CN112195107B - Erwinia glauca strain and application thereof - Google Patents

Abstract

The invention provides a strain of Erwinia closterium and a screening method and application thereof, wherein the taxonomy is named as Erwinia closterium HDR01, and the Erwinia closterium is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 20240. The invention adopts the steps of colloid part detoxification, dark culture, separation and purification and the like to screen and obtain the target Erwinia glauca mycelium, and the screening method is simple and efficient, simple and convenient to operate and low in pollution rate. The screened Eremothecium spongianum strain and the extract thereof have good capability of inhibiting microorganisms, are natural preservatives and have good application prospects.

Description

Erwinia glauca strain and application thereof

Technical Field

The invention relates to pseudomonads curvularia, in particular to pseudomonads curvularia and a construction method and application of the strain.

Background

The microorganisms such as bacteria are widely distributed in nature, food is inevitably polluted by different types and quantities of microorganisms, and when the environmental conditions are proper, the microorganisms can rapidly grow and reproduce to cause the food to be rotten and deteriorated, so that the nutrition and the sanitary quality of the food are reduced, and the health of human bodies can be harmed. With the development of society, the improvement of living standard of people, the food safety and health problems arouse the wide attention of people, and the food additive as the 'soul' of modern food industry also gradually arouses the attention of consumers. Commonly used chemical preservatives, such as potassium sorbate, sodium benzoate, although they counter the spoilage and off-taste problems of foods, are gradually discarded by consumers due to their side effects. In recent years, the concept of "clean diet" has been gradually recognized by people, the development trend of "clean diet" drives the development of products to return to nature, and the pressure of food manufacturers is doubled, and clean label components are searched for to prolong the shelf life and stability of foods.

The bioactive substances rich in the large edible fungi have important utilization value for maintaining human health, the components derived from the large edible fungi are very suitable for the dietary trend of cleanness and health, and the antibacterial activity of the large edible fungi is always valued by people for a long time. The mushroom glycolipid is a natural glycolipid mixture, has remarkable antifungal effect on common yeast and mould, and can be used as an antibacterial agent to be added into food; because the heat treatment of the beverage can cause the loss of nutritional value, vitamin content or mouthfeel, and the mushroom glycolipid also has better activity on gram-positive bacteria (including putrefying bacteria such as bacillus cereus, listeria and the like), can be used for prolonging the shelf life of the beverage and ensuring the microbial quality of the beverage, so that the mushroom glycolipid becomes a multifunctional substitute or supplement for other technologies such as heat treatment and the like.

The ear of Sarcodon glauca (Dacryapiaxspathularia) also called Osmanthus fragrans ear, Basidiomycetes, Araliaceae, has small fruiting body, spoon shape or antler shape, its upper part is irregular and split into fork shape, orange yellow, orange red after drying, and the color of the infertile part is light and smooth. The height of the fruiting body is 0.6-1.5cm, the thickness of the lower part of the handle is 0.2-0.3cm, fine villi exists, and the base part is chestnut brown to black brown and extends into cracks of the humus. The basidiomycetes has bifurcate, 2 spores, no color, smooth, no transverse septa in the initial stage, and 1-2 transverse septa in the later stage, i.e. 2-3 cells, oval shape near kidney, (8.9-12.8) μm x (3-4) μm, basidiomycetes, (28-38) μm x (2.4-2.6) μm. The coniferous trees such as the fir trees grow on the inverted rotten wood or the wood pile in spring to late autumn, and are widely distributed. It is edible, has small but fresh color fruiting body, and contains bioactive substances such as carotenoid.

In the prior art, pseudoglycolipid extraction from microorganisms such as microorganisms, bacteria, fungi. Anti-pathogenic bacteria of Anti-pathogenic fungi of mammalian macro-organism and monoclonal antibody of porcine immunoglobulin x-beta-pathogenic bacteria (Fries) against porcine so pathogenic bacteria of porcine reproductive and pathogenic bacteria of Pseudomonas spongiensis (Amar Kumar, et al, JETIR,6(1): 695-toxic 704) were studied, and the Anti-pathogenic bacteria of Pseudomonas spongiensis, Staphylococcus aureus, Proteus mirabilis, Bacillus subtilis and Salmonella typhi all have good Anti-bacterial effects. Patent CN103874411B provides a compound extracted from inula spongiosa strain FU50088, inula phloriza MUCL53180, inula gymnastica strain CBS173.60 or inula leucotricha strain MUCL53500, said compound having antiseptic effect and reducing microbial contamination. However, no research on the work of separating and purifying the Erythroseum spodophyllum species has been found. The acquisition of high-quality germplasm resources has very important significance for developing subsequent breeding and the work of separating and purifying related active substances, but no report of a separation method of the Erwinia tonkinensis is found at present. Meanwhile, in the common separation method, due to the small volume of the pseudoflower fungus of the spoon cover, wild materials of fruit bodies in the colloid part are difficult to obtain, the separation method is complex in technology, easy to be infected with mixed bacteria, high in operation difficulty and low in success rate.

Therefore, there is a need to develop a method for separating and purifying the Erythroseum spodophyllum strain with simple operation and high success rate, provide high quality germplasm resources, provide a natural preservative capable of replacing chemical additives, inhibit the growth of microorganisms in food and prolong the shelf life of the food without affecting human health.

Disclosure of Invention

Aiming at the defects, the invention finally obtains a strain of the pseudomonad perniciosa through the steps of pretreatment, colloid part culture, hypha separation and the like, and after the obtained pseudomonad perniciosa is cultured in a large scale, a biological active substance is obtained through extraction by a zymophyte liquid or a fruiting body extraction method, so that the biological active substance has better antibacterial property, can be used as a food antibacterial agent to be applied to food, inhibits the growth of microorganisms and improves the shelf life of the food.

