Journal of Food Processing and Preservation ISSN 1745-4549
ESSENTIAL OIL AND OLEORESINS OF BLACK PEPPER AS NATURAL FOOD PRESERVATIVES FOR ORANGE JUICE* jfpp_756
146..152
I.P.S. KAPOOR, BANDANA SINGH, SUNITA SINGH and GURDIP SINGH1 Chemistry Department, DDU Gorakhpur University, Gorakhpur 273009, India
1
Corresponding author. TEL: 91-551-2200745 (R) 2202856 (O); FAX: 91-551-2340459; EMAIL:
[email protected] *Part 70 Accepted for Publication April 15, 2012 doi:10.1111/j.1745-4549.2012.00756.x
ABSTRACT Essential oil and oleoresins (ethanol and ethyl acetate) of Piper nigrum L. collected by hydrodistillation and Soxhlet apparatus, respectively, were tested as natural food preservative in orange juice (Citrus reticulata). The physicochemical changes such as pH, ascorbic acid, total and reducing sugar, titratable acidity, nonenzymatic browning, percent weight loss (PWL), and microbial count were evaluated at fix time intervals of 7 days for 28 days. It has been observed that pepper oil and its ethyl acetate oleoresin have better preservatives effect in comparison with other samples.
PRACTICAL APPLICATIONS Essential oils and oleoresins derived from spices have a wide application in food flavoring and preservation as well as in fragrance industry. Moreover, they also possess antioxidant and antimicrobial activities. Essential oil and oleoresins extracted from black pepper are used in the preservation of orange juice, which is better and safer than synthetic preservatives. Thus, the reported work would have practical application in the food industry.
INTRODUCTION Attention of the scientific community worldwide is shifting toward spices and herbs to harness their natural food preservatives. Citrus reticulata is a native of China and is widely cultivated in all subtropical regions. Citrus is one of the most important fruit crops of the world because of its high nutritional value, huge fruit production and different processed products as described by Chaturvedi et al. (2001). Nagpur santara (Mandarins, C. reticulata Blanco family Rutaceae) is the important fruit crop of India next to mango and banana (Bhardwaz and Sen 2003). Orange juice is nutritious and refreshing because of its ascorbic acid content, sweet acidic taste, pleasing color, aromatic and healthful (Cook 1983; Ladaniya and Sonkar 1996). However, even after a few hours of extraction, the juice starts decaying and its color, taste, and aroma gets destroyed. This is due to heavy microbial loading and enzymatic activity, which spoils the sensory and nutritive qualities of juice, making it unfit for human consumption. The major causes of deterioration must be due to the growth of microbes, chemical reactions, structural changes and 146
storage conditions (Desrosier 1970). Therefore, some processing techniques, soon after the extraction of juices, are needed to preserve the freshness of juices. Preservation involves checking of the undesirable changes in fruits and vegetables. Refrigeration, sterilization, pasteurization, and addition of preservatives are some of the popular techniques used to attain microbiological stability and to preserve the fruit juice (Frazier 1966). Nowadays, there is an increasing trend of using packed or canned juices because they can be consumed at desire and are easy to carry. However, before packing, the juices are processed and some chemicals are added in order to preserve their quality. Some commonly used chemical preservatives in fruit are benzoic acid and its derivatives: salicylic acid, formic acid, formaldehyde, SO2, etc. (Ranganna 1986). However, the disputation over the safety of some chemical preservatives has promoted the search for their natural alternatives. As per our knowledge, there is no report on the preservation of orange juice by pepper essential oil and oleoresins (ethanol and ethyl acetate), which have remarkable antioxidant and antimicrobial activity (Singh et al. 2004; Kapoor
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I.P.S. KAPOOR ET AL.
et al. 2009). Hence, an attempt has been made to enhance the shelf life of orange fruit juice with natural additives derived from black pepper.
MATERIALS AND METHODS Extraction of Essential Oil and Oleoresins Piper nigrum L. (black pepper) fruits were purchased from the local market of Gorakhpur, India. Essential oil was extracted by hydrodistillation process using a Clevengertype apparatus in accordance with procedure as mentioned in the European Pharmacopeia (1983). The oleoresins were collected by Soxhlet extraction using solvents, viz. ethanol and ethyl acetate. The obtained essential oil (yield 2.6%) and oleoresins (yield 3.8 and 2.5%) were filled in bottles and stored in a refrigerator in the dark at 4C until use.
Sample Preparation Fresh, mature, uniform and fully ripped orange fruits were washed under running tap water, hand peeled, deseeded and the pulp is blended using electronic juicer under hygienic laboratory condition. Further extracted orange juice was filtered through a clean muslin cloth and divided into five equal batches (50 mL each). Pepper essential oil (10 mL) and each oleoresin dissolved separately in 90 mL of ethanol and were mixed homogeneously to extracted juice (50 mL) in plastic cups. The control sample contains only orange juice and control (I) was prepared by adding 100 mL of ethanol in orange juice.
