JP4799597B2 - Bifidobacterium - Google Patents

Description

  The present invention relates to a bacterium belonging to the genus Bifidobacterium having the ability to convert linoleic acid into cis-9, trans-11 type conjugated linoleic acid.

  Conjugated fatty acids are fatty acids in which adjacent carbons have a double bond with a single bond sandwiched between them. In particular, conjugated linoleic acid having one conjugated diene in a fatty acid molecule having 18 carbon atoms can have various physiological activities. It has been revealed.

  As a method for industrially producing conjugated linoleic acid, for example, an oil / fat containing linoleic acid or an alkali conjugation method in which free linoleic acid is used in an organic solvent such as ethylene glycol is known.

  The alkali conjugation method is usually performed by heating an unsaturated fatty acid and excess alkali to 150 ° C. or higher in an organic solvent. At this time, the conjugated fatty acid obtained is a mixture in which the position and arrangement of double bonds are different. For example, when linoleic acid is used as a raw material, a cis-9, trans-11 type or trans-9 is obtained. , Cis-11 type, trans-10, cis-12 type conjugated linoleic acid, and contains several other positional or geometric isomers.

  Therefore, the physiological action of the conjugated fatty acid is an action as a mixture of conjugated fatty acids, and if the specific conjugated fatty acid can be produced, the application value is high both academically and industrially. Further, in the alkali conjugation method, cyclization and other side reactions also occur, and problems such as a decrease in the yield of conjugated fatty acid and difficulty in purification have been pointed out.

  On the other hand, it has been reported that microorganisms or their enzymes produce conjugated fatty acids. For example, rumen bacteria produce conjugated fatty acids from polyunsaturated fatty acids (for example, see Non-Patent Document 1), Treponema (for example, see Non-Patent Document 2), Butyrivibrio fibrisolvens (for example, Non-patent document 3), Propionibacterium freudenreichii (see non-patent document 4, for example), over 10% of various lung pathogens (see non-patent document 5, for example) have linoleic acid isomerization activity. It has also been reported that conjugated linoleic acid produced by these microorganisms is mainly composed of cis-9 and trans-11 isomers.

  On the other hand, ruminants produce cis-9, trans-11 conjugated linoleic acid in their bodies, so dairy products and livestock meat contain a lot of cis-9, trans-11 conjugated linoleic acid. Therefore, cis-9, trans-11 type conjugated linoleic acid is considered to be a conjugated linoleic acid that is frequently consumed by humans in normal eating habits. Therefore, many studies have been conducted on the physiological effect of this isomer, and it has become clear that it has a protective action against various cancers (see, for example, Non-Patent Documents 6 and 7).

  As described above, when a microorganism is used, it can be expected that a product having a high cis-9, trans-11 type conjugated linoleic acid content, which is expected to have an anticancer action, can be obtained. However, no microorganism has yet been found that produces few by-products and produces a sufficient amount of conjugated linoleic acid.

  On the other hand, Bifidobacterium, which is a kind of intestinal bacteria, is also known as a microorganism having high utility value for food. In recent years, Bifidobacterium has been used in fermented milk and the like because it has settled in the human large intestine and exhibits various beneficial effects such as improved stool and suppression of intestinal rot.

  However, Bifidobacterium bacteria are obligately anaerobic and can be easily killed in the presence of oxygen or at low pH. Operation is required. For these reasons, there has been little research on substance production using Bifidobacterium, including conjugated linoleic acid production.

Shorland F.B., et al., Nature 175, p1129, 1955 Yokoyma, et al., J. Bacteriology 107, P519-527, 1971 Kepler C.R., et al., J. Biol. Chem. 242, p5686-92, 1967 Jiang J., Doctoralthesis, Swedish Uni.Uppsala, 1998 Jack C.I.A., et al., Clinica chlmica Acts 224, p139-4,1994 a Y.L., et al., Cancer Resarch 50, P1097-1101,1990 Ip C., et al., Cancer Research 51, P6118-6124

  As described above, the alkali conjugation method has a problem that a side reaction is likely to occur due to a reaction at a high temperature and it is difficult to obtain a specific conjugated fatty acid, and subsequent purification is complicated. In addition, in the conversion reaction by microorganisms, all-trans conjugated linoleic acid was also produced as a byproduct at the same time in conjugated linoleic acid, and the total amount of conjugated linoleic acid was not always satisfactory.

