CN111718925B

CN111718925B - Method for preparing tea dreg feed by fermenting mixed strains

Info

Publication number: CN111718925B
Application number: CN202010780949.3A
Authority: CN
Inventors: 黄炜乾; 蒋顺进; 张文; 周绍迁; 林万华; 张泳; 林昊
Original assignee: Fujian Xianyangyang Biotechnology Co ltd; Qingyuan Yisheng Natural Biological Research Institute Co ltd
Current assignee: Fujian Xianyangyang Biotechnology Co ltd; Qingyuan Yisheng Natural Biological Research Institute Co ltd
Priority date: 2020-08-06
Filing date: 2020-08-06
Publication date: 2023-06-13
Anticipated expiration: 2040-08-06
Also published as: CN111718925A

Abstract

The invention relates to the technical field of animal feeds, in particular to a method for preparing tea dreg feeds by fermenting mixed strains. The method comprises the following steps: s1.ARTP mutagenesis, domestication and screening of functional microorganisms; s2, mixed fermentation of the mutant strain. The invention aims at developing a functional bacterial tea biological feed prepared from the ground-derived green tea residues, adopts an ARTP mutagenesis and domestication screening method to select functional microorganisms, establishes a one-pot multi-bacterium mixed culture process, simultaneously combines proper auxiliary materials to promote the propagation of mixed functional microorganisms, and digests macromolecules in the tea residues by combining with the cooperation of exogenous enzymes to release active ingredients in the tea, further digs a raw material utilization space, fully strengthens community advantages in solid fermentation, provides high-content probiotics and functional active substances thereof, effectively realizes the utilization of tea residues resources, maintains animal intestinal health and brings benefits to healthy cultivation.

Description

Method for preparing tea dreg feed by fermenting mixed strains

Technical Field

The invention relates to the technical field of animal feeds, in particular to a method for preparing tea dreg feeds by fermenting mixed strains.

Background

The production of dry raw tea leaves in 2018 is over 260 ten thousand tons, wherein the proportion of green tea is over 65 percent, the green tea leaves are main production countries of global green tea, and the total yield is over 75 percent of the total yield of global green tea. As a traditional beverage, the edible part of green tea only accounts for a small part of tea leaves, more dry tea leaves enter tea deep processing enterprises along with the extension of tea industry chains at present, instant tea powder and tea concentrated solution are prepared by high-temperature water extraction, the total dry matter content of the existing tea leaves after deep processing is only about 3% of the dry weight of the tea leaves, and the active ingredient content is only less than 40%. The wet tea residue with approximately three times of tea raw materials is produced after tea processing, and contains more than 60% of tea nutrition components including a large amount of protein, fat, fiber, tea polyphenol, etc. Researches show that the green tea residue contains 17-19% of crude protein, 16-18% of crude fiber, 1-2% of tea polyphenol and 0.1-0.3% of caffeine, and furthermore, the composition of protein amino acid is rich, the ratio coefficient of amino acid reaches 57.51-68.01, and the green tea residue is better than that of conventional feeding corn and bran, is close to fish meal, has nutrition and functions, and has very high utilization and development values. At present, the utilization of tea residues is mainly based on low-valued fuel, fertilizer, feed or adsorption materials, and also comprises extraction research of some tea residue proteins, but the application of the tea residues is very limited in scale and industrialization, how to realize the recycling of the tea residues is more and more concerned, and the development of high-efficiency and valuable comprehensive schemes is urgently needed.

With the continuous advancement of development technology, researchers use a microbial fermentation mode to apply tea dreg feed to develop biological feed. Researches show that the nutritional ingredients of tea residues are increased after the tea residues are fermented by different microorganisms. For example, liu Shu (2001) uses tea residues as raw materials, and uses trichoderma, aspergillus and beneficial microorganisms to ferment, so that the content of crude protein and soluble substances is found to be obviously increased, and the nutrition content of the fermented tea residues completely meets the requirements of piglets for daily ration. Microorganisms reported to be used for fermentation of tea grounds include Aspergillus niger, penicillium, saccharomyces, rhizopus, aspergillus glaucus, bacteria, etc. Research into the development of tea residues into feeds has just been carried out, and there are problems in that, in addition to the nutrition of the matrix raw materials themselves, another important aspect is the kind and functionality of microorganisms. The selection of the fermentation strain is firstly in the catalogue of feed additives, but the core purpose of the strain is to pre-treat different raw materials and to metabolize the products in the process of 'curing' the feed, so that different substrate raw materials and different target selected strains are different. How to treat tea residues by utilizing a microbial fermentation method so as to improve the amino acid content and reduce the cellulose content in the tea residues, and is more suitable for feeding poultry, which is one aspect of current research needs to be overcome. Therefore, more strains capable of growing by using the tea leaves as carbon and nitrogen sources need to be selected according to the nutrition characteristics of the tea leaves, and the tea leaves solid state fermentation process is developed.

