A Hazard Analysis Critical Control Point Approach (HACCP ...177-198.pdf · (slow heating in an...

Food Microbiology, 1990,7,177-l 98

A Hazard Analysis Critical Control Point Approach (HACCP) to ensure the microbiological safety of sous vide processed meat/pasta product J P. Smith’*, C. Toupin2, B. Gagnon3, R. Voye?, P. P. Fiset4 and M. v’. Simpson’ ‘Department of Food Science and Agricultural Chemistry, McGill University, Ste Anne de Bellevue, Q&bee, 2Agriculture Canada Food Research and Development Center, Ste. Hyacinthe, Q&bee, and 3Agriculture Canada Food Production and Inspection Branch, Ottawa, Ontario, and 4La Tour Eiffel, Montreal, Que’bec, Canada

Received 5 December 1989

Increasing consumer demands for microwaveable, convenience foods with extended shelf life yet retaining %loser to fresh’characteristics, has resulted in thegrowth of sous vide or vacuum cooking processing technology. However, this new generation of minimally processed sous vide products are not shelf stable and pose a potential public health risk if subjected to temperature abuse at any stage of the product’s production, storage, distribution and marketing. To ensure the microbiological safety of sous vide products, a Hazard Analysis Critical Control Point (HACCP) approach is recommended at all stages of sous vide processing and distribution of end product. The HACCP approach is a preventive approach to microbiological quality control and is intended to prevent problems before they occur rather than finding them in the finished product. Hazard Analysis identifies the microbiological hazards and potential entry points of these hazards in the sous vide process. Critical Control Points to control the identified microbiological hazards include quality of raw ingredients, time1 temperature relationship, sanitation and packaging control and incorporation of additional barriers, such as pH and water activity (ad reduction, in the formulated product. The practical application ofHACCP to ensure the microbiological safety ofa sous vide processed meat/pasta product are given.

Introduction There has been a tremendous growth, in recent years, in the use of ‘sous vide’ processing (vacuum cooking) technology to extend the shelf life and keeping quality of fresh food. The growth of this technology, and new generation of food products, is in response to consumer needs for ready-to-eat, microwaveable, convenience foods with extended shelf life and yet retaining ‘closer to fresh’ charac-

*Corresponding author

0740-0020/90/030177 + 22 $02.00/O

teristics. With a current market of approximately $20 million per year, sales of sous vide processed products in Canada are projected to triple within the next 10 years, mainly at the expense of frozen and canned food products (Fiset, P. pers. commun.).

Sous vide processing consists of preparation (if necessary), packaging in heat- stable, air-impermeable bags under vacuum to remove all the air, sealing, cooking (pasteurization) to a time and temperature for a specific food, cooling,

0 1990 Academic Press Litited

178 J. P. Smith et 81.

FRESH, TOP-QUALITY INGREDIENTS

I BASIC PREPARATION

(composition of dish, i.e. seasonings, addition of ingredients and sauces under strict hygienic and quality control conditions)

PACKAGING (ingredients weighed, placed in a plastic dish and covered with a plastic seal, impermeable to air and contaminants)

AIR EXTRACTION AND HERMETIC SEALING (air removed with vacuum packaging machine

and produce is hermetically sealed)

PASTEURISATION (slow heating in an autoclave with

a precise electronic regulator)

QUICK-CHILLING (inside a quick chilling chamber)

STORAGE IN COLD CHAMBER (between 0 and 3°C)

I REHEATING

(for lrl-15 min in a boiling water bath or 4-5 min in the microwave)

SERVICE

Fig. 1. Steps in the sous vide (vacuum cooking) process.

and then storage under refrigeration. production, storage, distribution and The various steps involved in vacuum marketing. cooking or sous vide processing are Wyatt and Guy (1980) reported that shown in Fig. 1. Unlike conventional temperatures of supermarket display thermally processed foods, sous vide cases were consistently greater than products undergo only a minimal heat 74”C, with some as high as 176”C, while processing which does not produce a temperatures of home refrigerators shelf-stable product. Recently, concern ranged from l-7-20.2%. Therefore, the has been expressed about the public potential for temperature abuse-even health safety of such minimally processed mild temperature abuse - at some stage food, particularly if subjected to tempera- of the product’s life cycle, particularly ture abuse at some stage of the product’s during storage in the retail environment,

Microbiological safety of sous vide processed meat product 179

exists. Furthermore, since many of these products are marketed in packages that consumers view as trditionally contain- ing shelf stable items, there is a high risk for consumer temperature abuse, mishandling, and over extending the product’s shelf life (Corlett 1989).

In order to ensure the market growth of this new generation of minimally processed products, the onus lies with the processor to ensure the microbiological safety of sous vide processed products at all stages of processing, distribution, marketing and retailing up to the point of consumption by the consumer. While the traditional approach to microbiological quality control involves end product sampling and analyses, this approach has several disadvantages for sous vide processed products: (1) microbiological tests are laborious and time consuming and may generate results too late for corrective action to be taken for a specific batch of product, (2) the test methods employed may not be sensitive enough to detect low numbers of pathogen(s) present in sous vide products, and (3) microbiological test results provide no information concern- ing the sources of contamination/ microbial growth in the end product or the locations in the process for corrective action to prevent future microbial contamination/growth.

To overcome the limitations of conventional microbiological quality control and to ensure product safety, a Hazard Analysis Critical Control Point Approach (HACCP) is recommended at all stages of sous vide processing (Canadian Code of Manufacturing Practices for Pasteurized/ Modified Atmosphere Packaged/ Refriger- ated Food 1990). The HACCP approach is not a new concept and it has been used as a quality control tool in food processing plants (Bauman 1974, Petersen and Gun- nerson 1974, Warne et al. 1985) and also in food service and catering establish-

ments (Munce 1984, Bobeng and David 1977). The HACCP approach, which is essentially a preventative approach to quality control, particularly with regard to microbiological hazards, comprises the following inter-related stages: (1) preparation of a product description and flow process diagram for the manufacturing, storage and distribution of product, (2) assessment of the hazards associated with the purchase, processing, preparation, distribution and/or use of a given raw material or final product, (3) determination of critical control points to control the identified hazards, and (4) establish- ment of procedures to monitor critical control points.

