The Pharma Innovation Journal 2018; 7(4): 249-256

ISSN (E): 2277- 7695 ISSN (P): 2349-8242 NAAS Rating: 5.03 Process optimization of bael (Aegle marmelos) TPI 2018; 7(4): 249-256 © 2018 TPI polyphenols extract fortified stirred yoghurt www.thepharmajournal.com Received: 26-02-2018 Accepted: 30-03-2018 Urmila Choudhary and Amrita Poonia

Urmila Choudhary Dairy Technology Division, Abstract ICAR-National Dairy Research Bael (Aegle marmelos) being a natural antioxidant source has many health promoting effects. The aim of Institute, Karnal, Haryana this study was to formulate the functional stirred yoghurt with adding bael polyphenolic (BPP) extract and its process optimization by standardizing the levels of variables. In the current study, bael polyphenol Amrita Poonia extract (0.2-0.5%), fat (2.5-4.5%) and sucrose (10-15%) levels in stirred yoghurt were varied and Centre of Food Science and the assessment of product quality was characterised on the basis of antioxidant, textural and sensorial Technology, Institute of responses. Results show that the BPP extract, milk fat and sucrose affected the antioxidant activity as % Agricultural Sciences, Banaras DPPH inhibition and total phenolic content of the product, textural and sensory characteristics of product. Hindu University, , The BPP extract significantly affected the % DPPH inhibition and total phenolic content and increased Uttar Pradesh, with increasing concentration of BPP extract. The sensory characteristics (body & texture, flavour, colour and appearance) of BPP extract yoghurt were significantly affected by milk fat and BPP extract.

Keywords: bael polyphenol extract, yoghurt, antioxidant properties, textural properties, sensory characteristics

Introduction Yoghurt is popular milk based fermented product that contains the characteristic bacterial

cultures Lactobacillus bulgaricus and Streptococcus thermophilus and due to the nutritional value and health promoting effects, it can include in daily diet. The wide acceptability of yoghurt in various flavours makes it a good base for fortification with fruit extracts and natural antioxidants to make it a functional product with enhanced health benefits. The quality of yoghurt principally depends on the ingredients and milk composition. Previously, many

researchers observed the effect of ingredients such as effect of pectin and fruit concentrates dairy fat, protein and hydrocolloids on textural properties of yoghurt Ramaswamy and Basak (1992) [1]. Yoghurt is fortified with fruit extracts or specific antioxidant extracts for the enhancement of its antioxidant properties El-Assar et al. (2005) [3]. Fruit extracts were used in yoghurt as a functional ingredients to improve the kinetics and texture profile of yoghurt do [4] Espírito et al. (2012) . The bael a tropical and subtropical fruit, native to . It is a very hard fruit and the pulp of it has yellow or orange colour, possesses a slightly sweet taste and a characteristic pleasant flavour. The fruit is a good source of indigenous natural antioxidants and bioactive compounds containing relatively high content of dietary fiber, carotenoids, phenolics, , ascorbic acid and also strong antioxidant activities. The

main volatile compounds are monoterpenes and sesquiterpenes Charoensiddhi and Anprung [5] (2008) . The Bael is an important medicinal of India containing many functional and bioactive compounds such as carotenoids, phenolics, alkaloids, coumarines, flavonoids and terpenoids Nagaraju and Rao (1990) [6]. Many studies show that consuming different parts of bael such as pulp rich dishes is associated with lowering down risk of diabetes, cardiovascular

and anti-inflammatory cancer, hypoglycaemic, hypolipidemic and blood pressure diseases. Hence incorporating these bioactive compound rich extracts in other foods is a good idea. Biochemical compounds of bael , fruits and seeds have been used in several diseases like diabetes, cardiovascular and anti-inflammatory Maity et al. (2009) [7] and it also possess hypoglycaemic, hypolipidemic and blood pressure lowering property Lmbole et al. (2010) [8].

Correspondence The different parts of baelare used for various therapeutic purposes, such as antibacterial Raja [9] [10] Amrita Poonia et al. (2009) , antiviral Badam et al. (2002) , anti-inflammatory bowel disease Singh Centre of Food Science and (2009) [11], anticancer and chemopreventive agent Agrawal et al. (2010) [12], and it also Technology, Institute of prevents radiation-sickness and mortality Jagetia et al. (2004) [13]. Earlier, so many researchers Agricultural Sciences, Banaras worked on the optimization, processing and shelf life of yoghurt Singh et al. (2013), Cakmakci Hindu University, Varanasi, Uttar Pradesh, India ~ 249 ~ The Pharma Innovation Journal

