TW202214838A - Lactic acid bacteria and their acquisition method, and foods and drinks containing lactic acid bacteria - Google Patents

Abstract

An object of the invention is to provide a lactic acid bacteria that can grow well in a low pH and high sucrose environment. The solution is a lactobacillus species (Lactobacillus sp.) which is registered with the deposit number: NITE BP-03116 (identification mark: WR16-4).

Description

Lactic acid bacteria, method for obtaining the same, and food and drink containing lactic acid bacteria

The present invention relates to a novel lactic acid bacteria, a method for obtaining the same, and a lactic acid bacteria-containing food and drink containing the aforementioned lactic acid bacteria.

Generally, the optimal growth pH range of lactic acid bacteria is pH6-7, and generally speaking, the growth limit of the acid region is pH4, and the growth limit of the alkaline region is pH8. However, among the lactic acid bacteria, there are those that have excellent acid resistance and can still grow in a severe environment (about pH 3) where general lactic acid bacteria are not easy to grow (for example, Patent Documents 1 and 2). In addition, there are lactic acid bacteria having excellent salt tolerance that can grow in environments with high salt concentration such as miso and soy sauce (for example, Patent Documents 3 and 4). In this way, although there are a variety of lactic acid bacteria that can still grow under severe conditions among the lactic acid bacteria; and as far as the applicant of this case is aware, so far no one has reported that it can grow well under the severe environment of low pH (high acidity) and high sucrose concentration. of lactic acid bacteria present. Therefore, based on the existence of such lactic acid bacteria, the applicant of the present case concluded that, for example, the juice obtained by mixing and squeezing plants and sugar, or beverages based on this juice, or jams, fruit sauces and other acid values and sucrose can be easily High concentration, it is not easy to ferment with the materials fermented by general lactic acid bacteria, but can provide new fermented foods or fermented beverages, and research on lactic acid bacteria that can grow well in low pH environment and high sucrose environment as mentioned above, and finally successfully obtained the Lactic acid bacteria that grow well in harsh environments with low pH and high sucrose concentration. [Prior Art Literature] [Patent Literature] [Patent Document 1] Japanese Patent Application Laid-Open No. 2009-225792 [Patent Document 2] WO2013/001862 [Patent Document 3] Japanese Patent Laid-Open No. 2007-236344 [Patent Document 4] Japanese Patent Laid-Open No. 2018-42557

[The problem to be solved by the invention] The present invention aims to provide lactic acid bacteria that can grow well in a low pH and high sucrose environment. [Means of Solving Problems] The gist of the present invention will be explained with reference to the accompanying drawings. A lactic acid bacteria is characterized in that it belongs to Lactobacillus sp. which is registered with the deposit number: NITE BP-03116 (identification mark: WR16-4). Furthermore, the lactic acid bacteria according to claim 1, wherein the lactic acid bacteria have the nucleotide sequence of the 16S rDNA region shown in SEQ ID NO: 1. Also, the lactic acid bacteria according to claim 1, which are obtained from plant fermentation extracts. Also, the lactic acid bacteria of claim 2 are obtained from plant fermentation extracts. Also, the lactic acid bacteria according to claim 3, wherein the plant fermentation extract is a radish fermentation extract. Also, the lactic acid bacteria according to claim 4, wherein the aforementioned plant fermentation extract is a radish fermentation extract. Furthermore, the lactic acid bacteria according to claim 5, wherein the radish fermentation extract is obtained by mixing and fermenting radish, caster sugar and bacterial liquid. Furthermore, the lactic acid bacteria according to claim 6, wherein the radish fermentation extract is obtained by mixing radish, caster sugar and bacterial liquid, and fermenting them. Also, the lactic acid bacteria according to claim 7, wherein the bacterial liquid contains yeast. Furthermore, the lactic acid bacteria according to claim 8, wherein the bacterial liquid contains yeast. Furthermore, the lactic acid bacteria according