동결보호제 및 Sucrose 농도가 급속동결한 마우스 집합배의 생존성에 미치는 영향

신상태,Shin, Sang-tae 대한수의학회 1991 大韓獸醫學會誌 Vol.31 No.4

The effects of ethylene glycol, DMSO and glycerol as cryoprotectants and the effect of concentrations(0, 0.25, 0.5 and 1.0M) of sucrose in the diluent on the viability of the aggregated morulae frozen rapidly in liquid nitrogen$(LN_2)$ vapour were examined. The morulae were produced by aggregation of ICR and CBA mice embryos at 8-cell stage. Before freezing the embryos were equilibrated in 1.5M cryoprotectants+0.25M sucrose in oae-step or in 3.0M cryoprotectants+0.25M sucrose in two-steps. The embryos were pipetted into the freezing medium fraction of 0.25ml plastic straws. The straws were frozeu by directly transfer into $LN_2$ vapour(about lcm above $LN_2$) for 2 minutes, and then plunged into $LN_2$. After thawing the cryoprotectants were diluted with 0, 0.25, 0.5 or 1.0M sucrose solution. The post-thawed in vitro viability of the aggregated embryos was significantly dependent on the type and concentration of cryoprotectants in the freezing medium and also on the concentration of sucrose in the diluent. When the aggregated embryos were equilibrated in 1.5M cryoprotectants +0.25M sucrose in one-step and diluted with 0.5M sucrose after thawing, the survival rate of the embryo5 was significantly(p<0.05) higher in DMSO(62.5%) or ethylene glycol(52.2%) than in glycerol(33.3 %). In the case that the concentration of the cryoprotectants was raised to 3.0M in two-steps, thc higher survival rate of the embryos was obtained in ethylene glycol or glycerol than in DMSO followed by diluting them with 0.5 or 1.0M sucrose after thawing(p<0.01).

Preparation and Evaluation of Freeze-dried Solid Lipid Nanoparticles with Various Cryoprotectants

Li, Ri Hua,Seo, Seung-Yong,Eun, Jae-Soon,Lee, Mi-Kyung The Korean Society of Pharmaceutical Sciences and 2010 Journal of Pharmaceutical Investigation Vol.40 No.1

Solid lipid nanoparticles (SLNs) were freeze-dried to obtain a stable solid dosage form with the aid of various cryoprotectants such as trehalose, sucrose, glucose, fructose, and glycerol. Tricaprin(TC) and trilaurin(TL) were used as lipid matrices for SLNs and stabilizers were egg phosphatidylcholine and pegylated phospholipid. All cryoprotectants tested did not cause changes in mean particle size of SLNs when mixed with SLNs before freeze-drying. However, the mean particle sizes of reconstituted SLNs after freeze-drying were significantly different from those of the un-lyophilized original SLN dispersions depending on the types and concentration of cryoprotectants. Although the freeze-dried SLNs without any cryoprotectants were easily reconstituted by hand-shaking, the mean particle size drastically increased (> $8\;{\mu}m$ for TC SLNs and around $1\;{\mu}m$ for TL SLNs) compared to that of un-lyophilized original dispersion (97 nm for TC SLNs and 164 nm for TL SLNs). Trehalose and sucrose were the most effective additives to protect the SLNs during lyophilization. The reconstituted SLNs were physically stable for 24 hours when lyophilized with 12.5% trehalose, sucrose, glucose, fructose or glycerol.

pH 조절법으로 제조한 닭가슴살 수리미의 저장 중 품질특성에 미치는 냉동변성방지제 첨가 효과

진상근,김일석,최영준,양한술,박구부,Jin, Sang-Keun,Kim, Il-Suk,Choi, Yeung-Joon,Yang, Han-Sul,Park, Gu-Boo 한국축산식품학회 2007 한국축산식품학회지 Vol.27 No.3

