An artificial synthetic bacterial community for increasing the biomass of black soldier fly larvae cultured in pigeon manure and its application

Abstract

The present invention discloses an artificial synthetic bacterial community for increasing the biomass of black soldier fly larvae and its application. The Bacillus subtilis ZKY03 strain, the Bacillus subtilis ZKY04 strain, the Bacillus subtilis ZKY05 strain, and the Bacillus subtilis ZKY06 strain within the synthetic bacterial community have been deposited in GDMCC on September 7, 2022, and the ZKY03 deposit number is GDMCC NO: 62763, the ZKY04 deposit number is GDMCC NO: 62764, the ZKY05 deposit number is GDMCC NO: 62765, and the ZKY06 deposit number is GDMCC NO: 62766. This series of strains and the synthetic bacterial community constructed by them can increase the biomass of black soldier fly larvae. The addition of a single bacterium can increase the biomass by 9%-22% compared with the blank treatment, and the addition of a synthetic bacterial community constructed by 4 strains can increase the biomass by 33% compared with the blank treatment.

Description

Technical Field

[0001] The invention belongs to the field of probiotic preparations and relates to an artificially synthesized bacterial community for increasing the biomass of black soldier fly larvae and an application thereof. Background Art

[0002] The lack of protein sources has always been an important factor restricting the development of the breeding industry. The black soldier fly (Hermetia illucens) is a scavenging insect with a wide range of diets. It can be used to treat organic waste such as kitchen waste, livestock and poultry manure, etc., and produce fresh insects, insect protein powder or oil. The products are diverse and have a wide range of applications. They can be used as animal feed, pet food and pharmaceutical cosmetics. The waste after black soldier fly treatment can also be used in agricultural production as organic fertilizer. The market size is huge, and it is estimated that the global black soldier fly market will reach US$2.57 billion in 2030.

[0003] The annual output of livestock and poultry manure in my country is about 3.8 billion tons, and the pressure for harmless treatment is enormous [Liu Tianxu, Chen Zhaohui, Liu Jijun. Research Progress in Livestock and Poultry Manure Treatment [J]. Heilongjiang Animal Husbandry and Veterinary Medicine, 2022(17):26-30.DOI:10.13881 / j.cnki.hljxmsy.2022.01.0185.] According to incomplete statistics, my country produces 450 million squabs each year, and pigeon farming in Guangdong Province accounts for about half of the country's total farming volume, with an annual pigeon manure output of up to 1.7 million tons. At present, the utilization of livestock and poultry manure is mainly traditional fertilizer and energy utilization, but these methods have problems such as large capital investment, backward facilities, and serious secondary pollution. In recent years, insect transformation, oxidation ponds and other methods are green and efficient, economical and efficient, and the use of these methods to treat livestock and poultry manure has gradually become a mainstream trend [Peng Siyi, Pu Shihua, Jian Yue, Liu Zuohua, Huang Ping, Zeng Yaqiong. Research Progress on Resource Utilization Technology of Manure in Large-scale Farms [J / OL]. China Animal Husbandry Magazine: 1-12 [2022-09-23]. DOI: 10.19556 / j.0258-7033.20211125-05.]. Using black soldier flies to convert livestock and poultry manure into insect protein is an important way to utilize manure in a high-quality manner. In order to obtain more insect protein production, many scientific researchers have conducted research on the breeding conditions of black soldier flies. Using host-related microorganisms to promote the growth and development of black soldier fly larvae is one of the hot spots in the future research and utilization of black soldier flies.In 2011, Professor Yu Guohui, one of the inventors of this patent, first used the black soldier fly intestinal symbiotic Bacillus subtilis to add to chicken manure and found that the addition of a single Bacillus subtilis could significantly increase the weight of prepupae [Yu G, Cheng P, Chen Y. Inoculating poultry manure with companion bacteria influences growth and development of black soldier fly (Diptera: Stratiomyidae) larvae. Environ Entomol, 2011, 40: 30-35.]; in 2020, Kooienga found that the 11-day-old larvae fed with the Gainesville standard diet were supplemented with the Arthrobacter group. After 10 days of culture, the final insect weight of the 21-day-old mature larvae could be increased by about 6.7%-11% compared with the blank group [Kooienga EM, Baugher C, Currin M. Effects of Bacterial Supplementation on Black Soldier Fly Growth and Development at Benchtop and Industrial Scale. Front Microbiol, 2020, 11: 587979]; In 2020, Mazza used microorganisms isolated from black soldier fly eggs and intestines to co-culture with black soldier flies in chicken manure and found that host-related microorganisms can promote the growth and development of black soldier flies to varying degrees. The synthetic bacterial community constructed using the four best single bacteria (Kocuria marina, Lysinibacillus boronitolerans, Proteus mirabilis and Bacillus subtilis) was added to the 5-day-old larvae and chicken manure culture system. After 14 days of culture, the dry weight of the 19-day-old mature larvae increased by 28.6% compared with the blank [Mazza L, Xiao XP, Rehman K U. Management of chicken manure using black soldier fly (Diptera: Stratiomyidae) larvae assisted by companion bacteria. WASTE MANAGEMENT, 2020, 102: 312-318.].

