Reeta Davis

Melamine detection in infant formula powder using near- and mid-infrared spectroscopy.

Near- and mid-infrared spectroscopy methods (NIR, FTIR-ATR, FTIR-DRIFT) were evaluated for the detection and quantification of melamine in infant formula powder. Partial least-squares (PLS) models were established for correlating spectral data to melamine concentration: R(2) > 0.99, RMSECV ≤ 0.9, and RPD ≥ 12. Factorization analysis of spectra was able to differentiate unadulterated infant formula powder from samples containing 1 ppm melamine with no misclassifications, a confidence level of 99.99%, and selectivity > 2. These nondestructive methods require little or no sample preparation. The NIR method has an assay time of 1 min, and a 2 min total time to detection. The FTIR methods require up to 5 min for melamine detection. Therefore, NIR and FTIR methods enable rapid detection of 1 ppm melamine in infant formula powder.

Detection of E. coli O157:H7 from Ground Beef Using Fourier Transform Infrared (FT‐IR) Spectroscopy and Chemometrics

FT-IR spectroscopy methods for detection, differentiation, and quantification of E. coli O157:H7 strains separated from ground beef were developed. Filtration and immunomagnetic separation (IMS) were used to extract live and dead E. coli O157:H7 cells from contaminated ground beef prior to spectral acquisition. Spectra were analyzed using chemometric techniques in OPUS, TQ Analyst, and WinDAS software programs. Standard plate counts were used for development and validation of spectral analyses. The detection limit based on a selectivity value using the OPUS ident test was 10(5) CFU/g for both Filtration-FT-IR and IMS-FT-IR methods. Experiments using ground beef inoculated with fewer cells (10(1) to 10(2) CFU/g) reached the detection limit at 6 h incubation. Partial least squares (PLS) models with cross validation were used to establish relationships between plate counts and FT-IR spectra. Better PLS predictions were obtained for quantifying live E. coli O157:H7 strains (R(2)> or = 0.9955, RMSEE < or = 0.17, RPD > or = 14) and different ratios of live and dead E. coli O157:H7 cells (R(2)= 0.9945, RMSEE = 2.75, RPD = 13.43) from ground beef using Filtration-FT-IR than IMS-FT-IR methods. Discriminant analysis and canonical variate analysis (CVA) of the spectra differentiated various strains of E. coli O157:H7 from an apathogenic control strain. CVA also separated spectra of 100% dead cells separated from ground beef from spectra of 0.5% live cells in the presence of 99.5% dead cells of E. coli O157:H7. These combined separation and FT-IR methods could be useful for rapid detection and differentiation of pathogens in complex foods.

Conversion of grass biomass into fermentable sugars and its utilization for medium chain length polyhydroxyalkanoate (mcl-PHA) production by Pseudomonas strains

This study investigated the potential of grass biomass as a feedstock for mcl-PHA production. Pretreatments (2% NaOH at 120°C or hot water at 120°C) of perennial ryegrass were employed alone or in combination with sodium chlorite/acetic acid (SC/AA) delignification to evaluate the enzymatic digestibility and subsequent utilization of resultant sugars by Pseudomonas strains. NaOH pretreated sample had better digestibility than raw and hot water treated samples and this hydrolysate supported good growth of all tested strains with limited mcl-PHA (6-17% of cell dry mass (CDM)) accumulation. Digestibility of both untreated and pretreated samples was improved after SC/AA delignification and produced glucose (74-77%) rich hydrolysates. Tested strains accumulated 20-34% of CDM as PHA when these hydrolysates were used as sole carbon and energy source. CDM and PHA yields obtained for these strains when tested with laboratory grade sugars was similar to that achieved with grass derived sugars.

