TWI551684B

TWI551684B - Swiss lactic acid bacteria with high proteolytic activity

Info

Publication number: TWI551684B
Application number: TW100140985A
Authority: TW
Inventors: Taketo Wakai; Misaki Hatanaka; Yasunori Nakamura; Naoyuki Yamamoto
Original assignee: Asahi Group Holdings Ltd
Priority date: 2010-11-09
Filing date: 2011-11-09
Publication date: 2016-10-01
Also published as: CN103221531A; AU2011327288B2; CN103221531B; TW201305331A; JPWO2012063826A1; WO2012063826A1; JP5888701B2; AU2011327288A1; MY160992A

Description

具有高蛋白質分解活性之瑞士乳酸菌Swiss lactic acid bacteria with high protein decomposition activity

本發明係關於一種新穎之屬於瑞士乳酸菌之乳酸菌。又，本發明係關於一種使該乳酸菌醱酵所獲得之醱酵乳、以及含有該乳酸菌之組合物及飲食品。進而本發明係關於一種使用該乳酸菌之胺基酸及/或肽之製造方法。The present invention relates to a novel lactic acid bacterium belonging to the Swiss lactic acid bacteria. Further, the present invention relates to a fermented milk obtained by fermenting the lactic acid bacterium, a composition containing the lactic acid bacterium, and a food or drink. Further, the present invention relates to a process for producing an amino acid and/or a peptide using the lactic acid bacteria.

已知乳蛋白或食物蛋白中潛在有功能性肽，而正在進行藉由利用酵素分解或醱酵對乳蛋白或食物蛋白進行加工而加以利用之嘗試。例如，作為此種功能性肽，報告有三肽VPP(Valine-Proline-Proline，纈胺酸-脯胺酸-脯胺酸)及IPP(Isoleucine-Proline-Proline，異白胺酸-脯胺酸-脯胺酸)具有血管緊張素轉化酵素(ACE，Angiotensin Converting Enzyme)抑制作用，並顯示降血壓作用(專利文獻1~4及非專利文獻1)。It is known that there are potentially functional peptides in milk proteins or food proteins, and attempts are being made to utilize milk proteins or food proteins by utilizing enzyme decomposition or fermentation. For example, as such a functional peptide, a tripeptide VPP (Valine-Proline-Proline, lysine-proline-proline) and IPP (Isoleucine-Proline-Proline, iso-aracine-proline-- The proline acid has an inhibitory action by angiotensin converting enzyme (ACE) and exhibits a blood pressure lowering action (Patent Documents 1 to 4 and Non-Patent Document 1).

作為VPP及IPP以外之源自乳之功能性肽，報告有作為二肽之YP具有降血壓作用及抗焦慮效果(專利文獻5)。又，報告有作為二肽之IP、AP、EP、RP、QP、TP、MP及GP係難分解性，且具有ACE抑制活性之肽群(例如專利文獻6)。As a functional peptide derived from milk other than VPP and IPP, it has been reported that YP as a dipeptide has a blood pressure lowering action and an anxiolytic effect (Patent Document 5). In addition, a peptide group having ACE inhibitory activity, such as IP, AP, EP, RP, QP, TP, MP, and GP, which are dipeptides, is reported (for example, Patent Document 6).

另一方面，已知瑞士乳酸菌(L. helveticus)自先前起即用於乳製品之製造中，蛋白質分解活性較高。為了提供更容易攝取、易於加工之醱酵乳，報告有幾種蛋白質分解活性較高、肽產生性優異之L. helveticus株(專利文獻1~4及7、非專利文獻1)。On the other hand, it has been known that L. helveticus has been used in the manufacture of dairy products from the beginning, and the protein decomposition activity is high. In order to provide a lyophilized milk that is easier to ingest and easy to process, L. helveticus strains having high protein decomposition activity and excellent peptide production properties have been reported (Patent Documents 1 to 4 and 7, Non-Patent Document 1).

因蛋白質係由胺基酸所構成，故可藉由進一步分解而亦成為胺基酸之供給源。已知胺基酸於營養、風味以外亦具有各種功能。例如，已知支鏈胺基酸(BCAA，Branched Chain Amino Acid)包含於構成肌肉之蛋白質中，於運動時等肌肉中之BCAA被消耗而作為能量使用，因此BCAA之補充較為重要。又，丙胺酸(Ala)係作為肝臟之能量源而為人所知之糖原性胺基酸。由於消耗分解酒精時所產生之NADH(reduced nicotinamide adenine dinucleotide，還原型煙醯胺腺嘌呤二核苷酸)，故認為由於Ala之存在而進行肝臟中之分解反應。又，亦已知Ala於味覺方面引出美味，具有鹽醃、醋醃效果或刺激味等掩蓋效果。一般認為，離胺酸(Lys)係必需胺基酸之中最容易不足之胺基酸。必需以抗體或激素、酵素等原材料之形式攝取。又，報告有Lys亦與鈣吸收、膠原蛋白形成、骨組織之生產有關。絲胺酸(Ser)作為磷脂絲胺酸或其他胺基酸等生物體內之各種重要物質之前驅物較為重要。又，已知其降低血膽固醇，藉由使腦內之神經元活化等抑制腦細胞死亡而防止腦之老化，或成為皮膚之保濕成分。Since the protein is composed of an amino acid, it can be further decomposed to become a supply source of an amino acid. Amino acids are known to have various functions in addition to nutrition and flavor. For example, it is known that Branched Chain Amino Acid (BCAA) is contained in proteins constituting muscles, and BCAA in muscles such as exercise is consumed as energy, and therefore supplementation of BCAA is important. Further, alanine (Ala) is a glycogen-based amino acid which is known as an energy source of the liver. Since the NADH (reduced nicotinamide adenine dinucleotide) produced by the decomposition of alcohol is consumed, it is considered that the decomposition reaction in the liver is carried out due to the presence of Ala. Further, it is also known that Ala is delicious in terms of taste, and has a masking effect such as salting, vinegar pickling or irritating taste. It is generally considered that the amino acid which is the most easily deficient among the amino acid (Lys) is an essential amino acid. It must be taken in the form of antibodies or raw materials such as hormones and enzymes. Also, it is reported that Lys is also involved in calcium absorption, collagen formation, and bone tissue production. Serine is important as a precursor to various important substances in living organisms such as phospholipid serine or other amino acids. Further, it is known that it lowers blood cholesterol, prevents brain cell death by inhibiting brain cell death such as activation of neurons in the brain, or becomes a moisturizing component of the skin.

為生成此種胺基酸而積極地進行微生物之利用。然而，關於可作為微生物之醱酵物本身而攝取之胺基酸並無較多之例。乳酸菌中，為影響醃菜之風味而篩選向胺基酸之分解優異之乳酸菌，但並不知道為了以醱酵乳本身攝取功能性胺基酸及功能性肽而進行研究之例。The use of microorganisms is actively carried out for the production of such amino acids. However, there are not many examples of amino acids which can be taken as a fermentation product of microorganisms. In the case of the lactic acid bacteria, the lactic acid bacteria excellent in the decomposition of the amino acid are selected to affect the flavor of the pickles, but it is not known to study the intake of functional amino acids and functional peptides by the lactose itself.

[先前技術文獻][Previous Technical Literature] [專利文獻][Patent Literature]

[專利文獻1]歐州專利第1016709B號[Patent Document 1] European Patent No. 1016709B

[專利文獻2]國際公開WO 01/32836號[Patent Document 2] International Publication WO 01/32836

[專利文獻3]國際公開WO 2007/096510號[Patent Document 3] International Publication WO 2007/096510

[專利文獻4]歐州專利第1820850號[Patent Document 4] European Patent No. 1820850

[專利文獻5]歐州專利第0821968號[Patent Document 5] European Patent No. 0821968

[專利文獻6]歐州專利第1661909號[Patent Document 6] European Patent No. 1661909

[專利文獻7]日本專利特開平7-274949號公報[Patent Document 7] Japanese Patent Laid-Open No. Hei 7-274949

[非專利文獻][Non-patent literature]

[非專利文獻1]Nakamura,Y. et al.,J. Dairy Sci.第78卷第1253-1257頁，1995年[Non-Patent Document 1] Nakamura, Y. et al., J. Dairy Sci. Vol. 78, pp. 1253-1257, 1995

因此，本發明之目的在於提供一種具有高蛋白質分解活性，且肽生成能力及/或胺基酸生成能力優異之乳酸菌。又，本發明之目的在於提供一種高濃度地含有胺基酸及/或肽，且風味良好之醱酵乳。Accordingly, an object of the present invention is to provide a lactic acid bacterium which has high protein decomposition activity and is excellent in peptide-forming ability and/or amino acid-forming ability. Further, an object of the present invention is to provide a fermented milk which contains an amino acid and/or a peptide at a high concentration and which has a good flavor.

本發明者等人為解決上述課題而進行了努力研究，結果成功地自屬於瑞士乳酸菌之乳酸菌株中取得自乳蛋白生成胺基酸之能力及生成功能性肽之能力優異，且與先前之乳酸菌相比具有較高之蛋白質分解活性的菌株。又，獲得可使用該菌株獲得胺基酸及/或肽之含量較多之醱酵乳及組合物而製造胺基酸及/或肽之見解，以致完成本發明。The present inventors have made an effort to solve the above problems, and as a result, the lactic acid strain belonging to the Swiss lactic acid bacteria has been successfully obtained from the lactic acid bacteria of the Swiss lactic acid bacteria, and has excellent ability to produce an amino acid and a functional peptide, and is compatible with the prior lactic acid bacteria. More strains with higher proteolytic activity. Further, the knowledge that the amino acid and the peptide having a high content of amino acids and/or peptides can be obtained by using the strain to produce an amino acid and/or a peptide is obtained, so that the present invention has been completed.

即，本發明係包含如下內容。That is, the present invention includes the following.

[1]　一種屬於瑞士乳酸菌之乳酸菌，其特徵在於具有以下(a)~(d)中之至少1種性質：[1] A lactic acid bacterium belonging to the lactic acid bacteria of Switzerland, characterized by having at least one of the following properties (a) to (d):

(a)　藉由使獸乳醱酵而生成25 μmol/ml以上之游離胺基酸；(a) producing a free amino acid of 25 μmol/ml or more by fermenting the mammal;

(b)　藉由使獸乳醱酵而生成8.5 mg/ml以上之肽；(b) producing a peptide of 8.5 mg/ml or more by fermenting the animal's milk;

(c)　藉由使獸乳醱酵而生成總量為260 μg/ml以上之XP肽及/或XPP肽；(c) producing XP peptide and/or XPP peptide in a total amount of 260 μg/ml or more by fermenting the mammal;

(d)　使獸乳於30℃以上34℃以下醱酵所獲得之醱酵乳之單位酸度之IPP肽及VPP肽的量以VPP換算量計為6.0 mg/g以上。(d) The amount of the IPP peptide and the VPP peptide per unit acid of the fermented milk obtained by fermenting the animal's milk at 30 ° C or more and 34 ° C or less is 6.0 mg / g or more in terms of VPP.

[2]　如[1]之乳酸菌，其中游離胺基酸之15%以上為支鏈胺基酸。[2] The lactic acid bacterium according to [1], wherein more than 15% of the free amino acid is a branched amino acid.

[3]　如[1]或[2]之乳酸菌，其中游離胺基酸含有選自丙胺酸、離胺酸及絲胺酸之至少1種。[3] The lactic acid bacteria according to [1] or [2], wherein the free amino acid contains at least one selected from the group consisting of alanine, lysine, and serine.

[4]　如[1]至[3]中任一項之乳酸菌，其中生成60 μg/ml以上之YP肽。[4] The lactic acid bacterium according to any one of [1] to [3] wherein a YP peptide of 60 μg/ml or more is produced.

[5]　如[1]至[4]中任一項之乳酸菌，其為瑞士乳酸菌CM4株(台灣寄存編號CCRC 910111、日本寄存編號FERM BP-6060)之變異株。[5] The lactic acid bacteria according to any one of [1] to [4], which is a mutant strain of the Swiss lactic acid bacteria CM4 strain (Taiwan registration number CCRC 910111, Japanese registration number FERM BP-6060).

[6]　如[1]至[5]中任一項之乳酸菌，其為瑞士乳酸菌CP3232株(台灣寄存編號BCRC 910539、日本寄存編號FERM BP-11271)或其變異株。[6] The lactic acid bacteria according to any one of [1] to [5], which is a Swiss lactic acid bacteria CP3232 strain (Taiwan registered number BCRC 910539, Japanese registered number FERM BP-11271) or a variant thereof.

[7]　一種乳酸菌，其為瑞士乳酸菌CP3232株(台灣寄存編號BCRC 910539、日本寄存編號FERM BP-11271)。[7] A lactic acid bacterium which is a Swiss lactic acid bacteria CP3232 strain (Taiwan registration number BCRC 910539, Japanese registration number FERM BP-11271).

[8]　一種用於自獸乳生成胺基酸及/或肽之組合物，其特徵在於含有至少1種屬於瑞士乳酸菌之乳酸菌。[8] A composition for producing an amino acid and/or a peptide from a mammal, characterized in that it contains at least one lactic acid bacterium belonging to the Swiss lactic acid bacteria.

[9]　如[8]之組合物，其藉由使獸乳醱酵而生成25 μmol/ml以上之游離胺基酸。[9] The composition according to [8], which produces a free amino acid of 25 μmol/ml or more by fermenting the mammal.

[10]　如[9]之組合物，其中游離胺基酸之15%以上為支鏈胺基酸。[10] The composition according to [9], wherein 15% or more of the free amino acid is a branched amino acid.

[11]　如[9]或[10]之組合物，其中游離胺基酸含有選自丙胺酸、離胺酸及絲胺酸之至少1種。[11] The composition according to [9] or [10] wherein the free amino acid contains at least one selected from the group consisting of alanine, lysine, and serine.

[12]　如[8]至[11]中任一項之組合物，其中藉由使獸乳醱酵而生成8.5 mg/ml以上之肽。[12] The composition according to any one of [8] to [11] wherein a peptide of 8.5 mg/ml or more is produced by fermenting the mammal.

[13]　如[8]至[12]中任一項之組合物，其中藉由使獸乳醱酵而生成總量260 μg/ml以上之XP肽及/或XPP肽。[13] The composition according to any one of [8] to [12] wherein a total of 260 μg/ml or more of XP peptide and/or XPP peptide is produced by fermenting the mammal.

[14]　如[8]至[13]中任一項之組合物，其中藉由使獸乳醱酵而生成60 μg/ml以上之YP肽。[14] The composition according to any one of [8] to [13] wherein a YP peptide of 60 μg/ml or more is produced by fermenting the mammal.

[15]　如[8]至[14]中任一項之組合物，其中使獸乳於30℃以上34℃以下醱酵所獲得之醱酵乳之單位酸度之IPP肽及VPP肽的量為6.0 mg/g以上。[15] The composition according to any one of [8] to [14] wherein the amount of the IPP peptide and the VPP peptide of the acidity of the fermented milk obtained by fermenting the animal milk at 30 ° C or more and 34 ° C or less is 6.0. Above mg/g.

[16]　如[8]至[15]中任一項之組合物，其中乳酸菌為如[1]至[7]中任一項者。[16] The composition according to any one of [8] to [15] wherein the lactic acid bacteria are any one of [1] to [7].

[17]　一種醱酵乳，其特徵在於：藉由使用至少1種屬於瑞士乳酸菌之乳酸菌使獸乳醱酵而獲得，且含有以下(a)~(d)中之至少1中成分：[17] A fermented milk obtained by fermenting a mammal with at least one lactic acid bacterium belonging to the Swiss lactic acid bacterium, and comprising at least one of the following (a) to (d):

(a)　25 μmol/ml以上之游離胺基酸；(a) a free amino acid of 25 μmol/ml or more;

(b)　8.5 mg/ml以上之肽；(b) peptides of 8.5 mg/ml or more;

(c)　總量為260 μg/ml以上之XP肽及/或XPP肽；(c) XP peptide and/or XPP peptide in a total amount of 260 μg/ml or more;

(d)　6.0 mg/g以上之IPP肽及VPP肽之單位酸度之VPP換算量。(d) VPP conversion amount per unit acidity of IPP peptide and VPP peptide of 6.0 mg/g or more.

[18]　如[17]之醱酵乳，其為含有至少1種屬於瑞士乳酸菌之乳酸菌者。[18] The fermented milk of [17], which is a lactic acid bacteria containing at least one lactic acid bacteria belonging to Switzerland.

[19]　如[17]或[18]之醱酵乳，其為含有如[1]至[7]中任一項之至少1種乳酸菌者。[19] The fermented milk of [17] or [18], which is at least one lactic acid bacteria according to any one of [1] to [7].

[20]　如[17]至[19]中任一項之醱酵乳，其中游離胺基酸之15%以上為支鏈胺基酸。[20] The fermented milk of any one of [17] to [19], wherein more than 15% of the free amino acid is a branched amino acid.

[21]　如[17]至[20]中任一項之醱酵乳，其中游離胺基酸含有選自丙胺酸、離胺酸及絲胺酸之至少1種。[21] The fermented milk of any one of [17] to [20] wherein the free amino acid contains at least one selected from the group consisting of alanine, lysine, and serine.

[22]　如[17]至[21]中任一項之醱酵乳，其含有60 μg/ml以上之YP肽。[22] The fermented milk of any one of [17] to [21] which contains a YP peptide of 60 μg/ml or more.

[23]　如[17]至[22]中任一項之醱酵乳，其中醱酵係於30℃以上34℃以下之溫度下進行。[23] The fermented milk of any one of [17] to [22] wherein the fermentation is carried out at a temperature of from 30 ° C to 34 ° C.

