Microbial hydroxylation. I. Hydroxylation of aniline byAspergillus alliaceus,A. albertensis andA. terreus

Summary Six hundred and seventy microorganisms were screened for the ability to perform stereoselective aromatic hydroxylation reactions of industrial significance, using aniline as a model substrate. TLC and HPLC analyses with diode array detection were used to identify and characterize hydroxylase activities. Of 79 cultures belonging to the speciesAspergillus alliaceus, A. albertensis, andA. terreus, 26 strains produced 2-aminophenol. Thirty strains were able to hydroxylate aniline in thepara position. Five strains ofA. terreus produced an unidentified phenolic compound in high yield.The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.     

Ochratoxin production by the Aspergillus ochraceus group and Aspergillus alliaceus

Ochratoxin A is a toxic and carcinogenic fungal secondary metabolite; its presence in foods is increasingly regulated. Various fungi are known to produce ochratoxins, but it is not known which species produce ochratoxins consistently and which species cause ochratoxin contamination of various crops. We isolated fungi in the Aspergillus ochraceus group (section Circumdati) and Aspergillus alliaceus from tree nut orchards, nuts, and figs in California. A total of 72 isolates were grown in potato dextrose broth and yeast extract-sucrose broth for 10 days at 30 degrees C and tested for production of ochratoxin A in vitro by high-pressure liquid chromatography. Among isolates from California figs, tree nuts, and orchards, A. ochraceus and Aspergillus melleus were the most common species. No field isolates of A. ochraceus or A. melleus produced ochratoxin A above the level of detection (0.01 microg/ml). All A. alliaceus isolates produced ochratoxin A, up to 30 microg/ml. We examined 50,000 figs for fungal infections and measured ochratoxin content in figs with visible fungal colonies. Pooled figs infected with A. alliaceus contained ochratoxin A, figs infected with the A. ochraceus group had little or none, and figs infected with Penicillium had none. These results suggest that the little-known species A. alliaceus is an important ochratoxin-producing fungus in California and that it may be responsible for the ochratoxin contamination occasionally observed in figs.     

Phytase production byAspergillus ficuum on semisolid substrate

Summary Phytase production byAspergillus ficuum was studied using solid state cultivation on several cereal grains and legume seeds. The microbial phytase was used to hydrolyze the phytate in soybean meal and cotton seed meal. Wheat bran, soybean meal, cottonseed meal and corn meal supported good fungal growth and yielded a high level of phytase when an adequate amount of moisture was present. The level of phytase production on solid substrate was higher than that obtained by submerged liquid fermentation. Higher levels of phosphorus (more than 10 mg Pi/100 g substrate) in the growth medium (static culture) inhibited phytase synthesis, and the degree of phosphorus inhibition was less apparent in semisolid medium than in liquid medium. A static cultivation on semisolid substrate produced a higher level of phytase (2-20-fold) than that obtained by agitated cultivation. The minimal amount of water required for growth and enzyme production on those substrates was about 15%, while the optimum level for phytase production was between 25 and 35% and that for cell growth was above 50%. Optimum pH for phytase production was between 4 and 6.A ficuum grew well on raw (unheated) substrate containing a minimal amount of water and produced as much phytase as on heated substrate. About half of the phytic acid in soybean meal and cottonseed meal was hydrolyzed by treatment withA. ficuum phytase.     

Environmental assessment of yield improvements obtained by the use of the enzyme phospholipase in mozzarella cheese production

Background, aim and scope  Phospholipase is an enzyme which is able to increase the yield of cheese in, for instance, mozzarella production. Milk production is the most important source of environmental impacts in cheese production and it is obvious to assume that the milk saving that comes with the use of phospholipase reduces the overall environmental impacts of the final product. Production of industrial phospholipase is, however, also associated with environmental burdens and it is not known whether and to what extent the use of phospholipase is justified by overall environmental improvements. The aim of the present study is therefore to assess the environmental impacts that come with the use of industrial phospholipase in mozzarella production and compare with the savings that come with the avoided milk production. The study addresses mozzarella production in Denmark. Methods  LCA is used as analytical tool and environmental modelling is facilitated in SimaPro 7.1.8 LCA software. Yield improvements refer to full scale industrial application of phospholipase in cheese industry. The study is a comparative analysis and a marginal and market-oriented approach is taken. The study addresses contribution to global warming, acidification, nutrient enrichment, photochemical smog formation, energy consumption and use of agricultural land. Estimation of environmental impact potentials is based on Eco-indicator 95 v.2.1 equivalency factors. Toxicity is addressed by qualitative means. Results  The environmental impacts induced by phospholipase production are small compared with the savings obtained by reduced milk consumption for mozzarella production when all impact indicators are considered. Sensitivity analyses and data quality assessments indicate that this general outcome of the study is robust, although results at the more detailed level are the subject of much variation and uncertainty. Discussion  Transport of the enzyme from producer to mozzarella producer is insignificant and the general outcome of the study is considered applicable to other regions of the world where milk is produced in modern milk production systems. Conclusions  Use of phospholipase as a yield improvement factor is a means of reducing environmental impact of mozzarella production. Recommendations and perspectives  The total annual global warming mitigation potential of phospholipase used in production of mozzarella and other pasta filata products is in the order of 7 × 10⁸ kg CO₂ equivalents. The use of phospholipase is driven by overall cost savings and it is therefore recommended that the enzyme should be given attention as a cost-efficient means of reducing greenhouse gas emissions.     

