Ultrasound-assisted enzymatic synthesis of galacto-oligosaccharides using native whey with two commercial ß-galactosidases: Aspergillus oryzae and Kluyveromyces var lactis.

Native whey obtained during casein micelle microfiltration was used as a novel source to produce galacto-oligosaccharides (GOS). Since the presence of macromolecules and other interferers reduces biocatalyst performance, this work evaluated the effect of different ultrasound processing conditions on GOS synthesis using concentrated native whey. Ultrasonic intensities (UI) below 11 W/cm2 tended to increase the activity in the enzyme from Aspergillus oryzae for several minutes but accelerated the inactivation in that from Kluyveromyces lactis. At 40 °C, 40 % w/w native whey, 70 % wave amplitude, and 0.6 s/s duty-cycle, a UI of 30 W/cm2 was achieved, and the increased specific enzyme productivity was similar to the values obtained with pure lactose (∼0.136 g GOS/h/mgE). This strategy allows for obtaining a product containing prebiotics with the healthy and functional properties of whey proteins, avoiding the required purification steps used in the production of food-grade lactose.

Development of solid-state fermentation process of spent coffee grounds for the differentiated obtaining of chlorogenic, quinic, and caffeic acids.

BACKGROUND: Spent coffee grounds (SCGs) are a good source of chlorogenic acid (CGA), which can be hydrolyzed to quinic acid (QA) and caffeic acid (CA). These molecules have antioxidant and neuroprotective capacities, benefiting human health. The hydrolysis of CGA can be done by biotechnological processes, such as solid-state fermentation (SSF). This work evaluated the use of SSF with Aspergillus sp. for the joint release of the three molecules from SCGs. RESULTS: Hydroalcoholic extraction of the total phenolic compounds (TPCs) from SCGs was optimized, obtaining 28.9 ± 1.97 g gallic acid equivalent (GAE) kg-1 SCGs using 0.67 L ethanol per 1 L, a 1:9 solid/liquid ratio, and a 63 min extraction time. Subsequently, SSF was performed for 30 days, achieving the maximum yields for CGA, QA, and TPCs on the 16th day: 7.12 ± 0.01 g kg-1 , 4.68 ± 0.11 g kg-1 , and 54.96 ± 0.49 g GAE kg-1 respectively. CA reached its maximum value on the 23rd day, at 4.94 ± 0.04 g kg-1 . The maximum antioxidant capacity was 635.7 mmol Trolox equivalents kg-1 on the 14th day. Compared with unfermented SCGs extracts, TPCs and CGA increase their maximum values 2.3-fold, 18.6-fold for CA, 14.2 for QA, and 6.4-fold for antioxidant capacity. Additionally, different extracts' profiles were obtained throughout the SSF process, allowing us to adjust the type of enriched extract to be produced based on the SSF time. CONCLUSION: SSF represents an alternative to produce extracts with different compositions and, consequently, different antioxidant capacities, which is a potentially attractive fermentation process for different applications. © 2022 Society of Chemical Industry.

Modeling Tool for Studying the Influence of Operating Conditions on the Enzymatic Hydrolysis of Milk Proteins.

Systematic modeling of the enzymatic hydrolysis of milk proteins is needed to assist the study and production of partially hydrolyzed milk. The enzymatic hydrolysis of milk proteins was characterized and evaluated as a function of the temperature and protease concentration using Alcalase, Neutrase and Protamex. Modeling was based on the combination of two empirical models formed by a logarithmic and a polynomial equation to correlate the kinetic constants and the operating conditions. The logarithmic equation fitted with high accuracy to the experimental hydrolysis curves with the three proteases (R2 > 0.99). The kinetic constants were correlated with the operating conditions (R2 > 0.97) using polynomial equations. The temperature and protease concentration significantly affected the initial rate of hydrolysis, i.e., the kinetic constant a, while the kinetic constant b was not significantly affected. The values for the kinetic constant a were predicted according to the operating conditions and they were strongly correlated with the experimental data (R2 = 0.95). The model allowed for a high-quality prediction of the hydrolysis curves of milk proteins. This modeling tool can be used in future research to test the correlation between the degree of hydrolysis and the functional properties of milk hydrolysates.

