Preparation method of sea cucumber peptide wine

Abstract

The invention relates to a preparation method of sea cucumber peptide wine, which comprises the following steps: pretreating a raw material: removing salt, impure protein and fat of the raw material sea cucumber, and homogenizing the pretreated raw material sea cucumber to obtain sea cucumber pulp; enzymolysis: taking the sea cucumber pulp, adding protease for enzymolysis, and centrifuging to obtain supernate for extracting collagen and sea cucumber residue precipitate for extracting sea cucumber peptide; extraction and purification of collagen: carrying out impurity removal and desalination operation on the supernate for extracting the collagen to obtain the sea cucumber collagen; extracting the sea cucumber peptide: adding protease into the sea cucumber residue precipitate for extracting the sea cucumber peptide to carry out enzymolysis, and then carrying out an impurity removal step to obtain sea cucumber small molecule peptide enzymatic hydrolysate; and preparing the sea cucumber peptide wine: mixing the sea cucumber small molecule peptide enzymatic hydrolysate with the base wine, and then adding the sea cucumber collagen prepared in the previous step to obtain the sea cucumber peptide wine. The peptide wine has the unique flavor of the sea cucumber, and after the sea cucumber collagen is added, the flavor loss of a wine sample is small, and the stability is improved.

Description

Technical Field

[0001] The invention relates to the field of food technology, in particular to a method for preparing sea cucumber peptides, and specifically to a method for preparing sea cucumber peptide wine. Background Art

[0002] The nutritional value of sea cucumbers has attracted much attention. They are rich in active ingredients such as polysaccharides, proteins, bioactive peptides, collagen and saponins, among which the protein content accounts for more than 90%. Modern research shows that most of the proteins in sea cucumbers are degraded into polypeptides, especially small molecule peptides, after digestion and absorption, so that they can be efficiently absorbed and utilized by the human body. In addition, polypeptides or small molecule peptides are significantly better than original proteins in terms of antioxidant and immunomodulatory functions. Enzymatic hydrolysis has become the mainstream technology for the directional conversion of sea cucumber proteins into active peptides due to its mild reaction conditions, strong specificity and environmental friendliness.

[0003] The body wall of sea cucumber contains rich collagen, of which the body wall of sea cucumber contains more than 40% collagen. Collagen is generally extracted by acid extraction, water extraction, enzymatic extraction and other methods, and refined by salting out, dialysis and other technologies. The collagen in sea cucumber is type I fibrous collagen, which has a nano-scale mesh structure and is now widely used in medical dressings, drug carriers, food and light industries. Sea cucumber collagen (especially type I fibrous collagen) has a stable triple helix structure and a hydrophobic surface. The traditional acid extraction method (pH==2-3) may introduce free chloride ions or organic acids, destroying the flavor balance of the wine. In a high-concentration ethanol system, it is easy to irreversibly aggregate due to hydrogen bond destruction and hydrophobic effects, forming floccules or precipitation visible to the naked eye. This phenomenon directly leads to turbidity and stratification of the wine, which seriously damages the appearance stability of the product. Although the existing technology converts collagen into soluble peptides by enzymatic hydrolysis. However, if the undegraded macromolecular collagen is retained and added directly to the wine, additional solubilizers (such as phosphates) need to be introduced, which in turn increases the complexity of the process and may cause food safety disputes.

[0004] In recent years, based on the functional properties of sea cucumber peptides, the "sea cucumber peptide wine" formed by compounding with alcoholic beverages has become a hot spot in the research and development of functional beverages. However, there are two major technical bottlenecks in the existing preparation process: one is the lack of stability of the wine body. The sea cucumber peptide molecules contain hydrophobic amino acid residues, which are prone to turbidity and precipitation due to hydrogen bond breakage or hydrophobic aggregation at high ethanol concentrations; the second is the serious loss of characteristic flavors. Traditional enzymatic hydrolysis often uses high-temperature inactivation or long-term aging processes, which leads to the decomposition of sea cucumber characteristic umami peptides (such as glutamine dipeptide), and at the same time, fat-soluble aromatic substances (such as aldehydes and terpenes) volatilize and escape, resulting in residual fishy smell in the wine body and a bland flavor. Although the existing technology attempts to improve stability by adding gelatin or β-cyclodextrin encapsulation, the introduction of exogenous additives not only reduces the natural properties of the product, but also masks the true flavor of the sea cucumber peptide.

[0005] CN11 7586844A discloses a peptide wine for improving immunity and its production process. The technical solution discloses that the peptide wine is composed of base wine and sea cucumber peptide, and the mass ratio of the base wine to the sea cucumber peptide is 100:0.1-0.5. The preparation method of sea cucumber peptide includes: preparing sea cucumber powder; adding reducing protease and water to the sea cucumber powder, enzymatically filtering to obtain sea cucumber peptide, and mixing the obtained sea cucumber peptide with the base wine to obtain peptide wine. This technical solution is aimed at the problem of low content of small molecule peptides in peptide wine. It does not involve sea cucumber collagen, and cannot solve the problem of unstable body of peptide wine. CN11 1235205A discloses a method for preparing and using sea cucumber protein peptides, which comprises the following steps: (1) raw material pretreatment: remove the internal organs of fresh Icelandic red sea cucumbers, wash and dry them, and mince them into a homogenate for standby use; (2) enzymatic hydrolysis: add water to the homogenate in step (1) and measure the pH, then add protease to obtain a sea cucumber peptide hydrolysate; (3) refining and drying: after the sea cucumber peptide hydrolysate in step (2) is inactivated by boiling water bath, it is filtered, decolorized and spray dried to obtain sea cucumber protein peptides. However, the application does not disclose the technical solution for the combined use of sea cucumber peptides, base wine and sea cucumber collagen, nor can it solve the problem of unstable body and serious loss of characteristic flavor of sea cucumber peptide wine.

