Optimization of simultaneous saccharification and fermentation for the production of ethanol from lignocellulosic biomass

Simultaneous saccharification and fermentation (SSF) of alkaline hydrogen peroxide pretreated Antigonum leptopus (Linn) leaves to ethanol was optimized using cellulase from Trichoderma reesei QM-9414 (Celluclast from Novo) and Saccharomyces cerevisiae NRRL-Y-132 cells. Response surface methodology (RSM) and a three-level four-variable design were employed to evaluate the effects of SSF process variables such as cellulase concentration (20-100 FPU/g of substrate), substrate concentration (5-15% w/v), incubation time (24-72 h), and temperature (35-45 degrees C) on ethanol production efficiency. Cellulase and substrate concentrations were found to be the most significant variables. The optimum conditions arrived at are as follows: cellulase = 100 FPU/g of substrate, substrate = 15% (w/v), incubation time = 57.2 h, and temperature = 38.5 degrees C. At these conditions, the predicted ethanol yield was 3.02% (w/v) and the actual experimental value was 3.0% (w/v).     

Synthesis of structured triacylglycerols containing caproic acid by lipase-catalyzed acidolysis: optimization by response surface methodology.

Production in a batch reactor with a solvent-free system of structured triacylglycerols containing short-chain fatty acids by Lipozyme RM IM-catalyzed acidolysis between rapeseed oil and caproic acid was optimized using response surface methodology (RSM). Reaction time (t(r)), substrate ratio (S(r)), enzyme load (E(l), based on substrate), water content (W(c), based on enzyme), and reaction temperature (T(e)), the five most important parameters for the reaction, were chosen for the optimization. The range of each parameter was selected as follows: t(r) = 5-17 h; E(l) = 6-14 wt %; T(e) = 45-65 degrees C; S(r) = 2-6 mol/mol; and W(c) = 2-12 wt %. The biocatalyst was Lipozyme RM IM, in which Rhizomucor miehei lipase is immobilized on a resin. The incorporation of caproic acid into rapeseed oil was the main monitoring response. In addition, the contents of mono-incorporated structured triacylglycerols and di-incorporated structured triacylglycerols were also evaluated. The optimal reaction conditions for the incorporation of caproic acid and the content of di-incorporated structured triacylglycerols were as follows: t(r) = 17 h; S(r) = 5; E(l) = 14 wt %; W(c) = 10 wt %; T(e) = 65 degrees C. At these conditions, products with 55 mol % incorporation of caproic acid and 55 mol % di-incorporated structured triacylglycerols were obtained.     

Structural characterization of oligosaccharides and polysaccharides from apple juices produced by enzymatic pomace liquefaction

Eight apple pomace liquefaction juices were produced to characterize soluble cell wall material released by the action of pectolytic and cellulolytic enzyme preparations. Very high colloid values from 9.7 to 19.6 g/L were recovered from the juices by ethanol precipitation. The crude polysaccharides consisted mainly of galacturonic acid (49-64 mol %), arabinose (14-23 mol %), galactose (6-15 mol %), and minor amounts of rhamnose, xylose, and glucose. Separation of the polysaccharides by anion-exchange chromatography yielded one neutral, one slightly acidic, and one acidic polymer accounting for 60% of total colloids. Preparative size exclusion chromatography of the acidic fractions resulted in four polymers of different molecular weights and different sugar compositions. Among them, high molecular weight arabinans and rhamnogalacturonans as well as oligomeric fractions consisting of only galacturonic acid could be found. Linkage studies were performed on neutral fractions from anion-exchange chromatography and size exclusion chromatography. They revealed highly branched arabinans, xyloglucans, and mainly type I arabinogalactans.     

