JP2932130B2 - Production method of protein seasoning liquid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an enzyme preparation containing exo-type peptidase and a new protein seasoning liquid using the same, and more particularly to amino acids or bitterless oligopeptides that are protein degradation products as taste components. The present invention relates to a method for producing a salt-free protein seasoning liquid that can significantly improve the content of glutamic acid and the like, which are the main components of umami.

[0002]

2. Description of the Related Art Conventionally, a seasoning solution containing a protein decomposition product such as soy sauce or miso as a taste component has been known, but such a seasoning solution requires the use of a considerably large amount of salt. It causes various restrictions in terms of utilization.
That is, in general, soy sauce is produced as a raw fried soy sauce by inoculating a seed koji into a koji raw material mainly composed of steamed soybeans, producing a koji, mixing it with salt water, fermenting the resulting moromi, and fermenting the resulting moromi. During the preparation of the koji, the rot caused by propagation of microorganisms derived from the koji (such as various germs) is suppressed, fermentation of moromi,
Normally, high-concentration salt of about 23% is used so that aging can be performed well. On the other hand, while the taste peculiar to soy sauce is produced, the salt concentration of the product is set to a high concentration of 16 to 17%. It is the cause of being restricted in its use.

[0003] Further, conventionally, a method of producing an animal or plant extract by allowing a protein solubilizing enzyme to act on an animal or vegetable protein raw material has been known. However, when such a protein solubilizing enzyme is used, the resulting extract has bitterness. There are fatal drawbacks,
Use as a seasoning liquid is difficult. Although the above-mentioned bitterness can be eliminated by using an exo-type protease, it is very difficult to separate and purify the exo-type protease itself, and it has not yet been commercialized. There is no example used in the production of a seasoning liquid from A.

On the other hand, hydrolysates of animal and plant proteins (HAP,
HVP), a seasoning obtained by decomposing animal and plant proteins with hydrochloric acid, has been conventionally known, and its demand as a part of a natural seasoning (auxiliary seasoning) used in instant foods and the like has been gradually increasing. However, such HAP, H
As for VP, recently, there is a mutagen that is considered to be an organochlorine by-product generated when the above protein is hydrolyzed with hydrochloric acid, that is, a substance containing a substance that damages a gene and causes mutation in cells. It has been confirmed that its safety is becoming an issue.

Accordingly, an object of the present invention is to provide the above HVP, H
An object of the present invention is to provide a new seasoning liquid that is safe, salt-free, has no bitterness, and has excellent taste in place of AP.

[0006]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies on a seasoning solution comprising an enzymatic hydrolyzate of this species of animal, plant and vegetable protein material. A method has been developed to provide a good-quality seasoning solution containing an oligopeptide having good taste and no bitterness by using a solubilizing enzyme and a self-digesting solution of soy sauce koji. No. 20581).

Although the above-mentioned seasoning solution has a good taste, it has a high salt concentration and is similar to soy sauce in this respect.

[0008] However, in a subsequent study, when the above koji is immersed in an organic solvent such as ethyl alcohol and self-digested, there is no risk of spoilage even under salt-free conditions, and an exo-type which exhibits extremely excellent enzyme activity. A peptidase was obtained, which was effective for the production of the salt-free seasoning liquid of the above-mentioned purpose, and a new fact that the use of the salt-free seasoning liquid of the desired quality was obtained was discovered. In addition, the present inventors have found that the self-digestion by alcohol immersion can be applied not only to soy sauce koji, but also to solid cultures of other various microorganisms, and in such cases, exo can exhibit excellent enzyme activity. It has also been found that an autolysate containing mainly a peptidase is obtained, and a desired salt-free seasoning solution can be produced by utilizing the autolysate. The present invention has been completed based on such new findings.

That is, the present invention relates to Aspergillus spp.
From the group consisting of Lium, Rhizopus and Bacillus
Al solid culture koji of at least one microorganism selected
Immersed in coal and autolyzed at 15 ° C. or less in the absence of salt, obtained by solubilizing proteins in a salt-free enzyme preparation characterized by containing an exo-type peptidase enzyme, and animal and plant protein materials. The present invention relates to a method for producing a salt-free protein seasoning liquid, which comprises reacting an enzyme and then reacting the above enzyme agent.

The enzyme preparation of the present invention has excellent exo-type peptidase activity and is particularly suitable for producing a bitter-free seasoning liquid.

