MflTERIfiLS ffnd METHODS

MflTERIfiLS ffnd METHODS

CHAPTER - III MATERIALS AND METHODS This section deals with the methods and materials used in the preparation of Paneer together with standard analytical procedures. 3.1. COLLECTION OF RAW MATERIALS : (i) Milk: Proper amount of murrah buffalo milk was collected from instructional live stock farm N.D.U.A.T. Kumarganj, Faizabad. The skimmilk was obtained by separating whole milk at 40 C. (ii) Skim Milk Powder : The skim milk powder (spray dried) manufactured by a unit of P.C.D.F. Ltd., Lucknow (Parag) was obtained from local market of Faizabad city. (iii) Citric Acid : Citric acid (commercial grade) was purchased from local market of Faizabad city. 3.2. MANUFACTURE OF PANEER : The paneer was manufacture as per method suggested by Bhattacharya et al. (1971) and later on modified by Sachdeva (1983). 37

(I) STANDARDIZATION OF MILK : Murrah buffalo milk was standardized to 4.5, 5.0, 5.5 and 6.0 per cent milk fat with S.N.F. level of 9.0 per cent by adding fresh murrah buffalo skim milk. The additional requirement of S.N.F. content was made up through spray dried skim milk powder. (ii) HEATING AND COOLING OF MILK : The standardized murrah buffalo milk was subjected to three different heat treatments of 80, 90 and boiling temperature (100.17 C) with holding time of 5 minutes in the coagulating vessel. The milk was slowly stirred by a stainless steel ladle to avoid burning and to prevent skin formation during heating. The milk was removed from tl cooled qpto 70 C. (iii) ADDITION OF COAGULANT : Citric acid coagulant (1% solution) was added to milk at 70 C at the rate of 0.25, 0.35 and 0.45 per cent by weight of milk. Milk was kept on continuous agitation till clear whey (yellowish-greenish colour) separated out. (iv) DRAINING OF WHEY : The curd was left to settle down for 5-10 minute without agitation. The whey was then drained through a muslin cloth. In no case thejemperature-qf-w-hey was-allowed to fall below 63 C till the draining was over. - (v) PRESSING : After draining of whey, the solids collected in muslin cloth were tied and pressed for 30 minutes by applying a suitable pressure. 38

(vi) COOLING AND CUTTING : Pressed curd was dipped in chilled water (4-6 C) for two hours to get a firm curd. Thereafter, it was removed from the water and placed on wooden planks for 10-15 minutes to allow loose water, to drain and then cut into 200 gm blocks. (vii) PACKAGING: udps- The paneer blocks were then packaged in polythene sachet and thereafter paneer samples were stored at 8-10 C. PLAN OF STUDY : (A) Factors of the study : (I) Fat levels in milk : (i) 4.5% - (FO (ii) 5.0% - (F2) (iii) 5.5% - (F3) (iv) 6.0% - (F4) (II) Temperature of final heating of milk : (i) 80 C - (Tj) (ii) 90 C - (T2) (iii) Boiling temperature (100.17 C - (T3) (III) Levels of citric acid by weight of milk : (i) 0.25% - (Si) 39

(ii) 0.35% - (S2) (iii) 0.45% - (S3) (IV). Storage periods under refrigerated condition : (i) Zero days (fresh) - Pi (ii) Three days - P2 (iii) Six days - P3 B. PARAMETERS TO BE STUDIED : (i) (ii) (iii) Yield Recovery of total solids Sensory characteristics (a) (b) (c) (d) Flavour Body and texture Colour and appearance Overall acceptability Chemical characteristics : (a) (b) (c) (d) (e) Moisture Fat Protein Lactose Ash 40

