Phycocyanin bacterium enzyme synergistic treatment product with high functional activity and preparation method of phycocyanin bacterium enzyme synergistic treatment product

Abstract

The invention discloses a phycocyanin bacterium enzyme synergistic treatment product with high functional activity and a preparation method thereof, and belongs to the technical field of health food processing. The method comprises the following steps: firstly, carrying out enzymolysis on phycocyanin by using flavourzyme to obtain a phycocyanin enzymolysis product, preparing the enzymolysis product into a solution, inoculating lactobacillus plantarum into the solution for fermentation, and freeze-drying fermentation liquor to obtain the phycocyanin enzymolysis fermentation product subjected to bacterium-enzyme synergistic treatment. The method is simple in preparation process and short in overall time consumption, and the obtained product has antioxidant activity and anti-inflammatory activity superior to those of phycocyanin, corresponding phycocyanin fermentation products and phycocyanin enzymatic hydrolysate, has good flavor and color, and has good market application prospects. The invention not only provides a new product with high functional activity, but also provides a new thought and choice for expanded application of phycocyanin.

Description

Technical Field

[0001] The invention relates to the technical field of health food processing, and in particular to a phycocyanin bacterial enzyme synergistic treatment product with high functional activity and a preparation method thereof. Background Art

[0002] Phycocyanin (PC) is a bioactive substance extracted from natural algae such as Spirulina. Phycocyanin itself contains many nutrients and can be used to supplement the protein and vitamins needed by the human body. In addition, phycocyanin also has multiple biological functions such as anti-inflammatory, antioxidant, and anti-tumor. It can improve the body's immune ability and promote the regeneration of blood cells in the body, thereby improving the body's disease resistance. Although the most widely used application of phycocyanin is as a natural pigment, phycocyanin, as a plant protein, can also be eaten as a healthy food. At present, the research on phycocyanin is mostly focused on the extraction and preparation technology of high-purity phycocyanin and its stability. There are relatively few studies on the deep processing of phycocyanin and the functional characteristics of processed products.

[0003] Enzymatic hydrolysis is a common deep processing method. Enzymatic hydrolysis technology can effectively prevent the active substances in most substances from being destroyed, ensuring the efficacy and biological activity of the products. According to relevant reports, enzymatic hydrolysis of phycocyanin can make it easier for the human body to absorb and improve its utilization rate. Appropriate enzymatic hydrolysis can also reduce the unique fishy smell of algae. Zhang Ying et al. (Zhang Ying. Study on enzymatic hydrolysis of phycocyanin from Spirulina platensis and biological activity of enzymatic hydrolysis products [D]. Jinan: Shandong University, 2006) also improved the free radical scavenging ability and antihypertensive activity of the enzymatic hydrolysis products by enzymatic hydrolysis of spirulina phycocyanin crude extract with trypsin. However, the performance improvement effect of phycocyanin treated by enzymatic hydrolysis technology alone is still limited.

[0004] In addition to enzymolysis technology, fermentation is also a commonly used processing technology in food processing. During the fermentation process, bacteria synthesize vitamins and minerals, use various enzymes to hydrolyze to produce biologically active polypeptides, and remove some non-nutritional substances. The prior art has disclosed a scheme for the coordinated use of enzymolysis-fermentation technology to prepare functional products. For example, Chinese patent CN114521647 B discloses a kelp probiotic fermentation product with auxiliary antioxidant capacity, which is a product prepared by combining enzymolysis-fermentation technology. Specifically, the kelp raw material is first ground into powder, and then extracted and hydrolyzed by different polar solvents and protease to obtain kelp enzymolysis active substances and kelp full active substances; then the activated Lactobacillus reuteri, Pediococcus pentosaceus, and Lactobacillus acidophilus are inoculated in the kelp enzymolysis active substances and kelp full active substances liquid culture medium for fermentation, that is, kelp probiotic fermentation products with auxiliary antioxidant capacity can be prepared, and animal experiments show that the product has significant anti-aging effects. Therefore, it is feasible to prepare functional products by combining enzymolysis-fermentation technology.

