KR100693764B1 - Liquid stabilizer composition for probiotics - Google Patents

Abstract

A liquid stabilizer composition for probiotics is provided to be able to extend the expiration date by preventing lowering of microorganism activity and stability, and inhibit growth of harmful bacteria by adding stabilizer, thereby being able to prevent contamination of other strains and maintaining and improving functions of products. A liquid stabilizer composition for probiotics contains 10-40 wt.% of glycerol, 10 wt.% of arabic gum and 0.2-1.0 wt.% of citric acid in 0.1M phosphate buffer solution having a pH of 6.5-7.5.

Description

Liquid microbial stabilizer composition for probiotics {LIQUID STABILIZER COMPOSITION FOR PROBIOTICS}

Figures 1a to 1d is a bacterium of the genus Bacillus in a liquid microbial stabilizer containing 10, 20, 40% by weight glycerol in 0.1M phosphate buffer (pH 7.0) containing 10% by weight of arabic gum 5, 25, It is the result which showed the stability of the microbial cell over time by storing at 40 and 60 degreeC.

In the current agricultural and environmental fields, most of the microbial probiotics are in the form of powders, which are freeze-dried probiotics mixed with a suitable excipient. Such products may cause a decrease in efficacy due to mixing inconsistencies and a decrease in the activity of the probiotics and in addition to the dust when added to the feed. Powder type products are excellent in shelf life, but are not easy to handle and have disadvantages in activity compared to liquid products. However, in the case of liquid products, the stability is poor, so there is an urgent need for a technique capable of preserving the microorganisms for a long time with their inherent activity. Prior arts for liquid products are mainly made of technologies using synthetic preservatives, which are microbial growth inhibitors, but the synthetic preservatives themselves inhibit the growth of microorganisms, resulting in long-term stability and also causing residual problems. In addition, there is a significant difference in activity depending on the season due to the lack of resistance to high temperatures generated during the summer distribution process.

It is an object of the present invention to prevent the degradation and stability of microorganisms caused by the preparation of feed additive probiotics, etc. in the liquid phase to extend the shelf life, and also by adding a stabilizer that can suppress the growth of harmful bacteria other strains It is to provide a liquid microbial stabilizer composition for probiotics to prevent contamination of the product and to maintain and improve the function of the product.

The present invention is a microorganism of Bacillus sp. As a bacterium forming spores, Tryptic Soy Broth: Pancreatic digest of casein 17g / L, Enzymatic digest of soybean meal 3g / L, Dextrose 2.5g / L , Inoculated in sodium chloride 5g / L, Dipotassium phosphate 2.5g / L) and incubated at 37 ° C for 96 hours to form endogenous spores. After incubation, the supernatant was removed by centrifugation and washed (0.3% by weight of soluble starch, sodium chloride). 0.2 wt%) was washed two to three times to remove the culture medium components and the liquid microbial stabilizer was added to perform a stability test for each temperature. Probiotics that form spores in addition to the genus Bacillus include Clostridium butyricum and yeast ( Saccharomyces cerevisiae ).

The properties of the materials used as stabilizers are as follows.

Gum arbic

Arabic gum is a hemicellulose secreted from the branches of Acacia plants other than Acacia senegal. It is a white powder, soluble in water, and makes a colloidal solution. It is a complex structure in which L-arabinose, L-labose, and D-glucuronic acid are branched to D-galactan, which is a 1 → 3 bond, and is used to make tablets, emulsions, and grasses. Used.

Glycerol

A liquid belonging to an alcoholic group of organic compounds, which is transparent, sticky and sweet. The molecular formula is HOCH ₂ CHOHCH ₂ OH. Until 1948, it was obtained as a by-product of making soap from fats and oils of animals and plants, but has since been synthesized more using propylene or sugars. Products containing more than 95% by weight of glycerol are usually called glycerin. Glycerol has a wide range of uses, and it is a basic component of rubber or resin that makes protective coatings such as automotive enamels and outdoor paints. When nitrate (HNO ₃ ) and sulfuric acid (H ₂ SO ₄ ) are added, Nitroglycerin. It is also a constituent of monoglyceride and diglyceride emulsifiers used as softeners for confectionery and bread, plasticizers for shortening, and stabilizers for ice cream. In the pharmaceutical and cosmetics industry, glycerol is used to make skin lotions, mouthwashes, cough medicines, serums, vaccines and suppositories. It also plays an important role in protecting red blood cells, sperm, cornea, and other living tissues from freezing. At one time it was most commonly used as an antifreeze for cars, but now it uses methanol and ethylene glycol instead. Fats and oils are the main sources of carboxylic acid, which is combined with glycerol to form esters. The glycerol is removed and the remaining glycerol as a solution is purified by distillation after coagulation and precipitation of impurities and evaporation of water.

