A method for breeding black soldier fly to improve the bioconversion capacity of black soldier fly on agricultural waste

Abstract

The application discloses a black soldier fly breeding method for improving the biological conversion capacity of black soldier flies on agricultural wastes. Penicillium sclerotigenum The application discloses a black soldier fly breeding method for improving the biological conversion capacity of black soldier flies on agricultural wastes. The application discloses a black soldier fly breeding method for improving the biological conversion capacity of black soldier flies on agricultural wastes. The application discloses a black soldier fly breeding method for improving the biological conversion capacity of black soldier flies on agricultural wastes. The application discloses a black soldier fly breeding method for improving the biological conversion capacity of black soldier flies on agricultural wastes. The application discloses a black soldier fly breeding method for improving the biological conversion capacity of black soldier flies on agricultural wastes. The application discloses a black soldier fly breeding method for improving the biological conversion capacity of black soldier flies on agricultural wastes. The application discloses a black soldier fly breeding method for improving the biological conversion capacity of black soldier flies on agricultural wastes. The application discloses a black soldier fly breeding method for improving the biological conversion capacity of black soldier flies on agricultural wastes. The application discloses a black soldier fly breeding method for improving the biological conversion capacity of black soldier flies on agricultural wastes.

Description

Technical Field

[0001] The present invention relates to the technical field of organic waste resource utilization and black soldier fly breeding, and in particular to a black soldier fly breeding method for improving the black soldier fly's ability to bioconvert agricultural waste. Background Art

[0002] With the rapid development of agriculture and animal husbandry, large amounts of agricultural waste and livestock manure generated by the industry are posing severe challenges to the environment and resource utilization. Untreated livestock manure can easily breed pathogens, contaminate water sources, and release harmful gases that pollute the environment. Furthermore, manure, which is rich in organic matter and nutrients, is difficult to effectively utilize. Currently, waste treatment methods mainly include direct incineration, composting, and some resource utilization. However, these treatment methods generally suffer from low treatment efficiency, severe environmental pollution, and serious resource waste. How to achieve efficient degradation and resource utilization of agricultural waste and livestock manure has become an important issue that needs to be addressed.

[0003] The black soldier fly (Hermetia illucens), a recently emerging important resource insect, has garnered widespread attention for its unique ecological functions and efficient organic matter processing capabilities. Rich in high-quality protein, moderate amounts of fat, and various trace elements, black soldier fly larvae are not only recommended by the Food and Agriculture Organization (FAO) as ideal forage insects but are also considered an important source of protein for humans and livestock. As saprophytic insects, black soldier fly larvae exhibit unique advantages in degrading agricultural waste, efficiently processing a variety of organic wastes, such as food waste, crop straw, and livestock and poultry manure. However, despite significant progress in recent years in the bioconversion of agricultural wastes such as chicken manure using black soldier fly larvae, numerous deficiencies and controversies remain regarding carbon and nitrogen conversion, losses, and bioavailability. Further improving the bioconversion capacity of black soldier fly larvae for agricultural wastes such as manure and livestock and poultry manure remains a pressing technical challenge. Summary of the Invention

[0004] In order to realize the resource utilization of agricultural waste, the purpose of the present invention is to provide a black soldier fly breeding method for improving the biotransformation ability of black soldier flies on agricultural waste, by adding Penicillium sclerotiorum ( Penicillium sclerotigenum ) for black soldier fly breeding can effectively reduce fecal pollution, improve the growth of black soldier flies and waste treatment efficiency, and promote the sustainable development of agricultural waste.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides a method for breeding black soldier flies for improving the bioconversion ability of black soldier flies on agricultural waste, comprising the following steps:

[0007] When Penicillium sclerotiorum ( Penicillium sclerotigenum Inoculate black soldier fly larvae into agricultural waste from agricultural waste, carry out breeding, and collect insect sand and insect bodies;

[0008] The agricultural waste consists of livestock and poultry excrement, straw and aquatic waste.

[0009] In the above method, the aquatic waste is fish excrement and / or fish processing waste; preferably, the fish processing waste includes one or both of fish bones and fish viscera. More preferably, the agricultural waste is composed of chicken manure, stevia straw, and fish viscera; further preferably, the agricultural waste is composed of chicken manure, stevia straw, and fish viscera in a mass ratio of 2:1:1.

[0010] In the above method, the effective viable bacteria count of the Penicillium sclerotiorum in the agricultural waste is 0.78×10 8 ~1.12×10 8 CFU / g. As an example, the Penicillium sclerotiorum ( Penicillium sclerotigenum ) is Penicillium sclerotiorum CICC40923, which is available to the public from the China Industrial Culture Collection Center (CICC).

