MICROBIOLOGICAL QUALITY AND SAFETY IN DAIRY INDUSTRY

Module 3. Plant hygiene and sanitation

Lesson 15
EQUIPMENT HYGIENE

15.1 Introduction

Pathogenic microorganisms are the major safety concern for the food industry. The vast majority of outbreaks of food-related illness are due to microbial pathogens and their effective control depends on a thorough understanding of the growth conditions favoring particular organisms. This understanding can be used to minimize contamination of incoming raw materials, to inactivate bacteria during processing and prevent decontaminated food from becoming re-contaminated. It is also important to know where and how microorganisms can become established, if growth conditions are favorable.

Hygiene problems in equipment are caused when micro-organisms attach to the surfaces, survive on them and later become detached thus contaminating and reducing the quality of the product. This can be due to a poor hygienic design in cases where the machines cannot be cleaned properly. Constructions that cause problems include sites where soil, product debris and micro-organisms can accumulate, e.g. dead ends, sharp corners and low-quality seals and joints. Even if ‘dead’ areas have been ‘designed out’, some product will attach to equipment surfaces, despite the possibility of fast-moving liquids. Microbes may reside on such surfaces long enough to multiply, and contaminate the product. The problem is exacerbated when a process includes dead spaces where product can stagnate. Microbes may also penetrate through very small leaks. There is considerable evidence that they can pass through microscopic openings very rapidly and that pressure differences may retard, but not prevent, passage, even if the pressure-difference is as high as 0.5 bar. The bacterium Serratia marcescens may move at a speed of 160 mm per hour. Motile bacteria may propel themselves against the flow of liquid through a leak. Whether motile or not, they may also penetrate by forming a biology on the surface.

Good hygienic design of food processing equipment protects the product from contamination with substances harmful to consumer health and provides access for cleaning, maintenance and inspection. Factors reported to affect the hygiene level of food processing equipment, include hygienic design of the equipment, hygienic practices of personnel, cleaning and disinfection of the equipment, lubricants used in the equipment as well as lay-out of the processing, air-currents, type of food product and cleanliness of the processing environment.

15.2 General Aspects of Sanitary Construction and Design of Food Equipment

The surfaces of food equipment can be subdivided into two categories:

1. Food product contact surfaces, and
2. Non-product contact surfaces.

A food product contact surface is defined as a surface in "direct contact with food residue, or where food residue can drip, drain, diffuse, or be drawn". Because these surfaces, if contaminated, can directly result in food product contamination, rigid sanitary design criteria must be met. Non-product contact surfaces are those that are part of the equipment (e.g., legs, supports, housings) that do not directly contact food. As contamination of non-product contact surfaces can cause indirect contamination of the food product, these surfaces cannot be ignored with regard to sanitary design.

15.2.1 Food product contact surfaces

In terms of sanitary design, all food contact surfaces should be: Smooth, impervious, free of cracks and crevices, nonporous, nonabsorbent, non-contaminating, nonreactive, corrosion resistant, durable and maintenance free, nontoxic and cleanable. The food contact surfaces should comply with the following criteria:

15.2.1.1 Surface texture and/ or finish

If any surface is ground, polished, or textured in any way, it must be done so the final surface is smooth, durable, free of cracks and crevices and meets the other sanitary design requirements described above. Sanitary Standards require that ground or polished stainless steel surfaces meet ground surface, and unpolished surfaces meet a No. 2B or mill finish. The 3-A Sanitary Standards development group has recently adopted an industry recognized method for determining an acceptable food contact surface termed roughness average or Ra value. The Ra is determined using a sensitive instrument (termed a profilometer) which employs a diamond tipped stylus to measure peaks and valleys in a relatively smooth surface.

