Method for preparing tea residue feed by fermenting mixed strains
阅读说明：本技术 一种混合菌株发酵制备茶渣饲料的方法 (Method for preparing tea residue feed by fermenting mixed strains ) 是由 黄炜乾 蒋顺进 张文 周绍迁 林万华 张泳 林昊 于 2020-08-06 设计创作，主要内容包括：本发明涉及动物饲料技术领域,具体涉及一种混合菌株发酵制备茶渣饲料的方法。所述的方法包括如下步骤：S1.ARTP诱变及驯化筛选功能性微生物；S2.突变菌株的混合发酵。本发明以开发地源性的绿茶茶渣制备功能性的菌茶生物饲料为目的,采用ARTP诱变及驯化筛选的方法进行功能性微生物的选育,并建立“一罐多菌”混合培养工艺,同时配以合适的辅料组合,促进混合功能微生物的繁殖,结合外源酶的协同,消化茶渣中的大分子,释放茶中的活性成分,进一步挖掘原料利用空间,充分强化固体发酵中的群落优势,提供高含量的益生菌及其功能性活性物质,有效的实现茶渣资源化利用,维护动物肠道健康,为健康养殖带来益处。(The invention relates to the technical field of animal feed, in particular to a method for preparing tea residue feed by fermenting mixed strains. The method comprises the following steps: s1, performing ARTP mutagenesis, domestication and screening on functional microorganisms; s2, mixed fermentation of mutant strains. The invention aims to develop the ground-source green tea residues to prepare the functional bacterial tea biological feed, adopts the ARTP mutagenesis and domestication screening method to breed the functional microorganisms, establishes a one-pot multi-bacteria mixed culture process, is matched with a proper auxiliary material combination to promote the propagation of the mixed functional microorganisms, combines the cooperation of exogenous enzyme to digest macromolecules in the tea residues, releases active ingredients in the tea, further excavates the raw material utilization space, fully strengthens the community advantage in solid fermentation, provides high-content probiotics and functional active substances thereof, effectively realizes the resource utilization of the tea residues, maintains the intestinal health of animals, and brings benefits for the healthy culture.)
1.A method for preparing tea residue feed by fermenting mixed strains is characterized by comprising the following steps:
s1, ARTP mutagenesis and domestication screening of functional microorganisms: adopting ARTP to mutate bacillus pumilus (Bacillus pumilus) and Lactobacillus plantarum (Lactobacillus plantarum), inoculating the obtained mutant strain into a domestication culture medium, and screening to obtain the mutant strain which can tolerate high-concentration tea leaves by gradually increasing the content of the tea leaves in the domestication culture medium;
s2, mixed fermentation of mutant strains, namely uniformly mixing the mutant strains which are obtained in the step S1 and can tolerate the high-concentration tea residues to obtain the total number of bacterial colonies of 0.5 × 106-1.5×106cfu/g of mixed bacterial liquid; mixing the mixed bacterial liquid with a fermentation substrate according to a weight ratio of 4-6: 94-96 to obtain a mixed fermentation substrate; adding cellulase, hemicellulase, xylanase and pectinase into the mixed fermentation substrate, adjusting the water content of the mixed fermentation substrate to 35-45%, and fermenting at 20-30 deg.C for 7-10 days to obtain tea residue feed.
2. The method according to claim 1, wherein the mutant strain tolerant to high concentration of tea leaves is Bacillus pumilus (Bacillus pumilus) BP-09 and Lactobacillus plantarum (Lactobacillus plantarum) LP-08;
the Bacillus pumilus (Bacillus pumilus) BP-09 is preserved in Guangdong province microbial culture collection center (GDMCC) at 6-16 th month in 2020, and the preservation number is GDMCC No: 61062, respectively;
the Lactobacillus plantarum (Lactobacillus plantarum) LP-08 is preserved in Guangdong province microbial culture collection center (GDMCC) 6-16 th 2020, with the preservation number of GDMCC No: 61061.
