Method for improving conversion efficiency of kitchen waste by using black soldier fly feces pre-fermentation

Abstract

The present application belongs to the technical field of organic solid waste resource utilization and insect bioconversion, and relates to a method for improving conversion efficiency of kitchen waste by using pre-fermentation of Hermetia illucens feces. Unsterilized feces obtained after treating kitchen waste by the previous batch of Hermetia illucens is mixed with kitchen waste at a dry basis mass ratio of 1:1, the moisture content is adjusted to 75%, and after nitrogen replacement to form an anaerobic environment, pre-fermentation is carried out at 35 DEG C for 5 days, and no exogenous microbial inoculum is added in the whole process; the obtained pre-fermentation product is directly used as a Hermetia illucens larva feeding substrate without sterilization. The examples show that the method can improve the average larva weight and growth rate, reduce the feed conversion rate, and is beneficial to form a fermentation environment with lactic acid accumulation and reduced ethanol.

Description

Technical Field

[0001] This invention belongs to the field of organic solid waste resource utilization and insect biotransformation technology, specifically involving a method for pre-fermenting kitchen waste using black soldier fly frass and then using it for the transformation of black soldier fly larvae. Background Technology

[0002] Food waste is a significant component of urban organic solid waste, characterized by high moisture content, complex composition, and susceptibility to decay and spoilage. Black soldier fly larvae can convert food waste into insect biomass and residual organic fertilizer, combining volume reduction and resource recovery value, thus becoming one of the important technical pathways for the biological treatment of food waste.

[0003] To improve substrate palatability and nutrient utilization, existing technologies typically employ two approaches: one is to directly use food waste as a rearing substrate for black soldier fly larvae; the other is to ferment or pre-ferment the food waste before feeding. For the latter, exogenous microorganisms such as lactic acid bacteria, yeast, EM bacteria, or compound microbial agents are often used to pre-treat the food waste, aiming to alter the substrate's physicochemical state and microbial community structure.

[0004] However, the exogenous microbial agent route often faces problems such as increased agent costs, significant impact on microbial community stability due to raw material fluctuations and operating conditions, and uncertain compatibility with the digestive microecology of black soldier flies. Meanwhile, existing technologies also include schemes for the return, reprocessing, or reuse of insect excrement, but most only consider insect excrement as part of the material composition, failing to highlight the use of unsterilized insect excrement obtained from the previous batch of food waste treated by black soldier flies as a host-source microbial inoculum, and the direct participation of its retained active microorganisms in subsequent food waste pre-fermentation.

[0005] Therefore, it is still necessary to provide a method that does not rely on exogenous microbial agents, can utilize the microbial resources of the black soldier fly conversion system itself, and improve the ecological compatibility of food waste and black soldier fly digestion, thereby increasing the bioconversion efficiency. Summary of the Invention

[0006] The technical problem to be solved by this invention is: addressing the issues that existing pre-fermentation technologies for kitchen waste rely heavily on exogenous microbial agents, have high costs, and are not well-suited to the digestive ecology of black soldier fly larvae, this invention provides a method that uses unsterilized insect excrement obtained from the previous batch of kitchen waste treated by black soldier flies as a host-source microbial inoculum to perform anaerobic pre-fermentation of kitchen waste, and then directly uses the pre-fermentation products for black soldier fly larval transformation without sterilization.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: a method for improving the conversion efficiency of kitchen waste by pre-fermentation of black soldier fly frass, comprising the following steps: Collect the unsterilized insect excrement obtained during the collection of black soldier fly larvae after processing the previous batch of kitchen waste, and mix it thoroughly. The insect excrement and kitchen waste were mixed at a dry basis mass ratio of 1:1. The moisture content of the mixed substrate was adjusted to 75%. The mixture was placed in a sealed fermentation container. After nitrogen replacement to form an anaerobic environment, it was pre-fermented at 35°C for 5 days to obtain the pre-fermented product. No additional exogenous microbial agents are added during the pre-fermentation and subsequent biotransformation process, and the pre-fermentation product is directly used as a feeding substrate for black soldier fly larvae for biotransformation.

[0008] Preferably, the insect excrement is stored at 4°C for no more than 8 days and then restored to room temperature before use.

[0009] Preferably, the kitchen waste is treated by removing non-organic impurities, thawing, and re-mixing before mixing, and is in a homogeneous semi-solid state with uniform particle distribution.

