MICROBIAL FOOD SPOILAGE CONTENTS: Introduction Microbial Food Spoilage Factors Affecting Spoilage Microflora Microbial Spoilage – How Does It Manifest Itself? Foods Affected By Microbial Spoilage Spoilage Microorganisms Some of The More Important Moulds Responsible For Post-Harvest Diseases of Fruits Genera of Fungi Causing Important Spoilage of Fresh And Stored Vegetables Yeast Spoilage of Fresh Fruits And Vegetables Bacteria That Cause Soft Rot of Vegetables And Fruit Monitoring And Detection of Microbial Spoilage Microbial Indicators of Product Quality Prevention of Microbial Spoilage INTRODUCTION • Food spoilage can be defined as “any sensory change (tactile, visual, olefactory or flavour)

which the consumer considers to be unacceptable. • Spoilage may occur at any stage along the food chain. • Spoilage may arise from:

- insect damage - physical damage (bruising, freezing, drying, etc) - indigenous enzyme activity in the animal or plant tissue - chemical changes (usually involving oxygen)

• Spoilage is therefore complex, involving physical, chemical, biochemical and biological changes.

MICROBIAL FOOD SPOILAGE By bacteria, yeasts and moulds:

Bacteria

Gram -ve Gram +ve

Cocci Rods Non-spore Spore Formers Lactic Acid Formers Bacteria Coliform/ Pseudomonas Micrococcus Bacillus Lactobacillus Enteric Acinetobacter Brocothrix Clostridium Streptococcus Citrobacter Aeromonas Leuconostoc Serratia Alcaligenes Pediococcus Proteus Moraxella Escherichia Altermonas Enterobacter Flavobacterium Erwinia Klebsiella Hafnia Yeasts: Candida, Saccharomyces, Zygosaccharomyces, Torulospora, Rhodotolura, Pichia, Moulds: Aspergillus, Mucor, Penecillium, Rhizopus, Geotrichum, Botrytis, Cladosporium, Byssochlamys

FACTORS AFFECTING SPOILAGE MICROFLORA 1. Initial microbial flora 2. Food type/intrinsic properties: - pH - Aw (water activity) - nutrient content - antimicrobial constituents - Eh-Redox potential (oxidation-reduction potential) - biological structures. 3. Processing and storage conditions: - heating - acidification - reduced water activity - use of preservatives - chilled storage - storage atmosphere - combination of (above) treatments In general terms, organisms tolerant of/resistant to the preservation mechanisms are those that will cause subsequent spoilage i.e.

PRESERVATION MECHANISM ORGANISMS OF SIGNIFICANCE (TOLERANT) Heat processing Thermodurics : clostridia, bacilli, spores Pasteurisation Thermodurics: streptococci, lactobacilli, spores Chilled Storage (c. 5°C)

Psychrotrophs: pseudomonads, Enterobacteriaceae, lactic acid bacteria, some micrococci, flavobacteria Most yeasts incl. Candida, Rhodotolura, Torulopsis Most moulds

Frozen Storage (-18°C)

None grow, but Gram-positives and spores survive

Dried Foods (<25% water, Aw 0.6)

Osmophilic yeasts and moulds, e.g. Saccharomyces rouxii, Aspergillus spp.

Intermediate Moisture Foods (<50% water, Aw <0.85)

• Osmophilic yeasts and moulds • Staphylococci

Salt Osmophiles, micrococci, staphylococci Sugar Osmophiles Vacuum packing • Anaerobes, microaerophiles e.g. clostridia,

Enterobacteriaceae, lactobacilli, streptococci, some bacilli

• Some yeasts Carbon dioxide Lactobacilli, Brocothrix thermosphacta Preservatives - benzoate - sorbate - nitrites/nitrates

Yeasts, e.g. Zygosaccharomyces bailii Some Escherichia coli Lactobacillus acidophilus

- sulphur dioxide - chlorine - alcohol

Zygosaccharomyces spp. e.g. bysporus Pseudomonas fragi Saccharomyces cerevisiae

Irradiation • Sporeformers, Micrococcus radiodurans, streptococci • Moulds (Candida)

Acid • Most yeasts and moulds • Lactic acid bacteria

MICROBIAL SPOILAGE – HOW DOES IT MANIFEST ITSELF? 1. Visible growth – e.g. moulds, often produce large, pigmented colonies (visible bacterial

and yeast colonies are less common) 2. Gas production 3. Slime 4. Diffusable pigment and enzymes – which may produce softening and rotting (proteolysis) 5. Off-odours 6. Off-flavours There are a wide range of metabolites produced during microbial spoilage, including:

• Alcohols • Sulphur compounds • Ketones • Hydrocarbons • Fluorescent pigments • Organic acids • Esters • Carbonyls • Diamines

Flavour, aroma and textural changes on spoilage:

Flavour/Aroma Problem

Food Chemical Cause Organisms Involved

Nitrogenous (e.g. bad eggs)

Meat, eggs, fish Trimethylamines, ammonia, H2S

Pseudomonads Acinetobacters Moxarellas Clostridia

Souring Dairy, vacuum packed meats, beer, wine

Acids: acetic, lactic, citric, butryic

Lactic acid bacteria Brochothrix thermosphacta Bacillus sp. Butyric acid bacteria Acetobacter spp.

Alcoholic Fruit juices Mayonnaise-dressed salads

Ethanol Yeasts

Mustiness Bread, Cake Chloroanisoles Moulds Pig-sty Vegetables P-cresole, indole,

skatole Erwinia sp. Clostridia

Garlic Various Bis (methylthio)-methane Trimethylarsine

Unknown

Fruity Meat Esters of short chain fatty acids

Pseudomonas fragi

Potato-like Meat, eggs, milk 2-methoxy-3-isopropyl Pyrazine

Pseudomonads

Texture Problem

Food Chemical/Biochemical Cause

Organisms Involved

Slime e.g. meats confectionery

Polysaccharide production

Pseudomonas fragi Leuconostoc mesentroides Bacillus subtilis

Ropiness Bread, milk Polysaccharides Alcaligenes sp. Bittiness Milk Bacillus cereus Holes Hard cheese (gas production) Coliforms Softening/rotting Fruit and vegetables Pectinases

Cellulase Xylanase

Erwinia, Clostridia, Yeasts, Moulds

Curdling Milk Meat

Acid production Lactic acid bacteria

Visual Spoilage:

Problem Food Organism Gas formation Holes

Cheese

Coliforms

Bubbles Cottage cheese, coleslaw, etc Yeasts, lactic acid bacteria Fisheyes Olives Bloaters Cucumbers Surface growth Moulds

Many

Fungi

Surface Potato salad, etc Pichia membranaefacien Discolouration/growth Beetroot Bacillus spp. Cloudiness Beverages/brines Yeasts Discolouration Pigments

- flourescent

Meats/egg

Pseudomonas sp.

- pink Sauerkraut Rhodotorula sp. Red spot Cheese Lactobacillus plantarum Browning Brined vegetable Lactobacillus brevis Blackening Dairy products Pseudomonas nigrifaciens Greening Meats Lactobacillus viridescens FOODS AFFECTED BY MICROBIAL SPOILAGE • Sources of spoilage microorganisms include:

- Soil and water - Plants and plant products - Air and dust - Animal hides - Gastro intestinal tract of animals and humans - Food handlers - Food utensils and processing equipment

• Generally, spoilage is most rapid in proteinaceous chilled fresh foods (e.g red meats, poultry, fish and dairy products). Due to: - Highly nutritious - High moisture content/Aw - Relatively neutral pH

• Other fresh foods that may suffer significant spoilage are fruit and vegetables - Fruit = spoilt by yeast and moulds, as the low pH prevents bacterial growth - Vegetables = subject to both bacterial and fungal spoilage

• Suitably processed (and packaged) or ambient stable foods with a low moisture content will be expected to show little sign of microbial spoilage. As regards the latter, will be limited to yeasts or moulds.

SPOILAGE MICROORGANISMS Gram -ve Rod Shaped Bacteria • The most common spoilage microorganisms of fresh chilled produce • Min. growth temperatures are often 0-3°C and they grow rapidly at 5-10°C • Pseudomonas is the most common.

- They are widely distributed in the environment and may contaminate from many sources - Often may proliferate on inadequately cleaned surfaces - Able to utilise a wide range of materials as substrates for growth - May multiple relatively rapidly at chill temperatures and hence may come to dominate the

microflora of foods stored at low temperatures - Spoilage caused by:

Production of lipase or protease enzymes that result in food rot, putrid odours (sulphurous and nitrogenous compounds), off flavours and rancidity

Production of extracellular slime Development of visible, often pigmented growth (in thermally processed products with extended shelf life) extremely heat resistant

enzymes may cause long-term defects • Other Gram–ve rod-shaped bacteria may also grow at chill temperatures and have been

shown to contribute to the spoilage of chilled red meat, cured meats, poultry, fish, shellfish, milk and dairy products, e.g. Acinetobacter, Aeromonas, Alcaligenes, Alteromonas, Flavobacterium, Moraxella and Archromobacter. [Also Vibrio spp, which are halophilic (salt loving) and therefore may cause spoilage of sea fish and cured meats.]

