Application of 5-HMF in feed additives and feeds for promoting growth and improving immunity of yellowtail amberjack

Abstract

The present invention relates to the application of HMF in a feed additive for promoting growth and improving immunity of yellowtail amberjack, belonging to the field of feed additives, wherein the feed additive contains 5-HMF, i.e., 5-hydroxymethylfurfural. The present invention also provides a functional feed for yellowtail amberjack, wherein the feed contains 0.25-1% 5-HMF by weight, and the addition of 5-HMF to the feed can improve the intestinal tissue morphology and flora structure, increase the proportion of beneficial bacteria in the digestive tract, thereby significantly promoting the feeding and growth of yellowtail amberjack; and at the same time, can improve the antioxidant capacity and immunity of the liver of striped amberjack.

Description

Technical Field

[0001] The invention belongs to the field of feed additives, and specifically relates to application of 5-HMF in a feed additive and feed for promoting growth and improving immunity of yellowtail amberjack. Background Art

[0002] Yellowtail amberjack (Seriola lalandi) belongs to the Perciformes, Carangidae, Seriola genus. It is a globally distributed oceanic economic fish with long-distance migratory characteristics in the middle and upper layers of the ocean. Due to its fast growth rate, large size, rich nutrition, and good taste, the consumption demand is increasing worldwide, and it has become an excellent fish species for the development of deep-sea aquaculture in my country. In recent years, due to the intensive aquaculture and water pollution, aquatic diseases have occurred frequently, and the aquatic industry has suffered economic losses. Antibiotics are often used in aquaculture to prevent diseases, but due to drug residues and improper use, antibiotics can easily lead to damage to the quality of aquatic products, pollution of the environment, and even harm to human health. Many researchers are committed to seeking substances to replace antibiotics, and the promotion of green additives has become an inevitable trend in the development of aquaculture. In addition, there is currently no special compound feed for yellowtail amberjack farming on the market. It is urgent to develop special high-efficiency compound feed and green additives to promote the development of the aquaculture industry.

[0003] 5-Hydroxymethyl furfural (5-HMF) is a furfural compound with a furan ring structure produced by the dehydration of monosaccharide compounds such as glucose under high temperature or weak acid conditions. It is one of the typical products of caramelization reaction and Maillard reaction and is widely present in food, plants, and traditional Chinese medicine. In recent years, the biological functions of 5-HMF have been gradually discovered, mainly including antioxidant activity, improvement of blood rheology, and influence on glycyrrhizic acid metabolism. With the widespread application of modern science and technology in the field of traditional Chinese medicine research, 5-HMF has been deeply studied and understood. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a 5-HMF feed additive and feed for promoting growth and improving immunity of yellowtail amberjack. 5-hydroxymethyl furfural (5-HMF) has a promoting effect on feeding, growth and immune function of yellowtail amberjack.

[0005] The present invention is achieved through the following technical solutions:

[0006] The invention discloses an application of 5-HMF in a feed additive for promoting growth and improving immunity of yellowtail amberjack, wherein the feed additive contains 5-HMF.

[0007] The invention also provides a functional feed for yellowtail amberjack, wherein the feed contains 5-HMF, and the mass of the added 5-HMF is 0.25-1% of the weight of the feed.

[0008] Furthermore, the mass of the added 5-HMF is 0.5% of the weight of the feed.

[0009] The beneficial effects of the present invention compared with the prior art are as follows:

[0010] The invention adds 5-HMF to the feed, which can improve the intestinal tissue morphology and flora structure, increase the proportion of beneficial bacteria in the digestive tract, and thus significantly promote the feeding and growth (weight gain rate, specific growth rate and fatness) of the yellowtail; and at the same time, can improve the antioxidant capacity and immunity of the liver of the yellowtail. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Effects of adding different concentrations of 5-HMF to the feed on the intestinal morphology of yellowtail amberjack (HE staining). DETAILED DESCRIPTION

[0012] The present invention is further described in detail below with reference to the accompanying drawings and examples.

