JP2631202B2 - Peptide production method - Google Patents

Peptide production method

Info

Publication number
JP2631202B2
JP2631202B2 JP6271862A JP27186294A JP2631202B2 JP 2631202 B2 JP2631202 B2 JP 2631202B2 JP 6271862 A JP6271862 A JP 6271862A JP 27186294 A JP27186294 A JP 27186294A JP 2631202 B2 JP2631202 B2 JP 2631202B2
Authority
JP
Japan
Prior art keywords
enzyme
bitterness
peptide
peptidase
rhizopus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
JP6271862A
Other languages
Japanese (ja)
Other versions
JPH07274995A (en
Inventor
賢一 平野
浩史 伊藤
隆司 藤吉
哲郎 中村
幸孝 宿野部
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
AMANO SEIYAKU KK
YUKIJIRUSHI NYUGYO KK
Original Assignee
AMANO SEIYAKU KK
YUKIJIRUSHI NYUGYO KK
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by AMANO SEIYAKU KK, YUKIJIRUSHI NYUGYO KK filed Critical AMANO SEIYAKU KK
Priority to JP6271862A priority Critical patent/JP2631202B2/en
Publication of JPH07274995A publication Critical patent/JPH07274995A/en
Application granted granted Critical
Publication of JP2631202B2 publication Critical patent/JP2631202B2/en
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

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Classifications

    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02PCLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00Technologies relating to chemical industry
    • Y02P20/50Improvements relating to the production of bulk chemicals
    • Y02P20/52Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts

Landscapes

  • Meat, Egg Or Seafood Products (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Description

【発明の詳細な説明】DETAILED DESCRIPTION OF THE INVENTION

【0001】[0001]

【産業上の利用分野】本発明は、リゾプス属菌の生産す
るプロティナーゼ及びペプチダーゼを含む複合酵素を用
いて、苦味のないペプチドを製造する方法及びペプチド
から苦味を消去する方法に関するものである。本発明に
よって調製されたペプチドは苦味を有しないので、非常
に食べ易くなり、ペプチドが本来有する栄養価を充分に
利用することができる。したがって本発明は、食品、栄
養食品、医療、飼料、微生物工業等の各種工業において
広範且つ有利に利用することができる。
BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a peptide having no bitter taste and a method for eliminating bitter taste from the peptide by using a complex enzyme containing a proteinase and a peptidase produced by a genus Rhizopus. Since the peptide prepared according to the present invention has no bitterness, it is very easy to eat and can fully utilize the nutritional value originally possessed by the peptide. Therefore, the present invention can be widely and advantageously used in various industries such as food, nutritional food, medical care, feed, and microbial industry.

【0002】[0002]

【従来の技術】蛋白質を分解してペプチドとする方法に
おいて、酵素を用いる方法はマイルドな条件で操作が出
来る等の利点があるために工業的に多用されている。し
かしながら、このような方法によって製造したペプチ
ド、つまり酵素分解ペプチドには苦味が付随しており、
これを栄養食として投与しても極端な場合には幼児や病
人等によっては吐き出してしまう場合もあって、結局投
与することができないこととなり、ペプチドが本来有す
る栄養性その他の特性が利用できないことがしばしば生
じる。
2. Description of the Related Art In a method of decomposing a protein into a peptide, a method using an enzyme is widely used industrially because it has advantages such as operation under mild conditions. However, peptides produced by such a method, that is, enzymatically degraded peptides are accompanied by bitterness,
Even if this is administered as a nutritional diet, in extreme cases, it may be exhaled depending on the infant or sick person, etc., resulting in the inability to administer, and the inability to utilize the nutritional and other properties inherent in the peptide. Often occurs.

【0003】これを改良する目的で、例えば苦味を生じ
ないような酵素をスクリーニングする方法が従来より行
われており、例えばペニシリウム・シトリナム等ペニシ
リウム属由来の中性及び/又はアルカリ性プロテアーゼ
を利用する方法が本発明者らによって開発されている
(特開昭62−14796号)。
For the purpose of improving this, for example, a method of screening for an enzyme that does not cause bitterness has been conventionally performed, for example, a method using a neutral and / or alkaline protease derived from the genus Penicillium such as Penicillium citrinum. Have been developed by the present inventors (JP-A-62-14796).

【0004】しかしながら、本発明のようにリゾプス
(Rhizopus)属菌由来の酵素、しかもプロティナーゼと
ペプチダーゼを含む複合酵素を用いて苦味を消去したペ
プチドを製造する技術は従来全く知られておらず、新規
である。
[0004] However, the technique of producing a peptide having a bitter taste eliminated by using an enzyme derived from the genus Rhizopus and a complex enzyme containing a proteinase and a peptidase as in the present invention has not been known at all, and a novel technique has been proposed. It is.

【0005】[0005]

【発明が解決しようとする課題】本発明は、上記した技
術の現状に鑑みてなされたものであって、従来の酵素分
解ペプチドの致令的欠陥であるところの苦味を除去する
技術を開発する目的でなされたものである。
DISCLOSURE OF THE INVENTION The present invention has been made in view of the above-mentioned state of the art, and develops a technique for removing bitterness, which is a conventional defect of enzymatically degraded peptides. It was done for the purpose.

【0006】[0006]

【課題を解決するための手段】本発明は、上記目的を達
成するためになされたものであって、各方面から検討の
結果、反応条件がマイルドであり、比較的容易に分解度
をコントロールすることができて目的とするペプチドが
自由に得られる等の利点があることから、生物学的方
法、特に微生物を用いる方法に着目した。そこで各種の
微生物について鋭意スクリーニングを行った結果、ある
種のリゾプス属菌に苦味のないペプチドを生成する作用
があることを発見した(第1表)。
DISCLOSURE OF THE INVENTION The present invention has been made to achieve the above object, and as a result of investigations from various aspects, the reaction conditions are mild and the degree of decomposition can be controlled relatively easily. Therefore, the present inventors have paid attention to biological methods, particularly methods using microorganisms, because they have the advantage of being able to obtain the desired peptide freely. Accordingly, as a result of intensive screening of various microorganisms, it was found that certain Rhizopus bacteria have an action of producing a peptide having no bitterness (Table 1).

