CN104988102B - The cultural method of one bacillus amyloliquefaciens and its application - Google Patents

Abstract

The invention discloses a method for cultivating a strain of Bacillus amyloliquefaciens and its application, belonging to the technical field of microbes. A strain of Bacillus amyloliquefaciens PC2 has a preservation number of CCTCC NO: M2015435. Beneficial effects of the present invention: the fermented liquid of Bacillus amyloliquefaciens strain provided by the present invention has broad-spectrum antibacterial activity, can effectively control post-harvest tomato fruit rot pathogen, is for post-harvest storage and preservation of tomato, reduces post-harvest loss, and improves Product quality and safety provide an effective, non-toxic, non-residue, and environmentally friendly new approach to biological control. The bacterial strain fermentation medium of the present invention has simple requirements, low cost, simple cultivation method, simple preparation process of the involved fresh-keeping agent, simple use method, and is suitable for popularization and application in production.

Description

A method for cultivating Bacillus amyloliquefaciens and its application

technical field

The invention belongs to the technical field of microorganisms, and in particular relates to a method for cultivating a strain of Bacillus amyloliquefaciens and its application.

Background technique

Fruits and vegetables are important non-staple foods in people's lives, the second largest category of agricultural products after grain, and important raw materials for the food industry. Tomato is an important vegetable variety. There are many kinds of tomato postharvest storage diseases. Among them, Fusarium tomato fruit rot is a postharvest pathogenic fungus discovered in recent years. It mainly infects mature fruits and causes fruit soft rot. greater economic loss.

my country's fruit and vegetable storage and preservation technology is relatively backward compared with developed countries, and the main method is to spray chemically synthesized fungicides for fruit and vegetable preservation. Long-term use will not only produce drug resistance of pathogenic bacteria or spoilage bacteria, but also the chemical residues in the fruit pose a potential threat to human health. In addition, logistics methods such as controlled atmosphere storage and refrigeration are also important technologies for fruit and vegetable preservation, but their investment is large and their operating costs are high, so they cannot be widely used. Therefore, it is of great significance to seek a safe, efficient, convenient and low-cost post-harvest preservation technology for fruits and vegetables.

Microbial preservation is a safe, non-toxic and effective biological preservation technology with broad application prospects. Using antagonistic antibacterial preparations to preserve fruits and vegetables can reduce the adverse effects of chemically synthesized fungicides on human health, and effectively prevent the drug resistance of plant pathogens. The key core of microbial fresh-keeping technology is to obtain efficient, safe, broad-spectrum and stable antagonistic bacterial strains. At present, many antagonistic bacteria strains with great application potential have been screened at home and abroad, including bacteria, yeast, mold and so on. However, only a handful of them have been commercially produced and applied. Among them, the narrow antibacterial spectrum of the strains, high production costs, harsh application environment conditions, and unstable control effects have limited the production and application of a large number of antagonistic strains.

Bacillus amyloliquefaciens is a non-pathogenic bacterium that widely exists in the environment. It can produce a variety of rich antibacterial active substances in the metabolic process, and has a wide range of antibacterial and fungal activities. It has been obtained in agricultural production. Accredited by the European Food Safety Commission. At present, domestic research and application of it mainly focus on the disease resistance and growth promotion function of replacing synthetic pesticides in the production process of agricultural products for various bacterial and fungal diseases. For example: Bacillus amyloliquefaciens EA19 and its preparation for preventing and controlling wheat root diseases (CN200910273498.8), a strain of Bacillus amyloliquefaciens and its application (CN201410389174.1). In recent years, there have also been studies on post-harvest preservation of fruits and vegetables such as citrus, olive, fig, and Dendrobium candidum by using Bacillus amyloliquefaciens. At present, there are no domestic invention patents and literature related research and application reports on the use of Bacillus amyloliquefaciens to control postharvest Fusarium fruit rot in tomatoes.

Most of the culture medium of Bacillus amyloliquefaciens producing antibacterial active substances have relatively high nutritional requirements, such as NB medium, LB medium, etc. Therefore, obtaining antagonistic strains with simpler nutritional requirements can reduce production costs and promote the development and promotion of products related to antagonistic strains.

