Lactobacillus reuteri and uses thereof

Abstract

The application discloses lactobacillus reuteri and application thereof, the lactobacillus reuteri C3 preservation number is CCTCC NO: M 20252241. The lactobacillus reuteri has the effects of regulating intestinal flora, enhancing immunity, relieving diarrhea, improving allergic symptoms and inhibiting helicobacter pylori. Black soldier fly larva worm pulp feed fermented by the lactobacillus reuteri has the advantages of high protein and low anti-nutritional factors, and can replace soybean meal or fish meal in the preparation of livestock and poultry feed to a certain extent. The lactobacillus reuteri can efficiently utilize high-nitrogen wastewater, convert the wastewater into high-value bacterial protein, realize the resource utilization of waste, reduce the wastewater treatment cost and promote the development of circular economy.

Description

Technical Field

[0001] This invention relates to the field of bio-fermented feed technology, specifically to a type of Lactobacillus reuteri and its applications. Background Technology

[0002] Black soldier flies (Hermetia illucens) possess numerous advantages, including a wide range of food sources, large food intake, a moderate growth and development cycle, strong resilience, tolerance to oil and salt, high ecological safety, and rich nutritional value in their larvae. Their waste processing ability is remarkable; from egg to mature larva (optimal harvest time), it only takes 12-18 days. This means multiple batches can be raised continuously throughout the year, resulting in extremely high land and time utilization. The economic value of black soldier flies lies not only in the output of their larvae but also in their unique biotransformation ability, converting "negative value" waste into "high value" products. Black soldier fly larvae contain various fatty acids, organic acids, and functional enzymes; fully developed larvae contain approximately 40%-50% protein. This offers significant advantages for high-end aquaculture sectors that primarily feed on meat.

[0003] Currently, the main application of black soldier fly larvae is to directly prepare them as insect powder or insect pulp and add them directly to feed. However, the chitin and high fat content in black soldier fly larvae powder / pulp are anti-nutritional factors, which can only partially replace traditional feed. High or complete replacement will lead to a decline in the growth performance of the animals.

[0004] Lactobacillus reuteri is a common probiotic that can regulate intestinal flora balance, enhance immune function, inhibit the growth of pathogens, and promote nutrient absorption. Studies have shown that lactic acid bacteria fermentation can improve the digestibility of feed ingredients, reduce anti-nutritional factors, and increase protein utilization. Therefore, applying Lactobacillus reuteri to the fermentation of black soldier fly larvae larvae can decompose unfavorable components through microbial fermentation, improve the nutritional characteristics and palatability of the larvae ...

[0005] In conclusion, exploring the application potential of Lactobacillus reuteri has significant application value and promotional significance. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a *Lactobacillus reuteri* strain and its applications. This *Lactobacillus reuteri* is a probiotic derived from the chicken intestine, possessing functions such as regulating intestinal flora, enhancing immunity, relieving diarrhea, improving allergy symptoms, and inhibiting *Helicobacter pylori*. Black soldier fly larvae larvae feed fermented with *Lactobacillus reuteri* has the advantages of high protein and low anti-nutritional factors, and can, to a certain extent, replace soybean meal or fishmeal in livestock and poultry feed preparation. Simultaneously, *Lactobacillus reuteri* can efficiently utilize high-nitrogen wastewater, converting it into high-value microbial protein, achieving resource utilization of waste; wastewater treatment costs are reduced, promoting the development of a circular economy.

[0007] To achieve the above objectives, the technical solution designed by the present invention is as follows:

[0008] This invention provides a Lactobacillus reuteri, named C3, with the preservation number CCTCC NO: M 20252241.

[0009] The above-mentioned Lactobacillus reuteri C3 ( Lactobacillus reuteri (strain C3), deposited at the China Center for Type Culture Collection, accession number: CCTCC NO: M 20252241, deposit date: October 17, 2025, address: Wuhan University, Wuhan, China.

[0010] The present invention also provides the application of the above-mentioned Lactobacillus reuteri C3 in the preparation of black soldier fly larvae feed.

[0011] This invention also provides a method for preparing black soldier fly larvae feed, comprising the following steps:

[0012] 1) Preparation of black soldier fly larvae by-products;

[0013] 2) Weigh out the black soldier fly larvae by-products, water, and basic feed at a mass ratio of 1:4~7:15~20;

[0014] 3) First, mix the black soldier fly larvae by-products and the basic feed evenly, then add water and mix to obtain a mixture;

[0015] 4) Activate and culture the above-mentioned Lactobacillus reuteri C3 to obtain Lactobacillus reuteri C3 bacterial solution; wherein, the OD value of Lactobacillus reuteri C3 bacterial solution is 1~2;

[0016] 5) Add Lactobacillus reuteri C3 bacterial solution to the mixture; ferment to obtain black soldier fly larvae feed; wherein, the amount of Lactobacillus reuteri C3 bacterial solution added is 1~4mL per 100g of basic feed and insect pulp.

[0017] Furthermore, in step 1), the black soldier fly larvae byproduct is black soldier fly larvae larvae plasma or black soldier fly protein;

[0018] The larval larvae of the black soldier fly contain 10-15% chitin, 40-50% protein, and 30-40% oil.

