CN103865837A - Streptomyces with antagonistic effect on penicillium viridis - Google Patents

Abstract

The present invention relates to Streptomyces antagonistic to Penicillium viridans. The bacterial strain obtained by the present invention is preserved in a preservation unit designated by the State Intellectual Property Office. The preservation unit is the China Typical Culture Collection Center, and the preservation date is November 2013. On March 18, the deposit number is CCTCCNO: M2013575. The Streptomyces was identified as Streptomyces anandii, named Streptomyces anandii JXNU02, Latin name Streptomycesanandii JXNU02. Streptomyces anandui JXNU02 fermentation broth has a strong inhibitory effect on Penicillium viridans, and its performance is stable, which has good application value for the development of new biological preservatives.

Description

Streptomyces antagonistic to Penicillium viridans

technical field

The present invention relates to the technical field of microbes, in particular to a Streptomyces having an antagonistic effect on Penicillium viridans. the

Background technique

Penicillium viridicatum belongs to the Penicillium genus fungus, which often grows and reproduces during the ripening and storage of foods such as grains, fruits, vegetables, nuts and meat. Penicillium viridans is prone to produce a variety of mycotoxins during the growth and reproduction process, such as ochratoxin A (ochratoxin A, OTA). OTA has strong carcinogenicity and teratogenicity, and has serious damage to the kidney and liver. Therefore, the control of Penicillium viridans has been widely concerned by scholars.

 Because food is vulnerable to various harmful microorganisms and deteriorates during processing, storage, and consumption, food preservation and preservation has become an important issue in the food industry. With the improvement of people's living standards and the enhancement of environmental awareness, traditional chemical preservatives have been proven to be potentially harmful. Therefore, it is imperative to develop highly efficient, nontoxic and safe biological preservatives. the

Contents of the invention

The object of the present invention is to provide Streptomyces having antifungal effects, especially Streptomyces having antagonistic effects on Penicillium aureaurus. the

Realize the technical scheme of the present invention as follows: Streptomyces that antagonistic effect is arranged to Penicillium viridans described in the present invention is obtained through separation and preparation from the soil sample collected in the suburb of Nanchang City, Jiangxi Province, and its formal name used at school is Anand's chain Mold JXNU02 ( Streptomyces anandii JXNU02).

The Streptomyces bacterial strain that has antagonistic effect to Penicillium viridans described in the present invention has been preserved in the preservation unit designated by the State Intellectual Property Office, and the preservation unit is the Chinese Typical Culture Collection Center, and the preservation address is Wuhan, China, Wuhan University; The date of deposit is November 18, 2013, and the deposit number is CCTCC NO: M 2013575. the

  Morphological characteristics of Streptomyces anandella JXNU02 strain: 

The hyphae in the base are well developed, without septum and fracture; the aerial mycelia are grown in ISP2 medium (yeast extract 4 g, malt extract 10 g, glucose 4 g, agar 20 g, distilled water 1 L, pH 7.3) It grows well on the top, multi-branched, and can form spore chains. The spore filaments are helical, and the spores are oval and smooth (see Figure 1).

  Physiological and biochemical characteristics of Streptomyces anandii JXNU02 strain: 

Streptomyces anandella JXNU02 can hydrolyze starch, produce hydrogen sulfide, produce melanin from tyrosine, liquefy gelatin, reduce nitrate, decompose cellulose, peptonize milk; utilize glucose, Sucrose, D-fructose, L-arabinose, D-xylose, raffinose, inositol and D-mannitol, but can not utilize L-rhamnose; cell wall type belongs to type I, the whole cell hydrolyzate contains ribose, Characteristic sugars were not detected.

Determination of the antibacterial spectrum of Streptomyces anandii JXNU02 fermentation broth with antagonistic effect on Penicillium viridans. the

 The antibacterial spectrum of Streptomyces anandellai JXNU02 fermentation broth was determined by agarose diffusion method. The target bacteria used were Penicillium viridans, Penicillium citrinum, yeast, Candida albicans, rice blast fungus, Aspergillus niger, Mucor, cotton fusarium wilt, Fusarium oxysporum, rice sheath blight, Rhizopus oryzae, Sclerotinia, Staphylococcus aureus and Escherichia coli. The results showed that the fermentation stock solution of Streptomyces ananduii JXNU02 had inhibitory effects on the above 13 target bacteria. the

Effect of Streptomyces ananduii JXNU02 Fermentation Broth on Mycelia Growth of Penicillium aeruginosa

  Streptomyces anandii JXNU02 fermentation broth can completely inhibit the mycelial growth of Penicillium viridans, and the inhibition rate reaches 100%. The results are shown in Figure 2.

