Freeze tolerance and accumulation of cryoprotectants in the enchytraeid Enchytraeus albidus (Oligochaeta) from Greenland and Europe

The freeze tolerance and accumulation of cryoprotectants was investigated in three geographically different populations of the enchytraeid Enchytraeus albidus (Oligochaeta). E. albidus is widely distributed from the high Arctic to temperate Western Europe. Our results show that E. albidus is freeze tolerant, with freeze tolerance varying extensively between Greenlandic and European populations. Two populations from sub Arctic (Nuuk) and high Arctic Greenland (Zackenberg) survived freezing at −15 °C, whereas only 30% of a German population survived this temperature. When frozen, E. albidus responded by catabolising glycogen to glucose, which likely acted as a cryoprotectant. The average glucose concentrations were similar in the three populations when worms were frozen at −2 °C, approximately 50 μg glucose mg⁻¹ tissue dry weight (DW). At −14 °C the glucose concentrations increased to between 110 and 170 μg mg⁻¹ DW in worms from Greenland. The average glycogen content of worms from Zackenberg and Nuuk were about 300 μg mg⁻¹ DW, but only 230 μg mg⁻¹ DW in worms from Germany showing that not all glycogen was catabolised during the experiment. Nuclear magnetic resonance spectrometry (NMR) was used to screen for other putative cryoprotectants. Proline, glutamine and alanine were up regulated in frozen worms at −2 °C but only in relatively small concentrations suggesting that they were of little significance for freeze survival. The present study confirms earlier reports that freeze tolerant enchytraeids, like other freeze tolerant oligochaete earthworms, accumulate high concentrations of glucose as a primary cryoprotectant.     

Repeated freezing induces oxidative stress and reduces survival in the freeze-tolerant goldenrod gall fly,Eurosta solidaginis

Freeze tolerant insects must not only survive extracellular ice formation but also the generation of reactive oxygen species (ROS) during oxygen reperfusion upon thawing. Furthermore, diurnal fluctuations in temperature place temperate insects at risk of being exposed to multiple freeze–thaw cycles, yet few studies have examined metrics of survival and oxidative stress in freeze-tolerant insects subjected to successive freezing events. To address this, we assessed survival in larvae of the goldenrod gall fly Eurosta solidaginis, after being subjected to 0, 5, 10, 20, or 30 diurnally repeated cold exposures (RCE) to −18 °C or a single freeze to −18 °C for 20 days. In addition, we measured indicators of oxidative stress, levels of cryoprotectants, and total aqueous antioxidant capacity in animals exposed to the above treatments at 8, 32, or 80 h after their final thaw. Repeated freezing and thawing, rather than time spent frozen, reduced survival as only 30% of larvae subjected to 20 or 30 RCE successfully pupated, compared to those subjected to fewer RCE or a single 20 d freeze, of which 82% pupated. RCE had little effect on the concentration of the cryoprotectant glycerol (4.26 ± 0.66 μg glycerol·ng protein⁻¹ for all treatments and time points) or sorbitol (18.8 ± 2.9 μg sorbitol·mg protein⁻¹ for all treatments and time points); however, sorbitol concentrations were more than twofold higher than controls (16.3 ± 2.2 μg sorbitol·mg protein⁻¹) initially after a thaw in larvae subjected to a single extended freeze, but levels returned to values similar to controls at 80 h after thaw. Thawing likely produced ROS as total aqueous antioxidant capacities peaked at 1.8-fold higher than controls (14.7 ± 1.6 mmol trolox·ng protein⁻¹) in animals exposed to 5, 10, or 20 RCE. By contrast, aqueous antioxidant capacities were similar to controls in larvae subjected to 30 RCE or the single 20 d freeze regardless of time post final thaw, indicating these animals may have had an impaired ability to produce primary antioxidants. Larvae lacking an antioxidant response also had elevated levels of oxidized proteins, nearly twice that of controls (21.8 ± 3.2 mmol chloramine-T·mg protein⁻¹). Repeated freezing also lead to substantial oxidative damage to lipids that was independent of aqueous antioxidant capacity; peroxides were, on average, 5.6-fold higher in larvae subjected to 10, 20 or 30 RCE compared to controls (29.1 ± 7.3 mmol TMOP·μg protein⁻¹). These data suggest that oxidative stress due to repeated freeze–thaw cycles reduces the capacity of E. solidaginis larvae to survive freezing.     

