CN113373078B

CN113373078B - Compound microorganism and application thereof in combination with black soldier fly to transform edible fungus residues

Info

Publication number: CN113373078B
Application number: CN202110180947.5A
Authority: CN
Inventors: 熊强; 周燊; 张靖; 李新福
Original assignee: Nanjing Tech University
Current assignee: Nanjing Tech University
Priority date: 2021-02-09
Filing date: 2021-02-09
Publication date: 2022-07-12
Anticipated expiration: 2041-02-09
Also published as: CN113373078A

Abstract

The invention discloses a compound microorganism, which comprises any one or combination of more of klebsiella pneumoniae, pseudomonas anthracis, pseudomonas faecalis, and galactose saccharomycete geotrichum, and also discloses application of the compound microorganism in the conversion of edible fungus residues into biomass in combination with hermetia illucens. Compared with the prior art, the invention has the following advantages: the bacterial strains used by the method are all the original bacteria screened from the intestinal tracts of the hermetia illucens larvae, the hermetia illucens larvae after being fixedly planted by the original bacteria can better convert the edible fungus residues, and the highest biological conversion rate of the fungus residues can reach 17.47%.

Description

Compound microorganism and application thereof in combination with black soldier fly to transform edible fungus residues

Technical Field

The invention relates to the field of environmental protection and resource utilization, in particular to a compound microorganism and application thereof in combination with black soldier fly transformation of edible fungus residues.

Background

With the continuous development of the domestic fungus industry in China, related enterprises are increasing day by day, and the quantity of the produced domestic fungus dregs is also continuously expanded. According to statistics, the quantity of edible fungus dregs generated in China every year is up to thousands of tons, most of the fungus dregs are incinerated or randomly stacked, so that the environment is polluted, and pathogenic bacteria and the like which are harmful to human health are seriously generated. At present, most of utilization researches on edible fungus residues are concentrated in a single field and are not thorough enough, and the fungus residues cannot be comprehensively and effectively utilized. Therefore, how to treat the edible fungi residues in an environment-friendly and effective manner becomes a more and more serious problem. The edible fungus dregs have rich nutrients including N, P, K, C, coarse protein, organic compound, mycelium, etc. and thus have high utilization value. However, the mushroom dregs contain most of cellulose and hemicellulose, and these components are difficult to be directly utilized.

The hermetia illucens larvae is a saprophytic insect, has a very wide feeding range, can quickly convert organic wastes such as kitchen waste, livestock and poultry excrement, deteriorated vegetables and fruits, aged grains, food processing leftovers and the like into biomass of the hermetia illucens larvae, and is an important resource environmental insect. Researches show that the black soldier fly has excellent treatment effect on wastes such as kitchen waste, aged grain and the like, but when agricultural wastes such as mushroom dregs, straws, livestock and poultry manure and the like are treated, the black soldier fly is difficult to realize effective conversion of the black soldier fly, especially the agricultural wastes with low dry matter content, unbalanced C/N and high lignocellulose content.

A large number of microorganisms inhabit in the intestinal tracts of the hermetia illucens, the intestinal microorganisms and the insects show a close symbiotic relationship in a long-term evolution process, play important roles of providing nutrition, resisting colonization, participating in a multiple nutrition relationship, causing an insect immune response and the like, and have important influence on the growth and development and the life activities of hosts. The method comprises the steps of extracting microorganisms in the intestinal tracts of the hermetia illucens, carrying out enrichment culture, fixedly planting the microorganisms back into hermetia illucens larvae in a feeding mode, degrading and digesting edible fungus residues rich in cellulose by using the hermetia illucens larvae after strain fixedly planting, and hopefully realizing efficient conversion and utilization of the fungus residues.

Chinese patent 2020100791997 discloses a black soldier fly compound microbial preparation, a preparation method thereof and application thereof in transformation of bean dregs, wherein the target object is the bean dregs, all strains used in the patent are strains which are capable of degrading proteins and producing protease with high yield, and the strains can not effectively degrade the bean dregs through tests. The content of crude protein in the bean dregs is the largest and is about 28 percent; the content of cellulose in the fungus dregs is about 35 percent at most.

Therefore, a treatment system capable of efficiently converting the mushroom dregs is urgently needed at present.

Disclosure of Invention

The invention provides a compound microorganism aiming at treating fungus residues, which aims to solve the problem of recycling of edible fungus residues, realize the recycling of the wastes and provide the compound microorganism for treating the fungus residues.

The invention also aims to solve the technical problem of providing the application of the compound microorganism in the transformation of the edible fungus dregs by combining the hermetia illucens.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a compound microorganism comprises one or more of Klebsiella pneumoniae, Pseudocitrobacter anthracis, Pseudocitrobacter faecalis, and Geotrichum candidum galactosaccharomyces, wherein the microorganism is isolated from the intestinal tract of hermetia illucens.

Preferably, the Klebsiella pneumoniae is Klebsiella pneumoniae (Klebsiella pneumoniae), the strain number is Cellulase:001, and the preservation number is CCTCC NO: m2019615, which has been deposited in the China center for type culture Collection in 2019, 8, 7, month, and the deposition address is Wuhan university, Wuhan, China; the 16S rDNA sequence is shown in SEQ ID No. 1. The strain is screened from black soldier fly larva intestinal tracts in Nanjing industry university at 7, 8 and 7 months in 2019.

Preferably, the pseudomonas anthracis is pseudomonas antrropi, the strain number is Cellulase:002, and the preservation number is CCTCC NO: m2019887, which has been deposited in the China center for type culture Collection in 2019, 11/1, with the deposit address of Wuhan university in Wuhan, China; the 16S rDNA sequence is shown in SEQ ID No. 2. The strain is screened from black soldier fly larva intestinal tracts in Nanjing industry university in 2019, 10 months and 7 days.