[0005] However, there has been no report on the prior art of combining enzymatic hydrolysis and fermentation technology to process phycocyanin to obtain high-performance products. On this basis, if we can successfully explore an easy-to-operate and effective phycocyanin enzyme synergistic treatment scheme to prepare phycocyanin extended products with high functional activity, it will provide a new path for the functional application of phycocyanin and also provide new ideas for the in-depth development of phycocyanin. Summary of the invention

[0006] The purpose of the present invention is to solve the problems existing in the prior art and provide a high functional activity phycocyanin enzyme synergistic treatment product and a preparation method thereof. The obtained product has good antioxidant activity and anti-inflammatory effect and has good market application prospects.

[0007] In order to achieve the above technical objectives, the present invention is implemented by the following technical scheme: a method for preparing a high functional activity phycocyanin bacterial enzyme synergistic treatment product, comprising the following steps:

[0008] 1) preparing a phycocyanin aqueous solution;

[0009] 2) adding protease to the phycocyanin aqueous solution for enzymatic hydrolysis, centrifuging to obtain the supernatant, and freeze-drying to obtain a phycocyanin enzymatic hydrolysis product;

[0010] 3) centrifuging the lactic acid bacteria solution, discarding the supernatant, and diluting with sterile saline to obtain an inoculated bacterial solution;

[0011] 4) preparing an aqueous solution of phycocyanin hydrolysate, adding the inoculated bacterial solution prepared in step 3) to carry out fermentation, terminating the fermentation in a water bath, and freeze-drying to obtain a phycocyanin hydrolysate fermentation product treated with bacterial enzymes.

[0012] Furthermore, in step 1), the concentration of the prepared phycocyanin aqueous solution is 0.01-0.2 g / mL.

[0013] Furthermore, in step 2), the protease used is flavor protease, the enzyme addition amount is 8000-10000 U / g, the enzymolysis temperature is 50-60° C., and the enzymolysis time is 3-6 h.

[0014] Furthermore, the method for preparing a high-functional active phycocyanin bacterial enzyme co-processing product as described in claim 1 is characterized in that in step 3), the lactic acid bacteria used are Lactobacillus plantarum, and the Lactobacillus plantarum is diluted to OD600 = 0.3 with sterile saline.

[0015] Furthermore, in step 4), the bacterial liquid inoculation amount is 3%, the fermentation temperature is 37° C., and the fermentation time is 24 h.

[0016] Furthermore, in step 4), the fermentation is terminated by water bathing at 60° C. for 5 min.

[0017] The present application discloses a high-functional-activity phycocyanin hydrolysis fermentation product, which is prepared according to the above-mentioned preparation method. The antioxidant performance and anti-inflammatory activity of the phycocyanin hydrolysis fermentation product are better than those of untreated phycocyanin and the corresponding phycocyanin hydrolysis product and the corresponding phycocyanin fermentation product.

[0018] The present application also discloses a high functional activity phycocyanin hydrolysis and fermentation product, which contains the phycocyanin hydrolysis and fermentation product.

[0019] The beneficial effects of the present invention are:

[0020] 1. The present application adopts a bacterial enzyme synergistic treatment method to deeply process phycocyanin, the preparation process is simple, and the obtained bacterial enzyme synergistic treatment product has good antioxidant activity and anti-inflammatory effect, and the corresponding efficacy exceeds that of phycocyanin itself and the corresponding phycocyanin fermentation product, as well as the phycocyanin enzymatic hydrolysis product, and the performance is significantly improved;

[0021] 2. The phycocyanin hydrolyzed fermented product prepared in the present application has a unique flavor and color, and good palatability. After being formulated, it can be used to make phycocyanin hydrolyzed fermented beverages, oral liquids and other extended products;

