The use of fuzzy logic to determine the concentration of betel leaf essential oil and its potency as a juice preservative

The use of fuzzy logic to determine the concentration of betel leaf essential oil and its potency as a juice preservative

Food Chemistry 240 (2018) 1113–1120 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem The...

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Food Chemistry 240 (2018) 1113–1120

Contents lists available at ScienceDirect

Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

The use of fuzzy logic to determine the concentration of betel leaf essential oil and its potency as a juice preservative

MARK

Suradeep Basak Indian Institute of Technology Kharagpur, Agricultural and Food Engineering Department, Kharagpur 721302, West Bengal, India

A R T I C L E I N F O

A B S T R A C T

Keywords: Fuzzy logic Sensory evaluation Essential oil Apple juice Shelf life

The present study was attempted to determine organoleptically acceptable concentration of betel leaf essential oil (BLEO) in raw apple juice using fuzzy logic approach, and to evaluate the efficacy of the acceptable concentration in the juice under refrigerated storage. The presence of BLEO components in treated juice was confirmed by FTIR spectroscopy. Based on similarity values, the acceptable concentration in the juice was found to be 0.19 µl/ml of BLEO. Total antioxidant capacity of untreated juice was found to be 16% less than treated juice at the end of storage. The treated juice exceeded total aerobic plate count of 2 log10 (cfu/ml) on 15th day of storage. Based on safe limits of microbial load, the shelf life of treated juice was extended by 6 days as compared to untreated juice under refrigerated storage. BLEO contributes to green consumerism and its application as food preservative will add value to the product.

1. Introduction Raw apple juice (RAJ) is unfiltered, cloudy, high soluble solids, low acid (pH < 4.5), unpasteurized non-alcoholic beverage that has shorter shelf life of 2–3 weeks (Dock, 1999). Despite several foodborne illness associated with unpasteurized apple juice, it has always been preferred over thermally pasteurized apple juice by the consumers (Parish, 1997). Thermal processing is one of the most common practices, but it has adverse effects on sensory, nutritional and functional properties of food (Mañas & Pagán, 2005; Raso & Barbosa-Cánovas, 2003). The demand of minimally processed food has escalated with emergence of green consumerism, which in turn has promoted the use of naturally occurring antimicrobials (Juneja, Dwivedi, & Yan, 2012). Essential oils are secondary metabolites of plants which can be extracted from herbs and spices. Antimicrobial properties of essential oils can be attributed to the presence of bioactive phenolic compounds (Tepe, Daferera, Sokmen, Sokmen, & Polissiou, 2005). Basak and Guha (2015) have identified chavibetol, estragole, β-cubebene, chavicol, and caryophyllene as the major chemical compounds of betel leaf essential oil (BLEO) of the cultivar Tamluk Mitha. And estragole of the five BLEO components has restricted use in the European Union, whereas all five chemical compounds of BLEO have GRAS status in US FDA. Alfonzo et al. (2016) have investigated the biopreservative effect of lemon essential oil micro-emulsion while improving safety and sensory attributes of salted sardines. Use of essential oil is also limited due to its ability to make undesirable alteration in sensory attributes of food

E-mail address: [email protected] http://dx.doi.org/10.1016/j.foodchem.2017.08.047 Received 25 May 2017; Received in revised form 11 August 2017; Accepted 15 August 2017 Available online 16 August 2017 0308-8146/ © 2017 Elsevier Ltd. All rights reserved.

(Hyldgaard, Mygind, & Meyer, 2012). Therefore, detailed sensory evaluation of any product treated with essential oil forms the basis of its marketability. Sensory evaluation is a scientific method to decide acceptance and rejection of food by the evaluator upon consumption (Kemp, Hollowood, & Hort, 2009). Fuzzy logic is an important tool which can draw an important conclusion regarding acceptance, rejection, ranking, strong and weak attributes of food using vague and imprecise data in linguistic form filled in by single or multiple experts (Zimmermann, 1991). Fuzzy sets provide the mathematical method that represents the fuzziness of human expressions (Lazim & Suriani, 2009) using linguistic variables instead of numerical values (Zadeh & Kacprzyk, 1999). Comparison and choice is fundamental to consumers (Imm, Lee, & Lee, 2011) so is the market risk of any food products. Alongside organoleptic acceptability, the quality of any food products over storage period is determined by their physical and chemical characteristics, microbiological and toxicological safety, and sensory attributes, packaging and labelling (Molnár, 1995). From industrial point of view, consumer acceptability of BLEO treated unpasteurized apple juice and its potency in shelf life extension of the selected food product should be studied. In view of the above research gap, this study was taken up to conduct sensory analysis of raw apple juice treated with betel leaf essential oil using fuzzy logic followed by evaluation of antimicrobial efficacy of organoleptically acceptable concentration of BLEO in the unpasteurized juice during refrigerated storage at 4 °C.

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2. Materials and methods

attributes of raw apple juice in general was calculated using sum of sensory scores, triplets associated with sensory scales, and number of panellists as given below:

2.1. Essential oil

Quality attributes of RAJ in general=[{no. of panellist × (0 0 25)} + {no. of panellist × (25 25 25)} + {no. of panellist × (50 25 25)}

BLEO was extracted from fresh betel leaves of cv. Tamluk Mitha (Guha, 2007), and the essential oil based microemulsion (BLEO-ME) using Tween 20 (SRL Chem., India) as emulsifier and water as the continuous phase was formulated according to Basak and Guha (2017a).

