Quantification and reactivity of cellulose reducing ends : implication for cellulose

The primary purpose of this study was to (1) develop methods for the analysis of and (2) provide information on the chemical nature of reducing ends in typical cellulose substrates used for the study of cellulolytic enzymes. The studies were designed such that values obtained for cellulose substrates were compared with those obtained for a series of soluble cellooligosaccharides. The initial phase of the study tested the validity of using established colorimetric reducing sugar assays, developed for the measurement of reducing sugars in solution, for the quantification of reducing ends on insoluble substrates. The results demonstrate that published methods give widely differing values for the number of reducing ends per unit weight cellulose. The Cu⁺⁺-based assay, using bicinchoninic acid (BCA) as a color yielding chelator of Cu⁺, is shown to provide values that appear most consistent the properties of the substrates. A method was developed using the Cu⁺⁺-BCA reagent, following a mild sodium borohydride treatment, to provide an estimate of the number of solvent accessible reducing ends on insoluble substrates. The kinetics of sodium borohydride reduction of reducing ends on crystalline cellulose, amorphous cellulose and soluble cellooligosaccharides were compared in order to ascertain the relative reactivity of these reducing ends. The apparent second order rate constants for the reduction of reducing ends associated with the crystalline celluloses were significantly lower than those for the reduction of reducing ends associated with either the insoluble amorphous celluloses or the soluble cellooligosaccharides. These results indicate the reducing ends associated with crystalline celluloses are not extended out from the surface as though mimicking solution phase reducing ends. The relevance of this, as well as the other results, to the behavior of cellulolytic enzymes is discussed. The final phase of the study was the demonstration of both a reducing sugar-based and a viscositybased assay for the detection of a prototypical polysaccharide depolymerizing glycosyl hydrolase, polygalacturonase

Assaying the activities of Thermomonospora fusca E₅ and Trichoderma reesei CBHI cellulases bound to polystyrene

In this study the enzymatic activity of adsorbed Thermomonospora fusca E₅ and Trichoderma reesei CBHI cellulases were investigated using fluorescence techniques. In particular, cellulases were allowed to contact hydrophobic polystyrene surfaces under conditions of different solution concentrations, and adsorption times. Each of these variables is known to have a potential effect on enzyme structure and activity at an interface. Enzymatic activity was measured after partial elution of the adsorbed layer with both protein-free buffer and the surfactant, dodecyltrimethylammonium bromide. For E₅ at high concentration (0.5 mg/ml), adsorbed enzyme activity decreased about 20% in increasing adsorption time from 0.25 h to 24 h. At low concentration (0.001 mg/ml), adsorbed enzyme activity decreased by one order of magnitude during a 24 h period. CBHI layers lost activity only after a sufficiently long contact time with the surface, and this effect was not strongly dependent on enzyme concentrations in solution. These findings were explained with reference to structural changes undergone by adsorbed enzyme as a function of time and available interfacial area

Nutritional Quantification and Shelf Life Analysis of Non-thermal Processed Coconut Juice

Nonthermal processing methods are attracted by many food and beverage industry because it can kill the microbial contamination under mild temperatures used in thermal processing; They can sustain flavours, essential nutrients, and vitamins undergo minimal changes. The objective of this research was to evaluate nonthermal processing as the high current impulse generator (HCIG) and investigate the reduction the contaminated microorganism in coconut juice under the batch and continuous treatment of HCIG. The physical, biochemical and nutritional changes of treated coconut juice were also investigated. The application of the direct electricity through cathode of high current impulse generator (HCIG) in 1,170 chambers contained the contaminated coconut juice. Significantly reduction both Saccharomyces cerevisiae and Escherichia coli as 5-log were found when treatments were applied with impulses at 5.17 kA. Comparison of nutritional value of non-thermal processes before and after high current impulse was showed no significant differences between main nutritional values and free amino acid. For the continuous HCIG treatment under the treatment of 5.17 kA current with 9, 15 and 30 pulses with 5 L coconut juice at the flow rate of 1 L/min, results from initial concentration at 1.41 × 105 CFU/mL showed that S. cerevisiae reductions were found 78%, 66% and 96% as increasing number of pulses as 9, 15 and 30 pulses, respectively. The increment of microbial reduction with the increasing number of pulses was also detected as 78 %, 82 % and 96 % from 1.11 × 105 CFU/ml E. coli. Results revealed that the microbial reduction with HCIG under batch treatment were successful preserved the nutritional components of the coconut juice without significant physicochemical changes

Optimization and Characterization Freeze Dried Fish Protein Hydrolysate Production

