Construction of low-chitin enzymatic hydrolysate cricket raw material based on Trichoderma and copepod autolysins, its preparation method and application

Abstract

This invention discloses a method for preparing and applying a low-chitin enzymatic hydrolysed cricket feed based on Trichoderma and copepod autolysins, belonging to the field of aquaculture technology. This invention employs a combined enzymatic hydrolysis method using Trichoderma activation and copepod autolysins, which not only significantly reduces the chitin content in the cricket feed (to below 1%), but also optimizes the nutritional composition of the feed by introducing marine functional proteins from copepods. This not only replaces fishmeal in feed but also contributes to the growth and health of aquatic animals.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology, specifically to a method for preparing and applying a low-chitin enzymatic hydrolysis cricket raw material based on Trichoderma and copepod autolysins. Background Technology

[0002] Aquaculture provides a vital food source for humankind. With the rapid development of my country's aquaculture industry, the market demand for aquatic feed is gradually increasing. Promoting the sustainable development of aquaculture and ensuring future aquatic product yields can effectively safeguard food security. Scientifically and rationally formulated aquatic feed is of great significance for improving aquaculture efficiency, ensuring the health of farmed animals, and protecting the environment. Fishmeal, with its balanced amino acid and fatty acid composition, provides high-quality protein and essential minerals, and is widely recognized as the best protein source in aquatic feed, especially for carnivorous aquatic animals that require large amounts of fishmeal. Therefore, the sustainable development of aquaculture urgently requires finding high-quality protein sources that can replace fishmeal.

[0003] Insect protein has become increasingly popular in recent years, driven by global, particularly EU, demand for a low-carbon economy. Its production relies heavily on food waste and low-value feed ingredients, making it an environmentally friendly and circular economy with cost advantages. Among insect proteins, those from black soldier fly larvae, mealworms, housefly maggots, and crickets have been developed on a large scale, with silkworm pupa powder also being a significant factor in domestic production. In Europe, black soldier fly larvae powder is particularly sought after. While black soldier fly larvae powder boasts high protein content and a relatively balanced amino acid composition, it also has drawbacks such as a relatively high chitin content.

[0004] Among current potential insect-based feed ingredients, crickets possess unique advantages: high protein content (55-73%), low chitin content, and the inability of adult crickets to fly, thus minimizing environmental disturbance. However, even in crickets, chitin remains a significant nutritional resistance factor, with levels reaching as high as 8-10%. Aquatic animals lack chitinase in their intestines, making it generally difficult for them to digest high chitin content. Therefore, removing or reducing the chitin content in insect feed ingredients has been a major challenge hindering the application of insects in aquatic animal feed.

[0005] Currently, there are two main technical methods for removing chitin from feed ingredients: one is mechanical removal through insect exoskeletons, which is energy-intensive and results in a lower product yield; the other is enzymatic hydrolysis using commercial chitinases to degrade chitin, but these enzymes are currently very expensive, leading to high costs. Therefore, finding a more economical and feasible chitin removal technology from feed ingredients is an urgent problem to be solved. Summary of the Invention

[0006] The technical problem to be solved by this invention is to overcome the shortcomings of the prior art and provide a method for preparing and applying a low-chitin enzymatic hydrolysis cricket raw material based on Trichoderma and copepod autolysins. This invention integrates Trichoderma-activated chitin technology and innovatively utilizes copepod autolysins. By maintaining the activity of autolysins and coupling the raw material with enzymatic hydrolysis, the chitin content in aquatic feed with high cricket content is greatly reduced while introducing functional active ingredients from marine copepods, resulting in a significant improvement in feed efficacy.

[0007] The technical solution of this invention is as follows:

[0008] In a first aspect, the present invention provides a method for preparing a low-chitin enzymatic hydrolysis cricket raw material based on Trichoderma and copepod autolysins, comprising the following steps:

[0009] S1: Steam sterilize adult crickets, coarsely crush them into a slurry, sieve them, add Trichoderma harzianum and corn starch, carry out large-scale solid-state fermentation, control the temperature at 27~30℃, the relative humidity at 63~66%RH, and aerate for 45~50h to activate the chitin in the insect body.

