Influence of manufacturing variables on the characteristics and effectiveness of chitosan products. II. Coagulation of activated sludge suspensions

Chitosan samples manufactured under different conditions were compared for effectiveness of coagulating an activated sludge suspension grown on vegetable canning wastes. Computer analysis of data from Buchner funnel filterability tests resulted in quadratic polynomial equations describing the response curves for volume of filtrate versus dosage, expressed as g/liter chitosan/100 g sludge suspended solids (SSS). The quotient of the filtrate volume and dosage at the inflection points of the equations obtained for 10 test samples and 1 commercial chitosan sample were compared to evaluate the response (effectiveness) per unit amount for each chitosan product. The product made by a standard procedure (deproteinated with 3% NaOH at 100°C for 1 hr, demineralized with 1N HCL at ambient temperature for 30 min, and deacetylated with 50% NaOH at 145–150°C under N₂ for 5 or 15 min) gave the best performance as a coagulating agent for this activated sludge system. Other products, including the commercial preparation, required higher dosages to achieve the same effectiveness. Products deacetylated in the presence of sir rather than nitrogen decreased waste treatment effectiveness, which approximated the trends of reduced viscosity and molecular-weight distribution. The products containing minerals were less effective than products from which minerals had been removed prior to deacetylation, but they were more effective than the enzyme treated sample and the commercial product. In general, although chitosan products obtained after 15 min deacetylation were more effective than those receiving 5 min deacetylation, effectiveness did not correlate linearly with viscosity and molecular-weight distribution trends. However, chitosan products deacetylated for 15 min did show that the higher-molecular-weight products (0.65–1.1 × 10⁶) were more effective coagulating agents for activated sludge than the manufactured product having the lowest molecular weight (0.47 × 10⁶) and the commercial reference sample (0.56 × 10⁶). Thus, higher values for molecular weight were predictive of greater effectiveness for coagulation of activated sludge suspensions.     

Chitosan-based coagulating agents for treatment of cheddar cheese whey

Chitosan-Polyanion (Chi-Pol) complexes were used as coagulating agents for treating Cheddar cheese whey. Complexation and coagulation time played a significant role in adsorption, whereas polymer concentration was significant only for chitosan-alginate complexes. Complexes of chitosan with alginate (ALG), pectin (PEC), and carrageenan (CAR) used at 30 mg complex/L whey showed turbidity reductions of 40-43% and 65-72% after 1 and 39 h, respectively. At 10 mg/L, the percent reduction in turbidity after 1 and 39 h were 35-39% and 61-64%, respectively. No significant differences in turbidity reduction (P > 0.05) were observed when using complexes at different Chi-Pol monomeric mixing ratios (MR) except for Chi-Alg at 30 mg/L, wherein reduction at 0.2 was higher than 0.8 MR. Also, UV-vis spectroscopy suggested the preference of this complex for the absorption of specific whey protein fractions. This study successfully demonstrated the effectiveness of Chi-Pol complexes in flocculation of suspended solid wastes in cheese whey with over 70% protein recovery.     

Influence of manufacturing variables on the characteristics and effectiveness of chitosan products. I. Chemical composition,viscosity, and molecular-weight distribution of chitosan products

Ten chitosan products were manufactured from dry shrimp hulls under differing process conditions and compared to a commercially available product. Manufacturing variables tested were: alkali versus enzymatic deproteination; acid demineralization versus no treatment; air versus nitrogen atmosphere; 5 min vs. 15 min deacetylation period: and varying the particle size of the dry starting material. Deproteination by alkali of enzymatic extraction did not substantially affect the nitrogen and ash compositions of dry chitosan samples. However, the viscosity was reduced in samples deproteinated by enzymatic hydrolysis. Elimination of the demineralization step resulted in products having 31–36% ash, as expected. Some differences in viscosity were observed between deminiralized and undemineralized samples, but on important differences in the molecular-weight distribution of these samples were evident. Purging the reaction vessel with nitrogen resulted in chitosan preparations having higher viscosities and molecular-weight distributions than those prepared in an air atmosphere. The degradative effect of air became more proshrimp hulls to 1 mm prior to any treatment resulted in a chitosan product of both higher viscosity and molecular weight than when ground to either 2 or 6.4 mm. Viscosity was not always a direct indicator of molecular weight, for although the presence of colloidal particles increased the viscosity of some samples, the molecular-weight distribution after filtration was essentially the same as in other less viscous samples.     

