Effect of Penicillium roqueforti and incorporation of whey cheese on volatile profiles and sensory characteristics of mould‐ripened Civil cheese

In this study, four different types of mould‐ripened Civil cheese were manufactured. A defined (nontoxigenic) strain of a Penicillium roqueforti (SC 509) was used as secondary starter for the manufacture of mould‐ripened Civil cheese with and without addition of the whey cheese Lor; in parallel, secondary starter‐free counterparts were manufactured. A total of 83 compounds were identified. Ketones, alcohols and esters were the principal classes of volatile components. Principal component analysis of the headspace volatiles grouped cheeses by age and type. P. roqueforti inoculated cheese was clearly separated from the other cheeses at 180 days of ripening, and these cheeses were characterised with high levels of ketones (e.g., 2‐butanone, 2‐heptanone). Differences in the panel scores between the cheese samples were not significant during the first stage of ripening (up to 60 days); as ripening proceeded, these differences were become evident and P. roqueforti inoculated cheeses received higher scores than others. Addition of Lor in the manufacture of mould‐ripened Civil cheese caused lower points by the sensory panel, and the cheese inoculated with P. roqueforti and Lor‐free was the best type of mould‐ripened Civil cheese. The results showed that the use of P. roqueforti in the manufacture of mould‐ripened Civil cheese has significant impact on the volatile profiles and sensory attributes.     

Effects of Penicillium roqueforti and whey cheese on gross composition,microbiology and proteolysis of mould‐ripened Civil cheese during ripening

Four different types of mould‐ripened Civil cheese were manufactured. A defined (nontoxigenic) strain of a Penicillium roqueforti (SC 509) was used as the secondary starter with and without addition of the whey cheese (Lor); in parallel, secondary starter‐free counterparts were manufactured. Chemical composition, microbiology and proteolysis were studied during the ripening. The incorporation of whey cheese in the manufacture of mould‐ripened Civil cheese altered the gross composition and adversely affected proteolysis in the cheeses. The inoculated P. roqueforti moulds appeared to grow slowly on those cheeses, and little proteolysis was evident in all cheese treatments during the first 90 days of ripening. However, sharp increases in the soluble nitrogen fractions were observed in all cheeses after 90 days. Microbiological analysis showed that the microbial counts in the cheeses were at high levels at the beginning of ripening, while their counts decreased approximately 1–2 log cfu/g towards the end of ripening.     

Ultrasonic-assisted phosphate curing: a novel approach to improve curing rate and chicken meat quality

The effects of ultrasound-assisted phosphate curing (UPC), phosphate curing (PC), and deionised water curing (DC) on the quality of chicken breast meat were investigated. The analysis of tissue sections, low-field nuclear magnetic resonance (LF-NMR), and headspace solid-phase microextraction gas-phase mass spectrometry (HS-SPME-GC-MS) of water distribution, myofibrillary structure, and volatile components of the meat samples were performed to reveal the mechanism. Also, the curing rate, water holding capacity (WHC), and shear force of meat were assessed. The results revealed that ultrasonic-assisted salting improved the meat salting rate, WHC, and tenderness by mechanically destroying the muscle fibre structure, increasing the space between the muscle fibre bundles, promoting water migration and uniform distribution, and reducing water freedom in the treated samples. In addition, HS-SPME-GC-MS analysis revealed that PC significantly reduced the content of volatile flavour components in the chicken breast meat compared with DC (P < 0.05); however, the combination treatment with PC and ultrasonic curing could reconstruct the flavour loss caused by phosphate. Thus, our cumulative results suggest that the ultrasonic technology may effectively assist the processing technology in improving the quality of meat and reconstructing the flavour loss in meat products.     

Variations of volatile flavour compounds in Cordyceps militaris chicken soup after enzymolysis pretreatment by SPME combined with GC-MS,GC × GC-TOF MS and GC-IMS

Cordyceps militaris chicken soup has always been one of the most popular Chinese-style soups due to its high nutritional value and special flavour. In the present work, C. militaris chicken soup was stewed after enzymolysis pretreatment. Several advanced techniques including GC-MS, GC × GC-TOF MS and GC-IMS were utilised to investigate the volatiles of as-prepared C. militaris chicken soup. GC-MS and GC  ×GC-TOF MS analysis indicated that contents and kinds of the main compounds especially aldehydes and esters which have fatty and sweet aroma in C. militaris chicken soup were increased after enzymolysis process. GC-IMS results demonstrated that some volatile substances in chicken soup disappeared with the addition of C. militaris and concentrations of some volatile substances were increased after enzymolysis process. Enzymolysis pretreatment was confirmed to be an effective way to enhance the soluble protein dissolution rate and volatile flavour compounds of C. militaris chicken soup.     

