A method for increasing lauric acid content in hermetia illucens based on cyanobacterial resource and probiotics

Abstract

The application discloses a method for increasing lauric acid content in black soldier fly based on cyanobacteria resource and probiotics, and belongs to the field of cyanobacteria resource. The method for increasing lauric acid content in black soldier fly based on cyanobacteria resource and probiotics comprises the following steps: mixing cyanobacteria powder and wheat bran to obtain a feed matrix, then adjusting moisture, adding a probiotic suspension to obtain a feed, and finally feeding the black soldier fly with the feed; wherein the probiotic is one or more of bacillus subtilis, saccharomyces cerevisiae and lactobacillus plantarum. The method can promote the weight increase of black soldier fly larvae, has no obvious influence on the survival rate of the black soldier fly larvae, improves the dry matter reduction rate, feed conversion ratio and biological conversion rate, and improves the nutritional value of the black soldier fly larvae, especially the content of lauric acid.

Description

Technical Field

[0001] This invention relates to a method for increasing the lauric acid content in black soldier flies based on cyanobacterial resource utilization and probiotics, belonging to the field of cyanobacterial resource utilization. Background Technology

[0002] With the continuous growth of the global population and the acceleration of urbanization, cyanobacterial blooms caused by lake eutrophication have gradually become one of the major environmental problems affecting the quality of life and sustainable social development in my country. For lakes like Taihu Lake, which have multiple major drinking water sources, the most direct, safe, and effective prevention and control method is to harvest the cyanobacteria. However, the harvested cyanobacteria are prone to decay and contain various pollutants, such as microcystins. If they are not disposed of in a timely and effective manner and are simply dumped, they will cause secondary pollution to the environment.

[0003] In recent years, using insect biotransformation to treat organic waste has become a clean and efficient treatment method that has attracted widespread attention. Compared with other farmed insect species, black soldier flies have gained increasing attention due to their wide feeding range, short growth cycle, and lack of disease transmission. Using black soldier flies to treat cyanobacteria sludge not only facilitates the resource utilization of cyanobacteria sludge, but the harvested black soldier fly prepupae are also high in protein and fat, making them suitable for use as feed additives and in biodiesel production, showing broad application prospects. However, this method for treating cyanobacteria sludge suffers from drawbacks such as low black soldier fly larvae conversion rates and low material reduction rates, hindering the promotion and application of this technology.

[0004] Lauric acid is a natural antibacterial agent with a wide range of excellent properties, including widespread presence, antibacterial activity, non-induction of bacterial resistance, antiviral activity, anti-inflammatory effects, and antitoxin activity. Recent research indicates that lauric acid plays a positive role in promoting animal growth and preventing and treating animal diseases. Adding appropriate amounts of lauric acid to fish feed helps provide healthy, nutritious, and palatable aquatic products; its addition to low-protein diets can improve the growth performance and intestinal barrier function of weaned piglets, as well as their immune function. Glyceryl monolauric acid is also considered an effective feed supplement for broiler chickens. However, conventional feeds contain relatively low levels of lauric acid. Summary of the Invention

[0005] [Technical Issues]

[0006] Simply using black soldier flies to treat cyanobacteria has drawbacks such as low conversion rate of black soldier fly larvae and low reduction rate of cyanobacteria.

[0007] Conventional feeds contain low levels of lauric acid.

[0008] [Technical Solution]

[0009] To address the aforementioned problems, this invention utilizes black soldier fly biotransformation to treat harvested and dehydrated cyanobacteria sludge. By adding probiotics, the fermentation process of the substrate and the absorption of nutrients by the fly larvae are regulated, thereby improving the efficiency of black soldier fly biotransformation of cyanobacteria and enhancing the nutritional value of the fly larvae. This method not only efficiently treats cyanobacteria but also improves the efficiency of black soldier fly biotransformation of cyanobacteria and enhances the nutritional value of the fly larvae, particularly increasing the lauric acid content.

[0010] The first objective of this invention is to provide a method for increasing the lauric acid content in black soldier flies based on cyanobacteria resource utilization and probiotics, comprising the following steps:

[0011] Blue-green algae powder and wheat bran are mixed to obtain a feed matrix. The moisture content is then adjusted, and a probiotic suspension is added to obtain the feed. Finally, the feed is used to feed black soldier flies.

[0012] The probiotics are one or more of Bacillus subtilis, Saccharomyces cerevisiae, and Lactobacillus plantarum.

[0013] In one embodiment of the present invention, the preparation method of the cyanobacteria powder is as follows: fresh cyanobacteria are placed in an oven to dry and then ground to obtain cyanobacteria powder.

