doi: 10.1111/j.1471-0307.2009.00555.

ORIGINAL RESEARCH

The development of bur sweetened with aspartame

SUMIT ARORA*, HEMANT GAWANDE, VIVEK SHARMA, BALBIR KAUR WADHWA, VISHWAS GEORGE, GHANSHYAM S SHARMA and ASHISH KUMAR SINGH
Dairy Chemistry Division, National Dairy Research Institute, Karnal-132 001, Haryana, India

Sucrose was successfully replaced with the sweetener aspartame for the preparation of the indigenous dairy product bur. Analytical conditions were standardised for the solid phase extraction of aspartame and its degradation products from bur followed by their reverse phase HPLC. Recovery using this method was 9097%. Aspartame at a level of 0.065% of milk w w scored highest in terms of sweetness perception and resembled control bur in sweetness. Storage studies at 68C revealed that aspartamesweetened bur resembled the control bur in retaining the sensory prole, but showed an increase in acidity and microbial load and could not retain the texture. High-performance liquid chromatography analysis revealed no degradation of aspartame in bur, establishing its stability and hence its sweetness on storage. Keywords Aspartame, Bur, Stability, Colour, Texture, Analysis.

INTRODUCTION Sweeteners play an important role in our society, not only for diabetic patients but also for people using low-calorie foods. With increased consumer interest in reducing sugar intake, food products made with sweeteners rather than sugar have become more popular. The change in food legislation opens up a vast untapped market of sugar-free food products, including dairy products; recent change in legislation in India (PFA 2004) now permits the use of aspartame in sweets like halwa, khoa, bur, rasgolla, gulabjamun and other milk products as per the limits prescribed under proper label declarations. This can lead to partial or whole replacement of sucrose in sweetened dairy products like bur. Consumption of sweets is an integral part of Indian dietary system and diabetic-friendly traditional sweets is a new category for such products, the production of which is being contemplated by many enterprising manufacturers. Milk, a perishable commodity, in this way gets converted into value-added long-life products using processes such as heat and or acid coagulation, desiccation and fermentation (Vaswani 2002). Bur is one of the most popular khoa-based sweets, a product obtained from khoa and sugar having a mildly caramelised and pleasant avour. Replacement of sucrose with aspartame in bur may result in changes in its physicochemical attributes. Aspartame is a high-intensity sweetener which is added to about 1200 food products, including diabetic and dietetic ice cream, desserts, yogurt, ready-to-eat cheesecakes, frozen dairy frostings and

toppings, avoured and chocolate milk, skim milk and whey beverages (Rothwell 1985; Jenesen et al. 1989; Weider 1989; Beukema and Jelen 1990 and Keller et al. 1991a,b; Pinheiro et al. 2005). Longterm storage and heat stability are important factors for the use of intense sweeteners in many food products and beverages. Sweeteners have been found to have different responses to heat and pH (George et al. 2006). Since the application of sweeteners in indigenous dairy products is new, qualitative as well as quantitative information on sweeteners degradation in dairy systems is required. Aspartame loses sweetness with prolonged exposure to high temperatures and storage; it breaks down into aspartylphenylalanine and methanol, or in case of cyclisation into diketopiperazine (Jana et al. 1994). No work has been done on the estimation and stability of aspartame in indigenous dairy products. Therefore, the present study has been undertaken to develop a methodology for determining aspartame in bur and to identify the most appropriate inclusion rate for aspartame for an organoleptically acceptable product. M AT E R I A L S A N D M E T H O D S

*Author for correspondence. E-mail: sumitak123@yahoo.com 2009 Society of Dairy Technology

Preparation of bur Pooled buffalo milk and skim milk were collected from the experimental dairy, NDRI, Karnal. The sweetened bur was prepared according to the method of Bhatele (1983) with slight modication (Figure 1) for sweetener-containing product. A control sample was also prepared using sugar procured from the local market.
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Buffalo milk Filtration Desiccation in double-jacketed steam kettle using steam until semi-solid consistency is reached (steam pressure 12 kg/cm2) Cooling in tray for 15 min Khoa Addition of sugar (6%) or sweetener (aspartame 0.065%) dissolved in 25 mL of milk Further desiccation for 10 min Cooling to room temperature Packaging and storage (68 C)

Figure 1 Flowchart for manufacture of bur.