Unlike conventional quality control, the HACCP approach shifts the emphasis from microbiological testing of the final product to raw material and process control. The HACCP approach is therefore a much more forward looking approach and is intended to prevent problems before they occur rather than finding them in the finished product. The objectives of this paper are to briefly review each of the stages involved in the HACCP process and to discuss their practical application to ensure the microbiological safety of a sous vide processed meat/pasta product.

1. Product description and preparation of flow process diagram The first stage in the HACCP approach begins with a product description. This description should include information on raw ingredients and their commercial specifications used in the product formulation, details of product composition, including a, and pH, information on packaging materials/specifications. processing conditions and labelling instructions. An example of a product description for a sous vide processed meat/pasta product is shown in Table 1. The information will assist in the hazard

180 J. P. Smith et al.

0 . J3

cl D v El

OPERATION. An operation occurs when an object is intentionally changed from another object; or is arranged for another operation, transportation, inspection, or storage. An operation also occurs when information is given or received or when planning or calculating takes place. An operation symbol is also used to represent a person doing work.

TRANSPORTATION. A transportation occurs when an object is moved from one place to another, except when such movements are a part of the operation or are caused by the operator at the work station during an operation or inspection.

INSPECTION. An inspection occurs when an object is examined for identification or is verified for quality or quantity in any of its characteristics.

DELAY. A delay occurs to an object when conditions, except those which intentionally change the physical or chemical characteristics of the object, do not permit or require immediate performance of the. next planned action.

STORAGE. A storage occurs when an object is kept and protected against unauthorized removal.

COMBINED ACTIVITY. When it is desired to show activities performed either concurrently or by the same operator at.the same work station, the symbols for those activities are combined, as shown by the circle placed within the square to represent a combined operation and inspection.

Fig. 2. Symbols used in the construction of a flow process diagram.

analysis of the raw ingredients, processes or storage conditions which could result in a hazardous situation occurring and also to determine if suppliers of raw ingredients comply with commercial and/or regulatory specifications.

The next step is the production of a detailed flow diagram for the sous vide process. A flow process diagram is essentially a diagrammatic scheme of a plant’s operations from receipt of raw materials through processing to packaging, storage and distribution of finished product and should be constructed by production spe- cialists with expertise in these areas.

Each step in the process should be written down systematically and then recorded diagrammatically using the flow diagram symbols shown in Fig. 2 (Apple 1977). Even minor steps, such as delays in the production process, should be listed, because even though they may not be considered important, they could have an additive effect on the safety of the product at some other stage further on in the process. An example of a flow process chart, summarizing the operations for a sous vide processed meat/ pasta product, is shown in Fig. 3. Process flow charts have several important func-

Microbiological safety of sous vide processed meat product I731

Table 1. Product description for a sous vide processed meat/pasta product

Product description

Ingredients

Meat/pasta heat-and-serve product (28 day shelf life at refrigerated storage conditions)

Lean meat (1ean:fat 80:20), pasta, fresh mushrooms, tomato paste, spices (salt/pepper), water

Product compositiona

Packaging specifications

Processing conditions

Labelling specifications

Moisture (30%), fat cl%), protein (16%), carbohydrate (50%), ash (3%) Water activity (aJ0-98 pH 5.3

Tbermoformed polypropylene trays (10 mill polypropylene top web (2 mil) Net weight of packaged product 375 g

Pasteurize at 70°C for 45 min. Cool to 4°C within 2-3 h of processing. List of ingredients. Keep refrigerated at 4°C. Expiry date. Cooking/microwave instructions.

% Wet weight basis.

tions in food processing operations and can be used: (a) to analyze material movement and to show interrelationships between product lines, (b) to highlight ex- cessive transportation steps, (c) to predict potential ‘bottlenecks’ and delays in production, and (d) to optimize the use of space and equipment. However, the most important function of a flow diagram is that it facilitates the Hazard Analysis and Critical Control Point (HACCP) evaluation of the sous vide process.

2. Hazard analysis .

In order to establish the safety of sous vide products, a hazard analysis must be undertaken at each stage of a product’s transition from raw ingredients to finished product and distribution. Hazard analysis has been defined as ‘any system which analyses the significance of a hazard to consumer safety or product acceptability’ (Thorpe and Leaper 1988). A hazard has been defined as a ‘potential to cause harm to the consumer (safety) or the product (spoilage)’ (Thorpe and Leaper 1988). Since the hazard analysis must cover the entire flow process dia-

gram, it should be conducted by a team with expertise in procurement of raw materials, food processing operations, food packaging, food microbiology, food chemistry, toxicology, quality assurance and regulatory affairs. The objectives of the hazard analysis are: (a) to identify all the potential food hazards, specifically microbiological hazards, which are a threat to consumer safety, (b) to identify potentially hazardous foods or ingredients, and (cl to identify the process points in the sous vide process where a potential mirobiological hazard may exist (ICMSF 1988, Peterson and Gunnerson 1974). Each of these parts of the hazard analysis will be briefly reviewed.

(a) Identification of food hazards

The major categories of food hazards associated with food processing are sum- marized in Table 2 (Stauffer 1988). While the HACCP evaluation is intended to address all potential physical, chemical and microbiological hazards which compromise product safety, microbiological hazards (specifically food borne pathogens) pose the greatest threat to con-


0 1

9

0,

0 2

w

D 1

0 3

w

0 4

0 5

0 6

w

0 7

w

D 2

0 8

Receipt of raw materials (meat, vegetables. spices, packaging materials)

Transfer to controlled storage

Controlled storage

Transfer to production area

Formulation of raw ingredients

Transfer to filling area

Delay prior to filling/during filling

Filling/vacuum packaging of product

Transfer to retort

Filling of retort

Pasteurization/cooling of product

Removal of product from retort

Transfer to cooling area

Rapid cooling of product

Transfer to labelling area

Delay prior to/during labelling due to product buildup

Labeling

Fig. 3. A flow process diagram for a sous vide processed meat/pasta product.

sumer safety because of their ubiquitous nature, and must receive priority in any HACCP evaluation, and so will be the only hazards addressed in this paper.

Waites (1988) subdivided food borne pathogens into three main groups: those that present severe hazards, those that present moderate hazards with potentially extensive spread, and those that present moderate hazards with limited spread. Examples of food borne patho-

gens within each group are shown in Table 3 (Waites 1988).