to any one of claims 1 to 10 can be propagated in a low pH environment of pH 3.5 to 5.5, and can also be propagated in a high concentration sucrose environment of 30 to 60 wt % of sucrose. Also, the lactic acid bacteria according to any one of claims 1 to 10, which can multiply in a low pH environment of pH 3.5 to 5.5, and in a fructose and glucose high concentration environment with fructose concentration and glucose concentration of 10 to 30 wt% each It can also reproduce below. Also, as in the lactic acid bacteria of any one of claims 1 to 10, it can multiply in a low pH environment of pH 3.5 to 5.5, and can also reproduce in a high concentration sucrose environment of 30 to 60 wt% sucrose concentration; even in It can reproduce in the low pH environment of pH 3.5-5.5, and can also reproduce in the high-concentration environment of fructose and glucose with fructose concentration and glucose concentration of 10-30wt% respectively. Also, a method for obtaining lactic acid bacteria is characterized by: adding a plant fermentation extract to a sterilized liquid medium to obtain a culture solution, then multiplying and culturing the culture solution to obtain a growth culture solution, and then adding the growth culture solution to the culture solution. The sterilized separation medium is mixed and released to prepare a plate medium, and then the colonies appearing in the plate medium are fished out to obtain lactic acid bacteria that can multiply in a low pH environment and a high concentration sucrose environment. Also, the method for obtaining lactic acid bacteria according to claim 14, wherein the plant fermentation extract is a radish fermentation extract obtained by mixing and fermenting radish, caster sugar, and bacterial liquid. Also, the method for obtaining lactic acid bacteria according to claim 15, wherein the bacterial liquid contains yeast. Also, the method for obtaining lactic acid bacteria according to any one of claims 14 to 16, wherein the liquid medium is a mixed fermentation extract obtained by using either or both of vegetable fermentation extract and fruit fermentation extract, and It is obtained by mixing the boiled juice of wild grass and fine white sugar, and then adding antibiotics to the vegetable and fine white sugar mixture. Also, the method for obtaining lactic acid bacteria according to claim 17, wherein the antibiotic is cycloheximide. Moreover, the food-drinks containing lactic acid bacteria are characterized by containing the lactic acid bacteria in any one of Claims 1-10. Moreover, the food-drinks containing lactic acid bacteria are characterized by containing the lactic acid bacteria as claimed in claim 11. Moreover, the food-drinks containing lactic acid bacteria are characterized by containing the lactic acid bacteria as claimed in claim 12. Moreover, the food-drinks containing lactic acid bacteria are characterized by containing the lactic acid bacteria as claimed in claim 13. Moreover, the food-drinks containing lactic acid bacteria according to claim 19, wherein the food-drinks containing lactic acid bacteria are fermented food-drinks. Moreover, the food-drinks containing lactic acid bacteria according to claim 20, wherein the food-drinks containing lactic acid bacteria are fermented food-drinks. Moreover, the food-drinks containing lactic acid bacteria according to claim 21, wherein the food-drinks containing lactic acid bacteria are fermented food-drinks. Moreover, the food-drinks containing lactic acid bacteria according to claim 22, wherein the food-drinks containing lactic acid bacteria are fermented food-drinks. [Effect of invention] Since the lactic acid bacteria of the present invention can grow (reproduce) excellently in a low pH and high sucrose environment, for example, a juice obtained by mixing and squeezing a plant and sugar, a beverage based on the juice, or a jam can be easily made. , fruit sauce and other high acid value and sucrose concentration, it is not easy to ferment with the materials fermented by ordinary lactic acid bacteria, and can provide new fermented foods or fermented beverages.

