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The increased demand for cheese and the limited availability of calf rennet justifies the search for milk-clotting enzymes from alternative sources. Trypsin-like protease by Penicillium roqueforti was produced by solid-state fermentation using cocoa shell waste as substrate. The production of a crude enzyme extract that is rich in this enzyme was optimized using a Doehlert-type multivariate experimental design. The biochemical characterization showed that the enzyme has excellent activity and stability at alkaline pH (10-12) and an optimum temperature of 80°C, being stable at temperatures above 60°C. Enzymatic activity was maximized in the presence of Na+ (192%), Co2+ (187%), methanol (153%), ethanol (141%), and hexane (128%). Considering the biochemical characteristics obtained and the milk coagulation activity, trypsin-like protease can be applied in the food industry, such as in milk clotting and in the fabrication of cheeses.

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In the present paper is presented a review on the application of Doehlert design in the optimization of some of the steps of analytical procedures aimed the analysis of food samples. The theoretical principles and the main characteristics of this type of design are described. In addition, the main advantages and limitations of Doehlert design over other designs (Central Composite Design and Box-Behnken) and its application in the area of food analysis are discussed. Finally, to illustrate its potential, some examples of Doehlert design application in other areas of food chemistry without the purpose of analytical determination will be briefly presented.

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The degradation of dodecylpyridinium chloride (DPC) by SO4•- and HO• radicals, generated by UVA and thermal-activated persulfate (PS) was investigated. Temperatures of 30-50°C were used for the heat activation of PS. In the case of UVA/PS, the effects of [PS]0 and specific photon emission rate (EP,0) were studied through a Doehlert design coupled with statistical analysis and response surface methodology. The results showed high DPC removal (99.8%) and pseudo-first-order degradation rate (kobs = 0.0971 min-1) for [DPC]0 = 4.60 ± 0.11 mg L-1, [PS]0 = 7.75 mmol L-1, and EP,0 = 0.437 µmol photons L-1 s-1, with a major role of SO4•- radicals in comparison with HO•. The specific DPC degradation rate found under these conditions was higher than that observed for thermal activation at 50°C and [PS]0 = 5.5 mmol L-1 (kobs = 0.0712 min-1) over the same time, although complete DPC removal was also achieved in the latter. The positive effect of EP,0 on DPC degradation by the UVA/PS process depends on PS concentrations, with kobs values increasing linearly with [PS]0 in the range 7.75-10 mmol L-1, whereas lower EP,0 values can be compensated by increasing [PS]0 up to about 10 mmol L-1, without significant scavenging. The second-order rate constants of DPC with HO• and SO4•-, estimated by comprehensive kinetic modeling, were 8.26 × 109 and 4.44 × 109 L mol-1 s-1, respectively. Furthermore, higher [DPC]0 would negatively affect the DPC degradation rate by the UVA/PS process, while 62% DPC removal was obtained in WWTP water, which can be considered good given the complexity of the real matrix. Finally, our results shed light on the possibility of using available UVA radiation (4.5%) in solar irradiance on the Earth's surface, making this treatment process more sustainable and cost-effective.

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In this study, multivariate methodologies were applied in the optimization of a dispersive liquid-liquid microextraction (DLLME) method, aiming at the determination of Cu and Mn in coconut water samples by flame atomic absorption spectrometry. Some extractors (chloroform and CCl4), dispersants (ethanol, methanol and acetonitrile) and complexing agents (5-Br-PADAP and Dithzone) were previously tested in the extraction. A mixture design was used to optimize the component proportions formed by chloroform (10%), acetonitrile (76%), and 0.020% 5-Br-PADAP solution (14%). Doehlert design optimized the variables pH, NaCl, and buffer amounts for the extraction of both metals. The following analytical characteristics, respectively for Cu and Mn, were accessed: limit of quantification (4.83 and 3.32 µg L-1), enrichment factors (11 and 8 fold), and precision (6.6 and 6.0% RSD, n = 10). Addition/recovery tests of the analytes allowed to find values in the range of 96.5-120% for Cu and 99-107% for Mn.

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The degradation of three pesticides, azoxystrobin (AZO), difenoconazole (DFZ), and imidacloprid (IMD), commonly found in the tomato rinse water, was studied through UVC (251-257 nm) and UVC/H2O2 photolysis. The results showed that direct photolysis follows pseudo-first-order kinetics, with total AZO and IMD removals within 15 min, using 21.8 and 28.6 W m-2, respectively, while the highest percentage of DFZ degradation was 51.7% at 28.6 W m-2 UVC. The estimated quantum yields were 0.572, 0.028, and 0.061 mol Einstein-1 for AZO, DFZ, and IMD, respectively. With regard to UVC/H2O2, total pesticide removal was achieved after 10 min, while optimal treatment conditions in relation to the pesticide removal rates, estimated through the sequential Doehlert design, were about [H2O2]0 = 130 mg L-1 and 26 W m-2. Cytotoxicity and genotoxicity assays carried out with Allium cepa, for real industrial tomato rinse water sampled from washing belts did not show abnormalities during cell division, with total pesticides degradation after 15 min, demonstrating the potential application of the UVC/H2O2 process as a viable localized treatment with a focus on the possible reuse of treated water.

