Application of Catalytic Hydrogen Evolution in the Presence of Neonicotinoid Insecticide Clothianidin

Clothianidin, a new generation of pesticide, was determined in spiked tap water, apple juice, and soil by square-wave adsorptive stripping voltammetry. The method of determination is based on the hydrogen evolution reaction catalyzed by clothianidin at the hanging mercury drop electrode. The optimal signal was detected at −1.4 V versus Ag/AgCl in citrate buffer at pH 2.2. Various parameters such as pH, buffer concentration, frequency, amplitude, step potential, accumulation time, and potential were investigated to enhance the sensitivity of the determination. The optimal results were recorded at an accumulation potential of −0.35 V, accumulation time of 7 s, amplitude of 100 mV, frequency of 200 Hz, and step potential of 7 mV. The mechanism of catalytic hydrogen evolution was considered under experimental and theoretical conditions. This electroanalytical procedure enabled to determine clothianidin in the concentration range 9 × 10⁻⁹–4 × 10⁻⁶ mol L⁻¹ in supporting electrolyte and tap water, 1 × 10⁻⁷–4 × 10⁻⁶ mol L⁻¹ in diluted apple juice, and 2 × 10⁻⁷–1 × 10⁻⁶ mol L⁻¹ in soil. The detection and quantification limits in supporting electrolyte and diluted apple juice were found to be 2.6 × 10⁻⁹, 8.6 × 10⁻⁹ and 3 × 10⁻⁸, and 1 × 10⁻⁷ mol L⁻¹, respectively. A standard addition method was successfully used to determine clothianidin in spiked tap water, spiked apple juice, and spiked soil.     

Co-production of Lactic Acid and <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis> Cells from Whey Permeate with Nutrient Supplements

Lactic acid and cell production from whey permeate by Lactobacillus rhamnosus with different nutrient supplements were investigated in this study. Yeast extract was identified as the most effective nutrient affecting lactic acid production. Increase in inoculum size from 0.05% to 1% (v/v) resulted in a substantial increase in lactic acid productivity from 0.66 to 0.83 g L⁻¹ h⁻¹ (P < 0.001). The optimal temperature for lactic acid production was 37 °C, while the highest cell production was obtained at 42 °C. When whey permeate and yeast extract concentrations were 6.8% (w/v) and 3 g L⁻¹, respectively, lactic acid productivity reached 0.85 g L⁻¹ h⁻¹ after 48-h cultivation, which is 3.40 times of those without nutrient supplements.     

Optimization of Solid-Phase Microextraction Procedure Coupled to GC-ECD for Triazole Fungicides Determination in Juice Samples

A solid-phase microextraction (SPME) procedure followed by gas chromatography electron capture detection (GC/ECD) for the determination of triazole residues was developed. An experimental design with two steps was done. Firstly, a 2⁶⁻² fractional factorial design for screening several experimental variables (fiber-coating type, extraction temperature, extraction time, stirring rate, desorption temperature, and desorption time) was done. After, a two-factor central composite design for optimizing, the experimental conditions were carried out. The chosen experimental conditions were: fiber, PDMS/DVB; extraction time, 45 min; extraction temperature, 60 °C; desorption time, 3 min; desorption temperature, 260 °C, and stirring speed, 500 rpm. Using those conditions the limits of detection obtained for tetraconazole, myclobutanil, and diniconazole were in the order of few μg L⁻¹ in grape and apple liquid extracts. Recoveries were from 93.6% to 112.1%. Relative standard deviation ranged from 1.2% to 11.6% (apple) and 6.7 to 18.0% (grape). The method was applied to five grape samples and 13 apple samples collected in Navarra, Rioja, and Basque Country. Quantification was performed by the standard addition method. Three standard additions by duplicate covering adequate range concentration were used. Myclobutanil was found in three apple samples (110–122 μg L⁻¹) and diniconazole in one grape sample (9.4 μg L⁻¹).     