Preferably, the pseudomonas faecalis is Pseudomonas faecalis, the strain number is Cellulase 003, the preservation number is CCTCC NO: M2019888, the pseudomonas faecalis is preserved in China center for type culture collection in 11 months and 1 days in 2019, and the preservation address is Wuhan university in Wuhan, China; the 16S rDNA sequence is shown in SEQ ID No. 3. The strain is screened from black soldier fly larva intestinal tracts in Nanjing industry university in 2019, 10 months and 7 days.

Preferably, the geotrichum galactococcus is geotrichum galactococcus (Galactomyces geotrichum), the strain number is Cellulase:004, the preservation number is CCTCC NO: m2019889, which was deposited in the China center for type culture Collection on 1/11 in 2019 at the location of Wuhan university in Wuhan, China. The 16S rDNA sequence is shown in SEQ ID No. 4. The strain is screened from black soldier fly larva intestinal tracts in Nanjing industry university in 2019, 10 and 7 days.

Further preferably, the complex microorganism comprises Klebsiella pneumoniae (Klebsiella pneumoniae) Cellulase:001CCTCC NO: m2019615, pseudorhizobacter anthracotic Cellulase:002CCTCC NO: m2019887, Pseudomonas farealis Cellulase:003CCTCC NO: m2019888, Geotrichum galobaculum (Galactomyces geotrichum) Cellulase:004CCTCC NO: m2019889 any two or more of the four strains.

Most preferably, the complex microorganism comprises Klebsiella pneumoniae (Klebsiella pneumoniae) Cellulase:001CCTCC NO: m2019615 and Pseudomonas farealis Cellulase:003CCTCC NO: M2019888.

The compound microorganism of the invention has no special limitation on the mixture ratio of different strains, and preferably has the same cell number mixture ratio or is prepared by compounding bacterial suspensions with the same bacterial concentration according to the same volume.

A compound microorganism bacterium agent is prepared by inoculating the compound microorganism into beef extract peptone culture medium, and culturing to obtain bacterium suspension.

The beef extract peptone medium comprises 3g/L of beef extract, 10g/L of peptone, 5g/L of sodium chloride and 6.8-7.4 of pH; the components in the culture medium are uniformly stirred and then sterilized for 20min at 121 ℃; the culture is carried out under the conditions of 100-200 r/min at 35-40 ℃ for 20-30 h, preferably 150r/min at 37 ℃ for 24 h.

The application of the compound microorganism in the conversion of the edible fungus residues into the biomass by combining the hermetia illucens is also within the protection scope of the invention.

The application method comprises the steps of carrying out enrichment culture on the compound microorganisms, and then fixedly planting the compound microorganisms back into intestinal tracts of black soldier fly larvae in a feeding mode, so that the black soldier fly efficiently converts edible fungus residues into biomass.

The method specifically comprises the following steps:

(1) and (3) field planting of strains: inoculating the composite microorganism into a beef extract peptone culture medium for culture to obtain a bacterial suspension, or independently inoculating each microorganism in the composite microorganism into a beef extract peptone culture medium for culture and mixing to obtain a bacterial suspension, inoculating the bacterial suspension into sterilized concentrated feed, mixing uniformly to obtain a mixed material, and placing the mixed material into a feeding box; covering a gauze on the top of the feeding box, placing the hermetia illucens eggs on the gauze for incubation, directly dropping the incubated larvae into the mixed material through gaps of the gauze, and feeding to ensure that the compound microbial strains are fixedly planted in intestinal tracts of the hermetia illucens larvae;

(2) and (3) conversion of the edible fungus residues: inoculating the hermetia illucens larvae bred in the step (1) into edible fungus dregs containing a certain water content, culturing and converting, stopping conversion until the whole body of the larvae is light brown, namely before pupation, and separating the larvae from biomass.

In the step (1), the concentration of each component of the beef extract peptone medium is 3g/L of beef extract, 10g/L of peptone, 5g/L of sodium chloride and pH is 6.8-7.4; the components in the culture medium are uniformly stirred and then sterilized for 20min at 121 ℃; the culture is carried out under the conditions of 100-200 r/min at 35-40 ℃ for 20-30 h, preferably 150r/min at 37 ℃ for 24 h.

The bacterial suspension prepared in the step (1) has the total bacterial concentration of 10⁶CFU/mLThat is, when the bacteria are single strains, the concentration of the bacteria is 10⁶CFU/mL, when the bacteria are compounded by multiple strains, the total concentration of the bacteria in the bacterial suspension obtained after mixing is controlled to be 10⁶CFU/mL。

In the step (1), the sterilized concentrated feed is sterilized for 15min at 121 ℃.

In the step (1), the concentrated feed is a concentrated feed for young animals, preferably a feed for piglets.

In the step (1), the bacterial suspension is inoculated into the feed according to the inoculation amount of 50 percent (mL/g); the ratio of the worm eggs to the mixed material is 1.0 g/kg; the incubation is carried out for 4-5 days at 28 ℃; the breeding is carried out for 5-6 days under the conditions that the temperature is 28 ℃, the relative humidity is 60-80%, and the water content in the mixture is 60-80%.

In the step (2), the water content of the edible fungus residues is 60-80 wt%; the dosage of the black soldier fly larvae is 1.2g per 80g dry weight of edible fungus residues; the culture is carried out for 10-15 days at the temperature of 30 ℃ and the relative humidity of 60-80%.

In the step (2), the separated hermetia illucens larvae are dried at 65 ℃ for 48 hours and then are subjected to deep processing, such as extraction of high-value substances such as antibacterial peptide, chitosan and the like; the separated materials are treated in a centralized manner for 1-2 weeks in a natural composting manner and then can be used as high-quality organic fertilizer.

The hermetia illucens is a hermetia illucens Wuhan strain.

The edible fungus dregs are solid waste left after the edible fungus is cultivated, wherein the solid waste comprises about 15% of crude protein, about 9% of crude fat, about 34% of crude fiber, about 11% of crude ash and 30-40% of cellulose.