Citric acid

Citric acid is a colorless crystalline organic compound. It is a kind of carboxylic acid and exists in the tissues and body fluids of many animals and plants. It is one of a series of compounds that appear in the process of converting carbon dioxide and water through fat, protein, and physiological oxidation (TCA cycle). Citric acid is first isolated from lemon juice and is industrially produced by fermenting sugar cane or molasses with Aspergillus niger . Citric acid is used as a component of sugars, soft drinks and metal abrasives. It is used to enhance the stability of foods and other organic substances by inhibiting harmful effects of dissolved metal salts.

The microorganisms of the genus Bacillus used in this study are antagonists of harmful pathogens that cause various intestinal diseases as well as produce vitamins B ₁ , B _2, C and K. In addition, lactic acid bacteria and bifidus bacteria that are currently on the market reach the intestines, and special coatings and capsule products have to be considered because of gastric acid and various digestive enzymes. However, since these microorganisms are follicle forming bacteria, strong acids and digestive enzymes Once enough follicles can be tolerated, the activity is not lost until reaching the intestine, which is sufficient to prevent intestinal diseases and the like. Due to these characteristics, it is more stable microorganism in liquid than other lactic acid bacteria.

EXAMPLE

Hereinafter, the actual experimental example of the present invention will be described in detail.

Step 1: Bacillus polyfermenticus strains were inoculated in 100 ml of TSB medium tryptic soy broth and shaken at 37 ° C for 150 hours at 150 rpm to obtain a preculture.

Step 2: Incubate the preculture solution in 5L of the main culture medium, incubate for 60 hours at 37 ℃, agitation speed of 500rpm, aeration rate of 1vvm, pH 6.5-8.0, form enough endogenous spores, and centrifuge to recover the cells.

Step 3: Sterilize the wash solution containing 3% by weight of starch and 2% by weight of sodium chloride, sterilize and cool at 121 ° C for 30 minutes, mix the cells obtained above, wash the media components, and centrifuge again to obtain cells.

Step 4: The obtained cells are mixed with a liquid microbial stabilizer and left at each temperature to measure the number of cells by stability test.

Example 1

The Bacillus polypermanticus cells recovered by centrifugation contained 10, 20 and 40% by weight of glycerol in 0.1M phosphate buffer solution (pH 6.5-7.5) containing 10% by weight of arabic gum, respectively. The microbial stabilizer was mixed and stored at refrigerated, room temperature, 40 ° C. and 60 ° C. to observe changes in the number of bacteria. Examples and conditions and preparation methods of the liquid microbial agent obtained above are the same, but the comparative experiments prepared by diluting with 0.1M phosphate buffer solution without using the liquid microbial stabilizer under the same conditions are shown in the comparative experiments. 1> and <Table 1>.

