Determination of inulin in foods

A method was developed for determining fructan inulin in various foods (yogurts, honey cakes, chocolates). Warm water was applied for extraction of samples, and mono- and dissacharides were determined by a thin-layer chromatographic densitometric method. A portion of the test solution was hydrolyzed 30 min with 1% oxalic acid in a boiling water bath. Fructose was determined in the hydrolysate. The amount of inulin in a sample was calculated as the difference between the amount of fructose in the sample before and after hydrolysis. The fructose from sucrose formed during the hydrolysis was also considered. The mean recovery from yogurt fortified with 4% inulin was 95.5 +/- 4.5% (mean +/- standard deviation); from honey cakes extract fortified with 10% inulin, 97.3 +/- 5.5%; and from chocolate extract fortified with 30% inulin, 98.6 +/- 6.6% (6 replicates in all cases). Determination of glucose is not necessary for analyzing fructans with the composition expressed shortened to GFn-1 (G, glucose; F, fructosyl) with the average degree of polymerization 8 < or = n < or = 15.     

Cloning and functional characterization of a fructan 1-exohydrolase (1-FEH) in edible burdock (<Emphasis Type="Italic">Arctium lappa</Emphasis>L.)

Background  

We have previously reported on the variation of total fructooligosaccharides (FOS), total inulooligosaccharides (IOS) and inulin in the roots of burdock stored at different temperatures. During storage at 0°C, an increase of FOS as a result of the hydrolysis of inulin was observed. Moreover, we suggested that an increase of IOS would likely be due to the synthesis of the IOS by fructosyltransfer from 1-kestose to accumulated fructose and elongated fructose oligomers which can act as acceptors for fructan:fructan 1-fructosyltransferase (1-FFT). However, enzymes such as inulinase or fructan 1-exohydorolase (1-FEH) involved in inulin degradation in burdock roots are still not known. Here, we report the isolation and functional analysis of a gene encoding burdock 1-FEH.     

Ethylenediaminetetraacetic acid (EDTA) as an auxiliary tool in the electrospray ionization mass spectrometry analysis of native and derivatized β-cyclodextrins,maltoses, and fructans contaminated with Ca and/or Mg

The effect of Ca²⁺ (and Mg²⁺) and the disodium salt of ethylenediaminetetraacetic acid (EDTA), a well known Ca²⁺ (and Mg²⁺) chelating agent, on the volatilization/ionization of carbohydrates by using electrospray ionization mass spectrometry has been studied. Model compounds such as maltoses (maltose to maltoheptaose), β-cyclodextrins (β-cyclodextrin, methyl-β-cyclodextrin, heptakis(2,6-di-O-methyl)-β-cyclodextrin, heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin, and 2-hydroxypropyl-β-cyclodextrin) and fructans (sucrose, 1-ketose, nystose, and 1F-fructofuranosylnystose) were used.     

Chemo-enzymatic production of fuel ethanol from cellulosic materials utilizing yeast expressing β-glucosidases

Ethanol was produced in a considerably high yield by fermenting hydrolyzates from cellulosic materials by means of a recombinant laboratory yeast expressing β-glucosidases. Tissue paper, cotton, and sawdust were hydrolyzed by two-step sulfuric acid hydrolysis to give mixtures containing glucose, cellobiose, and higher cello-oligosacc arides. After the cellulosic material was partially hydrolyzed with 80% sulfuric acid, the hydrolysis was continued with 5% sulfuric acid. Except for non-carbohydrate components, all constitutents in the hydrolyzates were fermented by the yeast that was preincubated in the medium that the plasmid encoded by the β-glucosidases gene was kept in the muliplicated yeast. A solution containing 4% hydrolyzates from paper was fermented to give as high as 1.9% maximum ethanol concentration and 70% ethanol conversion. Cotton also gave a similar result. Sawdust was converted into ethanol in approx 22% conversion. Accordingly, it was revealed that the β-glucosidases-expressing yeast can ferment the cello-oligosaccharides obtained by hydrolysis of cellulosic materials into ethanol. In addition, a hydrolyzate containing a high glucose proportion gave a high ethanol concentration in a short time.     