Prior art relevant to the present invention one

Technical solution of prior art one

At present, chemical agents are mainly used to prevent and control tomato sickle fruit rot. Commonly used agents include 25% pyrimethanil WP 500-600 times liquid, 50% Garner Gold WP 500 times liquid, 70% thiophanate-methyl WP 1000-1200 times liquid, etc. Spray the whole leaf in the field, focusing on the fruit.

The shortcoming of prior art one

Although chemical control can effectively control the occurrence of diseases, chemical residues affect human health and pose food safety risks.

Contents of the invention

Aiming at the deficiencies of the prior art, the present invention provides a method for cultivating Bacillus amyloliquefaciens and its application, which can solve the problem of chemical residues produced by chemical agents for fresh-keeping fruits and vegetables, thereby affecting the health of the body.

In order to solve the above problems, the technical scheme adopted by the present invention is as follows, a strain of Bacillus amyloliquefaciens PC2, the preservation number is CCTCC NO: M2015435.

Another technical scheme that the present invention adopts is as follows, the method for bacterial strain PC2 antibacterial active substance fermentation culture, comprises the following steps:

Isolation and purification of S1 antagonistic strains

Beef extract peptone liquid medium and potato dextrose liquid culture were respectively used for enrichment culture at 28-37°C for 24-48 hours, and the dilution plate method was used to separate microorganisms. Bacteria and fungi were separated by beef extract peptone liquid medium and potato dextrose respectively. On liquid culture medium plates, place them in incubators at 30-37°C and 25-30°C for 2-3 days respectively. According to the characteristics of the colonies such as shape, color, and transparency, pick single colonies with obvious differences in shape for streaking and purification. Store at 4°C for later use;

Screening of S2 antagonistic strains

S2.1 Fermentation broth preparation

Inoculate the bacterial strain to be screened in potato glucose liquid medium, and ferment and culture in a shake flask at 120-180rpm at 30-37°C for 24-48h, take the culture broth and ferment liquid and freeze and centrifuge at 10000-12000rpm at 4°C for 10-20min, Collect the supernatant fermentation liquid and filter it through a 0.22 μm microporous membrane to obtain a sterile fermentation filtrate, and store it at 4°C for later use;

S2.2 Determination of Antagonism Spectrum

The antagonistic strains were screened by the Oxford cup double-layer plate method. Staphylococcus aureus was used as the indicator bacteria, and 10-15mL NA medium was used as the lower medium. After solidification, four sterilized Oxford cups were evenly placed 2.5cm from the center of the plate, and Pour 10-15 mL of LB medium mixed with Staphylococcus aureus on each plate as the upper medium; add 100-150 μL supernatant fermentation filtrate into the Oxford cup, culture at 30-37°C for 24-36 hours, and observe the inhibition zone Whether there is and measure its diameter;

Determination of Antagonism Spectrum in S3 Antagonistic Bacteria Ferment Broth

The antibacterial effects of antagonistic strains on Staphylococcus aureus, Escherichia coli, Micrococcus luteus, Bacillus subtilis, Saccharomyces cerevisiae, Rhodotorula, tomato fusarium fruit rot, tomato cotton rot and other strains were determined by Oxford cup double plate method , Bacteria were cultured at a constant temperature of 37°C, and fungi were cultured at a constant temperature of 28°C. After 24 hours, the presence or absence of the inhibition zone was observed and its diameter was measured. Each treatment was repeated three times;

Among the isolated strains, a strain PC2 with the strongest antibacterial activity and the broadest antibacterial spectrum was screened out.

Another technical scheme that the present invention adopts is as follows, the preparation method that contains bacterial strain PC2 bacteriostatic activity fermented liquid preservative, will prepare PC2 aseptic strain fermented liquid with distilled water and following composition by following concentration preparation: fermented liquid 30～ 50%, soybean protein isolate 3% to 5%, phytic acid 0.1% to 0.2%, sodium carboxymethyl cellulose 0.5% to 2%, calcium chloride 1% to 3%.

As preferred: 45% of fermented liquid, 4% of soybean protein isolate, 0.15% of phytic acid, 1.5% of sodium carboxymethyl cellulose, and 2.0% of calcium chloride.