為確認依上述分離方法取得之WR16-4株之特性，而使用一般乳酸菌進行根據與此等之比較的pH耐性試驗及蔗糖耐性試驗。 具體而言，作為WR16-4株之比較對象之乳酸菌，係使用乳酸菌當中屬週知之屬之種，且與一般乳酸菌相同，最佳生長pH區為pH6～7的乳酸桿菌(Lactobacillus)屬之Lactobacillus acidophilus JCM 1132 ^T、乳酸球菌(Lactococcus)屬之Lactococcus lactis subsp. lactis NBRC12007、白念珠菌(Leuconostoc)屬之Leuconostoc mesenteroides subsp. dextranicum NBRC100495 ^T此3種乳酸菌。 ＜各乳酸菌的前培養＞ 進行上述2種耐性試驗之際，對各乳酸菌進行如下之前培養。 (1) 以2倍濃縮調製圖9所示MRS培養基。此時，藉由添加4N氫氧化鈉溶液或4N鹽酸，而將WR16-4株用培養基調整為pH4.5、比較對象用培養基調整為pH6.5。 (2) 調製4wt%蔗糖溶液或4wt%葡萄糖溶液。 (3) 針對上述(1),(2)，分別進行使用高壓釜之濕熱滅菌(121℃,20分鐘)。 (4) 將經濕熱滅菌之(1)及(2)之溶液冷卻後，於無塵實驗台內以1：1(v/v)混合，調製成含有2wt%蔗糖之MRS培養基(pH4.5)或含有2wt%葡萄糖之MRS培養基(pH6.5)。 (5) WR16-4株係使用含有2wt%蔗糖之MRS培養基(pH4.5)，比較對象用則使用含有2wt%葡萄糖之MRS培養基(pH6.5)，以30℃、24～48小時之厭氧條件進行。 ＜pH耐性試驗＞ 如下進行經前培養之各乳酸菌的pH耐性試驗。 (1) 以2倍濃縮調製圖9所示MRS培養基。此時，藉由添加4N氫氧化鈉溶液或4N鹽酸，而調整為pH3,3.5,4,4.5,5,5.5,6,6.5。 (2) 調製4wt%蔗糖溶液。 (3) 針對上述(1),(2)，分別進行使用高壓釜之濕熱滅菌(121℃,20分鐘)。 (4) 將經濕熱滅菌之(1)及(2)之溶液冷卻後，於無塵實驗台內以1：1(v/v)混合，調製成含有2wt%蔗糖之MRS培養基(pH3～6.5)。 (5) 將經前培養之各乳酸菌株進行離心分離(15,000× g,10分鐘)而去除上清液後，以初始濃度成為A _660nm≒0.05～0.10的方式使其再懸浮於(3)中調製之培養基。 (6) 以成為200μl/well的方式，按每3well分注至96孔微孔盤後(n=3)，以30℃、厭氧條件進行培養。 (7) 使用微盤分析儀隨時間經過進行濁度測定。 圖1～3為表示比較對象用(Lactobacillus acidophilus JCM 1132 ^T(圖1)、Lactococcus lactis subsp. lactis NBRC12007 (圖2)、Leuconostoc mesenteroides subsp. dextranicum NBRC100495 ^T(圖3))之乳酸菌的濁度測定結果的圖表；圖4為表示WR16-4株之濁度測定結果的圖表。 在以上述2wt%蔗糖為碳源的pH耐性試驗中，如各乳酸菌的濁度測定結果所示，相對於比較對象用之乳酸菌在pH5～6.5之接近中性的酸性區生長良好，WR16-4株係於此等乳酸菌良好地生長之區域更靠酸性側的區域，具體而言為pH3.5～5之低pH區生長，經確認其在pH較此為高之接近中性的區域幾乎無法生長。 ＜蔗糖耐性試驗＞ 如下進行經前培養之各乳酸菌的蔗糖耐性試驗。 (1) 將添加有蔗糖的圖9所示MRS培養基調整成蔗糖濃度為5wt%, 10wt%, 20wt%, 30wt%, 40wt%, 50wt%, 60wt%, 70wt%後，對各者添加4N鹽酸而調整成pH4.5。 (2) 針對上述(1)，分別進行使用高壓釜之濕熱滅菌(121℃,20分鐘)。 (3) 將經前培養之各乳酸菌株進行離心分離(15,000 ×g,10分鐘)而去除上清液後，以初始濃度成為A _660nm≒0.05～0.10的方式使其再懸浮於(2)中調製之培養基。 (4) 以成為200μl/well的方式，按每3well分注至96孔微孔盤後(n=3)，以30℃、厭氧條件進行培養。 (5) 使用微盤分析儀隨時間經過進行濁度測定。 圖5～8為表示比較對象用(Lactobacillus acidophilus JCM 1132 ^T(圖5)、Lactococcus lactis subsp. lactis NBRC12007 (圖6)、Leuconostoc mesenteroides subsp. dextranicum NBRC100495 ^T(圖7))之乳酸菌的濁度測定結果的圖表；圖8為表示WR16-4株之濁度測定結果的圖表。 在上述初始pH4.5之酸性條件下的蔗糖耐性試驗中，如各乳酸菌的濁度測定結果所示，結果確認比較對象用之乳酸菌可於蔗糖濃度20wt%以下生長，但其生長狀態(繁殖率)低，在30wt%以上的高蔗糖環境則幾乎無法生長；相對於此，確認WR16-4株在蔗糖濃度約40wt%前顯示良好的生長狀態，且於60wt%之高蔗糖環境下仍可生長。 ＜果糖暨葡萄糖耐性試驗＞ 蔗糖為單糖之果糖與葡萄糖鍵結而成的雙糖類，由於確認WR16-4株如上述在高蔗糖環境下可生長，吾人試想其可能在果糖及葡萄糖各以單糖狀態存在的果糖暨葡萄糖高濃度環境下，亦可與蔗糖之情形同樣地生長，而與上述蔗糖耐性試驗同樣地如下進行果糖暨葡萄糖耐性試驗。 (1) 將各自單獨添加有蔗糖、果糖、葡萄糖之糖的圖10所示MRS培養基分別調整成糖濃度為20wt%後，對各者添加4N鹽酸而調整成pH4.5。 (2) 另外將添加有果糖及葡萄糖的圖10所示MRS培養基調整成果糖、葡萄糖各者的糖濃度為10wt%(合計20wt%)後，對各者添加4N鹽酸而調整成pH4.5。 (3) 針對上述(1),(2)，分別進行使用高壓釜之濕熱滅菌(121℃,20分鐘)。 (4) 將經前培養之WR16-4株進行離心分離(15,000× g,10分鐘)而去除上清液後，以初始濃度成為A _660nm≒0.05～0.10的方式使其再懸浮於調製之培養基。 (5) 以成為200μl/well的方式，按每3well分注至96孔微孔盤後(n=3)，以30℃、厭氧條件進行培養。 (6) 使用微盤分析儀隨時間經過進行濁度測定。 將在此初始pH4.5之酸性條件下之果糖暨葡萄糖耐性試驗的上述濁度測定的結果示於圖11。 如此圖11所示，WR16-4株單獨含有果糖、葡萄糖任一者時其繁殖率較低(為低生長狀態)；而含有果糖、葡萄糖兩者時，與含有蔗糖時相同，繁殖率較高(呈高生長狀態)，由此結果確認，WR16-4株具有蔗糖耐性及果糖暨葡萄糖耐性。 又，如前述，由於蔗糖為果糖與葡萄糖鍵結而成的雙糖類，因此，WR16-4株，與蔗糖之情形相同，例如在各含30wt%的果糖與葡萄糖(糖濃度的合計為60wt%)的高糖濃度環境亦可充分生長。 ＜WR16-4株之用途＞ WR16-4株可使用於含有乳酸菌之飲食品，具體而言為發酵飲食品。 諸如上述，WR16-4株由於為具有在低pH且在高蔗糖環境下可優良地繁殖之特徵的乳酸菌，而能夠容易地使例如植物與糖經混合並榨壓而得之汁液或以此汁液為基底之飲料，或者果醬、水果醬料等酸性值及蔗糖濃度高，不易以一般乳酸菌發酵的素材(酸性且高糖濃度萃取液)發酵，藉此可使前述萃取液含有有機酸，而能夠獲得增添酸味(減少甜味或增添爽口感)或防腐(不需要屬添加物之保存劑)等效果。 再者，WR16-4株由於為具有在低pH且高果糖暨葡萄糖環境下亦可優良地繁殖之特徵的乳酸菌，除上述外，亦可使用於採用含有果糖與葡萄糖此兩者的糖(高果糖漿、葡萄糖及果糖各精製糖混合而成者等)之飲食品。 又，以下示出上述酸性且高糖濃度萃取液之取得方法的一例。此外，萃取液之取得方法非限定於此。 (1) 將切好的蔬菜或水果與細白砂糖以等比率混合，添加菌液使其發酵。 (2) 發酵後，進行搾汁並回收汁液成分，使此回收液進一步發酵、熟成而得到發酵液A。 (3) 對野草之熬汁添加細白砂糖後對其添加發酵液A，使其發酵、熟成而得到發酵液B。 此發酵液A或發酵液B為酸性且高糖濃度萃取液。此外，此發酵液A及發酵液B將製成為飲料，藉由WR16-4株使其發酵，可增添酸味，且無需添加保存劑即可獲得防腐效果，可提供前所未有的飲料。 再者，WR16-4株由於如上述試驗結果所示，具有在一般乳酸菌之最佳pH環境的pH6～7之環境下幾乎無法生長之特性，欲使例如蔗糖濃度高且pH為中性附近的素材以WR16-4株與其他微生物(例如一般的乳酸菌或酵母等)發酵時，因WR16-4株在達到最佳pH前的期間無法生長，而不會妨害其他微生物的生長。從而，利用多種微生物之複合發酵時為有用者。 又，基於上述特性，就WR16-4株，藉由將素材的pH調整於中性附近，能以非加熱方式使發酵停止，對利用於無法加熱的發酵製品屬有用者。 此外，本發明非限於本實施例，各構成要件之具體構成可適宜設計。 [Mode for Carrying Out the Invention] A preferred embodiment of the present invention will be briefly described based on the drawings showing the operation of the present invention. The lactic acid bacteria of the present invention belong to the genus Lactobacillus sp., and are deposited under the deposit number: NITE BP-03116 (identification mark: WR16-4). Based on the sequence data (16S rDNA sequence) obtained from the lactic acid bacteria of the present invention, the result of homology search in the gene database of NCBI (National Center for Biotechnology Information) shows that the homology with the above sequence is the highest at 93% (refer to FIG. 12 ). As a result, the lactic acid bacteria of the present invention can be described as an unprecedented new species of lactic acid bacteria (a species belonging to the genus Lactobacillus). The lactic acid bacteria of the present invention have the characteristic that they are not easy to grow at pH 6-7, which is regarded as