Effect of repetitive ultraviolet irradiation on the physico-chemical properties and microbial stability of pineapple juice

INNFOO-01143; No of Pages 7 Innovative Food Science and Emerging Technologies xxx (2014) xxx–xxx

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Effect of repetitive ultraviolet irradiation on the physico-chemical properties and microbial stability of pineapple juice Rosnah Shamsudin a,⁎, Noranizan Mohd Adzahan b, Yap Pui Yee a, Atikah Mansor a a b

Department of Process and Food Engineering, Faculty of Engineering, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

a r t i c l e

i n f o

Article history: Received 21 November 2013 Accepted 6 February 2014 Available online xxxx Editor Proof Receive Date 19 March 2014 Keywords: Ultraviolet irradiation Juice Dimethyl dicarbonate

a b s t r a c t The study aims to investigate the effect of repetitive ultraviolet irradiation (UV–UV) and the combination effect with dimethyl dicarbonate (UV–DMDC–UV) on the physico-chemical properties and microbiological stability of pineapple juice. UV dosages of 10.76 mJ/cm2 percycle and 250 ppm of DMDC were used. There was a significant decrease in turbidity, total phenolic and vitamin C in the treated juices. The UV–UV reported a significant reduction of 1.91 log CFU/ml in total plate count and 1.4 log CFU/ml in yeast and mould. Post addition of DMDC into the UV irradiated juice (UV–UV–DMDC) showed reductions of 2.61 log CFU/ml for TPC and 4.87 log CFU/ml for YM. This study demonstrated the effectiveness of UV irradiation in preserving the nutritional quality and the addition of DMDC can have a combination effect with the UV irradiation of juice in terms of microbial reduction. However, the treatments were not sufficient to achieve adequate microbial reduction as required by the FDA. Industrial relevance: Dimethyl dicarbonate (DMDC) is one of the effective anti-microbial agents that can control a wide range of microorganisms which includes Escherichia coli 0157:H7 and yeast. The effect of dimethyl dicarbonate (DMDC) in reducing microbial counts was significant in this study. According to Threlfall and Morris (2002), DMDC is used to prevent fermentation in excessive yeast contamination in wine production. Moreover, Halim et al. (2012) stated that DMDC has shown promising results for microbial inactivation of fruit juices in a preliminary study in lab. Therefore, combination effect with additives (DMDC) may be able to increase the efficiency of the UV irradiation for microbial reduction in juice and longer the shelf life of juice. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Fruit juices are defined as unfermented but yet can be fermentable products collected from fresh, mature and healthy fruits (Anon, 2006). Such juices can be formed using a single strength of fruit or a number of fruits. Fruit juices are famous for their good food value and high mineral and vitamin content (Bates et al., 2001; Kabasakalis, Siopidou, & Moshatou, 2000). Recently, the trend of fruit juice consumption has been on the rise over the last few decades due to their diverse health benefits (Bates, Morris, & Crandall, 2001; Borenstein, Dai, Wu, Jackson, & Larson, 2005; Liu, 2003). Pineapple juice is famous for its very pleasing aroma and flavour (Rattanathanalerk, Chiewchan, & Srichumpoung, 2005). It is generally drinkable in single-strength, reconstituted or concentrated forms, and can be mixed with other juices to develop new flavours for beverages and other products due to its strong acid flavour (Carvalho, Castro, & Silva, 2008). The Morris pineapple variety has the advantages of producing fruits that are high in sugar (11– 18 °Brix) and are attractive for their golden yellow flesh colour (De ⁎ Corresponding author. E-mail address: [email protected] (R. Shamsudin).

Silva, Kadir, Aziz, & Kadzimin, 2008). According to the Malaysia Pineapple Industry Board (MPIB) (2011), the estimated production cost for the Morris variety is cheaper than other varieties such as Josephine. Hence, the Morris pineapple could have the potential to attract consumers and increase their preference for pineapple juice in the industry. Fruit juice, especially fresh or unpasteurized juice, has a high tendency for spoilage due to being unprotected by skin or a cell wall and by exposure to the air and microorganisms from the environment. According to Wood and Moellering (2003), microorganisms can adapt themselves to adverse environments which were previously harmful to them. Hence, due to microbial resistance, the juice industry has to constantly face this problem. From estimation by the United States Food and Drug Administration (FDA), there are 140 juice related illnesses that can be prevented yearly. Hence, the labelling rules for juice products have increased the awareness of consumers of the dangers of drinking untreated juice (FDA, 2001a). Further, Foley et al. (2002) explained that there are nearly 16,000 to 48,000 illness issues annually that arise due to unpasteurized juice consumption. Pasteurization is an effective technology used to satisfy safety requirements in the fruit juice industry. Thermal treatment can

http://dx.doi.org/10.1016/j.ifset.2014.02.005 1466-8564/© 2014 Elsevier Ltd. All rights reserved.