et al. (2014), Yadav et al. (2016) Mercan et al. (2017) [14, 15, 16, 17]. Keeping this in mind the present study was deals to Experimental design develop bael (Aeglemarmelos) polyphenols extract fortified In present study, optimization of bael polyphenol extract stirred yoghurt and its process optimization by standardizing fortified stirred yoghurt central composite rotatable design the levels of bael polyphenols (BPP) extracts, sucrose and (CCRD) based on response surface methodology (RSM) was milk fat and to study their effects on various parameters such used. Constraints and criteria for optimization of the three as antioxidant properties (% DPPH inhibition and total design factors are presented in Table 1 and the experimental phenolic content), textural properties (cohesiveness, design comprise of 20 trials as presented in Table 2. consistency and index of viscosity) and sensory Optimization was done for sensory, rheological and characteristics and also study their functional stability during antioxidant properties of yoghurt. Fitting of mathematical storage /the antioxidant loss and textural stability during models and finally selecting variable levels by optimizing the storage period. Bael gave some astringent taste therefore response was employed as per the method given by Khuri and sucrose was added to overcome this astringent taste to the Cornell (1987) [20]. A second-order polynomial model was product. fitted to study the relationship between the responses and the three factors (fat, bael polyphenol extract and sugar). For Materials and Methods these three variables, the equation could be given as: The research work was carried out at Centre of Food Science

and Technology, Institute of Agriculture Sciences, Banaras 2 Y = β0 + ∑βiXi+ ∑βijXiXj+ ∑βiiXi +ε (1) Hindu University, Varanasi.

Raw materials Where Y is the response, β0 is the model constant; βi is the Toned Milk (3.1% milk fat and 8.6% milk solids not fat) was linear effect of factor Xi; βijis the cross product of factor Xi and procured from milk booth of Varanasi market (Parag Dairy), Xjand βii is the quadratic effect of factor Xi; ε is the residual Uttar Pradesh, India. It was standardized to contain 4% milk error. The error includes experimental errors and lack of fit ® chosen for the model. The quality of fit of chosen model was fat. Freeze-dried starter yoghurt cultures (YoFlex culture) 2 [Direct Vat Set (DVS)] were purchased from Chr. Hansen evaluated by R , coefficient of determination. Design Expert (India) Pvt. Ltd., Mumbai, Maharashtra (India) in lyophilized 8.0.3 software was used to generate the design of the form. Cane sugar (sucrose) was procured from local market of experiments, to fit model by multiple regression and to Varanasi for the preparation of experimental bael analyze the response surfaces. The response surfaces were (Aeglemarmelos) polyphenols extract (BPE) fortified stirred drawn by plotting y as a function of two variables by keeping yoghurt. Gallic acid was purchased from Sigma-Aldrich the third variable constant. The regression analysis of the Chem. Co., USA. responses was conducted by fitting linear and quadratic models as suitable in the case of the respective responses. Preparation of aqueous extract of bael polyphenol Table 1: Constraints and criteria for optimization of BPE fortified Preparation of the baelpolyphenol aqueous extract was carried [18] stirred yoghurt out following the method described by Cossu et al. (2009) . The bael pulp (500 g) was mixed with 1000mL distilled Constraints Lower limit Upper limit water. The mixture was blended with mechanical stirrer (30 A: BPE (%) 0.2 0.5 min), subjected to boiling (30 min) and centrifuged at 4029 B: Sugar (%) 10 15 g×15 min (Sigma 3-30K, Sigma Laborzentrifugen, Germany). C: Milk fat (%) 2.50 4.50 The supernatant was collected, frozen and concentrated using % DPPH inhibition 63.75 89.94 lyophiliser (SRL. NO. 049, Khera Instruments Pvt. Ltd, TPC (mg/100g) 19.85 44.85 Consistency (g sec.) 298.45 387.65 Delhi). Concentrated polyphenol extract was stored at -20°C Cohesiveness (g) 6.07 7.89 until use. The total phenolic content of the extract was Index of Viscosity (g sec.) 5.70 9.26 determined using the Folin-Ciocalteu method by measuring Colour and appearance 6.8 8.5 absorbance at 760nm using UV-specrophotometer(UV-1800, Body and texture 6.0 8.5 [19] Shimadzu, Japan) Huang et al. (2005) . A calibration curve Sweetness 6.75 8.75 of gallic acid (0-120 µg/mL) was prepared, and the results Flavur 6.65 8.75 were expressed as µg Gallic acid equivalent (GAE) per mL. Mouthfeel 6 8

Table 2: Experimental design for optimization of bael polyphenols extract fortified (BPE) stirred yoghurt