The technical scheme of the invention is as follows:

in one aspect, the invention provides a false floral ear of spoon cover: (Dacryopinaxspathularia) The Erythroseum spongoides is Erythroseum spongoides HDR01 with the preservation number of CGMCC No.20240 which is preserved by China general microbiological culture Collection center (CGMCC No. 20240) ((DacryopinaxspathulariaHDR01）。

The invention relates to a spoon cover false lug HDR01 (DacryopinaxspathulariaHDR 01) deposited at the common microorganism center of the committee for culture collection of microorganisms (CGMCC) of china at 11.08/2020, having the address of No. 3, west road, north chen, yang ward, beijing, and the collection number of CGMCC No.20240, the institute for microbiology, china academy of sciences.

In another aspect, the present invention provides a fermented product of Erysiphe spongiensis.

Specifically, the preparation method of the fermentation product comprises the following steps: collecting the culture solution after fermentation culture, centrifuging to remove thallus and culture medium residue, collecting the supernatant of the fermentation liquid, and freeze drying to obtain the final product.

More specifically, the freeze drying condition is that the freezing temperature is-50 to-30 ℃, preferably-40 ℃, and the freezing time is 2 to 5 hours, preferably 3.5 hours; the drying temperature is 20-40 deg.C, preferably 40 deg.C, and the drying time is 25-35h, preferably 30 h.

In a further aspect, the present invention provides an extract of Erysiphe spongiensis.

Specifically, the extract comprises a ruscus aculeatus flavone extract or a ruscus aculeatus glycolipid extract.

In still another aspect, the present invention provides a method for isolating and purifying Erwinia glaucocalycinus.

The method for separating and purifying the Erythroseum sporum strains is a spore separation method, an auricularia separation method or a tissue separation method;

preferably, the tissue isolation method comprises the following steps:

(1) pretreatment: selecting fruiting bodies of the Erysiphe spongiosa strain collected in the current year, washing with deionized water to avoid damage of a colloid part, and washing with sterile pure water;

(2) cutting the sporocarp of the pseudomonad spongiosa strain pretreated in the step (1) into small gel sections under the aseptic condition, rinsing with alcohol, washing with aseptic pure water, and sucking off surface water;

(3) inoculating the colloid small segments which are dried by suction in the step (2) on a culture medium, and culturing in a dark place;

(4) after the culture in the step (3), selecting white hyphae growing from the colloid part, and inoculating the white hyphae on a culture medium for culture;

(5) and (4) performing morphological and genetic sequencing on the mycelium grown in the step (4), and separating, purifying and identifying to obtain the Erythroseum spongoides strain.

Specifically, in the pretreatment in the step (1), the deionized water is repeatedly washed for 5-20min, preferably 10-15min, and more preferably 10 min.

Specifically, in the pretreatment in the step (1), the deionized water is used for washing impurities such as floating dust, silt and wood dust on the surface layer.

Specifically, in the pretreatment of step (1), the sterile water can be repeatedly washed for 0-3 times, preferably 1-2 times.

Specifically, in the step (2), the aseptic cutting is performed by cutting the fruiting body into 0.05-0.3cm long colloidal fragments, preferably 0.1-0.2cm long colloidal fragments, and more preferably 0.1-0.15cm long colloidal fragments, using a sterile scalpel.

Specifically, in the step (2), the alcohol rinsing is performed by using 60-85% alcohol, preferably 65-80% alcohol, and more preferably 75% alcohol by volume percentage; the rinsing time is 20 to 60s, preferably 30 to 50s, and more preferably 45 s.

Specifically, in the step (2), the sterile water is washed for 3 to 8 times, preferably 5 to 6 times by using the sterile water.

Specifically, in the step (2), the step of sucking out the water on the surface layer is that the small colloid section is placed on a surface dish filled with sterile filter paper, and the water on the surface layer of the small colloid section is sucked out.

Specifically, in the step (3), the light-shielding culture condition is a culture temperature of 21 to 31 ℃, preferably 24 to 28 ℃, and more preferably 26 ℃; the relative humidity of the air is 65-75%, preferably 68-72%, and more preferably 70%; the culture time is 8-12 days, preferably 10 days.

Specifically, in the step (4), the inoculation is carried out by picking up white hyphae with a sterile inoculating needle.

Specifically, in the step (4), the culture conditions are a culture temperature of 21 to 31 ℃, preferably 24 to 28 ℃, and more preferably 26 ℃; the relative humidity of the air is 65-75%, preferably 68-75%, and more preferably 70-75%; the culture time is 13-17 days, preferably 15 days.

Specifically, the culture medium used in the step (3) and the step (4) is a wort solid culture medium or a PDA solid culture medium, and is preferably a wort solid culture medium.

More specifically, the wort solid medium comprises: wort 120-140g/L, agar 15-25g/L, and adjusting pH to 6.0.

Further specifically, the PDA solid culture medium comprises: peeled potato 180-220g/L, glucose 15-25g/L, agar 15-25 g/L.

In another aspect, the present invention provides a method for culturing Erysiphe spongiensis, wherein the culture method is fermentation culture or fruiting body culture.

The fermentation culture method comprises the following steps:

(1) activating strains: inoculating the strains in the preservation state to a culture medium for culturing, and gradually carrying out amplification culture to screen out strains with vigorous activity;

(2) seed culture: inoculating the screened strains in the step (1) into a culture medium for culturing to obtain a seed solution;

(3) introduction and fermentation: inoculating the seed liquid into a culture medium, and performing fermentation culture.

Specifically, step (1), step (2) and step (3) are performed under aseptic conditions.

Specifically, the strain activation in step (1) can be performed by a streaking method or a coating method.

Specifically, the strain activation medium in step (1) is a wort solid medium or a PDA solid medium, preferably a wort solid medium.

Specifically, the culture conditions in the step (2) are that the culture temperature is 21-35 ℃, preferably 25-32 ℃, and more preferably 30 ℃; the culture time is 2-4 days, preferably 3 days.

