JPH07236484A - Enzymatic preparation - Google Patents

Abstract

PURPOSE:To obtain an enzymatic preparation having peptidase activity and specifically eliminated acid-forming capability and an enzymatic preparation capable of imparting a fermented diary product with excellent taste and flavor by the decomposition of protein without applying acid taste to the product. CONSTITUTION:This enzymatic preparation having peptidase activity and specifically eliminated acid-forming capability is produced by treating cells of lactic bacteria selected from Lactobacillus helveticus, Lactobacillus bulgaricus, Lactobacillus lactis, Lactobacillus easel, Lactobacillus acidophilus, Lactobacillus gasseri, Streptococcus thermophilus, Streptococcus cremoris and Streptococcus lactis or mixed cells of two or more kinds of the above strains under a pressure of >=150MPa.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to enzyme preparations.
Specifically, the present invention relates to Lactobacillus helveti which may be used in the production of fermented dairy products.
cus: sometimes referred to as Lh below), Lactobacillus bulgaricus (hereinafter sometimes referred to as Lb), Lactobacillus lactis (L
actobacillus lactis: sometimes referred to as Ll below)
Lactobacillus casei (hereinafter sometimes referred to as Lc), Lactobacillus acidophilus (hereinafter sometimes referred to as La), Lactobacillus gasseri (Lacto)
bacillus gasseri: sometimes referred to as Lg hereinafter),
Streptococcus t
hermophilus: sometimes referred to as St hereinafter), Streptococcus cremoris:
(Hereinafter sometimes referred to as Sc) and Streptococcus lactis (hereinafter sometimes referred to as Sl), or a lactic acid bacterium mixed with two or more thereof. The present invention relates to an enzyme preparation comprising a body pressure-treated product, having peptidase activity, and having no acid-forming ability.

In the present specification, percentages are values by weight except for relative values of enzyme activity, unless otherwise specified.

[0003]

2. Description of the Related Art From ancient times, fermented milk (yogurt) or cheese has been produced from animal milk by utilizing the acid-producing ability and proteolytic activity of lactic acid bacteria. Particularly in the production of cheese, lactic acid bacteria-derived proteolytic enzyme added as a starter during ripening and rennet (coagulant enzyme) added to coagulate animal milk become proteins in animal milk. It acts to form a unique flavor and plays an important role in enhancing the taste of cheese.

On the other hand, since it usually takes 3 to 6 months to mature the tease, a lactic acid bacterium starter having a high proteolytic activity is used for the purpose of shortening the maturation period, or during the tease. A method for increasing the number of starter lactic acid bacteria is known [Netherland Milk and Dairy Journal, Volume 15,
Pages 151-164, 1961. Prior art 1 below
May be described as].

Further, a method of using cultured cells of Lc or cell-free extracts of Lc, Lh, Lb and Ll together with starter lactic acid bacteria [Milchwissensc
haft), Volume 36, Pages 140-142, 1981
Year. Milchwissenschaft, Volume 37, pages 325-326, 1987. Hereinafter, it may be referred to as "prior art 2"], a method of using a lysochy-treated cell of Sc [Journal of Dairy Research, Vol. 43, No. 30].
Pages 1-311, 1976. Hereinafter, it may be referred to as "prior art 3"], a method of using Sl cells deficient in lactose utilization [Australian Journal of Daily Technology (The Australian Journal of Dai
ry Technology), Volume 38, Pages 49-54, 19
1983. Hereinafter, it may be referred to as Prior Art 4], a method of using a heat-treated mixed lactic acid bacterium starter, Lh, Lb, Lt [Journal of Daily Research (J
ournal of Dairy Research), Volume 42, 313-3
26 pages, 1975. Mil Hibissen Shaft (Mil
chwissenschaft), Volume 42, Pages 83-88, 1
987. Hereinafter, it may be described as prior art 5.], a method of using after freeze-thawing [Mill Hibissen shaft
(Milchwissenschaft), Vol. 42, pp. 139-144, 1987. Hereinafter, it may be referred to as "conventional technique 6".