[0010] S2: Copepods caught in nearshore or salt fields are quick-frozen with liquid nitrogen and then stored at -50℃ to -30℃. Dithioerythritol is added to maximize the preservation of the activity of their natural autolytic enzyme system (including chitinase, protease, etc.) to obtain frozen copepods.

[0011] S3: Mix the activated cricket material obtained in step S1 with the frozen copepod obtained in step S2. Crush and stir the mixture directly while it is still frozen. After natural thawing, add water to make a slurry and construct a coupled enzymatic hydrolysis reaction system. Stir at 36~40℃ and pH=8~8.5 for 3~5 hours. Then, heat with steam to inactivate the enzyme and sterilize to obtain the enzymatically hydrolyzed cricket raw material. Cool and set aside for later use.

[0012] Preferably, in step S1, the steam sterilization temperature is 88~95℃ and the time is 9~12min.

[0013] Preferably, in step S1, the material is coarsely crushed into a slurry and then passed through a 15-25 mesh sieve.

[0014] Preferably, in step S1, the mass ratio of the sieved adult crickets, Trichoderma harzianum, and corn starch is 1000:(0.8~1.5):35.

[0015] Preferably, in step S2, the mass ratio of copepods quick-frozen with liquid nitrogen to dithioerythritol is 100:(1.8~2.2).

[0016] Preferably, in step S3, the mass ratio of the activated cricket material to the frozen copepods is 10:(0.8~1.2), and the mass ratio of the total mass of the activated cricket material and the frozen copepods to the mass of water is 100:(8.5~11).

[0017] Preferably, in step S3, the steam heating temperature is 88~95℃ and the time is 18~23min.

[0018] Secondly, the present invention provides a low-chitin enzymatic hydrolysed cricket raw material based on Trichoderma and copepod autolysins, which is prepared by the above-mentioned preparation method of the low-chitin enzymatic hydrolysed cricket raw material based on Trichoderma and copepod autolysins.

[0019] Thirdly, the present invention also provides an application of enzymatic hydrolysis of cricket raw materials based on Trichoderma and copepod autolysins to construct low-chitin content. After determining the actual moisture content of the enzymatic hydrolysis of cricket raw materials based on Trichoderma and copepod autolysins to construct low-chitin content, it is added to a commercial aquatic feed formula at a dry matter addition rate of 2-8%, and the protein replaces the fish meal in the formula to obtain cricket-based aquatic functional feed.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] 1. This invention promotes chitin degradation by combining the chitin-activating function of Trichoderma and the enzyme system of copepod autolysins, thereby reducing the chitin content in cricket-based aquatic feeds. Furthermore, through coupled enzymatic hydrolysis of Trichoderma and copepod autolysins, it introduces functional nutrients from marine copepods, increasing the proportion of cricket feed added to aquatic feeds. Simultaneously, it optimizes the feed's nutrient composition, thus improving feed efficiency. Through extensive experimental exploration and data accumulation, this invention has developed suitable autolysin activity preservation technology and application technology for low-chitin cricket protein feeds. This allows for the efficient replacement of fishmeal in feed with cricket protein, improving feed efficiency and maintaining the health and rapid growth of farmed animals.

[0022] 2. By using the enzymatically hydrolyzed cricket raw material of this invention to replace fishmeal in aquatic feed formulations, the weight gain rate, feed intake rate, and protein efficiency of farmed marine fish can be greatly improved, while the feed conversion ratio is significantly reduced. This invention has low equipment requirements, controllable costs, and is highly operable. Its application can reduce the amount of fishmeal used in fish compound feeds, resulting in significant economic and ecological benefits. Attached Figure Description

[0023] Figure 1 This refers to the survival rate of the experimental fish in each treatment group in Example 1 of this invention.

[0024] Figure 2This refers to the weight gain rate of the experimental fish in each treatment group in Example 1 of this invention; the data are expressed as mean ± standard error (n=4), and there are significant differences between data columns without the same letter. P <0.05).