Atmospheric pressure room temperature plasma (Atmospheric Room Temperature Plasma, ARTP) is known as the fourth state of matter other than gas, liquid, solid, and plasma of different thermodynamic states can be generated by changing the excitation regime and generator configuration. The plasma has the characteristics of extremely low ozone concentration and ultraviolet radiation intensity, high safety, environmental friendliness, quick mutagenesis and the like, and the normal-pressure room-temperature plasma mutagenesis operation is simple, the condition is mild, the mutation rate of the strain is high, and mutation points and spans are wide. The ARTP working gas source type, flow, discharge power, treatment time and other conditions are controllable, the mutation strength and mutation reservoir capacity of the strain can be greatly improved by changing the operation conditions of the instrument, and the ARTP has become a novel method for high-efficiency evolutionary breeding by combining pressure screening and high-throughput screening technologies. ARTP mutagenesis generally uses mortality as an index for screening mutagenesis conditions and the like, and the mortality is not too high or too low, and researches show that the better the mortality is, the better the mutagenesis effect is, and the better the mutagenesis conditions are. Chen Ruilong et al (2018) disclose mutagenesis breeding of high-yield bacterial strains of Lactobacillus plantarum and the preservation and fresh-keeping effect of the bacterial strains on meat balls, in particular to ARTP mutagenesis, methyl nitronitrosoguanidine (MNNG) mutagenesis and genome reorganization of Lactobacillus plantarum JL-A65 serving as an initial bacterial strain. The ARTP mutagenesis parameters were set as: carrier gas: high purity helium (99.99%); incident power: 200W; carrier gas flow rate: 10SLM; treatment temperature: 25 ℃; reflected power: 40W; distance between stage and radioactive source: 10.0mm; mutagenesis was performed at 15, 30, 45, 60, 75, 90 and 105s irradiation time, respectively. And then, performing primary screening and secondary screening on the strain subjected to the mutagenesis treatment by using an agar diffusion method to obtain a mutant strain with improved bacteriocin yield.

At present, the related report of tea dreg fermentation feed research by adopting an ARTP mutagenesis, domestication and screening method to select functional microorganisms and combining a method of mixing various strains, preferably combining auxiliary materials and fermenting in a synergistic way is not seen.

Disclosure of Invention

Aiming at the defects existing in the prior art, the technical problem to be solved by the invention is to establish a one-pot multi-fungus mixed culture process by adopting ARTP mutagenesis, domestication and screening of functional microorganisms, and simultaneously to be matched with proper auxiliary material combination and exogenous fungus enzymes for tea residue fermentation and high-value utilization of tea residue resources, so as to develop the fungus tea biological feed taking tea nutrition as a main component.

In order to solve the technical problems, the invention provides the following technical scheme:

a method for preparing tea dreg feed by fermenting mixed strains comprises the following steps:

s1.ARTP mutagenesis, domestication and screening of functional microorganisms: inoculating a mutant strain obtained by adopting ARTP to induce Bacillus pumilus (Bacillus pumilus) and lactobacillus plantarum (Lactobacillus plantarum) into a domestication culture medium, and screening to obtain the mutant strain tolerant to high-concentration tea residues by gradually increasing the tea residue content in the domestication culture medium;

s2, mixed fermentation of mutant strains: uniformly mixing the mutant strains which are obtained in the step S1 and are tolerant to high-concentration tea residues to obtain the bacterial colony total number of 0.5 multiplied by 10 ⁶ -1.5×10 ⁶ cfu/g of mixed bacterial liquid; mixing the mixed bacterial liquid and the fermentation substrate according to the weight ratio of 4-6:94-96, and obtaining a mixed fermentation substrate; adding cellulase, hemicellulase, xylanase and pectase into the mixed fermentation substrate, adjusting the water content of the mixed fermentation substrate to 35-45%, and fermenting at 20-30deg.C for 7-10d to obtain tea residue feed.

Preferably, the mutant strain tolerant to high-concentration tea residues is Bacillus pumilus BP-09 and Lactobacillus plantarum (Lactobacillus plantarum) LP-08;

the Bacillus pumilus BP-09 was deposited at the Guangdong province microbiological bacterial culture collection center (GDMCC) on 16 th month of 2020 under the accession number GDMCC No:61062;

the lactobacillus plantarum (Lactobacillus plantarum) LP-08 was deposited at the cantonese province microorganism strain collection (GDMCC) at 6/16/2020 under accession number GDMCC No:61061.

preferably, the conditions for the art 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 60s.

Preferably, the high concentration tea leaves in step S1 means that the domestication medium contains 20% tea leaf leaching solution.

Preferably, the ratio of the bacillus pumilus 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 1×10 ⁶ cfu/g。

Preferably, the weight ratio of the mixed bacterial liquid to the fermentation substrate is 5:95.

preferably, the fermentation substrate comprises tea residues, defatted rice bran and soybean meal powder, wherein the mass ratio of the tea residues to the defatted rice bran to the soybean meal powder is 7:2:1.

preferably, the final concentration of cellulase in the mixed fermentation substrate is 300 mu/g, the final concentration of hemicellulase is 300 mu/g, the final concentration of xylanase is 200 mu/g, and the final concentration of 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 8-9d.

