WO2004056980A1

WO2004056980A1 - Activating protease of oil grains and hydrolyzing vegetable proteins

Info

Publication number: WO2004056980A1
Application number: PCT/CN2003/001095
Authority: WO
Inventors: Lusheng Bao; Xuesong Chen
Original assignee: Beijing Food Research Institute
Priority date: 2002-12-23
Filing date: 2003-12-19
Publication date: 2004-07-08
Also published as: CN1212072C; AU2003292861A1; CN1509626A

Abstract

The invention relates to deep processing of vegetable proteins and vegetable protein products. It provides a process of activating protease which are contained in oil grains and hydrolyzing vegetable proteins, which is completely different from the conventional technology in activating and utilizing protease of the oil grains. The invention further provides a vegetable protein products produced by the processing, especially for instant soy milk powder as well as deodorized soy milk, not reducing high cost of using protease for hydrolyzing protein, but also removing the Soya-bean smell of soy milk etc., thereby improving the solubility of soy milk powder.

Description

Oil species activation of proteolytic enzymes and hydrolysis of plant proteins Technical field

The present invention relates to the field of deep processing of plant proteins and plant protein products, and in particular to the study of proteolytic enzymes contained in oil crops. The present invention also relates to a method for hydrolyzing plant protein in the seeds of oil crops, which adopts a process completely different from the prior art, does not use expensive protease preparations, but activates the oil crops themselves by appropriate Protease to complete the hydrolysis process. The invention also relates to products prepared by such a hydrolysis method, especially soy protein products. Background technique

Oilseed seeds are rich in protein and are the main source of plant protein essential for humans. The use of proteolytic enzymes in the process of plant protein processing and extraction can increase yields, change various physicochemical and biological properties of proteins, develop new products, and expand the scope of plant protein applications. This has been the consensus of the industry. The processing and extraction of plant proteins is a process of dissociating the protein macromolecules into small molecular substances. For example, by controlling in different hydrolysis stages, it can be partially decomposed into polypeptides, or completely hydrolyzed into small molecule amino acids. Among the three hydrolysis methods using acid, alkali, and enzyme, enzymatic hydrolysis is a relatively gentle and easy to control method. Therefore, when the hydrolysate is required to be a peptide, the enzymatic hydrolysis method is usually selected, that is, the appropriate protease preparation is used to make the protein molecules in It is cut into small molecules (peptides or amino acids) under certain conditions. Oil crops are the main source of plant protein. Proteolytic enzymes are widely used in deep processing and new product development. However, the cost of commercial protease preparations has limited the development of plant protein products. Cheap protease sources will have a profound impact on the development and application of plant proteins. Oilseed seeds are the main source of functional protein currently being developed. Plant physiology has inspired us that the germination process of seeds is the process in which various nutritional components (such as protein shields, sugars, and fats) are constantly broken down, transported to the growth point in the form of small molecules, and then synthesized into new tissues. Its sprouting The process is a process in which various enzymes are active. If these enzymes can be used in the food processing process, it will promote the technology of the food industry. Humans have invented a method for producing beer by using amylase in malt. Enzymes are possible. The invention is driven by this idea, and it is related to how to activate the protease.

On the other hand, the raw materials currently available for the preparation of plant proteins are generally low-temperature meals, or oilseeds are directly used, and high-temperature meals after high-temperature pressing are not used (the important use is feed). The method can activate the inactivated protease in the high-temperature meal, thus exploiting the utilization value of the high-temperature meal. Summary of the Invention

The inventors found that the proteolytic enzymes of oil crops can be activated under appropriate conditions under the conditions of the above-mentioned prior art. Therefore, a method for activating these proteolytic enzymes was proposed. It can replace the protease preparations currently used in industrial production for the hydrolysis of plant proteins.

On the basis of the above, the present inventor also proposed a method for hydrolyzing plant protein that is completely different from the traditional process, that is, the use of protease of the oil crop to hydrolyze the protein in the raw material, especially the activation and utilization of the oil crop The method of seed protease can reduce the high cost of proteolysis using protease preparation.

The invention also provides a plant protein product obtained by using the above hydrolysis method.

According to the first aspect of the present invention, a method for activating proteolytic enzymes in oil crop seeds is proposed, which comprises adding a slurry of proteolytic enzymes in which the proteolytic enzyme has been activated in advance as an activator to control ρΗ value is 6. 5 ~ 8. 5, keep stirring at 30-65 ° C.

The method of the present invention is applicable to seeds of various oil crops, including: soybeans, peanuts, oil seeds, sunflower seeds, cotton seeds, ¾fr, two, walnut kernel seeds, and the like.

The method of the present invention is also suitable for the cake meal of the above-mentioned raw material seeds after being pressed, including ordinary low-temperature meal and high-temperature meal currently mainly used as feed; the method of the present invention is also applicable to genetically modified products of these raw materials. Digestion of raw materials such as non-GMO and GMO soybeans.

According to the activation method of the present invention, it is first necessary to obtain an "activator" for starting an enzymatic hydrolysis reaction, which already contains a proteolytic enzyme that is activated by any feasible method. Under the action of the activator, the proteolysis in the raw material Enzymes can also be activated. Once the protease in the raw material is activated, it can be directly used as a new activator to activate the protease in the next batch of raw materials. In this way, the proteinase can be hydrolyzed by using the protease possessed by the raw material itself. It is necessary to additionally use protease preparations currently used in industrial production, which can reduce production costs.

According to a preferred solution of the present invention, the activator can be obtained in three ways:

-Exogenous protease activation method;

-Self-activation method;-

-Use any oil crop protease that has been activated to activate other oil crop proteases, ie, the cross-activation method.

1. Utilizing exogenous proteases, that is, a method for activating proteases in oilseed seeds and cakes using commercial protease preparations. The preferred activation method of the present invention further includes first preparing the activator: using a protease preparation under suitable activation (the temperature and threshold value required by different enzyme preparations are different, and only need to operate according to the requirements of the enzyme preparation product, Or just a little test) Maintain the stirring, observe the pH value of the reaction solution, add alkaline preparations to maintain the pH value when it starts to fall, or leave it to fall without controlling the pH value, and get the activator after 0.5 ~ 1.0 hours It can also be adjusted to neutrality by adding an alkaline agent, or as an activator without neutralization.

