Kluwer Academic Plenum Publishers New York, Boston, Dordrecht, London, Moscow

Kluwer Academic / Plenum Publishers New York, Boston, Dordrecht, London, Moscow

The author has made every effort to ensure the accuracy of the information herein. However, appropriate information sources should be consulted, especially for new or unfamiliar procedures. It is the responsibility of every practitioner to evaluate the appropriateness of a particular opinion in in the context

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Contents

Preface

xiii

B Spoilage

C Pathogens

62 D Control (raw, cured,

1 Meat and meat products

1 shelf-stable meats)

64 Control (Chinese sausages)

I Introduction

1 D Control (dry salami, A Deﬁnitions

2 e.g., Hungarian) 66 B Important properties

2 D Control (fermented, C Methods of processing and

high acid sausages) 67 preservation

3 VIII Dried meats

D Types of meat products 4 A Effects of processing on

II Initial microﬂora

4 microorganisms 68 A Ruminants

69 B Pigs

4 B Spoilage

10 C Pathogens

III Primary processing

15 D Control (dried meats) 69 A Ruminants

15 IX Cooked perishable uncured meats 71

B Pigs 23 A Effects of processing on C Spoilage

31 microorganisms 71 D Pathogens

72 E Control

32 B Spoilage

73 E Control (farm)

38 C Pathogens

38 D Control (cooked perishable F Control (transport and

uncured meats) 74 holding at abattoirs)

40 X Fully retorted shelf-stable

G Control (slaughter and

uncured meats 75

dressing of cattle and sheep) 41 A Effects of processing on H Control (slaughter and

microorganisms 75 dressing of pigs)

76 I Control (chilling (cattle,

42 B Spoilage

76 sheep, and pigs))

C Pathogens

43 D Control ( fully retorted J Control (storage and

shelf-stable uncured meats) 76 transport)

45 XI Cooked perishable cured meats 78 IV Carcass cutting and packaging

46 A Effects of processing on A Effects of processing on

microorganisms 78 microorganisms

79 B Spoilage

46 B Spoilage

80 C Pathogens

48 C Pathogens

50 D Control (cooked, perishable D Control (meat stored in air,

cured meats) 82 vacuum-packed or stored in

XII Shelf-stable cooked cured meats 83

modiﬁed atmospheres) 51 A Effects of processing on

V Frozen meat

52 microorganisms 83 A Effects of processing on

83 microorganisms

B Spoilage

83 B Spoilage

52 C Pathogens

52 D Control (shelf-stable cooked C Pathogens

53 cured meats) 84 D Control (frozen meats)

84 VI Raw comminuted meats

53 XIII Snails

84 A Effects of processing on

54 A Deﬁnition

B Production and processing 85 microorganisms

85 B Spoilage

54 C Pathogens

55 D Control (snails) 85 C Pathogens

55 XIV Froglegs

86 meats)

D Control (raw comminuted

A Deﬁnition

58 B Production and processing 86

VII Raw cured shelf-stable meats

87 A Effects of processing on

59 C Pathogens

D Control (froglegs) 87 microorganisms

59 References 59 References

CONTENTS

2 Poultry products

C Pathogens

209 D Control (aquaculture)

I Introduction

A Deﬁnitions 211 108 A Freezing process

V Frozen raw seafood

211 B Important properties

B Saprophytes and spoilage 212 C Method of processing

212 D Types of poultry products

C Pathogens

D Control (frozen raw seafood) 213

II Initial microﬂora (effect of VI Minced ﬁsh and surimi products 214 farm practices)

VII Cooked crustaceae (frozen or III Primary processing (whole birds

215 and parts)

chilled)

A Cooking, picking, and packaging 215 A Effects of processing on

B Saprophytes and spoilage 215 microorganisms

217 B Spoilage

C Pathogens

D Control (cooked C Pathogens

crustaceae, frozen or chilled) 220 D Control (primary processing,

whole birds and parts) 221 145 A Introduction

VIII Lightly preserved ﬁsh products

IV Frozen poultry products

B Saprophytes and spoilage 222 A Effects of processing on

223 microorganisms

C Pathogens

D Control (lightly preserved B Spoilage

ﬁsh products) 225 C Pathogens

D Control (frozen poultry product) 227 147

IX Semi-preserved ﬁsh products

A Introduction 227

V Perishable, cooked poultry

B Saprophytes and spoilage 227

products

228 A Effects of processing on

C Pathogens

D Control (semi-preserved microorganisms

ﬁsh products) 228 B Spoilage

X Fermented ﬁsh products 229

C Pathogens

A Control (fermented ﬁsh products) 230 D Control (perishable,

XI Fully dried or salted products 231

cooked poultry products)

A Control (fully dried or salted products)

VI Fully retorted (“botulinum-

cooked”) poultry products

A Control (fully retorted shelf-stable 232 A Introduction

XII Pasteurized products

232 poultry products)

B Saprophytes and spoilage 233

VII Dried poultry products

233 A Effects of processing on

C Pathogens

D Control (pasteurized ﬁsh products) 234 microorganisms

B Spoilage 235 156 A Processing

XIII Canned seafood

235 C Pathogens

B Control (canned seafood) 235 D Control (dried poultry products)

4 Feeds and pet foods 250

3 Fish and ﬁsh products

I Introduction 250 I Introduction

II Roughages

A Effects of processing on B Important properties

A Deﬁnitions

II Initial microﬂora

252 A Saprophytic microorganisms

B Spoilage

253 B Pathogens and toxicants

C Pathogens

D Control (roughages) 254

III Primary processing

III Animal by-products 256

A Finﬁsh of marine and A Effects of processing on freshwater origin

microorganisms 257 Control (ﬁnﬁsh of marine

258 and freshwater origin)