On the other hand, recently, attempts have been made to utilize pressure in food processing, and various reports have been made to clarify the influence on food flavor, physical properties and bactericidal effect on food microorganisms. The bactericidal effect on Lactobacillus casei in relation to lactic acid bacteria (antibacterial and antifungal, Vol. 18, p. 129, 1
990), a method of pressurizing fermented milk fermented with lactic acid bacteria and / or yeast to suppress post-acidity increase (Journal of Food Engineering, Vol. 39, No. 2, pp. 173-177, 1992 and Unexamined-Japanese-Patent No. 4-75555. It may be hereafter described as the prior art 7.) etc. can be illustrated.

[0007]

However, in the method of the prior art 1 described above, aging is accelerated by the proteolytic power of lactic acid bacteria, but at the same time, due to the strong acid-forming ability of lactic acid bacteria, excess aging during aging. Cause the formation of various acids,
It was a cause of lowering the pH of the dust and deteriorating the quality.

In order to solve this inconvenience, the methods of the above-mentioned prior arts 2 to 6 were adopted, but these methods are not sufficient in deactivation of the acid-forming ability and the proteolytic activity necessary for promoting aging. However, there were problems such as decrease at the same time, complicated processing operation, and strong generation of bitterness.

Further, although there is an example in which pressure treatment is carried out to maintain the quality of dairy products such as yogurt as in the prior art 7, certain functions of the enzyme preparation are eliminated and other functions are maintained. There is no example of using pressure treatment for the purpose of producing the enzyme preparation.

In view of the above-mentioned prior art, the inventors of the present invention have conducted diligent research on an enzyme preparation having a loss of acid-forming ability and having peptidase activity. The present invention has been completed by finding that it has an effect.

An object of the present invention is to provide an enzyme preparation which has a specific loss of acid-producing ability and has peptidase activity.

[0012] Another object of the present invention is to provide an enzyme preparation which can impart no sourness to fermented dairy products and can impart an excellent flavor due to protein decomposition.

[0013]

Means for Solving the Problems The present invention for solving the above-mentioned problems is based on Lactobacillus (Lactobacillus).
helveticus), Lactoba bulgaricus (Lactoba
cillus bulgaricus), Lactobacillus lactis (Lac
tobacillus lactis), Lactoba
cillus casei), Lactobacillus acidophilus (Lac
tobacillus acidophilus), Lactobacillus gasseri
(Lactobacillus gasseri), Streptococcus sur
Streptococcus thermophilus, Streptococcus cremoris and Streptococcus lactis
A lactic acid bacterium selected from the group consisting of
At least 150 of mixed lactic acid bacteria mixed with at least one species
It is an enzyme preparation which comprises a pressure-treated product of MPa, has peptidase activity, and specifically loses the acid-forming ability, and the aminopeptidase activity is at least 10 per 1 g of the dry weight of the enzyme preparation. It is also preferable that the unit is a unit and the pressure treatment pressure is 200 to 500 MPa.

Next, the present invention will be described in detail.

The enzyme preparation of the present invention comprises culturing lactic acid bacteria,
It is manufactured by collecting cells from the medium itself containing the cells or from the medium and pressurizing the cells. The lactic acid bacterium used in the enzyme preparation of the present invention is not particularly limited and may be any species as long as it is easily available.

The lactic acid bacterium can be cultured by a known method, and there is no particular limitation as long as the lactic acid bacterium grows well. That is, as the medium used for culturing the lactic acid bacterium, a known liquid medium in which lactic acid bacterium can grow well can be used, and glucose, lactose, sucrose and the like as carbon sources, yeast extract and meat extract as nitrogen sources, Examples thereof include liquid media consisting of monopotassium phosphate, dipotassium phosphate, sodium acetate, and the like as salts such as peptone, skim milk, reduced skim milk, and a milk medium in which the above nitrogen source is added.