According to this preferred method, soybeans, peanuts, rapeseeds, sunflower seeds, cotton seeds, walnut kernels, sesame seeds, almonds and other raw materials are soaked for 0 to 60 r, ground into water to form a pulp, or the meal of the above-mentioned crops after oil extraction Add water to the slurry directly, add one or more protease preparations in a container with automatic stirring, such as plant proteases: papain, bromelain; animal proteases; pepsin, trypsin; various microbial proteases: subtilisin 1398, etc., at its optimal H value and temperature, symptomatic, appropriate alkaline agents can be used during the reaction, preferably Na, K, Ca, Mg hydroxide or carbonate or citrate to maintain the reaction The pH of the solution is stable. 5 ~ After the pH value starts to fall 1.0 hr (the protease in the seeds of the crop is already activated) is ready for subsequent operations.

The slurry activated by the above method can be used as an activator to be added to the next batch of raw material slurry, and the added amount is generally 1/1 ⁵ to 1/5 (weight) of the next batch of raw material slurry. That is, only the exogenous enzyme is used as the "activator" in the hydrolysis of the first batch of raw materials, and then the materials after each batch of activated hydrolysis reaction can be used as activators and passed on from generation to generation.

2. Self-activation method: Stir the raw material slurry at 30 ~ 65 ° C, pH value 5.5 ~ 8.5, and stir for 2 ~ 6 hours. The protease can be activated to obtain the activator, and the pH value during the It is maintained by adding i ± p to the alkaline agent.

According to this preferred method, oil crop seeds (soaked or not soaked) are ground into a slurry (soaked for 0 ~ 60 hr, or until germination), and cakes made by low temperature leaching can be directly ground into a slurry. Keep it in a container with automatic stirring at 30 ~ 65 ° C, keep stirring, and when the pH value starts to drop, add alkaline agents (such as Na, K, Ca, Mg lye or carbonate or citric acid) Salt), maintaining the pH between 6.5-8.5, the protease in the raw material starts to activate in about 2-6 hr. At this time, the hydrolysis reaction can be continued, or it can be added to the subsequent raw material slurry as an activator to achieve the same transfer.

3. Mutual (cross) activation method, that is, the raw materials of the activator and the subsequent raw materials are different types. Using exogenous enzyme activation or automatic activation method, the slurry of any activated oil crop or the supernatant of the activated slurry after removing protein by precipitation can be used as an activator to activate any other oil crop. Protease, for example, activated soybean slurry or its supernatant can activate the protease in peanut, rapeseed, cottonseed, sunflower and other crop seeds and cake pulp. Similarly, peanut slurry can also activate soybean, rapeseed, etc. Protease. It can be understood that when the protease of each crop activates and hydrolyzes the protease of other crops, the speed will be different, and the end point of action will also be different. Therefore, the optimal hydrolysis t and hydrolysis end point can be selected by matching.

Use the activator obtained by any of the above three activation methods as the "starting source", and then use a method similar to fermentation and inoculation, and use each batch of activated materials as "seed liquid" to access the next batch of materials. By activating the protease in the next batch, the production can continue stably. In addition to the slow start-up activation response, the subsequent batch reaction rate is fast and stable. "Seed liquid" The addition amount is generally 1/15 1/5 of the next batch of raw material slurry. In the production of Mo, each batch of raw materials has slightly different enzyme activity and activation time due to different varieties and storage periods. The proportion of the activator added in batches can adjust the reaction time and maintain a stable flow rate, which is easy for those skilled in the art.

When the protein is hydrolyzed under acid, alkali or alkaline conditions, it appears that a large amount of insoluble protein is converted into soluble protein. After a lot of experiments, the inventor found that the protein extraction rate of the raw material was significantly improved by operating according to the above method of the present invention, thereby proving that the protease of the raw material itself was activated and the protein was hydrolyzed. The specific experimental methods and results can be found in the following experiments. Example 1. When performing the proteolysis according to the preferred method of the present invention, the amount of the activator is 1/15 to 1/5 of the raw material slurry, and the time required to activate the protease in the raw material is about 0.5 to 1.5 hours.

The experimental results of the inventors also show that the method of the present invention has the same effect when applied to ordinary soybeans and genetically modified soybeans, see Real-Risk Example 4. The enzyme activation time of transgenic soybean is slightly shorter than that of non-transgenic soybean. Other raw materials have slightly different enzyme activities due to the origin and variety.

The inventor's experiments also show that by adding "activators" to activate proteases, in addition to low-temperature meals, proteases in high-temperature meals that are currently considered to be unsuitable as raw materials for plant protein production can also be activated to achieve protein hydrolysis. Real-risk example 2 and real example 5.

The invention successfully realizes the activation of the protease of the oil crop, which means that in the process of enzymatic hydrolysis of the plant protein, at least the use of a high-cost protease preparation is no longer required, and thus the production cost of the plant protein product can be greatly reduced. At the same time, the inventors also found that by activating the self-protease for hydrolysis, the protein extraction rate was significantly improved, and the unpleasant "beany smell" in soybeans disappeared; the original seeds of sesame, peanut, rapeseed, etc. The aroma, spicy odor decreases or disappears completely with hydrolysis.

Therefore, according to another aspect of the present invention, a method for activating a proteolytic enzyme of a raw material itself by using the above method to implement proteolysis is proposed. That is, the present invention provides a method for hydrolyzing vegetable protein, using oil crop seeds as a raw material, and using the method described above, firstly activating the protease contained in the raw material itself to make it an "activator" in the raw material hydrolysis reaction, Add to first batch In the raw material slurry, maintain a pH value of 3.0 to 8.5 under continuous stirring, preferably a pH value of 6.5 to 8.5, a temperature of 15 to 65 ° C, and preferably 30 to 65 ° C. When the pH value starts to drop, add Na, K, Ca , Mg hydroxide or carbonate or alkaline agent such as citrate, maintain the pH in the optimal range. After the enzymatic hydrolysis of plant protein is completed, a part of the hydrolyzed slurry is retained as the "activator" for the hydrolysis of the next batch of raw materials. The new batch of slurry also maintains a pH value of 3.0 to 8.5 and a temperature of 15 to 65 ° under constant stirring. C. When the pH value starts to drop, add Na, K, Ca, Mg hydrogen compounds or carbonates or alkaline agents such as citrate to maintain the pH in the optimal range. Hydrolysis can be maintained for 1.5 to 60 hr according to the purpose of processing. After the reaction is terminated, 1/15 1/5 is left as "activator", and a new batch of materials is added to continue the hydrolysis reaction. The rest of the hydrolyzed materials can be processed in the next step to extract proteins, peptides and other products. Repeating the hydrolysis reaction in this way allows the hydrolysis process to be passed on, and continuous production can be maintained.

