7. CULTURES

7.1 General Functions of Cheese Cultures

Lactic acid bacteria and other microorganisms are present as 'contaminants' in cheese milk and further environmental contamination takes place during cheese manufacture. Provided the milk is not chilled, it is possible to make cheese without any additional cultures, but normal practice is to add domestic cultures for the manufacture of cheese from both raw and pasteurized milk. Culture, then, refers to prepared inocula of bacteria, yeast and moulds which are added to cheese milk and cheese. In the broadest terms cultures have two purposes in cheese making: (1) to develop acidity; and (2) to promote ripening. Lactic acid cultures contribute to both of these functions, while numerous special or secondary cultures are added to help with the second function.

Development of Acidity

Raw milk at warm temperature will support a variety of micro-organisms in succession as the pH changes over time (see illustration in Figure 7.1). In controlled conversion of milk to fermented dairy products, a primary component of fermentation is development of acidity by lactic acid bacteria. Acid development in cheese making is absolutely essential to cheese flavour, cheese texture and cheese safety. Acid is required to:

• Assist coagulation. Lower pH results in faster coagulation and in acid coagulated cheese is the only factor which induces coagulation.
• Promote syneresis. This is a most critical means of controlling moisture content. Acidity (specifically reduced pH) causes the protein matrix in the curd to contract and squeeze out moisture. That process of contraction is called syneresis.
• Prevent growth of pathogenic and spoilage bacteria. Proper rate and extent of acid development is the most important principle with respect to quality and safety of natural cheese. I would argue that with the exception of noncultured cheese varieties such as ricotta, proper culture growth and acid development is equal in importance to pasteurization with respect to safety.
• Develop cheese texture, flavour and colour. The following general associations are relevant to most cheese varieties.
• high pH produces soft, soapy, fruity and bitter cheese
• low pH produces cheese with brittle texture and mottled colour

Assist curing

• Growth factors produced by lactic cultures are required for other non-starter microorganisms which contribute to the desired flavour and body of cheese
• Enzymes (both lipases and proteases) produced by lactic cultures contribute to interior ripening of cheese and are important to both flavour and texture development.
• Special or secondary cultures are responsible for eye development, surface ripening etc. See Section 7.5.

7.2 General characteristics of lactic acid cultures

Lactic acid cultures are often called starters or referred to by the acronym 'LAB' which stands for lactic acid bacteria. The following lists identify and briefly describe some properties of LAB. LAB are:

• Non-motile gram+ bacteria
• Non spore forming
• Catalase and nitrate negative
• Micro-aerophilic which means they tolerate only low oxygen concentrations
• Not psychrotrophic which means that cold storage rapidly depletes their numbers and encourages the growth of spoilage bacteria as described in Chapter 4.
• Cocci (spherical cells) 1 µm in diameter OR rods (rod shaped cells) 1 µm wide and 2 to 3 µm long.

7.3 Classification of Lactic Acid Cultures

Classification of lactic cultures, is confusing, because many LAB have been renamed. Table 7.1 lists the old and new Latin names for some common lactic cultures.

It is helpful to categorize lactic cultures according to general technological and growth characteristics. From that perspective, LAB are grouped by four criteria, namely:

• Principal metabolites (end products of fermentation)
• Optimum growth temperatures: meso- versus thermophilic
• Starter composition:
• Pure defined strains
• Mixed defined strains
• Pure (single) strains
• Forms of inoculation

(I) Principal metabolites: homo- versus heterofermentative

Homofermentative means that lactic acid is the principal metabolite without production of gas (CO₂) and flavour compounds.

Heterofermentative means that lactic acid is the principal end product of fermentation but technologically significant amounts of one or more of the following metabolites are also produced.

• Carbon dioxide (CO₂ ) which causes the small gas holes in Havarti, Gouda and other cheeses. Gasiness in most cheese varieties is a defect.
• Short chain fatty acids such as acetic acid and propionic
• Acetaldehyde, a principal component of yoghurt flavour
• Diacetyl, a principal flavour note in sour cream, butter milk, Dutch cheese and Havarti cheese
• Ethyl alcohol

(II) Optimum growth temperatures: meso- versus thermophilic

Mesophilic cultures as the name implies prefer medium range temperatures, rather than cold temperatures (psychrophilic) or hot temperatures (thermophilic).

