JP7696593B2 - How to cultivate lactic acid bacteria - Google Patents

Description

The present invention relates to a medium for culturing lactic acid bacteria, a method for culturing lactic acid bacteria, etc.

Lactic acid bacteria have long been used in fermented foods such as yogurt, and in recent years have been found to have various functionalities, such as the suppression of allergies, and are widely used as familiar and useful bacteria. For this reason, methods for efficiently culturing lactic acid bacteria have been investigated.

To date, known methods for culturing lactic acid bacteria include a method for culturing Lactobacillus lactic acid bacteria, which comprises a step of culturing the bacteria in a medium containing grains (Patent Document 1). On the other hand, Patent Document 2 shows that the addition of more than a certain amount of oleic acid reduces the growth rate of the bacteria, resulting in a decrease in the number of bacteria at the end of the culture.

JP 2017-143802 A Patent No. 3650711

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 culture method, and a food, drink, pharmaceutical, cosmetic, or feed containing the lactic acid bacteria culture.

The inventors discovered 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 thus completed the present invention.

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 a calcium-containing water-insoluble composition, and 0.005 to 0.5% by weight of a manganese-containing microorganism, when the liquid medium is taken as 100% by weight.
[3] The medium for culturing lactic acid bacteria according to [1] or [2], which contains 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 an oil or fat.
[5] A method for culturing lactic acid bacteria, comprising culturing lactic acid bacteria in a medium according to any one of [1] to [4].
[6] The method for culturing the lactic acid bacterium described in [5], wherein the culture is carried out under aerobic conditions.
[7] A lactic acid bacteria culture obtained by the method described in [5] or [6].
[8] A food, drink, pharmaceutical, cosmetic or feed comprising the lactic acid bacteria culture according to [7].

The present invention provides a medium for culturing lactic acid bacteria that can culture lactic acid bacteria more efficiently than conventional media. In addition, the use of the medium activates the proliferation of lactic acid bacteria, allowing efficient lactic acid bacteria culture and providing a lactic acid bacteria culture at low cost. Furthermore, a safe lactic acid bacteria culture can be provided, which can be widely used for food and beverages, medicines, cosmetics, and feed.

1 shows (1) the number of bacteria in a culture and (2) a micrograph when brown sugar or sucrose was used as a carbon source in a medium for culturing lactic acid bacteria. This shows the number of bacteria in a culture when culture media with various brown sugar contents were used for lactic acid bacteria culture. 1 shows the number of bacteria and pH in the culture when culture media having various contents of a calcium-containing water-insoluble composition were used in lactic acid bacteria culture. This shows the number of bacteria in a culture when culture media with various manganese-containing yeast contents were used in lactic acid bacteria culture. This shows the number of bacteria in a culture when culture media with various oil and fat contents were used in lactic acid bacteria culture. The figure shows the number of bacteria in the culture when various oils and fats were used as medium components in lactic acid bacteria culture. This shows the number of bacteria in a culture of Lactobacillus putantalum or Lactobacillus paracasei when a medium not containing oyster shells, manganese-containing yeast and/or fats and oils was used as a medium component in lactic acid bacteria culture.

The medium for culturing lactic acid bacteria of the present invention contains brown sugar, a calcium-containing water-insoluble composition, and a manganese-containing microorganism. It is preferable that the medium further contains fats and oils. It is also preferable that the medium contains yeast extract, and may contain other general medium components.

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

The calcium-containing water-insoluble composition described in the present invention is not particularly limited as long as it contains calcium and is insoluble in tap water, and examples thereof include shells such as oysters and scallops, eggshells, bones, coral, limestone, dolomite, etc., but oyster shells or coral powder are preferred. The calcium-containing water-insoluble composition content is preferably 0.1 to 10 wt%, more preferably 0.2 to 8 wt%, and even more preferably 0.5 to 5 wt%, based on the liquid medium at the start of culture being 100 wt%, and the calcium content is preferably 0.05 to 5 wt%, more preferably 0.1 to 3 wt%, and even more preferably 0.2 to 2 wt%. By including the calcium-containing water-insoluble composition, as the culture medium becomes acidic during culture, the calcium-containing water-insoluble composition gradually dissolves, and the pH drop is alleviated, and the pH can be adjusted, thereby suppressing the inhibition of lactic acid bacteria growth associated with the pH drop.

The manganese-containing microorganism described in the present invention is not particularly limited as long as it is a microorganism that contains manganese, and examples thereof include yeast or lactic acid bacteria containing about 1 to 10% by weight of manganese, and for example, commercially available edible yeast containing about 4 to 8% by weight of manganese can be used. The manganese-containing microorganism content is preferably 0.005 to 0.5% by weight, more preferably 0.01 to 0.4% by weight, and even more preferably 0.015 to 0.2% by weight, when the liquid medium at the start of culture is taken as 100% by weight, and the manganese content is preferably 2.5 to 250 ppm, more preferably 5 to 200 ppm, and even more preferably 7.5 to 100 ppm.

