CN111718925A

CN111718925A - Method for preparing tea residue feed by fermenting mixed strains

Info

Publication number: CN111718925A
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: 2020-09-29
Anticipated expiration: 2040-08-06
Also published as: CN111718925B

Abstract

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.

Description

Method for preparing tea residue feed by fermenting mixed strains

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).

EXAMPLE 1 ARTP mutagenesis of the Strain

1. Activation of bacterial strains

Inoculating glycerol strains of different strains (bacillus pumilus and lactobacillus plantarum) to corresponding slant culture media, culturing at 30 ℃ for 24 hours, taking a ring of strains from the slant culture media after the culture is finished, streaking the strains to a fresh slant culture medium, and culturing at 30 ℃ for 16 hours, thereby further enhancing the activity of the strains, rejuvenating the strains and achieving the aim of activating the strains.

2. Determination of mutagenesis parameters of Strain ARTP

Adding sterile normal saline into the activated and cultured slant, eluting, preparing bacterial suspension, and controlling OD of the bacterial suspension_600nmThe value is between 0.5 and 0.7. 10 μ L of the bacterial suspension was uniformly applied to the surface of a metal slide, and after drying, the plate with the sample slide was transferred to an ARTP operating chamber with sterile forceps. By using high purityHelium is used as working gas of plasma to treat the bacterial slide, the power supply power is set to be 60W, the irradiation distance is 3mm, the temperature of the plasma is 26 ℃, the gas flow is 10L/min, different treatment groups are set, the treatment time of each group is respectively 0 (contrast), 30, 60, 90, 120, 150 and 180s, and each group is set to be repeated for three times. The treated slide was transferred to an EP tube containing 1mL of sterile physiological saline, and the microorganism attached to the slide was eluted into the sterile physiological 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, the flat plate is placed in an incubator at 30 ℃ for 48 hours for culture, counting is carried out, and the lethality is calculated according to the following method:

percent lethality ═ number of colonies not subjected to mutagenesis treatment-number of colonies subjected to mutagenesis treatment)/number of colonies not subjected to mutagenesis × 100%

By counting the fatality rate of each treatment group, the irradiation treatment time with the fatality rate of about 80% is selected for formal experiments, so that certain mutation abundance is ensured, and certain survival rate is provided.

As a result: the bacterial suspensions of the different strains were subjected to ARTP mutagenesis, and the lethality curves of the different strains were plotted against a strain which had not been treated with ARTP (treatment time 0s) (FIG. 1). As can be seen from fig. 1, the tolerance of different strains to ARTP is significantly different: (1) in the case of lactobacillus plantarum, the lethality rate is only 36.6% when the lactobacillus plantarum is treated by ARTP for 30s, the lethality rate is greatly increased to 76.8% when the treatment time is 60s, and the lethality rate is almost 100% when the treatment time is more than 90s, the lethality rate is more than 95% and the treatment time is 120 s. (2) The Bacillus pumilus which is also used as a prokaryote has better tolerance to ARTP, the lethality rate is only 24.6 percent when the Bacillus pumilus is treated for 60s under the same condition, the lethality rate is increased to 50.6 percent when the Bacillus pumilus is treated for 90s, the lethality rate is 80.1 percent when the Bacillus pumilus is treated for 120s, the cell survival rate is 3.6 percent when the Bacillus pumilus is treated for 150s, and the cell survival rate is basically 0 when the Bacillus pumilus is treated for 180 s.

Therefore, the subsequent screening is carried out while the mutagenesis effect is ensured and a certain cell survival rate is ensured, and the ARTP treatment is carried out under the condition that the lethality rate is about 80 percent, so that the ARTP treatment time of the lactobacillus plantarum is determined to be 60s, and the treatment time of the bacillus pumilus is determined to be 120 s.

