Cryoprotectants

Freeze dried surimi powder is a surimi type that has been processed through freeze drying. To utilize oversized catfish and to reduce surimi handling cost, oversized catfish has been used as raw material of surimi powder. The study aimed to determine the effect of defatting (NaHCO3 concentration and soaking duration), washingcycle,and dryoprotectant type on catfish surimi, as well as quality differences between surimi powder and wet surimi. With regard to defatting step, soaking in NaHCO3 0.75% for 10 min was found as the best treatment and resulting in a fat content of 1.52%. Moreover, one time of washing cycle was found as the most appropriate procedure to obtain a superior quality of surimi whiteness value 57.21%, water holding capacity 73.28%, salts soluble protein 7.17%, pH 6.69, and gel strength 482.3 g/cm2, folding value of 4.84, teeth cutting value of 8.26, respectively. Trehalose 6% was the most suitable dryoprotectant resulting in surimi powder with water holding capacity of 8.01 mL/g, gel strength 826.3 g/cm2, salt soluble protein 18.98%, density 4.06 mL/10 g, rehydration capacity 3.81, emulsion capacity 69.3%, emulsion stability 59.3%, foaming capacity 25.33% and foaming stability 9.40%. The microstructure profile of surimi powder added with trehalose had more compact tissues, consisted of no damage and clots, than in surimi treated with other dryoprotectants. However, surimi powder still had lower protein content than in wet surimi, and lower physical and chemical properties, particularly its teeth cutting and folding characteristics.

The cryoprotective effects of gelatin hydrolysate from blacktip shark (Carcharhinus limbatus) skin with different degrees of hydrolysis (DHs:5, 10, 20 and 30%) and commercial cryoprotectant (sucrose/sorbitol blend, 3:1) on surimi from threadfin bream (Nemipterus spp.) subjected to 3 and 6 freeze-thaw cycles were investigated. Ca 2+-ATPase, total sulfhydryl group content and solubility of all natural actomyosin (NAM) extracted from surimi decreased with increasing freeze-thaw cycles, while surface hydrophobicity and disulfide bond content increased (P < 0.05). Among all samples, the highest Ca 2+-ATPase activity with the coincidental lowest surface hydrophobicity was observed in surimi added with gelatin hydrolysate having 10%DH after repeated freeze-thawing (P < 0.05). On the other hand, surimi without cryoprotectant showed the lowest Ca 2+-ATPase and the highest surface hydrophobicity (P < 0.05). The similar solubility was obtained between samples added with commercial cryoprotectant and gelatin hydrolysate (10%DH) (P > 0.05). The results suggest that gelatin hydrolysate with 10%DH was able to prevent the denaturation of surimi protein comparably to commercial cryoprotectant. However, it exhibited the higher protective effect on denaturation of myosin heavy chain, especially globular head domain. Therefore, gelatin hydrolysate can be an alternative cryoprotectant with the lower sweetness in surimi products.