3. the method according to claim 1, wherein the conditions of ARTP mutagenesis in step S1 are as follows: the power of the power supply is 60W, the irradiation distance is 3mm, the temperature of the plasma is 26 ℃, the air flow is 10L/min, the ARTP treatment time of the bacillus pumilus is 120s, and the ARTP treatment time of the lactobacillus plantarum is 60 s.
4. The method according to claim 1, wherein the high concentration of tea leaves in step S1 is obtained by using 20% tea leaves extract in the acclimatization medium.
5. The method according to claim 2, wherein the ratio of the bacillus brevis BP-09 to the lactobacillus plantarum LP-08 in the mixed bacterial liquid is 1: 1.5.
6. the method according to claim 1, wherein the weight ratio of the mixed bacterial liquid to the fermentation substrate is 5: 95.
7. the method as claimed in claim 1, wherein the fermentation substrate comprises tea residue, defatted rice bran and soybean meal in a mass ratio of 7: 2: 1.
8. the method according to claim 1, wherein the mixed fermentation substrate has a final cellulase concentration of 300 μ/g, a final hemicellulase concentration of 300 μ/g, a final xylanase concentration of 200 μ/g, and a final pectinase concentration of 200 μ/g.
9. The method of claim 1, wherein the mixed fermentation substrate has a moisture content of 40-45%.
10. The method of claim 1, wherein the mixed fermentation substrate is fermented at a temperature of 25-30 ℃ for a period of 8-9 days.
Technical Field
The invention relates to the technical field of animal feed, in particular to a method for preparing tea residue feed by fermenting mixed strains.
Background
China is a big tea-producing country, the yield of the dried and raw tea leaves in 2018 is over 260 ten thousand tons, the proportion of green tea is over 65 percent, the tea leaves are main green tea-producing countries in the world, and the total yield exceeds 75 percent of the total yield of the green tea in the world. The edible part of the green tea serving as a traditional beverage only accounts for a small part of tea, and along with the extension of the tea industry chain at present, more dry tea leaves enter tea deep processing enterprises, and high-temperature water extraction is carried out to prepare instant tea powder and tea concentrated solution, while the total dry matter content of the tea deep processed at present is only about 3% of the dry weight of the tea leaves, and the content of effective components is only extracted to be less than 40%. After the tea is processed, wet tea residue with the tea raw material amount being nearly three times is produced, wherein the wet tea residue contains more than 60% of tea nutrient components, and contains a large amount of protein, fat, fiber, tea polyphenol and the like. Researches show that the green tea dregs contain 17-19% of crude protein, 16-18% of crude fiber, 1-2% of tea polyphenol and 0.1-0.3% of caffeine, and moreover, the green tea dregs are rich in protein amino acid composition, have amino acid specific value coefficient of 57.51-68.01, are better than conventional corn and bran for feeding, are close to fish meal, have nutrition and functions and have very high utilization and development values. At present, tea residue utilization is mainly performed on low-valued fuels, fertilizers, feeds or adsorption materials, and extraction research on tea residue protein is also included, but the tea residue utilization is very limited in scale and industrial application, so that the tea residue recycling is concerned more and more, and an efficient and valued comprehensive scheme is urgently needed to be developed.