[0025] Figure 3 This refers to the feeding rate of the experimental fish in each treatment group in Example 1 of this invention; the data are expressed as mean ± standard error (n=4), and there are significant differences between data columns without the same letter. P <0.05).

[0026] Figure 4 These are the feed conversion ratios of the experimental fish in each treatment group in Example 1 of this invention; the data are expressed as mean ± standard error (n=4), and there are significant differences between data columns without the same letter. P <0.05).

[0027] Figure 5 This refers to the protein efficiency of the experimental fish in each treatment group in Example 1 of this invention; the data are expressed as mean ± standard error (n=4), and there are significant differences between data columns without the same letter. P <0.05).

[0028] Figure 6 This refers to the survival rate of experimental fish in each treatment group in Example 2 of this invention; data are expressed as mean ± standard error (n=4), and there are significant differences between data columns without the same letter. P <0.05).

[0029] Figure 7 This refers to the weight gain rate of the experimental fish in each treatment group in Example 2 of this invention; the data are expressed as mean ± standard error (n=4), and there are significant differences between data columns without the same letter. P <0.05).

[0030] Figure 8 This refers to the feeding rate of the experimental fish in each treatment group in Example 2 of this invention; the data are expressed as mean ± standard error (n=4), and there are significant differences between data columns without the same letter. P <0.05).

[0031] Figure 9 These are the feed conversion ratios of the experimental fish in each treatment group in Example 2 of this invention; the data are expressed as mean ± standard error (n=4), and there are significant differences between data columns without the same letter. P <0.05).

[0032] Figure 10 This refers to the protein efficiency of the experimental fish in each treatment group in Example 2 of this invention; the data are expressed as mean ± standard error (n=4), and there are significant differences between data columns without the same letter.P <0.05).

[0033] Figure 11 This refers to the survival rate of the experimental fish in each treatment group in Example 3 of this invention; the data are expressed as mean ± standard error (n=4), and there are significant differences between data columns without the same letter. P <0.05).

[0034] Figure 12 This refers to the weight gain rate of the experimental fish in each treatment group in Example 3 of this invention; the data are expressed as mean ± standard error (n=4), and there are significant differences between data columns without the same letter. P <0.05).

[0035] Figure 13 This refers to the feeding rate of the experimental fish in each treatment group in Example 3 of this invention; the data are expressed as mean ± standard error (n=4), and there are significant differences between data columns without the same letter. P <0.05).

[0036] Figure 14 These are the feed conversion ratios of the experimental fish in each treatment group in Example 3 of this invention; the data are expressed as mean ± standard error (n=4), and there are significant differences between data columns without the same letter. P <0.05).

[0037] Figure 15 This refers to the protein efficiency of the experimental fish in each treatment group in Example 3 of this invention; the data are expressed as mean ± standard error (n=4), and there are significant differences between data columns without the same letter. P <0.05). Detailed Implementation

[0038] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] Example 1: Evaluation test of the effect of applying the enzymatic hydrolysis of cricket raw materials of the present invention in turbot farming.

[0040] 1. Experimental design, raw material preparation and experimental feed formulation (the basic feed formulation is a simulation of commonly used commercial feed formulations, and is not intended to limit the scope of protection of this invention. As long as the normal growth of farmed fish can be guaranteed, the nutritional methods of this invention can achieve the effects of this invention.)

[0041] S1: Sterilize adult crickets with steam at 90℃ for 10 minutes, coarsely crush them into a slurry, pass them through a 20-mesh sieve, add commercial Trichoderma harzianum at a ratio of 1 wt.‰, and add 3.5 wt.% corn starch, carry out large-scale solid-state fermentation, control the temperature at 28℃, the relative humidity at 65%, and aerate for 48 hours to achieve chitin activation in the insect body.

[0042] S2: Copepods (Dichroa febrifuga) caught in nearshore waters or salt fields are flash-frozen in liquid nitrogen and then stored at -40°C. 2 wt.% dithioerythritol is added evenly to maximize the preservation of the activity of their natural autolytic enzyme system (including chitinase, protease, etc.).

