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Leveraging pH Profiles to Direct Enzyme Production (Cellulase, Xylanase, Polygalacturonase, Pectinase, α-Galactosidase, and Invertase) by Aspergillus foetidus
Abstract
Aspergillus foetidus was found to have different optimal pH conditions for producing different carbohydrases including cellulase, xylanase, pectinase, α-galactosidase, polygalacturonase, and invertase. Designs to trigger these conditions sequentially using controlled pH gradients were evaluated for directing the culture through multiple production stages optimal for different enzymes. For production of enzyme mixtures with particularly high pectinase and α-galactosidase activities, for more effective hydrolysis of the complex carbohydrate in soybean meal, the best method tested was a pH gradient started at pH 7.0, decreased to pH 6.0 over 72 h, held constant for 24 h, and then decreased to pH 5 over 24 h.
... Fed-batch hull addition was considered for its potential effects of 1) increasing/ distributing the substrate and inducer availability to sustain longer active enzyme synthesis and 2) reducing the feedback repression by unconsumed hydrolytic products (sugars), assuming slower sugar generation and accumulation from hydrolysis of the hull added in a smaller quantity each time. The pH effects (initial and decreasing rate) were included in the study because A. niger had been reported to have different optimal pH for producing different carbohydrases (Sohail et al., 2009;Ajayi et al., 2018;Li et al., 2018;Elshafei et al., 2022). The objective of this work was to produce enzymes with high pectinase, α-galactosidase, cellulase, and sucrase in a single fermentation. ...
... per h, decreasing pH from 7 (initial) to 5.5 at 96 h. In previous studies, pH 5.5-6.5 was found to be more favorable for pectinase production by Aspergillus (Li et al., 2018;Li et al., 2020). F7 was therefore designed to maximize pectinase production by 96 h. ...
... F7 was therefore designed to maximize pectinase production by 96 h. However, the high pH was not optimal for synthesis of α-galactosidase, xylanase, and especially cellulase (Sohail et al., 2009;Ajayi et al., 2018;Li et al., 2018;2020;Elshafei et al., 2022). Higher pH drop rates were therefore used in F8 and F9, to lower the pH to about 4 in 96 h (F8) or 84 h (F9). ...
Enzyme production is critical and often costly for biorefinery. It is challenging to produce enzymes with not only high titers but also proper combinations of all required activities in a single fermentation. This work aimed at improving productivity and composition of the multiple enzyme activities required for hydrolysis of complex soybean carbohydrate in a single fermentation. A previously selected Aspergillus niger strain was used for its high carbohydrases and low protease production. Strategies of fed-batch substrate addition and programmed pH-decrease rates were evaluated. Cheap soybean hull (SH) was confirmed to induce production of all necessary carbohydrases. Surprisingly, fed-batch SH addition, originally thought to sustain substrate-inducer availability and reduce feedback repression by sugars, did not increase pectinase and cellulase production significantly and even lowered the α-galactosidase production, when compared with batch fermentation having the same total SH amount (all added initially). On the other hand, the pH-decrease rate could be effectively optimized for production of complex enzyme mixtures. The best fermentation was programmed to lower pH from 7 to 4 in 84 h, at a drop rate of .0357 per h. It produced the highest pectinase (19.1 ± .04 U/mL), α-galactosidase (15.7 ± .4 U/mL), and cellulase (.88 ± .06 FPU/mL). Producing these high enzyme activities in a single fermentation significantly improves the effectiveness and economics of enzymatic soy processing, which, e.g., can hydrolyze the 30%–35% carbohydrate in soybean meal to sugars, with minimal protein degradation, to generate high-value protein-rich products and a hydrolysate as fermentation feedstock.
... A previous strain-screening study established Aspergillus niger NRRL 322 as a high producer of all carbohydrases required to hydrolyze various soy carbohydrates, when growing on soy hull as the inducing substrate [16]. Further, the carbohydrases composition produced could be adjusted by altering the fermentation conditions, particularly the pH profile [17][18][19]. In this study, soy molasses processing was studied using the enzymes produced by A. niger NRRL 322 fermentations. ...
... More importantly, the ability gained from this work to estimate the desired α-galactosidase-to-sucrase ratio is valuable for guiding the fermentation for enzyme production. The carbohydrases composition produced by A. niger NRRL 322 growing on soy hull could be adjusted by altering the fermentation conditions, particularly the pH profile [17][18][19]. For example, the α-galactosidase-to-sucrase ratio in the enzyme broths used in this study (Table 1) varied from 1.2 to 6.0, although most were in the 2.4-2.9 range. ...
Soybean processing generates huge amounts of soy molasses that can support biorefinery but require development of waste-to-value conversion technologies. Here, soy molasses processing by Aspergillus niger enzymes was studied to optimize the conversion of oligosaccharides to monomeric sugars as ready fermentation feedstock. The effects of pH and temperature were first investigated using fixed enzyme composition and loading. pH, in the tested 3.0-6.5 range, significantly affected hydrolysis particularly in galactose release. The hydrolysis was fastest at pH 4.8 and 60 °C although the 48-h sugar (glucose, fructose, and galactose) yields were similar at pH 4.8 and 5.7, and 50 and 60 °C. Study was next made at these favorable pH and temperatures using different enzyme compositions and loadings. Glucose and fructose were effectively released, reaching ∼100 % yields in 24-48 h by most of the enzymes and loadings evaluated. Galactose production was less effective and varied significantly with the pH-temperature condition and enzyme loading and composition. Mechanistic evaluation suggested formation and accumulation of galactose disaccharide, whose slow hydrolysis was rate-limiting in the systems with complete glucose and fructose releases but low galactose yields. Model equations were developed to describe the kinetic sugar-release profiles and make technoeconomic analysis, which showed that a process of lower enzyme loading, while requiring longer duration, is more economical within the analyzed range of 5-50 (U α-galactosidase/g molasses). With 5 (U/g) loading, the total cost is about 30 % lower at 60 °C-pH 5.7 than 50 °C-pH 4.8. The α-galactosidase-to-sucrase ratio plays a less significant role in affecting the overall process cost.
... The requirement of multiple enzymes to hydrolyze all types of carbohydrates present in the biomass relied on effective biomass pretreatment and optimal mixtures of multiple enzyme activities. Usually, these enzymes are produced from different sources and then blended into cocktails and evaluated for hydrolysis effectiveness for particular biomass (LI et al., 2018). ...
... Enzyme hydrolysis has been accepted as the most environmentally friendly technology for the conversion of carbohydrate in biomass into monomeric sugars. An enzyme mixture having multiple activities is required to achieve more complete hydrolysis of the complex carbohydrate present in lignocellulosic biomass (RODRIGUES & ODANETH, 2021;LI et al., 2018;SUWANNARANGSEE et al., 2012). ...
This study evaluated the action of commercial and non-commercial cellulases and pectinases in the hydrolysis of soybean hulls (SH) and corn stover and cobs (CSC), the effect of temperature and agitation on the lignocellulosic substrate hydrolysis and the bromatological characteristics of hydrolyzed substrates. The effect of pretreatment on the hydrolysis of lignocellulosic residues and bromatological analysis were also evaluated. The highest hydrolytic activity occurred at 300 rpm for SH (47.95 and 51.43% for cellulase and pectinase, respectively) and at 350 rpm for CSC (26.05 and 9.23% for cellulase and pectinase, respectively). Non-commercial enzymes achieved 7.26-30% of the amount of hydrolysis obtained with commercial enzymes, on the same substrates. Pretreatment with 7.5% of NaOH and a particle size of the substrate of 0.5 mm significantly increased the hydrolysis of SH and CSC for both enzymes. The bromatological characteristics showed that soybean hulls hydrolyzed with both commercial cellulase and pectinase have potential for large-scale use in animal feed production.
... The effect of different extraction pH values (4.0, 4.5, 5.0, 5.5, and 6.0) on starch content is shown in Figure S3B. The KS content first increased and then decreased with the increase in pH, potentially because the optimal pH values of the three enzymes used in this experiment are different [20][21]. When the pH was 5.0, the comprehensive activity of the three enzymes was the highest, the reaction was the most thorough, and the impurities removal effect was the best. ...
