Salt-tolerant thermophilic brevibacillus parabrevis H166 as well as screening method and application thereof

Abstract

The invention discloses a salt-tolerant thermophilic brevibacillus parabrevis H166 and a screening method and application thereof, the salt-tolerant thermophilic brevibacillus parabrevis H166 is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation date is November 26, 2020, and the preservation number is CGMCC No.21234; the invention discloses a method for screening salt-tolerant thermophilic brevibacillus parabrevis H166. The method comprises the following steps: raw material collection, enrichment culture, dilution and coating, purification and preservation, plate hydrolysis test, strain extraction and identification, and preparation of a microbial inoculum: a, drawing of a strain growth curve, b, quantitative determination of enzyme activity, c, preparation of a first-stage seed culture solution, and d, preparation of a second-stage seed culture solution. The H166 salt-tolerant strain is derived from the kitchen waste and has high lipase activity, a microbial agent prepared from the H166 salt-tolerant strain can help to accelerate the aerobic fermentation process of the kitchen waste, and an efficient and environment-friendly microbial pretreatment mode is provided for rapid aerobic fermentation of the kitchen waste.

Description

Technical Field

[0001] The invention relates to the technical field of bacterial agent preparation, and in particular to a salt-resistant and thermophilic Bacillus parabrevis H166 and a screening method and application thereof. Background Art

[0002] The annual amount of food waste in my country is increasing year by year, and its treatment needs to follow the principles of reduction, resource utilization and harmlessness. However, food waste has the characteristics of high oil content and high salt content, which increases the difficulty of transportation and treatment. In recent years, the application prospect of microbial inoculation technology in aerobic fermentation of food waste is broad. Studies have shown that adding microbial agents during the aerobic fermentation of food waste can significantly degrade crude fat and crude fiber in food waste, increase crude protein content, etc., which is one of the effective ways to efficiently treat food waste and turn it into organic fertilizer. Therefore, it is necessary to further optimize the adaptability of microorganisms to enhance the role of microorganisms in the aerobic fermentation of food waste, accelerate the treatment of food waste and reduce environmental problems caused by accumulation.

[0003] Soil salinization is a global ecological problem. Affected by salinization, it is urgent to improve the current situation of land degradation and increase agricultural productivity. At present, the biological treatment of saline soil mainly relies on microbial fertilizers. The permeability and air permeability of saline soil are improved by preparing bacterial fertilizers with halophilic microorganisms to increase the yield of crops. Kitchen waste itself has the characteristics of high salt content. At present, the bacterial agents for kitchen waste are mainly exogenous microorganisms. It is rare to enrich and screen salt-tolerant strains from their own sources and prepare them into bacterial agents. If local salt-tolerant thermophilic microorganisms are enriched and screened from the mixed solution of kitchen waste and saline soil, the kitchen waste can be used as fertilizer in a more environmentally friendly and thorough manner, which may also be of great significance to solving the problem of soil salinization.

[0004] Most of the existing aerobic fermentation agents for food waste are exogenous microorganisms selected from outside the raw materials. Although they have a certain broad-spectrum suitability, it is difficult to predict the interaction between the strains in the agent and the indigenous microorganisms. They are not enough to accelerate the fermentation of special components such as high oil and high salt in food waste, which requires certain exploratory pre-tests during the inoculation process. At the same time, the cost of inoculation of the agent needs to be considered.

[0005] Therefore, in view of the shortcomings of the prior art, the present invention provides a salt-tolerant thermophilic Bacillus parabrevis H166 and a screening method and application thereof, aiming to screen out a salt-tolerant thermophilic strain from the composting process of food waste under salt stress conditions, prepare the bacterial agent into a bacterial agent and apply it to food waste to enhance the aerobic fermentation process of food waste. Summary of the invention

[0006] The purpose of the present invention is to provide a salt-resistant and thermophilic Bacillus parabrevis H166 and a screening method and application thereof, so as to fill the gap in the current technology.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A salt-tolerant and thermophilic Brevibacillus parabrevis H166 and a screening method thereof, wherein the salt-tolerant and thermophilic Brevibacillus parabrevis H166 is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, with a deposit date of November 26, 2020 and a deposit number of CGMCC No. 21234;

[0009] The screening method of the salt-tolerant and thermophilic Brevibacillus parabrevicaulis H166 specifically comprises the following steps:

[0010] (1) Raw material collection:

[0011] Collect food waste samples and saline soil at the same time, remove the larger particles, mix them evenly by quartering, put them into ziplock bags, and store them at 4°C for later use;

[0012] (2) Enrichment culture:

[0013] The kitchen waste collected in step (1) is thoroughly mixed with saline soil and sawdust, and placed in a container for culturing at a constant temperature of 50 to 70° C. for 28 to 32 days. During the culturing process, water is added to the container every 5 to 6 days to replenish water to prevent water loss from affecting the activity of microorganisms;

[0014] (3) Dilution coating:

[0015] Dilute the enriched sample solution to 10 -3 , 10 -4 , 10 -5 and 10 -6 , prepare 1-2% bromocresol purple filtered through a sterilizing filter for later use;

[0016] Prepare LB selection medium containing NaCl: weigh tryptone, yeast extract, and NaCl, put them into container 1, add a small amount of deionized water to mix the solutes, rinse with deionized water several times in small amounts to make up the volume, transfer all the solution to container 2, wrap it with sealing film and sterilize it at 121°C for 15-25 minutes, take out the culture medium and place it at room temperature to cool to 45-55°C, add bromocresol purple and cycloheximide dissolved in methanol, mix well, pour it into a plate in an ultra-clean bench, condense it into a solid plate, and set it aside;

[0017] Continue to operate in the clean bench and take 10 -3 , 10 -4 , 10-5 and 10 -6 The enriched sample dilution was spread on LB selection medium containing NaCl, cultured overnight at a constant temperature of 25-35°C, and the growth of each plate was observed;

[0018] (4) Purification and preservation:

[0019] Select plates with dispersed and uniform colony growth, pick single colonies with different colony morphology for streaking purification, and store the obtained single strains at -80°C using 40% glycerol. In addition, pick the purified strains into centrifuge tubes, add LB broth medium, culture at a constant temperature of 28-33°C for 20-28h, centrifuge at 10000xg for 5-7min, remove the supernatant and store at -20°C for extraction of strain DNA;

[0020] (5) Flat plate hydrolysis test:

[0021] Prepare protein, starch, and oil hydrolysis medium, sterilize the three hydrolysis medium, and pour them into plates. After the plates are condensed to a solid state, inoculate the isolated and purified strains into the three hydrolysis mediums respectively to observe whether a hydrolysis transparent circle appears. If so, it means that the substrate has been hydrolyzed and the strain has the ability to degrade the substrate. Otherwise, it does not appear.

[0022] (6) Strain extraction and identification:

[0023] The above-screened salt-tolerant strains were extracted, and universal primers 27F and 1492R were selected for PCR amplification. After the products were qualified by agarose gel electrophoresis, they were tested. In the hydrolysis medium test, it was observed that the strain H166 had a strong oil degradation ability. It was identified by sequencing that it was Brevibacillus Parabrevis H166, and the rRNA sequence similarity reached 98.90%;

[0024] (7) Preparation of bacterial agent:

[0025] a. Strain growth curve drawing:

[0026] Take a single colony and culture it in a centrifuge tube at 28-32℃ overnight until OD 600 The value is 0.8 to 1, and the LB medium is inoculated at a volume ratio of 1 to 2%. The culture medium is cultured on a constant temperature shaker at 28 to 32°C and 170 to 200 r / min for 16 to 22 hours. The culture medium is aspirated and placed in a 96-well plate to measure the OD 600 value;

[0027] b. Quantitative determination of enzyme activity:

[0028] After the strain H166 obtained by separation and purification culture was activated on LB medium, it was cultured at a constant temperature of 28-32°C for 10-16h, and a single bacterium was picked out in a 24-well cell culture plate, and cultured at 28-32°C and 170-200r / min for 10-16h, and then transferred to 24-well plates containing protease, lipase and amylase active fermentation broth, respectively, and cultured at 35-39°C and 180-230r / min for 24-26h, and the fermentation broth was centrifuged at 5000-6000rpm and 4-5°C for 10min, and the obtained supernatant was filtered to obtain the crude enzyme solution;

[0029] c. Primary seed culture medium:

[0030] The above strains were inoculated into a container containing LB liquid culture medium in a clean bench and cultured in a constant temperature shaker at 28-32°C and 170-200 r / min for 46-54 h. 600 When the value exceeds 3.0, the culture is stopped and the primary seed culture solution is obtained;

[0031] d. Secondary seed culture medium:

[0032] The primary seed culture solution was inoculated into a container containing LB liquid culture medium in a clean bench and cultured in a constant temperature shaker at 28-32°C and 170-200 r / min for 34-40 h. 600 When the value exceeds 3.0, the culture is stopped and the obtained secondary seed culture solution is the salt-tolerant and thermophilic Bacillus parabrevis H166.