Optimization of lycopene extraction from tomato cell suspension culture by response surface methodology

Radioisotope-labeled lycopene is an important tool for biomedical research but currently is not commercially available. A tomato cell suspension culture system for the production of radioisotope-labeled lycopene was previously developed in our laboratory. In the current study, the goal was to optimize the lycopene extraction efficiency from tomato cell cultures for preparatory high-performance liquid chromatography (HPLC) separation. We employed response surface methodology (RSM), which combines fractional factorial design and a second-degree polynomial model. Tomato cells were homogenized with ethanol, saponified by KOH, and extracted with hexane, and the lycopene content was analyzed by HPLC-PDA. We varied five factors at five levels: ethanol volume (1.33-4 mL/g); homogenization period (0-40 s/g); saturated KOH solution volume (0-0.67 mL/g); hexane volume (1.67-3 mL/g); and vortex period (5-25 s/g). Ridge analysis by SAS suggested that the optimal extraction procedure to extract 1 g of tomato cells was at 1.56 mL of ethanol, 28 s homogenization, 0.29 mL of KOH, 2.49 mL of hexane, and 17.5 s vortex. These optimal conditions predicted by RSM were confirmed to enhance lycopene yield from standardized tomato cell cultures by more than 3-fold.     

Preparation of glucosamine from exoskeleton of shrimp and predicting production yield by response surface methodology

Chitin was prepared from Persian Gulf shrimp (Metapenaeus monoceros), and then, the obtained chitin was hydrolyzed by hydrochloric acid solutions. The production yield of glucosamine hydrochloride from chitin was optimized, and the effect of three factors (acid concentration, acid to chitin ratio, and reaction time) was investigated. A Box-Behnken design by Minitab software created 12 reactions with different conditions. Each reaction was performed in two replicates. Response surface methodology was used for predicting the glucosamine preparation. The optimum conditions for glucosamine hydrochloride preparation were 30 and 37% hydrochloric acid, 9:1 (v/w) acid solution to solid ratio, and 4 h of reaction time. Time ratio and time acid concentrations were the effective factors on the yield.     

Evaluation of the simultaneous effects of processing parameters on the iron and zinc solubility of infant sorghum porridge by response surface methodology

The purpose of this study was to improve the micronutrient quality of indigenous African infant flour using traditional techniques available in the region. Response surface methodology was used to study the effect of duration of soaking, germination, and fermentation on phytate and phenolic compounds (PC), pH, viscosity, and the in vitro solubility (IVS) of iron and zinc in infant sorghum flour. The phytate and the PC concentrations of the flour were significantly modified as a result of the duration of germination and fermentation and their mutual interaction. These modifications were accompanied by a significant increase in % IVS Zn after 24 h of sprouting. Except for the interaction of soaking and fermentation, none of the processing parameters exerted a significant effect on the % IVS Fe. The viscosity of the porridge prepared with the flour decreased significantly with the duration of germination, making it possible to produce a porridge with high energy and nutrient density. The use of germination in combination with fermentation is recommended in the processing of cereals for infant feeding in developing countries.     

Enzymatically catalyzed synthesis of low-calorie structured lipid in a solvent-free system: optimization by response surface methodology

A kind of low-calorie structured lipid (LCSL) was obtained by interesterification of tributyrin (TB) and methyl stearate (St-ME), catalyzed by a commercially immobilized 1,3-specific lipase, Lipozyme RM IM from Rhizomucor miehei . The condition optimization of the process was conducted by using response surface methodology (RSM). The optimal conditions for highest conversion of St-ME and lowest content LLL-TAG (SSS and SSP; S, stearic acid; P, palmitic acid) were determined to be a reaction time 6.52 h, a substrate molar ratio (St-ME:TB) of 1.77:1, and an enzyme amount of 10.34% at a reaction temperature of 65 °C; under these conditions, the actually measured conversion of St-ME and content of LLL-TAG were 78.47 and 4.89% respectively, in good agreement with predicted values. The target product under optimal conditions after short-range molecular distillation showed solid fat content (SFC) values similar to those of cocoa butter substitutes (CBS), cocoa butter equivalent (CBE), and cocoa butters (CB), indicating its application for inclusion with other fats as cocoa butter substitutes.     

