KR0158049B1 - Novel bifidobacterium longum hspo which produce polysaccharide - Google Patents

Abstract

The present invention is a new lactic acid bacterium, Bifidobacterium longum HS90, which produces a large amount of viscous polysaccharides with excellent moisture retention (Bifidobacterium longum HS90) (Deposit Organization: Korean spawn association; Deposit date: July 1995, 26 (B) No. KFCC10864), more specifically, it can produce a highly moisturizing viscous polysaccharide, which can be used as a moisturizer of cosmetics and a stabilizer of food, and fermented milk using the strain itself as a fermentation strain. When preparing the high viscosity of the fermented milk and soften the tissue to obtain a rich fermented milk without body whey separation (body), Bifidobacterium long gum HS90 is a lactic acid bacteria in the Bifidobacterium.

Bifidobacterium longgum HS90 of the present invention is obtained by separating and identifying from feces of Korean infants, and can be used as a seed for fermented milk production, and by separating the viscous polysaccharides produced by the strain and food additives or cosmetics. Can be used as a moisturizer.

Description

Bifidobacterium long gum HS90 producing large amount of viscous polysaccharide with excellent moisture

1 is a graph showing the viscosity of skim milk culture broth.

2 is a graph showing the whey separation of skim milk culture.

3 is a graph showing whey separation according to culture temperature.

The present invention is a new lactic acid bacterium, Bifidobacterium longum HS90, which produces a large amount of viscous polysaccharides with excellent moisture retention (Bifidobacterium longum HS90) (Deposit Organization: Korean spawn association; Deposit date: July 1995, 26 (B) No. KFCC10864), more specifically, it can produce a highly moisturizing viscous polysaccharide, which can be used as a moisturizer of cosmetics and a stabilizer of food, and fermented milk using the strain itself as a fermentation strain. When preparing the high viscosity of the fermented milk and soften the tissue to obtain a rich fermented milk without body whey separation (body), Bifidobacterium long gum HS90 is a lactic acid bacteria in the Bifidobacterium.

Extracellular polysaccharides, which microorganisms deposit outside the cell wall, vary according to the type and physiological state of the microorganism and are secreted into the medium in the form of viscous substances or produced in the form of capsules connected to the cell wall or membrane. The polysaccharides produced by these microorganisms are biodegradable and nontoxic to humans, and according to the conditions, they have the functions of gel formation, surface tension control, water absorption, binding, adhesion, lubrication, and film formation. And the petroleum industry.

In recent years, with the rapid development of the industry, the use of polysaccharides has also become widespread, and is widely used in waste treatment agents, turbulence inhibitors, cleaning agents, biodegradable materials, natural materials, sutures, artificial biological components, sustained-release agents, vaccines, and the like. It shows the possibility of being used for treatment.

Polysaccharide producing bacteria known to date include Xanthomonas, Agrobacterium, Azotobacter, Bacillus, Pseudomonas, Arthrobacter, and Methylmonas Methylomonas, Chromobacterium, Alkaligenes, Sclerotium, Erwinia and Lactic Acid Bacteria, among which the polysaccharides produced are dextran, alginate, xanthan, Only gellan, pullulan, levan and the like are used industrially.

As such, the reason why the number of polysaccharides actually used in industrial industry is much smaller than that of the found polysaccharides is due to the common properties of the existing polysaccharides and their common properties. In addition, the exploration, application and development of new polysaccharides or their producing microorganisms require considerable time and effort.

The lactic acid bacteria reported in the past are produced by Streptococcus thermophilus, Lactobacillus vulgaris, Lactococcus lactis subsp. Cremoris, and their polysaccharide production is Streptococcus thermo Lactobacillus Lactobacillus Bulgaricus is 60-150mg / l, Lactococcus Lactose Subspecs Cremoris is 80-600mg / l, and the production of polysaccharides by lactic acid bacteria is applied to bacteria of Xanthomonas or Pseudomonas. Much less than. However, unlike bacteria such as Xanthomonas and Pseudomonas, lactic acid bacteria have been widely used for fermented foods for a long time and generally have the advantage of being a microorganism whose safety is recognized. Therefore, due to the safety of these lactic acid bacteria, polysaccharides produced by lactic acid bacteria are expected to be gradually used in the medical field, such as food, cosmetics, medicine.

