Detection of the cholera toxin-binding activity of kappa-casein macropeptide and optimization of its production by the response surface methodology

The cholera toxin (CT)-binding activity of purified kappa-casein macropeptide (CMP) from bovine kappa-casein was detected. In addition, a statistical model was developed to optimize the production of CMP. CMP was prepared by chymosin hydrolysis of kappa-casein and a subsequent 3% trichloroacetic acid treatment. CMP was further fractionated in an ion-exchange column by FPLC. CT binding activity was eluted at 0.18 M NaCl and was a single 8.9 kDa peptide without tyrosine and arginine residues. The CT binding activity was rapidly lost by a carbohydrase treatment. The conditions for CMP production with chymosin were optimized by using the response surface methodology (RSM). The estimated optimum levels of the factors were as follows: reaction temperature, 38.5 degrees C; pH, 6.44; and time, 35.9 min. A validation experiment was performed in which CMP was prepared under the predicted parameters, and it was ascertained that the estimated optimum conditions gave better production of CMP than any other conditions.     

Physicochemical properties of carbons prepared from pecan shell by phosphoric acid activation

Activated carbons were prepared from pecan shell by phosphoric acid activation. The pore structure and acidic surface groups of these carbons were characterized by nitrogen adsorption, Boehm titration and transmittance Fourier infrared spectroscopy (FTIR) techniques. The characterization results demonstrated that the development of pore structure was apparent at temperatures 250 degrees C, and reached 1130m(2)/g and 0.34cm(3)/g, respectively, at 500 degrees C. Impregnation ratio and soaking time at activation temperature also affected the pore development and pore size distribution of final carbon products. At an impregnation ratio of 1.5, activated carbon with BET surface area and micropore volume as high as 861m(2)/g and 0.289cm(3)/g was obtained at 400 degrees C. Microporous activated carbons were obtained in this study. Low impregnation ratio (less than 1.5) and activation temperature (less than 300 degrees C) are favorable to the formation of acidic surface functional groups, which consist of temperature-sensitive (unstable at high temperature) and temperature-insensitive (stable at high temperature) two parts. The disappearance of temperature-sensitive groups was significant at temperature 300 degrees C; while the temperature-insensitive groups are stable even at 500 degrees C. FTIR results showed that the temperature-insensitive part was mostly phosphorus-containing groups as well as some carbonyl-containing groups, while carbonyl-containing groups were the main contributor of temperature-sensitive part.     

Optimization of alkaline protease production by <Emphasis Type="Italic">Aspergillus clavatus</Emphasis> ES1 in <Emphasis Type="Italic">Mirabilis jalapa</Emphasis> tuber powder using statistical experimental design

Medium composition and culture conditions for the bleaching stable alkaline protease production by Aspergillus clavatus ES1 were optimized. Two statistical methods were used. Plackett-Burman design was applied to find the key ingredients and conditions for the best yield. Response surface methodology (RSM) including full factorial design was used to determine the optimal concentrations and conditions. Results indicated that Mirabilis jalapa tubers powder (MJTP), culture temperature, and initial medium pH had significant effects on the production. Under the proposed optimized conditions, the protease experimental yield (770.66 U/ml) closely matched the yield predicted by the statistical model (749.94 U/ml) with R (2)=0.98. The optimum operating conditions obtained from the RSM were MJTP concentration of 10 g/l, pH 8.0, and temperature of 30 degrees C, Sardinella heads and viscera flour (SHVF) and other salts were used at low level. The medium optimization contributed an about 14.0-fold higher yield than that of the unoptimized medium (starch 5 g/l, yeast extract 2 g/l, temperature 30 degrees C, and pH 6.0; 56 U/ml). More interestingly, the optimization was carried out with the by-product sources, which may result in cost-effective production of alkaline protease by the strain.     

