Application of lysyl-glutamate in promoting fish growth, improving flavor and inhibiting bacterial enteritis

Abstract

The present invention relates to the field of feed additives, and discloses a lysyl-glutamic acid dipeptide as an additive to promote fish growth, improve fish flavor and inhibit bacterial enteritis. The present invention is directed to the problems of decreased growth performance, loose and poor meat quality, and frequent bacterial diseases in current fish farming, and provides a novel feed additive that is simple in composition, can significantly improve the growth performance of Xiangyun crucian carp, improve muscle quality and flavor, reduce bacterial-induced intestinal inflammation, and significantly increase the economic benefits of fishermen's farming. Lysyl-glutamic acid dipeptide is included in the additive, which is a dipeptide substance combined with lysine and glutamic acid. Compared with other amino acids, lysyl-glutamic acid dipeptide can better maintain its structure in the high temperature of feed preparation, and is protected from high temperature damage.

Description

Technical Field

[0001] The present invention relates to the field of aquaculture, in particular to a use of a lysyl-glutamic acid dipeptide, and more particularly to a use of the lysyl-glutamic acid dipeptide in promoting fish growth, improving fish flavor and inhibiting bacterial enteritis. Background Art

[0002] Lysine, an essential amino acid for aquatic animals, promotes growth and development and enhances immunity. It can only be obtained from food and is often a limiting amino acid in aquatic feeds. Glutamic acid, an acidic amino acid, is an important raw material for protein synthesis in organisms. It is primarily involved in brain protein synthesis and sugar metabolism, promoting growth. It combines with ammonia in the body to form glutamine, reducing ammonia concentrations and thus having a detoxifying effect. However, the high temperatures generated during feed preparation can decompose glutamate, rendering it inactive.

[0003] In 1999, Liu Tao et al. published a study demonstrating that adding 1% glutamate to feed increased the average daily weight gain of weaned piglets, reduced diarrhea frequency, and improved feed efficiency. In 2007, Tan Chanyuan et al. published a study on the antitumor activity of lysyl-glutamate dipeptide, demonstrating that Lys-Glu exhibited significant dose-dependent inhibitory effects on cultured human intestinal cancer LOVO, human gastric cancer MKN45, and human liver cancer QGY7703 cells. In vivo tumor inhibition experiments demonstrated that lysyl-glutamate dipeptide inhibited the growth of cultured mouse liver cancer H22 cells, demonstrating that lysyl-glutamate dipeptide exhibited significant in vitro and in vivo antitumor activity. However, the use of lysyl-glutamate dipeptide in aquatic feed has not been reported. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings of the above-mentioned background technology and provide a use of lysyl-glutamate dipeptide, which includes but is not limited to promoting fish growth, improving fish flavor and inhibiting intestinal bacterial enteritis. Specifically, the use of the present invention is to use lysyl-glutamate dipeptide as a feed additive, growth promoter, meat quality improver or anti-inflammatory agent to improve fish meat quality and enhance the meat flavor of fish, and the feeding effect is more significant than that of ordinary feed.

[0005] Furthermore, in some embodiments of the present invention, the use is to improve the growth performance and nitrogen retention rate of fish.

[0006] Furthermore, in some embodiments of the present invention, the use is to increase the content of one or more of the fish flavor amino acids glycine, alanine and tyrosine.

[0007] Furthermore, in some embodiments of the present invention, the use is to increase the content of one or more of cytosine nucleotides, guanine nucleotides, hypoxanthine nucleotides and adenine nucleotides in fish.

[0008] Furthermore, in some embodiments of the present invention, the fish is Xiangyun crucian carp.

[0009] Furthermore, in some embodiments of the present invention, the feed uses fish meal, soybean meal, rapeseed meal and casein as protein sources, and uses fish oil and soybean oil as fat sources.

[0010] Furthermore, in some embodiments of the present invention, the feed contains fish meal, soybean meal, rapeseed meal, casein, fish oil, soybean oil, corn starch, flour, choline, multivitamins, sodium carboxymethyl cellulose, cellulose and lysyl-glutamate dipeptide.

