JPWO2006004105A1 - Method for producing peptide mixture, method for producing fermented milk containing antihypertensive peptide and method for producing antihypertensive peptide preparation - Google Patents

Abstract

Provided is a method for producing a peptide mixture containing IPP and / or VPP at a high concentration, fermented milk containing an antihypertensive peptide, and a preparation thereof with high efficiency. The method for producing a fermented milk containing a peptide mixture or an antihypertensive peptide of the present invention comprises a step of preparing a mixed raw material (a) comprising a protein containing an amino acid sequence of an antihypertensive peptide, a lactic acid bacteria assimilating sugar, and a lactic acid bacterium (1 -A), including stirring the mixed raw material (a), adjusting the pH with an alkaline agent and performing lactic acid fermentation to produce an antihypertensive peptide, etc. (1-B), and for the protein in the mixed raw material (a) The ratio of lactic acid bacteria assimilating sugar is 1.53 times or more by weight, and the pH adjustment in step (1-B) is controlled to 4.8 to 5.8.

Description

  The present invention relates to a method for producing a peptide mixture containing at least one of Ile Pro Pro and Val Pro Pro at a high concentration, a method for producing antihypertensive peptide-containing fermented milk, and a method for producing an antihypertensive peptide.

Angiotensin-converting enzyme (hereinafter abbreviated as ACE) is mainly present in lungs and vascular endothelial cells, and produces angiotensin II having a strong blood pressure increasing effect from angiotensin I, while degrading and inactivating antihypertensive peptides such as bradykinin. As a result, the blood pressure is increased. Therefore, a substance that inhibits this enzyme activity can be used as a substance that suppresses an increase in blood pressure.
In recent years, ACE inhibitory peptides (hereinafter abbreviated as ACEI peptides) have attracted attention as low-toxic and highly safe antihypertensive substances, and many natural or synthetic peptides having such actions have been reported. For example, the tripeptides Ile Pro Pro and Val Pro Pro (hereinafter abbreviated as IPP and VPP, respectively) are described as having ACE inhibitory activity in JP-A-3-120225 and JP-A-6-40944. .
Paragraph 0011 of JP-A-6-197786 discloses a method for producing an ACEI peptide in which lactic acid bacteria are cultured in a medium containing a protein having a sequence containing the sequences IPP and VPP. Here, it is described that various food materials are used as a medium for culturing lactic acid bacteria. Among these, an aqueous solution of skim milk powder containing about 35% by weight of protein and about 43 to 50% by weight of lactose has been generally used so far as being optimal for cultivation of lactic acid bacteria.
Further, anti-hypertensive action is also known for dipeptide Tyr-Pro (hereinafter abbreviated as YP). Paragraph 0016 of Japanese Patent Application Laid-Open No. 10-95736 includes media derived from various food materials and other media for lactic acid bacteria. However, the use of a medium supplemented with yeast extract, vitamins and minerals is described, but sugar is not mentioned.
The production of anti-hypertensive peptides such as the ACEI peptide by the conventional culture of lactic acid bacteria described above has the disadvantage that it takes time and the utilization efficiency of the protein as a raw material is low. Accordingly, there is a need for a production method capable of producing antihypertensive peptides with higher efficiency, and further a method for producing peptide mixtures containing IPP and / or VPP at a high concentration that can be expected to have other useful physiological activities such as antihypertensive action. It has been.

An object of the present invention is to provide a production method capable of producing a peptide mixture containing IPP and / or VPP at a high concentration, which can be expected to have other antihypertensive action and other useful physiological activities, with high efficiency.
Another object of the present invention is to provide a production method capable of producing fermented milk containing an antihypertensive peptide and an antihypertensive peptide preparation with high efficiency.