Factors Cons Coh IV % DPPH inhibition TPC C&A Flavour B & T Sweetness Mouthfeel Milk Fat Extract Sugar 3.50 0.35 12.50 352.65 6.84 7.85 78.19 38.85 7.40 7.25 7.00 7.75 8.00 1.82 0.35 12.50 350.67 7.05 6.78 76.35 32.15 8.50 6.80 6.00 7.60 7.00 4.50 0.20 15.00 385.87 7.89 7.86 70.94 19.85 6.80 7.50 7.25 8.55 6.00 3.50 0.60 12.50 298.45 7.52 7.59 89.94 44.85 7.40 8.75 7.50 7.50 8.00 4.50 0.50 15.00 337.76 7.60 8.89 88.46 42.05 7.25 8.50 7.75 8.55 7.50 2.50 0.20 10.00 310.54 6.40 7.09 70.19 29.85 7.30 6.85 6.00 7.00 8.00 2.50 0.20 15.00 340.75 6.80 7.55 70.78 28.15 7.90 7.00 6.50 8.25 7.50 3.50 0.35 12.50 327.45 6.50 7.89 74.78 33.84 7.40 7.25 7.00 7.25 8.00 3.50 0.35 12.50 328.45 6.59 7.82 74.68 33.25 7.79 7.25 7.00 7.25 7.50 3.50 0.35 16.70 387.65 6.09 9.26 75.98 34.85 7.60 7.50 7.00 8.75 7.00 3.50 0.35 8.29 298.67 6.77 5.70 72.94 28.05 7.58 7.20 7.25 6.75 8.00

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4.50 0.20 10.00 310.65 6.98 6.60 71.45 29.85 7.10 7.25 7.50 7.25 7.00 5.18 0.35 12.50 340.76 7.49 7.23 73.56 33.15 7.92 7.50 8.50 7.25 7.50 2.50 0.50 15.00 365.43 6.07 7.87 76.78 37.85 7.92 8.00 7.50 8.50 8.00 4.50 0.50 10.00 340.23 6.82 6.79 82.14 39.85 7.40 8.75 8.50 7.00 7.50 3.50 0.35 12.50 339.98 7.34 7.32 88.9 30.85 7.10 7.75 6.50 7.25 8.00 3.50 0.10 12.50 335.51 7.62 7.3 63.75 23.05 7.00 6.65 7.00 7.25 7.50 2.50 0.50 10.00 317.89 7.42 6.39 88.78 32.6 7.25 8.25 6.50 7.00 7.00 3.50 0.35 12.50 326.01 6.64 7.83 76.39 33.05 7.50 7.50 7.00 7.20 8.00 3.50 0.35 12.50 320.45 6.60 7.87 76.89 33.15 7.67 7.25 6.50 7.20 7.50 Cons: consistency, Coh: cohesiveness, IV: Index of viscosity C&A: colour and appearance, B&T: body and texture, TPC: total phenolic content