Please cite this article as: Shamsudin, R., et al., Effect of repetitive ultraviolet irradiation on the physico-chemical properties and microbial stability of pineapple juice, Innovative Food Science and Emerging Technologies (2014), http://dx.doi.org/10.1016/j.ifset.2014.02.005

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achieve a 5-log reduction in the number of most resistant pathogens as required by the FDA. Currently, pasteurization is the most widely used process in the Malaysian juice industry for effective microbial eradication. However, the application of thermal energy may cause undesirable effects on the flavour and nutritional content of the juice as well as the physicochemical properties (MosquedaMelgar, Raybaudi-Massilia, & Martín-Belloso, 2008). Nevertheless, the cost of the equipment can be too expensive especially for small operations. Ultraviolet irradiation is a non-thermal disinfection technology that is applied at a low temperature (Tran & Farid, 2004). It can potentially be used as an alternative to thermal pasteurization in the juice industry. Based on the work of Canitez (2002), this process does not create chemical residues. Moreover, it is a low-cost operation and is effective against many microorganisms (Bintsis, Tzanetaki, & Robinson, 2000). Ultraviolet-C is considered to be germicidal against microorganisms such as bacteria, viruses, protozoa, yeasts, moulds, and algae (Bintsis et al., 2000), where the highest effect is achieved between 250 and 270 nm. Koutchma, Froney, and Moraru (2009) stated that the most efficient inactivation can be obtained at 253.7 nm due to the maximum absorption of UV photons by the genetic materials of microorganisms at this specific wavelength. The efficiency of ultraviolet irradiation relies on the absorbance of the medium, flow rate of the fluid, moisture content, amount of solid particles and suspended materials, fluid thickness, reactor design, ultraviolet intensity which is interconnected to the exposure time, age of lamps used, type of microorganisms and growth phase of the organism, and initial microbial density (Begum, Hocking-Ailsa, & Miskelly, 2009; Bintsis et al., 2000; Caron, Chevrefils, Barbeau, Payment, & Pre'vost, 2007; Guerrero-Betran & Barbosa-Canovas, 2005; Hansen, 1976; Koutchma et al., 2009). However, the poor penetration property of ultraviolet light is the main limitation. Although research has proven that UV irradiation is able to provide a 5-log reduction of Escherichia coli O157:H7 in apple cider, very few studies have been carried out to determine the effect of UV when used in combination with preservatives that are commonly applied in juices. According to Tandon, Worobo, Churey, and Padilla-Zakour (2003), ultraviolet irradiation alone might not be effective enough against spoilage microorganisms such as yeast and moulds in apple cider. Dimethyl dicarbonate (DMDC) is one of the effective anti-microbial agents that can control a wide range of microorganisms which includes E. coli 0157:H7 and yeast. The effect of dimethyl dicarbonate (DMDC) in reducing microbial counts was significant in this study, although small. Based on Threlfall and Morris (2002), DMDC is used to prevent fermentation in excessive yeast contamination in wine production. DMDC is also permitted for use in single strength juices given that it also has an inhibitory action in moulds and bacteria (Fisher & Golden, 1998). DMDC has shown promising results for microbial inactivation of fruit juices in a preliminary study in lab, as well as in the published work of others (Halim et al., 2012). The legal limit for the usage of DMDC in non-alcoholic beverages and juice is 250 mg/L or 250 ppm. The usage of DMDC in 100% juice is approved by the FDA as of June 2000 (FDA, 2001b). Thus it is interesting to observe the effect of combination with UV. Previous studies have proven that one cycle of ultraviolet irradiation on pineapple juice is not sufficient to achieve a 5 log reduction of microorganisms as required by the Food and Drug Administration (FDA) (Chia, 2011a,b; Hamzah, 2009). For this reason, a repeat using two cycles of ultraviolet irradiation is suggested in this study. Moreover, a combination effect with additives (DMDC) may be able to increase the efficiency of the UV irradiation for microbial reduction in juice. Hence, the objective of this study is to determine the effect of two cycles of ultraviolet irradiation (UV–UV) and the combination effect of dimethyl dicarbonate with ultraviolet irradiation (UV–DMDC–UV) on the physico-chemical properties and microbiological stability of pineapple juice.

