The use of chitosan solutions for defatting and clarification of protein hydrolysates]

The possibility of the use of small amounts of chitosan for defatting and clarification of protein solutions prepared by enzymatic hydrolysis was tested. The following treatment conditions were shown to be optimal: chitosan concentration range, from 1.0 to 1.5 g per kg raw weight; pH of precipitation medium, from 8.0 to 8.5; and duration of incubation of protein hydrolysate solution with chitosan, less than 1 h. The hydrolysate defatting grade was found to depend on the degree of chitosan deacetylation. A possible mechanism of the chitosan-induced effects was suggested. The use of chitosan allows the mass fraction of enzyme protein hydrolysates to be reduced fourfold to fivefold.     

Optimum conditions for the precipitation of chitosan oligomers with DP 5–7 in concentrated hydrochloric acid at low temperature

Chitosan was partially hydrolysed with 35% hydrochloric acid for 2 h at 80°C and the hydrolysate stored at −20°C after dilution with water to precipitate higher molecular weight (MW) chitosan oligomers. When the hydrolysate was not diluted with water, no precipitate was formed but 7.3% chitosan oligomers were precipitated at a dilution ratio of 1.0 (ml water/ml hydrolysate). The time for precipitation was not significantly changed after storing the hydrolysate at −20°C for 1 day. In addition, the precipitation yield was not significantly influenced by the concentration of HCl used for the hydrolysis except at less than 5.0 (ml HCl/g chitosan). However, the yield of precipitated oligomers changed with partial hydrolysis time. For 0.5 and 2 h hydrolysis, 10.1 and 7.3% of the oligomers were precipitated, respectively, but only 3.1% of the oligomers were obtained after a 4 h reaction. When methanol was added to the hydrolysate, the precipitation yield increased up to 70% but the amounts of lower MW chitosan oligomers in the precipitated oligomers also increased with the increase of higher MW. The precipitated oligomers were mainly composed of pentamers and hexamers.     

Depolymerization of High-Molecular-Weight Chitosan by the Enzyme Preparation Celloviridine G20x

A low-molecular-weight water-soluble chitosan was obtained from high-molecular-weight crab chitosan using the enzyme preparation Celloviridine G20x. Optimum conditions for enzymatic hydrolysis were designed. The reaction should be performed for 4 h in a sodium-acetate buffer (pH 5.2) at 55°C and an enzyme to substrate ratio of 1 : 400. Fractional extraction of chitosan hydrolysate by aqueous ethanol (ethanol:distilled water) yielded fractions with molecular weights in the range 3.2–26.4 kDa.     

The effect of size and acetylation degree of chitosan derivatives on tobacco plant protection against Phytophthora parasitica nicotianae

Enzymatic degradation of chitosan polymer with Pectinex Ultra SPL was used to obtain derivatives with biological potential as protective agents against Phytophthora parasitica nicotianae (Ppn) in tobacco plants. The 24 h hydrolysate showed the highest Ppn antipathogenic activity and the chitosan native polymer the lowest. The in vitro growth inhibition of several Phytophthora parasitica strains by two chitosans of different DA was compared. While less acetylated chitosan (DA 1%) fully inhibited three P. parasitica strains at the doses 500 and 1000 mg/l the second polymer (DA 36.5%) never completely inhibited such strains. When comparing two polymers of similar molecular weight and different DA, again the highest antipathogenic activity was for the less acetylated polymer. However, degraded chitosan always showed the highest pathogen growth inhibition. Additionally, a bioassay in tobacco seedlings to test plant protection against Ppn by foliar application demonstrated that partially acetylated chitosan and its hydrolysate induced systemic resistance and higher levels of glucanase activity than less acetylated chitosan. Similarly, when treatments were applied as seeds coating before planting, about 46% of plant protection was obtained using chitosan hydrolysate. It was concluded that, while less acetylated and degraded chitosan are better for direct inhibition of pathogen growth, partially acetylated and degraded chitosan are suitable to protect tobacco against P. parasitica by systemic induction of plant resistance.     

