JPH0740923B2 - Novel lactic acid bacterium by cell conjugation, fermented milk using the same, and method for producing the same - Google Patents
Novel lactic acid bacterium by cell conjugation, fermented milk using the same, and method for producing the sameInfo
- Publication number
- JPH0740923B2 JPH0740923B2 JP2184469A JP18446990A JPH0740923B2 JP H0740923 B2 JPH0740923 B2 JP H0740923B2 JP 2184469 A JP2184469 A JP 2184469A JP 18446990 A JP18446990 A JP 18446990A JP H0740923 B2 JPH0740923 B2 JP H0740923B2
- Authority
- JP
- Japan
- Prior art keywords
- pediococcus
- lactic acid
- ferm
- acid bacterium
- sbt
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title claims description 130
- 241000894006 Bacteria Species 0.000 title claims description 80
- 235000014655 lactic acid Nutrition 0.000 title claims description 65
- 239000004310 lactic acid Substances 0.000 title claims description 65
- 235000015140 cultured milk Nutrition 0.000 title claims description 30
- 238000004519 manufacturing process Methods 0.000 title claims description 30
- 230000021615 conjugation Effects 0.000 title description 7
- 241000604136 Pediococcus sp. Species 0.000 claims description 41
- 241000192001 Pediococcus Species 0.000 claims description 37
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 18
- 239000008101 lactose Substances 0.000 claims description 18
- 239000000284 extract Substances 0.000 claims description 17
- 238000011160 research Methods 0.000 claims description 17
- 240000000599 Lentinula edodes Species 0.000 claims description 15
- 235000013618 yogurt Nutrition 0.000 claims description 14
- 241000186660 Lactobacillus Species 0.000 claims description 13
- 229940039696 lactobacillus Drugs 0.000 claims description 13
- 238000000855 fermentation Methods 0.000 claims description 9
- 230000004151 fermentation Effects 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 9
- 235000013336 milk Nutrition 0.000 claims description 9
- 239000008267 milk Substances 0.000 claims description 9
- 210000004080 milk Anatomy 0.000 claims description 9
- 239000007858 starting material Substances 0.000 claims description 8
- 244000057717 Streptococcus lactis Species 0.000 claims description 5
- 235000014897 Streptococcus lactis Nutrition 0.000 claims description 5
- 239000002994 raw material Substances 0.000 claims description 4
- 241000194024 Streptococcus salivarius Species 0.000 claims description 3
- 241000194041 Lactococcus lactis subsp. lactis Species 0.000 claims 1
- 235000014969 Streptococcus diacetilactis Nutrition 0.000 claims 1
- 239000002609 medium Substances 0.000 description 24
- 239000002253 acid Substances 0.000 description 16
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- 235000020183 skimmed milk Nutrition 0.000 description 13
- 241000194017 Streptococcus Species 0.000 description 12
- 238000012360 testing method Methods 0.000 description 11
- CVSVTCORWBXHQV-UHFFFAOYSA-N creatine Chemical compound NC(=[NH2+])N(C)CC([O-])=O CVSVTCORWBXHQV-UHFFFAOYSA-N 0.000 description 10
- 230000000813 microbial effect Effects 0.000 description 10
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 9
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- 229920001817 Agar Polymers 0.000 description 7
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- 102000004190 Enzymes Human genes 0.000 description 4
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- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
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- IQUPABOKLQSFBK-UHFFFAOYSA-N 2-nitrophenol Chemical compound OC1=CC=CC=C1[N+]([O-])=O IQUPABOKLQSFBK-UHFFFAOYSA-N 0.000 description 3
- 241000191998 Pediococcus acidilactici Species 0.000 description 3
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- ABIUHPWEYMSGSR-UHFFFAOYSA-N bromocresol purple Chemical compound BrC1=C(O)C(C)=CC(C2(C3=CC=CC=C3S(=O)(=O)O2)C=2C=C(Br)C(O)=C(C)C=2)=C1 ABIUHPWEYMSGSR-UHFFFAOYSA-N 0.000 description 3
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- 150000008163 sugars Chemical class 0.000 description 3
- 102000016911 Deoxyribonucleases Human genes 0.000 description 2
- 108010053770 Deoxyribonucleases Proteins 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 235000013365 dairy product Nutrition 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 229960003276 erythromycin Drugs 0.000 description 2
- 235000013861 fat-free Nutrition 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 230000001766 physiological effect Effects 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
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- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-SVZMEOIVSA-N (+)-Galactose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-SVZMEOIVSA-N 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 240000007124 Brassica oleracea Species 0.000 description 1
- 235000003899 Brassica oleracea var acephala Nutrition 0.000 description 1
- 235000011301 Brassica oleracea var capitata Nutrition 0.000 description 1
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- 240000008067 Cucumis sativus Species 0.000 description 1
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 241000194036 Lactococcus Species 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241000165031 Pseudomonas lactis Species 0.000 description 1
- 235000005733 Raphanus sativus var niger Nutrition 0.000 description 1
- 235000006140 Raphanus sativus var sativus Nutrition 0.000 description 1
- 240000001970 Raphanus sativus var. sativus Species 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 229910000831 Steel Inorganic materials 0.000 description 1
- 241000194020 Streptococcus thermophilus Species 0.000 description 1
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- 125000003118 aryl group Chemical group 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 102000005936 beta-Galactosidase Human genes 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
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- 235000019634 flavors Nutrition 0.000 description 1
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- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
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- 125000001475 halogen functional group Chemical group 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
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- 229920001220 nitrocellulos Polymers 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 235000008373 pickled product Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
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- 235000013580 sausages Nutrition 0.000 description 1
- 230000035807 sensation Effects 0.000 description 1
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- 230000001953 sensory effect Effects 0.000 description 1
- 235000020254 sheep milk Nutrition 0.000 description 1
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- 235000010378 sodium ascorbate Nutrition 0.000 description 1
- PPASLZSBLFJQEF-RKJRWTFHSA-M sodium ascorbate Substances [Na+].OC[C@@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RKJRWTFHSA-M 0.000 description 1
- 229960005055 sodium ascorbate Drugs 0.000 description 1
- PPASLZSBLFJQEF-RXSVEWSESA-M sodium-L-ascorbate Chemical compound [Na+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RXSVEWSESA-M 0.000 description 1
- 241000894007 species Species 0.000 description 1
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- 235000008939 whole milk Nutrition 0.000 description 1
- 210000002268 wool Anatomy 0.000 description 1
Landscapes
- Dairy Products (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
【発明の詳細な説明】 産業上の利用分野 本発明は、ペディオコッカス属（Pediococcus）に属す
る乳酸菌と、ストレプトコッカス属（Streptococcus）
またはラクトバチルス属（Lactobacillus）に属する乳
酸菌とを細胞接合して得られるペディオコッカス属に属
し、芳香生産性とラクトース発酵性を有する新規な乳酸
菌に関する。TECHNICAL FIELD The present invention relates to a lactic acid bacterium belonging to the genus Pediococcus and a genus Streptococcus.
Alternatively, the present invention relates to a novel lactic acid bacterium belonging to the genus Pediococcus obtained by cell-bonding a lactic acid bacterium belonging to the genus Lactobacillus and having aroma producing property and lactose fermentability.
本発明の新規な乳酸菌は、ペディオコッカス属の有する
芳香生産性とストレプトコッカス属またはラクトバチル
ス属の有するラクトース醗酵性とを有し、発酵乳の製造
に有用に利用することができる。INDUSTRIAL APPLICABILITY The novel lactic acid bacterium of the present invention has the aroma-producing property of the genus Pediococcus and the lactose-fermenting property of the genus Streptococcus or Lactobacillus, and can be effectively used for the production of fermented milk.
従来の技術 ペディオコッカス属に属する乳酸菌は、耐塩性、酸生産
性、芳香生産性、抗菌性等の性質を有し、これらの性質
を利用して醗酵ソーセージ、塩蔵食品の製造に用いられ
ている。しかし、ラクトース醗酵性に欠けたりあるいは
醗酵性の微弱な菌種、菌株が多く、そのため醗酵乳の製
造に積極的に用いることができず、芳香生産性の利点を
醗酵乳製造に有効に活用できなかった。Conventional technology Lactic acid bacteria belonging to the genus Pediococcus have properties such as salt resistance, acid productivity, aroma productivity, antibacterial properties, etc., and these properties are used to produce fermented sausages and salted foods. There is. However, there are many strains of lactic acid that lack fermentability or are weakly fermentative, and therefore many strains cannot be used actively in the production of fermented milk, and the advantage of aroma productivity can be effectively utilized in fermented milk production. There wasn't.