In order to enhance the firmness of fermented milk, conventionally, the total solid content of fermented milk is increased, or starch, carrageenan, guar gum, pectin, gelatin, or the like is added to the fermented milk, or lactic acid bacteria that produce viscous materials are used as seed. However, additives such as starch, carrageenan, or pectin are not preferred because they adversely affect the taste, aroma, and feel of fermented milk. Therefore, it is recommended to use lactic acid strains that gradually produce viscous substances. In particular, in the Netherlands and France, the addition of stabilizers is legally regulated, so lactic acid bacteria that produce viscous substances are used.

Viscous material in fermented milk combines lactobacillus and milk protein to alleviate whey separation, a major drawback of fermented milk products, increase viscosity and increase adhesiveness, thereby improving yogurt properties. Therefore, when the fermented milk product is manufactured using lactic acid bacteria that produce viscous substances, the tissue becomes soft and thick, and at the same time, it maintains the tissue against the impact of pumping, branding, filling process, temperature and physical impact during the manufacturing process. There is an advantage.

In the case of Bifidobacteria, which has been recognized for its usefulness as a human-derived intestinal microorganism, even if it produces little or no polysaccharides, its production is not used because it is very small.

Accordingly, the present inventors succeeded in separating and identifying a new Bifidobacterium producing a large amount of viscous polysaccharides from feces of Korean infants while studying to develop a new lactic acid bacterium, particularly a Bifidobacteria, which produces viscous substances.

An object of the present invention is to provide a Bifidobacterium long gum HS90, which produces a large amount of viscous polysaccharides.

In addition, an object of the present invention is to separate the polysaccharides produced by Bifidobacterium long gum HS90 to use them in food, cosmetics, drugs and the like.

In addition, an object of the present invention is to provide a fermented milk with a rich appearance without whey separation phenomenon by producing fermented milk using Bifidobacterium long gum HS90 itself as a seed. Bifidobacterium longgum HS90, a novel lactic acid bacterium that produces a large amount of viscous polysaccharides according to the present invention, is a strain of Bifidobacterium spp. Isolated from the feces of Korean infants.

[Isolation and Identification of Bifidobacterium Long Gum HS90]

This strain was isolated and identified from feces of Korean infants. The feces of healthy infants were taken immediately to a laboratory in blood-glucose-liver liquid medium without fluid paraffin-layered horse blood. This sample was continuously diluted with an anaerobic dilution solution, plated in NPNL agar medium, and placed in an anaerobic jar, replaced with carbon dioxide, and incubated at 37 ° C. for 48 hours. Sticky colonies were contacted with platinum from the resulting colonies and selected as a viscous-producing strain. After purely isolated strains were incubated for 24 hours at 37 ℃ in Biel liquid medium, and then re-inoculated with 12% sterile reduced skim milk powder containing 0.2% yeast extract and incubated at 37 ℃ for 24 hours and then the viscosity of the culture medium Confirmed.

[Characteristics of Bifidobacterium long gum HS90]

The properties of the Bifidobacterium long gum HS90 used in the present invention are as follows.

1) Morphology

Morphology of the bacteria when incubated at 37 ° C for 2 days in BL agar plate medium

① Cell size: 1.0 ~ 2.5㎛

② Cell shape: Bacillus, Y-shaped, club

③ Mobility: None

④ Spore Formability: None

⑤ Gram staining: positive

2) Form of the flora

Morphology of the bacteria when incubated at 37 ° C for 2 days in BL agar plate medium

① Shape: Round

② Size: 0.1 ~ 0.4㎝ (Diameter)

③ uplift: convex

④ Tint: Glossy Yellowish White

⑤ surface: smooth

3) Physiological Properties

Growth temperature: Growth temperature range: 25 ~ 44 ℃ Optimum temperature range: 35 ~ 42 ℃

