Enhancement of growth and chitosan production by Rhizopus oryzae in whey medium by plant growth hormones

The effect of some plant growth hormones, viz., gibberellic acid, indole-3-acetic acid, indole-3-butyric acid, and kinetin on chitosan production by Rhizopus oryzae in deproteinized whey was studied. Hormones, at different concentrations, increase the mycelial growth by 19-32%. However, increase in chitosan content of the mycelia was relatively small (1.7-14.3%) over the control. Maximum enhancement was observed with gibberellic acid. Fifty percent more chitosan could be obtained from 1L of whey containing 0.1mg/L gibberellic acid. Hormones, at higher dose, instead of stimulation inhibited both growth and mycelial chitosan content. This study showed that hormones have no influence on degree of deacetylation of chitosan but increase the quality of the chitosan by increasing weight average molecular weight and decreasing polydispersity. All the hormones had been found to enhance chitin deacetylase activity of R. oryzae by 1.067-1.267-fold and may be one of the reasons for increased chitosan production.     

里氏木霉与米根霉混合固态发酵产纤维素酶

以里氏木霉及米根霉单菌固态发酵为对象,考察不同混合发酵形式对里氏木霉与米根霉混合固态发酵产纤维素酶的影响。结果表明:同时接种里氏木霉与米根霉,试验考察的两菌种接种量比1∶1(以孢子个数计)及5∶1条件下,两菌未产生明显协同产酶作用。米根霉延时(24 h)接种且菌种量比5∶1以及米根霉延时(48 h)接种且菌种量比1∶1,2种发酵形式产酶情况类似,滤纸酶活(FPA)及羧甲基纤维素酶(CMCase)酶活相对米根霉单菌发酵有所提高,而β-葡萄糖苷酶(β-GA)酶活相对里氏木霉单菌固态发酵结束时分别增加4.66及4.40倍,可以发现两菌产生一定协同作用。在米根霉延时(48 h)接种且菌种量比5∶1的发酵形式下,FPA及CMCase在发酵第7天酶活分别达到44.04 IU/g、627.14 U/g(以1 g干曲计),分别是里氏木霉固态单菌发酵产酶达到稳定期时酶活的1.36和1.63倍,两菌产生了有效的协同作用。     

Structure, function, and phylogeny of the mating locus in the Rhizopus oryzae complex

The Rhizopus oryzae species complex is a group of zygomycete fungi that are common, cosmopolitan saprotrophs. Some strains are used beneficially for production of Asian fermented foods but they can also act as opportunistic human pathogens. Although R. oryzae reportedly has a heterothallic (+/-) mating system, most strains have not been observed to undergo sexual reproduction and the genetic structure of its mating locus has not been characterized. Here we report on the mating behavior and genetic structure of the mating locus for 54 isolates of the R. oryzae complex. All 54 strains have a mating locus similar in overall organization to Phycomyces blakesleeanus and Mucor circinelloides (Mucoromycotina, Zygomycota). In all of these fungi, the minus (-) allele features the SexM high mobility group (HMG) gene flanked by an RNA helicase gene and a TP transporter gene (TPT). Within the R. oryzae complex, the plus (+) mating allele includes an inserted region that codes for a BTB/POZ domain gene and the SexP HMG gene. Phylogenetic analyses of multiple genes, including the mating loci (HMG, TPT, RNA helicase), ITS1-5.8S-ITS2 rDNA, RPB2, and LDH genes, identified two distinct groups of strains. These correspond to previously described sibling species R. oryzae sensu stricto and R. delemar. Within each species, discordant gene phylogenies among multiple loci suggest an outcrossing population structure. The hypothesis of random-mating is also supported by a 50:50 ratio of plus and minus mating types in both cryptic species. When crossed with tester strains of the opposite mating type, most isolates of R. delemar failed to produce zygospores, while isolates of R. oryzae produced sterile zygospores. In spite of the reluctance of most strains to mate in vitro, the conserved sex locus structure and evidence for outcrossing suggest that a normal sexual cycle occurs in both species.     

