Oyster peptide and anthocyanin assembled compound as well as preparation method and application thereof

Abstract

The invention discloses an oyster peptide and anthocyanin assembled compound as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The preparation method of the oyster peptide and anthocyanin assembled compound comprises the steps that oyster peptide and anthocyanin are dissolved in water, the oyster peptide and anthocyanin assembled compound is obtained after reaction, and the mass ratio of the oyster peptide to the anthocyanin is 1: 4-4: 1. According to the invention, the oyster peptide-anthocyanin assembled compound is taken as a research object, and the effect of synergistically inhibiting pancreatic lipase activity of the assembled compound is discussed. Research results can provide theoretical basis and scientific guidance for clarification of a synergistic lipid-lowering action mechanism of oyster peptide and anthocyanin and development and utilization of related weight-losing and lipid-lowering functional foods.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to an oyster peptide and anthocyanin assembly complex and a preparation method and application thereof. Background Art

[0002] With the rapid increase in the number of obese people, obesity has become a global problem, and it is generally considered to be a risk factor for various diseases, such as diabetes and cardiovascular disease. The formation of obesity is largely caused by unhealthy eating habits and lifestyles, especially long-term excessive intake of high-fat diets, which are characterized by hyperlipidemia. Pancreatic lipase (PL) is a key enzyme in the process of fat digestion and absorption. Inhibiting the activity of pancreatic lipase can reduce the body's absorption of fat. Therefore, reducing fat digestion and absorption by inhibiting pancreatic lipase activity is an effective method to inhibit obesity and reduce hyperlipidemia. However, currently commonly used weight loss and lipid-lowering drugs in clinical practice, such as orlistat, liraglutide, statins, etc., have certain side effects on the human body, including gastrointestinal adverse reactions, oily stools, stool urgency and oily spots, etc., which limits their widespread application. Therefore, the development of new pancreatic lipase inhibitors that are natural, efficient, and non-toxic has become one of the research hotspots in recent years.

[0003] At present, common natural sources of pancreatic lipase inhibitors include active substances such as flavonoids, saponins, terpenes, and alkaloids. Anthocyanin is a flavonoid substance that is widely distributed in dark grains, berries, and vegetables, giving them red, blue, purple, and other colors. Studies have found that anthocyanins have multiple functions such as antioxidant, anti-inflammatory, hypoglycemic, and hypolipidemic. However, anthocyanins have poor stability, and their application is easily restricted by some physical or chemical factors, such as pH, oxygen, temperature, and metal ions. With the continuous deepening of research on marine biological resources, marine bioactive peptides have received widespread attention due to their good natural activity, safety, and high efficiency. Studies have shown that oyster peptides have multiple biological activities such as hypoglycemic, immunomodulatory, hypolipidemic, and anti-fatigue, but they have the problem of poor digestive stability, which greatly limits the activity of active peptides. In recent years, literature studies have reported that the interaction between flavonoids and proteins has the advantage of improving the stability of both and promoting their activity. Therefore, the synergistic assembly of oyster peptides and anthocyanins into a complex has the potential to protect the active structures of both and synergistically enhance the inhibitory activity of pancreatic lipase. However, there are few reports on the synergistic inhibition of pancreatic lipase activity by oyster peptides and anthocyanins and the exploration of the interaction between the two. Summary of the invention

[0004] The purpose of the present invention is to provide an oyster peptide and anthocyanin assembly complex and its preparation method and application. The present invention takes the oyster peptide-anthocyanin assembly complex as the research object and explores that the assembly complex has the effect of synergistically inhibiting the activity of pancreatic lipase. The research results can provide a theoretical basis and scientific guidance for clarifying the synergistic lipid-lowering mechanism of oyster peptides and anthocyanins and the development and utilization of related weight-loss and lipid-lowering functional foods.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] One of the technical solutions of the present invention is to provide a method for preparing an oyster peptide and anthocyanin assembly complex, comprising the following steps:

[0007] The oyster peptide and anthocyanin are dissolved in water, and after the reaction, an oyster peptide and anthocyanin assembly complex is obtained;

[0008] The amino acid sequence of the oyster peptide is VDLM, VAVL, VADL, VAFE, IGAL, LGA L, LGLE, ALPNSPF, AIPNSPF, AFGPGLE, DQPHFPF, GIQYPVVV, QEIPNSPF, EHVPGSPF, GFPPITGT, TFPVINPA, AIPVHDPSQ, APPPPPPMQ, DAHIPGSPF, PTAPGEYAVH, APPPPPPMQGQ, VVGLDKPMTVN, DIRIPVGPIFN, SAGPGDIGIAL and YFPTAPGEYAVH.

[0009] The mass ratio of the oyster peptide to the anthocyanin is 1:4 to 4:1.

[0010] Preferably, the reaction temperature is 25-50°C and the reaction time is 0.5-5h.

[0011] Preferably, the preparation steps of the oyster peptide include: adding water to oyster meat and homogenizing it, adjusting the pH to 9, adding alkaline protease for enzymolysis, inactivating the enzyme, centrifuging, and drying the supernatant to obtain the oyster peptide.

[0012] More preferably, the amount of alkaline protease added is 2% of the weight of oyster meat; the temperature of the enzymolysis is 50° C., and the time is 3 hours.

[0013] The second technical solution of the present invention: provides an oyster peptide and anthocyanin assembly complex prepared according to the above-mentioned preparation method of the oyster peptide and anthocyanin assembly complex.

[0014] The third technical solution of the present invention is to provide an application of the above-mentioned oyster peptide and anthocyanin assembly complex in the preparation of a drug for preventing or treating obesity or hyperlipidemia.