Has the beneficial effects that: compared with the prior art, the invention has the following advantages:

(1) the compound microorganisms are all the original bacteria screened from the intestinal tracts of the black soldier fly larvae, and the material environment after the fermentation of the original bacteria is more suitable for the growth and development of the black soldier fly larvae.

(2) The hermetia illucens and the microorganisms are used for jointly transforming the edible fungus residues, so that the edible fungus residues can be effectively transformed into useful biomass, and the utilization rate of the biomass is improved. The hermetia illucens has a strong immune function, is basically not affected by diseases and insect pests, and can inhibit the growth of pathogenic bacteria in the transformation process, so that the transformation process is harmless, and the treatment process period is short.

(3) The hermetia illucens feces left after the treatment can be used as high-quality organic fertilizer.

(4) After the treatment is completed, the hermetia illucens larvae can be used as an important source of high-quality protein, grease, chitosan and antibacterial peptide for resource maximum utilization, and the hermetia illucens larvae has considerable economic benefit, development value and good application prospect.

(5) Simple treatment process, low requirement on operating equipment, convenient management, less pollution in the production process and environmental friendliness.

Drawings

FIG. 1 is a photograph showing the Congo red staining of four cellulase-producing strains screened from intestinal tracts of hermetia illucens larvae.

Wherein: SX-1 represents Klebsiella pneumoniae Cellulase:001CCTCC NO: M2019615; CMC2 represents Pseudocitrobacter anthracis cellulolase: 002CCTCC NO: M2019887; CMC4 represents Pseudomonas faecalis Cellulase 003CCTCC NO: M2019888; CMC-3 represents Geotrichum galactococcus (Galactomyces geotrichum) Cellulase:004CCTCC NO: M2019889.

FIG. 2 Effect of rearing time on larval body fresh weight.

Detailed Description

The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for the purpose of illustration and should not be taken as a limitation of the invention.

The reagents, methods and apparatus employed in the present invention are all conventional in the art, unless otherwise indicated. In the examples: the edible fungus residues are sourced from Hua Green biotechnology limited company in Jiangsu province and are solid waste left after the cultivation of edible fungus flammulina velutipes, and the adjusting water content before the cultivation is 70 percent; the concentrated feed is Yilong 552 piglet feed and is purchased from a Sanhui feed raw material sales shop in Zaozhuang city of Shandong province; the commercial leaven is a beneficial biological feed leaven (which is compounded by various beneficial microbial floras such as lactobacillus, saccharomycetes, bacillus, actinomycetes and the like and metabolites thereof) and is purchased from a beneficial headquarter sales shop of Zhengzhou city in Henan province; the streptomyces termitomyces strain is purchased from Ningbo Ming boat Biotechnology Co., Ltd, and the strain number is BMZ 028768; the experiment was carried out in a constant temperature and humidity incubator, the temperature was controlled at 30 ℃ and the relative humidity was 70%.

Screening functional strains:

and (3) screening the strains in the intestinal tracts of the hermetia illucens larvae by using a selective culture medium, namely a carboxymethyl cellulose Congo red culture medium.

The concentration of each component in the carboxymethyl cellulose Congo red culture medium is 10g/L of sodium carboxymethyl cellulose, 10g/L of peptone, 4g/L of ammonium sulfate, 2g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate heptahydrate, 15g/L of agar powder, and the pH value is 7.0-7.2; the components in the culture medium are uniformly stirred and then sterilized for 20min at 121 ℃.

(1) Putting the hermetia illucens larvae subjected to body surface sterilization into 0.9% physiological saline, removing heads and tails, pulling out intestinal tracts of the hermetia illucens larvae, putting the intestinal tracts into a 1.5mL centrifugal tube filled with 0.9% physiological saline in advance, mashing the mixture into homogenate, and separating supernatant bacterial suspension containing intestinal microorganisms by a differential centrifugation method;

(2) diluting the supernatant with 0.9% physiological saline to 10%^-1～10^-6And six gradients, respectively coating 200 mu L of the six gradients on a carboxymethyl cellulose Congo red plate, culturing for 24 hours at 37 ℃, selecting a bacterial colony with a transparent ring, carrying out streaking separation culture on a beef extract peptone plate, and identifying the separated single bacterial strain as the cellulase-producing bacterial strain on the carboxymethyl cellulose Congo red plate.

(3) Screening out 8 strains producing cellulase, and respectively measuring the diameter of a hydrolysis transparent ring on a carboxymethyl cellulose Congo red flat plate and the diameter of a bacterial colony, wherein the ratio of the diameters is a DR value; and determining the cellulase activity of each strain by adopting a 3, 5-dinitrosalicylic acid (DNS) method; finally, 4 strains with the maximum enzyme activity are screened, and the result is shown in table 1, wherein the larger the DR value is, the stronger the cellulose degradation capability of the strain is, and the larger the corresponding enzyme activity is. The stained image is shown in FIG. 1. Wherein: SX-1 represents Klebsiella pneumoniae Cellulase:001CCTCC NO: m2019615; CMC2 represents the nucleic acid sequence of Pseudocitrobacter anthracis Pseudocitrobacter anthracropi Cellulase:002CCTCC NO: m2019887; CMC4 represents Pseudomonas faecalis Cellulase:003CCTCC NO: m2019888; CMC-3 represents a Geotrichum galactococcus (Galactomyces geotrichum) Cellulase:004CCTCC NO: m2019889.

Definition of cellulase activity units: the amount of enzyme required to react with sodium carboxymethylcellulose (CMC-Na) at pH6.0 per minute to produce 1. mu. mol of reducing sugar is U/mL.