The seasoning liquid of the present invention is of course free of bitterness, is salt-free and safe, can be concentrated to extract, and can be used in a wide variety of processed foods or food materials such as instant foods, It can be widely applied to various uses such as kneaded products, ham / sausage, pickles, dressings, sauces, beverages, etc., to improve the taste, and to supplement protein nutrition. In particular, the enzymatic preparation of the present invention from soy sauce koji has a rich body with an improved content of glutamic acid and the like, which is a main component of umami, by applying the enzyme preparation to animal and vegetable protein raw materials as shown in Examples described later. A salt-free seasoning liquid exhibiting a very excellent taste can be produced.

Hereinafter, the method for producing the enzyme of the present invention will be described in detail. The enzyme of the present invention is obtained from a soaked organic solvent of koji obtained by solid-culture of microorganisms. Here, typical examples of koji obtained by solid-culturing the above microorganisms include various types of soy sauce koji and miso koji that have been well known in the art, such as dark soy sauce koji, light mouth soy sauce koji, and tame soy sauce koji. It is a koji raw material mainly consisting of steamed soybeans and a koji mold as seed koji, namely Aspergillus oryzae, Aspergillus soya (As
pergillus sojae) and other microorganisms belonging to the genus Aspergillus, which are inoculated and cultivated. However, the present invention is not limited to this, and various other microorganisms that are conventionally well-known for food use, for example, Aspergillus niger, Penicillium (Penicill)
ium, typically Penicillium ci
Similarly, solid cultures of the genus Rhizopus (Rhizopus, Rizopus niveus used for tempeh, etc.) and Bacillus (Bacillus, Bacillus subtilis used for kneaded products, etc.), that is, those koji are also advantageously used. Available. The solid culture of the genus Bacillus also includes natto.
The solid medium for culturing these various microorganisms may be soybean or the like similar to soy sauce koji, or various nutrients that do not hinder the growth of each of these microorganisms, for example, various proteins, sugars,
Fibers and the like may be appropriately selected and contained to give characteristics derived from the raw materials.

The organic solvent immersed product of each koji is prepared by immersing each koji in an appropriate organic solvent solution. Here, as the organic solvent, usually, ethanol can be suitably used, but is not particularly limited thereto, and may be, for example, isopropanol. These organic solvents usually have a final concentration of about 5
It is suitable to use in the range of about 20%, preferably about 6% to 15%.

The koji is immersed in the organic solvent in an amount of about 1 to 3 times the weight of the koji, and is immersed at a temperature of 20 ° C. or less, usually 0 to 20 ° C.
The desired enzyme preparation of the present invention can be obtained by autolyzing at a temperature of preferably about 5 to 15 ° C. for about 5 to 20 days, preferably about 7 to 10 days. However, when the temperature condition is higher than 20 ° C., since the autolysis is carried out under salt-free conditions, the growth of spoilage microorganisms cannot be completely avoided, and a desired enzyme preparation cannot be obtained. Can be difficult.

Thus, an enzyme preparation containing the desired exo-type peptidase of the present invention as a main component can be obtained. Needless to say, this can be purified by performing post-treatments and the like according to various known methods, if necessary.

Next, the method for producing the seasoning solution of the present invention will be described in detail. The seasoning solution is prepared by allowing a protein-solubilizing enzyme to act on animal and plant protein raw materials and then reacting the enzyme agent of the present invention obtained as described above. It can be manufactured by doing.

Here, the animal and plant proteins used as raw materials include various proteins that contain proteins and are edible,
For example, vegetable edible raw materials such as cereals and corn meal typified by wheat gluten, seafood, meat, processing by-products such as fish, seafood, end meats generated during processing of meat, bonito and dried noodles, etc. Animal edible raw materials such as broth during the production can be exemplified. These are used in the form of an aqueous liquid suitable for the action of the enzyme, that is, an aqueous solution, an aqueous dispersion, a suspension, etc., and are particularly preferably prepared in the form of an alkaline solution to facilitate dissolution of proteins. Is desirable.

As the protein solubilizing enzyme which is one of the enzyme agents acting on the animal and plant protein raw materials, any of various known ones commonly used in this field can be used. Representative examples are endo-type proteases, such as proteases of various microbial origins such as bacteria, molds, yeasts and actinomycetes, proteases of animal origins such as rennin and pancreatin, papain, bromelain and fusin. And proteases of plant origin. Of course, one of them can be used alone, or two or more can be used in combination. As the protein solubilizing enzyme,
In addition, depending on the pH condition of the raw material solution, each of alkaline protease, neutral protease and acidic protease can be used. Further, depending on the type of each enzyme, the pH of the raw material solution can be adjusted by the activity of each enzyme. Can be adjusted to the pH at which is best exhibited. The amount of the enzyme to be used can be appropriately determined depending on the type of the enzyme to be used and the like, and is not particularly limited, but is usually about 50 per g of protein.
It is good to be selected from the range of 0 to 10000 units, preferably about 2000 to 8000 units. The above activity unit is 1 μm / min at pH 7.0 and 30 ° C. using milk casein as a substrate, particularly in the case of endo-type neutral protease.
g of tyrosine-equivalent titer to produce a decomposition product was calculated as one unit.

