Composition for inhibiting generation of histamine

Abstract

The invention provides an application of carrageenan oligosaccharide in preparation of a composition for inhibiting histamine generation. The result shows that the generation of histamine can be obviously inhibited by the treatment of the kappa-COS. And the inhibition action mechanism of the compound is further verified in a molecular docking manner. By combining with three amino acid residues on histidine decarboxylase through hydrogen bonds, an original catalysis cavity for catalyzing histidine is blocked, then histidine decarboxylation reaction is prevented, and finally the inhibition effect of histamine is achieved.

Description

Technical Field

[0001] The invention belongs to the field of food, and in particular relates to a composition for inhibiting the production of histamine. Background Art

[0002] Histamine, with the chemical name 2-(4-imidazolyl)ethylamine, molecular formula C2H9N3, molecular weight 111.0, is widely found in animals and plants. It is the most toxic biogenic amine and an endogenously produced biogenic monoamine, usually stored in mast cells of the skin and intestinal mucosal tissues. During the production, processing and storage process, histamine is also generated when proteins are decarboxylated by histidine decarboxylase produced by microorganisms.

[0003] Previous studies have shown that lactic acid bacteria and Lactobacillus plantarum have the ability to degrade histamine. However, the types of microorganisms involved in histamine degradation, the degradation efficiency of histamine by different microorganisms, the mechanism of action, and the feasibility of industrial application all need further research and exploration.

[0004] Currently, there is an urgent need to develop new methods and new functional substances for regulating or alleviating histamine in production. Summary of the invention

[0005] The present invention aims to solve one of the technical problems existing in the prior art to at least a certain extent. To this end, the present invention provides a composition for inhibiting the production of histamine.

[0006] According to one aspect of the present invention, there is provided use of carrageenan oligosaccharide in preparing a composition for inhibiting histamine production.

[0007] Preferably, the effective concentration of the carrageenan oligosaccharide is 0.1-1 g / L.

[0008] According to another aspect of the present invention, a method for inhibiting histamine production by histamine-producing bacteria is provided, wherein the histamine-producing bacteria are treated with carrageenan oligosaccharides.

[0009] Preferably, the effective concentration of the carrageenan oligosaccharide is 0.1-1 g / L.

[0010] The results of the present invention show that the treatment of κ-COS can significantly inhibit the generation of histamine. The inhibitory mechanism is further verified by molecular docking. The catalytic cavity of the original catalytic histidine is blocked by hydrogen bonding with the three amino acid residues on histidine decarboxylase, thereby preventing the histidine decarboxylation reaction, and finally achieving the inhibitory effect of histamine. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0012] Figure 1 1 is a graph showing the inhibition of histamine content by various groups of κ-COS provided according to an embodiment of the present invention.

[0013] Figure 2 It is a schematic diagram of molecular docking provided according to an embodiment of the present invention.

[0014] Figure 3 This is a diagram showing changes in gene expression during storage at 4° C. for κ-COS at different concentrations provided in an example of the present invention.

[0015] Figure 4 This is the effect of each group of κ-COS on the weight loss rate provided by the embodiments of the present invention.

[0016] Figure 5 This is the effect of each group of κ-COS on pH value provided by the embodiments of the present invention.

[0017] Figure 6 This is the effect of each group of κ-COS on color change provided by the embodiments of the present invention.

[0018] Figure 7 This is the effect of each group of κ-COS on the inhibition of bacterial colonies provided by the embodiments of the present invention.

[0019] Figure 8 This is the effect of each group of κ-COS on the total protein content provided by the embodiments of the present invention.

[0020] Fig. 9 1 is the effect of each group of κ-COS on the total thiol content (A) and the carbonyl content (B) provided according to the embodiments of the present invention. DETAILED DESCRIPTION

[0021] The following examples are provided to allow those skilled in the art to more clearly understand the present invention. It should be noted that the following examples do not limit the scope of protection claimed in the present invention and are only illustrative examples. The raw materials, reagents or devices mentioned in the following examples, unless otherwise specified, can be obtained from commercial sources or by known existing methods.

