Transposon tagging in diploid strawberry.

Fragaria vesca was transformed with a transposon tagging construct harbouring amino terminally deleted maize transposase and EGFP (Ac element), NPTII, CaMV 35S promoter (P35S) driving transposase and mannopine synthase promoter (Pmas) driving EGFP (Ds element). Of 180 primary transgenics, 48 were potential launch pads, 72 were multiple insertions or chimaeras, and 60 exhibited somatic transposition. T1 progeny of 32 putative launch pads were screened by multiplex PCR for transposition. Evidence of germ-line transposition occurred in 13 putative launch pads; however, the transposition frequency was too low in three for efficient recovery of transposants. The transposition frequency in the remaining launch pads ranged from 16% to 40%. After self-pollination of the T0 launch pads, putative transposants in the T1 generation were identified by multiplex PCR. Sequencing of hiTAIL-PCR products derived from nested primers within the Ds end sequences (either P35S at the left border or the inverted repeat at the right border) of T1 plants revealed transposition of the Ds element to distant sites in the strawberry genome. From more than 2400 T1 plants screened, 103 unique transposants have been identified, among which 17 were somatic transpositions observed in the T0 generation. Ds insertion sites were dispersed among various gene elements [exons (15%), introns (23%), promoters (30%), 3' UTRs (17%) as well as intergenically (15%)]. Three-primer (one on either side of the Ds insertion and one within the Ds T-DNA) PCR could be used to identify homozygous T2 transposon-tagged plants. The mutant collection has been catalogued in an on-line database.

Qualitative and quantitative analyses of ginkgo terpene trilactones by liquid chromatography/sonic spray ionization ion trap mass spectrometry.

A liquid chromatography/sonic spray ionization mass spectrometry method (LC/SSI-MS) was developed for qualitative and quantitative analyses of ginkgo terpene trilactones. Five ginkgo terpene trilactones were successfully protonated for qualitative and quantitative analyses under the study conditions. The typical ion adducts were identified as (M + H)+, (M + NH4)+, and (M + Na)+. The limits of detection were achieved between 2.5 and 10 ng with RSD of 0.173-4.82% and a linear range of 10-80 ng with R2 = 0.991-0.999. This method was used to identify and quantify ginkgo terpene trilactones in extractions of ginkgo biloba leaves obtained from three different extraction methods. This is the first completely validated LC/MS method for quantification of ginkgo terpene trilactones. The factors that contributed to reduce the errors of identification and quantification of ginkgo terpene trilactones are systematically reported, and the advantages and disadvantages of LC/MS method in quantitative analysis are also discussed.

Enrichment of cheeses manufactured from cow's and sheep's milk blends with sheep-like species-related alkylphenols.

Enhancement of concentrations of species-related sheep-like alkylphenols, p- and m-cresols and 3- and 4-ethylphenols, in experimental Manchego-type cheeses manufactured from cow's and sheep's milk blends (80:20) by using arylsulfatases was investigated. A food-grade arylsulfatase from Aspergillus oryzae (ATCC 20719) was produced using a stimulatory medium, and crude dried cells were used as the enzyme source. Exogenous arylsulfatases from Helix pomatia and A. oryzae were added to cheese curd, and the amounts of species-related alkylphenols were measured. Arylsulfatase from H. pomatia released limited amounts of alkylphenols in the cheese only when used at a high level. Arylsulfatase from A. oryzae released substantial amounts of alkylphenols during 2 months of ripening. The concentrations of alkylphenols in A. oryzae arylsulfatase-treated cheese were comparable to the previously reported levels present in aged Manchego-type cheeses manufactured from pure sheep's milk.

Antioxidant protection of bulk fish oils by dispersed sugars and polyhydric alcohols.

