KR20160118497A

KR20160118497A - Cryoprotectant with turanose

Info

Publication number: KR20160118497A
Application number: KR1020150046709A
Authority: KR
Inventors: 유상호; 이수용; 정창환; 한동주; 최성원
Original assignee: 세종대학교산학협력단
Priority date: 2015-04-02
Filing date: 2015-04-02
Publication date: 2016-10-12

Abstract

The present invention relates to a cryoprotectant containing a turanose that can inhibit microorganism death at the time of cooling and thawing as much as possible and can provide the same stabilizing effect even in a smaller amount than trehalose used as a conventional cold / thaw stabilizer, A synergistic effect can be achieved by mixing with an auxiliary additive such as skim milk.

Description

Cryoprotectant with turanose < RTI ID = 0.0 >

TECHNICAL FIELD The present invention relates to a cryopreservation agent containing a turanose, and more particularly, to a cryoprotectant containing a turanose that can inhibit microbial death upon cooling and thawing.

The best storage conditions for preserving microorganisms are freezing or lyophilization, and some microorganisms die during freezing and thawing. Microscopic analysis showed that the cause of death was cell membrane damage caused by thawing, rupture and the like, which is caused by changes in electrolyte concentration and formation of ice crystals during freezing.

In order to prevent the microorganisms from being killed during freezing and thawing, it is necessary to use antiseptic agent. Antiseptic agent binds with moisture at the time of freezing and inhibits the formation of ice crystals inside and outside the cell, thereby increasing the survival rate of microorganisms. For freezing storage of microorganisms, sugar alcohols such as sorbitol, glycol, glycerol, and mannitol are widely used as antidisaster agents.

On the other hand, if the dough is frozen, the fermentation time is prolonged, the dough becomes weak, and the bread is not formed. In addition, the flavor is stronger in the smell of yeast and the product value is lowered. This is because the yeast is killed by freezing, and the gluten is eluted and the gluten is weakened.

Therefore, the main factor affecting the quality of bread made from frozen dough is that yeast has the greatest activity and gas holding power. Materials known to contribute to the stability of cold and thawing are the disaccharides such as trehalose. Disaccharides such as trehalose are not toxic and widely used as natural cryopreservation agents. They act as stabilizers for cell membranes when dehydration occurs during freezing. The availability of trehalose as a cryopreservation agent is due to the mutual protective action with the lipid membrane and the stability of the unstable protein during the freezing-thawing process.

By the way, besides trehalose, there is no sugar currently used as a cryoprotectant. In order to meet the desire of various users to select a material, it is necessary to widen the spectrum of usable cryopreservatives. Therefore, research for the development of new cryopreservation agents is urgently required.

Korean Patent Laid-Open No. 10-2012-0022868 (published on March 12, 2012) discloses a combination of a stabilizing compound to improve the survival rate and stability of a freeze-dried lactic acid bacterium containing starch hydrolyzate and glutamate salt and / . Japanese Patent Registration No. 05592049 (registered on Aug. 20, 2014) includes "one or more cryoprotectants selected from the group consisting of guanosine-5'-monophosphate (GMP), inosine-5'- Quot; freeze-dried or lyophilized re-culture liquid "

The present invention provides a new material derived from saccharides that can prevent microbial death when freezing and thawing microorganisms as a cryopreserving agent.

The present invention provides a microorganism cryopreservation agent characterized by containing a turanose as an effective ingredient. At this time, the microorganism may be, for example, yeast or lactic acid bacteria.

Turanose is a reducing disaccharide that occurs naturally in bees, and represents a sweet taste equivalent to about half of sucrose. Toranoz is an analog of sucrose and has the chemical structure of 3-O-a-D-glucopyranosyl-D-fructose. Since Toranoze is not fermented by microorganisms inducing dental caries, it is used as a calorie-free sweetener. In the present invention, it is newly confirmed that the Toranoze having the above characteristics has the characteristics of a cold / thaw stabilizer such as trehalose.

On the other hand, the Toranoze of the present invention can be used in combination with other components that can be used as a cold / thaw stabilizer such as trehalose, thereby generating a synergistic effect. For example, the microbial cryopreservative of the present invention may further comprise skim milk. Further, ascorbic acid or a salt thereof may further be contained. Ascorbic acid or its salt is more effective when applied to lactic acid bacteria.

