Properties of biopolymers produced by transglutaminase treatment of whey protein isolate and gelatin

Byproduct utilization is an important consideration in the development of sustainable processes. Whey protein isolate (WPI), a byproduct of the cheese industry, and gelatin, a byproduct of the leather industry, were reacted individually and in blends with microbial transglutaminase (mTGase) at pH 7.5 and 45 °C. When a WPI (10% w/w) solution was treated with mTGase (10 U/g) under reducing conditions, the viscosity increased four-fold and the storage modulus (G′) from 0 to 300 Pa over 20 h. Similar treatment of dilute gelatin solutions (0.5–3%) had little effect. Addition of gelatin to 10% WPI caused a synergistic increase in both viscosity and G′, with the formation of gels at concentrations greater than 1.5% added gelatin. These results suggest that new biopolymers, with improved functionality, could be developed by mTGase treatment of protein blends containing small amounts of gelatin with the less expensive whey protein.     

Mechanical,barrier and morphological properties of pea starch and peanut protein isolate blend films

Mechanical, barrier and morphological properties of edible films based on blends of Pea starch (PS) and Peanut protein isolate (PPI) plasticized with glycerol (30%, w/w) were investigated. As PPI ratio in PS/PPI blends increased, the thickness of films decreased, the opacity slightly elevated and color intensified. The addition of PPI to the PS film significantly reduced tensile strength from 5.44 MPa to 3.06 MPa, but increased elongation from 28.56% to 98.12% with the incorporation of PPI into PS at 50% level. Film solubility value fell from 22.31% to 9.78% upon the incorporation of PPI ranged from 0 to 50% level. When PPI was added into PS film at 40% level, the WVP and WVTR of the films markedly dropped from 11.18% to 4.19% and 6.16 to 1.95%, respectively. Scanning electron microscopy (SEM) of the surface of films showed that many swollen starch granules were presented in the 100% PS film, while 100% PPI film was observed to have rougher surfaces with presence of pores or cavities. The PS/PPI blend films upon the incorporation of PPI at 20% and 50% level were not homogeneous. However, the smoother film surface was observed in PS/PPI blend films with the addition of PPI at 40% level. SEM image of the cross-sections of the films revealed that the 100% PS film showed a uniform and compact matrix without disruption, and pore formation and 100% PPI film displayed a smooth structure. Rougher and flexible network was shown in blend film with the addition of PPI reaching 40% level.     

Structure and properties of carboxymethyl cellulose/soy protein isolate blend edible films crosslinked by Maillard reactions

Edible films based on carboxymethyl cellulose (CMC) and soy protein isolate (SPI), compatibilized by glycerol, were prepared by solution casting. The effects of CMC content on blend structure, thermal stability, water solubility and water sorption, and mechanical properties were systematically investigated. Fourier transform infrared (FTIR) spectra showed that Maillard reactions occurred between CMC and SPI, and X-ray diffraction (XRD) scans indicated that the Maillard reactions greatly reduced the crystallinity of SPI. According to differential scanning calorimetry (DSC) analysis, CMC/SPI blends had a single glass transition temperature (T_g) between 75 and 100 °C, indicating that CMC and SPI form one phase blends. Increasing the CMC content improved the mechanical properties and reduced the water sensitivity of blend films. The results indicate that the structure and properties of SPI edible films were modified and improved by blending with CMC.     

Extraction and characterization of gelatin from camel skin (potential halal gelatin) and production of gelatin nanoparticles

Gelatin is used as an ingredient in both food and non-food industries as a gelling agent, stabilizer, thickener, emulsifier, and film former. Porcine skins, bovine hides, and cattle bones are the most common sources of gelatin. However, mammalian gelatins are rejected by some consumers due to social, cultural, religious, or health-related concerns. In the present study, gelatin was obtained from camel skin as an alternative source using a combination of processing steps. Central composite design combined with response surface methodology was used to achieve high gelatin yields under different extraction conditions: temperatures of 40, 60, and 80 °C; pH values of 1, 4, and 7; and extraction times of 0.5, 2.0, and 3.5 min. Maximum gelatin yield from camel skin (29.1%) was achieved at 71.87 °C and pH 5.26 after 2.58 min. The extracted gelatin samples were characterized for amino acid profile, foaming capacity, film formation, foam stability, and gel strength (Bloom value). Gelatin nanoparticles were produced, and their morphology and zeta potential were determined. Bloom value of the camel skin gelatin was 340 g. Amino acid analysis revealed that the extracted gelatin showed high glycine and proline contents. Analysis of camel skin gelatin nanoparticle and functional properties revealed high suitability for food and non-food applications, with potential use in the growing global halal food market.     