Beneficial effects of the present invention: the present invention relates to a bacterial strain that has an antagonistic effect on Penicillium viridans, and the bacterial strain is classified and identified as Streptomyces anandii JXNU02 ( Streptomyces anandii JXNU02); The antibacterial spectrum of Streptomyces JXNU02 fermentation broth was studied, and it was determined that it had inhibitory effects on various fungi and bacteria; The fermentation broth inhibited the mycelial growth of Penicillium viridans, and the inhibition rate reached 100%. These data will lay the foundation for the development and application of food biological preservatives in the future.

Description of drawings

Figure 1 Morphology of mycelia and spores of Streptomyces anandii JXNU02 (X10000). the

Figure 2 Effect of Streptomyces anandellai JXNU02 fermentation broth on the growth of Penicillium viridans mycelium (A is the fermentation broth, B is the blank control). the

Detailed ways

Below in conjunction with implementation example the present invention will be further described

Example 1 The soil samples collected from the suburbs of Nanchang City, Jiangxi Province were separated and cultured according to the following method to obtain Streptomyces anandella JXNU02, which can be obtained by means of shaker liquid fermentation culture, which can inhibit Penicillium viridans The role of fermentation broth.

(1) Screening and preservation of Streptomyces

Soil samples were collected from the suburbs of Nanchang City, Jiangxi Province. The soil samples were naturally air-dried, ground and sieved, and dry-heated at 121°C for 1 h. Weighed 2 g of soil into 18 ml of sterile physiological water, and shaken at 150 r/min for 15 min. , and then serially diluted, 10 ^-2 and 10 ^-3 soil suspensions were respectively spread on Gaoshi No. 1 medium containing 75 mg/L K ₂ Cr ₂ O ₇ (soluble starch 20 g, KNO ₃ 1 g, NaCl 0.5 g, K ₂ HPO ₄ 3H ₂ O 0.5 g, MgSO ₄ 7H ₂ O 0.5 g, FeSO ₄ 7H ₂ O 0.01 g, agar powder 20 g, distilled water 1 L, pH 7.4~7.6) on a plate, cultured at 28°C On the 7th to 14th day, colonies with different colony shapes were picked to separate and purify on Gaoshi No. 1 medium plate, and the purified culture was inoculated on Gaoshi No. 1 slant, and stored at 4°C for future use.

Antagonistic actinomycetes were initially screened by the plate confrontation method, with the center of the potato dextrose agar medium (potato 200g, glucose 20g, agar 20g, water 1 L) as the origin, mark "ten", and actinomycetes with a diameter of 5 mm The block was placed at a distance of 2.5 cm from the center of the plate, and after culturing at 28°C for 2 days, the Penicillium viridans block with a diameter of 5 mm was inoculated in the center of the plate, and the results were observed after culturing at 28°C for 5 days. the

The antagonistic actinomycetes were re-screened by the tube-and-disk method, and Penicillium viridiferus was used as the target bacteria. The strains that could produce the inhibition zone were the antagonistic actinomycetes, and the diameter of the inhibition zone was measured with a vernier caliper by the cross method. the

(2) Preparation of Streptomyces anandii JXNU02 fermentation broth

After inoculating Streptomyces anandellai on Gaoshi No. 1 plate and culturing for 7 days, four pieces of actinomycete cakes with a diameter of 5 mm were placed in the seed medium (22.5 g sucrose, 12.5 g soybean meal powder, K ₂ HPO ₄ . 3H ₂ O 0.2 g, NaCl 1 g, Na ₂ SO ₄ 0.1 g, FeSO ₄ 7H ₂ O 0.01 g, CaCO ₃ 2 g, distilled water 1 L, pH 7.2), liquid volume 50 mL/ 250 mL Erlenmeyer flask, Cultivate for 48 hours at 200 r/min and 28°C, which is the seed solution. Inoculate 50 mL/250 mL fermentation medium (10 g soluble starch, 15 g soybean cake powder, 0.3 g KH ₂ PO ₄ , 0.3 g MgSO ₄ 7H ₂ O, 1 L, pH 7.2), cultured at 200 r/min, 28°C for 5 days. The fermentation broth was centrifuged at 10,000 r/min for 20 min, the supernatant was collected, and sterilized by filtration with a 0.22 μm filter membrane, and the sterile fermentation broth was stored at 4°C for later use.