Subtyping of Listeria monocytogenes at the haplotype level by Fourier transform infrared (FT-IR) spectroscopy and multivariate statistical analysis

Listeria monocytogenes is a widespread foodborne pathogen that represents a major concern with respect to food safety. Rapid identification of this bacterium at a subspecies level is important to trace back an outbreak and improve risk-based inspection programs. A method for subtyping L. monocytogenes at the serotype and haplotype levels was developed using Fourier transform infrared (FT-IR) reflectance microscopy. Thirty strains of L. monocytogenes belonging to four different PCR serotypes (1/2a, 1/2b, 4b, and 4c) that had previously been characterized by Multilocus genotyping (MLGT) and Pulsed field gel electrophoresis (PFGE) assays were used in this study. The FT-IR based identification and classification was compared to the known MLGT and PFGE subtyping of the L. monocytogenes. Canonical variate analysis (CVA) of the spectra resulted in 96.6% correct identification of L. monocytogenes at the serotype level. Hierarchical cluster analysis (HCA) and CVA of the spectra showed 91.7% correct identification of strains at the haplotype level consistent with their MLGT groupings. FT-IR spectra of strains were also differentiated correctly in accordance with their PFGE haplotyping. Additionally, by using HCA of FT-IR spectra, each bacterium was differentiated at the strain level. Starting from a pure culture, this method enabled classification of L. monocytogenes at the serotype, haplotype, and/or strain level within 18 h, which is faster and potentially less expensive than the molecular methods and previous FT-IR methods. This is the first report of the identification of L. monocytogenes at the haplotype level using FT-IR.

Detection and differentiation of live and heat-treated Salmonella enterica serovars inoculated onto chicken breast using Fourier transform infrared (FT-IR) spectroscopy.

Aims:  To evaluate Fourier transform infrared (FT‐IR) techniques for detecting, quantifying, and differentiating viable and heat‐treated cells of Salmonella enterica serovars from chicken breast.

Production and characterization of bacterial polyhydroxyalkanoate copolymers and evaluation of their blends by fourier transform infrared spectroscopy and scanning electron microscopy.

Rhizobium meliloti produced a copolymer of short chain length polyhydroxyalkanoate (scl-PHA) on sucrose and rice bran oil as carbon substrates. Recombinant Escherichia coli (JC7623ABC1J4), bearing PHA synthesis genes, was used to synthesize short chain length-co-medium chain length PHA (scl-co-mcl-PHA) on glucose and decanoic acid. Fourier transform infrared spectroscopy (FTIR) spectra of the PHAs indicated strong characteristic bands at 1282, 1723, and 2934 cm−1 for scl-PHA and at 2933 and 2976 cm−1 for scl-co-mcl-PHA polymer. Differentiation of polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-hydroxyvalerate-P(HB-co-HV) copolymer was obseverd using FTIR, with absorption bands at 1723 and 1281 for PHB, and at 1738, 1134, 1215 cm−1 for HV-copolymer. The copolymers were analyzed by GC and 1H NMR spectroscopy. Films of polymer blends of PHA produced by R. meliloti and recombinant E. coli were prepared using glycerol, polyethylene glycol, polyvinyl acetate, individually (1:1 ratio), to modify the mechanical properties of the films and these films were evaluated by FTIR and scanning electron microscopy.

High cell density cultivation of Pseudomonas putida KT2440 using glucose without the need for oxygen enriched air supply.

High Cell Density (HCD) cultivation of bacteria is essential for the majority of industrial processes to achieve high volumetric productivity (g L−1 h−1) of a bioproduct of interest. This study developed a fed batch bioprocess using glucose as sole carbon and energy source for the HCD of the well described biocatalyst Pseudomonas putida KT2440 without the supply of oxygen enriched air. Growth kinetics data from batch fermentations were used for building a bioprocess model and designing feeding strategies. An exponential followed by linearly increasing feeding strategy of glucose was found to be effective in maintaining biomass productivity while also delaying the onset of dissolved oxygen (supplied via compressed air) limitation. A final cell dry weight (CDW) of 102 g L−1 was achieved in 33 h with a biomass productivity of 3.1 g L−1 h−1 which are the highest ever reported values for P. putida strains using glucose without the supply of pure oxygen or oxygen enriched air. The usefulness of the biomass as a biocatalyst was demonstrated through the production of the biodegradable polymer polyhydroxyalkanoate (PHA). When nonanoic acid (NA) was supplied to the glucose grown cells of P. putida KT2440, it accumulated 32% of CDW as PHA in 11 h (2.85 g L−1 h−1) resulting in a total of 0.56 kg of PHA in 18 L with a yield of 0.56 g PHA g NA−1. Biotechnol. Bioeng. 2015;112: 725–733.