Antimicrobial effect of oxidized cellulose salts

Antimicrobial properties of oxidized cellulose and its salts in linters (-L) and microsphere (-M) form (OKCEL H-L, OKCEL Zn-M, OKCEL ZnNa-L, OKCEL ZnNa-M and OKCEL Ag-L) were tested by a dilution method against a spectrum of microbial strains: Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Bacillus licheniformis, Aspergillus niger, Penicillium chrysogenum, Rhizopus oryzae, Scopulariopsis brevicaulis, Candida albicans and Candida tropicalis. OKCEL Ag-L exhibited antimicrobial activity in the range 0.1-3.5% w/v against all the bacteria and fungi involved in this study. Strong inhibition by OKCEL ZnNa-M was observed for Staphylococcus epidermidis, Bacillus licheniformis, Rhizopus oryzae, Candida albicans and Candida tropicalis in the range 0.5-2.0% w/v. Antimicrobial effects of oxidized cellulose and its salts in textile form were investigated by a diffusion and dilution method against the spectrum of above-cited microbial strains extended by Clostridium perfringens. Generally, OKCEL Ag-T, OKCEL Zn-T and OKCEL H-T showed high antimicrobial activity against populations of Pseudomonas aeruginosa, Bacillus licheniformis and Staphylococcus epidermidis. OKCEL Zn-T was the only sample suppressing the growth of species.     

Identification and characterization of filamentous fungi isolated from fermentation starters for Hong Qu glutinous rice wine brewing

Hong Qu glutinous rice wine is one of the most popular traditional rice wines in China. Traditionally, this wine is brewed from glutinous rice with the addition of wine fermentation starters (Hong Qu (also called red yeast rice) and White Qu). The objective of this study was to investigate the variability of filamentous fungi associated with traditional fermentation starters through a traditional culture-dependent method and a molecular identification approach. In this study, forty-three filamentous fungi were separated by traditional culture-dependent means (macro- and microscopic characteristics) from 10 fermentation starters and classified into 16 different species based on morphological examination and the internal transcribed spacer (ITS) sequences analysis. It was observed that the genus Aspergillus had the highest number (14 isolates) of isolates followed by Rhizopus (11 isolates), Monascus (5 isolates) and Penicillium (4 isolates). The species R. oryzae, A. niger, A. flavus and M. purpureus were frequently found in wine starter samples, among which R. oryzae was the most frequent species. The enzyme-producing properties (glucoamylase, α-amylase and protease) of all fungal isolates from different starters were also evaluated. A. flavus, R. oryzae and M. purpureus were found to be better glucoamylase producers. A. flavus, R. oryzae and A.oryzae exhibited higher activity of α-amylase. A. flavus and A. oryzae had higher protease activity. However, some fungal isolates of the same species exhibited a significant variability in the production levels for all determined enzyme activity. This study is the first to identify filamentous fungi associated with the starter of Hong Qu glutinous rice wine using both traditional and molecular methods. The results enrich our knowledge of liquor-related micro-organisms, and can be used to promote the development of the traditional fermentation technology.     

Efficient and selective microbial esterification with dry mycelium of Rhizopus oryzae

The use of dry mycelium of Rhizopus oryzae as biocatalyst for ester production in organic solvent has been studied. Mycelia with notable carboxylesterase activity were produced when different Tweens (20, 40, 60 and 80) were employed as main carbon source for the growth. Dry mycelium of four strains of Rhizopus oryzae proved effective for efficiently catalysing the synthesis of different flavour esters (hexylacetate and butyrate, geranylacetate and butyrate) starting from the corresponding alcohol and free acid, including acetic acid. The esterification of the racemic mixture of 2-octanol and butyric acid proceeded with high enantioselectivity (R-ester produced with enantiomeric excess > or =97%) when Rhizopus oryzae CBS 112.07 and Rhizopus oryzae CBS 260.28 were employed.     

Production of lactic acid and fungal biomass by Rhizopus fungi from food processing waste streams

This study proposed a novel waste utilization bioprocess for production of lactic acid and fungal biomass from waste streams by fungal species of Rhizopus arrhizus 36017 and R. oryzae 2062. The lactic acid and fungal biomass were produced in a single-stage simultaneous saccharification and fermentation process using potato, corn, wheat and pineapple waste streams as production media. R. arrhizus 36017 gave a high lactic acid yield up to 0.94-0.97 g/g of starch or sugars associated with 4-5 g/l of fungal biomass produced, while 17-19 g/l fungal biomass with a lactic acid yield of 0.65-0.76 g/g was produced by the R. oryzae 2062 in 36-48 h fermentation. Supplementation of 2 g/l of ammonium sulfate, yeast extract and peptone stimulated an increase in 8-15% lactic acid yield and 10-20% fungal biomass.     

