Method for reducing fishy smell of fish viscera protein peptide by using glucosamine to induce glycosylation reaction

Abstract

The invention belongs to the technical field of fish viscera processing, and particularly relates to a method for reducing fishy smell of fish viscera protein peptides by inducing glycosylation reaction through glucosamine, Maillard reaction can be rapidly induced through glucosamine, the bitter taste of fish viscera protein hydrolysate is reduced through Maillard peptides, the salty taste and the delicate taste are improved, and the fishy smell of fish viscera protein peptides is reduced. Meanwhile, bad flavor components are effectively reduced, and aroma substances are generated. Compared with reducing sugars such as xylose, ribose and glucose, the content of free amino acid in the glucosamine in the induced glycosylation process is changed to the greatest extent, and along with continuing of the Maillard reaction, the fluorescence intensity and the change degree of the glucosamine are higher than those of the xylose, the ribose and the glucose; the glucosamine can induce glycosylation more quickly, so that the Maillard reaction degree is deepened, and the fishy smell of the fish viscera enzymatic hydrolysate can be removed quickly and efficiently.

Description

Technical Field

[0001] The present invention belongs to the technical field of fish viscera processing, and particularly relates to a method for reducing the fishy smell of fish viscera protein peptides by inducing glycosylation reaction with glucosamine. Background Art

[0002] Ictalurus punctatus is one of the important varieties in aquaculture. Research shows that the protein and fat contents in the viscera of Ictalurus punctatus reach 9.03% and 44.5% respectively, having a good basis for reuse. However, at present, the processing of Ictalurus punctatus mainly focuses on the muscle, and the utilization degree of fish viscera is relatively low. It is difficult to accurately separate each organ in the fish viscera during the segmentation process, and it also has a strong fishy smell. Except for a small number of them being processed into animal feed, most of the fish viscera are discarded, causing great waste of resources. Therefore, how to efficiently process the large amount of viscera generated during the processing of Ictalurus punctatus and effectively control its fishy smell to achieve the high-value utilization of fish viscera has become an urgent problem to be solved at present.

[0003] Enzymolysis is an important utilization method for aquaculture by-products. The fish viscera protein hydrolysate obtained after enzymolysis of fish viscera has various biological activities, such as antioxidant, blood pressure lowering, blood sugar lowering, anticoagulant, antibacterial, etc., and can be used as potential functional food raw materials. However, the protein hydrolysate generated after protease hydrolysis of fish viscera has a strong fishy smell, including various volatile components formed by adsorption of the breeding environment and lipid oxidation. At present, the commonly used methods for deodorizing protein hydrolysates are physical methods such as activated carbon adsorption and biological methods such as microbial fermentation. The activated carbon commonly used in physical methods can partially eliminate the fishy smell, but the protein loss is large during the treatment process. Microbial fermentation can eliminate the fishy smell to a certain extent, but it will produce unpleasant special odors, and at the same time, the microbial fermentation conditions are not easy to accurately control. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a method for reducing the fishy smell of fish viscera protein peptides by inducing glycosylation reaction with glucosamine, which can effectively remove the fishy smell of fish viscera protein hydrolysate and improve the utilization of fish viscera.

[0005] In order to achieve the above object, the technical solution of the present invention is as follows.

[0006] A method for reducing the fishy smell of fish viscera protein peptides by inducing glycosylation reaction with glucosamine, comprising the following steps:

[0007] The fish viscera is enzymatically hydrolyzed with a flavor protease solution to obtain a fish viscera protein hydrolysate. The fish viscera protein hydrolysate and glucosamine are mixed at a mass ratio of 1:10 to 20, and subjected to the Maillard reaction for 15 min to 60 min. Glucosamine is used to accelerate the glycosylation modification process in the Maillard reaction to increase the degree of glycosylation modification, promote the release of fishy odor in the fish viscera enzymatic hydrolysate, and complete the removal of the fishy odor of the fish viscera.

[0008] In the present invention, by mixing the fish viscera enzymatic hydrolysate and glucosamine at a mass ratio of 1:10 to 20, during the Maillard reaction for 15 min to 60 min, as the Maillard reaction proceeds, the content of the low molecular weight peptide component (<1 kDa) increases, and the content of the high molecular weight peptide (>5 kDa) decreases, and the absorbance and fluorescence intensity gradually increase, indicating that glucosamine can rapidly induce glycosylation. After glycosylation modification is induced by glucosamine, the bad odor is reduced, and 26 aromatic compounds are formed. L-arginine and lysine are identified as the main glycosylation modification sites by LC-MS / MS, and aldehydes modify the peptide segments through the carbonyl-amino reaction, thereby effectively improving the deodorizing effect on the fish viscera protein hydrolysate.

[0009] In another preferred embodiment, the temperature of the Maillard reaction is 90 °C to 100 °C.