TABLE 1 degradation circle and enzyme activity of cellulolytic bacteria Congo red staining

Example 1:

a method for converting edible fungus dregs by using hermetia illucens combined with a compound microbial preparation comprises the following steps:

(1) mixing CCTCC NO: m2019615 is subjected to shake amplification culture with nutrient broth culture medium (beef extract 3g/L, peptone 10g/L, sodium chloride 5 g/L; pH of nutrient broth culture medium 7.0 + -0.2) at 150r/min for 24h at 37 deg.C;

(2) sterilizing the concentrated feed at 121 deg.C for 15 min;

(3) inoculating 50% (mL/g) of concentrate into concentrated feed to obtain 10⁶Uniformly mixing the bacterial liquid with the sterilized concentrated feed, placing the mixture into a feed box, placing the unhatched eggs on a gauze above the feed box, incubating at 28 ℃ for 4 days, dropping the unhatched eggs into the concentrated feed after the eggs are incubated, and continuously feeding for 5 days under the conditions that the temperature is 28 ℃, the relative humidity is 70%, and the water content in the mixture is 70%, so that the bacterial strains are fixedly planted in intestinal tracts of hermetia illucens larvae;

(4) inoculating 5-day-old hermetia illucens larvae in the step (3) into the edible fungus dregs with the adjusted water content (the water content is 70 wt%), inoculating 80g of materials (dry weight) to every 1.2g of insects, and uniformly scattering the hermetia illucens larvae on the surfaces of the materials;

(5) the experiment is carried out in a constant temperature and humidity incubator, the temperature is controlled to be 30 ℃, and the relative humidity is 70 percent, and the cultivation is carried out for 12 days;

(6) after the conversion is finished, separating the materials and the larvae, drying, weighing and calculating conversion parameters.

Examples 2 to 4:

the same as in example 1, except that the selection of the species was different, the selection of the specific species is shown in Table 2.

Example 5:

(1) 2 kinds of bacteria separated from the intestinal tracts of hermetia illucens, Klebsiella pneumoniae CCTCC NO: m2019615 and Pseudocitrobacter anthracis CCTCC NO: m2019887 is subjected to shake-bed expansion culture with nutrient broth culture medium (beef extract 3g/L, peptone 10g/L, sodium chloride 5 g/L; pH of nutrient broth culture medium 7.0 + -0.2), and cultured at 150r/min at 37 deg.C for 24 hr to make the bacteria concentration reach 10⁶CFU/mL；

(2) Sterilizing concentrated feed at 121 deg.C for 15 min;

(3) mixing 2 kinds of bacteria liquid according to a volume ratio of 1:1, inoculating 50% (mL/g) of total bacteria concentration to concentrated feed to be 10⁶Uniformly mixing the bacterial liquid with the sterilized concentrated feed and then placing the mixture into a feed box, placing the unhatched eggs on a gauze above the feed box, incubating the eggs at 28 ℃ for 4 days, dropping the eggs into the concentrated feed after the eggs are incubated, and continuously feeding the eggs for 5 days under the conditions that the temperature is 28 ℃, the relative humidity is 70%, and the water content in the mixture is 70%, so that the bacterial strains are fixedly planted in the intestinal tracts of the black soldier fly larvae;

(4) inoculating 5-day-old hermetia illucens larvae in the step (3) into edible fungus dregs (with the water content of 70 wt%), inoculating 80g of material (dry weight) to every 1.2g of larvae, and uniformly scattering the hermetia illucens larvae on the surface of the material;

(5) the experiment is carried out in a constant temperature and humidity incubator, the temperature is controlled to be 30 ℃, the relative humidity is 70 percent, and the cultivation is carried out for 12 days;

Examples 6 to 10:

the same as example 5, except that the selection of the species was different, the selection of the specific species is shown in Table 2.

Example 11:

(1) 3 kinds of bacteria separated from the intestinal tracts of hermetia illucens, Klebsiella pneumoniae CCTCC NO: m2019615, Pseudocitrobacter anthracis CCTCC NO: m2019887 and Pseudomonas putida CCTCC NO: m2019888 is subjected to shake-bed expansion culture with nutrient broth culture medium (beef extract 3g/L, peptone 10g/L, sodium chloride 5 g/L; pH of nutrient broth culture medium 7.0 + -0.2), and cultured at 150r/min at 37 deg.C for 24 hr to make the bacteria concentration reach 10⁶CFU/mL；

(2) Sterilizing the concentrated feed at 121 deg.C for 15 min;

(3) mixing 3 kinds of bacteria liquid according to the volume ratio of 1:1:1, inoculating 50% (mL/g) of total bacteria concentration to the concentrated feed to be 10⁶Uniformly mixing the bacterial liquid with the sterilized concentrated feed and then placing the mixture into a feed box, placing the unhatched eggs on a gauze above the feed box, incubating the eggs at 28 ℃ for 4 days, dropping the eggs into the concentrated feed after the eggs are incubated, and continuously feeding the eggs for 5 days under the conditions that the temperature is 28 ℃, the relative humidity is 70%, and the water content in the mixture is 70%, so that the bacterial strains are fixedly planted in the intestinal tracts of the black soldier fly larvae;

Examples 12 to 13:

the same as in example 11, except that the selection of the species was different, the selection of the specific species is shown in Table 2.

Example 14:

(1) 4 kinds of bacteria separated from the intestinal tracts of hermetia illucens, Klebsiella pneumoniae CCTCC NO: m2019615, Pseudomonas anthracis CCTCC NO: m2019887, Pseudomonas putida CCTCC NO: m2019888 and Geotrichum galactose yeast CCTCC NO: m2019889 is subjected to shake-bed expansion culture with nutrient broth culture medium (beef extract 3g/L, peptone 10g/L, sodium chloride 5 g/L; pH of nutrient broth culture medium 7.0 + -0.2), and cultured at 150r/min at 37 deg.C for 24 hr to make the bacteria concentration reach 10⁶CFU/mL；

(2) Sterilizing concentrated feed at 121 deg.C for 15 min;

(3) mixing 4 kinds of bacteria liquid according to the volume ratio of 1:1:1:1, inoculating 50% (mL/g) of total bacteria concentration to concentrated feed to be 10⁶Uniformly mixing the bacterial liquid with the sterilized concentrated feed and then placing the mixture into a feed box, placing the unhatched eggs on a gauze above the feed box, incubating the eggs at 28 ℃ for 4 days, dropping the eggs into the concentrated feed after the eggs are incubated, and continuously feeding the eggs for 5 days under the conditions that the temperature is 28 ℃, the relative humidity is 70%, and the water content in the mixture is 70%, so that the bacterial strains are fixedly planted in the intestinal tracts of the black soldier fly larvae;