+ {no. of panellist × (75 25 25)} + {no. of panellist × (100 25 0)}] /(Total no of panellist)

(2)

Similarly, triplets for all quality attributes, viz. colour (QC), flavour (QF), taste (QT) and mouthfeel (QM) of RAJ in general were calculated according to Eq. (2).

2.2. Preparation of raw apple juice Apples (cv. Red delicious) at commercial maturity were purchased and surface sterilization of the apples was performed according to Buchanan, Edelson, Miller, and Sapers (1999). Briefly, apples were dipped in 2 μl/ml sodium hypochlorite for 1 min, followed by rinsing them in autoclaved distilled water for another 1 min. Each apple was cut and made into pulpy juice using mixer grinder followed by filtration using two layers of muslin cloth to obtain raw apple juice. Based on the previous study by Basak and Guha (2015), 0.14, 0.19, 0.28, 0.37 and 0.56 μl/ml of BLEO in the juice were selected for sensory evaluation that corresponds to RAJ-S2, RAJ-S3, RAJ-S4, RAJ-S5 and RAJ-S6, respectively. RAJ-S1 was served as untreated sample throughout the evaluation process. A panel of 20 participants was selected and trained to perform the sensory evaluation.

2.3.4. Triplet for relative weightage of quality attributes Relative weightage of quality attributes were calculated so as to determine the triplets for overall sensory scores of the juice using Eq. (3):

Qsum = sum of first digit of triplets of QC ,QF ,QT and QM

(3)

Triplet for relative weightage of colour (QCrel) attribute was calculated using Eq. (4):

QCrel =

QC Qsum

(4)

Accordingly, relative weightage for all four quality attributes of raw apple juice was calculated.

2.3. Fuzzy logic analysis 2.3.5. Triplets for overall sensory scores of the juice samples The values of triplets for colour, flavour, taste and mouthfeel of all samples are tabulated in Table S1 (supplementary material). Similarly, the triplets for sensory score of quality attributes of RAJ in general was calculated based on the preferences of panel members (Table S2, supplementary material). Overall sensory scores for every sample were calculated according to Eq. (5):

2.3.1. Sensory evaluation All the selected panellists were trained to familiarize the quality attributes of raw apple juice, sensory score sheets and method of scoring the samples. Quality attributes of sensory analysis were colour, flavour, taste and mouthfeel. The fuzzy scale factors were “Not satisfactory”, “Fair”, “Medium”, “Good” and “Excellent”. According to the explanation provided by Das (2005), ranking of raw apple juice samples were done using triangular fuzzy membership distribution function. Sensory scores of the juice samples obtained using fuzzy scores given by the panellists were converted into triplets and similarity analysis were performed in order to rank the samples. A program was coded in Matlab® 2015a (The Mathworks™; McGarrity, 2008) to perform calculations involved in aforementioned steps. As shown in Fig. S1 (supplementary materials), the distribution pattern of 5-point sensory scales comprises “Not satisfactory/Not at all important, (0, 0, 25)”, “Fair/Somewhat important, (25, 25, 25)”, “Medium/Important, (50, 25, 25)”, “Good/Highly important, (75, 25, 25)” and “Excellent/Extremely important, (100, 25, 0)”. The first number in the triplets denote coordinate of the abscissa where the value of the membership function is 1, whereas the second and third number represents distance to left and right of the first number where the membership function is zero, respectively (Chakraborty, Das, & Das, 2011).

SO1 = (S1 C× QCrel) + (S1 F× QFrel) + (S1 T× QTrel) + (S1 M× QMrel ) (5) where, SO1 is the overall sensory score for RAJ-S1; S1C, S1F, S1T and S1M represent the triplets corresponding to the colour, flavour, taste and mouthfeel of RAJ-S1; QCrel, QFrel, QTrel and QMrel represents the triplets corresponding to the relative weightage of quality attributes of RAJ in general. Multiplication of triplet (a, b, c) with (d, e, f) was performed using the rule given in Eq. (6):

(a,b,c ) × (d,e,f ) = [(a × d ),(a × e + d × b),(a × f + d × c )]

Using the above triplet multiplication rule, overall sensory score for all RAJ samples were calculated. 2.3.6. Standard fuzzy scale and ranking of the samples Standard fuzzy scale following triangular distribution pattern of 6point sensory scale is shown in Fig. S2 (supplementary material). The linguistic expression of the standard fuzzy scale and values of membership functions for F1–F6 are mentioned in Table S3 (supplementary material).

2.3.2. Triplets for sensory score of samples Quality attributes of the samples in form of triplets were calculated using sum of sensory scores, triplets associated with sensory scale and number of panellists as given below:

2.3.7. Values of overall membership function of sensory scores on standard fuzzy scales Membership function of overall sensory scores of raw apple juice samples was calculated on standard fuzzy scale. According to Fig. S3 (supplementary material), when the value of abscissa as a, the value of membership function become 1, and when (a − b) < abscissa < (a + c), the value of membership function becomes 0. For the given value of x on abscissa, value of membership function Bx can be expressed as,

Quality attribute of RAJ samples=[{no. of panellist × (0 0 25)}+ {no. of panellist × (25 25 25)} + {no. of panellist × (50 25 25)}+ {no. of panellist × (75 25 25)} + {no. of panellist × (100 25 0)}] /(Total no of panellist)

(6)

(1)

Accordingly, values of triplets for colour, flavour, taste and mouthfeel of all samples were obtained using Eq. (1). 2.3.3. Triplets for panellists’ preference to importance of quality attributes Preference of individual panellist to the importance of quality

Bx = 1114

x −(a−b) for (a−b) < x < a b

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Bx =

(a + c )−x for a < x < (a + c ) c

BI =

(7)

[100(x −0.31)] 0.172

The value of membership function Bx at x = 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 for every samples were determined.

where x = (a + 1.75L)/(5.645L + a −3.012b ) Since, initial colour of raw apple juice was not monitored while extraction, a normalized browning index (ΔBI) was calculated as the difference between initial BI and the BI at a given time during storage for untreated and treated juice samples.

