Liberation Of Sugars Research Articles

Immobilization of an alkaline endopolygalacturonase purified from Bacillus paralicheniformis exhibits bioscouring of cotton fabrics.

Pectin degrading enzyme has been increasing interest in an industrial application as biocatalysts, such as juice, textile, and wine industry. Bacillus paralicheniformis CBS3, isolated from popular traditional Korean food (kimchi), produced a novel extracellular thermostable alkaline endopolygalacturonase (BPN3). In this study, BPN3 was purified to 22.04-fold with a recovery yield of 18.93% and specific activity of 2216.41 U/mg by gel filtration and anion exchange column chromatography. The molecular mass of BPN3 was approximately 53kDa as analyzed by SDS-PAGE and pectic zymography. The N-terminal sequence of BPN3 was AIPVILAX. BPN3 was stable over a broad pH range (8.14-11.47), was thermally stable at 50-60°C, and functioned optimally in pH 9.1at 60°C. BPN3 had Km and Vmax values of 0.039mg/mL and 747.9 ± 1.2 U mg- 1, respectively, whereas pectin from apple as substrate. BPN3 activity was remarkably affected by metal ions, modulators, and detergents. Digalacturonic acid (GA2) was the major oligosaccharide produced by hydrolysis of BPN3. Immobilized BPN3 was active over a pH range (8.1-11.5), temperature (50-60)°C, and remained stable with 63.34 and 43.41% of its relative activity during second and third cycle, respectively. Desized cotton exhibited highest reducing sugar liberation through optimized conditions of bioscouring. Bioscouring effectiveness of BPN3 was characterized by the comparison of weight loss for purified BPN3 with commercial pectinase and comparison of BPN3 with grey fabric. BPN3 was simple to purify, had high thermal stability, and was stable over a broad pH range that suggests its suitability for bioscouring application as an industrial catalyst.

Recycling of Dilute Deacetylation Black Liquor to Enable Efficient Recovery and Reuse of Spent Chemicals and Biomass Pretreatment Waste

Deacetylation/dilute alkaline pretreatment followed by mechanical refining (DMR) has been proven as an effective process for biomass sugar liberation without severe chemical modification to lignin. Previous research has been focused on optimizing deacetylation conditions, reducing energy consumptions in mechanical refining, and improving sugar yields and titers in enzymatic hydrolysis. To successfully commercialize this process, another critical challenge is to develop a robust process to balance water usage, recover spent chemicals and utilize waste carbons from the dilute deacetylation waste liquor. In this work, a new process modification and strategy is pioneered to recycle and reuse the weak black liquor (WBL) in order to reduce water, chemical, and energy usage while increasing both inorganic and organic contents in the WBLto facilitate downstream processing. Results suggest that the accumulation did not lower acetyl and lignin removal in alkaline pretreatment, resulting in comparable sugar yields in enzymatic hydrolysis. Sodium and potassium were found to be the two most important inorganic compounds in the recycled weak black liquor. Moreover, the accumulated sodium and phenolic compounds did not inhibit the downstream ethanol fermentation processes. Finally, techno-economic analysis (TEA) showed a decrease in the minimum ethanol selling price (MESP) by approximately 5 to 15 cents per gallon of ethanol resulting from the inclusion of the recycling of weak black liquor when compared to a conventional non-recycling process.

Microbial Metabolic Networks at the Mucus Layer Lead to Diet-Independent Butyrate and Vitamin B12 Production by Intestinal Symbionts.

ABSTRACTAkkermansia muciniphila has evolved to specialize in the degradation and utilization of host mucus, which it may use as the sole source of carbon and nitrogen. Mucus degradation and fermentation by A. muciniphila are known to result in the liberation of oligosaccharides and subsequent production of acetate, which becomes directly available to microorganisms in the vicinity of the intestinal mucosa. Coculturing experiments of A. muciniphila with non-mucus-degrading butyrate-producing bacteria Anaerostipes caccae, Eubacterium hallii, and Faecalibacterium prausnitzii resulted in syntrophic growth and production of butyrate. In addition, we demonstrate that the production of pseudovitamin B12 by E. hallii results in production of propionate by A. muciniphila, which suggests that this syntrophy is indeed bidirectional. These data are proof of concept for syntrophic and other symbiotic microbe-microbe interactions at the intestinal mucosal interface. The observed metabolic interactions between A. muciniphila and butyrogenic bacterial taxa support the existence of colonic vitamin and butyrate production pathways that are dependent on host glycan production and independent of dietary carbohydrates. We infer that the intestinal symbiont A. muciniphila can indirectly stimulate intestinal butyrate levels in the vicinity of the intestinal epithelial cells with potential health benefits to the host.

