如上所述，於專利文獻1及2、以及非專利文獻3及4中記載之微生物能夠生產PGA。然而，由於具有抗腫瘤活性或脂質代謝控制活性之如分子量超過2,000,000之PGA為高黏性，故而於桿菌屬細菌或其近緣種中，難以效率良好地生產高分子量之PGA。 另一方面，為了效率良好地生產PGA，需要減輕生產製程之負荷。此處，作為減輕生產製程之負荷之手段之一，考慮降低培養基之黏度。若黏度降低，則於培養步驟中，氧或基質之擴散效率提昇，從而可期待生產效率之提昇。進而，於分離回收步驟中，亦可期待過濾或離心分離等之效率提昇。 作為降低培養基之黏度之方法，可列舉以高濃度含有培養基中所含之氯化鈉等鹽之方法。然而，如上所述，若於桿菌屬細菌或其近緣種中，培養基中所含之氯化鈉之濃度上升，則所生產之PGA一般而言會低分子量化、或生產性降低。 因此，於桿菌屬細菌或其近緣種中，於高鹽濃度條件下，如分子量超過300,000之PGA之生產目前仍較為困難。 因此，本發明係關於提供一種減輕對PGA之生產製程之負荷、且能夠生產高分子量之PGA的PGA之生產方法。 又，本發明係關於提供一種具有高鹽濃度耐性、及於高鹽濃度條件下之高分子量之PGA生產能力之枯草桿菌。 本發明者等人為了提供上述PGA之生產方法及枯草桿菌，進行了努力研究。結果發現具有高鹽濃度耐性、及於高鹽濃度條件下之高分子量之PGA生產能力之枯草桿菌。並且發現：於高鹽濃度條件下培養該枯草桿菌時，能夠實現高分子量之PGA生產，並且能夠減輕對PGA之生產製程之負荷。 本發明係基於該等見解而完成者。 本發明之枯草桿菌具有高鹽濃度耐性、及於高鹽濃度條件下之高分子量之PGA生產能力。 因此，藉由培養本發明之枯草桿菌，能夠生產高分子量之PGA。進而，藉由於高鹽濃度條件下培養本發明之枯草桿菌，可於對PGA之生產製程不施加負荷之情況下效率良好地生產PGA。 於本說明書中，所謂「納豆菌」，意指根據菌學性質、及16S rRNA基因之鹼基序列之分析結果，可分類為枯草桿菌、且具有PGA生產能力之微生物。 本發明之枯草桿菌具有高鹽濃度耐性、及於高鹽濃度條件下之高分子量之PGA生產能力。因此，本發明之枯草桿菌被分類於納豆菌。於高鹽濃度條件下，本發明之枯草桿菌生產之PGA之分子量大於先前之納豆菌生產之PGA。進而，本發明之枯草桿菌與具有PGA生產能力之先前之納豆菌相比，對高濃度之鹽之耐性優異。 藉由於適當之條件下培養本發明之枯草桿菌，能夠生產高分子量之PGA。尤其是藉由於高鹽濃度條件下培養本發明之枯草桿菌，能夠於對PGA之生產製程不施加負荷之情況下生產高分子量之PGA。 又，亦可藉由將所獲得之PGA進行低分子化而調整至所需之分子量。例如，藉由於適當之條件下培養本發明之枯草桿菌，可生產高分子量之PGA，將其於酸性條件下藉由加熱處理、或使用PGA分解酵素之處理等而將所獲得之PGA進行低分子量化，從而將PGA之分子量調整至所需範圍。此處，於本說明書記載之「分子量」與「重量平均分子量」含義相同。又，鹽濃度之標記「%(w/v)」或「M」、培養基成分濃度之標記「%(w/v)」或「(g/L)」、及PGA濃度之標記「(g/L)」均為於室溫下之濃度。 以下，對本發明詳細地進行說明。 本發明之枯草桿菌係具有高鹽濃度耐性之枯草桿菌，且其係於將氯化鈉濃度調整為12%(w/v)(相當於2.05 M，室溫)以上之LB培養基中能夠增殖之枯草桿菌。 如後述實施例亦所示般，具有PGA生產能力之公知之納豆菌不具有對高鹽濃度之耐性，於氯化鈉濃度12%(w/v)以上之LB培養基中無法增殖。相對於此，本發明之枯草桿菌具有高鹽濃度耐性，即便於氯化鈉濃度12%(w/v)以上之LB培養基中亦能夠增殖。本發明之枯草桿菌能夠增殖之氯化鈉濃度之上限值於使用TSB培養基之條件下為16～17%(w/v)(相當於2.74～2.91 M)，於使用LB培養基之條件下為15%(w/v)(相當於2.57 M)。 此處，所謂「於調整為氯化鈉濃度12%(w/v)以上之LB培養基中能夠增殖」，意指所接種之細胞數於氯化鈉濃度12%(w/v)以上之條件下藉由培養會增加。再者，於本說明書中，關於「增殖」，可測定培養前後之培養液之吸光度(OD600)，根據吸光度之增大而相對地算出。 進而，本發明之枯草桿菌具有高分子量之PGA生產能力。具體而言，於在氯化鈉濃度10%(w/v)(相當於1.71 M，室溫)之高鹽濃度條件下培養時，具有分子量為300,000以上之PGA生產能力。於在氯化鈉濃度為10%(w/v)之條件下培養時，本發明之枯草桿菌生產之PGA之分子量更佳為500,000以上，更佳為1,000,000以上，更佳為2,000,000以上，更佳為5,000,000以上，更佳為10,000,000以上。又，其上限值通常為50,000,000。 再者，所謂「於高鹽濃度條件下具有分子量為300,000以上之PGA生產能力」，具體係指於使用含有氯化鈉10%(w/v)且含有增殖所需之營養源、礦物質之培養基進行培養時，生產分子量為300,000以上之PGA至少0.1 g/L/3天以上，較佳為0.5 g/L/3天以上，更佳為1.0 g/L/3天以上，更佳為5.0 g/L/3天以上。又，於使用之培養基中，可含有成為PGA基質之麩胺酸，亦可不含有。進而，於利用含有麩胺酸鈉・一水合物8%(w/v)(相當於1.37 M)之培養基進行培養之情形時，較佳為可生產10 g/L以上之PGA，於不含有麩胺酸之情形時，較佳為可生產0.3 g/L以上之PGA。 本發明之枯草桿菌較佳為具有包含序列編號7或8所示之鹼基序列之16S rRNA基因。或者本發明之枯草桿菌較佳為具有包含與序列編號7或8所示之鹼基序列之同一性較佳為99.75%以上、更佳為99.85%以上、更佳為99.90%以上之鹼基序列的16S rRNA基因。或者本發明之枯草桿菌較佳為具有包含於序列編號7或8所示之鹼基序列中缺失、置換、插入或附加較佳為1～3個、更佳為1個鹼基之鹼基序列的16S rRNA基因。 此處，序列編號7所示之鹼基序列為枯草桿菌KSM-FFA610株所具有之16S rRNA基因之鹼基序列。又，序列編號8所示之鹼基序列為枯草桿菌KSM-FFB553株所具有之16S rRNA基因之鹼基序列。 此處，於本發明中，鹼基序列之同一性可使用位於公開之資料庫NCBI(美國生物技術資訊中心(National Center for Biotechnology Information)；http://www.ncbi.nlm.gov/)之選項單「Nucleotide」內之「BLAST」中之「Basic BLAST」而算出。或者亦可藉由使用Genetyx-Win(基因資訊處理軟體，GENETYX製造)之同源性分析程式，將單元大小(Unit size)(k-tuple)設為6進行分析，從而算出鹼基序列之同源性。 本發明之枯草桿菌較佳為具有下述表1所示之菌學性質。
本發明之枯草桿菌較佳為下述所示之枯草桿菌(1)或(2)。 (1)表現出表1記載之菌學性質、且具有16S rRNA基因的枯草桿菌，該16S rRNA基因包含序列編號7所示之鹼基序列、與序列編號7所示之鹼基序列之同一性較佳為99.75%以上、更佳為99.85%以上、更佳為99.90%以上之鹼基序列、或於序列編號7所示之鹼基序列中缺失、置換、插入或附加較佳為1～3個、更佳為1個鹼基之鹼基序列。 (2)表現出表1記載之菌學性質、且具有16S rRNA基因的枯草桿菌，該16S rRNA基因包含序列編號8所示之鹼基序列、與序列編號8所示之鹼基序列之同一性較佳為99.75%以上、更佳為99.85%以上、更佳為99.90%以上之鹼基序列、或於序列編號8所示之鹼基序列中缺失、置換、插入或附加較佳為1～3個、更佳為1個鹼基之鹼基序列。 於本發明之枯草桿菌中，枯草桿菌KSM-FFA610株於2016年6月2日以寄存編號NITE BP-02276寄存於獨立行政法人製品評價技術基盤機構 特許微生物寄託中心(千葉縣木更津市上總鎌足2-5-8)。關於枯草桿菌KSM-FFA610株，本發明者等人於2012年6月左右於申請人之實驗室(日本栃木縣)獲取。 又，枯草桿菌KSM-FFA631株於2016年6月2日以寄存編號NITE BP-02277寄存於獨立行政法人製品評價技術基盤機構 特許微生物寄託中心(千葉縣木更津市上總鎌足2-5-8)。關於枯草桿菌KSM-FFA631株，本發明者等人於2012年6月左右於申請人之實驗室(日本栃木縣)獲取。 又，枯草桿菌KSM-FFB406株於2016年6月2日以寄存編號NITE BP-02278寄存於獨立行政法人製品評價技術基盤機構 特許微生物寄託中心(千葉縣木更津市上總鎌足2-5-8)。關於枯草桿菌KSM-FFB406株，本發明者等人於2013年6月左右於申請人之實驗室(日本栃木縣)獲取。 又，枯草桿菌KSM-FFB425株於2016年6月2日以寄存編號NITE BP-02279寄存於獨立行政法人製品評價技術基盤機構 特許微生物寄託中心(千葉縣木更津市上總鎌足2-5-8)。關於枯草桿菌KSM-FFB425株，本發明者等人於2013年6月左右於申請人之實驗室(日本栃木縣)獲取。 又，枯草桿菌KSM-FFB540株於2016年6月2日以寄存編號NITE BP-02280寄存於獨立行政法人製品評價技術基盤機構 特許微生物寄託中心(千葉縣木更津市上總鎌足2-5-8)。關於枯草桿菌KSM-FFB540株，本發明者等人於2013年7月左右於申請人之實驗室(日本栃木縣)獲取。 進而，枯草桿菌KSM-FFB553株於2016年6月2日以寄存編號NITE BP-02281寄存於獨立行政法人製品評價技術基盤機構 特許微生物寄託中心(千葉縣木更津市上總鎌足2-5-8)。關於枯草桿菌KSM-FFB553株，本發明者等人於2013年7月左右於申請人之實驗室(日本栃木縣)獲取。 再者，本發明之枯草桿菌均為野生型微生物，分類於枯草桿菌，且具有作為納豆菌特徵之PGA生產能力。 本發明之枯草桿菌可藉由以下方法、及將該等方法組合而進行，從而進行單離而獲取。 具體而言，藉由將市售之食品試樣、或土壤等環境試樣懸浮於生理鹽水，並將其塗抹於瓊脂培養基供於靜置培養，可獲取出現於瓊脂培養基上之微生物。再者，於醫藥品或食品用PGA中，較佳為將其分離源作為食品試樣。微生物之純化可列舉如下方法等：將瓊脂培養基上之微生物畫線塗抹於新的瓊脂培養基、或使用生理鹽水等適當之稀釋液將上述懸浮試樣稀釋之後塗抹於瓊脂培養基上，藉此使單一菌落出現。 作為效率良好地獲取枯草桿菌之方法，可列舉：因枯草桿菌為芽孢菌而預先將上述試樣進行熱處理之方法、利用糖等營養源之合成代謝性之差異之方法、確認菌落周圍之黏性物質之生產之方法等。又，作為具有高鹽濃度耐性之微生物之獲取方法，可列舉預先利用含有高濃度之鹽之瓊脂培養基將微生物進行單離之方法、利用含有高濃度之鹽之液體培養基選擇表現出良好之生長之微生物之方法等。 進而又，作為獲取PGA生產中無需麩胺酸之微生物之方法，可列舉：於未添加麩胺酸之瓊脂培養基中獲取形成黏稠之菌落之微生物之方法；於使用未添加麩胺酸之液體培養基之培養中，於培養液中獲取生產高分子量之PGA之微生物之方法等。 本發明之PGA之生產方法係使用上述本發明之枯草桿菌而進行PGA之生產。 如上所述，本發明之枯草桿菌與先前之納豆菌相比，具有對高濃度鹽之耐性。因此，為了使用本發明之枯草桿菌生產PGA，可使用高於通常之鹽濃度之培養基。 一般而言，高分子之電解質於水溶液中成為高分子之離子，引起與抗衡離子之解離。由於因該解離會產生較強之靜電場，故而因該靜電力而使抗衡離子於周邊凝集。其結果為，產生抗衡離子活量之明顯之降低。又，假定高分子離子單鏈之形態主要受靜電相互作用支配，藉由鹽濃度之增加會產生明顯之收縮(川口正剛，高分子，53卷，p.716-718，2004年)。因此，於作為高分子電解質之PGA於水溶液中之行為中，亦可藉由使培養基中之鹽濃度上升而使水溶液之黏度降低。又，於黏性較高而流動性較低之液體中，溶存之氧分子之移動效率會降低，故而為了確保好氧微生物之生長所需要之氧供給能，而必須進行更多之通氣攪拌。又，假定於伴隨此種較多之通氣攪拌之使用醱酵槽之培養系中，於培養液之黏度較高之情形時產生發泡，而使培養變得困難。進而又，假定流動性較低之液體試樣於製造步驟之移行效率較差，尤其是藉由離心分離之菌體去除、微濾、或超過濾等膜處理中之透過性明顯降低。因此，根據本發明之PGA之生產方法，能夠降低於水溶液中表現高黏性之高分子電解質即PGA之製造製程之負荷。 於使用本發明之枯草桿菌生產PGA時，於適當之培養基中培養本發明之枯草桿菌，並自培養基將生產於菌體外之PGA回收。 作為培養基，可使用包含甘油、葡萄糖、果糖、麥芽糖、蔗糖、木糖、甘露糖、半乳糖、澱粉等糖類作為用以生產PGA之碳源的培養基。又，可使用包含檸檬酸、乙酸等各種有機酸或其鹽、以及麩胺酸或其鹽等作為用以生產PGA之碳源的培養基。 於本發明之PGA之生產方法中，作為用以生產PGA之碳源，可使用上述碳源中之1種，亦可將2種以上組合使用。 於本發明之PGA之生產方法所使用之培養基中，亦可視需要含有各種大豆蛋白質等天然物、胺基酸、聚蛋白腖、胰蛋白腖、氯化銨、硫酸銨、硝酸銨或脲等氮源等。作為本發明中可使用之氮源，可使用上述氮源中之1種，亦可將2種以上組合使用。 本發明中使用之培養基可為合成培養基，亦可為天然培養基。 就使PGA之生產性進一步提昇之觀點而言，可於上述培養基中添加麩胺酸或其鹽。 培養基中之麩胺酸或其鹽之濃度可適當設定。例如，培養基中之麩胺酸或其鹽之濃度(麩胺酸換算)較佳為0.005 g/L以上較佳，更佳為0.05 g/L以上，更佳為0.1 g/L以上，更佳為0.5 g/L以上。又，關於其上限值，就避免培養基中之麩胺酸或其他培養基成分析出之觀點而言，較佳為600 g/L以下，更佳為500 g/L以下，更佳為400 g/L以下，更佳為300 g/L以下。 本發明之枯草桿菌即便於不存在麩胺酸之情況下，亦能夠將無機之氮源、及葡萄糖、甘油等除麩胺酸以外之物質作為碳源進行PGA之生產。 麩胺酸可藉由以生質為原料之醱酵法進行生產，可作為食品素材、或飼料而利用。認為此種於原料中不使用麩胺酸而能夠效率良好地生產有用之高分子素材即PGA的微生物就避免與糧食之競爭等觀點、或工業生產成本之觀點而言亦有益。 因此，利用不含麩胺酸而含有除麩胺酸以外之低價之氮源、及碳源之培養基培養本發明之枯草桿菌而生產PGA就不與糧食生產進行競爭、生產成本之觀點而言較佳。 培養基中所含之鹽之種類可適當設定。例如可列舉：作為1價之金屬鹽之氯化鈉、氯化鉀、或作為2價之金屬鹽之氯化鈣、氯化鎂、碳酸鈣、碳酸鎂、硫酸鈣、硫酸鎂等。其中，較佳為使用選自由氯化鈉、氯化鉀、氯化鈣、及氯化鎂所組成之群中之至少1種。 又，培養基中之鹽濃度可適當設定。 例如，於1價之金屬鹽之情形時，較佳為0.01 M以上，更佳為0.1 M以上，更佳為0.5 M以上，更佳為1.0 M以上。又，其上限值較佳為不阻礙細胞增殖或PGA生產之濃度，具體而言，更佳為2.5 M以下，更佳為2.0 M以下，更佳為1.75 M以下。 又，例如，於2價之金屬鹽之情形時，較佳為0.01 M以上，更佳為0.1 M以上，更佳為0.5 M以上，更佳為1.0 M以上。又，其上限值較佳為不阻礙細胞增殖或PGA生產之濃度，具體而言，較佳為2.0 M以下，更佳為1.75 M以下，更佳為1.5 M以下。 再者，使用之培養基、及培養後之培養液之黏度可藉由調整培養基中之鹽濃度而設為所需範圍。再者，於本發明中，培養基之黏度之測定方法可藉由適合非牛頓性液體之黏性測定之B型黏度計而進行。 上述枯草桿菌之培養條件可根據使用之枯草桿菌等適當選擇。具體而言，最佳溫度較佳為20℃以上，較佳為25℃以上，更佳為30℃以上。其上限值較佳為50℃，更佳為45℃，更佳為40℃。最佳pH值較佳為5以上，較佳為5.5以上，更佳為6.5以上。其上限值較佳為8，更佳為7.5，更佳為7。 又，培養時間為種菌接種後0.5天以上，較佳為1天以上，更佳為3天以上。培養方法並無特別限制，可列舉振盪培養、攪拌培養、通氣培養、靜置培養等。 將培養基中所累積之PGA進行回收時，必須去除已生產PGA之枯草桿菌之菌體。去除菌體之方法並無特別限制，可列舉：離心分離法、使用微濾或超過濾膜之去除法、使用絮凝劑之沈澱去除、透析法等。又，亦可將該等方法適當地組合而使用。 又，自培養液分離PGA之方法亦並無特別限制，可利用將所生產之物質進行單離、回收時所使用之通常之方法而進行。例如，藉由丙酮、甲醇、或乙醇等有機溶劑之沈澱、利用凝膠過濾管柱或離子交換管柱之層析區分、進而利用將pH值調整於PGA之等電點附近之酸沈澱之分離、電透析法等，可將目標PGA進行單離、回收。 本發明之枯草桿菌即便於高鹽濃度之條件下亦具有優異之PGA生產性，且能夠生產高分子量之PGA。本發明之PGA之生產量較佳為每1 L培養基為0.1 g/3天以上，更佳為0.5 g/3天以上，更佳為1.0 g/3天以上，更佳為5.0 g/3天以上。 於納豆菌標準株中PGA之生產性降低之氯化鈉濃度7.3%(w/v)(相當於1.25 M)之條件下培養本發明之枯草桿菌之情形時，於含有成為PGA基質之麩胺酸鈉・一水合物8%(w/v)之條件下，較理想為每1 L培養基為10 g/3天以上之生產量。 又，於納豆菌標準株中生長變得困難之氯化鈉濃度10.2%(w/v)(1.75 M相當)下進行培養之情形時，於含有成為PGA基質之麩胺酸鈉・一水合物8%(w/v)之條件下，較理想為每1 L培養基為0.5 g/3天以上之生產量。進而又，於納豆菌標準株中不期待PGA之生產之氯化鈉濃度7.3%(w/v)之條件下，且於不存在成為PGA基質之麩胺酸之條件下培養本發明之枯草桿菌之情形時，較理想為每1 L培養基為0.1 g/3天以上之生產量。 本發明之枯草桿菌於鹽濃度未達0～10%(w/v)之條件下生產高分子量之PGA。又，本發明之枯草桿菌即便於鹽濃度為10%以上(w/v)之條件下亦能夠生產同等之高分子量之PGA。 於在氯化鈉濃度為10%(w/v)以上之條件下培養本發明之枯草桿菌之情形時所生產之PGA之分子量為300,000以上，較佳為500,000以上，更佳為1,000,000以上，更佳為2,000,000以上，更佳為5,000,000以上，更佳為10,000,000以上。又，其上限值為50,000,000，較佳為40,000,000，更佳為35,000,000。 由本發明所生產之PGA可使用於化妝品、醫藥品、食品、水質淨化劑、保水材料、增黏劑等各種用途。 尤其是本發明之枯草桿菌被分類於納豆菌。並且，本發明之枯草桿菌所生產之PGA之分子量高於其他微生物生產之PGA。因此，本發明之枯草桿菌生產之PGA可較佳地使用於具有抗腫瘤活性或脂質代謝控制活性之化妝品、醫藥品、食品等用途。 關於上述實施形態，本發明進而揭示以下方法及枯草桿菌。 ＜1＞一種PGA生產方法，其係培養由寄存編號NITE BP-02276、寄存編號NITE BP-02277、寄存編號NITE BP-02278、寄存編號NITE BP-02279、寄存編號NITE BP-02280、或寄存編號NITE BP-02281所特定之枯草桿菌而生產PGA。 ＜2＞如上述＜1＞記載之方法，其中上述枯草桿菌具有於調整為氯化鈉濃度12%(w/v)(相當於2.05 M，室溫)以上、較佳為12%(w/v)以上且16～17%(w/v)以下、更佳為12%(w/v)以上且15%(w/v)以下之LB培養基中能夠增殖之高鹽濃度耐性，且於在氯化鈉濃度10%(w/v)(相當於1.71 M，室溫)之條件下培養時，具有重量平均分子量為300,000以上之PGA生產能力。 ＜3＞如上述＜1＞或＜2＞項記載之方法，其中於在氯化鈉濃度為10%(w/v)之條件下培養時，上述枯草桿菌生產之PGA之重量平均分子量為300,000以上，較佳為500,000以上，更佳為1,000,000以上，更佳為2,000,000以上，更佳為5,000,000以上，更佳為10,000,000以上，且較佳為50,000,000以下。 ＜4＞如上述＜1＞至＜3＞中任一項記載之方法，其中於在氯化鈉濃度10%(w/v)以上之條件下培養上述枯草桿菌時，上述枯草桿菌生產PGA 0.1 g/L/3天以上，較佳為0.5 g/L/3天以上，更佳為1.0 g/L/3天以上，更佳為5.0 g/L/3天以上。 ＜5＞如上述＜1＞至＜4＞中任一項記載之方法，其中上述枯草桿菌具有包含序列編號7或8所示之鹼基序列、與序列編號7或8所示之鹼基序列之同一性較佳為99.75%以上、更佳為99.85%以上、更佳為99.90%以上之鹼基序列、或於序列編號7或8所示之鹼基序列中缺失、置換、插入或附加較佳為1～3個、更佳為1個鹼基之鹼基序列的16S rRNA基因。 ＜6＞如上述＜1＞至＜5＞中任一項記載之方法，其中上述枯草桿菌表現出上述表1記載之菌學性質。 ＜7＞如上述＜1＞至＜6＞中任一項記載之方法，其係利用含有選自由甘油、葡萄糖、果糖、麥芽糖、蔗糖、木糖、甘露糖、半乳糖、澱粉、檸檬酸或其鹽、乙酸或其鹽、以及麩胺酸或其鹽所組成之群中之至少1種、較佳為選自由甘油、葡萄糖、麥芽糖、以及麩胺酸或其鹽所組成之群中之至少1種作為碳源的培養基培養上述枯草桿菌。 ＜8＞如上述＜1＞至＜7＞中任一項記載之方法，其係利用含有麩胺酸或其鹽之培養基培養上述枯草桿菌。 ＜9＞如上述＜8＞記載之方法，其中上述培養基中之麩胺酸或其鹽之濃度為0.005 g/L以上，較佳為0.05 g/L以上，更佳為0.1 g/L以上，更佳為0.5 g/L以上，且為600 g/L以下，較佳為500 g/L以下，更佳為400 g/L以下，更佳為300 g/L以下。 ＜10＞如上述＜1＞至＜7＞中任一項記載之方法，其係於不存在麩胺酸之情況下培養上述枯草桿菌。 ＜11＞如上述＜1＞至＜10＞中任一項記載之方法，其係利用含有選自由氯化鈉、氯化鉀、氯化鈣、氯化鎂、碳酸鈣、碳酸鎂、硫酸鈣、及硫酸鎂所組成之群中之至少1種鹽、較佳為選自由氯化鈉、氯化鉀、氯化鈣、及氯化鎂所組成之群中之至少1種鹽之培養基培養上述枯草桿菌。 ＜12＞如上述＜11＞記載之方法，其中上述培養基中之上述鹽之濃度為0.01 M以上且2.5 M以下。 ＜13＞如上述＜11＞或＜12＞記載之方法，其中上述鹽為1價之金屬鹽，且上述培養基中之上述鹽之濃度為0.1 M以上，更佳為0.5 M以上，更佳為1.0 M以上，又，較佳為2.0 M以下，更佳為1.75 M以下。 ＜14＞如上述＜11＞或＜12＞記載之方法，其中上述鹽為2價之金屬鹽，且上述培養基中之上述鹽之濃度為0.1 M以上，更佳為0.5 M以上，更佳為1.0 M以上，又，較佳為2.0 M以下，更佳為1.75 M以下，更佳為1.5 M以下。 ＜15＞如上述＜1＞至＜14＞中任一項記載之方法，其中上述枯草桿菌之培養時間為0.5天以上，較佳為1天以上，更佳為3天以上。 ＜16＞如上述＜1＞至＜15＞中任一項記載之方法，其係培養上述枯草桿菌，並以每1 L培養基為0.1g/3天以上、較佳為0.5g/3天以上、更佳為1.0g/3天以上、更佳為5.0g/3天以上生產PGA。 ＜17＞如上述＜16＞記載之方法，其中生產之上述PGA之重量平均分子量為300,000以上，較佳為500,000以上，更佳為1,000,000以上，更佳為2,000,000以上，更佳為5,000,000以上，更佳為10,000,000以上，又，為50,000,000以下，較佳為40,000,000以下，更佳為35,000,000以下。 ＜18＞一種枯草桿菌，其由寄存編號NITE BP-02276、寄存編號NITE BP-02277、寄存編號NITE BP-02278、寄存編號NITE BP-02279、寄存編號NITE BP-02280、或寄存編號NITE BP-02281所特定。 ＜19＞如上述＜18＞項記載之枯草桿菌，其於調整為氯化鈉濃度12%(w/v)以上、較佳為12%(w/v)以上且17%(w/v)以下、更佳為12%(w/v)以上且15%(w/v)以下之LB培養基中能夠增殖，且於在氯化鈉濃度10%(w/v)之條件下培養時，具有重量平均分子量為300,000以上之PGA生產能力。 ＜20＞如上述＜18＞或＜19＞記載之枯草桿菌，其於在氯化鈉濃度為10%(w/v)之條件下培養時，生產重量平均分子量為300,000以上、較佳為500,000以上、更佳為1,000,000以上、更佳為2,000,000以上、更佳為5,000,000以上、更佳為10,000,000以上，又，較佳為50,000,000以下之PGA。 ＜21＞如上述＜18＞至＜20＞中任一項記載之枯草桿菌，其於在氯化鈉濃度10%(w/v)以上之條件下培養時，生產PGA 0.1 g/L/3天以上，較佳為0.5 g/L/3天以上，更佳為1.0 g/L/3天以上，更佳為5.0 g/L/3天以上。 ＜22＞如上述＜18＞至＜21＞中任一項記載之枯草桿菌，其具有包含序列編號7或8所示之鹼基序列、與序列編號7或8所示之鹼基序列之同一性較佳為99.75%以上、更佳為99.85%以上、最佳為99.90%以上之鹼基序列、或於序列編號7或8所示之鹼基序列中缺失、置換、插入或附加較佳為1～3個、更佳為1個鹼基之鹼基序列的16S rRNA基因。 ＜23＞如上述＜18＞至＜22＞中任一項記載之枯草桿菌，其表現出上述表1記載之菌學性質。 ＜24＞一種PGA之分子量調整方法，其係將藉由如上述＜1＞至＜17＞中任一項記載之方法所生產之PGA進行低分子化，而調整為所需分子量。 [實施例] 以下，基於實施例對本發明進一步詳細地進行說明，但本發明並不限定於此。又，對於未記載製造商之試劑，可使用通常可獲取之試劑。 此處，將本實施例所使用之引子之鹼基序列示於表2。
試驗例1 芽孢形成微生物之獲取方法 以無菌方式採集市售之醃菜、味噌、醱酵調味劑、或納豆等食品試樣約5 g並置於容積15 mL之錐形管(製品編碼352096，BD(Becton Dickinson)Falcon製造)中，對該試樣加入2倍重量之1%(w/v)氯化鈉水溶液(殺菌處理劑)。將該等壓抵至觸控混合機(MT-31型，Yamato Scientific製造)之振動面，使之以均勻混合之樣態懸浮，將試樣於80℃下供於10分鐘之加熱處理。繼而，將該等試樣利用1%(w/v)氯化鈉水溶液(殺菌處理劑)適當地階段性地進行稀釋，將各者塗抹於表3～6所示之微生物檢測用培養基(LB瓊脂培養基，將氯化鈉調整為終濃度10%之LB瓊脂培養基(LB＋10%NaCl培養基)、改良GAM瓊脂培養基(商品名：「NISSUI」，日水製藥製造)、及M＋Yex瓊脂培養基)。 將該等瓊脂培養基於30℃下供於2～5天之靜置培養，並觀察瓊脂培養基上之微生物之增殖及形態。繼而，選擇複數個出現於該等各瓊脂培養基上之單一菌落，於與成功確認增殖者相同之瓊脂培養基上進行畫線塗抹，將出現之單一菌落作為純化株。進而，利用相同之瓊脂培養基使該純化株增殖，使所獲得之菌體懸浮於含有20%(w/v)甘油之LB液體培養基中，並於-80℃下進行冷凍保存。
試驗例2 枯草桿菌之選擇方法(1) 將試驗例1中獲取之菌株(-80℃冷凍保存試樣)利用殺菌過之白金環(製品編碼254410，Nunc製造)畫線塗抹於LB瓊脂培養基。將該等於30℃下供於1天之靜置培養，並利用目視確認各菌株之生長。 繼而，使用預先滅菌過之牙籤將於LB瓊脂培養基上所生長之各菌株接種於M＋Yex瓊脂培養基，並將其於30℃下供於1天之靜置培養，利用目視進行各菌株之生長確認。 繼而，將於M＋Yex瓊脂培養基上所生長之菌株分別接種於表7所示之M/葡萄糖合成代謝檢定培養盤、及表8所示之M/塔格糖合成代謝檢定培養盤，並於37℃下供於1～3天之靜置培養。 本試驗例中，利用目視觀察合成代謝檢定培養盤上之生長，選擇於M/葡萄糖合成代謝檢定培養盤有作為生長指標之菌落形成、且於M/塔格糖合成代謝檢定培養盤中無菌落形成的菌株作為枯草桿菌候補株。
試驗例3 枯草桿菌之選擇方法(2) 將利用1 mM TE緩衝液(pH值8.0)將試驗例1所製備之甘油保存試樣稀釋至30倍而得者作為模板，使用表2所示之引子27f及引子1525r進行PCR(Polymerase Chain Reaction，聚合酶鏈反應)，使16S rRNA基因區域約1.5 kb之DNA片段擴增。DNA聚合酶使用TaKaRa LA Taq(TAKARA BIO製造)。於使模板DNA於95℃下改性5分鐘之後，以於95℃下1分鐘、於55℃下30秒鐘、於72℃下2分鐘作為1個循環並進行30個循環，進而於72℃下保持恆溫2分鐘。 針對所獲得之16S rRNA基因區域約1.5 kb之DNA片段，使用表2所示之引子27f確定550 bp之DNA鹼基序列。 再者，於序列(sequence)分析試樣之製備時，使用Big Dye Terminator v3.1循環測序套組(Cycle Sequencing Kit)(Applied Biosystems製造)，並根據隨附操作說明(protocol)進行試樣製備。於分析前之試樣精製時，使用Montage SEQ kit(MILLIPORE製造)。繼而，關於所製備之序列試樣，使用DNA序列分析儀(商品名：ABI 3100 Genetic Analyzer，Applied Biosystems製造)進行序列分析，確定鹼基序列。 序列之同源性檢索係使用位於公開資料庫NCBI(美國生物技術資訊中心(National Center for Biotechnology Information)；http://www.ncbi.nlm.gov/)之選項單「Nucleotide」內之「BLAST」中之「Basic BLAST」，自BLAST程式選擇「nucleotide blast」。於檢索對象之資料庫中指定「Reference genomic sequences(refseq_genomics)，於選擇程式中指定「Highly similar sequences(megablast)」，進行同源性檢索。 根據所獲得之結果，作為於本試驗例中同源性最高之菌株，判定為枯草桿菌，且選擇確定上述序列之550 bp和與其相當之枯草桿菌標準株(Bacillus subtilis
