Some Pectinolytic and Cellulolytic Enzyme Activities of Fungi Causing Rots of Cocoyams

Representative isolates of Aspergillus niger, Botryodiplodia theobromae, Corticium rolfsii, Geotrichum candidum, Fusarium oxysporum and F solani recovered from rotten cocoyams were studied for the production of pectinolytic and cellulolytic enzymes. All the isolates elaborated high levels of hydrolase, lyase and pectinesterase in cocoyam tissue medium and lyase and pectinesterase in pectin medium. There were no significant differences in the overall levels of lyase and pectinesterase activities produced by all the isolates in both media. The level of hydrolase, lyase and pectinesterase activities individually produced by A niger, B theobromae and C rolfsii in both media was significantly higher than that of any other isolate. The highest hydrolase activity was produced by C rolfsii in cocoyam medium while A niger produced the highest lyase activity in pectin medium. Maximum pectinesterase activity was obtained from B theobromae and C rolfsii in pectin medium. All the test isolates produced cellulase in a medium containing carboxymethyl cellulose with C rolfsii showing significantly high activity followed by F oxysporum and A niger. © 1997 SCI.     

Pectinolytic and cellulolytic activities of heat resistant fungi and their macerating effects on mango and African mango

Five heat resistant fungi (HRF), Neosartorya fischeri, N fischeri var spinosa, N quadricincta, Paecilomyces varioti and Byssochlamys nivea, were studied for production of pectinolytic and cellulolytic activities. All isolates produced considerable hydrolase, lyase and pectinesterase activities. Hydrolase activities were significantly higher in fruit tissue (mango and African mango) media than in pectin medium (P < 0.01) when assayed by both cup plate and viscometric methods. Activities produced in both fruit media were comparable in N fischeri, N fischeri var spinosa and P varioti but not in N quadricincta and B nivea. All isolates produced greater lyase activities in pectin medium than in fruit tissue media except for N quadricincta, while the converse was the case for pectinesterase. P varioti did not utilise carboxymethyl cellulose or produce cellulase activity. Other isolates produced cellulase with B nivea producing the greatest activity. Each isolate caused considerable maceration of artificially inoculated mango and African mango fruits, which is not directly related to measurable pectinase or cellulase. The possibility of co‐operation between pectinase and cellulase activities in the disintegration of fruit tissues is discussed. © 1999 Society of Chemical Industry     

Optimization of enzymatic process parameters for increased juice yield from carrot (Daucus carota L.) using response surface methodology

The effects of enzyme concentration (50–650 mg/kg grated carrot), pectolytic and cellulolytic enzyme ratio (3:7–7:3), incubation time (30–150 min) and temperature (25–65 °C) on juice recovery and viscosity from grated carrot were studied. A central composite rotatable design (CCRD) was used in designing the treatment combinations of four variables at five levels. The process involved in treating the blanched grated carrot with mixture of crude pectolytic enzyme from Aspergillus foetidus and crude cellulolytic enzyme from Trichoderma ressi, keeping the samples at the desired time, followed by extraction of juice. Enzyme-treated grated carrot sample showed increased juice recovery as compared to control. A second-order response surface model adequately fitted the data. All the variables affected juice recovery and viscosity significantly. Enzyme concentration, pectolytic and cellulolytic enzyme ratio, incubation time and temperature had total and combined effect at linear, square and interactive level on both responses. The optimum condition was enzyme concentration, 210.7 mg/kg of grated carrot; pectolytic and cellulolytic enzyme ratio, 3.84:6.16; incubation time, 130 min and incubation temperature 47 °C. Under the optimal conditions, juice extracted from enzyme-treated grated carrot was 74.3% having juice viscosity 1.07 cP, corresponding to the increase in yield by 13.95% and decrease in viscosity by 0.45 cP.     

Enhanced Oil Recovery by Pre-treatment of Mustard Seeds Using Crude Enzyme Extract Obtained from Mixed-Culture Solid-State Fermentation of Kinnow (Citrus reticulata) Waste and Wheat Bran

