Inulinase Production by Agro-Industrial Residues: Optimization of Pretreatment of Substrates and Production Medium

In this work, production of inulinase was studied. Media formulation was optimized by experimental design and response surface techniques, as well as the pretreatment of the agro-industry residues used in the formulation of fermentation medium. Two agro-industry residues were investigated: sugarcane molasses (SCM) and corn steep liquor (CSL). Pretreatment with sulfuric acid was the most effective for clarification of SCM (pH 5.0, 24 h of resting time and final pH 4.0). Clarification of CSL was accomplished with phosphoric acid (pH 3.0, 24 h of resting time and final pH 5.5). A color reduction of approximately 70% was achieved for both substrates. The highest production of inulinase was obtained in a medium containing 100 g l⁻¹ of pretreated SCM, 100 g l⁻¹ of pretreated CSL and 6 g l⁻¹ of Prodex Lac (yeast hydrolysate), yielding 1,139 U ml⁻¹.     

Optimization and Modeling of Process Parameters for Lipase Production by Bacillus brevis

Statistical evaluation of fermentation conditions and nutritional factors by Plackett–Burman two-level factorial design followed by optimization of significant parameters using response surface methodology for lipase production by Bacillus brevis was performed in submerged batch fermentation. Temperature, glucose, and olive oil were found to be the significant factors affecting lipase production. Maximum lipase activity of 5.1 U ml⁻¹ and cell mass of 1.82 g l⁻¹ at 32 h were obtained at the optimized conditions of temperature, 33.7 °C; initial pH, 8; and speed of agitation, 100 rpm, with the medium components: olive oil, 13.73 ml l⁻¹; glucose, 13.98 g l⁻¹; peptone, 2 g l⁻¹; Tween 80, 5 ml l⁻¹; NaCl, 5 g l⁻¹; CH₃COONa, 5 g l⁻¹; KCl, 2 g l⁻¹; CaCl₂·2H₂O, 1 g l⁻¹; MnSO₄·H₂O, 0.5 g l⁻¹; FeSO₄·7H₂O, 0.1 g l⁻¹; and MgSO₄·7H₂O, 0.01 g l⁻¹. The lipase productivity and specific lipase activity were found to be 0.106 U (ml h)⁻¹ and 2.55 U mg⁻¹, respectively. Unstructured kinetic models and artificial neural network models were used to describe the lipase fermentation. The kinetic analysis of the lipase fermentation by B. brevis shows that lipase is a growth-associated product.     

Evaluation of Protease-producing Ability of Fish Gut Isolate Bacillus cereus for Aqua Feed

Extracellular protease production by Bacillus cereus isolated from the intestine of fish Mugil cephalus has been investigated in shake-flask experiment using different preparations of tuna-processing waste such as raw fish meat, defatted fish meat, alkali hydrolysate, and acid hydrolysate as nitrogen source. Among the tuna preparations tested, defatted fish meat supported the maximum protease production (134.57 ± 0.47 U ml⁻¹), and 3% concentration of the same was found to be optimum for maximizing the protease production (178.50 ± 0.28 U ml⁻¹). Effect of carbon sources on protease production in the optimized concentration of defatted tuna fish meat revealed that galactose aided the higher protease production (259.83 ± 0.04 U ml^–1) than the other tested carbon sources and a concentration of 1.5% galactose registered as optimum to enhance the protease production (289.40 ± 0.16 U ml⁻¹). The halotolerancy of B. cereus for protease production indicated that 3% of sodium chloride was optimum to yield maximum protease (301.63 ± 0.20 U ml⁻¹). Among the surfactants tested, protease production was high in Triton X 100-added medium (298.63 ± 0.12 U ml⁻¹) when compared to other surfactants, and its optimum concentration recorded was 0.8% (320.57 ± 0.17 U ml⁻¹) for more protease production. Partial characterization of crude enzyme revealed that pH 7.0 (278.90 ± 0.08 U ml⁻¹) and 60°C temperature (332.37 ± 0.18 U ml⁻¹) were optimum for better protease activity by B. cereus.     