2. Materials and methods 2.1. Preparation of pineapple juice Pineapple fruits (Ananas comosus L.) of the Morris variety at commercial maturity were purchased from a commercial farm in Selangor, Malaysia. After the fruits were washed, the skins were removed using a meat slicer (300SL, DEUGI, Italy). Then, the flesh of the fruits was cut into smaller pieces using a food slicer (ECA-201, EMURA, Japan). The juice was then produced using a supermasscolloider (ZA10-20J, MASAKO, Japan), an ultra-fine friction grinder. It was followed by filtering the juice through a bean grinder (MH-280, Taiwan). The juice was filtered again using a 500 micron aperture stainless steel screen (BS 410-1, ALPHA, England) prior to treatment. 2.2. Ultraviolet pasteurizer The filtered pineapple juice was treated using a CiderSure 3500-B laboratory unit (Macedon, New York). This equipment was already commercialized to treat juice or cider using ultraviolet irradiation (Koutchma, Keller, Chirtel, & Parisi, 2004; Matak et al., 2007; Shamsudin, Chai, Mohd Adzahan, & Wan Daud, 2013). This laboratory unit consists of electronic controls and a process tube, through which the fluid flows. The UV irradiation consists of eight lowpressure lamps that emit UV light at 254 nm. The sensors provide information for UV dosage calculations. The ultraviolet dosage applied in study was 10.76 mJ/cm2 per cycle which is the highest dose that can kill microbes. The thickness of the thin film was in the range 0.21 to 0.48 mm. The flow rate of the fruit juice was set to run automatically in the ultraviolet pasteurizer. The machine will auto select a flow rate that should give a higher microbial count reduction. For the repetitive ultraviolet irradiation, the ultraviolet irradiated juice was allowed to run through another cycle. The flow rate was shown on the touch screen panel of the machine. The irradiated juice that had gone through two cycles was used for the analyses. For the study of the effect of DMDC addition on the UV treatment, dimethyl dicarbonate (DMDC) from Velcorin with a maximum limit of 250 ppm was added into the juice immediately after the first cycle of UV irradiation (FDA, 2001b). Then, the juice was allowed to stand for 90 min at room temperature (±27 °C) to allow full hydrolysis in the juice before entering for the second cycle. From the flow rate display on the screen of the UV pasteuriser, the exposure time of the juice in the UV pasteurizer was calculated. The calculation for the ultraviolet dosage followed the method reported by Chia (2011a). With the consideration of the energy lost by heat and when the ultraviolet light passed through the quartz tube, the power density was calculated. By using the calculated power density, the sensor placement factor was determined. Lastly, the ultraviolet dosage was calculated by multiplying the irradiance of the juice by the exposure and sensor placement factor. 2.3. Physico-chemical analyses 2.3.1. Total soluble solids (TSS) The total soluble solid (TSS) level of the juice was determined using a digital refractometer (AR-2008, Kruss, Germany). The measured value was expressed as °Brix. A substantial amount of extracted juice was dropped onto the refractometer. The reading shown was the reading of the total soluble solids for the juice. Two replications of the treatment were conducted. For each replication, duplicate measurements were conducted and the results averaged. 2.3.2. Total phenolic The total phenolic content of the juice was measured using the Folin–Ciocalteau method as reported by Lukanin, Gunkp, Bryk, and

Please cite this article as: Shamsudin, R., et al., Effect of repetitive ultraviolet irradiation on the physico-chemical properties and microbial stability of pineapple juice, Innovative Food Science and Emerging Technologies (2014), http://dx.doi.org/10.1016/j.ifset.2014.02.005

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Nigmatullin (2003), with some modification, i.e. the juices were centrifuged at 5000 rpm for 5 min at 4 °C. An amount of 1 ml of clarified juice was mixed with 1 ml Folin–Ciocalteau reagent and 10 ml of 20% sodium carbonate solution. Then, the mixture was diluted to 100 ml with distilled water and mixed thoroughly. The absorbance of the mixture was measured using a UV spectrophotometer (Ultraspec3100pro, Amersham Biosciences, UK) at 765 nm after standing for 1 h at room temperature in the dark. Data for total phenolic content was obtained from the calibration curve prepared with Gallic acid at concentrations of 0, 50, 100, 150, 250, 500, 750, and 1000 mg/L. The results are expressed as Gallic acid equivalents (mg GAE/L). 2.3.3. Titratable acidity and pH A digital autotitrator (785 DMP Titrino, Metrohm, Switzerland) was used to measure the titratable acidity and the pH. An amount of 10 ml of juice was mixed with 40 ml distilled water. The electrode was inserted into the mixture and measurements taken under continuous stirring. The pH value was shown automatically once the drift set on the titrator was reached. The pH value was recorded immediately after it appeared on the panel screen. The titratable acidity of the juice was obtained by titration with 0.1 mol/L NaOH to the end point of pH = ±8.5. The result was expressed in millilitres (ml). Two replications of the treatment were conducted. For each replication, duplicate measurements were carried out and the results averaged. The titratable acidity content was expressed as the percentage of a citric acid reference by the following equation (Scott, Clavero, & Trolller, 2001): EPI  0:064  C30  10C Titratable acidityð% citric acidÞ ¼ 2C00