Optimisation of aspartame levels in bur Aspartame levels were optimised by sensory evaluation of bur on the basis of its acceptance by a panel of eight judges on a 9-point hedonic scale score card. The levels of aspartame tried for bur were in the range 0.0450.075% on 6% sucrose equivalence milk basis. Aspartame standards Nutrasweet powder (Nutrasweet Company, Augusta, GA, USA). Aspartame degradation products standards: 2, 5-diketopiperazine, L-phenylalanine (Sigma-Aldrich, Lovfs, MO, USA). The chemicals used in this study were of analytical reagent grade and all aqueous solutions were prepared using reverse-osmosis quality water produced by a Milli-RO 15 plus Milli-Q purication system (Millipore, Bedford, MA, USA). Equipment The vacuum ltration assembly was supplied by the Millipore Corporation Bedford, MA, USA; the solid phase extraction C18 cartridge by Supelco (Bellfonte, PA, USA); silica gel 60 F254 TLC aluminium sheets (20 20 cm) by E. Merck India Ltd (Mumbai, India), the solid phase extraction vaccum manifold by VisiprepTM DL 9Supelco; Bellfonte, PA, USA; and the HPLC System by Shimadzu (LC10A, detector UV, SPD-M10AV, Japan). Solid phase extraction (SPE) of aspartame from bur The sample preparation procedure (Figure 2) used for isolation of aspartame from bur was essentially based upon the method of BS EN: 12856 (1999) used for semisolid and solid products. A blank experiment was also performed simultaneously. High-performance liquid chromatography analysis High-performance liquid chromatography analysis of reference standards of aspartame, degradation
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products and sample isolates from bur were performed under the following set of standardised conditions:
Column stationary phase Phase UV detector wavelength Mobile phase (A) Mobile phase (B) Flow rate Run time Maximum pressure Actual pressure Shimpak C18, S-5 lm, 120A, 250 4.6 mm ID Reverse phase 200 nm 0.02 M phosphate buffer, pH 5.0: acetonitrile (97:3) 0.02 M phosphate buffer, pH 3.5: acetonitrile (80:20) 1 mL min 30 min 400 kgmf 80 kgmf

Gradient programming: 0% mobile phase B for rst 8 min; 0-100% B linear gradient from 8 to 13 min; 100% B from 13 to 25 min; 100-0% B linear gradient from 25 to 27 min; 0% B from 27 to 30 min. The actual pressure varied during programming as 80-160-80 Kgmf.

Standard solutions of aspartame and degradation products Ten milligram each of aspartame and its degradation products were dissolved in a 10 mL mixture of mobile phases A and B (1:1) to give stock standard solutions of concentration 1 mg mL. About 100 lL from each of the stock standard solution of concentration 1 mg mL were pipetted out into separate 10 mL volumetric asks and the volume made up to the mark with the mixture of mobile phases A and B (1:1), to give standard solutions of concentration 10 ng lL Also, 100 lL from each of the standard solutions (1 mg mL) were pipetted out into a 10 mL volumetric ask and volume made up to the mark with mixture of mobile phases A and B (1:1), to give a mixture of aspartame and degradation products in standard solutions of concentration 10 ng lL. A 5-point calibration curves were

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burfi (1.75 g) in 100 mL beaker

Dilution with HPLC grade water (50 mL)

Ultra-sonification (40C/20 min)

Carrez clarification (6 mL Carrez solution No.1 + 6 mL Carrez solution No.2)

Dilute upto the mark (100 mL)

Filtration (Whatman No. 1 filter paper)

Solid phase extraction (2 mL filtrtate eluted through C18 cartridge previously activated with 3 mL of methanol and 20 mL water; elution with 10 mL of mobile phase A followed by 10 mL Mobile phase B)

Eluant (volume made to 25 mL)

HPLC analysis

Figure 2 Flowchart for isolation of aspartame from bur.

