JPS62201570A - Preparation of dried cell of survival bifidus bacteria - Google Patents

Abstract

PURPOSE:To obtain dried cell of survival bifidus bacteria having excellent survival stability and useful for the production of yogurt, etc., by adding a superoxide dismutase to a liquid containing cells of bifidus bacteria during or after cultivation and freeze-drying the mixture. CONSTITUTION:The objective bacterial cell can be produced by adding a superoxide dismutase (preferably originated from Serratia marcescens) to a liquid containing cells of bifidus bacteria [Bifidobacterium longum (ATCC 15707), etc.] during or after cultivation. The amount of the superoxide dismutase is preferably 30-100nu/ml (based on the number of bacterial cells).

Description

Detailed Description of the Invention (Industrial Application Field) The present invention relates to a method for producing dried viable bifidobacteria cells,
S8, a dried bacterial cell that maintains the viable number of obligate anaerobic bifidobacteria for a long time! It is related to the method of construction.

(Prior art) Bifidobacterium (Bifidobacterium genus) has intestinal regulation effects such as suppressing putrefaction in the intestines, preventing the production of harmful substances, and preventing the absorption of harmful substances generated in the intestines. It is a useful bacterium that has the following properties, and its effectiveness is being proven.

For this reason, recently, beverages such as milk and yogurt containing F. fudus bacteria, or dried bacterial cell preparations of F. fudus bacteria, have become commercially available and are widely used for the purpose of maintaining and promoting health.

However, Bifidus lily, which is an obligate anaerobic plant, is extremely unstable in the presence of oxygen, and requires culture medium preparation, inoculation, cultivation, drying,
Dried bacterial cells are preserved during storage, even though storage conditions such as deaeration, light shielding, and low temperature are strictly controlled during storage, and storage conditions such as deaeration, light shielding, and low temperature are strictly controlled. It is extremely difficult to maintain a constant number of viable bacteria over a long period of time.

The difficulty is said to be due to the toxic effect of oxygen on the anaerobic bifidobacteria.The oxidation mechanism includes the catalase theory and the superoxide anion (02-> theory), but there is no established theory.9On the other hand, in recent years superoxide Oxide dismutase (hereinafter abbreviated as SOD) is used to inhibit autoxidation of oxidizing substances in foods (see Japanese Patent Publication No. 13147/1983)
, suppressing pigmentation on the skin as a component of cosmetic compositions (
JP-A No. 55-87712), anti-inflammatory agents for rheumatism and arthritis, and treatment of radiation damage (see JP-A-55-87712)
-63984, Japanese Patent Application Laid-Open No. 16685/1982), and a method for industrially producing SOD by culturing various filamentous fungi on a solid medium (Japanese Patent Laid-Open No. 58-16685).
7-36984), a method for efficiently purifying human-derived SOD (see JP-A-58-1S:+o9x), and a method for increasing the stability of SOD (JP-A-58-32826). A variety of findings have been published, including the following:

However, the use of SOD to promote the survival of anaerobic bacteria such as bifidobacteria has never been proposed.

(Problems to be Solved by the Invention) As mentioned above, the chemical mechanism of killing viable bacteria that occurs during storage of dried Bifidobacterium cells has been clarified. The main problem of the present invention is to find an improvement in storage stability based on this known mechanism.

(Means for Solving the Problems) The present invention aims to solve the problems described in the previous section. The inventors of the present invention have conducted repeated research to realize this objective, and upon discovering that the addition of SOD is extremely useful, they have conducted further research and discovered that the addition of SOD is extremely useful. We have developed a method of adding SOD to and drying and freezing.

That is, in the culture of bifidobacteria, SOD is added aseptically to a commonly used culture medium and cultured under anaerobic conditions in a conventional manner for 9 days, or SOD is added to a solution containing bacterial cells obtained without adding SOD. After adding aseptically, collect the bacteria by centrifugation, add skim milk powder or a known protective agent such as monosodium glutamate, and freeze/dry to obtain highly viable Bifidobacterium desiccated bacteria. A method for manufacturing a body, comprising:
The origin of OD may be any source distributed in living organisms such as animals, plants, and microorganisms. However, from the standpoint of mass production, those obtained from microbial sources that are relatively easy to industrially produce and are economically advantageous, such as those from the genus Aspergillus, Rhizopus, Trichoderma, or Serratia, are preferred.

(Effect) The effect of the present invention was confirmed by SOD titer measurement and viable cell count measurement.