  In order to solve such problems, the present invention aims at conjugating unsaturated fatty acids having at least two double bonds, and in particular, cis-9, trans-11 type having high physiological effect from linoleic acid. The object is to screen and obtain bacteria having the ability to selectively and efficiently produce only conjugated linoleic acid.

The Bifidobacterium according to the invention described in claim 1 is a Bifidobacterium having the ability to convert linoleic acid to cis-9, trans-11 type conjugated linoleic acid, Bifidobacterium breve FERM P-18459 strain capable of producing at least 0.5 mg / 5 ml of conjugated linoleic acid in the medium according to the steps (a) and (b) of is there.
(a) a step of pre-culturing in an MRS medium containing linoleic acid-BSA-complex for at least 24 hours;
(b) A step of inoculating 3.8% of the genus Bifidobacterium obtained in step (a) into a milk medium containing linoleic acid-BSA-complex and incubating with shaking for 144 hours;

  As described above, the present invention can conjugate an unsaturated fatty acid having at least two double bonds, in particular, only cis-9, trans-11 conjugated linoleic acid having a high physiological effect from linoleic acid. There is an effect that it can be obtained by screening bacteria having the ability to produce selectively and efficiently.

  As a result of diligent research to find a method for producing conjugated linoleic acid using Bifidobacterium, the present inventors have conjugated treatment ability to certain Bifidobacterium, that is, unsaturated fatty acids. It has been found that there is an ability to isomerize in a state in which the double bonds are conjugated to each other by moving the position of the double bond in the molecule.

  Since the Bifidobacterium bacterium of the present invention conjugates an unsaturated fatty acid having at least two double bonds, cis-9, trans-11 type conjugated linoleic acid is considered to have high physiological activity Can be produced selectively and in a high yield.

  In the present invention, the unsaturated fatty acid having at least two double bonds is not particularly limited, and for example, linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid and the like can be preferably used. In particular, when conjugated linoleic acid is produced using linoleic acid as a raw material, specific isomers that have been reported to have physiological activity are produced specifically and efficiently, and the amount of other isomers (by-products) is extremely small. Therefore, it is preferable.

  In addition, the unsaturated fatty acid may form a salt or an ester, and examples of the salt include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, and ammonium salt. Examples of esters that can be used include methyl esters, ethyl esters, and other lipids containing the above fatty acids (phospholipids, glycolipids, etc.), monoglycerides, diglycerides, triglycerides, and the like.

  Natural fats and oils can also be used as raw materials. For example, safflower oil, cottonseed oil, soybean oil, sunflower seed oil, corn oil, peanut oil, rice bran oil, linseed oil, cocoa Plant-derived natural fats and oils such as fats that contain a large amount of linolenic acid in the molecule include animal-derived natural fats and oils such as sardine oil, herring oil, cod oil, and these lipase degradation products. It can be used as a raw material.

  The Bifidobacterium of the present invention can produce a conjugated fatty acid by inoculating and culturing a medium containing an unsaturated fatty acid having at least two double bonds. A milk medium is preferably used as the medium for this purpose.

  Further, the Bifidobacterium genus bacterium of the present invention may be any bacterium that has the ability to isomerize to a state in which the double bonds are conjugated to each other by moving the position of the double bond in the unsaturated fatty acid molecule, Particularly preferred are those having the ability to conjugate linoleic acid, that is, the ability to efficiently produce cis-9, trans-11 conjugated linoleic acid having a high physiological effect from linoleic acid. Examples of those having a conjugating ability include Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium infantis or Bifidobacterium. Bifidobacterium pseudocatenulatum can be cited as a high yield, and Bifidobacterium breve is particularly preferred.

