KR0157757B1 - Separation method for bifidobacterium which has acid tolerance - Google Patents

Abstract

The present invention is inoculated with natural material in Bifidobacterium selective medium under anaerobic conditions, and re-inoculated the obtained culture medium to optimal Bifidobacterium selective medium to increase the number of viable cells to increase the viable cell number, and obtained reinforced culture The culture was inoculated in a strongly acidic Bifidobacterium selective medium and cultured, and the culture medium obtained in the acidic medium was inoculated in a slightly acidic Bifidobacterium selective medium and cultured, and the obtained culture was selected for optimal Bifidobacterium selection. Inoculate the medium and incubate, confirm the presence of vivid bacterium in the culture medium obtained, inoculate the culture medium into the Bifidobacterium selective medium containing a high concentration of bile, and culture the obtained broth containing low concentration bile Inoculation was carried out in the culture medium of gambling bacterium, and viability was obtained from the obtained culture solution. It relates to a method for separating acid and bile resistant Bifidobacterium comprising the step of separating the bacterium. When screening Bifidobacterium from natural products according to the method of the present invention, it is possible to effectively isolate strains having acid tolerance and resistance and bile resistance.

Description

Separation method of acid and bile resistant Bifidobacterium

The present invention relates to a process for preparing acid and bile resistant Bifidobacterium.

When living microorganisms are administered to humans or animals in the form of powders or fermented products, they promote the desired balance of their intestinal microorganisms and their properties and produce beneficial effects on the host. These are called probiotics (O'sullivan MG, et. al., Probiotic bacteria: myth or reality ?, Trends Food Sci. Technol., 31, pp. 309-314 (1992)).

Probiotics are not limited to lactic acid bacteria, but interest in the role of microorganisms as food additives has been continually observed since the first beneficial effect of lactic acid beverages was mentioned by Mechinicoff. There are various attempts to introduce the technology.

Bifidobacterium strain is one of the predominant bacterial flora of the human intestinal microorganisms and has been reported to have beneficial effects such as resistance to infection, anticancer effect, lowering blood cholesterol content and intestinal effect (Hentges DJ, Human intestinal microflora) in health and disease, Academic Press, pp. 241-261, (1983); Hosono A., Wardojo R. and Otani H., lnhibitory effects of lactic acid nacteria from fermented milk on the mutagenicities of volatile bitrosoamines, Agr. Biol. Chem. 54, 7, pp. 1639-1643 (1990)). Therefore, the development of excellent strains of lactic acid bacteria such as Bifidobacterium as probiotics has emerged as an important task (Klaenhammer TR, Microbial considerations in selection and preparation of Lactobacillus strains for use as dietary adjuncts., J. Dairy Sci. , 65, pp. 1339-1349 (1982)).

Increasingly, the application of various benefits of Bifidobacterium to the food industry is increasing. However, this strain is an absolute anaerobic bacterium and secreted from the stomach and small intestine where the pH is lowered to 3 or lower on an empty stomach when taken orally. It is pointed out that the growth rate is inhibited by digestive enzymes and bile acids, which reduce the number of viable bacteria reaching the large intestine and decrease the intestinal settlement rate (Mitsuoka T., Microbes in the intestine, Our lifelong partners, pp. 1-18 , 1989). In fact, it is reported that the biomass of Bifidobacterium that reaches and colonizes the large intestine when ingested commercially available bifidus fermented milk is not sufficient to have a beneficial effect on the host (Roy D. et al., Uncoupling of growth and acids production in Bifidobacterium spp.J. Dairy Sci., 73 pp. 1478-1484, (1990)). In addition, Verada et al. Proposed the need for selection of acid resistant strains in the production of fermented beverages (Berrada et al., Bifidobacterium from fermented milks, Survival during gastric transit, J. Dairy Sci. 14, pp. 409-413 (1991).

In an effort to improve Bifidobacterium, Korean Patent Publication No. 92-1375 (February 11, 1992) discloses the pulverization of pigs and cattle, suspended in physiological saline, and then smeared on BCP agar medium at 24 It was reported that new lactic acid bacteria were obtained by time incubation. However, the strains thus obtained are not yet sufficient in acid resistance and bile resistance, and there is a need to develop strains with improved intestinal fixation rate.

The present invention has been made in view of the problems of the prior art, and its object is to provide an excellent method for separating Bifidobacterium having acid resistance and bile resistance.

