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Lectures 15 Notes

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27 views22 pages

Lectures 15 Notes

TEST PREP

Uploaded by

Omogau Kekana
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© © All Rights Reserved
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Fermented and Microbial foods

WHAT IS IMPORTANT?
▪ Why fermentation?

▪ Yeasts and LAB involved (scientific names and properties/characteristics).

▪ Activities of LAB in food and their health promoting effects.

▪ Malolactic fermentation.

▪ Know different fermented food products, how they are produced (processes and
starter cultures used), what or which organisms cause their spoilage.
INTRODUCTION
• Discovery: before mankind knew of microbes, but witnessed effects of their activity.

• Foods are fermented for improvement of shelf life and safety of product.

• Produced by actions of LAB and fungi, mainly yeasts and to a lesser extent, moulds.

• Able to grow at low pH and reduced aw.

• LAB and facultative yeasts grow under anaerobic conditions and therefore normally occur together in
fermented foods.

• Either act together to produce a product, or one group plays a role as spoilage organism.
YEASTS
• Single celled, true fungi, reproducing either by budding or fission. A few can develop
the filamentous structure of a typical mould.

• Natural habitat: nutrient rich environments e.g. nectarines of plants, plant exudates,
decaying fruits and the body fluids of animals.

• 500 species divided into 60 genera, 33 Ascomycetes, 10 Basidiomycetes and 17


Deuteromycetes.

• Grow effectively aerobically, anaerobic growth impose more fastidious nutritional


requirements.

• Only a small number involved in food fermentation though large diversity of yeasts
exists.

• Either all Ascomycetes or members of the genus Candida.


YEASTS
• Saccharomyces cerevisiae : Found in fermented beverages and foods based on fruits and
vegetables. All strains ferment glucose, sucrose, maltose and raffinose, but not lactose.

• Schizosaccharomyces pombe: dominant in the production of traditional fermented beverages


produced from cereals e.g. maize and millet.

• Kluyvermyces marxianus: a number of varieties isolated from various fermented milks


• K. marxianus var. marxianus (=K. fragilis ) from koumiss and kefir
• K. marxianus var. bulgaricus from yoghurt
• K. marxianus var. lactis from buttermilk, Italian cheese and fermented milks from Manchuria. . They hydrolyse
lactose and ferment galactose.

• Zygosaccharomyces rouxii: Associated with the fermentation of plant products in which the
addition of salt is an integral part of the process.

• Hansenula anomala and Debaryomyces hansenii can grow in concentrated salt solutions. D.
hansenii: Isolated from brined meat products and fermented sausages.
YEASTS
• Pichia guilliermondii and Saccharomycopsis fibuligera: Isolated from tropical
fermented products.

• Geotrichum candidum: Isolated as part of the surface flora of fermented milk


products such as cheeses.

• Spoilage yeast: Zygosaccharomyces bailii.


• Grow at relatively low aw and low pH

• Resistant to preservatives such as sorbic, benzoic and ethanoic acids, sulphur


dioxide and ethanol.

• Causes spoilage of products stored in plastic packs and glass bottles causing
explosion of the container.

• Pasteurization and membrane filtration before filling, followed by stringent


hygiene to prevent post contamination is important.
LACTIC ACID BACTERIA
• Gram positive, non-sporeforming rods or cocci, aerotolerant anaerobes, catalase
and oxidase negative.

• Cellular energy derived from fermentation of carbohydrate to produce lactic


acid through one of the two pathways.

• Homofermentative pathway: lactic acid is produced as a single


product from glucose fermentation.

• Heterofermentative pathway: equimolar amounts of lactate,


ethanol/acetate and CO2 from glucose.

• Principal genera: Lactococcus, Leuconostoc, Pediococcus, Lactobacillus and


Streptococcus.
LACTIC ACID BACTERIA
• Lactobacilli subdivided into 3 groups:
• Obligate homofermenters (Lb. acidophilus, Lb. delbruckii and Lb. helveticus) ferment
hexoses almost exclusively to lactate but are unable to ferment pentoses.

• Facultative heterofermenters (Lb. plantarum, Lb. casei and Lb. sake) ferment hexoses
via the EMP pathway to lactate but have an inducible phosphoketolase which allows them
to ferment pentoses to lactate and acetate.

• Obligate heterofermenters (Lb. brevis, Lb. fermentum, Lb. kefir) use the
phosphoketolase pathway for hexose fermentation.

• Leuconostoc : Produce only D-lactate and are unable to produce ammonia from arginine.

• Pediococcus include species such as P. pentosaceus, and P. halophilus reclassified in the genus of
its own, Tetragenococcus halophilus.

