TERMINOLOGY RELATING TO PROTEIN INSTABILITY IN MILK

Milk flocculation and gelation describe the phenomena relating to protein instability that could lead to the rejection of milk for processing or to defects in long-life milks such as UHT milk. Since Milk SA has embarked on a comprehensive R & D programme to understand and control these conditions, it is important to understand what is referred to, and to define it in the context of practical perceptions and interpretations in the South African Dairy Industry. Other related terms that should also be explained for the sake of clarity, include terms such as clotting, coagulation, curdling, denaturation and precipitation.

Although the phenomena of flocculation and gelation are related, the mechanisms that drive them differ. In order to discuss the terminology, it is necessary to first say something about milk proteins. The major milk protein is casein (constituting about 80% of proteins in milk) and there are four major types of casein molecules: alpha-S₁, alpha-S₂, beta and kappa. As the caseins are secreted, they self-associate into aggregates called micelles in which the so-called “calcium insoluble” alpha and beta caseins are kept from precipitating by their association with kappa casein. In essence, kappa casein normally keeps these proteins in suspension and prevents them from spontaneously precipitating in the presence of the abundance of calcium in milk. This stability of the protein suspension in milk can be negatively affected by a range of factors, resulting in precipitation or coagulation. These factors are referred to in discussing the different terms:

Clotting: Clotting describes milk (and especially cream) that has soured and thickened to the point that it forms a semi-solid mass. It is, however, more correct to refer to clotting as “curdling” or “coagulating”, since the word “clotting” is usually associated with the process by which blood changes from a liquid to a solid, forming a blood clot. In Afrikaans the word “stolling” is used synonymously for the terms clotting, curdling and coagulation.
Coagulation: Coagulation has the same meaning as clotting when referring to milk that has thickened to the point that it becomes a semi-solid mass. It also describes the creation of a thicker, more viscous, texture when producing fermented dairy products such as yoghurt, in addition to the formation of curds (Afr. “wrongel”) in the cheese making process. Coagulation involves the disruption of the stable suspension of casein micelles in the milk resulting in precipitation of the protein. In fermented dairy products coagulation is caused by the production of lactic acid by starter bacteria. The starters lower the pH of the milk to below the iso-electric point of casein (pH 4.6) at which pH the casein precipitates, thus providing the typical viscous texture of the product.

In cheese making, coagulation of the casein in milk forms the basis for cheese curd formation. A proteolytic enzyme such as chymosin (rennet or rennin) cuts the kappa-casein molecule, converting it into para-kappa-casein and a smaller protein called macro-peptide. Para-kappa-casein then no longer has the ability to stabilize the casein micellar structure and the calcium-insoluble caseins precipitate, forming a coagulum or curd.
Curdling: The term “curdling” is synonymous with coagulation when milk is coagulated during cheese-making. The precipitated milk proteins (casein) form solid masses of protein called “curds”. The curdling can be caused by adding rennet, by adding any edible acidic substance such as lemon juice or vinegar or by adding starter bacteria that produce lactic acid. The curds are pressed and drained to varying degrees to obtain different styles of cheese
Denaturation: According to the Biology On-Line Dictionary, “denaturation” is a process in which the folding molecular structure of a protein is altered due to exposure to certain chemical or physical factors (e.g. heat, acid, solvents, etc.). The denatured proteins have altered characteristics, resulting in loss of solubility, aggregation and coagulation. In egg white, for example, heating causes polypeptide chains in the transparent albumen to unfold and form an opaque coagulum by the re-association of these molecules. Casein in milk is particularly susceptible to denaturation by the enzyme rennin and by low pH. The resulting coagulation is the first step in most cheese production processes.
Flocculation: The term flocculation, as used in the South African dairy industry, applies to the protein precipitate that is formed in the test tube when ethanol unstable milk is subjected to the Alizarol test (in Afrikaans the milk is said to “flokkuleer” due to the precipitated protein “flokkies” seen in the reaction mixture). The ethanol concentration in the test varies from 68% to 76% ethanol and milk that flocculates is then declared unfit for further heat processing and rejected. The reason for rejection of such milk by the processing dairy can be ascribed to technological problems during, or following, heat treatment. Ultra High Heat (UHT) treatment of ethanol unstable milk can result in problems such as burnt deposits and blockages in the processing equipment, which could result in biofilms if not cleaned and removed properly.