Cryoprotectant effect of trehalose and low-density lipoprotein in extenders for frozen ram semen

This study tested trehalose and low-density lipoprotein (LDL) as cryoprotectants in extenders for frozen ram semen. In the first experiment, the extenders were Tris, with 20% egg yolk (E1-1); E1-1 with 5% glycerol (E1-2); E1-1 with 100 mM trehalose (E1-3); and E1-1 with 100 mM trehalose and 5% glycerol (E1-4). Sperm motility and membrane integrity of the E1-2, E1-3 and E1-4 extenders were greater than for E1-1 (P < 0.05), but acrosome integrity following cryopreservation did not differ. In the second experiment, the extenders were Tris, with 20% egg yolk and 100 mM trehalose (E2-1); Tris with 8% LDL and 5% glycerol (E2-2); Tris with 8% LDL and 100 mM trehalose (E2-3); and Tris with 8% LDL, 100 mM trehalose and 5% glycerol (E2-4). Sperm membrane integrity was lowest for the E2-1 extender (P < 0.05), but similar for extenders including LDL. Sperm motility post-thawing was highest for E2-2 and E2-3 extenders (P < 0.05), but acrosome integrity did not differ. Thus, extenders including trehalose and LDL as cryoprotectants recorded a post-thawing ram sperm quality similar to that achieved when using conventional cryoprotectants.     

The effect of pH,bile and calcium on the adhesion ability of probiotic enterococci of animal origin to the porcine jejunal epithelial cell line IPEC-J2

Examination of adhesion ability using a quantitative assay based on radiolabelled bacteria showed that 10 Enterococcus strains exhibited adhesion ability from 2 to 4%. Enterococcus faecium EF2019 (isolate from rabbit faeces, deponed to Czech Culture of Microorganisms in Brno, CCM 7420) showed the highest adhesion ability (4.0 ± 0.4%). With regard to survival, all strains displayed good resistance towards 0.3% oxgall and HCl (pH 3.0). Pretreatment of strains with HCl (pH 3.0) significantly reduced their adhesion. Pretreatment of strains by oxgall significantly reduced the adhesion capacity of E. faecium EF2019, EF1839 and EF319 strains, while the adhesion ability of E. faecium EE3 (isolate from canine feed) slightly increased. Furthermore, addition of calcium (200 mmol/l) significantly increased (P < 0.001) the adhesion ability for all strains tested. The adhesion ability of the isolates from rabbits, EF1839 and EF529, as well as the isolate EE3 (strain from canine feed) increased from 2–3% up to 50–55% upon calcium addition. Despite, in general low adhesive properties, strains can survive passage through the gastrointestinal tract.     

Formulation of Pseudomonas chlororaphis strains for improved shelf life

Formulations of Pseudomonas strains with long-term shelf life are needed for commercial use in biological disease control and growth promotion in crops. In the present work Pseudomonas chlororaphis (Pc) 63-28 formulated with coconut fiber [moisture content (MC) of 80%], talc (MC 8%) or peat (MC 40%), with or without the addition of carboxymethylcellulose or xanthan gum, and formulations of Pc 63-28 and P. chlororaphis TX-1 in coconut fiber with water contents (v:v) of 75%, 45%, and 25%, were evaluated in terms of shelf life and cell viability. The shelf life of Pc 63-28 was longer when formulated in coconut fibre with a MC was 80% than in the other formulations and longer at 3 ± 1 °C compared to 22 ± 1 °C. Densities of viable Pc 63-28 cells in coconut fiber stored at 3 ± 1 °C did not decline significantly during 224 days when the MC was 80% and within 120 days at 75% MC. Densities of Pc TX-1 in coconut fiber of 75% MC did not decline within 60 days at 3 ± 1 °C. P. chlororaphis 63-28 survived longer in deionized water and buffer than in canola oil. Cells of Pc 63-28 cells formulated in coconut fibre of 80% MC after storage for 140 days at 3 ± 1 °C in coconut fiber improved hydroponic growth of hydroponic lettuce and better than cells freshly recovered from culture. We conclude that coconut fiber is a carrier of superior performance in maintaining shelf life of Pseudomonas strains. The observed shelf life would be sufficient for practical use of Pseudomonas strains as tools for disease control and growth promotion in crops.     