[0013] Example 1 Effects of different concentrations of 5-HMF on the proliferation of Caco-2 cells

[0014] 1. Cell Viability Experiment Design and Methods

[0015] In order to determine the appropriate concentration of feed additives, we first explored the toxic effects of different 5-HMF concentrations on cells. In this experiment, different concentrations of 5-HMF (0mMol, 1.6mMol, 3.2mMol, 6.3mMol) were selected to intervene in Caco-2 cells for 24h, and the cell viability was detected by MTT method. 0.01mg of the sample to be tested was added to 1ml DEME cell culture medium to prepare the mother solution to be tested. The effective concentration of 5-HMF in the mother solution was 6.3mmol / L. The preparation and component concentration of the test solution in each experimental group are as follows:

[0016] Table 1 Preparation and component concentration of test solution in different experimental groups

[0017]

[0018] Note: The concentrations of M1, M2, and M3 groups in this experiment correspond to the concentrations of M1, M2, and M3 groups set in the additive feeding experiment.

[0019] 2. Experimental cell culture and passaging

[0020] Caco-2 cells (human colorectal adenocarcinoma cells) were cultured in 5 mL of complete DMEM medium (containing 10% fetal bovine serum and 1% penicillin and streptomycin antibodies).25 Cells were cultured in flasks at 37°C with 5% CO 2 The culture medium was replaced every 2 days.

[0021] When cells grow to T 25 When the cells are about 90% full, the original medium in the culture bottle is aspirated, and the cells are gently rinsed with 2 mL of sterile PBS (1×). After aspirating the PBS, the cells are treated with trypsin (containing 0.25% EDTA) at 37°C for 3 min. After treatment, 2 mL of complete α-MEM medium is added to neutralize the cells to terminate the reaction. After centrifugation at 1000 g for 5 min, the supernatant is discarded, and the cells are resuspended with 1 mL of complete α-MEM medium. After gently blowing, 0.5 mL is added to a new flask containing 4.5 mL of complete α-MEM medium. 25 The cell culture flask was placed in an incubator for further culture.

[0022] 3. MTT assay to detect the effect of 5-HMF on Caco-2 cell activity

[0023] Select Caco-2 cells within the 5-20th generation, inoculate 1×104 cells per well into a 96-well plate and culture for 24 hours, then aspirate the original culture medium in the well and add 100uL of culture medium containing different concentrations of the test substance (5-HMF) (the test substance is dissolved in a culture medium without fetal bovine serum), the specific concentrations are 0μg / mL, 25μg / mL, 50μg / mL, 100μg / mL, 200μg / mL, 400μg / mL, culture in an incubator for 20 hours, add 10μL, 5mg / mL MTT to each well, incubate at 37°C for 4 hours, aspirate the culture medium in the well after incubation, add 150μLDMSO, shake the plate for 15 minutes and measure the absorbance at 490nm, the cell proliferation activity is calculated according to the following formula:

[0024] Cell proliferation activity = (A sample - A blank) / (A control - A blank) x 100%.

[0025] Where A blank is the absorbance of the sample at a concentration of 0 μg / mL

[0026] Culture medium and serum were from Biological Industries (Israel Beit Haemek Co., Ltd); MTT was from Sigma; Caco-2 cells were from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai).

[0027] 3. Results

[0028] As shown in Table 2, there were significant differences between the 5-HMF administration group and the control group (P < 0.05), but the cell proliferation rate of the highest concentration A3 group was still greater than 80%, indicating that it had no obvious toxic effect on cells and was suitable for use as a feed additive concentration.

[0029] Table 2 Effects of different concentrations of 5-HMF on the proliferation of Caco-2 cells

[0030]

[0031] Note: Different lowercase letters in superscripts indicate significant differences among different experimental groups (P<0.05).

[0032] Note: The concentrations of M1, M2, and M3 groups in this experiment correspond to the concentrations of M1, M2, and M3 groups set in the additive feeding experiment.

[0033] Example 2

[0034] The experiment was completed in the factory breeding workshop of Dalian Fugu Food Co., Ltd. from September to October 2021, with a test period of 42 days. The yellowtail amberjack used in the experiment came from the company's marine cage base, with uniform specifications and healthy 1-year-old fish, with an average body length of (17.35±0.51) cm and an average body weight of (82.33±2.75) g.

