KR0145384B1 - Reduction Whey Basics-Method for Preparation of Lactic Acid Bacteria Freeze-Dried Cells Using Lyophilized Protective Agent - Google Patents

Abstract

[Technical field to which the invention described in the claims belong]

The present invention relates to a lyophilization method of lactic acid bacteria.

[Technical Challenges to Invent]

Whey, which is a byproduct of cheese production, provides better stability and preservation during lyophilization compared to skim milk powder, which has been widely used as a lyophilizer in the past. It is an object of the present invention to provide a method for producing lactic acid bacteria lyophilized cells using a lyophilized protective agent of a new composition.

[Summary of the solution of the invention]

The present invention is a 1-20 parts by weight of polyol, 0.1-0.5M amino acid, 0.1-10 parts by weight of a polymeric material, 0.05-1 parts by weight of ascorbic acid, or a mixture thereof in a 5-20 parts by weight of reducing whey solution It provides a method for producing lactic acid bacteria lyophilized cells using a reduced whey base-freeze-drying protective agent comprising a step added as.

[Important Uses of the Invention]

Lactobacillus bacillus, lactic acid cocci, and lyophilised bacteria of Bifidobacteria can be prepared by the production method according to the present invention.

Description

Method for preparing lactic acid bacteria lyophilized cells using reduced whey base-freeze drying

The present invention uses a lyophilized protective agent during lyophilization of Lactobacillus, Lactobacillus (Enterococcus), and Bifidobacteria to maintain the biological stability of the strain and at the same time maintain the viability of the bacteria according to the preservation of the bacteria. A method for producing lyophilized cells, more specifically, 1-20 parts by weight of a polyol, 0.1-0.5M amino acids, 0.1-10 parts by weight of a polymer material, 0.05-1 parts by weight in a 5-20 parts by weight of reduced whey solution The present invention relates to a method for producing lactic acid bacteria lyophilized cells using a reduced whey base-freeze-drying protector comprising a process in which negative ascorbic acid or a mixture thereof is added as a lyophilization aid.

To preserve industrially useful microbial strains for a long time, there are two methods of freezing the cells and storing them at low temperature (below -20 ℃) (freezing preservation method) or lyophilized powders at room temperature or refrigerated conditions (freeze drying preservation method). have. These two methods have different characteristics, but above all, the freeze-drying preservation method is more advantageous than the freezing preservation method in terms of product preservation and transportation.

Conventionally, these techniques have been performed on a small scale for long-term preservation of experimental strains in the laboratory, but recently, as various products using lyophilized cells have been developed, the scale of production has been greatly expanded with the development of many technologies.

Examples of products using these freeze-dried cells include formal preparations in the pharmaceutical field. Examples of food concentrates include lactobacillus-containing health supplements and powder concentrates used as starters in fermented dairy products, and in the animal medicine field, animal feed additives. And probiotic.

On the other hand, the types of lactic acid bacteria used for this purpose include thermophilus, lactis, cremoris, and diacetyl lactis of the genus Streptococcus; Faecium and pecharis of the genus Enterococcus; Ashdophyllus, Plantarium, Casey, Bulgaricus, and Helvicitis of the genus Lactobacillus; And Bifidum, Longgum, Breve, and Infinantis in Bifidobacterium.

In order to prepare lyophilized cells of lactic acid bacteria, lactic acid bacteria were cultured at a high concentration in a suitable culture medium, and concentrated by 10 to 15 times higher than the original culture volume by centrifugation or membrane separation. Freeze and freeze-drying are carried out by adding appropriate substances to prevent the East Sea.

The greatest characteristic of the lyophilized cells thus produced must be able to maintain a certain level of viable cell count until the product is consumed by the consumer. However, the viability of the cells can vary greatly depending on the strain used to prepare the lyophilized cells, culture time and conditions, cell recovery conditions, and lyophilization conditions. It largely depends on the substance used for the purpose of preventing and the composition of the substance (Ref. Appl. Environ. Microbiol. 56: 3112 (1990)).