The culture temperature is 25 to around the optimum growth temperature of lactic acid bacteria.
The culture can be appropriately performed at 40 ° C. for a culture time of 5 to 24 hours. In order to neutralize the lactic acid produced during the culture and enhance the growth of the bacterium, the stirring culture can be performed while adding an alkaline agent such as a caseiso-da solution, an ammonia solution and a sodium carbonate solution.

In addition to the batch culture, the lactic acid bacterium may be cultured by fed-batch culture, continuous culture, dialysis culture or the like in order to culture the bacterial cells more efficiently.

After the completion of the culture, the bacterial cells are collected by a centrifuge or a membrane separator, washed and concentrated, and then the bacterial cells are suspended in a liquid containing no milk component and subjected to a pressure treatment. Pressurization is at least 150 MPa, preferably 200 by hydrostatic pressure.
It can be performed in the range of MPa to 500 MPa. Usually, the pressurizing temperature is 20 to 45 ° C. and the pressurizing time is about 1 to 30 minutes, but it is possible to increase the pressurizing temperature and shorten the pressurizing time or to lower the pressurizing temperature and pressurize time. Can be made longer, and the temperature and time can be changed appropriately. However, most desirably, the pressurizing temperature is 37 ° C. and the pressurizing time is about 10 minutes.

The bacterial cell suspension after pressure treatment can be used as an enzyme preparation in a liquid form as it is, or can be concentrated at a low temperature to give a concentrated enzyme preparation. These liquid enzyme preparations can be frozen, or can be powdered by spray-drying or freeze-drying. Furthermore, an enzyme preparation can be prepared by diluting or doubling with an excipient such as skim milk powder or lactose in order to adjust the enzyme titer.

Usually, when the enzyme preparation of the present invention is used for degrading a protein, the AP activity of at least 10 units can sufficiently achieve the purpose. Therefore, an appropriate activity unit may be selected depending on the purpose of use. It can be prepared.

Next, the present invention will be described in detail by showing test examples.

Test Example 1 This test was conducted to examine the effect of pressurizing pressure on enzyme activity.

1) Preparation of sample 3% of 500 ml of activated milk medium (milk medium containing 0.2% of yeast extract in 10% reduced skim milk) sterilized with Lh IAM-1042 strain for 15 minutes at 115 ° C. , And statically cultured at 37 ° C. for 16 hours. To this culture solution, 500 ml of a 2% sodium citrate solution was added, and the pH was adjusted to 7.0 with a 20% caseisodal solution, followed by centrifugation to collect the cells, and a 0.1 M phosphate buffer solution ( The cells were washed twice with pH 7.0) to prepare about 50 ml of a 2% (w / v) cell suspension. 20 ml of the cell suspension was filled in a sterile polyethylene bag, sealed with a heat sealer, and used for the test.

2) Test method The sample was stored in a high-pressure processing apparatus (manufactured by Mitsubishi Heavy Industries, Ltd., model MFP-7000) which was previously kept at 37 ° C., and the hydrostatic pressure was 0, 100, 150, 200, 250, 300. Four
Pressurization was performed at 00 and 500 MPa for 10 minutes, and after the pressure treatment, the cells were crushed by ultrasonic waves, and the enzyme activity was measured by the following method.

Aminopeptidase (hereinafter sometimes referred to as AP) activity The microbial cell suspension after ultrasonic treatment was diluted with 0.1 M phosphate buffer (pH 7.0) to give 0.2% ( A cell suspension of w / v) was prepared. On the other hand, a synthetic substrate Leu-pNA (manufactured by Kokusan Chemical Works) was dissolved in 0.1 M phosphate buffer (pH 7.0) to prepare a 2 mM Leu-pNA solution.

1 ml of the cell suspension and Leu-pNA
Mix 1 ml of the solution and react at 37 ° C for 5 minutes.
The reaction was stopped by adding 2 ml of a 0% acetic acid solution, and the amount of released p-nitroanilide was calculated from the absorption value at a wavelength of 410 nm.