[0004] The weight of black soldier fly larvae increases with age, but in the process of breeding black soldier flies, the weight gain of the larvae gradually slows down around the fourth instar (10-15 days old) [Ma Jiakang, Guo Haoran, Wang Lixin. Effects of fresh duck manure on the growth and development of black soldier fly larvae and feces conversion rate [J]. Journal of Anhui University of Science and Technology, 2016, 30(01): 12-18.], and the crude protein content and conversion efficiency continue to decrease [Wang Cong, Ye Xiaomei, Xi Yonglan, Du Jing, Kong Xiangping, Wang Li, Han Ting, Zhu Fei, Zhang Yingpeng. Study on the changes in nutrient composition and digestive enzyme activity in the body of black soldier fly larvae during the treatment of food waste [J / OL]. Journal of Environmental Entomology: 1-12 [2022-10-27].], harvesting at the fourth instar larvae can achieve high conversion efficiency while obtaining a large biomass and insect body protein, which is helpful for the feed industry to further utilize insect crude protein. At present, the research on using host-associated microorganisms to promote the growth and development of black soldier flies is mainly aimed at the mature larvae (17-22 days old) of black soldier flies. However, the low protein content and high fat content reduce the quality of the mature larvae as feed raw materials. The synthetic bacterial community provided by this patent can significantly increase the biomass accumulation and protein content of fourth-instar (12-day-old) black soldier fly larvae, thereby increasing the biomass of artificially cultivated black soldier flies while increasing the feed value of the cultivated larvae, and has excellent prospects for large-scale industrial application. Summary of the invention

[0005] The purpose of the present invention is to provide an artificially synthesized bacterial community for increasing the biomass of black soldier fly larvae.

[0006] An artificial synthetic bacterial community for increasing the biomass of 12-day-old black soldier fly larvae, consisting of Bacillus subtilis ZKY03, Bacillus subtilis ZKY04, Bacillus subtilis ZKY05, and Bacillus subtilis ZKY06. The above four strains have been deposited in Guangdong Provincial Microbiological Culture Collection Center on September 7, 2022, with the deposit number of ZKY03 being GDMCC NO: 62763, the deposit number of ZKY04 being GDMCC NO: 62764, the deposit number of ZKY05 being GDMCC NO: 62765, and the deposit number of ZKY06 being GDMCC NO: 62766.

[0007] The artificial synthetic bacterial community of the present invention is used to increase the biomass of 12-day-old black soldier fly larvae and the total insect protein yield of artificially cultured black soldier fly larvae, preferably used to increase the biomass of 12-day-old black soldier fly larvae cultured with pigeon manure.

[0008] The artificial synthetic bacterial flora of the present invention is used in preparing a product for increasing the biomass of 12-day-old black soldier fly larvae cultured artificially and the total insect protein yield, preferably in preparing a product for increasing the biomass of 12-day-old black soldier fly larvae cultured in pigeon manure.

[0009] A product for increasing the biomass and total protein yield of black soldier fly larvae is composed of a dilution of the artificial synthetic bacterial community fermentation liquid.

[0010] The product of the present invention is used in increasing the biomass of black soldier fly larvae and the total insect protein yield, and is preferably used in increasing the biomass of black soldier fly larvae cultured with pigeon manure and the total insect protein yield.

[0011] A method for breeding insects with higher protein content, wherein the product of the invention is added into pigeon droppings for feeding 4-day-old black soldier fly larvae and culturing them to 12-day-old.