B Spoilage

258 B Crustacea

C Pathogens

D Control (animal by-products) 261 Control (crustacea)

IV Fish meal

A Effects of processing on Control (mollusca)

C Mollusca

IV Aquaculture

265 A Initial microﬂora

B Spoilage

265 B Spoilage

C Pathogens

D Control (ﬁsh meal) 265

CONTENTS

vii

V Compounded feeds

D Control (fermented and A Effects of processing on

acidiﬁed vegetables) 306 microorganisms

IX Sprouts

B Spoilage

A Effects of harvesting, C Pathogens

transportation, processing, and D Control (compounded feeds)

VI Pet foods

B Saprophytes and spoilage 307 A Effects of processing on

308 microorganisms

C Pathogens

D Control (sprouts) 309 B Spoilage

X Mushrooms

A Effects of harvesting, D Control (pet foods)

C Pathogens

transportation, processing, and

References

storage on microorganisms 311 B Saprophytes and spoilage

5 312 Vegetables and vegetable

C Pathogens

D Control (mushrooms) 313

products

314 I Introduction

XI Cassava

A Effects of harvesting, A Deﬁnitions and important

transportation, processing, and properties

storage on microorganisms 314

II Initial microﬂora (including ﬁeld

B Control (cassava) 315

practices and harvest)

A Saprophytic microorganisms

B Pathogens

6 Fruits and fruit products 326

C Good agricultural practices

III Raw and minimally processed I Introduction 326 vegetables

A Deﬁnitions 326 A Effects of transportation,

B Important properties 326 processing, and storage on

C Methods of processing 326 microorganisms

D Types of ﬁnal products 327 B Saprophytes and spoilage

II Initial microﬂora (fresh fruits) 328

C Pathogens

III Primary processing 328

D Control (raw and minimally A Effects of processing on processed vegetables)

microorganisms 328

IV Cooked vegetables

329 A Effects of processing on

B Spoilage

334 microorganisms

C Pathogens

D Control (fresh fruits) 336 B Saprophytes and spoilage

IV Pre-cut (minimally processed)

C Pathogens

D Control (cooked vegetables)

A Effects of processing on

V Frozen vegetables

microorganisms 338 A Effects of processing on

339 microorganisms

B Spoilage

339 B Saprophytes and spoilage

C Pathogens

D Control (pre-cut C Pathogens

(minimally processed) fruit) 340 D Control (frozen vegetables)

V Frozen fruits 341 VI Canned vegetables

A Effects of processing on A Effects of processing on

microorganisms 341 microorganisms

341 B Saprophytes and spoilage

B Spoilage

342 C Pathogens

C Pathogens

D Control (frozen fruits) 342 D Control (canned vegetables)

VI Canned fruits 342 VII Dried vegetables

A Effects of processing on A Effects of processing on

microorganisms 342 microorganisms

343 B Saprophytes and spoilage

B Spoilage

343 C Pathogens

C Pathogens

D Control (canned fruits) 344 D Control (dried vegetables)

345 VIII Fermented and acidiﬁed vegetables 303

VII Dried fruits

A Effects of processing on A Effects of processing on

microorganisms 345 microorganisms

346 B Saprophytes and spoilage

B Spoilage

346 C Pathogens

C Pathogens

D Control (dried fruits) 347 D Control (dried fruits) 347

CONTENTS

VIII Fermented and acidiﬁed fruits

II Initial microﬂora 394

394 microorganisms

A Effects of processing on

A Fungi

398 B Spoilage

B Bacteria

III Primary processing 399

C Pathogens

A Effects of processing on D Control (fermented and

microorganisms 399 acidiﬁed fruits)

B Spoilage

IX Tomato products

C Pathogens and toxins 403 A Effects of processing on

D Control (cereals) 408 microorganisms

IV Flours, starches, and meals 409

A Effects of processing on C Pathogens

B Spoilage

microorganisms 409 D Control (tomato products)

B Saprophytes and spoilage 411

References

C Pathogens and toxins 411 D Control (ﬂours, starches and meals)

7 Spices, dry soups, and oriental

V Dough

ﬂavorings

A Effect of processing on microorganism

I Spices, herbs, and dry vegetable

B Spoilage

seasonings

C Pathogens and toxins 414 A Deﬁnitions

D Control (dough) 415 B Important properties

VI Breads

C Methods of processing and A Effects of processing on preservation

microorganisms 415 D Types of ﬁnal products

417 E Initial microﬂora

B Spoilage

C Pathogens and toxins 419 F Primary processing

D Control (breads) 419 G Processing

VII Pasta and noodles 421

H Control (spices, herbs, A Effects of processing on and dry vegetable seasonings)

microorganisms 421

II Dry soup and gravy mixes

421 A Deﬁnitions

B Spoilage

C Pathogens and toxins 422 B Initial microﬂora

D Control (pasta and noodles) 423 C Primary processing

VIII Breakfast cereals and snack foods 423

D Control (dry soup and gravy mixes) 374 A Effects of processing on

III Soy sauces

microorganisms 423 A Deﬁnition

424 B Important properties

B Spoilage

C Pathogens and toxins 424 C Methods of processing and

D Control (breakfast cereals, preservation

snack foods) 424 D Types of ﬁnal products

IX Pastries and ﬁlled products 425

A Effects of processing on F Primary processing

E Initial microﬂora

microorganisms 425 G Control

B Spoilage

IV Fish and shrimp sauces and pastes 382

426 A Deﬁnitions

C Pathogens

D Control (pastries and B Important properties

ﬁlled products) 426 C Methods of processing and

D Types of ﬁnal products

E Initial microﬂora

9 Nuts, oilseeds, and

F Primary processing

dried legumes 440

G Control

I Introduction 440

A Deﬁnitions 440 B Important properties

8 Cereals and cereal products

C Methods of processing 441 D Types of ﬁnal products

I Introduction

II Initial microﬂora 443

A Deﬁnitions

443 B Important properties

A Nuts

443 C Methods of processing

B Oilseeds

443 D Types of ﬁnal products

C Legumes

D Coffee

CONTENTS

III Primary processing

III Mayonnaise-based salads 493

A Effects of processing on A Deﬁnitions 493 microorganisms

B Important properties 493 B Spoilage

C Methods of processing and C Pathogens

preservation 493 D Control (primary processing

D Microbial spoilage and pathogens 494 of tree nuts, peanuts, coconut,

E Control (mayonnaise-based salads) 495 dried legumes, and coffee)