Optimization of the Production of Total Carotenoids by Sporidiobolus salmonicolor (CBS 2636) Using Response Surface Technique

This work aimed at evaluating the conditions of growth and the recovery of total carotenoids produced by Sporidiobolus salmonicolor (CBS 2636). Optimization of carotenoid production was achieved by experimental design technique. A Plackett–Burman design was used, followed by a complete second-order design, to optimize the concentration of total carotenoids in a conventional medium. Maximum concentration of 1,019 μg l⁻¹ of total carotenoids was obtained in a medium containing 40 g l⁻¹ glucose, 10 g l⁻¹ malt extract, and 14 g l⁻¹ peptone, at 180 rpm, 25 °C, and initial pH of 4.0. So far, no previous systematic study using microorganisms of the genus Sporobolomyces (formerly Sporidiobolus) for production of carotenoids has been reported. In this study, very good yields of carotenoids (1 mg l⁻¹) could be obtained after optimization of fermentation medium and operation conditions.     

Use of Byproducts of Food Industry for Production of Antimicrobial Activity by Bacillus sp. P11

Production of antimicrobial activity by Bacillus sp. P11 was tested on different byproducts of food industry, like fish meal, grape waste, an industrial fibrous soybean residue, soybean meal, and cheese whey. Bacillus sp. P11 produced the higher amount of antimicrobial activity on soybean meal, followed by fish meal and fibrous soybean residue. Soybean meal was the selected medium to determine the effect of three variables (temperature, initial pH, and substrate concentration) on bacteriocin activity by response surface methodology, using a 2³ factorial design. Statistical analysis showed good adequacy to the model (R ² of 0.8268). In the range studied, temperature and initial pH of the medium have a significant effect on bacteriocin production, and substrate concentration has no significant effect. Response surface data showed maximum bacteriocin production at initial pH between 7.0 and 8.5 and temperature between 39 and 42 °C. In the optimum conditions (initial pH 7.0 and 42 °C), production of bacteriocin activity by Bacillus sp. P11 was compared using a commercial medium (BHI broth) and soybean meal. Maximum activity achieved with the soybean meal-based medium was similar to that obtained with BHI, indicating that soybean meal may be a cost-effective substrate for production of antimicrobial activity by Bacillus sp. P11.     

Partial Purification and Characterization of Extracellular Fructofuranosidase with Transfructosylating Activity from <Emphasis Type="Italic">Candida</Emphasis> sp.

The present work was carried out with the aim to investigate some properties of an extracellular fructofuranosidase enzyme, with high transfructosylating activity, from Candida sp. LEB-I3 (Laboratory of Bioprocess Engineering, Unicamp, Brazil). The enzyme was produced through fermentation, and after cell separation from the fermented medium, the enzyme was concentrated by ethanol precipitation and than purified by anion exchange chromatography. The enzyme exhibited both fructofuranosidase (FA) and fructosyltransferase (FTA) activities on a low and high sucrose concentration. With sucrose as the substrate, the data fitted the Michaellis–Menten model for FA, showing rather a substrate inhibitory shape for fructosyltransferase activity. The K _m and v _max values were shown to be 13.4 g L⁻¹ and 21.0 μmol mL⁻¹ min⁻¹ and 25.5 g L⁻¹ and 52.5 μmol mL⁻¹ min⁻¹ for FA and FTA activities, respectively. FTA presented an inhibitory factor K _i of 729.8 g L⁻¹. The optimum conditions for FA activity were found to be pH 3.25–3.5 and temperatures around 69 °C, while for FTA, the optimum condition were 65 °C (±2 °C) and pH 4.00 (±0.25). Both activities were very stable at temperatures below 60 °C, while for FA, the best stability occurred at pH 5.0 and for FTA at pH  4.5–5.0. Despite the strong fructofuranosidase activity, causing hydrolysis of the fructooligosaccharides (FOS), the high transfructosilating activity allows a high FOS production from sucrose (44%).     