[24]　如[17]至[23]中任一項之醱酵乳，其係用作降血壓劑、腦功能改善劑、自主神經調節劑、副交感神經調節劑、交感神經調節劑、營養佐劑、能量補充劑、肌肉分解損傷抑制劑、肌肉增強劑、疲勞緩解劑、耐力改善劑、調味劑或酒精代謝劑者。[24] The fermented milk of any one of [17] to [23], which is used as a blood pressure lowering agent, a brain function improving agent, an autonomic nervous regulator, a parasympathetic regulating agent, a sympathetic regulating agent, a nutritional adjuvant. , energy supplements, muscle breakdown damage inhibitors, muscle enhancers, fatigue relievers, endurance improvers, flavoring agents or alcohol metabolizing agents.

[25]　一種飲食品，其特徵在於：含有如[1]至[7]中任一項之至少1種乳酸菌及/或其處理物、以及/或者如[17]至[24]中任一項之醱酵乳及/或其處理物。[25] A food or drink comprising at least one lactic acid bacterium according to any one of [1] to [7] and/or a treatment thereof, and/or any one of [17] to [24] The fermented milk and/or its treatment.

[26]　一種胺基酸及/或肽之製造方法，其特徵在於：使用如[1]至[7]中任一項之至少1種乳酸菌或如[8]至[16]中任一項之組合物使獸乳或乳蛋白醱酵，並自所獲得之醱酵物提取胺基酸及/或肽。[26] A method for producing an amino acid and/or a peptide, characterized by using at least one lactic acid bacterium according to any one of [1] to [7] or any one of [8] to [16] The composition ferments the animal's milk or milk protein and extracts the amino acid and/or peptide from the obtained extract.

[27]　如[26]之方法，其中胺基酸為支鏈胺基酸。[27] The method of [26], wherein the amino acid is a branched amino acid.

[28]　如[26]或[27]之方法，其中胺基酸含有選自丙胺酸、離胺酸及絲胺酸之至少1種。[28] The method according to [26] or [27] wherein the amino acid contains at least one selected from the group consisting of alanine, lysine, and serine.

[29]　如[26]至[28]中任一項之方法，其中肽為選自由XP肽及XPP肽所組成之群中之至少1種。[29] The method according to any one of [26] to [28] wherein the peptide is at least one selected from the group consisting of XP peptide and XPP peptide.

[30]　如[26]至[29]中任一項之方法，其中於30℃以上34℃以下之溫度下進行醱酵。[30] The method according to any one of [26] to [29] wherein the fermentation is carried out at a temperature of 30 ° C or more and 34 ° C or less.

[31]　一種方法，其係取得具有蛋白質分解活性之屬於瑞士乳酸菌之乳酸菌的方法，且其包括以下步驟：[31] A method for obtaining a lactic acid bacterium belonging to a Swiss lactic acid bacterium having proteolytic activity, and comprising the steps of:

(a)　製備瑞士乳酸菌CM4株(台灣寄存編號CCRC 910111、日本寄存編號FERM BP-6060)或瑞士乳酸菌CP3232株(台灣寄存編號BCRC 910539、日本寄存編號FERM BP-11271)之變異株之步驟；及(a) a step of preparing a variant strain of the Swiss lactic acid bacteria CM4 strain (Taiwan registered number CCRC 910111, Japanese registered number FERM BP-6060) or the Swiss lactic acid bacteria CP3232 strain (Taiwan registered number BCRC 910539, Japanese registered number FERM BP-11271);

(b)　自該變異株選拔具有以下(a)~(d)中至少1種性質之菌株之步驟：(b) a step of selecting a strain having at least one of the following properties (a) to (d) from the mutant strain:

(a)　藉由使獸乳醱酵而生成25 μmol/ml以上之游離胺基酸，(a) producing a free amino acid of 25 μmol/ml or more by fermenting the animal's milk,

(b)　藉由使獸乳醱酵而生成8.5 mg/ml以上之肽，(b) Producing a peptide of 8.5 mg/ml or more by fermenting the animal's milk,

(c)　藉由使獸乳醱酵而生成總量為260 μg/ml以上之XP肽及/或XPP肽，(c) Producing a total of 260 μg/ml of XP peptide and/or XPP peptide by fermenting the mammalian milk,

[32]　一種功能性飲食品之製造方法，其包括如下步驟：製備如[1]至[7]中任一項之至少1種乳酸菌及/或其處理物、以及/或者如[17]至[24]中任一項之醱酵乳及/或其處理物之步驟；及將該乳酸菌及/或其處理物以及/或者醱酵乳及/或其處理物調配至飲食品中之步驟。[32] A method for producing a functional food or drink, comprising the steps of: preparing at least one lactic acid bacterium according to any one of [1] to [7] and/or a treatment thereof, and/or as in [17] [24] The step of fermenting the milk and/or the processed product thereof, and the step of preparing the lactic acid bacteria and/or the processed material thereof and/or the fermented milk and/or the processed material thereof into the food or beverage.

根據本發明，提供一種具有高蛋白質分解活性，且肽生成能力及/或胺基酸生成能力優異之屬於瑞士乳酸菌之乳酸菌。本乳酸菌所生成之胺基酸及/或肽係具有各種功能者，例如IPP、VPP、YP等肽具有降血壓作用。又，藉由將本乳酸菌用於醱酵中，看獲得高濃度地含有此種具有各種功能之多種胺基酸及/或肽之醱酵乳。由於所獲得之醱酵乳之單位酸度之IPP及VPP肽的量較高，故而並不存在先前之由伴隨醱酵之進行而生成之乳酸所引起的酸度較高之問題。即，由於可製造相對於生成乳酸量含有大量胺基酸及/或肽之醱酵乳，故而可使用少量之醱酵乳，簡便地製造在維持功能性之情況下風味良好且容易飲用之形態的製品，並提供給消費者。According to the present invention, there is provided a lactic acid bacterium belonging to a Swiss lactic acid bacterium which has high proteolytic activity and is excellent in peptide-forming ability and/or amino acid-forming ability. The amino acid and/or peptide system produced by the present lactic acid bacteria has various functions, and peptides such as IPP, VPP, and YP have blood pressure lowering effects. Further, by using the lactic acid bacteria in the fermentation, it is possible to obtain a fermented milk containing a plurality of kinds of amino acids and/or peptides having various functions at a high concentration. Since the amount of IPP and VPP peptide per unit acidity of the obtained fermented milk is high, there is no problem that the acidity caused by the lactic acid which is formed by the progress of the fermentation is high. In other words, since a fermented milk containing a large amount of amino acid and/or a peptide with respect to the amount of lactic acid produced can be produced, a small amount of fermented milk can be used, and a product having a good flavor and easy to drink in the case of maintaining functionality can be easily produced. And available to consumers.

以下詳細地說明本發明。本申請案係主張2010年11月9日提出申請之日本專利申請案第2010-250754號之優先權者，包含上述專利申請案之說明書及/或圖式中記載之內容。The present invention will be described in detail below. The present application claims the priority of Japanese Patent Application No. 2010-250754, filed on Nov. 9, 2010, which is incorporated herein by reference.

本發明之屬於瑞士乳酸菌(Lactobacillus helveticus)之乳酸菌係藉由醱酵而自糖類產生乳酸之乳酸菌之1種，本說明書中亦稱為「本發明之乳酸菌」。本發明之乳酸菌之特徵在於具有高蛋白質分解活性，且胺基酸生成能力及/或功能性肽生成能力優異。具體而言，其係具有下述(a)~(d)之性質中之至少1種者：The lactic acid bacteria belonging to the lactic acid bacteria (Lactobacillus helveticus) of the present invention are one type of lactic acid bacteria which produce lactic acid from sugar by fermentation, and are also referred to as "lactic acid bacteria of the present invention" in the present specification. The lactic acid bacterium of the present invention is characterized by having high protein decomposition activity and excellent in amino acid-forming ability and/or functional peptide-producing ability. Specifically, it has at least one of the following properties (a) to (d):

(a)　藉由使獸乳醱酵而生成25 μmol/ml以上、較佳為30 μmol/ml以上之游離胺基酸；(a) producing a free amino acid of 25 μmol/ml or more, preferably 30 μmol/ml or more, by fermenting the mammal;

(b)　藉由使獸乳醱酵而生成8.5 mg/ml以上、較佳為10 mg/ml以上之肽；(b) producing a peptide of 8.5 mg/ml or more, preferably 10 mg/ml or more, by fermenting the mammal;

(c)　藉由使獸乳醱酵而生成總量為260 μg/ml以上、較佳為280 μg/ml以上、進而較佳為300 μg/ml以上之XP肽及/或XPP肽；(c) producing XP peptide and/or XPP peptide in a total amount of 260 μg/ml or more, preferably 280 μg/ml or more, and more preferably 300 μg/ml or more by fermenting the mammal;

上述(a)~(d)之性質可藉由該技術領域中公知之方法，以例如下述實施例中記載般進行判定。若簡單地進行說明，則可首先以乳蛋白(例如獸乳)作為基質而使屬於瑞士乳酸菌之乳酸菌於30~45℃、較佳為30~34℃下醱酵10~30小時，再藉由測定所獲得之乳蛋白醱酵物中所含之游離胺基酸或肽之量、酸度等而判定上述(a)~(d)之性質。The properties of the above (a) to (d) can be determined by the methods known in the art, for example, as described in the following examples. For simple explanation, the lactic acid bacteria belonging to the Swiss lactic acid bacteria can be firstly fermented for 10 to 30 hours at 30 to 45 ° C, preferably 30 to 34 ° C, using milk protein (for example, animal milk) as a substrate. The properties of the above (a) to (d) were determined by measuring the amount of the free amino acid or peptide contained in the obtained milk protein mash and the acidity.

關於(a)，胺基酸之量可使用公知之胺基酸分析裝置，例如高效液相層析儀進行測定。本發明之乳酸菌係藉由獸乳之醱酵而生成25 μmol/ml以上、較佳為30 μmol/ml以上之游離胺基酸者。又，較佳為，生成5 μmol/ml以上、更佳為6 μmol/ml或7 μmol/ml以上之支鏈胺基酸，且所生成之游離胺基酸中15%以上、更佳為19%或20%以上為支鏈胺基酸(纈胺酸、白胺酸、異白胺酸)。又，游離胺基酸較佳為含有選自丙胺酸、離胺酸及絲胺酸之至少1種2000 μg/ml以上，進而較佳為含有至少2種以上胺基酸各1500 μg/ml。Regarding (a), the amount of the amino acid can be measured using a well-known amino acid analyzer such as a high performance liquid chromatography. The lactic acid bacteria of the present invention are produced by the fermentation of animal milk to produce a free amino acid of 25 μmol/ml or more, preferably 30 μmol/ml or more. Further, it is preferred to form a branched amino acid of 5 μmol/ml or more, more preferably 6 μmol/ml or 7 μmol/ml or more, and more than 15%, more preferably 19, of the free amino acid formed. More than or more than 20% is a branched amino acid (proline, leucine, isoleucine). Further, the free amino acid preferably contains at least one selected from the group consisting of alanine, lysine, and serine at 2000 μg/ml or more, and more preferably at least two or more amino acids each at 1500 μg/ml.

關於(b)，肽之量可使用該技術領域中公知之方法，例如OPA(O-Phthaldialdehyde，鄰苯二甲醛)法(Church等人，J. Dairy Sci. 1983. 66:1219-1227)測定。本發明之乳酸菌係藉由獸乳之醱酵而生成8.5 mg/ml以上、較佳為10 mg/ml以上之肽者。With regard to (b), the amount of the peptide can be determined by a method known in the art, such as the OPA (O-Phthaldialdehyde, phthalaldehyde) method (Church et al., J. Dairy Sci. 1983. 66: 1219-1227). The lactic acid bacteria of the present invention produce a peptide of 8.5 mg/ml or more, preferably 10 mg/ml or more, by fermentation of animal milk.

關於(c)，XP肽係指包含Xaa-Pro(Xaa為任意胺基酸)之肽，XP肽中包含例如：YP(Tyr-Pro，酪胺酸-脯胺酸)、VP(Val-Pro，纈胺酸-脯胺酸)、IP(Ile-Pro，異白胺酸-脯胺酸)、AP(Ala-Pro，丙胺酸-脯胺酸)、EP(Glu-Pro，穀胺酸-脯胺酸)、RP(Arg-Pro，精胺酸-脯胺酸)、QP(Gln-Pro，穀胺醯胺-脯胺酸)、TP(Thr-Pro，蘇胺酸-脯胺酸)、MP(Met-Pro，甲硫胺酸-脯胺酸)及GP(Gly-Pro，甘胺酸-脯胺酸)。又，XPP肽係指包含Xaa-Pro-Pro(Xaa為任意胺基酸)之肽，XPP肽中包含例如：VPP(Val-Pro-Pro，纈胺酸-脯胺酸-脯胺酸)、IPP(Ile-Pro-Pro，異白胺酸-脯胺酸-脯胺酸)及LPP(Leu-Pro-Pro，白胺酸-脯胺酸-脯胺酸)。再者，本說明書中亦將該等XP肽及XPP肽分別稱為二肽及三肽，又，亦總稱為「功能性肽」。XP肽及/或XPP肽之量可使用公知之LC/MS(Liquid chromatography/Mass spectrometry，液相色譜質譜聯用)裝置進行測定。本發明之乳酸菌藉由獸乳之醱酵而生成總量為260 μg/ml以上、較佳為280 μg/ml以上、更佳為300 μg/ml以上之XP肽及/或 XPP肽。尤佳為生成60 μg/ml以上、更佳為65 μg/ml以上、進而較佳為70 μg/ml以上之YP肽。Regarding (c), the XP peptide refers to a peptide comprising Xaa-Pro (Xaa is any amino acid), and the XP peptide contains, for example, YP (Tyr-Pro, tyrosine-proline), VP (Val-Pro) , valine-proline, IP (Ile-Pro, isoleucine-proline), AP (Ala-Pro, alanine-proline), EP (Glu-Pro, glutamic acid - Proline), RP (Arg-Pro, arginine-proline), QP (Gln-Pro, glutamine-proline), TP (Thr-Pro, threonine-proline) , MP (Met-Pro, methionine-proline) and GP (Gly-Pro, glycine-proline). Further, the XPP peptide refers to a peptide comprising Xaa-Pro-Pro (Xaa is any amino acid), and the XPP peptide contains, for example, VPP (Val-Pro-Pro, valine-proline-proline), IPP (Ile-Pro-Pro, isoleucine-proline-proline) and LPP (Leu-Pro-Pro, leucine-proline-proline). In addition, in the present specification, these XP peptides and XPP peptides are also referred to as dipeptides and tripeptides, respectively, and are also collectively referred to as "functional peptides". The amount of XP peptide and/or XPP peptide can be measured by a known LC/MS (Liquid chromatography/Mass spectrometry) apparatus. The lactic acid bacteria of the present invention produce a total of 260 μg/ml or more, preferably 280 μg/ml or more, more preferably 300 μg/ml or more of XP peptide and/or XPP peptide by fermentation of animal milk. More preferably, it is a YP peptide which is 60 μg/ml or more, more preferably 65 μg/ml or more, and still more preferably 70 μg/ml or more.

關於(d)，酸度(醱酵乳中之酸濃度)可藉由公知之中和滴定法，使用例如公知之自動滴定裝置進行測定。又，IPP肽及VPP肽可藉由針對(c)所記載之方法進行測定。本發明之乳酸菌係使獸乳於30℃以上34℃以下醱酵所獲得之醱酵乳之單位酸度之IPP肽及VPP肽的量以VPP換算量計為5.5 mg/ml以上、較佳為6.0 mg/ml以上者。再者，上述量係將醱酵乳比重設為1.02而算出。又，本說明書中記載之IPP肽及VPP肽之量係以ACE抑制活性為基礎之VPP換算量，可藉由下述式求出：Regarding (d), the acidity (acid concentration in the fermented milk) can be measured by a known method and a titration method using, for example, a known automatic titrator. Further, the IPP peptide and the VPP peptide can be measured by the method described in (c). In the lactic acid bacteria of the present invention, the amount of the IPP peptide and the VPP peptide per unit acid of the fermented milk obtained by fermenting the animal milk at 30 ° C or more and 34 ° C or less is 5.5 mg/ml or more, preferably 6.0 mg in terms of VPP. /ml or more. Further, the above amount was calculated by setting the specific gravity of the fermented milk to 1.02. In addition, the amount of the IPP peptide and the VPP peptide described in the present specification is a VPP conversion amount based on the ACE inhibitory activity, and can be obtained by the following formula:

(VPP換算量μg/ml)=IPP量(μg/ml)×1.7+VPP量(μg/ml)(VPP conversion amount μg/ml)=IPP amount (μg/ml)×1.7+VPP amount (μg/ml)

本發明之乳酸菌係具有上述(a)~(d)中至少1種性質者，但較佳為顯示上述性質中之2種、3種或全部之乳酸菌。例如，本發明之乳酸菌為如下者：具有(a)、(b)、(c)或(d)之性質，或者具有(a)及(b)、(a)及(c)、(a)及(d)、(b)及(c)、(b)及(d)、或(c)及(d)之性質，或者具有(a)及(b)及(c)、(a)及(b)及(d)、(a)及(c)及(d)、(b)及(c)及(d)之性質，或者具有(a)~(d)之性質。The lactic acid bacteria of the present invention has at least one of the above properties (a) to (d), but preferably one, three or all of the above-described properties are lactic acid bacteria. For example, the lactic acid bacteria of the present invention are as follows: having the properties of (a), (b), (c) or (d), or having (a) and (b), (a) and (c), (a) And (d), (b) and (c), (b) and (d), or (c) and (d), or (a) and (b) and (c), (a) and The nature of (b) and (d), (a) and (c) and (d), (b) and (c) and (d), or the nature of (a) to (d).