[0010] In another preferred embodiment, the specific process for obtaining the fish viscera enzymatic hydrolysate is as follows:

[0011] The fish viscera minced meat is mixed with water in equal volume, the pH value is adjusted to neutral with an alkali solution, flavor protease is added, and enzymatic hydrolysis is carried out at 50 °C to 55 °C for 1 h to 7 h, and enzyme inactivation is carried out at boiling water for 15 min to 20 min, followed by centrifugation, and the supernatant is taken to obtain the fish viscera enzymatic hydrolysate.

[0012] In another preferred embodiment, the mass ratio of the fish viscera minced meat to the flavor protease is 1:50 to 100.

[0013] In another preferred embodiment, the centrifugation means centrifuging at 8000 g to 10000 g for 15 min to 20 min.

[0014] In another preferred embodiment, the mass percentage concentration of the flavor protease is 1% to 2%.

[0015] In another preferred embodiment, the alkali solution is a NaOH solution.

[0016] In another preferred embodiment, the specific process for obtaining the fish viscera minced meat is as follows:

[0017] The fish bladder and gallbladder are removed from the fish viscera, and then crushed to obtain the fish viscera minced meat.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] During the glycosylation induction process of glucosamine in the present invention, compared with reducing sugars such as xylose, ribose, and glucose, the content change of its free amino acids is the largest, indicating that the Maillard reaction induced by glucosamine has the most significant effect; and as the Maillard reaction continues, the fluorescence intensity and change degree of glucosamine are higher than those of xylose, ribose, and glucose, indicating that compared with other reducing sugars, glucosamine can induce glycosylation more quickly, thereby being able to better remove the fishy smell of fish viscera hydrolysate.

[0020] The Maillard products induced by glucosamine significantly reduced bitterness, increased saltiness and umami, and effectively reduced the bad flavor components of the sample and generated aroma substances. L-arginine and lysine were identified as the main modification sites by LC-MS / MS, indicating that the Maillard reaction consumes fishy substances while generating aroma substances, which can greatly improve the flavor and smell of protein hydrolysates. Description of the Drawings

[0021] Figure 1 It is a graph of the free amino acid content and molecular weight distribution results in fish viscera hydrolysate; among them, (a) is the graph of the free amino acid content results, and (b) is the graph of the molecular weight distribution results.

[0022] Figure 2 Free amino groups of Maillard reaction products induced by different sugars, where (a) is glucosamine, (b) is xylose, (c) is ribose, and (d) is glucose.

[0023] Figure 3 It is the ultraviolet absorption spectrum of Maillard reaction products of different sugars, where (a) is glucosamine, (b) is xylose, (c) is ribose, and (d) is glucose.

[0024] Figure 4 It is the fluorescence spectrum diagram of Maillard reaction products of different sugars at different reaction times, where (a) is glucosamine, (b) is xylose, (c) is ribose, and (d) is glucose.

[0025] Figure 5 It is the graph of the molecular weight distribution results of enzymatically hydrolyzed peptides at different times, where (a) is glucosamine, (b) is xylose, (c) is ribose, and (d) is glucose.

[0026] Figure 6 It is the sensory analysis diagram of Maillard reaction products after different reaction times, (a) is the bitter taste, (b) is the salinity, and (c) is the umami.

[0027] Figure 7Principal component analysis diagram of Maillard reaction products induced by glucosamine.

[0028] Figure 8 Venn diagram of volatile components after different Maillard reaction times induced by glucosamine.

[0029] Figure 9 Bar graph of the number of volatile compounds in Maillard reaction products induced by glucosamine at different reaction times. Detailed implementation manners

[0030] The present invention will be described in detail below with specific embodiments, but it should not be construed as a limitation of the present invention. The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0031] In order to improve the utilization rate of fish viscera and alleviate fishy smell, the Maillard reaction can be used to improve the flavor of protein hydrolysates. The Maillard reaction is also known as the carbonyl-amine reaction. The Maillard reaction between food-derived peptides and reducing sugars has the potential to eliminate fishy substances in fish viscera protein hydrolysates. On the one hand, fishy substances such as aldehydes (containing carbonyl groups) can undergo carbonyl-amine condensation reactions with peptides, thereby reducing the fishy smell of protein hydrolysates; on the other hand, the generated aroma components can mask the fishy smell. However, the influence mechanism of reaction time and reducing sugar types on the Maillard reaction results is not yet clear.

[0032] In the embodiments of the present invention, the viscera of channel catfish are used as raw materials, and after hydrolysis with flavor protease, Maillard reaction is induced by reducing sugars to explore the effects of different types of reducing sugars and reaction times on the structure, flavor and volatile compounds of protein hydrolysates. Through research, it is found that glucosamine can rapidly induce Maillard reaction, reduce the bitterness of fish viscera protein hydrolysates through Maillard peptides, improve saltiness and umami, and at the same time effectively reduce bad flavor components and generate aroma substances.

