Aspergillus Niger Van Tiegh Research Articles

Effect of Different Levels of Cider, Baker Yeast and Hamei on Pineapple Cider

Cider is an alcoholic beverage that is produced when fruit juices are fermented; it is made using the same techniques as wine. Cider occupies a special place among its more well-known alcoholic siblings since it is brewed like wine but mostly served as beer. Eight treatments were reproduced three times in the CRD (Complete Randomized Design) format of the experiment. The pineapples were fermented by using two different kinds of yeast, baker yeast (Saccharomyces cerevisiae) and Hamei (Bacteria (Bacillus sp.), fungi (Aspergillus niger van Tiegh.) and the yeast (Saccharomyces cerevisiae) for 30 days. Longer fermentation times resulted in higher levels of alcohol, acidity, and sensory characteristics, but lower levels of total soluble solids, pH, and specific gravity were observed. Cider is a fermented product, produced from fruit juices, which increases juice’s shelf life. In this experiment, Hamei and baker yeast were used to make pineapple cider. Treatment T5 (Pineapple pieces (700 g) + Sugar (300g) + Hamei (0.5g) + Water (1L) produced the best cider among all the treatments in terms of alcohol content (7.00), pH (3.90) and total soluble solids (4.90 Brix).

Mycoflora Associated with Barely Grains (Hordeum vulgare L.) in the Eastern Parts of Libya

During the harvest season (2019-2020), local variety of barley seeds (Hordeum vulgare L.) were collected from three different locations, Almerj, Gerdina, and Sultan; situated in the eastern part of Libya. The present experiment was performed to identify, and compare natural mycoflora associated with barley seeds among these locations, also to evaluate sodium hypochlorite’s application, as seed disinfectants against fungal contaminators. Barley seeds were surface disinfected with a 4% sodium hypochlorite for two minutes or washed only with deionized water (control) before plating on potato dextrose agar (PDA) medium. The following parameters were recorded and calculated: seed survival (G %), fungal frequency (F), isolation frequency (IF) and fungal relative abundance (RD). Four fungal species were identified as Aspergillus niger van Tiegh, A. flavus Link (ascomycetes), Rhizopus stolonifera (Ehrenb. :Fr.) Vuill (zygomycetes), and Bipolaris australiensis (Bugnicourt) (ascomycetes). The most predominant recovered species was A. niger followed by B. australiensis, R. stolonifera, and A. flavus, (18.5%), (9.67%) (2.84%), (4.65%), respectively. Results also showed Sultan had the highest seed germination, followed by Almerj and Gerdina, 64.5 %, 44.5%, and 20.5 %, respectively. Moreover, seed’s pretreatment with sodium hypochlorite and seed’s origin had no significant effect on frequency and relative abundance of fungi.

First Report of Aspergillus niger causing Black rot of Grapes in Pakistan.