On-line sterilization of cheese whey using ultraviolet radiation

The effectiveness of ultraviolet radiation for on-line sterilization of cheese whey was investigated. The effects of flow rate and residence time on the performance of three UV reactors having different gap sizes (18, 13, and 6 mm) were studied. Six flow rates and six residence times were tested with the three UV reactors. The cheese whey used in this study had a very high turbidity (4317 NTU), very poor transmittance in the UV radiation germicidal range ( approximately 0%), and high percentage of large solid particles ( approximately 20% > 100 microm). Although the cheese whey physical characteristics showed low probability of sterilization using UV radiation, the study showed that UV radiation can be used on-line to sterilize cheese whey if the proper reactor gap size and the appropriate residence time are used. There were combined effects of the flow rate and gap size. The cell removal efficiency increased with increases in residence time and decreases in the UV reactor gap size. Removal efficiency of 100% was not achieved in this study with the first UV reactor (18-mm gap size), whereas 100% removal efficiency was achieved with the second (13-mm gap size) and third (6-mm gap size) UV reactors at residence times of 2.0 and 0.5 h, respectively. The microbial decay rates achieved in this study were 4.94, 7.62, and 20.9 h(-)(1) using the first, second, and third UV reactor, respectively. Residence times of 3.3, 2.1, and 0.8 h would be required to completely destruct a microbial population of 5.95 x 10(6) cells/mL using the first, second, and third UV reactors, respectively. Although cheese whey sterilization using UV radiation seems to be a good alternative to pasteurization, increases in cheese whey temperature resulted in lamp fouling. If online sterilization is to be used, the fouling problem should be investigated and a maintenance scheme for the UV reactor should be developed.     

Production of Bacillus thuringiensis Berliner var. kurstaki Grown in Alternative Media

Agro - industrial residues and by - products available in southeastern Brazil were used as ingredients for low - cost culture media for liquid fermentation of Bacillus thuringiensis var. kurstaki. Highest spore yield was obtained with a medium containing cheese whey , soya bean milk and molasses (WSM) . Crystals and spores were produced in all media and potency of the final product was highest for nutrient broth + yeast extract medium (NBY) . There was no correlation between the number of spores in the fermented media and the potency of the preparations . Considering all three factors , the potencies , costs and yields of the final products , lowest relative cost was obtained with BMM medium ( Bombyx mori pupae + molasses) . NBY and WSM had intermediate relative cost approximately nine times higher than BMM . The cost analysis suggests that BMM medium should be preferred for local production of B. thuringiensis var . kurstaki in comparison to other media tested . The results also demonstrate the importance of considering yields , cost and potency of the B. thuringiensis preparations in selecting the production medium .     

Biodegradability evaluation of dairy effluents originated in selected sections of dairy production

Main goal of the study was present the results of some respirometric measurements of activated sludge biodegrading the substrate in the wastewater originated in selected sections of the dairy processing line. The following dairy production effluents were analyzed in the research: the pumping station wastewater (combined wastewater from all the sections of the dairy factory), the apparatus room wastewater, the butter section wastewater, the milk reception point wastewater, the cheese section wastewater and the cottage cheese section wastewater. Apart from that, sweet and sour whey, which are secondary products of hard cheese and cottage cheese production, respectively, was the subject of the research. The amount of organic matter being oxidized during a 5-day measurement session was calculated on 1g of the activated sludge biomass. The research was conducted at the temperature of 20 degrees C and 35 degrees C at the applied sludge loading rate of A'=0.2 g BOD g(-1) dry mass d(-1), which ensured complete biodegradation. The results indicated a correlation between a technological process of dairy processing, an ultimate outcome of which was the wastewater analyzed, and dairy wastewater biodegradability. The results confirmed that all dairy processing effluents can be treated together, with the exception of whey, whose complex biodegradation demands may cause too much burden to any wastewater treatment technological system and thus should be managed within a separate installation.     

