Production and characterization of polygalacturonase fromGeotrichum candidum

An extracellular polygalacturonase (EC 3.2.1.15) fromGeotrichum candidum ATCC 34614 grown onsauerkraut brine was produced and characterized. Polygalacturonic acid markedly increased the enzyme yield in the brine. The fungus produced the highest activity (290 U/l) in brine with 0.3% (w/v) polygalacturonic acid. The pH and temperature optima of the enzymes were 4.5 to 5.0 and 30°C, respectively. It was stable from pH 4.0 to 5.8 and at 30°C but lost its activity at higher temperatures. The K_m and V_max values for polygalacturonic acid were 4.2 mg/ml and 0.19mm galacturonic acid/min, respectively. The enzyme was not substrate inhibited.     

Ethanol production from spent cherry brine

Spent cherry brine is an acidic byproduct of maraschino cherry processing and typically consists of variable amounts of glucose and fructose of up to 11% fermentable solids, 0.5–1.5% CaCl₂, up to 0.4% sulfur dioxide, sorbitol, and lesser amounts of other cherry constituents. Disposal of brine represents a significant cost to processors because of its high biological oxygen demand. As an alternative, brine was tested as a substrate for ethanol production. Initially, the toxic level of sulfur dioxide was reduced by raising brine pH to 8.0 to precipitate calcium sulfite. Because alkalinization was subsequently found to result in a 10-fold reduction in phosphorous, brines were titrated with phosphoric acid to pH 6.0 prior to inoculation with Saccharomyces cerevisiae. All strains of Saccharomyces cerevisiae tested were able to ferment all lots of Ca(OH)₂-treated and phosphorous-enriched brines efficiently. One lot of brine containing 10% (w/v) fermentable sugar yielded 4.7% (w/v) ethanol in 4 days. Received 24 December 1996/ Accepted in revised form 14 April 1997     

Development of a submerged-liquid sporulation medium for the potential smartweed bioherbicide Septoria polygonorum

Submerged culture experiments were conducted in three phases to determine the optimal medium for rapidly producing conidia of the fungal bioherbicide Septoria polygonorum. In phase I, 47 crude carbon sources were evaluated to determine which would support sporulation. Under the conditions tested, pea brine (5–10% v/v) provided best conidiation. In phase II, a fractional factorial design was utilized to screen 38 different medium adjuncts in combination with pea brine for improved sporulation. MgSO₄ was the only factor that resulted in a significant improvement. In phase III, a central composite design with response surface methodology was used to optimize concentrations of these critical factors. The model predicted optimal sporulation in a medium composed of 8.88% v/v pea brine+0.1 molar MgSO₄ with an expected titer of 1.78×10⁸ conidia/ml. Actual mean titer attained with the model-derived medium was 1.15×10⁸ conidia/ml. No significant difference was observed in virulence of conidia produced on agar vs. the model-derived (liquid) medium.     

Yeast heterogeneity during spontaneous fermentation of black Conservolea olives in different brine solutions

Aims: To assess the yeast community structure and dynamics during Greek‐style processing of natural black Conservolea olives in different brine solutions. Methods and Results: Black olives were subjected to spontaneous fermentation in 6% (w/v) NaCl brine solution or brine supplemented with (i) 0·5% (w/v) glucose, (ii) 0·2% (v/v) lactic acid and (iii) both glucose and lactic acid. Yeast species diversity was evaluated at the early (2 days), middle (17 days) and final (35 days) stages of fermentation by restriction fragment length polymorphism and sequence analyses of the 5·8S internal transcribed spacer and the D1/D2 ribosomal DNA (rDNA) regions of isolates. Analysis revealed a relatively broad range of biodiversity composed of 10 genera and 17 species. In all treatments, yeasts were the main micro‐organisms involved in fermentation together with lactic acid bacteria that coexisted throughout the processes. Metschnikowia pulcherrima was the dominant yeast species at the onset of fermentation, followed by Debaryomyces hansenii and Aureobasidium pullulans. Species heterogeneity changed as fermentations proceeded and Pichia membranifaciens along with Pichia anomala evolved as the main yeasts of olive elaboration, prevailing at 17 and 35 days of the process. Molecular techniques allowed for the identification of five yeast species, namely A. pullulans, Candida sp., Candida silvae, Cystofilobasidium capitatum and M. pulcherrima, which have not been reported previously in black olive fermentation. Conclusions: By using molecular techniques, a rich yeast community was identified from Conservolea black olive fermentations. Metschnikowia pulcherrima was reported for the first time to dominate in different brines at the onset of fermentation, whereas Pichia anomala and P. membranifaciens evolved during the course. The addition of glucose and/or lactic acid perturbed yeast succession and dominance during fermentation. Significance and Impact of the Study: Yeasts have an important role in black olive fermentation and contribute to the development of the organoleptic characteristics of the final product. At the same time, certain species can cause significant spoilage. The present study adds to a better knowledge of yeast communities residing in olive fermentations towards a well‐controlled process with minimization of product’s losses.     