In particular, Bifidobacterium breve FERM P-18459 strain was pre-cultured in MRS medium containing linoleic acid-BSA-complex for at least 24 hours, and then the cultured Bifidobacterium genus was treated with linole. Production of conjugated linoleic acid having the ability to produce at least 0.5 mg / 5 ml of conjugated linoleic acid in the medium by inoculating 3.8 wt% into a milk medium containing acid-BSA-complex and incubating for 144 hours with shaking It is preferable because it is a strain with extremely high performance.
A strain having such excellent production ability can be used to ferment a fermented food (eg, fermented milk) after fermentation when a food material (eg, milk raw material) containing linoleic acid is fermented. Conjugated linoleic acid can be produced to such an extent that it can be expected to be suitable for food production workability.

  Among the Bifidobacterium bacteria of the present invention, Bifidobacterium breve is a strain isolated from feces and vagina of infants and mammals. As a strain, for example, Bifidobacterium breve FERM P-18459 is an excellent strain capable of efficiently producing cis-9, trans-11 type conjugated linoleic acid. Bifidobacterium breve FERM P-18459 has been deposited with the Patent Material Deposit Center, National Institute of Advanced Industrial Science and Technology as of August 14, 2001.

  Bifidobacterium infantis is a strain isolated from infant feces.

  Bifidobacterium bifidum is a strain isolated from the feces and vagina of adults, infants and mammals.

  Bifidobacterium pseudocatenulatum is a strain isolated from sewage, infant feces, and calf feces.

  In order to produce conjugated fatty acids in the present invention, bacteria of the genus Bifidobacterium, in particular Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium infantis or Bifidobacterium pseudocatenulatum As a treatment for conjugating unsaturated fatty acids using one or more Bifidobacterium selected from the above, the Bifidobacterium is cultured in a growth medium containing the unsaturated fatty acid as a raw material. To produce conjugated fatty acids directly, or by adding Bacteria belonging to the genus Bifidobacterium by any method, collecting and washing the cells (washed cells) to the solution containing the unsaturated fatty acid as a raw material It is possible to use any method such as a method for producing a conjugated fatty acid. Furthermore, in a solution containing unsaturated fatty acids, the reaction should be performed using not only living cells of Bifidobacterium, but also dead cells, cell extracts, and enzymes extracted from cells. Is also possible.

  As a medium for culturing Bifidobacterium, such as Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium infantis or Bifidobacterium pseudocatenuratam, A medium usually used for the growth of bacteria belonging to the genus Fidobacterium or a milk medium containing milk can be used, and it is particularly preferable to treat unsaturated fatty acids in a medium using milk.

  When a commonly used growth medium, such as MRS medium, is used, raw oils such as linoleic acid are not dispersed in the medium and the processing efficiency deteriorates. Therefore, lipid binding proteins such as BSA (bovine serum albumin) and surface activity Addition of agent and homogenization under severe conditions are required, but when milk medium is used, homogenization of raw material oil becomes relatively easy, workability and processing efficiency are improved, BSA etc. This is because an increase in cost due to the addition of can be suppressed. In addition, since lipid binding proteins and surfactants such as BSA affect the flavor, when using the present invention particularly in the production of foods containing conjugated fatty acids, the reaction can be carried out efficiently even without BSA. It is preferred to produce fermented milk using a viable milk medium.

  The reason why such excellent dispersibility can be obtained in the milk medium is not certain, although the influence of protein components contained in milk can be considered. In this specification, milk means raw milk of animal milk such as cow milk and goat milk, skim milk powder, whole milk powder, fresh cream, or various milk protein-containing substances of vegetable milk such as soy milk, almond milk and coconut milk. Point to.

  In addition, washed cells or powders of Bifidobacterium genus bacteria such as Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium infantis or Bifidobacterium pseudocatenuratam When the reaction is carried out using dead cell bodies, bacterial cell extracts, etc., preparation of washed cells, bacterial cell powders, dead cell bodies, and bacterial cell extracts may be carried out in accordance with conventional methods. For example, the washed cells can be obtained by washing the cultured cells with physiological saline or a buffer solution, and the cell powder can be obtained by a drying technique such as freeze drying or spray drying.