The SOD titer was measured using a reduction reaction of nitroblue tetrazolium (NBT) to formazan, which is a known method for quantifying the amount of 02- produced. (Reference Hisashi Nikaneda, Nobuo Ueda, Lipid peroxide experimental method p141-14G, Ishiyaku Publishing 83) That is, the specific method of this SOD titer measurement method is to add 0.05M to each of multiple test tubes. Sodium carbonate buffer (
1) 2.4+nN of 1110.2), 0.75M f'
J B T 0.1 + oJ, 3, (hM xanthine 0
．． 1+aN, 0.1ml of 3.0mM sodium ethylenenoaminetetraacetate, 0.1+aL of 0.15% bovine serum albumin, and a sample solution containing the appropriate amount of SOD (buffer without SOD in control tubes). , 1mj' and kept at 25°C for 10 minutes, xanthine oxidase 01, m(!) was added to start the reaction. After 20 minutes, 60mM copper chloride 0.11 was added to stop the reaction.
Measure the OD value at 5601616.

The SOD activity that suppressed the increase in OD value in the control by 50% in 20 minutes was defined as one unit.

The attached table exemplifies the SOD activity of microbial-derived enzyme preparations obtained by the present inventors under normal culture conditions.

Separate table (Note) All strains are archived strains of the applicant company.

The method for measuring the number of viable bacteria is to dilute BL bacteria in stages with the diluent of Hikama (Reference: Clinical Examination Vol. 18, p. 1163, "74"), and then dilute the BL cells to 0.
1162 was spread on a commercially available BL agar plate medium in a Conrano frame, cultured anaerobically at 37°C for 48 hours, and the colonies formed after the culture were counted and multiplied by the dilution factor to calculate the number of viable bacteria per sample. I took it.

The method of the present invention has many examples using a plurality of Bifidobacterial cells, and the number of viable bacteria retained varies from example to example. Therefore, Examples (1) to (5) are shown in the next section, and the first
Tables 5 to 5 show the number of viable bacteria determined by the above measurement method.

(Example) o f51 Example Skim milk powder 7%, Yeast extract 0.5%, Line 80
0.05%, L-cis? In the culture of Bifidobacterium containing 0.05% ne, etc., 2 g of the liquid culture solution normally used is heat sterilized and cooled, and then the SOD of Serratia marsens in the attached table (mentioned above) is added to the culture solution at 0.24% (50 U/+ne).
Bifidobacterium longum (＾T
CC15707) was inoculated and cultured with 10% Na2CO,
was added to maintain I'11 at 6.5 or higher, and anaerobic culture was performed at 37°C for 18 hours while passing CO2 and N2 gas. After the culture was completed, the bacterial cells were concentrated by centrifugation, and the anaerobic Casein 12%, milk I!, a well-known protective agent commonly used during freeze-drying of bacteria. Using 300 mJ of a solution containing 20% sodium glutamate and 2% sodium glutamate, the 5OD60. 24% and the above-mentioned bacterial cell concentrate 60% was freeze-dried by a conventional method.

The weight of the dried bacterial cells obtained as described above was about 1108, and as shown in Table 1, dried bacterial cells with clearly high survivability were obtained.

◎ Second Example tj Liquid culture solution 2e with the composition used in Example S1
Bifidobacterium longum ^TCC15707 was inoculated without adding SOD to the cells, and cultured anaerobically in the same manner as in the first example. 0.1 for liquid
4% (50υ/1trl) was added, the bacterial cells were concentrated by centrifugation, 60+oj of the concentrated bacterial cells were added to 300ml of a protectant solution with the same composition as in the first example, and the SOD of Trichoderma viride in the attached table was added. Added 10.14% of
Freeze-drying was performed by a conventional method.

The weight of the dried bacterial cells obtained above was about 110 g,
As shown in Table 2, dried bacterial cells with clearly excellent survivability were obtained.

◎ Example 13 Lactose 0.2%, peptone 1.0%, yeast extract 1.0%
After sterilizing and cooling the liquid culture solution 22, which is commonly used for bifidobacteria and contains meat extract 0.5%, Tween 800.1%, cysteine 0.05%, etc., it is shown in the attached table (
(mentioned above) The SOD of Norizopus oryzae was set to 0.
33% (50υ/111F) was added to inoculate Bifidobacterium bifigum with TCC11146, and during cultivation, the PII was maintained at 6.5 or higher with 10% N arc O-, and the temperature was maintained at 35°C while passing CO2 gas and N2 gas. , conduct anaerobic culture for 10 hours, concentrate the bacterial cells by centrifugation immediately after the completion of culture, and use 8!300+nl! of solution containing 25% of deflated milk powder, 1096 starch decomposition products, and 1% sodium glutamate as a protective agent. Add to this 60 ml of the above bifidus concentrate and Zops Oryze SO
0.33% of D was added to the liquid, and freeze-drying was performed by a conventional method.