The major microbiological hazard associated with sous vide processing is the growth of, and toxin production by, Clostridium botulinum types A, B and E spores. These sporeforming organisms pose the greatest threat to consumer safety due to the ability of this sporeforming pathogen to withstand the mild heat processing conditions of sous vide pro-


w Transfer to cartoning area

D 3 Delay prior to/during cat-toning

0 9 Cartoning

w Transfer to warehouse

v Storage in warehouse

is/t Transfer to loading area

D 4 Delay prior to/during loading

0 10 Loading of transport container

0 11 Transportation to retail outlet

v Storage in supermarket

SUMMATION OF EVENTS:

11 Operations,

9 transfers,

3 controlled storages,

4 major delays.

cessing, the complete or partial destruction of non-sporeforming competing mic- roflora and potential indicators of incipi- ent spoilage, and the presence of anaerobic packaging conditions condu- cive to the growth of, and toxin production by, C:botulinum in the processed product. Control of this pathogen is therefore critical to product safety in view of the fact that non-proteolytic strains of C. botulinurn are capable of growth and toxin production at temperatures as low as 35”C, while C. botulinurn type A and B spores can grow at temperatures of lO- 12°C (Palumbo 1986).

Non-sporeforming, food borne pathogens of public health significance in sous vide processed products include entero- pathogenic strains of Escherichia coli,

Salmonella, Staphylococcus, Aeromonas, Listeria and Yersinia species. Recent studies have shown that L. monocytogenes and Y. enterocolitica are capable of growth at refrigeration temperatures (<5”C) over an extended time period (7- 21 days) (Wyatt and Guy 1980, Palumbo 1986). They also reported that traditional food borne pathogens, such as Salmo- nella species and Staph. aureus, are capable of growth at temperatures 5- 12”C, i.e. conditions of slight temperature abuse.

While all of these non-sporeforming pathogens should be destroyed during thermal processing, they pose a potential threat to consumer safety if the raw ingredients are of poor microbiological quality or the product becomes heavily con-

184 J. P. Smith et 81.

Table 2. Food hazards (Stauffer 1999)

A.

B.

C.

D.

E.

F.

G.

Bacteria Clostridium botulinum Clostridium perfringens Salmonella Staphylococcus aureus

Molds Aspergillus flavus Penicillium cyclopium

Parasites Tape worms Trichinellae

H.

I.

J.

Radioactive isotopes Cesium 137 Iodine 131 Potassium 90

Extraneous matter Filth Glass splinters Peeling paint Tramp metal

Pests Birds Insects Rodents

K.

Naturally occurring toxins Ciguatera poisoning Gilseed toxins Nutritional deficiencies Instant formula exceptions Processed foods

Residues Antibiotics Chlorinated insecticides Grganophosphate insecticides

L.

M.

Regulatory hazards Label errors Short weights

Industrial chemicals Hexabromobiphenyl Polychlorinated biphenyls Vinyl chloride

Heavy metals Arsenic Cadmium Lead Mercury Selenium

Functional hazards Packaging defects Particle size deviations

Table 3. Groups of food borne pathogens (Waites 19331

Group 1. Severe Hazards

Brucella abortus Salmonella sendai Brucella melitensis Salmonella typhi Brucella suis Shigella spp. Clostridium botulinum Vibrio cholerae Mycobacterium bovis Hepatitis B virus Salmonella cholerae-suis Fish and shellfish toxins Salmonella paratyphi A Some mycotoxins

Group 2. Moderate hazards with potentially extensive spread

Pathogenic Escherichia coli Salmonella spp. Listeria monocytogenes Streptococcus pyogenes

Group 3. Moderate hazards with limited spread

Bacillus cereus B. licheniformis B. subtilis Campylyobacterjejuni Clostridiumperfringens Coxiella burnetii

Staphylococcus aureus Streptococcus zooepidemicus Trichinella spiralis Vibrio parahaemolyticus Yersinia enterocolitica


taminated during processing as a result evaluation of a product and its ingredi- of poor manufacturing practices and the ents according to their hazard character- pasteurization process is inadequate to istics, using ‘+’ for yes and ‘0’ for no. destroy the high microbial load of non- According to Bauman (19741, the hazard sporeforming pathogens. categories based on decreasing risk are:

(6) Identification of hazard categories

An important step in the hazard analysis is the identification of the ingredients’ and finished product’s hazard characteristics and identification of their appropri- ate hazard category. According to Peter- son and Gunnerson (1974), a product and its ingredients may be a significant health risk ifit has the following hazard characteristics.

Category I: Foods intended for use by infants, the aged and the infirm.

Category II: Foods with all three hazards (+++I.

Category III: Foods with two hazards t+o+, o++, ++oj.

Category IV: Foods with one hazard coo+, +oo, o+oj.

Category V: Foods with none of these hazards (000).

They contain sensitive ingredients and therefore potentially harmful organisms.

The manufacturing process does not contain a controlling processing step that does not effectively destroy all harmful micro-organisms and microbial toxins.

There is a potential for microbiological abuse (temperature abuse) in distribution or in consumer handling that could render the product harmful when consumed.

The risk characteristics and hazard categories of a sous vide meat pasta product and the ingredients used in its formulation are shown in Table 4. It is evident from Table 4 that a sous vide meat/pasta product has a high degree of risk due to the fact that they contain sensitive ingredients, they are subject to a minimal heat processing step, and there is a real potential for consumer abuse.

The combination of all these factors can be used to classify food/ingredients into hazard categories based on a risk assessment. A risk has been defined as the probability of hazard occurring (ICMSF 1988). A risk assessment is a yes/no

(c) Identification of critical operations

Having identified the hazard characteristics and categories of the raw ingredients and finished product, the next stage in the hazard analysis is to identify the critical operations in the sous vide pro-

Table 4. Risk assessment of a sow vide meat/pasta product

Risk characteristics

Item

Product Sous vide meat/pasta product (a, 0.98/pH 5.6)

Ingredients Fresh ground beef Pasta Fresh mushrooms Tomato paste Water Spices/sugar

Sensitive Microbes Abuse Hazard ingredient not destroyed potential category

+ + + II

+ + + II 0 + 0 IV + + 0 0 + 0 if + 0 0 Iv 0 0 0 V


cess. A critical operation is a point in a process where a potential microbiological hazard may exist or arise. Examples of potential critical operations for a sous vide process product are shown in Table 5. Many of the critical operations are obvious - e.g. quality and storage of raw ingredients, thermal processing - but others may be less apparent, such as prolonged delays between processing stages which could increase the risk of temperature abuse and microbial growth.

