Enzymatic hydrolysis of whey protein was carried out in an automatic titrator 718 Stat Titrino (Metrohm AG, Herisau, Switzerland) to a degree of hydrolysis 10% (DH 10%) with Alcalase. For this purpose, a solution containing 36 g of protein was prepared with distilled water to a final volume of 0.9 L. The process conditions were set to 50 ºC and the pH to 8, whereas the enzyme-substrate ratio was fixed to 0.55 (w/w). The DH was estimated with the pH-stat-method, as previously described [19 (link)]. After the desired DH was reached, the enzyme was deactivated at 100 °C for 5 min. The whey protein hydrolysate (WPH) solution was freeze-dried in a Labconco freeze drying system (Kansas City, MO, USA).
718 stat titrino
Manufactured by Metrohm
Sourced in Switzerland
The 718 Stat Titrino is a high-precision titration system designed for automated titration analysis. It offers reliable and reproducible results, featuring a compact and user-friendly design.
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7 protocols using 718 stat titrino
1
Enzymatic Hydrolysis of Whey Protein
2
Omega Oil Encapsulation Formulation
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The refined fish oil (Omega Oil 1812 TG Gold) was purchased from BASF Personal Care and Nutrition GmbH (Illertissen, Germany) and stored at −80 °C until use. The Tween-20 (T20) was obtained from Sigma-Aldrich (Darmstadt, Germany) and CITREM (GRINDSTED® CITREM LR 10 EXTRA MT) were provided by Danisco (Copenhagen, Denmark). The pullulan (P) was kindly donated by Hayashibara Co., Ltd. (Okayama, Japan). The glucose syrup (GS; DE38, C*Dry 1934) was supplied by Cargill Germany GmbH (Krefeld, Germany). The maltodextrin (MD; DE21) and whey protein concentrate (ca. 35 wt% protein content) were generously donated by Abbott Laboratories S.A. (Granada, Spain). The whey protein concentrate hydrolysate (WPCH) used as an emulsifier was produced in an automatic titrator (718 Stat Titrino; Metrohm AG, Herisau, Switzerland) using Alcalase 2.4 L to a degree of hydrolysis of 10% (DH10), as described by Rahmani-Manglano et al. [16 (link)]. Then, the hydrolysate (WPCH) was freeze dried and stored at 4 °C until further use. The rest of the reagents used for the analysis were of analytical grade.
3
Enzymatic Hydrolysis of Tenebrio molitor Meal
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The enzymatic hydrolysis of Tenebrio molitor meal was conducted in a jacketed reactor connected to an automatic titrator (718 Stat Titrino, Metrohm, Herisau, Switzerland). Briefly, the T. molitor hydrolysis was conducted at 50 °C and pH 8. Thirty g/L protein was dissolved in distilled water and Alcalase 2.4 L (EC 3.4.21.62) was added at the beginning of the reaction at a 3% enzyme-to-substrate (protein) ratio. The reaction continued until the degree of hydrolysis (DH), measured by the pH-stat method [52 ], was 20%. The resulting hydrolysate was then deactivated by heating the solution at 100 °C for 15 min, centrifuged at 5300× g for 15 min, and vacuum-filtered through an 8 µm cellulose filter. The supernatant was lyophilized (LyoMicron, Coolvacuum Technologies S.L., Barcelona, Spain) and the powdered product was stored at −20 °C. The nitrogen content of the obtained hydrolysate powder was determined in triplicate according to the Dumas method using a Flash 2000 CHNS/O elemental analyzer (Thermo Scientific, Waltham, MA, USA). Protein content was calculated assuming a nitrogen-to-protein factor of 5.6 [53 (link)], resulting in 68.47 ± 0.39 wt.%.
4
Enzymatic Hydrolysis Assays in Aqueous and Organic Media
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The hydrolysis activity in aqueous solution was determined by titration using an automatic titrator pHStat (Metrohm 718 Stat Titrino) [24 (link)]. The substrate emulsions consisted of 67 mM triacylglycerol, 3% (w/v) gum arabic, 2 mM CaCl2, 2.5 mM Tris-HCl pH 8.0 and 150 mM NaCl, dispersed in distilled water [25 (link)]. The enzyme was added to 20 mL of the emulsion under magnetic stirring (300 rpm) at 30°C and the reaction was followed for 5 min. One unit of hydrolytic activity in aqueous medium corresponds to the release of 1 μmol of fatty acid per minute, under the assay conditions.