• Overall, the group is not heat-resistant and can be readily removed by mild thermal treatments.

Coliform/Enteric Bacteria • Generally slower-growing at chill temperatures than Pseudomonas spp. but become more

significant than Pseudomonas as the temperature rises above 5°C (dominating between 8-15°C)

• But many strains are psychrotrophic – have been isolated from vacuum packed meats, poultry, cured meats, milk, dairy and egg products

• Widely disseminated in the environment, including animals. Associated with animals, slaughter and dressing

• Typical spoilage species are Citrobacter, Enterobacter, Escherichia, Klebsiella, Proteus, Serratia, Hafnia and Erwinia.

• Spoilage characterised by - the production of gas - acid - slime - ‘rope’ - bitter flavours - pigmented colonies - ‘unclean’/’medicinal’/’faecal’ odours

• Coliforms are very susceptible to heat. Their presence in heat processed foods is indicative of inadequate processing or post-process contamination.

Gram-Positive Sporeforming Bacteria • Of particular significance due to their ability to produce heat-resistant spores which can

survive many heating/pasteurisation processes and germinate under suitable conditions and grow in foods. They are therefore seen in the spoilage of canned foods.

• In general, growth tends to be slower than that of the Gram negative. • Minimum growth temperatures are often 0-5°C, although growth is often slow below 8°C. • Genera of importance are Bacillus and Clostridium species. Common in the environment;

spores may survive for considerable periods. Common spoilage is production of large quantities of gas which may result in pack or product blowing.

• Bacillus spp. – largely aerobic, some psychrotrophic strains (e.g. 5°C or less) in milk; produce enzymes which result in ‘sweet curdling’ and ‘bitty cream’

• B. subtilis – ‘ropy’ spoilage of bread • Some Bacillus spp. are acid-tolerant and spoil pickles (blackening by Bacillus nigrificans and

softening by pectolytic strains) • Alicyclobacillus (acid tolerant sporeformer) has been identified as causing spoilage of

aseptically packaged fruit juice (particularly orange) with a ‘disinfectant’ or ‘antiseptic’ taint. Is a thermophile (growth only seems to occur over 25°C) – spores survive pasteurisation and later germinate and gown under suitable conditions.

Lactic Acid Bacteria • Spoil foods by the fermentation of sugars to form lactic acid. Results in drop in pH and off-

flavours (souring) • Tend to grow slowly at chill temperatures and are easily out competed by other

microorganisms • Generally present in low numbers but in warm countries or where temperatures are poorly

controlled can cause problems. • Also known to grow under conditions of vacuum and modified atmosphere. Constitute major

spoilage microorganisms of vacuum packed meat and poultry (where Pseudomonas spp. are inhibited)

• May also spoil cured or fermented meat products where growth of other spoilage microorganisms is inhibited or prevented

• More acid tolerant than other spoilage bacteria (down to pH 3.6). Typical genera are Lactobacillus, Streptococcus, Leuconostoc (main cause of souring) and Pediococcus.

NB Some lactic acid bacteria are used in food manufacture (cheese, yoghurts, fermented meats). May also produce antimicrobial compounds.

Gram Positive Bacteria • Brocothrix thermosphacta – Gram +ve rod, may occasionally be present on fresh meats.

Modified atmosphere packaging etc may allow Br. thermosphacta to dominate the microflora. - Has been isolated from frankfurters, fermented and fresh sausages and cured meats. - Spoils with low numbers (000’s usually compared with 105+ normally with other spoilage

microorganisms) - In some products (e.g. vacuum packed sliced meats) may generate objectionable and

pungent ‘cheesy’ odour • Micrococcus spp. – able to grow in the present of salt

- Can spoil bacon, producing slime, souring or pigmented growth - Often predominate in freshly collected milk and as many strains are thermoduric can

survive pasteurisation and so cause spoilage Yeasts and Moulds • Not normally found on freshly collected food materials (except some plants) but are

extremely common in the environment and can cause contamination through airborne transmission.