発明が解決しようとする課題 本発明は、ペディオコッカス属に属する乳酸菌の芳香生
産性等の有用な性質を醗酵乳の製造に積極的に活用すべ
くなされたものである。すなわち、ペディオコッカス属
に属する乳酸菌に、ラクトバチルス属、ストレプトコッ
カス属に属する乳酸菌のラクトース醗酵性を細胞接合手
段によって付与して芳香生産性とラクトース醗酵性とを
有する新規乳酸菌々株を創製し、これを醗酵乳等の製造
に利用しようとするものである。DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention The present invention has been made to positively utilize useful properties such as aroma productivity of lactic acid bacteria belonging to the genus Pediococcus in the production of fermented milk. That is, to lactic acid bacteria belonging to the genus Pediococcus, Lactobacillus, lactose fermentability of lactic acid bacteria belonging to the genus Streptococcus is imparted by cell joining means to create a novel lactic acid bacteria strain having aroma productivity and lactose fermentability, This is intended to be used for the production of fermented milk and the like.
古くから大腸菌が接合によりその形質を伝達しあうこと
が知られており、また乳酸球菌においても同様の現象が
認められている〔Kondo,J.K.andL.L.Mckay:J.Dairy Sc
i.,68,2143（1985）〕。It has long been known that Escherichia coli transfers the trait by conjugation, and a similar phenomenon has been observed in lactococcus [Kondo, JKandL.L.Mckay: J. Dairy Sc.
i., 68, 2143 (1985)].
しかし、ペディオコッカス属の細菌に接合法を応用して
新しい性質を持った新菌株を作成し、これを醗酵乳製造
に利用しようとした研究はみられない。また、現今、微
生物株の形質転換の研究においてはプラスミドの抽出、
新プラスミド作成、親株への挿入による新株の作成とい
った遺伝子工学的手法によるものが主流を占めている
が、自然界においても生じている現象である微生物細胞
同士の接合を積極的に取上げ、新しい性質を持った新規
な菌株を作り出すことは、新菌株の安定性、人に与える
影響が把握できることによる安全性等に優れているもの
と考えることができる。However, no studies have been made to apply a conjugation method to a bacterium belonging to the genus Pediococcus to create a new strain having new properties and to utilize it for the production of fermented milk. At present, in the study of transformation of microbial strains, plasmid extraction,
The mainstream is genetic engineering techniques such as the creation of new plasmids and the creation of new strains by inserting them into parent strains. It can be considered that the production of the new strains possessed is excellent in stability and the like because the stability of the new strains and the influence on humans can be grasped.
課題を解決するための手段 すなわち、本発明は、ペディオコッカス属に属する乳酸
菌と、ストレプトコッカス属またはラクトバチルス属に
属する乳酸菌とを細胞接合して得られるペディオコッカ
ス属に属し、芳香生産性及びラクトース醗酵性を有する
新規な乳酸菌に関する。Means for solving the problem That is, the present invention, lactic acid bacteria belonging to the genus Pediococcus, and belonging to the genus Pediococcus obtained by cell-bonding lactic acid bacteria belonging to the genus Streptococcus or Lactobacillus, aroma productivity and The present invention relates to a novel lactic acid bacterium having lactose fermentability.
さらに、本発明は、この新規な乳酸菌を利用した醗酵乳
及びその製造法に関する。Furthermore, the present invention relates to fermented milk using this novel lactic acid bacterium and a method for producing the same.
本発明で使用するペディオコッカス属に属する乳酸菌
は、ペディオコッカス・アシディラクチシ（Pediococcu
s acidilactici）またはペディオコッカス・エスピー
（Pediococcus sp.）が用いられる。しかし、本発明者
らが市販の漬物製品から分離したペディオコッカス・エ
スピー（Pediococcus sp.）２−５株やペディオコッカ
ス・アシディラクチシ（Pediococcus acidilactici）NC
DO1859株を用いることが望ましい。特に、ペディオコッ
カス・エスピー２−５株は、その生理学的性状からペデ
ィオコッカス・ハロフィリス（Pediococcus halophili
s）あるいはペディオコッカス・アシディラクチシ（Ped
iococcus acidilactici）に類似した性状を示したが、
バージース マニュアル オブ システィマティック
バクテリオロジー（Bergey′s Manualof Systematic Ba
cteriology）第２巻 第1075、1079頁に記載される標準
株と次の点で明らかな類似点が見られなかった。すなわ
ち、ペディオコッカス・エスピー（Pediococcus sp.）
２−５株は 1.ペディオコッカス・アシディラクチシ（Pediococcus
acidilactici）とは糖の醗酵性は近いが、生産する乳酸
の施光性がペディオコッカス・アシディラクチシ（Pedi
ococcus acidilacitci）がDL型であるのに対しペディオ
コッカス・エスピー（Pediococcus sp.）２−５株はＬ
型である。また 2.ペディオコッカス・ハロフィリス（Pediococcus halo
philis）とは、乳酸の施光性はＬ型で一致するが、45℃
の発育性と、糖の醗酵性が相違する。The lactic acid bacteria belonging to the genus Pediococcus used in the present invention are Pediococcus acidilactis (Pediococcu
acidilactici) or Pediococcus sp. However, the present inventors have isolated Pediococcus sp. 2-5 strains and Pediococcus acidilactici NC separated from commercially available pickled products.
It is desirable to use the DO1859 strain. In particular, the Pediococcus sp. 2-5 strain has a physiological property, and thus Pediococcus halophili
s) or Pediococcus acidi lactis (Ped
iococcus acidilactici) showed similar properties,
Vergis Manual of Cysticatic
Bacteriology (Bergey's Manualof Systematic Ba
Cteriology) Volume 2 Nos. 1075 and 1079 showed no obvious similarities to the standard strains in the following points. That is, Pediococcus sp.
2-5 strains are 1. Pediococcus
The acid fermentability of sugar is similar to that of acidilactici, but the light-transmittance of lactic acid produced by Pediococcus acidilactis is similar to that of acidilactici.
ococcus acidilacitci) is DL type, while Pediococcus sp.
It is a type. See also 2. Pediococcus halo
philis) has the same L-type lactic acid illuminance, but at 45 ° C
And the fermentability of sugar are different.
したがって、ここではペディオコッカス・エスピー（Pe
diococcus sp.）と表記した。これら一連の菌株の菌学
的性質を第１表に示す。Therefore, here is Pediococcus sp.
diococcus sp.). The mycological properties of these strains are shown in Table 1.
第１表 A.形態的性状 （１）細胞の形 双球或いは四連の球菌 （直径0.6〜1.0ミクロン） （２）運動性 なし （３）グラム染色性 陽性 B.倍地上の生育状況 BL寒天培地（栄研）平板上で本菌株を塗布し、スチール
ウール法により37℃、48時間培養して不透明な定型的な
Ｓ型コロニー形態を示す。Table 1 A. Morphological properties (1) Cell morphology Double spheres or four streptococci (diameter 0.6 to 1.0 micron) (2) No motility (3) Gram stain positive B. Growth condition on double medium BL agar This strain was applied on a medium (Eiken) plate and cultured by the steel wool method at 37 ° C. for 48 hours to show an opaque and typical S-type colony morphology.
C.生理学的性質 （１）カタラーゼ − （２）でんぷんの分解 − （３）グルコースよりホモ乳酸醗酵によりＬ（−）乳酸
を生成し、ガスは発生しない。C. Physiological Properties (1) Catalase- (2) Degradation of starch- (3) L-(-) lactic acid is produced from glucose by homolactic fermentation, and no gas is generated.
（４）芳香生産性 さわやかな醗酵臭 ストレプトコッカス・ラクチスなどのやゝ思い感じの酪
臭と比較してさわやかな感じ。(4) Aroma productivity A refreshing fermentation odor A refreshing feeling compared to the odor of lactic acid such as Streptococcus lactis.
