Method for culturing lactic acid bacteria

Abstract

To provide a culture medium for culturing lactic acid bacteria that can culture efficiently lactic acid bacteria, a method for culturing lactic acid bacteria using the culture medium, cultured lactic acid bacteria obtained by the culturing method, and food and drink, pharmaceuticals, cosmetic preparations or feed containing the cultured lactic acid bacteria.SOLUTION: The inventors have discovered that a culture medium containing unrefined sugar, a calcium-containing water-insoluble composition, and manganese-containing microorganisms is used so that lactic acid bacteria can be cultured efficiently, thereby completing this invention.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present invention relates to a medium for culturing lactic acid bacteria, a method for culturing lactic acid bacteria, and the like.

Background Art

[0002] Lactic acid bacteria have been used in fermented foods such as yogurt since ancient times, and in recent years, it has been elucidated that they have various functions such as an allergy-suppressing effect, and they are widely used as useful bacteria in daily life. Therefore, methods for efficiently culturing lactic acid bacteria have been studied.

[0003] So far, as a method for culturing lactic acid bacteria, a method for culturing Lactobacillus lactic acid bacteria having a step of culturing in a medium containing grains (Patent Document 1) and the like are known. On the other hand, Patent Document 2 shows that the addition of oleic acids exceeding a certain amount reduces the growth rate of bacteria and causes a decrease in the number of bacteria at the end of culturing.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] The present invention provides a medium for culturing lactic acid bacteria capable of efficiently culturing lactic acid bacteria, a method for culturing lactic acid bacteria using the medium, a lactic acid bacteria culture obtained by the culturing method, and a food, beverage, pharmaceutical, cosmetic or feed containing the lactic acid bacteria culture.

Means for Solving the Problems

[0006] The inventors have found that lactic acid bacteria can be efficiently cultured by using a medium containing brown sugar, a calcium-containing water-insoluble composition, and a manganese-containing microorganism, and have completed the present invention.

[0007] That is, the present invention relates to the following aspects [1] to [8]. [1] A medium for culturing lactic acid bacteria, comprising brown sugar, a calcium-containing water-insoluble composition, and a manganese-containing microorganism. [2] The medium for culturing lactic acid bacteria according to [1], comprising 0.5 to 30% by weight of brown sugar, 0.1 to 10% by weight of the calcium-containing water-insoluble composition, and 0.005 to 0.5% by weight of the manganese-containing microorganism when the liquid medium is 100% by weight. [3] The medium for culturing lactic acid bacteria according to [1] or [2], comprising 5% by weight or more of brown sugar. [4] The medium for culturing lactic acid bacteria according to any one of [1] to [3], further comprising 0.01 to 8% by weight of oil and fat. [5] A method for culturing lactic acid bacteria, comprising culturing lactic acid bacteria in the medium according to any one of [1] to [4]. [6] The method for culturing lactic acid bacteria according to [5], comprising culturing under aerobic conditions. [7] A lactic acid bacteria culture obtained by the method according to [5] or [6]. [8] A food, drink, pharmaceutical, cosmetic, or feed comprising the lactic acid bacteria culture according to [7].

Effects of the Invention

[0008] According to the present invention, it is possible to provide a medium for culturing lactic acid bacteria that can culture lactic acid bacteria more efficiently than conventional media. Further, by using the medium, the growth of lactic acid bacteria is activated, so that lactic acid bacteria can be cultured efficiently, and a lactic acid bacteria culture can be provided at low cost. Furthermore, a safe lactic acid bacteria culture can be provided, which can be widely used for foods, drinks, pharmaceuticals, cosmetics, and feeds.

Brief Description of the Drawings

[0009]

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Mode for Carrying Out the Invention

[0010] The medium for lactic acid bacteria culture of the present invention contains brown sugar, a calcium-containing water-insoluble composition, and a manganese-containing microorganism. It is preferably further contained with oil and fat. It is preferably contained with yeast extract, and may also contain other general medium components.

[0011] The brown sugar described in the present invention is not particularly limited as long as it is made by boiling down the squeezed juice of sugarcane. The brown sugar content is preferably 0.5 to 30% by weight, more preferably 1 to 25% by weight, still more preferably 3 to 20% by weight, and particularly preferably 5% by weight or more when the liquid medium at the start of culture is 100% by weight. By containing brown sugar in the medium, the growth of lactic acid bacteria is activated.

