Received date: March 11, 2013; Accepted date: May 22, 2013; Published date: May 30, 2013
Citation: Shilpi A, Kumar P (2013) Effect of Yoghurt Cultures and Probiotic Cultures on Physicochemical and Sensory Properties of Mango Soy Fortified Probiotic Yoghurt (Msfpy). J Food Process Technol 4:239. doi:10.4172/2157-7110.1000239
Copyright: © 2013 Shilpi A, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Mango soy fortified probiotic yoghurt (MSFPY) was prepared by using blends of 78.3% toned milk, 14.5% soy milk and 7.2% mango pulp. Effect of yoghurt cultures Streptococcus thermophilus (ST), Lactobacillus bulgaricus (LB) and probiotic culture Bifidobacterium bifidus (BB), Lactobacillus acidophilus (LA) on physicochemical properties and sensory properties were studied using different inoculum levels as per Response Surface Methodology (RSM). The optimized concentration of cultures was found out to be 1.75%, 1.95%, 2.44% and 1.37% for ST, LB, BB and LA respectively which yielded an acceptable quality of MSFPY having acidity 0.73%, total solids 14.02%, syneresis 14.12% and scored 8.5 on hedonic rating.
Yoghurt; Soy; Probiotic; Fortification; Optimization; RSM
In recent years, there is an increase in consumer demand for a new range of dairy products, including yoghurts, which have functional properties and low fat content . As a result, yoghurt products with different flavors and colors especially designed for children have resulted in increasing the market share . Yoghurt is a coagulated dairy product obtained by the lactic acid fermentation of milk by bacteria i.e. Streptococcus thermophilus (ST), Lactobacillus delbrueckii ssp. bulgaricus (LB) [3,4]. Addition of these two cultures results in acidification of milk and synthesis of aromatic compounds [5,6].
Although microflora (Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus) have been found to be valuable for human as they help in maintaining health and nutrition, emphasis has been placed on developing of yoghurt containing probiotic cultures like Lactobacillus acidophilus (LA) and B. bifidus (BB) . The conception of probiotic came into focus in early 1900’s but the term “probiotic” was suggested by Lilly and Stillwell. Probiotic cultures are live microbial food ingredients that are beneficial for human health , which includes improvement of intestinal microbial balance which results in the inhibition of bacterial pathogens, reducing the risk of colon cancer, improving the immune system, lowering serum cholesterol levels , alleviation of lactose intolerance and nutritional enhancement .
Though yoghurt can be made from the milk of many animals, cow’s milk is the most commonly used. Soymilk is also a good alternative to dairy milk and is widely available in markets. In comparison to full fat cow’s milk, soymilk has a lower fat content, low saturated fat, no cholesterol and free of lactose [11,12]. In addition to this it has low carbohydrate content, provides a good source of protein, and contains higher amount of iron, thiamine, and niacin than cow’s milk. Soyyoghurt is one of the lactic fermented products that are obtained from soymilk . However, addition of soy milk produces a beany flavor. To mask the beany flavor of soy milk, sweeteners like mango pulp may be added as it provides sweetness to yoghurt  and increases its nutritional properties as it is high in β-carotene, vitamin B complex and also helps in improving dietary fiber and folic acid when added in yoghurt .
Quality of the yoghurt depends on the quality and composition of the applied bacterial cultures [16,17]. For obtaining therapeutic benefits, minimum level for probiotic bacteria in yoghurt suggested is 105-l06 viable cells per g of product [18,19]. In general, the properties of yoghurt such as acidity as well as the sensory properties which includes aroma compounds produced (diacetyl, acetaldehyde) and nutritional value are important features of the product. These aspects are altered by the chemical composition of the milk, the processing conditions, added flavors and the action of starter culture during the incubation period [20,21].
Selection of inoculum level in the blend is very important for preparation of mango soy fortified probiotic yoghurt (MSFPY) as it has influence on the acidity, whey separation of the product which are crucial aspects of quality in consumer acceptance of yoghurt. Therefore, this study was aimed to examine the effect of inoculum level of yoghurt bacteria (ST, LB) and probiotic bacteria (BB, LA) for optimum physicochemical and sensory characteristics. Response surface methodology was used for experimental design and data analysis.
Four freeze dried cultures (Streptococcus thermophilus NCDC 074 and Lactobacillus delbrueckii ssp. bulgaricus NCDC 009, Lactobacillus acidophilus NCDC 015, and Bifidobacterium bifidus NCDC 255) were obtained from National Dairy Research Institute, Karnal, India. Cultures were cultivated in 10% non-fat dry milk, autoclaved at 115°C for 15min, inoculated under sterile conditions, incubated at 42 ± 2°C and kept in refrigerator at 4 ± 1°C until used.
