JPH0622746A - Frozen dry substance on microorganism containing medium and its preparation - Google Patents

Abstract

PURPOSE: To obtain a freeze-dried material of storage-stable under a relatively simple storage condition by making a microorganism-containing medium containing a bacterium keeping viability and fertility contain a carbohydrate, gelatin and ascorbic acid at a specific ratio and freeze-drying the resultant medium.

CONSTITUTION: This freeze-dried material is obtained by culturing a bacterium such as a strain of Acidophilus or a strain of Bifidobacterium in a culturing solution constituted with milk powder and a suitable nutritive agent so as the bacterium to be a stationary growing phase, and adding a carbohydrate (preferably milk sugar, etc.), gelatin, ascorbic acid in a dried weight ratio of (16/1/0.1)-(16/1/1) (preferably mixing them so as a dried weight ratio of the bacterium culturing solution, carbohydrate, gelatin and ascorbic acid to be 2/16/1/0.2) and mixed, and subjecting the resultant mixture to freeze-drying.

COPYRIGHT: (C)1994,JPO

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a bacterium which has viability and reproductive ability even after being stored in a freeze-dried state for several years, and in particular, a strain of Acidophilus, Bifidobterium.
The present invention relates to a freeze-dried product of a microorganism-containing medium in a strain of Escherichia coli (acterium) and a strain of Escherichia coli and a method for producing the same.

[0002]

2. Description of the Related Art Conventionally, a freeze-drying method has been used as a method for storing various medicines, blood products, enzyme products, experimental materials, etc. for a long period of time. In performing the freeze-drying, in order to suppress the deterioration of the viability and fertility due to humidity, temperature, and oxygen in the atmosphere as much as possible,
Various additives such as auxiliary agents, dispersants, protective agents and carriers are administered to the microorganism-containing medium before freeze-drying, and the freeze-dried product can be stored safely not only at low temperature but also at room temperature. It is devised like this.

On the other hand, in recent years, enteric bacteria such as Escherichia coli, Bacteroides, Eubacterium, Bifidobacteria, and Lactobacillus, which play an auxiliary role in the digestion of food, have been mixed with lactic acid products such as yogurt to provide health-maintaining foods. Manufacturing is becoming common, and it is considered that microorganisms (bacteria) to be used can be freeze-dried under favorable conditions in manufacturing the health-maintaining food.

[0004]

However, in freeze-drying several kinds of microorganisms (bacteria) with highly different sensitivities in the same medium, various administrations are carried out in the microorganism-containing medium before freeze-drying for that purpose. The condition of additives such as auxiliary agent, dispersant, protective agent, carrier and the like is a problem.

The present invention has been made in view of the above problems, and an additive to be administered into a microorganism-containing medium used in the production of the freeze-dried product was examined to freeze-dry the microorganism (bacteria). However, after that, the freeze-dried product was kept for a few years under a relatively simple storage condition (shade-proof condition at 8 ° C or less) not only to ensure the viability and fertility, but also at room temperature. It is an object of the present invention to provide a freeze-dried product of a microorganism-containing medium and a method for producing the same, which allows the freeze-dried product to be processed into a commercially available product under the conditions (normal indoor conditions in the atmosphere).

[0006]

The present invention takes the following technical means in order to solve the above problems.

[0007] That is, bacteria capable of viability and reproduction even after several years of freeze-drying, especially Acidophilus (Acidophilu)
s) strain, Bifidobacterium (Bifidobacterium) strain, Escherichia coli (Escherichia coli) strains of the lyophilized product of the microorganism-containing medium, as the microorganism-containing medium,
Using a mixture of carbohydrate: gelatin: ascorbic acid in a dry weight ratio of 16: 1: 0.1 to 16: 1: 1, a freeze-dried product was produced by freeze-drying this medium, The feature is that the dried product can be stably stored for a long period of time.

It is possible to suspend the bacteria having the above-mentioned highly different sensitivities, freeze-dry them, and process them under normal temperature and humidity, and at least 3 at a temperature of 8 ° C or less in a dry state. In order to maintain a high degree of stability (compensation of viability and fertility) for more than a year, various auxiliary agents in the microorganism-containing medium before freeze-drying the freeze-dried product,
Additives such as a dispersant, a protective agent and a carrier are added.

In the present invention, the additives are investigated, and carbohydrates, gelatin, and ascorbic acid, which mainly act as an auxiliary agent or a protective agent, are combined in an appropriate dry weight ratio, and the additives of Examples 1 to 15 shown below are used. A freeze-dried product was prepared using a medium, and the long-term storage stability of the freeze-dried product was determined by the number of colony formation per unit immediately after freeze-drying (CFU /
g) and the number of colonies formed after several years (CFU / g) were compared, and the stability of retention was judged by the survival rate (%) of the bacteria in the freeze-dried product.