In June 2015 & 2016, a postharvest survey of table grapes (Vitis vinifera) cv. King's Ruby, was carried out in five different commercial fruit markets of Rawalpindi (33°38'19.2″N, 73°01'45.0″E) district, Punjab Province. Symptoms appeared as brownish lesions with black sporulation on grapes berries. The incidence of these symptoms on bunches ranged from 12 to 17% at all sites. Symptomatic tissue pieces were surface-sterilized with 0.1% sodium hypochlorite (NaOCl) for 30 seconds, rinsed three times with sterile distilled water, dried on filter paper for 45 seconds, and incubated on potato dextrose agar (PDA) at 25°C. After 3 days, dark brown to black mycelium were formed on PDA media. A total of 24 isolates were examined morphologically. The apex of the conidiophore was observed to be radiate. Vesicles were found to be spherical and covered with irregular metulae and phialides. Conidia were globose or subglobose measured (3.14 μm ± 2.24 in averaged diameter: n=50), dark brown to black, with roughened cell walls. The conidiophores were also smooth-walled, hyaline, and became melanized toward the vesicle. These characteristics of the fungus were similar to those described for Aspergillus niger van Tiegh (de Hoog et al. 2000). For molecular identification, the internal transcribed spacer (ITS) region, beta-tubulin (Bt) gene and partial RNA polymerase II largest subunit (RPB2) gene of representative isolate (Asp.n02) was amplified using primers ITS1/ITS4, BT2a/BT2b and RPB2-6F/RPB2-7R respectively (White et al., 1990; Glass & Donaldson, 1995; Liu et al. 1999). Sequences were deposited in GenBank (ITS, MN658871; Bt2, MT117924; and RPB2, MT318289). Based on BLAST analysis, sequences of the ITS region, Bt2 genes, and RPB2 gene showed 99 to 100% similarity of isolate Asp.n02 to Aspergillus niger (Accession Nos. MK307680.1, MN195121.1, MF078661.1 for ITS gene, MN567299.1, MK451029.1, MK451020.1 for Bt2 gene, and MK450788.1, MK450790.1 for RPB2 gene). To complete Koch's postulates, 10-µl aliquots of spore suspensions (106 spores/ml) of isolate: Asp.n 02 was pipetted onto three non-wounded and four wounded (5 mm diam) asymptomatic grape berries cv. King's Ruby (seven berries per isolate), Sterile distilled water was applied to asymptomatic berries similaries to serve as a negative control (Ghuffar et al. 2018; Jayawardena et al. 2018). Berries were incubated at 25 ± 2°C in sterile moisture chambers, and the experiment was conducted twice. Brownish lesions leading to black sporulation similar to the original symptoms were observed on both wounded and non-wounded inoculated berries after 3 days, whereas no symptoms were recorded on the negative control. The morphology of the fungus that was re-isolated from each of the inoculated berries was identical to that of the original cultures. Aspergillus niger was reported previously in Europe and Israel causing mycotoxin (Ochratoxin A) OTA production on Table grapes (Bau et al. 2006). To our knowledge, this is the first report of Aspergillus niger causing black rot of grapes in Pakistan. This finding will help to plan effective disease management strategies against the black rot of grapes in Pakistan.

Assessment of fungi incidence, seed germination and aflatoxin contamination of groundnut (Arachis hypogaea L.) from Lagos, Nigeria

Groundnut (Arachis hypogaea L.) is a legume of high nutritive and market value usually contaminated by aflatoxin. The evaluation of aflatoxin contamination of groundnut entering the food system through the markets is necessary to ascertain the quality of the groundnut sold and subsequently advice groundnut consumers. The study assessed the aflatoxin B1 (AfB1) contamination levels in groundnut sold in some markets in Lagos, Nigeria. Groundnut seeds were sampled from five Local Government Areas (L. G.A) of Lagos State namely: Agege, Eti-Osa, Ifako Ijaye, Ketu and Oshodi. The samples were packaged in envelopes and transported to the Mycotoxin laboratory of the Federal University of Agriculture, Makurdi, Nigeria for assessment of the fungi infections, seed germination and aflatoxin contamination levels. The experiment for the assessment of fungi infecting and germination of groundnut seeds was laid out in a completely randomized design in ten replicates. Detection of AfB1 present in the groundnut samples was done using Enzyme Linked Immunesorbent Assay (ELISA) method in three replicates. Result showed that Aspergillus flavus Link was the most frequently isolated fungi with 46 % incidence, Lasiodiplodia theobromae Pat. had 20 % incidence, Aspergillus niger van Tiegh with 11 % and Fusarium verticollioides Sacc. 7 % incidence. The moisture content of groundnut seeds ranged between 7.20 % and 6.53 %. Aflatoxin B1 concentrations in groundnut samples was highest in Eti Osa LGA (5.11 µg/kg) while Oshodi LGA had the least concentration (3.09 µg/kg). Groundnut samples from the five Local Government Areas of Lagos State were contaminated with AfB1 at concentration levels below the safe limits for human consumption and health of 20µg/kg. Management of fungi infection on the field is recommended to prevent field to store contamination of groundnut seeds.

Screening of Cowpea (Vigna unguiculata ( L.)Walp) Varieties for Resistance to Leaf Spot in Southern Guinea Savannah Agro- Ecology of Nigeria