Substances used for the purpose of preventing cell damage caused by freezing of the cells are called lyophilized protective agents, and many kinds of substances are known to have such freezing effect.

Lyophilized protective agents are generally used in several major protective media (hereinafter referred to as lyophilization aids), such as adonitol, inositol, sorbitol, mannitol, and glycerol, in order to maximize the effect of preventing and preserving the East Sea. Polyols such as; Amino acids such as glutamic acid and glycine; Polymers such as gelatin, cellulose, starch; Prepared by adding small amounts of glucose, sucrose, maltose, sugar such as corn syrup, DMSO, and PVP.

The most commonly used dispersion medium for the preparation of such lyophilized protective agent is skim milk powder.

Thamin and Robinson (Dairy Ind. Int'L. 41: 408, 1976) suggested that 9-12% reduced skim milk powder solution is suitable for freeze-drying of lactic acid bacteria, Valdez (Cryobiology 20: 560, 1983), etc. The effects of various protective substances on lyophilization of lactic acid bacteria were compared using the reduced skim milk powder solutions of 20% and 20% as the basic dispersion medium.

In addition, Ahn et al. Used reduced skim milk powder as a dispersion medium in the preparation of lyophilized bacteria of Saccharomyces cerevisiae strain in Korean Patent No. 92-8363.

In addition to these skim milk powders, horse serum, lactose, and starch have been reported as a dispersion medium, but their use is currently insignificant.

However, when industrially producing lyophilized cells of lactic acid bacteria using conventionally known lyophilized protective agents, no satisfactory results were obtained.

Therefore, the present inventors have studied a new composition of lyophilized protective agent, and as a result, whey, a by-product of cheese production, provides improved stability and preservation during lyophilization compared to skim milk powder which has been used as a dispersion medium. This was used as a dispersion medium to develop a lyophilized protective agent of a new composition.

It is an object of the present invention to freeze 1-20 parts by weight of polyol, 0.1-0.5M amino acid, 0.1-10 parts by weight of polymer, 0.05-1 parts by weight of ascorbic acid, or a mixture thereof in 5-20 parts by weight of reduced whey solution. It is to provide a method for producing lactic acid bacteria lyophilized cells using a reduced whey base-freeze-drying protective agent comprising a step added as a drying aid.

Hereinafter, the present invention will be described in detail.

In order to obtain the cells, the liquid medium (pH6.5) having the composition shown in Table 1 was sterilized for 15 minutes at 121 ℃, the sterilized medium was cooled to 37 ℃ and lactobacillus pullantanium concentration of 0.1% (V / V) The cells were incubated at 37 ° C. for 16 hours, and then centrifuged at a speed of 7,000 rpm for 20 minutes (Sogal RC-5B, GSA Rota) to recover the cells.

The recovered cells were then washed once with 0.1 M phosphate solution (pH 6.8), divided into two fractions, and one fraction was suspended in 50 g of 10% (w / v) reduced skim milk solution and the other fraction 50 g dml. After suspending in 10% (w / v) reducing whey solution, 1 g of each suspension was collected and used for determination of viable cell count, and the rest was frozen at -40 ° C.

The freeze-concentrated bacteria solution was then lyophilized (Labconco lyophilizer, USA) for 24 hours at a vacuum degree of 5 Torr or less, 1g of dry powder was collected and used for the measurement of viable cell count, and the rest was put in a sterile polyethylene bottle 30 The number of viable cells was measured at weekly intervals while being stored in the air at room temperature and kept at room temperature. The survival rate (1) and freezing rate (2) after freeze-drying were calculated by the following equation.

The viable cell count was measured by counting the number of colonies incubated at 37 ° C. for 2 days by plating 1 ml of the sample diluted in sterile saline using a 10-fold dilution method in MRS agar medium. Described.

As can be seen from Table 2, whey is not only about twice as effective in preventing freezing during freeze-drying as compared to conventionally used skim milk powder, and the survival rate after 4 weeks of preservation of lyophilized cells is also excellent in preserving freeze-dried cells. Is 0.8%, while whey is 12.3%, which is about 10 times better.