Intestinal and immunological effects of daily oral administration of Lactobacillus salivarius CECT5713 to healthy adults

There is an increasing interest in the intestinal and immunological effects of probiotics. The aim of the present study is to evaluate the tolerance and beneficial effects in healthy adults of the strain, Lactobacillus salivarius CECT5713 isolated from breast milk. A phase II, randomized, double-blinded, placebo-controlled human clinical trial was carried out in 40 healthy adults. The Probiotic group received a daily dose of 2 × 10⁸ CFU of L. salivarius CECT5713 in capsules during 4 weeks while volunteers of the control received only a placebo. Gastrointestinal and immunological parameters were analyzed. Results showed that L. salivarius CECT5713 was well tolerated and no adverse effects were detected. Consumption of the probiotic strain increased fecal lactobacilli counts (7.9 ± 0.1 vs. 7.05 ± 0.2 CFU/g feces, P = 0.001). Also, an improvement in the frequency of defecation (P = 0.04) was observed. Probiotic treatment induced significantly the percentage of NK cells and monocytes, as well as the plasmatic levels of immunoglobulins M, A and G, and the regulatory cytokine IL-10 (72.3 ± 11.7 in probiotic group vs. 27.3 ± 6.4 pg/mL in control group, P < 0.01). Thus, it can be concluded that daily administration of L. salivarius CECT5713 to healthy adults is safe and improve gut microbiota and different parameters related to immune response.     

Effect of probiotic L. plantarum IS-10506 and zinc supplementation on humoral immune response and zinc status of Indonesian pre-school children

A 90-day randomized, double-blind, placebo-controlled, pre-post trial was conducted in four groups of Indonesian children aged 12–24 months: placebo, probiotic, zinc, and a combination of probiotic and zinc (n = 12 per group). Microencapsulated Lactobacillus plantarum IS-10506 of dadih origin was supplemented at a dose of 10¹⁰ CFU/day as a probiotic. Zinc was supplemented as 20 mg zinc sulfate monohydrate (8 mg zinc elemental). Blood and stool samples were collected at baseline and at the end of the study period. Fecal sIgA was assessed by ELISA and serum zinc concentrations by ICP-MS. Fecal sIgA increased significantly in the probiotic group (30.33 ± 3.32 μg/g; p < 0.01) and in the combination probiotic and zinc group (27.55 ± 2.28 μg/g; p < 0.027), as compared with the placebo group (13.58 ± 2.26 μg/g). Changes in serum zinc concentrations in the combination probiotic and zinc group showed the highest elevation at the end of the study period. A combination of probiotic L. plantarum IS-10506 at a dose of 10¹⁰ CFU/day and 8 mg of elemental zinc supplementation showed a potential ability to improve the zinc status of pre-school children. Taken together, supplementation with the probiotic L. plantarum IS-10506 and zinc for 90 days resulted in a significantly increased humoral immune response, as well as improved zinc status, in young children.     

Antifungal potentiality of mycogenic silver nanoparticles capped with chitosan produced by endophytic Amesia atrobrunnea

