Selected arsenic species: As(III), As(V) and dimethylarsenic acid (DMAA) in Xerocomus badius fruiting bodies

The aim of the study was to determine the content of As(III), As(V) and DMAA (dimethylarsinic acid) in Xerocomus badius fruiting bodies collected from selected Polish forests from areas subjected to very low or high anthropopressure and some commercially available samples obtained from the Polish Sanitary Inspectorate. The arsenic species determination was provided by two independent HPLC–HG-AAS hyphenated systems. The results show high levels (up to 27.1, 40.5 and 88.3 mg kg⁻¹ for As(III), As(V) and DMAA, respectively) of arsenic and occurrence of different species in mushrooms collected from areas subjected to high anthropopressure and two commercially available samples. For mushroom samples collected from areas not subjected to high anthropopressure and two commercially available samples the arsenic species level was below 0.5 mg kg⁻¹ for each arsenic form. Therefore, the accumulation of arsenic by mushrooms may lead to high (toxic for humans) arsenic concentrations, and arsenic species levels should be monitored in mushroom foodstuffs.     

Comparative antioxidative characteristics of polysaccharide-enriched extracts from natural sclerotia and cultured mycelia in submerged fermentation of Inonotus obliquus

The potential antioxidant property of polysaccharide-enriched extracts from the natural fungal sclerotia and the cultured mycelia in submerged fermentation of Inonotus obliquus was evaluated using three antioxidant assays. The extracts from both the natural sclerotia and cultured mycelia including extra- and intra-cellular extracts were effective in scavenging hydroxyl radicals, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals, and in inhibiting lipid peroxidation. The content and type of polysaccharide present in the extracts from the natural sclerotia and the cultured mycelia were different. Both the extra- and intra-cellular extracts from the mycelia of I. obliquus with a higher polysaccharide content demonstrated a stronger free radical scavenger activity against hydroxyl radical and a greater inhibition of lipid peroxidation, shown as much lower IC50 values, than those from the natural sclerotia.     

Effect of nutritional and environmental conditions on the production of exo-polysaccharide of Agaricus brasiliensis by submerged fermentation and its antitumor activity

Mushrooms have become attractive as a functional food and as a source for the development of drugs and nutraceuticals. A. brasiliensis is considered as the best among them. There are several published works on the fruiting bodies of this mushroom, showing high antitumor activity. The mycelium polysaccharide and exo-polysaccharide (EPS) of this mushroom also demonstrated a strong antitumor action. However, there is little information available in the literature about the optimization of physical and chemical conditions for production of EPS by submerged fermentation (SmF). After initial screening from the five strains available in the LPB Culture Collection, A. brasiliensis LPB 03 was selected for EPS production in SmF. Studies on supplementation of a carbon source to the medium revealed that sucrose was most effective for EPS production. Yeast extract was the best for EPS among the inorganic and organic nitrogen sources tested. The factorial experiment demonstrated that a temperature of 30 °C and a pH of 6.1 were the best for the EPS production. Glucose 10 g/l, yeast extract 3 g/l, K₂HPO₄ 0.6 g/l and MgSO₄ 0.3 g/l were most suitable for the EPS production. Maximum EPS production was obtained in the static condition with manual agitation two times per day after a 1-week culture (382 mg/l), increasing by 78.62% more than that before optimization. In the bioreactor with aeration and agitation, the maximum EPS was produced at 321.2 mg/l. The EPS of A. brasiliensis produced in SmF showed strong inhibition against Sarcoma 180 in mice, reaching 72.19% inhibition compared with the control group. Furthermore, 50% of mice in the test group demonstrated total tumor regression.     

Extraction optimization of a hydrocolloid extract from cress seed (Lepidium sativum) using response surface methodology

Extraction conditions for maximum values of yield, viscosity and minimum protein content of hydrocolloid extract from Lepidium sativum seed were investigated using response surface methodology. A Central Composite Face Design (CCFD) with four independent variables: temperature (25–85 °C); pH (3–10); extraction time (10–25 min) and water to seed ratio (10:1–80:1) was used to study the response variables (yield, viscosity and protein content). Data analysis showed that all the variables significantly (p < 0.05) affected the extraction yield and viscosity, whereas the effect of water to seed ratio on protein content was not significant (p > 0.05). Applying a desirability function method the optimum parameters were: extraction temperature 35 °C, pH 10, water to seed ratio of 30:1 and an extraction time of 15 min. At this optimum point, apparent viscosity, yield and protein content were 0.2 Pa s, 6.46% and 0.57%, respectively. The experimental values were very close to the predicted values and were not statistically different (p > 0.05).     