[0022] 3. This application not only provides a product with high functional activity, but also provides a new innovative method for the deep processing and research of phycocyanin, and also provides a new option for the expanded application of phycocyanin;

[0023] 4. The present application utilizes Lactobacillus plantarum and flavor protease to synergistically process phycocyanin. Lactobacillus plantarum is a type of lactic acid bacteria, which has the function of inhibiting the growth of harmful microorganisms, extending the shelf life of food, and improving food texture and flavor. The present application utilizes Lactobacillus plantarum to further process the enzymatic hydrolysis products of phycocyanin to change the material composition, adjust the product flavor and improve the product functionality. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Flow chart of preparing phycocyanin enzymatic fermentation product for bacterial enzyme synergistic treatment of phycocyanin;

[0025] Figure 2 are scanning electron microscope images of different materials, wherein panel a is a scanning electron microscope image of phycocyanin (PC), and panel b is a scanning electron microscope image of phycocyanin enzymatic fermentation product (F-Fla-PC);

[0026] Figure 3 This is the result of the NO-release experiment of the phycocyanin product treated with bacterial enzymes;

[0027] Figure 4 The figure is a gel electrophoresis diagram (SDS-PAGE) of a phycocyanin solution, a phycocyanin solution hydrolyzed by a type of protease, and a phycocyanin solution co-treated with a type of protease and Lactobacillus plantarum, wherein panel a is a schematic diagram of the molecular weight results of phycocyanin hydrolysis products treated with different proteases, and panel b is a schematic diagram of the molecular weight results of phycocyanin hydrolysis fermentation products obtained by the bacterial enzyme co-treatment;

[0028] Figure 5 ABTS of phycocyanin fermentation product (F-PC) and phycocyanin fermentation product co-treated with Lactobacillus plantarum-flavor protease (F-Fla-PC) + Clearance statistics chart;

[0029] Figure 6 Infrared spectra of different phycocyanin treatment products. DETAILED DESCRIPTION

[0030] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the essence of the present invention, modifications and substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention.

[0031] Experimental materials: Phycocyanin was purchased from Zhejiang Binmei Company (phycocyanin E30; Zhejiang, China), flavor protease was purchased from Yuanye Biotechnology Co., Ltd. (Shanghai), and Lactobacillus plantarum was purchased commercially.

[0032] Example 1

[0033] This embodiment discloses a method for preparing phycocyanin enzymatic fermentation product by co-processing phycocyanin with bacterial enzymes, comprising the following steps:

[0034] 1) Weigh phycocyanin, add sterile distilled water (mass volume ratio of PC to sterile distilled water / w:v=1:50, g / mL), and stir until completely dissolved to obtain a phycocyanin solution;

[0035] 2) adding flavor protease to the phycocyanin solution (enzyme addition amount is 8000U / g), and performing enzymolysis at 50°C for 4h to obtain phycocyanin hydrolyzate;

[0036] 3) centrifuging the phycocyanin hydrolysate at 5000 rpm for 10 min, taking the supernatant and freeze-drying it to obtain a phycocyanin hydrolysate;

[0037] 4) Resuscitate Lactobacillus plantarum for three generations using MRS medium, centrifuge the bacterial solution (5000 rpm, 5 min), discard the supernatant, and dilute with sterile saline to OD600 = 0.3 for inoculation;

[0038] 5) adding equal amounts of phycocyanin and phycocyanin hydrolysate to sterile water, shaking until completely dissolved, adding the bacterial solution prepared in the previous step according to a 3% inoculation amount, fermenting at 37° C. for 24 h, and placing the resulting product in a water bath at 60° C. for 5 minutes to terminate the fermentation, thereby obtaining a phycocyanin fermentation broth and a bacterial enzyme-co-treated phycocyanin solution;

[0039] 6) The phycocyanin fermentation liquid and the phycocyanin solution treated with bacterial enzymes obtained in the previous step were frozen at -80°C and then dried to obtain a phycocyanin fermentation product (denoted as F-PC) and a phycocyanin enzymatic fermentation product treated with Lactobacillus plantarum-flavor protease (denoted as F-Fla-PC).