(f) Acidity (v) Microbiological characteristics : (a) (b) (c) Standard plate count Coliform count Yeast and mould count. (vii) Assessment of cost of production Number of Replication - 3 Total number of samples - 4x3x3x3x3 = 324 Design - Factorial completely Randomized design. 3.3. EXAMINATION OF MILK : (i) Sampling of milk : Preparation of sample for analysis was done as per method prescribed in lab. manual in agriculture chemistry (1960). Before the sampling, milk was warmed upto about 38 c. It was then, mixed thoroughly by pouring into a clean vessel until a homogenous mixture was obtained, the milk was, later on, cooled down to 15-20 c. (II) TOTAL SOLIDS : It was determined according to gravimetric method described in lab. Mann, in Agriculture chemistry (1960). A small flat bottomed dish of about 7 cm diametre was heat and dried, allowed to cool in a desiccator and weighed, pippeted 10 cj:,, of the-'well mixed sample into it and weighed as quickly as possible, Evaporated the milk till dryness on a water bath and dried to constant weight in a electric oven at 100 C. The 41

same was cooled in a desiccator and weighed. The total solids per cent was calculated as given formula : where, W3- W, T.S. content ( %) =... x 100 W2 - Wi Wj - Weight of porcelain dish w2 - w3 - Weight of porcelaindish + Milk Weight of porcelain dish + dried milk (iii) Fat : Fat per cent of milk sample was determined by Gerber's method as per IS: 1223 (Part I 1970). Ten C.C. sulphuric acid (1.820 S.G.), 10.75 ML milk sample and 1.0 ml amyl alcohol (0.825 s.g.) were mixed together in milk butyrometre and content was centrifuged in Gerber's centrifuge at a speed of 1100 r.p.m. for 4-5 min. Fat per cent was noted directly on butyrometre scale. 3.4. EXAMINATION OF PANEER : 3.4.1. Sampling of paneer : The procedure described for the sampling of chhana in I.C.A.R. Bulletin No. 70 (1951) was followed with slight modification. The requisite amount of paneer was taken from different parts of the entire mass with the help of stainless steel knife. These portions were pooled together graded and subsequently used for analysis. For microbial analysis, stored samples of paneer were opened into an inoculation chamber sterilized by U.V.-irradiation. All suitable conditions were maintained during sampling. 42

3.4.2. Sensory Evaluation : The paneer samples were evaluated organoleptically for different attributes viz, Flavour (odour and taste), body and texture, colour and appearance by the judges. A score card based on 100 point scale as described by Patil and Gupta (1986) was used with certain modifications. Marks allotted for each attributes are given below : Attributes Perfect score Flavour 50 Body and texture 35 Colour and appearance 15 The grading of paneer was done on the basis of total score obtained as below : Grade Total score Excellent 92-100 Good 82-91 Fair 72-81 Poor 61-71 Bad Below 60 acceptable. Paneer samples upto 'Fair' grade were considered as 3.4.3. Chemical Examination of Paneer : (i) Moisture : Moisture content of paneer was determined by modified gravimetric method adopted by Roy (1990) for filled paneer. A clean porcelain dish was weighed and about 2 gm. of finely grounded and homogenized sample of paneer was taken into a clean, dry, 43

tared solid dish. Then, 4 ml of hot distilled water (approximately 65 C) was mixed with the sample and the mixture spread uniformly in the dish with the help of a small glass rod. An additional 1 ml hot distilled water was used to wash off the particles of paneer from the glass rod into the dish. The dish was, thereafter, transferred to an oven at 100 ± 1 C and the contents were allowed to dry for 4 hours. The dish was then cooled in a desiccator and weighed accurately. The procedure of heating in the oven (for 30 min.) cooling and weighing was repeated till a constant weight was obtained. The moisture content was calculated by the following formula. Moisture (%) where as, W2- W3 -... x 100 W2-Wi Wi - W2 - w3 - Weight of dish Weight of dish + sample Weight of dish + dried paneer sample 2. Estimation of Total Solids Content of Paneer : The procedure for total solids analysis was same as for moisture described earlier. The following formula was used for calculating the total solids percentage of paneer. Total solid (%) = 100 - moisture (%) 3. Estimation of Fat Content of Paneer : Fat content of paneer was determined by Gerber's method as described in IS-1224 (Part II)- 1977 for analysis of cheese. 44