Effect of inulin on yeast inulinase production in sauerkraut brine

Kluyveromyces marxianus NRRL Y-1196 produced the highest inulinase activity (38 U/mg protein) of six yeasts examined after 24 h growth in sauerkraut brine in shaking flasks at 30°C with 0.3% inulin as an enzyme inducer. The enzyme was recovered by acetone fractionation, with a yield of 81%. It had maximum activity at pH 4.4 and 55°C with K _m values for inulin and sucrose of 3.92 mm and 11.9 mm, respectively. The yeast raised the pH from 3.4 to above 7.0, using all the lactic acid in the brine. Growth of K. marxianus in sauerkraut brine with a small amount of inulin may usefully decrease the BOD and concomitantly produce inulinase.The authors are with the Department of Food Science and Technology, Cornell University, Geneva, New York 14456, USA     

Growth of Pichia guilliermondii A9, an osmotolerant yeast, in waste brine generated from kimchi production

The possibility of culturing an osmotolerant yeast using waste brine from a kimchi factory as a substrate for the production of single cell protein was investigated. Pichia guilliermondii A9 was selected from 70 isolates of yeast demonstrating substantial growth in the waste brine. The growth of P. guilliermondii A9 in waste brine was not inhibited by NaCl concentrations of up to 10% (w/v). However, it was reduced drastically at concentrations greater than 12% (w/v). Approximately 90% of BOD was removed from the waste brine by culturing of P. guilliermondii A9 for 24 h. The maximum cell yield was 0.69 g of dry cells per liter, containing 40% of protein. When the waste brine was enriched with cabbage juice from waste cabbage, the final cell mass increased proportionally with the amount of added organic material. Salt stressed cells of P. guilliermondii A9 grown in waste brine are shown in scanning electron micrographs. In conclusion, the large amounts of waste brine generated from kimchi production could be used directly for the culture of the osmotolerant yeast P. guilliermondii A9.     

Production of extracellular emulsifying agent byPseudomonas aeruginosa UG1

Summary Twenty-three bacterial strains were isolated from oil-contaminated soil samples. Of these, 20 displayed some ability to effect oil dispersion and they were screened quantitatively for the ability to emulsify 0.5% (v/v) reference oil. One strain, identified asPseudomonas aeruginosa UG1, produced extracellular material that emulsified reference oil, hexadecane and 2-methylnaphthalene at concentrations as high as 6% (v/v) in nutrient broth. Emulsification activity increased during a 10 day incubation period at 30°C. The activity was not influenced by pH over the range 5 to 9. The emulsifying agent was precipitated by cold ethanol. The highest emulsifying activity was detected in the extracellular fraction precipitated between 30 and 50% (v/v) ethanol. A linear relationship was observed between emulsifier concentration (mg/ml) and emulsifying activity. Genetic analysis showed that thePseudomonas aeruginosa UG1 strain did not carry extrachromosomal plasmids, suggesting that the gene(s) coding for emulsifying activity was carried on the chromosome.     