Physicochemical and microbiological description of Caxiri – a cassava and corn alcoholic beverage

Caxiri is a fermented alcoholic beverage made from cassava, corn and sweet potatoes by indigenous people in Brazil. Saccharomyces cerevisiae Rhodotorula mucilaginosa, Lactobacillus fermentum, Bacillus subtilis and L. helveticus were the main microbial species detected. Maltose was the main carbohydrate found (19.12 g L^?1), and lactic acid (15.09 g L^?1) and ethanol (92.16 g L^?1) were also found in high concentrations. Gas chromatography‐flame ionisation detector was used to identify thirteen volatile compounds. Among these volatiles, the higher concentrations were decanoic acid (123.04 μg L^?1) for the acids, diethyl malate (88.32 μg L^?1) for the esters, furfural (109.31 μg L^?1) for the aldehydes, 2‐phenylethanol (1022.76 μg L^?1) for the alcohols and 1,1‐diethoxyethane (226.24 μg L^?1) for the others. This study contributes to increasing knowledge of the microbiota present in the alcoholic fermentation produced from cassava, corn and sweet potatoes.     

Micelle-Mediated Extraction and Flame Atomic Absorption Spectrometric Method for Determination of Trace Cobalt Ions in Beverage Samples

A new method has been developed for preconcentration of cobalt at trace levels in beverage samples using calcon carboxylic acid as chelating agent and cetyl pyridinium chloride as an auxiliary ligand and entrapped into Triton X-114 prior to its determination by flame atomic absorption spectrometry (FAAS). The main parameters affecting cloud point extraction (CPE) efficiency such as pH, concentration of the complexing agent, cationic and nonionic surfactant concentration, salt effect, the equilibrium time, and temperature were investigated and optimized. After optimization of the CPE conditions, a preconcentration factor of 60, an enhancement factor of 106, and a detection limit of 0.20 μg L⁻¹ by (R ² = 0.9978) were obtained from a calibration curve constructed in the range of 0.7–100 μg L⁻¹. The proposed preconcentration procedure was successfully applied to the determination of cobalt ions in some real samples including natural drinking water, tap water, and beer and wine samples. The accuracy and validity of the proposed CPE/FAAS method was tested by means of five repeated analysis of reference standard materials (TM-253, a low level fortified water standard for trace elements). A good agreement between analytical results (28.8 and 28.5 μg L⁻¹ with calibration curve and standard addition curve method, respectively) and certified value (27.9 μg L⁻¹) for Co (p < 0.05) were obtained and verified by means of calibration curve and standard addition curve method using CPE procedure.     

Determination of Melamine in Liquid Milk and Milk Powder by Titania-Based Ligand-Exchange Hydrophilic Interaction Liquid Chromatography

A simple and rapid high-performance liquid chromatography (HPLC) procedure for the analysis of melamine in liquid milk and milk powder has been developed. Decrease of acetonitrile percentage and phosphate buffer concentration in mobile phase, and lowering of buffer pH and column temperature would benefit the retention of melamine on titania. Taking advantage of the ligand-exchange and hydrophilic interaction mixed retention mode on bare titania column, neither complex pretreatment nor ion-pair reagent was required. The whole analysis for one sample including sample pretreatment and HPLC analysis could be accomplished within 30 min. The method presented good linearity (R ² = 0.9998) in a wide range of 0.02–10 μg mL⁻¹. The limit of detection (3σ) and limit of quantification (10σ) of the method were 6 and 20 μg L⁻¹, respectively, which were equivalent to 15 and 50 μg kg⁻¹ melamine in liquid milk, 60 and 200 μg kg⁻¹ melamine in milk powder, respectively. Such sensitivity could be compared with those obtained by HPLC with solid-phase extraction or HPLC coupled with tandem mass spectrometry and was adequate for the screening of melamine in tainted dairy products. The repeatability (RSDs) of the retention times and peak areas of 11 replicate detections of 1.0 μg mL⁻¹ melamine were 0.32% and 2.5%, respectively. The intermediate precision on three consecutive days (RSDs, n = 6) of the retention times and peak areas were 1.1% and 2.3%, respectively. The recovery of spiked melamine in dairy samples ranged from 95.2% to 105%. The simplicity, sensitivity and rapidity of the proposed method make it an effective alternative detecting technique for melamine.     