[0006] In addition, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the applicant studied a large number of documents and patents when making the present invention, but due to space limitations, not all details and contents are listed in detail. However, this does not mean that the present invention does not have the characteristics of these prior arts. On the contrary, the present invention already has all the characteristics of the prior art, and the applicant reserves the right to add relevant prior art to the background technology. Summary of the invention

[0007] Existing sea cucumber peptide wines are mainly obtained by compounding the enzymatic hydrolysate after sea cucumber hydrolysis with base wine or by alcohol extraction and soaking sea cucumbers. Directly mixing sea cucumber enzymatic hydrolysate with base wine (such as white wine and yellow wine) has the following defects: the molecular weight distribution of the enzymatic hydrolysate is wide (1-30kDa), that is, there are problems of inaccurate polypeptide quantification and unclear polypeptide molecules, and functional small molecule peptides (such as antioxidant peptides and anti-inflammatory peptides) are not effectively enriched; the polypeptide components in the enzymatic hydrolysate are complex, and it is difficult to accurately quantify active polypeptides (such as collagen peptides with a molecular weight of <10 kDa) by Kjeldahl nitrogen determination or HPLC, resulting in a lack of scientific basis for efficacy labeling. Peptides and tannins, polysaccharides and other components in the wine body are prone to hydrophobic aggregation, especially at low temperatures (<0℃) or long-term storage to produce flocculation precipitation, affecting product stability, acceptance and sensory quality. However, there are the following problems when extracting dried sea cucumbers directly by soaking them in high-concentration alcohol: the dense collagen fibers in the sea cucumber body wall hinder the penetration of the solvent, resulting in a peptide dissolution rate of less than 20%; the alcohol extract contains a large amount of lipids, pigments and undegraded collagen fiber fragments, which requires additional deodorization and decolorization processes, which are costly and easy to destroy the active ingredients. At present, how to achieve the long-term stability and flavor preservation of sea cucumber peptide wine has become a key technical problem restricting the upgrading of the industry.

[0008] In view of the shortcomings of the prior art, the first aspect of the present invention provides a method for preparing sea cucumber peptide wine, the preparation method comprising the following steps:

[0009] (1) Raw material pretreatment: removing salt, foreign protein and fat from the raw sea cucumber, and homogenizing the pretreated raw sea cucumber to obtain sea cucumber slurry;

[0010] (2) Enzymatic hydrolysis: taking sea cucumber slurry, adding protease for enzymatic hydrolysis, and centrifuging to obtain a supernatant for extracting collagen and a sea cucumber residue precipitate for extracting sea cucumber peptides;

[0011] (3) Extraction and purification of collagen: removing impurities and salt from the supernatant used to extract collagen to obtain sea cucumber collagen;

[0012] (4) Extraction of sea cucumber peptides: adding protease to the sea cucumber residue precipitate used for extracting sea cucumber peptides for enzymolysis, and then performing an impurity removal step to obtain a sea cucumber small molecule peptide enzymolysis solution;

[0013] (5) Preparing sea cucumber peptide wine: mixing the sea cucumber small molecule peptide hydrolysate with the base wine, and then adding the sea cucumber collagen prepared in step (3) to obtain sea cucumber peptide wine.

[0014] According to a preferred embodiment, the raw material pretreatment step includes: treating the raw sea cucumber with water, sodium hydroxide, ethyl acetate, and ethanol solution at low temperature to remove foreign proteins and fat.

[0015] According to a preferred embodiment, the concentration of sodium hydroxide is 0.1 mol / L; the concentration of ethanol is 15% to 25%.

[0016] According to a preferred embodiment, the protease used in the enzymatic hydrolysis step can be one or more of a composite protease, a neutral protease, an acidic protease, and an alkaline protease.

[0017] According to a preferred embodiment, the enzymatic hydrolysis step further comprises adding acetic acid solution to the sea cucumber slurry.

[0018] According to a preferred embodiment, the concentration of acetic acid is 0.2 to 0.8 mol / L. More preferably, the concentration of acetic acid is 0.2 to 0.5 mol / L. Particularly preferably, the concentration of acetic acid is 0.4 mol / L.

[0019] According to a preferred embodiment, the enzymolysis time can be 12 to 72 hours. More preferably, the enzymolysis time is 12 to 48 hours. Particularly preferably, the enzymolysis time is 12 hours.

[0020] According to a preferred embodiment, in the collagen extraction and purification step, the impurity removal of the supernatant includes centrifugation and filtration operations. Centrifugation and filtration are performed after the collagen extract is filtered in a homogeneous liquid state and then salting out. Sodium chloride is used in the salting out operation.