Temperature and pH stability of cellouronic acid

Cellouronic acid (CUA), (1 → 4)-β-d-polyglucuronate sodium salt, was prepared from regenerated cellulose by 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated oxidation in water at pH 10. Changes in chemical structure and degree of polymerization (DP) of CUA by treatment in water under various pH and temperature conditions were studied to evaluate the stability of CUA. No depolymerization occurred on CUA in water at pH 1.0–7.0 and room temperature, while clear depolymerization took place at pH 10 and 13 by β-elimination. When heated in water at >50 °C, CUA was depolymerized by hydrolysis at pH 1.0 and 4.8, and by both hydrolysis and β-elimination at pH 7.0. Kinetic studies showed that CUA depolymerization rate constant was roughly increased with increasing the pH or temperature. Especially, the depolymerization rate constant at pH 13 was approximately 128 and 55 times greater than those at pH 1.0 and 10, respectively, at 60 °C. Activation energies of hydrolysis and β-elimination of CUA were approximately 100 and 20 kJ mol⁻¹, respectively.     

Analysis of high-molecular-weight fructan polymers in crude plant extracts by high-resolution LC-MS

The main water-soluble carbohydrates in temperate forage grasses are polymeric fructans. Fructans consist of fructose chains of various chain lengths attached to sucrose as a core molecule. In grasses, fructans are a complex mixture of a large number of isomeric oligomers with a degree of polymerisation ranging from 3 to >100. Accurate monitoring and unambiguous peak identification requires chromatographic separation coupled to mass spectrometry. The mass range of ion trap mass spectrometers is limited, and we show here how monitoring selected multiply charged ions can be used for the detection and quantification of individual isomers and oligomers of high mass, particularly those of high degree of polymerization (DP > 20) in complex plant extracts. Previously reported methods using linear ion traps with low mass resolution have been shown to be useful for the detection of fructans with a DP up to 49. Here, we report a method using high-resolution mass spectrometry (MS) using an Exactive Orbitrap MS which greatly improves the signal-to-noise ratio and allows the detection of fructans up to DP = 100. High-sugar (HS) Lolium perenne cultivars with high concentrations of these fructans have been shown to be of benefit to the pastoral agricultural industry because they improve rumen nitrogen use efficiency and reduce nitrous oxide emissions from pastures. We demonstrate with our method that these HS grasses not only contain increased amounts of fructans in leaf blades but also accumulate fructans with much higher DP compared to cultivars with normal sugar levels.     

Determination of the hydrothermal degradation products of D-(U-14C) glucose and D-(U-14C) fructose by TLC

The hydrothermal degradation was examined using D-(U-¹⁴C) glucose and D-(U-¹⁴C) fructose. By thin layer chromatography with methylene chloride, tetrahydrofuran (THF), acetic acid −60∶20∶20 as a mobile phase it was, possible to separate and identify the carbohydrates and their reaction products, glyceraldehyde, dihydroxyacetone, methylglyoxal, glycolaldehyde, 5-hydroxymethylfurfural and furfural. Up to 99% of the initial activity was determined by scintillation counting of the TL-chromatograms. A reaction scheme for the hydrothermal degradation of glucose and fructose was obtained from these results.     

Enzymatic hydrolysis of ammonia-treated rice straw

Rice straw pretreated with liquid anhydrous ammonia was hydrolyzed with cellulase, cellobiase, and hemicellulase. Ammonia-processing conditions were 1.5 g of NH₃/g of dry matter, 85°C, and several sample moisture contents. There were four ammonia addition time (min)-processing time (min) combinations. Sugars produced were analyzed as reducing sugars (dinitrosalicylic acid method) and by high-performance liquid chromatography. Monomeric sugars increased from 11% in the nontreated rice straw to 61% of theoretical in treated rice straw (79.2% conversion as reducing sugars). Production of monosaccharides was greater at higher moisture content and was processing time dependent. Glucose was the monosaccharide produced in greater amounts, 56.0%, followed by xylose, arabinose, and fructose, with 35.8, 6.6, and 1.4%, respectively.     

