BIOCHEMICAL, RHEOLOGICAL, and SENSORY CHARACTERISTICS of NON-FAT SET YOGURT SUPPLEMENTED with A MIXTURE of HYDROCOLLOIDS

sensory characteristics


INTRODUCTION
Consumers' awareness of how diet effects the health and their demand for healthy foods has encouraged the food industry to produce healthy food products (Annunziata & Pascale, 2009). Nowadays, obesity is a major health problem in both Western and developing countries that may lead to chronic diseases (Losasso et al., 2012; Pieniak, Pérez-Cueto, & Verbeke, 2009). Limiting the intake of fat through lowering the fat content of foods is a way to avoid overconsumption of this ingredient in the diet, thereby formulating low-calorie products and preventing obesity (Modzelewska-kapitula & Klebukowska, 2009). Yogurt is a highly-consumed dairy product with textural and rheological characteristics, which are substantial for consumer acceptability (Paseephol, Small, & Sherkat, 2008;Vélez-Ruiz, 2019). The presence of fat in dairy products has a considerable effect on their physical, rheological, and textural properties. In addition, fat influences other characteristics; for instance, appearance, flavor, and mouth feel which affect overall acceptance of the product (Rybak, 2016). The separation of whey protein (syneresis) and variations in viscosity has become a topic of great concern in yogurts, particularly in low fat yogurts. Accordingly, the characteristics of low and non-fat yogurt are impacted by reducing fat content (Dai, Corke, & Shah, 2016). In other words, low-fat and non-fat yogurts have low total solids and exhibit several defects such as lack of flavor, weak body, poor texture and syneresis (Aziznia, Khosrowshahi, Madadlou, Rahimi, & Abbasi, 2009; Nguyen, Kravchuk, Bhandari, & Prakash, 2017). Several methods have been suggested to overcome these adverse effects of low and non-fat yogurt, including adding certain dairy ingredients and hydrocolloids, an appropriate choice of starter cultures, the addition of thickeners, enhanced total solids concentration, and modification of processing parameters. Carbohydratebased fat replacers can mimic the functional properties of fat in the product while reducing the caloric value of foods (Guven, Yasar, Karaca, & Hayaloglu, 2005). It has been reported that exopolysaccharide (EPS) produced by some starter cultures can affect the end product quality, including texture, sensory and waterholding capacity of yogurt (Han et al., 2016). The addition of thickeners (polysaccharides or gelatin) leads to new cross-links in the network and increases the rigidity of the gel and its water holding capacity. Also, adjusting the total solid and protein levels can increase apparent viscosity and viscoelasticity of yogurt up to two or three times. Different processing parameters, including heat treatment, homogenization, shearing and acidification can change the mechanical, texture attributes and microstructure of yogurt (Tan, 2019). Shokrollahi Yancheshmeh et al. worked on Vicia villosa, as a good source of protein, fiber, and minerals. They . The objective of this study was to elucidate the effect of the application of a mixture of some fat replacers including inulin, whey protein isolate, starch, and gelatin at various levels on physicochemical, rheological, and sensory properties of non-fat set yogurt during storage.

Study design and sample preparation
Six yogurt samples containing inulin, whey protein isolate, modified starch (0.3, 0.5, 1%), and gelatine (0.2%) in three replications were formulated using skim milk powder reconstituted in sterilized distilled water to obtain a solution of 12% (w/w) total solid non-fat (Table 1). The hydrocolloids were subjected to rehydration 24 h before adding to milk. Control samples with 12% (w/w) total solid, 3% milk solid not fat, and without the inclusion of stabilizers were prepared. The samples were exposed to heat treatment at 90°C for 15 min. After heat exposure, the samples cooled in an ice bath and inoculation of starter culture, according to the instruction of the manufacturer, at 42°C until pH 4.5±0.02 was performed. The ultimate samples were quickly cooled and kept at 5°C for 28 days. Biochemical parameters, including changes in pH, acidity, and redox potential were determined during fermentation. These parameters were recorded at 30-minute time intervals. Other features, including rheological properties, syneresis, and sensory characteristics were recorded every 7 h.