ð1Þ

where, EPI is the NaOH up to the end point (pH ± 8.5), C30 is the molarity of NaOH and COO is the sample volume in ml. 2.3.4. Colour The colour measurement was performed by using a Spectrophotometer UltrascanPro (D65, HunterLab, USA) with reference to illuminant D 65 and a 10° observer angle to obtain values of L*, a*, b*. The L* value measures the lightness and changes from 0 (black) to 100 (white). The a* value measures the redness where it changes from − a* (greenness) to + a* (redness). Lastly, b* measures the yellowness; − b*(blueness) to + b*(yellowness). A 50 ml sample of juice was placed in an optical cell with a path length of 20 mm (Hunter Lab setting) for the measurement. The values of a* and b* were used to calculate the parameters of the colour appearance: hue angle (h = pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi  2  tan − 1 b*/a*) and chroma a 2 þb (Bernalte, Sabio, Hernandez, & Gervasini, 2003). Two replications of the treatment were conducted. For each replication, duplicate measurements were conducted and the results averaged. 2.3.5. Turbidity A turbidimeter (TN-100, Eutech, Singapore) was used to measure the turbidity of the juice. It was operated by shining an incident light beam through a 10 ml sample of juice with a scattering angle of 90°. The turbidity value was measured using Nephelos Turbidity units (NTU). The turbidimeter was calibrated using standard solutions in the range 0.02, 20.0, 100 and 800 NTU. As the turbidity of the pineapple juice was higher than the measurement range of the turbidimeter of 1000 NTU, a dilution procedure was needed. Hence, a 2.5 ml sample of juice was diluted with 7.5 ml of distilled water for the measurement. Two replications of the treatment were conducted. For each replication, duplicate measurements were conducted and the results averaged. The true turbidity value of the original sample juice was calculated using the following equation (Canitez, 2002): TurbidityðNTUÞ ¼ ðSðB þ CÞÞ=C

ð2Þ

3

where S* = turbidity found in the diluted sample, B = volume of dilution water (ml), and C = volume of sample juice used for dilution (ml). 2.3.6. Vitamin C The vitamin C content was measured by manual titration with 0.1% 2,6-dichlorophenol indophenol as the titrant. A standard solution of 0.1% ascorbic acid solution was prepared. The amount of standard ascorbic solution used to titrate 0.1% 2,6-dichlorophenol indophenol (DCPIP) as the titrant was recorded to compare with the juice. Then, 1 ml of 0.1% 2,6-dichlorophenol indophenol was placed into a specimen tube and titrated with the pineapple juice drop by drop until the DCPIP was discoloured. The result was expressed as mg/100 g. Two replications of the treatment were conducted. For each replication, duplicate measurements were carried out and the results averaged. The concentration of ascorbic acid of the juice is calculated by using the formula given below: Concentration of ascorbic acid ¼

volume of 0:1% ascorbic acid solution ð3Þ volume of fruit juice   mg : 100 100 g

2.4. Microbiological studies For the microbiological analysis, total plate counts (TPC) were determined using a plate count agar (PCA) (Merck, Germany). On the other hand, yeast and mould counts were determined using Dichloran Rose Bengal Chloramphenicol agar (DRBC) (Condalab, Spain). For both tests, 0.1 ml of sample from each serial dilution (10− 1 to 10 − 8) was spread onto solidified agar. The PCA plates were incubated for 2 days at 35 ± 2 °C whereas the plates for yeast and mould counts were incubated for 5 days at 25 ± 2 °C. Colonies of the TPC and yeast and moulds were counted using a colony counter. The results are expressed as log CFU/ml. Two replications of the treatment were conducted and the results averaged. For each replication, duplicate measurements were carried out. 2.5. Statistical analysis For the physico-chemical and microbiological analyses, two replications of the treatments were conducted. For each replication, duplicate measurements were conducted and the results averaged. A one-way analysis of variance (ANOVA) was used to compare the experimental treatments, and differences among treatment means were determined by the Tukey test where values of p b 0.05 were defined as significant. All the statistical analyses were conducted using SPSS Version 20.0 software (SPSS Inc., USA). 3. Results and discussion 3.1. Physico-chemical properties 3.1.1. Total soluble solids The total soluble solids were expressed in °Brix. It is important to determine the juice flavour and maturity (Matthews, 1994). The results for this study are displayed in Table 1. There was no significant difference in total soluble solids for the untreated (11.55 ± 0.06 °Brix) juice compared with the two cycle ultraviolet irradiated (UV–UV) (11.50 ± 0.12 °Brix) and the dimethyl dicarbonate added (UV–DMDC–UV) (11.4 ± 0.08 °Brix) pineapple juice. Hence, this shows that ultraviolet irradiation or the addition of dimethyl dicarbonate did not affect the total soluble solids of the pineapple juice. This is comparable to the findings of Hamzah (2009) who used the same equipment to UV-irradiate (single cycle) pineapple juice and reported the same

Please cite this article as: Shamsudin, R., et al., Effect of repetitive ultraviolet irradiation on the physico-chemical properties and microbial stability of pineapple juice, Innovative Food Science and Emerging Technologies (2014), http://dx.doi.org/10.1016/j.ifset.2014.02.005

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Table 1 Effect of repetitive (2 cycles) ultraviolet irradiation and combination effect with DMDC on the physicochemical properties of pineapple juice. Parameters

Untreated

Total soluble solid (°Brix) pH Titratable acidity (% citric acid) Turbidity (NTU) Vitamin C (mg/100 g) Total phenolic content (mg GAE/L) Colour (L*) Colour (hue angle) Colour (chroma)