15.2.1.2 Construction and fabrication

Food equipment should be designed and fabricated in such a way that all food contact surfaces are free of sharp corners and crevices. All mating surfaces must also be continuous (e.g., substantially flush). Construction of all food handling or processing equipment should allow for easy disassembly for cleaning and inspection. Where appropriate (e.g., vessels, chambers, tanks), equipment should be self-draining and pitched to a drainable port with no potential hold up of food materials or solutions. Piping systems not designed for routine dis-assembly must be sloped to drain. Piping systems installed in modern food processing systems designed for cleaning-in-place (CIP), require special consideration and close monitoring with regard to drainage.

15.2.1.3 Internal angles

Internal angles should be coved or rounded with defined radii. Equipment standards specify appropriate radii for specific equipment applications and components. For example, radii requirements stated in the 3A sanitary standards

15.3 Functional Requirements of Equipments

Hygienic food processing equipment should be easy to maintain to ensure it will perform as expected to prevent microbiological problems. Therefore, the equipment must be easy to clean and protect the products from contamination. In the case of aseptic equipment, the equipment must be pasteurisable or sterilisable (depending on the application) and must prevent the ingress of micro-organisms (i.e. it must be bacteria tight). It must be possible to monitor and control all of its functions which are critical from a microbiological safety point of view.

15.3.1 Cleanability and decontamination

Cleanliness is a very important issue. Equipment which is difficult to clean will need procedures which are more severe, require more aggressive chemicals and longer cleaning and decontamination cycles. Results will be higher cost, reduced availability for production, reduced lifetime of the equipment, and more effluent.

15.3.2 Prevention of ingress of micro-organisms

Ingress of micro-organisms into products must be avoided in general. Usually, it is desirable to limit the number of micro-organisms in food products as much as possible to meet requirements of public health and required shelf life. Equipment intended for aseptic processes must additionally be impermeable to micro-organisms.

15.3.3 Prevention of growth of micro-organisms

Equipment that causes problems in milk processing and packaging include pipes (Joints & Bends), filling and packing machines, conveyors, plate heat exchangers and tanks with piping. Listeria monocytogenes has been found on equipment and process surfaces, which are difficult to clean. Such equipment can thus cause microbial contamination in food processing. Therefore the design of the equipment and process line in the food processing and packaging industry is important for preventing formation of the biofilm and so improving process and production hygiene. Furthermore, pathogens in biofilms have been found to be more resistant to many types of disinfectants.

15.3.3.1 Biofim on dairy equipments

Biofilms form in two stages. First, an electrostatic attraction occurs between the surface and the microbe. The process is reversible at this stage. The next phase occurs when the organism forms an extracellular polysaccharide, which firmly attaches the cell to the surface. The cell then multiplies, forming micro-colonies and, ultimately, the biofilm (Fig15.1). Biofilm development may take place on any type of surface and is difficult to prevent, if conditions sustain microbial growth. These films are very difficult to remove during cleaning operations. Microorganisms that appear to be more difficult to remove because of biofilm formation include the pathogens Staphylococcus aureus and Listeria monocytogenes. Current information suggests that heat treatment is more effective than the application of chemical sanitizers and Teflon appears to be easier to clear of biofilm than stainless steel.

Fig. 15.1 Mechanism of biofilm formation

15.3.3.2 Microorganisms in biofilm formation

Many organisms, including a number of pathogens (Klebsiella pneumoniae, Legionella pneumophila, Listeria monocytogenes, Salmonella typhimurium, Staphylococcus aureus and Yersinia enterocolitica) form biofilms, even under hostile conditions, such as the presence of disinfectants. Adverse conditions even stimulate microorganisms to grow in biofilms. Thermophilic bacteria (such as Streptococcus salivarius spp. thermophilus) can form a biofilm in the cooling section of a milk pasteurizer, sometimes within five hours, resulting in massive contamination of the pasteurized product (up to 10⁶ cells per mL). On metal (including stainless steel) surfaces, biofilms may also enhance corrosion, leading to the development of microscopic holes. Such pinholes allow the passage of microbes and thus may cause contamination of the product. Like other causes of fouling, biofilms will also affect heat-transfer in heat exchangers. On temperature probes, biofilms may seriously affect heat-transfer and thereby the accuracy of the measurement. Reducing the effectiveness of heat treatment may itself help to stimulate further bacterial growth.