[0014] In one embodiment of the present invention, the mass ratio of the cyanobacteria powder to wheat bran is 30-60:40-70.

[0015] In one embodiment of the present invention, adjusting the moisture content means that the feed moisture content should reach 65-80%, specifically by adding water to the feed matrix so that the mass of the water is 65-80% of the sum of the mass of the feed matrix and the mass of the water.

[0016] In one embodiment of the present invention, the probiotic suspension is an activated probiotic suspension, and the inoculation amount of probiotics is 10. 8 CFU / mL; the ratio of probiotic suspension to feed matrix is ​​3-4 mL: 100 g.

[0017] In one embodiment of the present invention, the black soldier fly is a black soldier fly larva.

[0018] In one embodiment of the present invention, the black soldier flies are fed at 30°C.

[0019] The second objective of this invention is to prepare black soldier flies with high lauric acid content using the method described herein.

[0020] The third objective of this invention is the application of the black soldier fly with high lauric acid content described herein in the preparation of livestock and poultry feed and aquatic feed.

[0021] In one embodiment of the present invention, the application involves drying black soldier flies with high lauric acid content to a moisture content of 8-12% to obtain feed.

[0022] [Beneficial Effects]

[0023] Black soldier fly larvae possess a high capacity for digestion and transformation, capable of converting complex organic matter in organic waste such as livestock and poultry manure, kitchen waste, straw, landfill leachate, and biogas residue into biomass they require, as well as relatively stable insect excrement and raw material residue. The method described in this invention for increasing the lauric acid content in black soldier flies based on cyanobacteria resource utilization and probiotics has the following advantages:

[0024] (1) It can degrade anti-nutritional factors in feed, which is beneficial to the utilization of the nutritional value of feed;

[0025] (2) It decomposes macromolecules and produces various metabolites, which promotes the digestion and absorption of nutrients by black soldier flies.

[0026] (3) After probiotics colonize the intestines of black soldier flies, they can stimulate the host's immune system, enhance the body's immunity, and maintain the balance of intestinal flora.

[0027] (4) It promotes the increase of black soldier fly larvae weight, has no significant effect on the survival rate of black soldier fly larvae, increases dry matter reduction rate, feed conversion ratio and biological conversion rate; and improves the nutritional value of black soldier fly larvae, especially the content of lauric acid. Attached Figure Description

[0028] Figure 1 This is a flowchart of the method of the present invention.

[0029] Figure 2 These are routine nutritional indicators for black soldier fly larvae.

[0030] Figure 3 The activity of lipase in different groups of insects.

[0031] Figure 4 The amylase activity of different groups of insects was measured.

[0032] Figure 5 The enzyme activity of proteases in different groups of insects.

[0033] Figure 6 The distribution of amino acids in different groups of insects.

[0034] Figure 7 The nutritional composition of larval dry matter under different proportions of cyanobacteria. Detailed Implementation

[0035] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0036] Test method:

[0037] 1. Determination of the weight of black soldier fly larvae:

[0038] Separate the frass and black soldier fly larvae and weigh them.

[0039] 2. Conversion rate determination

[0040] All larvae that have finished feeding are sieved out and placed in an oven along with everything else in the rearing box. They are dried at 105°C for 24 hours. They are weighed and recorded, and indicators such as material reduction rate, biological conversion rate, feed conversion rate and survival rate are calculated.

[0041] The dry matter loss rate (dry weight) is calculated as follows (1):

[0042]

[0043] The bioconversion rate (dry weight) is calculated as follows (2):

[0044]

[0045] The feed conversion ratio (dry weight) is calculated as follows (3) (initial insect weight 0.52g):

[0046]

[0047] Survival rate is calculated as follows (4):

[0048]

[0049] Raw materials used in the examples:

[0050] 8-day-old black soldier fly larvae: These were obtained by hatching black soldier fly eggs (Guangzhou Wuliang Biotechnology Co., Ltd.). Specifically, about 5g of black soldier fly eggs were placed in wheat bran with a humidity of 60% and hatched for 8 days, while maintaining an ambient temperature of 30℃ and an air humidity of 60%. The hatched black soldier fly larvae were used for subsequent rearing experiments.

[0051] Wheat bran: purchased from Huamo Biotechnology Co., Ltd.;

[0052] Fresh cyanobacteria: taken from Huangtiangang algae-water separation station in Wuxi City. After being harvested, the cyanobacteria were stored in a -20℃ refrigerator for subsequent experiments.

[0053] Preparation of cyanobacteria powder: Cyanobacteria are dried and ground to obtain cyanobacteria powder;

[0054] Bacillus subtilis powder, purchased from Beihai Yeshengwang Biotechnology Co., Ltd.

