Production of macerating enzymes of mandarin orange peel by fungal cultures

Summary Thirty-nine fungal cultures belonging to the genera of Aspergillus, Podospora, Sordaria, Cbaetomium, Iodophanus, Scleotinia, Coniella, Pellicularia and others, were examined for the production of enzymes which macerate the mandarin orange peel using a wheat bran as substrate. An isolated strain of Aspergillus niger was an excellent producer of macerating enzymes compared to other organisms tested. The peel of the mandarin orange could be macerated by the crude enzymes produced by isolated A. niger. The maceration of 1 g of peel/24 h yielded 0.57 g of reducing sugars. Expressed differently, 83% of solid peel materials were released from the peel into the water/24 h under the following conditions: a peel concentration of 8 g peel/l, a crude enzyme concentration of 1 g protein/l, a temperature of 40°C, a pH of 5, a 24 h incubation time and 120 rpm reciprocal shaking. The test of the macerating activity of commercially available hydrolases on the orange peel showed that the two samples of pectinase originating from A. niger had about the same activity as isolated A. niger whereas the two samples of cellulase originating from Trichoderma viride had remarkably lower activities than A. niger.     

Azetidine-2-carboxylic acid in the food chain

Azetidine-2-carboxylic acid (Aze) 1 is a non-protein amino acid present in sugar beets and in table beets (Beta vulgaris). It is readily misincorporated into proteins in place of proline 2 in many species, including humans, and causes numerous toxic effects as well as congenital malformations. Its role in the pathogenesis of disease in humans has remained unexplored. Sugar beet agriculture, especially in the Northern Hemisphere, has become widespread during the past 150 years, and now accounts for nearly 30% of the world’s supply of sucrose. Sugar beet byproducts are also used as a dietary supplement for livestock. Therefore, this study was undertaken as an initial survey to identify Aze-containing links in the food chain. Herein, we report the presence of Aze 1 in three sugar beet byproducts that are fed to farm animals: sugar beet molasses, shredded sugar beet pulp, and pelleted sugar beet pulp.     

Screening of Bacterial Cellulose-producing Acetobacter Strains Suitable for Agitated Culture

Extensive screening for cellulose-producing Acetobacter strains suitable for agitated culture was done by developing the screening conditions. A total of 2096 strains were isolated; isolation from fruits was particularly efficient. The cellulose productivities of 412 isolates were estimated by culturing in two different media under both shaken and static conditions. No correlation between the amounts of cellulose accumulated in shaken and static cultures was observed. Higher cellulose accumulation was obtained in the shaken cultures using a corn steep liquor/fructose-based medium than a conventional yeast extract/peptone/glucose-based one. Many isolates showed higher cellulose accumulation than well-known cellulose-producing strains. The producer that yielded the highest cellulose accumulation in shaken culture was selected and named Acetobacter sp. BPR 2001. Using this strain, cellulose was produced in a jar fermentor.     

Comparative evaluation of Aspergillus niger strains for endogenous pectin-depolymerization capacity and suitability for d-galacturonic acid production

Pectinaceous agricultural residues rich in d-galacturonic acid (d-GalA), such as sugar beet pulp, are considered as promising feedstocks for waste-to-value conversions. Aspergillus niger is known for its strong pectinolytic activity. However, while specialized strains for production of citric acid or proteins are well characterized, this is not the case for the production of pectinases. We, therefore, systematically compared the pectinolytic capabilities of six A. niger strains (ATCC 1015, ATCC 11414, NRRL 3122, CBS 513.88, NRRL 3, and N402) using controlled batch cultivations in stirred-tank bioreactors. A. niger ATCC 11414 showed the highest polygalacturonase activity, specific protein secretion, and a suitable morphology. Furthermore, d-GalA release from sugar beet pulp was 75% higher compared to the standard lab strain A. niger N402. Our study, therefore, presents a robust initial strain selection to guide future process improvement of d-GalA production from agricultural residues and identifies a high-performance base strain for further genetic optimizations.

     

Aspergillus niger I-1472 and Pycnoporus cinnabarinus MUCL39533, selected for the biotransformation of ferulic acid to vanillin, are also able to produce cell wall polysaccharide-degrading enzymes and feruloyl esterases

The filamentous fungal strains Aspergillus niger I-1472 and Pycnoporus cinnabarinus MUCL39533, previously selected for the bioconversion of ferulic acid to vanillic acid and vanillin respectively, were grown on sugar beet pulp. A large spectrum of polysaccharide-degrading enzymes was produced by A. niger and very few levels of feruloyl esterases were found. In contrast, P. cinnabarinus culture filtrate contained low amount of polysaccharide-degrading enzymes and no feruloyl esterases. In order to enhance feruloyl esterases in A. niger cultures, feruloylated oligosaccharide-rich fractions were prepared from sugar beet pulp or cereal bran and used as carbon sources. Number of polysaccharide-degrading enzymes were induced. Feruloyl esterases were much higher in maize bran-based medium than in sugar beet pulp-based medium, demonstrating the ability of carbon sources originating from maize to induce the synthesis of feruloyl esterases. Thus, A. niger I-1472 could be interesting to release ferulic acid from sugar beet pulp or maize bran.     