X-Prolyl dipeptidyl aminopeptidase (hereinafter sometimes referred to as PDA) activity The microbial cell suspension after ultrasonic treatment was treated with 0.1 M phosphate buffer (pH 7.0). It was diluted to prepare a 0.2% (w / v) cell suspension. On the other hand, the synthetic substrate Gly-Pro-pN
A (manufactured by Peptide Institute) was added to 0.1M phosphate buffer (pH
It was dissolved in 7.0) to prepare a 2 mM Gly-Pro-pNA solution.

1 ml of the cell suspension and Gly-Pro
-1 ml of pNA solution was mixed and allowed to react at 37 ° C for 5 minutes, and then 2 ml of 30% acetic acid solution was added to stop the reaction, and the amount of released p-nitroanilide was adjusted to a wavelength of 410 n.
It was calculated from the absorbance value of m.

Proline iminopeptidase (hereinafter PI
The cell suspension after sonication is diluted with 0.1 M phosphate buffer (pH 7.0), and 0.2% (w / v) suspension is added. A suspension was prepared. On the other hand, a synthetic substrate Pro-pNA (manufactured by Kokusan Chemical Works) was dissolved in 0.1 M phosphate buffer (pH 7.0) to prepare a 2 mM Pro-pNA solution.

1 ml of the cell suspension and Pro-pNA
Mix 1 ml of the solution and react at 37 ° C for 5 minutes.
The reaction was stopped by adding 2 ml of a 0% acetic acid solution, and the amount of released p-nitroanilide was calculated from the absorption value at a wavelength of 410 nm.

In measuring the activities of AP, PDA and PIP, the amount of enzyme required to release 1 μM p-nitroanilide in 1 minute at 37 ° C. was defined as 1 unit, and in the present specification, these enzymes are defined. Units of activity are listed according to this definition.

Β-Galactosidase activity The microbial cell suspension after ultrasonic treatment was diluted with 0.1 M phosphate buffer (pH 7.0) to obtain a cell suspension of 0.05% (w / v). A suspension was prepared. On the other hand, a synthetic substrate o-nitrophenyl-p-galactopyranoside (manufactured by Boehringer Mannheim) was dissolved in 0.1M phosphate buffer (pH 7.0) to obtain 4 mM o-nitrophenyl-p-galactopyramide. A noside solution was prepared.

2 ml of the cell suspension and 1 ml of o-nitrophenyl-p-galactopyranoside solution were mixed and mixed with each other to give 3
After reacting at 7 ° C for 15 minutes, 625 mM Na ₂ CO _{3 was} added.
The reaction was stopped by adding 2 ml of the solution, and the amount of liberated o-nitrophenol was calculated from the absorption value at a wavelength of 420 nm.

Β-galactosidase activity was 1 at 37 ° C.
The amount of enzyme required to release 1 μM o-nitrophenol per minute is defined as 1 unit, and the unit of β-galactosidase activity is described herein according to this definition.

Lactic acid producing activity 5 ml of cell suspension after ultrasonic treatment and 1.1% gluco-
Solution (45 ml) was mixed and reacted at 37 ° C. for 1 hour, and the reaction solution was titrated with a 0.1N NaOH solution to calculate the acid production amount by converting it to lactic acid.

The activity of the lactic acid-producing system was expressed as the mass of lactic acid produced per 1 g of the dry weight of the enzyme preparation in the reaction at 37 ° C. for 1 hour.

3) Test Results The results of this test are shown in Table 1. In addition, the enzyme activity is each activity in the case of no treatment (enzyme preparation dry weight 1
Acid-producing ability 120 mg / g, β-galactosidase activity 170 units, AP 76 units, PDA 70 units and PI
Relative value (%) when P10 unit is 100
Displayed in.