Storage The control and treated samples (having ethanol, volatile oil and oleoresins) were stored at refrigerated temperature (4 ⫾ 1C) for 28 days in laboratory condition. The effect of storage on shelf life and physicochemical changes such as pH, titratable acidity, ascorbic acid, total and reducing sugar, nonenzymatic browning (NEB), percent weight loss (PWL), and microbial analysis (total microbial count and yeast and mold count) were observed after an interval of 7 days for 28 days. Each analysis was carried out in triplicate on 0, 7th, 14th, 21st and 28th days.
BLACK PEPPER ESSENTIAL OILS AS FOOD PRESERVATIVES
Ascorbic Acid A 10 mL of juice sample was dissolved in 3% HPO3 to make volume 100 mL (Ranganna 1986). The filtrate was titrated with standard dye solution (sodium salt of 2, 6-dichlorophenylindophenol and sodium bicarbonate in distilled water) to get end-point (pink). The ascorbic acid content was calculated using the formula,
mg of ascorbic acid/100 mL of juice =
T × D × V × 100 W1 × W2
where T is the volume of dye solution used, D is the dye factor, V is the volume made up, W1 and W2 are the weight of filtrate and weight of juice sample taken for estimation.
Titratable Acidity Titratable acidity of juice samples was determined as percent citric acid by titrating against NaOH. A 2 g juice sample was dissolved in boiled distilled water and cooled to make the volume 100 mL with distilled water. The solution was filtered and 25 mL of this filtrate was titrated against 0.1 N NaOH using phenolphthalein as an indicator. The titratable acidity was calculated as:
% citric acid =
T × N × E × 100 W × 1, 000
where T is the volume of NaOH, N is the normality of NaOH, E is the equivalent weight of acid and W is the weight of juice sample taken.
Total and Reducing Sugars A 4.0 g of all the samples were weighed in a volumetric flask and dissolved in hot water, to make the volume 100 mL (five sets). To precipitate the protein present in the sample, 2 mL of 45% lead acetate was added in each set followed by the addition of 2 mL of 22% potassium oxalate along the side of the flask in order to check further precipitation. The precipitates were filtered and analyzed to determine the amount of sugar present in the five sets of juice samples. The total and reducing sugars present in the samples were determined by the same methods as reported earlier Kapoor et al. (2009).
pH Measurement The pH values of each orange juice samples were measured using a digital pH-meter (Century CP-931, Century Instruments Pvt. Ltd., Chandigarh, India) fitted with a standard glass electrode. The pH meter was calibrated with a buffer solution of pH 4 and 9.
NEB NEB was monitored using the method of Nagy et al. (1992) on a Hitachi-U-2000 spectrophotometer (Tokyo, Japan) against water at 420 nm by using 1 mL of orange juice samples diluted with 10 mL ethanol.
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PWL The weight loss of each sample was observed after an interval of 7 days for a period of 28 days according to standard procedure as mentioned in AOAC (1994). The weight of each sample was determined by electronic digital balance (Petit balance, Tokyo, Japan). Furthermore, to avoid any inconvenience of disorder during storage, weight loss determination is allotted a specific symbol. The PWL was calculated as:
PWL =
Wt of first interval − Wt of second interval ×100 Wt of first interval
microbial count and the only difference being that in place of dextrose-tryptone agar, a PDA medium was used. All the microbiological studies were done in triplicate and the count data are expressed as colony-forming units per milliliter.
Statistical Analyses All the analyses were done in triplicate to report the mean of result. The results were computed as mean ⫾ standard deviation and subjected to one-way analysis of variance to establish whether the differences in experimental results for different samples were significant or not. The statistical significance was found to be P ⱕ 0.05.
Microbial Analysis Microbiological studies were done by following two methods.
Total Microbial Count The total microbial count was done using (Marth 1978) method. A 5 g of juice sample was taken and diluted with 90 mL of Ringer’s solution (1:10). After homogenization, 1 mL of sample and 10 mL of dextrose-tryptone agar medium was transferred in sterilized Petri plate at 45C and mixed thoroughly. Furthermore, they were incubated at 37C to examine the number of colonies that appeared in all Petri dishes. The observations were recorded as the number of colonies present in 1 mL of juice sample (cfu/ mL).
