FST Lab report 3 - Milk PDF

Preview

Summary

Download FST Lab report 3 - Milk PDF

Description

Lab report 3: Milk Objectives: 1. To examine the effect of acid on milk protein coagulation. 2. To investigate the effect of salt on milk protein coagulation. 3. To determine changes in colour and flavour of sweetened condensed milk during heating.

Introduction: Milk is an oil-in-water emulsion where fat particles (globules) are dispersed in an aqueous (watery) environment. Milk is primarily composed of 87-88% of water. The structure of milk is a dispersion of milk fat globules (fat particles) and casein micelles (protein particles) in a continuous phase of water, sugar (lactose), whey proteins and minerals. (Fox et al. 2015) The major group of milk proteins, casein, are not soluble and exist in milk as small particles. (Kelly & Bach Larsen 2010) Casein is composed of three fractions, known as alpha casein (αs-), beta casein (β-) and kappa casein (κ-). Another protein component found in milk is the whey proteins. Whey proteins include α-lactalbumins and β-lactoglobulins. (Horne 2017) The αs- and β-casein fractions contain several phosphate groups which make them “calciumsensitive” and thus may coagulate with the addition of calcium. κ-casein contains only one phosphate group and are not “calcium-sensitive”. (Vaclavik & Christian 2014) αlactalbumins and β-lactoglobulins are sensitive to heat and will denature and precipitate during prolonged heating of milk. In milk, the casein fractions will associate with each other and with colloidal calcium phosphate to form casein micelles. Some of the processes that milk undergo particularly on milk proteins include coagulation, denaturation, heating of milk and non-enzymatic browning such as Maillard browning. Casein micelles are coagulated by addition of acid at pH 4.6-5.2. As micelles approach their isoelectric point, the charge and extent of hydration is reduced, and the κ-casein hair-like structure flatten, reducing steric hindrance. The micelles are no longer stable and hence aggregate together. (Lucey 2016) A factor that affects coagulation of casein micelles are by the addition of enzyme such as rennin. Rennin cleaves a specific bond in κ-casein and causes the charged, hydrophilic hair-like structures to be removed from the micelles. (Vaclavik & Christian 2014) Another factor that causes the coagulation and denaturation of casein micelles are by the addition of salt into milk and heated afterwards. Salt ions interact with electrical charges on the surface of milk proteins, resulting in minimum conflicting charges and favouring denaturation and coagulation of milk proteins. (Sikand, Tong & Walker 2013) Whey proteins are denatured upon prolonged heating. When the highly reactive free sulfhydryl group in β-lactoglobulins is exposed to heat, β-lactoglobulins will unfold and form disulphide bonds with any milk proteins containing disulphide bonds. (Kelly & Larsen 2010) This will contribute to a slightly “cooked” flavour of the milk. Another factor that affects the flavour and colour of the milk is such that during heating, non-enzymatic browning such as Maillard browning will occur between the coagulated whey proteins and lactose. This result in colour of milk to turn slightly brown and the flavour of milk to a “cooked” flavour. (Fox et al. 2015) The processes that milk undergo are important in the food industry as such, coagulation of protein micelles with rennet enzymes in milk is the primary step in the manufacture of cheese. The coagulation of milk involves two steps, the first involves enzymatically

hydrolyzing the micelle-stabilizing protein, κ-casein, by selected proteinase preparations called rennets. The second step of coagulation involves the coagulation of rennet altered micelles. (Fox et al. 2015) Heating is also a process that is involved in the rennet-induced coagulation whereby the heat treatment of milk is at 70 °C. Furthermore, the non-enzymatic browning, Maillard browning, is an important process in food industry which gives the slightly brown colour of cheese, such as cheddar cheese on pizza, when heated. A type of milk product is sweetened condensed milk. Sweetened condensed milk is a concentrated non-fat milk with approximately 60% of the water in milk being removed and have sugar levels of 40-45% in the finished product. (Horne 2017) Milk is evaporated under partial vacuum at temperature below boiling point of water to remove water and to concentrate proteins and fats. The evaporated milk is then added with sugar to produce sweetened condensed milk.