Effect of the molecular weight of chitosan on its antiviral activity in plants

The effect of the molecular weight of chitosan on its ability to suppress systemic infection of bean mild mosaic virus in bean (Phaseolus vulgaris L.) plants was studied. The enzymatic hydrolysate of low-molecular-weight chitosan was successively fractionated by ultrafiltration through membranes with decreasing pore size. In total, four chitosan fractions with a weight-average molecular weight varying from 1.2 to 40.4 kDa were obtained. It was shown that the treatments of bean plants with these fractions (chitosan concentration, 10 or 100 μg/ml) inhibited virus accumulation and systemic propagation. The degree of chitosan-induced antiviral resistance increased as the molecular weight of chitosan decreased. The monomers comprising the chitosan molecule—glucosamine and N-acetylglucosamine—exhibited no antiviral activity.     

Depolymerization of high-molecular-weight chitosan by the enzyme preparation Celloviridine G20x

A low-molecular-weight water-soluble chitosan was obtained from high-molecular-weight crab chitosan using the enzyme preparation Celloviridine G20x. Optimum conditions for the enzymatic hydrolysis were designed. The reaction should be performed for 4 h in a sodium-acetate buffer (pH 5.2) at 55 degrees C and the enzyme to substrate ratio of 1:400. Fractional extraction of chitosan hydrolysate by aqueous ethanol (ethanol: distilled water) yielded fractions with molecular weights in the range 3.2-26.4 kDa.     

Chickpea protein hydrolysate as a substitute for serum in cell culture

The growth of mammalian cells in vitro requires the use of rich culture media that are prepared by combining serum with specific nutrient formulations. Serum, the most expensive component of culture media, provides a complex mixture of growth factors and nutrients. Protein hydrolysates that can support in vitro cell growth and eliminate or reduce the need to use serum have been obtained from different sources. Here we describe the use of two food grade proteases to produce a chickpea protein hydrolysate that has been added to cell culture medium in order to determine whether it can be used as a substitute for serum. Medium containing the hydrolysate has been tested using two human cells lines: the monocytic THP-1 cell line which grows in suspension, and the epithelial Caco-2 cell line which grows as a monolayer. The chickpea protein hydrolysate was a good substitute for serum in the first case, but did not allow growth of Caco-2 cells. Supplementation of culture media with this inexpensive and safe hydrolysate would greatly reduce the cost of cell culture.     

Formation of biocompatible nanoparticles by self-assembly of enzymatic hydrolysates of chitosan and carboxymethyl cellulose

A simple preparation method for biocompatible nanoparticles in high concentration (0.5 wt %) by self-assembly of chitosan and carboxymethyl cellulose hydrolysates was developed. Chitosan and carboxymethyl cellulose were hydrolyzed beforehand with chitosanase and cellulase respectively to make fragments having lower molecular weights. Nanoparticles were spontaneously formed only by mixing the two hydrolysate solutions. The particle size distribution was relatively narrow, about 200 nm in mean size. The mean particle size decreased from 226 nm to 165 nm with decreasing molecular weight of chitosan hydrolysate from 9.5 to 6.8 kDa. The mixing ratio of chitosan and carboxymethyl cellulose hydrolysates also affected particle size. Changes in particle size are discussed in relation to a possible mechanism of polyionic complexation. The chitosan-carboxymethyl cellulose nanoparticles were stably suspended over 1 week even under low pH (pH 3.0), high ionic strength (NaCl 1 M), or low temperature (4 degrees C) conditions.     

Rhipicephalus sanguineus: amino acid composition of egg shell

Acid hydrolysis of the protein fraction of a batch of egg shells of Rhipicephalus sanguineus was followed by determination of the amino acids in the hydrolysate. Using thin layer chromatography, the amino acids—lysine, arginine, aspartic acid, serine, glycine, glutamic acid, alanine, threonine, valine, tyrosine, isoleucine, and leucine were identified. Alkaline hydrolysis of the fraction followed by TLC revealed the presence of tryptophane. Chitin was revealed utilizing a chitosan test.     