Effect of high-pressure treatment on the quality of prepared chicken breast

In this study, the effect of high-pressure processing (HPP) on the physico-chemical properties and microstructure of prepared chicken breast meat products was investigated. The result showed that the pH and TBARS values of the prepared chicken breast gradually increased, while the total volatile basic nitrogen value constantly decreased with the increase of pressure (0.1–500 MPa). In addition, the colour attributes (L* and a* values) and texture indexes (hardness and chewiness) significantly improved as the pressure increased. The results of low-field nuclear magnetic resonance demonstrated that the peak area of immobilised water (T₂₂) first enhanced and then declined with the increase of pressure, reaching the maximum value at 200 MPa. Moreover, the integrity of microstructure and protein secondary structure of prepared chicken breast were also disrupted. These findings could serve as a theoretical basis for a better application of HPP in prepared chicken breast meat products.     

Effect of Penicillium chrysogenum and Debaryomyces hansenii on the volatile compounds during controlled ripening of pork loins

During ripening of meat products such as dry-cured ham, the moulds and yeasts that proliferate on the surface may contribute to flavour development. However, their contribution to volatile components of dry-cured meat products is not known. One strain each of Penicillium chrysogenum and Debaryomyces hansenii, selected from dry-cured ham by their proteolytic activity, were tested to determine their effect on the volatile compounds during ripening. Sterile pork loins were inoculated and ripened for 106 days. Volatile compounds collected with a Solid Phase Micro-Extraction (SPME) fibre were analysed by GC/MS. Inoculation of pork loins with P. chrysogenum lead to a decrease in compounds attributed to lipid oxidation and to an increase of compounds derived from free amino acids. Inoculation with D. hansenii seemed to favour the formation of complex alcohols.     

武定鸡加工过程理化指标与特征风味变化研究

目的:研究云南武定鸡加工过程理化指标及挥发性风味物质差异,并对不同加工阶段水分和挥发性风味化合物的变化进行分析,为科学地控制云南武定鸡的加工过程提供依据。方法:测定了武定鸡加工过程中水分含量、色泽、剪切力、pH、脂肪含量和蛋白质含量的变化,并利用核磁共振(NMR)分析武定鸡原料肉、卤煮1 h、油炸1 min、及卤煮2 h四个加工工艺点中水分迁移的变化,同时利用气相-离子迁移谱(GC-IMS)技术测定不同加工阶段挥发性风味化合物的差异。结果:加工过程中,鸡肉的色泽、剪切力值、pH、蛋白质含量均呈上升趋势,在油炸阶段,肉样中脂肪含量最高。武定鸡经煮制加工后水分含量下降11.54%(P<0.05);不易流动水含量呈逐渐降低的趋势;与卤煮1 h阶段相比,自由水在油炸及卤煮2 h阶段分别增加了0.75%(P<0.05)与1.17%(P<0.05)。GC-IMS结果显示在卤制武定鸡的不同加工阶段挥发性风味物质差异明显,种类不断增加,苯甲醛和戊醛等化合物浓度逐渐提高,正己醇和丙醇等少数化合物浓度呈现降低的趋势。不同加工阶段挥发性风味化合物差异明显,武定鸡经加工后产生的挥发性风味化合物浓度更高。结论:武定鸡加工过程水分含量不断降低,水分状态不断变化,挥发性风味化合物浓度有所增加。综合各项理化指标来看,卤煮后的武定鸡不仅风味更佳,营养价值也有所提高。     

The development of free amino acids and volatile compounds in cheese ‘Oloumoucké tvarůžky’ (PGI) during ripening