Total Mercury, Inorganic Mercury and Methyl Mercury Determination in Red Wine

This study deals with the development of a method for total Hg, inorganic Hg and methylmercury (MeHg) determination in red wine by using flow injection-cold vapour generation–inductively coupled plasma mass spectrometry (FI-CVG-ICP-MS) and gas chromatography-ICP-MS (GC-ICP-MS). For Hg speciation analysis, a derivatization step was carried out using a 1% (m/v) sodium tetraphenylborate (NaBPh₄) solution, followed by extraction of Hg species and their quantification by GC-ICP-MS. The main parameters evaluated were the make-up gas flow rate, volume of the NaBPh₄ solution, time for derivatization reaction/analyte extraction and solvent used for Hg species extraction. Accuracy was evaluated by analyte recovery, whereas recoveries ranged from 99% to 104% for Hg(II) and MeHg. The limits of detection (LODs) for Hg(II) and MeHg were 0.77 and 0.80 μg L⁻¹, respectively. Wine from Argentina, Brazil, Chile and Uruguay were analysed. The wine samples were also acid digested for total Hg determination by FI-CVG-ICP-MS. The LOD of the method used for total Hg determination was 0.01 μg L⁻¹. The concentrations of Hg species in red wine measured by GC-ICP-MS were lower than the respective LODs. Only total Hg was detected in the analysed samples, where the highest concentration of Hg found was 0.55 ± 0.02 μg L⁻¹.     

Quantification of Nitrite/Nitrate in Food Stuff Samples Using 2-Aminobenzoic Acid as a New Amine in Diazocoupling Reaction

2-Aminobenzoic acid has been used as an amine in diazocoupling reaction to form an azo dye in the quantification of nitrite/nitrate at trace level. The formed azo dye has an absorption maximum at 550 nm in aqueous phase, and the resulted dye can be extracted into organic solvent to lower the detection limit. The method obeys Beer's law in the concentration range 0–10 μg of nitrite in 25 ml of aqueous solution with a molar absorptivity of 3.6 × 10³ L mol⁻¹ cm⁻¹ and 0–2 μg of nitrite in 5 ml of organic phase. The detection limit of the dye has been found to be 0.056 μg ml⁻¹. Nitrate is determined by reducing it to nitrite after passing through a copperized cadmium reductor column. The effect of interfering ions on the determination of nitrite/nitrate has been described. The developed method has been applied to determine the nitrite/nitrate trace level in vegetable, fruit juice, and milk powder samples.     

Quality of mashed potatoes: effect of adding blends of kappa-carrageenan and xanthan gum

Blends of kappa-carrageenan (K) and xanthan gum (X) were added to mashed potatoes. Product was tested by instrumental texture profile analysis and cone penetration tests, oscillatory and steady rheology, colour, drip loss, total soluble solids and sensory analyses. A central composite rotatable design was used to study the effects of variation in levels of K (1.5–4.5 g kg⁻¹) and X (0.5–2.5 g kg⁻¹) concentrations. Addition of K had a major impact on gel strength, viscoelastic behaviour, sensory attributes and overall acceptability, whereas addition of X influenced textural and steady properties and colour. Mainly, elastic modulus (G′) was strongly dependent on the K concentration in mashed potatoes containing amylose. As compared to mashed potatoes with 1.5 g kg⁻¹ added X, additional incorporation of 5.12 g kg⁻¹ K increased G′ approximately twofold, possibly due to exclusion effect of the swollen starch granules and synergistic effect of K and denatured protein. The function of X in the mashed potatoes may in fact be confined to that of a filler rather than a dynamic constituent, although it does affect yield stress behaviour. When K/X blends (each biopolymer at 1.5 g kg⁻¹) were included in the formulation, the product exhibited very acceptable sensory quality. K provided the appropriate texture, while X imparted creaminess and mouthfeel to the product.     