A new ultrasound-assisted enzymatic extraction (UAEE) method of starch from kiwifruit was established and optimized using response surface methodology (RSM). Under optimal conditions (the pectinase-to-cellulase-to-papain ratio = 1:2:1 g/kg, solid/liquid ratio = 1:6.68, extraction pH = 5.23, ultrasound power = 300 W, and extraction temperature = 52 °C), the kiwi starch (KS) yield was about 4.25%, and the starch content of KS was 873.23 mg/g. Compared to other extraction methods, UAEE can obtain KS with high yield and purity with a shorter extraction time and less solvent and enzyme. The extracted KS has a low gelatinization enthalpy (8.02 J/g) and a high peak viscosity (7933 cP), with obvious particle properties and low adhesion. In addition, KS is rich in polyphenols, has strong antioxidant activity, and has higher contents of amylose starch (30.74%) and resistant starch (60.18%). This study established a novel and highly efficient method for KS extraction and suggest several possible applications for KS in the food industry.
... Different cellulase enzymes can be produced for a wide range of pH (between 3.0-8.0). [14,32,33] Hence, the cellulases T A B L E 1 Actual values and levels for pH and sonication power (W ml À1 ), adopted in Doehlert design to investigate complementary effect of ultrasound-assisted extraction (UAE) exhibited good tolerance to pH variations in the extraction medium. However, using a neutral buffer solution prevents eventual equilibrium reaction dislocations because the solubility of the enzymes depends on polar interactions with the aqueous phase, which decreases near its isoelectric point. ...
In this study, the optimum conditions for the multi-enzymatic recovery of cellulases produced by Aspergillus niger were investigated using sugarcane bagasse. Two extraction methods were investigated: the two-stage solid–liquid extraction (SLE) followed by ultrasound-assisted extraction (UAE), and the single-stage SLE. The ultrasound effects were evaluated using a Doehlert design, in which the pH (5.0–9.0) and sonication power (0.8–2.0 W mL⁻¹) were independent variables. For the single-stage SLE, temperature (25–45°C), time (10–60 min), and pH (5.0–9.0) were analyzed using the Box–Behnken design. Both processes were monitored to evaluate FPase, CMCase, and β-glucosidase (U mL⁻¹) activities. The maximum enzymatic activities (EA) obtained for FPase, CMCase, and β-glucosidase in the SLE–UAE were 0.352, 0.321, and 1.412 U mL⁻¹, respectively. Unlike in most previous studies, sonication was insignificant (p < 0.05) with respect to the enzymatic complex within the evaluated ranges. Moreover, sonication changed the EA when lower than 1.2 and higher than 1.6 W mL⁻¹, mainly inhibiting the EA of β-glucosidase. The single-stage SLE was more effective than the two-stage SLE–UAE, and the maximum EA values for FPase, CMCase, and β-glucosidase were 0.354, 0.303, and 3.135 U mL⁻¹, respectively. The single-stage process was better because it consumed less energy, required simpler equipment, and provided higher efficiency in a shorter time. This study will improve diversified enzyme extraction from sugarcane bagasse, reduce enzyme production costs, and enhance bagasse utilization.
... It had 30.2 ± 3.9% total carbohydrate (12.8 ± 0.6% soluble, 17.4 ± 3.5% structural) consisting of 9.4 ± 1.2% glucose, 1.9 ± 0.4% xylose, 6.1 ± 1.3% galactose, 2.9 ± 0.2% arabinose, 4.5 ± 0.3% fructose, 2.1 ± 0.6% mannose, and 3.3 ± 0.5% galacturonic acid [15]. The enzyme broths were produced in this laboratory by 4 Aspergillus niger NRRL 322 fermentations and 1 Aspergillus foetidus NRRL 341 fermentation (typical fermentation procedures reported in [16,17]). A commercial cellulase enzyme (Genencor's SPEZYME-CP) was also used, which was measured (prior to use) to have, per mL, 147 FPU cellulase, 349 U xylanase, 7.3 U pectinase, 2.2 U α-galactosidase, and 0.2 U sucrase. ...
Carbohydrate is a major underutilized and undervalued resource in soybean. Its indigestibility concern causes devaluation of soy protein for some uses. Conversion of carbohydrate to monomeric sugars as fermentation feedstock can significantly increase soybean value but it involves multiple carbohydrates and multi-enzyme dependency and interactions. Sugar yields were measured for soybean flour processing using 10 different enzymes and the results modeled to gain insights into soybean carbohydrates and monomerization, including individual sugar distributions among different carbohydrates, sugar composition of each carbohydrate, and the optimal carbohydrases composition. The study is important for guiding future optimization of enzyme activities and process design.
... Generally, it is already accepted that enzyme production is largely influenced by the type of the producing strain as well as nutritional and environmental conditions. Therefore, it is important to optimize such conditions for each isolated strain individually (Li et al., 2018). Results for investigating kinetics of cell growth and β-galactosidase production using the initial un-optimized medium clearly showed that both cellular growth as well as enzyme production rates increased during the logarithmic growth phase, and then decreased upon entering the stationary phase. ...
β-Galactosidase is an industrially important enzyme with many applications in various health, diagnostic and food processing industries. The present work describes the isolation and identification of a new isolate capable of producing β-galactosidase. The work was further extended to optimize the production medium and investigate the kinetics of cell growth and enzyme production during batch cultivation in shake-flask and 5L-stirred tank bioreactor. The isolated strain was identified as Bacillus licheniformis using 16S rRNA and BLAST analysis. The final optimized production medium contained (g/L): yeast extract, 14.0; lactose, 8.0; NaCl, 16.0; (NH4)H2PO4, 0.1; MgSO4.7H2O, 0.1; K2HPO4, 0.1. The optimized medium afforded maximal β-galactosidase production of 116.4 U/mL with a specific yield coefficient (YP/X) of 27.6 × 10³ U/g cells. These results corresponds to an increase of about 4.4- and 2.8-folds, respectively, than the initial un-optimized production medium. Further improvement in the production process was achieved by cultivating cells in 5L-stirred tank bioreactor, which increased volumetric enzyme productivity and specific yield coefficient by 8.5- and 6.0-folds than the initial un-optimized production medium, respectively. A new β-galactosidase producing strain was identified and the volumetric as well as specific productivities were greatly enhanced through medium optimization and bioreactor cultivation.
The essential oil industry is agro-based and its consumption is increasing very fast. The need to improve its production technology is essential to enhancing the overall yield and quality. An environmentally friendly process is therefore sought which could be achievable via enzyme-based processes. This study produced xylanase and pectinase by solid-state fermentation and applied them in pre-treating milled onions (Allium cepa) for improved essential oil yield. The pre-treated milled onion bulbs were hydro distilled at about 100 oC and the essential oils obtained characterized using Gas Chromatography – Mass Spectrometry to identify the components. Response Surface Methodology was employed for optimizing the extraction parameters. Results showed that the use of enzymes for pre-treatment of the onions increased the essential oil yield. The xylanase and pectinase function optimally at pH 5.5 and 4.0 respectively and at a temperature of 60 oC. About 10 compounds were identified in the essential oil obtained from untreated onion with Oleic acid as the main component. The mixed enzyme-treated onions showed 24 compounds in the essential oil which is rich in S-propyl methane thiosulfonate. The response surface methodology gave the optimum extraction time and enzyme quantity for the extraction. Antioxidant activity results indicate that the oil has a protective effect against oxidative stress which is of medical importance. Variability of the components of the extracted essential oils with enzymes makes it expedient to choose a pre-treatment enzyme based on the component of interest.
