Abstract
Addition of methanol pre-extract of dehusked ragi powder (DRP) at the rate of 0.1%, 0.25% and 0.5% of methanol extract, the corresponding increase (over control) in phospholipids content observed was about 2.15, 4.53 and 8.15 mg 100 g−1 and water extractable phenolics content was about 0.52, 1.25 and 2.78 mg 100 g−1 of ghee respectively. The anti-oxygenic indexes calculated from the induction periods of ghee samples stored at 80 ± 2 °C in comparison with control were in order: 1.47(0.1% pre-extract) < 1.89(0.02% butylated hydroxyl anisole) < 1.93(0.25% pre-extract) < 2.75(0.5% pre-extract) suggesting that the phospholipids and the phenolic compounds of DRP transferred to ghee enhance its oxidative stability.
Introduction
Ghee is anhydrous milk fat and occupies a prominent place in the Indian diet. Ghee is manufactured by direct heating of cream or butter churned out of fresh or ripened cream or dahi obtained by fermentation of milk with bacteria native to milk or selected starter cultures (Srinivasan, 1976). Chemically ghee is a complex lipid of mixed glycerides together with a small amount of free fatty acids, phospholipids, sterols and their esters, fat soluble vitamins (A, D, E and K), carotenoids, carbonyl compounds, hydrocarbons, charred casein, moisture and traces of trace elements like copper and iron (Sharma, 1981). Because of low moisture content (0.5%), ghee has better shelf-life than other indigenous dairy products. However, it undergoes deterioration (through hydrolytic and/or oxidative routes) which spoils its appetising flavour, making it unpalatable and toxic (Rangappa & Achaya, 1974; Vashney, 1997).
Addition of synthetic anti-oxidants like butylated hydroxyl anisole (BHA), butylated hydroxyl toluene (BHT), propyl gallate (PG), dodecyl gallate (DOG) and tetra butyl hydroquinone (TBHQ) individually or in combination not exceeding 0.02% have been tried to enhance the shelf-life of ghee (Kuchroo & Narayanan, 1972; Chatterjee, 1977; Pande & Verma, 1989). However only BHA at 0.02% level is permitted under Prevention of Food Adultration Rules (PFA Rules, 1955). The continuous use of such synthetic chemicals may cause health hazards such as teratogenic and carcinogenic effects in laboratory animals and primates (Hathway, 1966; Maeura et al., 1984; Heijden et al., 1986; Van esch, 1986). Constant research endeavours have been going on to screen the edible plant materials (Sethi & Aggarwal, 1956; Patel & Rajorhia, 1979; Guleria et al., 1983; Parmar & Sharma, 1986, 1990; Loliger, 1992), that can be explored for extracting the anti-oxidant principles residing in them naturally.
The natural agricultural products, easy availability, edible nature (Solomon, 1953), nutritive, long keeping quality (Sastri, 1952; Solomon, 1953) and the information about chemical constituents (like polyphenolic compounds, phospholipids) of Ragi (Virupaksha et al., 1975; Ramachandra et al., 1977; Mahadevappa & Raina, 1978; Gopalan et al., 1993; Swaminathan, 1999) have led to explore its use as a possible anti-oxidant to enhance the shelf-life of ghee and to compare its effectiveness with the permitted synthetic anti-oxidant BHA.
Material and methods
Ghee prepared by creamery butter method (cream is separated from the milk, churned in a butter churn and heated to approximately 120 °C to evaporate the moisture. The result is ghee). Ragi grains were collected from local market. The husk was manually removed by rubbing them in between gunny bag. The dehusked ragi was then ground to powder form and it is designated as dehusked ragi powder (DRP). The DRP was analysed for moisture, protein and ash by following standard procedures (ISI, 1981). The lipid content was determined by extracting DRP with petroleum ether (40–60 °C) in a Soxhlet apparatus. The carbohydrate content was calculated by difference. Phospholipids of DRP were extracted by the method of Folch et al. (1957) and estimated by the method Bartlett (1959) as adopted for ghee by Rama Murthy & Narayanan (1966). Resolution and detection of phospholipids on thin layer chromatograms was carried out by the methods of Morrison et al. (1980) and Stahl (1969) respectively. Total phenolics of DRP were estimated by the method of Swain & Hills (1959) and the chromatograms developed an appropriate dip reagent (Barton et al., 1952).