Growth pH: Growth pH Range: 4.5 ~ 7.5 Optimum pH Range: 6.0 ~ 7.0

Effect of Oxygen: Organized Anaerobic

4) Catalase:-

5) Whether gas is generated:-

6) F6PPK (fructose-6-phosphate phosphoketolase): +

7) Growth at 20 ℃:-

8) Growth at 45 ℃:-

9) Indole Production:-

10) Lactic Acid Production: +

11) Acetic acid production: +

12) Sugar availability

Galactose: +

Fructose: +

Gluconate:-

Glycerol:-

Dextrin:-

Ducitol:-

Rafinos: +

Lactulose: +

Rhamnose:-

Ribose:-

Mannose: ±

Mannitol:-

Maltose: +

Melegitos:-

Melibiose:-

Raised: +

Cellobiose:-

Sorboth:-

Sorbitol:-

Adonitol:-

Aribinos: +

Amigdalin:-

Erythritol:-

Esculin: +

Lactose: +

Inulin:-

Sucrose: +

Starch: +

Xylose: +

Trehalose:-

Glucose: +

As described above, the Bifidobacterium longgum HS90 strain does not grow aerobically, is Gram positive, catalase negative, F6PPK positive, and generates acid from adonitol, ducitol, erythritol, glycerol, rhamnose, and sorbose. Since lactic acid and acetic acid are produced in 1.56: 1, and microscopically shows bacilli, Y-shaped, and club-like shapes, the new strain according to the present invention was recognized as lactic acid bacteria in Bifidobacterium.

Bifidobacterium longgum HS90 according to the present invention was well grown in 12% skim milk, BELL medium, and produced a large amount of viscous polysaccharides unlike general strains of Bifidobacterium spp.

Hereinafter, the present invention will be described in more detail with reference to the following non-limiting examples.

Example 1

[Strain separation]

The feces of healthy infants were immediately collected for defecation and transferred to a laboratory in fluid paraffin-laminated Biel liquid medium. The sample was diluted with an anaerobic dilution solution, plated in 0.1 ml plate of NPEL agar, and then put in an anaerobic fertilizer and incubated at 37 ° C. for 48 hours.

The cracks on the NEP agar medium were contacted with platinum, and the sticky cracks were selected and separated as viscous substance-generating strains. Purely isolated strains were incubated at 37 ° C. for 24 hours in Biel medium, and then re-inoculated with 12% sterile agar skim milk containing 0.2% yeast extract, and cultured at 37 ° C. for 24 hours, and the presence of viscous culture medium was confirmed. The isolated new strain produced a large amount of polysaccharides of 1142 mg / ml, which is 3 to 10 times the normal Bifidobacteria.

The composition of BIEL and ENP Agar medium used for this is as follows.

* Composition of Biel Medium

0.5 g of cysteine chloride

10 g of glucose

Jujube Extract 3g

Liver extract solution ¹⁾ 150ml

Phyton (BBL) 3g

Pyoteose Peptone No.3 10g

Solution A ²⁾ 10ml

Solution B ³⁾ 5ml

Starch (aqueous) 0.5g

Tween 80 1 g

Tryton 5g

5g yeast extract

Distilled Water 815ml

1) Liver extract solution: Put 10g liver powder in 170ml of distilled water, let stand for 1 hour at 50 ~ 60 ℃, boil for 10 minutes and filter it with filter paper.

