David C. Manns

Reduction of Patulin in Apple Cider by UV Radiation

The presence of the mycotoxin patulin in processed apple juice and cider presents a continual challenge to the food industry as both consumer health and product quality issues. Although several methods for control and/or elimination of patulin have been proposed, no unifying method has been commercially successful for reducing patulin burdens while maintaining product quality. In the present study, exposure to germicidal UV radiation was evaluated as a possible commercially viable alternative for the reduction and possible elimination of the patulin mycotoxin in fresh apple cider. UV exposure of 14.2 to 99.4 mJ/cm(2) resulted in a significant and nearly linear decrease in patulin levels while producing no quantifiable changes in the chemical composition (i.e., pH, Brix, and total acids) or organoleptic properties of the cider. For the range of UV doses tested, patulin levels decreased by 9.4 to 43.4%; the greatest reduction was achieved after less than 15 s of UV exposure. The method of UV radiation (the CiderSure 3500 system) is an easily implemented, high-throughput, and cost-effective method that offers simultaneous UV pasteurization of cider and juice products and reduction and/or elimination of patulin without unwanted alterations in the final product.

A core–shell column approach to a comprehensive high-performance liquid chromatography phenolic analysis of Vitis vinifera L. and interspecific hybrid grape juices, wines, and other matrices following either solid phase extraction or direct injection

Four high-throughput reverse-phase chromatographic protocols utilizing two different core-shell column chemistries have been developed to analyze the phenolic profiles of complex matrices, specifically targeting juices and wines produced from interspecific hybrid grape cultivars. Following pre-fractionation via solid-phase extraction or direct injection, individual protocols were designed to resolve, identify and quantify specific chemical classes of compounds including non-anthocyanin monomeric phenolics, condensed tannins following acid hydrolysis, and anthocyanins. Detection levels ranging from 1.2 ppb to 27.5 ppb, analyte %RSDs ranging from 0.04 to 0.38, and linear ranges of quantitation approaching five orders of magnitude were achieved using conventional HPLC instrumentation. Using C(18) column chemistry, the non-anthocyanin monomeric protocol effectively separated a set of 16 relevant phenolic compounds comprised flavan-3-ols, hydroxycinnamic acids, and flavonols in under 14 min. The same column was used to develop a 15-min protocol for hydrolyzed condensed tannin analysis. Two anthocyanin protocols are presented, one utilizing the same C(18) column, best suited for anthocyanidin and monoglucoside analysis, the other utilizing a pentafluorophenyl chemistry optimized to effectively separate complex mixtures of coexisting mono- and diglucoside anthocyanins. These protocols and column chemistries have been used initially to explore a wide variety of complex phenolic matrices, including red and white juices and wines produced from Vitis vinifera and interspecific hybrid grape cultivars, juices, teas, and plant extracts. Each protocol displayed robust matrix responses as written, yet are flexible enough to be easily modified to suit specifically tailored analytical requirements.

Patulin Reduction in Apple Juice from Concentrate by UV Radiation and Comparison of Kinetic Degradation Models between Apple Juice and Apple Cider

Patulin, a mycotoxin produced by several genera of fungi, including Byssochlamys, Aspergillus, and Penicillium, has been an important concern in apple cider and apple juice due to its toxicity and health consequences. In this study, the effects of UV on the patulin level, physical and chemical properties, and sensory attributes in apple juice from concentrate were investigated. Kinetic modeling of patulin reduction by UV radiation in apple juice from concentrate was calculated and compared with the degradation rate observed previously in apple cider. From an initial patulin contamination of approximately 1,000 ppb (μg/liter), the UV exposure, ranging from 14.2 mJ/cm(2) (one pass) to 99.4 mJ/cm(2) (seven passes), was successful in reducing patulin levels by 72.57% ± 2.76% to 5.14% ± 0.70%, respectively. Patulin reduction by UV radiation followed first-order kinetic modeling in a fashion similar to first-order microbial inactivation. An exponential correlation between UV exposure and the percentage of patulin remaining was observed, giving an r(2) value of 0.9950. Apple juice was repeatedly exposed to 14.2 mJ/cm(2) for each treatment, and patulin levels were significantly decreased when compared with the level obtained with the previous UV exposure treatment. While there were no significant differences in the percentages of titratable acidity and ascorbic acid (P > 0.05), there were minor yet random sampling differences in pH and degrees Brix (1 °Brix is 1 g of sucrose in 100 g of solution; the °Brix represents the soluble solids content of the solution as percentage by weight [%, wt/wt]) (P ≤ 0.05). A significant difference (P ≤ 0.05) in sensory perception for the finished apple juice was detected between the control and the full seven-pass UV radiation treatment using an experienced consumer panel and a triangle test. Patulin reduction by UV radiation from both the current study and a previous study involving apple cider was compared, which showed that both matrices strongly fit a first-order kinetic degradation model. However, the kinetic constant for degradation in apple juice was approximately 5.5 times greater than that observed in an apple cider matrix.