Antimicrobial activity of honey from the stingless bee Trigona carbonaria determined by agar diffusion,agar dilution,broth microdilution and time‐kill methodology

Aims: The aim of this study was to determine the spectrum of antimicrobial activity of 11 samples of stingless bee honey compared to medicinal, table and artificial honeys. Methods and Results: Activity was assessed by agar diffusion, agar dilution, broth microdilution and time‐kill viability assays. By agar dilution, minimum inhibitory concentration (MIC) ranges were 4% to >10% (w/v) for Gram‐positive bacteria, 6% to >16% (w/v) for Gram‐negative bacteria and 6% to >10% (w/v) for Candida spp. By broth microdilution, all organisms with the exception of Candida albicans and Candida glabrata were inhibited at ≤32% (w/v). Geometric MIC (w/v) means for stingless bee honeys ranged from 7·1% to 16·0% and were 11·7% for medicinal honey and 26·5% for table honey. Treatment of organisms with 20% (w/v) stingless bee honey for 60 min resulted in decreases of 1–3 log for Staphylococcus aureus, >3 log for Pseudomonas aeruginosa and <1 log for C. albicans. Similar treatment with each control honey resulted in decreases of <1 log for all organisms. Conclusions: Stingless bee honey has broad‐spectrum antibacterial activity although activity against Candida was limited. Stingless bee honey samples varied in activity and the basis for this remains to be determined. Significance and Impact of the Study: Stingless bee honey had similar activity to medicinal honey and may therefore have a role as a medicinal agent.     

Residual and ovicidal efficacy of essential oil‐based formulations in vitro against the donkey chewing louse Bovicola ocellatus

Essential oils have shown good experimental potential as novel veterinary ectoparasiticides. However, if they are to be used as veterinary products, they must be available in formulations that are suitable for practical application against specific ectoparasites. Here, the efficacies of formulations containing 5% (v/v) lavender or tea tree oil, in combination with two emulsifiers [a surfactant, 5% (w/v) N‐lauroylsarcosine sodium salt (SLS), and a soluble polymer, 5% (w/v) polyvinylpyrrolidone (PVP)], with or without 10% coconut oil, were tested in contact bioassays against the donkey chewing louse Bovicola ocellatus (Piaget) (Phthiraptera: Trichodectidae). Residual activity was quantified in open and closed containers; ovicidal efficacy was also examined. Exposure to either of 5% (v/v) lavender or tea tree oils with SLS or PVP resulted in louse mortality of 100%, but when coconut oil was included as an excipient, significantly lower efficacy was recorded. However, the formulations became significantly less effective after 2 h in open containers and 40 h in closed containers. The results confirm that the residual activity of essential oils is relatively transitory and the addition of 10% coconut oil does not prolong the period of insecticidal activity by slowing essential oil evaporation. Too short a period of residual activity is likely to be a significant impediment to the effective practical use of essential oils. However, unlike many synthetic pediculicides, the essential oils tested here were highly ovicidal, which suggests that prolonged residual activity may not be essential to kill newly hatched nymphs after treatment.     

Lactic fermentation during the storage of 'Alorena' cultivar untreated green table olives

The development of lactic fermentation processes for the storage of directly brined olives (Aloreña cultivar) was investigated by three procedures: (1) a modification of the traditional method with an initial brine containing 9% (w/v) NaCl and 0.2% (w/v) acetic acid; (2) induced lactic fermentation with 6% NaCl and 0.2% acetic acid; and (3) conservation in acidified brine containing 6% NaCl and 0.6% acetic acid. In all cases, strains of Lactobacillus plantarum and Pediococcus spp. were present in each, indicating the great tolerance of these micro-organisms to high levels of lactic and acetic acids. They also appeared in an altered sequence. Counts of Pediococcus remained moderate (higher than Lact. plantarum ) throughout the last part of the preservation period. A commercial starter improved colonization by Lact. plantarum. Yeasts coexisted with the lactic bacteria throughout the preservation period although their importance in the fermentation process was very limited. The brine characteristics obtained after fermentation were suitable for assured product preservation. There was no spoilage. These results encourage research on the mechanism of lactic acid bacteria inhibition in brines and the development of lactic fermentation processes for directly brined olives from other olive cultivars.     

