Hermetia illucens breeding method for improving biotransformation capability of Hermetia illucens on agricultural wastes

Abstract

The invention discloses a hermetia illucens breeding method for improving the biotransformation capacity of hermetia illucens on agricultural wastes. The hermetia illucens breeding method capable of improving the biotransformation capacity of the hermetia illucens on the agricultural waste comprises the following steps: inoculating larvae of the hermetia illucens into the agricultural waste added with Penicillium nucleatum, breeding, and collecting insect sand and insect bodies. The method disclosed by the invention can be used for improving the biotransformation capability of the hermetia illucens on the agricultural wastes, which is specifically embodied in the aspects of increasing the waste reduction rate, increasing the biotransformation rate, increasing the biomass transformation ratio of the substrate and the hermetia illucens prepupae, increasing the growth rate of the hermetia illucens, increasing the dry matter content and the protein content of hermetia illucens bodies and the like; the method provided by the invention can realize rapid and efficient degradation of wastes, reduces resource loss and environmental pollution, provides a brand new solution for waste treatment, and comprehensively improves the resource utilization efficiency.

Description

Technical Field

[0001] The invention relates to the technical field of resource utilization of organic waste 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, a large amount of agricultural waste and livestock and poultry manure generated by the animal husbandry industry are posing severe challenges to the environment and resource utilization. Untreated livestock and poultry manure is prone to breed pathogens, pollute water sources, and release harmful gases to pollute the environment. At the same time, manure rich in organic matter and nutrients is difficult to be effectively utilized. At present, the main methods of waste treatment include direct incineration, composting, and partial resource utilization. However, these treatment methods generally have problems such as low treatment efficiency, large environmental pollution, and serious waste of resources. How to achieve efficient degradation and resource utilization of agricultural waste and livestock and poultry manure has become an important issue that needs to be solved urgently.

[0003] As an important resource insect that has emerged in recent years, the black soldier fly (Hermetia illucens) has attracted widespread attention for its unique ecological functions and efficient organic matter processing capabilities. The black soldier fly larvae are rich in high-quality protein, moderate fat and a variety of trace elements. They are not only recommended by the Food and Agriculture Organization of the United Nations (FAO) as ideal fodder insects, but are also considered an important source of protein for humans and livestock. As a saprophytic insect, the black soldier fly larvae show unique advantages in degrading agricultural wastes and can efficiently process various organic wastes, such as kitchen waste, crop straw and livestock and poultry manure. However, although significant progress has been made in recent years in the research on the biotransformation of agricultural wastes such as chicken manure by black soldier fly larvae, there are still many deficiencies and controversies in terms of carbon and nitrogen conversion, loss and bioavailability. How to further improve the biotransformation ability of black soldier fly larvae in agricultural wastes such as manure and livestock and poultry manure is still a technical problem that needs to be solved urgently. 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 solution: 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: In the presence of Penicillium sclerotogenum ( Penicillium sclerotigenum) to inoculate black soldier fly larvae in agricultural wastes, culture them, and collect insect sand and insect bodies; The agricultural waste consists of livestock and poultry excrement, straw and aquatic waste.

[0006] In the above method, the aquatic waste is fish excrement and / or fish aquatic processing waste; preferably, the fish aquatic processing waste includes one or two of fish bones and fish viscera. More preferably, the agricultural waste consists of chicken manure, stevia straw and fish viscera; further preferably, the agricultural waste consists of chicken manure, stevia straw and fish viscera in a mass ratio of 2:1:1.

[0007] 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 sclerotogenum ( Penicillium sclerotigenum ) is Penicillium sclerotiorum CICC40923, which is available to the public from the China Industrial Culture Collection Center (CICC).

[0008] 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 bacterial liquid of the Penicillium sclerotogenum into an enzyme production medium for cultivation to obtain the bacterial agent; preferably, the cultivation conditions are as follows: the cultivation temperature is 26 ° C, the medium pH is 6.0, the seed liquid inoculation amount is 8%, the rotation speed is 130 r / min, and the cultivation time is 4 days. As an example, in terms of mass percentage, the enzyme production medium is composed of the following components: KH 2 PO 4 0.2%, (NH 4 ) 2 S0 4 0.14%,MgSO 4 7H 2 O 0.03%, CaCl 2 0.03%, FeSO 4 7H 2 O 0.0005%, MnSO 4 0.00016%, ZnSO 4 7H 2 O 0.00014%, CoCl 2 0.0002%, CMC-Na 0.5%, peptone 0.5%, pH 7.0.