Anaerobic digestion of cheese whey using up-flow anaerobic sludge blanket reactor

Anaerobic treatment of cheese whey using a 17·5-litre up-flow anaerobic sludge blanket reactor was investigated in the laboratory. The reactor was studied over a range of influent concentration from 4·5 to 38·1 g chemical oxygen demand per litre at a constant hydraulic retention time of 5 days. The reactor start-up and the sludge acclimatization were discussed. The reactor performance in terms of methane production, volatile fatty acids conversion, sludge net growth and chemical oxygen demand reduction were also presented in this paper. Over 97% chemical oxygen demand reduction was achieved in this experiment. At the influent concentration of 38·1 g chemical oxygen demand per litre, an instability of the reactor was observed. The results indicated that the up-flow anaerobic sludge blanket reactor process could treat cheese whey effectively.     

Characterization and blood coagulation evaluation of the water-soluble chitooligosaccharides prepared by a facile fractionation method

Water-soluble chitooligosaccharides have been reported to have specific biological activities. In this study, the chitosan samples with different degree of acetylation were used separately to prepare chitooligosaccharide (COS) and highly deacetylated chitooligosaccharide (HDCOS) through the nitrous acid depolymerization. Rather than using the conventional fractionation schemes commonly employed, such as dialysis and ultrafiltration which require a large amount of deionized water as well as a fair long dwell time, an unique fractionation scheme is explored to recover and desalt these nitrous-acid depolymerized chitosan with different molecular weights. This fractionation scheme is based on the differential solubility variation of depolymerized products within the aqueous solutions that contain various ratios of methanol. It was noted that chitosan with different molecular weight can be successfully recovered and fractionated with methanol added sequentially up to a volume of four times of original depolmerized product. In addition, chemical characterization of the fractionated water-soluble COS and HDCOS by 1H NMR spectroscopy and diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) indicated that the chitosan depolymerization reaction is greatly influenced by the degree of acetylation of the parental chitosan reactant. Moreover, the modified whole blood clotting time assay and the platelet coagulation test suggested that the 1:2 fractionated water-soluble COS and HDCOS obtained are much less procoagulant than their parental chitosan compound and can be of use in biomedical applications in which blood coagulation is not desired.     

Sterol Composition of the Strobilus of Equisetum arvense L.

“Process whey protein” was prepared by heating bovine milk whey protein isolate solution at neutral pH under salt-free conditions. The process whey protein solution, being clear, was heated at various pHs (2.0 to 11.0) and NaCl concentrations (0 to 200 mM), and the turbidity and gel properties of the products were then examined. For comparison, the properties of the whey protein isolate treated under the same conditions were measured. The whey protein isolate formed a transparent gel or sol below pH 3 and above pH 7 at low NaCl concentration after heating, but the process whey protein formed transparent gels and sols over a wider range of pH and NaCl concentrations than those of the whey protein isolate. More elastic, firmer, and denser gels were obtained from the process whey protein than from the whey protein isolate. The process whey protein provides a novel food material with useful properties.     

Neutrophils exhibit distinct phenotypes toward chitosans with different degrees of deacetylation: implications for cartilage repair

Introduction  

Osteoarthritis is characterized by the progressive destruction of cartilage in the articular joints. Novel therapies that promote resurfacing of exposed bone in focal areas are of interest in osteoarthritis because they may delay the progression of this disabling disease in patients who develop focal lesions. Recently, the addition of 80% deacetylated chitosan to cartilage microfractures was shown to promote the regeneration of hyaline cartilage. The molecular mechanisms by which chitosan promotes cartilage regeneration remain unknown. Because neutrophils are transiently recruited to the microfracture site, the effect of 80% deacetylated chitosan on the function of neutrophils was investigated. Most studies on neutrophils use preparations of chitosan with an uncertain degree of deacetylation. For therapeutic purposes, it is of interest to determine whether the degree of deacetylation influences the response of neutrophils to chitosan. The effect of 95% deacetylated chitosan on the function of neutrophils was therefore also investigated and compared with that of 80% deacetylated chitosan.     