(4) inoculating 5-day-old hermetia illucens larvae in the step (3) into edible fungus residues (with the water content of 70 wt%), inoculating 80g of material (dry weight) to every 1.2g of larvae, and uniformly scattering the hermetia illucens larvae on the surface of the material;

(5) the experiment is carried out in a constant temperature and humidity incubator, the temperature is controlled to be 30 ℃, the relative humidity is 70%, and the cultivation is carried out for 12 days;

Comparative example 1:

the method for transforming the black soldier fly larvae planted by the commercial leaven into the edible fungus residues comprises the following steps:

(1) sterilizing the concentrated feed at 121 deg.C for 15 min.

(2) Adding 50% (mL/g) of concentrate into sterilized concentrated feed to obtain 10⁶Uniformly mixing CFU/mL commercial leaven, adjusting the water content to 70%, uniformly paving the mixture in a feed box, placing unhatched eggs on a gauze above the feed box, incubating the eggs at 28 ℃ for 4 days, dropping the hatched eggs into concentrated feed after the eggs are incubated, and continuously feeding the eggs for 5 days under the conditions of 28 ℃, relative humidity of 70% and water content of 70% in the mixture so as to fixedly plant the strains in intestinal tracts of black soldier fly larvae;

(3) inoculating 5-day-old hermetia illucens larvae in the step (3) into the edible fungus dregs with the adjusted water content (the water content is 70 wt%), inoculating 80g of materials (dry weight) to every 1.2g of insects, and uniformly scattering the hermetia illucens larvae on the surfaces of the materials;

(4) the experiment is carried out in a constant temperature and humidity incubator, the temperature is controlled to be 30 ℃, the relative humidity is 70 percent, and the cultivation is carried out for 12 days;

(5) after the conversion is finished, separating the materials and the larvae, drying, weighing and calculating conversion parameters.

Comparative example 2:

as the termite intestinal flora is well known to have strong cellulose degradation capability, but the termite is a harmful insect, the termite intestinal flora is selected to be planted in the intestinal tract of the black water larva, and whether the termite intestinal flora can produce good effect is observed to be used as a comparison experiment.

The transformation of the edible fungus dregs into the hermetia illucens larvae after the hermetia illucens larvae are fixedly planted by the streptomyces termitomyces, and the transformation method comprises the following steps:

(1) sterilizing the concentrated feed at 121 deg.C for 15 min.

(2) Adding 50% (mL/g) of concentrate to sterilized concentrated feed to obtain 10⁶Uniformly mixing the bacterial liquid of the streptomyces termitomyces, uniformly spreading the bacterial liquid in a feed box after adjusting the water content to 70%, placing the unhatched eggs on a gauze above the feed box, incubating for 4 days at 28 ℃, dropping the eggs into concentrated feed after the eggs are incubated, continuously feeding for 5 days under the conditions of 28 ℃, relative humidity of 70% and water content of 70% in the mixture, and fixedly planting the bacterial strains in the intestinal tracts of the black soldier fly larvae;

(3) inoculating 5-day-old hermetia illucens larvae in the step (2) into the edible fungus dregs with the adjusted water content (the water content is 70 wt%), inoculating 80g of material (dry weight) to every 1.2g of insects, and uniformly scattering the hermetia illucens larvae on the surface of the material;

Comparative example 3:

the method for transforming the hermetia illucens larvae which are not subjected to bacterial strain planting into the edible fungus dregs comprises the following steps:

(1) sterilizing the concentrated feed at 121 deg.C for 15 min.

(2) Adding sterilized concentrated feed and sterile water to adjust water content to 70%, uniformly spreading in a feed box, placing unhatched eggs on a gauze above the feed box, dropping into the concentrated feed after the eggs are hatched, and continuously feeding for 5 days under the conditions of 28 ℃ and relative humidity of 70%;

Comparative example 4:

the comparative example adopts a non-planting microorganism mode, but the microorganism firstly ferments the mushroom dregs and then converts the black soldier fly.

A method for converting edible fungus dregs by using hermetia illucens and a compound microbial preparation comprises the following steps:

(1) sterilizing the edible fungus residue and concentrated feed in autoclave at 121 deg.C for 15 min;

(2) inoculating 50% (mL/g) of the fungus with the fungus residue of the edible fungus to the fungus residue of the edible fungus with the fungus concentration of 10⁶Placing CFU/mL commercial starter bacteria solution in a feed box, fermenting at 37 deg.C and 70% relative humidity for 5 days;

(3) simultaneously, adding a proper amount of sterile water into the concentrated feed, uniformly mixing to ensure that the water content of the concentrated feed is about 70 percent, placing a frame with a gauze at the top end of the concentrated feed, placing hermetia illucens eggs on the gauze, hatching the hermetia illucens eggs, dropping the hermetia illucens eggs into the material, and feeding the hermetia illucens larvae to a 5-day-old stage;

(4) inoculating 5-day-old hermetia illucens larvae into the fermented mushroom dregs obtained in the step (2) with the adjusted water content (the water content is 70 wt%), inoculating 80g of materials (dry weight) to every 1.2g of insects, and uniformly scattering the hermetia illucens larvae on the surfaces of the materials;

Comparative example 5:

the same as in comparative example 4, except that the selection of the strain was different, CCTCC NO: m2019615 and CCTCC NO: m2019888 was mixed at a ratio of 1:1:1 to replace the commercial starter. The effect of non-colonizing means with the most preferred combination of strains according to the invention on hermetia illucens transformation residues was examined.