Preferably, the tea residue is green tea residue.

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, namely bacillus pumilus and lactobacillus plantarum, and the combination of the two strains has positive synergistic effect. Based on the method, from ARTP mutagenesis and domestication oriented breeding of tea leaves, strains which can tolerate tea leaves with certain concentration and grow by taking the tea leaves as main nutrition sources are screened for mixed strain fermentation.

(2) The method provided by the invention establishes a one-pot multi-fungus mixed culture process by evaluating the possibility of mixed culture of different functional mutant strains, solves the problem of time and labor waste and money waste of single strain culture, can greatly increase the symbiotic characteristic of the mixed strains, simultaneously promotes the reproduction of mixed functional microorganisms by matching with proper auxiliary material combinations, and digests macromolecules in tea residues by combining with the cooperation of exogenous enzymes to release active ingredients in tea, further digs the utilization space of raw materials, fully strengthens the community advantage in solid fermentation, provides high-content probiotics and functional active substances thereof, effectively realizes the recycling utilization of tea residues, maintains the intestinal health of animals and brings benefits to healthy culture.

Drawings

FIG. 1 shows the effect of ARTP treatment time on survival of different strains.

Detailed Description

The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, and the experimental methods under the specific conditions not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

Fresh green tea leaves have a high moisture content (70.5% moisture content) and a high crude fiber content (24.1% crude fiber content on a dry basis), especially the most abundant fiber, which is usually cellulose combined with hemicellulose, pectin and lignin, and is difficult to be directly utilized by microorganisms. In order to accelerate the biological utilization of tea residues, a certain amount of cellulase, hemicellulase, pectinase and xylanase are selected, plant cell walls are opened as much as possible, and then proper auxiliary materials are matched, so that the microbial nutrition structure of the whole solid fermentation is improved and improved (carbon nitrogen ratio, quick-acting nitrogen, inorganic salt, vitamins and the like are improved), a foundation is created for the high-strength propagation of microorganisms with different functions, and the high-efficiency fermentation is carried out on the green tea residues 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.

Lactic acid bacteria: lactobacillus plantarum LP (Lactobacillus plantarum, 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 ATCC 25922), wellDiameter 6.0+ -0.2 mm.

2. Reagent(s)

The tea residue obtained by extracting green tea is provided by Fujian Xianyang biotechnology Co., ltd;

defatted rice bran, wheat bran, soybean meal, etc. are provided by guangdong major organisms, inc;

cellulases (200000. Mu.g), xylanases (200000. Mu.g), pectinases (30000. Mu.g), hemicellulases (100000. Mu.g) were all purchased from JEOSE Inc. of Zaozhuang;

yeast extract powder, yeast extract, beef extract, peptone, etc. are available from the division of bioengineering (Shanghai);

lactic acid bacteria culture Medium (MRS) and the like are purchased from Qingdao high-tech industrial park Haibo biotechnology Co., ltd;

atmospheric room temperature plasma mutagenesis instrument (ARTP) was purchased from the company of the biotechnology of notarsa qinghao;

the fermented breathing bag was purchased from wenzhou wound packing materials limited;

other reagents, consumables were purchased from the company division of bioengineering (Shanghai).

3. Culture medium

Bacterial culture (%): 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 121deg.C for 21min. The culture medium is mainly used for culturing spore bacteria.

MRS medium (%): peptone 1.0, beef extract 1.0, yeast extract 0.5, diammonium citrate 0.2, glucose 2.0, tween 80.1 ml, sodium acetate trihydrate 0.5, dipotassium phosphate trihydrate 0.2, magnesium sulfate heptahydrate 0.058, manganese sulfate monohydrate 0.025, naOH to adjust pH to 6.5, if solid culture medium is prepared, 2.0% agar powder is added, and sterilization is carried out at 121 ℃ for 21min. The culture medium is mainly used for culturing lactobacillus.

LB medium (%): tryptone 1.0, yeast extract 0.5,NaCl 1.0,NaOH, adjusting pH to 7.0, adding 2.0% agar powder if solid culture medium is prepared, and sterilizing at 121deg.C for 21min. The culture medium is mainly used for culturing bacteria.

Plate count medium (%): TSA medium+1.0% glucose, mainly used for mixed culture of strains and colony plate counting after solid fermentation.

Acclimatization medium (%): the green tea residue after hot water leaching is used as a main carbon source, bacterial strains capable of propagating and metabolizing in tea residue with certain concentration are screened, and the specific formula comprises the following components in percentage: 5.0 parts of tea residues, 0.5 parts of ammonium sulfate, 0.15 parts of monopotassium phosphate, 0.1 parts of anhydrous sodium acetate, 0.02 parts of magnesium sulfate, 0.005 parts of manganese sulfate, 0.005 parts of ferrous sulfate, 0.3 parts of calcium carbonate and pH of NaoH are adjusted to 6.5-7.0, and sterilization is carried out for 20min at 121 ℃. 2.0% of agar powder is added into the solid flat plate.