DSM 10株)之序列之同源性為98.9%以上之菌株作為枯草桿菌候補株。 試驗例4 於未添加麩胺酸之條件下生產PGA之枯草桿菌之選擇方法 利用殺菌過之白金環(製品編碼254410，Nunc製造)自試驗例2及3中推定為枯草桿菌之冷凍保存試樣採集冷凍菌體，將其接種於5 mL之LB液體培養基，於30℃下供於24小時之振盪培養。將其作為種菌培養液，於30 mL之未添加麩胺酸之PGA生產用培養基[培養基組成：7.5%葡萄糖、1.8%氯化銨、0.5%酵母萃取液、0.035%硫酸鎂・七水合物、0.005%硫酸錳・四-五水合物、100 mM之3-嗎啉基丙磺酸(3-Morpholinopropanesulfonic acid，利用氫氧化鉀調整為pH值7.0，同仁化學研究所製造)]上接種1%(v/v)，並將該培養基於37℃下供於72小時之振盪培養。 培養結束後，對培養液之上清液中所含之PGA利用下述測定例1所示之方法進行定量。其結果為，選擇於培養液上清液中檢測出源自PGA之具有UV210 nm之吸收之高分子物質之溶出組分之菌株作為於未添加麩胺酸之條件下能夠生產PGA之枯草桿菌候補株。 試驗例5 具有高鹽濃度耐性之枯草桿菌之選擇方法(1) 將於試驗例2及3中推定為枯草桿菌、進而於試驗例4中選作PGA生產枯草桿菌候補株之株之冷凍保存試樣、及利用與試驗例1相同之順序所製備之自獨立行政法人製品評價技術基盤機構所獲取之公知之納豆菌標準株(NBRC 16449株、NBRC 3336株、NBRC 3936株)之冷凍保存試樣以成為1×103
～1×104
cell/mL之方式接種於LB＋10%NaCl液體培養基，並於37℃下供於24小時之振盪培養。於該振盪培養之後，將培養液試樣利用1%(w/v)氯化鈉水溶液適當進行稀釋，並使用分光光度計(商品名：U-2900型；Hitachi High-Technologies製造)測定成為增殖指標之培養液之吸光度600 nm(OD600)。 其結果為，於納豆菌標準株中未見吸光度之增加。於本試驗條件中，選擇發現吸光度增加之菌株6株作為具有高鹽濃度耐性及於高鹽濃度條件下之高分子量之PGA生產能力之枯草桿菌株。 試驗例6 枯草桿菌候補株之生長極限鹽濃度之評價試驗法 將作為標準株之納豆菌NBRC 3336株、及枯草桿菌候補株利用與試驗例4相同之條件，使用LB液體培養基製備種菌培養。 繼而，製備將氯化鈉之終濃度設為10%(w/v)、12%(w/v)、13%(w/v)、14%(w/v)、15%(w/v)、及16%(w/v)之LB培養基，對其以初始之吸光度成為0.05之方式接種上述種菌培養液，並於37℃下供於2天之振盪培養。於該振盪培養中經時性地採集培養液，並將培養液試樣利用氯化鈉水溶液(與使用培養基相同濃度之氯化鈉水溶液)適當進行稀釋，使用分光光度計(U-2900型，Hitachi High-Technologies製造)測定成為增殖指標之培養液之吸光度600 nm(OD600)。 試驗例7 選擇之枯草桿菌之生長極限鹽濃度之確認試驗 將具有高鹽濃度耐性之枯草桿菌候補株使用LB＋10%NaCl液體培養基並利用與試驗例5相同之條件製備種菌培養。 繼而，對將氯化鈉之終濃度設為10%(w/v)、12%(w/v)、14%(w/v)、15%(w/v)、16%(w/v)、17%(w/v)、18%(w/v)、19%(w/v)、或20%(w/v)之TSB培養基(Trypticase Soy broth(胰蛋白酶大豆培養液)，Becton, and Dickinson Company製造)，以初始吸光度成為0.1之方式接種上述種菌培養液，並於37℃下供於2天之振盪培養。於進行振盪培養之後，於培養開始第2天採集培養液試樣，利用10%(w/v)氯化鈉水溶液適當進行稀釋，並使用分光光度計(U-2900型，Hitachi High-Technologies製造)測定成為增殖指標之培養液之吸光度600 nm(OD600)。 本試驗例中，將直至培養第2天之培養液之吸光度成為種菌培養接種時之2倍以上之鹽濃度條件判定為菌株之生長極限濃度。 試驗例8 選擇之枯草桿菌之生長最佳鹽濃度之確認試驗 將具有高鹽濃度耐性之枯草桿菌候補株使用LB＋10%NaCl液體培養基並利用與試驗例5相同之條件製備種菌培養。 繼而，製備不添加氯化鈉且終濃度設為1%(w/v)、2(w/v)、3(w/v)、4(w/v)、5(w/v)、6(w/v)、7%(w/v)、8%(w/v)、及10%(w/v)之TSB培養基，將其於96孔圓底微盤(型號3870-096，IWAKI製造)中各孔分注200 μL。對其以各孔之初始吸光度成為0.05之方式接種上述種菌培養液，並使用生物微盤讀取器(HiTS-S2型，SCINICS製造)，於37℃下供於24小時之振盪培養。 生物微盤讀取器係以150 rpm進行振盪，並藉由干涉濾光器以30分鐘間隔經時性地測定600 nm之吸光度(OD600)。根據所獲得之吸光度之值算出每單位時間之吸光度之增加，將其作為菌體增殖速度(ΔOD600/hr)，求出培養試驗中之最大菌體增殖速度。 本試驗例中，將菌體增殖速度(ΔOD600/hr)自最大值至(最大值－0.2)之鹽濃度判定為生長最佳鹽濃度。 實施例1 選擇之枯草桿菌之特徵 將利用試驗例1～8所示之方法獲取之具有高鹽濃度耐性且生產PGA之枯草桿菌株(枯草桿菌KSM-FFA610株、枯草桿菌KSM-FFA631株、枯草桿菌KSM-FFB406株、枯草桿菌KSM-FFB425株、枯草桿菌KSM-FFB540株、枯草桿菌KSM-FFB553株)之生長特性示於表9～13。
表9表示試驗例5之結果。 如表9所示，於利用含有高濃度之氯化鈉之LB液體培養基進行培養之情形時，作為對照之納豆菌標準株中，吸光度成為檢測極限以下，未確認到增殖。相對於此，於試驗例2～4所選擇之枯草桿菌株中，於試驗例5所示之試驗條件下發現有吸光度(OD600)超過0.5(相當於1×107
cell/mL)之菌株。 根據以上結果確認：本發明之枯草桿菌株為於納豆菌標準株無法增殖之鹽濃度下能夠增殖之高鹽濃度耐性株。
表10及11表示試驗例6之結果。 如表10所示，於培養第1天中，於氯化鈉終濃度10%(w/v)之條件下，作為對照之納豆菌標準株(NBRC 3336株)之吸光度為約0.5，相對於此，本發明之枯草桿菌株之吸光度均表示出超過2.0之數值。 又，於氯化鈉終濃度13%(w/v)之條件下，作為對照之納豆菌標準株未確認到菌體增殖，相對於此，本發明之枯草桿菌株之吸光度表現出超過0.5之數值。 根據以上結果確認：本發明之枯草桿菌候補株為具有高於納豆菌標準株之鹽濃度耐性之株。 如表11所示，於培養第2天中，於氯化鈉終濃度13%(w/v)之條件下，作為對照之納豆菌標準株未確認到菌體增殖，相對於此，本發明之枯草桿菌候補株之吸光度均表示出超過1.5之數值。 進而，本發明之枯草桿菌株於氯化鈉終濃度為14%(w/v)之條件下，吸光度表示出超過0.5之數值。 根據以上結果確認：本發明之枯草桿菌候補株為具有高於納豆菌標準株之鹽濃度耐性之株。
表12表示試驗例7之結果。 如表12所示，於使用TSB培養基之高鹽濃度生長試驗中，關於本發明之枯草桿菌株於培養第2天之吸光度，KSM-FFA631株及KSM-FFB406株於氯化鈉終濃度16%(w/v)之條件下、KSM-FFB425株、KSM-FFB540株及KSM-FFB553株於17%(w/v)之條件下、KSM-FFA610株於18%(w/v)之條件下為種菌培養接種時之2倍以上之值。 根據以上結果確認：本發明之枯草桿菌株於使用TSB培養基之生長極限鹽濃度之確認試驗中，氯化鈉之生長極限濃度為16～18%(w/v)。
表13表示試驗例8之結果。 如表13所示，本發明之枯草桿菌株於使用TSB培養基之生長最佳鹽濃度之試驗中，於氯化鈉濃度為無添加～終濃度6%(w/v)(室溫)之添加條件下，菌體增殖速度(ΔOD600/hr)為0.3～0.5之值。 根據以上結果確認：於本發明之枯草桿菌株之使用TSB培養基之選擇之枯草桿菌之生長最佳鹽濃度之確認試驗中，關於氯化鈉之生長最佳濃度，枯草桿菌KSM-FFA610株中為0～5%(w/v)，枯草桿菌KSM-FFA631株中為0～4%(w/v)，枯草桿菌KSM-FFB406株中為0～5%(w/v)，枯草桿菌KSM-FFB425株中為0～4%(w/v)，枯草桿菌KSM-FFB540株中為0～5%(w/v)，枯草桿菌KSM-FFB553株中為0～5%(w/v)。 實施例2 基於菌學、及16S rRNA基因之鹼基序列分析之菌種鑑定 對上述枯草桿菌株(枯草桿菌KSM-FFA610株、枯草桿菌KSM-FFA631株、枯草桿菌KSM-FFB406株、枯草桿菌KSM-FFB425株、枯草桿菌KSM-FFB540株、枯草桿菌KSM-FFB553株)之菌學性質進行了研究。將其結果示於表14。 進而，針對上述枯草桿菌株，藉由下述測定例4，基於16S rRNA基因之鹼基序列分析進行菌種鑑定。將其結果示於表15。
如表14所示，確認到上述枯草桿菌株全部具有枯草桿菌之菌學性質。
如表15所示，基於16S rRNA基因之鹼基序列之同源性分析之結果明確得知，上述菌株全部具有與枯草桿菌DSM 10株99.9%以上之同源性較高之16S rRNA基因之鹼基序列。 因此，關於上述枯草桿菌株，根據菌學性質、以及16S rRNA基因之鹼基序列之分析結果，判斷為枯草桿菌。 再者，枯草桿菌KSM-FFA610株於2016年6月2日以寄存編號NITE BP-02276寄存於獨立行政法人製品評價技術基盤機構 特許微生物寄託中心(千葉縣木更津市上總鎌足2-5-8)。 又，枯草桿菌KSM-FFA631株於2016年6月2日以寄存編號NITE BP-02277寄存於獨立行政法人製品評價技術基盤機構 特許微生物寄託中心(千葉縣木更津市上總鎌足2-5-8)。 又，枯草桿菌KSM-FFB406株於2016年6月2日以寄存編號NITE BP-02278寄存於獨立行政法人製品評價技術基盤機構 特許微生物寄託中心(千葉縣木更津市上總鎌足2-5-8)。 又，枯草桿菌KSM-FFB425株於2016年6月2日以寄存編號NITE BP-02279寄存於獨立行政法人製品評價技術基盤機構 特許微生物寄託中心(千葉縣木更津市上總鎌足2-5-8)。 又，枯草桿菌KSM-FFB540株於2016年6月2日以寄存編號NITE BP-02280寄存於獨立行政法人製品評價技術基盤機構 特許微生物寄託中心(千葉縣木更津市上總鎌足2-5-8)。 進而，枯草桿菌KSM-FFB553株於2016年6月2日以寄存編號NITE BP-02281寄存於獨立行政法人製品評價技術基盤機構 特許微生物寄託中心(千葉縣木更津市上總鎌足2-5-8)。 實施例3 於高濃度鹽添加條件下之PGA生產性評價(1) 以公知之納豆菌標準株(NBRC 3336株、及NBRC 16449株)作為對照，使用本發明之枯草桿菌株(KSM-FFA610株、KSM-FFA631株、KSM-FFB406株、KSM-FFB425株、KSM-FFB540株、及KSM-FFB553株)，對高鹽濃度條件下之PGA生產性進行評價。 使用試驗例1所示之上述菌株之冷凍保存試樣、及利用相同之順序製備之納豆菌標準株之冷凍保存試樣，以與試驗例4相同之培養條件使用LB液體培養基，於30℃下供於24小時之振盪培養。將其作為種菌培養液，於30 mL之PGA生產性評價培養基[培養基組成：8.0%葡萄糖、8.0%麩胺酸鈉・一水合物、1.25%酵母萃取液、1.0%硫酸銨、0.2%硫酸鎂・七水合物、0.003%硫酸錳・四-五水合物、0.7%磷酸氫二鉀、0.35%磷酸二氫鉀、以及7.3%氯化鈉(相當於1.25 M)或10.2%氯化鈉(相當於1.75 M)]上接種1%(v/v)。將該培養基於37℃下供於72小時之振盪培養。 培養結束後，利用下述測定例1記載之方法對培養液之上清液中所含之PGA進行定量。將其結果示於表16。
如表16所示，本發明之枯草桿菌株與納豆菌標準株相比，即便於高鹽濃度之條件下亦表現出優異之PGA生產性。又，即便於如納豆菌標準株無法生長而無法生產PGA之含有高濃度之氯化鈉之條件下，本發明之枯草桿菌株亦能夠生產PGA。 根據以上結果，判斷本發明之枯草桿菌為具有高鹽濃度耐性之枯草桿菌。 實施例4 於高濃度鹽添加條件下之PGA生產性評價(2) 使用本發明之枯草桿菌株KSM-FFB553株，於1價之金屬鹽為高濃度之條件下對PGA生產性進行評價。 藉由與試驗例4相同之方法製備種菌培養液，將其於30 mL之PGA生產性評價培養基[培養基組成：8.0%葡萄糖、8.0%麩胺酸鈉・一水合物、1.25%酵母萃取液、1.0%硫酸銨、0.2%硫酸鎂・七水合物、0.003%硫酸錳・四-五水合物、0.7%磷酸氫二鉀、0.35%磷酸二氫鉀、以及氯化鈉10.2%(相當於1.75 M)或氯化鉀11.2%(相當於1.5 M)]上接種1%(v/v)。將該等於37℃下供於72小時之振盪培養。 培養結束後，利用下述測定例1記載之方法對培養液上清液中所含之PGA進行定量。將其結果示於表17。
如表17所示，確認出本發明之枯草桿菌株即便於1價之金屬鹽為高濃度之條件下亦表現出優異之PGA生產性。 實施例5 於高濃度鹽添加條件下之PGA生產性評價(3) 使用本發明之枯草桿菌株KSM-FFB553株，於2價之金屬鹽為高濃度之條件下對PGA生產性進行評價。 藉由與試驗例4相同之方法製備種菌培養液，將其於30 mL之PGA生產性評價培養基[培養基組成：8.0%葡萄糖、8.0%麩胺酸鈉・一水合物、1.25%酵母萃取液、1.0%硫酸銨、0.2%硫酸鎂・七水合物、0.003%硫酸錳・四-五水合物、0.7%磷酸氫二鉀、0.35%磷酸二氫鉀、以及10.2%氯化鎂・六水合物(相當於0.5 M)或7.4%氯化鈣・二水合物(相當於0.5 M)]上接種1%(v/v)。將該等於37℃下供於72小時之振盪培養。 培養結束後，利用下述測定例1記載之方法對培養液之上清液中所含之PGA進行定量。將其結果示於表18。
如表18所示，確認出本發明之枯草桿菌株即便於2價之金屬鹽為高濃度之條件下亦表現出優異之PGA生產性。 實施例6 PGA之分子量之測定(1) 對本發明之枯草桿菌株(KSM-FFA610株、KSM-FFA631株、KSM-FFB406株、KSM-FFB425株、KSM-FFB540株、及KSM-FFB553株)所生產之PGA之分子量進行測定。 藉由與試驗例5相同之方法製備種菌培養液，將其於30 mL之生產性評價培養基[培養基組成：8.0%葡萄糖、8.0%麩胺酸鈉・一水合物、1.25%酵母萃取液、1.0%硫酸銨、0.2%硫酸鎂・七水合物、0.003%硫酸錳・四-五水合物、0.7%磷酸氫二鉀、0.35%磷酸二氫鉀、10.2%氯化鈉(相當於1.75 M)]上接種1%(v/v)。將該培養基於37℃下供於72小時之振盪培養。 培養結束後，利用下述測定例1記載之方法對培養液之上清液中所含之PGA之分子量進行測定。將其結果示於表19。
如表19所示，確認出本發明之枯草桿菌株即便於添加有如納豆菌標準株無法生長之高濃度之鹽之條件下，亦能夠生產高分子量之PGA。又，進而確認藉由使用本發明之高鹽濃度耐性株，能夠生產高分子量之PGA。 實施例7 PGA之分子量之測定(2) 使用實施例1所示之本發明之枯草桿菌株KSM-FFB553株，對於1價或2價之金屬鹽為高濃度之條件下生產之PGA之分子量進行評價。 藉由與試驗例4相同之方法製備種菌培養液，將其於30 mL之PGA生產性評價培養基[培養基組成：8.0%葡萄糖、8.0%麩胺酸鈉・一水合物、1.25%酵母萃取液、1.0%硫酸銨、0.2%硫酸鎂・七水合物、0.003%硫酸錳・四-五水合物、0.7%磷酸氫二鉀、0.35%磷酸二氫鉀、以及11.2%氯化鉀(相當於1.5 M)、10.2%氯化鎂・六水合物(相當於0.5 M)或7.4%氯化鈣・二水合物(相當於0.5 M)]上接種1%(v/v)。將其於37℃下供於72小時之振盪培養。 培養結束後，利用下述測定例1記載之方法對培養液之上清液中所含之PGA之分子量進行測定。將其結果示於表20。
如表20所示，確認出本發明之枯草桿菌株於1價或2價之金屬鹽為高濃度之條件下能夠生產高分子量之PGA。 實施例8 於麩胺酸未添加條件下之PGA生產性評價 使用本發明之枯草桿菌株(KSM-FFA610株、KSM-FFA631株、KSM-FFB406株、KSM-FFB425株、KSM-FFB540株、及KSM-FFB553株)，於高鹽濃度、且未添加麩胺酸之條件下對PGA生產性進行評價。 藉由與試驗例5相同之方法製備種菌培養液，將其於30 mL之PGA生產性評價培養基[培養基組成：8.0%甘油、0.5%酵母萃取液、1.0%硫酸銨、0.2%硫酸鎂・七水合物、0.003%硫酸錳・四-五水合物、0.7%磷酸氫二鉀、0.35%磷酸二氫鉀、7.3%氯化鈉(相當於1.25 M)]上接種1%(v/v)。將該培養基於37℃下供於72小時之振盪培養。 培養結束後，利用下述測定例1記載之方法對培養液之上清液中所含之PGA進行定量，並測定分子量。將其結果示於表21。
如表21所示，確認出本發明之枯草桿菌株即便於未添加麩胺酸之條件下亦能夠生產高分子量之PGA。 [測定例1]PGA之定量法、及分子量測定法 於PGA之定量、及分子量之測定時，使用高效液相層析裝置。 [HPLC裝置構成] 送液泵：L-6200型，日立製作所製造 自動取樣器：AS-4000型，日立製作所製造 管柱烘箱：L-5020型，日立製作所製造 UV檢測計：L-4250型，日立製作所製造 層析資料分析裝置：D-2500型，日立製作所 分析管柱使用排斥極限不同之親水性聚合物用凝膠過濾管柱TSKgel G6000PWXL(7.8 mm I.D.×30 cm，Tosoh製造)、及TSKgel G4000PWXL(7.8 mm I.D.×30 cm，Tosoh製造)。將該等串聯連接，並於分析管柱之正前面連接保護管柱TSK guardcolumn PWXL(6.0 mm I.D.×4.0 cm，Tosoh)而使用。 關於分析，將溶離液設為0.1 M硫酸鈉、流速1.0 mL/min、管柱溫度50℃，溶出波峰於檢測波長210 nm下進行測定。又，於樣品之預處理時，將經0.1 M硫酸鈉適當稀釋之培養液上清液試樣利用0.45 μm Durapore膜(型號MULTI SCREEN MNHV45，MILLIPORE製造)進行過濾器過濾。 於濃度檢定時，使用分子量880,000之PGA(Meiji Food Materia)製作校正曲線。又，於分子量檢定時，使用利用普魯蘭Shodex STANDARD P-82(昭和電工)預先求出重量平均分子量之各種分子量不同之聚麩胺酸(和光純藥工業162-21411及162-21401；SIGMA-ALDRICH P-4886及P-4761；Meiji Food Materia(分子量880,000))。 [測定例2]培養液上清液試樣中之高分子物質之鑑定方法 將於實施例4中所獲得之培養結束後之培養液試樣以14,800 rpm供於30分鐘之離心分離(himac CR21GIII型，日立工機製造)，並將經過菌體去除之上清液試樣回收。繼而，將該等上清液試樣1～10 mL移入聚丙烯製之容積50 mL離心管(型號227 261，greiner bio-one製造)，對該上清液試樣量加入2倍容積之乙醇進行倒置混合，其後於-30℃下恆溫放置一晩。其後，以3,000 rpm供於30分鐘之離心分離(himac CF7D2型，日立工機製造)，並回收沈澱組分。將所獲得之沈澱組分再次溶解於2 mL之蒸餾水中，再次製備上述藉由添加乙醇之沈澱組分，並將其回收。繼而，將所回收之試樣溶解於2 mL之蒸餾水中，並將其0.5 mL移入至螺旋蓋之試驗管(型號ST-13M，日本電子理化硝子製造)中，其後加入0.5 mL之濃鹽酸進行攪拌，其後封入氮氣，並於105～110℃下進行16小時加熱處理。加熱處理後，於氮氣氣流下蒸餾去除鹽酸及水分(約6小時)，並將所獲得之乾燥物作為水解試樣。 再者，作為PGA樣品，使用市售PGA(分子量880,000，Meiji Food Materia)，作為水解試樣之對照，使用L-麩胺酸(L-Glutamic acid)及D-麩胺酸(D-Glutamic acid)(和光純藥工業公司製造)。 繼而，將所獲得之水解試樣適當進行稀釋，利用全自動胺基酸分析裝置(L-8900型，Hitachi High-Technologies製造)進行試樣中之各種胺基酸分析、及麩胺酸之定量。又，使用L-麩胺酸測定套組(YAMASA醬油)，按照套組隨附之操作說明記載之方法，進行L-麩胺酸量之測定。藉由全自動胺基酸分析裝置之測定中，獲得光學活性異構物(D/L)之總量作為定量結果，將自該結果減去利用L-麩胺酸測定套組所獲得之定量結果而得之差量作為D-麩胺酸量。 關於測定之結果，自KSM-FFA610株、KSM-FFB425株、KSM-FFB540株、及KSM-FFB553株之培養液試樣所回收之高分子物質之麩胺酸之光學異構物比(D/L)分別為68/32、67/33、69/31、及67/33。 又，於上述藉由全自動胺基酸分析裝置之測定中，由於未檢測到除麩胺酸以外之胺基酸，故而培養上清液中之高分子物質判定為PGA。又，進而判斷：上述所選擇之本發明之具有高鹽濃度耐性之枯草桿菌株生產之PGA之D/L比與公知之納豆菌標準株生產之PGA之D/L比相等。 [測定例3]PGA溶液之黏度測定法 上述菌株中使用KSM-FFB553株，於PGA製備用培養基[培養基組成：8.0%葡萄糖、8.0%麩胺酸鈉・一水合物、0.5%酵母萃取液、1.0%硫酸銨、0.2%硫酸鎂・七水合物、0.003%硫酸錳・四-五水合物、0.7%磷酸氫二鉀、0.35%磷酸二氫鉀]中以1%(v/v)製備培養液試樣，由該培養液試樣，藉由利用酸沈澱之回收、繼而利用乙醇沈澱之精製回收、及冷凍乾燥而製備PGA乾燥粉末。繼而，將所獲得之PGA試樣(Mw5,000 k)以成為4%(w/w)、8%(w/w)之方式溶解於蒸餾水、及1.25 M氯化鈉水溶液中。將該等約40 mL以不產生氣泡之方式分別移入玻璃製螺旋管(型號No.7、或No.8，Maruemu製造)、或聚丙烯製之容積50 mL離心管(型號227 261，greiner bio-one製造)中，使用B型黏度計(TVB-15型，東機產業製造)，以試樣溫度20～25℃(室溫)、測定時間60秒(自動停止模式)、轉子旋轉速度60 rpm之條件下使用M2轉子進行測定。再者，關於上述測定條件下之測定值超過上限之試樣，將轉數適當變更為30 rpm、或將使用轉子適當變更為M3、M4進行測定。 關於測定之結果，於PGA4%(w/w)之試樣中，未添加鹽之試樣之黏度為380 mPa・s，相對於此，添加有鹽之試樣為60 mPa・s。又，於PGA8%(w/w)試樣中，未添加鹽之試樣之黏度為1,480 mPa・s，相對於此，添加有鹽之試樣為450 mPa・s。 根據上述測定結果，確認出藉由添加鹽之PGA試樣之黏度降低效果。 [測定例4]基於16S rRNA基因鹼基序列之菌種鑑定分析法 基於16S rRNA基因之鹼基序列之菌種鑑定係按照以下實驗順序而進行。 以與試驗例2相同之方式利用冷凍保存菌體製備PCR用模板試樣，使用表2所示之引子27f及引子1525r進行PCR，使16S rRNA基因區域約1.5 kb之DNA片段擴增。DNA聚合酶使用TaKaRa LA Taq(TAKARA BIO製造)。使模板DNA於95℃下改性5分鐘之後，以於95℃下1分鐘、於55℃下30秒鐘、於72℃下2分鐘作為1個循環並進行30個循環，進而於72℃下保持恆溫2分鐘。 針對所獲得之16S rRNA基因區域之DNA片段，使用表2所示之引子27f、引子f2L(-)、引子926f、引子rE1L、引子r2L'、及引子1525r分別作為序列用引子，進行DNA鹼基序列之分析。再者，於序列分析試樣之製備時，使用Big Dye Terminator v3.1循環測序套組(Cycle Sequencing Kit)(Applied Biosystems製造)，根據隨附操作說明進行試樣製備。於分析前之試樣精製時，使用Montage SEQ kit(MILLIPORE製造)。 關於所製備之序列試樣，使用DNA序列分析儀(商品名：ABI 3100 Genetic Analyzer，Applied Biosystems製造)進行序列分析，確定鹼基序列。 繼而，對所獲得之各鹼基序列使用GENETYX ATSQ ver2.01(GENETYX製造)進行單片段化。序列之同源性檢索係使用處於公開資料庫NCBI(美國生物技術資訊中心(National Center for Biotechnology Information)；http://www.ncbi.nlm.gov/)之選項單「Nucleotide」內之「BLAST」中之「Basic BLAST」，自BLAST程式選擇「nucleotide blast」。於檢索對象之資料庫中指定「Reference genomic sequences(refseq_genomics)，於選擇程式中指定「Highly similar sequences(megablast)」，選擇同源率最高之標準株。繼而，對於所選擇之基準株之16S rRNA基因序列及上述經過序列確定之具有高鹽濃度耐性之枯草桿菌候補株之16S rRNA基因序列，使用GENETYX Ver.13(GENETYX製造)，藉由「Nucleotide vs Nucleotide Homology」選項單實施鹼基序列對鹼基序列之同源性分析，算出同源性(%)。 對本發明以及其實施態樣一併進行了說明，但只要我等未特別指定，則於說明之任何細節部分均不欲對我等之發明進行限定，認為可於不違反隨附之申請專利範圍所示之發明之精神及範圍之情況下寬泛地進行解釋。 本申請案係主張基於2016年8月25日於日本提出專利申請之日本專利特願2016-165099之優先權，且係以其為參照將其內容作為本說明書之記載之一部分而引入本說明書中。