Controlling molecular weight and degree of deacetylation of chitosan by response surface methodology

Response surface methodology (RSM) was used for controlling molecular weight (MW) and degree of deacetylation (DOD) of chitosan in chemical processing. In a reduced model, MW of chitosan is y = 1736166.406 - 250.745X(1)X(2) - 265.452X(2)X(3), with R( 2) = 0.86, and DOD of chitosan is y = 30.6069 + 0.3396X(1) + 0.4948X(2) + 0.0094X(3)(2), with R( 2) = 0.89. MW of chitosan depends on the crossproduct of temperature and NaOH concentration and the crossproduct of NaOH concentration and time, and DOD depends linearly on temperature and NaOH concentration, and quadratically on time. Chitosan was widely depolymerized in a range from 1,100 kDa to 100 kDa and deacetylated from 67.3 to 95.7% by NaOH alkaline treatment. MW and DOD of chitosan were drastically decreased and increased, respectively, with increase of temperature, reaction time, and NaOH concentration. Furthermore, the rate of MW decrease and DOD increase of chitosan gradually decreased with prolonged reaction time.     

Modeling of lipase-catalyzed acidolysis of sesame oil and caprylic Acid by response surface methodology: optimization of reaction conditions by considering both acyl incorporation and migration

Lipase-catalyzed acidolysis in hexane to produce structured lipids (SLs) from sesame oil and caprylic acid was optimized by considering both total incorporation (Y1) and acyl migration (Y2). Response surface methodology was applied to model Y1 and Y2, respectively, with three reaction parameters: temperature (X1), reaction time (X2), and substrate molar ratio (X3). Well-fitting models for Y1 and Y2 were established after regression analysis with backward elimination and verified by a chi2 test. All factors investigated positively affected Y1. For Y2, X1 showed the greatest positive effect. However, there was no effect of X3. We predicted the levels of Y2 and acyl incorporation into sn-1,3 positions (Y3) based on Y1. The results showed that over the range of ca. 55 mol % of Y1, Y3 started to decrease, and Y2 increased rapidly, suggesting that Y1 should be kept below ca. 55 mol % to prevent decrease in quality and yield of targeted SLs.     

Identification of urolithin a as a metabolite produced by human colon microflora from ellagic acid and related compounds

Dietary ellagic acid and related polyphenols are metabolized in humans to dibenzopyran-6-one derivatives, and the microbial origin of these metabolites has been suggested. However, this has not been demonstrated so far. Fecal samples donated by six volunteers were incubated under anaerobic conditions, and aliquots were used to evaluate the fecal metabolism of ellagic acid, the ellagitannin punicalagin, and an ellagitannin rich extract from walnuts. The isoflavone daidzein was also incubated with the same fecal samples to follow the production of the microbial metabolites previously reported (dihydrogenistein, O-demethylangolensin, and equol) as a positive control of the system and to evaluate similarities between isoflavone and ellagic acid fecal flora metabolism. After fermentation the metabolite "urolithin A" (3,8-dihydroxy-6H-dibenzo[b,d]pyran-6-one) was produced from ellagic acid, punicalagin, and the ellagitannin extract in all the fecal cultures from different volunteers, but with very different production rates and concentrations. This large variability in the concentration of metabolite and kinetics of metabolite production is consistent with the large variability found in the excretion of these metabolites in urine in vivo after human consumption of ellagitannins, and with differences in the composition of the fecal microflora. No correlation between isoflavone and ellagic acid metabolism by fecal microflora was observed. The present study confirms the microbial origin of the recently reported in vivo generated hydroxy-6H-dibenzo[b,d]pyran-6-one derivatives in humans and is a further step in the study of the bioavailability and metabolism of ellagic acid and ellagitannins.     