Chemical analysis
Titratable acidity (TA) (as % lactic acid) was measured every half hour during fermentation and every 7 days during refrigerated storage and determined according to the method adopted by the Association of Official Analytical Chemists (AOAC) 947.05 using 0.1 M NaOH (AOAC 1999

Synersis measurement
Inoculated milk samples, prepared using the procedure described above, were fermented in test tubes with the same geometry and height at 42°C. The initial height of yogurt in the test tube and the height of the drained liquid were recorded during refrigerated storage. The degree of syneresis was represented as a percentage. % Syneresis= [height of separated serum/initial height of yogurt in tubes] × 100

Rheological measurements
To monitor the rheological characteristics of yogurts, dynamic oscillatory shear testing was performed using a rheometer (Anton Paar, MCR 301, Graz, Austria). The temperature was set to 4±0.01°C before running rheological experiments. For each sample, a frequency sweep was executed with a frequency range between 0.01 and 100 Hz at a constant strain of 0.5%. The rheological parameters measured were elastic modulus (G΄), viscous modulus (G˝), loss tangent (tan δ = G˝/G΄), complex modulus (G*) and crossover point calculated using the Rheoplus/32 software (version V3.21). Triplicate measurements were performed for each sample and the power law model satisfactory fitted the experimental data for each sample with a correlation coefficient (R 2 ) of at least 0.95. The strain sweep with strain varied from 0.01 to 1000% at a constant frequency of 1 Hz was done for each sample to define the linear viscoelastic range (LVE) and to determine above-mentioned moduli

Sensory evaluation
Sensory analysis was carried out by a panel of 30 assessors, all with previous experience in dairy products evaluation. The sensory properties included flavor, oral texture, appearance, non-oral texture (texture smoothness and scoopability), and overall acceptability. Each of these characteristics was scored on a five-point scale: 0= inconsumable; 1= unacceptable; 2= acceptable; 3= pleasant or satisfactory and 4= excellent. The samples were randomly numbered by three-digit coding and the sensory panel evaluated the coded yogurts. All sessions were carried out in a sensory laboratory with separate booths.

Statistical analysis
Analysis of variance (ANOVA) was conducted on the resulting data using Duncan's multiple range test to compare treatment means. The SPSS V 17 was used and the significance was defined at P < 0.05. The experiments were executed in triplicates.

Biochemical characteristics
As seen in Table 2, control samples presented the lowest pH during refrigerated storage. This can be ascribed to the greater level of lactose due to the additional amount of skim milk compared to other treatments. Addition of 1% inulin or 1% modified starch and 1% inulin had no significant effect on pH change at the end of fermentation. Guven et al. (2005) announced that the incorporation of inulin at different levels into low-fat set yogurt did not influence on pH of yogurts (  Control yogurts had the highest titratable acidity at the end of fermentation and during refrigerated storage due to the higher content of lactose and the generation of a higher amount of acid during fermentation. The lowest titratable acidity was observed in T5 and T6 during storage (Table 3). Paseephol et al. (2008) assessed the effect of inulin with different chain lengths on non-fat set yogurt and declared that the level and chain length of inulin had no effect on the titratable acidity of yogurt samples (Paseephol et al., 2008).  As shown in Table 4, at the end of fermentation, no significant difference regarding redox potential was observed among samples. The redox potential was increased in all of the samples during refrigerated storage. On day 28 of storage, control samples had the highest redox potential, while samples containing 1% whey protein showed the lowest values. There was no significant difference between the samples containing 1% inulin (T1and T2) and the ones containing 1% modified starch (T3 and T4). *Means shown with different small and capital letters represent significant differences (p <0.05) in the same columns (among the treatments) and rows (between the two day in each treatment), respectively. **B= control samples without hydrocolloids T1= 1% inulin, 0.5% starch, 0.3% whey protein and 0.2% gelatin T2= 1% inulin, 0.3% starch, 0.5% whey protein and 0.2% gelatin T3= 1% starch, 0.5% whey protein, 0.3% inulin and 0.2% gelatin T4= 1% starch, 0.5% inulin, 0.3% whey protein and 0.2% gelatin T5= 1% whey protein, 0.5% inulin, 0.3% starch and 0.2% gelatin T6= 1% whey protein, 0.5% starch, 0.3% inulin and 0.2% gelatin.  *Means shown with different small and capital letters represent significant differences (p <0.05) in the same columns (among the treatments) and rows (between the two day in each treatment), respectively. **B= control samples without hydrocolloids T1= 1% inulin, 0.5% starch, 0.3% whey protein and 0.2% gelatin T2= 1% inulin, 0.3% starch, 0.5% whey protein and 0.2% gelatin T3= 1% starch, 0.5% whey protein, 0.3% inulin and 0.2% gelatin T4= 1% starch, 0.5% inulin, 0.3% whey protein and 0.2% gelatin T5= 1% whey protein, 0.5% inulin, 0.3% starch and 0.2% gelatin T6= 1% whey protein, 0.5% starch, 0.3% inulin and 0.2% gelatin.
The highest pH drop rate during the storage was obtained in control yogurts, while the lowest was reported in T5 and T6. pH drop rate did not differ significantly in T1, T2, T3, and T4 which indicates that the addition of the hydrocolloids at these levels was not effective on the fermentation process during storage (Table 5). In a similar trend, Radi et al. (2009) reported that the incorporation of wheat-modified starch into low-fat yogurts had no impact on biochemical parameters in comparison with control samples (Radi et al., 2009). The highest titratable acidity increase rate, as well as redox potential increase rate, was observed in control yogurts. Whereas the lowest values were obtained in T5 and T6 which can be imputed to the buffering effect of whey protein in these treatments (Table 5).