As described above, the microorganisms described in Patent Documents 1 and 2 and Non-Patent Documents 3 and 4 can produce PGA. However, since PGA having an antitumor activity or a lipid metabolism controlling activity such as a molecular weight of more than 2,000,000 is highly viscous, it is difficult to efficiently produce a high molecular weight PGA in Bacillus bacteria or a related species thereof. On the other hand, in order to efficiently produce a PGA, it is necessary to reduce the load of the production process. Here, as one of means for reducing the load on the production process, it is considered to reduce the viscosity of the medium. If the viscosity is lowered, the diffusion efficiency of oxygen or a matrix is increased in the culturing step, and an increase in production efficiency can be expected. Further, in the separation and recovery step, efficiency improvement such as filtration or centrifugation can be expected. As a method of lowering the viscosity of the medium, a method of containing a salt such as sodium chloride contained in the medium at a high concentration can be mentioned. However, as described above, when the concentration of sodium chloride contained in the culture medium is increased in the Bacillus bacterium or its related species, the PGA produced generally has a low molecular weight or a decrease in productivity. Therefore, in the Bacillus bacterium or its related species, the production of PGA having a molecular weight of more than 300,000 is still difficult under high salt concentration conditions. Accordingly, the present invention is directed to a method of producing a PGA that reduces the load on the production process of a PGA and is capable of producing a high molecular weight PGA. Further, the present invention relates to a Bacillus subtilis which provides a high molecular weight PGA-producing ability with high salt concentration tolerance and high salt concentration. The inventors of the present invention have conducted intensive studies in order to provide the above-described production method of PGA and Bacillus subtilis. As a result, it was found that Bacillus subtilis having high salt concentration tolerance and high molecular weight PGA production ability under high salt concentration conditions. It has also been found that when the Bacillus subtilis is cultured under high salt concentration, high molecular weight PGA production can be achieved, and the load on the PGA production process can be reduced. The present invention has been completed based on these findings. The Bacillus subtilis of the present invention has high salt concentration tolerance and high molecular weight PGA production capacity under high salt concentration conditions. Therefore, a high molecular weight PGA can be produced by culturing the Bacillus subtilis of the present invention. Further, by culturing the Bacillus subtilis of the present invention under high salt concentration conditions, PGA can be efficiently produced without applying a load to the PGA production process. In the present specification, the term "Bacillus natto" means a microorganism which can be classified into Bacillus subtilis and has a PGA-producing ability based on the bacteriological property and the analysis result of the base sequence of the 16S rRNA gene. The Bacillus subtilis of the present invention has high salt concentration tolerance and high molecular weight PGA production capacity under high salt concentration conditions. Therefore, the Bacillus subtilis of the present invention is classified into natto. Under the condition of high salt concentration, the molecular weight of the PGA produced by the Bacillus subtilis of the present invention is greater than that of the PGA produced by the prior natto. Further, the Bacillus subtilis of the present invention is superior in resistance to a salt having a high concentration as compared with the prior natto bacteria having a PGA-producing ability. High molecular weight PGA can be produced by culturing the Bacillus subtilis of the present invention under appropriate conditions. In particular, by culturing the Bacillus subtilis of the present invention under high salt concentration conditions, it is possible to produce a high molecular weight PGA without applying a load to the PGA production process. Further, it is also possible to adjust to a desired molecular weight by lowering the obtained PGA. For example, by culturing the Bacillus subtilis of the present invention under appropriate conditions, a high molecular weight PGA can be produced, and the obtained PGA can be subjected to low molecular weight by heat treatment or treatment with PGA decomposing enzyme under acidic conditions. The molecular weight of the PGA is adjusted to the desired range. Here, the "molecular weight" and the "weight average molecular weight" described in the present specification have the same meanings. In addition, the salt concentration mark "% (w/v)" or "M", the medium component concentration mark "% (w/v)" or "(g/L)", and the PGA concentration mark "(g/ L)" is the concentration at room temperature. Hereinafter, the present invention will be described in detail. The Bacillus subtilis strain of the present invention has a high salt concentration tolerance Bacillus subtilis, and is capable of proliferating in an LB medium having a sodium chloride concentration adjusted to 12% (w/v) (corresponding to 2.05 M, room temperature) or more. Bacillus subtilis. As described in the examples below, the well-known natto bacteria having the PGA-producing ability do not have high salt concentration tolerance, and cannot proliferate in LB medium having a sodium chloride concentration of 12% (w/v) or more. On the other hand, the Bacillus subtilis of the present invention has high salt concentration tolerance and can proliferate even in an LB medium having a sodium chloride concentration of 12% (w/v) or more. The upper limit of the concentration of sodium chloride capable of proliferating by Bacillus subtilis of the present invention is 16 to 17% (w/v) (corresponding to 2.74 to 2.91 M) under the conditions of using TSB medium, and is under the condition of using LB medium. 15% (w/v) (equivalent to 2.57 M). Here, "proliferation in LB medium adjusted to a sodium chloride concentration of 12% (w/v) or more" means that the number of cells to be inoculated is greater than or equal to 12% (w/v) of sodium chloride concentration. The increase will be increased by cultivation. In the present specification, regarding "proliferation", the absorbance (OD600) of the culture solution before and after the culture can be measured, and the absorbance is relatively calculated based on the increase in absorbance. Further, the Bacillus subtilis of the present invention has a high molecular weight PGA production capacity. Specifically, when cultured under a high salt concentration condition of a sodium chloride concentration of 10% (w/v) (corresponding to 1.71 M, room temperature), the PGA has a molecular weight of 300,000 or more. When cultured under the condition of a sodium chloride concentration of 10% (w/v), the molecular weight of the PGA produced by the Bacillus subtilis of the present invention is more preferably 500,000 or more, more preferably 1,000,000 or more, still more preferably 2,000,000 or more, more preferably It is 5,000,000 or more, more preferably 10,000,000 or more. Also, the upper limit is usually 50,000,000. In addition, the term "the PGA production capacity having a molecular weight of 300,000 or more under high salt concentration conditions" means that the nutrient source and minerals required for proliferation are contained in 10% (w/v) containing sodium chloride. When the medium is cultured, the PGA having a molecular weight of 300,000 or more is produced at least 0.1 g/L/3 days or more, preferably 0.5 g/L/3 days or more, more preferably 1.0 g/L/3 days or more, more preferably 5.0. g/L/3 days or more. Further, the medium to be used may or may not contain glutamic acid which is a PGA matrix. Further, when culture is carried out using a medium containing 8% (w/v) of glutamate monohydrate (corresponding to 1.37 M), it is preferred to produce PGA of 10 g/L or more, without containing In the case of glutamic acid, it is preferred to produce a PGA of 0.3 g/L or more. The Bacillus subtilis of the present invention is preferably a 16S rRNA gene having a base sequence represented by SEQ ID NO: 7 or 8. Or the Bacillus subtilis of the present invention preferably has a base sequence having an identity with a nucleotide sequence represented by SEQ ID NO: 7 or 8, preferably 99.75% or more, more preferably 99.85% or more, still more preferably 99.90% or more. 16S rRNA gene. Or the Bacillus subtilis of the present invention preferably has a base sequence of 1 to 3, more preferably 1 base, which is deleted, substituted, inserted or affixed in the nucleotide sequence shown in SEQ ID NO: 7 or 8. 16S rRNA gene. Here, the nucleotide sequence shown in SEQ ID NO: 7 is the nucleotide sequence of the 16S rRNA gene of the Bacillus subtilis KSM-FFA610 strain. Further, the nucleotide sequence shown in SEQ ID NO: 8 is the nucleotide sequence of the 16S rRNA gene of the Bacillus subtilis KSM-FFB553 strain. Here, in the present invention, the identity of the base sequence can be used in the open database NCBI (National Center for Biotechnology Information; http://www.ncbi.nlm.gov/). It is calculated by "Basic BLAST" in "BLAST" in the menu "Nucleotide". Alternatively, the unit size (k-tuple) can be analyzed by using a homology analysis program of Genetyx-Win (gene information processing software, manufactured by GENETYX) to calculate the base sequence. Source. The Bacillus subtilis of the present invention preferably has the bacteriological properties shown in Table 1 below. The Bacillus subtilis of the present invention is preferably Bacillus subtilis (1) or (2) shown below. (1) Bacillus subtilis having the bacteriological property shown in Table 1 and having a 16S rRNA gene comprising the nucleotide sequence shown in SEQ ID NO: 7 and the nucleotide sequence sequence shown in SEQ ID NO: 7. Preferably, the base sequence of 99.75% or more, more preferably 99.85% or more, more preferably 99.90% or more, or the base sequence represented by SEQ ID NO: 7 is preferably 1 to 3 deleted, substituted, inserted or added. More preferably, it is a base sequence of 1 base. (2) Bacillus subtilis having the bacteriological property described in Table 1 and having a 16S rRNA gene comprising the nucleotide sequence of SEQ ID NO: 8 and the nucleotide sequence of SEQ ID NO: 8. Preferably, the base sequence of 99.75% or more, more preferably 99.85% or more, more preferably 99.90% or more, or the base sequence represented by SEQ ID NO: 8 is preferably 1 to 3 deleted, substituted, inserted or added. More preferably, it is a base sequence of 1 base. In the Bacillus subtilis of the present invention, the Bacillus subtilis KSM-FFA610 strain was deposited on the licensed microbiological center of the independent administrative agency product evaluation technology base institution on June 2, 2016 under the registration number NITE BP-02276 (Machiganjin City, Chiba Prefecture) Foot 2-5-8). The Bacillus subtilis KSM-FFA610 strain was obtained by the present inventors in the applicant's laboratory (Tochigi Prefecture, Japan) around June 2012. In addition, the Bacillus subtilis KSM-FFA631 strain was deposited on the licensed microbiological center of the independent administrative company's product evaluation technology base institution on June 2, 2016 (2-5-8, Kawasaki-shi, Chiba Prefecture). ). The Bacillus subtilis KSM-FFA631 strain was obtained by the present inventors in the applicant's laboratory (Tochigi Prefecture, Japan) around June 2012. In addition, the Bacillus subtilis KSM-FFB406 strain was deposited in the authorized microbial center of the independent administrative agency product evaluation technology base institution on June 2, 2016 (the number of the NAI BP-02278). ). The Bacillus subtilis KSM-FFB406 strain was obtained by the present inventors in the applicant's laboratory (Tochigi Prefecture, Japan) around June 2013. In addition, the Bacillus subtilis KSM-FFB425 strain was deposited on the licensed microbiological center of the independent administrative agency product evaluation technology base institution on June 2, 2016 (2-5-8, Kawasaki-shi, Chiba Prefecture). ). The Bacillus subtilis KSM-FFB425 strain was obtained by the present inventors in the applicant's laboratory (Tochigi Prefecture, Japan) around June 2013. In addition, the Bacillus subtilis KSM-FFB540 strain was deposited on the licensed microbiological center of the independent administrative company's product evaluation technology base institution on June 2, 2016 under the registration number NITE BP-02280 (2-5-8, Izumo, Izumi, Chiba Prefecture, Chiba Prefecture) ). The Bacillus subtilis KSM-FFB540 strain was obtained by the present inventors in the applicant's laboratory (Tochigi Prefecture, Japan) around