2.3.8. Similarity values of the juice samples After obtaining the membership function of each juice samples, similarity values were compared with the corresponding values of membership function of standard fuzzy scale. This is followed by calculation of similarity values of samples under F1 (Not satisfactory), F2 (Fair), F3 (Satisfactory), F4 (Good), F5 (Very good), and F6 (Excellent) were made using following equation:

2.5.2. pH and total soluble solids The pH value of the juice was measured using the pH meter (Toshcon Industries Pvt. Ltd., Ajmer, India). Hand held refractometer (Erma, Tokyo, Japan) was used to measure total soluble solids (TSS) of the juice.

Bx = 0 for all other values of x

Sm (F,B) =

F×BT maximum of ( F× F Tand B × BT)

2.5.3. Total antioxidant capacity Total antioxidant capacity (TAOC) of the juice samples were measured according to Arabshahi-Delouee and Urooj (2007). Briefly, 2.5 g of RAJ was mixed with methanol in ratio 1:8 (v/v) and incubated for at least 18 h at 4 °C. After incubation, 100 μl of sample was mixed with 1 ml of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate). The tubes were capped and incubated at 95 °C for 90 min. Samples were cooled at room temperature and absorbance was measured at 695 nm. TAOC of the samples were expressed as equivalents of α-tocopherol (extinction coefficient, ∊ = 4 × 103 M−1 cm−1).

(8) T

where, Sm is the similarity value for the sample; F × B is the product of matrix F with the transpose of matrix B; F × FT is the product of matrix F with the transpose of F; B × BT is the product of matrix B with its transpose. Therefore, for sample 1, Sm (F1, B1), Sm (F2, B1), Sm (F3, B1), Sm (F4, B1), Sm (F5, B1) and Sm (F6, B1) were calculated using the rules of matrix multiplication. 2.4. FTIR characterization

2.5.4. Microbial counts Microbial count in raw apple juice were enumerated by ten-fold dilution of juice samples in phosphate buffer (pH 7.4) and plated out in duplicate on plate count agar (PCA; HiMedia, India) for bacteria and potato dextrose agar (PDA; HiMedia, India) acidified with 10% tartaric acid for yeasts and moulds (Müller, Noack, Greiner, Stahl, & Posten, 2014) growth.

Fourier transform infra-red (FTIR) spectroscopic analysis of pure BLEO, BLEO-ME (50 μl/ml), raw apple juice (untreated and treated) samples were loaded on KBr discs and signals were recorded from wavenumber 400–4000 cm−1 at a resolution of 2 cm−1 by a Thermo Nexus-870 (USA). 2.5. Storage study

3. Results and discussion

As suggested by Sant’Ana et al., 2010, the polyethylene terephthalate (PET) bottles and caps were washed and sanitized in a 0.05% and 0.01% (v/v) of peracetic acid solution for 30 min, respectively followed by rinsing with 70% ethanol and dried under laminar air flow chamber. Raw apple juice was freshly prepared as mentioned earlier in Section 2.2. To every sterile PET bottle, 100 ml of RAJ treated with organoleptically acceptable concentration of BLEO was aseptically transferred. Untreated RAJ samples were used as control sets alongside treated juice samples during entire storage study under refrigerated (4 °C) conditions. PET bottles (untreated and treated) were randomly picked up for microbial enumeration, determination of L, a∗ and b∗ values, pH, total soluble solids (TSS) and total antioxidant capacity (TAOC) at every 3 days interval during storage.

3.1. Overall sensory scores of raw apple juice samples Sensory scores of raw apple juice samples as given by the panellists are shown in Table S1 (supplementary materials) and sum of panellists preferences to relative importance of quality attributes of raw apple juice in general are shown in Table S2 (supplementary materials). Triplets for overall sensory scores of all individual samples were obtained as given below: SO1 = (81.22, 50.59, 30.02) SO2 = (61.33, 42.42, 31.47) SO3 = (61.88, 42.92, 32.78) SO4 = (43.58, 33.72, 32.17) SO5 = (45.29, 33.29, 32.00) SO6 = (37.67, 28.24, 30.62)

2.5.1. Colour The colour parameters of the juice was measured using a portable Chromameter CR-400 (Konica Minolta Sensing, Inc., Osaka, Japan) in the L∗ (lightness), a∗ (red-green) and b∗ (yellow-blue) colour spaces at constant lighting conditions after standardizing the instrument with standard black and white plate. Total colour difference (ΔE∗) was calculated to quantify the overall colour difference of the juice samples with respect to the reference sample. It was calculated using the following equation (Hirschler, 2012):

ΔE =

Values of overall membership function of sensory scores on standard fuzzy scales Using Eq. (7), the value of Bx at x = 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 for individual juice samples was obtained as: B1 = (0, 0, 0, 0.1854, 0.3830, 0.5810, 0.7783, 1.0405, 0.7074, 0.3744) B2 = (0, 0.0255, 0.2613, 0.4970, 0.7328, 0.9686, 0.7246, 0.4068, 0.0890, 0) B3 = (0, 0.0242, 0.2572, 0.4902, 0.72315, 0.9561, 0.7523, 0.4472, 0.1421, 0) B4 = (0.0041, 0.3007, 0.5973, 0.8939, 0.8003, 0.4895, 0.1786, 0, 0, 0)

[(ΔL)2 + (Δa )2 + (Δb )2]

where, ΔL∗ = (L1∗ − L0∗); Δa∗ = (a1∗ − a0∗); and Δb∗ = (b1∗ − b0∗). Subscript ‘0’ and ‘1’ depicts the colour value of reference sample (zero day value of the sample) and the sample being analysed, respectively. The browning index (BI) represents the purity of brown colour and it was calculated according to Palou, López-Malo, Barbosa-Cánovas, Welti-Chanes, and Swanson (1999): 1115

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Table 1 Similarity values for raw apple juice under different scale factors.