Combined effect of alkali pretreatment and sodium chloride addition on the olive fermentation process

Green olives of the Tunisian variety "Meski" were treated according to a Spanish-style green olive preservation process by using an alkaline treatment (1.5, 2 and 2.5% (w/v) NaOH) to eliminate bitterness, combined with different brine concentrations (6, 9 and 12% (w/v) NaCl). A spontaneous fermentation by the environmental microflora took place. Results showed that 2% NaOH solution and 9% sodium chloride brine was an optimal combination inducing the best growth of Lactobacillus species (10(8) CFU/ml) and acidity of 0.726 g lactic acid/100 ml brine. In all trials and independently of the treatment, Lb. plantarum was the most dominant strain of Lactobacillus. Moreover, pretreatment with lye and lactic fermentation of olives contributed to coliform elimination.     

Bioconversion of butyronitrile to butyramide using whole cells of <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> PA-34

Butyramide is an important chemical commodity, which is used for the synthesis of hydroxamic acids and electrorheological fluids and for the preparation of β-amodoorganotin compounds. The nitrile hydratase (Nhase) of Rhodococcus rhodochrous PA-34 catalyzed the conversion of butyronitrile to butyramide. The maximum Nhase activity [18 U/mg dry cell weight (dcw)] of whole cells of R. rhodochrous PA-34 was observed at pH 7.0 with 10% (v/v) butyronitrile and 1 mg cells (dcw)/ml reaction mixture at 10°C. The cells of R. rhodochrous PA-34 retained almost 50% activity when incubated for 1 h in the presence of 85% (v/v) butyronitrile. A yield of 597 g of butyramide (6.8 M) was obtained using 60% (v/v) butyronitrile, 1 g cells (dry weight) in a 1-l batch reaction at 10°C for 6 h.     

1,8-cineole and castor oil in sodium lauryl ether sulphate disrupt reproduction and ovarian tissue of Rhipicephalus (Boophilus) microplus

Essential and fixed oils have been researched as alternatives to chemical acaricides. The activity of volatile compounds from essential oils (1,8-cineole, citral and eugenol) at 1.0% (w/v) and fixed oil (castor oil) at 0.3% (w/v) dissolved in 2.0% (v/v) dimethyl sulfoxide (DMSO) + 0.2% (w/v) Tween 80^® was assessed against Rhipicephalus microplus using immersion tests. 1,8-cineole (29.0%) and castor oil (30.2%) had the highest reproductive inhibition rate. A second experiment was performed to verify the effect of the 1,8-cineole (10.0% w/v) and, or castor oil (0.3% w/v) on tick reproduction using different solubilizing agents. The highest reproductive inhibition was observed for the combination of 1,8-cineole/castor oil (94.1%) and 1,8-cineole in 2.0% (w/v) sodium lauryl ether sulphate (SLES) (92.8%). A third experiment showed morphological changes in R. microplus oocytes at different stages of development, as well as in pedicel cells. The most intense effects were observed when ticks were immersed in the formulation containing 1,8-cineole (10.0% w/v) and castor oil (0.3% w/v) dissolved in 2% (w/v) SLES. These findings highlight the potential of this formulation as an alternative for managing cattle ticks as their cytotoxic effects can reduce R. microplus reproductive success.     

Solvent effect on enzyme-catalyzed synthesis of β-d-glucosides using the reverse hydrolysis method: Application to the preparative-scale synthesis of 2-hydroxybenzyl and octyl β-d-glucopyranosides

Almond β-d-glucosidase was used to catalyze alkyl-β-d-glucoside synthesis by reacting glucose and the alcohol in organic media. The influence of five different solvents and the thermodynamic water activity on the reaction have been studied. The best yields were obtained in 80 or 90% (v/v) tert-butanol, acetone, or acetonitrile where the enzyme is very stable. In this enzymatic synthesis under thermodynamic control, the yield increases as the water activity of the reaction medium decreases. Enzymatic preparative-scale syntheses were performed in a tert-butanol-water mixture which was found to be the most appropriate medium. 2-Hydroxybenzyl β-d-glucopyranoside was obtained in 17% yield using a 90:10 (v/v) tert-butanol-water mixture. Octyl-β-glucopyranoside was obtained in 8% yield using a 60:30:10 (v/v) tert-butanol-octanol-water mixture.     