Effect of temperature on the formation and inactivation of syringomycin E pores in human red blood cells and bimolecular lipid membranes

The effects of temperature on the formation and inactivation of syringomycin E (SRE) pores were investigated with human red blood cells (RBCs) and lipid bilayer membranes (BLMs). SRE enhanced the RBC membrane permeability of 86Rb and monomeric hemoglobin in a temperature dependent manner. The kinetics of 86Rb and hemoglobin effluxes were measured at different temperatures and pore formation was found to be only slightly affected, while inactivation was strongly influenced by temperature. At 37 degrees C, SRE pore inactivation began 15 min after and at 20 degrees C, 40 min after SRE addition. At 6 degrees C, below the phase transition temperature of the major lipid components of the RBC membrane, no inactivation occurred for as long as 90 min. With BLMs, SRE induced a large current that remained stable at 14 degrees C, but at 23 degrees C it decreased over time while the single channel conductance and dwell time did not change. The results show that the temperature dependent inactivation of SRE pores is due to a decrease in the number of open pores.     

Kinetics of folding of Escherichia coli OmpA from narrow to large pore conformation in a planar bilayer

The outer membrane protein of Escherichia coli, OmpA, is currently alleged to adopt two native conformations: a major two-domain conformer in which 171 N-terminal residues form a narrow eight beta-barrel pore and 154 C-terminal residues are in the periplasm and a minor one-domain conformer in which all 325 residues create a large pore. However, recent studies in planar bilayers indicate the conformation of OmpA is temperature-sensitive and that increasing temperature converts narrow pores to large pores. Here we examine the reversibility and kinetics of this transition for single OmpA molecules in planar bilayers of diphytanoylphosphatidylcholine (DPhPC). We find that the transition is irreversible. When temperatures are decreased, large pores close down, and when temperatures are stabilized they reopen in the large pore conformation, with gradually increasing open time. Large pores are converted to narrow pores only by denaturing agents. The transition from narrow to large pores requires temperatures >or= 26 degrees C and is a biphasic process with rates that rise steeply with temperature. The first phase, a flickering stepwise transition from a low-conductance to a high-conductance state requires approximately 7 h at 26 degrees C but only approximately 13 min at 42 degrees C, signifying an activation energy of 139 +/- 12 kJ/mol. This is followed by a gradual increase in conductance and open probability, interpreted as optimization of the large pore structure. The results indicate that the two-domain structure is a partially folded intermediate that is kinetically stable at lower temperatures and that mature fully folded OmpA is a large pore.     

Preparation and characterization of activated carbon from palm shell by chemical activation with K2CO3

Palm shell was used to prepare activated carbon using potassium carbonate (K2CO3) as activating agent. The influence of carbonization temperatures (600-1000 degrees C) and impregnation ratios (0.5-2.0) of the prepared activated carbon on the pore development and yield were investigated. Results showed that in all cases, increasing the carbonization temperature and impregnation ratio, the yield decreased, while the adsorption of CO2 increased, progressively. Specific surface area of activated carbon was maximum about 1170 m2/g at 800 degrees C with activation duration of 2 h and at an impregnation ratio of 1.0.     

Pore characteristics of nontypeable Haemophilus influenzae outer membrane protein P5 in planar lipid bilayers

The structure of outer membrane protein P5 of NTHi, a homolog of Escherichia coli OmpA, was investigated by observing its pore characteristics in planar lipid bilayers. Recombinant NTHi P5 was overexpressed in E. coli and purified using ionic detergent, LDS-P5, or nonionic detergent, OG-P5. LDS-P5 and OG-P5 could not be distinguished by their migration on SDS-PAGE gels; however, when incorporated into planar bilayers of DPhPC between symmetric aqueous solutions of 1 M KCl at 22 degrees C, LDS-P5 formed narrow pores (58 +/- 6 pS) with low open probability, whereas OG-P5 formed large pores (1.1 +/- 0.1 nS) with high open probability (0.99). LDS-P5 narrow pores were gradually and irreversibly transformed into large pores, indistinguishable from those formed by OG-P5, at temperatures >or=40 degrees C; the process took 4-6 h at 40 degrees C or 35-45 min at 42 degrees C. Large pores were stable to changes in temperatures; however, large pores were rapidly converted to narrow pores when exposed to LDS at room temperatures, indicating acute sensitivity of this conformer to ionic detergent. These studies suggest that narrow pores are partially denatured forms and support the premise that the native conformation of NTHi P5 is that of a large monomeric pore.     

High surface area activated carbon prepared from cassava peel by chemical activation

Cassava is one of the most important commodities in Indonesia, an agricultural country. Cassava is one of the primary foods in our country and usually used for traditional food, cake, etc. Cassava peel is an agricultural waste from the food and starch processing industries. In this study, this solid waste was used as the precursor for activated carbon preparation. The preparation process consisted of potassium hydroxide impregnation at different impregnation ratio followed by carbonization at 450-750 degrees C for 1-3 h. The results revealed that activation time gives no significant effect on the pore structure of activated carbon produced, however, the pore characteristic of carbon changes significantly with impregnation ratio and carbonization temperature. The maximum surface area and pore volume were obtained at impregnation ratio 5:2 and carbonization temperature 750 degrees C.     

Production of schizophyllan using Schizophyllum commune NRCM

In the present work, four strains were screened for schizophyllan production, of which Schizophyllum commune NRCM was selected for further work. The fermentation was carried out for 168 h at 28+/-2 degrees C on an orbital shaker at 180 rpm. In the first step, one factor-at-a-time method was used to investigate the effect of media constituents such as carbon and nitrogen sources on schizophyllan production. Subsequently in the second step, concentration of the medium components was optimized using Response Surface Method (RSM). The yield increased from 3.25+/-0.72 g/l in the unoptimized media to 8.03+/-1.12 g/l in the medium optimized by RSM.     

Activated carbons prepared from phosphoric acid activation of grain sorghum.

The production of activated carbons from grain sorghum with phosphoric acid activation has been studied by means of two processes, i.e., one-stage and two-stage. The former comprises simultaneous carbonization and activation after impregnation; the latter, the carbonization of the precursor at 300 degrees C for 15 min, followed by the activation of the resultant char after impregnation with phosphoric acid. The preparation conditions, e.g., activation duration, phosphoric acid concentration, and activation temperature, have been varied to determine the optimal processing conditions. The optimal activation conditions for the highest surface areas have been determined to be 600 and 500 degrees C with a phosphoric acid concentration of 35% for the one-stage and two-stage processes, respectively. The two-stage process has been found to greatly enhance the porosity development, especially the microporosity.     

Process parameters study of α-amylase production in a packed-bed bioreactor under solid-state fermentation with possibility of temperature monitoring

Production of α-amylase in a laboratory-scale packed-bed bioreactor by Bacillus sp. KR-8104 under solid-state fermentation (SSF) with possibility of temperature control and monitoring was studied using wheat bran (WB) as a solid substrate. The simultaneous effects of aeration rate, initial substrate moisture, and incubation temperature on α-amylase production were evaluated using response surface methodology (RSM) based on a Box-Behnken design. The optimum conditions for attaining the maximum production of α-amylase were 37°C, 72% (w/w) initial substrate moisture, and 0.15 L/min aeration. The average enzyme activity obtained under the optimized conditions was 473.8 U/g dry fermented substrate. In addition, it was observed that the production of enzyme decreased from the bottom of the bioreactor to the top.     

Optimization of key process variables for enhanced hydrogen production by Enterobacter aerogenes using statistical methods

The individual and mutual effects of glucose concentration, temperature and pH on the hydrogen production by Enterobacter aerogenes were investigated in a batch system. A Box-Behnken design and response surface methodology (RSM) were employed to determine the optimum condition for enhanced hydrogen production. The hydrogen production rate was investigated by simultaneously changing the three independent variables, which all had significant influences on the hydrogen production rate. The maximum hydrogen production rate of 425.8 ml H(2)(g dry cell h)(-1) was obtained under the optimum condition of glucose concentration 118.06 mM, temperature 38 degrees C and pH 6.13. The experimental results showed that the RSM with the Box-Behnken design was a useful tool for achieving high rate of hydrogen production by E. aerogenes.     

Optimization of gelatine extraction from grass carp (Catenopharyngodon idella) fish skin by response surface methodology

To establish the optimum gelatine extraction conditions from grass carp fish skin, response surface methodology (RSM) was adopted in this study. The effects of concentration of HCl (%, A), pre-treatment time (h, B), extraction temperature ( degrees C, C) and extraction time (h, D) were studied. The responses were yield (%) and gel strength (g). A=1.19%, B=24 h, C=52.61 degrees C and D=5.12h were determined as the optimum conditions while the predicted responses were 19.83% yield and 267 g gel strength. Gelling and melting points were 19.5 degrees C and 26.8 degrees C, respectively. Moreover, grass carp gelatine showed high contents of imino acids (proline and hydroxyproline) 19.47%. RSM provided a powerful tool to optimize the extraction parameters and the results may be adapted for industrial extraction of gelatine from grass carp fish skins.     

