio, obviously reduce the feed coefficient, reduce the feed input cost and improve the cultivation economic benefit.
Sequence listing
<110> fresh water fishery research center of China aquatic science institute
JIANGSU SUWEI MICROBIOLOGY RESEARCH Co.,Ltd.
<120> Bacillus Zhangzhou strain and method for fermenting mulberry leaf powder by using same
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<170> SIPOSequenceListing 1.0
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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 (7)
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 slurry;
(3) And (3) sterilization: adjusting the pH value of the mulberry leaf pulp obtained in the step (2), and sterilizing;
(4) Fermentation: adopts Zhangzhou sporeFermenting the sterilized mulberry leaf pulp by bacillus JSSW-BP 44; 1-2mL viable bacteria per 50mL mulberry leaf pulp is inoculated with 10 7 The fermentation condition of the bacillus Zhangzhou suspension with CFU/mL is that the material-water ratio is 5.0% -6.0%, the liquid filling amount of a fermentation bottle is 10.0% -12.0%, the fermentation time is 59-62h, and the fermentation temperature is 33-35 ℃;
the bacillus Zhangzhou is preparedBacillus zhangzhouensis) JSSW-BP44 classified and named as bacillus Zhangzhou @Bacillus zhangzhouensis) The preservation number is CCTCC NO:2019989, the date of preservation is 2019, 12 months and 2 days, and the Chinese typical culture collection is provided with the following addresses: university of martial arts in chinese;
(5) And (3) drying: and (5) freeze-drying after fermentation is finished, so that a finished product of the fermented mulberry leaf powder can be obtained.
2. The method for preparing the fermented mulberry leaf powder according to claim 1, wherein: in the step (1), the fresh mulberry leaves are fresh mulberry leaves harvested every 4 months of the year; drying at 60-65 deg.c to constant weight, and with water content of 2.0-4.0%; pulverizing folium Mori at 20000r/min-24000r/min for 2-10min, and sieving with 60 mesh sieve to obtain folium Mori powder.
3. The method for preparing the fermented mulberry leaf powder according to claim 1, wherein: and (3) adding 940-950mL 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 slurry.
4. The method for preparing the fermented mulberry leaf powder according to claim 1, wherein: and (3) adjusting the pH of the mulberry leaf pulp to 7.0-8.0 by adopting a sodium hydroxide solution and a hydrochloric acid solution with the mass concentration of 20-30%, and then autoclaving for 30min at 121 ℃.
5. The method for preparing the fermented mulberry leaf powder according to claim 1, wherein: the temperature of freeze drying in the step (5) is-40 to-60 ℃, the vacuum degree is 30-40Pa, and the drying time is 24-36h.
6. The method for preparing the fermented mulberry leaf powder according to claim 1, wherein: the 16S rRNA gene sequence of the bacillus Zhangzhou JSSW-BP44 is shown as SEQ ID No. 1.
7. The application of the fermented mulberry leaf powder prepared by the method of claim 1, which is characterized in that: the fermented mulberry leaf powder is used as a raw material to be applied to the preparation of aquatic feed.
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