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Factors affecting Alicyclobacillus acidoterrestris growth and guaiacol production and controlling apple juice spoilage by lauric arginate and ϵ-polylysine
LWT - Food Science and Technology 119 (2020) 108883
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Factors affecting Alicyclobacillus acidoterrestris growth and guaiacol production and controlling apple juice spoilage by lauric arginate and ϵpolylysine
T
Xiaohuan Hua, En Huangb,∗, Sheryl A. Barringera, Ahmed E. Yousefa,c a
Department of Food Science and Technology, The Ohio State University, 2015 Fyffe Rd., Columbus, OH, 43210, USA Department of Environmental and Occupational Health, University of Arkansas for Medical Sciences, 4301 West Markham Street, # 820, Little Rock, AR, 72205, USA c Department of Microbiology, The Ohio State University, 484 West 12th Avenue, Columbus, OH, 43210, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: Alicyclobacillus acidoterrestris Guaiacol Lauric arginate ϵ-polylysine
Growth of Alicyclobacillus acidoterrestris in fruit juices has been associated with the release of off-odor compounds, such as guaiacol. Guaiacol release was quantified using Selected Ion Flow Tube Mass Spectrometry (SIFT-MS) and method's minimum detection level was 3.6 ppb. The amount of guaiacol released was dependent on the amount of vanillin added to the medium. Sucrose-supplemented yeast starch glucose broth (SS-YSG broth; pH 3.7) and apple juice were spiked with vanillin (10 mg/L), inoculated with A. acidoterrestris and incubated at different temperatures (20°C–45 °C) for up to 528 h. At the temperatures that permitted growth, the bacterium grew slower in apple juice than in SS-YSG broth. Bacterial growth and guaiacol production were prevented in apple juice and inhibited in SS-YSG, when the inoculated products were incubated at 20 °C and 25 °C. When Alicyclobacillus was inoculated in SS-YSG adjusted to pH 2.7–6.7 and incubated at 37 °C, growth of the bacterium and guaiacol production were suppressed at pH 6.7 for up to 354 h and delayed at pH 2.7. Lauric arginate and ϵpolylysine inactivated Alicyclobacillus cells with no guaiacol production in both SS-YSG and apple juice. However, ϵ-polylysine was more effective than lauric arginate when the SS-YSG (pH 3.7) was inoculated with Alicyclobacillus spores.
1. Introduction Alicyclobacillus acidoterrestris is a thermophilic, acidophilic, sporeforming microorganism. The bacterium can survive over a wide temperature range, and it grows well at 42 °C–60 °C (Chang & Kang, 2004). Spores of the bacterium can survive commercial pasteurization of fruit juices; subsequently, these spores may germinate and spoil the juice by producing an odorous compound, guaiacol, when the population reaches 105 CFU/mL (Chang & Kang, 2004; Spinelli, Sant'Ana, Rodrigues-Junior, & Massaguer, 2009). It is estimated that 11.4% of apple juice concentrate in Argentina is contaminated with Alicyclobacillus (Oteiza, Ares, Sant'Ana, Soto, & Giannuzzi, 2011). A. acidoterrestris can produce guaiacol at 25 or 45 °C, but the bacterium grows slower at 25 than at 45 °C in Bacillus acidoterrestris medium (Witthuhn, Smith, Cameron, & Venter, 2013). The bacteria also can grow in media with pH 2.5 to 6.0 (Chang & Kang, 2004). Guaiacol is presumed to be the main factor that leads to the smoky or medicinal off-flavor in juice spoiled by Alicyclobacillus spp. The odor
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threshold of guaiacol in orange, apple, and noncarbonated fruit juices is about 2 ppb (Chang & Kang, 2004; Pettipher, Osmundson, & Murphy, 1997). Guaiacol is produced by a biological conversion of ferulic acid to vanillic acid and then decarboxylation to guaiacol (Chang & Kang, 2004). Vanillin, vanillic acid and ferulic acid, which are naturally present in some fruits and juice products, are three important precursors of guaiacol, but only vanillin or vanillic acid can be decomposed by Alicyclobacillus spp. directly without participation of other bacteria (Witthuhn, Merwe, Venter, & Cameron, 2012). Higher concentrations of substrates (vanillin or vanillic acid) tend to correlate with higher final amounts of guaiacol by A. acidoterrestris in Bacillus acidoterrestris broth (Witthuhn et al., 2012). In this investigation, generally-recognized as safe (GRAS) antimicrobial agents were investigated as potential treatment for controlling the growth of A. acidoterrestris and release of guaiacol in fruit juices. Lauric arginate is a new natural cationic surface-active molecule which has a broad antimicrobial activity (Rinrada, Aran, & Catherine, 2014). Its antimicrobial efficacy has been tested in meat products
Corresponding author. E-mail addresses: [email protected] (X. Hu), [email protected] (E. Huang), [email protected] (S.A. Barringer), [email protected] (A.E. Yousef).
https://doi.org/10.1016/j.lwt.2019.108883 Received 12 August 2019; Received in revised form 5 November 2019; Accepted 24 November 2019 Available online 24 November 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
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were incubated at 37 °C for 2 days with agitation at 200 rpm. When a mixed culture of the two strains was used, equal volumes of the overnight culture were combined and an aliquot of the mixture (e.g., 1 mL) was used to inoculate the test medium.
(Benli, Sanchez-Plata, & Keeton, 2011) but not in juices. According to the U.S. Food and Drug Administration (FDA), lauric arginate is a GRAS compound and it can be used in beverages up to 200 ppm (Food and Drug Administration, 2005). Similarly, ε-polylysine is a GRAS antimicrobial food additive and it can be used in fruit-flavored drink up to 250 ppm (Food and Drug Administration, 2004 and 2010). The compound has antimicrobial activity against yeasts, molds, Gram-positive and Gram-negative bacteria (Chang, Lu, Park, & Kang, 2010). ε-polylysine can inhibit bacterial growth, mainly through electrostatic adsorption to the bacterial cell surface and its cationic properties which can destroy cell membrane and finally leads to cell death (Chang et al., 2010; Shima, Matsuoka, Iwamoto, & Sakai, 1984; Yoshida & Nagasawa, 2003). Both ε-polylysine (300 mg/L) and lauric arginate (200 mg/L) have been reported to effectively reduce Salmonella population on inoculated chicken carcasses (Benli et al., 2011). Cai et al. (2015) reported that ε-polylysine inhibited the growth of the vegetative cells of A. acidoterrestris but its effect on bacterial spores was not studied. One of the objectives of this study was to determine the factors that are conducive to growth of A. acidoterrestris and release of guaiacol in liquid media. These factors were medium type (microbiological medium vs. apple juice), medium pH (2.7–6.7), storage temperature (20–45 °C), and vanillin concentration. The main objective was to control juice spoilage using two emerging antimicrobial additives, lauric arginate, and ε-polylysine. Quantification of synthesized guaiacol was accomplished using Selected Ion Flow Tube Mass Spectrometry (SIFT-MS), an analytical system for detection and quantification of volatile molecules at parts per billion (ppb) levels (Smith & Španěl, 2005).
2.4. Preparing Alicyclobacillus acidoterrestris spore suspensions Spore suspensions of Alicyclobacillus ATCC 49025 and OSYE were prepared as described previously (Palop et al., 2000). Cell suspensions were spread onto the sporulation agar medium, and the plates were incubated at 45 °C for 4 days. The growth (cells and spores) on the agar medium was removed with a sterile microscope slide and transferred into a 2-ml centrifuge tube with sterilized water to a final volume of 1 ml. The suspension was centrifuged (15 min, 16,000 x g, 4 °C), the supernatant was decanted and the sediment was re-suspended in 50% (v/v) aqueous ethanol and held for 30 min, to destroy non-sporulated cells. The suspension was centrifuged, the supernatant was discarded and the sedimented spores were re-suspended in sterile water; this process was repeated three times. The final sediment was re-suspended in sterile distilled water, to a final volume of 1 mL, and heated at 80 °C for 10 min. Spore count in the suspension was determined, and the suspension was held at 4 °C until use. 2.5. Guaiacol production by Alicyclobacillus at different concentrations of vanillin Samples of SS-YSG broth (pH 3.7) were inoculated with an inoculum made of equal volumes of A. acidoterrestris ATCC 49025 and OSYE cell suspensions. Vanillin (Sigma-Aldrich Co.) stock solution was added to the inoculated media to achieve the following final concentrations: 0.25, 0.5, 1.0, and 2.0 mg/L. Three replicates of this treatment were prepared. Broth, with or without added vanillin, was incubated at 37 °C, and tested after 24 h, when guaiacol reached its highest concentration. The minimum concentration of guaiacol in the headspace that was significantly different from the background (p < 0.05), and the corresponding vanillin concentration were recorded. The amounts of guaiacol released in the headspace, as a function of vanillin concentration, were determined as described below. The limit of detection and limit of quantification for guaiacol were determined to be 3.6 ppb.