Effect of fatty acids on aflatoxin production byAspergillus parasiticus

The effect of saturated and unsaturated fatty acids on aflatoxin production was studied in a synthetic medium. The aflatoxin production decreased (10-75%) in the presence of lauric acid and palmitic acid but the addition of behenic and sebacic acid stimulated aflatoxin production by 125-541%. Linolenic and linoleic acids effected aflatoxin production and mycelium growth. An 34-fold increase in aflatoxin production was observed with 50 mM linoleic acid. An inverse relationship was observed between aflatoxin production and mycelium mass, irrespective of the nature of the fatty acid.     

Effect of tweens on the production of ergot alkaloids byAspergillus fumigatus

Tween 80, which caused increased biomass formation, also produced the highest increase in the uptake rate of all components of the medium. The fatty acid components of the respective Tweens,i.e. palmitic acid (Tween 40), stearic acid (Tween 60), and oleic acid (Tween 80), have no effect either on alkaloid production or on substrate uptake. The fatty acid composition was different in the cell membrane of the culture supplemented with Tween 60 and facilitated the transport of metabolites into the cells.     

Stimulation of protease production byAspergillus oryzae with oils in continuous culture

Summary The effect of soy sauce oil and various other oils on protease production by Aspergillus oryzae NISL 1913 was studied in chemostat cultures (dilution rate=0.02 h^–1). Soy sauce oil was consumed as a carbon source by the cells and also accelerated protease production. When soy sauce oil was used as sole carbon source, the specific protease production rate was 2.89 protease units·(mg dry weight of mycelium)^–1·h^–1, which was threefold higher than that with starch. The specific protease production rate with linoleic acid, oleic acid, Tween 80 and soybean oil exhibited similar values to that with soy sauce oil but the fatty acids with carbon chains shorter than six, such as caproic acid and acetic acid, did not stimulate protease production. The oils did not cause an increase in other exocellular enzymes such as -amylase, indicating that the protease production was selectively stimulated by the oils. Offprint requests to: Y. Fukushima     

Effect of Dithiocarbamates on Citric acid production byAspergillus niger

Dithiocarbamates were found to enhance the production of citric acid under solid-state fermentation conditions by Aspergillus niger. Maximum increase was observed with tetramethylthiuram disulfide (TMTD). Percent increases observed were 74.2% with 2.5 micrograms/mL of TMTD, 19.6% with 2.5 micrograms/mL of sodium dimethyldithiocarbamate and 33.1% with 0.6 micrograms/mL of zinc dimethyldithiocarbamate.     

Influence of environmental conditions on the production of pectinesterase and polygalacturonase byAspergillus niger

Summary A pectinesterase- and polygalacturonase-producer strain ofAspergillus niger showed a diauxic growth when cultivated in a medium containing pectin as the carbon source. Diauxie was suppressed by adding yeast extract to the medium. The specific production of polygalacturonase was decreased by increasing the initial concentration of pectin, while that of pectinesterase was enhanced with up to 15 g pectin/l. Yeast extract increased the production of biomass but decreased that of the enzymes. The temperature of incubation (25 to 40°C) did not affect the production of biomass or that of polygalacturonase, but production of pectinesterase was markedly affected, 30°C being the optimal temperature.