Xylan hydrolysis in zinc chloride solution

Xylan is the major component of hemicellulose, which consists of up to one-third of the lignocellulosic biomass. When the zinc chloride solution was used as a pretreatment agent to facilitate cellulose hydrolysis, hemicellulose was hydrolyzed during the pretreatment stage. In this study, xylan was used as a model to study the hydrolysis of hemicellulose in zinc chloride solution. The degradation of xylose that is released from xylan was reduced by the formation of zinc-xylose complex. The xylose yield was >90% (w/w) at 70°C. The yield and rate of hydrolysis were a function of temperature and the concentration of zinc chloride. The ratio of zinc chloride can be decreased from 9 to 1.3 (w/w). At this ratio, 76% of xylose yield was obtained. When wheat straw was pretreated with a concentrated zinc chloride solution, the hemicellulose hydrolysate contained only xylose and trace amounts of arabinose and oligosaccharides. With this approach, the hemicellulose hydrolysate can be separated from cellulose residue, which would be hydrolyzed subsequently to glucose by acid or enzymes to produce glucose. This production scheme provided a method to produce glucose and xylose in different streams, which can be fermented in separated fermenters.     

Hydrolysis of starch or pullulan by glucoamylase or pullulanase immobilized on poly(N-isopropylacrylamide) gel

Starch or pullulan was hydrolyzed using glucoamylase or pullulanase immobilized on N-isopropylacrylamide gel. The gel used is temperature sensitive; its mesh size becomes smaller at higher temperatures (30 °C) and larger at lower temperatures (20 °C). The molecular weight distribution of starch is wide and it consists of high-molecular-weight amylopectin, amylose and glucose. From the change in the chromatograms for the substrate and products, it was found that the hydrolysis rate at 30 °C was faster than that at 20 °C for amylose, though it was the reverse for amylopectin. This finding suggests that the smaller molecular sized amylose can enter the gel phase at both temperature, while the larger molecular sized amylopectin can hardly do so; only the end group, which can partly enter the gel phase at 20 °C (larger mesh size), was hydrolyzed. Further, several molecular weight pullulans (monodisperse) were hydrolyzed and the experimental chromatograms for substrate and products confirm the hydrolysis mechanism estimated. Received: 14 July 1998 Accepted in revised form: 26 August 1998     

The enzymatic hydrolysis of pretreated agricultural residues and the fermentation of the liberated sugars to ethanol

Agricultural residues were pretreated by steam explosion and the cellulosic component of these substrates were converted to ethanol using a combined enzymatic hydrolysis and fermentation (CHF) process. The enzymatic hydrolysis was carried out using culture filtrates ofTrichoderma harzianum E58 while the liberated sugars were fermented to ethanol byS. cerevisiae. Initially, pretreatment conditions were optimized to ensure that the substrates were readily hydrolyzed and fermented. The agricultural residues were steamed for various times between 30 and 120 s at approximately 240‡C prior to rapid decompression (explosion) in a small masonite-type gun. The various substrates were selectively extracted by water and alkali to see whether the enzymatic hydrolysis and fermentability of the substrates were enhanced. A comparison between the overall conversion of wheat and barley straw was made since these are the two most readily available agricultural residues in Canada. Steam explosion did not affect the hexosan content of the residues, although the pentosan content of the substrates decreased with increasing duration of steaming. The hexosan (cellulose) content of wheat straw was 50.7% of the total substrate while a slightly higher 52.9% cellulose content was detected in the barley straw. Wheat straw was more efficiently hydrolyzed after it had been steamed for 90 s while optimum hydrolysis of the barley straw was detected after 60 s. Steam exploded wheat and barley straw that was subsequently extracted with water was readily hydrolyzed to their component sugars.S. cerevisiae could almost quantitatively convert these sugars to ethanol. This indicated that water washing not only enhanced the enzymatic hydrolysis of the steam exploded substrates, it also removed inhibitory material that restricted the growth of S.cerevisiae. Maximum hydrolysis (78.5%) and ethanol yields (10 mg/mL) were obtained when wheat straw was steamed for 90 s. Slightly lower hydrolysis (76.0%) and ethanol yields (9.5 mg/mL) were obtained with barley straw that had been steamed for 120 s.     