According to the method of the present invention, the protein is extracted from the low-temperature meal. Compared with the traditional method of extracting water, the protein extraction rate can be improved by at least 8-12%, because the hydrolysis of the method of the present invention can make the original water-insoluble protein Hydrolyzed to soluble protein.

The method of the invention has no special requirements on the quality of water and does not require softening treatment.

As mentioned earlier, the test results found that the hydrolysis reaction using the method of the invention can: 1. remove the original odor of the seeds; 2. increase the protein extraction rate by 8 to 12 percentage points; 3. reduce the viscosity of the material, which is beneficial to In the subsequent pulp and slag separation process, the cylinder separation equipment can increase the concentration of the material after concentration. Through the above hydrolysis reaction, many physical and chemical properties of the protein are changed, and in particular, corresponding polypeptides can be generated, thereby providing a low-cost method for producing a plant protein polypeptide. It is worth noting that the polypeptide obtained by the method of the present invention significantly reduces or even eliminates the bitterness caused by the conventional method, and has various beneficial effects on plant protein processing.

Therefore, by using the hydrolysis method provided by the present invention, it is possible to prepare plant protein products that are currently available or have not yet been developed, such as preparing plant protein polypeptides without bitter taste.

On the basis of the above technology, the invention ¾ found that the solution method of the invention can be used to produce instant soy milk powder and soy milk products with good solubility, better flavor and taste.

The instant soybean milk powder of the present invention basically uses soybean as the main material, so that the ground slurry can be used in soybeans. Hydrolysis occurs under the action of the soybean protease contained in it, and the hydrolyzed soybean milk is subjected to enzyme inactivation and spray drying, wherein the hydrolysis is achieved by using the aforementioned method for hydrolyzing plant proteins. When the instant soybean milk powder is manufactured by the method of the present invention, after the hydrolysis reaction is completed, an appropriate amount of soybean paste is retained and added to the next batch of materials as

"Activator", as mentioned in the previous paragraph 4, the amount of "activator" may be 1/15 to 1/5 of the raw material (based on the slurry after refining), and may preferably be 1/10 of the amount of raw material. . The hydrolysis reaction time of each batch of raw materials is preferably in the range of 1 to 2.5 hours. The soybean paste that has completed the hydrolysis reaction is separated to remove residues, so that the H value of the soybean milk obtained by filtration is reduced to the range of 7. 5-5. 6 and the enzyme is immediately heated to destroy . In addition to inactivating various harmful enzymes, the inactivation process also includes inactivating activated proteases. Enzyme can be boiled for 5 minutes at normal pressure; the enzyme can also be killed at 121 ° C for 30 seconds under high pressure. It is then spray-dried and can be concentrated and spray-dried if necessary. Spray drying conditions are the same as traditional conditions. The concentration operation can be concentrated in a vacuum by a usual method.

In the above process, it was found that the hydrolyzed bean dregs were loose and dry and easy to filter. With this hydrolysis method, the solids concentration of the concentrate can reach more than 30%, which is conducive to improving the spray drying efficiency, and can improve the bulk density and dispersion of the finished product. These are all unattainable in the prior art.

According to the method of the present invention, the degree of hydrolysis has a significant effect on the solubility of soy milk powder. By selecting an appropriate degree of hydrolysis, the soy milk powder can almost achieve the effect of dissolving the milk powder. This true dissolution is fundamentally different from the visual "dissolution" often achieved in soy milk powder production by adding sugar to increase the water content. Moreover, there is no bitterness caused by hydrolysis of exogenous enzyme preparations using soybean protease. The degree of hydrolysis can be controlled by 5 watts. If calculated as NaOH, the alkali consumption should be controlled within the range of 0 to 3. 0% of the weight of soybeans. Both can improve the solubility of soybean milk powder. In general, the degree of hydrolysis is high, the amount is large, the solubility is good, and the polypeptide content in the product is high, but the product flavor and taste will be different.

When the activated peanut paste is used as an "activator" and added to the soybean raw material slurry, or when the "activator" is used, the peanut proteinase is divided into ^, and peanut milk powder with a more unique flavor can be obtained.

In addition to producing soy milk powder with good instant solubility, the inventors have also discovered that using the protease-activated and hydrolyzed vegetable protein technology of the present invention can produce a soy milk product with excellent deodorization effect. That is, the present invention also provides a deodorized soybean milk obtained by using soybean as a main ingredient and hydrolyzing soybean slurry by the above method. In the process of producing soy milk by using the method of the present invention, 6 to 8 · times water is added after the soybean absorbs water and is saturated. Grind into soybean paste, add the "activator" made by any of the methods described above at a ratio of 1/15 1/5, and react under the conditions of 30 ~ 65 ° C, pH value 6.5 ~ 8.5, and vigorous stirring The reaction is completed in about 45 to 90 minutes, and the amount of machinery is about 0.5 to 1.0% of the weight of soybean. After the reaction is completed, 1/15 to 1/5, preferably 1/10, is left in the reaction tank as the next batch of raw materials. The "activator", the rest are processed into deodorized soybean milk products by heating (95 ~ 10 (TC, 5 minutes or 121 ° C, 30 seconds), homogenization, flavoring, etc .. The purpose of the enzyme inactivation is in addition to inactivating urine In addition to harmful enzymes such as enzymes and antitrypsin, activated proteases must be inactivated, otherwise excessive hydrolysis will also produce a series of negative effects.

In the above process, soybean milk can be used for hydrolysis after the residue has been separated, or soybean paste can be directly used for hydrolysis first, and then the residue is separated and removed.

There is also a kind of sour milk that is very popular with consumers in the soy drink market, but it also has a flavor similar to fermented tofu that is difficult for some consumers to accept. This has become the biggest obstacle to the sour milk market. The deodorized soybean milk produced by the present invention is inoculated and fermented in accordance with a conventional method, and the sour milk obtained has no such odor, and the flavor and taste of the sour milk are greatly improved.