Aqueous medium enzymatic preparation of <Emphasis Type="SmallCaps">l</Emphasis>-alpha glycerylphosphorylcholine optimized by response surface methodology

The ability of Rhizopus chinensis lipase (Thermo 4S-3) to catalyze the deacylation of l,2-diacyl-sn-glycero-3-phosphocholines (sn-1,2-PC) for l-alpha glycerylphosphorylcholine (l-α-GPC) preparation was investigated. Response surface methodology (RSM), based on a modified central composite rotatable design, was employed to examine the effects of substrate concentrations, temperature, enzyme loading, and dosage of CaCl₂ on the l-α-GPC yield. RSM analysis indicated good correlation between experimental and predicated values. The optimal condition was confirmed as follows: reaction time, 3.5 h; temperature, 43 °C; enzyme loading, 28.2 U mL⁻¹; substrate concentration, 51.5 mg mL⁻¹; and dosage of CaCl₂, 1.9 mg mL⁻¹. Under these conditions, the l-α-GPC yield increased by 96.8%, which was close to the amount predicted by the model.     

Electrochemical Determination of Phenothrin in Agricultural Formulations, Vegetables, and Storage Bags of Wheat and Rice by Differential Pulse Adsorptive Stripping Voltammetry (DP-AdSV)

An electroanalytical method has been developed for the determination of the pesticide phenothrin by differential pulse adsorptive stripping voltammetry on a hanging mercury drop electrode in universal buffer as supporting electrolyte. The best adsorption conditions were found to be pH 6.0, an accumulation potential of −0.6 V, and an accumulation time of 75 s. Effects of stirring rate, scan rate, pulse amplitude, and purge time were examined for the optimization of instrumental conditions. Calibration curve is linear in the range 2 × 10⁻⁹ to 2 × 10⁻⁷ mol l⁻¹ with a detection limit of 1.9 × 10⁻¹⁰ mol l⁻¹. The correlation coefficient and relative standard deviation were 0.995% and 1.1%. The method is applied to the determination of the phenothrin in agricultural formulations, vegetables, and storage bags of wheat and rice under Food Corporation of India’s storage system.     

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.     

Feather meal: a previously unrecognized route for reentry into the food supply of multiple pharmaceuticals and personal care products (PPCPs)

Antimicrobials used in poultry production have the potential to bioaccumulate in poultry feathers but available data are scarce. Following poultry slaughter, feathers are converted by rendering into feather meal and sold as fertilizer and animal feed, thereby providing a potential pathway for reentry of drugs into the human food supply. We analyzed feather meal (n = 12 samples) for 59 pharmaceuticals and personal care products (PPCPs) using EPA method 1694 employing liquid chromatography tandem mass spectrometry (LC/MS/MS). All samples tested positive and six classes of antimicrobials were detected, with a range of two to ten antimicrobials per sample. Caffeine and acetaminophen were detected in 10 of 12 samples. A number of PPCPs were determined to be heat labile during laboratory simulation of the rendering process. Growth of wild-type E. coli in MacConkey agar was inhibited by sterilized feather meal (p = 0.01) and by the antimicrobial enrofloxacin (p < 0.0001) at levels found in feather meal. Growth of a drug-resistant E. coli strain was not inhibited by sterilized feather meal or enrofloxacin. This is the first study to detect antimicrobial residues in feather meal. Initial results suggest that more studies are needed to better understand potential risks posed to consumers by drug residues in feather meal.     

Effect of pH and Temperature on the Activity of Enzymatic Extracts from Pineapple Peel

This study was carried out to characterize a crude extract from pineapple peel after precipitation by three methods with the aim of obtaining an enzymatic extract from agro-industrial waste. The characterization of these extracts involved the determination of both protein content and specific protease activities. The effects of pH and temperature on specific protease activity and on the stability of the extracts were also evaluated. The optimal values of specific activity for the crude extract (CE) were pH 6.0 (5.76 U mg⁻¹ protein) and 7.0 (5.71 U mg⁻¹ protein) and a temperature of 70 °C (16 U mg⁻¹ protein). The average values for the relative specific activity were 17.4% (pH 3.0 to 9.0) and 42.7% (at 30, 50, and 70 °C). The ethanolic extract had the highest specific activity (10.7 U mg⁻¹ protein) in comparison to the best results obtained for the isoelectric precipitation (7.7 U mg⁻¹ protein) and the ammonium sulfate precipitation (4.7 U mg⁻¹ protein). Moreover, the ethanolic extract was more stable than the CE, retaining 60.9% and 53.7% of the initial specific activity during the evaluation of the stability at different pH and temperature values, respectively. The optimal values of pH and temperature were almost the same for the crude and the ethanolic extracts. In addition, the ethanolic extract was more stable than the CE in the experimental conditions tested in this work.     