[0033] A method for reducing the fishy smell of fish viscera protein peptides by inducing glycosylation reaction with glucosamine will be specifically described below.

[0034] The materials in the following embodiments are specifically as follows:

[0035] The channel catfish viscera are the channel catfish viscera with the swim bladder and gallbladder removed, and among them, the protein content of the channel catfish viscera is 9.03%.

[0036] Glucosamine (purity > 98%), Shenzhen Inno Food Ingredients Co., Ltd.; Xylose, Shenzhen Inno Food Ingredients Co., Ltd.; Ribose, Shenzhen Inno Food Ingredients Co., Ltd.; Glucose, Shenzhen Inno Food Ingredients Co., Ltd.; Flavor protease, Amano Enzyme Inc. (Nagoya, Japan); o-Phthalaldehyde, Shanghai Tengzhun Biotechnology Co., Ltd.; Formic acid, Chengdu Kelong Chemical Co., Ltd.; Sodium dodecyl sulfate, Guangzhou Saiguo Biotechnology Co., Ltd.; Sodium tetraborate decahydrate, Chengdu Kelong Chemical Co., Ltd.; Sodium chloride, Shanghai Tengzhun Biotechnology Co., Ltd.; Leucine, Merck Chemical Technology (Shanghai) Co., Ltd.; Acetonitrile, Chengdu Kelong Chemical Co., Ltd.; Sodium hydroxide, Chengdu Kelong Chemical Co., Ltd.; Trichloroacetic acid, Chengdu Kelong Chemical Co., Ltd.; N-Acetyl-L-cysteine, Chengdu Kelong Chemical Co., Ltd.

[0037] 1. Sample treatment

[0038] The viscera tissue of catfish was crushed to obtain fish viscera meat paste.

[0039] The fish viscera meat paste was mixed with deionized water in equal mass, and the pH value was adjusted to 7.0 with NaOH. Then, it was hydrolyzed with 1% flavor protease by mass percentage at 50 °C for 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h. After hydrolysis, the enzyme was inactivated in a boiling water bath; centrifuged at 10000 g for 15 min to separate the upper layer of fish oil, and the fish viscera protein hydrolysate was obtained.

[0040] Glucosamine, glucose, xylose, and ribose were added to the fish viscera protein hydrolysate at a mass ratio of 1:10, and then Maillard reactions were carried out at 100 °C for 5 min, 30 min, 45 min, 60 min respectively. After that, it was transferred to an ice bath for cooling. The Maillard peptides after the reaction were freeze-dried and stored at -18 °C for subsequent analysis. The fish viscera protein hydrolysate without Maillard reaction, that is, the enzymatically hydrolyzed protein peptide, was selected as the control group.

[0041] 2. Determination of free amino group content

[0042] The hydrolysis degree was determined by the o-phthalaldehyde method to measure the free amino groups of the hydrolysate.

[0043] The o-phthalaldehyde reagent was composed of 50 mM, 10 mL of o-phthalaldehyde, 50 mM, 10 mL of N-acetyl-L-cysteine, 20%, 5 mL of SDS, and 75 mM, pH 9.5 borate buffer; the prepared o-phthalaldehyde reagent was stored in the dark for 1 hour before use.

[0044] During the determination, 10 μL of the different samples obtained in 1 above were mixed with 1.2 mL of the o-phthalaldehyde reagent respectively, and the absorbance was measured at 340 nm after reacting at room temperature for 10 min.

[0045] Measure the absorbance values of the reaction solution at different reaction times respectively; measure the absorbance values of a series of concentrations of leucine standard solutions to draw a standard curve of free amino group concentration, and then calculate to draw a curve showing the change trend of degree of hydrolysis with hydrolysis time.

[0046] The degree of hydrolysis DH(%) is calculated as follows:

[0047]

[0048] In the formula: h is the number of partially hydrolyzed peptide bonds per gram of protein, with the unit of mmol / g; h tot is the total number of peptide bonds per gram of protein, with the unit of mmol / g; [NH2] is the concentration of free amino groups in the enzymolysate, with the unit of mmol / mL; [NH2] tot is the total content of free amino groups, with the unit of mmol / mL; V1 is the volume of the sample enzymolysate, with the unit of mL; m1 is the mass of the sample, with the unit of g.

[0049] Measure h tot Method: Accurately pipette 0.2 g of enzymolyzed protein peptide into a hydrolysis tube, add 15 mL of 6 mol / L hydrochloric acid solution, then seal the hydrolysis tube after repeating the evacuation 3 times, place it in an electric heating blast constant temperature oven at 110 °C ± 1 °C for hydrolysis for 22 h, filter and wash, transfer the water washing solution into a 50 mL volumetric flask, make up the volume, and measure its absorbance at 340 nm by the o-phthalaldehyde method.