[0043] S3: The activated cricket material obtained in step S1 is mixed with the frozen copepods obtained in step S2 at a mass ratio of 10:1. The mixture is directly crushed and stirred before thawing. After natural thawing, 10 wt.% water is added to form a slurry to construct a coupled enzymatic hydrolysis reaction system. The mixture is stirred at 39℃ and pH=8.3 for 3 hours. Then, the mixture is heated with steam at 90℃ for 20 minutes to inactivate the enzyme and sterilize it, thus obtaining the enzymatically hydrolyzed cricket raw material, which is then cooled for later use.

[0044] According to GB / T 38479-2021 "Determination of Chitosan Content by High Performance Liquid Chromatography", the chitin content in the enzymatically hydrolyzed cricket raw material was determined to be 0.87% (as a percentage of dry matter weight).

[0045] The experimental feed formulations are shown in Table 1, with fishmeal, soybean protein concentrate, soybean meal, corn gluten meal, and millet flour as the main protein sources. The crude protein content was approximately 50%, and the crude fat content was approximately 11%. The control group did not contain enzymatically hydrolyzed cricket feed, while the four experimental feed groups contained different amounts (calculated as a percentage of dry matter) of enzymatically hydrolyzed cricket feed. The feed groups were designated as A (control group), B (1% added), C (2% added), D (4% added), and E (8% added), respectively.

[0046] Table 1. Formula and crude composition (% dry matter) of the experimental feed

[0047]

[0048] The amino acid composition of the experimental feed is shown in Table 2:

[0049] Table 2 Amino acid composition of experimental feed (g / 100g feed, dry matter)

[0050]

[0051] 2. Experimental fish and aquaculture management

[0052] This experiment used juvenile turbot with an initial weight of 9g. Before the formal experiment, the experimental fish were kept at 25m... 2The fish were temporarily kept in cement tanks for two weeks to acclimatize to the experimental environment. Before the formal experiment began, the experimental fish were randomly divided into 20 polyethylene tanks (height: 100cm; diameter: 230cm), with 4 replicates per group and 45 fish per tank. Indoor flow-through culture was used, with deep well water as the culture medium. The fish were artificially fed twice daily until full. The culture period was 8 weeks. The culture experiment was conducted under natural photoperiod and ambient temperature. During the feeding and culture experiment, the water temperature ranged from 16-20℃; salinity from 29-31; pH from 7.2-8.4; and dissolved oxygen from 6-8 mg·L⁻¹. -1 Half an hour after each feeding, use a siphon to remove any remaining feed and feces.

[0053] 3. Sample collection and index determination

[0054] At the beginning and end of the culture experiment, turbot were starved for 24 hours, and their initial and final average weights were recorded. Survival rate and weight gain rate were calculated. Feed intake rate and feed conversion ratio were calculated based on daily feed consumption. Protein efficiency was calculated based on the measured nutrient content. The calculation formulas are as follows:

[0055] Weight gain rate (%) = 100 × ( W t – W 0) / W 0, where W 0 - Initial average weight of turbot, g; W t - Final average weight of turbot, g.

[0056] Food intake rate (%) = 100 × F c / [0.5×( W t + W 0)× t ], where F c -Food intake, g; W 0 - Initial average weight of turbot, g; W t - Final average weight of turbot, g; t - Number of feeding days, d.

[0057] Feed conversion ratio = F c / ( W t – W 0), where F c -Food intake, g; W 0 - Initial average weight of turbot, g; W t - Final average weight of turbot, g.

[0058] Protein efficiency (%) = 100 × ( Wt – W 0) / ( F c × W p ) ,in W 0 - Initial average weight of turbot, g; W t - Final average weight of turbot, g; F c -Food intake, g; W p -Feed protein content,%.

[0059] 4. Experimental Statistical Methods

[0060] One-way ANOVA was performed on the experimental data using SPSS 16.0, and pairwise comparisons were conducted using the Tukey method. Data are expressed as mean ± standard error (n=4). P <0.5 indicates a significant difference.

[0061] 5. Experimental Results

[0062] This experiment mainly evaluates the application effect of the invention based on survival rate, weight gain rate, feed intake rate, feed conversion ratio, and protein efficiency.