Sugarcane molasses and yeast powder used in the Fructooligosaccharides production by Aspergillus japonicus-FCL 119T and Aspergillus niger ATCC 20611

Different concentrations of sucrose (3–25% w/v) and peptone (2–5% w/v) were studied in the formulation of media during the cultivation of Aspergillus japonicus-FCL 119T and Aspergillus niger ATCC 20611. Moreover, cane molasses (3.5–17.5% w/v total sugar) and yeast powder (1.5–5% w/v) were used as alternative nutrients for both strains’ cultivation. These media were formulated for analysis of cellular growth, β-Fructosyltransferase and Fructooligosaccharides (FOS) production. Transfructosylating activity (U _t ) and FOS production were analyzed by HPLC. The highest enzyme production by both the strains was 3% (w/v) sucrose and 3% (w/v) peptone, or 3.5% (w/v) total sugars present in cane molasses and 1.5% (w/v) yeast powder. Cane molasses and yeast powder were as good as sucrose and peptone in the enzyme and FOS (around 60% w/w) production by studied strains.     

Enhanced phytase production from Achromobacter sp. PB‐01 using wheat bran as substrate: Prospective application for animal feed

This article deals with the optimization of the various parameters for production of phytase using Achromobacter sp. PB‐01 in submerged fermentation (SmF). A semisynthetic medium containing ingredients of phytase screening media (PSM) supplemented with 2% (w/v) sucrose, 1% (w/v) peptone, and 10% (w/v) wheat bran was found to be the best production medium among the various combinations tried. Among various surfactants added to SmF, Triton X‐100 (0.1%) exhibited a 16% increase in phytase activity. An overall 11.2 fold enhancement in enzyme activity (0.79 U/mL→8.84 U/mL) was attained when SmF was carried out using 0.5% (v/v) inoculum of a 15 h old culture of Achromobacter sp. PB‐01 at an initial pH of 5.5, temperature 30°C and allowed to grow for 48 h. Presence of accessory hydrolytic enzymes in the crude extract further added value as feed additive by mediating efficient degradation of non‐starch polysaccharides (NSP). In addition, we also investigated the efficacy of phytase on different agro‐industrial residues using in vitro experiments that simulated the conditions of the digestive tract. Results indicate that phytase from our source hydrolyze phytate efficiently with the concomitant liberation of inorganic phosphate, protein, reducing sugar, and calcium. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Production of an extracellular polysaccharide with emulsifier properties by Penicillium citrinum

Penicillium citrinum produced a glycolipid with emulsifier properties during cultivation on mineral medium with 1% (v/v) olive oil as carbon source. The emulsifier production was growth-associated and reached maximal activity at 60 h of cultivation. The production yield (Y _p/s) was 0.54 and the best emulsifying activity was observed for xylene and diesel oil when compared to other carbohydrates tested. The emulsifier was shown to be stable to a wide range of pH and temperature values and was shown to contain D-galactose, D-glucose and D-xylose (8.2:1.0:5.3) with a total carbohydrate content of 43%. The presence of salts stimulated the emulsification activity, suggesting potential for its application in industrial waste or marine remediation.     

Characterization of alkaline proteases from a novel alkali-tolerant bacterium Bacillus patagoniensis

