Journal of Food Engineering 54 (2002) 715 www.elsevier.

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A new test method for in-place cleanability of food processing equipment

T. B en ezech *, C. Leli evre, J.M. Membr e, A.-F. Viet, C. Faille
INRA Laboratoire de Ge nie des Proce de s et Technologie Alimentaires, 369 rue Jules-Guesde, B.P. 39, 59651 Villeneuve dAscq, France Received 3 June 2001; accepted 14 September 2001

Abstract A practical and quantitative method for assessing complex food equipment cleanability is described. After soiling a positive displacement pump by a composite model food made of custard and Bacillus cereus spores isolated from a food processing line, a mild cleaning-in-place procedure was carried out using basic detergents such as sodium hydroxide and nitric acid. After cleaning, surfaces potentially in contact with the contaminated food were overlaid with nutrient agar containing a tetrazolium salt. Residual contaminants appeared as small red colonies and contamination levels could be dened. A non-parametric statistical analysis was performed to compare the dierent areas in the pump and three cleanability levels were dened. Geometry appeared to be one of the main factors in hygiene, emphasised by the way the equipment is connected to the CIP circuit. 2002 Elsevier Science Ltd. All rights reserved.
Keywords: In place cleanability assessment; Food industries; Bacillus spores; Hygienic design; Pump

1. Introduction Liquid food treatments which are intended to inactivate unwanted micro-organisms (pasteurisation and sterilisation) can adversely aect avour and damage many nutrients. This trend appears to conict with the new consumer demands for fresher foods of high organoleptic and nutritional quality, which suggests lighter preservation methods. Of course, low heat treatment conditions imply that hygienic processing requirements must be correctly and consistently met (Moster & Lelieveld, 2000). Since 1998 in Europe, the design of food processing equipment must meet the Machinery Directive (European Council Directive 98/ 37/EC on machinery, 1998). The Directive includes a short section dealing with hygiene and design requirements which states that machinery intended for the preparation and processing of foods must be designed and constructed so as to avoid health risks and this section consists of hygiene rules that must be observed. These rules concern the suitability and cleanability of materials in contact with food. Recent international

Corresponding author. Fax: +33-3-20-43-64-26. E-mail address: benezech@lille.inra.fr (T. B en ezech).

standards such as ISO 14159 for food, cosmetics and pharmaceutical industries (International Organisation for standardisation (ISO), 1999) and CEN EN 1672-2 for food industries (Comit e Europ een de Normalisation (CEN), 1997) give details on hygiene requirements for processing machines associated with a risk assessment procedure. Within the requirements set out in the standard EN 1672, it was noticed that cleanability and/or capability of being disinfected should be checked by means of visual inspection (on drawing(s) or machineries) and/or a practical test, microbiological test or functional test. In addition, according to ISO 14159, most closed product processing machinery are considered cleanable if the cleaning procedure eciency can be veried by means of a practical test for the entire plant or its individual components. Machines designed to be pasteurised, sterilised or for aseptic production, usually for closed product processing, shall require practical testing. However, no practical way of testing is either indicated or referred to. A new standard prEN 197-042 specic to liquid pumps is under preparation by CEN to dene design rules to ensure hygiene in use (Comit e Europ een de Normalisation (CEN), 1999). Pumps complying with this standard shall achieve or have veried one of the specied categories of cleanability which shall be

0260-8774/02/$ - see front matter 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 0 - 8 7 7 4 ( 0 1 ) 0 0 1 7 1 - 6