The weight of the dried bacterial cells obtained in the above manner was approximately 110 g, and as shown in Table 3, dried bacterial cells with clearly good survival stability were obtained.

◎ Fourth Example Liquid culture solution with the composition used in the third example 2f!
was inoculated with Bifidobacterium bifidum ATCC 11146 without adding SOD and cultured anaerobically.
f-8OD 'eH culture solution 6% ('1017/1a
e) was added, and the bacterial cells were concentrated by centrifugation, and 60 mff1 of this bacterial cell concentrate: 300 + nl of the same protective agent solution as in the third example was added, and freeze-dried by a conventional method.

The weight of the dried bacterial cells obtained in the above manner was about 110 g, and as shown in Table 4, the survival stability was clearly high, and one dried bacterial cell was obtained.

◎ Fifth Example As in the fourth example, without adding SOD, Bifidobacterium bifidum ΔTCC11146 was inoculated, cultured anaerobically, and centrifuged to concentrate the bacterial cells. +n e was mixed with 300 + n l of a protective agent solution having the same composition as in the third example, and 4.1% of the mixed solution (90 υ / 1
61! ) and freeze-dried by a conventional method.

The weight of the dried bacterial cells obtained in the above manner was approximately 1108, and as shown in Table 5, dried bacterial cells with clearly excellent survival stability were obtained.

Table 1: Bifidobacterium longum ^TCC15
707 viable count Table 2: Bifidobacterium longum^TCC15707 viable count Table 3: Bifidobacterium bifidum^TCC1114G viable count Table 4: Bifidobacterium bifidum^TCC1114
6 Viable Bacteria Count Table 5: Bifidobacterium Bifidum ATCC 11146 Viable Bacterial Count Applicant Shin Nippon Chemical Co., Ltd. Procedure Amendment Date February 20, 1986 3. Relationship with the person making the amendment case Applicant 4. Address of the agent: Taisho Seimei Building 6, 15-19 Tsubaki-cho, Nakamura-ku, Nagoya City Number of inventions increased by the amendment 7.11 Il Correction subject Description 8. Contents of the amendment February 18, 1988 as attached Contents 1 of the amendment to the patent application submitted on the date, Part 8, No. 9'r1, lines 2 and 3, the following is added:

[Bifidobacteria used in the present invention include, for example, Bifidobacterium longum (B, Iongum), Bifidobacterium infantis (B, 1nfanL)
is), Bifidobacterium breve (B, bre
ve), Bifidobacterium bifidum (B, bi
Any strain belonging to Phytobacterium may be used.

SOD of these Bifidobacterium culture cells
The effective amount of addition is 10u/ regardless of the origin of SOD generation.
ml! tI L500t/ /ml tear; 4fy'lil
30υ/1ml in consideration of quality and flavor! Or 100 Ll/ml! degree is desirable. ” or later
Written amendment of the above procedure dated March 24, 1986, Mr. Commissioner of the Patent Office, 1 ('+
1□ 1. Indication of the case rf1 1986-34121, Title of the invention Method for producing dried viable bifidobacterium cells 3.1'll Person who makes the correction Name of relationship to the applicant case Title Shin Nippon Chemical Industry Co., Ltd. 4 , Agent 7, Subject of amendment Akira+g 8, Contents of amendment As attached.

Japanese Patent Application No. 1983-34121 Amendment Contents 1, page 17, lines 9 to 11 of the Memorandum, the text "Measure 01.mJ of oxidase..." was replaced with "Add 0.1J' of oxidase and react. Start.

After 20 minutes, 0.1 me of 60+aM copper chloride is added to stop the reaction, and the OD value at 560 nm is measured. ” he corrected.

2. In Book #I, page 9, line 14, the text ``Serratia marcescens'' is corrected to ``Serratia marcenscens''.

3. Specification page 110 No. 11↑Ryo 1ko “S OD 60
．． 24%" should be corrected to "s OD 0.24%."

that's all

Claims (1)

[Claims] A method for producing dried bacterial cells of viable bifidobacteria, which comprises adding superoxide dismutase to a liquid containing bacterial cells during or after culturing bifidobacteria, and drying and freezing the mixture.