JP5218041B2 - Rice lactic acid fermented food and drink and method for producing the same - Google Patents

Description

      The present invention is suitable for fermentation of rice, and Lactobacillus sp. The present invention relates to a lactic acid bacterium belonging to, a food and drink of rice containing the lactic acid bacterium, and a method for producing the same.

  Lactic acid bacteria are deeply involved in the production of fermented milk, traditional fermented foods such as pickles, miso, and soy sauce, and are attracting attention for their physiological function (called probiotics) via the intestinal tract. In addition to restoring the balance of intestinal bacteria and improving symptoms such as diarrhea and constipation, it is also known for its effects such as enhancing immune function and reducing excessive blood cholesterol level. Development of yogurt and fermented milk utilizing the function of such lactic acid bacteria has been carried out, but even if plant-based raw materials such as rice are inoculated with lactic acid bacteria derived from fermented milk, growth may be slow and fermentation may not proceed. Many. This is because lactic acid bacteria are inherently highly auxotrophic, and plant materials alone cannot satisfy the nutrients necessary for the growth of lactic acid bacteria. However, lactic acid bacteria inhabit traditional fermented foods such as pickles, miso, and soy sauce. Among these lactic acid bacteria, there are lactic acid bacteria that grow only with plant materials.

  Regarding soy milk, (patent document 1) shows lactic acid bacteria suitable for soy milk. Regarding rice, for example, (patent document 2) and (patent document 3), saccharified brown rice and rice bran are used for lactic acid fermentation. Attempts have been made, but brown rice and rice bran are richer in nutrients than milled rice and are suitable for lactic acid fermentation, but have a strong odor, poor texture and poor palatability. In polished rice, even if it uses the disclosed lactic acid bacteria, it does not ferment and selection of the lactic acid bacteria suitable for rice fermentation of polished rice is not performed. In (Patent Document 4), steamed rice is used as a raw material, sake lees are produced by a general koji making method using koji mold, prepared into a high sugar-containing saccharified solution, and the protein content by adding soy protein After increasing the amount, the method of sterilization, inoculating lactic acid bacteria and fermenting is described, but this also solves the protein necessary for the growth of lactic acid bacteria by adding soy protein, for the production of ordinary fermented milk Lactic acid bacteria to be used are selected, and it is difficult to say that they are suitable for rice fermentation.

(Patent Document 5) describes a method of simultaneously performing saccharification of rice and lactic acid fermentation by lactic acid bacteria having starch degradability, but fermentation requires addition of nutrients other than rice such as skim milk and yeast extract. . In other cases, ingredients other than rice that are necessary for the growth of lactic acid bacteria are added to the fermentation of rice, and the existing lactic acid bacteria still leave a lot of room for improvement in order to prepare the intended rice food and drink. The current situation is.
JP-A-2005-224224 JP-A-8-289341 JP-A-60-94057 JP 2003-116502 A JP-A-7-255438

  The problem to be solved by the present invention is that it is suitable for rice fermentation and has characteristics when fermenting rice (survival of lactic acid bacterial strains) for the purpose of obtaining food and drink with the functionality of lactic acid bacteria. It is to select a lactic acid strain having a high quality, flavor, and palatability) and to newly provide a rice bran food that can be ingested inexpensively and daily using the lactic acid strain.

The present invention has been made in order to solve the above-mentioned problems, and the present inventors newly set the following criteria and performed selection work when screening the target lactic acid bacteria. .
That is, the present inventors, as a lactic acid bacterium having a long dietary experience and high safety, among the lactic acid bacteria derived from traditional fermented foods, (1) high resistance to gastric acid, (2) good growth at low pH, (3) High resistance to bile (4) As a result of repeated research on selection of strains with good growth in rice or saccharified rice and excellent flavor and taste after fermentation, Lactobacillus as strains meeting these conditions sp. FPL1 (this strain was deposited as NITE AP-691, a patent microorganisms deposit center NITE AP-691, an independent administrative agency), was found from raccoon pickles. The mycological properties of this strain are as follows.