【0007】[0007]

【表1】 第 1 表 ─────────────────────────── 菌 株 苦味生成 ─────────────────────────── Rhizopus pseudochinesis IAM 6042 − Rhizopus formosaensis No.3540 − Rhizopus peka No.3626 − Rhizopus hangchow No.3545 − Rhizopus javanicus No.3553 − Rhizopus niveus No.3593 − Aspergillus oryzae IAM 2686 + Aspergillus niger IAM 2020 + Bacillus cereus IAM 1229 + Bacillus subtilis IAM 1163 + ───────────────────────────[Table 1] Table 1 生成 Bacterial strain generation ──────────── ─────────────── Rhizopus pseudochinesis IAM 6042 − Rhizopus formosaensis No. 3540 − Rhizopus peka No. 3626 − Rhizopus hangchow No. 3545 − Rhizopus javanicus No. 3553 − Rhizopus niveus No. 3593 − Aspergillus oryzae IAM 2686 + Aspergillus niger IAM 2020 + Bacillus cereus IAM 1229 + Bacillus subtilis IAM 1163 + ───────────────────────────

【0008】そして上記作用は1種の酵素ではなく複合
酵素が関与していること、そして更に苦味のないペプチ
ドを直接的に蛋白質から生成する作用のほかに、他の酵
素によって生成した苦味を有するペプチドから苦味を除
去する作用もある、という新規にして有用な知見も得
た。
[0008] The above-mentioned action involves not a single enzyme but a complex enzyme. Further, in addition to the action of directly producing a peptide having no bitterness from a protein, it has a bitter taste generated by another enzyme. A new and useful finding that it also has the action of removing bitterness from peptides was obtained.

【0009】そしてこれらの新知見に基づき更に検討の
結果、上記酵素がプロティナーゼとペプチダーゼを含む
複合酵素であり、またこれらの酵素を産生して苦味を除
去しうる微生物がリゾプス・シュードキネシス(Rhizop
us pseudochinesis)、リゾプス・フォルモサエンシス
(R. formosaensis)、リゾプス・ペーカ(R. peka)、
リゾプス・ハンショウ(R. hangchow)、リゾプス・ジ
ャバニクス(R. javanicus)、リゾプス・ニベウス(R.
niveus)等のリゾプス属菌であることも併せ確認した
(第2表)。
As a result of further studies based on these new findings, the above enzyme is a complex enzyme containing proteinase and peptidase, and a microorganism capable of producing these enzymes and eliminating bitterness is Rhizops pseudokinesis (Rhizop pseudokinase).
us pseudochinesis), Rhizopus formosaensis (R. formosaensis), Rhizopus peka (R. peka),
R. hangchow, R. javanicus, R. niveus
niveus) and the like (Table 2).

【0010】[0010]

【表2】 第 2 表 ─────────────────────────── 菌 株 苦味生成 ─────────────────────────── Rhizopus pseudochinesis IAM 6042 − Rhizopus formosaensis No.3540 − Rhizopus peka No.3626 − Rhizopus hangchow No.3545 − Rhizopus javanicus No.3553 − Rhizopus niveus No.3593 − Rhizopus arrhizus IAM 6056 + Rhizopus delmer IAM 6062 + Rhizopus oryzae IAM 6060 + ───────────────────────────[Table 2] Table 2 生成 Bacterial strain generation ──────────── ─────────────── Rhizopus pseudochinesis IAM 6042 − Rhizopus formosaensis No. 3540 − Rhizopus peka No. 3626 − Rhizopus hangchow No. 3545 − Rhizopus javanicus No. 3553 − Rhizopus niveus No. 3593 − Rhizopus arrhizus IAM 6056 + Rhizopus delmer IAM 6062 + Rhizopus oryzae IAM 6060 + ───────────────────────────

【0011】本発明は、これらの新規知見を基礎とし、
更に研究の結果完成されたものであって、リゾプス属菌
が生成する複合酵素(プロティナーゼ、ペプチダーゼ)
によるペプチドからの苦味除去システムに関するもので
ある。
The present invention is based on these new findings,
A complex enzyme (proteinase, peptidase) produced by Rhizopus sp.
And a system for removing bitterness from peptides.

【0012】本発明に係る複合酵素剤を製造するには、
リゾプス属菌を培養し、培養物から酵素を取得すればよ
い。これらの処理は常法によって行えばよく、例えばリ
ゾプス属菌の培養は、ふすま等を用いる固体培養法ある
いは液体培養法といった糸状菌において常用される培養
技術が適宜用いられる。
To produce the complex enzyme preparation of the present invention,
Rhizopus bacteria may be cultured and the enzyme may be obtained from the culture. These treatments may be carried out by a conventional method. For example, for the cultivation of Rhizopus bacteria, a culture technique commonly used for filamentous fungi such as a solid culture method using bran or a liquid culture method is appropriately used.

【0013】培養によって産生された酵素は常法によっ
て取得することができる。つまり酵素が菌体内に産生蓄
積される場合には菌体を分離した後これを破壊した後、
また菌体外酵素の場合には培養液から酵素を抽出し、常
法にしたがった塩析、限外濾過、逆滲透膜、分子篩、イ
オン交換樹脂等の処理を行って精製する。
[0013] The enzyme produced by the culture can be obtained by a conventional method. In other words, when the enzyme is produced and accumulated in the cells, the cells are separated and destroyed,
In the case of an extracellular enzyme, the enzyme is extracted from the culture solution and purified by subjecting to salting out, ultrafiltration, reverse osmosis membrane, molecular sieve, ion exchange resin, etc. according to a conventional method.