Example 2 acclimatization screening of mutant strains

Directly inoculating the bacterial suspension treated by ARTP into an acclimatization culture medium, culturing for 72h at 30 ℃ and 220rpm in a triangular flask with the liquid loading of 50mL, and inspecting the growth and reproductive capacity of the strain in the culture medium taking tea residue as a main carbon source. And the tea residue content in the domestication culture medium is gradually increased, and the plate separation is combined, so that excellent strains which have strong growth capacity and can tolerate and utilize high-concentration tea residues are obtained and stored for later use. The method comprises the following specific steps:

(1) domestication of mutant strain by low-concentration tea leaves

Directly transferring the bacterial suspension treated by the ARTP to an acclimation culture medium for culturing, observing the color and odor change of fermentation liquor by taking an untreated bacterial strain as a contrast, and counting bacterial colonies of the fermentation liquor.

The results show that: the mutant strains of different types can propagate in different degrees in a culture medium containing 5.0% of tea leaves as a unique carbon source, and can normally have certain metabolic activity, while the original strains cannot normally grow under the same culture medium condition. From the growth aspect, the Bacillus pumilus mutant bacteria grow best, and the Lactobacillus plantarum mutant bacteria grow second; from the aspect of metabolite activity, the Bacillus pumilus embodies a certain pair G⁺(Micrococcus luteus) while Lactobacillus plantarum has G⁺And G^-The bacteriostatic activity of (Escherichia coli) is relatively fuzzy, the boundary is not clear, the bacteriostatic activity is possibly related to organic acid produced by lactic acid bacteria, and the bacteriostatic performance of low-concentration organic acid is not thorough (Table 1).

TABLE 1 acclimatization and culture characteristics of ARTP mutant bacteria in culture medium containing green tea leaves

Type of Strain	pH	Colony count (CFU/mL)	G⁺Activity (mm)	G^-Activity (mm)
					Bacillus pumilus	7.19±0.05	3.9±0.05×10⁷	11.2±0.1	ND
Lactobacillus plantarum	4.10±0.05	8.9±0.05×10⁶	10.2±0.1	10.3±0.1

Note: ND means no bacteriostatic activity was detected.

(2) Rescreening of high tea-residue concentration tolerant mutant strains

Meanwhile, bacterial liquids obtained by domestication culture are continuously coated on domestication culture medium plates containing 10%, 15% and 20% of tea residue leaching liquor respectively, and bacterial colonies capable of growing in high-concentration tea residues are picked for shake flask culture.

The results show that: different strains have bacterial colonies which can grow in 20% tea residue leaching liquor, and the liquid fermentation bacterial concentration of the strains is basically consistent with the domestication culture result of low-concentration tea residue, which shows that the high-concentration tea residue nutrition environment basically does not inhibit the growth of mutant strains, but the capacities of different strains for synthesizing active ingredients are obviously stimulated, and the bacteriostatic activity and the acid production capacity are improved to a certain degree (Table 2).

Overall, Bacillus pumilus for G⁺The antibacterial activity of the compound is lower, and the highest antibacterial activity is 12 +/-0.1 mm. The lactic acid bacteria mutant strain shows good acid production capability, the pH is 4.01(LP-08 strain) at the lowest, and good lactic acid bacteria fermentation characteristics are shown.

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

Note: ND means no bacteriostatic activity was detected.

Selecting strains with best biomass and metabolite activity, namely bacillus pumilus BP-09 and lactobacillus plantarum LP-08, to perform shake flask re-screening, and performing activity antibacterial activity detection by adopting an agar diffusion method.

The results show that: bacillus pumilus BP-09 is only for G⁺The strain has obvious bacteriostatic effect and can form clear and definite bacteriostatic circles; and Lactobacillus plantarum LP-08 vs G⁺And G^-The bacterial strains all have certain inhibition effect, but the inhibition zone has low transparency, which is basically consistent with the result of the tolerance screening.

