[0021] The technical solutions of the present invention will be described in detail below in conjunction with embodiments.
[0022] A kind of Penicillium oxalicum that degrades organic acid odor, which is Penicillium oxalicum DH-1, which has been deposited in the China Center for Type Culture Collection on April 16, 2018, and the deposit number is CCTCC NO: M 2018201; Address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.
[0023] The Penicillium oxalicum DH-1 of the present invention is separated and screened from piled dairy cow farming manure, and the specific steps are as follows:
[0024] (1) Take 0.2 g of cow feces accumulation and inoculate it in 100 mL of liquid screening medium. The configuration steps of the liquid screening medium are as follows: first prepare a basal medium. The basal medium is based on 20 g/L glucose and 20 tryptone. g/L, yeast extract 10g/L, dry cow dung 5 g/L, adjust the pH to 6.5~7.5, add water to make the volume, and sterilize it; then add acetic acid, propionic acid, 0.1 g/L of n-butyric acid, isobutyric acid, n-valeric acid, and isovaleric acid, as well as 0.06 g/L of indole and 0.06 g/L of skatole, to obtain a liquid screening medium, wherein the above-mentioned acetic acid, propionic acid, The mass concentrations of n-butyric acid, isobutyric acid, n-valeric acid, isovaleric acid, indole, and skatole are the mass concentrations of each component relative to the liquid screening medium; then add chloromole at a volume ratio of 0.1% Antibiotics such as chloramphenicol, kanamycin, streptomycin, erythromycin, and ampicillin were obtained, and the pH was adjusted to 7.0. The above-mentioned chloramphenicol, kanamycin, streptomycin, erythromycin, and ampicillin The volume ratios of equal antibiotics are the volume ratios of antibiotics to the liquid screening medium; the above liquid screening medium is placed at 30℃, 200 r·min -1 Culture on a shaker for 8 days;
[0025] (2) Take 100 μL of the bacterial solution obtained in step (1) and inoculate it in another liquid screening medium (the liquid screening medium configuration method is completely the same as the liquid screening medium configuration method in step (1) Same), place the liquid screening medium at 30°C, 200 r·min -1 Shaker culture for 8 days;
[0026] (3) Repeat the culture operation of step (2) 2~3 times;
[0027] (4) Take another 100 μL of the bacterial solution obtained after the last culture and inoculate it in the acclimation medium for acclimation culture. The configuration steps of the acclimation medium are as follows: first prepare a basic medium, which is 20g/L glucose , Tryptone 20g/L, Yeast extract 10g/L, 5 g/L dry cow dung, adjust the pH to 6.5~7.5, add water to the volume and sterilize it; then add acetic acid, Propionic acid, n-butyric acid, isobutyric acid, n-valeric acid, and isovaleric acid are respectively 0.15 g/L, and 0.1 g/L indole and 0.1 g/L skatole. Adjust the pH to 7.0 to obtain the acclimation medium; Then at 30℃, 200 r·min -1 Incubate on a shaker for 8 days, repeat the above-mentioned domestication and culture operation 2~3 times, in which the mass concentrations of the above-mentioned acetic acid, propionic acid, n-butyric acid, isobutyric acid, n-valeric acid, isovaleric acid, indole, and skatole are in each group Relative to the mass concentration of the acclimation medium; while repeating the acclimation culture operation, gradually increase the concentration of various organic acids added to 0.2g/L, and the concentration of indole and skatole to 0.15 g/L;
[0028] (5) Take 100 μL of the bacterial solution obtained after the last domestication and culture, and spread it evenly on the agar solid screening medium containing 10 g/L after dilution, and incubate at 30°C for 3-8 days;
[0029] (6) Pick a single colony from the cultured medium in step (5) and proceed to the colony separation step, where the colony separation step is: the single colony picked out is streaked and separated on agar solid selection medium containing 10 g/L Cultivate the colony at 30°C; then subject the grown single colony to the colony isolation step, repeat the colony isolation step 2 to 3 times to obtain a monoclonal colony. The agar solid screening medium refers to adding 20 g/L of agar powder to the above liquid screening medium, wherein the mass concentration of the agar powder is the mass concentration of the agar powder relative to the agar solid screening medium, and the obtained agar solid Screening medium
[0030] (7) The separated and purified monoclonal colonies are inoculated into 50 mL liquid YPD medium. The configuration steps of liquid YPD medium are as follows: according to glucose 20g/L, tryptone 20g/L, yeast extract 10g/L, adjust pH To 6.5~7.5, add water to a constant volume and sterilize it; take 5μL of culture solution to prepare scanning electron microscope samples, and the mycelial morphology of the monoclonal colony at 2000 times, 5000 times, and 10000 times are respectively Such as figure 1 , figure 2 , image 3 As shown, it can be seen that the conidiophore is broom-like, with conidia arranged on the top, and the shape conforms to the characteristics of Penicillium.
[0031] The monoclonal colony grows rapidly on the PDA nutrient culture plate (ie, the existing and common potato dextrose agar medium), and mature velvet plaque can be obtained by culturing at 30°C for 3 days. At the initial stage of culture, the color of the plaque surface is white Yes, as the culture time increases, it gradually turns greenish green, and the fungus has a septum in its hyphae.