Five cowpea varieties (Vigna unguiculata (L.) Walp) namely UAM 09 1055-6, UAM 09 1051-1, IT 99k-573-1-1, IT 90k-277-2 and IT 99k-573-2-1 were investigated for fungi associated with the seed, leaf spot disease incidence and severity in Makurdi (07o 45’- 7o 50’N and 08o 45’ - 08o 50’E ; 98 m)and Otobi (7°07’ - 7°11'N and 8° 05- 8°10'E) in Benue State, Nigeria under natural infection. The effect ofleaf spot incidence and severity on the growth and yield of cowpea varieties were also evaluated. Fungi associated with the seeds and leaf spot lesions were isolated and identified. Diseased leaf samples from the study locations were found to be infected with Fusarium verticillioides, Curvularia lunata, Aspergillus tamarii Kite, Lasiodiplodia theobromae, Aspergillus flavus Link and Aspergillus niger van Tiegh, Pythium spp, Fusarium solani, Macrophomina phaseolina and Phoma sp. Otobi field had significantly higher incidence and severity of leaf spot disease than the Makurdi field. Cowpea variety IT 99k-573-1-1, IT 99k-573-2-1 and IT 90k-277-2 were classified as moderately resistant in Makurdi with a mean incidence of 22.23%, 16.97% and 16.67% respectively while varieties UAM 09 1051-1 and UAM 09 1055-6 were classified as Moderately susceptible and Highly susceptible with mean leaf spot incidence of 41.67% and 99.17% respectively. In Otobi, all the cowpea varieties screened were classified as Susceptible to leaf spot incidence with the exception of variety IT 90k-277-2 which was classified as moderately susceptible to leaf spot disease.
 Key words: Cowpea, leaf spot, screening, resistant, susceptible.

Isolasi dan Karakterisasi Jamur Pendegradasi Katekin dari Seresah Pinus

Isolation of catechin-degrading fungus from pine litter samples was done using minimal medium that containing catechin as sole carbon and energy source. A total of 53 isolates were chosen to represent different colonial types of catechin degrading-fungus. The isolates were screened for their ability to degrade catechin in three stages. The first stage of screening was based on their ability to grow on solid medium containing 2 mM, and as a result, 28 isolates were selected. The second stage of screening on the same medium but containing 4 mM of catechin resulting in 14 selected isolates. The third stage screening was based on their mean growth rate constant (k), instantaneous growth rate constant (m) and generation time (g) on minimal medium containing 4 mM catechin. The result showed that four isolates (D9, K2, K11, and S11) were the best catechin degradator. Further growth kinetic study (k, m ,and g) of selected isolates indicated that D9, K2, and S11 grew well on the medium containing 40 mM, but K11 was inhibited by concentration of higher than 10 mM. Catechin biodegradation process was determined by following the decrease of catechin concentration on liquid medium. It was found that isolate K2 had higher ability to degrade catechin than the isolate K11. Finally, the four selected isolates from the third stage were characterized in terms of macroscopic, microscopic and phenotypic characters and identified. The result of the study showed that the isolates D9, K2 and S11 were identified as member of Aspergillus niger group. The isolate D9 was very similar to isolate S11, while the isolate K2 was found to be the most similar with Aspergillus niger van Tiegh. IFO 6341. The isolate K11 was assigned to be member of the genus Trichoderma.

Evaluation of the aflatoxin contamination and proximate composition of groundnut (<i>Arachis hypogea</i> L.) infected by <i>Aspergillus</i> spp.

Aflatoxins B1 concentration and the proximate composition of groundnuts consumed in six Local Government Areas (LGAs) of Benue State in the Southern Guinea Savannah agro- ecological zone of Nigeria were evaluated. Seed health testing was conducted and associated fungi were identified. Aflatoxin B1 (AfB1) produced by Aspergillus flavus were detected and the concentrations quantified using the Enzyme Linked Immunosorbent Assay (ELISA) method. Proximate content of seeds was determined using standard methods of Official Analytical Chemists. Aspergillus flavus Link and Aspergillus niger van Tiegh were the associated fungi found in contaminated groundnut seeds with 15.67 % and 7.67 % occurrence respectively. A combination of both fungi had 4.67 % occurrence. The proximate content of groundnut seed samples ranged from 0.35 % to 40.21 % across the samples analyzed. The proximate analysis indicated that groundnut samples from Agatu LGA had significantly (P 򪪪 0.05) higher protein and fat content of 29.11 % and 40.21 % respectively compared with samples from other LGAs samples. Groundnut samples from Makurdi LGA had significantly (P 򪪪 0.05) higher ash content while samples from Ohimini recorded significantly (P 򪪪 0.05) higher crude fibre. The highest moisture content (9.37 %) was from groundnut seeds from Otukpo LGA. The contamination of the groundnut samples by AfB1 resulted in a significant (P 򪪪 0.05) reduction in the nutritional quality of the groundnut seeds.Keywords: Aflatoxin B1, groundnut, seeds, quality, contamination

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In Vitro Evaluation of Selected Plant extracts and Chemicals against Pathogenic Fungi isolated from Momordica Charantia L.