Selected sugars (fructose, sucrose, or raffinose) and polyhydric alcohols (sorbitol or mannitol) were equilibrated directly with bulk fish oil (10% by weight, excess) and exposed to fluorescent lighting (2550 Lx) for 24 h at 5 degrees C. Data for room temperature-equilibrated samples revealed that polyols functioned as antioxidants in fish oil. Increased times and temperatures of equilibration (to 90-110 degrees C, 1-2 mmHg, to 2 h) greatly enhanced the antioxidant activity of polyols in fish oil exposed to light. Under accelerated oxidation conditions (60 degrees C) in the dark, dispersed sorbitol in bulk fish oil greatly suppressed the peroxide value, primarily by chelating transition metals, while fructose showed a limited antioxidant activity. Sugars with a lower molecular weight and smaller numbers of equatorial OH groups exhibited a higher rate of permeation of sugars into fish oil triacylglycerols and hence rendered greater antioxidant activities. The treatment of bulk fish oils with polyols and then using the oils in the preparation of emulsions greatly reduced their antioxidant activities as compared to those observed for treated bulk oils. The introduction of polyols dissolved in propylene glycol into bulk fish oils at 90 degrees C (0.025% polyol, 0.25 h of equilibration) provided a similar antioxidant activity to that imparted by the introduction of polyols into the oil by equilibrating excess polyols (10% by weight) with them at 90-110 degrees C for 2 h. However, regardless of the method of the introduction of polyols to bulk fish oil, an elevated temperature (90 degrees C) exposure during fish oil treatment was required to induce a notable antioxidant activity.

Controlling acrylamide in French fry and potato chip models and a mathematical model of acrylamide formation: acrylamide: acidulants, phytate and calcium.

We previously reported that in potato chip and French fry models, the formation of acrylamide can be reduced by controlling pH during processing steps, either by organic (acidulants) or inorganic acids. Use of phytate, a naturally occurring chelator, with or without Ca++ (or divalent ions), can reduce acrylamide formation in both models. However, since phytate itself is acidic, the question remains as to whether the effect of phytate is due to pH alone or to additional effects. In the French fry model, the effects on acrylamide formation of pH, phytate, and/or Ca++ in various combinations were tested in either blanching or soaking (after blanching) steps. All treatments significantly reduced acrylamide levels compared to control. Among variables tested, pH may be the single most important factor for reducing acrylamide levels, while there were independent effects of phytate and/or Ca++ in this French fry model. We also developed a mathematical formula to estimate the final concentration of acrylamide in a potato chip model, using variables that can affect acrylamide formation: glucose and asparagine concentrations, cut potato surface area and shape, cooking temperature and time, and other processing conditions.

Chemical intervention strategies for substantial suppression of acrylamide formation in fried potato products.

Prototype processes were developed for the substantial suppression of acrylamide formation (40-95% compared to untreated controls) in cut surface fried potato products using potato chips (crisps) as the primary model. The most efficacious procedures employed sequentially both surface preparation and subsequent acrylamide precursor complexation and/or competitive inhibition processing steps. Surface preparation processing involved either various low-temperature (50-75 degrees C) aqueous (5-30 min) or ca. 80% ethanol blanch solutions for various times (1-5 min) combined with aqueous leaching steps (1-10 min) to reduce concentration of acrylamide precursors in the critical frying zone of cut potato surfaces. Acrylamide precursor complexation and/or competitive inhibition processing strategies included immersion exposure of prepared cut potato surfaces to solutions or dispersions of various combinations of either calcium chloride, phytic acid, chitosan, sodium acid pyrophosphate, or N-acetylcysteine.

Characterization of the antioxidant activity of sugars and polyhydric alcohols in fish oil emulsions.