Twenty‐two free amino acid (FAA) concentrations were observed during manufacture (7 days) and ripening period (42 days follow‐up) of Olomoucké tvar??ky (PGI, smear acid cheese). During the ripening period, the amounts of volatile compounds and selected sensory attributes were also analysed. The free amino acids were determined by means of ion‐exchange chromatography, and the volatile compounds were detected and identified using HS‐SPME coupled with GC/MS method. The development of the individual FAA content positively correlated with the ripening period (r = 0.7734–0.9229; P < 0.01). Forty‐six volatile compounds (14 alcohols; 10 esters; 7 ketones; 5 acids; 4 aldehydes; 3 sulphur compounds; 2 phenol compounds; and 1 terpene) were identified. Concentrations of several compounds increased (e.g. 3‐methylbutanal, 2‐butanol, 2‐butanone; P < 0.05) or decreased (e.g. dimethyldisulphide, methanethiol, 2‐phenylethylacetate, methylbutyric acid; P < 0.05) over cheese ageing. The results gave information about the development of sensory active substances and its precursors in Olomoucké tvar??ky during ripening. In conclusion, we found that free amino acid concentrations and sulphur compounds are positive with improved flavour.     

Chicken breast quality – normal,pale, soft and exudative (PSE) and woody – influences the functional properties of meat batters

Three classes (normal, PSE‐like and woody, thirty for per class) of fresh chicken breast meats were selected from a Chinese processing plant. Then, three classes of chicken breast meats were used to prepare meat batters and meatballs. This study investigated the effects of three classes of chicken breast meat on the physicochemical properties, water distribution, protein secondary structures and microstructures of meatballs. PSE‐like and woody meatballs both had lower water holding capacity and textural properties than normal meatballs. Furthermore, the free water mobility and proportion in PSE‐like and woody meatballs were increased (P < 0.05). According to near‐infrared spectroscopy results, the intensity of the 1940‐nm bands of PSE‐like and woody classes both increased. Both PSE‐like and woody meatballs formed more aggregated gel matrices than normal class. PSE‐like and woody classes had higher α‐helix and lower β‐structure contents (P < 0.05). Overall, compared with normal meatballs, PSE‐like and woody meatballs showed inferior functional properties.     

Differentiation of yeasts growing on dry-cured Iberian ham by mitochondrial DNA restriction analysis, RAPD-PCR and their volatile compounds production

The efficiency of mitochondrial DNA (mtDNA) restriction analysis, RAPD-PCR and volatile compounds analysis to differentiate yeast biotypes involved in flavour development of dry-cured Iberian ham throughout the ripening process is evaluated. For this purpose, 86 yeasts isolated from Iberian hams in the main ripening stages at different industries of the four Protected Designations of Origin of this product, were used. The combination of mtDNA restriction analysis and RAPD-PCR using the primer (GACA)₄ showed a higher variability in the yeast species detected than obtained using only mtDNA restriction analysis. Only two species, Debaryomyces hansenii and Candida zeylanoides, were identified throughout the whole ripening process and a wide diversity of biotypes was found in these two species, with those of D. hansenii predominating. Clear differences between biotypes were detected in the generation of volatile compounds, with the biotype C2-2 of D. hansenii showing the highest concentrations of volatiles. The combined use of mtDNA restriction analysis and RAPD-PCR distinguishes yeast biotypes with different production of volatile compounds. In addition, analysis of the production profile of volatile compounds is needed to differentiate yeast strains of the same biotype recovered at different stages of ripening. Thus, the combination of these three methods could be very useful to select or monitor yeasts as starter cultures in dry-cured meat products.     

Chemical and microbiological parameters and sensory attributes of a typical Sicilian salami ripened in different conditions

