LAUSR

BACKGROUND Corn processing byproducts corn steep liquor (CSL), and thin stillage (TS) were evaluated as growth media for recombinant Lactococcus lactis modified to produce antifreeze proteins (AFPs) that could have important food and non-food applications. The AFP III sequence from ocean pout was cloned into a shuttle vector to make an expression vector that facilitated the production of recombinant AFP III in L. lactis. Light CSL from yellow dent corn and thin stillage from the industrial corn bioethanol process were optimized as fermentation media with a combination of the following additives and trace elements: disodium-β-glycerophosphate (DG), tryptone (T), ascorbic acid (AA), iron (Fe), zinc (Zn), and magnesium (Mg). The growth of wild-type and recombinant L. lactis strains were compared over a 72-h period in 96-well plates and 250-mL shake flasks. RESULTS The corn coproducts media consisting of 50% (v/v) light steep in water supplemented with DG-5 g L-1 , T- 5 g L-1 , AA- 0.5 g L-1 , and Zn- 4 ppm resulted in best growth and was considered as the best-optimized media. The addition of additives and trace elements better supported the growth of both wild-type and recombinant L. lactis strains compared to control media without any additives. Respective fermentation supernatants were frozen to -20 °C, and the time to supercool and freeze was compared. A distinct supercooling effect was observed for the supernatants from recombinant strains thus, extending the time and temperature of supercooling and freezing. The maximum time of supercooling extended was 17.55± 4.45 min for thin stillage followed by M17 media (17.25 ± 4.45 min), Kent Corporation CSL (10.80 ± 2.12 min), and yellow dent CSL (6.9 ± 0.85 min) when fermented with recombinant L. lactis strains. CONCLUSION The supplemented corn coproduct-based media enhanced the growth of both wild-type and recombinant L. lactis strains. These optimized media can replace or supplement more expensive media (for example, M17), potentially reducing cost. The fermentation supernatants exhibited longer times to supercool, and freeze compared to control supernatants, indicating potential use as antifreeze compounds in frozen food and non-food applications. This article is protected by copyright. All rights reserved.