於本發明中，只要為顯示上述性質中之至少1種之屬於瑞士乳酸菌的乳酸菌，則包含於本發明之乳酸菌中。作為具有此種性質之較佳之瑞士乳酸菌，可列舉本發明者自以瑞士乳酸菌CM4株(台灣寄存編號CCRC 910111、日本寄存編號FERM BP-6060，專利文獻1)為母株所獲得之變異株中單獨選拔之瑞士乳酸菌CP3232株。再者，瑞士乳酸菌CM4株及CP3232株係由本申請人於作為基於用於專利微生物寄存之布達佩斯條約(Budapest Treaty)的國際寄存當局之獨立行政法人產業技術綜合研究所專利生物寄存中心(日本茨城縣築波市東1丁目1番地1中央第6郵政編碼305-8566)，分別於平成9年(1997年)8月15日及平成22年(2010年)8月4日寄存為FERM BP-6060及FERM BP-11271。已確認瑞士乳酸菌CP3232株如實施例中記載般具有所有上述(a)~(d)之性質。其中，瑞士乳酸菌CM4株已於1998年9月16日寄存於食品工業發展研究所，寄存編號為CCRC 910111；瑞士乳酸菌CP3232株已於2012年1月12日寄存於食品工業發展研究所，寄存編號為BCRC 910539。In the present invention, a lactic acid bacterium belonging to the Swiss lactic acid bacteria which exhibits at least one of the above properties is included in the lactic acid bacterium of the present invention. The preferred strain of the lactic acid bacterium of the present invention is the mutant strain obtained by the present inventors from the lactic acid bacteria CM4 strain (Taiwan registered number CCRC 910111, Japanese registered number FERM BP-6060, Patent Document 1). Individually selected Swiss lactic acid bacteria CP3232 strain. In addition, the Swiss lactic acid bacteria CM4 strain and the CP3232 strain are used by the applicant as a patented biological storage center of the Institute of Industrial Technology and Technology, an independent administrative agency based on the Budapest Treaty for the Collection of Patented Microorganisms (Ibaraki Prefecture, Japan) It is registered as FERM BP-6060 on August 15th, 2011 (August 15th) and August 4th (2010) in Heisei City, 1st Floor, 1st Floor, 1st Floor, Tsukuba City, 1st Floor, 305-8566) FERM BP-11271. It has been confirmed that the Swiss lactic acid bacteria CP3232 strain has all of the properties (a) to (d) described above as described in the examples. Among them, the Swiss lactic acid bacteria CM4 strain was deposited in the Food Industry Development Research Institute on September 16, 1998, the registration number is CCRC 910111; the Swiss lactic acid bacteria CP3232 strain was deposited in the Food Industry Development Institute on January 12, 2012, the registration number For BCRC 910539.

又，瑞士乳酸菌CP3232株如上所述係源自作為母株之瑞士乳酸菌CM4株之變異株。因此，瑞士乳酸菌CM4株及瑞士乳酸菌CP3232株之變異株亦具有上述(a)~(d)中任一性質之可能性較高。源自此種瑞士乳酸菌CP3232株或瑞士乳酸菌CM4株之變異株只要具有上述性質，則亦包含於本發明中。Further, the Swiss lactic acid bacteria CP3232 strain was derived from the mutant strain of the Swiss lactic acid bacterium CM4 strain as a parent strain as described above. Therefore, the mutant strains of the Swiss lactic acid bacteria CM4 strain and the Swiss lactic acid bacteria CP3232 strain are also highly likely to have any of the above properties (a) to (d). The mutant strain derived from such a Swiss lactic acid bacteria CP3232 strain or the Swiss lactic acid bacteria CM4 strain is also included in the present invention as long as it has the above properties.

於本發明中，「變異株」係指自母株所獲得之任意株。具體而言，係指藉由自母株自然突然變異或利用由化學或物理變異原導致之誘導變異而人工提高突然變異之頻率之方法、或藉由特異性突然變異誘導技術(例如基因重組)所獲得之株。對藉由此種方法所生成之微生物個體重複進行選擇、分離，將有用之微生物個體進行育種，藉此可獲得具有目標性質之變異株。In the present invention, "mutant strain" means any strain obtained from a mother strain. Specifically, it refers to a method of artificially increasing the frequency of sudden mutations by spontaneous mutation of the mother strain or by using induced mutations caused by chemical or physical mutations, or by specific sudden mutation induction techniques (eg, genetic recombination). The strain obtained. The microorganisms produced by such a method are repeatedly selected and separated, and the useful microorganism individual is cultured, whereby a mutant having a target property can be obtained.

此處所謂之變異株，包括CM4株、CP3232株或以該變異株為母株所獲得之株。即，對CM4株進行數次變異處理並選擇之變異株亦包含於CM4株之變異株中。Here, the mutant strain includes a CM4 strain, a CP3232 strain, or a strain obtained by using the mutant strain as a parent strain. That is, the mutant strain which was subjected to several variations of the CM4 strain and selected was also included in the mutant strain of the CM4 strain.

例如，源自瑞士乳酸菌CM4株之變異株、瑞士乳酸菌CP3232株及其變異株可根據藉由聚合酶鏈反應(PCR，polymerase chain reaction)法所得之乳酸菌之基因組DNA之放大片段的分子量分佈而容易地與其他乳酸菌株相區別。若簡單地進行說明，則係針對目標乳酸菌製備DNA試劑，藉由使用具有例如序列編號1及2中所示之序列之引子的PCR法進行基因放大，並分析所獲得之片段之電泳圖案，藉此可判斷是否為源自瑞士乳酸菌CP3232株或瑞士乳酸菌CM4株之變異株。但確認是否為變異株之方法並不限定於該方法，亦存在即便於未檢測出上述PCR片段之情形時，亦為源自瑞士乳酸菌CM4株之變異株、瑞士乳酸菌CP3232株之變異株之情況，可藉由針對其他基因片段之檢測或菌學性質等該技術領域中公知之方法而確認是否為變異株。For example, the mutant strain derived from the Swiss lactic acid bacterium CM4 strain, the Swiss lactic acid bacteria CP3232 strain, and the variant thereof can be easily obtained from the molecular weight distribution of the amplified fragment of the genomic DNA of the lactic acid bacteria obtained by the polymerase chain reaction (PCR) method. The ground is distinguished from other lactic acid strains. For simplicity of explanation, a DNA reagent is prepared for a target lactic acid bacterium, and gene amplification is carried out by using a PCR method having primers having, for example, the sequences shown in SEQ ID NOs: 1 and 2, and the electrophoresis pattern of the obtained fragment is analyzed. This can be judged whether it is a mutant strain derived from the Swiss lactic acid bacteria CP3232 strain or the Swiss lactic acid bacteria CM4 strain. However, the method of confirming whether or not the mutant strain is a strain is not limited to the method, and there is also a case where the mutant strain of the lactic acid bacteria CM4 strain and the mutant strain of the Swiss lactic acid bacteria CP3232 strain are derived even when the PCR fragment is not detected. Whether or not a mutant strain can be confirmed by a method known in the art such as detection of other gene fragments or bacteriological properties.

可藉由判斷此種變異株是否具有上述(a)~(d)之性質而取得可用作本發明之乳酸菌之菌株。A strain which can be used as the lactic acid bacteria of the present invention can be obtained by judging whether or not such a mutant has the properties of the above (a) to (d).

本發明之乳酸菌可藉由使用乳酸菌之培養中通常使用之培養基，於適當條件下進行培養而製備。培養中使用之培養基只要為含有碳源、氮源、無機鹽類等，並可有效地進行乳酸菌之培養者，則可使用天然培養基、合成培養基之任一者，只要為業者，即可為所使用之菌株適當選擇適合之公知之培養基。作為碳源，可使用乳糖、葡萄糖、半乳糖、赤糖蜜等，作為氮源，可使用酪蛋白之水解物、乳清蛋白水解物、大豆蛋白水解物等有機含氮物。又，作為無機鹽類，可使用磷酸鹽、鈉、鉀、鎂等。作為適於乳酸菌之培養之培養基，可列舉例如：MRS液體培養基、GAM培養基、BL培養基、日本鰻肝臟培養基(Briggs Liver Broth)、獸乳、脫脂乳、乳性乳清等。較佳為使用含有滅菌脫脂乳粉之乳培養基。The lactic acid bacteria of the present invention can be produced by culturing under appropriate conditions using a medium which is usually used in the culture of lactic acid bacteria. The medium to be used in the culture may be any one of a natural medium and a synthetic medium, as long as it is a carbon source, a nitrogen source, an inorganic salt, or the like, and can be efficiently cultured. The strain to be used is appropriately selected from a suitable medium. As the carbon source, lactose, glucose, galactose, brown molasses or the like can be used, and as the nitrogen source, an organic nitrogen-containing substance such as a hydrolysate of casein, a whey protein hydrolyzate or a soy protein hydrolyzate can be used. Further, as the inorganic salt, phosphate, sodium, potassium, magnesium or the like can be used. Examples of the medium suitable for culturing the lactic acid bacteria include MRS liquid medium, GAM medium, BL medium, Briggs Liver Broth, animal milk, skim milk, milk whey, and the like. It is preferred to use a milk medium containing sterilized skim milk powder.

又，本發明之乳酸菌之培養係於20℃至50℃、較佳為25℃至42℃、更佳為28℃至37℃下於厭氧條件下進行。溫度條件可藉由恆溫槽、覆套式加熱器、夾套等進行調整。又，厭氧條件下係指乳酸菌可增殖之程度之缺氧環境下，例如，可藉由使用厭氧腔室、厭氧箱或裝有脫氧劑之密閉容器或者袋子等，或者僅藉由密閉培養容器而製造厭氧條件。培養之形式為靜置培養、振盪培養、槽培養等。又，培養時間可設置為3小時至96小時。培養開始時之培養基之pH值較佳為維持於4.0~8.0。Further, the culture of the lactic acid bacteria of the present invention is carried out under anaerobic conditions at 20 ° C to 50 ° C, preferably 25 ° C to 42 ° C, more preferably 28 ° C to 37 ° C. The temperature conditions can be adjusted by a thermostatic bath, a mantle heater, a jacket, and the like. Further, under anaerobic conditions, it refers to an anoxic environment in which the lactic acid bacteria can proliferate, for example, by using an anaerobic chamber, an anaerobic tank or a closed container or bag containing a deoxidizing agent, or simply by sealing The container is cultured to produce anaerobic conditions. The culture form is static culture, shaking culture, tank culture, and the like. Also, the culture time can be set to 3 hours to 96 hours. The pH of the medium at the start of the culture is preferably maintained at 4.0 to 8.0.

對本發明之乳酸菌之具體製備例簡單地進行說明。例如，於使用瑞士乳酸菌CP3232株之情形時，於滅菌乳培養基(含有9.00%(w/w)還原脫脂乳粉之乳培養基等)中以約5%之比例將乳酸菌進行植菌，並於28~37℃下經過一晚(約18~28小時)進行培養。較佳為重複進行該培養操作。A specific preparation example of the lactic acid bacteria of the present invention will be briefly described. For example, in the case of using the Swiss lactic acid bacteria CP3232 strain, the lactic acid bacteria are sterilized in a sterilized milk medium (milk medium containing 9.00% (w/w) reduced skim milk powder, etc.), and at 28%, and at 28 The culture was carried out overnight (about 18 to 28 hours) at ~37 °C. Preferably, the culturing operation is repeated.

培養後，可直接使用所獲得之培養物，亦可進而視需要進行利用離心分離等之部分純化及/或利用過濾等之固液分離或滅菌操作。After the culture, the obtained culture can be used as it is, or a part of purification by centrifugation or the like and/or solid-liquid separation or sterilization using filtration or the like can be carried out as needed.

又，只要具有目標性質，則可使用對本發明之乳酸菌進行處理所獲得之乳酸菌之處理物，又，亦可對乳酸菌之處理物進行進一步處理。將此種處理之例記載於下文中。Further, as long as it has a target property, the treated product of the lactic acid bacteria obtained by treating the lactic acid bacteria of the present invention may be used, and the treated product of the lactic acid bacteria may be further processed. Examples of such treatments are described below.

可藉由使用乳酸菌之菌體及/或處理物使生乳、脫脂乳或豆乳醱酵而製備醱酵物。例如，將乳酸菌或經其他處理之乳酸菌接種於生乳、脫脂乳或豆乳等中，並於該技術領域中公知之乳酸菌醱酵條件(與上述乳酸菌培養之條件大致相同)下進行醱酵。所獲得之醱酵物可直接使用，或亦可進行過濾、滅菌、稀釋、濃縮等其他處理。The fermented product can be prepared by fermenting raw milk, skim milk or soymilk using the bacteria and/or treatment of the lactic acid bacteria. For example, lactic acid bacteria or other treated lactic acid bacteria are inoculated into raw milk, skim milk, soy milk, etc., and fermentation is carried out under the conditions of lactic acid bacteria fermentation conditions (substantially the same as those of the above lactic acid bacteria culture) known in the art. The obtained fermented material can be used directly, or can be subjected to other treatments such as filtration, sterilization, dilution, concentration, and the like.

可藉由使乳酸菌之菌體及/或處理物懸浮或稀釋於適當之溶劑中而製備成懸濁物或稀釋物。作為可使用之溶劑，例如可列舉：水、生理鹽水、磷酸緩衝生理鹽水(PBS，Phosphate buffered saline)等。The suspension or dilution can be prepared by suspending or diluting the cells and/or treatments of the lactic acid bacteria in a suitable solvent. Examples of the solvent that can be used include water, physiological saline, and PBS (Phosphate buffered saline).

可藉由對乳酸菌之菌體及/或處理物進行滅菌處理而製備成滅菌處理物。對乳酸菌之菌體及/或處理物進行滅菌處理時，例如，可進行過濾滅菌、放射性殺菌、過熱式殺菌、加壓式殺菌等公知之滅菌處理。The sterilized product can be prepared by sterilizing the cells and/or the treated material of the lactic acid bacteria. When the bacteria and/or the treated material of the lactic acid bacteria are sterilized, for example, a known sterilization treatment such as filtration sterilization, radioactive sterilization, superheat sterilization, or pressurized sterilization can be performed.

又，可藉由對乳酸菌之菌體及/或處理物進行加熱處理而製備成加熱處理物。製備加熱處理物時，使乳酸菌之菌體及/或處理物歷經固定時間，例如約10分鐘~1小時(例如約10~20分鐘)，進行高溫處理(例如80~150℃)。Further, the heat-treated product can be prepared by heat-treating the cells and/or the treated material of the lactic acid bacteria. When the heat-treated product is prepared, the cells and/or the treated material of the lactic acid bacteria are subjected to high-temperature treatment (for example, 80 to 150 ° C) for a fixed period of time, for example, for about 10 minutes to 1 hour (for example, about 10 to 20 minutes).

可藉由將乳酸菌之菌體及/或處理物破碎、粉碎或磨碎而製備破碎物、無細胞萃取物。例如，此種破碎可藉由物理破碎(攪拌、過濾器過濾等)、酵素溶解處理、化學品處理或者自溶處理等而進行。The crushed material and the cell-free extract can be prepared by crushing, pulverizing or grinding the cells and/or the treated material of the lactic acid bacteria. For example, such crushing can be carried out by physical crushing (stirring, filter filtration, etc.), enzyme dissolution treatment, chemical treatment, or autolysis treatment.

可藉由使用適當之水性溶劑或有機溶劑對乳酸菌之菌體及/或處理物進行萃取而獲得萃取物。作為萃取方法，只要為使用水性溶劑或有機溶劑作為萃取溶劑之萃取方法則並無特別限制，可列舉將上述乳酸菌或對乳酸菌進行有其他處理之處理物於水性或有機溶劑(例如水、甲醇、乙醇等)中浸漬、攪拌或回流之方法等公知之方法。The extract can be obtained by extracting the cells and/or treatments of the lactic acid bacteria using a suitable aqueous solvent or organic solvent. The extraction method is not particularly limited as long as it is an extraction method using an aqueous solvent or an organic solvent as an extraction solvent, and examples thereof include the above-mentioned lactic acid bacteria or a treatment for treating lactic acid bacteria in an aqueous or organic solvent (for example, water, methanol, or the like). A known method such as a method of immersing, stirring, or refluxing in ethanol or the like.

又，可將乳酸菌之菌體及/或處理物乾燥而製成粉狀物(粉末)或粒狀物。作為具體之乾燥方法，並無特別限制，例如可列舉：噴霧乾燥、轉筒乾燥、真空乾燥、冷凍乾燥等，可將該等單獨或組合使用。此時，亦可視需要而添加通常使用之賦形劑。Further, the cells and/or the treated material of the lactic acid bacteria can be dried to obtain a powder (powder) or a granular material. The specific drying method is not particularly limited, and examples thereof include spray drying, drum drying, vacuum drying, freeze drying, and the like, and these may be used singly or in combination. At this time, an excipient which is usually used may be added as needed.

再者，本發明之乳酸菌可為濕潤菌體或亦可為乾燥菌體。Furthermore, the lactic acid bacteria of the present invention may be wet cells or dry cells.

上述處理可進行單一處理，或者亦可將複數個處理適當組合進行。於本發明中，亦可與乳酸菌同樣地使用此種處理物。The above processing may be performed in a single processing, or a plurality of processing may be appropriately combined. In the present invention, such a treatment product can also be used in the same manner as the lactic acid bacteria.

上文中所獲得之乳酸菌及/或處理物可單獨地或與其他成分一同用作用以自獸乳生成胺基酸及/或肽之組合物。即，本發明提供一種含有至少1種屬於瑞士乳酸菌之乳酸菌，且用以生成胺基酸及/或肽之組合物。The lactic acid bacteria and/or treatments obtained above may be used alone or in combination with other ingredients to form a composition for the production of amino acids and/or peptides from animal milk. That is, the present invention provides a composition comprising at least one lactic acid bacterium belonging to the lactic acid bacteria of Switzerland and used to form an amino acid and/or a peptide.