The fats and oils described in the present invention are not particularly limited, but are preferably vegetable fats and oils, more preferably vegetable fats and oils containing oleic acid, linoleic acid, α-linolenic acid, conjugated linoleic acid, etc., and may be esters of the above fatty acids. Examples include sesame oil, salad oil, white bean oil, corn oil, soybean oil, rapeseed oil (canola oil), rice oil, rice bran oil, wheat germ oil, camellia oil, safflower oil, coconut oil (palm kernel oil), cottonseed oil, sunflower oil, perilla oil, linseed oil, olive oil, peanut oil, almond oil, avocado oil, hazelnut oil, walnut oil, grapeseed oil, seaberry oil, borage seed oil, etc. The fats and oils can be contained in an amount of preferably 0.01 to 8% by weight, more preferably 0.02 to 6% by weight, and even more preferably 0.05 to 3% by weight, based on the liquid medium at the start of culture being 100% by weight.

The yeast extract used in the present invention is not particularly limited, and commercially available yeast extracts can be used. The yeast extract can be contained in an amount of preferably 0.1 to 10% by weight, more preferably 0.2 to 8% by weight, and even more preferably 0.5 to 5% by weight, based on 100% by weight of the liquid medium at the start of culture.

The method for culturing lactic acid bacteria of the present invention is not particularly limited as long as it is a method for culturing using the above-mentioned medium for culturing lactic acid bacteria, but in detail, it is preferable to sterilize a liquid medium containing the above-mentioned components, cool it, inoculate the lactic acid bacteria, and culture it under aerobic conditions such as aeration and stirring, shaking, etc. The culture temperature and number of days may be general culture conditions, for example, 20 to 40°C, 1 to 50 hours, preferably 25 to 38°C, 10 to 40 hours. The pH at the start of the culture may be unadjusted, but may be adjusted to about pH 6.0 to 8.0. The amount of inoculation during culture is not particularly limited as long as it is a general inoculation amount, and examples thereof include 0.1 to 10% by weight, preferably 0.5 to 5% by weight.

The type of lactic acid bacteria is not particularly limited, and any common lactic acid bacteria may be used, such as bacteria belonging to the genera Lactobacillus, Lactococcus, Streptococcus, Bifidobacterium, Leuconostoc, Pediococcus, etc. Representative examples of bacteria include Lactobacillus plantarum and Lactobacillus paracasei, and one or more types may be used.

The lactic acid bacteria culture of the present invention is obtained by the above-mentioned culture method, and may be sterilized after the completion of the culture, and the sterilization conditions are not particularly limited as long as they are common methods, but 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.

The lactic acid bacteria culture of the present invention can be obtained using a medium using natural products or food raw materials, and can be used safely, so that it can be added to various products. There are no particular limitations on the foods and beverages to which it can be added, and it can be used in various foods and beverages such as beverages, foods, seasonings, functional foods, and supplements, as well as medicines, quasi-drugs, cosmetics, feed, etc. The content of the lactic acid bacteria culture in various products is not particularly limited, but is preferably 0.1 to 50% by weight, more preferably 0.5 to 30% by weight, and even more preferably 1 to 20% by weight.

The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following examples. In the present invention, all percentages are by weight unless otherwise specified.

(Consideration of carbon source)
50 mL of liquid medium consisting of 13% brown sugar (from Iejima), 3% yeast extract (Meat P1G, manufactured by Asahi Beer Foods Co., Ltd.), 3% oyster shell powder (containing 40% calcium) as a calcium-containing water-insoluble composition, 0.2% olive oil as fats and oils, 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 baffled Erlenmeyer flask 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.

50 mL of each of the liquid media shown in Table 1 was placed in a 200 mL baffled Erlenmeyer flask and autoclaved at 121° C. for 15 minutes. The seed culture was inoculated into the cooled liquid medium at 1% and cultured with shaking at 35° C. and 120 rpm for 21 hours. After the culture was completed, the culture was sterilized at 90° C. for 10 minutes to obtain each lactic acid bacteria culture.
The decaglycerol monooleate used was Sunsoft Q-17S (manufactured by Taiyo Kagaku Co., Ltd.).

(evaluation)
For each lactic acid bacteria culture, pH measurement and bacterial count were performed using a bacterial counting chamber. The results are summarized in Table 1 and Fig. 1 (1), and micrographs are shown in Fig. 1 (2).

From Table 1 and Figure 1, it was found that the use of brown sugar as a medium component significantly increased the number of bacteria compared to sucrose. Also, from Figure 1 (2), the bacterial cells obtained using the brown sugar medium were spherical and looked like dots, whereas the bacterial cells obtained using the sucrose medium were long and rod-shaped. This is thought to be because the inclusion of brown sugar activated the growth of lactic acid bacteria, causing repeated division in a short period of time before the bacterial cells could grow large.
Therefore, it was found that brown sugar is an important carbon source for the efficient cultivation of lactic acid bacteria.

(Consideration of brown sugar content)
Using the liquid media shown in Table 2, the same treatment as in Example 1 was carried out to obtain each lactic acid bacteria culture.

The evaluation was carried out in the same manner as in Example 1, and the results are shown in Table 2 and FIG.