• Optimum growth range for mesophyllic cultures is 30 - 35C.
• Acid production is slow or absent at temperatures less than 20C.
• Growth is inhibited at temperatures greater than 39C.
• Generally any cheese which does not require high temperatures to dry the curd will utilize mesophilic cultures. These include Cheddar, soft ripened cheese, most fresh cheese, and most washed cheese.
• Include both homo- and heterofermentative cultues (Table 7.1)

Thermophilic cultures are defined by their ability to grow at temperatures above 40C. With respect to cheese making their important characteristics are:

• Optimum growth in the range of 39-50C
• Survive 55C or higher
• Minimum growth temperature is about 20C below which cell counts decrease rapidly, so, bulk thermophilic cultures should not be stored at temperatures <20C.
• Thermophilic starters are normally mixtures of cocci and rod cultures which at the time of inoculation are about equal in numbers. That is, the initial inoculum is 50% cocci and 50% rods.
• Rod/cocci blends grow together in a relationship referred to as 'mutualism' where the overall growth rate and acid production is faster than either culture on its own. The rods produce amino acids and peptides which stimulate the growth of cocci, and the cocci produce formic acid which is required by rods.
• The balance between the rods and cocci can be controlled by temperature and pH
• The cocci prefer higher temperatures (optimum about 46C) than the rods (optimum about 39C).
• The rods are more acid tolerant than the cocci, so, normally the cocci develop the initial acidity and out grow the rods. But, as the acidity increases the rods begin to grow faster than the cocci.
• Some thermophilic rod cultures have the ability to ferment galactose as well as glucose which is desirable in some cheese, especially Mozzarella.
• Although yoghurt cultures which include both rod and cocci, produce acetaldehyde which is the principal component of the characteristic yoghurt flavour, none of the thermophilic LAB are considered heterofermentative (Table 7.1).

(III) Starter composition:

• Pure defined cultures are single strain cultures selected from natural mixed populations for specific properties such as proteolytic characteristics or resistance to phage (bacterial viruses).
• May be rotated to avoid phage infection
• Have the advantages of uniform rate of acid development and uniform flavour profiles
• A mixed defined culture is a blend of single strain cultures.
• May be rotated to avoid phage infection
• Has the advantages of uniform rate of acid development and uniform flavour profiles
• Mixed cultures are nonspecific blends of cultures, some what like a natural eco system
• Normally have complex systems of phage resistance
• Mixed mesophilic starters are still common, but thermophilic starters are usually mixed defined cultures.
• Disadvantage is nonuniform rates of acid development from vat to vat and nonuniform flavour profiles

(IV) Forms of Inoculation

Cultures can be carried and prepared for cheese milk inoculation in one of three general formats:

• Traditional starters which need several scale up transfers. This system requires some microbiological facilities and expertise and is only feasible for very large plants or perhaps for smaller plants which use mixed strain cultures.
• Bulk set culture. In this system, the culture supplier does all the purification and transfer work, and delivers a bulk set culture which is used to inoculate a bulk culture, which in turn is used to inoculate the cheese milk. Bulk cultures are the norm in medium to large plants because the cost savings are significant.
• Direct to the vat cultures require no scale up at the cheese plant. Concentrated cultures ready to inoculate the cheese milk are supplied directly by the culture supplier.

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7. 4 Summary: technological properties of lactic acid cultures

In addition to properties mentioned above, the following lists includes other technological properties of importance to cheese making. Note that many of these technological characteristics are encoded on extra-chromosomal genetic material called plasmids. Plasmids have the disadvantage of being unstable so characteristics encoded on plasmids are also unstable. The advantage is that plasmids can be transferred to other bacteria so microbiologists can readily transfer technological properties from one LAB to another.