[0040] Related performance tests:

[0041] 1. Product structure characterization

[0042] Take the phycocyanin solution prepared in step 1) and the phycocyanin solution treated with bacterial enzymes prepared in step 5), and pre-freeze them in a -80°C refrigerator for 30 minutes. Encapsulate the pre-frozen samples with tin foil, and place the pre-frozen samples in a freeze dryer for 4 days (keep away from light during the freeze drying process). The freeze-dried samples were respectively mounted on aluminum columns, and a 10 nm thick gold layer was sprayed with a spraying device. On this basis, the morphological characteristics of different component proteins were detected with a scanning electron microscope at an accelerating voltage of 3 kV and different magnifications (1000×, 2000×, 5000× and 10000×).

[0043] Scanning electron microscopy images of phycocyanin (PC) and phycocyanin enzymatic fermentation product (F-Fla-PC) Figure 2 As shown in the figure, it can be seen that after the synergistic treatment of flavor protease and Lactobacillus plantarum, the morphology of phycocyanin changes from the initial lamellar crystals to a dense granular structure.

[0044] 2. NO inhibition test

[0045] (1) Mouse macrophage cell line RAW264.7 cells (cell concentration 50×10 4 cells / mL, 200 μL per well) were inoculated in a 96-well plate, and different concentrations of F-PC and F-Fla-PC (mass concentrations of 50, 100, 200, 400, 800, and 1600 μg / mL) were added at the same time, and treated with lipopolysaccharide (LPS); after LPS (1 μg / mL) and the test substance acted together for 24 hours, the relative concentration of NO was detected by the nitrite concentration (nitrite is a stable product of NO and is an indicator for detecting NO).

[0046] (2) Griess reagent is prepared by mixing reagent A and reagent B in a 1:1 volume ratio;

[0047] Reagent A: 100 mL distilled deionized water, 2.77 mL phosphoric acid, 2 g sulfonamide;

[0048] Reagent B: 100 mL distilled deionized water, 0.2 g naphthylethylenediamine hydrochloride;

[0049] (3) 5×LPS: Add 200 μL of serum-free medium containing HEPES to the original bottle of 1 mg LPS, mix thoroughly and dispense into small centrifuge tubes.

[0050] (4) 1×LPS: dilute 5×LPS with distilled deionized water or serum-free culture medium at a ratio of 5×LPS: diluent = 1:4.

[0051] (5) 150 μL of cell culture medium was mixed with 100 μL of pre-cooled Griess reagent (1% sulfonamide and 0.1% naphthylethylenediamine hydrochloride dissolved in phosphoric acid), followed by incubation at room temperature for 30 min. The absorbance was measured at a wavelength of 540 nm (a cell-free culture medium was used as a blank control in each experiment), and the NO inhibition percentage was then determined.

[0052] Test results such as Figure 3 As shown in the figure, it can be seen that the phycocyanin F-PC treated with simple fermentation has no obvious effect on the relative concentration of NO produced by LPS-induced RAW264.7 cells; however, the phycocyanin F-Fla-PC treated with enzymatic hydrolysis and fermentation is different. As the concentration of F-Fla-PC in the cell culture medium gradually increases, F-Fla-PC has a significant inhibitory effect on the production of NO by LPS-induced RAW264.7 cells, thereby exerting an anti-inflammatory effect.