（５）各種炭水化物の分解性 第３表参照 本発明者は、この菌株を新菌株と判断し、工業技術院微
生物工業技術研究所に寄託した〔寄託番号 微工研菌寄
第10977号（FERM P−10977）〕。(5) Degradability of various carbohydrates See Table 3 The present inventor judged this strain to be a new strain and deposited it at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology [Deposit No. P-10977)].
また、ペディオコッカス・アシディラクチシNCDO 1859
株は、National Collection of Dairy Organisms（Read
ing,England）に寄託されており、公知株であって、第
３者は自由に分譲を受けることができる。Also, Pediococcus acidi lactis NCDO 1859
Strains from the National Collection of Dairy Organisms (Read
ing, England) and is a publicly known strain, and the third party can freely receive the distribution.
一方、乳酸菌は、ストレプトコッカ・サブスピーシズ・
ラクティス（Streptococcus subsp.lactis）、ストレプ
トコッカス・サリヴァリウス・サブスピーシズ・サーモ
フィルス（Streptococcus salivarius subsp.thermophi
lus）、ラクトバチルス・デルブルキ・サブスピーシズ
・ブルガリクス（Lactobacillus delburuseckii subsp.
bulgaricus）等がラクトース醗酵性が高いことや接合の
しやすさからみて本発明において用いられる。これらの
乳酸菌は市販されており、容易に入手できるものであ
る。On the other hand, lactic acid bacteria are Streptococcus subspecies
Lactis (Streptococcus subsp.lactis), Streptococcus salivarius subsp.thermophi
lus), Lactobacillus delburuseckii subsp.
bulgaricus) and the like are used in the present invention in view of their high lactose fermentability and ease of conjugation. These lactic acid bacteria are commercially available and are easily available.
接合は、従来行われているどのような手段によっても行
うことができるが、スミスら（Smith,M.D.et al.）の方
法〔J.Bacteriol.（144）457（1980）〕に準じて行うこ
とが望ましい。その方法を示すと次のとおりである。The joining can be performed by any conventional means, but can be performed according to the method of Smith, MD et al. [J. Bacteriol. (144) 457 (1980)]. desirable. The method is as follows.
DNaseを含む変法Elliker培地（MEB培地）で受容菌（ペ
ディオコッカス属）ならびに供与菌（ストレプトコッカ
ス属、ラクトバチルス属）を継代培養し、交配用試験管
には受容菌と供与菌を、またそれらの対照用試験管を用
意し、各試験管からの菌液を滅菌メンブランフィルター
で濾過する。そのフィルターを予め調製したMEB寒天培
地に置き、さらに同様の寒天培地を重層する。培養を行
って交配後、フィルターとフィルター上の寒天をMEB培
地で混合、洗浄する。次にその菌液をラクトース、指示
薬ならびに抗生物質含有の選択培地に塗抹し、培養後、
黄変したコロニー（すなわち、Lac+変異株と考えられ
る）を釣菌し、交配株を得る。Subculture of recipient bacteria (Pediococcus spp.) And donor strains (Streptococcus spp., Lactobacillus spp.) In modified Elliker medium (MEB medium) containing DNase, and the recipient strain and donor strain are placed in a mating test tube. Also, prepare test tubes for these controls, and filter the bacterial solution from each test tube with a sterilized membrane filter. The filter is placed on the MEB agar medium prepared in advance, and the same agar medium is overlaid. After culturing and mating, the filter and agar on the filter are mixed with MEB medium and washed. Next, the bacterial solution is smeared on a selective medium containing lactose, an indicator and an antibiotic, and after culturing,
Yellowed colonies (ie, suspected to be Lac + mutants) are picked to obtain crosses.
指示薬としては、ブロムクレゾール パープル（BCP）
等が使用され、抗生物質としてはペニシリン、エリスロ
マイシン等が使用される。Bromcresol purple (BCP) as an indicator
Etc. are used, and penicillin, erythromycin, etc. are used as antibiotics.
得られる交配株は、受容菌（ペディオコッカス属）の形
態学的特徴を有し、これにラクトース醗酵性が付与され
ており、交配株を単独で醗酵乳スターターとして用いあ
るいはラクトバチルス・デルブルキ・サブスピーシズ・
ブルガリクス、ストレプトコッカス・サリヴァリウス・
サブスピーシズ・サーモフィルス等の醗酵乳スターター
と共に醗酵乳の製造に利用することができる。The resulting mating strain has the morphological characteristics of the recipient bacterium (Pediococcus spp.) And is imparted with lactose fermentability, and thus the mating strain is used alone as a fermented milk starter or Lactobacillus delburki. Sub species
Bulgarix, Streptococcus salivarius
It can be used for the production of fermented milk together with the fermented milk starter such as Subspecies Thermophilus.
このようにすると、ラクトースの醗酵性を高め、ペディ
オコッカス属のもつさわやかな芳香性のあるヨーグルト
臭を製品に付与することができ、しかも製品の組織はな
めらかなものとなる。By doing so, it is possible to enhance the fermentability of lactose, impart a refreshing aromatic yogurt odor of the genus Pediococcus to the product, and make the structure of the product smooth.
実施例１（新菌株の創製） （１）親株及びその性質 第２表に本実施例の接合に使用した受容菌及び供与菌
と、そのラクトース醗酵性及び抗生物質耐性を示す。Example 1 (Creation of a new strain) (1) Parent strain and its properties Table 2 shows the recipient strain and donor strain used for the conjugation of this Example, and their lactose fermentability and antibiotic resistance.
第３表にこれら菌株の性質を示す。 Table 3 shows the properties of these strains.
（２）菌株の接合 第２表に示した受容菌及び供与菌をそれぞれDNase10μg
/mlを含有する変法Elliker培地（MEB培地〔ラクトース
又はグルコース0.5％、酵母エキス0.5％、トリプトン２
％、NaCl0.4％、酢酸ナトリウム0.15％、アスコルビン
酸ナトリウム0.05％（pH6.8）を121℃、15分間滅菌して
調製された培地〕に接種し、30℃で12時間培養した。 (2) Conjugation of strains Recipients and donors shown in Table 2 were DNase 10 μg each
modified Elliker medium (MEB medium [lactose or glucose 0.5%, yeast extract 0.5%, tryptone 2
%, NaCl 0.4%, sodium acetate 0.15%, sodium ascorbate 0.05% (pH 6.8) were sterilized at 121 ° C. for 15 minutes to prepare a medium], and cultured at 30 ° C. for 12 hours.
受容菌2mlと供与菌1mlとを交配用試験管にとり、また対
照用試験管には受容菌2mlまたは供与菌1mlを個別に注入
した。各試験管から菌液1mlを滅菌ニトロセルロースフ
ィルターで濾過した。このフィルターを予め調製したME
B寒天培地に置き、さらにその上を同じ寒天培地で重層
する。これを30℃で48時間培養して交配を行い、フィル
ターとフィルター上の寒天とをMEB培地と混合洗浄す
る。次に、この菌液0.2mlをラクトース、指示薬（BCP）
及びペニシリン0.00108g/またはエリスロマイシン0.0
2g/を含有する選択培地に塗抹し、30℃で５日間培養
し、Lac+考えられる黄変したコロニーを釣菌した。2 ml of the recipient strain and 1 ml of the donor strain were taken in a tube for mating, and 2 ml of the recipient strain or 1 ml of the donor strain were individually injected into the control test tube. 1 ml of the bacterial solution was filtered from each test tube with a sterile nitrocellulose filter. This filter was prepared in advance
Place on B agar and overlay with the same agar. This is cultured at 30 ° C. for 48 hours for mating, and the filter and agar on the filter are mixed with the MEB medium and washed. Next, 0.2 ml of this bacterial solution is used for lactose and indicator (BCP)
And penicillin 0.00108 g / or erythromycin 0.0
The cells were smeared on a selective medium containing 2 g /, and cultured at 30 ° C. for 5 days to pick Lac + possible yellowish colonies.