3. Determination of critical control points An important stage of the HACCP evaluation is the identification of critical control points (CCP) in the process to control

identified microbiological hazards to ensure product safety. Several defi- nitions for critical control point exist. The National Advisory Committee on Micro- biological Criteria for Foods (1989) defined Critical Control Points as any point or procedure in a specific food system where loss of control may result in an unacceptable health risk. Bobeng and David (19771, defined Critical Control Points as the points in a process which eliminate or reduce a microbiological hazard from occurring. Bauman (19741, defined Critical Control Points as those processing factors where loss of control would result in an unacceptable food safety risk. Baird-Parker and Hayes (1990) defined a Critical Control Point (CCP) as a location, practice, procedure

Table 5. Control and Critical Points for a sous vide processed meat/pasta product

Critical control points

Raw Sanitation Time ingredient equipment/ temperature Additional

Critical operations control personnel relationship Packaging ‘barriers’

Receipt of raw ingredients X

Controlled storage of raw X X ingredients

Formulation X X X X

Delay prior to filling X X

Filling of product X X X

PasteurizationCooling X X in retort

Rapid cooling of product on X X leaving retort

Delay prior to labelling X

Labeling X X

Delay prior to cartoning X

Car-toning X X

Delay prior to loading X container

Loading of container X X

Transportation to retail X outlet

Storage in retail outlet X X


Table 6. Sensitivity categories for food ingredients (Stauffer 1966)

I. Ingredients for special groups Infants IIlfmn Geriatric

II. Susceptible ingredients

III. Insensitive ingredients

Dairy products Em Fish Meat soup

Flour Shortening SOY Starch

IV. Ingredients free of pathogens Baking Soda Citric Acid Food colors Monosodium Glutamate Salt Sugar

or stage in the food production, distribution and use chain which can be used to control the risk (probability of occur- rence) of an identified hazard to an acceptable level, i.e. safe level. Critical Control Points can take a variety of forms such as raw ingredient quality, heat processes, chemical and physical changes to the food, such as acidification, reduction of a, and addition of preservatives and specific hygiene practices. Examples of CCPs, either alone or in combination with each other, which reduce the risk of contamination by, and survival and pro- liferation of, the identified microbial hazards are: quality of raw ingredients, sanitation (personnel/equipment), time/ temperature relationship, packaging and levels of additional barriers.

It is evident from Table 5 that more than one CCP may influence product safety at a specific critical operations during the process. For example, during the formulation stage, the microbiological quality of the raw ingredients, equipment and personal sanitation, the time sensitive ingredients are held at a particular temperature and levels of additional barriers used in the final product formulation are all important critical control points

affecting the microbiological safety of the end product. Each critical control point must therefore be monitored on a regular basis as loss of control of any one of these critical control points could have an additive effect on the microbial load of the finished product and result in a hazardous situation occurring. Specific control options for each critical control point and their contribution to microbiological safety of sous vide processed meat/pasta product are briefly discussed.

(a) Quality of raw ingredients

Raw ingredients are the primary vectors of harmful or potentially harmful micro- organisms in all processed food products. According to Stauffer (1988) food ingredients can be arranged into categories on the basis of their sensitivity to microbial contamination (Table 6). While certain raw ingredients are free or essentially free of micro-organisms, care must be taken with the purchase and storage of more sensitive ingredients, particularly those of animal origin such as meat, fish and poultry which may be contaminated with pathogenic organisms (Stauffer 1988). The hazard analysis should identify all the ingredients at risk in the for-


mulation and identify the food borne pathogens commonly associated with each raw ingredient. To minimize the potential risks from hazardous micro- organisms, procurement of raw materials of the best possible microbiological quality is essential. Physical inspection of sensitive raw ingredients for visible signs of contamination, e.g. hair, dirt, slime should be done to ensure the microbiological quality of raw ingredients. Tem- perature checks should also be done on all refrigerated/frozen product to ensure that the product has not been subjected to any temperature abuse during transportation to the plant. Laboratory testing of ingredients should be done on a regular basis by food processors and ingredient suppliers to ensure ingredients conform to regulatory and commercial microbiological specifications.

(b) Sanitation (equipment and personnel)

An important critical control point in the sous vide process is sanitation. This CCP is important since the effectiveness of the pasteurization treatment will depend on the microbial load of the product. Bryan (1974) reported that two factors contribu- ting to food borne disease outbreaks were inadequate cleaning of equipment and cross-contamination of products. Cross- contamination of in-process product from raw product can be avoided through good manufacturing practices. Procedures should be established by each processor for cleaning and sanitizing of storage facilities, food contact surfaces, tools and utensils and processing equipment. In addition, detailed schedules specifying methods of disassembling equipment, methods and frequency of cleaning, and tests for cleaning efficiency should all be clearly specified. It is also imperative to prevent/minimize cross-contamination of the in-process and finished product from raw ingredients. Raw vegetables, fish,

meat, etc. should be segregated in specific storage areas and cleaned/prepared in separate processing areas. Furthermore, traffic patterns should be established to control movement of food products between processing areas of the plant to prevent cross contamination and build up of product. Buildings and processing equipment should be well designed to facilitate cleaning operations and to ensure a forward and regulated flow in the process operations from arrival of raw ingredients to shipping of finished product.

In addition to plant and equipment sanitation, employee hygiene and good handling practices are important in pre- venting inoculation of food materials with pathogenic bacteria. Bryan (1974) reported that infected persons practicing poor personal hygiene contributed to 151 of 725 food poisoning outbreaks reported during 1961-1972. It is the repsonsibility of management to ensure that employees be properly attired during food handling/ processing, to minimize product contamination. Any person suffering from a food borne illness should not be working in any food handling or preparation area. All employees must feel a sense of personal responsibility for the microbiological safety of food products. Adequate and continuing education of personnel in food and personal hygiene should be an important priority of both employer and employee.

