JP4966679B2 - Lactic acid bacteria and method for making a koji using the same - Google Patents
Lactic acid bacteria and method for making a koji using the same Download PDFInfo
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Description
本発明は、乳酸菌およびこれを用いる製麹方法に関する。 The present invention relates to a lactic acid bacterium and a koji making method using the same.
味噌等の醸造食品の製造に用いられる麹は、通常、連続通風した環境下で、米等の固体培地に種麹を接種し、培養することで製造される（特開平８−２２８７６４号公報）。 The koji used for the production of brewed foods such as miso is usually produced by inoculating and cultivating a seed medium in a solid medium such as rice in an environment of continuous ventilation (Japanese Patent Laid-Open No. 8-228764). .
ところで、味噌の雑菌の中で、混入防止対策が最も困難な細菌類はBacillus（バチルス）属である。Bacillus属は有胞子桿菌に分類され、土壌など自然界に普遍的に存在していることから、ほとんどの食品がこの菌の混入する機会に晒されている。しかもBacillus属は生育環境が悪化すると耐熱性の芽胞を形成することがあり、それ故Bacillus属の混入は加熱処理した食品でも変敗、腐敗のリスクを排除できず、食品を保蔵する上で耐熱性芽胞対策が最重要課題となっている。 By the way, among miscellaneous bacteria of miso, the most difficult to prevent contamination is Bacillus genus. The genus Bacillus is classified as a spore-forming gonococcus and is ubiquitous in nature, such as soil, so most foods are exposed to the contamination of this bacterium. In addition, Bacillus sp. May form heat-resistant spores when the growth environment deteriorates.Therefore, contamination of Bacillus sp. Can not eliminate the risk of deterioration and spoilage even in heat-treated foods. Sexual spore countermeasures are the most important issue.
味噌はそのままの状態では、高濃度食塩（12％前後）、弱酸性（pH5.0付近）、0.75前後の中間的水分活性（Ａｗ）の環境下にあることから、Bacillus属が混入しても栄養細胞型では、増殖することなくやがて菌体膨隆、伸張など変形を来たして死滅後自己消化を起こす。また、芽胞細胞型においては増殖が阻止されたままで終始する。しかし、味噌が、各種ソースなどの味噌加工品の加工素材として用いられる場合は、低食塩、高水分の環境になる機会が多いため、Bacillus属の耐熱性芽胞の存在により味噌加工品の変敗、腐敗を招来するおそれがある。 In the state of miso as it is, it is in an environment of high-concentration salt (around 12%), weak acidity (around pH 5.0), and intermediate water activity (Aw) around 0.75. The vegetative cell type eventually undergoes self-digestion after death due to deformation such as bulging and stretching without growing. Moreover, in the spore cell type, the growth is stopped throughout. However, when miso is used as a raw material for processed miso products such as various sauces, there are many opportunities for low salt and high moisture environments, so the deterioration of processed miso products due to the presence of heat-resistant spores of the genus Bacillus There is a risk of corruption.
ところで、味噌製造工程でのBacillus属の主たる汚染源は麹である。従来Bacillus属の混入防止については、製造設備機器の徹底した洗浄と、工場内の清掃区域並びに非清掃区域との隔離等の対策を講じてきたが十分とは言い難かった。
そこで本発明は、上記課題を解決すべくなされたものであり、その目的とするところは、特に製麹工程でBacillus属菌数の低減化が可能な乳酸菌およびこれを用いた製麹方法を提供する。
By the way, the main contamination source of the genus Bacillus in the process of producing miso is koji. In the past, Bacillus genus contamination prevention measures have been taken, such as thorough cleaning of manufacturing equipment and isolation between clean and non-clean areas in the factory, but it has not been sufficient.
Therefore, the present invention has been made to solve the above-mentioned problems, and the object of the present invention is to provide a lactic acid bacterium capable of reducing the number of Bacillus bacteria in the cocoon making process and a cocoon making method using the lactic acid bacterium. To do.
本発明は次の乳酸菌に係る。
すなわち、ラクトバチルス・ファーメンタム（Lactobacillus fermentum）FERMP−２１０７４の乳酸菌。
上記の乳酸菌を固体培地に麹菌と共に接種して培養することによって、Bacillus属を低減した麹を製造することができる。
The present invention relates to the following lactic acid bacteria.
That is, a lactic acid bacterium of Lactobacillus fermentum FERMP-21074 .