This research reports the fabrication of silver nanoparticles (AgNPs) from endophytic fungus, Amesia atrobrunnea isolated from Ziziphus spina-christi (L.). Influencing factors for instance, thermal degree of incubation, media, pH, and silver nitrate (AgNO₃) molarity were optimized. Then, the AgNPs were encapsulated with chitosan (Ch-AgNPs) under microwave heating at 650 W for 90 s. Characterization of nanoparticles was performed via UV–visible (UV–vis) spectrophotometer, Fourier-transform infrared spectrophotometer (FTIR), zeta potential using dynamic-light scattering (DLS), and field-emission-scanning electron microscope (FE-SEM). Anti-fungal activity of Ch-AgNPs at (50, 25, 12.5, 6.25 mg/L) was tested against Fusarium oxysporum, Curvularia lunata, and Aspergillus niger using the mycelial growth inhibition method (MGI). Results indicated that Czapek-dox broth (CDB) with 1 mM AgNO₃, an acidic pH, and a temperature of 25–30 °C were the optimum for AgNPs synthesis. (UV–vis) showed the highest peak at 435 nm, whereas Ch-AgNPs showed one peak for AgNPs at 405 nm and another peak for chitosan at 230 nm. FTIR analysis confirmed that the capping agent chitosan was successfully incorporated and interacted with the AgNPs through amide functionalities. Z-potential was −19.7 mV for AgNPs and 38.9 mV for Ch-AgNPs, which confirmed the significant stability enhancement after capping. FES-SEM showed spherical AgNPs and a reduction in the nanoparticle size to 44.65 nm after capping with chitosan. The highest mycelial growth reduction using fabricated Ch-AgNPs was 93% for C. lunata followed by 77% for A. niger and 66% F. oxysporum at (50 mg/L). Biosynthesis of AgNPs using A. atrobrunnea cell-free extract was successful. Capping with chitosan exhibited antifungal activity against fungal pathogens.     

Development to the blastocyst stage of immature pig oocytes arrested before the metaphase-II stage and fertilized in vitro

In vitro fertilization (IVF) and embryonic development of mature and meiotically arrested porcine oocytes were compared in the present study. After in vitro maturation (IVM) of cumulus-oocyte complexes for 48 h, 75.4% of them extruded a visible polar body (PB). Most of the oocytes with a first polar body (PB+ group) were at the metaphase-II (M-II) stage (91.4%). Most of the oocytes without a visible polar body (PB− group) appeared to be arrested at the germinal vesicle (GV) (41.6%) and metaphase-I (M-I) (34.0%) stages. After IVF of oocytes (day of IVF = Day 0), there was no difference between PB⁺ and PB⁻ groups in rates of sperm penetration, mono-spermy, however oocyte activation rate after penetration was greater in the PB+ than in the PB− group (P < 0.05). On Day 2, there was no difference between rates of embryos cleaved at the 2–4 cell stages in PB+ and PB− groups (42.1 ± 48.8% and 33.6 ± 2.1%, respectively). On Day 4, the rate of PB+ embryos developing beyond the 4-cell stage was greater than that of PB− embryos (P < 0.05, 31.7 ± 3.9% and 14.1 ± 1.5%, respectively), and PB+ embryos had more cells than the PB− embryos (P < 0.05, 8.3 ± 0.4 and 6.0 ± 0.8 cells, respectively). On Day 6, a greater proportion of PB+ embryos developed to the blastocyst stage than did PB− embryos (P < 0.05, 34.6 ± 2.4% and 20.7 ± 2.8%, respectively). However, when the GV oocytes of the PB− group were not included in recalculations, there was no difference in blastocyst rates between M-I arrested and M-II oocytes (35.3 and 34.6%, respectively). The number of blastomere nuclei in embryos obtained from the PB+ group (52.0 ± 2.5) was greater than that from the PB− group (P < 0.05, 29.1 ± 2.8). The proportion of degenerated parts in the blastocysts, as determined by morphological appearance, was the same in the PB+ and PB− groups. Although the quality of PB+ embryos was enhanced as compared with that of the PB− group, the proportion of inner cell mass and trophectoderm cells in PB+ and PB− blastocysts did not differ (1:1.9 and 1:2.2, respectively). Chromosome analysis revealed that PB+ blastocysts had more diploidy (P < 0.05, 69.7%) than did PB− blastocysts (44.0%), whereas PB− blastocysts had more triploid cells (P < 0.05, 34.0%) than did PB+ oocytes (8.4%). These results indicate that pig oocytes arrested before the M-II stage (M-I oocytes) undergo cytoplasmic maturation during maturation culture and have the same ability to develop to blastocysts after IVF as M-II oocytes, but some of them resulted in degeneration or delayed development with poor embryo quality.     