As is clear from Table 1, the acid-forming ability is 1
Gradually decreased by pressure treatment of 50 MPa or more (15
45% at 0 MPa, 9% at 200 MPa), 250MP
It disappeared completely in a.

On the other hand, β-galactosidase, AP and P
The activity of DA did not decrease at all under pressure treatment up to 400 MPa and tended to decrease slightly at 500 MPa, which were 92%, 91% and 96%, respectively. In addition, the activity of PIP does not decrease at all under pressure treatment up to 300 MPa,
The value was 94% at 400 MPa and 81% at 500 MPa. Therefore, the pressurizing condition is at least 150 MPa,
It has been found that it is preferably 200 to 500 MPa.

From these results, in the enzyme preparation of the present invention, not only the activity of the main proteolytic enzyme, AP, is not decreased, but also the other proteolytic enzyme, PD.
It was confirmed that the activities of A and PIP were not reduced. Further, in the enzyme preparation of the present invention, it was found that the acid-producing ability surely disappears, but the activity of an enzyme such as β-galactosidase does not disappear, and thus the enzyme preparation of the present invention is desired. It was proved that only the acid-forming ability of P.

ATCC 801 obtained from ATCC
8, common method (supervised by Michio Ozaki, "Lactic Acid Bacteria Manual", No. 6
Pp. 20, Asakura Shoten, 1992), the same test was carried out for a total of 3 strains of Lh, R-1 separated from raw milk and C-1 separated from commercial cheese, but the same results as above were obtained. Was obtained.

[0043]

[Table 1] Test Example 2 This test was carried out to compare the enzyme preparation of the present invention with that prepared by the method by heat treatment (conventional method).

The pressure treatment is performed at a pressure of 300 MPa and 37 ° C.
An enzyme preparation was produced by the same method as in Test Example 1 except that the treatment was performed at the temperature of 10 minutes, and each enzyme activity was measured by the same method as in Test Example 1. On the other hand, in the method of Test Example 1, a 2% bacterial cell suspension was prepared and heat-treated at 59 ° C. for 18 seconds and at 69 ° C. for 18 seconds to prepare an enzyme preparation by a conventional method, and each enzyme was similarly prepared. The activity was measured.

The results of this test are shown in Table 2. The enzyme activity was the same as in Test Example 1 in the case of no treatment (acid-producing ability of 115 g per 1 g of the dry weight of the enzyme preparation).
mg, β-galactosidase activity 160 units, AP75
The unit, the PDA 70 unit, and the PIP 11 unit) were each expressed as a relative value (%) when set to 100.

As is clear from Table 2, in the enzyme preparation produced by the heat treatment at 59 ° C. for 18 seconds, the activity of the lactic acid-producing system was reduced but 50% remained, resulting in insufficient deactivation. And the activities of AP and PIP, which have an important influence on the maturation of cheese, are reduced to 60% and 20%, respectively.
Not desirable. Further, in the enzyme preparation produced by heating at 69 ° C. for 18 seconds, the ability to produce lactic acid completely disappeared, but AP and PIP activities also disappeared at the same time, and the activities of β-galactosidase and PDA were also slightly reduced. 20
% And 30% remained.

On the other hand, it was confirmed that in the enzyme preparation of the present invention (pressure treatment), the enzyme activity of the lactic acid producing system was completely disappeared and other enzyme activities were not reduced at all. From this result, the enzyme preparation of the present invention was compared with the enzyme preparation produced by the conventional method by heat treatment, only the enzyme system involved in the production of lactic acid was selectively inactivated, and other enzyme It was demonstrated that the activity of P. The enzyme preparations produced by changing the other conventional methods and the pressure treatment conditions were also tested, but almost the same results were obtained.

[0048]

[Table 2] Test Example 3 This test was conducted to examine the acid-producing ability and AP activity of lactic acid bacteria other than Lh.