[0015] The fourth technical solution of the present invention is to provide an application of the above-mentioned oyster peptide and anthocyanin assembly complex in the preparation of lipid-lowering food.

[0016] The beneficial technical effects of the present invention are as follows:

[0017] The present invention prepares oyster peptides by enzymatic hydrolysis, explores the in vitro lipid-lowering activity of oyster peptides and anthocyanins, and compounds oyster peptides and anthocyanins at different mass ratios to study their synergistic lipid-lowering effects. The in vitro pancreatic lipase activity inhibition model and the ZIP model and HSA model in the Synergy Finder software were used to screen out the best synergistic region, and the Compusyn synergistic calculation model was used to analyze the activity enhancement effect of oyster peptides and anthocyanins. At the same time, the enzyme inhibition kinetic model was used to study the inhibition type of pancreatic lipase of oyster peptides, anthocyanins and their complexes, and multispectroscopy was used to study the interaction mechanism of oyster peptides and anthocyanins. The results show that oyster peptides and anthocyanins have good pancreatic lipase inhibitory activity, among which oyster peptide-anthocyanin complexes of different ratios all show a certain synergistic effect, and the synergistic effect is the strongest when oyster peptide: anthocyanin = 1:1. Enzyme inhibition kinetic analysis shows that the inhibition modes of oyster peptides, anthocyanins and their complexes on pancreatic lipase include mixed inhibition and competitive inhibition. Analysis of the multispectroscopy experimental results showed that oyster peptides and anthocyanins interacted to form a complex, exposing tyrosine and tryptophan residues in the complex, reducing hydrophobicity and increasing polarity. The oyster peptide and anthocyanin assembly complex provided by the present invention provides a new direction for the treatment and prevention of obesity and hyperlipidemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 These are the results of the inhibitory activity measurement of oyster peptides and anthocyanins on pancreatic lipase in Example 2, wherein A is the inhibitory activity of oyster peptides on pancreatic lipase, and B is the inhibitory activity of anthocyanins on pancreatic lipase.

[0019] Figure 2 This is a synergistic score diagram of the lipid-lowering effect of oyster peptides and anthocyanins under the HSA model and the ZIP model based on Example 2, wherein A is the overall synergistic score of the ZIP model when the concentration of oyster peptides and anthocyanins is 0.4-3.2 mg / mL, B is the overall synergistic score of the ZIP model when the concentration of oyster peptides and anthocyanins is 0.8-3.2 mg / mL, C is the overall synergistic score of the HSA model when the concentration of oyster peptides and anthocyanins is 0.4-3.2 mg / mL, and D is the overall synergistic score of the HSA model when the concentration of oyster peptides and anthocyanins is 0.8-3.2 mg / mL.

[0020] Figure 3The graph shows the inhibitory activity of oyster peptides and anthocyanins at different mass ratios on pancreatic lipase in Example 2, wherein A is oyster peptide: anthocyanin = 4:1, B is oyster peptide: anthocyanin = 2:1, C is oyster peptide: anthocyanin = 1:1, D is oyster peptide: anthocyanin = 1:2, and E is oyster peptide: anthocyanin = 1:4.

[0021] Figure 4 The combined index (CI) and dose reduction index (DRI) of the inhibitory effect of oyster peptides and anthocyanins in different mass ratios on pancreatic lipase in Example 2 are shown in FIG. 1 A is the combined index diagram of the pancreatic lipase inhibitory effect of oyster peptide: anthocyanin = 4:1, 2 A is the combined index diagram of the pancreatic lipase inhibitory effect of oyster peptide: anthocyanin = 2:1, 3 A is the combined index diagram of the pancreatic lipase inhibitory effect of oyster peptide: anthocyanin = 1:1, 4 A is the combined index diagram of the pancreatic lipase inhibitory effect of oyster peptide: anthocyanin = 1:2, 5 B is the combined index diagram of the pancreatic lipase inhibitory effect of oyster peptide: anthocyanin = 1:4, 1 B is the dose reduction index diagram of oyster peptide: anthocyanin = 4:1. 2 B is the dose reduction index diagram of oyster peptide: anthocyanin = 2:1. 3 B is the dose reduction index diagram of oyster peptide: anthocyanin = 1:1, 4 B is the dose reduction index diagram of oyster peptide: anthocyanin = 1:2. 5 This is a dose reduction index graph for oyster peptide: anthocyanin = 1:4.

[0022] Figure 5 Graphs showing the reversible inhibitory effects of oyster peptides, anthocyanins and their synergistic complexes on pancreatic lipase in Example 2, wherein A is a graph showing the reversible inhibitory effects of oyster peptides on pancreatic lipase, B is a graph showing the reversible inhibitory effects of anthocyanins on pancreatic lipase, and C is a graph showing the reversible inhibitory effects of oyster peptide-anthocyanin synergistic complexes on pancreatic lipase.

[0023] Figure 6 It is a Lineweaver-Burk curve diagram of the inhibitory effect of oyster peptides, anthocyanins and their synergistic complexes on lipase in Example 2, wherein A is a Lineweaver-Burk curve diagram of the inhibitory effect of oyster peptides on lipase, B is a Lineweaver-Burk curve diagram of the inhibitory effect of anthocyanins on lipase, and C is a Lineweaver-Burk curve diagram of the inhibitory effect of oyster peptide-anthocyanin synergistic complex on lipase.

