JP6283481B2 - Lactic acid bacteria culture method - Google Patents

Description

  The present invention relates to a culture method for culturing lactic acid bacteria using a medium containing a solid substance, and a food material obtained by this culture method.

  Examples of foods using lactic acid bacteria include fermented milk and lactic acid bacteria beverages. Fermented milk and lactic acid bacteria beverages are produced by inoculating and sterilizing sterilized liquid raw materials with lactic acid bacteria, and the culture method of lactic acid bacteria in these fermented milk and lactic acid bacteria beverages corresponds to liquid culture.

JP 2004-057020 A

  When a lactic acid bacterium is purely cultured using a liquid medium, the pH of the liquid medium is lowered by lactic acid produced by the lactic acid bacterium. Compared with other microorganisms, lactic acid bacteria grow well even under relatively low pH conditions. However, when the production of lactic acid increases and the pH of the liquid medium decreases to around 4, the lactic acid bacteria Its own growth is also suppressed. Therefore, in a general method for cultivating lactic acid bacteria using a liquid medium, there is a limit to the concentration of live lactic acid bacteria actually obtained (the number of live lactic acid bacteria per unit amount of the medium).

  Therefore, an object of the present invention is to provide a method for culturing lactic acid bacteria that can provide a higher concentration of lactic acid bacteria than when a liquid medium is used, and a food material obtained by this method.

The method for cultivating lactic acid bacteria according to the present invention comprises inoculating a lactic acid bacterium into a medium containing skim milk powder and wheat flour, setting the water content of the medium after inoculation with the lactic acid bacterium to more than 40% and less than 65%. Culturing at an appropriate growth temperature range for 12 hours or more, and the lactic acid bacteria are Bulgaria bacteria .

Moreover, the method for cultivating lactic acid bacteria according to the present invention comprises inoculating lactic acid bacteria on a medium containing skim milk powder and wheat flour, the water content of the medium after inoculation with lactic acid bacteria is less than 65%, and in an optimum growth temperature range of lactic acid bacteria. It is cultured for 48 hours or more, and the lactic acid bacteria are Bulgaria bacteria .

  According to the method for cultivating lactic acid bacteria according to the present invention, a liquid medium (generally containing water is about 90%. In the present invention, “%” means “% by weight” unless otherwise specified. It is possible to obtain a higher concentration of live lactic acid bacteria than in the case of using.

The graph which shows a time-dependent change of the living microbe density | concentration of lactic acid bacteria in Example 1 The graph which shows the time-dependent change of pH in Example 1. The graph which shows the time-dependent change of the lactic acid density | concentration in Example 1. Graph showing lactic acid production after 24 hours, 48 hours and 72 hours from the start of culture in Example 1 The graph which shows the time-dependent change of the living bacteria density | concentration of lactic acid bacteria in Example 2, and pH The graph which shows a time-dependent change of the lactic acid concentration of the lactic acid bacteria in Example 2. Graph showing lactic acid production after 24 hours, 48 hours and 72 hours from the start of culture in Example 2

  The method for cultivating lactic acid bacteria according to the present embodiment comprises inoculating lactic acid bacteria in a medium containing solid matter, cultivating lactic acid bacteria in the optimum growth temperature range with the water content of the medium after inoculation with lactic acid bacteria being not more than a certain percentage. It is.

  A mixture of skim milk powder and wheat flour is used as the solid added to the medium according to the present embodiment. The kind of wheat flour is not particularly limited, and any of weak flour, medium flour, strong flour and the like may be used. Even if only wheat flour is used as the medium, it is possible to culture lactic acid bacteria. However, when wheat flour and nonfat dry milk are used in combination, the production of lactic acid by lactic acid bacteria is suppressed. The viable cell concentration is further increased (the viable cell count of lactic acid bacteria is further increased). Although the mixing ratio of skim milk powder and wheat flour is not particularly limited, it may be mixed such that the weight ratio of skim milk powder and wheat flour is, for example, 1: 0.1 to 10, preferably 1: 0.5 to 5, More preferably, it is 1: 0.5-1.5, More preferably, it is 1: 1.

  A medium containing skim milk powder and wheat flour is inoculated with lactic acid bacteria pre-cultured in a predetermined medium, and the water content of the medium after inoculation with lactic acid bacteria (ratio of the weight of water to the weight of the medium) is adjusted. A liquid medium can be used for preculture of lactic acid bacteria. The culture solution obtained in the pre-culture is added to the medium, and a predetermined amount of water is added to adjust the water content of the medium after inoculation with lactic acid bacteria. Here, the moisture content of the medium refers to the sum of the amount of moisture initially contained in skim milk powder and wheat flour and the amount of water added for moisture content adjustment. Although the content of water in the medium after inoculation with lactic acid bacteria is adjusted to less than 65%, it is actually adjusted to 35% or more and less than 65%. At this time, if the content of water in the medium after inoculation with lactic acid bacteria is less than 35%, it becomes difficult for the water to reach the medium, making it difficult to make the medium and water uniform. On the other hand, when the water content of the medium after inoculation with lactic acid bacteria is 65% or more, the increase in the number of viable bacteria of lactic acid bacteria with respect to the culture method using a liquid medium becomes small.