[0012] The calcium-containing water-insoluble composition described in the present invention contains calcium and is not particularly limited as long as it is insoluble in tap water. Examples include shells such as oyster shells and scallop shells, eggshells, bones, corals, limestone, dolomite, etc., but oyster shell powder or coral powder is preferred. When the liquid medium at the start of cultivation is taken as 100% by weight, the content of the calcium-containing water-insoluble composition preferably contains 0.1 to 10% by weight, more preferably 0.2 to 8% by weight, and still more preferably 0.5 to 5% by weight. As the calcium content, it preferably contains 0.05 to 5% by weight, more preferably 0.1 to 3% by weight, and still more preferably 0.2 to 2% by weight. By including the calcium-containing water-insoluble composition, as the culture solution becomes acidic during cultivation, it gradually dissolves, alleviates the decrease in pH, enables pH adjustment, and suppresses the growth inhibition of lactic acid bacteria associated with the decrease in pH.

[0013] The manganese-containing microorganism described in the present invention is not particularly limited as long as it is a microorganism containing manganese. Examples include yeasts or lactic acid bacteria containing about 1 to 10% by weight of manganese. For example, commercially available edible yeast containing about 4 to 8% of manganese can be used. When the liquid medium at the start of cultivation is taken as 100% by weight, the content of the manganese-containing microorganism preferably contains 0.005 to 0.5% by weight, more preferably 0.01 to 0.4% by weight, and still more preferably 0.015 to 0.2% by weight. As the manganese content, it preferably contains 2.5 to 250 ppm, more preferably 5 to 200 ppm, and still more preferably 7.5 to 100 ppm.

[0014] The fats and oils described in the present invention are not particularly limited, but vegetable fats and oils are preferred, and vegetable fats and oils containing oleic acid, linoleic acid, α-linolenic acid, conjugated linoleic acid, etc. are more preferred, and esters of the above fatty acids may also be used. For example, sesame oil, salad oil, sardine oil, corn oil, soybean oil, rapeseed oil (canola oil), rice bran oil, rice bran oil, wheat germ oil, camellia oil, safflower oil, palm kernel oil, cottonseed oil, sunflower oil, perilla oil, linseed oil, olive oil, peanut oil, almond oil, avocado oil, hazelnut oil, walnut oil, grape seed oil, seaberry oil, borage seed oil, etc. can be exemplified. When the liquid medium at the start of culture is 100% by weight, the fats and oils can preferably contain 0.01 to 8% by weight, more preferably 0.02 to 6% by weight, and even more preferably 0.05 to 3% by weight.

[0015] The yeast extract described in the present invention is not particularly limited, and commercially available yeast extracts can be used. When the liquid medium at the start of culture is 100% by weight, the yeast extract can preferably contain 0.1 to 10% by weight, more preferably 0.2 to 8% by weight, and even more preferably 0.5 to 5% by weight.

[0016] The method for culturing lactic acid bacteria of the present invention is not particularly limited as long as it is a method of culturing using the above-mentioned medium for culturing lactic acid bacteria. Specifically, the liquid medium containing the above components is sterilized, cooled, inoculated with lactic acid bacteria, and then cultured under aerobic conditions such as aeration stirring and shaking. The culture temperature and the number of culture days may be general culture conditions. For example, it can be cultured at 20 to 40 °C for 1 to 50 hours, preferably at 25 to 38 °C for 10 to 40 hours. The pH at the start of culture may not be adjusted, but it may also be adjusted to about pH 6.0 to 8.0. The inoculum amount at the time of culture is not particularly limited as long as it is a general inoculum amount. For example, 0.1 to 10% by weight, preferably 0.5 to 5% by weight can be exemplified.

[0017] The types of lactic acid bacteria are not particularly limited, and any common lactic acid bacteria may be used. For example, bacteria belonging to the genera Lactobacillus, Lactococcus, Streptococcus, Bifidobacterium, Leuconostoc, Pediococcus, etc. can be exemplified. Representative bacteria include Lactobacillus plantarum, Lactobacillus paracasei, etc. One or more of them can be used.

[0018] The lactic acid bacteria culture of the present invention is obtained by the above-mentioned culture method, and may be sterilized after the culture is completed. The sterilization conditions are not particularly limited as long as they are common methods. For example, heating at 60 to 120 °C for 1 to 30 minutes or at 80 to 100 °C for 5 to 20 minutes is preferred. The form may be liquid, but it may also be powdered by air drying, spray drying, vacuum and / or freeze drying, etc.

[0019] The lactic acid bacteria culture of the present invention can be obtained using a medium containing natural products or food raw materials, and can be used safely, so it can be added to various products. The food and drink products to be added are not particularly limited, but in addition to various food and drink products such as beverages, foods, seasonings, functional foods, supplements, etc., it can also be used in pharmaceuticals, quasi-drugs, cosmetics, feeds, etc. The content of the lactic acid bacteria culture in various products is not particularly limited, but 0.1 to 50% by weight is preferred, 0.5 to 30% by weight is more preferred, and 1 to 20% by weight is even more preferred.