Gelation: Gelation is a major factor limiting the shelf life of UHT milk. The defect manifests in the formation of a semi-solid to solid coagulum or gel in the milk over time. The gel which forms is a three-dimensional protein matrix initiated by interactions between the whey protein β-lactoglobulin and the к-casein of the casein micelle during the high heat treatment. Significant factors which influence the onset of gelation include the nature of the heat treatment, proteolysis during storage (by milk plasmin or bacterial proteolytic enzymes), milk composition and quality, seasonal milk production factors and storage temperature.
Precipitation: When the casein in milk precipitates it implies that the casein micelles suspended in the milk have become insoluble. If ethanol unstable milk, for example, is subjected to the Alizarol test the insoluble casein forms small particles (Afr. “flokkies”) in the reagent mixture and on the inside surface of the test-tube. The protein is then said to have precipitated or flocculated. In the isolation of specific protein moieties such as enzymes or peptides, such proteins, or derivatives of proteins, can be precipitated using reagents such as trichloracetic acid or hydrochloric acid.
Sweet curdling: The coagulation of casein by rennet during cheese making can be described as “sweet curdling” (Afr. “soetstolling”) since the pH of the coagulum is still above 6. Bacteria growing in milk can also produce enzymes that are capable of the sweet curdling of milk. The spores of bacteria, such as Bacillus cereus, can survive heat treatment of milk. On germination in the processed product, the active cells may produce proteolytic enzymes that can cause sweet curdling in pasteurised, ESL and UHT milk. This can be regarded as one of the causes of gelation. Bacillus cereus is ubiquitous in the environment and raw milk is frequently contaminated with low numbers of spores at the farm from soil, bedding material and milking equipment. There is a seasonal variation in the occurrence of B. cereus, with higher spore counts in milk during the grazing season due to contamination of the teats by soil.
Ropiness: Ropiness in milk is a change from milk’s normal consistency to a condition in which long slimy threads are observed when the milk is poured. This is attributed to the production of carbohydrate (polysaccharide) derivatives by bacteria. The organisms which usually produce ropiness in milk are those possessing a large capsule, such as Alcaligenes viscosus and members of the Escherichia-Aerobacter group. The capsular material produced by many bacteria consists of complex polysaccharides identified as polymers of simple monosaccharides. This defect is sporadically encountered on a seasonal basis in heated milk contaminated by such organisms in South Africa. Since the above organisms are Gram negative and killed by heat treatment, one would not expect to find them in aseptically packaged milk such as ESL and UHT milk. If found in the latter milks it could be attributed to spore forming organisms and be a causative factor in gelation.

Conclusion. All the terms referred to above, with the exception of ropiness and possibly denaturation, basically imply the destabilisation of the micelle suspension in milk resulting in the precipitation of the protein. It might, however, be pragmatic when using the above terminology, to use each term in the context that it is familiar in the South African dairy industry or in every-day language. To the cheese maker the phenomenon is usually known as coagulation. In the production of fermented milk products or when milk sours naturally, it is said to have curdled. When the protein precipitates in the Alizarol test it is said to have flocculated and when it precipitates in UHT milk forming a gel it is referred to as gelation. One would normally not refer to milk as having clotted since such a term is usually associated with blood. It however is not wrong to refer to sour cream as having clotted, but once again this is a matter of semantics or popular parlance.

Reference:

Jooste, P.J. & Du Plessis, C., 2017. Terminology relating to protein instability in milk. Milk SA DRDC Minutes 18 April

2017, Pretoria.