The production of fish protein hydrolysate from underutilized fish species is attracting the industrial interest for increasing the rich protein values. The central composite design was used to optimize the degree of enzymatic hydrolysis of freeze-dried fish protein hydrolysate (FPH) production by the application of commercial Alcalase on the recovery mixed small fish protein. The effects of time, temperature, pH and enzyme concentration on the degree of hydrolysis (DH) of five strains of fish as Rastrelliger brachysoma (short-bodied mackerel), Rastrelliger kanagurta (Indian mackerel), Leiognathidae (Ponyfish), Amblygaster leiogaster (Smooth belly sardinella) and Selaroides leptolepis (yellow-stripe scad) were experimented. Result showed that the FSH production was optimized at 2.85%v/w enzyme concentration at 61 °C, pH 8.50 for 27 min with 89.42% DH. Mathematical model was proposed and validated under the optimum condition. The high proportion (46.43%) of smaller molecular weight <1 kDa was found in hydrolysate. Freeze-dried fish protein hydrolysate was produced and revealed that three predominant amino acids as glutamine, lysine and alanine. Based on amino acid compositions, the waste fish hydrolysate showed nutritional value and high potential for the applications of feed supplementation

Statistical Approach of Nutrient Optimization for Microalgae Cultivation

The Plackett-Burman Design (PBD) was applied to study fresh water microalgae cultivation using Chlorella sp. TISTR 8411 to select the influential nutrient factors for biomass and lipid production. The PBD for 13 trials from 11 nutrient factors with 3 levels was studied in the mixotrophic cultivation at 28 0C under 16:8 light and dark photoperiods over 7 days of cultivation time. Two influential factors were chosen as glucose and cobalt chloride hexahydrate to further design via Box-Behnken Design (BBD) in order to optimize the cultivation of this microalgae for biodiesel production. The 17 trials of 3 factors and 3 levels of BBD experimental design technique were applied with varying factors of glucose (20-40 g/L), cobalt chloride hexahydrate (0.01-0.04 mg/L) and light intensity (4,500-7,500 Lux) under 16:8 light and dark photoperiods over 7 days of cultivation time at 28 0C. Result showed that Chlorella sp. TISTR 8411 cultivation yield 0.52 g/L biomass and 0.31 g/L lipid production resulting in approximately 60% of lipid production when cultivated in 20.05 g/L glucose, 0.04 mg/L CoCl26H2O under light intensity of 4,614 Lux with the supplementation of 4.38 g/L NaHCO3 coupled with 1 g/L of both NaNO3 and KH2PO4. Under statically mixotrophic cultivation, result indicated that Chlorella sp. TISTR 8411 had potential to produce high lipid content for biodiesel application and biomass production for nutraceutical application. Further experiment with the longer cultivation period up to 2 weeks would implement not only for monitoring the growth kinetics but also evaluating the suitable type of fatty acid production

คุณสมบัติทางเคมีกายภาพโปรตีนไข่ขาวไฮโดรไลเซตแห้งแช่เยือกแข็งที่ย่อยด้วยเอนไซม์นิวเทรสPhysicochemical Characteristics of Freeze Dried Egg White Protein Hydrolyzed by Neutrase