pH 조절법을 활용하여 닭가슴살을 이용한 수리미 제조 시 냉동변성방지제의 첨가에 따른 수리미의 냉동 저장 중 품질 특성을 파악하기 위해 C(명태수리미, 2회수세, 4% 설탕, % sorbitol and 0.3% polyphosphate 첨가), T1(닭가슴살수리미, pH 11.0, 5% sorbitol and 0.3% polyphosphate 첨가), T2(닭가슴살수리미, pH 11.0, 4% sugar, 5% sorbitol and 0.3% polyphosphate 첨가) 및 T3(닭가슴살수리미, pH 11.0, 2% salt, 4% sugar, 5% sorbitol and 0.3% polyphosphate 첨가)으로 분류하여 실험한 결과, 대조구에서 높은 조단백질 함량 및 낮은 수분함량을 나타내었다. 또한 닭가슴살을 활용한 수리미는 냉동변성방지제의 첨가 수준이 증가할수록 낮은 수분함량을 나타내었다. 물리적 특성 측정 결과, 대조구에서 높은 pH값을 보인 반면, T3에서 높은 보수력을 나타내었다. 처리구별 콜라겐 함량은 차이를 보이지 않았으나 대조구와 T3에서 높은 근원섬유단백질 함량을 나타내었다. 전단가는 대조구에 비해 닭가슴살을 활용한 수리미에서 높게 나타났다. 겔 특성 중 파괴강도는 모든 처리구에서 저장기간이 증가할수록 증가하며, 대조구에서 다른 처리구들에 비해 높은 값을 보였다. 또한 닭가슴살을 활용한 수리미는 T3에서 높은 파괴강도 값을 보여준다. 변형값, 겔강도, 젤리강도 및 접기실험 결과, 대조구와 T4에서 높게 나타난 반면, T1 및 T2에서 낮은 결과를 보였다. 수리미의 표면 색 측정 결과, 대조구에 비해 닭가슴살을 활용한 수리미에서 높은 명도 값을 보이며, T3에서 가장 높게 나타났다. 백색도는 T1에서 가장 낮은 값을 나타낸 반면, T3에서 가장 높게 나타났다. 색을 포함한 모든 관능평가 항목에서 대조구와 T3에서 T1과 T2에 비해 높은 선호도를 보였다. 또한 소금과 설탕을 첨가하지 않은 T1에 비해 첨가량이 증가할수록 높게 나타났다. 따라서 수리미의 품질 특성을 결정하는 물리 화학적 특성을 고려해보면, 대조구에서 높은 조단백질 함량, pH, 겔 특성을 보인 반면, T3에서 높은 보수력, 겔 특성, 명도, 백색도 및 기호도를 보였다. 특히 대조구에 비해 T3에서 높은 명도 및 백색도를 보여 수리미로서의 품질 특성이 뛰어난 것으로 판단된다. 또한 기호도 역시 높게 나타나 닭가슴살을 활용한 수리미 제조가 가능하며, 소금을 포함한 냉동변성방지제를 첨가한 T3가 수리미의 냉동 저장 중 물리 화학적 특성에 미치는 효과를 종합해 볼 때 품질저하방지에 가장 효과적이었다. This study was conducted to determine the effect of pH adjustment and the addition of cryoprotectants on the quality characteristics of chicken breast surimi. We prepared surimi from Alaska pollack, as a the control, by two time washing times and the addition of cryoprotectants. Different preparations of surimi were manufactured by adjusting to pH 11.0 and the addition of different addition cryoprotectants during frozen storage (T1 : 5% sorbitol and 0.3% polyphosphate, T2: 4% sugar, 5% sorbitol and 0.3% polyphosphate, and T3: 2% salt, 4% sugar, 5% sorbitol and 0.3% polyphosphate). The moisture content was significantly lower in the control and T3 samples. The crude protein content was increased with storage times. The crude protein was higher in the control. The water-holding capacity, myofibrillar protein and shear force were significantly higher in T3 than other surimi samples. All gel characteristics were significantly higher in the control and T3 than other surimi samples. pH 11.0 adjusted chicken breast surimi had greater lightness than the control, and T3 samples had the highest lightness and whiteness. Sensory evaluations were significantly higher in the control and T3 than the other samples. The gel, and physical characteristics and sensory evaluation of T3 were similar to the control. T3 samples had superior color and pH than the control Alaska pollack surimi.

Effects of Meiotic Stages, Cryoprotectants, Cooling and Vitrification on the Cryopreservation of Porcine Oocytes

Huang, Wei-Tung,Holtz, Wolfgang Asian Australasian Association of Animal Productio 2002 Animal Bioscience Vol.15 No.4