[0021] According to a preferred embodiment, in the extraction and purification step of collagen, the amount of protease added is 0.01% to 0.1% by weight of the sea cucumber pulp added. More preferably, the amount of protease added is 0.01% by weight of the sea cucumber pulp added.

[0022] According to a preferred embodiment, the extraction and purification of collagen further comprises adding an alkaline regulator to adjust the pH to 6 to 7. The alkaline regulator is preferably sodium hydroxide. The concentration of sodium hydroxide is preferably 1 mol / L.

[0023] According to a preferred embodiment, in the step of extracting sea cucumber peptides, enzymatic hydrolysis is performed in a ratio of sea cucumber residue precipitate: water = 1:10 (mass ratio).

[0024] According to a preferred embodiment, in the step of extracting sea cucumber peptides, the amount of protease added is 1% to 3% of the mass of the sea cucumber residue precipitate.

[0025] According to a preferred embodiment, in the step of extracting sea cucumber peptides, the enzymatic hydrolysis temperature is 40-60° C. More preferably, the enzymatic hydrolysis temperature is 55° C.

[0026] According to a preferred embodiment, in the step of extracting sea cucumber peptides, the enzymatic hydrolysis pH is 6 to 8. More preferably, the enzymatic hydrolysis pH is 7.5.

[0027] According to a preferred embodiment, in the step of extracting sea cucumber peptides, the enzymatic hydrolysis time is 2 to 12 hours. More preferably, the enzymatic hydrolysis time is 2 hours.

[0028] According to a preferred embodiment, the sea cucumber small molecule peptide hydrolysate refers to a peptide liquid obtained after filtration in which the small molecule peptides account for more than 90%, wherein the molecular weight of the small molecule peptides is less than 10 kDa.

[0029] According to a preferred embodiment, the collagen is a white viscous net-like substance obtained after desalting.

[0030] According to a preferred embodiment, the standing temperature of the prepared sea cucumber peptide wine is -15 to 0°C.

[0031] According to a preferred embodiment, both sea cucumber peptides and sea cucumber collagen are derived from raw sea cucumbers.

[0032] The second aspect of the present invention provides sea cucumber peptide wine prepared according to the method for preparing sea cucumber peptide wine according to the first aspect of the present invention.

[0033] The third aspect of the present invention provides a method for preparing sea cucumber collagen, comprising the following steps:

[0034] (1) Raw material pretreatment: removing salt, foreign protein and fat from the raw sea cucumber, and homogenizing the pretreated raw sea cucumber to obtain sea cucumber slurry;

[0035] (2) Enzymatic hydrolysis: Take sea cucumber pulp, add protease for enzymatic hydrolysis, and centrifuge to obtain a supernatant for extracting collagen;

[0036] (3) Extraction and purification of collagen: The supernatant used for extracting collagen is subjected to impurity and salt removal operations to obtain sea cucumber collagen.

[0037] The fourth aspect of the present invention provides sea cucumber collagen obtained according to the method for preparing sea cucumber collagen according to the third aspect of the present invention.

[0038] A fifth aspect of the present invention provides a method for preparing sea cucumber peptides, comprising the following steps:

[0039] (1) Raw material pretreatment: removing salt, foreign protein and fat from the raw sea cucumber, and homogenizing the pretreated raw sea cucumber to obtain sea cucumber slurry;

[0040] (2) Enzymatic hydrolysis: taking sea cucumber slurry, adding protease for enzymatic hydrolysis, and centrifuging to obtain sea cucumber residue precipitate for extracting sea cucumber peptides;

[0041] (3) Extraction of sea cucumber peptides: Add protease to the sea cucumber residue precipitate used for extracting sea cucumber peptides for enzymatic hydrolysis, and then perform a decontamination step to obtain a sea cucumber small molecule peptide enzymatic hydrolyzate.

[0042] The sixth aspect of the present invention provides a sea cucumber peptide obtained according to the method for preparing the sea cucumber peptide according to the fifth aspect of the present invention.

[0043] The seventh aspect of the present invention provides the preparation method of the sea cucumber peptide wine provided by the first aspect of the present invention, the sea cucumber peptide wine provided by the second aspect of the present invention, the preparation method of sea cucumber collagen provided by the third aspect of the present invention, the sea cucumber collagen provided by the fourth aspect of the present invention, the preparation method of sea cucumber peptide provided by the fifth aspect of the present invention, and the use of sea cucumber peptide in food provided by the sixth aspect of the present invention.

[0044] Technical effects of the present invention:

[0045] The present invention aims at the problem that the existing sea cucumber peptide wine has unstable body, and provides a peptide wine with better stability obtained by mixing sea cucumber collagen extracted from sea cucumber with peptide wine and filtering. The sea cucumber peptide wine prepared by the present invention contains the unique flavor of sea cucumber, and after adding sea cucumber collagen to the sea cucumber peptide wine, the flavor loss of the wine is small.