In summary, it can be seen that the key of the present invention is to propose a plant protein processing idea that is completely different from the prior art, that is, to try to activate the proteolytic enzyme contained in the raw material itself, and then to play a role in the autoprotease. Hydrolysis reaction occurred. Compared with the records and uses of the prior art, the present invention stands at a completely new angle to study the physiological characteristics of oil crops, and successfully applies this result to the deep processing of plant proteins, which not only saves a large number of enzyme preparations, but also It greatly reduces the traditional process flow and supporting equipment, thereby reducing investment and production costs, and at the same time solving difficult problems such as soybean deodorization and solubility of soybean milk powder that have been difficult to solve for many years with traditional processing ideas. Therefore, the inventors are convinced that the implementation and promotion of the present invention will open up a broad new space for the research and processing of plant proteins, especially the theoretical research of soybeans and the production and development of products. Specific implementation plan

The following describes in detail the implementation process of the present invention and the unexpected and obvious obvious effects brought by the specific tests and examples, in order to help readers better understand and appreciate the essence of the present invention. It is innovative and innovative, but it cannot constitute any limitation on the implementable scope of the present invention. Any modification and modification to the technical solution of the present invention made by those skilled in the art under the teaching and inspiration of this specification shall belong to the present The protection scope of the invention claims.

Explanation: In the following experimental examples of the present invention,

"Solid matter extraction rate" = solids content in slurry / total amount of raw materials X 100%;

"Protein extraction rate" = protein content in the slurry / total protein content in the raw material X 100% (here the protein extraction rate only represents the effect under the production process conditions, and does not represent the NSI value of soybean protein)

'' Experimental example 1.

100 g of soybeans were saturated with water by soaking and ground into 800 ml of soybean paste. 0.1 g of papain (600,000 viable units / g) was added, kept at 42 ° C for constant stirring, and continuously added dropwise with a 0.1 N NaOH solution to maintain its _PH value stable at 7.0 to 7. 2. The reaction was stopped when ^? Wei solution reached 50 ml. Take 100 ml and add it to the next batch of the same soybean paste, and react under the same conditions. After that, 100 ml of the finished soybean paste was taken as an "inoculation solution" each time and added to the next batch, and a total of 12 batches of delivery reactions were performed.

After each batch of soybean paste, soybean dregs were separately removed, and the protein and solid content in the soybean milk were determined. Compared with the non-hydrolysis reaction. The results are shown in Table 1:

Table 1

As can be seen from the results in the table above: When the reaction proceeds to the twelfth batch, solids and protein are extracted The rate did not decrease, indicating that the enzyme activity was stable, and the added papaya protein ^ ί 10 " ¹² g left, it is impossible to maintain the same enzyme activity, so it is presumed that the protein alcohol present in soybean must be activated and the effect Experimental example 2. Effect of protease auto-activated hydrolysis on protein extraction rate in low temperature soybean meal

100g of low temperature soybean meal and 9 times the weight of tap water are ground into soybean paste, and the same amount of soybean paste is separately processed as follows:.

1. Incubate at 5ITC and continuously stir for 1 hour;

2.Adjust the pH value to 8. 0, 50 ° C, keep stirring, and continuously stir for 1 hour;

3. Keep the temperature at 50 ° C and keep stirring. When the pH value starts to drop, add 0. IN NaOH solution dropwise to maintain the pH value of 7. 0 7. 2. The pH value will not drop for about 4.5 hours.

The above three groups of samples were separated and removed by the same method, and the protein extraction rate (protein in the extract / protein in soybean meal) and the pH value of 4.5 ^ soluble protein in the supernatant after the precipitation of bovine acid protein (Kelvin) Determination of nitrogen method), the results are shown in Table 2:

Table 2

It can be seen from Table 2 that the protein extraction rate of the automatic hydrolysis method is much higher than that of the other two methods, and the soluble protein is also significantly improved under the condition of pH 4.5, and the increased part is the soluble polysaccharide produced by hydrolysis. Experimental example 3. Experiment of automatic hydrolysis and passage hydrolysis of peanut

Soak 100 g peanuts overnight with 850 ml tap water. The temperature was kept constant at 50 ° C. After 4 hours, the pH value began to decrease, and the solution was neutralized with a 0.1 IN NaOH solution to maintain the pH value of 7.0 to 7. 5 and the reaction was stopped when the liquid reached 50 ml after 1.5 hours. . Add 100 ml to the next batch of the same peanut paste and continue the reaction at the same temperature. Five batches were transferred in this manner, and the protein extraction rate (extracted protein / peanut, W / W) was measured. Reaction time

Extraction protein extraction rate H value 4.5 soluble protein

(Consumption 50 ml of alkali

Unhydrolyzed extraction 24.2% 2.8%

Automatic hydrolysis 5 hr 30 min 28. 6% 7. 4% Second generation 2 hr 10 rain 27. 9% 7. 5%

3rd generation 1 hr 33 min 28.1% 7.2%

4th generation 1 hr 34 min 28.3% 7.3%

Fifth generation 1 hr 40 min 27. 7% 7.1%

λ 3 It can be seen that the proteolytic enzymes in peanuts can be automatically activated, and can transfer and activate the proteases in the next batch of materials. After hydrolysis, the protein extraction rate is significantly improved. After acid precipitation, the soluble protein increased significantly. Example! ^ Example 4. Protease automatic activation and passage activation of transgenic soybeans

The GM soybean imported from the United States was directly crushed and passed through a 60 mesh screen. Take 100 g of this soybean flour and 900 ml of tap water and mix to form a suspension. Adjust the pH to 7. 0 ~ 7. 5 with 0.1 N NaOH and stir continuously in a water bath at 50 ~ 60 ° C. After 2 hr 30 min, the pH value began to decrease, and the reaction was continued for 1 hr 45 min. When the alkaline solution reached 30 ml, it stopped. Take 100 ml of this milky product: ^ Orally put in the second portion of the same soybean flour suspension, and process again according to the above conditions. The reaction was stopped when the amount reached 30 ml in 2 hr 15min.