The secret life of the giant Australian cuttlefish Sepia apama (Cephalopoda): Behaviour and energetics in nature revealed through radio acoustic positioning and telemetry (RAPT)

Sepia apama were tagged with acoustic transmitters and monitored on their native House Reef, Boston Bay, South Australia, with a radio acoustic positioning telemetry (RAPT) system. Cuttlefish were tagged with position-only and intra-mantle jet pressure transmitters. New data analyses were developed to handle problem data that arise with an uneven reef environment. Maximum range for the cuttlefish varied from 90 m to 550 m. Cuttlefish home range was between 5300 m² and 23,700 m². S. apama were found to be diurnal as average distance travelled was higher in the day than at night, and cuttlefish were active for 32 days, but only 18 nights. After the cuttlefish settled into reef crevices, activity spectrum and positioning analysis showed foraging behaviour at only 3.7% per day and 2.1% per night. Cuttlefish were found to spend more than 95% of the day resting, which suggests that their bioenergetics are more akin to those of octopus than of squid. The cuttlefish combination of predator avoidance, efficient foraging and quiescent lifestyle allows energy to be channelled into growth and fulfillment of the live-fast-die-young cephalopod philosophy.     

Effect of acetylation and succinylation on physicochemical properties and structural characteristics of oat protein isolate

The effects of acetylation and succinylation on physicochemical properties and structural characteristics of oat protein isolate (OPI) were investigated. The degree of N-acylation rapidly increased prior to O-acylation, due to higher reactivity of ε-amino groups than hydroxyl groups. The acylation was able to decrease zeta potential of OPI at neutral pH, and succinylated OPI had lower zeta potential than acetylated OPI. The surface hydrophobicity (H₀) of OPI was changed significantly by acylation treatment, which varied with the type and level of applied anhydrides. The succinylation led to a remarkable increase in the molecular weight of OPI determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The secondary structure and tertiary conformation of proteins in the OPI was analyzed by Fourier transform infrared (FTIR) and intrinsic fluorescence spectroscopy. The acylation could result in the transformation of β-sheet to α-helix and random coil, and less compact tertiary conformation, especially succinylation.     

A gelatin film procedure for the localization of proteolytic activity in Leucochloridiomorpha constantiae (Trematoda) adults

A gelatin film procedure was used to localize proteolytic activity in the lumen of the intestinal ceca of Leucochloridiomorpha constantiae (Trematoda) adults. Slides were coated with a 7·5% gelatin solution and then fixed in 10% neutral buffered formalin (NBF). Cryostat sections of isolated worms or those attached to the bursa of Fabricius of the domestic chick were affixed to the gelatin film. Experimental and control slides were incubated in a humid chamber for 30 min at 37·5 and 4°C, respectively. Slides were again fixed in NBF, and then stained for protein with mercuric bromphenol blue (MBB). In experimental slides, the lumen of the intestinal ceca was lysed and did not stain, whereas worm and host tissue and the gelatin were protein-positive. Control sections stained uniformly positive for protein. In this procedure tissue is retained on the slide and proteolytic activity can be correlated with a tissue site on the same slide.     

Experimental study of enriched frozen diet on digestive enzymes and growth of juvenile cuttlefish Sepia officinalis L. (Mollusca Cephalopoda)

Hatchlings cuttlefish were reared in the laboratory from hatching until 30 days old, fed with live shrimp, frozen shrimp or fish oil-enriched frozen shrimp. Survival of cuttlefish fed with oil-enriched frozen shrimp was better than in animals receiving live shrimp. However, there was no difference with cuttlefish fed with frozen shrimp, even if survival of those receiving oil-enriched frozen shrimp was always higher all along the experiment. Lower survival in animals fed with live shrimp represented the problem of using such food and confirms the necessity to elaborate an artificial food. Utilization of artemia was detrimental to growth and induced low values of instantaneous growth rate (IGR) and conversion rate even after feeding cuttlefish with shrimp. Nevertheless, growth parameters evolutions generally corresponded to those observed by other researchers. The profile noticed at the end of the experiment is typically observed when cuttlefish acquire their adult digestive system. Main differences were observed between groups fed with live shrimp or oil-enriched frozen shrimp. Enrichment did not induce same growth as in cuttlefish receiving live prey. However, at 20 and 25 days after hatching (DAH), in cuttlefish fed with oil-enriched frozen shrimp, ration was lower for the same growth than in other groups.These data showed capacity of juvenile cuttlefish to adjust their digestive enzyme activities according to the diet and the stage of development. Indeed, chymotrypsin was strongly influenced by enrichment, while other enzymes showed difference between live and frozen preys. Trypsin exhibited regulation by diet after 20 DAH. Freezing seemed to delay development as acid phosphatases, characteristic of first stages of cuttlefish, had lower activity in cuttlefish fed with live shrimp at 10 DAH. Moreover, influence of the stage of development was strong as activities between 20 and 30 DAH were different in all groups. This was in relation with evolution of the digestive system. These data illustrated the difficulty to elaborate optimal diet as digestive system evolves.     