Yeast and Mold Count Yeasts and molds were counted using the acidified potato dextrose agar (PDA) according to Standard Methods for Examination of Dairy Products (Speck 1976). All the experimental procedures were the same as the total
RESULTS AND DISCUSSION pH It is evident from Fig. 1 that the pH value of all the samples under refrigerated condition significantly increased during storage. Maximum pH value was found in control followed by control (I) and minimum was noted in essential oil containing orange juice. The increase in pH during storage may be due to decrease in acidity and increase in total sugar content (Baruah and Mohan 1985).
Acidity Change in titratable acidity of orange juice upon storage at 4 ⫾ 1C with various treatments for 28 days is shown in Fig. 2. From the plot, it is clear that under storage condition, firstly a decrease in acidity occurs after 14 days and then acidity increases slightly after 21 and 28 days. Similar observations have been recorded for kinnow by Panesar et al. (2000) and for orange juice (Goyle and Ojha 1998). In different samples of pepper oil and its ethyl acetate oleoresin,
5
4.8
Control 4.6 pH
Control 1 Ethanol oleo.
4.4
Eth. acetate oleo. Pepper oil
4.2
4 0 days
7 days
14 days
Storage days
148
21 days
28 days
FIG. 1. EFFECT OF PEPPER ESSENTIAL OIL AND OLEORESIN ON pH OF ORANGE JUICE DURING STORAGE
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Ascorbic acid g/100 mL
25
control
20
Contro 1 Ethanol oleo. Eth. acetate oleo.
15
Pepper oil
10
FIG. 2. EFFECT OF PEPPER ESSENTIAL OIL AND OLEORESIN ON ASCORBIC ACID OF ORANGE JUICE DURING STORAGE
0 days
7 days
14 days
21 days
28 days
Storage period
having an orange juice has significantly less (P ⱕ 0.05) titratable acidity value than the control and other samples. The decline in acidity could be due to conversion of acid into sugar and salts Ruttner et al. (1975)
ascorbic acid by enzyme ascorbicnase (Dash and Das 1967). It is observed that ascorbic acid content is more in fruit juice having pepper oil and ethyl acetate oleoresin.
NEB Ascorbic Acid (Vitamin C) Evaluation of Vitamin C is as an index of the nutrient quality of fruits because as compared with other nutrients, it is much more sensitive to various mode of degradation in food processing and storage as reported by Ozkan et al. (2004). Under storage condition, Vitamin C decline significantly in all treatment (Fig. 3). Our observations are well correlated with that of Kaanane et al. (1988) and Lee and Chen (1998), who describe ascorbic acid degradation in orange juice. According to Garcia et al. (1998), this reduction may be due to high-respiration rate in the juice samples. Furthermore, decrease in ascorbic acid content might be due to oxidation of ascorbic acid to dehydro-
It is a well-known fact that during storage of orange juice browning increases, which causes loss of food value (Tally and Porter 1968). Increase in browning during storage of juice was also reported by (Clegg 1964) and attributed to the degradation of ascorbic acid, thus, leading to formation of brown pigment. From Fig. 4, it is clear that the essential oil containing juice sample have less NEB than the rest of the treated samples.
PWL The maximum weight loss was observed in the control, whereas the lowest was noted in essential oil containing
% Titratable acidity
0.25
Control 0.2
Control 1 Ethanol oleo. Eth. acetate oleo.
0.15
pepper oil 0.1
FIG. 3. EFFECT OF PEPPER ESSENTIAL OIL AND OLEORESIN ON TITRATABLE ACIDITY OF ORANGE JUICE DURING STORAGE
0.05
0 days
7 days
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14 days
21 days
28 days
Storage days
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OD at 440 nm
0.35 Control
0.3
Control 1 Ethanol oleo. 0.25
Eth. acetate oleo. pepper oil
0.2
FIG. 4. EFFECT OF PEPPER ESSENTIAL OIL AND OLEORESIN ON NON-ENZYMATIC BROWNING OF ORANGE JUICE DURING STORAGE
0.15 0 days
7 days
14 days
21 days
28 days
Storage days
25
Control
% weight loss
20
Control1 Ethanol oleo.
15
Eth. acetate oleo. 10
pepper oil
5
0
0 days
7 days
14 days
21 days
FIG. 5. EFFECT OF PEPPER ESSENTIAL OIL AND OLEORESINS ON PERCENT WEIGHT LOSS OF ORANGE JUICE DURING STORAGE
28 days
Storage days
juice sample (Fig. 5). The possible reason may be that essential oil served as a semipermeable membrane around juice surface, which resulted in the reduction of evaporation process as mentioned by Kapoor et al. (2009). Gradual increase in PWL was observed during storage.
sion of polysaccharides (starch) into monosaccharides and water loss. The results are the same as reported by Gosh and Sen (1984) on sweet orange and Kapoor et al. (2008) on pineapple juice. Orange juice containing essential oil and ethyl acetate oleoresin of pepper maintains lower sugar content during storage.