Penicillium echinulatum secretome analysis reveals the fungi potential for degradation of lignocellulosic biomass.

BackgroundThe enzymatic degradation of lignocellulosic materials by fungal enzyme systems has been extensively studied due to its effectiveness in the liberation of fermentable sugars for bioethanol production. Recently, variants of the fungus Penicillium echinulatum have been described as a great producer of cellulases and considered a promising strain for the bioethanol industry.ResultsPenicillium echinulatum, wild-type 2HH and its mutant strain S1M29, were grown on four different carbon sources: cellulose, sugar cane bagasse pretreated by steam explosion (SCB), glucose, and glycerol for 120 h. Samples collected at 24, 96, and 120 h were used for enzymatic measurement, and the 96-h one was also used for secretome analysis by 1D-PAGE LC–MS/MS. A total of 165 proteins were identified, and more than one-third of these proteins belong to CAZy families. Glycosyl hydrolases (GH) are the most abundant group, being represented in larger quantities by GH3, 5, 17, 43, and 72. Cellobiohydrolases, endoglucanases, β-glycosidases, xylanases, β-xylosidases, and mannanases were found, and in minor quantities, pectinases, ligninases, and amylases were also found. Swollenin and esterases were also identified.ConclusionsOur study revealed differences in the two strains of P. echinulatum in several aspects in which the mutation improved the production of enzymes related to lignocellulosic biomass deconstruction. Considering the spectral counting analysis, the mutant strain S1M29 was more efficient in the production of enzymes involved in cellulose and hemicellulose degradation, despite having a nearly identical CAZy enzymatic repertoire. Moreover, S1M29 secretes more quantities of protein on SCB than on cellulose, relevant information when considering the production of cellulases using raw materials at low cost. Glucose, and especially glycerol, were used mainly for the production of amylases and ligninases.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0476-3) contains supplementary material, which is available to authorized users.

Evaluation of different lignocellulosic biomass pretreatments by phenotypic microarray-based metabolic analysis of fermenting yeast

Advanced generation biofuel production from lignocellulosic material (LCM) was investigated. A range of different thermo-chemical pre-treatments were evaluated with different LCM. The pre-treatments included; alkaline (5% NaOH at 50°C), acid (1% H2SO4 at 121°C) and autohydrolytical methods (200°C aqueous based hydrothermal) and were evaluated using samples of miscanthus, wheat-straw and willow. The liberation of sugars, presence of inhibitory compounds, and the degree of enhancement of enzymatic saccharification was accessed. The suitability of the pre-treatment generated hydrolysates (as bioethanol feedstocks for Saccharomyces cerevisiae) was also accessed using a phenotypic microarray that measured yeast metabolic output. The use of the alkaline pre-treatment liberated more glucose and arabinose into both the pre-treatment generated hydrolysate and also the hydrolysate produced after enzymatic hydrolysis (when compared with other pre-treatments). However, hydrolysates derived from use of alkaline pre-treatments were shown to be unsuitable as a fermentation medium due to issues with colloidal stability (high viscosity). Use of acid or autohydrolytical pre-treatments liberated high concentrations of monosaccharides regardless of the LCM used and the hydrolysates had good fermentation performance with measurable yeast metabolic output. Acid pre-treated wheat straw hydrolysates were then used as a model system for larger scale fermentations to confirm both the results of the phenotypic microarray and its validity as an effective high-throughput screening tool.

Liberation of fermentable sugars from soybean hull biomass using ionic liquid 1-butyl-3-methylimidazolium acetate and their bioconversion to ethanol.