[0046] Generally speaking, collagen is used to filter impurities, especially in the biomedical and food industries. First, it can be used as a filter membrane to separate and purify impurities in solutions, but the operation cost is high; secondly, the porous structure, electrostatic effect, and hydrophobic effect of collagen can adsorb impurities in solutions, including heavy metal ions; in the food industry, collagen is used to clarify beverages, the most common of which is beer, by adsorbing suspended particles and colloidal substances to remove impurities. In the filtration of liquor, especially high-alcohol liquor, it is less used due to the deformation caused by ethanol. However, the present invention unexpectedly found that the problem of peptide wine loss can be effectively reduced by controlling the temperature and adsorption time, and the adsorption placement is conducive to filtration treatment. This operation further expands the application of collagen in the liquor industry. On the other hand, collagen fibers have electrostatic and hydrophobic effects. The present invention adds collagen for adsorption and filtration operations, thereby improving the taste of the sea cucumber peptide wine and enhancing its stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The turbidity change results of the wine samples provided by the present invention measured under storage conditions of room temperature, 4°C, and -15°C over time;

[0048] Figure 2 The comparison results of the sedimentation and filtration effects of different temperatures and additives provided by the present invention;

[0049] Figure 3 The comparison results of the gloss loss phenomenon of the sea cucumber wine provided by the present invention after being treated with sea cucumber collagen at -15°C;

[0050] Figure 4 The sea cucumber peptide wine samples of Examples 2 to 3 and Comparative Examples 1 to 6 provided by the present invention were placed and processed, and then flavor sensory scoring results were performed. DETAILED DESCRIPTION

[0051] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that these descriptions are exemplary only and are not intended to limit the scope of the present disclosure. In the following detailed description, for ease of explanation, many specific details are set forth to provide a comprehensive understanding of the embodiments of the present disclosure. However, it is obvious that one or more embodiments can also be implemented without these specific details. In addition, in the following description, the description of the known technology is omitted to avoid unnecessary confusion of the concept of the present disclosure. In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be clearly and completely described below. If the specific conditions are not indicated in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the reagents or instruments used are not indicated by the manufacturer, they are all conventional products that can be purchased commercially. All terms (including technical and scientific terms) used herein have the meanings commonly understood by those skilled in the art, unless otherwise defined. It should be noted that the terms used here should be interpreted as having the meanings consistent with the context of this specification, and should not be interpreted in an idealized or overly rigid manner.

[0052] The proteases used in the following examples can be commercially available proteases, such as composite proteases, neutral proteases, acidic proteases, alkaline proteases, and the like.

[0053] According to the configuration requirements of liquor products, it is generally necessary to configure two gradients of 42% vol and 52% vol. The present invention divides the samples of the same batch into 3 bottles, and the 3 bottles are placed in 3 different environments such as natural light room temperature (the average indoor temperature is 20°C), 4°C, and -15°C. Observe the clarity of the wine every 24 hours, and take samples to measure the turbidity. Put the target wine in the sample pool, measure the turbidity value after cleaning the outer wall of the sample pool, measure multiple times, and the fluctuation range of the measured value is within 0.05NTU for more than 3 consecutive times. The average value of the 3 times is the turbidity of the wine sample to be tested.

[0054] The present invention placed the prepared sea cucumber wine samples at room temperature, 4°C, and -15°C, and measured their turbidity for 7 consecutive days. The results are as follows: Figure 1 shown. Figure 1 In the graph, the horizontal axis is time (h) and the vertical axis is turbidity (NTU). Figure 1The results showed that the turbidity of 42%vol and 52%vol at room temperature was less than 0.5NTU, indicating that the sea cucumber wine samples of the two alcohol contents were stable at room temperature. Under 4℃, the turbidity of 42%vol sea cucumber wine was less than 0.5NTU within 5 days, and the wine was relatively stable. The turbidity of 52%vol sea cucumber wine rose to 1.15NTU on the 3rd day, and the turbidity of the wine after the 3rd day basically remained around 1.15NTU, with little change. Under -15℃, the turbidity of 42%vol sea cucumber wine rose to more than 2NTU on the 3rd day, and then changed little; while the turbidity of 52%vol sea cucumber wine increased the fastest, and on the 3rd day, its turbidity rose to more than 3NTU.

[0055] Figure 1 The results further showed that under the same time conditions (such as the 3rd day), the lower the temperature (such as -15℃ vs. 4℃ or room temperature; 4℃ vs. room temperature), the more significant the increase in turbidity of sea cucumber wine. The turbidity growth rate of high alcohol content (52%vol) at low temperature is significantly higher than that of low alcohol content (42%vol).

[0056] On the other hand, the wines in the initial period were transparent and clear; at 3 days, the wines of 42%vol sea cucumber wine were clear and transparent, and 52%vol sea cucumber wine had a slight loss of gloss; at 5 days, 42%vol sea cucumber wine had a slight loss of gloss, and 52%vol sea cucumber wine had a significant loss of gloss. That is, under refrigerated or frozen conditions, sea cucumber peptide wine is relatively unstable, and no precipitation occurs in the samples, but the sea cucumber wines of the two gradients of 42%vol and 52%vol both show different degrees of gloss loss.

[0057] Based on the above research, the present invention proposes that the wine should be placed for 5 days before adsorption filtration, so that better clarification and transparency can be achieved. In addition, the following examples and comparative examples use 52% vol sea cucumber peptide wine as the test material to reflect the uniqueness of the technical effect of the present invention.

[0058] The present invention extracts collagen and sea cucumber peptides from sea cucumbers, and uses the processed collagen as a filter for sea cucumber peptide wine, thereby finally obtaining a sea cucumber peptide wine with good stability and obvious sea cucumber flavor. The specific operation is shown in the following embodiments.