Use of Metals and Anion Species with Chemometrics Tools for Classification of Unprocessed and Processed Coconut Waters

Coconut water is a natural isotonic, nutritive, and low-caloric drink. Preservation process is necessary to increase its shelf life outside the fruit and to improve commercialization. However, the influence of the conservation processes, antioxidant addition, maturation time, and soil where coconut is cultivated on the chemical composition of coconut water has had few arguments and studies. For these reasons, an evaluation of coconut waters (unprocessed and processed) was carried out using Ca, Cu, Fe, K, Mg, Mn, Na, Zn, chloride, sulfate, phosphate, malate, and ascorbate concentrations and chemometric tools. The quantitative determinations were performed by electrothermal atomic absorption spectrometry, inductively coupled plasma optical emission spectrometry, and capillary electrophoresis. The results showed that Ca, K, and Zn concentrations did not present significant alterations between the samples. The ranges of Cu, Fe, Mg, Mn, PO₄³⁻, and SO₄²⁻ concentrations were as follows: Cu (3.1–120 μg L⁻¹), Fe (60–330 μg L⁻¹), Mg (48–123 mg L⁻¹), Mn (0.4–4.0 mg L⁻¹), PO₄³⁻ (55–212 mg L⁻¹), and SO₄²⁻ (19–136 mg L⁻¹). The principal component analysis (PCA) and hierarchical cluster analysis (HCA) were applied to differentiate unprocessed and processed samples. Multivariated analysis (PCA and HCA) were compared through one-way analysis of variance with Tukey–Kramer multiple comparisons test, and p values less than 0.05 were considered to be significant.     

Recovery of Native Potato Protein Comparing Expanded Bed Adsorption and Ultrafiltration

Obtaining native protein from potato fruit water (PFW) acceptable for food consumption was attempted by comparing expanded bed adsorption (EBA) and ultrafiltration (UF).The methods were assessed on their process performance and the product quality. Extractable tuber proteins were recovered from lab-prepared PFW either by adsorption to an EBA column using a mixed mode resin (0.31 L) or by batch concentration in an UF (10 kDa MWCO, 0.093 m²) unit and then freeze dried. The yields on protein and esterase activity were higher (p < 0.05 and p < 0.01; Mann–Whitney U-test) in UF (3.2 g l⁻¹ PFW and 3.17 kU l⁻¹ PFW) than in EBA (1.8 and 1.21). The performance difference was also reflected in process productivity for esterase activity which was fivefold better (p < 0.01) in UF (4.30 kU h⁻¹) than with EBA (0.84) due to the higher enzyme retention; protein productivities were the same. The content of solanidine glycoalkaloids was depleted to moderate levels but came out unaffected by the processing method: EBA 286 ppm, UF 213 ppm. The low levels of chlorogenic acid in all EBA preparations were negatively correlated to high brightness score (L* = 73.8%), a favorable attribute in food-quality proteins. Both methodologies produced native preparations of comparable protein content (75%). EBA processing, however, increased the fraction of the patatin protein which may offer advantages in food applications.     