Table 2 Similarity values of individual quality attribute of raw apple juice samples.

Sensory Scale

RAJ-S1

RAJ-S2

RAJ-S3

RAJ-S4

RAJ-S5

RAJ-S6

Sensory Scale

Not satisfactory, F1 Fair, F2 Satisfactory, F3 Good, F4 Very good, F5 Excellent, F6

0 0.0341 0.3160 0.7502 0.8317 0.2680

0.0051 0.2141 0.7379 0.9052 0.3433 0.0179

0.0048 0.2080 0.7192 0.9065 0.3816 0.0281

0.0716 0.5388 0.8951 0.4273 0.0357 0

0.0565 0.4807 0.8937 0.4777 0.0455 0

0.1059 0.6297 0.8084 0.2278 0 0

Bold values represents the maximum similarity value.

B5 = (0, 0.2403, 0.5407, 0.8412, 0.8527, 0.5402, 0.2277, 0, 0, 0) B6 = (0.0199, 0.3741, 0.7283, 0.9240, 0.5974, 0.2710, 0, 0, 0, 0) 3.2. Similarity values of the juice samples and their ranking Similarity (Sm) values were calculated using the values of membership function of standard fuzzy scale and overall membership function values of sensory scores according to Eq. (8). As shown in Table 1, the Sm values of RAJ-S1 under ‘not satisfactory’ category was 0, while the same sample had Sm value of 0.0341, 0.3160, 0.7502, 0.8317 and 0.2680 under ‘Fair, Satisfactory, Good, Very good and Excellent’ category, respectively. Hence, overall quality of RAJ-S1 can be considered as ‘very good’ because the highest Sm value belongs to this category. Interestingly, RAJ-S3 was found to have slightly higher Sm value of 0.9065 under ‘good’ category as compared to Sm value of 0.9052 for RAJ-S2 under same category. On the other hand, RAJ-S4, RAJ-S5 and RAJ-S6 were placed under ‘satisfactory’ category with Sm value of 0.8951, 0.8937 and 0.8084, respectively. Based on Sm values, the order of ranking of raw apple juice samples was found to be:

RAJ−S1 > RAJ−S3 > RAJ−S2 > RAJ−S4 > RAJ−S5 > RAJ−S6 As far as general perception of raw apple juice is concerned it was found that Sm values of flavour, mouthfeel and taste are highest under ‘highly important’ category, whereas colour has highest Sm value under ‘important’ category. The order of ranking of quality attributes of raw apple juice in general as mentioned in Table S4 (supplementary materials) was:

Flavour> Mouthfeel > Taste>Colour A comparable Sm values for flavour, taste and mouthfeel of RAJ-S1 under ‘very good’ category was observed, whereas colour had highest Sm value under ‘good’ category. This satisfied the ranking of quality attributes of raw apple juice in general. Now, for BLEO treated raw apple juice, RAJ-S2 and RAJ-S3 was found to have all four quality attributes under ‘good’ category, whereas quality attributes of remaining samples were ranked accordingly to Sm values as given in Table 2. RAJ-S3 was found to have a minor increment in Sm value (0.7767) of taste as compared to RAJ-S2 (Sm value = 0.7743). On the contrary, Sm values of remaining quality attributes viz. flavour colour and mouthfeel were found to be comparatively higher in RAJ-S2. It can also be observed that Sm value of mouthfeel, flavour and colour for RAJ-S3 under ‘very good’ and ‘excellent’ category are on the higher side as compared to RAJ-S2 for the same. It can be suggested that increase in concentration of BLEO by 0.05 μl/ml increased the acceptability due to tinge of BLEO essence in RAJ-S3 over RAJ-S2. It is evident that highest concentration of BLEO has imparted a ‘fair’ taste in RAJ-S6 as opposed to ‘satisfactory’ taste for RAJ-S4 and RAJ-S5. The bitter and pungent taste at concentration greater than or equal to 0.28 µl/ml of BLEO has resulted in unacceptability of the juice. Therefore, raw apple juice treated with any concentration less than or equal to 0.19 µl/ml of BLEO will be organoleptically acceptable. Although overall quality of RAJ-S1 (untreated) sample was unanimously preferred by the panellists, however RAJ-S2 and RAJ-S3 fared well among treated juice and was placed under ‘good’ category. RAJ-S3

Colour

Flavour

Taste

Mouthfeel

Sm value of the quality attribute of RAJ-S1 Not satisfactory, F1 0.0128 Fair, F2 0.1912 Satisfactory, F3 0.5526 Good, F4 0.7519 Very good, F5 0.4406 Excellent, F6 0.0738