[0010] Preferably, the mixed substrate is maintained in a closed manner for anaerobic fermentation after nitrogen replacement.

[0011] Preferably, in step S3, 8-day-old black soldier fly larvae are used for biotransformation, where 8 days refers to the larvae obtained after black soldier fly eggs are cultured in wheat bran substrate for 8 days; more preferably, 200 black soldier fly larvae are inoculated for every 100 g of pre-fermented dry matter; the biotransformation conditions are 30°C and 70% relative humidity, and harvesting is carried out after 50% of the larvae have entered the prepupal stage, wherein the criterion for determining the prepupal stage is that the body color of the black soldier fly larvae gradually changes from milky white to dark brown or black.

[0012] Beneficial effects First, this invention utilizes the insect excrement produced by the black soldier fly conversion system itself as the host-source microbial inoculum, eliminating the need for additional exogenous bacterial agents, thereby reducing pretreatment costs and simplifying the process.

[0013] Secondly, this invention involves short-term pre-fermentation of unsterilized insect excrement obtained from the previous batch of food waste treated by black soldier flies with the food waste under specific moisture content, temperature and anaerobic conditions. This is beneficial for forming a fermentation environment characterized by lactic acid accumulation and reducing the accumulation of adverse metabolites such as ethanol.

[0014] Third, the examples show that, compared with the sterilized insect excrement pre-fermentation control, when using the method of the present invention, the average weight of black soldier fly larvae reached 0.260 g, which was 17.66% and 32.70% higher than the 65℃ sterilization group and the 121℃ sterilization group, respectively; the larval growth rate reached 1.291, which was 23.03% and 32.97% higher, respectively; and the feed conversion ratio was 2.162, which was 8.47% and 14.49% lower, respectively.

[0015] Fourth, the embodiments also show that the method of the present invention is beneficial to increase the protein content of the insect body and reduce the fat content, while increasing the activity of acidic protease and cellulase in the larval intestine, thereby improving the utilization of protein and complex carbohydrates in kitchen waste by black soldier flies.

[0016] Fifth, the examples further demonstrate that when using the method of the present invention, the relative abundance of lactic acid bacteria in the pre-fermented substrate is high, the diversity of the larval gut microbiota is high, and functional bacteria such as Paenibacillus maintain a high abundance, indicating that the method is beneficial to maintaining a relatively stable substrate-gut microecology. Attached Figure Description

[0017] Figure 1 Figures showing the growth performance of black soldier flies after pre-fermentation in different treatment groups: (a) growth curve; (b) average weight of flies at harvest; (c) survival rate. Figure 2 Figure showing the nutritional composition of black soldier fly larvae in different treatment groups; Figure 3 Figures showing the nutrient composition of substrates before and after pre-fermentation for different treatment groups: (a) carbohydrate content; (b) protein content; (c) fat content; Figure 4 Figures showing the content of substrate metabolites before and after pre-fermentation in different treatment groups: (a) lactic acid content; (b) acetic acid content; (c) ethanol content; Figure 5 Figure 1 shows the intestinal digestive enzyme activity of black soldier fly larvae after pre-fermentation in different treatment groups. Figure 6 Diagrams showing the microbial community structure of substrates after pre-fermentation for different treatment groups: (a) phylum-level chord diagram; (b) genus-level heatmap; Figure 7 Figures showing the gut microbiota analysis of black soldier fly larvae after pre-fermentation in different treatment groups: (a) ACE index at OTU level; (b) Shannon index at OTU level; (c) genus level heatmap. Detailed Implementation

[0018] The present invention will be further described below with reference to embodiments and comparative examples, but the scope of protection of the present invention is not limited to the following embodiments. Unless otherwise specified, the raw materials, equipment, reagents and detection methods used are all conventionally selected in the art.

[0019] In this instruction manual, "insect excrement" refers to the residual substrate obtained from the feeding system after black soldier fly larvae have fed on kitchen waste during the larval collection stage; "host-derived microbial inoculum" refers to the active microbial community retained in the insect excrement that can participate in the subsequent pre-fermentation of kitchen waste; and "prepupal stage" refers to the stage in which the larvae's body color darkens, their feeding decreases significantly, and they exhibit migration behavior away from the feed.

[0020] Unless otherwise stated, the mass ratio of insect excrement to kitchen waste mentioned in this instruction manual is on a dry basis; "8-day-old black soldier fly larvae" refers to larvae obtained after black soldier fly eggs are cultured in wheat bran substrate for 8 days.