Materials and methods: Part 1- Effect of acid on milk protein coagulation. Materials: UHT full cream milk (40 mL), vinegar (20 mL), pH meter, distilled water (for rinsing pH meter probe), glass rod (x1). Methods: 1. The pH and initial appearance of the 40mL of UHT full cream milk contained inside a 100mL beaker was recorded. 2. The pH of vinegar in another beaker was recorded. 3. The vinegar was added into the milk and the mixture was stirred until the vinegar was well mixed in the milk. 4. The pH of the mixture was measured. 5. The mixture was left to stand for 30 minutes and the observation was recorded. Part 2- Effect of salt on milk protein coagulation. Materials: UHT full cream milk (40 mL) (x2), sodium chloride (NaCl) (10 g), magnetic stirring hot plate, pH meter, distilled water (for rinsing pH meter probe), glass rod (x1), time (x1). Methods: 1. 10g of sodium chloride was added into the 40mL UHT full cream milk. The milk was well stirred using a glass rod until the sodium chloride had completely dissolved in the milk. The pH was recorded, and the appearance of the milk was observed. 2. The milk was heated with a hot plate (medium heat No. 5) until it boils. The milk was left to boil for 5 minutes before it was removed from the heat. 3. The pH of the milk was checked after it had cooled to room temperature. The observation was recorded. 4. Steps 1-3 was repeated for control using another 40mL of milk without the addition of sodium chloride.

Part 3- Changes of milk during heating. Materials: Sweetened condensed milk (~40 mL), universal bottles (10 mL, x4), test tube rack (x1), timer (x1), 90°C water bath (x2), pH meter (x1), distilled water (x1), glass pipette (x1), Colourflex spectrophotometer. Methods: 1. 10mL of sweetened condensed milk was pipetted into a universal bottle and the universal bottle was closed with a lid. 2. The initial pH of the sweetened condensed milk was recorded, and the cap of this sample was labelled. The colour and flavour of the sample was determined. The colour of the milk was determined using a Colourflex spectrophotometer. The flavour of the milk was determined by sniffing the milk. 3. Steps 1-2 was repeated with 2mL of sweetened condensed milk diluted with 4mL of water in a universal bottle using glass pipette. 4. All the 2 universal bottles were put in a rack and incubated in a 90 °C water bath for 90 minutes. 5. The sample was removed from the water bath. 6. The changes in pH, colour and flavour was recorded when the sample was cooled to room temperature.

Results: Table 1. pH values and observations in terms of precipitation and flowability of different types of conditions of milk in experiment Part 1 and Part 2. Component pH Observations Milk 6.29 No precipitation, good flowability Vinegar

1.29

No precipitation, good flowability

Milk + vinegar

3.19

Formation of curd, poor flowability

Milk + salt (before heating)

5.16

No Precipitation, good flowability

Milk + salt (after heating)

4.80

Formation of curd, poor flowability

Milk (after heating)

6.18

Less precipitation, formation of plastic-like curd, reduced flowability

Table 2. The pH, L*, a*, b* values and flavour of condensed milk that was diluted, undiluted and both heated. Component pH L a b Flavour Condensed Sweet, milky milk 6.12 80.22 -1.93 13.27 (diluted)

Condensed milk (undiluted) Condensed milk (diluted + heated) Condensed milk (undiluted + heated)

Sweet, milky 6.25

81.93

-1.48

7.77

5.97

74.62

-1.82

8.68

5.17

56.91

-1.15

22.76

Cooked flavour

Cooked flavour

Discussion: In part 1, the pH of milk is 6.29 and the pH of vinegar is 1.29. When vinegar was added into milk, the overall pH of the milk mixture decreased to 3.19. This is due to the higher concentration of hydrogen ions, H+, in vinegar compared to milk and thus, the addition of vinegar into milk decreased the pH value of milk as there was an increase in hydrogen ions concentration. (Alexander, Donato & Dalgleish 2007) Furthermore, in part 1 vinegar was added into milk and the formation of curd occurred, the mixture was poor in flowability. This is because vinegar that was added into milk lowered the pH of milk to 3.19 which has reached the isoelectric point of milk at pH 4.6. That said, when the pH of milk has reached isoelectric point, coagulation of casein micelles in milk occur and form curd. This is due to the decline of net negative charge of κ-casein as it approaches isoelectric point and causes an increase in electrostatic interactions and reduce in electrostatic repulsion. This then increases the hydrophobic interactions between casein micelles. As a result, chains and clusters formed linked together to form a three-dimensional network which is the curd in the milk. (Lucey 2017) In part 2, the pH of milk and salt, sodium chloride, mixture before heating is 5.16. However, when the mixture was heated, the pH decreased to 4.80. The addition of salt into milk will increase the level of total ionic calcium level in the serum phase of the milk. This causes the pH level of milk to decrease because there is a strong interrelationship between the concentration of ionic calcium and its pH. (Huppertz & Fox 2006) Additionally, there was no precipitation in mixture of milk and salt before heating however, formation of curd and poor flowability of mixture of milk and salt after heating could be observed. This is because adding salt into milk will increase its level of ionic calcium in the serum phase of the milk. This decreases the net-negative charge of the casein micelles, particularly the κ-casein. As a result, the electrostatic interactions increase, and the electrostatic repulsion is reduced between the casein micelles. Hence, the κ-casein become less stable and favour aggregation and coagulation of milk. This is also supported by the fact that stability of casein micelles to prevent aggregation is dependent on the negative charge and steric repulsion of casein micelles. (Lucey 2017) Another factor that causes the formation of curd in mixture of milk and salt after heating is due to whey proteins such as βlactoglobulins that have sulfhydryl groups attached to it was exposed to heat and cause βlactoglobulins to unfold. The unfolded β-lactoglobulins will form disulphide bonds with any milk proteins containing disulphide bonds. (Kelly & Larsen 2010) As a result, the whey proteins are denatured and form precipitate during heating.