Antifungal lactic acid bacteria with potential to prolong shelf-life of fresh vegetables

AIM: The aim of this study was to isolate and identify antifungal lactic acid bacteria from fresh vegetables, and evaluate their potential in preventing fungal spoilage of vegetables. METHODS AND RESULTS: Lactic acid bacteria from fresh vegetables were enriched in MRS (de Man Rogosa Sharpe) broth and isolated by plating on MRS agar. All the isolates (359) were screened for activity against Aspergillus flavus of which 10% showed antifungal activity. Potent antifungal isolates were identified by phenotypic characters and confirmed by partial 16S rRNA gene sequencing. These were screened against additional spoilage fungi viz. Fusarium graminearum, Rhizopus stolonifer, Sclerotium oryzae, Rhizoctonia solani, Botrytis cinerea and Sclerotinia minor by overlay method. Most of the isolates inhibited wide range of spoilage fungi. When fresh vegetables were inoculated with either cell suspension (10(4) cells ml(-1)) or cell-free supernatant of Lact. plantarum, followed by application of vegetable spoilage fungi (A. flavus and F. graminearum, R. stolonifer, B. cinerea each with 10(4) conidia ml(-1)) the vegetable spoilage was significantly delayed than control. CONCLUSIONS: Fresh vegetables constitute a good source of lactic acid bacteria with ability to inhibit wide range of spoilage fungi. Such bacteria can be applied to enhance shelf-life of vegetables. In the present study, we report for the first time the antifungal activity of Weissella paramessenteroides and Lact. paracollinoides isolated from fresh vegetables, against wide range of food spoilage fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: Fresh vegetables can be used as a source of antifungal lactic acid bacteria. Their exploitation as biopreservative will help in prolonging shelf-life of fresh vegetables.     

l-leucine aminopeptidase production by filamentous Aspergillus fungi

AIMS: To screen various filamentous fungi belonging to Aspergillus spp. producing leucine and methionine aminopeptidases. METHODS AND RESULTS: Twenty-eight Aspergillus strains representing 14 species within the genus were screened for L-leucine aminopeptidase (LAP) production in two media in shake flask fermentation. Two Aspergillus sojae (NRRL 1988 and NRRL 6271) and one Aspergillus oryzae (NRRL 6270) strains were selected as the best producers for further studies. The peak LAP activities were 2.61, 2.59 and 1.30 IU ml(-1) for the three fungi on days 2, 5 and 4 respectively. In addition to LAP, L-methionine aminopeptidase (MAP) activity was also detected. Apart from submerged fermentation, the highest LAP yields by solid-state fermentation were 11.39, 17.40 and 13.02 IU g(-1) dry matter for the above fungi. The temperature and pH optimum of the enzyme was found to be in the range of 65-75 degrees C at pH 8.0-9.0 for all three fungi. Metal ions, Co(2+) and Fe(2+) in 2 mmol l(-1) concentration apparently enhanced the relative enzyme activity and heat stability. CONCLUSIONS: Two A. sojae (NRRL 1988 and NRRL 6271) and one A. oryzae (NRRL 6270) strains were found to be the best producers of LAP and MAP. The preliminary characterization studies revealed that the enzyme is considerably thermostable and belongs to the class metalloenzymes. SIGNIFICANCE AND IMPACT OF THE STUDY: A good number of aspergilli were screened and the ability of the fungal aminopeptidase to release a particular N-terminal amino acid along with its high thermal stability, makes them interesting for controlling the degree of hydrolysis and flavour development for a wide range of substrate.     

Production, purification and characterization of chitosanase produced by Gongronella sp. JG

AIMS: To optimize the production condition of chitosanases of Gongronella sp. JG and to characterize the major chitosanase. METHODS AND RESULTS: In the optimized medium and culturing condition, strain JG produced 800 micromol min(-1) l(-1) chitosanase activity at 72 h. The major chitosanase - csn1 was purified through three chromatography steps: CM (carboxymethyl)-Sepharose fast flow (FF), Sephacryl S200, SP (sulfopropyl)-Sepharose FF. The molecular weight and the pI value of csn1 were about 90,000 Da and 5 x 8, respectively. Its specific activity was 82 micromol min(-1) mg(-1). The optimal reaction pH for csn1 was between 4 x 6 and 4 x 8. The optimal reaction temperature was 50 degrees C. The half-life of csn1 at 50 degrees C was estimated to be about 65 min. Mn(2+) was a strong stimulator of csn1 activity, both at 1 and 10 mmol l(-1). csn1 showed its highest activity with chitosan of 85% degree of deacetylation, but did not hydrolyse colloidal chitin and carboxylmethyl cellulose. In 20 mmol l(-1) sodium acetate buffer (pH 4 x 8) and at 50 degrees C, the K(m) of csn1 was calculated to be 4 x 5 mg ml(-1). CONCLUSIONS: The production condition of chitosanases by Gongronella JG was optimized and the major chitosanase, csn1, was characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: The present work for the first time reported the production, purification and characterization of chitosanases produced by fungus of Gongronella sp. These results provided us more information on fungal chitosanases.     