The fungal system holds morphological plasticity and metabolic versatility which makes it unique. Fungal habitat ranges from the Arctic region to the fertile mainland, including tropical rainforests, and temperate deserts. They possess a wide range of lifestyles behaving as saprophytic, parasitic, opportunistic, and obligate symbionts. These eukaryotic microbes can survive any living condition and adapt to behave as extremophiles, mesophiles, thermophiles, or even psychrophile organisms. This behaviour has been exploited to yield microbial enzymes which can survive in extreme environments. The cost-effective production, stable catalytic behaviour and ease of genetic manipulation make them prominent sources of several industrially important enzymes. Pectinases are a class of pectin-degrading enzymes that show different mechanisms and substrate specificities to release end products. The pectinase family of enzymes is produced by microbial sources such as bacteria, fungi, actinomycetes, plants, and animals. Fungal pectinases having high specificity for natural sources and higher stabilities and catalytic activities make them promising green catalysts for industrial applications. Pectinases from different microbial sources have been investigated for their industrial applications. However, their relevance in the food and textile industries is remarkable and has been extensively studied. The focus of this review is to provide comprehensive information on the current findings on fungal pectinases targeting diverse sources of fungal strains, their production by fermentation techniques, and a summary of purification strategies. Studies on pectinases regarding innovations comprising bioreactor-based production, immobilization of pectinases, in silico and expression studies, directed evolution, and omics-driven approaches specifically by fungal microbiota have been summarized.
White-rot fungi represent an outstanding alternative for valorizing fruit residues due to their ability to decompose structural carbohydrates and other bioactive compounds abundant in fruit waste. Enzymes produced by these fungi are responsible for the biological pretreatment process carried out by oxidases and hydrolases, resulting in the degradation of structural carbohydrates and lignin. This work reviews and compares reported data on Solid-State Fermentation (SSF) and Submerged Fermentation (SmF) using various strains from the genus Pleurotus for the biological pretreatment of fruit residues. SSF could be the best cultivation strategy due to its similarity to the natural growth conditions of fungi on wood substrates. Nonetheless, SmF has advantages if the process is to be scaled at industrial level. Further research will be required to determine adequate growth conditions of Pleurotus spp. on fruit residues, including the study of relevant parameters of the process (i.e., fungi strains, substrates, additives).
The potential of cellulolytic enzymes has been widely studied and explored for bioconversion processes and plays a key role in various industrial applications. Cellulase, a key enzyme for cellulose-rich waste feedstock-based biorefinery, has increasing demand in various industries, e.g., paper and pulp, juice clarification, etc. Also, there has been constant progress in developing new strategies to enhance its production, such as the application of waste feedstock as the substrate for the production of individual or enzyme cocktails, process parameters control, and genetic manipulations for enzyme production with enhanced yield, efficiency, and specificity. Further, an insight into immobilization techniques has also been presented for improved reusability of cellulase, a critical factor that controls the cost of the enzyme at an industrial scale. In addition, the review also gives an insight into the status of the significant application of cellulase in the industrial sector, with its techno-economic analysis for future applications. The present review gives a complete overview of current perspectives on the production of microbial cellulases as a promising tool to develop a sustainable and greener concept for industrial applications.
The enzymatic treatment of defatted soy flour (SF) to reduce indigestible carbohydrates can result in undesirable protein loss. Here protein loss was minimized with quantitation of the effects of ionic strength (IS), protease activity, and SF toasting. At the enzyme processing condition (25% w/v SF, 50 °C, pH 4.8, 48 hours), protein loss increased linearly with the IS in enzyme broths (EB); e.g., contacting untoasted SF with water or heat‐deactivated EB showed protein loss of 28% in water but up to 40% when IS was increased in the range of 0.04–0.19 M. Protein loss also increased with protease in EB (nondeactivated): after adjusted for IS‐related loss, approximately 10% and 25% additional protein loss occurred in EB of 73 and 490–557 U/(g SF) protease, respectively. SDS‐PAGE results showed that proteolysis was not extensive, mainly on β‐conglycinin α'/α and glycinin acidic 37‐kDa subunits; and most of the proteolytic products could be recovered by heat‐induced precipitation. SF toasting effects were studied, particularly at 2‐hours 160°C, with material balances, protein distributions, and monosaccharide yields in hydrolysates. Overall, protein loss was minimized to 5.2% and the conversion of carbohydrate to monomeric sugars increased to 89.2%.
Enzymes play a significant role in several biotechnology-based industries for making the process cost effective. These enzymes are predominantly obtained from microbes (bacteria, fungi, and microalgae), plants and animals for serving intended applications. Recently, biofuels are advocated as clean and green, alternative source of energy to meet the growing demand of fossil fuels. However, biofuel production is not commercially scalable since lignocellulose biomass (LCB)-converting enzymes/processes are not cost effective. Several commercial enzymes including cellulase, xylanase, laminarinase and other ligninolytic enzymes are used to implement a synergistic action in the breakdown of LCB structure into pentose or hexose sugars, and other co-products. Enzyme dosage optimisation during the biomass processing and co-product production are considered amongst the major challenges in this biorefinery pathway. This chapter covers application of commercial enzyme preparations in LCB processing, and factors affecting co-product production in a biorefinery set-up to address processing challenges.
α-Galactosidase, (E.C. 3.2.1.22) is an exoglycosidase that target galactooligosaccharides such as raffinose, melibiose, stachyose and branched polysaccharides like galactomannans and galacto- glucomannans by catalysing the hydrolysis of α-1,6 linked terminal galactose residues. The enzyme has been isolated and characterized from microbial, plant and animal sources. This ubiquitous enzyme possesses physiological significance and immense industrial potential. Optimization of the growth conditions and efficient purification strategies can lead to a significant increase in the enzyme production. To boost commercial productivity, cloning of novel α-galactosidase genes and their heterologous expression in suitable host has gained popularity. Enzyme immobilization leads to its greater reutilization, superior thermostability, pH tolerance and increased activity. The enzyme is well explored in food industry in the removal of raffinose family oligosaccharides (RFOs) in soymilk and sugar crystallization process. It also improves animal feed quality and biomass processing. Applications of the enzyme is in the area of biomedicine includes therapeutic advances in treatment of Fabry disease, blood group conversion and removal of α-gal type immunogenic epitopes in xenotransplantation. With considerable biotechnological applications, this enzyme has been vastly commercialized and holds greater future prospects.
Inosine monophosphate (IMP) was considered to be an important component of meat flavor and an important indicator for evaluating the quality of meat products. This paper developed a novel dual-enzyme biosensor for the quantitative detection of inosinic acid to assess meat quality. Using the conductivity of MXene materials and the ability of Au@Pt nanoflowers to catalyze the oxidation of H2O2, a dual-enzyme biosensor was assembled and prepared for sensitive and rapid detection of IMP. The MXene-Ti3C2Tx material had 2D nanostructure similar to that of graphene, as well as metal conductivity and good biocompatibility. MXene was used as a carrier for 5′-nucleotidase and xanthine oxidase, with good biological environment and stable microenvironment. Bimetallic nanoflowers with a core-shell structure had better ability to catalyze H2O2 than the single metal. The double-enzyme hydrolyzed IMP to produce the H2O2. The Au@Pt nanoflowers of the sensor can catalyze the decomposition of H2O2, which causes electron transfer to produce current change. Therefore, the content of IMP is indirectly obtained by monitoring the current change. The results showed that the linear range of the double-enzyme biosensor was 0.04~17 g L⁻¹, the correlation coefficient was 0.9964, and the detection limit was 2.73 ng mL⁻¹. The biosensor had great reproducibility and stability. Compared with high-performance liquid chromatography, biosensors could quickly and accurately detect the content of inosine monophosphate in meat, and provided a better method to detect the quality index of meat products.