Examples

[0020] Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited by the following examples. In the present invention, % is all by weight unless otherwise specified.

Examples

[0021] (Examination of carbon source) 50 mL of a liquid medium composed of 13% brown sugar (produced in Iejima Island), 3% yeast extract (Mist P1G, manufactured by Asahi Breweries Foods Co., Ltd.), 3% oyster shell powder (containing 40% calcium) as a calcium-containing water-insoluble composition, 0.2% olive oil as an oil, 0.05% manganese-containing yeast (HIGH MANGANESE YEAST (containing 5% manganese), manufactured by Seti Co., Ltd.) and 81.75% tap water was placed in a 200 mL Erlenmeyer flask with baffles and autoclaved at 121 °C for 15 minutes. Lactobacillus plantarum NBRC15891 was inoculated into the cooled liquid medium and cultured with shaking at 35 °C for 20 hours to obtain a seed culture solution.

[0022] 50 mL each of the liquid media described in Table 1 were placed in 200 mL Erlenmeyer flasks with baffles and autoclaved at 121 °C for 15 minutes. 1% of the above seed culture solution was inoculated into the cooled liquid media and cultured with shaking at 35 °C and 120 rpm for 21 hours. After the culture was completed, sterilization treatment was performed at 90 °C for 10 minutes to obtain each lactic acid bacteria culture. In addition, decaglycerin monooleate used was Sansoft Q-17S (manufactured by Taiyo Chemical Co., Ltd.).

[0023] (Evaluation) For each lactic acid bacteria culture, pH measurement and measurement of the number of bacteria using a bacterial counting board were performed. The results were summarized in Table 1 and (1) of Figure 1, and the micrographs were shown in (2) of Figure 1.

[0024] [Table 1]

[0025] From Table 1 and Figure 1, it was found that by using brown sugar as a medium component, the number of bacteria increased significantly compared to sucrose. Also, from (2) of Figure 1, the bacterial cells obtained using the brown sugar medium were spherical, looking like dots, while the bacterial cells obtained using the sucrose medium were elongated rod-shaped. This is considered to be because the growth of lactic acid bacteria was activated by containing brown sugar, and division was repeated in a short time before the bacterial cells became large. Therefore, it was found that brown sugar is important as a carbon source for the medium components in efficiently culturing lactic acid bacteria.

Example

[0026] (Examination of brown sugar content) Using the liquid medium described in Table 2, each lactic acid bacteria culture was obtained by treating in the same manner as in Example 1.

[0027] Evaluation was carried out in the same manner as in Example 1, and the results were summarized in Table 2 and Figure 2.

[0028]

Table 2

[0029] From Table 2 and Figure 2, it was found that up to a brown sugar content of 12%, the number of lactic acid bacteria increased with the increase in brown sugar. When the sugar concentration in the medium is too high, the osmotic pressure becomes high. Usually, in an environment under high osmotic pressure, it is known that microorganisms are stressed and their growth is suppressed. However, it was surprising that even at a high brown sugar content of 20%, the growth of lactic acid bacteria was hardly suppressed.

Example

[0030] (Examination of the content of calcium-containing water-insoluble composition) Using the liquid medium described in Table 3, each lactic acid bacteria culture was obtained by treating in the same manner as in Example 1. Incidentally, oyster shell powder was used as the calcium-containing water-insoluble composition.

[0031] Evaluation was carried out in the same manner as in Example 1, and the results were summarized in Table 3 and Figure 3.

[0032]

Table 3

[0033] From Table 3 and Figure 3, it was found that by using oyster shell powder as a medium component, the number of bacteria increased significantly compared to the non-added case, and furthermore, the higher the content of oyster shell powder, the more the number of lactic acid bacteria increased. Also, the higher the content of oyster shell powder, the higher the pH at the end of the culture, and the decrease in pH due to the culture was suppressed. That is, by including a calcium-containing water-insoluble composition, as the culture solution became acidic during the culture, it gradually dissolved, and the decrease in pH was alleviated, suppressing the growth inhibition of lactic acid bacteria associated with the decrease in pH. Therefore, it was found that a calcium-containing water-insoluble composition is important as a medium component for efficiently culturing lactic acid bacteria.

Example

[0034] (Examination of the content of manganese-containing microorganisms) Using the liquid medium described in Table 4, each lactic acid bacteria culture was obtained by treating it in the same manner as in Example 1.

[0035] It was evaluated in the same manner as in Example 1, and the results were summarized in Table 4 and Figure 4.

