Biosynthesis of protein by microscopic fungi in solid state fermentation. II. Optimization of aspergillus oryzae A. or. 11 cultivation for protein enrichment of starchy raw materials

Study was made of the effect of medium humidity, source and dose of nitrogen, dose of phosphorus, dose of inoculum and aeration on protein biosynthesis by strain Aspergillus oryzae A. or. 11 in solid state fermentation. It was found that for a maximal protein yield the medium ought to contain about: 60% of water, 36% of starchy raw materials d. m. (e.g. 28.8% of coarse rye meal and 7.2% of beet pulp), 0.85% of nitrogen sources (1.6% of (NH₄)₂SO₄ and 1.1% of urea), and 0.35% of phosphorus source (1.3% of KH₂PO₄). pH of medium should be near 6.5. The dose of inoculum should not be lower than 10⁸ spores/100 g medium. The duration of culture ought to be 20–22 h at 30–35°C, with aeration at least 40 dm³ of air/h × 1000 g medium. Under these conditions the protein yield is about 6.0–6.3 g/100 g of starting medium d.m. at the cost of utilization of about 25 g total carbohydrates.     

Biosynthesis of protein by microscopic fungi in solid state fermentation. III. The effect of different cultivation methods and various media on protein biosynthesis by Aspergillus oryzae A. or. 11

The effect of the culture method in solid medium and the influence of starch-containing raw materials on the yield of fungal protein biosynthesis were studied. Three procedures (laboratory bioreactor method, tray method and pile method) gave satisfactory results. Protein yields amounted to 5.0–5.9 g/100 g of starting medium d.m. upon utilization of 20.9–24.1 g carbohydrates. The procedure involving the use of a fermenter with a mixer afforded protein yields by about 50% lower as compared with the three above-mentioned procedures, and therefore it requires technical improvement. As a result of fungal culture using various starch-containing raw materials, the protein content in post-culture products increased by 46–88%, as compared with starting medium. The contents of 13 amino acids (including some exogenous ones which increased by 52–82%) in post-culture products substantially rose (by 34–63%). The post-culture products exhibited proteolytic activity (1260–3500 HU/g of d.m.). The kind of the source of starch evidently influenced protein biosynthesis. Media based on potatoes afforded the greatest increases in protein (5.8–5.9 g/100 g of starting medium d.m.), and those containing coarse rye meal and milling by-products — the smallest ones (4.5–4.6 g/100 g of starting medium d.m.).     

Production of Aspergillus terreus alpha-L-rhamnosidase by solid state fermentation

AIMS: The study of production of Aspergillus terreus CECT 2663 alpha-L-rhamnosidase in solid state fermentation using wheat bran, washed sugar cane bagasse and polyurethane foam as substrates or supports for the enzyme production. METHODS AND RESULTS: Cultures were carried out in Petri dishes under controlled temperature and humidity. Naringin or rhamnose were the enzyme inducers and carbon sources. The enzyme activity to inducer ratio was appreciably greater when using sugar cane bagasse or polyurethane foam than wheat bran. The influence of inoculum size, inducer, airflow, humidity and temperature were determined. Under optimum conditions, about four units of enzyme per ml nutrient solution were obtained after 4-6 d. CONCLUSIONS: The activity to inducer ratio was higher, and the cultivation time was shorter in solid state fermentation than those observed in submerged cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: Solid cultures, using naringin as inducer, can be appropriate alpha-L-rhamnosidase production.     

Biosynthesis of pectinase by fungi Bjerkandera and Coriolus by solid phase fermentation]

Production of an extracellular pectinase by wood-rot fungi of the genus Bjerkandera and Coriolus was studied. The active producers B. adusta 40 and C. versicolor 24 were selected. The dynamics of production of pectinase and effects of temperature, initial pH, humidity of the medium and addition of nitrogen sources on the biosynthesis of pectinase were studied.     