【0014】このようにして得られた酵素はそのまま
で、あるいは乾燥した後、酵素剤として使用する。また
場合によっては、精製することなく粗製酵素ないし粗製
酵素液として、粗製のまま酵素剤として使用してもよ
い。必要ある場合には他の酵素を添加したり、あるいは
酵素製剤上容認される成分を常法にしたがって添加配合
することも可能である。またこれとは逆に各酵素を各々
分離しておき、必要に応じてこれを併用してもよい。
The enzyme thus obtained is used as it is or after drying, as an enzyme preparation. In some cases, the enzyme may be used as a crude enzyme or a crude enzyme solution without purification, or as a crude enzyme. If necessary, another enzyme can be added, or a component acceptable in an enzyme preparation can be added and blended according to a conventional method. Conversely, each enzyme may be separated from each other, and these may be used in combination as needed.

【0015】本発明の酵素剤は、蛋白質を分解して苦味
を生成しない特徴を有しているが、その特徴を明確にす
る為に酵素剤中に含まれるプロティナーゼ及びペプチダ
ーゼを各々精製しその特性について検討を行なった。な
お、用いた酵素粉末のペプチダーゼはプロティナーゼ1
単位当り24.3単位混在していた。
The enzyme preparation of the present invention has a characteristic that it does not decompose proteins to produce bitterness, but in order to clarify the characteristics, proteinase and peptidase contained in the enzyme preparation are respectively purified and their properties are determined. Was examined. The peptidase of the enzyme powder used was proteinase 1
24.3 units were mixed per unit.

【0016】(1)プロティナーゼの特性 酵素粉末を酢酸緩衝液(pH4.0)に溶解後、ペプチ
ダーゼを失活させる為に50℃で30分間加温を行な
い、その後透析を行なった。リン酸緩衝液(pH7.
0)にて緩衝化したDEAEセファロースに吸着させ、
洗浄後NaClにて濃度勾配法で溶離し、活性画分を分
取し分子量10,000分画の限外濾過膜にて濃縮し精
製した。この標品の夾雑ペプチダーゼはプロティナーゼ
1単位当り0.016単位であり約1500倍純度が上
がった。
(1) Characteristics of Proteinase After dissolving the enzyme powder in acetate buffer (pH 4.0), the mixture was heated at 50 ° C. for 30 minutes to inactivate the peptidase, and then dialyzed. Phosphate buffer (pH 7.
Absorb to DEAE Sepharose buffered in 0),
After washing, the fraction was eluted with NaCl by a concentration gradient method, and the active fraction was collected, concentrated and purified by an ultrafiltration membrane having a molecular weight of 10,000. The contaminating peptidase of this sample was 0.016 units per 1 unit of proteinase, and the purity was increased about 1500 times.

【0017】得られた標品を用いた酸カゼインの分解を
行い、ペプチダーゼ活性を多く含むカビ起源の市販酵素
剤である「プロテアーゼP」(天野製薬(株)製)と、
分解物の分子量分布の比較を行なった。分子量分布の結
果を図1に示した。この図面から明らかなように、ペプ
チダーゼを含まない本発明の酵素剤に含まれるプロティ
ナーゼ(B)は、ペプチダーゼを多く含む市販酵素剤
(A)より蛋白質をより低分子化する作用が強いことが
判る。
The obtained sample is used to decompose acid casein, and a protease-derived commercially available enzyme agent having a high peptidase activity, "Protease P" (manufactured by Amano Pharmaceutical Co., Ltd.);
The molecular weight distribution of the decomposition products was compared. The results of the molecular weight distribution are shown in FIG. As is apparent from this drawing, the proteinase (B) contained in the enzyme preparation of the present invention containing no peptidase has a stronger effect of lowering the molecular weight of the protein than the commercially available enzyme preparation (A) containing more peptidase. .

【0018】(2)ペプチダーゼの特性 酵素粉末を精製水に溶解し透析をした後、リン酸緩衝液
(pH8.0)にて緩衝化したDEAEセファロースカ
ラムに吸着させ、洗浄後NaClにて濃度勾配法で溶離
を行なった。活性画分を分取し、その後プロティナーゼ
を失活させる為にpH9.0で50℃、30分加温処理
を行い、透析後エバポレーターで濃縮し精製した。この
標品の夾雑ペプチダーゼはプロティナーゼ1単位当り換
算で約3000倍純度が上がった。
(2) Characteristics of Peptidase The enzyme powder was dissolved in purified water, dialyzed, adsorbed on a DEAE Sepharose column buffered with a phosphate buffer (pH 8.0), washed, and concentrated with NaCl. Elution was performed by the method. The active fraction was collected and then heated at 50 ° C. for 30 minutes at pH 9.0 to inactivate the proteinase, and after dialysis, concentrated by an evaporator and purified. The purity of the contaminating peptidase of this sample was increased by about 3000 times in terms of 1 unit of proteinase.

【0019】このペプチダーゼ(リゾプス・ハンショウ
No.3545菌ペプチダーゼ)の酵素的性質に関し
て、至適pH、pH安定性、至適濃度、温度安定性につ
いて検討を行い、図2の結果を得た。この結果から明ら
かなように、この酵素は、pH及び温度安定性にすぐ
れ、至適pH及び至適温度域も広くて、使用しやすい酵
素であり工業的用途に適していることが判る。
With respect to the enzymatic properties of this peptidase (Rhizopus hansha No. 3545 peptidase), the optimum pH, pH stability, optimum concentration, and temperature stability were examined, and the results shown in FIG. 2 were obtained. As is clear from these results, this enzyme is excellent in pH and temperature stability, has a wide optimum pH and temperature range, is easy to use, and is suitable for industrial use.

【0020】次に、得られた標品を用いて、苦味の消去
実験を行なった。市販酵素剤である「プロテアーゼA」
(天野製薬(株)製)」を用いて酸カゼインを分解し、
苦味を生成させた後、本標品を4000単位添加し45
℃で3時間反応した結果、ペプチダーゼ無添加では(+
2)の苦味であったのが本ペプチダーゼを添加した場合
は苦味が無くなった。
Next, an experiment for eliminating bitterness was carried out using the obtained sample. "Protease A", a commercially available enzyme preparation
(Manufactured by Amano Pharmaceutical Co., Ltd.) "
After generating bitterness, add 4000 units of this sample and add 45 units.
C. for 3 hours. As a result, without adding peptidase, (+
When the present peptidase was added, the bitterness of 2) disappeared.