A study was carried out on a typical Sicilian salami prepared from meat of the local Nero Siciliano pig in order to characterize this typical product. One formulation of salami was divided in two batches and ripened in two different environments, a traditional sicilian room (TR) and a controlled industrial ripening room (RR). Microbiological and physico-chemical analysis were performed on raw mixture and after 7 and 90 days of ripening. Sensory analysis was carried out on salami at the end of ripening, and flavour compounds were extracted by simultaneous distillation–extraction and analysed by gas chromatography/mass spectrometry. Commercial salami prepared from meat from white pig were purchased locally and used as comparative samples. The experimental salami at the end of ripening was characterized by a high level of fat and low level of moisture. Fatty acid analysis showed that experimental salami contained a higher percentage of oleic acid, vaccenic acid and palmitic acid and a lower percentage of stearic acid and linoleic acid, when compared to commercial salami (P<0.05). No significant differences were found in fatty acid composition of the experimental salami between the two types of ripening. Instrumental analysis of flavour volatile compounds in the experimental salami demonstrated that traditionally ripened salami contained the most volatiles, especially aldehydes (8217 vs. 3104 ng g⁻¹, P<0.05). Sensory analysis showed no significant differences as a consequence of different ripening conditions for firmness, saltiness, acidity, cohesiveness and elasticity. In contrast, there were significant differences for hardness and rancidity, which were higher in TR salami compared with RR and commercial salami. Lactic acid bacteria and Micrococcaceae counts were higher in controlled ripened salami although the hygienic quality of both products was satisfactory. The use of a controlled room for the ripening of this typical salami seems to be a potential technological improvement to maintain an all year around production of this salami, that otherwise cannot be produced in the summer period due to the higher environmental temperatures. However, the non traditionally ripened product showed some chemical differences that were not evidenced by sensory analysis.     

Influence of exopolysaccharide‐producing cultures on the volatile profile and sensory quality of low‐fat Tulum cheese during ripening

The objective of this investigation was to compare the composition and changes in the concentration of volatiles in low‐fat and full‐fat Tulum cheeses during ripening. Tulum cheese was manufactured from low‐ or full‐fat milk using exopolysaccharide (EPS)‐producing or non‐EPS‐producing starter cultures. A total of 82 volatile compounds were identified belonging to the following chemical groups: acids (seven), esters (21), ketones (14), aldehydes (six), alcohols (14) and miscellaneous compounds (20). The relative amounts of acids, alcohols and aldehydes increased in the cheeses made with EPS‐producing cultures during 90 days of ripening. Differences were found in the volatile profile of full‐fat Tulum cheese compared with the low‐fat variant, especially after 90 days of ripening. Exopolysaccharide‐producing cultures changed the volatile profile, and the EPS‐producing cultures including Streptococcus thermophilus + Lactobacillus delbrueckii subsp. bulgaricus + Lactobacillus helveticus (LF‐EPS2) produced cheese with higher levels of methyl ketones and aldehydes than the non‐EPS cultures. In the sensory analysis, full‐fat Tulum cheeses and the cheese produced with the EPS‐producing culture containing Lb. helveticus (LF‐EPS2) were preferred by the expert panel. It was concluded that the use of EPS‐producing starter cultures in the manufacture of low‐fat Tulum cheese had the potential to improve the flavour.     

Influence of ripening temperature on the volatiles profile and flavour of Cheddar cheese made from raw or pasteurised milk

Cheddar cheeses were made from raw (R1, R8) or pasteurised (P1, P8) milk and ripened at 1°C (P1, R1) or 8°C (P8, R8). Volatile compounds were extracted from 6 month-old cheeses and analysed, identified and quantified by gas chromatography-mass-spectrometry. A detailed sensory analysis of the cheeses was performed after 4 and 6 months of ripening. The R8 cheeses had the highest and P1 the lowest concentrations of most of the volatile compounds quantified (fatty acids, ketones, aldehydes, esters, alcohols, lactones and methional). The R8 and P8 cheeses contained higher levels of most of the volatiles than R1 and P1 cheeses. Ripening temperature and type of milk influenced most of the flavour and aroma attributes. Principal component analysis (PCA) of aroma and flavour attributes showed that P1 and R1 had similar aroma and flavour profiles, while R8 had the highest aroma and flavour intensities, highest acid aroma and sour flavour. The age of cheeses influenced the perception of creamy/milky and pungent aromas. PCA of the texture attributes separated cheeses on the basis of ripening temperature. The R8 and P8 cheeses received significantly higher scores for perceived maturity than P1 and R1 cheeses. The P1 and R1 cheeses had similar values for perceived maturity. In a related study, it was found that concentrations of amino acids and fatty acids were similar in R1 and P1 during most of the ripening period, and R1 and P1 cheeses had low numbers of non-starter lactic acid bacteria (NSLAB). The panel found that ripening temperature, type of milk and age of cheeses did not influence the acceptability of cheese. It is concluded that NSLAB contribute to the formation of volatile compounds and affect the aroma and flavour profiles and the perceived maturity of Cheddar cheese.     