  As a method for obtaining dead cells, in addition to a method of allowing cell wall lytic enzyme to act, a method of treatment at low osmotic pressure, a method of freezing and thawing, a method of high pressure treatment, a method of crushing treatment, a method of heat treatment, etc. It is done. Especially, the method of heat-processing and self-melting a microbial cell is preferable at the point which can process a lot of microbial cells without incurring cost. The bacterial cell extract can be obtained as a centrifugal supernatant after adding a suitable solvent to the washed bacterial cells, bacterial cells, dead cells and the like obtained as described above.

  For the production of conjugated fatty acids using Bifidobacterium bacteria such as Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium infantis or Bifidobacterium pseudocatenuratam More specific examples of the conjugation treatment method include the following (a) to (e).

(a) A method of conjugating unsaturated fatty acids in the form of fermentation production by culturing Bifidobacterium in growth medium or milk medium (fermented milk)
(b) Method for conjugating unsaturated fatty acids with washed cells of Bifidobacterium
(c) Method for conjugating unsaturated fatty acids using Bifidobacterium spp.
(d) Method for conjugating unsaturated fatty acids using dead Bifidobacterium spp.
(e) Method for conjugating unsaturated fatty acids using Bifidobacterium

Hereinafter, the processing methods (a) to (e) will be described in detail.
(a) Method of conjugating unsaturated fatty acids in the form of fermentation production by culturing Bifidobacterium in growth medium or fermented milk:
When conjugation is performed in a growth medium or fermented milk, the basic operation may be performed by a commonly performed technique, but it is preferable to use the following as a starter.

  First, in order to efficiently induce the conversion ability in cell preparation, unsaturated materials such as linoleic acid were referred to the literature (Yoshiko Omura, Graduate School of Agricultural Science, Kyoto University, 1999 Biotechnology Society Abstract, p191). Fatty acids were added to a growth medium for lactic acid bacteria such as MRS (LACTOBACILLI MRS BROTH, manufactured by DIFCO) medium. This concentration is 0 to 0.2%, particularly preferably 0.05 to 0.1%.

  In this case, the culture is preferably stopped at pH 3.5 to 5.5, particularly preferably 4.5 to 5.5. If the culture is carried out to pH 3.5 or less, the conjugation ability may be reduced due to the overgrowth of the bacteria, and if the pH is 5.5 or more, a sufficient amount of cells for conjugation cannot be obtained.

  The culture medium cultured as described above is preferably used as a starter, and the starter inoculation amount is preferably 0.5 to 8%, particularly preferably about 1 to 4%, of the fermented milk preparation medium. The raw material is preferably about 0.02 to 0.8% in the medium, particularly preferably 0.1 to 0.2%. The culture temperature is about 20 to 40 ° C, preferably 28 to 37 ° C.

  In order to obtain the target conjugated fatty acid more efficiently, the culture is preferably stopped at pH 3.5 to 5.5, particularly preferably at pH 4.5 to 5.5. Similarly, the target conjugated fatty acid can be obtained by neutralization culture.

(b) Method for conjugating unsaturated fatty acids with Bifidobacterium genus washings:
What washed the microbial cell culture | cultivated by the same culture conditions as the time of the starter preparation of the processing method (a) with the physiological saline was collect | recovered, and it suspended in the buffer solution. The buffer solution for suspending the cells and the washing cell reaction are carried out using an aqueous solution under conditions capable of maintaining an appropriate pH. For example, a 0.1 to 1.0 M phosphate buffer is preferred, and the pH is 5.0 to 7.5, preferably 6.0 to 7.0. The cell concentration of the cell suspension is 0.025 to 0.25% by wet weight, and preferably 0.025 to 0.1%. The raw material is a mixed solution of bovine serum albumin (BSA) as the raw material at the time of addition, and the raw material concentration is preferably added to the reaction solution by about 0.1 to 4.0%, particularly preferably 0.3 to 1.0%.