Purification and characterization of protease from a newly isolated Rhizopus oryzae

A newly isolated Rhizopus oryzae was found to exhibit some unusual phenomenon of secreting alkaline protease which was purified and characterized. The molecular weight was determined to be 28,600 dalton in gel electrophoresis. The enzyme is stable in the pH range from 3 to 11 and most active at pH 8. The temperature optimum of this thermostable biocatalyst is at 60 °C. The enzyme is sensitive to metal chelators, most of the metal ions (excepting a few monovalent cations) and inhibitor like PMSF. This indicates that the protease of isolated Rhizopus oryzae falls under alkaline serine group.     

Kinetic studies of Rhizopus oryzae lipase using monomolecular film technique

Rhizopus oryzae lipase (ROL) was found to be a true lipase. This enzyme presents the interfacial activation phenomenon. The N-terminal amino acid sequence of ROL was compared to those of rhizopus lipases. Purified ROL possesses the same N-terminal sequence as the mature Rhizopus niveus lipase (RNL). This sequence was found in the last 28 amino acids of the propeptide sequence derived from the cDNA of Rhizopus delemar lipase (RDL). Using the baro-stat method, we have measured the hydrolysis rate of dicaprin films by ROL as a function of surface pressure. Our results show that Rhizopus oryzae lipase is markedly stereoselective of the sn-3 position of the 2,3 enantiomer of dicaprin. Polyclonal antibodies (PAB) directed against ROL have been produced and purified by immunoaffinity. The effects of these PAB on the interfacial behavior of ROL were determined. The immunoblot analysis with polyclonal antibodies anti-ROL (PAB anti-ROL) and various lipases shows a cross-immunoreactivity between the lipase from the rhizopus family (Rhizopus delemar lipase and Rhizopus arrhizus lipase).     

Comparison of fumaric acid production by Rhizopus oryzae using different neutralizing agents

Fumaric acid fermentation in a 10-L bubble column fermenter using different neutralizing agents [CaCO(3), Ca(OH)(2), NaHCO(3)] by Rhizopus oryzae ATCC 20344 was examined. It was found that in the fermentation using CaCO(3 )as the neutralizing agent the highest fumaric acid weight yield and volumetric productivity were obtained, 53.4% and 1.03 g/L x h(-1) respectively. In the NaHCO(3) case, the fumaric acid weight yield and volumetric productivity were 33.7% and 0.69 g/L x h(-1), respectively, much lower than the CaCO(3) case. However, the NaHCO(3) alternative has advantages of cell reuse and simple downstream processing because of the high solubility of sodium fumarate. These advantages may offset the disadvantages of using NaHCO(3) as the neutralizing agent, and the overall fumaric acid weight yield and volumetric productivity will increase.     

Efficient and direct fermentation of starch to ethanol by sake yeast strains displaying fungal glucoamylases

Aspergillus oryzae glucoamylases encoded by glaA and glaB, and Rhizopus oryzae glucoamylase, were displayed on the cell surface of sake yeast Saccharomyces cerevisiae GRI-117-UK and laboratory yeast S. cerevisiae MT8-1. Among constructed transformants, GRI-117-UK/pUDGAA, displaying glaA glucoamylase, produced the most ethanol from liquefied starch, although MT8-1/pUDGAR, displaying R. oryzae glucoamylase, had the highest glucoamylase activity on its cell surface.     

Conversion of zearalenone to zearalenone glycoside by Rhizopus sp

The microbial conversion of zearalenone by various species of fungi was studied. Among them, Rhizopus sp. was the sole fungus which produced a new metabolite from zearalenone in addition to alpha- and beta-zearalenol. The structure of the new metabolite was determined to be zearalenone 4-beta-D-glucopyranoside on the basis of mass, infrared, and nuclear magnetic resonance spectroscopies. The results suggest that the mycelium of Rhizopus sp. catalyzes the glycosidation at the C-4 position of zearalenone.     

Conversion of zearalenone to zearalenone glycoside by Rhizopus sp.