本發明之組合物係可藉由使獸乳醱酵而生成25 μmol/ml以上、較佳為30 μmol/ml以上之游離胺基酸者。較佳為所生成之游離胺基酸之15%以上為支鏈胺基酸。或者進而，本發明之組合物係可藉由使獸乳醱酵而生成8.5 mg/ml以上、較佳為10 mg/ml以上之肽者。或者進而，本發明之組合物係可藉由使獸乳醱酵而生成總量為260 μg/ml以上、較佳為280 μg/ml、更佳為300 μg/ml以上之XP肽及/或XPP肽者。或者進而，本發明之組合物係使獸乳於30℃以上34℃以下醱酵所獲得之醱酵乳之單位酸度之IPP肽及VPP肽的量以VPP換算量計為6.o mg/g以上者。The composition of the present invention can produce a free amino acid of 25 μmol/ml or more, preferably 30 μmol/ml or more, by fermenting the mammal. Preferably, 15% or more of the free amino acid produced is a branched amino acid. Alternatively, the composition of the present invention can produce a peptide of 8.5 mg/ml or more, preferably 10 mg/ml or more, by fermenting the mammal. Alternatively, the composition of the present invention can produce a total of 260 μg/ml or more, preferably 280 μg/ml, more preferably 300 μg/ml or more of XP peptide and/or by fermenting the mammal. XPP peptide. Alternatively, the composition of the present invention is such that the amount of the IPP peptide and the VPP peptide per unit acid of the fermented milk obtained by fermenting the animal milk at 30 ° C or more and 34 ° C or less is 6.0 mg/g or more in terms of VPP. By.

本發明之組合物係含有上述乳酸菌及/或處理物作為有效成分者，可含有1種乳酸菌及/或處理物，亦可含有複數種不同之乳酸菌及/或處理物，進而亦可組合含有經不同處理之複數種乳酸菌處理物。再者，本發明之組合物中較佳為含有1×10⁷個/ml以上之乳酸菌之菌體。The composition of the present invention may contain one type of lactic acid bacteria and/or a processed substance, and may contain a plurality of different lactic acid bacteria and/or processed materials, and may also contain a combination of the above-mentioned lactic acid bacteria and/or processed materials. A plurality of lactic acid bacteria treatments treated differently. Further, the composition of the present invention is preferably a bacterial cell containing 1 × 10 ⁷ /ml or more of lactic acid bacteria.

又，本發明之組合物中除有效成分乳酸菌以外，只要不妨礙目標作用，則亦可單獨地或組合複數種而添加該技術領域中公知之添加劑及賦形劑。又，本發明之組合物亦可含有促進乳酸菌之醱酵之添加物(例如麩胺酸或葡萄糖等糖)。本發明之組合物之形態並無特別限制，可製成懸濁液、顆粒、粉末、膠囊等。本發明之組合物中之有效成分(乳酸菌)之含量根據其形態不同而不同，乳酸菌之量通常為0.0001~99質量%，較佳為0.001~80質量%，更佳為0.001~75質量%之範圍。又，本發明之組合物中所含之乳酸菌為約10⁷個/g~約10¹²個/g。Further, in the composition of the present invention, in addition to the active ingredient lactic acid bacteria, additives and excipients known in the art may be added singly or in combination of plural kinds as long as the target action is not inhibited. Further, the composition of the present invention may further contain an additive (for example, saccharide or glucose such as glucose) which promotes fermentation of lactic acid bacteria. The form of the composition of the present invention is not particularly limited, and it can be prepared into a suspension, a granule, a powder, a capsule or the like. The content of the active ingredient (lactic acid bacteria) in the composition of the present invention varies depending on the form thereof, and the amount of the lactic acid bacteria is usually 0.0001 to 99% by mass, preferably 0.001 to 80% by mass, more preferably 0.001 to 75% by mass. range. Further, the lactic acid bacteria contained in the composition of the present invention is from about 10 ⁷ /g to about 10 ¹² /g.

藉由使用本發明之乳酸菌或組合物使獸乳醱酵，可獲得高濃度地含有胺基酸及/或肽之醱酵乳組合物。因此，本發明提供一種醱酵乳，其特徵在於：藉由使用至少1種屬於瑞士乳酸菌之乳酸菌使獸乳醱酵而獲得，且含有以下(a)~(d)中至少1種成分：By fermenting the animal milk using the lactic acid bacteria or composition of the present invention, a fermented milk composition containing an amino acid and/or a peptide at a high concentration can be obtained. Accordingly, the present invention provides a fermented milk obtained by fermenting a mammal with at least one lactic acid bacterium belonging to the Swiss lactic acid bacterium, and containing at least one of the following (a) to (d):

(a)　25 μmol/ml以上、較佳為30 μmol/ml之游離胺基酸(較佳為其中15%以上為支鏈胺基酸)；(a) 25 μmol/ml or more, preferably 30 μmol/ml of free amino acid (preferably 15% or more of which is a branched amino acid);

(b)　8.5 mg/ml以上、較佳為10 mg/ml以上之肽；(b) a peptide of 8.5 mg/ml or more, preferably 10 mg/ml or more;

(c)　總量為260 μg/ml以上、較佳為280 μg/ml以上、更佳為300 μg/ml以上之XP肽及/或XPP肽；(c) XP peptide and/or XPP peptide in a total amount of 260 μg/ml or more, preferably 280 μg/ml or more, more preferably 300 μg/ml or more;

(d)　6.0 mg/g以上之IPP肽及VPP肽之單位酸度的VPP換算量。(d) VPP conversion amount per unit acidity of IPP peptide and VPP peptide of 6.0 mg/g or more.

本發明之醱酵乳可藉由使用本發明之乳酸菌或組合物並依據該技術領域中公知之方法進行醱酵處理而製備。醱酵處理可藉由於獸乳中添加至少1種乳酸菌並於適當條件下培養而進行。作為獸乳，可使用牛乳、山羊乳、馬乳等源自哺乳動物之乳汁，或該等之脫脂乳、還原乳、煉乳等加工乳，可使用1種乳汁，亦可組合使用複數種乳汁或加工乳。乳之固形物成分濃度並無特別限定，例如，使用脫脂乳之情形時之非脂乳固形物成分濃度為3~15質量%左右，較佳為6~15質量%。獸乳亦可於醱酵處理前進行殺菌處理。獸乳中亦可添加促進乳酸菌醱酵之添加物(例如麩胺酸或葡萄糖等糖)。或者，亦可自獸乳單離乳蛋白成分，並以此種乳蛋白成分為基質進行醱酵。The fermented milk of the present invention can be prepared by subjecting the lactic acid bacteria or composition of the present invention to a fermentation treatment according to a method known in the art. The fermentation treatment can be carried out by adding at least one lactic acid bacteria to the animal milk and culturing under appropriate conditions. As the animal milk, milk derived from a mammal such as cow's milk, goat's milk or horse's milk, or processed milk such as skim milk, reduced milk or condensed milk may be used, and one type of milk may be used, or a plurality of types of milk may be used in combination or Processing milk. The concentration of the solid content of the milk is not particularly limited. For example, when the skim milk is used, the concentration of the non-fat milk solid content is about 3 to 15% by mass, preferably 6 to 15% by mass. The animal milk can also be sterilized before the fermentation. Additives that promote lactic acid bacteria fermentation (such as sugars such as glutamic acid or glucose) may also be added to the animal milk. Alternatively, the milk protein component may be separated from the animal milk, and the milk protein component may be used as a substrate for fermentation.

添加於獸乳中之乳酸菌較佳為使用經預培養之乳酸菌作為醱酵劑。又，添加之乳酸菌量並無限定，通常以乾燥乳酸菌體換算計為0.005~10質量%，較佳為0.05~5質量%。使用之乳酸菌可直接為乳酸菌培養物，亦可為將培養之乳酸菌藉由過濾或離心分離而自培養基分離者，亦可為自培養基分離後進行冷凍或冷凍乾燥而保存者。The lactic acid bacteria added to the animal milk preferably use a pre-cultured lactic acid bacterium as a bacterium. In addition, the amount of the lactic acid bacteria to be added is not limited, and is usually 0.005 to 10% by mass, preferably 0.05 to 5% by mass in terms of dry lactic acid bacteria. The lactic acid bacteria to be used may be directly cultured as a lactic acid bacteria, or may be isolated from the culture medium by filtration or centrifugation of the cultured lactic acid bacteria, or may be stored by freezing or freeze-drying after separation from the culture medium.

醱酵處理之條件與上述乳酸菌之培養條件大致相同，於20℃至50℃、較佳為25℃至42℃、更佳為28℃至37℃下並於厭氧條件下進行。再者，於下述實施例中，本發明之乳酸菌於30~34℃(約32℃)下特別多地生成支鏈胺基酸及功能性肽。因此，尤佳為於30~34℃下進行醱酵處理。溫度條件可藉由恆溫槽、覆套式加熱器、夾套等進行調整。醱酵處理可藉由靜置培養、振盪培養、槽培養等形式進行。又，醱酵時間可設為3小時~96小時，較佳為12~36小時。較佳為一面測定醱酵槽之pH值及酸度一面進行醱酵，pH值較佳為維持於4.0~8.0。The conditions of the fermentation treatment are substantially the same as those of the above lactic acid bacteria, and are carried out under anaerobic conditions at 20 ° C to 50 ° C, preferably 25 ° C to 42 ° C, more preferably 28 ° C to 37 ° C. Further, in the following examples, the lactic acid bacteria of the present invention particularly produced branched-chain amino acids and functional peptides at 30 to 34 ° C (about 32 ° C). Therefore, it is especially preferable to carry out the fermentation treatment at 30 to 34 °C. The temperature conditions can be adjusted by a thermostatic bath, a mantle heater, a jacket, and the like. The fermentation treatment can be carried out by means of static culture, shaking culture, tank culture, and the like. Further, the fermentation time can be set to 3 hours to 96 hours, preferably 12 to 36 hours. Preferably, the fermentation is carried out while measuring the pH and acidity of the fermentation tank, and the pH is preferably maintained at 4.0 to 8.0.

以如上所述之方式獲得之醱酵乳只要含有所需成分，則可於醱酵乳中進行處理，又亦可於醱酵乳之處理物中進行進一步處理。將此種處理之例記載於下文中。The fermented milk obtained as described above may be treated in the fermented milk as long as it contains the desired ingredients, or may be further processed in the treatment of the fermented milk. Examples of such treatments are described below.

可藉由使醱酵乳及/或醱酵乳處理物懸浮或稀釋於適當之溶劑中而製備成懸濁物或稀釋物。作為可使用之溶劑，例如可列舉：水、生理鹽水、磷酸緩衝生理鹽水(PBS)等。Suspensions or dilutions can be prepared by suspending or diluting the fermented milk and/or fermented milk treatment in a suitable solvent. Examples of the solvent that can be used include water, physiological saline, and phosphate buffered saline (PBS).

可藉由對醱酵乳及/或醱酵乳處理物進行滅菌處理而製備成滅菌處理物。對醱酵乳及/或醱酵乳處理物進行滅菌處理時，例如可進行過濾滅菌、放射性殺菌、過熱式殺菌、加壓式殺菌等公知之滅菌處理。The sterilization treatment can be prepared by sterilizing the fermented milk and/or the fermented milk treatment. When sterilizing the fermented milk and/or the fermented milk processed product, for example, a known sterilization treatment such as filter sterilization, radioactive sterilization, superheat sterilization, or pressurized sterilization can be performed.

可藉由對醱酵乳及/或醱酵乳處理物進行加熱處理而製備成加熱處理物。製備加熱處理物係歷經固定時間，例如約10分鐘~1小時(例如約10~20分鐘)，進行高溫處理(例如80~150℃)。The heat treatment can be prepared by heat-treating the fermented milk and/or the fermented milk treatment. The heat treatment material is prepared for a fixed period of time, for example, for about 10 minutes to 1 hour (for example, about 10 to 20 minutes), and subjected to high temperature treatment (for example, 80 to 150 ° C).

又，可藉由對醱酵乳及/或醱酵乳處理物進行過濾或離心分離而製備成其上清液(乳清)。Further, the supernatant (whey) can be prepared by filtering or centrifuging the fermented milk and/or the fermented milk treated product.

進而可使醱酵乳及/或醱酵乳處理物乾燥而製成粉狀物(粉末)或粒狀物。作為具體乾燥方法，並無特別限制，例如可列舉：噴霧乾燥、轉筒乾燥、真空乾燥、冷凍乾燥等，可將該等單獨地或組合使用。此時，亦可視需要而添加通常使用之賦形劑。Further, the fermented milk and/or the fermented milk processed product can be dried to prepare a powder (powder) or a granule. The specific drying method is not particularly limited, and examples thereof include spray drying, drum drying, vacuum drying, freeze drying, and the like, and these may be used singly or in combination. At this time, an excipient which is usually used may be added as needed.

上述處理可進行單一處理，或者亦可將複數種處理適當組合而進行。本發明中亦包含此種醱酵乳處理物。The above treatment may be carried out in a single treatment, or a plurality of treatments may be appropriately combined. Such a fermented milk processed product is also included in the present invention.

本發明之醱酵乳或處理物由於高濃度地含有各種胺基酸及/或肽，因此可用於利用其胺基酸及/或肽之性質之用途中。例如，胺基酸與風味、能量源、精神穩定作用、成長促進作用等有關，因此可將本發明之醱酵乳用作調味劑、營養佐劑、能量補充劑。又，含有纈胺酸、白胺酸及異白胺酸之支鏈胺基酸(BCAA)為必需胺基酸，可用於增強肌肉或預防身體疲勞。又，認為藉由丙胺酸(Ala)之存在而促進肝臟中之分解反應。又，亦已知Ala於味覺方面可引出美味，具有鹽醃、醋醃效果或刺激味等掩蓋效果。一般認為，離胺酸(Lys)係必需胺基酸中最容易不足之胺基酸，必需以抗體或激素、酵素等原材料之形式攝取。又，報告有Lys亦與鈣吸收、膠原蛋白形成、骨組織之生產有關。絲胺酸(Ser)作為磷脂絲胺酸或其他胺基酸等生物體內之各種重要物質之前驅物較為重要。又，已知其降低血膽固醇，藉由使腦內之神經元活化等抑制腦細胞死亡而防止腦之老化，或形成肌膚之保濕成分。因此，本發明之醱酵乳或處理物可用作調味劑、營養佐劑、能量補充劑、腦功能改善劑、自主神經調節劑、副交感神經調節劑、交感神經調節劑、肌肉分解損傷抑制劑、肌肉增強劑、疲勞緩解劑、耐力改善劑、酒精代謝劑等。進而VPP肽及IPP肽具有ACE抑制活性及鬆弛效果，LPP肽具有ACE抑制活性，YP肽具有降血壓作用及抗焦慮作用，VP、IP、AP、EP、RP、QP、TP、MP及GP肽為生物體內非分解性，具有ACE抑制活性，因此，本發明之醱酵乳或處理物可用作降血壓劑及壓力緩和劑。Since the fermented milk or the treated material of the present invention contains various amino acids and/or peptides at a high concentration, it can be used in applications in which the properties of the amino acids and/or peptides are utilized. For example, the amino acid is related to flavor, energy source, mental stabilizing action, growth promoting action, and the like, and thus the fermented milk of the present invention can be used as a flavoring agent, a nutritional adjuvant, and an energy supplement. Further, a branched amino acid (BCAA) containing lysine, leucine and isoleucine is an essential amino acid which can be used to strengthen muscles or prevent physical fatigue. Further, it is considered that the decomposition reaction in the liver is promoted by the presence of alanine (Ala). Further, it is also known that Ala can be tasted in terms of taste, and has a masking effect such as salting, vinegar pickling or irritating. It is considered that the amino acid which is most easily deficient in the amino acid (Lys) essential amino acid must be ingested as a raw material such as an antibody or a hormone or an enzyme. Also, it is reported that Lys is also involved in calcium absorption, collagen formation, and bone tissue production. Serine is important as a precursor to various important substances in living organisms such as phospholipid serine or other amino acids. Further, it is known that it lowers blood cholesterol, prevents brain aging by inhibiting brain cell death by activating neurons in the brain, or forms a moisturizing component of the skin. Therefore, the fermented milk or the treatment of the present invention can be used as a flavoring agent, a nutritional adjuvant, an energy supplement, a brain function improving agent, an autonomic nervous regulator, a parasympathetic regulating agent, a sympathetic regulating agent, a muscle decomposition damage inhibitor, Muscle enhancer, fatigue reliever, endurance improver, alcohol metabolism agent, and the like. Furthermore, VPP peptide and IPP peptide have ACE inhibitory activity and relaxation effect, LPP peptide has ACE inhibitory activity, YP peptide has hypotensive effect and anxiolytic effect, VP, IP, AP, EP, RP, QP, TP, MP and GP peptide It is non-decomposable in vivo and has ACE inhibitory activity. Therefore, the fermented milk or the treatment of the present invention can be used as a blood pressure lowering agent and a stress relieving agent.

又，亦可自所獲得之醱酵乳或其處理物，使用公知之分離、純化法，將各種胺基酸及/或肽成分或者含有該等成分之餾分純化。Further, various amino acid and/or peptide components or fractions containing the components may be purified from the obtained fermented milk or a processed product thereof by a known separation and purification method.

本發明之醱酵乳或其處理物為含有上述(a)~(d)成分中之至少1種成分(例如胺基酸、肽)者，亦可組合含有複數種成分。又，於醱酵乳或其處理物中亦可含有醱酵處理中使用之乳酸菌。The fermented milk of the present invention or the treated product thereof may contain at least one component (for example, an amino acid or a peptide) of the above components (a) to (d), or may contain a plurality of components in combination. Further, the lactic acid bacteria used in the fermentation treatment may be contained in the fermented milk or the processed material thereof.

又，於本發明之醱酵乳中，只要不妨礙目標作用，則亦可單獨地或組合複數種而添加下述添加劑、其他公知之降血壓劑、壓力緩和劑、營養佐劑、能量補充劑、肌肉增強劑、調味劑等。Further, in the fermented milk of the present invention, as long as the target action is not inhibited, the following additives, other known blood pressure lowering agents, pressure alleviators, nutritional adjuvants, energy supplements, and the like may be added singly or in combination of plural kinds. Muscle enhancers, flavoring agents, etc.

