Rapid Detection of Alicyclobacillus acidoterrestris in fruit juice matrices using a real-time PCR assay Shima Shayanfar* 1 , Suresh D. Pillai 1 , Christina Harzman 2 1 National Center for Electron Beam Research, Department of Nutrition & Food Science, Texas A&M University, USA 2 BIOTECON Diagnostics GmbH, Hermannswerder 17, 14473 Potsdam, Germany * shayanfar@tamu.edu Background  Alicyclobacillus acidoterrestris - Gram + acid tolerant (optimum growth pH 2.2 – 5.8)  Spore forming  Aerobic, acidophilic, & thermophilic microorganism  Survive typical pasteurization procedures employed in juice industry  Growth at 20-70 °C (with the optimum temperature 42–60 °C)  Results in formation of gauiacol that contributes to an unpleasant ‘hammy smell” in fruit juice  Conventional plating methods to screen for the presence of this spoilage organism can take as long as 48 hours to a few weeks.  Real-time -PCR is a rapid method to detect A. acidoterrestris at a concentration 10 3 - 10 4 cells tracking DNA  The PCR-based methods employed up to now require enriching the juice samples were up to 48 hours prior to PCR analysis  It is hypothesized that commercial real-time PCR based kits available in the market can detect A. acidoterrestris at lower concentration and in shorter time.  We tested 14 different fruit juice and puree samples that were spiked with A.acidoterresteris spores (10 CFU/ml) and un-spiked samples •Alicyclobacillus acidoterrestris isolate (ATCC 49025) diluted with Alicyclobacillus acidoterrestris Thermophilic broth (BAT) broth to achieve 10 CFU/ml. •14 different fruit juice/puree matrices with different Brix and pH values (Table 1) •The 14 different samples were inoculated with 10 CFU/ml of A. acidoterrestris, and the samples were incubated in a shaking incubator at 48 ºC. •A commercial guaiacol detection kit (Kyokuto Pharmaceutical Co., Ltd., Japan) used for confirming guaiacol formation in inoculated samples • Total community DNA was extracted from juice samples using foodproof® ShortPrep II kit (BIOTECON Diagnostics) • Alicyclobacillus target DNA sequences detected using foodproof® Alicyclobacillus Detection 5’Nuclease Kit ( BIOTECON Diagnostics) • The effect of juice matrix, incubation time and the possible interaction between the juice matrix and incubation time evaluated using ANOVA using Design-Expert™ (Stat-Ease, MN). •Significant differences (p< 0.01) between the treatments were analyzed using by Student's t-test Objectives  Evaluating the possibility of using commercial PCR based kits to detect Alicyclobacillus acidoterrestris in different juice matrices  Studying the effect of Brix and incubating time in rapid detection of Alicyclobacillus acidoterrestris, using RT-PCR  Evaluating the possibility of detecting Alicyclobacillus acidoterrestris, at low initial concentration of 10 CFU/ml in juice Significance and commercial value  Saves time and personnel costs for detection of Alicyclobacillus acidoterrestris  Rapid detection enabling product separation from production  Economic savings from reduced product recalls  Protection of brand equity Experimental Approach Table 1. List of the different fruit juice and puree matrices used in the study. Juice Tomato Juice Tomato Sauce Tomato Paste Ketchup Apple Juice Filtered Apple Juice unfiltered Apple puree Apple Juice Concentrate Orange Juice Filtered Orange Juice with Pulp Orange Juice Concentrate Peach Juice Peach Puree Brix 6 9 26 27 23 22 