In the method of the present invention, first, the above-mentioned solubilizing enzyme agent is allowed to act on the above-mentioned animal or plant protein raw material. This is generally accomplished by applying the appropriate amount of the enzyme to the starting material, usually about 4 hours.
0-65 ° C, preferably about 50-60 ° C, about 1-5
It is carried out by acting for about a time, thus hydrolyzing as much protein as possible to an oligopeptide having an average amino acid chain length of about 3 to 15 (soluble at a final concentration of trichloracetic acid of 0.17 mol). it can.

In the method of the present invention, once the above-mentioned hydrolysis is completed, the enzyme once used is deactivated, and thereafter, an enzyme agent mainly comprising exo-type peptidase by autolysis of the koji of the present invention (self-digested liquid of koji) Act. The pH of the solution on which the enzyme agent acts depends on the type of the raw material and the conditions under which the protein solubilizing enzyme has acted, and the like, but generally exhibits an acidic or neutral property suitable for expressing the activity of the enzyme agent. No special adjustment is required. Of course, if desired, the pH can be adjusted to a condition suitable for the action of the enzyme used in this step. The amount of the autolysed juice of the koji can be determined as appropriate, but is usually selected from the range of about 0.1 to 2 g per gram of animal or plant protein material. In addition, the conditions under which the koji self-digesting solution is applied are generally preferably about 40 to 45 ° C. and about 1 to 5 hours. Thus, a seasoning liquid which is free from bitterness and has excellent taste can be produced.

According to the method of the present invention, as described above, a protein solubilizing enzyme is first allowed to act on a raw material solution, and then an autolysate of koji is allowed to act thereon. In one step, both enzymes can act on the raw materials, respectively, to obtain a desired seasoning solution, and such a method is also included in the present invention.

The seasoning liquid of the present invention thus obtained can be used as it is for various uses, and if necessary, is subjected to post-treatments and the like according to various known methods to obtain a product suitable for various uses. You can also.

As the above-mentioned post-processing means, for example, the following means and combinations thereof can be appropriately adopted.

(1) Solids and the like are removed by a solid-liquid separation process such as centrifugation or filtration.

(2) Heat treatment (usually at 70 to 100 ° C. for 5 hours)
To about 30 minutes) to deactivate the enzyme used.

(3) Purification such as treatment with activated carbon is performed to remove unnecessary odors and the like.

(4) Concentrate to a solids concentration of about 30 to 70%.

(5) A powdery seasoning is prepared by a drying means such as spray drying.

[0029]

EFFECT OF THE INVENTION The autolysate obtained by using the koji organic solvent according to the present invention contains, as a main component, an exo-type peptidase which is particularly suitable for producing a seasoning solution having no bitterness. Thus, the desired protein hydrolysis can be efficiently performed in a short time.

According to the method of the present invention using the above enzyme, the content of glutamic acid or the like, which is a main component of umami, is high in a short time (usually within half a day), free of bitterness, salt-free and safe,
It is possible to produce a seasoning liquid of good quality which has a rich and very excellent taste.

[0031]

EXAMPLES Hereinafter, reference examples and examples will be given to explain the present invention in further detail.

The components of the seasoning liquid in each example were analyzed by the following methods.

(1) T. N (total nitrogen, g / dl) Kjeldahl method.

(2) F. N (formol nitrogen, g / d
1) By formol titration.

(3) Salt Content According to the Mohr method.

[0036]

Reference Example 1 Production of Soy Sauce Koji Ethyl Alcohol Self-Digesting Solution 1 kg of concentrated soy sauce koji was immersed in twice the amount of 20% ethyl alcohol and left at 5-10 ° C. for 7 days to obtain a koji auto-digesting solution. .

[0037]

[Test Example 1] Characteristics of autolyzed digestion liquid of soy sauce koji with alcohol The activity expression of enzymes in koji in alcohol soaked with koji according to the present invention is compared with that of ordinary soy sauce koji (in the presence of salt) as follows. Examined.

That is, 5 g of soy sauce koji (dark soy sauce koji) was homogenized in water to 100 ml, and the resulting solution was homogenized with a saline solution (final concentration 16%) or an ethyl alcohol solution (final concentration 15%). An enzyme solution was prepared, and each of them was allowed to act on each substrate solution containing a predetermined concentration of sodium chloride or alcohol, and each enzyme activity was examined.
Each enzyme activity was determined and indicated by the following method.