Preparation of bael polyphenol extract-fortified stirred UV-1800spectrophotometer (Shimadzu, Japan) against the yoghurt (BPE stirred yoghurt) blank. Gallic acid (0 - 800 mg/L) was used to produce The milk (4% milk fat) was heated to 90oC for 5 min., cooled standard calibration curve. The total phenolic content was to 32oC and DVS starter culture at the rate of 0.03% was expressed in mg of Gallic acid equivalents (GAE) / g of added. The mixture was incubated at 37oC until to get pH of extract. 4.5 within 5-6 hours and product was stored overnight at 4- 7oC to stop the fermentation process. The coagulum was Textural properties of yoghurt broken by gentle stirring using hand blender and mixed with This study was performed using texture analyzer (Model TA- lyophilized BPE powder (0.2-0.5%) (w/w) and sugar (10- XT plus, Stable Micro Systems, GodalmingUK) using 35 mm 15%) for preparation of stirred yoghurt fortified with bael back extrusion rig as the probe with 5 kg load cell. The data polyphenol extract. The yoghurt was filled in sterile plastic was analysed by using texture expert software (version TEE containers with lids (Tarson 100 mL) and stored at 7-8oC. The 322 4.0.13.0). Analysis was done at 10oC. The results were yoghurt prepared without polyphenol extract was taken as observed as maximum positive force as firmness (g), control. maximum negative force as cohesiveness (g), mean positive area as consistency (gs) and mean negative area as index of Radical Scavenging Activity (DPPH inhibition) viscosity (gs). Texture analyzer settings for analysis of bael Determination of antioxidant activity of sample was done by polyphenol extract fortified stirred yoghurt were set to 1, 1-Diphenyl-2-picryl-hydrazyl (DPPH) inhibition method measure force in compression. The pre-test, test and post test Nishino et al. (2000) [21]. Sample (200 mg) was taken in speeds were set at 1.0, 1.0 and 10.0 mm/s, respectively. centrifuge tube (in replications). In blank, distilled water (0.2 Penetration distance of probe was set to be 30 mm. mL) was taken instead of sample. DPPH (8mg/100 mL ethanol) (1mL) solution was added to the sample and tubes Sensory characteristics were left for 30 min (vortexed in between). Tubes were then The freshly prepared BPE stirred yoghurt was evaluated for centrifuged at 4000 rpm for 10 min. Supernatant (0.5 mL) was their sensory characteristics viz., color and appearance, body poured in fresh tubes and 1.0 mL ethanol to dilute the content. and texture, flavour, sweetness and mouthfeel scores based on Absorbance was measured at 517 nm by using UV- 9-point hedonic sensory score card (1=dislike extremely; 1800spectrophotometer (Shimadzu, Japan) against the 9=like extremely). Sensory evaluation was done using 8 ethanol. experienced panellists drawn from the Centre of Food Science and Technology, Banaras Hindu University, Varanasi, Uttar % DPPH inhibition = (AB-AS/AS) × 100 Pradesh (India) at room temperature. Where, AB = OD for blank, AS = OD for sample Results and Discussion Total Phenolic Content Determination Table 2 shows the actual values of the three factors obtained Determination of total phenolic content of sample was done from experimental design of CCRD along with corresponding by Folin-Ciocalteu Reagent method. Sample (200 mg) was rheological, sensorial and antioxidant properties scores of taken in centrifuge tube (in replications). Distilled water (0.3 BPE stirred yoghurt. Table 3 presents regression coefficients mL) was added and made volumeup to 0.5mL. In blank, and ANOVA of fitted quadratic models for textural, sensorial distilled water (0.2 mL) was taken instead of sample. Folin- and antioxidant properties scores of BPE stirred yoghurt. A Ciocalteau reagent (0.2 N) (2.5 mL) was added to the sample. quadratic model was fitted by numerical optimization After 1 min, Na2CO3 (1 mL of 7.5%) was added to the sample command of Design Expert software to optimize the levels of and tubes were left for 1 h (vortexed in between). Tubes were three factors (fat, bael polyphenol extract and sugar). The then centrifuged at 4000 rpm for 10 min. Supernatant (0.5 effect of changes in the levels of the factors on the mL) was poured in fresh tubes and diluted 10 times. rheological, sensorial and antioxidant properties scores are Absorbance was measured at 760 nmusing 1 cm cuvette by represented in (Figure 1 and 2).

Table 3: Regression coefficient and ANOVA of fitted quadratic models for textural, antioxidant characteristics and sensorial attributes of BPE stirred yoghurt

Responses Cons Coh IV % DPPH inhibition TPC C&A Flavour B&T Sweetness Mouthfeel Factors Intercept 332.39 6.75 7.76 78.23 33.83 7.49 7.36 6.83 7.31 7.84 Model F-value 3.46 3.48 16.39 3.64 10.77 3.87 10.86 11.02 13.09 3.21 Milk fat (A) 1.70NS 0.24* 0.15 NS 0.13 NS 0.35 NS -0.20* 0.22** 0.64*** 0.00NS -0.12NS Extract (B) -3.57NS -0.02NS 0.10 NS 7.09*** 5.95*** 0.10NS 0.62*** 0.28** 0.03NS 0.17NS Sugar (C) 21.98*** -0.03NS 0.83*** -0.03 NS 0.53 NS 0.06NS 0.03NS 0.00NS 0.66*** -0.16NS

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AB -6.32NS -0.09NS 0.2 NS 0.45 NS 2.47* 0.10NS 0.01NS 0.00NS -0.06NS 0.31* AC -0.62NS 0.33* 0.18 NS 2.15 NS -1.42NS -0.21* 0.01NS -0.31* 0.01NS -0.19NS BC -7.54NS -0.23NS 0.23* -0.72 NS 2.39* 0.03NS -0.11NS 0.00NS 0.06NS 0.31* A2 5.34NS 0.16NS -0.25** -0.72 NS -0.44 NS 0.19* -0.003NS 0.14NS 0.09NS -0.24* B2 -4.81NS 0.26* -0.09 NS -0.05 NS 0.02 NS -0.16* 0.19* 0.14NS 0.07NS -0.06NS C2 4.44NS -0.14NS -0.08 NS -0.90 NS -0.86 NS -0.03NS 0.07NS 0.09NS 0.20** -0.15NS R2 0.76 0.76 0.94 0.77 0.91 0.78 0.91 0.91 0.92 0.74 ADV 6.70 6.64 14.42 7.06 12.77 8.48 12.44 12.28 13.14 7.05 Lack of fit 0.12 0.30 0.19 0.71 0.60 0.41 0.22 0.24 0.34 0.12 NS: not significant, *: Significant (p<0.05), **: Significant (p<0.01), ***: Significant (p<0.001)