Breeding and growth of Rhizopus in raw cassava by solid state fermentation

Nineteen Rhizopus strains were selected and tested for their growth capacity on raw cassava starch and their ability to produce amylase when grown on solid-state fermentations. Only three strains grew significantly on this natural substrate. Glucoamylase production was higher on raw cassava than on cooked cassava. After 48 h of fermentation, the protein content of cassava was increased from 1.75% to 11.3%. The byproducts of fermentation were fumaric acid, lactid acid and ethanol.     

黑曲霉固态发酵生产单宁酶的条件优化

研究采用响应面法优化黑曲霉固态发酵生产单宁酶的培养条件。应用Plackett—Burman试验筛选出重要影响因子：五倍子粉含量、（NH4）2SO4浓度以及接种孢子量,最陡爬坡试验逼近最大响应区域。应用Box．Behnken响应面试验对重要影响因子进一步优化。得到最佳培养条件：每250mL三角瓶中装入1．0g五倍子粉、4．4g稻壳和0．5g麸皮、液固比（mL／g）2：1且营养盐溶液组成为（NH4）2s0421g/L、MgSO4·7H2O1g／L、NaCl1g／L,培养基pH自然,接种5．7×10^7个孢子后在30℃温度下培养4d。在此条件下,单宁酶产量从40U／g提高到114U／g,3次重复验证性试验平均值为115U／g,验证了模型的可靠性。     

Culture requirements for the production of protease by Aspergillus oryzae in solid state fermentation

Summary A number of culture conditions for protease production by Aspergillus oryzae NRRL 2160 on solid substrates were investigated. The pH of the medium and the substrate markedly affected protease production. High protease yield was obtained when the fungus was cultivated for 72–96 h on rice hulls: rice bran (7:3), at an initial pH of 7.0. Maximal protease production was achieved at an initial moisture content of 35–40%, corresponding to a water activity range of 0.982–0.986. Casein and gluten were effective inducers. Polyethylene bags proved to be promising containment systems for solid state cultivation. Offprint requests to: A. M. R. Pilosof     

Protease-conidia relationships of Aspergillus niger grown in solid state fermentation

Protease activity of Aspergillus niger growing on solid substrate correlated well with conidia formation (R: 0.91–0.96) for initial moisture contents of 38–48% (wet basis), initial pH 5.4 and 6 and temperature (29–37 °C ). However, conidia/protease ratio varied with most of these conditions and by NaCl addition indicating only a partial association between them.     

A process for protein enrichment of cassava by solid substrate fermentation in rural conditions

An artisanal static process for protein enrichment of cassava by solid-state fermentation, developed in laboratory and tested on pilot units in Burundi (Central Africa), provides enriched cassava containing 10.7% of dry matter protein versus 1% before fermentation. Cassava chips, processed into granules of 2-4-mm diameter, are moistened (40% water content) and steamed. After cooling to 40 degrees C, cassava is mixed with a nutritive solution containing the inoculum (Rhizopus oryzae, strain MUCL 28627) and providing the following per 100 g dry matter: 3.4 g urea, 1.5 g KH(2)PO(4), 0.8 g MgSO(4).7H(2)O, and 22.7 g citric acid. For the fermentation, cassava, with ca. 60% moisture content, is spread in a thin layer (2-3 cm thick) on perforated trays and slid into an aerated humidified enclosure. The incubation lasts +/- 65 h. The production of protein enriched cassava is 3.26 kg dry matter/m(2) tray. The effects of the variation of the nutritive solution composition and the inoculum conservation period on the protein production are equally discussed.     

Air pressure pulsation solid state fermentation of feruloyl esterase by Aspergillus niger

Air pressure pulsation solid state fermentation (APP-SSF) was applied to produce feruloyl esterase (FAE) by Aspergillus niger. With the optimization of some variables by orthogonal design, the optimal condition obtained was 0.2 MPa (gauge pressure) of high pressure intensity, 30 min of low pressure duration and 20s of high pressure duration. Based on the optimized condition, the APP-SSF achieved the reasonable enzyme yield of 881 mU/g at 48 h, which was 58% more than that by static solid state fermentation (static SSF) at 72 h. By comparison of two fermentation methods in temperature, O(2) and CO(2) concentration, and respiration intensity, it was concluded that APP-SSF enhanced heat and mass transfer of fermentation system and strengthened the metabolism of microorganisms. The APP-SSF had a greatly positive effect on FAE production by A. niger, by enhancing mass and heat transfer and activating growth and metabolism.     