本發明之醱酵乳之形態並無特別限制，例如，可製成醱酵處理後所獲得之醱酵乳原樣之懸濁液形態，以及藉由該技術領域中公知之方法進行處理所獲得之片劑、膠囊劑、顆粒劑、散劑、粉劑、糖漿劑、乾糖漿劑等形態。醱酵乳較佳為製成容易經口投予或攝取之形態。再者，懸濁液等液體可為於即將投予或攝取之前懸浮於水或其他適當之介質中之形態，又，於片劑、顆粒劑之情形時，亦可以眾所周知之方法塗佈其表面。The form of the fermented milk of the present invention is not particularly limited, and for example, a suspension form of the lyophilized milk obtained after the fermentation treatment, and a tablet obtained by treating by a method known in the art, Capsules, granules, powders, powders, syrups, dry syrups and the like. The fermented milk is preferably in a form that is easily administered or ingested orally. Further, the liquid such as the suspension may be in the form of being suspended in water or other suitable medium immediately before administration or ingestion, and in the case of tablets or granules, the surface may be coated by a well-known method. .

上述形態之醱酵乳可於藉由醱酵處理所獲得之醱酵乳中調配通常使用之賦形劑、崩解劑、黏合劑、濕潤劑、穩定劑、緩衝劑、潤滑劑、防腐劑、界面活性劑、甜味料、調味劑、芳香劑、酸味料、著色劑等添加劑，並依據常用方法而製造。例如，於醱酵乳以醫藥或增進健康目的使用之情形時，可調配藥學上所容許之載體或添加劑。作為此種藥學上所容許之載體及添加物之例，可列舉：水、藥學上所容許之有機溶劑、膠原蛋白、聚乙烯醇、聚乙烯吡咯烷酮、羧乙烯聚合物、海藻酸鈉、水溶性葡聚糖、水溶性糊精、羧甲基澱粉鈉、果膠、三仙膠、***膠、酪蛋白、明膠、瓊脂、甘油、丙二醇、聚乙二醇、凡士林、石蠟、硬脂醇、硬酯酸、人類血清白蛋白、甘露醇、山梨糖醇、乳糖、作為醫藥添加物所容許之界面活性劑等。The fermented milk of the above form can be formulated with the excipients, disintegrants, binders, wetting agents, stabilizers, buffers, lubricants, preservatives, interfacial activities commonly used in the fermented milk obtained by the fermentation treatment. Additives such as a sweetener, a flavoring agent, a flavoring agent, a flavoring agent, a coloring agent, and the like, and are manufactured according to a usual method. For example, when the fermented milk is used for medical or health-promoting purposes, a pharmaceutically acceptable carrier or additive may be added. Examples of such pharmaceutically acceptable carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium alginate, and water-soluble. Dextran, water-soluble dextrin, sodium carboxymethyl starch, pectin, tricyanidin, acacia, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, hard An ester acid, human serum albumin, mannitol, sorbitol, lactose, a surfactant which is allowed as a pharmaceutical additive, and the like.

進而，於本發明之醱酵乳中亦可使醫藥、飲食品、飼料之製造中所使用之各種添加劑或其他各種物質共存。作為此種物質或添加劑，可列舉：各種油脂(例如，大豆油、玉米油、紅花油、橄欖油等植物油，牛油、鯡魚油等動物油脂)、天然藥(例如蜂王漿、人蔘等)、胺基酸(例如麩醯胺、半胱胺酸、白胺酸、精胺酸等)、多元醇(例如乙二醇、聚乙二醇、丙二醇、甘油、糖醇，作為糖醇之例有山梨糖醇、赤藻糖醇、木糖醇、麥芽糖醇、甘露醇等)、天然高分子(例如***膠、瓊脂、水溶性玉米蛋白纖維、明膠、三仙膠、酪蛋白、麩質或麩質水解物、卵磷脂、澱粉、糊精等)、維生素(例如維生素C、維生素B群等)、礦物(例如鈣、鎂、鋅、鐵等)、食物纖維(例如甘露聚糖、果膠、半纖維素等)、界面活性劑(例如甘油脂肪酸酯、山梨醇脂肪酸酯等)、純化水、賦形劑(例如葡萄糖、玉米澱粉、乳糖、糊精等)、穩定劑、pH值調節劑、抗氧化劑、甜味料、呈味成分、酸味料、著色料及香料等。Further, in the fermented milk of the present invention, various additives or various other substances used in the manufacture of medicines, foods, and feeds may be coexistent. Examples of such a substance or an additive include various oils and fats (for example, vegetable oils such as soybean oil, corn oil, safflower oil, and olive oil, animal fats such as butter and salmon oil), natural medicines (for example, royal jelly, human cockroaches, etc.). Amino acids (such as branamine, cysteine, leucine, arginine, etc.), polyols (such as ethylene glycol, polyethylene glycol, propylene glycol, glycerol, sugar alcohols, as an example of sugar alcohols) Sorbitol, erythritol, xylitol, maltitol, mannitol, etc.), natural polymers (eg gum arabic, agar, water soluble zein fiber, gelatin, sanmon gum, casein, gluten or bran) Hydrolysate, lecithin, starch, dextrin, etc.), vitamins (such as vitamin C, vitamin B group, etc.), minerals (such as calcium, magnesium, zinc, iron, etc.), dietary fiber (such as mannan, pectin, Hemicellulose, etc.), surfactant (such as glycerin fatty acid ester, sorbitan fatty acid ester, etc.), purified water, excipients (such as glucose, corn starch, lactose, dextrin, etc.), stabilizers, pH adjustment Agent, antioxidant, sweetener, flavoring ingredient, sour material, Materials and spices.

又，本發明之醱酵乳中，作為上述有效成分以外之功能性成分或添加劑，例如可調配：牛磺酸、麩胱苷肽、肉鹼、肌酸、輔酶Q、葡糖醛酸、葡糖醛酸內酯、辣椒萃取物、生薑萃取物、可可萃取物、瓜拿納萃取物、藤黃果萃取物、茶胺酸、γ-胺基丁酸、辣椒素、辣椒素酯、各種有機酸、類黃酮類、多酚類、兒茶素類、黃嘌呤衍生物、低聚果糖等難消化性寡糖、聚乙烯吡咯烷酮等。該等添加劑之調配量係視添加劑之種類及所需攝取量而適當決定。Further, in the fermented milk of the present invention, as a functional component or an additive other than the above-mentioned active ingredient, for example, taurine, glutathione, carnitine, creatine, coenzyme Q, glucuronic acid, glucose may be formulated. Aldolonic acid lactone, capsicum extract, ginger extract, cocoa extract, guarana extract, Garcinia Cambogia extract, theanine, γ-aminobutyric acid, capsaicin, capsaicin ester, various organic Acid, flavonoids, polyphenols, catechins, xanthine derivatives, oligofructose and other indigestible oligosaccharides, polyvinylpyrrolidone, and the like. The blending amount of these additives is appropriately determined depending on the kind of the additive and the required intake amount.

本發明之醱酵乳之投予或攝取對象為脊椎動物，具體而言，為哺乳動物，例如人、靈長類(猿、黑猩猩等)、家畜動物(牛、馬、豬、羊、鳥等)、寵物用動物(狗、貓等)、實驗用動物(小白鼠、大白鼠等)、進而爬行動物及鳥類。尤其是希望攝取胺基酸及/或肽之對象，例如以高血壓患者、顯示由壓力導致之症狀之人、運動前後之人等作為對象較佳。The fermented milk of the present invention is administered or vaccinated to a vertebrate, specifically, a mammal, such as a human, a primate (猿, chimpanzee, etc.), a livestock animal (cattle, horse, pig, sheep, bird, etc.). Animals for pets (dogs, cats, etc.), experimental animals (white mice, rats, etc.), reptiles and birds. In particular, it is preferred that the subject to take up the amino acid and/or the peptide is, for example, a hypertensive patient, a person who exhibits symptoms caused by stress, a person before and after exercise, or the like.

本發明之醱酵乳之投予或攝取量根據對象之年齡及體重、投予/攝取路徑、投予/攝取次數、投予目的等而異，為能夠達成目標作用，可根據業者之斟酌而於較廣之範圍內進行變更。醱酵乳中所含之有效成分之含有比例並無特別限定，結合製造之容易性或較佳之一日投予量等而適當調節即可。由於本發明之醱酵乳安全性較高，故而亦可進一步增加攝取量。平均1日之攝取量可1次攝取，亦可分為數次攝取。又，其投予或攝取之頻率亦並無特別限定，可視投予/攝取路徑、對象之年齡及體重、目標效果之各種條件而適當選擇。The dosage or intake of the fermented milk of the present invention varies depending on the age and weight of the subject, the route of administration/intake, the number of administrations/intakes, the purpose of administration, and the like, and can be achieved in accordance with the discretion of the practitioner. Make changes within a wider range. The content ratio of the active ingredient contained in the mashed milk is not particularly limited, and may be appropriately adjusted in accordance with the easiness of production or a preferred one-day dosage. Since the fermented milk of the present invention has high safety, it is possible to further increase the intake. The average intake per day can be taken once, and can be divided into several intakes. Further, the frequency of administration or ingestion is not particularly limited, and may be appropriately selected depending on various conditions of the administration/intake route, the age and weight of the subject, and the target effect.

本發明之醱酵乳之投予/攝取路徑並無特別限定，較佳為經口投予或經口攝取。例如，可調配於飲食品或飼料中，或者製成片劑或顆粒劑等，經口投予或攝取。The administration/intake route of the fermented milk of the present invention is not particularly limited, and it is preferably oral administration or oral ingestion. For example, it can be formulated into foods or foods, or made into tablets or granules, etc., orally or ingested.

本發明之醱酵乳亦可與其他醫藥、治療或預防法等並用。此種其他醫藥可與本發明之醱酵乳共同製成一種製劑，又，亦可為單獨之製劑而同時或隔開間隔投予。The fermented milk of the present invention can also be used in combination with other medicines, treatments or prevention methods. Such other medicines may be formulated together with the fermented milk of the present invention, or may be administered as separate preparations at the same time or at intervals.

如上所述，本發明之醱酵乳可用作降血壓劑、腦功能改善劑、自主神經調節劑、副交感神經調節劑、交感神經調節劑、營養佐劑、能量補充劑、肌肉分解損傷抑制劑、肌肉增強劑、疲勞緩解劑、耐力改善劑、調味劑或酒精代謝劑等。As described above, the fermented milk of the present invention can be used as a blood pressure lowering agent, a brain function improving agent, an autonomic nervous regulator, a parasympathetic regulating agent, a sympathetic adjusting agent, a nutritional adjuvant, an energy supplement, a muscle decomposition damage inhibitor, Muscle enhancer, fatigue reliever, endurance improver, flavoring agent or alcohol metabolism agent.

本發明之乳酸菌及醱酵乳由於安全性較高且長期持續攝取較為容易，故亦可用於飲食品及飼料中。本發明之乳酸菌為大量生成具有各種功能之胺基酸及/或肽者，又本發明之醱酵乳為高濃度地含有此種胺基酸及/或肽者。進而，本發明之乳酸菌之相對於酸度之功能性肽之生成量較高，可抑制酸味，添加於各種飲食品中亦不妨礙飲食品本身之風味。因此，本發明之乳酸菌及醱酵乳可添加於各種飲食品中持續地攝取，期待各種效果。The lactic acid bacteria and the fermented milk of the present invention are also easy to be used for long-term continuous intake, and therefore can be used in foods and drinks and feed. The lactic acid bacteria of the present invention are those which produce a large amount of amino acids and/or peptides having various functions, and the fermented milk of the present invention contains such amino acids and/or peptides at a high concentration. Further, the amount of the functional peptide of the lactic acid bacteria of the present invention with respect to the acidity is high, and the sourness can be suppressed, and the flavor of the food or beverage itself is not hindered by being added to various foods and drinks. Therefore, the lactic acid bacteria and the fermented milk of the present invention can be continuously added to various foods and drinks, and various effects are expected.

因此，本發明之飲食品含有本發明之至少1種乳酸菌及/或其處理物、以及/或者本發明之醱酵乳及/或其處理物。於本發明中，飲食品中亦包含飲料。本發明之飲食品中除藉由胺基酸及/或肽謀求促進健康之健康飲食品、功能性飲食品、特定保健用飲食品等之外，亦包含可調配本發明之乳酸菌及醱酵乳之所有飲食品。Therefore, the food or drink of the present invention contains at least one lactic acid bacterium of the present invention and/or a treated product thereof, and/or the fermented milk of the present invention and/or a processed product thereof. In the present invention, the food and beverage also contains a beverage. In addition to the amino acid and/or peptide, the food or drink of the present invention contains healthy lactic acid foods, functional foods, and foods for specific health care, and the lactic acid bacteria and the fermented milk of the present invention. All food and drink.

作為本發明之飲食品，尤佳為功能性飲食品。本發明之「功能性飲食品」係指對於生物體具有固定之功能性之飲食品，例如包括：包括特定保健用飲食品(包括條件性特保[特定保健用食品])及營養功能飲食品之保健功能飲食品、特殊用途飲食品、營養輔助飲食品、健康輔助飲食品、補充品(例如，片劑、包衣錠、糖衣錠、膠囊及液劑等各種劑形者)及美容飲食品(例如節食飲食品)等所有所謂健康飲食品。As a food or drink of the present invention, a functional food or drink is particularly preferred. The "functional foods and drinks" of the present invention are foods and drinks having a fixed function for a living body, and include, for example, foods and beverages including specific health care (including conditional special care [special health foods]) and nutritional function foods and drinks. Health-care foods, foods, special-purpose foods, nutritional supplements, health-assist foods, supplements (for example, tablets, coated tablets, sugar-coated tablets, capsules, and liquids) and beauty foods and drinks ( For example, dieting foods and drinks, etc., all so-called healthy foods and drinks.

作為飲食品之具體例，可列舉：經管經腸營養劑等流食、壓片點心、片劑、咀嚼片、片劑、粉劑、散劑、膠囊劑、顆粒劑及飲劑等製劑形態之健康飲食品及營養輔助飲食品；綠茶、烏龍茶及紅茶等茶飲料、清涼飲料、果凍飲料、運動飲料、乳飲料、碳酸飲料、蔬菜飲料、果汁飲料、醱酵蔬菜飲料、醱酵果汁飲料、醱酵乳飲料、醱酵乳(優酪乳、固體酸乳酪)、醱酵乳飲料(殺菌)、乳酸菌飲料、濃縮型飲料、濃縮固形物、乳飲料(咖啡牛乳等)、飲料粉、可可飲料、牛乳以及純化水等飲料；黃油、果醬、香松及人造黃油等塗抹類；蛋黃醬、起酥油、乳蛋糕乳脂、調味汁類、麵包類、米飯類、面類、麵食、味噌湯、豆腐、酸乳酪、湯或醬料類、糕點(例如，餅乾或曲奇類、巧克力、糖果、蛋糕、冰淇淋、口香糖、含片)等。Specific examples of the food and drink include healthy foods and beverages in the form of a liquid such as a enteral nutrient, a tablet, a chewable tablet, a tablet, a powder, a powder, a capsule, a granule, and a drink. And nutritional supplement foods; tea drinks such as green tea, oolong tea and black tea, refreshing drinks, jelly drinks, sports drinks, milk drinks, carbonated drinks, vegetable drinks, fruit drinks, fermented vegetable drinks, fermented fruit drinks, fermented milk drinks, Beverages (yoghurt, solid yogurt), yoghurt beverage (sterilization), lactic acid bacteria beverage, concentrated beverage, concentrated solids, milk beverage (coffee milk, etc.), beverage powder, cocoa beverage, milk and purified water Butter, jam, pine and margarine; mayonnaise, shortening, custard, sauce, bread, rice, noodles, pasta, miso soup, tofu, yoghurt, soup or sauce Materials, pastries (for example, biscuits or cookies, chocolate, candy, cakes, ice cream, chewing gum, lozenges), etc.

關於本發明之飲食品，除本發明之乳酸菌及/或醱酵乳以外，亦可調配該飲食品之製造中所使用之其他食品原材料、各種營養素、各種維生素、礦物、食物纖維、各種添加劑(例如呈味成分、甜味料、有機酸等酸味料、穩定劑、香料)等，並依據常用方法而製造。In addition to the lactic acid bacteria and/or fermented milk of the present invention, other food materials, various nutrients, various vitamins, minerals, dietary fibers, and various additives used in the manufacture of the foods and drinks may be blended (for example, the food and beverage of the present invention). It is a flavoring ingredient, a sweetener, a sour material such as an organic acid, a stabilizer, a perfume, etc., and is produced according to a usual method.

於本發明之飲食品中，乳酸菌、醱酵乳或該等之處理物之調配量可考慮飲食品之形態或追求之食味或食感而由業者適當決定。如此，藉由將本發明之飲食品以可管理所需攝取量之形態飲用或食用，而提供使用該飲食品之降血壓、壓力緩和、營養補充、能量輔助、肌肉增強、腦功能改善、自律神經調節、副交感神經調節、交感神經調節、肌肉分解損傷抑制、肌肉增強、疲勞感之減輕、耐力之改善、酒精代謝等方法。In the food or drink of the present invention, the blending amount of the lactic acid bacteria, the fermented milk or the processed materials can be appropriately determined by the manufacturer in consideration of the form of the food or drink or the taste or texture desired. Thus, by drinking or eating the food or drink of the present invention in a form that can manage the desired intake, blood pressure reduction, stress relief, nutritional supplementation, energy assistance, muscle enhancement, brain function improvement, and self-discipline are provided using the food or drink. Neuromodulation, parasympathetic regulation, sympathetic regulation, inhibition of muscle breakdown injury, muscle enhancement, reduction of fatigue, improvement of endurance, alcohol metabolism, etc.