[0033] Preferably, in step (5), the protein hydrolysis medium is prepared by: peptone, beef extract, NaCl, casein, CaCl2, L-tyrosine, agar, distilled water, pH adjusted to 7.0-7.2, sterilized at 121°C for 20 min, cooled to 55-65°C, and adding sterilized Tween 80.

[0034] Preferably, in step (5), the preparation method of the starch hydrolysis medium is: soluble starch, peptone, NaCl, agar, triphenyl blue, distilled water, pH 7.0-7.2, and sterilization at 121°C for 20 min.

[0035] Preferably, in step (5), the preparation method of the oil hydrolysis medium is: oil, 1.6% neutral red, peptone, sodium chloride, beef extract, agar, distilled water, pH adjusted to 7.0-7.2, and sterilized at 121°C for 20 minutes.

[0036] Preferably, the preparation method of the protease in step b in step (7) comprises: taking a crude enzyme solution, adding a 0.5% casein solution pre-incubated at 36-38° C., bathing at 36-38° C. for 25-35 min, then adding a 10% TCA solution to terminate the reaction, centrifuging at 12000 r / min for 5-8 min, taking a supernatant, and determining the tyrosine content;

[0037] Preferably, the preparation method of the lipase described in step b in step (7) is as follows: polyvinyl alcohol solution and olive oil are mixed at a ratio of 3:1 (V / V), ultrasonically treated for 5 to 8 minutes to prepare a milky white polyvinyl alcohol emulsion, which is the substrate solution; 4 mL of the substrate solution is taken, 5 mL of phosphate buffered saline is added, and 1 mL of crude enzyme solution is added after being bathed at 38 to 42°C for 5 to 6 minutes. After mixing, the reaction is carried out at a constant temperature for 12 to 18 minutes, and 15 mL of 95% ethanol is added to terminate the reaction, and the amount of enzyme required for the fatty acid is determined.

[0038] Preferably, the preparation method of the amylase described in step b in step (7) is as follows: 0.1 mL of crude enzyme solution is taken, added to 2 mL of 1% soluble starch solution, extracted and incubated in a 36-38° C. water bath for 25-35 min, shaken once every 5 min, then cooled to room temperature after boiling in a water bath for 5-6 min, 3 mL of 3,5-dinitrosalicylic acid reagent is added, and then rapidly cooled to room temperature after boiling in a water bath for 5-6 min again, constant volume is taken, and OD value is measured.

[0039] Due to the application of the above technical solution, the present invention has the following beneficial effects compared with the prior art:

[0040] 1. The salt-tolerant strain H166 of the present invention is derived from kitchen waste and has high lipase activity. The bacterial agent prepared from the strain can help accelerate the aerobic fermentation process of kitchen waste, thus providing an efficient and environmentally friendly microbial pretreatment method for realizing rapid aerobic fermentation of kitchen waste;

[0041] 2. The H166 bacterial agent of the present invention helps to increase the maximum temperature of the food waste pile and prolong the high temperature period of the pile. The significant change in the temperature of the pile helps thermophilic microorganisms maintain a high activity, thereby promoting the composting process;

[0042] 3. The H166 bacterial agent of the present invention improves the efficiency of microorganisms in utilizing organic matter and the decomposition efficiency, making the fermentation effect better. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, some of the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be made based on these drawings without paying any creative work.

[0044] Figure 1 is a growth curve diagram of strain H166 in Example 1 of the present invention;

[0045] Figure 2 This is a temperature change diagram of the pile after inoculation of H166 bacterial agent in Example 1 of the present invention;

[0046] Figure 3 This is a graph showing the change in moisture content of the pile after inoculation of the H166 bacterial agent in Example 1 of the present invention;

[0047] Figure 4 This is a graph showing the change in organic matter content in the pile after inoculation of the H166 bacterial agent in Example 1 of the present invention;

[0048] Figure 5 This is a graph showing the GI changes of the pile after inoculation of the H166 bacterial agent in Example 1 of the present invention. DETAILED DESCRIPTION

[0049] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, a specific implementation scheme is now described in detail.

[0050] The present invention is further described below in conjunction with the examples, but the present invention is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to the different requirements of specific use, and the implementation conditions not indicated are conventional conditions in the industry. The technical features involved in each embodiment of the present invention can be combined with each other as long as they do not conflict with each other.