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.     

The Influence of Oxygen Volumetric Mass Transfer Rates on Cyclodextrin Glycosyltransferase Production by Alkaliphilic Bacillus circulans in Batch and Fed-Batch Cultivations

In this work, the influence of oxygen mass transfer rates on the production of cyclodextrin glycosyltransferase (CGTase) by the alkaliphilic bacterium Bacillus circulans ATCC 21783 was investigated. Experimental design and response surface methodology were applied to optimize agitation speed and air flow rate in batch cultivations, in order to identify their significant effects and interactions with the synthesis of CGTase. Results were expressed as the volumetric mass transfer rates of oxygen (k_la, [per hour]). The maximal CGTase productivity of 155 U mL⁻¹ h⁻¹ was achieved with k_la of 48 h⁻¹. CGTase production was also studied in fed-batch cultures using the optimized parameters obtained in the batch experiments. The maximal CGTase productivity on fed-batch cultivations was 137 U mL⁻¹ h⁻¹ with feeding rates of starch at 0.17 g L⁻¹ h⁻¹.     

Occurrence of aflatoxin M1 in raw, pasteurized and UHT milk commercialized in Esfahan and Shahr-e Kord, Iran

Between September 2006 and September 2007, 236 samples of raw (n = 140), pasteurized (n = 48) and UHT (n = 48) milk were collected from supermarkets and from bulk milk tanks of eight dairy plants in the cities of Esfahan and Shahr-e Kord, Iran. All samples were analyzed for aflatoxin M₁ (AFM₁) contamination by ELISA and 213 (90.3%) were positive with mean concentrations 65 ng.l⁻¹. These concentrations are lower than the standards of Codex Alimentarius and FDA (500 ng.l⁻¹), but 119 samples (55.9%) had higher concentrations than the maximum tolerance accepted by some European countries (50 ng.l⁻¹). Mean concentrations of AFM₁ in raw, pasteurized and UHT milk were 68, 56, and 65 ng.l⁻¹, respectively. Mean concentrations of AFM₁ in autumn and winter samples were significantly higher (P < 0.05) than those of spring and summer but differences between AFM₁ concentrations of spring and summer samples were not significantly different. Concentrations of AFM₁ in milk from Shahr-e Kord were significantly lower (P ≤ 0.05) than those from Esfahan.     

Lipase-catalyzed acylation of <Emphasis Type="SmallCaps">l</Emphasis>-carnitine with conjugated linoleic acid in [Bmim]PF<Subscript>6</Subscript> ionic liquid

Improving significantly the acylation reaction of l-carnitine with conjugated linoleic acid catalyzed by lipase needs to select an efficient and suitable reaction medium that is not only polar but also hydrophobic. [Bmim]PF₆ which is satisfied with the above two requirements was applied as the reaction medium. The optimal reaction conditions were: Novozyme 435 as the catalyst, 5:1 of molar ratio of fatty acid to l-carnitine, 40 g L⁻¹ lipase, 0.22 a_W of initial water activity, 125 g L⁻¹ molecular sieves (they were added within 0–3 h of reaction time), 60 °C of reaction temperature, 200 rpm of rotation speed, 32 h of reaction time. The overall conversion could reach 98.27%. The conversion in [Bmim]PF₆ was 2.13 and 1.56 times higher than that in acetonitrile and in solvent-free system, respectively. The lipase in the present work could be used eight times. [Bmim]PF₆ as the reaction medium was better than acetonitrile and solvent-free system.     

Evaluation of Functional Properties in Protein Hydrolysates from Bluewing Searobin (Prionotus punctatus) Obtained with Different Microbial Enzymes