Simultaneous and Direct Determination of As, Bi, Pb, Sb, and Se and Co, Cr, Cu, Fe, and Mn in Milk by Electrothermal Atomic Absorption Spectrometry

Tungsten permanent modifier with coinjection of Pd(NO₃)₂ and W–Ru permanent modifiers are proposed for the direct and simultaneous determination of As, Bi, Pb, Sb, and Se (group 1) and Co, Cr, Cu, Fe, and Mn (group 2), respectively, in milk by graphite furnace atomic absorption spectrometry. The performance of modifiers was evaluated by means of thermal behavior of analytes, sensitivity, atomic signal profile, repeatability, graphite tube lifetime, and background intensity. An air-assisted pyrolysis step was necessary to quantitative elimination of the organic matter. After methods optimization, 14 commercial milk samples were analyzed. The found concentrations of As, Bi, Pb, Sb, Se, Co, and Cr were lower than their limit of detection (2.13, 2.21, 1.49, 1.63, 2.05, 1.0, and 1.2 μg L⁻¹, respectively). Concentrations of Cu, Fe, and Mn were in the 1.58–5.74 μg L⁻¹, 9.79–49.3 μg L⁻¹, and 2.25–4.08 μg L⁻¹ intervals, respectively. The limits of detection for Cu, Fe, and Mn were 1.7, 5.3, and 2.0 μg L⁻¹, respectively. The accuracy of methods was checked after analysis of two milk standard materials. Results for Cr, Cu, Fe, Mn, Pb, and Mn were in agreement with certified values of SRMs at the 95% confidence level. Accuracy was also evaluated by addition–recovery tests and recoveries in the 86–127% range were obtained for all elements. The use of pretreat platform of graphite tubes with W or W–Ru allowed enlarging the lifetime of atomizer in 750 heating cycles.     

Evaluation of potential side activities of commercial enzyme preparations used in winemaking

Twenty‐one commercial enzyme preparations used in winemaking were characterised for the α‐L‐rhamnosidase, α‐L‐arabinosidase, β‐D‐xylosidase, ?β‐D‐galactosidase, β‐D‐glucosidase, esterase, protease, cinnamoyl esterase and laccase activities. A new rapid fluorimetric method to assay esterase activity was developed. Enzyme preparations differed for the level of each enzyme activity assayed rather than for the type of enzymatic activity detected. High levels of protease, glycosidase, esterase and cinnamoyl esterase activity were found among enzyme preparations for different technological applications. A drastic reduction in the level of cinnamoyl esterase was observed in commercial grape juice, pH 3.6, total acidity 5.3 g L^?1_, sugar 170 g L^?1. Protease activity was only weakly reduced, from 10 to 20%, in commercial grape juice. β‐glucosidase activity levels are reduced in the presence of increasing concentration of glucose but are still present at the higher glucose concentration (100 g L^?1). The extent of the reduction observed was dependent on the enzymatic preparations tested.     

Isolation and characterization of protein fractions from deoiled rice bran

Rice bran contains underutilised protein materials. Sequential extraction of rice bran protein (RBP) from defatted rice bran was conducted based on the differences in their solubility. Three extraction methods were investigated. Method 1 involved the isoelectric and acetone precipitation using water, 50 g kg⁻¹ NaCl, 0.02 mol L⁻¹ NaOH and 70% ethanol as extracting solvents for albumin (pH 4.1), globulin (pH 4.3), glutelin (pH 4.8) and prolamin, respectively. Method 2 adopted dialysis and sequential extraction was carried out with 20 g kg⁻¹ NaCl, 70% ethanol, 0.1 mol L⁻¹ acetic acid and 0.1 mol L⁻¹ NaOH solution as extracting solvents. Method 3 combined dialysis, isoelectric and acetone precipitation for the extraction. Based on the yields and data obtained from sodium dodecyl sulphate polyacrylamide gel electrophoresis, size-exclusion chromatography and differential scanning calorimetry, method 3 was chosen for the isolation and characterization of RBPs. Rice bran protein fraction (RBPF)—albumin, globulin, glutelin and prolamin were obtained in good yields. Denaturation temperature and enthalpy values of denaturation of RBPF vary. Highest phytate content was found in albumin and lowest in prolamin. The highest antioxidative and hemagglutinating activities were observed in albumin.     