Total and Reducing Sugars The total and reducing sugars have been found to be increased (Table 1). This may be due to some acid converted into sugars during respiration of microorganisms, conver-
Microbiological Examinations The results of microbial analyses, i.e. total microbial count, yeast and mold count are reported (Table 2). In all the TABLE 1. EFFECT OF PEPPER ESSENTIAL OIL AND OLEORESINS ON TOTAL AND REDUCING SUGARS OF “ORANGE” JUICE
Storage days Total sugars*, %
Reducing sugar*, %
Sample
7
14
21
28
7
14
21
28
Control Control (I) Ethanol oleoresin Ethanolacetate oleoresin Pepper oil
7.5 7.3 6.7 6.1 6.2
8.2 7.5 7.4 6.9 6.4
8.9 8.0 7.6 7.4 7.2
9.1 8.7 8.2 7.8 7.6
5.3 5.2 5.0 4.3 4.4
6.6 6.3 6.1 5.8 6.2
6.9 6.5 6.2 6.1 6.8
7.2 6.9 6.8 6.6 6.8
* Data are the means of three runs.
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TABLE 2. EFFECT OF PEPPER ESSENTIAL OIL AND OLEORESINS ON THE MICROBIAL STATUS OF “ORANGE” JUICE
Storage days Total microbial count (cfu/ mL)* Sample
7
14 2
1.7 ¥ 10 1.45 ¥ 102 1.3 ¥ 102 1.2 ¥ 102 1.0 ¥ 102
Control Control (I) Ethanol oleoresin Ethanol acetate oleoresin Pepper oil
21 2
2.9 ¥ 10 2.5 ¥ 102 2.95 ¥ 102 3.4 ¥ 102 3.2 ¥ 102
28 3
6.2 ¥ 10 3.4 ¥ 103 1.08 ¥ 103 1.02 ¥ 103 1.5 ¥ 103
6.5 ¥ 103 6.0 ¥ 103 1.8 ¥ 103 2.4 ¥ 103 2 ¥ 103
21
28
Yeast and mold count (cfu/mL)* 7
14 2
1.87 ¥ 10 1.45 ¥ 102 1.6 ¥ 102 2.5 ¥ 102 1.2 ¥ 102
Control Control (1) Ethanol oleoresin Ethanol acetate oleoresin Pepper oil
2
2.37 ¥ 10 1.85 ¥ 102 1.65 ¥ 102 3.4 ¥ 102 3.25 ¥ 102
3
1.3 ¥ 10 1.14 ¥ 103 1.02 ¥ 103 6.4 ¥ 102 1.5 ¥ 103
1.6 ¥ 103 1.2 ¥ 103 1.1 ¥ 103 0.24 ¥ 102 2.0 ¥ 103
* Data are the means of three runs.
samples, microbial loading had increased with increasing storage time. However, beneficial effects of addition of pepper oil and oleoresins on the microbial growth were observed. These additives were able to delay the growth of microorganisms in juices. The ethyl acetate oleoresin was found to be most effective in controlling microbial growth. Other oleoresins and essential oil were also found to be very effective. The activity of essential oil is due to the presence of higher percentage of monoterpene hydrocarbons, which may be responsible for controlling the microbial growth as explained by Ahmed et al. (1993); whereas, the oleoresins contains a pungent alkaloid piperine, with many phenolic amides (Ilondu and Iloh 2007), which acts synergistically. Piperine is a bioactive compound and has been reported to be the major contributors to the antimicrobial activity of oleoresins. Kapoor et al. (2009) also mentioned the synergistic action of various phenolic compounds, piperine, flavonides and several other constituents, which inhibits the growth of these microbes (present in pepper oil and oleoresin). The antimicrobial activities of pepper essential oil and its acetone extract against several species of fungi were also reported by Singh et al. (2004). Nevertheless, it is difficult to give a definite explanation for all results recorded. Although, major components of essential oil and oleoresins of P. nigrum L. are considered to be responsible for their activities.
CONCLUSIONS The present study revealed that black pepper volatile oil and oleoresins (ethyl acetate) are responsible for controlling the microbial growth, which is becoming a threat to human health. Thus, it can be concluded that the addition of pepper oil and its oleoresins may help to preserve the
orange juice by delaying the growth of spoilage microbes. The shelf life of orange juice has been found to be increased. These results can serve as an important platform for the development of effective natural preservatives.
ACKNOWLEDGMENTS Authors are thankful to the Head, Chemistry Department, DDU Gorakhpur University, Gorakhpur for providing laboratory facilities. Financial assistance by Council of Science & technology, Uttar Pradesh and University Grants Commission for providing Emeritus Fellow to Dr. Gurdip Singh is highly appreciated.
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