Effect of the molecular weight of chitosan on its antiviral activity in plants

The effect of the molecular weight of chitosan on its ability to suppress systemic infection of bean mild mosaic virus in bean (Phasoleus vulgaris L.) plants was studied. The enzymatic hydrolysate of low-molecular-weight chitosan was successively fractionated by ultrafiltration through membranes with decreasing pore size. In total, four chitosan fractions with a weight-average molecular weight varying from 1.2 to 40.4 kDa were obtained. It was shown that the treatments of bean plants with these fractions (chitosan concentration, 10 or 100 microg/ml) inhibited virus accumulation and systemic propagation. The degree of chitosan-induced antiviral resistance increased as the molecular weight of chitosan decreased. The monomers comprising the chitosan molecule-glucosamine and N-acetylglucosamine--exhibited no antiviral activity.     

Chitosan production from hemicellulose hydrolysate of corn straw: impact of degradation products on Rhizopus oryzae growth and chitosan fermentation

Aims: To examine the potential use of hemicellulose hydrolysate (HH) for the production of chitosan by Rhizopus oryzae and investigate the influence of contents in HH on mycelia growth and chitosan synthesis. Methods and Results: Compared to xylose medium, HH enhanced mycelia growth, chitosan content and production of R. oryzae by 10·2, 64·5 and 82·1%, respectively. During sulfuric acid hydrolysis of corn straw, sugars (glucose, galactose, etc) and inhibitors (formic acid, acetic acid and furfural) were generated. Acetic acid (2·14 g l^?1) and formic acid (0·83 g l^?1) were stimulative, while furfural (0·55 g l^?1) was inhibitory. Inhibitors, at different concentrations, increased the mycelia growth and chitosan production by 24·5–37·8 and 60·1–207·1%. Conclusions: HH of corn straw is a good source for chitosan production. Inhibitors in HH, at proper concentrations, can enhance chitosan production greatly. Significance and Impact of the Study: This work for the first time reported chitosan production from HH. Chitosan production can be greatly enhanced by cheap chemicals such as inhibitors in HH.     

Enzymatic hydrolysate of geniposide directly acts as cross-linking agent for enzyme immobilization

Enzyme immobilization is a routine biotechnology of many industries such as pharmaceutical, chemical and food. Among the different techniques of enzyme immobilization, cross-linking methods are often used. Geniposide is a natural product extracted from gardenia and its hydrolysate genipin is one of green cross-linking agent for enzyme immobilization, but the environmental pollution and cost of the genipin extraction process have become the main obstacle to its wide application. Enzyme β-glucosidase was immobilized on chitosan by self-catalysis and further used to hydrolyze geniposide. The laccase was immobilized on Nano-SiO₂ through the hydrolysate of geniposide directly acts as cross-linking agent. The simplification of the extraction steps overcomes the obstacles to the widespread use of genipin. Compared with the free laccase, the Nano-SiO₂@laccase exhibited better pH stability and thermal stability. The Nano-SiO₂@laccase was used to degrade Bisphenol A (BPA) and the biodegradation efficiency of the Nano-SiO₂@laccase was 84.3 % after 10 cycles of reusing.     

Protein hydrolysate from visceral waste proteins of Catla (Catla catla): optimization of hydrolysis conditions for a commercial neutral protease

Protein hydrolysate was prepared from visceral waste proteins of an Indian freshwater major carp, Catla catla. Hydrolysis conditions (viz., time, temperature and enzyme to substrate level) for preparing protein hydrolysates from the fish visceral waste proteins using in situ pH of the visceral mass were optimized by response surface methodology (RSM) by employing a factorial design. The regression coefficient close to 1.0, observed during both experimental and validation runs, indicated the validity of prediction model. An enzyme to substrate level of 1.25 % (v/w), temperature of 55 degrees C and a hydrolysis time of 165 min were found to be the optimum conditions to obtain a higher degree of hydrolysis of >48% using multifect-neutral. The amino acid composition of the protein hydrolysate prepared using the optimized conditions revealed that the protein hydrolysate was similar to FAO/WHO reference protein. The chemical scores computed indicated methionine to be the most limiting amino acid. The protein hydrolysate has the potential for application as an ingredient in balanced fish diets.     