July 2013. Furthermore, the Bacillus subtilis KSM-FFB553 strain was deposited on the licensed microbiological center of the independent administrative company's product evaluation technology base institution on June 2, 2016 under the registration number NITE BP-02281 (2-5-8, Izumo, Izumi, Chiba Prefecture, Chiba Prefecture) ). The Bacillus subtilis KSM-FFB553 strain was obtained by the present inventors in the applicant's laboratory (Tochigi Prefecture, Japan) around July 2013. Further, the Bacillus subtilis of the present invention is a wild type microorganism, is classified into Bacillus subtilis, and has a PGA production capacity characteristic of natto. The Bacillus subtilis of the present invention can be obtained by combining the following methods and combining the methods. Specifically, microorganisms appearing on an agar medium can be obtained by suspending a commercially available food sample or an environmental sample such as soil in physiological saline and applying it to an agar medium for static culture. Further, in the PGA for pharmaceuticals or foods, it is preferred to use the separation source as a food sample. The purification of the microorganism may be carried out by applying a microbial line on the agar medium to a new agar medium, or by diluting the suspension sample with a suitable diluent such as physiological saline, and then applying it to the agar medium, thereby making the single sample Colonies appear. Examples of the method for efficiently obtaining Bacillus subtilis include a method in which the sample is heat-treated in advance by Bacillus subtilis, a method of utilizing a difference in anabolic properties of a nutrient source such as sugar, and confirmation of viscosity around the colony. The method of production of substances, etc. Moreover, as a method of obtaining a microorganism having high salt concentration tolerance, a method of separating a microorganism by using an agar medium containing a salt having a high concentration in advance, and selecting a liquid medium containing a salt having a high concentration to exhibit good growth is selected. Microbial methods, etc. Further, as a method for obtaining a microorganism which does not require glutamic acid in the production of PGA, a method of obtaining a microorganism which forms a viscous colony in an agar medium to which glutamic acid is not added is used; and a liquid medium containing no glutamic acid is used. In the culture, a method of producing a microorganism having a high molecular weight PGA is obtained in a culture solution. The production method of the PGA of the present invention is carried out by using the above-described Bacillus subtilis of the present invention to produce PGA. As described above, the Bacillus subtilis of the present invention has resistance to a high concentration of salt as compared with the prior natto. Therefore, in order to produce PGA using the Bacillus subtilis of the present invention, a medium higher than the usual salt concentration can be used. In general, a polymer electrolyte becomes a polymer ion in an aqueous solution, causing dissociation from a counter ion. Since a strong electrostatic field is generated by the dissociation, the counter ion is agglomerated around the periphery due to the electrostatic force. As a result, a significant reduction in the counter ion activity is produced. Further, it is assumed that the form of the single-chain of the polymer ion is mainly dominated by the electrostatic interaction, and the contraction of the polymer ion causes a significant shrinkage (Kawaguchi Masahiro, Polymer, Vol. 53, p. 716-718, 2004). Therefore, in the behavior of PGA as a polymer electrolyte in an aqueous solution, the viscosity of the aqueous solution can be lowered by increasing the salt concentration in the medium. Further, in a liquid having a high viscosity and a low fluidity, the efficiency of movement of dissolved oxygen molecules is lowered. Therefore, in order to secure the oxygen supply energy required for the growth of aerobic microorganisms, more aeration stirring must be performed. Further, it is assumed that in the culture system using the fermentation tank with such a large amount of aeration and agitation, foaming occurs when the viscosity of the culture solution is high, and the culture becomes difficult. Further, it is assumed that the liquid sample having a low fluidity is inferior in the production process, and in particular, the permeability in the film treatment by centrifugation, microfiltration, or ultrafiltration is remarkably lowered. Therefore, according to the production method of the PGA of the present invention, it is possible to reduce the load of the manufacturing process of the PGA which is a polymer electrolyte which exhibits high viscosity in an aqueous solution. When the PGA is produced using the Bacillus subtilis of the present invention, the Bacillus subtilis of the present invention is cultured in a suitable medium, and the PGA produced outside the bacteria is recovered from the culture medium. As the medium, a medium containing glycerin, glucose, fructose, maltose, sucrose, xylose, mannose, galactose, starch or the like as a carbon source for producing PGA can be used. Further, various organic acids such as citric acid and acetic acid or salts thereof, and glutamic acid or a salt thereof can be used as a medium for producing a carbon source of PGA. In the production method of the PGA of the present invention, one type of the above-mentioned carbon sources may be used as the carbon source for producing the PGA, or two or more types may be used in combination. In the medium used in the production method of the PGA of the present invention, various natural substances such as soybean protein, amino acid, polypeptone, tryptone, ammonium chloride, ammonium sulfate, ammonium nitrate or urea may be contained as needed. . As the nitrogen source which can be used in the present invention, one of the above-mentioned nitrogen sources may be used, or two or more types may be used in combination. The medium used in the present invention may be a synthetic medium or a natural medium. From the viewpoint of further improving the productivity of PGA, glutamic acid or a salt thereof can be added to the above medium. The concentration of glutamic acid or a salt thereof in the medium can be appropriately set. For example, the concentration of glutamic acid or a salt thereof in the medium (calculated as glutamic acid) is preferably 0.005 g/L or more, more preferably 0.05 g/L or more, still more preferably 0.1 g/L or more, more preferably It is 0.5 g/L or more. Further, the upper limit thereof is preferably 600 g/L or less, more preferably 500 g/L or less, still more preferably 400 g, from the viewpoint of avoiding analysis of glutamic acid or other medium in the medium. Below /L, it is preferably less than 300 g/L. The Bacillus subtilis of the present invention can produce PGA by using an inorganic nitrogen source and a substance other than glutamic acid such as glucose or glycerin as a carbon source even in the absence of glutamic acid. The glutamic acid can be produced by a fermentation method using biomass as a raw material, and can be used as a food material or a feed. It is considered that such a microorganism which can efficiently produce a useful polymer material, that is, PGA, which does not use glutamic acid as a raw material, is advantageous in terms of avoiding competition with food, or industrial production cost. Therefore, the production of PGA by the cultivation of the Bacillus subtilis of the present invention using a medium containing no glutamic acid and a low-cost nitrogen source other than glutamic acid and a carbon source does not compete with food production and production costs. Preferably. The type of the salt contained in the medium can be appropriately set. For example, sodium chloride, potassium chloride, or a divalent metal salt, calcium chloride, magnesium chloride, calcium carbonate, magnesium carbonate, calcium sulfate, magnesium sulfate, or the like may be mentioned as the monovalent metal salt. Among them, at least one selected from the group consisting of sodium chloride, potassium chloride, calcium chloride, and magnesium chloride is preferably used. Further, the salt concentration in the medium can be appropriately set. For example, in the case of a monovalent metal salt, it is preferably 0.01 M or more, more preferably 0.1 M or more, still more preferably 0.5 M or more, still more preferably 1.0 M or more. Further, the upper limit is preferably a concentration which does not inhibit cell proliferation or PGA production, and is more preferably 2.5 M or less, more preferably 2.0 M or less, still more preferably 1.75 M or less. Further, for example, in the case of a divalent metal salt, it is preferably 0.01 M or more, more preferably 0.1 M or more, still more preferably 0.5 M or more, and still more preferably 1.0 M or more. Further, the upper limit thereof is preferably a concentration which does not inhibit cell proliferation or PGA production, and specifically, is preferably 2.0 M or less, more preferably 1.75 M or less, still more preferably 1.5 M or less. Further, the medium used and the viscosity of the culture solution after the culture can be set to a desired range by adjusting the salt concentration in the medium. Further, in the present invention, the method for measuring the viscosity of the medium can be carried out by a B-type viscometer suitable for the viscosity measurement of a non-Newtonian liquid. The culture conditions of the above Bacillus subtilis can be appropriately selected depending on the Bacillus subtilis used or the like. Specifically, the optimum temperature is preferably 20 ° C or higher, preferably 25 ° C or higher, more preferably 30 ° C or higher. The upper limit thereof is preferably 50 ° C, more preferably 45 ° C, still more preferably 40 ° C. The optimum pH is preferably 5 or more, preferably 5.5 or more, more preferably 6.5 or more. The upper limit thereof is preferably 8, more preferably 7.5, still more preferably 7. Further, the culture time is 0.5 days or more after inoculation of the inoculum, preferably 1 day or longer, more preferably 3 days or longer. The culture method is not particularly limited, and examples thereof include shaking culture, stirring culture, aeration culture, and static culture. When the PGA accumulated in the medium is recovered, it is necessary to remove the cells of the BGA-producing Bacillus subtilis. The method for removing the cells is not particularly limited, and examples thereof include a centrifugal separation method, a removal method using a microfiltration or an ultrafiltration membrane, a precipitation removal using a flocculant, a dialysis method, and the like. Further, these methods may be used in combination as appropriate. Further, the method for separating the PGA from the culture solution is not particularly limited, and can be carried out by a usual method used for separating and recovering the produced material. For example, separation by an organic solvent such as acetone, methanol, or ethanol, separation by chromatography using a gel filtration column or an ion exchange column, and further separation of an acid precipitate by adjusting the pH to the isoelectric point of the PGA. , electrodialysis, etc., the target PGA can be separated and recovered. The Bacillus subtilis of the present invention has excellent PGA productivity even under conditions of high salt concentration, and is capable of producing a high molecular weight PGA. The production amount of the PGA of the present invention is preferably 0.1 g/3 days or more per 1 L of the medium, more preferably 0.5 g/3 days or more, still more preferably 1.0 g/3 days or more, more preferably 5.0 g/3 days. the above. When the Bacillus subtilis of the present invention is cultured under the condition that the productivity of PGA is reduced by 7.3% (w/v) (corresponding to 1.25 M) in the natto standard strain, the glutamine containing the PGA matrix is contained. Under the conditions of sodium octahydrate monohydrate (w/v), it is preferred to produce 10 g/3 days or more per 1 L of the medium. In the case where the sodium glutamate standard strain is grown at a concentration of 10.2% (w/v) (1.75 M equivalent), which is difficult to grow, the sodium glutamate monohydrate which is a PGA matrix is contained. Under the condition of 8% (w/v), it is desirable to produce 0.5 g/3 days or more per 1 L of the medium. Further, the Bacillus subtilis of the present invention is cultured under the condition that the sodium chloride concentration of the production of PGA is not expected to be 7.3% (w/v) in the natto standard strain, and in the absence of glutamic acid which is a PGA matrix. In the case of the case, it is preferably 0.1 g/3 days or more per 1 L of the medium. The Bacillus subtilis of the present invention produces a high molecular weight PGA at a salt concentration of less than 0 to 10% (w/v). Further, the Bacillus subtilis of the present invention can produce an equivalent high molecular weight PGA even under a salt concentration of 10% or more (w/v). When the Bacillus subtilis of the present invention is cultured under the condition that the sodium chloride concentration is 10% (w/v) or more, the molecular weight of the PGA produced is 300,000 or more, preferably 500,000 or more, more preferably 1,000,000 or more. Preferably, it is 2,000,000 or more, more preferably 5,000,000 or more, and still more preferably 10,000,000 or more. Further, the upper limit is 50,000,000, preferably 40,000,000, more preferably 35,000,000. The PGA produced by the present invention can be used for various purposes such as cosmetics, pharmaceuticals, foods, water purification agents, water retention materials, and tackifiers. In particular, the Bacillus subtilis of the present invention is classified into natto. Further, the PGA produced by the Bacillus subtilis of the present invention has a higher molecular weight than the PGA produced by other microorganisms. Therefore, the PGA produced by the Bacillus subtilis of the present invention can be preferably used for cosmetics, pharmaceuticals, foods and the like having antitumor activity or lipid metabolism control activity. In the above embodiment, the present invention further discloses the following methods and Bacillus subtilis. <1> A PGA production method, which is constructed by a registration number NITE BP-02276, a registration number NITE BP-02277, a registration number NITE BP-02278, a registration number NITE BP-02279, a registration number NITE BP-02280, or a registration number. PGA is produced by Bacillus subtilis specific to NITE BP-02281. <2> The method according to the above <1>, wherein the Bacillus subtilis is adjusted to have a sodium chloride concentration of 12% (w/v) or more (corresponding to 2.05 M, room temperature) or more, preferably 12% (w/ v) high salt concentration tolerance capable of proliferating in LB medium of 16% or more (w/v) or less, more preferably 12% (w/v) or more and 15% (w/v) or less, and When cultured under the conditions of a sodium chloride concentration of 10% (w/v) (corresponding to 1.71 M, room temperature), the PGA has a weight average molecular weight of 300,000 or more. <3> The method according to the above <1> or <2> wherein the PGA produced by the Bacillus subtilis has a weight average molecular weight of 300,000 when cultured under a sodium chloride concentration of 10% (w/v). The above is preferably 500,000 or more, more preferably 1,000,000 or more, still more preferably 2,000,000 or more, still more preferably 5,000,000 or more, still more preferably 10,000,000 or more, and more preferably 50,000,000 or less. The method according to any one of the above aspects, wherein the Bacillus subtilis produces PGA 0.1 when the Bacillus subtilis is cultured under conditions of a sodium chloride concentration of 10% (w/v) or more. g/L/3 days or more, preferably 0.5 g/L/3 days or more, more preferably 1.0 g/L/3 days or more, more preferably 5.0 g/L/3 days or more. The method according to any one of the above aspects, wherein the Bacillus subtilis has a base sequence comprising SEQ ID NO: 7 or 8, and a base sequence represented by SEQ ID NO: 7 or 8. The identity is preferably 99.75% or more, more preferably 99.85% or more, more preferably 99.90% or more, or a deletion, substitution, insertion or addition in the nucleotide sequence shown in SEQ ID NO: 7 or 8. Preferably, the 16S rRNA gene is preferably 1 to 3, more preferably 1 base base sequence. The method according to any one of the above aspects, wherein the Bacillus subtilis exhibits the bacteriological properties described in Table 1 above. The method according to any one of the items <1> to <6> wherein the method comprises using a substance selected from the group consisting of glycerin, glucose, fructose, maltose, sucrose, xylose, mannose, galactose, starch, citric acid or At least one selected from the group consisting of a salt, acetic acid or a salt thereof, and glutamic acid or a salt thereof, preferably at least one selected from the group consisting of glycerin, glucose, maltose, and glutamic acid or a salt thereof The above B. subtilis was cultured in a medium as a carbon source. The method of any one of the above-mentioned <1> to <7>, wherein the Bacillus subtilis is cultured in a medium containing glutamic acid or a salt thereof. <9> The method according to the above <8>, wherein the concentration of the glutamic acid or a salt thereof in the medium is 0.005 g/L or more, preferably 0.05 g/L or more, more preferably 0.1 g/L or more. More preferably, it is 0.5 g/L or more, and is 600 g/L or less, preferably 500 g/L or less, more preferably 400 g/L or less, still more preferably 300 g/L or less. The method according to any one of the items <1> to <7>, wherein the Bacillus subtilis is cultured in the absence of glutamic acid. The method of any one of the above-mentioned <1> to <10> which is selected from the group consisting of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, calcium carbonate, magnesium carbonate, calcium sulfate, and The Bacillus subtilis is cultured in at least one salt of the group consisting of magnesium sulfate, preferably selected from the group consisting of at least one salt of a group consisting of sodium chloride, potassium chloride, calcium chloride, and magnesium chloride. <12> The method according to the above <11>, wherein the concentration of the salt in the medium is 0.01 M or more and 2.5 M or less. The method according to the above <11>, wherein the salt is a monovalent metal salt, and the concentration of the salt in the medium is 0.1 M or more, more preferably 0.5 M or more, more preferably 1.0 M or more, more preferably 2.0 M or less, more preferably 1.75 M or less. The method according to the above <11>, wherein the salt is a divalent metal salt, and the concentration of the salt in the medium is 0.1 M or more, more preferably 0.5 M or more, more preferably 1.0 M or more, more preferably 2.0 M or less, more preferably 1.75 M or less, still more preferably 1.5 M or less. The method of any one of the above-mentioned <1> to <14>, wherein the culture time of the Bacillus subtilis is 0.5 days or longer, preferably 1 day or longer, more preferably 3 days or longer. The method according to any one of the above aspects, wherein the Bacillus subtilis is cultured in an amount of 0.1 g/3 days or more, preferably 0.5 g/3 days or more per 1 L of the medium. More preferably, the PGA is produced in an amount of 1.0 g/3 days or more, more preferably 5.0 g/3 days or more. <17> The method according to the above <16>, wherein the PGA produced has a weight average molecular weight of 300,000 or more, preferably 500,000 or more, more preferably 1,000,000 or more, still more preferably 2,000,000 or more, still more preferably 5,000,000 or more, more Preferably, it is 10,000,000 or more, and further, it is 50,000,000 or less, preferably 40,000,000 or less, more preferably 35,000,000 or less. <18> A Bacillus subtilis, which is registered number NITE BP-02276, registered number NITE BP-02277, registered number NITE BP-02278, registered number NITE BP-02279, registered number NITE BP-02280, or registered number NITE BP- 02281 is specific. <19> The Bacillus subtilis described in the above <18>, which is adjusted to have a sodium chloride concentration of 12% (w/v) or more, preferably 12% (w/v) or more and 17% (w/v). The following, more preferably 12% (w/v) or more and 15% (w/v) or less of LB medium can be proliferated, and when cultured under the conditions of a sodium chloride concentration of 10% (w/v), The weight average molecular weight is a PGA production capacity of 300,000 or more. <20> The Bacillus subtilis described in <18> or <19>, which has a weight average molecular weight of 300,000 or more, preferably 500,000 when cultured under the conditions of a sodium chloride concentration of 10% (w/v). More preferably, the above is more preferably 1,000,000 or more, more preferably 2,000,000 or more, still more preferably 5,000,000 or more, more preferably 10,000,000 or more, still more preferably 50,000,000 or less. The Bacillus subtilis according to any one of the above <18> to <20>, which is produced under the conditions of a sodium chloride concentration of 10% (w/v) or more, and produces PGA 0.1 g/L/3. Above day, it is preferably 0.5 g/L/3 days or more, more preferably 1.0 g/L/3 days or more, more preferably 5.0 g/L/3 days or more. The Bacillus subtilis according to any one of the above items <18> to <21> which has the nucleotide sequence represented by SEQ ID NO: 7 or 8, and the same as the nucleotide sequence shown in SEQ ID NO: 7 or 8. Preferably, the base sequence of 99.75% or more, more preferably 99.85% or more, and most preferably 99.90% or more, or the base sequence represented by SEQ ID NO: 7 or 8 is preferably deleted, substituted, inserted or added. A 16S rRNA gene of 1 to 3, more preferably 1 base, base sequence. <23> The Bacillus subtilis described in any one of <18> to <22> which exhibits the bacteriological properties described in Table 1 above. <24> A method for adjusting the molecular weight of the PGA, which is obtained by subjecting the PGA produced by the method according to any one of the above <1> to <17> to a low molecular weight, and adjusting the molecular weight to a desired molecular weight. [Examples] Hereinafter, the present invention will be described in further detail based on examples, but the present invention is not limited thereto. Further, for reagents not described in the manufacturer, generally available reagents can be used. Here, the base sequence of the primer used in the present Example is shown in Table 2. Test Example 1 Method for obtaining spore forming microorganisms A sample of about 5 g of commercially available pickles, miso, decoctant, or natto was collected in a sterile manner and placed in a conical tube having a volume of 15 mL (product code 352096, BD) (Becton Dickinson) manufactured by Falcon), 2 times by weight of a 1% (w/v) aqueous sodium chloride solution (sterilization treatment agent) was added to the sample. These were pressed against a vibrating surface of a touch mixer (Model MT-31, manufactured by Yamato Scientific), suspended in a uniformly mixed state, and the sample was subjected to heat treatment at 80 ° C for 10 minutes. Then, these samples were appropriately diluted in stages using a 1% (w/v) sodium chloride aqueous solution (sterilization treatment agent), and each was applied to the microorganism detection medium (LB shown in Tables 3 to 6). The agar medium was adjusted to a final concentration of 10% LB agar medium (LB + 10% NaCl medium), modified GAM agar medium (trade name: "NISSUI", manufactured by Nissui Pharmaceutical Co., Ltd.), and M+Yex agar medium). The agar culture medium was allowed to stand at 30 ° C for 2 to 5 days, and the proliferation and morphology of the microorganisms on the agar medium were observed. Then, a plurality of single colonies appearing on each of the agar mediums were selected and subjected to line drawing on the same agar medium as the successfully confirmed proliferation, and a single colony appeared as a purified strain. Further, the purified strain was propagated by the same agar medium, and the obtained cells were suspended in an LB liquid medium containing 20% (w/v) glycerol, and stored frozen at -80 °C. Test Example 2 