[0032] Take 20 mg of the dried monoclonal mycelium and grind it into powder with liquid nitrogen. According to the operating instructions of the kit (Ezup column fungal genomic DNA extraction kit purchased from SangonBiotech), the genomic DNA is extracted, and the DNA is finally precipitated. Dissolve it in 100 μL TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0), dilute the genomic DNA to 20 mg/L, and take 1 μL as a template for ITS-rDNA amplification. The primer pairs used are: ITS1: 5 ’ -TCCGTAGGTGAACCTGCGG-3 ’ And ITS4: 5 ’ -TCCTCCGCTTATTGATATGC -3 ’. The PCR reaction mixture is prepared as follows: template DNA 1 μL, PCR Taq mix 25 μL, each of the upstream and downstream primers 1.0 μL (20 μM), add ddH 2 O to 50 μL. The PCR program runs as follows: 94°C pre-denaturation for 1 min; 55°C annealing for 1 min; 72°C extension for 51 min; after 32 cycles, 72°C reaction for 10 min to amplify the incomplete sequence. The PCR amplification product is purified by the PCR cleaning kit, and then sequenced by a professional biological company, and analyzed by Shenggong Biotech Co., Ltd. The obtained sequence is analyzed by Blastn using network tools, and genetic tree analysis is completed by the bioinformatics software Mega6 . The nucleotide sequence of the sequenced ITS-rDNA is: the effective sequence length is 564 bp, as shown in SEQ ID NO. 1 in the sequence table. The sequence was compared with the ITS-rDNA sequence in NCBI database and GenBank. Penicillium oxalicum clone GDPo03 (NCBI accession number is MH766384.1) The similarity is the highest, and the homology is as high as 99%, indicating that the bacteria is a strain of Penicillium oxalicum, so it is named Penicillium oxalicum ( Penicillium oxalicum ) DH-1.
[0033] The method for amplifying and cultivating Penicillium oxalicum DH-1 that degrades organic acid odors of the present invention uses a liquid amplifying medium for amplifying culture. The configuration steps of the liquid amplifying medium are: first preparing a basic medium, The basic medium is based on glucose 5-20g/L, tryptone 5-20g/L, yeast extract 5-15g/L, dry cow manure 2-10 g/L, adjust the pH to 6.5-7.5, add water to make the volume, Manufactured by sterilization; then add acetic acid, propionic acid, n-butyric acid, isobutyric acid, n-valeric acid, isovaleric acid each 0.02~0.3 g/L, and 0.02~0.1 g/L indino Indole and 0.02~0.1 g/L skatole, adjust the pH to 6.5~7.5 to obtain a liquid amplification medium, in which the above-mentioned acetic acid, propionic acid, n-butyric acid, isobutyric acid, n-valeric acid, isovaleric acid, indole, The mass concentration of skatole is the mass concentration of each component relative to the liquid amplification medium; then the liquid amplification medium is inoculated with the above-mentioned Penicillium oxalicum DH-1, which degrades organic acid odor, 1.0 x 10 6 ~5.0 x 10 6 Each/ml Penicillium oxalicum spore suspension is added to the liquid medium at a volume ratio of 0.5-3.0% of the spore suspension to the mixed liquid of the spore suspension and the liquid amplification medium, and the culture temperature is 28-35°C. The liquid amplification medium of the present invention is also called: liquid selection medium. After the liquid amplification medium is amplified and cultured, the penicillium oxalicum that degrades organic acid odors of the present invention can also be further screened. After 8 days of culture in a liquid amplification medium shaker, all the culture broth obtained after the liquid amplification medium culture is inoculated into 1000 ml liquid amplification medium for amplification culture. The liquid amplification medium formula is the same as the above liquid amplification medium The formula is the same, shake culture for 24~300h, regularly sample 1ml, take the supernatant after centrifugation, and measure the concentration of organic acids and other acid odors by HPLC. The results are as follows Figure 4 , Figure 5 Shown. by Figure 5 It can be seen that skatole and indole are basically degraded within 200 hours; Figure 4 It can be seen that in the experimental group (DH-1-skatole and DH-1-indole), the strong odor of n-butyric acid was completely degraded within 6 days, and the remaining organic acids were completely degraded within 4-8 days.
[0034] Further, after the liquid amplification medium is used for the amplification culture, the solid amplification medium is used for the amplification culture, and the solid amplification medium is 5-10% grass meal and 5-15% bran according to the weight ratio of each raw material. 5-15% of soybean meal, 5-10% of corn meal, 10-20% of wood chips, 2-5% of dry cow dung, 0.5-2% of microcrystalline fiber, and 40-50% of water are mixed together; The medium is inoculated with the culture solution obtained by culturing the above-mentioned liquid amplification medium; the inoculation amount of the culture solution is 5-10% of the total volume of the culture solution and the solid amplification medium, and every 10-20% during the culture process Stir once every minute, and the culture temperature is 28-35°C.