11.55 3.89 0.67 1441.25 19.17 825 39.51 87.51 17.70

± ± ± ± ± ± ± ± ±

2 cycles of ultraviolet irradiation (UV–UV) 0.06a 0.01a 0.02a 43.45a 1.67 a 8.12a 0.47a 0.45a 0.47a

11.50 3.90 0.66 1194.00 15.43 716.79 39.10 86.02 16.13

± ± ± ± ± ± ± ± ±

0.12a 0.01a 0.01 a 17.74b 0.97 b 1.37b 0.40a 1.50a 0.97a

2 cycles of ultraviolet irradiation with DMDC addition (UV–DMDC–UV) 11.40 3.88 0.66 1389.00 13.46 698.93 38.44 86.75 16.76

± ± ± ± ± ± ± ± ±

0.08a 0.03a 0.01a 88.88b 0.79b 6.53c 0.45a 1.22 a 0.28a

Values followed by the same letter within the same row are not significantly different from each other (p b 0.05).

soluble solid values (fresh juice, 13.2 ± 1.98 °Brix; UV irradiated juice, 13.2 ± 1.91 °Brix). 3.1.2. Total phenolic content Juice is high in phenolic content. The beneficial effects resulting from phenolic compounds have been attributed to their antioxidant activity (Heim, Tagliaferro, & Bobilya, 2002). Table 1 illustrates the results of this study. Ultraviolet irradiated juice (UV–UV) (716.79 ± 1.37 mg GAE/L) and dimethyl dicarbonate added juice (UV–DMDC– UV) (698.93 ± 6.53 mg GAE/L) showed a significant decreased in total phenolic content compared to the untreated juice (825.00 ± 8.12 mg GAE/L). According to Noci et al. (2008), ultraviolet treated apple juice did show a significant reduction in total phenolic content. In addition, the reduction was significantly lower as compared to the thermally treated juice (Chia, 2011a,b). According to Chia (2011a), by applying a 53.42 mJ/cm2 UV dosage in one cycle on pineapple juice, the total phenolic content for fresh juice, 643.63 ± 2.26 mg GAE/L was reduced to 537.10 ± 0.88 mg GAE/L. On the other hand, the phenolic content was reduced to 419.96 ± 1.66 mg GAE/L when the juice was treated with thermal pasteurization. A contrasting observation was reported for clarified pineapple juice as mentioned by Goh, Noranizan, Leong, Sew, and Sobhi (in press) who applied ultraviolet irradiation to clarified pineapple juice at 7.5 mJ/cm2. They mentioned that there was no significant difference between the total phenolic content of fresh, ultraviolet treated and thermally treated (97 °C for 5 min) juice. The difference between the results shown might be due to the different techniques of processing, time or temperature. On the other hand, the addition of dimethyl dicarbonate showed a significant decrease (p b 0.05) in total phenol content when compared to the juices treated with ultraviolet irradiation alone. This might be due to the dimethyl dicarbonate that reacts with polyphenols in food (Bizri & Wahem, 1994; Ough, 1993). 3.1.3. pH There was no significant difference in pH value of the untreated juice (3.89 ± 0.01) compared to the ultraviolet irradiated juice (UV–UV) (3.90 ± 0.01) and dimethyl dicarbonate added juice (UV–DMDC–UV) (3.88 ± 0.03) as shown in Table 1. There was also no significant effect of the two cycle ultraviolet irradiation (UV–UV) on the pineapple juice which was the same as compared to the single cycle ultraviolet irradiated pineapple juice (fresh juice, 3.56 ± 0.08; UV irradiated juice, 3.54 ± 0.99) (Hamzah, 2009). Similar results have been reported by Bhat, Ameran, Han, Karim, and Liong (2011) who found that there was no significant effect on the pH value of starfruit juice treated with ultraviolet radiation. 3.1.4. Titratable acidity In this study, the titratable acidity was not significantly different between the untreated juice (0.67 ± 0.02% of citric acid) and the ultraviolet irradiated (UV–UV) (0.66 ± 0.01% of citric acid) as well as the dimethyl dicarbonate added juice (UV–DMDC–UV) (0.66 ± 0.01%