Selection method of Bacillus subtilis (1) The strain obtained in Test Example 1 (-80 ° C cryopreserved sample) was applied to an LB agar medium by a sterilized platinum ring (product code 254410, manufactured by Nunc). This was allowed to stand at 30 ° C for 1 day of static culture, and the growth of each strain was visually confirmed. Then, each strain grown on the LB agar medium was inoculated on the M+Yex agar medium using a pre-sterilized toothpick, and it was allowed to stand for one day at 30 ° C, and the growth of each strain was visually confirmed. Then, the strains grown on the M+Yex agar medium were inoculated separately into the M/glucose anabolic assay plate shown in Table 7, and the M/tagatose anabolic assay plate shown in Table 8, and at 37 ° C. Serve for 1 to 3 days of static culture. In this test example, the growth on the anabolic assay plate was visually observed, and the M/glucose anabolic assay plate was selected to have colony formation as a growth indicator and was aseptically dropped in the M/tagatose anabolic assay plate. The formed strain was used as a candidate strain of Bacillus subtilis. Test Example 3 Method for selecting Bacillus subtilis (2) The glycerin-preserved sample prepared in Test Example 1 was diluted to 30 times with 1 mM TE buffer (pH 8.0), and the template was used as shown in Table 2. The primer 27f and the primer 1525r were subjected to PCR (Polymerase Chain Reaction) to amplify a DNA fragment of about 16 kb in the 16S rRNA gene region. The DNA polymerase was TaKaRa LA Taq (manufactured by TAKARA BIO). After modifying the template DNA at 95 ° C for 5 minutes, the cycle was carried out at 95 ° C for 1 minute, at 55 ° C for 30 seconds, at 72 ° C for 2 minutes as one cycle and for 30 cycles, and further at 72 ° C. Keep under constant temperature for 2 minutes. A DNA fragment of 550 bp was determined using the primer 27f shown in Table 2 for the DNA fragment of about 1.5 kb in the obtained 16S rRNA gene region. Further, in the preparation of a sequence analysis sample, a Big Dye Terminator v3.1 Cycle Sequencing Kit (manufactured by Applied Biosystems) was used, and sample preparation was carried out according to the accompanying operation protocol (protocol). . When the sample was purified before the analysis, Montage SEQ kit (manufactured by MILLIPORE) was used. Then, with respect to the prepared sequence sample, sequence analysis was carried out using a DNA sequence analyzer (trade name: ABI 3100 Genetic Analyzer, manufactured by Applied Biosystems) to determine the base sequence. The sequence homology search uses "BLAST" in the menu "Nucleotide" of the open database NCBI (National Center for Biotechnology Information; http://www.ncbi.nlm.gov/) "Basic BLAST" in the middle, select "nucleotide blast" from the BLAST program. "Reference genomic sequences (refseq_genomics) are specified in the database of the search target, and "Highly similar sequences (megablast)" is specified in the selection program to perform homology search. According to the obtained results, as the strain having the highest homology in the test example, it was determined to be Bacillus subtilis, and the 550 bp of the above sequence and the Bacillus subtilis standard strain corresponding thereto were selected and determined ( Bacillus subtilis A strain having a homology of 98.9% or more of the sequence of DSM strain 10 was used as a candidate strain of Bacillus subtilis. Test Example 4 Selection method of Bacillus subtilis producing PGA without adding glutamic acid A sterilized platinum ring (product code 254410, manufactured by Nunc) was used as a cryopreservation sample presumed to be Bacillus subtilis in Test Examples 2 and 3. The frozen cells were collected, inoculated into 5 mL of LB liquid medium, and cultured at 30 ° C for 24 hours with shaking. This was used as an inoculum culture solution in 30 mL of glutamic acid-free PGA production medium [media composition: 7.5% glucose, 1.8% ammonium chloride, 0.5% yeast extract, 0.035% magnesium sulfate, heptahydrate, 0.005% manganese sulfate, tetra-pentahydrate, 100 mM 3-morpholinopropanesulfonic acid (pH adjusted to 7.0 by potassium hydroxide, manufactured by Toray Chemical Research Institute) was inoculated with 1% ( v/v), and the medium was cultured at 37 ° C for 72 hours with shaking. After the completion of the culture, the PGA contained in the supernatant of the culture solution was quantified by the method shown in the following Measurement Example 1. As a result, a strain in which a dissolution component of a polymer substance having an absorption of UV 210 nm derived from PGA was detected in a culture supernatant was selected as a candidate for Bacillus subtilis capable of producing PGA without adding glutamic acid. Strain. Test Example 5 Method for selecting Bacillus subtilis having high salt concentration tolerance (1) A cryopreservation test in which plants of B. subtilis were estimated in Test Examples 2 and 3 and selected as PGA producing Bacillus subtilis candidate strain in Test Example 4 The frozen preservation sample of the well-known natto standard strain (NBRC 16449 strain, NBRC 3336 strain, NBRC 3936 strain) obtained from the independent administrative company product evaluation technical base mechanism prepared in the same order as in Test Example 1 To become 1×10 3 ~1×10 4 The cell/mL method was inoculated into LB + 10% NaCl liquid medium, and cultured at 37 ° C for 24 hours with shaking. After the shaking culture, the culture solution sample was appropriately diluted with a 1% (w/v) sodium chloride aqueous solution, and measured to be proliferated by using a spectrophotometer (trade name: U-2900 type; manufactured by Hitachi High-Technologies). The absorbance of the indicator culture solution is 600 nm (OD600). As a result, no increase in absorbance was observed in the natto standard strain. Under the test conditions, 6 strains of strains with increased absorbance were selected as strains of P. aeruginosa having high salt concentration tolerance and high molecular weight PGA production capacity under high salt concentration conditions. Test Example 6 Evaluation of the growth limit salt concentration of the B. subtilis candidate strain The test method was used to prepare the inoculum culture using the LB liquid medium under the same conditions as in Test Example 4 as the natto NBRC 3336 strain of the standard strain and the B. subtilis candidate strain. Then, the final concentration of sodium chloride was set to 10% (w/v), 12% (w/v), 13% (w/v), 14% (w/v), 15% (w/v). And 16% (w/v) of LB medium, which was inoculated with the above-mentioned inoculum culture medium at an initial absorbance of 0.05, and cultured at 37 ° C for 2 days with shaking. The culture solution was collected over time in the shaking culture, and the culture solution sample was appropriately diluted with an aqueous sodium chloride solution (sodium chloride aqueous solution having the same concentration as the use medium), and a spectrophotometer (U-2900 type, Hitachi High-Technologies manufactured) The absorbance of the culture solution to be a proliferation index was measured at 600 nm (OD600). Test Example 7 Confirmation Test for Growth Limit Salt Concentration of Selected Bacillus Subtilis The Bacillus subtilis candidate strain having high salt concentration tolerance was prepared by using the LB + 10% NaCl liquid medium and using the same conditions as in Test Example 5. Then, the final concentration of sodium chloride was set to 10% (w/v), 12% (w/v), 14% (w/v), 15% (w/v), 16% (w/v). ), 17% (w/v), 18% (w/v), 19% (w/v), or 20% (w/v) TSB medium (Trypticase Soy broth, Becton) The inoculum culture solution was inoculated in such a manner that the initial absorbance was 0.1, and it was cultured at 37 ° C for 2 days with shaking. After the shaking culture, the culture liquid sample was collected on the second day from the start of the culture, and appropriately diluted with a 10% (w/v) sodium chloride aqueous solution, and a spectrophotometer (U-2900 type, manufactured by Hitachi High-Technologies) was used. The absorbance of the culture solution to be a proliferation index was measured at 600 nm (OD600). In the test example, the growth limit concentration of the strain was determined as the salt concentration condition of the culture solution until the second day of culture was twice or more the concentration of the inoculum culture inoculation. Test Example 8 Confirmation test for the optimum growth salt concentration of the selected B. subtilis The Bacillus subtilis candidate strain having high salt concentration tolerance was prepared by using the LB + 10% NaCl liquid medium and using the same conditions as in Test Example 5. Then, the preparation was carried out without adding sodium chloride and the final concentrations were set to 1% (w/v), 2 (w/v), 3 (w/v), 4 (w/v), 5 (w/v), 6 (w/v), 7% (w/v), 8% (w/v), and 10% (w/v) TSB medium, which was placed on a 96-well round bottom microplate (model 3870-096, IWAKI) Each well was dispensed with 200 μL. The inoculum culture solution was inoculated in such a manner that the initial absorbance of each well was 0.05, and the culture was shaken at 37 ° C for 24 hours using a biological microdisk reader (HiTS-S2 type, manufactured by SCINICS). The biomicrodisk reader was oscillated at 150 rpm and the absorbance at 600 nm (OD600) was measured over time at 30 minute intervals by an interference filter. The increase in absorbance per unit time was calculated from the obtained absorbance value, and the maximum cell growth rate in the culture test was determined as the cell growth rate (ΔOD600/hr). In the test example, the salt concentration of the cell growth rate (ΔOD600/hr) from the maximum value to (maximum value -0.2) was determined as the optimum growth salt concentration. Example 1 Characteristics of selected Bacillus subtilis The Bacillus subtilis strain (Bacillus subtilis KSM-FFA610 strain, Bacillus subtilis KSM-FFA631 strain, and subtilis grass) having high salt concentration tolerance and producing PGA obtained by the methods shown in Test Examples 1 to 8 was used. The growth characteristics of Bacillus sp. KSM-FFB406 strain, Bacillus subtilis KSM-FFB425 strain, Bacillus subtilis KSM-FFB540 strain, and Bacillus subtilis KSM-FFB553 strain are shown in Tables 9-13. Table 9 shows the results of Test Example 5. As shown in Table 9, when culturing was carried out using an LB liquid medium containing a high concentration of sodium chloride, the absorbance was below the detection limit in the natto standard strain as a control, and no proliferation was observed. On the other hand, in the test strains selected in Test Examples 2 to 4, the absorbance (OD600) was found to be more than 0.5 (equivalent to 1 × 10) under the test conditions shown in Test Example 5. 