Optimization of growth of Lactobacillus acidophilus FTCC 0291 and evaluation of growth characteristics in soy whey medium: a response surface methodology approach

Four strains of probiotics were evaluated for their alpha-galactosidase activity. Lactobacillus acidophilus FTCC 0291 displayed the highest specific alpha-galactosidase activity and was thus selected to be optimized in soy whey medium supplemented with seven nitrogen sources. The first-order model showed that meat extract, vegetable extract, and peptone significantly (P < 0.05) influenced the growth of L. acidophilus. The second-order polynomial regression estimated that maximum growth was obtained from the combination of 7.25% (w/v) meat extract, 4.7% (w/v) vegetable extract, and 6.85% (w/v) peptone. The validation experiment showed that response surface methodology was reliable with a variation of only 1.14% from the actual experimental data. Increased utilization of oligosaccharides and reducing sugars contributed to increased growth of L. acidophilus in the soy whey medium. This was accompanied by increased production of short-chain fatty acids and a decrease in pH.     

Production of a novel 9,12-dihydroxy-10(E)-eicosenoic acid from eicosenoic acid by Pseudomonas aeruginosa PR3

Microbial conversions of unsaturated fatty acids often generate polyhydroxy fatty acids, giving them new properties such as higher viscosity and reactivity. A bacterial strain Pseudomonas aeruginosa (PR3) has been intensively studied to produce mono-, di-, and trihydroxy fatty acids from different 9-cis-monoenoic fatty acids such as oleic acid, ricinoleic acid, and palmitoleic acid. However, from the results and the postulated similar metabolic pathways involved in these transformations, it was assumed that the enzyme system involved in transformation of the monoenoic fatty acid by strain PR3 could utilize fatty acids with different chain lengths and locations of the double bond. In this study was used as a substrate for bioconversion by strain PR3 eicosenoic acid (C20:1, ω-9) containing a singular cis double bond at different positions from the carboxyl end as oleic acid, and it was confirmed that PR3 could produce a novel 9,12-dihydroxy-10(E)-eicosenoic acid (DED) with 6.2% yield from eicosenoic acid. The structure of DED was confirmed using GC-MS, FTIR, and NMR analyses. DED production was maximized at 72 h after the substrate was added to the 24 h culture. Some other nutritional factors were also studied for optimal production of DED.     

Phytate degradation by lactic acid bacteria and yeasts during the wholemeal dough fermentation: a 31P NMR study

myo-Inositol hexaphosphate (IP6) is the main source of phosphorus in cereal grains, and therefore, in bakery products. Different microorganisms such as yeasts and lactic acid bacteria have phytase enzymes able to hydrolyze IP6 during the wholemeal breadmaking. In this paper, the phytase activity of Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus curvatus, and Saccharomyces cerevisiae strains, isolated from southern Italian sourdoughs, is assayed using the (31)P NMR technique. The sourdough technology based on the use of lactic acid bacteria in the breadmaking is finally suggested.     

The application of thermal desorption GC/MS with simultaneous olfactory evaluation for the characterization and quantification of odor compounds from a dairy

Few analytical methods exist that combine chemical and sensory analysis of odorous compounds in whole air. Volatile organic compounds were collected by sampling air downwind from a small dairy through sorbent tubes of Tenax TA and Carboxen 569. Samples were analyzed by thermal desorption into a cryotrap and subsequent gas chromatographic separation, followed by simultaneous olfactometry and mass spectrometry. Because compounds are concentrated during sampling, sensory analysis encountered compounds at a concentration 40 times that in air, making this a useful method for identifying trace compounds participating in odor. Twenty odorous and nonodorous compounds were identified and quantified, including straight-chain and aromatic hydrocarbons, chlorinated compounds, alcohols, ketones, aldehydes, and organic acids, at air concentrations of 0.55-320.20 microg/m(3). Compound peaks were characterized by odors ranging from offensive to pleasant, demonstrating the integrative nature of olfaction. This method could be useful in studying many kinds of odors in air.     