Measurement of total fructan in foods by enzymatic/spectrophotometric method: collaborative study

An AOAC collaborative study was conducted to evaluate the accuracy and reliability of an enzyme assay kit procedure for measuring oligofructans and fructan polysaccharide (inulins) in mixed materials and food products. The sample is extracted with hot water, and an aliquot is treated with a mixture of sucrase (a specific sucrose-degrading enzyme), alpha-amylase, pullulanase, and maltase to hydrolyze sucrose to glucose and fructose, and starch to glucose. These reducing sugars are then reduced to sugar alcohols by treatment with alkaline borohydride solution. The solution is neutralized, and excess borohydride is removed with dilute acetic acid. The fructan is hydrolyzed to fructose and glucose using a mixture of purified exo- and endo-inulinanases (fructanase mixture). The reducing sugars produced (fructose and glucose) are measured with a spectrophotometer after reaction with para-hydroxybenzoic acid hydrazide. The samples analyzed included pure fructan, chocolate, low-fat spread, milk powder, vitamin tablets, onion powder, Jerusalem artichoke flour, wheat stalks, and a sucrose/cellulose control flour. Repeatability relative standard deviations ranged from 2.3 to 7.3%; reproducibility relative standard deviations ranged from 5.0 to 10.8%.     

Comparison of antibody and albumin catalyzed hydrolysis of steroidalp-nitrophenylcarbonates

A monoclonal antibody (MAb) was produced against thep-nitrophenylphosphate derivative of 3α,5β-lithocholic acid, a transition-state analog for hydrolysis of a steroidalp-nitrophenylcarbonate. The indicated reaction was catalyzed by this Ab with kinetic constants k_cat = 4.0 × 10^-2min and K_m = 3.3 μM at pH 9.0 and 35°C. The Ab also hydrolyzed the isomericp-nitrophenylcarbonate of 3β,5β-lithocholic acid with k_cat = 8.4 × 10^-2/min and K_m = 1.0 μM. Bovine serum albumin (BSA) was found to catalyze the same reactions with similar turnover rates and Michaelis constants of 15 and 14 μM, respectively. Although the BSA-catalyzed reaction was only weakly inhibited by the phosphate ester TSA (IC₅₀ ca. 40 μM), the Ab-catalyzed reaction was completely inhibited at less than 1 μM of the TSA. The relative rates and efficiencies of the MAbcatalyzed and BSA-catalyzed reactions are discussed in the context of the hydrophobic sites and intrinsic reactivity of the protein surfaces, and the induction of groups on the Ab to enhance the enzymatic function.     

Comparison of α- and β-trifluoromethylsubstituted acrylic acids in their reactions with thiols

α-(Trifluoromethyl)acrylic acid (1) and γ,γ,γ-trifluorocrotonic acid (2) add AcSH (exothermally and at 100 °C, respectively) in the absence of a catalyst to form products of β-thiolation, which can be easily hydrolyzed to the corresponding β-mercaptoalkanoic acids. Thiols also add regiospecifically to acids1 (in the absence of a catalyst) and2 only in the presence of trifluoromethanesulfonic acid as the catalyst) when heated. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 6, pp. 1230–1232, June, 1997.     