Polyols have been incorporated into fish oil emulsions as a means for the inhibition of lipid oxidation and suppression of fishy flavor. However, the role of sugars and polyhydric alcohols as antioxidants has not been clearly established. Selected polyols were evaluated for their performance as antioxidants and modifiers of oxidation pathways in a model system. Oil/water (O/W) emulsions were prepared with freshly steam-deodorized menhaden oil. A layer of emulsion in aluminum pans held at 5 degrees C was exposed to 2550 lx fluorescent lights for 24 h before peroxide values and volatile flavor compounds were analyzed by GC headspace entrainment procedure. Antioxidant activity was confirmed for fructose, sucrose, raffinose, sorbitol, or mannitol when incorporated at 16% of the aqueous phase into model fish oil-in-water emulsions. Peroxide values were suppressed 10-18% in treated samples compared to control samples. Viscosity data did not exclude possible contributions from a restricted oxygen diffusion mechanism in the antioxidant activity, but revealed that emulsion viscosity did not govern fish oil oxidation rates. Combining polyols with phenolic antioxidants (alpha-tocopherol, BHT, or TBHQ) frequently diminished the antioxidant activity compared to that for individual phenolic antioxidants, which was interpreted as indicating that the H-donating activity of phenolic antioxidants was hindered by the H-bonding activity of polyols. A viscosity-based inhibition of the retroaldol conversion of (E,Z)-2,6-nonadienal to (Z)-4-heptenal with a high fructose concentration (67%) was attributed to a restriction of molecular mobility of reactants, but the conversion was only slightly inhibited by the concentration of fructose (16%) used in experimental emulsions. The data supported a hypothesis that either or both free radical scavenging and transition state metal chelation activities were provided by polyols in fish oil emulsions. Also, polyols retarded the water-requiring retroaldol decomposition of (E,Z)-2,6-nonadienal to (Z)-4-heptenal in the model systems and the reaction may be involved in some suppression of fishy flavors in emulsions.

A method for extraction and quantification of Ginkgo terpene trilactones.

A method was developed for the extraction and quantification of pharmacologically active terpene trilactones (ginkgolides, bilobalide) from the tissues of Ginkgo biloba L. and pharmaceutical ginkgo products by RP-HPLC, based on the theory of terpene trilactones ionization. Four ginkgolides (GA, GB, GC, GJ) and bilobalide (BB) from both the ginkgo leaves and commercially available ginkgo extracts were quantitatively extracted by using this method. The recovery rate of the method was 97.5-100% with RSD of 1.2-2.8%. The detection limit was 0.05-0.1 microg, and the linear range was 0.1-12 microg. This detection limit represents a marked improvement over previously reported methods, suggesting the new method is a viable technique for routine analysis of ginkgo terpene trilactones in natural and commercial samples. The method reported by van Beek et al. in 1991 (van Beek, T. A.; Scheeren, H. A.; Rantio T.; Melger, W. C.; Lelyveld, G. P. J. Chromatogr. 1991, 543, 375-387.) was used as a reference method to monitor the accuracy of extraction and analysis in this study. SSI-MS technique was used to identify isolated target components. Carbohydrase treatment and solubility of terpene trilactones in various solvents were also discussed.

Musty Aroma Compounds Produced by Selected Molds and Actinomycetes on Agar and Whole Wheat Bread.

Musty aroma compounds produced by cultures of Streptomycetes odorifer , Streptomycetes griscus , Penicillium roqueforti , Aspergillus flavus , Aspergillus niger , and Botrytis cineria when grown on agar and whole wheat bread were isolated and identified using headspace entrainment and GC-MS analysis. Actinomycete cultures produced the most intense musty aromas, which were attributed to the presence of 2-methylisoborneol and geosmin, whereas P. roqueforti and B. cineria cultures produced an overall musty-fruity odor quality caused by the combination of 2-methylisoborneol and 8-carbon alcohols and ketones. Several musty compounds in the cultures were not identified including an intensely musty, cat-like aroma compound produced by A. flavus . Seven musty aroma-type categories are proposed to assist in defining musty taints produced by microorganisms in food and feedstuffs.

Sensory Characteristics of Reduced Nitrite Bacon Manufactured by the Wisconsin Process.