[0053] 3. Molecular weight determination of phycocyanin hydrolyzed fermentation products

[0054] 10mg / mL phycocyanin solution of different components, phycocyanin hydrolysate and phycocyanin hydrolysate fermentation solution were mixed with loading buffer solution in a volume ratio of 5:1, heated in a 90℃ water bath for 15min, and then placed in ice water for cooling. According to the molecular weight of phycocyanin, a separation gel with a mass concentration of 12% and a concentrated gel with a mass concentration of 5% were prepared. Accurately weigh 3.02g Tris, 18.8g glycine and 1g SDS powder and dissolve them in 800mL deionized water, dilute to 1L, and prepare 1×Tris-glycine electrophoresis buffer. Pour the prepared electrophoresis buffer into the electrophoresis tank, add 3μL Marker and 20μL samples of different components to the loading well of the concentrated gel. Adjust the voltage to 90V, electrophoresed until the protein sample just enters the separation gel interface, adjust the voltage to 120V, and continue electrophoresis until bromophenol blue reaches about 1cm above the bottom of the separation gel to stop the experiment. The gel was stained in Coomassie Brilliant Blue solution. After 30 minutes, the stained gel was immersed in destaining solution and placed in a speed-controlled shaker at 75 r / min overnight until clear protein bands appeared on the gel. The image was obtained by a fluorescent imaging blotting system.

[0055] The gel electrophoresis of phycocyanin solution, enzymatic phycocyanin solution and phycocyanin solution treated with protease and lactobacillus is shown in Figure 4 As shown, wherein panel a shows the molecular weight results of phycocyanin (PC), phycocyanin hydrolysis products of acid protease (ACi-PC), phycocyanin hydrolysis products of alkaline protease (Alk-PC), phycocyanin hydrolysis products of bromelain (Bro-PC), phycocyanin hydrolysis products of composite protease (Com-PC), phycocyanin hydrolysis products of flavor protease (Fla-PC) and phycocyanin hydrolysis products of papain (Pap-PC); panel b shows the molecular weight results of PC, ACi-PC, Alk-PC, Bro-PC, Com-PC, Fla-PC and The molecular weight results of the phycocyanin fermentation product F-PC obtained by fermentation treatment with Pap-PC and the fermentation product treated with bacterial enzymes (respectively recorded as F-ACi-PC, F-Alk-PC, F-Bro-PC, F-Com-PC, F-Fla-PC and F-Pap-PC); It can be seen from the two small figures that the enzymatic hydrolysis treatment changes the structure of the macromolecular phycocyanin and the molecular weight is significantly reduced. It can be seen from the electrophoresis results that phycocyanin is split into multiple bands, which are mainly composed of α subunits and β subunits. The enzymatic hydrolysis and fermentation treatment destroy the subunit structure, forming smaller molecular weight bands, and the fermentation treatment further decomposes the phycocyanin and releases more free amino acids.

[0056] 4. Effects of enzymatic hydrolysis and bacterial enzyme synergistic treatment on the amino acid composition of phycocyanin

[0057] 1) Preparation of amino acid standard solution

[0058] Accurately weigh appropriate amounts of 21 amino acid standards, add 0.1M HCl water to dissolve and dilute to 1 mL, vortex to mix, and obtain standard stock solutions.

[0059] Preparation of internal standard: Take appropriate amount of L-Trp-d5, L-Gln-d5, L-Lys-d4, and prepare 100μg / mL with 25mM TCA aqueous solution. In this embodiment, accurately pipette 50μL of different samples, add 91μL of water, 100μL of 0.15% DOC to mix, add 4μL of 100μg / mL of three internal standard solutions (Lys-d4 / Try-d5 / Gln-d4), mix and ultrasonicate for 10min (5℃, 40KHz), add 5μL of 10 M trichloroacetic acid (TCA) to mix, freeze the sample and let it settle for 10min, centrifuge at 4℃, 14000rcf for 10min, take 50μL of supernatant, add 350μL of water, vortex to mix, and pass through biotage 0.2μm PTFE filter.