Lb, Ll, Lc, La, L instead of Lh
g, St, Sc and Sl lactic acid bacteria were used, Sc and Sl were cultured at 25 ° C., acid-producing ability and AP activity were measured, and pressure was 300 MPa and temperature was 37 ° C. for 10 minutes. Except for the above, the acid-producing ability and AP activity of the enzyme preparation prepared from various lactic acid bacteria by pressure treatment were tested by the same method as in Test Example 1.

The results of this test are shown in Table 3. The enzyme activity was the same as that of Test Example 1 in the case of no treatment (Lb: acid production capacity 105 mg / AP51 unit per 1 g dry weight of enzyme preparation, Ll: acid production capacity 83 mg per 1 g dry weight of enzyme preparation). And AP 48 units, Lc
Is 77 mg of acid-producing ability and AP unit of 42 units per 1 g of the dry weight of the enzyme preparation, La is 70 mg of acid-producing ability and 35 units of AP per 1 g of the dry weight of enzyme preparation, and Lg is 89 mg and 40 units of AP-producing ability per 1 g of the dry weight of enzyme preparation.
St is 42 mg of acid-forming ability per 1 g of dry weight of the enzyme preparation.
And AP 60 units, Sc is 30 mg acid production capacity per 1 g dry weight of the enzyme preparation and AP 47 units, Sl is an acid production capacity 33 mg per 1 g dry weight of the enzyme preparation and AP 34 units) relative values (%), respectively. Displayed in.

As is clear from Table 3, the enzyme preparation prepared from each lactic acid bacterium tested lost the acid-producing ability and maintained the AP activity as in Test Example 1. From this result, it was confirmed that the enzyme preparation of the present invention can be prepared using various lactic acid bacteria as raw materials.

[0052]

[Table 3] Reference Example 1 50 kg of raw cow milk was sterilized at 60 ° C. for 10 minutes, and then 30
Cooled to ° C. Commercially available cheese starter for this pasteurized milk
100 g of the enzyme preparation obtained in Example 1 was added to 1 kg of bulk starter prepared using CH-01 (manufactured by Hansen) and mixed well, and fermented at 30 ° C. for 2 hours. Next, calf rennet (manufactured by Hansen) was added at 0.03% and kept for 30 minutes to generate a card. This card was cut into pieces of about 10 mm square and gently stirred for 1 hour while heating at 38 ° C. Whey separated from card
The card was squeezed to remove the ash, and about 400 g of 10 blocks were manufactured. This was immersed in a 20% saline solution at 10 ° C. for 15 hours, then the surface was dried, filled in a polyethylene bag and vacuum-packed. After aging for 2 months in a thermostatic chamber at 15 ° C, no excessive acid formation was observed, and good taste and texture were obtained.

Reference Example 2 Except that 100 g of the enzyme preparation obtained in Example 1 was changed to 20 g of the enzyme preparation obtained in Example 6 and that it was aged in a thermostatic chamber at 15 ° C. for 1 month. A cheese was manufactured by the same method as in Reference Example 1. The taste and texture of the cheese were good, and excessive acid formation was not observed.

Reference Example 3 Except that 100 g of the enzyme preparation obtained in Example 1 was changed to 10 g of the enzyme preparation obtained in Example 9 and that it was aged in a thermostatic chamber at 15 ° C. for 1.5 months. Produced a cheese by the same method as in Reference Example 1. The taste and texture of the cheese were good, and excessive acid formation was not observed.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0056]

【Example】

Example 1 Lh IAM-1042 strain was inoculated at a ratio of 3% into 50 l of activated milk medium sterilized at 90 ° C for 10 minutes, and statically cultured at 37 ° C for 16 hours. This culture broth was mixed with 50 l of a 2% sodium citrate solution and the pH was adjusted to 7.0 with a 20% caseisodal solution, and then the culture broth was centrifuged to collect bacterial cells. It was washed twice with 1M phosphate buffer (pH 7.0). About 5 l of a 2% (w / v) cell suspension was prepared,
A pressure treatment was performed at 400 MPa and 30 ° C. for 15 minutes to produce about 1.5 l of a liquid enzyme preparation.