[0059] Example 1

[0060] This embodiment provides a method for preparing sea cucumber peptide. The preparation method comprises the following steps:

[0061] Raw material pretreatment: Take 1000g of fresh sea cucumber, wash and remove the salt of the product, then add 0.1mol / L sodium hydroxide, ethyl acetate and 15% ethanol solution to treat the sea cucumber to remove foreign protein and fat, and set aside.

[0062] Enzymatic hydrolysis: Add water to the pretreated sea cucumber at a mass volume ratio of 1:10 (kg / L), that is, add 10L of water, homogenize to obtain sea cucumber pulp, take the sea cucumber pulp, add 0.4mol / L acetic acid solution, add pepsin at 0.01% of the weight of the sea cucumber raw material, enzymatic hydrolysis for 12h, centrifuge, and obtain supernatant and sea cucumber residue precipitation. Take the supernatant, centrifuge, filter, add 1mol / L sodium hydroxide to adjust the pH to 6, and add sodium chloride for salting out, stand and filter to obtain sea cucumber collagen, mix the filtered sea cucumber collagen at a wet weight and ice water volume ratio of 1:30 (that is, use 30L of water precooled to 0°C to rinse every 1kg of wet collagen tissue), and set aside.

[0063] Extraction of sea cucumber peptides: Take the above sea cucumber residue precipitate, add 10g of composite protease for enzymolysis, the enzymolysis temperature is 55°C, pH 7.5, and the enzymolysis is carried out in a warm and shaking water bath for 2h. After the enzymolysis is completed, heat at 95°C for 15min to kill the enzyme, and then centrifuge, coarse filter, and fine filter with a 1kDa membrane to obtain a sea cucumber small molecule peptide enzymolysis solution. The enzymatic activity of the protease used in this embodiment is preferably 100 units / mg.

[0064] Example 2

[0065] This embodiment provides a method for preparing sea cucumber peptide wine.

[0066] Specifically, the sea cucumber small molecule peptide hydrolysate provided in Example 1 is mixed according to 3% of the weight of the base wine (that is, the amount of the sea cucumber small molecule peptide hydrolysate added is 3% of the mass of the base wine), and the mixed solution is allowed to stand at -15°C for 5 days according to the alcohol content of 52%vol. After standing, sea cucumber collagen is added at 0.4%0 (mass volume ratio, i.e., 0.4 grams per liter of mixed solution) of the volume of the mixed solution, and the mixture is stirred evenly and filtered to obtain a finished sea cucumber peptide wine.

[0067] Example 3

[0068] This embodiment provides another method for preparing sea cucumber peptide wine.

[0069] Specifically, the sea cucumber small molecule peptide hydrolysate provided in Example 1 is mixed according to 3% of the weight of the base wine (that is, the amount of the sea cucumber small molecule peptide hydrolysate added is 3% of the mass of the base wine), and the mixed solution is allowed to stand at -5°C for 5 days according to the alcohol content of 52%vol. After standing, sea cucumber collagen is added at 0.4%0 (mass volume ratio, i.e., 0.4 grams per liter of mixed solution) of the volume of the mixed solution, and the mixture is stirred evenly and filtered to obtain a finished sea cucumber peptide wine.

[0070] Comparative Example 1

[0071] Raw material pretreatment: Take 1000g of fresh sea cucumber, wash and remove the salt of the product, then add 0.1mol / L sodium hydroxide, ethyl acetate and 15% ethanol solution to treat the sea cucumber to remove foreign protein and fat, and set aside.

[0072] Enzymatic hydrolysis: Add water to the pretreated sea cucumber at a mass-to-volume ratio of 1:10 (kg / L), i.e., add 10 L of water, homogenize to obtain sea cucumber slurry, take the sea cucumber slurry, add 0.4 mol / L acetic acid solution, add 0.01% pepsin by weight of the sea cucumber, enzymatically hydrolyze for 12 hours, centrifuge, and obtain the supernatant and sea cucumber residue precipitate.

[0073] Extraction of sea cucumber peptides: Take the above sea cucumber residue precipitate, add 10g of composite protease for enzymolysis, the enzymolysis temperature is 55℃, pH 7.5, and the enzymolysis is kept in a shaking water bath for 2h. After the enzymolysis is completed, heat at 95℃ for 15min to inactivate the enzyme, then centrifuge, coarse filter, and fine filter with a 1kDa membrane to obtain the sea cucumber small molecule peptide enzymolysis solution.

[0074] Furthermore, the step of preparing sea cucumber peptide wine is: mixing the sea cucumber small molecule peptide hydrolysate prepared in the comparative example according to 3% of the weight of the base wine (that is, the addition amount of the sea cucumber small molecule peptide hydrolysate is 3% of the mass of the base wine), according to the alcohol content of 52% vol, the mixed solution is allowed to stand at -15°C for 5 days, and filtered to obtain the sea cucumber peptide wine.

[0075] Comparative Example 2

[0076] The raw material pretreatment, enzymatic hydrolysis and sea cucumber peptide extraction steps of this comparative example are the same as those of comparative example 1.

[0077] The steps of preparing sea cucumber peptide wine are as follows: the sea cucumber small molecule peptide hydrolysate prepared in the comparative example is mixed according to 3% of the weight of the base wine (that is, the addition amount of the sea cucumber small molecule peptide hydrolysate is 3% of the mass of the base wine), and the mixture is placed at -5°C for 5 days and filtered to obtain the sea cucumber peptide wine.

