Effect of Different Milk Composition on Physico-Chemical Characteristics of Set Type Yoghurt

The gross composition of milk may affect the textural, compositional and microbiological properties of yoghurt. Sahiwal, Holstein Friesian milk procured from Dairy Animal Training and Research Center, B-block University of Veterinary and Animal Sciences, Pattoki and Nili Ravi buffalo milk procured from Nili Ravi buffalo Research Institute (BRI), Pattoki. All other ingredients purchased from Sigma Aldrich Germany through local suppliers. Milk was standardized to 15% total solids with 3.5% fat. Skim Milk Powder (SMP) was used for the standardization of the yogurt. Milk was pasteurized at 82 o C for 5 minutes and cool down to 43 o C and at this temperature starter culture was added. Yogurt batch incubated at 43 o C for 3 to 3.5 h. During this period pH was monitored regularly and as the pH drops to 4.6, the batch was shifted to blast chilling at 1-2 o C and then stored at 4 o C. Sensory evaluation of all yogurt samples was carried out on a 9-point hedonic scale. All the tests were carried out on day 0, 7, 14 and 21. The collected data was investigated through ANOVA technique under complete randomized design (CRD) using SAS 9.1 software. The results showed that yoghurt made from Nili Ravi buffalo milk (T o ) showed a significant ( p ≥ 0.5) overall acceptability as compared to other treatments. Yoghurt made from Nili Ravi buffalo milk (T o ) were followed by T 3 , T 4 , T 1 and T 5 respectively. The Nili Ravi buffalo milk showed higher acceptability to the consumers.


INTRODUCTION
P akistan is the 4 th largest milk producing country in the globe; most of the milk is produced by cattle and Nili Ravi buffaloes, with some contribution from other milk breeds as well. Milk production in Pakistan has been increased by more than 40% in the last 10 years with a total production of 54,328 thousand tons. The Nili Ravi buffalo milk and cow milk contribution in the total milk production in Pakistan is 60.9% and 35.7%, respectively (GOP, 2018-19). Despite of gradual increase in demand of milk and milk products the dairying community of Pakistan is facing multiple problems. Globally, surplus milk is converted into numerous value-added products, but only few of these milk products are prepared in Pakistan.
Nili Ravi buffalo contributes approximately 60% of the total milk produced in Pakistan (GOP, 2018-19). The main reasons for popularity of Nili Ravi buffalo milk is due to its higher fat content (~6.5%) as well as solids-notfat (~10.5%). The local culture of making Lassi, tea and dahi using Nili Ravi buffalo milk renders it a good choice. Nili Ravi buffalo milk is particularly rich in vitamin A and lacks in carotene. This is the reason of whiter color of Nili Ravi buffalo milk as compared to cattle milk. Nili Ravi buffalo milk is highly suitable for the manufacturing

O n l i n e F i r s t A r t i c l e
of large number of value-added dairy products including fermented milk products such as yoghurt, lassi and cheese (Moioli et al., 2006). In current industrial process of procuring the fermented products, cow milk is predominant world-wide. Variety of fermented milk products including fruit, flavored, plain yoghurt, whipped, drinking type, smoked, dried, strained and frozen products are prepared from cow milk globally. The total solids contents of cow milk are less than Nili Ravi buffalo milk therefore to have proper curd consistency of fermented products, cow's milk is generally fortified with skim milk powder (White et al., 2008).
Fermented milk products that are made from various milk sources, originated in the Middle East, perhaps before the Phoenician era, and spread throughout Europe. Human diet could be supplemented with fermented dairy products that can eventually provide essential nutrients vital for growth and better health (Serhan et al., 2016). Haj et al. (2007) reported that acidifying the milk through fermentation is the oldest methods of preservation of milk. Fermentation can be attained through chemical and microbiological methods. The variation in fermentation processes results in the production of various fermented dairy products such as keifr, kumiss, acidophilus milk, yoghurt etc.
Yogurt is a coagulated milk, mostly obtained by the action of two bacteria, which are Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus (Trachoo, 2002). Yogurt is made of milk, considered as a complete food. It is mostly consumed by infants or elder people. It contains all nutrition required by the people, along with this also contain live microbes (Ayar et al., 2006).
Yoghurt having health benefits like enhancing lactose digestion by maldigesters, protection against gastrointestinal upsets, lowering the risk of cancer, improving immune response, decreasing the blood cholesterol, calcium and iron, helping the body to assimilate protein, diarrhoea protection, longevity and control. It also helps in maintenance of GIT (gastrointestinal) microflora as well (Andronoiu et al., 2011;Vahedi et al., 2008;Iwalokun and Shittu, 2007;Foda et al., 2007;Aly et al., 2004). Other health benefits of yoghurt include improvement in the bowel movement, enhancement of GIT (gastrointestinal) function, consumption of different constituents in the GIT. These are most likely due to the action of gut microflora and also improving immune response (Adolfsson et al., 2004). Consumers (children, young and aged person) mostly demand novel product with the formulation of nutrition in plain yoghurt.