[0021] Example 1: Inoculation of pre-fermented kitchen waste with unsterilized insect excrement for black soldier fly bioconversion. Black soldier fly eggs were placed in wheat bran with a moisture content of 75% and cultured for 8 days at 30℃ and 70% relative humidity to obtain 8-day-old black soldier fly larvae.

[0022] Kitchen waste from the school cafeteria was collected, and after manually removing obvious non-organic impurities, it was mixed thoroughly and stored at -20℃ for later use. Before the experiment, it was thawed naturally at room temperature and mixed again. Additionally, insect excrement obtained during the collection of insects from the previous batch of black soldier fly-treated kitchen waste was collected, thoroughly mixed, and stored at 4℃ for a short period of 8 days. Before use, it was brought to room temperature. The basic physicochemical parameters of the kitchen waste, insect excrement, and pre-fermentation system are shown in Table 1.

[0023] Table 1 Basic physicochemical parameters of raw materials and pre-fermentation system Unsterilized insect excrement was mixed with the kitchen waste at a dry basis mass ratio of 1:1. The moisture content of the mixed substrate was adjusted to 75%. The mixture was then placed into a polyethylene bottle with a screw cap, purged with nitrogen, and sealed. The nitrogen flow rate was 1 L·min^-1, and the purging time was 5 min. Anaerobic pre-fermentation was carried out at 35℃ for 5 days to obtain the pre-fermented product. The pre-fermented product was directly used as a rearing substrate for black soldier fly larvae without further sterilization or exogenous inoculation.

[0024] Polyethylene plastic containers (170 mm × 120 mm × 70 mm) were used as rearing containers. 100 g of pre-fermented product (dry matter) was added to each container, and 200 8-day-old black soldier fly larvae were inoculated. The containers were cultured at 30℃ and 70% relative humidity. Larval growth was observed daily during the rearing period, and the substrate was gently turned periodically. Each treatment group had three replicates. Every two days, 20 larvae were randomly removed, cleaned, dried, weighed, and returned to their original rearing containers to record the growth curve. When 50% of the larvae entered the prepupal stage (i.e., when the larvae's body color gradually changed from milky white to dark brown or black), the larvae were separated from the remaining substrate, counted, and weighed.

[0025] Larval survival rate (SR) by N end / N start Calculations; Larval growth rate (GR) is calculated based on Wgain / D; Feed conversion ratio (FCR) is calculated based on W initial / W gain Calculation; Bioconversion Rate (BCR) in Wprepupa / W initial Calculation: The Waste Reduction Index (WIR) is calculated as (Winitial - Wresidue) / D, where Nend is the number of surviving larvae at the end of the experiment, Nstart is the number of larvae introduced at the beginning of the experiment, Wgain is the larval weight gain, D is the number of development days, and W... initial W represents the total weight of the substrate added at the start of the experiment. prepupa W represents the total weight of larvae harvested at the end of the experiment. residue This represents the remaining matrix weight.

[0026] Comparative Example 1 After pasteurizing the insect excrement at 65°C for 60 minutes, it was mixed with kitchen waste, pre-fermented, and then used to feed black soldier fly larvae, following the same method as in Example 1. This comparative example was set up to remove as many microorganisms as possible while preserving the original nutrient composition of the insect excrement to a greater extent.

[0027] Comparative Example 2 The insect excrement was sterilized by high-pressure steam at 121°C for 30 minutes at a pressure of 0.25 MPa. Then, it was mixed with kitchen waste, pre-fermented, and black soldier fly larvae were fed to it, following the same method as in Example 1. This comparative example was set up to completely inactivate the microorganisms in the insect excrement, in order to distinguish between the effects of active microorganisms and the influence of non-microbial factors brought about by the sterilization treatment.

[0028] Table 2 Key process parameters for Example 1 and comparative examples Detection and Data Processing Methods The protein content of the rearing substrate and insects was determined by the Kjeldahl method; the fat content was determined by the Soxhlet extraction method; and the carbohydrate content was determined by the phenol-sulfuric acid method.

[0029] The lactic acid content in the pre-fermented substrate was determined by high performance liquid chromatography; the acetic acid and ethanol content were determined by gas chromatography.

