Validation of aseptic packing machines

Recontamination is responsible for a large percentage of spoilage of canned products1. In addition, it is more and more recognised that, in practise, recontamination with pathogens may be a frequent and important cause of outbreaks of foodborne disease2. Wrapping of food is hence a really tricky operation, particularly in the case that a sterilised product is aseptically wrapped into a decontaminated sealed container, in order to give a commercially sterile food. Adequate cleanability and sterilisability of the filler and the filling area, decontamination of the packing material and bacteria-tightness of the packing machine are six necessary conditions for safe aseptic wrapping of food.

With the contributing factor of lacking scientific interest, there are not many articles in literature on experimental validation of cleanability, sterilisability and bacteria tightness of food processing equipment. The European Hygienic Engineering and Design Group (EHEDG) developed six test methods (Docs 2,4,5,7,15,19) and also published a document (Doc.21) on challenge tests for packing machines. The title of EHEDG test-methods are shown in Table 1.

Table 1 EHEDG Test-methods

Decontamination of packing material in aseptic systems could be a matter of concern. Because of the various chemicals used and the various technological solutions adopted, it is not possible to use only one test method for every application, and generally, the validation procedure must be developed case by case. In this paper, a test-method for the assessment of decontamination of the external surfaces of internally pre-sterilised spout-less pouches is described.

The real case

The advantages of aseptic processing in terms of reduced processing time, longer shelf life and improved quality are widely known. However, the almost traditional bag in box aseptic technology is currently limited to liquid product applications, due to the small one inch spout aperture3. The elimination of the spout allows wrapping of particulates foods and also allows significant saving in the use of plastic packaging material together with less costs and lower environmental impact. The considered packing machine consists of:

• a chamber for sprinkling of H2O2/water solution on the external surfaces of internally γ-ray pre-sterilised spout-less sealed pouches
• a chamber in which activation of the H2O2/water solution occurs by means of warm air and hence the external surfaces of the sealed pouches are decontaminated
• a chamber for pouches cutting, filling and sealing

The last chamber is sterilised by means of steam, and the bacteria-tightness is ensured by overpressure of sterile air. The test-method described in the following is one part of a whole validation procedure that includes validation of cleanability, sterilisability and bacteria-tightness of the machine. The procedure also includes a preliminary check of compliance of the packing machine with the available regulations, standards and guidelines (materials, hygienic design, monitoring and control), based on a specifically developed check-list.

Literature survey

With regard to sterilisation by hydrogen peroxide, the use of spores of the following bacillus strains were proposed:

• Bacillus subtilis var. globigii (NCIB 8058, ATCC 9372, NCA 7552): in 30 per cent H2O2/water solution at 30°C it is much more resistant compared to other Bacilli. In 25.8 per cent H2O2/water solution, at 24°C, its Decimal Reduction Time (D-value) is equal to two minutes, 0.92 minutes at 40°C and, based on zvalue calculated at 30°C, 5.5 seconds at 80°C4. According to the Elopak method No. 644, 95-08-11, ‘Filler Sterility Test’5, five decimal reductions (5-D) are at least acceptable.
• Bacillus subtilis SA22 (NCA 72-52; DSM ATCC 4181): in 25.8 per cent H2O2/water solution at 24°C, D=7.3 minutes, compared to two minutes of B. subtilis var. globigii and 1.5 minutes for B. stearothermophilus4. In 29.5 per cent H2O2/water solution at 65°C, D=0.05 minutes6. The method proposed by Cerny7 and recommended by the EHEDG Doc.215 for validation of sterilisability of the inner surface of packing material, considers acceptable the application of at least 4-D. The VDMA 2006 / N.148, with regard to external sterilisation of containers by H2O2, considers acceptable the application of at least 3−D. Whereas the VDMA 2003 / N. 89, with regard to sterilisability of the sterile zone in machine interior, considers acceptable the application of at least 4−D in all critical areas.
• Bacillus subtilis A: it is recommended for treatment with H2O2 and UV10 and also for mixtures of H2O2 and peracetic acid11. In the BOSCH Machine Pre-sterilisation procedures5 samples inoculated with 104, 105 and 106 spores are used (the application of at least 5−D is required).
• Geobacillus stearothermophilus (ATCC 7953/12980; NCTC 10003/10007; DSM 494/22/5934; CIP 52.81): in 30 per cent H2O2 /water solution at 30°C it is much less resistant compared to B. subtilis var. globigii and B. subtilis A, whereas at 87.8°C its resistance is slightly less than that of B. subtilis var. globigii, and greater than that of B. subtilis A13. In 28.5 per cent H2O2 /water solution at 24°C its resistance is lower than that of B. subtilis SA22 and scarcely lower than that of B. subtilis var. globigii5. In Oxonia® at room temperature, it has lower heat resistance than B. subtilis A and about the same resistance of B. subtilis var. globigii11. In H2O2 vapour, however, it has the highest resistance12.