Four lactic acid bacteria strains (Lactobacillus paracasei, Leuconostoc mesenteroides, Lactobacillus rhamnosus GG and Lactobacillus plantarum), possessing β-glucosidase enzyme activity, were investigated for tofu whey fermentation based on the capacity of bioconversion of isoflavones. The results suggested that the mixture of L. rhamnosus GG and L. paracasei had great potential for efficient enrichment of bioactive isoflavone aglycones and the percentage composition (daidzein, genistein)increased from 4.6 to 93.78% after fermentation of tofu whey. At the same time, the viable cell counts of the mixture of L. rhamnosus GG and L. paracasei increased to 9.13 log 10 CFU/mL while pH value dropped to 4.48 and titratable acidity was 0.28%. Headspace solid-phase microextraction method followed by gas chromatography-mass spectrometry analysis indicated that the odor compounds, particularly the aldehydes and alcohols, were metabolized to trace or undetected levels after fermentation. This research provided not only a new way to develop a functional probiotic beverage enriched in isoflavone aglycones, but also provided a potentially valuable new use for soy whey protein.
Digestion is a very complex process involving many different enzymes expressed by the human cells and by the microbial community in the digestive tract. Digestive problems such as lactose intolerance and poor digestion of vegetable oligosaccharides affect a great part of the human population, causing discomforts due to their fermentation by gas-producing microorganisms. Although the treatment may involve the supplementation with digestive enzymes, such as lactase (beta-galactosidase) and alpha-galactosidase, respectively, the industrial processing of food products is another alternative. Gluten intolerant and celiac individuals could potentially be benefited by the administration of peptidases or the consumption of peptidase-treated food, however, this is not yet considered a treatment option that substitutes the complete avoidance of gluten. Enzymes have been applied in food processing for various purposes including the removal of undesired components. Besides the galactosidases that remove specific saccharides, another example is the use of l-asparaginase to avoid the formation of acrylamide, a possible carcinogen. In this chapter, the application of digestive enzymes in food bioprocessing will be reviewed, from traditional applications of alpha- and beta-galactosidases, to potential applications of proteases and lipases. Examples of commercial products and of the most recent and relevant patents in this area will be included.
Soybean carbohydrate is often found to limit the use of protein in soy flour as food and animal feed due to its indigestibility to monogastric animal. In the current study, an enzymatic process was developed to produce not only soy protein concentrate and soy protein isolate without indigestible carbohydrate but also soluble reducing sugar as potential fermentation feedstock. For increasing protein content in the product and maximizing protein recovery, the process was optimized to include the following steps: hydrolysis of soy flour using an Aspergillus niger enzyme system; separation of the solid and liquid by centrifugation (10 min at 7500×g); an optional step of washing to remove entrapped hydrolysate from the protein-rich wet solid stream by ethanol (at an ethanol-to-wet-solid ratio (v/w) of 10, resulting in a liquid phase of approximately 60 % ethanol); and a final precipitation of residual protein from the sugar-rich liquid stream by heat treatment (30 min at 95 °C). Starting from 100 g soy flour, this process would produce approximately 54 g soy protein concentrate with 70 % protein (or, including the optional solid wash, 43 g with 80 % protein), 9 g soy protein isolate with 89 % protein, and 280 ml syrup of 60 g/l reducing sugar. The amino acid composition of the soy protein concentrate produced was comparable to that of the starting soy flour. Enzymes produced by three fungal species, A. niger, Trichoderma reesei, and Aspergillus aculeatus, were also evaluated for effectiveness to use in this process.
Soy protein is a well-known nutritional supplement in proteinaceous food and animal feed. However, soybeans contain complex carbohydrate. Selective carbohydrate removal by enzymes could increase the protein content and remove the indigestibility of soy products for inclusion in animal feed. Complete hydrolysis of soy flour carbohydrates is challenging due to the presence of proteins and different types of non-structural polysaccharides. This study is designed to guide complex enzyme mixture required for hydrolysis of all types of soy flour carbohydrates. Enzyme broths from Aspergillus niger, Aspergillus aculeatus and Trichoderma reesei fermentations were evaluated in this study for soy carbohydrate hydrolysis. The resultant hydrolysate was measured for solubilized carbohydrate by both total carbohydrate and reducing sugar analyses. Conversion data attained after 48 h hydrolysis were first fitted with models to determine the maximum fractions of carbohydrate hydrolyzable by each enzyme group, i.e., cellulase, xylanase, pectinase and α-galactosidase. Kinetic models were then developed to describe the increasing conversions over time under different enzyme activities and process conditions. The models showed high fidelity in predicting soy carbohydrate hydrolysis over broad ranges of soy flour loading (5-25%) and enzyme activities: per g soy flour, cellulase, 0.04-30 FPU; xylanase, 3.5–618 U; pectinase, 0.03–120 U; and α-galactosidase, 0.01–60 U. The models are valuable in guiding the development and production of optimal enzyme mixtures towards hydrolysis of all types of carbohydrates present in soy flour and in optimizing the design and operation of hydrolysis reactor and process.
The filamentous fungi A. fumigatus produced high levels of invertase when cultured for 96 hr at 30°C and pH 5 in Czapek Dox supplemented with fruit peel waste as substrate. Enhanced production occurred on addition of sucrose and yeast extract as nutritional factors. The crude extract was purified to 4.73 fold with recovery of 4.91% by DEAE-column chromatography and the molecular weight was estimated to be 67 KDa by SDS-PAGE. The enzyme activity was stimulated by Na+ and Ca2+ and inhibited by Zinc. The enzyme hydrolyzed sucrose exhibiting Vmax value of 27.53 U/mg and Km value of 0.28 mg/ml. The activity of the invertase was found to be stable at 50°C for 30 minutes at pH 6.
Agro-industrial residues are primarily composed of complex polysaccharides that strengthen microbial growth for the production of industrially important enzymes. Pectinases are one of the most widely disseminated enzymes in bacteria, fungi and plants. Czapeck media supplemented with orange waste peel as carbon source under submerged fermentation process Aspergillus niger presenting the preeminent enzymatic production. On partial optimization culture showed the maximum enzyme yield (117.1 ± 3.4 μM/mL/min) at 30 °C in an orange waste peel medium having pH 5.5 and substrate concentration (4%) after 5th day of fermentation. The produced enzyme was purified by ammonium sulfate precipitation and ion exchange chromatography. A purification fold of 5.59 with specific activity and % recovery of 97.2 U/mg and 12.96% was achieved respectively after gel filtration chromatographic technique. The molecular weight of purified pectinase from A. niger was 30 kDa evidenced by SDS-PAGE. Pectinase activity profile showed purified enzyme was optimally active at pH = 7 and 55 °C. The maximum production of pectinase in the presence of cheaper substrate at low concentration makes the enzyme useful in industrial sectors especially for textile and juice industry.
Pectinase enzyme finds extensive application in food and beverage industries. Pectinase production was studied in solid-state fermentation process using wheat bran and sugarcane bagasse as substrates utilizing Aspergillus niger. Optimization of media and fermentation conditions for maximum production of pectinase was carried out by one at a time procedure. Various combination of substrates were tried to achieve maximum pectinase production. The mixed substrates consisting of 90% of wheat bran and 10% of sugarcane bagasse gives maximum pectinase yield during the fermentation period of 96 h. The optimum temperature was found to be 40°C and optimum pH was found to be 5.The kinetics of pectinase production by solid state fermentation using Aspergillus niger was studied. The kinetic parameters value were found to be K m = 294.12 and V max = 2.33 h ml/U.
Soybean contains a high concentration of carbohydrates that consist mainly of non-starch polysaccharides (NSP) and oligosaccharides. The NSP can be divided into insoluble NSP (mainly cellulose) and soluble NSP (composed mainly of pectic polymers, which are partially soluble in water). Monogastric animals do not have the enzymes to hydrolyze these carbohydrates, and thus their digestion occurs by means of bacterial fermentation. The fermentation of soybean carbohydrates produces short chain fatty acids that can be used as an energy source by animals. The utilization efficiency of the carbohydrates is related to the chemical structure, the level of inclusion in the diet, species and age of the animal. In poultry, soluble NSP can increase digesta viscosity, reduce the digestibility of nutrients and depress growth performance. In growing pigs, these effects, in particular the effect on gut viscosity, are often not so obvious. However, in weaning piglets, it is reported that soy oligosaccharides and soluble NSP can cause detrimental effects on intestinal health. In monogastrics, consideration must be given to the anti-nutritive effect of the NSP on nutrient digestion and absorption on one hand, as well as the potential benefits or detriments of intestinal fermentation products to the host. This mirrors the needs for i) increasing efficiency of utilization of fibrous materials in monogastrics, and ii) the maintenance and improvement of animal health in antibiotic-free production systems, on the other hand. For example, ethanol/water extraction removes the low molecular weight carbohydrate fractions, such as the oligosaccharides and part of the soluble pectins, leaving behind the insoluble fraction of the NSP, which is devoid of anti-nutritive activities. The resultant product is a high quality soy protein concentrate. This paper presents the composition and chemical structures of carbohydrates present in soybeans and discusses their nutritive and anti-nutritive effects on digestion and absorption of nutrients in pigs and poultry.
