Lesson 20. ACCELERATED CHEESE RIPENING

Module 5. Application of biotechnology in dairying

Lesson 20
ACCELERATED CHEESE RIPENING

20.1 Introduction

Ripening of cheese refers to physical and biochemical changes that take place in cheese when it is being held under specific and controlled conditions. Ripening process is considered as the most crucial step in cheese making as it has greater influence on the development of characteristic cheese flavor. Most varieties of cheeses require 6-12 months of ripening period depending on the type of cheese and/or conditions of ripening. Two important parameters that influence cheese ripening are temperature and relative humidity.

20.2 Ripening Changes

A series of microbiological and biochemical changes that occur during ripening process are responsible for converting fresh, bland cheese curd into a final mellow, waxy product having a characteristic taste, aroma and texture. Ample information with regard to contribution of milk components to maturation is available in most varieties of the cheeses due to break down of components by enzymes derived from rennet, milk and added cheese starters to control the overall intensity of flavor.

20.2.1 Physical changes

Gradual change occurs from rubbery curd of green cheese to the mellow, waxy ripened product, due to proteolysis and velvetiness of cheese due to lipolysis.

20.2.2 Chemical changes

The major chemical changes occurring during ripening are
(i) glycolysis i.e. conversion of residual lactose into lactic acid and metabolism of lactate and citrate yielding end products such as diacetyl, acetyl methyl carbinol etc;
(ii) proteolysis, wherein proteins are broken down to amino acids and further to simpler compounds like amines (Histamine etc.); and
(iii) lipolysis which involves hydrolysis of milk fats to fatty acids, further leading to the formation of keto-acids etc.,

Among these, proteolysis is considered to be the most important change occurring in cheese and often used as ripening index of cheese.

20.3 Approaches for Accelerating Cheese Ripening

The ripening of cheese is a slow and consequently an expensive process as it involves heavy expenditure on refrigeration and labour apart from resulting in loss of product due to microbial damage. Attempts are being made to reduce ripening period by accelerating the bio-chemical changes. Two important approaches employed or tried by the cheese industry for accelerating the cheese ripening process include (1) Technological and (2) Bio-technological.

20.3.1 Technological approaches

Technological approaches involve the manipulation of curing conditions such as elevated temperatures or by increasing the moisture content of cheeses and making use of these as cheese slurries.

Cheese is ripened at elevated temperature i.e. at 15°-20°C for about 2-3 weeks depending upon the required flavor intensity and on the type of cheese before transferring to traditional normal temperature of ripening. As a result of elevated temperature, bacterial growth is stimulated resulting in increased release of enzymes for bringing about enhanced proteolytic and lipolytic changes. However, controlling the temperature is very critical as even 1°C rise in normal ripening temperature results in improper balance between proteolysis and lipolysis.

Cheese slurries are prepared by raising the moisture content in the product by adding at various stages as per the convenience and incubating at higher temperatures (30°C) for few days (7-10 days). The slurries containing 40% total solids are being reported to have developed strong flavor in days rather than in months. However, these methods suffer due to lack of control and often the overall flavor scores of such cheeses were depressed by a higher incidence of off flavors. Moreover, with cheese-slurry method the contamination with undesirable organisms is quite possible because of exposure to air, or due to aeration during incubation. In view of such drawbacks, development of flavor through natural and rapid methods by biotechnological approaches for accelerating the ripening process is most promising.

20.3.2 Biotechnological approaches

Biotechnological approaches involve addition of exogenous enzymes (proteinases, peptidases, and lipases), use of attenuated starter cultures, adjunct cultures, or genetically modified lactic cultures.

20.3.2.1 Exogenous microbial enzymes

Addition of exogenous enzymes enhances the enzyme pool and thus can help in accelerating the ripening changes. These include addition of ß-galactosidases, proteolytic enzymes, lipolytic enzymes or mixture of these.

20.3.2.1.1
β-galactosidase

β-galactosidase hydrolyses the lactose in milk yielding glucose and galactose. Glucose is better utilized by the microorganisms than other carbohydrates and thus helps in rapid proliferation of microbes. β-galactosidase helps in accelerating the cheese ripening by reducing the cheddaring time due to faster development of acidity due to enhanced growth and activity of microbes and reduces ripening period by providing proteinases indirectly due to increased population of starters. The basis of the increased flavor may have been due to more rapid proteolysis in cheeses caused by either higher proteinase content contributed by increased populations of starter bacteria or by contaminating proteinases of β-galactosidase preparation. However, clear distinction is still under investigation.

20.3.2.1.2 Proteinases

Proteinases include chymosin, natural proteinases present in milk, and proteinases produced from starter and non-starter microorganisms. Proteinases and peptidases break down proteins to peptides and further to free amino acids. Proteinases reduce the ripening period due to availability of increased substrate resulting from casein hydrolysis for microbial peptidases, thus accelerating production of flavor precursors and flavor compounds.