Cryopreservation is the process of freezing and storing biological material at ultra-low temperatures
(such as -196⁰C) in liquid nitrogen (LN2) for unlimited periods. Thanks to the application
of this biotechnology, all biological activities including the biochemical reactions
leading to cell death and DNA degradation discontinue. From this point of view, cryopreservation
biotechnology focuses on preservation of cells that have many applications in the
fields of human and veterinary medicine, agriculture and aquaculture. In addition, this form
of biotechnology also has many applications in biomedical research, specifically in the areas
of immunology, virology, neurobiology, toxicology and the pharmaceutical industry.
Cryopreservation Biotechnology in Biomedical and Biological Sciences describes principles and applications
of cryopreservation biotechnology in different research areas and includes seven
chapters that have been written by experts in their research fields. The book chapters are
divided into four sections.
Section I, "Mechanism of Cryopreservation" contains one chapter entitled "Cryoprotectants
and their usage in cryopreservation process", which describes the importance and mechanism
of the cryopreservation process and also its comparison to vitrification. In addition, the
functions and physical and chemical properties of cryoprotectants are discussed.
Section II, "Application of Cryopreservation in Human Medicine Researches" is divided into
four chapters. The first chapter, "Clinical outcomes of assisted reproductive techniques using
cryopreserved gametes and embryos in human medicine", provides valuable information
regarding fertility treatments in human reproductive medicine, focusing on
cryopreservation of spermatozoa, oocytes and embryos. The second chapter, "Cryopreservation
of platelets: advances and current practices" focuses on the long-term preservation of
platelets, which is necessary for the coagulation of blood and stopping bleeding. The third
chapter, "Cryopreservation of preantral follicles", provides valuable information regarding
the structure and development of ovarian follicles. Beside this, advantages and necessities of
using oocytes in preantral follicles for the aim of cryopreservation are explained in humans
and mammals. The fourth chapter, "Vitrification: Fundamental principles and its application
for cryopreservation of human reproductive cells", is related to mechanisms of cryopreservation
in terms of application of this technique in human medicine with emphasis on
reproductive cells. In this way, mechanisms underlying the problem of the intra- and extracellular
ice formation, the role of cryoprotectants, stages of the warming and cooling process
and use of this biotechnology in the field of IVF industry are explained.
Section III, "Application of Cryopreservation in Veterinary Medicine Research", contains one
chapter entitled "Biological signals of sperm membrane resistance to cryoinjury in boars",
discusses biological mechanisms before the cryopreservation/vitrification process causing
cryoinjuries in sperm cells in boars.
Section IV, "Application of Cryopreservation in Agricultural Research", contains one chapter
entitled "Cryopreservation protocols for grapevine shoot tips", which presents valuable information
regarding cryopreservation of shoot tips of grapevines, which is an economically
important agricultural product.
This book covers different applications of cryopreservation biotechnology. I hope that this
book will be helpful for researchers studying cryobiology and related issues. I would like to
thank all the authors for their distinguished contributions, IntechOpen Publishing Company,
and its Author Service Manager Ms. Dolores Kuzelj for her help in publishing this book.

Ethylene vinyl alcohol copolymer (EVOH) films containing green tea extract were successfully produced by
extrusion. The films were brown and translucent, and the addition of the extract increased the water and oxygen barrier at low
relative humidity but increased the water sensitivity, the glass transition temperature, and the crystallinity of the films and improved
their thermal resistance. An analysis by HPLC revealed that the antioxidant components of the extract suffered partial degradation
during extrusion, reducing the content of catechin gallates and increasing the concentration of free gallic acid. Exposure of the films
to various food simulants showed that the liquid simulants increased their capacity to reduce DPPH• and ABTS•+ radicals. The
release of green tea extract components into the simulant monitored by HPLC showed that all compounds present in the green tea
extract were partially released, although the extent and kinetics of release were dependent on the type of food. In aqueous food
simulants, gallic acid was the main antioxidant component released with partition coefficient values ca. 200. In 95% ethanol (fatty
food simulant) the K value for gallic acid decreased to 8 and there was a substantial contribution of catechins (K in the 1000 range) to
a greatly increased antioxidant efficiency. Kinetically, gallic acid was released more quickly than catechins, owing to its faster
diffusivity in the polymer matrix as a consequence of its smaller molecular size, although the most relevant effect is the plasticization
of the matrix by alcohol, increasing the diffusion coefficient >10-fold. Therefore, the materials here developed with the combination
of antioxidant substances that constitute the green tea extract could be used in the design of antioxidant active packaging for all type
of foods, from aqueous to fatty products, the compounds responsible for the protection being those with the higher compatibility
with the packaged product.

Cryoprotectants are basically some chemical compounds which prevent cells or tissues from damage due to freezing. Mostly vitrification and thawing process are mostly used in cryopreservation. Now a day’s cryopreservation is becoming an apex technology for Health sector, as many organ and tissue transplant methods has been developed in recent time. Organs or tissues need to preserve properly before implantation, hence cryoprotectants plays a key role modern cryonics. This article seeks to provide answers for how cryoprotectants works and prevent tissue damage from shrinking ice crystals during freeze drying or vitrification. The various animal tissues, organs, lymphocytes, cartilages, bone marrow, proteins can be stored by using proper cryoprotectants. Optimization of cryoprotectants is a challenging task, as elevated concentration may cause cytotoxicity. This article also highlights the implication, challenges and recent advances of cryoprotectants along with its types. The importance of cryobank system and its importance were also being emphasized in this article. Further, importance of cryoprotectants in nano suspension preparation was also been discussed in this article.

