DNA and Protein Analysis throughout the Industrial Refining Process of Sugar Cane

Abstract

The amount, nature, and fate of DNA and protein in the major purification fractions generated during industrial scale refining of sugar cane into raw sugar by the diffuser and tandem roller mills was determined. The presence and size of sugar cane DNA were estimated using PCR and sugar cane specific primers that amplified fragments of various sizes from different segments of the repetitive intergenic region (IGS) of the 25S rDNA. Both the maximum fragment size capable of amplification and the amount of DNA decreased as refining progressed, indicating sequential degradation during the milling. However, PCR still detected minute quantities of sugar cane DNA in raw sugar (<10e-3 ppm). Using a bicinchoninic acid assay on trichloroacetic acid precipitated sodium dodecylsulfate-extracts, protein was found in all mill fractions and decreased from 4500 to 10 ppm as sugar cane was refined to raw sugar. Analysis of these extracts by one-and two-dimensional gel electrophoresis suggested a gradual degradation of proteins during refining. Shotgun proteomic analyses identified complex populations of sugar cane proteins, or peptides thereof, in all mill fractions, but the population complexity decreased during processing. Retail-purchased refined cane sugar showed no detectable protein or DNA (<2 ppm protein; 0.001 ppm DNA).

Lack of Detection of Bt Sugarcane Cry1Ab and NptII DNA and Proteins in Sugarcane Processing Products Including Raw Sugar

“…These results obtained by TaqMan assay are consistent with the findings of Cullis et al (2014) who also found a dramatic reduction in total sugarcane DNA quantity upon production of the clarified juice, the common starting material for production of raw sugar and ethanol production. No heterologous DNA was detected by PCR amplification in the final products raw sugar or flegma (the starting material for ethanol production).…”

“…First, published studies of total DNA and protein loss during stalk processing to refined sugar showed levels of <1 pg total DNA/g refined sugar and ~1 μg total protein/g refined sugar (Cullis et al, 2014). Given these extremely low detection levels for total DNA and protein, it is expected that the small quantities of heterologous DNA and newly expressed protein would also be no detectable.…”

“…Samples from leaves and bagasse were further ground to a homogeneous powder and protein extraction was performed as described by Cullis et al (2014) with minor modifications. Lyophilized samples from individual tubes combined into a 15-mL polypropylene tube and prepared as solutions, where former solid samples (leaves, bagasse, and filter cake) were dissolved at a 3% (w/v) in water.…”

“…OECD states that the extracted juice has high water content (about 85%) and contains mainly sucrose and reducing sugars (RSs) like glucose and fructose and that its protein content is negligible, around 0.2% of the dry matter (OECD, 2011). Additionally, sugarcane processing involves harsh conditions known to precipitate and denature protein and DNA, leading to the removal of detectable intact plant DNA and protein in raw and refined sugar (Cullis et al, 2014). …”

“…Therefore, the processes of extraction, raw sugar production, and refining involves multiple steps involving conditions known to denature, precipitate, and eliminate DNA and protein macromolecules found in low concentrations in sugarcane stalks (Cheavegatti-Gianotto et al, 2011; Cullis et al, 2014). As a result, OECD states that sugar is a very purified substance as raw sugar is typically 97–98% sucrose, whereas refined sugar purity is about 99.93% sucrose.…”