Different factors may affect the sensitivity of porcine oocytes during cryopreservation. The effect of two methods (cooling and vitrification), four cryoprotectants [glycerol (GLY), 1, 2-propanediol (PROH), dimethyl sulfoxide (DMSO) or ethylene glycol (EG)] and two vitrification media (1 M sucrose (SUC)+8 M EG; 8 M EG) on the developmental capacity of porcine oocytes at the germinal vesicle (GV) stage or after IVM at the metaphase II (M II) stage were examined. Survival was assessed by FDA staining, maturation and cleavage following IVF and IVC. A toxicity test for different cryoprotectants (GLY, PROH, DMSO, EG) was conducted at room temperature before cooling. GV and M II-oocytes were equilibrated stepwise in 1.5 M cryoprotectant and diluted out in sucrose. The survival rate of GV-oocytes in the GLY group was significantly lower (82%, p<0.01) than that of the other group (92 to 95%). The EG group achieved a significantly higher maturation rate (84%, p<0.05) but a lower cleavage rate (34%, p<0.01) than the DMSO group and the controls. For M II-oocytes, the survival rates for all groups were 95 to 99% and the cleavage rate of the GLY group was lower than the PROH-group (21 vs 43%, p<0.01). After cooling to $10^{\circ}C$, the survival rates of GV-oocytes in the cryoprotectant groups were 34 to 51%, however, the maturation rates of these oocytes were low (1%) and none developed after IVF. For M II-oocytes, the EG group showed a significantly higher survival rate than those of the other cryoprotectant groups (40% vs 23-26%, p<0.05) and the cleavage rates of PROH, DMSO and EG group reached only 1 to 2%. For a toxicity test of different vitrification media, GV and M II-oocytes were equilibrated stepwise in 100% 8 M EG (group 1) and 1 M SUC + 8 M EG (group 2) or equilibrated in sucrose and then in 8 M EG (SUC+8 M EG, group 3). For GV-oocytes, the survival, maturation and cleavage rates of Group 1 were significantly lower than those in group 2, 3 and control group (p<0.05). For M II-oocytes, there were no differences in survival, maturation and cleavage rates between groups. After vitrification, the survival rates of GV and M II-oocytes in group 2 and 3 were similarly low (4-9%) and none of them matured nor cleaved after in vitro maturation, fertilization and culture. In conclusion, porcine GV and M II-oocytes do not seem to be damaged by a variety of cryoprotectants tested, but will succumb to a temperature decrease to $10^{\circ}C$ or to the process of vitrification, regardless of the cryoprotectant used.

소 수정란의 간이 동결기법 개발에 관한 연구 I. 내동제의 종류, 농도 및 동결방법이 체외발생율에 미치는 영향

김상근,남윤이,현병화,석호봉 한국동물번식학회 1997 Reproductive & developmental biology Vol.21 No.2

The studies on the carried out to investigate the effective concentration of cryoprotectant agents and sucrose by one-step straw method of bovine embryos. The follicular oocytes were cultured in TCM-199 medium containing 10 IU/ml PMSG(Sigma, USA), 10 IU/ml hCG(Sigma, USA), 1$\mu\textrm{g}$/ml $\beta$-estradiol(Sigma, USA) and 10% FCS for 24~48 hrs in incubator with 5% CO2 in air at 38.5$^{\circ}C$ and then matured oocytes were again cultured for 12~18 hrs with motile capacitated sperm by preincubation of heparin. The bovine embryos following dehydration by cryoprotective agents and various concentrations of sucrose were directly plunged into liquid nitrogen and thawed in 3$0^{\circ}C$ water. Survival and in vitro developmental rate was defined as devellpmental rate on in vitro culture or FDA-test. The results are smmarized as followes : 1. The high in vitro developmental rates of bovine frozen embryos after rapidly thawed in freezing medium was attained 2.0M glycerol, 2.0M DMSO, 1M or 2.0M propanediol. 2. The high in vitro developmental rates of bovine frozen embryos after rapidly thawed in freezing medium was obtained single cryoprotectant(6.7~17.4%) than mixed cryoprotectants(6.7~16.7%). 3. In vitro developmental rate of bovine embryos after rapid frozen-thawing in the freezing medium added 0.25M and 0.50M sucrose were higher cleavage rate than those of sucrose concentration of 0.75M and 1.00M. 4. The freezing methods on in vitro developemental rates of bovine embryos was attained slow freezing method(9.70~15.6%) higher than rapid freezing method(9.4~13.3%).