Fifty grams of DRP was soaked overnight with 250 mL of methanol after thorough shaking. Methanol extract was filtered through Whatman filter paper No. 1. Methanol extract so obtained was concentrated under vacuum in rotary flash vacuum evaporator at the temperature of 55 ± 1 °C. Solvent was evaporated in vacuum oven at 50 ± 2 °C and 600 mmHg vacuum. Out of this, 1.25 mL methanol extract was dissolved in ghee at 60 °C and volume was made to 25 mL in a volumetric flask with ghee. This was designated as methanol pre-extract.
The methanol pre-extract of DRP added to ghee at the rate of 0.0% (T0) (control), 0.1% (T1), 0.25% (T2) and 0.5% (T3) (v/v) levels. The samples were kept at 40 ± 2 °C for 30 min, then temperature was raised to 50 ± 2 °C to 30 min and then temperature was raised upto 120 °C and allowed to cool at 60 °C. For comparison of above treatments, BHA was added to ghee at the rate of 0.02% (T4) (v/w) in each trial. Ghee samples with and without additives were analysed for moisture and free fatty acids (ISI, 1981), phospholipids (Rama Murthy & Narayanan, 1966) and water-extractable phenolic compounds (Swain & Hills, 1959). For testing the oxidative stability, ghee samples were stored in an oven at 80 ± 2 °C and the peroxide development was monitored (ISI, 1981) at an interval of 48 h. The effectiveness of the additives in term of anti-oxidative indexes was calculated as suggested by Pruthi et al. (1970). The data were statistically analysed using randomised block design (Snedecore & Cochran, 1967).
Results and discussion
The data on the phospholipids content of ghee samples with or without additives (Table 1) were observed in order 3.99 (T0) < 4.15 (T4) < 6.14 (T1) < 8.52 (T2) < 12.14 (T3). There was a gradual increase in phospholipids content with increase addition of methanol pre-extract of DRP over control or BHA added samples of ghee. A critical evaluation of data indicated that when pre-extract was added at the rate of 0.1%, 0.25% and 0.5% of methanol extract, the corresponding increase (over control) in phospholipids content observed was about 2.15, 4.53 and 8.15 mg 100 g−1 ghee respectively. The rise in the concentration of phospholipids treated ghee samples is because of the transfer of phospholipids of DRP during course of heating. The DRP contained on dry matter basis 0.17% phospholipids. Mahadevappa & Raina (1978) reported total phospholipids content in ragi to the extent of 0.10–0.11%. The phospholipids extracted from DRP (chloroform:methanol) when subjected to thin layer chromatography on silica gel G, resolved into five different fractions.
Phospholipids, water-extractable phenolics, induction period and anti-oxygenic indexes of ghee as affected by addition of methanol pre-extract of dehusked ragi powder at various levels (%, v/v) and BHA
| Treatment | Phospholipids* (mg 100 g−1) | Water-extractable phenolics* (mg 100 g−1) | Induction period* (h) | Anti-oxygenic indexes* |
|---|---|---|---|---|
| Control (T0) | 3.99 | 0.32 | 103.7 | – |
| 0.1% (T1) | 6.14 | 0.52 | 152.5 | 1.47 |
| 0.25% (T2) | 8.52 | 1.25 | 195.5 | 1.93 |
| 0.5% (T3) | 12.14 | 2.78 | 278.7 | 2.75 |
| 0.02% BHA (T4) | 4.15 | 0.36 | 191.5 | 1.89 |
| Treatment | Phospholipids* (mg 100 g−1) | Water-extractable phenolics* (mg 100 g−1) | Induction period* (h) | Anti-oxygenic indexes* |
|---|---|---|---|---|
| Control (T0) | 3.99 | 0.32 | 103.7 | – |
| 0.1% (T1) | 6.14 | 0.52 | 152.5 | 1.47 |
| 0.25% (T2) | 8.52 | 1.25 | 195.5 | 1.93 |
| 0.5% (T3) | 12.14 | 2.78 | 278.7 | 2.75 |
| 0.02% BHA (T4) | 4.15 | 0.36 | 191.5 | 1.89 |
Average of four replications.
Significant at 5% level for all the treatments.
