BIOTECHNOLOGICAL APPLICATIONS TO DERIVE ADDED-VALUE INGREDIENTS FROM DAIRY PRODUCTS AND TO ASSESS EFFICIENCY OF ELECTROMICROBIAL PROTEIN PRODUCTION

Abstract

Lactose intolerance is a problem that affects 70% of the global population and, along with shifting consumer trends towards higher fat dairy products, is a major culprit behind the decreased demand for skim milk and its products. The declining consumer demand has led to negative financial consequences for producers and also results in deleterious environmental effects caused by skim milk waste. Fermentation was evaluated as a method for removal of lactose from skim milk, with the hopes of generating added-value ingredients, creating functional fermented skim milk beverages and alleviating the damages from food waste. Three separate anaerobic skim milk fermentation experiments were conducted, beginning with the inoculation of skim milk with 5X106 CFU/ml of four separate yeast species, Brettanomyces bruxellensis, Brettanomyces claussenii, Kluyveromyces marxianus and Saccharomyces cerevisiae, with and without the addition of an exogenous lactase enzyme. General fermentation performance of these yeast was screened, as well as their respective abilities to produce ethanol and galactose. Of particular interest was the presumed ability of B. claussenii to selectively ferment glucose, leaving behind galactose, a low glycemic index sugar, upon enzymatic hydrolysis of lactose. Samples were drawn every other day for cell count determinations as well as density measurements, which served as estimates of sugar consumption by the yeast throughout the fermentation. HPLC quantification of residual fermentable sugars (lactose, glucose, galactose), organic acids, and ethanol determined that, with lactase, highest ethanol production of 2.5% was achieved by B. claussenii, whose fermentation performance mirrored that of a very established lactose utilizing yeast, K. marxianus. When lactase was added, some galactose was retained by B. claussenii, but these values were much lower than expected, and highly variable across replicates. As such, the above framework was extended to an acidified skim milk substrate as a means of replicating an environment where more stable residual galactose levels had previously been observed. Acidification of the skim milk to pH 3.45 helped reduce excessive protein coagulation observed in the previous experiment, and galactose recovery from the skim milk was much higher and more consistent than in previous non-acidified skim milk fermentations. Assuming that initial galactose content represents half the initial lactose content, galactose recovery from acidified skim milk fermentations by B. claussenii was about 65%. The effect of changing initial inoculum concentrations of B. claussenii (5X106, 5X107 and 5X108CFU/ml) on fermentation time and galactose yields in non-acidified skim milk was investigated in a third and final fermentation experiment. The initial solids content of the substrate was also adjusted to 50g/L and 100g/L, respectively. Once again, low and variable galactose outputs were observed in this experiment. However, high galactose recoveries of 68% were achieved in the non-acidified skim milk substrate containing 100g/L starting solids content, and pitched with 5X107CFU/ml. With increased stabilization of galactose yields there is hope for the production of valuable ingredients from skim milk. Further improvement of organoleptic properties would also increase the promise of a stand-alone functional beverage from skim milk fermentation by B. claussenii. This research also provides valuable insights into the fermentation conditions that dictate sugar consumption or repression by B. claussenii. While the fermentation mechanism of this yeast has not been fully established, a putative β-galactosidase-expressing gene from B. claussenii was recently identified by members of the Alcaine Research Group. β-galactosidase enzymes are required for lactose removal during the fermentation of dairy products and byproducts from dairy processing, whose disposal is highly polluting. As such, a second component of this research was centered around characterizing the temperature and pH values conducive to optimal hydrolytic activity of this β-galactosidase. First, S. cerevisiae PGY453, a non-β-galactosidase-expressing yeast, was transformed with constructs containing a β-galactosidase-expressing LAC4 gene insert (p425GPD-LAC4

p416GPD). For temperature characterization, the β-galactosidase enzyme was isolated from the transformant, incubated for 1 hour at 20℃, 30℃, 40℃, 50℃, 60℃ and combined with a pH 7 phosphate-citrate buffer mixed with an ONPG lactose analog. Resulting mean β-galactosidase activities were highest at 30℃, a common fermentation temperature for many dairy products. For pH characterizations, the isolated enzyme was incubated at 40℃ for 1 hour prior to adding buffers mixed with ONPG at pH 3, 4, 5, 6, 7 and 8. Optimal β-galactosidase activities were achieved at pH 6 and 7, suggesting applications of this enzyme from B. claussenii in milk, as well as permeates from dairy processing. As a complement to the above research, particularly applied to upcycling waste in order to improve sustainability in the dairy industry, another component of this research was conducted in collaboration with the Biological and Environmental Engineering Department at Cornell. Specifically, the aim of this project was to theoretically assess the maximum energetic efficiencies of electromicrobial production of protein, a technology combining renewable electricity and carbon and nitrogen-fixing microbial metabolism. This is an emerging protein production scheme given the projected doubling by mid 21st century in protein demand and the major environmental toll of traditional protein production. Results from this analysis show that microorganisms that fix CO2 and nitrogen and use H2-oxidation or extracellular electron uptake to produce reducing equivalents can generate amino acid molecules with energy efficiencies as low as 64 MJ kg-1. This value is roughly ten times more efficient than the next most efficient energy estimates for electromicrobial protein production.