Purification and characterization of glucoamylase produced by Aspergillus niger in solid state fermentation

Glucoamylases produced by Aspergillus niger grown on wheat bran in solid cultures were purified. Four different forms, GA I, GA I', GA II and GA III, were found having apparent molecular weights of 112 000, 104 000, 74 000 and 61 000 Da respectively. The enzymes are glycoproteins with a carbohydrate content of 16%, and optimal activity at 60C and pH 4.4. Activity was strongly inhibited by Hg²⁺ while Mn²⁺ and Fe²⁺ were stimulatory. The K_m values for the degradation of starch and maltose were 3.5 and 7.8 mg ml^-1, respectively.     

Modification of wheat straw lignin by solid state fermentation with white-rot fungi

The potential of crude enzyme extracts, obtained from solid state cultivation of four white-rot fungi (Trametes versicolor, Bjerkandera adusta, Ganoderma applanatum and Phlebia rufa), was exploited to modify wheat straw cell wall. At different fermentation times, manganese-dependent peroxidase (MnP), lignin peroxidase (LiP), laccase, carboxymethylcellulase (CMCase), avicelase, xylanase and feruloyl esterase activities were screened and the content of lignin as well as hydroxycinnamic acids in fermented straw were determined. All fungi secreted feruloyl esterase while LiP was only detected in crude extracts from B. adusta. Since no significant differences (P > 0.05) were observed in remaining lignin content of fermented straw, LiP activity was not a limiting factor of enzymatic lignin removal process. The levels of esterified hydroxycinnamic acids degradation were considerably higher than previous reports with lignocellulosic biomass. The data show that P. rufa, may be considered for more specific studies as higher ferulic and p-coumaric acids degradation was observed for earlier incubation times.     

Evaluation of filamentous fungi and inducers for the production of endo-polygalacturonase by solid state fermentation

Aspergillus oryzae CCT 3940, Aspergillus awamori NRRL 3112 and a Trichoderma sp.) were compared for their capacity to produce endo-polygalacturonase (endo-PG) in solid state fermentation. Maximum pectinolytic activity was reached in 72 h of growth, the best two fungal strains being A. niger T0005007-2 and A. oryzae CCT 3940. Three types of commercial purified pectin and four of unprocessed pectin (tangerine, orange, Tahiti lime and sweet lime rind) were used to assess the effect of pectin on the production of endo-PG by A. niger T0005007-2. Maximum pectinolytic activity was achieved using 6 and 10% (w/w) of purified pectin as inducer. Depending on the origin of the commercial pectin used as inducer, maximum endo-PG levels varied from 223 to 876 units per gram of dry medium (one endo-PG unit (U) was defined as the quantity of enzyme which caused a reduction in viscosity of 50% in a 1% w/v solution of pectin in 30 min), indicating that care should be taken when choosing this component of the medium. When the crude pectins were used as inducers at the same concentration as purified pectin, maximum endo-PG activities were 250-300 units/g. However, by increasing the amount of Tahiti lime rind to 50% (w/w) maximum endo-PG was 919 U/g, thus opening up the possibility of a low cost medium for endo-PG production.     