With the continuous advance of development technologies, researchers utilize a microbial fermentation mode to apply tea residue feed to develop biological feed. Researches show that the nutritional ingredients of the tea leaves are increased after the tea leaves are fermented by different microorganisms. For example, Liushu, etc. (2001) uses tea leaves as raw materials, and fermentation is carried out by using trichoderma, aspergillus and beneficial microorganisms, so that the content of crude protein and soluble substances is obviously increased, and the nutrient content of the tea leaves completely meets the requirement of piglets for matching with daily ration. Microorganisms reported to be used for fermentation of tea leaves include Aspergillus niger, Penicillium, Saccharomyces, Rhizopus, Aspergillus glaucus, bacteria and the like. The research for developing the tea dregs into the feed is just started, and some problems still exist, besides the nutrition of the matrix raw materials, another very important aspect is the type and the functionality of the microorganism. The selection of the fermentation strain should be in the catalog of feed additive varieties firstly, but the core purpose of the strain for fermenting the feed is the pretreatment aiming at different raw materials and the metabolite in the process of feed curing, so different substrate raw materials and different target-selected strains are different. How to treat the tea residues by using a microbial fermentation method to improve the amino acid content in the tea residues and reduce the cellulose content, and the method is more suitable for feeding poultry, which is one aspect that needs to be overcome in the current research. Therefore, more strains capable of growing by using the tea leaves as a carbon-nitrogen source need to be screened according to the nutritional characteristics of the tea leaves, and a tea leaf solid state fermentation process needs to be developed.
Atmospheric Room Temperature Plasma (ARTP) is called the fourth state of substances except gas, liquid and solid, and plasmas in different thermodynamic states can be generated by changing the excitation mode and the generator structure. The plasma has the characteristics of extremely low ozone concentration and ultraviolet radiation intensity, high safety, environmental friendliness, rapid mutagenesis and the like, and the normal-pressure room-temperature plasma mutagenesis is simple to operate, mild in condition, high in strain mutation rate, and wide in mutation point position and span. The ARTP working gas source type, flow, discharge power, processing time and other conditions are controllable, the intensity of strain mutation and the mutation library capacity can be greatly improved by changing the operating conditions of the instrument, and the ARTP becomes a new method for efficient evolution breeding by combining pressure screening and high-throughput screening technologies. In ARTP mutagenesis, the lethality rate is generally used as an index for screening mutagenesis conditions and the like, and the lethality rate is not too high or too low, and studies show that the closer the lethality rate is to 90%, the better the mutagenesis effect is, and the better the mutagenesis conditions are. An article of Chenhelong and the like (2018) discloses mutation breeding of a lactobacillus plantarum bacteriocin high-yield strain and an antiseptic and fresh-keeping effect of the lactobacillus plantarum bacteriocin high-yield strain on meatballs, and particularly, the lactobacillus plantarum JL-A65 is used as an initial strain, and ARTP mutagenesis, Methylnitronitrosoguanidine (MNNG) mutagenesis and genome reorganization are carried out on the lactobacillus plantarum JL-A65. The ARTP mutagenesis parameters were set to: carrier gas: high purity helium (99.99%); incident power: 200W; flow rate of carrier gas: 10 SLM; treatment temperature: 25 ℃; reflected power: 40W; distance between objective table and radioactive source: 10.0 mm; the mutagenesis was performed with 15, 30, 45, 60, 75, 90 and 105s of irradiation time, respectively. And then carrying out primary screening and secondary screening on the mutagenized strains by using an agar diffusion method to obtain the mutant strains with the increased bacteriocin yield.
At present, no relevant report is found for the research of tea residue fermented feed by adopting an ARTP mutagenesis and domestication screening method to carry out the breeding of functional microorganisms and combining a method of mixing various strains, preferably combining auxiliary materials and carrying out bacterium-enzyme synergistic fermentation.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to solve the technical problems that ARTP is adopted to mutate, domesticate and screen functional microorganisms, a one-pot multi-bacterium mixed culture process is established, and meanwhile, proper auxiliary material combination and exogenous bacterium enzyme are matched for high-value utilization of tea residue fermentation and tea residue resources, so that 'bacterium tea' biological feed taking tea nutrition as a main component is developed.