Production and analysis of ochratoxin A produced by Aspergillus ochraceus ITEM 5137 in submerged culture

A rapid simple and economical method was described for the determination of ochratoxin A produced by Aspergillusochraceus ITEM 5117 grown in a biofermenter in submerged culture. The ochratoxin A was determinate with RP-HPLC-FLD (reverse phase high performance liquid chromatography) with direct injection in the HPLC apparatus using a C18 column. The mycotoxin was completely resolved by using the mixture acetonitrile, water and acetic acid (49:49:2 v/v) as the mobile phase with a flow rate of 1.0 mL/min. Mean recoveries of ochratoxin A ranged from 95.36% to 103.15%. The limit of detection for ochratoxin A in medium was found to be 1 μg L⁻¹.     

Optimisation of supercritical fluid extraction of flavonoids from Pueraria lobata

Supercritical carbon dioxide extraction was employed to extract flavonoids from Pueraria lobata. The optimal conditions for flavonoid extraction were determined by response surface methodology. Box–Behnken design was applied to evaluate the effects of three independent variables (pressure, temperature and co-solvent amount) on the flavonoid yield of P. lobata. Correlation analysis of the mathematical-regression model indicated that a quadratic polynomial model could be employed to optimise the supercritical carbon dioxide extraction of flavonoids. From response surface plots, pressure, temperature and co-solvent amount exhibited independent and interactive effects on the extraction of flavonoids. The optimal conditions to obtain the highest flavonoid yield of P. lobata were a pressure of 20.04 MPa, a temperature of 50.24 °C and a co-solvent amount of 181.24 ml. Under these optimal conditions, the experimental values agreed with the predicted values, using analysis of variance, indicating a high goodness of fit of the model used and the success of response surface methodology for optimising supercritical carbon dioxide extraction of flavonoids from P. lobata.     

Conidial production by Penicillium nalgiovense for use as starter cultures in dry fermented sausages by solid state fermentation

Penicillium nalgiovense is the typical species used as starter culture on the casing of dry fermented sausages and it has been often produced by solid state fermentation (SSF). Soy beans, maize kernels and wheat bran (WB) at 50% humidity were tested as substrates in SSF for conidial production and viability in P. nalgiovense. Among them WB was the best substrate for conidial production and viability. Thus, WB proved to be a suitable and convenient choice for conidial production by P. nalgiovense in SSF. By analysing conidial production on different membranes in malt extract agar (MEA) and WB no differences were observed between different treatments for the same substrate. Therefore, the practical choice of the inert support seems to be filter paper. A natural substrate like WB together with membranes as support helped the production of viable conidia by SSF as starters for dry fermented sausages.     

Optimization of nutrient-induced germination of Bacillus sporothermodurans spores using response surface methodology

Spores of Bacillus sporothermodurans are known to be contaminant of dairy products and to be extremely heat-resistant. The induction of endospore germination before a heat treatment could be an efficient method to inactivate these bacteria and ensure milk stability. In this study, the nutrient-induced germination of B. sporothermodurans LTIS27 spores was studied. Testing the effect of 23 nutrient elements to trigger an important germination rate of B. sporothermodurans spores, only d-glucose, l-alanine, and inosine were considered as strong independent germinants. Both inosine and l-alanine play major roles as co-germinants with several other amino acids. A central composite experimental design with three factors (l-alanine, d-glucose, and temperature) using response surface methodology was used to optimize the nutrient-induced germination. The optimal rate of nutrient-induced germination (100%) of B. sporothermodurans spores was obtained after incubation of spore for 60 min at 35 °C in presence of 9 and 60 mM of d-glucose and l-alanine, respectively. The results in this study can help to predict the effect of environmental factors and nutrients on spore germination, which will be beneficial for screening of B. sporothermodurans in milk after induction their germination. Moreover, the chosen method of optimization of the nutrient-induced germination was efficient in finding the optimum values of three factors.     