[0022] Example 1

[0023] Obtaining or preparing carrageenan oligosaccharides

[0024] Carrageenan Oligosaccharide, English name: Carrageenan Oligosaccharide, Trade name:

[0025] Structural formula:

[0026]

[0027] Carrageenan oligosaccharides are a series of oligosaccharides with different relative molecular weights and compositions obtained by degrading carrageenan using bio-enzyme degradation technology. Carrageenan oligosaccharides have a skeleton structure formed by alternating 1,3-β-D-galactose and 1,4-α-D-galactose. Depending on whether galactose contains internal ethers and the number and connection positions of sulfate groups on galactose, carrageenan oligosaccharides can be divided into seven types, among which the most common are κ-, ι- and λ-types. Compared with carrageenan, carrageenan oligosaccharides have a smaller molecular weight, better solubility, improved stability and safety, and because the active groups on the molecular chain are fully exposed, their original activity is improved, and even new activity is generated.

[0028] Enzymatic degradation by κ-carrageenase is one of the main ways to prepare κ-carrageenan oligosaccharides (κ-COS). Carrageenase produces even-numbered carrageenan oligosaccharides by breaking κ-1,4 glycosidic bonds. Usually, after characterizing the relevant enzymatic properties, oligosaccharides are prepared by thermostatic reactor under the optimal reaction conditions of the enzyme. Prepare a 1% carrageenan polysaccharide solution, add the enzyme solution and carrageenan substrate to the thermostatic reactor at a ratio of 1:3, and perform hydrolysis reaction under optimal conditions for 24 hours. After the reaction time is over, the degradation solution is inactivated at 100°C for 10 minutes, cooled to room temperature, and centrifuged at 6000rpm for 15 minutes to remove the undegraded polysaccharide fragments. Add 1 volume of anhydrous ethanol to precipitate the macromolecular polysaccharide, collect the supernatant by centrifugation, repeat this operation until no precipitate is generated, take the supernatant and concentrate it by rotary evaporation at 60°C, and then obtain powdered carrageenan oligosaccharides by vacuum freeze drying.

[0029] Example 2

[0030] Extraction and analysis of histamine

[0031] Using distilled water as solvent, κ-COS experimental groups with concentrations of 100, 200, 500 mg / L and 1 g / L were prepared. Fresh Spanish mackerel were selected and cleaned, impurities, scales and internal organs were removed, and treated with different concentrations of κ-COS solution for 5 minutes. The control group was immersed in the same volume of distilled water. After completion, the samples were dried at room temperature and stored at 4°C and a relative humidity of 80±2%.

[0032] Weigh 2g of fish sample into a 50ml centrifuge tube, add 10ml of 0.4mol / L perchloric acid solution, vortex extract for 2 minutes, centrifuge at 4000r / min for 5min, transfer the supernatant into a 25ml brown volumetric flask, repeat the above operation once and dilute to the mark with 0.4mol / L perchloric acid solution. Measure 1ml of sample solution and add it to a 5ml centrifuge tube, add 100μl of 2mol / L sodium hydroxide solution, 300μl of saturated sodium bicarbonate solution and 2mL of 10mg / mL dansyl chloride solution in sequence, cover the lid and react at 40℃ in the dark for 40min, shake once every 5min. After the reaction is completed, add 100μL of concentrated ammonia water to terminate the derivatization reaction and let it stand for 30min. Dilute it to 5mL with methanol, mix well, pass through a 0.45μm organic filter membrane, and detect it on the liquid phase. The liquid phase conditions are shown in Table 1.

[0033] Table 1. Liquid phase gradient elution program

[0034]

[0035] The histamine content in each test group was Figure 1 The horizontal axis (X axis) represents time, with days as the unit, from 0 to 8 days. The vertical axis (Y axis) represents histamine content (HIS content), with the unit of mg / kg. Figure 1 There are five groups of samples with different concentrations, namely: 0 mg / mL (black squares); 100 mg / mL (red dots); 200 mg / mL (blue triangles); 500 mg / mL (green inverted triangles); and 1 g / L (purple diamonds).