A next-generation marker genotyping platform (AmpSeq) in heterozygous crops: a case study for marker-assisted selection in grapevine

Marker-assisted selection (MAS) is often employed in crop breeding programs to accelerate and enhance cultivar development, via selection during the juvenile phase and parental selection prior to crossing. Next-generation sequencing and its derivative technologies have been used for genome-wide molecular marker discovery. To bridge the gap between marker development and MAS implementation, this study developed a novel practical strategy with a semi-automated pipeline that incorporates trait-associated single nucleotide polymorphism marker discovery, low-cost genotyping through amplicon sequencing (AmpSeq) and decision making. The results document the development of a MAS package derived from genotyping-by-sequencing using three traits (flower sex, disease resistance and acylated anthocyanins) in grapevine breeding. The vast majority of sequence reads (⩾99%) were from the targeted regions. Across 380 individuals and up to 31 amplicons sequenced in each lane of MiSeq data, most amplicons (83 to 87%) had <10% missing data, and read depth had a median of 220–244×. Several strengths of the AmpSeq platform that make this approach of broad interest in diverse crop species include accuracy, flexibility, speed, high-throughput, low-cost and easily automated analysis.

Bactericidal thurincin H causes unique morphological changes in Bacillus cereus F4552 without affecting membrane permeability

Thurincin H is an antilisterial bacteriocin produced by Bacillus thuringiensis SF361. It exhibits inhibitory activity against a wide range of Gram-positive foodborne pathogens and spoilage bacteria including Listeria monocytogenes, B. cereus, and B. subtilis. This hydrophobic, anionic bacteriocin folds into a hairpin structure maintained by four pairs of unique sulfur to α-carbon thioether bonds. As its hydrophobicity and structure are quite different from most archived bacteriocins, this study aimed to elucidate its mode of action and compare it with the mechanisms of other well-characterized bacteriocins. The results indicated that, although bactericidal to B. cereus F4552, thurincin H did not lead to optical density reduction or detectable changes in cell membrane permeability. B. cereus F4552 imaged by scanning electron microscopy after treatment with thurincin H at 32 × MIC showed regular rod-shaped cells, while only cells treated with thurincin H at the elevated levels of 256 × MIC showed loss of cell integrity and rigidity. Both concentrations caused greater than 99% of cell viability reduction. In contrast, nisin caused significant cell membrane permeability at concentration as low as 2 × MIC. These results indicated a difference in the mode of action for thurincin H compared with the generalized pore-forming mechanism of many lantibiotics, such as nisin.

Impact of Processing Parameters on the Phenolic Profile of Wines Produced from Hybrid Red Grapes Maréchal Foch, Corot noir, and Marquette

Phenolic extraction in hybrid and interspecific wine grape cultivars is poorly understood, especially in terms of the impact of fermentation and enological conditions on condensed tannins and anthocyanins. Following fractionation via solid-phase extraction and high-performance liquid chromatography, phenolic profiles of must and wine from red hybrid grape cultivars Maréchal Foch, Corot noir, and Marquette were examined to assess the impact of enzyme and tannin addition, cold soak, and hot press during vinification. Across cultivars, hot press treatments resulted in the greatest extraction of condensed tannin, anthocyanin, and other monomeric phenolic compounds in musts, and treatments that increased skin contact time or cellular degradation during fermentation produced higher concentrations of tannins, anthocyanins, and flavonols. However, these increases were transient, evincing incomplete carryover into finished wines. Depending on initial must extraction, diglucoside forms of anthocyanins were either selectively extracted or selectively retained throughout fermentation when compared to their monoglucoside counterparts. Typical of hybrid grapes, tannin concentrations across cultivars were low, even under hot press conditions. For condensed tannins and anthocyanins, a cultivar-specific, stable-state concentration and phenolic profile emerged regardless of fermentation conditions. Due to the high levels of diglucoside anthocyanins and low levels of condensed tannins, it is expected that the color development and profile in these wines produced from hybrid grape cultivars will be dictated by the monomeric anthocyanins and their potential role in copigmentation processes involving other monomeric phenolic species, as opposed to the formation of polymeric color pigments.

Next Generation Mapping of Enological Traits in an F2 Interspecific Grapevine Hybrid Family.

In winegrapes (Vitis spp.), fruit quality traits such as berry color, total soluble solids content (SS), malic acid content (MA), and yeast assimilable nitrogen (YAN) affect fermentation or wine quality, and are important traits in selecting new hybrid winegrape cultivars. Given the high genetic diversity and heterozygosity of Vitis species and their tendency to exhibit inbreeding depression, linkage map construction and quantitative trait locus (QTL) mapping has relied on F1 families with the use of simple sequence repeat (SSR) and other markers. This study presents the construction of a genetic map by single nucleotide polymorphisms identified through genotyping-by-sequencing (GBS) technology in an F2 mapping family of 424 progeny derived from a cross between the wild species V. riparia Michx. and the interspecific hybrid winegrape cultivar, ‘Seyval’. The resulting map has 1449 markers spanning 2424 cM in genetic length across 19 linkage groups, covering 95% of the genome with an average distance between markers of 1.67 cM. Compared to an SSR map previously developed for this F2 family, these results represent an improved map covering a greater portion of the genome with higher marker density. The accuracy of the map was validated using the well-studied trait berry color. QTL affecting YAN, MA and SS related traits were detected. A joint MA and SS QTL spans a region with candidate genes involved in the malate metabolism pathway. We present an analytical pipeline for calling intercross GBS markers and a high-density linkage map for a large F2 family of the highly heterozygous Vitis genus. This study serves as a model for further genetic investigations of the molecular basis of additional unique characters of North American hybrid wine cultivars and to enhance the breeding process by marker-assisted selection. The GBS protocols for identifying intercross markers developed in this study can be adapted for other heterozygous species.

