阅读说明：本技术 一种虾天然防黑变剂及其制备方法和用途 (Natural anti-blackening agent for shrimps as well as preparation method and application thereof ) 是由 曹敏杰 杜雪莉 章骞 杨汝晴 翁凌 陈玉磊 孙乐常 张凌晶 刘光明 于 2021-09-09 设计创作，主要内容包括：本发明涉及一种虾天然防黑变剂及其制备方法和用途,制备方法包括以下步骤：粗提：绿豆浸泡后捣碎,进行离心处理取上清液,得到粗提液；热处理：将所述粗提液加热后,冷却离心取上清液,得到粗溶液；超滤膜分离：将所述粗溶液采用截留分子量为11-16KDa的超滤膜进行分离,收集透过液；将透过液用截留分子量为4-6KDa的超滤膜进行分离,收集未透过超滤膜的液体,得到浓缩液；干燥制粉：将所述浓缩液干燥制粉,即为虾天然防黑变剂。本发明所述虾天然防黑变剂可以显著延长虾黑变的时间,满足虾运输时间的要求,保证虾的品质和价值,具有极强的市场运用前景。(The invention relates to a natural anti-blackening agent for shrimps as well as a preparation method and application thereof, wherein the preparation method comprises the following steps: crude extraction: soaking semen Phaseoli Radiati, mashing, centrifuging, and collecting supernatant to obtain crude extractive solution; and (3) heat treatment: heating the crude extract, cooling, centrifuging and taking supernatant to obtain a crude solution; and (3) ultrafiltration membrane separation: separating the crude solution by adopting an ultrafiltration membrane with the molecular weight cutoff of 11-16KDa, and collecting a permeate; separating the permeate with ultrafiltration membrane with molecular weight cutoff of 4-6KDa, and collecting the liquid which does not permeate the ultrafiltration membrane to obtain concentrated solution; drying to prepare powder: and drying the concentrated solution to prepare powder, thus obtaining the natural shrimp blackening preventing agent. The natural shrimp blackening preventing agent can obviously prolong the shrimp blackening time, meets the requirement of shrimp transportation time, ensures the quality and value of the shrimps, and has strong market application prospect.)

1. A preparation method of a natural shrimp blackening preventing agent is characterized by comprising the following steps:

crude extraction: soaking semen Phaseoli Radiati, mashing, centrifuging, and collecting supernatant to obtain crude extractive solution;

and (3) heat treatment: heating the crude extract at 80-100 deg.C for 30-60min, cooling, centrifuging, and collecting supernatant to obtain crude solution;

and (3) ultrafiltration membrane separation: separating the crude solution by adopting an ultrafiltration membrane with the molecular weight cutoff of 11-16KDa, and collecting a permeate; separating the permeate with ultrafiltration membrane with molecular weight cutoff of 4-6KDa, and collecting the liquid which does not permeate the ultrafiltration membrane to obtain concentrated solution;

drying to prepare powder: and drying the concentrated solution to prepare powder, thus obtaining the natural shrimp blackening preventing agent.

2. The method for preparing the natural shrimp blackening preventing agent as claimed in claim 1, wherein: in the crude extraction step, the mung beans are soaked in water with the volume of 3-5 times for 8-10h, then are mashed, the gravity acceleration for centrifugal treatment is 8000-12000g, and are centrifuged for 10-30min to obtain supernatant.

3. The method for preparing the natural shrimp blackening preventing agent as claimed in claim 1, wherein: in the heat treatment step, the gravity acceleration of the centrifugal treatment is 8000-12000g, and the centrifugation is carried out for 10-30min to obtain the supernatant.

4. The method for preparing the natural shrimp blackening preventing agent according to any one of claims 1 to 3, wherein: in the step of ultrafiltration membrane separation, the crude solution is separated by adopting an ultrafiltration membrane with the molecular weight cutoff of 14-16KDa, and a permeate is collected; separating the permeate with ultrafiltration membrane with cut-off molecular weight of 5-6KDa, and collecting the liquid which does not permeate the ultrafiltration membrane to obtain concentrated solution.

5. The method for preparing the natural shrimp blackening preventing agent according to any one of claims 1 to 3, wherein: the dry powder preparation is freeze-drying powder preparation or spray-drying powder preparation.

6. A natural anti-blackening agent for shrimps, which is prepared by the preparation method of the natural anti-blackening agent for shrimps as claimed in any one of claims 1 to 5.