2. Materials and methods 2.1. Media Yeast Starch Glucose (YSG) broth was used to culture and enumerate Alicyclobacillus strains (Goto et al., 2002). YSG broth was prepared to contain 0.2% yeast extract (IBI Scientific, IA, USA), 0.2% soluble starch (J. T. Barker, NJ, USA) and 0.1% glucose (Becton, Dickinson and Company, MD, USA). The medium was adjusted to pH 3.7, using 6M HCl, before autoclaving. YSG agar was prepared by adding 1.5% agar to YSG broth. Sucrose-supplemented YSG broth (SSYSG) was prepared from acidified YSG broth by adding sucrose to 8% level, to simulate fruit juice products (pH 3.7). Sporulation agar medium (Palop, Álvarez, Raso, & Condón, 2000) was prepared by mixing the following two solutions at equal volume after being sterilized separately. The first solution was prepared by mixing 1 g yeast extract, 0.2 g (NH4)2SO4, 0.25 g CaCl2, 0.5 g MgSO4, 1 g glucose, 0.6 g KH2PO4 with 500 mL distilled water, and adjusting the pH to 4.0 with 6 M HCl. The second solution was prepared by dissolving 20 g agar in 500 mL distilled water.
2.6. Measurement of guaiacol by Selected Ion Flow Tube Mass Spectrometry Selected Ion Flow Tube Mass Spectrometry (SIFT-MS; Voice 200; Syft Technologies Ltd., Christchurch, New Zealand) was used previously to detect volatiles in food (e.g., Dryahina, Smith, & Španěl, 2018; Huang & Barringer, 2011). We adopted SIFT-MS methods for guaiacol measurement under Selected Ion Monitoring mode. Guaiacol reacted with the precursor ion, NO+, to exclusively produce the positively-charged guaiacol ion, C7H8O2+, with m/z of 124. The internal standard, 2-ethoxyphenol, reacted with precursor ion O2+ to produce C8H10O2+, with m/z of 138. Volatile concentrations were measured for 60 s and averaged. The guaiacol concentration in the headspace of a medium was standardized using the concentration of the internal standard, 2-ethoxyphenol.
2.2. Bacterial strains A. acidoterrestris ATCC 49025 and A. acidoterrestris OSYE were tested in this study. The strain OSYE was isolated from spoiled, thermallyprocessed, commercial juice and provided by a food processor (Yousef, 2015). Morphology of A. acidoterrestris OSYE was consistent with that of the ATCC 49025 strain, and its ability to produce guaiacol in juice was verified. Stocks of these strains were held in YSG broth with 40% glycerol at −80 °C.
2.7. Alicyclobacillus growth and guaiacol production in different media at different temperatures Aliquots (100 mL, each) of SS-YSG broth (pH 3.7) or apple juice (Old Orchard, LLC Co, Sparta, MI, USA) were mixed with 2-ethoxyphenol (final concentration, 1.068 μg/mL) as an internal standard, vanillin (Sigma-Aldrich) at a final concentration of 10 mg/L, as a substrate for guaiacol, and 1 mL mixture of the overnight culture of A. acidoterrestris ATCC 49025 and OSYE, into a 500-mL sterilized Pyrex glass bottle. In order to test the influence of incubation temperature,
2.3. Culturing Alicyclobacillus acidoterrestris Portions (loop-full) of frozen stocks of A. acidoterrestris, ATCC 49025 or OSYE were streaked onto YSG agar plates and incubated at 37 °C for 2 days. An isolated colony was then transferred into a 50-mL centrifuge tube containing 30 mL YSG broth. The contents of the centrifuge tubes 2
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bottles were held at 20, 25, 37, or 45 °C and sampled at various incubation intervals; a bottle and its contents represent one sample. To analyze volatiles in the headspace, bottles were held during the test in a water bath at the appropriate temperature (37 °C) for 1 h to volatilize guaiacol and 2-ethoxyphenol. Concentration of these compounds in the bottle headspace was measured using SIFT-MS, as described in section 2.6. At every incubation temperature, samples were tested at 4-time intervals. The sampling times varied because cell growth was substantially different at different temperature and media. After testing for guaiacol, Alicyclobacillus population in the bottle contents was determined as follows. Decimal dilutions were prepared and spread-plated onto YSG agar. Inoculated plates were incubated at 37 °C for 2 days before enumeration. Fig. 1. Relationship between vanillin concentration in sucrose-supplemented yeast starch glucose broth, pH 3.7, and amount of guaiacol (ppb) released in the headspace of Alicyclobacillus acidoterrestris culture. Guaiacol concentration was determined using Selected Ion Flow Tube Mass Spectrometry. The experiment was repeated three times and averages ( ± standard deviation) are reported.
2.8. Alicyclobacillus growth and guaiacol production in SS-YSG broth at different pH values SS-YSG broth was adjusted to different pH values (2.7, 3.7, 4.7, and 6.7) with 6 M HCl. The broth was prepared and inoculated as described previously (section 2.7), and incubated at 37 °C. For every pH level, guaiacol concentration in the headspace and bacterial counts were tested for up to six incubation intervals. The sampling times varied because cell growth was substantially different at different medium pH. Replicate samples were tested at every time interval. The experiment was repeated 3 times.
antimicrobial compound added). No growth was observed in the noninoculated apple juice. During testing of every batch, guaiacol concentrations in the headspace, as well as bacterial counts, were determined as described previously. 2.11. Statistical analysis
2.9. Minimum inhibitory concentrations of antimicrobial compounds against Alicyclobacillus strains
One-way analysis of variance (ANOVA) and Tukey's post hoc analysis (R Studio Statistical Software, RStudio, Inc, Richmond Hill, ON, Canada) were used to determine statistical difference between treatments. p value of 0.05 was used for indicating significant difference.
A commercial preparation of lauric arginate (CytoGuard™ LA 20, 10% lauric arginate; A&B Ingredients, Inc., Fairfield, NJ, USA) and, ϵpolylysine (95% purity; Siveele, Breda, The Netherlands) were tested in this study. These two antimicrobial agents were dissolved in water and sterilized by passing through a 0.22 μm membrane filter. Antimicrobial efficacy of these agents was tested by measuring their minimum inhibitory concentrations (MICs) against A. acidoterrestris ATCC 49025 and OSYE using the method of Clinical and Laboratory Standards Institute (CLSI, 2006). Briefly, a series of lauric arginate dilutions were prepared using distilled water, then 50 μL of these solutions were dispensed in wells of a 96-well plate and mixed with 50 μL 2X concentration of SS-YSG broth (pH 3.7). Overnight culture of A. acidoterrestris ATCC 49025 or OSYE (1 μL) was added to each well. The final concentrations of lauric arginate in wells were 2.35, 4.7, 9.4, and 18.8 μg/mL. Similarly, ϵ-polylysine was diluted and tested as described for lauric arginate treatment to produce final concentrations of 18.6, 37.1, 74, and 149 μg/mL. Concentrations of lauric arginate and ϵpolylysine suitable for testing their MIC were determined based on preliminary experiments. The 96-well plates were incubated at 37 °C for 24 h and bacterial growth was measured as absorbance readings at 600 nm using a microtiter plate reader.
