Cryo-storage of porcine hides at the industrial scale for tissue engineering and regenerative medicine application.

BACKGROUND: The industrial scale cryo-storage of raw tissue materials requires a robust, low-cost and easy-to-operate method that can facilitate the down-stream process. OBJECTIVE: The study was aimed to develop the multifunctional protective solutions (MPS) for transportation at ambient conditions and also subsequent cryo-storage below -20 degree C of raw porcine hides for tissue engineering and regenerative medicine. MATERIALS AND METHODS: Protective solutions with antimicrobial activity and proteinase-inhibiting activity were developed and tested for its efficacy in preserving the extracellular matrix of porcine dermis from microbial spoilage, proteolytic degradation, freeze damage and excessive dehydration during shipping and cryo-storage. The MPSs contained phosphate-buffered saline with ethylene diamine tetra acetic acid (EDTA) added as chelator and proteinase inhibitor, as well as glycerol or maltodextrin (M180) as cryoprotectants. RESULTS: MPSs prepared with EDTA and glycerol or M180 had significant antimicrobial activity and proteinase-inhibiting activity during the period of shipping and handling. Glycerol and M180 prevented eutectic salt precipitation and excessive freeze dehydration upon cryo-storage of porcine hides. Without glycerol or M180, hides could be freeze-dehydrated to the low hydration at ~0.4 g/g dw, and formed irreversible plications after freezing. A critical hydration (0.8~0.9 g/g dw) was observed for the extracellular matrix of porcine dermis, and dehydration to a lower level could impose enormous stress and potential damage. The soaking of porcine hides in MPSs decreased water content as glycerol and M180 entered into dermis. Upon equilibration, the glycerol content in the tissue was about 94% of the incubating glycerol solution, but the M180 content in the tissue was only about 50% of the incubating M180 solution, indicating that M180 did not get into the entire aqueous domain within dermis. MPSs reduced ice formation and increased the unfrozen water content of porcine raw hides upon cryo-storage. CONCLUSION: MPSs prepared with EDTA and glycerol or M180 have antimicrobial activity and proteinase-inhibiting activity, which can be used for transportation and cryo-storage of raw hides at the industrial scale. Glycerol at 7.5% w/v and M180 at 20% w/v were sufficient to prevent freeze damage and excessive freeze dehydration. Doi.org/10.54680/fr24310110312.

Viscoelastic properties of decellularized and freeze-dried human dermis between i909c and 40cC.

BACKGROUND: Human donor skin is processed to make the acellular dermis matrix (ADM) for tissue repair and regeneration. There is no data on the viscoelastic properties of ADM at room and subzero temperatures. OBJECTIVE: The work evaluated the temperature dependence of viscoelastic properties of freeze-dried ADM. MATERIALS AND METHODS: Donor skin was de-epidermized, de-cellularized and freeze-dried with trehalose as the lyo-protectant. Glass transition of freeze-dried ADM was measured by differential scanning calorimeter (DSC), and viscoelastic properties were examined by dynamic mechanical analyzer (DMA). RESULTS: At the low moisture range (1.4 +/- 0.5%), the glass transition temperature (Tg) of freeze-dried ADM was 90 degree C to 100 degree C. As the moisture content increased, the Tg decreased steadily. At the high moisture range (10.8 +/- 2.9%), the Tg was 40 degree C to 60 degree C. There were large donor-to-donor variations in viscoelastic properties of freeze-dried ADM as demonstrated by the changes in storage modulus (G'), loss modulus (G") and damping factor tan delta (G"/G'). However, the trends of the temperature dependence for G', G" and tan delta were similar among all 8 donors. For each donor, changes in G' and G" were relatively small between -90 degree C and 40 degree C, and G' was at least one order of magnitude greater than G". Two viscoelastic relaxations were observed in freeze-dried ADM, one at -20 degree C and the other at -60 degree C respectively. CONCLUSION: Freeze-dried ADM was protected in the glassy carbohydrate matrix. DMA observed two viscoelastic relaxations (i.e., alpha process at -20 degree C and beta process at -60 degree C). Overall changes in G' and G'' of freeze-dried ADM were relatively small within one order of magnitude between -90 degree C and 40 degree C. https://doi.org/10.54680/fr24110110612.