In order to solve the technical problems, the invention provides the following technical scheme:
a method for preparing tea residue feed by fermenting mixed strains comprises the following steps:
s1, ARTP mutagenesis and domestication screening of functional microorganisms: adopting ARTP to mutate bacillus pumilus (Bacillus pumilus) and Lactobacillus plantarum (Lactobacillus plantarum), inoculating the obtained mutant strain into a domestication culture medium, and screening to obtain the mutant strain which can tolerate high-concentration tea leaves by gradually increasing the content of the tea leaves in the domestication culture medium;
s2, mixed fermentation of mutant strains, namely uniformly mixing the mutant strains which are obtained in the step S1 and can tolerate the high-concentration tea residues to obtain the total number of bacterial colonies of 0.5 × 106-1.5×106cfu/g of mixed bacterial liquid; mixing the mixed bacterial liquid with a fermentation substrate according to a weight ratio of 4-6: mixing 94-96 to obtain a mixed fermentation substrate; adding cellulase, hemicellulase, xylanase and pectinase into the mixed fermentation substrate, adjusting the water content of the mixed fermentation substrate to 35-45%, and fermenting at 20-30 deg.C for 7-10 days to obtain tea residue feed.
Preferably, the mutant strain tolerant to high-concentration tea residues is Bacillus pumilus (Bacillus pumilus) BP-09 and Lactobacillus plantarum (Lactobacillus plantarum) LP-08;
the Bacillus pumilus (Bacillus pumilus) BP-09 is preserved in Guangdong province microbial strain collection center (GDMCC) at 6-16 th 2020, and the preservation number is GDMCC No: 61062, respectively;
the Lactobacillus plantarum (Lactobacillus plantarum) LP-08 is deposited in Guangdong province microorganism strain collection center (GDMCC) 6 and 16 in 2020, with the deposit number of GDMCC No: 61061.
preferably, the conditions for ARTP mutagenesis described in step S1 are: the power of the power supply is 60W, the irradiation distance is 3mm, the temperature of the plasma is 26 ℃, the air flow is 10L/min, the ARTP treatment time of the Bacillus pumilus is 120s, and the ARTP treatment time of the Lactobacillus plantarum is 60 s.
Preferably, the high concentration tea-leaves in step S1 is that the acclimation medium contains 20% tea-leaves leaching solution.
Preferably, the ratio of the bacillus brevis BP-09 to the lactobacillus plantarum LP-08 in the mixed bacterial liquid is 1: 1.5.
more preferably, the total number of colonies in the mixed bacterial liquid is 1 × 106cfu/g。
Preferably, the weight ratio of the mixed bacterial liquid to the fermentation substrate is 5: 95.
preferably, the fermentation substrate comprises tea leaves, defatted rice bran and soybean meal, and the mass ratio of the tea leaves to the defatted rice bran to the soybean meal is 7: 2: 1.
preferably, the final concentration of the cellulase in the mixed fermentation substrate is 300 mu/g, the final concentration of the hemicellulase is 300 mu/g, the final concentration of the xylanase is 200 mu/g, and the final concentration of the pectinase is 200 mu/g.
Preferably, the water content of the mixed fermentation substrate is 40-45%, the fermentation temperature is 25-30 ℃, and the fermentation time is 8-9 days.
Preferably, the tea leaves are green tea leaves.
Compared with the prior art, the invention has the following beneficial effects:
(1) the method provided by the invention selects safe strains which accord with the national feed microorganism catalogue, and the safe strains are bacillus pumilus and lactobacillus plantarum, and the combination of the two strains has positive synergistic effect. On the basis, starting from ARTP mutagenesis and domestication oriented breeding of tea dregs, strains which can tolerate the tea dregs with certain concentration and can grow by taking the tea dregs as a main nutrient source are screened for mixed strain fermentation.