[0051] Example 1

[0052] See attached Figure 1-5 , this embodiment provides a salt-tolerant and thermophilic Bacillus parabrevis H166 and a screening method and application thereof, wherein the salt-tolerant and thermophilic Bacillus parabrevis (Brevibacillus Parabrevis) H166 is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with a deposit date of November 26, 2020 and a deposit number of CGMCC No.21234;

[0053] The screening method of the salt-tolerant and thermophilic Brevibacillus parabrevicaulis H166 specifically comprises the following steps:

[0054] (1) Raw material collection:

[0055] Food waste samples were collected from 5 random points at the Dongshan food waste treatment base of Suzhou Hanbo Environmental Technology Co., Ltd. using an "S" type. At the same time, saline soil was collected from 5 random points at the Zhoushan coast using an "S" type. Larger particles were removed and the soil was passed through a 2 mm sieve. After that, it was mixed by quartering and packed into self-sealing bags and stored at 4 °C for later use.

[0056] (2) Enrichment culture:

[0057] 60 g of the kitchen waste collected in step (1), 25 g of saline soil and 15 g of sawdust were fully mixed, placed in a 500 mL conical flask and cultured in a constant temperature incubator at 60° C. for 30 days, with 3 replicates. During the culture process, water was added to the container every 5 days to replenish moisture to prevent moisture loss from affecting microbial activity;

[0058] (3) Dilution coating:

[0059] Dilute the enriched sample solution to 10 -3 , 10 -4 , 10 -5 and 10 -6 , prepare 1% bromocresol purple filtered through a membrane sterilizing filter (0.45 μm pore size) for later use;

[0060] Prepare LB selection medium containing 1% NaCl: weigh 10g of tryptone, 5g of yeast extract, and 10g of NaCl, put them into a 500mL beaker, add a small amount of deionized water, mix the solute with a glass rod, rinse with deionized water several times, transfer to a 1L volumetric flask and make up to volume, transfer all the solution to a 1L conical flask, wrap it with sealing film, sterilize it at 121℃ for 20min, take out the culture medium and place it at room temperature. When it cools to about 50℃, add 1% bromocresol purple and 100mg of cycloheximide dissolved in methanol, mix well, pour it into a plate in a clean bench, condense it into a solid plate, and set it aside;

[0061] Continue to operate in the clean bench and take 100 μL of 10 -3 , 10 -4 , 10 -5 and 10 -6 The enriched sample dilution was spread on LB selection medium containing 1% NaCl, with 3 replicates for each treatment, and cultured overnight in a 30°C constant temperature incubator to observe the growth of each plate;

[0062] (4) Purification and preservation:

[0063] Select plates with dispersed and uniform colony growth, pick single colonies with different colony morphology for streaking purification, and store the obtained single strains at -80°C using 40% glycerol. In addition, use a sterilized toothpick to pick the purified strains into a 1.5mL centrifuge tube, add 600μLLB broth medium, culture on a 30°C constant temperature shaker for 24h, centrifuge at 10000xg for 5min, remove the supernatant and store at -20°C for strain DNA extraction;

[0064] (5) Flat plate hydrolysis test:

[0065] Prepare protein, starch, and oil hydrolysis medium, sterilize the three hydrolysis medium, and pour them into plates. After the plates are condensed to a solid state, inoculate the isolated and purified strains into the three hydrolysis mediums respectively to observe whether a hydrolysis transparent circle appears. If so, it means that the substrate has been hydrolyzed and the strain has the ability to degrade the substrate. Otherwise, it does not appear.

[0066] Wherein, the preparation method of protein, starch and oil hydrolysis medium is as follows:

[0067] ① Protein hydrolysis medium: peptone 10g, beef extract 5g, NaCl 5g, casein 5g, CaCl2 0.1g, L-tyrosine 0.1g, agar 20g, distilled water 1L, pH adjusted to 7.0-7.2, sterilized at 121℃ for 20min, cooled to 60℃, and added 1% sterilized Tween 80;

[0068] ② Starch hydrolysis medium: soluble starch 2g, peptone 10g, NaCl 5g, agar 20g, 0.05-0.1g of triphenyl blue, distilled water 1L, pH 7.0-7.2;

[0069] ③ Oil hydrolysis medium: 10 mL oil, 1 mL 1.6% neutral red, 10 g peptone, 5 g sodium chloride, 5 g beef extract, 20 g agar, 1 L distilled water, pH adjusted to 7.0-7.2, sterilized at 121°C for 20 min;