TABLE 2 Change in feed reduction, bioconversion and insect weight gain after feeding

Note: the calculation method of the biotransformation efficiency refers to the formula: biotransformation efficiency ═ W₂-W₁)/(M₀-M₁) X 100%, wherein W₂Total dry weight (g), W, of larvae after rearing₁Dry weight (g), M of 5-day-old larvae added before rearing₀Is the total dry weight (g) of the initial mushroom dreg feed, M₁The total dry weight (g) of the residual mushroom dreg feed after the feeding is finished.

Material reduction rate (M)₀-M₁)/M₀X 100%, wherein M₀Is the total dry weight (g) of the initial mushroom dreg feed, M₁The total dry weight (g) of the residual mushroom dreg feed after the feeding is finished.

The initial pellet feed of the above examples and comparative examples had a total dry weight of 80 g.

Compared with examples 1-14, comparative examples 1, 2 and 3 respectively adopt hermetia illucens larvae which are fixedly planted by a commercial starter microbial agent and a streptomyces termitomyces microbial agent and are not fixedly planted by a strain to be converted into edible fungus dregs. To ensure that the experimental data was valid, the comparative example was operated under the same ambient conditions as the other examples.

Compared with the comparative ratio 3, namely the hermetia illucens larvae which are not subjected to bacterial strain planting are used for converting the edible fungus residues, the conversion effect of the edible fungus residues of other groups is better, and the reduction rate of materials, the biological conversion rate of the larvae and the average weight of the larvae can be improved to a certain extent. However, the effect of the combination of different strains on the experimental results was different (see table 1). Compared with the comparative example 1, the transformation effect of the transformation group of the hermetia illucens larvae which are fixedly planted by the commercial leavening agent is inferior to that of the experimental group for screening the strains for permanent planting from the intestinal tracts of the hermetia illucens larvae. In addition, in the comparative example 2, the strain with high cellulase yield from the intestinal tract of the termite is adopted, the streptomyces termitomyces performs field planting on the hermetia illucens larvae, and then the fungus dregs are transformed, so that the transformation efficiency is not ideal. In comparative examples 4 and 5, the commercial leaven and the mixed microbial inoculum of the best effect in example 6 were used to ferment the mushroom dregs, and then black soldier fly larvae were used to transform the dregs, and the fermentation effect was found to be lower than that of the method using the strain for permanent planting. In general, the composite microbial preparation prepared from the bacterial strains screened from the intestinal tracts of the hermetia illucens larvae has a more obvious improvement on the conversion rate of edible fungus residues, and is better than the results of commercial starter experiments and better than the results of edible fungus residues which are not subjected to bacterial strain field planting and are subjected to field planting conversion by using exogenous bacteria. Wherein the Klebsiella pneumoniae CCTCC NO: m2019615 and Pseudomonas putida CCTCC M2019888 NO: after the field planting experiment is carried out according to the proportion of 1:1, the biotransformation rate can reach the highest.

In addition, changes in the fresh weight of the grenades of hermetia illucens larvae were recorded every other day during the course of each example, and the results are shown in table 3 and fig. 2. As can be seen from the trend of the change in the figure, the black soldier fly larvae grew better in all the examples than in the comparative example, and the growth cycle was shorter than in the comparative example. The growth cycle for example 6 was around 12 days, the remaining examples were after 15 days, and the comparative examples were all longer.

TABLE 3 fresh weight of worms varied with time mg/worm

The reasons for this may be: (1) intestinal tracts of the hermetia illucens larvae after the bacterial strains are planted can better decompose and utilize organic matters in the feed; (2) the streptomyces termitorum is from intestinal tracts of termites, and strains in a commercial starter are not hermetia illucens intestinal strains, so that the streptomyces termitorum is not suitable for growing in the hermetia illucens intestinal tracts and is not beneficial to the transformation of hermetia illucens bodies to materials; (3) the bacterial strains of the compound microbial preparation are all from the hermetia illucens, and the hermetia illucens have stronger adaptability to the hermetia illucens, so that the hermetia illucens are more beneficial to the growth of the hermetia illucens and the conversion of feed.

The invention provides a compound microorganism and an idea and a method for applying the compound microorganism in the transformation of edible fungus dregs by combining hermetia illucens, and particularly provides a plurality of methods and ways for realizing the technical scheme, wherein the methods and the ways are only preferred embodiments of the invention. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be construed as the protection scope of the present invention. All the components not specified in the present embodiment can be realized by the prior art.

Sequence listing

<110> Nanjing university of industry

<120> compound microorganism and application thereof in combination with black soldier fly transformation of edible fungus residues

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1439

<212> DNA

<213> Klebsiella pneumoniae (Klebsiella pneumoniae)