By inoculating and cultivating the above lactic acid bacterium together with gonococcus in a solid medium, it is possible to produce cocoons with reduced genus Bacillus.
本発明に係る乳酸菌は、特に製麹過程においてBacillus属の低減化が図れ、この麹を利用した味噌等の醸造品、さらにはこの醸造品を用いた加工食品の変敗、腐敗を防止できる。 The lactic acid bacterium according to the present invention can reduce the genus Bacillus, particularly in the koji making process, and can prevent the deterioration and spoilage of brewed products such as miso using this koji, as well as processed foods using this brewed product.
以下本発明の好適な実施の形態を添付図面に基づき詳細に説明する。
本発明では、製麹工程でのBacillus属菌数の低減化を目的に、製麹時に抗菌性乳酸菌を利用するにあたっては、製麹環境に適応し、蒸米上で増殖旺盛な乳酸菌であることが望ましいと考え、抗菌性乳酸菌の分離源を使用目的の生育環境に近い米麹に求めた。実際には、長野県内の数箇所の味噌工場から収集した米麹を分離源とし、分離した乳酸菌について、Bacillus属に対する抗菌性の検索を行い、またその利用について検討した。
Preferred embodiments of the present invention will be described below in detail with reference to the accompanying drawings.
In the present invention, in order to reduce the number of Bacillus bacteria in the koji making process, when using antibacterial lactic acid bacteria at the time of koji making, the lactic acid bacteria must adapt to the koji making environment and grow vigorously on steamed rice. We thought that it was desirable, and asked the rice bran close to the intended growth environment for the source of antibacterial lactic acid bacteria. In fact, we used the rice bran collected from several miso factories in Nagano Prefecture as a source of separation, and we searched for antibacterial activity against the genus Bacillus and examined its use.
１．試験方法
１）米麹中からの乳酸菌の分離
長野県内の味噌工場から収集した米麹を分離源とし、BCP加プレートカウント寒天培地にカビサイジン（100mg力価/l）とアジ化ナトリウム（100PPM）を加えた分離培地を用い、重層法にて30℃、３日間培養後のBCPが変色したコロニーを乳酸菌として分離した。分離した乳酸菌は、純粋分離を繰り返した後、Difco Lactobacilli MRS Broth（以下MRS）に寒天を1.5wt％加えた高層培地で保存し、以後の試験に用いた。
1. Test method 1) Separation of lactic acid bacteria from rice bran Using rice bran collected from a miso factory in Nagano Prefecture as the source of separation, kavisaidin (100 mg titer / l) and sodium azide (100PPM) were added to a BCP-added plate count agar medium. Using the added separation medium, colonies in which BCP was discolored after culturing at 30 ° C. for 3 days were separated as lactic acid bacteria by the multilayer method. The isolated lactic acid bacteria were subjected to pure separation and then stored in a high-layer medium in which 1.5 wt% of agar was added to Difco Lactobacilli MRS Broth (hereinafter referred to as MRS), and used for subsequent tests.
２）抗菌性試験
米麹中より分離した乳酸菌の抗菌性試験は、一般抗菌薬感受性試験法に準じた方法によるカップ（円筒）法を用い（図１）、味噌から分離のコロニーの形状および色が異なるBacillus属５株（Ａ〜Ｅ）を指示菌とし、カップ周辺のBacillus属の生育阻止円の有無で判定した。すなわち、標準寒天平板培地（グルコース１％添加）を30℃で２時間表面乾燥させ、それに味噌から分離したBacillus族の培養液（普通ブイヨン培地で培養）を0.1〜0.2ml塗沫後、直ちに殺菌済みのプラスチックカップ（直径１cm、高さ１cm）を差し込み、そのカップ内に、米麹より分離の乳酸菌をMRS培地で30℃、72時間培養後遠心分離して、pH6.8前後に調整した上澄液を0.1〜0.2ml添加し、30℃、72時間後のカップ周辺のクリアゾーンを観察した。
2) Antibacterial test The antibacterial test of lactic acid bacteria isolated from rice bran uses the cup (cylindrical) method according to the general antimicrobial susceptibility test method (Fig. 1). 5 strains (A to E) of different genus Bacillus were used as indicator bacteria, and the presence or absence of a growth inhibition circle of Bacillus genus around the cup was determined. That is, a standard agar plate medium (with 1% glucose) was surface-dried at 30 ° C for 2 hours, and 0.1 to 0.2 ml of Bacillus culture broth (cultured in normal broth medium) separated from miso was smeared immediately. Insert a plastic cup (1 cm in diameter and 1 cm in height) into the cup, and cultivate lactic acid bacteria isolated from rice bran in MRS medium at 30 ° C for 72 hours and then centrifuge to adjust the pH to around 6.8. 0.1 to 0.2 ml of the supernatant was added, and a clear zone around the cup after 30 hours at 30 ° C. was observed.
