The efficacy of various fractions of Peanut Skin Extract (PSE) in combination with sodium polyphosphate (SPP; BekaPlus^TM FS, BK Giulini) were tested in apple juice medium (AJM, a model juice system) against the economically important spoilage yeasts Zygosaccharomyces bailii and Zygosaccharomyces bisporus.

Z. bailii (ATCC 60483) or Z. bisporus (Y108, an industrial isolate) were inoculated in AJM or AJM containing SPP, PSE or combinations of SPP and PSE, then incubated at 30°C for up to 120 h (5 d). Both whole PSE and 9 individual HPLC-separated PSE fractions were tested. A Bioscreen C automated turbidimeter determined the impact of PSE on yeast growth. After 120 h, samples were taken from each Bioscreen well, serially diluted and plated onto Yeast Mold agar for enumeration. Initial experiments with Z. bailii were used to determine the minimum inhibitory concentrations (MICs) of SPP and whole PSE. A single sub-MIC concentration of SPP (0.013%) was then evaluated in combination with a concentration of whole PSE that was not inhibitory alone, yet yielded complete inhibition in the presence of 0.013% SPP. This concentration, 2 mg/mL, was chosen as a "benchmark" level for further testing of PSE fractions with and without 0.013% SPP against both Z. bailii and Z. bisporus. This rational testing approach enabled us to minimize variables, focus our screening approach and avoid wastage of valuable PSE samples.

Twenty-four treatments were tested (in duplicate) including: no-antimicrobial control (AJM only), AJM plus 0.013% SPP and the following PSE treatments with and without 0.013% SPP: whole PSE, nine HPLC-separated fractions derived from whole PSE and designated fractions A-I, and a catechin control. Uninoculated AJM served as an uncontaminated control.

This screen was replicated in 3 separate experiments and data were analyzed using SAS software. Three variables were analyzed: treatment, time, and treatment as a function of time (treatment*time). A Tukey pairwise comparison indicated that whole PSE and the fractions were significantly different from the control.

Endpoint data were analyzed to determine whether the PSE and SPP were killing the cells or keeping them static. The CFU/mL was determined after 120 h of treatment. Based on the significant P-values for both yeasts (P-values <0.0001), it was determined that the treatments had an effect on growth.

A "reconstituted" whole PSE comprised of known levels of each HPLC-separated fraction was formulated and compared to "natural" whole PSE, in which the levels of each component remain unknown. The comparison tested the hypothesis that the reconstituted whole PSE, containing normalized levels of each fraction, might be more inhibitory at lower concentrations than the natural whole PSE, whose activity might be dependent on one or more dominant fractions. However, our results indicated that both PSEs had identical activities against both spoilage yeasts with and without SPP.

The physical mode of action of whole PSE and select fractions against Z. bailii, with and without SPP was investigated at various timepoints during exposure in AJM using a dye exclusion assay and flow cytometric analysis, which was conducted in parallel with optical density and cell enumeration measurements, made over a 24 h period. Controls included AJM-only and SPP-only treatments, to evaluate the impact of the cell cycle and SPP alone on membrane permeability. Although cell membranes became transiently permeable to propidium iodide at early points during the growth curve without PSE or SPP treatment, these treatments resulted in additional permeabilization of the cell membrane, which serves a primary barrier function for the cell.

Our data demonstrated increased inhibitory action of combined PSE and SPP suggests growth suppression results from the combined effects of two agents capable of permeabilizing the cell membrane. Based primarily on studies involving the effects of SPP on Gram-negative bacteria, the observed cell permeabilization is likely related to the chelation of structurally, nutritionally or metabolically essential cations. This chelation could therefore impact cell physiology (cell membrane permeabilization), availability of required metals (siderophore-like activity), or enzyme systems dependent on these metals as cofactors. It is possible that these antimicrobials also act at other points within the cell, but additional assay approaches, including genomic-based methods may be required for such determinations.

Although clear (and significant) effects/differences were seen between PSE-only and combined PSE-SPP treatments, whether these are additive or synergistic requires additional analyses. The Fractional Inhibitory Concentration (FIC) method integrates data from the MICs of two antimicrobials alone and from their combination to derive a point value indicative of one of three outcomes for antimicrobial interactions: antagonistic effects, additive effects or synergistic effects. A FIC value (1.6) was determined for the effect of whole PSE on Z. bailii , indicating an additive effect.

While the combination of various PSE treatments (whole PSE, fractions F, G, H, I) with SPP resulted in full growth suppression, final cell counts after 120 h indicated a maximum 3 log reduction of viable cells as compared to the control culture (whole PSE), with other treatments yielding ~1 - 1.5 log reductions. These data indicate that while not all combinations are lethal to these spoilage yeasts, this approach is a promising method for suppressing the growth of contaminants, if present. This conclusion is fairly conservative, given that natural contaminants will likely be present at comparatively low levels, compared to the challenge level used here of 105 CFU/300 μL AJM (roughly a contamination level of 108 CFU per 12 oz container of a soda or juice beverage, an extremely unlikely contamination level for a processor following Good Manufacturing Practices).

The effects of the addition of PSE and SPP to full-strength apple juice and to AJM were analyzed using a Hunter L*a*b* system. Statistical analysis indicated that there was a significant difference between the colors within the groups. To evaluate the potential for negative organoleptic impact of the antimicrobial treatments on both apple juice and AJM, aroma was analyzed using gas chromatography upon the addition of SPP and PSE to both apple juice and AJM. No off aromas were detected in either liquid upon the addition of these compounds, suggesting that the organoleptic properties of the beverage or medium would not be affected by the antimicrobial treatments.

Together, our data suggest that the combined use of PSE and SPP is a promising natural approach for preserving juices or potentially other beverages at risk for spoilage by Zygosaccharomyces spp.