阅读说明：本技术 一种高通量筛选抑霉菌性乳酸菌的方法 (Method for high-throughput screening of mould-inhibiting lactic acid bacteria ) 是由 贾彩凤 冉琼 鲍子旗 刘玉婷 *** 李超文 潘建泽 于 2019-11-14 设计创作，主要内容包括：本发明公开了一种高通量筛选抑霉菌性乳酸菌的方法,步骤如下：(1)乳酸菌种子液的制备；(2)霉菌孢子悬液的制备；(3)在多孔板中加入固体培养基、上述乳酸菌种子液(样品孔)或空白的MRS培养基(对照孔)进行培养；(4)然后每孔加入霉菌孢子悬液共培养,比较样品孔和对照孔的霉菌生长情况,筛选具有抑霉菌活性的乳酸菌。本发明在多孔板(如24孔板或者48孔板)上进行抑霉菌性乳酸菌的筛选,排除了由于霉菌菌丝及孢子在平板上大面积生长、繁殖对抑菌效果造成的干扰；本发明所述方法能够同时对几十株菌进行筛选,提高了筛选效率和准确度。(The invention discloses a method for high-throughput screening of mould-inhibiting lactic acid bacteria, which comprises the following steps: (1) preparing lactobacillus seed liquid; (2) preparing a mould spore suspension; (3) adding solid culture medium, the lactobacillus seed solution (sample well) or blank MRS culture medium (control well) into a multi-well plate for culturing; (4) then adding mould spore suspension into each hole for co-culture, comparing the mould growth conditions of the sample hole and the control hole, and screening the lactic acid bacteria with mould inhibiting activity. The method screens the mould-inhibiting lactic acid bacteria on a porous plate (such as a 24-pore plate or a 48-pore plate), and eliminates the interference on the antibacterial effect caused by the large-area growth and propagation of mould hyphae and spores on a flat plate; the method can screen dozens of strains at the same time, and improves the screening efficiency and accuracy.)

1. A method for screening mould inhibiting active lactic acid bacteria at high flux, which is characterized by comprising the following steps:

(1) preparing a lactobacillus seed solution: inoculating the lactobacillus colony into a culture medium for culturing to obtain lactobacillus seed liquid;

(2) preparation of a mould spore suspension: inoculating mould on a solid culture medium for culturing to prepare mould spores, and then washing with sterile water containing tween-80 to prepare mould spore suspension;

(3) and (3) detection of antibacterial activity: and (2) adding a solid culture medium, the lactic acid bacteria seed liquid prepared in the step (1) and the mould spore suspension prepared in the step (2) into a perforated plate, then carrying out co-culture, comparing the growth condition of mould, and carrying out primary screening on lactic acid bacteria with mould inhibiting activity.

2. The method according to claim 1, characterized in that it comprises in particular the steps of:

(1) preparing a lactobacillus seed solution: selecting a single lactobacillus colony from a lactobacillus plate, and inoculating the single lactobacillus colony into an MRS culture medium for culture to obtain a lactobacillus seed solution;

(2) preparation of a mould spore suspension: inoculating mould on a potato glucose solid culture medium inclined plane, culturing to prepare mould spores, washing the inclined plane by using sterile water containing 0.01-0.05% Tween-80 by volume fraction, and uniformly mixing on a vortex oscillator to prepare uniformly dispersed mould spore suspension;

(3) and (3) detection of antibacterial activity: adding a culture medium and the lactobacillus seeds prepared in the step (1) into a perforated plate for culturing; and (3) adding the mould spores prepared in the step (2), then carrying out co-culture, comparing the growth condition of the mould, and carrying out primary screening on the lactic acid bacteria with mould inhibiting activity.

3. The method according to claim 2, wherein in step (1), the composition of the MRS medium per liter of the seed medium comprises: 8-12 g of peptone, 8-12 g of beef extract, 3-7 g of yeast powder, 15-25 g of glucose, 0.08-0.12 g of magnesium sulfate, 3-7 g of sodium acetate, 1.5-2.5 g of ammonium citrate, 1.5-2.5 g of dipotassium hydrogen phosphate, 0.04-0.06 g of manganese sulfate and 800.8-1.2 g of tween, and the pH is adjusted to 6.8-7.4.