A. Morphological characteristics Cell morphology: Neisseria gonorrhoeae motility: None Spore presence: None Gram staining: Positive

B. Physiological properties (positive: +, negative:-, weakly positive: w)
Catalase −
Gas production −
Growth at 15 ° C +
Growth at 45 ° C-
Fermentation type Homofermentation lactic acid rotatory DL
Peptidoglycan type DAP
Saltiness and salt tolerance Growth at 0-8% +
Growth at 9%-

C. Carbohydrate fermentability (positive: +, negative:-, weakly positive: w)
Arabinose +
Ribose +
Xylose −
Gluconic acid −
Glucose +
Fructose +
Galactose +
Mannose +
Rhamnose −
Cellobiose +
Lactose +
Maltose +
Melibiose +
Sucrose +
Raffinose +
Salicin +
Trehalose +
Merichitose −
Mannitol +
Sorbitol +
Starch −
Inulin −
Glycerol +

D. Genetic Characteristics The 500 bp base sequence of 16SrDNA using MicroSeq 500 16SrDNA Bacterial Sequencing Kit showed a high homology of 99% or more to Lactobacillus pentosus and 16SrDNA of Lactobacillus plantarum. Using this base sequence, a homology search was performed to create a molecular phylogenetic tree using the top 10 strains with the highest homology and the neighbor binding method. FIG. 1 shows that the 16S rDNA of this strain is also Lactobacillus pentosus and It was shown to form a cluster with Lactobacillus plantarum 16SrDNA and closely related.

E. Gastric acid resistance The gastric acid resistance test was performed as follows. 0.5 ml of lactic acid bacteria culture solution (GYP broth 30 ° C. 24 hr) is added to 4.5 ml of artificial gastric juice (100 mM HCl / KCl buffer pH 2.0 with 0.04% Pepsin), and left at 37 ° C. for 2 hours. The number of bacteria after contact with the artificial gastric juice was measured using MRS agar, and the survival rate was calculated. By this method, this strain showed a high survival rate among food-derived lactic acid bacteria (120 strains), and when the number of viable bacteria after inoculation was observed over time, it hardly decreased as shown in FIG.

F. Bile tolerance The bile tolerance test was performed as follows. Inoculate GYP broth containing 0, 0.1, 0.2, 0.3, 0.4% bile powder, and culture for 24 hours at 37 ° C, using a microplate reader The 630 nm OD value was measured. As the bile concentration increased, the growth of the bacteria worsened, but as shown in FIG. 3, the strain showed growth even under a bile concentration of 0.4%.

G. Fermentability of rice The fermentability of rice was examined using the top 10 strains showing gastric acid resistance and bile resistance from the aforementioned gastric acid resistance and bile resistance tests. The test was conducted as follows. Add 0.2 g of Uchichi rice flour (upper flour), 0.2 g of glucose, and 20 ml of deionized water to a 50 ml medium bottle, sterilize, add 20 μl of lactic acid bacteria culture solution that has been pre-cultured for 24 hours in GYP broth, and add 30 Fermentation was carried out at 0 ° C. for 2 days, and pH measurement and sensory evaluation (smell, taste) by three members of the Fukui Food Processing Laboratory were carried out. As a result, as shown in Table 1, five strains containing this strain were excellent in overall evaluation. In addition, the evaluation criteria of a flavor are as follows.

◎; very good, ○; good, △; not good, ×; bad

上記の結果で評価の高かった本菌株を含む５株について、さらに米糖化液を用いて再度発酵性を検討した。米糖化液による発酵製試験は以下の通りに実施した。精白米１５０ｇに水３７０ｍｌを加え炊飯後、約６０℃の温湯６００ｍｌを加え、さらに市販麹１５０ｇを加え、よく混ぜ、インキュベーターにて５５℃、１４時間保温し、甘酒（米糖化液）を調製した。これをミキサーでピューレ状にし８０．０ｇずつ滅菌済みポリ容器にいれ、ＧＹＰ液体培地にて２４時間前培養を行った乳酸菌培養液を５０μｌ添加し、３０℃で２日間発酵させ、ｐＨ、酸度、乳酸菌生菌数、福井県食品加工研究所所員３名による官能評価（臭い、食味）を実施した。乳酸菌数は炭酸カルシウム加ＭＲＳ寒天培地（Ｄｉｆｃｏ社製）を用い、混釈平板培養により計数測定した。３０℃４８時間培養した後、コロニー数をカウントした。表２に示すように添加した乳酸菌は米糖化液中で速やかに増加し、２４時間後には１０^９個／ｇに達し、表３に示すように乳酸生成、ｐＨ低下が観察され、本菌株を含む選択株に優位性が認められた。官能評価の結果は表４に示すように、２４時間後、４８時間後とも、本菌株が食味評価が高く、嗜好性が優れていた。なお、風味の評価基準は以下の通りである。