The present inventors have made various studies in order to solve the above problems, and instead of using an aqueous solution of skim milk powder that has been considered to be optimal in culturing lactic acid bacteria, a specific medium is employed, and the medium and specific conditions are used. It was found that the production amount of a peptide mixture containing anti-hypertensive peptide and IPP and / or VPP at a high concentration can be greatly improved by combining with the fermentation operation below, and the present invention was completed.
Specifically, according to the present invention, (i-1) a protein having at least one amino acid sequence of Ile Pro Pro and Val Pro Pro, (ii) a lactic acid bacteria assimilating sugar, and (iii) a mixed raw material containing lactic acid bacteria (a Peptide containing at least one of Ile Pro Pro and Val Pro Pro by lactic acid fermentation while stirring the mixed raw material (a) and adjusting the pH using an alkaline agent. A step (1-B) for producing a mixture, and in the step (1-A), in the mixed raw material (a), the content ratio of (ii) lactic acid bacteria assimilating sugar to (i-1) protein is: Production of a peptide mixture containing at least one of Ile Pro Pro and Val Pro Pro at a high concentration, which is 1.53 times or more by weight and is carried out while controlling the pH adjustment in the step (1-B) to 4.8 to 5.8 Law is provided.
According to the present invention, (i-2) a step of preparing a mixed raw material (a) containing an antihypertensive peptide amino acid sequence, (ii) lactic acid bacteria assimilating sugar, and (iii) lactic acid bacteria (1- A) and a step (1-B) of lactic acid fermentation while stirring the mixed raw material (a) and adjusting the pH using an alkaline agent to produce an antihypertensive peptide, the step (1-A) ) In the mixed raw material (a), the content ratio of (i) lactic acid bacteria assimilating sugar to (i-2) protein is 1.53 times or more by weight, and pH adjustment in the step (1-B) A method for producing fermented milk containing an antihypertensive peptide, which is carried out while controlling 4.8 to 5.8, is provided.
Furthermore, according to this invention, the manufacturing method of an antihypertensive peptide formulation including the process (2-A) which isolate | separates whey from fermented milk obtained by the manufacturing method of the said antihypertensive peptide containing fermented milk is provided.

  A method for producing a peptide mixture containing IPP and / or VPP at a high concentration of the present invention, a method for producing fermented milk containing an antihypertensive peptide and a method for producing an antihypertensive peptide preparation include a medium containing a high concentration of lactic acid bacteria-assimilating sugar, In particular, because it employs a process that combines a fermentation operation performed while controlling the pH within a specific range, a peptide mixture containing a high concentration of IPP and / or VPP useful for various physiological activities and a fermentation containing an antihypertensive peptide Milk and preparations can be produced with high efficiency, and are very useful in the field of functional foods such as pharmaceuticals and foods for specified health use.

It is a graph which shows the result of having measured the IPP and VPP peptide density | concentration in each heating temperature when the heat sterilization processing time by the ultra-high temperature heat sterilization method performed in Example 9 was 30 seconds. It is a graph which shows the result of having measured the IPP and VPP peptide density | concentration in each heating temperature when the heat sterilization processing time by the batch sterilization method performed in Example 9 was 10 minutes.

The present invention will be described in detail below.
The method for producing a peptide mixture and fermented milk containing IPP and / or VPP at a high concentration of the present invention includes a step (1-A) of preparing a specific mixed raw material (a).
The mixed raw material (a) includes (i-1) a protein having an amino acid sequence of IPP and / or VPP and (i-2) a protein having an amino acid sequence of an antihypertensive peptide such as an ACEI peptide. Examples of these proteins (hereinafter referred to as (i) protein) include proteins containing the sequences IPP, VPP, YP or combinations thereof as the amino acid sequence of the antihypertensive peptide. Specific examples include various animal and vegetable proteins such as milk protein, corn protein, wheat protein, and soy protein.
The mixed raw material (a) contains a specific proportion of (ii) lactic acid bacteria assimilating sugar. As said (ii) lactic acid bacteria assimilation sugar, lactose, glucose, galactose, or a mixture thereof can be mentioned.

The content ratio of (ii) lactic acid bacteria assimilating sugar to (i) protein in the mixed raw material (a) is 1.53 times or more by weight. (ii) A highly efficient antihypertensive peptide can be produced by containing lactic acid bacteria assimilating sugar at this ratio and performing fermentation in combination with the operation described below. The non-fat milk solid content in the mixed raw material (a) is preferably 3 to 15% by weight.
In particular, when the non-fat milk solid content in the mixed raw material (a) is 9 to 15% by weight, the content ratio of (ii) lactic acid bacteria assimilating sugar to protein is 1.53 to 3.9 times by weight. The amount is preferably high because the amount of the peptide mixture containing anti-hypertensive peptide and IPP and / or VPP at a high concentration relative to the non-fat milk solid content subjected to fermentation is high. Further, when the non-fat milk solid content in the mixed raw material (a) is more than 6% by weight and less than 9% by weight, the content ratio of (ii) lactic acid bacteria assimilating sugar to protein (i) It is preferable that the amount is 1.53 to 4.62 times because the production efficiency of the antihypertensive peptide and the peptide mixture containing IPP and / or VPP at a high concentration relative to the non-fat milk solid content subjected to fermentation is increased. Furthermore, when the non-fat milk solid content in the mixed raw material (a) is 3 to 6% by weight, the content ratio of (ii) lactic acid bacteria-assimilating sugar to (i) protein is 1.53 to 9.30 times by weight, In particular, 1.90 to 9.30 times the amount of antihypertensive peptide and IPP and / or peptide mixture containing IPP and / or VPP at a high concentration with respect to the non-fat milk solid content subjected to fermentation is not high, Since the production amount of high blood pressure peptide, IPP and / or VPP becomes high, it may be preferable.

