title: Distinctive proteolytic activity of cell envelope proteinase of Lactobacillus helveticus isolated from airag, a traditional Mongolian fermented mare's milk.
abstract: Airag is a traditional fermented milk of Mongolia that is usually made from raw mare's milk. Lactobacillus helveticus is one of the lactic acid bacteria most frequently isolated from airag. In this study, we investigated the genetic and physiological characteristics of L. helveticus strains isolated from airag and clarified their significance in airag by comparing them with strains from different sources. Six strains of L. helveticus were isolated from five home-made airag samples collected from different regions of Mongolia. The optimal temperature for acidification in skim milk was 30 to 35°C for all the Mongolian strains, which is lower than those for the reference strains (JCM 1554 and JCM 1120(T)) isolated from European cheeses. All of the strains had a prtH1-like gene encoding a variant type of cell envelope proteinase (CEP). The CEP amino acid sequence in Snow Brand Typeculture (SBT) 11087 isolated from airag shared 71% identity with PrtH of L. helveticus CNRZ32 (AAD50643.1) but 98% identity with PrtH of Lactobacillus kefiranofaciens ZW3 (AEG40278.1) isolated from a traditional fermented milk in Tibet. The proteolytic activities of the CEP from SBT11087 on artificial substrate (N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide) and pure casein were measured using an intact-cell degradation assay. The activity of the CEP from SBT11087 was observed to be weak and exhibited a lower optimal temperature (40°C) than those from the reference strains (45-50°C). The specificity of the SBT11087 CEP for αS1-casein was typical of the CEPs previously reported in L. helveticus, as determined through the degradation profiles obtained through gel electrophoresis and mass spectrometry analyses. In contrast, the degradation profile of β-casein revealed that the CEP of SBT11087 primarily hydrolyzes its C-terminal domain and hydrolyzed nine of the 16 cleavage sites shared among the CEPs of other L. helveticus strains. Thus, the CEP of SBT11087 is distinct from those from previously reported L. helveticus strains in terms of its optimal temperature and its degradation of β-casein. Therefore, the Mongolian L. helveticus strains differ from other strains of the species in different collections and are specifically suited for the natural lactic acid bacterial population in airag.
authors.affiliation: Milk Science Research Institute, Megmilk Snow Brand Co., Ltd., Minamidai 1-1-2, Kawagoe, Saitama 350-1165, Japan.
authors.forename: Mari
authors.initials: M
authors.lastname: Miyamoto
authors.affiliation: Milk Science Research Institute, Megmilk Snow Brand Co., Ltd., Minamidai 1-1-2, Kawagoe, Saitama 350-1165, Japan.
authors.forename: Hiroshi M
authors.initials: HM
authors.lastname: Ueno
authors.affiliation: Milk Science Research Institute, Megmilk Snow Brand Co., Ltd., Minamidai 1-1-2, Kawagoe, Saitama 350-1165, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Watanabe
authors.affiliation: Milk Science Research Institute, Megmilk Snow Brand Co., Ltd., Minamidai 1-1-2, Kawagoe, Saitama 350-1165, Japan.
authors.forename: Yumi
authors.initials: Y
authors.lastname: Tatsuma
authors.affiliation: Milk Science Research Institute, Megmilk Snow Brand Co., Ltd., Minamidai 1-1-2, Kawagoe, Saitama 350-1165, Japan.
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Seto
authors.affiliation: Animal Food Function, Graduate School of Environmental and Life Science, Okayama University, Tsushimanaka 3-1, Kita-ku, Okayama-shi, Okayama 700-8530, Japan.
authors.forename: Taku
authors.initials: T
authors.lastname: Miyamoto
authors.affiliation: Milk Science Research Institute, Megmilk Snow Brand Co., Ltd., Minamidai 1-1-2, Kawagoe, Saitama 350-1165, Japan. Electronic address: h-nakajima@wayo.ac.jp.
authors.forename: Hadjime
authors.initials: H
authors.lastname: Nakajima
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D003611:Dairy Products / Q000382:microbiology*; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005285:Fermentation; D006736:Horses; D052198:Lactobacillus helveticus / Q000201:enzymology* / Q000302:isolation & purification*; D008892:Milk / Q000382:microbiology*; D008986:Mongolia; D059748:Proteolysis; D017386:Sequence Homology, Amino Acid
chemical_list: D002364:Caseins; D010450:Endopeptidases
keywords: Airag; Cell envelope proteinase; Cleavage site specificity; Lactobacillus helveticus