[0055] Saccharomyces cerevisiae (Angel Biotech Feed Additive Livestock and Poultry Yeast): Fermented yeast powder, purchased from Angel Yeast Co., Ltd.;

[0056] Lactobacillus plantarum: 10 billion CFU / g, bacterial powder, purchased from Zhenjiang Tianyi Biotechnology Co., Ltd.;

[0057] Bacillus subtilis suspension: Prepare a seed culture solution with 5g yeast powder, 10g peptone, 10g sodium chloride, 1000mL water, and pH 7.0-7.2. Dispense the prepared seed culture solution into Erlenmeyer flasks, seal with sealing film, wrap with newspaper, and sterilize in an autoclave at 121℃ for 20min. Inoculate the bacterial powder at a 2% inoculation rate into Erlenmeyer flasks containing the seed culture medium, with a volume of 100 / 250mL. Shake at 130r / min and 37℃ for 24h.

[0058] Saccharomyces cerevisiae suspension: One loop of the Saccharomyces cerevisiae strain preserved on slant culture was inoculated into YEPD (100 mL water, 2% peptone, 1% yeast extract, 2% glucose, pH 4.8) liquid shake flask medium for activation. The culture was then incubated at 28℃ and 180 rpm for 20 h to revive the strain. The activated liquid culture was then inoculated into the experimental medium at an inoculum size of 2%, and incubated at 28℃ for 20 h using a shaker at 180 rpm.

[0059] Lactobacillus plantarum suspension: First, prepare MRS medium (10.0g peptone, 10.0g beef extract, 5.0g yeast extract, 5.0g glucose, 1.0g Tween 80, 2.0g diammonium citrate, 5.0g anhydrous sodium acetate, 2.0g K2HPO4, 0.05g MnSO4·7H2O, 0.2g MgSO4·7H2O, 1.0L distilled water, pH 6.80, sterilized at 121℃ for 20min); then, inoculate 2% of the medium into 100mL of sterilized MRS liquid medium and incubate at 30℃ for 20h for later use.

[0060] Unless otherwise specified, the solvent used in the solutions mentioned in the examples and comparative examples is water, and unless otherwise specified, the percentages refer to mass percentages.

[0061] Example 1

[0062] A method for increasing the lauric acid content in black soldier flies based on cyanobacteria resource utilization and probiotics includes the following steps:

[0063] Mix 50g of cyanobacteria powder (dry matter) and 50g of wheat bran to obtain a feed matrix. Then add 233.3g of distilled water to achieve a moisture content of 70%, and then inoculate with 3.3mL of a 10% concentration. 8 A CFU / mL suspension of Bacillus subtilis was used to obtain feed; the feed was then used to feed 200 8-day-old black soldier fly larvae, which were cultured in an incubator at 30°C; this group was named K.

[0064] Example 2

[0065] The Bacillus subtilis in Example 1 was replaced with Saccharomyces cerevisiae, while everything else remained the same as in Example 1. This group was named Group J.

[0066] Example 3

[0067] The Bacillus subtilis in Example 1 was replaced with Lactobacillus plantarum, while everything else remained the same as in Example 1. This group was named Group Z.

[0068] Example 4

[0069] The 3.3 mL Bacillus subtilis suspension in Example 1 was adjusted to 1.1 mL, with a concentration of 10. 8 CFU / mL Bacillus subtilis suspension, 1.1 mL of 10 8 CFU / mL of Saccharomyces cerevisiae suspension and 1.1 mL of 10 8 A mixed solution of *Lactobacillus plantarum* suspension at cfu / mL, otherwise consistent with Example 1, is named Group H.

[0070] Comparative Example 1

[0071] The Bacillus subtilis strain from Example 1 was omitted, and only 3.3 mL of distilled water was added. Everything else remained the same as in Example 1, and this group was named the CK group.

[0072] Performance tests were conducted on Examples 1-4 and Comparative Example 1. Specifically, when 50% of the experimental groups showed prepupae, black soldier flies were washed, weighed, and their fresh weight was measured. At the same time, some worms and their excrement were dried and their moisture content was measured. Four to five worms were selected from each group, their intestines were removed, and a portion of their base was taken. The worms were frozen in liquid nitrogen and then transferred to a -80°C freezer for storage. The worm excrement and worm bodies were collected in sealed bags and stored in a -80°C freezer.

[0073] The results are as follows:

[0074] The effects of adding probiotics on the growth and development of black soldier flies are shown in Table 1.