【0021】以上記したように、本発明の酵素剤に含ま
れるプロティナーゼは蛋白質を低分子化する作用が強
く、また同時に含まれるペプチダーゼは苦味を消去する
作用が強いという特色を持っており、その両者の作用で
蛋白質を高度に分解しても従来検討されてきた酵素剤に
比較して、苦味の生成が無い理由であろうと考えられ
る。
As described above, the proteinase contained in the enzyme preparation of the present invention has a strong action of lowering the molecular weight of a protein, and the peptidase contained at the same time has a strong action of eliminating bitterness. It is considered that even if the protein is highly degraded by the action of both, there is no generation of bitterness compared to the enzyme agent which has been conventionally studied.

【0022】本発明に係る複合酵素剤を蛋白質に作用さ
せれば、苦味のない蛋白分解物つまりペプチドを直接得
ることができる。処理対象蛋白質としては、カゼインそ
の他の乳蛋白、魚肉蛋白、畜肉蛋白等の動物性蛋白;大
豆蛋白、小麦蛋白、米蛋白、落花生蛋白、菜種蛋白、ヒ
マワリ蛋白等の植物性蛋白;酵母蛋白等の微生物蛋白そ
の他すべての蛋白が単用又は併用できる。これらの蛋白
質は、変性したものでも未変性のものでもよいし、精製
されたものでも粗製のものでもよく、また更に不純物を
多く含んだ例えば豚肉の煮汁のようなものでもよい。
When the complex enzyme preparation according to the present invention is applied to a protein, a protein degradation product having no bitterness, that is, a peptide can be directly obtained. Proteins to be processed include animal proteins such as casein and other milk proteins, fish proteins, and animal meat proteins; vegetable proteins such as soy protein, wheat protein, rice protein, peanut protein, rapeseed protein, sunflower protein; yeast proteins, etc. Microbial proteins and all other proteins can be used alone or in combination. These proteins may be denatured or undenatured, may be purified or crude, and may further contain impurities, such as pork broth.

【0023】また、本発明に係る酵素剤は、これを、他
の酵素によって製造した苦味を有するペプチドに作用さ
せれば、これから苦味を除去することができる。酵素剤
を作用させる条件については格別の限定はなく、通常、
蛋白ないしペプチド濃度として約1〜30%、温度は3
0〜58℃程度が適当であって、酵素濃度は酵素力価及
び基質濃度等によって適宜選択すればよい。
Further, the enzyme agent according to the present invention can remove bitter taste from the enzyme agent by acting it on a peptide having bitter taste produced by another enzyme. There are no particular restrictions on the conditions under which the enzyme agent acts, and usually,
The protein or peptide concentration is about 1 to 30% and the temperature is 3
The temperature is suitably about 0 to 58 ° C., and the enzyme concentration may be appropriately selected depending on the enzyme titer, the substrate concentration and the like.

【0024】[0024]

【実施例】以下、本発明を試験例、製造例及び実施例に
より更に詳しく説明する。
The present invention will be described in more detail with reference to Test Examples, Production Examples and Examples.

【0025】試験例1 (蛋白質の部分分解における苦味生成の比較実験)5%
酸カゼイン溶液(pH7.0)に市販酵素剤(天野製薬
(株)製)及び本発明の菌株より得られた粗製酵素を添
加し、45℃で1時間反応させた分解率15%の部分分
解溶液を得、苦味を官能試験して第3表の結果を得た。
分解率は反応液を酢酸でpH4.6に調整し可溶画分を
ケルダール定量し、全体に対する比較として表わした。
又、苦味の判定はカフェインによる苦味を指標として、
カフェイン濃度0.10%の苦味を(+5)、0.08
%を(+4)、0.06%を(+2)、0.02%を
(+1)、そして苦味無しを(0)と表示した。
Test Example 1 (Comparative experiment of bitterness generation in partial decomposition of protein) 5%
A commercially available enzyme preparation (manufactured by Amano Pharmaceutical Co., Ltd.) and a crude enzyme obtained from the bacterial strain of the present invention were added to an acid casein solution (pH 7.0), and the mixture was reacted at 45 ° C. for 1 hour. The solution was obtained and subjected to a sensory test for bitterness to obtain the results shown in Table 3.
The decomposition rate was adjusted to pH 4.6 with acetic acid, and the soluble fraction was subjected to Kjeldahl quantification, and expressed as a comparison with the whole.
In addition, the determination of bitterness is based on the bitterness due to caffeine as an index.
Caffeine concentration 0.10% bitterness (+5), 0.08
% As (+4), 0.06% as (+2), 0.02% as (+1) and no bitterness as (0).

【0026】第3表の酸カゼインの部分分解物の苦味試
験の結果からも明らかなように、市販酵素剤での酸カゼ
イン分解液は苦味があったが、本発明の菌株の産生する
酵素での分解液は、苦味が無かった。
As is clear from the results of the bitterness test of the partially decomposed product of acid casein in Table 3, the acid casein decomposed solution with a commercially available enzyme preparation had a bitter taste, but the enzyme produced by the strain of the present invention did not. The decomposition solution had no bitterness.