Experimental site
The research was carried out in postgraduate laboratory of Department of Dairy Technology and Central Laboratory Complex (CLC), Ravi campus, University of Veterinary and Animal Sciences, Pattoki, District Kasur, Pakistan.

Procurement of milk
Sahiwal, Holstein Friesian (HF) milk was procured from Dairy Animal Training and Research Center, B-block University of Veterinary and Animal Sciences, Pattoki and Nili Ravi Nili Ravi buffalo milk procured from Buffalo Research Institute (BRI), Pattoki. All other ingredients were purchased from Sigma Aldrich Germany through local suppliers. The experiment and analysis of the samples were carried out in the Department of Dairy Technology and CLC, A-Block, University of Veterinary and Animal Sciences, Pattoki.

Procurement of ingredients
Starter culture, Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus, was purchased from Danisco, Sweden through local suppliers. All other ingredients (stabilizers) were procured from local market, Lahore.

Preparation of starter culture
The freeze-dried culture was propagated by inoculation into fresh milk which was heated at 90°C for 3 minutes. The inoculated milk incubated at 45°C until pH 4.6 is reached and then Stored overnight at 4°C.

Preparation of yogurt
Standardized milk was used having 15% total solids and 3.5% fat. Skim Milk Powder (SMP) was used for the standardization of the yogurt. Milk was pasteurized at 82 o C for 5 minutes and cool down to 43 o C and at this temperature starter culture was added. Yogurt batch incubated at 43 o C for 3 to 3.5 h. During this period pH was monitored regularly and as the pH drops to 4.6, the batch was shifted to blast chilling at 1-2 o C and then stored at 4 o C.

Experimental design
A total of 6 treatments were prepared and 100% Nili Ravi buffalo milk yogurt was used as control (T o ). T 1 was prepared from Sahiwal milk (100%), T 2 was prepared from 100% Holstein Friesian milk, T 3 was prepared from 50% Nili Ravi and 50% Sahiwal milk, T 4 was prepared from 50% Nili Ravi and 50% Holstein Friesian milk while O n l i n e

F i r s t A r t i c l e
T 5 was prepared from 50% Sahiwal and 50% Holstein Friesian milk. Yogurt was stored at 4 o C for three weeks and analyzed at day 0, 7, 14 and 21.

Physico-chemical determination
Physico-chemical characteristics of all the treatments were carried out for protein, total solids, fat content, pH and titratable acidity according to the standard methods of Association of Analytical Chemists (AOAC, 1999).

Textural properties
Textural properties of all the treatments were determined for the following parameters.

Gel firmness
Gel firmness of yogurt was determined at 4-6ºC by penetration measurements (Texture Analyzer, LFRA-4500, Brookfield, Inc., USA) equipped with a 4.5 kg load cell. The apparatus was adjusted to the following conditions: cylindrical probe (38mm in diameter); penetration speed 1mm/s; penetration distance 30mm into surface. The peak force measured in grams (Aly et al., 2004).

Monitoring of viscosity during yogurt manufacturing
Yogurt viscosity was measured by using Brookfield viscometer model BM type. Three readings from every sample taken and an average value was recorded. The readings taken at 10ºC, which is the temperature at which the yogurt is most likely to be consumed. The spindle speed adjusted keeping in view to the thickness level of the sampled yogurt. In this experiment, the standard combination used, speed 12 (revolutions/second) and spindle number 4. To calculate the final viscosity in centipoises, a factor of 500 is taken to multiply the obtained figure.

Monitoring of pH during yogurt manufacturing
The milk pH during the process of gelatin was recorded on regular basis by immersing the glass electrode of the pH meter (H19020 microprocessor bench pH meter; Hanna Instruments, Woonsocket, RI, USA) in the milk. The pH meter standardized with buffers at 45°C before use.

Syneresis
Syneresis measured according to the method described by Aly et al., (2004). Yogurt mix (25g) weighed in a centrifuge tube and the samples was then be reared together and the set gels were then be stored at 4ºC for 24 h. Samples of yogurt weighed and centrifuged for 10 min at 3500xg and at 4ºC. The whey extracted from the sample of yogurt de-caned off and the rest of the yogurt reweighed. The WHC is defined as weight (g) of the remaining (or drained) yogurt out of the total weight (100g) of the yogurt (before centrifugation).

Water holding capacity
The water-holding capacity of yogurt was determined according to the technique recommended by Aly et al. (2004). 20g of a sample of yogurt (Y) centrifuged at 1250 x g for 10 min at 4°C. The whey that is extracted (W) removed and weighed. The water-holding capacity (WHC, g.kg −1 ) calculated as:

Sensory evaluation
Sensory evaluation of all yogurt samples was carried out on a 9-point hedonic scale by a panel of 10 semi trained judges. Sensory evaluation was carried out on day 0, 7, 14 and 21.

Statistical analysis
The collected data under investigation was analyzed through ANOVA technique under complete randomized design (CRD) using SAS 9.1 software. Duncan multiple range (DMR) test applied to find the difference between means. The data obtained in triplicates and each treatment was also triplicated.