Effect of antioxidant procyanidin b2 (pcb2) on ovine oocyte developmental potential in response to in vitro maturation (ivm) and vitrification stress.

BACKGROUND: It was demonstrated that external stress, such as in vitro maturation (IVM) and vitrification process can induce significantly reduced development capacity in oocytes. Previous studies indicated that antioxidants play a pivotal part in the acquisition of adaptation in changed conditions. At present, the role of the natural potent antioxidant PCB2 in response to IVM and vitrification during ovine oocyte manipulation has not been explored. OBJECTIVE: To investigate whether PCB2 treatment could improve the developmental potential of ovine oocytes under IVM and vitrification stimuli. MATERIALS AND METHODS: The experiment was divided into two parts. Firstly, the effect of PCB2 on the development of oocytes during IVM was evaluated. Un-supplemented and 5 ug per mL PCB2-supplemented in the IVM solution were considered as control and experimental groups (C + 5 ug per mL PCB2). The polar body extrusion (PBE) rate, mitochondrial membrane potential (MMP), ATP, reactive oxygen species (ROS) levels and early apoptosis of oocytes were measured after IVM. Secondly, we further determine whether PCB2 could improve oocyte quality under vitrification stress. The survival rate, PBE rate and early apoptosis of oocytes were compared between fresh group, vitrified group and 5 ug per mL PCB2-supplemented in the IVM solution after vitrification (V + 5 ug per mL PCB2). RESULTS: Compared to the control group, adding PCB2 significantly increased PBE rate (79.4% vs. 62.8%, P < 0.01) and MMP level (1.9 +/- 0.08 vs. 1.3 +/- 0.04, P < 0.01), and decreased ROS level (47.1 +/- 6.3 vs. 145.3 +/- 8.9, P < 0.01). However, there was no significant difference in ATP content and early apoptosis. Compared to the fresh group, vitrification significantly reduced oocytes viability (43.0% vs. 90.8%, P < 0.01) as well as PBE rate (24.2% vs. 60.6%, P < 0.05). However, 5 ug per mL PCB2-supplemention during maturation had no effect on survival, PBE or early apoptosis in vitrified oocytes. CONCLUSION: PCB2 could effectively antagonise the oxidative stress during IVM and promote oocyte development. DOI: 10.54680/fr23210110412.

Pregnancy of cryopreserved ovine embryos at different developmental stages.

BACKGROUND: Developmental stage and cryopreservation method have significant impact on the pregnancy rate after transfer of embryos produced in vivo. OBJECTIVE: To determine the pregnancy outcomes from ovine embryos cryopreserved at different developmental stages. MATERIALS AND METHODS: Embryos at different developmental stages were obtained from donor ewes through simultaneous estrus treatment and laparoscopic artificial insemination. Embryos, either cryopreserved via vitrification or slow freezing method, were implanted into recipient ewes. The pregnancy rate was determined 35 days after transfer. RESULTS: The pregnancy rate of developing embryos increases after transfer from the morula stage, early blastocyst to expanded blastocyst stages (64.9%, 73.9% and 81.3%, respectively). However, cryopreservation significantly decreases the pregnancy rate of embryos at all three developmental stages, and there is no significant difference among developmental stages (43.9%, 43.7%, 52.9%, respectively). There is also no significant difference in the pregnancy rate between slowly-frozen embryos and vitrified embryos. CONCLUSION: The pregnancy outcomes of embryo transfer is better at the expanded blastocyst stage than at earlier stages. However, no difference is observed in the pregnancy rate of embryos at different developmental stage after cryopreservation, either by slow freezing and vitrification. Cryopreservation methods for ovine embryos, both slow freezing and vitrification, need further improvement. doi.org/10.54680/fr22510110512.

PERSPECTIVE: Cryopreservation of Human Oocytes and the 'Carryover' Effect on Early Embryo Development.