4. The method according to claim 1 or 2, wherein in the step (1), the temperature of the culture is 30-37 ℃; and/or the culture time is 10-36 h.

5. The method according to claim 1 or 2, wherein in the step (2), the temperature of the culture is 25-30 ℃; and/or the culture time is 3-10 days.

6. The method according to claim 1 or 2, wherein in the step (2), the mold is one or more of penicillium, aspergillus flavus, aspergillus niger, cladosporium, mucor, and rhizopus.

7. The method according to claim 1 or 2, wherein in the step (3), the culture medium in the multi-well plate is prepared by adding 1.0-2.0% of agar and 0.05-0.2M K on the basis of MRS liquid culture medium₂HPO₄Adjusting the pH value to 6.5-7.5; and/or the volume of the culture medium in the multi-hole plate is 100-300 uL.

8. The method according to claim 1 or 2, wherein in the step (3), the volume of the culture medium inoculated with the lactic acid bacteria seed solution in the multi-well plate is 0.3-1.2 uL; and/or the conditions for culturing the lactic acid bacteria in the multi-hole plate are as follows: culturing at 30-37 ℃ for 12-72 h.

9. The method according to claim 1 or 2, wherein in step (3), the amount of inoculated mold spores in the multi-well plate is 50-100 uL per well; and/or the final concentration of the mould spores in the porous plate is 0.2-5 multiplied by 10⁴one/mL.

10. The method according to claim 1 or 2, wherein in step (3), the co-culturing conditions of the lactic acid bacteria and the mold spores are as follows: culturing at 25-30 ℃.

Technical Field

The invention belongs to the technical field of microorganism screening, and relates to a method for screening fungistatic lactic acid bacteria in a high-throughput manner.

Background

In recent years, food safety issues have received much attention all over the world, and mold contamination has led to spoilage of various foods, causing significant economic losses. In addition, some molds can produce mycotoxins in food products, posing a threat to human health. Therefore, the search for more natural biological preservatives is one of the main research directions for the development of food preservatives at home and abroad at present.

Bioprotection, defined as the inhibition of one organism against another, has received widespread attention in recent years. Lactic acid bacteria are naturally present in many foods and have been used for a long time in fermented foods, and the use of lactic acid bacteria for bioprotection has important application prospects.

However, the method for screening for fungistatic lactic acid bacteria in the prior art is generally a double-layer agar method. The method can only screen one by one, and has the disadvantages of high cost and workload of reagents, consumables and the like, difficulty in screening large batches of strains at the same time, incapability of quickly obtaining results and low efficiency.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a method for screening the mould-inhibiting lactic acid bacteria at a high flux, which aims to solve the problem of low working efficiency of lactic acid bacteria screening and provides a method for screening the mould-inhibiting lactic acid bacteria at a high flux. Specifically, a porous plate method is used for high-throughput screening of lactic acid bacteria, and the screening method is suitable for penicillium, aspergillus flavus, aspergillus niger, cladosporium, mucor, rhizopus and other molds.

The invention provides a method for screening mould-inhibiting lactic acid bacteria in high flux, which comprises the steps of preparing lactic acid bacteria seed liquid, inoculating the seed liquid into a perforated plate for culture, preparing mould spores, carrying out bacteriostatic activity detection after the co-culture of lactic acid bacteria and mould is finished, and screening the lactic acid bacteria with the mould-inhibiting activity.

Specifically, the method comprises the following steps:

(1) preparing a lactobacillus seed solution: inoculating the lactobacillus colony into a culture medium for culturing to obtain lactobacillus seed liquid;

(2) preparation of a mould spore suspension: inoculating mould into a solid culture medium for culturing to prepare mould spores, and then washing with sterile water containing tween-80 to prepare mould spore suspension;

(3) and (3) detection of antibacterial activity: and (2) adding a solid culture medium, the lactic acid bacteria seed liquid prepared in the step (1) and the mould spore suspension prepared in the step (2) into a perforated plate, then carrying out co-culture, comparing the growth condition of mould, and carrying out primary screening on lactic acid bacteria with mould inhibiting activity.