[0011] In the above method, the Penicillium sclerotogenum is added in the form of a bacterial agent, and the preparation method of the bacterial agent comprises the following steps: inoculating the seed culture liquid of the Penicillium sclerotogenum into an enzyme production medium for culturing to obtain the bacterial agent; preferably, the culturing conditions are as follows: culture temperature of 26 ° C, culture medium pH 6.0, seed liquid inoculation amount of 8%, rotation speed of 130 r / min, and culture time of 4 days. As an example, the enzyme production medium is composed of the following components in terms of mass percentage: KH2PO4 0.2%, (NH4)2S04 0.14%, MgSO4·7H2O 0.03%, CaCl2 0.03%, FeSO4·7H2O 0.0005%, MnSO4 0.00016%, ZnSO4·7H2O 0.00014%, CoCl2 0.0002%, CMC-Na 0.5%, peptone 0.5%, pH 7.0.

[0012] In the above method, before inoculating the black soldier fly larvae, the carbon-nitrogen ratio of the agricultural waste is adjusted to 25-30 (e.g., 25). The carbon-nitrogen ratio can be adjusted using carbon-containing materials, such as sawdust.

[0013] In the above method, before inoculating the black soldier fly larvae, the moisture content of the agricultural waste is adjusted to 70% to 75% (eg, 70%).

[0014] In the above method, the black soldier fly larvae are black soldier fly larvae of several days old, such as 2 to 3 days old.

[0015] In the above method, as an example, 3 g of the black soldier fly larvae are inoculated into every 2000 g of substrate.

[0016] In the above method, as an example, the cultivation is carried out in an open environment at a constant temperature of 30°C and a constant humidity of 30% in the dark.

[0017] In the above method, the improvement of the bioconversion ability of black soldier flies on agricultural waste is reflected in at least one of the following aspects (A1) to (A6):

[0018] (A1) Increase waste reduction rates;

[0019] (A2) Improve biotransformation rate;

[0020] (A3) improving the conversion ratio of substrate to black soldier fly prepupae biomass;

[0021] (A4) increasing the growth rate of black soldier flies;

[0022] (A5) increasing the dry matter content of black soldier flies;

[0023] (A6) Increase the protein content of black soldier flies.

[0024] The present invention has the following significant effects in the treatment of agricultural waste and livestock and poultry manure:

[0025] (1) Improve the degradation rate of agricultural waste and livestock and poultry manure, and achieve significant reduction effects;

[0026] (2) Improve the survival rate, growth rate and weight of black soldier fly larvae;

[0027] (3) The preparation process of the microbial agent is simple and efficient, and has the potential for commercial application;

[0028] (4) Realize the economic and environmental benefits of waste resource utilization.

[0029] High efficiency and environmental adaptability of the microbial agent: The microbial agent prepared using optimized culture conditions has high enzyme activity and good environmental adaptability. It can maintain stable performance under a variety of conditions and is suitable for complex and changeable waste treatment environments, solving the problem of poor environmental adaptability of traditional microbial agents.

[0030] The method of the present invention can achieve rapid and efficient degradation of waste, reduce resource loss and environmental pollution, provide a new solution for waste treatment, and comprehensively improve resource utilization efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG1 shows the single-factor optimization results of time (a), temperature (b), pH (c), and rotation speed (d) in the microbial agent culture condition optimization experiment in Example 1 of the present invention.

[0032] Figure 2 The dry matter content and protein content of the insect body of each experimental group in Example 2 of the present invention. DETAILED DESCRIPTION

[0033] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0034] Unless otherwise specified, the methods used in the following examples are all conventional methods and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0035] The sources of the raw materials in the following examples are as follows: Penicillium sclerotiorum ( Penicillium sclerotigenum ) was purchased from the China Industrial Culture Collection (CICC); strain number: CICC40923; platform resource number: 1511C0005000005715; characteristics: the colony is gray-green and rope-shaped; the conidia are nearly spherical, smooth, and 3-3.5 μm.

[0036] Livestock and poultry manure was sourced from the Chenguang Group Company in Quzhou County, Hebei Province, with fresh chicken manure as the primary raw material. Stevia straw and fish offal were also prepared as supplementary ingredients. All ingredients were blended according to dry matter weight ratio. Black soldier fly eggs were purchased from Zhengzhou Kuocheng Ecological Agriculture Technology Co., Ltd. The straw was pre-ground in a grinder until it was finely chopped without large branches. Fish offal remained unprocessed.