  Such mixed raw material (a) is prepared by adding (ii) lactic acid bacteria assimilating sugar to a milk material such as skim milk powder, whole milk powder, reduced milk, cow milk, condensed milk or a mixture thereof, or a solvent such as water. Can be obtained by adding the milk material and (ii) lactic acid bacteria assimilating sugar.

In the production method of the present invention, it is preferable to perform a heating step such as a sterilization treatment or a sterilization treatment prior to the step (1-B) of subjecting the mixed raw material (a) to lactic acid fermentation described below. For example, when neutralizing the lactic acid bacteria by controlling the pH to about 5.0 to 5.5 or less, the growth of the lactic acid bacteria is dominant and the growth of the contaminating microorganisms is suppressed. However, even in such a case, it is preferable that the mixed raw material (a) is sterilized by heat treatment under conditions such as those employed for ordinary fermented foods. Since the sterilization treatment is less affected by the heat history, the quality of the mixed raw material (a) is less deteriorated, fermentation by lactic acid bacteria proceeds, and a desired antihypertensive peptide or fermented milk containing the peptide can be obtained efficiently.
On the other hand, when high hygienic quality is required for the obtained fermented milk or the like, that is, when it is required to strictly and completely control the growth of contaminating microorganisms, the mixed raw material (a) is used in step (1). It is desirable to sterilize prior to -B).

  Examples of the sterilization method include a batch sterilization method and a continuous sterilization method. The batch sterilization method is a method in which all of the mixed raw materials are enclosed in a pressure vessel and heat-treated under high pressure and high temperature conditions. As sterilization conditions, for example, the temperature is preferably set to 115 ° C. or less, usually about 100 to 115 ° C., and the sterilization time is set to 20 minutes or less, preferably 10 minutes or less, more preferably about 5 to 10 minutes. It is desirable. In this case, if the time to reach the target sterilization temperature is long, or if it takes a long time to cool to the target temperature from the end of the sterilization process, it becomes difficult to manage the heat history in the culture medium, and the change in the components proceeds. It becomes easy. As a result, fermentation by lactic acid bacteria is difficult to proceed, and there is a risk that the amount of the desired peptide accumulated in the culture solution will be reduced.

On the other hand, the continuous sterilization method includes an indirect heating method and a direct heating method, and it is particularly beneficial to use an ultra-high temperature heat sterilizer (UHT). In the case of the indirect heating method, a plate heater type and a tube type can be used. In the case of the direct heating method, a steam injection type and a steam infusion type can be used. In addition, when an ultra-high temperature heat sterilizer (UHT) is used, it can be rapidly cooled by a plate heat exchanger after the heat treatment, and management control of the heat history can be easily performed.
Sterilizing the mixed raw material (a) by continuous sterilization method maintains the quality of the mixed raw material (a) during subsequent lactic acid fermentation, so that fermentation by lactic acid bacteria is easy to proceed, and higher efficiency than batch sterilization method. Thus, it becomes possible to accumulate the antihypertensive peptide in the culture solution. As the sterilization conditions by the continuous sterilization method, for example, the temperature is preferably controlled to 140 ° C. or less, usually about 115 to 140 ° C., and the sterilization time is preferably controlled to 60 seconds or less, particularly about 2 to 30 seconds.

The production method of the present invention includes a step (1-B) of subjecting the mixed raw material (a) to lactic acid fermentation while stirring and adjusting the pH to 4.8 to 5.8 using an alkaline agent.
Unlike normal stationary culture, the agitation can be performed by continuous or intermittent agitation and fermentation. The conditions include, for example, production of protein particles and whey containing antihypertensive peptides. It is preferable to set the stirring conditions according to the stirring device such that the particle size of the obtained protein particles is 5 to 40 μm. At this time, a stirring method in which air entrainment does not occur as much as possible is particularly preferable.