Specifically, the method comprises the following steps:

(1) preparing a lactobacillus seed solution: selecting a single lactobacillus colony from a lactobacillus plate, and inoculating the single lactobacillus colony into an MRS culture medium for culture to obtain a lactobacillus seed solution;

(2) preparation of a liquid of a fungal spore suspension: inoculating mould on a potato glucose solid culture medium inclined plane, culturing to prepare mould spores, washing the inclined plane by using sterile water containing Tween-80 with the volume fraction of 0.01-0.05%, and uniformly mixing on a vortex oscillator to prepare uniformly dispersed mould spore suspension.

(3) And (3) detection of antibacterial activity: adding a culture medium and the lactobacillus seed solution prepared in the step (1) into a perforated plate for culturing; and (3) adding the mould spore suspension prepared in the step (2), then carrying out co-culture, comparing the growth condition of the mould, and carrying out primary screening on the lactic acid bacteria with mould inhibiting activity.

In the step (1), the MRS culture medium comprises per liter of seed culture medium: 8-12 g of peptone, 8-12 g of beef extract, 3-7 g of yeast powder, 15-25 g of glucose, 0.08-0.12 g of magnesium sulfate, 3-7 g of sodium acetate, 1.5-2.5 g of ammonium citrate, 1.5-2.5 g of dipotassium hydrogen phosphate, 0.04-0.06 g of manganese sulfate and 800.8-1.2 g of tween, and the pH is adjusted to 6.8-7.4.

Preferably, the composition of the MRS medium comprises per liter of seed medium: 10g of peptone, 10g of beef extract, 5g of yeast powder, 20g of glucose, 0.1g of magnesium sulfate, 5g of sodium acetate, 2g of ammonium citrate, 2g of dipotassium hydrogen phosphate, 0.05g of manganese sulfate and 801g of tween, and the pH value is adjusted to 7.0.

In the step (1), the culture temperature is 30-37 ℃; preferably, it is 30 ℃.

In the step (1), the culture time is 10-36 h; preferably, the time is 16-24 h.

In the step (1), the culture is preferably anaerobic static culture.

In the step (2), the mould is one or more of penicillium, aspergillus flavus, aspergillus niger, cladosporium, mucor, rhizopus and the like; preferably, it is Penicillium, Aspergillus niger.

In the step (2), the potato glucose solid culture medium comprises the following formula: 200g of potato leachate, 20g of glucose, 15-20 g of agar and 1000mL of water, and the pH value is not adjusted.

In the step (2), the washing times are 2-5 times; preferably, the number of times is 3 to 5.

In the step (2), the culture temperature is 25-30 ℃; preferably, it is 30 ℃.

In the step (2), the culture time is 3-10 days; preferably, the time is 5-7 d.

In the step (2), the culture is preferably a static culture.

In the step (3), the culture medium in the multi-well plateBased on MRS culture medium, 1.0-2.0% agar and 0.05-0.2M K are added₂HPO₄And adjusting the pH value to 6.5-7.5.

Preferably, the culture medium in the multi-well plate is prepared by adding 1.5% agar and 0.1MK on the basis of MRS culture medium₂HPO₄The pH was adjusted to 7.0.

In the step (3), the volume of the culture medium in the perforated plate is 100-300 uL; preferably 200uL, after the culture medium is completely solidified, adding the lactobacillus seed solution.

In the step (3), the inoculation amount of the lactobacillus in the porous plate is 0.3-1.2 uL per hole; preferably, it is 0.75 uL.

In the step (3), the conditions for culturing the lactobacillus in the multi-well plate are as follows: culturing for 12-72 h at 30-37 ℃; preferably, the culture is performed for 48 hours at 30 ℃.

In the step (3), the inoculation amount of the mold spores in the porous plate is 50-100 uL per hole; preferably, it is 100 uL.

In the step (3), the final concentration of the mould spores in the porous plate is 0.2-5 multiplied by 10⁴Per mL; preferably, it is 1X 10⁴one/mL.