[0037] Example 1: Optimization of bacterial culture conditions

[0038] 1. Preparation of microbial agents

[0039] 1. Strain activation: Penicillium sclerotiorum ( Penicillium sclerotigenum ) and inoculate into MEA medium. Weigh 33 g of medium, heat to a boil and dissolve in 1000 ml of distilled water. Aliquot and sterilize by autoclaving at 121°C for 15 minutes. Incubate at 28°C for 1–2 days until the strain is activated and colonies are clearly growing.

[0040] 2. Liquid Seed Preparation: Take a small amount of the activated strain and inoculate it into a liquid seed culture medium. Place the inoculated liquid culture medium in a constant temperature shaker at 120 rpm and 30°C for 24-48 hours. Check the bacterial concentration and turbidity of the liquid culture medium. Once the bacterial culture reaches the appropriate growth stage, use it as a seed culture medium.

[0041] 3. Liquid inoculum preparation: Add liquid seed to the enzyme-producing medium at an inoculum rate of 8%. Fermentation should be performed in a constant temperature shaker at 28°C and 120 rpm. Incubate for 24–48 hours, monitoring bacterial growth periodically. After incubation, collect the bacterial culture for subsequent processing.

[0042] MEA culture medium: purchased from Qingdao Haibo Biological Co., Ltd., Malt Extract Agar Medium, product number: HB8892.

[0043] The liquid seed culture medium was composed of 10 g glucose, 5 g peptone, 5 g yeast extract, 1 g potassium dihydrogen phosphate, 0.2 g magnesium sulfate, 20 g sodium chloride, and 1000 mL distilled water, with the pH adjusted to 7.0.

[0044] The enzyme production culture medium is: KH2PO4 2 g, (NH4)2S04 1.4 g, MgSO4·7H2O 0.3 g, CaCl2 0.3 g, FeSO4·7H2O 0.005 g, MnSO4 0.0016 g, ZnSO4·7H2O 0.0014 g, CoCl2 0.002 g, CMC-Na 5 g, peptone 5 g, distilled water 1000 mL, pH 7.0, sterilized at 121 ℃ for 30 min.

[0045] 2. Single factor optimization experiment

[0046] Following the principle of step-by-step optimization, with enzyme activity as the sole evaluation variable, single-factor optimization experiments were conducted on the cultivation conditions of Penicillium sclerotiorum, including incubation time, temperature, pH, and rotation speed. Initial conditions were set as: 28°C, pH 6.5, inoculum size 8%, and rotation speed 120 rpm.

[0047] The single factor optimization experimental method is as follows:

[0048] (1) Optimization of culture time: Under the initial conditions, the culture time was set as the dependent variable, which was 2 days, 3 days, 4 days, 5 days and 6 days respectively. The enzyme activity values ​​of the strains under different culture times were measured to screen the optimal culture time. The results are as follows Figure 1aAs shown in the figure, the incubation time has a significant impact on the activity values ​​of the four enzymes. When the incubation time is short or long, the activity values ​​of the four enzymes are all low. When the incubation time is 4 days, the activity of the three enzymes is better, and the protease activity is better on the 3rd day. However, considering all factors, the optimal incubation condition is 4 days.

[0049] (2) Optimization of culture temperature: Based on the optimal culture time, the culture temperature was set as the dependent variable, which was 22℃, 24℃, 26℃, 28℃ and 30℃ respectively. The enzyme activity values ​​at different culture temperatures were measured to screen the appropriate culture temperature. Figure 1b As shown in the figure, high temperature has a certain influence on the enzyme activity values ​​of this strain. The overall trend of the four enzyme activities first increases and then decreases with increasing temperature. Therefore, considering all factors, the subsequent culture conditions were set to 26°C for 4 days.

[0050] (3) Optimization of culture pH: Under the optimal culture time and culture temperature conditions, adjust the culture medium pH to 5.5, 6.0, 6.5, 7.0 and 7.5, measure the enzyme activity under different pH conditions, and screen the appropriate culture pH value. Figure 1c As shown in the figure, with the increase of pH, the activity of bacterial protease, CMCase and β-Gase first increased and then decreased, reaching the maximum value at pH 6.0.

[0051] (4) Speed ​​optimization: Keep the optimal culture time, culture temperature and pH value unchanged, adjust the speed, set to 100 r / min, 110 r / min, 120 r / min, 130 r / min and 140 r / min, measure the effect of different speeds on enzyme production capacity, and screen the optimal speed. Figure 1d As shown in the figure, overall, the rotation speed as a single factor has a significant effect on the enzyme activity of the strain. The rotation speed of 130 r / min can effectively promote the enzyme activity.