[0019] Furthermore, the black soldier fly protein is prepared by the following steps:

[0020] a. Crush the black soldier fly larvae into a paste using a grinder.

[0021] b. The insect slurry is added to a stirred and heated reactor for alkaline hydrolysis. After filtration, the chitinous shell is removed to obtain insect protein. The alkaline solution of the alkaline hydrolysis is a sodium hydroxide solution with a mass fraction of 0.1~1.0%.

[0022] Furthermore, the sodium hydroxide solution has a mass fraction of 0.5%.

[0023] Furthermore, in step 2),

[0024] The ingredients of the basic feed, by weight percentage, include 67% corn flour, 20% soybean meal, 8% fish meal, 1.2% limestone powder, 1.5% dicalcium phosphate, 0.3% salt, 1% broiler premix and 1% vegetable oil.

[0025] Furthermore, in step 5), the amount of Lactobacillus reuteri C3 bacterial solution added is 2 mL per 100 g of basic feed and insect slurry.

[0026] The present invention also provides the application of the above-mentioned Lactobacillus reuteri in the preparation of fermented products of black soldier fly larvae.

[0027] This invention also provides a method for preparing a fermented product of black soldier fly larvae, comprising the following steps:

[0028] 1) Crush fresh black soldier fly larvae into a paste and autoclave.

[0029] 2) Activate the above-mentioned Lactobacillus reuteri C3, culture it, and obtain Lactobacillus reuteri C3 bacterial culture;

[0030] 3) Add the above-mentioned Lactobacillus reuteri C3 bacterial solution to the insect slurry, ferment, and obtain the fermented product of black soldier fly larvae.

[0031] Furthermore, in step 1), the insect slurry contains 10-15% chitin, 40-50% protein, and 30-40% oil.

[0032] In step 3), 10-30 mL is added to every 1 kg of insect slurry;

[0033] Fermentation temperature is 20~30℃, fermentation time is 2~4 days.

[0034] The present invention also provides a black soldier fly compound feed, the compound feed comprising a basic feed and the above-mentioned black soldier fly larvae fermented product, wherein the weight ratio of the black soldier fly larvae fermented product to the basic feed is 1:18~20.

[0035] The present invention also provides an application of the above-mentioned Lactobacillus reuteri C3 in the synthesis of protein in the treatment of high-nitrogen wastewater.

[0036] The beneficial effects of this invention are:

[0037] 1. The present invention isolates a probiotic strain of Lactobacillus reuteri from the chicken intestine, which has the effects of regulating intestinal flora, enhancing immunity, relieving diarrhea, improving allergy symptoms, and inhibiting Helicobacter pylori.

[0038] 2. The Lactobacillus reuteri fermentation of the present invention can improve the amino acid composition of feed, increase the proportion of growth-promoting and weight-gain-promoting amino acids, and improve the nutritional value of feed;

[0039] 3. The abundance of probiotics in the feed fermented with Lactobacillus reuteri of the present invention is significantly increased, which is beneficial to the intestinal health of animals, enhances the biological value of the feed, maintains the stability and quality of meat without affecting the fat and protein content of the meat, and at the same time improves the growth rate of chickens and improves meat quality.

[0040] 4. The compound feed of the present invention can replace 5% of the basic feed, significantly improve the growth rate of broilers, promote healthy intestinal development, and does not affect meat quality.

[0041] 5. The Lactobacillus reuteri of the present invention can efficiently utilize high-nitrogen wastewater, converting the wastewater into high-value bacterial protein, thereby realizing the resource utilization of waste; the wastewater treatment cost is reduced, promoting the development of a circular economy.

[0042] This invention provides an innovative technical approach for the high-value utilization of black soldier fly resources, the sustainable development of livestock and poultry feed, and the resource utilization of waste, and has significant economic, social, and ecological benefits. Attached Figure Description

[0043] Figure 1 A schematic diagram of Gram staining of Lactobacillus reuteri in chicken intestines;

[0044] Figure 2 A diagram illustrating the preparation process of black soldier fly larvae larval plasma;

[0045] Figure 3 A schematic diagram showing the pH value of the fermentation product after different fermentation days;

[0046] Figure 4 Abundance diagrams of community structure for basic feed, black soldier fly larvae slurry mixed feed 3, and black soldier fly larvae slurry fermented feed 7;

[0047] Figure 5 Weight measurement charts for broiler chickens fed with different feeds;

[0048] In the figure, a indicates no significant difference between groups, and b indicates a significant difference between groups;

[0049] Figure 6 Chart showing the weight of chicken breasts and legs of broilers fed different feeds;

[0050] In the figure, a indicates no significant difference between groups, and b indicates a significant difference between groups;

[0051] Figure 7 The images show the effects of feeding broiler chicken breasts and legs with mixed feed 3 and fermented feed 7, respectively.

[0052] In the diagram, A shows the effect of feeding broiler chicken breasts and legs with mixed feed 3.

