Utilization of Cassava Fibrous Residue for the Production of Glucose and High Fructose Syrup

Abstract

Cassava (Manihot esculenta Crantz) is one of the largest sources of starch and sago. The cassava fibrous residue (CFR) contains 61%-63% starch and other polysaccharides and causes major disposal problems. Due to its high starch content, it can be utilized to obtain value-added products. Work was therefore undertaken to obtain glucose syrup by hydrolyzing starch and cellulose with various enzyme treatments, followed by conversion to high fructose syrup. Standardization of various conditions for hydrolysis was determined. A combination of α-amylase and glucoamylase (T1 & T2) resulted in 52.88–54.24% conversion of CFR to glucose. The yield could be enhanced to 58.70–60.00% by adding cellulase enzyme complex, Accellerase 1000, in combination with α-amylase and glucoamylase. Cellulase activity helped to reduce viscosity of the CFR slurry and also improved the starch hydrolysis by α-amylase and glucoamylase. The most suitable enzyme treatment was found to be simultaneous hydrolysis of CFR with glucoamylase, Dextrozyme GA and cellulase enzyme Accellerase 1000 at pH 4.5 and 60°C for 48h, followed by a one hour liquefaction with α-amylase, Liquezyme X (T5). Because of the high fiber content, it was not possible to handle a slurry containing more than 15% of the material, as per the reports of earlier workers. However, hydrolysis of CFR at different substrate concentrations in the present study indicate that hydrolysis of a higher substrate concentration, i.e., 25%, was possible with this enzyme treatment. Production of high fructose syrup from higher slurry concentrations reduces the cost of concentrating glucose syrup. Results obtained for saccharification and isomerization of two different samples of cassava fibrous residue indicated that the yield of glucose and fructose depended on the starch content in the initial raw material.

Introduction

High fructose syrup (HFS) is a common sweetener for the food and beverage industries, owing to its high sweetness, non- crystalline nature, and its ability to enhance fruit and spice flavor while preserving freshness by maintaining consistent moisture.^1,2 HFS is made by partial isomerization of the glucose in a starch hydrolysate, which contains about 94% D-Glucose on a dry basis. It is commercially produced mainly from corn starch. To cope with increasing demand for HFS, alternative substrates need to be identified. Byproducts from crops like sugarcane, cereals, and various tuber crops are good sources of fiber, which could be converted to value-added products with commercial use. Conversion of these wastes into such economic products could transform them into a valuable resource, alleviate food and energy shortages, and reduce pollution-load.³ These residues contain polysaccharides like starch, cellulose, hemicelluloses, and pectin, in addition to nitrogenous compounds and minerals.^4,5 Cassava (Manihot esculenta Crantz), a sturdy perennial crop grown in many parts of Asia, Africa, and South America, is also one of the major tuber crops of the world. On dry weight basis, cassava contains about 80%–82% starch, of which 55%-60% is recoverable as starch. The starch finds use in a large range of industries. Four types of waste are produced in the manufacture of starch from cassava: outer skins, inner rinds, waste water, and fibrous residues.⁶ The outer skin, inner rinds and waste water, are used for production of biogas, single-cell proteins and yeast single-cell proteins, respectively.^7
–9 The cassava fibrous waste generated in the manufacture of starch and sago from cassava chips/tubers, however, poses problems for disposal, especially in medium- and large-scale industries.¹⁰ In India, more than 500 cottage and small industries crush over 5,000 tonnes of cassava per day during harvest season for the manufacture of starch and sugar, thereby generating 1,000 tonnes of dry residue equivalent per day.¹¹ In the process of producing starch, about 20% of waste fibrous residue is obtained. Currently the waste fibrous residue is is not utilized efficiently even though it contains more than 50% starch.