About 5 strains including this strain which was highly evaluated by the above results, fermentability was examined again using a rice saccharified solution. The fermentation test using rice saccharified solution was performed as follows. After adding 370 ml of water to 150 g of polished rice and cooking rice, add 600 ml of hot water at about 60 ° C., add 150 g of commercial rice cake, mix well, and incubate at 55 ° C. for 14 hours to prepare amazake (rice saccharified solution). . This was pureed with a mixer and placed in a sterilized plastic container in an amount of 80.0 g, 50 μl of lactic acid bacteria culture solution pre-cultured for 24 hours in GYP liquid medium was added, fermented at 30 ° C. for 2 days, pH, acidity, The number of living lactic acid bacteria and sensory evaluation (smell, taste) by three Fukui Food Processing Research Institute members were carried out. The number of lactic acid bacteria was counted by pour plate culture using a calcium carbonate-added MRS agar medium (Difco). After culturing at 30 ° C. for 48 hours, the number of colonies was counted. Table the added lactic acid bacteria as shown in 2 rapidly increases in US saccharified solution reached 10 ⁹ / g after 24 hours, lactic acid generated as shown in Table 3, pH drop was observed, this strain Superiority was found in the selected strains. As shown in Table 4, the results of the sensory evaluation were high in taste evaluation and excellent in palatability after 24 hours and 48 hours. In addition, the evaluation criteria of a flavor are as follows.

◎; very good, ○; good, △; not good, ×; bad

  Lactobacillus sp. FPL1 has good growth in rice or saccharified rice, excellent flavor and taste after fermentation, gastric acid resistance and bile resistance, and can be expected to survive after ingestion. Is selected as a strain that can be provided as a food (liquid, coagulated, pasty, frozen, dried). Therefore, Lactobacillus sp. The production characteristics of the rice fermented food and drink using the FPL1 strain will be described in detail with examples, but the present invention is not limited to these.

  That is, the present invention relates to Lactobacillus sp. Suitable for rice fermentation by adding sugar and water that can be used for lactic acid bacteria to rice. It is related with the food-drinks characterized by including at least one of the lactic acid bacteria which belong to this, the lactic acid bacteria containing material, and its processed material. Examples of the lactic acid bacteria-containing material include lactic acid bacteria suspensions, lactic acid bacteria cultures, lactic acid bacteria culture solutions obtained by removing solids from lactic acid bacteria cultures, fermented rice drinks, and yogurt-like foods.

  Examples of processed products include coagulated products, concentrates, pasted products, dried products, liquid products, diluted products, and frozen products. As lactic acid bacteria, live cells, wet cells, dry cells and the like can be used as appropriate.

  According to the present invention, lactic acid fermentation can be performed using raw materials obtained by adding rice cake to rice and saccharifying, or by adding a minimum amount of sugar to rice flour, and efficiently producing lactic acid-fermented foods and drinks for rice. It can be performed. For saccharification of rice, an enzyme agent such as amylase can be used in addition to rice bran, and in this case, lactic acid fermentation can be performed using only rice as a raw material.

  In addition, by adding lactic acid bacteria and fermenting, the growth of deteriorating microorganisms such as heat-resistant spore-forming Bacillus bacteria derived from the raw rice is suppressed, so that the storage stability of the product can be improved.

  And, by using lactic acid bacteria that have gastric acid resistance and bile resistance and can be expected to survive after ingestion, it becomes possible to produce health-oriented foods and drinks that have health functions and contribute to the expansion of sluggish consumption of rice. be able to.