Toned milk containing 3% fat (Verka, Sangrur, India) and soybean were purchased from the local market. Cans of mango pulp (Kaytis, India) having 18% total solids, were purchased from the local market. Soymilk containing 8.2% total solids was prepared using wet grinding technique.
MSFPY was prepared using mixture of 78.3% toned milk, 14.5% soy milk and 7.2% mango pulp as suggested by Kumar and Mishra . The blend of milk and soymilk was pasteurized at 95°C for 5 min and cooled to 45°C. Mango pulp was then added into the mixture and blended thoroughly. Inoculation was done with different inoculums levels of yoghurt and probiotic bacteria and incubated at 42 ± 1°C for 4h. After incubation, the samples were kept under refrigeration at 8°C for 4h before performing analysis.
Different ratios of four cultures i.e. Streptococcus thermophilus (ST), Lactobacillus delbrueckii ssp. bulgaricus (LB), Lactobacillus acidophilus (LA) and Bifidobacterium bifidus (BB) were used for studying the combined effect of the independent variables i.e. ST, LB, BB and LA on the response variables i.e. physicochemical (acidity, syneresis, total solids) and sensory properties of MSFPY.
Analysis of physicochemical characteristics
Physicochemical properties of MSFPY such as acidity , syneresis  and total solids  were determined. Titratable acidity (%) was measured by titrating 10 g of sample with 0.1 N NaOH using phenolphthalein as indicator. In syneresis, 40 grams of sample was weighed and centrifuged for 10 min at 1000rpm and the supernatant recovered was weighed. For finding out total solids, 2gms of Zinc oxide was weighed in a beaker and kept in hot air oven at a temperature of 100 ± 2°C for 1 hour followed by cooling. 5 grams of yoghurt was then added into the beaker and stirred to mix zinc oxide and then kept in water bath at a temperature of 90°C for 30 min with frequent mixing. Then the beaker was placed in hot air oven at 100 ± 2°C for 3 hours. After cooling in desiccators, the weight of the beaker was taken and calculated the total solids.
Twenty six yoghurt samples comprising of 25 samples of MSFPY and one MSFY as control were prepared and kept at 8 ± 1°C before sensory evaluation. A sensory panel of 10 trained judges was formed from the Department of Food Engineering and Technology, S.L.I.E.T Longowal. The panelists rated the samples for different quality attributes on 9-point Hedonic scale for their appearance, color, body and texture and flavor.
Optimization of inoculum level, used for the preparation of MSFPY was done by RSM. The experimental plan consists of a four-factored (n = 4) factorial design with five levels. Levels of variation and codes of the independent variables of ST (X1), LB (X2), BB (X3) and LA (X4) for the preparation of MSFPY are presented in table 1.
|Variables||Codes||Levels (coded and real values)|
Table 1: Levels and codes of the independent variables for addition of inoculums of ST, LB, BB and LA.
As per central composite rotatable design (CCRD), thirty one experiments were performed with varying levels of cultures as shown in table 2. Response surface methodology  (RSM) was used to study the process and optimization of inoculum level of probiotic and yoghurt culture.
|No. of expts.||ST||LB||BB||LA|
|Real value (%)||Coded value (X1)||Real value(%)||Coded value (X2)||Real value(%)||Coded value (X3)||Real value (%)||Coded value (X4)|
Table 2: Real and coded values for independent variables proportions of Streptococcus thermophillus (X1), Lactobacillus bulgaricus (X2), Bifidobacterium bifidus (X3) and Lactobacillus acidophilus (X4) for the preparation of MSFPY.
A mathematical function, f, was assumed for describing the relationship between each of the response variables, Yi and the factors Xi, such as
Yi = f ( X1, X2, X3......) (1)
A second order polynomial equation of the following form was assumed to relate Ykij and Xi
where βko, βki, βkii, βkij are the regression coefficients and εkij is pure error. X’s are the coded independent variables related to real variables linearly. This equation was used to study the linear, quadratic and interactive effects of independent variables on the chosen response. Analysis for variance (Anova) was performed on experimental data to determine the effect of the independent variables, i.e. inoculum rate of ST, LB, BB and LA on the physicochemical and sensory properties of MSFPY. Statistical software, Design Expert (DX-6) have been used for the selection of appropriate model and the development of response surface models and multiple regression analysis was performed considering a full second order polynomial (SOP) model.