[0060] 2) LC-MS detection of amino acid content

[0061] This experiment uses LC-ESI-MS / MS (UHPLC-Qtrap) to perform qualitative and quantitative detection of the target substances in the sample. The specific parameters are as follows:

[0062] Chromatographic conditions: AdvanceBio MS Spent Media, (2.1*50mm, 2.7μm), column temperature 40°C, injection volume 1μL. Mobile phase A (0.1% formic acid, 10mM ammonium formate, 95% aqueous solution), mobile phase B (0.1% formic acid, 10mM ammonium formate, 95% acetonitrile solution). The injection volume was set to 20μL.

[0063] Substitute the chromatographic peak area of ​​the target substance in the sample into the linear equation of the calibration curve to calculate the concentration of the sample to be tested, and calculate the actual content of the target substance in the sample based on the sampling amount.

[0064] The calculation formula is as follows:

[0065] Sample amino acid content (ng / mg) = (C*V*DF) / W

[0066] Wherein, C is the concentration measured on the LC-MS machine, V is the fixed volume, DF is the dilution factor, and W is the sample weight.

[0067] The amino acid contents in different samples are shown in Table 1:

[0068] Table 1. Analysis of amino acid content in different samples

[0069]

[0070]

[0071] It can be seen from the above table that enzyme treatment and flavor protease-fermentation combined treatment can significantly promote the exposure of aromatic amino acids and sulfur-containing amino acids (such as methionine Met and tyrosine Tyr) of phycocyanin. The content of such amino acids in phycocyanin treated with bacterial enzymes is significantly increased compared with that in phycocyanin. These amino acids are positively correlated with the antioxidant activity of the protein.

[0072] It is reported that the increase of hydrophobic amino acids such as alanine Ala, valine Val, isoleucine Ile and methionine Met is beneficial to improve the free radical scavenging activity of proteins. In addition, amino acids with aromatic side chains (such as tyrosine Tyr and phenylalanine Phe) can also provide protons to electron-deficient free radicals, further enhancing the free radical scavenging performance of ABTS. The content of such amino acids in phycocyanin treated with bacterial enzymes is significantly increased compared with phycocyanin.

[0073] In animals, arginine Arg participates in nitrogen cycle and amino acid metabolism, and plays an important role in the development and function of the reproductive system. In addition, arginine can regulate immune function and anti-oxidative stress response. Glutamate Glu is an important neurotransmitter, involved in the regulation of various physiological functions, and glycine Gly participates in the synthesis of neurotransmitters and energy metabolism. The content of such amino acids in phycocyanin treated with bacterial enzymes is also significantly increased compared with phycocyanin, which will lay the foundation for the functional application of phycocyanin products treated with bacterial enzymes in the future.

[0074] 5. Determination of ABTS free radical scavenging ability

[0075] Preparation of storage solution: Weigh ABTS (2,2′-hydrazine-bis-3-ethylbenzothiazoline-6-sulfonic acid) powder and dissolve it in deionized water to prepare an ABTS solution with a concentration of 7.4 mmol / L. Weigh a certain amount of potassium persulfate powder and add it to the prepared ABTS solution to make the concentration of potassium persulfate 2.6 mmol / L. After it is completely dissolved, wrap it with tin foil, protect it from light for 12 hours, and then store it in a 4°C refrigerator for later use.

[0076] Preparation of PBS buffer: Accurately weigh a certain amount of disodium hydrogen phosphate and sodium dihydrogen phosphate powder, and add deionized water to prepare solutions with a concentration of 0.2 mol / L. Take 4.75 mL of sodium dihydrogen phosphate solution and 20.25 mL of disodium hydrogen phosphate solution and add them to a 500 mL volumetric flask, then add 4.383 g of sodium chloride, and finally add deionized water to make the volume 500 mL, so as to obtain a PBS buffer solution with a concentration of 0.01 mol / L.

[0077] Preparation of working solution: dilute the prepared storage solution with PBS buffer so that the absorbance of the diluted working solution at 734nm is 0.7±0.02. The diluted working solution should be stored away from light and stored in a refrigerator at 4℃ for no more than 3 days.