[0063] like Figure 1 As shown, during the experiment, there was no significant difference in the final survival rate among the five groups, all exceeding 95%. Regarding weight gain rate, as... Figure 2 As shown, the weight gain rate of the 4% enzymatically hydrolyzed cricket raw material group (Group D) using the present invention was significantly higher than that of the control group (Group A) and the 1% enzymatically hydrolyzed cricket raw material group (Group B). P <0.05), and higher than the 2% enzymatic hydrolyzed cricket raw material group (Group C) and the 8% enzymatic hydrolyzed cricket raw material group (Group E) ( P >0.05).

[0064] In this invention, such as Figure 3 As shown, the food intake rates of groups C and D were significantly higher than those of the control group (group A). P <0.05), while there was no significant difference in feeding rate among the groups (groups B, C, D, and E) that added enzymatically hydrolyzed cricket feed. P >0.05). For example Figure 4 As shown, the feed conversion ratio of group D was significantly lower than that of groups A and B. P <0.05), while there was no significant difference between it and groups C and E ( P >0.05). For example Figure 5 As shown, the protein efficiency of group D was significantly higher than that of the other groups ( P <0.05).

[0065] In summary, the enzymatically hydrolyzed cricket raw material obtained through this embodiment, when added to turbot feed at a ratio of 2-4% (by dry matter weight) as a substitute for fishmeal during the rearing period, can improve feed intake and promote growth. The effect is best at a dosage of 4%, which can significantly reduce the feed conversion ratio and improve protein efficiency.

[0066] Example 2: Evaluation test of the effect of applying the enzymatic hydrolysis of cricket raw materials of the present invention in the culture of redfin pufferfish.

[0067] 1. Experimental design, raw material preparation and experimental feed formulation (the basic feed formulation is a simulation of commonly used commercial feed formulations, and is not intended to limit the scope of protection of this invention. As long as the normal growth of farmed fish can be guaranteed, the nutritional methods of this invention can achieve the effects of this invention.)

[0068] S1: Sterilize adult crickets with steam at 91℃ for 9.5 minutes, coarsely crush them into a slurry, pass them through a 20-mesh sieve, add commercial Trichoderma harzianum at a ratio of 1.2wt.‰, and add 3.5wt.% corn starch for large-scale solid-state fermentation. Control the temperature at 29.5℃, the relative humidity at 66%, and aerate for 46 hours to activate the chitin in the insect body.

[0069] S2: Copepods (Dichroa febrifuga) caught in nearshore or salt fields are flash-frozen in liquid nitrogen and then stored at -45°C. 1.9 wt.% dithioerythritol is added evenly to maximize the preservation of the activity of their natural autolytic enzyme system (including chitinase, protease, etc.).

[0070] S3: The activated cricket material obtained in step S1 is mixed with the frozen copepods obtained in step S2 at a mass ratio of 10:0.9. The mixture is directly crushed and stirred before thawing. After natural thawing, 9 wt.% water is added to form a slurry to construct a coupled enzymatic hydrolysis reaction system. The mixture is stirred at 40℃ and pH=8 for 3.6 hours. Then, the mixture is heated with steam at 92℃ for 19 minutes to inactivate the enzyme and sterilize it, thus obtaining the enzymatically hydrolyzed cricket raw material, which is then cooled for later use.

[0071] According to GB / T 38479-2021 "Determination of Chitosan Content by High Performance Liquid Chromatography", the chitin content in the enzymatically hydrolyzed cricket raw material was determined to be 0.82% (as a percentage of dry matter weight).

[0072] The experimental feed formulations are shown in Table 3, with fishmeal, soybean protein concentrate, krill meal, corn gluten meal, and high-gluten flour as the main protein sources. The crude protein content was approximately 48%, and the crude fat content was approximately 12%. The control group did not contain enzymatically hydrolyzed cricket feed, while the four experimental feed groups contained different amounts (calculated as a percentage of dry matter) of enzymatically hydrolyzed cricket feed. The feed groups were designated as A (control group), B (1% added), C (2% added), D (4% added), and E (8% added), respectively.