Specifically, the inoculation in the step (3) is carried out according to an inoculation amount of 10%.

Specifically, the fermentation culture conditions in step (3) are a culture temperature of 21 to 35 ℃, preferably 25 to 32 ℃, and more preferably 30 ℃.

Specifically, the fermentation culture mode in the step (3) is fed-batch fermentation or continuous fermentation.

Further specifically, the fed-batch fermentation is as follows: the fermentation method comprises the steps of putting culture solution into a liquid fermentation tank at one time, discharging raw materials at one time after fermentation is finished, repeating the processes of feeding, sterilizing, inoculating, fermenting and the like after discharging, and intermittently or continuously supplementing fresh culture medium in batch fermentation. The supplementary raw materials can be complete materials, and can also be a nitrogen source, a carbon source and the like.

More specifically, the fed-batch fermentation time is 23-27 days, preferably 25 days; the feeding is that fresh culture media with 20 percent of initial culture volume are respectively supplemented on the 5 th day, the 9 th day, the 13 th day, the 17 th day and the 21 st day of fermentation culture.

More specifically, the fed-batch fermentation aims at prolonging the synthesis time of metabolites to achieve maximum titer; after the feed has stopped, the fermentation broth is further incubated in order to free glucose.

Further specifically, the continuous fermentation is as follows: a fermentation method comprising continuously feeding a fresh culture solution into a liquid fermentation tank and continuously discharging the culture solution. Its advantages are high utilization rate of equipment, stable quality of product, easy automatic control and easy pollution to bacteria.

More specifically, the continuous fermentation time is 13-17d, and preferably 15 d.

Specifically, the culture medium in the step (2) and the step (3) is a wort culture medium, and the wort culture medium comprises: the wort is 120-140g/L, and the pH is adjusted to 6.0.

The method for culturing the fruit body comprises the following steps:

inoculating the separated strain of the pseudomonascus glauca or fermentation liquor containing fermentation hypha into a fruiting body culture medium for culture, wherein the fruiting body culture medium comprises cottonseed hulls, wheat bran, rotten wood chips and hydrated lime.

Specifically, the culture condition is that the culture temperature is 23-33 ℃, preferably 26-30 ℃, and more preferably 28 ℃; the relative humidity of the air is 50-80%, preferably 55-75%, and more preferably 60-70%; the culture time is 18-27 days, preferably 20-25 days; ventilating and cooling to culture.

Specifically, the fruiting body culture medium is a bag material culture medium, and the preparation method of the fruiting body bag material culture medium comprises the following steps: adding pure water into cottonseed hulls, wheat bran, wood dust of rotten wood (smashed) and hydrated lime according to the mass ratio of 60:20:18:2 to adjust the water content of the materials to 70-75%, adjusting the pH value to about 6.5, subpackaging the materials into bags with 400 g/bag, sterilizing at 121 ℃ and 0.1MPa for 120min at high temperature and high pressure to obtain a fruiting body bag material culture medium.

In another aspect, the invention provides a preparation method of an Erysiphe spongiensis extract, wherein the preparation method is a fermentation broth extraction method, a fermentation mycelium pellet extraction method or a fruiting body extraction method.

The extraction method of the fermentation liquor comprises the following steps:

(1) coarsely filtering the fermentation liquor to obtain coarse filtrate;

(2) adjusting the pH of the coarse filtrate obtained in the step (1) to be alkaline by using an alkaline adjusting solution, centrifuging to remove thalli, and collecting supernatant;

(3) adjusting the pH of the supernatant obtained in the step (2) to acidity by using an acidic adjusting solution, separating out a precipitate, centrifuging to remove the supernatant, washing the precipitate by using water, neutralizing the pH, and repeating the step for 2-3 times;

(4) and (5) drying.

Specifically, the alkaline conditioning solution in the step (2) is NaOH, and the concentration is 0.5-1.5mol/L, preferably 1 mol/L.

Specifically, the pH in step (2) is adjusted to 7.5 to 8.5, preferably to pH 8.

Specifically, the centrifugal speed in the step (2) is 8000r/min, and the centrifugal time is 10 min.

Specifically, the acidic conditioning solution in the step (3) is HCl, and the concentration is 1.7-2.3mol/L, preferably 2 mol/L.

Specifically, the pH in step (3) is adjusted to 2.5 to 3.5, preferably to pH 3.

Specifically, the centrifugal rotating speed in the step (3) is 6000r/min, and the centrifugal time is 15 min.

Specifically, the drying in step (4) is spray drying or freeze drying.

The method for extracting the fermentation mycelium pellets comprises the following steps:

(1) drying the mycelium pellet, crushing and sieving, and collecting powder;

(2) adding a mixed solution of chloroform, methanol and water to dissolve the powder obtained in the step (1), filtering and collecting filtrate;

(3) adding pure water for extraction, standing for layering, and collecting lower-layer liquid;

(4) concentrating and drying.

Specifically, the sieving in the step (1) is a 50-70 mesh sieve, preferably a 60 mesh sieve.

Specifically, the feed-to-liquid ratio of the powder to the mixed solution of chloroform, methanol and water in step (2) was 1mg:20 mL.

Specifically, the volume ratio of chloroform to methanol to water in the step (2) is 1:2: 0.8.

Specifically, the feed-liquid ratio of the powder to pure water in step (3) is 1mg:2 mL.

Specifically, the extraction time in the step (3) is 20-40min, preferably 30 min.

Specifically, the drying in step (4) is spray drying or freeze drying.

The fruit body extraction method comprises the following steps:

(1) drying the fruiting body, pulverizing, sieving, and collecting powder;

(2) adding a mixed solution of chloroform, methanol and water to dissolve the powder obtained in the step (1), filtering and collecting filtrate;

(3) adding pure water for extraction, standing for layering, and collecting lower-layer liquid;

(4) concentrating and drying.