7 Cell/mL) strain. From the above results, it was confirmed that the Bacillus subtilis strain of the present invention is a high salt concentration resistant strain capable of proliferating at a salt concentration at which the natto standard strain cannot proliferate. Tables 10 and 11 show the results of Test Example 6. As shown in Table 10, the absorbance of the natto standard strain (NBRC 3336 strain) as a control was about 0.5 at the final concentration of sodium chloride at 10% (w/v) on the first day of culture, relative to Thus, the absorbance of the strain of Bacillus subtilis of the present invention all showed a value exceeding 2.0. Further, under the condition that the final concentration of sodium chloride was 13% (w/v), the growth of the bacteria was not confirmed as a natto standard strain as a control, whereas the absorbance of the strain of the present strain of the present invention showed a value exceeding 0.5. . From the above results, it was confirmed that the B. subtilis candidate strain of the present invention is a strain having a salt concentration tolerance higher than that of the natto standard strain. As shown in Table 11, in the second day of culture, under the conditions of a final concentration of sodium chloride of 13% (w/v), the growth of the bacteria was not confirmed as a natto standard strain as a control, whereas the present invention The absorbance of the B. subtilis candidate strains all showed values exceeding 1.5. Further, the B. subtilis strain of the present invention has a final concentration of sodium chloride of 14% (w/v), and the absorbance indicates a value exceeding 0.5. From the above results, it was confirmed that the B. subtilis candidate strain of the present invention is a strain having a salt concentration tolerance higher than that of the natto standard strain. Table 12 shows the results of Test Example 7. As shown in Table 12, in the high salt concentration growth test using TSB medium, the absorbance of the Bacillus subtilis strain of the present invention on the second day of culture, the KSM-FFA631 strain and the KSM-FFB406 strain were 16% of the final concentration of sodium chloride. Under the conditions of (w/v), KSM-FFB425 strain, KSM-FFB540 strain and KSM-FFB553 strain under conditions of 17% (w/v) and KSM-FFA610 strain at 18% (w/v) More than twice the value of inoculation for inoculum culture. From the above results, it was confirmed that the growth limit concentration of sodium chloride in the confirmation test for the growth limit salt concentration of the TSB medium of the present invention was 16 to 18% (w/v). Table 13 shows the results of Test Example 8. As shown in Table 13, the B. subtilis strain of the present invention was added in the test for the optimum salt concentration for growth using TSB medium, and the sodium chloride concentration was added to the final concentration of 6% (w/v) (room temperature). Under the conditions, the cell proliferation rate (ΔOD600/hr) is a value of 0.3 to 0.5. Based on the above results, it was confirmed that in the confirmation test for the optimum salt concentration for growth of Bacillus subtilis using the TSB medium in the strain of Bacillus subtilis of the present invention, the optimal concentration of sodium chloride growth was in the Bacillus subtilis KSM-FFA610 strain. 0 to 5% (w/v), 0 to 4% (w/v) in Bacillus subtilis KSM-FFA631 strain, 0 to 5% (w/v) in Bacillus subtilis KSM-FFB406 strain, Bacillus subtilis KSM- The FFB425 strain was 0 to 4% (w/v), the Bacillus subtilis KSM-FFB540 strain was 0 to 5% (w/v), and the Bacillus subtilis KSM-FFB553 strain was 0 to 5% (w/v). Example 2 Identification of strains based on bacteriology and base sequence analysis of 16S rRNA gene to the above-mentioned Bacillus subtilis strains (Bacillus subtilis KSM-FFA610 strain, Bacillus subtilis KSM-FFA631 strain, Bacillus subtilis KSM-FFB406 strain, Bacillus subtilis KSM) The bacteriological properties of the -FFB425 strain, Bacillus subtilis KSM-FFB540 strain, and Bacillus subtilis KSM-FFB553 strain were studied. The results are shown in Table 14. Further, with respect to the above-mentioned B. subtilis strain, strain identification was carried out based on the base sequence analysis of the 16S rRNA gene by the following measurement example 4. The results are shown in Table 15. As shown in Table 14, it was confirmed that all of the above-mentioned B. subtilis strains have the bacteriological properties of Bacillus subtilis. As shown in Table 15, the results of the homology analysis based on the nucleotide sequence of the 16S rRNA gene clearly show that all of the above strains have a base of the 16S rRNA gene having a higher homology with 99.9% or more of the B. subtilis DSM 10 strain. Base sequence. Therefore, the B. subtilis strain was judged to be Bacillus subtilis based on the bacteriological property and the analysis result of the base sequence of the 16S rRNA gene. In addition, the Bacillus subtilis KSM-FFA610 strain was deposited with the authorized microbiological center of the independent administrative agency product evaluation technology base institution on June 2, 2016, under the registration number NITE BP-02276 (2-5- 8). In addition, the Bacillus subtilis KSM-FFA631 strain was deposited on the licensed microbiological center of the independent administrative company's product evaluation technology base institution on June 2, 2016 (2-5-8, Kawasaki-shi, Chiba Prefecture). ). In addition, the Bacillus subtilis KSM-FFB406 strain was deposited in the authorized microbial center of the independent administrative agency product evaluation technology base institution on June 2, 2016 (the number of the NAI BP-02278). ). In addition, the Bacillus subtilis KSM-FFB425 strain was deposited on the licensed microbiological center of the independent administrative agency product evaluation technology base institution on June 2, 2016 (2-5-8, Kawasaki-shi, Chiba Prefecture). ). In addition, the Bacillus subtilis KSM-FFB540 strain was deposited on the licensed microbiological center of the independent administrative company's product evaluation technology base institution on June 2, 2016 under the registration number NITE BP-02280 (2-5-8, Izumo, Izumi, Chiba Prefecture, Chiba Prefecture) ). Furthermore, the Bacillus subtilis KSM-FFB553 strain was deposited on the licensed microbiological center of the independent administrative company's product evaluation technology base institution on June 2, 2016 under the registration number NITE BP-02281 (2-5-8, Izumo, Izumi, Chiba Prefecture, Chiba Prefecture) ). Example 3 Evaluation of PGA productivity under conditions of high-concentration salt addition (1) Using the well-known natto standard strain (NBRC 3336 strain and NBRC 16449 strain) as a control, the strain of Bacillus subtilis (KSM-FFA610 strain of the present invention) was used. KSM-FFA631 strain, KSM-FFB406 strain, KSM-FFB425 strain, KSM-FFB540 strain, and KSM-FFB553 strain) were evaluated for PGA productivity under high salt concentration conditions. The cryopreserved sample of the above-described strain shown in Test Example 1 and the cryopreserved sample of the natto standard strain prepared in the same order were used, and the LB liquid medium was used under the same culture conditions as in Test Example 4 at 30 ° C. For 24 hours of shaking culture. As an inoculum culture solution, 30 mL of PGA productivity evaluation medium [media composition: 8.0% glucose, 8.0% sodium glutamate monohydrate, 1.25% yeast extract, 1.0% ammonium sulfate, 0.2% magnesium sulfate)・Heptahydrate, 0.003% manganese sulfate, tetra-pentahydrate, 0.7% dipotassium hydrogen phosphate, 0.35% potassium dihydrogen phosphate, and 7.3% sodium chloride (equivalent to 1.25 M) or 10.2% sodium chloride (equivalent Inoculate 1% (v/v) on 1.75 M)]. The medium was cultured at 37 ° C for 72 hours with shaking. After the completion of the culture, the PGA contained in the supernatant of the culture solution was quantified by the method described in the following Measurement Example 1. The results are shown in Table 16. As shown in Table 16, the Bacillus subtilis strain of the present invention exhibited superior PGA productivity even under conditions of high salt concentration as compared with the natto standard strain. Further, the P. subtilis strain of the present invention can produce PGA even under conditions in which a high concentration of sodium chloride is not produced in a PGA, such as a natto standard strain. Based on the above results, it was judged that the Bacillus subtilis of the present invention is Bacillus subtilis having high salt concentration tolerance. Example 4 PGA productivity evaluation under high-concentration salt addition conditions (2) PGA productivity was evaluated using the B. subtilis strain KSM-FFB553 strain of the present invention under conditions in which a monovalent metal salt was present at a high concentration. The inoculum culture solution was prepared in the same manner as in Test Example 4, and was cultured in 30 mL of PGA productivity evaluation medium [media composition: 8.0% glucose, 8.0% sodium glutamate monohydrate, 1.25% yeast extract, 1.0% ammonium sulfate, 0.2% magnesium sulfate, heptahydrate, 0.003% manganese sulfate, tetra-pentahydrate, 0.7% dipotassium hydrogen phosphate, 0.35% potassium dihydrogen phosphate, and 10.2% sodium chloride (equivalent to 1.75 M ) or 1% (v/v) of potassium chloride 11.2% (equivalent to 1.5 M). This was incubated at 37 ° C for 72 hours of shaking. After the completion of the culture, the PGA contained in the culture supernatant was quantified by the method described in the following Measurement Example 1. The results are shown in Table 17. As shown in Table 17, it was confirmed that the Bacillus subtilis strain of the present invention exhibited excellent PGA productivity even under the condition that the monovalent metal salt was at a high concentration. Example 5 PGA productivity evaluation under high-concentration salt addition conditions (3) PGA productivity was evaluated using the B. subtilis strain KSM-FFB553 strain of the present invention under conditions in which a divalent metal salt was present at a high concentration. The inoculum culture solution was prepared in the same manner as in Test Example 4, and was cultured in 30 mL of PGA productivity evaluation medium [media composition: 8.0% glucose, 8.0% sodium glutamate monohydrate, 1.25% yeast extract, 1.0% ammonium sulfate, 0.2% magnesium sulfate, heptahydrate, 0.003% manganese sulfate, tetra-pentahydrate, 0.7% dipotassium hydrogen phosphate, 0.35% potassium dihydrogen phosphate, and 10.2% magnesium chloride hexahydrate (equivalent to 0.5%) or 7.4% calcium chloride dihydrate (equivalent to 0.5 M) was inoculated with 1% (v/v). This was incubated at 37 ° C for 72 hours of shaking. After the completion of the culture, the PGA contained in the supernatant of the culture solution was quantified by the method described in the following Measurement Example 1. The results are shown in Table 18. As shown in Table 18, it was confirmed that the B. subtilis strain of the present invention exhibited excellent PGA productivity even under the condition that the divalent metal salt was at a high concentration. Example 6 Measurement of molecular weight of PGA (1) For the strain of Bacillus subtilis of the present invention (KSM-FFA610 strain, KSM-FFA631 strain, KSM-FFB406 strain, KSM-FFB425 strain, KSM-FFB540 strain, and KSM-FFB553 strain) The molecular weight of the produced PGA was measured. The inoculum culture solution was prepared by the same method as in Test Example 5, and was cultured in a production evaluation medium of 30 mL [media composition: 8.0% glucose, 8.0% sodium glutamate monohydrate, 1.25% yeast extract, 1.0). % ammonium sulfate, 0.2% magnesium sulfate, heptahydrate, 0.003% manganese sulfate, tetra-pentahydrate, 0.7% dipotassium hydrogen phosphate, 0.35% potassium dihydrogen phosphate, 10.2% sodium chloride (equivalent to 1.75 M)] Inoculate 1% (v/v). The medium was cultured at 37 ° C for 72 hours with shaking. After the completion of the culture, the molecular weight of the PGA contained in the supernatant of the culture solution was measured by the method described in the following Measurement Example 1. The results are shown in Table 19. As shown in Table 19, it was confirmed that the B. subtilis strain of the present invention can produce a high molecular weight PGA even under the condition that a high concentration of a salt which cannot be grown by a natto standard strain is added. Further, it was confirmed that a high molecular weight PGA can be produced by using the high salt concentration resistant strain of the present invention. Example 7 Measurement of Molecular Weight of PGA (2) Using the strain of Bacillus subtilis KSM-FFB553 of the present invention shown in Example 1, the molecular weight of PGA produced under conditions of high concentration of a monovalent or divalent metal salt was carried out. Evaluation. The inoculum culture solution was prepared in the same manner as in Test Example 4, and was cultured in 30 mL of PGA productivity evaluation medium [media composition: 8.0% glucose, 8.0% sodium glutamate monohydrate, 1.25% yeast extract, 1.0% ammonium sulfate, 0.2% magnesium sulfate, heptahydrate, 0.003% manganese sulfate, tetra-pentahydrate, 0.7% dipotassium hydrogen phosphate, 0.35% potassium dihydrogen phosphate, and 11.2% potassium chloride (equivalent to 1.5 M 1% (v/v) was inoculated with 10.2% magnesium chloride, hexahydrate (corresponding to 0.5 M) or 7.4% calcium chloride and dihydrate (corresponding to 0.5 M). This was cultured at 37 ° C for 72 hours with shaking. After