Biosynthesis of polyhydroxyalkanoates co-polymer in E. coli using genes from Pseudomonas and Bacillus

Expression of Pseudomonas aeruginosa genes PHA synthase1 (phaC1) and (R)-specific enoyl CoA hydratase1 (phaJ1) under a lacZ promoter was able to support production of a copolymer of Polyhydroxybutyrate (PHB) and medium chain length polyhydoxyalkanoates (mcl-PHA) in Escherichia coli. In order to improve the yield and quality of PHA, plasmid bearing the above genes was introduced into E. coli JC7623, harboring integrated β-ketothiolase (phaA) and NADPH dependent-acetoacetyl CoA reductase (phaB) genes from a Bacillus sp. also driven by a lacZ promoter. The recombinant E. coli (JC7623ABC1J1) grown on various fatty acids along with glucose was found to produce 28–34% cellular dry weight of PHA. Gas chromatography and 1H Nuclear Magnetic Resonance analysis of the polymer confirmed the ability of the strain to produce PHB-co-Hydroxy valerate (HV)-co-mcl-PHA copolymers. The ratio of short chain length (scl) to mcl-PHA varied from 78:22 to 18:82. Addition of acrylic acid, an inhibitor of β-oxidation resulted in improved production (3–11% increase) of PHA copolymer. The combined use of enzymes from Bacillus sp. and Pseudomonas sp. for the production of scl-co-mcl PHA in E. coli is a novel approach and is being reported for the first time.

Differentiation of live, dead and treated cells of Escherichia coli O157:H7 using FT-IR spectroscopy

Aims:  To apply specific collection techniques and spectroscopy to differentiate between live and dead Escherichia coli O157:H7 cells, as well as cells subjected to various inactivation treatments, including heat, salt, UV, antibiotics and alcohol.

Fourier Transform Infrared (FT-IR) Spectroscopy Coupled with Filtration and Immunomagnetic Separation for the Detection of Escherichia coli O157:H7 in Ground Beef

The majority of Escherichia coli O157:H7 outbreaks are associated with ground beef. To detect this pathogen, separation techniques were tested with E. coli O157:H7 in ground beef followed by FT-IR analyses. Ground beef samples were inoculated with various levels of live and heat treated E. coli O157:H7 cells and the bacteria were extracted by filtration or immunomagnetic separation (IMS). Spectra were collected and detection limits were established by discriminant analysis of the 1800-800 cm-1 region and comparison to standard plate counts. The detection limit for the Filtration-FT-IR and IMS-FT-IR assays was 105 CFU/g. Partial least squares model established significant linear relationships between plate counts and spectra [R ≥ 0.99]. Discriminant analysis and canonical variate analysis of the spectra differentiated live and heat treated cells of E. coli O157:H7. Validation experiments using ground beef inoculated with fewer cells (101- 102 CFU/g) reached the detection limit within a six hour incubation. A portable IR sensor was also used to detect E. coli O157:H7 in ground beef, and the detection limit was 107 CFU/g. The total time to detection for Filtration-FT-IR and IMS-FT-IR were one hour and 3.75 h, respectively which is faster than conventional plate count methods (48h) and conventional IMS methods (48h). The FT-IR methods developed are potentially rapid and simple protocols that could be further developed for the detection of different species of pathogenic bacteria in complex food systems.