Cultures

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

Graph of natural fermentation of raw milkRaw milk at warm temperature will support a variety of micro-organisms in succession as the pH changes over time (see illustration on the right). 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.

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 Raw milk quality.
  • Cocci (spherical cells) 1 µm in diameter OR rods (rod shaped cells) 1 µm wide and 2 to 3 µm long.

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 

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 

(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. 

Table 7.1: Some lactic acid bacteria commonly used in cheese making.

 

Old Name

New Name

Comments

Mesophilic Cultures

Streptococcus cremoris

Streptococcus lactis

Lactococcus lactis ssp cremoris

Lactococcus lactis ssp lactis

  • As a mixed blend these two form the most common mesophilic and homofermentative culture.
  • Used for many low temperature varieties; fresh cheese, Cheddar, American varieties etc.

Leuconostoc citrovorum

Leuconostoc lactis

Leuconostoc mesenteroides spp cremoris

Leuconostoc lactis

  • Heterofermentative cultures; ferment citrate; produce both CO2 and diacetyl.
  • Often mixed with L. lactis ssp cremoris / lactis for traditional butter and butter milk.
  • May be used for cheese with small holes.

Streptococcus diacetylactis

Lactococcus lactis ssp lactis biovar diacetylactis

  • Hetero culture; ferments citrate; produces both CO2 and diacetyl
  • Mixed with homofermentative lactococci for cheese with small holes

Thermophilic Cultures

Streptococcus thermophilus

Lactobacillus helveticus

Streptococcus thermophilus

Lactobacillus helveticus

  • Commonly used coccus/rod blend for high temperature varieties, Swiss and Italian
  • L. helveticus galactose positive, used to reduce browning in Moz, and to promote proteolysis in Cheddar

Lactobacillus bulgaricus

Lactobacillus delbrueckii ssp bulgaricus

  • Commonly blended with S. salivarius. ssp thermophilus for yoghurt
  • Alternative to L. helveticus in high temperature cheese

Lactobacillus lactis

Lactobacillus delbrueckii ssp lactis

  • Alternative to L. helveticus and L. bulgaricus where low acid is preferred as in mild and probiotic  yoghurts
 

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.

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.

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. 

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