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.     

Statistical optimization of a high maltose-forming,hyperthermostable and Ca2+-independent alpha-amylase production by an extreme thermophile Geobacillus thermoleovorans using response surface methodology

AIM: Statistical optimization for maximum production of a hyperthermostable, Ca2+-independent and high maltose-forming alpha-amylase by Geobacillus thermoleovorans. METHODS AND RESULTS: G. thermoleovorans was cultivated in 250 ml flasks containing 50 ml of chemically defined glucose-arginine medium (g l(-1): glucose 20; arginine 1.2; riboflavin 150 microg ml(-1); MgSO4. 7H2O 0.2; NaCl 1.0; pH 7.0). The medium was inoculated with 5 h-old bacterial inoculum (1.8x10(8) CFU ml(-1)), and incubated in an incubator shaker at 70 degrees C for 12 h at 200 rev min(-1). The fermentation variables optimized by 'one variable at a time' approach were further optimized by response surface methodology (RSM). The statistical model was obtained using central composite design (CCD) with three variables: glucose, riboflavin and inoculum density. An over all 24 and 70% increase in enzyme production was attained in shake flasks and fermenter because of optimization by RSM, respectively. A good coverage of interactions could also be explained by RSM. The end products of the action of alpha-amylase on starch were maltose (62%), maltotriose (31%) and malto-oligosaccharides (7%). CONCLUSIONS: RSM allowed optimization of medium components and cultural parameters for attaining high yields of alpha-amylase, and further, a good coverage of interactions could be explained. The yield of maltose was higher than maltotriose and malto-oligosaccharides in the starch hydrolysate. SIGNIFICANCE AND IMPACT OF THE STUDY: By applying RSM, critical fermentation variables were optimized rapidly. The starch hydrolysate contained a high proportion of maltose, and therefore, the enzyme can find application in starch saccharification process for the manufacture of high maltose syrups. The use of this enzyme in starch saccharification eliminates the addition of Ca2+.     

Utilization of Response Surface Methodology in Optimization of Proline Extraction from Castanea sativa Honey

Proline constitutes approximately 85 % of the amino acid composition of honey. Therefore, the quantitative determination of this amino acid in honey samples is used by many national/international authorities to evaluate the quality of honey types. In this study, it was aimed to achieve maximum proline amino acid extraction from honey samples whose botanical origins were confirmed by melissopalynological analysis. For this reason, based on three different spectrophotometric methods used in the literature for proline analysis, proline extraction was optimized with the Response Surface Method (RSM) and Box-Behnken experimental design. Three independent variables were determined as treatment time (2, 6, and 10 min), treatment temperature (22, 46, and 70 °C), and cooling time (5, 25, and 45 min). As a result of the optimization, it was seen that only significantly effective independent variable on the proline content of honey was the processing temperature. The optimum conditions obtained as a result of the RSM were found to be 2 min for the treatment time, 70 °C for the treatment temperature and 45 min for the cooling time. The composite desirability of the optimum conditions (R²) was found to be 1.00. It was determined that the method proposed by International Honey Commission (IHC) is efficient for proline analysis, but it provides more proline extraction by reducing of time from 10 min to 2 min in hold time in boiling water bath only during the extraction step. As a result, the conditions to be used in order to achieve maximum proline extraction with different spectrophotometric methods were determined and optimum values were determined. In addition, since the botanical origin of honey samples significantly affects the proline content of honey, it can be suggested that this study be optimized for different monofloral honey samples as well.     