Potential of Aspergillus oryzae CFTRI 1480 for producing proteinase in high titres by solid state fermentation

Aspergillus oryzae CFTRI 1480, an isolate from a spoiled moist sample of casein, produced 59,105 units of an extracellular proteinase/g dry mouldy bran (DMB) at 72 h in an arbitrarily formulated wheat bran medium in a solid state fermentation system. The enzyme production was significantly affected by mineral salt content and pH of the liquid used for moistening the wheat bran. Enzyme titres were enhanced 1.34-fold with the addition of 0.4% corn starch. Optimization of key parameters, i.e., initial moisture content, age and size of inoculum, increased the enzyme production to 191,869 units/g DMB and reduced the fermentation time to 48 h. Such high titres in a simple medium, surpassing most of the literature reports, indicate the industrial importance of the culture. The properties of acetone-precipitated enzyme, viz, the optimum pH of 10.0, more than 95% activity between pH 7.0 and 10.0, temperature optimum at 55° C and more than 90% activity between 10 and 27°C, are similar to those of commercially available fungal proteinases employed in animal feed, leather and other industries. Correspondence to: B. K. Lonsane     

A simple protocol for extraction and detection of pyrazines produced by fungi in solid state fermentation

Summary A protocol for the extraction and detection of pyrazines produced by fungi in Solid State Fermentation that gave good recovery of these compounds was devised together with specific techniques for detection by Gas Chromatography (GC). The protocol involved solid-liquid and liquid-liquid extractions, concentration of the extracts and GC analysis. Extraction yields were measured from a model system in which synthetic pyrazines were added. Extracts were analysed with a gas chromatograph equipped with a thermo-ionic detector to allow specific pyrazine detection.     

Extraction and purification of amyloglucosidase produced by solid state fermentation with Aspergillus niger

Summary Aqueous two-phase extraction was employed, as a new approach, for the recovery of amyloglucosidase (AMG) after solid state fermentation. In an aqueous two-phase system of PEG 6000 and potassium phosphate, 95% of the AMG was recovered in the bottom salt phase, with all visible particles from solid fungal bran in the interface, giving a purification factor of 11. Affinity purification of AMG was also done by adsorption onto crosslinked starch, in the presence of PEG or salt. Both were found to enhance the adsorption. In the presence of PEG 6000 (10% w/w), two fold increase in AMG adsorption, 85% of AMG recovery and 6.0 fold of purification were achieved/The purified AMG was found homogeneous by SDS polyacrylamide gel electrophoresis.     

Biomass estimation in solid state fermentation I. Manual biochemical methods

Summary We examined the changes in three biomass constituents (glucosamine, ergosterol, total sugar), sucrose consumption and conidia formation, during cultivation of Beauveria bassiana on agar plates or clay granules. We showed that glucosamine can be considered a good biomass indicator on condition that the media had the same constituents but not necessarily the same C/N ratio. Total sugar was not constant during the different growth phases and cannot accurately represent biomass changes. The ergosterol amounts changed during the different growth phases but, for a fixed process, it can be a good indicator of conidiation. Good correlations were obtained between glucosamine and sucrose consumption allowing biomass yield calculations.Offprint requests to: C. Desgranges     

Xylanase and pectinase production by Aspergillus awamori on grape pomace in solid state fermentation

The feasibility of using grape pomace for the production of xylanase and exo-polygalacturonase by Aspergillus awamori in solid state fermentation has been evaluated. Solid state fermentation experiments indicated that the particle size did not influence the enzyme production. The addition of extra carbon sources and the initial moisture content of the grape pomace were found to have a marked influence on the enzymes yields. Xylanase and exo-PG activities were high at 65% (w/w) initial moisture content and glucose supplementation.     

Amylolysis of raw corn by Aspergillus niger for simultaneous ethanol fermentation

The novelty of this approach was hydrolysis of the raw starch in ground corn to fermentable sugars that are simultaneously fermented to ethanol by yeast in a non-sterile environment. Thus, the conventional cooking step can be eliminated for energy conservation. A koji of Aspergillus niger grown on whole corn for 3 days was the crude enzyme source. A ratio of 0.2 g dry koji/g total solids was found sufficient. Optimum pH was 4.2. Ethanol concentration was 7.7% (w/w) in the aqueous phase with 92% raw starch conversion. Agitation increased rate. Sacharification was the rate-limiting step. The initial ethanol concentration preventing fermentation was estimated to be 8.3% by weight.