[SOLVED] Food Microbiology and Safety

Food Microbiology and Safety
AProf Helen Billman-Jacobe

Intended learning outcomes

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The intended learning outcomes of this lecture are
-to describe the process for producing a ripened hard cheese
-to understand the use of starter cultures for primary and secondary inoculation in food fermentation.
-problems that can occur with starter cultures -strategies to avoid problems with starter cultures

Dairy fermentations: cheese
Cheddar cheese production
originally from village of Cheddar, Somerset, SW England
made by coagulation of casein in milk with lactic acid produced by LAB
with rennin or rennet
collecting the coagulated casein for further processing
Australia produced 336,700 tonnes of cheese in 2016/17 About 33% milk production goes into cheese-making
Of which about 55% is cheddar cheese
About $1.8 billion in sales on the domestic market

Cheddar Cheese production
Cheddar is an example of ripened, hard cheese
It is made from whole milk
It contains < 39% moisture, 48% fat, orange-yellow (annatto)It is ripened to give desired flavour (numerous compounds)Cheddar processPasteurised, natural colour added, cooled and starter cultures are added Incubated 30C until acidity decreases then rennin/rennet added Allowed to coagulate (30 min), then cut into cubesCooked at 38 C, whey drained away, cheddaring to lose wheyMilled, salted, put in form, pressed 16hr to drain wheyDried for 5 days, packed, cured at 4.4C for 2-12 months Cheddar Cheese: starter cultureMixed strains of Lactococcus lactis ssp. cremoris or lactis. Leuconostoc may be added in some cases for flavourThe starter culture is usually added as frozen concentrates Growth parameters 30C, mesophilic, cells grow, convert lactose to lactic acid lactic acid production usually about 60 min at this level, coagulation by rennin/rennet occurs quickly then incubated for further 30 min for coagulation during curing, cells die and release enzymes into the cheese Cheddar cheese rennin vs rennetRennin and rennet are often confused but they play a similar role in cheese makingRennin is an enzyme mixture found in the stomach of cows, sheep and goatsRennin curdles milk by transforming the soluble milk protein caesinogen into insoluble casein.In young animals who are feeding on milk, rennin is an important first step in digestions milk.Traditionally, rennin from animals was used for cheese makingToday, most cheese is made with enzymes produced by microorganismsAspergillus oryzae is one of the fungi that commercially produce rennet for industrial cheese manufacture Production of cheeses using a secondary culture, e.g. mould Penicillium camemberti and Penicillium roqueforti are filamentous fungi (moulds) Secondary fungal cultureBlue vein cheese First stages similar to cheddar, with bacterial starter cultureCamembert cheeseSecond inoculation with filamentous fungus Penicillium camemberti on the surface. Aerobic growth to form the white rind.Second inoculation with filamentous fungus Penicillium roqueforti, and cheese is spiked to allow in air (aerobic fungus) > veins

Starter culture PROBLEMS
Strain antagonism
mixed cultures either different growth rates, or secretion of inhibitors (acids, bacteriocins)
Loss of desired trait
e.g. loss of plasmid borne gene
Cell death and injury
mainly mishandling of purchased cultures (temperature abuse, etc.)
Inhibitors in raw materials e.g. antibiotics, disinfectant Bacteriophages infect and destroy cells

Bacteriophages
Typical head-tail phage

Bacteriophage (phage) is a virus that infects bacteria. Very common in nature. Cell lysis, virus release
From: http://www.nature.com/nrd/journal/v14/n8/fig_tab/nrd4695_F1.html

Bacteriophages
Can only replicate in bacteria (not animals/plants)
Usually very host specific, often closely related strains of
same species
Inactivated by heat, pH, disinfectants, desiccation, but phage can persist in moist environments.
Can act very quickly (life cycle of ~ 5-10 minutes)
Can reach enormous numbers in environment (of
fermentation facility), e.g. >108 phage/ml
How to deal with this problem??

Starter cultures and bacteriophages:
If one routinely uses pure cultures of bacteria for inoculation of fermented foods, then you run a risk of contamination with bacteriophages
These expand in numbers with every new batch of fermented food, and contaminate the entire manufacturing plant: machinery, floors, pipes, etc.
Typically, the inoculation of a new batch of raw material (e.g. milk), where there are high levels of bacteriophage from the machinery or work environment, will lead to lysis of the starter culture and failure of the fermentation
The batch has to be discarded, usually at considerable cost. Imagine a 100,000 litres of milk to be discarded

Starter cultures and bacteriophages:
Most starter cultures are purchased from specialist sources
They are frozen or freeze-dried concentrates with high cell
densities, and can be used directly
They are designed to be bacteriophage resistant and to maintain consistent genetic characteristics, in order that the fermentation product is of consistent quality.
For interest:
small large
https://www.cheeselinks.com.au/product/blue-camembert-fetta-kit/ http://www.cheesemaking.com.au/product-category/cheese-yoghurt-and-other-ripening-cultures/

Bacteriophages, and how to deal with them:
Regular cleaning/disinfection in fermenter facility
No residual fluids (milk, etc.) in pipes/fermenter that would allow
bacteria and phage to grow or contaminate next batch
Aseptic technique, heat treat fermenter tanks/pipes before use
Use phage resistant starter cultures, and change/rotate starter culture strains regularly
Monitor plant for phage active against starter culture strains
If confirm a phage damaged culture, do intensive clean

In summary;
We have looked at cheddar cheese as an example of a dairy fermentation that results in a hard cheese and how the primary starter culture is important in influencing the characteristics of the cheese.
Secondary inoculation with other bacteria or moulds result in different styles of cheese for instance camembert and stilton.
Starter cultures used for either primary or secondary inoculation need to be of high quality and obtained from a reliable source.
Bacteriophage are a significant risk to starter cultures and can result in failure of fermentation

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