[0011] Furthermore, in some embodiments of the present invention, the mass ratio of fish meal, soybean meal, rapeseed meal, casein, fish oil, soybean oil, corn starch, flour, choline, multivitamin, sodium carboxymethyl cellulose and cellulose in the feed is 700-850:1250-1450:900-1100:400-500:150-210:150-210:1050-1300:600-800:25-35:150-210:150-210:380-480.

[0012] Furthermore, in some embodiments of the present invention, 0.4-2.0% of lysyl-glutamate dipeptide is added to the feed, for example, 0.4%, 0.8%, 1.2%, 1.6%, or 2.0% of lysyl-glutamate dipeptide is added.

[0013] Preferably, in some embodiments of the present invention, 1.6-2.0% of lysyl-glutamic acid dipeptide is added to the feed. The inventors conducted nutritional breeding experiments on juvenile Xiangyun crucian carp using the prepared feed and found that adding 1.6% and 2.0% lysyl-glutamic acid dipeptide to the feed can significantly promote the growth performance of Xiangyun crucian carp and improve its feed utilization efficiency.

[0014] Compared with the prior art, the advantages of the present invention are as follows:

[0015] (1) In the present invention, lysyl-glutamic acid dipeptide is used as a growth promoter and added to the Xiangyun crucian carp feed in the form of an additive. As the addition ratio increases, it can be observed that the weight gain rate, specific growth rate and feed efficiency of the Xiangyun crucian carp are significantly increased, and are all higher than those of the control group (the addition amount of lysyl-glutamic acid dipeptide is 0%).

[0016] (2) The lysyl-glutamic acid dipeptide additive in the present invention is used as a meat quality improver to increase the content of flavor amino acids and nucleotides in fish muscle. As the addition ratio of lysyl-glutamic acid dipeptide increases, the content of flavor amino acids glycine, alanine, and tyrosine, and flavor nucleotides cytosine nucleotide, guanine nucleotide, hypoxanthine nucleotide, and adenine nucleotide in Xiangyun crucian carp muscle increases, and all of them are higher than those in the group without lysyl-glutamic acid dipeptide addition.

[0017] (3) The lysyl-glutamate dipeptide additive in the present invention is used as an inflammatory inhibitor to inhibit the intestinal inflammatory response of fish induced by bacteria. With the addition of lysyl-glutamate dipeptide in the feed, the gene expression level of inflammatory factors in the intestine of Xiangyun crucian carp showed a gradual downward trend, and the expression of inflammatory genes in the control group was significantly higher than that in the lysyl-glutamate dipeptide-added group.

[0018] (4) The lysyl-glutamate dipeptide in the present invention is used as a feed additive to improve the growth performance and disease resistance of Xiangyun crucian carp, which is more significant than the feeding effect of ordinary feed. Therefore, the lysyl-glutamate dipeptide additive improves the economic benefits of Xiangyun crucian carp farming and has important scientific significance and practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1-Figure 5 It is the expression level of intestinal inflammatory genes in Xiangyun crucian carp 48 hours after poisoning. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. Additional aspects and advantages of the present invention will be partially given in the following description, partially become apparent from the following description, or be understood through practice of the present invention. It should be understood that the following description is only used to explain the present invention and is not intended to limit the present invention.

[0021] As used herein, the terms "comprises," "including," "having," "containing" or any other variations thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a listed element is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0022] The conjunction "consisting of" excludes any unspecified element, step, or component. If used in a claim, this phrase renders the claim closed, excluding materials other than those described, except for conventional impurities associated therewith. When the phrase "consisting of" appears in a clause of the body of a claim, rather than immediately following the subject matter, it limits only the elements described in that clause; other elements are not excluded from the claim as a whole.

