JPH07313140A - Production of frozen or lyophilized lactobacillary cell - Google Patents

Abstract

PURPOSE:To obtain the subject cells with high viability and low level damage and death during their freezing or lyophilizing process, by adding, as a protective agent, partial hydrolyzates of the polysaccharides in devil's tongue powder to a dispersion of lactobacillery cells. CONSTITUTION:Partial hydrolyzates of the polysaccharides in devil's tongue powder are added at >=0.5 (pref. 2-10)wt.% to a dispersion of lactobacillary cells followed by homogeneous mixing and then freezing or lyophilization. The average molecular weight of the partial hydrolyzates is pref. 2000-15000 dalton or so.

Description

Detailed Description of the Invention

[0001]

INDUSTRIAL APPLICABILITY The present invention uses a partial hydrolyzate of a polysaccharide contained in konjak flour as a protective agent to reduce damage and death of lactic acid bacterial cells during freeze-thawing and freeze-drying. And a method for producing a frozen or freeze-dried lactic acid fungus body having a high survival rate.

In this specification, percentages are values by weight, except for the survival rate of lactic acid bacteria, unless otherwise specified.
Microorganisms belonging to Bifidobacterium are sometimes referred to as bifidobacteria, lactic acid bacteria other than bifidobacteria are referred to as lactic acid bacteria, and bifidobacteria and lactic acid bacteria are collectively referred to as lactic acid bacteria.

[0003]

BACKGROUND OF THE INVENTION Lactic acid bacteria are widely known as one of the useful intestinal bacteria, and there are many reports on the physiological significance of bifidobacteria, which produce organic acids such as lactic acid and acetic acid in the intestine. And the effect of suppressing the growth of harmful bacteria,
The production of vitamins and activation of immunity have been revealed.

Therefore, various foods containing bifidobacteria, such as yogurt, confectionery, and beverages, have been developed for the purpose of maintaining health by ingesting bifidobacteria. When adding Bifidobacteria to these foods, pre-cultured Bifidobacteria are processed and added,
The processing requires a great deal of labor and time, and there is a technically difficult point in maintaining a high survival rate in the processing.

On the other hand, lactic acid bacteria and the like have been used as a starter for a very large number of dairy products such as yogurt and cheese since ancient times, and in recent years, like Bifidobacteria, they are expected to have an intestinal regulating action. It has been added to various foods. Conventionally, these lactic acid bacteria and the like obtain their bacterial cells by culturing them in milk or the like, but this requires a lot of labor and time, and unless careful attention is paid to the quality control of the bacterial strains. There was a problem of not becoming.

Therefore, by using viable cells such as bifidobacteria or lactic acid bacteria which have been frozen or lyophilized in advance, foods containing bifidobacteria or lactic acid bacteria, such as dairy products such as yogurt and cheese, can be easily prepared. It can be manufactured.
However, the preparation of frozen or lyophilized lactic acid bacteria having the bacterial concentration required for the production of these products must prevent damage and / or killing of the cells during thawing or lyophilization. Was extremely difficult.

In the past, many cryoprotectants and freeze-dried protectants have been developed to solve these problems. These substances include skim milk, sodium glutamate, gelatin and sucrose (Japanese Patent Publication No. 53-8792), phenylalanine, histidine, citric acid, succinic acid, tartaric acid and alkali carbonate (Japanese Patent Laid-Open No. 265085). Glucose, trehalose, skimmed milk powder, ascorbic acid soda, etc. are also known [CRYOBIOLOGY, No. 26.
Vol., Pp. 149-153, 1989].

However, even with these conventional techniques, the damage or death of the cells during freezing and freeze-drying is great, and it is difficult to prepare a high-concentration and stable frozen or freeze-dried cell. there were. In particular, in the state where the bacterial cell dispersion liquid and the protected material that the bacteria utilize are mixed at the time of preparing the bacterial cells, if the freezing work is not carried out at the lowest possible temperature and rapidly, it is produced by the active metabolism of the bacterial cells. The pH of the bacterial cell dispersion liquid to be frozen is lowered by the acid used, and the survival rate is significantly lowered. Furthermore, the pH of the microbial cell dispersion at the time of freezing has a significant effect on the damage or death of the microbial cells at the time of thawing or lyophilization, so pay sufficient attention to the pH of the microbial cell dispersion before freezing,
In some cases, it was necessary to neutralize with an alkali or the like.