The importance of hydrolysis is widely recognized in the conversion of residual plant biomass considered “waste” into various value-added products such as biofuels, chemicals, cheap carbon and energy sources for fermentation, improved animal feeds and human nutrients.^12
–14 Unlike chemical methods, enzymatic hydrolysis operates under very mild conditions, does not produce undesirable products, and is also environmentally friendly. A major limitation of enzymatic hydrolysis, however, is the poor yield of glucose. Cassava fibrous material poses a problem of environmental pollution due to its high Biological Oxygen Demand (BOD) loads. Because of the difficulty in the disposal of solid residues, it also leads to serious environmental problems.¹⁵ The utilization of waste to obtain value-added products would relieve pollution as well as improve process economics. Various schemes had been suggested for the utilization of cassava fibrous residue (CFR) to obtain value-added products and were critically analyzed for practicability and commercial feasibility by Ghildyal and Lonsane.¹⁶ They found that the production of confectioner's syrup, potable alcohol, and the use of the waste as substitute to wheat bran in solid state fermentations show considerable promise. The residue, if effectively used, could lead to any one of a number of value-added products, such as enzyme (α-amylase), lactic acid and ethanol. Owing to the high starch content and nutrient content of these wastes, cassava fibrous waste can serve as an important substrate for production of various bioproducts, i.e. amylase enzyme, fructose syrup, glutamic acid, xanthan gum, etc.^11,15,17,18 Solid substrate fermentation of CFR for bioethanol production was also investigated by various researchers.^19
–21 Dual or multi-enzyme systems were used to optimize ethanol yield. The scope of using CFR for microbial production of lactic acid was also studied.^22,23

The possibility of hydrolyzing starch and cellulose from fibrous waste with various enzyme processes to obtain glucose syrup for subsequent conversion to HFS has been investigated. Work was therefore undertaken to find the most suitable enzyme treatments for the saccharification process for HFS production using cassava residue as the raw material.

Materials and Methods

Source of Substrate

CFR was collected from the cassava starch industries of Tamil Nadu, India, where the CFR is left over as a solid waste after the extraction of starch. Because of its high water content (70%–80%) and the presence of large amounts of starch, the residues were sun-dried and then oven-dried at 80°C for 24 hr to prevent microbial deterioration. They were then finely ground in a blender, and the unsieved material was subjected to enzymatic hydrolysis.

Enzymes

Liquezyme X^® (thermostable α-amylase; EC 3.2.1.1; hereinafter Liquezyme), derived from a genetically modified strain of Bacillus licheniformis with an activity of 200 kilo novo units (KNU) per gram, and Dextrozyme GA^® (glucoamylase; EC 3.2.1.3; hereinafter Dextrozyme), produced from a genetically modified strain of an Aspergillus sp. with an activity of 270 amyloglucosidase (AGU) units per gram, were purchased from M/s Novozymes A/s, Denmark. Stargen^™ 001enzyme (hereinafter Stargen), which contains α-amylase from Aspergillus kawachi and a glucoamylase from A. niger and had activity of ≥456 GSHU/g (where GSHU=granular starch hydrolyzing units), and an Accellerase^™ 1000 cellulase enzyme (hereinafter Accellerase) complex containing multiple enzyme activities including exoglucanase, endoglucanase, hemicellulase and beta-glucosidase derived from a genetically modified strain of Trichoderma reesei with declared endoglucanase activity of 2,500 CMC U/g (carboxymethyl cellulose units) and beta-glucosidase activity of 400 pNPG U/g (p-nitrophenyl beta-D-glucoside), were supplied by M/s Genencor International Inc. USA^. Sweetzyme T^® (immobilized glucose isomerase; EC 5.3.1.5; hereinafter Sweetzyme) with an activity of 350 IGIU per gram (where IGIU=Immobilized glucose isomerase units) was purchased from M/s Novo Nordisk Biochem., USA.

Analytical Methodologies

Initial biochemical profile

Starch and total sugars

The initial starch and total sugar content of CFR was determined by the titrimetric method of Moorthy and Padmaja.²⁴ Soluble sugars were extracted from CFR (four replicates and duplicate analysis on each replicate) using 80% ethanol (1:20 w/v). The alcoholic sugar filtrate was hydrolyzed with concentrated hydrochloric acid (1.0 ml) for 30 min, converting the non-reducing sugars to reducing sugars. The residue containing starch was hydrolyzed with 20 ml 2 N Hydrochloric acid for 20 min at 100 °C to completely hydrolyze the root starch to glucose. The acid-hydrolyzed sugar and starch were separately titrated against potassium ferricyanide using methylene blue indicator. The total sugar and starch content were computed from the titer value.