0 0.0095 0.1608 0.4818 0.7932 0.4532

0 0.0130 0.1783 0.5034 0.7938 0.4346

0 0.0159 0.1937 0.5236 0.7949 0.4163

Sm value of the quality attribute of RAJ-S2 Not satisfactory, F1 0.0169 Fair, F2 0.2237 Satisfactory, F3 0.6114 Good, F4 0.7532 Very good, F5 0.3924 Excellent, F6 0.0483

0.0174 0.2313 0.6302 0.7593 0.3722 0.0385

0 0.1341 0.5045 0.7743 0.5234 0.1048

0 0.0773 0.3778 0.7264 0.6477 0.1723

Sm value of the quality attribute of RAJ-S3 Not satisfactory, F1 0.0141 Fair, F2 0.2044 Satisfactory, F3 0.5776 Good, F4 0.7433 Very good, F5 0.4329 Excellent, F6 0.0825

0.0135 0.2066 0.5932 0.7575 0.4138 0.0605

0.0031 0.1509 0.5267 0.7767 0.4857 0.0828

0 0.0771 0.3664 0.7033 0.6638 0.1995

Sm value of the quality attribute of RAJ-S4 Not satisfactory, F1 0.0215 Fair, F2 0.2491 Satisfactory, F3 0.6405 Good, F4 0.7364 Very good, F5 0.3605 Excellent, F6 0.0396

0.0848 0.4742 0.8149 0.5467 0.1104 0

0.1389 0.6902 0.8356 0.3243 0.0215 0

0.0383 0.3910 0.8325 0.6360 0.1381 0

Sm value of the quality attribute of RAJ-S5 Not satisfactory, F1 0.0096 Fair, F2 0.1761 Satisfactory, F3 0.5361 Good, F4 0.7416 Very good, F5 0.4647 Excellent, F6 0.1006

0.0520 0.4636 0.8432 0.5272 0.0911 0

0.0593 0.5298 0.8787 0.4606 0.0483 0

0.0480 0.4455 0.8574 0.5641 0.0912 0

Sm value of the quality attribute of RAJ-S6 Not satisfactory, F1 0.0242 Fair, F2 0.2661 Satisfactory, F3 0.6652 Good, F4 0.7325 Very good, F5 0.3293 Excellent, F6 0.0316

0.1322 0.7309 0.8124 0.2370 0.0015 0

0.1802 0.8001 0.7923 0.2063 0 0

0.0552 0.5071 0.8829 0.4631 0.0452 0

Bold values represents the maximum similarity value.

has additional benefits of being green and clean food product despite lower organoleptic acceptability due to presence of bioactive chemical components in form of BLEO. 3.3. FTIR characterization Fig. 1 depicts FTIR spectra of BLEO, BLEO based microemulsion, treated and untreated raw apple juice. In general, BLEO exhibit characteristic peaks at 3419 (hydroxyl group, H bonded eOH stretch), 3076 (vinylidene eCH stretch), 2954 (methyl eCH asymmetric stretch), 2929 and 2864 (methylene eCH asymmetric and symmetric stretch, respectively), 1639 cm−1 (alkenyl C]C stretch or conjugated ketone group), 1614, 1595 and 1510 cm−1 represents C]CeC aromatic ring stretch, 1365 (phenol or tertiary alcohol eOH bend), 1267 and 1240 cm−1 (aromatic ethers, aryl–O–stretch), 1174, 1149, 1128, 1032, 993, 966 cm−1 represents aromatic CeH in plane bend and 823, 762, 711 cm−1 represents aromatic CeH out of plane bend, and finally 637 and 591 cm−1 could be OeH out of plane bend (Fig. 1(a)). The obtained functional groups and bond vibration are in agreement with functional groups of chemical compounds of BLEO reported earlier by Basak and Guha (2015). For BLEO based microemulsion (Fig. 1(b)), 1116

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Fig. 1. FTIR spectra of (a) BLEO, (b) microemulsified BLEO (50 μl/ ml), (c) BLEO treated raw apple juice (0.19 μl/ml), and (d) untreated raw apple juice.

peak of H bonded eOH stretch shifted to 3419–3417 cm−1 and peak at 3076 and 2954 cm−1 of vinylidene eCH stretch and methyl eCH asymmetric stretch was found to be missing in BLEO-ME. Also, peak at 2929 and 2864 for methylene eCH asymmetric and symmetric stretch were shifted to 2925 and 2880 cm−1, respectively. An additional peak at 2104 cm−1 was observed in BLEO-ME that could possibly represent terminal alkyne groups present. FTIR spectra of treated and untreated raw apple juice (Fig. 1(c) and (d), respectively) shared every characteristic peaks except an additional peak at 1344 cm−1 that suggested the presence of primary or secondary or phenol or tertiary alcohol (OeH in-plane bend or eOH bend) in juice treated with BLEO-ME. Presence of the functional group in treated raw apple juice could be due to reduced ingredient interaction of apple juice and essential oil components of BLEO-ME that probably helped it retain its bioactivity mostly unaltered. Previous report by Basak and Guha (2017b) suggested no significant alteration in antifungal potential of microemulsified BLEO on growth of P. expansum in raw apple juice with respect to in vitro growth medium. Ma, Davidson, and Zhong (2016) have also reported to have similar antifungal potential of free and microemulsified cinnamon bark essential oil against Salmonella enterica and Escherichia coli O157:H7.