3. Results and discussion 3.1. Guaiacol production at different concentrations of vanillin The correlation between the substrate (vanillin) concentration and guaiacol production by A. acidoterrestris was linear, in the range of 0.1–2 mg/L vanillin (Fig. 1). The analytical method used (SIFT-MS technology) reliably allowed for the detection of 3.6 ppb of guaiacol in the headspace when 0.1 mg/L vanillin was added to the medium, which was the limit of quantification. In a previous study, A. acidoterrestris produced 62 and 171 mg/L guaiacol when the concentration of vanillin was at 100 and 1000 mg/L, respectively (Witthuhn et al., 2012). 3.2. The influence of temperature on bacterial growth and guaiacol production in SS-YSG at pH 3.7 and in commercial apple juice When SS-YSG broth (pH 3.7) was used as a medium, the incubation temperature in the range of 20 °C and 45 °C affected the time A. acidoterrestris took to reach its stationary phase (18.5 to > 327 h), but it did not influence the final population size (6.5–7.2 log CFU/mL) considerably (Fig. 2). The higher the temperature, the faster was the growth of the bacterium in this medium. Growth of A. acidoterrestris in apple juice was affected similarly by the incubation temperature; however, juice samples held only at 37 and 45 °C supported the growth of the bacterium (Fig. 2). Under growth-permitting conditions, A. acidoterrestris grew much faster in SS-YSG broth than in apple juice; for example, at 45 °C, maximum population was attained after 18.5 h in SSYGS and 76 h in apple juice. A. acidoterrestris is a thermophilic bacterium and its optimum growth temperature ranges from 42 to 60 °C (Chang & Kang, 2004). Other researchers also reported that A. acidoterrestris strains grew slower at 25 than 45 °C in Bacillus acidoterrestris broth (Witthuhn et al., 2013). A. acidoterrestris isolate 2498 grew at 25, 35, and 44 °C but
2.10. Control of Alicyclobacillus growth and guaiacol production in SS-YSG broth and apple juice with antimicrobial compounds The effect of the antimicrobial compounds on Alicyclobacillus growth and guaiacol production was tested as follows. Bottles containing sterilized SS-YSG broth (pH 3.7) or shelf stable apple juice were spiked with sterilized ϵ-polylysine or lauric arginate, at their MIC levels, 1 mL of vanillin solution (final concentration, 10 mg/mL), and 1 mL overnight culture (cells) or 0.1 mL spore suspension of A. acidoterrestris ATCC 49025 or OSYE. Final concentration of cells or spores, in the mixture, was 103–104 CFU/mL and final volume was ~100 mL. The inoculated bottles were incubated at 37 °C for 48 h for SS-YSG broth and 72 h for apple juice. In every batch, there were three side-by-side treatments: lauric arginate, ϵ-polylysine and control (i.e., no 3
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Fig. 3. Guaiacol production by Alicyclobacillus acidoterrestris ATCC 49025 and OSYE mixed populations in sucrose-supplemented yeast starch and glucose (SSYSG, pH 3.7) broth and apple juice, as influenced by incubation temperature. Panel A, SS-YSG broth; panel B, apple juice. Symbols: 20 °C (circle); 25 °C (diamond); 37 °C (triangle); 45 °C (square). No guaiacol was detected in juice at 20 °C or 25 °C (Panel B) for up to 326 h; data not shown.
Fig. 2. Growth of Alicyclobacillus acidoterrestris ATCC 49025 and OSYE mixed populations (Log CFU/mL) in sucrose-supplemented yeast starch and glucose (SS-YSG, pH 3.7) broth and apple juice, as influenced by incubation temperature. Panel A, SS-YSG broth; panel B, apple juice. Symbols: 20 °C (circle); 25 °C (diamond); 37 °C (triangle); 45 °C (square). No growth was observed in juice at 20 °C or 25 °C (Panel B) for up to 528 h; data not shown.
when incubation temperature increased from 32 to 50 °C (Chang, Park, & Kang, 2015). Others also reported that in YSG broth, A. acidoterrestris can produce guaiacol at 25 or 45 °C, and longer time was needed to produce guaiacol at 25 than 45 °C (Witthuhn et al., 2013). On the contrary, other researchers found no guaiacol formation at 25 °C, in a different growth medium with different Alicyclobacillus strains, during 48 h of incubation (Chang et al., 2015). Therefore, storage temperature, medium constituents, and bacterial strain are important factors controlling the growth of A. acidoterrestris and the production of guaiacol.
it didn't grow at 4 °C in apple or orange juice (Pettipher et al., 1997). There are ω-alicyclic fatty acids in the cytoplasmic membrane of A. acidoterrestris, which protect the bacteria from high temperature and low pH conditions (Chang & Kang, 2004). In apple juice, there was no bacterial growth at 20 and 25 °C (Fig. 2). Yeast extract, glucose and starch in the YSG broth are important nutrients which support the growth of bacteria. Therefore, in a favorable media, such as YSG broth, ambient temperature (e.g., 25 °C) can only delay bacterial growth, while in nutrient-limited media, such as apple juice, storage at room temperature can inhibit the growth of the bacterium. Guaiacol production is highly related to the bacterial population size (Figs. 2 and 3). In apple juice, no guaiacol production occurred at 20 and 25 °C, because there was no bacterial growth. There was guaiacol production in SS-YSG broth at all temperatures tested (Fig. 3). Temperature did not influence the maximum amount of guaiacol produced in this medium, but it did impact the time for guaiacol to reach its highest concentration. There was no significant difference (p < 0.05) in the maximum amount of guaiacol produced in SS-YSG broth and juice samples, at temperatures that permitted bacterial growth (Fig. 3). Bacteria grew faster at 37 or 45 °C than at 20 or 25 °C, leading to different times-to-guaiacol-production under different temperature conditions. In a previous study, guaiacol formation accelerated
3.3. Impact of pH on bacterial growth and guaiacol production during incubation at 37 °C High pH (6.7) inhibited the growth of Alicyclobacillus in sucrosesupplemented YSG for up to 354 h, thus no guaiacol was produced (Fig. 4). Low pH (2.7) delayed bacterial growth and guaiacol production was delayed similarly. Among pH values tested, bacterial growth in the exponential phase was the fastest at pH 3.7 and 4.7, with corresponding fast guaiacol production. Cultures with growth-permitting pH reached similar final microbial counts and maximum guaiacol concentrations at the stationary phase. According to other researchers, guaiacol can be produced by Alicyclobacillus isolate 1016 in a pH range of 3.5–4.5 and Alicyclobacillus isolate 1101 in a pH range of 3.5–5.0 (Chang et al., 2015). The maximum concentration of guaiacol was similar but required different time to reach their maximum at different 4
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tested and growth media used. 3.4. Control of Alicyclobacillus by selected antimicrobial compounds The efficacy of lauric arginate and ϵ-polylysine against Alicyclobacillus was determined using broth dilution approach and results were reported as minimum inhibitory concentrations (MIC). Lauric arginate (MIC 9.4 μg/mL) was much more effective than ϵpolylysine (MIC 74 μg/mL) against both Alicyclobacillus strains in SSYSG (pH 3.7) at 37 °C. In a subsequent experiment these antimicrobials were added, at their MIC levels, to SS-YSG broth (pH 3.7) or apple juice, and cells or spores of the two Alicyclobacillus strains were inoculated in both media. Relatively small inocula of Alicyclobacillus strains (103–104 CFU or spore/mL) were tested to mimic natural contamination levels in juice. Results are summarized in Table 1 and an example of detailed data is shown in Fig. 5. In most experiments, addition of the antimicrobial compounds decreased Alicyclobacillus populations to counts below the detection limit (10 CFU/mL) of the enumeration method, and no guaiacol was produced in the media (Table 1). Lauric arginate and ϵ-polylysine had bactericidal or bacteriostatic effects on cells and spores of A. acidoterrestris ATCC 49025 and OSYE in apple juice during incubation at 37 °C. Similarly, these two antimicrobials inhibited cell growth in SS-YSG broth at pH 3.7 (Table 1). However, A. acidoterrestris OSYE population increased in SS-YSG broth (37 °C, pH 3.7) that was inoculated with bacterium spore suspension and treated with lauric arginate (Fig. 5). This observation may indicate that spores of A. acidoterrestris OSYE were resistant to this additive in SS-YSG broth; these spores germinated successfully, produced sizable cell populations, and released guaiacol in the medium. The increased efficacy of lauric arginate against OSYE spores in apple juice when compared to its activity in SS-YSG broth could be explained by the synergistic effect between apple juice (low nutrient and inhibitory polyphenol compound) (Kahle, Kraus, & Richling, 2005) and the antimicrobial effect of lauric arginate. Lauric arginate has a cationic head from L-arginine and a nonpolar tail from lauric acid. It is presumed that lauric arginate adheres to cell membrane phospholipids and crosses the membrane into the cytoplasm, where it disrupts the intercellular environmental and inhibits metabolic processes (Rodríguez, Seguer, Rocabayera, & Manresa, 2004). Similarly, ϵ-polylysine is a cationic antimicrobial agent. It is hypothesized that the cationic ϵ-polylysine disturbs the charge across the cytoplasmic membrane causing damage to the cell (Shima et al., 1984). It should be cautioned, however, that turbidity was observed in lauric arginate-treated SS-YSG broth and ϵ-polylysine-treated apple juice; on the contrast, we didn't find turbidity in lauric arginate-treated apple juice or ϵ-polylysine-treated SS-YSG broth. Turbidity may have been caused by the interaction between antimicrobial agents and media
Fig. 4. Growth (Log CFU/mL) and guaiacol production (ppb) by Alicyclobacillus acidoterrestris ATCC 49025 and OSYE mixed populations during incubation at 37 °C in sucrose-supplemented yeast starch and glucose (SS-YSG) broth which was adjusted to different pH values. Panel A, population; panel B, guaiacol. Symbols: pH 2.7 (triangle); pH 3.7 (square); pH 4.7 (diamond); pH 6.7 (circle). No guaiacol was detected at pH 6.7 (Panel B) for up to 354 h; data not shown.