Worldwide women are increasingly facing the issue of delayed child-bearing and fertility decline. Oocyte cryopreservation provides an option for fertility preservation, especially for women with diseases and other special needs to conceive babies later. In this review we examine the effect of oocyte cryopreservation on early development of human embryos. Databases (Medline, PubMed and Web of Science) were searched for relevant clinical studies published between 1999 and 2020. A total of 27 studies on oocyte cryopreservation and embryo development were identified, and data in those studies are retrieved for meta-analysis on the outcomes of oocyte survival, fertilization and early embryo development. In comparison to the slow freezing technique, vitrification yields significantly better oocyte survival (84.7% ± 0.6% vs 58.0% ± 0.5%), and subsequently higher rates of fertilization (65.5% ± 0.9% vs 40.0% ± 0.6%), cleavage (58.8% ± 0.9% vs 34.6% ± 0.8%), as well as embryo implantation (5.9% ± 0.3% vs 2.9% ± 0.2%). This analysis reveals a negative 'carryover' effect of oocyte cryopreservation on early development of embryos after oocyte fertilization (i.e., cleavage and implantation). This 'carryover' effect is greater for slowly-frozen oocytes than for vitrified oocytes, and may represent subtle functional or molecular alterations that are not severe enough to affect cell survival and fertilization, but sufficient to impair later development. The nature of the 'carryover' effect is unknown. Hypothermia, membrane ion channels, bioenergy metabolism and epigenetic modifications are likely involved. In conclusion, oocyte cryopreservation can negatively affect early development of human embryos. Future studies should go beyond oocyte survival and look further into the effects on epigenetic changes.

[Effects of intraoperative graft flow measurements on the early mid-term outcomes after off-pump coronary artery bypass grafting].

OBJECTIVE: To investigate and analyze the relationship between intraoperative graft flow measurements and the early mid-term outcomes after off-pump coronary artery bypass grafting (OPCAB). METHODS: Patients who underwent isolated OPCAB in the Department of Cardiac Surgery of Peking University People's Hospital from January 2013 to June 2016 were included. Perioperative characteristics, graft flow measurements and postoperative follow-up outcomes were retrospectively collected. Comparison was made between flow measurements of grafts and the early mid-term outcomes. Flow measurements of grafts included the mean flow (MF) and the pulsatility index (PI). The early outcomes included peri-operative myocardial infarction (PMI), use of an intra-aortic balloon pump (IABP), reoperation for all causes, new-onset atrial fibrillation and in-hospital or 30-day mortality. RESULTS: A total of 463 patients were included in the study. Mean age was (62.80±8.36) years, and 24.8% were females. The total number of grafts was 1 435, which averaged 3.10 grafts per patient. The MF and PI were separately (32.34±14.45) mL/min and 2.87±0.92. Of all the patients, 23(5%) had PMI, and 11 used IABP. Observed in-hospital or 30-day mortality was 0.86% (4 patients). Compared with non-PMI group, the MF was lower and the PI was higher in the PMI group (P<0.05). However, the differences of other early outcomes had no statistical significance between the PMI group and the non-PMI group. The lower MF (Wald=5.684, P=0.017, 95%CI: 0.894-0.989) and the higher PI (Wald=9.040, P=0.003, 95%CI: 1.252-2.903) were risk factors of PMI in multivariable Logistic regression modeling. The longest follow-up time was 37 months, and 7 patients died. The differences of graft flow measurements between the surviving group and the nonsurvivors had no statistical significance, but overall mid-term survival was lower in patients with poor left internal mammary artery (LIMA) to left anterior descending artery (LAD) graft flow (MF<10 mL/min; OR=9.6, P<0.05). CONCLUSION: Intraoperative graft flow parameters during OPCAB can predict the early mid-term outcomes. The lower MF and the higher PI should increase the rate of PMI. A lower flow of LIMA to LAD graft (<10 mL/min) should increase the rate of midterm mortality, but further research will be needed to confirm and explore the findings.

Associations between parent-reported sleep duration and adiposity in Chinese early adolescents.

BACKGROUND: Short sleep duration has recently been found to be associated with obesity in children, but findings involving adolescents have been less consistent, and some have mentioned gender differences. OBJECTIVES: To investigate the association between parent-reported sleep duration and adiposity in early adolescence (10-12 years old) and to explore gender differences within this population. METHODS: Participants were 1309 fifth-grade students (685 boys) from 10 primary schools in Shanghai, China. Body mass index (BMI), waist-height ratio (WHeR) and body fat percentage (BF%) were assessed. Sleep and other potential contributors were recorded by parents or self-reported. RESULTS: Compared with adolescents in the longest sleep group (greater than or equal to +1 SD, ≥10.05 h), those in the shortest sleep group (less than -1 SD, <8.89 h) had significantly higher BMI, WHeR and BF%. Sleep was found to be closely related to increased adiposity in girls who were in the shortest and shorter sleep group (

Effect of inorganic phosphate supplementation on egg production in Hy-Line Brown layers fed 2000 FTU/kg phytase.