Modulation of the glycerol and ethanol syntheses in the yeast Saccharomyces kudriavzevii differs from that exhibited by Saccharomyces cerevisiae and their hybrid

In the last years there is an increasing demand to produce wines with higher glycerol levels and lower ethanol contents. The production of these compounds by yeasts is influenced by many environmental variables, and could be controlled by the choice of optimized cultivation conditions. The present work studies, in a wine model system, the effects of temperature, pH and sugar concentration on the glycerol and ethanol syntheses by yeasts Saccharomyces cerevisiae T73, the type strain of Saccharomyces kudriavzevii IFO 1802^T, and an interspecific hybrid between both species (W27), which was accomplished by the application of response surface methodology based in a central composite circumscribed design. Results show that carbon flux could be especially directed towards glycerol synthesis instead of ethanol at low pH, high sugar concentrations and low temperatures. In general, the non-wine yeast S. kudriavzevii produced higher glycerol levels and lower ethanol content than wine strains S. cerevisiae T73 and the hybrid W27, with specific and different glycerol production profiles as a function of temperature and pH. These results were congruent with the higher glycerol-3-phosphate dehydrogenase activities estimated for this species, chiefly at low temperatures (14 °C), which could explain why S. kudriavzevii is a cryotolerant yeast compared to S. cerevisiae.     

Optimisation of the solvent extraction of bioactive compounds from Parkia speciosa pod using response surface methodology

Central composite design was employed to optimise the buffer-to-solids ratio (X₁: 20–50 ml/g), incubation temperature (X₂: 35–55 °C) and time (X₃: 100–200 min), obtaining extracts from Parkia speciosa pod with high total phenolic and flavonoid contents and high antioxidant activities. Analysis of variance showed that the contribution of a quadratic model was significant for the responses. An optimisation study using response surface methodology was performed and 3D response surfaces were plotted from the mathematical models. The optimal conditions based on combination responses were: X₁ = 20 ml/g, X₂ = 35–36 °C and X₃ = 100–102 min. These optimum conditions yielded total phenolic contents of 664–668 mg gallic acid equivalents/100 g, total flavonoid contents of 47.4–49.6 mg pyrocatechol equivalents/100 g, %DPPH_sc of 81.2–82.1%, %ABTS_sc of 78.2–79.8% and FRAP values of 3.2–3.3 mM. Close agreement between experimental and predicted values was found. This methodology could be applied in the extraction of bioactive compounds in the natural product industry.     

Influencing cocoa flavour using Pichia kluyveri and Kluyveromyces marxianus in a defined mixed starter culture for cocoa fermentation

The potential impact of aromatic and pectinolytic yeasts on cocoa flavour was investigated using two defined mixed starter cultures encompassing strains of Pichia kluyveri and Kluyveromyces marxianus for inoculating cocoa beans in small scale tray fermentations. Samples for microbial and metabolite analysis were collected at 12–24 hour intervals during 120 h of fermentation. Yeast isolates were grouped by (GTG)₅-based rep-PCR fingerprinting and identified by sequencing of the D1/D2 region of the 26S rRNA gene and the actin gene. Pulsed Field Gel Electrophoresis (PFGE) was conducted on isolates belonging to the species P. kluyveri and K. marxianus to verify strain level identity with the inoculated strains. Furthermore, Denaturing Gradient Gel Electrophoresis (DGGE) was performed to follow yeast and bacterial dynamics over time including the presence of the bacterial inoculum consisting of Lactobacillus fermentum and Acetobacter pasteurianus. Yeast cell counts peaked after 12 h of fermentation with the predominant species being identified as Hanseniaspora opuntiae and Hanseniaspora thailandica. P. kluyveri and K. marxianus were found to compose 9.3% and 13.5% of the yeast population, respectively, after 12 h of fermentation whilst PFGE showed that ~ 88% of all P. kluyveri isolates and 100% of all K. marxianus isolates were identical to the inoculated strains. Despite never being the dominant yeast species at any stage of fermentation, the un-conched chocolates produced from the two inoculated fermentations were judged by sensory analysis to differ in flavour profile compared to the spontaneously fermented control. This could indicate that yeasts have a greater impact on the sensory qualities of cocoa than previously assumed.     