Multi-strain mixed medium (%): bean pulp powder 2.0, corn starch 1.5, brown sugar powder 2.0, glucose 0.5, yeast extract 0.5, peptone 0.5, cane molasses 1.0, sodium acetate 0.5, diammonium citrate 0.2, ammonium sulfate 0.1, monopotassium phosphate 0.15, light calcium carbonate 0.3, magnesium sulfate 0.02, manganese sulfate 0.005, tween 80.1, naOH for adjusting pH to 6.5-7.0, and sterilizing at 121 ℃ for 20min for later use. The method is mainly used for mixed culture of multiple strains.

Tea dreg 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 uniformly stirring the materials together with tea residues and auxiliary materials after mixing, wherein the amount of each fermentation bag is 10kg.

Phosphate buffer: 3.4g KH was added ₂ PO ₄ Dissolved in 50mL of distilled water, adjusted to pH 7.2 with 1mol/L NaOH, and finally the volume was adjusted to 100mL with distilled water. Sterilizing at 121deg.C for 15 min, and storing at 4deg.C.

4. Detection method

Determination of total acid content: the determination is carried out by referring to an acid-base titration method in national standard GB/T12456-2008 'determination of total acids in food'.

Crude protein content: reference is made to the Kjeldahl nitrogen determination method of GB/T6432-2018 determination of crude protein in feed.

Acid soluble protein content: the content of acid soluble protein in GB/T22492-2008 soybean peptide powder is referred to.

Crude fiber content: reference is made to the filtration method of GB/T6434-2006 "determination of crude fibre content in feed".

Amino acid content: reference is made to GB/T18246-2000 determination of amino acids in feed.

5. Plate colony count

1.0g of solid fermentation sample (or 1mL of fermentation broth sample) was weighed and added to 9.0mL of phosphate buffer, followed by addition of 2 drops of Tween 80, thus obtaining a concentration of 10 ^-1 Is a solution of (a) and (b). Shaking at 150rpm for 5-10min, and gradient diluting with phosphate buffer solution to obtain 10 ^-3 ，10 ^-5 And 10 ^-7 The dilutions of different gradients were spread on 2 plate counting dishes, incubated at 30℃for 72 hours, total colony counts were calculated, and differential counts were performed for the different colony types.

6. Analysis of bacteriostatic Activity

The antibacterial activity of the metabolites was detected by modified agar diffusion.

Reference is made to: hao Xiangmei, wang Yuhang, wang Yuan, etc. several methods for detecting the fungal inhibition of lactic acid bacteria compare [ J ]. Food research and development 2020 (9).

EXAMPLE 1 ARTP mutagenesis of strains

1. Strain activation

Inoculating glycerol bacteria of different strains (Bacillus pumilus and Lactobacillus plantarum) into corresponding slant culture medium, culturing at 30deg.C for 24 hr, and culturing at 30deg.C for 16 hr after culturing, thereby further strengthening strain activity and rejuvenating strain to achieve strain activation.

2. Determination of ARTP mutagenesis parameters of Strain

Adding sterile physiological saline into the activated and cultured inclined plane, eluting to prepare bacterial suspension, and controlling bacterial suspension OD _600nm The value is between 0.5 and 0.7. Taking 10 mu L of bacterial suspension, uniformly coating the bacterial suspension on the surface of a metal slide, and transferring a flat plate with the sample slide to an ARTP operation bin by using a sterilizing forceps after drying. The method comprises the steps of treating a bacteria slide by using high-purity helium as working gas of plasma, setting power supply to be 60W, irradiating the bacteria slide at a distance of 3mm, and enabling the temperature of the plasma to be 26 ℃ and the airflow to be the same as the working gas of the plasmaThe amount was 10L/min, different treatment groups were set, and the treatment time of each group was 0 (control), 30, 60, 90, 120, 150, 180s, three replicates per group. The treated slide was transferred to an EP tube containing 1mL of sterilized normal saline, and the microorganisms attached to the slide were eluted into the sterilized normal saline by shaking for 60 seconds to form a bacterial suspension. The bacterial suspension is properly diluted and then coated on a corresponding flat plate, and is placed in a 30 ℃ incubator for culturing for 48 hours, and then counting is carried out, and the calculated mortality is as follows:

mortality% = (number of non-mutagenized colonies-number of mutagenized colonies)/number of non-mutagenized colonies × 100%

And (3) through statistics of the mortality of each treatment group, a formal experiment is carried out by selecting irradiation treatment time with the mortality of about 80%, so that a certain mutation richness is ensured, and a certain survival rate is provided.

