Cheese and Fermented Mites EXERCISE No. 8 DATE nok TITLE: Chemical analysis of cheese INTRODUCTION FSSA has laid down standards for several varieties of cheeses. especially for ‘moisture and fat on dry matter, The progress of cheese ripening can be ‘monitored through chemical analysis of cheese. Sampling of cheese A representative sample of the cheese can be obtained by following one of the methods of sampling [depending upon size, shape, mass and type of cheese] stated below: (A) Sampling by cutting a sector Using a knife with a sharp blade, two random cuts are made radially proceeding from the centre of the cheese. The size of the sector thus obtained shall be such that after removal of the non-edible surface layer (1-2 mm thick), the weight of the edible part should be not less than 100 g. (8) Sampling by means of a cheese trier A cheese trier is used in taking a cheese sample from a block for analysis. The trier is driven obliquely into the surface of cheese towards the centre once or several times of a point at least 10-20 cm. from the edge of the cheese. Each plug extends half way through the cheese. The plugs are drawn one from top and other from the bottom of cheese. From the borings or boring thus obtained a part of at least 2 cm length is cut-off together with the crust and is used to close the hole in the cheese. The remaining portion of the boring constitutes the sample. Great care should be taken in closing the bore holes especially in case of the large cheeses and if possible, they should be pasted over with some compound agreed for this purpose. This method is suited for the sampling of cheese like Cheddar. When the cheese is delivered in drums, cases or other larger containers or is formed in large compact block, sampling may be eamed out by driving tier a7Cheese and Fermented Milks obliquely through the content of the container from the top to the bottom. This method is suited for sampling of Processed Cheese, The plugs are cut into small pieces by pushing them quickly through a 1/16" mesh screen into the sampling jar. The sample is mixed thoroughly and the jar is kept tightly closed except for the instant when a portion of the sample is removed for weighing. It is necessary to take several plugs from different parts of the cheese in order to obtain a representative sample. Samples are analyzed as ‘soon as possible. If it is necessary to hold them, they are kept at approximately 40°F, (C) Sampling by taking whole cheese as a sample This method is used in case of cheese packed in small size containers. While making choice between methods (A) and (B), it should be taken into account that method (A) is often more exact and method (B) is easier especially in case of large hard cheeses. Preparation of Composite Sample Taking approximately equal amount of cheese from each of the containers selected, collect about 300 g of the material which shall be mixed. Samples to be used for Chemical Analysis should be passed quickly through a grater by grinding them quickly in a mortar and retuming them to the sample container or by cutting them into small pieces with a sharp knife in the container. DETERMINATION OF MOISTURE IN CHEESE Moisture in cheese is essential for the biological activity of microorganisms and enzymes for chemical and biochemical reactions to proceed for proper body and textural properties and as a solvent for as a carrier for generated flavouring compounds}. Some of the water present in cheese evaporates during ripening While some amount of moisture becomes initially associated with various cheese components. The association may be either physical or chemicalCheese and Fermented Milks FSSA Requirements of cheese (Hard variety), Processed cheese, Processed cheese Spread, Soft and Extra hard cheese Characteristics | Natural [Processed|Processed] Soft | Extra Cheese | Cheese | Cheese | cheese | hard Spread cheese Moisture % bymass | wax 39| Max.47 | Max.60 | Max. 80 | Max. 36 Milk Fat (OM) % by | Min.48 | Min. 40 | Min.40 | Min.20 | Min. 32 mass \Salt (NaCl) % by mass | Max. 3.0| Max. 3.0 | Max. 3.0 - - In cheese moisture is present in three different states: (a) Free moisture which can be