３）分類、同定試験
抗菌性が認められた乳酸菌と思われる菌株を、生理的手法と分子生物学的手法によって同定試験を行った。
イ．生理的諸性質
乳酸菌実験マニュアルに従って、グラム染色、カタラーゼ反応、形態観察、生育温度、耐塩性等を調べた。炭素源の資化性試験はAPI 50 CH（日本ビオメリュー社製）を用いて行った。
ロ．16SrRNAの塩基配列の解析
16SrRNA遺伝子の塩基配列解析および相同性検索を常法により行った。
3) Classification and identification test Identification tests were carried out on physiological strains and molecular biology methods for bacterial strains that appeared to have antibacterial properties.
A. Physiological properties According to the lactic acid bacteria experiment manual, Gram staining, catalase reaction, morphology observation, growth temperature, salt tolerance, etc. were examined. The carbon source assimilation test was conducted using API 50 CH (manufactured by Biomeryu Japan).
B. Analysis of 16S rRNA nucleotide sequence
Base sequence analysis and homology search of 16S rRNA gene were performed by conventional methods.
４）抗菌性乳酸菌接種による製麹
イ．フラスコでの製麹
蒸米100gを500mlの三角フラスコに入れ、120℃、５分間殺菌後、麹汁斜面培地で純粋培養した麹菌胞子の懸濁液を約10６（cfu/g）と、Bacillus cereus JCM2152T(普通ブイヨン培地で培養)を約10５（cfu/g）とを接種し、乳酸菌接種区分ではさらに抗菌性を示したNo.7、No.8、No.12株の培養液（MRS培地）を約10６（cfu/g）を接種し、また対照区では分離株で抗菌性を示さなかった菌株の中からNo.11（未同定）を選び、同様に接種した。それを30℃の恒温室で43時間製麹を行った。
ロ.小型通風製麹機での製麹
蒸米10Kgに常法通り種麹菌を種付けし、味噌から分離したBacillus Ｄ株の培養液を約10４（cfu/g）接種（噴霧）した対照区と、さらにそこへ乳酸菌No.12の培養液を約10５（cfu/g）噴霧した試験区を設け、小型通風製麹機を用い43時間製麹を行った。
4) Koji making by inoculation with antibacterial lactic acid bacteria. Fermentation with Flask 100 g of steamed rice was put into a 500 ml Erlenmeyer flask, sterilized at 120 ° C for 5 minutes, and then a suspension of Neisseria gonorrhoeae spores that were purely cultured in a broth slant medium was about 10 6 (cfu / g), and Bacillus cereus JCM2152 T (cultured in normal bouillon medium) was inoculated with about 10 5 (cfu / g), and the culture solution (MRS) of No.7, No.8 and No.12 strains that showed further antibacterial activity in the lactic acid bacteria inoculation category The medium was inoculated with about 10 6 (cfu / g), and No. 11 (unidentified) was selected from the isolates that did not show antibacterial activity in the control group and inoculated in the same manner. It was smelted for 43 hours in a constant temperature room at 30 ° C.
B. Smoldering with a small ventilating machine A control group inoculated (sprayed) with approximately 10 4 (cfu / g) of Bacillus D strain culture seeded with 10 kg of steamed rice as usual and seeded with Bacillus D strain isolated from miso Further, a test area was sprayed with about 10 5 (cfu / g) of the culture solution of lactic acid bacteria No. 12 there, and the plate was made for 43 hours using a small ventilation machine.
５）乳酸菌およびBacillus属の生菌数の測定
乳酸菌の生菌数の測定は、１）の分離培地と同じ培地を用い、重層法にて30℃、３日間培養後のBCPが変色したコロニー乳酸菌として測定した。
Bacillus属の生菌数は、標準寒天培地にカビサイジン（100mg力価/ｌ）を加えた培地を用いて常法どおり行った。
６）麹のプロテアーゼ活性と乳酸量の測定
麹のプロテアーゼ活性は蔭山変法で、乳酸量はBarker,Summerson法で行った。
5) Measurement of the number of living bacteria of lactic acid bacteria and genus Bacillus The number of living bacteria of lactic acid bacteria was measured using the same medium as the separation medium of 1), and colony lactic acid bacteria whose BCP had been discolored after culturing at 30 ° C for 3 days by the multilayer method. As measured.