[0023] When amount, concentration or other value or parameter is represented with range, preferred range or the range that a series of upper preferred value and lower preferred value limit are expressed, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value and any range lower limit or preferred value, no matter whether this range is disclosed separately.For example, when disclosing scope "1 to 5", described scope should be interpreted as including scope "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5" etc.When numerical range is described in this article, unless otherwise stated, otherwise this scope is intended to include its end value and all integers and fractions within this range.

[0024] The singular includes plural references unless the context clearly dictates otherwise. "Optional" or "either" means that the subsequently described event or incident can or cannot occur, and that the description includes instances where the event occurs and instances where it does not.

[0025] Approximating terms in the specification and claims are used to modify a quantity to indicate that the invention is not limited to that specific quantity and includes acceptable modifications close to that quantity that do not result in a change in the relevant basic function. Accordingly, the use of "about," "approximately," or the like to modify a numerical value indicates that the invention is not limited to that exact numerical value. In some instances, approximating terms may correspond to the precision of the instrument used to measure the value. In the specification and claims of this application, range definitions may be combined and / or interchanged, and unless otherwise indicated, such ranges include all subranges contained therein.

[0026] The indefinite articles "a" and "an" before the elements or components of the present invention do not limit the quantity requirement (i.e. the number of times they appear). Therefore, "a" or "an" should be interpreted as including one or at least one, and elements or components in the singular also include plural forms, unless the quantity clearly refers to only the singular form.

[0027] In addition, the terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples" described below mean that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic descriptions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the technical features involved in the various embodiments of the present invention may be combined with each other as long as they do not conflict with each other.

[0028] The Xiangyun crucian carp feed additive lysyl-glutamic acid dipeptide in the specific embodiment of the present invention was purchased from Green Leaf Biotechnology Co., Ltd., and the Xiangyun crucian carp used in the experiment was from Hunan Fisheries Science Research Institute. 0%, 0.4%, 0.8%, 1.2%, 1.6% and 2.0% of lysyl-glutamic acid dipeptide were added to the Xiangyun crucian carp basic feed to form 6 different feeds, which were fed to the Xiangyun crucian carp respectively.

[0029] Example 1

[0030] Effect of Lysyl-Glutamic Acid Dipeptide on the Growth Performance of Xiangyun Crucian Carp

[0031] The Xiangyun crucian carp feed used in the present invention is added with 0%, 0.4%, 0.8%, 1.2%, 1.6% and 2.0% of lysyl-glutamic acid dipeptide, and the Xiangyun crucian carp indoor breeding experiment is carried out using the following process.

[0032] (1) The Xiangyun crucian carp feed of the present invention comprises fish meal, soybean meal, rapeseed meal, and casein as the main protein sources, and fish oil and soybean oil as the fat sources. Six feeds (see Table 1) were designed to contain lysyl-glutamic acid dipeptide at contents of 0%, 0.4%, 0.8%, 1.2%, 1.6%, and 2.0% in the basal feed. All components in the feed were separated using a 40-mesh sieve, stirred in a feed stirrer for 20 minutes, and pelleted using a laboratory granulator to obtain 2.0 mm feeds. The feeds were dried in a blast dryer and stored sealed at -20°C.

[0033] Table 16 feed formulas and basic composition

[0034]

[0035] (2) The breeding experiment was carried out in the nutritional breeding laboratory of Changsha College. Before the start of the breeding experiment, the Xiangyun crucian carp was placed in 4 glass fiber tanks (1500L) for two weeks. A recirculating water breeding system was used. During the temporary breeding period, the Xiangyun crucian carp was fed at 8:30 and 14:30 every day. The feed fed was the control group feed (i.e., feed containing 0% lysyl-glutamic acid dipeptide).

[0036] (3) After the growth of the crucian carp stabilized, i.e., after the temporary rearing was completed, the crucian carp were divided into tanks. 450 crucian carp (11.77 ± 0.04 g) of similar size and shape were weighed in batches and randomly distributed into 18 100 L fiberglass tanks. The experiment consisted of 6 treatments, 3 replicates per treatment, and 25 crucian carp per replicate. During the experiment, the crucian carp were fed at 8:30 and 14:30 every day until they appeared to be full. The water flow in the culture system was 1300 mL / min, the ammonia nitrogen level was maintained below 0.1 mg / kg, the pH was 6.5-7.0, and the light period was from 8:00 to 20:00. The culture experiment lasted for 60 days.