the completion of the culture, the molecular weight of the PGA contained in the supernatant of the culture solution was measured by the method described in the following Measurement Example 1. The results are shown in Table 20. As shown in Table 20, it was confirmed that the Bacillus subtilis strain of the present invention can produce a high molecular weight PGA under the condition that the monovalent or divalent metal salt is at a high concentration. Example 8 Evaluation of PGA productivity under conditions in which glutamic acid was not added The Bacillus subtilis strain of the present invention (KSM-FFA610 strain, KSM-FFA631 strain, KSM-FFB406 strain, KSM-FFB425 strain, KSM-FFB540 strain, and KSM-FFB553 strain) The PGA productivity was evaluated under conditions of high salt concentration and no addition of glutamic acid. The inoculum culture solution was prepared in the same manner as in Test Example 5, and was cultured in 30 mL of PGA productivity evaluation medium [media composition: 8.0% glycerol, 0.5% yeast extract, 1.0% ammonium sulfate, 0.2% magnesium sulfate, seven) Hydrate, 0.003% manganese sulfate, tetra-pentahydrate, 0.7% dipotassium hydrogen phosphate, 0.35% potassium dihydrogen phosphate, and 7.3% sodium chloride (corresponding to 1.25 M) were inoculated with 1% (v/v). The medium was cultured at 37 ° C for 72 hours with shaking. After the completion of the culture, the PGA contained in the supernatant of the culture solution was quantified by the method described in the following Measurement Example 1, and the molecular weight was measured. The results are shown in Table 21. As shown in Table 21, it was confirmed that the Bacillus subtilis strain of the present invention can produce a high molecular weight PGA even under the condition that glutamic acid is not added. [Measurement Example 1] A quantitative method of PGA and a molecular weight measurement method In the measurement of PGA and the measurement of molecular weight, a high performance liquid chromatography apparatus was used. [HPLC apparatus configuration] Liquid delivery pump: L-6200 type, automatic sampler manufactured by Hitachi, Ltd.: AS-4000 type, column oven manufactured by Hitachi, Ltd.: L-5020 type, UV detector manufactured by Hitachi, Ltd.: L-4250 type, Hitachi Manufactured Chromatography Data Analysis Device: Model D-2500, Hitachi Co., Ltd. uses a gel filtration column TSKgel G6000PWXL (7.8 mm ID × 30 cm, manufactured by Tosoh) and TSKgel for the hydrophilic polymer with different rejection limits. G4000PWXL (7.8 mm ID × 30 cm, manufactured by Tosoh). The cells were connected in series and connected to the protective column TSK guardcolumn PWXL (6.0 mm ID × 4.0 cm, Tosoh) directly in front of the analysis column. For the analysis, the dissolution solution was set to 0.1 M sodium sulfate, the flow rate was 1.0 mL/min, the column temperature was 50 ° C, and the elution peak was measured at a detection wavelength of 210 nm. Further, in the pretreatment of the sample, the culture supernatant sample appropriately diluted with 0.1 M sodium sulfate was subjected to filter filtration using a 0.45 μm Durapore membrane (model MULTI SCREEN MNHV45, manufactured by MILLIPORE). At the concentration check, a calibration curve was prepared using a PGA (Meiji Food Materia) having a molecular weight of 880,000. Further, at the molecular weight check, polyglutamic acid having various molecular weights different in weight average molecular weight was obtained in advance using Pulley Shodex STANDARD P-82 (Showa Denko) (Wako Pure Chemical Industries 162-21411 and 162-21401; SIGMA) - ALDRICH P-4886 and P-4761; Meiji Food Materia (molecular weight 880,000)). [Measurement Example 2] Method for identifying high molecular substance in culture supernatant sample The culture solution sample after completion of the culture obtained in Example 4 was subjected to centrifugation at 19,800 rpm for 30 minutes (himac CR21GIII) Type, manufactured by Hitachi Machine Co., Ltd.), and the supernatant sample removed by the removal of the cells is recovered. Then, transfer 1 to 10 mL of the supernatant sample to a 50 mL centrifuge tube (Model 227 261, manufactured by Greiner Bio-one) made of polypropylene, and add 2 volumes of ethanol to the supernatant sample. Inverted mixing was carried out, followed by placing a crucible at a constant temperature of -30 °C. Thereafter, it was centrifuged at 3,000 rpm for 30 minutes (himac CF7D2 type, manufactured by Hitachi Instruments), and the precipitated component was recovered. The obtained precipitated component was redissolved in 2 mL of distilled water, and the above-mentioned precipitated component by adding ethanol was again prepared and recovered. Then, the recovered sample was dissolved in 2 mL of distilled water, and 0.5 mL of the sample was transferred to a test tube (model ST-13M, manufactured by Japan Electronic Physicochemical Glass) of a screw cap, and then 0.5 mL of concentrated hydrochloric acid was added. Stirring was carried out, and then nitrogen gas was sealed and heat-treated at 105 to 110 ° C for 16 hours. After the heat treatment, hydrochloric acid and water (about 6 hours) were distilled off under a nitrogen gas stream, and the obtained dried product was used as a hydrolysis sample. Further, as a PGA sample, a commercially available PGA (molecular weight 880,000, Meiji Food Materia) was used as a control for hydrolyzing a sample, and L-Glutamic acid and D-Glutamic acid were used. ) (manufactured by Wako Pure Chemical Industries, Ltd.). Then, the obtained hydrolyzed sample was appropriately diluted, and various amino acid analysis and glutamic acid quantification in the sample were carried out using a fully automatic amino acid analyzer (Model L-8900, manufactured by Hitachi High-Technologies). . Further, the L-glutamic acid measurement kit (YAMASA soy sauce) was used, and the amount of L-glutamic acid was measured according to the method described in the operating instructions attached to the kit. The total amount of optically active isomers (D/L) is obtained as a quantitative result by measurement in a fully automated amino acid analyzer, and the amount obtained by using the L-glutamic acid assay kit is subtracted from the result. As a result, the difference was obtained as the amount of D-glutamic acid. As a result of the measurement, the optical isomer ratio of glutamic acid of the polymer substance recovered from the culture liquid sample of KSM-FFA610 strain, KSM-FFB425 strain, KSM-FFB540 strain, and KSM-FFB553 strain (D/) L) are 68/32, 67/33, 69/31, and 67/33, respectively. Further, in the measurement by the fully automatic amino acid analyzer described above, since the amino acid other than glutamic acid was not detected, the polymer substance in the culture supernatant was judged to be PGA. Further, it was judged that the D/L ratio of the PGA produced by the above-mentioned selected strain of the strain having high salt concentration of the present invention was equal to the D/L ratio of the PGA produced by the known natto standard strain. [Measurement Example 3] Viscosity measurement method of PGA solution KSM-FFB553 strain was used for the above strain, and the medium for PGA preparation [media composition: 8.0% glucose, 8.0% sodium glutamate monohydrate, 0.5% yeast extract, Preparation of 1% (v/v) in 1.0% ammonium sulfate, 0.2% magnesium sulfate, heptahydrate, 0.003% manganese sulfate, tetra-pentahydrate, 0.7% dipotassium hydrogen phosphate, 0.35% potassium dihydrogen phosphate] The liquid sample was prepared from the culture liquid sample by recovery by acid precipitation, followed by purification by ethanol precipitation, and freeze-drying to prepare a PGA dry powder. Then, the obtained PGA sample (Mw 5,000 k) was dissolved in distilled water and 1.25 M sodium chloride aqueous solution in a manner of 4% (w/w) or 8% (w/w). The approximately 40 mL was transferred to a glass spiral tube (Model No. 7, or No. 8, manufactured by Maruemu) or a polypropylene 50 mL centrifuge tube (model 227 261, greiner bio) without generating bubbles. In the -one manufacturing method, a B-type viscometer (TVB-15 type, manufactured by Toki Sangyo Co., Ltd.) is used, and the sample temperature is 20 to 25 ° C (room temperature), the measurement time is 60 seconds (automatic stop mode), and the rotor rotation speed is 60. The measurement was carried out using an M2 rotor under rpm conditions. In addition, in the sample whose measurement value exceeds the upper limit under the above-mentioned measurement conditions, the number of revolutions is appropriately changed to 30 rpm, or the rotor is appropriately changed to M3 or M4. As a result of the measurement, in the sample of PGA 4% (w/w), the viscosity of the sample to which no salt was added was 380 mPa·s, whereas the sample to which the salt was added was 60 mPa·s. Further, in the PGA8% (w/w) sample, the viscosity of the sample in which no salt was added was 1,480 mPa·s, whereas the sample to which the salt was added was 450 mPa·s. From the above measurement results, the viscosity reduction effect by the PGA sample to which the salt was added was confirmed. [Measurement Example 4] The strain identification method based on the base sequence of the 16S rRNA gene based on the nucleotide sequence of the 16S rRNA gene base sequence was carried out in the following experimental sequence. A PCR template sample was prepared by cryopreservation of the cells in the same manner as in Test Example 2, and PCR was carried out using primer 27f and primer 1525r shown in Table 2 to amplify a DNA fragment of about 16 kb in the 16S rRNA gene region. The DNA polymerase was TaKaRa LA Taq (manufactured by TAKARA BIO). The template DNA was modified at 95 ° C for 5 minutes, and then subjected to 1 cycle at 95 ° C for 1 hour at 55 ° C for 30 minutes at 72 ° C for 30 cycles, and further at 72 ° C. Keep at a constant temperature for 2 minutes. For the DNA fragment of the obtained 16S rRNA gene region, primers 27f, primer f2L (-), primer 926f, primer rE1L, primer r2L', and primer 1525r shown in Table 2 were used as primers for sequence, and DNA bases were used. Analysis of the sequence. Further, in the preparation of the sequence analysis sample, a Big Dye Terminator v3.1 Cycle Sequencing Kit (manufactured by Applied Biosystems) was used, and sample preparation was carried out in accordance with the accompanying operation instructions. When the sample was purified before the analysis, Montage SEQ kit (manufactured by MILLIPORE) was used. With respect to the prepared sequence sample, sequence analysis was carried out using a DNA sequence analyzer (trade name: ABI 3100 Genetic Analyzer, manufactured by Applied Biosystems) to determine the base sequence. Then, each of the obtained base sequences was subjected to single fragmentation using GENETYX ATSQ ver2.01 (manufactured by GENETYX). The homology search of the sequence uses "BLAST" in the menu "Nucleotide" of the open database NCBI (National Center for Biotechnology Information; http://www.ncbi.nlm.gov/) "Basic BLAST" in the middle, select "nucleotide blast" from the BLAST program. "Reference genomic sequences (refseq_genomics) is specified in the database of the search target, and "Highly similar sequences (megablast)" is specified in the selection program, and the standard strain having the highest homology rate is selected. Then, GENETYX Ver. 13 (manufactured by GENETYX) was used for the 16S rRNA gene sequence of the selected reference strain and the sequence-determined 16S rRNA gene sequence of the Bacillus subtilis candidate strain having high salt concentration tolerance, by "Nucleotide vs The Nucleotide Homology tab performs homology analysis of the base sequence to the base sequence, and calculates homology (%). The present invention and its implementation are described in detail, but as long as I have not specifically specified, it is not intended to limit the invention in any detail, and it is considered that the patent application scope may not be violated. The spirit and scope of the invention as illustrated is broadly explained. The present application claims priority to Japanese Patent Application No. 2016-165099, the entire disclosure of which is hereby incorporated herein in .