The number of viable bacteria of the genus Bacillus was determined in the usual manner using a medium obtained by adding kabacidine (100 mg titer / l) to a standard agar medium.
6) Determination of protease activity and amount of lactic acid in cocoon Protease activity in cocoon was performed by the modified Hiyama method, and the amount of lactic acid was measured by the Barker and Summerson method.
２.実施結果
１）抗菌性乳酸菌の分離株と同定
製麹工程においてBacillus属などの汚染防止に対して、抗菌性乳酸菌を利用する場合は、その乳酸菌が製麹初期において、蒸米上で旺盛に増殖することが必須条件となる。そこで抗菌性乳酸菌の分離源を使用目的の生育環境に近い米麹中に求め、分離およびBacillus属に対する抗菌性菌の検索を行った（前記カップ法）。
その結果、表１および図１にみられるように分離株No.7、8、12が、味噌より分離のBacillus属５株に対して抗菌性を示した。図１において、No.12と表記したカップは、分離株No.12を接種した株であり、カップ内およびその周辺にクリアゾーンが見られ、Bacillus属を低減し、Bacillus属に対する抗菌性を有していることがわかる。なお、図１におけるカップＣには、分離株で抗菌性を示さなかった菌株を接種した対照区であり、クリアゾーンを示さず、Bacillus属に対する抗菌性を示していない。また、同様のカップ法によって、No.7、およびNo.8の菌株の抗菌性も確認された。
2. Implementation results 1) Isolation and identification of antibacterial lactic acid bacteria When antibacterial lactic acid bacteria are used to prevent contamination of the genus Bacillus in the koji making process, the lactic acid bacteria are vigorously grown on steamed rice at the initial stage of koji making. Proliferation is a prerequisite. Therefore, a source of antibacterial lactic acid bacteria was obtained in rice bran close to the intended growth environment, and isolation and antibacterial bacteria against the genus Bacillus were searched (the cup method).
As a result, as shown in Table 1 and FIG. 1, isolates Nos. 7, 8, and 12 showed antibacterial activity against 5 strains of Bacillus isolated from miso. In FIG. 1, the cup labeled No. 12 is a strain inoculated with isolate No. 12, with clear zones in and around the cup, reducing the genus Bacillus and having antibacterial properties against the genus Bacillus. You can see that In addition, the cup C in FIG. 1 is a control group inoculated with a strain that did not show antibacterial activity as an isolate, does not show a clear zone, and does not show antibacterial activity against the genus Bacillus. In addition, the antibacterial properties of No. 7 and No. 8 strains were also confirmed by the same cup method.
表１
Table 1
抗菌性物質生産性を有する乳酸菌として取得したこの３株について、16SrRNAの塩基配列解析後、相同性検索を行った。その結果、No.7は、Leuconostoc citreum ATCC49370と相同値が99.5％、No.8は、Weissella cibaria LMG17699と相同値が99.7％、No.12は、Lactobacillus fermentum YB5と相同値が99.6％であった。これらの乳酸菌は独立行政法人 産業技術研究所 特許生物寄託センターに寄託してあり、受託番号は、No.7が、FERM P-21075、No.8が、FERM P-21076、No.12が、FERM P-21074である。 The three strains obtained as lactic acid bacteria having antibacterial substance productivity were subjected to homology search after analyzing the base sequence of 16S rRNA. As a result, No. 7 had a homology value of 99.5% with Leuconostoc citreum ATCC49370, No. 8 had a homology value of 99.7% with Weissella cibaria LMG17699, and No. 12 had a homology value of 99.6% with Lactobacillus fermentum YB5. . These lactic acid bacteria are deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, and the accession numbers are No. 7, FERM P-21075, No. 8, FERM P-21076, No. 12, FERM P-21074.
次に確認のため炭素源の資化性等の表現形質を基準株と比較した結果を表２に示した。
表２
Next, for comparison, the results of comparison of phenotypic characteristics such as assimilability of the carbon source with the reference strain are shown in Table 2.