Both samples were separated and removed in time, and the solids and protein extraction rates were measured, compared with the extraction rates of soy beans, refining, and separation according to the conventional method. Calculated based on solids / soybean weight, extracted protein / total protein in soybean, the results are shown in Table 4:

Table 4

The results in Table 4 indicate that transgenic soybean proteases can also be activated automatically and can be passaged Activated, and it shows that unsoaked, directly crushed soybean flour can also achieve the purpose of activating protease. Then the conventional method and the first-generation transgenic soybean hydrolyzed soybean milk were vacuum concentrated to measure the solids concentration and viscosity, respectively. The results are shown in Table 5:

table 5

Experimental Example 5. Hydrolysis of high-temperature soybean meal and boiling enzyme

100 g of high-temperature defatted soybean meal was soaked and softened, and then ground into soybean paste; 100 g of soybeans were soaked for 8 hr, ground into soybean paste, and boiled for 5 minutes, and then cooled to normal temperature. Both samples were adjusted to a pH of about 7.0, and kept stirring in a 55 ° C water bath to observe the change in pH. After 8 hr, both the _P H value no changes (at any time during make-up water to maintain the original concentration), to each of the extracted isolated lOOtnl slag bean extract, which is measured with a solid content rate of protein extraction. Then add 100 ml of activated "activator" of the same concentration, and continue the reaction under the same conditions. The pH value began to drop quickly, and 4% Na ₂ C 0 ₃ solution was continuously added to maintain H stable at 7.5, and the reaction was stopped when 50ml of each alkaline solution was consumed, and the soybean milk obtained by deslagging was used to measure the solids and protein extraction rates, respectively. The results are shown in Table 6:

Table 6

This test shows that high-temperature soybean meal and heated boiled soybean paste have no protease activity and are difficult to automatically activate, but can still be activated after adding an activated soy protease "seed solution". However, compared with low-temperature soybean meal (also time-consuming), the solid and protein extraction rates were significantly reduced; the boiling soybean paste extraction rate did not change significantly (compared to Table 1). Experimental example 6 Protease autoactivation in seeds of various oil species

Ear flower kernel, ¾ ^ kernel, rapeseed (mustard type), kernel ^ 100g each add 900ml tap water and mashed with tissue ^ machine into a thin paste, all of which are white emulsion. And with 0.1 IN NaOH to adjust the pH to about 7.5, keep warm and stir in a 5 (TC water bath, when the pH value drops, add lye to maintain the pH value of 7.5. When the emulsion is reduced due to water evaporation, make up water to maintain The volume of the material liquid. Record the time when the pH value starts to fall. The time it takes for the pH value to stop changing, and the amount of power. Observe the phenomenon. Table 7:

Table 7

In the table: 7j solution time = time from pH value drop to pH stop change.

The enzymes of the above several crop seeds have vitality and can be automatically activated, but the activity and hydrolysis reaction are far lower than those of soybean and peanut. Another common phenomenon is that after hydrolysis, the original aroma and spicy odor of the seeds are significantly reduced or even completely disappeared. The sunflower seed was hydrolyzed, and the protein precipitated ^^ Qing-^ ^ showed a dark green color of chlorogenic acid. It was shown that proteases can be activated in the seeds of the above several oilseeds. Experimental Example 7 Control test of solubility of soybean milk powder produced by the method of the present invention and commercially available soybean milk powder Take 10.0 g of each of the soybean milk powder produced by the method of the present invention and a commercially available sample, add 80 ml of warm water at 50 ° C, and stir. Incubate in a water bath at 50 ° C for 10 min. After centrifugation (3000 rpm, 10 min), discard the upper solution, and dry the precipitate and centrifuge tube to constant weight (105 Ό). Calculate the proportion of insoluble matter in the sample and compare the solubility of each sample. See Table 8.

The commercial products A and B in the table are 60% in quantity and have milk ingredients (package labeling). Two samples of the present invention (hydrolyzed and unhydrolyzed soy milk powder) were added without soluble ingredients such as sugar and milk.

Table 8

Protein content in soy milk (milk) powder

Unhydrolyzed soy milk powder 46.3% 92.3%

Hydrolyzed soybean milk powder 46.3%-50% 2.1%

Commercial soy milk powder A> 16% (package label) 2. 0%

Commercial soy milk powder B> 16% (package label) 33.1% Experimental Example 8: Comparison of Soy Milk Soy Flavors Made by Automatically Activated Protease Hydrolysis

Take 2 Kg of soybeans and soak overnight, grind it with 16 Kg of tap water with a grinding wheel to make bean paste, warm to 55 ° (and stir with an electric mixer. Weigh 12 g of NaOH and 10 g of Ca (0H) ₂ and 400 ml of water to form a suspension. Use this suspension to adjust the pH value of soybean paste to 7.5. When the pH value starts to drop, continue to add the mixed lye to maintain the pH basically unchanged. After the lye is added and the pH drops to 7.0, use immediately The centrifuge was separated and removed by a small centrifuge, and the obtained soybean milk was immediately boiled for 5 min to obtain 18 Kg of soybean milk (protein concentration 3.52%). 45 Og of sucrose was added and homogenized (20/5 MPa).

Soak with 2 Kg of soybean overnight, add 14 Kg of tap water to the same mill, and directly separate soybean milk 15.'5 (protein concentration 3.45%), add 775g of sucrose, and homogenize after boiling. This slurry was used as a control sample.

The sweetness of these two batches is similar.

A certain brand of soy milk was purchased from a supermarket (produced for an imported production line). The process is: peeling, alkaline dry refining, vacuum flash deodorization, and protein content> 3.5 ° /. .

Doudou than tasting method, 12 participants (6 male and 6 female). The results are shown in Table 9:

Table 9

Example 1

lKg soybeans are soaked in tap water (temperature below 60 ° C, time 4 ~ 60 hr, can be arbitrarily combined), after water absorption reaches saturation, force ~ 8 Kg 7j to grind soybean paste, add bromelain according to its optimum pH value Perform the enzymatic hydrolysis reaction under the optimal temperature conditions, and add a mixed solution consisting of k NaOH and 2% Ca (OH) _{2 to} maintain a stable pH value. When the amount of soybeans is 0.5 ~ 1.0% by weight, the final 4L reaction will be completed, and this batch of material will be used as the enzyme activator for the next batch of soybean paste. The above process can also use any other protease preparation, such as trypsin, papain, 1398 and the like.