In part 3, pH of sweetened condensed milk decreased from 6.25 to 5.17 after heating. This is because during heating, water in sweetened condensed milk was ionized and forms hydrogen ions into the serum phase of the milk. The process was sped up by heating as water molecules absorbed energy and the ionization of water occur rapidly. This resulted in an increase in hydrogen ions concentration in milk which lowers the pH value. (Alexander, Donato & Dalgleish 2007) Besides that, unheated sweetened condensed milk was initially whitish-yellow however, its colour changed to a brownish-yellow colour, slightly caramel, after heating. This can be seen that Maillard reaction took place in the sweetened condensed milk during heating. During heating, the reducing sugars, lactose, react with amino groups on milk proteins via condensation. The condensation reaction leads to formation of Amadori compounds which then break down into numerous fission products of sugar-amino compounds. The condensation of amino compounds and sugar fragments are then polymerized into proteins and brown pigments called melanoidins. (Kumar et al. 2017) On top of that, the flavour of unheated sweetened condensed milk changed from sweet and milky to a “cooked flavour” after heating. Part of this is due to the Maillard reaction that took place during heating. However, the change into the “cooked” flavour of heated sweetened condensed milk was due to the formation of hydrogen sulphide, H2S. During heating, βlactoglobulins which contains sulfhydryl groups underwent denaturation which causes the sulfhydryl groups to decompose. The decomposed sulfhydryl groups lead to the formation of hydrogen sulphide, H2S, which is mainly responsible for the “cooked” flavour of heated sweetened condensed milk. (Fox et al. 2015)

Discussion questions: Part 2: 1. What effect did vinegar have on the milk? Explain your answer. (3 marks) -Since the pH of milk is 6.29 and pH of vinegar is 1.29, the addition of vinegar into milk had decreased the overall pH of the mixture to 3.19. The isoelectric point of casein in milk was reached when the pH milk of is 4.6 and that said, the overall pH of the mixture of vinegar and milk is 3.19 and it has reached the isoelectric point. This is when coagulation of casein in milk occurs. This is because the net negative charge of casein micelles, κ-casein particularly, declines as it approaches the isoelectric point and there are increased electrostatic interactions and reduced electrostatic repulsion, which increases the hydrophobic interactions between the casein micelles. This eventually leads to the formation of chains and clusters that are linked together to form a three-dimensional network. (Lucey 2017) As a result, the casein micelles to become less stable and aggregate together which form curd in milk. 2. Explain the curdling process that may happen when salt is heated with milk. (3 marks) -Adding salt into milk will increase the level of total and ionic calcium in the serum phase of the milk which decreases the net-negative charge of casein micelles, κ-casein particularly. (Huppertz & Fox 2006) Since the net-negative charge of κ-casein decreases, this causes an increase in electrostatic interactions and a reduce in electrostatic repulsion of the casein micelles. This causes the κ-casein to be less stable and favour aggregation and coagulation. Since the stability of casein micelles to prevent aggregation are influenced by a combination