Strain sweep test
Storage modulus (G′) and loss modulus (G″) on day 0 have been shown inFig.
(1). According to the viscoelastic properties, all samples indicated weak gel behavior with storage modulus (G′) higher than loss modulus (G″  As can be seen in Table 6, treatments are significantly different regarding yield stress (τf). Samples containing 1% modified starch showed the highest values followed by T5, T6, and control samples. The lowest τf was reported in yogurts containing 1% inulin. According to Heydari et al. (2009), who evaluated the effect of prebiotics addition on probiotic yogurt, samples with 3% starch showed the highest τf value, which is in consistency with our finding. By increasing the modified starch level, a part of starch would be adsorbed on the casein micelle surface through electrostatic repulsion and the hydrophilic chains of polymer that forms a thick adsorption layer resulting in the decline of zeta potential and increasing the stability of the yogurt system (Cui, Lu, Tan, Wang, & Li, 2014).
Assessing crossover point (G′=G″) indicated that T3 had the highest value, followed by T5, T6, control, T2, and T1. Monitoring viscoelastic range (LVE) shows that the greatest value belongs to T6, followed by T5, T3, T4, control, T2, and T1 (Table 6). It is worth noting that G′ and G″ on day 28 and during refrigerated storage were enhanced in all treatments compared to day 0 (Fig. 2).

Figure 2
Storage and loss moduli in yogurts as a function of strain on days 0 and 28. Fig. 3 illustrates the changes of storage modulus (G′) and loss modulus (G″) in a frequency range of 0.628-314 rad/s. In all treatments, G′ was higher than G″, indicative of a more elastic feature of the samples than a viscous feature. Fig. 3 shows that T5 and T6 had the greatest G′ compared to other treatments, while T1 and T2 demonstrated the lowest values. Generally, n, b, and tan are altered with frequency ( ) according to the power law model (G′= k n ).

Figure 3
Storage and loss moduli in yogurts as a function of frequency on day 0.
According to Table 7, there is a significant difference among treatments in respect of the factors 'nʼ and 'kʼ. The highest values were recorded in T5 and T6 while the lowest values were observed in T1 and T2. Furthermore, T5 and T6 had the highest 'k' followed by control yogurt, T3, T4, T2, and T1, respectively. The higher 'k' factor is an indication of a strong gel structure, whereas with increasing 'n' factor; samples exhibit characteristics of a gel with higher sensitivity to mechanical stresses (Steffe, 1996).
In treatments of control, T1 and T2, the syneresis was increased till day 21 but declined from day 21 to 28. In treatments of T3, T4, T5, and T6, no syneresis was observed at the end of fermentation and during storage. In the samples containing 1% whey protein, syneresis decreased due to the reduction of casein to whey protein ratio. Accordingly, Remeuf et al. (2003) announced that by increasing the whey protein level in yogurt, gel strength would increase and subsequently, a decrease in syneresis would be observed (Remeuf, Mohammed, Sodini, & Tissier, 2003). Puvanenthiran et al. (2002) reported that by reducing the ratio of the casein to whey protein, the protein network became finer, the size of the aggregates became smaller, the pores smaller, and the network of cross-links denser, which entraps water leading to lower whey drainage (Puvanenthiran, Williams, & Augustin, 2002). It was pointed that starch can absorb water and reduce the whey separation in yogurt (Radi et al., 2009). In T1 and T2, syneresis was lower compared to control yogurts but was not reduced entirely. This is in agreement with the data obtained by Heydari et al. (2011) and Vasiljevic et al. (2007) that ascribed this phenomenon to the presence of long chain polysaccharides. These polysaccharides could interfere with the development of a three-dimensional casein structure, leading to the formation of a weaker gel with less water retention (