第４表にLac+変異株の出現頻度を示した。それぞれの受
容菌、供与菌およびそれらの交配で得られた交配株を表
示し、交配株の出現頻度を表わしている。出現頻度は供
与菌数当りのLac+変異株数で表わしており、8.3×10-4
から2.8×10-6の範囲であった。Table 4 shows the appearance frequency of Lac + mutants. The respective recipient strains, donor strains, and the hybrid strains obtained by crossing them are displayed, and the appearance frequency of the hybrid strains is shown. The frequency of appearance is expressed as the number of Lac + mutant strains per number of donor strains, 8.3 × 10 -4
To 2.8 × 10 -6 .
（３）新菌株及びその性質 第５表には、受容菌、供与菌ならびにそれらの交配で得
られたLac+変異株について、選択マーカーとして用いた
ラクトース醗酵性及び抗生物質耐性の安定性、また分類
上の性質の一部として45℃および6.5％食塩含有培地で
の生育性と各種糖類の醗酵性を検討した結果を示した。
なおこれらの性質は植え継ぎ前と、20回の連続した植え
継ぎ後に調べた。その結果いずれの性質も安定であっ
た。 (3) New strains and their properties Table 5 shows the stability of lactose fermentation and antibiotic resistance used as selection markers for recipient strains, donor strains and Lac + mutant strains obtained by crossing them. As a part of the taxonomic properties, the results of examining the growth ability at 45 ℃ and 6.5% salt-containing medium and the fermentability of various sugars are shown.
These properties were examined before transplanting and after 20 consecutive transplants. As a result, all properties were stable.
交配株は、45℃と6.5％食塩存在下での生育性は受容菌
と同一の性質を持っていた。また第５表に示した、受容
菌と供与菌について調べた糖類の醗酵性について交配株
についても調べ、受容菌と相違のみられた糖４種類の結
果を示した。糖の醗酵性について、交配株にはラクトー
スの醗酵性のみ獲得したもの（185−PM−52、185−PM−
18、185−PM−72）、受容菌と供与菌との中間型を示す
もの（25−EM−KM）、受容菌の性質の一部を欠落したも
の（25−PM−KM、25−PM−72）の３種類があった。The hybrid strain had the same properties as the recipient strain in terms of viability at 45 ° C and in the presence of 6.5% sodium chloride. Further, the fermentability of the sugars examined in the recipient and donor bacteria shown in Table 5 was also examined in the crossed strains, and the results of four kinds of sugars which were different from the recipient bacteria were shown. Regarding the fermentability of sugar, the mating strain obtained only the fermentability of lactose (185-PM-52, 185-PM-
18, 185-PM-72), those showing an intermediate form between the recipient and donor bacteria (25-EM-KM), those lacking some of the properties of the recipient bacteria (25-PM-KM, 25-PM There were three types of -72).
第６表に脱脂乳培地における生酸性（酸度）と芳香生産
性（クレアチンテスト）及び加糖脱脂乳培地の蛋白質分
解性（遊離チロシン含有量）を示した。 Table 6 shows the raw acidity (acidity) and aroma productivity (creatine test) in skim milk medium, and the proteolytic property (free tyrosine content) in sweetened skim milk medium.
生酸性については、いずれの交配株も受容菌に比べてか
なり高い酸の生成を示した。クレアチンテストにおいて
は、25−EM−KM株は特に発色が強く、さわやかなヨーグ
ルトのような芳香を有し、芳香生産性が優れた菌株であ
った。この菌株は蛋白質分解性も強く、脱脂乳培地を24
時間以内に凝固した。In terms of bioacidity, all the hybrids showed significantly higher acid production than the recipient strain. In the creatine test, the 25-EM-KM strain was a strain with particularly strong color development, a refreshing yogurt-like aroma, and excellent aroma productivity. This strain is also highly proteolytic and can
Solidified within hours.
第７表にラクトース分解酵素活性を調べた結果を示す。
方法は、菌株をトルエン処理し、基質としてオルソニト
ロフェノールガラクトピラノサイド（ONPG）もしくはオ
ルソニトロフェノールガラクトピラノサイド−６フォス
フェイト（ONPG−6P）を用いてβ−ガラクトシダーゼも
しくはフォスフォβ−ガラクトシダーゼ活性を調べた。
数値は反応時間と菌体蛋白質当りのそれらの基質より遊
離したオルソニトロフェノールのモル数を示す。 Table 7 shows the results of examining the lactose-degrading enzyme activity.
The method was to treat the strain with toluene and use β-galactosidase or phosphoβ-galactosidase activity using ortho-nitrophenol galactopyranoside (ONPG) or ortho-nitrophenol galactopyranoside-6 phosphate (ONPG-6P) as a substrate. I checked.
The numerical values indicate the reaction time and the number of moles of orthonitrophenol released from their substrate per cell protein.
受容菌には両酵素の活性は見られなかったが、交配株は
いずれか一方、あるいは両酵素の活性を持っていた。こ
れらの酵素活性の発現は供与菌から受容菌への伝達によ
るものと推測される。Recipients did not show activity of both enzymes, but the hybrid strain had activity of either one or both enzymes. It is presumed that the expression of these enzyme activities is due to the transfer from the donor bacterium to the recipient bacterium.
また第１〜９図に示すように交配によって得られる乳酸
菌はその形態において受容菌のペディオコッカス属のそ
れと同じであり、また各表に示す菌学的性質を示してい
る。そして、この点を考慮して実施例の結果をみてみる
と、受容菌のペディオコッカス属の乳酸菌が供与菌のス
トレプトコッカス属またはラクトバチルス属の乳酸菌か
らラクトース醗酵性、蛋白分解性等の性質を伝達され、
この点において新規な乳酸菌が創製されたものと考え
る。 Further, as shown in FIGS. 1 to 9, the lactic acid bacteria obtained by mating are the same in form as those of the recipient genus Pediococcus, and show the mycological properties shown in each table. Then, looking at the results of the examples in consideration of this point, lactic acid bacteria of the recipient bacterium Pediococcus genus is a lactose bacterium of the genus Streptococcus genus or Lactobacillus genus lactose fermentability, such as proteolytic properties. Transmitted,
In this respect, it is considered that a new lactic acid bacterium was created.
そしてこれらをPediococcus sp.185−PM−52 SBT 3325
〔微工研菌寄第10955号（FERM P−10955）〕、Pediococ
cus sp.185−PM−18 SBT 3326〔微工研菌寄第10956号
（FERM P−10956）〕、Pediococcus sp.185−PM−72 SB
T 3327〔微工研菌寄第10957号（FERMP−10957）〕、ped
iococcus sp.25−PM−KM SBT 3328〔微工研菌寄第10958
号（FERM P−10958）〕、Pediococcus sp.25−PM−72 S
BT 3329〔微工研菌寄第10959号（FERM P−10959）〕、P
ediococcus sp.25−EM−KMSBT 3330〔微工研菌寄第1096
0号（FERM P−10960）〕と命名し、上記の寄託番号で工
業技術院微生物工業技術研究所に寄託した。And these are Pediococcus sp.185-PM-52 SBT 3325
[Microtech Lab, No. 10955 (FERM P-10955)], Pediococ
cus sp.185-PM-18 SBT 3326 [Microtechnical Research Institute No. 10956 (FERM P-10956)], Pediococcus sp.185-PM-72 SB
T 3327 [Microtechnology Research Institute, Bacteria No. 10957 (FERMP-10957)], ped
iococcus sp.25-PM-KM SBT 3328
(FERM P-10958)], Pediococcus sp. 25-PM-72 S
BT 3329 [Microtechnology Research Institute, Microbial Research No. 10959 (FERM P-10959)], P
ediococcus sp. 25-EM-KMSBT 3330
No. 0 (FERM P-10960)], and deposited at the Institute of Microbial Technology, Institute of Industrial Science and Technology with the above deposit number.
実施例２ Pediococcus sp.25−EM−KM SBT 3330の酸生
成 脱脂乳を水に８％に溶解し、95℃で15分間加熱殺菌した
ものに、Pediococcus sp.25−EM−KM SBT 3330を２％接
種し30℃に培養した時の酸生成は次の第８表に示すとお
りであった。また無菌添加のコントロールと、親株であ
るPediococcus sp.2−5EとStreptococcus lactis subs
p.lactis KMの数植を共に示す。Example 2 Acid Generation of Pediococcus sp.25-EM-KM SBT 3330 Skim milk was dissolved in water at 8% and sterilized by heating at 95 ° C for 15 minutes. %, The acid production when inoculated and cultured at 30 ° C. was as shown in Table 8 below. In addition, aseptic control and parent strain Pediococcus sp.2-5E and Streptococcus lactis subs
Along with a number plant of p.lactis KM.