removed (evaporated) by Oven Drying procedure. (b) Moisture, associated with the lipids as interstitial moisture which can also be removed by normal Oven Drying procedure. (c) Moisture which is tightly or internally bound to milk proteins. This moisture cannot be removed by Oven Drying procedure. Procedure 4. Weigh accurately about 2.0 g of well mixed cheese sample directly into flat bottom aluminum or stainless steel moisture dish (previously cleaned and dried) with a glass rod. Add 5 mi hot distilled water and disperse it to fine particles. Put the dish on water bath til contents are almost dried. Transfer the dish in hot air oven maintained at 102°C for 90 min. Cool it in a desiccators and weigh it. eaeen Repeat the process of heating the dish in the oven for 30 min. and cooling till the difference between two successive weighing is less than 1 mg. 7. Record the lowest weight and calculate the Moisture and /or Total Solids. 49and Fermented Milks Cheese DETERMINATION OF FAT CONTENT OF CHEESE BY GERBER METHOD By addition of Gerber HSO, to the cheese and on vigorous agitation the acid ests the protein and thereby release the fat entrapped between the proteins. However, the digestion also generates heat which helps to convert the fat in liquid form, so its viscosity also decreases. The acid increases the sp. 9 difference between fat (0.93) and rest of the mixture (1.2). So on centrifugation, the acid mixture will be thrown towards the periphery while the fat (lighter Portions) moves towards the centre. 'so-amyl alcohol acts as surfactant and gives clear suspension of fat column from rest of the mixture. It extracts the moisture entrapped by fat and so gives a clear (yellowish) fat column. It also solubilizes the phospholipid and hence helps to have a clear fat test. Procedure 1. Weigh accurately 3 g well mixed cheese in a dry butyrometer cups. Insert the bulb of the butyrometer and secure it tightly 2. Insert the opposite end, add 7 mi of hot distilled water and without stoppering shake gently to make a homogeneous fluid. 3. Add more than § mi of Gerber H2SO4 slowly with help of 10 mi automatic tit measure. Add 1 ml iso-amyl alcohol with the help of 1 mt automatic tilt measure. 5. Shake the butyrometer without stoppering gently till all the content is dissolved. 6. Add the rest of the Gerber H2SOx and required quantity of distilled water to bring the fat within column, ‘Stopper it tightly and give inversion to mix the content. 8. Immediately transfer the butyrometer to Gerber centrifuge machine and centrifuge for § min at 1100 rpm. Remove it and keep in water bath at 651 °C for 3 min, 9. Record the % fat. soCheese and Fermented Milks DETERMINATION OF SODIUM CHLORIDE IN CHEESE 'n testing cheese for salt, a direct titration approach cannot be used because of the interfering nature of proteins. A modified Volhard test is employed in which the cheese is digested by being boiled in the presence of nitric acid and Potassium permanganate prior to titration with the silver ions. The principle in the salt test is that the standard amount of 0.1 N Silver Nitrate, added in excess, reacts with the available NaCl as: NaCl + AgNO3 —> NaNO; + AgCI Then the AgNOs not used up in the first reaction is back titrated in the presence of a Ferric Ammonium Sulphate indicator with 0.1 N Potassium thiocyanate as: AgNO; + KSCN —> AgSCN + KNOs When all the remaining AgNO3 reacts with the KSCN, the yellow ferric indicator teaches a reddish brown end point. Once the amount of excess standard AgNO3 is determined, it is a simple chore to find, by subtraction and use of chemical equivalents, the amount which reacted with the sodium chloride. The above method was recently simplified by introducing nitrobenzene to coat the silver chloride precipitate and to create a more distinct end-point capable of more easily detecting 0.03 % NaCl in cheese. It eliminated the tedious filtration step without affecting either the sensitivity or the accuracy of the method. Modified Volhard Test using Nitrobenzene 1. Weigh 3 g of cheese into a 300 mi conical flask, 2. Then add 25 mi of 0.1 N AgNO» from a burette. This is more than adequate to combine with the available chloride. 