The hydrolytic activity in organic medium was determined by adding 5 mL of a reaction medium containing 4.9 mL of n-heptane, 70 mM triolein and 2% (v/v) distilled water to a 25-mL Erlenmeyer flask. The reaction was started by the addition of either free (Fr-LipG9) or immobilized LipG9 (Im-LipG9). The mixture was then incubated in an orbital shaker at 200 rpm and 40°C. At fixed intervals, 100-μL samples of the mixture were collected and analyzed for residual free fatty acids by the Lowry-Tinsley method [26 (link)]. One unit of hydrolytic activity in organic medium corresponds to the release of 1 μmol of fatty acid per minute, under the assay conditions.
The hydrolytic activity in organic medium was determined by adding 5 mL of a reaction medium containing 4.9 mL of n-heptane, 70 mM triolein and 2% (v/v) distilled water to a 25-mL Erlenmeyer flask. The reaction was started by the addition of either free (Fr-LipG9) or immobilized LipG9 (Im-LipG9). The mixture was then incubated in an orbital shaker at 200 rpm and 40°C. At fixed intervals, 100-μL samples of the mixture were collected and analyzed for residual free fatty acids by the Lowry-Tinsley method [26 (link)]. One unit of hydrolytic activity in organic medium corresponds to the release of 1 μmol of fatty acid per minute, under the assay conditions.
5
Optimizing Penicillin G Production
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The
pH of the fermentation broth was adjusted to 7.0 in a stirred glass
reactor kept at 4 °C. The pH was then adjusted to the desired
values using an automatic system (718 STAT Titrino, Metrohm). The
PenG concentration was determined as described previously.
pH of the fermentation broth was adjusted to 7.0 in a stirred glass
reactor kept at 4 °C. The pH was then adjusted to the desired
values using an automatic system (718 STAT Titrino, Metrohm). The
PenG concentration was determined as described previously.
6
Enzymatic Hydrolysis of Whey Protein
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Enzymatic hydrolysis of whey protein (WP) was carried out in an automatic titrator 718 Stat Titrino (Metrohm AG, Herisau, Switzerland) to a degree of hydrolysis 10% (DH 10) with alcalase. For this purpose, a solution containing 36 g of protein was prepared with distilled water to a final volume of 0.9 L. The process conditions were set to 50 °C and the pH to 8, and the enzyme-substrate ratio was fixed to 0.55 (w/w). The degree of hydrolysis was estimated with the pH-stat-method, as described by Camacho et al. [18 (link)]. The hydrolysis reaction took ca. 1.5 h, and the enzyme was deactivated at 100 °C for 5 min. The whey protein hydrolysate (WPH) solution was stored at −20 °C until further use.
7
Pectin Methylesterase Activity Assay
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PME was assayed using the method reported by Vervoort et al. (2011) . First, 1 mL juice was added to 30 mL of a 0.35% (w/v) apple pectin solution, containing 0.117 M NaCl. The pH of the mixture was maintained constant by addition of 0.01 N NaOH using an automatic pH-stat titrator (718 STAT titrino, Metrohm, Herisau, Switzerland). The PME activity was determined by the amount of enzyme required to release 1 μmol of carboxyl group per min during the pectin hydrolysis as a function of time at pH 7.0 and 22 C.
The PME activity of each sample was measured in triplicate. Relative residual activities of PPO, POD and PME were evaluated as:
% 𝑅𝑒𝑠𝑖𝑑𝑢𝑎𝑙 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = 𝐸𝑛𝑧𝑦𝑚𝑒 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑎𝑓𝑡𝑒𝑟 𝑡𝑟𝑒𝑎𝑡𝑚𝑒𝑛𝑡 𝐸𝑛𝑧𝑦𝑚𝑒 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑖𝑛 𝑡ℎ𝑒 𝑢𝑛𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑗𝑢𝑖𝑐𝑒 × 100% (Eq. 6)
The PME activity of each sample was measured in triplicate. Relative residual activities of PPO, POD and PME were evaluated as:
% 𝑅𝑒𝑠𝑖𝑑𝑢𝑎𝑙 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = 𝐸𝑛𝑧𝑦𝑚𝑒 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑎𝑓𝑡𝑒𝑟 𝑡𝑟𝑒𝑎𝑡𝑚𝑒𝑛𝑡 𝐸𝑛𝑧𝑦𝑚𝑒 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑖𝑛 𝑡ℎ𝑒 𝑢𝑛𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑗𝑢𝑖𝑐𝑒 × 100% (Eq. 6)
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