The purpose of the present invention as described above, by inoculating the natural product in the Bifidobacterium selection medium under anaerobic conditions, and re-inoculated the obtained culture medium in the optimum Bifidobacterium selection medium to increase the viable cell number The cultured cultures were inoculated in a strongly acidified Bifidobacterium selective medium, and the culture medium obtained in the acidic medium was inoculated in an optimal Bifidobacterium selective medium and cultured. The presence of terium was confirmed, the culture was inoculated in a bifidobacterium selective medium containing a high concentration of bile, and the obtained culture was inoculated in a bipyrobacterium selective medium containing a low concentration of bile, followed by culture. Acid and bile resistant BP comprising the step of separating the viable Bifidobacterium from It can be achieved by a method of separating tumefaciens.

As the selection medium used for culturing Bifidobacterium, various kinds of known media may be used. Representative examples include BS (Mitsuoka T., A color atlas of anaerobic bacteria 2nd ed., Tokyo (1984)), NPNL (Teraguchi S. et al., Japanese Journal of Bacteriology, 33, 753 (1978)), or GL ( Hirokazu l. Et al., Bifidobacteria Microflora, 12, 39 (1993)). However, TP medium developed by the applicant is most preferred for the purposes of the present invention. TP medium is composed of trypticase, proteus peptone, ammonium sulfate, potassium, phosphate, cysteine, transition galactooligosaccharide and propion chloride.It is a selective medium of Bifidobacterium and inhibits the growth of various bacteria and Bifidobacterium strains. Has been shown to promote the proliferation of erythematosus (Lee, et al., Korean Food Science Society 26 (5), 526-531, (1994)).

Bifidobacterium is a microorganism widely distributed in nature and can be obtained from various kinds of natural products. Representative examples of the animal feces, milk, the large intestine of the animal, etc. can be exemplified, and excellent strains can also be isolated from human feces.

In the present invention, first, the natural material containing Bifidobacterium is inoculated in a selective medium and cultured. At this time, Bifidobacterium is an absolute anaerobic bacterium, so it should be maintained in anaerobic conditions. Therefore, it is preferable to inoculate a tube filled with a medium immediately after collecting natural products.

As such, the natural material is inoculated into Bifidobacterium selective medium and then incubated at about 37 ° C. for about 12 hours. The incubation time is not specified, but the incubation proceeds for the time necessary for the propagation of the inoculated Bifidobacterium and the growth of various bacteria.

The strain is enriched by inoculating the medium containing the Bifidobacterium thus obtained in an optimal selection medium and subcultured. That is, it is preferable to carry out the subculture 6 times, and the growth of the Bifidobacterium strain can be achieved by such an enrichment culture. The culture conditions also proceed to about 37 ° C. in anaerobic atmosphere and the time is maintained for about 12 hours per subculture.

Enriched cultures are inoculated in a strongly acidified Bifidobacterium selective medium and incubated at about 37 ° C. for about 2 hours to 3 hours. To this end, the selective medium must first be adjusted to strong acidity, so that the pH is adjusted to about 1.5 to 2.5 by the addition of acid (HCl, etc.) to the selective medium. Under strong acid conditions, the growth of virtually all bacteria is suppressed, and in particular, all bacteria that are weak in acid resistance are killed.

Prior to inoculation of the obtained culture medium into a slightly acid-adjusted selection medium, it is preferable to cultivate the strain in an optimal medium, that is, inoculation into a weakly acidic medium because the cell count is reduced and viability is reduced by culturing in a strong acidic medium. It is necessary to increase the number of cells and enhance the activity by inoculating the optimal medium (about 1% level) and incubating for about 12 hours.

The culture medium incubated in the optimum medium is inoculated again in a weakly acidic selective medium and incubated at about 37 ° C. for about 5 to 10 hours. At this time, the pH of the medium is adjusted to about 3.5 to 4.5 by the addition of HCl.

After culturing strong acid and weak acid conditions by the above steps, the culture medium is inoculated into an optimum medium and incubated for about 12 hours at 37 ° C. again to proliferate and activate bacteria, and then confirm the presence of Bifidobacterium surviving. do. For confirmation, gram staining can be used to observe the form (curved form of gram-positive bacillus), or F6PPK experiments can be performed using the Scardovi method (Scardovi V. et al., The fructose-6-phosphate shunt as a peculiar pattern of hexose degradation in the genus Bifidobacterium.Ann. Microbial.Enzimol., 15, pp. 19-29 (1965)).

The culture medium in which the presence of the Bifidobacterium strain is confirmed is inoculated in a Bifidobacterium selective medium containing high concentration of bile (37 ° C., 2-3 hours). At this time, the concentration of bile in the medium is adjusted to about 1 to 2%. As oxgall power, 1% bile solution corresponds to 10% bile concentration. Before successively applying bile stress to the resulting culture, it is desirable to incubate (about 12 hours) at about 37 ° C. to inoculate the optimal selection medium for the growth and activity of the cells.