496 IV Tree nut, peanut, and coconut

IV Margarine

A Deﬁnitions 496

processing

B Important properties 497 A Effects of processing on

C Methods of processing and microorganisms

preservation 497 B Spoilage

D Microbial spoilage and pathogens 500 C Pathogens

E Control (margarine) 502 D Control (tree nut, peanut and

V Reduced-fat spread 504

coconut processing)

A Deﬁnitions 504

V Oilseed products

B Important properties 505 A Effects of processing on

C Methods of processing and microorganisms

preservation 505 B Spoilage

D Microbial spoilage and pathogens 506 C Pathogens

E Control (reduced-fat spread) 507 D Control (oilseed products)

508 VI Legume products

VI Butter

A Deﬁnitions 508 A Effects of processing on

B Important properties 509 microorganisms

C Methods of processing and B Spoilage

preservation 509 C Pathogens

D Microbial spoilage and pathogens 510 D Control (legume products)

E Control (butter) 513

VII Water-continuous spreads 515 References

VII Coffee products

VIII Miscellaneous products 516

References

10 Cocoa, chocolate, and confectionery

12 Sugar, syrups, and honey 522

I Introduction

I Introduction 522

A Deﬁnitions

II Cane sugar

B Important properties

A Initial microﬂora 522

II Initial microﬂora

B Effects of processing on

III Primary processing

microorganisms 523 A Effects of processing on

525 microorganisms

C Spoilage

526 B Methods of processing

D Pathogens

E Control (cane sugar) 527

IV Processed products

III Beet sugar

A Effects of processing on A Initial microﬂora 528 microorganisms

B Effects of storage and processing B Spoilage

on microorganisms 528 C Pathogens

530 D Control (cocoa,

C Spoilage

531 chocolate and confectionery)

D Pathogens

E Control (beet sugar) 531

References

F Microorganisms in reﬁned sugar capable of spoiling other food

IV Palm sugar

11 Oil- and fat-based foods

A Initial microﬂora 533 B Effects of processing on

I General introduction

microorganisms 533

II Mayonnaise and dressings

533 A Deﬁnitions

C Spoilage

533 B Important properties

D Pathogens

E Control (palm sugar) 534 C Methods of processing and

V Syrups

preservation

A Initial microﬂora 534 D Microbial spoilage and pathogens

B Effect of processing on E Control (mayonnaise and

microorganisms 535 dressings)

C Spoilage

CONTENTS

C Primary processes of processing E Control (syrups)

D Pathogens

or product water 584

VI Honey

D Control (process or product water) 585 A Initial microﬂora

IV Bottled water 587

B Effect of processing on A Deﬁnitions 587 microorganisms

B Initial microﬂora 587 C Spoilage

C Primary processing 587 D Pathogens

C Effects of processing on E Control (honey)

C Pathogens

D Spoilage

590 E Control (natural mineral water)

13 590 Soft drinks, fruit juices,

concentrates, and fruit 592 preserves

References

15 Eggs and egg products 597

I Introduction

A Foods covered 597 544 B Important properties

I Introduction

A Deﬁnitions 597 C Initial microﬂora

B Important properties 597

C Types of products 601 A Mycotoxins

II Potential food safety hazards

II Initial microﬂora 602

B Bacterial pathogens

A Transovarian transmission 602 C Viruses

B Contamination in the cloacae 606 D Parasites

C Contamination in the production

environment 607 A Preservative resistant yeasts

III Spoilage

III Shell eggs

B Filamentous fungi (molds)

A Effect of initial processing 608 C Bacteria

614 A Heat processing

B Spoilage

C Pathogens

IV Processing

D Control (shell eggs) 616 B Chilled storage

A Effects of processing on D Stabilization of concentrated

IV Liquid eggs

microorganisms 617 fruit products

C Preservatives

625 E Stabilization of fruit preserves

B Spoilage

625 F Combination of pasteurization

C Pathogens

D Control (liquid eggs) 627 and preservatives

V Dried eggs

G Alternative non-thermal A Effects of processing on methods

microorganisms 629 Control (soft drinks,

631 carbonated and non-carbonated)

B Spoilage

631 Control (fruit juice and

C Pathogens

D Control (dried eggs) 632

Preface

The second edition of Microbiology of Foods 6: Microbial Ecology of Food Commodities was written by the ICMSF, comprising 16 scientists from 11 countries, plus consultants and other contributors to chapters.

The intention of the second edition was to bring the ﬁrst edition (published in 1996) up to date, taking into account developments in food processing and packaging, new products, and recognition of new pathogens and their control acquired since the ﬁrst edition.

The overall structure of the chapters has been retained, viz each covers (i) the important properties of the food commodity that affect its microbial content and ecology, (ii) the initial microﬂora at slaughter or harvest, (iii) the effects of harvesting, transportation, processing, and storage on the microbial content, and (iv) an assessment of the hazards and risks of the food commodities and (v) the processes applied to control the microbial load.