Solid-state fermentation (SSF) of kinnow (Citrus reticulata) waste supplemented with wheat bran was used for production of cellulase, protease and pectinase with individual and mixed cultures of Trichoderma reesei and Aspergillus niger. Mustard seeds were pre-treated with crude filtrate extract (CFE) obtained from A. niger and T. reesei independently, and combination of the two cultures, prior to solvent extraction. Mixed-culture fermentation resulted in higher enzyme activity for filter paper cellulase (FPU), carboxymethyl cellulase (CMCase), β-glucosidase, and exoploygalacturonase (exo-PG) in comparison to fermentation with individual cultures. This study indicated that pre-treatment using crude enzyme obtained with mixed cultures enhanced the oil recovery by 11% as compared to control where no enzyme was used. Mustard seeds pre-treated with CFE obtained from mixed cultures having ratio of 2:1:1 for exo-polygalacturonase, FPU, and protease resulted in highest oil recovery. About 7–10% more oil was recovered when mustard seeds were pre-treated with CFE obtained from individual cultures, compared with control. Enzymatic pre-treatment also improved some of the quality attributes of mustard oil. To the best of our knowledge, this is the first study where mixed-culture SSF was attempted to produce enzyme consortium for pre-treatment of mustard seeds for enhanced oil recovery.     

Kinetics of Growth and Inhibition of Listeria monocytogenes in the Presence of Antioxidant Food Additives

Listeria monocytogenes strain Scott A in Tryptose Broth was treated with 100-300 ppm butylated hydroxyanisole (BHA), 300-700 ppm butylated hydroxytoluene (BHT) and 10-30 ppm tertiary butylhydroquinone (TBHQ). Resulting growth curves were fitted using the logistic model, and growth parameters [lag period (LP), generation time (GT), and maximum growth (MG)] were calculated. BHA and BHT inhibited Listeria monocytogenes by increasing LP and GT and decreasing MG. Extent of inhibition was concentration-dependent for cultures with BHA, but not with BHT. TBHQ at 10-30 ppm increased LP but did not affect other parameters. LP increased exponentially with increased BHA or TBHQ in Listeria culture. Concentrations of additive required to increase LP by one order of magnitude were 240 ppm for BHA and 26 ppm for TBHQ.     

Evaluation of plant oils for suppression of crown rot disease and improvement of shelf life of banana (Musa spp. AAA subgroup,cv. Robusta)

Fourteen plant oils were evaluated to control the crown rot disease caused by Lasiodiplodia theobromae and Colletotrichum musae. Five of these, viz. Ocimum sanctum, Cymbopogan citratus, C. martinii, C. nardus and Pelargonium graveolens oils completely arrested the mycelial growth of both test pathogens at their lowest concentration compared to other oils. Besides, these plant oils have also inhibited the activity of cellulolytic and pectinolytic enzymes produced by these pathogens effectively under in vitro condition. The treatment of banana fruit var. Robusta (Cavendish‐AAA) with oils of O. sanctum, C. citratus, C. nardus and C. martinii not only reduced the crown rot severity significantly, but also increased the shelf life of banana fruits. However, under low‐temperature storage (14 °C) condition, O. sanctum oil increased the shelf life of banana fruits up to 48 days without affecting their organoleptic properties. Hence, O. sanctum oil could be used as an alternative to chemical fungicides for the management of crown rot disease.     

Influence of white light,near-UV irradiation and other environmental conditions on production of aflatoxin B1 by Aspergillus flavus and ochratoxin A by Aspergillus ochraceus

The effects of illumination, near-ultraviolet, incubation temperature pH and some minor elements on the growth rate and production of aflatoxin B₁ by A. flavus and ochratoxin A by A. ochraceus were investigated. Aflatoxin B₁ and ochratoxin A production was considerably higher in the light than in the dark. The greatest aflatoxin B₁ and ochratoxin A production was occurred after 11 days of fermentation with light- and dark-grown cultures at 25 °C. The mycelial dry weight was also greater in the light than in the dark for both A. flavus and A. ochraceus. Exposure of conidia to near-UV irradiation increased mycelial dry weight and mycotoxins by both fungi more than white light. The greatest aflatoxin B₁ and ochratoxin A was at 25 °C with UV-grown culture (24 h exposure) producing a mean of 400 and 260 μg/50 ml of medium, respectively. The maximum aflatoxin B₁ and ochratoxin A yield was obtained at pH 5.5 and with increasing the initial pH to near neutrality, both mycotoxins yield decreased. Iron, cupper and zinc were observed to stimulate aflatoxin B₁ and ochratoxin A production and enhanced the growth rate of both A. flavus and A. ochraceus.     