Effect of composition of ingredients on antioxidant DPPH of the product increases. Chouchouli et al. (2013) [22] activities (% DPPH inhibition) of BPE fortified stirred observed that grape seed extract fortified yoghurts exhibited yoghurt significantly higher radical scavenging capacity and ferric ion Antioxidant activity of BPE fortified stirred yoghurt varied reducing power (FRAP) than the respective controls. from 63.75% to 89.94% DPPH inhibition (Table 1). Model F Kaaraslan et al. (2011) [23] added grape extract and callus to value was significant (p<0.05), lack of fit was not significant improve the antioxidant potential of yoghurt. Similarly, Kale and ADV was more than 4 (Table 3), well fitted the model. At et al. developed pome yoghurt with improved antioxidant linear level, BPE had very high positive significant (p<0.001) potential Kale (2007) [24]. effect indicating that as the level of milk fat increases %

Design-Expert® Software

Index of viscosity A 9.26 B Design-Expert® Software 5.7 Cohesiveness 7.89 X1 = B: Extract X2 = C: Sugar 6.07 Actual Factor 9.3 X1 = A: Milk Fat A: Milk Fat = 3.50 X2 = C: Sugar

Actual Factor 8.4 B: Extract = 0.35

7.5 7.5

7.125 6.6 6.75

6.375 5.7 Index of viscosity

6 Cohesiveness 4.50 15.00 0.50 15.00 13.75 0.42 4.00 12.50 0.35 13.75 3.50 C: Sugar 11.25 0.27 B: Extract 12.50 10.00 0.20 3.00 A: Milk Fat 11.25 C: Sugar 10.00 2.50

Design-Expert® Software Design-Expert® Software Total phenolic content Total phenolic content 44.85 44.85 D C 19.85 19.85 X1 = B: Extract X1 = A: Milk Fat X2 = C: Sugar X2 = B: Extract Actual Factor 45 Actual Factor 45 A: Milk Fat = 3.50 C: Sugar = 12.50 39.5 39.5

34 34

28.5 28.5

23

23 Total phenolic content

Total phenolic content 15.00 0.50 0.50 4.50 13.75 0.42 0.42 4.00 12.50 0.35 0.35 3.50 C: Sugar 11.25 0.27 B: Extract B: Extract 0.27 3.00 A: Milk Fat 10.00 0.20 0.20 2.50

Fig 1: Response plots showing effect of composition of ingredients viz., BPE, sugar and milk fat on A) cohesiveness B) index of viscosity C) TPC D) TPC of BPE fortified stirred yoghurt

Effect of composition of ingredients on total phenolic that at higher level there were increase in the TPC of the content of BPE fortified stirred yoghurt product and vice versa (Fig. 1c). Also, BPE and sugar have TPC of BPE fortified (BPE) stirred yoghurt varied from 19.85 positive significant (p<0.05) effect on the product quality % to 44.85 % (Table 1). Model F value was significant (Fig. 1d). Sun-Waterhouse et al. (2012) [31] reported that (p<0.05), lack of fit was not significant and ADV was more addition of apple polyphenol extract at the rate of 0.75% in than 4 (Table 3), well fitted the model. At linear level, BPE yoghurt on post fermentation, the actual total extractable had very high positive significant (p<0.001) effect indicating polyphenol extract was 60% of theoretical value of the total that as the level of milk fat increases TPC of the product apple polyphenol extract added to the low molecular pectin increases. At interaction level, milk fat and BPE have positive yoghurt. significant (p<0.05) effect on the quality of product indicating ~ 252 ~ The Pharma Innovation Journal