of negative charge and steric repulsion of casein micelles. (Lucey 2017) Furthermore, adding salt into milk will also decrease its pH level as there is a strong interrelationship between the concentration of ionic calcium and its pH. (Huppertz & Fox 2006) As the pH of milk declines and approaches its isoelectric point of 4.6, aggregation and coagulation of milk is likely to occur as κ-casein are less stable and will leave the casein micelles exposing α s- and β-casein fractions that are hydrophobic. This causes the α s- and β-casein to aggregate and coagulate the milk. The increase in temperature during heating will increase the kinetic energy of casein micelles which give rise to the frequency of collision and the aggregation of casein micelles. During heating, whey proteins such as β-lactoglobulins are exposed to heat and thus the highly reactive free sulfhydryl group in β-lactoglobulins will cause them to unfold and form disulphide bonds with any milk proteins containing disulphide bonds. (Kelly & Larsen 2010) As a result, the whey proteins are denatured and form precipitate during heating. Hence, milk with addition of salt and are heated will contain precipitate in it. 3. Give one example of dairy product that is important to have curd formation. (1 mark) -Cheese Part 3: 1. What are the changes in terms of pH, colour and flavour of heated sweetened condensed milk? Explain your answer. (4 marks) -The pH of heated sweetened condensed milk decreased slightly after heating, from a pH of ~6.25 to a pH of ~5.17. The colour of sweetened condensed milk changed from a whitishyellow colour to a slightly caramel colour that is brownish-yellow after heating. The flavour of heated sweetened condensed milk gives off a “cooked flavour” meanwhile the unheated sweetened condensed milk has a sweet milky flavour. The pH of sweetened condensed milk decreased after heating due to the increase in ionization of water which increases the concentration of hydrogen ions, H+, in the milk. Thus, when the concentration of hydrogen ions increases, the pH value of milk decreases. The colour of sweetened condensed milk changes from a whitish-yellow colour to a slightly caramel colour of brownish-yellow, because of Maillard browning that took place during heating. (Nieuwenhuijse 2011) During heating of sweetened condensed milk, reducing sugars in milk, lactose, react with amino acids from milk proteins via condensation. The condensation reaction leads to formation of Amadori compounds which then break down into numerous fission products of sugar-amino compounds. The condensation of amino compounds and sugar fragments are then polymerized into proteins and brown pigments called melanoidins. (Kumar et al. 2017) The colour of sweetened condensed milk after heating can also be determined by a Colourflex spectrophotometer. The heated sweetened condensed milk was determined to have a value of 56.91 on L*, a negative value of -1.15 on a* which indicates a green colour and a positive value of 22.76 on b* which indicates a yellow colour and these indicates a brownish-yellow colour of the heated sweetened condensed milk. The flavour of heated sweetened condensed milk has a “cooked flavour” partially due to Maillard reaction but mainly because of denaturation of β-lactoglobulins which contains sulfhydryl groups that may decompose due to exposure to heat. The decomposed sulfhydryl groups lead to the formation of hydrogen

sulphide, H2S, which is mainly responsible for the “cooked” flavour of heated sweetened condensed milk. (Fox et al. 2015) 2. Name a compound that may attribute to the cooked flavour of heated milk. (2 marks) -Hydrogen sulphide, H2S

Conclusion: In conclusion, the pH of milk will decrease when added with vinegar. This is because vinegar has higher concentration of hydrogen ions in it and it will dissociate in the milk when vinegar is added into it. Concentration of hydrogen ions in mixture of milk and vinegar will then increase which decreases the pH value of the mixture. Furthermore, curd or precipitate is formed in milk when vinegar was added. This is because addition of vinegar has decreased the pH value of milk and vinegar mixture. Thus, the lowered pH value has approached isoelectric point of milk at pH 4.6 and coagulation of casein micelles will occur. When the net-negative charge of κ-casein decreases, there is an increase in electrostatic interactions, reduce in electrostatic repulsion and increase in hydrophobic interactions between casein micelles. As a result, the casein micelles aggregates and this is the curd formed when milk coagulates. Moreover, the pH of mixture of milk and salt will decrease after heating it. This is because the addition of salt into milk will increase the level of total ionic calcium level in the serum phase of the milk. As a result, the pH level of milk decreases because there is a strong interrelationship between the concentration of ionic calcium and its pH. Besides that, formation of curd in mixture of milk and salt can be seen after it is heated. Net-negative charge of the casein micelles will decrease, particularly the κ-casein. As a result, the electrostatic repulsion will reduce, and the electrostatic interactions will increase between the casein micelles. Hence, the stability of κ-casein reduces and favour aggregation and coagulation of milk. This is also true as the stability of casein micelles are based on the negative charge and steric repulsion of casein micelles. Additionally, the pH of sweetened condensed milk will decrease after heating. As water...