Generation of non-protein nitrogen and volatile compounds by Penicillium chrysogenum Pg222 activity on pork myofibrillar proteins

A non-toxigenic strain of proteolytic Penicillium chrysogenum Pg222 isolated from dry-cured ham was tested for its ability to generate non-protein nitrogen (NPN) and volatile compounds from muscle myofibrillar proteins. The activity of mold led to higher accumulation of non-protein and amino acidic nitrogens than control samples. Volatile compound analysis revealed the presence of branched compounds, such as 3-methylbutanal and 3-methylbutanol only in samples inoculated with the mold. Similarly, compounds such as ethanol, propanol and 2-methoxy ethanol were detected only in inoculated samples at all sampling time. 3- and 2-methylpentane, benzoic and acetic acids, 2-butanone and 2-ethylhexanol, pyridine and 3-carene were detected occasionally, but only in the Pg222 batch. The proteolytic activity of P. chrysogenum Pg222 lead to accumulation of soluble NPN compounds, in addition to the generation of volatile compounds of great interest for dry-cured meat products. Therefore, this mold could be appropriate to be used as a non-toxigenic starter culture during the ripening of dry-cured meat products to stimulate proteolysis and flavour development.     

Potent odorants formed by lipid peroxidation as indicators of the warmed-over flavour (WOF) of cooked meat

Hexanal (I), 1-octen-3-one (II), (E,E)-2,4-nonadienal (III), (E,E)-2,4-decadienal (IV) andtrans-4,5-epoxy-(E)-2-decenal (V) were quantified in cooked meat (beef, pork, chicken) in which the warmed-over flavour (WOF) was observed after storage for 2 days at 4°C. Odour activity values of the compounds on the basis of odour threshold values in water revealed that compounds I and V contributed most strongly to WOF. During the storage, free iron but not metmyoglobin catalysed the increase of compounds I, II, IV and V in models containing the lipids of beef meat.     

Analysis of volatile compounds in L. edodes blanched by hot water and microwave

The comprehensive flavour characterisation and volatile compounds of raw L. edodes, hot water blanching (HB) sample and microwave blanching (MB) sample were comparatively analysed by electronic nose technology and headspace solid‐phase micro‐extraction combined with gas chromatography‐mass spectrometry (HS‐SPME‐GC‐MS). Results indicated that volatile components in L. edodes markedly changed after HB and MB. Volatile compounds of raw L. edodes consisted mainly ketones, sulphide and alcohols, and 3‐octanone, as well as 1‐octen‐3‐one, were the major compounds. The content of ketones and sulphides in blanched samples markedly decreased, while the relative content of aldehydes, hydrocarbons and esters increased, which became the major volatile compounds of treatment samples. In addition, the percentage contents of esters, alcohols and sulphides in MB samples were significantly (P < 0.05) higher than that in HB samples, especially 1‐octen‐3‐ol, which contributes more to mushroom flavour. Therefore, MB is a better pretreatment method of L. edodes processing and cooking according to the results of experiment.     

Effect of ripening temperature on the growth and significance of non-starter lactic acid bacteria in Cheddar cheese made from raw or pasteurised milk

Two cheese-making trials were conducted, each involving four cheeses, two made from raw milk (R1, R8) and two from pasteurised milk (P1, P8), and ripened at 1°C (R1, P1) or 8°C (R8, P8). The 1-day-old R1 and R8 cheese in trials 1 and 2 contained ∼10⁴ non-starter lactic acid bacteria (NSLAB) g⁻¹. In trial 1, no NSLAB were detected in 1-day-old P1 and P8 cheeses while those in trial 2 contained 10² cfu g⁻¹. In both trials, the maximum differences between the number of NSLAB in the cheeses ripened at 1 or 8°C were observed at 4 months, when the number of NSLAB in cheeses ripened at 8°C were 3 log cycles higher than in those ripened at 1°C. At the end of ripening (6-months), the number of NSLAB in P8 and R8 were ∼2 log cycles higher than in P1 and R1 cheeses, respectively. Primary proteolysis in the cheeses was markedly affected by ripening temperature, but not by pasteurisation of the cheese milk. Urea-polyacyrlamide gel electrophoretograms and reverse-phase (RP)-HPLC of the water-soluble fraction showed differences between cheeses made from raw or pasteurised milk and between cheeses ripened at 1 or 8°C. The concentration of amino acids and fatty acids were in the order R8>P8>R1>P1. Commercial graders awarded highest flavour scores to the R1 cheeses during gradings at 4, 5 and 6 months. A sensory panel found that most flavour and aroma attributes and maturity were in the order of R8>P8>R1=P1. The results of this study suggest that NSLAB play an important role in the development of flavour in Cheddar cheese by contributing to the production of amino acids and fatty acids.     