(c) Timeltemperature relationship

It is evident from Table 5 that time- temperature is one of the most important CCPs influencing product safety. The time/temperature relationship, i.e. the time a product is held at a specified temperature, is critical to minimize microbial growth during product preparation, storage and distribution and in the retail environment. All sensitive ingredients used in sous vide products should be

Microbiological safety of sous vide processed meat product k39

stored under strict temperature controlled conditions. Longree (1972) recommended optimal refrigerated storage temperatures by food CategoryAairy product and eggs 2 to 4”C, meat and poultry -1 to 2”C, fish - 1 to 0°C and fruits and vegetables 2-4”C. Refrigerated storage time limits should be specified for all potentially hazardous raw ingredients and a first-in, first-out system of inven- tory control should be used. Refrigerated storage areas should be well designed to permit a free flow of cold air between and around ingredients/products and be equipped with a mechanical refrigeration system capable of maintaining ingredient/product temperature at - 1 to 4°C under normal conditions of outside humidity, temperature and peak loading capacity. The general principles outlined above are also applicable to storage, distribution, and retail display of end product. If a breakdown occurs in any of the refrigerated units, product temperature should be checked and if within acceptable limits (<4”C), the product should be transferred to another refrigerated unit. If product temperature is significantly above the acceptable range established for products, expert advice should be sought from the manufacturer, process authority or from regulatory agency officials.

The temperature of’ all preparation/ packaging areas should be maintained at 10°C or below to minimize the growth of food borne pathogens prior to heat processing. If there is a delay at any processing operation, e.g. product build up at filling operations or pasteurization, the product should be diverted and stored at ~4% until product build up decreases and normal flow is resumed.

The time/temperature relationship is also critical to maximize microbial destruction during pasteurization. Longree (1972) recommended that food should be heated to 74-77”C, in order to destroy

vegetative cells of common pathogens that cause food borne illness. Sous vide products should therefore be heat processed until all parts of the food are at least 60°C or above and held at this temperature for a specified time period, as determined by a process authority. The maximum time a product will be held at temperature will depend on the types and numbers of micro-organisms contami- nating the product, the heat resistance of each organism, the physicochemical nature of the food product, and the desired organoleptic quality of the final product. In most cases, a thermal process is designed to achieve a 12-13 log cycle of a target organism with the highest D value. The most common organism used is Streptococcus faecalis which has a D value of 2.95min at 70°C (Rosset and Poumeyrol1986). Therefore, thermal in- activation of this organism would ensure the destruction of vegetative cells of non- spore forming pathogens such as L. monocytogenes and Y. enterocolitica (Rosset and Poumyrol 1986). The thermal processing requirement for each product should be carried out during the product development phase in order to verify the efficacy of the heat process. Operation of the heat processing equipment, thermal process calculations of the lethal potential of the pasteurization treatment, and adequacy of the pasteurization treatment should all be carried out by appropriately trained personnel. Any process deviations should be brought to the immediate attention of competent process authority for immediate corrective action. It is important for all thermal process parameters to be reassessed on a continuous basis if changes are made to formulation/portioning/filling weights of product.

While the pasteurization treatment should inactivate all vegetative cells of non-sporeforming food borne pathogens, it will have little effect on the C. botuli-


num spores. However, whenever a product can tolerate a more severe heat process, the product could be processed at temperatures sufficient to inactivate less heat resistant spores C. botulinum type E which have a D value of O-33 min at 90°C. Processing at this temperature will have no effect on C. botulinurn type A and B spores which have D values of 200 min at 90°C (Basset and Poumeyrol 1986). Con- trol of these organisms can be achieved through proper refrigeration and/or the use of additional barriers which are discussed below.

In addition to heating, cooling should be carried out as quickly and efficiently as possible to bring the temperature of the warmest point of food from 60°C and above to less than 7.2% within l-5 h and to 4°C within 3 h. This is essential to prevent the growth of C. botulinurn species or any other food borne pathogen which may have survived thermal processing. The processed product should be stored at 4°C or less and maintained at that temperature throughout the distribution chain until consumed.

(d) Additional barriers

The growth of food borne pathogens can be inhibited if proper temperature control (0 to 2°C) can be maintained throughout all stages of processing, storage and distribution. However, refrigeration by itself cannot be guaranteed as an adequate barrier for the microbiological safety of sous vide products, or any other minimally processed food product, and additional barriers may be necessary to ensure the public health safety of the end product (Palumbo 1986). The combined use of several barriers or preservative factors is best explained by the ‘hurdle concept’ of Leistner (1978) which states that ‘several barriers, even if any of them individually cannot inhibit microbial growth, will nevertheless prevent microbial growth if the barriers are incor-

porated into a food in sufficient number and height’. Barriers which could be used in conjunction with temperature to inhibit surviving micro-organisms, specifically C. botulinurn in sous vide products, include water activity (~1, pH, and preservatives. Combinations of a, and pH reduction have proved effective in controlling growth of C. botulinurn type E in caviar stored at room temperature while combinations of heat treatment, smoking and brine have been used to control growth of C. botulinum type E in smoked fish (Hauschild and Hilsheimer 1979, Christiansen et al. 1968). The effectiveness of additional barriers in sous vide products should be evaluated using inoculated pack studies during the product development stage in order to establish the levels of barrier(s) to ensure both safety and organoleptic quality of the product. This testing should be done under temperature abuse conditions in order to demonstrate the effectiveness of the additional barriers to prevent growth of, and toxin production by, C. botulinum. Product a,, pH and level of preservative must be monitored on a continual basis to ensure that this critical control point is within commercial and regulatory specifications.

(e) Packaging

Packaging is an important critical control point and plays an essential role in pro- tecting the food from external contamination before, during and after thermal processing. The packaging material itself should not be a source of microbial contamination and/or lack protective characteristics, particularly under thermal process conditions. Upon receipt, all packaging materials should be tested visually for their integrity and physical cleanliness and stored in a clean area at a specified temperature/relative humidity in accordance with the manufacturer’s


specifications. Specifications (structural, chemical and microbiological) of packaging materials and the final paackage should be defined and monitored for each batch of product. If possible, packages should be chosen to avoid confusion with those used for shelf stable products. Monitoring procedures should be applied to ensure filling temperatures/weights, sealing pressures/temperatures comply with both commercial and regulatory specifications. Careful control of filling temperature is critical to prevent microbial growth prior to pasteurization while correct filling weights ensure adequate heat distribution and proper processing of product. Packaging integrity should be monitored routinely in order to prevent contamination by micro- organisms prior to and after thermal processing and during storage, distribution and sale. Improper labeling may also increase the risk for consumer abuse and the growth of potentially hazardous bacteria. Each package should be promi- nently labeled ‘keep refrigerated’ or ‘keep under refrigeration’ and also contain a ‘use by’ date or a ‘sell by’ date. The consumer should also be advised of proper handling and preparation procedures to ensure product safety.