On the other hand, the present invention provides a microorganism freezing method comprising adding a turanozide to a culture medium containing microorganisms, followed by freezing. In this case, the microorganism freezing method may further include freeze-drying after freezing. It has been confirmed through the present invention that the microorganisms killed during freezing and thawing can be effectively prevented from being killed when frozen is added with Toranoz.

Meanwhile, in the microorganism freezing method of the present invention, the turanose is preferably added in an amount of 2 to 12% (w / v) in the medium.

The present invention can provide the same stabilizing effect even in a smaller amount than trehalose used as a conventional cold / thaw stabilizer, and can achieve a synergistic effect by mixing with an auxiliary additive such as skim milk have.

Fig. 1 shows an FE-SEM photograph of the surface of the yeast by the Toranoz concentration. (2), (2), (3), (4) and (4) are Toranos, Toranos and Toranos, respectively. It is a photograph of yeast when 8% halose is added.

Hereinafter, the present invention will be described in more detail with reference to the following Examples and Experimental Examples. However, the scope of the present invention is not limited to the following embodiments and experimental examples, and includes modifications of equivalent technical ideas.

[ Example 1: of yeast Viable cell count Evaluation of stability of cold and thawing through confirmation - Toranoz Added group]

1. Culture of yeast

The raw yeast Saccharomyces S. cerevisiae ) was dissolved in 1 mL of YPD broth and then diluted with a suitable dilution (10 ⁵ -10 ⁷ -fold dilution). 100 占 퐇 of the diluted live yeast solution was spread on a plate and cultured at 30 占 폚 for 72 hours. Single colonies were collected and cultured in 5 mL of YPD broth at 30 ° C for 48 hours. The cultured cells were recovered by centrifugation and washed twice with sterilized distilled water.

2. Lyophilization of yeast

To prepare a medium for evaluation of cold and thaw stability, the amount of Toranoz was divided into 5 groups of 0 to 12% (w / v). The control group was experimented with trehalose 8% (w / v). The collected cells were redissolved in a medium containing turanose or trehalose, which was a cold / thawing stabilizer, and then frozen at -80 ° C and lyophilized for 72 hours. The dried cells were ground in a mortar and sieved through a 100 mesh sieve and homogenized.

3. Viable cell count Evaluation of stability of cold and thawing through confirmation

The viable counts of the samples before and after freeze drying were compared and compared. The sample was spread on a YPD plate and cultured at 30 ° C for 72 hours, and the number of colonies was confirmed. The bacterial counts were expressed as CFU / mL, and the dry weight was measured using a water content meter for unit conversion with CFU / g. The results are shown in Table 1 below.

Amount of cold and thaw stabilizer added Before lyophilization After lyophilization Survival rate CFU / g % Toranoz 0% (5.87 + - 0.01) x 10 < ⁷ > (6.00 + - 0.14) x 10 < ⁵ > 1.02 + 0.04 Toranoz 2% (4.03 + - 0.01) x 10 < ⁷ > (4.30 + - 0.42) x 10 ⁵ 1.07 + - 0.12 Toranoz 4% (2.31 + - 0.01) x 10 < ⁷ > (1.14 + - 0.42) x 10 < ⁶ > 4.94 + 0.04 Toranoz 8% (1.96 + 0.00) x 10 ⁷ (2.16 0.50) x 10 ⁶ 10.97 + 0.13 Toranos 12% (1.64 + - 0.01) x 10 < ⁷ > (2.34 ± 0.15) x 10 ⁶ 14.29 ± 1.44 Trehalose 8% (2.38 + 0.02) x 10 < ⁷ > (1.29 0.50) x 10 ⁶ 5.41 0.30

As shown in Table 1, the survival rate was 1.02% when no turanose was added, and the survival rate when the 2% turanose was added was about 1.07%, indicating that the survival rates of the two groups were almost the same . However, as the amount of Toranoz added, the survival rate also increased proportionally. In addition, the survival rate was found to be 5.41% in the case of trehalose 8%, but the survival rate in the case of Turaanoz 8% was 10%, which is almost twice as high as that in Toranoz.