Prediction of the Limiting Flux and Its Correlation with the Reynolds Number during the Microfiltration of Skim Milk Using an Improved Model.

Limiting flux (JL) determination is a critical issue for membrane processing. This work presents a modified exponential model for JL calculation, based on a previously published version. Our research focused on skim milk microfiltrations. The processing variables studied were the crossflow velocity (CFV), membrane hydraulic diameter (dh), temperature, and concentration factor, totaling 62 experimental runs. Results showed that, by adding a new parameter called minimum transmembrane pressure, the modified model not only improved the fit of the experimental data compared to the former version (R2 > 97.00%), but also revealed the existence of a minimum transmembrane pressure required to obtain flux (J). This result is observed as a small shift to the right on J versus transmembrane pressure curves, and this shift increases with the flow velocity. This fact was reported in other investigations, but so far has gone uninvestigated. The JL predicted values were correlated with the Reynolds number (Re) for each dh tested. Results showed that for a same Re; JL increased as dh decreased; in a wide range of Re within the turbulent regime. Finally, from dimensionless correlations; a unique expression JL = f (Re, dh) was obtained; predicting satisfactorily JL (R2 = 84.11%) for the whole set of experiments.

Recent advances and perspectives of ultrasound assisted membrane food processing.

Power ultrasound (US) transmits substantial amounts of small mechanical movements serving for particle detaching in membrane filtrations. This topic has been reviewed in recent years mainly focused on the mechanisms by which the flux is improved under specific processing conditions. US also been shown to improve food quality by changing physical properties and modifying the activity of enzymes and microorganisms. Surprisingly, limited information exists regarding on how the application of US results in terms of process and quality during membrane filtration of complex matrices such as liquid foods. This review highlights the recent advances in the use of US in membrane filtration processes focused in the manufacturing of foodstuffs and food ingredients, and perspectives of novel hybrid membrane-US systems that may be quite interesting for this field. The application of US in food membrane processing increases the flux, but the lack of standardization regarding to experimental conditions, make suitable comparisons impossible. In this sense, careful attention must be paid regarding to the ultrasonic intensity (UI), the membrane configuration and type of transducers and volume of the treated solution. Dairy products are the most studied application of US membrane food processing, but research has been mainly focused on flux enhancement; hitherto there have been no reports of how operational variables in these processes affect critical aspects such as quality and food safety. Also, studies performed at industrial scale and economical assessments are still missing. Application of US combined with membrane operations such as reverse osmosis (RO), forward osmosis (FO) and enzyme membrane bioreactors (EMBR) may result interesting for the production of value-added foods. In the perspective of the authors, the stagnation of the development of acoustic filtration systems in food is due more to a prejudice on this subject, rather than actual impedance due to the lack of technological development of transducers. This later has shown important advances in the last years making them suitable for tailor made applications, thus opening several research opportunities to the food engineering not yet explored.

Calculation of statistic estimates of kinetic parameters from substrate uncompetitive inhibition equation using the median method.

We provide initial rate data from enzymatic reaction experiments and tis processing to estimate the kinetic parameters from the substrate uncompetitive inhibition equation using the median method published by Eisenthal and Cornish-Bowden (Cornish-Bowden and Eisenthal, 1974; Eisenthal and Cornish-Bowden, 1974). The method was denominated the direct linear plot and consists in the calculation of the median from a dataset of kinetic parameters Vmax and Km from the Michaelis-Menten equation. In this opportunity we present the procedure to applicate the direct linear plot to the substrate uncompetitive inhibition equation; a three-parameter equation. The median method is characterized for its robustness and its insensibility to outlier. The calculations are presented in an Excel datasheet and a computational algorithm was developed in the free software Python. The kinetic parameters of the substrate uncompetitive inhibition equation Vmax , Km and Ks were calculated using three experimental points from the dataset formed by 13 experimental points. All the 286 combinations were calculated. The dataset of kinetic parameters resulting from this combinatorial was used to calculate the median which corresponds to the statistic estimator of the real kinetic parameters. A comparative statistical analyses between the median method and the least squares was published in Valencia et al. [3].