[0012] As a preferred embodiment of the present invention, the culture condition is culturing at 28° C. for 8 days.

[0013] Beneficial effects:

[0014] The present invention provides an artificially synthesized bacterial community composed of a specific strain. After diluting the fermented liquid of the bacterial community, pigeon manure is added to feed black soldier fly larvae, which can significantly increase the biomass and insect protein content of 12-day-old black soldier fly larvae. Since black soldier flies are artificially bred using livestock and poultry feces or other organic wastes, and the black soldier fly larvae are mainly used for their insect protein when they are applied to feed for livestock and poultry and aquaculture, the method of the present invention can obtain a large number of black soldier fly larvae with higher insect protein content than other days, and greatly increase the total protein harvest of the larvae harvested at this age. Therefore, the artificially synthesized bacterial community of the present invention has excellent large-scale industrial application prospects in improving the yield and quality of artificially bred black soldier flies using organic wastes, and using them as feed raw materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Phylogenetic tree of strains ZKY03, ZKY04, ZKY05, and ZKY06 and related strains based on 16S rDNA gene sequences;

[0016] Figure 2 Phylogenetic tree of strains ZKY03, ZKY04, ZKY05, and ZKY06 and related strains based on gyrB gene sequences;

[0017] Figure 3 Results of kinship identification of ZKY03, ZKY04, ZKY05 and ZKY06

[0018] A: Phylogenetic identification of ZKY03 and ZKY04; B: Phylogenetic identification of ZKY03 and ZKY05;

[0019] C: Phylogenetic identification of ZKY03 and ZKY06; D: Phylogenetic identification of ZKY04 and ZKY05;

[0020] E: Phylogenetic identification of ZKY04 and ZKY06; F: Phylogenetic identification of ZKY05 and ZKY06;

[0021] Figure 4 ZKY03, ZKY04, ZKY05, ZKY06 single bacteria and synthetic bacteria colony insect weight gain

[0022] Figure 5 Residue weight of single bacteria and synthetic bacteria of ZKY03, ZKY04, ZKY05, ZKY06

[0023] Figure 6 Bioconversion rate of single bacteria and synthetic bacterial groups of ZKY03, ZKY04, ZKY05 and ZKY06

[0024] Figure 7 Effect of synthetic bacterial flora and deletion of strains on insect weight gain

[0025] Figure 8 Weight of residues after synthetic bacterial flora and strain deletion

[0026] Fig. 9 Bioconversion rate of synthetic bacterial flora and deleted strains

[0027] Fig.10 Total protein of single bacteria and synthetic bacteria

[0028] Biomaterial Deposit Certificate

[0029] Bacillus subtilis str. ZKY03, deposited in Guangdong Microbiological Culture Collection Center (GDMCC), deposit date is September 2, 2022, deposit location is 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, deposit number is GDMCCNO: 62763;

[0030] Bacillus subtilis str. ZKY04, deposited in Guangdong Microbiological Culture Collection Center (GDMCC), deposit date is September 2, 2022, deposit location is 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, deposit number is GDMCCNO: 62764;

[0031] Bacillus subtilis str.ZKY05, deposited in Guangdong Microbiological Culture Collection Center (GDMCC), deposit date is September 2, 2022, deposit location is 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, deposit number is GDMCCNO:62765;

[0032] Bacillus subtilis str.ZKY06, deposited in Guangdong Microbiological Culture Collection Center (GDMCC), the deposit date is September 2, 2022, the deposit location is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCCNO:62766. DETAILED DESCRIPTION

[0033] Example 1: Isolation, purification and partial physicochemical properties of ZKY03, ZKY04, ZKY05 and ZKY06 (I) Isolation, purification and storage of ZKY03, ZKY04, ZKY05 and ZKY06

[0034] Use culture medium: LB (g / L): tryptone 10; yeast extract 5; sodium chloride 10; pH 7.0-7.2. Solid culture medium: add 2% agar to liquid culture medium. Sterilize at 115℃ for 20min;

[0035] 2216E (g / L): Bacteriological peptone 5, FePO4·2H2O 0.1, agar 18, pH 7.6-7.8, sterilization at 115℃ for 20min.