5 Kg of soybeans are soaked and ground into soybean paste under the same conditions as above, and heated to 30-65 V in a container with an electric stirrer, using the same ¾ ^ solution as a neutralizer, and the soybean paste H value is 6. 5 8.5, add the first batch of soybean paste (activator) hydrolyzed by protease, start the agitator to perform the enzymatic hydrolysis reaction, and when the pH value starts to drop, add the above lye to maintain the pH value unchanged. According to the purpose of processing Different, the enzymatic hydrolysis reaction time can be controlled from 1 to 30 hr. After termination, take 1/10 of this batch of soybean paste and add the next batch of soybean paste to continue the same reaction. This can be continued continuously, and continuous production can be formed in industrial production. In addition to using one batch of enzyme preparations, each batch of raw materials can use the previous batch of enzymatic hydrolysis reaction solution as an activator.

After the enzymolysis reaction, the viscosity of soybean paste is reduced, which facilitates the separation of pulp and dregs. It can be separated in one go by ordinary centrifugal tamping machine .: Dried soybean dregs, the moisture content of soybean dregs is 80%, and the viscosity of soybean milk is greatly reduced by 4 氐, which can be concentrated. It maintains good fluidity with a solid content of 18 to 26% and a viscosity of less than 200 cp. The viscosity of the reference soymilk solids reached 14 to 16% when the viscosity was hundreds of cp. This method can also improve the solids and protein extraction rate. The solids extraction rate (for soybeans) can reach 58 to 63% (the control sample is 48 to 52%), and the protein extraction rate (for soybeans) can reach 30-36% ( The control sample is 24 to 26%), and a series of changes have occurred in the properties of the extracted soybean protein shield. 25 to 30% of the total extracted protein can be converted to acidic conditions; Example 2

5 ~ 8 1 Kg of soybeans after soaking (soaking conditions are the same as in Example 1) or directly adding water to soy paste without soaking (the dryness of the soybean paste shall be iiJ 60 ~ 100 mesh). Within the range of 5 under the electric stirring, keep the temperature 30 ~ 65. C. Perform an automatic enzymatic hydrolysis reaction. After 2.5 to 6 hr, the pH value of soybean paste began to decrease. The alkaline solution in Example 1 was used as a neutralizing agent, and the H value was maintained dropwise. The reaction stops when the amount of energy reaches about 1.0% of the weight of the soybean.

Take 10 Kg of soybeans and grind them into soybean paste with or without soaking. Add the soybean paste with the above enzymolysis reaction and carry out the enzymolysis reaction under the same conditions of the first batch. Depending on the purpose of the enzymatic hydrolysis, the reaction time is controlled from 1 to 30 hr. Then take 1/10 soybean paste and add the next batch of soybean paste to carry out the same reaction, so that it can continue continuously. Except The activation period of the first batch of reactions is about 2 to 6 hr, and the activation period of each batch of materials in the future is about 1 hr.

The separation effect and extraction rate of the soybean paste digested by this method are the same as in Example 1.

From the second batch of materials, the bean paste can be unheated, or it can be cooled to 30 ~ 65 ° C after boiling, and the enzymes can be activated again. The enzymatic hydrolysis reaction can be performed in soybean paste or in separated soybean milk. The obtained soy milk has lost the original smell of soy, and has produced flavor and sweetness. It becomes deodorized soy milk, and then concentrated and spray-dried to obtain a soy milk powder with good solubility (the soy milk powder is not added with milk and sugar, etc. Ingredients), its solubility can reach the level of sugary soy milk powder currently on the market.

Example 3

The hulled and red-covered peanuts are ground into a paste by soaking or without soaking and adding water, and the same enzymolysis reaction is performed according to the methods of Examples 1 and 2, respectively, and the same effect can be obtained. The reaction process, temperature and H value are the same as those in Examples 1 and 2.

Peanut paste reaction can also use mash-activated soybean paste or separated beans as the "activator". The reaction effect and reaction speed are not affected.

If soybeans are activated with activated peanut paste, the enzymatic hydrolysis of soybeans will be faster.

The enzymatic hydrolysis reaction in peanut paste is carried out until the pH value no longer drops, and the pH value is adjusted to 4.5, and the centrifugal separation can separate oil, acid precipitated protein and soluble protein, that is, this method can form three extractions at a time Methods of main ingredients. Example 4

Low temperature soybean meal or peanut meal produced by low temperature leaching method, milled with water to make a paste, the same as in Examples 1, 2 ^ cattle reaction: temperature 30 ~ 65 ° C, pH value 6. 5 ~ 8. 5, uninterrupted stirring , Automatic activation time is 3.5 to 6 hr; activator activation time is 0.5 to 1 hr.

After the low temperature soybean meal and peanut meal are digested by the method of the present invention, the protein extraction rate is increased by 8-12 percentage points, the viscosity is reduced, and the solids concentration after concentration is up to 36%, and other degradation products can be obtained for many months by controlling the reaction time . The isolated protein made by this method has better solubility. Example 5

The rapeseed and its low-temperature meal were subjected to the same enzymatic hydrolysis reaction as described in Example 1, 2, and 4. The results of the reaction, in addition to the increased protein extraction rate, reduced the content of glucosinolate in rapeseed and the content of mustard essential oil produced by the reaction. With the extension of the reaction time, the spicy taste decreases obviously until it disappears.

Example 6

1 Kg of soybeans were soaked with ordinary tap water until it became saturated with water, and the weight was increased to 2.3 Kg, then 6 liters of tap water was added, ground into a bean paste with a small grinding wheel, transferred to a stainless steel barrel with a stirring and holding device, and the stirrer was started. And holding at 45 ~ 55 ° C, the initial pH of soybean paste was 6.84, H value began to decrease after 3 hr 20 min, when the pH of soybean paste dropped to about 6.4, the residue was separated to obtain soybean milk 7. 5 Liter, 7.4% solids concentration, immediately heated to boiling and maintained for 5 minutes, homogeneous (20 / 5MPa), concentrated in batches using a vacuum thin-film evaporator, the solids concentration was 14%, and then dried with a small spray dryer ( Inlet air temperature 195 Outlet temperature 78 ~ 82 ° C, centrifuge 12000rpm), 475 g of soybean flour was obtained. This soybean flour can be completely dissolved in warm water at 50 ° C, no precipitates and floating matters are formed, and a small amount is not completely dissolved under the microscope Of debris.