  The mixing ratio of BSA is preferably about 1/5 of the raw material. The temperature during the conversion reaction with the washed cells is 20 to 52 ° C, preferably 32 to 37 ° C. The appropriate time for generating the conversion is 1 to 96 hours, preferably 24 to 72 hours. In addition, the microbial cell cultured while neutralizing pH can be used similarly.

(c) Method for conjugating unsaturated fatty acids using Bifidobacterium spp.
Bifidobacterium bacterial powder is obtained, for example, by pulverizing Bifidobacterium bacterial cells obtained by the same method as the culture conditions for preparing the starter in treatment method (a) by dry treatment. I can do it. As the drying method, freeze drying, spray drying, or the like can be used. The conversion reaction after the preparation of the bacterial powder may be performed in the same manner as the reaction conditions for the washing cell reaction in the treatment method (b).

(d) Method for conjugating unsaturated fatty acids using dead Bifidobacterium spp .:
Bifidobacterium bacterial cells can be obtained, for example, by destroying the cell walls of Bifidobacterium bacterial cells obtained by the same method as the culture conditions for preparing the starter in treatment method (a). . Cell wall destruction is treated with cell wall disrupting enzyme, microbial cells are suspended in a solvent and treated at low osmotic pressure, freeze-thaw method, high-pressure treatment method, crushing treatment method, heat treatment method, etc. Can be used. The conversion reaction after preparation of the cell wall disrupting solution may be performed in the same manner as the reaction conditions for the washing cell reaction in the treatment method (b).

(e) Method for conjugating unsaturated fatty acid using Bifidobacterium genus extract:
Examples of Bifidobacterium bacterial cell extracts include Bifidobacterium washed cells, cell powders, dead cells, etc. obtained by the same method as in the treatment methods (b) to (d). It can be obtained by extracting with an appropriate solvent and removing the residue by centrifugation. The reaction after preparing the bacterial cell extract may be carried out in the same manner as the reaction conditions for the washing bacterial cell reaction in the treatment method (b).

  1 type or 2 types selected from Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium infantis or Bifidobacterium pseudocatenuratam, etc. (a) to (d) above The fatty acid composition of the reaction solution or culture solution obtained by the above Bifidobacterium can calculate the amount of each fatty acid based on the ratio of the area values of the internal standard substance and each fatty acid by gas chromatography analysis. For example, when conjugated linoleic acid is targeted, gas chromatography may be performed under the conditions shown in Table 1.

  The conjugated fatty acid obtained by the present invention can be administered in the form of pharmaceuticals, foods, cosmetics and the like. For example, when used in the form of a pharmaceutical or nutritional supplement that promotes the physiological effect of conjugated linole, it should be administered orally as a solid preparation such as a capsule, granule, tablet, powder, or a liquid preparation such as a syrup. Can do. Moreover, even if it is not an oral administration agent, it can also administer in parenteral forms, such as an injection, a skin external preparation, and a rectal administration agent.

  At the time of manufacture of each preparation, excipients such as lactose, starch, crystalline cellulose, calcium lactate, magnesium aluminate metasilicate, and anhydrous silicic acid, binders such as sucrose, hydroxypropylcellulose, polyvinylpyrrolidone, carboxymethylcellulose, carboxymethylcellulose A disintegrating agent such as calcium, a lubricant such as magnesium stearate, talc, monoglyceride and sucrose fatty acid ester, and other components acceptable as pharmaceuticals and foods may be appropriately used.

  Further, when the same physiological effect is expected and used in the form of a general food (in the form of “obvious food”), the conjugated fatty acid obtained by the method of the present invention is used as it is or after appropriate purification treatment as a fat or tablet. What is necessary is just to add to food / beverage products, such as confectionery, fermented milk, rice cake, a seasoning, and sprinkles, and to manufacture using a conventional method. When used as a fermented food, it can be produced by adding a fatty acid having two or more double bonds in the fermentation raw material and fermenting (culturing) it with fermenting bacteria. In particular, fermented milk obtained by fermenting milk containing linoleic acid is preferable because it can easily contain a specific and large amount of cis-9, trans-11 conjugated linoleic acid as described above.