of citric acid) as shown in Table 1. This indicates that ultraviolet irradiation or the addition of dimethyl dicarbonate did not affect the quality of juice in terms of colour where the untreated pineapple juice retained the same colour similar to the untreated juice. A similar result was found by Mohd Adzahan, Sharizah, Sew, and Roselina (2011) and Chia (2011b) who stated that there was no significant change in pineapple juice after ultraviolet treatment. 3.1.5. Colour The measurement of colour is important for the quality assessment of juice. The L value measures the lightness of the juice whereby values nearer to zero indicate a darker juice (0 = black, 100 = white). In this study, there was no significant change in L value between the untreated juices (39.51 ± 0.47) and the ultraviolet irradiated juice (UV–UV) (39.10 ± 0.40) or the dimethyl dicarbonate added and ultraviolet irradiation treated juice (UV–DMDC–UV) (38.19 ± 0.31). According to Chia (2011a) who applied one cycle of ultraviolet radiation to the juice, the ultraviolet irradiated juice can retain its lightness properties similar to the untreated juice due to the fact that no substantial amount of heat was applied to the juice during the ultraviolet irradiation. Hence, from this study it can be said that the two cycles of ultraviolet irradiation still maintain the colour quality of the juice. On the other hand, the hue angle and chroma are colour properties that are related to the a*(redness) and b*(yellowness). Chroma (saturation) can be defined as the strength or dominance of the hue. It is the colourfulness relative to the brightness of another colour that appears white under similar viewing conditions. There was no significant difference in the chroma between the untreated juice (17.70 ± 0.47) and the ultraviolet irradiated (UV–UV) (16.13 ± 0.97) or the DMDC added and ultraviolet irradiation treated juice (UV–DMDC–UV) (16.76 ± 0.28). The slight decrease in chroma for the ultraviolet irradiated juice showed a less intense colour than the untreated juice. Chia (2011a) reported that there was no significant variation in the chroma of pineapple juice after ultraviolet irradiation. According to Crisosto, Bremer, Ferguson, and Crisosto (2010), the hue angle illustrates a pure spectrum colour at the dominant wavelength. In this study, there was no significant change among all the juices: the untreated juice (87.51 ± 0.45); two cycle ultraviolet irradiated juice (UV–UV) (86.02 ± 1.50); the DMDC added and two cycle of ultraviolet irradiation treated juice (UV–DMDC–UV) (86.75 ± 1.22). This result showed that ultraviolet irradiation and the addition of dimethyl dicarbonate were not affecting the colour quality of the juice. However, the slight decrease of hue is indicated by the greater red (a*) and less yellow (b*) colour of the samples (Esteve & Frigola, 2007). As far as is known, no study has been published on the effect of dimethyl dicarbonate on the colour of the juice. However, according to Chen (2011), dimethyl dicarbonate is colourless and hydrolyses quickly. Hence, it might not cause any effect on the colour on the juice. From this study, the result showed that ultraviolet irradiation as well as the addition of dimethyl dicarbonate did not have any effect on the colour of the juice. This finding was similar to Tran and Farid (2004)

Please cite this article as: Shamsudin, R., et al., Effect of repetitive ultraviolet irradiation on the physico-chemical properties and microbial stability of pineapple juice, Innovative Food Science and Emerging Technologies (2014), http://dx.doi.org/10.1016/j.ifset.2014.02.005

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who also found that the colour of orange juice was not impacted by ultraviolet light of 73.8 mJ/cm2. 3.1.6. Turbidity Turbidity can be defined as a measurement of the degree to which light is scattered by suspended particles and soluble solids in water. The effectiveness of ultraviolet disinfection systems is directly related to turbidity and total suspended solids (EPA, 1999; Mahmoud & Ghaly, 2004). Based on the study result (Table 1), there was a significant decrease (p b 0.05) in the turbidity of the two cycle UV irradiated juice (UV–UV) (1194 ± 17.74 NTU) and the DMDC addition into the UVirradiated (UV–DMDC–UV) (1389 ± 88.88 NTU) pineapple juice respectively compared to the untreated juice (1441.25 ± 43.45 NTU). There have been only a few studies into the effect of ultraviolet irradiation and the addition of dimethyl dicarbonate on the turbidity of juice. However, according to Canitez (2002), one of the possible reasons that cause a decrease in turbidity in pineapple juice after ultraviolet irradiation is the result of reduced yeast and mould counts in the pineapple juice. Yeast and mould can contribute to the turbidity of fluids and if there is a decrease in number then the turbidity is speculated to demonstrate a decrease. Besides that, Digiacomo and Gallagher (1959) had studied that spoilage by yeast and bacteria has induced sediment and turbidity in soft drink that contain juice. A comparison of the single cycle and the two cycle ultraviolet irradiation of the juice shows that the turbidity of the two cycle juice is lower than that of the single cycle juice (Canitez, 2002). This should provide a more efficient treatment for microbial reduction as more microorganisms can be directly exposed to the ultraviolet light. On the other hand, in addition of dimethyl dicarbonate to the ultraviolet irradiated juice (UV–DMDC–UV), the turbidity of the juice was non-statistically higher than the ultraviolet irradiated juice (UV–UV). This is due to the higher level of microorganisms in the juice as shown in Figs. 1 and 2. 3.1.7. Vitamin C With regard to the level of vitamin C, a significant decrease was recorded for the two cycle ultraviolet irradiated (UV–UV) juice (15.43 ± 0.97 mg/100 g) and the dimethyl dicarbonate added (UV–DMDC–UV) juice (13.46 ± 0.79 mg/100 g) where the vitamin C content for the untreated juice is 19.17 ± 1.67 mg/100 g as shown in Table 1. There was around 19.5% and 29.8% of vitamin C loss for the juice after two cycles of ultraviolet irradiation (UV–UV) and the addition of dimethyl dicarbonate in the ultraviolet irradiated juice (UV–DMDC–UV) respectively. From Bendich, Machlin, Scandurra, Burton, and Wayner (1986), vitamin C is a heat-sensitive bioactive compound that has a crucial role in human health and performs as an antioxidant. A similar result was reported by Bhat et al. (2011) who noticed a significant reduction in vitamin C for starfruit juice after ultraviolet treatment. According to

Fig. 2. Microbiological analyses on the combination effect of dimethyl dicarbonate with 2 cycle ultraviolet irradiation (pre, intermediate, post-addition).