[0070] (6) Strain extraction and identification:

[0071] The above-screened salt-tolerant strains were extracted using the Bomed Bacterial Genomic DNA Rapid Extraction Kit (refer to the kit manual for specific operation steps), and universal primers 27F and 1492R were selected for PCR amplification. After the products were tested by 1% agarose gel electrophoresis and qualified, they were sent to Guangdong Meige Gene Technology Co., Ltd. for sequencing and identification of the genus;

[0072] In the hydrolysis medium test, it was observed that the strain H166 had a strong oil-dissolving ability. It was identified by sequencing as Brevibacillus Parabrevis H166, with an rRNA sequence similarity of 98.90%.

[0073] (7) Preparation of bacterial agent:

[0074] a. Strain growth curve drawing:

[0075] Take a single colony and culture it in a 1 mL centrifuge tube in a 30°C constant temperature incubator overnight until the OD 600 The value was 0.8-1, and 1% (100 μL) was inoculated into new LB medium, and cultured on a constant temperature shaker at 30°C and 180 r / min for 18 h. 200 μL of the culture solution at 0, 2, 4, 6, 8, 10, 12, 14, 16 and 18 h was taken and placed in a 96-well plate to measure the OD 600 Value (When OD 600 When the value is greater than 1, perform multiple dilution and then measure, and multiply by the dilution multiple when calculating);

[0076] b. Quantitative determination of enzyme activity:

[0077] The strain H166 obtained by separation and purification was activated on LB medium and cultured in a constant temperature incubator at 30°C for 12 hours. A single bacterium was picked and placed in a 24-well cell culture plate and cultured at 30°C and 180 r / min for 12 hours. 1% was transferred to a 24-well plate containing 2 mL of protease, lipase and amylase active fermentation broth, respectively. Three replicates were set for each enzyme active fermentation broth, and cultured at 37°C and 200 r / min for 24 hours. The fermentation broth was centrifuged at 5000 rpm and 4°C for 10 minutes. The obtained supernatant was filtered through a 0.22 μm microfiltration membrane to obtain a crude enzyme solution.

[0078] Wherein, the preparation method of protease, lipase and amylase is as follows:

[0079] ① Protease: Pipette 1 mL of crude enzyme solution, add 2 mL of 0.5% casein solution pre-incubated at 37°C, incubate at 37°C for 30 min, then add 3 mL of 10% TCA solution to terminate the reaction, centrifuge at 12000 r / min for 5 min, and then pipette 1 mL of supernatant. Use the blank control without 0.5% casein to determine the tyrosine content (1 enzyme activity unit: the amount of enzyme required to hydrolyze casein to generate 1 μg of tyrosine in 1 min at 37°C);

[0080] ② Lipase: Mix 40g / L polyvinyl alcohol solution with olive oil at a ratio of 3:1 (V / V), and ultrasonically treat for 6 minutes to prepare a milky polyvinyl alcohol emulsion, which is the substrate solution. Pipette 4mL of substrate solution, add 5mL of phosphate buffered saline solution, and add 1mL of crude enzyme solution after 5min in a 40℃ water bath. Mix well and react at constant temperature for 15min. Add 15mL of 95% ethanol to terminate the reaction. Add 15mL of 95% ethanol for preheating and then add crude enzyme solution for 15min as blank control. Use 0.05mol / L NaOH standard solution for titration (1 enzyme activity unit: the amount of enzyme required to produce 1μmol of titratable fatty acid per minute at pH=7.5 and 40℃);

[0081] ③Amylase: Pipette 0.1mL crude enzyme solution, add it to 2mL of 1% soluble starch solution, incubate in a 37℃ water bath for 30min, shake once every 5min, cool to room temperature after boiling in a water bath for 5min, add 3mL 3,5-dinitrosalicylic acid reagent, boil in a water bath for 5min again, cool to room temperature quickly, dilute to 25mL, and measure OD value;

[0082] The enzyme activities of protease, lipase and amylase are shown in Table 1;

[0083] Table 1

[0084] Enzyme activity Vitality value (U / μL) Protease 16.78 Lipase 0.63 Amylase 41.62

[0085] c. Primary seed culture medium:

[0086] The above strains were inoculated into a 250 mL conical flask containing 100 mL LB liquid medium in a clean bench and cultured in a constant temperature shaker at 30 °C and 180 r / min for 48 h. The OD of the bacterial solution was 600 When the value exceeds 3.0, the culture is stopped and the primary seed culture solution is obtained;

[0087] d. Secondary seed culture medium:

[0088] Inoculate 5 mL of the primary seed culture solution into a 250 mL conical flask containing 100 mL of LB liquid medium in a clean bench and culture in a constant temperature shaker at 30 °C and 180 r / min for 36 h. 600 When the value exceeds 3.0, the culture is stopped and the obtained secondary seed culture solution is the salt-tolerant and thermophilic Bacillus parabrevis H166.