[0075] Table 1. Insect development status

[0076]

[0077] Note: *p<0.05; **p<0.01

[0078] As can be seen from Table 1:

[0079] (1) In Examples 1-4, the average weight of larvae in the probiotic-inoculated groups was significantly greater than that in the control group (p<0.05); compared with the CK group, the average weight of larvae in the K, J, Z, and H groups increased by 3.85g, 4.42g, 4.9g, and 3.46g, respectively. This shows that the addition of probiotics promotes the increase in the weight of black soldier fly larvae.

[0080] (2) Adding probiotics to ferment the feed had no significant effect on the survival rate of black soldier fly larvae, and the survival rates of the treatment groups were not significantly different (p>0.05). The survival rate of group K was the highest, reaching 99.33%, followed by group H at 98.83%, and group CK had the lowest survival rate at only 96.5%. This result may be related to the characteristic that probiotics can enhance the body's immunity and thus reduce the probability of disease occurrence.

[0081] (3) The dry matter reduction rate of black soldier fly larvae in the experimental group with added probiotics was higher than that in the control group (p<0.05), especially in the experimental group with added mixed probiotics (Group H), the dry matter reduction rate reached 55.78%.

[0082] (4) Feed conversion ratio and bioconversion rate can reflect the larvae’s utilization of feed; the bioconversion rate of the larvae in the treatment group after adding probiotics showed a significant upward trend compared with the CK group which only added ordinary feed (p<0.05), especially the experimental group with added Bacillus subtilis (K group) and the experimental group with added mixed probiotics (H group), whose bioconversion rates reached 9.04% and 9.06%, respectively.

[0083] By measuring conventional nutritional indicators such as moisture, protein, fat, and ash in the prepupae of black soldier flies in each group, the changes in the nutritional composition of black soldier flies after the addition of probiotics were determined. The results are as follows: Figures 2-5 As shown.

[0084] As can be seen from Table 2:

[0085] (1) There was no significant difference in the nutritional composition of the insects after the addition of probiotics compared with the control group (CK) (p>0.05). The water content was the highest in the nutritional composition of black soldier fly prepupae. The water content of the black soldier fly prepupae in the experimental groups (K, J, Z, and H) with added probiotics was not significantly different from that in the control group (CK). The protein content of the black soldier fly larvae raised in different experimental groups ranged from 10.9% to 11.53%, with a fluctuation range of less than 1%. Among them, the protein content of the insects after the addition of probiotics was increased compared with the CK group (10.9%).

[0086] (2) In terms of fat content, the fat content of the control group CK was 5.52%, while the fat content of the K, J, Z and H groups was 5.74%, 6.12%, 6.3% and 6.12% respectively, all of which were higher than that of the CK group.

[0087] from Figures 3-5 It can be seen that:

[0088] There were significant differences in lipase and amylase activities between the experimental group with added probiotics and the control group without added probiotics (p < 0.05), but there was no significant difference in amylase activity before and after adding probiotics (p > 0.05).

[0089] Compared with the CK group, the enzyme activities of the experimental groups (K group, J group, Z group, and H group) with added probiotics were all increased. Among them, the lipase activity of the K group was the highest (reaching 16.87 μmol / min / g), while the amylase and protease activities of the H group were the highest, at 1.15 mg / min / g and 54.73 nmol / min / g, respectively.

[0090] Tables 2-4 and Figure 6 The results are for the tests of saturated and unsaturated fatty acids.

[0091] Table 2 Saturated Fatty Acids

[0092]

[0093]

[0094] Note: *p<0.05**p<0.01

[0095] Table 3 Monounsaturated fatty acids

[0096]

[0097] Note: *p<0.05**p<0.01

[0098] Table 4 Polyunsaturated Fatty Acids

[0099]

[0100] Note: *p<0.05**p<0.01

[0101] As can be seen from Tables 2-4:

[0102] Compared to the control group, the experimental groups with added probiotics (groups K, J, Z, and H) showed the most significant increase in lauric acid content, with increases of 5.71%, 5.87%, 5.33%, and 5.37%, respectively.

[0103] Regarding unsaturated fatty acids, the addition of probiotics leads to a decrease in the content of monounsaturated and polyunsaturated fatty acids in black soldier flies. The total unsaturated fatty acid content in the CK group was 36.21%. After the addition of different probiotics, the content decreased to 32.70% in the J group, 32.24% in the K group, 32.53% in the H group, and 32.99% in the Z group.

[0104] High lauric acid content is beneficial for black soldier fly larvae to be used as feed protein, increasing larvae weight and improving the lauric acid content in feed, which is more conducive to the growth and development of farmed animals.

