JPS6322179A - Production of survivable and acid-resistant dried bifidus cell - Google Patents

Abstract

PURPOSE:To produce dried cell of bifidus bacteria capable of stably keeping the number of living cells even after a long-term storage and having high resistance to acid, by freeze-drying concentrated liquid of bifidus bacteria in the presence of a conventional protecting agent added with a carbonate. CONSTITUTION:Concentrated liquid of cultured bifidus bacteria is freezedried by conventional method using a conventional protecting agent. In the above process, the protecting agent is added with about 1-7.5% carbonate such as sodium bicarbonate, magnesium carbonate, etc.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention relates to a method for producing dried bifidus cells, which maintains the number of viable bacteria stably even during long-term storage and which has high resistance to acids. It concerns an efficient manufacturing method.

(Conventional technology) Bifidobacteria are considered to be useful intestinal bacteria.

However, since it is an obligate anaerobic bacterium, it easily dies in the presence of oxygen, and the number of viable bacteria decreases significantly even in low pH conditions.

For this reason, when preparing dried Bifidobacterium cells, they should always be kept as close to air as possible, and deaerated during the holding process.
Despite strict control of manufacturing conditions such as light shielding and low temperatures, bacteria tend to die, and it is difficult to maintain a constant number of viable bacteria over a long period of time in dried bacterial powder.

In general, skim milk powder, various saccharides, amino acids, etc. are used as protective agents in the freeze-drying of bifidobacteria, and these exhibit excellent effects as protective agents.

However, in this case, even if the mixed solution is kept at a low temperature, a decrease in pl+ is usually observed due to the acid produced by the metabolism of active bifidobacteria.

The degree of this pH decrease varies depending on the number of surviving bifidobacteria, temperature, and time, but in mass production, working time becomes shorter, so an excessive decrease in pt+ may be reached. When such a bifidobacteria solution is freeze-dried, the freezing rate is 10
The freezing yield is reduced due to serious drying problems.

Furthermore, the survivability and acid resistance of dried bacterial cells during storage are clearly inferior.

(Problems to be Solved by the Invention) The present invention maintains a good anaerobic condition by generating carbon dioxide from the protective agent using the acid produced by the Bifidobacterium when a Bifidobacteria solution and a protective agent are mixed. with pl+
The biggest problem is to find ways to improve the survival of dried microbial cells by reducing the damage caused by freezing and drying.

(Means for Solving the Problems) The present invention aims to solve the problem α mentioned above.

The inventors of the present invention have conducted extensive research to realize this objective, and have discovered that carbonates are extremely useful.

That is, substances that generally have this effect include metal oxides such as sodium and calcium aluminum;
Possible hydroxides.

When freeze-drying a concentrated Bifidobacterium solution, the present inventors added 0.1 to 10% of various salts to a mixed liquid containing skim milk powder, sugars, amino acids, etc. as a protective agent, and heated the mixture at 37°C.
After allowing it to stand for 1 hour, the number of viable bifidus bacteria was determined from the freeze-dried bacterial powder.

The salts used are as follows.

Sodium acetate, sodium bicarbonate, sodium carbonate,
Sodium phosphate, disodium phosphate, sodium sulfate, calcium hydroxide, calcium hydrogen phosphate, calcium carbonate, magnesium sulfate, magnesium carbonate, magnesium oxide, aluminum sulfate, aluminum chloride, aluminum silicate, artificial Carlus salt (F59 version) Japanese Pharmacopoeia Commentary D).

As a result, as shown in the Examples, it was confirmed that among the carbonates that are generally widely used as agents for neutralizing hydrochloric acid in gastric juice, sodium bicarbonate (m-carbonate) and magnesium carbonate are particularly effective.

The mechanism by which sodium bicarbonate and magnesium carbonate are effective is presumed to be as follows. That is, these carbonates are relatively difficult to dissolve in cold water, but this slightly dissolved portion becomes weakly alkaline as a result of dissociation and generates carbon dioxide gas. For this reason, the acid produced by the metabolism of Bifidobacterium is neutralized, and the carbonic acid is protected by SO, which is effective for the survival of Bifidobacterium and improves the storage stability and acid resistance of dried Bifidobacterium cells. It seems like you'll get something great to do with it.

Calcium carbonate and sodium carbonate are also widely used as antacids, but calcium carbonate is the
As shown in the table, the solubility is too low compared to sodium bicarbonate and magnesium carbonate, so it is less effective because it does not sufficiently prevent the pH drop.On the other hand, sodium carbonate, on the other hand, has too much solubility. , ρ1] is too high (pit 11.6), so it is presumed that there is almost no effect.

A preferred embodiment of the invention is described in the following section.

(Example) Example-1 Skim milk powder 7%, yeast extract O05%, Tween 800.
The culture solution containing 0.05% L-cysteine and 0.0596 L-cysteine was heat sterilized and cooled. This was inoculated with Bifidobacterium longum AT CC15707. 10% Na during culture
.