[0052] Experimental Results and Analysis: The results showed that the enzyme activity was relatively stable under initial conditions. Single-factor optimization experiments yielded the following optimal conditions: 26°C, pH 6.0, a rotational speed of 130 r / min, and a 4-day growth period. This single-factor optimization experiment successfully enhanced the enzyme production capacity of the strain, providing a scientific basis for the production of microbial inoculants. The optimized microbial inoculant has significant application value in agricultural waste treatment and resource utilization.

[0053] Example 2: Application of dual-degradation bacterial agent in the conversion of agricultural waste by black soldier flies

[0054] The black soldier fly eggs were placed in an artificial climate chamber at a constant temperature of 30°C and a relative humidity of 30% for hatching for 2 to 3 days. After the eggs showed obvious fluidity, they were transferred to coarse wheat bran for culture.

[0055] The Penicillium sclerotiorum ( Penicillium sclerotigenum The bacterial suspension was inoculated into a culture box containing 2000 g of a substrate mixture of chicken manure, stevia straw, and fish offal at a mass ratio of 2:1:1. Treatment group T was designated. Liquid bacterial suspension was weighed to 1-3% of the total substrate mass and diluted with sterile saline. The different bacterial suspensions were then evenly sprayed onto the substrate surface before inoculation with insects. Using the plate count method, the total viable count of Penicillium sclerotiorum in the agricultural waste was controlled to be 0.78 × 10 8 ~1.12×10 8 CFU / g. Feeding conditions used sawdust to adjust the substrate's carbon-nitrogen ratio to 25 and its moisture content to 70%. 3g of black soldier fly eggs were added for further feeding. The cells were placed in an artificial climate chamber, kept at a constant temperature of 30°C and a constant humidity of 30%, and kept in the dark. A control group, containing sterile water instead of the bacterial solution, was designated CK. Three replicates were set for each group.

[0056] When 50% of the larvae entered the prepupal stage, the excrement was separated. The insect bodies and the insect litter were sieved using a filter, and the insect litter and the insect bodies were sampled and weighed.

[0057] The optimization effect of adding Penicillium sclerotigenum on the efficiency of black soldier fly larvae in treating organic waste was evaluated by calculating indicators such as waste reduction rate (WR), substrate to black soldier fly (BSF) prepupae biomass conversion ratio (SBC), bioconversion rate (BR), and growth rate. The formula is as follows:

[0058] WR (%) = consumed dry substrate weight (g) / initial dry substrate weight (g) × 100%;

[0059] SBC (%) = prepupal weight (g) / initial dry substrate weight (g) × 100%;

[0060] BR (%) = total biomass of fresh larvae (g) / added fresh substrate (g) × 100%;

[0061] Growth rate (g / d) = (fresh weight of larvae after feeding - initial fresh weight of larvae) / number of days of feeding.

[0062] Table 1 shows the addition of Penicillium nucleatum ( Penicillium sclerotigenum) treatment group and the control group without addition of biotransformation capacity results.

[0063]

[0064] As shown in Table 1, compared with the control group (CK) without the addition of microbial agents, the waste reduction rate increased after the addition of microbial agents to agricultural waste (T treatment), showing a good effect. The waste reduction rate of the T treatment group reached 49.71±3.51%, which was significantly higher than the 20.14±2.72% of the CK group. This indicates that the addition of Penicillium sclerotiorum ( Penicillium sclerotigenum ) significantly improved the waste reduction effect. The bioconversion rate of the T treatment group was about 25.4% higher than that of the CK group, indicating that the Penicillium sclerotigenum The addition of ) enhanced the conversion efficiency of waste to larval biomass. Substrate-to-prepupal biomass conversion (SBC) and growth rate were also significantly improved.

[0065] After separation, the black soldier fly samples were cleaned to remove surface impurities. The samples were then oven-dried at 65°C to a constant weight. The dried samples were ground into a uniform powder using a mortar and pestle and placed in a sealed container for later use. The dry matter ratio and protein content of the samples were then determined. The nutritional effects of the addition of Penicillium nucleatum on the black soldier fly samples were evaluated.