The enzyme activity of this enzyme preparation was measured by the same method as in Test Example 1, and as a result, no acid-forming ability was observed.
AP activity was 76 units per gram dry weight of enzyme preparation.

Example 2 Changing Lh to C-1 strain, 300 MPa, 37 ° C.
About 1.5 liters of a liquid enzyme preparation was prepared by the same method as in Example 1 except that pressure treatment was performed for 10 minutes.
Lyophilization was performed by a conventional method to obtain about 12 g of a powdery enzyme preparation.

The enzyme activity of this enzyme preparation was measured by the same method as in Test Example 1, and as a result, no acid-forming ability was observed.
AP activity was 60 units per gram dry weight of enzyme preparation.

1 part (weight) of this enzyme preparation was triturated with 3 parts (weight) of commercially available lactose (manufactured by Mirai Co.) to give about 4 parts of enzyme preparation.
5 g was produced.

Example 3 Changing Lh to ATCC8018 and 200MP
a, about 1.5 liters of the liquid enzyme preparation was prepared by the same method as in Example 1 except that pressure treatment was performed at 30 ° C. for 30 minutes.
Was prepared and stored frozen in a freezer at -25 ° C.

After storage at -25 ° C for 6 months, the enzyme activity of this enzyme preparation was measured by the same method as in Test Example 1. As a result, no acid-forming ability was observed, and the AP activity was the enzyme preparation dry matter. It was 45 units per gram of weight.

Example 4 Changing Lh to R-1 strain and applying pressure treatment to 250MP
a, about 1.5 l of enzyme preparation was prepared by the same method as in Example 1 except that it was carried out at 45 ° C. for 5 minutes.

The enzyme activity of this enzyme preparation was measured by the same method as in Test Example 1. As a result, no acid-forming ability was observed, and the AP activity was 53 units per 1 g of the dry weight of the enzyme preparation.

Example 5 Lh was changed to Ll and the pressure treatment was 350 MPa,
About 1.5 liters of enzyme preparation was prepared by the same method as in Example 1 except that it was carried out at 35 ° C. for 10 minutes.

The enzyme activity of this enzyme preparation was measured by the same method as in Test Example 1. As a result, no acid-forming ability was observed, and the AP activity was 48 units per 1 g of the dry weight of the enzyme preparation.

Example 6 Lh was changed to Lb and 1 at 350 MPa and 37 ° C.
About 1.5 l of a liquid enzyme preparation was produced by the same method as in Example 1 except that pressure treatment was performed for 0 minutes, and freeze-dried by a conventional method to obtain about 18 g of a powdery enzyme preparation. It was

The enzyme activity of this enzyme preparation was measured by the same method as in Test Example 1, and as a result, no acid-forming ability was observed.
AP activity was 51 units per gram dry weight of enzyme preparation.

10 g of this enzyme preparation was dispersed with 20 g of commercially available reduced skim milk powder (manufactured by Morinaga Milk Industry Co., Ltd.) to produce an enzyme preparation.

Example 7 Lh was changed to Lc, and 350 MPa and 5 ° C. at 5
Approximately 1.5 liters of the liquid enzyme preparation was prepared in the same manner as in Example 1 except that pressure treatment was performed for 25 minutes.
It was frozen and stored in a freezer at ℃.

After storing at -25 ° C for 6 months, the enzyme activity of this enzyme preparation was measured by the same method as in Test Example 1. As a result, no acid-forming ability was observed, and the AP activity was found to be the dry matter of the enzyme preparation. It was 42 units per gram of weight.

Example 8 Changing Lh to La and applying a pressure treatment of 400 MPa,
About 1.5 liters of enzyme preparation was prepared by the same method as in Example 1 except that it was performed at 25 ° C. for 25 minutes.

The enzyme activity of this enzyme preparation was measured by the same method as in Test Example 1. As a result, no acid-forming ability was observed, and the AP activity was 35 units per 1 g of the dry weight of the enzyme preparation.