Encapsulation of lactate dehydrogenase in carbon nanotube doped alginate–chitosan capsules

Alginate–chitosan shell–core (AC) capsules doped with carbon nanotubes (CNTs) were prepared for lactate dehydrogenase (LDH, EC 1.1.1.27) encapsulation to convert pyruvic acid to lactic acid coupling with the oxidation of NADH to NAD⁺. LDH was entrapped within the liquid core of the capsules and the CNTs were incorporated in the alginate or chitosan matrices or both. The physical properties of the capsules and the immobilized LDH activity were investigated. The AC capsules doped with CNTs showed better mechanical strength than that without CNTs. The LDH loading efficiency of the AC capsules with CNTs (10 mg/mL) doped in both the shell and the core was 30.7% higher than that without CNTs. The optimal pH value for the bioconversion catalyzed by immobilized LDH was 7.0, lower than that by free LDH (7.5). The optimal temperature was 35 °C for both immobilized and free LDH. Operational stability of the immobilized LDH was greatly improved by doping CNTs in AC capsules. The results showed that this method was efficient for enzyme encapsulation in the biotechnology applications.     

Culture parameters affecting xylitol production by Debaryomyces hansenii immobilized in alginate beads

Yeast immobilization offers operational advantages such as high cell concentration, and some drawbacks related to cell leaking and restricted mass transfer inside particles. The influence of bead size, chitosan, bead charge, volume of liquid media, and the use of corncob hydrolyzates and vinasses as culture medium were analyzed on xylitol production by Debaryomyces hansenii immobilized in alginate beads. The results showed a profuse growth of free cells, accounting 75–95% of total biomass, but electron micrographs revealed the generation of a dense biofilm with hyphal morphology at the bead surface and a very low intraparticular growth. Xylitol production was not affected by the size of particle; however chitosan had a negative effect. The use of corn cob as carbon source and twofold diluted vinasses as economic nutrients incremented xylitol concentration to 13.7 g L^?1 (Y_P/S = 0.56 g g^?1; Q_P = 0.29 g L^?1 h^?1). The best conditions corresponded to high bead charges and intermediate liquid volumes (44 g Na-alginate and 110 mL liquid medium). These results showed the feasibility of employing these cheap substrates, reflected the importance of the microaerobical conditions, and pointed to the favorable effect of cell immobilization on the metabolism of xylitol production.     

Effect of cooling rate and equilibration time on pre-freeze and post-thaw survival of buck sperm

Survival of buck sperm is affected due to duration and temperature of stages of refrigerated or frozen storage. This study investigated interactive effect of cooling rates (moderate; MC and rapid cooling; RC); and equilibration times (0, 2, 4 and 8 h) on survival before freezing at 4 °C and post-thaw quality of buck sperm. Semen was collected (three Beetal bucks; replicates = 6), pooled and diluted with Tris-citrate extender. Pooled semen samples were subjected to either RC (−2.2 °C/min) or MC (−0.3 °C/min) from 37 °C to 4 °C in separate aliquots and further equilibrated at 4 °C for 8 h. Semen was frozen using standard procedure after completion of each equilibration period i.e. 0, 2, 4 and 8 h. Semen was evaluated for motility, viability, plasma membrane integrity (PMI) and normal apical ridge (NAR) before freezing and after thawing. The survival time (time for survival above threshold limit i.e. 60%) at 4 °C, of motility and PMI was observed 5 and 6 h respectively in RC group while >8 h in MC group. Rate of decline (slope) in motility and viability was higher (P < 0.05) in RC overtime during equilibration at 4 °C while PMI and NAR declined at equal rate in both cooling groups. Post-thaw motility and NAR were higher (P < 0.05) in MC when equilibrated for 2–8 h while viability and PMI of RC was observed equal to MC group. In conclusion, survival of buck sperm is higher when cooled with moderate rate. However, RC can maintain post-thaw sperm viability and PMI equal to MC when equilibrated for 2–8 h. The methods should be explored to maintain motility and NAR during rapid cooling of buck sperm.     