[0037] (4) Record the water temperature of the fish tanks, the amount of feed and food intake of each fish tank every day.

[0038] (5) After the breeding period, the crucian carp were fasted for 24 hours, all the fish in each tank were removed, and the experimental fish were anesthetized with 50 mg / L MS-222 (ethyl m-aminobenzoate methanesulfonate). The fish were then weighed per tank to calculate the growth performance indicators such as the final weight, weight gain rate, specific growth rate, and feed efficiency of the crucian carp. Five fish were randomly selected from each repetition to measure body length, weight, and visceral weight, and to test their body fatness and visceral-to-body ratio. The formulas involved in the experiment are as follows:

[0039] Weight gain rate, % = (Wf-Wi) / Wi×100

[0040] Specific growth rate, % = [ln(Wf) - ln(Wi)] / d × 100

[0041] Feed efficiency, % = (Wf-Wi) / FI×100

[0042] Fullness, g / cm 3 =W / L 3 ×100

[0043] Visceral to body ratio, % = Wv / W×100

[0044] Wherein, Wi and Wf are the initial and final body weights (g) of the experimental fish, respectively; d is the culture days (60 days); FI represents the feed intake of the experimental fish; Wv, W and L represent the visceral mass (g), body mass (g) and body length (cm) of a single fish, respectively.

[0045] All data in this example were statistically analyzed using SPSS 19.0 software, and data are expressed as mean ± standard error. Each relevant indicator was first tested for homogeneity of variance. After homogeneity of variance was confirmed, one-way ANOVA was performed. If significant differences were found between experimental groups, Duncan's method was used for multiple comparisons. P < 0.05 indicated a significant difference.

[0046] The effects of lysyl-glutamate dipeptide on the growth performance of Xiangyun crucian carp are shown in Table 2 below. With increasing levels of lysyl-glutamate dipeptide supplementation, the weight gain rate, specific growth rate, and feed efficiency of the experimental fish all showed a significant upward trend. In particular, the 1.60% and 2.00% dipeptide supplementation groups achieved significantly better growth performance than the control group and other supplementation groups. Furthermore, lysyl-glutamate dipeptide did not significantly affect the fatness of the Xiangyun crucian carp, but the visceral-to-body ratio of the experimental fish showed a downward trend with the addition of lysyl-glutamate dipeptide to the feed. These results indicate that the addition of lysyl-glutamate dipeptide to the feed can significantly improve feed utilization efficiency, promote growth performance, and reduce visceral fat content in Xiangyun crucian carp. The best growth performance was achieved in the 1.60% and 2.00% dipeptide supplementation groups.

[0047] Table 2 Effects of lysyl-glutamic acid dipeptide on growth performance of Xiangyun crucian carp

[0048]

[0049] Note: Data are expressed as mean ± standard error. The same lowercase letters or no letters in the same column indicate no significant difference (P>0.05). Different lowercase letters in the same column indicate significant difference (P<0.05).

[0050] Example 2

[0051] Experimental study on the effect of lysyl-glutamic acid dipeptide on the flavor of Xiangyun crucian carp muscle

[0052] The experimental materials and methods, experimental location, and feeding management plan in this example were the same as those described in Example 1. After the 60-day breeding experiment, three fish of approximately average weight were randomly selected from each tank. The back muscles of the Xiangyun crucian carp were removed on ice, and the muscle flavor free amino acid and nucleotide content were determined using fresh muscle samples. The determination method is as follows:

[0053] Accurately weigh 2-4 g (accurate to 0.0001 g) of fish muscle sample into a 10 mL volumetric flask. Dissolve and adjust to volume with 0.02 mol / L hydrochloric acid. Ultrasonicate the solid sample for 20 minutes and centrifuge at 6000 rpm for 5 minutes. Collect the supernatant for purification.