• Slow growers in comparison to bacteria and so are often out-competed

• Seldom responsible for the spoilage of fresh proteinaceous material • Yeasts and moulds more resistant to low temperature, low pH, lower Aw values and the

presence of preservatives than bacteria. • Most are not heat resistant (except mould Byssochlamys – may produce relatively heat-

resistant ascospores) • Chilled foods subject to spoilage include fruit and vegetables, unprotected meat carcasses,

yoghurt, cheese, fermented meat products and salads • If carcass surface dries out, this inhibits bacteria and permits mould growth (visible

mycelial growth described as ‘whiskers’, ‘black spot’ or ‘white spot’). • Chill foods of low pH inhibit bacteria but may allow mould/yeasts to develop (yoghurt,

fruit/mayonnaise-based salads, fermented meat products, fruit juices, etc). • Species involved are detailed above. • Fungal spoilage may be characterised by

- Highly visible, often pigmented growth - Slime - Fermentation of sugars to form acid, gas or alcohol - Off odours/off flavours

• Yeasts and moulds may be added as part of food manufacture, e.g. Penicillium camembertii SOME OF THE MORE IMPORTANT MOULDS RESPONSIBLE FOR POST-HARVEST DISEASES OF FRUITS

Genus Spoilage Problems Alternaria Brown to black spots on apples, stone fruits1 and figs; stem-end and black

rot of citrus fruits [ 1Stone fruits include cherries, peaches, nectarines, apricots and plums]

Aspergillus Black rot on peaches, nectarines, apricots, citrus fruits and figs Botryodiplodia Cushion or crown rot of bananas; ripe rot of pawpaws Botrytis Grey mould rot of apples, pears, raspberries, strawberries, grapes, figs,

blueberries, citrus and stone fruits Cladosporium Restricted rot with grey black core on stone fruits, olive-green growth on

raspberries, black rot on grapes and spotting of figs. Colletotrichum Brown to black spots (anthracnose) on citrus fruits, avocados, mangoes,

pawpaws and papayas Diplodia Stem-end rot of citrus fruit, avocados, mangoes and papayas; watery, tan-

brown rot of peaches Fusarium Brown rot of citrus fruit and pineapple; soft rot of figs Geotrichum Sour rot of citrus fruits and peaches Gloeosporium Anthracnose, black rot and lesion rot of bananas; eye rot of pome fruits2

[2 Pome fruits include apples, pears and quince] Monilinia Brown rot of stone fruits Mucor Soft rot of strawberries, pears Nigrospora Soft, watery (squirter) rot of the pulp of bananas Penicillium Blue and green mould rots of citrus fruits; blue mould rot of apples,

grapes, pears, stone fruits and figs; brown rot of pineapples Phomopsis Stem-end rot of citrus fruits and avocados Phytophthora Brown rot of apples and citrus fruits, leathery rot of strawberries Rhizopus Watery, soft rot of apples, pears, stone fruits, grapes, strawberries,

avocados and figs Trichoderma Cocoa-brown to green rot of citrus fruits

GENERA OF FUNGI CAUSING IMPORTANT SPOILAGE OF FRESH AND STORED VEGETABLES

Examples of Commodities Most Affected

Genus Type of Spoilage

Most vegetables especially carrot, lettuce, celery, cabbage

Botrytis Grey mould rot

Most vegetables. Especially carrot, lettuce, legumes, Brassica spp.

Sclerotinia Watery soft rot

Legumes, carrot, Brassica spp. Rhizopus Soft rot Tomato, cucumber, asparagus, potato

Fusarium Dry rots

Tomato, potato, carrot Phytophthora Brown rots (blight) Tomato, potato, beetroot Phoma Dry brown, black rots Cucumber, legumes, potato Pythium Cottony leak

Onion, Brassica spp. Peronospora Downy mildews Tomato, Brassica spp Alternaria Firm, black rots YEAST SPOILAGE OF FRESH FRUITS AND VEGETABLES Dates Saccharomyces spp.

Hanseniaspora valbvensis Candida quilliermandii

Figs Hanseniaspora uvarum (Klorkera apiculata) Hanseniaspora valbvensis Torulopsis stellata Saccharomyces cerevisiae Candida krusei

Strawberries

Kloekera apiculata

Tomatoes

Hanseniaspora uvarum (K. apiculata) Pichia kluvveri Nematospora corvli

Legumes Coffee berries Citrus fruits Nuts

Nematospora corvli

Pineapple Candida spp. Rhubarb Trichosporon cutaneum

YEAST SPOILAGE OF PROCESSED FRUITS AND VEGETABLES a. Salt-brined Vegetables Sauerkraut