[0078] Preparation of sample solution: Accurately weigh a certain amount of phycocyanin fermentation product (F-PC) and phycocyanin fermentation product (F-Fla-PC) co-treated with flavor protease and Lactobacillus plantarum, add deionized water to fully dissolve, and prepare solutions with concentrations of 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL and 1 mg / mL, respectively. Take out a certain amount of each solution and adjust its pH to 2.5 to measure its ABTS free radical scavenging activity.

[0079] The experimental steps are as follows:

[0080] A 样品 : Mix 3 mL of working solution and 0.45 mL of sample solution evenly, incubate in a 37°C water bath for 5 min, and measure the absorbance at 734 nm.

[0081] A 样品干扰 : Mix 3 mL of PBS buffer solution and 0.45 mL of sample solution evenly, incubate in a 37°C water bath for 5 min, and measure its absorbance at 734 nm.

[0082] A 对照 : Mix 3 mL of working solution with 0.45 mL of PBS buffer, incubate in a 37°C water bath for 5 min, and measure its absorbance at 734 nm.

[0083] The ABTS free radical scavenging activity was calculated by the formula:

[0084] ABTS free radical scavenging rate % = (A 对照 -A 样品 +A 样品干扰 ) / A 对照

[0085] Test results see Figure 5From the figure, it can be seen that the ABTS free radical scavenging ability of F-Fla-PC after bacterial enzyme synergistic treatment is significantly improved compared with untreated and single fermentation phycocyanin, and it is dose-dependent within a certain range. The free radical scavenging rate ability reflects the antioxidant capacity of the product to a certain extent, so this product has excellent antioxidant effect.

[0086] 6. Structural characterization of products

[0087] The effect of bacterial enzyme synergistic treatment on the existence state of phycocyanin and the interaction force of each component were studied by Fourier transform infrared spectrophotometer.

[0088] The components used in the experiments were prepared by freeze drying in a Christ Alpha 1-4 freeze dryer (Christ, ALPHA 1-4 / 2-4LD plus, Shanghai). -1 The FT-IR spectra of phycocyanin (PC), flavor protease hydrolyzed phycocyanin (Fla-PC), Lactobacillus plantarum fermented phycocyanin (F-PC), and bacterial enzyme synergistic phycocyanin hydrolyzed fermentation (F-Fla-PC) were obtained within the wavenumber range of Figure 6 ), it can be seen that the treated product has similar infrared absorption peaks to phycocyanin, but the absorption intensities are different. The main absorption peaks are located in the CH out-of-plane bending vibration region of 995-985 and the double bond stretching vibration region of 1500-1690. The absorption peaks of different phycocyanin treated products have different degrees of red and blue shifts.

[0089] 7. Analysis of volatile flavor substances of products

[0090] Gas Chromatography-Mass Spectrometry (GC-MS) Analysis The samples were examined by GC-MS (7890b GC system, 5977A MSD).

[0091] Gas chromatography conditions: inlet temperature 250°C; carrier gas He (99.999%): constant flow; column flow rate 1 ml / min; column outlet pressure 0 psi, injection volume 1.0 l; split ratio 10:1.

[0092] The temperature program of the FFAP column (60 m×0.25 mm×0.25 μm) was as follows: initially 50°C, held for 2 minutes; heated to 230°C at a rate of 6°C / min, held for 45 minutes, for a total of 78 minutes.

[0093] Mass spectrometry conditions: electron impact (EI) source, electron energy 70 eV; ion source 230°C; quadrupole temperature 150°C; transmission line temperature 245°C; full scan mode for characterization; scanning mass range m / z: 35-450 amu; solvent delay: 4.98-5.3 minutes.

[0094] Identification: Mass spectra were retrieved using NIST 2020, and matches greater than or equal to 700 were retained for mass spectrometry characterization (MS) and retention index (RI) for qualitative analysis.

