JP2622864B2 - Production of fermented lactic acid food and curdling enzyme composition - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing a lactic acid-fermented food using milk as a raw material and a novel milk-clotting enzyme composition.

[Conventional technology]

Cheese and fermented milk are typical as lactic acid fermented foods using milk as a raw material, but in the process of producing cheese, the milk is coagulated using a curdling enzyme called rennet, and whey separation is performed. The point is different from fermented milk.

As a curdling enzyme, calf rennet has been most commonly used since ancient times, but in recent years, enzyme preparations derived from microorganisms, for example, those produced by fungi of the genus Mucor have come into practical use. The characteristic of cheese is that not only protein and milk fat are concentrated by whey separation, but also αs ₁ -casein is decomposed by the casein decomposing action of this clotting enzyme and the synergistic action of the microorganism used as a starter. Therefore, the amount of water-soluble nitrogen is large, and part of the nitrogen is hydrolyzed to amino acids, so that it is digested and absorbed well and has excellent flavor (amino acids are also produced by lactic acid fermentation. Very little compared to those due to milk enzymes). However, when cheese is produced using bacteria as a starter, the hydrolysis of proteins by the above-described milk-clotting enzymes produces bitter peptides, which adversely affects the flavor and requires a long time for ripening. There is. In Camembert cheese, β-casein is also decomposed, but this is not due to the milk-clotting enzyme but to the enzyme produced by the mold that grows in the ripening step.

In the case of fermented milk as well, the type and amount of amino acids and other water-soluble nitrogen generated during the lactic acid fermentation process have some effect on the umami of the product. There is no grant. Therefore, in the case of fermented milk, its flavor is almost determined by lactic acid, acetaldehyde, diacetyl, acetone, lower fatty acids, etc. generated by lactic acid fermentation, and in addition, differences in physical properties such as liquid or solid, sweeteners, and juice In many cases, differences in flavor due to flavors and the like are characteristic. In the case of pasty fermented milk, agar or gelatin may be used for solidification, but these are merely gelling agents and have little to do with the flavor.

[Problems to be solved by the invention]

An object of the present invention is to provide a fermented lactic acid food product which contains more amino acids and other water-soluble proteins than conventional fermented cheese yam milk and has an excellent flavor.

Another object of the present invention is to provide a method for producing the above lactic acid-fermented food in a short period of time, and a milk-clotting enzyme used for the method.

Still another object of the present invention is to provide a method for producing pasty fermented milk having good properties and excellent umami without using agar or gelatin.

[Means for solving the problem]

The present invention relates to Penicilliu casei columns.
m caseicolum) by made of the enzyme mixture is salted out with ammonium sulfate solution produced 30% to 90% saturation extracellularly, .alpha.s ₁ with milk clotting activity - Casein decomposition and β-
It has a casein decomposition effect and has an optimum pH (casein) of 6.0, a stable pH range of 5.0 to 8.5, an optimum temperature of 50 ° C, and a molecular weight of 18,0.
A milk-clotting enzyme composition characterized by containing a neutral protease that is 00 and a method for producing a cheese or fermented milk in which both αs ₁ -casein and β-casein are degraded using the same. Things.

First, the milk-clotting enzyme composition of the present invention (hereinafter sometimes referred to as milk-clotting enzyme) will be described. This enzyme is an enzyme mixture containing a neutral protease and an acid protease as main components. The protease group is extracted by culturing Penicillium casei column in a wheat bran medium or the like and extracting the culture with a phosphate buffer or the like. If the extract is salted out with an ammonium sulfate solution having a saturation of 30% or more and 90% or less, an enzyme mixture purified to a degree usable as a milk-clotting enzyme can be obtained. The salted out product can be used as a milk-clotting enzyme, for example, by mixing lactose with several percent and freeze-drying.

Milking activity is exhibited by both acidic and neutral proteases in the milk-clotting enzyme composition of the present invention.

In the case of neutral protease, the temperature dependency is not so much observed up to around 40 ° C., but at higher temperatures, the activity increases with increasing temperature. The pH dependence is remarkably observed in the weakly acidic region, and the activity increases almost linearly with decreasing pH in a range up to about pH 5.8. The ratio of curdling activity / protease activity is lower than in calf rennet and mucor rennet. However, while β-casein was not degraded by any conventional rennet, this milk-clotting enzyme is largely characterized by having a β-casein degrading action.

When using this enzyme to make cheese, raw milk,
There are no restrictions on the conditions for lactic acid fermentation. Lactic acid fermentation is carried out by the same method as in the conventional production method, and when it is time to add rennet, this curdling enzyme is added. The resulting curd has lower shear stress, firmness and cohesiveness than the curd for traditional Gouda cheese making, which is one of the characteristics of this curdling enzyme. After performing whey separation by a conventional method, the obtained green curd is transferred to an aging step.