この結果、本発明の新菌株Pediococcus sp.25−EM−KM
SBT 3330は親株のPediococcus sp.2−5Eにくらべて酸生
成が著しく高く、この性質は親株のStreptococcus lact
is subsp.lactis KMに由来すると思われる。 As a result, the novel strain Pediococcus sp. 25-EM-KM of the present invention
SBT 3330 has a significantly higher acid production than the parent strain Pediococcus sp. 2-5E, and this property indicates that the parent strain Streptococcus lact
It is probably derived from is subsp.lactis KM.
実施例３ Pediococcus sp.25−EM−KM SBT 3330の芳香
生産性、酸度、蛋白分解性 脱脂乳を10％および14％に溶解し、95℃15分間殺菌した
ものに、Pediococcus sp.25−EM−KM SBT 3330を２％接
種し、経時的に芳香生産性（クレアチンテスト）、酸
度、蛋白分解性（遊離チロシン量）測定した。Example 3 Pediococcus sp.25-EM-KM SBT 3330 Aroma Productivity, Acidity, and Proteolytic Property Non-fat milk was dissolved in 10% and 14% and sterilized at 95 ° C. for 15 minutes, and then Pediococcus sp.25-EM. 2% of -KM SBT 3330 was inoculated, and aroma productivity (creatine test), acidity, and protein degradability (free tyrosine amount) were measured over time.
その結果を第９表に示す。The results are shown in Table 9.
この結果、本発明の新菌株Pediococcus sp.25−EM−KM
SBT 3330は、コントロールにくらべて芳香、酸度、蛋白
分解性が著しく向上した。 As a result, the novel strain Pediococcus sp. 25-EM-KM of the present invention
SBT 3330 has significantly improved aroma, acidity, and proteolytic activity compared to the control.
実施例４ Pediococcus sp.185−PM−52 SBT 3325（FER
M P−10955）等の酸生成 脱脂乳を10％に水に溶解し中試験管に分注したものを95
℃、15分間殺菌し、Pediococcus sp.185−PM−52 SBT 3
325（FERM P−10955）、Pediococcus sp.185−PM−72 S
BT 3327（FERM P−10957）、Pediococcus sp.25−PM−K
M SBT 3328（FERM P−10958）及びPediococcus sp.25−
EM−KM SBT 3330（FERM P−10960）、をそれぞれ２％接
種して30℃に培養して酸度の上昇を調べた。Example 4 Pediococcus sp.185-PM-52 SBT 3325 (FER
MP-10955), etc. Acid generation Non-fat milk dissolved in 10% water and dispensed into a medium test tube
Sterilized for 15 minutes at ℃, Pediococcus sp.185-PM-52 SBT 3
325 (FERM P-10955), Pediococcus sp.185-PM-72 S
BT 3327 (FERM P-10957), Pediococcus sp.25-PM-K
M SBT 3328 (FERM P-10958) and Pediococcus sp. 25-
EM-KM SBT 3330 (FERM P-10960) was inoculated in an amount of 2% and cultured at 30 ° C. to examine the increase in acidity.
その結果を第10表に示す。The results are shown in Table 10.
さらに、本発明は、このようにして創製した新規な乳酸
菌を醗酵スターターとして用いた乳酸飲料及びその製造
法に関する。 Furthermore, the present invention relates to a lactic acid beverage using the novel lactic acid bacterium thus created as a fermentation starter and a method for producing the same.
本発明における新規な乳酸菌は、前述したペディオコッ
カス属（Pediococcus）に属する乳酸菌と、ストレプト
コッカス属（Streptococcus）またはラクトバチルス属
（Lactobacillus）に属する乳酸菌とを細胞接合して得
られる及びペディオコッカス属に属し、芳香生産性及び
ラクトース醗酵性を有する乳酸菌である。The novel lactic acid bacterium of the present invention is obtained by cell-bonding a lactic acid bacterium belonging to the genus Pediococcus described above and a lactic acid bacterium belonging to the genus Streptococcus or Lactobacillus and the genus Pediococcus. Is a lactic acid bacterium that has aroma-producing property and lactose-fermenting property.
このような乳酸菌には、ペディオコッカス属に属し、芳
香生産性及びラクトース醗酵性を有する乳酸菌には、前
述したように、ペディオコッカス・エスピー（Pediococ
cus sp.）185−PM−52 SBT 3325〔微工研寄託番号 微
工研菌寄第10955号（FERM P−10955）〕、ペディオコッ
カス・エスピー（Pediococcus sp.）185−PM−18 SBT 3
326〔微工研寄託番号 微工研菌寄第10956号（FERM P−
10956）〕、ペディオコッカス・エスピー（Pediococcus
sp.）185−PM−72 SBT 3327〔微工研寄託番号 微工研
菌寄第10957号（FERM P−10957）〕、ペディオコッカス
・エスピー（Pediococcus sp.）25−PM−KM SBT 3328
〔微工研寄託番号 微工研菌寄第10958号（FERM P−109
58）〕、ペディオコッカス・エスピー（Pediococcus s
p.）25−PM−72 SBT 3329〔微工研寄託番号 微工研菌
寄第10959号（FERM P−10959）〕あるいはペディオコッ
カス・エスピー（Pediococcus sp.）25−EM−KM SBT 33
30〔微工研寄託番号 微工研菌寄第10960号（FERM P−1
0960）〕等を挙げることができる。Such a lactic acid bacterium belongs to the genus Pediococcus, and a lactic acid bacterium having an aroma-producing property and a lactose-fermenting property includes, as described above, Pediococcus sp.
cus sp.) 185-PM-52 SBT 3325 [Ministry of Industrial Research Deposit No. Microorganisms Research Institute No. 10955 (FERM P-10955)], Pediococcus sp. 185-PM-18 SBT 3
326 [Micromachine Research Deposit No.
10956)], Pediococcus sp.
sp.) 185-PM-72 SBT 3327 [Ministry of Industrial Deposit No. 10957 (FERM P-10957)], Pediococcus sp. 25-PM-KM SBT 3328
[Ministry of Industrial Research Deposit No. Microorganisms Research Institute No. 10958 (FERM P-109
58)], Pediococcus s
p.) 25-PM-72 SBT 3329 [Ministry of Industrial Science Deposit No. Microorganisms Research Institute No. 10959 (FERM P-10959)] or Pediococcus sp. 25-EM-KM SBT 33
30 (Micromachine Research Deposit No., Micromachine Research Center No. 10960 (FERM P-1
0960)] and the like.
醗酵乳としては、醗酵乳飲料、ヨーグルト等を例示する
ことができ、その製造法は通常知られている醗酵乳の製
造法を用いることができる。Examples of the fermented milk include fermented milk drinks, yogurt, and the like, and the production method thereof can be the commonly known production method of fermented milk.
すなわち、牛乳、羊乳、全乳、脱脂乳、還元乳等の通常
用いられている乳原料を混合して醗酵乳原料を調製し、
これを加熱殺菌し、冷却を行う。これに前記した本発明
の新規な乳酸菌単独、またはこのような乳酸菌と、ラク
トバチルス・デルブルキ・サブスピーシズ・ブルガリク
ス、ストレプトコッカス・サリヴァリウス・サブスピー
シズ・サーモフィルス等の乳酸菌とを併用して乳酸菌ス
ターターとして用い醗酵を行う。醗酵は、30〜45℃で６
〜42時間程度行うとよい。このようにして調製された醗
酵乳は、通常のスターターを用いて調製された醗酵乳に
くらべて一味ちがった爽やかな芳香と組織とを有してい
る。That is, milk, sheep milk, whole milk, skim milk, to prepare a fermented milk raw material by mixing commonly used milk raw materials such as reduced milk,
This is sterilized by heating and cooled. A novel lactic acid bacterium of the present invention described above alone, or such a lactic acid bacterium is used as a lactic acid bacterium starter in combination with a lactic acid bacterium such as Lactobacillus delburki subspecies bulgaricus and Streptococcus salivarius subspecies thermophilus. Perform fermentation. Fermentation is 6 at 30-45 ℃
It is good to do it for about 42 hours. The fermented milk thus prepared has a refreshing aroma and texture which is different from that of the fermented milk prepared by using an ordinary starter.