Optimized hydrolysis of lignocellulosic waste biomass is essential to achieve the liberation of sugars to be used in fermentation process. Ionic liquids (ILs), a new class of solvents, have been tested in the pretreatment of cellulosic materials to improve the subsequent enzymatic hydrolysis of the biomass. Optimized application of ILs on biomass is important to advance the use of this technology. In this research, we investigated the effects of using 1-butyl-3-methylimidazolium acetate ([bmim][Ac]) on the decomposition of soybean hull, an abundant cellulosic industrial waste. Reaction aspects of temperature, incubation time, IL concentration, and solid load were optimized before carrying out the enzymatic hydrolysis of this residue to liberate fermentable glucose. Optimal conditions were found to be 75°C, 165 min incubation time, 57% (mass fraction) of [bmim][Ac], and 12.5% solid loading. Pretreated soybean hull lost its crystallinity, which eased enzymatic hydrolysis, confirmed by Fourier Transform Infrared analysis. The enzymatic hydrolysis of the biomass using an enzyme complex from Penicillium echinulatum liberated 92% of glucose from the cellulose matrix. The hydrolysate was free of any toxic compounds, such as hydroxymethylfurfural and furfural. The obtained hydrolysate was tested for fermentation using Candida shehatae HM 52.2, which was able to convert glucose to ethanol at yields of 0.31. These results suggest the possible use of ILs for the pretreatment of some lignocellulosic waste materials, avoiding the formation of toxic compounds, to be used in second-generation ethanol production and other fermentation processes. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:312-320, 2016.

Optimising the (Microwave) Hydrothermal Pretreatment of Brewers Spent Grains for Bioethanol Production

For the production of bioethanol from lignocellulosic biomass, it is important to optimise the thermochemical pretreatment which is required to facilitate subsequent liberation of monomeric sugars. Here, we report optimisation of pretreatment conditions for brewers spent grains (BSG) with the main objectives of (1) working at commercially relevant high solids content, (2) minimising energy and chemical inputs, and (3) maximising downstream sugar yields. Studies indicated there to be a play-off between pretreatment solids content, the usage of an acid catalyst, and pretreatment temperature. For example, yields of 80–90% theoretical glucose could be obtained following pretreatment at 35% w/v solids and 200°C, or at 140–160°C with addition of 1% HCl. However, at very high solids loadings (40–50% w/v) temperatures of 180–200°C were necessary to attain comparable sugar yields, even with an acid catalyst. The feasibility of producing bioethanol from feedstocks generated using these protocols was demonstrated (but not optimised) at laboratory scale.

A novel expansin protein from the white-rot fungus Schizophyllum commune.

A novel expansin protein (ScExlx1) was found, cloned and expressed from the Basidiomycete fungus Schizophylum commune. This protein showed the canonical features of plant expansins. ScExlx1 showed the ability to form “bubbles” in cotton fibers, reduce the size of avicel particles and enhance reducing sugar liberation from cotton fibers pretreated with the protein and then treated with cellulases. ScExlx1 was able to bind cellulose, birchwood xylan and chitin and this property was not affected by different sodium chloride concentrations. A novel property of ScExlx1 is its capacity to enhance reducing sugars (N-acetyl glucosamine) liberation from pretreated chitin and further added with chitinase, which has not been reported for any expansin or expansin-like protein. To the best of our knowledge, this is the first report of a bona fide fungal expansin found in a basidiomycete and we could express the bioactive protein in Pichia pastoris.

Optimization of wastewater microalgae saccharification using dilute acid hydrolysis for acetone, butanol, and ethanol fermentation

Exploring and developing sustainable and efficient technologies for biofuel production are crucial for averting global consequences associated with fuel shortages and climate change. Optimization of sugar liberation from wastewater algae through acid hydrolysis was determined for subsequent fermentation to acetone, butanol, and ethanol (ABE) by Clostridium saccharoperbutylacetonicum N1-4. Acid concentration, retention time, and temperature were evaluated to determine optimal hydrolysis conditions by assessing the sugar and ABE yield as well as the associated costs. Sulfuric acid concentrations ranging from 0 to 1.5M, retention times of 40–120min, and temperatures from 23°C to 90°C were combined to form a full factorial experiment. Acid hydrolysis pretreatment of 10% dried wastewater microalgae using 1.0M sulfuric acid for 120min at 80–90°C was found to be the optimal parameters, with a sugar yield of 166.1g for kg of dry algae, concentrations of 5.23g/L of total ABE, and 3.74g/L of butanol at a rate of USD $12.54 per kg of butanol.