Example 9 Lh was changed to St, and 500 MPa and 3 ° C. at 30 ° C.
About 1.5 liters of a liquid enzyme preparation was produced by the same method as in Example 1 except that pressure treatment was performed for 1 minute, and freeze-dried by a conventional method to obtain about 19 g of a powdery enzyme preparation. .

The enzyme activity of this enzyme preparation was measured by the same method as in Test Example 1. As a result, no acid-forming ability was observed.
AP activity was 60 units per gram dry weight of enzyme preparation.

10 g of this enzyme preparation was triturated with 30 g of commercially available whey protein (manufactured by Mirai Co.) to prepare an enzyme preparation.

Example 10 Lh was changed to Lg and 500 MPa, 1 at 45 ° C.
Approximately 1.5 liters of the liquid enzyme preparation was prepared in the same manner as in Example 1 except that pressure treatment was performed for 25 minutes.
It was frozen and stored in a freezer at ℃.

After storage at -25 ° C for 6 months, the enzyme activity of this enzyme preparation was measured by the same method as in Test Example 1. As a result, no acid-forming ability was observed, and AP activity was the dry matter of the enzyme preparation. It was 40 units per gram of weight.

Example 11 The enzyme preparation was carried out in the same manner as in Example 1 except that Lh was changed to Sc, culture was carried out at 25 ° C., and pressure treatment was carried out at 250 MPa and 20 ° C. for 30 minutes. About 1.5 l of product was produced. The enzyme activity of this enzyme preparation was measured by the same method as in Test Example 1. As a result, no acid-forming ability was observed, and the AP activity was 4 per 1 g of the dry weight of the enzyme preparation.
It was 7 units.

Example 12 The enzyme preparation was carried out in the same manner as in Example 1 except that Lh was changed to Sl, culture was carried out at 25 ° C., and pressure treatment was carried out at 250 MPa and 30 ° C. for 20 minutes. About 1.5 l of product was produced. The enzyme activity of this enzyme preparation was measured by the same method as in Test Example 1. As a result, no acid-forming ability was observed, and the AP activity was 34 units per 11 g of the dry weight of the enzyme preparation.

[0081]

INDUSTRIAL APPLICABILITY As described above in detail, the present invention relates to an enzyme preparation having peptidase activity and inability to produce an acid. The effects of the present invention are as follows. Is. 1) The enzyme preparation of the present invention has peptidase activity,
In addition, since it has no acid-producing ability, when it is used for producing a fermented dairy product, no acid is produced and a product with excellent flavor can be obtained. 2) The enzyme preparation of the present invention can be produced by a simple operation.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location C12R 1: 225) (C12N 9/52 C12R 1:23) (C12N 9/52 C12R 1: 245) (C12N 9/52 C12R 1:46) (72) Inventor Katsue Anzou 5-1-83 Higashihara, Zama City, Kanagawa Prefecture Morinaga Milk Industry Co., Ltd.

Claims (3)

[Claims] 1. A Lactobacillus helveticus (Lacto)
bacillus helveticus), Lactobacillus bulgaricus
(Lactobacillus bulgaricus), Lactobacillus lactis, Lactobacillus casei
(Lactobacillus casei), Lactobacillus acidophilus, Lactobacillus
Gasseri (Lactobacillus gasseri), Streptococcus thermophilus, Streptococcus cremoris
s) and Streptococcus lactis
Lactic acid bacteria selected from the group consisting of lactis) or a mixture of two or more kinds of lactic acid bacteria mixed with at least 150 MPa under pressure, and having peptidase activity and specifically An enzyme preparation that has lost the ability to generate acid. 2. The enzyme preparation according to claim 1, wherein the aminopeptidase activity is at least 10 units per gram dry weight of the enzyme preparation. 3. The enzyme preparation according to claim 1 or 2, wherein the pressure treatment pressure is 200 to 500 MPa.