 term  Condition Comparative example 10 wt% Glycerol + 10 wt% Arabic Big Gum 20 wt% Glycerol + 10 wt% Arabic Big Gum 40 wt% Glycerol + 10 wt% Arabic gum Number of bacteria (CFU / ml) Survival rate (%) Number of bacteria (CFU / ml) Survival rate (%) Number of bacteria (CFU / ml) Survival rate (%) Number of bacteria (CFU / ml) Survival rate (%)  0 Day 5 ℃ 1.85 × 10 ⁹ 100.0 1.67 × 10 ⁹ 100.0 1.78 × 10 ⁹ 100.0 1.84 × 10 ⁹ 100.0 25 ℃ 1.74 × 10 ⁹ 100.0 1.90 × 10 ⁹ 100.0 1.53 × 10 ⁹ 100.0 1.46 × 10 ⁹ 100.0 40 ℃ 1.81 × 10 ⁹ 100.0 1.89 × 10 ⁹ 100.0 1.80 × 10 ⁹ 100.0 1.63 × 10 ⁹ 100.0 60 ℃ 1.83 × 10 ⁹ 100.0 1.29 × 10 ⁹ 100.0 1.73 × 10 ⁹ 100.0 1.79 × 10 ⁹ 100.0  7 Day 5 ℃ 7.86 × 10 ⁸ 42.5 1.73 × 10 ⁹ 103.6 1.95 × 10 ⁹ 109.6 2.01 × 10 ⁹ 109.2 25 ℃ 6.32 × 10 ⁸ 36.3 1.99 × 10 ⁹ 104.7 1.75 × 10 ⁹ 114.4 1.67 × 10 ⁹ 114.4 40 ℃ 6.58 × 10 ⁸ 36.4 1.50 × 10 ⁹ 79.4 1.84 × 10 ⁹ 102.2 1.76 × 10 ⁹ 108.0 60 ℃ 1.03 × 10 ² 0.0 1.25 × 10 ⁶ 0.097 4.03 × 10 ⁸ 23.3 1.27 × 10 ⁹ 77.9  14 Day 5 ℃ 3.57 × 10 ⁸ 19.3 1.77 × 10 ⁹ 106.0 1.97 × 10 ⁹ 110.7 2.16 × 10 ⁹ 117.4 25 ℃ 2.84 × 10 ⁸ 16.3 1.93 × 10 ⁹ 101.6 1.73 × 10 ⁹ 113.1 1.73 × 10 ⁹ 118.5 40 ℃ 2.89 × 10 ⁸ 16.0 1.57 × 10 ⁹ 83.1 1.90 × 10 ⁹ 105.6 1.69 × 10 ⁹ 103.7 60 ℃ N.D 0.0 1.02 × 10 ⁴ 0.0 9.90 × 10 ⁵ 0.06 3.95 × 10 ⁸ 24.2  21 Day 5 ℃ 8.79 × 10 ⁷ 4.8 1.63 × 10 ⁹ 97.6 2.03 × 10 ⁹ 114.0 2.19 × 10 ⁹ 119.0 25 ℃ 9.53 × 10 ⁷ 5.5 1.46 × 10 ⁹ 76.8 1.84 × 10 ⁹ 120.3 1.63 × 10 ⁹ 111.6 40 ℃ 9.26 × 10 ⁷ 5.1 1.52 × 10 ⁹ 80.4 1.93 × 10 ⁹ 107.2 1.71 × 10 ⁹ 104.9 60 ℃ N.D 0.0 2.35 × 10 ² 0.0 5.9 × 10 ² 0.0 1.35 × 10 ⁸ 7.5  28 Day 5 ℃ 4.87 × 10 ⁷ 2.6 1.57 × 10 ⁹ 94.0 1.99 × 10 ⁹ 111.8 2.21 × 10 ⁹ 120.1 25 ℃ 5.21 × 10 ⁷ 3.0 1.39 × 10 ⁹ 73.1 1.77 × 10 ⁹ 115.7 1.83 × 10 ⁹ 125.3 40 ℃ 3.89 × 10 ⁷ 2.1 1.47 × 10 ⁹ 77.8 1.83 × 10 ⁹ 101.7 1.75 × 10 ⁹ 107.4 60 ℃ N.D 0.0 1.09 × 10 ² 0.0 5.9 × 10 ² 0.0 7.63 × 10 ⁷ 4.3  35 Day 5 ℃ 1.35 × 10 ⁷ 0.7 1.50 × 10 ⁹ 89.8 1.93 × 10 ⁹ 108.4 2.27 × 10 ⁹ 123.4 25 ℃ 1.28 × 10 ⁷ 0.7 1.33 × 10 ⁹ 70.0 1.53 × 10 ⁹ 100.0 1.79 × 10 ⁹ 122.6 40 ℃ 7.54 × 10 ⁶ 0.4 1.38 × 10 ⁹ 73.0 1.80 × 10 ⁹ 100.0 1.67 × 10 ⁹ 102.5 60 ℃ N.D 0.0 1.07 × 10 ² 0.0 5.83 × 10 ² 0.0 3.67 × 10 ⁷ 2.1

N.D: Not detected

In the phosphate buffer solution, the viability of the bacteria decreased rapidly with the storage temperature and the period of time, whereas the group treated with the liquid microorganism stabilizer of the present invention showed a very stable result, and 10 wt% arabic gum and glycerol 20 In the case of more than 1% by weight, there was no decrease in the number of bacteria at the storage temperature of 5 to 40 ℃. In the experimental example in which 40% by weight of glycerol was administered, it exhibited stability of about 24% even for 2 weeks at 60 ° C., thus preventing damage due to high temperature that may occur during summer transportation. On the other hand, in the other experimental examples, there was no stability at 60 ° C. The higher the concentration of glycerol, the higher the stability.

