Temperature, soluble solids and pH effect on Alicyclobacillus acidoterrestris viability in lemon juice concentrate

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Author: Maldonado,M.C; Belfiore C and Navarro AR

Alicyclobacillus in lemon juice

TEMPERATURE, SOLUBLE SOLIDS AND PH EFFECT ON ALICYCLOBACILLUS ACIDOTERRESTRIS VIABILITY IN LEMON JUICE CONCENTRATE

Maldonado, María C.*, Belfiore Carolina, Navarro Antonio R.

Instituto de Biotecnología. Facultad de Bioquímica, Química y Farmacia. UNT. Ayacucho 465. (4000) Tucumán. Argentina.

* Author for correspondence. Tel: 54-381-4247752 ext.7002 E-mail: cristimaldone@hotmail.com

1. Introduction

Alicyclobacillus acidoterrestris, a thermoacidophilic, non pathogenic, spore-forming bacterium, has been detected in several spoiled commercial pasteurized fruit juices. Wisotzey et al. (1992) characterized this genus by Ω-alicyclic fatty acids as the major natural membrane lipid component. A.acidoterrestris spore germination and growth to 106 cfu/ml under acidic conditions have been reported in orange juice stored at 44°C for 24h (Pettipher et al. 1997) and they concluded that 105- 106 cells/ml in apple and orange juices formed enough guaiacol (ppb) to produce sensory taint. Splittstoesser et al. (1994) reported growth inhibition of acidic spore-forming bacillus spores with a high content of total SS. Several authors (Gocmen et al., 2005; Jensen and Whitfield, 2003) reported the presence the guaiacol in fruits juices. Visual detection of spoilage is very difficult because A.acidoterrestris does not produce gas during growth and incipient swelling of containers does not occur. Besides, it does not represent a hazzard for human health since it is a non pathogenic microorganism (Orr et al. 2000). Due to these facts, spoilage can occur without visible changes (Walls and Chuyate, 1998) during retail storage of the product. Concerning heat resistence, Pontius et al. (1998) concluded that the type of organic acid (malic, citric, and tartaric) did not significantly affect D-values. Silva et al. (1999) studied the thermal inactivation of A.acidoterrestris spores under different temperature, soluble solids and pH conditions in a solution that simulated a fruit system. There are few published data and no systematic work that simultaneously investigated the influence of pH, SS and temperature (T) on the heat resistance of A.acidoterrestris spores. The main objective of this work was to investigate the effect of pH, soluble solids and temperature on D-values of A.acidoterrestris in clarified and non clarified lemon juice concentrates, because there are no lemon juice data in the references.

2. Materials and methods

2.1. Microorganism.

The highly heat resistant A. acidoterrestris used in this work was supplied by CIATIac (Centro de investigación y asistencia técnica a la industria) and it was kept in orange serum agar (OSA) (Murray et al., 2007) at 4°C. The strain was confirmed for biochemical tests.

2.2. Bacterial activation and spore production.

Cells were activated in orange serum medium (OS) in g/l: tryptein: 10; dextrose: 4; yeast extract 3; K2HPO4: 2.5; orange juice: 200 ml; pH 5.5 at 42ºC for 24 h. The microorganism was spread in an orange serum agar medium (OSA) and incubated for 48h at 42ºC. At the end of this period the cells were transferred to a tube with sterile distilled water and Tween 80 until reaching a DO = 0.250 at 540 nm and they were kept at 6º C for a week. The spores thus obtained were used for the tests performed.

