Use of high pressure homogenization in bacterial inactivation

High pressure homogenization has been of growing interest as a nonthermal technology for the inactivation of microorganisms in fruit and vegetable juices. Cells of Escherichia coli and Listeria innocua, used as surrogates for foodborne pathogens, were inoculated into apple or carrot juice (~7 log₁₀ CFU/ml) containing 0 or 10 IU/ml nisin and subjected to 350 to 0 MPa high pressure homogenization. At 50 MPa homogenization pressure intervals, juice samples were collected, immediately cooled to \u3c10°C, and then serially diluted and plated on nonselective recovery media. Following incubation, survivors were enumerated. As processing pressure increased, inactivation of E. coli increased, and a \u3e5 log reduction of cells was achieved following exposure to pressures in excess \u3e250 MPa. In contrast, little inactivation was observed for L. innocua with pressure \u3c250 MPa and up to 350 MPa processing pressure was required to achieve an equivalent 5 log inactivation. The addition of 10 IU nisin, together with high pressure homogenization, did not exhibit significant additional E. coli inactivation, but interactions were observed with L. innocua. Results indicate that high pressure homogenization processing is a promising technology to achieve pathogen decontamination in fruit and vegetable juices

Production of a functional human milk oligosaccharide, 2'-fucosyllactose, using microbial cell factories

Human breast milk is the gold standard for infant nutrition. In human milk, the oligosaccharides may protect babies by acting as decoy receptors for pathogens. Also, human milk oligosaccharides (HMOs) enhance the proliferation of probiotics to strengthen the host immune system. Among HMOs, 2’-fucosyllactose (2-FL), composed of L-fucose and D-lactose, is known to possess an anti-infection capability against many harmful organisms such as Campylobacter jejuni, enteropathogenic Escherichia coli, and even Norovirus. Consequently, great quantities of 2-FL are being demanded for food applications and thorough investigation of its biological properties. The current synthetic methods of 2-FL including chemical production and enzymatic catalysis are complicated, expensive, and thus impractical for a large-scale synthesis. In my research study, an alternative route to produce 2-FL using a microbial cell factory was devised. First, E. coli, as a host strain to produce 2-FL, was engineered to overexpress genes in the metabolic pathway of GDP-L-fucose, a donor of L-fucose, and harbor the fucosyltransferase enzyme (FucT2) which catalyzes the transfer of L-fucose onto a lactose molecule enabling 2-FL production. Second, the production conditions of 2-FL in the engineered E. coli were optimized in order to pinpoint the appropriate conditions promoting the efficient and consistent synthesis of 2-FL. Third, the key enzyme in 2-FL production, FucT2, from different sources was compared in search for the FucT2 leading to an enhanced synthesis of 2-FL. Fourth, the possibility to produce 2-FL was demonstrated in a different host, Saccharomyces cerevisiae, which is a generally recognized as safe (GRAS) organism. Through metabolic engineering, the minimum three components required to construct a 2-FL-producing S. cerevisiae consisting of GDP-L-fucose production, lactose internalization, and functional expression of FucT2 were confirmed. Additionally, the gene target perturbation, deletion of GDA1, which resulted in an improved production of GDP-L-fucose was identified. GDP-L-fucose is a beneficial sugar nucleotide which serves as a precursor to innumerable compounds aside from 2-FL. From this study, the feasibility and scalability of employing the microbial cell factory approach for 2-FL and GDP-L-fucose production were manifested

Whole cell biosynthesis of a functional oligosaccharide, 2′-fucosyllactose, using engineered Escherichia coli

BACKGROUND: 2'-Fucosyllactose (2-FL) is a functional oligosaccharide present in human milk which protects against the infection of enteric pathogens. Because 2-FL can be synthesized through the enzymatic fucosylation of lactose with guanosine 5′-diphosphate (GDP)-l-fucose by α-1,2-fucosyltransferase (FucT2), an 2-FL producing Escherichia coli can be constructed through overexpressing genes coding for endogenous GDP- l-fucose biosynthetic enzymes and heterologous fucosyltransferase. RESULTS: The gene for FucT2 from Helicobacter pylori was introduced to the GDP- l-fucose producing recombinant E. coli BL21 star(DE3) strain. However, only small amount of 2-FL was produced in a batch fermentation because the E. coli BL21star(DE3) strain assimilated lactose instead of converting to 2-FL. As an alternative host, the E. coli JM109(DE3) strain which is incapable of assimilating lactose was chosen as a 2-FL producer. Whole cell biosynthesis of 2-FL from lactose was investigated in a series of batch fermentations using various concentrations of lactose. The results of batch fermentations showed that lactose was slowly assimilated by the engineered E. coli JM109(DE3) strain and 2-FL was synthesized without supplementation of another auxiliary sugar for cell growth. A maximum 2-FL concentration of 1.23 g/l was obtained from a batch fermentation with 14.5 g/l lactose. The experimentally obtained yield (g 2-FL/g lactose) corresponded to 20% of the theoretical maximum yield estimated by the elementary flux mode (EFM) analysis. CONCLUSIONS: The experimental 2-FL yield in this study corresponded to about 20% of the theoretical maximum yield, which suggests further modifications via metabolic engineering of a host strain or optimization of fermentation processes might be carried out for improving 2-FL yield. Improvement of microbial production of 2-FL from lactose by engineered E. coli would increase the feasibility of utilizing 2-FL as a prebiotic in various foods

Quality of carrots as affected by pre- and postharvest factors and processing

The aim of this review is to provide an update on factors contributing to quality of carrots, with special focus on the role of pre- and postharvest factors and processing. The genetic factor shows the highest impact on quality variables in carrots, causing a 7–11-fold difference between varieties in content of terpenes, β-carotene, magnesium, iron and phenolics as well as a 1–4-fold difference in falcarindiol, bitter taste and sweet taste. Climate-related factors may cause a difference of up to 20-fold for terpenes, 82% for total sugars and 30–40% for β-carotene, sweet taste and bitter taste. Organic farming in comparison with conventional farming has shown 70% higher levels for magnesium and 10% for iron. Low nitrogen fertilisation level may cause up to 100% increase in terpene content, minor increase in dry matter (+4 to +6%) and magnesium (+8%) and reduction in β-carotene content (−8 to −11%). Retail storage at room temperature causes the highest reduction in β-carotene (−70%) and ascorbic acid (−70%). Heat processing by boiling reduces shear force (−300 to −1000%) and crispiness (−67%) as well as content of phenolics (−150%), terpenes (−85%) and total carotenes (−20%) and increases the risk of furan accumulation. Sensory and chemical quality parameters of carrots are determined mainly by genetic and climate-related factors and to a minor extent by cultivation method. Retail temperature and storage atmosphere as well as heating procedure in processing have the highest impact in quality reduction. © 2013 Society of Chemical Industr

Accepted for the Council:

electronic copy of this thesis for form and content and recommend that it be accepte