동결보호제의 종류에 따른 냉동보관자가골의 골형성능에 대한 연구

박현욱(Hyun-Wook Park),이백수(Baek-Soo Lee) 대한구강악안면외과학회 2005 대한구강악안면외과학회지 Vol.31 No.3

Purpose: Several cryoprotectants are in use to help the survival of cells during cryopreservation of bone in maxillofacial region. Among them, Me2SO(dimethyl sulfoxide), EG(ethylene glycol), sucrose were used for experimentally created defects with accompanying cryopreserved bone graft in the rabbit model. The aim of this study is to analyze the effect of above mentioned agents on bone formation using histologic and histomorphometrical methods, thus to provide experimental support for clinical application of these agents. Materials and methods: Nine rabbits were used as experimental animals. Surgical defects were created on the distal femoral heads and mesial tibial heads of each animal using trephine drill(5mm diameter and 5mm length). The harvested bones were cryopreserved in -80℃ refrigerator for one week. The defects were filled with cryopreserved bone with cryoprotectants as experimental groups and cryopreserved bone without cryoprotectant as control. Then, the animals were sacrificed at 1, 2, and 3 weeks after surgery. With Goldner’s modified Masson trichrome staining and semiautomatic image analysis system, we observed the change of the cells and bone formation. Results: After bone graft, bone formation and active remodeling process were examined in all experimental groups and the control. But the intensity of such activities of the control were somewhat weaker than that of the experiments. Especially Me2SO+sucrose group was the best in bone formation and bone remodeling. Me2SO group was more than that of EG group in bone fomation. Sucrose seems to be helpful in survival of the bone cell. Histologic findings showed superior bony quantity and quality in experimental groups than that in control. Conclusions: The data from this study provides the basis for future studies for evaluating the effect of cryoprotectants in the cryopreservation of bone and clinical study for predictable use of these agents.

Rheological properties of washed and unwashed tilapia (Oreochromis mossambicus) fish meat: effect of sucrose and sorbitol

Lakshmi Narasimha Murthy,Girija Gajanan Phadke,Vijayakumar Siddaiah,Rajanna Karani Boraiah 한국식품과학회 2017 Food Science and Biotechnology Vol.26 No.5

In the present study, the dynamic viscoelastic behavior (DVB) and flow behavior of fresh tilapia (Oreochromis mossambicus) meat containing cryoprotectants were evaluated with and without water washing. The DVB profile of washed meat with 4% sucrose and sorbitol indicated the maximum structure buildup reaction up to 56.8 C; thereafter, hydrophobic interactions leading to decreased gelation were suppressed. In both the samples, there was no clear indication of the sol–gel transition temperature. In flow-profile measurements, the presence of cryoprotectants gave rise to the minimum thixotropic area, indicating a low level of impairment in structure. The shear-stress sweep of water-washed tilapia proteins added with cryoprotectants did not reveal significant changes at 28 and 40 C. In texture-profile analysis, the hardness values were lower in fresh meat than cooked meat. The findings of this study will be helpful in the formulation and design of various mince-based products and in determining the appropriate use of cryoprotectants and water washing in the processing of minced meat.

해양 규조류 Nitzschia sp.의 초저온동결보존을 위한 보존제의 영향 분석

이인혜,전지영,김경미,강명석,Lee, In Hye,Jeon, Ji Young,Kim, Kyeung Mi,Kang, Myung suk 한국식물생명공학회 2018 식물생명공학회지 Vol.45 No.4

21세기 들어 전 세계는 환경파괴에 따른 지구 온난화로 인하여 생물 다양성이 지속적으로 감소하였다. 생물다양성의 감소는 생태계의 자정 및 복원능력 등을 악화 시켜 환경오염이 더욱 심화 되었고 나아가 생물의 생존을 위협하고 있다. 우리나라는 현재 나고야 의정서로 인해 국내 유전자원을 효율적으로 발굴 보호해야 하는 중요한 시점에 있다. 본 연구에서 사용하는 미세조류는 유용 생물자원으로 활용도가 높으나 동결보존하는 방법이 개발되어 있지 않아 장기보존에 어려움이 있다. 대부분의 원핵생물이나 균류 등의 미생물은 종에 관계없이 glycerol 을 이용하여 저온동결보존이 가능하나 미세조류는 동결보존제의 종류와 농도, 유리화 과정 및 해동방법 등에 따라 세포 재생률에 차이가 있기 때문에 구체적인 연구가 필요하다. 미세조류 중 규조류는 전 세계적으로 발견되며 규산질로 구성된 cell wall 은 이미 산업적으로 많이 이용되고 있다. 본 연구에서는 해양 규조류 Nizschia frustulum과 Nitzschia amabilis를 이용하여 장기 동결보존을 위한 기초연구를 수행하였다. 동결보존제로는 세포막 침투성 보존제인 glycerol, DMSO, methanol을 각각 5, 10, 15% 농도로 제조하여 실험하였고 메탄올의 경우 시약 특성상 5, 10, 12% 농도로 실험하였으며 미세조류는 N. frustulum과 N. amabilis 두 종을 각각 $10^2$, $10^3$, $10^4cells\;ml^{-1}$로 희석하여 사용하였다. 실험에 사용한 미세조류 두 종 모두 메탄올 12%에서 가장 높은 생존율을 보였으며 N. frustulum은 $6.94{\pm}0.31%$, N. amabilis는 $8.85{\pm}0.16%$로 나타났다. 동결 후 해동한 미세조류를 3주간 재배양한 결과에서는 N. frustulum이 재배양 초기 농도보다 약10배 증가하였고 N. amabilis는 약12배 증가한 결과를 나타내었다. 본 연구에서는 유용생물자원인 미세조류 Nizschia sp.에 한하여 적합한 동결보존제를 탐색하였으며 이 자료를 바탕으로 더 많은 동결보존제에 대한 효과와 다양한 미세조류 종에 적합한 동결보존 기법 연구가 필요한 것으로 사료된다. Biodiversity has continued to degrade in the $21^{st}$ century due to global warming occasioned by destruction of the environment around the world.. The Nagoya protocol places Korea in a unique position to effectively develop and protect its domestic genetic resources. Microalgae under study in this research contains large amount of antioxidant substances such as beta carotene and astaxanthin, that can be used as biological resource owing to the large amounts of biomass that can be secured through photosynthesis. However, it is difficult to preserve it since cryopreservation method used for long-term preservation is yet to be developed. A basic study for long term cryopreservation was carried out on Nizschia frustulum and Nitzschia amabilis which belong to marine diatoms. As cryoprotectants (CPAs), glycerol, DMSO, and methanol which penetrate into cells were prepared at 5%, 10%, and 15% concentrations each, in case of methanol, it was tested at concentrations of 5%, 10% and 12% by its nature. Two kinds of microalgae, N. frustulum and N. amabilis, were diluted with $10^2$, $10^3$ and $10^4cells\;ml^{-1}$, respectively. The highest survival rate was shown at12% concentration of methanol, and the figures were $6.94{\pm}0.31%$ in N. frustulum and $8.85{\pm}0.16%$ in N. amabilis. As a result of 3 weeks cultivation of thawed microalgae after freezing, the result is shows that N. frustulum increased about 10 times faster and N. amabilis increased about 12 times the original concentration.