Phytase has long been used to decrease the inorganic phosphorus (Pi) input in poultry diet. The current study was conducted to investigate the effects of Pi supplementation on laying performance, egg quality and phosphate-calcium metabolism in Hy-Line Brown laying hens fed phytase. Layers (n = 504, 29 weeks old) were randomly assigned to seven treatments with six replicates of 12 birds. The corn-soybean meal-based diet contained 0.12% non-phytate phosphorus (nPP), 3.8% calcium, 2415 IU/kg vitamin D3 and 2000 FTU/kg phytase. Inorganic phosphorus (in the form of mono-dicalcium phosphate) was added into the basal diet to construct seven experimental diets; the final dietary nPP levels were 0.12%, 0.17%, 0.22%, 0.27%, 0.32%, 0.37% and 0.42%. The feeding trial lasted 12 weeks (hens from 29 to 40 weeks of age). Laying performance (housed laying rate, egg weight, egg mass, daily feed intake and feed conversion ratio) was weekly calculated. Egg quality (egg shape index, shell strength, shell thickness, albumen height, yolk colour and Haugh units), serum parameters (calcium, phosphorus, parathyroid hormone, calcitonin and 1,25-dihydroxyvitamin D), tibia quality (breaking strength, and calcium, phosphorus and ash contents), intestinal gene expression (type IIb sodium-dependent phosphate cotransporter, NaPi-IIb) and phosphorus excretion were determined at the end of the trial. No differences were observed on laying performance, egg quality, serum parameters and tibia quality. Hens fed 0.17% nPP had increased (P < 0.01) duodenum NaPi-IIb expression compared to all other treatments. Phosphorus excretion linearly increased with an increase in dietary nPP (phosphorus excretion = 1.7916 × nPP + 0.2157; R2 = 0.9609, P = 0.001). In conclusion, corn-soybean meal-based diets containing 0.12% nPP, 3.8% calcium, 2415 IU/kg vitamin D3 and 2000 FTU/kg phytase would meet the requirements for egg production in Hy-Line Brown laying hens (29 to 40 weeks of age).

BLACAT1 is negatively associated with prognosis in patients with NSCLC and inhibits cell progression, metastasis and epithelial-mesenchymal transition through down-regulating Wnt/ß-catenin signaling pathway.

OBJECTIVE: To evaluate the clinical significance and molecular mechanism of bladder cancer-associated transcript 1 (BLACAT1) in non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: Overall, 156 NSCLC cancer patients were recruited and divided into high and low BLACAT1 level group by the median value of BLACAT1 expression. The associations of BLACAT1 expression with the clinicopathological features and prognosis were evaluated. A series of in vitro assays were performed to explore the role of BLACAT1 on NSCLC progression and metastasis. RESULTS: Patients with high BLACAT1 expression had shorter overall survival and progression-free survival than those with low BLACAT1 expression. Multivariate analyses showed that BLACAT1 was an independent prognostic factor of survival in NSCLC patients. In vitro assays showed that the downregulation of BLACAT1 significantly suppressed cell progression, migration, and invasion. The epithelial-mesenchymal transition was also inhibited when BLACAT1 was silenced, indicated by an increase in E-cadherin expression and a decrease in vimentin expression by mediating Wnt/ß-catenin signaling pathway. CONCLUSIONS: BLACAT1 should be a potential prognostic biomarker and therapeutic target for NSCLC.

[Investigation of dose-dependent association between bedtime routines and sleep outcomes in infants and toddlers].