Synthesis of a trigalacturonic acid analogue mimicking the expected transition state in the glycosidases

A trigalacturonic acid analogue carrying a cyclohexene framework in place of the central pyranose ring was synthesized as a molecular probe for the mechanistic investigation of endo-polygalacturonase 1 (endo-PG 1). Preliminary enzymatic studies revealed that this analogue inhibited endo-PG 1 activity by about 30% at 0.3 mM concentration.     

Induction of Gram-negative bacterial growth by neurochemical containing banana (Musa x paradisiaca) extracts

Bananas contain large quantities of neurochemicals. Extracts from the peel and pulp of bananas in increasing stages of ripening were prepared and evaluated for their ability to modulate the growth of non-pathogenic and pathogenic bacteria. Extracts from the peel, and to a much lesser degree the pulp, increased the growth of Gram-negative bacterial strains Escherichia coli O157:H7, Shigella flexneri, Enterobacter cloacae and Salmonella typhimurium, as well as two non-pathogenic E. coli strains, in direct relation to the content of norepinephrine and dopamine, but not serotonin. The growth of Gram-positive bacteria was not altered by any of the extracts. Supplementation of vehicle and pulp cultures with norepinephrine or dopamine yielded growth equivalent to peel cultures. Total organic analysis of extracts further demonstrated that the differential effects of peel and pulp on bacterial growth was not nutritionally based, but due to norepinephrine and dopamine. These results suggest that neurochemicals contained within foodstuffs may influence the growth of pathogenic and indigenous bacteria through direct neurochemical-bacterial interactions.     

Biological control of cucumber and sugar beet damping-off caused by Pythium ultimum with bacterial and fungal antagonists

AIMS: Five bacterial strains belonging to Bacillus subtilis, Pseudomonas fluorescens and Ps. corrugata and two fungal strains belonging to Trichoderma viride and Gliocladium virens were evaluated for their efficacy in controlling sugar beet and cucumber damping-off caused by Pythium ultimum. METHODS AND RESULTS: The in vitro antagonistic activity of bacteria against various Pythium spp. was evaluated with dual cultures in various media. Pseudomonas strains inhibited the pathogen better than Bacillus strains. To identify potentially useful antagonist combinations, dual compatibility of antagonists was also evaluated, based on growth in two liquid media containing substrate previously used by other antagonists. Four pairs of bacteria were selected. Sugar beet damping-off biocontrol was attempted with bacterial seed treatments (individually and in pairs). Cucumber damping-off biocontrol was attempted with bacterial seed treatments and bacterial and fungal compost treatments. In sugar beet, satisfactory biocontrol was only achieved with Pseudomonas antagonists. Antagonist combinations did not show any superior biocontrol ability to individual antagonists and compatibility of bacteria in vitro did not correlate with compatibility in vivo. Bacterial seed treatments and fungal compost treatments failed to control cucumber damping-off. Better biocontrol in cucumber was achieved when bacterial antagonists were applied by drenching or by coating seed with bacteria in a peat carrier. CONCLUSIONS: Pseudomonas antagonists were superior to Bacillus antagonists in controlling damping-off in cucumber and sugar beet. Pseudomonas peat inocula maintained a good shelf-life 2 years after preparation. SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas peat formulations have the potential for development into commercial biopesticides.     

Growth tolerance of Zygomycetes Mucor indicus in orange peel hydrolysate without detoxification

The capability of two zygomycetes strains, Mucor indicus and an isolate from tempeh (Rhizopus sp.), to grow on orange peel hydrolysate and their tolerance to its antimicrobial activity, was investigated. Both fungi, in particular M. indicus, tolerated up to 2% d-limonene in semi-synthetic media during cultivation in shake flasks, under aerobic as well as anaerobic conditions. The tolerance of M. indicus was also tested in a bioreactor, giving rise to varying results in the presence of 2% limonene. Furthermore, both strains were capable of consuming galacturonic acid, the main monomer of pectin, under aerobic conditions when no other carbon source was present. The orange peel hydrolysate was based on 12% (dry w/v) orange peels, containing d-limonene at a concentration of 0.6% (v/v), which no other microorganism has been reported to be able to ferment. However, the hydrolysate was utilised by M. indicus under aerobic conditions, resulting in production of 410 and 400 mg ethanol/g hexoses and 57 and 75 mg fungal biomass/g sugars from cultivations in shake flasks and a bioreactor, respectively. Rhizopus sp., however, was slow to germinate aerobically, and neither of the zygomycetes was able to consistently germinate in orange peel hydrolysate, under anaerobic conditions. The zygomycetes strains used in the present study demonstrated a relatively high resistance to the antimicrobial compounds present in orange peel hydrolysate, and they were capable of producing ethanol and biomass in the presence of limonene, particularly when cultivated with air supply.     