In 1980s, control of food safety was largely by inspection and compliance with hygiene regulations, together with end-product testing. Microorganisms in Foods 2: Sampling for Microbiological Analysis: Principles and Speciﬁc Applications (2nd ed. 1986) put such testing on a sounder statistical basis through sampling plans, which remain useful when there is no information on the conditions under which a food has been produced or processed, e.g. at port-of-entry. At an early stage, the Commission recognized that no sampling plan can ensure the absence of a pathogen in food. Testing foods at ports of entry, or elsewhere in the food chain, cannot guarantee food safety.

This led the Commission to explore the potential value of HACCP for enhancing food safety, partic- ularly in developing countries. Microorganisms in Foods 4: Application of the Hazard Analysis Critical Control Point (HACCP) System to Ensure Microbiological Safety and Quality (1988) illustrated the procedures used to identify the microbiological hazards in a practice or a process, to identify the critical control points at which those hazards could be controlled, and to establish systems by which the effectiveness of control could be monitored. Recommendations are given for the application of HACCP from production/harvest to consumption, together with examples of how HACCP can be applied at each step in the food chain.

Effective implementation of HACCP requires knowledge of the hazardous microorganisms and their response to conditions in foods (e.g. pH, a w , temperature, preservatives). The Commission concluded that such information was not collected together in a form that could be assessed easily by food industry personnel in quality assurance, technical support, research and development, and by those in food inspection at local, state, regional or national levels. Microorganisms in Foods 5: Characteristics of Microbial Pathogens (1996) is a thorough, but concise, review of the literature on growth, survival, and death responses of foodborne pathogens. It is intended as a quick reference manual to assist making judgements on the growth, survival, or death of pathogens in support of HACCP plans and to improve food safety.

The second edition of Microorganisms in Foods 6: Microbial Ecology of Food Commodities (2004) is intended for those primarily in applied aspects of food microbiology. For 17 commodity areas, it describes the initial microbial ﬂora and the prevalence of pathogens, the microbiological consequences of processing, typical spoilage patterns, episodes implicating those commodities with foodborne illness, and measures to control pathogens and limit spoilage. Those control measures are presented in a standardized format, and a comprehensive index has been added.

xiv

PREFACE

The second edition of Microorganisms in Foods 6: Microbial Ecology of Food Commodities has been written following Microorganisms in Foods 7: Microbiological Testing in Food Safety Management (2002). The latter illustrates how systems such as HACCP and GHP provide greater assurance of safety than microbiological testing, but also identiﬁes circumstances where microbiological testing still plays

a useful role in systems to manage food safety. It continues to address the Commission’s objectives to: (a) assemble, correlate, and evaluate evidence about the microbiological safety and quality of foods; (b) consider whether microbiological criteria would improve and assure the microbiological safety of particular foods; (c) propose, where appropriate, such criteria; (d) recommend methods of sampling and examination; (e) give guidance on appraising and controlling the microbiological safety of foods. It introduces the reader to a structured approach for managing food safety, including sampling and microbiological testing. The text outlines how to meet speciﬁc food safety goals for a food or process using Good Hygienic Practice (GHP) and the HACCP system. Control measures as used in GHP and HACCP are structured into three categories: those that inﬂuence the initial level of the hazard, those that cause reduction, and those that may prevent increase, i.e. during processing and storage. In Microorganisms in Foods 6, a control section following each commodity group uses this structured approach.

Microorganisms in Foods 5, 7, and the second edition of Microorganisms in Foods 6 (2005) are intended for anyone using microbiological testing and/or engaged in setting Microbiological Criteria, whether for the purpose of Governmental Food Inspection and Control or in Industry. The contents are essential reading for food processors, food microbiologists, food technologists, veterinarians, public health workers and regulatory ofﬁcials. For students in Food Science and Technology, they offer a wealth of information on Food Microbiology and Food Safety Management, with many references for further study.

PREFACE

Editorial committee

T. A. Roberts (Joint Chairman)

J. I. Pitt (Joint Chairman)

J.-L. Cordier

L. G. M. Gorris

L. Gram

K. M. J. Swanson

R. B. Tompkin

ICMSF Members during preparation of the second edition of Microbiology of Foods 6: Microbial Ecology of Food Commodities