3.4. Storage study 3.4.1. Colour Total colour difference (ΔE∗) of untreated as well as treated juice samples significantly (P < 0.0001) increased from 0.42 ± 0.06 and 0.28 ± 0.02 to 1.86 ± 0.09 and 1.35 ± 0.08 during 3 and 15 days after storage, respectively. Similarly, significant (P < 0.0001) increase in ΔBI values from 0.67 ± 0.02 and 0.32 ± 0.06 to 3.2 ± 0.11 and 2.1 ± 0.11 was observed during 3 and 15 days after storage of untreated and treated juice samples, respectively. The increase in browning can be due to oxidation of naturally occurring phenolic components in apple by polyphenol oxidase that remained active because the juice was not exposed to any thermal treatment prior to storage (Ndiaye, Xu, & Wang, 2009). Perhaps, presence of polyphenols in BLEO might have provided an added benefit to the treated juice to have lower ΔE∗ and ΔBI values as compared to untreated juice, which has only indigenous phenolic compounds of apple (Tomás-Barberán & Espín, 2001). Other reason for increase in ΔBI value can be direct photo-oxidation of phenolic compounds present in both untreated and treated juice samples (Manzocco, Quarta, & Dri, 2009). Variation in ΔE∗ and ΔBI values as given in Table 3, suggested that concentration of BLEO selected in the present study does not have much impact on prevention of browning, but it can slow down the reaction in the treated juice as compared to untreated juice during storage.

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(500 µM) free radicals was found to be 11.29 µg/ml (unpublished data). The value of TAOC suggested high antioxidant potency of BLEO, which was further elaborated by lower IC50 value of the essential oil. It is evident that terpene, terpenoid and phenolic compounds of essential oil of betel leaf alongside natural antioxidants in the juice might have provided an enhanced electron donating capability to convert reactive free radicals into stable non-reactive products in treated raw apple juice during storage.

Table 3 Total colour difference (ΔE*) and normalized browning index (ΔBI) of raw apple juice during storage at 4 °C. DAS+

(ΔE*)#

(ΔBI)¶

Untreated 3 6 9 12 15

Treated

fg

g

0.42 ± 0.06 0.86de ± 0.07 1.33bc ± 0.03 1.52ab ± 0.08 1.86a ± 0.09

0.28 ± 0.02 0.51eg ± 0.11 0.70def ± 0.03 1.02cd ± 0.06 1.35bc ± 0.08

Untreated gh

0.67 ± 0.02 1.25ef ± 0.03 1.87cd ± 0.01 2.39b ± 0.10 3.19a ± 0.11

Treated 0.32h ± 0.06 0.64gh ± 0.11 1.04fg ± 0.01 1.55de ± 0.07 2.10bc ± 0.11

3.4.4. Microbial counts The growth of microbial population in untreated and treated raw apple juice during storage at 4 °C is represented in Fig. 2. The initial total aerobic plate count (TAPC) and yeast and mould (YM) count of raw apple juice was enumerated to be 1.77 ± 0.05 log10 (cfu/ml) and 2.23 ± 0.04 log10 (cfu/ml), respectively. Initial microbial load in freshly extracted apple juice can be supported by a few previous studies. Among those studies, Beech (1958) has reported initial yeast populations of raw apple juice produced in a clean pilot plant and commercial plant varied from 5.48–5.79 log10 (cfu/ml) and 5.83–6.11 log10 (cfu/ml), respectively. Vasavada and Heperkan (2002) have also cited initial yeast levels of 3 log10 (cfu/ml) to 5 log10 (cfu/ml) for juices produced from healthy apples. Similarly, Miller and Keller (2005) have also reported TAPC and YM population of unsorted tree harvested fruit apple cider or juice ranged from 1.90–3.40 log10 (cfu/ml) and 1.99–3.32 log10 (cfu/ml), respectively. They also reported that cider or juice extracted from poorer quality, ground harvested apples have substantially higher TAPC and YM count ranging from 4.19–5.43 log10 (cfu/ml) and 3.84–5.23 log10 (cfu/ml), respectively. In the present study, low initial bacterial load of the juice could be due to pre-treatment of the apples with sodium hypochlorite as suggested by Buchanan et al. (1999). TAPC of untreated juice constantly increased during storage and exceeded the permissible limit of 2 log10 (cfu/ml) count (EFSA, 2005) on day 9, followed by TAPC of treated juice on day 15. On the contrary, initial microbial load of unpasteurized apple juice on PDA plate was dominated by yeast population. However, lowest YM count of untreated and treated juice was observed at day 6 with 1.98 log10 (cfu/ ml) and 1.63 log10 (cfu/ml), respectively. The difference in YM count of untreated and treated can be attributed to the fact that rate of inhibition using sub-lethal concentration (¼ MIC = 0.19 μl/ml) of BLEO was slow due to heavy initial load of inherent microbial population in freshly extracted unpasteurized apple juice. Therefore, low concentration of the essential oil imparted lower toxicity on the inherent microorganisms in the juice samples that eventually led to constant increase in TAPC and YM count at par with untreated juice. According to the basic aspects of hurdle technology (Leistner, 2000), the sub-lethal concentration of BLEO in the present study can be opted for effective preservation of the juice in combination with other preservation factors viz. mild temperature, pH, and water activity. Previously, Tyagi, Gottardi, Malik, and Guerzoni (2013) have suggested the preservation of a mixed fruit juice using combination of Mentha

Values are mean ± S.E. Means represented by the same letter in untreated and treated column of same parameter are not significantly different according to ANOVA and Tukey’s multiple comparison tests. + DAS: Days after storage. # Values are compared with reference sample (zero day value of untreated sample). ¶ Values are based on difference between initial browning index and browning index on the observation.