  After adjusting the water content of the medium, the lactic acid bacteria are cultured for a predetermined time in the optimum growth temperature range or in a temperature range near the optimum growth temperature range. The optimal growth temperature range of lactic acid bacteria refers to a temperature range in which lactic acid bacteria actually cultured (used) grow well. And the optimal growth temperature range of lactic acid bacteria should just be set so that it may be set to 30-45 degreeC, for example, Preferably it is 30-43 degreeC, More preferably, it is 33-43 degreeC, More preferably, it is 35-40 degreeC. . In addition, the temperature range near the optimum growth temperature range is not the optimum growth temperature for the strain actually used, but refers to the temperature range in which culture can be performed practically. Specifically, the optimum growth temperature The lower limit value of the region is −5 ° C. or more and less than the lower limit value of the optimum growth temperature range, or is higher than the upper limit value of the optimum growth temperature region and is the upper limit value of the optimum growth temperature range + 5 ° C. or less. In addition, the culture | cultivation time which concerns on this embodiment is 12 hours or more, for example.

  The water content and the culture time according to the present embodiment can be properly used as follows according to the purpose of the culture.

  First, when the water content in the medium after inoculation with lactic acid bacteria is 45% or more and less than 65%, preferably more than 45% (more than 45%) and less than 63%, the culture time should be 12 hours or more and 48 hours or less As a result, the number of viable bacteria of lactic acid bacteria per unit weight of the medium can be increased as compared with the case of culturing in a liquid medium. Within the range of these conditions, the water content of the medium after inoculation with lactic acid bacteria is preferably 50% or more and 60% or less, and the culture time is more preferably 12 hours or more and 36 hours or less, and 55% or more and 58% or less. More preferably, the time is 12 hours or more and 36 hours or less. In this case, the water content of the medium is 50% or more and 60% or less, preferably 55% or more and 58% or less, and the culture time is about 24 hours, thereby obtaining the culture method according to the present embodiment. Since the number of living lactic acid bacteria per unit weight of the medium can be increased up to about 10 times that when cultured in a liquid medium, the number of lactic acid bacteria can be increased more efficiently.

  Next, when the content of water in the medium after inoculation with lactic acid bacteria is 35% or more and less than 65%, preferably 35% or more and less than 63%, when culturing in a liquid medium by setting the culture time to 48 hours or more In comparison with the above, the number of living lactic acid bacteria per unit weight of the medium can be increased, and the number of living lactic acid bacteria per unit weight of the medium can be increased as much as possible. Within the range of these conditions, the water content of the medium after inoculation with lactic acid bacteria is 35% or more and 60% or less, more preferably 48 hours or more, 37% or more and 55% or less, and the culture time is 48 hours or more. More preferably, it is 37% or more and 50% or less, and culture time is particularly preferably 48 hours or more. In this case, the water content of the medium is 35% to 60%, preferably 37% to 55%, more preferably 37% to 50%, and the culture time is 60 hours or more. Since the number of living lactic acid bacteria per unit weight of the medium obtained by the culture method according to the embodiment can be increased to about 10 times that when cultured in a liquid medium, the number of lactic acid bacteria can be increased more efficiently. Can do. And the culture medium obtained by the culture | cultivation method which concerns on this embodiment by making the moisture content of the said culture medium into 37% or more and less than 45%, Preferably 37% or more and 40% or less, and making culture | cultivation time 60 hours or more The number of viable lactic acid bacteria per unit weight can be maximized.

  As described above, in the method for culturing lactic acid bacteria according to the present embodiment, a medium containing skim milk powder and wheat flour and having a significantly reduced water content compared to a liquid medium is used. By reducing the water content of the medium to approximately less than 63%, the production of lactic acid by lactic acid bacteria is suppressed. By suppressing the production of lactic acid, the rate of decrease in the pH of the medium becomes slower than that in the liquid medium. As a result, even when the lactic acid bacteria are propagated to a certain extent, the growth of the lactic acid bacteria is hardly suppressed, and the number of living lactic acid bacteria can be increased as compared with the liquid medium. At this time, the lactic acid bacteria include Bulgarian bacteria (Lactobacillus delbrueckii subsp. Bulgaricus), Helveticus bacteria (Lactobacillus helveticus), Thermophilus (Streptococcus thermophilus), Cremoris (Lactococcus lactis subsp. Cremoris), Lactococcus lactis. ), Preferably Lactobacillus gasseri, more preferably Bulgaria, Thermofilus, and Gasseri, more preferably Bulgaria, and Thermophilus, and particularly preferably Bulgaria. In the present invention, the concept of lactic acid bacteria includes microorganisms capable of preparing fermented products such as bifidobacteria and propionic acid bacteria.