[0054] A C18 cleanup column (SHISEIDO, 4.6 mm*250 mm*5 μm) was activated with 5 mL of methanol and 5 mL of water, respectively. 2.5 mL of sample was added, followed by 1.5 mL of 0.02 mol / L hydrochloric acid. 100 μL of the sample after column was accurately taken into a 15 mL centrifuge tube and placed in a vacuum drying oven at 60°C for 2 h (to completely dry out the solvent). The centrifuge tube was filled with nitrogen, and 50 μL of derivatization reagent (ethanol:phenyl isothiocyanate:water:triethylamine=7:1:1:1) (prepared before use and filled with nitrogen) was accurately added. Derivatization was carried out at room temperature for 30 min. 0.45 mL of mobile phase A (0.1 mol / L anhydrous sodium acetate:acetonitrile=97:3) was added, mixed, and passed through a 0.45 μm organic membrane liquid chromatograph (Agilent, 1260).

[0055] All data in this example were statistically analyzed using SPSS 19.0 software, and data are expressed as mean ± standard error. Each relevant indicator was first tested for homogeneity of variance. After homogeneity of variance was confirmed, one-way ANOVA was performed. If significant differences were found between experimental groups, Duncan's method was used for multiple comparisons. P < 0.05 indicated a significant difference.

[0056] The contents of flavor free amino acids and nucleotides in the muscles of Xiangyun crucian carp of each group are shown in Table 3.

[0057] Table 3 The amount of flavor free amino acids and nucleotides in the muscles of Xiangyun crucian carp in each group

[0058]

[0059] Note: Data are expressed as mean ± standard error. The same lowercase letters or no letters in the same column indicate no significant difference (P>0.05). Different lowercase letters in the same column indicate significant difference (P<0.05).

[0060] As can be seen from Table 3, with the increase of glutathione content in the feed, the content of flavor amino acids glycine, alanine, and tyrosine in the muscle showed a gradual upward trend; at the same time, after feeding the experimental fish with glutathione added to the feed, the content of cytosine nucleotide, guanine nucleotide, hypoxanthine nucleotide, and adenine nucleotide in the muscle flavor nucleotides also showed a certain upward trend compared with the group without glutathione added, especially guanine nucleotide, which was significantly higher in the 0.40%, 0.80%, 1.20%, and 2.00% addition groups than in the control group, and reached the maximum in the 2.00% addition group. The above results show that adding an appropriate amount of lysylglutamate dipeptide to the feed can increase the content of flavor substances in the muscle of Xiangyun crucian carp and improve the flavor quality of Xiangyun crucian carp.

[0061] Example 3

[0062] Effect of Lysyl-Glutamic Acid Dipeptide on Intestinal Inflammatory Factors in Xiangyun Carp after Poisoning

[0063] The experimental materials and methods, experimental location, and feeding management plan in this example are the same as those described in Example 1. After the 60-day breeding experiment, after the sampling of Example 1 and Example 2 is completed, 10 experimental fish are taken from each tank and returned to the original experimental tank for the challenge experiment. Each experimental fish is intraperitoneally injected with 0.1 mL of Aeromonas hydrophila solution for infection. The challenge dose is 10 8 CFU / mL. 48 h after challenge, three fish were randomly selected from each tank, dissected on ice, and their intestinal tissues were quickly frozen in liquid nitrogen and then transferred to a -80°C freezer for genetic testing and analysis.