pH values (Chang et al., 2015). Longer time was needed at pH 3.5 than at pH 4 or 4.5 for Alicyclobacillus isolate 1016 and 1101 to produce guaiacol, and no guaiacol production occurred at pH 5 (Chang et al., 2015). Discrepancies between results of this study and those reported previously may be attributed to difference in Alicyclobacillus strains
Table 1 Difference* between the initial (0 h) and final (48 h for SS-YSG broth at pH 3.7 and 72 h for juice) bacterial counts and guaiacol concentration, as determined by SIFT-MS, for Alicyclobacillus acidoterrestris ATCC 49025 and OSYE during incubation at 37 °C, in the presence of lauric arginate and ε-polylysine. Medium/Inoculum type/Alicyclobacillus strain
Change in bacterial population (log CFU/mL) ** Control
Juice/Spores/ATCC Juice/Spores/OSYE Juice/Vegetative cells/ATCC Juice/Vegetative cells/OSYE SS-YSG broth/Spores/ATCC SS-YSG broth/Spores/OSYE SS-YSG broth/Vegetative cells/ATCC SS-YSG broth/Vegetative cells/OSYE
ab
2.9 1. 9bc 1. 6bc 2.1abc 1.3c 3.2a 2.2abc 2.6ab
Change in guaiacol concentration (ppb)***
Lauric arginate
ε-polylysine
−2.1 −1.7defg −1.8defg −1.3defgh −1.0de 2.3abc −1.8defg −2.2efgh
−2.1 −0.91d −1.6defgh −1.1de −2.6gh −2.4fgh −1.4defg −3.1h
ed
defgh
Control AB
72 90A 33C 50C 69B 75AB 73AB 85AB
Lauric arginate
ε-polylysine
ND ND ND ND ND 48 ND ND
ND ND ND ND ND ND ND ND
*Measurements for population or concentration with the same letter are not significantly different. Three replicates for each group were executed. **Change in population = Final log CFU/mL– initial log CFU/mL; negative sign indicates that population decreases during the treatment, compared to initial population. *** Change in concentration = Final concentration (ppb) – initial concentration (ppb). 5
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Fig. 5. Alicyclobacillus acidoterrestris OSYE growth (Log CFU/mL) and guaiacol production (ppb) during incubation at 37 °C in sucrose-supplemented yeast starch glucose (SS-YSG, pH 3.7) broth which was spiked with antimicrobial agents. Panel A, bacterial cells (No guaiacol was detected in the antimicrobial treated groups, data not shown); panel B, bacterial spores (No guaiacol was detected in the polylysine-treated group, data not shown). Dotted line indicates limit of detection for the enumeration method. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Declaration of competing interest
components. Turbidity was observed immediately after adding the antimicrobial compounds, which ruled out the possibility of bacterial growth due to microbial contamination. Other researchers reported that cationic lauric arginate interacted with food components such as anionic polysaccharides and increased product turbidity or formed a sediment layer in a concentration-dependent manner (Loeffler et al., 2014). Similarly, Chang, McLandsborough, and McClements (2014) reported that cationic ϵ-polylysine formed insoluble precipitates with anionic components in food matrix, leading to an increase in product cloudiness or the formation of sediments. Turbidity if not addressed by optimizing antimicrobial formulation might have a negative effect on juice product acceptability.
None. Acknowledgment This research was partially supported by funding from the Center for Advanced Processing and Packaging Studies (CAPPS), a NSF IUCRC founded center, The Ohio State University, Columbus, Ohio, USA. References Benli, H., Sanchez-Plata, M. X., & Keeton, J. T. (2011). Efficacy of ε-polylysine, lauric arginate, or acidic calcium sulfate applied sequentially for Salmonella reduction on membrane filters and chicken carcasses. Journal of Food Protection, 74, 743–750. Cai, R., Yuan, Y., Wang, Z., Guo, C., Liu, B., Pan, C., & Yue, T. (2015). Effects of preservatives on Alicyclobacillus acidoterrestris growth and guaiacol production. International Journal of Food Microbiology, 214, 145–150. Chang, S. S., & Kang, D. H. (2004). Alicyclobacillus spp. in the fruit juice industry: History, characteristics, and current isolation/detection procedures. Critical Reviews in Microbiology, 30, 55–74. Chang, S., Lu, W. W., Park, S. H., & Kang, D. H. (2010). Control of foodborne pathogens on ready-to-eat roast beef slurry by ε-polylysine. International Journal of Food Microbiology, 141, 236–241. Chang, Y., McLandsborough, L., & McClements, D. J. (2014). Antimicrobial delivery systems based on electrostatic complexes of cationic ϵ-polylysine and anionic gum Arabic. Food Hydrocolloids, 35, 137–143. Chang, S., Park, S. H., & Kang, D. H. (2015). Effect of extrinsic factors on the production of guaiacol by Alicyclobacillus spp. Journal of Food Protection, 78, 831–835. CLSI -Clinical and Laboratory Standards Institute (2006). Method for dilution antimicrobial susceptibility tests for bacterial that grow aerobically. Wayne, PA: Approved standard
4. Conclusions In conclusion, strains of A. acidoterrestris that produce guaiacol in fruit juices can grow under a broad range of temperature and acidic pH values. Both strains can utilize vanillin in the juice as a substrate for production of the odorous guaiacol. Control of these strains in juice may be accomplished by addition of antimicrobial food additives. Although lauric arginate and ϵ-polylysine effectively control the two problematic strains of A. acidoterrestris, effect of these additives on juice quality should be investigated and thus concentrations applied should be optimized.
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acidocaldarius in water, various buffers, and orange juice. Journal of Food Protection, 63, 1377–1380. Pettipher, G., Osmundson, M., & Murphy, J. (1997). Methods for the detection and enumeration of Alicyclobacillus acidoterrestris and investigation of growth and production of taint in fruit juice and fruit juice containing drinks. Letters in Applied Microbiology, 24, 185–189. Rinrada, P., Aran, H., & Catherine, C. (2014). Effect of lauric arginate, nisin Z, and a combination against several food-related bacteria. International Journal of Food Microbiology, 188, 135–146. Rodríguez, E., Seguer, J., Rocabayera, X., & Manresa, A. (2004). Cellular effects of monohydrochloride of l-arginine, Na-lauroyl ethylester (LAE) on exposure to Salmonella typhimurium and Staphylococcus aureus. Journal of Applied Microbiology, 96, 903–912. Shima, S., Matsuoka, H., Iwamoto, T., & Sakai, H. (1984). Antimicrobial action of εpolylysine. Journal of Antibiotics, 37, 1449–1455. Smith, D., & Španěl, P. (2005). Selected ion flow tube mass spectrometry (SIFT-MS) for on-line trace gas analysis. Mass Spectrometry Reviews, 24, 661–700. Spinelli, A. C. N., Sant'Ana, A. S., Rodrigues-Junior, S., & Massaguer, P. R. (2009). Influence of different filling, cooling, and storage conditions on the growth of Alicyclobacillus acidoterrestris CRA7152 in orange juice. Applied and Environmental Microbiology, 75, 7409–7416. Witthuhn, R., Merwe, E., Venter, P., & Cameron, P. (2012). Guaiacol production from ferulic acid, vanillin and vanillic acid by Alicyclobacillus acidoterrestris. International Journal of Food Microbiology, 157, 113–117. Witthuhn, R., Smith, Y., Cameron, M., & Venter, P. (2013). Guaiacol production by Alicyclobacillus and comparison of two guaiacol detection Methods. Food Control, 30, 700–704. Yoshida, T., & Nagasawa, T. (2003). ε-Polylysine, microbial production, biodegradation and application potential. Applied Microbiology and Biotechnology, 62, 21–26. Yousef, A. E. (2015). Personal communication. USA: Fruit juice processor.