[0053] B is an image showing the effect of feeding broiler chicken breasts and legs with fermented feed.

[0054] Figure 8 A schematic diagram showing the fat and protein content of broilers fed with different feeds;

[0055] Figure 9 This is a diagram illustrating the preparation process of black soldier fly protein.

[0056] Figure 10 A schematic diagram showing the protein content after hydrolysis with sodium hydroxide solution or water of different concentrations;

[0057] Figure 11 Weight measurements of broilers fed with different protein diets;

[0058] In the figure, a indicates no significant difference between groups, and b indicates a significant difference between groups;

[0059] Figure 12 A comparison chart of the weight of chicken breasts and legs in broilers fed with different protein diets;

[0060] In the figure, a indicates no significant difference between groups, and b indicates a significant difference between groups; Figure 13 A schematic diagram showing the pH value of the fermentation product checked by different strains at different fermentation days;

[0061] Figure 14 Figure 3 shows the detection of oil and protein content in insect pulp and fermented product 3 of black soldier fly larvae insect pulp.

[0062] Figure 15pH values ​​of fermented insect slurry products with different moisture contents after different weeks are shown in the graph.

[0063] Figure 16 Weight measurement chart for comparing broiler chickens fed with feed and compound feed;

[0064] Figure 17 A comparison chart showing the weight of chicken breasts and legs in broilers fed with feed and compound feed.

[0065] Figure 18 A comparison chart showing the jejunal length of broilers fed with a feed and a compound feed;

[0066] Figure 19 A photograph of the jejunum of broilers fed with feed and compound feed for comparison;

[0067] In the figure, A is a photograph of the jejunum of broiler chickens fed with a control feed;

[0068] B is a picture of the jejunum of broiler chickens fed with compound feed;

[0069] Figure 20 A comparison chart showing the protein content synthesized by Lactobacillus reuteri inoculated into basal culture medium and chicken manure wastewater, respectively;

[0070] In the figure, A is a schematic diagram of the protein content synthesized by Lactobacillus reuteri inoculation basal culture medium;

[0071] B is a schematic diagram showing the protein content synthesized from chicken manure wastewater inoculated with Lactobacillus reuteri. Detailed Implementation

[0072] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0073] Example 1: Isolation and Identification of Lactobacillus reuteri from Chicken Intestine

[0074] 1. Separation and morphological identification

[0075] Sacrifice healthy broiler chickens, separate the ileum, and cut off a segment (approximately 2-3 cm) using a sterile scalpel. Make a longitudinal incision and gently rinse with sterile saline to remove contents and residual food, thus removing bacteria. Place the intestinal tissue segment into a sterile homogenizing bag or tube. Add a specific volume (e.g., 9 mL) of sterile phosphate-buffered saline (PBS) or physiological saline to the bag. Homogenize at high speed for 1-2 minutes using a beater homogenizer, or grind in a sterile mortar to prepare an intestinal tissue homogenate. Perform a 10-fold serial dilution (e.g., 10... - ¹, 10 - ², 10 - ³, 10 -4 10 -5 ).

[0076] MRS medium was used, which is the classic and preferred selective medium for isolating lactic acid bacteria (especially Lactobacillus). Its low pH (approximately 5.7) and high concentrations of carbon and nitrogen sources inhibit most contaminating bacteria and promote the growth of Lactobacillus. 100 μL of each medium was spread onto MRS agar plates. The plates were incubated anaerobically at 37°C for 48–72 h. After incubation, single colonies were observed on the plates.

[0077] Gram staining: Lactobacillus is a Gram-positive bacterium and should appear purple under a microscope ( ). Figure 1 ).

[0078] 2. Molecular identification

[0079] Genomic DNA extraction: High-quality total DNA is extracted from freshly cultured bacterial cultures using commercially available bacterial genomic DNA extraction kits.

[0080] 16S rRNA gene sequencing: PCR amplification: PCR amplification of the bacterial 16S rRNA gene was performed using universal primers. The universal primers are:

[0081] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', as shown in SEQ ID NO.2

[0082] 1492R: 5'-GGTTACCTTGTTACGACTT-3', as shown in SEQ ID NO.3;

[0083] Purification and sequencing: The PCR products were purified and sent to a sequencing company for sequencing.

[0084] Sequence analysis and alignment: The obtained 16S rRNA gene sequence is shown in SEQ ID NO.1:

[0085]

[0086] Compare with the database at (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0087] Sequence alignment with the type strain showed a sequence similarity >99.9%, identifying it as *Lactobacillus reuteri*. Lactobacillus reuter i strain), strain numbered C3.

[0088] The above-mentioned Lactobacillus reuteri C3 ( Lactobacillus reuteri (strain C3), deposited at the China Center for Type Culture Collection, accession number: CCTCC NO: M 20252241, deposit date: October 17, 2025, address: Wuhan University, Wuhan, China.

[0089] Example 2

[0090] The preparation method of fermented feed 1 containing black soldier fly larvae includes the following steps:

[0091] 1. Preparation of Black Soldier Fly Powder

[0092] Fresh black soldier fly larvae were pulverized into insect pulp using a grinder. Figure 2 The chitin content was found to be 10-15%, the protein content 40-50%, and the fat content 30-40%.