4. Monitoring of critical control points For each CCP in the processing operation, detailed monitoring procedures for control options influencing product safety should be determined and written down. Such information should include methods of monitoring, frequency of monitoring, acceptable limits or specifications for specific control options, and corrective action if a CCP is out of specification. Sampling plans should be developed according to ICMSF (1986) guidelines based on the hazards a food contains and the intended end use of the product. An

example of a sampling plan based on the hazard characteristics of ingredients/ finished product is shown in Table 7. It is evident from this plan that the more hazards a food contains, the greater the frequency and stringency of sampling. Wherever possible, CCPs should be monitored continuously, e.g. critical temperatures during processing should be monitored with a temperature recording device or temperature/time integrator. It is important that all monitoring devices be efficient and well designed, checked regularly and accurately calibrated. When monitoring by automatic methods is not possible, monitoring should be done by appropriately trained personnel involved with processing operations. Moni- toring may involve visual observations (e.g. visual inspection of meat, cleanliness of work surfaces), physical measurements (filling temperature/weights, temperatures of coldrooms/work areas), and chemical measurements (e.g. Q, pH). Examples of monitoring procedures at specific critical control points for a sous vide processed meat/pasta product are shown in Table 8. Results of monitoring must be recorded to serve a documen- tation that the process is under control at all time. Several methods can be used to record observations, e.g. use of control charts, which can be used to visualize when a critical control point parameter is out of specification. Once the HACCP plan is put into operation, it should be continuously checked to ensure that each step is being carried out, that results are being recorded, and that prompt action is taken when control limits for control options are out of specification. The seriousness of a failure to control the identified CCP must then be determined. A concern has been defined as ‘an expression of the seriousness of a failure to control a critical control point, derived from knowledge of a hazard and the risk of it occurring’ (Thorpe and Leaper 1988). According to

Tabl

e 7.

Exa

mpl

es

of m

icro

biol

ogic

al

sam

plin

g pl

ans

depe

ndin

g on

deg

ree

of h

ealth

ha

zard

Cond

itions

in

which

th

e fo

od

is ex

pect

ed

to b

e ha

ndled

an

d co

nsum

ed

afte

r sa

mpl

ing,

in

the

usua

l co

urse

of

eve

nts’

Degr

ee

of c

once

rn

relat

ive

Cond

itions

re

duce

.to

utili

ty

and

healt

h ha

zard

de

gree

of

con

cern

Co

nditio

ns

caus

e no

ch

ange

in

conc

ern

Cond

itions

m

ay

incre

ase

conc

ern

No d

irect

he

alth

haza

rd

Utilit

y (e

.g.

shel

f Iif

e an

d sp

oilag

e)

Incr

ease

sh

elf

life

Case

1

3-cla

ss

n =

5, c

= 3

b

No c

hang

e Ca

se

2 3-

class

n

= 5,

c =

2

Redu

ce

shel

f life

Ca

se

3 3-

class

n

= 5,

c =

1

Heal

th

haza

rd

Low,

ind

irect

(indic

ator

or

gani

sms)

Re

duce

ha

zard

Ca

se

4 3-

class

n

= 5,

c =

3

No c

hang

e Ca

se 5

3-

class

n

= 5,

c =

2

Incr

ease

ha

zard

Ca

se

6 3-

class

n

= 5,

c =

1

Mod

erat

e,

dire

ct,

limite

d sp

read

Ca

se 7

Ca

se 6

Ca

se 9

3-

class

n

= 5,

c =

2

3-cla

ss

n =

5, c

=

1 3-

class

n

= 10

, c

= 1

Mod

erat

e,

dire

ct,

pote

ntial

ly ex

tens

ive

Case

10

Ca

se

11

Case

12

sp

read

a-

class

n

= 5,

c =

0

a-cla

ss

n =

10,

c =

0 2-

class

n

= 20

, c

= 0

Seve

re,

dire

ct

Case

13

Ca

se

14

Case

15

2-

class

n

= 15

, c

= 0

2-cla

ss

n =

30,

c =

0 a-

class

n

= 60

, c

= 0

aMor

e st

ringe

nt

sam

pling

pla

ns w

ould

gene

rally

be

use

d fo

r se

nsitiv

e fo

ods

desti

ned

for

susc

eptib

le

popu

lation

s. by

= n

umbe

r of

sam

ple

units

pe

r lo

t. c

= m

axim

um

allow

able

num

ber

of s

ampl

e un

its y

ieldin

g an

uns

atisf

acto

ry

resu

lt.

Sour

ce:

ICM

S (1

986)

.

Table

8.

M

onito

ring

of C

ritica

l Co

ntro

l Po

ints

fo

r a

sow

vide

mea

t/pas

ta

prod

uct

Crit

ical

op

erat

ion

Pote

ntia

l ha

zard

/risk

C

ritic

al

Con

trol

Poin

t(s)

Prev

enta

tive,

co

ntro

l an

d m

onito

ring

proc

edur

es

Rec

eipt

of r

aw i

ngre

dien

ts

(fres

h m

eat)

Pres

ence

of s

poila

ge/p

atho

geni

c ba

cter

ia,

e.g.

Sal

mon

ella

, Li

ster

ia

mon

ocyt

ogen

es a

nd C

lost

ridiu

m

botu

linum

.

Con

trolle

d st

orag

e of

raw

in

gred

ient

s G

row

th

of s

poila

ge/p

atho

geni

c ba

cter

ia.

. C

ross

-con

tam

inat

ion

of ra

w

ingr

edie

nts.

Form

ulat

ion

Mic

robi

al

load

of

raw

ing

redi

ents

.

Del

ay p

rior

to f

illin

g

Con

tam

inat

ion

by e

quip

men

U

hand

lers

. G

row

th

of s

poila

ge/p

atho

geni

c ba

cter

ia.

Gro

wth

of

sur

vivi

ng

path

ogen

s af

ter

proc

essi

ng,

parti

cula

rly

unde

r te

mpe

ratu

re

abus

e co

nditi

ons.

Mic

robi

olog

ical

qu

ality

of

raw

in

gred

ient

s.

Tem

pera

ture

co

ntro

l.

Sani

tatio

n of

sto

rage

co

nditi

ons.

Mic

robi

olog

ical

qu

ality

of

raw

in

gred

ient

s.

Equi

pmen

t an

d pe

rson

nel

sani

tatio

n.

Tem

pera

ture

co

ntro

l.

Form

ulat

ion

cont

rol.

Cro

ss c

onta

min

atio

n G

row

th

of s

poila

ge/p

atho

geni

c ba

cter

ia.