Optimisation of solid state fermentation of potato peel for the production of cellulolytic enzymes

This paper analyses the effects of water content, temperature and time on the kinetic activity of cellulolytic enzymes produced during the solid state fermentation of potato peel, using Aspergillus niger. Three main analytical steps – analysis of variance, regression analysis and plotting of response surface – were performed to obtain an optimum condition for enzymatic activity. The statistical results indicated that the best activity time for enzyme CMCase (carboxymethylcellulase) is 82.88 h, with water content of 51.48% and temperature of 29.46 °C; for FPase (filter paperase), the best activity time is 80.62 h, water content of 50.19% and temperature at 30.00 °C; for xylanase, time is 81.92 h, water content is 50.72% and temperature is 28.85 °C. Pareto charts have shown that all variables were significant in enzymatic activity for CMCase and xylanase. On the other hand, FPase shows that time and temperature have significant effect for this response variable.     

Inactivation of Serratia liquefaciens on dry-cured ham by high pressure processing

To quantify the inactivation of Serratia liquefaciens exerted by high pressure processing (HPP), slices of dry-cured ham were inoculated and processed combining different levels of technological parameters: pressure (347–852 MPa), time (2.3–15.8 min) and temperature (7.6–24.4 °C) according to a central composite design. Bacterial inactivation, as logarithmic reduction, indicated that S. liquefaciens was relatively sensitive to HPP. Six log reductions were achieved in a total of 10 trials combining pressures of 600 MPa or above with different holding times and temperatures. The inactivation of S. liquefaciens was analysed through the multiple regression analysis to generate a second order polynomial equation. Pressure and time were the two factors which significantly determined the inactivation of S. liquefaciens on dry-cured ham. Temperature did not significantly affect the lethality of the process. The response surface methodology was used to determine optimum process conditions to maximize the inactivation of S. liquefaciens in the experimental range tested. The maximum inactivation of S. liquefaciens in dry-cured ham was achieved by combining a pressure of 650 MPa with a holding time of 8 min. Combinations above these values (i.e. 750 MPa for 13 min) would not significantly improve the lethality of the process.     

Biochemical intervention of ergothioneine-rich edible mushroom (Flammulina velutipes) extract inhibits melanosis in crab (Chionoecetes japonicus)

We investigated the effects of decreasing phenoloxidase (PO) activity and prophenoloxidase (proPO) gene expression on the inhibition of postharvest melanosis formation in the red queen crab, Chionoecetes japonicus. The cDNA of proPO from hemocytes of C. japonicus was partially cloned and sequenced. Immersion of live crabs in a 1.0% ergothioneine (ESH)-rich mushroom extract (Flammulina velutipes; ME) solution resulted in significant inhibition of haemolymph PO activity and a reduction of the proPO gene expression in hemocytes that consequently controlled melanosis in the crabs during ice storage. Treatments with a 0.05% w/v sodium sulphite solution or a 0.05% w/v 4-hexyl-1,3-benzenediol solution had similar positive effects as the treatment with a 1.0% ME solution in vivo. In vitro experiments showed that authentic l-(+)-ESH inhibited PO activity and decreased proPO gene expression in crab hemocytes. Thus, the application of ESH-rich ME can be a novel alternative to synthetic melanosis-inhibiting agents to control postharvest melanosis in crabs.     

Fruit wine produced from cagaita (Eugenia dysenterica DC) by both free and immobilised yeast cell fermentation

The aims of this work were to elaborate a fruit wine from cagaita (Eugenia dysenterica DC) pulp and to compare the fermentations conducted with free and with Ca-alginate immobilised cells. Two strains of Saccharomyces cerevisiae (UFLA CA11 and CAT-1) were tested and four fermentation batches were performed, in triplicate, at 22 °C for 336 h: UFLA CA11 in free and immobilised cells and CAT-1in free and immobilised cells. Fermentation time and ethanol production were influenced by the yeast strain and by the cell status, with immobilised cells of UFLA CA11 and CAT-1 fermenting faster (4 days and 8 days, respectively) than UFLA CA11 and CAT-1 free cells (10 days and 12 days, respectively). Ethanol content (g/L) was slightly higher when the fermentation was conducted with free cells (94.63 and 94.94 for UFLA CA11 and CAT-1, respectively) than with immobilised cells (86.82 and 87.21 for UFLA CA11 and CAT-1, respectively). The beverage from CAT-1 free cells showed the highest concentration of higher alcohols (82,086.12 ??/L), whereas the lowest concentration (37,812.17 ??/L) was found in the beverage from immobilised UFLA CA11. The ethyl ester concentration ranged from 1511.42 ??/L (CAT-1 free cells) to 2836.34 ??/L (UFLA CA11 free cells). According to the sensory evaluation, the fruit wine acceptability was greater than 70% for colour, flavour and taste for all cagaita beverages.     