Crude fiber content

The crude fiber content of CFR was determined by Association of Official Analytical Chemists (AOAC) method.²⁵

Pentosans

For estimation of pentosans, the powdered sample was treated overnight with 0.2 N sodium hydroxide solution, and the filtrate was used for the determination of pentoses by the cysteine-carbazole method of Dische and Borenfreund as modified by Chen and Anderson.^26,27

Reducing sugars

The reducing sugar content was determined by the Nelson-Somogyi method.^28,29

Glucose

The glucose content of the aliquots drawn from each treatment was determined by the glucose oxidase- peroxidase method using the glucose (GO) assay kit (Sigma, Missouri, USA). The kit contained glucose oxidase-peroxidase reagent (product code G 3660), O-dianisidine reagent (product code D 2679) and glucose standard (product code G 3285).³⁰

Fructose

The fructose content was estimated by the cysteine-carbazole method of Dische and Borenfreund as modified by Chen and Anderson.^26,27

Enzymatic hydrolysis of CFR to glucose

The enzymatic hydrolysis of CFR to glucose was performed using the various enzymes described herein:

Enzyme pretreatment with two enzymes, α-amylase and glucoamylase

Treatment 1

CFR (10% w/v) was adjusted to pH 5.5 and kept in a thermostatic water bath (Julabo SW21) at 90°C for 10 min to attain equilibrium. 0.05 % (v/w) Liquezyme was added, mixed and incubated for 1.0 h at 90°C. The temperature of the liquefied slurry was then brought down to 60°C and pH was adjusted to 4.0 using dilute hydrochloric acid. 0.1% (v/w) Dextrozyme was added and incubation continued at 60°C for 48 h.

Treatment 2

CFR (10% w/v) was adjusted to pH 4.5 and gelatinized for 15 min, with stirring in a water bath at 95°C. The slurry was cooled to room temperature (30±1 °C) and 0.75% v/w Stargen was added and mixed. Incubation continued for 48 h at room temperature.

Enzyme pretreatment with three enzymes, α-amylase and glucoamylase and cellulase – Sequential hydrolysis by enzymes

Treatment 3

10% (w/v) of CFR (sample 1) was hydrolyzed with 5% (v/w) Accellerase for 24 h at pH 4.5 and at 60°C. The pH was adjusted to 5.5 and 0.05 % (v/w) Liquezyme was added, mixed and incubated for 1.0 h at 90°C. The temperature of the liquefied slurry was then brought down to 60°C, and pH was adjusted to 4.0 using dilute hydrochloric acid. 0.1% (v/w) Dextrozyme was added and incubation continued at 60°C for 48 h.

Treatment 4

10% (w/v) of CFR (sample 1) was hydrolyzed with 5.0% (v/w) Accellerase for 24h at pH 4.5 and at 60°C. The slurry was then gelatinized for 15 min, and cooled to room temperature (30±1 °C). 0.75% (v/w) Stargen was added and mixed, and incubation continued for 48 h at room temperature.

Simultaneous hydrolysis by Dextrozyme and Accellerase

Treatment 5

CFR (10% w/v) was adjusted to pH 5.5 and kept in a thermostatic water bath (Julabo SW21) at 90 °C for 10 min to attain equilibrium. 0.05 % v/w Liquezyme was added, mixed and incubated for 1.0 h at 90°C. The temperature of the liquefied slurry was then brought down to 60°C, and pH was adjusted to 4.0 using dilute hydrochloric acid. 0.1% (v/w) Dextrozyme and 5.0 % (v/w) Accellerase were added, and incubation continued at 60°C for 48 h.

Treatment 6

CFR (10% w/v) was adjusted to pH 4.5 and gelatinized for 15 min, with stirring in a water bath at 95°C. The slurry was cooled to room temperature (30±1 °C) and 0.75% (v/w) Stargen and 5.0% (v/w) Accellerase were added and mixed. Incubation continued for 48 h at 60°C.

Enzyme treatment with single enzyme, cellulase

Treatment 7

CFR (10% w/v) (sample 1) was adjusted to pH 4.5 and gelatinized for 15 min, followed by hydrolysis with 5.0% (v/w) of Accellerase for 48h at pH 4.5 and at 60°C.

Reducing sugar and glucose content were determined in the aliquots from all the treatments (duplicate samples) as described in Treatment 1.

Isomerization to Fructose

Glucose syrup produced from CFR after various enzyme treatments was filtered and concentrated to 40% (w/v). The pH was adjusted to 7.5, and the mixture kept in a thermostatic water bath at 60°C. After attaining the equilibrium temperature, 50 mg Sweetzyme/g glucose was added and incubated at 60°C for 24 h.