Physico-chemical properties
Highest protein, total solids (TS) and fat content were recorded at day 1 st and gradual reduction was observed with the passage of storage period as shown in Table I. The maximum protein (p<0.001) contents were obtained in 1 st day and the least in 21 st day. Fat and protein largely affected by microbes. During storage fat and protein decreases as microbial action increases. Table II shows pH and acidity of yoghurt at different storage days prepared from different milk sources.

Textural properties
The microstructure and the rheological properties of set yogurts are considerably critical to product quality and shelf life. Syneresis, serum release from the gel matrix, is regarded as a technological defect in set yogurts. Serum release, known as syneresis, is considered as one of the most important parameters indicating the quality of yogurt during storage. Gel firmness, viscosity, syneresis and water holding capacity of yoghurt at different storage days prepared from different sources of milk are shown in

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with storage period and highest firmness was observed in at day 21 of storage period in all the treatments. It was also observed that Highest firmness was observed in T 0 (100%) Nili Ravi buffalo milk followed by T 1 and T 5 .

Sensory properties
Sensory properties such as appearance, texture, taste, smell are shown in Table IV. Table V shows the overall acceptability of different milk sources.

Physico-chemical properties
Reduction in protein may be due to proteolysis activity as lactic acid bacteria hydrolyze milk protein and produces amino groups (Unal and Akalin, 2012). These results are in line with literature, lactose converted into galactose to glucose and decreasing lactose contents, so TS was decreased in all the treatments with storage period. Another study showed that some amino groups make interaction with lactose so same trend was observed in decreasing TS contents. Ca, P and Mg decreased as storage period increased.
Acidity decreases towards the storage period as shown in Tables III and V. It may be due to the microbial action. As Streptococcus thermophilus and Lactobacillus bulgaricus are the major source of decreasing acidity and conversion of lactose to lactic acid as well. She said that there was slight increase in the acidity of yoghurt during storage period. Acidity decreases from 0.9 to 1.7% during the storage of commercial yoghurt. This was due to the microbial action and non-availability of blast chiller room. They reported that there was not storage effect on TS and SNF of yoghurt, and these values decreased as storage period prolongs. This might be due to the relation between lactose, amino acid and other components. This all happens due the microbial action and different chemical reactions.

Textural properties
Undoubtedly, the tendency to exhibit syneresis also depends on the changing in pH, which affects the gel structure, which is a casein micelle network containing heat-denatured whey proteins bound to the surface of the casein micellesIn milk, the integrity of casein micelles is controlled by a localized balance between hydrophobic interaction and electrostatic repulsion (Horne, 1998;Lucey, 2002). As the pH of milk decreases during fermentation, the CCP within casein micelles is solubilized, especially at pH<6.0, and it is completed by pH~5.0, which leads to the partial rearrangement of the internal structure of casein micelle (Lucey, 2002). As the pH of milk approaches the isoelectric point (i.e., pH< 5.0), electrostatic repulsion decreases, which facilitates enhanced casein-casein attractions due to increased hydrophobic interactions.
These factors increase bond formation/strength and thus increase gel stiffness. Continuing to grow the lactic acid bacteria and also to produce lactic acid through the storage is responsible for the reduction in the pH.
Viscosity of the yoghurt increased with storage period in all the treatments and highest viscosity was observed in T o followed by T 1 and T 5 . The viscosity may be related with gel firmness. Syneresis of the samples and control is shown in Table III. It is evident that syneresis increased with the passage of time in all the treatments including control. In Table III, it is shown that water holding capacity was decreased up to storage period of 14 days and decreased on 21 days. Highest water holding capacity was observed in T 0 followed by T 1 and T 5 . As considered by many researchers syneresis is one of the most important parameters indicating the quality of yogurt during storage and consumer satisfaction (Shakerian et al., 2015).

Sensory properties
Appearance of yoghurt is increased with storage period in all the treatments. Appearance of yoghurt made from 100% Nili Ravi buffalo milk was higher throughout the storage period as compared to other treatments followed by T 1 and T 5 , respectively. Similarly, texture was also increased with the passage of storage in all the treatments. Higher texture was observed in T 1 and T 5 , respectively. Score for smell was also found increased in all the treatments throughout storage period in all the treatments. Highest score was found in T o followed by T 1 and T 5 . Overall acceptability is shown in Table V which shows that overall acceptability of yoghurt made from different combination of milk sources was found increased with the passage of storage period. Overall acceptability of T o was higher followed by T 1 and T 5 .

CONCLUSION
Yoghurt made from different sources of milk showed that using 100% Nili Ravi buffalo milk has good physicochemical, textural and sensory properties as compared to yoghurt made from other sources of milk or mixed milk. The results in comparison shows that highest score was observed in T o (100% Nili Ravi buffalo milk) followed by T 3 (50% Nili Ravi buffalo and 50% Sahiwal Cow milk), T 4 (50:50 of Nili Ravi buffalo and Friesian), T 1 (100% Sahiwal milk), T 5 (50:50 of Sahiwal and Friesian) and T 2 respectively. Blends of cow and Nili Ravi buffalo milk have also good effects on overall performances and result of yoghurt.