[0009] Further, the mechanism of damage or killing of the bacterial cells differs between thawing and freeze-drying, and therefore an effective protectant against freeze-drying cannot always be an effective protectant against melting. On the contrary, an effective protective agent against melting is
Not necessarily an effective protectant against freeze-drying,
For the preparation of frozen cells or freeze-dried cells, there is a disadvantage that separate protective agents have to be investigated.

Furthermore, in the process of producing frozen or freeze-dried cells, the rate of freezing the cell dispersion liquid has a great influence on the damage or death of the cells. That is, it is desirable to lower the liquid temperature as quickly as possible, but in the case of manufacturing on an industrial scale, there were many problems that had to be overcome in carrying out rapid freezing.

As described above, conventionally, there is no general-purpose protective agent capable of preventing damage or death of microbial cells in the process of freezing or lyophilizing lactic acid bacteria, and its development has been long-awaited.

On the other hand, the present inventors have invented a water-soluble dietary fiber which is a partial hydrolyzate of a polysaccharide contained in konjak flour and has an average molecular weight of 2,000 to 15,000 daltons. Filed (Japanese Patent Laid-Open No. 2-222659)
Issue Bulletin. (Hereinafter referred to as prior application), and regarding the use of this water-soluble dietary fiber, peritoneal dialysate (JP-A-4-154725).
Japanese Laid-Open Patent Publication No. 5-246860) and a colon cancer preventive agent (JP-A-5-246860
Patent invention) was completed and a patent application has already been filed.

[0013]

In view of the above-mentioned prior art, the present inventors have earnestly studied a general-purpose protective agent capable of preventing damage or death of bacterial cells in the steps of freezing and freeze-drying lactic acid bacteria. As a result of conducting a search for the use of the water-soluble dietary fiber, it is a partial hydrolyzate of a polysaccharide contained in konjak flour and has an average molecular weight of 2,000 to
The present inventors have completed the present invention by finding that the fraction of 15,000 daltons can prevent the damage or killing of bacterial cells in the process of freezing or lyophilizing lactic acid bacteria.

It is an object of the present invention to provide a method for producing frozen or freeze-dried lactic acid fungal cells which has a high survival rate with less damage or death of the cells in the freezing or freeze-drying step.

[0015]

Means for Solving the Problems The present invention for solving the above problems provides a lactic acid bacterial cell dispersion containing at least 0.5%.
A method for producing a frozen or freeze-dried lactic acid fungus body characterized by adding a partial hydrolyzate of a polysaccharide contained in konjak flour at a concentration of (weight) and uniformly mixing and freeze or freeze-drying. The partial hydrolyzate of the polysaccharide contained in the konjak flour has an average molecular weight of 2,000 to 15,000 daltons, and the partial hydrolyzate of the polysaccharide contained in the konjak flour is 2 to 10% (weight. ) And lactic acid bacteria are added to the Bifidobacterium (B
a microorganism belonging to ifidobacterium, Lactobacillus (Lac
Streptococcus, a microorganism belonging to
Microorganisms belonging to eptococcus and Enterococcus (Ent)
A desirable mode is a microorganism selected from the group consisting of microorganisms belonging to E. ococcus) or a mixed microorganism of any of these.

Next, the present invention will be described in detail.

A partial hydrolyzate of a polysaccharide contained in konjak flour used in the present invention, having an average molecular weight of 2,0.
The fraction of 00 to 15,000 daltons (hereinafter sometimes referred to as the hydrolyzate) is obtained by the method of the above-mentioned prior application.
It can be manufactured as follows.

Commercially available konjac flour (fine powder, medium powder or rough powder), konjac flour decolorized and deodorized with glucomannan or alcohol separated from konjac flour, etc. can be used as a starting material.