[0009] In the above method, before inoculating black soldier fly larvae, the carbon-nitrogen ratio of agricultural waste is adjusted to 25-30 (such as 25). The carbon-nitrogen ratio can be adjusted using carbon-containing materials, such as sawdust.

[0010] In the above method, before inoculating the black soldier fly larvae, the moisture content of the agricultural waste is adjusted to 70% to 75% (such as 70%).

[0011] 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.

[0012] In the above method, as an example, 3 g of the black soldier fly larvae are inoculated into every 2000 g of substrate.

[0013] In the above method, as an example, the cultivation is carried out in an open environment protected from light at a constant temperature of 30°C and a constant humidity of 30%.

[0014] In the above method, the improvement of the bioconversion ability of black soldier flies on agricultural waste is embodied in at least one of the following aspects (A1) to (A6): (A1) Increase waste reduction rates; (A2) Improve bioconversion 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.

[0015] The present invention has the following significant effects in the treatment of agricultural waste and livestock and poultry excrement: (1) Improve the degradation rate of agricultural waste and livestock and poultry manure, and achieve significant reduction effects; (2) Improve the survival rate, growth rate and weight of black soldier fly larvae; (3) The preparation process of the microbial agent is simple and efficient, and has the potential for commercial application; (4) Realize economic and environmental benefits from waste resource utilization.

[0016] High efficiency and environmental adaptability of microbial agents: The microbial agents prepared using optimized culture conditions have high enzyme activity and good environmental adaptability. They can maintain stable performance under a variety of conditions and are suitable for complex and changeable waste treatment environments, solving the problem of poor environmental adaptability of traditional microbial agents.

[0017] 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

[0018] FIG1 is a single factor optimization result 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.

[0019] Figure 2 It is the insect body dry matter content and insect body protein content of each experimental group in Example 2 of the present invention. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0021] The methods used in the following examples, unless otherwise specified, are all conventional methods, carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0022] The sources of the raw materials in the following examples are as follows: Penicillium sclerotigenum ) was purchased from China Industrial Culture Collection Center (CICC); strain number CICC40923; platform resource number: 1511C0005000005715; characteristics: colonies are gray-green and rope-shaped; conidia are nearly spherical, smooth, and 3-3.5μm.

[0023] Livestock and poultry manure was sourced from the Chenguang Group Company in Quzhou County, Hebei Province, with fresh chicken manure as the main raw material. At the same time, stevia straw and fish viscera were prepared as auxiliary additives. All raw materials were proportioned according to the dry matter weight ratio. Black soldier fly eggs were purchased from Zhengzhou Kuocheng Ecological Agriculture Technology Co., Ltd. The straw needed to be crushed into small crumbs without large branches by a grinding machine in advance. Fish viscera did not need to be processed.

[0024] Example 1: Optimization of bacterial culture conditions 1. Preparation of microbial agents 1. Strain activation: Penicillium nucleatum ( Penicillium sclerotigenum ), inoculate into MEA medium. Weigh 33g of medium, heat and boil to dissolve in 1000ml of distilled water, divide into portions, sterilize at 121℃ for 15 minutes, and set aside. Culture at 28℃ for 1-2 days until the strain is activated and colonies grow obviously.

[0025] 2. Liquid seed preparation: Take a small amount of the activated strain and transfer it to the liquid seed culture medium. Place the inoculated liquid culture medium in a constant temperature shaker and shake at 30°C at a speed of 120 r / min for 24 to 48 hours. Check the bacterial concentration and turbidity of the liquid culture medium. When the bacterial liquid reaches the appropriate growth stage, use it as seed liquid for later use.

[0026] 3. Preparation of liquid bacterial agent: Add liquid seeds to the enzyme-producing culture medium at an inoculation rate of 8%. Fermentation is carried out in a constant temperature shaker at 28°C and 120 r / min. The culture time is 24 to 48 hours, and the growth of the bacterial liquid is checked regularly. After the culture is completed, the bacterial liquid is collected for subsequent treatment.

[0027] MEA culture medium: purchased from Qingdao Haibo Biological Co., Ltd., Malt Extract Agar Medium, product number: HB8892.

[0028] The liquid seed culture medium was: 10 g glucose, 5 g peptone, 5 g yeast extract, 1 g potassium dihydrogen phosphate, 0.2 g magnesium sulfate, 20 g sodium chloride, 1000 mL distilled water, and the pH was adjusted to 7.0.