27 40 10 11 43 12 17 pH 4.15 4.24 3.69 3.69 3.8 3.73 4.08 4.00 4.06 4.03 4.09 3.75 4.08 10 ml BAT broth + 1 ml juice + 10 CFU/ml Alicyclobacillus acidoterrestris 37 ºC 10 min 80 ºC DNA extraction 24h 48h RT-PCR Ct curves on RT-PCR K agar at 37ºC for 3-5 Days Gauicacol Detection Results Source Sum Squares df Mean Square F Value P-value Juice 6280.43 12 523.37 13.06 < 0.0001 Incubation time 2866.86 1 2866.86 71.53 < 0.0001 Juice X Incubation time 1148.67 10 114.87 2.87 0.0035 Table 3 - Analysis of variance (ANOVA) for the effect of juice matrices, incubation time and their mutual effect on detecting A. acidoterrestris by RT-PCR(Aliased) Analysis of variance table Ct values for Alicyclobacillus spp. Ct Values for A. acidoterrestris Juice Back- ground 24h 48h Back- ground 24h 48h Apple juice 27.1 a ±3.9 29.2 a ±2.2 12.4 b ±2.7 35.5 a ±0.9 26.6 a ±3.4 10.4 b ±2.1 Cloudy Apple 37.0 a ±3.9 26.6 b ±2.2 13.6 c ±2.2 35.1 a ±0.7 23.9 b ±8.1 11.0 c ±0.3 Orange Juice no pulp 40.0 a ±5.5 22.5 b ±3.1 19.3 b ±2.2 35.2 a ±0.1 26.1 a ±8.3 17.8 b ±7.7 Orange Juice with pulp 38.2 a ±3.1 31.8 a ±2.4 22.7 b ±2.2 UD 26.7 a ±6.7 20.6 a ±6.4 Peach Nectar 49.4 a ±5.5 19.8 b ±2.2 14.3 b ±2.2 35.4 a ±0.6 17.0 b ±2.1 11.6 b ±0.2 Tomato Juice 37.6 a ±2.4 26.8 b ±1.7 15.7 c ±1.7 33.4 a ±1.7 26.1 a ±3.8 14.1 b ±6.6 Apple Concentrate 33.8 a ±3.9 47.7 a ±3.9 40.2 a ±3.1 32.3 a ±0.0 34.9 a ±1.51 34.8 a ±1.7 Apple puree UD* 34.0 a ±2.2 21.4 b ±2.2 UD 29.0 a ±4.80 20.3 a ±9.9 Ketchup 46.8 a ±5.5 46.3 a ±5.5 39.3 a ±2.7 UD 35.7 a ±4.4 35.3 a ±0.5 Orange Concentrate 30.8 a ±3.1 33.9 a ±2.2 34.3 a ±2.2 UD 36.2 a ±2.4 34.7 a ±2.7 Peach Puree UD UD 27.8±2.7 UD 38.0 a ±1.6 25.3 a ±2.1 Tomato Paste 39.0 a ±0.8 29.6 a ±11.6 23.4 a ±13.7 UD 25.6 a ±6.7 18.9 a ±10.2 Tomato Sauce UD 31.7 a ±3.1 16.3 b ±2.7 37.0 a ±0.2 30.4 a ±3.8 16.0 b ±3.0 Undetected Detected Table 2 - The effect of enrichment time on detecting Alicyclobacillus and acidoterrestris genome in different juice matrices by RT-PCR Real time-PCR Growth on K agar Guiacol Detection Juice (hour) 24h 48h 24h 48h Apple juice 24h - + - + Cloudy Apple 24h + + + + Orange Juice no pulp 24h - + - + Orange Juice with pulp 48h - + - + Peach Nectar 24h + + + + Tomato Juice 24h + + + + Apple Concentrate NA + + + + Apple puree 24 h - + - + Ketchup 48h - - - - Orange Concentrate NA - - - - Peach Puree 48h - + - + Tomato Paste 24h + + + + Tomato Sauce 24h + + + + Table 4 – Comparison of detection of A. acidoterrestris in different fruit matrices by real-time PCR and conventional plating method using K agar and confirmation of guaiacol formation Conclusions  Real-time PCR can be used for rapid detection of A. acidoterrestris at low contamination ( 10 CFU/mL) levels within 24 hours of enrichment  The juice matrix and enrichment time influence the detection of A. acidoterrestris by real-time PCR  Real-time PCR results are significantly influenced by the interaction between juice matrix and enrichment times  A. acidoterresteris’ growth is inhibited if the juice has a Brix value > 20.  Real-time PCR is not capable of detecting A.acidoterrestris in juice matrices with Brix values > 20 .  To detect A. acidoterrestris in purees and other high viscous products, the samples have to be diluted to Brix values < 20 to facilitate detection by real-time PCR. Figure 1 – Detection of A. acidoterrestris after 24 and 48 hours of incubation by RT-PCR * UD = Undetermined View publication stats View publication stats