Chairman

M. B. Cole

Secretary

M. van Schothorst (retired 2003) L. Gram (from 2003)

Treasurer

J. M. Farber

Members

R. L. Buchanan

J.-L. Cordier

S. Dahms

R. S. Flowers

B. D. G. M. Franco

L. G. M. Gorris

J.-L. Jouve

F. Kasuga

A. M. Lammerding

Z. Merican

J. I. Pitt (to 2002)

M. Potter

K. M. J. Swanson

P. Teufel

R. B. Tompkin (to 2002)

Consultants

J. Braeunig (2000)

M. Germini (2003)

L. G. M. Gorris (2000)

F. Kasuga (2002–03)

H. Kruse (2000)

X. Lui (2003)

J. I. Pitt (2003)

M. Potter (2002–03)

T. A. Roberts (2001–03)

R. Stephan (2003)

K. M. J. Swanson (2000)

R. B. Tompkin (2003)

M. Zwietering (2003) M. Zwietering (2003)

PREFACE

Contributors and reviewers

J. Greig (Can) T. Nesbakken (Norway)

R. Stephan (Switz)

2 Poultry

F. Kasuga (Japan)

J. E. L. Corry (UK) T. Humphrey (UK)

3 Fish

F. Kasuga (Japan)

Q. L. Yeoh (Malaysia)

4 Feeds

B. Veldman (Neth)

F. Driehuis (Neth)

C. Jakobsen (Den)

5 Vegetables

M. L. Tortorello (USA)

M. Kundura (USA)

6 Fruit

7 Spices

8 Cereals T. Smith (USA) S. Hood (USA)

9 Nuts

10 Cocoa

11 Oils & fats

R. van Santen (Neth)

G. Naaktgeboren (Neth)

12 Sugar

L. Eyde (Aus)

13 Soft drinks

C. Stewart (Aus) K. Deibel (USA)

14 Water

15 Eggs

R. Buchner (USA)

J. E. L. Corry (UK) T. Humphrey (UK)

16 Milk

J. Braunig (Ger) P. Hall (USA)

17 Fermented beverages

A. Lillie (Den) P. Sigsgaard (Den)

18 Index

J. Eyles (Aus)

1 Meat and meat products

I Introduction

Red meat is derived from a number of animal species (e.g. cattle, sheep, goat, camel, deer, buffalo, horse, and pig). Total world production of red meats and quantities in international trade can be obtained from http://apps.fao.org/page/collections?subset=agriculture, a part of http://www.fao.org.

Red meat has the potential to carry pathogenic organisms to consumers. In the past, the main public health problem was caused by the classical zoonoses, i.e. diseases or pathogens that can be transmitted from animals to human beings, such as bovine tuberculosis, and also produce pathological changes in animals. However, the measures introduced by classical meat inspection (inspection, palpation, and incision) have proved highly effective against them. Thus, tuberculosis shows very typical changes of the lymph nodes (granulomatous lymphadenitis); they can be reliably detected by incision of the nodes during meat inspection. However, today, the main problem is latent zoonoses. These pathogens occur as a reservoir in healthy animals, where they produce no pathological conditions or changes. However, they can contaminate the food chain in meat production, for instance during slaughtering. The slogan “healthy animals, healthy food” is not true from this point of view. Strict maintenance of good practices of slaughter hygiene in meat production is of central importance, because microbiological hazards are not eliminated in the slaughtering process. Bacteria able to cause food-borne disease, and which can constitute a hazard in at least some meat products, include Salmonella spp., thermophilic Campylobacter spp., enterohemorrhagic Escherichia coli (e.g. serogroup O157; EHEC), some serovars of Yersinia enterocolitica, Listeria monocytogenes, Clostridium perfringens, Staphylococcus aureus, Cl. botulinum, and Bacillus cereus. Meats are also subject to microbial spoilage by a range of microorganisms including Pseudomonas spp., Shewanella, Enterobacteriaceae, Brochothrix thermosphacta, lactic acid bacteria (LAB), psychrotrophic clostridia, yeasts, and molds.

In recent years, bovine spongiform encephalopathy (BSE) (“mad cow disease”) has attracted public health attention. The ﬁrst cases of BSE were reported in Great Britain in November 1986. It appears probable that the disease can be transmitted to humans by food. The prions that cause the disease are very resistant to chemical and physical inﬂuences, i.e. to heat, UV, and ionizing radiations and disinfectants. Prions are sensitive to certain alkaline substances and moist heat under high pressure. An effective disinfectant measure is steam sterilization at 133 ◦

C and 3 bar pressure for 20 min. On the basis of current knowledge, the cause of the BSE epidemic was animal feed (meat- and bone-meal and the like) containing brain, eyes or spinal cord of infected animals, and other tissues that had been inadequately heated during the production process.

To protect human health, the use of certain bovine organs (so-called speciﬁed risk materials: brain, eyes, spinal cord, spleen, thymus (sweetbread), bovine intestines of cattle >6 months old, visible lymph and nerve tissue, as well as lymph nodes) is prohibited for manufacturing foodstuffs, gelatine, tallow, drugs or cosmetics. More information and actual data can be obtained from the following web-sites: http://www.oie.int/eng/en index.htm; http://www.who.int/mediacentre/factsheets/fs113/en/; http://www.defra.gov.uk/animalh/bse/index.html; http://www.aphis.usda.gov/oa/bse/; http://www.tseandfoodsafety.org/; http://www.unizh.ch/pathol/neuropathologie/.

This chapter, however, mainly describes the microorganisms that contaminate red meats and meat products, and factors and operations that increase or decrease the numbers or spread of microorganisms

2 MICROORGANISMS IN FOODS 6

during processing, storage, and distribution. It also contains sections on the microbiology of froglegs and snails as foods.

A Deﬁnitions Red meat is primarily the voluntary striated skeletal muscular tissue of “red” meat animals. The muscle

is made up of contractile myoﬁbrillar proteins, soluble sarcoplasmic proteins (e.g. glycolytic enzymes and myoglobin) and low molecular weight soluble organic and inorganic compounds. Connective tissue is in intimate association with muscle cells and can constitute up to 30% of total muscle protein. Fat cells occur subcutaneously and both within and surrounding the muscle. Within a muscle, fat cells are located in the perimysial space. Up to one-third of the weight of some muscles may be fat. Muscle tissues also contain 0.5–1% phospholipid.

Meat as legally deﬁned commonly includes various organs (“variety meats” or “offals”). The organs and other parts of the carcass that are regarded as edible vary between countries. The heart has some similarities to skeletal muscle and is composed of striated involuntary muscle, connective tissue, and some lipid. The liver contains uniform liver cells with a network of blood vessels and epithelial-lined sinusoids. In the kidney, there is a meshwork of connective tissue that supports renal tubules, small veins, and arteries.

B Important properties Meat has a high water and protein content, is low in carbohydrates and contains a number of low molec-

ular weight soluble constituents (Table 1.1). The vitamin content (µg/g) of muscle is approximately: thiamine, 1; riboﬂavin, 2; niacin, 45; folic acid, 0.3; pantothenic acid, 10; B 6 , 3; B 12 , 0.02 and biotin,

0.04 (Schweigert, 1987). The concentrations of vitamins vary with species, age, and muscle. Pork muscle has 5–10 times more thiamine than is found in beef or sheep muscle. Vitamins tend to be higher in organs (e.g. liver and kidney) than in muscle.