[0036]

Table 4

[0037] From Table 4 and Figure 4, it was found that by using manganese-containing yeast as a medium component, the number of bacteria increased significantly compared to the non-added case, and furthermore, the higher the content of manganese yeast, the more the number of lactic acid bacteria increased. Therefore, it was found that a manganese-containing microorganism is important as a medium component for efficiently culturing lactic acid bacteria.

Example

[0038] (Examination of the oil and fat content) Using the liquid medium described in Table 5, each lactic acid bacteria culture was obtained by treating it in the same manner as in Example 1.

[0039] It was evaluated in the same manner as in Example 1, and the results were summarized in Table 5 and Figure 5.

[0040]

Table 5

[0041] From Table 5 and Figure 5, it was found that by using oil and fat as a medium component, the number of bacteria increased slightly compared to the case without addition.

Example

[0042] (Examination of oil and fat types) Using the liquid medium described in Table 6, the same treatment as in Example 1 was carried out to obtain each lactic acid bacteria culture. In addition, conjugated linoleic acid was used from Ciel Ace (manufactured by Nisshin Oillio Group).

[0043] Evaluation was carried out in the same manner as in Example 1, and the results were summarized in Table 6 and Figure 6.

[0044]

Table 6

[0045] From Table 6 and Figure 6, it was found that lactic acid bacteria can be cultured using various oils and fats.

Example

[0046] (Examination of medium components) 50 mL each of the liquid media described in Table 7 were placed in 200 mL baffled Erlenmeyer flasks, and autoclaved at 121 °C for 15 minutes. To the cooled liquid media, the seed culture solution described in Example 1 or Lactobacillus paracasei NBRC3533 similarly seed-cultured was inoculated at 1% each, and cultured with shaking at 35 °C and 120 rpm for 21 hours. After completion of the culture, sterilization treatment was carried out at 90 °C for 10 minutes to obtain each lactic acid bacteria culture.

[0047] Evaluation was carried out in the same manner as in Example 1, and the results were summarized in Table 7 and Figure 7.

[0048]

Table 7

[0049] From Table 7 and Figure 7, it was found that in any of the lactic acid bacteria, the use of a medium containing oyster shell and manganese-containing yeast significantly increased the number of lactic acid bacteria.

Example

[0050] (Powderization) 1 L of a liquid medium consisting of 8% brown sugar, 3% yeast extract, 3.6% coral powder (containing 39% calcium, Okinawa coral) as a calcium-containing water-insoluble composition, 0.2% olive oil as an oil, 0.1% manganese-containing yeast (containing 5% manganese), and 85.1% tap water was placed in a 3-L Erlenmeyer flask with baffles and autoclaved at 121°C for 15 minutes. Lactobacillus plantarum NBRC14711 was inoculated into the cooled liquid medium and cultured with shaking at 33°C for 20 hours to obtain a seed culture solution.

[0051] 2 L of the same liquid medium as in the seed culture was placed in each of 6 3-L Erlenmeyer flasks with baffles and autoclaved at 121°C for 15 minutes. The seed culture solution was inoculated at 1% into the cooled liquid medium and cultured with shaking at 33°C and 90 rpm for 20 hours. After the culture was completed, it was sterilized at 90°C for 10 minutes to obtain 12.4 kg of a lactic acid bacteria culture. The lactic acid bacteria culture was powdered using a spray dryer to obtain 977 g of lactic acid bacteria culture powder (moisture 3.2%, cell count 1.1×10 11 cells / g).

Claims

1. A culture medium for culturing lactic acid bacteria, comprising brown sugar, a calcium-containing water-insoluble composition, and manganese-containing microorganisms, wherein when the liquid medium is 100% by weight, it contains 3 to 10% by weight of the calcium-containing water-insoluble composition.

2. The culture medium for culturing lactic acid bacteria according to Claim 1, which contains 0.5 to 30% by weight of brown sugar and 0.005 to 0.5% by weight of manganese-containing microorganisms when the liquid medium is 100% by weight.

3. The culture medium for culturing lactic acid bacteria according to Claim 1 or 2, which contains 5% by weight or more of brown sugar.

4. The culture medium for culturing lactic acid bacteria according to any one of Claims 1 to 3, further containing 0.01 to 8% by weight of oil and fat.

5. A method for culturing lactic acid bacteria, which comprises culturing lactic acid bacteria in the culture medium according to any one of Claims 1 to 4.

6. The method for culturing lactic acid bacteria according to Claim 5, which is cultured under aerobic conditions.

7. A lactic acid bacteria culture obtained by the method according to Claim 5 or 6.

8. A food or drink, pharmaceutical, cosmetic or feed containing the lactic acid bacteria culture according to Claim 7.

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