Inhibition of Citrus Fungal Pathogens by Using Lactic Acid Bacteria

Abstract: The effect of lactic acid bacteria (LAB) on pathogenic fungi was evaluated and the metabolites involved in the antifungal effect were characterized. Penicillium digitatum (INTA 1 to INTA 7) and Geotrichum citri-aurantii (INTA 8) isolated from decayed lemon from commercial packinghouses were treated with imazalil and guazatine to obtain strains resistant to these fungicides. The most resistant strains (4 fungal strains) were selected for evaluating the antifungal activity of 33 LAB strains, among which only 8 strains gave positive results. The antifungal activity of these LAB strains was related to the production of lactic acid, acetic acid, and phenyllactic acid (PLA). A central composite design and the response surface methodology were used to evaluate the inhibitory effect of the organic acids produced by the LAB cultures. The antifungal activity of lactic acid was directly related to its concentration; however, acetic acid and PLA showed a peak of activity at 52.5 and 0.8 mM, respectively, with inhibition rates similar to those obtained with Serenade^® (3.0 ppm) imazalil (50 ppm) and guazatine (50 ppm). Beyond the peak of activity, a reduction in effectiveness of both acetic acid and PLA was observed. Comparing the inhibition rate of the organic acids, PLA was about 66- and 600-fold more effective than acetic acid and lactic acid, respectively. This study presents evidences on the antifungal effect of selected LAB strains and their end products. Studies are currently being undertaken to evaluate the effectiveness in preventing postharvest diseases on citrus fruits.     

Effect of macerate enzymes on the yield,quality, volatile compounds and rheological property of prickly pear juice

Pectinase and cellulase enzymes were used to investigate efficacy for improving juice yield, stability and quality from prickly pear fruit. Pectinase improved the yield, stable color, color‐assayed as release of anthocyanins or carotinoids and clarity of the juice. A significant increase in the effectiveness of pectinase was observed as the concentration was increased from 0.05 to 0.50% v/w. However, at concentration >0.25% v/w they tended to impart a bitter flavor in the juice. Among three concentrations of pectinase and cellulase, pectinase at 0.50% v/w produced higher yield, a sediment‐free clear juice and high‐quality juice. The results indicated that depectinated clarified prickly pear juice behaves as a Newtonian fluid. It was found that the activation energy (Ea) for viscous flow was in the range of 5.02×10³–20.06×10³ kJ/mol depending on the concentrations of pectinase and cellulase enzyme treatment of prickly pear juice, in contrast to 22.15×10³ kJ/mol in untreated juice. Volatile compound concentrations of twelve compounds were not affected by pectinase and cellulase treatment. Overall the quality of prickly pear juice was better in pectinase‐treated juice compared with untreated and cellulase‐treated juice.     

Prolonging Storage Time of Baby Ginger by Using a Sand‐Based Storage Medium and Essential Oil Treatment

Wilt and rot occur readily during storage of baby ginger because of its tender skin and high moisture content (MC). A storage medium, which consisted of sand, 20% water, and 3.75% super absorbent polymers delayed weight loss and loss of firmness at 12 °C and 90% relative humidity. Microorganisms were isolated and purified from decayed rhizomes; among these, 3 fungi were identified as pathogens. The results of 18S rDNA sequence analysis showed that these fungi belonged to Penicillium, Fusarium, and Mortierella genera. The use of essential oil for controlling these pathogens was then investigated in vitro. Essential oils extracted from Cinnamomum zeylanicum (cinnamon) and Thymus vulgaris (thyme) completely inhibited the growth of all of the above pathogens at a concentration of 2000 ppm. Cinnamon oil showed higher antifungal activity in the drug sensitivity test with minimal fungicidal concentration (<500 ppm against all pathogens). In the in vivo test, cinnamon fumigation at a concentration of 500 ppm reduced infection rates of Penicillium, Fusarium, and Mortierella by 50.3%, 54.3%, and 60.7%, respectively. We recommended cinnamon oil fumigation combined with medium storage at 12 °C as an integrated approach to baby ginger storage.     

Coconut husk extract: antibacterial properties and its application for shelf-life extension of Asian sea bass slices

Antibacterial properties of ethanolic coconut husk extracts (ECHE) prepared using varying ethanol concentrations as the extracting media against Pseudomonas aeruginosa, Escherichia coli, Vibrio parahaemolyticus, Listeria monocytogenes and Staphylococcus aureus was studied. ECHE60, prepared using 60% ethanol, had similar minimum inhibitory concentration and minimum bactericidal concentration to those extracted using 80% and 100% ethanol (P ˂ 0.05). When Asian sea bass (Lates calcarifer) slices were treated with ECHE60 at 200 and 400 ppm, the quality changes were monitored in comparison with the control (without treatment) during 12 days of refrigerated storage (4 °C). The shelf life of sea bass treated with 400 ppm ECHE60 was extended to 9 days, whereas the control had a shelf life of 3 days. Lipid oxidation was lowered in ECHE60 treated samples than the control during storage. Therefore, ECHE60 could inhibit both spoilage and pathogenic bacteria and also extended the shelf life of sea bass slices.     