Aspergillus niger was used for cellulase production in submerged (SmF) and solid state fermentation (SSF). The maximum production of cellulase was obtained after 72 h of incubation in SSF and 96 h in Smf. The CMCase and FPase activities recorded in SSF were 8.89 and 3.56 U per g of dry mycelial bran (DBM), respectively. Where as in Smf the CMase & FPase activities were found to be 3.29 and 2.3 U per ml culture broth, respectively. The productivity of extracellular cellulase in SSF was 14.6 fold higher than in SmF. The physical and nutritional parameters of fermentation like pH, temperature, substrate, carbon and nitrogen sources were optimized. The optimal conditions for maximum biosynthesis of cellulase by A. niger were shown to be at pH 6, temperature 30 °C. The additives like lactose, peptone and coir waste as substrate increased the productivity both in SmF and SSF. The moisture ratio of 1:2 (w/v) was observed for optimum production of cellulase in SSF.
The total water-soluble and water-insoluble non-starch polysaccharides (NSP) were determined in seven soyabean meals processed in Australia, three soyabean meals processed in the U.S.A., and one sunflower meal processed in Australia. Sunflower meal had a higher content of total NSP than any of the soyabean meals, due mainly to increased concentrations of cellulose and xylose in the insoluble NSP and uronic acids in the soluble NSP. Galactose and fucose concentrations were much greater in the insoluble NSP of soyabean meals. Soyabean meals processed in the U.S.A. had lower concentrations of total NSP, cellulose and xylose than equivalent meals processed in Australia. Broiler chickens fed diets containing soyabean meal as the sole dietary protein concentrate grew significantly more poorly than broilers fed isoenergetic and isonitrogenous diets in which approximately 25% of the soyabean meal was replaced with sunflower meal. There was a significant (P < 0.05) negative correlation (r2 = 0.57) between the water-soluble xylose content of the soyabean meals and the improvement in growth observed with sunflower meal supplementation. Multi-enzyme preparations designed to act on soyabean meal NSP substrates failed to induce any improvement in the growth of broilers fed soyabean meal diets. The poor performance of broilers fed the soyabean meal diets did not appear to be related to inadequate processing or to the NSP content or composition of the soyabean meals. Measurement of free sugars in the supernatant of the digesta in the ileum indicated that the stachyose derived from the oligosaccharides of soyabean meals appeared to exert an antinutritive effect when soyabean meal was present at high concentrations as the sole protein concentrate in broiler diets.
Response surface methodology was used for optimization of polygalacturonase (PG) and pectinesterase (PE) production in submerged fermentation by A.niger. A Central Composite Experimental Design was applied, consisting of 22 experiments, including eight central points. Variables studied were: fermentation time (24 to 120 h), pH (3.5 to 6.5) and initial concentration of pectin (5 to 20 g/l). Maximum PE production was 220 U/l, after 74 h of culture, in a medium containing 20 g/l of pectin (pH 6.5). The optimal conditions for PG production were pH: 4.1, 20 g/l of pectin and 94 h of fermentation with a maximum value of 1032 U/l. Under these conditions, the PE production was low (15 U/l). A liquid extract with high PG activity and low PE activity could be suitable to be used in food processing in order to reduce the production of methanol.
Twenty laboratories participated in a collaborative investigation of assays for endo-1,4-β-xylanase activity based on production of reducing sugars from polymeric 4-O-methyl glucuronoxylan. The substrates and methods already in use in the different laboratories were first recorded and the apparent activities obtained using these methods in the analysis of a distributed enzyme sample were compared. The standard deviation of the results reported in this analysis was 108% of the mean. Significant reduction in interlaboratory variation was obtained when all the participants used the same substrate for activity determination, each with their own assay procedure. The level of agreement was further improved when both the substrate and the method procedure were standardized.In a round robin testing of a single substrate and method, including precise instructions for enzyme dilution, the standard deviation between the results after the rejection of two outliers was 17% of the mean. This figure probably reflects the inherently poor reproducibility of results when using only partially soluble, poorly defined and rather impure polymeric substrates. The final level of variation was however low enough to allow meaningful comparison of results obtained in different laboratories when using the standardized assay substrate and method procedure.Fifteen laboratories also participated in preliminary testing of an assay based on the release of dyed fragments from 4-O-methyl glucuronoxylan dyed with Remazol Brilliant Blue dye. High values of the coefficients of correlation indicated good linearity between the amount of dyed fragments released and enzyme concentration. The relative standard deviations of the results obtained by fifteen laboratories were about 30% for an optimum range of xylanase activity in the reaction mixture.
Proteolytic degradation by host proteases is one of the key issues in the application of filamentous fungi for non-fungal protein production. In this study the influence of several environmental factors on the production of extracellular proteases of Aspergillus niger was investigated systematically in controlled batch cultures. Of all factors investigated in a series of initial screening experiments, culture pH and nitrogen concentration in particular strongly affected extracellular protease activities. For instance, at a culture pH of 4, protease activity was higher than at pH 5, and protease activity increased with increasing concentrations of ammonium as nitrogen source. Interestingly, an interdependence was observed for several of the factors studied. These possible interaction effects were investigated further using a full factorial experimental design. Amongst others, the results showed a clear interaction effect between nitrogen source and nitrogen concentration. Based on the observed interactions, the selection of environmental factors to reduce protease activity is not straightforward, as unexpected antagonistic or synergistic effects occur. Furthermore, not only were the effects of the process parameters on maximum protease activity investigated, but five other protease-related phenotypes were studied as well, such as maximum specific protease activity and maximum protease productivity. There were significant differences in the effect of the environmental parameters on the various protease-related phenotypes. For instance, pH significantly affected final levels of protease activity, but not protease productivity. The results obtained in this study are important for the optimization of A. niger for protein production.
The use of biomass to provide energy has been fundamental to the development of civilisation. In recent times pressures on the global environment have led to calls for an increased use of renewable energy sources, in lieu of fossil fuels. Biomass is one potential source of renewable energy and the conversion of plant material into a suitable form of energy, usually electricity or as a fuel for an internal combustion engine, can be achieved using a number of different routes, each with specific pros and cons. A brief review of the main conversion processes is presented, with specific regard to the production of a fuel suitable for spark ignition gas engines.
Soybean is well known for its high-value oil and protein. Carbohydrate is, however, an underutilized major component, representing almost 26-30% (w/w) of the dried bean. The complex soybean carbohydrate is not easily hydrolysable and can cause indigestibility when included in food and feed. Enzymes can be used to hydrolyze the carbohydrate for improving soybean processing and value of soybean products. Here the enzyme-based processing developed for the following purposes is reviewed: hydrolysis of different carbohydrate-rich by/products from soybean processing, improvement of soybean oil extraction, and increase of nutritional value of soybean-based food and animal feed. Once hydrolyzed into fermentable sugars, soybean carbohydrate can find more value-added applications and further improve the overall economics of soybean processing.