[0036] Three black soldier fly larvae cultured in chicken manure were selected, placed in a sterile culture dish, and the surface was cleaned with 5 mL of sterile water. Then, the larvae were placed in another culture dish and the surface was disinfected with 75% ethanol. Then, the larvae were washed with sterile water for 3 times. The sterile water of the third wash was coated and cultured as a control. The intestines were dissected under sterile conditions, and placed in a sterilized glass homogenizer. 0.2 mL of sterile water was added for grinding as the stock solution, and then 1.8 mL of sterile water was added to make a 10-diluted bacterial suspension. Then, 1.5 cm × 15 cm test tubes were used to dilute the dilutions of different concentrations. The appropriate dilutions were coated on LB or 2216E plates and placed in a 28°C constant temperature incubator for 3 days. Bacteria with different morphologies were selected, purified and cultured on new LB plates, and stored at -20°C and -80°C in the form of bacterial liquid with 30% glycerol.

[0037] (II) Determination of some physical and chemical properties of ZKY03, ZKY04, ZKY05 and ZKY06

[0038] Protease-producing strain screening culture medium: bacteriological peptone 10g, beef extract 3g, sodium chloride 5g, agar 15g, dilute to 500mL with pure water, sterilize at 115℃ for 20min; skim milk powder 4.5g, dilute to 500mL with pure water, sterilize at 110℃ for 15min separately, mix the two after the temperature drops to about 50℃.

[0039] Cellulase-producing strain screening medium: 0.5g potassium hydrogen phosphate, 0.25g magnesium sulfate, 2g sodium carboxymethyl cellulose, 0.2g Congo red, 2g bacteriological peptone, 15g agar powder, natural pH, pure water to 1L.

[0040] Lipase-producing strain screening medium: 0.2 g ammonium sulfate, 0.05 g sodium chloride, 0.05 g magnesium sulfate heptahydrate, 0.1 g disodium hydrogen phosphate, 4 mL substrate solution, 0.02 g bromocresol purple, adjust pH to 7.0, 15 g agar powder, dilute to 1 L with pure water, sterilize at 115°C for 20 min. (Substrate solution: olive oil and 2% polyvinyl alcohol 1:3 (V / V) mixed, stand at 4°C for 1 h, and then homogenize at high speed for 3 min to obtain).

[0041] Organophosphorus decomposing strain screening medium: 10g glucose, 0.5g yeast powder, 0.1g calcium chloride, 0.25g magnesium sulfate heptahydrate, 15g agar, dilute to 1L with pure water, sterilize at 115℃ for 20min. Wipe the egg shell with alcohol cotton balls, extract the egg yolk from the egg with a 10ml syringe (sterilized for standby use) and place it in a sterilized empty test tube, add an equal amount of 0.9% physiological saline (sterilized) and shake well to make egg yolk liquid, draw 6ml of egg yolk liquid and add it to the culture medium cooled to 60℃, shake well and pour it into a plate.

[0042] Amylase-producing strain screening culture medium: 0.5 g beef extract, 10 g yeast powder, 20 g dipotassium hydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 10 g soluble starch, 15 g agar powder, dilute to 1 L with pure water, and sterilize at 115°C for 20 min.

[0043] Chitosanase-producing strain screening medium: Liquid A: 1% colloidal chitosan, pH 5.5; Liquid B: 0.2g ammonium sulfate, 0.2g potassium dihydrogen phosphate, 0.05g magnesium sulfate heptahydrate, 0.1g yeast powder, 15g agar powder, adjust the pH to 7.0. Liquid A and liquid B are sterilized at 115℃ for 20min and then mixed in equal volumes. 1% colloidal chitosan: 1g chitosan is dissolved in 25mL of 1% acetic acid solution, an appropriate amount of alkali solution is added, the pH value is adjusted to between 5.5-5.8, and deionized water is added to 100mL.

[0044] Chitinase-producing strain screening medium: 10g colloidal chitin, 5g peptone, 15g agar powder, dilute to 1L with pure water, pH natural. w=1% colloidal chitin solution: 10g chitin powder plus 100mL concentrated hydrochloric acid, stir evenly, add pure water to wash repeatedly until neutral, and finally re-dissolve with PBS (pH=7.0) buffer, store in a refrigerator away from light for future use.

[0045] The test results are shown in Table 1.

[0046] Table 1 Determination of some physical and chemical properties of ZKY03, ZKY04, ZKY05 and ZKY06

[0047]

[0048] Note: “-” indicates negative, and “+” indicates positive.