---

Influence de la composition du milieu et les conditions des cultures sur la production de pectinestérase et polygalacturonase par Aspergillus niger

Résumé Aspergillus niger que produit pectinestérase et polygalacturonase, quand est developpé dans un milieu avec pectine comme source de carbone a presenté le phenomene de diauxie. Cela ne succedait pas quand le milieu avait aussi extrait de levure dans sa composition. L'effect de la concentration initiale de pectine sur la production specifique des enzymes des cellules (unité d'enzyme g^–1 de cellules poid sec) a été différent selon la phase du developpement de la culture, sugérant une rélation étroite parmi la synthèse des enzymes et l'état physiologique des cellules. L'augmentation de la concentration de pectine a troublé beaucoup la production spécifique de polygalacturonase. Au contraire, jusqu'à une concentration de 15 g/l a augmenté la synthèse de pectinestérase. La grandeur et structure des pellets ont changé avec la variation de la concentration de pectine, ces modifications peuvent avoir eu influence sur les valeurs des coefficients de transfert de masse et en conséquence sur la cinétique de production des enzymes. L'augmentation de la concentration iniciale d'extrait de levure dans le milieu que contenait pectine comme source de carbone a augmenté la production de biomasse, mais a baisse la synthèse des enzymes surtout celle de pectinestérase. Températures parmi 25°C et 40°C n'ont pas modifié la production de biomasse et non plus polygalacturonase. Au contraire la production de pectinesterase a été bien différente selon la température utilisé ayant un optimum de 30°C.

     

Cowpeas as growth substrate do not support the production of aflatoxin byAspergillus sp

A number of 21Aspergillus sp. strains isolated from cowpeas from Benin (Africa) were characterized by RAPD methodology. Seven of these strains grouped withA. flavus in the dendrogram generated with the RAPD data. Only three were able to produce aflatoxin in significant amounts. Twelve other isolates grouped withA. parasiticus. All of these strains except 3 produced aflatoxin. Two additional strains neither fit with theA. flavus group, nor theA. parasiticus group according to their RAPD pattern. Both did not produce aflatoxin in measurable amounts. Generally the aflatoxin positive strains produced high amounts of aflatoxin after growth on YES medium. However after growth on cowpea based medium aflatoxin biosynthesis was strongly ceased, albeit the growth of the colony was only partly reduced. This was true for media made either with the whole cowpea seed or with cowpea seed without seed coat. Interestingly when the cowpea medium was heat sterilized the fungus was able to produce high amounts of aflatoxin. This, however, was not the case after the use of gamma irradiation as sterilization method for the medium. The expression of thenor- 1 gene, which is one of the early genes involved in aflatoxin biosynthesis, was significantly repressed after growth on gamma irradiated cowpea medium in contrast to YES medium. This study was part of the project “Capability Building for Research and Quality Assurance in Traditional Food Processing in West Africa”     

Recent improvements in the production of antibody-secreting hybridoma cells

The field of cell fusion technology has exploded over the past ten years, revolutionizing biology and medicine. Today, monoclonal antibodies specific for almost any antigen can be generated, setting the stage for many different applications. Continued research that focuses on refining the technology and developing innovative techniques will enable the field of cell fusion technology to reach its full potential.     

Effect of cadmium and copper on the production of citric acid byAspergillus niger

Copper and cadmium inhibited the growth as well as citric acid production (depending on the heavy metal concentrations) by citric-acid-producingAspergillus niger. Activity of citrate synthase was connected with citrate synthesis in the absence as well as in the presence of heavy metals. The activity of aconitase, and both NAD- and NADP-isocitrate dehydrogenases was strongly inhibited by copper. The contents of DNA and proteins in the cells decreased but the contents of lipids and polysaccharides increased considerably in the presence of both heavy metals.     

Influence of medium design on lovastatin and mevastatin production byAspergillus terreus strains

In order to investigate the influence of medium design on lovastatin and mevastatin production byAspergillus terreus strains, several nitrogen complex sources, such as vegetal flour and peptones of different origin (animal and vegetal) were tested, together with the addition of methionine, an aminoacid that is directly involved in lovastatin biosynthetic pathway. Soybean peptone generally allowed the best lovastatin yields to be achieved (250–280 mg l^?1), particularly in the presence of soybean and peanut flours. For mevastatin, the best results (300–320 mg l^?1) were obtained at 7 days fermentation with modified base medium (CLD), and at 14 days with standard medium (STD), not being possible in this case to associate the best yield with a defined flour and/or peptone. The results show that lovastatin production is influenced by the presence of soybean peptone and by the addition of methionine; instead, the production of mevastatin appears more strictly strain-associated and not directly dependent on the complex ingredients employed.     