Bacillus pumilus (Bacillus pumilus) BP-09 is preserved in Guangdong province microorganism culture collection center (GDMCC) at 6-16 months in 2020, with the preservation number of GDMCC No: 61062. the preservation address is No. 59 building 5 of No. 100 college of Jiedui Zhonglu, Guangzhou city.

Lactobacillus plantarum LP-08 was deposited at 16.6.2020 in Guangdong province collection center for microbial cultures (GDMCC) with the deposit number GDMCC No: 61061. the preservation address is No. 59 building 5 of No. 100 college of the first Lizhong Lu of Guangzhou city.

Example 3 liquid culture of Mixed mutant strains

(1) Bacteriostatic analysis of single strain fermentation product

Before multi-strain mixed culture, the inhibition effect of metabolites of different strains on other strains needs to be considered, the strains obtained by screening are separately cultured by adopting a mixed fermentation culture medium, fermentation liquor supernatant is centrifugally collected, and the inhibition effect among the strains is detected according to an agar diffusion method.

The results show that: the strains have no mutual inhibition effect basically.

(2) Mixed culture analysis of multiple strains

On the basis of pure culture of a single strain and strains of the same type, different types of strains are designed to be cultured in the same fermentation system according to physiological and metabolic characteristics of different strains, one-pot multi-strain culture is realized, and the viable count and the metabolite activity of mixed culture conditions are inspected. The method comprises the following specific steps:

scraping a large ring in activated slant (18 × 180mm) of different bacteria respectively, inoculating to corresponding slant of eggplant bottle, culturing at 30 deg.C for 24 hr, adding 50mL sterile water into each eggplant bottle, eluting, preparing bacterial suspension, OD_600nmControlling the concentration to be between 0.5 and 0.6, namely the slant seed liquid.

Mixing the eluted bacterial suspensions, inoculating the total amount of the mixed bacterial suspensions to a 50L mixed culture tank, and carrying out expanded culture on the strains, wherein the liquid volume of the culture tank is 30L. The culture temperature is as follows: 30 ℃, the pot pressure is 0.05-0.08Mpa, the primary stirring is 250rpm, the dissolved oxygen naturally drops to 0, the pH naturally drops, 20% ammonia water is fed to control the pH to be about 5.0, the ventilation volume is 15L/min, and the period is as follows: and (5) 24 h. Sampling every two hours to detect the bacteriostatic activity of the fermentation broth (G)⁺And G^-) And the total number of colonies.

The results showed that the total number of Colonies (CFU) was about 1.0 × 10 at the initial (0 h fermentation)⁷The strain enters a fermentation system at 0-6h, the strain is in an adaptation period, the concentration of reducing sugar in the culture system is not reduced, the mixed strain does not consume the reducing sugar, the mixed strain keeps the concentration of the reducing sugar in the system by hydrolyzing complex glycogen through amylase in the adaptation period, the strain enters a logarithmic growth phase after fermenting for 6-8h, the colony number is increased sharply, fermentation foam is increased sharply, the consumption of the reducing sugar is increased correspondingly, the amount of the reducing sugar is reduced sharply along with the propagation of the mixed strain, the amount of the reducing sugar is not detected basically after about 16h, the growth of the foam is stopped basically, and the colony is cultured for 20hThe number reaches the maximum, and the CFU is 3.5 × 10¹⁰The results show that the mixed bacteria can be well adapted to the mixed culture system and can fully utilize the mixed culture components to propagate and metabolize the bacteria.