the optimum growth environment for general lactic acid bacteria, and can grow well in an environment of pH lower than this (eg, pH 3.5-5.5). Furthermore, the lactic acid bacteria of the present invention have the feature that they can grow well in low pH (eg, pH 5 or less) and high sucrose (eg, 30 wt% or more) environments where general lactic acid bacteria are difficult to grow. In this way, the lactic acid bacteria of the present invention have the characteristics of being able to reproduce well in a low pH and high sucrose environment, and can easily make, for example, a juice obtained by mixing plants and sugar and squeezing or a beverage based on the juice, Or jams, fruit sauces, etc. have high acidity and sucrose concentration, and are not easily fermented with materials fermented by ordinary lactic acid bacteria. [Examples] Specific examples of the present invention will be described based on the drawings. The lactic acid bacteria in this example has the nucleotide sequence of the 16S rDNA region shown in SEQ ID NO: 1, and belongs to the Lactobacillus sp. of lactic acid bacteria. The cell shape of the lactic acid bacteria in this example is short rod-shaped, and the colony state is white, and can produce exopolysaccharide (sticky substance). The lactic acid bacteria of this example (hereinafter referred to as strain WR16-4) will be described in detail below. <About the strain of the WR16-4 strain> Based on the 16S rDNA data of the WR16-4 strain, a microorganism identification test was carried out, and the strain of the WR16-4 strain was investigated. In addition, the microbiological identification inspection is entrusted to FASMAC Co., Ltd. The results of microbial identification and inspection showed that the strain with the highest homology value was Lactobacillus kefiri (ATCC=35441) (homology value 93.14%). <About the novelty of the WR16-4 strain> Based on the 16S rDNA data of the WR16-4 strain, a homology search was performed in the gene database of NCBI, and the novelty of the WR16-4 strain was investigated. Figure 12 is a list of data showing high homology to the sample-derived sequence in this homology search. As shown in Fig. 12, the homology with the highest WR16-4 strain is only about 93%. Generally speaking, if there is no species with a homology value of 98.7% or more, it can be judged as a new species, so this WR16-4 strain can be regarded as a new type (new species) of lactic acid bacteria. <Isolation source of WR16-4 strain> The WR16-4 strain uses a plant fermentation extract, specifically, a radish fermentation extract as an isolation source. The radish fermented extract, which is an isolation source, is obtained by mixing and fermenting radish, caster sugar, and bacterial liquid; It is obtained by fermenting yeast in it, extracting juice to obtain a juice component, and further fermenting and aging the juice component. In addition, the plant as an isolation source may be a vegetable other than radish. <Isolation method of WR16-4 strain> The WR16-4 strain was isolated from the above-mentioned radish fermented extract, which could not be isolated by a general lactic acid bacteria isolation method, but was isolated by the following isolation method discovered by the present applicant. (1) 80 μl of the above-mentioned radish fermentation extract was added to 10 ml of the liquid medium which had been wet-sterilized (121° C., 15 minutes), and the mixture was incubated at 25° C. for 6 days to obtain a culture solution. (2) 80 μl of the obtained culture solution was added to 10 ml of the above-mentioned liquid culture medium, and the culture solution was again incubated at 25° C. for 3 to 4 days. (3) Proliferation culture in which the above-mentioned 2 treatment was repeated several times (8 times in this Example) was performed to obtain a growth culture medium. (4) 1 ml of the obtained growth medium was mixed with 15 ml of the separation medium which had been wet-sterilized (121° C., 15 minutes), and then cooled and solidified to prepare a plate. (5) The plate was incubated at 25°C for 10 days. (6) The colonies appearing on the above-mentioned plates were fished out to obtain the WR16-4 strain. In addition, the above-mentioned liquid medium is a mixed fermented extract obtained by using either or both of vegetable fermented extract and fruit fermented extract. After mixing the sugar mixture and adding antibiotics to it, it is obtained by mixing the above-mentioned mixed fermented extract and the boiled juice of various weeds at 1:1 with fine white sugar. Weed and fine white sugar mixture Mixed with 1g/L cycloheximide solution at 10:90:1. In addition, the above-mentioned medium for separation was obtained by mixing the medium A and medium B shown in the table below by wet sterilization.