Discolouration

Saccharomyces cerevisiae Torulopsis holmii Candida krusei

“Bloaters” Brettanomyces spp. T. holmii Hansenula subpelliculosa Sacch. rosei Sacch. rouxii

Cucumbers

Surface films Debaryomyces spp. Olives Softening Rhodotorula glutinis

Rh. minuta Rh. rubra Sacch. oleaginosus Sachh. kluvveri

Gas Pockets Hansenula anomala

Gas formation Zygosaccharomyces bailii

b. Acetic Acid Preserves Onions Gherkins Red beetroot Red cabbage Piccalilli Sauces

Off odours Surface films

Pichia membranaefaciens

c. Mayonnaise-based Salads Coleslaw

Off odour Gas production

Sacch. exiguus Sacch. dairensis

Potato salad Gas production Sacch. exiguus Sacch. dairensis Pichia membranaefaciens

Mixed vegetable and frusalads

Gas production Off odour

Saccharomyces spp.

BACTERIA THAT CAUSE SOFT ROT OF VEGETABLES AND FRUIT

Temperatures for Growth (°C)

Bacterium Min Opt Max Produce Affected Erwinia carotovora subsp. atroseptica

3 27 35 Most vegetables, particularly potatoes, some fruit

Erwinia carotovora subsp. carotovora

6 28-30 37-42 Most vegetables and somefruit

Erwinia chrysanthemi 6 34-37 >45 Pineapple Pseudomonas marginalis >0.2 25-30 >41 Many vegetables Pseudomonas viridiflava - - - Beans Psedomonas cichorii - c. 30 >41 Chicory, endive, cabbage,

lettuce

Pseudomonas cepacia >4 30-35 40-41 Onion Pseudomonas gladioli pv. Allicola

>4 30-35 40-41 Onion

Bacillus polymyxa 5-10 - 35-40 Potato, pepper Bacillus subtilis 5-20 - 45-55 Potato, tomato Clostridium puniceum 7 - 39 Potato Low temperature clostridia 0-4 9-22 17-30 Potato MONITORING AND DETECTION OF MICROBIAL SPOILAGE • Direct microbiological methods • Chemical/physical/physiochemical methods • ‘Acceptability’ criteria include sensory determinations – colour , texture, odour, flavour and

general appearance. MICROBIAL INDICATORS OF PRODUCT QUALITY • Microbial product quality or shelf-life indicators are organisms and/or their metabolic

products whose presence in given foods at certain levels may be used to assess existing quality or, better, to predict shelf-life. These may be: - the spoilage organisms themselves, or - their metabolic products

Microbial Indicators that are Highly Correlated with Food Quality: Organisms Products Acetobacter Bacillus Clostridium Lactic acid bacteria Leuconostoc mesenteroides Yeasts Pseudomonas putrefaciens Flat-sour spores Byssochlamys spp. Zygosaccharomyces bailii

Fresh cider Bread dough Hard cheeses Beers, wines Sugar (during refinery) Fruit juice concentrates Butter Canned vegetables Canned fruits Mayonnaise, salad dressing

Microbial Metabolites that Correlate with Food Quality: Metabolite Food Product Cadaverine and putresaine Diacetyl Histamine Lactic acid Trimethylamine (TMA) Total volatile bases (TVB) Total volatile nitrogen (TVN) Volatile fatty acids

Vacuum packaged beef Frozen juice concentrate Canned tuna Canned vegetables Fish Seafish Seafish Butter

Use Of Total Viable Count (TVC) • Total viable count is of greater value in assessing quality than predicting shelf-life. • A ‘Spoilage spectrum’ has been developed by Jay (1992)

Range A (TVC 103-106 cfu/g) o Microbial spoilage is generally not recognised (with the possible exception of milk)

Range B (TVC 106-107 cfu/g) o Some food products show incipiency o Vacuum packed meats often display objectionable odours and may be spoiled

Range C (TVC 107-108 cfu/g) o Off odours are generally associated with aerobically stored meat and some

vegetables Range D (TVC 5x107-108 cfu/g)

o Almost all food products display obvious signs of spoilage o Slime will be common on aerobically stored meats

Range E (TVC in excess of 5x108 cfu/g) o Definite structural change in product occurs

PREVENTION OF MICROBIAL SPOILAGE • Use of quality raw materials • Correct storage for the food type • Allocation of appropriate shelf-life • HACCP and other quality systems • Hygiene of processing environment • Use of predictive methods • Training and education Note: Above text derived from notes provided by Dr P Voysey, Campden & Chorleywood Food Research Association