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Isolation and Identification of Lactic Microbiota from Kimchi, Produced in Iran, Based on Biochemical and Molecular Methods

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Isolation and Identification of Lactic Microbiota from Kimchi, Produced in Iran, Based on Biochemical and Molecular Methods ﻓﺼﻠﻨﺎﻣﻪ ﻋﻠﻮم و ﺻﻨﺎﻳﻊ ﻏﺬا ﻳﻲ ﺷﻤﺎره 54، دوره 13، ﻣﺮداد 1395 ﺟﺪاﺳﺎزي و ﺷﻨﺎﺳﺎﻳﻲ ﻓﻠﻮر ﻻﻛﺘﻴﻜﻲ ﻛﻴﻤﭽﻲ ﺗﻮﻟﻴﺪ ﺷﺪه در اﻳﺮان ﺑﺮ ﭘﺎﻳﻪ روش ﻫﺎي ﺑﻴﻮﺷﻴﻤﻴﺎﻳﻲ و ﻣﻮﻟﻜﻮﻟﻲ ∗ ﻓﺮﻳﺪه ﻃﺒﺎﻃﺒﺎﻳﻲ ﻳﺰدي1 ، ﻋﻠﻴﺮﺿﺎ وﺳﻴﻌﻲ2 ، ﺑﻬﺮوز ﻋﻠﻴﺰاده ﺑﻬﺒﻬﺎﻧﻲ 2 2 -1 داﻧﺸﻴﺎر و ﻋﻀﻮ ﻫﺌﻴﺖ ﻋﻠﻤﻲ ﮔﺮوه ﻋﻠﻮم و ﺻﻨﺎﻳﻊ ﻏﺬاﻳﻲ، داﻧﺸﻜﺪه ﻛﺸﺎورزي، داﻧﺸﮕﺎه ﻓﺮدوﺳﻲ ﻣﺸﻬﺪ -2 داﻧﺸﺠﻮي دﻛﺘﺮي ﻋﻠﻮم و ﺻﻨﺎﻳﻊ ﻏﺬاﻳﻲ، داﻧﺸﻜﺪه ﻛﺸﺎورزي، داﻧﺸﮕﺎه ﻓﺮدوﺳﻲ ﻣﺸﻬﺪ ( ﺗﺎرﻳﺦ درﻳﺎﻓﺖ: 17/5/93 ﺗﺎرﻳﺦ ﭘﺬﻳﺮش: 93/7/1) ﭼﻜﻴﺪه ﻛﻴﻤﭽﻲ ﻳﻚ اﺻﻄﻼح ﻋﻤﻮﻣﻲ ﺑﺮاي ﺳﺒﺰﻳﺠﺎت ﺗﺨﻤﻴﺮي اﺳﺖ . ﻫﺪف از اﻧﺠﺎم اﻳﻦ ﻣﻄﺎﻟﻌﻪ ﺷﻨﺎﺳﺎﻳﻲ ﻓﻠﻮر ﻻﻛﺘﻴﻜﻲ ﻛﻴﻤﭽﻲ ﺑﺮ ﭘﺎﻳﻪ روشﻫﺎي ﺑﻴﻮﺷﻴﻤﻴﺎﻳﻲ و ﻣﻮﻟﻜﻮﻟﻲ ﺑﻮد. در اﻳﻦ ﭘﮋوﻫﺶ ﭘﺲ از اﻧﺠﺎم ﻛﺸﺖ ﻫﺎي ﻣﺘﻮاﻟﻲ ﺑﺮ روي ﻣﺤﻴﻂ ﻫﺎي اﺧﺘﺼﺎﺻﻲ و ﻣﺸﺎﻫﺪه ﻣﻴﻜﺮوﺳﻜﻮﭘﻲ 85 ﺟﺪاﻳﻪ ﻛﻪ ﺑﺎ اﻧﺠﺎم آزﻣﺎﻳﺶ ﻫﺎي اوﻟﻴﻪ ﺑﻪ ﻧﻈﺮ ﻣﻲ رﺳﻴﺪ ﺟﺰء ﺑﺎﻛﺘﺮي ﻫﺎي اﺳﻴﺪ ﻻﻛﺘﻴﻚ ﺑﺎﺷﻨﺪ، اﻧﺘﺨﺎب ﺷﺪه و ﺑﺮاي ﮔﺮوه ﺑﻨﺪي آن ﻫﺎ، آزﻣﻮن ﻫﺎي ﻓﻴﺰﻳﻮﻟﻮژﻳﻜﻲ، ﺑﻴﻮﺷﻴﻤﻴﺎﻳﻲ و ﺗﺨﻤﻴﺮ 10 ﻧﻮع ﻛﺮﺑﻮﻫﻴﺪرات ﻫﺎي ﻣﺨﺘﻠﻒ اﻧﺠﺎم ﭘﺬﻳﺮﻓﺖ . ﺑﺮ ﻣﺒﻨﺎي ﺗﺴﺖ ﻫﺎي ﺑﻴﻮﺷﻴﻤﻴﺎﻳﻲ و ﺗﺨﻤﻴﺮ ﻗﻨﺪ 85 اﻳﺰوﻟﻪ ﺑﻪ 10 ﮔﺮوه ﺗﻘﺴﻴﻢ ﺑﻨﺪي ﺷﺪﻧﺪ. از ﻫﺮ ﮔﺮوه ﭼﻨﺪ اﻳﺰوﻟﻪ اﻧﺘﺨﺎب و ژن ﻧﺎﺣﻴﻪ 16S rRNA آن ﻫﺎ ﺑﻪ ﻛﻤﻚ ﭘﺮاﻳﻤﺮ ﻫﺎي ﻋﻤﻮﻣﻲ و ﺑﺎ ﺗﻜﻨﻴﻚ PCR ﺗﻜﺜﻴﺮ داده ﺷﺪ . ﻧﺘﺎﻳﺞ ﻧﺸﺎن داد ﻛﻪ ﺗﻨﻮع ﺑﺎﻛﺘﺮيﻫ ﺎي اﺳﻴﺪ ﻻﻛﺘﻴﻚ ﻛﻴﻤﭽﻲ ﺷﺎﻣﻞ ﻻﻛﺘﻮﺑﺎﺳﻴﻠﻮس ﭘﻼﻧﺘﺎروم (17/41%)، ﻻﻛﺘﻮﺑﺎﺳﻴﻠﻮس ﻓﺮﻣﻨﺘﻮم (41/9%)، اﻧﺘﺮوﻛﻮﻛﻮس ﻓﺎﺳﻴﻮم (%7/06)، اﻧﺘﺮوﻛﻮﻛﻮس ﻓﻜﺎﻟﻴﺲ (52/3%)، ﻟﻮﻛﻮﻧﻮﺳﺘﻮك ﺳﻴﺘﺮﺋﻮم (33/2%)، ﻟﻮﻛﻮﻧﻮﺳﺘﻮك ﻣﺰﻧﺘﺮوﺋﻴﺪوس (52/3%)، ﭘﺪﻳﻮﻛﻮﻛﻮس ﭘﻨﺘﻮزاﺳﺌﻮس (23/8%) و وﻳﺴﻼ ﺳﻴﺒﺎرﻳﺎ (70/24%) ﺑﻮد. ﺑﻪ ﻃﻮر ﻛﻠﻲ اﻳﻦ ﻓﺮاوردهي ﺗﺨﻤﻴﺮي داراي ﺗﻨﻮع ﻓﺮاواﻧﻲ در ﻓﻠﻮر ﻣﻴﻜﺮوﺑﻲ ﺧﻮد ﻣﻲﺑﺎﺷ ﺪ ﻛﻪ از ﻣﻴﻜﺮوﻓﻠﻮر ﻃﺒﻴﻌﻲ ﻣﻮﺟﻮد در ﻣﻮاد ﺧﺎم ﻣﻮرد اﺳﺘﻔﺎده ﻧﺸﺄت ﻣﻲ ﮔﻴﺮد. ﺑﺎ ﺗﻮﺟﻪ ﺑﻪ اﻳﻦ ﻛﻪ ﺑﺎﻛﺘﺮي ﻫﺎي اﺳﻴﺪ ﻻﻛﺘﻴﻚ ﻣﻲ ﺗﻮاﻧﻨﺪ ﺑﺎ ﺗﻮﻟﻴﺪ اﺳﻴﺪﻫﺎ و آﻧﺰﻳﻢ ﻫﺎي ﻣﺨﺘﻠﻒ ﻧﻘﺶ ﻣﻬﻤﻲ را در اﻳ ﺠﺎد ﻃﻌﻢ و ﺑﺎﻓﺖ ﻣﻄﻠﻮب اﻳﻔﺎ ﻧﻤﺎﻳﻨﺪ ، ﺑﻨﺎﺑﺮاﻳﻦ ﻣﻲ ﺗﻮان از ﺳﻮﻳﻪ ﻫﺎي ﺟﺪا ﺷﺪه از اﻳﻦ ﻣﺤﺼﻮل در ﺻﻨﻌﺖ ﻏﺬا ﺑﻬﺮه ﺑﺮد . Downloaded from fsct.modares.ac.ir at 15:48 IRST on Thursday September 30th 2021 ﻛﻠﻴﺪ واژﮔﺎن: ﻛﻴﻤﭽﻲ، ﻓﻠﻮر ﻻﻛﺘﻴﻜﻲ، ﺗﺴﺖ ﻫﺎي ﺑﻴﻮﺷﻴﻤﻴﺎﻳﻲ ، ﺗﻮاﻟﻲ ﻳﺎﺑﻲ ژن S rRNA 16 ∗ ﻣﺴﺌﻮل ﻣﻜﺎﺗﺒﺎت : [email protected] 1 ﻓﺮﻳﺪه ﻃﺒﺎﻃﺒﺎﻳﻲ ﻳﺰدي و ﻫﻤﻜﺎران ﺟﺪاﺳﺎزي و ﺷﻨﺎﺳﺎﻳﻲ ﻓﻠﻮر ﻻﻛﺘﻴﻜﻲ ﻛﻴﻤﭽﻲ ﺗﻮﻟﻴﺪ ﺷﺪه در... 1- ﻣﻘﺪﻣﻪ ﺑﺎﻛﺘﺮي ﻫﺎي اﺳﻴﺪ ﻻﻛﺘﻴﻚ در ﺗﻮﻟﻴﺪ ﻏﺬاﻫﺎي ﺗﺨﻤﻴﺮي ﺑﻬﺮه ﺑـﺮده اﺳﺖ. اﻳﻦ اﻣﺮ ﺑﻪ ﺧﺎﻃﺮ ﺗﻮاﻧﺎﻳﻲ اﻳﻦ ﺑﺎﻛﺘﺮي ﻫﺎ در اﻳﺠـﺎد ﻋﻄـﺮ و ﻛﻴﻤﭽﻲ1 ﻳﻚ ﺧﻮراك ﺗﺨﻤﻴﺮﺷﺪه ﺳﻨﺘﻲ ا اﺳﺖ ﻛﻪ از ﺳـﺒﺰﻳﺠﺎت ﻃﻌﻢ و ﻣﻬﺎر ﭘﺎﺗﻮژن ﻫﺎ و ﻣﻴﻜﺮوارﮔﺎﻧﻴﺴﻢ ﻫﺎي ﻓﺎﺳﺪﻛﻨﻨﺪه ﻣﻲ ﺑﺎﺷﺪ و ادوﻳﻪﺟﺎت ﮔﻮﻧﺎﮔﻮن و اﻟﺒﺘﻪ ﺑﻴﺸﺘﺮ اوﻗﺎت ﺑﺎ ﻧﻮﻋﻲ ﻛﻠﻢ ﭼﻴﻨﻲ ﺑﻪ .[2] ﻧﺎم ﺑﺎﺋﭽﻮ و ﻓﻠﻔﻞ ﻗﺮﻣﺰ درﺳﺖ ﻣﻲ ﺷـﻮد . ﻛﻠﻤـﻪ ي ﻛﻴﻤﭽـﻲ ﻳـﻚ ﻗﺒﻞ از ﻇﻬـﻮر و ﮔـﺴﺘﺮش روش ﻫـﺎي ﻣﻮﻟﻜـﻮﻟﻲ، روش اﺻـﻠﻲ اﺻﻄﻼح ﻋﻤﻮﻣﻲ ﺑﺮاي ﺳﺒﺰﻳﺠﺎت ﺗﺨﻤﻴﺮي اﺳـﺖ ﻛـﻪ از واژه ي ﺷﻨﺎﺳﺎﻳﻲ ﺑﺎﻛﺘﺮي ﻫﺎي اﺳﻴﺪ ﻻﻛﺘﻴﻚ ﺑﺮ ﻣﺒﻨـﺎي ﺧـﻮاص ﻓﻨـﻮﺗﻴﭙﻲ ﭼﻴﻨﻲ ﭼﻴﻤﭽﺎ 2 ﺑﻪ ﻣﻌﻨﻲ ﺳﺒﺰي ﺷﻮر ﮔﺮﻓﺘﻪ ﺷﺪه اﺳﺖ . ﺑﻪ ﺻﻮرت ﺑــﺎﻛﺘﺮيﻫــﺎ ﺑــﻮد، ﻛــﻪ ﺷــﺎﻣﻞ وﻳﮋﮔــﻲ ﻫــﺎي ﻣﻮرﻓﻮﻟــﻮژﻳﻜﻲ، ﺳﻨﺘﻲ ﻣﻘﺪار زﻳﺎدي ﻛﻴﻤﭽﻲ ﺑﻪ ﺻﻮرت ﺳﺎﻻﻧﻪ ﺗﻮﻟﻴﺪ ﻣﻲ ﺷﻮد ﻛﻪ ﻓﻴﺰﻳﻮﻟﻮژﻳﻜﻲ و ﺑﻴﻮﺷﻴﻤﻴﺎﻳﻲ ﻣﻲ ﺑﺎﺷﺪ [3]. از آﻧﺠﺎﻳﻲ ﻛﻪ ﻛـﺎرﺑﺮد ﺑﻴﺸﺘﺮ در ﻓﺼﻞ زﻣﺴﺘﺎن ﻛﻪ دﺳﺘﺮﺳﻲ ﺑﻪ ﺳﺒﺰﻳﺠﺎت ﺗـﺎزه ﻛـﺎﻫﺶ روشﻫﺎي ﻓﻨﻮﺗﻴﭙﻲ در ﺷﻨﺎﺳﺎﻳﻲ ﺑﺎﻛﺘﺮي ﻫـﺎ ﺑـﺴﻴﺎر وﻗـﺖ ﮔﻴـﺮ و ﻣﻲ ﻳﺎﺑﺪ ﻣﺼﺮف ﻣﻲ ﮔﺮدد. ﻛﻴﻤﭽﻲ در واﻗﻊ ﻧـﻮﻋﻲ ﺷـﻮري ﺗﻬﻴـﻪ ﻃﻮﻻﻧﻲ اﺳﺖ و ﻧﺘﺎﻳﺞ ﺣﺎﺻﻞ ﻋﻤﻮﻣﺎ از دﻗﺖ ﺑـﺎﻻﻳﻲ ﺑﺮﺧـﻮردار