Three different types of proteinases namely acid proteinases, alkaline proteinases or neutral proteinases can be added. Acid and alkaline proteinases produce weak, soft body with crumbly texture, while neutral proteinases produce good quality cheese. The proteinases derived from Aspergillus spp. Pseudomonas fluroscens, Micrococcus caseolyticus, Bacillus subtilis etc have been tested. Although the ripening period can be reduced by the use of proteinases by 35 to 50%, it results in development of bitterness which is undesirable and requires more attention.

Peptidase extract alone results only a minor effect on flavor intensity because no additional proteolysis can be achieved to provide more substrate peptides. If only proteinases are added, then more peptides are accumulated resulting bitterness. Instead of using proteinases or peptidases alone, a combination of proteinases and peptidase is being increasingly used. When they are added in combination, proteinases form peptides and peptidases utilize these peptides forming free amino acids.

20.1

20.3.2.2 Lipases


Lipolysis is desirable in strongly flavored cheeses such as Italian and Blue veined cheeses but not in delicately flavored varieties like cheddar.

20.3.2.3 Mixtures of enzymes

For proper and balanced flavor development in cheese it is preferred to use a mixture of enzymes as they enhance the rate of multiple reactions aimed at increasing or modifying particular aspects of cheese ripening instead of individual enzymes which can control only one particular type of reaction during ripening. Few examples for commercially available cheese enzyme mixtures are Accelase™, Neutrase™, DCA 50 etc.

20.3.2.4 Methods of enzyme additions

Enzymes are added to milk or to cheese curd at different stages. Addition of enzymes to milk in solution form helps in uniform mixing and final homogenous distribution of enzymes in curd. It also ensures maximum interaction between coagulated particles and enzymes. The limitation of such additions is the loss of about 60-90% of enzymes in whey as they are water soluble contaminating the whey and thus making it unsuitable for other purposes. In addition, this protocol often results in poor gel strength and cause difficulties during working of the cheese curd for expulsion of moisture due to early breakdown of casein. Alternatively enzymes are mixed with salt and sprinkled on the surface of cheese curd after milling, wherein salt acts as vehicle. However, the major limitations of salt enzyme mixtures that the salt dissolves and penetrates the curd more rapidly than the enzymes and leaving the enzymes to remain on surface and developing ‘Hot spots’. The other defects are mottled wavy colour defects. Moreover, this method is not suitable for surface-salted cheeses.

20.3.2.5 Microencapsulation

Another promising method of exploiting enzyme technology for accelerated cheese ripening includes encapsulation of enzymes. In this method, enzyme of interest is encapsulated in some type of artificial sacs usually with the object of protecting it from particular environment. The sac dissolves at the required target site and stage, and releases the enzyme for its designated action. In cheese manufacture, microencapsulation helps in entrapment of more enzymes in cheese curd. Various substances have been used for the encapsulation of enzymes but most widely used ones are liposomes. These are artificial sacs/capsules in micro vesicle form consisting of a central aqueous core surrounded by one or more eccentric layers of fat. Liposomes are formed from phosphotidyl choline. These are stable in milk for long time enough to be included in curd and being pressed along with curd. Size of liposomes is ~1 micron. When added to milk, because of its particulate nature, majority of them are retained and enmeshed during coagulation in curd matrix and thus enzyme loss in whey is minimized. The encapsulated enzymes are released subsequently due to the dissolution of capsule by acid environment or other causes and the liberated enzymes cause biochemical changes. This method has several advantages such as simplicity in addition, reduction in loss of enzyme in whey (only less than 10% of enzyme is lost), maintaining flavor balance as there will be regulated seepage of enzyme from capsule into cheese, absence of body and textural defects as enzymes are uniformly distributed in addition to providing better and good mouthfeel as it contains high fat due to presence of capsules.

20.3.2.6 Manipulation of starters

Many of starter organisms produce proteinases and peptidases. For accelerated cheese ripening, addition of increased volumes of starter culture or enhancing their growth is necessary for producing more proteinases or peptidases. But increasing the starter culture would affect the manufacturing of cheese and produce “atypical” cheese by increased production of acid and hence modification of cheese starters is necessary. The concept is based on the augmentation of normal starter inoculum with starter preparations which have been treated in such a way so as to prevent them from metabolizing and producing acid during cheese making but yet have their important degradative enzymes left intact.

20.3.2.7 Attenuated starters

Attenuated starters are the modified lactic acid bacteria that lack the ability of producing sufficient levels of acid during the cheese making but provide active enzymes which can play a significant role in maturation and flavor development in cheese. However, attenuated starters are always added to cheese milk along with regular starter cultures. Various approaches for the production of attenuated starters are heat or freeze-shocked cells, solvent treated cells, natural mutant starters, lysozyme treatment, spray drying etc.