Effect of composition of ingredients on consistency of BPE had high negative significant (p<0.01) effect indicating that as fortified stirred yoghurt the level of milk fat increases index of viscosity of the Consistency refers to thickness of a fluid and it varied from product decreases and vice versa. High-heat treatment causes 298.45 to 387.65 g sec. for BPE fortified stirred yoghurt high degrees of whey protein denaturation (>50%), which is (Table 1). Model F value was significant (p<0.05), lack of fit associated with a marked increase in complex viscosity was not significant and adequate precision value (ADV) was Damin et al. (2009) [28]. more than 4 (Table 3), well fitted the model. At linear level, sugar had very high positive significant (p<0.001) effect Effect of composition of ingredients on colour and indicating that as the level of sugar increases consistency of appearance of BPE fortified stirred yoghurt the product increases. Similarly, Yadav et al. (2016) [16] Colour and appearance of the product ranged from 6.8 to 8.5 observed that the consistency of the grape peel extract (Table 1). Model F value was signifacant (p<0.05), lack of fit fortified yoghurt was increased with increasing concentration was not significant and adequate precision value (ADV) was of extract, milk fat and sugar at linear and interaction level. more than 4 (Table 3), well fitted the model. At linear level, milk fat had negative significant (p<0.05) effect indicating Effect of composition of ingredients on cohesiveness of that as the level of milk increases colour and appearance of BPE fortified stirred yoghurt the product decreases. At interaction level, milk fat and sugar Cohesiveness reflects the maximum capacity of deformation have negative significant (p<0.05) effect on the quality of the of a sample before break and it varied between 6.07 and 7.89 product indicating that at higher level there were decrease in g (Table 1). Model F value was significant (p<0.05), lack of the colour and appearance of the product and vice versa (Fig. fit was not significant and ADV was more than 4 (Table 3), 2a). At quadratic level, milk fat had positive significant well fitted the model. At linear level, milk fat had positive (p<0.05) effect indicating that as the level of milk fat and BPE significant (p<0.05) effect indicating that as the level of milk increases colour and appearance of the product increases. fat increases cohesiveness of the product increases (Fig. 1a). While, BPE had negative significant (p<0.05) effect on the At interaction level, milk fat and sugar have positive quality of the product. Addition of lyophilized bael extract to significant (p<0.05) effect on the quality of product indicating yoghurt gives slight yellowish colour to it which was more that at higher level there were increase in the cohesiveness of acceptable to panellists than plain yoghurt. Similar results the product and vice versa. At quadratic level, BPE had were found when date palm extract was fortified in yoghurt positive significant (p<0.05) effect on the product quality Kadam et al. (2010) [29]. indicating that at higher level there were increase in the cohesiveness of the product. Starch contributes to greater Effect of composition of ingredients on flavour of BPE cohesiveness of all forms of yoghurt. Addition of starch acts fortified stirred yoghurt as a stabilizer and resulted in increased cohesiveness of Flavour of the product ranged from 6.0 to 8.5 (Table 1). mango soy fortified yoghurt Lejko et al. (2007) [25] and Kumar Model F value was signifacant (p<0.05), lack of fit was not & Mishra (2004) [26] Increased protein interactions and significant and adequate precision value (ADV) was more protein–protein bonds increases the elastic character of the gel than 4 (Table 3), well fitted the model. At linear level, milk making yoghurt less susceptible to rupture Lankes et al. fat and BPE had positive significant (p<0.01) and (p<0.001), (1998) [27]. respectively effect indicating that as the level of milk increases flavour of the product increases. At quadratic level, Effect of composition of ingredients on index of viscosity BPE had positive significant (p<0.05) effect indicating that as of BPE fortified stirred yoghurt the level of BPE increases colour and appearance of the Index of viscosity of BPE fortified stirred yoghurt ranged product increases. Sensory scores increased with level of between 5.70 and 9.26 g sec. (Table 1). Model F value was stabilizer up to 0.4%, but addition of 0.6% resulted in lowered significant (p<0.05), lack of fit was not significant and ADV score because of development of unacceptable flavour at was more than 4 (Table 3), well fitted the model. At linear higher concentrations. Sensory scores increased with level of level, sugar had very high positive significant (p<0.001) effect stabilizer up to 0.35%, but addition of 0.5% resulted in an indicating that as the level of sugar increases index of undesirable flavour, especially in stored yoghurt Mehanna and viscosity of the product increases. At interaction level, BPE Mehanna (1989) [30]. Yadav et al.(2016) [16] also found that the and sugar had positive significant (p<0.05) effect indicating flavour score was increased with increasing conentration of that as the level of BPE and sugar increases index of viscosity milk fat and grape polyphenol extract. of the product increases (Fig. 1b). At quadratic level, milk fat

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Colour and appearance Body and texture The Pharma8.5 Innovation Journal 8.5 6.8 6

X1 = A: Milk Fat X1 = A: Milk Fat X2 = C: Sugar X2 = C: Sugar Design-Expert® Software 8.5 Design-Expert® Software Actual Factor Actual Factor B: Extract = Colour0.35 and appearance BodyB: Extract and texture = 0.35 8.5 8.5 8.5 8.125 6.8 6

X1 = A: Milk Fat X1 = A: Milk Fat 7.875 X2 = C: Sugar 7.75 X2 = C: Sugar 8.5 Actual Factor Actual Factor B: Extract = 0.35 B: Extract = 0.35 8.5 7.375 7.25 8.125

7.875 7 7.75 6.625

7.375 7.25

Colour and appearance 15.00

6 andBody texture 7 6.625 13.75 4.50 4.50 Colour and appearance 15.00 6

andBody texture 15.00 12.50 4.00 4.00 13.75 13.75 3.50 3.50 C: Sugar 4.50 12.50 4.50 11.25 15.00 12.50 3.00 4.00 3.00 4.00 A: Milk Fat 13.75 11.25 C: Sugar 3.50 C: Sugar 3.50 10.0011.25 2.50 A: Milk Fat 12.50 10.00 2.50 3.00 11.25 3.00 A: Milk Fat C: Sugar 10.00 2.50 A: Milk Fat 10.00 2.50