Influence of temperature and pH on the nonenzymatic reduction of triphenyltetrazolium chloride

The effects of temperature and pH on the nonenzymatic (chemical) reduction of triphenyltetrazolium chloride (TTC) to triphenyl formazan (TF) in cheese whey and municipal solid waste compost samples were studied. Ten different incubation temperatures and 13 pH levels were tested. The study showed that the TTC could be reduced nonenzymatically at high temperatures and/or under alkaline pH conditions. The nonenzymatic TTC reduction was observed at pH values greater than 9.5 and 11.0 for the cheese whey and compost, respectively. The TTC chemical reduction rate followed the same trend in both media. The TF content increased with increasing the pH value, reaching its maximum at a pH of 12, then decreased and was not detected at a pH of 13. The TTC was also reduced nonenzymatically at temperatures higher than 70 and 85 degrees C for cheese whey and compost, respectively. Evaporation did not seem to have any significant effect on the TTC chemical reduction since less than 3% of water content was lost at a temperature of 100 degrees C. It was noticed that the TF yield in cheese whey samples was higher than that in compost samples. This was due to the higher moisture content of cheese whey and the presence of copper in the compost samples, which reacted chemically with the TF causing reduction in the red color. For a given incubation period, the effect of pH on the TTC chemical reduction was more significant than the effect of incubation temperature (at a 2 h incubation period, 57.5% and 17.9% of the TTC were chemically reduced at a pH of 12 compared to 10.9% and 7.7% at an incubation temperature of 100 degrees C, for cheese whey and compost, respectively). Among the six metals tested (Ca, Cu, K, Na, Ni, and Zn) only Cu affected the color intensity of the TF. The activation energy of the TTC chemical reduction was 168,808 and 239,102 J/mol in cheese whey and municipal solid compost, respectively. For dehydrogenase activity measurement, the pH of the samples and the incubation temperature should not be higher than 9 and 60 degrees C in order to ensure that the TTC reduction is caused only by the biochemical reaction. Measuring the color intensity of TF in waste samples that contain copper could give misleading results as a result of the formation of formazan copper complex, which reduces the red color.     

Characterization of a Chitosanase from Aspergillus fumigatus ATCC13073

Chitosanase II was purified from the culture filtrate of Aspergillus fumigatus ATCC13073. The purified enzyme had a molecular mass of 23.5 kDa. The N-terminal amino acid sequence of chitosanase II was identical to those of other Aspergillus chitosanases belonging to glycoside hydrolase family 75. The optimum pH and temperature were pH 6.0 and 40 °C. Chitosanase II hydrolyzed 70% deacetylated chitosan faster than fully deacetylated chitosan. Analysis of the degradation products generated from partially N-acetylated chitosan showed that chitosanase II split GlcN-GlcN and GlcNAc-GlcN bonds but not GlcNAc-GlcNAc or GlcN-GlcNAc, suggesting that it is a subclass I chitosanase. It degraded (GlcN)(6) to produce (GlcN)(3) as main product and small amounts of (GlcN)(2) and (GlcN)(4). Reaction rate analyses of mono-N-acetylated chitohexaose suggested that the (+3) site of chitosanase II recognizes the GlcNAc residue rather than the GlcN residue of its substrate.     