In one of these approaches i.e. heat-shocking of starter cells by sublethal heat treatment at 60°C or 70°C for few seconds substantially delays lactic acid production of mixed strain mesophilic starter cultures or thermophilic streptococci and lactobacilli, while only marginally reducing their proteinase or peptidase activity. Several studies have shown that addition of heat shocked cells significantly reduces the period of cheese ripening and intensifies the flavor production. But heating procedure was found to be critical since slightly higher temperatures caused dramatic inactivation of proteinases and some of peptidases. The cost is prohibitively high, as higher volumes of heat shocked cells (>109 cfu/g) is necessary for bringing about desirable effect.

Freezing and thawing of lactic acid bacteria kill them without inactivating their enzymes. For this kind of attenuation, concentrated bacterial cells are subjected to freezing at –20°C overnight and rapidly thawing the cells to 40°C. The other methods of attenuation are freeze drying or spray drying of concentrated starter cultures. However, these methods have not gained much commercial value.

In lysozyme treated cells, the lysozyme hydrolyses ß 1-4 linkage of peptidogylcan of bacterial cell wall i.e. linkage between N-acetyl Muramic acid and N- acetyl Glucosamine, resulting in weakening of cell wall followed by lysis of cell. Lysozyme treatment has been used to prevent acid production by starters. However, the enzyme is too expensive to apply to large scale cheese making and the results with cheddar cheese suggested that typical flavor was only marginally increased even by increased starter proteinase and peptidase activity equivalent to 1010 cells/g cheese.

In case of solvent treated cells, treatment of starter cell suspension with organic solvents results in activation of some membrane bound proteolytic enzymes. Now it is possible to generate cells which do not produce acid but have 10 times greater peptidase activity than normal cells. But this approach suffers due to prohibition by regulatory authorities across the globe.

20.3.2.8 Use of mutant starter strains

It consists of lactase negative (Lac-) or proteinases negative (Prt-) or lactase negative proteinases positive (Lac- Prt+) derivatives which have also been found to significantly influences ripening changes in cheeses. These mutant strains can be obtained either by selection from natural pool of mutants or through induction of mutation using x-rays or chemicals such as ethidium bromide, nitrosoguanidine (NTG) etc., The Lac- strains of LAB with higher proteinases and or peptidase activity are the most promising ones. Extensive research on the use of these mutant strains by and large has been highly promising till date, but their commercial use is yet to be explored.

20.3.2.9 Adjunct starters

This approach relies heavily on the use of lactobacilli which intensify the cheese flavor production when used in combination with lactococci. It is believed that addition of small volumes of these adjunct lactobacilli modify the proteolysis resulting in higher concentration of amino acids. The lactobacilli can be mesophilic or thermophilic strains. In comparison with mesophilic strains, the thermophilic lactobacilli die out rapidly during cheese making, lyse and release intracellular proteolytic enzymes to produce higher amounts of free amino acids. Thermpohilic lactobacilli do not grow in cheeses like cheddar, but when added with other cheese starters intensify the flavor production.

20.3.2.10 Genetic engineering

The decoding of complete genome sequence of Lactococcus lactis ssp. lactis IL 1403 strain has raised the hopes of exploiting the genetic engineering of starter cultures as the best options available for finding a viable, sustainable and promising tool for accelerating the cheese ripening. Most of the strains of starter cultures used in cheese industries can be modified by DNA recombination mechanisms. It is possible to produce starter cultures expressing proteinases and many intracellular enzymes which play significant role in secondary proteolysis during cheese ripening. The plasmid controlled characters like lactose utilization; protein metabolism and citrate metabolism can be transferred to suitable recipient strains. For example, more proteolysis producing strain can be allowed to take up lactose utilizing plasmid (if it is lac-) and thus two characters can be combined.

Genetic engineering research is also capable of delivering the technology to make cultures tailored to specific cheese flavor and texture profiles. However, identifying the key enzymes and their role in ripening the cheese is yet to be established and in the absence of such knowledge, the exploitation of genetic engineering for acceleration of cheese ripening will be a limiting factor, in spite enormous potential of this technology. In future, if this technique is specially developed by the dairy researchers to be safe and food compatible, it will play a vital role in proving the efficacy of this metabolic, rather than enzymatic approach.

Suggested Readings

Chapman, H.R., & Sharpe, M.E. (1990). Microbiology of cheese. In R.K. Robinson (Ed.), Dairy microbiology (vol.2). London: Elsevier Applied Science Publishers.

Barry A. Law Dr. A. Y. Tamime. 2010. Technology of cheese making. Second edition Wiley publications

Internet resources

http://en.wikipedia.org/wiki/Cheese_ripening

Last modified: Thursday, 1 November 2012, 9:19 AM