M7-A7. Dryahina, K., Smith, D., & Španěl, P. (2018). Quantification of volatile compounds released by roasted coffee by selected ion flow tube mass spectrometry. Rapid Communications in Mass Spectrometry, 32, 739–750. Food and Drug Administration (2004). Agency response letter GRAS Notice No. GRN 000135. https://www.accessdata.fda.gov/scripts/fdcc/index.cfm?set=GRASNotices &id=135, Accessed date: 23 January 2019. Food and Drug Administration (2005). Agency response letter GRAS notice No. GRN 000164. https://www.accessdata.fda.gov/scripts/fdcc/index.cfm?set=GRASNotices &id=164, Accessed date: 23 January 2019. Food and Drug Administration (2010). GRAS notices. GRN No. 336. http://www. accessdata.fda.gov/scripts/fdcc/?set=GRASNotices&id=336, Accessed date: 23 January 2019. Goto, K., Matsubara, H., Mochida, K., Matsumura, T., Hara, Y., Niwa, M., et al. (2002). Alicyclobacillus herbarius sp. nov, a novel bacterium containing omega-cycloheptane fatty acids, isolated from herbal tea. International Journal of Systematic and Evolutionary Microbiology, 52, 109–113. Huang, Y., & Barringer, S. (2011). Monitoring of cocoa volatiles produced during roasting by selected ion flow tube-mass spectrometry (SIFT-MS). Journal of Food Science, 76, C279–C286. Kahle, K., Kraus, M., & Richling, E. (2005). Polyphenol profiles of apple juices. Molecular Nutrition & Food Research, 49, 797–806. Loeffler, M., McClements, D. J., McLandsborough, L., Terjung, N., Chang, Y., & Weiss, J. (2014). Electrostatic interactions of cationic lauric arginate with anionic polysaccharides affect antimicrobial activity against spoilage yeasts. Journal of Applied Microbiology, 117, 28–39. Oteiza, J. M., Ares, G., Sant'Ana, A. S., Soto, S., & Giannuzzi, L. (2011). Use of a multivariate approach to assess the incidence of Alicyclobacillus spp. in concentrate fruit juices marketed in Argentina, Results of a 14-year survey. International Journal of Food Microbiology, 151, 229–234. Palop, A., Álvarez, I., Raso, J., & Condón, S. (2000). Heat resistance of Alicyclobacillus
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Factors affecting Alicyclobacillus acidoterrestris growth and guaiacol production and controlling apple juice spoilage by lauric arginate and ϵpolylysine
T
Xiaohuan Hua, En Huangb,∗, Sheryl A. Barringera, Ahmed E. Yousefa,c a
Department of Food Science and Technology, The Ohio State University, 2015 Fyffe Rd., Columbus, OH, 43210, USA Department of Environmental and Occupational Health, University of Arkansas for Medical Sciences, 4301 West Markham Street, # 820, Little Rock, AR, 72205, USA c Department of Microbiology, The Ohio State University, 484 West 12th Avenue, Columbus, OH, 43210, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: Alicyclobacillus acidoterrestris Guaiacol Lauric arginate ϵ-polylysine
Growth of Alicyclobacillus acidoterrestris in fruit juices has been associated with the release of off-odor compounds, such as guaiacol. Guaiacol release was quantified using Selected Ion Flow Tube Mass Spectrometry (SIFT-MS) and method's minimum detection level was 3.6 ppb. The amount of guaiacol released was dependent on the amount of vanillin added to the medium. Sucrose-supplemented yeast starch glucose broth (SS-YSG broth; pH 3.7) and apple juice were spiked with vanillin (10 mg/L), inoculated with A. acidoterrestris and incubated at different temperatures (20°C–45 °C) for up to 528 h. At the temperatures that permitted growth, the bacterium grew slower in apple juice than in SS-YSG broth. Bacterial growth and guaiacol production were prevented in apple juice and inhibited in SS-YSG, when the inoculated products were incubated at 20 °C and 25 °C. When Alicyclobacillus was inoculated in SS-YSG adjusted to pH 2.7–6.7 and incubated at 37 °C, growth of the bacterium and guaiacol production were suppressed at pH 6.7 for up to 354 h and delayed at pH 2.7. Lauric arginate and ϵpolylysine inactivated Alicyclobacillus cells with no guaiacol production in both SS-YSG and apple juice. However, ϵ-polylysine was more effective than lauric arginate when the SS-YSG (pH 3.7) was inoculated with Alicyclobacillus spores.
1. Introduction Alicyclobacillus acidoterrestris is a thermophilic, acidophilic, sporeforming microorganism. The bacterium can survive over a wide temperature range, and it grows well at 42 °C–60 °C (Chang & Kang, 2004). Spores of the bacterium can survive commercial pasteurization of fruit juices; subsequently, these spores may germinate and spoil the juice by producing an odorous compound, guaiacol, when the population reaches 105 CFU/mL (Chang & Kang, 2004; Spinelli, Sant'Ana, Rodrigues-Junior, & Massaguer, 2009). It is estimated that 11.4% of apple juice concentrate in Argentina is contaminated with Alicyclobacillus (Oteiza, Ares, Sant'Ana, Soto, & Giannuzzi, 2011). A. acidoterrestris can produce guaiacol at 25 or 45 °C, but the bacterium grows slower at 25 than at 45 °C in Bacillus acidoterrestris medium (Witthuhn, Smith, Cameron, & Venter, 2013). The bacteria also can grow in media with pH 2.5 to 6.0 (Chang & Kang, 2004). Guaiacol is presumed to be the main factor that leads to the smoky or medicinal off-flavor in juice spoiled by Alicyclobacillus spp. The odor
∗
threshold of guaiacol in orange, apple, and noncarbonated fruit juices is about 2 ppb (Chang & Kang, 2004; Pettipher, Osmundson, & Murphy, 1997). Guaiacol is produced by a biological conversion of ferulic acid to vanillic acid and then decarboxylation to guaiacol (Chang & Kang, 2004). Vanillin, vanillic acid and ferulic acid, which are naturally present in some fruits and juice products, are three important precursors of guaiacol, but only vanillin or vanillic acid can be decomposed by Alicyclobacillus spp. directly without participation of other bacteria (Witthuhn, Merwe, Venter, & Cameron, 2012). Higher concentrations of substrates (vanillin or vanillic acid) tend to correlate with higher final amounts of guaiacol by A. acidoterrestris in Bacillus acidoterrestris broth (Witthuhn et al., 2012). In this investigation, generally-recognized as safe (GRAS) antimicrobial agents were investigated as potential treatment for controlling the growth of A. acidoterrestris and release of guaiacol in fruit juices. Lauric arginate is a new natural cationic surface-active molecule which has a broad antimicrobial activity (Rinrada, Aran, & Catherine, 2014). Its antimicrobial efficacy has been tested in meat products
Corresponding author. E-mail addresses: [email protected] (X. Hu), [email protected] (E. Huang), [email protected] (S.A. Barringer), [email protected] (A.E. Yousef).
https://doi.org/10.1016/j.lwt.2019.108883 Received 12 August 2019; Received in revised form 5 November 2019; Accepted 24 November 2019 Available online 24 November 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
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were incubated at 37 °C for 2 days with agitation at 200 rpm. When a mixed culture of the two strains was used, equal volumes of the overnight culture were combined and an aliquot of the mixture (e.g., 1 mL) was used to inoculate the test medium.
(Benli, Sanchez-Plata, & Keeton, 2011) but not in juices. According to the U.S. Food and Drug Administration (FDA), lauric arginate is a GRAS compound and it can be used in beverages up to 200 ppm (Food and Drug Administration, 2005). Similarly, ε-polylysine is a GRAS antimicrobial food additive and it can be used in fruit-flavored drink up to 250 ppm (Food and Drug Administration, 2004 and 2010). The compound has antimicrobial activity against yeasts, molds, Gram-positive and Gram-negative bacteria (Chang, Lu, Park, & Kang, 2010). ε-polylysine can inhibit bacterial growth, mainly through electrostatic adsorption to the bacterial cell surface and its cationic properties which can destroy cell membrane and finally leads to cell death (Chang et al., 2010; Shima, Matsuoka, Iwamoto, & Sakai, 1984; Yoshida & Nagasawa, 2003). Both ε-polylysine (300 mg/L) and lauric arginate (200 mg/L) have been reported to effectively reduce Salmonella population on inoculated chicken carcasses (Benli et al., 2011). Cai et al. (2015) reported that ε-polylysine inhibited the growth of the vegetative cells of A. acidoterrestris but its effect on bacterial spores was not studied. One of the objectives of this study was to determine the factors that are conducive to growth of A. acidoterrestris and release of guaiacol in liquid media. These factors were medium type (microbiological medium vs. apple juice), medium pH (2.7–6.7), storage temperature (20–45 °C), and vanillin concentration. The main objective was to control juice spoilage using two emerging antimicrobial additives, lauric arginate, and ε-polylysine. Quantification of synthesized guaiacol was accomplished using Selected Ion Flow Tube Mass Spectrometry (SIFT-MS), an analytical system for detection and quantification of volatile molecules at parts per billion (ppb) levels (Smith & Španěl, 2005).