[0093] 2) Basic feed formulation

[0094] Table 1 Basic Feed Formulation

[0095]

[0096] 3) Activate and culture Lactobacillus reuteri C3 to obtain Lactobacillus reuteri C3 bacterial solution; wherein, the OD value of Lactobacillus reuteri C3 bacterial solution is 1~2;

[0097] 4) Weigh out the black soldier fly larvae by-products, water, and basic feed according to the formula in item 1 of Table 2 below;

[0098] Table 2

[0099]

[0100] 5) First, mix the black soldier fly larvae by-products and the basic feed evenly, then add water and mix to obtain a mixture;

[0101] 6) Add Lactobacillus eeleris C3 bacterial solution to the mixture according to the formula in serial number 1 in Table 2; ferment for 1 day, 2 days, 3 days and 4 days at room temperature (20~30℃) to obtain black soldier fly larvae larvae fermented feed 1~4 respectively.

[0102] Examples 6-9

[0103] The fermented feeds of black soldier fly larvae prepared in Examples 6-9 are basically the same as those prepared in Example 1, except for steps 4) and 6).

[0104] 4) Weigh out the black soldier fly larvae by-products, water, and basic feed according to the formula in item 2 of Table 2 below;

[0105] 6) Add Lactobacillus eeleris C3 bacterial solution to the mixture according to the formula in serial number 2 in Table 2; ferment for 1 day, 2 days, 3 days and 4 days at room temperature (20~30℃) to obtain 5~8 black soldier fly larvae slurry fermented feed.

[0106] Examples 10-14

[0107] The fermented feeds of black soldier fly larvae prepared in Examples 10-14 are basically the same as those prepared in Example 1, except for steps 4) and 6).

[0108] 4) Weigh out the black soldier fly larvae by-products, water, and basic feed according to the formula in item 3 of Table 2 below;

[0109] 6) Add Lactobacillus eeleris C3 bacterial solution to the mixture according to the formula in serial number 3 in Table 2; ferment for 1 day, 2 days, 3 days and 4 days at room temperature (20~30℃) to obtain black soldier fly larvae larvae fermented feed, and obtain 9~12 black soldier fly larvae larvae fermented feed.

[0110] Comparative Examples 1-4

[0111] The preparation method of black soldier fly larvae larval insect pulp mixed feed 1-4 includes the following steps:

[0112] 1. Preparation of Black Soldier Fly Powder

[0113] Fresh black soldier fly larvae were crushed into insect pulp using a pulverizer. The chitin content was found to be 10-15%, the protein content 40-50%, and the oil content 30-40%.

[0114] 2) The basic feed formula is the same as above;

[0115] 3) Weigh out the black soldier fly larvae by-products, water, and basic feed according to the formula in Table 3 below;

[0116] Table 3

[0117]

[0118] 4) First, mix the black soldier fly larvae by-products and the basic feed evenly, then add water and mix to obtain a mixture; place the mixture at room temperature (20~30℃) for 1 day, 2 days, 3 days and 4 days respectively to obtain mixed feed 1~4.

[0119] 1. Check the pH value of the fermented product according to the formulas in Tables 2 and 3 for different fermentation days.

[0120] like Figure 3 As shown: pH testing revealed that the pH values ​​of all formulations with added *Lactobacillus reuteri* showed a decreasing trend during fermentation. The pH decreasing trends were similar for formulation 2 (containing 2 mL of *Lactobacillus reuteri*) and formulation 3 (containing 4 mL of *Lactobacillus reuteri*). Therefore, formulation 2 (containing 2 mL of *Lactobacillus reuteri*) exhibited similar pH values ​​(pH 5.0) after 3 and 4 days of fermentation. Preliminary evaluation of the fermentation effect using the pH decrease rate showed no significant difference in pH values ​​after 3 and 4 days. Therefore, the fermented feed 7 obtained after 3 days of fermentation using the raw materials from formulation 2 is preferred.

[0121] II. Testing was conducted on the basic feed, the black soldier fly larvae larvae slurry mixed feed 3, and the black soldier fly larvae slurry fermented feed 7.

[0122] 1. Amino acid composition analysis:

[0123] The amino acid composition of the basal feed, the black soldier fly larvae slurry mixture 3, and the fermented black soldier fly larvae slurry 7 was analyzed by HPLC. After fermentation with Lactobacillus reuteri, the contents of valine, isoleucine, tyrosine, and lysine increased. The efficacy analysis of the amino acids is as follows:

[0124] Valine: Promotes weight gain, improves feed efficiency (reduces feed conversion ratio), and maintains normal feed intake.

[0125] Isoleucine: Increases feed intake and promotes growth.

[0126] Tyrosine: Promotes weight gain and improves feed efficiency.

[0127] Lysine: Promotes growth, increases weight gain, and improves feed efficiency.

[0128] Table 4

[0129]

[0130] As shown in Table 4, fermentation with Lactobacillus truncatum can improve the amino acid composition of feed, increase the proportion of some growth-promoting and weight-gain-promoting amino acids, and improve the nutritional value of feed.