[0050] 3. Color analysis

[0051] Prepare the different samples obtained in 1 into solutions with a concentration of 20 mg / mL, and further evaluate the color of the Maillard reaction peptides using the CIELab scale, including the CIELab parameters L* (brightness value), a* (redness value), and b* (yellowness value) of the Maillard reaction peptides, which are determined by an NR10QC color difference meter (3nh + TILO Group). Calculate the total color difference (ΔE) according to the following formula:

[0052]

[0053] Among them, ΔL* represents the brightness difference, Δa* represents the redness difference, and Δb* represents the yellowness difference.

[0054] 4. Ultraviolet-visible spectroscopy analysis and fluorescence spectroscopy analysis

[0055] To study the reaction degree of Maillard peptides, a UV-visible spectrophotometer (UV-8000A) was used to scan the samples with different reaction times in 1 within the range of 200 nm to 500 nm, and the absorbances of all samples were analyzed and compared. The fluorescence spectrum of Maillard peptides (dry matter 5 mg / mL) was determined using a fluorescence spectrophotometer (F93). The excitation wavelength was fixed at 347 nm, and the emission wavelength range was selected as 400 nm to 600 nm.

[0056] 5. Molecular weight distribution analysis

[0057] The molecular weight distribution of the reaction products was determined using size exclusion chromatography. The molecular weight distribution of Maillard peptides was analyzed using an Ultimate 3000 high-performance liquid chromatograph equipped with a Phenomenex BioSP SECS2000 column. The eluent was an aqueous acetonitrile solution (30%) containing 0.1% trifluoroacetic acid (v / v). The injection volume was 10 μL, the flow rate was 0.5 mL / min, and the detection wavelength was 214 nm. The chromatographic data were analyzed using Chromeleon software, and the peptide size distribution was calculated by fitting the standard sample and the elution time equation. The following standards were used: Trp (204 Da), GLV (287 Da), SGNIGFPGK (1114 Da), insulin (5700 Da), and myoglobin (17600 Da) to obtain the MW calibration curve LogMW = -0.6086t + 6.9381, where MW is the molecular weight (Da) and t is the elution time (min).

[0058] 6. Sensory evaluation

[0059] Ten professional sensory panelists, five males and five females, with an average age of 22 - 25 years, were trained. The panelists were required to have no taste disorders and to receive regular sensory experiment training in a sensory laboratory. The training lasted for 3 weeks, with 3 training sessions per week and each session lasting 20 min. They were asked to distinguish and re-rank 5 basic taste solutions with different concentrations. During the training process, the panelists were provided with five basic tastes to describe the taste characteristics of the samples within the range of 0 to 15. The following taste reference substances were used during training: 30 mmol / L sodium chloride solution presenting a salty taste, 20 mmol / L citric acid solution presenting a sour taste, 50 mmol / L sucrose solution presenting a sweet taste, 10 mmol / L quinine sulfate solution presenting a bitter taste, and 30 mmol / L sodium glutamate solution presenting a umami taste for taste training.

[0060] Before sensory evaluation, the research team conducted intensity grading training on a 15-cm continuous line scale using reference solutions: quinine sulfate (0.1 g / L and 0.2 g / L corresponding to 5 and 10 respectively on the 15-cm continuous line scale), sodium chloride (1 g / L and 2 g / L corresponding to 5 and 10 respectively on the 15-cm continuous line scale), and monosodium glutamate (2.5 g / L and 5.0 g / L corresponding to 5 and 10 respectively on the 15-cm continuous line scale). Under room temperature conditions, the samples containing 2% Maillard reaction peptides were placed in black cups with plastic lids, coded with three-digit numbers, presented in random order, and scored by sensory evaluators by drawing lines.

[0061] 7. Gas Chromatography-Mass Spectrometry (GC-MS) Analysis

[0062] Weigh 8 mL of the sample and transfer it into a 15-mL extraction bottle. Add 2.5 g of NaCl and a magnetic stir bar, then quickly seal it. Age the SPME extraction fiber head in the GC-MS injection port at 250 °C until there are no impurity peaks. Place the sample bottle on the solid-phase microextraction device, set the temperature to 70 °C, and the rotation speed to 1000 rpm; preheat the sample bottle on the extraction device for 15 min; insert the SPME extraction head through the bottle cap into the headspace of the sample, push out the fiber head, with the extraction head about 1.0 cm above the upper surface of the sample, and perform headspace extraction for 40 min; retract the fiber head and pull out the extraction head from the sample bottle; then insert the extraction head into the GC-MS injection port, push out the fiber head, and desorb at 250 °C for 3 min for injection analysis.