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The fungal kingdom has been widely studied as a source of bioactive compounds of interest to the pharmaceutical and food industry. This paper studies the production of natural red pigments by Fusarium solani BRM054066 in the submerged fermentation system, using Doehlert experimental design to determine optimal cultivation conditions. The chemical composition of the red pigment was determined by Nuclear Magnetic Resonance spectroscopy (NMR) and Ultra-Performance Liquid Chromatography coupled to Mass Spectrometry (UPLC-MS). Antioxidant activity was assessed by the ability to sequester of free radical DPPH. In the analysis of anti-inflammatory activity, murine peritoneal macrophages activated by LPS were used, and the gene expression of TNF-α, IL-1ß, IL-6, IL-10 and IL-17 was determined using qPCR. As a result, it was found that agitation at 200 rpm and glucose concentration ≥ 20 g/L promote the best results in the production of red pigment. The chemical compounds identified were two naphthoquinones, fusarubin and dihydrofusarubin, and an anthraquinone, a bostrycoidin, being fusarubin the majority compound. The red pigment showed antioxidant activity by scavenge 50% of the DPPH radical, in a concentration of 24 µg/mL. The pigment also showed an effective anti-inflammatory capacity by reducing the overexpression of the pro-inflammatory cytokines TNF-α, IL-1ß and IL-6 and promoting the production of anti-inflammatory IL-10 and IL-17, in murine macrophages activated by LPS (p < 0.05). According to the results, the fungus F. solani BRM054066, under optimized conditions of cultivation, proved to be a promising source of biologically active natural pigments with wide industrial applicability.

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The anti-cancer drug Flutamide (FLUT) is widely used and is of great environmental concern. The solar photo-Fenton (SPF) process can be an effective treatment for the removal of this type of micropollutant. The use of a single addition of 5 mg L-1 of Fe2+ and 50 mg L-1 of H2O2 achieved 20% primary degradation and only 3.05% mineralization. By using three additions of 5 mg L-1 Fe2+, with an initial H2O2 concentration of 150 mg L-1, 58% primary degradation was achieved, together with 12.07% mineralization. Consequently, thirteen transformation products (TPs) were formed. The SPF process was further combined with adsorption onto avocado seed activated carbon (ASAC) as an environmentally friendly approach for the removal of remained FLUT and the TPs. Doehlert design was used to assess the behavior of 13 TPs by optimizing the contact time and the adsorbent mass load. The optimal conditions for removal of FLUT and the TPs were 14 mg of ASAC and a contact time of 40 min. Remained FLUT and the TPs were totally removed using the adsorption process. The mechanisms of adsorption of FLUT and the TPs were strongly influenced by their polarity and π-π interactions of the TPs onto ASAC.

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Conventional wastewater treatments are not efficient in removing parabens, which may thus end up in surface waters, posing a threat to aquatic biota and human health. As an alternative treatment, persulfate (PS)-driven advanced oxidation technologies have gained growing attention for removing these pollutants. In this study, the degradation of propylparaben (PrP) by UVA- and zero-valent iron (ZVI)-activated persulfate was investigated. The effects of initial PS concentration ([PS]0) and irradiance or ZVI concentration were explored using the Doehlert experimental design. For the UVA-activated system, the specific PrP degradation rate (k) and percent removal were consistently higher for increasing [PS]0 and irradiance, varying in the ranges 0.0053-0.0192 min-1 and 37.9-77.3%, respectively. In contrast, extremely fast PrP degradation was achieved through the ZVI/PS process (0.3304 < k < 0.9212 min-1), with removal percentages above 97.5%; in this case, paraben degradation was hindered for a ZVI dosage beyond 40 mg L-1. Regarding toxicity, ECOSAR predictions suggest that the degradation products elucidated by LC-MS/MS are less toxic than PrP toward fish, daphnid, and green algae. In addition, both processes showed to be strongly dependent on the water matrix, being ZVI/PS more impacted for a MBR effluent, although its performance was much better than that exhibited by the UVA-driven process (t1/2 of 65.4 and 276.1 min, respectively).

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Doehlert design and desirability function were used in the optimization of an ultrasound assisted dissolution method of fish fillet samples with tetramethyl ammonium hydroxide (TMAH) for the determination of Ca, Fe, Zn and Mg by flame atomic absorption spectrometry. The variables values after optimization were 620 µL (volume of TMAH), 25 min (sonication time) and 46 °C (bath temperature). The quantification limits for Ca, Mg, Fe and Zn were 56, 107, 23 and 2.9 µg g-1, respectively. Accuracy was evaluated by comparing the results generated from the analysis of the samples treated by the proposed method and by wet digestion. Additionally, accuracy for Fe and Zn was assessed by the analysis of certified reference materials Tort-3 (Lobster hepatopancreas), ERM-CE 278 (mussel tissue) and Dolt-4 (Fish liver). There were no significant differences between the results obtained at a 95% confidence level when results were evaluated by t-test application.

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Tannase can be used in different industrial sectors such as in food (juices and wine) and pharmaceutical production (trimethoprim) because it catalyses the hydrolysis of hydrolysable tannins. The aim of the current study is to assess the tannase found in the crude extract of Saccharomyces cerevisiae CCMB 520, and to set its catalytic and thermodynamic properties. The enzyme was optimally active at pH 6.0 and temperature 30 °C. Tannase was activated by Na+, Ca2+, K+ at 5 × 10-3 mol/L. The half-life at 30 °C was 3465.7 min. The activation energy was 40.32 kJ/mol. The Gibbs free energy, enthalpy and entropy at 30 °C were 85.40, 48.10 and -0.12 kJ/mol K, respectively. Our results suggest that the tannase found in the crude extract of S. cerevisiae is an attractive enzyme for industrial applications, such as for beverage manufacturing and gallic acid production, due its catalytic and thermodynamic properties (heat-stable and resistant to metal ions).

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