Hydrolysis of CFR at different substrate concentrations by simultaneous reaction of glucoamylase and cellulase and subsequent conversion to HFS

Different concentrations of CFR [10% (C1), 15% (C2), 20% (C3), and 25% (C4) w/v] were added to 250 ml conical flasks, pH adjusted to 5.5 and kept in a thermostatic water bath (Julabo SW21) at 90°C for 10 min to attain equilibrium. Liquezyme (0.05 % v/w) was added, mixed and incubated for 1.0 h at 90°C. The temperature of the liquefied slurry was then brought down to 60°C, and the pH was adjusted to 4.0 using dilute hydrochloric acid. Dextrozyme (0.1% v/w) and Accellerase (5% v/w) were added and incubated at 60°C for 48 h (as in Treatment 5, which was identified as the optimum treatment). Aliquots were drawn at different time intervals for glucose estimation. Glucose content was determined by Sigma glucose (GO) assay kit. Glucose syrup was filtered, purified and evaporated to 40% w/v syrup. The pH was adjusted to 7.5 and kept in a thermostatic water bath at 60°C. After attaining the equilibrium temperature, 50 mg of Sweetzyme/g glucose was added and incubated at 60°C for 24 h. Aliquots were drawn from each treatment and the fructose content was estimated by the cysteine-carbazole method.²⁷

Studies on the comparative production of HFS from two different samples of CFR

Two different samples (sample 1 and sample 2) of CFR (25% w/v or 250 g/1000ml) at pH 5.5 were liquefied by Liquezyme (0.05% v/w) at 90°C for 1.0 h. The temperature of the liquefied slurry was then brought down to 60°C, and the pH was adjusted to 4.0 using dilute hydrochloric acid. Dextrozyme (0.1% v/w) and Accellerase (5.0% v/w) were added and incubated at 60°C for 48 h (as in Treatment 5). Glucose syrup was filtered, purified and evaporated to 40% w/v syrup. The pH was adjusted to 7.5 and the mixture kept in a thermostatic water bath at 60°C. After attaining the equilibrium temperature, 50 mg of Sweetzyme/g glucose was added and incubated at 60°C for 24 h.

Statistical analysis

The data were analyzed using the software package GenStat to perform the Analysis of Variance (ANOVA).³¹ Four replicates were maintained for experimental studies, and duplicate analyses were performed on each replicate. Statistical analysis was done using one-way analysis of variants (ANOVA) for comparison of mean values among different treatments. All pairwise comparison of mean values of different treatments was analyzed with Duncan's multiple range test (direction=ascending; PROB=0.05). Two-way ANOVA was done for analyzing the effect of substrate and reaction time on the rate of glucose production. The treatments are considered significantly different at 5% level of significance (p<0.05).

Results and Discussion

Initial Biochemical Profile

The biochemical composition of dried cassava residue is given in Table 1. Starch was the major component, and it was in the range of 58.81–64.00% (w/w). The second major compound was crude fiber, and it varied in the range of 13.53%–15.67% (w/w). The values were somewhat consistent with the report of Kunhi et al.⁴ The chemical constituents of the dried cassava residue are reported to vary with the type and maturity of the tubers processed, the variety, processing conditions etc.^4,32,33 It is interesting to note that the residue originally contains 75–87 ppm of hydrocyanic acid but more than 99% of this was reported to be destroyed during saccharification.³² The nutrient and starch profile of CFR from cassava starch industries vary because of the lack of standard procedure. This variability in nutrient and starch content is attributed to several factors such as cassava tuber washing process, the ratio of tuber to water used in the extraction process, and the quality of raspers used for starch extraction.⁶ In general, a starch content of 55%-65% (dry weight basis) is found in CFR surveyed from several factories in India.^6,11 The starch granules are located in the root cells which remained unruptured during the rasping process and hence are not easily recoverable.

Table 1.