本發明之飲食品可藉由業者可利用之任意適當方法而製造。例如，可將本發明之乳酸菌及/或醱酵乳(處理物)製備成液體狀、凝膠狀、固體狀、粉末狀或顆粒狀後，再將其調配入飲食品中。或者亦可使本發明之乳酸菌及/或醱酵乳(處理物)直接混合或溶解於飲食品之原料中。乳酸菌及/或醱酵乳(處理物)亦可塗佈、包覆、滲透或附於飲食品上。乳酸菌及/或醱酵乳(處理物)可均勻地分散於飲食品中，亦可分佈不均。可調劑為裝有乳酸菌及/或醱酵乳(處理物)之膠囊等。可將乳酸菌及/或醱酵乳(處理物)以可食性膜或食用被膜劑等包裹。又，亦可於本發明之乳酸菌及/或醱酵乳(處理物)中添加適合之賦形劑等後成形為片劑等形狀。本發明之飲食品可進一步加工，此種加工品亦包含於本發明之範圍中。The food or beverage of the present invention can be produced by any suitable method available to the manufacturer. For example, the lactic acid bacteria and/or the fermented milk (treated product) of the present invention can be prepared into a liquid form, a gel form, a solid form, a powder form or a granulated form, and then blended into a food or drink. Alternatively, the lactic acid bacteria and/or the fermented milk (treated product) of the present invention may be directly mixed or dissolved in a raw material of a food or beverage. Lactic acid bacteria and/or fermented milk (treated material) may also be coated, coated, infiltrated or attached to food or beverage. Lactic acid bacteria and/or fermented milk (treated material) can be uniformly dispersed in foods and drinks, and may be unevenly distributed. The adjustable agent is a capsule containing lactic acid bacteria and/or fermented milk (treated material). The lactic acid bacteria and/or the fermented milk (treated material) may be wrapped in an edible film or an edible film or the like. In addition, a suitable excipient or the like may be added to the lactic acid bacteria and/or fermented milk (treated product) of the present invention, and then formed into a shape such as a tablet. The food or beverage of the present invention can be further processed, and such processed products are also included in the scope of the present invention.

於本發明之飲食品之製造中，亦可使用飲食品中慣用之各種添加物。作為添加物，並無限定，可列舉：顯色劑(亞硝酸鈉等)、著色料(梔子色素、紅102等)、香料(橙子香料等)、甜味料(甜菊甙、阿斯巴甜等)、防腐劑(乙酸鈉、山梨酸等)、乳化劑(軟骨素硫酸鈉、丙二醇脂肪酸酯等)、抗氧化劑(EDTA二鈉、維生素C等)、pH值調整劑(檸檬酸等)、化學調味料(肌苷酸鈉等)、增黏劑(三仙膠等)、膨脹劑(碳酸鈣等)、消泡劑(磷酸鈣)等、黏合劑(聚磷酸鈉等)、營養強化劑(鈣強化劑、維生素A等)、賦形劑(水溶性糊精等)等。進而，亦可進一步添加亞洲蔘萃取物、刺五加萃取物、桉樹萃取物、杜仲茶萃取物等功能性原材料。In the manufacture of the food or beverage of the present invention, various additives conventionally used in foods and drinks can also be used. The additive is not limited, and examples thereof include a color developer (such as sodium nitrite), a coloring material (such as scorpion dye and red 102), a fragrance (such as orange flavor), and a sweetener (stevia sinensis and aspartame). Sweet, etc.), preservatives (sodium acetate, sorbic acid, etc.), emulsifiers (sodium chondroitin sulfate, propylene glycol fatty acid esters, etc.), antioxidants (disodium EDTA, vitamin C, etc.), pH adjusters (citric acid, etc.) ), chemical seasonings (sodium inosine, etc.), tackifiers (Sanxian gum, etc.), bulking agents (calcium carbonate, etc.), defoamers (calcium phosphate), etc., adhesives (sodium polyphosphate, etc.), nutrition A strengthening agent (calcium fortifier, vitamin A, etc.), an excipient (water-soluble dextrin, etc.), and the like. Further, functional raw materials such as Asian anthraquinone extract, Acanthopanax senticosus extract, eucalyptus extract, and Eucommia tea extract may be further added.

本發明之飲食品如上所述，具有由胺基酸及/或肽引起之各種功能，再者安全性較高而無需擔心副作用。又，本發明之乳酸菌及/或醱酵乳可抑制酸度，風味較佳，添加於各種飲食品中亦不妨礙該飲食品之風味。所獲得之飲食品容易長期持續攝取，期待長時間地發揮各種功能。As described above, the food or drink of the present invention has various functions caused by an amino acid and/or a peptide, and further has high safety without worrying about side effects. Further, the lactic acid bacteria and/or the fermented milk of the present invention can suppress the acidity and have a good flavor, and are added to various foods and drinks without impeding the flavor of the food or drink. The obtained foods and drinks are easy to be ingested for a long period of time, and it is expected to exhibit various functions for a long time.

進而，本發明之乳酸菌及/或醱酵乳不僅可調配入人用飲食品中，亦可調配入家畜(牛、豬等)、賽馬、寵物(狗、貓等)之動物之飼料中。關於飼料，除對象為人以外，與飲食品大致相同，因此關於上述飲食品之記載對於飼料亦可同樣適用。Further, the lactic acid bacteria and/or the fermented milk of the present invention can be incorporated into human food and beverage products, and can be blended into feeds of animals such as livestock (bovine, pig, etc.), horse racing, pets (dogs, cats, etc.). Regarding the feed, the food is substantially the same as the food and drink, and therefore the description of the food or drink is also applicable to the feed.

[實施例][Examples]

以下，基於實施例進一步具體說明本發明，但本發明並不限定於該等實施例。Hereinafter, the present invention will be specifically described based on examples, but the present invention is not limited to the examples.

[實施例1]　L. helveticus CP3232株之鑑定[Example 1] Identification of L. helveticus CP3232 strain

將L. helveticus CM4株(台灣寄存編號CCRC 910111、日本寄存編號FERM BP-6060)進行育種，自所獲得之變異株中獲得顯示實施例2~5中所示之性質的新穎之L. helveticus CP3232株。The L. helveticus CM4 strain (Taiwan registration number CCRC 910111, Japanese registration number FERM BP-6060) was bred, and the novel L. helveticus CP3232 showing the properties shown in Examples 2 to 5 was obtained from the obtained mutant strain. Strain.

將L. helveticus CP3232株於MRS瓊脂培養基中於37℃下厭氧培養24小時，研究所獲得之群落之形態觀察學性質及生理學性質等。又，確定16S rRNA之序列，並確認與其他瑞士乳酸菌(模式菌株，type strain)具有99.9%之同源性。藉此可知，所獲得之菌株確實屬於L. helveticus，並為新穎之菌株。The L. helveticus CP3232 strain was anaerobic cultured on MRS agar medium at 37 ° C for 24 hours, and the morphological observation properties and physiological properties of the obtained community were studied. Further, the sequence of 16S rRNA was determined and confirmed to have 99.9% homology with other Swiss lactic acid bacteria (type strain). From this, it can be seen that the obtained strain does belong to L. helveticus and is a novel strain.

[實施例2]　醱酵乳之製備[Example 2] Preparation of fermented milk

將對9.00%(w/w)還原脫脂乳粉進行95℃達溫殺菌再冷卻至15℃者用作乳培養基。於乳培養基中將含有乳酸菌之醱酵乳以5%之比例接種，並於37℃下培養24小時。進而，重複該操作，並將所獲得之醱酵乳用作醱酵劑。本培養係於乳培養基中添加醱酵劑3%，並於32℃或37℃下培養24小時。測定醱酵乳之pH值及酸度。酸度係使用平沼自動滴定裝置COMTITE-450(平沼產業)進行測定。作為乳酸菌，係使用L. helveticus CP3232株、及比較用L. helveticus CP3264株(自抗高血壓製品分離)、L. helveticus CM4株(台灣寄存編號CCRC 910111、日本寄存編號FERM BP-6060)、L. helveticus CP1092株、L. helveticus CP1100株、L. helveticus CP1081株及L. helveticus CPN4株(日本專利特開平7-123977號公報)。The 9.00% (w/w) reduced skim milk powder was subjected to temperature sterilization at 95 ° C and then cooled to 15 ° C for use as a milk medium. The lactic acid bacteria-containing fermented milk was inoculated at a ratio of 5% in a milk medium, and cultured at 37 ° C for 24 hours. Further, the operation was repeated, and the obtained fermented milk was used as a fermenting agent. The culture was added with 3% fermenting agent in the milk medium, and cultured at 32 ° C or 37 ° C for 24 hours. Determine the pH and acidity of the fermented milk. The acidity was measured using a flat-automatic titrator COMTITE-450 (Pinguma Industry). As the lactic acid bacteria, L. helveticus CP3232 strain, and comparative L. helveticus CP3264 strain (separated from antihypertensive preparation), L. helveticus CM4 strain (Taiwan registration number CCRC 910111, Japanese registration number FERM BP-6060), L are used. Helveticus CP1092 strain, L. helveticus CP1100 strain, L. helveticus CP1081 strain, and L. helveticus CPN4 strain (Japanese Patent Laid-Open No. Hei 7-123977).

將其結果示於表1中。結果為，IPP之單位重量之ACE抑制活性為VPP之1.7倍，因此，將IPP量及VPP量藉由下式表示為VPP換算量：The results are shown in Table 1. As a result, the ACE inhibitory activity per unit weight of IPP was 1.7 times that of VPP. Therefore, the amount of IPP and the amount of VPP were expressed as VPP by the following formula:

VPP換算量(μg/ml)=IPP量(μg/ml)×1.7+VPP量(μg/ml)VPP conversion amount (μg/ml) = IPP amount (μg / ml) × 1.7 + VPP amount (μg / ml)

又，「酸度」表示醱酵乳中所含之酸(乳酸)之濃度。如表1中所示，於藉由L. helveticus CP3232株之醱酵所獲得之醱酵乳中，IPP肽及VPP肽之量相對於所生成之乳酸(即酸度)以VPP換算量計為6.2 mg/g，顯示單位酸度之肽量較高。醱酵乳比重係設為1.02而算出。Further, "acidity" means the concentration of the acid (lactic acid) contained in the fermented milk. As shown in Table 1, in the fermented milk obtained by fermentation of L. helveticus CP3232 strain, the amount of IPP peptide and VPP peptide was 6.2 mg in terms of VPP relative to the amount of lactic acid produced (ie, acidity). /g, which shows a higher amount of peptide in unit acidity. The fermented milk specific gravity was calculated to be 1.02.

[實施例3]醱酵乳上清液中之游離胺基酸之測定[Example 3] Determination of free amino acid in mashed milk supernatant

取實施例2中所獲得之醱酵乳10000 g並離心分離10分鐘，將上清液以蒸餾水適當稀釋後，以0.2 μM之膜濾器進行過濾，並藉由高效液相層析儀(HPLC，島津製作所)之胺基酸自動分析計(C-R7A/LC-10A)進行測定。分析條件如下。10000 g of the fermented milk obtained in Example 2 was centrifuged for 10 minutes, and the supernatant was appropriately diluted with distilled water, filtered through a 0.2 μM membrane filter, and subjected to high performance liquid chromatography (HPLC, Shimadzu). The amino acid automatic analyzer (C-R7A/LC-10A) of the manufacturer was measured. The analysis conditions are as follows.

管柱：Shim-pack Amino-Li(100 mmL×6.0 mmI.D.)Column: Shim-pack Amino-Li (100 mmL × 6.0 mmI.D.)

氨捕集器：Shim-pack ISC-30Li(50 mmL×4.0 mmI.D.)Ammonia trap: Shim-pack ISC-30Li (50 mmL × 4.0 mmI.D.)

管柱溫度：38~58℃Column temperature: 38~58°C

反應溫度：65℃Reaction temperature: 65 ° C

流動相：島津胺基酸分析用流動相套組Li型Mobile phase: mobile phase set Li-type for analysis of simian amino acid

反應液：島津胺基酸分析用反應液套組OPA試劑Reaction solution: reaction solution for the analysis of oxazinamide acid OPA reagent

流速：0.4 ml/min。Flow rate: 0.4 ml/min.

為檢測脯胺酸，於反應液中添加次氯酸鈉。最終以分光螢光檢測器(Ex：348 nm，Em：450 nm)測定與鄰苯二甲醛發生反應之胺基酸之螢光顯色。作為標準物質，使用濃度已知之胺基酸混合標準型(H)算出含量。To detect the proline, sodium hypochlorite was added to the reaction solution. Finally, the fluorescence coloration of the amino acid reacted with o-phthalaldehyde was measured by a spectroscopic fluorescence detector (Ex: 348 nm, Em: 450 nm). As a standard substance, the content was calculated using an amino acid mixed standard type (H) having a known concentration.

將其結果示於圖1及2中。圖1之A係表示使各乳酸菌於32℃下醱酵24小時所獲得之醱酵乳上清液中所含之各胺基酸量(nmol/ml)的結果，B係表示使各乳酸菌於37℃下醱酵24小時所獲得之醱酵乳上清液中所含之胺基酸量(nmol/ml)的結果。又，圖2之A係表示使各乳酸菌於32℃或37℃下醱酵24小時所獲得之醱酵乳上清液中所含之游離胺基酸總量(μmol/ml)，B係表示使各乳酸菌於32℃或37℃下醱酵24小時所獲得之醱酵乳上清液中所含之支鏈胺基酸量(μmol/ml)。根據該等結果可知，L. helveticus CP3232株具有較高之蛋白質分解活性，24小時之醱酵中生成25 mol/ml以上之游離胺基酸。尤其是於37℃下之醱酵中生成29.9 μmol/ml之游離胺基酸，於32℃下之醱酵中生成33.1 μmol/ml之游離胺基酸(圖2A)。又，顯示所生成之游離胺基酸中所含之支鏈胺基酸量較多，24小時之醱酵中所生成之游離胺基酸中15%以上為支鏈胺基酸。具體而言，於37℃下之醱酵中約19.4%為支鏈胺基酸，於32℃下之醱酵中約21.5%為支鏈胺基酸(圖2B)。The results are shown in Figures 1 and 2. Fig. 1A shows the results of the amount of each amino acid (nmol/ml) contained in the fermented milk supernatant obtained by fermenting each lactic acid bacterium at 32 ° C for 24 hours, and the B system shows that each lactic acid bacterium is in 37 The amount of amino acid (nmol/ml) contained in the fermented milk supernatant obtained by fermentation for 24 hours at °C. Further, A of Fig. 2 shows the total amount of free amino acid (μmol/ml) contained in the fermented milk supernatant obtained by fermenting each lactic acid bacterium at 32 ° C or 37 ° C for 24 hours, and the B system indicates that The amount of branched amino acid (μmol/ml) contained in the fermented milk supernatant obtained by lactic acid bacteria at 32 ° C or 37 ° C for 24 hours. According to these results, the L. helveticus CP3232 strain has a high proteolytic activity, and a free amino acid of 25 mol/ml or more is produced in the fermentation for 24 hours. In particular, 29.9 μmol/ml of free amino acid was produced in the fermentation at 37 ° C, and 33.1 μmol/ml of free amino acid was produced in the fermentation at 32 ° C ( FIG. 2A ). Further, it is shown that the amount of the branched amino acid contained in the produced free amino acid is large, and 15% or more of the free amino acid produced in the fermentation for 24 hours is a branched amino acid. Specifically, about 19.4% of the fermentations at 37 ° C were branched-chain amino acids, and about 21.5% of the fermentations at 32 ° C were branched-chain amino acids ( FIG. 2B ).

[實施例4]醱酵乳上清液中之肽量(OPA法)[Example 4] Peptide amount in fermented milk supernatant (OPA method)

將實施例2中所獲得之醱酵乳上清液中之肽量依據Church等人(J. Dairy Sci. 1983. 66:1219-1227.)之方法進行測定。若簡單地進行說明，則係將100 mM四硼酸鈉溶液25 ml、20%SDS(Sodium dodecyl sulfate，十二烷基硫酸鈉)2.5 ml、2-巰基乙醇0.1 ml混合，並於其中添加使對苯二甲醛溶解於甲醇中之4重量%之溶液1 ml，最終以蒸餾水製備成50 ml，從而將其製成OPA試劑。取實施例2中所獲得之醱酵乳10000 g並離心分離10分鐘，將培養上清液適當稀釋而製成分析試樣。將OPA試劑200 μl與分析試樣10 μl混合並攪拌十分鐘，此後於室溫下放置5分鐘，測定340 nm之吸收。使用胰化蛋白腖作為標準物質。The amount of the peptide in the fermented milk supernatant obtained in Example 2 was measured in accordance with the method of Church et al. (J. Dairy Sci. 1983. 66: 1219-1227.). For simple explanation, mix 25 ml of 100 mM sodium tetraborate solution, 2.5 ml of 20% SDS (Sodium dodecyl sulfate), 0.1 ml of 2-mercaptoethanol, and add it to the solution. The phthalic acid was dissolved in 1 ml of a 4% by weight solution in methanol, and finally 50 ml was prepared in distilled water to prepare an OPA reagent. 10000 g of the fermented milk obtained in Example 2 was taken and centrifuged for 10 minutes, and the culture supernatant was appropriately diluted to prepare an analysis sample. 200 μl of the OPA reagent was mixed with 10 μl of the analysis sample and stirred for ten minutes, after which it was allowed to stand at room temperature for 5 minutes, and the absorption at 340 nm was measured. Trypsin was used as a standard substance.

將其結果示於圖3中。根據該結果，顯示L. helveticus CP3232株具有較高之蛋白質分解活性，藉由醱酵而生成大量肽。具體而言，於24小時之醱酵中生成8.5 mg/ml以上之肽，於32℃之醱酵中生成11.1 mg/ml之肽，於37℃之醱酵下生成9.6 mg/ml之肽(圖3)。The results are shown in Fig. 3. Based on the results, it was revealed that the L. helveticus CP3232 strain has high proteolytic activity, and a large amount of peptide is produced by fermentation. Specifically, a peptide of 8.5 mg/ml or more was produced in 24 hours of fermentation, and 11.1 mg/ml of the peptide was produced in the fermentation at 32 ° C, and a peptide of 9.6 mg/ml was produced at 37 ° C ( image 3).