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identied by the manufacturer in the instruction handbook, and may be related to specic types of processes and cleaning procedures. Four cleanability categories are identied in the standard, based on the potential use of the pump in factories. Only one category species a level of remaining micro-organisms after cleaning dened by a relevant test to be chosen. This should be assessed by applying tests established by the manufacturer or by a laboratory and shall comprise a visible soil test and a micro-organism test. The standard EN 12462 referring to the standard EN 1672-2, indicates that the cleanability of pumps used in biotechnological processes is one major element of the performance criteria to be taken into account. As explained in the standard, any prescribed cleanability threshold value should be based on the required safety level and can be the detection limit of an approved test method (e.g. European Hygienic Equipment Design Group (EHEDG)). Therefore, there is an obvious need for the creation of standardised cleanability test methods. Over recent last years, a research programme funded by the European commission (AIR 1-CT92-0091) was undertaken to develop test protocols on a practical basis to assess the cleanability of equipment intended for food production (Holah, 2000). A standardised cleanability test method (Timperley et al., 2000), recently published by the EHEDG is based on the comparison, in laboratory conditions, of the cleanability of a test item (closed processing equipment) with that of a straight piece of pipe with a dened surface nish. A previous version of the Timperley et al. (2000) procedure is therefore quoted in the standard NF EN 1296-X 42 120, as an example of the application of a relevant methodology (Comit e Europ een de Normalisation (CEN), 1995). The chosen soil was made of a suspension of Bacillus stearothermophilus spores in soured milk, spores being a tracer of the milk soil as demonstrated by Galesloot, Radema, Kooy, and Hup (1967) and Carpentier and Kobilinsky (1993). The presence of the soil after cleaning is detected after direct growth of the remaining micro-organisms on the surfaces. Four successive steps are required and could be summarised as follows: 1. soiling of the surface of the equipment to be tested together with a standard component (a length of pipe); 2. cleaning the surface of the soiled equipment under appropriate conditions; 3. detecting the remaining soil and, when possible, quantifying it and; 4. comparing with the standard component. Other practical methods have been proposed based on the use of mixed soil containing a milk product and smark, relevant bacteria such as Bacillus spores (Hu nner, & B Faille, Ro en ezech, 1999; Holah, 2000) for assessing the cleanability of pieces of equipment such as

at surfaces and pipes. Indeed Bacillus species are important as food-spoilage organisms, and can be isolated from a great range of food products such as spices, vegetables, meat, milk and dairy products (Goepfert, Spira, & Kim, 1972; Johnson, 1984). B. cereus is noteworthy a predominant organism that determines the keeping quality of pasteurised milk products (Stewart, 1975). Such a risk remains today as reported by Te Giel, Beumer, Leijendekkers, and Rombouts (1996). Food contamination of these thermo-resistant microorganisms may originate from raw materials, but also from fouled processing equipment surfaces (Wong, 1998). Indeed, spores of Bacillus species such as B. cereus or B. thuringiensis were demonstrated to be highly nner, 1990, smark & Ro adherent to various surfaces (Hu 1992; Faille, Dennin, Bellon-Fontaine, & B en ezech, 1999). The aim of this study was to propose a detailed test method to assess the cleanability (soiling and cleaning ability) of food processing equipment designed for specic heat, chemical or physical treatment to free the machinery from relevant micro-organisms. A B. cereus strain isolated from a dairy processing line and demonstrated to be a relevant test organism in dening the potential risk of contamination in dairy processing lines (Faille, Fontaine, & B en ezech, 2001) was proposed. Spore adhesion was obtained by circulating a contaminated dairy desert throughout the equipment to be tested. A positive displacement pump was chosen as an example of complex food processing machinery used for the processing of viscous and/or fragile products (eggs, custard, stirred yoghurt). The design of such an apparatus could be considered as complex due to the presence of various hydraulic diameters, moving parts, dierent materials and the various ways it can be installed in processing lines.

2. Materials and methods 2.1. Equipment and test rig The progressive-cavity pump used to carry out the cleanability test method (Fig. 1) was chosen for its high occurrence in the food industry. It is mainly composed of a rotor and a stator with two internal spiral bearings. In any cross-section, the rotor is in contact with the stator. The rotational movement causes a shift, along the axis from the inlet to the discharge, of the sealed cells marked o by the rotor and stator. Body and suction ports are made of 316 L stainless steel and the stator of chlorosulfonated polyethylene (CSPE)-based synthetic rubber. Two ways of connecting the pump to the CIP circuit were tested using two dierent pump bodies with an axial exit pipe or a tangential exit pipe. Exit pipes thus

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Fig. 1. General drawings of a progressive cavity pump: 1 axial connection, 2 tangential connection.