plotted for aspartame and its degradation products. Curves were prepared representing 50, 100, 150, 200 and 250 ng concentration and their corresponding peak areas. The correlation coefcient of 0.99 for aspartame as well as its degradation products showed the linearity of the system. Recovery procedures of aspartame and its degradation products were performed at 800 and 625 ppm respectively in bur.

and texture prole analysis were performed as described below: Sensory evaluation A sensory panel consisting of eight trained panellists drawn from the faculty and students of the Dairy Chemistry Division, National Dairy Research Institute, Karnal, evaluated the samples of bur. The panellists were served with 40 50 g of the product at 30C (room temperature). They evaluated samples for various attributes, namely sweetness, colour and appearance, body and texture, and overall acceptability using a nine-point hedonic scale (ranging from 9like extremely to 1-dislike extremely) (Amerine et al. 1965). Between evaluations of the samples the panellists rinsed their mouths with de-ionised water to remove any lingering sweet taste. Subjects refrained from drinking, smoking, or consuming anything except water for 30 min prior to each panel session. This method does not, of course, reect actual consumer perception, but it does strongly indicate attributes which a good quality product should possess. Colour measurement The colour of the bur was measured using a Colourex (Hunter lab, Reston, VA, USA). The light source was a xenon ash lamp. Data was received through the software in terms of L* (Lightness) ranging Zero (black) to 100 (white), a* (Redness) ranging from +60 (red) to )60 (green) and b* 129

Sample preparation and HPLC analysis The sample preparation was standardised to 1.75 g of bur to ensure completely fat free extraction through C18 cartridges. The amounts of Carrez solutions 1 and 2 required for the precipitation of proteins in bur were standardised as 6 mL each. Elution was done with 10 mL of mobile phase A followed by 10 mL of mobile phase B for efcient recovery of aspartame and its degradation products Storage and analysis of dairy products Control and sweetened samples of dairy products (with the best selected sweetener level) were stored at refrigerated temperature (68C) and samples were analysed at 0, 3 and 7 days of storage for the following analytical parameters: titratable acidity and pH by IS: SP-18 Part XI (1981), microbiological examination by IS: SP18 Part I (1980) using Plate Count Agar; incubation of the plates was carried out at 37C for 48 h. Sensory evaluation, colour measurement
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(Yellowness) +60 (yellow) to )60 (blue) of the International colour system. Texture prole analysis Instrumental texture prole analysis (TPA) was done to characterise hardness, adhesiveness, springiness, cohesiveness, gumminess and chewiness. The samples of bur were cut into standard cubes (25 mm3). Texture prole analysis tests were performed using the Texture Analyzer TA-xT2i (Stable MicroSystem, Godalming, Surrey, UK) and the instrument was tted with a 25 kg load cell.

substantially below the acceptable daily intake (ADI) of 50 mg kg body weight day (Butchko et al. 2002).

Analysis of bur during storage

Sensory prole Sensory evaluation during storage (Table 2) revealed that aspartame-sweetened- bur resembled the control in retaining the sensory prole (sweetness, colour and appearance, body and texture, and overall acceptability) at all periods of storage. However, aspartame-sweetened bur ranked lower (P < 0.05) than the control. The higher scores in control bur could be attributed to sucrose. Sucrose helps to form a network and create a binding effect in the product. Hence, the inclusion of sugar improved the body and texture of the product. Sugar also added to the caramelised colour of the control product. On the other hand, aspartame had insufcient water-binding capacity, which resulted in a product with a slightly lower score. Titratable acidity and pH There was no apparent difference in initial titratable acidity of the control and aspartame-sweetened bur (Table 3). A signicant increase (P < 0.05) in acidity was observed in aspartamesweetened bur and the control at all periods of storage. The titratable acidity was signicantly higher (P < 0.05) in aspartame-sweetened bur than the control during storage. This difference in titratable acidity might be due to the preservative effect of sucrose, which led to the retarded microbial growth (Table 3) in control samples resulting in low acidity. The pH of aspartame-sweetened bur and control decreased (P > 0.05) during storage (Table 3). The pH of control and aspartamesweetened bur were similar initially as well as at different periods of storage. Textural parameters Aspartame-sweetened bur like the control could not retain most textural attributes (Table 4) hardness, adhesiveness, springiness and chewiness except for cohesiveness, at all periods of