Another technical scheme adopted by the present invention is as follows. The method of using the antistaling agent is to select tomato fruits with no damage in appearance, consistent maturity and size, and disinfect the surface with 1-2% sodium hypochlorite solution for 0.5-2 minutes, and then use aseptic Wash with water several times and dry in the air; use a sterilized toothpick to evenly stab four 2cm deep wounds on the equatorial surface of the fruit, immerse in the prepared fresh-keeping solution for 25-45s, and then immerse in the prepared spores of Fusarium tomato fruit rot to suspend solution (10 ⁶ pieces/mL) for 30s, or directly spray the preservative solution on the fruit surface.

Preservation information: A Bacillus amyloliquefaciens PC2 was deposited in the China Center for Type Culture Collection of Wuhan University on July 7, 2015, with the preservation number CCTCC NO:M2015435.

The beneficial effects of the present invention are as follows:

1. In order to overcome the food safety risks brought about by the chemical control methods of postharvest fusarium fruit rot in tomatoes, a strain of Bacillus amyloliquefaciens with simpler nutritional requirements and a wide antagonistic spectrum is provided, and the fermentation liquid of the strain is stable in heat and pH , Strong UV stability;

2. Provide a method for cultivating Bacillus amyloliquefaciens bacteriostatically active fermentation broth. The method is simple and convenient, the medium formula is cheap and easy to obtain, suitable for large-scale fermentation production, and reduces production costs;

3. Provide the method for controlling post-harvest fusarium fruit rot of tomato by the strain. The method is simple to operate, does not require special equipment, and is safe, effective and convenient.

In summary, the fermented liquid of Bacillus amyloliquefaciens strains provided by the present invention has broad-spectrum antibacterial activity, can effectively control post-harvest tomato fruit rot pathogens, store and keep fresh tomatoes after harvest, reduce post-harvest loss, and improve product quality. Quality and Safety provides an effective, non-toxic, non-residue, and environmentally friendly new approach to biological control.

The bacterial strain fermentation medium of the present invention has simple requirements, low cost, simple cultivation method, simple preparation process of the involved fresh-keeping agent, simple use method, and is suitable for application and popularization in production.

Description of drawings

Fig. 1 is the bacteriostasis effect figure of bacterial strain PC2 aseptic fermented liquid to tomato Fusarium fruit rot pathogen;

Figure 2 is an effect diagram of the colony morphology of antagonistic bacteria PC2;

Fig. 3 is the morphological effect diagram of Gram staining of antagonistic bacteria PC2;

Fig. 4 is a schematic diagram of the phylogenetic tree of strain PC2 based on 16S rDNA.

detailed description

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be described in further detail below with reference to the accompanying drawings and examples.

Example:

1 Isolation and purification of antagonistic strains

The bee grain was cultured with beef extract peptone liquid medium and potato glucose liquid culture respectively based on 28-37°C for 24-48 hours, and the dilution plate method was used to separate microorganisms, and the bacteria and fungi were separated using beef extract peptone liquid medium ( NA) and potato dextrose liquid medium (PDA) plates, respectively placed in 30 ~ 37 ℃ and 25 ~ 30 ℃ incubator culture 2 ~ 3d. According to the characteristics of the colony's shape, color, transparency, etc., single colonies with obvious morphological differences were picked for streaking and purification, and the slopes were stored at 4°C for later use. A total of 56 bacterial strains were obtained.

2 Screening of antagonistic strains

2.1 Fermentation broth preparation

Inoculate the bacterial strain to be screened in potato glucose liquid medium, and ferment and culture in a shake flask at 120-180rpm at 30-37°C for 24-48h, take the culture broth and ferment liquid and freeze and centrifuge at 10000-12000rpm at 4°C for 10-20min, The supernatant fermentation broth was collected and filtered through a 0.22 μm microporous membrane to obtain a sterile fermentation filtrate, which was stored at 4°C for future use.

2.2 Antagonism Spectrum Determination

The antagonistic strains were screened by the Oxford cup double-layer plate method. Staphylococcus aureus was used as the indicator bacteria, and 10-15mL NA medium was used as the lower medium. After solidification, four sterilized Oxford cups were evenly placed 2.5cm from the center of the plate, and Pour 10-15 mL of LB medium mixed with Staphylococcus aureus on each plate as the upper medium. Add 100-150 μL of the supernatant fermentation filtrate into the Oxford cup, incubate at 30-37°C for 24-36 hours, observe the presence or absence of the inhibition zone and measure its diameter. A total of 17 antagonistic strains with strong antibacterial activity against Staphylococcus aureus were obtained.