Specifically, the sieving in the step (1) is a 50-70 mesh sieve, preferably a 60 mesh sieve.

Specifically, the feed-to-liquid ratio of the powder to the mixed solution of chloroform, methanol and water in step (2) was 1mg:20 mL.

Specifically, the volume ratio of chloroform to methanol to water in the step (2) is 1:2: 0.8.

Specifically, the ratio of the powder to the pure water in the step (3) is 1mg:2 mL.

Specifically, the extraction time in the step (3) is 20-40min, preferably 30 min.

Specifically, the drying in step (4) is spray drying or freeze drying.

In still another aspect, the present invention provides a food, a health product, a daily chemical product, a packaging material, an additive, a pharmaceutical preparation and a medical device comprising the Erwinia glauca, the fermented product of the Erwinia glauca or the extract of the Erwinia glauca.

In still another aspect, the present invention provides a use of Erwinia spongiensis, a fermented product of Erwinia spongiensis or an extract of Erwinia spongiensis for the preparation of foods, health products, daily chemicals, packaging materials, additives, pharmaceutical preparations and medical devices having a function of inhibiting microorganisms.

Specifically, the microorganism in the microorganism is inhibited by bacteria, mold and/or yeast.

More specifically, the bacteria include bacillus subtilis, bacillus cereus, propionibacterium acnes, clostridium perfringens, clostridium sporogenes, enterococcus faecalis, listeria welshimeri, listeria monocytogenes, corynebacterium mutans, lactobacillus plantarum, staphylococcus aureus, and the like; the mould comprises aspergillus fumigatus, aspergillus niger, pure rhizopus flavus, mucor densus, garcinia cambogia, pyricularia oryzae, candida albicans, candida glabrata and the like; the yeast includes Saccharomyces cerevisiae, Zygosaccharomyces Bayer, Zygosaccharomyces bisporus, Zygosaccharomyces floribularis, Dekkera naeslundii, Zygosaccharomyces rouxii, etc.

Compared with the prior art, the invention has the advantages that:

(1) the invention obtains a strain of Eremothecium spodophyllum HDR01 by separation and screening, and the preservation unit is as follows: china general microbiological culture Collection center; the preservation number is: CGMCC No. 20240.

(2) The method adopts the method of performing detoxification culture on the colloid part and then separating and purifying the target Erwinia closterium mycelium, the colloid part is separated to control the sterilization time of 75 percent alcohol, and the problem of mixed bacteria pollution of wild Erwinia closterium in the direct separation process is effectively avoided through dark culture.

(3) The culture medium adopted by the invention is combined with the growth characteristics of wild type Erysiphe spongiensis, simulates nutrition and environment required by the growth of the fruiting body of the strain, is beneficial to the germination and growth of the mycelium, and effectively solves the problem that the mycelium of the gum part can not grow in the process of transferring the fruiting body.

(4) The pseudomonad glauca and the pseudomonad glauca fermentation liquid or extract obtained by separation and screening have good functions of inhibiting microorganisms, resisting bacteria, preventing corrosion and prolonging the shelf life of food, and is a natural preservative.

Deposit description

And (3) classification and naming: false lug of spoon cover

Latin name:Dacryopinaxspathularia

the biological material of the reference: HDR01

The preservation organization: china general microbiological culture Collection center

The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)

And (4) storage address: xilu No. 1 Hospital No. 3, the institute of microbiology, China academy of sciences, Beijing, Chaoyang

The preservation date is as follows: 2020, 08 and 11 months

Registration number of the preservation center: CGMCC No. 20240.

Drawings

FIG. 1 is a diagram of the sporophytes of Eremothecium sporum.

FIG. 2 is a graph of microbial inhibition of apple juice.

FIG. 3 is a graph of Aspergillus niger antagonism experiment.

FIG. 4 is an electron micrograph of mycelium.

Detailed Description

The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.

EXAMPLE 1 Medium preparation

1. Wort solid medium: collecting wort (130 g of 101701 liquid malt extract from Huaga food science and technology (Shanghai) Ltd.) and agar (20 g of 10208ES60 from Shanghai assist in Sheng Biotechnology Ltd.), adjusting pH to 6.0, adding appropriate amount of pure water to a constant volume of 1L, 121 deg.C, 0.1MPa, and sterilizing at high temperature and high pressure for 20 min.

PDA solid medium: peeling potato 200g, adding 500mL pure water, boiling, filtering, collecting supernatant, adding glucose 20g and agar 20g, adding pure water to constant volume of 1L, 121 deg.C, 0.1MPa, and sterilizing at high temperature and high pressure for 20 min.

3. Wort medium: adding 130g of wort into pure water to reach a constant volume of 1L, adjusting pH to 6.0, 121 deg.C and 0.1MPa, sterilizing at high temperature and high pressure for 20min, and making into culture medium.

4. Preparing a sporophore culture medium: adding pure water into cottonseed hulls, wheat bran, wood dust of rotten wood (smashed) and hydrated lime according to the mass ratio of 60:20:18:2 to adjust the water content of the materials to 70-75%, adjusting the pH value to about 6.5, subpackaging the materials into bags with the weight of 400 g/bag, sterilizing at 121 ℃ and 0.1MPa for 120min at high temperature and high pressure, and preparing a sporophore culture medium.

EXAMPLE 2 isolation and purification of the Strain

1. Material

Erichthys sp: collected from rotten wood in the west Taihu lake.

