Optimization of caseinolytic and coagulating activities of Solanum tuberosum rennets for cheese making.

BACKGROUND: In recent years, the rising global demand for cheese, the high cost and limited supply of calf rennet, and consumer choices have increased research into new alternatives to animal or recombinant chymosins for cheese making. Plant proteases with caseinolytic activity (CA) and milk-clotting activity (MCA) have been proposed as alternatives for milk clotting to obtain artisanal cheeses with new organoleptic properties. They have been named vegetable rennets (vrennets). The aim of this study was to evaluate the performance of two Solanum tuberosum aspartic proteases (StAP1 and StAP3) as vrennets for cheese making and to obtain a statistical model that could predict and optimize their enzymatic activity. RESULTS: To optimize the CA and MCA activities, a response surface methodology was used. Maximum values of CA and MCA for both enzymes were found at pH 5.0 and 30-35 °C. Analysis of the degradation of casein subunits showed that it is possible to tune the specificity of both enzymes by changing the pH. At pH 6.5, the αS - and ß- subunit degradation is reduced while conserving a significant MCA. CONCLUSION: The statistical models obtained in this work showed that StAP1 and StAP3 exert CA and MCA under pH and temperature conditions compatible with those used for cheese making. The casein subunit degradation percentages obtained also allowed us to select the best conditions for the degradation of the κ-casein subunit by StAPs. These results suggest that StAP1 and StAP3 are good candidates as vrennets for artisan cheese making. © 2023 Society of Chemical Industry.

Milk adulteration with acidified rennet whey: a limitation for caseinomacropeptide detection by high-performance liquid chromatography.

BACKGROUND: High-performance liquid chromatography (HPLC) is widely employed to determine the caseinomacropeptide (CMP) index and to detect milk tampering with rennet whey. Prior to HPLC analysis, CMP is subject to a trichloracetic acid isolation, causing further soluble proteins in the sample to precipitate. On this basis, we aimed to determine whether rennet whey acidification could adversely affect the HPLC sensitivity with respect to detecting this peptide. RESULTS: As hypothesized, the CMP index from milk with added acidified rennet whey was, on average, half that quantified from milk with added rennet whey. Moreover, the quantum satis of acidified whey added to milk sufficient to demonstrate a HPLC CMP > 30 mg L-1 was 94% greater than that required for this threshold to be reached with rennet whey. CONCLUSION: Milk tampering with acidified rennet whey may limit the analytical sensitivity of the reversed-phase HPLC employed for the screening of CMP and, ultimately, disguise the fraudulent addition of whey to milk. © 2017 Society of Chemical Industry.

Cloning, expression and optimized production in a bioreactor of bovine chymosin B in Pichia (Komagataella) pastoris under AOX1 promoter.

The codon sequence optimized bovine prochymosin B gene was cloned under the control of the alcohol oxidase 1 promoter (AOX1) in the vector pPIC9K and integrated into the genome of the methylotrophic yeast Pichia (Komagataella) pastoris (P. pastoris) strain GS115. A transformant clone that showed resistance to over 4 mg G418/ml and displayed the highest milk-clotting activity was selected. Cell growth and recombinant bovine chymosin production were optimized in flask cultures during methanol induction phase achieving the highest coagulant activity with low pH values, a temperature of 25°C and with the addition of sorbitol and ascorbic acid at the beginning of this period. The scaling up of the fermentation process to lab-scale stirred bioreactor using optimized conditions, allowed to reach 240 g DCW/L of biomass level and 96 IMCU/ml of milk-clotting activity. The enzyme activity corresponded to 53 mg/L of recombinant bovine chymosin production after 120 h of methanol induction. Western blot analysis of the culture supernatant showed that recombinant chymosin did not suffer degradation during the protein production phase. By a procedure that included high performance gel filtration chromatography and 3 kDa fast ultrafiltration, the recombinant bovine chymosin was purified and concentrated from fermentation cultures, generating a specific activity of 800 IMCU/Total Abs(280 nm) and a total activity recovery of 56%. This study indicated that P. pastoris is a suitable expression system for bioreactor based fed-batch fermentation process for the efficient production of recombinant bovine chymosin under methanol-inducible AOX1 promoter.