• Lactose metabolism. Most but not all LAB are able to metabolize lactose.
• Galactose metabolism. The ability to ferment lactose is important for late acid development in Italian cheese and to control browning on Mozzarella cheese.
• Proteolytic characteristics which determine cheese flavour development.
• Resistance to phage (bacterial viruses).
• The ability to metabolize citrate which is associated with flavour development (diacetyl or butter milk flavour) and gas formation.
• Production of bacteriocins, that is, antibiotics produced by bacteria against other bacteria.
• Resistance to bacteriocins
• Antibiotic resistance.

7.5 Secondary Cultures

In addition to lactic acid cultures many special or secondary cultures are used to promote specific ripening (both flavour and texture) characteristics.

• Large holes: Propioni bacterium freudenreichii subsp. shermaniee
• White moulds: Penicillium camembertii, P. caseiocolum, and P. candidum
• Blue/green moulds: Penicillium roqueforti, Penicillium glaucum
• Smears:
• yeasts and moulds
• Various coryneform bacteria including Brevibacterium linens, several species of micrococci, and several species of Staphylocci
• Ripening adjuncts:
• Bacterial or yeast cultures added in addition to the regular lactic acid cultures
• Attenuated cultures which are not intended to grow but only to contribute their enzymes
• Species of Lactobacilli and pediococci which are intended to grow during cheese ripening and contribute enzymes

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7.6 Culture Production, Distribution and Storage

Commercial culture preparation

Genetic techniques offer much opportunity to develop cultures with specific technological characteristics. However, at the commercial level, culture preparation is relatively simple.

• Lactic cultures are grown in buffered media to facilitate maximum growth without acid inhibition
• The cells are concentrated by centrifugation
• The cell concentrate is fast frozen or freeze dried (lyophilized). Frozen (-40C) or lyophilized cultures can be stored for several months without substantial loss of activity. Lyophilized cultures usually require a longer "lag time", i.e. time between inoculation and rapid cell growth.

Culture Practice in the Cheese Plant

Direct to the vat cultures need only be stored under prescribed conditions and opened and delivered to the vat under aseptic conditions. The following comments relate to the preparation of bulk culture at the cheese plant.

• Culture preparation should take place in a separate culture room which is kept at positive air pressure with hepa-filtered air (0.2 µm filter).
• All surfaces in the culture room must be of a material that can be sterilized.
• Use sterile pipettes and sanitize surfaces and equipment with 200 ppm chlorine.
• Alternative culture media are:
• Milk, but care must be taken to avoid rancid milk, mastitic milk, milk containing antibiotics, and milk with high bacteria counts.
• 10 -12% reconstituted skim milk powder is adequate provided that the powder is tested and certified antibiotic free.
• Whey and reconstituted whey powder may be used, but may not achieve the same cell counts as skim milk (due to less buffer capacity).
• A number of commercially prepared culture media are available. Most of these are based on milk protein powders.
• Culture media may be buffered with phosphates to increase cell counts but some cultures particularly Lactobacillus. bulgaricus appear to be inhibited by phosphates.
• Addition of phosphates also confers phage resistance because phosphates bind calcium, and phage require calcium to attach themselves to the bacterial cells.
• Calcium reduced skim milk powder and addition of anhydrous ammonia have also been used to inhibit phage in bulk cultures
• Culture media should be heated (at >88C for 1 h) to destroy bacteria and some inhibitory substances. Heating also reduces the redox potential (lowers oxygen concentration) which encourages the growth of LAB.
• Optimum pH endpoint before cooling is between 4.5 and 5.0. At pH less than 4.5 some cultures will pass from growth (log) phase to stationary phase and will be less active when added to the cheese vat.
• Cell count can be increased by:
• Internal pH control using buffered media
• External pH control by adding sodium hydroxide or ammonium hydroxide to maintain pH at 5.0 - 5.5.
• Generally cultures should be cooled to 4C after the desired minimum pH and cell counts are obtained. However, the optimum storage temperature depends on the particular culture. Consult with the culture supplier. For example, some thermophilic cultures should not be cooled below 20C. Storage time should be as short as possible, but I am aware of plants which successfully use a single bulk set culture for a week before making a new batch.