Influence of whole microalgal cell immobilization and organic solvent on the bioconversion of androst-4-en-3,17-dione to testosterone by Nostoc muscorum

The use of free, immobilized and reused immobilized cells of the microalga Nostoc muscorum was studied for bioconversion of androst-4-en-3,17-dione (AD) to testosterone in hexadecane. Among polymers such as agar, agarose, κ-carrageenan, polyacrylamide, polyvinyl alcohol, and sodium alginate that were examined for cell entrapment, sodium alginate with a concentration of 2% (w/v) proved to be the proper matrix for N. muscorum cells immobilization. The bioconversion characteristics of immobilized whole algal cells at ranges of temperatures, substrate concentrations, and shaking speeds were studied followed by a comparison with those of free cells. The conditions were 30 °C, 0.5 g/L, and 100 rpm, respectively. The immobilized N. muscorum showed higher yield (72 ± 2.3%) than the free form (24 ± 1.3%) at the mentioned conditions. The bioconversion yield did not decrease during reuse of immobilized cells and remained high even after 5 batches of bioreactions while Na-alginate 3% was used; however, reuse of alginate 2% beads did not give a satisfactory result.     

Reduction of fecal shedding of enterohemorrhagic Escherichia coli O157:H7 in lambs by feeding microbial feed supplement

Enterohemorrhagic Escherichia coli O157:H7, an emerging food-borne pathogen, has been implicated in several outbreaks in the US. Ruminants, including cattle, sheep and deer are reservoirs of E. coli O157:H7 and fecal shedding of the pathogen forms the vehicle of entry into the human food chain. We studied the efficacy of Lactobacillus acidophilus, Streptococcus faecium, a mixture of L. acidophilus and S. faecium and a mixture of L. acidophilus, S. faecium, Lactobacillus casei, Lactobacillus fermentum and Lactobacillus plantarum in reducing fecal shedding of E. coli O157:H7 by sheep experimentally infected with the pathogen prior to administration with the microbials. Following oral inoculation with 10¹⁰ CFU of E. coli O157:H7, 30 Suffolk ram lambs were blocked by body weight (six blocks of five lambs each) and lambs within the block randomly assigned to five groups. The lamb groups were fed daily for 7 weeks a basal diet without microbial supplement (control) or the basal diet with L. acidophilus or with S. faecium or with a mixture of L. acidophilus and S. faecium or with a mixture of L. acidophilus, S. faecium, L. casei, L. fermentum and L. plantarum. The microbial supplements contained stabilized live naturally occurring bacteria and were mixed with the diet at the rate of 6.0×10⁶ CFU per kilogram of diet. Fecal samples were collected weekly and analyzed for E. coli O157:H7 using modified tryptic soy broth with novobiocin as a pre-enrichement broth and cefixim-tellurite sorbitol MacConkey agar (CT-SMAC) as a selective media. E. coli O157:H7 was confirmed by its reaction with O157 and H7 antisera. E. coli O157:H7 was shed continuously and in varying numbers in the feces throughout the 7-week experimental period by all five groups. However, lambs administered a mixture of L. acidophilus, S. faecium, L. casei, L. fermentum and L. plantarum shed significantly lower (P=0.0211) average number of E. coli O157:H7 (2.3 log₁₀ CFU per gram of feces per week) than the other lamb groups over the entire experimental period. S. faecium supplemented lambs were comparable (P=0.0884) to lambs fed a mixture of L. acidophulus and S. faecium in fecal shedding of the pathogen (3.5 versus 4.4 log₁₀ CFU per gram of feces) but significantly lower (P=0.0001) than the control lambs (5.6 log₁₀ CFU per gram of feces) and those supplemented with L. acidophilus (5.5 log₁₀ CFU per gram of feces). Average daily gain (ADG) and gain to feed ratio (G:F) were significantly improved (P=0.0145) by the mixed culture microbials (163.0 g and 0.33 for the control, 186.4 g and 0.37 for L. acidophulus, 168.2 g and 0.36 for S. faecium, 213.6 g and 0.46 for L. acidophulus and S. faecium, and 219.1 g and 0.44, respectively for L. acidophilus, S. faecium, L. casei, L. fermentum and L. plantarum supplemented lambs. The study indicates that supplementing lambs infected with E. coli O157:H7 with S. faecium or a mixture of S. faecium, L. acidophilus, L. casei, L. fermentum and L. plantarum in the diet can reduce total number of E. coli O157:H7 shed in the feces and improve animal meat production performance as well.     