Chemical Analysis of the CFR

	CASSAVA FIBROUS RESIDUE
CONSTITUENTS (G/100G CFR)	SAMPLE 1 % (W/W)	SAMPLE 2 % (W/W)
Starch	58.81±0.97	64.00±0.99
Crude fiber	15.67±0.39	13.53±0.33
Reducing sugars	0.52±0.05	0.45±0.04
Pentosans, expressed as xylose	1.90±0.03	2.11±0.02
Moisture	12.50±0.23	10.45±0.10

Enzymatic hydrolysis of CFR to glucose

Cassava fibrous residue was hydrolyzed with either two enzymes (α-amylase and glucoamylase) or a group of three enzymes (α-amylase, glucoamylase and cellulase) by different procedures of treatment depending on the type of enzymes and their working conditions. In Treatment 1 (T1), enzyme-enzyme processing with α-amylase and glucoamylase was used. In Treatment 2 (T2), a commercial enzyme blend containing α-amylase and glucoamylase with optimum activity at 30°C was used. In treatments 3 to 6, a cellulase enzyme complex was used to supplement the enzymes used in T1 and T2. Sequential hydrolysis of enzymes was done in treatments T3 and T4, whereas simultaneous hydrolysis of enzymes was done in treatments T5 and T6. In Treatment 7, Accellerase enzyme alone was used.

Comparing the enzyme treatments T1 and T2, percent conversion of CFR to glucose based on dry weight and starch content was found to be higher in the case of T1 (Table 2). About 95.34% and 96.95% starch in CFR was hydrolyzed to glucose and reducing sugars, respectively, using T1 enzymes. About 98%-99% conversion of starch present in the residue to reducing sugars was reported by Kunhi et al.⁴ The Liquezyme used in the study was an endoamylase while Dextrozyme was a glucoamylase (T1). Although high extent of conversion of starch to glucose was observed in T1, the total reaction time for T1 was 49 h and the temperature of the system had to be maintained at 90°C for 1 h and at 60°C for 48 h, making the process energy intensive. However, in the case of T2, hydrolysis of gelatinized starch in the residue was done at room temperature (30±1°C) using Stargen, indicating considerable energy could be saved.

Table 2.

Effect of enzyme treatments on hydrolysis of CFR (sample 1)

ENZYME TREATMENTS	REDUCING SUGARS (G/100 G CFR)	GLUCOSE YIELD (G/100 G CFR)	RESIDUAL CRUDE FIBER (%)^**	DRY WT. OF RESIDUE LEFT OVER AFTER HYDROLYSIS (G/100G CFR HYDROLYZED)
T1	56.23±0.91^b (96.95±1.58)^*	54.24±0.25^c (93.51±0.43)^*	15.24^g (2.74)	29.42^d
T2	55.44±1.28^b (95.58±2.23)^*	52.88±0.21^b (91.17±0.36)^*	15.11^f (3.57)	30.12^e
T3	63.40±1.26^cd	59.80±0.39^e	10.28^e (34.40)	22.90^c
T4	61.80±1.31^c	58.70±0.34^d	10.16^d (35.16)	21.66^b
T5	65.70±0.92^e	60.00±0.33^e	9.66^b (38.35)	20.60^a
T6	64.90±2.22^de	59.87±0.29^e	9.85^c (37.14)	21.60^b
T7	25.30±1.27^a	21.73±0.15^a	9.14^a (41.67)	64.40^f
LSD (5%)	2.015	0.4275	0.0533	0.3210

p<0.001; Mean±SD from four replicates; Means with the same superscript within a column do not differ significantly

Based on starch in CFR; ^**Crude fiber content in the residue left after enzyme hydrolysis under various treatments; Figures in parentheses indicate the percentage degradation of crude fiber (computed as: [residual crude fiber (%)÷original crude fiber (%)] x 100)

Presence of high fiber content necessitates the use of complementary enzymes such as cellulase in hydrolysis and saccharification.^33

–36 The results showed that the combination of three enzymes provided more degradation of CFR than two enzymes In both sequential and simultaneous hydrolysis (T3 to T6), about 58.70% to 60.00% of CFR on a material basis was converted to glucose. Reducing sugar content was also increased to 61%-65% in treatments T3 to T6 (Table 2). However, in sequential systems (T3 and T4), an additional 24 h is necessary to complete the reaction compared with simultaneous hydrolysis (T5 and T6). Simultaneous hydrolysis of glucoamylase and complementary enzyme cellulase (T5 and T6) was found to be more advantageous considering the time required for hydrolysis. With the slurry of CFR containing more than 15% solids, the mass becomes very viscous at gelatinization temperature because of the high fiber content, and thus simultaneous Stargen- and Accellerase-mediated hydrolysis (T6) could not be tried for higher slurry concentrations. Based on these results, simultaneous hydrolysis of Dextrozyme and Accellerase 1000 treatment (T5) was selected as the most optimum treatment for CFR hydrolysis. This treatment (T5) was used for further experimental studies such as effect of time and different substrate concentration on hydrolysis.