Accordingly, the present invention uses 1-20 parts by weight of polyol, 0.1-0.5M amino acid, 0.1-10 parts by weight of a polymeric material using 5-20 parts by weight of the reduced whey solution as a dispersion medium, which is determined to have an excellent anti-frost effect as described above. , 0.05-1 parts by weight of ascorbic acid, or a mixture thereof, is added to provide a reduced whey base-freeze-drying protector.

The lyophilized protective agent according to the present invention is selected from citric acid, sodium hydroxide, sodium monophosphate, sodium diphosphate, potassium monophosphate, potassium diphosphate, sodium carbonate, sodium bicarbonate, calcium carbonate, and mixtures thereof. It is preferred that the pH is adjusted to 5.0-9.0 using a pH adjuster.

Polyols used in the present invention include glycerol, adonitol, sorbitol, and mixtures thereof.

In addition, amino acid materials used in the present invention include glycine, glutamic acid, and mixtures thereof.

In addition, the polymer material used in the present invention includes gelatin, carboxy methyl cellulose, cellulose, starch, arginic acid, agar, guar, and mixtures thereof.

As described above, the present invention was able to significantly reduce the death rate due to the preservation of the bacteria as well as to prevent the freezing effect of the bacteria by using a new lyophilized protective material that was not used conventionally during the lyophilization of lactic acid bacteria.

On the other hand, the new lyophilized protective material according to the present invention was excellent in not only Lactobacillus but also various other types of lactic acid bacteria, it was also possible to enhance the efficacy by adding several lyophilized protective agents.

Such a method for preparing lactic acid bacteria lyophilized cells according to the present invention will be described in detail as follows.

Example 1

Inoculated with Enterococcus pescium bacteria in a sterilized liquid medium having the composition of Table 1 at a concentration of 0.1% (v / v) for 24 hours at 37 ℃ centrifugation for 20 minutes at a speed of 7,000rpm (fire RC- 5B, GSA rota) to recover the cells.

The recovered cells were washed once with 0.1 M phosphate solution (pH 6.8), and the concentrated bacterial solution obtained was divided into two fractions, one of which was suspended in 50 g of 10% (w / v) reduced skim milk powder solution. Fractions were suspended in 50 g of 10% (w / v) reducing whey solution.

Then, 1 g of each suspension was collected and used for determination of viable cell count, and the rest was frozen at -40 ° C and lyophilized (Labconco lyophilizer, USA) for 24 hours under vacuum of 5 Torr. After that, 1 g of dry powder was collected and used for measuring the number of viable cells, and the remainder was put in a sterile polyethylene bottle and stored in a state exposed to air at 30 ° C. in a thermostat, and the number of viable cells was measured at one week intervals.

The viable cell count measurement method and the survival rate calculation in each were as described above, and the results are shown in Table 3 below.

As can be seen from the table, Enterococcus lyophilized cells prepared using whey as a lyophilized protective agent were found to be superior to the survival rate as well as preservation after lyophilization than when using skim milk powder as a protective agent. Therefore, it can be seen that the lyophilized protective agent according to the present invention can be used to prepare various kinds of lactic acid bacteria lyophilized cells.

Example 2

The Lactobacillus plantarium bacteria solution was inoculated in a sterile medium having the composition of Table 1 and incubated at 37 ° C. for 16 hours. Then, the Lactobacillus plantarium concentrated bacteria solution was prepared in the same manner as in Example 1.

The prepared concentrate was suspended in a lyophilized protective agent prepared by adding lactose, sucrose, and glucose at a concentration of 1% (w / v) as a lyophilization aid to a 10% (w / v) reducing whey solution. Lyophilization and other experiments were carried out in the same manner as in Example 1, and the effect of each sugar as a lyophilization aid is described in Table 4 below.

Whey solution without any sugar

As can be seen from Table 4, even if various sugars were added as a lyophilization aid to the whey solution used as the dispersion medium, there was no significant difference in the survival rate after freeze-drying or survival after 4 weeks of storage.