Production of isomaltulose using Erwinia sp. D12 cells: Culture medium optimization and cell immobilization in alginate

Isomaltulose is a structural isomer of sucrose commercially used in food industries. Glucosyltransferase produced by Erwinia sp. D12 catalyses an intramolecular transglucosylation of sucrose giving isomaltulose. An experimental Design and Response Surface Methodology were applied for the optimization of the nutrient concentration in the culture medium for enzyme production in shaken flasks at 200 rpm and 30 °C. A higher production of glucosyltransferase (7.47 Uml⁻¹) was observed in the culture medium containing sugar cane molasses (160 gl⁻¹), bacteriological peptone (20 gl⁻¹) and yeast extract Prodex Lac SD^® (15 gl⁻¹) after 8 h, at 30 °C. The highest production of glucosyltransferase in the 6.6-l bioreactor (14.6 Uml⁻¹) was obtained in the optimized culture medium after 10 h at 26 °C. When Erwinia sp. D12 cells were immobilized in sodium alginate, it was verified that sodium alginate solution A could be substituted by a cheaper one, sodium alginate solution B. Using a 40% cell suspension and 2% sodium alginate solution B for cell immobilization in a packed-bed reactor, 64.1% conversion of sucrose to isomaltulose was obtained. The packed-bed reactor with immobilized cells plus glutaraldehyde and polyethylenimine solutions remained in a pseudo-steady-state for 180 h.     

Cold tolerance of the maize orange leafhopper,Cicadulina bipunctata

Cicadulina bipunctata was originally distributed in tropical and subtropical regions of the Old World. This leafhopper recently expanded its distribution area to southern parts of temperate Japan. In this study, factors affecting the overwintering ability of C. bipunctata were examined. A series of laboratory experiments revealed that cold acclimation at 15 °C for 7 days enhanced the cold tolerance of C. bipunctata to the same level as an overwintering population, adult females were more tolerant of cold temperature than adult males, and survival of acclimated adult females was highly dependent on temperature from −5 to 5 °C and exposure duration to the temperature. The temperature of crystallization of adult females was approximately −19 °C but temperatures in southern temperate Japan rarely dropped below −10 °C in the winter, indicating that overwintering C. bipunctata adults in temperate Japan are not killed by freezing injury but by indirect chilling injury caused by long-term exposure to moderately low temperatures. An overwintering generation of C. bipunctata had extremely low overwinter survival (<1%) in temperate Japan; however, based on winter temperature ranges, there are additional areas amenable to expansion of C. bipunctata in temperate Japan.     

Forming nanoparticles of α-amylase and embedding them into solid surfaces

In the current work nanoparticles (NPs) of α-amylase were generated in an aqueous solution using high-intensity ultrasound, and were subsequently immobilized on polyethylene (PE) films, or polycarbonate (PC) plates, or on microscope glass slides. The α-amylase NPs coated on the solid surfaces have been characterized by ESEM, TEM, FTIR, XPS and AFM. The substrates immobilized with α-amylase were used for hydrolyzing soluble potato starch to maltose. The amount of enzyme introduced in the substrates, leaching properties, and the catalytic activity of the immobilized enzyme were compared. The catalytic activity of the amylase deposited on the three solid surfaces was compared to that of the same amount of free enzyme at different pHs and temperatures. α-Amylase coated on PE showed the best catalytic activity in all the examined parameters when compared to native amylase, especially at high temperatures. When immobilized on glass, α-amylase showed better activity than the native enzyme over all pH and temperature values studied. However, the immobilization on PC did not improve the enzyme activity at any pH and any temperature compared to the free amylase. The kinetic parameters, K_m and V_max were also calculated. The amylase coated PE showed the most favorable kinetic parameters (K_m = 5 g L⁻¹ and V_max = 5E−07 mol mL⁻¹ min⁻¹). In contrast, the anchored enzyme-PC exhibited unfavorable kinetic parameters (K_m = 16 g L⁻¹, V_max = 4.2E−07 mol mL⁻¹ min⁻¹). The corresponding values for amylase-glass were K_m = 7 g L⁻¹, V_max = 1.8E−07 mol mL⁻¹ min⁻¹, relative to those obtained for the free enzyme (K_m = 6.6 g L⁻¹, V_max = 3.3E−07 mol mL⁻¹ min⁻¹).     