were horizontally (axial) or side (tangential) oriented. Tangential connection allows the pump to be self drained. The pump was installed in a soiling circuit (Fig. 2). The model food as the soiling suspension was maintained at 50 C in a tank and circulated in the loop consisting of the pump to be tested, stainless steel 304 L pipes (0.15 m length, 0.38 m diameter and an average

roughness of 0.4 lm) and a three way seat valve (Alpha Laval, Sweden) for the CIP connection. Between the dierent ttings, rubber gaskets (Nitrile) specic to the food industry, were used. The cleanability of the dierent parts of the equipment tested was compared to results obtained for the 304 L pipes used as references. After soiling the test equipment, the three-way valve was opened to by-pass and clean in place the tested

Fig. 2. Soiling [1] and CIP [2] circuits; A: agitator; CP: centrifugal pump; FI: ow indicator; PS: pressure sensor; TH: tank heating; TI: temperature indicator; V: valves (Ma: manual; 2W: 2 ways; 3W: 3 ways; MD: motorised drive; PR: pressure relief); VP: volumetric pump.

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pump (Fig. 2). An automatic CIP unit was used. Simple laboratory made software, was run on an IPC 486 computer connected to a module, containing 16 automatic switches (action in the centrifugal pump and the CIP valves). In addition, the signals of dierent sensors (temperature, ow-rate and pressure) were acquired by means of a Hewlett Packard 39970 data acquisition switch unit. 2.2. Testing protocol 2.2.1. Soil and soil detection B. cereus CUETM 98/4 (Collection Unit e Eco Toxicologie Microbienne, Villeneuve dAscq, France) was used throughout this study. This strain was isolated from a dairy processing line. Spores were obtained as previously described (Faille, Lebret, Gavini, & Maingonnat, 1997). Commercially available custard containing fat, starch, sucrose, cocoa and skimmed milk was used. Around 105 CFU ml1 of B. cereus spores were added to the custard. The TTC-nutrient agar was made of 13 g l1 nutrient broth (Biorad, France) and 15 g l1 agar type E (Biokar Diagnostics) supplemented by 0:1 w/w 2,3,5 triphenyltetrazoliumchloride (TTC). This soluble non-coloured salt which is an electron acceptor, will be reduced in the bacterial cells to formazan which is non-soluble and red. The colonies will be coloured red and easily counted. 2.2.2. Test method During the soiling step, 12 kg of the contaminated custard were poured into the tank 60 1 C and circulated by means of the pump through connecting pipes for 1 h at a ow rate of 300 l h1 . The custard was then removed and the CIP was connected to the rig. The power of the tested pump was set at its maximum ow rate of 600 l h1 and the following steps were completed: 1. Rinsing with cold tap-water for 2 min, at a ow velocity of 0:5 m s1 . A pipe was removed to evaluate the soiling rate (soiling control or SC) and an unsoiled reference pipe (deposit control or DC) was set into the test rig. 2. Rinsing with cold tap-water for 6 min, at a ow velocity of 0:5 m s1 . 3. Circulating 0.2% NaOH at 60 C for 10 min, at a ow velocity of 1.5 m s1 . 4. Rinsing with cold tap-water for 6 min, at a ow velocity of 0:5 m s1 . 5. Circulating 0.2% HNO3 at 60 C for 10 min, at a ow velocity of 1.5 m s1 . 6. Rinsing with cold tap-water for 6 min, at a ow velocity of 0.5 m s1 .