Davey et al. (2000), the decrease in the vitamin C content can be attributed to the oxidation process together with the activities of ascorbate oxidase and peroxidase enzymes. However, a study by Chia (2011a) found that ultraviolet irradiated juice retained ascorbic acid more efficiently than a thermal process which gives benefit to the overall juice quality. She reported a 9.91% vitamin C loss after a single cycle of ultraviolet irradiation and a 37.83% vitamin C loss for pasteurized juice. Hence, even though the vitamin loss in this study was higher than when applying a single cycle of ultraviolet irradiation, the quality of the juice is still better than that of pasteurized juice. 3.2. Microbiological activities The effects of two cycles of ultraviolet irradiation (UV–UV) of the juice and the addition of dimethyl dicarbonate in between the two cycles of ultraviolet irradiation (UV–DMDC–UV) of the juice on the total plate counts and yeast and mould counts are shown in Fig. 1. The ultraviolet irradiation applied was 10.76 mJ/cm2 per cycle on average. The study showed a significant reduction (p b 0.05) in total plate count (TPC) for the treated juice. The total plate count (TPC) for the untreated juice was 6.06 ± 0.10 log CFU/ml. The total plate count was reduced to 4.15 ± 0.07 log CFU/ml after two cycles of ultraviolet irradiation (UV–UV) and to 4.33 ± 0.59 log CFU/ml after adding dimethyl dicarbonate in between the two cycles of ultraviolet irradiation (UV– DMDC–UV). The reductions in the total plate counts (TPC) were up to 1.91 log CFU/ml and 1.73 log CFU/ml respectively. A significant reduction (p b 0.05) was also observed in the yeast and mould counts. The yeast and mould counts for the untreated, two cycle ultraviolet irradiated (UV–UV) and dimethyl dicarbonate added (UV– DMDC–UV) juice were 5.74 ± 0.14 log CFU/ml, 4.34 ± 0.26 log CFU/ml

Fig. 1. Effect of 2 cycle ultraviolet irradiation (UV–UV) and addition of dimethyl dicarbonate in between 2 cycle ultraviolet irradiated (UV–DMDC–UV) juice on total plate counts and yeast and mould.

Please cite this article as: Shamsudin, R., et al., Effect of repetitive ultraviolet irradiation on the physico-chemical properties and microbial stability of pineapple juice, Innovative Food Science and Emerging Technologies (2014), http://dx.doi.org/10.1016/j.ifset.2014.02.005

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R. Shamsudin et al. / Innovative Food Science and Emerging Technologies xxx (2014) xxx–xxx

and 4.70 ± 0.17 log CFU/ml respectively. The reductions of yeast and mould counts were up to 1.4 log CFU/ml and 1.04 log CFU/ml for the ultraviolet irradiated (UV–UV) and the dimethyl dicarbonate added (UV–DMDC–UV) juice correspondingly. Both of the treatments did not kill the microorganisms as effectively as required to achieve the 5 log reduction mandated by the FDA. This might be due to the fibre and cells in the juice that can act as a protective barrier between the ultraviolet light and the microbes (Keyser, Muller, Cilliers, Nel, & Gouws, 2008). Further, Tahiri, Makhlouf, Paquin, and Fliss (2006) reported that the sugar concentration (°Brix) can protect the microorganisms from the high pressure treatment which may be considered as a limiting factor for the ultraviolet irradiation process. In addition, Tran and Farid (2004) declared that there was a maximum of a 3 log reduction of aerobic bacteria, yeasts and moulds by applying an ultraviolet dosage of 12–147.6 mJ/cm 2 . Yeast and mould exhibit a higher resistance compared to bacteria under ultraviolet irradiation mainly due to their DNA structure as they are known to possess a reduced pyrimidine nucleoside (cytosine and thymine) base. Furthermore, the chemical composition and thickness of the cell wall of yeast and mould are different from bacteria which can make them resistant to ultraviolet light (Bhat et al., 2011). Hamzah (2009) reported that by applying a single cycle ultraviolet irradiation, the log reductions were around 1.7 log CFU/ml in the total plate count (TPC) and 0.04 log CFU/ml in the yeast and mould count. Hence, this shows that the two cycles of ultraviolet irradiation can be more effective for microbial reduction. On the other hand, dimethyl dicarbonate should be an effective antimicrobial agent especially on yeast and mould. According to Quicho (2005), dimethyl dicarbonate (75 and 150 ppm) in conjunction with ultraviolet irradiation showed more than a 6 log reduction in a population of E. coli ATCC 25922 in apple cider. However, for this study, the result was not as expected. This might due to a few reasons. Dimethyl dicarbonate is only effective on microorganisms that are in a vegetative state. Spores are a dormant structure that consists of a hardened coat that encompasses the specific remnants of the vegetative-state which is needed for the organism to re-instate germination. Hence, the spore state offers protection from chemical and physical agents which are lethal to vegetative forms. In addition, dimethyl dicarbonate gives a rapid decomposition in aqueous systems where the rate of degradation is somewhat fast. Once dimethyl dicarbonate has decomposed, there is no more sterilising effect. So, there are difficulties for the action of residual dimethyl dicarbonate on mould spores as the spores begin to germinate 1 to 2 h after becoming exposed to a liquid food (Richard, 2010). In addition, the initial amount of microorganisms might become a factor that limits the sterilising effect of dimethyl dicarbonate.