Objective: To investigate the current bedtime routine among Chinese children less than 3 years of age and explore its dose-dependent association with sleep duration and sleep quality. Method: Healthy full-term born children aged 0-35 months were selected by stratified cluster random sampling method from 8 provinces in China following the "Hospital of Province-City-County" sampling technical route during 2012-2013.Brief Infant Sleep Questionnaire(BISQ) was used to assess sleep conditions of these children.Children's personal and family information was obtained by Shanghai Children's Medical Center Socio-demographic Questionnaire.Both of these questionnaires were filled in by parents. The effects of bedtime routine on children's sleep duration and quality were analyzed by multivariate analysis of variance. Result: The children's average age was(12±10) months(n=1 304), of whom 689 were males (52.8%, 689/1 304). There were 48.5%(632/1 304)of the parents reported that their children had not established regular sleep routines. There was a consistent dose-dependent association between bedtime routine and sleep duration, as well as other indicators for sleep quality (all P<0.05). The more regular the sleep routines, the longer the sleep duration, the earlier the children went to sleep, the shorter the sleep onset latency, the fewer the nighttime wakeup and the shorter the nighttime waking.The nighttime sleep duration was significantly longer for those with a bedtime routine 'every night' than those who 'never' had a bedtime routine (9.5(95%CI: 9.4-9.6)vs. 8.9(95%CI: 8.6-9.3)h, t=3.345, P=0.001). Compared with children who never had bedtime routines, children with regular bedtime routines had fewer night wakeup (1.3(95%CI: 1.2-1.4) vs. 2.4( 95%CI: 2.0-2.9), t=3.182, P=0.001) and shorter night waking duration(16.6(95%CI: 14.6-18.8) vs. 59.2 (95%CI: 47.0-72.7)min, t=6.383, P<0.01). Conclusion: The percentage of children who have established regular bedtime routine is low in China. There is significant dose-dependent association between regular bedtime routine and sleep outcomes, especially sleep quality. The more regular the sleep routines, the better the sleep quality.

Protein inactivation in amorphous sucrose and trehalose matrices: effects of phase separation and crystallization.

Trehalose is the most effective carbohydrate in preserving the structure and function of biological systems during dehydration and subsequent storage. We have studied the kinetics of protein inactivation in amorphous glucose/sucrose (1:10, w/w) and glucose/trehalose (1:10, w/w) systems, and examined the relationship between protein preservation, phase separation and crystallization during dry storage. The glucose/trehalose system preserved glucose-6-phosphate dehydrogenase better than did the glucose/sucrose system with the same glass transition temperature (Tg). The Williams-Landel-Ferry kinetic analysis indicated that the superiority of the glucose/trehalose system over the glucose/sucrose system was possibly associated with a low free volume and a low free volume expansion at temperatures above the Tg. Phase separation and crystallization during storage were studied using differential scanning calorimetry, and three separate domains were identified in stored samples (i.e., sugar crystals, glucose-rich and disaccharide-rich amorphous domains). Phase separation and crystallization were significantly retarded in the glucose/trehalose system. Our data suggest that the superior stability of the trehalose system is associated with several properties of the trehalose glass, including low free volume, restricted molecular mobility and the ability to resist phase separation and crystallization during storage.

Protein stability in the amorphous carbohydrate matrix: relevance to anhydrobiosis.

The formation of intracellular glass is proposed to be relevant to protein stabilization and survival of anhydrobiotic organisms in the dry state. The stability of proteins in the amorphous carbohydrate matrix and its relevance to seed survival have been investigated in the present study. Glucose-6-phosphate dehydrogenase (G6PDH) was preserved in the amorphous glucose/sucrose (1:10, w/w) matrix by freeze-drying. The stability of freeze-dried G6PDH was examined at temperatures above and below the glass transition temperature (Tg). The rate of G6PDH inactivation in the amorphous carbohydrate matrix deviated significantly from the Arrhenius kinetics, and conformed to the Williams-Landel-Ferry (WLF) relationship. The temperature dependence of G6PDH inactivation in two sets of samples with different Tg values was compared. Identical temperature dependence of G6PDH inactivation was observed after temperature normalization by (T-Tg). Seed survival of Vigna radiata Wilczek (mung bean) showed a similar WLF kinetics at storage temperatures T > or = Tg. In situ protein stability in mung bean embryonic axes was studied using differential scanning calorimetry (DSC). Thermal stability of seed proteins exhibited a strong dependence on the Tg of intracellular glass. These results indicate an important role of the glassy state in protein stabilization. Our data suggest an association between protein stability in intracellular glass and seed survival during storage.

Studies of the genotoxicity of glycidyl methacrylate (GMA).