[0036] from Figure 1 It can be seen that the HIS content of samples with different concentrations gradually increased over time. Among them, the HIS content of the 0 mg / mL group (black squares) increased the fastest, reaching about 120 mg / kg on day 8. The 100 mg / mL group (red dots) was second, about 100 mg / kg on day 8. The HIS content of the remaining concentration groups also increased, but the growth rate was slower. As time went on, the HIS content of all groups showed a significant upward trend from day 6 to day 8, especially the low concentration groups (0 mg / mL and 100 mg / mL), which grew faster. There was almost no significant difference between the groups with different concentrations in the early stage (0-4 days), but as time went on, the effect of concentration on HIS content began to appear. Figure 1 The variation trend of HIS content over time under different concentration conditions is shown, indicating the use of carrageenan oligosaccharides in preparing a composition for inhibiting histamine production.

[0037] Example 3 Isolation of histamine-producing bacteria

[0038] Select fresh Spanish mackerel and wash it to remove impurities, scales and internal organs. After completion, dry the samples at room temperature and store them at 4°C and a relative humidity of 80±2%. Weigh 25 grams of fish sample and put it into a conical flask containing 125 ml of saline to prepare gradient dilutions. These dilutions are then cultured on HBI medium plates and cultured at 30°C for 72 hours. Pick out purple colonies for isolation and purification. The purified strains were then cultured in TSB, and after culturing at 30°C for 48 hours, a bacterial culture medium containing 20% ​​glycerol was prepared. These culture media were then frozen at -80°C for storage. For further analysis, 2 ml of activated bacterial solution was extracted from the TSB medium and frozen, and then shipped to Shanghai Shenggong Biotechnology Co., Ltd. to determine the complete genome of the strain.

[0039] The expression sequence of histidine decarboxylase was predicted according to the NCBI database, and the three-dimensional structure of the protein was obtained by homology modeling. The software Schrodinger Maestro was used to perform molecular docking of histidine decarboxylase with histidine and κ-COS, and the active site with the lowest binding energy was selected as the binding site of the histidine decarboxylase flavor small molecule. The three-dimensional structure formed by the binding was observed using the PyMOL molecular graphics system ( Figure 2 ).

[0040] Figure 2 A and Figure 2 B shows a three-dimensional structure of histidine decarboxylase, with the surface rendered in purple to show the overall shape and surface features of the molecule. Figure 2 B is the binding of κ-COS to histidine decarboxylase, Figure 2 A is the binding of histidine to histidine decarboxylase. Figure 2 C is a magnified detail of κ-COS binding to histidine decarboxylase, showing the interaction of specific amino acid residues with small molecules. It can be seen that some amino acid residues (such as Glu 95, HID 120 and HID 232) interact with small molecules through hydrogen bonds (dashed lines with bond length information). The hydrogen bond length is indicated as and etc., indicating the strength of these interactions.

[0041] from Figure 2 C, some key amino acid sites in histidine decarboxylase (Glu 95, HID 120, HID 232) form hydrogen bonds with the green κ-COS, indicating that these sites are very important for the binding of small molecules. Figure 2The binding mode between a histidine decarboxylase and κ-COS is shown, highlighting the binding pocket of the protein and its hydrogen bonding interaction with the ligand. This helps to understand the mechanism of protein-ligand interaction.

[0042] Example 4 Fluorescence PCR analysis of the mechanism of histamine inhibition.

[0043] The expression of the histidine decarboxylase gene cluster begins with the hdcA gene, followed by the hdcP, hdcRS, and finally the hdcB gene. Related studies have shown that the combination of hdcP and hdcA is a typical decarboxylation pathway in bacteria, while the hdcB and hdcRS genes are not essential in the histamine synthesis process. Therefore, the present invention selected the hdcA and hdcP genes to explore the regulatory effect of COS on histamine synthesis ( Figure 3 ). Figure 3 The results show the changes in gene expression levels in different treatment groups (0 mg / L and 1 g / L). In the 0 mg / L treatment group, the relative expression rates of hdcA and hdcP genes were close to 1, indicating that the expression of these two genes was high under this condition. In the 1 g / L treatment group, the relative expression rate of hdcA gene decreased significantly, close to 0, and the expression rate of hdcP gene also decreased significantly, but still maintained a certain level. 1 g / L COS significantly reduced the expression of hdcA and hdcP genes, especially the expression of hdcA gene was almost completely inhibited. This shows that COS inhibits the expression of genes related to histamine production, thereby reducing the histamine content in tissues.

[0044] The inhibitory mechanism of κ-COS on histamine was detected by fluorescence PCR technology. After screening out histamine-producing strains, different concentrations of bacterial liquid were prepared. The histamine-producing bacteria were inoculated into LB broth medium containing κ-COS and 0.5% histidine and incubated at 37°C for 24 hours; the sample without κ-COS was used as a control. After 12 hours of culture, the total RNA of the bacteria was isolated using the RNAprep pure cell / bacteria kit. The first-strand complementary DNA (cDNA) was synthesized using the FastKing RT kit reverse transcription system. The total volume of PCR was 20μL, including 10μL of genomic DNA removal product, 2μL of 10×King RT Buffer, 1μL of FastKing RT Enzyme Mix, 2μL of FQ-RT primer Mix, and 5μL of RNase-Free ddH2O. The reaction procedure was as follows: 42°C for 3 minutes, 42°C for 15 minutes, and 95°C for 3 minutes. SuperReal PreMix Plus (SYBR Green) was used for relevant gene expression analysis according to the instructions. The total volume of PCR was 25 μL, including 12.5 μL of 2×SYBR Green I Mix, 0.75 μL of each primer (10 μM), 2 μL of cDNA template, and 9 μL of RNase-Free ddH2O. The PCR amplification conditions were 95°C for 15 min, 95°C for 10 s, 45°C for 30 s, and 72°C for 20 s, for 40 cycles. The expression of 16S rRNA was detected as an internal control. Each analysis was performed three times. -△△Ct The relative quantification of specific genes was analyzed to detect their ability to inhibit histamine production.

[0045] Example 5 Weight loss, pH value and color change

[0046] Using distilled water as solvent, κ-COS experimental groups with concentrations of 100, 200, 500 mg / L and 1 g / L were prepared. Fresh Spanish mackerel were selected and cleaned, impurities, scales and internal organs were removed, and treated with different concentrations of κ-COS solution for 5 minutes. The control group was immersed in the same volume of distilled water. After completion, the samples were dried at room temperature and stored at 4°C and a relative humidity of 80±2%.

[0047] Three groups of fish were selected from each experimental group, and the weight loss was measured every other day ( Figure 4 ). The weight loss calculation was performed according to the following formula: weight loss rate (%) = [(initial weight - final weight) / initial weight] × 100.

[0048] The fish sample (10 g) and 100 mL of distilled water were homogenized with a homogenizer for 2 min. The homogenized solution was filtered and the pH value of the filtrate was measured with a glass electrode pH meter ( Figure 5 ).

[0049] The color change of the fish samples was measured every two days using a colorimeter. The color was expressed in terms of lightness (L*), redness (a*), and yellowness (b*). Figure 6 Finally, the brightness difference is calculated by the following formula to analyze the total color change: ΔE*=[(L0*-L*) 2 +(a0*-a*) 2 +(b0*-b*) 2 ] 1 / 2

[0050] Where L0*, a0*, and b0* represent the initial color values ​​on day 0. Figure 6 The X-axis represents time (in days), from 0 days to 8 days. The Y-axis represents the ΔE value, which is an indicator of color change and is used to indicate the degree of color change. The larger the ΔE value, the more obvious the color change.

[0051] Figure 6 There are five treatment groups with different concentrations, namely: 0mg / L (black squares); 100mg / L (red dots); 200mg / L (blue triangles); 500mg / L (green inverted triangles); 1g / L (purple diamonds). As time goes by, the ΔE values ​​of each group gradually increase, indicating that the color has changed, and the color change becomes more and more obvious as time goes on. The effects of different concentrations of treatment on the ΔE value are different: the color change of the 0mg / L group is the most significant, and the ΔE value reaches the highest on the 8th day, about 18. The 100mg / L group is second, and the ΔE value is also high. The ΔE values ​​of the 500mg / L and 1g / L groups are relatively low, indicating that the high concentration treatment has a certain inhibitory effect on the color change. Starting from the 4th day, the change rate of the ΔE value of each group accelerated, and the color change became more obvious. Figure 6 The trend of color change (ΔE) under different concentration treatments is shown. As time goes by, the color change becomes more and more significant, and the high concentration treatment (500 mg / L and 1 g / L) has a certain inhibitory effect on the color change, while the color change of the low concentration treatment group (0 mg / L and 100 mg / L) is more significant.