2.4. Preparing Alicyclobacillus acidoterrestris spore suspensions Spore suspensions of Alicyclobacillus ATCC 49025 and OSYE were prepared as described previously (Palop et al., 2000). Cell suspensions were spread onto the sporulation agar medium, and the plates were incubated at 45 °C for 4 days. The growth (cells and spores) on the agar medium was removed with a sterile microscope slide and transferred into a 2-ml centrifuge tube with sterilized water to a final volume of 1 ml. The suspension was centrifuged (15 min, 16,000 x g, 4 °C), the supernatant was decanted and the sediment was re-suspended in 50% (v/v) aqueous ethanol and held for 30 min, to destroy non-sporulated cells. The suspension was centrifuged, the supernatant was discarded and the sedimented spores were re-suspended in sterile water; this process was repeated three times. The final sediment was re-suspended in sterile distilled water, to a final volume of 1 mL, and heated at 80 °C for 10 min. Spore count in the suspension was determined, and the suspension was held at 4 °C until use. 2.5. Guaiacol production by Alicyclobacillus at different concentrations of vanillin Samples of SS-YSG broth (pH 3.7) were inoculated with an inoculum made of equal volumes of A. acidoterrestris ATCC 49025 and OSYE cell suspensions. Vanillin (Sigma-Aldrich Co.) stock solution was added to the inoculated media to achieve the following final concentrations: 0.25, 0.5, 1.0, and 2.0 mg/L. Three replicates of this treatment were prepared. Broth, with or without added vanillin, was incubated at 37 °C, and tested after 24 h, when guaiacol reached its highest concentration. The minimum concentration of guaiacol in the headspace that was significantly different from the background (p < 0.05), and the corresponding vanillin concentration were recorded. The amounts of guaiacol released in the headspace, as a function of vanillin concentration, were determined as described below. The limit of detection and limit of quantification for guaiacol were determined to be 3.6 ppb.
2. Materials and methods 2.1. Media Yeast Starch Glucose (YSG) broth was used to culture and enumerate Alicyclobacillus strains (Goto et al., 2002). YSG broth was prepared to contain 0.2% yeast extract (IBI Scientific, IA, USA), 0.2% soluble starch (J. T. Barker, NJ, USA) and 0.1% glucose (Becton, Dickinson and Company, MD, USA). The medium was adjusted to pH 3.7, using 6M HCl, before autoclaving. YSG agar was prepared by adding 1.5% agar to YSG broth. Sucrose-supplemented YSG broth (SSYSG) was prepared from acidified YSG broth by adding sucrose to 8% level, to simulate fruit juice products (pH 3.7). Sporulation agar medium (Palop, Álvarez, Raso, & Condón, 2000) was prepared by mixing the following two solutions at equal volume after being sterilized separately. The first solution was prepared by mixing 1 g yeast extract, 0.2 g (NH4)2SO4, 0.25 g CaCl2, 0.5 g MgSO4, 1 g glucose, 0.6 g KH2PO4 with 500 mL distilled water, and adjusting the pH to 4.0 with 6 M HCl. The second solution was prepared by dissolving 20 g agar in 500 mL distilled water.
2.6. Measurement of guaiacol by Selected Ion Flow Tube Mass Spectrometry Selected Ion Flow Tube Mass Spectrometry (SIFT-MS; Voice 200; Syft Technologies Ltd., Christchurch, New Zealand) was used previously to detect volatiles in food (e.g., Dryahina, Smith, & Španěl, 2018; Huang & Barringer, 2011). We adopted SIFT-MS methods for guaiacol measurement under Selected Ion Monitoring mode. Guaiacol reacted with the precursor ion, NO+, to exclusively produce the positively-charged guaiacol ion, C7H8O2+, with m/z of 124. The internal standard, 2-ethoxyphenol, reacted with precursor ion O2+ to produce C8H10O2+, with m/z of 138. Volatile concentrations were measured for 60 s and averaged. The guaiacol concentration in the headspace of a medium was standardized using the concentration of the internal standard, 2-ethoxyphenol.
2.2. Bacterial strains A. acidoterrestris ATCC 49025 and A. acidoterrestris OSYE were tested in this study. The strain OSYE was isolated from spoiled, thermallyprocessed, commercial juice and provided by a food processor (Yousef, 2015). Morphology of A. acidoterrestris OSYE was consistent with that of the ATCC 49025 strain, and its ability to produce guaiacol in juice was verified. Stocks of these strains were held in YSG broth with 40% glycerol at −80 °C.
2.7. Alicyclobacillus growth and guaiacol production in different media at different temperatures Aliquots (100 mL, each) of SS-YSG broth (pH 3.7) or apple juice (Old Orchard, LLC Co, Sparta, MI, USA) were mixed with 2-ethoxyphenol (final concentration, 1.068 μg/mL) as an internal standard, vanillin (Sigma-Aldrich) at a final concentration of 10 mg/L, as a substrate for guaiacol, and 1 mL mixture of the overnight culture of A. acidoterrestris ATCC 49025 and OSYE, into a 500-mL sterilized Pyrex glass bottle. In order to test the influence of incubation temperature,
2.3. Culturing Alicyclobacillus acidoterrestris Portions (loop-full) of frozen stocks of A. acidoterrestris, ATCC 49025 or OSYE were streaked onto YSG agar plates and incubated at 37 °C for 2 days. An isolated colony was then transferred into a 50-mL centrifuge tube containing 30 mL YSG broth. The contents of the centrifuge tubes 2
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bottles were held at 20, 25, 37, or 45 °C and sampled at various incubation intervals; a bottle and its contents represent one sample. To analyze volatiles in the headspace, bottles were held during the test in a water bath at the appropriate temperature (37 °C) for 1 h to volatilize guaiacol and 2-ethoxyphenol. Concentration of these compounds in the bottle headspace was measured using SIFT-MS, as described in section 2.6. At every incubation temperature, samples were tested at 4-time intervals. The sampling times varied because cell growth was substantially different at different temperature and media. After testing for guaiacol, Alicyclobacillus population in the bottle contents was determined as follows. Decimal dilutions were prepared and spread-plated onto YSG agar. Inoculated plates were incubated at 37 °C for 2 days before enumeration. Fig. 1. Relationship between vanillin concentration in sucrose-supplemented yeast starch glucose broth, pH 3.7, and amount of guaiacol (ppb) released in the headspace of Alicyclobacillus acidoterrestris culture. Guaiacol concentration was determined using Selected Ion Flow Tube Mass Spectrometry. The experiment was repeated three times and averages ( ± standard deviation) are reported.
2.8. Alicyclobacillus growth and guaiacol production in SS-YSG broth at different pH values SS-YSG broth was adjusted to different pH values (2.7, 3.7, 4.7, and 6.7) with 6 M HCl. The broth was prepared and inoculated as described previously (section 2.7), and incubated at 37 °C. For every pH level, guaiacol concentration in the headspace and bacterial counts were tested for up to six incubation intervals. The sampling times varied because cell growth was substantially different at different medium pH. Replicate samples were tested at every time interval. The experiment was repeated 3 times.
antimicrobial compound added). No growth was observed in the noninoculated apple juice. During testing of every batch, guaiacol concentrations in the headspace, as well as bacterial counts, were determined as described previously. 2.11. Statistical analysis
2.9. Minimum inhibitory concentrations of antimicrobial compounds against Alicyclobacillus strains
One-way analysis of variance (ANOVA) and Tukey's post hoc analysis (R Studio Statistical Software, RStudio, Inc, Richmond Hill, ON, Canada) were used to determine statistical difference between treatments. p value of 0.05 was used for indicating significant difference.