[0089] Application of H166 bacterial agent in food waste composting:

[0090] (1) Composting test:

[0091] Kitchen waste was collected from Suzhou Hanbo Environmental Technology Co., Ltd. using an "S" type sampling method. After manual sorting to remove impurities and quartering, it was crushed and dehydrated and passed through a 2 mm sieve. It was mixed evenly with commercial sawdust at a moisture content of 60% and a C / N ratio of 20, and then added to a 50L closed foam box composting reactor system for composting. The aeration volume was 6 min -1 L -1 , aeration for 20 min every 30 min, inoculation of 5‰ H166 bacterial agent (T1), and addition of 5‰ sterile water as blank control (CK), a 15-day aerobic fermentation test, temperature measurement every 24 h, and samples collected every 3 days for physical and chemical determination;

[0092] (2) Determination of physical and chemical indicators:

[0093] ①Temperature: electronic thermometer;

[0094] ② Moisture content: Weigh 5g (accurate to 0.0001g) of compost sample in a dry aluminum box, dry at 105℃ for 12h, record the net weight of the aluminum box, the total weight of the fresh sample and the aluminum box, and the total weight of the dry sample and the aluminum box, and calculate according to the formula: moisture content = (fresh sample weight - dry sample weight) / fresh sample weight × 100%;

[0095] ③ Organic matter content: Take 15.00 mg (accurate to 0.01 mg) of dried sample and put it in the tin foil paper specially used for elemental analyzer, wrap it, press it into tablets, and then measure it on the analyzer for 6 minutes, record the data, and calculate it according to the formula: Organic matter content = TC × 1.724;

[0096] ④ Seed germination index: add 50mL of distilled water to a 150mL conical flask with glass beads, sterilize at 121℃ for 20min, weigh 10g of fresh compost sample, add it to the conical flask, oscillate at 25℃, 180r / min for 1h, transfer to a 50mL centrifuge tube, centrifuge at 3000r / min for 20min, filter the supernatant into a 10mL centrifuge tube, take 5mL of the percolate and add it to a 9.0cm Petri dish covered with 2 layers of filter paper, use distilled water as a blank control, select 20 full Zhongnong No. 20 cucumber seeds and arrange them evenly in the Petri dish, and after constant temperature culture at 30℃ for 48h, determine the germination rate and total root length of the cucumber seeds, and calculate according to the formula: GI = (A1A2 / B1B2) × 100%, where A is the compost extract, B is distilled water, 1 represents the seed germination rate, and 2 represents the total root length of the seeds;

[0097] (3) Test results:

[0098] During aerobic fermentation, temperature, moisture content, organic matter content and GI are important indicators for measuring the reduction of composting materials. Figure 2It can be seen that compared with CK without inoculation, inoculation of 5‰ H166 bacterial agent helps to increase the maximum temperature of the food waste pile and prolong the duration of the high temperature period of the pile. The significant change in the pile temperature helps thermophilic microorganisms maintain a high activity, thereby promoting the composting process; Figure 3 It can be seen that although the moisture content of each treatment dropped to about 45% in the end, the moisture content of the treatment inoculated with 5‰ H166 inoculant decreased more than that of CK; Figure 4 It can be seen that the reduction in organic matter content in the treatment inoculated with 5‰ H166 bacterial agent was significantly greater than that in CK, indicating that the efficiency of organic matter utilization by microorganisms in this treatment was higher. Figure 5 It can be seen that the treatment inoculated with 5‰ H166 bacterial agent reached the maturity standard first, indicating that the fermentation effect was better;

[0099] In summary, the H166 salt-tolerant strain of the present invention is derived from kitchen waste and has a high lipase activity. The bacterial agent prepared from the bacterial agent can help accelerate the aerobic fermentation process of kitchen waste, and provides an efficient and environmentally friendly microbial pretreatment method for realizing rapid aerobic fermentation of kitchen waste; the H166 bacterial agent of the present invention helps to increase the maximum temperature of the kitchen waste pile and prolong the high temperature period of the pile. The significant change in the temperature of the pile helps thermophilic microorganisms to maintain a high activity, thereby promoting the composting process; the H166 bacterial agent of the present invention improves the efficiency of microorganisms in utilizing organic matter and the decomposition efficiency, so that the fermentation effect is better; it is of great significance to realize rapid aerobic fermentation through an environmentally friendly biological treatment method.