Results: bacterial suspensions of different strains were subjected to ARTP mutagenesis, and the mortality curves of the different strains were plotted against the strains not subjected to ARTP treatment (treatment time 0 s) (FIG. 1). As can be seen from fig. 1, the different types of strains have a significant difference in their tolerance to ARTP: (1) In the case of lactobacillus plantarum, the mortality rate is only 36.6% for 30s, and is greatly increased to 76.8% for 60s, whereas when the treatment time exceeds 90s, the mortality rate exceeds 95% for 120s, and almost 100%. (2) The Bacillus pumilus which is also used as a prokaryote has better tolerance to ARTP, the death rate is only 24.6 percent after being treated for 60 seconds under the same conditions, the death rate is increased to 50.6 percent after being treated for 90 seconds, the death rate is 80.1 percent after being treated for 120 seconds, and the cell survival rate is 3.6 percent after being treated for 180 seconds and is basically 0 when the treatment time reaches 150 seconds.

Therefore, the subsequent screening was performed with a certain cell viability while ensuring the mutagenesis effect, and ARTP treatment was performed under conditions of a mortality rate of about 80%, so that the ARTP treatment time of Lactobacillus plantarum was determined to be 60s and the treatment time of Bacillus pumilus was determined to be 120s.

EXAMPLE 2 domestication screening of mutant strains

Taking the bacterial suspension subjected to ARTP treatment, directly inoculating the bacterial suspension into an acclimation culture medium, culturing for 72 hours at the temperature of 30 ℃ and 220rpm with the liquid loading amount of 50mL of 250mL triangular flask liquid, and examining the growth and reproductive capacity of the bacterial strain in the culture medium taking tea residues as a main carbon source. And the tea dreg content in the domestication culture medium is gradually increased, and the strain is obtained by combining with plate separation, so that the excellent strain with strong growth capacity and capable of tolerating and utilizing high-concentration tea dreg can be stored for later use. The method comprises the following steps:

(1) Domestication of mutant strain by low-concentration tea residue

The bacterial suspension after ARTP treatment is directly transferred to a domestication culture medium for culture, untreated bacterial strains are used as a control, the color and smell changes of the fermentation broth are observed, and the colony of the fermentation broth is counted.

The results show that: different types of mutant strains can be propagated in different degrees in a culture medium with 5.0% tea residues as the only carbon source, and can normally perform certain metabolic activities, and the original strains can not normally grow under the same culture medium conditions. In terms of growth, the Bacillus pumilus mutant strain grows best and the Lactobacillus plantarum mutant strain grows next time; bacillus pumilus exhibits a certain G-pair in terms of metabolite activity ⁺ (Micrococcus luteus) and Lactobacillus plantarum have G at the same time ⁺ And G ^- The bacteriostatic activity of (E.coli), but the zone of inhibition was relatively blurry and the boundaries were not clear, which may be related to the organic acids produced by lactic acid bacteria, and the bacteriostatic properties of low concentrations of organic acids were often not very thorough (Table 1).

TABLE 1 domestication culture characteristics of ARTP mutant in tea residue containing Medium

Type of strain	pH	Colony count (CFU/mL)	G ⁺ Activity (mm)	G ^- Activity (mm)
					Bacillus pumilus	7.19±0.05	3.9±0.05×10 ⁷	11.2±0.1	ND
Lactobacillus plantarum	4.10±0.05	8.9±0.05×10 ⁶	10.2±0.1	10.3±0.1

Note that: ND indicates no bacteriostatic activity was detected.

(2) Re-screening of high tea dreg concentration tolerant mutant

Meanwhile, bacterial solutions obtained by domestication culture are respectively coated on domestication culture medium plates containing 10%, 15% and 20% tea dreg leaching solutions, and bacterial colonies which can grow in high-concentration tea dreg are picked for shake flask culture.

The results show that: the bacterial strains of different types all have bacterial colonies which can grow in 20% tea dreg leaching liquor, and the bacterial concentration of the liquid fermentation is basically consistent with the domestication culture result of the tea dreg of low concentration, which shows that the growth of mutant strains is basically not inhibited by the nutritional environment of the tea dreg of high concentration, but the capability of synthesizing active ingredients of the variant strains of different types is obviously stimulated, and the bacteriostatic activity and the acid production capability are improved to a certain extent (Table 2).

Overall, the Bacillus pumilusBacillus pair G ⁺ The antibacterial activity of the composition is relatively low, and the highest antibacterial activity is 12+/-0.1 mm. The mutant strain of the lactobacillus has better acid-producing ability and the minimum pH value of 4.01 (LP-08 strain), and has better fermentation characteristic of the lactobacillus.

Table 2 screening of mutant strains for tolerance to high concentration tea leaves (part)

Note that: ND indicates no bacteriostatic activity was detected.

The bacillus pumilus BP-09 and the lactobacillus plantarum LP-08 with the best biomass and good metabolite activity are selected for shake flask rescreening, and the agar diffusion method is adopted for activity bacteriostasis activity detection.

The results show that: bacillus pumilus BP-09 against G only ⁺ The strain has obvious antibacterial effect and can form a clear and definite inhibition zone; and Lactobacillus plantarum LP-08 to G ⁺ And G ^- The strain has a certain inhibition effect, but the transparency of the inhibition zone is low, which is basically consistent with the result of the tolerance screening.