2. Strain separation and purification step

(1) Pretreatment of Erysiphe spongiensis: selecting whole strain of fruiting body of Ruscus aculeatus collected in the same year, repeatedly soaking and washing with deionized water for 10min, cleaning surface dust, silt, wood dust, etc., avoiding gum damage, and washing with sterile pure water for 1-2 times;

(2) cutting the sporocarp of the pseudomonad strain preprocessed in the step (1) into small sections of colloid with the length of 0.1-0.15cm by using a sterile scalpel, putting the small sections into 75% alcohol for rinsing for 45s by using tweezers, taking out the small sections of colloid, repeatedly washing for 5-6 times by using sterile pure water, putting the small sections of colloid on a surface dish padded with sterile filter paper, and sucking out surface water;

(3) inoculating the colloid small segments with water absorbed in the step (2) on a wort culture medium, and culturing in a dark place, wherein the culture temperature is controlled to be 26 ℃, the relative air humidity is controlled to be 70%, and the time is 10 days;

(4) and after culturing for 10 days, removing foreign bacteria with obvious colors on the culture medium, selecting a colloid part to grow white hypha, inoculating the white hypha on the wort culture medium by using a sterile inoculating needle, and standing at the temperature of 26 ℃ and the relative air humidity of 70-75% for 15 days to obtain the mycelium.

EXAMPLE 3 isolation and purification of the Strain

1. Material

Erichthys sp: collected from rotten wood in the west Taihu lake.

2. Strain separation and purification step

(1) Pretreatment of Erysiphe spongiensis: selecting the whole strain of the pseudolarix japonicus fruiting body collected in the current year, repeatedly soaking and washing with deionized water for 5min, and cleaning impurities such as floating dust, silt, wood dust and the like on the surface layer, and simultaneously avoiding the damage of a colloid part;

(2) cutting the sporocarp of the pseudomonad spongiensis strain pretreated in the step (1) into small sections of colloid with the length of 0.05-0.1cm by using a sterile scalpel, putting the small sections into 60% alcohol for rinsing for 20s by using tweezers, repeatedly washing the small sections of colloid for 3-4 times by using sterile pure water after taking out the small sections of colloid, putting the small sections of colloid on a surface dish padded with sterile filter paper, and sucking out surface water;

(3) inoculating the dried colloid segments obtained in the step (2) on a wort culture medium, and culturing in a dark place, wherein the culture temperature is controlled to be 21 ℃, the relative air humidity is controlled to be 65%, and the time is 8 d;

(4) and after 8 days of culture, removing obviously colored mixed bacteria on the culture medium, selecting a colloid part to grow white hypha, selecting the white hypha without bacteria and mixed bacteria pollution on the periphery, selecting the white hypha by using a sterile inoculating needle, inoculating the white hypha on a malt wort culture medium, and standing at the temperature of 21 ℃ and the relative humidity of air of 65% for 13 days to obtain the mycelium.

EXAMPLE 4 isolation and purification of the Strain

1. Material

Erichthys sp: collected from rotten wood in the west Taihu lake.

2. Strain separation and purification step

(1) Pretreatment of Erysiphe spongiensis: selecting whole strain of Pseudolarix japonicus fruiting body collected in the same year, repeatedly soaking and washing with deionized water for 20min, cleaning surface layer floating dust, silt, sawdust, etc., avoiding colloid damage, and washing with sterile pure water for 3 times;

(2) cutting the sporocarp of the pseudomonad spongiensis strain pretreated in the step (1) into small sections of colloid with the length of 0.2-0.3cm by using a sterile scalpel, putting the small sections into 80% alcohol by using tweezers to rinse for 60s, taking out the small sections of colloid, repeatedly washing the small sections of colloid for 7-8 times by using sterile pure water, putting the small sections of colloid on a surface dish padded with sterile filter paper, and sucking out surface water;

(3) inoculating the dried colloid segments obtained in the step (2) on a wort culture medium, and culturing in a dark place, wherein the culture temperature is controlled to be 31 ℃, the relative air humidity is controlled to be 75%, and the time is 12 d;

(4) culturing for 12 days, removing obviously colored mixed bacteria on the culture medium, selecting white hypha growing on the colloid part, inoculating the white hypha on the wort culture medium by using a sterile inoculating needle, and standing at the temperature of 31 ℃ and the relative humidity of air of 75% for 17 days to obtain the mycelium.

EXAMPLE 5 identification of the strains

The mycelium obtained in example 2 was subjected to morphological and genetic sequencing to identify the isolated and purified strain as Erwinia glauca.

(1) Morphological Observation of the Strain

The method comprises the following steps: taking the mycelium of the Erysiphe spongiensis cultured on a wort culture medium flat plate, and carrying out macroscopic observation on the color and morphological characteristics of the colony.

As a result: colony morphology: the hyphae in the early and late stages are white, the aerial hyphae are not developed, and no spores are produced on the conventional culture medium.

The electron micrograph of the mycelium is shown in FIG. 4.

(2) Genetic sequencing of strains

Inoculating the screened strain HDR01 on a PDA plate, selecting a fungal mycelium growing for 7d, extracting genomic DNA by an SDS method, amplifying by universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGC-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'), handing an amplification product to the Shanghai biological engineering limited company for ITS DNA sequencing, comparing a sequence of an in-vivo transcription ribosome spacer DNA (ITS gene) in the mycelium obtained by a sequence shown in SEQ ID NO:3 with a sequence in Genebank by NCBI Nucleotide BLAST, showing 99% sequence similarity with a collected strain of Erysiphelus spongiensis, and determining that the strain is Erysiphe spongiosa (Dacryapiaxpatula sporulata).

EXAMPLE 6 cultivation of the Strain

1. Fermentation culture-fed batch of strain

(1) Activating strains: streaking the strains in the preservation state to a wort solid culture medium for culture, and gradually carrying out amplification culture to screen out strains with vigorous activity;

(2) seed culture: inoculating the screened strains in a culture medium on a sterile operating platform, and fermenting and culturing for 3 days at 30 ℃;

(3) introduction and fermentation: 5mL of the seed solution was introduced into 50mL of the medium on a sterile table, shake-fermented at 30 ℃ for 25 days, and the medium was added in a fed-batch manner. On days 5, 9, 13, 17 and 21 of the culture, 10mL of fresh medium was added.