In order to confirm the characteristics of the WR16-4 strain obtained by the above-described isolation method, a pH tolerance test and a sucrose tolerance test based on comparison with these were conducted using general lactic acid bacteria. Specifically, the lactic acid bacteria used as the comparison target of the WR16-4 strain were Lactobacillus species belonging to a well-known genus among lactic acid bacteria, and the optimum growth pH range was the same as that of general lactic acid bacteria. acidophilus JCM 1132 ^T , Lactococcus lactis subsp. lactis NBRC12007 of the genus Lactococcus, and Leuconostoc mesenteroides subsp. dextranicum NBRC100495 ^T of the genus Candida albicans. <Pre-culture of each lactic acid bacteria> When performing the above-mentioned two kinds of resistance tests, each of the lactic acid bacteria was subjected to the following pre-culture. (1) The MRS medium shown in Fig. 9 was prepared at a 2-fold concentration. At this time, by adding 4N sodium hydroxide solution or 4N hydrochloric acid, the medium for WR16-4 strain was adjusted to pH 4.5, and the medium for comparison object was adjusted to pH 6.5. (2) Prepare 4wt% sucrose solution or 4wt% glucose solution. (3) Moist heat sterilization using an autoclave (121° C., 20 minutes) was performed for the above (1) and (2), respectively. (4) After cooling the solutions of (1) and (2) sterilized by moist heat, mix them at a ratio of 1:1 (v/v) in a dust-free laboratory bench to prepare MRS medium (pH 4.5) containing 2wt% sucrose ) or MRS medium (pH 6.5) containing 2 wt% glucose. (5) MRS medium (pH 4.5) containing 2wt% sucrose was used for the WR16-4 strain, and MRS medium (pH 6.5) containing 2wt% glucose was used for the comparison object. oxygen conditions. <pH tolerance test> The pH tolerance test of each lactic acid bacteria precultured was performed as follows. (1) The MRS medium shown in Fig. 9 was prepared at a 2-fold concentration. At this time, pH was adjusted to 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5 by adding 4N sodium hydroxide solution or 4N hydrochloric acid. (2) Prepare a 4wt% sucrose solution. (3) Moist heat sterilization using an autoclave (121° C., 20 minutes) was performed for the above (1) and (2), respectively. (4) After cooling the solutions of (1) and (2) sterilized by moist heat, mix them at a ratio of 1:1 (v/v) in a dust-free laboratory bench to prepare MRS medium (pH 3-6.5) containing 2wt% sucrose ). (5) After centrifuging (15,000×g, 10 minutes) of each lactic acid strain previously cultured to remove the supernatant, it was resuspended in (3) so that the initial concentration would be A _660nm ≒ 0.05-0.10 Modulated medium. (6) After 200 μl/well was dispensed into a 96-well microplate every 3 wells (n=3), the cells were cultured at 30° C. under anaerobic conditions. (7) Turbidity measurements were performed over time using a microplate analyzer. Figures 1 to 3 show the turbidity measurement results of lactic acid bacteria for comparison (Lactobacillus acidophilus JCM 1132 ^T (Figure 1), Lactococcus lactis subsp. lactis NBRC12007 (Figure 2), Leuconostoc mesenteroides subsp. dextranicum NBRC100495 ^T (Figure 3)) Figure 4 is a graph showing the turbidity measurement results of the WR16-4 strain. In the above pH tolerance test using 2 wt% sucrose as the carbon source, as shown by the turbidity measurement results of each lactic acid bacteria, compared with the lactic acid bacteria used for the comparison object, they grew well in the pH 5-6.5 near-neutral acidic region. WR16-4 These strains grow well in the region where the lactic acid bacteria grow well in the region on the acidic side, specifically in the low pH region of pH 3.5 to 5, and it has been confirmed that it is almost impossible to grow in the region where the pH is higher and close to neutral. grow. <Sucrose tolerance test> The sucrose tolerance test of each lactic acid bacteria precultured was performed as follows. (1) After adjusting the MRS medium shown in FIG. 9 to which sucrose was added so that the sucrose concentration was 5wt%, 10wt%, 20wt%, 30wt%, 40wt%, 