[0010]

The freeze-dried product of the present invention comprises carbohydrates, gelatin and ascorbic acid in a dry weight ratio of 16: 1: 0.1 to 16: 1: as auxiliary agents or protective agents in a microorganism-containing medium.
Since it is input in 1, due to their interaction,
This lyophilized product is long-term safe by sufficiently suppressing deterioration of viability and fertility due to humidity, temperature and oxygen in the atmosphere not only at low temperatures (4 to 8 ° C) but also at room temperature. It is possible to preserve sex.

[0011]

EXAMPLES Examples of the freeze-dried product of the present invention and a method for producing the same will be described below with reference to tables and graphs. still,
Table 1 shows a list of the culture media used in Examples 1 to 15.

[0012]

[Table 1]

The bacterium used in this example is Acidophilus
philus), a type of Lactobacillus
gasseri), a type of Bifidobacterium (Bifidobacterium), which is a type of Bifidobacterium lon
gum) and 3 strains of Escherichia coli
It is a seed.

Example 1 First, each bacterium was placed in a bacterial culture solution (I) (composed of whole milk or milk powder and an appropriate nutrient) until each bacterium became a stationary growth phase. Incubate.

Next, a culture solution 250 containing the bacterium
A suspension medium (microorganism-containing medium) is prepared by mixing 0 ml of the additive (II), which is a combination of lactose, gelatin and ascorbic acid.

The method for preparing the additive (II) is as follows.
0 g of lactose (DAB 9) is dissolved in 1500 ml of purified water (DAB 9) with heating, and 50 g of Bloom 200 gelatin (DAB 9) is dissolved with heating (at 37 ° C.).

On the other hand, 23 g of ascorbic acid (DAB 9) was dissolved in 240 ml of purified water (DAB 9) and neutralized (pH 6.0) with 5 g of 10% weight / volume sodium hydroxide.
Then, the neutralized solution of ascorbic acid is added to the mixed solution of lactose and gelatin to prepare a mixed solution of three kinds, and this mixed solution is used as an additive (II).

Then, the microorganism-containing medium is freeze-dried under a constant pressure to prepare a freeze-dried product.

Further, the freeze-dried product obtained by the freeze-drying is crushed under normal indoor conditions with a sieve for dry particles having a mesh size of 0.75 mm, and then various crushed lyophilized products are crushed. Mix and homogenize in a gyro wheel mixer and fill into, for example, hard gelatin plug-on capsules to form capsules.

Then, the freeze-dried product in the form of the capsule is treated at low temperature (4 ° C to 8 ° C) and normal temperature (20 ° C to 2 ° C).
5 ° C.) and high temperature (30 ° C. and 40 ° C.) are set for storage for 3 years or more in a storage room (of course, humidity is constant and light is shielded), and then a storage stability test is performed.
However, because of the continuous test, there are several capsules that are taken out of the storage room during storage.

The above-mentioned storage stability test uses a usual method in microbiology, and the freeze-dried product stored under each condition for a specific period is taken out from the storage room and diluted with a fixed experimental medium to prepare a dilution series. After culturing, the cells are cultured on a gel-like experimental medium for an appropriate period of time, and thereafter, the number of colonies formed (CFU / g) per unit is counted. By plotting this number on a logarithmic scale, the exponential survival rate of the microorganism (bacteria) is converted, and from this result, the deterioration of the viability / fertility of the bacterium can be known.

As a control, the one subjected to the storage stability test as described above immediately after freeze-drying (0 days of storage) is used.

<< Embodiment 2 >> In this embodiment, the above-mentioned embodiment 1 is used.
Sucrose was used instead of lactose. Other operations are the same as those in the first embodiment.

<< Embodiment 3 >> In this embodiment, the above-mentioned Embodiment 1 is used.
Instead of 800 g of lactose, 300 g of lactose and 500 g
Of sucrose was used. Other operations are the same as those in the first embodiment.

<Fourth Embodiment> In the present embodiment, the above-mentioned first embodiment is used.
Instead of 800 g of lactose, 300 g of lactose and 500 g
Was used as a mixture with milk powder. Other operations are the same as those in the first embodiment.

<Fifth Embodiment> In the present embodiment, the above-mentioned first embodiment is used.
As the additive of the above, the one to which ascorbic acid was not added was used. Therefore, neutralization treatment with sodium hydroxide is not required. Other operations are the same as those in the first embodiment.