From the conditions of thalli in different culture periods, when fermentation is carried out for 0h, the number of long rods and short bacilli in a visual field is basically consistent, spores are not formed basically, and the thalli are deeply dyed, which indicates that the activity of the mixed bacterial strain is possible; microscopic examination of 12h of culture shows that the number of colonies in the whole visual field is obviously increased, the number of brevibacterium is increased, and a small amount of spores begin to appear, wherein the number of the long bacillus is changed from slender to thick, and the number of the strains mainly in a pair or a splayed shape is more, which indicates that the mixed strain is in a vigorous breeding growth phase; and (3) sampling before the culture is finished, further increasing the number of thalli, particularly obviously increasing the number of spores, and meanwhile, occasionally observing individual brevibacterium in a visual field, staining, wherein the proportion of colony types in the visual field is approximately equal to the ratio of spores: bacillus 9: 7.

in addition, in view of the metabolic characteristics of the functional strains themselves, the bacteriostatic properties of the supernatants at each period of the mixed culture were measured by the agar diffusion method, and the results are shown in table 3 below.

TABLE 3 bacteriostatic properties under mixed culture of functional strains

In terms of bacteriostatic properties, the bacteriostatic activity throughout the sampling period is expressed as G⁺The bacteriostatic activity is dominant, and in the early stage of mixed bacteria culture, along with the increase of the number of bacillus pumilus and the increase of foam in the fermentation process, the bacteriostatic activity of the supernatant of the fermentation liquor is correspondingly increased and reaches the maximum value in 12 hours, G⁺The diameter of the inhibition zone reaches 22.2 +/-0.2 mm, and the proliferation of other types of strains, G, is carried out along with the reduction of fermentation foam⁺The bacteriostatic properties were also slightly reduced from their G⁺Connection of bacteriostatic Properties to the foam, G⁺The bacteriostatic active ingredient may be lipopeptide (such as surfactin, iturin, etc.). Is different from G⁺Bacteriostatic activity, G^-The bacteriostatic activity was barely observed until 12h after the culture, andthe zone of inhibition is also not clear and transparent.

In general, different types of strains co-inhabit well under this same system. Moreover, the good antibacterial activity exhibited by the mixed culture of the strains brings benefits for subsequent solid fermentation and animal breeding application.

Example 4 fungal enzyme synergistic fermentation of tea leaves

The solid fermentation tea residue test steps are as follows:

the fermentation substrate is composed of wet green tea leaves and auxiliary materials, wherein the tea leaves: degreasing rice bran: bean pulp 7: 2: 1. meanwhile, in order to better promote the conversion of the microorganisms to the tea residues, enzyme synergistic fermentation is selected, wherein the final concentration of cellulase 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.

The liquid mixed bacterial liquid (the mixed bacterial liquid of the Bacillus pumilus BP-09 and the Lactobacillus plantarum LP-08, the total number of initial colonies is about 1 × 10⁶cfu/g; wherein, the bacillus pumilus BP-09: lactobacillus plantarum LP-08 ═ 1: 1.5) inoculating, wherein the inoculation amount is 5: 95, while adjusting the water content to ensure that the matrix disperses upon contact and is optimally kneaded into a mass without dripping water (water content of about 40-45%). Then the mixed substrate is put into a fermentation bag with a one-way valve and fermented for 7-10 days at normal temperature (25-30 ℃) until the sweet and sour flavor of the distiller's yeast is achieved, which indicates that the fermentation is complete and sufficient. After fermentation, the indexes of total viable count, total acid, crude protein, acid soluble protein, crude fiber, lysine, threonine, methionine and the like of a fermentation sample are mainly measured, and the detection method is executed by referring to national standard and industrial standard methods.

The bacterial enzymes are fermented for 9 days in a synergistic way, and the detection results are shown in table 4.

TABLE 4 fungus enzyme synergistic fermentation of tea leaves

Note: the detection data of the fermentation index are all measured by wet weight.

The results in table 4 show that the tea leaves were fermented using the mixed bacterial liquid:

(1) from the water content before and after fermentation, the water 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 the 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 into the mixed bacteria before fermentation was 1 × 10⁶cfu/g, the strain can firstly utilize digestible carbon and nitrogen sources to carry out basic metabolism (aerobic and anaerobic), particularly auxiliary materials such as soybean meal, amino acid nitrogen and the like in the strain are most beneficial to the growth of the strain (the strain is increased by two orders of magnitude), and meanwhile, synthetic metabolites such as organic acid, enzyme and the like of the strain further assist in hydrolyzing complex substrates, so that the further growth and propagation of the strain are promoted, and the adaptability and the growth characteristics of the better tea residue matrix of BP-09 and LP-08 strains are reflected.