The enzyme used for the hydrolysis is one that hydrolyzes glucomannan, for example, Aspergillus nigar or Trichoderma niger.
Trichoderma viride, cellulase of Penicillium notatum, microorganisms belonging to the genus Streptomyces, Rhizopus niveus, or tuber of Bacillus circulans or konjac. Intramolecular β- of glucomannan
Among enzymes that hydrolyze (endo-type) a glucoside bond, it is particularly preferable to use a cellulase derived from a microorganism belonging to the genus Aspergillus.

The pH and reaction time of the enzymatic reaction have a great influence on the yield and molecular weight of the reaction product. That is, since the commercially available enzyme preparation is a mixture of exo-1,4-β-D-glucanase, β-glucosidase, etc., which is an exo-type enzyme that hydrolyzes endo-type and terminal β-glucoside bonds, the exo-type contained in the enzyme is included. PH and reaction time of the reaction solution that allows the enzymatic reaction to be performed under conditions that reduce the activity of the enzyme, and minimizes the release of monosaccharides and oligosaccharides, for example, 4 to 16 hours at 40 ° C or 4 hours at 55 ° C. Hold.

Then, the enzyme is inactivated by heating, the supernatant is collected, and as it is or with an ion exchange resin or activated carbon, desalting, decoloring, deodorizing, concentrating, and further drying,
It can be powdered.

The hydrolyzate produced as described above has an average molecular weight of 2,000 to 15,000 daltons.

The Bifidobacterium used in the present invention is
A microorganism belonging to the genus Bifidobacterium, for example, Bifidobacterium longum
(Bifidobacterium longum), Bifidobacterium infantis, Bifidobacterium adrescentis
olescentis), Bifidobacterium breve (Bifido)
bacterium breve) and the like, all of which are commercially available or can be easily obtained from depository institutions.

The lactic acid bacteria and the like used in the present invention are
Microorganisms that are generally classified as lactic acid bacteria, such as those belonging to the genus Lactobacillus [for example, Lactobacillus acidophilus (Lactobacillus a
cidophilus), Raltobacillus casei (Lactobacillus
casei), Raltobacillus bulgaricus (Lactobacill
us bulgaricus), etc., Streptococc
us) Microorganisms belonging to the genus [eg Streptococcus
Thermophilus (Streptococcus thermophilus), Streptococcus lactis
Etc.], a microorganism belonging to the genus Lactococcus [Lactococcus lactis (Lactococcus l
actis), Lactococcus p.
lantarum), etc.], a microorganism belonging to the genus Enterococcus [eg, Enterococcus faecium]
(Enterococcus faecium), Enterococcus faecalis, etc.] and the like, all of which are commercially available or easily available from depository institutions.

The cells of lactic acid bacteria can be produced by a conventional method. For example, glucose, yeast extract, one or more of the lactic acid bacteria are usually cultured at 25 to 45 ° C. for 4 to 24 hours in a liquid medium containing a peptone or the like, and cells are collected from the culture solution. Wash to obtain wet cells.

The obtained wet bacterial cells are usually dispersed in water at a concentration of 0.5 to 10% (dispersion concentration of wet bacterial cells varies depending on the purpose of preservation of bacterial cells, mass production of bacterial cells, etc.), The hydrolyzate is added to the cell dispersion liquid at a concentration of 0.5 to 25%, preferably 2 to 10%, and mixed uniformly. In the dispersion concentration range of the microbial cells, the concentration of the hydrolyzate added is irrelevant to the dispersion concentration of the microbial cells, and the protective effect is exerted depending on the concentration in the microbial cell dispersion.

When the pH of this mixed solution is acidic,
With a pH adjusting agent such as sodium hydroxide, sodium carbonate or potassium hydroxide, the pH is adjusted to be approximately neutral, preferably 6 to 8.
Adjust H.

Freezing or lyophilization of this mixed solution can be carried out by a conventional method.
When freeze-drying at −160 ° C. or lower (using liquid nitrogen or the like), the shelf temperature can be 35 ° C. or lower at a vacuum of about 50 to 400 hPa.

In the present invention, by adding and mixing the hydrolyzate as described above, damage or death of lactic acid bacteria during freezing or lyophilization is reduced, and a high concentration of lactic acid bacteria with a high survival rate is obtained. Frozen cells or freeze-dried cells can be obtained. The method of the present invention can be applied to bacteria other than lactic acid bacteria, yeast, etc., as in the case of lactic acid bacteria.