Partial Synthesis of CoαCoβ-Dicyano-176-Norcobinamide

Summary. Recent interest in norvitamin B₁₂-derivatives, homologues of complete vitamin B₁₂-derivatives, lacking the methyl group at carbon 176, stems from the identification of the corrinoid cofactor of the tetrachloroethene reductive dehalogenase of Sulfurospirillum multivorans as 176-nor-pseudovitamin B₁₂. Here we report the partial synthesis of the corrinoid Co_αCo_β-dicyano-176-norcobinamide by condensation of cobyric acid and 2-aminoethanol. In addition, the partial synthesis of crystalline Co_α-aquo-Co_β-cyanocobyric acid by acid catalyzed hydrolysis of vitamin B₁₂ is detailed, improving the method and the isolation procedure worked out earlier by Bernhauer et al. The solution structure of Co_αCo_β-dicyano-176-norcobinamide was studied by spectroscopy and was compared with that of the homologue Co_αCo_β-dicyanocobinamide. The title compound, Co_αCo_β-dicyano-176-norcobinamide, represents the dicyano-form of a potential biosynthetic precursor of the 176-nor-B₁₂-derivatives, such as 176-nor-pseudovitamin B₁₂.     

High solids simultaneous saccharification and fermentation of pretreated wheat straw to ethanol

Wheat straw was pretreated with dilute (0.5%) sulfuric acid at 140°C for 1 h. Pretreated straw solids were washed with deionized water to neutrality and then stored frozen at –20°C. The approximate composition of the pretreated straw solids was 64% cellulose, 33% lignin, and 2% xylan. The cellulose in the pretreated wheat straw solids was converted to ethanol in batch simultaneous saccharification and fermentation experiments at 37°C using cellulase enzyme fromTrichoderma reesei (Genencor 150 L) with or without supplementation with β–glucosidase fromAspergillus niger (Novozyme 188) to produce glucose sugar and the yeastSaccharomyces cerevisiae to ferment the glucose into ethanol. The initial cellulose concentrations were adjusted to 7.5, 10, 12.5, 15, 17.5, and 20% (w/w). Since wheat straw particles do not form slurries at these concentrations and cannot be mixed with conventional impeller mixers used in laboratory fermenters, a simple rotary fermenter was designed and fabricated for these experiments. The results of the simultaneous saccharification and fermentation (SSF) experiments indicate that the cellulose in pretreated wheat straw can be efficiently fermented into ethanol for up to a 15% cellulose concentration (24.4% straw concentration).     

Partial Synthesis of CoαCoβ-Dicyano-176-Norcobinamide

Recent interest in norvitamin B₁₂-derivatives, homologues of complete vitamin B₁₂-derivatives, lacking the methyl group at carbon 176, stems from the identification of the corrinoid cofactor of the tetrachloroethene reductive dehalogenase of Sulfurospirillum multivorans as 176-nor-pseudovitamin B₁₂. Here we report the partial synthesis of the corrinoid Co_αCo_β-dicyano-176-norcobinamide by condensation of cobyric acid and 2-aminoethanol. In addition, the partial synthesis of crystalline Co_α-aquo-Co_β-cyanocobyric acid by acid catalyzed hydrolysis of vitamin B₁₂ is detailed, improving the method and the isolation procedure worked out earlier by Bernhauer et al. The solution structure of Co_αCo_β-dicyano-176-norcobinamide was studied by spectroscopy and was compared with that of the homologue Co_αCo_β-dicyanocobinamide. The title compound, Co_αCo_β-dicyano-176-norcobinamide, represents the dicyano-form of a potential biosynthetic precursor of the 176-nor-B₁₂-derivatives, such as 176-nor-pseudovitamin B₁₂.     