การคัดเลือกโปรตีนไข่ขาวไฮโดรไลเสตไปประยุกต์ใช้ในอุตสาหกรรมอาหารนั้น สมบัติทางเคมีกายภาพด้านอุณหภูมิและค่า pH ส่งผลต่อสมบัติเชิงหน้าที่ทางด้านความสามารถในการละลาย การเกิดโฟม และอิมัลซิไฟเออร์ ถือว่าเป็นปัจจัยสำคัญในการคัดเลือกไปใช้ในอุตสาหกรรม การวิจัยนี้เพื่อศึกษาผลของการย่อยสลายโปรตีนไข่ขาวด้วยเอนไซม์นิวเทรสที่ความเข้มข้นเอมไซม์ร้อยละ 1 (v/v) ที่อุณหภูมิ 45, 50 และ 55 องศาเซลเซียส และ pH 5, 6 และ 7 ในช่วงระยะเวลา 30 นาที ถึง 6 ชั่วโมงผลการศึกษาพบว่าเอนไซม์นิวเทรสสามารถย่อยสลายเพิ่มขึ้นเมื่อระยะเวลาเพิ่มขึ้นโดยมีระดับการย่อยสลายเหมาะสมสูงสุดเท่ากับ 51 เปอร์เซ็นต์ ที่อุณหภูมิ 50 องศาเซลเซียส และ pH 7.0 ซึ่งระดับการย่อยสลายขึ้นอยู่กับความเป็นกรด-ด่าง อุณหภูมิและระยะเวลาของการย่อยสลายอย่างมีนัยสำคัญ การวิเคราะห์การกระจายน้ำหนักโมเลกุลของโปรตีนไฮโดรไลเสตในช่วง1–100 กิโลดาลตัน พบว่ามีสัดส่วนของโปรตีนขนาดเล็กสูงกว่าโปรตีนไข่ขาวที่ไม่ผ่านการย่อยสลายด้วยเอนไซม์ มีสัดส่วนขนาด30–100, 10–30, 5–10, 3–5, 1–3 และน้อยกว่า 1 กิโลดาลตัน 11.60, 9.90, 12.04, 8.54 และ 8.81 เปอร์เซ็นต์ ตามลำดับการศึกษาสมบัติการละลาย การเกิดโฟม ความเสถียรของโฟม และอิมัลซิไฟเออร์ที่ระดับ pH 3.6, 7.6 และ 9.0 ของโปรตีนไข่ขาวไฮโดรไลเซตด้วยเอนไซม์นิวเทรสที่อุณหภูมิ 50 องศาเซลเซียส pH 7 ที่ผ่านการทำแห้งแบบแช่เยือกแข็ง พบว่าสมบัติในการเกิดโฟม ความเสถียรของโฟม และสมบัติอิมัลซิไฟเออร์ขึ้นอยู่กับค่าความเป็นกรด-ด่าง โปรตีนไข่ขาวไฮโดรไลเซตที่มีโปรตีนขนาดเล็กเพิ่มขึ้นส่งผลให้ความสามารถในการละลายและเกิดโฟมเพิ่มขึ้น แต่ความสามารถในการเป็นอิมัลซิไฟเออร์ลดลงThe effects of temperature and pH on the physicochemical attributes as solubility, foaming and emulsifying properties of egg white protein hydrolysate are important functional quantities for selecting applications in food industries. In this research, 1% (v/v) Neutrase was used to hydrolyze egg white protein under various temperatures (45, 50 and 55°C) and pHs (5, 6 and 7) over increasing hydrolysis times (30 min to 6 hours). The effects of Neutrase hydrolysis showed that degree of hydrolysis (DH) increased with increasing hydrolysis times resulting in 51% DH at the optimum temperature of 50°C, pH 7.0. Significantly differences of DH were detected in different pH levels, temperatures and hydrolysis times. The molecular distributions of protein hydrolysate in the ranges of 1–100 kDa at optimum hydrolysis condition were reported. Result showed the smaller size proteins in range of 30–100, 10–30, 5–10, 3–5, 1–3 and &lt;1 kDa of 11.60, 9.90, 12.04, 8.54 and 8.81%, respectively. The freeze dried yield of egg white protein hydrolysate at 50°C, pH 7.0 was further investigated for solubility in pH values, 3.6, 7.6 and 9.0 with the foaming capacity and stability associated to those pH values. The emulsifying activity index of various pHs was also explained. These results indicated that the foaming of egg white hydrolysate increased as increasing solubility; however, emulsifying properties were decreased as decreasing solubility

Renewable Biodiesel Production from Oleaginous Yeast Biomass Using Industrial Wastes

The accumulation lipid from oleaginous microorganisms is recognized as a second generation fuel. Biooil is known to as intracellular product of oily yeast utilizing various carbon substrates and converting different quantities of lipids in the form of triacylglycerols. This second generation fuel can be used to make biodiesel via a transesterification process. This study investigated the morphological characteristics of eight strains of Thai oleaginous yeasts via microscopy and analyzed the fatty acid profiling of yeasts cultured in three carbon sources: glucose, sugar cane molasses and crude glycerol in order to estimate biodiesel properties. To approach this goal, batch fermentations were used to culture eight yeast strains, Rhodosporidium toruloides TISTR 5123, TISTR 5154, TISTR 5149, Yarrowia lipolytica TISTR 5054, TISTR 5151, TISTR 5621, Rhodotorula glutinis TISTR 5159 and Rhodotorula graminis TISTR 5124 for 96 h under 30°C at 250 rpm. Result revealed that eight yeast strains contained significant amounts of fatty acids and lipids and accumulated mainly palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C 18:1) and linoleic acid (C18:2), and they are suitable for the production of biodiesel. Fatty acid productions and profiles indicated that these yeast strains can be potentially used as the triacylglycerols producers for biodiesel production

Statistical design and optimization of nutritional value production by an oleaginous yeast

Abundant by-products from sugar mills as industrial-waste molasses can be used as a carbon source in yeast culture media. Yarrowia lipolytica is an interesting yeast used as a candidate for cultivation in molasses medium. Here, we used response surface methodology to derive a statistical model for the individual and interactive effects of pH, temperature, and shaking speeds on the production of yeast cells. Cultivation conditions of yeast were optimized using Design Expert based on a 23 factorial central composite design (CCD) for maximum yeast cell production. Optimal conditions for maximum Y. lipolytica 5151 cell masses were as follows: pH, 6.45; temperature, 30°C; Shaking speed, 165 rpm. The Design Expert represented the maximal numerical solution with a predicted cell mass production level at 8.96 g/L. The experimental production of Y. lipolytica 5151 cell mass yielded 8.27 g/L that is 7.67% deviated from the model. Whereas, the model of TISTR 5621 was not adequate for prediction. Yeasts cultured under statistic prediction provide 55.94% and 51.25% of total protein. Amino acid content and vitamin B1 (1.06 mg and 1.47 mg per 100 g of dried Y. lipolytica 5151 and 5621, respectively) provided the relevant information for an alternative supplement in aquatic feed