【0027】[0027]

【表3】 第 3 表 ─────────────────────────── 酵 素 剤 苦 味 ─────────────────────────── プロテアーゼA +2 プロテアーゼP +3 プロテアーゼS +2 プロテアーゼN +1 プロレザー +1 パンクレアチンF +3 トリプシン +3 ブロメライン +2 パパインW−40 +1 (本発明の菌株) Rhizopus pseudochinesis 0 Rhizopus formosaensis 0 Rhizopus peka 0 Rhizopus hangchow 0 Rhizopus javanicus 0 Rhizopus niveus 0 ───────────────────────────[Table 3] Table 3 酵 Enzyme Bitterness ─────────── ──────────────── Protease A +2 Protease P +3 Protease S +2 Protease N +1 Proleather +1 Pancreatin F +3 Trypsin +3 Bromelain +2 Papain W-40 +1 (strain of the present invention) Rhizopus pseudochinesis 0 Rhizopus formosaensis 0 Rhizopus peka 0 Rhizopus hangchow 0 Rhizopus javanicus 0 Rhizopus niveus 0 ───────────────────────────

【0028】試験例2 (蛋白質の高度分解における苦味生成の比較試験)5%
酸カゼイン及び5%大豆蛋白溶液(pH7.0)に市販
酵素剤及び本発明により得られた粗製酵素を蛋白質1g
当りプロティナーゼ活性で300単位添加し、45℃で
16時間反応した。苦味を官能試験して第4表の結果を
得た。
Test Example 2 (Comparative test of bitterness generation in advanced protein degradation) 5%
A commercially available enzyme preparation and the crude enzyme obtained according to the present invention in 1 g of protein were added to acid casein and a 5% soybean protein solution (pH 7.0).
Then, 300 units were added per proteinase activity and reacted at 45 ° C. for 16 hours. Table 4 shows the results of the sensory test for bitterness.

【0029】第4表の各種蛋白質の高度分解における苦
味生成の比較実験結果からも明らかなように、市販酵素
剤による分解液は苦味があったが、本発明の酵素による
分解液は苦味が酸カゼイン、大豆蛋白とも無かった。
As is clear from the results of the comparative experiments of bitterness generation in the advanced degradation of various proteins shown in Table 4, the degradation solution by the commercially available enzyme preparation had a bitter taste, whereas the degradation solution by the enzyme of the present invention had a bitter taste. There was neither casein nor soy protein.

【0030】[0030]

【表4】 第 4 表 ───────────────────────────────── 酵 素 剤 乳蛋白(酸カゼイン) 大豆蛋白 ───────────────────────────────── プロテアーゼA +1 +1 プロテアーゼP +2 +2 プロテアーゼS +4 +2 プロテアーゼN +4 +2 プロレザー +4 +2 パンクレアチンF +2 +4 トリプシン +4 +4 ブロメライン +4 +4 パパインW−40 +4 +4 (本発明の菌株) Rhizopus pseudochinesis 0 0 Rhizopus formosaensis 0 0 Rhizopus peka 0 0 Rhizopus hangchow 0 0 Rhizopus javanicus 0 0 Rhizopus niveus 0 0 ─────────────────────────────────[Table 4] Table 4-Enzyme agent Milk protein (acid casein) Soybean Protein ───────────────────────────────── Protease A + 1 + 1 Protease P + 2 + 2 Protease S + 4 + 2 Protease N + 4 + 2 Proleather +4 +2 Pancreatin F +2 +4 Trypsin +4 +4 Bromelain +4 +4 Papain W-40 +4 +4 (strain of the present invention) 0 ─────────────────────────────────

【0031】試験例3 (苦味の消去実験)5%酸カゼイン(pH7.0)に
「プロテアーゼA」、「プロテアーゼP」、「プロテア
ーゼS」、「プロレザー」及び「パンクレアチンF」
(各々天野製薬(株)製)を酸カゼイン1g当りプロテ
ィナーゼ活性で200単位添加し、45℃で16時間反
応した。反応液の苦味を官能試験した結果、(+5)か
ら(+2)の苦味であった。各々の分解液に本発明の酵
素をペプチダーゼ活性として、酸カゼイン1g当り20
00単位、4000単位、8000単位添加し、45℃
にて3時間反応した結果、第5表に示した様に顕著な苦
味の消去が認められた。
Test Example 3 (Elimination test of bitterness) 5% acid casein (pH 7.0) was added to "Protease A", "Protease P", "Protease S", "Proleather" and "Pancreatin F".
200 units of each (manufactured by Amano Pharmaceutical Co., Ltd.) were added at a proteinase activity per 1 g of acid casein, and reacted at 45 ° C. for 16 hours. As a result of a sensory test of the bitterness of the reaction solution, the bitterness was from (+5) to (+2). The enzyme of the present invention was added to each digested solution as a peptidase activity, and the enzyme was added in an amount of 20 / g of acid casein.
00 units, 4000 units, 8000 units added, 45 ° C
As a result, a significant elimination of bitterness was observed as shown in Table 5.

【0032】[0032]

【表5】 第 5 表 ───────────────────────────────── 本発明のペプチダーゼ添加量 無 添 加 2000単位 4000単位 8000単位 ───────────────────────────────── プロテアーゼA +2 +1 0 0 プロテアーゼP +4 +3 +1 0 プロテアーゼS +5 +4 +3 +2 プロレザー +5 +4 +3 +2 パンクレアチンF +2 +1 +1 0 ─────────────────────────────────[Table 5] Table 5-The amount of the peptidase of the present invention not added 2000 Unit 4000 units 8000 units プ ロ テ ア ー ゼ Protease A +2 +10 0 Protease P +4 +3 +10 Protease S +5 +4 +3 +2 Pro Leather +5 +4 +3 +2 Pancreatin F +2 +1 +1 0 ─────────────────────────────── ──

【0033】〔製造例1〕ふすま180gに水を150
ml添加し殺菌後、Rhizopus pseudochinesis IAM 6042
の種麹を接種し25℃で4日間培養した。培養後得られ
た麹に20mM、pH7、リン酸バッファーを500m
l加え、低温室にて一夜抽出を行い300mlの粗製酵
素液を得た。遠心分離後、凍結乾燥を行い酵素粉末3.
6gを得た。この粉末の酵素活性はプロティナーゼ12
5u/gであり、ペプチダーゼは37500u/gであ
った。
[Production Example 1] 150 g of water was added to 180 g of bran.
After adding and sterilizing, add Rhizopus pseudochinesis IAM 6042
Was inoculated and cultured at 25 ° C. for 4 days. 20 mM, pH 7, phosphate buffer is added to the koji obtained after the culturing, 500 m
l, and the mixture was extracted overnight in a low-temperature room to obtain 300 ml of a crude enzyme solution. After centrifugation, freeze-drying and enzyme powder 3.
6 g were obtained. The enzymatic activity of this powder is proteinase 12
5 u / g and peptidase 37500 u / g.