[0035] The volume used for factory-scale workshop breeding is 3m 3 The round glass tank was fed with compound pellet feed (Hayashikan Industry Co., Ltd., Japan). Before the experiment, the fish were temporarily kept for 7 days, during which they were fed with basic feed, i.e., compound pellet feed (Hayashikan Industry Co., Ltd., Japan). The fish were fed twice a day, at 8:00 and 16:00, respectively, at 2% of their body weight. The fish were fasted for 24 hours before the experiment. The experimental fish were randomly divided into 4 groups (M0 group, M1 group, M2 group, M3 group), with 3 replicates in each group and 30 fish in each replicate. The experimental group feed was to be added with different concentrations of 5-HMF, which was evenly covered on the surface of the feed by spraying, and then dried at low temperature to attach 5-HMF to the basic feed. The M1 group, M2 group, and M3 group were fed with experimental feeds with a mass ratio of 0.25%, 0.5%, and 1% of the basic feed, respectively. The feeding amount was 2-3% of body weight, and the fish were cultured in flowing water with a daily water replacement rate of 200-300%. The water environment conditions during the entire breeding process are: water temperature is 13-24℃, salinity is 31-32, and dissolved oxygen is >7mg / L.

[0036] After the experiment, 6 yellowtail amberjacks were sampled from each parallel of the control group and the experimental group, and 18 were sampled from each experimental group. After anesthesia with MS-222, the body surface was wiped with 75% alcohol cotton, and the fish weight and body length were measured and recorded. Blood samples were collected from the tail vein using a 2ml syringe, and the blood was placed at 4°C, centrifuged at 4000r / min for 10min, and the supernatant was taken and stored at -80°C for the determination of serum biochemical indicators. After dissection, liver and digestive tract (stomach, pyloric caeca, intestine) samples were taken, and the residual contents in the digestive tract were discharged, rinsed with pre-cooled sterile saline, and stored in liquid nitrogen.

[0037] 1. Growth performance measurement

[0038] The growth parameters were calculated according to the following formula:

[0039] Weight gain ratio (WGR, %) = [(W t –W 0 ) / W 0 ]×100;

[0040] Specific growth rate (SGR, % / d) = [(lnW t -lnW 0 ) / t]×100;

[0041] Fullness (Condition factor, CF, g / cm 3 )=W t / L 3 .

[0042] In the above formula: W 0 , W t are the average fish weight (wet weight, g) at the beginning and end of the experiment respectively; t is the number of experimental days (d); L is the body length (cm).

[0043] The effects of adding different concentrations of 5-HMF to the feed on the growth performance of yellowtail amberjack fry are shown in the table. As shown in Table 3, after 42 days of feeding, the weight gain rate and specific growth rate of the M2 and M3 groups were significantly higher than those of the M0 group (P < 0.05); the fatness of the M2 group was significantly higher than that of the M0 group (P < 0.05).

[0044] Table 3 Effects of different concentrations of 5-HMF on growth indicators of yellowtail amberjack juveniles

[0045]

[0046] Note: Different lowercase letters in superscripts indicate significant differences among different experimental groups (P < 0.05)

[0047] 2. Determination of liver antioxidant capacity

[0048] The antioxidant enzyme detection kit used in the experiment was purchased from Nanjing Jiancheng Bioengineering Institute. The activities of liver glutathione peroxidase (GSH-PX), superoxide dismutase (SOD) and catalase (CAT) were determined according to the instructions, and the malondialdehyde (MAD) concentration was determined.

[0049] The effects of different concentrations of 5-HMF added to feed on the antioxidant function of the liver of yellowtail amberjack fry are shown in Table 4. Adding 5-HMF to feed can significantly improve the antioxidant capacity of the liver of yellowtail amberjack fry. Compared with the control group, GSH in the experimental group increased significantly (P<0.05); the MAD level in the M0 group was significantly higher than that in the M2 and M3 groups (P<0.05). The SOD and CAT activities in the M2 and M3 groups were significantly higher than those in the M0 group (P<0.05).

[0050] Table 4 Effects of different concentrations of 5-HMF on the antioxidant capacity of the liver of yellowtail amberjack juveniles

[0051]

[0052] Note: Different lowercase letters in superscripts indicate significant differences among different experimental groups (P<0.05).

[0053] 3 Serological index determination

[0054] The activities of immunoglobulin M (IgM), lysozyme (LZM), alkaline phosphatase (AKP), alanine aminotransferase (GPT), aspartate aminotransferase (GOT) and acid phosphatase (ACP) were detected using kits produced by Nanjing Jiancheng Bioengineering Institute, and the detection method was performed according to the instructions of the kits.