Anaerobic culture was carried out at 37° C. for 8 hours while adding CO3 to maintain the pit at 6.0 or higher and passing C2 gas and N2 gas. After completion of the culture, a concentrated bacterial solution was prepared by centrifugation using ITI.

To this, add a protectant solution containing 25% skim milk powder, 10% starch decomposition product, and 1% sodium glutamate to a pH of 7.0.
After 4 hours, sodium bicarbonate or magnesium carbonate, or a mixture of both, was added and stirred well.

If the amount of sodium bicarbonate or magnesium carbonate added to the protectant solution is less than 0.5%, it will not be effective, and if it is more than 10%, it will be harmful.
The increase in IT is large. For this reason, economic efficiency and taste: i
fa! The addition amount was selected from 1% to 7.5%.

The concentrated j7 bacteria solution was added to the protective agent solution thus prepared and mixed.

Next, each of these was divided into halves, and one portion was immediately freeze-dried, and the other portion was left to stand at 37° C. for 1 hour, and then freeze-dried.

In addition, the antacid effect, freeze-drying yield, storage stability of dried bacterial cells, and acid resistance were compared using a control sample to which no sodium bicarbonate or magnesium carbonate was added.

As a result, as shown in Tables 2 and 3, the effects of sodium bicarbonate and magnesium carbonate were clearly observed.

Table 3: Acid resistance (Example-1) Chejing acid resistance test method Artificial gastric juice (#Mother extract 1.25g, peptone 2.5g,
Lactose 0.5g, Tween 80 Q, 5g, L Shitin 0.05. , sodium chloride 1.08 in distilled water 500m
7 tons of dried bacterial cells (sterilized after dissolving in step 1), IN HO2
"l'pH3,01: WI491Jz.

Cook this for 1 hour at 37℃ for 3IL! Leave it for a while and immediately adjust the pH to 6.
, to 0? I4 was adjusted and the number of viable bacteria was measured.

Example-2 A concentrated bacterial solution was prepared using a liquid medium with the same composition as used in Example-1! ! s! did.

Add to this 12% skim milk powder, 5% sugar, 1.2% gelatin,
p of protectant solution containing 1.0% sodium glutamate
After adjusting H to 7.0, sodium bicarbonate and magnesium carbonate were added and well dispersed.

The above-mentioned concentrated bacterial solution was added and mixed well, and the effects of sodium bicarbonate and magnesium carbonate were investigated in the same manner as in Example-1.

As a result, as shown in Tables #S4 and 5, the effects of sodium bicarbonate and magnesium carbonate were clearly observed.

tpJSl&;Acid resistance (Example-2) This acid resistance test method Artificial gastric juice (yeast extract 1.25g, peptone 2.5g,
Lactose 0.5g, Tween 800.5g.

L-cysteine 0.05g, sodium chloride 1. Dissolve Og in distilled water (sterilized) and add the dried bacterial cells to INH.
(I adjusted i!l to pl+ 3.0 with J'.

This was left at 37°C for 1 hour and 3 hours, and immediately p++ 6
．． The number of viable bacteria was measured.

(Actions and Effects of the Present Invention) As clarified from the above explanation, the present invention provides a protective agent that is generally used for freeze-drying Bifidobacterium, and that the acid produced by Bifidobacterium causes a decrease in pl+. The addition of carbonate to the protective agent improves the survival of dried microorganisms, which reduces freezing and drying damage due to poor anaerobic conditions and reduces the survival and acid resistance of dried bacterial cells during storage. This invention makes it possible to produce a dried bifidobacterium with excellent properties and acid resistance, making it an extremely useful invention.

Applicant Shin Nippon Chemical Co., Ltd. Procedural Amendment 1. Indication of the case: Japanese Patent Application No. 1645222/1983, title of the invention: Viability and at-acid bifidobacterium dried bacterial cell production method 3, person making the amendment Relationship with the case: Applicant name Title: Shin Nippon Chemical Co., Ltd. 4, Agent 6 , the number of inventions increased by the amendment 7, the subject of the amendment, paragraph 1 of the amendment to the specification, the phrase ``calcium aluminum'' in line 13 of page 4 of the specification is corrected to ``calcium, aluminum.''

2. In the specification ejS, page 6, lines 10-11, the phrase "excellent acid resistance 1 rub" is corrected to "excellent acid resistance."

3. On page 8, lines 7-8 of the specification, the phrase "protectant solution containing 1% sodium glutamate" is corrected to "protectant solution containing 196 sodium glutamate."

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Claims (1)

[Claims] Bifidobacteria drying with excellent survivability and acid resistance is achieved by adding carbonates such as sodium bicarbonate and magnesium carbonate to a known protective agent commonly used for concentrated bifidobacteria culture liquids and freeze-drying the mixture. Bacterial cell production method.