Development of a Homologous Expression System for and Systematic Site-Directed Mutagenesis Analysis of Thurincin H, a Bacteriocin Produced by Bacillus thuringiensis SF361

ABSTRACT Thurincin H is an antimicrobial peptide produced by Bacillus thuringiensis SF361. With a helical back bone, the 31 amino acids of thurincin H form a hairpin structure maintained by four pairs of very unique sulfur-to-α-carbon thioether bonds. The production of thurincin H depends on a putative gene cluster containing 10 open reading frames. The gene cluster includes three tandem structural genes (thnA1, thnA2, and thnA3) encoding three identical 40-amino-acid thurincin H prepeptides and seven other genes putatively responsible for prepeptide processing, regulation, modification, exportation, and self-immunity. A homologous thurincin H expression system was developed by transforming a thurincin H-deficient host with a novel expression vector, pGW133. The host, designated B. thuringiensis SF361 ΔthnA1 ΔthnA2 ΔthnA3, was constructed by deletion of the three tandem structural genes from the chromosome of the natural thurincin H producer. The thurincin H expression vector pGW133 was constructed by cloning the thurincin H native promoter, thnA1, and a Cry protein terminator into the Escherichia coli-B. thuringiensis shuttle vector pHT315. Thirty-three different pGW133 variants, each containing a different point mutation in the thnA1 gene, were generated and separately transformed into B. thuringiensis SF361 ΔthnA1 ΔthnA2 ΔthnA3. Those site-directed mutants contained either a single radical or conservative amino acid substitution on the thioether linkage-forming positions or a radical substitution on all other nonalanine amino acids. The bacteriocin activities of B. thuringiensis SF361 ΔthnA1 ΔthnA2 ΔthnA3 carrying different pGW133 variants against three different indicator strains were subsequently compared.

Shelf-Life Evaluation of Natural Antimicrobials for Concord and Niagara Grape Juices

This study was conducted to evaluate the effectiveness of natural antimicrobials for shelf-life extension of cold-filled still and carbonated Concord and Niagara grape juices, which have traditionally been preserved with chemical preservatives. Commercial juices were inoculated with a spoilage yeast cocktail of Dekkera, Kluveromyces, Brettanomyces, and Zygosaccharomyces at 10(2) and 10(4) CFU/ml. The following agents were added to still juices: no preservative (negative control), 0.05% potassium sorbate plus 0.05% sodium benzoate (positive control), 0.1 or 0.2% cultured dextrose, 250 ppm of dimethyldicarbonate (DMDC), 10 or 20 ppm of natamycin, and 250 ppm of DMDC plus 5 or 10 ppm of natamycin. Carbonated juice was treated with the negative control, positive control, and 250 ppm of DMDC plus 10 ppm of natamycin. Microbial stability of samples was assessed every 2 weeks during 6 months of storage at 21°C by yeast enumeration and measurement of turbidity, pH, and °Brix. Juices were deemed spoiled when yeast counts exceeded 10(6) CFU/ml. Cultured dextrose was not effective at levels tested in both types of juice. The most promising results were obtained with DMDC and natamycin combination treatments in still Niagara juice and in carbonated Concord and Niagara juices. In these treatments, shelf-life extension similar to that of the positive control (153 to 161 days) was achieved while maintaining similar turbidity, pH, and °Brix. Spoiled juices had lower pH and °Brix values and higher turbidity due to microbial activity and increased in microbial levels.

Reaction Kinetics of Monomeric Anthocyanin Conversion to Polymeric Pigments and Their Significance to Color in Interspecific Hybrid Wines

The color stability of red wines produced from interspecific hybrid grapes, which is partially dependent on anthocyanin diglucosides, is not well understood. In this study, the rate of decrease of monomeric anthocyanins as they polymerized to polymeric pigments due to the presence of excess catechin and acetaldehyde was measured in model wine using HPLC. Colorimetry was used to measure L*, a*, and b* values, hue angle, and change in color (ΔE). Concentrations of individual diglucosides decreased more slowly than monoglucosides. When monoglucosides and diglucosides were combined, the reaction rate of monoglucosides was slower than that of monoglucosides alone. Hue angles described transitions from red to red-orange, orange, or orange-yellow as anthocyanin-specific changes occurred. The evolution in color represents dynamic reactions between anthocyanins, catechin, and acetaldehyde. Consequently, wines containing high concentrations of diglucosides, such as those produced from interspecific hybrid grapes, will form less polymeric pigment than wines containing primarily monoglucosides.