[0100] The above-mentioned embodiment only expresses one implementation mode of the present invention, and its description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

Claims

1. A salt-tolerant and thermophilic Bacillus parabrevis H166 and a screening method thereof, characterized in that: The salt-tolerant and thermophilic Brevibacillus Parabrevis H166 is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with a deposit date of November 26, 2020 and a deposit number of CGMCC No. 21234; The screening method of the salt-tolerant and thermophilic Brevibacillus parabrevicaulis H166 specifically comprises the following steps: (1) Raw material collection: Collect food waste samples and saline soil at the same time, remove the larger particles, mix them evenly by quartering, put them into ziplock bags, and store them at 4°C for later use; (2) Enrichment culture: The kitchen waste collected in step (1) is thoroughly mixed with saline soil and sawdust, and placed in a container for culturing at a constant temperature of 50 to 70° C. for 28 to 32 days. During the culturing process, water is added to the container every 5 to 6 days to replenish water to prevent water loss from affecting the activity of microorganisms; (3) Dilution coating: Dilute the enriched sample solution to 10 -3 , 10 -4 , 10 -5 and 10 -6 , prepare 1-2% bromocresol purple filtered through a sterilizing filter for later use; Prepare LB selection medium containing NaCl: weigh tryptone, yeast extract, and NaCl, put them into container 1, add a small amount of deionized water to mix the solutes, rinse with deionized water several times in small amounts to make up the volume, transfer all the solution to container 2, wrap it with sealing film and sterilize it at 121°C for 20 minutes, take out the culture medium and place it at room temperature to cool to 45-55°C, add bromocresol purple and cycloheximide dissolved in methanol, mix well, pour it into a plate in an ultra-clean bench, condense it into a solid plate, and set it aside; Continue to operate in the clean bench and take 10 -3 , 10 -4 , 10 -5 and 10 -6 The enriched sample dilution was spread on LB selection medium containing NaCl, cultured overnight at a constant temperature of 25-35°C, and the growth of each plate was observed; (4) Purification and preservation: Select plates with dispersed and uniform colony growth, pick single colonies with different colony morphology for streaking purification, and store the obtained single strains at -80°C using 40% glycerol. In addition, pick the purified strains into centrifuge tubes, add LB broth medium, culture at a constant temperature of 28-33°C for 20-28h, centrifuge at 10000xg for 5-7min, remove the supernatant and store at -20°C for extraction of strain DNA; (5) Flat plate hydrolysis test: Prepare protein, starch, and oil hydrolysis medium, sterilize the three hydrolysis medium, and pour them into plates. After the plates are condensed to a solid state, inoculate the isolated and purified strains into the three hydrolysis mediums respectively to observe whether a hydrolysis transparent circle appears. If so, it means that the substrate has been hydrolyzed and the strain has the ability to degrade the substrate. Otherwise, it does not appear. (6) Strain extraction and identification: The above-screened salt-tolerant strains were extracted, and universal primers 27F and 1492R were selected for PCR amplification. After the products were qualified by agarose gel electrophoresis, they were tested. In the hydrolysis medium test, it was observed that the strain H166 had a strong oil degradation ability. It was identified by sequencing that it was Brevibacillus Parabrevis H166, and the rRNA sequence similarity reached 98.90%; (7) Preparation of bacterial agent: a. Strain growth curve drawing: Take a single colony and culture it in a centrifuge tube at 28-32℃ overnight until OD 600 The value is 0.8 to 1, and the LB medium is inoculated at a volume ratio of 1 to 2%. The culture medium is cultured on a constant temperature shaker at 28 to 32°C and 170 to 200 r / min for 16 to 22 hours. The culture medium is aspirated and placed in a 96-well plate to measure the OD 600 value; b. Quantitative determination of enzyme activity: After the strain H166 obtained by separation and purification culture was activated on LB medium, it was cultured at a constant temperature of 28-32°C for 10-16h, and a single bacterium was picked out in a 24-well cell culture plate, and cultured at 28-32°C and 170-200r / min for 10-16h, and then transferred to 24-well plates containing protease, lipase and amylase active fermentation broth, respectively, and cultured at 35-39°C and 180-230r / min for 24-26h, and the fermentation broth was centrifuged at 5000-6000rpm and 4-5°C for 10min, and the obtained supernatant was filtered to obtain the crude enzyme solution; c. Primary seed culture medium: The above strains were inoculated into a container containing LB liquid culture medium in a clean bench and cultured in a constant temperature shaker at 28-32°C and 170-200 r / min for 46-54 h. 600 When the value exceeds 3.0, the culture is stopped and the primary seed culture solution is obtained; d. Secondary seed culture medium: The primary seed culture solution was inoculated into a container containing LB liquid culture medium in a clean bench and cultured in a constant temperature shaker at 28-32°C and 170-200 r / min for 34-40 h. 600 When the value exceeds 3.0, the culture is stopped and the obtained secondary seed culture solution is the salt-tolerant and thermophilic Bacillus parabrevis H166.