SAKE BREWING FROM LIQUEFIED-RICE WITH IMMOBILISED FUNGAL MYCELIA AND IMMOBILISED YEAST CELLS

Sake brewing from liquefied-rice solution with immobilised fungal mycelia and immobilised yeast cells was investigated. Rice was liquefied by α-amylase in order to improve fluidity. Aspergillus oryzae was used for the production of saccharifying enzymes, and Saccharomyces cerevisiae for fermentation. The saccharifying enzyme productivity of immobilised fungal mycelia grown in highly aerobic conditions was much higher than that of fungal mycelia grown in liquid culture. Furthermore, saccharifying enzyme production was stimulated by the use of liquefied-rice treated with protease. The enzyme activity of immobilised mycelia was 4.5 times higher than that of rice-koji. The saccharifying enzyme was produced 10 times over a period of about 30 d at 41°C using protease treated liquefied-rice. Ethanol production was carried out with immobilised yeast using liquefied-rice containing saccharifying enzyme extracted from immobilised fungal mycelia. 19.0% (v/v) ethanol was obtained after incubation at 15°C for 5 d.     

Effect of substrate and fermentation conditions on pectinase and cellulase production by <Emphasis Type="Italic">Aspergillus niger</Emphasis> NCIM 548 in submerged (SmF) and solid state fermentation (SSF)

The present study deals with the optimization of substrate and fermentation conditions for the production of both pectinase and cellulase by Aspergillus niger NCIM 548 under same fermentation conditions in submerged fermentation (SmF) and solid state fementation (SSF) using a central composite face centered design of response surface methodology (RSM). As per statistical design, the optimum conditions for maximum production of pectinase (1.64 U/mL in SmF and 179.83 U/g in SSF) and cellulase (0.36 U/mL in SmF and 10.81 U/g in SSF) were, time 126 h, pH 4.6, and carbon source concentration 65 g/L in SmF and were time 156 h, pH 4.80, and moisture content 65% in SSF. The response surface modeling was applied effectively to optimize the production of both pectinase and cellulase by A. niger under same fermentation conditions to make the process cost-effective in both submerged and solid state fermentation using agro industrial wastes as substrate.     

Establishment of a stable Amaranthus tricolor callus line for production of food colorant

This study aimed at demonstrating the effects of fermentation time (24, 48, 72, 96, and 120 h) and water activity (0.943, 0.970, and 0.985) on the production of cellulolytic enzymes by solid-state fermentation of purple mombin (Spondias purpurea L.) residue using Aspergillus niger. The fermentation was carried out at 35°C and the enzyme production was measured as endoglucanase and total cellulose activities. The optimum condition for endoglucanase was water activity 0.974 and 93.8 h of fermentation, reaching a production of 3.21 U/g of residue; whereas for total cellulase it was 0.958 and 79.4 h achieving 12.1 U/g of residue. Fermentation time had a greater effect on the endoglucanase activity, while water activity had a more significant influence on the total cellulase activity. Endoglucanase had optimum activity at temperature of 50°C and pH 5.0. Although cellulase total optimum activity was also at pH 5.0, the maximum activity was at 60°C.     

Batch kinetics and modelling of ethanolic fermentation of whey

The fermentation of whey by Kluyveromyces marxianus strain MTCC 1288 was studied using varying lactose concentrations at constant temperature and pH. The increase in substrate concentration up to a certain limit was accompanied by an increase in ethanol formation, for example, at a substrate concentration of 10 g L^?1, the production of ethanol was 0.618 g L^?1 whereas at 50 g L^?1 it was 3.98 g L^?1. However, an increase in lactose concentration to 100 g L^?1 led to a drastic decrease in product formation and substrate utilization. The maximum ethanol yield was obtained with an initial lactose concentration of 50 g L^?1. A method of batch kinetics was utilized to formulate a mathematical model using substrate and product inhibition constants. The model successfully simulated the batch kinetics observed at S₀ = 10 and 50 g L^?1 but failed in case of S₀ = 100 g L^?1 because of strong substrate inhibition.     