As the agitation method, a stirring blade type and an air lift type can be used. However, in the case of the air lift type, since the generation of shearing force is low, the protein particles are hardly sheared and the particles are not easily refined. In addition, the generation of bubbles is remarkable, and fermentation control may be difficult. Therefore, it is desirable to use a stirring blade. There are various types of stirring blades, such as a paddle type, a propeller type, a helical ribbon type, and a turbine type. Any format can be used as long as the whole culture solution can be continuously and thoroughly mixed during the culture period. In particular, in the case of the turbine type and the paddle type, since the shearing force against the protein particles is large, it is easy to form protein particles having a small particle size. In order to increase the strip efficiency, a baffle plate can be installed in the fermentation bed in accordance with the selection of the shape of the stirring blade. Further, since the shearing force also affects the tank size and the rotational speed, it is preferable to set the conditions in consideration of these.
Usually, when the stirring speed is too high, the particle size of the protein particles becomes too small and becomes less than 5 μm. If the stirring speed is too slow, the particle size of the protein particles becomes too large and becomes larger than 40 μm.

  The pH can be adjusted using the alkaline agent by monitoring the pH in the medium and providing a device or the like that can add an appropriate amount of the alkaline agent when the pH drops below a predetermined value. Although it does not specifically limit as said alkali agent, Sodium hydroxide aqueous solution etc. can be used preferably.

  In the production method of the present invention, fermented milk containing an antihypertensive peptide or a peptide mixture containing IPP and / or VPP at a high concentration can be produced. The antihypertensive peptide can include a peptide selected from the group consisting of tripeptide IPP, tripeptide VPP, dipeptide YP, and mixtures thereof. The content ratio of the antihypertensive peptide, IPP and / or VPP can be 10-30 mg / 100 ml in the total amount of fermented milk. In particular, when the concentration is high, the non-fat milk solid content can be adjusted to 20 to 30 mg / 100 ml by adjusting the non-fat milk solid content to 9 to 15% by weight. As a result, it can be made 10 to 20 mg / 100 ml.

The method for producing an antihypertensive peptide preparation of the present invention includes a step (2-A) of separating whey from fermented milk obtained by the production method.
The separation step (2-A) can be performed by at least one of centrifugation and squeeze filtration. The centrifugation can be performed using a centrifuge, for example, at a rotational speed of about 2000 to 10,000 rpm. On the other hand, the said press filtration can be performed on the pressurization conditions of 2-8 kg / cm < ² > using a press filter.
The whey separated by the separation step (2-A) can be used as it is as the antihypertensive peptide preparation of the present invention, but can also be prepared by further formulation treatment as necessary. Specifically, it can be formulated by performing steps such as concentration, drying, desalting treatment, addition of additives, tableting, and the like. Although the form of the formulation obtained by the formulation is not particularly limited, it can be in the form of an injection, or a tablet, granule, powder, solution, suspension or the like for oral administration.

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited thereto.
The breakdown of non-fat milk solids (SNF) in the examples is as follows: SNF 1% by weight lactose 0.57% by weight, protein 0.43% by weight, SNF 2% by weight lactose 1.13% by weight, protein 0.87% by weight SNF 3% by weight lactose 1.7%, protein 1.3% by weight, SNF 6% by weight lactose 3.4% by weight, protein 2.6% by weight, SNF 9% by weight lactose 5.1% by weight, protein 3.9% by weight %, SNF 12% by weight is lactose 6.8% by weight, protein 5.2% by weight, SNF 15% by weight is lactose 8.5% by weight, protein 6.5% by weight.

Example 1
(Preparation of starter)
9 kg of skim milk powder was dissolved in 91 kg of water to make a total amount of 100 kg. This was sterilized at 115 ° C. for 20 minutes and then cooled to 37 ° C. In addition to this, Lactobacillus helveticus CM-4 strain (National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (Chuo 1-1-1, Higashi 1-1-1, Tsukuba, Japan), Deposit Number BP-6060, Deposit Date 1997 August 15) was inoculated so that the bacterial count was 2.0 × 10 ⁷ cells / ml, and cultured at 37 ° C. for 24 hours to prepare a starter (lactic acid bacterial count 5 × 10 ⁸ cells / ml).
(Adjustment of medium)
Nonfat dry milk was added to ion-exchanged water so that the final concentration of SNF was 9% by weight. Furthermore, lactose was added so that the total content of lactose in skim milk powder was 10% by weight, and the total amount was 1000 kg. This was sterilized at 95 ° C and then cooled to 37 ° C. Therefore, the content ratio of lactose to protein in the obtained medium is about 2.56 times by weight.