Flow velocities were indicated for the connecting pipes and the entry and exit of the tested pump. The pump was then completely dismantled, drained and all parts to be tested were protected with aluminium foil to prevent airborne contamination. All the relevant surfaces were covered with or partially immersed in the TTC-nutrient-agar. The volume of the molten agar poured was chosen to obtain a mould of a representative part of the internal surface. These test items were incubated for 4 h at room temperature. Removal of the solid TTC-nutrient-agar from pipes and pump pieces was done carefully in order to avoid smearing the young colonies growing on the agar surface in contact with the tested surfaces and to avoid damaging the moulds. These were then placed in vessels, covered by an aluminium foil to prevent any agar drying out or contamination and further incubated for 16 h at 37 C. The small, red colonies grown on the agar surface were counted on photographs, taken using a digital Olympus camedia C-2020 Z camera. At least four trials were required to properly analyse the results. The mohnopump was tested four times with an axial CIP connection or six times with a tangential CIP connection mainly due to some diculties in handling the agar moulds. 2.3. Evaluation of the results The equipment was arbitrarily divided into elementary areas. The partition presented in Fig. 6 was based on the functional specicity such as the stator (ST), gasket (BB), pipe entry (EP) and exit (EXP) and CIP connection pipe (EPCIP ). In addition, larger areas were divided into several parts such as 3 for the rotor (RO, AX and RS) and 6 from the motor to the stator/rotor area for the pump body BC; Ba1 ; Ba2 ; Ba3 ; Bb1 ; Bb2 . For each part of the equipment tested, the number of colony forming units per square centimetre (cfu cm2 ) was determined after cleaning except for the soiling control (SC). When it was not possible to count Bacillus colonies due to extensive damage of the mould, an arbitrary value was given. This value was calculated as follows: the median value obtained from the other repetitions for the same area was multiplied by the median value from all counts of the same trial divided by the overall median value of all the repetitions. S-plus software (Seattle, USA) was used to perform the statistical analysis. Hartigan and Wongs clustering method (1979) allowed us to identify groups of areas according to their hygienic level (Hartigan & Wong, 1979). A partition of the observations with n groups that minimised the within-cluster sum of squares, was obtained. The number of groups, n was found to be equal to 2 or 3 according to the data. Without assuming normal distribution, the Friedman multiple comparisons test (Conover, 1980) was then performed to conrm the

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hygienic groups obtained. In addition, box plot diagrams (Tukey, 1990) were produced: the box contains 50% of the data, the horizontal line within the box corresponding to the median value. The vertical lines are an index of the data variability.

3. Results The number of colonies counted on the moulds may vary greatly between areas (from 1 to more than 40 cfu cm2 ). Given that the largest dierence between soiling control (SC) and cleaning control (CC) pipe data was found to be of this order of magnitude, the cleaning of some areas in the pump appeared to be as contaminated as the non-cleaned reference pipe. Apart from any difculty in the cleaning ability, this could be due to a higher amount of spores deposited during soiling. The observation of no spore deposition on the deposit control (DC) pipe, allowed us to conclude on the absence of surface contamination during cleaning. On the pump moulds, such a cfu variation was illustrated in Fig. 3(a) (c). A wide variability was observed on the pump body moulds. The largest amount of colonies was found to be close to the CIP connection (Fig. 3(a) and (b)). Conversely, the stator mould (Fig. 3(c)) showed an homogeneous repartition of the colonies. According to Holah (2000), detection of B. thuringiensis by TTC on at surfaces probably covers a range of 1100 cfu cm2 above which colonies become undistinguishable (i.e. 2 log orders). Holah (2000) considered such a range as sucient to both determine cleanability dierences by means of colony counts, or by visual assessment. On non-at surfaces, the enumeration of B. cereus of more than 40 cfu cm2 was found to be uncertain. This cfu count was thus arbitrarily given when the number of cfu was actually uncountable. This could be considered as a limit of the use of this technique, as a great number of elementary surfaces cannot be enumerated. The variability between trials was easily visualised throughout box plot charts. Data obtained for the two CIP connections tested are shown in Figs. 4 and 5. When the box plot chart was only based on three legible data sets, no vertical lines were indicated as shown in Fig. 4 ( Ba3 ; Bb1 ; Bb2 , CE, ST and AX). It is noticeable that the variability of the attachment of the spores to the soiling control pipe (SC) was found to be dierent between the data presented in Figs. 4 and 5. However, in both situations, the soiling control median cfu values were similar and less than 10 cfu cm2 . The SC variability when testing the CIP axially connected pump, was found to be close to the variability of less hygienic areas. In addition, a high surface contamination along with a high variability was observed for the pump body when axially connected to the CIP circuit. A slight

Fig. 3. Examples of the grown colonies on the incubated TTC-nutrientagar moulds (axial CIP connection); (a) Pump body; (b) CIP connection; (c) Stator.

