A method for extracting low-smelling and bitter codfish bone collagen peptides by complex enzyme hydrolysis

Abstract

The present invention provides a method for extracting low-smelling and bitter codfish bone collagen peptide by using a composite enzyme, comprising the following steps: (1) taking fresh codfish bones, adding deionized water and lipase to carry out enzymatic hydrolysis reaction; (2) washing the fish bones after the reaction, freezing, thawing, repeating the process 2-3 times, and then crushing them into particles with a particle size of ≤5 mm, filtering and drying; (3) adding the composite enzyme into deionized water, adjusting the pH to 6-7 with citric acid, adding the crushed fish bone powder, and carrying out enzymatic hydrolysis reaction in a vacuum state for 6-8 hours; (4) after inactivating the enzyme in a boiling water bath, separating the supernatant and the precipitate from the obtained enzymatic hydrolyzate, and concentrating the supernatant in a vacuum and drying to obtain the codfish bone collagen peptide. The cod bone collagen peptide extracted by the method disclosed in the present invention has a high content of active peptides, with total nitrogen at 13.7-14.2%, oligopeptides at 78.3-79.7%, and protein hydrolysates with a relative molecular mass of less than 1000u accounting for 97.6-98.9%. The extracted cod bone collagen peptide has no obvious bitterness and fishy smell, which is beneficial to improving the application range of the cod bone collagen peptide.

Description

Technical Field

[0001] The invention relates to a method for extracting low-smelly and bitter cod bone collagen peptides by compound enzyme hydrolysis, and belongs to the field of enzymatic extraction of collagen peptides. Background Art

[0002] During the processing of fish, a large amount of processing by-products (fish bones, fish skin, viscera and fish scales, etc.) will inevitably be produced, accounting for about 40% to 55% of the weight of the raw materials. These aquatic products are often discarded as waste or processed into animal roughage for reuse, which greatly wastes these by-product resources. The main components of fish bones are protein and ash. Taking cod bones as an example, the ash and protein account for 52.64% and 35.78% respectively, and the fat content is only 1%. Cod bones are a high-quality protein source processing raw material. It is of great significance to use cod bones to extract collagen peptides.

[0003] The existing methods for extracting fish bone collagen peptides generally use enzymatic hydrolysis, and the enzymes used include lipase, neutral protease or alkaline protease, so that enzymatic hydrolysates containing a large number of polypeptides of different lengths and nutrients such as amino acids can be obtained, effectively improving the utilization value of fish bones. However, there are the following two problems: (1) After enzymatic hydrolysis of fish bones, many bitter peptides will be generated, forming a strong bitter taste, which has become a problem that cannot be ignored in the development and utilization of protein resources; (2) Fish bones themselves have a fishy smell, which makes some people dislike it, reducing the scope of utilization. Summary of the invention

[0004] The invention provides a method for extracting codfish bone collagen peptide with low fishy and bitter taste by using composite enzyme hydrolysis, which solves the problem that bitterness and fishy smell exist in the existing extraction process of codfish bone collagen peptide, thus limiting the application scope thereof.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0006] A method for extracting low-smelly and bitter cod bone collagen peptides by complex enzyme hydrolysis, comprising the following steps:

[0007] (1) Take fresh cod bones, remove the fish meat on them, and then add deionized water and lipase to perform enzymatic hydrolysis;

[0008] (2) The cod bones after the reaction are washed, frozen at -30--40℃, thawed, repeated 2-3 times, and then crushed into particles with a particle size of ≤5mm, soaked in 50-60℃ water for 10-12h, filtered, and dried to a moisture content of 30-40%;

[0009] (3) Add the complex enzyme into deionized water, adjust the pH to 6-7 with citric acid, then add the crushed cod bone powder, and perform enzymatic hydrolysis in a vacuum state for 6-8 hours, wherein the weight ratio of the complex enzyme is 7:1:2 for papain: kiwi protease: eucalyptus honey;

[0010] (4) After enzymatic hydrolysis, the enzyme is inactivated in a boiling water bath, and the obtained enzymatic hydrolyzate is separated into a supernatant and a precipitate. The supernatant is vacuum concentrated and dried to obtain cod bone collagen peptide.