Design-Expert® Software Design-Expert® Software Mouthfeel Design-Expert® Software 8 Design-Expert®Mouthfeel Software Mouthfeel 8 8 Mouthfeel 6 8 6 6 X1 = A: Milk Fat 6 X2 = B: ExtractX1 = A: Milk Fat X1 = B: Extract X2 = B: Extract X1 = B: Extract X2X2 = = C: C: Sugar Sugar Actual FactorActual Factor C: Sugar = 12.50C: Sugar = 12.50 8 8 ActualActual Factor Factor A:A: Milk Milk Fat Fat = 3.50= 3.50 8 8 7.725 7.725 7.725 7.45 7.725 7.45 7.45 7.175 7.45 7.175 7.175

Mouthfeel 6.9 7.175 Mouthfeel 6.9

Mouthfeel 6.9 Mouthfeel 6.9 0.50 15.00 0.50 4.50 0.50 0.42 13.75 4.00 15.00 0.42 0.50 4.50 0.35 3.50 12.50 0.42 13.75 0.35 4.00 0.42 B: Extract 0.27 3.00 11.25 0.27 0.35 A: Milk Fat C: Sugar B: Extract 0.20 2.50 3.50 12.50 0.35 10.00 0.20 B: Extract 0.27 3.00 11.25 0.27 A: Milk Fat C: Sugar B: Extract 0.20 2.50 10.00 0.20

Fig 2: Response plots showing effect of composition of ingredients viz., BPE, sugar and milk fat on A) color and appearance B) Body and texture C) Mouthfeel D) Mouthfeel fortified stirred yoghurt

Effect of composition of ingredients on body and texture than 4 (Table 3), well fitted the model. At linear level, sugar of BPE fortified stirred yoghurt had very high positive significant (p<0.001) effect indicating Body and texture of the product ranged from 6.75 to 8.75 that as the level of milk increases sweetness of the product (Table 1). Model F value was signifacant (p<0.05), lack of fit increases. At quadratic level, sugar had positive significant was not significant and adequate precision value (ADV) was (p<0.01) effect indicating that as the level of sugar increases more than 4 (Table 3), well fitted the model. At linear level, sweetness of the product increases. BPE and milk fat had positive significant (p<0.01) and (p<0.001), respectively effect indicating that as the level of Effect of composition of ingredients on mouthfeel of BPE milk increases flavour of the product increases. At interaction fortified stirred yoghurt level, milk fat and sugar had negative significant (p<0.05) Mouthfeel of the product ranged from 6.0 to 8.0 (Table 1). effect indicating that as the level of milk fat and sugar Model F value was signifacant (p<0.05), lack of fit was not increases colour and appearance of the product decreases significant and adequate precision value (ADV) was more (Fig. 2b). Kumar and Mishra (2004) [26] found that body and than 4 (Table 3), well fitted the model. At interaction level, texture were significantly affected by the amount of stabilizer milk fat and BPE had positive significant (p<0.05) effect added to yoghurt such as gelatin and starch. The structure of indicating that as the level of milk fat and BPE increases fruit yoghurt can be improved by using stabilizing agents, mouthfeel of the product increases (Fig 2c). While, BPE and such as starches, gelatin or pectin. sugar also had positive significant (p<0.05) effect on the quality of the product as indicating that as the level of BPE Effect of composition of ingredients on sweetness of BPE and sugar increases mouthfeel score of the product (Fig.2d). fortified stirred yoghurt At quadratic level, milk fat had positive significant (p<0.05) Sweetness of the product ranged from 6.65 to 8.75 (Table 1). effect indicating that as the level of milk fat increases Model F value was signifacant (p<0.05), lack of fit was not mouthfeel score of the product increases. significant and adequate precision value (ADV) was more

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Table 4: Actual response values observed against predicted response 8. Lmbole VB, K, Kumar U, Bhatt SP, Gajera V. values for BPE stirred yoghurt Phytopharmacological properties of Aegle Marmelo sas a