[0131] 2. Microbial abundance detection:

[0132] Microbial community genomic DNA was extracted from the feed using a rapid DNA rotation extraction kit. The abundance of community structure was analyzed using Trimmomatic quality control within the standard protocol of the Illumina MiSeq platform (Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China)).

[0133] The test results belong to horizontal analysis, such as Figure 4 As shown: The intestinal probiotics of fermented feed 7 mainly include Lactobacillus such as Lactobacillus reuteri, with a colony abundance of 57.28%, which is higher than 23.69% in the basal feed group and 21.28% in mixed feed 3.

[0134] 3. Feed used in broiler chicken experiments

[0135] Select 1-day-old AA white-feathered broiler chickens. For the first week, they were mixed-species rearing to ensure acclimatization and to cull weaker chickens. Starting in the second week, they were divided into groups of 20 chickens each. Group 1 was fed a basal feed, Group 2 a mixed feed (3), and Group 3 a fermented feed (7). Feeding and watering were provided daily, and the broiler chickens' growth was observed. After 6 weeks of this feeding program, the chickens were euthanized, and the growth performance of each group was assessed.

[0136] 3.1 Weight Inspection

[0137] like Figure 5 As shown: At 1 week of age, the weights of the three groups of chickens were 130±6g, 130±5g, and 130±8g, respectively, with no significant difference among the three groups (P>0.05). At 6 weeks of age, the weights of the three groups of chickens were 2452±136g, 2496±141g, and 2588±143g, respectively. The weights of the two feed-fed groups were slightly higher than those of the feed-fed group 1, but the difference was not significant (P>0.05). The weights of the three feed-fed groups were significantly higher than those of the feed-fed groups 1 and 2 (P<0.05). This indicates that crushed black soldier fly larvae pulp can be used as a basic protein substitute in chicken feed without affecting chicken growth. Lactobacillus reuteri fermented black soldier fly larvae pulp feed can promote broiler growth.

[0138] 3.2 Chicken leg and chicken breast meat testing

[0139] like Figures 6-7 As shown, at 6 weeks of age, the weights of chicken breasts in the three groups were 530±60g, 531±55g, and 567±46g, respectively. There was no significant difference in chicken breast weight between the two feed groups and the first feed group (P>0.05). The weights of chicken breasts in the three feed groups were significantly higher than those in the first and second feed groups (P<0.05). This indicates that crushed black soldier fly larvae pulp can be used as a basic protein substitute in chicken feed without affecting the weight of chicken breasts and legs. Fermented black soldier fly larvae pulp feed using *Lactobacillus reuteri* can promote the growth of chicken breasts and legs.

[0140] 3.3 Meat quality testing

[0141] The fat content in chicken breast and chicken leg was determined by Soxhlet extraction, and the protein content in chicken breast and chicken leg was determined by Kjeldahl method.

[0142] like Figure 8 The results showed that there was no significant difference in the fat content of chicken breasts and legs among the three types of feed, and no significant difference in the protein content of chicken breasts and legs among the three types of feed.

[0143] Example 15

[0144] like Figure 9 The method for preparing black soldier fly protein shown includes the following steps:

[0145] a. Crush the black soldier fly larvae into a paste using a grinder.

[0146] b. Add the insect slurry and sodium hydroxide solution or water to a stirred and heated reactor according to the proportions in Table 5. Perform alkaline hydrolysis for 6 hours at a temperature of 80℃ and a stirring rate of 30 rpm. After filtration, remove the chitinous shell to obtain insect proteins 1-3 and control proteins.

[0147] Table 5

[0148]

[0149] 2. Protein content:

[0150] The protein content was determined using the Kjeldahl method, and samples were taken every hour to determine the protein concentration.

[0151] like Figure 10 As shown, the protein content of insect protein 2 is close to that of insect protein 3, with protein concentrations of 45.4±6g and 46±6g, respectively, both higher than that of insect protein 1 and the control group. Therefore, a 0.5% NaOH solution and a reaction time of 6 hours were selected as the optimal conditions for preparing insect protein.

[0152] Example 16

[0153] A method for preparing fermented protein feed from black soldier fly larvae includes the following steps:

[0154] 1) Weigh out the black soldier fly protein, water, and basic feed according to Table 6 below;

[0155] Table 6

[0156]

[0157] 2) First, mix the black soldier fly protein and the basic feed evenly, then add water and mix to obtain a mixture;

[0158] 3) Activate and culture Lactobacillus reuteri C3 to obtain Lactobacillus reuteri C3 bacterial solution; wherein, the OD value of Lactobacillus reuteri C3 bacterial solution is 1~2;

[0159] 5) Add Lactobacillus reuteri C3 bacterial solution to the mixture; ferment for 3 days at room temperature (20~30℃) to obtain black soldier fly protein fermented feed.