There are no Bacilli spores used as biological indicators with a certificate of resistance to treatment with H2O2. Their resistance depends on the operating conditions that may be various (dip or fill, spray, spray and condensation, at different concentration and temperature, followed by sterile rinse or treatment with hot air at different temperature) and in addition, of course, it depends on the different strain. The available data on resistance to treatment with H2O2 of spores of C. botulinum are very poor and mostly for dipping treatment at room temperature. The only research on post-treatment with hot air, allowing direct comparison with G. stearothermophilus, reports that G. stearo – thermophilus spores are at least 3.3 times more resistant than those of the more resistant strain of C. botulinum13. Using sporicidal H2O2 +air (55°C−85°C), 4−D reduction of spores of G. stearothermophilus could be considered as equal to more than 12−D for the most resistant spores of C. botulinum. In cold Oxonia® (dipping), the spores of G. stearothermophilus have resistance at least 4.5 times greater compared to the more resistant spores of C. botulinum11. Moreover at the present time, the spores of G. stearothermophilus are used as a biological indicator to validate the sterilising effect of H2O2 in the vapour phase14.

In addition, it is preferable the use of spores of G. stearothermophilus, compared to other Bacilli, since its thermophilic character allows to inoculate and manipulate samples without the need of nominally aseptic conditions.

The test method

According to the above reported literature survey, the following procedure was developed:

1. Locate critical areas of the pouches (for instance four spots), depending on the H2O2 and hot air fluxes. Check the possibility to apply rectangular strips (5×70 millimetres) of plastic laminate and codify their position with letters

2. Cut a sufficient number (for instance 50) of strips from pouch samples. Apply on a face of the strips a double-sided tape. With indelible pen, divide the other side of the samples with vertical marks so as to have, starting from the left, a 5×10 millimetre area to be used for manipulation and three 5×20 millimetre adjacent areas

3. Using a micro-pipette Eppendorf, inoculate the three 20 millimetre areas of 42 samples with 0.1ml of a hydro-alcoholic suspension (ethanol 40 − 70 per cent) at three different dilutions, approximately 104, 105 and 106 cfu/ml spores of Geobacillus stearo – thermophilus (ATCC 7953 = NCA 1518 = DSM 59344). Allow complete drying of the inoculated samples and store them in open Petri plates in a dryer for at least 16 hours before using. Extract the samples to be used and close and codify their plates

4. Arrange 150 sterile tubes with suitable nutrient and 200EU/ml of catalase

5. Attach with the tape the inoculated strips on four critical spots of 10 numbered pouches, taking care not to touch the surface inoculated. Start the cycle of H2O2 sprinkling and hot air. Take the samples, taking care not to touch the inoculated surface, cut the three parts with different inoculation and insert each one in a tube encoded with the number of the pouch, the letter of the critical point and the level of inoculation

6. Repeat the previous step for two pouches on which not inoculated strips were glued

7. Insert in the remaining six tubes the six part of the two strips not subjected to sterilisation.

8. Set the tubes at 55°C for three days (72 hours)

9. Express the results as maximum completely inactivated inoculum for each critical point of each pouch.

The result is to be considered acceptable if in all the inoculated samples there was at least the complete inactivation of 104 cfu. The test of the not inoculated and treated should be negative, whereas those inoculated and untreated should be positive. The whole procedure should be repeated at least two times (with more repetitions in case of inconsistent results).

Conclusions

Reliable test methods are necessary to evaluate and validate cleaning, pre-sterilisation and decontamination processes of aseptic packing machines and packing materials. The proposed method allows convenient and reliable assessment of effectiveness of a decon – tamination process based on synergistic effect of hydrogen peroxide and hot air.