7. The natural shrimp blackening preventing agent as set forth in claim 6, wherein: the natural shrimp blackening preventing agent contains components capable of inhibiting the activity of serine protease, and has a molecular weight of 8-9 KDa.

8. Use of the natural shrimp blackening preventing agent according to claim 6 or 7 for the preservation of shrimp.

9. A method for keeping shrimps fresh by using the natural anti-blackening agent for shrimps as claimed in claim 6 or 7, which is characterized in that: the method comprises the following steps: dissolving the natural shrimp blackening preventing agent in water to obtain a blackening preventing solution; immersing shrimps in the anti-blackening solution for soaking treatment; then taking out the shrimps and draining off water.

10. A method of preserving shrimp as claimed in claim 9, wherein: dissolving the natural shrimp anti-blackening agent in water to obtain an anti-blackening solution with the concentration of 0.1-0.3 wt%; immersing the shrimps in the anti-blackening solution, soaking at 0-4 ℃ for 20-30min, taking out, draining, and finally storing at 0-4 ℃ for later use.

Technical Field

The invention relates to the technical field of shrimp preservation, in particular to a natural anti-blackening agent for shrimps as well as a preparation method and application thereof.

Background

In southern areas of China, such as Hainan province, Guangdong province, Guangxi province, Fujian province and the like, shrimps have become the pillar industry of aquaculture due to the fact that the water area is vast and the climate is appropriate. The prawn is rich in nutrition, thin in shell and fertile, delicious in taste, low in fat content, high in protein content, good in nutrition balance, rich in vitamins and minerals, and is popular with consumers.

However, the shrimps are easy to blacken after death, and lose the nutritional value and the economic value. The phenomenon of blackening after death of shrimps is mainly due to some enzymatic reactions caused by the presence of polyphenol oxidase. Blackening generally begins at the head and gradually spreads to the prephoretic foot and then to the sub-crustal part to spread to the whole body. After the shrimps die, the phenol oxidase zymogen is activated, and the activated polyphenol oxidase still has certain activity even if being stored at a lower temperature. Under the condition of oxygen, polyphenol oxidase reacts with tyrosine and phenolic substances in shrimp bodies to generate a large amount of melanin, and most of the melanin can be combined with protein and is difficult to remove. For this reason, in the prior art, shrimp treatment is carried out using specific preservatives. For example, the invention patent of application publication No. CN107156263A discloses a fresh-keeping agent for seawater shrimps and a using method thereof, aiming at the problems that the fresh-keeping agent obtained by directly compounding compounds has potential harm to human health and the fresh-keeping effect of natural fresh-keeping agents is not ideal enough, the fresh-keeping agent from natural plants is provided, and the fresh-keeping agent can effectively delay the blackening of seawater shrimps and the increase of volatile basic nitrogen and prolong the fresh-keeping period of the seawater shrimps. The preservative for seawater shrimps comprises the following raw materials in parts by weight: 3-5 parts of traditional Chinese medicine powder, 5-8 parts of ferment powder, 2-5 parts of soybean powder, 30-45 parts of maltodextrin, 0.5-1.5 parts of xanthan gum, 1-2 parts of honey, 0.01-0.05 part of edible essence and 150-200 parts of water. The anti-blackening effect of the seawater shrimps preserved at low temperature can be maintained for 7 days. However, the method has complex components and higher use cost, the anti-blackening effect needs to be improved, and the method is not easy to apply on a large scale.

The invention patent of application publication No. CN 106259829A discloses a natural anti-blackening agent, which comprises the following components in parts by weight: 0.5-0.8 part of albizia julibrissin seed freeze-dried powder, 100-160 parts of deionized water and 1.2-3 parts of natural thickening agent; also relates to a preparation method and application of the natural anti-blackening agent. The natural anti-blackening agent takes pure natural albizia julibrissin seeds as raw materials, and has obvious anti-blackening and fresh-keeping effects on the south America white shrimps. The anti-blackening effect of the low-temperature preserved shrimps can be kept for 10-14 days. However, the method needs low-temperature plasma treatment, adopts albizia julibrissin seeds as raw materials, has high use cost, and is not easy to apply on a large scale.

Disclosure of Invention

The invention aims to overcome the problems of the existing shrimp preservative and provide a natural shrimp blackening preventing agent which is prepared from mung beans serving as raw materials by a special extraction process, has a remarkable effect on the preservation of shrimps and is suitable for prawns, south American white shrimps, crystal shrimps, arctic shrimps and the like.