[0160] Comparative Example 5

[0161] A method for preparing black soldier fly protein-mixed feed includes the following steps:

[0162] 1) Weigh out the black soldier fly protein, water, and basic feed according to Table 7 below;

[0163] Table 7

[0164]

[0165] 2) First, mix the black soldier fly protein and the basic feed evenly, then add water and mix to obtain a mixture; ferment at room temperature (20~30℃) for 3 days to obtain the mixed feed.

[0166] I. Testing was conducted on the basic feed, insect protein mixed feed, and black soldier fly protein fermented feed:

[0167] 1. Amino acid composition analysis:

[0168] The amino acid composition of three groups of fermented feeds was analyzed using HPLC. After fermentation with *Lactobacillus reuteri*, the proportions of valine, isoleucine, and lysine increased. The efficacy analysis of the amino acids is as follows:

[0169] Valine: Promotes weight gain, improves feed efficiency (reduces feed conversion ratio), and maintains normal feed intake.

[0170] Isoleucine: Increases feed intake and promotes growth.

[0171] Lysine: Promotes growth, increases weight gain, and improves feed efficiency.

[0172] Table 8

[0173]

[0174] As shown in Table 7, fermentation with Lactobacillus reuteri can improve the amino acid composition of feed, increase the proportion of some growth-promoting amino acids, and improve the nutritional value of feed.

[0175] 2. Feed used in broiler chicken experiments

[0176] One-day-old AA white-feathered broiler chickens were selected. During the first week, they were mixed-species rearing to ensure acclimatization and to cull weaker chickens. Starting in the second week, they were divided into groups of 20 chickens each. Group 1 was fed a basal feed, Group 2 a mixed feed, and Group 3 a fermented insect protein feed. Feeding and watering were provided daily, and the broiler chickens' growth was observed. After 6 weeks, the chickens were euthanized, and the growth performance of each group was assessed.

[0177] 2.1 Weight Inspection

[0178] like Figure 1 At week-old age, the weights of the three groups of chickens were 135±5g, 135±5g, and 133±6g, respectively, with no significant difference among the three groups (P>0.05). At 6 weeks of age, the weights of the three groups of chickens were 2352±115g, 2489±143g, and 2513±165g, respectively. The weights of chickens fed protein-rich diets in groups 2 and 3 were significantly higher than those fed diet 1 (P<0.05). This indicates that insect protein can be used as a basic alternative protein in chicken feed. Lactobacillus reuteri fermented insect protein feed can promote the growth of broilers.

[0179] 2.2 Chicken thigh and chicken breast

[0180] like Figure 12 As shown, at 6 weeks of age, the weights of chicken breasts in the three groups were 515±55g, 542±34g, and 559±42g, respectively. The weight of chicken breasts in the three feed groups was significantly higher than that in feed groups 1 and 2 (P<0.05). The weights of chicken legs in the three groups were 346±23g, 378±23g, and 399±22g, respectively. The weight of chicken legs in the three feed groups was significantly higher than that of chicken breasts in feed groups 1 and 2 (P<0.05). This indicates that the Lactobacillus reuteri fermented insect protein diet can promote the growth of chicken breasts and legs.

[0181] Examples 17-19: Preparation of fermented black soldier fly larvae larvae slurry products

[0182] 1. Preparation of Black Soldier Fly Powder

[0183] Fresh black soldier fly larvae were crushed into insect pulp using a pulverizer. The chitin content was found to be 10-15%, the protein content 40-50%, and the oil content 30-40%. The pulp was then sterilized by high pressure to obtain sterilized insect pulp.

[0184] 2. Preparation and Detection of Insect Pulp Fermentation Products

[0185] The raw materials were prepared according to the formula in serial number 2 of Table 9, and fermented sequentially for 1 day, 2 days and 3 days at room temperature (20~30℃) to obtain black soldier fly larvae larvae fermentation products 1~3 respectively.

[0186] Table 9

[0187]

[0188] Comparative Examples 6-8

[0189] The methods used in Comparative Examples 6-8 are basically the same as those used in Examples 17-19, except that:

[0190] The raw materials were prepared according to the formula in serial number 3 of Table 9, and fermented sequentially for 1 day, 2 days and 3 days at room temperature (20~30℃) to obtain black soldier fly larvae larvae fermentation products D1~D3 respectively.

[0191] Comparative Examples 9-11

[0192] The methods of Comparative Examples 9-11 are basically the same as those of Examples 17-19, except that:

[0193] The raw materials were prepared according to the formula in No. 4 of Table 9, and fermented sequentially for 1 day, 2 days and 3 days at room temperature (20~30℃) to obtain black soldier fly larvae larvae fermentation products D4~D6 respectively.

[0194] 1. Check the pH value of the fermented product according to the formula in Table 9 and the fermentation time.

[0195] like Figure 13 As shown: pH testing of the products revealed a decreasing trend in pH value during the microbial fermentation of insect slurry. Among them, the pH value of the fermented product with added Lactobacillus reuteri in Formula 2 decreased at the fastest rate, reaching 4.7 after 3 days, which was significantly lower than the pH value of Formula 1 (6.0), Formula 3 (with added yeast) (5.1), and Formula 3 (with added lactic acid bacteria) (5.0). Therefore, the fermented insect slurry product obtained by adding Lactobacillus reuteri and fermenting for 3 days was selected.