[ Example 2: of yeast Viable cell count Evaluation of stability of cold and thawing through confirmation - Toranoz + Skim milk Added group]

1. Culture of yeast

2. Lyophilization of yeast

To prepare a medium for evaluation of cold and thaw stability, the amount of Toranoz was divided into 5 groups of 0 to 12% (w / v). The control group was experimented with trehalose 8% (w / v). In addition, 5% (w / v) skim milk was added to each medium as a stabilizer. The recovered cells were redissolved in a medium containing turanose or trehalose as a cooling and thawing stabilizer, respectively, and then frozen at -80 ° C and lyophilized for 72 hours. The dried cells were ground in a mortar and sieved through a 100 mesh sieve and homogenized.

The viable counts of the samples before and after freeze drying were compared and compared. The sample was spread on a YPD plate and cultured at 30 ° C for 72 hours, and the number of colonies was confirmed. The number of bacteria in the experiment was expressed as CFU / mL, and the dry weight was measured using a water content meter for unit conversion with CFU / g. The results are shown in Table 2 below.

Amount of cold and thaw stabilizer added Before lyophilization After lyophilization Survival rate CFU / g % Toranoz 0% + skim milk 5% (2.34 + 0.02) x 10 < ⁷ > (4.17 + - 0.49) x 10 < ⁶ > 17.29 ± 1.42 Toranoz 2% + skim milk 5% (2.16 +/- 0.17) x 10 < ⁷ > (5.93 + - 0.40) x 10 < ⁶ > 27.53 + - 2.78 Toranoz 4% + skim milk 5% (1.55 + - 0.01) x 10 < ⁷ > (6.63 + - 0.40) x 10 < ⁶ > 42.94 + - 3.77 Toranoz 8% + skim milk 5% (1.57 + 0.02) x 10 < ⁷ > (7.40 +/- 0.96) x 10 < ⁶ > 47.25 + - 5.66 Toranoz 12% + skim milk 5% (1.08 + - 0.01) x 10 < ⁷ > (6.80 + 0.26) x 10 < ⁶ > 62.97 + - 5.50 Trehalose 8% + skim milk 5% (1.40 0.00) x 10 ⁷ (6.07 + - 0.42) x 10 < ⁶ > 43.42 + 2.03

As shown in Table 2, the survival rate of the group to which 5% skim milk was added without adding the turanose was 17.29%, and the survival rate of the group to which 2% of turanose and 5% of skim milk were added was about 27.53% And the survival rate was increased by about 10% when Toranoz was added. It was also found that the survival rate was increased with increasing amount of Toranoz.

On the other hand, in case of trehalose 8%, the survival rate was 43.42%, and the survival rate was similar to that of 4% of Toranoz. This means that as much as 1/2 of the amount of Toranozo compared to trehalose shows the same survival rate as that of trehalose.

[ Example 3: Lactic acid bacteria Viable cell count Evaluation of stability of cold and thawing through confirmation - Toranoz + Skim milk Added group]

1. Culture of lactic acid bacteria

Lactobacillus, Lactobacillus paracasei paracasei ) were cultured in 5 mL MRS broth at 37 ° C for 48 hours. The cultured cells were recovered by centrifugation and washed twice with sterilized distilled water.

2. Lyophilization of lactic acid bacteria

To prepare the medium for the evaluation of cold and thaw stability, the amount of the turanose was divided into four groups of 0 to 12% (w / v). The control group was experimented with trehalose 8% (w / v). In addition, 6% (w / v) skim milk and 4% (w / v) sodium ascorbate were added to each medium as a stabilizer. The recovered cells were redissolved in the respective cold / thawing stabilizer-added medium, frozen at -80 ° C for 2 weeks, and then lyophilized for 48 hours. The dried cells were ground in a mortar and sieved through a 100 mesh sieve and homogenized.

The number of viable cells of the sample before freeze-drying and the freeze-dried sample was confirmed and compared. The samples were incubated in an MRS liquid medium at 37 DEG C for 48 hours, and then transferred to a plate and cultured to confirm the number of colonies. The bacterial counts were expressed as CFU / mL, and the dry weight was measured using a water content meter for unit conversion with CFU / g. The results are shown in Table 3 below.