10 ml of sulphuric acid (sp. gr. 1.825) and same quantity of. lukewarm water was taken in to a clean cheese butyrometre and thenf5 3 gms of homogenized paneer (grinded) and 1 ml of Amyl alcohol (sp. gr. 0.825) was also added to the same butyrometre. The butyrometre was stoppered and then the contents mixed well and digested with occasional shaking until the coagulum was dissolved. The butyrometre was then placed into the water bath maintained at temperature of 70 C for 5 minutes. The butyrometre was then placed in the Gerber's centrifuge and rotated for about 5 minutes at a speed of 1000 to 1200 r.p.m. The centrifuge was then stopped and butyrometre was taken out from centrifuge and transferred to water bath maintained at a temperature of 70 C for 5 minutes. The reading was, thus recorded, on graduated stem of butyrometre. 4. Estimation of Protein Content of Paneer : Protein content of paneer was determined by Kjeldahl method as described by Davis and Macdonald (1953). Two gms of paneer sample was taken in a clean dry kjeldahl flask. To it 20 ml pure nitrogen free sulphuric acid, 10 gm of pure potassium sulphate crystals and 2 gm pure copper sulphate crystals were added. The flask was then transferred to digestion chamber for digestion of contents. At the digestion, when the content of the flask became carbon free, the flask was allowed to cool down. The content was then dissolved in 400 ml of distilled water and transferred to distillation flask. Some pieces of glass beads were also added to distillation flask to avoid bumping on heating. Fifty ml N/10 sulphuric acid was taken into 500 ml beaker and added 3-4 drops of methyl red indicator. The beaker was then placed below condenser to collect the condensate. The tip of condensor 45

was dipped into the content of beaker. 90 ml of 40 per cent sodium. hydroxide solution was then added to the distillation flask and the distillation head was fixed on distillation flask and condensor. The distillation process was continued until about 300 ml. distillate was collected in the beaker. Following the usual precautions the beaker was removed from assembly. The excess acid was titrated back with N/10 sodium hydroxide solution. In this way the actual amount of N/10 sulphuric acid used for neutralization of ammonia was determined. The percentage of protein in paneer sample was calculated by following way. 1 ml. of N/10 H2S04 = 0.0014 gm N2 Amount of N/10 H2S04 (ml) Neutralized x 0.0014 Nitrogen (%) in sample =------------------------------------------------------- x 100 Weight of paneer taken Protein (%) in sample = Nitrogen (%) x 6.38 5. Estimation of Lactose Content of Paneer : The lactose content of paneer was estimated by Fehling's solution method as described in lab. Mann, in Agriculture chemistry (1960) with the some modification. Ten grams of paneer weighed and transferred into the pastel and mortar and finely grounded with about 25 ml of lukewarm water. It was transferred into a beaker. Again 30 to 40 ml lukewarm water was taken into mortar to clean it and its pastel and poured into the same breaker. Allowed to the protein particle settle down and it was then filtered through a filter paper. The volume of filtrate was made upto 100 ml. The filtration was carried out by taking the lactose solution in the burette. 10 ml of fehling's solution (5 ml of each A + B) was taken 46

into a conical flask. Few pieces of glass beads were also added to reducing bumping. Then content of the conical flask were allowed to boil. The lactose solution was now added gradually when the contents became slightly reddish. Few drops of methyline blue indicator was added into the flask and titration was continued until a brick red precipitate was obtained. The percentage of lactose content in paneer was calculated with the help of the following formula : 0.0678 x 100 x 100 Lactose (%) =... Xx 10 whereas : X = Volume of lactose solution (filtrate) needed for reducing 10 ml of Fehling's solution. 6. Estimation of Ash Content of Paneer : Ash content of paneer was determined as per the method described for chhana (IS 1962-1980). Five grams of air dried paneer sample was accurately weighed in silica dish. The dish was heated gently on a flame at first and then strongly in muffle furnace at 550 ± 20 C till the grey ash resulted. The dish with ash was cooled in a desicator and weighed again. The ash content was calculated by the following formula. W2-Wi Ash % =... x 100 W where as, W = Weight of sample Wi = Weight of silica dish W2 = Weight of silica dish + ash. 47