[0073] Table 3. Formula and crude composition (% dry matter) of the experimental feed

[0074]

[0075] The amino acid composition of the experimental feed is shown in Table 4:

[0076] Table 4 Amino acid composition of experimental feed (g / 100g feed, dry matter)

[0077]

[0078] 2. Experimental fish and aquaculture management

[0079] This experiment used juvenile redfin pufferfish with an initial weight of 12g. Before the formal experiment, the experimental fish were kept at 25m... 2 The fish were temporarily kept in cement tanks for 10 days to acclimatize to the experimental environment. Before the formal experiment began, the experimental fish were randomly divided into 20 polyethylene tanks (height: 100cm; diameter: 230cm), with 4 replicates per group and 35 fish per tank. Indoor flow-through culture was used, with deep well water as the culture medium. The fish were artificially fed twice daily. The culture period was 8 weeks. The culture experiment was conducted under natural photoperiod and ambient temperature. During the feeding and culture experiment, the water temperature ranged from 19-22℃; salinity from 29-31; pH from 7.2-8.4; and dissolved oxygen from 6-8 mg·L⁻¹. -1 Half an hour after each feeding, use a siphon to remove any remaining feed and feces.

[0080] 3. Sample collection and index determination

[0081] At the beginning and end of the culture experiment, the redfin pufferfish were starved for 24 hours, and the initial and final average weights were recorded. The survival rate and weight gain rate were calculated. The feed intake rate and feed conversion ratio were calculated based on the daily feed intake. The protein efficiency was calculated based on the measured nutrient content. The calculation formulas are the same as in Example 1.

[0082] 4. Experimental Statistical Methods

[0083] One-way ANOVA was performed on the experimental data using SPSS 16.0, and pairwise comparisons were conducted using the Tukey method. Data are expressed as mean ± standard error (n=4). P <0.5 indicates a significant difference.

[0084] 5. Experimental Results

[0085] This experiment mainly evaluates the application effect of the invention based on survival rate, weight gain rate, feed intake rate, feed conversion ratio, and protein efficiency.

[0086] like Figure 6As shown, during the experiment, there was no significant difference in the final survival rate among the five groups, all exceeding 95%. Figure 7 As shown, among the five experimental groups, groups D and E had the highest weight gain rates, which were significantly higher than those of the control group A. P <0.05). Meanwhile, groups B and C showed an upward trend compared to the control group.

[0087] In this invention, such as Figure 8 As shown, there were significant differences in feeding rates among the five groups. All treatment groups (groups B, C, D, and E) with added enzymatically hydrolyzed cricket feed showed significantly increased feeding rates, indicating a better feeding-inducing effect. P <0.05). For example... Figure 9 As shown, the feed conversion ratios of groups A and B were significantly higher than those of group D. P <0.05), while the feed conversion ratios of groups C and E were not significantly different from those of other groups ( P >0.05). For example Figure 10 As shown, the protein efficiency of group D was significantly higher than that of the control group A. P <0.05).

[0088] In summary, the enzymatically hydrolyzed cricket raw material obtained through this embodiment, when added to the feed of redfin pufferfish during the rearing period at a ratio of 2-8% (by weight of dry matter) to replace fishmeal, can improve feed intake and promote growth. The effect is best at a dosage of 4%, which can significantly reduce the feed conversion ratio and improve protein efficiency.

[0089] Example 3: Evaluation Test of the Effect of the Enzymatic Hydrolysis of Cricket Raw Material of the Present Invention in Pearl Giant Grouper Farming

[0090] 1. Experimental design, raw material preparation and experimental feed formulation (the basic feed formulation is a simulation of commonly used commercial feed formulations, and is not intended to limit the scope of protection of this invention. As long as the normal growth of farmed fish can be guaranteed, the nutritional methods of this invention can achieve the effects of this invention.)

[0091] S1: Sterilize adult crickets with steam at 95℃ for 12 minutes, coarsely crush them into a slurry, pass them through an 18-mesh sieve, add commercial Trichoderma harzianum at a ratio of 1.5wt.‰, and add 3.5wt.% corn starch, carry out large-scale solid-state fermentation, control the temperature at 27℃, the relative humidity at 63%, and aerate for 45 hours to activate the chitin in the insect body.