• Streptococci comprise 3 distinct groups, namely: Lactococcus, Enterococcus and Streptococcus.

• Bifidobacterium are included by some authors among LAB though different both
phylogenetically and biochemically.
LAB ACTIVITIES IN FOOD
• LAB inhibits other microbes thereby improving keeping quality and safety of food products.

• Factors contributing to microbial inhibition:


• Production of lactic acid leading to a drop in pH.
• Production of bacteriocins, H2O2, ethanol and diacetyl.
• Nutrient depletion.
• Low redox potential.

Bacteriocins
• Bacterial proteins active against species of closely related to producing strain.
• Natural food preservative produced by food grade microbes.
• Nisin: Produced by certain strains of Lactococcus lactis. Have broad spectrum against G+. G- whose membranes have
been damaged by thermal shock are also sensitive. Bacterial spores are sensitive and nisin is used to inhibit their
outgrowth in heat processed products.

Hydrogen peroxide
• Produced by LAB in the presence of O2. It potentiates the lactoperoxidase antimicrobial system which catalyse
oxidation of thiocyanate by H2O2 to produce hypothiocyanate which kills G- and inhibits G+.

Ethanol
• Produced by heterofermentative LAB and contributes to the inhibition of competitors.

NB: Contribution of other factors is negligible when compared to ability to produce


lactic acid up to around 100 millimolar and pH in range 3.5 to 4.5.
HEALTH PROMOTING EFFECTS OF LAB
Probiotics: live microorganisms which when administered in adequate amounts confer a health
benefit on the host.

• Lactose intolerance
• Individuals lacking the enzyme β-galactosidase which hydrolyses lactose into glucose and galactose.

• The lactose passes to small intestine undigested and is then attacked by resident population of lactose
fermenting microbes producing abdominal discomfort, flatulence and diarrhoea.

• Adverse effects less severe with consumption of viable starter cultures in yoghurt.

• The starter cultures use their β-galactosidase activity to assist in hydrolysis of lactose.

• Inhibition of pathogens
• LAB colonising the gut mask the potential attachment sites in the gut for pathogenic microorganisms.

• Immune system stimulation


• LAB activate macrophages and lymphocytes, improve levels of IgA and the production of gamma interferon.

• They also have antitumor activity where the activity of enzymes such as β-glucuronidase, azoreductase and
nitroreductase (convert procarcinogens to carcinogens)in faeces are reduced.
MALOLACTIC FERMENTATION
• LAB decarboxylate a strong acid, L-malic acid, to produce a weak acid, L-lactate.
Encouraged in wines from cool regions with high acidity.

• Provides bacteriological stability to the bottled product and, modify and improve
the body and flavour of wine.

• Encouraged naturally by leaving the new wine on yeast sediment for longer than
usual.

• Produced from commercial starter cultures of Oenecoccus oeni, formerly


Leuconostoc oenos
FERMENTED MILKS
YOGHURT
Milk mix (skim or whole +fresh) :supplement the solid content to enhance final texture of the product
improves milk as growth medium for starter culture by killing competing microflora, inactivates
Heat treatment: immunoglobulins, expulsion of O2 to produce a microaerophilic environment, release of
stimulatory levels of sulfhydryl groups. It also promotes interaction between whey or serum
proteins and casein, which increase yoghurt viscocity, stabilize the gel and limit syneresis
(separation of whey)
Homogenise :reduce the size of fat globules improving the product’s stability, increases milk’s viscosity and
also makes the product more whiter

Cool : Ensures that starter cultures are not killed by high temperature

Inoculate with starter: bulk tank


Lb. delbrueckii subp. bulgaricus, S. thermophilus
(stirred) and retail pots (set yoghurt) :

Incubate for 4h : Starter bacteria ferment lactose to lactic acid reducing the pH from 6.3-6.6 to 3.8-4.2

Cool(15-20oC)

Add fruits and/or flavourings

Pack at 4oC Dispatch at 2-4oC


YOGHURT
• Spoilage
• Yoghurt should contain >108 cfu/g of starter organism, < 1 coliform g-1, < 1
mould g-1 and <10 yeasts g-1

• However, yoghurts containing fruits may have up to 100 yeasts and still remain of
satisfactory quality

• Acidoduric yeasts eg. Kluyveromyces fragilis, S. cerevisiae

• Moulds e.g. Mucor, Rhizopus, Aspergillus, Alternaria, Penicillium


Acidophilus milk
• Produced from UHT processed milk (previously tyndalization was used).

