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Bacillus licheniformis HYT-9 with broad-spectrum bacteriostasis activity and preparation method and application of bacterial agent of bacillus licheniformis HYT-9

A technology of Bacillus licheniformis and HYT-9, applied in the field of microorganisms, can solve the problems of narrow antibacterial spectrum and low yield of antibacterial active metabolites, and achieve high antibacterial activity, strong tolerance, and stable properties

Active Publication Date: 2019-05-21
青岛红樱桃生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the existing Bacillus licheniformis has the following defects: one is that the yield of antibacterial active metabolites is not high, and the other is that the antibacterial spectrum is narrow

Method used

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  • Bacillus licheniformis HYT-9 with broad-spectrum bacteriostasis activity and preparation method and application of bacterial agent of bacillus licheniformis HYT-9
  • Bacillus licheniformis HYT-9 with broad-spectrum bacteriostasis activity and preparation method and application of bacterial agent of bacillus licheniformis HYT-9
  • Bacillus licheniformis HYT-9 with broad-spectrum bacteriostasis activity and preparation method and application of bacterial agent of bacillus licheniformis HYT-9

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1: Screening, isolation and purification of HYT-9 bacterial strain

[0026] Add the collected rotten milk sample into 50 mL of sterile water, treat it at 80°C for 120 minutes, take 5 mL and inoculate it into fresh LB for self-enrichment culture, repeat this process 4-5 times, and transfer the final enrichment culture solution Spread it on the LB solid medium plate containing Escherichia coli indicator bacteria, place it in a constant temperature incubator at 37°C for overnight culture, select a single colony with different morphological characteristics of the inhibition zone and inoculate it into the fresh LB medium, and carry out a variety of induction For the antibacterial verification of the pathogenic strains, the colony with a larger inhibition zone and the broadest spectrum was selected, and the strain with better antibacterial effect was named HYT-9, which was preserved in glycerol tubes and LB solid slant medium.

Embodiment 2

[0027] Embodiment 2: Identification of HYT-9 bacterial strain

[0028] 1. Morphological, physiological and biochemical characteristics of the strain

[0029] The colony morphology of strain HYT-9 on LB solid plate is as follows: figure 1 Shown: large colonies, milky white, irregular edges, bulges, vacuoles, translucent, moist, fluid around. Observed under the optical microscope, its morphology is short rod-shaped, and it is a Gram-positive bacterium with spores, such as figure 2 shown. When LB was used as the medium, the optimum culture temperature was 37°C, and spores began to be produced after 30 hours of culture. It belongs to aerobic bacteria and can utilize protein, starch and various sugars.

[0030] 2. Identification of strain 16S rDNA

[0031] (1) Extract the genome of Bacillus licheniformis HYT-9, and use primers (27F: AGAGTTTGATCMTGGCTCAG; 1492R: TACGGYTACCTTGTTACG ACTT) for 16s rDNA amplification:

[0032] The reaction system is: PCR upstream primer (25pmol / ...

Embodiment 3

[0040] Embodiment 3: the cultivation of bacterial strain

[0041] 1. Strains and vectors

[0042] Bacillus licheniformis HYT-9 (hereinafter referred to as HYT-9); T-vector, Escherichia coli DH5α, purchased from Invitrogen.

[0043] The primer sequences are as follows:

[0044] 27F: 5'-AGAGTTTGATCMTGGCTCAG-3';

[0045] 1492R: 5'-TACGGYTACCTTGTTACG ACTT-3';

[0046] M13F(-47): 5'-CGCCAGGGTTTTTCCCAGTCACGAC-3';  

[0047] M13R(-48): 5'-AGCGGATAACAATTTCACACAGGA-3'.

[0048] 2. Reagents and media

[0049] Plasmid extraction kits, fragment purification and recovery kits, restriction enzymes, etc. were purchased from Bao Biological Engineering (Dalian) Co., Ltd.; ampicillin was purchased from Sangon Bioengineering (Shanghai) Co., Ltd.

[0050] Bacillus fermentation medium: 50-80 g / L soybean meal, 60-100 g / L corn starch, 2-4 g / L disodium hydrogen phosphate, 1-2 g / L sodium carbonate, natural pH.

[0051] When the above medium is solid, add 2% agar powder.

[0052] 3. Training me...

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Abstract

The invention discloses bacillus licheniformis HYT-9 with broad-spectrum bacteriostasis activity and a preparation method and application of a bacterial agent of the bacillus licheniformis HYT-9. Thebacillus licheniformis is named HYT-9, and the preservation number of the bacillus licheniformis is CGMCC NO. 17225. The bacteriostatic test shows proves that the bacillus licheniformis has significant bacteriostatic activity for multiple pathogenic bacteria, especially staphylococcus aureus, escherichia coli, streptococcus, vibrio alginolyticus, vibrio splendidus, pseudomonas and the like, and can be taken as a substitute product of antibiotics and applied to the fields of feed additives, antibacterial medicinal preparations, health care products, preservatives and the like. After fermentation in a tank of 15 L, the escherichia coli is taken as an indicator bacterium, doxycycline hydrochloride is taken as a contrast, and the bacteriostatic titer of fermented liquid of the bacillus licheniformis reaches 100 thousand ppm. The bacillus licheniformis has a wide market application prospect.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, and in particular relates to a bacillus licheniformis HYT-9 with broad-spectrum antibacterial activity and a preparation method and application thereof. Background technique [0002] With the overuse and misuse of antibiotics, drug-resistant strains are increasing, resulting in superbugs that many drugs cannot treat. Take methicillin-resistant staphylococcus (MRSA) as an example. In the early 1980s, it was effective in 95% of patients; by 1996, it was ineffective in 72% of patients; and it is now basically ineffective. Moreover, drug-resistant Staphylococcus aureus (MRSA, VRSA, etc.) can achieve cross-infection between livestock and poultry and humans, and many MRSA strains that cause human infections are finally found to be derived from animals. The long-term abuse of antibiotics in animal husbandry has caused serious harm: leading to the decline of animal immunity, accelerating the emer...

Claims

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Application Information

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IPC IPC(8): C12N1/20A23K10/18A01N63/02A01P1/00A01P3/00C12R1/10
Inventor 肖志壮方安然薛海曌
Owner 青岛红樱桃生物技术有限公司
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