<400> 1

ctgtgcggca gctacacatg cagtcgagcg gtagcacaga gagcttgctc tcgggtgacg 60

agcggcggac gggtgagtaa tgtctgggaa actgcctgat ggagggggat aactactgga 120

aacggtagct aataccgcat aacgtcgcaa gaccaaagtg ggggaccttc gggcctcatg 180

ccatcagatg tgcccagatg ggattagcta gtaggtgggg taacggctca cctaggcgac 240

gatccctagc tggtctgaga ggatgaccag ccacactgga actgagacac ggtccagact 300

cctacgggag gcagcagtgg ggaatattgc acaatgggcg caagcctgat gcagccatgc 360

cgcgtgtgtg aagaaggcct tcgggttgta aagcactttc agcggggagg aaggcgacaa 420

ggttaataac cttggcgatt gacgttaccc gcagaagaag caccggctaa ctccgtgcca 480

gcagccgcgg taatacggag ggtgcaagcg ttaatcggaa ttactgggcg taaagcgcac 540

gcaggcggtc tgtcaagtcg gatgtgaaat ccccgggctc aacctgggaa ctgcattcga 600

aactggcagg ctagagtctt gtagaggggg gtagaattcc aggtgtagcg gtgaaatgcg 660

tagagatctg gaggaatacc ggtggcgaag gcggccccct ggacaaagac tgacgctcag 720

gtgcgaaagc gtggggagca aacaggatta gataccctgg tagtccacgc cgtaacgatg 780

tcgatttgga ggttgtgccc ttgaggcgtg gcttccggag ctaacgcgtt aaatcgaccg 840

cctggggagt acggccgcaa ggttaaaact caaatgaatt gacgggggcc cgcacaagcg 900

gtggagcatg tggtttaatt cgatgcaacg cgaagaacct tacctggtct tgacatccac 960

agaacttacc agagatggat tggtgccttc gggaactgtg agacaggtgc tgcatggctg 1020

tcgtcagctc gtgttgtgaa atgttgggtt aagtcccgca acgagcgcaa cccttatcct 1080

ttgttgccag cggttcggcc gggaactcaa aggagactgc cagtgataaa ctggaggaag 1140

gtggggatga cgtcaagtca tcatggccct tacgaccagg gctacacacg tgctacaatg 1200

gcatatacaa agagaagcga cctcgcgaga gcaagcggac ctcataaagt atgtcgtagt 1260

ccggattgga gtctgcaact cgactccatg aagtcggaat cgctagtaat cgtagatcag 1320

aatgctacgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac catgggagtg 1380

ggttgcaaaa gaagtaggta gcttaacctt cgggagggcg ctaccacttt gtatactgg 1439

<210> 2

<211> 1447

<212> DNA

<213> Pseudocitrobacter anthracis (Pseudocerobacter anthracropi)

<400> 2

ctgttgcggc aggcctacac atgcaagtcg aacggtaaca gaaagcagct tgctgcattt 60

gctgacgagt ggcggacggg tgagtaatgt ctgggaaact gcctgatgga gggggataac 120

tactggaaac ggtagctaat accgcataac gtcgcaagac caaagagggg gaccttcggg 180

cctcttgcca tcggatgtgc ccagatggga ttagctagta ggtggggtaa tggctcacct 240

aggcgacgat ccctagctgg tctgagagga tgaccagcca cactggaact gagacacggt 300

ccagactcct acgggaggca gcagtgggga atattgcaca atgggcgcaa gcctgatgca 360

gccatgccgc gtgtatgaag aaggccttcg ggttgtaaag tactttcagc gaggaggaag 420

gtgttgtggt taataaccac agcaattgac gttactcgca gaagaagcac cggctaactc 480

cgtgccagca gccgcggtaa tacggagggt gcaagcgtta atcggaatta ctgggcgtaa 540

agcgcacgca ggcggtttgt taagtcagat gtgaaatccc cgggctcaac ctgggaactg 600

catttgaaac tggcaagctt gagtctcgta gaggggggta gaattccagg tgtagcggtg 660

aaatgcgtag agatctggag gaataccggt ggcgaaggcg gccccctgga cgaagactga 720

cgctcaggtg cgaaagcgtg gggagcaaac aggattagat accctggtag tccacgctgt 780

aaacgatgtc gacttggagg ttgtgccctt gaggcgtggc ttccggagct aacgcgttaa 840

gtcgaccgcc tggggagtac ggccgcaagg ttaaaactca aatgaattga cgggggcccg 900

cacaagcggt ggagcatgtg gtttaattcg atgcaacgcg aagaacctta cctggtcttg 960

acatccacag aacttagcag agatgctttg gtgccttcgg gaactgtgag acaggtgctg 1020

catggctgtc gtcagctcgt gttgtgaaat gttgggttaa gtcccgcaac gagcgcaacc 1080

cttatccttt gttgccagcg gttaggccgg gaactcaaag gagactgcca gtgataaact 1140

ggaggaaggt ggggatgacg tcaagtcatc atggccctta cgaccagggc tacacacgtg 1200

ctacaatggc atatacaaag agaagcgacc tcgcgagagc aagcggacct cataaagtat 1260

gtcgtagtcc ggattggagt ctgcaactcg actccatgaa gtcggaatcg ctagtaatcg 1320

tggatcagaa tgctacggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca 1380

tgggagtggg ttgcaaaaga agtaggtagc ttaaccttcg ggagggcgct taccactttg 1440

atcgtgc 1447

<210> 3

<211> 1446

<212> DNA

<213> Pseudocitrobacter faecalis (Pseudomonas faecalis)