7.7 Bacteriophage (bacterial viruses)

Bacteriophage are the stuff of a cheese maker's nightmare. Like all viruses, bacteriophage (hence forth abbreviated to phage) are parasites, that is, part of their life cycle is dependent on the host bacteria. Here's a few facts about their characteristics and how they can be controlled.

• Extracellular phage, that is, phage particles existing separate from their bacterial hosts are called mature or resting particles.
• Resting particles are sperm shaped, < 1 micron in length.
• Resting particles consist entirely of DNA (genetic material) and protein. The basic construction is a DNA core enclosed in a protein sheath.
• The basic life cycle, called the lytic cycle, is:
• The phage attaches itself to the bacterial cell wall by its tail, bores a hole in the wall with the help of enzymes and injects its DNA into the cell. The protein sheath remains outside the cell.
• From the moment of invasion the bacteria begins to reproduce phage DNA and protein in addition to its own.
• Nucleic acid and protein strands assemble themselves into new phage particles which eventually lyse the cell (break it open) to release the phage particles into the medium. A new generation of resting phage are now available to repeat the lytic cycle
• Sometimes infection occurs without lysis resulting in a lysogenic culture where infected cells survive and reproduce infected daughter cells. Therefore, cheese cultures can exist in one of three states with respect to phage sensitivity:

(1) Insensitive due to inherent or acquired resistance.

(2) Phage carrier (lysogenic). In this state the bacteria are resistant to another phage infection

(3) Phage sensitive in which case the phage will grow quickly and may terminate the culture. Culture growth will stop when phage levels reach 10³ to 10⁷ per ml.

• Phage have a short latent period (reproduce as quickly as every 30 to 50 min) and a large burst size (each lysed cell will release 50 to 100 new phage).
• Phage are quite strain specific which is the reason for culture rotation. As many as 10 different cultures may be rotated on a daily basis.
• Culture failure due to phage can be recognized by normal acid development initially followed by a decrease or termination of culture growth at a later stage. This is different than inhibition due to antibiotics which can be recognized by no or slow initial growth; if inhibition is not severe, culture growth and acid development by resistant strains or mutants may increase with time.

Summary of phage control measures

• Use aseptic techniques with proper culture room.
• Rotate cultures daily and/or use defined phage resistant strains.
• Use phage resistant media for culture preparation.
• Use direct-to-vat culture to avoid contamination during transfers.
• Use a mixed strain culture of two closely related strains.
• Remove and dispose of whey daily
• Routinely check for presence of phage using a culture activity test with the culture currently in use and some whey from the most recent vat

Figure 7.1 Natural Fermentation of Raw Milk

8. COAGULATION

8.1 Milk Structure

Chapter 4 provided an introduction to milk chemistry. Now we look briefly at milk physics to help understand how milk coagulation works. Refer to Figure 8.1 and review the following facts:

• Milk is an emulsion with fat particles (globules) dispersed in an aqueous (watery) environment.
• The fat globules do not coalesce and form a separate layer (oil off or churn) because they are protected by a membrane layer which keeps the fat particles separate from the water phase.
• The principal group of milk proteins, the caseins, are not soluble in water and exist in milk as small particles (<300 nm) called micelles.

We can now define the following terms:

Milk: 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.

Milk Plasma: what is left after you separate the fat globules; equivalent to skim milk for practical purposes.

Milk Serum: what is left after you take away both fat globules and casein micelles; equivalent to cheese whey for most practical purposes

Milk permeate: what is left after you take away fat globules, casein micelles, and whey proteins.

Coagulation is what happens when the casein micelles stick together. Because casein particles are hydrophobic (they hate water) their natural tendency is to aggregate (clump together). In normal milk, aggregation is prevented by two factors. If one of these factors is eliminated the micelles will aggregate and form a gel something like jello.