[0092] S2: Copepods (Dichroa febrifuga) caught in nearshore waters or salt fields are flash-frozen in liquid nitrogen and then stored at -30°C. 2.2 wt.% dithioerythritol is added evenly to maximize the preservation of the activity of their natural autolytic enzyme system (including chitinase, protease, etc.).

[0093] S3: The activated cricket material obtained in step S1 is mixed with the frozen copepods obtained in step S2 at a mass ratio of 10:0.8. The mixture is directly crushed and stirred before thawing. After natural thawing, 9 wt.% water is added to form a slurry to construct a coupled enzymatic hydrolysis reaction system. The mixture is stirred at 40℃ and pH=8.5 for 4.2 hours. Then, the mixture is heated with steam at 90℃ for 20 minutes to inactivate the enzyme and sterilize it, thus obtaining the enzymatically hydrolyzed cricket raw material, which is then cooled for later use.

[0094] According to GB / T 38479-2021 "Determination of Chitosan Content by High Performance Liquid Chromatography", the chitin content in the enzymatically hydrolyzed cricket raw material was determined to be 0.91% (as a percentage of dry matter weight).

[0095] The experimental feed formulations are shown in Table 5, with fishmeal, soybean protein concentrate, shrimp meal, soybean meal, corn gluten meal, and high-gluten flour as the main protein sources. The crude protein content was approximately 48%, and the crude fat content was approximately 12%. The control group did not contain enzymatically hydrolyzed cricket feed, while the four experimental feed groups contained different amounts (calculated as a percentage of dry matter) of enzymatically hydrolyzed cricket feed. The feed groups were designated as A (control group), B (1% added), C (2% added), D (4% added), and E (8% added), respectively.

[0096] Table 5. Formula and crude composition (% dry matter) of the experimental feed

[0097]

[0098] The amino acid composition of the experimental feed is shown in Table 6:

[0099] Table 6 Amino acid composition of experimental feed (g / 100g feed, dry matter)

[0100]

[0101] 2. Experimental fish and aquaculture management

[0102] This experiment used juvenile pearl grouper with an initial weight of 20g. Before the formal experiment, the experimental fish were kept at 25m... 2 The fish were temporarily kept in cement tanks for 10 days to acclimatize to the experimental environment. Before the formal experiment began, the experimental fish were randomly divided into 20 polyethylene tanks (height: 100cm; diameter: 230cm), with 4 replicates per group and 35 fish per tank. Indoor flow-through culture was used, with deep well water as the culture medium. The fish were artificially fed twice daily. The culture period was 8 weeks. The culture experiment was conducted under natural photoperiod and ambient temperature. During the feeding and culture experiment, the water temperature ranged from 22-24℃; salinity from 29-31; pH from 7.2-8.4; and dissolved oxygen from 6-8 mg·L⁻¹. -1 Half an hour after each feeding, use a siphon to remove any remaining feed and feces.

[0103] 3. Sample collection and index determination

[0104] At the beginning and end of the culture experiment, the pearl grouper were starved for 24 hours, and the initial and final average weights were recorded. The survival rate and weight gain rate were calculated. The feed intake rate and feed conversion ratio were calculated based on the daily feed intake. The protein efficiency was calculated based on the measured nutrient content. The calculation formulas were the same as in Example 1.

[0105] 4. Experimental Statistical Methods

[0106] One-way ANOVA was performed on the experimental data using SPSS 16.0, and pairwise comparisons were conducted using the Tukey method. Data are expressed as mean ± standard error (n=4). P <0.5 indicates a significant difference.

[0107] 5. Experimental Results

[0108] This experiment mainly evaluates the application effect of the technology of this invention based on survival rate, weight gain rate, feed intake rate, feed conversion ratio, and protein efficiency.

[0109] like Figure 11 As shown, during the experiment, there was no significant difference in the final survival rate among the five groups, all exceeding 95%. Regarding weight gain rate, as... Figure 12 As shown, the weight gain rate of the 4% enzymatically hydrolyzed cricket raw material group (Group D) using the present invention was significantly higher than that of Groups A and B, while Groups C and E showed no significant difference compared to other groups. P >0.05).