Statistical analysis In all experiments, one-way two-way analysis of variance (ANOVA) with a subsequent least signicant difference (LSD) test was applied for multiple sample comparison. This was done to test for any signicant differences (P < 0.05) in the mean values of all the groups as described by Snedecor and Cochran (1989).
R E S U LT S A N D D I S C U S S I O N

Optimisation of aspartame levels in bur Sensory evaluation revealed that aspartame when used at the levels of 0.055, 0.06 and 0.07% in bur (Table 1) resulted in signicantly lower sensory scores (P < 0.05) than the control. Aspartame at the level of 0.065% in bur scored highest in terms of sweetness perception and resembled the control. However, colour and appearance, body and texture, and overall acceptability scores were lower (P < 0.05) in aspartame- sweetened bur than in the control. This is because sugar (sucrose) not only contributes towards the sweetness but also helps in development of desirable avour and characteristic colour through maillard browning and caramelisation. At higher levels of aspartame (0.07%) there was persistence of sweetness for longer periods, which impartean unusual taste to the bur. Hence, the aspartame level of 0.065% in bur was nally selected for preparation of sweetened bur and its subsequent analysis. At this level, the actual level of consumption of aspartame in bur is well within acceptable limits i.e.

Table 1 Optimisation of aspartame level in bur Aspartame level (%) Characteristics Sweetness Colour and appearance Body & texture Overall acceptability Control 8.2 8.0 8.0 8.0 0.16a 0.92a 0.31a 0.32a 0.055 6.9 7.1 6.0 6.3 0.44b 0.23b 0.39b 0.26b 0.06 6.8 7.2 6.3 6.5 0.29b 0.31b 0.37b 0.32b 0.065 7.7 7.2 6.5 7.1 0.18a 0.32b 0.31b 0.35c 0.07 6.3 7.2 6.4 7.0 0.39b 0.2b 0.26b 0.33c

Mean in each row with different superscripts (a, b, c) were signicantly different (LSD test, P < 0.05) from each other. Data are presented as mean SEM (n = 8).

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Table 2 Sensory prole of bur during storage bur storage (days) Sensory parameters Sweetness Control Aspartame Colour and appearance Control Aspartame Body and texture Control Aspartame Overall acceptability Control Aspartame 0 8.1 0.13Aa 7.9 0.16Aa 8.1 0.22Aa 7.4 0.18Ba 8.0 0.18Aa 6.5 0.2Ba 8.1 0.12Aa 7.7 0.16Ba 3 8.0 0.18Aa 7.8 0.17Aa 7.8 0.12Aa 7.2 0.18Ba 7.7 0.16Aa 6.7 0.16Ba 8.0 0.18Aa 7.5 0.21Ba 7 8.0 0.18Aa 7.8 0.12Aa 7.7 0.35Aa 7.1 0.33Ba 7.7 0.31Aa 7.0 0.26Ba 7.5 0.34Aa 7.0 0.27Ba

Mean in each row with different superscripts (a) was signicantly different (LSD test, P < 0.05) from each other. Mean in a column with different superscripts (A, B) were signicantly different (LSD test, P < 0.05) from each other. Data are presented as mean SEM (n = 8).

Table 3 Titratable acidity and pH of bur during storage Storage period (days) bur Titratable acidity (% LA) Control Aspartame pH Control Aspartame 0 0.36 0.00Aa 0.37 0.00Aa 6.45 0.01Aa 6.43 0.00Aa 3 0.38 0.00Ab 0.40 0.00Bb 6.43 0.00Aa 6.41 0.00Aa 7 0.37 0.00Ab 0.43 0.00Bc 6.42 0.00Aa 6.40 0.01Aa

Mean in each row with different superscripts (a, b, c) were signicantly different (LSD test, P < 0.05) from each other. Mean in a column with different superscripts (A, B) were signicantly different (LSD test, P < 0.05) from each other. Data are presented as mean SEM (n = 8).