Preparation of active corn peptides from zein through double enzymes immobilized with calcium alginate–chitosan beads

Double enzymes (alcalase and trypsin) were effectively immobilized in a composite carrier (calcium alginate–chitosan) to produce immobilized enzyme beads referred to as ATCC. The immobilization conditions for ATCC were optimized, and the immobilized enzyme beads were characterized. The optimal immobilization conditions were 2.5% of sodium alginate, 10:4 sodium alginate to the double enzymes, 3:7 chitosan solution to CaCl₂ and 2.5 h immobilization time. The ATCC beads had greatly enhanced stability and good usability compared with the free form. The ATCC residual activity was retained at 88.9% of DH (degree of hydrolysis) after 35 days of storage, and 36.0% of residual activity was retained after three cycles of use. The beads showed a higher zein DH (65.8%) compared with a single enzyme immobilized in the calcium alginate beads (45.5%) or free enzyme (49.3%). The ATCC kinetic parameters V_max and apparent K_m were 32.3 mL/min and 456.62 g⁻¹, respectively. Active corn peptides (CPs) with good antioxidant activity were obtained from zein in the ethanol phase. The ATCC might be valuable for preparing CPs and industrial applications.     

Lead biosorption efficiency of Levilactobacillus brevis MZ384011 and Levilactobacillus brevis MW362779: A response surface based approach

Lead (Pb) is a substantial contaminant in the environment and a potent toxin for living organisms. Current study describes probiotic characteristics of Pb-biosorbing lactic acid bacteria (LAB), and response surface methodology (RSM) based optimization of physical conditions for maximum Pb biosorption. A total of 18 LAB, isolated from carnivore feces (n = 8) and human breast milk (n = 9), along with one reference strain Lactobacillus acidophilus ATCC4356 were included in the study. Pb biosorption was strain specific. Eight strains, demonstrating ≥ 70 % lead biosorption, were selected for further testing. The lactobacillus-Pb complex was found to be stable and strains had a negative surface charge. The strains displayed good probiotic properties with the survival rate of 71–90 % in simulated gastric environment, 36–69 % in intestinal condition (1.8 % bile salts) and 55–72 % hydrophobicity. On the basis of excellent probiotic ability, Levilactobacillus brevis MZ384011 and Levilactobacillus brevis MW362779 were selected for optimization of physical conditions of Pb biosorption through RSM. Maximum biosorption was observed at pH 6 in 60 min at a cell density of 1 g/L. L. brevis MZ384011 and L. brevis MW362779 are recommended for experimentation on Pb toxicity amelioration and safety evaluation in in-vivo setting.     

Electrochemical analysis of autodisplayed adrenodoxin (Adx) on the outer membrane of E. coli

In this work, adrenodoxin (Adx) was expressed on the outer membrane of E. coli by autodisplay and then the iron–sulfur cluster was incorporated into apo-Adx by an anaerobic reconstitution process. For the determination of the redox potentials of the iron–sulfur clusters of the autodisplayed Adx, E. coli cells with autodisplayed Adx were immobilized on a gold electrode modified with a self-assembled monolayer of mercaptoundecanoic acid (MUA). From the repeated cyclic voltammetry (CV) analysis, the E. coli (10 mM HEPES buffer, pH 7.0) with autodisplayed Adx showed significant changes in shape with an oxidation peak at + 0.4 V (vs. Ag/AgCl) and a reduction peak at − 0.3 V (vs. Ag/AgCl) after the reconstitution process for the incorporation of the iron–sulfur cluster. From the repeated CV analysis in the reduction and oxidation potential ranges, the iron–sulfur clusters of the autodisplayed Adx were observed to undergo reversible redox reactions via direct electron transfer to the MUA-modified gold electrode.