(2) According to the method provided by the invention, a one-pot multi-bacterium mixed culture process is established by evaluating the possibility of mixed culture of mutant strains with different functions, so that the problems of time and labor waste and money waste in single strain culture are solved, the symbiosis characteristic of the mixed strains can be greatly increased, the propagation of mixed functional microorganisms is promoted by matching with a proper auxiliary material combination, macromolecules in tea leaves are digested by combining the cooperation of exogenous enzymes, active ingredients in the tea are released, the utilization space of raw materials is further excavated, the community advantage in solid fermentation is fully enhanced, high-content probiotics and functional active substances thereof are provided, the resource utilization of the tea leaves is effectively realized, the intestinal health of animals is maintained, and the benefit is brought to healthy culture.
Drawings
FIG. 1 shows the effect of ARTP treatment time on survival of various strains.
Detailed Description
The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions, unless otherwise specified.
Fresh green tea leaf has high water content (70.5%) and high crude fiber content (24.1% crude fiber content by dry weight), wherein the content of fiber is most abundant, and cellulose is usually combined with hemicellulose, pectin and lignin, so that the fresh green tea leaf is difficult to be directly utilized by microorganisms. In order to accelerate the biological utilization of the tea residues, a certain amount of cellulase, hemicellulase, pectinase and xylanase are selected, plant cell walls are opened as far as possible, and then proper auxiliary materials are matched to improve and enhance the microbial nutrition structure (carbon-nitrogen ratio, quick-acting nitrogen, inorganic salt, vitamins and the like) of the whole solid fermentation, so that a foundation is created for the high-strength propagation of microbes with different functions, and the green tea residues are efficiently fermented by combining a bacterial enzyme synchronous fermentation mode, so that the bioavailability of the tea residues is improved.
1. Bacterial strain
(1) Original bacteria
Bacillus species: bacillus pumilus BP (BP).
Lactic acid bacteria: lactobacillus plantarum LP (LP).
(2) Bacteria detection
G+The strain is Micrococcus luteus ATCC 4698(Micrococcus luteus ATCC 4698), G-The strain is Escherichia coli ATCC25922(Escherichia coli ATCC25922), and the pore diameter is 6.0 +/-0.2 mm.
2. Reagent
The tea residue obtained after green tea extraction is provided by Fujian Xianhuyang Biotech limited;
defatted rice bran, soybean meal, etc. are provided by Guangdong-Hold Dabiol GmbH;
cellulase (200000 μ/g), xylanase (200000 μ/g), pectinase (30000 μ/g), and hemicellulase (100000 μ/g) were obtained from Jenno Bio-enzyme Co., Ltd, Zaozhuang;
yeast extract powder, yeast extract, beef extract, peptone, etc. are available from Biotechnology engineering (Shanghai) GmbH;
lactic acid bacteria culture Medium (MRS) and the like are purchased from Haibo biotechnology limited of Qingdao high-tech industrial garden;
atmospheric room temperature plasma mutagenesis (ARTP) was purchased from Qingtianmu Biotech limited, without a tin source;
the fermentation breathing bag was purchased from Wenzhou Chuangjia packaging materials, Inc.;
other reagents and consumables were purchased from Biotechnology (Shanghai) GmbH.
3. Culture medium
Bacterial culture medium (%): beef extract 0.5, peptone 1.0, sodium chloride 0.5, NaOH to adjust pH to 7.0, adding 2.0% agar powder if preparing solid culture medium, and sterilizing at 121 deg.C for 21 min. The culture medium is mainly used for culturing the spore bacteria.
MRS medium (%): 1.0 part of peptone, 1.0 part of beef extract, 0.5 part of yeast extract, 0.2 part of diammonium hydrogen citrate, 2.0 parts of glucose, 800.1 mL of tween, 0.5 part of sodium acetate trihydrate, 0.2 part of dipotassium hydrogen phosphate trihydrate, 0.058 part of magnesium sulfate heptahydrate, 0.025 part of manganese sulfate monohydrate and NaOH for adjusting the pH to 6.5, if a solid culture medium is prepared, adding 2.0% agar powder, and sterilizing at 121 ℃ for 21 min. The culture medium is mainly used for culturing lactic acid bacteria.