Sani

tatio

n.

Tem

pera

ture

co

ntro

l.

Visu

al i

nspe

ctio

n of

mea

t fo

r di

rt,

hair,

sl

ime.

If m

eat

disc

olor

ed/

smel

ls,

do m

icro

biol

ogic

al

anal

ysis

. N

ote

on p

rodu

ctio

n ca

rd.

If pr

oduc

ts o

ut o

f com

mer

cial

sp

ecifi

catio

n,

retu

rn

to s

uppl

ier.

Mea

sure

te

mpe

ratu

re

of fr

esh

mea

t. C

heck

tem

pera

ture

/rela

tive

hum

idity

of

sto

rage

con

ditio

ns.

Adju

st

tem

pera

ture

s.

Not

e ph

ysic

al c

lean

lines

s of

sto

rage

ar

eas/

sepa

ratio

n of

raw

fro

m

proc

esse

d pr

oduc

t. Vi

sual

ins

pect

ion

of ra

w

ingr

edie

nts.

M

easu

re

tem

pera

ture

of

tem

pere

d m

eat.

Che

ck p

hysi

cal

clea

nine

ss o

f wor

k ar

eas.

Not

e pe

rson

nel

hygi

ene

of a

ll fo

od

hand

lers

. En

sure

all

food

han

dler

s pr

oper

ly

attir

ed

to h

andl

e fo

od.

Che

ck t

empe

ratu

re

of a

ll pr

oces

sing

are

as (

10 f

1°C

). C

heck

fo

rmul

atio

n,

flnal

a,

and

pH o

f pr

oduc

t. If

any

chan

ges

in

form

ulat

ion

notif

y pr

oces

sing

au

thor

ity.

Che

ck p

hysi

cal

clea

nlin

ess

of

area

s w

here

bui

ld u

p of

pro

duct

oc

curs

. Not

e te

mpe

ratu

re

of

prod

uct.

Not

e tim

e/le

ngth

of

del

ay.

If pr

olon

ged

dela

ys a

ntic

ipat

ed,

stor

e pr

oduc

t at

l-2

%.

Con

tinue

d

Tabl

e 8.

C

ontin

ued

Crit

ical

op

erat

ion

Pilli

ng o

f pro

duct

Pote

ntia

l ha

zard

/risk

Gro

wth

of

pat

hoge

nic

bact

eria

pr

ior

to p

roce

ssin

g.

Und

erpr

oces

sing

of

pro

duct

an

d su

rviv

al

of n

on-s

pore

for

min

g pa

thog

enic

ba

cter

ia.

Crit

ical

C

ontro

l Po

int(s

)

Equi

pmen

t/per

sonn

el

sani

tatio

n.

Tem

pera

ture

co

ntro

l. Pa

ckag

ing/

fillin

g/se

alin

g op

erat

ion

Prev

enta

tive,

co

ntro

l an

d m

onito

ring

proc

edur

es

Che

ck p

hysi

cal

clea

nlin

ess

of

fillin

g eq

uipm

ent/a

rea.

C

heck

hy

gien

e pr

actic

es o

f foo

d ha

ndle

rs.

Ensu

re a

ll ra

w p

rodu

ct

is

phys

ical

ly r

emov

ed f

rom

pro

cess

ed

prod

uct.

Che

ck t

empe

ratu

re

of

fillin

g ar

ea (

10 +

1°C

). C

heck

in

tegr

ity

of p

acka

ging

m

ater

ials

. C

heck

fill

wei

ght/v

acuu

m/p

acka

ge

head

spac

e. I

f pr

oduc

t ov

erfil

led,

ad

just

fill

ers.

If

impr

oper

ly

seal

ed

or to

o hi

gh r

esid

ual

Oz,

rese

t va

cuum

/hea

t se

al s

ettin

g.

Not

e on

pr

oduc

tion

card

. N

otify

pa

ckag

ing

supe

rvis

or o

f var

iatio

n fro

m

stan

dard

s.

Past

euriz

atio

n of

pro

duct

/ co

olin

g Su

rviv

al

and

grow

th

of s

pore

and

Ti

me/

tem

pera

ture

of

ther

mal

no

n-sp

ore

form

ing

path

ogen

ic

proc

essi

ng/c

oolin

g.

bact

eria

. Pa

ckag

ing

inte

grity

.

Che

ck p

roce

ssin

g tim

e/

tem

pera

ture

an

d ac

cura

cy o

f th

erm

omet

ers.

C

heck

tem

pera

ture

at

cen

tre o

f pro

duct

dur

ing

heat

ing.

Ve

rify

ther

mal

pr

oces

sing

ca

lcul

atio

n.

Not

e on

pro

duct

ion

card

. N

otify

th

erm

al

proc

essi

ng

auth

ority

de

viat

ions

fro

m

esta

blis

hed

proc

essi

ng p

roce

dure

s.

Che

ck p

acka

ging

in

tegr

ity

prio

r to

pa

ckag

ing.

C

heck

for

bul

ging

pa

ckag

es.

Rem

ove

from

bat

ch.

Cont

inued

Tabl

e 8.

Con

tinue

d

Crit

ical

op

erat

ion

Rap

id c

oolin

g of

pro

duct

Pote

ntia

l ha

zard

/risk

Gro

wth

of

sur

vivi

ng

path

ogen

ic

bact

eria

. C

onta

min

atio

n of

pro

duct

by

sp

oila

ge/p

atho

geni

c ba

cter

ia.

Crit

ical

C

ontro

l Po

int(s

)

Tim

e/te

mpe

ratu

re.

Pack

agin

g co

ntro

l.

Prev

enta

tive,

co

ntro

l an

d m

onito

ring

proc

edur

es

Che

ck in

tern

al

tem

pera

ture

of

pr

oduc

t du

ring

cool

ing

to 4

°C.

Rec

ord

time

to r

each

thi

s te

mpe

ratu

re.

If th

e tim

e ta

ken

to

reac

h th

is t

empe

ratu

re

is g

reat

er

than

30

min

out

side

spe

cific

atio

n re

cord

on

prod

uctio

n ca

rd a

nd

notif

y pr

oduc

tion

supe

rvis

or.

Che

ck p

acka

ging

in

tegr

ity

and

do

rand

om

leak

tes

ts o

n se

lect

ed

sam

ples

. O

bser

ve f

or le

akag

e/

spilla

ge.