This cause means that the addition of more sugar has no effect because the whey itself already contains a large amount of lactose (more than 60% of the total solid content).

Therefore, while conventionally used skim milk powder requires the addition of sugar as a lyophilization aid, the present invention does not require the addition of sugar as a lyophilization aid.

Example 3

Lactobacillus plantarium concentrate prepared in the same manner as in Example 2, glycerol and 5% (1%) adonitol in 10% (w / w) reduced whey solution as a lyophilization aid Suspended in the lyophilized protective agent prepared by the addition of the respective freeze-dried to determine the effect of the polyol on the cryopreservation of lactic acid bacteria, the results are shown in Table 5 below.

The control is whey solution with no addition

As can be seen from Table 5, polyol materials such as glycerol or adonitol had a clear effect on the survival rate after lyophilization compared to the control group when lyophilizing the lactic acid bacteria, and especially adonitol is a control and also in the cell preservation Compared with the survival effect was about 2 times.

Example 4

Lactobacillus plantarium concentrate prepared in the same manner as in Example 2 was suspended in 10% (w / w) reduced whey solution, each containing glycine and glutamic acid at a concentration of 0.2 M as a lyophilization aid, followed by lyophilization. The experiment was carried out in the same manner as in Example 2. Through this experiment was determined the cryopreservation effect of amino acids, the results are shown in Table 6 below.

The control is whey solution with no addition

As can be seen from Table 6, it was confirmed that the freeze-drying effect and preservation effect of amino acids, especially glutamic acid during lyophilization of lactic acid bacteria.

Example 5

The concentrated Lactobacillus plantarium solution prepared in the same manner as in Example 2 was suspended in a reducing whey solution to which 0.5% (w / w) ascorbic acid was added as a lyophilization aid and then lyophilized. It carried out in the same manner as in Example 2. The results are shown in Table 7 below.

The control is whey solution with no addition

As can be seen from Table 7, the freeze-drying effect of ascorbic acid was not significantly different compared to the control, but it was proved to be very good in the preservation of the cells, which is due to the antioxidant effect of ascorbic acid in the air It may be because it was prevented from being damaged by oxygen.

Example 6

The Lactobacillus plantarium concentrate culture solution prepared in the same manner as in Example 2 was prepared by adding 10% (1% (w / w) of adonitol, 0.2M glutamic acid, and 0.5% (w / w) of ascorbic acid. w / w) suspended in reducing whey solution and then lyophilized.

As a result, Lactobacillus freeze-dried product was confirmed that the survival of 88.2% was produced, and the survival rate was 49.2% after the lyophilisate was stored for 4 weeks in the air at 30 ℃ thermostat. .

Claims (5)

1-20 parts by weight of polyol, 0.1-0.5 M of amino acid, 0.1-10 parts by weight of polymeric material, 0.05-1 part by weight of ascorbic acid, or a mixture thereof is added to 5-20 parts by weight of reducing whey solution as a lyophilization aid. A method for producing lactic acid bacteria lyophilized cells using a reduced whey base-freeze-drying protective agent comprising a step. The method of claim 1, wherein the lyophilized protective agent is selected from citric acid, sodium hydroxide, sodium monophosphate, dibasic sodium phosphate, potassium monophosphate, dibasic potassium phosphate, sodium carbonate, sodium bicarbonate, calcium carbonate, and mixtures thereof A method for producing lactic acid bacteria lyophilized cells using a reduced whey base-freeze drying protector, characterized in that the pH is adjusted to 5.0-9.0 using one pH adjusting agent. The method of claim 1, wherein the polyol is selected from glycerol, adonitol, sorbitol, and mixtures thereof. The method of claim 1, wherein the amino acid material is selected from glycine, glutamic acid, and mixtures thereof. The method of claim 1, wherein the polymer material is selected from gelatin, carboxy methyl cellulose, cellulose, starch, arginic acid, agar, guar, and mixtures thereof. Production method of the cells.