[0030] For the analysis of larval gut microbiota, 15 larvae were randomly collected and starved for 24 hours without feed. After surface disinfection with 75% ethanol and washing with sterile water, the intestinal tissue was dissected and collected. DNA was extracted using the DNeasy PowerSoil® kit, and the V3-V4 region of the 16S rRNA gene was sequenced using the Illumina high-throughput sequencing platform. The amplification primers were 338F and 806R.

[0031] All experimental groups were preferably set up with 3 parallel replicates. One-way ANOVA was used to compare the significance of differences in the means of different treatment groups. p<0.05 was considered to be significant. Figures 1 to 4Different lowercase letters indicate significant differences (p<0.05).

[0032] Table 3. Biotransformation performance of black soldier flies in different treatment groups The results showed that Example 1, which used unsterilized insect excrement in pre-fermentation, exhibited superior growth performance in black soldier fly larvae compared to the two sterilized insect excrement controls. Black soldier fly larvae growth performance is shown in [the table below]. Figure 1 The specific results are shown in Table 3.

[0033] As shown in Table 3, the average weight of black soldier fly larvae in Example 1 reached 0.260 g, which was 17.66% and 32.70% higher than that of Comparative Example 1 and Comparative Example 2, respectively; the larval growth rate reached 1.291, which was 23.03% and 32.97% higher, respectively; and the feed conversion ratio was 2.162, which was 8.47% and 14.49% lower than that of Comparative Example 1 and Comparative Example 2, respectively. The survival rate of each group remained above 94%, indicating that the method of the present invention does not have an adverse effect on larval survival.

[0034] Further testing showed that the protein content of the black soldier fly larvae in Example 1 was 28.74%, higher than that of Comparative Example 1 (26.51%) and Comparative Example 2 (25.88%), representing increases of 8.44% and 11.08%, respectively; the fat content was 45.68%, lower than that of Comparative Example 1 (47.88%) and Comparative Example 2 (48.73%), representing decreases of 4.59% and 6.25%, respectively. The nutritional composition of the larvae is shown below. Figure 2 This indicates that using unsterilized insect excrement for pre-fermentation is beneficial for increasing the protein content of insects and reducing their fat content.

[0035] See below for substrate nutrient composition before and after pre-fermentation in different treatment groups. Figure 3 .Depend on Figure 3 It is evident that the pre-fermentation and larval transformation processes are accompanied by changes in the main nutrient components of the substrate, such as carbohydrates, proteins, and fats.

[0036] Regarding the major metabolites of the pre-fermented substrate, the lactic acid content in Example 1 reached 10.67 g / L, which was 6.16% and 13.68% higher than that in Comparative Example 1 and Comparative Example 2, respectively. The acetic acid content in Example 1, Comparative Example 1, and Comparative Example 2 was 3.77 g / L, 3.15 g / L, and 1.20 g / L, respectively, and the ethanol content was 3.79 g / L, 8.56 g / L, and 12.56 g / L, respectively. The pH values ​​before and after pre-fermentation were 6.24 / 4.42, 6.28 / 4.66, and 6.26 / 4.75, respectively, indicating that Example 1 was more conducive to the formation of a fermentation environment with lactic acid accumulation and ethanol reduction. See [link to specific results] for details. Figure 4 And Table 4.

[0037] Table 4. Major metabolites and pH of pre-fermented substrates in different treatment groups Regarding the activity of digestive enzymes in the larval gut, in Example 1, the acidic protease activity was 45.73 U / g, which was 2.58 times and 3.38 times that of Comparative Example 1 (17.69 U / g) and Comparative Example 2 (13.53 U / g), respectively; the cellulase activity was 922.38 U / g, which was higher than that of Comparative Example 1 (899.65 U / g) and Comparative Example 2 (659.11 U / g); the amylase activities were 36.82 U / g, 34.59 U / g, and 33.88 U / g, respectively; and the lipase activities were 954.64 U / g, 1066.18 U / g, and 1068.49 U / g, respectively. See the detailed results below. Figure 5 As shown in Table 5, and in conjunction with Table 3, this change corresponds to an increase in larval growth rate and a decrease in feed conversion rate.