The results of physicochemical parameters i.e. acidity, total solids and syneresis obtained are given in table 3.
|Expt no.||Acidity (%)||TS (%)||Syneresis (%)||Sensory|
Table 3: Effect of culture addition on the physicochemical properties of MSFPY.
Effect of cultures on physicochemical properties of MSFPY
Acidity: Different experimental runs were performed according to CCRD and the results obtained were modeled according to a polynomial quadratic equation to identify the variables that affected significantly or non- significantly. The analysis of variance (ANOVA) was determined and is given in table 4. Determination coefficient (R2=0.91) illustrates that only 9% of the total variations are not described by the model. Adjusted determination coefficient (adjusted R2=0.89) is also high, which indicates the model is highly significant.
|Source||Sum of Squares||DF||Mean square||F-value||Prob>F|
|Lack of Fit||4.219E-003||19||2.220E-004||0.74||0.7112ns|
|Adj R-squared||0.90||Adeq precision||21.4|
Table 4: ANOVA showing the variables as a linear, quadratic and interaction terms on acidity of MSFPY.
The response surface equation for acidity obtained after eliminating the non-significant terms is:
Acidity = 0.90 + 0.06ST - 0.24LB - 0.0053BB - 0.06LA + 0.04LB2 - 0.04STLB + 0.10LBLA (3)
The probability value F is less than 0.05 which indicates that the model is significant. The first-order term coefficients in the equation indicated that the acidity of the MSFPY increased with increase in ST. According to Krueder et al.  lactic acid bacteria responsible milk acidification to pH 4.8 is predominantly thermophillic cocci. The acidification ability of S. thermophillus strains is a function of their carbohydrate metabolism. Negative sign of coefficients of linear term indicated decrease in acidity on addition of LB, BB and LA. According to Reyhan and Ufuk , using starter cultures with lesser amount of L. bulgaricus produces yoghurt with decreased acidity and has lesser chance of post acidification. Marshall and Tamime  have reported that ST and LB are fast acid producers as compared to LA and BB. According to Dave and Shah , the bifidobacteria under anaerobic conditions would decrease the pH of the sterile reconstituted skim milk. Shah  in his studies reported a decrease in pH values of commercial yoghurts containing L. acidophilus and Bifidobacterium bifidus during storage. The interaction term of ST and LB were highly significant having while the interaction terms of LB and BB were found to be significant. As desirable, the lack-of-fit for acidity is in significant. The 3-D graphs for acidity as a function of ST and LB, LB and LA are shown in figure 1.
Total solids: The total solids of MSFPY ranged from 13.16 to 14.98%. The second-order polynomial model fitted suitably with the observed data with a high correlation coefficient (R2) 0.73. The analysis of variable (ANOVA) after omitting the non-significant terms is given in table 5. The 3D plots for total solids as a function of LA and BB, ST and LA are shown in figure 2.
|Source||Sum of Squares||DF||Mean square||F-value||Prob>F|
|Lack of Fit||0.52||15||0.03||2.13||0.1798ns|
|Adj R-squared||0.87||Adeq precision||16.29|
Table 5: ANOVA table showing the variables as a linear, quadratic and interaction terms on total solids of MSFPY.
The response surface equation after removing the non-significant terms, for total solids is given as follows:
Total Solids =9.55+0.19ST+1.89LB+2.4479BB+2.76LA-0.32LB2-0.40BB2-0.65LA2-0.36LBBB-0.82BBLA (4)
The coefficients of the first-order term in the equation showed that the total solids of the MSFPY increased with the concentration of ST, LB, BB and LA. . In a research done by Akter et al. , the total solids and solids-not-fat, protein, carbohydrates content of dahi samples were significantly increased due to the increased level of sugar and culture. Purwandari et al.  in his study reported that S. thermophillus helps in the developement of yoghurt texture through exopolysaccharide (EPS) production which tends to increase the total solids. According to Haddadin , ST and LB have been reported to secrete exopolysaccharide which increases the total solids of the product. The negative coefficient of the interaction term (BB and LA) indicated a decrease in total solids of MSFPY with the addition of these variables. These results are similar with the findings of Istikhar  that the natural yoghurts contain higher acidities and total solids as compared to probiotic yoghurts.