in the total plate count as well as the yeast and mould counts although there was no statistical difference between the three treatments. The microbial reduction for the post ultraviolet irradiation (UV–UV–DMDC) on the total plate count and yeast and mould counts were 2.61 log CFU/ml and 4.87 log CFU/ml, respectively. This is a similar result to Quicho (2005) who reported that post ultraviolet irradiation on apple cider had a significantly higher reduction in microorganisms. The study result proposed that dimethyl dicarbonate can enhance the microorganism reduction with a greater efficiency on yeast and mould counts. The microbial killing effect of dimethyl dicarbonate is due to the inhibition of enzymes where protein modification takes place through a reaction of the nucleotide groups such as amines or thiols that can react with dicarbonate. The changes of active site blocking and inhibition of enzymes can cause the cell to die especially in yeast cells (Ough, 1993). From this study, although it showed that repetitive ultraviolet irradiation (UV–UV) as well as the addition of dimethyl dicarbonate cannot satisfy the FDA requirement for microbial reduction, the study did show a better microbial reduction compared to previous studies especially with the addition of dimethyl dicarbonate.

4. Conclusions In general, based on the physico-chemical analyses (turbidity, colour (L*, Hue angle and chroma), total soluble solids, pH, total phenolic content, titratable acidity and vitamin C), either the ultraviolet irradiated (UV–UV) or the combined dimethyl dicarbonate (UV–DMDC–UV) juice maintained similar characteristics as the untreated juice. The loss of vitamin C that was observed in the treated juice (UV–UV and UV–DMDC–UV) was considered minimal (b30%) as compared to the pasteurization of normal juice (N38%). Hence, ultraviolet irradiated juice definitely provides a better alternative for juice treatment in order to preserve its attributes (colour, flavour and odour). Further, dimethyl dicarbonate did not cause any negative effect (apart from the total phenolic content) in the juice. From the aspect of microbial reduction, ultraviolet irradiation alone showed a low microbial reduction (b2 log reduction) which might be due to an insufficient ultraviolet exposure as well as the blockage of ultraviolet irradiation by the existence of particles and fibres in the juice. On the other hand, dimethyl dicarbonate added after the ultraviolet irradiation (UV–UV–DMDC) showed a much better microbial reduction especially for yeast and mould. However, the microbial reduction was still not sufficient as the reductions of microbial activity were 2.61 and 4.87 for the total plate count and yeast and mould counts respectively. Hence, to increase the efficiency of the ultraviolet irradiation treatment, it is suggested to use a higher UV dosage.

3.3. Repetitive study for microbiological activities The microbiological study results shown did not meet the requirement of the FDA which is a 5 log reduction. Therefore, a repetitive study on the microbiological activity was conducted by changing the sequence of dimethyl dicarbonate addition. Three sequences of dimethyl dicarbonate addition into two cycles of ultraviolet irradiation juice were conducted: (i) Pre addition of dimethyl dicarbonate (DMDC– UV–UV), (ii) Intermediate addition of dimethyl (UV–DMDC–UV) and (iii) Post addition of dimethyl dicarbonate (UV–UV–DMDC). Fig. 2 shows the results for the addition of dimethyl dicarbonate prior to ultraviolet irradiation (DMDC–UV–UV) (4.26 ± 0.43 log CFU/ml for total plate count; 2.41 ± 1.63 log CFU/ml for yeast and mould counts), intermediate addition between two cycles of ultraviolet irradiation (UV–DMDC–UV) (4.23 ± 0.34 log CFU/ml for total plate count; 2.37 ± 1.62 log CFU/ml for yeast and mould counts) and an addition post-ultraviolet irradiation (UV–UV–DMDC) (3.75 ± 0.44 log CFU/ml for total plate count; 1.35 ± 1.67 log CFU/ml for yeast and mould counts). Post ultraviolet irradiation (UV–UV–DMDC) presented the best reduction

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Please cite this article as: Shamsudin, R., et al., Effect of repetitive ultraviolet irradiation on the physico-chemical properties and microbial stability of pineapple juice, Innovative Food Science and Emerging Technologies (2014), http://dx.doi.org/10.1016/j.ifset.2014.02.005