Effect of chitin sources on production of chitinase and chitosanase byStreptomyces griseus HUT 6037

The advantage of usingStreptomyces griseus HUT 6037 in the production of chitinase or chitosanase is that the organism is capable of hydrolyzing amorphous or crystal-line chitin and chitosan according to the type of the substrate used. We investigated the effects of the enzyme induction time and chitin sources, CM-chitosan and deacetylated chitosan (degree of deacetylation 75–99%), on production of chitosanase. We found that this strain accumulated chitosanase when cells were grown in the culture medium containing chitosanaceous substrates instead of chitinaceous substrates. The highest chitosanase activity was obtained at 4 days of cultivation with 99% deacetylated chitosan. Soluble chitosan (53% deacetylated chitosan) was found to induce chitinase as well as chitosanase. The specific activities of chitinase and chitosanase were 0.91 and 1.33 U/mg protein at 3 and 5 days, respectively. From the study of the enzymatic digestibility of various degrees of deacetylated chitosan, it was found that (GlcN)₃, (GlcN)₄ and (GlcN)₅ were produced during the enzymatic hydrolysis reaction. The results of this study suggested that the sugar composition of (GlcN)₃ was homogeneous and those of (GlcN)₄ and (GlcN)₅ were heterogeneous.     

Effect of molecular weight of chitosan with the same degree of deacetylation on the thermal, mechanical, and permeability properties of the prepared membrane

Different molecular weight, 90% deacetylated chitosans were obtained by ultrasonic degradation on 90% deacetylated chitosan at 80 °C for various times.

Ninety percent deacetylated chitosan was prepared from alkali treatment of chitin that was obtained from red shrimp waste. Number average-, viscosity average- molecular weights were measured by gel permeation chromatography and the viscometric method, respectively. Degree of deacetylation was measured by the titration method. Enthalpy, maximum melting temperature, tensile strength and elongation of the membranes, flow rate of permeates and water are properties measured to elucidate the effect of molecular weight of chitosan on the above thermal, mechanical, and permeation properties, respectively of the prepared membranes. Results show tensile strength, tensile elongation, and enthalpy of the membrane prepared from high molecular weight chitosans were higher than those from low molecular weight. However, the permeability show membranes prepared from high molecular weight chitosans are lower than that from those of low molecular weight.     

Anaerobic digestion of cheese whey using an upflow anaerobic sludge blanket reactor: III. Sludge and substrate profiles

Anaerobic treatment of cheese whey using a 17·5 litre upflow anaerobic sludge blanket reactor was investigated in the laboratory over a range of influent concentration from 4·5 to 38·1 g COD litre⁻¹ at a constant hydraulic retention time of 5 days. The results indicated that two sludge distribution regions, a sludge bed and a sludge blanket, as well as two distinct reaction phases, acidogenic and methanogenic, were formed. However, as the substrate loading was increased, the acidogenic region extended into the methanogenic region in the upper portion of the reactor until the whole region was acidogenic, leading to the failure of the reactor.     

Einwirkung des immobilisierten Präparates „Sirenin”︁ aus Bacillus mesentericus auf Milch als Substrat

Comparative investigations for the possibilities of milk coagulation by the action of soluble and immobilized (on DEAE-cellulose by adsorption) bacterial enzyme preparation “syrenin” in periods from 30–120 minutes at 5°C are carried out. The milk clotting activities of both preparations are equalized after 40 minutes. The laboratory experiments for producing a bulgarian white brine cheese by the immobilized “syrenin” show a tendency to decrease the losses of substance into the whey and to increase the curdle yields in comparison with these made by soluble preparation.     

Emulsions Based on the Interactions Between Lactoferrin and Chitosans

In this work, purification of lactoferrin from whey was performed with high recovery rate. Lactoferrin was then exploited in the preparation of food emulsions. Two tertiary emulsions, formed by olive oil, lecithin, chitosan, and lactoferrin, were compared: both the emulsions showed similar turbidity and stability. In the secondary emulsion formed by oil/lecithin/chitosan, the pH was increased to 9 before addition of lactoferrin. Then, lactoferrin was added, and the pH was stabilized above pH 9. Lactoferrin was found in amounts of 1 to 2.5 mg/ml in the multiple experiments. A fraction of the added lactoferrin was also present in a milky layer above the emulsion layer. This was, to our knowledge, the first study of emulsions made exploiting the interactions between lactoferrin and chitosan. It was noted that chitosan droplets remained soluble, although the hydrocolloid solubility occurs at pH lower than 5.9. These results showed the feasibility of manufacturing lactoferrin-based emulsions as functional foods.     