Isolation of mutants of Talaromyces emersonii CBS 814.70 with enhanced cellulase activity

By a combination of genetic mutation and modification of growth medium the cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4 etc.] activity of culture filtrates of Talaromyces emersonii CBS 814.70 has been increased four-fold to approximately 2 U ml^?1 and a productivity of 20–25 Ul^?1h^?1. At 50°C this system was completely stable for at least 24 h. At 60°C in static reaction mixtures 19% of the original activity was lost compared with 21% when mixtures were shaken. At 70°C the loss of activity after 4 h was 64% without shaking and 70% when shaken. The cellulase system from Trichoderma reesei was decidedly less stable than that of Talaromyces emersonii under each of the above conditions. The ability of each enzyme system, separately and together, to digest beet pulp was investigated.     

Screening for alkaline keratinolytic activity in fungi isolated from soils of the biosphere reserve “Parque Costero del Sur” (Argentina)

A screening was carried out on 69 fungal strains isolated from alkaline-calcareous, neutral and alkaline-sodic soils, as well as from their associated plant material, to determine their ability to grow at alkaline pH. A total of 32 fungi were selected for their ability to produce alkaline keratinase activity in submerged shaken cultures supplemented with soybean meal (SM) and tryptone and on cow hair (CH) under solid state fermentation conditions. Although several fungal strains produced keratinolytic activity on both SM and CH, they differed in the levels detected. Among them, Aspergillus niger, Cladosporium cladosporioides, Metarrhizium anisopliae, Neurospora tetrasperma and Westerdikella dispersa were the best producers, with levels higher than 1.2 U ml⁻¹. Different fungal species are here reported for the first time for their ability to produce keratinolytic activity at alkaline pH.     

Anaerobic biodegradation of sugar beet pulp

Sugar beet pulp is a by-product of sugar production and consists mainly of cellulose, hemicellulose and pectin. Its composition is suitable for biological degradation. A possible alternative for the utilization of this material (besides cattle feeding) can be anaerobic methanogenic degradation. It has an additional advantage – biogas production. Beet pulp was treated by a two-step anaerobic process. The first step consisted of hydrolysis andacidification. The second step was methanogenesis. In this paper, observation ofthe process of anaerobic degradation and determination of optimal parameters is discussed. A laboratory-scale model for sugar beet pulp anaerobic biodegradation was operated. Results of model performance have shown very good pulp digestion characteristics. In addition, high efficiency removal of organic matter was achieved. Methane yield was over 0.360 m³ kg^-1 dried pulp and excess sludge production was 0.094 g per gram COD added.     

Chelating agents improve enzymatic solubilization of pectinaceous co-processing streams

This study investigates the hypothesis that loosening of the egg-box structure by presence of divalent ion chelating agents during enzymatic degradation of homogalacturonan (HG) can improve enzymatic polysaccharide solubilization on pectinaceous, agro-industrial co-processing streams. The influence of different levels of ethylene-diaminetetraacetic acid (EDTA), citric acid, oxalic acid, and phosphate was assessed in relation to enzymatic solubilization of isopropanol precipitatable oligo- and polysaccharides from sugar beet pulp, citrus peel, and two types of potato pulp. The two types of potato pulp were FiberBind 400, a dried commercial potato pulp product, and PUF, a dried calcium reduced product, respectively. The enzymatic treatment consisted of 1% (w/w) of substrate treated with pectin lyase from Aspergillus nidulans and polygalacturonase from A. aculeatus [each dosed at 1.0% (w/w) enzyme/substrate] at 60 °C, pH 6.0 for 1 min. Characterization of the released fractions demonstrated a significantly improved effect of chelating agents for polysaccharide solubilization from FiberBind 400, PUF, and citrus peel, whereas only low amounts of polysaccharides were solubilized from the sugar beet pulp. The results substantiated the importance of chelating agents during enzymatic extraction of pectinaceous polysaccharides. Lower levels of chelating agents were required for the calcium-reduced potato pulp substrate (PUF) indicating the significance of calcium cross-linking in HG in relation to the enzymatic solubilization yields. The effect of the chelating agents correlated to their dissociation constants (pK_a values) and calcium binding constants and citric acid and EDTA exerted highest effects. Maximum polysaccharide yield was obtained for FiberBind 400 where the enzymatic treatment in presence of citric acid yielded 22.5% (w/w) polysaccharides of the initial substrate dry matter.     