Experimental allergic encephalomyelitis. Isolation of basic proteins and polypeptides from central nervous tissue

1. Basic protein (mol.wt. 16500) and polypeptides (mol.wt. 3500) were isolated from bovine spinal cord by a procedure involving defatting, acid extraction of the defatted material and repeated chromatography on Sephadex G-50. Similar fractions were isolated from guinea-pig brain. 2. These fractions produced experimental allergic encephalomyelitis in guinea pigs. 3. The polypeptides appeared to be derived from a basic protein of myelin as a result of the action of an acid proteinase during extraction with acid. Similar proteolysis might also occur in the isolation of other biologically active polypeptides from acetone-dried powders of nervous tissue. The activity of the acid proteinase was lowered by defatting with chloroform-methanol. 4. Peptides from tryptic digests of encephalitogenic polypeptides and protein were also encephalitogenic, which suggests that the encephalitogenic determinant may be quite a short sequence of amino acids. 5. These encephalitogenic polypeptides are further examples of antigens of low molecular weight.     

In vitro antioxidant and antibacterial properties of hydrolysed proteins of delimed tannery fleshings: comparison of acid hydrolysis and fermentation methods

Proteins in delimed tannery fleshings were fermentatively hydrolysed using Enterococcus faecium NCIM5335 and also hydrolysed using mild organic acids (formic acid and propionic acid). The liquor portion containing hydrolysed proteins was spray dried, in both the cases, to obtain a powder. The spray dried powder was evaluated for in vitro antioxidant activities with respect to scavenging different free radicals and antibacterial properties against nine different pathogens. Fermentation and acid hydrolysates scavenged 83 and 75.3% of 2,2-azino-bis-3-ethyl-benzthiazoline-6-sulphonic acid (ABTS) radicals, respectively, at a protein concentration of 0.25 mg. Further, fermentation hydrolysate showed higher 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity of 59% as compared to 56% scavenging by acid hydrolysate at a protein concentration of 5 mg. Acid hydrolysate exhibited lesser (82.3%) peroxy radical scavenging compared to hydrolysate from fermentation (88.2%) at a protein concentration of 10 mg. However, acid hydrolysate exhibited higher (89.2%) superoxide anion scavenging while its fermentation counterpart showed lower activity (85.4%) at 2.5 mg hydrolysate protein. Well as superoxide anion scavenging properties. All the in vitro antioxidant properties exhibited dose dependency. Fermentation hydrolysate exhibited maximum antagonistic activity against Salmonella typhi FB231, from among host of pathogens evaluated. Both the hydrolysates have potential to be ingredients in animal feeds and can help reduce oxidative stress in the animals.     

Interaction of Protein Particles with Lipids in Soybean Milk

The protein in soybean milk exists as 11S and 7S globulins, and the particles formed from them. The lipid content and composition in the protein fractions and effects of defatting on the form of the protein particles were investigated. The size-distribution of protein particles in both raw and heated soybean milk (soymilk) was not influenced by defatting with hexane, but the number of large particles were slightly increased. The protein particles from raw and heated soymilk samples contained 60% and 3% of the total lipid, respectively. Almost all neutral lipid in the particles of raw soymilk was moved to a floating fraction by heating, but a half of the phospholipids was retained in the particles. The protein components from the hexane-defatted meal were similar to those from whole meal, but those from the C-M-de-fatted meal contained remarkably little β-conglycinin. C-M-de-fatting (Removal of polar lipids) caused a reduction in the particulate fraction, and the addition of phospholipids (lecithin) promoted the formation of protein particles.     