Enzymatic hydrolysis of proteins from low commercial value fish could be produced for uses like functional ingredients in a wide and always increasing zone of application in different food products. The objective of this work was to evaluate the functional properties and the amino acid profile of enzymatic hydrolysates from Bluewing searobin (Prionotus punctatus), using two microbial enzymes, Alcalase and Flavourzyme. The enzymatic hydrolysate obtained through the addition of the enzyme Alcalase reached the maximum solubility (42%) at pH 9, water holding capacity (WHC) of 2.4 g_water  g_protein ⁻¹, 4.5 g_oil g_protein ⁻¹ of oil holding capacity (OHC) and an emulsifying activity index (EAI) of 54 m² g_solids ⁻¹ at pH 3. On the other hand, the hydrolysate obtained from Flavourzyme attained 38% of solubility at pH 9, 3.7 g_water  g_protein ⁻¹ and 5.5 g_oil g_protein ⁻¹ for the holding capacities, and an EAI of 71 m² g_solids ⁻¹ at pH 11. The hydrolysate with Flavourzyme produced best results for WHC, OHC, and EAI because it had solubility lower than the hydrolysate of Alcalase. The hydrolysate produced by Alcalase had a higher amino acid content compared with Flavourzyme’s hydrolysate. However, both showed a good essential amino acid amounts. In general, these results indicate the potential utilization of the hydrolysate from Bluewing searobin in food formulations for the direct human consumption.     

Production of Cell Membrane-Bound α- and β-Glucosidase by Lactobacillus acidophilus

Hydrolytic enzymes, viz. α- and β-glucosidase, were produced from indigenous isolate, Lactobacillus acidophilus, isolated from fermented Eleusine coracana. Production of these enzymes was enhanced by optimizing media using one factor at a time followed by response surface methodology. The optimized media resulted in a 2.5- and 2.1-fold increase in α- and β-glucosidase production compared with their production in basal MRS medium. Localization studies indicated 80% of the total activity to be present in the cell membrane-bound fraction. Lack of sufficient release of these enzymes using various physical, chemical, and enzymatic methods confirmed their unique characteristic of being tightly cell membrane bound. Enzyme characterization revealed that both α- and β-glucosidase exhibited optimum catalytic activity at 50 °C and pH 6.0 and 5.0, respectively. K _m and V _max of α-glucosidase were 4.31 mM and 149 μmol min⁻¹ mL⁻¹ for p-nitrophenyl-α-d-glucopyranoside as substrate and 3.8 mM and 120 μmol min⁻¹ mL⁻¹ for β-glucosidase using p-nitrophenyl-β-d-glucopyranoside as the substrate.     

Sucrose Biotransformation to Fructooligosaccharides by <Emphasis Type="Italic">Aspergillus</Emphasis> sp. N74 Free Cells

Fructooligosaccharide production with the fructosyltransferase from free cells of the native strain Aspergillus sp. N74 at laboratory level was evaluated. The biomass of the native strain Aspergillus sp. N74 was produced in a sucrose fermentation medium and was employed in the enzymatic reaction in solutions of sucrose and phosphate buffer, where pH, temperature, and initial sucrose concentration effect were evaluated. Fructooligosaccharides and reaction subproducts were identified and quantified by high-performance liquid chromatography. The enzyme produced by the strain Aspergillus sp. N74 possessed hydrolytic and transfructosylating activities that changed with process conditions. The best transfructosylating condition was obtained at 80 min reaction time at pH 5.5, 60 °C, and initial sucrose concentrations higher than 550 g L⁻¹, with fructooligosaccharide production of about 50% w/w (based on initial sucrose concentration) and conversion selectivity higher than 90%. In addition, the transfructosylating and hydrolytic activities ratio was of 20. The high transfructosylating activity showed by the fructosyltransferase produced from the native strain Aspergillus sp. N74 suggest considering it as an alternative for the scale-up production of fructooligosaccharides by means of the whole microorganism at bench and pilot plant levels.     

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.     

Effect of chlorine dioxide on ripening of ‘Xiaobai’ apricots

‘Xiaobai’ apricots were treated with chlorine dioxide at rates of 0, 10, 100, 150, 1000 nl l⁻¹ at 25 °C for 10 h. Fruits harvested at two developmental stages were used. The onset of ethylene production was delayed and respiration rate was reduced following ClO₂ treatment. Ethylene production was efficiently inhibited by 1000 nl l⁻¹ ClO₂. ClO₂ treatment resulted in less firmness and titratable acidity loss during shelf life at 25 °C. Decay development in apricots was decreased when fruits were treated with ClO₂ after harvest. It is suggested that after harvest, ClO₂ treatment is efficient for extending the shelf life and maintaining the quality of ‘Xaobai’ apricots.