In Vitro Digestibility of Dockounou, a Traditional Plantain Derivate Dish of C?te d’Ivoire

The aim of this study was to identify sugar products from dockounou digestion. Tests of digestibility on various dockounou using a digestive juice of young snails namly Archachatina ventricosa and experimental acid hydrolysis were used. The results had showed that boiled dockounou were more hydrolysis than the backed one. Specifically, the rice boiled dockounou was more hydrolyzed than the maize boiled dockounou. Both rice boiled an baked dockounou are more hydrolyzed than the maize dockounou did. The sugars liberated for rice backed dockounou range from 0 to 0.80 µmol thereby it’s 0 to 2.40 µmol for maize one. These results are significantly differents (p≤0.05). Enzymatic or acid hydrolysis products of boiled and backed dockounou are only the glucose. The intensity of this reducing sugar liberation depends on the time. This information shows, in the whole, that dockounou is an energetic dishe for children neither for diabetics.

Biohydrogen production from ricebran using Clostridium saccharoperbutylacetonicum N1-4

Treated ricebran hydrolysate was fermented anaerobically using Clostridium saccharoperbutylacetonicum N1-4 at an initial pH of 6 ± 0.2 and an operating temperature of 30 °C for production of hydrogen. The effects of different pretreatment methods on the liberation of sugar from 100 g of ricebran per litre of medium (distilled water) were investigated. In addition, the effects of the pretreatment method on ricebran hydrolysates of different initial ricebran concentrations on liberated sugar as well as the effects of the initial inoculum concentration, ricebran (substrate) concentration, and FeSO4·7H2O concentration on the yield as well as the productivity of hydrogen were investigated. The combination of enzymatic hydrolysis and a boiling pretreatment method produced the most fermentable sugar, 29.03 ± 0.0 g/L from 100 g of ricebran per litre of medium (distilled water), while the amount of sugar liberated by ricebran hydrolysates of different initial ricebran concentrations upon pretreatment monotonically increased with the initial ricebran concentration. The increment in substrate, inoculum, and FeSO4·7H2O concentrations had a significantly positive effect (p < 0.05) on both the yield and productivity of hydrogen. The maximum hydrogen gas yield (YP/S) and productivity of 3.37 mol-H2 per mol-sugar consumed and 7.58 mmol/(L h), respectively, were obtained from ricebran hydrolysate with a 100 g/L ricebran concentration (equivalent to 28.59 ± 1.27 g sugar/L). In other experiments, 0.03 g/L FeSO4·7H2O and 1.5 g/L inoculum resulted in the best hydrogen gas yield and productivity from ricebran hydrolysates.

Comparison of Bifidobacterium breve strain Yakult transcriptomes in germ-free mice with those in fecal cultures

Bifidobacteria are beneficial to human health, but the mechanism remains unknown. We employed oligonucleotide microarrays to identify the Bifidobacterium breve strain Yakult (BbrY) genes up-regulated specifically in mouse intestine. Based on BbrY transcriptional responses in germ-free mice and in fecal cultures, k-means clustering picked up 93 genes that were up-regulated in the mouse intestine and thereafter Venn analysis to exclude genes that were up-regulated in both the mouse intestine and the fecal culture classified 45 genes as up-regulated specifically in the mouse intestine. Most of those genes are involved in sugar transport or sugar liberation, although the functions of several genes are unknown. Most of these genes are clustered on the BbrY genome and appear to be organized into operons. Expressions of several genes were further investigated by real time PCR, revealing that their expression profiles were identical in the mouse cecum and colon. The up-regulation of genes involved in sugar liberalization and uptake suggests that BbrY could possibly maintain energy homeostasis inside the mouse intestine, which contains low quantities of readily fermentable sugars.