3 Determination of antagonistic spectrum in fermentation broth of antagonistic bacteria

The antibacterial effects of antagonistic strains on Staphylococcus aureus, Escherichia coli, Micrococcus luteus, Bacillus subtilis, Saccharomyces cerevisiae, Rhodotorula, tomato fusarium fruit rot, tomato cotton rot and other strains were determined by Oxford cup double plate method . Bacteria were cultured at a constant temperature of 37°C, and fungi were cultured at a constant temperature of 28°C. After 24 hours, the existence of the inhibition zone was observed and its diameter was measured. Each treatment was repeated three times.

Among the 17 isolated strains, one strain PC2 had the strongest antibacterial activity and the broadest antibacterial spectrum. Table 1 is the bacteriostatic spectrum of the PC2 bacterial strain wherein, wherein the bacteriostatic zone to tomato Fusarium fruit rot is 21.5mm (see Fig. 1).

Antibacterial activity of table 1 bacterial strain PC2

4 Identification of antagonistic strain PC2

PC2 strain was identified as Bacillus amyloliquefaciens by morphological characteristics, physiological and biochemical characteristics and 16S rDNA sequence analysis.

After PC2 was cultured on the PDA plate for 36 hours, a single colony was formed, which was nearly round to oval, with irregular edges and nicks, wrinkled surface, straw hat-like bulge in the center, translucent, and the colony was milky white (Figure 2); when the liquid medium was still There is biofilm formation. Microscopically, the cells were short rod-shaped or oval, Gram-positive, and spore-forming (Figure 3).

Table 2 Physiological and biochemical characteristics of strain PC2

Note: "+" means the reaction is positive, and "-" means the reaction is negative.

Fig. 4 is a phylogenetic tree of PC2, it can be seen that PC2 and 3 strains of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) gather in the same branch. Combined with morphological, physiological and biochemical characteristics, the strain PC2 was identified as Bacillus amyloliquefaciens.

Stability of antibacterial activity of fermentation broth of 5 strains

5.1 Thermal stability

Take 10mL of sterile fermentation filtrate into a sterile stoppered test tube, put it in a water bath at 40°C, 60°C, 80°C, and 100°C for 0.5h, 1h, 2h, and 3h, respectively, and sterilize it by autoclaving at 121°C for 20min. After returning to room temperature, tomato fusarium fruit rot was used as an indicator bacterium, and the antibacterial activity of the fermentation filtrates of each treatment was detected by the Oxford cup double-layer plate method, and the thermal stability of the antibacterial substances was reflected by the average diameter of the inhibition zone. Each treatment was performed in triplicate, and the fermentation filtrate without heat treatment (at room temperature of 25° C.) was used as a blank control.

Table 3 is the result of thermal stability of the fermentation broth of the strains. It can be seen that the thermal stability of the fermentation broth is good, and its antibacterial activity still maintains 71.1% of the original value after being treated at 121°C for 20 minutes.

Table 3 Inhibition zone (mm) of aseptic fermented liquid treated with different temperatures on tomato Fusarium fruit rot

5.2 Acid-base stability

Take 10 mL of sterile fermentation filtrate into a sterile stoppered test tube, and adjust to different pH (pH1, pH3, pH5, pH8, pH10, pH12, pH14) with 1.0mol/L HCl solution or 1.0mol/L NaOH solution respectively. After standing at 25°C for 2 hours, adjust the pH of the initial fermentation filtrate to 7.0, and quantify it with sterile water to ensure that the concentration of antibacterial substances in each treatment is the same. Using Fusarium tomato fruit rot as the indicator bacteria, the Oxford cup double-layer plate method was used to detect the antibacterial activity of the fermentation filtrate of each treatment, and the average diameter of the antibacterial zone reflected the acid-base stability of the antibacterial substances. Each treatment was performed in triplicate, and the original fermentation filtrate (pH7.0) was used as the control.

Table 4 is the results of the acid-base stability of the fermentation broth of the strains. It can be seen that the fermentation broth has strong acid stability, especially under the condition of strong acid (pH1-4), the antibacterial activity remains basically unchanged.