[0024] Figure 7It is the ultraviolet absorption spectrum of the synergistic complex of oyster peptide and anthocyanin in Example 2 and the infrared spectrum of oyster peptide, anthocyanin and their synergistic complex, wherein A is the ultraviolet absorption spectrum after the synergistic complex of oyster peptide and anthocyanin, and B is the infrared spectrum of oyster peptide, anthocyanin and their synergistic complex.

[0025] Figure 8 These are the fluorescence spectrum and synchronous fluorescence spectrum of the interaction between oyster peptide and anthocyanin in Example 2, wherein A is the fluorescence spectrum of the interaction between oyster peptide and anthocyanin, B is the synchronous fluorescence spectrum of the interaction between oyster peptide and anthocyanin when △λ=15nm, and C is the synchronous fluorescence spectrum of the interaction between oyster peptide and anthocyanin when △λ=60nm. DETAILED DESCRIPTION

[0026] Now, various exemplary embodiments of the present invention are described in detail, and this detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention. It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention.

[0027] In addition, for the numerical range in the present invention, it is understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] The anthocyanins used in the embodiment of the present invention are mulberry anthocyanin extracts, and the mass fraction of anthocyanins in the anthocyanin extracts used is 36.0% based on cyanidin-3-O-glucoside.

[0031] Example 1

[0032] Preparation of oyster peptides:

[0033] The viscera-free Hong Kong oyster meat was thawed in running water, and water was added to the slurry at a solid-liquid ratio of 1:3 (g:mL), and then homogenized at 8000rpm for 2min, the pH was adjusted to 9, 2wt.% alkaline protease was added to the homogenized oyster meat, and enzymatic hydrolysis was performed in a 50°C water bath for 3h, the enzyme was inactivated in a boiling water bath for 15min, and the mixture was cooled to room temperature in an ice water bath and centrifuged (4°C, 8000r / min) for 15min. The supernatant was collected by filtration with absorbent gauze, and the oyster peptide was obtained after freeze-drying for later use.

[0034] It was determined that the amino acid sequence of the oyster peptide prepared in Example 1 was VDLM, VAVL, VADL, VAFE, IGAL, LGAL, LGLE, ALPNSPF, AIPNSPF, AFGPGLE, DQPHFPF, GIQYPVVV, QEIPNSPF, EHVPGSPF, GFPPITGT, TFPVINPA, AIPVHDPS Q, APPPPPPMQ, DAHIPGSPF, PTAPGEYAVH, APPPPPPMQGQ, VVGLDKPMTVN, DIRIPVGPIFN, SAGPGDIGIAL and YFPTAPGEYAVH.

[0035] Example 2

[0036] 1. Determination of the inhibitory activity of oyster peptides and anthocyanins on pancreatic lipase

[0037] The sample oyster peptide and anthocyanin were dissolved in ultrapure water according to the gradient concentration to prepare a series of sample solutions with a concentration gradient. 100 μL of sample solution and 100 μL of lipase solution (0.35 U / mL) were mixed in a 2 mL centrifuge tube, and Tris-HCl buffer solution (pH = 8.0) was added to 350 μL, and the mixture was incubated at 37°C for 10 min. Then, 50 μL of 1 mg / mL P-npp substrate was added and the mixture was incubated at 37°C for 20 min. Finally, 200 μL of the reaction solution was taken in a 96-well plate, and the absorbance value was measured at 405 nm, and the inhibition rate was calculated according to formula (1). The experiment set up a sample group, a sample blank group, a control group, and a blank group.

[0038]

[0039] In formula (1): A 1 is the absorbance of the sample group

[0040] A 2 is the absorbance of the sample blank group

[0041] B 1 The absorbance of the control group

[0042] B 2 The absorbance of blank group

[0043] 2. Preparation method of the composite with synergistic lipid-lowering effect

[0044] Ultrapure water was used to prepare oyster peptide and anthocyanin solutions with a concentration of 3.2 mg / mL, and the sample solutions were respectively pipetted into 2 mL centrifuge tubes to make the mass ratio of oyster peptide: anthocyanin in the mixed solution = 4:1, 2:1, 1:1, 1:2, 1:4. After sufficient oscillation and mixing, the mixture was reacted at 25°C and 1000 r / min for 2 h to obtain the oyster peptide-anthocyanin complex.

[0045] 3. Analysis of the synergistic effect of oyster peptides and anthocyanins in inhibiting pancreatic lipase activity

[0046] 3.1 Screening of synergistic concentration ranges for oyster peptides and anthocyanins in inhibiting pancreatic lipase

[0047] The sample dose synergistic matrix of oyster peptides and anthocyanins was designed, and the sample concentrations corresponding to the matrix were combined for lipase inhibition activity determination. The highest single drug (HSA) and zero interaction potency (ZIP) synergistic scoring models of Synergy Finder software were used to analyze the synergistic lipid-lowering ability of the two at different concentrations. The Synergy score (δ) was calculated using the HSA model and the ZIP model, and δ>10, δ<-10 and -10<δ<10, respectively corresponding to the synergistic, antagonistic and additive effects between sample combinations.

[0048] 3.2 Optimization of the synergistic ratio of oyster peptides and anthocyanins in inhibiting pancreatic lipase

[0049] According to the region with the highest synergy screened out by Synergy Finder, Compusyn software was used to further explore the synergistic effect of oyster peptide-anthocyanin complexes (4:1, 2:1, 1:1, 1:2, 1:4) with different mass ratios in this region. CI was determined according to the median effect principle proposed by Chou and Talalay, and the interaction strength and properties between the single components of the complex were quantitatively judged by the CI value. CI < 1 is considered to be a synergistic effect, CI = 1 is considered to be an additive effect, and CI > 1 is considered to be an antagonistic effect; the lower the CI value, the stronger the synergistic potential of the combination.