Vitamin E is one of the most powerful antioxidants for prevention of cell damage resulting from cryopreservation, but its efficacy for cryopreserving brown trout sperm is still unclear. In this work, the protective effect of vitamin E on quality, fertilizing capacity, and DNA damage of brown trout (Salmo trutta macrostigma) sperm after cryopreservation was evaluated. Sperm samples were diluted at the ratio of 1:10 with three different extenders (E): (E-I): 300 mM glucose, 10% egg yolk; (E-II): 33.3 mM glucose, 5.1 mM NaCl, 0.5 mM NaH-CO 3, , 15% DMA; and (E-III): 61.6 mM NaCl, 134.2 mM KCl, 1.9 mM CaCl 2 , 0.8 mM MgCl 2 , 2.3 mM NaHCO 3 in distilled water. Each extender was supplemented with 10% DMSO and different concentrations of vitamin E at 0.1, 0.5, and 1.0 mM. Spermatozoa frozen without vitamin E (0 mM, control) and fresh sperm were also used. After dilution, the sperm was aspirated into 0.25 mL straws, frozen 3 cm above the liquid nitrogen (LN 2) surface, and plunged into the LN 2. Cell motility, viability, fertilization, and eyeing were determined in postthawed samples. DNA damage was determined by the comet assay after cryopreservation. Supplementation of 1 mM vitamin E to all extenders exhibited the best cryoprotective effect in terms of sperm motility, duration of motility, viability, fertility, and DNA integrity against cryopreservation damage, compared with 0.1, 0.5, and control group (0 mM) (p < 0.05). The highest post-thaw motility (62.4%-0.36%), fertilization (48.2-0.84), and the lowest DNA damage (7.245%) were obtained with the extender-II including 1.0 mM vitamin E (p < 0.05). Consequently, vitamin E positively affected the motility parameters, fertility, and DNA integrity, and the results suggest the addition of extenders with vitamin E as an antioxidant for the cryopreservation of brown trout sperm.

BACKGROUND: Cryopreservation of sperm from different fish species requires different protocols. Therefore, it is necessary to perform studies to establish reliable procedures for each species. OBJECTIVE: Experiments were designed to analyse the effect of different types of cryoprotectants on post-thaw motility, viability and fertility as well as cryoresistance of cryopreserved Nile tilapia (Oreochromis niloticus) sperm. MATERIALS AND METHODS: Sperm samples were diluted with an ionic extender containing glycerol (Gly), methanol (MeOH) and dimethyl sulfoxide (DMSO) at ratios of 5, 10 and 15 % respectively. Diluted samples were aspirated into 0.25 ml French straws and frozen 3 cm above the surface of liquid nitrogen (LN) in a styrofoam box and stored in a LN tank. DNA damage was evaluated with the comet assay technique following cryopreservation. RESULTS: Supplementation of extender with 10% glycerol gave the highest motility rate compared with the other cryoprotectant groups (P<0.05). Differences in terms of post-thaw motility duration, cell viability and fertilization rates were not significant among treatments (P>0.05). Although Gly gave the best score (5.0 ± 0.1%, P>0.05) at the concentration of 10%, 5% Me2SO caused significant DNA damage (15.0 ± 1.0%, P<0.05) with the comet test. CONCLUSION: Gly or MeOH are more suitable cryoprotectants than DMSO for the cryopreservation of Nile tilapia sperm.