Table 4 The inhibition zone (mm) of acid-base treatment aseptic fermented liquid to tomato Fusarium fruit rot

5.3 UV Stability

Take 10mL of sterile fermentation filtrate in a sterile stoppered test tube, and irradiate for 10, 20, 30, 40, 50, and 60 min under a 15W ultraviolet lamp at a distance of 30 cm. Using tomato cotton rot as indicator bacteria, the Oxford cup double-layer plate method was used to detect the antibacterial activity of the fermentation filtrates of each treatment, and the average diameter of the antibacterial zone reflected the ultraviolet stability of the antibacterial substances. Each treatment was performed in triplicate, and the fermentation filtrate without UV treatment was used as a blank control.

Table 5 is the UV stability results of the strain fermentation broth. It can be seen that the ultraviolet irradiation stability of the fermentation broth is strong, and the ultraviolet irradiation for 10-60 minutes has no significant effect on the antibacterial activity of the fermentation broth.

Table 5 Inhibition zone (mm) of ultraviolet irradiation treatment of sterile fermented liquid on tomato Fusarium fruit rot

6 Determination of the control effect of PC2-containing fermented broth preservative on tomato postharvest rot

6.1 Preparation of antistaling agent: Prepare the PC2 sterile strain fermentation liquid prepared in the above test with distilled water and the following ingredients at the following concentrations: 30-50% fermentation liquid, 3%-5% soybean protein isolate, 0.1%- phytic acid 0.2%, sodium carboxymethylcellulose 0.5% to 2%, calcium chloride 1% to 3%. The preferred formula: 45% of fermented liquid, 4% of soybean protein isolate, 0.15% of phytic acid, 1.5% of sodium carboxymethylcellulose, and 2.0% of calcium chloride.

6.2 Preservation test: Select tomato fruits with no damage in appearance, consistent maturity and size, disinfect the surface with 1-2% sodium hypochlorite solution for 0.5-2 minutes, wash them with sterile water several times, and dry them in the air. Use a sterilized toothpick to evenly injure four wounds about 2 cm deep on the equatorial surface of the fruit, immerse them in the above-prepared preservative solution for 25-45 seconds, and then immerse them in the prepared tomato Fusarium fruit rot spore suspension (10 ⁶ pcs/mL) 30s, or directly spray the preservative solution on the surface of the fruit, and the control group is only immersed in the spore suspension of fruit rot fungus, 10 fruits per treatment. After being processed, pack into cartons, put into a humidity of 95%, store in an environment with a temperature of 25°C, and count the fruit rot index and lesion diameter after one week.

Table 6

It can be seen from Table 6 that no matter whether the composite preservative of the present invention adopts fruit dipping or spraying mode, it can effectively control the occurrence of post-harvest rot diseases of tomatoes, reduce fruit rot index and lesion diameter, and prolong the storage period.

Those skilled in the art will appreciate that the embodiments described here are to help readers understand the implementation method of the present invention, and it should be understood that the protection scope of the present invention is not limited to such specific statements and embodiments. Those skilled in the art can make various other specific modifications and combinations based on the technical revelations disclosed in the present invention without departing from the essence of the present invention, and these modifications and combinations are still within the protection scope of the present invention.

Claims (3)

1. A strain of Bacillus amyloliquefaciens PC2, characterized in that the preservation number of said strain of Bacillus amyloliquefaciens PC2 is CCTCC NO: M2015435. 2. a preparation method of a preservative containing bacterial strain PC2 antibacterial activity fermented liquid, is characterized in that, described bacterial strain PC2 is a strain bacillus amyloliquefaciens PC2 described in claim 1, will prepare PC2 aseptic strain The fermented liquid is prepared with distilled water and the following ingredients at the following concentrations: 30-50% fermented liquid, 3%-5% soybean protein isolate, 0.1%-0.2% phytic acid, 0.5%-2% sodium carboxymethyl cellulose, Calcium chloride 1% to 3%. 3. The preparation method according to claim 2, characterized in that 45% of fermented liquid, 4% of soybean protein isolate, 0.15% of phytic acid, 1.5% of sodium carboxymethylcellulose, and 2.0% of calcium chloride.