Table 2
基準株は、No.7に対し、Leuconostoc citreum JCM9698Tを、No.8に対し、Weissella cibaria JCM12495T を、No.12に対しては、Lactobacillus fermentum JCM1173T を各々用いた。No.7は、基準株と比べGalactoseを資化し、Amygdalineの資化は極微弱であった。その他の炭素源の資化性は基準株と一致した。No.8は、全ての炭素源の資化性が基準株と一致していた。No.12については、今回用いた基準株と比べ、L-Arabinose、D-Xylose、D-Mannose、Trehalose、5-keto-gluconateを資化した。しかし、これらの炭素源の資化は、API 50 CHL Mediumプロトコルの陽性率表にあるLactobacillus fermentum種が示す陽性比率（L-Arabinose 31％、D-Xylose 50％、D-Mannose 50%、Trehalose 16％、5-keto-gluconate 1%）の各々の範囲内にあり、菌株内のバリエーションと考えられた。このNo.12は中でもまれに5-keto-gluconateを資化していた。
また、No.7、No.8、No.12はいずれもグラム染色において陽性を示し、カタラーゼ試験では陰性を示した。
Type strain, compared No.7, a Leuconostoc citreum JCM9698 T, to No.8, the Weissella cibaria JCM12495 T, for the No.12, it was used respectively Lactobacillus fermentum JCM1173 T. No. 7 assimilated Galactose compared to the reference stock, and Amygdaline was very weak. The availability of other carbon sources was consistent with the reference stock. In No. 8, the assimilation of all carbon sources was consistent with the reference stock. For No. 12, L-Arabinose, D-Xylose, D-Mannose, Trehalose, and 5-keto-gluconate were assimilated compared to the reference strain used this time. However, the utilization of these carbon sources is due to the positive ratios (L-Arabinose 31%, D-Xylose 50%, D-Mannose 50%, Trehalose 16%) shown in the positive rate table of API 50 CHL Medium protocol. %, 5-keto-gluconate 1%), and it was considered a variation within the strain. This No. 12 rarely utilized 5-keto-gluconate.
No. 7, No. 8, and No. 12 were all positive in Gram staining and negative in the catalase test.
また、分離株の耐塩性と生育温度範囲を調べたところ、No.7、8が食塩7.5wt％まで生育でき中程度の耐塩性菌と考えられ、No.12は５wt％までの生育であり耐塩性はほとんど認められなかった。生育温度は、No.7が15〜35℃、No.8は15〜40℃の範囲であったが、No.12は20〜50℃の比較的高温度域でも生育し、製麹時の蒸米の冷却温度が高めでも対応可能な菌と思われる。
以上、分離株の16SrRNA遺伝子シーケンスの解析と表現形質から、No.7は、Leuconostoc citreum （以下Leu. Citreum L7）、No.8は、Weissella cibaria (以下W. cibaria L8)、No.12は、Lactobacillus fermentum（以下Lb. fermentum L12）とそれぞれ同定された。
In addition, when the salt tolerance and growth temperature range of the isolate were examined, No.7 and 8 were considered to be moderate salt-tolerant bacteria capable of growing up to 7.5 wt% of salt, and No.12 was grown up to 5 wt%. Almost no salt tolerance was observed. The growth temperature was 15 to 35 ° C for No.7 and 15 to 40 ° C for No.8, but No.12 grew even at a relatively high temperature range of 20 to 50 ° C. It seems to be a fungus that can handle even when the cooling temperature of steamed rice is high.
From the analysis and phenotype of the 16S rRNA gene sequence of the isolate, No.7 is Leuconostoc citreum (hereinafter Leu. Citreum L7), No.8 is Weissella cibaria (hereinafter W. cibaria L8), and No.12 is It was identified as Lactobacillus fermentum (hereinafter Lb. fermentum L12).
２）フラスコ製麹での抗菌性乳酸菌によるBacillus cereusの抑制効果
味噌より分離したBacillus５株に対して、抗菌性が認められた乳酸菌Leu. Citreum L7（No.7）、W. cibaria L8（No.8)、Lb. fermentum L12（No.12）各々について、製麹中でのBacillus cereus JCM2152T(以下B.cereus)に対する抗菌作用を、製麹時に他の菌の影響がないフラスコ製麹で検討した。
2) Inhibitory effect of Bacillus cereus by antibacterial lactic acid bacteria in flask koji Lactic acid bacteria Leu. Citreum L7 (No.7), W. cibaria L8 (No. 8) For each of Lb. fermentum L12 (No. 12), antibacterial activity against Bacillus cereus JCM2152 T (hereinafter referred to as B. cereus) during koji making was examined with flask koji making no influence of other bacteria during koji making. did.