Despite having high protein and carbohydrate, soybean flour utilization is limited to partial replacement of animal feed to date. Enzymatic process can be exploited to increase its value by enriching protein content and separating carbohydrate for utilization as fermentation feedstock. Enzyme hydrolysis with fed-batch and recycle designs were evaluated here for achieving this goal with high productivities. Fed-batch process improved carbohydrate conversion, particularly at high substrate loadings of 250-375 g/L. In recycle process, hydrolysate retained a significant portion of the limiting enzyme α-galactosidase to accelerate carbohydrate monomerization rate. At single-pass retention time of 6 h and recycle rate of 62.5%, reducing sugar concentration reached up to 120 g/L using 4 ml/g enzyme. When compared with batch and fed-batch processes, the recycle process increased the volumetric productivity of reducing sugar by 36% (vs. fed-batch) to 57% (vs. batch) and that of protein product by 280% (vs. fed-batch) to 300% (vs. batch).
Soybean hull consists mainly of three major plant carbohydrates, i.e., cellulose, hemicellulose and pectin. It is inexpensive and a good potential substrate for carbohydrase production because it is capable of inducing a complete spectrum of activities to hydrolyze complex biomass. Aspergillus is known for carbohydrase production but no studies have evaluated and compared, among Aspergillus species and strains, the soybean hull induced production of various carbohydrases. In this study, A. aculeatus, A. cinnamomeus, A. foetidus, A. phoenicis and 11 A. niger strains were examined together with T. reesei Rut C30, another known carbohydrase producer. The carbohydrases evaluated included pectinase, polygalacturonase, xylanase, cellulase, α-galactosidase and sucrase. Growth morphology and pH profiles were also followed. Among Aspergillus strains, morphology was found to correlate with both carbohydrase production and pH decrease profile. Filamentous strains gave higher carbohydrase production while causing slower pH decrease. The enzyme broths produced were also tested for separation of soy flour carbohydrate and protein. Defatted soy flour contains about 53% protein and 32% carbohydrate. The enzymatic treatment can increase protein content and remove indigestible oligo-/poly-saccharides, and improve use of soy flour in feed and food. Protease production by different strains was therefore also compared for minimizing protein degradation. A. niger NRRL 322 and A. foetidus NRRL 341 were found to be the most potent strains that produced maximal carbohydrases and minimal protease under soybean hull induction.
Soy protein is a valuable nutritional supplement for food and animal feed. While protein constitutes ~50 % of defatted soy flour (SF), it coexists with complex carbohydrates (30–35 %) which may have anti-nutritional effects. An enzymatic process can remove the carbohydrate and produce protein-enriched soy products. The hydrolysate with monomerized carbohydrates is valuable fermentation feedstock. In this study, Aspergillus niger and Trichoderma reesei enzymes were compared for use in the process. Effects of pH (3.2–6.4), temperature (40–60 °C), enzyme-to-SF ratio (0–2 ml/g) and SF loading (150–350 g/l) were evaluated for the enzymatic conversion of SF carbohydrate to reducing sugar (Y
RS) and total soluble carbohydrate (Y
TC) in the hydrolysate. Effects of these single factors and interactions between factors were investigated. Optimal pH and temperature were similar for both enzymes: pH 4.8 and 50–51 °C for Y
TC, and pH 5.1–5.2 and 48–51 °C for Y
RS. The two enzymes also gave similar protein contents in resultant soy protein concentrates, i.e., 74–75 % with 2 ml/g enzyme broth and 150 g/l SF, which were higher than the 64–68 % protein in commercial concentrates. A. niger enzyme was significantly more effective in carbohydrate conversion, achieving Y
RS = 75 % and Y
TC = 78 % with 2 ml/g enzyme and 150 g/l SF, higher than the Y
RS (30 %) and Y
TC (64 %) obtained with T. reesei enzyme. Monomerization was essentially complete in hydrolysate produced with A. niger enzyme.
Being one of the major oilseed crops throughout America and Asia, soybean production has increased rapidly due to the rising demand for oil and protein. Carbohydrates are also major components in soybean. During the soybean processing for producing oil and protein products, large amounts of carbohydrate-rich byproducts or waste are generated. Soy protein products such as soybean meal, soy protein concentrate and soybean milk also contain carbohydrates that have anti-nutritional concerns and decrease the value of these products. Removing these carbohydrates from soy protein products and finding valuable uses for these carbohydrates and other carbohydrate-rich waste/byproducts are highly desirable. Here, the various carbohydrate-rich waste byproducts generated from soybean processing and their compositions are described. Recent developments on their use as fermentation feedstocks for production of biofuels, enzymes and a variety of specialty chemicals are then reviewed and summarized. This review can facilitate knowledge and technology integration for development of a soy-based biorefinery platform.
Polygalacturonase and pectinase activities reported in the literature were measured by several different procedures. These procedures do not give comparable results, partly owing to the complexity of the substrates involved. This work was aimed at developing consistent and efficient assays for polygalacturonase and pectinase activities, using polygalacturonic acid and citrus pectin, respectively, as the substrate. Different enzyme mixtures produced by Aspergillus niger and Trichoderma reesei with different inducing carbon sources were used for the method development. A series of experiments were conducted to evaluate the incubation time, substrate concentration, and enzyme dilution. Accordingly, for both assays the recommended (optimal) hydrolysis time is 30min and substrate concentration is 5g/L. For polygalacturonase, the sample should be adjusted to have 0.3-0.8U/mL polygalacturonase activity, because in this range the assay outcomes were consistent (independent of dilution factors). Such a range did not exist for the pectinase assay. The recommended procedure is to assay the sample at multiple (at least 2) dilution factors and determine, by linear interpolation, the dilution factor that would release reducing sugar equivalent to 0.4g/L d-galacturonic acid, and then calculate the activity of the sample accordingly (dilution factor×0.687U/mL). Validation experiments showed consistent results using these assays. Effects of substrate preparation methods were also examined.
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The soybean [Glycine max (L.) Merr.] is grown worldwide for its high protein and oil contents. Characterization of soybean seed components lends itself to understanding how soybean production can meet the needs of a growing world population. For this article, literature was reviewed and condensed to create a well-rounded picture of the current understanding of structural, functional, and nutritional properties of soybean components. Natural variation in soybean protein, lipid, and carbohydrate components, as well as the minor constituents phytic acid and isoflavones, are mentioned. Environment- or genetic-induced shifts in natural variation are described with respect to nutrition and functional improvements in soybean.
The enzyme induction utility of soybean hulls (SBH), consisting in excess of 50 wt% non-starch polysaccharides (NSP) cellulose, hemicellulose, and pectin, was studied through cultivation of the carbohydrase-producing fungus Trichoderma reesei Rut C-30. Shake flask systems of T. reesei were grown in a medium consisting of defatted soybean flour as a nitrogen source and SBH, some of which were untreated and others pretreated by liquid hot water, alkaline, and supercritical carbon dioxide, as carbon source. Cellulase, xylanase, and polygalacturonase activities were measured for the systems, and the natural hull systems were found to yield optimum enzyme production. Controlled batch fermentation experiments were carried out to compare enzyme production resulting from media with Avicel® (FMC BioPolymer, Philadelphia, PA, USA) versus natural SBH with and without soybean flour as the nitrogen source. Soybean hull fermentations were also performed at several pH levels to observe the effects on enzyme production. Soybean hulls induced comparable levels of cellulase, and higher levels of xylanase and polygalacturonase, than Avicel®. With SBH, cellulase and xylanase production were enhanced at higher pH levels (6.0), and polygalacturonase was enhanced at lower pH levels (4.0–4.5). Enzyme production was largely unaffected by the presence of soybean flour as the nitrogen source.
Enzymatic hydrolysis is the unit operation in the lignocellulose conversion process that utilizes enzymes to depolymerize lignocellulosic biomass. The saccharide components released are the feedstock for fermentation. When performed at high-solids loadings (>= 15% solids, w/w), enzymatic hydrolysis potentially offers many advantages over conversions performed at low- or moderate-solids loadings, including increased sugar and ethanol concentrations and decreased capital and operating costs. The goal of this review is to provide a consolidated source of information on studies using high-solids loadings in enzymatic hydrolysis. Included in this review is a brief discussion of the limitations, such as a lack of available water, difficulty with mixing and handling, insufficient mass and heat transfer, and increased concentration of inhibitors, associated with the use of high solids, as well as descriptions and findings of studies that performed enzymatic hydrolysis at high-solids loadings. Reactors designed and/or equipped for improved handling of high-solids slurries are also discussed. Lastly, this review includes a brief discussion of some of the operations that have successfully scaled-up and implemented high-solids enzymatic hydrolysis at pilot- and demonstration-scale facilities.