[0011] Furthermore, preferably: the lipase reaction in step (1) is specifically as follows: the lipase is a microbial lipase with an enzyme activity of 60,000 u / g, the amount of lipase added is 0.3-0.5%, the reaction temperature is 55-65°C, the pH is 6.5-7.5, and the reaction time is 20-30 min.

[0012] Furthermore, preferably, the added amount of the complex enzyme is 0.1-0.3%.

[0013] Furthermore, preferably: the enzymatic activity of the papain is 200,000-300,000 u / g, with a slight papaya smell; the enzymatic activity of the kiwifruit protease is 5000 u / g, with a fresh kiwifruit fragrance; the eucalyptus pollen content in the eucalyptus honey is ≥50%, fructose and glucose are ≥60 g / 100g, and the amylase value is ≥10 mL / (g·h), with a stimulating and refreshing fragrance.

[0014] Furthermore, preferably: the vacuum degree of the vacuum reaction in step (3) is -75--101.325KPa.

[0015] Furthermore, preferably: a citric acid extraction step is added before step (2).

[0016] Furthermore, preferably: the citric acid extraction is specifically as follows: using 0.3 mol / L citric acid, a solid-liquid ratio of 1:20 (m:V), a temperature of 5-10°C, soaking for 2-3 days, centrifuging, adding ice cubes to the supernatant to form an ice-water mixture, keeping the ice-water mixture in a state of standing for 20-30 minutes, and filtering; adding the filtrate to the ice-water mixture again, keeping the ice-water mixture in a state of standing for 20-30 minutes, and filtering; repeating the above steps until there is no obvious sour taste in the filtrate, adding the obtained filtrate to step (3), and adding 0.1% of the complex enzyme to react together.

[0017] Beneficial effects of the present invention:

[0018] The present invention adopts the method of microbial lipase composite high temperature reaction, which can effectively remove and decompose fat in fish bones and reduce bitter substances produced by fat oxidation. At the same time, the low-temperature freezing, soaking and drying methods can effectively form pores on the fish bones that are conducive to composite enzyme hydrolysis, providing a favorable space for subsequent enzymatic hydrolysis. At the same time, high-temperature soaking can effectively reduce the content of water-soluble calcium ions in the bones, reduce the subsequent combination of calcium ions and collagen peptides, and improve the yield.

[0019] In the process of preparing the cod bone collagen peptide, the invention adopts a composite enzyme of papain, kiwi protein and eucalyptus honey, which promote each other and can effectively reduce the bitterness of the collagen peptide.

[0020] Kiwi protease and papain are both highly efficient proteases that complement each other, cover a wider range of peptide bonds, accelerate the hydrolysis of collagen, and cut it into small molecular peptide chains, thereby reducing the generation of bitter components. The two enzymes complement each other during the action process. Papain has the ability to cut terminal peptides, while kiwi protease plays a promoting role in the protein degradation process, jointly improving the efficiency of protein degradation.

[0021] Eucalyptus honey contains a variety of sugars, minerals, vitamins and active enzymes, which has a certain seasoning effect, improving the taste of collagen, increasing its sweetness, and thus reducing the perception of bitterness; at the same time, the sugar in eucalyptus honey can also interact with the bitter peptide chains in collagen to form a stable complex, reducing the perception of bitterness; eucalyptus honey can not only neutralize bitterness, but also stabilize the activity of enzymes, prolong the action time of enzymes, and improve the extraction efficiency of collagen peptides.

[0022] The composite enzyme of papain, kiwifruit protein and eucalyptus honey can effectively reduce the fishy smell while reducing the bitter taste.

[0023] Papain and kiwi protease have a strong ability to decompose protein and can effectively decompose the protein decomposition products in the fishy smell, thereby reducing the fishy smell.