ﺷﺪه از ﺳﺒﺰي ﻫﺎي ﻣﺨﺘﻠﻒ اﺳﺖ [1]. اﻳﻦ ﻣﺤﺼﻮل ﻓﻌﺎﻟﻴﺖ ﻫﺎي ﻧﻤﻲﺑﺎﺷﺪ و ﻧﻤﻲﺗﻮاﻧﺪ ﺑﻪ ﻃﻮر ﻛﺎﻣﻞ و دﻗﻴﻖ ﺑﺎﻛﺘﺮيﻫﺎ را در ﺳﻄﺢ اﻳﻤﻨﻲ را اﻓﺰاﻳﺶ داده، روﻧﺪ ﭘﻴﺮ ﺷـﺪن را ﻛـﺎﻫﺶ ﻣـﻲ دﻫـﺪ و از ﮔﻮﻧﻪ ﺗﻔﻜﻴﻚ ﻧﻤﺎﻳﺪ، ﻟﺬا ﺑﻬﺘﺮ اﺳﺖ ﺟﻬﺖ ﺷﻨﺎﺳﺎﻳﻲ ﺑﺎﻛﺘﺮي ﻫـﺎ از ﻳﺒﻮﺳﺖ ﺟﻠﻮﮔﻴﺮي ﻣﻲ ﻛﻨﺪ، ﻋﻼوه ﺑﺮ اﻳﻦ ﻛﻴﻤﭽـﻲ ﻣ ﻜﻤـﻞ ﻃﻌـﻢ روشﻫﺎي ﻣﻮﻟﻜﻮﻟﻲ اﺳﺘﻔﺎده ﺷﻮد . واﻛﻨﺶ زﻧﺠﻴـﺮه اي ﭘﻠـﻲ ﻣـﺮاز ﺳﺒﺰﻳﺠﺎت ﻧﻴﺰ ﺑﻮده و ﻫﻤﺎﻧﻨﺪ ﻳﻚ ﻏﺬاي ﭘﺮوﺑﻴﻮﺗﻴـﻚ ﻋﻤـﻞ ﻣـﻲ (PCR) ﺑﺮاي ﺷﻨﺎﺳﺎﻳﻲ ﺳﺮﻳﻊ و دﻗﻴﻖ ﺑﺎﻛﺘﺮي ﻫﺎي اﺳﻴﺪ ﻻﻛﺘﻴﻚ ﻛﻨﺪ. ﺑﻪ دﻟﻴﻞ ارزش ﻏﺬاﻳﻲ ﺑﺎﻻ، ﻛﻴﻤﭽﻲ ﻣـﻲ ﺗﻮاﻧـﺪ ﻣﻨﺒـﻊ ﺑـﺴﻴﺎر در ﻃﻲ دو د ﻫﻪ اﺧﻴﺮ ﭘﻴﺸﺮﻓﺖ ﻓﺮاواﻧﻲ داﺷﺘﻪ اﺳﺖ. اﻣﺮوزه روش ﺧﻮب و ﻣﻨﺎﺳﺒﻲ از ﭘﺮوﺗﺌﻴﻦ، ﻣﻮاد ﻣﻌﺪﻧﻲ، اﺳـﻴﺪ ﻫـﺎي ﭼـﺮب و ﺗﻮاﻟﻲ ﻳﺎﺑﻲ ژن 16S rRNA ﺑﻪ ﻋﻨﻮان ﻳﻜـﻲ از ﭘﺮﻛـﺎرﺑﺮدﺗﺮﻳﻦ اﺳﻴﺪﻫﺎي آﻣﻴﻨﻪ ﺿـﺮوري ﺑﺎﺷـﺪ [1]. ﻏـﺬاﻫﺎي ﺗﺨﻤﻴـﺮ ﺷـﺪه در روشﻫﺎي ﻣﻮﺟـﻮد در ﺷﻨﺎﺳـﺎﻳﻲ ﺟﻤﻌﻴـﺖ ﻣﻴﻜﺮوﺑـﻲ ﻏـﺬاﻫﺎي ﺑﺴﻴﺎري از ﻛﺸﻮرﻫﺎ، ﺑﻪ ﻋﻨـﻮان اﺟـﺰاي ﺿـﺮوري رژﻳـﻢ ﻏـﺬاﻳﻲ ﺗﺨﻤﻴﺮي ﻛﺎرﺑﺮد دارد [3]. ﻣﺤﺴﻮب ﻣﻲ ﺷﻮﻧﺪ. ﺑﺮاﺳﺎس ﺗﺤﻘﻴﻘـﺎت ﻛـﻴﻢ و ﭼﺎﻧـﮓ (2005) ﺑـﺮ روي ﻛﻴﻤﭽـﻲ، ﺗﺨﻤﻴﺮ ﻣﻮاد ﻏﺬاﻳﻲ ﺳـﻨﺘﻲ ﻋﻤـﺪﺗﺎ ﺗﻮﺳـﻂ ﻣﻴﻜﺮوارﮔﺎﻧﻴـﺴﻢ ﻫـﺎي ﺑﺎﻛﺘﺮي ﻫـﺎي اﺳـﻴﺪ ﻻﻛﺘﻴـﻚ دﺧﻴـﻞ در ﺗﺨﻤﻴـﺮ ﺑـﻪ ﺟـﻨﺲ ﻫـﺎي ﻃﺒﻴﻌﻲ ﻛﻪ در ﻣﻮاد ﺧﺎم ﺣـﻀﻮر دارﻧـﺪ ﺻـﻮرت ﻣـﻲ ﮔﻴـﺮد ﻛـﻪ ﻟﻮﻛﻮﻧﻮﺳﺘﻮك، ﻻﻛﺘﻮﺑﺎﺳﻴ ﻠﻮس و وﻳﺴﻼ ﺗﻌﻠﻖ داﺷـﺘﻨﺪ . ﻫﻤﭽﻨـﻴﻦ ﺑﺎﻛﺘﺮيﻫﺎي اﺳـﻴﺪ ﻻﻛﺘﻴـﻚ ﻫـﻢ ﺟﺰﺋـﻲ از اﻳـﻦ ﻓﻠـﻮر ﻃﺒﻴﻌـﻲ اﻳﻦ ﭘﮋوﻫﺶ ﻧﺸﺎن داد ﻛﻪ وﻳﺴﻼ ﻛﻮرﻳﻨﺴﻴﺲ در ﻫﻤﻪ ي ﻧﻤﻮﻧﻪ ﻫﺎ ﻣﺤﺴﻮب ﻣﻲﺷﻮﻧﺪ. ﺑﺎﻛﺘﺮيﻫﺎي اﺳـﻴﺪ ﻻﻛﺘﻴـﻚ ﮔـﺮوه ﺑـﺰرگ و ﺣﻀﻮر داﺷﺖ و ﺑﻪ ﻋﻨﻮان ﮔﻮﻧﻪ ي ﻏﺎﻟﺐ ﻣﻌﺮﻓﻲ ﺷﺪ. در ﻣﺠﻤـﻮع ﻣﺘﻨﻮﻋﻲ از ﺑﺎﻛﺘﺮيﻫﺎ را ﺷﺎﻣﻞ ﻣﻲﺷﻮد، اﻳﻦ ﺑﺎﻛﺘﺮي ﻫﺎ ﮔﺮم ﻣﺜﺒﺖ، ﺑﺎ در ﻧﻈﺮ ﮔﺮﻓﺘﻦ ﭘﮋوﻫﺶ ﻫﺎي ﻣﺨﺘﻠﻒ ﻛﻪ ﺑﺮ روي ﻛﻴﻤﭽﻲ اﻧﺠﺎم Downloaded from fsct.modares.ac.ir at 15:48 IRST on Thursday September 30th 2021 ﻓﺎﻗﺪ ﺗﻮاﻧﺎﻳﻲ ﺗﻮﻟﻴﺪ اﺳﭙﻮر، ﻛﺎﺗﺎﻻز ﻣﻨﻔﻲ، ﻣﻘـﺎوم ﺑـﻪ اﺳـﻴﺪ، ﺑـﻲ - ﺷﺪه اﺳﺖ، ﺑﻴﺸﺘﺮﻳﻦ ﺑﺎﻛﺘﺮي ﻫﺎي اﺳـﻴﺪ ﻻﻛﺘﻴـﻚ ﻛـﻪ در ﺗﺨﻤﻴـﺮ ﻫﻮازي اﺧﺘﻴﺎري و از ﻟﺤﺎظ ﻇﺎﻫﺮي اﻛﺜﺮا ﻣﻴﻠﻪاي ﻳﺎ ﻛﺮوي ﻫﺴﺘﻨﺪ ﻛﻴﻤﭽــﻲ ﻧﻘــﺶ دا رﻧــﺪ ﺑــﻪ ﺗﺮﺗﻴــﺐ از ﺟــﻨﺲ ﻫــﺎي وﻳــﺴﻼ، ﻛﻪ ﺗﻮاﻧﺎﻳﻲ ﺗﻮﻟﻴﺪ اﺳﻴﺪ ﻻﻛﺘﻴﻚ را ﺑﻪ ﻋﻨﻮان ﻣﺤﺼﻮل ﻋﻤﺪه ﻧﻬﺎﻳﻲ ﻟﻮﻛﻮﻧﻮﺳــﺘﻮك، ﻻﻛﺘﻮﺑﺎﺳــﻴﻠﻮس، ﭘــﺪﻳﻮﻛﻮﻛﻮس و اﻧﺘﺮوﻛﻮﻛــﻮس ﺗﺨﻤﻴــﺮ دارا ﻣــﻲﺑﺎﺷــﻨﺪ. ﺑــﺎﻛﺘﺮيﻫــﺎي اﺳــﻴﺪ ﻻﻛﺘﻴــﻚ ﻳﻜــﻲ از ﻫـﺴﺘﻨﺪ [4-6]. ﻣﻄﺎﻟﻌــﺎت ﻣﺨﺘﻠــﻒ اﻧﺘـﺸﺎر ﻳﺎﻓﺘــﻪ در ﺧــﺼﻮص ﻓﺮاوان ﺗﺮﻳﻦ ﮔﺮوه ﺑﺎﻛﺘﺮي ﻫﺎي ﻣﺮﺗﺒﻂ ﺑـﺎ اﻧـﺴﺎن ﻫـﺴﺘﻨﺪ [1 و 2]. ﻛﻴﻤﭽﻲ، ﺑﺮﺧـﻲ از اﺧﺘﻼﻓـﺎت ﻣﺮﺑـﻮط ﺑـﻪ ﺳـﺎﺧﺘﺎر و دﻳﻨﺎﻣﻴـﻚ آن ﻫﺎ ﺑﻪ ﻃﻮر ﮔﺴﺘﺮده در اﻛﻮﺳﻴﺴﺘﻢ ﻫﺎي ﻣﺨﺘﻠﻒ وﺟﻮد دارﻧـﺪ و