[0095] Quantification: Calculate the concentration of each compound in the sample using the internal standard method.

[0096] The GC-MS analysis results and flavor analysis results of F-Fla-PC are shown in Table 2:

[0097] Table 2. GC-MS analysis and flavor analysis results of F-Fla-PC

[0098]

[0099]

[0100]

[0101]

[0102] As shown in the table above, the volatile flavor substances of phycocyanin co-treated with flavor protease and Lactobacillus plantarum include 15 kinds of alcohols, 10 kinds of aldehydes, 6 kinds of ketones, 10 kinds of ester compounds, and some acid hydrocarbons, which together constitute the special flavor of the product.

[0103] In summary, the use of flavor protease-Lactobacillus plantarum to synergistically process phycocyanin can not only make the product have good antioxidant activity and anti-inflammatory effects, but the phycocyanin hydrolyzate fermentation product also has a unique flavor and color, and has excellent comprehensive performance, making it suitable for promotion as a health product.

[0104] The above shows and describes the basic principles, main features and advantages of the present invention. However, the above is only a specific embodiment of the present invention, and the technical features of the present invention are not limited thereto. Any other implementation methods derived by any technician in the field without departing from the technical solution of the present invention should be included in the patent scope of the present invention.

Claims

1. A method for preparing a high functional activity phycocyanin enzyme synergistic treatment product, characterized in that: The steps include: 1) preparing a phycocyanin aqueous solution; 2) adding protease to the phycocyanin aqueous solution for enzymatic hydrolysis, centrifuging to obtain the supernatant, and freeze-drying to obtain a phycocyanin enzymatic hydrolysis product; 3) centrifuging the lactic acid bacteria solution, discarding the supernatant, and diluting with sterile saline to obtain an inoculated bacterial solution; 4) preparing an aqueous solution of phycocyanin hydrolysate, adding the inoculated bacterial solution prepared in step 3) to carry out fermentation, terminating the fermentation in a water bath, and freeze-drying to obtain a phycocyanin hydrolysate fermentation product treated with bacterial enzymes.

2. The method for preparing a phycocyanin enzyme synergistic treatment product with high functional activity according to claim 1, characterized in that: In step 1), the concentration of the prepared phycocyanin aqueous solution is 0.01-0.2 g / mL.

3. The method for preparing a phycocyanin enzyme synergistic treatment product with high functional activity according to claim 1, characterized in that: In step 2), the protease used is flavor protease, the enzyme addition amount is 8000-10000 U / g, the enzymolysis temperature is 50-60° C., and the enzymolysis time is 3-6 hours.

4. The method for preparing a phycocyanin enzyme synergistic treatment product with high functional activity according to claim 1, characterized in that: In step 3), the lactic acid bacteria used is Lactobacillus plantarum, and the Lactobacillus plantarum is diluted with sterile saline to OD600=0.

3.

5. The method for preparing a phycocyanin bacterial enzyme synergistic treatment product with high functional activity according to claim 1, characterized in that: In step 4), the bacterial liquid inoculation amount is 3%, the fermentation temperature is 37° C., and the fermentation time is 24 h.

6. The method for preparing a phycocyanin enzyme synergistic treatment product with high functional activity according to claim 1, characterized in that: In step 4), the fermentation was terminated by placing the mixture in a water bath at 60° C. for 5 min.

7. A high functional activity phycocyanin enzymatic hydrolysis fermentation product, characterized in that: It is prepared according to the preparation method described in any one of claims 1 to 6, and the antioxidant performance and anti-inflammatory activity of the phycocyanin hydrolysis and fermentation product are better than those of untreated phycocyanin and the corresponding phycocyanin hydrolysis products and the corresponding phycocyanin fermentation products.

8. A high functional activity phycocyanin enzymatic hydrolysis and fermentation product, characterized in that: The fermented product contains the phycocyanin enzymatic fermentation product according to claim 7.

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