その結果を図２に示した。製麹時に10６（cfu/g）レベル接種した４株の乳酸菌区分は、何れも１９時間後には10８〜10９（cfu/g）に達した。麹菌とB.cereusのみ接種の対照区は、製麹中のB.cereus菌数が接種菌数に近い10５（cfu/g）前後の高いレベルを維持したままであった。また、No.11の抗菌性を有しない乳酸菌を接種した区分もB.cereus菌数は対照区と同様に高いレベルを維持していた。それに対し、Leu. Citreum L7、W. cibaria L8、Lb. fermentum L12の抗菌性乳酸菌を接種した区分は明らかにB.cereusの増殖を抑制した。中でもW. cibaria L8、Lb. fermentum L12接種区分にそれが顕著で、両者とも製麹１９時間後にはB.cereusは検出されなかった。Leu. Citreum L7接種区分は、B.cereus 菌数が初発より１/1000程度の減少があった。
このフラスコ製麹実施結果により比較的高温でも生育可能なことが明らかとなったLb. fermentum L12を用いて通風製麹を行った。
The results are shown in FIG. The four strains of lactic acid bacteria that were inoculated at a level of 10 6 (cfu / g) at the time of koji making reached 10 8 to 10 9 (cfu / g) after 19 hours. In the control group inoculated with koji mold and B. cereus alone, the number of B. cereus bacteria in the koji making remained high at around 10 5 (cfu / g), which was close to the number of inoculated bacteria. In addition, the number of B. cereus bacteria in the group inoculated with No. 11 non-antibacterial lactic acid bacteria was maintained at a high level as in the control group. In contrast, Leu. Citreum L7, W. cibaria L8, and Lb. fermentum L12 inoculated with antibacterial lactic acid bacteria clearly inhibited the growth of B. cereus. In particular, this was remarkable in the inoculation sections of W. cibaria L8 and Lb. fermentum L12, and B. cereus was not detected after 19 hours of the preparation. In the Leu. Citreum L7 inoculation category, the number of B. cereus bacteria was reduced by about 1/1000 from the first occurrence.
Ventilation ironmaking was performed using Lb. fermentum L12, which was found to be able to grow even at a relatively high temperature from the results of this flask making.
３）小型通風製麹機でのLb. fermentum L12によるBacillusの抑制効果
通常の製麹条件に近い小型通風製麹機を用い、蒸米10Kgを製麹してLb. fermentum L12接種によるBacillus属の抑制効果を検討した。
その製麹経過を図３（ａ）〜（ｄ）に示した。製麹時にLb. fermentum L12を10５（cfu/g）レベル接種した区分の生酸菌数（乳酸菌）は、１９時間後には10７（cfu/g）レベルに達していた（図３（ａ））。対照区は乳酸菌を接種していないが、製麹作業工程から混入した乳酸菌と思われる生酸菌が10５（cfu/g）程度まで増殖がみられたものの、Bacillusの菌数は、接種菌数に近い10４（cfu/g）付近のままであった。これに対し、Lb. fermentum L12を接種した区分は、１９時間後にBacillusの菌数が数個以内（10倍希釈）に激減していた。今回のLb. fermentum L12の接種菌数が10５（cfu/g）レベルであったので、この接種菌数を10６（cfu/g）レベルに増加させれば、さらにBacillus属の抑制効果が期待できる。
3) Inhibitory effect of Bacillus by Lb. fermentum L12 on small ventilation machine Suppressing Bacillus genus by Lb. fermentum L12 inoculation with 10g of steamed rice using a small ventilation machine close to normal conditions The effect was examined.
The iron making process is shown in FIGS. The number of live acid bacteria (lactic acid bacteria) in the section inoculated with 10 5 (cfu / g) of Lb. fermentum L12 at the time of koji making reached 10 7 (cfu / g) after 19 hours (FIG. 3 (a )). Although the control group did not inoculate lactic acid bacteria, the number of Bacillus bacteria was inoculated, although there was growth of about 10 5 (cfu / g) of raw acid bacteria that seemed to be lactic acid bacteria mixed from the koji making process. It remained around 10 4 (cfu / g) which was close to the number. On the other hand, in the group inoculated with Lb. fermentum L12, the number of Bacillus bacteria was drastically reduced within several hours (diluted 10 times) after 19 hours. Since the number of inoculated bacteria of Lb. fermentum L12 this time was 10 5 (cfu / g) level, if this number of inoculated bacteria is increased to 10 6 (cfu / g) level, the inhibitory effect of Bacillus genus will be further increased. I can expect.