Next, the present invention will be described in detail by showing test examples.

Test Example 1 This test was carried out in order to investigate the effect of the hydrolyzate on the pH, freezing rate and presence / absence of a dispersion of lactic acid bacteria.

1) Preparation of Samples Peptone (manufactured by Difco) 1%, yeast extract (manufactured by Difco) 1%, glucose (manufactured by Difco) 1%, amino acid mixture (manufactured by Kanto Kagaku. 20 essential amino acids) 1% each
Bifidobacterium longum (Bifidobacterium longum) A in a liquid medium (pH 6.5) containing 0.5%
Inoculated with TCC15707 (obtained from ATCC), 37
The culture was carried out at 0 ° C for 12 hours. After the completion of the culture, the culture solution was collected, washed with water, and collected again to obtain a wet bifidobacteria.

A sample in which the wet cells were dispersed in a 10% solution of the hydrolyzate (average molecular weight 3,000 daltons) produced by the same method as in Reference Example 1 at a cell solid content of 12% (sample 1), a sample in which wet bacterial cells were similarly dispersed in a solution containing 10% of the same hydrolyzate, 5% of commercially available skimmed milk powder and 2% of glutamate soda (manufactured by Kanto Kagaku) (Sample 2) ) And the same wet cells were similarly dispersed in a solution containing skim milk powder and 5% sodium glutamate (control).

2) Test Method Regarding pH Each sample was adjusted to pH 4 to 10 shown in Table 1, rapidly frozen using liquid nitrogen, then thawed at room temperature, and the survival rate was tested by the method described below.

Freezing rate Each sample prepared to pH 7.0 was rapidly frozen using liquid nitrogen, and a sample slowly frozen in a freezer at -30 ° C was thawed at room temperature and then prepared by the method described below. The residual rate was tested.

Regarding the presence or absence of standing, each sample prepared at pH 7.0 was prepared by allowing it to stand at 20 ° C. for 3 hours and not standing it, rapidly freezing using liquid nitrogen, and then thawing at room temperature. The survival rate was tested by the method described below. The pH of the stationary sample was measured by a conventional method using a pH meter before freezing.

Survival rate The number of viable bacteria before and after freezing of each sample was measured by a conventional method, and the survival rate was calculated from the following formula. Survival rate (%) = (the number of viable cells per 1 g after freeze-thawing / the number of viable cells per 1 g before freezing) x 100

3) Test Results The results of this test are shown in Table 1, Table 2 and Table 3. As is clear from the survival rate according to the pH before freezing in Table 1, Samples 1 and 2 in which the hydrolyzate alone and the hydrolyzate and the known protective agent were used in combination used only the known protective agent. It was confirmed that the survival rate was higher than that of the control at any pH, and in particular, the survival rate of Sample 2 in which the hydrolyzate was used in combination with a known protective agent was high.

As is clear from the survival rate according to the freezing rate in Table 2, the hydrolyzate alone and the hydrolyzate and the known protective agent were used even under the condition of slow freezing where bifidobacteria were easily damaged. The samples 1 and 2 used in combination had a high survival rate.

As is clear from the survival rate in Table 3 with or without standing, the hydrolyzate alone or the hydrolyzate and a known protective agent are used together even if it takes some time before freezing. No decrease in the survival rate was observed in Samples 1 and 2 that were tested. In particular, Sample 2 before freezing showed a significantly higher survival rate than the control, although the pH was almost the same as the control before freezing.

From the above results, the method of the present invention was proved to be extremely effective for freeze-thawing lactic acid bacteria. still,
The lactic acid bacteria and the hydrolyzate were tested with different types, but almost the same results were obtained.

[0042]

[Table 1]

[0043]

[Table 2]

[0044]

[Table 3] Test Example 2 This test was performed to examine the effect of freeze-drying on the hydrolyzate different from Test Example 1.

1) Preparation of sample The hydrolyzate (average molecular weight of 7,000 daltons) obtained in the same manner as in Reference Example 2 was used, and a solution containing 2% each of sodium glutamate and sodium aspartate was used. Samples 3 and 4 were prepared by the same method as in Test Example 1 except for the above.