15.3.3.3 Effect of cleaning and sanitation chemicals on biofilm microflora

Biofilms may be much more difficult to remove than ordinary soil. If the cleaning procedure used is not capable of removing the biofilm completely, decontamination of the surface by either heat or chemicals may fail, since a biofilm dramatically increases the resistance of the embedded organisms. It is therefore imperative that product contact-surfaces are well cleaned before disinfection. the effects of a variety of cleaning and sanitizing chemicals on L. monocytogenes which was allowed to attach to stainless steel and plastic material used in conveyor belts over a period of 24 hours. They found that sanitizers alone had little effect on the attached organisms, even when the exposure time was increased to 10 minutes. Unattached cells, on the other hand, showed a 5-log reduction in numbers within 30 seconds. In general, acidic quaternary ammonium compounds, chlorine dioxide and peracetic acid were the most effective sanitizers for eliminating attached cells. Least effective were chlorine, iodophors and neutral quaternary ammonium compounds. When the attached organisms were exposed to cleaning compounds prior to treatment with sanitizers, the bacteria were readily inactivated.

15.3.4 Compatibility with other requirements

A design with excellent hygienic characteristics but lacking the ability to perform its functional duties is of no use; hence a designer may have to compromise. Such action, however, will have to be compensated by more intensive cleaning and decontamination procedures and these must be documented so that the users are aware of the nature of the compromise. The cleanability of the equipment, including the CIP where appropriate, must be demonstrated.

15.3.5 Validation of the hygienic design of equipment

Irrespective of the amount of know-how and experience with hygienic design which is applied when designing and fabricating, practice has shown that inspection, testing and validation of the resulting design to check if the requirements are met is very important. In critical cases it may be necessary to check the hygiene level as part of the maintenance procedures. The designer has to make sure that relevant areas are accessible for inspection and/or validation. Acceptance for food processing equipment used in some European countries is based upon "cleanability" testing performed in European Hygienic Design Group (EHEDG) laboratories. The organization like International standard organization (ISO), International Dairy Federation (IDF) and Codex Alimentarius commission (CAC) are generally involved in development of equipment hygiene standards. Some of these organizations have symbol or insignia use authorization programs that require third party verification of compliance with the appropriate standard or guideline.

15.4 Hygiene of Personnel Working with Equipment

An important factor affecting on hygienic status of dairy processing equipment is the hygienic practices of people working with the equipment. Many researchers have published information on improper hygienic practices in food handling and outbreaks have been reported. Contamination of foods by food handlers has been identified as one of the most important causes of food borne outbreaks. Personnel are both reservoirs and vectors of micro-organisms. The level and risk of contamination from personnel is difficult to measure as it depends on various factors such as the different activities and the range of personnel movement patterns during the working day as well as the perceptions and attitudes of the personnel.

15.5 Equipment Hygiene Monitoring

Hygiene detection of the food processing equipment and surfaces is one important part of the quality control. By sampling the surfaces it can be possible to prevent the contamination of milk and milk products, helps in tracing the source of contamination and optimization of cleaning process. Quantitative estimation of micro-organisms from surfaces is very difficult because of strong microbial adherence and because the cells grow in layers, forming biofilms. The threshold of detection of adhering micro-organisms can vary according to the enumeration technique employed and some techniques underestimate the number of micro-organisms on a surface.

The problems associated with identifying biofilm microorganisms is repeatability and reproducibility. The methods currently available mostly used in surface hygiene monitoring are cultivation methods based on surface agar contact method (swab, rinse, adhesive tape method and Vacuum method) and agar contact method (Replicate organisms direct agar contact (RODAC), agar slice and direct surface agar plating method) the ATP bioluminescence method (measurements directly from the surface or from swabbed sample).