storage. Gumminess was not retained by the aspartame-sweetened bur whereas the control retained gumminess upto 3rd day of storage. Chewiness was retained by the aspartame-sweetened bur upto the 3rd day of storage whereas it was not retained by the control even up to the 3rd day of storage. These changes in the textural attributes of both the bur samples were due to loss of moisture during storage. Aspartame-sweetened bur ranked signicantly lower (P < 0.05) than the control for various textural attributes except cohesiveness initially, as well as at different periods of storage. These differences in textural attributes are due to the presence of sucrose in the control which contributes towards total solids and hence the hardness. Gupta et al. (1990) and Suresh and Jha (1994) reported that the increased hardness of khoa (base material for bur) correlated highly with the total solids. Jha (2003) reported that sugar had a positive linear effect on adhesiveness of Doda bur (a milk wheat based sweetmeat). However, Gupta et al.
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(1990) noticed no signicant effect of compositional factors on adhesiveness of khoa. Colour measurement The changes in lightness l* (Black white) and b* (yellowishness blueness) values were not signicant during storage for both control and sweetened bur (Table 5). The lightness was signicantly less (P < 0.05) in control samples than aspartamesweetened bur during the entire period of storage, which was due to caramelisation in the presence of sucrose. The changes in a* (redness greenness) values were signicant (P < 0.05) during storage for both types of bur. Our results are in accordance with the results of Gothwal and Bhavdasan (1991), who reported an increase in browning as a result of storage in khoa.

Microbiological examination Total plate counts were higher (P < 0.05) in the aspartame-sweetened product (7.37 103 cfu mL) than their corresponding control (5.05
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Table 4 Texture parameters of bur during storage bur storage (days) Parameters Hardness (N) Control Aspartame Adhesiveness (N) Control Aspartame Springiness (mm) Control Aspartame Cohesiveness Control Aspartame Gumminess Control Aspartame Chewiness (N.mm) Control Aspartame 0 62.60 0.20Aa 44.56 3.69Ba 0.38 0.06Aa 0.27 0.06Ba 1.69 0.09Aa 1.59 0.043Ba 0.19 0.007Aa 0.18 0.001Aa 11.68 0.10Aa 8.17 1.01Ba 20.23 0.99Aa 12.77 2.15Ba 3 68.83 2.52Ab 57.40 4.05Bb 0.34 0.06Ab 0.23 0.06Bb 1.47 0.06Ab 1.34 0.006Bb 0.18 0.02Aa 0.16 0.00Aa 12.57 1.09Aa 9.28 0.97Bb 18.34 1.54Ab 12.28 1.94Ba 7 89.51 4.48Ac 65.38 3.95Bc 0.28 0.02Ac 0.10 0.01Bc 1.10 0.045Ac 1.03 0.095Bc 0.17 0.0Aa 0.16 0.02Aa 15.42 0.76Ab 10.47 1.02Bc 16.87 1.35Ac 10.67 2.34Bb

Mean in each row with different superscripts (a, b, c) were signicantly different (LSD test, P < 0.05) from each other. Mean in a column with different superscripts (A, B) were signicantly different (LSD test, P < 0.05) from each other. Data are presented as mean SEM (n = 8).

Table 5 Colour parameters of bur during storage in IU (International Units) Storage period (days) bur L* (light ness) Control Aspartame a* (redness) Control Aspartame b*(yellowness) Control Aspartame 0 66.21 0.75Aa 70.16 0.09Ba 7.63 0.21Aa 7.28 0.21Aa 30.44 0.60Aa 29.65 0.19Aa 3 65.79 1.03Aa 69.20 0.13Ba 8.41 0.40Aab 7.86 0.48Aab 29.51 0.27Aa 28.85 0.14Aa 7 65.12 0.03Aa 68.45 0.13Ba 8.75 0.31Ab 8.82 0.20Ab 29.08 0.06Aa 28.24 0.10Aa

Mean in each row with different superscripts (a, b) were signicantly different (LSD test, P < 0.05) from each other. Mean in a column with different superscripts (A, B) were signicantly different (LSD test, P < 0.05) from each other. Data are presented as mean SEM (n = 8).