scattering of data was observed for areas such as BC, EP, EPCIP with a maximum of 10 cfu cm2 . The tangential connection drastically changed the results in terms of variability and signicance of the number of remaining spores after cleaning (Fig. 5): the highest data variability, of up to 18 cfu cm2 was observed for Bb2 , located at the CIP connection and the mean cfu cm2 values for all the areas remained under 10 cfu cm2 . The results of grouping the dierent areas together (Hartigan & Wong (1979) clustering method) are presented in Figs. 6 and 7 (3 groups) and (2 groups). The Friedman multiple comparison test P-values were found to be 0.07 for the tangential CIP and 0.006 for the axial one. This means that the groups (colours) dened were highly signicantly dierent. In Fig. 6, for the axially CIP connected pump, most of the pump elements appeared to be at a poor level, only one area being close to the levels of the pump entry (BC), the stator/rotor (ST, RO), exit pipe (EXP) and the rotating parts (RS, AX and RO) being at a moderate level. According to Fig. 4, there is a gap between areas at a poor hygienic level and those at a moderate one, thus emphasising the poor hygienic level of the dark grey areas. The highest hygienic areas were found to be the ones allowing the pump to be connected to the circuit (EP, EPCIP ). In Fig. 7, the tangentially CIP connected

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Fig. 4. Box plot of the cleanability data for the axial CIP connection.

Fig. 5. Box plot of the cleanability data for the tangential CIP connection.

pump, all the pump elements appeared to be at a high hygienic level, some of them clearly more hygienic: a large part of the body (BC, Ba1 ; Ba2 ; Ba3 ), the stator/ rotor area, the rotating parts and as in Fig. 6 the connecting pipes EP and EPCIP .

4. Discussion Cleanability tests are often suggested in recent standards without any detail being provided. Only the EN

12462 for biotechnology industries gives general elements on how to evaluate cleanability and explains that any prescribed threshold value obtained from an approved test should be based on a required safety level. This required safety level should be dened by the removal rate of a relevant bacteria. In this work, B. cereus CUETM 98/4 was isolated from milk products, so that the risk could be addressed of the processing of a milky product to be pasteurised or sterilised. The chosen strain had been previously demonstrated by Faille et al. (2001) to adhere to any kind of surfaces (polymer or stainless

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Fig. 6. Grouping of the cleanability data for the horizontal CIP connection: good (light grey), moderate (grey) and low (dark grey).

Fig. 7. Result of the grouping of the cleanability data for the tangential CIP connection: good (light grey), moderate (grey).

steel), the soiling level depending on the suspending media. For example, custard as a suspending medium was shown to reduce by 100 fold the number of adherent spores when compared to media known to enhance the smark & spore adhesion strength such as saline (Hu nner, 1992; Faille et al., 1999). Consequently, a high Ro level of spores in custard (105 cfu ml1 ) allowed a high level of adherent spores. A mild cleaning procedure was chosen to allow comparison of the hygienic status of the dierent areas. Nevertheless, the cleaning conditions were based on those in use in industrial practice: sodium hydroxide followed by nitric acid (Grassho, 1995). In spite of the dierent experimental methods applied, Bird and Bartlett (1995); Jeurnink and Brinkmann (1994) and tscher, Henck, and Gallmann (1994) agreed that 0.5 Lo 1% NaOH was undoubtedly the optimum concentration for milk deposit removal. To obtain milder cleaning conditions, only NaOH 0.2% and HNO3 0.1% were chosen together with reasonable cleaning time (10 min) and temperature (60 C). One of the main factors explaining the cleanability is the material. The axially CIP connected pump did not

show any particular dierence between its ability to be cleaned from that of polymers and stainless steel. The CSPE stator was shown to be at a moderate hygienic level and not at a low one as was most of the pump. The relatively good cleaning of this part could be explained by the contact when cleaning between the rotor and the stator. Shear forces at the wall are much higher than those generated by the detergent uid ow. In addition, smark & as shown recently by some authors (Hu nner, 1992 and Faille et al., 1999) hydrophobicity of Ro the material largely explains spore adhesion strength. The water sessile drop technique (Van Oss, 1994) used to evaluate water anity did not show any dierence in the hydrophobicity for stainless steel and CSPE, with a water contact angle below 60 for both materials. It must be noticed that the polymer tested here was a new one and the above conclusions could be slightly modied after any ageing eect such as abrasion or scratching. The entrapped micro-organisms would in this case be highly protected from any sanitising process. Indeed, Holah and Thorpe (1990) on domestic sink materials pointed out the role of ageing on bacterial retention. Changes in surface nish due to abrasion did enhance