[0110] In this invention, such as Figure 13 As shown, the food intake rate of group D was significantly higher than that of groups A and B. P <0.05). For example... Figure 14 As shown, the feed conversion ratio of group D was significantly lower than that of the other groups ( P <0.05). Regarding protein efficiency, such as... Figure 15 As shown, the protein efficiency of group D was only significantly higher than that of group A. P <0.05), with no significant difference between it and groups B, C, and D.

[0111] In summary, the enzymatically hydrolyzed cricket feed obtained in this embodiment, when added at a ratio of 4% (by weight of dry matter) to the feed of pearl grouper during the rearing period as a substitute for fishmeal, can promote growth and feed utilization. While the addition doses of 2% and 8% also had a positive effect on weight gain and feed intake, there was no significant difference compared to the control group without added enzymatically hydrolyzed cricket feed.

[0112] Although the present invention has been described in detail with reference to the accompanying drawings and preferred embodiments, the invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the invention should also be covered within the protection scope of the invention. Therefore, the protection scope of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing a low-chitin enzymatic cricket feedstock based on Trichoderma and copepod autolytic enzymes, characterized in that, Includes the following steps: S1: Steam sterilize adult crickets, coarsely crush them into a slurry, sieve them, add Trichoderma harzianum and corn starch, and carry out large-scale solid-state fermentation. Control the temperature at 27~30℃, the relative humidity at 63~66%RH, and aerate for 45~50h to activate the chitin in the insect body. The mass ratio of sieved adult crickets, Trichoderma harzianum and corn starch is 1000:(0.8~1.5):

35. S2: Copepods were flash-frozen in liquid nitrogen and then stored at -50℃ to -30℃. Dithiocarbazin was added to obtain frozen copepods. The mass ratio of the quick-frozen copepods to dithiocarbazin was 100:(1.8~2.2). The copepods were *Zygophora spp.* S3: Mix the activated cricket material obtained in step S1 with the frozen copepods obtained in step S2. Crush and stir the mixture directly while it is still frozen. After natural thawing, add water to make a slurry and construct a coupled enzymatic hydrolysis reaction system. Stir at 36~40℃ and pH=8~8.5 for 3~5 hours. Then, heat with steam to inactivate the enzyme and sterilize to obtain the enzymatically hydrolyzed cricket raw material. Cool it for later use. The mass ratio of activated cricket material to frozen copepods is 10:(0.8~1.2), and the mass ratio of the total mass of activated cricket material and frozen copepods to water is 100:(8.5~11).

2. The method for preparing chitin-reduced enzymatic cricket feedstock based on Trichoderma and copepod autolytic enzymes according to claim 1, characterized in that, In step S1, the steam sterilization temperature is 88~95℃ and the time is 9~12min.

3. The method for preparing chitin-reduced enzymatic cricket feedstock based on Trichoderma and copepod autolytic enzymes according to claim 1, characterized in that, In step S1, the material is coarsely crushed into a slurry and then passed through a 15-25 mesh sieve.

4. The method for preparing chitin-reduced enzymatic cricket feedstock based on Trichoderma and copepod autolytic enzymes according to claim 1, characterized by, In step S3, the steam heating temperature is 88~95℃ and the time is 18~23min.

5. Construction of low-chitin enzymatic cricket feed based on Trichoderma and copepod autolytic enzymes, characterized by, The cricket raw material was prepared by the method described in any one of claims 1-4, which uses Trichoderma and copepod autolysins to construct low-chitin enzymatic hydrolysis.

6. Use of a low-chitin enzymatic cricket feed based on Trichoderma and copepod autolytic enzymes, characterized in that, After determining the actual moisture content of the enzymatically hydrolyzed cricket raw material with low chitin content constructed based on Trichoderma and copepod autolysins as described in claim 5, it is added to the aquatic feed formula at a dry matter addition rate of 2-8%, replacing the fish meal in the formula with protein, to obtain cricket-based aquatic functional feed.

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