[0078] Comparative Example 3

[0079] The raw material pretreatment, enzymatic hydrolysis and sea cucumber peptide extraction steps of this comparative example are the same as those of comparative example 1.

[0080] The steps of preparing sea cucumber peptide wine are as follows: the sea cucumber small molecule peptide hydrolysate prepared in the comparative example is mixed according to 3% of the weight of the base wine (that is, the amount of the sea cucumber small molecule peptide hydrolysate added is 3% of the mass of the base wine), and the mixture is allowed to stand at -15°C for 5 days according to the alcohol content of 52%vol. After standing, the prepared activated carbon (powdered) is added at 0.4‰ (mass volume ratio, that is, 0.4 grams are added per liter of wine) of the volume of the sea cucumber peptide wine, and the sea cucumber peptide wine is filtered to obtain the sea cucumber peptide wine.

[0081] Comparative Example 4

[0082] The raw material pretreatment, enzymatic hydrolysis and sea cucumber peptide extraction steps of this comparative example are the same as those of comparative example 1.

[0083] The steps of preparing sea cucumber peptide wine are as follows: the sea cucumber small molecule peptide hydrolysate prepared in the comparative example is mixed according to 3% of the weight of the base wine (that is, the amount of the sea cucumber small molecule peptide hydrolysate added is 3% of the mass of the base wine), and the mixture is allowed to stand at -5°C for 5 days according to the alcohol content of 52%vol. After standing, the prepared activated carbon (powdered) is added at 0.4‰ (mass volume ratio, that is, 0.4 grams are added per liter of wine) of the volume of the sea cucumber peptide wine, and the sea cucumber peptide wine is filtered to obtain the sea cucumber peptide wine.

[0084] Comparative Example 5

[0085] The raw material pretreatment, enzymatic hydrolysis and sea cucumber peptide extraction steps of this comparative example are the same as those of comparative example 1.

[0086] The steps of preparing sea cucumber peptide wine are as follows: the sea cucumber small molecule peptide hydrolysate prepared in the comparative example is mixed according to 3% of the weight of the base wine (that is, the amount of the sea cucumber small molecule peptide hydrolysate added is 3% of the mass of the base wine), and the mixture is allowed to stand at -15°C for 5 days according to the alcohol content of 52%vol. After standing, diatomaceous earth (powdered) is added at 0.4‰ of the volume of the sea cucumber peptide wine (mass volume ratio, that is, 0.4 grams are added per liter of wine), and the sea cucumber peptide wine is filtered.

[0087] Comparative Example 6

[0088] The raw material pretreatment, enzymatic hydrolysis and sea cucumber peptide extraction steps of this comparative example are the same as those of comparative example 1.

[0089] The steps of preparing sea cucumber peptide wine are as follows: the sea cucumber small molecule peptide hydrolysate prepared in the comparative example is mixed according to 3% of the weight of the base wine (that is, the amount of the sea cucumber small molecule peptide hydrolysate added is 3% of the mass of the base wine), and the mixture is allowed to stand at -5°C for 5 days according to the alcohol content of 52%vol. After standing, diatomaceous earth (powdered) is added at 0.4%o (mass volume ratio, that is, 0.4 grams are added per liter of wine) of the volume of the sea cucumber peptide wine, and the sea cucumber peptide wine is filtered to obtain the sea cucumber peptide wine.

[0090] Figure 2 The results are a comparison of sedimentation and filtration effects treated with different temperatures and additives. Figure 2The results show that under low temperature conditions of -15°C, when no stabilizing substance is added to the sea cucumber peptide wine (Comparative Example 1), the degree of light loss is most obvious; after adding sea cucumber collagen (Example 2) or activated carbon / diatomaceous earth (Comparative Examples 3 and 5), it is reduced to slight, indicating that low temperature accelerates protein aggregation and causes a decrease in transmittance, but adding stabilizing substances or adsorbents can alleviate it. Under -5°C conditions, the control of light loss at -5°C is better than that at -15°C when the stabilizing substance is added in the same amount (Example 2 and Example 3), indicating that the increase in temperature helps to inhibit excessive aggregation of colloids. Sea cucumber collagen has excellent temperature adaptability. Specifically, slight or no light loss is achieved at -15°C (Example 2) and -5°C (Example 3), which is significantly better than the unadded group (Comparative Examples 1 and 2). Compared with sea cucumber collagen, the limitations of adsorbents (activated carbon / diatomaceous earth) are more obvious. Activated carbon (Comparative Examples 3 and 4) and diatomaceous earth (Comparative Examples 5 and 6) only reduce light loss to "slight" at both temperatures, and do not achieve the effect of sea cucumber collagen. The adsorbent reduces scattering centers by physically adsorbing impurities, but has no direct regulation on protein-ethanol interactions, so its effect is limited. No obvious suspended matter or precipitation appeared in all samples, and the difference in light loss reflected the presence of submicron aggregates.

[0091] Therefore, 0.4% o sea cucumber collagen was selected to be added to sea cucumber peptide wine to achieve the best stability of the wine.