[0055] The effects of adding different concentrations of 5-HMF to the feed on the serum immune indexes of yellowtail amberjack fry are shown in Table 5. Compared with the M0 group, feeding different concentrations of 5-HMF feed improved the immune ability of the experimental fish to varying degrees. The results of immune indexes showed that the IgM and AKP in the yellowtail amberjack fry in the M2 and M3 groups were significantly higher than those in the M0 group (P<0.05); the LZM and ACP activities in the M2 group were significantly higher than those in the M0 group (P<0.05); the GPT in the M0 group was significantly lower than that in the experimental group (P<0.05); the GOT activities in the M1 and M3 groups were significantly higher than those in the M0 group (P<0.05).

[0056] Table 5 Effects of different concentrations of 5-HMF on serum immune indexes of yellowtail amberjack juveniles

[0057]

[0058] Note: Different lowercase letters in superscripts indicate significant differences among different experimental groups (P<0.05).

[0059] 4 Intestinal tissue morphology analysis

[0060] The intestinal samples fixed with Bouin's solution were taken, dehydrated with 75%, 80%, 95%, and 100% graded alcohol, transparentized with xylene, routinely embedded in paraffin and sliced ​​(5 μm thick), stained with hematoxylin-eosin (HE), and sealed with neutral gum. The samples were observed under an OLYMPUS microscope and photographed. Image J software was used to measure the thickness of the intestinal muscle layer, the height of the villi, and the number of goblet cells.

[0061] The effects of adding different concentrations of 5-HMF to feed on the intestinal tissue morphology of yellowtail amberjack fry are shown in Table 6. The staining results are shown in Figure 1 The thickness of the myometrium and the height of the villi in the M2 group were significantly higher than those in the M0 group (P<0.05); there was no significant difference in the number of goblet cells between the 5-HMF group and the control group.

[0062] Table 6 Effects of different concentrations of 5-HMF on intestinal tissue morphology of yellowtail amberjack juveniles

[0063]

[0064]

[0065] Note: Different lowercase letters in superscripts indicate significant differences among different experimental groups (P<0.05).

[0066] 5 Extraction and high-throughput sequencing of total microbial DNA

[0067] The collected samples were extracted from the genomic DNA using a DNA extraction kit (MagPure Soil DNAKF Kit), and then the DNA concentration was detected and separated using Nano Drop 2000 and agarose gel electrophoresis. Using genomic DNA as a template, PCR was performed using barcoded specific primers and Tks Gflex DNA Polymerase (Takara), and primers 343F (5'-TACGGRAGGCAGCAG-3') and 798R (5'-AGGGTATCTAATCCT-3') were used to amplify the 16S V3-V4 region. After the amplified sequence was qualified by agarose gel electrophoresis, it was handed over to Qingdao Ouyi Biotechnology Co., Ltd. for high-throughput sequencing using the Illumina MiSeq PE300 platform.

[0068] The diversity index of the digestive tract microorganisms of yellowtail amberjack juveniles supplemented with different concentrations of 5-HMF was analyzed, and the results are shown in Table 7. The Coverage index indicates the sequencing depth of the sample and also indicates the coverage of the sample. The Coverage index in the 12 treatment groups was above 0.99, indicating that the probability of not being detected in the sample was low. The Shannon index and the Simpson index reflect the species diversity of the community. Under the same species richness, the greater the uniformity of each species in the community, the greater the diversity of the community. The Shannon index in the pyloric caeca of the M2 group was significantly higher than that of the other groups (P<0.05), while the Simpson index was significantly lower than that of the other groups (P<0.05). The Chao index reflects the species richness of the community in the sample, which simply refers to the number of species in the community without considering the abundance of each species in the community. The Chao1 index of the intestinal tract of the M2 group was significantly higher than that of the other groups (P<0.05).

[0069] Table 7 Effects of different concentrations of 5-HMF on the alpha diversity index of the digestive tract of yellowtail amberjack juveniles

[0070]

[0071] Note: Different lowercase letters in superscripts indicate significant differences among different experimental groups (P<0.05).

[0072] Note: Groups M0S, M0P, and M0G are the stomach, pyloric caeca, and intestinal samples of the control group; M1S, M1P, and M1G are the stomach, pyloric caeca, and intestinal samples with an addition amount of 0.25%; M2S, M2P, and M2G are the stomach, pyloric caeca, and intestinal samples with an addition amount of 0.5%; and M3S, M3P, and M3G are the stomach, pyloric caeca, and intestinal samples with an addition amount of 1%.