103 cfu mL) throughout the storage period as there was no preservation protection effect of aspartame, which ultimately lead to higher microbial counts. There was an increase (P < 0.05) in the total plate counts in both the control and the aspartame-sweetened bur during storage. Stability of aspartame Aspartame, diketopiperazine and L-phenylalanine gave kmax at 200 nm. Figure 3 represents the HPLC chromatogram of these three components at 200 nm under the standardised analytical conditions. The degradation products are eluted by 132
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mobile phase A and the main sweetener aspartame by mobile phase B. Regression equations and correlation coefcient were also obtained. The correlation coefcient of 0.99 for aspartame as well as its degradation products showed the linearity of the system. Their detection limits were in the range of 1030 ng at 200 nm. Lawrence and Charbonneau (1988) also reported similar observations for aspartame. However, no information was available in the literature regarding the detection limits of these degradation products of the sweetener. Recovery of the method was 9097%.

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Figure 3 High-performance liquid chromatography (HPLC) chromatogram of aspartame and its degradation products standards.

Table 6 Stability of aspartame in bur during storage 0 day Product (level added in ppm) bur (650) Recovery ppm 627.64 Recovery (%) 96.56 3 day Recovery ppm 622.70 Recovery (%) 95.80 7 day Recovery ppm 613.79 Recovery (%) 94.43

Data are presented as mean SEM (n = 8).

Stability of aspartame in bur during storage Table 6 shows the stability of aspartame in bur. It is evident from the table that the aspartame levels remained unchanged up to 7 days of storage in bur, establishing thereby the stability of aspartame during storage. High-performance liquid

chromatography chromatograms obtained at 0, 3 and 7 days of storage for bur also supported this observation as there was no peak other than aspartame in these chromatograms (Figure 4). This established that aspartame was not degraded during storage in the bur samples under investigation.

(a)

Aspartame

(b)

Aspartame

(c)

Aspartame

Figure 4 High-performance liquid chromatography (HPLC) chromatograms of sample isolates of aspartame-sweetened bur during storage. (a) 0 day; (b) 3rd day; (c) 7th day.

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This might be due to the pH of bur (6.5) and its low moisture content (aw = 0.764). Aspartame decomposes in liquids during prolonged storage. In avoured milk on the 5th and 7th days of storage aspartame recovery was 75% and 62%, thereby indicating 25% and 38% loss, respectively (Arora et al. 2008). Beukema and Jelen (1990) also reported a loss of 13.2% of the aspartame in whey beverage with pH 4.3. Nevertheless, Bell and Labuza (1994) observed that decreasing the pH from 6.7 to 6.4 doubled the stability of aspartame in sterilised avoured dairy beverages sweetened with aspartame. Arman and Temiz (1988) evaluated aspartame degradation in reduced moisture toothpaste at pH 6.05. They found a half life of 114 days at 40C for a system with water activity (aw) determined experimentally to be 0.76. Aspartame is unstable if subjected to prolonged heating and therefore cannot be used in baking or cooking unless added at the end of the cooking process (Kroger et al. 2006), as has been done in the preparation of sweetened bur in the present study. CONCLUSIONS Sensory prole during storage studies at 68C revealed that aspartame-sweetened bur resembled the control bur in retaining sweetness. The optimum level of aspartame was found to be 0.065% in bur on the basis of sensory evaluation. However, aspartame-sweetened bur ranked lower than the control in sensory and textural attributes, whereas the reverse was true for lightness, acidity and microbial load. A solid phase extraction (SPE) method was standardised for the isolation of aspartame from bur followed by HPLC. High-performance liquid chromatography analytical conditions were standardised for the separation of aspartame and its degradation product diketopiperazine and L-phenylalanine over a C18 column at UV 200 nm. Recovery of the method was 9097%. The results of the present investigation have established the successful use of aspartame in the preparation of the indigenous dairy product bur. ACKNOWLEDGEMENTS
The authors would like to express appreciation to NutraSweet Company, USA for supplying ingredients for this study.

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