Mate (<Emphasis Type="Italic">Ilex paraguariensis</Emphasis>) as dietary additive for broilers: performance and oxidative stability of meat

Aqueous extract of mate (dried leaves of Ilex paraguariensis) added to drinking water for broilers for the last 14 days prior to slaughter did not affect performance at 25 days of age, but improved oxidative stability of the chicken meat. Oxidative stability of precooked breast meat made from control meat (CON) and from meat of broilers raised on water with mate added was investigated during chill storage for up to 7 days. The use of mate showed no influence on the content of lipids in chicken breast meat; however, lipid oxidation measured as thiobarbituric acid–reactive substances (TBARS) was significantly lower for meat from broilers raised on water with mate extracts in different concentrations (MA0.1, MA0.5, and MA1.0 corresponding to 0.1, 0.5, and 1.0% of mate dried leaves). The relative effect was largest at 1 day of storage with more than 50% reduction on TBARS; the result was still significant after 3 days, but almost vanished after 7 days, when oxidative rancidity was very high in all samples. In meat from broilers raised on water with mate extract, vitamin E was protected during cooking, although in the very rancid meat balls at 7 days of storage, the protection almost disappeared. Nevertheless, mate can be an interesting natural alternative to be used in chicken diets to improve lipid stability of the meat.     

Effect of high-pressure treatment on microbiology,proteolysis, lipolysis and levels of flavour compounds in mature blue-veined cheese

The effect of high-pressure (HP) treatment (400 MPa, 600 MPa) on ripening of mature 42-day-old Irish blue-veined cheese was studied. Counts of non-starter lactic acid bacteria, lactococci, yeasts, moulds, enterococci and total aerobic bacteria significantly decreased due to HP, with moulds being most sensitive and 600 MPa the most effective treatment. The levels of pH 4.6-soluble nitrogen and (12%) trichloroacetic acid-soluble nitrogen increased immediately after both HP treatments; however, after 28 days of storage, values were lower in HP-treated cheeses than in the control cheese. Urea-polyacrylamide gel electrophoresis showed increased breakdown of β-casein due to HP treatment at both 400 MPa and 600 MPa. Levels of free fatty acids were lower in HP-treated cheese than in the control, but not significantly so, and no significant changes could be observed in the level of flavour compounds of blue-veined cheese. Overall, HP treatment of blue-veined cheese reduced microbiological activity and decelerated proteolysis, with no statistically significant effects on development of flavour compounds.Industrial relevanceHigh-pressure treatment has been studied for the past 100 years; nevertheless, it was not applied in dairy industry, until recently, for a cheese spread. In this study, HP-induced inactivation of microbes and enzymes, which could arrest the ripening of high-quality mature (i.e., ripened) Irish farmhouse blue-veined cheese and thus extend shelf-life at optimal quality, was examined.     

Effects of different thawing methods on the quality of chicken breast

Freezing and thawing technologies are widely used in meat industry. This study aimed to investigate the effects of freezing and different thawing methods (Control group, CG; water immersing thawing, WT; low temperature combined with high‐humidity thawing, LT; combined thawing 1 (WT 15 min, then LT), CT1; combined thawing 2 (LT 2 h, then WT), CT2) on the quality of chicken breast. The results showed that all thawing methods significantly increased shear force value, the gap between muscle fibres, the contents of malondialdehyde (MDA) and carbonyl compounds, meanwhile, decreased the pH, CIE a* value, total protein solubility and sarcoplasmic protein solubility, compared with CG (P < 0.05). Among different thawing methods, LT was most effective in decreasing thawing loss but it had the highest contents of MDA and carbonyl compounds (P < 0.05). CT1 significantly decreased shear force value and MDA content (P < 0.05). Overall, freezing and thawing process had a detrimental effect on the quality of chicken breast, and the extent was pronounced with different thawing methods. On the basis of shortening thawing time and keeping meat quality, the results of this study suggested that CT1 could be chosen as the better thawing method.