(Fermentation by neutralization culture)
In the neutralization culture apparatus, 60 kg of the starter was inoculated into the medium and fermented for 24 hours. As the fermentation progressed, the pH decreased, but when the pH reached 5.0 or less, neutralization was performed by gradually dropping 20 wt% sodium hydroxide until the pH returned to 5.0, and the pH was maintained at 5.0 until the end of the fermentation. . Moreover, it stirred on the conditions shown in Table 1 using the stirring apparatus of the turbine type blade during the fermentation period.
The amount of ACEI peptide (VPP, IPP) in the obtained fermented product was measured by high performance liquid chromatography (manufactured by HITACHI). Moreover, the particle size of the protein in the fermented product was analyzed by a particle size distribution measuring device (manufactured by HORIBA). The whey collection rate was determined by removing the card fraction by centrifugation at 3000 rpm for 10 minutes using a continuous centrifuge (manufactured by Hitachi, Ltd., 20PR52), collecting the whey fraction, This ratio was measured. The results are shown in Table 1.
From Table 1, it was found that by carrying out neutralization operation and strong stirring operation, the particle size of the protein particles was reduced, and the amount of ACEI peptide produced and the whey recovery rate were increased compared to those without neutralization operation.

Example 2
(Preparation of medium)
Nonfat dry milk was added to ion-exchanged water so that the final concentration of SNF was the concentration (% by weight) shown in Table 2. Furthermore, lactose was added so that the lactose content ratio combined with lactose in skim milk powder was 10% by weight, and the total amount was 1000 kg respectively. These were sterilized at 95 ° C. and then cooled to 37 ° C. to prepare a medium. Therefore, the content ratio of lactose to protein in the obtained medium is about 33.3 times for SNF 1% by weight, about 11.49 times for SNF 2% by weight, about 7.69 times for SNF 3% by weight, SNF 6% by weight is about 3.85 times, SNF 9% by weight is about 2.56 times, SNF 12% by weight is about 1.92 times, and SNF 15% by weight is about 1.54 times.
(Fermentation by neutralization culture)
In the neutralization culture apparatus, 60 kg of the starter prepared in Example 1 was inoculated into each medium and fermented for 24 hours. The pH decreased as the fermentation progressed, but when the pH reached 5.0 or less, neutralization was performed by gradually dropping 20 wt% sodium hydroxide until the pH returned to 5.0, and the pH was maintained at 5.0 until the end of the fermentation. . Further, during the fermentation period, stirring was continued at 150 rpm using a turbine-type blade stirrer so that the particle size of protein particles in the fermentation medium after neutralization culture was 8 to 40 μm. The amount of ACEI peptide (VPP, IPP) in the obtained fermented product was measured by high performance liquid chromatography. The results are shown in Table 2. Table 3 shows the whey collection rate.

Comparative Example 1
Stirring and neutralization were not performed, and lactose was not added. The same procedure as in Example 2 was performed, fermentation was performed, and the amount of ACEI peptide (VPP, IPP) and whey recovery rate were the same as in Example 2. Measured. The results are shown in Table 2 and Table 3, respectively. In the obtained fermented product, a hard curd was formed, and the particle size became a large value exceeding 40 μm.
In addition, the content ratio of lactose to the protein in the medium used in Comparative Example 1 was not added with lactose, so that SNF 1 wt% was about 1.30 times, SNF 2 wt% was about 1.30 times, SNF 3% by weight approximately 1.30 times, SNF 6% by weight approximately 1.30 times, SNF 9% by weight approximately 1.30 times, SNF 12% by weight approximately 1.30 times, SNF 15% by weight Is about 1.30 times.

Comparative Example 2
The procedure was the same as in Example 2 except that the stirring and neutralization operations were not performed. Fermentation was performed, and the amount of ACEI peptide (VPP, IPP) and whey recovery rate were measured in the same manner as in Example 2. The results are shown in Table 2 and Table 3, respectively. In the obtained fermented product, a hard curd was formed, and the particle size became a large value exceeding 40 μm.
In addition, the content ratio of lactose to the protein in the medium used in Comparative Example 2 is the same as in Example 2.