[實施例5]功能性肽之定量[Example 5] Quantification of functional peptides

取實施例2中所獲得之醱酵乳10000 g並離心分離10分鐘，再將上清液以蒸餾水適當稀釋，此後以0.2 μM之膜濾器進行過濾，並以LC/MS進行分析。LC/MS分析係依據Inoue等人(J. Biosci. Bioeng.,2009,108,111-115.)之方法實施。即，於LCMS-2010A系統(島津製作所)中使用RP-aqueous Column(野村科學)作為分離管柱進行分析。以合成肽作為標準物質算出含量。合成肽係使用(¹³C₅)Val-(¹³C₅)Pro-Pro、Ile-(¹³C₅)Pro-Pro、Glu-Pro、Gln-Pro(以上自Scrum公司購入)、Leu-Pro-Pro、Tyr-Pro、Val-Pro、Ile-Pro、Ala-Pro、Arg-Pro、Thr-Pro、Met-Pro、Gly-Pro(以上自Bachem公司購入)。10000 g of the fermented milk obtained in Example 2 was taken and centrifuged for 10 minutes, and the supernatant was appropriately diluted with distilled water, and then filtered with a 0.2 μM membrane filter, and analyzed by LC/MS. LC/MS analysis was carried out according to the method of Inoue et al. (J. Biosci. Bioeng., 2009, 108, 111-115.). That is, RP-aqueous Column was used as a separation column in the LCMS-2010A system (Shimadzu Corporation) for analysis. The content was calculated using a synthetic peptide as a standard substance. Synthetic peptides were used ( ¹³ C ₅ )Val-( ¹³ C ₅ )Pro-Pro, Ile-( ¹³ C ₅ )Pro-Pro, Glu-Pro, Gln-Pro (supplied from Scrum Corporation), Leu-Pro- Pro, Tyr-Pro, Val-Pro, Ile-Pro, Ala-Pro, Arg-Pro, Thr-Pro, Met-Pro, Gly-Pro (above purchased from Bachem).

將其結果示於圖4及5中。根據圖4之A及B中所示之結果可知，L. helveticus CP3232株具有較高之蛋白質分解活性，於24小時之醱酵中生成各種功能性肽。報告有，圖4中所列舉之功能性肽中，VPP及IPP具有ACE抑制活性及鬆弛效果，LPP具有ACE抑制活性，YP具有降血壓作用及抗焦慮作用，VP、IP、AP、EP、RP、QP、TP、MP及GP為生物體內非分解性，且具有ACE抑制活性。L. helveticus CP3232株尤其生成大量之VPP、IPP、YP、QP及TP。The results are shown in Figures 4 and 5. According to the results shown in A and B of Fig. 4, the L. helveticus CP3232 strain has high proteolytic activity, and various functional peptides are produced in the fermentation for 24 hours. It is reported that among the functional peptides listed in Figure 4, VPP and IPP have ACE inhibitory activity and relaxation effect, LPP has ACE inhibitory activity, YP has hypotensive effect and anxiolytic effect, VP, IP, AP, EP, RP , QP, TP, MP and GP are non-decomposable in vivo and have ACE inhibitory activity. The L. helveticus CP3232 strain in particular produces a large amount of VPP, IPP, YP, QP and TP.

又，根據圖5之A及B中所示之結果可知，L. helveticus CP3232株具有較高之蛋白質分解活性，於24小時之醱酵中生成總量為260 μg/ml以上之功能性肽(XP+XPP)。尤其是於32℃下之醱酵中生成318 μmol/ml之功能性肽(圖5A)，於37℃下之醱酵中生成282 μmol/ml之功能性肽(圖5B)。Further, according to the results shown in A and B of Fig. 5, L. helveticus CP3232 strain has a high proteolytic activity, and a functional peptide having a total amount of 260 μg/ml or more is produced in 24 hours of fermentation ( XP+XPP). In particular, a functional peptide of 318 μmol/ml was produced in the fermentation at 32 ° C (Fig. 5A), and a functional peptide of 282 μmol/ml was produced in the fermentation at 37 ° C (Fig. 5B).

[實施例6]醱酵乳之製備及肽量之測定[Example 6] Preparation of mashed milk and determination of peptide amount

與實施例2同樣，作為乳酸菌，係使用L. helveticus CP3232株、及比較用L. helveticus CNCM I-3435株(專利文獻4)、L. helveticus FERM BP-5445株(專利文獻7)、及L. helveticus CM4株(台灣寄存編號CCRC 910111、日本寄存編號FERM BP-6060)，測定培養後之醱酵乳之pH值及酸度、以及所生成之IPP肽及VPP肽之量。In the same manner as in the case of the lactic acid bacteria, L. helveticus CP3232 strain, and comparative L. helveticus CNCM I-3435 strain (patent document 4), L. helveticus FERM BP-5445 strain (patent document 7), and L were used. Helveticus CM4 strain (Taiwan registration number CCRC 910111, Japanese registration number FERM BP-6060), the pH and acidity of the fermented milk after culture, and the amount of the produced IPP peptide and VPP peptide were measured.

將其結果示於表2中。結果與實施例2同樣將IPP量及VPP量表示成VPP換算量。又，「酸度」表示醱酵乳中所含之酸(乳酸)之濃度。如表2中所示，於藉由L. helveticus CP3232株醱酵所獲得之醱酵乳中，IPP肽及VPP肽之量相對於所生成之乳酸(即酸度)以VPP換算量計為7.0 mg/g，顯示單位酸度之肽量較高。醱酵乳比重係設為1.02而算出。The results are shown in Table 2. As a result, in the same manner as in the second embodiment, the amount of IPP and the amount of VPP were expressed as VPP converted amounts. Further, "acidity" means the concentration of the acid (lactic acid) contained in the fermented milk. As shown in Table 2, the amount of the IPP peptide and the VPP peptide in the fermented milk obtained by the fermentation of the L. helveticus CP3232 strain was 7.0 mg/v in terms of VPP with respect to the produced lactic acid (ie, acidity). g, showing a higher amount of peptide per unit acidity. The fermented milk specific gravity was calculated to be 1.02.

[實施例7]醱酵乳上清液中之游離胺基酸之測定[Example 7] Determination of free amino acid in mashed milk supernatant

使用實施例6中所獲得之醱酵乳，與實施例3同樣地針對游離胺基酸進行測定。將其結果示於圖6及7中。The free amino acid was measured in the same manner as in Example 3 using the fermented milk obtained in Example 6. The results are shown in Figures 6 and 7.

圖6之A表示使各乳酸菌於32℃下醱酵24小時所獲得之醱酵乳上清液中所含之各胺基酸量(nmol/ml)的結果，B表示使各乳酸菌於37℃下醱酵24小時所獲得之醱酵乳上清液中所含之胺基酸量(nmol/ml)的結果。又，圖7之A表示使各乳酸菌於32℃或37℃下醱酵24小時所獲得之醱酵乳上清液中所含之游離胺基酸總量(μmol/ml)，B表示使各乳酸菌於32℃或37℃下醱酵24小時所獲得之醱酵乳上清液中所含之支鏈胺基酸量(μmol/ml)。根據該等結果可知，L. helveticus CP3232株具有較高之蛋白質分解活性，於24小時之醱酵中生成25 mol/ml以上之游離胺基酸。尤其是於37℃下之醱酵中生成28.1 μmol/ml之游離胺基酸，於32℃下之醱酵中生成32.4 μmol/ml之游離胺基酸(圖7A)。又，顯示所生成之游離胺基酸中所含之支鏈胺基酸量較多，於24小時之醱酵中所生成之游離胺基酸中15%以上為支鏈胺基酸。具體而言，於37℃下之醱酵中約20%為支鏈胺基酸，於32℃下之醱酵中約21%為支鏈胺基酸(圖7B)。Fig. 6A shows the results of the amount of each amino acid (nmol/ml) contained in the fermented milk supernatant obtained by fermenting each lactic acid bacterium at 32 ° C for 24 hours, and B indicates that each lactic acid bacterium was subjected to 37 ° C at 37 ° C. The amount of amino acid (nmol/ml) contained in the fermented milk supernatant obtained by fermentation for 24 hours. Further, A of Fig. 7 shows the total amount of free amino acid (μmol/ml) contained in the fermented milk supernatant obtained by fermenting each lactic acid bacterium at 32 ° C or 37 ° C for 24 hours, and B indicates each lactic acid bacterium. The amount of branched amino acid (μmol/ml) contained in the fermented milk supernatant obtained by fermentation at 32 ° C or 37 ° C for 24 hours. According to these results, the L. helveticus CP3232 strain has a high proteolytic activity, and a free amino acid of 25 mol/ml or more is produced in the fermentation for 24 hours. In particular, 28.1 μmol/ml of free amino acid was produced in the fermentation at 37 ° C, and 32.4 μmol/ml of free amino acid was produced in the fermentation at 32 ° C ( FIG. 7A ). Further, it was shown that the amount of the branched amino acid contained in the produced free amino acid was large, and 15% or more of the free amino acid produced in the fermentation for 24 hours was a branched amino acid. Specifically, about 20% of the fermentations at 37 ° C were branched-chain amino acids, and about 21% of the fermentations at 32 ° C were branched-chain amino acids ( FIG. 7B ).

[實施例8]醱酵乳上清液中之肽量(OPA法)[Example 8] Peptide amount in fermented milk supernatant (OPA method)

與實施例4同樣地測定實施例6中所獲得之醱酵乳上清液中之肽量。將其結果示於圖8中。根據該結果顯示，L. helveticus CP3232株具有較高之蛋白質分解活性，藉由醱酵生成大量肽。具體而言，於24小時之醱酵中生成8.5 mg/ml以上之肽，於32℃之醱酵中生成10.4 mg/ml之肽，於37℃之醱酵中生成9.1 mg/ml之肽(圖8)。The amount of the peptide in the fermented milk supernatant obtained in Example 6 was measured in the same manner as in Example 4. The results are shown in Fig. 8. According to the results, the L. helveticus CP3232 strain has a high proteolytic activity, and a large amount of peptide is produced by fermentation. Specifically, a peptide of 8.5 mg/ml or more was produced in 24 hours of fermentation, and a peptide of 10.4 mg/ml was produced in the fermentation at 32 ° C, and a peptide of 9.1 mg/ml was produced in the fermentation at 37 ° C ( Figure 8).

[實施例9]功能性肽之定量[Example 9] Quantification of functional peptide

與實施例5同樣地以LC/MS分析實施例6中所獲得之醱酵乳，並測定功能性肽量。將其結果示於圖9及10中。根據圖9之A及B中所示之結果可知，L. helveticus CP3232株具有較高之蛋白質分解活性，於24小時之醱酵中生成各種功能性肽。L. helveticus CP3232株尤其生成大量之VPP、IPP、YP、QP及TP。The fermented milk obtained in Example 6 was analyzed by LC/MS in the same manner as in Example 5, and the amount of functional peptide was measured. The results are shown in Figures 9 and 10. According to the results shown in A and B of Fig. 9, the L. helveticus CP3232 strain has a high proteolytic activity, and various functional peptides are produced in the fermentation for 24 hours. The L. helveticus CP3232 strain in particular produces a large amount of VPP, IPP, YP, QP and TP.

又，根據圖10之A及B中所示之結果可知，L. helveticus CP3232株具有較高之蛋白質分解活性，於24小時之醱酵中生成總量為260 μg/ml以上之功能性肽(XP+XPP)。尤其是於32℃下之醱酵中生成280 μmol/ml之功能性肽(圖10A)，於37℃下之醱酵中生成295 μmol/ml之功能性肽(圖10B)。Further, according to the results shown in A and B of Fig. 10, L. helveticus CP3232 strain has a high proteolytic activity, and a functional peptide having a total amount of 260 μg/ml or more is produced in 24 hours of fermentation ( XP+XPP). In particular, a functional peptide of 280 μmol/ml was produced in the fermentation at 32 ° C (Fig. 10A), and a functional peptide of 295 μmol/ml was produced in the fermentation at 37 ° C (Fig. 10B).

[參考例1]L. helveticus CP3232株及其變異株之鑑定方法[Reference Example 1] L. helveticus CP3232 strain and its mutant strain identification method

於實施例1中所獲得之乳酸菌L. helveticus CP3232株及其變異株藉由如實施例2~9中記載之方法進行測定，此外結合聚合酶鏈反應(PCR)法之識別，藉此可容易地與其他乳酸菌株區別。又，亦可藉由PCR法簡便地判定是否為作為L. helveticus CP3232株母株之L. helveticus CM4株的變異株。The lactic acid bacteria L. helveticus CP3232 strain obtained in Example 1 and its variant strain were measured by the methods as described in Examples 2 to 9, and in addition to the identification by the polymerase chain reaction (PCR) method, thereby being easily The ground is distinguished from other lactic acid strains. Further, it is also possible to easily determine whether or not it is a mutant strain of the L. helveticus CM4 strain which is a mother strain of L. helveticus CP3232 strain by a PCR method.

若對一例簡單地進行說明，則針對目標乳酸菌製備DNA試劑，並藉由使用以下引子之PCR法進行基因放大，以0.9%瓊脂糖凝膠進行電泳並分析其泳動圖案，藉此可判定是否為L. helveticus CP3232株之變異株，及是否為L. helveticus CM4株之變異株。When an example is briefly described, a DNA reagent is prepared for a target lactic acid bacterium, and the gene amplification is carried out by a PCR method using the following primers, and electrophoresis is carried out on a 0.9% agarose gel to analyze the migrating pattern, thereby determining whether or not L. helveticus CP3232 strain variant, and whether it is a variant of L. helveticus CM4 strain.

引子：Introduction:

agccacttcctccgattacag(序列編號1)、及gctattttagcagcgattcg(序列編號2)。Agccacttcctccgattacag (SEQ ID NO: 1) and gctattttagcagcgattcg (SEQ ID NO: 2).

於本實施例中，使用複數之L. helveticus菌株實施上述PCR法。將其結果示於圖11中。於圖11中各區帶如下。In the present example, the above PCR method was carried out using a plurality of L. helveticus strains. The result is shown in FIG. The zones in Figure 11 are as follows.

區帶1：L. helveticus CP3232株Zone 1: L. helveticus CP3232 strain

區帶2：L. helveticus CM4株Zone 2: L. helveticus CM4 strain

區帶3：L. helveticus CP3231株Zone 3: L. helveticus CP3231 strain

區帶4：L. helveticus CP3264株Zone 4: L. helveticus CP3264 strain

區帶5：L. helveticus CP1081株Zone 5: L. helveticus CP1081 strain

區帶6：L. helveticus CP209株Zone 6: L. helveticus CP209 strain

區帶7：L. helveticus CP210株Zone 7: L. helveticus CP210 strain

區帶8：L. helveticus CP293株Zone 8: L. helveticus CP293 strain

區帶9：L. helveticus CP617株Zone 9: L. helveticus CP617 strain

區帶10：L. helveticus CP39株Zone 10: L. helveticus CP39 strain

區帶11：L. helveticus CP790株Zone 11: L. helveticus CP790 strain

區帶12：L. helveticus JCM1120株Zone 12: L. helveticus JCM1120 strain

M：λ噬菌體DNA限制酵素HindIII分解物。M: λ phage DNA restriction enzyme HindIII decomposition product.

如圖11中所示，CM4變異株L. helveticus CP3232株、CP3231株及母株CM4株(區帶1~3)中可確認特徵性DNA片段，其他L. helveticus株無法確認到該片段。作為DNA標記物，同時對λ噬菌體DNA之限制酵素HindIII分解物進行電泳，測定特徵性DNA片段之分子量，結果為約2.2 kb。As shown in Fig. 11, characteristic DNA fragments were confirmed in the CM4 mutant L. helveticus CP3232 strain, CP3231 strain, and parental strain CM4 strain (zones 1 to 3), and the other L. helveticus strain could not confirm the fragment. As a DNA marker, the restriction enzyme HindIII decomposition product of λ phage DNA was simultaneously subjected to electrophoresis, and the molecular weight of the characteristic DNA fragment was measured and found to be about 2.2 kb.

再者，L. helveticus CP3232株與CP3231株、母株CM4株之識別亦可藉由實施例4及8中記載之使用OPA試劑之醱酵乳中的肽量分析而選擇L. helveticus CP3232株。即，若觀察以OPA試劑測定依據實施例2製備之醱酵乳中之肽量之值，則L. helveticus CP3231株及CM4株分別為7.5 mg/ml及4.5 mg/ml，與此相對，L. helveticus CP3232株顯示11.1 mg/ml之較高值而可容易地識別。Further, the L. helveticus CP3232 strain, the CP3231 strain, and the parent strain CM4 strain were identified by the peptide amount analysis in the fermented milk using the OPA reagent described in Examples 4 and 8, and the L. helveticus CP3232 strain was selected. That is, if the value of the amount of the peptide in the fermented milk prepared according to Example 2 was measured by the OPA reagent, the L. helveticus CP3231 strain and the CM4 strain were 7.5 mg/ml and 4.5 mg/ml, respectively, whereas L. The helveticus CP3232 strain shows a higher value of 11.1 mg/ml and can be easily identified.

[參考例2]L. helveticus CP3232株及其變異株之鑑定方法[Reference Example 2] L. helveticus CP3232 strain and its mutant strain identification method

以與參考例1相同之方式，試驗是否能夠藉由聚合酶鏈反應(PCR)法將實施例1中所獲得之乳酸菌L. helveticus CP3232株及其變異株與其他乳酸菌株(L. helveticus CNCM I-3435株(專利文獻4)及L. helveticus FERM BP-5445株(專利文獻7))加以區別。In the same manner as in Reference Example 1, it was tested whether the lactic acid bacteria L. helveticus CP3232 strain obtained in Example 1 and its variant strain and other lactic acid strains (L. helveticus CNCM I) can be obtained by polymerase chain reaction (PCR). -3435 strain (Patent Document 4) and L. helveticus FERM BP-5445 strain (Patent Document 7)) were distinguished.