なお、そのときの米麹のプロテアーゼ活性は、Lb. fermentum L12を接種した区分の方がむしろ高めであり、この接種した乳酸菌による麹菌への生育阻害は認められなかった（図３（ｂ））。このように抗菌性乳酸菌を接種した米麹が、プロテアーゼ活性が若干高い傾向にあるが、これはBacillus属の麹菌の生育阻害を、抗菌性乳酸菌によって抑えたためと考えられる。
また、製麹中の乳酸量は、Lb. fermentum L12接種区分でも最大値が15mg/100g程度で対照区と大差なく、抗菌作用が期待できるほどのものではなかった。したがって、製麹中のpHの推移も両者間に大きな相違はみられなかった（図３（ｃ）、（ｄ））。
In addition, the protease activity of the rice bran at that time was rather higher in the section inoculated with Lb. fermentum L12, and growth inhibition to the koji mold by the inoculated lactic acid bacteria was not observed (FIG. 3 (b)). . The rice bran inoculated with antibacterial lactic acid bacteria as described above tends to have a slightly higher protease activity, which is thought to be because the inhibition of growth of Bacillus genus bacilli was suppressed by the antibacterial lactic acid bacteria.
In addition, the amount of lactic acid in the koji was about 15 mg / 100 g in the Lb. fermentum L12 inoculation category, which was not much different from the control group, and was not so high that antibacterial action could be expected. Therefore, there was no significant difference between the pH during the iron making (FIGS. 3 (c) and 3 (d)).
図４は、Lb. fermentum L12を接種した通風製麹中における抗菌作用の様子を経時的にみたものである。製麹中に麹を手入れ作業ごとにサンプリングし、B.cereus JCM2152Tを指示菌として、麹の抗菌性を調べたところ１９時間目の麹には明らかな阻止円が認められた。しかし２７時間目以降の麹にはそれがみられなかった。乳酸菌が生産する抗菌性物質に乳酸などの有機酸、過酸化水素、ジアセチルおよび低分子のタンパク質性の物質（バクテリオシン）などが知られている。このことと、製麹中のプロテアーゼ活性が２７時間目前後に急伸していることを考え合わせると、Lb. fermentum L12から生産されたタンパク質性の抗菌物質が、麹のプロテアーゼによって分解を受けたためではないかと思われる。
しかしながら、製麹過程における細菌の増殖は、引き込み後、比較的初期の１８時間付近で最高菌数に達し、以後製麹後半のＡｗ（水分活性）の低下により細菌の増殖は停止に向かうことから、製麹前半にLb. fermentum L12の抗菌性が作用していれば、Bacillus属の増殖の抑制が十分可能と考えられる。
FIG. 4 shows a time course of antibacterial activity in the ventilated koji seeded with Lb. fermentum L12. Samplings were sampled at every care during the koji making, and B.cereus JCM2152 T was used as an indicator to examine the antibacterial properties of the koji. However, it was not seen in the kites after the 27th hour. As antibacterial substances produced by lactic acid bacteria, organic acids such as lactic acid, hydrogen peroxide, diacetyl, and low molecular weight protein substances (bacteriocin) are known. Considering this and the fact that the protease activity during koji making increased rapidly around the 27th hour, it is not because the proteinous antibacterial substance produced from Lb. fermentum L12 was degraded by the koji protease. I think.
However, the growth of bacteria in the koji making process reaches the maximum number of bacteria in the relatively early 18 hours after entrainment, and then the growth of bacteria tends to stop due to a decrease in Aw (water activity) in the latter half of the koji making. If the antibacterial activity of Lb. fermentum L12 acts on the first half of the koji, it is considered possible to sufficiently suppress the growth of the genus Bacillus.
なお、本発明に係る乳酸菌は、製麹のみでなく、他の用途にも利用できることはもちろんである。 Of course, the lactic acid bacteria according to the present invention can be used not only for ironmaking but also for other purposes.
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