Meat is a nutritious substrate with an a w (0.99) suitable for the growth of most microorganisms. Growth is primarily at the expense of low molecular weight materials (carbohydrates, lactate, and amino acids). Microbial proteolysis of structural proteins occurs at a very late stage of spoilage (Dainty et al., 1975).

Table 1.1 Approximate composition of adult mammalian muscle after rigor mortis

Component % Wet weight Water

Lipid Glycogen a

2.5 0.1 Glucose a,b and glycolytic intermediates a 0.2 Lactic acid a 0.9 Inosine monophosphate b 0.3 Creatine b 0.6 Amino acids b 0.35 Dipeptides (carnosine and anserine) b 0.35

pH a (5.5) Lawrie (1985). a

b Varies between muscles and animals. Varies with time after rigor mortis.

3 During death of the animal when the oxygen supply to the muscle is cut off, anaerobic glycolysis

MEAT AND MEAT PRODUCTS

of stored glycogen to lactic acid lowers the pH. Post-mortem glycolysis continues as long as glycogen is available or until a pH is reached which inhibits the glycolytic enzymes. In typical muscles this pH is 5.4–5.5. In some muscles (e.g. beef sternocephalicus muscle), glycolysis ceases at a pH near 6 even though considerable glycogen remains. The ultimate pH varies between muscles of the same animal and between animals, and is determined by the glycogen content of the muscle and the accessibility of glycogen to glycolysis. The pH of post-rigor muscle can vary from 5.4–5.5 (lactate content close to 1%) to 7.0 (very little lactate present). The lactate content of muscle is inversely proportional to its pH. On the surfaces of beef and sheep carcasses, the availability of oxygen permits aerobic metabolism to continue, and much of the exposed surface tissue has a pH >6 (Carse and Locker, 1974), which facilitates microbial growth.

In the live animal, the glycogen concentration of muscle averages 1%, but varies considerably. Glycogen in pig muscle is readily depleted by starvation and moderate exercise, whereas glycogen in the muscles of cattle is more resistant to starvation and exercise. In both species, pre-slaughter stress (e.g. excitement and cold) depletes muscle glycogen. Glycogen is more concentrated in liver (2–10%) than in muscle, and its content is also affected by pre-slaughter conditions. A low concentration of glycogen in muscles results in a high ultimate pH, which gives rise to “dark-cutting” beef or dark, ﬁrm and dry meat (DFD).

The amount of glucose in post-rigor muscle varies with pH (Newton and Gill, 1978) being virtually absent in muscle of pH > 6.4. In normal-pH (5.5–5.8) muscle, glucose is present at about 100–400 µg/g (Gill, 1976). Liver has a high glucose content (3–6 mg/g), which appears to be independent of pH (Gill, 1988).

By the time the ultimate pH is reached, adenosine triphosphate has largely broken down to inosine monophosphate (IMP). During the storage of meat, IMP and inosine continue to degrade to hypox- anthine, ribose, and ribose phosphate. Ribose, inosine, and IMP can be used as energy sources by a number of fermentative Gram-negative bacteria, and ribose by Broch. thermosphacta, and a number of lactic acid bacteria.

Fatty tissue contains less water than muscle, has a pH near neutrality with little lactate, and contains low molecular weight components (glucose and amino acids) from serum (Gill, 1986). Consequently, microbial growth on fat is slower than on the surface of muscle.

C Methods of processing and preservation Animals are raised on farms where some are grazed and some are raised under intensive or almost

industrial conditions. The microﬂora in the intestinal tract or on the external surfaces of the animals may vary with the systems of animal production (e.g. more fecal material on the hides of feed-lot cattle). Animals may be slaughtered when young (e.g. calves at 3–4 weeks of age), or when 1 or 2, or several, years old (e.g. cattle and sheep). At the abattoir, the skin of cattle and sheep is removed, the skin of pigs is usually scalded (although it is removed in some plants), then the intestinal tract and viscera are removed. The carcass may then be washed, where regulations permit it, or not, and then chilled.

Spoilage organisms grow rapidly on meat, which is a highly perishable commodity. Thus, trade in meat, even at the local level, depends on some degree of preservation that controls the spoilage flora. The most important means of preservation are chilling or freezing, cooking (includes canning), curing, drying, and packaging. Packaging affords extension of shelf-life. Several procedures to reduce microbial growth are often combined. Chilled temperature storage enables fresh meat to be held for only a limited time before spoilage ensues. However, by vacuum-packaging chilled meat in films of low permeability to gases, or by packaging in modified atmospheres, storage-life may be extended for up to at least 12 weeks.

4 MICROORGANISMS IN FOODS 6

D Types of meat products Red meats are traded as chilled or frozen carcasses, large primal pieces or retail size portions, chilled or

frozen offals, chilled vacuum-packed meat, dried meats, fermented meat, raw or cooked cured products, cooked uncured meat and cooked canned products.

II Initial microﬂora

A Ruminants At birth, the digestive tract of a ruminant is physiologically that of a monogastric. The rumino-reticulum

complex develops quickly between 2 and 6 weeks of age when the animals are fed roughage. Initially, large numbers of E. coli, Cl. perfringens and streptococci are in the gut and are shed in feces (10 7 –

10 8 cfu Cl. perfringens/g, 10 9 cfu E. coli/g). After about 2 weeks, Cl. perfringens declines to about

10 4 cfu/g and E. coli to ca. 10 6 cfu/g at about 3 months of age. When comparing fecal excretion of coliforms, the mean count for eight calves between 3 and 8 weeks of age was log 10 7.2 cfu/g and for

adult cows was log 10 4.9 cfu/g (Howe et al., 1976).