Modification of Immobead 150 support for protein immobilization: Effects on the properties of immobilized Aspergillus oryzae β‐galactosidase

We studied the modification of Immobead 150 support by either introducing aldehyde groups using glutaraldehyde (Immobead‐Glu) or carboxyl groups through acid solution (Immobead‐Ac) for enzyme immobilization by covalent attachment or ion exchange, respectively. These two types of immobilization were compared with the use of epoxy groups that are now provided on a commercial support. We used Aspergillus oryzae β‐galactosidase (Gal) as a model protein, immobilizing it on unmodified (epoxy groups, Immobead‐Epx) and modified supports. Immobilization yield and efficiency were tested as a function of protein loading (10–500 mg g^?1 support). Gal was efficiently immobilized on the Immobeads with an immobilization efficiency higher than 75% for almost all supports and protein loads. Immobilization yields significantly decreased when protein loadings were higher than 100 mg g^?1 support. Gal immobilized on Immobead‐Glu and Immobead‐Ac retained approximately 60% of its initial activity after 90 days of storage at 4°C. The three immobilized Gal derivatives presented higher half‐lifes than the soluble enzyme, where the half‐lifes were twice higher than the free Gal at 73°C. All the preparations were moderately operationally stable when tested in lactose solution, whey permeate, cheese whey, and skim milk, and retained approximately 50% of their initial activity after 20 cycles of hydrolyzing lactose solution. The modification of the support with glutaraldehyde provided the most stable derivative during cycling in cheese whey hydrolysis. Our results suggest that the Immobead 150 is a promising support for Gal immobilization. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:934–943, 2018     

Presence of adropin,nesfatin-1, apelin-12, ghrelins and salusins peptides in the milk,cheese whey and plasma of dairy cows

Biological fluids (milk and serum/plasma) and cheese whey milk-derived fluid contain numerous molecules, especially amino acids and proteins. Therefore, the purpose of this study was to find out whether cheese whey (n:6), cow milk (n:6) and its blood (n = 6) have adropin, nesfatin-1, apelin-12, ghrelins and salusin peptides. Adropin, nesfatin-1, apelin-12 concentrations were measured by ELISA, whereas ghrelin and salusin concentrations were measured by EIA methods. It was found that adropin, nesfatin-1, apelin-12, des-acylated ghrelin and salusins in cheese whey were higher than in the corresponding milk peptides and plasma of dairy cows, with the exception of salusin alpha and acylated ghrelin in milk being the same than that of the corresponding cheese whey concentration and plasma of dairy cows. A correlation was also found between milk peptides and cheese whey, as also with plasma of dairy cows. The data suggest that peptides in cow milk might be an important and nutritious food for (neonatal) calves and human diet due to their biological and physiological properties.     

Purification and characterization of a novel chitinase from Burkholderia cepacia strain KH2 isolated from the bed log of Lentinus edodes,Shiitake mushroom

One of the chitinases secreted in the culture filtrate of a gram-negative bacteria, Burkholderia cepacia strain KH2, which was isolated from the bed log of Lentinus edodes, Shiitake mushrooms, was purified by DEAE Sepharose CL-6B chromatography, followed by Sephacryl S-100 HR gel filtration. The purified enzyme was homogenous, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), with an estimated molecular weight of 34,000 and an isoelectric point (pI) of 5.9. The enzyme was stable at pH values of 4.0-6.0, and at temperatures up to 50 degrees C; the optimum pH and temperature were 4.5 and 50 degrees C, respectively. The enzyme exhibited higher activities toward chitosan 7B, a 62% deacetylated chitosan, than toward the highly deacetylated chitosan substrates. The enzyme was observed to drastically hydrolyze partially deacetylated chitin substrates, with the subsequent formation of N-acetylchitooligosaccharides [(GlcNAc) (n), n=2-7]. Separation and quantification of the hydrolysis products of (GlcNAc) (n), n52-6, by HPLC showed the splitting into (GlcNAc)(n), n=3-6. Activity toward N-acetylchitobiose was not detected. Oligomers with a higher number of units than the starting substrate were also detected, which indicate transglycosylation activity.