2.3. Experimental design. The D-value of A. acidoterrestris as a function of temperature (82-95º C), SS (50; 68; 9.8 and 6.2 ºBrix) and pH (2.28-4) was determined by heating lemon juice. Heating periods were chosen taking into account the time spent in the pasteurization process at 82º C and the longest time at which spores survived (95º C). The SS values correspond to clarified lemon juice concentrate (50 ºBrix), non clarified lemon juice concentrate (68 ºBrix), beverages manufactured with clarified (sodas), and seasonings manufactured with non clarified lemon juice concentrate (9.8 and 6.2 ºBrix respectively). D values were determined by placing 5ml of clarified and non clarified lemon juice concentrate in glass tubes (15 x 97mm), seeding with 50ul inoculum and heating. SS were adjusted with sterile distilled water and pH values with NaOH 40%. After setting the temperature, the tubes were placed and stabilized in a thermostated bath for 2 minutes, necessary time for the temperature to be homogenous in the juice sample. Time was clocked at the end of this period (30sec, 45sec, 1min, 2.5min, 5min, 10min, 15min, 20min and 30min). The tubes were withdrawn and put in an ice bath to stop the thermal treatment. Survival count was carried out by placing 1ml of the appropriate dilution in 10 ml OSA medium. The inoculated plates were incubated at 42ºC for 48h and microorganisms were counted in ufc/ml.

2.4. Data evaluation.

Survivor curves were plotted as the log of survivors per milliliter versus time in minutes. D values (the time necessary to reduce the viable spore population by 90% at a given temperature) were determined as the negative reciprocal of the survivor curve slope. D values for a replicate trial were averaged and decimal reduction time curves (DRTC) were plotted (logarithm of the average D values versus temperature). The reaction rate constant k was calculated as k= 2.303/D. Two predictive models were created by using the Mathematics 4 software to describe and visually illustrate the effect of pH and temperature interactions on heat resistance. The statistical model generated a predictive equation which takes into account pH and temperature interaction on D values. The equations are as follows: D= 46.7716(pH) - 0.450125(pH)(T) (clarified juice). D= -1747.24-13.9718(pH2) - 0.218849(T2) + 98.053(pH) + 37.6104(T) (non clarified juice).

3. Results and discussion Low water activity, high sugar concentration, and limited O2 dilution capacity -all physicochemical characteristics of the juice concentrate- are important growth inhibitors for most altering and pathogenic microorganisms. Heating at 82ºC to 96ºC for 2 minutes is enough to destroy filamentous fungi and yeast and prevent possible contamination (McIntyre et al. 1995). Our results proved that Alicyclobacillus spores can survive at these temperatures in lemon juice. When temperature was raised from 82º C to 95º C, D values decreased from 17.36 min to 6.20 min in clarified juice with pH 2.28 (Table 1). In non clarified juice D values were 15.50 min at 82ºC and 8.55 min at 95ºC (Table 2). Lemon juice is generally pasteurized at 82º C for 2 min; our results and those reported for apple, white and dark grape juice (Splittstoesser et al. 1994), orange and apple juice (Pettipher et al. 1997) and a fruit juice model (Pontius et al. 1998) evidenced that Alicyclobacillus spores can survive the pasteurization process. This study confirms previous findings in other systems and the novelty lies in using lemon juice matrices, where the conditions the microorganism has to resist are much more adverse.