15.5.1 Monitoring techniques and microbiological criteria

15.5.1.1 Swab contact method

In swab contact method, a cotton swab moistened with the quarter strength ringers solution is applied over a known area of the equipment and in the process all the micro-organisms adhered to the surface of the equipments are transferred to the swab. After proper mixing, the swab suspension is diluted and plated on different agar plates containing tryptone dextrose agar, violet red bile agar and potato dextrose agar etc. followed by incubation at respective recommended temperature - time combination. The counts of the colony developed in the respective plates were counted. Generally, two specified areas of 50 cm² are randomly selected on the test equipment. This method can be applied to any type of equipment, the recovery of the surface bacteria is only moderate (52-90%).

This method has now been modified and simplified by combining with membrane filtration technique and place it in enriched broth for qualitative identification of microorganisms (Fig. 15.2). This swab suspension is filtered through cellulose acetate filter (0.45 µm) and then filter membrane is layered on a selective medium.

Fig. 15.2. Swab technique for qualitative identification of hygiene

15.5.1.2 Surface rinse method

It is a very effective and useful method for enumeration of microorganisms on the surface of the equipment. This method is mostly used for microbiological examination of bottles, cans, farm dairy equipment and pipelines of the processing machines. In this method, a standard amount of 20 mL for milk bottles and 500 mL for milk cans, of sterile quarter strength ringer’s solution is poured into the containers, mixed or shaken several times so that the diluents or rinse comes in contact with whole surface of the container followed by colony count of rinse is determined on specific media. This is method has a moderate recovery (70%) and good repeatability.

15.5.1.3 Agar contact method

a) RODAC

This method is most popular as surface sampling technique and can give not only approximate number of the organisms but also their types. In this method, plastic strips of specific size (25cm²) are filled enough molten agar medium to allow a convex surface to form. After solidification of the medium, the agar surface is gently pressed against the test surface of the equipment, removed and covered with the lid and subsequently incubated. This test is not suitable for heavily contaminated surfaces. Another drawback with this method is that it cannot distinguish single surface bacteria and clusters of microorganisms. The possible maximum accurate count is limited to 200 colonies/ plate. The recovery is very high (80%) and reproducibility is excellent.

b) Agar slice method

The efficacy of this method is comparable to RODAC method. The specified molten agar medium is filled in a sterile syringe and is allowed to solidify. The solidified medium is pushed out and allowed to make contact with the test surface. The portion of the agar that comes in contact with the surface is sliced off and transferred to petri dish or subsequent incubation and colonies are counted. The recovery with this method is 72-75%.

c) Direct surface agar plating method

This method, the molten agar medium directly poured onto the surface. Due to the direct contact of the surface to the medium, the colonies are developed after appropriate incubation and counted. The recovery is fairly high (80%). The main draw backs are that does not work with certain equipments like pipelines and cans.

15.5.1.4 Microbiological criteria for dairy equipment

The microbial count of the surface studied is calculated as per the following formula:

Count/ ft-2 = 9.29 X count/ 100 cm²
Or
Count/ 100 cm2 = 0.108 X count/ ft^-2

The results and their interpretation for finding the microbiological status of the contact surface in question have been summarized in Table 15.1 and 15.2

Table 15.1 Microbiological criteria for sanitary condition of dairy equipment

The suggested microbiological standards for the surface counts of dairy equipment are give in Table 15.2. Where large surfaces are available the area swabbed (or rinsed or contacted) is 900 cm².