Comparative Example 3
The neutralization operation was not performed, and lactose was not added. The same operation as in Example 2 was performed, fermentation was performed, and the amount of ACEI peptide (VPP, IPP) and whey recovery were measured in the same manner as in Example 2. . The results are shown in Table 2 and Table 3, respectively. In the obtained fermented product, the protein was chopped and the particle size became a small value of 40 μm or less.
The content ratio of lactose to the protein in the medium used in Comparative Example 3 is the same as in Comparative Example 1.

Comparative Example 4
The procedure was the same as in Example 2 except that lactose was not added, fermentation was performed, and the amount of ACEI peptide (VPP, IPP) and whey recovery were measured in the same manner as in Example 2. The results are shown in Table 2 and Table 3, respectively. In the obtained fermented product, the protein was chopped and the particle size became a small value of 40 μm or less.
The content ratio of lactose to the protein in the medium used in Comparative Example 4 is the same as in Comparative Example 1.

Example 3
The procedure was the same as in the case of 9% by weight of SNF in Example 2 except that the pH maintained in the neutralization operation was 5.5, and a fermented product was obtained and the amount of ACEI peptide (VPP, IPP) and whey recovery were measured. . The results are shown in Table 4. In the obtained fermented product, the protein was chopped and the particle size became a small value of 40 μm or less.

Comparative Examples 5-7
A fermentation product was obtained in the same manner as in Example 2 except that the pH maintained in the neutralization operation was 4.0 (Comparative Example 5), 4.5 (Comparative Example 6) or 6.0 (Comparative Example 7). VPP, IPP) and whey recovery were measured. The results are shown in Table 4. In the obtained fermented product, the protein was chopped and the particle size became a small value of 40 μm or less.

Example 4, Comparative Examples 8 and 9
In the case of 9% by weight of SNF in Example 2 except that the pH maintained in the neutralization operation is 4.0 (Comparative Example 8), 5.0 (Example 4) or 6.0 (Comparative Example 9) and the fermentation time is 48 hours The same operation as above was performed, and fermentation by neutralization culture was performed. The amount of ACEI peptide (VPP, IPP) during the fermentation period was measured. The results are shown in Table 5. In the obtained fermented product, the protein was chopped and the particle size became a small value of 40 μm or less.

Example 5
The fermented product was obtained in the same manner as in Example 2 except that the lactose content in the medium and the lactose content relative to the protein (weight ratio) were as shown in Table 6, respectively. The amount of peptide (VPP, IPP) was measured over time. The results are shown in Table 7. In the obtained fermented product, the protein was chopped and the particle size became a small value of 40 μm or less. The whey recovery rate was good at over 70%.

Example 6
A medium was prepared by adding to skim milk powder 9% by weight (breakdown: lactose 5.1% by weight, milk protein 3.9% by weight) and lactose 4% by weight in ion-exchanged water. A starter prepared by the same procedure as in Example 1 was added to this medium so as to be 3% by weight, so that the total amount was 2.3 liters. This mixture was fermented at 37 ° C. for 24 hours. Although the pH decreased as the fermentation progressed, a neutralization operation was carried out in which 20% by weight of sodium hydroxide was added dropwise at 5 ml / min, and the pH was maintained at 5.0 ± 0.05 until the end of the fermentation. During the fermentation period, the medium was continuously stirred at 150 rpm by a stirring device having a turbine type stirring blade. When the amount of dipeptide YP in the obtained fermented product was measured by LC / MS, it was 7.7 mg / 100 ml. In the obtained fermented product, protein was fragmented, and the particle size was a small value of 40 μm or less. The whey recovery rate was 75%, which was good.

Comparative Example 10
Except that lactose was not added to the medium and neutralization and stirring were not performed, the same procedure as in Example 7 was performed to obtain a fermented product. The amount of dipeptide YP was measured and found to be 1.2 mg / 100 ml. In the obtained fermented product, a hard curd was formed, and the particle size became a large value exceeding 40 μm. The whey recovery rate was poor at 30%.