[0052] Example 6 Inhibition of bacterial colony analysis

[0053] Using distilled water as solvent, κ-COS experimental groups with concentrations of 100, 200, 500 mg / L and 1 g / L were prepared. Fresh Spanish mackerel were selected and cleaned, impurities, scales and internal organs were removed, and treated with different concentrations of κ-COS solution for 5 minutes. The control group was immersed in the same volume of distilled water. After completion, the samples were dried at room temperature and stored at 4°C and a relative humidity of 80±2%.

[0054] 10 g of fish meat sample was extracted and homogenized in 90 ml of sterile saline (0.9% NaCl) in a homogenizer for 2 minutes. TVC analysis was performed on plate count agar, and the plates were incubated at 30°C for 48 hours. Psychrophilic bacteria counts were analyzed using the spread plate method on plate count agar. The plates were incubated at 4°C for 240 hours. All results are expressed as log CFU / g ( Figure 7 ). Figure 7 In A, the X-axis represents time (in days), from 0 to 8 days. The Y-axis represents the total colony count (TVC, in Log10 CFU / g), where CFU stands for colony forming unit and is used to quantify the number of bacteria in a sample. Figure 7 A shows five treatment groups with different concentrations, namely: 0 mg / L (black squares); 100 mg / L (red dots); 200 mg / L (blue triangles); 500 mg / L (green inverted triangles); 1 g / L (purple diamonds). As time goes by, the total colony count (TVC) of each concentration group gradually increases, indicating that the number of bacteria continues to increase over time. Treatments with different concentrations have different effects on the growth of colony counts: the bacteria in the 0 mg / L group grew the fastest, and the TVC value was about 9 log CFU / g on the 8th day. The bacteria in the high-concentration treatment groups (500 mg / L and 1 g / L) grew more slowly, and the TVC value was about 7.5 log CFU / g on the 8th day, which shows that high-concentration treatment has a certain inhibitory effect on bacterial growth.

[0055] Figure 7 In B, the X-axis also represents time (in days), from 0 to 8 days. The Y-axis represents the number of bacteria of a specific type (PB, in Log10 CFU / g). The same five different concentration treatment groups show the growth trend of the number of bacteria over time. As time goes by, the PB values ​​in all treatment groups gradually increase, indicating that the number of bacteria of a specific type also increases over time. The bacteria in the low concentration groups (0 mg / L and 100 mg / L) grew faster, with a PB value of approximately 5 logCFU / g on the 8th day, while the bacteria in the high concentration treatment groups (500 mg / L and 1 g / L) grew slower, reaching approximately 4.5 logCFU / g on the 8th day.

[0056] In general, the total bacterial colony count (TVC) and the number of specific types of bacteria (PB) increased over time. The high-concentration treatments (500 mg / L and 1 g / L) performed better in inhibiting bacterial growth, and the bacterial growth rate was slower than that of the low-concentration treatment groups (0 mg / L and 100 mg / L). These data indicate that the high-concentration treatment may have an inhibitory effect on bacterial growth, while the low-concentration treatment has a faster bacterial growth.

[0057] Example 7 Protein oxidation analysis

[0058] Protein oxidation was evaluated by measuring protein content, free thiol and carbonyl content. Using distilled water as solvent, κ-COS experimental groups with concentrations of 100, 200, 500 mg / L and 1 g / L were prepared. Fresh Spanish mackerel were selected and cleaned, impurities, scales and viscera were removed, and treated with different concentrations of κ-COS solution for 5 minutes. The control group was immersed in the same volume of distilled water. After completion, the samples were dried at room temperature and stored at 4°C and a relative humidity of 80±2%.