Bacillus pumilus BP-09 was deposited at the Guangdong province microbiological bacterial collection center (GDMCC) on 16 th month of 2020 under the accession number GDMCC No:61062. the preservation address is building 5 of Guangzhou city first-violent Zhonglu No. 100 college No. 59.

Lactobacillus plantarum (Lactobacillus plantarum) LP-08 was deposited at 16 months 6 in 2020 with the cantonese institute of microbiological bacterial strain (GDMCC) accession No. GDMCC No:61061. the preservation address is building 5 of Guangzhou city first-violent Zhonglu 100 # college 59.

EXAMPLE 3 liquid culture of Mixed mutant Strain

(1) Antibacterial analysis of single strain fermentation products

Before mixed culture of multiple strains, it is necessary to examine the inhibition of metabolites of different strains on other strains, and the strains obtained by the screening are respectively cultured individually by adopting a mixed fermentation medium, and fermentation broth supernatants are collected by centrifugation, and inhibition among the strains is detected according to an agar diffusion method.

The results show that: there was substantially no mutual inhibition between the strains.

(2) Mixed culture analysis of multiple strains

On the basis of pure culture of single strain and same type of strain, different types of strains are cultured in the same fermentation system according to physiological and metabolic characteristics of different strains, so that one-pot multi-strain culture is realized, and the number of viable bacteria and activity of metabolic products under mixed culture conditions are examined. The method comprises the following specific steps:

scraping a large ring in the activated inclined planes (18X 180 mm) of different bacteria respectively to graft to the inclined planes of corresponding eggplant bottles, culturing at 30 ℃ for 24h, eluting each eggplant bottle with 50mL of sterile water, preparing bacterial suspension and OD _600nm Controlling the temperature between 0.5 and 0.6, namely the inclined seed liquid.

After the eluted bacterial suspension was mixed, the whole amount was inoculated into a 50L mixed culture tank, and the strain was subjected to expansion culture in a tank of 30L. Culture temperature: the temperature is 30 ℃, the tank pressure is 0.05-0.08Mpa, the primary stirring is carried out at 250rpm, the dissolved oxygen naturally drops to 0, 20% ammonia water is fed into the tank after the pH naturally drops to control the pH to about 5.0, the ventilation rate is 15L/min, and the period is: 24h. Samples were taken every two hours to detect the bacteriostatic activity (G) ⁺ And G ^- ) And total number of colonies.

The results show that: about 1.0X10 total colony Count (CFU) at initial (fermentation 0 h) ⁷ The strain/mL enters a fermentation system, 0-6h is in a adaptation period, the concentration of reducing sugar in the culture system is not reduced, the method is not that the mixed strain does not consume reducing sugar, on the contrary, the mixed strain keeps the concentration of reducing sugar in the system by synthesizing a series of amylases through hydrolysis of complex glycogen, the strain enters a logarithmic growth period after 6-8h of fermentation, the colony count is sharply increased, fermentation foam is sharply increased, the consumption of reducing sugar is correspondingly increased, the amount of reducing sugar is sharply reduced along with the propagation of the mixed strain, the amount of reducing sugar is not basically detected in about 16h, the growth of foam is basically stopped, the colony count is cultivated for 20h to be maximum, and CFU is 3.5x10 ¹⁰ the/mL shows that the mixed bacteria can be very well adapted to a mixed culture system,and can make full use of the mixed culture components to carry out the propagation and metabolism of the thalli.

When the bacteria are fermented for 0h from the conditions of the bacteria in different culture periods, the quantity of the long rod and the short rod bacteria in the visual field is basically consistent, the formation of spores is basically avoided, and the bacteria are deeply dyed, so that the activity of the mixed bacterial strain is indicated to be possible; the culture for 12 hours shows that the colony number in the whole visual field is obviously increased, the number of the short bacilli is increased, and a small amount of spores begin to appear, wherein the long bacilli are thickened from slender and mainly grow in opposite or splayed strains, which indicates that the mixed strains are in vigorous propagation growth period; sampling before the end of culture, the number of the bacterial cells is further increased, particularly the number of spores is obviously increased, meanwhile, individual short bacilli are occasionally seen in the field of view, the bacterial colony types in the field of view are stained, and the proportion of the bacterial colony types in the field of view is approximately the spores: bacillus=9: 7.

in addition, in view of the metabolic characteristics of the functional strain itself, the bacteriostasis of the supernatant of each period of mixed culture was examined by the agar diffusion method, and the results are shown in Table 3 below.