Proteome analysis of sugar beet (Beta vulgaris L.) elucidates constitutive adaptation during the first phase of salt stress

Salinity is one of the major stress factors responsible for growth reduction of most of the higher plants. In this study, the effect of salt stress on protein pattern in shoots and roots of sugar beet (Beta vulgaris L.) was examined. Sugar beet plants were grown in hydroponics under control and 125 mM salt treatments. A significant growth reduction of shoots and roots was observed. The changes in protein expression, caused by salinity, were monitored using two-dimensional gel-electrophoresis. Most of the detected proteins in sugar beet showed stability under salt stress. The statistical analysis of detected proteins showed that the expression of only six proteins from shoots and three proteins from roots were significantly altered. At this stage, the significantly changed protein expressions we detected could not be attributed to sugar beet adaptation under salt stress. However, unchanged membrane bound proteins under salt stress did reveal the constitutive adaptation of sugar beet to salt stress at the plasma membrane level.     

Purification and conservation of infective sugar beet mosaic virus

Sugar beet mosaic virus (SBMV) was precipitated by polyethyleneglycol (PEG) 6000 from the cell sap of infected sugar beet leaves. After centrifugation and addition of dextrane T 10 the virus was lyophilized. Its infectious activity was demonstrated by mechanical transmission toChenopodium quinoa Willd. and to sugar beet. Stability of infectious activity of the lyophilized virus was verified.     

Formation of organic acids by fungi isolated from the surface of stone monuments

Capacity of the fungi isolated from the surface of stone monuments for acid formation was studied in cultures under various carbon sources and cultivation conditions. The composition of organic nutrients was adjusted according to the results of investigation of the surface layers from the monuments in urban environment. The primary soil formed at the surface of the stone monuments under urban conditions was shown to contain a variety of carbon and nitrogen sources and is a rich substrate for fungal growth. Oxalic acid was produced by fungi grown on media with various concentrations of sugars, sugar alcohols, and organic acids. Malic, citric, fumaric, and succinic acids were identified only at elevated carbohydrate concentrations, mostly in liquid cultures. Oxalic acid was the dominant among the acids produced by Aspergillus niger at all experimental setups. Unlike A. niger, the relative content of oxalic acid produced by Penicillium citrinum decreased at high carbohydrate concentrations.     

Distribution of a novel enzyme of sialidase family among native filamentous fungi

Sialidases (neuraminidases, EC 3.2.1.18) are widely distributed in biological systems but there are only scarce data on its production by filamentous fungi. The aim of this study was to obtain information about sialidase distribution in filamentous fungi from non-clinical isolates, to determine availability of sialidase gene, and to select a perspective producer. A total of 113 fungal strains belonging to Ascomycota and Zygomycota compassing 21 genera and 51 species were screened. Among them, 77 strains (11 orders, 14 families and 16 genera) were able to synthesize sialidase. Present data showed a habitat-dependent variation of sialidase activity between species and within species, depending on location. Sialidase gene was identified in sialidase-positive and sialidase-negative strains. .Among three perspective strains, the best producer was chosen based on their sialidase production depending on type of cultivation, medium composition, and growth temperature. The selected P. griseofulvum Р29 was cultivated in 3L bioreactor at 20 °C on medium supplemented with 0.5% milk whey. The results demonstrated better growth and 2.3-fold higher maximum enzyme activity compared to the shaken flask cultures. Moreover, the early occurring maximum (48 h) is an important prerequisite for future up scaling of the process.     

Hydrolysis of orange peel by a pectin lyase-overproducing hybrid obtained by protoplast fusion between mutant pectinolytic Aspergillus flavipes and Aspergillus niveus CH-Y-1043

Pectin lyases cleave the internal glycosidic bonds of pectin by β-elimination, producing non-saturated galacturonic oligomers. Genetic improvement of pectin lyase-overproducing strains is still necessary to improve industrial processes based on this enzyme. In the present study hybrids were obtained by protoplast fusion between mutant pectinolytic Aspergillus flavipes and Aspergillus niveus CH-Y-1043 strains. Prototrophic segregants showed different isoenzymatic profiles and produced increased levels of pectin lyase in cultures containing lemon peel as a sole carbon source. Hybrid HZ showed an increase of 450% and 1300% in pectin lyase production compared with that of A. niveus CH-Y-1043 and A. flavipes, respectively. Pectin lyase produced by the hybrid HZ was partially purified and used for the hydrolysis of orange peel. Pectin lyase was able to hydrolyze 56% of orange peel biomass. However, addition of 2 RFU and 20 U of endo- and exo-polygalacturonase, respectively, induced the hydrolysis of 92% of orange peel solids. In conclusion HZ is a pectin lyase-overproducing hybrid with potential applications in the pectin industry.