Tables 3 and 4 show the effect of SS on D values for both juices. A. acidoterrestris was less resistant in clarified juice (50º Brix) than in non clarified juice (68º Brix). This results coincide with the results reported for Silva et al. (1999). The D values were 25.81 min and 50.50 min for clarified and non clarified juice respectively at 82º C and pH 2.8. Similar behavior was observed in juice with pH 3.5 and pH 4 at the temperature studied (Tables I and II). The same thing does not hold true with pH 2.28 lemon juice: differences in D values between clarified and non clarified juice were not significant. In this case, pH effect would play a more important role than SS on Alicyclobacillus viability. To confirm that bacillus behavior in juice with higher pH than 2.28 will depend on SS concentration, clarified and non clarified juices with pH 2.8 were adjusted at the same SS value (9.8º Brix corresponding to the ones measured in lemon soda) with sterile distilled water and D was calculated. No observations were made that confirmed that D is influenced by SS. D values for the aciduric sporeforming bacilli were almost the same even though the juices differed in pH and SS concentration. The effect of these variables on aciduric bacilli resistance is unknown although spores are often more resistant when heated in solutions of higher pH and Brix (Splittstoesser et al. 1994). Since the industrial pasteurization process currently used does not eliminate A.acidoterrestris spores, it was important to understand the behavior of the bacillus in goods manufactured with clarified (sodas) and non clarified juice concentrates (seasonings). Hence, SS and pH characteristics of these products were imitated and the systems were heated to 82º C and 95º C for different time periods (Table III y IV). The results show that Alicyclobacillus survives well in the systems studied. Splittstoesser et al. (1994) found that when commercial juice beverages were inoculated with Alicyclobacillus spores, the results differed depending on the type of juice; apple and tomato juice consistently supported growth, whereas grape juice at both pH 2.9 and 3.3 did not permit it. Figures 1 and 2 show the model of A. acidoterrestris in clarified and non clarified lemon juice. All the interactions in the equation employed had a statistically significant effect (p< 0.05) on the D value. Fig. 1 shows that as pH values increase in clarified juice so does D, whereas D values diminish when temperature rises. In non clarified juice (Fig.2), maximum resistance to A.acidoterrestris occurs at pH 3 to 3.5 at 90ºC approximately. Due to the differences observed between predicted and measured D-values in fruit products, the model obtained cannot be used directly to estimate heat resistance in real fruit systems, but it is useful for predicting the trends and relative changes in D-values brought about by temperature and pH variations. The results of this work show that A.acidoterrestris survives industrial pasteurization (82ºC for about 2 min). Besides, at 95ºC, a much higher temperature than the one used in the hot-fill-hold process, the D value for A.acidoterrestris was 6 to 8.5 min. When the product is held for only 2 min at hot-fill processing temperature, spoilage is likely to occur if the raw product is contaminated even with a modest A.acidoterrestris spore population. To check the problem, lemon juice manufacturers should make sure that spore population is sufficiently low by increasing process temperature and/or hold time or adding preservatives to control this organism.

REFERENCES 1- Gocmen D, Elston A, Williams T, Parish M and Rouseff RL ( 2005) Identification of medicinal off-flavours generated by Alicyclobacillus species in orange juice using GC-olfactometry and GC-MS . Letters in Applied Microbiology 40 , 173-177. 2- Jensen N and Whitfield FB (2003). Role of Alicyclobacillus acidoterrestris in the development of a disinfectant taint in shelf-stable fruit juice. Letters in Applied Microbiology 36, 9-14. 3- McIntyre S, Ikawa JY, Parkinson N, Haglund J and Lee J (1995). Characteristics of an acidophilic Bacillus strain isolated from shelf-stable juices. Journal of Food Protection. 58(3), 319-321. 4- Murray M, Gurtler J, Ryu J, Harrison M and Beuchat L (2007). Evaluation of direct plating methods to enumerate Alicyclobacillus in beverages. International Journal of Food Microbiology 115(1): 59-69. 5- Orr RV, Shewfelt RL, Huang CJ, Tefera S and Beuchat LR (2000). Detection of guaiacol produced by Alicyclobacillus acidoterrestris in apple juice by sensory and chromatographic analyses, and comparison with spore and vegetative cell populations. Journal of Food Protection 63(11), 1517-1522. 6- Pettipher GL, Osmundson ME and .Murphy J.M (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 Microbioliology 24, 185-189. 7- Pontius AJ, Rushing JE and Foegeding PM (1998). Heat resistance of Alicyclobacillus acidoterrestris spores as affected by various pH values and organic acids. Journal of Food Protection 61(1), 41-46. 8- Silva FM, Gibbs P, Vieira MC and Silva CLM (1999). Thermal inactivation of Alicyclobacillus acidoterrestris spores under different temperature, soluble solids and pH conditions for the desing of fruit processes. International Journal of Food Microbiology 51, 95-103. 9- Splittstoesser DF, Lee CY and Churey JJ (1998). Control of Alicyclobacillus in the juice industry. Dairy Food and Environmental Sanitation 8(9), 585-587. 10- Splittstoesser DF, Churey JJ and Lee CY (1994). Growth characteristics of aciduric sporeforming bacilli isolated from fruit juices. Journal of Food Protection 57(12), 1080-1083. 11- Walls I and Chuyate R (1998) Alicyclobacillus historical perspective and preliminary characterization study. Dairy Food and Environmental Sanitation 18(1), 1-5. 12- Wisotzkey JD, Jurtshuk P, Fox GE, Deinhard G and Poralla K (1992) Comparative sequence analysis on the 16S rRNA (rDNA) of Bacillus acidocaldarius, Bacillus acidoterrestris, and Bacillus cycloheptanicus and proposal for creation of a new genus, Alicyclobacillus gen.nov. International Journal of Systematic Bacteriology 42(2): 263-269.