【0034】〔製造例2〕グルコース0.5%、大豆蛋
白2.0%、ポリペプトン0.2%、酵母エキス0.2
%、リン酸二カリウム0.1%、硫酸マグネシウム0.
05%の組成の培地100mlにRhizopus javanicus N
o.3553の胞子を接種し30℃で3日間振とう培養を行な
った。遠心分離により菌体分離をし、酵素溶液を得た。
この溶液の酵素活性はプロティナーゼ2.4u/ml、
ペプチダーゼ20.4u/mlであった。
[Production Example 2] 0.5% glucose, 2.0% soybean protein, 0.2% polypeptone, 0.2% yeast extract
%, Dipotassium phosphate 0.1%, magnesium sulfate 0.
Rhizopus javanicus N is added to 100 ml of a medium having a composition of 05%.
o.3553 spores were inoculated and shake-cultured at 30 ° C. for 3 days. The cells were separated by centrifugation to obtain an enzyme solution.
The enzyme activity of this solution is 2.4 u / ml of proteinase,
The peptidase was 20.4 u / ml.

【0035】〔製造例3〕ふすま1800gに水を15
00ml添加し殺菌後、Rhizopus hangchow No.3545の
種麹を接種し30℃で3日間培養した。得られた麹に2
0mM、pH7リン酸バッファーを10リットル加え低
温室にて一夜抽出を行い7350mlの粗製酵素液を得
た。遠心分離後、ホローファイバー(旭化成(株)AI
L−1010)を用いて濃縮を行なった後、凍結乾燥を
し酵素粉末15.5gを得た。この粉末の酵素活性はプ
ロティナーゼ2100u/gであり、ペプチダーゼは9
3500u/gであった。また、活性収率は各々68
%、61%であった。
Production Example 3 Water was added to 1800 g of bran with 15
After sterilization by adding 00 ml, seed koji of Rhizopus hangchow No. 3545 was inoculated and cultured at 30 ° C. for 3 days. 2 in the obtained koji
10 liters of 0 mM, pH 7 phosphate buffer was added, and the mixture was extracted overnight in a low temperature room to obtain 7350 ml of a crude enzyme solution. After centrifugation, hollow fiber (Asahi Kasei Corporation AI
L-1010), followed by lyophilization to obtain 15.5 g of enzyme powder. The enzymatic activity of this powder was 2100 u / g of proteinase, and the peptidase was 9
It was 3500 u / g. The activity yield was 68
%, 61%.

【0036】〔実施例1〕 「酸カゼインの分解」5%酸カゼイン溶液(pH7)9
0mlに「製造例3」で得た酵素粉末640mgを10
mlの精製水に溶解した酵素液を添加し(酸カゼイン1
g当りプロティナーゼ活性で300単位)、45℃にて
16時間静置反応した。反応後100℃で10分間加熱
した後、遠心分離を行い清澄な溶液を得た。この溶液に
ついて苦味を官能試験で測定したところ、苦味は無かっ
た。またファルマシア社製FPLCシステム(カラム:
スーパーロース12)を用いて、分子量分布を分析した
結果を図3に示したが、ほとんど分子量1000以下の
ペプチド及びアミノ酸であった。
[Example 1] "Decomposition of acid casein" 5% acid casein solution (pH 7) 9
640 mg of the enzyme powder obtained in "Production Example 3"
An enzyme solution dissolved in ml of purified water is added (acid casein 1).
(300 units of proteinase activity per gram) and allowed to stand at 45 ° C. for 16 hours. After heating at 100 ° C. for 10 minutes after the reaction, centrifugation was performed to obtain a clear solution. When the bitterness of this solution was measured by a sensory test, no bitterness was found. Pharmacia FPLC system (column:
The result of analyzing the molecular weight distribution using Superose 12) is shown in FIG. 3, and almost all peptides and amino acids having a molecular weight of 1,000 or less were obtained.

【0037】〔実施例2〕 「大豆蛋白の分解」5%大豆蛋白溶液(pH7)100
0mlに「製造例3」で得た酵素粉末7.1gを添加
し、45℃にて17時間撹拌しつつ反応した。反応後1
00℃で10分間加熱した後遠心分離を行い清澄な溶液
を得、ついで凍結乾燥を行い42.5gの微黄色の粉末
を得た。この粉末について苦味を官能試験した結果、苦
味は無かった。また「実施例1」と同様に分子量分布を
分析した結果を図4に示したが、ほとんど分子量100
0以下のペプチド及びアミノ酸であった。
[Example 2] "Decomposition of soybean protein" 5% soybean protein solution (pH 7) 100
To 0 ml, 7.1 g of the enzyme powder obtained in "Production Example 3" was added, and the mixture was reacted while stirring at 45 ° C for 17 hours. After reaction 1
After heating at 00 ° C. for 10 minutes, the solution was centrifuged to obtain a clear solution, and then freeze-dried to obtain 42.5 g of a slightly yellow powder. As a result of a sensory test of the bitterness of the powder, no bitterness was found. FIG. 4 shows the result of analyzing the molecular weight distribution in the same manner as in “Example 1”.
0 or less peptides and amino acids.

【0038】〔実施例3〕 「肉煮汁の分解」豚肉の煮汁(蛋白質濃度10%)10
00mlに「製造例3」で得られた酵素粉末を480m
g添加した後、55℃にて4時間反応した。反応後酵素
の失活と殺菌の目的で100℃で10分間加熱後、官能
試験したところ苦味は感じられなかった。
Example 3 "Decomposition of meat broth" Boiled pork (protein concentration 10%) 10
480 m of the enzyme powder obtained in "Production Example 3"
After adding g, the mixture was reacted at 55 ° C. for 4 hours. After the reaction, the mixture was heated at 100 ° C. for 10 minutes for the purpose of deactivating and sterilizing the enzyme, and a sensory test showed no bitterness.