The ratio of water-soluble nitrogen to total nitrogen exceeds 20% even at the green card stage (when using the conventional milk-clotting enzyme,
This value is 10% or less), and the casein decomposition during aging is extremely rapid, so that even when aging on short days, a much higher aging effect is achieved than when long-term aging is performed using conventional milk-clotting enzymes. can do.

In the obtained cheese, not only αs ₁ -casein but also β-casein is decomposed, and the ratio of water-soluble nitrogen to total nitrogen is 30% or more even in a short-term aging product of about 15 days. High amino acid release, for example, glutamic acid content is 100m
g / 100 g or more, and leucine content is 150 mg / 100 g or more.
In addition, a large amount of hydrophobic amino acids (isoleucine, leucine, tyrosine, phenylalanine, tryptophan, proline, etc.), which are the main constituent amino acids of the bitter peptide, are released, and the bitter peptide produced during the casein hydrolysis process remains intact. It can be seen that the hydrolysis occurs.
Thereby, the cheese produced by the production method of the present invention has a feature that almost no bitterness is felt. Thus, this cheese is of a unique and excellent flavor different from any of the conventional manufacturing methods.

When producing fermented milk using the enzyme of the present invention, raw milk and lactic acid fermentation are also not limited. When the enzyme is added, it is appropriate that lactic acid fermentation proceeds and the coagulation of milk protein by lactic acid starts. If it is too early, whey off may occur and the properties of the product may deteriorate. Since the same problem occurs even if the amount of enzyme added is too large, it is desirable that the amount of enzyme be at most about one-half of the amount of enzyme that coagulates unfermented milk in 5 minutes. In addition, as the amount of enzyme added increases, the fluidity of the product decreases due to the curdling action, and the product becomes a so-called hard yogurt. Therefore, the amount of enzyme added is adjusted in consideration of the intended product properties. Immediately after the addition of the enzyme, the fermentation may be terminated by immediately filling the container and continuing the fermentation in a stationary state, and then cooling and terminating the fermentation. After application, the container may be filled. In any case, in order to cause casein hydrolysis by the added enzyme, it is desirable to keep the enzyme operable for at least 4 to 8 hours after the addition of the enzyme.

The effect of the addition of the curdling enzyme of the present invention, which hydrolyzes casein in a short time, is remarkably exhibited even in the case of producing fermented milk having a short fermentation time. That is, as long as the treatment with the added enzyme is performed for at least 3 hours, both αs ₁ -casein and β-casein are decomposed, and the ratio of water-soluble nitrogen to total nitrogen exceeds 25% (in the case of conventional fermented milk, , This value is less than about 20% and no degradation of β-casein occurs). As in the case of cheese, the amount of released amino acids related to umami is large, and the bitter peptide produced in the course of casein hydrolysis is well hydrolyzed so that little bitterness is felt.

〔Example〕

Example 1 A penicillium casei column was inoculated into a wheat bran medium and cultured at 20 ° C. for 7 days. The culture was extracted with a phosphate buffer at pH 6.5, and a 30% to 90% saturated ammonium sulfate salting-out fraction was collected from the extract. The saltout showed curdling activity, which was inhibited by pepstatin and EDTA. This confirmed that at least an acidic protease (aspartic protease) and a neutral protease were present. Table 1 shows the protease activity and milk-clotting activity of the enzyme solution of the 30-90% saturated ammonium sulfate salting-out fraction, and the effects of the addition of EDTA and hepstatin.
Table 2 shows that the crude enzyme solution was prepared using Sephadex G100,
The main characteristics of the enzyme solution when purified by gel filtration using DEAE Sephadex and Sephadex G75 to obtain a neutral protease fraction were shown.

In the following examples, the unpurified ammonium sulfate salting-out fraction was used as a milk-clotting enzyme.

Table 2 Properties of neutral protease Optimum pH (casein) 6.0 Stable pH range 5.0-8.5 Optimum temperature 50 ° C Inhibitor EDTA molecular weight 18000 Milking activity Medium Moderate Example 2 Nonfat milk solid content by adding skim milk to milk Was adjusted to 10.5%, sterilized at 100 ° C for 30 minutes, and cooled to 40 ° C. 2% of each of the previously prepared and stored starters of Streptococcus thermophilus and Lactobacillus bulgaricus, and a milk-clotting enzyme solution of Penicillium casei column (an amount sufficient to coagulate unfermented milk in 5 minutes) About 1/5 amount),
After mixing and filling in a container, the mixture was fermented at 40 ° C. for 5 hours. When the amount of lactic acid reached 0.75%, the mixture was cooled to 10 ° C. or lower to obtain a pasty yogurt. When the degradation state of casein was examined by electrophoresis, both αs ₁ -casein and β-casein were degraded. The ratio of water-soluble nitrogen to total nitrogen was 35%.