[0196] II. Detection of insect slurry and fermented black soldier fly larvae slurry products 3

[0197] 1. Detection of oil and protein content in fermented products of insect pulp and black soldier fly larvae.

[0198] The oil content of insect slurry and fermented product 3 of black soldier fly larvae slurry was determined by Soxhlet extraction, and the protein content of insect slurry and fermented product 3 of black soldier fly larvae slurry was determined by Kjeldahl nitrogen determination.

[0199] like Figure 14 As shown: the proportion of oil in insect slurry fermentation product 3 decreased, while the proportion of protein showed no significant difference.

[0200] 2. Study on the moisture content and stability of insect slurry fermentation products.

[0201] The insect slurry fermentation product 3 was dried to different degrees, with its moisture content controlled at <10%, <20%, <30%, and <40%. The insect slurry fermentation products 3 with different moisture contents (<10%, <20%, <30%, <40%) were placed at room temperature for one month, and the pH value and odor were measured weekly. It was found that the stability was good after one month when the moisture content was below 10% (Table 10 and...). Figure 15 ).

[0202] Table 10

[0203]

[0204] Example 20: Black soldier fly larvae fermentation product 3 used in experiments with yellow-feathered chickens.

[0205] The insect pulp fermentation product 3 of Lactobacillus reuteri was mixed with the basic feed to obtain the compound feed.

[0206] Table 11

[0207]

[0208] I. Feed used in broiler chicken experiments

[0209] One-month-old yellow-feathered chickens were selected and divided into groups of 20 chickens each. One group was fed a control diet, while the other two groups were fed a compound feed. Chickens were euthanized after one month of rearing, and their growth performance was assessed.

[0210] 1. Weight Inspection

[0211] like Figure 16 As shown: At 1 month of age, the weights of the two groups of yellow-feathered chickens were 356±26g and 350±23g, respectively, with no significant difference (P>0.05). At 2 months of age, the weights of the two groups of chickens were 673±61g and 738±56g, respectively. The weight of the yellow-feathered chickens fed the compound feed was significantly higher than that fed the first group (P<0.05). This indicates that the compound feed can be used as a basic protein substitute in chicken feed. Lactobacillus reuteri fermented insect pulp products can promote the growth of broilers.

[0212] 2. Chicken breast and chicken leg testing

[0213] like Figure 17 As shown: At 2 months of age, the weights of chicken breasts in the two groups were 211±15g and 222±15g, respectively. The weight of the yellow-feathered chicken breasts in the compound feed group 2 was significantly higher than that in the compound feed group 1. The weights of chicken legs in the two groups were 132±19g and 162±13g, respectively. The weight of the yellow-feathered chicken legs in the compound feed group was significantly higher than that in the control feed group. This indicates that the Lactobacillus reuteri fermented insect pulp product can promote the growth of chicken breasts and legs.

[0214] 3. Gut health testing

[0215] At 2 months of age, the jejunal lengths of the two groups of chickens were 56±4 cm and 65±4 cm, respectively. The jejunal length of the yellow-feathered chickens fed the compound feed was significantly longer than that of the chickens fed the control feed. This indicates that the Lactobacillus reuteri fermented insect pulp product can promote intestinal health in chickens.

[0216] Example 21: Lactobacillus reuteri synthesizes protein from high-nitrogen wastewater

[0217] 1. Protein synthesis in basal culture medium inoculated with Lactobacillus reuteri

[0218] Take 1000ml of basal culture medium, sterilize it at 65℃ for 3 hours, transfer it to a fermenter, and inoculate it with 10ml of Lactobacillus reuteri culture, wherein the total number of Lactobacillus reuteri is 10. 10 ~10 11 / ml, sealed for fermentation, controlled at a temperature of 25~30℃, fermentation cycle of 7 days, with pH ≤5 at the end of fermentation; take the remaining fermentation liquid and stir thoroughly to fully stir up the lactic acid bacteria that have settled at the bottom, then filter through a 400-mesh filter using a pump to obtain bacterial protein. Concentrate the bacterial protein under reduced pressure to obtain an active bacterial protein paste, then wash with sterile water, filter, and obtain approximately 0.8g of pure active bacterial protein paste.

[0219] The reagents and dosages used to prepare the above basic culture medium are as follows: peptone 0.5 g / L, yeast extract 0.25 g / L, NaHCO3 2.6 g / L, NH4Cl 1 g / L, NaCl 0.1 g / L, MgCl 0.1 g / L, CaCl2 0.05 g / L, K2HPO4 0.4 g / L, trace element solution 1 mL / L, and vitamin solution 1 mL / L.

[0220] The trace element solution composition is as follows: FeCl·2H₂O 2000 mg / L, H₃BO₃ 50 mg / L, ZnC 250 mg / L, CuCl₂ 30 mg / L, MnCl₂4H₂O 50 mg / L, CoCl₂·6H₂O 50 mg / L, (NH₄)₆Mo₇O 24 50mg / L, AlCl350mg / L, NiCl250mg / L, H2SeO349mg / L.