TABLE 3 antibacterial Properties of functional strains under Mixed culture

In terms of bacteriostasis, the bacteriostasis activity of the whole sampling period is G ⁺ The antibacterial activity is mainly that in the earlier stage of mixed bacteria culture, along with the increase of the quantity of bacillus pumilus and the increase of foam in the fermentation process, the antibacterial activity of the supernatant of the fermentation liquor is correspondingly increased, and the maximum G is reached at 12h ⁺ The diameter of the inhibition zone reaches 22.2+/-0.2 mm, other strains proliferate along with the reduction of fermentation foam, and G ⁺ The antibacterial performance is slightly reduced, from G ⁺ Relation of bacteriostatic Properties to foam, G ⁺ The antibacterial active ingredient of (a) may be lipopeptides ingredient (such as surfactant, iturin, etc.). Different from G ⁺ Bacteriostatic activity, G ^- The antibacterial activity is barely seen until after 12 hours of culture, and the antibacterial zone is not clear and transparent.

In general, different types of strains can be well co-dwelling in this same system. Moreover, the good antibacterial activity exhibited by the mixed culture of the strain brings benefits to the subsequent solid fermentation and animal culture application.

Example 4 bacterial enzyme synergistic fermentation of tea leaves

The solid fermentation tea dreg test steps are as follows:

the fermentation substrate consists of wet green tea residues and auxiliary materials, wherein the tea residues are as follows: defatted rice bran: bean pulp = 7:2:1. meanwhile, in order to better promote the transformation of microorganisms to tea residues, enzyme synergistic fermentation is selected, wherein the final concentration of cellulase is 300 mu/g, the final concentration of hemicellulose enzyme is 300 mu/g, the final concentration of xylanase is 200 mu/g, and the final concentration of pectinase is 200 mu/g.

Adopts liquid mixed bacterial liquid (namely mixed bacterial liquid of bacillus pumilus BP-09 and lactobacillus plantarum LP-08), and the total number of initial bacterial colonies is about 1 multiplied by 10 ⁶ cfu/g; wherein, the bacillus pumilus BP-09: lactobacillus plantarum LP-08=1: 1.5 Inoculating, wherein the inoculation amount is 5 according to the weight ratio of the mixed bacterial liquid to the fermentation substrate: 95 meters, and simultaneously, the water content is adjusted to ensure that the matrix is dispersed once touching, and is kneaded into a mass by hand without dripping water to be optimal (the water content is about 40-45%). And then the mixed substrate is put into a fermentation bag with a one-way valve, and is fermented for 7-10 days at normal temperature (25-30 ℃) until the substrate has sweet and sour distiller's yeast flavor, which indicates that the fermentation is complete and full. After fermentation, the indexes such as total viable count, total acid, crude protein, acid soluble protein, crude fiber, lysine, threonine and methionine of the fermentation sample are mainly measured, and the detection method is implemented by referring to national standard and industry standard methods.

The detection results are shown in Table 4 after the bacterial enzymes are fermented cooperatively for 9 days.

Table 4 bacterial enzyme synergistic fermentation tea residue

Note that: the detection data of the fermentation index are calculated by wet weight.

The results in Table 4 show that the mixed bacterial liquid was used for tea dreg fermentation:

(1) From the aspect of the moisture content before and after fermentation, the moisture content after fermentation is slightly reduced compared with that before fermentation, which shows that the mixed functional strain of BP-09 and LP-08 can effectively grow in a fermentation substrate and produce gas or heat in the whole fermentation process.

(2) From the total number of colonies, the total number of colonies inoculated with the mixed bacteria before fermentation was 1X 10 ⁶ cfu/g, the strain can firstly utilize digestible carbon and nitrogen sources to perform basic metabolism (aerobic before anaerobic), in particular to auxiliary materials, namely bean pulp, wherein amino acid nitrogen and the like are most beneficial to the growth of the strain (the strain is increased by two orders of magnitude), meanwhile, anabolic products such as organic acid, enzyme and the like are further assisted to hydrolyze complex substrates, further growth and propagation of the strain are promoted in turn, and the adaptability and growth characteristics of tea dreg matrixes of BP-09 and LP-08 strains are reflected.

(3) From the protein content, the crude protein of the matrix after fermentation is increased, wherein the acid soluble protein is obviously increased. The increase of the total crude protein content may be related to moisture, gas production metabolism and the like, and after all, the addition of auxiliary materials is beneficial to microorganism propagation and also beneficial to microorganism metabolism. In addition, BP-09 and LP-08 strains also accelerate the degradation of insoluble proteins in the matrix, and the conversion and synthesis of non-protein nitrogen into soluble small molecule proteins or peptides, increasing acid soluble proteins by 83.6%.

(4) From the total acid after fermentation, the total acid content is greatly improved, which shows that BP-09 and LP-08 strains can adapt to certain acid environment and have better acid production characteristics, particularly the LP-08 strain utilizes a matrix to carry out acid metabolism under anaerobic condition, and the strain is used as fermented feed, and certain acidity value is not only favorable for mildew prevention of the fermented matrix, but also has certain phagostimulant effect as biological feed, thus reflecting better application characteristics.