7. Determination of Acidity in Paneer : Acidity of paneer was determined as per the procedure adopted by Rajorhia et al. (1984). Two gram of finely broken paneer sample taken in a pastle morter with 3 ml of hot distilled water and diluted by another 17 ml of hot distilled water washing off the adherants of the pastle. Ten ml of the 0.1 N sodium hydroxide was added to dissolve the contents. Excess alkali was titrated against 0.1 N hydrochloric acid using 1 ml of 0.5 per cent phenolphathalein indicator. Acidity was expressed as lactic acid and calculated by using the following formula : where as, 10-V Lactic acid (%) = ------x 0.9 W V = Volume of 0.1 N HC1 required for titration W = Weight of sample taken. 3.4.4. Microbial Examination of Paneer : (i) Methods of preparation of dilution : The paneer suspension for microbiological analysis was prepared in accordance with the following steps. (a) One gram of paneer was weighed on a wax paper, using a spatula that had been dipped into alcohol and flamed. (b) Placed 1 ml of alcohol in the mortar and ignited until the alcohol burnt, stirred in around with pastle so that all the inner surface of the mortar was in touch by the burning alcohol. 48

(c) Allowed one minute for the inner surface of the mortar to cool slightly and poured 1 ml of sterile solution of 0.9 per cent sodium chloride in the mortar. One gram of paneer, already weighed, was added and grind it with the pastle. Gradually 8 ml solution of 0.9 per cent sodium chloride was added as the grinding progressed. Thus, the finished suspension of 1:10 obtained was resulted in smooth milky and free from lumps. Further dilutions were made from this solution. The dilution's upto 1:105 were made in dilution bottles. General precautions were taken during the preparation of dilutions to avoid contamination. Under this examination following microbial tests were conducted according to the technique described in standard methods for the examination of Dairy products (1978). Standard Plate Count: It was done to asses the total number of living bacteria in the sample's of paneer. The nutrient medium for growing bacteria was plate count agar. Plates of 1:1000, 10,000 and 1: 100,000 were prepared. A proper amount of dilution was transferred in duplicates sterilized petriplates. Ten ml of melted plate count agar was poured in each petriplate. After thorough mixing, the plate were left for some time on the bench for solidification of the medium. All essential precautions were kept to avoid external contaminating during plating. The inverted plates were placed in incubator maintained at 37 ± 1 C for 48 hours. After the incubation period, the colonies made by bacteria on the nutrient medium of the plates were counted with the help of colony counter. The number of bacteria was calculated by multiplying the dilution factor with number of colonies counted. 49

Coliform Count : In paneer, coliform count, violet red agar (Hi media) is used for enumeration. Plates of 1;10 and 1: 101 dilutions were prepared. A suitable amount of dilution was transferred in duplicate sterilized petriplate. Then, 10 ml. melted nutrient medium was poured in each petriplate. After gently shaking the plates were left for some time on the bench for solidification of medium. The inverted plates were placed in incubator maintained at 37±1 C for 48 hours. After incubation period the bacterial colonies were counted with the help of colony counter and multiplied by respective dilution factor to enumerate the total number of bacteria in paneer per gram. Yeast and Mould Count : For yeast and mould count 1:10, 1 :101, and 1:102 dilution of paneer suspension were transferred in duplicate plates. 10 ml melted potato dextrose agar medium was poured in each petriplate. After mixing the plate were left for some time on the bench for solidification of medium. The inverted plates were placed in incubator maintained at 22±1 C for three four days. After the incubation period, the colonies of yeast and mould counted with the help of colony counter. The total number of yeast and mould per gram of paneer was calculated by multiplying of respective dilution factor with the colonies counted. 3.5 COST OF PRODUCTION : The cost of production of paneer was calculated on the basis of existing prices at fat and solid not fat (S.N.F.) suggested by Singh and Kalara (1979). 50

The cost of production of paneer was calculated on the prepare of 1 kg paneer from different level of fat. The cost of different items are given as below : 1. Fat Rs. 80.00 2. S.N.F. Rs. 53.33 3. Heating medium Rs. 1.25/hour 4. Coagulant (citric acid)- Rs. 468/kg. 5. Labour charges Rs. 45/ day 6. Packaging material Rs. 0.40/pack 3.6. STATISTICAL ANALYSIS : Calculation of means, standard error and analysis of variance as described by Snedecor and cochran (1968). 51