The following experiments were conducted to evaluate the genotoxic effects of GMA (glycidyl methacrylate) on mammalian and human cells. (1) Using the absorption spectrum shift method in vitro, we observed that the maximums of calf thymus DNA and GMA were shifted toward longer wavelengths (a change of more than 15 nm) and the absorbance decreased after incubation at room temperature for 15 min or more. The result indicates that binding of DNA and GMA had occurred. The binding force is strong, not affected by the addition of concentrated sodium chloride solution, and only slightly decreased by the addition of 8 M urea solution. Therefore the bond between DNA and GMA might be covalent. (2) In cell cultures, unscheduled DNA synthesis (UDS) in human and/or rat lymphocyte was induced and DNA semiconservative replication was inhibited by GMA at concentrations of less than 5.2 mM. (3) Sperm abnormality tests and assays of UDS in germ cells of male mice were conducted to study the in vivo genotoxicity of GMA. The results revealed that GMA could damage DNA, increase sperm abnormality frequency, and reduce the number of sperm cells.

Effect of dextran molecular weight on protein stabilization during freeze-drying and storage.

The effect of dextran molecular weight on structural stability of freeze-dried products and protein stability in amorphous matrices was investigated during storage at elevated temperatures. Glucose-6-phosphate dehydrogenase (G6PDH) was freeze-dried in 10% (w/v) dextrans of five molecular weights (12 kD, 42 kD, 71 kD, 512 kD and 2000 kD) to residual water content of 0.027 ( 0.004 g/g dry mass. The molecular weight of dextrans affected the glass transition temperature (Tg) of freeze-dried products and the recovery of enzyme activity after freeze-drying. As the molecular weight of dextrans increased from 12 kD to 2000 kD, the Tg increased from 100 degrees C to 120 degrees C, whereas the recovery of protein activity decreased from 85 +/- 4 % to 70 +/- 5%. The inactivation of freeze-dried protein during storage followed a bi-phasic first-order kinetics. The stability of amorphous matrices and protein increased significantly as the molecular weight increased from 12 kD to 512 kD. However, at a higher molecular weight (2000 kD), the stability was reduced. In a separate experiment, the stability of dried dextran/protein samples was studied during heating from 30 degrees C to 99 degrees C at 0.2 degrees C/min and subsequent incubation at 99 degrees C. Dextran with an average molecular weight of 512 kD was again found to provide the best protection. Mechanisms that cause the differences in protein stability among different molecular weight dextrans remain unclear.

Dielectric relaxation of water and water-plasticized biomolecules in relation to cellular water organization, cytoplasmic viscosity, and desiccation tolerance in recalcitrant seed tissues.

To understand the relationship between the organization of cellular water, molecular interactions, and desiccation tolerance, dielectric behaviors of water and water-plasticized biomolecules in red oak (Quercus rubra) seeds were studied during dehydration. The thermally stimulated current study showed three dielectric dispersions: (a) the relaxation of loosely-bound water and small polar groups, (b) the relaxation of tightly-bound water, carbohydrate chains, large polar groups of macromolecules, and (c) the "freezing in" of molecular mobility (glassy state). Seven discrete hydration levels (water contents of 1.40, 0.55, 0.41, 0.31, 0.21, 0.13, and 0.08 g/g dry weight, corresponding to -1.5, -8, -11, -14, -24, -74, and -195 MPa, respectively) were identified according to the changes in thermodynamic and dielectric properties of water and water-plasticized biomolecules during dehydration. The implications of intracellular water organization for desiccation tolerance were discussed. Cytoplasmic viscosity increased exponentially at water content < 0.40 g/g dry weight, which was correlated with the great relaxation slowdown of water-plasticized biomolecules, supporting a role for viscosity in metabolic shutdown during dehydration.

Protein modification by Amadori and Maillard reactions during seed storage: roles of sugar hydrolysis and lipid peroxidation.