[0066] Figure 2 The changes in dry matter and protein content of black soldier flies under different treatments (CK and T) are shown. The dry matter content of the control group (CK) was 21.28 ± 0.68. The addition of the microbial inoculant significantly increased this content, increasing by 34.68% compared to the control group. Regarding protein content, the addition of the microbial inoculant further increased the protein content of the treatment group (T), reaching 36.33 ± 0.78. This marked a significant increase of 29.66% compared to the control group. In summary, treatment (T) significantly increased the dry matter and protein content of black soldier flies, demonstrating a clear advantage over the control group. This demonstrates that the microbial inoculant has a positive effect in optimizing insect growth and nutrient accumulation.

[0067] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, can implement the present invention in a wider range under equivalent parameters, concentration and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principle of the present invention, the application is intended to include any variation, purposes or improvements of the present invention, including departing from the disclosed scope in the application and the changes made with conventional techniques known in the art.

Claims

1. A method for breeding black soldier flies to improve their ability to bioconvert agricultural waste, characterized in that: The steps include: When Penicillium sclerotiorum ( Penicillium sclerotigenum Inoculate black soldier fly larvae into agricultural waste from agricultural waste, carry out breeding, and collect insect sand and insect bodies; The agricultural waste consists of livestock and poultry manure, straw and aquatic waste; The effective viable bacterial count of the Penicillium sclerotiorum in the agricultural waste was 0.89×10 8 ~1.02×10 8 CFU / g.

2. The method for breeding black soldier flies for improving their ability to bioconvert agricultural waste according to claim 1, characterized in that: The aquatic waste is fish excrement and / or fish and aquatic product processing waste.

3. The method for breeding black soldier flies for improving their ability to bioconvert agricultural waste according to claim 2, wherein: The fish aquatic product processing waste includes one or both of fish bones and fish viscera.

4. The method for breeding black soldier flies for improving the bioconversion ability of black soldier flies on agricultural waste according to claim 1 or 2, characterized in that: The agricultural waste consists of chicken manure, stevia straw and fish viscera.

5. The method for breeding black soldier flies for improving their ability to bioconvert agricultural waste according to claim 4, characterized in that: The agricultural waste consists of chicken manure, stevia straw and fish viscera in a mass ratio of 2:1:

1.

6. The method for breeding black soldier flies for improving the bioconversion ability of black soldier flies on agricultural waste according to any one of claims 1-2, characterized in that: The Penicillium sclerotogenes is added in the form of a bacterial agent. The preparation method of the bacterial agent comprises the following steps: inoculating the seed bacterial liquid of the Penicillium sclerotogenes into an enzyme production culture medium for culturing to obtain the bacterial agent.

7. The method for breeding black soldier flies for improving their ability to bioconvert agricultural waste according to claim 6, characterized in that: The culture conditions are as follows: culture temperature of 26°C, culture medium pH of 6.0, seed liquid inoculation amount of 8%, rotation speed of 130 r / min, and culture time of 4 days.

8. The method for breeding black soldier flies for improving the bioconversion ability of black soldier flies on agricultural waste according to any one of claims 1-2, characterized in that: Before the inoculation of black soldier fly larvae, the carbon-nitrogen ratio of the agricultural waste is adjusted to 25-30.

9. The method for breeding black soldier flies for improving their ability to bioconvert agricultural waste according to any one of claims 1 to 2, characterized in that: Before the inoculation of black soldier fly larvae, the moisture content of the agricultural waste is adjusted to 70% to 75%.

10. The method for breeding black soldier flies for improving the bioconversion ability of black soldier flies on agricultural waste according to any one of claims 1 to 2, characterized in that: The black soldier fly larvae are black soldier fly larvae of several days old.

11. The method for breeding black soldier flies for improving their ability to bioconvert agricultural waste according to claim 10, characterized in that: The black soldier fly larvae are 2 to 3 days old.

12. The method for breeding black soldier flies for improving their ability to bioconvert agricultural waste according to any one of claims 1 to 2, characterized in that: Inoculating 3 g of the black soldier fly larvae per 2000 g of substrate; and / or, The culture was carried out in an open environment at a constant temperature of 30°C and a constant humidity of 30% in the dark.

13. The method for breeding black soldier flies for improving their ability to bioconvert agricultural waste according to any one of claims 1 to 2, characterized in that: The improvement of the bioconversion ability of black soldier flies on agricultural waste is reflected in at least one of the following aspects (A1) to (A6): (A1) Increase waste reduction rates; (A2) Improve biotransformation rate; (A3) improving the conversion ratio of substrate to black soldier fly prepupae biomass; (A4) increasing the growth rate of black soldier flies; (A5) increasing the dry matter content of black soldier flies; (A6) Increase the protein content of black soldier flies.

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