NMR and homology modeling studies of copper(II)-halocyanin from Natronobacterium pharaonis bacteria

Halocyanin from the haloalkaliphilic archaean Natronobacterium pharaonis is a peripheral membrane type 1 blue copper protein with a single polypeptide chain of 163 amino acid residues. Halocyanin participates as putative electron carrier protein associated to an electron acceptor role for a terminal oxidase and has the lowest redox potential value reported to date for a BCP. NMR studies and homology modeling calculations were performed to evaluate the electronic properties of Cu(II)-halocyanin from Natronobacterium pharaonis. The copper coordination site properties of Cu(II)-halocyanin are discussed. The ¹H NMR spectra, isotropic chemical shifts and relaxation times for halocyanin are compared with those of other BCPs such as azurin, amicyanin, plastocyanin and stellacyanin. The wild-type Cu(II)-halocyanin presents almost the same ¹H NMR spectra in comparison with Cu(II)-plastocyanin as expected from a similar coordination symmetry. However, minor differences were found. In order to get some insight on these differences, a computational model for Cu(II)-halocyanin from N. pharaonis was built. Model is based on sequential homology of halocyanin with two different families of proteins: plastocyanins and pseudoazurins. Homology modeling was performed using two different structural templates and copper ion was added for further refinement of the coordination site. Proposed structure was in good agreement with NMR experimental information and is the first three-dimensional model reported to date of an halocyanin. Small differences were found in the copper coordination site with respect to other BCP with known structure. This work is also an interesting example of expertise-driven homology modeling across different protein families.     

Assessment of the exposure pathway in the uptake and distribution of americium and cesium in cuttlefish (Sepia officinalis) at different stages of its life cycle

Laboratory radiotracer experiments were performed to study the uptake, assimilation and retention of americium (²⁴¹Am) and cesium (¹³⁴Cs) by the common cuttlefish Sepia officinalis. Uptake and loss kinetics of the radionuclides were measured following exposure through sediments, seawater and food at different stages of the animal's life cycle. Sediment was found to be a minor uptake pathway for both radionuclides in juveniles. Following a short seawater exposure, cuttlefish accumulated ²⁴¹Am and ¹³⁴Cs, but only to a limited extent (whole-body CF < 2). Among the cuttlefish organs, branchial hearts and their appendages displayed the highest degree of uptake for ²⁴¹Am (CF = 42 and 16, respectively), but these tissues contained low percentage of total ²⁴¹Am due to their relatively small contribution to whole organism weight. The major fraction of incorporated radionuclides was associated with muscular tissues (viz. 65% and 82% of total ²⁴¹Am and ¹³⁴Cs, respectively). Whole-body loss of ²⁴¹Am and ¹³⁴Cs was relatively rapid (T_b½ = 14 and 6 days, respectively). After dietary exposure, around 60% and 30% of ingested ²⁴¹Am was assimilated into the tissues of juvenile and adult cuttlefish, respectively. However, assimilated ²⁴¹Am was more strongly retained in adults than in juveniles (T_b½ = 28 vs. 5 days, respectively), suggesting that different mechanisms govern ²⁴¹Am elimination at both ages. Ingested ¹³⁴Cs was assimilated to a similar extent in juveniles (29%) and adults (23%), but the depuration rate was four times faster in adults. Our results strongly suggest that these two radionuclides follow different excretion pathways and that the mechanisms can vary with age for a given radionuclide.     