Example 7

2.0 Kg of soybeans soaked in tap water at room temperature for 14 hr and 300 g of red-covered peanuts soaked under the same conditions were ground together with 6 liters of tap water to form a paste, which was stirred and hydrolyzed at 50 ° C in the same hydrolysis reaction apparatus as in Example 1. 75。 The initial pH value of the material 6.75. After about 2 hr 45 min, the pH value started to drop. Use a mixed lye (NaOH: Ca (OH) ₂ = 1: 1, mixed concentration 5%) dropwise to maintain the same pH. When the amount of alkali consumed is 10 g, stop dropping the alkali, continue the reaction for about 45rain, take out 90% of the material and immediately separate the residue, and boil for 5 rain to destroy the enzyme. Leave 1/10 of the material in the tank and add a batch of the same soybean and peanut paste to continue the reaction. After 30 minutes, the pH value starts to drop. Immediately add the same mixed lye to maintain the pH value. After 10 g of mixed alkali is added, continue to react for 30 minutes. Also take 90% of the material to separate the slag and the remaining 10% for the next batch of reactions. This process can be repeated. The reaction time from the second batch of materials is about 1 hr 20 mm (this time may vary depending on the raw materials). The pulp obtained in each batch was processed according to the method of Example 6. Get soybean flour. This soy flour can be completely dissolved in warm water at 50 ° C. The appearance is similar to fresh soymilk, no beany smell, no floating matter and precipitation.

Example 8

Soak soybeans in 2.3 Kg for 14 hr, grind them into soybean paste with 7 liters of tap water and add the activated soybean paste. Perform hydrolysis reaction at 5 ° C under electric stirring, and use 4% Na0H solution dropwise to maintain pH 7.5. When the amount of salt reaches 750 ml, continue the reaction for 10-30 minutes, and remove the residue. About 9 liters of soybean milk is obtained, 10 g of emulsifier (monoglyceride, sucrose ester, etc.) is added, mixed and boiled for 5 minutes to kill the enzyme, and then Homogeneous (20 / 5.0 MPa) to obtain delicate and bitter-free soy milk. Concentrated in vacuum and spray-dried to obtain soy milk powder. This soy milk powder is soluble in cold water, warm water, and hot water. After dissolution, the appearance is as delicate as milk, visually without The undissolved particles float and sink, the particles are not seen under the microscope, and the taste is not bitter from the hydrolysis of the protease preparation. Example 9

Soak 100 g soybeans at room temperature for 8 ~ 14 hr, add 800ml tap water to grind soy paste, heat in a water bath at about '42 ° C, adjust the pH value to about 7.0 with 0.1 M NaOH, electric stirring (100-150 rpm), add 0 lg Bromelain or papain (600,000 units / g), gradually dropping the p NaOH solution to maintain 7.0, after about 2 ~ 2.5 hr, when the consumption of NaOH solution reaches 15 ~ 25 ml, the pH value drops rapidly, indicating that soybean The protease has been activated and this batch of soybean paste is used as an "activator".

Another 1000 g of soybeans were soaked (room temperature, 8 ~ 14 hr), and 7 liters of tap water was added to grind the soybean paste, and the above-mentioned hydrolyzed soybean paste was added, kept at 42 ° C and stirred, and the G.1M NaOH solution was continuously added dropwise. Maintaining pH 7.0, after about 1 ~ 1.5 hours, salty amount 150 ~ 200ml, the reaction was stopped, and 90% soybean paste was taken out for separation and residue removal. 8500ml of soybean milk was obtained, with a solid concentration of 7.3% and a protein concentration of 3. '4 ₀ The resulting soybean milk was naturally cooled after boiling for 3 minutes. Add 210g of sucrose (sucrose concentration 2.5%) and dissolve it homogeneously (20 ~ 25 / 5.0 Mpa) to obtain a fishy taste-free soy milk with a sweetness equivalent to 5% sucrose ordinary soy milk. Degree increased.

1000 ml of soybean paste remain in the reaction tank, and then add 1 Kg of soybean paste to the same concentration of soybean paste, Follow the same reaction again, stop the reaction after 1 hr, and perform the same processes as slag removal and boiling. Leave 1000 ml of soybean paste in the reaction tank as "activator" and add new soybean paste. Repeatedly, a continuous production can be formed.

Example 10

1 Kg of soybeans were soaked (room temperature, 8 ~ 14 hr), 8000 ml of water was ground into soybean paste, kept at 50 C, stirred with an electric stirrer, and added dropwise with NaOH solution (4% concentration) to a pH value of 7.0. After 3.5 hours, the pH of soybean paste began to drop, and the aOH solution was added dropwise to stabilize the pH at 7.0, and then the NaOH amount was 200 ml over about 45 minutes to stop the reaction. Take out about 9000 ml of soybean paste to remove residue, inactivate enzymes, and homogenize to obtain deodorized soybean milk equivalent to 2.5% sucrose sweetness. The remaining 1000 ml of the hydrolyzed soybean paste in the reaction container was used as an "activator", and then added 2.3 Kg of soaked soybeans and 8000 mj of ground soybean paste. The hydrolysis reaction was continued in the same manner, and it took ¾011 to 200 ml. At this time, the reaction was stopped. This batch of hydrolysis reaction took 1 hr.

The same effect can be achieved by repeating the above operations, except that the first batch of reaction takes about 4 hr, and the second batch takes about 1 hr afterwards, and the deodorization effect can be achieved. Example 11

The soybean paste prepared according to the methods of Examples 9 and 10 is heated to boil to destroy the enzyme, and the temperature is reduced to 55 ° C. Then, 1/10 of any batch of soybean paste that has been hydrolyzed in Examples 9 and 10 is added as an "activator". Performing the hydrolysis reaction under the same conditions as above can achieve the same effect: removing the fishy smell and improving the yield of protein and solids. The disadvantage is that the enzymatic soybean paste is boiled, and the time for proteol to be "activated" again is extended from 1 hr to 2.5 hr, indicating that the inactive soybean protease can be activated again. Example 12

900 g of soybeans and 100 g of unred-skinned peanuts. According to the method of Example 10 and operating under the same conditions, deodorized soy milk with peanut flavor can be obtained. The other effects are exactly the same as in Example 10, except that the hydrolysis reaction rate and enzyme activation rate are the same. Both are faster than using soy alone, which is beneficial to shorten the production cycle. The aroma of peanuts weakens more slowly than the removal of beany smell. Therefore, peanut flavored soy milk can be obtained. Example 13

At present, in the production of various fermented soy milk, no matter what kind of bacteria is used and the addition of various additives, the smell of sour milk similar to the fermentation of tofu cannot be removed, which has become the biggest obstacle to the development of sour milk. The method of the present invention can prepare soymilk raw soymilk.