A control sample was prepared in the same manner as in Test Example 1, except that a protective solution containing 8% sucrose, 2% sodium glutamate and 2% sodium aspartate was used.

2) Test Method The same method as in Test Example 1 was used except that each sample was freeze-dried at a vacuum degree of 100 to 200 hPa and a temperature of 30 ° C. or lower. However, the survival rate of lactic acid bacteria in the freeze-dried sample was calculated from the following formula. Survival rate (%) = [the number of viable cells per 1 g after freeze-drying x solid content of solution before freezing (%) / number of viable cells per 1 g before freezing x 10
0] × 100

3) Test Results The results of this test are shown in Table 4, Table 5 and Table 6. As is clear from the survival rate according to the pH before freezing in Table 4, the hydrolyzate alone and the sample 3 and the sample 4 in which the hydrolyzate and the known protective agent are used in combination use only the known protective agent. It was recognized that the survival rate was higher at any pH than the control, and particularly the survival rate of Sample 4 in which the hydrolyzate was used in combination with a known protective agent was high.

As is clear from the survival rate according to the freezing rate in Table 5, the hydrolyzate alone or the hydrolyzate and a known protective agent were used even under the condition of slow freezing where bifidobacteria were easily damaged. The samples 3 and 4 used together had a high survival rate.

As is clear from the survival rate in Table 6 with or without standing, the hydrolyzate alone and the hydrolyzate and the known protection were observed even when it took some time to freeze. Samples 3 and 4 in which the agent was used in combination did not show a significant decrease in the survival rate. On the other hand, in the control, the survival rate was drastically reduced by standing, and almost no viable bacteria were present.

From the above results, the method of the present invention was proved to be extremely effective in freeze-drying lactic acid bacteria. The lactic acid bacteria and the hydrolyzate were tested with different types, but almost the same results were obtained.

[0052]

[Table 4]

[0053]

[Table 5]

[0054]

[Table 6] Test Example 3 This test was conducted to examine the effect of the hydrolyzate on freeze-thawing of various lactic acid bacteria.

1) Preparation of Samples Peptone (manufactured by Difco) 1%, yeast extract (manufactured by Difco) 1%, glucose (manufactured by Difco) 1%, amino acid mixture (manufactured by Kanto Kagaku. 20 essential amino acids) 1% each
0.5%, monopotassium phosphate (manufactured by Nacalai Tesque) and dipotassium phosphate (manufactured by Nacalai Tesque) 0.2% each in a liquid medium (pH 6.5), Streptococcus thermophilus (Streptococcus). thermophilus)
ATCC 19258 (obtained from ATCC), Lactobacillus bulgaricus ATC
C11842 (obtained from ATCC), Lactobacillus
Lactobacillus acidophilus ATCC
4356 (obtained from ATCC) and Enterococcus
Enterococcus faecium IFO3128 (I
(Obtained from FO) separately and inoculated at 37 ° C for 12
Incubated for hours. After the completion of the culture, the culture solution was collected, washed with water, and collected again to obtain wet microbial cells of each lactic acid bacterium.

A sample in which the wet cells were dispersed in a 10% solution of the hydrolyzate (average molecular weight 3,000 daltons) produced by the same method as in Reference Example 1 at a cell solid content of 12% ( Sample 5) A sample (control) was prepared by similarly dispersing the same wet microbial cell in a solution containing skim milk powder and 5% each of glutamate soda (control). The sample was slowly frozen and rapidly cooled in the same manner as in Test Example 1 below. Frozen Also, as in Test Example 1, a sample that was allowed to stand before freezing was prepared.

2) Test Method The same method as in Test Example 1 was used.

3) Test Results The results of this test are shown in Tables 7 and 8.
As is clear from the survival rate according to the freezing rate in Table 7, even under conditions of slow freezing in which lactic acid bacteria are easily damaged,
Sample 5, which was produced by the method of the present invention, was found to have a higher survival rate for all bacterial species than the control.

As is clear from the survival rate depending on the presence or absence of standing in Table 8, the sample 5 produced by the method of the present invention was treated with any of the bacteria even when it took some time to freeze. Regarding the species, no significant decrease in the survival rate was observed as compared with the control.