Experimental design and response surface modeling applied for the optimisation of pectin hydrolysis by enzymes from A. niger CECT 2088

Optimisation of pectin hydrolysis using pectolytic enzymes produced by Aspergillus niger CECT 2088 was carried out. The effect of enzyme concentration (0.018–0.072%, w/v), substrate concentration (0.004–0.896%, w/v), pH (4.49–5.91), temperature (32.2–67.8 °C) and reaction time (18.2–71.8 min) on the enzymatic process was studied. The experiments were arranged according to a central composite statistical design. Response surface methodology (RSM) was used to assess factor interactions and empirical models regarding four product response variables (i.e., reactor conversion, reducing sugar concentration, endopectolytic productivity and enzymatic depolymerization productivity). Multi-response optimisation was also performed on the reactor and endopectolytic productivity data set of the factorial design. The highest reactor conversion (61%) and endopectolytic productivity (7.2 cSt mg⁻¹ ml⁻¹) were achieved with 1 h reaction time at 46 °C and pH 4.8 at a substrate/enzyme ratio of 11.6.     

Response surface optimisation of aqueous enzymatic oil extraction from bayberry (Myrica rubra) kernels

Aqueous enzymatic extraction (AEE) of oil from Myrica rubra kernels was performed. The four AEE parameters namely mixed enzyme (cellulose/neutral protease = 1/2, w/w) amount, liquid/solid ratio, extraction time, and temperature, were optimised by response surface methodology. The statistical analysis indicated that the enzyme amount, liquid/solid ratio, time, and the quadratics of liquid/solid ratio, and enzyme amount, as well as the interactions between time and temperature, showed significant effects on oil yield. The optimal extraction conditions for oil yield were mixed enzyme amount, liquid/solid ratio, time, temperature as 3.17% (w/w), 4.91 ml/g, 4 h and 51.6 °C. Under the optimum conditions, the experimental oil extraction yield was 31.15%. The GC–MS analysis showed that the oil was abundant in the unsaturated fatty acids (9-octadecenoic acid and 9,12-octadecadienoic acid accounting for more than 80%), and the AEE was more efficient method to extract polyunsaturated fatty acid than the organic solvent process.     

Mycobial enhancement of ergothioneine by submerged cultivation of edible mushroom mycelia and its application as an antioxidative compound

Ergothioneine (2-mercaptohistidine trimethylbetaine, ESH) in mushroom is one of bioactive and functional components. In the present study, a method of producing ESH within a short period and with high productivity was established by applying submerged fermentation of edible Shiitake mushroom (Lentinula edodes) mycelia. High-resolution mass spectrometry and online flow injection analysis of 2,2-diphenyl-1-picrylhydrazyl scavenging activity clearly confirmed that ESH was the most potent antioxidant in the mycelial extract. Among several mushroom mycelia, Shiitake produced the highest amount of ESH (0.60 mg/g dry weight (DW)). Optimisation of the culture medium was performed to achieve higher ESH production. The monosaccharide content in the medium, particularly fructose in combination with aspartic acid, was one of the factors that enhanced ESH production by 3.15-fold (1.89 mg/g DW). Individual supplementation of 2 mM methionine as an amino acid precursor yielded a significantly higher amount of ESH (3.45 mg/g DW) compared to that of the control medium (1.85 mg/g DW). The mycelial extracts containing ESH were effective in retarding the formation of secondary lipid oxidation products in the yellowtail red meat model during chilled storage as well as in the oxidation of linoleate and bleaching of beta-carotene in an oil-in-water emulsion system.