50wt%, 60wt%, and 70wt%, 4N hydrochloric acid was added to each of them. And adjusted to pH4.5. (2) For the above (1), moist heat sterilization using an autoclave (121° C., 20 minutes) was performed, respectively. (3) After centrifugation (15,000 × g, 10 minutes) of each lactic acid strain previously cultured to remove the supernatant, it was resuspended in (2) so that the initial concentration would be A _660nm ≒ 0.05 to 0.10 Modulated medium. (4) After 200 μl/well was dispensed into 96-well microplates every 3 wells (n=3), the cells were cultured at 30°C under anaerobic conditions. (5) Turbidity measurements were performed over time using a microplate analyzer. 5 to 8 show the turbidity measurement results of lactic acid bacteria for comparison (Lactobacillus acidophilus JCM 1132 ^T ( FIG. 5 ), Lactococcus lactis subsp. lactis NBRC12007 ( FIG. 6 ), and Leuconostoc mesenteroides subsp. dextranicum NBRC100495 ^T ( FIG. 7 )). Figure 8 is a graph showing the turbidity measurement results of the WR16-4 strain. In the above-mentioned sucrose tolerance test under the acidic condition of initial pH 4.5, as shown in the turbidity measurement results of each lactic acid bacteria, it was confirmed that the lactic acid bacteria used for the comparison could grow at a sucrose concentration of 20 wt% or less, but the growth state (reproduction rate) ) is low, and it can hardly grow in a high sucrose environment of more than 30 wt%; in contrast, it is confirmed that the WR16-4 strain shows a good growth state before the sucrose concentration of about 40 wt%, and can still grow in a high sucrose environment of 60 wt% . <Fructose and glucose tolerance test> Sucrose is a disaccharide composed of monosaccharides, fructose and glucose. Since it was confirmed that the WR16-4 strain can grow in a high sucrose environment as described above, we imagined that it may be possible to use a single monosaccharide in fructose and glucose. In the high concentration environment of fructose and glucose in the sugar state, growth can be performed in the same manner as in the case of sucrose, and the fructose and glucose tolerance test was performed as follows in the same manner as the above-mentioned sucrose tolerance test. (1) The MRS medium shown in FIG. 10 to which sucrose, fructose, and glucose sugar were individually added were adjusted to a sugar concentration of 20 wt %, respectively, and then adjusted to pH 4.5 by adding 4N hydrochloric acid. (2) The MRS medium shown in FIG. 10 to which fructose and glucose were added was adjusted to have a sugar concentration of 10 wt % (total 20 wt %) of fructose and glucose, and then 4N hydrochloric acid was added to each to adjust to pH 4.5. (3) Moist heat sterilization using an autoclave (121° C., 20 minutes) was performed for the above (1) and (2), respectively. (4) The precultured WR16-4 strain was centrifuged (15,000 × g, 10 minutes) to remove the supernatant, and then resuspended in the prepared medium so that the initial concentration would be A _660nm ≒ 0.05-0.10 . (5) After 200 μl/well was dispensed into 96-well microplates every 3 wells (n=3), the cells were cultured at 30°C under anaerobic conditions. (6) Turbidity measurements were performed over time using a microplate analyzer. The results of the above-mentioned turbidity measurement of the fructose-glucose tolerance test under the acidic conditions of the initial pH 4.5 are shown in FIG. 11 . As shown in Fig. 11 , when the strain WR16-4 contains either fructose or glucose alone, the reproduction rate is low (low growth state); when both fructose and glucose are contained, the reproduction rate is higher than when sucrose is contained (in a high growth state), it was confirmed from the results that the WR16-4 strain had sucrose tolerance and fructose and glucose tolerance. Also, as described above, since sucrose is a disaccharide in which fructose and glucose are bonded, the WR16-4 strain, as in the case of sucrose, contains, for example, 30 wt % of fructose and glucose each (the total sugar concentration is 60 wt %). ) in a high sugar concentration environment can also fully grow. <Use of the WR16-4 strain> The WR16-4 strain can be used for food and drink containing lactic acid bacteria, specifically, fermented food and drink. As mentioned above, since the WR16-4 strain is a lactic acid bacterium having the characteristics of being able to reproduce well at a low pH and in a high sucrose