<Embodiment 6> In this embodiment, the above-mentioned embodiment 1 is used.
As the additive of (3), the one to which gelatin was not added was used. Other operations are the same as those in the first embodiment.

<Embodiment 7> In the present embodiment, the above-mentioned Embodiment 1 is used.
Add ascorbic acid and gelatin as additives, and replace 800 g of lactose with
A mixture of 300 g lactose and 500 g milk powder was used.
Since ascorbic acid is not added, neutralization treatment with sodium hydroxide is not required. Other operations are the same as those in the first embodiment.

<Embodiment 8> In the present embodiment, the above-mentioned Embodiment 1 is adopted.
270 instead of 800 g lactose and 50 g gelatin
Lactose (g) and 17 g of gelatin were used. Other operations are the same as those in the first embodiment.

<< Embodiment 9 >> In this embodiment, the above-mentioned Embodiment 1 is used.
As an additive for the above, no ascorbic acid was added, and a trace amount of lactose and gelatin was used instead of 800 g of lactose and 50 g of gelatin. Since ascorbic acid is not added, neutralization treatment with sodium hydroxide is not required. Other operations are the same as those in the first embodiment.

Example 10 In this example, 16 g of lactose and 50 g of gelatin in Example 1 were used instead of 16 g of lactose.
00 g of lactose and 100 g of gelatin were used. Other operations are the same as those in the first embodiment.

Example 11 In this example, as the additive of Example 1 above, ascorbic acid was not added, and 1600 g of lactose and 100 g of lactose were used instead of 800 g of lactose and 50 g of gelatin. Of gelatin was used. Since ascorbic acid is not added, neutralization treatment with sodium hydroxide is not required. Other operations are the same as those in the first embodiment.

Example 12 In this example, for the strain of Lactobacillus gasseri and the strain of Bifidobacterium longum, the additive used in Example 5, that is, Regarding the strain of Escherichia coli (Escherichia coli) using the additive of Example 1 without ascorbic acid (neutralization treatment with sodium hydroxide is not necessary, of course),
The additives used in Example 1 were used. Other operations are the same as those in the first embodiment.

Example 13 In this example, a strain of Lactobacillus gasseri, a strain of Bifidobacterium longum, and a strain of Escherichia coli were used.
Three strains of (Escherichia coli) were mixed to prepare a suspension in the medium used in Example 1 above. Other operations are the same as those in the first embodiment.

Example 14 In this example, the three strains were mixed and a suspension was prepared in the medium used in Example 5. Other operations are the same as those in the first embodiment.

Example 15 In this example, a bacterial culture solution is used.
As (I), the whole milk or the one not containing milk powder is used, and the additive (II) prepared by the following method in the culture solution (I)
Were mixed to prepare a suspension medium (microorganism-containing medium). Other operations were the same as in Example 1, and 200 mg of the freeze-dried product was filled in the plug-on type capsule and stored.

The additive (II) was prepared by first dissolving 1200 g of lactose (DAB 9) in 2300 ml of purified water (DAB 9) while heating and adding gelatin of Bloom 200 (DAB 9) to the solution.
9) Dissolve 75 g with heating (at 25 ° C to 37 ° C).

On the other hand, 13 g of ascorbic acid (DAB 9) was dissolved in 250 ml of purified water (DAB 9) and neutralized (pH 6.0 or more) with 3 g of sodium hydroxide. The neutralization solution is added to the mixed solution of lactose and gelatin to prepare three types of mixture.

Next, the results of the storage stability test in Examples 1 to 15 will be described in detail.

The results of Examples 1 to 4 and Example 7 are shown in Table 2 below. The table shows the results after three years of storage, and the storage temperature conditions are low temperature (4 ° C), normal temperature (under room temperature), and high temperature (30 ° C), respectively.

[0041]

[Table 2]

As can be seen from Table 2 above, a microorganism-containing medium in which a bacterial culture solution: carbohydrate: gelatin: ascorbic acid was mixed at a dry weight ratio of 8: 16: 1: 0.5 (Example 1, 2 and 3) have a very high survival rate after storage for 3 years, especially Lactobacillus gasseri (Lactobacillus) at 4 ° C.
gasseri) and Bifidobacterium Long Bomb (Bifidobac
The survival rate of the terium longum) strain is as high as 80%.