[0029] The enzyme production medium is: KH 2 PO 4 2 g, (NH 4 ) 2 S0 4 1.4 g MgSO 4 7H 2 O 0.3 g, CaCl 2 0.3 g, FeSO 4 7H 2 O 0.005 g, MnSO 4 0.0016 g, ZnSO 4 7H 2 O 0.0014 g, CoCl 2 0.002 g, CMC-Na 5 g, peptone 5 g, distilled water 1000 mL, pH 7.0, sterilize at 121 ℃ for 30 min.

[0030] 2. Single factor optimization experiment According to the principle of step-by-step improvement, the enzyme activity value was used as the only evaluation variable to conduct single factor optimization experiments on the culture conditions of Penicillium nucleatum, including culture time, culture temperature, culture pH value, and rotation speed. The initial conditions were set as: 28℃, pH value 6.5, inoculation size 8%, and rotation speed 120 r / min.

[0031] The single factor optimization experimental method is as follows: (1) Cultivation time optimization: Under the initial conditions, the cultivation 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 at different cultivation times were measured to screen the optimal cultivation time. Figure 1a As shown in the figure, the culture time has a great influence on the activity values ​​of the four enzymes. When the culture time is short or long, the activity values ​​of the four enzymes are low. When the culture time is 4 days, the activity of the three enzymes is better, and the protease activity is better on the 3rd day, but considering comprehensively, the best culture condition is 4 days.

[0032] (2) Optimization of culture temperature: Based on the optimal culture time, the culture temperature was set as the dependent variable, which was 22°C, 24°C, 26°C, 28°C and 30°C. The enzyme activity values ​​at different culture temperatures were measured to screen the appropriate culture temperature. Figure 1b As shown in the figure, the effect of high temperature on the enzyme activity value of the strain is somewhat different. The overall trend of the four enzyme activities first increased and then decreased with the increase of temperature. Therefore, the subsequent culture conditions were set to 26 ℃ for 4 days based on comprehensive considerations.

[0033] (3) Optimization of culture pH: Under the optimal culture time and temperature conditions, the pH value of the culture medium was adjusted to 5.5, 6.0, 6.5, 7.0 and 7.5, and the enzyme activity under different pH conditions was measured to 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.

[0034] (4) Speed ​​optimization: Keep the optimal culture time, culture temperature and pH value unchanged, adjust the speed, set it 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.

[0035] Experimental results and analysis: The experimental results show that the enzyme activity is relatively stable under the initial conditions. The following optimal conditions were obtained through single-factor optimization experiments: The optimal conditions are recommended as follows: 26 ℃, pH 6.0, rotation speed 130 r / min, 4 d. Through the single-factor optimization experiment, the enzyme production capacity of the strain was successfully improved, providing a scientific basis for the production of microbial agents. The optimized microbial agent has significant application value in agricultural waste treatment and resource utilization.

[0036] Example 2: Application of dual-degradation bacterial agent in the transformation of agricultural waste by black soldier flies 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 incubation for 2 to 3 days. After obvious fluidity appeared, the eggs were transferred to coarse wheat bran for cultivation.

[0037] The Penicillium sclerotiorum ( Penicillium sclerotigenum ) The bacterial solution was inoculated into a culture box containing 2000g of a substrate mixed with chicken manure, stevia straw, and fish viscera in a mass ratio of 2:1:1. The treatment group was set as T. The liquid bacterial solution was weighed according to 1-3% of the total substrate mass and diluted with sterile saline. Then, the different bacterial solutions were evenly sprayed on the substrate surface before inoculating insects. Using the plate count method, the total viable count of the Penicillium nucleatum solution in the agricultural waste was controlled to be 0.78×10 8 ~1.12×10 8 CFU / g. The feeding conditions were adjusted to a substrate carbon-nitrogen ratio of 25 and a moisture content of 70% using sawdust. 3g of black soldier fly eggs were added for further feeding, placed in an artificial climate chamber, and cultured in an open environment away from light at a constant temperature of 30°C and a constant humidity of 30%. The control group was treated with sterile water instead of bacterial solution as CK. Three replicates were set for each group.

[0038] When 50% of the larvae enter the prepupa stage, the insect excrement is separated. The insect body and the insect sand are separated by sieving with a filter, and the insect sand and the insect body are sampled and weighed.