A commercial preparation of lauric arginate (CytoGuard™ LA 20, 10% lauric arginate; A&B Ingredients, Inc., Fairfield, NJ, USA) and, ϵpolylysine (95% purity; Siveele, Breda, The Netherlands) were tested in this study. These two antimicrobial agents were dissolved in water and sterilized by passing through a 0.22 μm membrane filter. Antimicrobial efficacy of these agents was tested by measuring their minimum inhibitory concentrations (MICs) against A. acidoterrestris ATCC 49025 and OSYE using the method of Clinical and Laboratory Standards Institute (CLSI, 2006). Briefly, a series of lauric arginate dilutions were prepared using distilled water, then 50 μL of these solutions were dispensed in wells of a 96-well plate and mixed with 50 μL 2X concentration of SS-YSG broth (pH 3.7). Overnight culture of A. acidoterrestris ATCC 49025 or OSYE (1 μL) was added to each well. The final concentrations of lauric arginate in wells were 2.35, 4.7, 9.4, and 18.8 μg/mL. Similarly, ϵ-polylysine was diluted and tested as described for lauric arginate treatment to produce final concentrations of 18.6, 37.1, 74, and 149 μg/mL. Concentrations of lauric arginate and ϵpolylysine suitable for testing their MIC were determined based on preliminary experiments. The 96-well plates were incubated at 37 °C for 24 h and bacterial growth was measured as absorbance readings at 600 nm using a microtiter plate reader.
3. Results and discussion 3.1. Guaiacol production at different concentrations of vanillin The correlation between the substrate (vanillin) concentration and guaiacol production by A. acidoterrestris was linear, in the range of 0.1–2 mg/L vanillin (Fig. 1). The analytical method used (SIFT-MS technology) reliably allowed for the detection of 3.6 ppb of guaiacol in the headspace when 0.1 mg/L vanillin was added to the medium, which was the limit of quantification. In a previous study, A. acidoterrestris produced 62 and 171 mg/L guaiacol when the concentration of vanillin was at 100 and 1000 mg/L, respectively (Witthuhn et al., 2012). 3.2. The influence of temperature on bacterial growth and guaiacol production in SS-YSG at pH 3.7 and in commercial apple juice When SS-YSG broth (pH 3.7) was used as a medium, the incubation temperature in the range of 20 °C and 45 °C affected the time A. acidoterrestris took to reach its stationary phase (18.5 to > 327 h), but it did not influence the final population size (6.5–7.2 log CFU/mL) considerably (Fig. 2). The higher the temperature, the faster was the growth of the bacterium in this medium. Growth of A. acidoterrestris in apple juice was affected similarly by the incubation temperature; however, juice samples held only at 37 and 45 °C supported the growth of the bacterium (Fig. 2). Under growth-permitting conditions, A. acidoterrestris grew much faster in SS-YSG broth than in apple juice; for example, at 45 °C, maximum population was attained after 18.5 h in SSYGS and 76 h in apple juice. A. acidoterrestris is a thermophilic bacterium and its optimum growth temperature ranges from 42 to 60 °C (Chang & Kang, 2004). Other researchers also reported that A. acidoterrestris strains grew slower at 25 than 45 °C in Bacillus acidoterrestris broth (Witthuhn et al., 2013). A. acidoterrestris isolate 2498 grew at 25, 35, and 44 °C but
2.10. Control of Alicyclobacillus growth and guaiacol production in SS-YSG broth and apple juice with antimicrobial compounds The effect of the antimicrobial compounds on Alicyclobacillus growth and guaiacol production was tested as follows. Bottles containing sterilized SS-YSG broth (pH 3.7) or shelf stable apple juice were spiked with sterilized ϵ-polylysine or lauric arginate, at their MIC levels, 1 mL of vanillin solution (final concentration, 10 mg/mL), and 1 mL overnight culture (cells) or 0.1 mL spore suspension of A. acidoterrestris ATCC 49025 or OSYE. Final concentration of cells or spores, in the mixture, was 103–104 CFU/mL and final volume was ~100 mL. The inoculated bottles were incubated at 37 °C for 48 h for SS-YSG broth and 72 h for apple juice. In every batch, there were three side-by-side treatments: lauric arginate, ϵ-polylysine and control (i.e., no 3
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Fig. 3. Guaiacol production by Alicyclobacillus acidoterrestris ATCC 49025 and OSYE mixed populations in sucrose-supplemented yeast starch and glucose (SSYSG, pH 3.7) broth and apple juice, as influenced by incubation temperature. Panel A, SS-YSG broth; panel B, apple juice. Symbols: 20 °C (circle); 25 °C (diamond); 37 °C (triangle); 45 °C (square). No guaiacol was detected in juice at 20 °C or 25 °C (Panel B) for up to 326 h; data not shown.
Fig. 2. Growth of Alicyclobacillus acidoterrestris ATCC 49025 and OSYE mixed populations (Log CFU/mL) in sucrose-supplemented yeast starch and glucose (SS-YSG, pH 3.7) broth and apple juice, as influenced by incubation temperature. Panel A, SS-YSG broth; panel B, apple juice. Symbols: 20 °C (circle); 25 °C (diamond); 37 °C (triangle); 45 °C (square). No growth was observed in juice at 20 °C or 25 °C (Panel B) for up to 528 h; data not shown.
when incubation temperature increased from 32 to 50 °C (Chang, Park, & Kang, 2015). Others also reported that in YSG broth, A. acidoterrestris can produce guaiacol at 25 or 45 °C, and longer time was needed to produce guaiacol at 25 than 45 °C (Witthuhn et al., 2013). On the contrary, other researchers found no guaiacol formation at 25 °C, in a different growth medium with different Alicyclobacillus strains, during 48 h of incubation (Chang et al., 2015). Therefore, storage temperature, medium constituents, and bacterial strain are important factors controlling the growth of A. acidoterrestris and the production of guaiacol.
it didn't grow at 4 °C in apple or orange juice (Pettipher et al., 1997). There are ω-alicyclic fatty acids in the cytoplasmic membrane of A. acidoterrestris, which protect the bacteria from high temperature and low pH conditions (Chang & Kang, 2004). In apple juice, there was no bacterial growth at 20 and 25 °C (Fig. 2). Yeast extract, glucose and starch in the YSG broth are important nutrients which support the growth of bacteria. Therefore, in a favorable media, such as YSG broth, ambient temperature (e.g., 25 °C) can only delay bacterial growth, while in nutrient-limited media, such as apple juice, storage at room temperature can inhibit the growth of the bacterium. Guaiacol production is highly related to the bacterial population size (Figs. 2 and 3). In apple juice, no guaiacol production occurred at 20 and 25 °C, because there was no bacterial growth. There was guaiacol production in SS-YSG broth at all temperatures tested (Fig. 3). Temperature did not influence the maximum amount of guaiacol produced in this medium, but it did impact the time for guaiacol to reach its highest concentration. There was no significant difference (p < 0.05) in the maximum amount of guaiacol produced in SS-YSG broth and juice samples, at temperatures that permitted bacterial growth (Fig. 3). Bacteria grew faster at 37 or 45 °C than at 20 or 25 °C, leading to different times-to-guaiacol-production under different temperature conditions. In a previous study, guaiacol formation accelerated
3.3. Impact of pH on bacterial growth and guaiacol production during incubation at 37 °C High pH (6.7) inhibited the growth of Alicyclobacillus in sucrosesupplemented YSG for up to 354 h, thus no guaiacol was produced (Fig. 4). Low pH (2.7) delayed bacterial growth and guaiacol production was delayed similarly. Among pH values tested, bacterial growth in the exponential phase was the fastest at pH 3.7 and 4.7, with corresponding fast guaiacol production. Cultures with growth-permitting pH reached similar final microbial counts and maximum guaiacol concentrations at the stationary phase. According to other researchers, guaiacol can be produced by Alicyclobacillus isolate 1016 in a pH range of 3.5–4.5 and Alicyclobacillus isolate 1101 in a pH range of 3.5–5.0 (Chang et al., 2015). The maximum concentration of guaiacol was similar but required different time to reach their maximum at different 4