[0092] Figure 3 These are the comparative results of the gloss loss phenomenon of sea cucumber wine after being treated with sea cucumber collagen at -15℃. Figure A shows that 52%vol sea cucumber wine obviously lost gloss at -15℃; Figure B shows that sea cucumber wine with 0.4‰ sea cucumber collagen added was placed at -15℃ for 5 days, and there was no obvious gloss loss after adsorption, and the 42%vol wine was transparent, and the 52%vol wine was light yellow; Figure C shows that 52%vol sea cucumber wine had no obvious gloss loss after adsorption after being placed at -15℃ for 5 days. Figure 3 The results show that adding common adsorption media for liquor, such as activated carbon (powdered) and diatomaceous earth (powdered), is helpful in preventing the occurrence of gloss loss and suspension. However, the flavor of liquor samples treated with activated carbon and diatomaceous earth is greatly lost, while the flavor of liquor samples treated with sea cucumber collagen is relatively intact.

[0093] Example 4

[0094] In this example, the sea cucumber peptide wine samples of Examples 2 to 3 and Comparative Examples 1 to 6 were placed and treated, and then the flavor sensory scores were performed, wherein the total score was 100 points, 90 to 100 points were excellent, 80 to 89 points were good, and 70 to 79 points were medium. The results are as follows: Figure 4 shown.

[0095] Specifically, the scoring criteria for sea cucumber flavor are as follows:

[0096] Aroma (0-20 points): aroma intensity, complexity, and pleasantness.

[0097] Appearance (0-20 points): color, transparency, viscosity, gloss, etc. of the wine.

[0098] Taste (0-30 points): thickness, layering, taste balance, and unique flavor.

[0099] Aftertaste (0-15 points): the persistence and variability of flavor.

[0100] Coordination (0-15 points): The balance and harmony of the overall flavor.

[0101] Figure 4 These are the flavor evaluation results of 52%vol sea cucumber peptide wine after treatment with different temperatures and additives. Figure 4 The results show that the flavor scores of Examples 2 and 3 are the highest, with Example 2 being 95. The flavor of the wine after adding sea cucumber collagen is as follows: strong wine aroma, mellow, fresh and fragrant, rich taste layers, slightly sweet, sweet aftertaste, and light yellow. The flavor is long-lasting and harmonious. The score of Example 3 is 96. The flavor of the wine after adding sea cucumber collagen is as follows: strong wine aroma, mellow, fresh and fragrant, slightly sweet, sweet aftertaste, and light yellow. The flavor is harmonious and well balanced. The flavor scores of Comparative Examples 1 and 2 without additives are the lowest, with Comparative Example 1 being 84, with obvious gloss loss and poor smoothness. Comparative Example 2 is 86, with slight gloss loss, thin taste and poor smoothness. Comparative Examples 3 and 4 add activated carbon, and their freshness is weak or lacking, the sense of layers is poor, and the refreshingness is lacking. Comparative Examples 5 and 6 add diatomaceous earth, and their flavor is weak, the sense of layers is poor, and the refreshingness is lacking.

[0102] Example 5

[0103] In this example, the sea cucumber peptide wine samples of Examples 2 to 3 and Comparative Examples 1 to 6 were processed and then subjected to flavor substance detection. Specifically, the wine sample was added with pure water, the alcohol content was adjusted to 10°, quantified to 5 mL, and then 10 μL of 2-octanol (internal standard, 0.0822 μg / mL) and 3 g of sodium chloride were added for flavor substance detection. This example uses headspace solid-phase micro-extraction-GC-MS (HS-SPME-GC-MS) to determine the flavor substances.

[0104] According to this embodiment, the GC conditions are set as follows: HP-INNOWAX (60m×0.25mm×0.25mm) chromatographic column; carrier gas is high-purity helium (He, 99.999%), flow rate 1mL / min, no split; hydrogen flow rate 30mL / min; air flow rate 450mL / min; vaporization chamber temperature 250℃; detector temperature 250℃; injection volume 1μL; electron ionization source (EI); ion source temperature 230℃; scanning range 20~450eV, detection voltage 350V, preheating at 60℃ for 10min, and then keeping at 60℃ for 30min. The program temperature is 40℃ for 5min; rise to 100℃ at 4℃ / min; rise to 230℃ at 6℃ / min and keep for 10min. MS conditions: mass spectrometer interface temperature was 250°C, ion source temperature was 230°C, electron ionization (EI) source, electron energy was 70 eV, and the scanning range was 30-350 m / z.

[0105] Qualitative and quantitative methods: The detected unknown compounds were matched with the National Institute of Standards and Technology (NIST) library 2020 by computer retrieval, and substances with a matching degree of >80% were selected as active ingredients, and screened in combination with the retention index (RI); the concentration of the corresponding substance was calculated according to the internal standard method using 2-octanol as the internal standard.

[0106] In this embodiment, samples under -15°C (ie, Example 2, Comparative Example 1, Comparative Example 3, Comparative Example 5) were tested, and the results are shown in Table 1.