Del

ay p

rior

to la

belin

g G

row

th

of s

urvi

ving

pa

thog

enic

ba

cter

ia.

Tim

e/te

mpe

ratu

re.

Labe

ling

Gro

wth

of

sur

vivi

ng

path

ogen

ic

bact

eria

. C

onsu

mer

ab

use

of p

rodu

ct.

Tim

e/te

mpe

ratu

re.

Labe

ling

inst

ruct

ions

.

Car

toni

ng

Gro

wth

of

sur

vivi

ng

path

ogen

ic

bact

eria

. C

ross

-con

tam

inat

ion.

Tim

e/te

mpe

ratu

re.

Pack

gagi

ng.

Che

ck p

rodu

ct

tem

pera

ture

at

re

gula

r in

terv

als.

If

prod

uct

build

up

exc

essi

ve, r

emov

e to

re

frige

rate

d st

orag

e un

til

prod

uct

build

up

decr

ease

s. N

ote

on

prod

uctio

n ca

rd.

Che

ck p

rodu

ct

tem

pera

ture

as

ab

ove.

Che

ck la

bellin

g in

stru

ctio

ns

for

stor

age

cond

ition

s ‘K

eep

refri

gera

ted’

. C

heck

use

by

date

and

coo

king

/mic

row

ave

inst

ruct

ions

.

Che

ck t

empe

ratu

re

of c

arto

ning

ar

ea (

10°C

) and

pot

entia

l si

gns

of

prod

uct

build

up/

tem

pera

ture

ab

use.

C

heck

wei

ghts

/inte

grity

of

se

cond

ary

cont

aine

rs

and

labe

ling

inst

ruct

ions

on

ext

erna

l pa

ckag

es.

Cont

inued

Tabl

e 8.

C

ontin

ued

Crit

ical

op

erat

ion

Del

ay p

rior

to lo

adin

g co

ntai

ner

Load

ing

of tr

ansp

ort

cont

aine

r

Pote

ntia

l ha

zard

/risk

Gro

wth

of

sur

vivi

ng

path

ogen

ic

bact

eria

.

Cro

ss-c

onta

min

atio

n of

pro

duct

. G

row

th

of s

urvi

ving

pa

thog

enic

ba

cter

ia.

Crit

ical

C

ontro

l Po

int(s

)

Tim

e/te

mpe

ratu

re.

Sani

tatio

n.

Tim

e/te

mpe

ratu

re.

Prev

enta

tive,

co

ntro

l an

d m

onito

ring

proc

edur

es

Che

ck t

empe

ratu

re

of lo

adin

g do

ck a

rea/

tem

pera

ture

of

pro

duct

. N

ote

on p

rodu

ctio

n ca

rd.

Che

ck p

hysi

cal

clea

nlin

ess

of

trans

port

cont

aine

r. C

heck

ref

riger

ated

te

mpe

ratu

re

of

cont

aine

r/loa

ding

of

con

tain

ers/

air

circ

ulat

ion.

Tran

spor

tatio

n to

ret

ail

Gro

wth

of

sur

vivi

ng

path

ogen

ic

outle

t ba

cter

ia.

Tim

e/te

mpe

ratu

re.

Stor

age

in r

etai

l ou

tlet

Gro

wth

of

sur

vivi

ng

path

ogen

ic

bact

eria

. Ti

me/

tem

pera

ture

.

Mon

itor

time/

tem

pera

ture

in

dica

tors

on

pac

kage

s. C

heck

te

mpe

ratu

re

of p

rodu

ct

at s

elec

ted

area

s in

tra

nspo

rt co

ntai

ner.

Keep

al

l chi

ll ch

ecks

. Not

e on

pro

duct

ion

reco

rd.

Advi

se t

rans

porte

r of

te

mpe

ratu

re

abus

e/pr

oduc

t/out

of

te

mpe

ratu

re

spec

ifica

tion.

Mon

itor

time/

tem

pera

ture

on

pac

kage

s. P

erfo

rm

rand

om

tem

pera

ture

ch

eck

on

prod

uct,

if pr

oduc

t te

mpe

ratu

re

grea

ter

than

4°

C re

com

men

ded

tem

pera

ture

, no

tify

stor

e. D

o m

icro

biol

ogic

al

anal

ysis

on

rand

om

sam

ples

. Ad

vise

ret

ail

outle

t of

pro

per

stor

age

cond

ition

s.


these authors, there are four main levels of concern:

High Concern: An expert judgment that without control there is a life threa- tening risk;

Medium Concern: An expert judgment that there is a threat to consumer or to the product which must be controlled;

Low Concern: An expert judgment that there is little threat to the consumer or the product. It may still be advantageous to control;

No Concern: An Expert judgment that there is no threat to the consumer.

In any HACCP system, it is prudent to recognize that no system can give 100% security. Occasionally, products which pose a public health threat may appear on the market. It is essential for the industry to respond quickly to such threats and to implement a rapid and effective product recall plan. Each com- pany should have a detailed product recall plan. This includes the keeping of accurate process control and monitoring records for each batch of product. In addition, proper preparation also includes identifying all internal and external personnel involved in the recall, their func- tions and responsibilities and channels and means of communication. Mock recall plans should be.carried out on a regular basis to ensure the effectiveness of the plan in case of an emergency and also to modify and improve the plan where possible.

Conclusion While the HACCP concept is commonly used by the food canning industry to

References

ensure the microbiological safety of low acid canned food products, the technique is either used sparingly or not at all by other food processing industries in the control of food products. Despite its limited use, the HACCP approach has several advantages over the traditional approach of end product sampling for microbiological quality control, namely that

(a) it is forward looking and does not rely on after the fact testing to resolve a problem, (b) it is flexible and can be integrated with all stages of ingredient storage/manufacturing /distribution / marketing and in the retail handling, (c) it documents all operations and procedures and ensures that product is produced within strict control limits, and (d) it integrates several areas of expertise from within a production plant. With increasing consumer demands for

convenience foods, the market potential for sous vide products is excellent. While no system is 100% foolproof, implemen- tation of the HACCP approach and employee training in HACCP principles will result in a higher degree of con& dence in product safety than is possible using traditional approaches to microbiological control and ensure the con- tinued market growth generation of minimally products.

of this new processed food

Acknowledgments The authors wish to thank Agriculture Canada and the Natural Sciences and En@- neering Research Council of Canada (NSERC) for funding this study.

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