[0105] Example 5: Optimization of Cyanobacteria Dosage

[0106] Under conditions of 30℃ and 70% humidity, cyanobacteria and wheat bran were mixed into a feed with a moisture content of 70%. Each feed portion consisted of 100 grams (dry matter), and 200 eight-day-old black soldier flies were inoculated with the feed. The percentages of cyanobacteria dry matter were 0%, 30%, 40%, 50%, 60%, 70%, and 80%, respectively, and were denoted as P0, P1, P2, P3, P4, P5, and P6.

[0107] The test results are shown in Table 5 and Figure 7 .

[0108] Table 5. Development and substrate transformation of black soldier fly larvae under different proportions of cyanobacteria.

[0109]

[0110] Table 5 shows that as the proportion of cyanobacteria increases, the development of black soldier flies slows down, with the developmental period increasing from 16.20 days to 20.00 days; the larval survival rate also decreases from 97.67% to 69.67%. The substrate dry matter reduction rate is relatively less affected by the increase in the proportion of cyanobacteria, decreasing from 53.09% to 41.69%. With the increase in the proportion of cyanobacteria, the bioconversion rate decreases from 5.34% to 3.07%. This means that substrates with a higher proportion of cyanobacteria produce less larval dry matter for the same dry matter mass. In summary, the conversion efficiency of black soldier flies decreases with the increase in the proportion of cyanobacteria. When the proportion of cyanobacteria exceeds 70%, the conversion efficiency deteriorates significantly, resulting in less larval dry matter production.

[0111] Figure 7 The nutritional composition of larval dry matter under different proportions of cyanobacteria. From Figure 7 It can be seen that, in terms of the nutritional composition of larvae, the crude protein content in the larvae first increases and then decreases as the proportion of cyanobacteria in the substrate increases, reaching its highest level of 40.92% at 50%. The crude fat content increases with the proportion of cyanobacteria, which may be related to the decrease in wheat bran content. The proportion of crude ash also increases accordingly with the increase in the proportion of cyanobacteria.

[0112] Based on the above analysis of the conversion effect and nutritional composition of black soldier flies, it can be seen that when using cyanobacteria and wheat bran as substrates, the overall conversion effect first increases and then decreases as the proportion of cyanobacteria increases, with the best effect observed at 50%. Excessively high proportions of cyanobacteria have a limiting effect on the conversion and nutritional composition of black soldier flies.

[0113] In summary, this invention analyzed the growth, nutritional indicators, enzyme activity, amino acid distribution, fatty acid distribution, and fatty acid synthesis pathway enzyme activity of black soldier flies. The results showed that using probiotics to enhance the bioconversion of solid waste by black soldier flies not only regulates the fermentation process of the substrate and decomposes macromolecular nutrients, but also regulates the absorption of nutrients from the substrate by the flies, thus enhancing the bioconversion performance of black soldier flies and improving their nutritional value, particularly increasing the content of lauric acid. In this invention, adding probiotics to the feed decreased the content of unsaturated fatty acids in the flies, but significantly increased the content of lauric acid. Compared to the control group, the lauric acid content in groups K, J, Z, and H increased by 5.71%, 5.87%, 5.33%, and 5.37%, respectively.

[0114] In actual production, in order to improve the efficiency of black soldier fly larvae in converting cyanobacteria and to promote the subsequent use of black soldier flies as a feed additive for livestock and poultry, it is necessary to add the probiotics described in this invention.

[0115] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for increasing the lauric acid content in black soldier fly larvae based on cyanobacteria resource utilization and probiotics, characterized in that, Includes the following steps: Blue-green algae powder and wheat bran are mixed to obtain a feed matrix. The moisture content is then adjusted, and a probiotic suspension is added to obtain the feed. Finally, the feed is used to feed black soldier flies. The preparation method of the blue-green algae powder is as follows: fresh blue-green algae are dried and ground to obtain blue-green algae powder. The mass ratio of blue-green algae powder to wheat bran is 30-60:40-70. The moisture content adjustment refers to the feed's humidity reaching 65-80%. The black soldier flies used are black soldier fly larvae. The black soldier flies are fed at 30°C. The probiotic is Bacillus subtilis; The probiotic suspension mentioned is an activated Bacillus subtilis suspension with a concentration of 10. 8 CFU / mL; the ratio of probiotic suspension to feed matrix is ​​3-4 mL: 100 g.

2. The application of the black soldier fly with high lauric acid content prepared by the method of claim 1 in the preparation of livestock and poultry feed and aquatic feed.

3. The application according to claim 2, characterized in that, The application involves drying black soldier flies with high lauric acid content to a moisture content of 8-12% to obtain feed.

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