Invasive serotypes of salmonellae, such as Salmonella Typhimurium and S. Enteritidis, are more difﬁcult to control in the live animal than serovars occasionally found in feed. In the ﬁrst few days of life, young ruminants are more susceptible to salmonellae. Calves dosed with S.Typhimurium prior to

3 days of age were more easily infected, and excreted salmonellae for longer periods and in greater numbers, than calves inoculated at 18 days (Robinson and Loken, 1968). At slaughter, salmonellae were also detected more frequently in mesenteric and cecal lymph nodes from the younger animals. Young calves that are surplus to dairy farm requirements may be sold through markets and dealers to rearing farms. In England, salmonellae have been found in 3.7% of environmental samples taken at calf markets and in 20.6% of swab samples from vehicles used to transport calves (Wray et al., 1991). Salmonellae have also been detected on the walls (7.6% of swabs) and ﬂoors (5.3% of swabs) at dealers’ premises (Wray et al., 1990). The mixing of young susceptible calves and their subsequent transport to rearing farms disseminates salmonellae. On arrival at rearing farms, the prevalence of salmonellae in calf feces is relatively low but can increase rapidly. When fecal samples were taken from 437 calves within 2 days of arrival at a rearing farm, salmonellae were detected in 5.3% (Hinton et al., 1983). After about

2 weeks on the farm, salmonellae were found in 42.2% of 491 animals sampled. The shedding rate of salmonellae peaked at 2–3 weeks and then declined; this is possibly associated with the development of a more adult-type intestinal ﬂora.

The high concentration of volatile fatty acids and the pH of the fluid in the developed rumen of the well-fed animal provide some protection to infection with salmonellae and verotoxin-producing E. coli (often of the serogroup O157; VTEC) (Chambers and Lysons, 1979; Mattila et al., 1988). Viable cells of these organisms disappear from rumen fluid at a rate faster than expected from wash-out. Starved or intermittently fed ruminants are more susceptible to infection as salmonellae and VTEC O157 can then grow in the rumen. This probably influences the percentage of infected animals on farms during periods of low feed intake (e.g. drought, mustering, shearing or dipping and high stocking densities). On farms, the prevalence of salmonellae in the intestinal tract varies (Edel and Kampelmacher, 1971). Outbreaks of clinical bovine salmonellosis tend to show seasonal patterns. In the UK, most incidents of bovine salmonellosis occur in summer–autumn and peak near the end of the grazing season (Williams, 1975). Peaks of clinical salmonellosis in sheep in New Zealand during summer–autumn have been associated with movement and congregation of sheep for shearing and dipping.

In a study of the prevalence of salmonellae in cow–calf operations (Dargatz et al., 2000), of 5 049 fecal samples collected from 187 beef cow–calf operations, salmonellae were recovered from 1 or more

5 fecal samples collected on 11.2% (21 of 187) of the operations. Overall 78 salmonellae representing 22

MEAT AND MEAT PRODUCTS

serotypes were isolated from 1.4% (70 of 5 049) of samples, and multiple serotypes from eight samples from a single operation. The ﬁve most common serotypes were S. Oranienburg (21.8% of isolates) and S. Cerro (21.8%), followed by S. Anatum (10.3%), S. Bredeney (9.0%) and S. Mbandaka (5.1%).

Although it is broadly accepted that human salmonellosis is derived from foods, especially meat and poultry, ﬁrm proof is elusive. Sarwari et al. (2001) concluded from US data for 1990–1996, that there was

a signiﬁcant mismatch between the distribution of Salmonella species isolated from animals at the time of slaughter and that of isolates found in humans. This questions the validity of assumptions that raw animal products are the primary source for human salmonellosis, or whether there are methodological reasons for the difference.

The increased susceptibility to infection resulting from changes in the rumen can also affect the prevalence of salmonellae in cattle and sheep during transport from farm to slaughter, or in long transport from farm to farm when feeding patterns and type of feed are changed. Frost et al. (1988) reported a high prevalence of salmonellae in the mesenteric lymph nodes and rumen ﬂuid of adult cattle during the ﬁrst 18 days of entering a feed-lot from a market. After 80 days in the feed-lot, there was little evidence of salmonellae infection. Some of the deaths of sheep during sea-shipment from Australia to Singapore and the Middle East have been due to salmonellosis, which was associated with empty gastrointestinal tracts, loss of appetite and poor adjustment from grazing green pastures to dry feed.

Although healthy cattle may excrete thermophilic campylobacters in their feces, numbers are gen- erally low (NACMCF, 1995). While thermophilic campylobacters are frequently found in the lower intestinal tract of ruminants (prevalence range 0–54%), it is usually present in numbers <1000/g. The organism occurs more frequently and in higher numbers in the feces of very young calves (<3–4 weeks old). It can be present in small numbers (<100/g) in the rumen, where it is probably only part of the transient ﬂora.

Streams, ﬁelds, wild-life and other livestock are all likely to be sources of salmonellae and C. jejuni. The opportunity for animal-to-animal spread is increased in intensively reared animals. Salmonellae contaminated feeds can be a source of infection. Jones et al. (1982) reported an infection of cattle on three dairy farms that was directly attributable to consumption of a vegetable fat supplement contaminated with S. Mbandaka.

L. monocytogenes can exist as a saprophyte in the plant–soil ecosystem, and clinical outbreaks of listeriosis in cattle and sheep have long been linked with feeding silage of inferior quality. L. monocytogenes has been reported in the feces of apparently normal cattle in many countries, whether animals were examined on the farm or at slaughter (Table 1.2). On Danish farms, where there was a high occur-

rence of the organism in dairy cows, it was commonly found in the feed (silage from different crops, and alkalized straw). Silage and decaying vegetable material can contain large numbers of Listeria spp. The higher incidence in Danish cattle than in Danish pigs has been associated with feeding wet plant material to cattle and providing dry feed to pigs (Skovgaard and Norrung, 1989).