[0024] Papain is derived from papaya and has a certain papaya fragrance, which can help cover up the fishy smell; kiwi protease is derived from kiwi fruit and has a fresh kiwi fragrance, which helps reduce the fishy smell and makes the smell more refreshing; eucalyptus honey has a unique cool fragrance, which can stimulate the taste buds and reduce people's perception of the fishy smell. When these three ingredients are used at the same time, their fragrance and function can complement each other to jointly cover up and reduce the fishy smell. The fruity aroma of papaya and kiwi fruit and the cool fragrance of eucalyptus honey can jointly form a more pleasant taste and smell; the fragrances of different ingredients can complement each other to form a more harmonious aroma, which can better cover up the fishy smell; the cool feeling of eucalyptus honey and the fresh fruity aroma of kiwi protease can enhance the overall flavor experience and make the fish bone protein peptide taste more delicious.

[0025] The cod bone collagen peptide extracted by the method of the present invention has a high content of active peptides, a total nitrogen (on a dry basis) of 13.7-14.2%, an oligopeptide (on a dry basis) of 78.3-79.7%, and a protein hydrolyzate with a relative molecular mass of less than 1000u accounting for 97.6-98.9%. At the same time, the cod bone collagen peptide extracted by the present invention has no obvious bitterness and fishy smell, which is conducive to improving the application range of the cod bone collagen peptide and improving its use value. DETAILED DESCRIPTION

[0026] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] The following raw materials used in the present invention are as follows:

[0028] The cod bones were frozen and stored for no more than one week, and the protein content was 36.12%;

[0029] The lipase is a microbial lipase made from Aspergillus niger through fermentation, extraction and purification, with an enzyme activity of 60,000 u / g.

[0030] The enzyme activity of papain is 260,000 u / g, with a slight papaya smell;

[0031] Kiwi protease is extracted from kiwi fruit, with an enzyme activity of 5000u / g and a kiwi fragrance.

[0032] Eucalyptus honey contains 52% eucalyptus pollen, 76g / 100g fructose and glucose, and 11mL / (g·h) amylase value. It has a stimulating and refreshing fragrance.

[0033] Citric acid uses food grade citric acid with an active ingredient content of 99%.

[0034] Example 1

[0035] A method for extracting low-smelly and bitter cod bone collagen peptides by complex enzyme hydrolysis, comprising the following steps:

[0036] (1) Take fresh cod bones, remove the fish meat on them, and then add deionized water and lipase for enzymatic hydrolysis. The amount of lipase added is 0.3%, the reaction temperature is 60°C, the pH is 7.5, the reaction time is 30 min, and the volume ratio of cod bones to deionized water is 1:3.

[0037] (2) The cod bones after the reaction are washed, dehydrated, frozen at -30°C, and thawed, repeated twice, and then crushed into particles with a particle size of ≤5 mm, soaked in 60°C water for 10 hours, filtered, and dried to a moisture content of 30%;

[0038] (3) Add the complex enzyme to deionized water, adjust the pH to 6.5 with citric acid, then add the crushed cod bone powder, and perform enzymatic hydrolysis in vacuum at room temperature for 6 hours. The volume ratio of deionized water to the crushed cod bone powder is 1:3, the weight ratio of the complex enzyme is papain: kiwi protease: eucalyptus honey is 7:1:2, the amount of the complex enzyme added is 0.2%, and the vacuum degree of the vacuum reaction is -100 KPa;

[0039] (4) After enzymatic hydrolysis, the enzyme was inactivated in a boiling water bath, and the obtained enzymatic hydrolyzate was centrifuged at 8000 r / min for 20 min to separate the supernatant and the precipitate. The supernatant was vacuum concentrated and dried to obtain cod bone collagen peptide.

[0040] Example 2

[0041] A method for extracting low-smelly and bitter cod bone collagen peptides by complex enzyme hydrolysis, comprising the following steps:

[0042] (1) Take fresh cod bones, remove the fish meat on them, and then add deionized water and lipase for enzymatic hydrolysis. The amount of lipase added is 0.4%, the reaction temperature is 60°C, the pH is 7.5, the reaction time is 30 min, and the volume ratio of cod bones to deionized water is 1:3.