2) Solution A:

25 g of potassium phosphate (monobasic)

25 g of potassium diphosphate

250ml of distilled water

3) Solution B:

0.5 g of iron sulfate (FESO ₄ · 7H ₂ O)

10 g of magnesium sulfate (MgSO ₄ .7H ₂ O)

0.33 g of manganese sulfate (MnSO ₄ 4H ₂ O)

0.5 g sodium chloride

250ml of distilled water

* ENP & L Agar Badge

Biel Badge 100ml

Agar 1.5g

NP & L solution ¹⁾ 5ml

1) nP & L solution

6 g of lithium chloride (LiCl)

Nalidixic acid 0.03 g

0.2 g neomycin sulfate

Paromomycin 0.4 g

100ml of distilled water

Example 2

[Viscosity of skim milk culture solution]

To the sterilized medium by adding 0.2% yeast extract to 12% reduced skim milk, 1% of the strain Bifidobacterium longgum HS90 and Bifidobacteria strain (M) which does not produce a viscous substance were inoculated 1% and pH 4.5 at 37 ° C. After culturing until the viscosity of the culture was measured. As a result, the change in viscosity according to the shear rate is shown in FIG.

It can be seen from FIG. 1 that the Bifidobacterium longgum HS90 according to the present invention produced a large amount of viscous polysaccharides, which resulted in a much higher viscosity of the culture medium than that of the normal Bifidobacteria.

Example 3

[Whey Separation Test of skim milk culture]

Inoculated 1% of strain Bifidobacterium longgum HS90 and strain (M) which do not produce a viscous substance, respectively, in sterilized medium by adding 0.2% yeast extract to 12% reduced skim milk and incubated to pH 4.5 at 37 ° C. The culture was then centrifuged to determine the degree of whey separation. As a result, the degree of whey separation according to the centrifugal force is shown in FIG.

2 shows that whey separation of fermented milk is greatly alleviated when Bifidobacterium longgum HS90 according to the present invention is used as a fermentation strain.

Example 4

[Whey Separation Test by Culture Temperature]

1% of Bifidobacterium longgum HS90, which produces viscous substances, was inoculated into a sterilized medium by adding 0.2% yeast extract to 12% reduced skim milk, and incubated to pH 4.5 at 32 ° C, 37 ° C and 40 ° C, respectively. After the culture was centrifuged to determine the degree of whey separation. As a result, the degree of whey separation according to the centrifugal force is shown in FIG.

Example 5

[Skin conductivity test]

To isolate the viscous polysaccharides produced by this strain to determine the moisturizing effect on the skin and to measure the electrical conductivity, and compared with purified water and hyaluronic acid, the results are shown in Table 1 below.

As can be seen from Table 1, the viscous polysaccharide produced by Bifidobacterium long gum HS90 exhibits an excellent skin moisturizing effect comparable to that of hyaluronic acid.

As can be seen from the above embodiment, the new lactic acid bacteria Bifidobacterium longgum HS90 according to the present invention produces a large amount of viscous polysaccharides, thus increasing the viscosity of the fermented milk when preparing the fermented milk using the strain as a fermentation strain. It can be used to obtain high quality fermented milk with body without whey separation. In addition, this strain produces viscous polysaccharides with good moisturizing properties, so it can be separated and used as a moisturizer for food and stabilizer of food. .

Claims (5)

Bifidobacterium longum HS90 (Bifidobacterium longum HS90), a new lactic acid bacterium in Bifidobacterium, which produces a large amount of viscous polysaccharides with excellent moisture retention. 26 July; Accession No .: KFCC10864) Bifidobacterium longum HS90 (Bifidobacterium longum HS90) (depositment institution: Korean spawn association; deposit date: July 26, 1995; accession number: No. KFCC10864) A method for producing lactic acid bacteria fermented milk, characterized in that the fermentation strain. Viscosity improvement of fermented milk, characterized by using viscous polysaccharides produced by Bifidobacterium longum HS90 (depositing institution: Korean spawn association; deposit date: July 26, 1995; accession number: KFCC10864) Way. Separation of whey from fermented milk characterized by using viscous polysaccharides produced by Bifidobacterium longum HS90 (depositing institution: Korean spawn association; deposit date: July 26, 1995; accession number: KFCC10864) How to alleviate the phenomenon. Bifidobacterium longum HS90 (deposit institution: Korean spawn association; deposit date: July 26, 1995; accession number: No. KFCC10864) of cosmetics characterized in that it uses the viscous polysaccharide produced by the moisturizing agent Manufacturing method.