Amount of cold and thaw stabilizer added Before lyophilization After lyophilization Survival rate CFU / g % Toranoz 0% + Skim Milk 6% + Sodium Ascorbate 4% 3.08 ± 0.70 × 10 ¹¹ 6.17 ± 0.25 × 10 ¹⁰ 20.02 + - 0.82 Toranoz 4% + Skim Milk 6% + Sodium Ascorbate 4% 7.41 ± 0.18 × 10 ¹⁰ 5.03 ± 0.24 × 10 ¹⁰ 67.86 + - 2.80 Toranoz 8% + Skim Milk 6% + Sodium Ascorbate 4% 6.60 ± 0.27 × 10 ¹⁰ 3.73 ± 0.41 × 10 ¹⁰ 56.40 + - 4.08 Toranoz 12% + Skim Milk 6% + Sodium Ascorbate 4% 5.15 ± 0.65 × 10 ¹⁰ 2.83 ± 0.60 × 10 ¹⁰ 54.40 + - 5.49 Trehalose 8% + Skim Milk 6% + Sodium ascorbate 4% 6.50 ± 0.37 × 10 ¹⁰ 4.27 ± 0.54 × 10 ¹⁰ 65.50 ± 6.76

As shown in Table 3, when only skim milk and sodium ascorbate were added without adding turanose, the survival rate was 20.02%. However, the survival rate of the Toranoz or Trehalose added group was more than doubled. In particular, the survival rate of Toranoz 4% added group was 67.86%, indicating that the survival rate was the highest among the added groups.

The survival rate of 4% Turaanose was similar to the survival rate of 8% Terahalose. This indicates that even at half the concentration of trehalose, the survival rate of Turaanoz can be similar to that of trehalose it means.

[ Example 4: Evaluation of stability of cold and thawing through confirmation of restoration ability of yeast cells - Toranoz Added group]

1. Culture of yeast

2. Lyophilization of yeast

To prepare a medium for evaluation of cold and thaw stability, the amount of Toranoz was divided into 5 groups of 0 to 12% (w / v). The control group was experimented with trehalose 8% (w / v). The recovered cells were redissolved in a medium containing turanose or trehalose as a cooling and thawing stabilizer, respectively, and then frozen at -80 ° C and lyophilized for 72 hours. The dried cells were ground in a mortar and sieved through a 100 mesh sieve and homogenized.

3. Evaluation of stability of cold and thawing through confirmation of cell restoration

The restoration of the cells was carried out by surface observation using a scanning electron microscope, and the method was as follows. The cells were filtered using a 0.2 μm polycarbonate filter for scanning electron microscopy and then pretreated.

For the experiment, the lyophilized cells were diluted to 1% with sterile distilled water. The cells were filtered using a 0.2 μm polycarbonate filter. And the mixture was placed in 2% glutaraldehyde and subjected to primary fixation at 4 ° C for 2 hours. The cells were washed three times with 100 mM phosphate buffer solution at 4 ° C for 10 minutes and then subjected to secondary fixation with 1% osmium tetroxide at 4 ° C for 2 hours. Thereafter, the cells were rinsed twice with sterilized distilled water at room temperature and dehydrated with 30 to 100% ethanol. The pretreated samples were surface coated with platinum at a thickness of 18 nm and then observed with a scanning electron microscope.

The experimental results are shown in Fig. Fig. 1 shows an FE-SEM photograph of the surface of the yeast by the Toranoz concentration. (2), (2), (3), (4) and (4) are Toranos, Toranos and Toranos, respectively. It is a photograph of the yeast when 8% halose is added.

As shown in FIG. 1, when no turanose was added, it was found that the cells were shrunk considerably because the restoration ability of the cells was low. However, as the amount of the Toranoze was increased, the restoring force of the cells increased and the shrinkage of the cells was lowered. On the other hand, it was confirmed that the restorations of Toranoz 8% and Trehalose 8% were similar.

Claims

A microorganism cryopreservation agent characterized by containing turanose as an active ingredient.

The method according to claim 1,
The micro-
A microbial cryopreservation agent characterized by being a yeast or a lactic acid bacterium.

The method according to claim 1,
The microorganism freeze-
Wherein the microbial cryopreservation agent further comprises skim milk.

The method of claim 3,
The microorganism freeze-
Characterized in that it further comprises ascorbate or a salt thereof.

The method of freezing microorganisms according to any one of claims 1 to 3, wherein the microorganism freezing method comprises adding Toranoz into a medium containing microorganisms, followed by freezing.

6. The method of claim 5,
After the freezing,
And freeze-drying the microorganism.

6. The method of claim 5,
The Toranoz,
And 2 to 12% (w / v) of the culture medium is added to the microorganism.