LB medium (%): tryptone 1.0, yeast extract 0.5, NaCl 1.0, NaOH adjusted pH to 7.0, if solid medium is prepared, add 2.0% agar powder, and sterilize at 121 deg.C for 21 min. The culture medium is mainly used for culturing bacteria.
Plate count medium (%): TSA medium + 1.0% glucose, was used mainly for mixed culture of strains and colony plate count after solid fermentation.
Acclimatization medium (%): the method comprises the following steps of taking the green tea leaves after hot water extraction as a main carbon source, screening strains capable of reproducing and metabolizing in tea leaves with certain concentration, and adopting a specific formula which comprises (%): 5.0 parts of tea leaves, 0.5 part of ammonium sulfate, 0.15 part of monopotassium phosphate, 0.1 part of anhydrous sodium acetate, 0.02 part of magnesium sulfate, 0.005 part of manganese sulfate, 0.005 part of ferrous sulfate, 0.3 part of calcium carbonate and NaoH, adjusting the pH value to 6.5-7.0, and sterilizing at 121 ℃ for 20 min. 2.0% agar powder was added to the solid plate.
Multi-strain mixed medium (%): 2.0 parts of soybean meal, 1.5 parts of corn starch, 2.0 parts of brown sugar powder, 0.5 part of glucose, 0.5 part of yeast extract, 0.5 part of peptone, 1.0 part of cane molasses, 0.5 part of sodium acetate, 0.2 part of diammonium citrate, 0.1 part of ammonium sulfate, 0.15 part of monopotassium phosphate, 0.3 part of light calcium carbonate, 0.02 part of magnesium sulfate, 0.005 part of manganese sulfate, 800.1 parts of tween, and the pH value of NaOH is adjusted to 6.5-7.0, and the pH value is sterilized at 121 ℃ for 20min for later use. The method is mainly used for mixed culture of multiple strains.
Tea-leaf solid fermentation medium (%): 0.1 part of ammonium sulfate, 0.02 part of magnesium sulfate, 0.15 part of monopotassium phosphate, 0.5 part of light calcium carbonate and 0.6 part of cane molasses, and the mixture is uniformly stirred with the tea leaves and auxiliary materials, wherein the packaging amount of each fermentation bag is 10 kg.
Phosphate buffer solution: mixing 3.4g KH2PO4Dissolved in 50mL of distilled water, adjusted to pH 7.2 with 1mol/L NaOH and finally brought to a volume of 100mL by adding distilled water. Autoclaving at 121 deg.C for 15 min, and storing at 4 deg.C.
4. Detection method
Determination of the total acid content: the determination is carried out by an acid-base titration method according to the national standard GB/T12456-2008 'determination of total acid in food'.
Crude protein content: refer to GB/T6432-2018 Kai Kjeldahl method for determination of crude protein in feed.
Acid soluble protein content: refer to the determination of the content of acid soluble protein in GB/T22492-.
Crude fiber content: refer to GB/T6434-2006 filtration method of content determination of crude fiber in feed.
Amino acid content: reference is made to GB/T18246-.
5. Plate colony counting
1.0g of the solid fermentation sample (or 1mL of the fermentation broth sample) was weighed into 9.0mL of phosphate buffer, and then 2 drops of Tween 80 were added, to obtain a concentration of 10-1The diluted solution of (1). Shaking at 150rpm for 5-10min, and diluting with phosphate buffer solution to obtain 10-3，10-5And 10-7The dilution gradient of (2) plates are respectively coated with the diluents with different gradients, the plates are cultured at 30 ℃ for 72 hours, the total colony count is calculated, and different colony types are classified and counted.
6. Analysis of bacteriostatic Activity
And detecting the bacteriostatic activity of the metabolite by adopting an improved agar diffusion method.
Reference documents: several methods for detecting the fungal inhibition of lactic acid bacteria were compared [ J ] food research and development, 2020(9).