Synthesis of conjugated linoleic acid by human-derived Bifidobacterium breve LMC 017: utilization as a functional starter culture for milk fermentation

This study was performed to discover bifidobacteria isolated from human intestines that optimally convert linoleic acid (LA) to conjugated linoleic acid (CLA) and to optimize the culture conditions of milk fermentation. One hundred and fifty neonatal bifidobacteria were screened for CLA-producing ability, and Bifidobacterium breve LMC 017 was selected as it showed about 90% conversion of free LA in MRS broth. The selected strain showed resistance at 0.5% LA in microaerophillic conditions. When monolinolein (LA 90%) was used as a substrate for CLA production, the conversion rate was lower compared to free LA, but the growth rate was unaffected during the milk fermentation. There was no significant difference in CLA production between aerobic and anaerobic conditions, and little decline in CLA was shown after the maximal CLA level had been reached. CLA production increased by 80% with 24 h of incubation in milk containing additional skim milk (5%), where the proteins may have facilitated the production of CLA by enhancing the interaction of substrate with the bacteria. CLA production did not decline after 9 h of fermentation and an additional 12 weeks of storage with other commercial starters. This demonstrates the possibility of using this strain as a costarter in the production of CLA-enriched yogurt.     

Production of fibrinolytic enzyme from Bacillus amyloliquefaciens by fermentation of chickpeas, with the evaluation of the anticoagulant and antioxidant properties of chickpeas

To develop safe and cheap thrombolytic agents, a fibrinolytic enzyme productive strain of LSSE-62 was isolated from Chinese soybean paste. This strain was identified as Bacillus amyloliquefaciens by 16S rDNA sequence analysis. Nucleotide and amino acid sequence analysis showed that this fibrinolytic enzyme was identical to subtilisin DJ-4. Chickpeas were used as the substrate for fibrinolytic enzyme production from B. amyloliquefaciens in solid-state fermentation. Under the optimized conditions (34 °C and 50% initial moisture content), the fibrinolytic activity of fermented chickpeas reached 39.28 fibrin degradation units (FU)/g. Additionally, the fermented chickpeas showed anticoagulant activity, and the purified anticoagulant component showed higher anticoagulant activity than heparin sodium. After fermentation, the total phenolic and total flavonoid contents increased by 222 and 71%, respectively, and then the antioxidant activities were improved significantly. This study provided a novel method for the preparation of multifunctional food of chickpeas or raw materials for the preparation of functional food additives and potential drugs.     

Production of pantothenic acid and thiamine by Azotobacter vinelandii in a chemically defined medium and a dialysed soil medium

The production of pantothenic acid and thiamine by Azotobacter vinelandii was studied in a chemically defined medium and a dialysed soil medium amended with different amounts of carbohydrate and NH _inf4 ^sup+ . Pantothenic acid and thiamine were produced in large amounts in the culture medium containing 2.0–5.0% glucose and 0.1–0.3% NH₄Cl. The results showed that the liberation of these vitamins was increased under adiazotrophic conditions and with an excess of C source.Group of Environmental Microbiology, Department of Microbiology, Faculty of Pharmacy and Institute of Water Research, University of Granada, 18071 Granada, Spain.     

Simultaneous determination of phenolic compounds and saponins in quinoa (Chenopodium quinoa Willd) by a liquid chromatography-diode array detection-electrospray ionization-time-of-flight mass spectrometry methodology