Seasonal modulation of plasma antifreeze protein levels in Atlantic (Anarhichas lupus) and spotted wolffish (A. minor)

The Atlantic and spotted wolffish (Anarhichas lupus and A. minor, respectively) inhabit the cold waters of the northeast Atlantic Ocean. Although both species experience subzero water temperatures during winter, the Atlantic wolffish, which occupies shallower waters than the spotted wolffish, faces the greater threat of coming into contact with ice and freezing. This laboratory study was designed to determine whether these species differed in their abilities to resist freezing by examining the seasonal changes in blood plasma freezing points, antifreeze protein (AFP) activity and Na⁺ and Cl⁻ concentrations when exposed to seasonally cycling water temperatures and photoperiod. The plasma of both species showed distinct seasonal cycles in all parameters with the highest values occurring during the winter. However, of the two species, only the Atlantic wolffish produced sufficient AFP to protect the fish down to the freezing point of seawater (− 1.80 °C). The levels of AFP in the spotted wolffish were too low to impart any significant improvement in their resistance to freezing (approximately − 0.8 °C).When wolffish were maintained in warm water under a seasonally changing photoperiod, the amplitude of the seasonal cycle in AFP activity was greatly reduced, indicating that low water temperatures are necessary to maximize plasma AFP levels. However, despite being maintained in warm water, plasma levels of AFP activity began to increase over summer values at the same time of year as did the fish exposed to seasonally changing water temperatures. This suggests that photoperiod plays a major role in the timing of the annual AFP cycle.     

Development of a bioassay from isolated digestive gland cells of the cuttlefish Sepia officinalis L. (Mollusca Cephalopoda): effect of Cu, Zn and Ag on enzyme activities and cell viability

The purpose of this study was to establish a bioassay from isolated digestive gland cells of the cuttlefish Sepia officinalis in order to observe the effect of heavy metals on digestive enzyme activities. Digestive cells were isolated using a pronase enzyme that was removed by several washings of the cell suspension. Cell viability was tested by the MTT assay (3-4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium) and microscopic analysis. The results showed that isolated digestive cells could be maintained 24 h with preservation of whole digestive functionality, measured in terms of MTT test. In fact, the viability was maintained at a high level during 24 h and the intra- and extracellular digestive enzyme activities became stabilised rapidly. Furthermore, suspension cells responded to calcium ionophore and 8-Bromo-cAMP by an unspecific secretion of extracellular digestive enzyme, trypsin, which demonstrated that isolated digestive cells were functional. Using the bioassay, ecotoxicological studies showed that heavy metals could have effects on digestive enzyme activities after 24 h of an incubation time of the metal with the cells. In fact, zinc and silver affected trypsin and/or cathepsins specific activity of the cells. On the contrary, copper had no effect on digestive enzyme activities. Zinc, which is a trace element in all living animals, generated two different responses of cathepsins and cell viability. At a low concentration (0.02 μM), it increased viability and cathepsins specific activity, whereas at a high concentration (0.02 mM), zinc inhibited the cathepsins specific activity with an inhibition of cathepsins. For silver, whatever the tested concentration (0.02 mM or 0.02 μM), it has no impact on digestive gland isolated cell viability. Nevertheless, heavy metal induced high disturbance of enzymatic systems.     

Caerulein precursor fragment (CPF) peptides from the skin secretions of Xenopus laevis and Silurana epitropicalis are potent insulin-releasing agents

Peptidomic analysis of norepinephrine-stimulated skin secretions of the tetraploid clawed frog Xenopus laevis (Pipidae) led to the identification of 10 peptides with the ability to stimulate the release of insulin from the rat BRIN-BD11 clonal β cell line. These peptides were purified to near homogeneity and structural characterization showed that they belong to the magainin (2 peptides), peptide glycine-leucine-amide (PGLa) (1 peptide), xenopsin precursor fragment (1 peptide), and caerulein precursor fragment (CPF) (6 peptides) families. CPF-1, CPF-3, CPF-5 and CPF-6 were the most potent producing a significant (P < 0.05) increase in the rate of insulin release at concentration of 0.03 nM. CPF-7 (GFGSFLGKALKAALKIGANALGGAPQQ) produced the maximum stimulation of insulin release (571 ± 30% of basal rate at 3 μM). In addition, CPF-SE1 (GFLGPLLKLGLKGVAKVIPHLIPSRQQ), previously isolated from skin secretions of the tetraploid frog Silurana epitropicalis, produced a significant (P < 0.05) increase in the rate of insulin release at 0.03 nM with a 514 ± 13% increase over basal rate at 3 μM. No CPF peptide stimulated release of the cytosolic enzyme, lactate dehydrogenase from BRIN-BD11 cells at concentrations up to 3 μM indicating that the integrity of the plasma membrane had been preserved. The mechanism of action of the CPF peptides involves, at least in part, membrane depolarization and an increase in intracellular Ca²⁺ concentration. The CPF peptides show potential for development into agents for the treatment of Type 2 diabetes.     