environment, it is possible to easily make, for example, a juice obtained by mixing plants and sugar and squeezing or using the same. Beverages as the base, or jams, fruit sauces, etc. have high acidity and sucrose concentration, and are not easily fermented with materials fermented by ordinary lactic acid bacteria (acidic and high-sugar concentration extracts). The effects of adding sourness (reducing sweetness or adding refreshing taste) or antiseptic (no need for preservatives that are additives) can be obtained. Furthermore, since the WR16-4 strain is a lactic acid bacterium that has the characteristics of being able to reproduce well in a low pH and high fructose-glucose environment, in addition to the above, it can also be used for the use of sugar (high) containing both fructose and glucose. Fructose syrup, glucose and fructose refined sugar mixed with food and beverages, etc.). In addition, an example of the acquisition method of the said acidic and high sugar-concentration extract liquid is shown below. In addition, the acquisition method of an extract is not limited to this. (1) Mix the chopped vegetables or fruits with caster sugar in equal proportions, add bacterial liquid to ferment. (2) After fermentation, juice extraction is performed to recover the juice components, and the recovered liquid is further fermented and aged to obtain fermentation liquid A. (3) Fermentation liquid A is added to the boiled juice of wild grass, and then fermentation liquid A is added to obtain fermentation liquid B. This fermentation broth A or fermentation broth B is an acidic and high sugar concentration extract. In addition, the fermented liquid A and the fermented liquid B will be made into beverages, and the WR16-4 strain will be used to ferment them, so that the sour taste can be added, and the preservative effect can be obtained without adding a preservative, and an unprecedented beverage can be provided. Furthermore, the WR16-4 strain has the characteristic that, as shown in the above test results, it can hardly grow in the environment of pH 6 to 7, which is the optimum pH environment of general lactic acid bacteria, so it is necessary to make, for example, the sucrose concentration high and the pH near neutral. When the material is fermented with other microorganisms (such as general lactic acid bacteria or yeast) with the WR16-4 strain, the WR16-4 strain cannot grow until the optimum pH is reached, and the growth of other microorganisms will not be hindered. Therefore, it is useful in the case of complex fermentation using a plurality of microorganisms. In addition, based on the above-mentioned properties, the WR16-4 strain can stop fermentation by non-heating by adjusting the pH of the material to near neutrality, and is useful for use in fermented products that cannot be heated. In addition, the present invention is not limited to this embodiment, and the specific structure of each component can be appropriately designed.

Fig. 1 is a graph showing the results of a pH resistance test of Lactobacillus acidophilus JCM 1132 ^T in this example. Fig. 2 is a graph showing the results of the pH tolerance test of Lactococcus lactis subsp. lactis NBRC12007 in this example. Fig. 3 is a graph showing the results of a pH resistance test of Leuconostoc mesenteroides subsp. dextranicum NBRC100495 ^T in this example. [ Fig. 4] Fig. 4 is a graph showing the results of a pH tolerance test of the WR16-4 strain in this example. Fig. 5 is a graph showing the results of a sucrose tolerance test of Lactobacillus acidophilus JCM 1132 ^T in this example. Fig. 6 is a graph showing the results of a sucrose tolerance test of Lactococcus lactis subsp. lactis NBRC12007 in this example. Fig. 7 is a graph showing the results of a sucrose tolerance test of Leuconostoc mesenteroides subsp. dextranicum NBRC100495 ^T in this example. Fig. 8 is a graph showing the results of the sucrose tolerance test of the WR16-4 strain in this example. Fig. 9 is a table showing the composition of the MRS medium in the sucrose tolerance test of this example. Fig. 10 is a table showing the composition of the MRS medium in the fructose and glucose tolerance test of the present example. Fig. 11 is a graph showing the results of the fructose and glucose tolerance test of the WR16-4 strain in this example. Fig. 12 is a list of data showing the results of homology search in the gene database (NCBI) of the present invention.