The obtained culture solution is again inoculated in a Bifidobacterium selective medium containing a low concentration of bile and cultured. At this time, the concentration of bile is adjusted to about 0.5 to 1% and incubated at about 37 ° C. for 5 to 7 hours.

The culture solution thus obtained is inoculated in an optimal Bifidobacterium selective medium and cultured (about 12 hours at about 37 ° C.) to increase the growth and activity of the cells. Dilute this culture according to the method of Mitsuoka (Mitsuoka T., Recent trends in research on intestinal flora, Bifidobacterium Microflora, 1, pp. 3-4, (1982)) to spread the diluted solution to the optimal medium and to 37 ° C under anaerobic conditions. Survival Bifidobacterium is isolated by incubation for 48 to 72 hours.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail based on an Example.

Example 1

Separation of Bifidobacterium from Natural Products

50 healthy adults and 20 infants were provided with a screw cap tube filled with TP, a reinforcing culture medium, and about 0.5 g of fecal inoculated into the medium was transferred to a laboratory. The composition of TP medium is shown in Table 1 below.

1) TOS: transgalactooligosaccharide (manufactured by Yakult, Japan)

※ The final pH is adjusted to 7.0

The fecal inoculated medium was incubated at 37 ° C. for 12 hours, re-inoculated with fresh TP medium, and subcultured at 37 ° C. six times. After passage, a small amount of the culture medium was taken, stained with gram, and observed under a microscope. As a result, the number of Bifidobacterium was increased. After the pH was adjusted to 2 by adding 4N HCl to the sterilized TP medium, the incubated culture was added and incubated at 37 ° C. for 2 hours. After the incubation, the culture solution was inoculated in optimal TP medium at 1% level and incubated at 37 ° C. for 12 hours.

The culture was inoculated in TP medium adjusted to pH 4 by the addition of HCl and incubated at 37 ° C. for 6 hours. The culture solution thus obtained was inoculated in optimal TP medium and incubated at 37 ° C for 12 hours. The presence of bifidobacterium was confirmed by gram staining of the culture and observing under a microscope. The sample was inoculated in TP medium containing 1.5% bile and incubated for 2 hours at 37 ° C for the sample confirmed the presence of Bifidobacterium by the above method. The resulting culture was inoculated at 1% level in TP medium without bile and incubated at 37 ° C. for 12 hours. Again, the culture solution was inoculated in TP medium containing 1% bile and incubated at 37 ° C. for 6 hours, and then the culture solution was inoculated in optimal TP medium and incubated at 37 ° C. for 12 hours.

The obtained culture solution was diluted to 10 ⁻⁸ using an anaerobic bacteria dilution solution according to the Mitsoka method, and then 100 μl of each diluted solution was plated on TP medium. The composition of the anaerobic diluent is shown in Table 2 below.

1) Salt solution: prepared by dissolving 0.47 g of K ₂ HPO ₄ , 1.18 g of NaCl, 1.20 g of (NH ₄ ) ₂ SO ₄ , 0.12 g of CaCl ₂ and 0.25 g of MgSO ₄ H ₂ O in 100 ml of distilled water.

This medium was prepared by replacing oxygen with nitrogen in an anaerobic jar (using a Gas Pack system and Anaerobic glove box) to form an anaerobic environment, and then incubating at 37 ° C. for 48 to 72 hours.

Example 2

Confirmation of Bifidobacterium (F6PPK Test)

In a solid TP medium cultured in the anaerobic jar of Example 1, a single colony presumed to be Bifidobacterium was taken, and gram staining was performed first, and then observed under a microscope to confirm that it was a Bifidobacterium strain. F6PPK experiment was performed by Scardovi's method with the fungus inoculated by activating the strain in BHI liquid and solid medium. The reaction solution in yellow was negative, and the reaction solution in purple was positive.

Example 3

Determination of Acid Tolerance of Bifidobacterium

In Example 1, after activating the Bifidobacterium strains taken in each culture step in the MRS liquid medium having the composition shown in Table 3, 1 to the MRS liquid medium adjusted to pH 7.5 and 4.5 using 4N HCl. Inoculated at the% level. Absorbance (Spectronic 20, Miton Roy C0.) Was measured at 620 nm for 24 hours at 3 hour intervals after inoculation. Each test was repeated three times. The results are shown in Table 4 below.

Table 4 shows the growth rate of the bacteria as a relative value by measuring the absorbance for the development of the growth curve, with 0.0-0.3 being induction, 0.3-0.9 being logarithmic, 0.9 The above shows a stopper. Strain samples HJ, Sl, and SJ isolated and separated in Example 1 according to the method of the present invention to the control strains l1 and J1 (strains isolated from TP-selective strain without stress from feces) taken directly from feces as a general separation method. It can be seen that it has a high acid resistance.