[0063] The chromatographic column is DB-WAX (30.0 m × 250 μm, 0.25 μm); the initial temperature is maintained at 40 °C for 5 min, then increased to 120 °C at a rate of 5 °C / min, and then increased to 230 °C at a rate of 10 °C / min and maintained for 5 min; the injection port temperature is 250 °C; the transfer line temperature is 240 °C; the carrier gas is He; the carrier gas flow rate is 1.0 mL / min; without splitting.

[0064] Mass spectrometry conditions: EI source; electron energy 70 eV; ion source temperature 230 °C; quadrupole 150 °C; scanning mode is Scan; the scanning mass range is 20 - 500 Da.

[0065] Qualitative analysis: Identify the detected components using the MS database NIST11 and retention time; subtract column bleed peaks, etc. from the database screening results.

[0066] Quantitative analysis: The area normalization method, that is, the percentage of the peak area of the identified component in the sum of the peak areas of all identified components is used as the quantitative result. The formula is as follows:

[0067]

[0068] Where: Ci is the content of a certain identified component, with the unit of %; Ai is the peak area of a certain identified component; A 1+ A2 ··· + A i ··· + A n Among them, 1, 2 ··· i ··· n represent the number of identified components.

[0069] 8. Liquid Chromatography - Mass Spectrometry (LC - MS) Analysis

[0070] Sample Pretreatment: 200 μg of each sample was taken out, thoroughly redissolved with 0.1% TFA / H2O, and after 10K ultrafiltration, the samples were desalted using a C18 desalting column. The specific process includes: filtering the sample through a 10K ultrafiltration membrane and collecting the filtrate; desalting the filtrate using a C18 desalting column; adding 200 μL of methanol to activate the C18 desalting column; adding 200 μL of 0.1% TFA / ddH2O for chromatographic column equilibration; loading the sample; washing with 200 μL of 0.1 wt% TFA / ddH2O; adding 200 μL of 80 wt% ACN / 0.1 wt% TFA for elution; collecting the eluate and freeze - drying for standby.

[0071] LC - MS / MS Detection Conditions: High - performance liquid chromatograph: Dionex U3000; Chromatographic column: C18, 3 μm, 75 μm × 15 cm; Mobile phase: A: 0.1 wt% Formic acid in water; B: 0.1 wt% Formic acid in Acetonitrile; Mass spectrometer: Thermo Scientific Q Exactive; Spray voltage: 3.8 kV; Capillary temperature: 320 °C; Resolution settings: First - stage 70,000 @ m / z 200, Second - stage 17,500 @ m / z 200; Parent ion scan range: m / z 300 - 1400; Daughter ion scan range: m / z 100; MS1 AGC: 3e6, Ion injection time: 60 ms; MS2 AGC: 5e4, Ion injection time: 80 ms; Ion screening window: 3.0 m / z; Fragmentation mode: HCD, Energy NCE 27; Data - dependent MS / MS: Top 20; Dynamic exclusion time: 15 s.

[0072] Data Analysis: Software: PEAKS 8.5; Database: Ictalurus punctatus protein database downloaded from the NCBI website, species number 7998.

[0073] 9. Statistical Analysis

[0074] Each group of experiments was repeated 3 times, and the results were expressed as mean ± standard deviation. ANOVA was performed using SPSS 22.0, and Duncan's post hoc multiple comparison was used (P < 0.05 indicates significant difference, P > 0.05 indicates no significant difference). Principal component analysis (PCA) was performed using The Unscrambler X 10.4, and the analysis results were visualized.

[0075] 10. Results and Analysis

[0076] 1) Physicochemical determination of enzymatic hydrolysis

[0077] Determination of free amino acid content

[0078] During the enzymatic hydrolysis reaction, proteins and peptides are degraded into free amino acids and small peptides under the action of flavor protease. Therefore, the change in free amino concentration can reflect the degree of enzymatic hydrolysis. The results are shown as Figure 1 shown in a of [reference]. As the reaction time extended, the content of free amino acids in the fish viscera enzymatic hydrolysate also increased significantly (P < 0.05). In the time range of 1 h to 7 h, the content of free amino acids gradually increased, reaching a maximum of 117.96 mmol / L. In addition, the increase degree slowed down slightly after 5 h, indicating that in an environment of 50 °C, fish viscera proteins were continuously degraded under the catalysis of flavor protease, and the contents of free amino groups and small peptides continuously increased, and the degradation rate gradually became gentle.