  Raw rice can be used, such as glutinous rice, glutinous rice, pigmented rice, etc., and can be used as brown rice, but usually it can be used after cooking polished rice. Moreover, it can also be used by adding water to powdered rice flour such as upper fresh flour and rice bran powder and heating it. Lactic acid bacteria require sugars such as glucose for growth, which can be used by causing rice straw or saccharifying enzymes to act on rice to partially saccharify rice starch to produce maltose or glucose. Of course, it is also possible to add sugars such as glucose to the rice. Conventionally, saccharification of rice has been widely carried out using sweet sake using koji, and the method of allowing this lactic acid strain to act on the sweet sake can perform lactic acid fermentation with good reproducibility and is the most suitable method.

  Pre-culture of lactic acid strains for food and beverage production involves collecting only cultured cells by centrifugation, etc., removing and using the medium components, yeast extract as a food additive that can be used as food, Even a liquid medium consisting only of glucose can be grown sufficiently. It is also possible to add the fermented lactic acid of rice as a starter as it is. The addition amount can be used in the range of 1 / 10,000 to 1/10 of the raw material.

  The growth temperature of the present lactic acid bacteria is 15 ° C. to 40 ° C., and the optimum temperature is 30 ° C., and lactic acid fermentation can be performed under this temperature condition. The culture time is 16 to 48 hours at 30 ° C. After the fermentation, the number of lactic acid bacteria can be maintained even after about one month by refrigeration (5 ° C.) storage. Since there is concern about yeast contamination during lactic acid fermentation, it is desirable to heat sterilize the raw material before adding lactic acid bacteria. In order to maintain the lactic acid live bacteria in the product, refrigeration, freezing, lyophilization and the like are effective after completion of fermentation. However, when sterilization is acceptable, heat sterilization may be performed after completion of lactic acid fermentation.

  When provided as a beverage, the viscosity of the product can be adjusted by adjusting the amount of water added. By adding a gelling agent such as agar at the time of addition of lactic acid bacteria, it can also be provided as a yogurt-like coagulation. Moreover, it can be provided as a frozen type dessert by freezing, and it can be pulverized by lyophilization and used for supplements.

Test example 1

Wash 300g (2 go) of polished rice (2 rice), soak in 740 mL of water, cook with an electric rice cooker, add about 600 mL of water and 220 g (2 go) of commercial rice cake, stir, and then add water to add 2L It was. The top was covered with wrap and saccharified for 24 hours in an incubator kept at 55 ° C. to prepare amazake. This is homogenized with a home mixer, made into a paste, sterilized by heating in a warm bath (center temperature 85 ° C. for 30 minutes), cooled to about 30 ° C. with water, and then precultured for this strain (GYP broth 30 ° C. 24 ° C.) 2 mL (1/1000 volume) of time culture), dispense 30 mL each into a sterilized sample tube, and leave in an incubator kept at 20 ° C., 30 ° C., 40 ° C., pH, acidity, number of lactic acid bacteria Was measured. The acidity was expressed by converting 2 mL of a sample with 0.1 N sodium hydroxide and converting it to an amount corresponding to lactic acid. The number of lactic acid bacteria was counted and measured by pour plate culture using a calcium carbonate-added GYP agar medium. After culturing at 30 ° C. for 48 hours, the number of colonies was counted. As shown in FIGS. 4, 5, and 6, the increase in the number of bacteria, the decrease in pH, and the increase in acidity were observed at the optimum temperature of 30 ° C., but even at 20 ° C. and 40 ° C., after 24 hours. The number of bacteria reached 10 ⁸ CFU / g or more.

Test example 2

Amazake prepared in the same way as in Test Example 1 was homogenized with a home mixer, pasted, pasteurized, sterilized by heating (center temperature 85 ° C for 30 minutes), cooled to about 30 ° C with water, and then 200 mL each in a medium bottle. Into three bottles, 40 mL of the pre-culture solution of this strain (GYP broth, 30 ° C., 24 hour culture: 2.5 × 10 ⁹ CFU / ml) is centrifuged at 2000 rpm × 10 min to remove the medium components, and 2 with sterile physiological saline. 0.2 ml (0.01%), 2 ml (0.1%), and 20 ml (1%) of the cell solution suspended in 40 ml of sterilized physiological saline after washing twice, respectively, and kept at 30 ° C. The lactic acid bacteria count was counted. The number of lactic acid bacteria was counted and measured by pour plate culture using a calcium carbonate-added GYP agar medium. After culturing at 30 ° C. for 48 hours, the number of colonies was counted. As shown in FIG. 7, even when the amount of the initial lactic acid bacterium added was different, it reached nearly 10 ⁹ CFU / g in 24 hours and showed good fermentability. In Test Example 2, in consideration of food production, the medium component was removed by centrifugation and only the cells were added. However, it showed sufficient growth and was judged to be an industrially available strain. .