(5) From the crude fiber content, the difference between the crude fiber content before and after fermentation is obvious, which is related to the exogenously added enzyme preparation and also closely related to the growth metabolism of the strains BP-09 and LP-08. The good tea dreg tolerance and growth characteristics of the BP-09 strain also fully show the degradation capability of crude fibers of BP-09, and meanwhile, the organic acid fermentation characteristics of the LP-08 strain also create conditions for metabolism of complex matrixes, which are the basis of fermenting tea dreg matrixes by mixed strains, and the crude fiber content is reduced by 51.6% under the synergistic conditions of bacteria and enzymes.

(6) From the content of amino acids, lysine, threonine and methionine in essential amino acids before and after fermentation are multiplied by 7.7 times, 8.0 times and 3.3 times respectively. The three amino acids are taken as limiting amino acids in animal nutrition, directly influence the absorption and utilization of other amino acids by animals, and fully reflect the good tea dreg fermentation metabolism capability of LP-08 and BP-09 strains.

In conclusion, the preferred safe strains according with the national feed microorganism catalogue of the invention are bacillus pumilus and lactobacillus plantarum, and some of the two strains have positive synergistic effect. The bacillus pumilus has stronger enzyme production capability, can produce various enzymes including protease, glycosidase, amylase, lipase and the like, can metabolize complex carbon and nitrogen sources, secrete various bioactive substances such as cyclic lipopeptides, bacteriocins, antibacterial peptides and the like, is very favorable for degrading complex nitrogen-containing compounds in green tea residues, and utilizes partial carbohydrate, so that the amino acid content in the feed is improved; the lactobacillus plantarum not only can secrete some enzymes, but also can metabolize and produce various organic acids and lactic acid bacteria, and the lactobacillus plantarum is greatly helpful for improving the quality of feed. Based on the method, strains which can tolerate tea residues with certain concentration and grow by taking the tea residues as main nutrition sources are screened from ARTP mutagenesis and domestication directional breeding of the tea residues, and a one-pot multi-bacterium mixed culture process is established by evaluating the possibility of mixed culture of different functional strains, so that the problem of time and labor waste and money waste in single strain culture is solved, the symbiotic characteristic of the mixed strains is greatly increased, meanwhile, the proper auxiliary material combination is matched, the propagation of mixed functional microorganisms is promoted, the cooperation of exogenous enzymes is combined, macromolecules in the tea residues are digested, active ingredients in the tea are released, the utilization space of raw materials is further excavated, community advantages in solid fermentation are fully enhanced, high-content probiotics and functional active substances thereof are provided, the utilization of tea residues is effectively realized, the intestinal health of animals is maintained, and the benefit is brought to healthy culture.

Finally, it should be noted that the above description is only for illustrating the technical solution of the present invention, and not for limiting the scope of the present invention, and that the simple modification and equivalent substitution of the technical solution of the present invention can be made by those skilled in the art without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. The method for preparing tea dreg feed by fermenting the mixed strain is characterized by comprising the following steps of:

s1, selecting Bacillus pumilus BP-09 and lactobacillus plantarum (Lactobacillus plantarum) LP-08 as mutant strains for tolerating high-concentration tea leaves,

the Bacillus pumilus BP-09 was deposited at the Guangdong province microbiological bacterial culture collection center (GDMCC) on 16 th month of 2020 under the accession number GDMCC No:61062,

the lactobacillus plantarum (Lactobacillus plantarum) LP-08 was deposited at the cantonese province microorganism strain collection (GDMCC) at 6/16/2020 under accession number GDMCC No:61061;

s2, uniformly mixing the mutant strains which are tolerant to the high-concentration tea residues in the step S1 to obtain the bacterial colony total number of 0.5 multiplied by 10 ⁶ -1.5×10 ⁶ The mixed bacterial liquid of cfu/g, wherein the ratio of bacillus pumilus BP-09 to lactobacillus plantarum LP-08 in the mixed bacterial liquid is 1:1.5; mixing the mixed bacterial liquid and the fermentation substrate according to the weight ratio of 4-6:94-96, wherein the fermentation substrate consists of wet green tea residues, defatted rice bran and soybean meal powder, so as to obtain a mixed fermentation substrate; adding cellulase, hemicellulase, xylanase and pectase into the mixed fermentation substrate, adjusting the water content of the mixed fermentation substrate to 35-45%, and fermenting at 20-30deg.C for 7-10d to obtain tea residue feed.

2. The method of claim 1, wherein the weight ratio of the mixed bacterial liquid to the fermentation substrate is 5:95.

3. the method according to claim 1, wherein the fermentation substrate consists of wet green tea residue, defatted rice bran and soybean meal powder in a mass ratio of 7:2:1.

4. the method of claim 1, wherein the final concentration of cellulase in the mixed fermentation substrate is 300U/g, the final concentration of hemicellulase is 300U/g, the final concentration of xylanase is 200U/g, and the final concentration of pectinase is 200U/g.

5. The method of claim 1, wherein the mixed fermentation substrate has a moisture content of 40-45%.

6. The method according to claim 1, wherein the fermentation temperature of the mixed fermentation substrate is 25-30 ℃ for 8-9d.