3. Introduce 10 ml of halogen free HNO3 and 50 mi of distilled water into the flask. Heat the mixture in the flask to boiling under a hood. 4, As it boils, add 15 mi of fresh 5 % Potassium Permanganate (KMnO,) solution in three § mi portions. Add each successive § ml portion after the purple colour changes to yellow. stO00 0ZUu CC Cheese and Fermented Milks 5. Cool the digested yellowish solution to room temperature and add 2 mi of Nitrobenzene and 2 mi of saturated ferric ammonium sulphate Separately with immersion pipettes, 8. Tivate the contents directly with 0.1 N KSCN to a brickred end-point, while stirring. Decantation, filtration and volume adjustment are not necessary. The percentage of salt can be calculated by substituting the values in the following equations: mil of 0.1 N AgNO3 X Milliequivatent of 0.1 N NaCl X 100 Weight of Sample or (25 - ml of 0.1.N KSCN) x 0.00585 X 100 30 The approximate time for duplicate samples is 30 min, a reagent blank may be Tun with 3 g sucrose to destroy the excess permanganate. DETERMINATION OF TOTAL AND SOLUBLE PROTEIN IN CHEESE Total protein refers to all the protein in cheese regardless of its form. This value 's significant nutitionally. Cheddar cheese contains 23-25 % total protein, Soluble protein refers only to that cheese protein which becomes soluble in an aqueous solution. This is of some importance in gauging the cheese ripening fale. A high soluble protein value generally means a soft, well ripened cheese, because insoluble protein acts as a structural framework. During ripening the Protease, peptidase, decarboxylase and deamidase enzymes hydrolyze insoluble di calcium paracaseinate to soluble mono calcium paracaseinate, and free Paracasein and then to peptones, peptides, amino acids. All such reactions contribute to the soluble protein of cheese. In Cheddar cheese, the soluble protein (as % of total nitrogen) ranged from 18.1% (at 42 days) to 38.4% (510 days) of ripening respectively. 82Cheese and Fermented Milks TOTAL PROTEIN IN CHEESE BY KJELDAHL METHOD Procedure 1. Weigh accurately 1.5 g cheese and place it in a 300/800 mi Kjeldahl flask Add 10 g KzSOx + 0.2 g CuSO, as a catalyst with 25 mi concentrated H2SO.. 2. Digest this mixture under moderate heat for 2-2.5 h. As the contents begin to settle out, turn the flask 180°, After digestion cool the flask in air. 3. Dilute the contents with distilled water. 4. Add 100 mi of 45-50 % sodium hydroxide, to neutralize the digestion acid. 5. Connect the Kjeldahl flask to the stil, swirl its contents very carefully and immediately turn on heat source, water inlet in the condenser. 6. Distill untit 150-200 mi distillate is collected in saturated boric acid (50 mi) having mixed indicator (methyl red + methylene blue). 7. Titrate the content with 0.1 N HSO«, 8. Run a blank without sample and subtract from experimental sample reading (BR). Calculation 0.14 x (BR — Blank reading) Total N(%) = Weight of sample Where BR — Burette reading for sample SOLUBLE NITROGEN CONTENT IN CHEESE BY KJELDAHL METHOD Procedure 1. Place 3 g cheese in a porcelain mortar. To the mortar add a small amount of Sharpe's extraction solution, tempered to 50°C and grind the cheese to form a thick paste. Continue to add more solution until paste is liquid. 2. Transfer this dilute suspension to a 100 mi volumetric flask. Rinse the mortar with additional portions of the solution until the total volume reaches the 100 mi mark on the flask. 3. Insert the volumetric flask in a 50°C water bath and, with occasional shaking, maintain it at this temperature for 1 h. Filter the solution through 53ll Cheese and Fermented Milks Whatman No.40 filter paper and pipette 50 mi of the fitrate into a Kjeldahl flask. 