3.4.2. pH and total soluble solids During refrigerated storage of 15 days, no significant variation was observed in the pH of untreated (P = 0.338) as well as treated (P = 0.614) raw apple juice samples. TSS of untreated juice decreased significantly (P = 0.002). However, the change in TSS of treated juice was not significant (P = 0.012) during storage (Table 4). The reduction in TSS of untreated raw apple juice during refrigerated storage can be correlated to the breakdown of polysaccharides into monosaccharide in presence of spoilage microorganisms and utilization of the available monosaccharide by the same for further growth and proliferation in the untreated juice (Bhardwaj & Pandey, 2011).

3.4.3. Total antioxidant capacity As mentioned in Table 4, TAOC was found to have decreased from 10.12 ± 0.06 to 2.46 ± 0.15 mM α-tocopherol/g of untreated juice sample, whereas TAOC in treated raw apple juice was reduced from 10.92 ± 0.05 to 4.39 ± 0.03 mM α-tocopherol/g treated juice sample at 0 and 15 days after storage, respectively. As suggested by Van der Sluis, Dekker, Skrede, and Jongen (2002), the antioxidant activity imparted by fresh raw apple juice is due to naturally occurring polyphenols, which provides only 10% of antioxidant activity of the fresh apple, if the juice is obtained by pulping and pressing. No significant change in °Brix, pH and concentrations of polyphenols in pasteurized apple juice during storage at 4 °C was also observed by Van Der Sluis, Dekker, and Van Boekel (2005). On the contrary, present study deals with unpasteurized and unfiltered apple juice that had active oxidizing enzymes, which were perhaps responsible for degradation of TAOC of the juice during storage even at refrigerated condition. TAOC of pure BLEO was found to be 362.9 ± 1.24 mM α-tocopherol/g of pure BLEO and the concentration of BLEO required to scavenge 50% of DPPH

Table 4 Comparison of pH, total soluble solids (TSS) and total antioxidant capacity (TAOC) of untreated and treated raw apple juice during refrigerated storage at 4 °C. DAS+

pH

TSS (°Brix)

Untreated 0 3 6 9 12 15

4.36 4.35 4.33 4.33 4.32 4.31

± ± ± ± ± ±

0.01 0.02 0.02 0.02 0.02 0.02

Treated 4.35 4.35 4.34 4.33 4.33 4.32

± ± ± ± ± ±

TAOC (mM α-tocopherol/g sample)

Untreated 0.02 0.02 0.02 0.01 0.01 0.01

a

15.57 15.50a 15.47a 15.47a 14.37b 14.27b

± ± ± ± ± ±

Treated 0.03 0.06 0.03 0.03 0.03 0.03

a

15.57 15.57a 15.57a 15.50a 15.43a 15.37a

± ± ± ± ± ±

Untreated 0.03 0.03 0.03 0.06 0.03 0.03

b

10.12 ± 0.06 6.96d ± 0.13 5.74e ± 0.02 4.86fg ± 0.03 3.14h ± 0.14 2.46i ± 0.15

Treated 10.92a ± 0.05 7.74c ± 0.08 6.47d ± 0.23 5.26ef ± 0.17 4.53g ± 0.04 4.39g ± 0.03

Values are mean ± S.E. Means represented by the same letter in untreated and treated column of same parameter are not significantly different according to ANOVA and Tukey’s multiple comparison tests. + DAS: Days after storage.

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Fig. 2. Microbial count (a) TAPC and (b) YM count of untreated and BLEO treated (0.19 μl/ml) unpasteurized raw apple juice during storage at 4 °C.