Overseas storage information 1. Japan ; NITE-IPOD ; 2020/02/05 ; NITE BP-03116

Claims (26)

A lactic acid bacteria is characterized in that it belongs to Lactobacillus sp. which is registered with the deposit number: NITE BP-03116 (identification mark: WR16-4). The lactic acid bacteria according to claim 1, wherein the lactic acid bacteria have the base sequence of the 16S rDNA region shown in SEQ ID NO: 1. The lactic acid bacteria of claim 1, which are obtained from plant fermentation extracts. The lactic acid bacteria of claim 2, which are obtained from plant fermentation extracts. The lactic acid bacteria of claim 3, wherein the aforementioned plant fermentation extract is a radish fermentation extract. The lactic acid bacteria of claim 4, wherein the aforementioned plant fermentation extract is a radish fermentation extract. The lactic acid bacteria according to claim 5, wherein the radish fermentation extract is obtained by mixing and fermenting radish, caster sugar and bacterial liquid. The lactic acid bacteria according to claim 6, wherein the radish fermentation extract is obtained by mixing and fermenting radish, caster sugar and bacterial liquid. The lactic acid bacteria of claim 7, wherein the bacterial liquid contains yeast. The lactic acid bacteria of claim 8, wherein the bacterial liquid contains yeast. The lactic acid bacteria according to any one of claims 1 to 10 can multiply in a low pH environment of pH 3.5 to 5.5, and can also reproduce in a high concentration of sucrose with a sucrose concentration of 30 to 60 wt %. The lactic acid bacteria according to any one of claims 1 to 10 can be propagated in a low pH environment of pH 3.5 to 5.5, and also in a fructose and glucose high concentration environment with fructose concentration and glucose concentration of 10 to 30 wt% each. Breedable. The lactic acid bacteria according to any one of claims 1 to 10 can multiply in a low pH environment of pH 3.5 to 5.5, and can also reproduce in a high concentration of sucrose with a sucrose concentration of 30 to 60 wt%; even at pH 3.5. It can multiply in a low pH environment of 5-5.5, and can also reproduce in a high-concentration environment of fructose and glucose with fructose concentration and glucose concentration of 10-30wt%. A method for obtaining lactic acid bacteria, which is characterized in that: adding a plant fermentation extract to a sterilized liquid culture medium to obtain a culture solution, then multiplying and culturing the culture solution to obtain a growth culture solution, and then mixing the growth culture solution with the sterilized culture solution. The separation medium is mixed and released to make a plate medium, and then the colonies appearing in the plate medium are fished out to obtain lactic acid bacteria that can multiply in a low pH environment and a high concentration sucrose environment. The method for obtaining lactic acid bacteria according to claim 14, wherein the aforementioned plant fermentation extract is a radish fermentation extract obtained by mixing radish, caster sugar, and bacterial liquid and fermenting it. The method for obtaining lactic acid bacteria according to claim 15, wherein the bacterial liquid contains yeast. The method for obtaining lactic acid bacteria according to any one of claims 14 to 16, wherein the liquid medium is a mixed fermented extract obtained by using either or both of vegetable fermented extracts and fruit fermented extracts, and mixed fermented extracts with weeds. It is obtained by mixing boiled juice and caster sugar to obtain a mixture of vegetables and caster sugar and then adding antibiotics to it. The method for obtaining lactic acid bacteria according to claim 17, wherein the antibiotic is cycloheximide. A food and drink containing lactic acid bacteria is characterized by containing the lactic acid bacteria according to any one of claims 1 to 10. A food and drink containing lactic acid bacteria is characterized by containing the lactic acid bacteria as claimed in claim 11. A food and drink containing lactic acid bacteria is characterized by containing the lactic acid bacteria as claimed in claim 12. A food and drink containing lactic acid bacteria is characterized by containing the lactic acid bacteria as claimed in claim 13. The food-drinks containing lactic acid bacteria according to claim 19, wherein the food-drinks containing lactic acid bacteria are fermented food-drinks. The food-drinks containing lactic acid bacteria according to claim 20, wherein the food-drinks containing lactic acid bacteria are fermented food-drinks. The food-drinks containing lactic acid bacteria according to claim 21, wherein the food-drinks containing lactic acid bacteria are fermented food-drinks. The food-drinks containing lactic acid bacteria according to claim 22, wherein the food-drinks containing lactic acid bacteria are fermented food-drinks.

Images