(3) Determination of the antibacterial spectrum of Streptomyces anandii JXNU02 fermentation broth

Suspend the slant-activated culture of the target bacteria with an appropriate amount of sterile saline to obtain a cell suspension, and control the cell concentration to 1X10 ⁶ CFU/mL. Take 15 μl of the bacterial suspension and add it to 7 mL of medium pre-cooled to about 50°C, mix thoroughly, and spread on the agar bottom plate (15 mm in diameter) poured with 20 mL of 2%. After coagulation, place a sterile Oxford cup, add 200 μl of fermentation broth, incubate at 37°C for 24 hours (bacteria) or 28°C for 48 hours (fungi), and measure the diameter of the inhibition zone, which is the activity of the antibiotic.

(4) Effect of Streptomyces anandii JXNU02 fermentation broth on the growth of Penicillium viridans mycelia

Measure 1 mL of Streptomyces anandii JXNU02 fermentation broth and normal saline (blank control) respectively, add 10 mL of potato dextrose agar medium pre-cooled to about 50 °C, mix well, pour into a petri dish, and then Inoculate the Penicillium viridans with a diameter of 5 mm in the center of the plate, and observe the results after culturing at 28°C for 5 days.

the

CGCGCGGCGCGTGCTTACCATGCAGTCGAACGATGAACCACTTCGGTGGGGATTAGTGGCGAACGGGTGAGTAACACGTGGGCAATCTGCCCTGCACTCTGGGACAAGCCCTGGAAACGGGGTCTAATACCGGATATGAGCCGCCCAGGCATCTGGGTGGCTGTAAAGCTCCGGCGGTGCAGGATGAGCCCGCGGCCTATCAGCTTGTTGGTGAGGTAACGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGAGAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCGCGTCGGTTGTGAAAGCCCGGGGCTTAACCCCGGGTCTGCAGTCGATACGGGCAGGCTAGAGTTCGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCGATACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGCACTAGGTGTGGGCAACATTCCACGTTGTCCGTGCCGCAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACATACACCGGAAAACCCTGGAGACAGGGTCCCCCTTGTGGTCGGTGTACAGGTGGTGCATGGCTGTCG TCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCCGTGTTGCCAGCAGGCCCTTGTGGTGCTGGGGACTCACGGGAGACCGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGGTACAATGAGCTGCGATACCGCGAGGTGGAGCGAATCTCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGTCGGTAACACCCGAAGCCGGTGGCCCAACCCCTTGTGGGAGGGAGCTGTCGAATGGTGTAAGCTGCAGGTCC。

the

Claims (3)

1. A Streptomyces antagonistic to Penicillium viridans, characterized in that it is named after Streptomyces anandii JXNU02 ( Streptomyces anandii JXNU02), and the bacterial strain has been preserved in a depository unit designated by the State Intellectual Property Office, The depository unit is China Center for Type Culture Collection, the deposit date is November 18, 2013, and the deposit number is CCTCC NO: M 2013575. 2. the Streptomyces bacterial strain that has antagonistic effect to Penicillium viridans as claimed in claim 1, is characterized in that, its 16S rRNA gene sequence is: CGCGCGGCGCGTGCTTACCATGCAGTCGAACGATGAACCACTTCGGTGGGGATTAGTGGCGAACGGGTGAGTAACACGTGGGCAATCTGCCCTGCACTCTGGGACAAGCCCTGGAAACGGGGTCTAATACCGGATATGAGCCGCCCAGGCATCTGGGTGGCTGTAAAGCTCCGGCGGTGCAGGATGAGCCCGCGGCCTATCAGCTTGTTGGTGAGGTAACGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGAGAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCGCGTCGGTTGTGAAAGCCCGGGGCTTAACCCCGGGTCTGCAGTCGATACGGGCAGGCTAGAGTTCGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCGATACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGCACTAGGTGTGGGCAACATTCCACGTTGTCCGTGCCGCAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACATACACCGGAAAACCCTGGAGACAGGGTCCCCCTTGTGGTCGGTGTACAGGTGGTGCATGGCTGTCG TCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCCGTGTTGCCAGCAGGCCCTTGTGGTGCTGGGGACTCACGGGAGACCGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGGTACAATGAGCTGCGATACCGCGAGGTGGAGCGAATCTCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGTCGGTAACACCCGAAGCCGGTGGCCCAACCCCTTGTGGGAGGGAGCTGTCGAATGGTGTAAGCTGCAGGTCC。 3. the Streptomyces bacterial strain that has antagonism to Penicillium viridans as claimed in claim 1, is characterized in that, it is used for the control of Penicillium viridans.