Primary structures of skin antimicrobial peptides indicate a close,but not conspecific,phylogenetic relationship between the leopard frogs Lithobates onca and Lithobates yavapaiensis (Ranidae)

The phylogenetic relationship between the relict leopard frog Lithobates (Rana) onca (Cope, 1875) and the lowland leopard frog Lithobates (Rana) yavapaiensis (Platz and Frost, 1984) is unclear. Chromatographic analysis of norepinephrine-stimulated skin secretions from L. onca led to the identification of six peptides with antimicrobial activity. Determination of their primary structures indicated that four of the peptides were identical to brevinin-1Ya, brevinin-1Yb, brevinin-1Yc and ranatuerin-2Ya previously isolated from skin secretions of L. yavapaiensis. However, a peptide belonging to the temporin family (temporin-ONa: FLPTFGKILSGLF.NH₂) and an atypical member of the ranatuerin-2 family containing a C-terminal cyclic heptapeptide domain (ranatuerin-2ONa: GLMDTVKNAAKNLAGQMLDKLKCKITGSC) were isolated from the L. onca secretions but were not present in the L. yavapaiensis secretions. Ranatuerin-2ONa inhibited the growth of Escherichia coli (MIC = 50 μM) and Candida albicans (MIC = 100 μM ) and showed hemolytic activity (LC₅₀ = 90 μM) but was inactive against Staphylococcus aureus. The data indicate a close phylogenetic relationship between L. onca and L. yavapaiensis but suggest that they are not conspecific species.     

A recombinant immunosuppressive protein from Pimpla hypochondriaca (rVPr1) increases the susceptibility of Lacanobia oleracea and Mamestra brassicae larvae to Bacillus thuringiensis

The precise mechanisms underlying Bacillus thuringiensis-mediated killing of pest insects are not clear. In some cases, death may be due to septicaemia caused by Bt and/or gut bacteria gaining access to the insect haemocoel. Since insects protect themselves from microbes using an array of cellular and humoral immune defences, we aimed to determine if a recombinant immunosuppressive wasp venom protein (rVPr1) could increase the susceptibility of two pest Lepidoptera (Lacanobia oleracea and Mamestra brassicae) to Bt. Bio-assays indicated that injection of 6 μl of rVPr1 into the haemocoel of both larvae caused similar levels of mortality (less than 38%). On the other hand, the LD_30-40 of Bt for M. brassicae larvae was approximately 20 times higher than that for L. oleracea larvae. Furthermore, in bio-assays where larvae were injected with rVPr1, then fed Bt, a significant reduction in survival of larvae for both species occurred compared to each treatment on its own (P < 0.001); and for L. oleracea larvae, this effect was more than additive. The results are discussed within the context of insect immunity and protection against Bt.     

Haemolymph protein reserves of diapausing and nondiapausing codling moth larvae, Cydia pomonella (L) (Lepidoptera: Tortricidae)

Haemolymph from diapausing codling moth larvae contain a dominant soluble protein, which is of minor significance in the haemolymph of nondiapausing larvae. This haemolymph protein is accumulated during the ultimate larval instar and its concentration is maintained throughout diapause development. Larvae induced into a diapause-like condition following topical treatment with a juvenile hormone mimie stored the haemolymph protein.     

Detecting and differentiating Theileria sergenti and Theileria sinensis in cattle and yaks by PCR based on major piroplasm surface protein (MPSP)

Theileria sergenti and Theileria sinensis are closely related members of benign Theileria species found in cattle and yaks in China. They are morphologically indistinguishable. A polymerase chain reaction (PCR) targeting major piroplasm surface protein of T. sergenti and T. sinensis was developed in this study. The newly developed oligonucleotide primer set was able to specifically amplify the DNA of T. sinensis and in conjunction with primers for T. sergenti and these two species could be detected and distinguished. Specificity testing also revealed that there was no cross-reaction with the other tick-borne diseases Theileria annulata, Babesia ovata, Anaplasma marginale as well as bovine white blood cells. Phylogenetic analysis based on the MPSP gene sequences confirmed the specificity of PCR assays. The sensitivity of the methods was 0.1 pg DNA for the T. sergenti PCR and 1 pg DNA for T. sinensis PCR. Two hundred and thirty-six field blood samples from of cattle and yaks were collected from five different geographical regions in China where benign Theileria species have been found. T. sergenti was found in all five provinces but was absent from one county in Gansu Province. T. sinensis was only found in Gansu Province. In both counties in Gansu where the parasites co-existed, mixed infections were detected. Our results indicate that the PCR methods developed in this study are suitable for the detection and differentiation of T. sergenti and T. sinensis.