• Milk homogenized and cooled to 37-40oC before addition of 2-5% starter (Lb. acidophilus).

• Incubated for 24h, ending with a product with 0.7% acidity.

• Drawbacks:
• Unlike yoghurt, it has a sour acidic taste.
• Shelf life of a week at 5oC due to poor survival of Lb. acidophilus in acidic conditions.

• Non fermented sweet acidophilus milk is produced by addition of starter culture to pasteurized milk
without incubation.

Bioyoghurts
• Produced in an attempt to combine qualities of acidophilus milk and yoghurt.

• Contain organisms able to colonize gut such as Lb. acidophilus and Bifidobacterium spp., and S. thermophilus
to provide characteristic yoghurt flavor.
Kefir and koumiss
• Both produced by mixed LAB and alcoholic fermentation.

• Kefir
• Microflora added as kefir grains with smooth outer surface containing LAB and inside
rough surface containing a mixture of yeast and LAB.

• LAB used in production: Lactobacillus kefiranofaciens and L. kefir

• Yeasts: Candida kefir, Saccharomyces cerevisiae and S. exiguus

• Flavour compounds are ethanol, lactic acid, carbon dioxide, acetaldehyde and diacetyl.

• Koumiss
• Fizzy, greyish white drink produced traditionally from mare’s milk (modified cow’s milk).
• Produced by activities of Lb. delbrueckii subsp. bulgaricus and a number of lactose
fermenting yeasts.
KEFIR PRODUCTION
Homogenize milk

Heat treatment (85-95oC for 3-10 min)

Cool to 22oC

Add 5% kefir grains

Incubate for 8-12 h

Slow cool to 8oC over 10-12h

Final product: 0.8 % acidity; 0.01 -1% alcohol content


CHEESE
CHEESE
• Valuable means of conserving nutrients in milk

• Practised on relatively small scale accounting for rich diversity (78 types of blue and 36
Camamberts). Table 9.7 A & M

• Most popular is cheddar cheese with a smooth texture and good keeping qualities.

• Keeping qualities vary with type but are always better than those of milk.

• Yeasts and moulds are of concern in cheese spoilage.

• Controlled procedures for exclusion of air such as waxing or by vacuum packaging.


Milk
Cheese
Pasteurization: Destroys potentially harmful bacteria

Cool milk to fermentation temperature: 29-31oC for cheddar, Stilton, Leister, Wensleydale
Addition of starter culture as concentrated cultures (WHY??) at 106-107 cfu/ml
Convert lactose to lactic acid{shelf life, safety,flavour}; Weakens casein and release
Mesophillic: Lactococcus lactis
calcium from it : aids chymosin action; Moisture expulsion and curd shrinkage
Thermophillic: Lb. helveticus, Lb. casei, Lb. lactis, Lb. bulgaricus, S. thermophilus

Incubate for 45 min: Starter multiply to 108-109 cfu/g and produce 0.6-0.7% acidity

Curdling: Add rennet & incubate for 30-45 min Coagulates milk: chymosin/rennin cleaves k-casein forming
cross links between micelles to form network

Whey expulsion: Curd cut into 1 cm slices


Soft: allowed to drain naturally; Hard: heated and more whey is drained
Scaling: heat curds to 38-42oC or higher Sprayed with mould spores, washed in alcohol or covered in
herbs: Starters, proteases and peptidases release free amino acids
which contribute to flavour
Cheddaring: compress curd to remove extra whey
Penicillium roquefortii: promote blue veining in blue cheese such as
Stilton
Propionic acid: encourage hole formation
P. camembertii: ripening of soft-cheeses such as camembert and brie
Mill curd into small chips &
Mould into shapes Ripening/maturation @ 10oC for 5 months for cheddar
add salt (1.5-2%) for cheddar
Flavour develops, cheese becomes firmer and drier, special
characteristics such as holes, blue veining and crust formation
BACTERIOPHAGES IN CHEESE MAKING
• Common when starter cultures containing a single or few strains
• Continuous use of same starter culture

• Problems of bacteriophages
• Slows or stops acidification: Financial losses to producer; Increased risk of pathogens

• Methods for control or prevention of bacteriophages


• Observation of rigorous hygiene

• Rotation of starter cultures with differing phage susceptibilities

• Propagation of starters in phage inhibitory media containing phosphate salts to chelate Mg2+
and Ca2+ ions
• Intrinsic methods of LAB resistance to phage infection
• Restriction/modification of non-host DNA

• Inhibition of phage adsorption by alteration or masking specific receptors on cell surface

• Reduction of burst size (number of phages released per infected cell)

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