The inventor researches and discovers that the head of the shrimp has polyphenol oxidase capable of blackening the shrimp, and the polyphenol oxidase contains Cu in an active center²⁺The metalloenzyme of (1). In live shrimp, polyphenol oxidase exists as Prophenoloxidase (proPO), which is not active. When the shrimp is dead, the pancreas of the shrimp is ruptured, the enzyme balance in the shrimp is broken, the prophenoloxidase is activated into active polyphenoloxidase, the monophenolic substances in the body are catalyzed to be converted into melanin, the blackening of the shrimp is caused, and the sensory product is causedThe quality is reduced, the nutrition is lost, and the market value is reduced. The inventors considered that the problem of blackening of shrimps could be fundamentally solved by finding a substance capable of inhibiting pro-phenoloxidase from being activated into polyphenol oxidase.

How to find such a substance is the key to the present invention. The inventors have found that the prophenoloxidase activating system is a complex system involving Serine Protease (SP) and performs a precise cascade reaction to activate prophenoloxidase (ProPO) to Polyphenoloxidase (PO), and the activation mechanism is shown in fig. 1. Therefore, inhibition of serine protease activity, and thus inhibition of prophenoloxidase activation to polyphenoloxidase, is expected to inhibit blackening of shrimp from the source.

When the inventor researches the deep processing of mung beans in early days, mung bean trypsin (CN 102295700A) is obtained through separation and purification, and the Mung Bean Trypsin (MBTI) belongs to a Bowman-Birk type serine protease inhibitor, has the molecular weight of 8883Da and has high stability to digestion of heat, acid, alkali and enzyme. Further intensive studies have revealed that Mung Bean Trypsin (MBTI) has a strong inhibitory effect on serine protease, i.e., the activity of myofibril-binding serine protease is reduced to about 10% at an extremely low concentration of 5 ng/mL. Therefore, the invention improves the prior art, creatively separates the concentrated solution containing the serine protease inhibitor with higher concentration from the mung bean by crude extraction, heat treatment and ultrafiltration membrane separation, and prepares the concentrated solution into powder by drying, and has simple method, high yield and obvious effect on preventing the shrimp from blackening.

In the preparation method of the natural shrimp blackening preventing agent, crude extraction is used for extracting active components from mung beans, and heat treatment is used for removing impurity proteins through thermal denaturation, because a target object serine protease inhibitor has strong heat resistance. The separation of the ultrafiltration membrane is used for purifying and enriching to obtain the high-concentration natural shrimp blackening preventing agent, and the drying and powdering is used for drying the concentrated solution of the natural shrimp blackening preventing agent to prepare powder which is convenient to store and use.

The specific scheme is as follows:

a preparation method of a natural shrimp blackening preventing agent comprises the following steps:

crude extraction: soaking semen Phaseoli Radiati, mashing, centrifuging, and collecting supernatant to obtain crude extractive solution;

and (3) heat treatment: heating the crude extract at 80-100 deg.C for 30-60min, cooling, centrifuging, and collecting supernatant to obtain crude solution;

and (3) ultrafiltration membrane separation: separating the crude solution by adopting an ultrafiltration membrane with the molecular weight cutoff of 11-16KDa, and collecting a permeate; separating the permeate with ultrafiltration membrane with molecular weight cutoff of 4-6KDa, and collecting the liquid which does not permeate the ultrafiltration membrane to obtain concentrated solution;

drying to prepare powder: and drying the concentrated solution to prepare powder, thus obtaining the natural shrimp blackening preventing agent.

Further, in the crude extraction step, the mung beans are soaked in water with the volume of 3-5 times for 8-10h, then are mashed, and are centrifuged at the gravitational acceleration of 8000-12000g for 10-30min to obtain supernatant.

Further, in the heat treatment step, the gravity acceleration of the centrifugal treatment is 8000-12000g, and the centrifugation is carried out for 10-30min to take the supernatant.

Further, in the step of ultrafiltration membrane separation, the crude solution is separated by adopting an ultrafiltration membrane with the molecular weight cutoff of 14-16KDa, and a permeate is collected; separating the permeate with ultrafiltration membrane with cut-off molecular weight of 5-6KDa, and collecting the liquid which does not permeate the ultrafiltration membrane to obtain concentrated solution.

Further, the dry powder preparation is freeze-drying powder preparation or spray-drying powder preparation.

The invention also discloses a natural shrimp blackening preventing agent prepared by the preparation method of the natural shrimp blackening preventing agent.

Furthermore, the natural shrimp blackening preventing agent contains a component capable of inhibiting the activity of serine protease, and the molecular weight of the natural shrimp blackening preventing agent is 8-9 KDa.