VTEC is a group of E. coli that produces one or more verocytotoxins (VT) also known as Shiga toxins (STX). This group of bacteria has many synonyms. In the United States and to a varying extent in Europe, the notation Shiga-toxin producing E. coli (STEC) is used. The term, EHEC was originally used to denote VTEC causing hemorrhagic colitis (HC) in humans; later EHEC has been used as a synonym for VTEC in the medical domain in some European countries (SCVPH, 2003).

VTEC are frequently present in the feces of calves, cattle, buffaloes, sheep and goats (Mohammad et al., 1985; Suthienkul et al., 1990; Beutin et al., 1993; Clarke et al., 1994). These VTEC strains belong to a large number of serotypes. Some (e.g. O5:NM, O8:H9, O26:H11 and O111:NM) may cause diarrhea or dysentery with attaching–effacing lesions in calves (Moxley and Francis, 1986; Schoonderwoerd et al., 1988; Wray et al., 1989). However, VTEC has emerged as a pathogen that can cause food-borne

6 MICROORGANISMS IN FOODS 6

Table 1.2 Listeria monocytogenes in red-meat animals Species

Reference Cattle

Sample site

25 25 van Renterghem et al., 1991

Denmark

75 52 Skovgaard and Morgen, 1988

New Zealand

15 0 Lowry and Tiong, 1988

Yugoslavia

52 19 Buncic, 1991

52 29 Buncic, 1991 Cattle

I.R.P.N. a Yugoslavia

Feces 30 11 Johnson et al., 1990 Cattle (dairy)

6.7 Husu, 1990 Feces

40 3 Siragusa et al., 1993 Cattle (beef)

USA

8 0 Loncarevic et al., 1994 Cattle

Lymph nodes

B&H

33.3 Weber et al., 1995 Cattle

2 Iida et al., 1998 Cattle (dairy)

Content of large intestine

Japan

6 Unnerstad et al., 2000 Cattle

29 31 Fenlon et al., 1996 Sheep

Feces

Scotland

20 0 Lowry and Tiong, 1988 Sheep

Feces

New Zealand

8 Adesiyun and Krishnan, 1995 Pig

25 20 van Renterghem et al., 1991

Denmark

1.7 Skovgaard and Norrung, 1989

Hungary

25.6 Ralovich, 1984.

97 3 Buncic, 1991 Tonsils

Yugoslavia

45 Buncic, 1991 Pig

Yugoslavia

21 5 Loncarevic et al., 1994 Pig

Lymph nodes

B&H

5 Adesiyun and Krishnan, 1995 Pig

Rectal swabs

Trinidad

34 5.9 Weber et al., 1995 Pig

Feces

Germany

0.8 Iida et al., 1998 Pig

Content of large intestine

Japan

50 12 Autio et al., 2000 Horse

Tonsils

Finland

4.8 Weber et al., 1995 a I.R.P.N., Internal retropharyngeal nodes.

infections and severe and potentially fatal illness in humans. VTEC are the cause of human gastroenteritis that may be complicated by hemorrhagic colitis (HC) or hemolytic-uremic syndrome (HUS).

VTEC strains causing human infections belong to a large, still increasing number of O:H serotypes.

A review of the world literature on isolation of non-O157 VTEC (by K.A. Bettelheim) is available on the MicroBioNet website (http://www.sciencenet.com.au). Most outbreaks and sporadic cases of HC and HUS have been attributed to O157:H7 VTEC strains. However, especially in Europe, infections with non-O157 strains, such as O26:H11 or O26:H−, O91:H−, O103:H2, O111:H−, O113:H21, O117:H7, O118:H16, O121:H19, O128:H2 or O128:H−, O145:H−, and O146:H21 are frequently associated with severe illness in humans.

Pathogenicity of VTEC is associated with several virulence factors. The main factor is the ability to form different types of exotoxins (verotoxins). They can be subdivided into a Verotoxin 1 group (Stx1) and a Verotoxin 2 group (Stx2). Characterization of the stx1 and stx2 genes revealed the existence of different variants in both Stx groups. At present, three stx1 subtypes (stx1, stx1c, and stx1d) and several stx2 gene variants have been described (e.g. stx2, stx2c, stx2d, stx2e and stx2f ). Apart from the capability to produce verotoxins, these pathogroups may possess accessory virulence factors such as intimin (eae), VTEC auto-agglutinating adhesin (saa) or enterohemolysin (ehxA). Characterization of eae genes revealed the existence of different eae variants. At present, 11 genetic variants of the eae gene have been identiﬁed and are designated with letters of the Greek alphabet. It is believed that different intimins may be responsible for different host- and tissue cell tropism.

E. coli O157 is found in the feces of cattle and sheep (Table 1.3) and of water buffalo (Dorn and Angrick, 1991). It has been isolated from healthy cattle, from dairy and beef cattle and from pasture-fed and feed-lot cattle (Tables 1.3 and 1.4). In some studies, the highest prevalence appears to occur in young calves shortly after weaning (Meng et al., 1994). Although individual animal infection with

7 Table 1.3 Escherichia coli O157:H7 in the feces of cattle and sheep

MEAT AND MEAT PRODUCTS

Country Animal

Reference Germany

No. samples

% Positive

Dairy cow 47 < 2 Montenegro et al., 1990 Germany

0.9 Montenegro et al., 1990 Scotland

Bull

0.4 b Synge and Hopkins, 1992 Scotland

Cattle a 1 247

< 0.2 Synge and Hopkins, 1992 Spain

Sheep

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