[0049] (III) Molecular identification using 16S rDNA and gyrB genes

[0050] The present application has carried out 16S rDNA gene sequence (SEQ ID NO.1) and gyrB gene sequence on Bacillus subtilis ZKY03 strain, Bacillus subtilis ZKY04 strain, Bacillus subtilis ZKY05 strain and Bacillus subtilis ZKY06 strain.

[0051] For 16S rDNA sequence amplification, sequence alignment and phylogenetic tree construction, please refer to the inventor's authorized invention patent "A broad-spectrum antifungal plant endophytic Bacillus subtilis and its application" (ZL200910214430.2). The phylogenetic tree constructed based on the publicly published sequences of Bacillus subtilis strains is shown in the attached figure. Figure 1 ZKY03, ZKY04, ZKY05 and ZKY06 belong to the Bacillus subtilis group and cannot be accurately identified to species by 16S rDNA sequence.

[0052] 2 Phylogenetic analysis based on gyrB gene blast results

[0053] The gyrB sequences were obtained from the genome sequences of ZKY03, ZKY04, ZKY05 and ZKY06. Multiple comparisons and phylogenetic analysis were performed using Mega7 software using the published gyrB gene sequences of other strains of the Bacillus subtilis group. The phylogenetic tree constructed using the neighbor method is shown in the attached figure. Figure 2 The results showed that ZKY03, ZKY04, ZKY05 and ZKY06 were all Bacillus subtilis.

[0054] Example 2: Effects of adding single bacteria and synthetic bacterial flora on black soldier fly larvae

[0055] (I) Phylogenetic identification of ZKY03, ZKY04, ZKY05, and ZKY06

[0056] The present application has carried out kinship identification on ZKY03, ZKY04, ZKY05 and ZKY06 strains. The results are shown in Table 2 and Appendix. Figure 3 .

[0057] Table 2. Kinship identification results

[0058]

[0059] Note: “+” indicates fusion, and “-” indicates non-fusion.

[0060] (II) Effect of adding single bacteria and 4 strains of bacteria on insect weight gain

[0061] 1. Strain activation medium (LB medium, g / L):

[0062] Tryptone 10; yeast extract 5; sodium chloride 10; pH 7.0-7.2 (solid culture medium plus 2% agar in liquid culture medium), sterilize at 115℃ for 20min.

[0063] 2. Fermentation medium (NB medium, g / L):

[0064] Bacteriology: peptone 10; beef extract 3; sodium chloride 5; pH 7.0-7.2.

[0065] 3. Microbial culture

[0066] Take the strain stored at -20℃, streak it on LB solid culture medium using the plate streak method, and culture it in a 375 constant temperature incubator overnight. The next day, pick a single colony and put it in 5mL LB liquid culture medium, place it in a 37℃ shaker, and culture it at 180rpm overnight. The next day, inoculate it into 200mL NB liquid culture medium at a 1% inoculum size, place it in a 37℃ shaker, and culture it at 180rpm for 48h until spores are fully formed.

[0067] 4. Dilution coating count

[0068] Take 500 μL of the shaken bacterial solution after 48 hours of fermentation and put it into a 10 mL centrifuge tube containing 4500 μL of sterile water, shake it for 15 seconds to mix it, and then add it to the centrifuge tube. -1 Bacterial liquid, from 10 -1 Take 500 μL of the mixed bacterial solution and place it in a new 10 mL centrifuge tube containing 4500 μL of sterile water. Vortex for 15 seconds to mix. -2 Bacterial liquid, obtain 10 -3 ,10 -4 ,10 -5 ,10 -6 Bacterial liquid, from 10 -4 ,10 -5 ,10 -6 Take 100 μL of bacterial solution and spread it on LB solid medium. Repeat three times for each concentration. Place it in a 37℃ constant temperature incubator and incubate it for 12 hours. Then count and calculate the number of viable bacteria. The calculation method is as follows:

[0069] Number of viable bacteria per milliliter of sample = (C / V)*M

[0070] C is the average colony count at a certain dilution concentration, V is the volume of the diluent used during coating, and M represents the dilution multiple.

[0071] The results are shown in Table 3

[0072] Table 3 Dilution coating results

[0073]

[0074] 5. Black soldier fly cultivation

[0075] The black soldier fly eggs produced on the same day were incubated in dry bran at 28°C for 4 days, and artificial feed with a water content of 70% (volume ratio, bran: chicken feed: corn flour = 5:3:2) was added. After 4 days of incubation, 100 black soldier fly eggs were taken for use.