Optimization of the extraction of anthocyanin from Bokbunja (Rubus coreanus Miq.) marc produced during traditional wine processing and characterization of the extracts

The extraction of anthocyanin from Bokbunja (Rubus coreanus Miq.) marc generated during traditional wine processing was optimized using response surface methodology (RSM). A face-centered cube design (FCD) consisting of 17 experimental runs, including five replicates at the center point, was used to investigate the effects of the three variables (solid-liquid ratio, time, and temperature) on anthocyanin extraction, and the results showed that the relationship between the three variables and the total anthocyanin content followed a quadratic model (R2=0.8853). In addition, the RSM analysis predicted that the optimum conditions for extraction consisted of a solid-liquid ratio of 20, a time of 60min, and a temperature of 60 degrees C. Verification tests performed under these optimum conditions gave 34.7+/-1.4mg/100g of anthocyanin, which was close to predicted value of 37.2mg/100g. Additionally, analysis of water extracts prepared using the predicted optimum conditions revealed that the carbohydrates (sugar and pectin) in Bokbunja marc underwent significant variation toward the formation of by-products (glycerol and uronic acids) during yeast fermentation, and that the amount of anthocyanin produced was reduced 10-fold when compared to the original extraction. Further, the results of HPLC-PDA-MS/MS analysis of the anthocyanins extracted from Bokbunja marc revealed the presence of six anthocyanin components, which were tentatively identified as cyanidin 3-O-sambubioside, cyanidin 3-O-xylosylrutinoside, cyanidin 3-O-rutinoside, pelargonidin 3-O-rutinoside, delphinidin 3-O-rutinoside-?, and delphinidin 3-O-glucuronide.     

Exploiting the combined effects of high pressure and moderate heat with nisin on inactivation of Clostridium botulinum spores

In the present work, we studied the combined effects of pressure (300.0-700.0 MPa), temperature (30-70 degrees C) and the presence of nisin (0-333 IU/ml) on the inactivation of Clostridium botulinum 33A spores at various pressure holding times (7.5-17.5 min). Moreover, response surface methodology (RSM) was employed and a quadratic equation for HPP and nisin-induced inactivation was built with RSM. By analyzing the response surface plots and their corresponding contour plots as well as solving the quadratic equation, the experimental values were shown to be significantly in good agreement with predicted values because the adjusted determination coefficient (R(Adj)(2)) was 0.9261 and the level of significance was P<0.0001. The optimum process parameters for a six-log cycle reduction of C. botulinum spores were obtained as: pressure, 545.0 MPa; temperature, 51 degrees C; pressure holding time, 13.3 min; and nisin concentration, 129 IU/ml. The adequacy of the model equation for predicting the optimum response values was verified effectively for 10 test points. Compared to conventional high pressure processing (HPP) techniques, the main process advantages of HPP-nisin combination sterilization in the UHT milk are, lower pressure, natural preservative (nisin), and temperature in a shorter treatment time.     

细菌纤维素发酵培养基的优化及超微观结构分析

为了提高细菌纤维素的产量, 本研究对一株氧化葡糖杆菌菌株J2液体发酵生产细菌纤维素的培养基进行了优化, 并对其代谢的细菌纤维的超微观结构进行了观察。运用Plackett-Burman试验设计法对8个相关影响因素的效应进行了评价, 筛选出了有显著效应的3个因素: 酵母膏、ZnSO4、无水乙醇, 其他5个因素的影响未达到显著水平(P>0.05)。然后采用Box-Behnken的中心组合试验设计和响应面分析方法(RSM)确定了上述三个因素的最佳浓度, 并且以棉纤维为对照, 运用扫描电镜观察了细菌纤维素的超微观结构, 结果表明: 菌株J2利用优化后的发酵培养基生产细菌纤维素的产量为11.52 g/100 mL, 是优化前的1.35倍, 电镜照片显示细菌纤维素微纤维丝直径<0.1 mm, 比棉纤维细很多, NaOH处理可以除去纤维网络结构中的菌体。     

Optimization of enzymatic hydrolysis of visceral waste proteins of Catla (Catla catla) for preparing protein hydrolysate using a commercial protease

Protein hydrolysate was prepared from visceral waste proteins of Catla (Catla catla), an Indian freshwater major carp. Hydrolysis conditions (viz., time, temperature, pH and enzyme to substrate level) for preparing protein hydrolysates from the fish visceral waste proteins were optimized by response surface methodology (RSM) using a factorial design. Model equation was proposed with regard to the effect of time, temperature, pH and enzyme to substrate level. An enzyme to substrate level of 1.5% (v/w), pH 8.5, temperature of 50 degrees C and a hydrolysis time of 135 min were found to be the optimum conditions to obtain a higher degree of hydrolysis close to 50% using alcalase. 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 can well be used to meet the amino acid requirements of juvenile common carp and hence has the potential for application as an ingredient in balanced fish diets.