From the above results, it was proved that the method of the present invention is extremely effective in freeze-thawing various lactic acid bacteria. The lactic acid bacteria and the hydrolyzate were tested with different types, but almost the same results were obtained.

[0061]

[Table 7]

[0062]

[Table 8] Test Example 4 This test was performed in order to examine the effect on freeze-thaw by the average molecular weight of the partial hydrolyzate of the polysaccharide contained in konjak flour.

1) Preparation of sample The same method as in Test Example 3 except that a partial hydrolyzate of a polysaccharide contained in konjak flour of various average molecular weights produced by the same method as in Reference Examples 2 to 4 was used. Frozen cells of various lactic acid bacteria were prepared by.

2) Test Method The same method as in Test Example 1 was used.

3) Test Results The results of this test are shown in Table 9. As is clear from Table 9, the hydrolyzate (average molecular weight 2,000
(0 to 15,000 daltons) showed a remarkably high survival rate against any lactic acid bacterium and was confirmed to have a freeze-thaw protection effect on lactic acid bacterium. The hydrolyzate produced by other methods and other lactic acid bacteria were also tested, but almost the same results were obtained.

[0066]

[Table 9] Test Example 5 This test was conducted to examine the effect of the partial hydrolyzate of the polysaccharide contained in konjak flour on freeze-drying by the average molecular weight.

1) Preparation of sample The same method as in Test Example 2 except that a partial hydrolyzate of a polysaccharide contained in konjak flour of various average molecular weights produced by the same method as in Reference Examples 2 to 4 was used. Freeze-dried cells of various lactic acid bacteria were prepared by.

2) Test Method The same method as in Test Example 2 was used.

3) Test Results The results of this test are shown in Table 10. Table 10
As is clear from the above, the hydrolyzate (average molecular weight 2,
000 to 15,000 Daltons) showed a remarkably high survival rate against any lactic acid bacterium, and was confirmed to have a freeze-drying protective effect on lactic acid bacterium. The hydrolyzate produced by other methods and other lactic acid bacteria were also tested, but almost the same results were obtained.

[0070]

[Table 10] Test Example 6 This test was conducted to examine the effective amount of the hydrolyzate.

1) Preparation of sample Same as Test Example 1 except that the hydrolyzate produced by the same method as in Reference Example 1 was used at various ratios shown in Table 11 to the bacterial cell dispersion liquid. Frozen cells of various lactic acid bacteria were prepared by the method.

2) Test Method The same method as in Test Example 1 was used.

3) Test Results The results of this test are shown in Table 11. Table 11
As is clear from the above, when the ratio of addition of the hydrolyzate to the bacterial cell dispersion liquid is less than 0.5%, no increase in the survival rate due to freeze-thawing of lactic acid bacteria is observed, and the addition ratio is 2%.
If it exceeds 5%, the survival rate is not increased with the increase of the addition rate.

On the other hand, when the addition ratio of the hydrolyzate to the bacterial cell dispersion liquid is 0.5% to 25%, the survival rate is increased due to the increase of the addition ratio, particularly in the range of 2 to 10%. A high survival rate was observed in. Therefore, the addition ratio of the hydrolyzate to the bacterial cell dispersion liquid is 0.5 to 25.
%, Preferably 2 to 10%.

The hydrolyzate produced by other methods and other lactic acid bacteria were also tested, but almost the same results were obtained.

[0076]

[Table 11] Reference Example 1 Cellulase derived from a microorganism belonging to the genus Aspergillus (trade name: Cellulase A "Amano", product of Amano Pharmaceutical Co., Ltd., fibrin saccharification power: 1 in 80 l of a phosphate buffer (pH 6.5))
67 g (5,000 U / g) was added, the mixture was heated to 40 ° C., and 4.0 kg of commercially available konjak powder was added thereto, and the mixture was stirred and dissolved. After holding this mixed solution at 40 ° C. for 12 hours to carry out an enzyme reaction, the reaction mixed solution is heated to 100 ° C.,
The enzymatic reaction was stopped by holding for 10 minutes or at 100 ° C.
Centrifuge the reaction mixture at 6,000 G,
The supernatant is used as a cation exchange resin (Dowex 50w × 8.H +
Type) 1,000 ml column and anion exchange resin (D
owex 1x8. (OH-type) was passed through a 4,000 ml column for desalting, concentrated, and spray-dried to obtain about 1,250 g of the hydrolyzate. The average molecular weight of the obtained hydrolyzate was 3,000 daltons.