암모니아 Soaking 방법을 이용한 섬유소계 바이오매스의 전처리 특성

Liberation of fermentable sugars from lignocellulosic biomass is one of the key challenges in production ofcellulosic ethanol. Aqueous ammonia cleaves ether and ester bonds in lignin carbohydrate complexes. It is an effectiveswelling reagent for lignocellulosic biomass. The aqueous ammonia pretreatment selectively reduces the lignin contentof biomass. However, at high temperatures, this process solubilizes more than 50% of the hemicellulose in the biomass.Here we conducted a SAA(Soaking in Aqueous Ammonia) process by moderate reaction temperatures at atmosphericpressure using various lignocellulosicbiomass. The optimum condition of this process was 15 wt% of aqueous ammoniaat 50 of reaction time during 72 hr. The delignification was up to 60% basis on initial biomass and the enzymatic digest-ibility was 60~90% for agricultural biomass, respectively. Key words: Biomass Pretreatment, SAA, Enzymatic Hydrolysis, Bio Ethanol

Two-stage pretreatment of rice straw using aqueous ammonia and dilute acid

Liberation of fermentable sugars from recalcitrant lignocellulosic biomass is one of the key challenges in production of cellulosic ethanol. Here we developed a two-stage pretreatment process using aqueous ammonia and dilute sulfuric acid in a percolation mode to improve production of fermentable sugars from rice straw. Aqueous NH3 was used in the first stage which removed lignin selectively but left most of cellulose (97%) and hemicellulose (77%). Dilute acid was applied in the second stage which removed most of hemicellulose, partially disrupted the crystalline structure of cellulose, and thus enhanced enzymatic digestibility of cellulose in the solids remaining. Under the optimal pretreatment conditions, the enzymatic hydrolysis yields of the two-stage treated samples were 96.9% and 90.8% with enzyme loadings of 60 and 15FPU/g of glucan, respectively. The overall sugar conversions of cellulose and hemicellulose into glucose and xylose by enzymatic and acid hydrolysis reached 89.0% and 71.7%, respectively.

Cellulolytic Enzymes Production by Solid State Culture

Problem Statement: Great interest in the use of lignocellulosic biomass is increasing in order to diminish the accumulation of residues, such as pecan nut shells. One of the alternatives is the fungal degradation of these residues. Approach: The capacity of Trichoderma (coded as T1, T2 and T3) strains to produce cellulase and xylonite was evaluated. Results: Pecan nut shell fibers were used as sole carbon source. The fiber characterization study showed that cellulose levels were of 0.1% while hemicellulose was up to 25 %. Three Trichoderma strains were used on solid fungal cultures using the fibers as sole carbon and inductor source for the production of cellulolytic enzymes. The behavior of the sugars liberated by the fungi showed that the strain T2 is able to accumulate more monomeric reducing sugars than the other two strains, this could be attributed at this strain has a higher sugar liberation rate and slower sugar consumption rate. This strain also expressed more cellulase and xylanase activity. The low quantity of cellulose registered in the fibers can still be used to induce cellulase activity. Conclusion: The T2 strain had the highest level of enzymatic activity both cellulase and xylanase.

Evaluation of Different Teas against Starch Digestibility by Mammalian Glycosidases

Current work investigated the ability of different tea (green, oolong and black teas) in inhibiting human salivary alpha-amylase (HSA) and mammalian alpha-glucosidase (AGH). The inhibitory profiles were correlated to their major polyphenol content (theaflavins and catechins). The fully fermented black tea was demonstrated to be most potent in inhibiting HSA and AGH (IC50 of 0.42 to 0.67 and 0.56 to 0.58 mg of tea leaves/mL respectively). Its capability in retarding the digestion of a real food system (rice noodle) was further elucidated with an in vitro digestion study. Results indicated that black tea was able to retard starch digestion moderately, thereby allowing a gradual reduction of sugar liberation. Polyphenolic profile analysis suggested that the oxidized catechins, theaflavins, may be responsible for its activity. We have found that refractive index (RI) measurement is a rapid, direct, and highly convenient method for quantifying the degree of enzymatic starch digestion and kinetics. The RI method has good linearity range, limit of detection (0.1596 mg/mL, maltose equivalent) and limit of quantitation (0.6312 mg/mL) and was successfully applied in our study.