Results presented in Table 2 also show that 38.35% crude fiber degradation was achieved in T5 enzyme treatment, and the residue left over after hydrolysis was 20.60%. Polysaccharide content of CFR other than starch is about 20%–24%, of which crude fiber accounts for 13% to 16%. Crude fiber consists of mainly cellulose associated with other structural polysaccharides, particularly hemicelluloses and a structural non-carbohydrate, lignin. The usefulness of cellulose and other cell wall polysaccharides is dependent upon their hydrolysis into simple sugars.³⁷ There are essentially three types of enzymes in the cellulase system: β-1,4-endoglucanase (EC 3.2.1.4), which cleaves internal β-1,4-glycosidic bonds; cellobiohydrolase (EC 3.2.1.9), which releases cellobiose from the non-reducing end of cellulose; and β-glucosidase (EC 3.2.1.21), which hydrolyzes cellobiose to glucose.^38,39 The three enzymes act in synergy to degrade cellulose. Hemicellulase breaks down hemicellulose, which is a polysaccharide composed of a broad range of simple sugar monomers including glucose, xylose, arabinose, galactose, mannose and rhamnose. Three types of cellulases, namely, endoglucanases, exoglucanases and β-glucosidases, and hemicellulases, particularly xylanases, are required for the hydrolysis of lignocelluloses.⁴⁰ The Accellerase enzyme complex contains multiple enzyme activities, mainly exoglucanase, endoglucanase, beta-glucosidase and hemi-cellulase. Hemicellulases present in the Accellerase degrade hemicelluloses to expose starch and cellulose for further hydrolysis by amylases and cellulases, respectively. Hydrolytic efficiency of a multi-enzyme complex for lignocellulose saccharification depends both on properties of individual enzymes and their ratio in multi-enzyme cocktail.⁴¹ Enzyme cocktails have been developed by mixing Trichoderma reesei cellulase with other enzymes (xylanases, pectinases and Bgl), and these cocktails were tried to hydrolyze various feedstocks.^42
–44

In T7, where Accellerase alone was used, there was a higher percentage degradation of crude fiber (41.67%). Nevertheless, this treatment left almost 64.40% CFR unhydrolyzed (Table 2). Even though Accellerase enzyme is a cellulase enzyme complex for lignocellulosic biomass hydrolysis, it is reported to contain amylase as a coactivity, which aids in liquefaction and viscosity reduction.⁴⁵ Hence it was inferred that starch present in the CFR was also hydrolyzed to a limited extent by the action of Accellerase. Once the cellulosic fiber structure is disrupted, the trapped starch becomes available for amylolytic hydrolysis. However, a major part of the CFR remained unutilized, as T7 did not have any starch hydrolyzing enzyme component other than the mild coactivity in Accellerase.

Glucose yield from CFR after various enzyme treatments is shown in Table 2. Glucose yield from T1 and T2 was lower than from T3, T4, T5, or T6, and it was observed that maximum yield was obtained from the T5 enzyme treatment. In the enzyme treatments T1 and T2, only starch-hydrolyzing enzymes were used. However, in the case of enzyme treatments T3 to T6, cellulase enzyme was used complementary to starch-hydrolyzing enzymes. The use of complementary enzymes in the process of hydrolysis of CFR is an alternative to increase the biodegradation of CFR. Haska and Ohta reported that addition of cellulase at the initial stage of the hydrolysis process resulted in an increase in the ability of raw-starch-digesting amylase obtained from Penicillium brunneum No. 24 to digest sago starch granules.³⁴ Mainly glucose was produced by this process.

Isomerization of glucose syrup produced from CFR to high fructose syrup

The glucose yield in the hydrolysate was important, as only the glucose was isomerized by Sweetzyme to fructose. The glucose syrup solution resulting from enzyme treatments T1 to T6 was then isomerized to high fructose syrup. The results indicated that no significant differences were noticed in the percent conversion to fructose; the conversion value was 38%–39% (Table 3). However, the fructose yield was higher in T5 and T6 with a slight reduction in T3. Khalid reported a composition of 60.7% glucose, 36.9% fructose and 2.4% maltose in the high fructose syrup obtained from cassava starch.⁴⁶ Chen and Chang reported a composition of 50% glucose, 42% fructose and 3% maltose in the high fructose syrup obtained from rice flour.⁴⁷

Table 3.