  The present invention further provides a food or drink containing a conjugated fatty acid obtained by the above-described method according to the present invention.

  Fermented milk refers to fermented milk specified by a ministerial ordinance, milk-containing beverages such as dairy lactic acid bacteria beverages, dairy drinks containing sterilized fermented milk, and kefir Etc. During fermentation, other fungi such as Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus zeae, Lactobacillus johnsonii, Lactobacillus delbruchy subspecies. Bulgaricus, Lactobacillus delbrukki Subspecies. Lactobacillus genus bacteria such as Delbrukki, Streptococcus bacterium such as Streptococcus thermophilus, Lactococcus lactis subspecies. Lactis, Lactococcus lactis Subspecies. Enterococcus genus such as Lactococcus spp. Bacteria etc. can be used.

  Also, Bifidobacterium bacteria such as Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium animaris, yeast and other microorganisms may be used. These can be used alone or in combination of two or more.

  In addition, other food materials, that is, various sugars, emulsifiers, thickeners, sweeteners, acidulants, fruit juices, and the like may be appropriately blended with these foods. Specifically, sugars such as sucrose, isomerized sugar, glucose, fructose, palatinose, trehalose, lactose, xylose, sorbitol, xylitol, erythritol, lactitol, palatinit, reduced starch syrup, reduced maltose starch syrup, sucrose fatty acid Examples thereof include emulsifiers such as esters, glycerin fatty acid esters, and lecithin, and thickening (stabilizing) agents such as carrageenan, guar gum, xanthan gum, pectin, and locust bean gum. In addition, various vitamins such as vitamin A and vitamin B and minerals such as calcium, iron, manganese, and zinc may be blended.

  When used in the form of these pharmaceuticals and foods, the conjugated fatty acid obtained by the method of the present invention can be appropriately blended. In addition, when appealing the physiological effect of conjugated fatty acids, an appropriate amount is added so that the effect can be obtained and an amount such as overdose is not caused, and an amount of intake of about 10 mg to 1000 mg / day is expected. It ’s fine.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not restrict | limited to these Examples at all.

Example 1: Screening for Bifidobacterium producing conjugated linoleic acid 50 mg of linoleic acid and 10 mg of BSA were dissolved in 1 mL of 100 mM phosphate buffer (pH 6.5) to prepare a linoleic acid-BSA-complex solution in advance. did. After adding 200 μL of this linoleic acid-BSA-complex solution to 15 mL of MRS (LACTOBACILLI MRS BROTH, manufactured by DIFCO) medium, each strain was inoculated with 15 strains of the genus bifidobacterium and cultured with shaking at 35 ° C., 120 rpm for 48 hours, and Bifidobacterium. A genus bacterial culture was prepared.

  100 μL of linoleic acid-BSA-complex solution (5 mg of linoleic acid content) and previously prepared in 10% skim milk (glucose 1%, soybean peptide 0.1% added) medium dispensed 5 mL into a 15 mL test tube (with cap) 200 μL of the Bifidobacterium culture solution was added and the cap was closed. This medium was shake-cultured at 35 ° C. and 120 rpm for 144 hours to prepare fermented milk. 1 mg of internal standard substance (HEPTADECANOIC ACID) was added to the obtained fermented milk, extracted by the Bligh-Dyer method, methyl esterified (4% hydrochloric acid in methanol for 30 minutes), and analyzed by gas chromatography The fatty acid analysis was performed. The results are shown in Table 2 below.

  The peak of conjugated linoleic acid was judged and analyzed based on the retention time of a standard product (CLA80 manufactured by Linol Oil). As a result, Bifidobacterium breve FERM P-18459, Bifidobacterium infantis ATCC15702, Bifidobacterium bifidum FERM BP-791, and Bifidobacterium pseudocatenulatum ATCC27919 produced conjugated linoleic acid.