Table. 15.2 Bacteriological standards for sterility of dairy equipment

15.5.1.5 ATP bioluminescence test

ATP bioluminescence is biochemical which measures the presence of adenosine triphosphate (ATP) by its reaction with the luciferin-luciferase complex. It can be incorporated in the estimation of microbial load of a food sample, equipment surfaces and water sample. The bioluminescent reaction requires ATP, luciferin, and firefly luciferase - an enzyme that produces light in the tail of the firefly. During the reaction, luciferin is oxidized and emits light. A luminometer measures the light produced, which is proportional to the amount of ATP present in the sample. The ATP content of the sample can be correlated with the number of microorganisms present because all microbial cells have a specific amount of ATP. An automated luminometer can detect the presence of yeast, mould, or bacterial cells in liquid samples in three minutes (Figs. 15.3 and 15.4).

Fig. 15.3 ATP bioluminescence assay principle



Fig. 15.4 Step wise procedure for ATP bioluminescence assay

A computer-interfaced luminometer, which employs customized software, a printer, and an automatic sampler, can analyze samples with a sensitivity of one microorganism per 200 mL. Use of this method has increased because ATP bioluminescence test can release product in less than 24 hours. A major advantage of this test is that ATP from tissue exudates can be detected, whereas other tests do not offer this feature. Furthermore, this test identifies dirty equipment. However, this test has also limitation in the sense that cleaning compound residues can quench the light reaction to prevent proper response from the assay system. The results were expressed as log10 Relative Light Unit (RLU) released by the total ATP.

The higher the amount of ATP on the surface or food samples, the higher the light output expressed in RLU. According to recommendations of the ATP-bioluminescence equipment manufacturer, measurements lower than 150 RLU were considered clean, from 151 to 300 RLU were considered suspect, and values higher than 301 RLU were considered inadequate hygienic conditions.

15.6 Assessment of Efficacy of Detergent and Sanitizer by Capacity and Suspension Test

15.6.1 Capacity test

Capacity test involves taking a definite quantity of test solution (sanitizer) & definite number of cells. The action of sanitizer depends on its concentration & the period of contact with organism. The shorter the period to destroy the test organism in a particular concentration, the better would be the efficiency of detergent/ sanitizer. Suspension test is based on the enumeration of survivors of the test culture after a definite period of contact with sanitizer.

Procedure

1. Take 20 mL Sanitizer , inoculate 1 mL inoculum (washed suspension of culture O.D.0.3)
2. After 1 min take out 0.1 mL of mixture & transfer in 5 mL nutrient broth(N.B.)
3. Add 0.1 mL inoculums immediately so that amount remain same, take 10 increments
4. Incubate all samples and one positive blank (from 21 mL add 1 mL in N. B.) at 37^oC for 48 hrs
5. Observe growth in nutrient broth tubes
6. Plating and count colonies

15.6.2 Suspension test

In these tests, a sample of the bacterial culture is suspended into the disinfectant solution and after exposure it is verified by subculture whether this inoculum is killed or not. Suspension tests are preferred to carrier tests as the bacteria are uniformly exposed to the disinfectant. There are different kinds of suspension tests: the qualitative suspension tests, the test for the determination of the phenol coefficient and the quantitative suspension tests. Initially this was done in a qualitative way. A loopful of bacterial suspension was brought into contact with the disinfectant and again a loopful of this mixture was cultured for surviving organisms. Results were expressed as ‘growth’ or ‘no growth’. In quantitative methods, the number of surviving organisms is counted and compared to the original inoculum size. By subtracting the logarithm of the former from the logarithm of the latter, the decimal log reduction or microbicidal effect (ME) is obtained. An ME of 1 equals to a killing of 90% of the initial number of bacteria, an ME of 2 means 99% killed. A generally accepted requirement is an ME that equals or is greater than 5: at least 99.999% of the germs are killed. Even though these tests are generally well standardized, their approach is less practical.

Procedure

1. Take 100 mL of disinfectant and inoculate 1 mL inoculums.
2. From step 1 take 1mL and inoculate in petriplate after 0, 1, 2, 5, 10, 15 minutes
3. Incubate plate at 37^oC for 48 hrs and count colonies