Influence of processing steps and fruit maturity on volatile concentrations in juices from clementine, mandarin, and their hybrids

During growth, ripening, and post-harvest, the quality of fruits can be modified by various stresses. Ample maturation allows prolonged storage and increases the quality of citrus fruits. The eight major volatile compounds in fresh juices from mandarin (Citrus reticulata Blanco var. Willow Leaf), clementine (Citrus reticulata × Citrus sinensis var. Commune), and their six cross-pollinated hybrids were quantified by gas chromatography. Mandarin juice contained the highest volatile concentration (2177.6 ± 110.6 mg L⁻¹), whereas the total aromatic compounds of the hybrid and clementine juices were lower (1060.6 ± 81.6–1724.2 ± 138.5 mg L⁻¹ and 864.8 ± 67.8 mg L⁻¹, respectively). Limonene was the major compound (667.3 ± 53.7–1259.9 ± 98.4 mg L⁻¹) in all juices. We observed significant differences according to the process of juice extraction used (with and without peel). The relative volatile concentration was higher when the juice was extracted with peel, and the juicing process induced differences in the relative volatile concentrations of between 20 and 40%. The levels of volatile compounds in the juice changed according to the degree of fruit maturity. The concentrations of the eight major juice components increased during ripening and until the commercial maturity stage when the juice was obtained from peeled fruits, whereas their concentrations increased until the post-maturation stage when the juices were produced from whole fruits. These two factors determined the contributions of these compounds to the characteristic aromas of Citrus juices.     

Determination of Histamine in Some Foods by Isotachophoretic Method with Simple Sample Preparation

A one-dimensional capillary isotachophoretic method in cationic system of the separation has been applied for histamine determination in food samples. The proposed electrolyte system consisted of 0.01 M potassium hydroxide with l-valine to pH = 9.9 as the leading electrolyte and 0.02 M 2-amino-2-hydroxymethyl-propane-1,3-diol adjusted to pH = 8.3 with 0.1 M hydrochloric acid as terminating electrolyte. Proposed method was characterized by linearity range 5–50 mg L⁻¹ and R ² = 0.9982, accuracy (recoveries ranged from 95% to 102%), detection (2.10 mg L⁻¹), and quantification (7.01 mg L⁻¹) limits. The sample preparation for proposed electrophoretic method included only simple extraction with trichloroacetic acid with filtration and derivatisation stage are avoided. The histamine concentration was determined in meat (turkey, chicken, beef and pork) and meat products (ripened sausage and dry-cured ham), fish (smoked salmon and mackerel), and different kind of mildew and mold ripened cheeses samples. The histamine content ranged from not detected level for fresh meat to 29.63 mg 100 g⁻¹ for cheese samples. The reversed phase HPLC was applied as reference method and the F-Snedecor test and the t test were employed to compare the precision and accuracy of the both methods. Positive correlations were found between the two analytical methods for histamine determination in food products. The obtained results indicate that the proposed electrophoretic method is simple, precise, accurate, and convenient.     

Speciation of Organic and Inorganic Selenium in Selenium-enriched Eggs by Hydride Generation Atomic Fluorescence Spectrometry

A new method was established for the speciation of Se by hydride generation-atomic fluorescence spectrometry. Effective separation of organic and inorganic Se in Se-enriched eggs was achieved by precipitating albumen with trichloroacetic acid. The detection limit for Se was 0.07 μg L⁻¹. The proposed method was successfully applied for the determination of organic and inorganic Se as well as total Se in Se-enriched eggs with the recovery of 90.1–112% and the relative standard deviation (n = 5) of 0.6–3.4%. Se in Se-enriched eggs presents mainly in organic forms, the content of which was three to seven times that of inorganic Se.     