Fructose production from CFR after different enzyme pretreatments

TREATMENTS	FRUCTOSE YIELD (G/100 G)	PERCENT CONVERSION TO FRUCTOSE¹
T1	21.10±0.15^b	38.98±0.27^b
T2	20.21±0.16^a	38.11±0.31^a
T3	23.44±0.18^d	39.13±0.30^b
T4	22.60±0.21^c	38.46±0.35^a
T5	23.85±0.13^e	39.60±0.20^c
T6	23.80±0.17^e	39.80±0.30^c
LSD(5%)	0.2501	0.4345

p<0.001; Mean±SD from four replicates; Means with the same superscript within a column do not differ significantly; ¹Based on initial glucose content

Hydrolysis of CFR at different substrate concentrations by simultaneous reaction of glucoamylase and cellulase and subsequent conversion in to HFS

The concentration of CFR was varied from 10% to 25%, and simultaneous hydrolysis was done using a glucoamylase (0.1% v/w) and cellulase (5% v/w) enzyme cocktail after liquefying with α-amylase. The data presented in Table 4 indicate that as the substrate concentration was increased, the amount of glucose production also increased. About 59%–60% conversion to glucose on a material basis was achieved in all the substrate concentrations after 48 h hydrolysis. However, the amount of glucose produced was less from lower substrate concentrations. Thus lower substrate concentration leads to a lower concentration of glucose, and a higher quantity of water needs to be evaporated to raise glucose concentration to 40% DS, a requirement for isomerization processes. Production of HFS from higher slurry concentration reduces the expense of concentration the glucose syrup. According to Kunhi et al., starch present in CFR can be converted to reducing sugars by an enzyme-enzyme process.⁴ They reported that only 10% suspension of the material could be handled, as the slurry became highly viscous above that concentration. But in our study, attempts were made to increase the amount of CFR that can be handled for saccharification. About 59.41% conversion to glucose was obtained when 25% cassava fibrous slurry was hydrolyzed. Utilization of cellulase enzyme could release more reducing sugars and glucose from CFR and also help in decreasing the material's viscosity, which may be due to disruption of various cell matrix components and consequent exposure of starch for rapid thinning action by amylases. Srikanta et al reported a novel technique for acid hydrolysis of starch in CFR using a substrate concentration of 30% in shallow trays in a horizontal cooker.⁴⁸ However, the low pH necessitates pH adjustments during the isomerization step, adding to the cost of the process.

Table 4.

Hydrolysis of CFR at different substrate concentrations

SUBSTRATE CONC. (%W/V)	GLUCOSE CONTENT (G/100 ML)¹	PERCENT CONVERSION TO GLUCOSE (ON DRY MATERIAL BASIS)
10 (C1)	6.05±0.02^a	60.51±0.25^b
15 (C2)	9.03±0.05^b	60.22±0.33^b
20 (C3)	11.91±0.04^c	59.53±0.19^a
25 (C4)	14.85±0.07^d	59.41±0.27^a
LSD (5%)	0.0733	0.4073

p<0.001; Mean±SD from four replicates; Means with the same superscript within a column do not differ significantly; ¹After 48h of enzyme action

The time course production of glucose from different substrate concentrations of CFR hydrolysis is presented in Table 5. The rate of hydrolysis increases with time in all substrate concentrations. Maximum amount of glucose was released from 25% slurry at 48 h incubation. About 43%–45% conversion to glucose was achieved in 6 h, after which the reaction rate slows down further. When saccharification was done for 24 h, around 51%–52% conversion of CFR to glucose was achieved, which increased to 59%–60% after 48 h hydrolysis. It was also observed that the glucose yield at various reaction times was almost similar in different substrate concentrations of CFR.

Table 5.