Role of Fungi on Oil Quality of Stored Seeds of Sesame, Rape and Linseed

Deteriorative efficacy of storage fungi through change in the quality of different edible oils was studied. Maximum loss of oil in both rape and linseed was recorded with A. fumigatus and in black cultivar of sesame with A. niger. Refractive indices of oil decreased in most of the cases with concomitant increase in free fatty acids (FFA). The deteriorated oil samples showed change in color, saponification value and iodine value with longer incubation which depended partly on the fungus involved and partly on the type of substrate. Both A. niger and A. fumigatus produced higher amount of lipase than others. Production of lipase enzyme and mycelia were always higher on emulsified oil than on seedmeal media.     

Submerged culture conditions for mycelial yield and polysaccharides production by Lyophyllum decastes

The effects of various carbon and nitrogen sources, their concentrations, initial pH and fermentation duration on the production of mycelia in terms of dry weight, exo-polysaccharide (EPS) and inner polysaccharide (IPS) by Lyophyllum decastes, a culinary-medicinal mushroom, were investigated in shake-flask cultures. Lactose, glucose and fructose were the top three best carbon sources for mycelial growth with corresponding yields of 6.73 g/l, 6.36 g/l and 6.10 g/l, respectively. Glucose was the best for production of EPS and IPS with 1.65 g/l and 317 mg/g dry mycelia, respectively. Maltose also performed well for EPS production. Yeast extract was the best nitrogen source for the production of mycelia (7.03 g/l) and IPS (325 mg/g dry mycelia), whereas EPS was improved further by increasing the yeast extract concentration (2.46 g/l at 2%). Similarly, initial pH 7 and 8 were best for polysaccharides production (EPS 1.73 g/l and IPS 320 mg/g) and mycelial growth (7.10 g/l), respectively. Maximum mycelial growth peaked at 15 days of cultivation whereas polysaccharides peaked at 10 days, and then tapered off. A concentration of glucose 3% and yeast extract 1% (mycelial yield and IPS) were found to be a suitable condition for submerged culture.     

The effect of pepsin pretreatment of herbage on the prediction of dry matter digestibility from solubility in fungal cellulase solutions

Cellulase preparations from different fungi differed markedly in their ability to solubilise herbage and cellulose; T. viride cellulase was the most active, solubilising 70% of cellulose paper in 24 h. The correlation of cellulase solubility with the in vivo and in vitro dry matter digestibility of grasses, and with the in vitro digestibility of legumes was markedly improved by pretreatment of the herbage with acid pepsin. Using the two stage technique, closely similar regression lines were obtained for predicting the in vitro digestibility of both grasses and legumes. Use of the pepsin treatment also enabled a less active Basidiomycete cellulase to be used with results very similar to those obtained with the T. viride enzyme. The technique is proposed as a more rapid, convenient and precise method of predicting digestibility than the usual in vitro procedure.     

Effect of molasses on the production and activity of dye-decolorizing peroxidase from Geotrichum candidum Dec1

The production of dye-decolorizing peroxidase (DyP) was investigated by cultivating Geotrichum candidum Dec1 using molasses as a carbon source. Molasses at concentrations greater than 10 g·l⁻¹ was found to increase the decolorization activity of the culture broth toward dye, reactive blue 5 mainly because the amount of enzyme produced was enhanced. However, complete inhibition of DyP activity by molasses was observed at the concentration of 20 g·l⁻¹, indicating that the inhibitory effect of molasses on the culture broth activity to decolorize the dye was involved. When the culture broth was diluted 25 times, the dye-decolorizing activity was 7 times as much as that of non-diluted culture broth. The molasses fractions separated by gel chromatography (300–400 ml and 400–500 ml fractions) completely inhibited the purified DyP. On the basis of these results, we propose a scheme to control both positive and negative effects of molasses on the dye decolorization process.     

Biomass and Patulin Production by Byssochlamys nivea in Apple Juice as Affected by Sorbate, Benzoate, SO2 and Temperature

The effects of potassium sorbate, sodium benzoate, SO₂ and incubation temperature on biomass and patulin production by Byssochlamys nivea in apple juice were determined. Growth at 21, 30 and 37°C over a 25-day incubation period was significantly retarded by 75 ppm SO₂, 150 ppm potassium sorbate and 500 ppm sodium benzoate. Biomass accumulated to approximately 500 mg/100 ml in control samples of apple juice. Patulin was produced in the highest concentrations at 21°C after 20 days incubation. After reaching a maximum concentration at 30 and 37°C, a rapid decline in patulin content was observed. Patulin production was also observed at 12°C. On the basis of concentration, SO₂ had the most significant effect on the rate of biomass and patulin production by B. nivea followed by potassium sorbate and sodium benzoate, respectively.