FIGURE LEGENDS Figure 1: Statistically generated Model of A.acidoterrestris in clarified lemon juice using the Mathematics 4 program. Figure 2: Statistically generated model of A.acidoterrestris in non clarified lemon juice using the Mathematics 4 program.

Table I: D values, reaction rate constants (K) and Td values at different pH and temperature for A. acidoterrestris in clarified lemon juice concentrates

T(Cº) pH K(min-1) D(min) Td(min) 2.28 - 0.13 17.36 6.36 82ºc 2.80 - 0.09 25.81 113.79 3.50 - 0.07 33.66 141.51 4.00 - 0.10 21.95 93.95 2.28 - 0.13 18.06 55.46 86ºC 2.80 - 0.10 22.01 82.90 3.50 - 0.03 68.95 293.47 4.00 - 0.06 35.16 129.76 2.28 - 0.30 7.60 25.98 92ºC 2.80 - 0.15 15.35 76.98 3.50 - 0.13 16.87 86.22 4.00 - 0.09 23.19 122.57 2.28 - 0.37 6.2 28.43 95ºC 2.80 - 0.20 11.32 56.68 3.50 - 0.18 12.63 60.34 4.00 - 0.23 9.72 48.18

Table II: D values, reaction rate constants (K) and Td values at different pH and temperature for A. acidoterrestris in non clarified lemon juice concentrates

T(Cº) pH K(min-1) D(min) Td(min)

2.28 - 0.15 15.50 66.53 82ºc 2.80 - 0.04 50.50 229.82 3.50 - 0.06 38.00 181.05 4.00 - 0.08 27.48 113.66 2.28 - 0.16 14.54 63.13 86ºC 2.80 - 0.07 31.67 126.39 3.50 - 0.02 95.15 360.00 4.00 - 0.04 58.15 260.56 2.28 - 0.26 8.81 37.88 92ºC 2.80 - 0.06 39.30 179.18 3.50 - 0.04 59.50 310.00 4.00 - 0.03 85.29 451.85 2.28 - 0.27 8.55 33.36 95ºC 2.80 - 0.10 22.03 108.52 3.50 - 0.13 17.22 81.64 4.00 - 0.09 23.33 119.87

Table III: D values, reaction rate constants (K) and Td values at different ºBrix for A. acidoterrestris in clarified lemon juice-pH 3.5

ºBrix T(Cº) K(min-1) D(min) Td(min) 82 - 0.20 11.23 51.73 9.8 86 - 0.22 10.54 49.90 92 - 0.24 9.47 48.90 95 - 0.27 8.55 33.95 82 - 0.13 17.35 60.36 50 86 - 0.12 18.06 59.60 92 - 0.30 7.60 25.98 95 - 0.37 6.20 28.43 6.2 82 - 0.17 13.21 63.58 95 - 0.24 9.38 48.88

Table IV: D values, reaction rate constants (K) and Td values at different ºBrix for A. acidoterrestris in non clarified lemon juice-pH 2.45

ºBrix T(Cº) K(min-1) D(min) Td(min) 82 - 0.1377 16.72 87.14 9.8 86 - 0.2034 11.32 58.99 92 - 0.2177 10.58 55.12 95 - 0.2306 9.98 52.03 82 - 0.1486 15.50 66.53 50 86 - 0.1584 14.54 63.13 92 - 0.2614 8.81 37.88 95 - 0.2694 8.56 33.40 6.2 82 - 0.1292 17.82 85.15 95 - 0.2483 9.44 45.26