The invention also protects the application of the natural shrimp blackening preventing agent in shrimp preservation.

The invention also provides a method for keeping shrimps fresh, which adopts the natural anti-blackening agent for shrimps and comprises the following steps: dissolving the natural shrimp blackening preventing agent in water to obtain a blackening preventing solution; immersing shrimps in the anti-blackening solution for soaking treatment; then taking out the shrimps and draining off water.

Further, dissolving the natural shrimp blackening preventing agent in water to obtain a blackening preventing solution with the concentration of 0.1-0.3 wt%; immersing the shrimps in the anti-blackening solution, soaking at 0-4 ℃ for 20-30min, taking out, draining, and finally storing at 0-4 ℃ for later use.

Has the advantages that:

(a) the natural shrimp blackening preventing agent prepared from mung beans contains a high-concentration serine protease inhibitor, has good thermal stability and pH stability, is stored for a long time, and can keep the activity for a long time.

(b) The natural shrimp blackening preventing agent has a remarkable effect on inhibiting shrimp blackening, solves the problem that the shrimp blackening brings inconvenience to preservation, storage, transportation and processing, improves the quality of shrimp products, prolongs the shelf life, and has high popularization and application values.

(c) The natural shrimp blackening preventing agent developed by taking mung beans as raw materials is low in cost and can be applied in a large scale.

Drawings

In order to illustrate the technical solution of the present invention more clearly, the drawings will be briefly described below, and it is apparent that the drawings in the following description relate only to some embodiments of the present invention and are not intended to limit the present invention.

FIG. 1 is a diagram showing the mechanism of pro-phenoloxidase activation involving serine proteases provided by the present invention.

Detailed Description

Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the present invention, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available. In the following examples, "%" means weight percent, unless otherwise specified.

Example 1: natural anti-blackening agent for shrimps and preparation thereof

Step one, crude extraction: soaking semen Phaseoli Radiati in 3 times volume of distilled water for 10 hr, mashing, centrifuging at 8000g (gravity acceleration) for 30min, and collecting supernatant as coarse extractive solution;

step two, heat treatment: heating the crude extractive solution at 80 deg.C for 60min, immediately cooling to room temperature, centrifuging at 8000g for 30min, and collecting supernatant as crude solution;

thirdly, ultrafiltration membrane separation: separating the crude solution by adopting an ultrafiltration membrane with the molecular weight cutoff of 16KDa, collecting a permeated solution, separating and concentrating the permeated solution by adopting an ultrafiltration membrane with the molecular weight cutoff of 4KDa, and collecting an unpermeated solution to obtain a concentrated solution of the natural shrimp blackening preventing agent;

and fourthly, drying and pulverizing, namely spray drying the concentrated solution of the natural shrimp anti-blackening agent to prepare powder, thus obtaining the finished product of the natural shrimp anti-blackening agent.

Example 2: natural anti-blackening agent for shrimps and preparation thereof

Step one, crude extraction: soaking semen Phaseoli Radiati in 5 times volume of distilled water for 8 hr, mashing, centrifuging at 12000g for 10min, and collecting supernatant as coarse extractive solution;

step two, heat treatment: heating the crude extractive solution at 100 deg.C for 30min, immediately cooling to room temperature, centrifuging at 12000g for 10min, and collecting supernatant to obtain crude solution;

thirdly, ultrafiltration membrane separation: separating the crude solution by adopting an ultrafiltration membrane with the molecular weight cutoff of 15KDa, collecting a permeated solution, separating and concentrating the permeated solution by adopting an ultrafiltration membrane with the molecular weight cutoff of 6KDa, and collecting an unpermeated solution to obtain a concentrated solution of the natural shrimp blackening preventing agent;

and fourthly, drying and pulverizing, namely spray drying the concentrated solution of the natural shrimp anti-blackening agent to prepare powder, thus obtaining the finished product of the natural shrimp anti-blackening agent.

Example 3: natural anti-blackening agent for shrimps and preparation thereof

Step one, crude extraction: soaking semen Phaseoli Radiati in 4 times volume of distilled water for 9h, mashing, centrifuging at 10000g for 20min, and collecting supernatant as coarse extractive solution;

step two, heat treatment: heating the crude extractive solution at 90 deg.C for 45min, immediately cooling to room temperature, centrifuging at 10000g for 20min, and collecting supernatant to obtain crude solution;

thirdly, ultrafiltration membrane separation: separating the crude solution by adopting an ultrafiltration membrane with the molecular weight cutoff of 11KDa, collecting a permeated solution, separating and concentrating the permeated solution by adopting an ultrafiltration membrane with the molecular weight cutoff of 4KDa, and collecting an unpermeated solution to obtain a concentrated solution of the natural shrimp blackening preventing agent;

and fourthly, drying and pulverizing, namely freeze-drying the shrimp natural anti-blackening agent concentrated solution to prepare the shrimp natural anti-blackening agent finished product.