Responses Predicted Observed potential medicinal tree: an overview. International Consistency (gs) 315.37 320.79±0.44 Journal of Pharmaceutical Sciences Review and Cohesiveness (g) 7.10 7.82±0.52 Research. 2010; 5(2):67-72. Index of viscosity (gs) 6.55 7.24±0.57 9. Raja SB, Murali MR, Malathi GK, Anbarasu K, Devaraj % DPPH inhibition 82.84 83.70±0.78 SN. Effect of aqueous extract of Aeglemarmelos fruit on TPC (mg/100g) 39.82 40.55±0.34 adherence and β-lactam resistance of Enteropathogenic Colour and appearance 7.61 7.55±0.44 Escherichia coli by down regulating outer membrane Flavour 8.54 8.45±0.52 protein C. American Journal of Infectious Diseases. Body and texture 8.43 8.50±0.57 2009; 5:154-162. Sweetness 6.91 7.00±0.78 10. Badam L, Bedekar SS, Sonawane KB, Joshi SP. In vitro Mouth feel 7.78 7.75±0.34 antiviral activity of bael (Aeglemarmelos Corr) upon human coxsackieviruses B1–B6. Journal of Conclusion Communication Disorders. 2002; 34:88-99. Addition of BPE powder in yoghurt increased its antioxidant 11. Singh PBV. Potential effect of Aeglemarmelos in DNBS potential in proportion to the level of BPE powder added. induced inflammatory bowel disease in experimental Texture profile of product was acceptable up to 0.50% level animals.http://www.pharmainfo.net/pppc03/potentialeffe of fortification of BPE powder. There was non significant ct-aegle-marmelos-dnbs-induced-inflammatory-bowel- difference in the predicted and observed values using t-test disease-experimenta lanimals. (2009) Accessed April 28 (Table 4). Optimized BPE fortified stirred yoghurt had 315.37 2012. g. sec consistency, 7.10 g cohesiveness, 6.55 g. sec. index of 12. Agrawal A, Verma P, Goyal P. Chemomodulatory effects viscosity, 82.84% DDPH inhibition antioxidant activity and of Aeglemarmelos against DMBA-induced skin 39.82 mg/100 g total phenolic content with the desirability of tumorigenesis in Swiss albino mice. Asian Pacific 0.78. The prepared product had good organoleptic properties Journal of Cancer Prevention. 2010; 11:1311-1314. when the product was prepared using optimized solution 13. Jagetia GC, Venkatesh P, Baliga MS. Fruit extract of having 0.5% BPE, 15% sugar and 4.5% milk fat. Aeglemarmelos protects mice against radiation-induced lethality. Integrative Cancer Therapies, 2004; 3:323-332. Acknowledgement 14. Singh R, Kumar R, Venkateshappa R, Mann B, Tomar, We gratefully acknowledge Late Dr. Alok Jha, Former SK. Studies on physico-chemical and antioxidant Regional Representative of International Livestock Research properties of strawberry polyphenol extract–fortified Institute for South Asian based in New Delhi, and stirred Dahi. International Journal of Dairy Technology. Coordinator of Centre of Food Science and Technology, 2013; 66(1):103-108. Banaras Hindu University (Varanasi), for his guidance in 15. Cakmakci S, Tahmas‐Kahyaoglu D, Erkaya T, Cebi K, this research work. 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Genetic honey addition on physicochemical, microbiological and analysis of Egyptian date (Phoenix dactylifera L.) sensory properties of set‐type yoghurt. International accessions using AFLP markers. Genetic Resources and Journal of Dairy Technology. 2017; 70(2):245-252. CropEvolution. 2005; 52(5):601-607. DOI:10.1111/1471-0307.12332 4. Espirito do Santo AP, Perego P, Converti A, Oliveira 18. Cossu M, Juliano CCA, Pisu R, Alamanni MCP. Effects MN. Influence of milk type and addition of passion fruit of enrichment with polyphenolic extracts from Sardinian peel powder on fermentation kinetics, texture profile and on physico-chemical, antioxidant and bacterial viability in probiotic yoghurts. LWT-Food microbiological properties of yoghurt. Italian Journal of Science and Technology. 2012; 47(2):393-399. Food Science. 2009; 21(4):447-459. 5. Charoensiddhi S, Anprung P. Bioactive compounds and 19. Huang D, Ou B, Prior RL. The chemistry behind volatile compounds of Thai bael fruit (Aeglemarmelos antioxidant capacity assays. Journal of Agricultural and (L.) Correa) as a valuable source for functional food Food Chemistry. 2005; 53(6):1841-1856. ingredients. International Food Research Journal. 2008; 20. Khuri AI, Cornell JA. Response surfaces designs and 15(3):287-295. analysis. Marcel Decker, New York, 1987. 6. Nagaraju N, Rao KN. A survey of plant crude drugs of 21. Nishino T, Shibahara-Sone H, Kikuchi-Hayakawa H, Rayalaseema, Andhra Pradesh. Indian Journal of Lshikawa F. Transit of radical scavenging activity of Ethnopharmacology. 1990; 29(2):137-158. milk products prepared by Millard reaction and 7. Maity P, Hansda D, Bandyopadhyay U, Mishra DK. Lactobacillus caseii strain shirota fermentation through Biological activity of crude extracts and chemical the hamster intestine. Journal of Dairy Science. 2000; constituents of bael, Aeglemarmelos (L.) Corr. Indian 83(5):915-922. 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