Phospholipids, water-extractable phenolics, induction period and anti-oxygenic indexes of ghee as affected by addition of methanol pre-extract of dehusked ragi powder at various levels (%, v/v) and BHA
| Treatment | Phospholipids* (mg 100 g−1) | Water-extractable phenolics* (mg 100 g−1) | Induction period* (h) | Anti-oxygenic indexes* |
|---|---|---|---|---|
| Control (T0) | 3.99 | 0.32 | 103.7 | – |
| 0.1% (T1) | 6.14 | 0.52 | 152.5 | 1.47 |
| 0.25% (T2) | 8.52 | 1.25 | 195.5 | 1.93 |
| 0.5% (T3) | 12.14 | 2.78 | 278.7 | 2.75 |
| 0.02% BHA (T4) | 4.15 | 0.36 | 191.5 | 1.89 |
| Treatment | Phospholipids* (mg 100 g−1) | Water-extractable phenolics* (mg 100 g−1) | Induction period* (h) | Anti-oxygenic indexes* |
|---|---|---|---|---|
| Control (T0) | 3.99 | 0.32 | 103.7 | – |
| 0.1% (T1) | 6.14 | 0.52 | 152.5 | 1.47 |
| 0.25% (T2) | 8.52 | 1.25 | 195.5 | 1.93 |
| 0.5% (T3) | 12.14 | 2.78 | 278.7 | 2.75 |
| 0.02% BHA (T4) | 4.15 | 0.36 | 191.5 | 1.89 |
Average of four replications.
Significant at 5% level for all the treatments.
The water extractable phenolic compounds were also found to increase significantly (P < 0.05) in samples added with methanol pre-extract of DRP (Table 1). The water extractable phenolics in ghee with BHA were 0.36 mg 100 g−1 whereas 2.78 mg 100 g−1 in ghee treated with the maximum level of methanol pre-extract of DRP. The observed increase in the concentration of water extractable phenolics of the treated samples of ghee is also because of the transfer of phenolics from the grain powder. DRP on an average on dry matter basis contained 2.55% total phenolics. The total phenolics in ragi were reported to vary from 0.3% to 2.4% by Ramachandra et al. (1977), Bagepalli et al. (1982), Rao & Deosthale (1988) and Gopalan et al. (1993). Seventeen different phenolic compounds were detected in methanol extract of DRP.
To see the initiation of peroxide development, peroxide value was measured. The peroxide development was faster in control samples than those with additives (Fig. 1). The induction periods (time taken in hours to reach a peroxide value to 5 as millieqivalent of peroxide oxygen per kilogram of ghee) increase with the addition of DRP (Table 1). The effectiveness is further observed when the anti-oxidative indexes (ratio of induction period of treated sample to induction period of control sample) are calculated (Table 1). The anti-oxygenic indexes for treatments were in order: 1.47 (T1) < 1.89 (T4) < 1.93 (T2) < 2.75 (T3) over the control samples of ghee.
Peroxide value of ghee as affected by the addition of methanol pre-extract of DRP.
The results of peroxide values, induction periods and the anti-oxygenic indexes suggest that addition of DRP enhance the stability of ghee against autoxidation. The results also reveal that addition of methanol pre-extract of DRP at a level of 0.25% and above have higher effect than the addition of permitted level of BHA.
It is evident from the results that the protective action of DRP could be attributed to the transfer of phospholipids and phenolic compounds present in DRP. Phospholipids and phenolic compounds are the potential agents in preventing the autoxidation of fat through various mechanisms (Stuckey, 1962; Bhatia et al., 1978; Dugan, 1980). These two classes of compounds are also known to have synergestic effect (Brandt et al., 1973) in protecting the fat.
The DRP also contained on an average 10.05% moisture, 2.00% lipids, 6.10% protein, 79.65% carbohydrates and 2.20% ash. These results are in general agreement with those reported by Mahadevappa & Raina (1978) and Swaminathan (1999). Addition of methanol pre-extract of DRP to ghee did not influence the moisture, free fatty acids, colour, flavour and granulation characteristics of ghee. It can therefore be concluded that addition of methanol pre-extract of DRP at a level of 0.25% (v/v) and above can protect ghee against oxidative rancidity for periods longer than BHA.