【0039】〔実施例4〕 「苦味の消去」5%酸カゼイン溶液(pH7)90ml
に「プロテアーゼP−1」(天野製薬(株)製)90m
gを10mlの精製水に溶解した溶液を添加し(酸カゼ
イン1g当りプロティナーゼ活性で200単位)、45
℃にて16時間静置反応した。反応後「製造例3」で得
られた酵素粉末を190mg添加し(酸カゼイン1g当
りペプチダーゼ活性として4000単位)、45℃で5
時間更に反応した。反応後加熱処理を行なった後、苦味
を官能試験した。その結果、本発明の酵素添加前の溶液
は+4の苦味であったが、酵素添加処理後は苦味は無か
った。
[Example 4] "Elimination of bitterness" 90 ml of 5% acid casein solution (pH 7)
"Protease P-1" (manufactured by Amano Pharmaceutical Co., Ltd.) 90 m
g was dissolved in 10 ml of purified water (200 units of proteinase activity per gram of acid casein), and
The reaction was allowed to stand at 16 ° C. for 16 hours. After the reaction, 190 mg of the enzyme powder obtained in "Production Example 3" was added (4000 units as peptidase activity per 1 g of acid casein), and the mixture was added at 45 ° C for 5 minutes.
The time further reacted. After the heat treatment after the reaction, the bitterness was subjected to a sensory test. As a result, the solution of the present invention before the addition of the enzyme had a bitterness of +4, but no bitterness after the enzyme addition treatment.

【0040】なお本明細書において、酵素活性及び分子
量分布の測定は、それぞれ次のようにして行った。酵素活性測定法 1.プロティナーゼ 0.75%ミルクカゼイン(メルク社製)を基質として
37℃で反応し、0.4モルのトリクロロ酢酸を反応液
と等量添加し、濾紙で濾過後(東洋濾紙No.131)濾液に
ついてフォーリン試薬で発色させた。濾液1ml当り6
0分間に100マイクログラムのチロシン相当量の発色
量を1単位とした。 2.ペプチダーゼ 0.125mMロイシン・グリシル・グリシンを基質と
し(pH7.0)、30℃にて反応しニンヒドリン法に
て行なった。1分間に1マイクロモルのアミノ酸生成量
を1単位とした。
In the present specification, the measurement of the enzyme activity and the molecular weight distribution were performed as follows. Enzyme activity measurement method Reaction was performed at 37 ° C. using proteinase 0.75% milk casein (manufactured by Merck) as a substrate, 0.4 mol of trichloroacetic acid was added in the same amount as the reaction solution, and the mixture was filtered through filter paper (Toyo Filter Paper No. 131). Color was developed with Foreign reagent. 6 per ml of filtrate
The amount of color development equivalent to 100 micrograms of tyrosine per 0 minute was defined as 1 unit. 2. Peptidase was reacted at 30 ° C. using 0.125 mM leucine / glycyl / glycine as a substrate (pH 7.0), and the reaction was carried out by the ninhydrin method. One micromol of amino acid produced per minute was defined as one unit.

【0041】分子量分布の測定法 ファルマシア社製「スパーロース12」ゲル濾過用カラ
ムを用いた。なお分子量の標準としては、25000は
キモトリプシノーゲン、12300はチトクロームC、
6500はトリプシンインヒビターをまた1450はバ
シトラシンを用いた。
Measurement method of molecular weight distribution A "Sparose 12" gel filtration column manufactured by Pharmacia was used. As the molecular weight standards, 25000 is chymotrypsinogen, 12300 is cytochrome C,
6500 used trypsin inhibitor and 1450 used bacitracin.

【0042】[0042]

【発明の効果】本発明は、リゾプス属菌が産生するプロ
ティナーゼ及びペプチダーゼを有効成分とする酵素剤を
用いることによってはじめて成功したものであって、本
発明により各種蛋白質から苦味のないペプチドを直接製
造することができるほか、他の酵素によって生成した苦
味を有するペプチドから苦味を除去することもできる。
このように本発明によれば苦味のないペプチドを効率よ
く調製することができるので、栄養性その他の有用性を
有するペプチドであっても苦味を有するが故に自由に使
用することができなかった飲食品、経口投与剤、栄養
剤、飼餌料等にも広く利用することができ、ペプチドの
用途が飛躍的に拡大される。
Industrial Applicability The present invention has been successfully achieved for the first time by using an enzymatic agent containing proteinase and peptidase produced by Rhizopus sp. As active ingredients. According to the present invention, a peptide having no bitterness is directly produced from various proteins. In addition to removing bitterness from peptides having bitterness produced by other enzymes.
As described above, according to the present invention, a peptide having no bitterness can be efficiently prepared, so that even a peptide having nutritional properties or other usefulness has a bitter taste and cannot be used freely because of its bitterness. Products, oral preparations, nutrients, feeds, etc., and the use of peptides is dramatically expanded.

【0043】その具体例としては、医療食、経腸栄養
剤、乳児食、低アレルギー育児粉乳、バランス栄養食、
健康食品、機能性食品といったヒト用の栄養源のほか、
飼料、餌料、培地用ペプトンといったヒト以外の栄養源
が挙げられる。したがって本発明によれば、ペプチドを
投与しても、乳幼児や病弱者が苦味の故にこれを吐き出
してしまうというようなこともなくなり、人類の健康や
医療に本発明は大いに寄与するものである。
Specific examples thereof include medical food, enteral nutrition, infant food, hypoallergenic infant formula, balanced nutrition food,
In addition to human nutrition sources such as health foods and functional foods,
Non-human nutrients such as feed, feed, and media peptones. Therefore, according to the present invention, even if a peptide is administered, infants and the disabled will not exhale due to bitterness, and the present invention greatly contributes to human health and medical care.