[0050] 4. Study on the enzyme inhibition kinetics of oyster peptides, anthocyanins and their synergistic complexes

[0051] The concentration of p-NPP was set at 1 mg / mL, and the experimental conditions were with and without the addition of oyster peptide (0, 0.8, 2 mg / mL), anthocyanin (0, 1.6, 3.2 mg / mL) and complex (0, 2, 2.4 mg / mL). The absorbance values ​​at different enzyme concentrations (0.05, 0.15, 0.25, 0.35, 0.45 U / mL) were measured. The enzyme concentration was used as the abscissa and the reaction rate (v) was used as the abscissa to draw a curve to determine the reversible effect of the inhibitor on lipase.

[0052] The concentration of pancreatic lipase solution was set at 0.35 U / mL. The experimental conditions were without addition and addition of oyster peptide (0, 0.6, 1 mg / mL), anthocyanin (0, 0.8, 2 mg / mL) and complex (0, 1.2, 2 mg / mL). The absorbance values ​​at different p-NPP concentrations (0.6, 0.8, 1, 1.2, 1.4 mg / mL) were measured. The Lineweaver-Burk curve was drawn with the reciprocal of the substrate concentration (1 / [S]) as the abscissa and the reciprocal of the reaction rate (1 / v) as the ordinate. The inhibition type of the inhibitor on pancreatic lipase was determined according to the Lineweaver-Burk equation.

[0053] 5. Study on the interaction between oyster peptides and anthocyanins

[0054] 5.1 UV-Vis absorption spectrum analysis

[0055] A fixed concentration of oyster peptide (1 mg / mL) was mixed with different concentrations of anthocyanins (0.1 mg / mL-1 mg / mL) to prepare samples. The samples were oscillated at 298 K for 2 h, and the UV-visible absorption spectra of oyster peptide and the complex were measured using a UV-visible spectrophotometer in the wavelength range of 190-600 nm.

[0056] 5.2 Fourier transform infrared spectroscopy (FT-IR) analysis

[0057] 1 mg of vacuum freeze-dried oyster peptides, anthocyanins and complex samples were accurately weighed, mixed with 100 mg of potassium bromide, ground and pressed into tablets, and the infrared spectrum was measured by potassium bromide tablet method with a scanning wavelength of 4000-400 cm-1 and 32 scans.

[0058] 5.3 Fluorescence spectroscopy analysis

[0059] Intrinsic fluorescence: Referring to the method in 1.3.2, the samples were prepared by mixing a fixed concentration of oyster peptide (1 mg / mL) with different concentrations of anthocyanins (0.04 mg / mL-0.6 mg / mL) and oscillating the samples at 298 K for 2 h, so that the final concentration of oyster peptide in each mixture was accurately 0.5 mg / mL, the emission signal collection range of the sample was 300-500 nm, the excitation wavelength was 280 nm, and the excitation and emission slit widths were both 5.0 nm.

[0060] Synchronous fluorescence: The sample concentration remains unchanged, and the sample is oscillated at 298K for 2h. The synchronous fluorescence spectra are recorded when the emission wavelength is 280-400nm and Δλ is 15nm and 60nm respectively.

[0061] 6. Statistical Analysis

[0062] All experiments were repeated 3 times, and the experimental results were expressed as mean ± standard deviation. SPSS 27.0 software was used to process the experimental data and perform one-way ANOVA. Duncan analysis was used for multiple comparisons, and P < 0.05 indicated significant differences between groups. Origin Pro 2021 was used to draw images.

[0063] VII. Results and Analysis

[0064] 7.1 Inhibitory activity of oyster peptides and anthocyanins on pancreatic lipase

[0065] The results of the inhibitory activity of oyster peptides and anthocyanins on pancreatic lipase are shown in Figure 1 , wherein A is the inhibitory activity of oyster peptide on pancreatic lipase, and B is the inhibitory activity of anthocyanin on pancreatic lipase. Figure 1 As shown in Figure A, oyster peptides have a certain inhibitory ability on pancreatic lipase. As the concentration of oyster peptides increases, the inhibitory ability on pancreatic lipase also increases, showing a dose-concentration effect. Its IC 50 The value is 1.35±0.02mg / mL. Figure 1 As shown in Figure B, with the increase of anthocyanin concentration, the inhibitory activity against pancreatic lipase also increased. Its IC 50 The value was 3.54±0.06mg / mL. Both of them had certain inhibitory activity on pancreatic lipase.

[0066] 7.2 Synergistic lipid-lowering effects of oyster peptide-anthocyanin complex

[0067] The present invention simultaneously adopts two synergistic calculation models, HSA and ZIP, to evaluate the synergistic effect of oyster peptides and anthocyanins in inhibiting pancreatic lipase, and screens the synergistic concentration range thereof. Figure 2This is a synergistic score diagram of the lipid-lowering effect of oyster peptides and anthocyanins based on the HSA model and the ZIP model in Example 2, wherein A is the overall synergistic score of the ZIP model when the concentration of oyster peptides and anthocyanins is 0.4-3.2 mg / mL, B is the overall synergistic score of the ZIP model when the concentration of oyster peptides and anthocyanins is 0.8-3.2 mg / mL, C is the overall synergistic score of the HSA model when the concentration of oyster peptides and anthocyanins is 0.4-3.2 mg / mL, and D is the overall synergistic score of the HSA model when the concentration of oyster peptides and anthocyanins is 0.8-3.2 mg / mL. Figure 2 From A and C, we can see that when the concentration of oyster peptides and anthocyanins is 0.4-3.2 mg / mL, the overall synergistic score (δ) of the ZIP model is 7.899, and the overall synergistic score (δ) of the HSA model is 11.412, indicating that there is a synergistic effect between the two within this concentration range. Figure 2 As shown in Figures B and D, when the concentration of oyster peptides and anthocyanins is in the range of 0.8 mg / mL to 3.2 mg / mL, the ZIP synergy score (δ) is 11.208 (δ>10), and the HSA synergy score (δ) is 17.781 (δ>10), at which time the two achieve a strong synergistic effect. In summary, both the ZIP model and the HSA model show that oyster peptides and anthocyanins have a synergistic inhibitory effect on pancreatic lipase, and when the concentration is 0.8 mg / mL to 3.2 mg / mL, oyster peptides and anthocyanins have a significant synergistic inhibitory and synergistic effect on pancreatic lipase.