  Since the culture obtained by the method for cultivating lactic acid bacteria according to this embodiment is a mixture of skim milk powder and wheat flour, it can be used as a food material (raw material) as it is. Specifically, it can be used as a raw material for baked confectionery using wheat flour or the like as a raw material. When the culture obtained by the culturing method according to the present embodiment is blended (added) as all or part of the raw material, the acidity of lactic acid produced by lactic acid bacteria, the special flavor of fermentation of lactic acid bacteria, and the lactic acid bacteria ( Functionality due to the action of probiotics etc.) can be imparted to the actually produced food.

  In addition, instead of pre-culturing lactic acid bacteria using a liquid medium, lactic acid bacteria may be pre-cultivated by using the culture method according to the present embodiment using a medium containing solid matter. In this case, what is necessary is just to inoculate lactic acid bacteria by mixing the culture containing the solid substance obtained by preculture with a new culture medium. However, as in the above example, it is easier to adjust the water content of the medium after inoculation with lactic acid bacteria, so it is preferable to pre-culture lactic acid bacteria using a liquid medium.

  Examples in which the method for culturing lactic acid bacteria according to the present invention is specifically described below.

<Preculture medium>
MRS liquid medium (Becton, Dickinson and Company) was used as the pre-culture medium. The composition of the MRS medium is as shown in Table 1 below.

<Main culture medium>
As a medium for main culture, 5 g of skim milk powder (Meiji Co., Ltd.) and 5 g of strong powder (Imazu Co., Ltd.) are collected in a plastic tube and sterilized by irradiation with 10 kGy of γ-rays in a sealed state. It was used.

<Pre-culture method>
Lactic acid bacteria (Lactobacillus delbrueckii subsp. Bulgaricus, isolated from Meiji Bulgaria yogurt plain) frozen in stock were inoculated into 5 ml of MRS liquid medium and statically cultured overnight at 37 ° C. What diluted this culture solution 100 times with physiological saline (preculture solution after dilution) was used for inoculation of lactic acid bacteria in the main culture.

<Main culture method>
Add 0.1 g of the diluted preculture and a predetermined amount of sterilized water to the main culture medium after sterilization, mix with a spatula sterilized with flame until uniform, and then continue at 37 ° C for a predetermined time. The culture was stationary. The relationship between the amount of sterilized water added and the amount of water contained in the medium after adjustment with sterilized water is shown in Table 2 below. In Table 2 below, a medium having a moisture content of 91% corresponds to a conventional liquid medium (fat dry milk medium, reduced skim milk). In preparing a medium having a moisture content of 37 to 63%, the amount of sterilized water added is the actual amount of sterilized water added to a mixture of 5 g of skim milk powder and 5 g of strong powder. Represents the ratio of the total amount of 0.9 g of water originally contained in a mixture of 5 g of skim milk powder and 5 g of strong powder and the addition amount of sterilized water to the total amount of the medium.

<Sampling method>
Using an electronic balance in a clean bench, 1 g of the cultured medium was weighed, placed in a polyethylene bag with 9 g of physiological saline, and uniformly suspended using a stomacher (Interscience Minimix CC) ( 10 seconds at 4 strokes / sec). Using this suspension, the number of viable lactic acid bacteria, pH, and lactic acid concentration were measured.

<Measurement method of viable count>
The suspension obtained by sampling was diluted with a predetermined amount of physiological saline, 10 μl of this diluted solution was plated on MRS agar medium, and the number of colonies was counted. From the counted number of colonies and the dilution ratio of the suspension, the number of living lactic acid bacteria per gram of the medium was determined.

<Measurement method of lactic acid concentration>
About the suspension obtained by sampling, the lactic acid density | concentration was measured using the biosensor (Oji Scientific Instruments Co., Ltd., BF-5). In this measurement, a calibration curve was prepared using pure water, 1, 2, and 5 mM lactic acid standard solutions. The suspension obtained by sampling was appropriately diluted so that the lactic acid concentration was within the range of the calibration curve, and the lactic acid concentration was measured.