【図面の簡単な説明】[Brief description of the drawings]

【図1】酸カゼイン分解物の分子量分布を図示したもの
であるが、(A)はプロテアーゼPによる分解物、
(B)は本発明のプロティナーゼによる分解物の分子量
分布を図示したものである。
FIG. 1 is a diagram illustrating the molecular weight distribution of an acid casein hydrolyzate, wherein FIG.
(B) illustrates the molecular weight distribution of the degradation product of the proteinase of the present invention.

【図2】Rhizopus hangchow No.3545菌由来のペプチダ
ーゼの酵素的性質を図示したものである。
FIG. 2 illustrates the enzymatic properties of a peptidase from Rhizopus hangchow No. 3545.

【図3】酸カゼイン分解物の分子量分布を図示したもの
である。
FIG. 3 illustrates the molecular weight distribution of the acid casein hydrolyzate.

【図4】大豆蛋白質分解物の分子量分布を図示したもの
である。
FIG. 4 illustrates the molecular weight distribution of a soybean protein degradation product.

───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 FI 技術表示箇所 // A23L 1/313 A23L 1/313 (C12N 9/48 C12R 1:845) (72)発明者 宿野部 幸孝 埼玉県川越市古谷上6083−8、B2− 205 (56)参考文献 特開 昭48−52967(JP,A)──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. 6 Identification code Agency reference number FI Technical display location // A23L 1/313 A23L 1/313 (C12N 9/48 C12R 1: 845) (72) Inventor Yukitaka Sukunobe 6083-8 Furuya, Kawagoe-shi, Saitama B2-205 (56) References JP-A-48-52967 (JP, A)

Claims (2)

(57)【特許請求の範囲】(57) [Claims] 【請求項1】 リゾプス属菌が生産し、苦味のないペプ
チドを生成することのできる、プロティナーゼ及び下記
の理化学的性質を有するペプチダーゼを含む複合酵素を
有効成分とするペプチド製造用酵素剤を蛋白質に作用さ
せること、を特徴とする苦味のないペプチドを製造する
方法。 ペプチダーゼの理化学的性質 1.苦味のないペプチドを生成する。 2.至適pHは6〜8である。 3.pH7.0、60分の至適温度は35〜47℃であ
る。 4.37℃、60分、pH5以上で80%以上の残存活
性を示す。 5.pH7.0、60分、50℃以下で90%以上の残
存活性を示す。
1. An enzyme preparation for peptide production comprising a complex enzyme containing a proteinase and a peptidase having the following physicochemical properties as an active ingredient, which can be produced by a Rhizopus bacterium and can produce a peptide having no bitterness. And producing a peptide having no bitterness. Physicochemical properties of peptidase Produces a non-bitter peptide. 2. The optimum pH is 6-8. 3. The optimum temperature at pH 7.0 for 60 minutes is 35 to 47 ° C. 4. Shows 80% or more residual activity at 37 ° C., 60 minutes, pH 5 or more. 5. It shows a residual activity of 90% or more at pH 7.0 for 60 minutes at 50 ° C. or less.
【請求項2】 リゾプス属菌が生産し、苦味のないペプ
チドを生成することのできる、プロティナーゼ及び下記
の理化学的性質を有するペプチダーゼを含む複合酵素を
有効成分とするペプチド製造用酵素剤を苦味ペプチドに
作用させ、苦味を消去すること、を特徴とする苦味のな
いペプチドを製造する方法。 ペプチダーゼの理化学的性質 1.苦味のないペプチドを生成する。 2.至適pHは6〜8である。 3.pH7.0、60分の至適温度は35〜47℃であ
る。 4.37℃、60分、pH5以上で80%以上の残存活
性を示す。 5.pH7.0、60分、50℃以下で90%以上の残
存活性を示す。
2. An enzyme preparation for producing a peptide, comprising a complex enzyme containing a proteinase and a peptidase having the following physicochemical properties as an active ingredient, which is capable of producing a peptide having no bitterness, which is produced by a Rhizopus bacterium. A method for producing a peptide having no bitterness, wherein the peptide has no bitterness. Physicochemical properties of peptidase Produces a non-bitter peptide. 2. The optimum pH is 6-8. 3. The optimum temperature at pH 7.0 for 60 minutes is 35 to 47 ° C. 4. Shows 80% or more residual activity at 37 ° C., 60 minutes, pH 5 or more. 5. It shows a residual activity of 90% or more at pH 7.0 for 60 minutes at 50 ° C. or less.
JP6271862A 1994-10-12 1994-10-12 Peptide production method Expired - Lifetime JP2631202B2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP6271862A JP2631202B2 (en) 1994-10-12 1994-10-12 Peptide production method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
JP6271862A JP2631202B2 (en) 1994-10-12 1994-10-12 Peptide production method

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
JP1257720A Division JPH0753106B2 (en) 1989-10-04 1989-10-04 Enzyme preparation for peptide production

Publications (2)

Publication Number Publication Date
JPH07274995A JPH07274995A (en) 1995-10-24
JP2631202B2 true JP2631202B2 (en) 1997-07-16

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001069949A (en) * 1999-09-06 2001-03-21 Nippon Meat Packers Inc Decomposed pork and food containing the same
US20050053705A1 (en) * 2003-09-04 2005-03-10 Kraft Foods Holdings, Inc. Soluble soy protein with superior functional properties
BRPI0820304A2 (en) * 2007-11-07 2017-05-23 Mead Johnson Nutrition Co process to reduce bitterness and improve the taste of protein-free and hydrolyzed infant formulas
JP5458536B2 (en) * 2008-09-17 2014-04-02 不二製油株式会社 Method for producing lactic acid and additive for lactic acid fermentation
CN115141693A (en) * 2022-08-02 2022-10-04 王启含 Beer preparation method based on active polypeptide

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5116506B2 (en) * 1971-11-10 1976-05-25

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