Basak, S., & Guha, P. (2017b). Use of predictive model to describe sporicidal and cell viability efficacy of betel leaf (Piper betle L.) essential oil on Aspergillus flavus and Penicillium expansum and its antifungal activity in raw apple juice. LWT - Food Science and Technology, 80, 510–516. Basak, S., & Guha, P. (2017a). Betel leaf (Piper betle L.) essential oil microemulsion: Characterization and antifungal activity on growth, and apparent lag time of Aspergillus flavus in tomato paste. LWT - Food Science and Technology, 75, 616–623. Beech, F. W. (1958). The yeast flora of apple juices and ciders. Journal of Applied Bacteriology, 21(2), 257–266. Bhardwaj, R. L., & Pandey, S. (2011). Juice blends—A way of utilization of under-utilized fruits, vegetables, and spices: A review. Critical Reviews in Food Science and Nutrition, 51(6), 563–570. Buchanan, R. L., Edelson, S. G., Miller, R. L., & Sapers, G. M. (1999). Contamination of intact apples after immersion in an aqueous environment containing Escherichia coli O157:H7. Journal of Food Protection, 62(5), 444–450. Chakraborty, D., Das, S., & Das, H. (2011). Aggregation of sensory data using fuzzy logic for sensory quality evaluation of food. Journal of Food Science and Technology, 50, 1–9. Das, H. (2005). Sensory evaluation using fuzzy logic. Food processing operations analysis (pp. 384–402). New Delhi: Asian Books Private Limited. Dock, L. L. (1999). Development of thermal and non-thermal preservation methods for producing microbilogically safe apple cider. West Lafayette IN, USA: Purdue University. EFSA (2005). Commission Regulation (EC) No 2073/2005 of 15th November 2005 on microbiological criteria for foodstuffs. Official Journal of the European Union, L338, 1–26. Guha, P. (2007). Extraction of essential oil: An appropriate rural technology for minimizing wastage of surplus betel leaves. Agricultural Mechanization in Asia, Africa and Latin America, 38(4), 47–50. Hirschler, R. (2012). Whiteness, yellowness and browning in food colorimetry. In J. L. Caivano, & M. del P. Buera (Eds.), Color in food: Technological and psychophysical aspects (pp. 93–104). Boca Raton, USA: CRC Press. Hyldgaard, M., Mygind, T., & Meyer, R. L. (2012). Essential oils in food preservation: Mode of action, synergies, and interactions with food matrix components. Frontiers in Microbiology, 3, 1–24. Imm, B. Y., Lee, J. H., & Lee, S. H. (2011). Sensory quality index (SQI) for commercial food products. Food Quality and Preference, 22(8), 748–752. Juneja, V. K., Dwivedi, H. P., & Yan, X. (2012). Novel natural food antimicrobials. Annual Reviews in Food Science and Technology, 3, 381–403. Kemp, S., Hollowood, T., & Hort, J. (2009). Sensory evaluation: A practical handbook (1st ed.). Chichester, United Kingdom: Wiley-Blackwell. Lazim, M. A., & Suriani, M. (2009). Sensory evaluation of the selected coffee products using fuzzy approach. World Academy of Science, Engineering and Technology, 3(2), 717–720. Leistner, L. (2000). Basic aspects of food preservation by hurdle technology. International Journal of Food Microbiology, 55, 181–186. Ma, Q., Davidson, P. M., & Zhong, Q. (2016). Antimicrobial properties of microemulsions formulated with essential oils, soybean oil, and Tween 80. International Journal of Food Microbiology, 226, 20–25. Mañas, P., & Pagán, R. (2005). Microbial inactivation by new technologies of food preservation. Journal of Applied Microbiology, 98(6), 1387–1399. Manzocco, L., Quarta, B., & Dri, A. (2009). Polyphenoloxidase inactivation by light exposure in model systems and apple derivatives. Innovative Food Science and Emerging Technologies, 10(4), 506–511. McGarrity, S. (2008). Introduction to object-oriented programming in MATLAB®. Retrieved October 27, 2016, from < www.mathworks.com > . Miller, A. J., & Keller, S. E. (2005). Microbiological safety of fresh citrus and apple juices. In J. R. Gorny, A. E. Yousef, & G. M. Sapers (Eds.), Microbiology of fruits and vegetables (pp. 211–230). Boca Raton, Florida: CRC Press. Molnár, P. J. (1995). A model for overall description of food quality. Food Quality and Preference, 6(3), 185–190. Müller, A., Noack, L., Greiner, R., Stahl, M. R., & Posten, C. (2014). Effect of UV-C and UV-B treatment on polyphenol oxidase activity and shelf life of apple and grape juices. Innovative Food Science & Emerging Technologies, 26, 498–504. Ndiaye, C., Xu, S. Y., & Wang, Z. (2009). Steam blanching effect on polyphenoloxidase, peroxidase and colour of mango (Mangifera indica L.) slices. Food Chemistry, 113(1), 92–95. Palou, E., López-Malo, A., Barbosa-Cánovas, G. V., Welti-Chanes, J., & Swanson, B. G. (1999). Polyphenoloxidase Activity and Color of Blanched and High Hydrostatic

piperita essential oil and mild thermal treatment had minimum alteration in organoleptic properties viz. colour and flavour. On the other hand, present study showed that colour and flavour are in same category for all organoleptically rejected samples (RAJ-S4, RAJ-S5 and RAJS6), but the taste of RAJ-S6 changed from remaining two rejected samples. This study justified the need of detailed sensory analysis of essential oil treated food products followed by its potential as food preservative during storage along with proper threshold for organoleptically acceptable concentration. 4. Conclusion The present study investigated the impact of BLEO on sensory attributes of raw apple juice, and also its efficacy on the storability of the juice. Fuzzy logic approach using similarity analysis provided an insight into variation in consumer acceptability with respect to colour, flavour, taste and mouthfeel of raw apple juice under the influence of essential oil of betel leaf. Based on similarity values, the juice treated with 0.19 µl/ml of BLEO was found to have highest organoleptic acceptability among all other BLEO treatments. The organoleptically acceptable sub-lethal concentration of BLEO was effective to extend the shelf life of raw apple juice by 6 days as compared to the untreated raw apple juice samples under refrigerated conditions. Such extension of shelf life was determined on the basis of safe limit of microbial load of the juice. This study suggested the potency of BLEO as raw apple juice preservative. As the sensory barrier restricted luxurious application of BLEO in the juice, the future study on use of BLEO at sub-lethal concentration along with other hurdle techniques is necessary to achieve longer shelf life so as to have economic feasibility at industrial scale. Acknowledgement The author is grateful to Indian Institute of Technology Kharagpur for financial assistance, infrastructure and facilities to conduct the research. The author is also thankful to Prof. P. Guha, Agricultural and Food Engineering Department, IIT Kharagpur for his supervision and support. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2017.08.047. References Alfonzo, A., Martorana, A., Guarrasi, V., Barbera, M., Gaglio, R., Santulli, A., et al. (2016). Effect of the lemon essential oils on the safety and sensory quality of salted sardines (Sardina pilchardus Walbaum 1792). Food Control, 73, 1265–1274. Arabshahi-Delouee, S., & Urooj, A. (2007). Antioxidant properties of various solvent extracts of mulberry (Morus indica L.) leaves. Food Chemistry, 102(4), 1233–1240. Basak, S., & Guha, P. (2015). Modelling the effect of essential oil of betel leaf (Piper betle L.) on germination, growth, and apparent lag time of Penicillium expansum on semisynthetic media. International Journal of Food Microbiology, 215, 171–178.

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