Comparative study of amylolytic enzymes production byAspergillus niger in liquid and solid-state cultivation

Summary Amylolytic enzymes produced by a strain ofAspergillus niger cultivated on cassava starch in liquid or solid culture were found to be mainly glucoamylases. For the same initial amount of substrate, the glucoamylase activity increased even after 60 h of culture on solid medium whereas it decreased in liquid culture. Some characteristics of the amylases produced in both culture conditions were compared. The pH optima for enzymes produced in solid and liquid cultures were 4.5 and 5.0 respectively. Glucoamylase synthetized in solid cultures was significantly more thermostable than that from liquid culture and was maximally active at 70°C compared to 50°C for the enzyme from liquid cultures. The Km values expressed as mg soluble starch/100 ml were 0.1% for crude enzyme from solid culture and 0.057% for crude enzyme from liquid culture.     

High yield of mannosylglycerate production by upshock fermentation and bacterial milking of trehalose-deficient mutant <Emphasis Type="Italic">Thermus thermophilus</Emphasis> RQ-1

A production process, using upshock fermentation and osmotic downshock, for the effective production/excretion of mannosylglycerate (MG) by the trehalose-deficient mutant of the strain Thermus thermophilus RQ-1 has been developed. In the first phase of fed-batch fermentation, the knockout mutant was grown at 70°C on a NaCl-free medium. After the culture reached the end of the exponential growth phase, upshift in temperature and NaCl concentration was applied. The temperature was increased to 77°C, and NaCl was added up to 3.0% and kept constant during the second phase of fermentation. Although this shift in cultivation parameters caused a dramatic drop of cell density, a significant improvement in accumulation of MG up to 0.64 μmol/mg protein compared to batch fermentations (0.31 μmol/mg protein) was achieved. A total yield of 4.6 g MG/l of fermentation broth was obtained in the dialysis bioreactor with a productivity of 0.29 g MG l⁻¹ h⁻¹. The solute was released from the harvested biomass by osmotic downshock using demineralized water at 70°C. More than 90% of the intracellularly accumulated solute was recovered from the water fraction. The process was very efficient, as hyperosmotic shock, release of the solute, and reiterative fed-batch fermentation could be repeated at least four times.     

Culture condition for production of glucoamylase from rice bran byAspergillus terreus

Summary Optimal conditions for the production of glucoamylase from rice bran usingAspergillus terreus in stationary culture were a medium containing 20 g rice bran/l, 0.3% (w/v) (NH₄)₂SO₄ and 0.2% (w/v) peptone at 30°C with an initial pH of 3.0. Enzymatic activity was maximal after 4 d. Glucose was the major reducing sugar produced by hydrolysis of starch. Carbohydrates favouring induction of glucoamylase were, in order: maltose, starch, cellobiose, lactose, glucose, fructose and galactose. Amino acids, in particular glycine, lysine, isoleucine and histidine, were vital for glucoamylase synthesis. Tween 80 and Triton X-100 enhanced the growth but suppressed glucoamylase synthesis.

---

Conditions de culture pour la production de glucoamylase à partir de son de riz parAspergillus terreus

Résumé Les conditions optimales pour la production de glucoamylase à partir de son de riz en utilisantAspergillus terreus en culture en état stationnaire, consistent en un milieu contenant 20 g de son de riz par litre, 0.3 % (poids/vol.) de (NH₄)₂ SO₄ et 0.2 % (poids/vol.) de peptone, à 30 °C avec un pH initial de 3.0. L'activité enzymatique est maximum après 4 jours. Le glucose est le principal sucre réducteur produit par hydrolyse de l'amidon. Les hydrates de carbone qui favorisent l'induction de la glucoamylase, sont, dans l'ordre: le maltose, l'amidon, la cellobiose, le lactose, le glucose, le fructose et le galactose. Les acides aminés, en particulier la glycine, la lysine, l'isoleucine et l'histidine sont vitales pour la synthèse de glucoamylase. Le tween 80 et le triton X-100 augmentent la croissance mais suppriment la synthèse de glucoamylase.

     

Influence of carbon and nitrogen sources on glucoamylase production byAspergillus in batch process

Summary Glucoamylase production kinetics was greatly affected by medium composition. While maltose and cassava flour induced glucoamylase synthesis, fructose clearly repressed it, reaching a maximum enzyne activity value (A_m) of only 6% of those obtained with the former carbon sources. Among the nitrogen sources the best result was obtained with urea, reaching A_m values of about 40% higher than those obtained with ammonium sulphate.     