Sensory analysis
Sensory evaluation data for oral texture, non-oral texture (texture smoothness and scoopability), flavor, appearance, and overall acceptability are shown in Table 8. T5 and T6 received the highest score, and T2 obtained the lowest score regarding oral texture at the end of fermentation and there was no significant difference between these treatments and T4 as well as control samples. It can be elucidated that higher protein content improves the texture of non-fat yogurt, but greater concentrations of inulin or starch compared to whey protein have no effect on the oral texture of the samples. Likewise, Radi et al. (2009) implied that increasing the starch level from 1.6% to 3.2% improved the sensory characteristics of low-fat yogurt (Radi et al., 2009). On day 28, the highest and the lowest scores in this regard were attributed to T6 and T2. In a study, the effect of inulin and agav fructans addition on microstructural, rheological, and sensory characteristics of reduced-fat stirred yogurt was investigated (Crispin-Isidro et al., 2015). It was reported that inulin at the level of 4% could mimic the sensory perception of the full-fat yogurt; while in the present study utilization of 1% inulin in non-fat yogurt had no remarkable effect on product acceptability. In control samples, the oral texture acceptability were reduced on day 28 compared to day 0, which can be ascribed to the increment of acidity and low pH values in these products. Treatments T1 and T2 received the lowest score on day 0 concerning non-oral texture. These two treatments showed high syneresis values that justify their low acceptability by panelists. Control samples had the highest acceptability from this point of view. On day 28, T5 and T6 showed the highest acceptability in terms of non-oral texture, which was consistent with the higher values of rheological parameters (storage, loss, and complex moduli), while T1 and T2 received the lowest acceptability in this context. The highest flavor acceptability on days 0 and 28 were recorded in T5 and T6 and the lowest was observed in T3 and T4. T1 and T2 were more favorable by the assessors on day 28 compared to day 0 because of the impact of inulin on masking the sour taste of acid (Meyer & Blaauwhoed, 2009). The least acceptable samples in respect of the appearance on day 0 were T1 and T2 because of higher syneresis values in these samples, while no significant difference was observed between other treatments, as well as control samples. On day 28, the highest scores were ascribed to T3, T4, T5, and T6 followed by control yogurts and the lowest ones were observed in T1 and T2. Regarding overall acceptability, the highest scores on day 0 were reported in T5 and T6 and the lowest scores were obtained in the case of T1 and T2. Pang et al. (2016) examined the effect of whey protein along with xanthan, starch, and carrageenan on rheological and sensory properties of yogurt and reported that yogurt samples containing whey protein were more acceptable in comparison with other treatments (Pang, Deeth, Prakash, & Bansal, 2016). On day 28, T5 and T6 received the highest scores followed by control sample and T1, T2, T3, and T4 received the lowest ones.

CONCLUSIONS
In this study, the effect of the mixture of hydrocolloids on biochemical, rheological, and sensory characteristics of non-fat set yogurt was investigated. The results revealed that a higher concentration of whey protein compared to modified starch and inulin prevents the increase of acidity and pH decrease and subsequently defects of yogurt texture and sensory properties of non-fat yogurt during refrigerated storage. Moreover, 1% whey protein improved the rheological characteristics of yogurt samples. The highest values of G′ were obtained in T5 and T6 (maximum whey protein content) on day 0. G′ and G″ on day 28 and during refrigerated storage were enhanced in all treatments compared to day 0. Sensory evaluation data for oral texture, non-oral texture (texture smoothness and scoop ability), flavor, appearance, and overall acceptability revealed that T5 and T6 obtained the highest score and T2 received the lowest score regarding oral texture at the end of fermentation. T5 and T6 were the most acceptable on days 0 and 28 of storage. Treatments containing 1% whey protein and 1% modified starch showed no syneresis at the end of the fermentation and during the storage period, while 1% inulin could not inhibit the syneresis completely. It can be concluded that the best mixture of hydrocolloids as fat replacer regarding biochemical, rheological, and sensory properties is 1% whey protein, 0.5% starch, 0.3% inulin, and 0.2% gelatin.