Effect of reaction time on the enzymatic hydrolysis of CFR at different substrate concentrations

	GLUCOSE YIELD (G/100G)
SUBSTRATE (% W/V)	6H	12H	24H	48H	MEAN
10 (C1)	45.81	48.66	52.23	60.46	51.79^b
15 (C2)	45.79	48.51	52.18	60.22	51.68^b
20 (C3)	45.05	47.82	51.99	59.54	51.10^a
25 (C4)	42.51	46.92	51.37	59.41	50.05^a
Mean	44.79	47.98	51.94	59.90

p<0.001; LSD (5%) 0.2183(substrate) 0.2183(time) 0.4366(substrate time); Means with the same superscript do not differ significantly

The fructose yield in the isomerized syrups from different substrate concentrations of CFR was 24.05%−25.37%, with a percentage conversion of 39.76%–42.72% to fructose ( Table 6 ). It was found that the yield of fructose from different concentrations of CFR slurries was almost similar. The percentage conversion and fructose yield was slightly less in the isomerized syrup from lower substrate concentrations. One of the requisites for isomerization is that the concentration of glucose in the saccharified hydrolysate should be 40% (w/v). The production of high fructose syrup proved technically feasible; however the concentration of the saccharified hydrolysate with low glucose content was found to be a major problem. Using 10% and 15% CFR slurries, the initial glucose content of the hydrolysate was 6.05% and 9.03% respectively (Table 4). Using higher slurry concentrations, the cost associated with concentration of saccharified hydrolysate to raise the glucose level to 40% can be considerably reduced. Results obtained for saccharification and isomerization of CFR indicate that from an economic point of view, CFR at high concentration (25%) should be hydrolyzed by the simultaneous action of glucoamylase and cellulase prior to isomerization.

Table 6.

Isomerization of glucose syrup produced from CFR slurries of different substrate concentrations

SUBSTRATE CONC. (% W/V)	PERCENT CONVERSION TO FRUCTOSE¹	FRUCTOSE YIELD (G/100 G)
10 (C1)	39.76±0.26^a	24.05±0.13^a
15 (C2)	41.01±0.19^a	24.70±0.13^a
20 (C3)	42.46±0.25^b	25.29±0.15^b
25 (C4)	42.72±0.19^b	25.37±0.09^b
LSD (5%)	0.3469	0.1956

p<0.001; Mean±SD from four replicates; Means with the same superscript within a column do not differ significantly; ¹Based on initial glucose content

Comparative data on production of HFS from two different samples of CFR

Table 7 gives data on the production of glucose and fructose syrup from CFR used at a higher slurry concentration of 25% w/v. It was found that an initial liquefaction of CFR at pH 5.5 with Liquezyme at 90°C for 1 h, followed by simultaneous hydrolysis using Dextrozyme and Accellerase at pH 4.5 and at 60°C for 48 h (same as T5, identified as the optimum treatment) resulted in about 68.49% and 73.87% conversion to glucose respectively from samples 1 and 2. When 250 g of CFR was hydrolyzed, the amount of glucose produced from sample 1 and sample 2 was 149.83g and 165.38g, respectively, whereas the percentage conversion to glucose was 59.93% and 66.15% respectively. It was found that yield of glucose was higher from sample 2. Similarly the amount of fructose produced from sample 1 and sample 2 was 63.40g and 71.28g, with a fructose yield of 25.36% and 28.51%, respectively. Percent conversion to fructose based on glucose content after isomerization of 40% w/v glucose syrup from CFR was 42%–43%. The lower starch content in the initial raw material of sample 1 CFR (58%) led to the lower yield of glucose and fructose during saccharification and isomerization as compared to sample 2 of CFR (64%).

Table 7.

Data on hydrolysis of CFR to glucose syrup and its further conversion to high fructose syrup

DETAILS	CFR SAMPLE 1	CFR SAMPLE 2
Substrate concentration (% w/v)	25	25
Percent moisture in the waste	12.50	10.45
Weight of dried CFR taken for study (g)	250	250
Dry weight of the CFR (g)	218.75	223.88
Weight of dry starch present in the residue (g)	145	160
Weight of crude fiber present in the residue (g)	37.5	32.5
Quantity of glucose produced (g)	149.83	165.38
Percentage conversion to glucose
i) on material basis	59.93	66.15
ii) on dry weight basis	68.49	73.87
Quantity of fructose produced (g)	63.40	71.28
Percent conversion to fructose after isomerization based on glucose content	42.31	43.10
Fructose Yield (g/100g CFR)	25.36	28.51

Footnotes

Acknowledgments

Regy Johnson acknowledges a Research Fellowship by the Council of Scientific and Industrial Research (CSIR), Govt. of India. The authors are grateful to the Director, CTCRI for the facilities provided for the study.

Author Disclosure Statement

The authors report no competing financial interests.

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