[0221] Vitamin solution composition: Biotin 50mg / L, Folic acid 20mg / L, Vitamin B6 100mg / L, Vitamin B2 50mg / L, Vitamin B1 50mg / L, Niacin 50mg / L, Alpha-lipoic acid 50mg / L, Vitamin B5 50mg / L.

[0222] 2. Lactobacillus reuteri synthesizes protein using chicken manure wastewater.

[0223] Take 1000g of fresh chicken manure wastewater (nitrogen content approximately 1%) as raw material, sterilize it at 65℃ for 3 hours, put it into a fermentation tank, and inoculate it with 10ml of Lactobacillus reuteri culture. The total lactic acid bacteria count is 10. 10 ~10 11 / ml sealed fermentation, controlled at a temperature of 25~30℃, fermentation cycle of 7 days, with pH ≤5 at the end of fermentation; take the remaining fermentation liquid and stir thoroughly to fully stir up the lactic acid bacteria that have settled at the bottom, pump it out, and filter it through 80 mesh, 200 mesh, and 400 mesh successively to obtain bacterial protein. Concentrate the bacterial protein under reduced pressure to obtain active bacterial protein paste, then wash it with sterile water, filter it, and obtain approximately 7.6g of pure active bacterial protein paste.

[0224] like Figure 20 As shown, Lactobacillus reuteri can efficiently utilize high-nitrogen wastewater to synthesize bacterial protein, thus possessing the value of waste resource utilization.

[0225] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A type of Lactobacillus reuteri ( Lactobacillus reuteri strain C3, its accession number is: CCTCC NO: M 20252241.

2. The application of Lactobacillus reuteri C3 as described in claim 1 in the preparation of black soldier fly larvae feed.

3. A method for preparing feed for black soldier fly larvae, characterized in that: Includes the following steps: 1) Preparation of black soldier fly larvae by-products; wherein, the black soldier fly larvae by-products are black soldier fly larvae larvae plasma or black soldier fly protein; The black soldier fly larvae larvae's insect pulp contains 10-15% chitin, 40-50% protein, and 30-40% oil. The black soldier fly protein is prepared by the following steps: a. Crush the black soldier fly larvae into a paste using a grinder. b. The insect slurry is added to a stirred and heated reactor for alkaline hydrolysis. After filtration, the chitinous shell is removed to obtain insect protein. The alkaline solution of the alkaline hydrolysis is a sodium hydroxide solution with a mass fraction of 0.1-1.0%. 2) Weigh out the black soldier fly larvae by-products, water, and basic feed at a mass ratio of 1:4~7:15~20; 3) First, mix the black soldier fly larvae by-products and the basic feed evenly, then add water and mix to obtain a mixture; 4) Activate and culture the Lactobacillus reuteri C3 as described in claim 1 to obtain Lactobacillus reuteri C3 bacterial solution; wherein the OD value of the Lactobacillus reuteri C3 bacterial solution is 1~2; 5) Add Lactobacillus reuteri C3 bacterial solution to the mixture; ferment to obtain black soldier fly larvae feed; wherein, the amount of Lactobacillus reuteri C3 bacterial solution added is 1~4mL per 100g of basic feed and insect pulp.

4. The preparation method according to claim 3, characterized in that: The sodium hydroxide solution has a mass fraction of 0.5%.

5. The preparation method according to claim 3, characterized in that: In step 2), the raw materials of the basic feed include, by weight percentage, 67% corn flour, 20% soybean meal, 8% fish meal, 1.2% limestone powder, 1.5% dicalcium phosphate, 0.3% salt, 1% broiler premix and 1% vegetable oil.

6. The preparation method according to claim 3, characterized in that: In step 5), the amount of Lactobacillus reuteri C3 bacterial solution added is 2 mL per 100 g of basic feed and insect slurry.

7. The use of Lactobacillus reuteri as described in claim 1 in the preparation of fermented products from black soldier fly larvae.

8. A method for preparing a fermented product of black soldier fly larvae, characterized in that: Includes the following steps: 1) Crush fresh black soldier fly larvae into a paste and autoclave. 2) Activate and culture the Lactobacillus reuteri C3 as described in claim 1 to obtain Lactobacillus reuteri C3 bacterial culture; 3) Add the above-mentioned Lactobacillus reuteri C3 bacterial solution to the insect slurry, ferment, and obtain the fermented product of black soldier fly larvae.

9. The preparation method according to claim 8, characterized in that: In step 1), the insect slurry contains 10-15% chitin, 40-50% protein, and 30-40% oil. In step 3), 10-30 mL of bacterial solution is added to every 1 kg of insect slurry; the fermentation temperature is 20-30℃, and the fermentation time is 2-4 days.

10. A compound feed for black soldier fly larvae, characterized in that: The compound feed includes a basic feed and a fermented product of black soldier fly larvae prepared by the method of claim 8, wherein the weight ratio of the fermented product of black soldier fly larvae to the basic feed is 1:18~20.

11. The application of Lactobacillus reuteri C3 as described in claim 1 in the synthesis of protein using high-nitrogen wastewater.

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