Five plant extracts and two chemicals were evaluated against radial growth of five pathogenic fungi isolated from fresh vegetables of two varieties of Momordica charantia L. The isolated fungi were Aspergillus niger Van Tiegh, Curvularia brachyspora Boedijn, Fusarium Link, Rhizopus stolonifer (Ehrenb.:Fr.) Vuill and Trichoderma viride Pers.. Five plant extracts namely Allium sativum L., Azadirachta indicia A. Juss., Citrus limon (L.) Burm. f, Mangifera indica L. and Psidium guajava L. were evaluated for inhibiting growth of above mentioned fungi associated with the vegetable. Out of the five plant extracts, A. sativum showed complete radial growth inhibition of A. niger, C. brachyspora, Fusarium sp. and R. stolonifer at 15 and 20% concentrations. Azadirachta indica showed complete radial growth inhibition of C. brachyspora and Fusarium sp. Rhizopus stolonifer was completely inhibited by 15 and 20% concentration owing to leaf extract of A. indica and P. guajava. The same fungus was completely inhibited by M. indica at 20% concentration. Fusarium sp. was completely inhibited by C. limon at 20% concentration. Two chemicals, sodium bicarbonate and sodium chloride were used against the test pathogens and sodium bicarbonate showed complete inhibition of radial growth of A. niger, C. brachyspora, Fusarium sp. and R. stolonifer at 20% concentration. The last one also inhibited by the chemical at 15% concentration. Sodium chloride showed highest 95% inhibition of radial growth of T. viride at 20% concentration.Journal of Bangladesh Academy of Sciences, Vol. 41, No. 1, 11-16, 2017

Evaluation of the Virulence of Three Isolates of Aspergillus niger van Tiegh. on Dioscorea Species

Evaluation of the Virulence of Three Isolates of Aspergillus niger van Tiegh. on Dioscorea Species Three isolates of Aspergillus niger van Tiegh. from Nigerian yam Dioscorea rotundata were assessed for their virulence on Nigerian varieties of white, water and yellow/bitter Dioscorea species (Yams). The first isolate labelled An2 had been assessed in a previous study along with nine others from a range of geographical locations globally and hosts, for their virulence on Brazilian D. alata, Jamaican D. cayenensis, Nigerian and Ghanaian D. rotundata and French D. opposita. It was found to be most aggressive. The second isolate labelled An3 had also been assessed in a previous study on three improved clones from the International Institute of Tropical Agticulture, Ibadan, Gene Bank namely Dioscorea rotundata TDr 179, Dioscorea alata TDa 297 and Dioscorea esculenta (IITA). It was found to be equally virulent on all three clones. The third isolate labelled An5 was got ftom a rotting tuber in a yam lot from a Yam Market in Abeokuta, in the South West region of Nigeria. Isolates An2, An3 and An5 were assessed in this study on Dioscorea rotundata varieties Efuru and Apepe, Dioscorea alata variety Agbodo and Dioscorea. cayenensis variety Alo, varieties popular within the South Western region of Nigeria. An2 and An5 were similar in virulence on D. cayenensis variety Alo. The combined virulence of An2 and An5 were not significantly different (P=0.05) on D. rotundata variety Efuru and D. cayenensis variety Alo. An2, An3 and An5 were not significantly different in combined virulence on D. rotundata variety Apepe and D. alata variety Agbodo. A cluster analysis also revealed a close relationship among the three isolates. A molecular genetic analysis of isolates of A. niger from Nigerian Dioscorea varieties is necessary to confirm host specificity.