Reference Example 2 About 2,250 g of the hydrolyzate was obtained in the same manner as in Reference Example 1, except that the time of the enzymatic reaction with cellulase was 8 hours. The average molecular weight of the obtained hydrolyzate was 7,000 daltons.

Reference Example 3 About 2,900 g of the hydrolyzate was obtained in the same manner as in Reference Example 1, except that the time of the enzymatic reaction with cellulase was 20 hours. The average molecular weight of the obtained hydrolyzate was 1,800 daltons.

Reference Example 4 About 1,050 g of the hydrolyzate was obtained in the same manner as in Reference Example 1, except that the enzymatic reaction time with cellulase was 2 hours. The average molecular weight of the obtained hydrolyzate was 16,000 daltons.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0081]

【Example】

Example 1 Peptone (manufactured by Difco) 1%, yeast extract (manufactured by Difco) 1%, glucose (manufactured by Difco) 1%, amino acid mixture (manufactured by Kanto Kagaku. 1% each of 20 essential amino acids)
Liquid medium (pH 6.5) 300m containing 0.5%
l, Bifidobacter longum (Bifidobacter
ium longum) ATCC 15707 (obtained from ATCC)
3% inoculation with the preculture liquid of
Bifidobacteria solution 300 with 1.43 × 10 ⁹ CFU / ml
ml was obtained. This bacterial solution is cooled in a refrigerator and 300 ml
In a centrifuge tube, and use a centrifuge (Tomi Seiko Co., Ltd.) to
Centrifugation was carried out at 000 × g for 20 minutes, and the supernatant was removed by decantation. 300 for wet bacterial cells
Sterile water (4 ° C.) was added to disperse the wet cells, the same centrifugation was performed again, and the supernatant was similarly removed to obtain about 0.5 g of wet cells.

To the wet cells, 30 ml of a 10% solution of the hydrolyzate (average molecular weight 3,000) produced by the same method as in Reference Example 1 was added to disperse the cells.
P with 0% sodium hydroxide (Nacalai Tesque) solution
The H was adjusted to 7.0 to prepare a bacterial cell dispersion liquid.

This dispersion was filled into 10 ml vials (manufactured by Nichiden Rikagaku Co., Ltd.) of 3 ml each, and three of them were soaked in liquid nitrogen for 2 minutes for quick freezing to obtain rapidly frozen bifidobacteria. Further, another three vials were left in a freezer (manufactured by Sanyo Co., Ltd.) at -30 ° C for 1 hour for slow freezing to obtain slow-frozen bifidobacteria.

The frozen cells thus obtained were allowed to thaw at room temperature for 30 minutes to thaw them, and the survival rate was measured by the same method as in Test Example 1. As a result, in the rapidly frozen vial bottle, 95.3%,
It was 85.2% in the slowly frozen vial, maintaining a high survival rate.

Example 2 Wet cells of bifidobacteria obtained in the same manner as in Example 1
To 5 g, 20 ml of a 5% solution of a hydrolyzate (average molecular weight 7,000) produced by the same method as in Reference Example 2 was added, wet bacterial cells were dispersed, and 10% sodium hydroxide (manufactured by Nacalai Tesque, Inc.) The pH was adjusted to 7.0 with the solution to prepare a bacterial cell dispersion liquid.

This dispersion was poured into a tray, cooled to −40 ° C. using a freeze dryer (Edwards Co., Ltd.) and frozen, and then freeze-dried under the conditions of 30 ° C. and a vacuum degree of 10 Pa, About 1.5 g of freeze-dried cells was obtained.

The survival rate of the obtained freeze-dried microbial cells was measured by the same method as in Test Example 1, and the result was 30.7%, and the high survival rate was maintained.