Bacon with a culture of lactic acid-forming bacteria, Pediococcus acidilactici , plus 0.7% sucrose and 40 or 80 mg sodium nitrite/kg (Wisconsin Process), and control bacon with 120 mg sodium nitrite/kg but no lactic acid bacteria and sucrose, were produced at three commercial bacon plants under production conditions. The bacon was stored under refrigeration for 5 to 8 wk, then subjected to sensory analyses by an experienced sensory panel. Quantitative descriptive visual analysis was performed on uncooked as well as cooked samples, and the cooked samples were served for quantitative descriptive sensory analysis. Results indicated that the test bacon with reduced amounts of sodium nitrite was as acceptable as the control bacon with no sugar and lactics, with the 80 mg/kg nitrite-bacon being the most preferred of all. These results and the results of botulinal challenge and nitrosamine tests indicate that the test process can be a satisfactory alternative to processing bacon by the conventional procedure with 120 mg sodium nitrite/kg.

Depletion of Sorbate from Different Media During Growth of Penicillium Species.

Penicillium cyclopium , Penicillium roqueforti , Penicillium viridicatum , Penicillium puberulum , Penicillium cyclopium (atypical strain), Penicillium crustosum and Penicillium lanoso-viride were isolated from spoiled cheese. These molds grew and depleted sorbate from media when the chemical was present initially at a concentration of up to 3,000, 10,000, 6,000, 12,000, 12,000, 7,000 and 3,000 ppm, respectively. A combination of paper chromatography and spectrophotometry was used to determine amounts of residual sorbate. Seventy-one to 100% of sorbate present initially was depleted from media by the various molds during 4-20 days of incubation at 21°C and 22-48 days at 4°C. The substrate influenced growth of mold and depletion of sorbate, but uniform behavior was not observed for all the Penicillium species studied. For example, presence of 3,000 ppm of sorbate plus 1% casein in the medium inhibited P. cyclopium and P. lanoso-viride but not the other five species. Concentration of sorbate (3,000 - 9,000 ppm) plus temperature (4, 12, 21 °C) were important for inhibitory action of the preservative on P. cyclopium , P. viridicatum , P. crustosum and P. lanoso-viride but not P. puberulum , P. cyclopium (atypical strain) which grew at 4 °C and depleted sorbate when the initial concentration was up to 9,000 ppm and P. roqueforti which grew at up to 6,000 ppm at the same temperature.

Microflora and Flavor of Wild Rice Fermented Under Different Conditions.

Changes in number and types of microorganisms in fermenting wild rice were studied. The effect of various microorganisms on keeping quality of wild rice during fermentation and on flavor of wild rice was also determined. Addition of microbial cultures and/or nutrient solutions did not increase the storage life of fermenting wild rice held at 21 or 5°C. Refrigeration of rice greatly increased the effective keeping time of the unprocessed grain. Periodic addition of a 0.1% (w/v) (NH4)2SO4 solution apparently decreased the acceptable storage life of refrigerated (5°C) rice. Although unprocessed rice could not be kept beyond 10-14 d at 21 °C without obvious changes in organoleptic quality, rice stored at 5°C remained acceptable for 7 weeks. Steaming wild rice for 15 min before fermentation generally caused a marked reduction in microbial load during the first few days of storage. Rice inoculated with Streptomyces sp. in a circulating nutrient medium to produce an "earthy" flavor was rapidly spoiled by microorganisms indigenous to rice. Cultures of Pseudomonas perolens and Pseudomonas taetrolens inoculated into rice stored at 5°C gave only mild methoxylated-pyrazine (green-earthy) flavors to resulting processed rice. In most treatments, bacteria and molds generally increased during the first week of fermentation, maintained a rather constant number for most of the remainder of the fermentation, and often decreased near the end of storage. Gram-negative, rod-shaped bacteria were the microorganisms most commonly isolated from fermenting wild rice. Many psychrotrophic bacteria were isolated from refrigerated (5°C) rice. Low-temperature storage greatly increased the keeping quality of rice, but unacceptable organoleptic changes eventually occurred at this temperature. Many bacteria isolated from wild rice were facultatively anaerobic Enterobacteriaceae . Although rice contained large populations of various types of microorganisms, the potentially hazardous Bacillus cereus and Pseudomonas aeruginosa were not found.