[0076] 6. Add bacterial solution and larvae

[0077] Dilute the counted bacterial solution to 1*10 7 CFU / mL, thaw pigeon droppings at -80℃, weigh 105g and add it to a plastic bottle with a diameter of 10cm and a height of 12cm, add 75mL of water, and for single bacteria treatment, add 20mL of 1*10 7 CFU / mL of bacterial solution to make the final bacterial count 2*10 8 CFU / 200g, the moisture content of pigeon feces is 70%, and the 4-strain treatment is 4 strains of bacteria, each adding 5mL at a concentration of 1*10 7 CFU / mL of bacterial solution to make the final bacterial count 2*10 8 CFU / 200g, the moisture content of pigeon manure is 70%. After stirring evenly, the weight is weighed as the total weight of the initial system. The initial matrix weight is obtained by subtracting the bottle weight from the total weight of the initial system. Add 100 4-day-old black soldier fly larvae, seal with 40-mesh non-woven fabric, and culture in a 28°C culture room for 8 days.

[0078] 7. Weight of insect bodies and residues and bioconversion rate

[0079] After 8 days of cultivation, the final system weight and the total weight of the collected insects were weighed, the larvae were separated from the pigeon feces, the surface of the insects was washed with tap water, the surface of the insects was wiped dry with a paper towel and the moisture was counted and weighed to obtain the total weight of the collected insects. The weight of the residue was obtained by subtracting the total weight of the collected insects and the weight of the bottle from the total weight of the final system. The total weight of the collected insects / initial matrix weight was used as the bioconversion rate. Data were sorted using Excel, and data were analyzed and plotted using R (the same below);

[0080] See attached for insect weight Figure 4Compared with the blank treatment, the addition of the four strains of bacteria alone and the mixture of the four strains of bacteria can promote the weight gain of black soldier fly larvae. The average weight of the blank treatment was 0.1722±0.0062g / larvae, the average weight of the ZKY03 treatment was 0.1879±0.0121g / larvae, the average weight of the ZKY04 treatment was 0.1923±0.0198g / larvae, and the average weight of the ZKY03 treatment was 0.2062±0.0041g / larvae. The average weight of the insects treated with ZKY03 was 0.1944±0.0056g / individual, and the average weight of the insects treated with the four strains of bacteria was 0.2286±0.0035g / individual; ZKY03, ZKY04, ZKY05, ZKY06 and the four strains of bacteria increased by 9%, 12%, 20%, 13% and 33% respectively compared with the blank treatment. Therefore, adding a single bacterium can increase the weight gain of the insects to a certain extent, while the synthetic bacterial community of the four strains of bacteria can more significantly increase the weight gain of the insects.

[0081] See attached for the weight of the residue Figure 5 The residue of the blank treatment was 171.98g±1.55g, and the residue of synthetic bacterial group 4 weighed the least, which was 163.30g±1.08g, significantly lower than the blank treatment; the residue weights of ZKY03, ZKY04, ZKY05 and ZKY06 were 166.99g±1.77g, 164.42g±0.42g, 164.37g±1.16g and 165.91g±0.89g, respectively, all significantly lower than the blank treatment;

[0082] Bioconversion rate see attached Figure 6 The bioconversion rate of the blank treatment was 8%, and the bioconversion rates of ZKY03, ZKY04, ZKY05 and ZKY06 were 9%, 9%, 10% and 10% respectively. The synthetic bacterial community 4 had the highest bioconversion rate of 11%. Except for ZKY03, the other treatments were significantly better than the blank treatment.

[0083] The results of insect body weight, residue weight and bioconversion rate consistently showed that synthetic bacterial community 4 can significantly increase the biomass of 12-day-old black soldier fly larvae in the pigeon manure culture system and promote the conversion and degradation of pigeon manure. In order to further determine whether the 4 strains within the synthetic bacterial community are all essential strains, further synthetic bacterial community strain deletion experiments were carried out.