Using any of the methods of Examples 10, 11, 12 to adjust the degree of hydrolysis of the hydrolysis reaction, the salty amount reached between 1 to 2% of the weight of the soybean, and the pH was terminated when the pH dropped to 6. 5-7. 0, Immediately heat and inactivate the enzyme and homogenize, and then ferment according to the conventional inoculation method to remove odors and improve the taste and taste of sour milk.

Claims

Claim

1. A method for activating a proteolytic enzyme in an oil crop seed, which comprises adding a proteolytic enzyme in which a proteolytic enzyme has been activated in advance as an activator to control a pH of the raw material slurry at 6. 5-8 5. Keep stirring at a temperature of 30 to 65 ° C.

2. The method according to claim 1, wherein the oil crops include: soybeans, peanuts, rapeseeds, sunflower seeds, cotton seeds, sesame, almond, and walnut kernel seeds; cakes of these raw seeds after oil extraction Meal; genetically modified products of these ingredients.

3. The method according to claim 1, further comprising first preparing the activator: using a protease preparation to maintain agitation under appropriate activation conditions, and observing a decrease in the pH value of the reaction solution, 0.5 to 1.0 hours later The activator is obtained.

4. The method according to claim 3, wherein the protease preparations used include animal protease, plant protease, and microbial protease preparation.

5. The method according to claim 1, further comprising first preparing the activator: stirring the raw material slurry at a temperature of 30 to 65 ° C and a pH of 6. 5 to 8. 5 for 2 to 6 hours.

6. The method according to claim 3 or 5, in the process of preparing the activator, observe that the pH value of the anti-symptomatic solution starts to decrease and add an alkaline agent to maintain the pH of the reaction solution.

7. The method according to claim 1, wherein the activator can be derived from an oil crop different from the raw material.

8. The method according to any one of claims 1 to 5, wherein the added amount of the activator is 1/15 to 1/5 of the weight of the raw material slurry, and the time required to activate the proteolytic enzyme in the raw material slurry is 0. . 5 ~ 1.5 hours.

9. The method according to claim 6, wherein the alkaline agent is selected from the group consisting of sodium, bell, calcium or magnesium hydroxide, carbonate or citrate.

10. A method for hydrolyzing vegetable protein, using oil crop seeds as a raw material, using the method according to any one of claims 1 to 5, firstly activating a proteolytic enzyme contained in the raw material itself, so that the raw material is subjected to the action of the proteolytic enzyme The hydrolysis reaction occurs, the stirring process is maintained with stirring, the temperature is controlled from 15 to 65 'C, the pH value is controlled from 3.0 to 8.5, and the hydrolysis time is from 1.5 to 60 hours.

11. The method of claim 10, comprising: using any protease preparation to first hydrolyze the raw material spring, observe that the pH value of the reaction solution begins to decrease, and then obtain the activator after 0.5 to 1.0 hours, and then The activator is added to the first batch of raw material slurry to be hydrolyzed to perform an enzymatic hydrolysis reaction. After the enzymatic hydrolysis reaction is completed, a part of the hydrolyzed slurry is retained as the next batch of raw material enzymatic activator, so that the hydrolysis process is passed on.

12. The method according to claim 11, wherein the protease preparations used include animal proteinases, plant proteases, and microbial protease preparations.

13. The method according to claim 10, comprising: stirring the raw material slurry under the conditions of 30 to 65 ° C and a pH value of 5.5 to 8.5 for 2 to 6 hours, during which the pH value is added by adding an alkaline agent To maintain the plant's own protease, and then use the hydrolyzed material as an activator to add the first batch of raw material slurry to be hydrolyzed for enzymatic hydrolysis reaction. After the enzymatic hydrolysis reaction is completed, a part of the hydrolyzed slurry is retained as the next The batch of raw material enzymatic hydrolysis activator allows the hydrolysis process to be passed on.

14. The method according to any one of claims 10 to 13, wherein the kinds of raw materials for preparing the activator slurry and the kinds of raw materials used for hydrolysis may be different.

15. The method according to claim 13, wherein the alkaline agent is selected from the group consisting of hydroxide, carbonate or citrate of sodium, potassium, calcium or magnesium.

16. The method according to claim 11 or 13, wherein the added amount of the activator is 1/15 to 1/5 of the weight of the subsequent raw material slurry.

17. A vegetable protein product prepared by the method according to any one of claims 10 to 16.

18. The plant protein product according to claim 17, which is a plant protein polypeptide or an isolated protein.

19. An instant soybean milk powder, which is mainly made of soybeans, and is prepared by hydrolyzing the raw materials by the method according to any one of claims 10-16.

20. The method for producing instant soymilk powder according to claim 19, comprising hydrolyzing the refined pulp under the action of a soy protein hydrolase enzyme contained in the soybean itself, wherein the hydrolysis process is performed by using the methods in claims 10-16 Either method is completed, and the hydrolyzed soybean milk is subjected to enzyme inactivation and spray drying.

21. The manufacturing method according to claim 20, wherein an alkali can be added to control the pH during the hydrolysis, and the hydrolysis reaction is completed when the salt content is 0 to 3.0% by weight of the raw material.

22. A soy milk, which is basically made of soybeans, and is made from soybean milk obtained by hydrolyzing soybean slurry by the method of any one of claims 10-16.

23. The method for producing soy milk according to claim 22, comprising saturating soybeans with water absorption and grinding 6 to 8 times water to grind soybean paste, and hydrolysis occurs under the action of soybean protease contained in the soybean itself, wherein the hydrolysis process uses claim 10 The method of any one of ~ 16 is completed, and the pH value is added during the hydrolysis process, and the alkali consumption is about 0.5 to 1. 0% of the weight of the soybean; after the reaction is completed, the soybean milk is left as an activator. The remaining hydrolysate is heated to destroy the enzyme.

24. A sour milk is obtained by inoculating and fermenting the soybean milk according to claim 22.