[0039] The optimization effect of the efficiency of black soldier fly larvae in treating organic waste after adding Penicillium sclerotigenum was evaluated by calculating the 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: WR (%) = consumed dry substrate weight (g) / initial feed dry substrate weight (g) × 100%; SBC (%) = prepupa weight (g) / initial feed dry substrate weight (g) × 100%; BR (%) = total biomass of fresh larvae (g) / added fresh substrate (g) × 100%; Growth rate (g / d) = (fresh weight of worms after feeding - initial fresh weight of larvae) / number of days of feeding.

[0040] Table 1 shows the addition of Penicillium nucleatum ( Penicillium sclerotigenum ) treatment group and the control group without addition of biotransformation capacity results.

[0041]

[0042] As shown in Table 1, compared with the control group (CK) without adding microbial agents, the waste reduction rate increased after adding microbial agents to agricultural waste (T treatment), showing good results. 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 ) can enhance the conversion efficiency of waste to larval biomass. The conversion ratio of substrate to black soldier fly prepupae (SBC) and growth rate (Growth rate) also showed significant improvement.

[0043] The separated black soldier fly body samples were cleaned and the surface impurities were removed. The samples were placed in a 65°C oven and dried to constant weight. The dried samples were ground into 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 body were determined. The nutritional effect of the black soldier fly body after adding Penicillium nucleatum was evaluated.

[0044] Figure 2 The changes in the dry matter and protein content of black soldier flies under different treatment groups (CK and T) were shown. The dry matter content of the control group (CK) was 21.28±0.68. After adding the microbial agent, the dry matter content increased significantly, which was 34.68% higher than that of the control group. In terms of protein, after adding the microbial agent, the protein content of the treatment group (T) was further increased to 36.33±0.78. Compared with the control group, the protein content of the treatment group increased by 29.66%, which was a significant effect. In summary, treatment (T) significantly increased the dry matter and protein content of black soldier flies, which had obvious advantages over the control group. It shows that the microbial agent has a positive effect on optimizing insect growth and nutrient accumulation.

[0045] 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, concentrations 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 changes, uses or improvements to 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 for improving the bioconversion ability of black soldier flies on agricultural waste, characterized in that: The steps include: In the presence of Penicillium sclerotogenum ( Penicillium sclerotigenum ) to inoculate black soldier fly larvae in agricultural wastes, culture them, and collect insect sand and insect bodies; The agricultural waste consists of livestock and poultry excrement, straw and aquatic waste.

2. The method for breeding black soldier flies for improving the bioconversion ability of black soldier flies on agricultural waste according to claim 1, characterized in that: The aquatic waste is fish excrement and / or fish aquatic product processing waste; preferably, the fish aquatic product processing waste includes one or both of fish bones and fish viscera.

3. 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 stalks and fish viscera; preferably, the agricultural waste consists of chicken manure, stevia stalks and fish viscera in a mass ratio of 2:1:

1.

4. 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 3, characterized in that: The effective viable count of the Penicillium sclerotiorum in the agricultural waste was 0.89×10 8 ~1.02×10 8 CFU / g.

5. 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 4, characterized in that: The sclerotinia Penicillium is added in the form of a bacterial agent, and the preparation method of the bacterial agent comprises the following steps: inoculating the seed bacterial liquid of the sclerotinia Penicillium into an enzyme production culture medium for culturing to obtain the bacterial agent; preferably, the culture conditions are as follows: culture temperature is 26°C, culture medium pH is 6.0, seed liquid inoculation amount is 8%, rotation speed is 130 r / min, and culture time is 4 d.

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 to 5, characterized in that: Before the inoculation of black soldier fly larvae, the carbon-nitrogen ratio of the agricultural waste is adjusted to 25-30.

7. 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 6, characterized in that: Before the inoculation of black soldier fly larvae, the moisture content of the agricultural waste is adjusted to 70% to 75%.

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 to 7, characterized in that: The black soldier fly larvae are several days old black soldier fly larvae, preferably 2 to 3 days old.

9. 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 8, characterized in that: Inoculate 3 g of the black soldier fly larvae per 2000 g of substrate; and / or, The culture is carried out in an open-air manner at a constant temperature of 30°C and a constant humidity of 30% in the dark.

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 9, characterized in that: The improvement of the bioconversion ability of black soldier flies on agricultural waste is embodied in at least one of the following aspects (A1) to (A6): (A1) Increase waste reduction rates; (A2) Improve bioconversion 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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