將藉由使用引子之PCR法進行基因放大並進行電泳之結果示於圖12中。於圖12中，各區帶如下：The results of gene amplification and electrophoresis by PCR using primers are shown in Fig. 12. In Figure 12, the zones are as follows:

區帶1：L. helveticus CP3232株Zone 1: L. helveticus CP3232 strain

區帶2：L. helveticus CM4株Zone 2: L. helveticus CM4 strain

區帶3：L. helveticus CNCM I-3435株Zone 3: L. helveticus CNCM I-3435 strain

區帶4：L. helveticus FERM BP-5445株Zone 4: L. helveticus FERM BP-5445 strain

如圖12中所示，於作為CM4變異株之L. helveticus CP3232株及母株CM4株(區帶1及2)中確認特徵性DNA片段，其他L. helveticus株中未確認該片段。As shown in Fig. 12, a characteristic DNA fragment was confirmed in L. helveticus CP3232 strain and mother strain CM4 strain (zones 1 and 2) which are CM4 mutant strains, and this fragment was not confirmed in other L. helveticus strains.

本說明書中引用之所有刊物、專利及專利申請案係將其全文作為參考而組入本說明書中。All publications, patents and patent applications cited in this specification are hereby incorporated by reference in their entirety.

[產業上之可利用性][Industrial availability]

根據本發明，提供一種具有高蛋白質分解活性，且肽生成能力及/或胺基酸生成能力優異之屬於瑞士乳酸菌之乳酸菌。本乳酸菌所生成之胺基酸及/或肽具有各種功能，例如IPP、VPP、YP等肽具有降血壓作用。又，藉由將本乳酸菌用於醱酵，可獲得高濃度地含有此種具有各種功能之多種胺基酸及/或肽，且風味良好之醱酵乳。因此，本發明可用於醫藥、飲食品、促進健康等領域中。According to the present invention, there is provided a lactic acid bacterium belonging to a Swiss lactic acid bacterium which has high proteolytic activity and is excellent in peptide-forming ability and/or amino acid-forming ability. The amino acids and/or peptides produced by the present lactic acid bacteria have various functions, and peptides such as IPP, VPP, and YP have blood pressure lowering effects. Further, by using the lactic acid bacteria for fermentation, it is possible to obtain a fermented milk having a high concentration of such a plurality of amino acids and/or peptides having various functions and having a good flavor. Therefore, the present invention can be used in the fields of medicine, food and drink, and promotion of health.

[寄存編號][registration number]

台灣寄存編號BCRC 910539、日本寄存編號FERM BP-11271(Lactobacillus Helveticus(瑞士乳酸菌)CP3232株，台灣於2012年1月12日寄存、日本於平成22年(2010年)8月4日寄存)Taiwan registration number BCRC 910539, Japanese registration number FERM BP-11271 (Lactobacillus Helveticus (Swiss lactic acid bacteria) CP3232 strain, Taiwan was deposited on January 12, 2012, Japan was deposited on August 4, 2010 (2010))

台灣寄存編號CCRC 910111、日本寄存編號FERM BP-6060(Lactobacillus Helveticus(瑞士乳酸菌)CM4株，台灣於1998年9月16日寄存、日本於平成9年(1997年)8月15日寄存)Taiwan registration number CCRC 910111, Japanese registration number FERM BP-6060 (Lactobacillus Helveticus CM4 strain, Taiwan was deposited on September 16, 1998, Japan was deposited on August 15, 1997 (1997))

[序列表自由內容][sequence table free content]

序列編號1及2：DNA(合成寡核苷酸)Sequence numbers 1 and 2: DNA (synthetic oligonucleotides)

<110>　具有高蛋白質分解活性之瑞士乳酸菌<110> Swiss lactic acid bacteria with high protein decomposition activity

<120>　日商可爾必思股份有限公司<120> Japanese Business Co., Ltd.

<130>　PH-5013TW<130> PH-5013TW

<140>　100140985<140> 100140985

<141>　2011-11-09<141> 2011-11-09

<150>　2010-250754<150> 2010-250754

<151>　2010-11-09<151> 2010-11-09

<160>　2<160> 2

<170>　PatentIn第3.4版<170> PatentIn version 3.4

<210>　1<210> 1

<211>　21<211> 21

<212>　DNA<212> DNA

<213>　人工序列<213> Artificial sequence

<220><220>

<223>　合成寡核苷酸<223> Synthetic oligonucleotide

<400>　1<400> 1

<210>　2<210> 2

<211>　20<211> 20

<212>　DNA<212> DNA

<213>　人工序列<213> Artificial sequence

<220><220>

<223>　合成寡核苷酸<223> Synthetic oligonucleotide

<400>　2<400> 2

圖1係表示使各乳酸菌於32℃(A)或37℃(B)下醱酵24小時所獲得之醱酵乳上清液中所含之各胺基酸量(nmol/ml)的圖表。Fig. 1 is a graph showing the amount of each amino acid (nmol/ml) contained in the fermented milk supernatant obtained by fermenting each lactic acid bacterium at 32 ° C (A) or 37 ° C (B) for 24 hours.

圖2係表示使各乳酸菌於32℃或37℃下醱酵24小時所獲得之醱酵乳上清液中所含之游離胺基酸總量(μmol/ml)(A)及支鏈胺基酸量(μmol/ml)(B)的圖表。Figure 2 is a graph showing the total amount of free amino acid (μmol/ml) (A) and branched-chain amino acid contained in the fermented milk supernatant obtained by fermenting each lactic acid bacterium at 32 ° C or 37 ° C for 24 hours. A graph of the amount (μmol/ml) (B).

圖3係表示使各乳酸菌於32℃或37℃下醱酵24小時所獲得之醱酵乳上清液中之肽量(mg/ml)的圖表。Fig. 3 is a graph showing the amount of peptide (mg/ml) in the fermented milk supernatant obtained by fermenting each lactic acid bacterium at 32 ° C or 37 ° C for 24 hours.

圖4係使各乳酸菌於32℃(A)或37℃(B)下醱酵24小時所獲得之醱酵乳上清液中所含之功能性肽量(μg/ml)的圖表。Fig. 4 is a graph showing the amount of functional peptide (μg/ml) contained in the fermented milk supernatant obtained by fermenting each lactic acid bacterium at 32 ° C (A) or 37 ° C (B) for 24 hours.

圖5係表示使各乳酸菌於32℃(A)或37℃(B)下醱酵24小時所獲得之醱酵乳上清液中所含之XP肽及XPP肽之量(μg/ml)的圖表。Fig. 5 is a graph showing the amount of XP peptide and XPP peptide (μg/ml) contained in the fermented milk supernatant obtained by fermenting each lactic acid bacterium at 32 ° C (A) or 37 ° C (B) for 24 hours. .

圖6係表示使各乳酸菌於32℃(A)或37℃(B)下醱酵24小時所獲得之醱酵乳上清液中所含之各胺基酸量(nmol/ml)的圖表。Fig. 6 is a graph showing the amount of each amino acid (nmol/ml) contained in the fermented milk supernatant obtained by fermenting each lactic acid bacterium at 32 ° C (A) or 37 ° C (B) for 24 hours.

圖7係表示使各乳酸菌於32℃或37℃下醱酵24小時所獲得之醱酵乳上清液中所含之游離胺基酸總量(μmol/ml)(A)及支鏈胺基酸量(μmol/ml)(B)的圖表。Figure 7 is a graph showing the total amount of free amino acid (μmol/ml) (A) and branched-chain amino acid contained in the fermented milk supernatant obtained by fermenting each lactic acid bacterium at 32 ° C or 37 ° C for 24 hours. A graph of the amount (μmol/ml) (B).

圖8係表示使各乳酸菌於32℃或37℃下醱酵24小時所獲得之醱酵乳上清液中之肽量(mg/ml)的圖表。Fig. 8 is a graph showing the amount of peptide (mg/ml) in the fermented milk supernatant obtained by fermenting each lactic acid bacterium at 32 ° C or 37 ° C for 24 hours.

圖9係表示使各乳酸菌於32℃(A)或37℃(B)下醱酵24小時所獲得之醱酵乳上清液中所含之功能性肽量(μg/ml)的圖表。Fig. 9 is a graph showing the amount of functional peptide (μg/ml) contained in the fermented milk supernatant obtained by fermenting each lactic acid bacterium at 32 ° C (A) or 37 ° C (B) for 24 hours.

圖10係表示使各乳酸菌於32℃(A)或37℃(B)下醱酵24小時所獲得之醱酵乳上清液中所含之XP肽及XPP肽之量(μg/ml)的圖表。Fig. 10 is a graph showing the amount of XP peptide and XPP peptide (μg/ml) contained in the fermented milk supernatant obtained by fermenting each lactic acid bacterium at 32 ° C (A) or 37 ° C (B) for 24 hours. .

圖11係表示藉由聚合酶鏈反應(PCR)法將源自各種乳酸菌株之基因組DNA放大之片段的電泳圖案。Figure 11 is a diagram showing an electrophoresis pattern of a fragment amplified from genomic DNA of various lactic acid strains by polymerase chain reaction (PCR).

圖12係表示藉由聚合酶鏈反應(PCR)法將源自各種乳酸菌株之基因組DNA放大之片段的電泳圖案。Figure 12 is a diagram showing an electrophoresis pattern of a fragment amplified from genomic DNA of various lactic acid strains by polymerase chain reaction (PCR).

(無元件符號說明)(no component symbol description)

Claims

一種屬於瑞士乳酸菌之乳酸菌，其特徵在於具有以下(a)、(b)及(c)之性質或者(a)、(b)及(d)之性質，且該乳酸菌為瑞士乳酸菌CM4株(台灣寄存編號CCRC 910111、日本寄存編號FERM BP-6060)之變異株、瑞士乳酸菌CP3232株(台灣寄存編號BCRC 910539、日本寄存編號FERM BP-11271)或其變異株：(a)藉由使獸乳醱酵而生成25μmol/ml以上之游離胺基酸；(b)藉由使獸乳醱酵而生成8.5mg/ml以上之肽；(c)藉由使獸乳醱酵而生成總量為260μg/ml以上之XP肽及/或XPP肽；(d)使獸乳於30℃以上34℃以下醱酵所獲得之醱酵乳之單位酸度之IPP肽及VPP肽的量以VPP換算量計為6.0mg/g以上。 A lactic acid bacterium belonging to the lactic acid bacteria of Switzerland characterized by having the following properties (a), (b) and (c) or the properties of (a), (b) and (d), and the lactic acid bacteria is a lactic acid bacteria CM4 strain (Taiwan) The variant strain of the registration number CCRC 910111, Japanese registration number FERM BP-6060, the Swiss lactic acid bacteria CP3232 strain (Taiwan registration number BCRC 910539, Japanese registration number FERM BP-11271) or its variant: (a) by making the colander Fermentation produces 25 μmol/ml or more of free amino acid; (b) produces 8.5 mg/ml or more of the peptide by fermenting the animal's milk; (c) produces a total amount of 260 μg by fermenting the animal's milk. XP peptide and/or XPP peptide of more than ml; (d) The amount of IPP peptide and VPP peptide of the acidity of the fermented milk obtained by fermenting the animal's milk at 30 ° C or more and 34 ° C or less is 6.0 mg in terms of VPP /g or more.

如請求項1之乳酸菌，其中游離胺基酸之15%以上為支鏈胺基酸。 The lactic acid bacterium according to claim 1, wherein 15% or more of the free amino acid is a branched amino acid.

如請求項1或2之乳酸菌，其生成60μg/ml以上之YP肽。 A lactic acid bacterium according to claim 1 or 2 which produces a YP peptide of 60 μg/ml or more.

一種乳酸菌，其係瑞士乳酸菌CP3232株(台灣寄存編號BCRC 910539、日本寄存編號FERM BP-11271)。 A lactic acid bacterium which is a strain of Swiss lactic acid bacteria CP3232 (Taiwan registered number BCRC 910539, Japanese registered number FERM BP-11271).

一種至少1種如請求項1之屬於瑞士乳酸菌之乳酸菌的用途，其係用以製造自獸乳生成胺基酸及/或肽之組合物。 A use of at least one lactic acid bacterium belonging to the lactic acid bacteria of Switzerland according to claim 1 for producing a composition derived from animal milk to produce an amino acid and/or a peptide.

如請求項5之用途，其中藉由使獸乳醱酵而生成25μmol/ml以上之游離胺基酸。 The use of claim 5, wherein a free amino acid of 25 μmol/ml or more is produced by fermenting the mammal.

如請求項6之用途，其中游離胺基酸之15%以上為支鏈胺基酸。 The use of claim 6, wherein more than 15% of the free amino acid is a branched amino acid.

如請求項5至7中任一項之用途，其中藉由使獸乳醱酵而生成8.5mg/ml以上之肽。 The use according to any one of claims 5 to 7, wherein the peptide of 8.5 mg/ml or more is produced by fermenting the mammal.

如請求項5至7中任一項之用途，其中藉由使獸乳醱酵而生成總量260μg/ml以上之XP肽及/或XPP肽。 The use according to any one of claims 5 to 7, wherein a total of 260 μg/ml or more of XP peptide and/or XPP peptide is produced by fermenting the mammal.

如請求項5至7中任一項之用途，其中藉由使獸乳醱酵而生成60μg/ml以上之YP肽。 The use according to any one of claims 5 to 7, wherein a YP peptide of 60 μg/ml or more is produced by fermenting the mammal.

如請求項5至7中任一項之用途，其中使獸乳於30℃以上34℃以下醱酵所獲得之醱酵乳之單位酸度之IPP肽及VPP肽的量以VPP換算量計為6.0mg/g以上。 The use of any one of the items 5 to 7, wherein the amount of the IPP peptide and the VPP peptide of the acidity of the fermented milk obtained by fermenting the animal milk at 30 ° C or more and 34 ° C or less is 6.0 mg in terms of VPP. /g or more.

一種醱酵乳，其特徵在於：藉由使用至少1種屬於瑞士乳酸菌之乳酸菌使獸乳醱酵而獲得，該乳酸菌為瑞士乳酸菌CM4株(台灣寄存編號CCRC 910111、日本寄存編號FERM BP-6060)之變異株、瑞士乳酸菌CP3232株(台灣寄存編號BCRC 910539、日本寄存編號FERM BP-11271)或其變異株，且含有以下(a)、(b)及(c)之成分或者(a)、(b)及(d)之成分：(a)25μmol/ml以上之游離胺基酸，(b)8.5mg/ml以上之肽，(c)總量為260μg/ml以上之XP肽及/或XPP肽，(d)6.0mg/g以上之IPP肽及VPP肽之單位酸度之VPP換算量。 A fermented milk obtained by fermenting a mammalian lactic acid bacteria by using at least one lactic acid bacteria belonging to the Swiss lactic acid bacteria, which is a Swiss lactic acid bacteria CM4 strain (Taiwan registered number CCRC 910111, Japanese registered number FERM BP-6060) The mutant strain, the Swiss lactic acid bacteria CP3232 strain (Taiwan registration number BCRC 910539, Japanese registration number FERM BP-11271) or a variant thereof, and contains the following components (a), (b) and (c) or (a), (b) And (d) components: (a) free amino acid of 25 μmol/ml or more, (b) peptide of 8.5 mg/ml or more, (c) XP peptide and/or XPP peptide of 260 μg/ml or more in total (d) A VPP conversion amount of the unit acidity of the IPP peptide and the VPP peptide of 6.0 mg/g or more.

如請求項12之醱酵乳，其為含有至少1種屬於瑞士乳酸菌之乳酸菌者。 The fermented milk of claim 12, which contains at least one species belonging to Swiss lactic acid The lactic acid bacteria of bacteria.

如請求項12或13之醱酵乳，其為含有如請求項1至4中任一項之至少1種乳酸菌者。 The fermented milk of claim 12 or 13 which is at least one lactic acid bacteria according to any one of claims 1 to 4.

如請求項12或13之醱酵乳，其中游離胺基酸之15%以上為支鏈胺基酸。 The fermented milk of claim 12 or 13, wherein more than 15% of the free amino acid is a branched amino acid.

如請求項12或13之醱酵乳，其含有60μg/ml以上之YP肽。 The fermented milk of claim 12 or 13 which contains a YP peptide of 60 μg/ml or more.

如請求項12或13之醱酵乳，其中醱酵係於30℃以上34℃以下之溫度下進行。 The fermented milk of claim 12 or 13 wherein the fermentation is carried out at a temperature above 30 ° C and below 34 ° C.

如請求項12或13之醱酵乳，其係用作降血壓劑、腦功能改善劑、自主神經調節劑、副交感神經調節劑、交感神經調節劑、營養佐劑、能量補充劑、肌肉分解損傷抑制劑、肌肉增強劑、疲勞緩解劑、耐力改善劑、調味劑或酒精代謝劑。 The fermented milk of claim 12 or 13 is used as a blood pressure lowering agent, a brain function improving agent, an autonomic nervous regulator, a parasympathetic regulating agent, a sympathetic regulating agent, a nutritional adjuvant, an energy supplement, and a muscle decomposition damage inhibition. Agent, muscle enhancer, fatigue reliever, endurance improver, flavoring agent or alcohol metabolism agent.

一種飲食品，其特徵在於：含有如請求項1至4中任一項之至少1種乳酸菌及/或其處理物、以及/或者如請求項12至18中任一項之醱酵乳及/或其處理物。 A food or drink comprising at least one lactic acid bacterium according to any one of claims 1 to 4 and/or a treatment thereof, and/or the fermented milk of any one of claims 12 to 18 and/or Its treatment.

一種胺基酸及/或肽之製造方法，其特徵在於：使用如請求項1至4中任一項之至少1種乳酸菌使獸乳或乳蛋白醱酵，並自所獲得之醱酵物提取胺基酸及/或肽。 A method for producing an amino acid and/or a peptide, characterized in that at least one lactic acid bacterium according to any one of claims 1 to 4 is used to ferment animal milk or milk protein, and an amine is extracted from the obtained mash Base acid and / or peptide.

如請求項20之方法，其中胺基酸為支鏈胺基酸。 The method of claim 20, wherein the amino acid is a branched amino acid.

如請求項20或21之方法，其中肽為選自由XP肽及/或XPP肽所組成之群中之至少1種。 The method of claim 20 or 21, wherein the peptide is at least one selected from the group consisting of XP peptides and/or XPP peptides.

如請求項20或21之方法，其中醱酵係於30℃以上34℃以下之溫度下進行。The method of claim 20 or 21, wherein the fermentation is carried out at a temperature of from 30 ° C to 34 ° C.