Example 3 Peptone (manufactured by Difco) 1%, yeast extract (manufactured by Difco) 1%, glucose (manufactured by Difco) 1%, amino acid mixture (manufactured by Kanto Kagaku. 20 essential amino acids each) 1%
0.5%, monopotassium phosphate (manufactured by Nacalai Tesque) and dipotassium phosphate (manufactured by Nacalai Tesque) in 300 ml of a liquid medium (pH 6.5) each containing 0.2%,
Lactobacillus acid
3% of the preculture liquid of ophilus ATCC4356 (obtained from ATCC) was inoculated and cultured at 37 ° C. for 12 hours to obtain 300 ml of Lactobacillus bacterial liquid.

This bacterial solution was cooled in a refrigerator to 300 ml.
In a centrifuge tube, and use a centrifuge (Tomi Seiko Co., Ltd.) to
Centrifugation was carried out at 000 × g for 20 minutes, and the supernatant was removed by decantation. 300 for wet bacterial cells
Sterile water (4 ° C.) was added to disperse the wet cells, and the same centrifugation was performed again to remove the supernatant in the same manner to obtain about 0.6 g of wet cells.

10 parts of the hydrolyzate (average molecular weight 3,000) produced by the same method as in Reference Example 1 was added to the wet cells.
% Solution 30 ml was added to disperse the cells, and the pH was adjusted to 7.0 with a 10% sodium hydroxide (Nacalai Tesque) solution.
To prepare a bacterial cell dispersion liquid.

This dispersion was filled into 10 ml vials (manufactured by Nichiden Rikagaku Co., Ltd.) of 3 ml each, and the three vials were immediately immersed in liquid nitrogen for 2 minutes and frozen to obtain frozen cells. On the other hand, another three vials were left at room temperature for 3 hours and then frozen in the same manner to obtain frozen bacterial cells.

The frozen cells thus obtained were left to thaw at room temperature for 3 hours to be thawed, and the survival rate was measured by the same method as in Test Example 1. As a result, in the rapidly frozen vial, it was 93.0%, which was slow. It was 83.7% in the frozen vial, and the high survival rate was maintained.

[0093]

INDUSTRIAL APPLICABILITY As described above in detail, the present invention uses the hydrolyzate as a protective agent during freezing or lyophilization to produce frozen or lyophilized lactic acid fungal cells having a high survival rate. The effects obtained by the method of the present invention are as follows.

1) Frozen cells or freeze-dried cells of lactic acid bacteria that are less damaged or killed in the freezing or freeze-drying step and have a high survival rate can be obtained.

2) Frozen cells or freeze-dried cells of lactic acid bacteria having a high survival rate even at a slow freezing rate can be obtained.

3) Frozen or freeze-dried cells of lactic acid bacteria having a high survival rate can be obtained even if it takes time to freeze after producing wet bacterial cells of lactic acid bacteria.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Sayuri Miyaura 5-1-83 Higashihara, Zama City, Kanagawa Prefecture Morinaga Milk Industry Co., Ltd., Institute of Nutritional Sciences (72) Akemi Zaitsu 5-1-183 Higashihara, Zama City, Kanagawa Prefecture Dairy Co., Ltd. Nutritional Research Institute

Claims (4)

[Claims] 1. A partial hydrolyzate of a polysaccharide contained in konjak flour is added to a lactic acid bacterial cell dispersion at a concentration of at least 0.5% (by weight), and the resulting mixture is uniformly mixed and then frozen or lyophilized. A method for producing a frozen or freeze-dried lactic acid fungus body, which comprises: 2. The method for producing a frozen or freeze-dried lactic acid fungus body according to claim 1, wherein the partial hydrolyzate of the polysaccharide contained in the konjak flour has an average molecular weight of 2,000 to 15,000 daltons. 3. The frozen or freeze-dried lactic acid fungus body according to claim 1 or 2, wherein the partial hydrolyzate of the polysaccharide contained in the konjak flour is added at a concentration of 2 to 10% (by weight). Manufacturing method. 4. The lactic acid bacteria are Bifidobacterium (Bif
Lactobacillus (Lacto)
bacillus, a microorganism belonging to Streptococcus (Strep
tococcus) and Enterococcus (Enter
The method for producing frozen or freeze-dried lactic acid fungal cells according to claim 1, which is a microorganism selected from the group consisting of microorganisms belonging to ococcus) or a mixed microorganism thereof.