Glucoamylase production byAspergillus awamori on rice flour medium and partial characterization of the enzyme

Summary Aspergillus awamori ATCC 22342 was selected from 12 strainsof Aspergillus spp.and Rhizopus spp. as the best producer of amylase. Optimal growth conditions for the enzyme production in shake flasks were provided by: a medium containing 60 g/1 rice flour, 0.075% (w/v) NaNO₂ and 0.075% (v/v) corn-steep liquor, a temperature of 30° C and initial pH value of 6.5. The enzyme was characterized as a glucoamylase with a molecular weight of 49,000. Maximum enzyme activity occurred at 45 C and pH 5.8. The enzyme was stable at 40° C and lost 70 and 90% of activity when heated for 30 min at 50 and 60°C, respectively. Thermal inactivation was slowed in the presence of starch. Michaelis-Menten constants for soluble starch and dextrin were estimated as 12.5 and 33.3 mg/ml, respectively. This enzyme may be used for the production of glucose-rich syrups from rice starch.

---

Producción de glucoamilasa por Aspergillus awamori en harina de arroz y caracterización parcial del enzima

Resumen Aspergillus awamori ATCC 22342 se seleccionó entre 12 cepas deAspergillus spp. y deRhizopus spp. como el mejor productor de amilasa. La condiciones óptimas de crecimiento para la producción del enzima en frascos de agitación fueron las siguientes: un medio con la composicion siguiente: 60 g/1 de harina de arroz, 0.075% (m/v) NaNO₂ y 0.075% (v/v) de extracto de maíz (corn steep liquor); una temperatura de 30°C y un pH inicial de 6.5. El enzima fue caracterizado como una glucoamilasa de peso molecular 49,000. La máxima actividad enzimática se obtuvo a 45°C con un pH de 5.8. El enzima era estable a 40 C pero perdió un 70 y un 90% de su actividad cuando se calentó durante 30 min a 50 y 60° C respectivamente. La inactivación térmica fue más lenta en presencia de almidón. Las constantes de Michaelis-Menten para almidón soluble y para dextrina se estimaron como 12.5 y 33.3 mg/ml respectivamente. Este enzima puede utilizarse para la producción de jarabes ricos en glucosa a partir de almidón de arroz.

---

Production de glucoamylase par Aspergillus awamori cultivé sur milieu à la farine de riz et caractérisation partielle de l'enzyme

Résumé Aspergillus awamori ATCC 22342 a été sélectionné parmi 12 souches d'Aspergillus spp. et deRhizopus spp. comme étant le meilleur producteur d'amylase. Les conditions optimales de croissance pour la production d'enzyme en fioles agitées sont: un milieu contenant 60 g/1 de farine de riz, 0.075% (w/v) de NaNO₂ et 0.075% (v/v) de liqueur de corn steep, une température de 30° C et un pH initial de 6.5. L'enzyme a été caractérisé comme étant une glucoamylase de poids moléculaire 49,000. L'activité maximum de l'enzyme se situe à 45°C et pH 5.8. L'enzyme est stable à 40°C et perd 70 et 90% de son activité par chauffage pendant 30 min à 50 et à 60°C, respectivement. L'inactivation thermique est ralentie en présence d'amidon. Les constantes de Michaelis-Menten pour l'amidon soluble et pour la dextrine ont été éstimées, respectivement, à 12.5 et 33.3 mg/ml. Cet enzyme peut être utilisé pour la production de sirops riches en glucose à partir d'amidon de riz.

     

Comparison of citric acid production byAspergillus niger immobilized in gels and cryogels of polyacrylamide

Aspergillus niger was immobilized in cryogels and in conventional gels of polyacrylamide. The growth of cells entrapped in two kinds of gels and the production of citric acid by the immobilized cells were investigated and compared. Cells immobilized in cryogels were more suitable for citric acid production.     

Statistical optimization of medium components for the production of xylanase byAspergillus niger KK2 in submerged cultivation

Medium composition was optimized for the production of xylanase byAspergillus niger KK2 using statistical experimental designs. Corn steep liquor (CSL) and industrial yeast extract (IYE) were the most important factors affecting xylanase activity. The medium that produced the optimum conditions for the production of xylanase contained 3% rice straw, 1% wheat bran, 6.3% CSL, 0.15% IYE, and 0.5% KH₂PO₄. After 4 days of cultivation under optimized conditions in a 2.5-L stirred tank reactor the activity and productivity of xylanase were 620 IU/mL and 6,458 IU/L.h, respectively. The highest xylanase activity obtained using the optimized medium was 80% greater than the activity obtained using basal medium. The xylanase activity predicted by a polynomial model was 670 IU/ml.