For comparison, a yogurt was prepared in the same manner as above except that no curdling enzyme was added.

The following sensory tests were performed on the above two types of yogurt.

Subjects: 20 males and females aged 21 to 45, 10 males and 10 females Method: Sample each sample and answer questions.

 The test results were as follows.

Example 3 After adding and dissolving skim milk powder and sucrose to milk, adjusting the concentration so that non-fat milk solid content is 10% and sucrose is 8%, sterilization is performed at 100 ° C. for 30 minutes. And cooled to 40 ° C. The same starter and milk-clotting enzyme solution as used in Example 1 (about 1/5 of the amount that can coagulate unfermented milk in 5 minutes) was added thereto, mixed, and filled in a container.
Ferment at 4 ° C for 4 hours.
The mixture was cooled to 0 ° C. or lower to obtain a pasty yogurt. When the degradation state of casein was examined by electrophoresis, both αs ₁ -casein and β-casein were degraded. The ratio of water-soluble nitrogen to total nitrogen was 32%.

The same sensory test as in Example 2 was performed on the above two types of yogurt. The results were as follows:

Example 4 Milk having a solid content of non-fat milk of 8.1% is heat-sterilized at 65 ° C. for 30 minutes, inoculated with 3% of a 1: 1 mixed starter of Streptococcus lactis and Streptococcus cremoris, and fermented to an acidity of 0.02%. Thereafter, rennet was added, and thereafter, cutting, whey-off, and pressing were carried out by a conventional method, and finally aging was performed for 90 days to produce Gouda cheese.

In the above production method, the case where the milk-clotting enzyme of the present invention was used as the rennet (Example of the present invention), the case where calf rennet was used (Comparative Example 1), and the case where rucol rennet was used (Control Example 2) Table 3 and Table 4 show the results of examining the composition of the obtained cheese. The number of days in Table 4 indicates the number of aging days.

When the degradation state of casein was examined by electrophoresis for 90 days of ripening, in the case of the present invention, both αs ₁ -casein and β-casein were decomposed and the ratio of water-soluble nitrogen to total nitrogen was 60%. However, in Comparative Example 1,
In the case of _No. 2, only αs ₁ -casein was slightly decomposed, and β
-Casein was hardly degraded.

Table 3 Composition (%) of fresh cheese Sample water protein lipid ash Example of the present invention 37.1 24.6 34.3 5.5 Control 1 40.6 23.2 32.5 5.0 Control 2 39.8 23.7 32.6 4.3 [Effect of the Invention] The cheese and fermented milk according to the present invention have α as described above.
Since both s ₁ -casein and β-casein have been decomposed, they are rich in water-soluble nitrogen such as amino acids, and have a much stronger umami taste than conventional products. Moreover, there is no bitterness due to bitter peptides, which is common in conventional well-ripened cheeses, and is also likely to occur when milk is treated with protease.
Excellent overall flavor. Such an excellent lactic acid fermented product, the production method using the curd enzyme of the present invention is in a very short time, and in the case of fermented milk, in the form of paste-like fermented milk with a unique tongue texture not found in conventional products, It can be easily manufactured.

Claims (3)

(57) [Claims] [1] In producing cheese from milk, Penicillium caseic column is used.
olum) is an enzyme produced outside the cells which is salted out with a 30% to 90% saturated ammonium sulfate solution to form a curd, separated by whey, and then aged, αs ₁ -A method for producing cheese in which both casein and β-casein have been degraded. 2. A method for producing fermented milk using milk as a raw material, comprising the steps of: Penicillium caseic column;
olum) is an enzyme produced extracellularly, salted out with a 30% to 90% saturated ammonium sulfate solution, and has milk-clotting activity, αs ₁ −
A method for producing fermented milk, wherein a substance having a casein-decomposing action and a β-casein-decomposing action is added at any stage of lactic acid fermentation to cause additional fermentation and milk coagulation. 3. Penicilliu casei column
m caseicolum) by made of the enzyme mixture is salted out with ammonium sulfate solution produced 30% to 90% saturation extracellularly, .alpha.s ₁ with milk clotting activity - Casein decomposition and β-
It has a casein decomposition effect and has an optimum pH (casein) of 6.0, a stable pH range of 5.0 to 8.5, an optimum temperature of 50 ° C, and a molecular weight of 18,0.
A milk-clotting enzyme composition comprising a neutral protease that is 00.