Optimization of Process Parameters for Foaming of Bael (Aegle marmelos L.) Fruit Pulp

Foam expansion and foam stability of the bael (Aegle marmelos L.) fruit pulp foam was studied. Foams were prepared from various pulp concentrations (PC) by adding different concentration of glycerol monostearate (GMS) and methyl cellulose (MC) at different whipping time (WT). Response surface methodology was used to predict the foam stability and expansion. Thirty experiments were carried out using a central composite rotatable design with four independent variables (GMS, MC, PC, and WT). Second-order polynomial equations were developed after removing insignificant terms for predicting foam expansion (R ² = 0.85) and stability (R ² = 0.95). The optimum conditions achieved after the numerical and graphical optimization for maximum foam expansion and stability was: GMS (3.10 g/100 g pulp), MC (0.32 g/100 g pulp), PC (13.2°Bx), and WT (2 min). The desirability of 0.712 was achieved at this optimum point. The predicted values of foam density and foam drainage volume were 0.658 g cm⁻³ and 1.75 mL, respectively, at optimum parameters while the experimental values were 0.635 ± 0.02 g cm⁻³ and 1.75 ± 0.12 mL, respectively.     

Determination of Nitrite and Nitrate in Brazilian Meats Using High Shear Homogenization

Nitrate and nitrite are usually added to processed meat products to protect against the growth of microorganisms. Two sample preparation methodologies using either manual grinding (with a mortar and pestle) or mechanical high shear homogenization were investigated and compared. The results showed that high shear homogenization was the most suitable for the extraction of nitrite and nitrate from ham, salami, and bacon samples, achieving high extraction recoveries (>98%) together with low relative standard deviations (RSDs) for the samples analyzed. Analyses were performed using capillary electrophoresis. A running buffer consisting of 60 mmol L⁻¹ tetraborate and 0.2 mmol L⁻¹ cetyltrimethylammonium bromide enabled separation of the analytes in <5 min. In validation experiments, good repeatability was obtained for both migration times (<0.8% RSD) and peak areas (<1.1% RSD). Analytical curves for nitrite and nitrate were linear (r > 0.998) in the 0.2- to 2.5-mg L⁻¹ and 0.5- to 5-mg L⁻¹ concentration ranges, respectively. The limits of detection were 0.15 mg L⁻¹ for nitrite and 0.17 mg L⁻¹ for nitrate. The method developed was applied to the analysis of different kinds of meats (sausage, ham, salami, bacon, and others) produced in Brazil. The ranges of concentration found were 17.3–46.4 mg kg⁻¹ (nitrite) and 69.9–198.1 mg kg⁻¹ (nitrate). The contents of nitrate and nitrite in the samples were below the Brazilian legislation limit values (150 and 300 mg kg⁻¹ for nitrite and nitrate, respectively).     

Optimization of the culture medium composition using response surface methodology for new recombinant cyprosin B production in bioreactor for cheese production

The optimization of culture medium composition was carried out for improvement the recombinant cyprosin B production, an enzyme with high milk-clotting activity. Response surface methodology (RSM) was applied to evaluate the effect of variables namely glucose, yeast extract (YE) and bactopeptone present in the culture medium, used for recombinant cyprosin B production by transformed Saccharomyces cerevisiae BJ1991 strain in shake-flask and bioreactor culture conditions. The central composite experimental design (CCD) was adopted to derive a statistical model for optimizing the composition of the fermentation medium. The optimal concentration estimated for each variable related to a theoretical maximum of cyprosin B activity (488 U mL⁻¹) was 30 g L⁻¹ glucose, 15 g L⁻¹ YE and 27 g L⁻¹ bactopeptone. The optimized medium composition, based on empirical model, led to a cyprosin B activity of 519 U mL⁻¹, which corresponds to an increase of 46%. The fermentation using optimized culture medium in a 5-L bioreactor allowed a significant increase in biomass (82%) and recombinant cyprosin B production (139%). The improvement in the recombinant cyprosin B production after optimization process can be considered adequate for large-scale applications, and the clotting activity of cyprosin B account for their use in industrial cheese making.