[0068] 7.3 Analysis of synergy of oyster peptide-anthocyanin complex

[0069] 7.3.1 Inhibitory effect of oyster peptide-anthocyanin synergistic complexes in different proportions on pancreatic lipase

[0070] Figure 3 The results of the inhibitory activity of oyster peptides and anthocyanins in different mass ratios on pancreatic lipase in Example 2 are shown in Figure 2, wherein A is oyster peptide: anthocyanin = 4:1, B is oyster peptide: anthocyanin = 2:1, C is oyster peptide: anthocyanin = 1:1, D is oyster peptide: anthocyanin = 1:2, and E is oyster peptide: anthocyanin = 1:4. Figure 3 It can be seen that the synergistic complexes of oyster peptides and anthocyanins in different mass ratios (4:1; 2:1; 1:1; 1:2; 1:4) have good inhibitory ability against pancreatic lipase, and with the increase of concentration, the inhibition rate gradually increases, showing a dose-dependent manner. The order of the inhibitory ability of oyster peptides and anthocyanins in different mass ratios against pancreatic lipase is (oyster peptides: anthocyanins) 4:1 (IC 50 =1.80±0.02mg / mL)>2:1(IC 50 =2.00±0.06mg / mL)>1:1(IC 50 =2.11±0.03mg / mL)>1:2(IC50 =2.56±0.04mg / mL)>1:4(IC 50 =2.69±0.08mg / mL), among which oyster peptide: anthocyanin = 4:1 had the strongest inhibitory effect on pancreatic lipase. 50 The values ​​were not higher than those of oyster peptides alone (1.35 mg / mL), but were significantly stronger than those of anthocyanins alone (3.54 mg / mL) (P<0.05). By further comparing and analyzing the amounts of peptides and anthocyanins used when the oyster peptide and anthocyanin synergistic complex exerted pancreatic lipase inhibitory activity at different mass ratios, it was found that when the pancreatic lipase inhibition rate was the same, the amounts of oyster peptides and anthocyanins used in the synergistic complex were significantly lower than those of oyster peptides and anthocyanins used alone to exert the same pancreatic lipase inhibitory effect. When the synergistic complexes with a mass ratio of oyster peptides to anthocyanins of 4:1, 2:1, 1:1, 1:2, and 1:4 reached the half-maximal inhibitory concentration, the amount of anthocyanins in the complexes was 0.36, 0.67, 1.06, 1.71, and 2.15 mg / mL, respectively, which was significantly reduced compared with the amount of anthocyanins used alone (P<0.05), and decreased by 89.83%, 81.07%, 70.06%, 51.69%, and 39.27%, respectively. When the complexes with a mass ratio of 1:1, 1:2, and 1:4 reached the half-maximal inhibitory concentration, the amount of oyster peptides in the complexes was 1.06, 0.85, and 0.54 mg / mL, respectively, which was significantly reduced compared with the amount of oyster peptides used alone (P<0.05), and decreased by 21.48%, 37.04%, and 60.00%, respectively. In summary, the dosage of oyster peptides and anthocyanins in the complexes (1:1, 1:2, 1:4) was significantly reduced compared with that used alone (P<0.05), which has the potential to reduce dosage, improve stability, and enhance activity.

[0071] 7.3.2 Analysis of the combined index (CI) and dose reduction index (DRI) of oyster peptides and anthocyanins in different ratios for synergistic inhibition of pancreatic lipase