A new liquid chromatography methodology coupled to a diode array detector and a time-of-flight mass spectrometer has been developed for the simultaneous determination of phenolic compounds and saponins in quinoa (Chenopodium quinoa Willd). This method has allowed the simultaneous determination of these two families of compounds with the same analytical method for the first time. A fused-core column C18 has been used, and the analysis has been performed in less than 27 min. Both chromatographic and electrospray ionization time-of-flight mass spectrometry parameters have been optimized to improve the sensitivity and to maximize the number of compounds detected. A validation of the method has also been carried out, and free and bound polar fractions of quinoa have been studied. Twenty-five compounds have been tentatively identified and quantified in the free polar fraction, while five compounds have been tentatively identified and quantified in the bound polar fraction. It is important to highlight that 1-O-galloyl-β-D-glucoside, acacetin, protocatechuic acid 4-O-glucoside, penstebioside, ethyl-m-digallate, (epi)-gallocatechin, and canthoside have been tentatively identified for the first time in quinoa. Free phenolic compounds have been found to be in the range of 2.746-3.803 g/kg of quinoa, while bound phenolic compounds were present in a concentration that varies from 0.139 and 0.164 g/kg. Indeed, saponins have been found to be in a concentration that ranged from 5.6 to 7.5% of the total composition of whole quinoa flour.     

Optimization of simultaneous flavanol, phenolic acid, and anthocyanin extraction from grapes using an experimental design: application to the characterization of champagne grape varieties

Optimization of polyphenol extraction from grape skin, seed, and pulp was performed on Vitis vinifera L. cv. Pinot Noir, by response surface methodology using a Doehlert design. An acidified mixture of acetone/water/methanol was the best solvent for simultaneous extraction of major polyphenol groups from all berry parts, while optimum extraction times and solid-to-liquid ratios varied according to the part. The determined composition from the model agreed with independent experimental results. Analysis of the three Champagne grape varieties showed that proanthocyanidins were the major phenolic compounds in each part (60-93%). The total berry proanthocyanidin content was highest in Pinot Meunier (11 g kg(-1)) and lowest in Chardonnay (5 g kg(-1)), but Pinot Meunier pulp contained lower amounts of proanthocyanidins and phenolic acids (210 and 127 mg kg(-1) berry, respectively) than that of the other two varieties. The berry anthocyanin content was equivalent in both Pinot Noir and Pinot Meunier (632 and 602 mg kg(-1), respectively).     

Release kinetics of volatile organic compounds from roasted and ground coffee: online measurements by PTR-MS and mathematical modeling

The present work shows the possibilities and limitations in modeling release kinetics of volatile organic compounds (VOCs) from roasted and ground coffee by applying physical and empirical models such as the diffusion and Weibull models. The release kinetics of VOCs were measured online by proton transfer reaction-mass spectrometry (PTR-MS). Compounds were identified by GC-MS, and the contribution of the individual compounds to different mass fragments was elucidated by GC/PTR-MS. Coffee samples roasted to different roasting degrees and ground to different particle sizes were studied under dry and wet stripping conditions. To investigate the accuracy of modeling the VOC release kinetics recorded using PTR-MS, online kinetics were compared with kinetics reconstituted from purge and trap samplings. Results showed that uncertainties in ion intensities due to the presence of isobaric species may prevent the development of a robust mathematical model. Of the 20 identified compounds, 5 were affected to a lower extent as their contribution to specific m/z intensity varied by <15% over the stripping time. The kinetics of these compounds were fitted using physical and statistical models, respectively, the diffusion and Weibull models, which helped to identify the underlying release mechanisms. For dry stripping, the diffusion model allowed a good representation of the release kinetics, whereas for wet stripping conditions, release patterns were very complex and almost specific for each compound analyzed. In the case of prewetted coffee, varying particle size (approximately 400-1200 microm) had no significant effect on the VOC release rate, whereas for dry coffee, the release was faster for smaller particles. The absence of particle size effect in wet coffee was attributed to the increase of opened porosity and compound diffusivity by solubilization and matrix relaxation. To conclude, the accurate modeling of VOC release kinetics from coffee allowed small variations in compound release to be discriminated. Furthermore, it evidenced the different aroma compositions that may be obtained depending on the time when VOCs are recovered.