• The first stabilizing factor is a 'hairy' layer of surface active protein, called kappa-casein (-casein), on the surface of the micelle. This layer helps prevent the micelles from getting close enough to stick together.
• The second factor is a negative charge on the micelles. At the pH of milk the micelles are negatively charged so they repel each other.

So, basically there are two ways to coagulate milk; one is to remove the hairy layer from the micelles. That's called enzymic coagulation. The other is to neutralize the negative charge on the micelle. That can be accomplished by acidification or a combination of high temperature and acidification.

8.2 Enzymic Coagulation of Milk

The three stages of enzymic coagulation

(1) Primary Stage

In the first stage, the enzyme (rennet) cuts off a specific fragment of one of the caseins, namely, -casein. At the natural pH of milk, about 80% of -casein must be cleaved to permit aggregation of the micelles to proceed.

(2) Secondary Stage

The next stage is the physical process of aggregation of casein particles (micelles) to form a gel. After losing its water soluble tail, -casein can no longer keep the casein particles separated, so they begin to form chains and clusters. The clusters continue to grow until they form a continuous, three dimensional network which traps water inside, and forms a gel, something like Jell-o.

(3) The third stage refers to an ongoing development of the gel network. For some cheese the gel is cut as soon as it is firm enough to do so. For others, like soft ripened cheese, cutting is delayed while the gel continues to become firmer.

Effects of processing parameters on enzymic coagulation

Because rennet coagulation takes place in stages, it is necessary to understand the effect of processing on each stage. We will focus mainly on only the first and second stages.

Effect of pH. Lower pH increases enzyme activity and neutralizes charge repulsion between micelles. Therefore, both primary and secondary stages of coagulation proceed more quickly at lower pH.

Effect of Calcium . Calcium is not required for the primary stage (i.e., enzyme hydrolysis of -casein) but is essential to aggregation of the casein micelles. At low levels of calcium the primary phase goes to completion. Subsequently, instantaneous coagulation can be induced by adding sufficient calcium chloride.

Effect of temperature. The optimum coagulation temperature for most cheese is 30-32C, the exception is Swiss which is set at 37C.

• At temperature less than 30C the gel is weak and difficult to cut without excessive yield loss due to fines.
• At temperatures less than 20C coagulation does not occur, but the primary stage goes to completion and the milk will then coagulate quickly when warmed.

Effects of heat treatments.

• Mild heat treatment such as pasteurization decreases the rate of the secondary stage. During heat treatment calcium and phosphate move from soluble to colloidal (insoluble) form, so there is less calcium available to assist with coagulation. This effect is reversed by cold storage or the addition of calcium chloride
• Heat treatment in excess of pasteurization results in increased clotting time and a weak gel. High heat treatments cause absorption of whey proteins onto the casein particles. The casein particles are then unable to form a strong gel.

Effects of Homogenization: The following effects occur if the cheese milk is homogenized in its entirety. As noted in Chapter 5, some of these results may be different if only the cream is homogenized and then added back to the skim milk. Homogenization primarily affects the secondary phase of aggregation. Some cheese quality effects are also noted.

• Reduced aggregation of casein particles
• Decreased syneresis
• Finer gel network due to smaller fat globules
• Improved texture of soft cheese
• Fat recovery (i.e., percent transfer from milk to cheese) is increased (Note: the same is true for acid and heat/acid coagulated cheese).
• Hard cheese becomes rubbery
• Makes cheese whiter because the yellow fat is masked by the artificial protein membranes on the homogenized fat globules.

Coagulating Enzymes

The traditional enzyme is rennet (chymosin) which is derived from the abomasum of the milk fed calf. The practice of cheese making probably began when somebody discovered that milk stored in bags made from calf stomachs formed a sweet curd.

Other proteases which have been used for cheese making include:

• Pepsins from the pig, cow and chicken
• Microbial proteases (Mucor miehi, Mucor pusillus, and Endothia parasitica).
• Synthetic chymosin by recombinant DNA techniques using strains of Eshericia coli or Klaveromyces lactis or Aspergillus niger as host organism is now available. The transferred genetic material exists in the host cell in the form of a plasmid and is used as a template for the production of an enzyme identical to chymosin.