さらに、本発明では、生シイタケエキス、酵母抽出物等
を培地に添加すると、乳酸菌の発育が促進され、酸生成
量を増加することができる。Furthermore, in the present invention, the addition of raw shiitake extract, yeast extract and the like to the medium promotes the growth of lactic acid bacteria and can increase the amount of acid production.
また、必要に応じて甘味料、香料、着色料等を添加して
もよい。In addition, sweeteners, flavors, colorants and the like may be added if necessary.
本発明の醗酵乳を製造するに至った経緯について、ヨー
グルトを例に挙げて詳細に説明する。The process of producing the fermented milk of the present invention will be described in detail by taking yogurt as an example.
まず、本発明の乳酸菌ペディオコッカス・エスピー25−
EM−KM SBT 3330とその受容菌であるペディオコッカス
・エスピー２−5E及び供与菌であるストレプトコッカス
・ラクチス・サブスピーシズ・ラクチスKMとをそれぞれ
種々の濃度（8,10,12,14及び16％）の還元脱脂乳の培地
に２％接種し、30℃で０〜120時間培養した。その10％
還元脱脂乳培地における培養72時間目の酸の生成及び芳
香の生産をそれぞれ滴定酸度及びクレアチン試験で測定
した。その結果を第11表に示す。First, the lactic acid bacterium Pediococcus sp. 25- of the present invention
EM-KM SBT 3330 and its recipient, Pediococcus sp. 2-5E, and the donor strain, Streptococcus lactis subspecies lactis KM, at various concentrations (8, 10, 12, 14, and 16%), respectively. 2% of the reduced skim milk culture medium was inoculated and cultured at 30 ° C. for 0 to 120 hours. 10%
Acid production and aroma production at 72 hours of culture in reduced skim milk medium were measured by titratable acidity and creatine test, respectively. The results are shown in Table 11.
この表からみられるように、本発明の乳酸菌を用いる
と、ラクトース醗酵性のない受容菌にくらべて酸の生成
はすぐれまた芳香産生は受容菌及び供与菌のいずれより
も著しく優れたものとなっている。 As can be seen from this table, when the lactic acid bacterium of the present invention is used, the production of acid is superior to that of the non-lactose-fermenting recipient bacterium, and the aroma production is significantly superior to both the recipient bacterium and the donor bacterium. There is.
また、本発明の乳酸菌の生酸性を高めるために、培地に
グルコースを添加した。その10％還元脱脂乳培地に、グ
ルコース0.5％を添加し、120時間培養し、それぞれの滴
定酸度とpHの変化を調べた。その結果を第10図に示す。Further, glucose was added to the medium in order to enhance the biogenicity of the lactic acid bacterium of the present invention. Glucose 0.5% was added to the 10% reduced skim milk medium and cultured for 120 hours, and changes in titratable acidity and pH were examined. The results are shown in Fig. 10.
この図にみられるように、本発明の乳酸菌を用いると、
グルコース無添加の場合（Ａ）もグルコース添加の場合
（Ｂ）も酸生成速度は、供与菌より遅かった。As shown in this figure, when the lactic acid bacterium of the present invention is used,
The acid generation rate was slower than that of the donor bacterium both in the case where glucose was not added (A) and in the case where glucose was added (B).
そこで、還元脱脂乳の固形分含量を上記したように８〜
16％の範囲にして固形分含量の増加による生酸量を測定
した。その結果を第11図に示す。受容菌（ａ）、本発明
乳酸菌（ｂ）及び供与菌（ｃ）のいずれの場合も固形分
含量の増加に従って生酸性は増加しているが顕著な増加
は認められなかった。Therefore, the solid content of the reduced skim milk is 8 to 8% as described above.
The amount of raw acid was measured by increasing the solid content in the range of 16%. The results are shown in Fig. 11. In all cases of the recipient bacterium (a), the lactic acid bacterium of the present invention (b) and the donor bacterium (c), the bioacidity increased as the solid content increased, but no remarkable increase was observed.
さらに本発明の乳酸菌の酸生成速度と酸生成量を増加さ
せるために、従来、乳酸菌に対して発育促進効果がある
といわれているトマト、キャベツ、キュウリ、ダイコ
ン、シイタケの抽出エキス（これらはいずれもその生鮮
品に水を加えジューサー・ミキサーで抽出したもの）、
豆乳及び酵母エキスをオートクレーブで滅菌するかある
いはフィルター滅菌し、これをそれぞれ0.5％、10％還
元脱脂乳に添加し、24時間培養し、その生酸性を測定し
た。この結果を第12図に示す。オートクレーブ滅菌した
場合（Ａ）もフィルター滅菌した場合（Ｂ）もいずれも
対照の無添加の場合にくらべて酵母エキス、シイタケエ
キスを添加した場合の生酸性がかなり増加した。特に、
フィルター滅菌したシイタケエキスは生酸性を向上さ
せ、その添加量が多いほど酸度が上昇した。Furthermore, in order to increase the acid production rate and the amount of acid production of the lactic acid bacterium of the present invention, tomato, cabbage, cucumber, Japanese radish, and shiitake extract extract, which are conventionally said to have a growth-promoting effect on lactic acid bacterium (all of which are Also added water to the fresh product and extracted with a juicer mixer),
Soybean milk and yeast extract were sterilized by an autoclave or filter sterilized, added to 0.5% and 10% reduced skim milk, respectively, and cultured for 24 hours, and the raw acidity was measured. The results are shown in FIG. In both cases of autoclave sterilization (A) and filter sterilization (B), the raw acidity was significantly increased when yeast extract and shiitake extract were added, as compared with the case where no control was added. In particular,
The filter sterilized shiitake extract improved the raw acidity, and the higher the amount added, the higher the acidity.
生産性の向上がみられたシイタケ抽出エキスのフィルタ
ー滅菌物についてその生酸性の経時変化を測定した。14
％還元脱脂乳培地にシイタケ抽出エキスを0.5％、1.0％
及び1.5％添加し、その滴定酸度を測定した。その結果
を第13図に示す。いずれの添加量の場合も24時間以内に
酸度が0.8〜1.0％に達した。The time-dependent changes in the raw acidity of the filter sterilized extract of Shiitake extract, which showed improved productivity, were measured. 14
% Shiitake extract extract in reduced skim milk medium 0.5%, 1.0%
And 1.5% were added and the titratable acidity was measured. The result is shown in FIG. The acidity reached 0.8-1.0% within 24 hours at any addition amount.
また、クレアチン試験を行ったところ、培養16時間以降
発色がみられ、また官能的にも明らかな香気が認められ
た。In addition, when a creatine test was performed, color development was observed after 16 hours of culture, and a scent with a clear sensory sensation was observed.
従って、本発明の乳酸菌を用いて醗酵乳を製造すること
ができ、さらにこの際、シイタケ抽出エキスを用いると
乳酸菌の生酸性を向上し、芳香のある醗酵乳を製造する
ことができるということが判明した。Therefore, it is possible to produce fermented milk using the lactic acid bacterium of the present invention, and further, in this case, it is possible to improve the bioacidity of the lactic acid bacterium by using a shiitake extract and produce fermented milk with aroma. found.
次に、本発明の醗酵乳の製造に関する実施例を挙げ、本
発明を具体的に説明する。Next, the present invention will be specifically described with reference to Examples relating to the production of fermented milk of the present invention.
実施例５ 牛乳90kg（固形分量7.4kg、脂肪量3.2kg、水分量79.5k
g）と脱脂粉乳6.8kg（固形分量6.6kg、水分量0.2kg）と
をよく混合し、ヨーグルトミックスを調製し、これを95
℃で15分間加熱殺菌し、35〜32℃に冷却した。別に、生
シイタケミキサーで破砕して搾汁し、ザイツ又はミリポ
アなどの濾過殺菌器で濾過殺菌してシイタケエキス0.7k
g（固形分量0.1kg、水分量0.6kg）を調製した。このヨ
ーグルトミックスにシイタケエキス及びペディオコッカ
ス・エスピー25EM KM SBT−3330（FERM−10960）2.5kg
（固形分量0.25kg、水分量2.25kg）を添加して容器に分
注し、30℃で16時間醗酵させてヨーグルトを得た。Example 5 Milk 90 kg (solid content 7.4 kg, fat content 3.2 kg, water content 79.5 k)
g) and skim milk powder 6.8 kg (solid content 6.6 kg, water content 0.2 kg) are mixed well to prepare a yogurt mix.