[0084] (III) Effect of insect weight gain after deletion of synthetic bacterial strains

[0085] The method is the same as (II), and the amount of synthetic bacteria added ensures that the final bacterial count is 2*10 8 CFU / 200g, the amount of each strain added in the flora is the same, and the composition of the synthetic flora is shown in the table:

[0086] Table 4 Composition of synthetic bacterial flora

[0087]

[0088] The results of insect body weight are shown in Table 4 and Appendix Figure 7 The results showed that the body weight of the combination of 2 strains was 0.1843-0.1983g, while the body weight of the combination of 3 strains was 0.1942-0.2054g, and the combination of 4 strains had the best effect, with a body weight of 0.2060g.

[0089] The amount of residues and bioconversion rate are shown in the attached Figure 8 And attached Fig. 9 As shown, the residue amounts of all treatments were significantly lower than those of the blank treatment, while the bioconversion rates were significantly higher than those of the blank treatment except for treatments 414 and 423, while there were no significant differences in the residue amounts and bioconversion rates among the other treatments.

[0090] Combining the results of insect weight, residue and bioconversion rate, it was found that in the pigeon manure culture system of black soldier flies, the synthetic bacterial community 4 composed of 4 strains of bacteria can significantly promote the accumulation of biomass of 12-day-old larvae, while the deletion of strains in sequence will reduce its promotion effect to varying degrees. (IV) Effect of adding single bacteria and synthetic bacterial communities on the proportion and total amount of crude protein in insect bodies

[0091] 1. Drying of insect bodies

[0092] The 12-day-old insect bodies after adding single bacteria and synthetic bacteria were taken and dried at 105°C to constant weight. The weights before and after drying were recorded and the moisture content was calculated.

[0093] 2. Determination of crude protein in insect bodies

[0094] The method for determining crude protein in insect bodies is carried out in accordance with GB / T 6432-2018 (Kjeldahl method for determination of crude protein in feed), and the crude protein content and total protein content are calculated.

[0095] Total protein g / 100 heads = M*(1-R)*C

[0096] M is the total weight of 12-day-old larvae harvested in the corresponding treatment, R is the moisture content of the corresponding larvae, and C is the percentage of crude protein content in the dry weight of the corresponding larvae.

[0097] The total protein content of the insect body is shown in the attached Fig.10 The treatment with the highest total protein content was the addition of synthetic bacterial colony 4, which increased the total protein content by 41.3% compared with the blank treatment. By adding synthetic bacterial colonies, the total protein content of black soldier fly larvae can be significantly increased, thereby improving economic benefits.

Claims

1. An artificially synthesized bacterial community for increasing the biomass of 12-day-old black soldier fly larvae, characterized in that By Bacillus subtilis ( Bacillus subtilis ) ZKY03, Bacillus subtilis ZKY04 ( Bacillus subtilis ), Bacillus subtilis ( Bacillus subtilis ) ZKY05, Bacillus subtilis ( Bacillus subtilis ) ZKY06. The above four strains have been deposited in Guangdong Provincial Microbiological Culture Collection Center on September 7, 2022. The deposit number of ZKY03 is GDMCC NO: 62763, the deposit number of ZKY04 is GDMCC NO: 62764, the deposit number of ZKY05 is GDMCC NO: 62765, and the deposit number of ZKY06 is GDMCC NO: 62766.

2. Use of the artificial synthetic bacterial flora according to claim 1 in increasing the biomass of 12-day-old black soldier fly larvae and the total insect protein yield.

3. The use according to claim 2, characterized in that The use of the artificial synthetic bacterial community described in claim 1 in increasing the biomass and total insect protein yield of 12-day-old black soldier fly larvae cultured in pigeon manure.

4. Use of the artificial synthetic bacterial flora according to claim 1 in the preparation of a product for increasing the biomass of 12-day-old black soldier fly larvae and the total insect protein yield.

5. The use according to claim 4, characterized in that Use of the artificial synthetic bacterial flora described in claim 1 in the preparation of a product for increasing the biomass and total insect protein yield of 12-day-old black soldier fly larvae cultured in pigeon manure.

6. A product for increasing the biomass of black soldier fly larvae, characterized in that The invention is composed of the diluted solution of the artificial synthetic bacterial fermentation liquid described in claim 1.

7. Use of the product according to claim 6 in increasing the biomass of black soldier fly larvae and the yield of insect protein.

8. A method for cultivating insects with higher protein content, characterized in that The product according to claim 6 is added to pigeon droppings for feeding 4-day-old black soldier fly larvae and cultured until they are 12 days old.

9. The method according to claim 8, characterized in that The culture condition is 28° C. for 8 days.

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