[0043] (2) The cod bones after the reaction were washed, frozen at -40°C, and thawed, repeated twice, and then crushed into particles with a particle size of ≤5 mm, soaked in 50°C water for 10 hours, filtered and dried to a moisture content of 40%;

[0044] (3) Add the complex enzyme to deionized water, adjust the pH to 6.5 with citric acid, then add the crushed cod bone powder, and perform enzymatic hydrolysis in vacuum at room temperature for 8 hours. The volume ratio of deionized water to the crushed cod bone powder is 1:3, the weight ratio of the complex enzyme is papain: kiwi protease: eucalyptus honey is 7:1:2, the amount of the complex enzyme added is 0.1%, and the vacuum degree of the vacuum reaction is -100 KPa;

[0045] (4) After enzymatic hydrolysis, the enzyme was inactivated in a boiling water bath, and the obtained enzymatic hydrolyzate was centrifuged at 8000 r / min for 20 min to separate the supernatant and the precipitate. The supernatant was vacuum concentrated and dried to obtain cod bone collagen peptide.

[0046] Example 3

[0047] A method for extracting low-smelly and bitter cod bone collagen peptides by complex enzyme hydrolysis, comprising the following steps:

[0048] (1) Take fresh cod bones, remove the fish meat on them, and then add deionized water and lipase for enzymatic hydrolysis. The amount of lipase added is 0.5%, the reaction temperature is 60°C, the pH is 7.5, the reaction time is 30 min, and the volume ratio of cod bones to deionized water is 1:3.

[0049] (2) The cod bones after the reaction are washed, frozen at -30°C, thawed, repeated twice, and then crushed into particles with a particle size of ≤5 mm, soaked in 60°C water for 10 hours, filtered and dried to a moisture content of 40%;

[0050] (3) Add the complex enzyme to deionized water, adjust the pH to 6.5 with citric acid, then add the crushed fish bone powder, and perform enzymatic hydrolysis under vacuum at room temperature for 7 hours. The volume ratio of deionized water to crushed cod bone powder is 1:3, the weight ratio of the complex enzyme is papain: kiwi protease: eucalyptus honey is 7:1:2, the amount of the complex enzyme added is 0.3%, and the vacuum degree of the vacuum reaction is -100 KPa;

[0051] (4) After the enzyme is inactivated in a boiling water bath, the resulting enzymatic solution is centrifuged at 8000 r / min for 20 min to separate the supernatant and precipitate. The supernatant is vacuum concentrated and dried to obtain cod bone collagen peptide.

[0052] Example 4

[0053] The method is basically the same as Example 1, except that a citric acid extraction step is added before step (2).

[0054] Citric acid extraction is specifically as follows: using 0.3 mol / L citric acid, a solid-liquid ratio of 1:20 (m:V), a temperature of 5°C, soaking for 3 days, 8000 r / min, centrifugation for 20 minutes, adding ice cubes to the supernatant to form an ice-water mixture, keeping the ice-water mixture in a state of standing for 20 minutes, and filtering; adding the filtrate to the ice-water mixture again, keeping the ice-water mixture in a state of standing for 20 minutes, and filtering; repeating the above steps until there is no obvious sour taste in the filtrate, adding the obtained filtrate to step (3), and adding 0.1% of the complex enzyme to react together.

[0055] Example 5

[0056] The method is basically the same as Example 1, except that a citric acid extraction step is added before step (2).

[0057] Citric acid extraction is specifically as follows: using 0.3 mol / L citric acid, a solid-liquid ratio of 1:20 (m:V), a temperature of 10°C, soaking for 2 days, 8000 r / min, centrifugation for 20 minutes, adding ice cubes to the supernatant to form an ice-water mixture, keeping the ice-water mixture in a state of standing for 30 minutes, and filtering; adding the filtrate to the ice-water mixture again, keeping the ice-water mixture in a state of standing for 30 minutes, and filtering; repeating the above steps until there is no obvious sour taste in the filtrate, adding the obtained filtrate to step (3), and adding 0.1% of the complex enzyme to react together.