Syneresis: The analysis of variance (ANOVA) after deleting the non-significant terms are given in table 6. The determination coefficient (R2 = 0.96) indicates that variance of only 4% are not explained by the model. The high value of the adjusted determination coefficient (adjusted R2 = 0.94) indicates that the model is highly significant.
|Source||Sum of Squares||DF||Mean square||F-value||Prob>F|
|Lack of Fit||7.45||13||0.57||0.55||0.8270ns|
|Adj R-squared||0.95||Adeq precision||30.3|
Table 6: ANOVA showing the variables as a linear, quadratic and interaction terms on syneresis of MSFPY.
The probability value F (shown in Table 7) is less than 0.05 which indicates that the model is significant.
|Source||Sum of Squares||DF||Mean square||F-value||Prob>F|
|Lack of Fit||0.40||14||0.028||0.88||0.6102ns|
|Adj R-squared||0.9228||Adeq precision||22.91|
Table 7: ANOVA showing the variables as a linear, quadratic and interaction terms on sensory evaluation of MSFPY.
Syneresis =36.31-10.86ST-19.28LB+16.21BB-6.68LA+3.58LT2+6.90LB2-5.76BB2-5.41LA2-7.13LBBB+5.38LBLA+9.29BBLA (5)
It was observed from the response surface equation that on increasing the concentration of ST, LB and LA the syneresis of MSFPY decreased while the addition of BB resulted in increase in syneresis. According to Thanut et al. , syneresis could be influenced by solids content and the type of starter culture used for the preparation of yoghurt. The increased total solid content of MSFPY due to the addition of soy might be responsible for decreased wheying off. The increased total solid increases yoghurt gel strength and thereby increases the density and reduces the pore size, with the result water is bound more firmly which increases the firmness of the yoghurt . The quadratic terms of ST and LB signified positive effect on syneresis while BB and LA had a negative effect. The 3-D graphs of syneresis as a function of BB and LB, LA and LB and LA and BB are shown in figure 3.
Sensory evaluation: The analysis of variance (ANOVA) after considering the significant terms is given in table 7.
Sensory = 0.94+2.89ST+4.66LB-1.13BB+3.16LA-1.01ST2-1.31LB2+0.50BB2+0.09LBBB-1.20LBLA-1.20BBLA (6)
Response surface equation depicted that on increasing the concentration of ST, LB and LA, the MSFPY was more acceptable to the sensory panel while increase in BB had a deleterious effect as the flavor and aroma of the dairy products cultured with Bifidobacteria are reportedly milder . The interaction terms of LB and BB indicated that increase in their levels had a positive effect on the flavor of yoghurt and this result is in accordance with the findings of Marshall and Arbuckle  who in their research reported that L. bulgaricus is responsible for the production of acetaldehyde which is the most important aroma compound in yogurt in the first 1 to 2h of incubation. LA either with LB or BB had an adverse effect on sensory properties of MSFPY as also reported by Shah , the addition of LA and BB resulted in increase in acidity of the product which was not acceptable. Also stated by Donkor et al. , probiotics are responsible for increasing the organic acid concentrations (lactic and acetic acid) and proteolysis during fermentation and storage of set yogurts. Also, addition of LA and BB had a negative effect on syneresis which may influence the overall acceptability of the product. The R2 is also high (0.95), which indicates a high significance of the model (Figure 4).
Optimization; The values for acidity, pH, total solids and syneresis as proposed by the final quadratic model along with their corresponding observed values, are shown in table 8. On comparing, an excellent agreement between the predicted and experimental data can be observed. The analysis of optimum as obtained by differentiation of the quadratic model, for achieving optimum acidity, total solids, syneresis and sensory properties was A=1.75, B=1.95, C=2.44, D=1.37. The predicted optimal acidity, total solids, syneresis and sensory properties corresponding to these values were 0.72%, 13.95%, 14.27%, 8.66 respectively.
|Variable||%age||Acidity (%)||Total solids (%)||Syneresis (%)||Sensory|
Table 8: Predicted values vs experimental values for optimum acidity, total solids, syneresis and sensory of MSFPY.
Additional experiments using these optimized inoculums levels were performed in triplicates to verify the accuracy of the model for predicting optimum acidity, total solids and syneresis and sensory properties. These triplicate experiments yielded an acidity, total solids, syneresis and sensory scores of 0.73%, 14.02%, 14.12% and 8.50 respectively. The good compliance between the experimental and predicted values confirms the reliability of the model as well as the existence of optimal point.
Effect of yoghurt cultures and probiotic cultures on physicochemical and sensory properties of MSFPY was studied by using different inoculums levels as per RSM. The optimum values of yoghurt bacteria namely ST and LB and probiotic bacteria namely BB and LA were 1.75%, 1.95%, 2.44% and 1.37% respectively which yielded MSFPY of good and acceptable quality.