The non-enzymatic modifications of proteins through Amadori and Maillard reactions play an important role in the loss of seed viability during storage. In the present study, the contribution of sugar hydrolysis and lipid peroxidation to Amadori and Maillard reactions, and to seed deterioration was investigated in mung-bean (Vigna radiata Wilczek). The contents of glucose and lipid peroxidation products in seed axes increased significantly during storage. The accumulation of Amadori products in seed axes was correlated to the lipid peroxidation, whereas the accumulation of Maillard products was closely correlated to sugar hydrolysis. The rate of accumulation of Maillard products was not well correlated to the content of Amadori products in both seed axes and protein/glucose model system, reflecting the complex nature of Amadori and Maillard reactions. The content of Amadori products in seed axes increased during the early stages of seed ageing, whereas the content of Maillard products increased steadily during the entire period of storage. The accumulation of Maillard products in seed axes was associated with the decline of seed vigour. These data suggest that, during seed ageing, sugar hydrolysis and lipid peroxidation are coupled with non-enzymatic protein modification through Amadori and Maillard reactions.

Desiccation tolerance of recalcitrant Theobroma cacao embryonic axes: the optimal drying rate and its physiological basis.

Recalcitrant seed axes were reported to survive to lower water contents under fast-drying conditions. The present study was to examine the hypothesis that drying rate and dehydration duration could interact to determine desiccation tolerance through different physico-chemical mechanisms. The effect of drying rate on desiccation tolerance of Theobroma cacao seed axes at 16 degrees C was examined. Rapid-drying at low relative humidity (RH) and slow-drying at high RH were more harmful to cocoa axes, because electrolyte leakage began to increase and axis viability began to decrease at high water contents. Maximum desiccation tolerance was observed with intermediate drying rates at RH between 88% and 91%, indicating the existence of an optimal drying rate or optimal desiccation duration. This maximum level of desiccation tolerance for cocoa axes (corresponding to a critical water potential of -9 MPa) was also detected using the equilibration method, in which axes were dehydrated over a series of salt solutions or glycerol solutions until the equilibrium. These data confirmed that the physiological basis of the optimal drying rate is related to both mechanical stress during desiccation and the length of desiccation duration during which deleterious reactions may occur. The optimal drying rate represents a situation where combined damages from mechanical and metabolic stresses become minimal.

Tyrosinase reaction and subsequent chitosan adsorption for selective removal of a contaminant from a fermentation recycle stream.

In the industrial production of penicillin V, the phenoxyacetate precursor is added to the fermentor to direct biosynthesis. When used for producing semisynthetic penicillins, the penicillin V is often hydrolyzed to 6-aminopenicillanic acid with the regeneration of the phenoxyacetate precursor. To reduce raw-material as well as waste-disposal costs, it is desirable to recycle the phenoxyacetate precursor. Unfortunately, the recycle stream is generally contaminated by the p-hydroxylated derivative of this precursor. We examined a two-step approach to eliminate this contaminant. In the first step the tyrosinase enzyme was used to selectively convert the p-hydroxyphenoxyacetate contaminant to a reactive intermediate-presumably its quinone. In the second step, the tyrosinase-generated reactive intermediate was allowed to react with and strongly bind to chitosan. In contrast, the phenoxyacetate precursor was neither oxidized by tyrosinase nor bound to chitosan. When concentrated phenoxyacetate solutions were tested, the combination of tyrosinase and chitosan effectively converted low levels of the p-hydroxyphenoxyacetate contaminant and removed its products from solution, while the concentration of the phenoxyacetate precursor was unaffected.

Tyrosinase-containing chitosan gels: A combined catalyst and sorbent for selective phenol removal.

There are a series of examples in which phenols appear as contaminants in process streams and their selective removal is required for waste minimization. For the selective removal of a phenol from a mixture, we are exploiting the substrate specificity of the enzyme tyrosinase to convert phenols into reactive o-quinones which are then adsorbed onto the amine-containing polymer chitosan. To effectively package the enzyme and sorbent, tyrosinase was immobilized between two chitosan gel films. The entrapment of tyrosinase between the films led to little loss of activity during immobilization, while tyrosinase leakage during incubation was limited. The chitosan gels rapidly adsorb the tyrosinase-generated product(s) of phenol oxidation while the capacity of the gels is substantially greater than the capacity of chitosan flakes. The performance of tyrosinase-containing chitosan gels significantly depends on the ratio of tyrosinase-to-chitosan. High tyrosinase-to-chitosan ratios result in less efficient use of tyrosinase, presumably due to suicide inactivation. However, the efficiency of chitosan use increases with increased tyrosinase-to-chitosan ratios. (c) 1996 John Wiley & Sons, Inc.