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bacterial retention. The greater the degree of surface irregularities, the greater the protection against shear forces during cleaning programmes. Recently, Strog ards, berg, and Wirtanen (1999a,b) came to simiSimola, Sjo lar conclusions on food processing equipment. Physical deterioration was shown on the aged materials tested in industrial processes e.g. ethylene propylene diene monomer rubber (EPDM), polytetrauoroethylene (PTFE), causing signicant reduction in cleanability. Conversely, no dierence was observed in the cleanability of new EPDM, PTFE and stainless steel when CIP parameters typical for dairies were used. In this work, for the most hygienic situation (tangential CIP connection), the stator/rotor area appeared to be completely cleaned. This would improve the hygienic status even after any ageing of the CSPE. Cleanability dierences seemed to be mainly based on the geometry and therefore on the mechanical eect of the detergent ow in the diering parts of the pump. According to Grassho (1992), of greatest importance is the uid ow in connection with cleaning of dead spaces. Grassho (1980, 1983) found that the uid exchange (rinsing behaviour) and the local wall shear stress (cleaning behaviour) decreased very rapidly with increasing stagnant dead space depth. Thus, Ba1 Ba3 areas being poorly hygienic, could not really be considered as dead areas but in our cleaning conditions only as shadow areas (Fuggle et al., 1998). The detergent ow velocity of 1.5 m s1 in the connection pipes based on a long industrial practice (International Association of r Normung, Milk Food, 1996; Deutsches Institut fu 1988) is clearly insucient to eciently ush out all of the Ba areas. However, in the axial CIP connected pump, the BC area close to the pump entry was found to be more cleanable than the immediate areas of the body probably due to the mechanical action of the CIP ow entering the pump. This is a good point for the cleaning of the mechanical shaft seal area, identied as a critical area by Fuggle et al. (1998). No particular contamination was thus observed in this area despite the diculties in testing such a narrow place. The hygienic status of the body of the pump appeared to be highly dependent on the connection to the CIP circuit. The tangential connection would allow the detergent to ow through the pump body in a centrifugal pattern and signicantly enhanced the cleaning eciency, the various areas then being characterised by a high or very high hygienic level (cfu count below 18 per cm2 ). As far as we know, no work has yet been done on the eect of an axial exit on the ow pattern in a pipe. Legrand et al. (1991) described the swirling decaying ow induced by a tangential inlet in an annular space. They concluded that the application of a tangential component to the axial movement leads to a signicant increase in the entrance length. This phenomenon was associated with an increase in momentum transfer and values of the friction

factor were greater in the swirling ow thus induced especially at high Reynolds number values. In this work, cleaning was achieved at a Reynolds number of 10,800 in the pump body, being above the range studied by Legrand et al. (1991) (up to 3800). It is worth stating that the specic perturbation induced by the tangential outlet, would, in our work, explain this clear increase in the Bacillus spores removal.

5. Conclusion The use of spores of a relevant B. cereus strain as micro-organism indicator in combination with the use of TTC agar was demonstrated to give sensitive and accurate results when testing the cleanability of a positive displacement pump, a widely used piece of complex equipment. The use of custard for soiling made the method even more realistic: besides other criteria, a micro-organisms attachment to equipment surfaces is dependent on environmental conditions. The possibility of localising and counting the remaining contamination permitted an accurate evaluation of the cleaning process of the critical points. Any prescribed threshold value for the cleanability should be based on a required safety level. The risk here is addressed to the processing of milky products to be pasteurised or sterilised based on the micro-organism choice. A statistical data analysis based on a non-parametric approach allowed us to pinpoint the importance of the design of the connection of any equipment in the food processing line. Geometry appeared here to be a determining factor in the hygienic status of the food processing equipment. The tangential CIP connection was shown to enhance the cleanability of the equipment as a whole and not only of the immediate areas. This emphasised the fact that any equipment testing should take into account how the equipment is connected to the CIP loop in its industrial environment. This point is considered as a major one in the EHEDG recommendations. However, such a technique could not be used on a routine basis. The handling of nutrient agar when testing less accessible areas, e.g. small and/or hidden areas but potentially in contact with food, is sometimes a critical task. Nevertheless, food processing equipment hygiene should be rapidly improved in the near future in a practical way. Acknowledgements This work was supported by UNIR (Ultrapropre Nutrition Industrie Recherche) program, involving food companies, the French Ministry of Agriculture, and the French Ministry of Research.

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