[0038] Table 5. Intestinal digestive enzyme activities of larvae in different treatment groups Microbial community analysis showed that the pre-fermented substrate of Example 1 had a relatively high abundance of lactic acid bacteria, a high diversity of larval gut microbiota, and a high abundance of functional bacteria such as Paenibacillus. Furthermore, the relative abundances of *Lactiplantibacillus*, *Companilactobacillus*, and *Levilactobacillus* in the pre-fermented substrate of Example 1 were 28.46%, 25.42%, and 18.16%, respectively, compared to 21.32%, 10.16%, and 8.21% in Comparative Example 2. The Shannon index of the larval gut in Example 1 was 1.57, higher than 0.79 in Comparative Example 1 and 1.34 in Comparative Example 2, while the Simpson indices were 0.37, 0.68, and 0.38, respectively. The relative abundances of *Providencia* in Example 1, Comparative Example 1, and Comparative Example 2 were 5.32%, 11.43%, and 8.30%, respectively, while *Paenibacillus* was 3.93% in Example 1 and 2.48% in Comparative Example 2. These results indicate that host-derived microorganisms retained in unsterilized insect feces can participate in pre-fermentation and further influence the larval gut microecological structure and nutrient utilization process. The substrate and larval gut microbiota structures are shown in the following figures. Figure 6 and Figure 7 .

[0039] Furthermore, the survival rates (SR) of Example 1, Comparative Example 1, and Comparative Example 2 were 0.9525, 0.9450, and 0.9425, respectively; the bioconversion rates (BCR) were 0.4614, 0.3967, and 0.3665, respectively; and the waste reduction indices (WIR) were 2.6511, 2.5889, and 2.2928, respectively. The specific results are shown in Table 6.

[0040] Table 6. Biotransformation supplementation indicators of black soldier fly larvae in different treatment groups The results above show that the unsterilized insect excrement obtained from the previous batch of black soldier fly treatment of kitchen waste can establish a substrate environment with lactic acid accumulation, ethanol reduction, and better compatibility with the intestinal microecology of larvae under specific anaerobic pre-fermentation conditions, thereby improving the biotransformation performance of kitchen waste.

[0041] This invention is not limited to the embodiments described above. Any equivalent substitutions, improvements, and adjustments made within the spirit and principles of this invention should fall within the protection scope of this invention.

Claims

1. A method for improving the conversion efficiency of kitchen waste by pre-fermenting black soldier fly frass, characterized in that, Includes the following steps: S1. Collect the unsterilized insect excrement obtained during the collection of the previous batch of processed kitchen waste from black soldier fly larvae and mix it thoroughly. S2. Mix the insect excrement obtained in step S1 with kitchen waste at a dry basis mass ratio of 1:1, adjust the moisture content of the mixed substrate to 75%, put it into a sealed fermentation container, and after nitrogen replacement to form an anaerobic environment, pre-ferment at 35℃ for 5 days to obtain the pre-fermented product. S3. No additional exogenous microbial agents are added during step S2 and subsequent biotransformation, and the pre-fermentation product is directly used as a feeding substrate for black soldier fly larvae for biotransformation.

2. The method according to claim 1, characterized in that, The insect excrement collected in step S1 is mixed and stored at 4°C for no more than 8 days, and then brought back to room temperature before use.

3. The method according to claim 1 or 2, characterized in that, The kitchen waste mentioned in step S2 is treated by removing non-organic impurities, thawing, and re-mixing before mixing, and is in a homogeneous semi-solid state with uniform particle distribution.

4. The method according to any one of the preceding claims, characterized in that, The mixed substrate described in step S2 is maintained in a closed manner for anaerobic fermentation after nitrogen replacement.

5. The method according to any one of the preceding claims, characterized in that, The pre-fermentation product obtained in step S2 is used directly for biotransformation in step S3 without sterilization after pre-fermentation.

6. The method according to any one of the preceding claims, characterized in that, In step S3, 8-day-old black soldier fly larvae are used. The 8-day-old refers to the larvae obtained after black soldier fly eggs are placed in wheat bran substrate and cultured for 8 days.

7. The method according to claim 6, characterized in that, In step S3, 200 black soldier fly larvae are inoculated with every 100 g of pre-fermented product of dry matter.

8. The method according to any one of the preceding claims, characterized in that, The biotransformation conditions in step S3 are 30°C and 70% relative humidity.

9. The method according to any one of the preceding claims, characterized in that, In step S3, a polyethylene plastic container is used as the rearing container, and the container opening is covered with gauze to maintain ventilation and prevent the larvae from escaping.

10. The method according to any one of the preceding claims, characterized in that, Step S3 continues until 50% of the larvae enter the prepupal stage, after which they are harvested. The criterion for determining the prepupal stage is that the body color of the black soldier fly larvae gradually changes from milky white to dark brown or black.

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