Novel Angiotensin I-Converting Enzyme Inhibitory Peptides Found in a Thermolysin-Treated Elastin with Antihypertensive Activity

Angiotensin I-converting enzyme (ACE) inhibitory activity was generated from elastin and collagen by hydrolyzing with thermolysin. The IC₅₀ value of 531.6 µg/mL for ACE inhibition by the elastin hydrolysate was five times less than 2885.1 µg/mL by the collagen hydrolysate. We confirmed the antihypertensive activity of the elastin hydrolysate in vivo by feeding spontaneously hypertensive rats (male) on a diet containing 1% of the elastin hydrolysate for 9 weeks. About 4 week later, the systolic blood pressure of the rats in the elastin hydrolysate group had become significantly lower than that of the control group. We identified novel ACE inhibitory peptides, VGHyp, VVPG and VYPGG, in the elastin hydrolysate by using a protein sequencer and quadrupole linear ion trap (QIT)-LC/MS/MS. VYPGG had the highest IC₅₀ value of 244 µM against ACE and may have potential use as a functional food.     

Induced Resistance to Erwinia carotovora ssp. atroseptica, Through the Treatment of Surface Wounds of Potato Tubers with Elicitors

We have investigated the ability of mixed oligogalacturonides, a cell wall hydrolysate from Phytophthora sojae Hildebrand, and chitosan oligomers to induce resistance to Erwinia carotovora ssp. atroseptica in potato tubers (cv. Pentland Squire). Natural infection by Erwinia in stored potatoes generally originates in the surface tissues which have a markedly different biochemical composition to the perimedullary tissue. We therefore investigated the effect of different elicitors in inducing resistance in 2 mm deep wounds made on the surface of potato tubers. When wounded in this way and stored at 20°C and 100% relative humidity, tubers developed a significant level of natural resistance within 24 h. When the potatoes were treated with oligogalacturonides, or Phytophthora cell wall hydrolysate at the time of wounding and challenged with inoculum 8 h later, a significant reduction in disease occurred. Treatment with the chitosan elicitor had no effect on resistance.     

CFD assessment of the effect of convective mass transport on the intracellular clearance of intracellular triglycerides in macrosteatotic hepatocytes

Donor livers available to transplant for patients with end-stage liver disease are in severe shortage. One possible avenue to expand the donor pool is to recondition livers that would be otherwise discarded due to excessive fat content. Severely steatotic livers (also known as fatty livers) are highly susceptible to ischemia-reperfusion injury and as a result, primary liver non-function post-transplantation. Prior studies in isolated perfused rat livers suggest that “defatting” may be possible in a timeframe of a few hours; thus, it is conceivable that fatty liver grafts could be recovered by machine perfusion to clear stored fat from the organ prior to transplantation. However, studies using hepatoma cells and adult hepatocytes made fatty in culture report that defatting may take several days. Because cell culture studies were done in static conditions, we hypothesized that the defatting kinetics are highly sensitive to flow-mediated transport of metabolites. To investigate this question, we experimentally evaluated the effect of increasing flow rate on the defatting kinetics of cultured HepG2 cells and developed an in silico combined reaction-transport model to identify possible rate-limiting steps in the defatting process. We found that in cultured fatty HepG2 cells, the time required to clear stored fat down to lean control cells can be reduced from 48 to 4–6 h by switching from static to flow conditions. The flow required resulted in a fluid shear of .008 Pa, which did not adversely affect hepatic function. The reaction-transport model suggests that the transport of l-carnitine, which is the carrier responsible for taking free fatty acids into the mitochondria, is the key rate-limiting process in defatting that was modulated by flow. Therefore, we can ensure higher levels of l-carnitine uptake by the cells by choosing flow rates that minimize the limiting mass transport while minimizing shear stress.     

Immobilization of ovomucoid on chitosan

The inhibitory activity of ovomucoid from duck egg white, immobilized on chitosan with the use of glutaraldehyde or carbodiimide as cross-linking agents, was studied. Glutaraldehyde proved to be a more preferable cross-linking agent than carbodiimide. When chitosan is used as a protein carrier, the possibility of shifting the pH optimum of these compounds should be taken into account.