In the step (3), the co-culture conditions of the lactic acid bacteria and the mould spores are as follows: culturing at 25-30 ℃; preferably: and (3) standing and culturing at 30 ℃.

The co-culture time of the lactic acid bacteria and the mould spores is different according to the growth speed of different moulds, and is generally 2-7 days.

According to the invention, the multi-hole plate method is used for detecting the mould inhibition effect of the lactic acid bacteria, and because each hole is mutually separated, the influence of the growth and spread of mould hyphae on the observation of the mould inhibition effect of different lactic acid bacteria can be eliminated. Meanwhile, due to the separation of the holes, the bacteriostatic effect of the lactic acid bacteria on different moulds can be simultaneously detected in different holes of the porous plate, so that the high-throughput screening of the bacteriostatic activity of the lactic acid bacteria is realized.

In one embodiment, the method comprises the steps of:

(1) preparing a lactobacillus seed solution: picking single lactobacillus colony from a lactobacillus plate, inoculating the single lactobacillus colony into 15mL of MRS culture solution with the liquid loading of a screw test tube of 15mL, and performing static culture at 30 ℃ for 10-36 h to obtain lactobacillus seed solution.

(2) Preparation of mold spore suspension (indicator bacterium liquid): inoculating the mould to a potato glucose solid culture medium, standing for 3-10 days to prepare mould spores, washing with sterile water containing 0.01% Tween-80 by volume fraction, and uniformly mixing on a vortex oscillator to prepare mould spore suspension.

(3) And (3) detection of antibacterial activity: adding a solid culture medium into a multi-hole plate, adding the lactobacillus seed solution (sample hole) or a blank MRS culture medium (control hole) after solidification, and culturing for a period of time; and then adding mould spores suspended in soft agar into each hole, culturing for 48-120 h at 30 ℃, comparing the mould growth conditions of the sample hole and the control hole, and screening the lactic acid bacteria with the mould inhibiting activity.

The invention also provides the mould inhibiting lactic acid bacteria obtained by screening by the method.

Wherein the bacteriostatic lactobacillus is probiotic bacteria, including Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus rhamnosus, etc. The described fungistatic lactic acid bacteria can ferment saccharide, and the product mainly is a kind of spore-free gram-positive bacteria of lactic acid, including more than 200 kinds of bacteria of Lactobacillus, Streptococcus, Leuconostoc, Bifidobacterium and Pediococcus, etc. The shape can be divided into cocci and bacilli.

The invention uses a perforated plate to detect the mould inhibition activity of the lactobacillus, adds a blank MRS culture medium without the lactobacillus to a negative control hole, and uses the hole added with the lactobacillus strain as a sample hole. After co-culturing, the growth of mold in the sample wells and the control wells were compared to determine the inhibitory activity of lactic acid bacteria against mold.

The invention has the beneficial effects that: the invention screens the mould-inhibiting lactic acid bacteria on a porous plate (such as a 24-pore plate or a 48-pore plate), and eliminates the interference on the bacteriostatic effect caused by the large-area growth and reproduction of mould hypha and spores on the flat plate due to the separation of the pores. Because of the difference in the mold inhibition effect of different species of lactic acid bacteria, even the same species but different strains, the screening work of the mold inhibition activity of lactic acid bacteria is large and complicated. By using the perforated plate method, the mould inhibiting activity of dozens of lactic acid bacteria can be screened on one plate at the same time, and the screening efficiency and accuracy are improved.

Drawings

FIG. 1: the experimental result of the high-throughput screening of the Aspergillus flavus inhibiting lactic acid bacteria in the embodiment 1 of the invention is shown.

FIG. 2: the experimental result of screening penicillium-inhibiting lactic acid bacteria by the high-throughput method in example 2 of the invention is shown.

FIG. 3: the experimental result of the high-throughput method for screening the cladosporium fulvum-inhibiting lactic acid bacteria in example 3 of the invention is shown.

Detailed Description

The present invention is further described in detail with reference to the following detailed description and the accompanying drawings, and the changes and advantages that can be made therein by those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention and are protected by the appended claims. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.