Example 7
Prepare a medium by adding 9% by weight of skim milk powder (breakdown: lactose: 5.1% by weight, milk protein: 3.9% by weight) and lactose, glucose, galactose, sucrose or maltose in Table 7 to ion-exchanged water. did. A starter prepared by the same procedure as in Example 1 was added to this medium so as to be 3% by weight. This mixture was fermented at 37 ° C. for 24 hours. Although the pH decreased as the fermentation progressed, a neutralization operation was carried out in which 20% by weight of sodium hydroxide was added dropwise at 5 ml / min, and the pH was maintained at 5.0 ± 0.05 until the end of the fermentation. During the fermentation period, the medium was continuously stirred at 150 rpm by a stirring device having a turbine type stirring blade. The amount of ACEI peptide (VPP, IPP) in the obtained fermented product was measured by high performance liquid chromatography. The results are shown in Table 8. In the obtained fermented product, the particle size of the protein became a small value of 40 μm or less. The whey recovery rate was 70% or higher.

Example 8
By the same operation as in Example 1, 100 kg of neutralized cultured fermented milk was obtained. Using a continuous centrifuge (trade name H-923, manufactured by Kokusan Co., Ltd.), insoluble protein was separated at a rotation speed of 13000 rpm and a liquid feed speed of 150 ml / min, and the water in the clarified liquid was removed using a vacuum concentrator. Until concentrated.
Sodium lactate contained in 30 kg of this concentrate was removed using an electrodialyzer (trade name TS-2-10, manufactured by Tokuyama Soda Co., Ltd.). 10 kg of this desalted concentrated solution was pulverized using a spray dryer (trade name: FGA-8, manufactured by Ogawara Chemical Co., Ltd.). When the amount of ACEI peptide (VPP, IPP) in the obtained powder was measured by high performance liquid chromatography, it was 3.6 mg / g. To 2 kg of this powder, 1.0 kg of crystalline sorbitol (Merck) and 0.08 kg of sugar ester (Daiichi Kogyo Seiyaku Co., Ltd., trade name “DK Ester F-20W”) were added and mixed. This mixture was tableted using a tableting machine (trade name HT-P18, manufactured by Hata Iron Works Co., Ltd.) to produce tablet confectionery (total weight 2 g / tablet). The amount of ACEI peptide (VPP, IPP) in one tablet of the obtained tablet confectionery was analyzed by high performance liquid chromatography, and it was 4.7 mg.

Example 9
It was added to ion-exchanged water so that skim milk powder 3% and lactose 10.6% were added to make 1000 kg. These were sterilized by a plate heater type ultra high temperature heat sterilization method (UHT). The sterilization temperature was 115, 120, 130, and 140 ° C., and the sterilization time was 30 seconds. After the heat sterilization treatment, the plate was quickly cooled to 37 ° C. using a plate heater type heat exchanger. On the other hand, the above raw materials were sterilized by batch sterilization at 105, 110, 115, and 120 ° C. for 10 minutes.
3% starter was added to these, and neutralization culture was started. The method of fermentation by neutralization culture and the measurement of IPP and VPP peptide concentrations in the fermented product were in accordance with Example 2. FIG. 1 shows the result of setting the UHT sterilization time to 30 seconds, and FIG. 2 shows the result of setting the sterilization time by the batch sterilization method to 10 minutes. In the graph, ◯ represents the VPP peptide concentration, and Δ represents the IPP peptide concentration.
From the results shown in FIGS. 1 and 2, it was found that the IPP and VPP peptide concentrations were high at 115 to 140 ° C. for UHT and 105 to 115 ° C. for batch sterilization.

Claims (22)