Effects of Calcium Fertilization on the Susceptibility of Dioscorea Species to the Yam Storage Pathogens Aspergillus Niger van Tiegh and Botryodiplodia Theobromae Pat

The effects of soil amendment with basal dose of nitrogen (60 kg ha- 1), phosphorus (30 kg ha-1) and potassium (75 kg ha-1) for Yams Dioscorea species in South West Nigeria and calcium carbonate were assessed on yield and susceptibility to two storage rot pathogens, in Dioscorea alata TDa 92-2, D. rotundata TDr 131 and D. esculenta TDe 89-1. The study site at the Teaching and Research Farms of the Federal University of Agriculture, Abeokuta, had been monocropped to yams without fertilizer for three years before the study. A 3 by 5 factorial desgn, with the Dioscorea species as main plots and 0 kg ha-1, 2 kg ha-1, 4 kg ha-1, 6 kg ha-1 and 8 kg ha-1 calcium carbonate as subplot treatments, was used in each of two years. Aspergillus niger van Tiegh. and Botryodiplodia theobromae Pat. were the two yam storage pathogens used. Pre-planting soil analysis in the two years revealed calcium, magnesium and potassium below the critical levels required for Dioscorea species in South Western Nigeria. Calcium fertilizer enhanced resistance and reduced weight loss in yam ‘’sections;’’ of D. rotundata TDr 131 and Dioscorea alata TDa 92-2 to the pathogens after long-terms storage of over four months in both years. Response of whole tubers incubated for an additional (after the four months) period of ten weeks and six weeks in years one and two respectively, was not significantiy (P=0.05) different from the control. Futher work to determine endogenous or physiological reasons for this is necessary. D. esculenta TDe 89-1 was more susceptible to the rot pathogens than the other two species. D. esculenta TDe 89-1 was also not significantly different in infection or weight loss than the control in the two years, necessitating a need to ascertain optimum fertilizer placement methods for this species in Nigeria. There were no differences in tuber number or tuber weight per plant at the treatment levels.

Mycoflora of Cotton Plant (Gossypium hirsutum L.) - with three New Records of Deuteromycetes from Bangladesh

Fungal diseases of cotton plant (Gossypium hirsutum L.) were studied during July, 2011 to June, 2012. Eleven cotton varieties were selected for the experiment. Alternaria alternata (Fr.) Keissler, Aspergillus niger van Tiegh, Colletotrichum gossypii Sowthworth, Colletotrichum dematium (Pers.) Grove, Curvularia clavata Jain, Fusarium sp., Gleotrichum sp., Hyalopycnis sp., Pestalotiopsis guepinii (Desm.) Stay, and a sterile fungus were isolated from infected leaves of cotton varieties CB6 and CB11. Colletotrichum gossypii was isolated from both the varieties and C. dematium was exclusively isolated from CB6. Varieties CB6 and CB11 were more susceptible than other varieties. Cotton varieties CB1, CB2, and CB3 showed negligible infection on leaves and bolls. Colletotrichum gossypii and Hyalopycnis sp. are new record for Bangladesh. Moreover, association of C. dematium, Gleotrichum sp., P. guepinii and Hyalopycnis sp. with cotton is being reported first time. The three fungi Colletotrichum. gossypii, C. dematium and Hyalopycnis sp. are described and illustrated in this account as new Deuteromycetous records for Bangladesh.Journal of Bangladesh Academy of Sciences, Vol. 39, No. 2, 213-221, 2015

<i>In vitro</i> screening of selected herbicides on rhizosphere mycoflora from yellow pepper (<i>Capsicum annum</i> L var. Nsukka yellow) seedlings in Nsukka, Enugu state, Nigeria

In vitro screening of five selected herbicides at different concentrations on rhizosphere mycoflora from yellow pepper ( capsicum annum L var. Nsukka yellow) seedlings at Nsukka were investigated. The herbicides employed for this study were Paraquat, Glyphosate, Primextra, Atrazine and Linuron. The isolated rhizosphere mycoflora organisms screened were Rhizopus stolonifer (Ehren ex. Fr) Lind, Trichoderma harmatum Bain aggr; Aspergillus niger Van Tiegh; Sclerotium rolfsii Sacc; Penicillium sp; Alternaria sp; Fusarium solani (Mart) Sacc and Rhizoctonia sp. The results showed that all the herbicides at different concentrations of 0.5 μg/ml a.i, 1.0 μg/ml a.i, 2.0 μg/ml a.i, 5.0 μg/ml a.i and 10.0 μg/ml a.i inhibited the mycelial growth of the isolated rhizosphere fungi. Growth inhibition of the rhizosphere fungi increased with increasing concentrations of each herbicide. Paraquat, Glyphosate and Atrazine showed higher inhibition potency on all the fungal mycelial growth than Primextra and Linuron. The significance of these results is discussed. KEYWORDS : In vitro screening, herbicides, Rhizosphere mycoflora, yellow pepper seedlings.