Example 1
As Example 1, the water content of the medium was adjusted to 37%, 45%, 55%, 58% and 63%, and lactic acid bacteria were cultured. For comparison between the culture medium of the present invention and a conventional liquid culture medium, the water content of the culture medium was adjusted to 91% as a control (liquid culture medium), and lactic acid bacteria were cultured. The changes over time in the concentration of viable lactic acid bacteria (the number of viable lactic acid bacteria per gram of the medium), pH, and lactic acid concentration are shown in FIGS. In addition, FIG. 4 shows the amount of lactic acid produced after 24 hours, 48 hours and 72 hours from the start of culture.

  As shown in FIG. 1, in a sample in which the moisture content of the medium is more than 45% (more) and less than 63%, that is, a sample having a moisture content of the medium of 55% or 58%, 12 hours after the start of culture. The viable bacterial concentration of lactic acid bacteria was about 10 times higher than that of the sample having 91% moisture content in the medium (when lactic acid bacteria were cultured using a liquid medium). Moreover, as shown in FIGS. 2-4, it was confirmed that the production | generation of the lactic acid by a lactic acid bacteria is suppressed and the fall of the pH of a culture medium becomes slow as the moisture content of a culture medium decreases.

  Moreover, it was confirmed that in the sample having a water content of less than 63%, the concentration of lactic acid bacteria increased as the culture time increased. In particular, in the 37% sample with the smallest water content in the culture medium, the viable lactic acid bacteria concentration increased monotonously, and the highest viable lactic acid bacteria concentration among all the samples could be obtained.

(Example 2)
As Example 2, the solids used in the culture medium were skim milk powder only, thin powder only, medium powder only, strong powder only, a mixture of skim milk powder and soft powder, a mixture of skim milk powder and medium power powder, a mixture of skim milk powder and strong powder. The lactic acid bacteria were cultured. In this example, the water content of the medium was adjusted to 55%.

  FIG. 5A shows the change over time in the concentration of live lactic acid bacteria (the number of viable bacteria per gram of the medium) and pH, and FIG. 5B shows the change over time in the concentration of lactic acid. In addition, FIG. 6 shows the amount of lactic acid produced after 24 hours, 48 hours and 72 hours from the start of culture.

  As shown in FIGS. 5A and 5B, compared to a sample using only skim milk powder, thin powder, or medium power powder in the medium, either soft powder, medium powder, or strong powder, and skim milk powder were used in the medium. In the sample, a high concentration of lactic acid bacteria was obtained. In addition, as shown in FIG. 6, in comparison with a sample using only one of the flour, the medium flour and the strong powder in the medium, the sample using either the flour, the medium flour or the strong powder and skim milk powder, It was confirmed that the amount of lactic acid produced was suppressed.

(Example 3)
As Example 3, the culture of lactic acid bacteria according to the present embodiment obtained in Example 2 (the solid used for the medium is only skim milk powder, only soft powder, and a mixture of skim milk and soft powder) in a predetermined amount. The baked confectionery was prepared by blending and using a conventionally known general method. These baked goods had a sour taste due to lactic acid produced by lactic acid bacteria and a unique flavor due to fermentation of lactic acid bacteria, and a unique taste was felt. Furthermore, in these baked confectionery, functionality by the action of lactic acid bacteria could be expected. It was confirmed that the culture of lactic acid bacteria according to this embodiment is useful as a food material such as baked goods and bread.

  The present invention can be used as a method for culturing lactic acid bacteria and a method for producing food materials such as baked goods and bread.

Claims (5)

A method for culturing lactic acid bacteria,
Inoculate lactic acid bacteria into a medium containing skim milk powder and wheat flour,
The water content of the medium after inoculation with lactic acid bacteria is 45% or more and less than 65%,
Culturing for 12 hours or more in the optimum growth temperature range of the lactic acid bacteria ,
The method for cultivating lactic acid bacteria, wherein the lactic acid bacteria are Bulgarian bacteria .   The method for cultivating lactic acid bacteria according to claim 1, wherein the water content of the medium after inoculation with lactic acid bacteria is 50% or more and 60% or less. A method for culturing lactic acid bacteria,
Inoculate lactic acid bacteria into a medium containing skim milk powder and wheat flour,
The water content of the medium after inoculation with lactic acid bacteria is less than 65%,
Incubate in the optimal growth temperature range of the lactic acid bacteria for 48 hours ,
The method for cultivating lactic acid bacteria, wherein the lactic acid bacteria are Bulgarian bacteria .   The method for cultivating lactic acid bacteria according to claim 3, wherein the water content of the medium after inoculation with lactic acid bacteria is 35% or more and 60% or less. The water content of the medium after inoculation with lactic acid bacteria is 35% to 60%,
The method for cultivating lactic acid bacteria according to claim 3, wherein culturing is performed for 60 hours or more in an optimum growth temperature range of the lactic acid bacteria.

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