[0073] The microbial phyla and relative abundance were statistically analyzed at the phylum level. The results showed that adding different concentrations of 5-HMF to the feed had no significant difference in the number of microbiota in the digestive tract samples of yellowtail amberjack fry. The top five microbiota abundances in the digestive tract samples were Bacteroidetes, Firmicutes, Proteobacteria, Actinobacteria, and Desulfobacterota. The relative abundances of Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria in each group were all above 90%, but the proportions in each group were different.

[0074] The microbial phyla and relative abundance were statistically analyzed at the genus level. The results showed that the addition of different concentrations of 5-HMF to the feed had similar effects on the bacterial flora in the digestive tract of yellowtail amberjack fry, which was mainly composed of Bacteroides, Muribaculaceae, Lachnoclostridium, Lactobacillus, Parabacteroides, Ileibacterium, Lachnospiraceae_NK4A136_group, Bifidobacterium, Faecalibaculum, etc. The genera with high relative abundance and commonality in each group were Bacteroides, Muribaculaceae, Lachnoclostridium and Lactobacillus. The percentage of each genus is shown in Table 8.

[0075] Table 8 Percentage of bacterial flora composition in the digestive tract of yellowtail amberjack juveniles Note: Groups M0S, M0P, and M0G are the stomach, pyloric caeca, and intestinal samples of the control group; M1S, M1P, and M1G are the stomach, pyloric caeca, and intestinal samples with an addition amount of 0.25%; M2S, M2P, and M2G are the stomach, pyloric caeca, and intestinal samples with an addition amount of 0.5%; and M3S, M3P, and M3G are the stomach, pyloric caeca, and intestinal samples with an addition amount of 1%.

[0076]

[0077] 6. Data Statistics and Analysis

[0078] The raw image data files obtained by high-throughput sequencing were converted into raw sequencing sequences in FASTQ format by base recognition analysis. After a series of shearing, de-impurity, splicing, quality control and chimera removal, the valid sequences were obtained. According to the similarity of the sequences, the sequences were classified into multiple classifiable operational units (OTUs), and the sequences with a similarity of ≥97% were classified as one OTU unit. The QIIME software package was used to select the sequence with the highest abundance in each OTU as the representative sequence of the OTU, and all representative sequences were compared and annotated with the Silva (version 123) database. The species comparison and annotation used the RDP classifier software, and the annotation results with a confidence interval greater than 0.7 were retained. Tax4fun (0.3.1) was used to compare and analyze the KEGG pathways involved in microbiome genes.

[0079] Excel 2016 was used for data processing, and SPSS 26.0 software was used for statistical analysis. Paired T test was used to analyze the differences of the same samples at different concentrations. One-way ANOVA was used for different types of samples at the same concentration, and Duncan multiple comparisons were performed for differences between groups. The difference was considered significant when the significant difference level was P < 0.05. All values ​​were expressed as "mean ± standard deviation" (Mean ± SD).

[0080] The OTUs of the digestive tract flora of yellowtail amberjack juveniles were analyzed for functional prediction in the KEGG database by adding different concentrations of 5-HMF to the feed, and the functional pathways with significant differences between groups were screened at the Level 3 level according to the Kruskal-Wallis algorithm (P < 0.05). The main signaling pathways involved in the flora genes in various tissues of the digestive tract were the same, but there were certain differences in gene abundance. According to the number of OTUs annotations of the digestive tract flora, the differential pathways were mainly concentrated in carbohydrate metabolism, amino acid metabolism, and energy metabolism.

[0081] In summary, adding 0.5% 5-HMF to the experimental feed can improve the intestinal morphology and growth performance of yellowtail amberjack, and increase serum immune indicators and liver antioxidant capacity. From the changes in intestinal microorganisms of yellowtail amberjack fry, Firmicutes and Bacteroidetes are absolutely dominant in the intestinal flora of each group of fry, and the intestinal flora of each group of fry has the same dominant genera. In addition, the main functions of the genes involved in the digestive tract flora of yellowtail amberjack fry in each group are similar.

Claims

The use of 1.5-HMF in the preparation of a feed additive capable of improving the antioxidant capacity of the liver of yellowtail amberjack and improving intestinal health is characterized in that: The feed additive contains 5-HMF, namely 5-hydroxymethylfurfural, and is used for the purpose of improving the antioxidant capacity of the liver of yellowtail amberjack and improving the intestinal morphology of the yellowtail amberjack including the intestinal wall thickness and villus height.

Images