[0079] 2) Molecular weight analysis

[0080] The change in the molecular weight distribution of the sample can show the reaction degree of flavor protease hydrolyzing fish viscera. Therefore, size exclusion chromatography was used to analyze the molecular weight distribution of the enzymatic hydrolysis samples at different reaction times. The results are shown as Figure 1 shown in b of [reference]. As the reaction time extended, the peptide component with a molecular weight > 3 kDa gradually decreased, from 5.67% to 3.49%, while the proportion of the peptide component with a molecular weight < 1 kDa increased, from 77.81% to 84.65%. At the same time, as the reaction time extended, the change in the peptide segment content in each molecular weight range also became gentle after 5 h. This indicates that fish viscera proteins have been hydrolyzed into a large amount of small peptides and free amino acids. By comprehensively detecting the free amino acid content of the enzymatic hydrolysis samples at different reaction times, the optimal hydrolysis time can be initially determined to be 5 h.

[0081] 3) Color change of samples in the Maillard reaction induced by reducing sugar

[0082] During the Maillard reaction, substances such as melanoidins generated in the reaction process often exhibit brown or even black colors. Therefore, the color change of the sample can indicate the degree of the Maillard reaction. The color changes of collagen glycopeptide at different reaction times are shown in Table 1. Generally speaking, the longer the reaction time, the more significant the change in the total color difference (ΔE*). As the reaction proceeds, compared with the control group, the a* value of the Maillard reaction product shows a slightly decreasing trend, that is, towards green, decreasing from 5.03 to 3.31. At the same time, as the heating time prolongs, the b* value increases, rising from 0.27 to 1.72, indicating that the color of the Maillard reaction sample becomes more light yellow.

[0083] Table 1 Color changes of Maillard reaction products

[0084]

[0085] Note: "-" indicates the absence of this item.

[0086] 4) Determination of free amino acid content in Maillard reaction samples induced by four reducing sugars

[0087] To a certain extent, the change in the free amino acid content in the sample can reflect the degree of the Maillard reaction. Therefore, the free amino acid content of different reaction samples was determined. The results are as Figure 2 shown. As the reaction time prolongs, the free amino content in the Maillard reaction sample induced by glucosamine first increases and then decreases. At 15 min, the free amino group increases from 89.41 mmol / L to 97.37 mmol / L. This is because glucosamine is introduced, and then glucosamine cross-links with the enzymolysis solution, resulting in a significant decrease in the free amino content (P < 0.05) to 65.67 mmol / L. For xylose and ribose, they decrease from 89.48 mmol / L to 87.42 mmol / L and 80.43 mmol / L at 15 min respectively, and then decrease rapidly until 30 min and then gradually slow down, finally decreasing to 53.25 mmol / L and 59.68 mmol / L at 60 min respectively. In addition, the free amino content in the Maillard reaction sample induced by glucose gradually decreases, from 89.48 mmol / L to 59.78 mmol / L, because of the cross-linking reaction between sugar and the enzymolysis solution, resulting in a decrease in the free amino content. In summary, the change in the free amino acid content in the Maillard reaction sample induced by glucosamine is the largest, indicating that the Maillard reaction induced by glucosamine has the most significant effect.

[0088] 5) Spectral analysis of Maillard reaction samples induced by reducing sugars

[0089] UV scanning analysis: The products at different stages of the Maillard reaction have different absorbances. Therefore, the UV scanning spectrum in a specific wavelength range can be used to monitor the degree of the Maillard reaction. The results are as Figure 3As shown, in the ultraviolet scan at wavelengths of 320 - 420 nm, the absorbance value of the Maillard reaction peptide gradually increases with the prolongation of the reaction time. Moreover, the Maillard reaction samples induced by glucosamine, xylose, ribose, and glucose all have characteristic peaks near 225 nm and 260 nm. The absorbance value of the Maillard reaction peptide is higher than that of the control group, and the absorbance of the Maillard reaction peptide is 60 min > 45 min > 30 min > 15 min. This indicates that more intermediate products are generated with the prolongation of the reaction time, and the degree of the Maillard reaction deepens.

[0090] Fluorescence spectroscopy analysis: The substances that can serve as precursors of brown compounds formed in the early stage of the Maillard reaction belong to fluorescent compounds. Therefore, fluorescence spectroscopy can be used to evaluate the fluorescent products formed during the Maillard reaction. The fluorescence scanning spectra of the Maillard reaction products obtained at different reaction times are as Figure 4 shown. Generally speaking, the Maillard reaction sample induced by glucosamine has a characteristic peak near 580 nm, while the Maillard reaction samples induced by xylose, ribose, and glucose have two characteristic peaks near 425 nm and 530 nm. Compared with the control group, each Maillard reaction peptide sample shows higher fluorescence intensity in the range of 400 - 550 nm, indicating that fluorescent products are formed during the Maillard reaction, and the longer the reaction time, the greater the fluorescence intensity. It shows that the changing trend of the fluorescence intensity of Maillard products at different reaction times is 60 min > 45 min > 30 min > 15 min > 0 min, that is, the degree of the Maillard reaction gradually deepens with the prolongation of the reaction time. At the same time, among the Maillard reaction samples induced by reducing sugars, the fluorescence intensity and the degree of change in the glucosamine group are higher than those of the other groups, which indicates that glucosamine can induce glycosylation more rapidly compared to other reducing sugars.