[0107] Table 1

[0108]

[0109] Ethyl acetate is one of the main aroma components of liquor; ethyl butyrate increases the complexity of fruity aroma; ethyl lactate gives the wine a soft feeling; ethyl caprylate enhances the layers of floral and fruity aroma; butyl caproate enhances the complexity of aroma; isopentyl caproate is a characteristic aroma component; hexyl caproate increases the sense of freshness; ethyl decanoate enhances the thickness of the wine; ethyl tetradecanoate enhances the roundness of the wine; ethyl palmitate increases the density of the taste; isobutanol can enhance the sense of layers; isopentanol needs to be balanced with esters; n-hexanol enhances the sense of freshness; a small amount of nonanal enhances the sense of aging; and benzaldehyde enhances the aroma of fruit core.

[0110] The results of the present invention show that the main flavor of the sea cucumber peptide wine treated with sea cucumber collagen is rich and mellow in fruity aroma and has strong persistence. The flavors of comparative examples 3 and 5 are more inclined to the characteristics of the base wine after the fruity aroma is missing.

[0111] Compared with Comparative Examples 3 and 5, the sea cucumber peptide wine treated with sea cucumber collagen has a distinct sea cucumber flavor, but the unpleasant feeling (fishy smell, amine taste, etc.) is greatly reduced. After drinking, there is a light taste of sea salt in the mouth and tongue, which is slightly sweet.

[0112] It should be noted that the above-mentioned specific embodiments are exemplary, and those skilled in the art can come up with various solutions inspired by the disclosure of the present invention, and these solutions also belong to the disclosure scope of the present invention and fall within the protection scope of the present invention. Those skilled in the art should understand that the present invention specification and its drawings are illustrative and do not constitute a limitation of the claims. The scope of protection of the present invention is defined by the claims and their equivalents. The present invention specification contains multiple inventive concepts, such as "preferably" and "according to a preferred embodiment", both of which indicate that the corresponding paragraph discloses an independent concept, and the applicant reserves the right to file a divisional application based on each inventive concept. Throughout the text, the features guided by "preferably" are only an optional method and should not be understood as being required. Therefore, the applicant reserves the right to abandon or delete the relevant preferred features at any time.

Claims

1. A method for preparing sea cucumber peptide wine, characterized in that: The following steps are involved: Raw material pretreatment: removing salt, impurity protein and fat from the raw sea cucumbers, and homogenizing the pretreated raw sea cucumbers to obtain sea cucumber slurry; Enzymatic hydrolysis: taking sea cucumber pulp, adding protease for enzymatic hydrolysis, and centrifuging to obtain a supernatant for extracting collagen and a sea cucumber residue precipitate for extracting sea cucumber peptides; Extraction and purification of collagen: removing impurities and salt from the supernatant used to extract collagen to obtain sea cucumber collagen; Extraction of sea cucumber peptides: adding protease to the sea cucumber residue precipitate used for extracting sea cucumber peptides for enzymatic hydrolysis, and then performing an impurity removal step to obtain a sea cucumber small molecule peptide enzymatic hydrolyzate; Preparation of sea cucumber peptide wine: mixing the sea cucumber small molecule peptide hydrolysate with the base wine, and then adding the sea cucumber collagen prepared in the above steps to obtain the sea cucumber peptide wine.

2. The preparation method according to claim 1, characterized in that: The protease used in the enzymatic hydrolysis step can be one or more of a composite protease, a neutral protease, an acidic protease, and an alkaline protease.

3. The preparation method according to claim 1 or 2, characterized in that: In the extraction and purification steps of collagen, the added protease is 0.01% to 0.1% of the weight of the sea cucumber pulp.

4. The preparation method according to claim 1 or 2, characterized in that: In the step of extracting sea cucumber peptides, the amount of protease added is 1% to 3% of the mass of the sea cucumber residue precipitate.

5. The preparation method according to claim 1, characterized in that: The sea cucumber small molecule peptide hydrolysate refers to a peptide liquid obtained after filtration, in which the small molecule peptides account for more than 90%, wherein the molecular weight of the small molecule peptides is less than 10kDa.

6. The sea cucumber peptide wine prepared according to the method for preparing sea cucumber peptide wine according to any one of claims 1 to 5.

7. A method for preparing sea cucumber collagen, characterized in that: The following steps are involved: Raw material pretreatment: removing salt, impurity protein and fat from the raw sea cucumbers, and homogenizing the pretreated raw sea cucumbers to obtain sea cucumber slurry; Enzymatic hydrolysis: Take sea cucumber pulp, add protease for enzymatic hydrolysis, and centrifuge to obtain the supernatant for extracting collagen; Extraction and purification of collagen: The supernatant used for extracting collagen is subjected to impurity and salt removal operations to obtain sea cucumber collagen.

8. Sea cucumber collagen prepared by the method for preparing sea cucumber collagen according to claim 7.

9. A method for preparing sea cucumber peptide, characterized in that: The following steps are involved: Raw material pretreatment: removing salt, impurity protein and fat from the raw sea cucumbers, and homogenizing the pretreated raw sea cucumbers to obtain sea cucumber slurry; Enzymatic hydrolysis: Take sea cucumber pulp, add protease for enzymatic hydrolysis, and centrifuge to obtain sea cucumber residue precipitate for extracting sea cucumber peptides; Extraction of sea cucumber peptides: Add protease to the sea cucumber residue precipitate used for extracting sea cucumber peptides for enzymatic hydrolysis, and then perform a decontamination step to obtain a sea cucumber small molecule peptide enzymatic hydrolyzate.

10. The sea cucumber peptide prepared by the method for preparing sea cucumber peptide according to claim 9.

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