[0064] The inflammatory factors selected for detection in this example include tumor necrosis factor α1 (TNF-α1), interferon γ2 (IFN-γ2), interleukin 1β (IL-1β), interleukin 6 (IL-6), and interleukin 12p40 (IL-12p40). Table 4 shows the primer sequences used for quantitative analysis of the above inflammatory factors. First, total RNA from intestinal tissue was extracted using TRlzol reagent. Subsequently, the RNA quality and concentration were tested using spectrophotometry and agarose gel electrophoresis. The RNA was then reverse transcribed into a complementary DNA strand using the PrimeScrip RT kit (TaKaRa, Dalian, China). Real-time quantitative fluorescent PCR was performed on the relevant genes using a CFX96 Real-time PCR instrument (Bio-Rad, USA). The reaction system was as follows: 2 μL of cDNA, 0.8 μL of upstream and downstream primers, 10 μL of SYBR Premix ExTaq (TaKaRa), and 6.4 μL of double-distilled water. The cycling reaction conditions were as follows: denaturation reaction at 95°C for 3 minutes, 40 cycles of 95°C for 10 seconds, 60°C for 20 seconds, and 72°C for 10 seconds. β-actin was used as an internal reference gene to detect the relative expression of the target gene. Relative quantification was performed using the comparative CT value method (2 -ΔΔCt Law).

[0065] All data in this example were statistically analyzed using SPSS 19.0 software, and data are expressed as mean ± standard error. Each relevant indicator was first tested for homogeneity of variance. After homogeneity of variance was confirmed, one-way ANOVA was performed. If significant differences were found between experimental groups, Duncan's method was used for multiple comparisons. P < 0.05 indicated a significant difference.

[0066] The expression levels of intestinal genes in each treatment group of Xiangyun crucian carp after challenge were as follows Figure 1 shown.

[0067] Depend on Figure 1 As can be seen, with increasing levels of glutathione in the diet, the expression levels of TNF-α1, IFN-γ2, IL-1β, and IL-6 genes in the intestines of Xiangyun crucian carp showed a gradual downward trend. TNF-α1 and IL-6 were significantly lower in the 1.6% glutathione supplementation group than in the control group, while IFN-γ2 and IL-1β were significantly lower in the 1.6%-2.0% supplementation group than in the unsupplemented group. The relative expression of IL-12p40 was also significantly downregulated in the 0.4%-1.6% glutathione supplementation group compared to the control group. These results indicate that glutathione can significantly reduce the expression levels of inflammatory factors in the intestines of Xiangyun crucian carp after challenge, preventing excessive intestinal inflammatory responses and improving the disease resistance of the experimental fish.

[0068] A comprehensive analysis of Xiangyun crucian carp growth performance, meat flavor, and intestinal inflammatory cytokine expression after challenge with a toxic substance showed that the addition of lysyl-glutamate dipeptide to feed improved growth performance, flavor accumulation, and suppressed intestinal inflammatory responses. Overall, supplementing Xiangyun crucian carp with 1.6%-2.0% lysyl-glutamate dipeptide resulted in optimal growth, flavor, and health.

[0069] It will be easily understood by those skilled in the art that the above description is merely an embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of a lysyl-glutamate dipeptide in the preparation of a fish feed additive, characterized in that the fish is Xiangyun crucian carp; The feed contains fish meal, soybean meal, rapeseed meal, casein, fish oil, soybean oil, corn starch, flour, choline, multivitamin, sodium carboxymethyl cellulose, cellulose and lysyl-glutamic acid dipeptide; The mass ratio of fish meal, soybean meal, rapeseed meal, casein, fish oil, soybean oil, corn starch, flour, choline, multivitamin, sodium carboxymethyl cellulose and cellulose in the feed is 700-850:1250-1450:900-1100:400-500:150-210:150-210:1050-1300:600-800:25-35:150-210:150-210:380-480; 0.4-2.0% of lysyl-glutamic acid dipeptide is added to the feed.

2. The use of the lysyl-glutamate dipeptide according to claim 1 in the preparation of a fish feed additive, characterized in that 0.4%, 0.8%, 1.2%, 1.6% or 2.0% of the lysyl-glutamate dipeptide is added to the feed.

3. Use of the lysyl-glutamate dipeptide according to claim 1 in the preparation of a fish feed additive, characterized in that 1.6-2.0% of the lysyl-glutamate dipeptide is added to the feed.

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