Summary The extracellular proteolytic activity produced by a moderately alkaliphilic bacterium, Bacillus patagoniensis PAT 05^T, was characterized. This strain, grown in a highly alkaline and saline medium, produced important levels of proteolytic activity. SDS-PAGE and zymogram analyses revealed two proteolytic active bands. Through isoelectricfocusing (IEF)-zymogram, an active band with alkaline pI and two slighter active bands with acid pI values were detected. The alkaline active enzyme in the IEF was purified and characterized. It showed a molecular mass of 29.4 kDa and its pI value was >‰10.3. Proteolytic activity of the culture supernatant showed an optimal temperature of approximately 60 °C and a plateau of maximum activity between pH 9.0 and 12.0. Such activity was not affected by H₂O₂ (10% v/v), 1,10-phenanthroline (10 mM), Triton X-100 (1% v/v) and Tween 20 (1% v/v), under the assay conditions. More than 80% of the activity was retained in 10 mM EDTA, 73% in 1 % (w/v) SDS and 63% in 2 M NaCl. The enzyme was inhibited by PMSF, indicating serine-protease activity. The proteolytic activity of the crude supernatant was thermosensitive with a half-life of 2.3 min at 70 °C, while high activity was detected at moderate temperatures. Considering PAT 05^T proteolytic activity characteristics, such as high optimum pH, high stability and residual activity in presence of oxidant, surfactant and chelating agents, this strain could be a potential source of enzymes for use as additives in detergent formulations or in the leather industry.     

Purification and Characterization of a Thermo- and Organic Solvent-Tolerant Alkaline Protease from Bacillus sp. JER02

Bacillus sp. JER02 is a bacterial strain that can be grown in a medium containing organic solvents and produce a protease enzyme. JER02 protease was purified with a yield of 31.9% of total protein and 328.83-fold purification. K _m and V_max of this protease were established as 0.826 µM and 7.18 µmol/min, respectively. JER02 protease stability was stimulated about 80% by cyclohexane. It exhibited optimum temperature activity at 70°C. Furthermore, this enzyme was active in a wide range of pH (4-12) and showed maximum activity at pH 9.0. The nonionic detergents Tween-20 and Triton X-100 improved the protease activity by 30 and 20%, respectively. In addition, this enzyme was shown to be very stable in the presence of strong anionic surfactants and oxidizing agents, since it retained 77%, 93%, and 98% of its initial activity, after 1 hr of incubation at room temperature with sodium dodecyl sulfate (SDS), sodium perborate (1%, v/v) and H₂O₂ (1%, v/v), respectively. Overall, the unique properties of the Bacillus sp. JER02 protease suggested that this thermo- and detergent-stable, solvent-tolerant protease has great potential for industrial applications.     

Effects of neem leaf extract on production of aflatoxins and activities of fatty acid synthetase,isocitrate dehydrogenase and glutathione S-transferase in Aspergillus parasiticus

The relationship between the activities of 3 cytosolic enzymes with aflatoxin biosynthesis in Aspergillus parasiticus cultured under different conditions has been investigated in order to find out the role of each enzyme in aflatoxin biosynthesis. Basically the activity of isocitrate dehydrogenase (IDH) was higher in non-toxigenic strains as compared to its counterpart toxigenic fungi (p < 0.05). In contrast, the activities of fatty acid synthase (FAS) as well as glutathione S-transferase (GST) were higher (P < 0.05) in toxigenic strains than that of the non-toxigenic fungi. Aflatoxin production was inhibited in fungi grown in presence of various concentrations of neem leaf extract. Aflatoxin was at its lowest level (>90% inhibition) when the concentration of neem extract was adjusted to 50% (v/v). No significant changes in FAS and IDH activities were observed when aflatoxin synthesis was under restraints by neem (Azadirachta indica) leaf extract. During a certain period of time of culture growth, when aflatoxin production reached to its maximum level, the activity of FAS was slightly induced in the toxigenic strains fed with a low concentration (1.56% v/v) of the neem leaf extract. At the time (96 h) when aflatoxin concentration reached to its maximum levels, the activity of GST in the toxigenic fungi was significantly higher (i.e., 7–11 folds) than that of non-toxigenic strains. The difference was highest in mycelial samples collected after 120 h. However unlike FAS and IDH, GST was readily inhibited (67%) in mycelia fed with 1.56% v/v of the neem extract. The inhibition reached to maximum of 80% in samples exposed to 6.25–12.5% of the extract. These results further substantiate previous finding that there is a positive correlation between GST activity and aflatoxin production in fungi.This revised version was published online in October 2005 with corrections to the Cover Date.