[0091] 6) Molecular weight distribution of Maillard reaction samples induced by reducing sugars

[0092] The change in the molecular weight distribution of the sample can show the degree of the Maillard reaction induced by reducing sugars. Therefore, size exclusion chromatography was used to analyze the molecular weight distribution of the enzymatically hydrolyzed peptides at different times. The results are as Figure 5As shown in the figure, as the reaction time prolongs, the proportion of low-component peptides with a molecular weight less than 1 kDa increases, while the proportion of peptides with a molecular weight greater than 3 kDa decreases. At the same time, as the reaction time prolongs, the change in the content of peptide segments in each molecular weight range also becomes gentle. The amino acids and N-terminal amino acids of low-molecular-weight polypeptides are more active and prone to polymerization and cross-linking reactions. In the samples of Maillard reaction induced by glucosamine, the proportion of low-component peptides with a molecular weight less than 1 kDa increased from 86.29% to 91.34%, and the proportion of peptides with a molecular weight greater than 3 kDa decreased from 4.79% to 1.32%. Moreover, after 15 minutes of reaction, the proportion in each molecular weight range basically no longer changed, which may be due to thermal degradation and cross-linking during the Maillard reaction. To sum up, during the Maillard reaction, the inducing effect of glucosamine is the best. Therefore, the Maillard samples induced by glucosamine are selected for subsequent analysis.

[0093] 7) Sensory evaluation of Maillard reaction samples induced by glucosamine

[0094] Quantitative descriptive analysis (QDA) was performed on Maillard reaction samples induced by glucosamine at different reaction times, which can present the flavor changes of enzymatically hydrolyzed peptides during the Maillard reaction. The results are as Figure 6 shown. As the reaction time prolongs, the bitterness intensity of Maillard reaction samples induced by glucosamine decreases, and the score drops from 7.5 to 5.0 (P < 0.05). The saltiness intensity increases, from 2.0 to 3.4 (P < 0.05), and the umami intensity increases, from 3.6 to 4.5 (P > 0.05). This indicates that the Maillard reaction can improve the flavor of fish viscera hydrolysates, enhance their saltiness and umami, and reduce bitterness.

[0095] 8) Gas-phase detection results of Maillard reaction samples induced by glucosamine

[0096] The PCA plot of Maillard reaction samples induced by glucosamine respectively describes 85% and 10% of the total variance of factor 1 (PC1) and factor 2 (PC2) ( Figure 7 ). The results show that as the Maillard reaction time prolongs, the interval between the 15-minute reaction sample and the 0-minute sample is relatively large, the intervals between the 30-minute, 45-minute, and 60-minute samples are relatively small, and they are both relatively large compared with the 15-minute and 0-minute samples. This indicates that the Maillard reaction improves the flavor of the hydrolysate, and there are obvious differences between Maillard peptides and enzymatically hydrolyzed peptides.

[0097] In addition, a total of 9 volatile compounds were detected, mainly including aldehydes, alcohols, esters, acids, ketones, furans, alkanes, aromatic hydrocarbons, and pyrazines, such as Figure 8。30, 33, 38, 35, and 28 volatile compounds were identified in the samples at reaction times of 0 min, 15 min, 30 min, 45 min, and 60 min, respectively. Among them, there were 10 common components, mainly alcohols, aldehydes, aromatic hydrocarbons, etc. There were only 2 volatile components unique to the 0 min reaction sample, while the number of volatile components unique to the 15 min, 30 min, 45 min, and 60 min reaction samples reached 11, 13, 10, and 8, respectively. As shown in Figure 9 , it indicates that unique volatile components are produced during the Maillard reaction, thereby promoting changes in its flavor.

[0098] As the reaction time extends, the contents of alcohols, ketones, and esters first increase and then decrease, and the content of aldehydes generally shows a decreasing trend. Among them, there are only 3 kinds of aldehyde substances in the enzymolysis solution, which increase to 6 kinds at 15 min of reaction and drop to 2 kinds at 60 min. At the same time, the contents of fishy substances such as hexanal, 2-heptenal, trans-2,4-decadienal, trans-2-octenal, and 1-octen-3-ol first increase and then decrease with the extension of the reaction time, and no longer exist after 30 min, indicating that with the progress of the Maillard reaction, the structure of fish visceral protein peptides degrades, exposing and releasing fishy substances, and the longer the reaction time, the more thoroughly the fishy substances are released. Therefore, the type and number of aldehyde substances first increase and then decrease, and the contents of some fishy substances also decrease after 30 min. While the contents of fragrant substances such as benzaldehyde, 3-methylbutyraldehyde, benzyl alcohol, phenethyl alcohol, dimethyl phthalate, and heptyl formate gradually increase with the extension of the reaction time, which indicates that fishy substances are gradually released and volatilized during the Maillard reaction, and their contents gradually decrease. At the same time, the reaction also produces unique fragrant substances, as shown in Table 2. Therefore, the Maillard reaction improves the flavor of the hydrolysate.