[0058] Determination of the content of cod bone collagen peptide

[0059] The contents of related substances of the cod bone collagen peptides prepared in Examples 1 to 5 were determined by the method of GB / T22729-2008 marine fish oligopeptide powder, as shown in Table 1.

[0060] Extraction rate (%) = protein content in cod bone collagen peptide / protein content in fresh cod bone raw material * 100

[0061] The protein content was determined according to the method specified in GB / T 5009.5.

[0062] Table 1 Determination results of related substances of cod bone collagen peptides prepared in different examples

[0063]

[0064] It can be seen from Table 1 that the total nitrogen (on a dry basis) of the cod bone collagen peptide extracted by the extraction method of the present invention is 13.7-14.2%, the oligopeptide (on a dry basis) is 78.3-79.7%, and the proportion of protein hydrolyzate with a relative molecular mass of less than 1000u is 97.6-98.9%.

[0065] The principle of using ice-water mixture to purify and remove citric acid is that the solubility of citric acid in water is much greater than that in ice, while the solubility of protein in ice-water mixture is very small. Therefore, ice-water mixture can be used to remove citric acid, thereby achieving the purpose of purifying protein and removing citric acid.

[0066] At the same time, the method of using citric acid and ice water mixture can effectively improve the extraction rate. Compared with Example 1, the extraction rate is increased by more than 50%.

[0067] Sensory evaluation of fishy and bitter taste:

[0068] 10 sensory assessors who have received certain sensory assessment training were selected to form an assessment team (5 men and 5 women, aged 20-35 years old). 1g of cod bone collagen peptide obtained in different embodiments was taken and added to 50mL of deionized water to prepare cod bone collagen peptide solution, and then sensory scoring was performed. The sensory assessors fasted for two hours before the assessment. At the beginning of the sensory assessment, the assessors first rinsed their mouths with ultrapure water, taking 2-3 mL of sample each time and holding it in the mouth for 10 s to allow the sample solution to be fully dispersed throughout the oral cavity, mainly allowing the root of the tongue to feel the taste, and spit out the sample and rinse the mouth with clean water. The interval between the two samples for bitterness assessment was 5-10 minutes to avoid mutual influence between samples. The fishy smell and bitterness were scored on a 10-point scale and divided into five levels according to the degree of fishy smell or bitterness (8 < no or slight fishy smell (or bitterness) ≤ 10; 6 < light fishy smell (or bitterness) ≤ 8; 4 < moderate fishy smell (or bitterness) ≤ 6; 2 < strong fishy smell (or bitterness) ≤ 4; 0 < extremely strong fishy smell (or bitterness) ≤ 2). The higher the score, the better the overall flavor. The scoring criteria are shown in Table 2. Each score was scored by an assessor individually, and the specific results are shown in Table 3.

[0069] Table 2 Sensory evaluation criteria for fishy and bitter taste of cod bone collagen peptide

[0070]

[0071] Table 3 Sensory evaluation results of fishy and bitter taste of cod bone collagen peptide

[0072]

[0073] As can be seen from Table 3, through sensory evaluation, the cod bone collagen peptide of the present invention has relatively low fishy smell and bitterness, and has almost no obvious fishy smell and bitterness.

[0074] Comparative Example 1-3 Effects of different cod bone processing methods on the extraction of cod bone collagen peptides

[0075] Comparison Example

[0076] As with Example 1, I do not want to explain it in detail.

[0077] Comparative Example 1

[0078] The method is basically the same as Example 1, except that the cod bones after enzymatic hydrolysis in step (2) are not frozen but directly crushed.

[0079] Comparative Example 2

[0080] The method is basically the same as Example 1, except that: in step (2), the cod bones after enzymatic hydrolysis are not soaked in hot water after being crushed, but are directly dehydrated and dried.

[0081] Comparative Example 3

[0082] The process is basically the same as Example 1, except that in step (3), a vacuum reaction is not used but a normal pressure reaction is used.

[0083] The extraction rates of different comparative examples were determined, and the specific results are shown in Table 4.