In addition, lactose was used alone as the above-mentioned carbohydrate (Example 1), sucrose was used alone (Example 2), or lactose and sucrose were mixed (Example 3). Although the survival rate is high, powdered milk was used (Example 4).
It can be seen that the survival rate of the product is not so high, and that the survival rate is considerably reduced when gelatin or ascorbic acid is not used (Example 7).

The results of Examples 5 and 6 and Examples 8 and 9 are shown in Table 3 below. The table shows the results after 1, 2, and 3 years of storage, and the storage temperature conditions are low (4 ° C).
C-8 ° C), normal temperature (20 ° C-25 ° C), high temperature (3
0 ° C).

[0045]

[Table 3]

As can be seen from Table 3 above, the survival rate was low when any of carbohydrates, gelatin and ascorbic acid was lacking (Examples 5, 6 and 9), and also Examples 1 to 3 and 3 above. As can be seen by comparing only the data after a year, it can be said that if the dry weight ratio of the microorganism-containing medium is not appropriate (Example 8), the survival rate, especially at room temperature or high temperature, is considerably reduced.

The results of Examples 10 and 11 are shown in Table 4 below. In addition, the table shows Escherichia col (Escherichia col
The results are only for the strain i) and the experimental results after 1, 2 and 3 years of storage, and the storage temperature conditions are low (4
° C to 8 ° C) and room temperature (20 ° C to 25 ° C).

[0048]

[Table 4]

As discussed in Table 4 above, the Escherichia coli strain was a mixture of carbohydrate: gelatin at a dry weight ratio of 16: 1 (Example 10).
In that case, the higher the ratio of carbohydrate and gelatin to the bacterial culture (bacterial culture: carbohydrate: gelatin is about 2: 16: 1), the higher the survival rate (immobilized for 3 years at low temperature storage). Of 100%).

The above Escherichia coli
It can also be seen that the strain of E. coli is highly dependent on ascorbic acid, and that storage in a medium that does not use ascorbic acid (Example 11) results in extremely low survival rate.

The results of Examples 12 to 14 are shown in Table 5 below. The table shows the results after 1, 2, and 3 years of storage, and the storage temperature conditions are low temperature (4 ° C to 8 ° C),
Normal temperature (20 ° C to 25 ° C).

[0052]

[Table 5]

As can be seen from Table 5 above, what can be said in Examples 1 to 11 is that Lactobacillus gasseri (L
Actobacillus gasseri) strain, Bifidobacterium longum
Escherichia coli strain (Bifidobacterium longum)
The same can be said when the above additives are added to a cell culture solution containing a mixture of (hia coli) strains, and in particular, ascorbic acid is less sensitive to less sensitive bacteria and less sensitive. It can be seen that the large bacteria should be increased.

Therefore, the highly sensitive Escherichia coli (Escheric
It can be said that the weight ratio of ascorbic acid should be appropriately adjusted within the range of 0.1 to 1.0 in consideration of the strain of H. coli.

Next, the results of Example 15 are shown in FIG.
The figure shows the results of the strain of Lactobacillus gasseri, which is a graph comparing the survival rates depending on the storage temperature, after 1, 2 and 3 years of storage. The storage temperature conditions are low temperature (4 ° C), normal temperature (under room temperature), and high temperature (30 ° C and 40 ° C).

As can be seen from FIG. 1, depending on the type of bacteria, there is a case in which the survival rate of the freeze-dried product is better when whole milk or milk powder is not added to the strain culture medium (I) for treatment.
Carbohydrate: gelatin: ascorbic acid dry weight ratio of 1
If the culture medium input at 6: 1: 0.1 to 16: 1: 1 (content in the internal phase) is used, the viability and reproductive ability of the bacteria are reduced even when freeze-dried and stored for a long time. I know I won't.

From the above examples, a graph summarizing the difference in stability test depending on the type of additive (II) is shown in FIG.
In addition, FIG. 2 shows the results for Escherichia coli strains, and the results of continuous tests for 1 to 2 years of storage.

As can be seen from FIG. 2, when carbohydrates, gelatin, and ascorbic acid were added as the microorganism-containing medium, the interaction showed almost unchanged survival rate several years after freeze-drying.

From the above results, the strains of Lactobacillus gasseri and the strains of Bifidobacterium longum, which are highly different in sensitivity,
When freeze-drying a strain of Escherichia coli in the same medium, the freeze-dried product was used for several years,
Relatively simple storage conditions (shading and humidity protection below 8 ° C)
In order to ensure the viability and fertility, various auxiliary agents to be administered in the microorganism-containing medium before lyophilization, dispersants, protective agents, as additives such as carriers (II), Carbohydrate: Gelatin: Ascorbic acid in a dry weight ratio of 1
It was found that it is sufficient to add at 6: 1: 0.1 to 16: 1: 1, and it has been found that it is particularly preferable to use lactose, sucrose, or a mixture of two kinds as the carbohydrate.