  In particular, Bifidobacterium breve FERM P-18459 has 11.4% of linoleic acid added (5 mg) converted to conjugated linoleic acid, and most of the conjugated linoleic acid produced is cis-9, trans-11 type conjugated linoleic acid. It was an acid. However, conjugated linoleic acid producing activity was not observed in Bifidobacterium breve YIT 4064 and Bifidobacterium breve YIT 4065 even in the same Bifidobacterium breve, and it was confirmed that the conjugated linoleic acid producing activity was limited to a special strain among Bifidobacterium breve. It was.

Example 2: Production of conjugated linoleic acid in washing cell reaction 50 mg of linoleic acid and 10 mg of BSA were dissolved in 1 mL of 100 mM phosphate buffer (pH 6.5) to prepare a linoleic acid-BSA-complex solution in advance.

  The linoleic acid-BSA-complex solution was added to 15 mL of MRS medium (LACTOBACILLI MRS BROTH, manufactured by DIFCO) at a rate of 0.07% and then inoculated with Bifidobacterium breve FERM P-18459, respectively, at 35 ° C., 120 rpm, 48 hours. Cultured with shaking. The culture broth was centrifuged to collect the cells and washed twice with physiological saline to obtain washed cells.

  Add 100 μL of linoleic acid-BSA-complex solution and 0.9 mL of 100 mM phosphate buffer (pH 6.5) to the washed cells, replace the gas phase with nitrogen gas, seal the tube, and keep the test tube in anaerobic condition. The reaction was carried out at 37 ° C. and 120 rpm for 72 hours.

  1 mg of internal standard substance (HEPTADECANOIC ACID) was added to the resulting reaction solution, extracted by the Bligh-Dyer method, methyl esterified (4% hydrochloric acid in methanol for 30 minutes), and analyzed by gas chromatography. The fatty acid analysis was performed.

  As a result, in Bifidobacterium breve FERM P-18459, 0.9% of the linoleic acid in the added substrate was converted to cis-9, trans-11 type conjugated linoleic acid.

Example 3: Preparation of fermented food containing conjugated linoleic acid using Bifidobacterium breve FERM P-18459 In a medium containing 10% skim milk powder, 1% glucose and 0.1% soy peptide, 0.1% or 1.0 linoleic acid as a substrate % Was added and homogenized at 150 kg / cm ² , and then sterilized at 115 ° C. for 10 minutes in an autoclave to prepare a medium for preparing fermented foods.

  Bifidobacterium breve FERM P-18459 was inoculated into 15 mL of MRS (LACTOBACILLI MRS BROTH, manufactured by DIFCO) medium, and cultured with shaking at 35 ° C. and 120 rpm for 48 hours to prepare a Bifidobacterium bacterium culture solution. 6 mL of this culture solution was inoculated into 150 mL of the previously prepared medium for preparing fermented food, and after replacing the gas phase with nitrogen gas, it was anaerobically cultured at 35 ° C. and 120 rpm for 72 hours to prepare fermented food.

  As a result of performing fatty acid analysis in the same manner as in Example 1 for the obtained reaction solution, it was found that a part of the added linoleic acid was converted to conjugated linoleic acid. All of the conjugated linoleic acid produced was cis-9, trans-11 type conjugated linoleic acid.

  Moreover, when sensory evaluation of the prepared fermented food was performed, the thing equivalent to the thing without linoleic acid addition was obtained.

Claims (1)

Bifidobacterium having the ability to convert linoleic acid to cis-9, trans-11 type conjugated linoleic acid,
Bifidobacterium breve FERM P-18459 strain having the ability to produce at least 0.5 mg / 5 ml of conjugated linoleic acid in the medium by the following steps (a) and (b). Fidobacterium spp.
(a) a step of pre-culturing in an MRS medium containing linoleic acid-BSA-complex for at least 24 hours;
(b) A step of inoculating 3.8% of the genus Bifidobacterium obtained in step (a) into a milk medium containing linoleic acid-BSA-complex and incubating by shaking for 144 hours