Total tract digestibility in Atlantic salmon and ileal digestibility in chicken were assessed from diets with different soyabean products (hulled, toasted, extracted, SBM; reduced oligosaccharide content, ROM; ethanol-extracted protein concentrate, SPC; isolated protein, ISP). The concentration of dietary fibre was highest in SBM and ROM, while it was low in ISP. In vitro viscosity was also higher in SBM than in the other soyabean products. The diets for the salmon and chickens were based on the same feed ingredients, with the exception that fish meal provided half the crude protein in the salmon diets. For each species, the diets were isonitrogenous, contained similar amounts of fat (fish oil), and were balanced with dextrin, thus substituting soyabean non-starch polysaccharides (NSP) and other non-proteinous components by dextrin.In the salmon, total tract digestibility of nitrogen and dry matter were lower (p
To study the incomplete enzymatic extractability of proteins and carbohydrates of thermally treated soybean meals, one unheated and three heat-treated soybean meals were produced. To obtain truly enzyme-resistant material, the meals were extracted by a repeated hydrolysis procedure using excessive concentrations of different combinations of commercial protease and carbohydrase preparations. The water extractability of protein from the different meals varied considerably (13−67%). For all soybean meals, enzymatic treatment extracted most of the original protein (89−94%). Carbohydrase preparations did not improve protein extraction. High-humidity heat treatment led to a more effective enzymatic extraction, which seemed to correlate with the extent of protein denaturation. Results with purified proteins indicated that the soybean meal matrix affects the enzymatic extraction of protein from the meals. Interactions between protein and other components (e.g., cellulose) may explain the incomplete enzymatic extractability of protein from the meals. Keywords: Soybean meal; heat treatment; hydrolysis; extraction; enzymatic residue; protease; carbohydrase; composition; protein; carbohydrate; amino acid
The success and sustainability of aquaculture depends on minimising the operational cost of feed that in general comprises 50–60% of the total cost in intensive farming. The major feed ingredient, fish meal, is expensive and there is increasing competition with other livestock industries for the available static supply of fish meal. Hence, the incorporation of plant-derived materials in fish feeds is receiving increasing attention. One of the main constraints in the utilisation of plant ingredients in aquaculture is the presence of indigestible carbohydrates, which consist primarily of non-starch polysaccharides (NSPs). These form a part of the cell wall structure of cereals and legumes. The presence of NSPs in the diet interferes with feed utilisation and adversely affects performance of the animal. Supplementation of NSP-degrading enzymes in feed mitigates the adverse effects of NSPs. The effects of NSPs in pigs and poultry have been widely studied; however little information exists for fish. This review synthesizes the available information on fish and highlights the knowledge gaps. It is hoped that this review will provide a momentum to the research on the roles of NSPs in fish nutrition and physiology and on the efficient use of NSP-degrading enzymes.
Synergistic enzyme system for the hydrolysis of alkali-pretreated rice straw was optimised based on the synergy of crude fungal enzyme extracts with a commercial cellulase (Celluclast™). Among 13 enzyme extracts, the enzyme preparation from Aspergillus aculeatus BCC 199 exhibited the highest level of synergy with Celluclast™. This synergy was based on the complementary cellulolytic and hemicellulolytic activities of the BCC 199 enzyme extract. A mixture design was used to optimise the ternary enzyme complex based on the synergistic enzyme mixture with Bacillus subtilis expansin. Using the full cubic model, the optimal formulation of the enzyme mixture was predicted to the percentage of Celluclast™: BCC 199: expansin=41.4:37.0:21.6, which produced 769 mg reducing sugar/g biomass using 2.82 FPU/g enzymes. This work demonstrated the use of a systematic approach for the design and optimisation of a synergistic enzyme mixture of fungal enzymes and expansin for lignocellulosic degradation.
Soybean [Glycine max (L.) Merril] carbohydrates make up approximately 35% of soybean (SB) seed and 40% of soybean meal (SBM) dry matter (DM). Approximately half of these carbohydrates are nonstructural in nature, including low molecular weight sugars, oligosaccharides, and small amounts of starch, while the other half are structural polysaccharides, including a large amount of pectic polysaccharides. The small amounts of free galactose, glucose, fructose, and sucrose make up the low molecular weight sugars. Galacto-oligosaccharides (raffinose, stachyose, and verbascose) comprise approximately 5% of the SB DM, while starch represents less than 1%. The structural carbohydrates can be divided into cotyledon meal and hull polysaccharides. The primary cotyledon meal polysaccharides are arabinogalactan and an acidic polysaccharide that is similar to pectin, whereas the hull contains pectin, hemicelluloses, and cellulose. A portion of SBM carbohydrates are digested and another portion fermented in the animal's gastrointestinal tract. The low molecular weight sugars and starch are essentially 100% digestible by nonruminants, while the oligosaccharides are not digested by gastrointestinal enzymes, but are fermented by bacterial populations in the ileum and colon of pigs. This fermentation may result in both positive and negative effects. The nutritional significance of the structural SB carbohydrates in nonruminant animal diets remains virtually unknown. Many factors affect the nutritional value of SB carbohydrates, including cultivar and genotype, processing, and addition of exogenous enzymes. A better understanding of the nutritive contribution of SB carbohydrates is necessary as regards their impact on animal nutrition broadly defined.
In this work soy and wheat bran were employed as raw materials for the production of pectinases by Aspergillus niger through solid-state fermentation. Several fermentation and recovery parameters were studied. The kinetics of enzyme synthesis was investigated in the range from 13 to 96 h with moisture contents varying from 25% to 70% (w/w). A medium moisture content of 40% and a fermentation time of 22 h were selected, as these conditions resulted in high pectolytic activity and enhanced polygalacturonase productivity. In order to optimise the recovery step, the best combination of temperature of extraction, contact time and solvent type was investigated. Acetate buffer (pH 4.4), 35°C and 30 min provided the best recovery. The present results show that optimising the extraction conditions is a simple way of obtaining more concentrated enzyme extracts and could be a useful instrument to extract more selectively a desired biomolecule from fermented solids.
The regulation of the synthesis of invertase in Aspergillus niger with different carbohydrates was studied. In order to investigate the mechanism of induction, we worked with conidia protoplasts and permeabilied mycelium and used actinomycin D, cycloheximide and cAMP.The synthesis of invertase in A. niger was induced by β-fructofuranoside substrates such as raffinose, sucrose and turanose. Inulin also produced synthesis, suggesting that the β link and the fructose located at the end of the molecule are involved in the induction mechanism.Glucose and fructose, which are products of the action of invertase, are repressors of the synthesis and act at the level of the translation of the genetic message. The induction of invertase is different from that of yeasts and other filamentous fungi. A mechanism of induction of the synthesis of invertase in A. niger with the participation of cAMP is proposed.
Fungal cellulases are well-studied enzymes and are used in various industrial processes. Much of the knowledge of enzymatic depolymerization of cellulosic material has come from Trichoderma cellulase system. Species of Trichoderma can produce substantial amounts of endoglucanase and exoglucanase but very low levels of b-glucosidase. This deficiency necessitates screening of fungi for cellulytic potential. A number of indigenously isolated fungi were screened for cellulytic potential. In the present study, the kinetics of cellulase production from an indigenous strain of Aspergillus niger MS82 is reported. Product formation parameters of endoglucanase and beta-glucosidase (Qp + Y(p/s)) indicate that A.niger MS82 is capable of producing moderate to high levels of both endoglucanase and beta-glucosidase when grown on different carbon containing natural substrates, for example, grass, corncob, bagasse along side purified celluloses. Furthermore, it was observed that the production of endoglucanase reaches its maximum during exponential phase of growth, while b-glucosidase during the Stationary phase. Enzyme production by solid-state fermentation was also investigated and found to be promising.Highest production of cellulase was noted at pH 4.0 at 35 degrees C under submerged conditions. Growth and enzyme production was affected by variations in temperature and pH.