Further, by appropriately adjusting the conditions of the additive (II), it becomes possible to process into a commercially available product even at room temperature (normal indoor conditions in the atmosphere). It will also be available as a drug.

[0061]

INDUSTRIAL APPLICABILITY In the present invention, when a freeze-dried product of a microorganism-containing medium is produced, carbohydrate, gelatin, and ascorbic acid are used as the microorganism-containing medium in a dry weight ratio of 1: 1.
Since it is input at 6: 1: 0.1 to 16: 1: 1,
Due to their interactions (mainly carbohydrate acts as an auxiliary or protective agent, gelatin acts as a dispersant, and ascorbic acid acts as an antioxidant), this lyophilizate is not only cold but at room temperature, and in some cases at high temperature. Even under the humidity,
The deterioration of viability and fertility due to temperature and atmospheric oxygen can be sufficiently controlled, and long-term safe preservation can be achieved. Therefore, this freeze-dried product can be used as a drug.

[Brief description of drawings]

FIG. 1 is a graph showing the results of stability tests depending on the difference in storage temperature.

FIG. 2 is a graph showing the results of stability tests depending on the medium of the lyophilized product.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12R 1:01)

Claims (14)

[Claims] 1. A freeze-dried product of a microorganism-containing medium containing a bacterium that retains viability and fertility, wherein the dry weight ratio in the medium is 16: 1: 0.1 to 16: 1: 1 (internal A lyophilized product of a microorganism-containing medium, characterized in that a mixture of carbohydrates, gelatin and ascorbic acid having a phase content) is added, and the medium is lyophilized. 2. The microorganism-containing medium according to claim 1, wherein the bacterium is a strain of Acidophilus, a strain of Bifidobacterium, or a strain of Escherichia coli. Lyophilized product. 3. The microbial-containing medium of the freeze-dried product of bacteria is a mixture of a bacterial culture solution having a dry weight ratio of 8: 16: 1: 0.5 (content in the internal phase), carbohydrate, gelatin, and ascorbic acid. The freeze-dried product of the microorganism-containing medium according to claim 1 or 2. 4. The microbial-containing medium of the lyophilized product of the bacterium is a mixture of a bacterial culture solution having a dry weight ratio of 2: 16: 1: 0.2 (content in the internal phase), carbohydrate, gelatin, and ascorbic acid. The freeze-dried product of the microorganism-containing medium according to claim 1 or 2. 5. The freeze-dried product of the microorganism-containing medium according to claim 1, wherein the carbohydrate comprises one substance or a mixture of a plurality of substances. 6. The freeze-dried product of the microorganism-containing medium according to claim 1, wherein the carbohydrate is lactose. 7. The freeze-dried product of the microorganism-containing medium according to claim 1, wherein the carbohydrate is sucrose. 8. The freeze-dried product of the microorganism-containing medium according to claim 1, wherein the carbohydrate is a mixture of lactose and sucrose. 9. Carbohydrate: gelatin: ascorbic acid are mixed at a dry weight ratio of 16: 1: 0.1 to 16: 1: 1 and added to a bacterial culture to prepare a microorganism-containing medium. Next, a method for producing a freeze-dried product of a microorganism-containing medium, which comprises freeze-drying the medium. 10. Bacterial culture: carbohydrate: gelatin: ascorbic acid in a dry weight ratio of 8: 16: 1: 0.5.
The method for producing a freeze-dried product of a microorganism-containing medium, the method comprising mixing the microorganisms to prepare a microorganism-containing medium, and then freeze-drying the medium. 11. Bacterial culture: carbohydrate: gelatin: ascorbic acid in a dry weight ratio of 2: 16: 1: 0.2.
The method for producing a freeze-dried product of a microorganism-containing medium, the method comprising mixing the microorganisms to prepare a microorganism-containing medium, and then freeze-drying the medium. 12. The carbohydrate in the medium is one substance or a mixture of a plurality of substances, wherein the carbohydrate is one of the substances.
1. A method for producing a freeze-dried product of a microorganism-containing medium according to any one of 1. 13. The method for producing a freeze-dried product of a microorganism-containing medium according to any one of claims 9 to 12, wherein the carbohydrate in the medium is a mixture of lactose and sucrose. 14. A drug comprising the freeze-dried product of the microorganism-containing medium according to any one of claims 1 to 8 as an active ingredient.