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

(4) The total acid content is greatly improved from the total acid after fermentation, which shows that both the BP-09 and LP-08 strains can adapt to a certain acidic environment and have better acid production characteristics, particularly the LP-08 strain utilizes a substrate to carry out acid metabolism under the anaerobic condition and is used as a fermented feed, a certain acidity value is favorable for mildew prevention of the fermented substrate, and the strain also has a certain food calling effect as a biological feed, thereby reflecting better application characteristics.

(5) The content of crude fiber is obviously different before and after fermentation according to the content of the crude fiber, and the content of the crude fiber is related to exogenously added enzyme preparations and is also closely related to the growth metabolism of the strains BP-09 and LP-08. The good tea residue tolerance and growth characteristics of the BP-09 strain also fully show the degradation capability of crude fiber of the BP-09, and meanwhile, the organic acid fermentation characteristic of the LP-08 strain also creates conditions for metabolism of a complex matrix, which is the basis that the mixed strain can ferment the tea residue matrix, and the content of the crude fiber is reduced by 51.6 percent under the synergistic condition of bacteria and enzyme.

(6) From the amino acid content, lysine, threonine and methionine in the essential amino acids before and after fermentation were all increased by a factor of 7.7, 8.0 and 3.3, respectively. The three amino acids are used as limiting amino acids in animal nutrition, directly influence the absorption and utilization of other amino acids by animals, and fully embody the good tea residue fermentation metabolic capacity of LP-08 and BP-09 bacterial strains.

In conclusion, the safe strains conforming to the national feed microorganism catalogue are the bacillus pumilus and the lactobacillus plantarum, and the combination of the bacillus pumilus and the lactobacillus plantarum has positive synergistic effect. The bacillus pumilus has strong enzyme production capacity, can produce various enzymes including protease, glycosidase, amylase, lipase and the like, can metabolize a composite carbon-nitrogen source, and secretes various bioactive substances such as cyclic lipopeptide, bacteriocin, antibacterial peptide and the like, so that the bacillus pumilus is very favorable for degrading complex nitrogen-containing compounds in green tea dregs, and utilizes part of carbohydrate, thereby improving the content of amino acid in feed; the lactobacillus plantarum can not only secrete some enzymes, but also metabolize to generate various organic acids and lactein, which is greatly helpful for improving the quality of the feed. On the basis, the method starts from ARTP mutagenesis and domestication and directed breeding of the tea residue, screens the tea residue which can tolerate a certain concentration, the method can establish a one-pot multi-bacterium mixed culture process by taking tea dregs as strains growing mainly as nutrient sources and evaluating the possibility of mixed culture of strains with different functions, thereby not only solving the problems of time and labor waste and expense of single strain culture, but also greatly increasing the symbiosis characteristic of mixed strains, meanwhile, the propagation of mixed functional microorganisms is promoted by matching with a proper auxiliary material combination, the coordination of exogenous enzyme is combined, macromolecules in the tea residues are digested, active ingredients in the tea are released, the raw material utilization space is further excavated, the colony advantages in solid fermentation are fully enhanced, high-content probiotics and functional active substances thereof are provided, the tea residues are effectively recycled, the health of animal intestinal tracts is maintained, and benefits are brought to healthy cultivation.

Finally, it should be noted that the above-mentioned contents are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, and that the simple modifications or equivalent substitutions of the technical solutions of the present invention by those of ordinary skill in the art can be made without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1.A method for preparing tea residue feed by fermenting mixed strains is characterized by comprising the following steps:

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.