ﺟﻤﻌﻴﺖ را آﺷﻜﺎر ﻧﻤﻮده اﺳﺖ . اﻳﻦ ﻣﻄﺎﻟﻌﺎت ﺑـﺮ روي ﻛﻴﻤﭽـﻲ ﻣﻌﻤـﻮﻻً در ﻏـﺬاﻫﺎﻳﻲ ﻫــﻢ ﭼـﻮن ﻓـﺮاورده ﻫــﺎي ﻟﺒﻨـﻲ، ﮔﻮﺷــﺖ، ﺗﻮﻟﻴﺪي در ﻣﻘﻴﺎس آزﻣﺎﻳﺸﮕﺎﻫﻲ ﻳﺎ ﻛﻴﻤﭽـﻲ ﺗﺠـﺎري ﺧﺮﻳـﺪاري ﺳﺒﺰي ﻫﺎي ﺗﺨﻤﻴﺮي، ﺧﻤﻴﺮ ﺗﺮش، ﺳﻴﻼژ و ﻧﻮﺷﻴﺪﻧﻲ ﻫﺎي اﻟﻜﻠـﻲ ﺷﺪه از ﺗﻮﻟﻴﺪﻛﻨﻨﺪﮔﺎن ﻣﺨﺘﻠﻒ ﺑﻪ اﺟﺮا درآﻣﺪه اﺳـﺖ . ﻋـﻼوه ﺑـﺮ ﻳﺎﻓﺖ ﻣﻲ ﺷﻮﻧﺪ. ﺑﺸﺮ آﮔﺎﻫﺎﻧﻪ ﻳﺎ ﻧﺎآﮔﺎﻫﺎﻧـﻪ ﻃـﻲ ﻫـﺰاران ﺳـﺎل از اﻳﻦ، ﻛﻴﻤﭽﻲ ﻳﻚ اﺻﻄﻼح ﻋﻤـﻮﻣﻲ ﺑـﺮاي ﮔﺮوﻫـﻲ از ﻏـﺬاﻫﺎي ﺗﺨﻤﻴﺮﺷﺪه ﺑﺮ ﭘﺎﻳﻪ ي ﺳﺒﺰي ﻫﺎي ﻣﺨﺘﻠﻒ ﻣﻲ ﺑﺎﺷﺪ ﻛﻪ در ﻓﺮآوري Kimchi .1 آن ﻣﻤﻜﻦ اﺳﺖ ﺑﻴﺶ از ﺻﺪ ﻧﻮع ﺳﺒﺰي ﻣﻮرد اﺳﺘﻔﺎده ﻗﺮار ﮔﻴﺮد 2. Chimchae 2 ﻓﺼﻠﻨﺎﻣﻪ ﻋﻠﻮم و ﺻﻨﺎﻳﻊ ﻏﺬا ﻳﻲ ﺷﻤﺎره 54، دوره 13، ﻣﺮداد 1395 [7] . ﻟﺬا ﺗﻔﺎوت ﻫﺎي ﻣﻮﺟﻮد در اﻳـﻦ ﻧﺘـﺎﻳﺞ، ﭼﻨـﺪان ﺗﻌﺠـﺐ آور ﻧﺒﻮده و ﻣﻲ ﺗﻮاﻧﺪ ﺑﺮ اﺳﺎس ﺗﻔـﺎوت در ﺷـﺮاﻳﻂ ﻓـﺮآوري و ﻧـﻮ ع ﺳﺒﺰي ﻣﻮرد اﺳﺘﻔﺎده، ﺗﻮﺟﻴﻪ ﺷﻮد . ﻫﺪف از اﻳﻦ ﭘﮋوﻫﺶ ﺷﻨﺎﺳﺎﻳﻲ و ﺟﺪاﺳـﺎزي ﻓﻠـﻮر ﻻﻛﺘﻴﻜـﻲ ﻛﻴﻤﭽﻲ ﺑﺮ اﺳﺎس روش ﻫﺎي ﺑﻴﻮﺷﻴﻤﻴﺎﻳﻲ و ﻣﻮﻟﻜﻮﻟﻲ ﻣﻲ ﺑﺎﺷـﺪ ، ﺗﺎ در ﻧﻬﺎﻳﺖ ﺑﺎ ﺗﻮﺟﻪ ﺑـﻪ اﻳـﻦ ﻛـﻪ ﺑـﺎﻛﺘﺮي ﻫـﺎي اﺳـﻴﺪ ﻻﻛﺘﻴـﻚ ﻣﻲ ﺗﻮاﻧﻨﺪ ﺑﺎ ﺗﻮﻟﻴﺪ اﺳﻴﺪﻫﺎ و آﻧﺰﻳﻢ ﻫﺎي ﻣﺨﺘﻠﻒ ﻧﻘﺶ ﻣﻬﻤﻲ را در اﻳﺠﺎد ﻃﻌﻢ و ﺑﺎﻓﺖ ﻣﻄﻠﻮب اﻳﻔﺎ ﻧﻤﺎﻳﻨﺪ، ﺑﺘﻮان از ﺳﻮﻳﻪ ﻫﺎي ﺟﺪا ﺷﺪه از اﻳﻦ ﻣﺤﺼﻮل در ﺻﻨﻌﺖ ﻏﺬا ﺑﻬﺮه ﺑﺮد. -2 ﻣﻮاد و روش ﻫﺎ -1-2 ﺗﻬﻴﻪ ﻛﻴﻤﭽﻲ ﻣﻮاد اوﻟﻴﻪ ﺗﻮﻟﻴﺪ ﻛﻴﻤﭽﻲ ﻛﻪ ﺷﺎﻣﻞ ﻛﻠﻢ ﭼﻴﻨـﻲ، ﺗـﺮب ﺳـﻔﻴﺪ، ﭘﻮدر ﻓﻠﻔﻞ ﻗﺮﻣﺰ، زﻧﺠﺒﻴﻞ، ﻋﺼﺎره ﻣﺎﻫﻲ و ﻣﻴﮕﻮ، ﺳـﻴﺮ، ﭘﻴﺎزﭼـﻪ، ﺗﺮه ﻓﺮﻧﮕﻲ، ﺷﻜﺮ، آرد ﺑﺮﻧﺞ، آرد ﮔﻨـﺪم و ﻧﻤـﻚ از ﺑـﺎزار ﻫـﺎي ﺷﻜﻞ 1 ﻣﺮاﺣﻞ آزﻣﺎﻳﺸﺎت ﻣﺮﺑﻮط ﺑﻪ ﺷﻨﺎﺳﺎﻳﻲ اﻳﺰوﻟﻪ ﻫﺎي ﻣﺘﻌﻠﻖ ﺑﻪ ﻛﻴﻤﭽﻲ ﺑﺎ ﺗﻜﻴﻪ ﺑﺮ روش ﻫﺎي ﻣﻮﻟﻜﻮﻟﻲ PCR 16 S rRNA ﻣﺤﻠﻲ در ﺷﻬﺮ ﻣﺸﻬﺪ ﺧﺮﻳﺪاري ﺷﺪ . ﺟﻬﺖ ﺗﻬﻴﻪ ﺷﻴﺮاﺑﻪ ﻛﻴﻤﭽـﻲ 120 ﮔﺮم آرد ﺑﺮﻧﺞ و آرد ﮔﻨﺪم ﭘـﺲ از ﻣﺨﻠـﻮط ﺷـﺪن ﺑـﺎ آب، -2-2 ﺟﺪاﺳﺎزي ﺑﺎﻛﺘﺮي ﻫﺎي اﺳﻴﺪ ﻻﻛﺘﻴﻚ ﺷﻜﺮ و ﻧﻤﻚ ﺣﺮارت داده ﺷﺪ ﺗﺎ ژﻻﺗﻴﻨﻪ ﺷﻮ د، ﺳـﭙﺲ 30ﮔـﺮم ده ﮔﺮم از ﻧﻤﻮﻧﻪ ﺑﻪ 90 ﻣﻴﻠﻲ ﻟﻴﺘﺮ از آب ﭘﭙﺘﻮن 1/0% ﻣﻨﺘﻘـﻞ ﺷـﺪ زﻧﺠﺒﻴﻞ، 30 ﮔﺮم ﭘﻴﺎزﭼﻪ، 50 ﮔﺮم ﺳﻴﺮ و 50ﮔﺮم ﺗﺮه ﻓﺮﻧﮕـﻲ را (ﻣﺮك، آﻟﻤﺎن ) و ﻋﻤﻞ ﻫﻤـﻮژﻧﻴﺰه ﺷـﺪن اﻧﺠـﺎم ﭘـﺬﻳﺮﻓﺖ (ﻣـﺪل ﻛﺎﻣﻼ آﺳﻴﺎب ﻛﺮده، ﺑﻪ ﻫﻤﺮاه ﺗﺮﺑﭽﻪ ﻧﻤﻚ ﺳﻮد ﺑﻪ آرد ﭘﺨﺘﻪ ﺷﺪه Seawar d، آﻟﻤﺎن ) . ﻛﺸﺖ ﺳﻄﺤﻲ از رﻗﺖ ﻫﺎي ﺗﻬﻴـﻪ ﺷـﺪه ﺑـﺮ اﺿﺎﻓﻪ ﺷﺪ . ﻛﺎﻫﻮ ﻫﺎ ﺑﺎ ﺗﻮﺟﻪ ﺑﻪ اﻧﺪازه ﺑﻪ ﭼﻨﺪ ﻗﺴﻤﺖ ﺗﻘﺴﻴﻢ ﺷﺪه روي ﻣﺤﻴﻂ ﻛﺸﺖ MRS Agar در ﺳﻪ ﺗﻜﺮار اﻧﺠﺎم ﺷـﺪ؛ ﺑـﻪ و ﺑﻪ ازاي ﻫﺮ ﺻﺪ ﮔﺮم ﻛﺎﻫﻮ ﻫﻔﺖ ﮔﺮم ﻧﻤﻚ ﺑﻴﻦ ﺑﺮگ ﻫـﺎي آن اﻳﻦ ﺗﺮﺗﻴﺐ ﻛﻪ روي ﻫﺮ ﭘﻠﻴﺖ 0/1 ﻣﻴﻠﻲ ﻟﻴﺘﺮ از ﻫﺮ رّﻗﺖ رﻳﺨﺘﻪ و ﭘﺎﺷﻴﺪه و ﺑﻪ ﻣﺪت ﺷﺶ ﺳﺎﻋﺖ ﺗﺤﺖ ﺗﻴﻤﺎر ﻧﻤﻜـﻲ ﻧﮕـﻪ داﺷـﺘﻪ ﺑﻪ ﻛﻤﻚ اﺳﭙﺮﻳﺪر ﭘﺨﺶ ﺷﺪ . در دو دﻣﺎي 30 و 45 درﺟﻪ ﺳﺎﻧﺘﻲ Downloaded from fsct.modares.ac.ir