[ Example  2]

Based on the above results, considering the economic feasibility, the product was produced with a liquid microbial stabilizer containing 20 wt% glycerol in 0.1M phosphate buffer (pH 6.5 ~ 7.5) containing 10 wt% arabic gum at the pilot scale. Occurred and the causative organism was genus fungus. For this reason, in order to find a way to prevent contamination, the presence of citric acid as a preservative was stored at 37 ℃ to check whether the bacteria were contaminated. Experimental example containing 0.2, 0.4, 0.6, 0.8 and 1.0 wt% citric acid in a liquid microbial stabilizer containing 20 wt% glycerol in 0.1M phosphate buffer solution (pH 6.5-7.5) containing 10 wt% arabic gum Was stored at room temperature and observed for changes in the number of bacteria and contamination of the fungus. The results are shown in Table 2.

Hours 0 7 14 21 28 35  Citric Acid 0% by weight Number of bacteria (CFU / ml) 1.76 × 10 ⁹ 1.79 × 10 ⁹ 1.73 × 10 ⁹ 1.74 × 10 ⁹ 1.69 × 10 ⁹ 1.72 × 10 ⁹ Survival rate (%) 100.0 101.7 98.3 98.9 96.0 97.7  Citric acid 0.2% by weight Number of bacteria (CFU / ml) 1.67 × 10 ⁹ 1.75 × 10 ⁹ 1.68 × 10 ⁹ 1.64 × 10 ⁹ 1.59 × 10 ⁹ 1.54 × 10 ⁹ Survival rate (%) 100.0 104.8 100.6 98.2 96.2 92.2  0.4 wt% citric acid Number of bacteria (CFU / ml) 1.82 × 10 ⁹ 1.87 × 10 ⁹ 1.79 × 10 ⁹ 1.83 × 10 ⁹ 1.74 × 10 ⁹ 1.72 × 10 ⁹ Survival rate (%) 100.0 102.7 98.4 100.5 95.6 94.5  Citric acid 0.6 wt% Number of bacteria (CFU / ml) 1.54 × 10 ⁹ 1.59 × 10 ⁹ 1.47 × 10 ⁹ 1.49 × 10 ⁹ 1.48 × 10 ⁹ 1.45 × 10 ⁹ Survival rate (%) 100.0 103.2 95.5 96.8 96.1 94.2  0.8 wt% citric acid Number of bacteria (CFU / ml) 1.69 × 10 ⁹ 1.64 × 10 ⁹ 1.72 × 10 ⁹ 1.59 × 10 ⁹ 1.63 × 10 ⁹ 1.57 × 10 ⁹ Survival rate (%) 100.0 97.0 101.8 94.1 96.4 92.9  Citric Acid 1.0 wt% Number of bacteria (CFU / ml) 1.56 × 10 ⁹ 1.63 × 10 ⁹ 1.59 × 10 ⁹ 1.57 × 10 ⁹ 1.48 × 10 ⁹ 1.42 × 10 ⁹ Survival rate (%) 100.0 104.5 101.9 100.6 94.9 91.0

As shown in the above results, a decrease in the number of bacteria was found in the experimental example containing 20 wt% glycerol and citric acid added at each concentration, but showed high stability of more than 90% as a whole. And no mold contamination was observed at all concentrations used in the experiment. However, in the comparative example without addition of citric acid, mold contamination was found. As a result, a liquid microbial stabilizer was prepared by adding 10-40 wt% glycerol, 10 wt% arabic gum, and 0.2-1.0 wt% citric acid to 0.1M phosphate buffer adjusted to pH 6.5-7.5 to increase the stability of microorganisms. The high stability of about 90% was maintained even during the storage period of 35 days.

As described above, the liquid microorganism stabilizer composition according to the present invention prevents the degradation and stability of the microorganisms caused by the preparation of the probiotic for feed additives, etc. in a liquid state, and maintains the intrinsic activity and survival rate of the microorganisms high. The shelf life of probiotics can be extended, and there is an effect of preventing contamination of other strains. In addition, citric acid, which is used as a preservative to prevent mold contamination in the liquid microorganism stabilizer of the present invention, is unlikely to be harmful to other antibiotics or preservatives in that it is abnormally fermented in fish intestines, antibiotic-resistant strains, and water quality due to the residual of antibiotics or preservatives. There is an excellent effect that does not cause problems such as contamination.

Claims (1)

A liquid microbial stabilizer composition for probiotics, characterized in that 10-40% by weight of glycerol, 10% by weight of arabic gum and 0.2-1.0% by weight of citric acid are contained in 0.1M phosphate buffer (pH 6.5-7.5).

Images