[0072] The dose reduction index (DRI) is an index used to measure how many times the dose of each substance is reduced when two or more active substances are used synergistically at the same level of action compared to when they are used alone. The larger the DRI, the greater the dose reduction under the same therapeutic effect; while the combination index (CI) is used to evaluate the synergistic effect between substances. The smaller the CI value, the stronger the synergistic effect between the two. The combination index (CI) and the dose reduction index (DRI) of the inhibitory effect of oyster peptides and anthocyanins in different mass ratios on pancreatic lipase in Example 2 are shown in Figure 2. 1 A is the combined index diagram of the pancreatic lipase inhibitory effect of oyster peptide: anthocyanin = 4:1, 2A is the combined index diagram of the pancreatic lipase inhibitory effect of oyster peptide: anthocyanin = 2:1, 3 A is the combined index diagram of the pancreatic lipase inhibitory effect of oyster peptide: anthocyanin = 1:1, 4 A is the combined index diagram of the pancreatic lipase inhibitory effect of oyster peptide: anthocyanin = 1:2, 5 B is the combined index diagram of the pancreatic lipase inhibitory effect of oyster peptide: anthocyanin = 1:4, 1 B is the dose reduction index diagram of oyster peptide: anthocyanin = 4:1. 2 B is the dose reduction index diagram of oyster peptide: anthocyanin = 2:1. 3 B is the dose reduction index diagram of oyster peptide: anthocyanin = 1:1, 4 B is the dose reduction index diagram of oyster peptide: anthocyanin = 1:2. 5 The dose reduction index of oyster peptide: anthocyanin = 1:4. Figure 4 It can be seen that the combined index (CI) of the synergistic complex of oyster peptides and anthocyanins at different mass ratios gradually decreases with the increase of concentration, and the corresponding CI of the complexes with different mass ratios (4:1; 2:1; 1:1; 1:2; 1:4) is less than 1 when Fa≥0.75, Fa≥0.97, Fa≥0.65, Fa≥0.7, and Fa≥0.55, respectively, indicating that oyster peptides and anthocyanins at different mass ratios have a certain synergistic effect. Combined with the analysis of the dose reduction index (DRI) graph, when oyster peptide: anthocyanin = 1:1, 1:2, 1:4, the dose reduction index (DRI) of oyster peptides and anthocyanins is greater than 1, and increases with the increase of the inhibitory effect (Fa), indicating that the combined use at these ratios can reduce the dosage of oyster peptides and anthocyanins. The CI values ​​of the complexes of oyster peptides and anthocyanins at different mass ratios are shown in Table 1. It can be seen from Table 1 that when oyster peptide: anthocyanin = 1:1, and Fa = 50 / 75 / 90 / 95, the corresponding CI values ​​are 1.08547, 0.88844, 0.73393, and 0.64752, which are significantly lower than other ratios, indicating that compared with other ratios, the synergistic inhibitory effect of oyster peptides and anthocyanins on pancreatic lipase is the strongest at this ratio.

[0073] In summary, oyster peptides and anthocyanins in different mass ratios (4:1; 2:1; 1:1; 1:2; 1:4) all have certain synergistic effects, among which oyster peptide: anthocyanin = 1:1 has the strongest synergistic effect. At the same time, it is speculated from the above results that the interaction between oyster peptides and anthocyanins when they work together may be affected by their compound ratio, which will affect the binding site of peptides and anthocyanins, and then affect the structure and properties of oyster peptide-anthocyanin complexes, resulting in different inhibitory abilities against pancreatic lipase.

[0074] Table 1 CI values ​​of oyster peptide-anthocyanin synergistic complexes with different mass ratios

[0075]

[0076] 7.4 Enzyme inhibition kinetics analysis

[0077] Enzyme inhibition types can be divided into reversible and irreversible types based on how the inhibitor binds to the enzyme binding site. Figure 5 The graphs are the reversible inhibitory effects of oyster peptides, anthocyanins and their synergistic complexes on pancreatic lipase in Example 2, wherein A is the reversible inhibitory effect of oyster peptides on pancreatic lipase, B is the reversible inhibitory effect of anthocyanins on pancreatic lipase, and C is the reversible inhibitory effect of oyster peptide-anthocyanin synergistic complex on pancreatic lipase. Figure 5 It can be seen that the straight lines of inhibitor samples with different concentrations: oyster peptides, anthocyanins and their synergistic complexes all pass through the origin approximately, and their slopes gradually decrease with increasing concentrations, indicating that the inhibition types of oyster peptides, anthocyanins and their complexes on pancreatic lipase are all reversible inhibition. The Lineweaver-Burk double reciprocal curve can be used to determine the type of inhibition of the inhibitor on the enzyme. Figure 6 The Lineweaver-Burk curves of the lipase inhibition effect of oyster peptides, anthocyanins and their synergistic complexes in Example 2, wherein A is the Lineweaver-Burk curve of the lipase inhibition effect of oyster peptides, B is the Lineweaver-Burk curve of the lipase inhibition effect of anthocyanins, and C is the Lineweaver-Burk curve of the lipase inhibition effect of the oyster peptide-anthocyanin synergistic complex. Figure 6 It can be seen that the double reciprocal curves of oyster peptides, anthocyanins and their complexes show a good linear relationship, such as Figure 6 As shown in A, with the increase of oyster peptide concentration, V m Reduce, K m The double reciprocal curve intersects at a point in the second quadrant, indicating that the inhibition type of oyster peptides on pancreatic lipase is non-competitive and competitive mixed inhibition, that is, oyster peptides can bind to pancreatic lipase through competitive inhibition and can also bind to pancreatic lipase-substrate complex through non-competitive inhibition. At the same time, the inhibition kinetic parameters of oyster peptides, anthocyanins and their synergistic complexes on pancreatic lipase are shown in Table 2. As shown in Table 2, the inhibition constant (K i :0.4582mg / mL) is less than the inhibition constant (K is :7.8049mg / mL), indicating that oyster peptides bind more tightly to pancreatic lipase than to the enzyme-substrate complex, that is, competitive inhibition plays a dominant role in mixed inhibition. Figure 6 As shown in Figure B, with the increase of anthocyanin concentration, V m Reduce, K mThe double reciprocal curve intersects at a point in the second quadrant, indicating that the inhibition type of anthocyanins on pancreatic lipase is also a mixed type of non-competitive and competitive inhibition, that is, anthocyanins can bind to both pancreatic lipase and pancreatic lipase-substrate complex; at the same time, the K i and K is They are 1.2114 mg / mL and 7.1436 mg / mL respectively, K i <K is This indicates that anthocyanins bind more tightly to pancreatic lipase than to the enzyme-substrate complex, and competitive inhibition plays a dominant role in mixed inhibition. Figure 6 As shown in Figure C, with the increase of the concentration of oyster peptide-anthocyanin complex, K m Increase, V m The double reciprocal curves intersect at a point on the Y axis, indicating that the complex has a competitive inhibition on lipase.