Effect of different cryoprotectants on acid/bile tolerance, β-galactosidase activity, and cholesterol assimilation properties of freeze dried Pediococcus acidilactici, Lactobacillus plantarum, and Lactobacillus salivarius were evaluated. The retention of probiotic properties and viability of the above three cultures freeze dried with lactose, skimmed milk, and maltodextrin were found to be significant (P ≤ 0.05). In another approach, lactic acid bacteria (LAB)

This paper describes the main thermodynamic concepts related to the construction of supplemented phase (or state) diagrams (SPDs) for aqueous solutions containing vitrify- ing agents used in the cryo- and dehydro-preservation of natural (foods, seeds, etc.) and syn- thetic (pharmaceuticals) products. It also reviews the empirical and theoretical equations em- ployed to predict equilibrium transitions (ice freezing, solute solubility) and

To date, traditional cryopreservation techniques have not been amenable to zebrafish (Danio rerio) embryos, due in part to their large yolky eggs, which have a low surface to volume ratio and limited permeability to water and cryoprotectants. However, recent vitrification and ultra-rapid warming studies in mice have demonstrated successful preservation by dehydrating 85% or more of their total water content. We hypothesized that this approach may help overcome the barriers to embryo cryopresrvation among D. rerio. The purpose of this study was to determine the osmotic tolerance limit of D. rerio embryos under conditions relevant to cryopreservation. We found that embryos undergoing gastrulation (30%-70% epiboly) were particularly sensitive to osmotic dehydration/rehydration. By contrast, a subset of embryos dehydrated during or after segmentation (20-22 somite, prim 5) survived 3 hours in a 2 M sucrose solution but exhibit developmental delay, edema and trunk necrosis 2-4 days post-treatment.

The study investigated the effects of internal (DMSO, 1,2-propanediol, glycerol, ethylene glycol, methanol, N,N-dimethylacetamide) and external cryoprotectants (glucose, sucrose) on the viability and on morphometric parameters of zebrafish embryos. From the tested internal cryoprotectants, DMSO had the lowest toxicity, followed by 1,2-propanediol and glycerol. The external cryoprotectants were less toxic then the internal ones. Early ontogenetic stages were more sensible to cryoprotectant exposure than advanced stages. Two-step incubation procedures in increasing concentrations of internal and external cryoprotectants were superior to multiple-step exposure procedures. All tested vitrification solutions exceeded the tolerance limit of embryos. The tolerance of zebrafish embryos to cryoprotectants was highly variable in a concentration range causing approximately 50% embryo mortality. The width of the perivitelline space showed significant morphometrical changes due to cryoprotectant exposure. In the germinative tissue non-significant changes occurred. The yolk did not change morphometrically after exposure to internal cryoprotectants and showed no sign of dehydration after exposure to external cryoprotectants. Based on these results the study comes to the following conclusions: as yolk dehydration was impossible and as vitrification solutions were over the tolerance limit it seems unlikely that successful vitrification of zebrafish embryos can be achieved. Under these considerations slow freezing methods would be a better option as lower cryoprotectant concentrations can be used and embryos can be dehydrated during freezing.

Background: The risk of multiple pregnancies, often present in programs of In Vitro Fertilization (IVF), is an important force for embryo cryopreservation. On the other hand, ethical restriction and assurance of potential fertility following chemo/radio therapy has led scientists to focus on female gamete preservation. Objective: Optimizing vitrification protocol by using less concentrated cryoprotectants (CPAs) in order to decrease CPAs toxicity. Materials and Methods: Mouse Metaphase-II (M-II) oocytes and four cell-stage embryos were collected. Oocytes Survival, Fertilization and Developmental Rates (SRs, FRs, DRs) were recorded after cryotop-vitrification/warming. As well as comparing fresh oocytes and embryos, the data obtained from experimental groups (exp.) applying 1.25, 1.0, 0.75 molar (M) CPAs were analyzed in comparison to those of adopting 1.5 M CPAs [largely-used concentration of Ethylen Glycol (EG) and Dimethyl-sulphoxide (DMSO)]. Results: The data of oocytes exposed to...

Ciência Rural, Santa Maria, v.38, n.7, p.1972-1977, out, 2008 ISSN 0103-8478 ... Paula Barros TerracianoI,IV* Ivan Cunha Bustamante-FilhoI, II Ludmila do Vale MiquelitoI Tamarini Rodrigues ArlasIII,IV Fabiana CastroIII Rodrigo Costa MattosIII,IV Eduardo Pandolfi PassosI Eneder ...