4, Digest the contents and distill in the same way as for total protein. Calculation a9 x OR ~ Blank reading % Soluble Nitrogen = 0.; Waa Maturity Index = — % Soluble Nx 100 % Total N ‘The Maturity Index of cheese may range from 4-6 % for fresh, 18 % at 2 months, 25 % at 4 months, 28 % at 6 months, and 32 % at 8 months of age. DETERMINATION OF TITRATABLE ACIDITY IN CHEESE Procedure 1. Weigh accurately about 3 g of cheese directly into a conical flask 2. Add 20 mi hot distilled water and make a fine paste with help of a glass rod. 3. Add 4-5 drops of phenolphthalein indicator and mix it. 4. Titrate against standard 0.1 N NaOH till faint pink colour appears and persist for few seconds. Calculation 9xAXxN Titratable acidity (% Lactic Acid) = Ww Where, A= Burette reading, N = Normality of NaOH W = Weight of the sample DETERMINATION OF pH OF CHEESE Procedure 1. To prepare a cheese sample, mince a small cubic portion of cheese (i.e. 20 g) finely and pack if into the small sample cup of a pH meter.Cheese and Fermented Mitks 2. A clean thermometer bulb is inserted into and removed fromm the cheese at appropriate places to create entry points for the fragile electrodes ard px (Never dilute the cheese with water, as this disturbs the salt equikorium and affects the pH) 3. Before testing, tum on the pH meter (for warming up) for about 5-15 mun, ‘and check its operating accuracy against two standard buffer solutions in @ pH range from 4 - 6 with a sensitivity of 0.1 pH unit. 4. insert the electrodes and automatic temperature probe into the softened cheese, Conduct the pH measurement in the manner determined by the type of potentiometer available. If it lacks an automatic temperature adjuster, set the machine manually for the temperature of the product. 5, After each determination, move the sample cup around and test another area of the same cheese. Make three determinations in this manner to establish the correct pH. 6. Between the samples rinse the sample cups well. DETERMINATION OF TOTAL VOLATILE FATTY ACIDS(TVFA) IN CHEESE Volatile fatty acids of cheese are those acids that are formed during cheese ripening and recovered by steam distillation. In Cheddar cheese, the volatile fatty ‘acid values, using a standard expression, range from 10 to 120 mi of N/10 acid per 100 g cheese. They increase with age of cheese. Raw milk cheese shows significantly more volatile fatty acids than pasteurized milk cheese of the same age. Volatile fatty acids increases are irregular, as the microorganisms often use selected fatty acids as sources of energy during ripening. The volatile fatty acids levels in Cheddar cheese are not directly related to their typical cheese flavour, although both advance with the increasing age of the cheese. Principle A fixed weight of cheese, solubilized in warm, 10 % ‘sulfuric acid is mixed with magnesium sulphate and water, and steam distilled. The steam which comes cover contains Volatile fatty acids in a concentration directly related to their solubility and volatility. The volatile acids in the condensed aqueous solution are titrated against a standard sodium hydroxide solution. 35Cheese and Fermented Milks Procedure 1. Place 10 g of cheese in a mortar. To the cheese add a small quantity of 10 % H2SOq and grind the mixture. 2. Continue to add more HzSO, until the cheese is in a complete emulsion. Then transfer the emulsion to an 800 mI Kjeldahl flask, 3. Wash the mortar with the 10 % HSO, until a total of 50 mi, including that for emulsification purposes, has been used. 4. Shake the Kjeldahl flask while adding approximately 35 g of MgSOg, @ few glass beads and exactly 250 ml of distilled water. 5. Place the flask on the Kjeldahl distillation apparatus and turn on the heat. 8. Collect exactly 280 mi of distillate and remove the flask for alkali titration using N/10 NaOH and phenolphthalein, 7. Insert a funnel fitted with No.2 Whatman Filter paper into a small Erlenmeyer flask and place it under the condensing pipe to catch the insoluble acids at the end of distillation. Wash the condenser pipes with 25 ml neutral alcohol. 8. The sum of the separate titrations of the water solution and of the alcohol rinse is the total volatile acidity of the cheese. 8. The final TVFA values are expressed as ml 0.1 N NaOH required per 100 g of cheese. Gmpes\ien of Shaw> Extyvachon Solulven C Kosikawski, 1379) cl< 6013 = aes Acetic aciel = 595 ml Sedium acetate, 3hie 136-4 g Sedsum chloide ut a Gletum chloxide cauhy dyous) 3-9 a Drs bed molex: Vol de male opt ooo me. Evlvachsn. Sol on . ae ml stock sel” and make velume i ne sal. wth sled water and pice epipigs