[0078] Table 2 Kinetic parameters of inhibition of pancreatic lipase by oyster peptides, anthocyanins and their synergistic complexes

[0079]

[0080]

[0081] 7.5 UV-Vis absorption spectrum analysis

[0082] Figure 7 A is the UV absorption spectrum of oyster peptides and anthocyanins after complexation (a→h: anthocyanins = 0, 0.1, 0.2, 0.4, 0.6, 0.7, 0.8, 1 mg / mL), Figure 7 As shown in Figure A, oyster peptides have absorption peaks near 200nm and 280nm, and with the increase of the concentration of synergistic anthocyanins, its maximum absorption peak gradually increases, indicating that there is an interaction between oyster peptides and anthocyanins. The formation of their conjugated system causes the π→π electron pair energy level transition, which leads to an increase in the intensity of their ultraviolet absorption. At the same time, the position of the maximum absorption peak undergoes a certain degree of red shift, indicating that after the synergistic anthocyanins, the aromatic amino acid microenvironment of oyster peptides changes, tyrosine and tryptophan residues are exposed, the hydrophobicity decreases, and the polarity increases, which further confirms that oyster peptides and anthocyanins interact with each other.

[0083] 7.6 Fourier Transform Infrared Spectroscopy (FT-IR) Analysis

[0084] Fourier transform infrared spectroscopy (FT-IR) is widely used to analyze the secondary structure changes of proteins or active peptides. FT-IR can provide a deep understanding of the interaction between proteins or active peptides and different ligands. -1The resulting absorption peak is called the amide A band, which mainly reflects the stretching vibration of NH and OH, and is between 1600 and 1700 cm -1 The absorption peak in the region is called amide I band, which mainly reflects C=O stretching; <1548cm -1 The absorption peak produced is called amide II band, which mainly reflects CN stretching and NH bending. The infrared spectrum of anthocyanin mainly reflects the benzene ring, oxygen-containing heterocycle, sugar and hydroxyl, and methoxy in its structure. Figure 7 From B, we can see that anthocyanin is at 3233cm -1 There is a strong and broad absorption peak near 2924cm, which is speculated to be due to the stretching vibration of the aromatic ring in the anthocyanin structure and the multiple hydroxyl groups on the sugar group. The hydroxyl groups between anthocyanin molecules are connected by hydrogen bonds, making the absorption peak waveform at this location wider; 2924cm -1 The absorption peak near 1639cm is due to the stretching vibration of CH in the methoxy group; -1 The absorption peak at 1506cm is speculated to be caused by the stretching vibration of the carbonyl group (C=O) in the anthocyanin molecular structure; -1 、1450cm -1 The characteristic vibration absorption peak at 1331cm reflects the basic skeleton of the benzene ring in the anthocyanin structure, which is speculated to be caused by the skeleton vibration of the aromatic ring and oxygen-containing heterocycle of anthocyanin benzopyran; -1 、1279cm -1 、1192cm -1 The absorption peak near 3402cm corresponds to the stretching vibration of the carbon-oxygen bond (COC) in the carbon ring. -1 Mainly OH and NH stretching characteristic peaks, 2930cm -1 The absorption peak near 1651cm is due to the stretching vibration of CH in the methoxy group. -1 The absorption peak at 1410cm is caused by the stretching vibration of the carbonyl group (C=O) in the oyster peptide structure. -1 The absorption peak at 1032cm is caused by the stretching vibration of CN and NH bonds. -1 The absorption peak at corresponds to the stretching vibration of -CO-.

[0085] Compared with the single oyster peptide, the absorption peak of the amide A band of the synergistic complex of oyster peptide and anthocyanin was blue-shifted (from 3402 cm -1 Move to 3368cm -1 ), indicating that the oyster peptides and anthocyanins may interact through hydrogen bonding and hydrophobic interaction. Amide I band (1600-1700 cm -1) is related to the stretching vibration of C=O on the main chain of the polypeptide or the hydrogen bond coupled with COO-. The amide II band is mainly responsible for the bending of the NH bond and the stretching vibration of CN. The amide I band and amide II band of the complex have a certain vibration shift compared with the single peptide, and the characteristic peak of the complex moves to the low wave number direction. After the oyster peptide and anthocyanin are combined, the peak of the amide I band moves from 1651cm -1 Blue shift to 1639cm -1 , indicating that the secondary structure of oyster peptides changed after synergizing with anthocyanins, which is most likely due to the interaction between oyster peptides and anthocyanins through electrostatic and hydrophobic interactions.

[0086] In summary, after the synergistic action of oyster peptides and anthocyanins, the two undergo hydrogen bonding and hydrophobic interactions through their C=O, CN and NH groups, leading to the rearrangement of the carbonyl hydrogen bond network structure of oyster peptides, which helps to improve the stability of the oyster peptide-anthocyanin complex.