Adding value to the Brazilian sisal: acid hydrolysis of its pulp seeking production of sugars and materials

The present work is inserted into the broad context of the upgrading of lignocellulosic fibers. Sisal was chosen in the present study because more than 50% of the world’s sisal is cultivated in Brazil, it has a short life cycle and its fiber has a high cellulose content. Specifically, in the present study, the subject addressed was the hydrolysis of the sisal pulp, using sulfuric acid as the catalyst. To assess the influence of parameters such as the concentration of the sulfuric acid and the temperature during this process, the pulp was hydrolyzed with various concentrations of sulfuric acid (30–50%) at 70 °C and with 30% acid (v/v) at various temperatures (60–100 °C). During hydrolysis, aliquots were withdrawn from the reaction media, and the solid (non-hydrolyzed pulp) was separated from the liquid (liquor) by filtering each aliquot. The sugar composition of the liquor was analyzed by HPLC, and the non-hydrolyzed pulps were characterized by viscometry (average molar mass), and X-ray diffraction (crystallinity). The results support the following conclusions: acid hydrolysis using 30% H₂SO₄ at 100 °C can produce sisal microcrystalline cellulose and the conditions that led to the largest glucose yield and lowest decomposition rate were 50% H₂SO₄ at 70 °C. In summary, the study of sisal pulp hydrolysis using concentrated acid showed that certain conditions are suitable for high recovery of xylose and good yield of glucose. Moreover, the unreacted cellulose can be targeted for different applications in bio-based materials. A kinetic study based on the glucose yield was performed for all reaction conditions using the kinetic model proposed by Saeman. The results showed that the model adjusted to all 30–35% H₂SO₄ reactions but not to greater concentrations of sulfuric acid. The present study is part of an ongoing research program, and the results reported here will be used as a comparison against the results obtained when using treated sisal pulp as the starting material.     

Pretreatment of Rice Straw by a Hot-Compressed Water Process for Enzymatic Hydrolysis

Hot-compressed water (HCW) is among several cost-effective pretreatment processes of lignocellulosic biomass for enzymatic hydrolysis. The present work investigated the characteristics of HCW pretreatment of rice straw including sugar production and inhibitor formation in the liquid fraction and enzymatic hydrolysis of pretreated material. Pretreatment was carried out at a temperature ranging from 140 to 240 °C for 10 or 30 min. Soluble oligosaccharides were found to constitute almost all the components of total sugars in the liquid fraction. The maximal production of total glucose at 180 °C and below accounted for 4.4–4.9% of glucan in raw material. Total xylose production peaked at 180 °C, accounting for 43.3% of xylan in raw material for 10-min pretreatment and 29.8% for 30-min pretreatment. The production of acetic acid increased at higher temperatures and longer treatment time, indicating more significant disruption of lignocellulosic structure, and furfural production achieved the maximum (2.8 mg/ml) at 200 °C for both 10-min and 30-min processes. The glucose yield by enzymatic hydrolysis of pretreated rice straw was no less than 85% at 180 °C and above for 30-min pretreatment and at 200 °C and above for 10-min pretreatment. Considering sugar recovery, inhibitor formation, and process severity, it is recommended that a temperature of 180 °C for a time of 30 min can be the most efficient process for HCW pretreatment of rice straw.     

Controlled hydrolysis of cheese whey proteins using trypsin and α-chymotrypsin

This study examined the production of protein hydrolysates with controlled composition from cheese whey proteins. Cheese whey was characterized and several hydrolysis experiments were made using whey proteins and purified β-lactoglobulin, assubstrates, and trypsin and α-chymotrypsin, as catalysts, at two tem peratures and several enzyme concentrations. Maximum degrees of hydrolysis obtained experimentally were compared to the theoretical values and peptide compositions were calculated. For trypsin, 100% of yield was achieved; for α-chymotrypsin, hydrolysis seemed to be dependent on the oligopeptide size. The results showed that the two proteases could hydrolyze β-lactoglobulin. Trypsin and α-chymotrypsin were stable at 40°C, but a sharp decrease in the protease activity was observed at 55°C.