[0084] Table 4 Determination results of total nitrogen content in cod bone treatment methods

[0085]

[0086] As shown in Table 4, the extraction method of the present invention is repeatedly frozen and thawed to form a gap on the surface of cod bones, so that the enzymatic solution is more likely to enter the fish bone surface in the subsequent enzymolysis process, improve the enzymolysis rate, and the extraction rate is improved by 30.5%, thereby increasing the yield. The hot water dissolution after crushing can effectively reduce the calcium ions in the solution in the subsequent enzymolysis process, causing calcium ions to combine with collagen, precipitate, and reduce the problem of yield. The vacuum enzymolysis process is adopted to make the enzymatic solution more likely to enter the cod bones, effectively improve the enzymolysis efficiency, and the yield is improved by 53.1%.

[0087] Comparative Examples 3-8 Effects of different complex enzyme compositions on the fishy smell and bitterness of cod bone collagen peptide

[0088] Comparison Example

[0089] As with Example 1, I do not want to explain it in detail.

[0090] Comparative Examples 3-8 The compositions of different complex enzymes were adjusted, as shown in Table 5.

[0091] Table 5 Composition of complex enzymes in different comparative examples

[0092]

[0093] The fishy smell and bitterness sensory evaluation method of Example 1 was used to evaluate the fishy smell and bitterness of the cod bone collagen peptides prepared in Comparative Examples 4-9. The specific results are shown in Table 6.

[0094] Table 6 Sensory evaluation results of fishy and bitter taste of different comparative ratios

[0095]

[0096] As can be seen from Table 6, the present invention uses a composite enzyme of papain, kiwi protein and eucalyptus honey, which promote each other and can effectively reduce the bitterness and fishy smell of collagen peptides. In particular, eucalyptus honey has an important effect on fishbone collagen peptides. The main reason is that eucalyptus honey has a unique cool fragrance, which can stimulate the taste buds and reduce people's perception of fishy smell. At the same time, the cool feeling of eucalyptus honey and the fresh fruity aroma of kiwi proteinase can enhance the overall flavor experience, making the taste of fishbone protein peptides more delicious.

[0097] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for extracting low-smelling and bitter cod bone collagen peptides by complex enzyme hydrolysis, characterized in that: The following steps are involved: (1) Take fresh cod bones, remove the fish meat on them, and then add deionized water and lipase to perform enzymatic hydrolysis; (2) The fish bones after the reaction are washed, frozen at -30-40°C, thawed, repeated 2-3 times, and then crushed into particles with a particle size of ≤5 mm, soaked in 50-60°C water for 10-12 hours, filtered, and dried to a moisture content of 30-40%; (3) Add the complex enzyme into deionized water, adjust the pH to 6-7 with citric acid, then add the crushed cod bone powder, and perform enzymatic hydrolysis in a vacuum state for 6-8 hours, wherein the weight ratio of the complex enzyme is 7:1:2 for papain: kiwi protease: eucalyptus honey; (4) After enzymatic hydrolysis, the enzyme is inactivated in a boiling water bath, and the obtained enzymatic hydrolyzate is separated into a supernatant and a precipitate. The supernatant is vacuum concentrated and dried to obtain cod bone collagen peptide.

2. The method according to claim 1, characterized in that: The lipase reaction in step (1) is specifically as follows: the lipase is microbial lipase, the enzyme activity is 60,000 u / g, the amount of lipase added is 0.3-0.5%, the reaction temperature is 55-65°C, the pH is 6.5-7.5, and the reaction time is 20-30 min.

3. The method according to claim 1, characterized in that: The added amount of complex enzyme is 0.1-0.3%.

4. The method according to claim 1, characterized in that: The papain has an enzyme activity of 200,000-300,000 u / g and has a slight papaya smell; the kiwifruit protease has an enzyme activity of 5,000 u / g and has a kiwifruit fragrance; the eucalyptus honey contains eucalyptus pollen ≥50%, fructose and glucose ≥60 g / 100g, and an amylase value ≥10 mL / (g·h), and has a stimulating and refreshing fragrance.

5. The method according to claim 1, characterized in that: The vacuum degree of the vacuum reaction in step (3) is -75--101.325 KPa.

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