2. Fermentation culture-continuous feeding of strain

(1) Activating strains: streaking the strains in the preservation state to a wort solid culture medium for culture, and gradually carrying out amplification culture to screen out strains with vigorous activity;

(2) seed culture: inoculating the screened strains in a culture medium on a sterile operating platform, and fermenting and culturing for 3 days at 30 ℃;

(3) introduction and fermentation: 5mL of the seed solution was introduced into 50mL of the medium on a sterile table and fermented continuously at 30 ℃ for 15 days.

3. Culturing strain sporophore

Inoculating the separated Erythroseum spodophyllum strains into a bag material of a fruiting body culture medium, standing at 28 deg.C and relative air humidity of 60-70%, ventilating, cooling in the shade, culturing for 20-25 days, and allowing fruiting body to grow on the bag material. FIG. 1 is a fruiting body map of strain HDR 01.

EXAMPLE 7 cultivation of the Strain

1. Fermentation culture-fed batch of strain

(1) Activating strains: streaking the strains in the preservation state to a wort solid culture medium for culture, and gradually carrying out amplification culture to screen out strains with vigorous activity;

(2) seed culture: inoculating the screened strains in a culture medium on a sterile operation platform, and fermenting and culturing for 2 days at 21 ℃;

(3) introduction and fermentation: 5mL of the seed solution was introduced into 50mL of the medium on a sterile table, shake-fermented at 21 ℃ for 23 days, and the medium was added in a fed-batch manner. On days 5, 9, 13, 17 and 21 of the culture, 10mL of fresh medium was added.

2. Fermentation culture-continuous feeding of strain

(1) Activating strains: streaking the strains in the preservation state to a wort solid culture medium for culture, and gradually carrying out amplification culture to screen out strains with vigorous activity;

(2) seed culture: inoculating the screened strains in a culture medium on a sterile operating platform, and fermenting and culturing for 2 days at 21 ℃;

(3) introduction and fermentation: 5mL of the seed solution was introduced into 50mL of the medium on a sterile table and fermented continuously at 21 ℃ for 13 days.

3. Culturing strain sporophore

Inoculating the separated Erythroseum spodophyllum strains into a bag material of a fruiting body culture medium, placing at 23 deg.C and air relative humidity of 50-55%, ventilating, cooling in the shade, culturing for 18-20 days, and growing fruiting bodies on the bag material.

EXAMPLE 8 cultivation of the Strain

1. Fermentation culture-fed batch of strain

(1) Activating strains: streaking the strains in the preservation state to a wort solid culture medium for culture, and gradually carrying out amplification culture to screen out strains with vigorous activity;

(2) seed culture: inoculating the screened strains in a culture medium on a sterile operating platform, and fermenting and culturing for 4 days at 35 ℃;

(3) introduction and fermentation: 5mL of the seed solution was introduced into 50mL of the medium on a sterile table, shake-fermented at 35 ℃ for 27 days, and the medium was added in a fed-batch manner. On days 5, 9, 13, 17 and 21 of the culture, 10mL of fresh medium was added.

2. Fermentation culture-continuous feeding of strain

(1) Activating strains: streaking the strains in the preservation state to a wort solid culture medium for culture, and gradually carrying out amplification culture to screen out strains with vigorous activity;

(2) seed culture: inoculating the screened strains in a culture medium on a sterile operating platform, and fermenting and culturing for 3 days at 35 ℃;

(3) introduction and fermentation: 5mL of the seed solution was introduced into 50mL of the medium on a sterile table and fermented continuously at 35 ℃ for 17 days.

3. Culturing strain sporophore

Inoculating the separated Erythroseum spodophyllum strains into a bag material of a fruiting body culture medium, placing at the temperature of 33 ℃, keeping the relative humidity of air at 75-80%, ventilating, cooling in the shade, culturing for 25-27 days, and growing the fruiting bodies on the bag material.

Example 9 preparation of extract of Strain

(1) Preparing a strain fermentation liquor extract:

1) coarsely filtering the feed liquid obtained by continuous fermentation in the embodiment 6 to obtain coarse filtrate;

2) adjusting the pH of the crude filtrate to 8 by using 1mol/L NaOH, and centrifuging for 10min at 8000r/min to remove thalli;

3) then adjusting pH to 3 with 2mol/L HCl, separating out precipitate, centrifuging for 15min at 6000r/min by a centrifuge, removing supernatant, washing with water for multiple times, neutralizing pH, and repeating the steps for 2-3 times;

4) and (5) freeze drying.

(2) Preparation of fermented mycelium pellet extract:

1) cleaning the mycelium pellets obtained by continuous fermentation in the embodiment 6, putting the mycelium pellets into a drying oven for drying until the weight of the mycelium pellets is constant, putting the mycelium pellets into a crusher for crushing, sieving the crushed powder with a 60-mesh sieve, and collecting the powder;

2) adding a mixed solution of chloroform, methanol and water (volume ratio is 1:2: 0.8) with the volume of 20 times to dissolve the powder, filtering and collecting filtrate;

3) adding 2 times of pure water for extraction for 30min, standing for layering, and collecting the lower layer liquid;

4) concentrating, and freeze drying.

(3) Preparation of a strain fruiting body extract:

1) removing roots of the sporophores obtained by culturing the sporophores in the embodiment 6, cleaning the sporophores, putting the sporophores into a drying box, drying the sporophores to constant weight, putting the dried sporophores into a crusher for crushing, sieving the crushed powder with a 60-mesh sieve, and collecting the powder;

2) adding a mixed solution of chloroform, methanol and water (volume ratio is 1:2: 0.8) with the volume of 20 times to dissolve the powder, filtering and collecting filtrate;

3) adding 2 times of pure water for extraction for 30min, standing for layering, and collecting the lower layer liquid;

4) concentrating, and freeze drying.