2. The salt-tolerant and thermophilic Bacillus parabrevicaulis H166 and the screening method thereof according to claim 1, characterized in that: In step (5), the preparation method of the protein hydrolysis medium is: peptone, beef extract, NaCl, casein, CaCl2, L-tyrosine, agar, distilled water, pH adjusted to 7.0-7.2, sterilized at 121°C for 20 minutes, cooled to 55-65°C, and sterilized Tween 80 was added.

3. The salt-tolerant and thermophilic Bacillus parabrevicaulis H166 and the screening method thereof according to claim 1, characterized in that: In step (5), the preparation method of the starch hydrolysis medium is: soluble starch, peptone, NaCl, agar, triphenyl blue, distilled water, pH 7.0-7.2, and sterilization at 121°C for 20 minutes.

4. The salt-tolerant and thermophilic Bacillus parabrevicaulis H166 and the screening method thereof according to claim 1, characterized in that: In step (5), the preparation method of the oil hydrolysis medium is: oil, 1.6% neutral red, peptone, sodium chloride, beef extract, agar, distilled water, pH adjusted to 7.0-7.2, and sterilized at 121°C for 20 minutes.

5. The salt-tolerant and thermophilic Bacillus parabrevicaulis H166 and the screening method thereof according to claim 1, characterized in that: The preparation method of the protease described in step b described in step (7) is as follows: aspirate the crude enzyme solution, add a 0.5% casein solution pre-incubated at 36-38° C., incubate in a water bath at 36-38° C. for 25-35 minutes, then add a 10% TCA solution to terminate the reaction, centrifuge at 12000 r / min for 5-8 minutes, aspirate the supernatant, and determine the tyrosine content.

6. The salt-tolerant and thermophilic Bacillus parabrevicaulis H166 and the screening method thereof according to claim 1, characterized in that: The preparation method of the lipase described in step b described in step (7) is as follows: polyvinyl alcohol solution and olive oil are mixed at a ratio of 3:1 (V / V), and ultrasonic treatment is performed for 5 to 8 minutes to prepare a milky white polyvinyl alcohol emulsion, which is the substrate solution. 4 mL of the substrate solution is taken, 5 mL of phosphate buffered saline is added, and 1 mL of crude enzyme solution is added after being heated in a water bath at 38 to 42° C. for 5 to 6 minutes. After mixing, the reaction is carried out at a constant temperature for 12 to 18 minutes, and 15 mL of 95% ethanol is added to terminate the reaction. The amount of enzyme required for the fatty acid is determined.

7. The salt-tolerant and thermophilic Bacillus parabrevicaulis H166 and the screening method thereof according to claim 1, characterized in that: The preparation method of the amylase described in step b described in step (7) is as follows: 0.1 mL of crude enzyme solution is taken, added to 2 mL of 1% soluble starch solution, extracted and incubated in a 36-38° C. water bath for 25-35 min, shaken once every 5 min, then cooled to room temperature after boiling in a water bath for 5-6 min, 3 mL of 3,5-dinitrosalicylic acid reagent is added, and then rapidly cooled to room temperature after boiling in a water bath for 5-6 min again, constant volume is obtained, and the OD value is measured.

8. Use of the salt-tolerant and thermophilic Bacillus parabrevis H166 as claimed in any one of claims 1 to 7 and the salt-tolerant and thermophilic Bacillus parabrevis H166 obtained by screening the same in composting.

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