(i-1) a step of preparing a mixed raw material (a) containing a protein having at least one amino acid sequence of Ile Pro Pro and Val Pro Pro, (ii) lactic acid bacteria assimilating sugar, and (iii) lactic acid bacteria (1) -A)
A step (1-B) of producing a peptide mixture containing at least one of Ile Pro Pro and Val Pro Pro by subjecting the mixed raw material (a) to lactic acid fermentation while adjusting the pH using an alkaline agent. Including
In the step (1-A), the content ratio of the assimilable lactic acid bacteria to the (i-1) protein in the mixed raw material (a) is 1.53 times or more by weight, and the step (1 A method for producing a peptide mixture containing at least one of Ile Pro Pro and Val Pro Pro at a high concentration, wherein the pH adjustment in -B) is controlled to 4.8 to 5.8.   (i-1) The production method according to claim 1, wherein the protein is one or more selected from the group consisting of milk protein, corn protein, wheat protein and soybean protein.   The mixed raw material (a) comprises (i-1) a protein containing milk material selected from the group consisting of skim milk powder, whole milk powder, reduced milk, cow milk, condensed milk and a mixture thereof as a material containing protein. Manufacturing method.   The non-fat milk solid content in the mixed raw material (a) is 9 to 15% by weight, and the mixed raw material (a) contains (ii) lactic acid bacteria assimilating sugar to (i-1) protein. The production method according to claim 3, wherein the weight ratio is 1.53 to 3.9 times.   The non-fat milk solid content in the mixed raw material (a) is more than 6% by weight and less than 9% by weight, and (ii) lactic acid bacteria assimilation property for (i-1) protein in the mixed raw material (a) The production method according to claim 3, wherein the sugar content is 1.53 to 4.62 times by weight.   The non-fat milk solid content in the mixed raw material (a) is 3 to 6% by weight, and the mixed raw material (a) has a content ratio of (ii) lactic acid bacteria assimilating sugar to (i-1) protein The production method according to claim 3, wherein the amount is 1.53 to 9.3 times by weight.   The process according to claim 1, further comprising a step of sterilizing the mixed raw material (a) prepared in the step (1-A) under a condition of 140 ° C or lower by a continuous sterilization method before the step (1-B).   The process according to claim 1, further comprising a step of sterilizing the mixed raw material (a) prepared in the step (1-A) by a batch sterilization method at 115 ° C or lower before the step (1-B).   The process according to claim 1, wherein the alkaline agent used in the step (1-B) is sodium hydroxide.   The process according to claim 1, wherein in the step (1-B), the mixed raw material (a) is stirred under the condition that the average particle size of the protein in the mixed raw material (a) is 5 to 40 µm. (i-2) a step (1-A) of preparing a mixed raw material (a) containing a protein having an amino acid sequence of an antihypertensive peptide, (ii) lactic acid bacteria assimilating sugar, and (iii) lactic acid bacteria,
Lactic acid fermentation while stirring the mixed raw material (a) and adjusting the pH using an alkaline agent to produce an antihypertensive peptide (1-B),
In the step (1-A), in the mixed raw material (a), the content ratio of the (i) lactic acid bacteria assimilating sugar to the (i-2) protein is 1.53 times or more by weight, and the step (1 A method for producing fermented milk containing an antihypertensive peptide, wherein the pH adjustment in -B) is controlled at 4.8 to 5.8.   The method according to claim 11, wherein (i-2) the protein is selected from the group consisting of milk protein, corn protein, wheat protein, soy protein, and a mixture thereof.   The method according to claim 11, wherein the antihypertensive peptide comprises a peptide selected from the group consisting of tripeptide Ile Pro Pro, tripeptide Val Pro Pro, dipeptide Tyr Pro, and mixtures thereof.   12. The mixed raw material (a) comprises (i-2) a milk material selected from the group consisting of skim milk powder, whole milk powder, reduced milk, cow milk, condensed milk and a mixture thereof as the material containing protein. Manufacturing method.   The non-fat milk solid content in the mixed raw material (a) is 9 to 15% by weight, and the mixed raw material (a) has a content ratio of (ii) lactic acid bacteria assimilating sugar to (i-2) protein The production method according to claim 14, wherein the amount is 1.53 to 3.9 times by weight.   The non-fat milk solid content in the mixed raw material (a) is more than 6% by weight and less than 9% by weight, and (ii) lactic acid bacteria assimilation property for (i-2) protein in the mixed raw material (a) The method according to claim 14, wherein the sugar content is 1.53 to 4.62 times by weight.   The non-fat milk solid content in the mixed raw material (a) is 3 to 6% by weight, and the mixed raw material (a) has a content ratio of (ii) lactic acid bacteria assimilating sugar to (i-2) protein The method according to claim 14, wherein the amount is 1.53 to 9.3 times by weight.   The process according to claim 11, comprising a step of sterilizing the mixed raw material (a) prepared in the step (1-A) under a condition of 140 ° C or lower by a continuous sterilization method before the step (1-B).   The process according to claim 11, further comprising a step of sterilizing the mixed raw material (a) prepared in the step (1-A) by a batch sterilization method at 115 ° C or lower before the step (1-B).   The process according to claim 11, wherein the alkaline agent used in the step (1-B) is sodium hydroxide.   The process according to claim 11, wherein in the step (1-B), the mixed raw material (a) is stirred under the condition that the average particle size of the protein in the mixed raw material (a) is 5 to 40 µm. The manufacturing method of an antihypertensive peptide formulation including the process (2-A) which isolate | separates whey from the fermented milk obtained by the manufacturing method of Claim 11.

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