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4 protocols using lactose

1

Recombinant Lipase LipA PSA01 Production

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Accurel MP1004, a porous polypropylene homopolymer powder with a particle size lesser than 0.4 mm, was purchased from Membrana GmbH (Obenburg, Germany). We purchased p-nitrophenyl palmitate (pNPP) and oleic acid (95%) from Sigma Aldrich (St Louis, Mo, USA). Tripalmitin was acquired from Spectrum Chemicals Mfg. Corp. (Gardena, CA, USA), and the coconut and Spanish extra virgin olive oils were bought from a local market. The reagents Bradford dye, acrylamide, and bisacrylamide were purchased from Biorad (Richmond, CA, USA).
We used the autoinducing medium for growth and expression of the recombinant lipase LipA PSA01: tryptone, yeast extract, lactose, glucose, glycerol, lactose, potassium dihydrogen phosphate, disodium hydrogen phosphate, ammonium chloride, and magnesium sulfate were purchased from PanReac AppliChem (Barcelona, Spain). All solvents (hexane, ethanol, acetone, 2-propanol, ethyl acetate, and methanol) were acquired from JT Baker (Phillipsburg, NJ, USA). Gas chromatography standards and other chemicals used in this study (sodium, sodium hydroxide, TRIS, acetic acid, HEPES) were analytical reagents obtained from Sigma Aldrich (St Louis, Mo, USA).
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2

Lactose Fermentation and Nitrate Reduction Assays

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Two concentrations of lactose and potassium nitrate were tested for a pool of strains in preliminary tests.
lactose fermentation was tested in a modified API 50CH medium containing the following: lactose 5 or 20 g.L−1 (Panreac, Lyon, France), tryptone 10 g.L−1, yeast extract 5 g.L−1, K2HPO4, 0.25 g.L−1, MnSO4 0.05 g.L−1, and bromocresol purple 0.17 g.L−1. The medium was inoculated using 1% (v/v) of 48-h cultures in YEL, and incubated at 30 °C under anaerobiosis (using the Anaerocult A system, Merck, Darmstadt, Germany). The production of acid from lactose was determined from the colour change of bromocresol purple from purple to yellow after 2, 5, and 7 days of incubation.
Nitrate reductase activity was detected by means of the Griess reagent (Biomérieux, Marcy l'Etoile, France) after incubation of cultures at 30 °C under microaerophilic conditions (air atmosphere without agitation) in a broth containing potassium nitrate, 0.5 or 1.5 g.L−1 (VWR International, Fontenay-sous-Bois, France), tryptone (Biokar Diagnostics, Allone, France) 10 g.L−1, yeast extract (Biokar Diagnostics) 5 g.L−1, and glucose (Grosseron, Saint-Herblain, France) 1 g.L−1, according to Dalmasso et al. (2011 (link)). The results of the tests were read after 2 and 5 days of incubation.
All the tests were carried out in triplicate.
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3

Porcine Collagen Extraction and Characterization

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Porcine collagen was supplied by Tenerias Omega (Navarre, Spain). The treatments of porcine skin to obtain native collagen were carried out according to the method of Andonegi et al. [45 (link)]. Soy protein isolate (SPI), PROFAM 974, with 90% protein on a dry basis, was supplied by ADM Protein Specialties Division (Amsterdam, Netherlands). A commercial fish gelatin, with the quality standard for edible gelatin (1999/724/CE), was also employed. Glycerol, used as plasticizer, and lactose and citric acid, used as cross-linking agents, were obtained from Panreac (Barcelona, Spain). Finally, agar was extracted from Gelidium sesquipedale.
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4

Cod Fish Gelatin-based Bioactive Film

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A commercial cod fish gelatin type A was employed in this study. It has bloom 200, 11.06% moisture and 0.147% ash. Fish gelatin was kindly supplied by Weishardt International (Liptovsky Mikulas, Slovakia) and meets the quality standard for edible gelatin (1999/724/CE). Glycerol and lactose (Panreac, Barcelona, Spain) were used as plasticizer and cross-linking agent, respectively. Tetrahydrocurcumin was gifted by Sabinsa Corporation (East Windsor, New Jersey, USA) and was used as bioactive agent.
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