A cellulolytic enzyme was isolated from a commercial cellulase preparation form Aspergillus niger. A yield of about 50mg of enzyme was obtained per 100g of commerial cellulase. The isolated enzyme was homogeneous in the ultracentrifuge at pH 4.0 and 8.0, and in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis but showed one major and two minor bands in disc gel electrophoresis. No carbohydrate was associated with the protein. Amino acid analysis revealed that the enzyme was rich in acidic and aromatic amino acids. Data from the amino acid composition and dodecyl sulphate/polyacrylamide-gel electrophoresis indicated a molecular weight of 26000. The purified enzyme was active towards CM-cellulose, but no activity towards either cellobiose or p-nitrophenyl beta-D-glucoside was detected under the assay conditions used. The pH optimum for the enzyme was pH 3.8-4.0, and it was stable at 25 degrees C over the range pH 1-9; maximum activity (at pH 4.0) was obtained at 45 degrees C. The cellulase was more stable to heat treatment at pH 8.0 than at 4.0. Kinetic studies gave pK values between 4.2 and 5.3 for groups involved in the enzyme-substrate complex.
Defatted untoasted soybean cotyledons and hulls were fractionated as water solutes (WSc and WSh) and water unextractable (WUc and WUh). Further fractionation of WUc through deproteinization yielded the isolation of a water unextractable solid (WUS) fraction that was mainly composed (molar percent) of galactose (28.1%), glucose (27.8%), arabinose (13.3%), and uronic acids (17.6%), which accounted for 76% of the water insoluble polysaccharides in soybean cotyledons (WUc). The cell wall (WUS) was sequentially fractionated with chelating agents (chelating agent soluble solids, ChSS) and a gradient of agents (dilute alkali, DASS; 1 M alkali, 1MASS; and 4M alkali, 4MASS), which gave a final cellulosic residue. The ChSS and DASS extracts were characterized as pectin-rich fractions, whereas 1MASS and 4MASS were hemicellulose- and cellulose-rich fractions. Incubation in vitro of the WUc fraction with pectinase, cellulase, and xylanase resulted in the release of low amounts (not more than 5% bound basis) of monosaccharides, mostly uronic acids, xylose, and arabinose. Protein extraction hardly increased this release after enzymatic incubation (<7%). However, progressive fractionation of the cell wall matrix markedly increased the release of monosaccharides from pectin- (ChSS and DASS) and hemicellulose-rich fractions (1MASS and 4MASS). Significant degradation of cellulose (up to 20%) was achieved only after complete protein, pectin, and hemicellulose extraction.
Lignocellulosic biomass can be utilized to produce ethanol, a promising alternative energy source for the limited crude oil. There are mainly two processes involved in the conversion: hydrolysis of cellulose in the lignocellulosic biomass to produce reducing sugars, and fermentation of the sugars to ethanol. The cost of ethanol production from lignocellulosic materials is relatively high based on current technologies, and the main challenges are the low yield and high cost of the hydrolysis process. Considerable research efforts have been made to improve the hydrolysis of lignocellulosic materials. Pretreatment of lignocellulosic materials to remove lignin and hemicellulose can significantly enhance the hydrolysis of cellulose. Optimization of the cellulase enzymes and the enzyme loading can also improve the hydrolysis. Simultaneous saccharification and fermentation effectively removes glucose, which is an inhibitor to cellulase activity, thus increasing the yield and rate of cellulose hydrolysis.
The influence of carbon and nitrogen sources on the production of cellulases was investigated. The enzyme production was variable according to the carbon source. Levels of beta-cellobiohydrolase (CBH) were minimal in the presence of even low concentrations of glucose. Enzyme production was stimulated by other carbohydrates. The enzyme is subject to carbon source control by easily metabolizable sugars. Wheat bran and cellulose were the most effective promoters of beta-cellobiohydrolase and filter paperase (FPase) activities respectively, followed by rice bran. Exogenously supplied glucose inhibited the synthesis of the enzyme in cultures of A. niger growing on wheat bran. In defined medium with cellobiose, the cellobiohydrolase titres were 2- to 110-fold higher with cells growing on monomeric sugars and 1.5 times higher than cells growing on other disaccharides. It appeared that synthesis of beta-cellobiohydrolase varied under an induction mechanism, and a repression mechanism which changed the rate of synthesis of beta-cellobiohydrolase and FPase in induced over non-induced cultures. In this organism, substantial synthesis of beta-cellobiohydrolase can be induced by cellobiose, cellodextrin, cellulose or cellulose and hemi-cellulose containing substrates which showed low volumetric substrate uptake rate. The organism required limiting concentration of carbon, nitrogen or phosphorous for production of beta-cellobiohydrolase and FPase. During growth of A. niger on wheat bran, maximum volumetric productivities (Qp) of beta-cellobiohydrolase and FPase were 39.6 and 32.5 IU/lh and were significantly higher than the values reported for some other potent fungi and bacteria. The addition of actinomycin D (a repressor of transcription) and cycloheximide, (a repressor of translation) completely repressed CBH/FPase biosynthesis, suggested that the regulation of CBH synthesis in this organism occurs at both transcriptional and translational level. Thermodynamic studies revealed that the culture exerted protection against thermal inactivation when exposed to different fermentation temperatures.
Studies were carried out on the production of pectinases using deseeded sunflower head by Aspergillus niger DMF 27 and DMF 45 in submerged fermentation (SmF) and solid-state fermentation (SSF). Higher titres of endo- and exo-pectinases were observed when medium was supplemented with carbon (4% glucose for SmF and 6% sucrose for SSF) and nitrogen (ammonium sulphate, 0.3% for both SmF and SSF) sources. Green gram husk proved to be relatively a better supplement to attain higher yield of endo-pectinase (11.7 U/g) and exo-pectinase (30.0 U/g) in solid-state conditions. Maximum production of endo-pectinase (19.8 U/g) and exo-pectinase (45.9 U/g) by DMF 45 were recorded in SSF when compared to endo-pectinase (18.9 U/ml) and exo-pectinase (30.3 U/ml) by DMF 27 in SmF under optimum process conditions.
Comparisons were made for alpha-galactosidase production using red gram plant waste (RGPW) with wheat bran (WB) and other locally available substrates using the fungus Aspergillus oryzae under solid-state fermentation (SSF). RGPW proved to be potential substrate for alpha-galactosidase production as it gave higher enzyme titers (3.4 U/g) compared to WB (2.7 U/g) and other substrates tested. Mixing WB with RGPW (1:1, w/w) resulted enhanced alpha-galactosidase yield. The volume of moistening agent in the ratio of 1:2 (w/v), pH 5.5 and 1 ml (1 x 10(6) spores) of inoculum volume and four days incubation were optimum for alpha-galactosidase production. Increase in substrate concentration (RGPW+WB) did not decrease enzyme yield in trays.
The work is intended to achieve optimum culture conditions of alpha-galactosidase production by a mutant strain Aspergillus foetidus ZU-G1 in solid-state fermentation (SSF). Certain fermentation parameters involving moisture content, incubation temperature, cultivation period of seed, inoculum volume, initial pH value, layers of pledget, load size of medium and period of cultivation were investigated separately. The optimal cultivating conditions of alpha-galactosidase production in SSF were 60% initial moisture of medium, 28 degrees C incubation temperature, 18 h cultivation period of seed, 10% inoculum volume, 5.0 approximately 6.0 initial pH of medium, 6 layers of pledget and 10 g dry matter loadage. Under the optimized cultivation conditions, the maximum alpha-galactosidase production was 2 037.51 U/g dry matter near the 144th hour of fermentation.