A CONVENIENT DOMINANT SELECTION MARKER FOR GENE TRANSFER IN INDUSTRIAL STRAINS OF SACCHAROMYCES YEAST: SMRI ENCODED RESISTANCE TO THE HERBICIDE SULFOMETURON METHYL

The SMR1-410 gene of S. cerevisiae, encoding resistance to the herbicide sulfometuron methyl (SM), was used as a dominant selection marker in yeast replicating and yeast integrating vectors for the transformation of wild type strains of baking, brewing (ale and lager), distilling, wine and sake Saccharomyces yeasts. Transformation of lithium treated cells by a YEp vector resulted in transformation frequencies ranging from 200 to 8,000 transformants per 10 ug of DNA. Utilizing a yeast integrating vector with SMR1–410 as the only yeast DNA sequences, it was demonstrated that a single copy of SMR1–410 is sufficient to confer stably inherited SM resistance. Thus the SMR1–410 sequence has the unique ability to act as a selectable marker and to also provide a site for chromosomal integration. Since transformants were resistant to levels at least seven fold higher than wild type strains the resistance phenotype was clearly expressed and easily scored in all industrial strains tested. Unlike other selection markers derived from mammalian or bacterial cells, SMR1–410 is derived from S. cerevisiae. Thus industrial utilization of this marker as a means of genetically improving food and beverage strains of Saccharomyces yeasts by recombinant DNA technology is enhanced, as government regulatory agencies are likely to view it in a more favourable light.     

Evaluation of potential human health risk associated with consumption of edible products from livestock fed ration supplemented with Red Lake Diatomaceous Earth

ABSTRACT

Red Lake Diatomaceous Earth (DE) is a naturally occurring blend of diatomaceous earth and calcium bentonite that can be used as an anti-caking agent in animal feed and contains naturally occurring dioxins. A quantitative risk assessment was conducted to assess potential human health risk associated with consumption of edible tissues from livestock exposed to dioxins via feed containing Red Lake DE. Empirical data characterising the transfer of dioxins to eggs and other tissues in chickens demonstrate that resulting concentrations in eggs are lower than those found in the general food supply. These data also provided product-specific input for a risk assessment conducted both with default parameters and with media-specific input from the feed study. Results demonstrate that exposure to dioxins in edible tissue from livestock that consumed Red Lake DE in feed would not be associated with an increased risk to humans. Findings from this assessment highlight the utility and importance of accounting for bioavailability as part of health-based risk assessment and provide information critical to risk managers in determining the safe use of Red Lake DE as an anticaking agent in livestock and pet feed.     

Screening of whey protein isolate hydrolysates for their dual functionality: Influence of heat pre-treatment and enzyme specificity

Heat pre-treated and non heat pre-treated whey protein isolate (WPI) were hydrolysed using α-chymotrypsin (chymotrypsin), pepsin and trypsin. The in vitro antioxidant activity, ACE-inhibition activity and surface hydrophobicities of the hydrolysates were measured in order to determine if peptides with dual functionalities were present. Dual functional peptides have both biological (e.g. antioxidant, ACE-inhibition, opioid activities) and technological (e.g. nanoemulsification abilities) functions in food systems. Heat pre-treatment marginally enhanced the hydrolysis of WPI by pepsin and trypsin but had no effect on WPI hydrolysis with chymotrypsin. With the exception of the hydrolysis by trypsin, heat pre-treatment did not affect the peptide profile of the hydrolysates as analysed using size exclusion chromatography, or the antioxidant activity (P > 0.05). Heat pre-treatment significantly affected the ACE-inhibition activities and the surface hydrophobicities of the hydrolysates (P < 0.05), which was a function of the specificity of the hydrolysing enzyme. Extended hydrolysis (up to 24 h) had no significant effect on the DH and the molecular weight profiles (P > 0.05) but in some instances caused a reduction in the antioxidant activity of WPI hydrolysates. The chymotrypsin hydrolysate showed a broad MW size range, and was followed by pepsin and then trypsin. The bioactivities of the hydrolysates generally decreased in the order; chymotrypsin > trypsin > pepsin. This study showed that by manipulating protein conformation with pre-hydrolysis heat treatment, combined with careful enzyme selection, peptides with dual functionalities can be produced from WPI for use as functional ingredients in the manufacture of functional foods.     

Explaining tolerance for bitterness in chocolate ice cream using solid chocolate preferences

Chocolate ice cream is commonly formulated with higher sugar levels than nonchocolate flavors to compensate for the inherent bitterness of cocoa. Bitterness, however, is an integral part of the complex flavor of chocolate. In light of the global obesity epidemic, many consumers and health professionals are concerned about the levels of added sugars in foods. Once a strategy for balancing undesirable bitterness and health concerns regarding added sugars has been developed, the task becomes determining whether that product will be acceptable to the consumer. Thus, the purpose of this research was to manipulate the bitterness of chocolate ice cream to examine how this influences consumer preferences. The main goal of this study was to estimate group rejection thresholds for bitterness in chocolate ice cream, and to see if solid chocolate preferences (dark vs. milk) generalized to ice cream. A food-safe bitter ingredient, sucrose octaacetate, was added to chocolate ice cream to alter bitterness without disturbing other the sensory qualities of the ice cream samples, including texture. Untrained chocolate ice cream consumers participated in a large-scale sensory test by indicating their preferences for blinded pairs of unspiked and spiked samples, where the spiked sample had increasing levels of the added bitterant. As anticipated, the group containing individuals who prefer milk chocolate had a much lower tolerance for bitterness in their chocolate ice cream compared with the group of individuals who prefer dark chocolate; indeed, the dark chocolate group tolerated almost twice as much added bitterant in the ice cream before indicating a significant preference for the unspiked (control) ice cream. This work demonstrates the successful application of the rejection threshold method to a complex dairy food. Estimating rejection thresholds could prove to be an effective tool for determining acceptable formulations or quality limits when considering attributes that become objectionable at high intensities.     