Post-harvest deterioration of banana fruits and its control using fungicides

Post-harvest deterioration is the most important cause of loss in banana production and this is mainly as a result of microbial invasion of the fruits. This research was therefore carried out to identify and control the fungal organism responsible for post-harvest deterioration of banana fruits. Mancozeb, carbendazim, propiconazole and SAAF at tested concentration were used as antifungal agents and the susceptibility of four, of the isolated major pathogenic fungi to them was observed in culture. The tested organisms were Lasiodiplodia theobromae Pat.Griffth and Maubl, Fusarium moniliformae Sheld, Fusarium sp. and Aspergillus niger van Tiegh. Maximum disease control of 98.76 per cent and 98.67 per cent of fruit rot was observed in propiconazole and SAAF treated fruits, respectively followed by carbendazim (96.79%) under storage condition up to eating ripe stage.

Mycoflora associated with the leaves of Senna alata (L.) Roxb.

Fungi associated with leaves of Senna alata (L.) Roxb. (Cassia alata L.) and their pathogenic potentiality have been discussed. A total of 8 species of fungi belonging to 8 genera of Deuteromycetes were found to be associated with S. alata. The fungi were Acromoniella sp., Arthrinium saccharicola Stevenson, Aspergillus niger Van Tiegh, Cladosporium cladosporioides (Fresen.) de Vries, Colletotrichum gloeosporioides (Penz.) Sacc., Curvularia lunata (Wakker) Boedijn, Nigrospora sphaerica (Sacc.) Mason, Pestalotiopsis guepinii (Desm.) Stay and unidentified Hyphomycetes, C. gloeosporioides and P. guepinii were found to be pathogenic to S. alata. DOI: http://dx.doi.org/10.3329/jbas.v37i2.17568 Journal of Bangladesh Academy of Scnces, Vol. 37, No. 2, 249-252, 2013

Report of Association of Mycoflora with Leaves And Stems of Leea Macrophylla Roxb. Ex Harnem from Bangladesh

Fifteen species of fungi belonging to ten genera were found to be associated with Leea macrophylla Roxb. ex Harnem. The species are Alternaria tenuissima (Kunze ex Pers.) Wiltshire, Aspergillus flavus Link, Aspergillus fumigatus, Aspergillus niger van Tiegh, Chaetomium Kunze, Colletotrichum musae (Berk. & Curt.) Arx, Colletotrichum dematium (Pres. Ex Fr.) Grove, Curvularia pallescens Boedijn,, Fusarium moniliforme Shelden, Fusarium sp., Melasmia Lev, Penicillium sp., Rhizopus sp., Trichoderma harzianum Pers. and Trichoderma viride Pers. This is the first record of association of the fungi with Leea macrophylla from Bangladesh. All the identified fungi were tested for their pathogenic potentiality. Colletotrichum dematium, Curvularia pallescens, and Fusarium sp. produced symptoms on artificially inoculated leaves of Leea macrophylla. DOI: http://dx.doi.org/10.3329/jbas.v36i2.12972 Journal of Bangladesh Academy of Sciences, Vol. 36, No. 2, 257-262, 2012

Association of Fungi with Breadfruit (Artocarpus altilis Fosb.)

Nine species of fungi belonging to eight genera of Deuteromycetes were found associated with leaves and fruits of breadfruit, Artocarpus altilis. The fungi were Alternaria alternata (Fries) Keissler, Aspergillus niger Van Tiegh, Aspergillus flavus Link, Colletotrichum gloeosporioides (Penz.) Sacc, Curvularia lunata (Wakker) Boedijn., Penicillium sp. Pteroconium state of Apiospora camptospora Penz. & Sacc. and Spicaria sp., and Zygosporium oscheoides Mont.. This is the first report of association of A. alternata, C. gloeosporioides., Curvularia lunata, Penicillium sp. Pteroconium state of A. camptospora, Spicaria sp. and Z. oscheoides, with breadfruit. Colletotrichum gloeosporioides was the predominating fungus recorded on all infected matured leaves showing brown lesion and anthracnose symptom, and also pathogenic to breadfruit plant. DOI: http://dx.doi.org/10.3329/jbas.v36i1.10930 Journal of Bangladesh Academy of Sciences, Vol. 36, No. 1, 143-146, 2012