Comparative study of corn stover pretreated by dilute acid and cellulose solvent‐based lignocellulose fractionation: Enzymatic hydrolysis, supramolecular structure, and substrate accessibility

Liberation of fermentable sugars from recalcitrant biomass is among the most costly steps for emerging cellulosic ethanol production. Here we compared two pretreatment methods (dilute acid, DA, and cellulose solvent and organic solvent lignocellulose fractionation, COSLIF) for corn stover. At a high cellulase loading [15 filter paper units (FPUs) or 12.3 mg cellulase per gram of glucan], glucan digestibilities of the corn stover pretreated by DA and COSLIF were 84% at hour 72 and 97% at hour 24, respectively. At a low cellulase loading (5 FPUs per gram of glucan), digestibility remained as high as 93% at hour 24 for the COSLIF-pretreated corn stover but reached only approximately 60% for the DA-pretreated biomass. Quantitative determinations of total substrate accessibility to cellulase (TSAC), cellulose accessibility to cellulase (CAC), and non-cellulose accessibility to cellulase (NCAC) based on adsorption of a non-hydrolytic recombinant protein TGC were measured for the first time. The COSLIF-pretreated corn stover had a CAC of 11.57 m(2)/g, nearly twice that of the DA-pretreated biomass (5.89 m(2)/g). These results, along with scanning electron microscopy images showing dramatic structural differences between the DA- and COSLIF-pretreated samples, suggest that COSLIF treatment disrupts microfibrillar structures within biomass while DA treatment mainly removes hemicellulose. Under the tested conditions COSLIF treatment breaks down lignocellulose structure more extensively than DA treatment, producing a more enzymatically reactive material with a higher CAC accompanied by faster hydrolysis rates and higher enzymatic digestibility.

Sessions 3 and 8: Pretreatment and Biomass Recalcitrance: Fundamentals and Progress

Overcoming lignocellulosic biomass recalcitrance followed by enzymatic hydrolysis of structural polymeric carbohydrates (i.e., cost-efficient liberation of fermentable sugars) is perhaps the most challenging technical and economic barrier to success of biorefineries [1–3]. Lignocellulosic biomass is a natural composite having three main biopolymers (cellulose, hemicellulose, and lignin) intertwined chemically and physically. The central role of biomass pretreatment has been indicated by the fact that 12 oral presentations and 76 posters were given on the subject at this symposium. Pretreatment is among the most costly steps in biochemical conversion of biomass [4, 5], accounting for up to 40% of the total processing cost [6]. Also, it affects the costs of other operations including size reduction prior to pretreatment and enzymatic hydrolysis and fermentation after pretreatment. Pretreatment can also strongly influence downstream costs involving detoxification if inhibitors were generated, enzymatic hydrolysis rate and enzyme loading, mixing power, product concentration, product purification, power generation, waste treatment demands, and other process variables [4]. Although a number of pretreatment methods have been proposed and investigated on the laboratory scale and even pilot plant scale during the past several decades, much of the data in the literature have been obtained and reported under different standards, leading to confusion on the part of academic researchers and potential industrial investors alike. In order to address and resolve this confusion, Prof. Bruce Dale of Michigan State University gave a brief opening to both sessions entitled “Raising the Bar for Pretreatment Research.” Since overall sugar yields are one of the most important factors for the evaluation of Appl Biochem Biotechnol (2009) 153:80–83 DOI 10.1007/s12010-009-8610-3

Pectinase production by Bacillus subtilis and its potential application in biopreparation of cotton and micropoly fabric

An alkaline and thermostable pectinase production from Bacillus subtilis SS was optimized under submerged fermentation and its application was tested in textile industry for desizing and bioscouring of cotton and micropoly fabrics. Desizing of fabric was the best with 5 U/g pectinase treatment for 120 min at pH 9.5 and 65 °C. Under optimized conditions of bioscouring, desized cotton showed highest reducing sugar liberation and weight loss than desized micropoly. Along with enzyme, addition of chelating (EDTA) and wetting agent markedly enhanced the weight loss compared to single use of enzyme or EDTA alone. Agitation (50 ± 2) enhanced the weight loss values of cotton (1.9%) and micropoly fabric (1.7%) at pH 9.5 after treatment time of 2 h. Bioscouring of fabrics with pectinase resulted in enhancement of various physical properties of fabrics viz. whiteness (1.2%), tensile strength (1.6%) and tearness (3.0%) over conventionally alkaline scoured fabrics.

Enzymatic treatment of linen

In this work, the effect of cellulase treatment was evaluated by means of phenol-sulphuric acid method. This method was performed by determining sugar liberation in the treatment bath with the amount expressed in glucose equivalence. As compared with the conventional method, the measurement of amount of sugar liberated gave a more reliable and accurate result than the weight loss method. It was found that although weight loss of cellulose became negligible when the treatment was done under agitation-free condition, the amount of sugar liberated was still readily measurable.