계육 Surimi에 Trehalose와 Oligosaccharide의 냉동변성 방지효과

이성기,민병진 한국가금학회 2002 韓國家禽學會誌 Vol.29 No.1

계육 surimi에서 trehalose와 oligosaccharide의 단백질 냉동변성 방지효과를 구명하기 위하여 본 실험을 실시하였다. 기계발골노계육에 0.5% 소금물(고기:용액, 1:4)로 2회, 증류수로 1회 수세하여 surimi를 제조하였다. Trehalose와 oligosaccharide를 각각 8%씩 혼합한 후 $-18^{\circ}C$에서 10주간 냉동저장하였다. 모든 Surimi의 적색도(a*)는 냉동기간동안 감소하였다. 냉동기간중에 trehalose, oligosaccharide, 무첨가 순으로 명도가 높고 적색도와 황색도가 낮아 색택 안정성이 있는 것으로 나타났다. 냉동기간중 surimi의 gel 강도(압착력, 경도, 탄성, 점착성)는 감소하였지만, trehalose와 oligosaccharide 첨가가 무침가구에 비해 유의적으로 높은 수준을 유지하였다. 냉동기간중에 부착성은 trehalose 첨가가 가장 높았다. 따라서 이들 당류가 계육 surimi에 단백질 냉동변성방지제로 효과가 인정되었으며, trehalose는 감미도가 기존 설탕보다 낮고 색깔 및 물성에서 oligosaccharide 보다 더 우수한 것으로 판단되었다. Cryoprotective effects on chicken surimi during storage were investigated. Chicken surimi from mechanically deboned spent layer meat was prepared with 4 volumes of 0.5% NaCl washing, and then blended with or without cryoprotectants (8% trehalose, 8% oligosaccharide) prior to frozen storage at $-18^{\circ}C$ to 10 weeks Redness (a) of all surimi decreased during storage. Color stability increased during storage when lightness increased but redness decreased. At this Point, surimi maintained a better color quality as followed order; trehalose > oligosaccharide ) non-additive. Gel strength such as compressive force, hardness, adhesiveness and gumminess tended to decrease during frozen storage. Cryoprotectants provided significantly better textural properties than non-auditive. Surimi with trehalose showed the highest adhesiveness. In conclusion, trehalose and oligosaccharide seemed to be good cryoprotectants of chicken surimi. Especially, trehalose resulted in better cryoprotectant than oligosaccharide because of better color stability, better textural properties, and lower sweet characteristics.

Effects of Cryoprotectants on the Quality Property of Chicken Surimi from Spent Layer Meat during Frozen Storage

Sung Ki Lee,Byung Jin Min 한국식품과학회 2002 Food Science and Biotechnology Vol.11 No.5