<Protease activity> The neutral protease was as described above. In the case of an acidic protease, the titer was determined in the same manner as the neutral protease except that the pH was 3.0.

<Carboxypeptidase (ACP) activity> Carbobenzoxy-glutamyl-tyrosine (Cbz
-Glu-Tyr) as a substrate at 30 ° C. for 1 hour for 60 minutes.
The activity of producing μg of tyrosine is defined as one unit.

<Leucine aminopeptidase (LAP)
Activity> Leucyl-glycyl-glycine (Leu-Gly)
-Gly) as a substrate, the activity of releasing 1 μmol of amino acid (calculated as glutamic acid) per minute at 30 ° C. is 1
Unit.

<Glutaminase activity> Using L-glutamic acid as a substrate, the unit is the number of mg of glutamic acid generated in 60 minutes at 37 ° C.

The results obtained are shown in Table 1 below.

[0044]

[Table 1]

In Table 1, each enzyme activity is expressed as a relative activity based on water (100), and each enzyme activity (unit / g) in the standard is shown in parentheses below the above standard value. It is displayed with a.

From the above Table 1, it is clear that the alcohol-soaked product of soy sauce koji according to the present invention has remarkably improved activity of any enzyme in comparison with the case using salt. In particular, the glutaminase activity is only 5% in the case of the use of salt, whereas it is actually 95% in the case of the use of the alcohol according to the present invention, which is substantially free from inhibition or reduction of the activity. It is clear that this is an effective thing which is very useful for producing a seasoning liquid having excellent taste.

[0047]

Reference Example 2 Production of autolyzed isopropyl alcohol of Rhizopus sp. Rhizopus sp. Koji was prepared as follows. That is, 50% of corn protein granules, 42% of wheat corn and 8% of corn starch (however, corn starch was liquefied with a part of water and liquefied by enzyme).
A koji raw material obtained by adding an equal amount of water to 20 kg of a raw material consisting of the following components was sterilized by pressurized steam, cooled, inoculated with Rhizopou niveus as a seed fungus, and inoculated at 30 to 35 ° C.
For 2 days.

Next, 30 liters of a 20% isopropyl alcohol solution was added to the koji and the mixture was allowed to stand at 5 to 10 ° C. for 10 days to obtain an autolysate of Rhizopus sp.

[0049]

Example 1 Production of the seasoning liquid of the present invention from wheat gluten 10 kg of wheat gluten (protein content: about 25%), 32 g of caustic soda (granules), a protein solubilizing enzyme agent (Orientase 22B, Ueda Chemical Industry Co., Ltd.) 68 g of bacterial alkaline protease) and 10 liters of water
A raw material-enzyme mixture of 0.5 was prepared.
After leaving the enzyme to act on the raw wheat gluten for 9 hours,
The enzyme was inactivated by heating at 0 ° C. for 15 minutes.

Next, 2.5 kg of the enzyme preparation (soy sauce koji ethyl alcohol autolysis solution) obtained in Reference Example 1 was added to the obtained liquid.
And reacted at 45 ° C. for 4 hours.
The mixture was heated for 0 minutes to obtain a seasoning solution (stock solution).

To the obtained stock solution, 2 kg of activated carbon and 1 kg of perlite as a filter aid were added, and the mixture was filtered using a small filter press to obtain 14.5 liters of a filtrate.
T. of this one N was 2.8%.

Next, the filtrate was concentrated in vacuo to obtain 4.8 liters of a concentrated seasoning solution. This is T.A. N8.4
%, F.I. N2.7%, pH 6.7, and its salt content was 0.08%.

From the above, it is clear that the use of soy sauce koji self-digested with alcohol can provide a seasoning liquid of excellent quality.

[0054]

Example 2 Production of Seasoning Solution of the Present Invention from Corn Meal 60 kg of corn meal stock solution (protein content about 15%), 300 ml of caustic soda solution (40%) and 250 g of protein solubilizing enzyme agent (Orientase 22B) were mixed. A raw material-enzyme mixture (pH 9.5) was prepared by allowing the mixture to stand at 60 ° C. for 3 hours to allow the enzyme to act on cornmeal, and then to 45 ° C.
The autolysis solution of Rhizopus genus koji obtained in Reference Example 2 with isopropyl alcohol was added to the cooling liquid at 10.8%.
kg, and reacted at 45 ° C. for 3 hours.
For 15 minutes to obtain a seasoning solution (stock solution).