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tested and growth media used. 3.4. Control of Alicyclobacillus by selected antimicrobial compounds The efficacy of lauric arginate and ϵ-polylysine against Alicyclobacillus was determined using broth dilution approach and results were reported as minimum inhibitory concentrations (MIC). Lauric arginate (MIC 9.4 μg/mL) was much more effective than ϵpolylysine (MIC 74 μg/mL) against both Alicyclobacillus strains in SSYSG (pH 3.7) at 37 °C. In a subsequent experiment these antimicrobials were added, at their MIC levels, to SS-YSG broth (pH 3.7) or apple juice, and cells or spores of the two Alicyclobacillus strains were inoculated in both media. Relatively small inocula of Alicyclobacillus strains (103–104 CFU or spore/mL) were tested to mimic natural contamination levels in juice. Results are summarized in Table 1 and an example of detailed data is shown in Fig. 5. In most experiments, addition of the antimicrobial compounds decreased Alicyclobacillus populations to counts below the detection limit (10 CFU/mL) of the enumeration method, and no guaiacol was produced in the media (Table 1). Lauric arginate and ϵ-polylysine had bactericidal or bacteriostatic effects on cells and spores of A. acidoterrestris ATCC 49025 and OSYE in apple juice during incubation at 37 °C. Similarly, these two antimicrobials inhibited cell growth in SS-YSG broth at pH 3.7 (Table 1). However, A. acidoterrestris OSYE population increased in SS-YSG broth (37 °C, pH 3.7) that was inoculated with bacterium spore suspension and treated with lauric arginate (Fig. 5). This observation may indicate that spores of A. acidoterrestris OSYE were resistant to this additive in SS-YSG broth; these spores germinated successfully, produced sizable cell populations, and released guaiacol in the medium. The increased efficacy of lauric arginate against OSYE spores in apple juice when compared to its activity in SS-YSG broth could be explained by the synergistic effect between apple juice (low nutrient and inhibitory polyphenol compound) (Kahle, Kraus, & Richling, 2005) and the antimicrobial effect of lauric arginate. Lauric arginate has a cationic head from L-arginine and a nonpolar tail from lauric acid. It is presumed that lauric arginate adheres to cell membrane phospholipids and crosses the membrane into the cytoplasm, where it disrupts the intercellular environmental and inhibits metabolic processes (Rodríguez, Seguer, Rocabayera, & Manresa, 2004). Similarly, ϵ-polylysine is a cationic antimicrobial agent. It is hypothesized that the cationic ϵ-polylysine disturbs the charge across the cytoplasmic membrane causing damage to the cell (Shima et al., 1984). It should be cautioned, however, that turbidity was observed in lauric arginate-treated SS-YSG broth and ϵ-polylysine-treated apple juice; on the contrast, we didn't find turbidity in lauric arginate-treated apple juice or ϵ-polylysine-treated SS-YSG broth. Turbidity may have been caused by the interaction between antimicrobial agents and media
Fig. 4. Growth (Log CFU/mL) and guaiacol production (ppb) by Alicyclobacillus acidoterrestris ATCC 49025 and OSYE mixed populations during incubation at 37 °C in sucrose-supplemented yeast starch and glucose (SS-YSG) broth which was adjusted to different pH values. Panel A, population; panel B, guaiacol. Symbols: pH 2.7 (triangle); pH 3.7 (square); pH 4.7 (diamond); pH 6.7 (circle). No guaiacol was detected at pH 6.7 (Panel B) for up to 354 h; data not shown.
pH values (Chang et al., 2015). Longer time was needed at pH 3.5 than at pH 4 or 4.5 for Alicyclobacillus isolate 1016 and 1101 to produce guaiacol, and no guaiacol production occurred at pH 5 (Chang et al., 2015). Discrepancies between results of this study and those reported previously may be attributed to difference in Alicyclobacillus strains
Table 1 Difference* between the initial (0 h) and final (48 h for SS-YSG broth at pH 3.7 and 72 h for juice) bacterial counts and guaiacol concentration, as determined by SIFT-MS, for Alicyclobacillus acidoterrestris ATCC 49025 and OSYE during incubation at 37 °C, in the presence of lauric arginate and ε-polylysine. Medium/Inoculum type/Alicyclobacillus strain
Change in bacterial population (log CFU/mL) ** Control
Juice/Spores/ATCC Juice/Spores/OSYE Juice/Vegetative cells/ATCC Juice/Vegetative cells/OSYE SS-YSG broth/Spores/ATCC SS-YSG broth/Spores/OSYE SS-YSG broth/Vegetative cells/ATCC SS-YSG broth/Vegetative cells/OSYE
ab
2.9 1. 9bc 1. 6bc 2.1abc 1.3c 3.2a 2.2abc 2.6ab
Change in guaiacol concentration (ppb)***
Lauric arginate
ε-polylysine
−2.1 −1.7defg −1.8defg −1.3defgh −1.0de 2.3abc −1.8defg −2.2efgh
−2.1 −0.91d −1.6defgh −1.1de −2.6gh −2.4fgh −1.4defg −3.1h
ed
defgh
Control AB
72 90A 33C 50C 69B 75AB 73AB 85AB
Lauric arginate
ε-polylysine
ND ND ND ND ND 48 ND ND
ND ND ND ND ND ND ND ND
*Measurements for population or concentration with the same letter are not significantly different. Three replicates for each group were executed. **Change in population = Final log CFU/mL– initial log CFU/mL; negative sign indicates that population decreases during the treatment, compared to initial population. *** Change in concentration = Final concentration (ppb) – initial concentration (ppb). 5
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Fig. 5. Alicyclobacillus acidoterrestris OSYE growth (Log CFU/mL) and guaiacol production (ppb) during incubation at 37 °C in sucrose-supplemented yeast starch glucose (SS-YSG, pH 3.7) broth which was spiked with antimicrobial agents. Panel A, bacterial cells (No guaiacol was detected in the antimicrobial treated groups, data not shown); panel B, bacterial spores (No guaiacol was detected in the polylysine-treated group, data not shown). Dotted line indicates limit of detection for the enumeration method. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Declaration of competing interest
components. Turbidity was observed immediately after adding the antimicrobial compounds, which ruled out the possibility of bacterial growth due to microbial contamination. Other researchers reported that cationic lauric arginate interacted with food components such as anionic polysaccharides and increased product turbidity or formed a sediment layer in a concentration-dependent manner (Loeffler et al., 2014). Similarly, Chang, McLandsborough, and McClements (2014) reported that cationic ϵ-polylysine formed insoluble precipitates with anionic components in food matrix, leading to an increase in product cloudiness or the formation of sediments. Turbidity if not addressed by optimizing antimicrobial formulation might have a negative effect on juice product acceptability.
None. Acknowledgment This research was partially supported by funding from the Center for Advanced Processing and Packaging Studies (CAPPS), a NSF IUCRC founded center, The Ohio State University, Columbus, Ohio, USA. References Benli, H., Sanchez-Plata, M. X., & Keeton, J. T. (2011). Efficacy of ε-polylysine, lauric arginate, or acidic calcium sulfate applied sequentially for Salmonella reduction on membrane filters and chicken carcasses. Journal of Food Protection, 74, 743–750. Cai, R., Yuan, Y., Wang, Z., Guo, C., Liu, B., Pan, C., & Yue, T. (2015). Effects of preservatives on Alicyclobacillus acidoterrestris growth and guaiacol production. International Journal of Food Microbiology, 214, 145–150. Chang, S. S., & Kang, D. H. (2004). Alicyclobacillus spp. in the fruit juice industry: History, characteristics, and current isolation/detection procedures. Critical Reviews in Microbiology, 30, 55–74. Chang, S., Lu, W. W., Park, S. H., & Kang, D. H. (2010). Control of foodborne pathogens on ready-to-eat roast beef slurry by ε-polylysine. International Journal of Food Microbiology, 141, 236–241. Chang, Y., McLandsborough, L., & McClements, D. J. (2014). Antimicrobial delivery systems based on electrostatic complexes of cationic ϵ-polylysine and anionic gum Arabic. Food Hydrocolloids, 35, 137–143. Chang, S., Park, S. H., & Kang, D. H. (2015). Effect of extrinsic factors on the production of guaiacol by Alicyclobacillus spp. Journal of Food Protection, 78, 831–835. CLSI -Clinical and Laboratory Standards Institute (2006). Method for dilution antimicrobial susceptibility tests for bacterial that grow aerobically. Wayne, PA: Approved standard
4. Conclusions In conclusion, strains of A. acidoterrestris that produce guaiacol in fruit juices can grow under a broad range of temperature and acidic pH values. Both strains can utilize vanillin in the juice as a substrate for production of the odorous guaiacol. Control of these strains in juice may be accomplished by addition of antimicrobial food additives. Although lauric arginate and ϵ-polylysine effectively control the two problematic strains of A. acidoterrestris, effect of these additives on juice quality should be investigated and thus concentrations applied should be optimized.
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