Requirements of suitable coagulating enzymes

• Suitable ratio of clotting to proteolytic activity (C/P). This ratio is dependent on the specificity of the enzyme for the Phe₁₀₅-Met₁₀₆ bond of -casein. Most rennet substitutes are more proteolytic than rennet (i.e., low C/P) and cause diminished yields of casein and fat, and bitterness during ripening

• Proteolytic specificity. Structure and flavour of ripened cheese depends on the type of proteolysis caused by the coagulant during cheese curing. The exception is in cheese such as Swiss or Parmesan where most of the rennet activity is destroyed by the high cooking temperature. During ripening chymosin breaks down one of the caseins, namely _s1-casein much more than other caseins.



• High pH optimum. Rennet activity is stable and able to coagulate milk at the normal pH of milk although its activity increases with decreasing pH. Most pepsins and microbial proteases are denatured at the pH of milk which has been a major difficulty in developing rennet substitutes.

• Denaturation temperature is important for two reasons:

• Ripening due to coagulating enzymes is not desirable in cooked cheese such as Swiss and Italian types. Rennet is eliminated during the high temperature cook in these cheeses but microbial coagulants are not.
• The coagulant must be eliminated by pasteurization to prevent proteolysis in products made from whey. Some microbial rennets survive pasteurization.

• Distribution between curd and whey. Only 0-15% of rennet remains in the curd, but small amounts of residual rennet are significant to ripening of aged cheese. The most important factors which determine rennet retention are:

• Cooking treatments.
• As noted above, rennet does not survive in high temperature cooked cheese varieties.
• In low cooked cheese such as Cheddar, variations in cooking temperature and time influence rennet activity during aging.
• The pH at draining. Rennet is more soluble at low pH and, therefore, the amount retained in the curd increases with decreasing pH at draining. Retention of microbial rennets in the curd is independent of pH at draining.
• Changing rennet sources may also influence rennet retention and cheese ripening. Different rennets with the same coagulating properties may have different thermal tolerances and different proteolytic characteristics.

• Standard and consistent activity. Single strength rennet is standardized so that 200 ml coagulates 1,000 kg of milk in 30 - 40 min. Typical commercial rennet preparations are about 50% chymosin (calf rennet) and 50% bovine pepsin, so there is much opportunity for variation. Commercial calf rennet preparations are about 94-96% chymosin. Using recombinant rennet it should be possible to produce commercial rennet preparations which are more consistent with respect to all of the properties listed above, including proteolytic specificity and heat tolerance.

8.3 Acid coagulation

Acid milk gels can be formed by lactic bacteria or the use of acidifying agents such as glucono-delta-lactone (GDL is slowly hydrolysed to gluconic acid in the presence of water). Acid coagulation is used in the production of cottage cheese, bakers cheese and quark as well as other fermented milk products such as yoghurt, commercial butter milk, kefir etc. In the case of cottage cheese and quark a small amount of chymosin may be used (2 ml/1,000 hl) to make the curd more elastic and less subject to breakage (dusting).

8.4 Heat-Acid coagulation

This process permits recovery of caseins and whey proteins in a single step. The basic principle is that whey proteins which are normally acid stable, become sensitive to acid coagulation after heat treatment. This principle is exploited in the manufacture of ricotta cheese, Paneer and Channa, and in the manufacture of "co-precipitated" milk protein concentrates. The basic process for heat-acid coagulation is:

• Heat milk or milk-whey blends to at least 80C for at least five minutes to completely denature (unfold) the whey proteins and encourage association of whey proteins with casein micelles.
• Continue heating and acidify slowly with gentle agitation. The caseins and whey proteins will coagulate together and form either sinking or floating curds.

Figure 8.1. Structural elements of milk. After Walstra and Jenness, 1984. Dairy Chemistry and Physics, Wiley & Sons, N.Y.