In vitro antifungal activity and chemical composition of Warionia saharae essential oil against 3 apple phytopathogenic fungi

Essential oil of aerial parts of Warionia saharae was obtained by hydrodistillation and analyzed by GC and GC-MS. Thirty-nine compounds were identified, accounting for 93.2% of the total oil. β-Eudesmol (34.9%), nerolidol-E (23%), and linalool (15.2%) were the most abundant components. The antifungal activity of the W. saharae oil was tested by poisoned food (PF) technique and the volatile activity (VA) assay against 3 phytopathogenic causing the deterioration for apple. The results indicated that the W. saharae oil inhibited significantly the mycelial growth of all strains tested (p<0.05). The minimum inhibitory concentration against Alternaria sp. was 2 μL/mL air in VA assay, whereas >2 μL/mL in PF technique for all strains. Fungal spore production was completely inhibited at 1 μL/mL air for Alternaria sp. and at 2 μL/mL air for Penicillium expansum and Rhizopus stolonifer.     

Detection of <Emphasis Type="Italic">Campylobacter</Emphasis> colonies using hyperspectral imaging

The presence of Campylobacter in foods of animal origin is the leading cause of bacterially induced human gastroenteritis. Isolation and detection of Campylobacter in foods via direct plating involves lengthy laboratory procedures including enrichments and microaerobic incubations, which take several days to a week. The incubation time for growing Campylobacter colonies in agar media usually takes 24–48 h. Oftentimes the problem is the difficulty of visually differentiating Campylobacter colonies from non-Campylobacter contaminants that frequently grow together with Campylobacter on many existing agars. In this study, a new screening technique using non-destructive and non-contact hyperspectral imaging was developed to detect Campylobacter colonies in Petri dishes. A reflectance spectral library of Campylobacter and non-Campylobacter contaminants was constructed for characterization of absorption features in wavelengths from 400 to 900 nm and for developing classification methods. Blood agar and Campy-Cefex agar were used as culture media. The study found that blood agar was the better culture medium than Campy-Cefex agar in terms of Campylobacter detection accuracy. Classification algorithms including single-band thresholding, band-ratio thresholding and spectral feature fitting were developed for detection of Campylobacter colonies as early as 24 h of incubation time. A band ratio algorithm using two bands at 426 and 458 nm chosen from continuum-removed spectra of the blood agar bacterial cultures achieved 97–99% of detection accuracy. This research has profound implications for early detection of Campylobacter colonies with high accuracy. Also, the developed hyperspectral reflectance imaging protocol is applicable to other pathogen detection studies.     

Comparison of personal, indoor, and outdoor exposures to hazardous air pollutants in three urban communities

Two-day average concentrations of 15 individual volatile organic compounds (VOCs) were measured concurrently in (a) ambient air in three urban neighborhoods, (b) air inside residences of participants, and (c) personal air near the breathing zone of 71 healthy, nonsmoking adults. The outdoor (O), indoor (I), and personal (P) samples were collected in the Minneapolis/St. Paul metropolitan area over three seasons (spring, summer, and fall) in 1999 using charcoal-based passive air samplers (3M model 3500 organic vapor monitors). A hierarchical, mixed-effects statistical model was used to estimate the mutually adjusted effects of monitor location, community, and season while accounting for within-subject and within-time-index (monitoring period) correlation. Outdoor VOC concentrations were relatively low compared to many other urban areas, and only minor seasonal differences were observed. A consistent pattern of P > I > O was observed across both communities and seasons for 13 of 15 individual VOCs (exceptions were carbon tetrachloride and chloroform). Results indicate that ambient VOC measurements at central monitoring sites can seriously underestimate actual exposures for urban residents, even when the outdoor measurements are taken in their own neighborhoods.     

Influence of aqueous food simulants on potential nanoparticle detection in migration studies involving nanoenabled food-contact substances

Research focused on assessing potential consumer exposure to nanoparticles released from nano-enabled food-contact materials (FCMs) has often reached conflicting conclusions regarding the detection of migrating nanoparticles. These conflicting conclusions, coupled with the potential for nanoparticles to be unstable in certain food simulants, has necessitated a closer look at the role played by food simulants recommended for use in nanoparticle migration evaluation. The influence of aqueous food simulants on nanoparticles under migration evaluation conditions is reported herein. The stability of silver nanoparticles (AgNP) spiked into three food simulants (water, 10% ethanol and 3% acetic acid) was investigated using asymmetric flow field-flow fractionation (AF4), ultrafiltration, electron microscopy (EM), and single-particle inductively coupled plasma mass spectrometry (sp-ICP-MS). While 3% acetic acid induced significant oxidative dissolution of AgNP to silver ions, there were very minor to no changes in the physicochemical properties of AgNP in water and 10% ethanol.