  Wash 750 g (5 go) of polished rice (uruchi rice), soak in 1.85 L of water, cook with an electric rice cooker, add about 1.5 L of water and 550 g (5 go) of commercial rice cake, stir, and further water To a total volume of 5L. The top was covered with wrap and saccharified for 24 hours in an incubator kept at 55 ° C. to prepare amazake. This is homogenized with a home mixer, made into a paste, sterilized by heating in a warm bath (center temperature 85 ° C. for 30 minutes), cooled to about 30 ° C. with water, and then precultured for this strain (GYP broth 30 ° C. 24 ° C.) 5 mL (1/1000 volume) was added, and lactic acid fermentation was performed at 30 ° C. for 24 hours to obtain a rice lactic acid fermentation liquid.

  Amazake prepared in the same manner as in Example 1 was homogenized with a home mixer, made into a paste, pasteurized, sterilized by heating (center temperature 85 ° C for 30 minutes), cooled to about 30 ° C with water, Add 5 mL (1/1000 volume) of the culture solution (GYP broth cultured at 30 ° C. for 24 hours), add 20 g of agar previously dissolved in 500 mL of water, mix quickly, homogenize, and dispense 50 ml into a paper cup container. Then, it was covered and lactic acid fermented at 30 ° C. for 24 hours to obtain a rice lactic acid fermented coagulum.

It is a figure which shows the molecular phylogenetic tree by the neighborhood joint method of this strain and related bacteria by the 500 bp base sequence of 16SrDNA regarding the genetic characteristic of this strain. It is a graph which shows the viability by artificial gastric juice regarding the gastric acid tolerance of this strain. It is a graph which shows the relationship between bile density | concentration and the proliferation of a microbe regarding the bile tolerance of this strain. It is a graph which shows the change of the number of lactic acid bacteria when this strain is added to rice saccharified liquid and it puts on predetermined | prescribed temperature. It is a graph which shows the change of pH when this strain is added to rice saccharified liquid and it puts on predetermined | prescribed temperature. It is a graph which shows the change of acidity (lactic acid conversion value) when this strain is added to a rice saccharified solution and placed at a predetermined temperature. It is a graph which shows the change of the number of lactic acid bacteria when changing the inoculation amount of this strain, adding to rice saccharified liquid, and putting it at 30 degreeC.

Claims (6)

Lactobacillus sp. Which is a lactic acid bacterium suitable for rice fermentation by adding sugar and water that can be used for lactic acid bacteria to rice. A food or drink comprising FPL1 (NITE P-691) , the lactic acid bacterium-containing product, and a processed product thereof. Lactobacillus sp. Which is a lactic acid bacterium suitable for rice fermentation by adding sugar and water that can be used for lactic acid bacteria to rice. FPL1 (NITE P-691) , this lactic acid bacteria containing material, The manufacturing method of the food-drinks characterized by including at least one of the processed material. Lactobacillus sp., Which is a lactic acid bacterium that has gastric acid resistance and bile resistance and has a high survival rate when an oral administration composition is prepared. A rice food or drink characterized by containing at least one of FPL1 (NITE P-691) , the lactic acid bacteria-containing product, and a processed product thereof. The food or drink according to any one of claims 1 to 3, wherein the lactic acid bacterium-containing material is at least one selected from a lactic acid bacterium suspension, a lactic acid bacterium culture, and a lactic acid bacterium culture solution. The treated product is at least one selected from a coagulated product, a concentrate, a pasted product, a dried product, a liquid product, a diluted product, and a frozen product, according to any one of claims 1 to 4. Food and drink. Lactobacillus sp. FPL1 (NITE P-691).

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