Cited references

1. A. Stersky, E. Todd and H. Pivnick, Food poisoning associated with post-process leakage (PPL) in canned foods. J. Food Prot. 43 (1980), 465–476

2. Reij, M.W., Den Aantrekker, E.D. (2004), Recontamination as a source of pathogens in processed foods. International Journal of Food microbiology, 91, 1-11

3. Borgese, R. (2009). Ricerca e innovazione tecnologica nella progettazione di linee di lavorazione per vegetali. PhD Thesis. Università digli Studi di Parma

4. Toledo, R., Escher, F. & Ayres, J. (1973). Sporicidal properties of hydrogen peroxide against food spoilage organisms. Appl. Microbiol., 26, 592–597

5. EHEDG (2001). Challenge tests for the evaluation of hygienic characteristics of packing machines for liquid and semi-liquid products. Trends in Food Science & Technology, 12, 244–248

6. Leaper, S. (1984). Comparison of the resistance to hydrogen peroxide of wet and dry spores of bacillus subtilis sa22. International Journal of Food Science & Technology, 19 (6), 695 – 702

7. Cerny, G. (1992). Testing of aseptic machines for efficiency of sterilization of packaging materials by means of hydrogen peroxide. Packaging Technology and Science, 5, 77–81

8. VDMA (2006). Verband deutscher maschinenund anlagenbau E.V. (German engineering federation). Food processing machinery and packaging machinery. Document no. 14 (English revised edition: July 2007) code of practice. Testing hygienic filling machines of VDMA class v (aseptic filling machines). external sterilization of packaging materials

9. VDMA (2003). Verband deutscher maschinenund anlagenbau E.V.. (German engineering federation). Food processing machinery and packaging machinery. Document no. 8 (English edition: March 2004) code of practice. testing aseptic plants: Sterilizing the sterile zone in machine interior

10. Reidmiller, J., Baldeck, J., Rutherford, G. & Marquis, R. (2003). Characterization of uv-peroxide killing of bacterial spores. Journal of food protection, 66 (7) July 2003:1233-1240. Characterization of UV-peroxide killing of bacterial spores. Journal of Food Protection, 66 (7), 1233–1240

11. Blakistone, B., Chuyate, R., Kautter, D.J., Charbonneau, J. & Suit, K. (1999). Efficacy of oxonia active against selected spore formers. Journal of Food Protection, 62(3), 262–267

12. McDonnell, G., Grignol, G. & Antloga, K. (2002). Vapour phase hydrogen peroxide decontamination of food contact surfaces. Dairy Food Environ. Sanit., 22, 868 873

13. Ito, K., Denny, C., Brown, C., Yao, M. & Seeger, M. (1973). Resistance of bacterial spores to hydrogen peroxide. Food Technology, 27 (11), 58–66

14. Kokubo, M., Inoue, T. & Akers, J. (1998). Resistance of common environmental spores of the genus bacillus to vapor hydrogen peroxide. J. Pharm. Sci. Technol., 52, 228–231

General references

• Anon (2001). February 27 2001 proposed rules. Federal Register , Volume 66, Number 39
• Anon (2005). Clostridium spp in foodstuffs. The EFSA Journal, 199, 1–65.
• Bernard, D.T., Gavin, A., Scott, V.N., Chandarana, D.I., Arndt, G. & Shafer, B. (1993). Establishing the Aseptic Processing and Packaging Operation In: Principles of Aseptic Processing and Packaging Food Processors Inst; 2nd edition. The Food Processors Institute.
• EHEDG (1993). A method for the assessment of bacteria tightness of food processing equipment. Trends in Food Science & Technology, 4, 190–192.
• Ito, K. & Stevenson, K. (1984). Sterilization of packaging materials using aseptic systems. Food Technology, 38 (3), 60–62.

About the Author

Giampaolo Betta is currently Professor in charge of Food Science and Technology at the faculty of Engineering of the University of Parma. Since 2005, he has been a member of the Food Technology Unit of the Industrial Engineering Department of that university, where its research work is focused on hygienic design of food equipments, validation of food equipments and modelling of thermal processes. He received a Mechanical Engineering Master Degree in 2004 and a PhD in Food Science and Technology in 2009. Since 2007, he has been Chairman of the Italian Section of the European Hygienic Engineering and Design Group.