EXAMPLE 10 Strain fermentation broth product preparation

The preparation method of the fermentation liquor product comprises the following steps: collecting the fermentation culture solution obtained by continuous fermentation in the embodiment 6, centrifuging at 8000rpm for 10min, removing thallus and culture medium residues, placing the collected fermentation solution after wall breaking and centrifugation in a freeze dryer, freezing at-40 deg.C for 3.5h, drying at 30 deg.C for 30h to obtain lyophilized powder, and obtaining the fermentation solution product.

Experimental example 1 bacterial Strain bacteriostasis test

1. Fermentation liquor of Pleurotus spodophyllus Bodinieri to fecal coccobacillusEnterococcus faecalis) Detection of MIC values

(1) Preparing a fermentation liquor mother liquor of the Erhua fungus with the spoon cover: 25.6g of the fermentation liquid product prepared in the embodiment 10 is accurately weighed, pure water is added to the fermentation liquid product to be constant volume to 1L, and mother liquor (25.6 g/L) is obtained for later use.

(2) Preparation of a liquid of the coprococcus faecalis: a single colony on an overnight-cultured LB agar plate at 37 ℃ was picked with an inoculating loop in sterile 0.85% physiological saline to be calibrated to 0.5 McLeod standard, with about 1X 10 of the number of colonies⁸CFU/mL, and then diluting 100 times to obtain about 1 × 10 bacteria-containing number⁶CFU/mL of bacterial liquid for later use.

(3) Diluting the fermentation liquor mother liquor of the Eremothecium spongoides by 100 times to obtain an antibacterial solution with the concentration of 256 mug/mL. Taking a sterile 96-well plate, adding 200 mu L of mother liquor into a first well, respectively adding 100 mu L of LB culture medium into a second well to a tenth well, sucking 100 mu L of LB culture medium from the first well, adding into the second well, uniformly mixing, sucking 100 mu L of LB culture medium into a third well, and repeating the steps, sucking 100 mu L of LB culture medium into the tenth well and discarding. The drug concentration in each well is as follows: 256. 128, 64, 32, 16, 8, 4, 2, 1 and 0.5 mu g/mL, 200 mu L of the bacterial liquid is added into the eleventh hole, and 200 mu L of the LB culture medium is added into the twelfth hole.

(4) Then 100. mu.L of the bacterial suspension was added to each of the 1 to 10 wells so that the final concentration of the bacterial suspension in each tube was about 5X 10⁵CFU/mL, and the drug concentrations from the 1 st to the 10 th wells are 128, 64, 32, 16, 8, 4, 2, 1, 0.5 and 0.25. mu.g/mL respectively. And (3) placing the inoculated 96-well plate in an incubator at 37 ℃ for culture, and observing the growth condition of the bacterial liquid for 24 hours. The minimum concentration of 1-10 wells without visible colony growth is the MIC value of the Pseudocercosporus sporum fermentation liquid to the coprococcus faecalis.

2. Fermentation of spoon-cover false earsLiquid pair of Aspergillus nigerAspergillusniger) Detection of MIC values

(1) Preparing a fermentation liquor mother liquor of the Erhua fungus with the spoon cover: 25.6g of the fermentation liquid product prepared in the embodiment 10 is accurately weighed, pure water is added to the fermentation liquid product to be constant volume to 1L, and mother liquor (25.6 g/L) is obtained for later use.

(2) Preparation of aspergillus niger spore suspension: inoculating Aspergillus niger to PDA plate, culturing at 28 deg.C for 5-7 days, eluting fresh Aspergillus niger on the plate with 0.85% physiological saline to obtain spore suspension with a bacterial count of 1 × 10 and a calibration of 0.5 McLeod⁸CFU/mL, and then diluting 100 times to obtain about 1 × 10 bacteria-containing number⁶CFU/mL of bacterial liquid for later use.

(3) Diluting the fermentation liquor mother liquor of the Eremothecium spongoides by 100 times to obtain an antibacterial solution with the concentration of 256 mug/mL. Taking a sterile 96-well plate, adding 200 mu L of mother liquor into a first well, respectively adding 100 mu L of LB culture medium into a second well to a tenth well, sucking 100 mu L of LB culture medium from the first well, adding into the second well, uniformly mixing, sucking 100 mu L of LB culture medium into a third well, and repeating the steps, sucking 100 mu L of LB culture medium into the tenth well and discarding. The drug concentration in each well is as follows: 256. 128, 64, 32, 16, 8, 4, 2, 1 and 0.5 mu g/mL, 200 mu L of the bacterial liquid is added into the eleventh hole, and 200 mu L of the LB culture medium is added into the twelfth hole.

(4) Then 100. mu.L of the bacterial suspension was added to each of the wells from 1 to 10, so that the final concentration of the bacterial suspension in each tube was about 5X 10⁵CFU/mL, and the drug concentrations from the 1 st to the 10 th wells are 128, 64, 32, 16, 8, 4, 2, 1, 0.5 and 0.25. mu.g/mL respectively. And (3) placing the inoculated 96-well plate in an incubator at 37 ℃ for culture, and observing the growth condition of the bacterial liquid for 24 hours. The minimum concentration of 1-10 holes without visible colony growth is the MIC value of the Erichthys spongiensis fermentation liquid to the Aspergillus niger.