[0059] 2.0 g of fish meat sample was mixed with 50 ml of 5% SDS solution in Tris buffer (100 mM, pH 8.0) and stirred at 12,000 rpm for 60 seconds using a homogenizer. After cooling the sample, the protein content in the filtrate was determined by measuring the absorbance at 280 nm using a spectrophotometer with bovine serum albumin as the standard ( Figure 8 ). Figure 8 In the figure, the X-axis represents time (in days), from 0 days to 8 days. The Y-axis represents protein content (in mg / g). There are five treatment groups with different concentrations, namely: 0 mg / L (black squares); 100 mg / L (red dots); 200 mg / L (blue triangles); 500 mg / L (green inverted triangles); 1 g / L (purple diamonds).

[0060] The protein content of each group showed a downward trend over time. From day 0 to day 8, the protein content of each treatment group gradually decreased, which indicated that there might be protein degradation or loss.

[0061] Different concentrations of treatment had different effects on the rate of protein content decline: the high concentration group (500 mg / L and 1 g / L) showed a slower protein content decline trend, while the low concentration group (0 mg / L and 100 mg / L) had a faster decline in protein content.

[0062] On the 8th day, the protein content of 0 mg / L and 100 mg / L dropped to nearly 100 mg / g, while the protein content of 500 mg / L and 1 g / L was higher, close to 140 mg / g. This indicates that the high concentration treatment may have a stronger protective effect on protein, delaying protein degradation. Figure 8 The results show the trend of protein content over time, and different treatment concentrations have different effects on protein content. The high concentration treatment group (500 mg / L and 1 g / L) can effectively delay the decrease of protein content, while the low concentration group has faster protein degradation.

[0063] Free sulfhydryl and carbonyl groups were detected using kits produced by Merck and Solebol ( Fig. 9 ). Fig. 9In A: The X-axis represents time (in days), from 0 to 8 days. The Y-axis represents the total thiol group content of the protein (in μmol / g protein). Points of different colors and shapes represent treatment groups with different concentrations, namely: 0 mg / L (black squares), 100 mg / L (red dots), 200 mg / L (blue triangles), 500 mg / L (green inverted triangles) and 1 g / L (purple diamonds).

[0064] The total thiol group content gradually decreased in each treatment group over time. This suggests that during the treatment process, the thiol groups of the protein may be gradually oxidized or react with other molecules. At 8 days, the 0 mg / L and 100 mg / L treatment groups showed the lowest total thiol group content, while the thiol group content of the higher concentration groups (such as 200 mg / L, 500 mg / L, 1 g / L) was relatively high, indicating that the high concentration treatment has a better protective effect on the thiol group.

[0065] Fig. 9 In B, the X-axis is also time (in days), from 0 days to 8 days. The Y-axis represents the carbonyl group content of the protein (in μmol / g protein). Over time, the carbonyl group content of all treatment groups gradually increased, indicating that the protein may have been oxidized during the treatment process, resulting in the accumulation of carbonyl groups. The 0 mg / L group (black squares) showed the most significant increase in carbonyl groups, while the carbonyl group increase rate of the high concentration group was slower, indicating that the high concentration treatment may have a certain inhibitory effect on protein oxidation.

[0066] In summary, Fig. 9 A indicates that the sulfhydryl groups of proteins are gradually reduced over time, which may be related to oxidation or other reactions. Fig. 9 B shows that the carbonyl groups of proteins gradually increase over time, further supporting the oxidation process of proteins. The high concentration groups (such as 500 mg / L and 1 g / L) showed a protective effect on proteins in these two indicators, delaying the loss of sulfhydryl groups and the generation of carbonyl groups. Overall, Fig. 9 The changing trends of the sulfhydryl and carbonyl group contents of proteins over time under different concentration treatments were shown, indicating that high concentration treatment had a certain inhibitory effect on protein oxidation.

[0067] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of carrageenan oligosaccharide in preparing a composition for inhibiting histamine production.

2. The use according to claim 1, characterized in that The effective concentration of the carrageenan oligosaccharide is 0.1-1 g / L.

3. A method for inhibiting histamine production by histamine-producing bacteria, characterized in that: The histamine-producing bacteria are treated with carrageenan oligosaccharides.

4. The method according to claim 3, characterized in that The effective concentration of the carrageenan oligosaccharide is 0.1-1 g / L.

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