[0087] 7.7 Fluorescence spectroscopy analysis

[0088] The fluorescence spectrum of the interaction between oyster peptide and anthocyanin in Example 2 is shown in Figure 8 A (a→g: anthocyanin = 0, 0.4×10 -1 , 0.8×10 -1 , 0.1, 0.2, 0.4, 0.6 mg / mL). Figure 8 As shown in Figure A, after adding different concentrations of anthocyanins, the fluorescence intensity of oyster peptides decreased significantly. When the concentration of anthocyanins was 0.6 mg / mL, its fluorescence intensity dropped from 648.566 au to 335.452 au, indicating that the addition of anthocyanins can quench the endogenous fluorescence of oyster peptides, and the quenching degree increases with the increase of anthocyanin concentration. When the concentration of anthocyanins was 0.6 mg / mL, the fluorescence quenching rate could reach 48.28%. This may be because the aromatic ring of anthocyanins and the hydrophobic amino acid residues (tyrosine and tryptophan residues) of oyster peptides combine to reduce the hydrophobicity of the microenvironment around the amino acids. And it can be observed from the figure that after adding different concentrations of anthocyanins, the maximum absorption peak showed a significant red shift (from 351 nm to 378 nm), which indicates that more Trp and Tyr residues are exposed to the solution, which enhances the hydrophilicity around these two amino acids, weakens the hydrophobicity, and enhances the polarity. This result is consistent with the above-mentioned ultraviolet absorption spectrum.

[0089] In order to further determine the effect of the synergistic complex of anthocyanins on the conformation of oyster peptides, synchronous fluorescence spectroscopy was used to explore the changes in the conformation of oyster peptides, which can be used to understand the changes in the microenvironment around tryptophan and tyrosine from a microscopic perspective. When the difference between the excitation light wavelength and the emission light wavelength (Δλ) is 15nm, synchronous fluorescence can reflect the characteristics of tyrosine residues; when Δλ is 60nm, synchronous fluorescence mainly reflects the characteristics of tryptophan residues. The synchronous fluorescence spectrum of the interaction between oyster peptides and anthocyanins in Example 2 when Δλ=15nm is shown in Figure 2. Figure 8 Medium B (a→g: anthocyanin = 0, 0.4×10 -1 , 0.8×10 -1 , 0.1, 0.2, 0.4, 0.6 mg / mL); Synchronous fluorescence spectra of oyster peptides interacting with anthocyanins at △λ = 60 nm are shown in Figure 8 C (a→g: anthocyanin = 0, 0.4×10 -1 , 0.8×10 -1 , 0.1, 0.2, 0.4, 0.6 mg / mL). Figure 8 As shown in Figures B and C, the fluorescence intensity of oyster peptides gradually decreased with the increase of anthocyanin concentration, and the maximum emission wavelength of tyrosine slightly red-shifted (from 295nm to 297nm); while the position of the maximum emission wavelength of tryptophan was significantly red-shifted (from 292nm to 305nm), indicating that the synergy of anthocyanins reduced the hydrophobicity of the microenvironment around the tyrosine and tryptophan residues of oyster peptides and increased polarity. In addition, when Δλ=15nm, as the concentration of anthocyanins increased, the fluorescence intensity of the characteristic peak of oyster peptides continued to quench from 290.963au to 8.215au, and when the concentration of anthocyanins was 0.6mg / mL, the fluorescence quenching rate was 97.18%; when Δλ=60nm, the fluorescence intensity of the characteristic peak of oyster peptides continued to quench from 229.987 to 47.358, and when the concentration of anthocyanins was 0.6mg / mL, the fluorescence quenching rate was 79.41%. It can be seen that the polarity near the tyrosine (Tyr) and tryptophan (Trp) residues changes, the hydrophobicity weakens, and the quenching effect of anthocyanins on the Tyr residue of oyster peptide (Δλ=15nm) is stronger than that on the Trp residue (Δλ=60nm), which may be because the interaction site between oyster peptide and anthocyanin is closer to the Tyr residue. In summary, after the synergistic effect of anthocyanins and oyster peptides, the two interact with each other to cause the conformation of oyster peptides to change, increase the polarity of the amino acid microenvironment, and weaken the hydrophobicity.

[0090] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a complex of oyster peptide and anthocyanin, characterized in that: The following steps are involved: The oyster peptide and anthocyanin are dissolved in water, and after the reaction, an oyster peptide and anthocyanin assembly complex is obtained; The amino acid sequence of the oyster peptide is VDLM, VAVL, VADL, VAFE, IGAL, LGA L, LGLE, ALPNSPF, AIPNSPF, AFGPGLE, DQPHFPF, GIQYPVVV, QEIPNSPF, EHVPGSPF, GFPPITGT, TFPVINPA, AIPVHDPSQ, APPPPPPMQ, DAHIPGSPF, PTAPGEYAVH, APPPPPPMQGQ, VVGLDKPMTVN, DIRIPVGPIFN, SAGPGDIGIAL and YFPTAPGEYAVH. The mass ratio of the oyster peptide to the anthocyanin is 1:4 to 4:

1.

2. The method for preparing the oyster peptide and anthocyanin assembly complex according to claim 1, characterized in that: The reaction temperature is 25-50°C and the reaction time is 0.5-5h.

3. The method for preparing the oyster peptide and anthocyanin assembly complex according to claim 1, characterized in that: The preparation steps of the oyster peptide include: adding water to oyster meat and homogenizing, adjusting the pH to 9, adding alkaline protease for enzymolysis, inactivating the enzyme, centrifuging, and drying the supernatant to obtain the oyster peptide.

4. The method for preparing the oyster peptide and anthocyanin assembly complex according to claim 3, characterized in that: The amount of alkaline protease added is 2% of the weight of the oyster meat; the temperature of the enzymolysis is 50° C. and the time is 3 hours.

5. An oyster peptide and anthocyanin assembled complex obtained according to the method for preparing an oyster peptide and anthocyanin assembled complex according to any one of claims 1 to 4.

6. Use of the oyster peptide and anthocyanin assembled complex described in claim 5 in the preparation of a drug for preventing or treating obesity or hyperlipidemia.

7. Use of the oyster peptide and anthocyanin assembly complex described in claim 5 in the preparation of lipid-lowering food.

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