Example 4: application of natural shrimp blackening preventing agent in shrimp preservation

1) Dissolving the natural shrimp anti-blackening agent prepared in the example 1 in sterile water to prepare a 0.1% natural anti-blackening solution;

2) selecting fresh shrimps, cleaning, soaking in the natural anti-blackening solution at 0-4 deg.C for 30min

3) Fishing out the shrimps, placing the shrimps on a gauze for draining, and storing the drained shrimps in a low-temperature environment of 0-4 ℃.

The refrigerated shrimps which are not treated by the natural anti-blackening agent (fresh shrimps are directly refrigerated at 0-4 ℃ after being washed and drained) have blackening on the 5 th day, and the refrigerated shrimps treated by the natural anti-blackening agent in the embodiment 4 of the invention have blackening on the 15 th day.

Example 5: application of natural shrimp blackening preventing agent in shrimp preservation

1) Dissolving the natural shrimp anti-blackening agent prepared in the example 2 in sterile water to prepare a 0.3% natural anti-blackening solution;

2) selecting fresh shrimps, cleaning, soaking in the natural anti-blackening solution for 20min at 4 DEG C

3) Fishing out the shrimps, placing the shrimps on a gauze for draining, and storing the drained shrimps in a low-temperature environment of 0-4 ℃.

The refrigerated shrimps which are not treated by the natural anti-blackening agent (fresh shrimps are directly refrigerated at 0-4 ℃ after being washed and drained) have blackening on the 5 th day, and the refrigerated shrimps treated by the natural anti-blackening agent in the embodiment 5 of the invention have blackening on the 16 th day.

Example 6: application of natural shrimp blackening preventing agent in shrimp preservation

1) Dissolving the natural shrimp anti-blackening agent prepared in the example 3 in sterile water to prepare a 0.2% natural anti-blackening solution;

2) selecting fresh shrimps, cleaning, soaking in the natural anti-blackening solution for 25min at 4 DEG C

3) Fishing out the shrimps, placing the shrimps on a gauze for draining, and storing the drained shrimps in a low-temperature environment of 0-4 ℃.

The refrigerated shrimps which are not treated by the natural anti-blackening agent (fresh shrimps are directly refrigerated at 0-4 ℃ after being washed and drained) have blackening on the 5 th day, and the refrigerated shrimps treated by the embodiment 6 of the invention have blackening on the 16 th day.

Comparative example 1

Referring to example 1, except that soybeans are used for replacing mung beans, finished shrimp natural anti-blackening agents are obtained according to the same method and are applied to shrimp fresh-keeping treatment, the treatment method is the same as example 4, and the effect of the finished shrimp natural anti-blackening agents extracted from soybeans is equivalent to that of fresh shrimps which are directly refrigerated at 0-4 ℃ after being washed and drained, namely that the shrimps are refrigerated to become black on day 5.

Comparative example 2

The high-purity mung bean trypsin inhibitor is obtained by referring to CN 102295700A and is used for keeping shrimps fresh, and the method has multiple steps, long flow, high production cost and low product yield, so that the use cost is high, and the method has no practical significance as a shrimp preservative.

On the other hand, reagents such as sulfuric acid, alkali, Tris-HCl and the like are used in the preparation process of the method, and the residual reagents can extremely inhibit the fresh keeping of the prawns.

Finally, the method adopts NaCl gradient salt leaching, so that the salt content of the product is high, the taste of the shrimps is affected when the product is applied to the fresh keeping of the shrimps, and the freshness of the shrimps is reduced.

Therefore, the invention forms a set of simple and convenient processing method through crude extraction, heating and membrane treatment, simultaneously avoids introducing extra reagents, ensures that the activity of the current product is not lost by controlling the specific conditions of the steps, combines membrane separation to carry out high-efficiency separation and concentration, has low salt content of the product, and is very suitable for keeping shrimps fresh.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various features described in the above embodiments may be combined in any suitable manner without departing from the scope of the invention. The invention is not described in detail in order to avoid unnecessary repetition.

In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.