[0099] Table 2 Volatile Compounds in Maillard Reaction Products at Different Reaction Times

[0100]

[0101]

[0102]

[0103]

[0104] Note: "-" indicates not detected.

[0105] 9) Liquid Chromatography-Mass Spectrometry Analysis of Samples with Glucosamine-Induced Maillard Reaction

[0106] LC-MS / MS analysis can determine the glycosylation modification sites of Maillard reaction peptides. The main information of the identified peptides is shown in Table 3. Generally speaking, lysine and arginine are the main modification sites, that is, these two amino acids represent the potential glycosylation sites of food-derived peptides. It is worth noting that in addition to glucosamine modification, modifications induced by valeral and hexanal were also found in the samples. This may be due to the unique structural features of valeral and hexanal with extremely high reactivity. Glycosylation modification can have a positive impact on the taste properties of protein hydrolysates from food sources. According to human sensory evaluation, glycosylation modification plays a key role in the change of the taste characteristics of the samples. Therefore, the possible mechanism for the significant saltiness enhancement effect may be due to the glucosamine-induced Maillard reaction to produce glycosylated protein peptides. At the same time, aldehyde modification also indicates that the fishy substances in the Maillard reaction crosslink with proteins, reducing the bad flavor of the protein hydrolysate and generating flavor substances. Therefore, the Maillard reaction peptides induced by glucosamine can significantly reduce the bitterness of the samples, enhance saltiness and umami.

[0107] Table 3 LC-MS / MS identification of modified polypeptides and their modification sites

[0108]

[0109]

[0110] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they know the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

[0111] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these changes and modifications.

Claims

1. A method for reducing the fish visceral protein peptide fishy smell by using glucosamine to induce glycosylation reaction, characterized in that, It includes the following steps: The fish viscera are enzymolyzed with a flavor protease solution to obtain a fish viscera protein hydrolysate; The fish viscera protein hydrolysate is mixed with glucosamine in a mass ratio of 1:10 - 20, and undergoes a Maillard reaction for 15 min - 60 min. Glucosamine is used to glycosylate L-arginine and lysine in the fish viscera protein hydrolysate, accelerating the Maillard reaction rate and promoting the release of the fishy smell in the fish viscera protein hydrolysate, thus completing the removal of the fishy smell of the fish viscera protein hydrolysate.

2. The method for reducing the fishy smell of fish visceral protein peptides by using glucosamine to induce glycosylation reaction according to claim 1, wherein The temperature of the Maillard reaction is 90°C - 100°C.

3. The method for reducing the fishy smell of fish visceral protein peptides by using glucosamine to induce glycosylation reaction according to claim 1, characterized in that, The specific process for obtaining the fish viscera protein hydrolysate is as follows: The fish viscera minced meat is mixed with water in an equal volume, the pH value is adjusted to neutral with an alkali solution, flavor protease is added, enzymolysis is carried out at 50°C - 55°C for 1 h - 7 h, the enzyme is inactivated in boiling water for 15 min - 20 min, and then centrifuged to remove the upper layer of fish oil to obtain the fish viscera protein hydrolysate.

4. The method for reducing the fishy smell of fish visceral protein peptides by inducing glycosylation reaction with glucosamine according to claim 3, characterized in that The mass ratio of the fish viscera minced meat to the flavor protease in the flavor protease solution is 1:50 - 100.

5. The method for reducing the fish visceral protein peptide fishy smell by using glucosamine to induce glycosylation reaction according to claim 3, wherein The centrifugation means centrifuging at 8000g - 10000g for 15 min - 20 min.

6. The method for reducing the fishy smell of fish visceral protein peptides by using glucosamine to induce glycosylation reaction according to claim 3, characterized in that, The mass percentage concentration of the flavor protease solution is 1% - 2%.

7. The method for reducing the fishy smell of fish visceral protein peptides by inducing glycosylation reaction with glucosamine according to claim 3, characterized in that, The alkali solution is a NaOH solution.

8. The method for reducing the fishy smell of fish visceral protein peptides by using glucosamine to induce glycosylation reaction according to claim 3, characterized in that The specific process for obtaining the fish viscera minced meat is as follows: After removing the swim bladder and gallbladder from the fish viscera, it is pulverized to obtain the fish viscera minced meat.

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