It heat-sterilized at 15 degreeC for 15 minutes, and cooled to 35-32 degreeC. Separately, crush with a fresh shiitake mixer and squeeze juice, filter sterilize with a filter sterilizer such as Zeitz or Millipore and shiitake extract 0.7k
g (solid content 0.1 kg, water content 0.6 kg) was prepared. To this yogurt mix, shiitake extract and Pediococcus sp. 25EM KM SBT-3330 (FERM-10960) 2.5 kg
(Solid content 0.25 kg, water content 2.25 kg) was added and dispensed into a container, and fermented at 30 ° C. for 16 hours to obtain yogurt.
得られたヨーグルトは、酸度が0.9％で通常のヨーグル
トスターターを用いて調製したヨーグルトにくらべて一
味ちがった爽やかな芳香と組織とを有し、食味が良好で
あった。The obtained yogurt had an acidity of 0.9%, had a refreshing aroma and texture that were slightly different from those of yogurt prepared using a normal yogurt starter, and had a good taste.
実施例６ 還元脱脂乳12kg、ショ糖8kgに水を加えて全量を100kgと
し、これらをよく混合し、ヨーグルトミックスを調製
し、95℃で15分間加熱殺菌し、35〜32℃に冷却した。こ
れに、実施例５で得られたシイタケエキスを0.5〜1.0kg
加え、さらにペディオコッカス・エスビー25−EM−KM S
BT 3330（FERM−10960）2kgを加え、容器に分注し、30
℃で16〜18時間醗酵させてヨーグルトを得た。Example 6 To 12 kg of reduced skim milk and 8 kg of sucrose, water was added to bring the total amount to 100 kg, and these were mixed well to prepare a yogurt mix, which was sterilized by heating at 95 ° C for 15 minutes and cooled to 35 to 32 ° C. 0.5 to 1.0 kg of the shiitake extract obtained in Example 5 is added to this.
In addition, Pediococcus SB 25-EM-KM S
Add 2 kg of BT 3330 (FERM-10960), dispense into containers, and
Fermentation was carried out at ℃ for 16 to 18 hours to obtain yogurt.
得られたヨーグルトは爽やかな芳香とショ糖による適度
の甘味を有し、組織がなめらかなものとなった。The obtained yogurt had a refreshing aroma and an appropriate sweetness due to sucrose, and had a smooth texture.
発明の効果 本発明によると、ペディオコッカス属に属する乳酸菌
に、ストレプトコッカス属またはラクトバチルス属に属
する乳酸菌のラクトース醗酵性を細胞接合によって付与
するので、ペディオコッカス属に属し、芳香生産性とラ
クトース醗酵性とを併有する乳酸菌を提供することがで
きる。Effects of the Invention According to the present invention, lactic acid bacteria belonging to the genus Pediococcus impart lactose fermentability of lactic acid bacteria belonging to the genus Streptococcus or Lactobacillus by cell conjugation, and therefore belong to the genus Pediococcus, and produce aroma and lactose. A lactic acid bacterium having both fermentability can be provided.
そして、この新規な乳酸菌をスターターとして用いて醗
酵乳を製造すると製品にさわやかな芳香を付与し、組織
をなめらかにし、食味の良い醗酵乳を製造することがで
きる。When fermented milk is produced by using this novel lactic acid bacterium as a starter, a refreshing aroma can be imparted to the product, the texture of the product can be smoothed, and fermented milk with a good taste can be produced.
しかも、醗酵乳製造のさい、シイタケエキスを醗酵促進
剤として添加すると本発明の乳酸菌の生酸性を向上し短
時間のうちに適度の酸味のある醗酵乳を製造することが
できる。Moreover, during production of fermented milk, by adding shiitake extract as a fermentation accelerator, the fermented acidity of the lactic acid bacterium of the present invention can be improved, and fermented milk having an appropriate sourness can be produced in a short time.
第１〜９図は、本発明における生物の形態を示す顕微鏡
写真である。すなわち、第１図はペディオコッカス・ア
ジディラクチシ（pediococcus acidilactici）NCDO 18
59P株の形状を、第２図は、ラクトバチルス・デルブル
キ サブスピーシズブルガリクス（Lactobacillus delb
urueckii subsp.bulgaricus）7235株の形状を、第３図
は両者を接合して創製された新菌株の形状を示す顕微鏡
写真である。 第４図はペディオコッカス・エスピー（Pediococcus s
p.）２−5Pの形状を、第５図はストレプトコッカスラク
チス サブスピーシズ ラクチス（（Streptococcus la
ctis subsp.lactis）KMの形状を、第６図は両者を接合
して創製された新菌株の形状を示す顕微鏡写真である。 第７図はペディオコッカス・エスピー（Pediococcus s
p.）２−5Eの形状を、第８図は、ストレプトコッカス・
ラクチス KMの形状を、第９図は、両者を接合して創製
された新菌株（以下、本発明乳酸菌という）の形状を示
す顕微鏡写真である。 10μｍが4cmに拡大されている。 第10図は、培地にグルコースを添加して乳酸菌を培養し
たときの生酸量を示す図である。 （Ａ）はグルコース無添加、（Ｂ）はグルコース添加を
それぞれ示す。 は受容菌による酸度を、 はpHをそれぞれ示す。 は供与菌による酸度を、 はpHをそれぞれ示す。 は本発明乳酸菌による酸度を、 はpHをそれぞれ示す。第11図は、培地に還元脱脂乳をそ
の固形分量を変えて添加したときの受容菌（Ａ）、本発
明乳酸菌（Ｂ）及び供与菌（Ｃ）の生酸量の経時変化を
示す。 第12図は、培地に乳酸菌の発育促進効果があるといわれ
ている物質を添加したときの本発明の乳酸菌の乳酸生産
量を示す図である。 （Ａ）はオートクレーヴ処理を、（Ｂ）はフィルター滅
菌処理をそれぞれ示す。 第13図は、培地にシイタケ抽出エキスを濃度を変えて添
加したときの本発明乳酸菌の生産量の経時変化を示す。 は無添加、 はシイタケ抽出エキス0.5％添加、 は1.0％添加、 は1.5％添加の場合を示す。1 to 9 are micrographs showing the morphology of living things in the present invention. That is, Fig. 1 shows the pediococcus acidilactici NCDO 18
The shape of the 59P strain is shown in Fig. 2. Lactobacillus delb
urueckii subsp. bulgaricus) 7235 strain, and Fig. 3 is a micrograph showing the shape of a new strain created by joining the two. Figure 4 shows Pediococcus s.
p.) The shape of 2-5P is shown in Fig. 5, and Streptococcus lactis Subspecies lactis ((Streptococcus la
ctis subsp.lactis) KM, and FIG. 6 is a micrograph showing the shape of a new strain created by joining the two. Figure 7 shows Pediococcus s.
p.) The shape of 2-5E is shown in Fig.8.