Example 4

Determination of Acid Tolerance of Bifidobacterium

0.05N sodium phosphate and dibasic were added to 0.05% of cysteine.HCl, followed by substitution with nitrogen gas to make a basic medium. The base medium was inoculated at 1% level in selected strains in sterilized culture by adjusting the pH to 7, 3 and 2 using 4N HCl.

At the start of inoculation, 100 µl of the culture solution for 0.5, 1 and 2 hours was taken and diluted in the anaerobic dilution solution by the method of Mitsoka, and then plated on BL solid medium (see Table 5) for 36-48 hours anaerobicly in the anaerobic vessel. After incubation, the number of viable cells was measured. Acid resistance of each strain was significantly affected by the change of viable cell number by pH. Each experiment was conducted in two replicates. The results are shown in Table 6 below.

※ The final pH is set at 7.5.

1) Preparation of liver extract (liver extract): 10g soy sauce powder in 170ml distilled water and leached in a 50-60 ℃ constant temperature water bath for 1 hour, and then heated to 100 ℃ to filter by filter paper.

2) A solution: KH ₂ PO ₄ 25g + K ₂ HPO ₄ 25g + distilled water 250ml

3) B solution: MgSO ₄ 7H ₂ O 10g + FeSO ₄ 7H ₂ O 0.5g + NaCl 0.5g + MnSO ₄ 0.337g + Distilled water 250ml

Example 5

Bile Resistance Test of Bifidobacterium

0.05N sodium phosphate and dibasic were added to 0.05% of cysteine.HCl, and then replaced with nitrogen gas to make a basic medium. Bile was added to the base medium so that 0%, 0.5% and 1% were filled in a tube that could be sealed with an aluminum cap, and then sterilized.

Strains selected from the culture and the control strains were inoculated at 1% level and then cultured in an incubator at 37 ℃. Take 100 µl of the culture solution for 1, 3, 8, and 12 hours at the start of the culture, dilute it in the anaerobic dilution solution according to the method of Mitsoka, and smear it on a BL solid medium and incubate anaerobicly for 36 to 48 hours in an anaerobic vessel. The number was measured. The bile resistance of each strain was compared with the change in viable cell counts. The results are shown in Table 7 below.

Example 6

Culture test in milk medium

The strains and control strains selected in Example 1 were inoculated at a 1% level in milk medium prepared by adding 0.05% cysteine-HCl to 10% skim milk. After inoculation, the culture medium was taken every 12 hours while incubating for 72 hours at 37 ° C. incubator, plated on BL solid medium, and cultured anaerobicly to measure the number of viable cells. The results are shown in Table 8 below.

As can be seen from the test results, when screening Bifidobacterium from natural products according to the method of the present invention, it is possible to effectively isolate strains having acid resistance (acid tolerance and acid resistance) and bile resistance. In particular, the strain isolated according to the present invention exhibits acid tolerance and acid reaistance in strong acidity.

In addition, in experimental results cultured in milk medium, even if the survival environment worsens due to the accumulation of degradation products in the embryo as the cells grow, the strains isolated according to the present invention (HJ, Sl, SJ) were taken directly from the feces by the general separation method. It can be seen that the viability is higher than that of l1 and j1.

As evident from the above description, the strain of the present invention can easily obtain strains having acid and bile resistance, especially among Bifidobacterium which has a beneficial effect on the human body. Since the strain isolated according to the method of the present invention orally ingested by humans exhibits severe pH conditions and rapid pH changes in the digestive tract and resistance to various digestive enzymes, and has a high intestinal fixation rate, The strain isolated according to the method of the present invention can be used as a seed of various bifidus fermented products.

Claims (3)

Natural products were inoculated in Bifidobacterium selective medium under anaerobic conditions, and the obtained culture was reinoculated into optimal Bifidobacterium selective medium to increase the number of viable cells by fermentation, and the obtained culture was strongly acidified. Inoculated and incubated in a Bifidobacterium selective medium, and the culture medium obtained from the acidic medium was inoculated into a Bifidobacterium selection medium adjusted to weak acidity, and the obtained culture was inoculated in an optimal Bifidobacterium selection medium. And cultured by confirming the presence of viable Bifidobacterium in the culture medium obtained, inoculating the culture medium into Bifidobacterium selective medium containing high concentration of bile, and cultivating the obtained culture medium with Bifidobacterium containing low concentration of bile. Inoculated and cultured in selective medium, surviving Bifidobacterium from the culture medium obtained Comprising the step of separating acid and bile-magnetic separation method of Bifidobacterium. The method of claim 1 wherein the natural product is human feces. The method according to claim 1 or 2, wherein the Bifidobacterium selective medium is TP medium.