From Table 2 and Figure 2, it was found that the number of lactic acid bacteria increased with an increase in brown sugar content up to 12%. If the sugar concentration in the medium is too high, the osmotic pressure increases, and it is known that microorganisms are usually stressed and their growth is suppressed in an environment of high osmotic pressure, but it was surprising that even with a high brown sugar content of 20%, the growth of lactic acid bacteria was hardly suppressed at all.

(Study on the content of calcium-containing water-insoluble composition)
Using the liquid media shown in Table 3, the same treatment as in Example 1 was carried out to obtain each lactic acid bacteria culture.
In addition, oyster shell powder was used as the calcium-containing water-insoluble composition.

The evaluation was carried out in the same manner as in Example 1, and the results are shown in Table 3 and FIG.

From Table 3 and Figure 3, it was found that the use of oyster shell powder as a medium component significantly increased the number of bacteria compared to when no oyster shell powder was added, and the number of lactic acid bacteria increased as the oyster shell powder content increased. In addition, the higher the oyster shell powder content, the higher the pH at the end of the culture, and the less the pH decreased during the culture.
In other words, by including the calcium-containing water-insoluble composition, the calcium-containing water-insoluble composition gradually dissolved as the culture medium became acidic during cultivation, and the decrease in pH was mitigated, thereby suppressing the inhibition of lactic acid bacteria growth that accompanies 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.

(Study of manganese-containing microorganism content)
The liquid media shown in Table 4 were used and treated in the same manner as in Example 1 to obtain each lactic acid bacteria culture.

The evaluation was carried out in the same manner as in Example 1, and the results are summarized in Table 4 and FIG.

From Table 4 and FIG. 4, it was found that the use of manganese-containing yeast as a medium component significantly increased the number of bacteria compared to when no manganese yeast was added, and furthermore, the higher the manganese yeast content, the greater the number of lactic acid bacteria.
Therefore, it was found that manganese-containing microorganisms are important as a medium component for efficiently culturing lactic acid bacteria.

(Study of fat and oil content)
Using the liquid media shown in Table 5, the same treatment as in Example 1 was carried out to obtain each lactic acid bacteria culture.

The evaluation was carried out in the same manner as in Example 1, and the results are summarized in Table 5 and FIG.

From Table 5 and FIG. 5, it was found that the use of fats and oils as a medium component slightly increased the number of bacteria compared to when no fats and oils were added.

(Consideration of types of oils and fats)
The liquid media shown in Table 6 were used and treated in the same manner as in Example 1 to obtain each lactic acid bacteria culture.
The conjugated linoleic acid used was CL Ace (manufactured by Nisshin Oillio Group, Ltd.).

The evaluation was carried out in the same manner as in Example 1, and the results are summarized in Table 6 and FIG.

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

(Study of medium components)
50 mL of each of the liquid media shown in Table 7 was placed in a 200 mL baffled Erlenmeyer flask and autoclaved at 121° C. for 15 minutes. The cooled liquid media was inoculated with 1% of the seed culture liquid described in Example 1 or the seed culture of Lactobacillus paracasei NBRC3533 similarly cultured, and cultured with shaking at 35° C. and 120 rpm for 21 hours. After the culture was completed, the culture was sterilized at 90° C. for 10 minutes to obtain each lactic acid bacteria culture.

The evaluation was carried out in the same manner as in Example 1, and the results are summarized in Table 7 and FIG.

From Table 7 and FIG. 7, it was found that for any type of lactic acid bacteria, the number of lactic acid bacteria was significantly increased by using a medium containing oyster shells and manganese-containing yeast.

(Powderization)
1L of 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 fats and oils, 0.1% manganese-containing yeast (containing 5% manganese) and 85.1% tap water was placed in a 3L baffled Erlenmeyer flask 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.

2 L of the same liquid medium as the seed culture was placed in six 3 L baffled Erlenmeyer flasks and autoclaved at 121° C. for 15 minutes. 1% of the seed culture was inoculated into the cooled liquid medium and cultured with shaking at 33° C. and 90 rpm for 20 hours. After the culture was completed, the mixture was sterilized at 90° C. for 10 minutes to obtain 12.4 kg of 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 content: 3.2%, cell count: 1.1×10 ¹¹ cells/g).

Claims (8)

The culture medium for culturing lactic acid bacteria contains brown sugar, a calcium-containing water-insoluble composition, and a manganese-containing microorganism, and contains 3 to 10% by weight of the calcium-containing water-insoluble composition when the liquid medium is taken as 100% by weight . The 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 taken as 100% by weight. The medium for culturing lactic acid bacteria according to claim 1 or 2, containing 5% by weight or more of brown sugar. The medium for culturing lactic acid bacteria according to any one of claims 1 to 3, further comprising 0.01 to 8% by weight of an oil or fat. A method for culturing lactic acid bacteria, comprising culturing lactic acid bacteria in a medium according to any one of claims 1 to 4. The method for culturing the lactic acid bacteria according to claim 5, wherein the culture is carried out under aerobic conditions. A lactic acid bacteria culture obtained by the method according to claim 5 or 6. A food, drink, medicine, cosmetic, or feed comprising the lactic acid bacteria culture according to claim 7.