FIG. 9 is a micrograph showing the shape of Lactis KM, and FIG. 9 shows the shape of a new strain created by joining the two (hereinafter referred to as the lactic acid bacterium of the present invention). 10 μm is expanded to 4 cm. FIG. 10 is a graph showing the amount of raw acid when lactic acid bacteria were cultured by adding glucose to the medium. (A) shows the addition of glucose and (B) shows the addition of glucose. Is the acidity of the recipient bacteria, Indicates pH, respectively. Is the acidity of the donor bacterium, Indicates pH, respectively. Is the acidity of the lactic acid bacterium of the present invention, Indicates pH, respectively. FIG. 11 shows the changes over time in the amount of raw acid of the recipient bacterium (A), the lactic acid bacterium of the present invention (B) and the donor bacterium (C) when reduced skim milk was added to the medium while changing its solid content. FIG. 12 is a graph showing the amount of lactic acid produced by the lactic acid bacterium of the present invention when a substance said to have a lactic acid bacterium growth promoting effect is added to the medium. (A) shows an autoclave process, (B) shows a filter sterilization process, respectively. FIG. 13 shows the time-dependent change in the production amount of the lactic acid bacterium of the present invention when the Shiitake extract was added to the medium at various concentrations. Is additive-free, Is added 0.5% of shiitake extract, Is 1.0% added, Indicates the case of 1.5% addition.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 ＦＩ 技術表示箇所 //(Ｃ１２Ｎ 1/21 Ｃ１２Ｒ 1:01） (Ｃ１２Ｎ 15/00 Ｃ１２Ｒ 1:46） (Ｃ１２Ｎ 15/00 Ｃ１２Ｒ 1:225） Ｃ１２Ｒ 1:46） (Ｃ１２Ｎ 15/00 Ｚ Ｃ１２Ｒ 1:225） 微生物の受託番号 ＦＥＲＭ Ｐ−１０９５６ 微生物の受託番号 ＦＥＲＭ Ｐ−１０９５７ 微生物の受託番号 ＦＥＲＭ Ｐ−１０９５８ 微生物の受託番号 ＦＥＲＭ Ｐ−１０９５９ 微生物の受託番号 ＦＥＲＭ Ｐ−１０９６０ 微生物の受託番号 ＦＥＲＭ Ｐ−１０９７７ (56)参考文献 特開 昭63−102686（ＪＰ，Ａ) 国際公開89／01970（ＷＯ，Ａ)─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 6 Identification number Office reference number FI technical display location // (C12N 1/21 C12R 1:01) (C12N 15/00 C12R 1:46) (C12N 15 / 00 C12R 1: 225) C12R 1:46) (C12N 15/00 Z C12R 1: 225) microbial accession number FERM P-10956 microbial accession number FERM P-10957 microbial accession number FERM P-10958 microbial accession FERM P-10959 microbial accession number FERM P-10960 microbial accession number FERM P-10977 (56) Reference JP-A-63-102686 (JP, A) International Publication 89/01970 (WO, A)
Claims (8)
diococcus acidilactici）またはペディオコッカス・エ
スピー（Pediococcus sp.）と、ストレプトコッカス・
ラクチス・サブスピーシズ・ラクチス（Streptococcus
lactis subsp.lactis）、ストレプトコッカスサリヴァ
リウス・サブスピーシズ・サーモフィラス（Streptococ
cus salivarius subsp thermophilus）または、ラクト
バチルス・デルブリキ・サブスピーシズ・ブリガリクス
（Lactobacillus delbureckii subsp.bulgaricus）とを
細胞接合して得られるペディオコッカス属に属し、芳香
生産性及びラクトース発酵性を有する乳酸菌。1. A Pediococcus acidi lactis (Pe
diococcus acidilactici) or Pediococcus sp. and Streptococcus
Lactis subspecies lactis (Streptococcus
lactis subsp.lactis), Streptococcus salivarius subspecies thermophilus (Streptococ
lactic acid bacterium belonging to the genus Pediococcus obtained by cell-conjugating cus salivarius subsp thermophilus) or Lactobacillus delbureckii subsp. bulgaricus and having aroma-producing and lactose-fermenting properties.
s sp.）185−PM−52 SBT 3325〔微工研菌寄第10955号
（FERM P−10955）〕、ペディオコッカス・エスピー（P
ediococcus sp.）185−PM−18 SBT 3326〔微工研菌寄第
10956号（FERM P−10956）〕、ペディオコッカス・エス
ピー（Pediococcus sp.）185−PM−72 SBT 3327〔微工
研菌寄第10957号（FERM P−10957）〕、ペディオコッカ
ス・エスピー（Pediococcus sp.）25−PM−KM SBT 3328
〔微工研菌寄第10958号（FERM P−10958）〕、ペディオ
コッカス・エスピー（Pediococcus sp.）25−PM−72 SB
T 3329〔微工研菌寄第10959号（FERM P−10959）〕及び
ペディオコッカス・エスピー（Pediococcus sp.）25−E
M−KM SBT 3330〔微工研菌寄第10960号（（FERM P−109
60）〕よりなる群から選択される乳酸菌である請求項
（１）記載の乳酸菌。2. Pediococcu sp.
s sp.) 185-PM-52 SBT 3325 [Microbiology Research Institute, Microbiology No. 10955 (FERM P-10955)], Pediococcus sp.
ediococcus sp.) 185-PM-18 SBT 3326
10956 (FERM P-10956)], Pediococcus sp. (Pediococcus sp.) 185-PM-72 SBT 3327 [Microtechnical Research Institute, No. 10957 (FERM P-10957)], Pediococcus sp. Pediococcus sp.) 25−PM−KM SBT 3328
[Microtechnology Research Institute Bacteria No. 10958 (FERM P-10958)], Pediococcus sp. 25-PM-72 SB
T 3329 [Microtechnology Research Institute, No. 10959 (FERM P-10959)] and Pediococcus sp. 25-E
M-KM SBT 3330 [Ministry of Industrial Research, Microbiology No. 10960 ((FERM P-109
60)] The lactic acid bacterium according to claim 1, which is a lactic acid bacterium selected from the group consisting of:
びラクトース発酵性を有する請求項（１）記載の乳酸菌
を含有し、発酵が行われている発酵乳。3. Fermented milk, which belongs to the genus Pediococcus and has aroma-producing and lactose-fermenting properties, which contains the lactic acid bacterium according to claim 1 and is fermented.
乳。4. The fermented milk according to claim 3, which is yogurt.
びラクトース発酵性を有する請求項（１）記載の乳酸菌
をスターターとして乳原料に接種し、発酵を行うことを
特徴とする発酵乳の製造方法。5. Production of fermented milk, characterized in that the lactic acid bacterium according to claim 1 which belongs to the genus Pediococcus and has aroma productivity and lactose fermentability is inoculated into a milk raw material as a starter and fermented. Method.
酵を行う請求項（５）記載の発酵乳の製造方法。6. The method for producing fermented milk according to claim 5, wherein the extract of shiitake mushroom is added to the milk raw material to perform fermentation.
（Pediococcus sp.）25−EM−KM SBT3330〔微工研菌寄
第10960号（FERM P−10960）〕である請求項（５）また
は（６）記載の製造方法。(7) The lactic acid bacterium is Pediococcus sp. (Pediococcus sp.) 25-EM-KM SBT3330 [Micromachinery Research Institute of Microbiology No. 10960 (FERM P-10960)] (5) or (6) ) The manufacturing method described.
（７）のいずれかに記載の製造方法。8. Fermented milk is yogurt (5).
The manufacturing method according to any one of (7).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2184469A JPH0740923B2 (en) | 1989-09-12 | 1990-07-12 | Novel lactic acid bacterium by cell conjugation, fermented milk using the same, and method for producing the same |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP23672389 | 1989-09-12 | ||
| JP1-236723 | 1989-09-12 | ||
| JP2184469A JPH0740923B2 (en) | 1989-09-12 | 1990-07-12 | Novel lactic acid bacterium by cell conjugation, fermented milk using the same, and method for producing the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH03175978A JPH03175978A (en) | 1991-07-31 |
| JPH0740923B2 true JPH0740923B2 (en) | 1995-05-10 |
Family
ID=26502515
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2184469A Expired - Fee Related JPH0740923B2 (en) | 1989-09-12 | 1990-07-12 | Novel lactic acid bacterium by cell conjugation, fermented milk using the same, and method for producing the same |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0740923B2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2681764B1 (en) * | 1991-10-01 | 1993-12-24 | Michel Renard | IMPROVEMENT TO THE PROCESS FOR OBTAINING FIRMED MILK FOR THE MANUFACTURE OF A YOGURT OR YOGHURT-LIKE COAGULUM. |
| ES2557160T3 (en) | 2006-10-23 | 2016-01-22 | Nestec S.A. | Modulation of aroma and flavor through biotransformation in dairy products |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0262516A2 (en) * | 1986-09-29 | 1988-04-06 | Miles Inc. | Genetic transformation of lactic acid bacteria |
-
1990
- 1990-07-12 JP JP2184469A patent/JPH0740923B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH03175978A (en) | 1991-07-31 |
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