at 15:48 IRST on Thursday September 30th 2021 ﺷﺪﻧﺪ و ﺳﭙﺲ ﺳﻪ ﺑﺎر آﺑﻜﺸﻲ ﺷﺪﻧﺪ . ﺑﻌﺪ ﻣﺨﻠﻮط اﻓﺰودﻧﻲ ﻫـﺎ و ﮔﺮاد ﺑﻪ ﻣﺪت 48 ﺳﺎﻋﺖ در ﺷﺮاﻳﻂ ﺑﻲ ﻫﻮازي ﮔﺮﻣﺨﺎﻧﻪ ﮔﺬاري ﺗﺮب ﺳﻔﻴﺪ را ﺑﻪ ﻛﺎﻫﻮ اﺿﺎﻓﻪ ﻛﺮده و ﺗﻚ ﺗﻚ ﺑﺮگ ﻫﺎ ﻛﺎﻣﻼ ﺑـﻪ ﺷﺪ ﺗﺎ ﺷﺮاﻳﻂ ﺑﺮاي رﺷﺪ ﺑﺎﻛﺘﺮي ﻫﺎي ﻧﺎﻣﻄﻠﻮب ﺳﺨﺖ ﺗﺮ ﮔـﺮدد آن آﻏﺸﺘﻪ ﺷﺪ . ﻛﺎﻫﻮ را ﺑﻪ ﻫﻤﺮاه ﻋﺼﺎره ﺑﺎ ﻧﺴﺒﺖ 60 ﺑـﻪ 40 در [9] . از ﭘﻠﻴﺖ ﻫﺎي داراي ﺑﺎﻻﺗﺮﻳﻦ رّﻗﺖ، ﻛﻠﻨﻲ ﻫـﺎﻳﻲ ﻛـﻪ از ﻧﻈـﺮ ﺷﻴﺸﻪ ﻗﺮار داده ﺷﺪ و ﺗﺨﻤﻴﺮ آن در دﻣﺎي 20درﺟـﻪ ﺳـﺎﻧﺘﻴﮕﺮاد ﺷـﻜﻞ ﻇـﺎﻫﺮي، ﺣﺎﺷـﻴﻪ ي ﻛﻠﻨــﻲ، رﻧـﮓ و ﺳـﺎﻳﺮ وﻳﮋﮔـﻲ ﻫــﺎي ﺻﻮرت ﮔﺮﻓﺖ ﺗﺎ pH آن ﺑﻪ ﻛﻤﺘﺮ از 4/2 ﺑﺮﺳﺪ [1 و 8] . ﺟﻬﺖ ﻣﻮرﻓﻮﻟﻮژﻳﻜﻲ ﻣﺘﻔﺎوت ﺑﻮدﻧﺪ، اﻧﺘﺨﺎب ﺷﺪه و ﺳﭙﺲ ﻫﺮ ﻛﺪام در ﺑﺮرﺳﻲ ﻓﻠﻮر ﻻﻛﺘﻴﻜﻲ ﻛﻴﻤﭽﻲ ، ﻧﻤﻮﻧﻪ ﺑﺮداري در ﺷﺮاﻳﻂ اﺳﺘﺮﻳﻞ ﭘﻠﻴﺖ ﺟﺪاﮔﺎﻧﻪ ﻛﺸﺖ ﺧﻄﻲ داده ﺷﺪﻧﺪ و ﭘﺲ از ﭼﻨﺪ ﺑﺎر ﻛـ ﺸ ﺖ اﻧﺠﺎم ﭘﺬﻳﺮﻓﺖ ﺻﻮرت ﮔﺮﻓﺖ . ﺧﻄﻲ ﻛﻠﻨﻲ ﻫﺎي ﺗﻚ از ﻫﺮ اﻳﺰوﻟﻪ ﺑﻪ دﺳﺖ آﻣﺪ . ﺑـﺮاي ﻧﮕﻬـﺪاري آزﻣﺎﻳﺸﺎﺗﻲ ﻛﻪ ﺑﺮاي ﺷﻨﺎﺳﺎﻳﻲ ﻓﻠﻮر ﻻﻛﺘﻴﻜـﻲ ﻛﻴﻤﭽـﻲ ﺑـﺎ ﻛﻤـﻚ اﻳﺰوﻟﻪ ﻫﺎ ﺑﻪ ﻣﺪت ﻃﻮﻻﻧﻲ، اﻳﺰوﻟﻪ ﻫﺎ در MRS broth ﺣـﺎوي روشﻫﺎي ﻣﻮﻟﻜﻮﻟﻲ ﻣﺒﺘﻨﻲ ﺑﺮ ﻛﺸﺖ ﺻﻮرت ﮔﺮﻓﺘﻪ ﺑ ـﻪ ﺻـﻮرت ﮔﻠﻴــﺴﺮول V/V) %15) دردﻣــﺎي 80 -درﺟــﻪ ﺳــﺎﻧﺘﻲ ﮔــﺮاد ﻛﻠﻲ در ﺷﻜﻞ 1، آورده ﺷﺪه اﺳﺖ .
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