Studies on the fungal flora of Garri, processed cassava (<i>Manihot esculentus crantz</i>) in Nsukka area of southeastern Nigeria

In Nsukka area of southeastern Nigeria, garri is usually displayed in the open market for sale which no doubt exposes the food stuff to dust particles from moving vehicles, wind and other sources. Thus, fungal spores from the air and soil environment could serve as major sources of fungal contamination of this product. Two garri types (white and yellow) were therefore sampled from six popular markets in the area which included Ogige, Nkwo-Ibagwa, Orie-Orba, Orie-Igboeze, Eke- Ozzi and Obollo-Afor markets. These samples were investigated for the presence of various species of fungi. Fungal isolates observed were: Aspergillus foetidus Thom and Raper, Aspergillus niger van Tiegh, Aspergillus sp., Penicillium sp., Rhizopus sp. and Candida sp. The fungal isolates occurred at varying frequencies, with A. foetidus being the most frequently isolated in both white and yellow garri while A. niger had the least occurrence in both white and yellow garri. Comparatively, white garri had higher frequency of occurrence of fungal organisms than yellow garri. Food processors are therefore, advised to always clean and sanitize equipment coming in contact with food to reduce microbial contamination and also to avoid other unhygienic handling of locally marketed foods.Key words: Garri, Processed Cassava, Fungal Flora, Marihot esculemta

Engineering of a fungal β-galactosidase to remove product inhibition by galactose

Beta-galactosidase is an enzyme administered as a digestive supplement to treat lactose intolerance, a genetic condition prevalent in most world regions. The gene encoding an acid-stable beta-galactosidase potentially suited for use as a digestive supplement was cloned from Aspergillus niger van Tiegh, sequenced and expressed in Pichia pastoris. The purified recombinant protein exhibited kinetic properties similar to those of the native enzyme and thus was also competitively inhibited by its product, galactose, at application-relevant concentrations. In order to alleviate this product inhibition, a model of the enzyme structure was generated based on a Penicillium sp. beta-galactosidase crystal structure with bound beta-galactose. This led to targeted mutagenesis of an Asp(258)-Ser-Tyr-Pro-Leu-Gly-Phe amino acid motif in the A. niger van Tiegh enzyme and isolation from the resultant library of a mutant beta-galactosidase enzyme with reduced sensitivity to inhibition by galactose (K (i) of 6.46 mM galactose, compared with 0.76 mM for the wildtype recombinant enzyme). The mutated enzyme also exhibited an increased K (m) (3.76 mM compared to 2.21 mM) and reduced V (max) (110.8 micromol min(-1) mg(-1) compared to 172.6 micromol min(-1) mg(-1)) relative to the wild-type enzyme, however, and its stability under simulated fasting gastric conditions was significantly reduced. The study nevertheless demonstrates the potential to rationally engineer the A. niger van Tiegh enzyme to relieve product inhibition and create mutants with improved, application-relevant kinetic properties for treatment of lactose intolerance.

A novel acid-stable, acid-active β-galactosidase potentially suited to the alleviation of lactose intolerance

Extracellular beta-galactosidase produced by a strain of Aspergillus niger van Tiegh was purified to homogeneity using a combination of gel filtration, ion-exchange, chromatofocusing, and hydrophobic interaction chromatographies. The enzyme displayed a temperature optimum of 65 degrees C and a low pH optimum of between 2.0 and 4.0. The monomeric glycosylated enzyme displayed a molecular mass of 129 kDa and an isoelectric point of 4.7. Protein database similarity searching using mass spectrometry-derived sequence data indicate that the enzyme shares homology with a previously sequenced A. niger beta-galactosidase. Unlike currently commercialised products, the enzyme displayed a high level of stability when exposed to simulated gastric conditions in vitro, retaining 68+/-2% of original activity levels. This acid-stable, acid-active beta-galactosidase was formulated, along with a neutral beta-galactosidase from Kluyveromyces marxianus DSM5418, in a novel two-segment capsule system designed to ensure delivery of enzymes of appropriate physicochemical properties to both stomach and small intestine. When subjected to simulated full digestive tract conditions, the twin lactase-containing capsule hydrolyzed, per unit activity, some 3.5-fold more lactose than did the commercial supplemental enzyme. The acid-stable, acid-active enzyme, along with the novel two-segment delivery system, may prove beneficial in the more effective treatment of lactose intolerance.