6 kg of activated carbon and 3 kg of perlite as a filter aid were added to the obtained stock solution of seasonings, and the mixture was filtered using a small filter press to obtain 43 liters of a filtrate. T. of this one N was 3.0%.

Next, the above filtrate was concentrated in vacuo to obtain 14.5 liters of a concentrated seasoning liquid. This is T.A. N8.8
%, F.I. N2.9%, pH 6.8, and its salt content was 0.06%.

Further, the obtained seasoning liquid had a high amount of glutamic acid, which is a main component of the taste component, and had excellent taste.

[0058]

Example 3 Production of Seasoning Solution of the Present Invention from Skipjack Extract 20 kg of skipjack extract (protein content about 40%), 400 ml of aqueous caustic soda (40%) and a protein solubilizing enzyme agent (Orientase 90N, Ueda Chemical Industry Co., Ltd.) ), A bacterial neutral protease) was mixed with 50 g to prepare a raw material-enzyme mixture having a pH of 8.5, which was allowed to stand at 60 ° C for 3 hours to allow the enzyme to act on the skipjack extract, and then cooled to 45 ° C. And
To the obtained liquid, 6 kg of the enzyme preparation (soy sauce koji ethyl alcohol self-digesting liquid) obtained in Reference Example 1 was added, reacted at 45 ° C. for 3 hours, and then heated at 90 ° C. for 15 minutes to obtain a seasoning liquid (stock solution). ) Got.

To the obtained stock solution, 1.5 kg of activated carbon and 1.5 kg of perlite as a filter aid were added, and the mixture was filtered using a small filter press to obtain 38 liters of a filtrate. T. of this one N was 3.6%.

Next, the above filtrate was concentrated in vacuo to obtain 18.5 liters of a concentrated seasoning liquid. This is T.A. N7.4
%, F.I. N 3.2%, pH 6.5, salt content 2.02
%, Which was excellent in taste.

The amino acid analysis of the seasoning solution of the present invention obtained as described above was performed using an amino acid analyzer (manufactured by Hitachi, Ltd.).
835 type high-speed amino acid analyzer).

The obtained results are shown in T.W. Table 2 below shows the amount of amylic acid per 1.0 N (mg / dl).

[0063]

[Table 2]

From Table 2, it can be seen that the seasoning liquid of the present invention has an amino acid composition corresponding to the constituent amino acid composition of the skipjack extract.
In particular, it is found that the content of histidine, taurine, glutamic acid and the like is high, and that it has good umami.

[0065]

Example 4 Production of the Seasoning Solution of the Present Invention from Salmon Fish Approximately 3.2 kg of salmon fish was dismantled, and only the fleshy portion was crushed with a mixer to obtain a crushed product having a protein content of about 20%.

A mixture of 2 kg of the crushed salmon fish body, 600 ml of an aqueous solution of caustic soda (5N), 400 ml of water and 11 g of a protein solubilizing enzyme agent (Orientase 22B) was mixed.
A raw material-enzyme mixture of 7.7 was prepared, and this was
After allowing the enzyme to act on the salmon fish by allowing to stand for a period of time, the mixture was cooled to 45 ° C, and 400 g of the enzyme preparation (self-digested soy sauce koji ethyl alcohol) obtained in Reference Example 1 was added to the obtained liquid.
For 3 hours, and then heated at 90 ° C. for 15 minutes to obtain a seasoning solution (stock solution).

To the obtained stock solution, 200 g of activated carbon and 200 g of perlite as a filter aid were added, and the mixture was filtered using Nutsue to obtain 2.1 liter of a filtrate. T. of this one N was 1.9%.

Next, the filtrate was concentrated in vacuo to obtain a concentrated seasoning liquid (0.6 liter). This is T.A. N6.5
%, F.I. N2.7%, pH 6.8, salt content 0.04
%, Which was excellent in taste.

With respect to the seasonings of the present invention obtained in each of the above Examples, their taste was evaluated by eight panelists in three stages according to the following criteria, and each sensory test was performed.

Score 1: no umami score 2: a little umami score 3: a strong umami result The results (shown by the total score of the eight people) are as shown in Table 3 below. It is clear that it is strong.

[0071]

[Table 3]

Claims (2)

(57) [Claims] 1. The genus Aspergillus, Penicillium, Li
Selected from the group consisting of the genus Zorpus and the genus Bacillus.
Even without immersing one microorganism of solid culture koji alcohol
And self-digested at 15 ° C or less in the absence of salt .
A salt-free enzyme preparation comprising an exo-type peptidase enzyme. 2. A method for producing a salt-free protein seasoning solution, which comprises reacting a protein-solubilizing enzyme with an animal or plant protein raw material and then reacting the enzyme agent according to claim 1.