The microbial conversion of zearalenone by various species of fungi was studied. Among them, Rhizopus sp. was the sole fungus which produced a new metabolite from zearalenone in addition to alpha- and beta-zearalenol. The structure of the new metabolite was determined to be zearalenone 4-beta-D-glucopyranoside on the basis of mass, infrared, and nuclear magnetic resonance spectroscopies. The results suggest that the mycelium of Rhizopus sp. catalyzes the glycosidation at the C-4 position of zearalenone.     

GC-MS based metabolite profiling of rice Koji fermentation by various fungi

In this study, Aspergillus kawachii, Aspergillus oryzae, and Rhizopus sp., were utilized for rice Koji fermentation, and the metabolites were analyzed in a time-dependent manner by gas chromatography-mass spectrometry. On Principal Component Analysis, the metabolite patterns were clearly distinguished based on the fungi species. This approach revealed that the quantities of glucose, galactose, and glycerol gradually increased as a function of fermentation time in all trials rice Koji fermentation. The time-dependent changes of these metabolites showed significant increases in glucose in the A. oryzae-treated rice, and in glycerol and galactose in the A. kawachii-treated rice. In addition, glycolysis-related enzyme activities were correlated with the changes in these metabolites. The results indicate that time-dependent metabolite production has the potential to be a valuable tool in selecting inoculant fungi and the optimal fermentation time for rice koji.     

Effect of cadmium on growth of potentially pathogenic soil fungi

The effect of Cd on the mycelial growth of some potentially pathogenic soil fungi was investigated. Sixty-four strains from twenty-five fungal species were tested for their susceptibility to Cd. Final colony diameter, radial growth rate and final dry mass of mycelium (for Cd: 1–200 ppm) were measured. EcD₅₀ values were calculated from these parameters. The intra- and interspecific variability in the results obtained is presented. Among the keratinolytic fungal group, the genera Arthrographis, Trichophyton and Chrysosporium (including the Chrysosporium anamorph of Aphanoascus reticulisporus) were the most resistant to Cd. T. mentagrophytes was the most sensitive of all Trichophyton species tested. Among the nonkeratinolytic fungal group, the genera Pseudallescheria, Absida and Rhizopus were highly resistant to Cd. Strains of Aspergillus were more resistant to Cd than strains of Penicillium. Zygomycetes were more resistant to Cd than Ascomycetes with Fungi Imperfecti. Nonkeratinolytic fungi showed higher resistance to Cd than keratinolytic fungi. The two last differences resulted from the extremely high EcD₅₀ values for R. oryzae and A. corymbifera. This revised version was published online in June 2006 with corrections to the Cover Date.     

Physical properties of fungal chitosan

Fungi are promising alternative sources of chitosan. This study evaluated the physical properties of fungal chitosan from Absidia coerulea (AF 93105), Mucor rouxii (Ag 92033), and Rhizopus oryzae (Ag 92033). FT-IR and X-ray diffraction of the extracted products showed typical chitosan peak distributions which confirmed the extracted products to be chitosan. All of their glucosamine contents and degrees of deacetylation (DD) were over 80%, not showing obvious differences respectively. However, differences had been observed in their molecular weight (M_w), ranging from 6.6  to 560 kDa. The results of this study demonstrated that different fungi could produce different M_w chitosan with high DD and high purity.     

Diversity of facultatively anaerobic microscopic mycelial fungi in soils

The numbers of microscopic fungi isolated from soil samples after anaerobic incubation varied from tens to several hundreds of CFU per one gram of soil; a total of 30 species was found. This group is composed primarily of mitotic fungi of the ascomycete affinity belonging to the orders Hypocreales (Fusarium solani, F. oxysporum, Fusarium sp., Clonostachys grammicospora, C. rosea. Acremonium sp., Gliocladium penicilloides, Trichoderma aureoviride, T. harzianum, T. polysporum, T. viride. T. koningii, Lecanicillum lecanii, and Tolypocladium inflatum) and Eurotiales (Aspergillus terreus, A. niger, and Paecilomyces lilacimus), as well as to the phylum Zygomycota, to the order Mucorales (Actinomucor elegans, Absidia glauca, Mucor circinelloides, M. hiemalis, M. racemosus, Mucor sp., Rhizopus oryzae, Zygorrhynchus moelleri, Z. heterogamus, and Umbelopsis isabellina) and the order Mortierellales (Mortierella sp.). As much as 10-30% of the total amount of fungal mycelium remains viable for a long time (one month) under anaerobic conditions.