3. The results of the measurements are shown in Table 1 below.

TABLE 1 Microtoena pseudolaris MIC test results

Category	Taxonomy	MIC(mg/L)
			Bacteria	Enterococcus faecalis（ATCC29212）	50
Molds	Aspergillusniger（ATCC16404）	6.2

Experimental example 2 inhibition of microorganisms in beverage

Water-based beverages such as apple juice, lemon juice, orange juice, coconut juice, sugarcane juice, flavored fruit juice beverages, energy beverages, tea beverages, and the like, are susceptible to the growth of spoilage microorganisms such as yeasts, acetobacter, and staphylococcus at normal temperature. 20g of apple juice is selected as an experimental material, 1g of the inula cappa fermentation broth product prepared in example 10 is added into an experimental group, 1g of pure water is added into a control group, the mixture is placed at 27 ℃ for 60 days, and the total number of bacterial colonies in the mixture is monitored. The monitoring result is shown in fig. 2, in the apple juice beverage added with the fermentation liquid of the pseudomonad spongiopsis, the total number of bacterial colonies is remarkably lower than that of a control group, and the control group is maintained to be not more than 10 for a long time, which shows that the fermentation liquid of the pseudomonad spongiopsis strain provided by the invention can obviously inhibit the total number of microbial colonies and prolong the shelf life.

Experimental example 3 antagonistic experiment

1 taking Aspergillus niger colonies to inoculate a PDA flat plate, culturing in a constant temperature box with the temperature of 30 ℃ and the RH of 60 percent until the Aspergillus niger grows over the whole flat plate for standby.

2, taking a little of the fermentation liquor of the Eremothecium spongoides, performing suction filtration to remove mycelium pellets, taking 10g of filtrate, 50g of liquid PDA and 2g of agar to prepare a culture medium containing the fermentation liquor, sterilizing at 121 ℃, and preparing a detection plate; in addition, a control group of PDA plate without a key cover and a false flower fungus fermentation liquid is prepared.

3 cutting small square blocks with the same size at the same position from the center of the Aspergillus niger full-grown flat plate by using a scalpel, respectively placing the small square blocks upside down between the detection flat plate and the control flat plate, and culturing in a constant temperature box at 30 ℃ and 60% RH.

As shown in FIG. 3, after several days of observation, the Aspergillus niger grows on the whole PDA plate within 4 days in the control group (left plate), and the Aspergillus niger does not grow for 30 days in the detection plate (right plate) added with the fermentation liquid of the Erythrostoma sporum, which indicates that the fermentation liquid of the Erythrostoma sporum has obvious inhibition effect on the growth of the Aspergillus niger.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

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<120> Erwinia glauca strain and application thereof

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Claims (7)

1. The Erythroseum spodophyllum strain is characterized in that the Erythroseum spodophyllum HDR01 is preserved in China general microbiological culture Collection center (CGMCC) at 11/08 in 2020, and the preservation number is CGMCC No. 20240.

2. A fermented product of pseudomonad curvosa according to claim 1, characterized in that it is prepared by the following method: collecting the culture solution after fermentation culture, centrifuging to remove thallus and culture medium residue, collecting the supernatant of the fermentation liquid, and freeze drying to obtain the final product.

3. A food product, a health product, a daily chemical product, a packaging material, an additive, a pharmaceutical preparation and a medical device comprising the pseudomonarda spongiensis strain of claim 1 or the fermented product of claim 2.

4. Use of the strain of pseudomonad glauca according to claim 1 or the fermented product according to claim 2 for the preparation of food products, health products, daily chemicals, packaging materials, additives, pharmaceutical preparations and medical devices having a microbial inhibitory function.

5. The use according to claim 4, wherein the microorganism of the inhibited microorganisms is Aspergillus niger and/or Bacillus faecalis.

6. A method for culturing Erysiphe spongiensis according to claim 1, wherein the culture method is fermentation culture or fruiting body culture;

the fermentation culture steps are as follows:

(1) activating strains: inoculating the strains in the preservation state to a culture medium for culturing, and gradually carrying out amplification culture to screen out strains with vigorous activity, wherein the culture medium in the step is a wort solid culture medium or a PDA solid culture medium;

(2) seed culture: inoculating the screened strains in the step (1) into a culture medium for culturing to obtain a seed solution, wherein the culture medium in the step is a wort culture medium;

(3) introduction and fermentation: inoculating the seed liquid into a culture medium, and performing fermentation culture, wherein the culture medium in the step is a wort culture medium;

the sporophore culturing steps are as follows:

inoculating the separated strain of the pseudomonascus glauca or fermentation liquor containing fermentation hypha into a fruiting body culture medium for culture, wherein the fruiting body culture medium comprises cottonseed hulls, wheat bran, rotten wood chips and hydrated lime.

7. A method for preparing an extract of Erysiphe spongiensis according to claim 1, wherein the method comprises a fermentation broth extraction method, a fermentation mycelial group extraction method or a fruit body extraction method;

the method for extracting the fermentation liquor comprises the following steps:

(1) coarsely filtering the fermentation liquor to obtain coarse filtrate;

(2) adjusting the pH of the coarse filtrate obtained in the step (1) to be alkaline by using an alkaline adjusting solution, centrifuging to remove thalli, and collecting supernatant;

(3) regulating the pH of the supernatant obtained in the step (2) to acidity by using an acidic regulating solution, separating out a precipitate, centrifuging to remove the supernatant, washing the precipitate by using water, neutralizing the pH, and repeating the step for 2-3 times;

(4) drying;

the method for extracting the fermentation mycelium pellets comprises the following steps:

(1) drying the mycelium pellets, crushing and sieving the dried mycelium pellets, and collecting powder;

(2) adding a mixed solution of chloroform, methanol and water to dissolve the powder obtained in the step (1), filtering and collecting filtrate;

(3) adding pure water for extraction, standing for layering, and collecting lower-layer liquid;

(4) concentrating and drying;

the method for extracting the fruit body comprises the following steps:

(1) drying the fruiting body, pulverizing, sieving, and collecting powder;

(2) adding a mixed solution of chloroform, methanol and water to dissolve the powder obtained in the step (1), filtering and collecting filtrate;

(3) adding pure water for extraction, standing for layering, and collecting lower-layer liquid;

(4) concentrating and drying.

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