Application of the median method to estimate the kinetic constants of the substrate uncompetitive inhibition equation.

In 1974, Eisenthal and Cornish-Bowden published the direct linear plot method, which used the median to estimate the Vmax and Km from a set of initial rates as a function of substrate concentrations. The robustness of this non-parametric method was clearly demonstrated by comparing it with the least-squares method. The authors commented that the method cannot readily be generalized to equations of more than two parameters. Unfortunately, this comment has been misread by other authors. Comments such as "this method cannot be extended directly to equations with more than two parameters" were found in some publications. In addition, recently, the most drastic comment was published: "this method cannot be applied for the analysis of substrate inhibition." Given all of these presumptions, we have been motivated to publish a demonstration of the contrary: the median method can be applied to more than two-parameter equations, using as an example, the substrate uncompetitive inhibition equation. A computer algorithm was written to evaluate the effect of simulated experimental error of the initial rates on the estimation of Vmax, Km and KS. The error was assigned to different points of the experimental design. Four different KS/Km ratios were analyzed with the values 10, 100, 1000 and 10,000. The results indicated that the least-squares method was slightly better than the median method in terms of accuracy and variance. However, the presence of outliers affected the estimation of kinetic constants using the least-squares method more severely than the median method. The estimation of KS using the median method to estimate 1/KS was much better than the direct estimation of KS, causing a negative effect of non-linearity of KS in the kinetic equation. Considering that the median method is free from the assumptions of the least-squares method and the arbitrary assumptions implicit in the linearization methods to estimate the kinetic constants Vmax, Km and KS from the substrate uncompetitive inhibition equation, the median method is highly superior to all published methods, including the non-linear regression by least squares. We concluded that the median method can be applied to the substrate uncompetitive inhibition equation and other equations with more than two parameters. In addition, as we can project, the median method is the most reliable and robust method for the estimation of kinetic parameters from enzyme kinetic models.

Mechanisms of mineral membrane fouling growth modulated by pulsed modes of current during electrodialysis: evidences of water splitting implications in the appearance of the amorphous phases of magnesium hydroxide and calcium carbonate.

Experiments revealed the fouling nature evolutions along different electrodialysis (ED) trials, and how it disappears when current pulsation acts repetitively on the interfaces of ion-exchange membranes (IEMs). Fouling was totally controlled on the diluate side of cation-exchange membrane (CEM) by the repetitive pulsation frequency of the higher on-duty ratios applied. They created steady water splitting proton-barriers that neutralized OH(-) leakage through the membrane, decreasing the interfacial pH, and fouling of the concentrate side. The anion-exchange membrane (AEM) on the diluate side was similarly protected, but it was fouled once water splitting OH(-) generation became either intense enough or excessively weak. Interestingly, amorphous magnesium hydroxide (AMH) stemmed on the CEM-diluate side from brucite under intense water splitting OH(-) generation, and/or strong OH(-) leakage electromigration through the membrane. Water dissociation and overlimiting current regimes triggered drastic water molecule removal from crystal lattices through an accelerated cascade water splitting reaction. Also, amorphous calcium carbonate (ACC) appeared on CEM under intense water splitting reaction, and disappeared once intense OH(-) leakage was allowed by the water splitting proton-barrier dissipation. Our findings have implications for membrane fouling control, as well as for the understanding of the growth behavior of CaCO3 and Mg(OH)2 species on electromembrane interfaces.