<400> 3

cgtgcggcag gcctacacat gcaagtcgaa cggtaacaga aagcagcttg ctgcattcgc 60

tgacgagtgg cggacgggtg agtaatgtct gggaaactgc ctgatggagg gggataacta 120

ctggaaacgg tagctaatac cgcataacgt cgcaagacca aagaggggga ccttcgggcc 180

tcttgccatc ggatgtgccc agatgggatt agctagtagg tggggtaatg gctcacctag 240

gcgacgatcc ctagctggtc tgagaggatg accagccaca ctggaactga gacacggtcc 300

agactcctac gggaggcagc agtggggaat attgcacaat gggcgcaagc ctgatgcagc 360

catgccgcgt gtatgaagaa ggccttcggg ttgtaaagta ctttcagcga ggaggaaggt 420

gttgtggtta ataaccacag caattgacgt tactcgcaga agaagcaccg gctaactccg 480

tgccagcagc cgcggtaata cggagggtgc aagcgttaat cggaattact gggcgtaaag 540

cgcacgcagg cggtttgtta agtcagatgt gaaatccccg ggctcaacct gggaactgca 600

tttgaaactg gcaagcttga gtctcgtaga ggggggtaga attccaggtg tagcggtgaa 660

atgcgtagag atctggagga ataccggtgg cgaaggcggc cccctggacg aagactgacg 720

ctcaggtgcg aaagcgtggg gagcaaacag gattagatac cctggtagtc cacgctgtaa 780

acgatgtcga cttggaggtt gtgcccttga ggcgtggctt ccggagctaa cgcgttaagt 840

cgaccgcctg gggagtacgg ccgcaaggtt aaaactcaaa tgaattgacg ggggcccgca 900

caagcggtgg agcatgtggt ttaattcgat gcaacgcgaa gaaccttacc tggtcttgac 960

atccacagaa cttagcagag atgctttggt gccttcggga actgtgagac aggtgctgca 1020

tggctgtcgt cagctcgtgt tgtgaaatgt tgggttaagt cccgcaacga gcgcaaccct 1080

tatcctttgt tgccagcggt taggccggga actcaaagga gactgccagt gataaactgg 1140

aggaaggtgg ggatgacgtc aagtcatcat ggcccttacg accagggcta cacacgtgct 1200

acaatggcat atacaaagag aagcgacctc gcgagagcaa gcggacctca taaagtatgt 1260

cgtagtccgg attggagtct gcaactcgac tccatgaagt cggaatcgct agtaatcgtg 1320

gatcagaatg ctacggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccatg 1380

ggagtgggtt gcaaaagaag taggtagctt aaccttcggg agggcgctta ccactttgat 1440

cagtgc 1446

<210> 4

<211> 1346

<212> DNA

<213> Geotrichum galactococcus (Galactomyces geotrichum)

<400> 4

cgggcatggt ctaaggtata agcattatac agtgaaactg cgaatggctc attaaatcag 60

ttatcgttta tttgatatta cattactact tggataaccg tggtaattct agagctaata 120

catgctaaaa cggccgggtt tccggctggt atttattaga taaaaaacca atgccttcgg 180

gctctatggt gaatcataat aacttgtcga atcgcatggc cttgtgctgg cgatggttca 240

ttcaaatttc tgccctatca actttcgatg gtaggataga ggcctaccat ggttttaacg 300

ggtaacgggg aatcagggtt cgattccgga gagggagcct gagaaacggc taccacatcc 360

aaggaaggca gcaggcgcgc aaattaccca atcctgacac agggaggtag tgacaataaa 420

taacgatacg gggcctatta ggtctcgtaa ttggaatgag aacaatttaa ataccttaac 480

gaggaacaat tagagggcaa gtctggtgcc agcagccgcg gtaattccag ctctgatagt 540

atatattaaa gttgttgcag ttaaaaaagc tcgtagttga aacttgggtg cgtaggggcg 600

gtctctttta gagtactacc ctgaaacatc tttctttggt gtaaactctt tattcactta 660

aggagtgtaa accaaacatt tactttgaaa aaattagagt gttcaaagca ggcctttgct 720

cgaatatatt agcatggaat aatagaatag gacgtatggt tctattttgt tggtttctag 780

gaccgtcgta atgattaata gggacggtcg ggggcatcag tattcagttg tcagaggtga 840

aattcttgga tttactgaag actaactact gcgaaagcat ttgccaagga cgttttcatt 900

aatcaagaac gaaagttagg ggatcgaaga cgatcagata ccgtcgtagt cttaaccgta 960

aactatgccg actagggatc ggagggcgtt ataataacct ctccggcacc ttacgagaaa 1020

tcaaagtttt tgggttctgg ggggagtatg gttgcaaggc tgaaacttaa aggaattgac 1080

ggaagggcac caccaggagt ggagcctgcg gcttaatttg actcaacacg gggaaactca 1140

ccaggtccag acacaataag gattgacaga ttgagagctc tttcatgatt ttgtgggtgg 1200

tggtgcatgg ccgttcttag ttggtggagt gatttgtctg cttaattgcg ataacgaacg 1260

agaccttaac ctgctaaata gctgtaacaa tagattattg ttttgacagc ttcttaggag 1320

ggactatcga tttcagtccg aggatt 1346

Claims

1. A complex microorganism selected from one of the following combinations:

the combination is as follows: CCTCC NO: m2019615 and CCTCC NO: m2019887;

combining two: CCTCC NO: m2019615 and CCTCC NO: m2019888;

combining three components: CCTCC NO: m2019887 and CCTCC NO: m2019888;

and (4) combining: CCTCC NO: m2019887 and CCTCC NO: m2019889;

and (5) combining: CCTCC NO: m2019615, CCTCC NO: m2019888, and CCTCC NO: m2019889;

the CCTCC NO: m2019615 represents Klebsiella pneumoniae (K.pneumoniae)Klebsiella pneumoniae) The strain number is Cellulase:001, and the preservation number is CCTCC NO: m2019615, which was deposited with the China center for type culture Collection on 7/8.2019;

the CCTCC NO: m2019887 is Pseudocitrobacter anthracis: (A)Pseudocitrobacter anthropi) The strain number is Cellulase:002, the preservation number is CCTCC NO: m2019887, which was deposited with the China center for type culture Collection on 1/11 of 2019;

the CCTCC NO: m2019888 is Pseudocitrobacter faecalis: (Pseudocitrobacter faecalis) The strain number is Cellulase:003, the preservation number is CCTCC NO: M2019888, and the strain is preserved in the China center for type culture Collection in 2019, 11 and 1 months;

the CCTCC NO: m2019889 is Geotrichum galactococcus (C.) (Galactomyces geotrichum) The strain number is Cellulase:004, the preservation number is CCTCC NO: m2019889, deposited at the China center for type culture Collection on 1/11 of 2019.

2. A complex microorganism bacterium agent, characterized in that the complex microorganism of claim 1 is inoculated into a beef extract peptone medium to culture the resulting bacterial suspension.

3. The compound microbial inoculant according to claim 2, wherein the concentration of each component of the beef extract peptone medium is 3g/L of beef extract, 10g/L of peptone, 5g/L of sodium chloride, and the pH value is 6.8-7.4; the culture is carried out under the conditions of 100-200 r/min and at the temperature of 35-40 ℃ for 20-30 h.

4. The use of the composite microorganism of claim 1 in combination with hermetia illucens to convert edible fungus dregs into biomass.

5. The use of claim 4, wherein the composite microorganisms are fed and then colonized in the intestinal tracts of hermetia illucens to efficiently convert hermetia illucens to biomass.

6. Use according to claim 5, characterized in that it comprises the following steps:

7. The use of claim 6, wherein in the step (2), the water content of the edible fungus residue is 60-80 wt%.