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Ultrasonic Doppler: a possible method for non-invasive sterility control
Ultrasonic Doppler: a possible method for non-invasive sterility control Harriet Gestrelius In order to improve the total quality control (TQC) of the production and packaging of food, new non-destructive methods for in-line self-inspection are needed. It was investigated whether inducing acoustic streaming in the product, by transmitting an ultrasonic beam into the packages, could be used as a nondestructive method for detecting microbial growth in aseptic milk packages. It was shown that the velocity spectrum of the induced streaming, measured by the Doppler shift of reflected sound, decreased in packages with microbial growth. Keywords: non-destructive control; GMP; HACCP
testing
of aseptic
milk;
ultrasonic
Doppler;
INTRODUCTION Total quality control (TQC) is one of the most important issues in the production and packaging of food today. Quality cannot be attained by simply checking the end-product, on the contrary it must be worked into the company in a number of different ways ranging from management policies and reorganization of responsibilities to the introduction of improvement teams. Most quality programmes aimed at applying good manufacturing practice (GMP) or hazard analysis critical control points (HACCP) procedures try to readjust the quality control focus from inspection of the end product made by QC specialists to in-line selfinspection of the various processes. Although the introduction of such a scheme will reduce the need for a comprehensive inspection of end products, testing of critical parameters such as product safety will remain. This is in keeping with a growing trend to let product safety responsibility remain with the producer. Such a notion re-evaluates the importance of inspection, as the issue of product liability and responsibility requires assembling of data for evidence that will hold in court,
Tetra Pak Processing Systems AB, Lund, Sweden. Presented at the third EFFoST conference ‘Food Control - On-Line Control for Improved Quality’, 26-29 September 1993. Porto, Portugal 0956-7135/94/02/0103-03
@ 1994 Butterworth-Heinemann
Ltd
acoustic
streaming,
in-line
quality
and not only for the quality assurance itself (Ishikawa, 1985). A decrease in product defect rate per se does imply larger samples in order to control quality and in some cases, e.g. baby food and other special dietetic products (SDPs), the entire production may need to be checked, if it is technically possible. Thus, the value of non-destructive testing of safety parameters, in which the product and packages are left intact and at full market value, will increase as customer demand for safety increases. In the case of aseptic packaging, one important aspect of product safety can be defined as ‘the absence of microorganisms capable of reproducing in the food under normal non-refrigerated conditions of storage and distribution’ (FDA, 1993). In order to be able to continuously improve the processes of sterilization and aseptic packaging while maintaining a high quality (close to zero-defect) product to the consumers, it is important to have a non-destructive testing method that detects the presence or growth of microorganisms as part of the in-line self-inspection quality scheme. Preferably, the method shall be rapid enough to permit testing of many packages and sensitive enough to provide results after a short period of incubation. The only non-destructive commercial test today is the Electester (Tuomo Halonen Oy, Finland), which is used for milk products in carton packages and is based on the detection of viscosity changes caused by spoilage of the product. There is a need to develop other Food Control 1994 Volume 5 Number 2
103
Ultrasonic Doppler for sterility control: H. Gestrelius
methods, Some ideas on such non-destructive methods for spotting growth of microorganisms are given below. A non-destructive method for detecting microorganisms in aseptic packages should preferably have a high sensitivity (in order to detect low concentrations of microorganisms early), but low selectivity (it should preferably detect any microorganism). Basically, the ideal test would be one that directly detected any living microorganism but as this is still not possible, the next best thing is to measure chemical and/or physical properties that are secondary to growth of microorganisms. Chemical changes may range from alterations in pH and oxygen content to the presence of specific molecules. An example of a new possibility based on chemical properties is the development of a ‘smart package’ with selective sensors incorporated into the package material itself. Traditionally, non-destructive tests have been based on the physical changes in the texture of the product (viscosity, presence of bubbles and coagula, etc.) that are secondary to growth of microorganisms. Although this approach is predominant in methods based on the physical properties, there are some interesting new experiments being done to measure the small amounts of heat that microorganisms produce while growing (Meijer et al., 1993). In an attempt to find better ways to non-destructively detect bacterial growth, it was decided to test whether a method based on ultrasound Doppler could be employed to spot infected packages. This test was carried out as a joint project between Tetra Pak and Lund Institute of Technology (Gestrelius et al., 1993).
METHODS
AND MATERIALS
The material used was UHT treated milk with 3% fat packaged in Tetra Brik Aseptic (375 ml) packages with headspace and pulltab. The metal pulltab opener, which measures 15 x lOmm, was used as a window for transmitting ultrasound waves into (and receiving reflected ultrasound waves from) the sealed packages. The packages were inoculated with either Stuphylococcus epidermidis, which is known to induce coagulation fast, or Bacillus subtilis A, which only induces slow and small changes in the texture. The ultrasound beam transmitted into the liquid induces a streaming motion known as acoustic streaming (Lighthill, 1978). In addition to characteristics of the beam, the velocity of this streaming is dependent of the viscosity, speed of sound and ultrasound absorption of the liquid. When moving particles (or gas bubbles) reflect sound, the frequency of the sound is changed due to the Doppler effect. Hence, the velocity of the induced acoustic streaming in the liquid is proportional to the frequency change Af of the reflected ultrasonic waves, while the amplitude of the reflected waves depends on the acoustic properties and the amount of particles (or bubbles) in the liquid. By measuring the amplitude of the different frequency changes, a spectrum diagram like Figure I could be constructed. The obtained spectrum can be analysed in various ways to obtain information about the liquid (see Discussion), but in this work the analysis was limited to finding the maximum frequency change, which is proportional to the maximum streaming velocity. A maximum 104
Food Control 1994 Volume 5 Number 2
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Frequency change [Hz]
Figure 1 Spectrum obtained with the ultrasound streaming method from milk. The frequency change of the reflected ultrasonic waves is proportional to the velocity of the induced acoustic streaming in the liquid, while the amplitude of the reflected waves depends on the acoustic properties and the amount of particles in the liquid
frequency change of 200 Hz corresponds to a maximum streaming velocity of approximately 30 mm s-l. The measurement time was around lOs, during which 13 spectra were acquired and averaged. RESULTS Staphybcoccns
epidemidis
As can be seen from Figure 2, a clear decrease in streaming velocity (decrease in maximum frequency change) could be seen 4-5 days after inoculation. This development over time is compared with the observations (by destructive methods) that the bacterial content flattened out at around 108c.f.u. ml-’ after 2 days and that the milk had coagulated around day 5. However, no packages opened on day 4 had coagulated. These observations suggested that, in the case of S. the ultrasound method does detect epidermidis, infected packages. The method also spots the infected packages before any visible coagulation of the milk could be observed. -20 7
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Frequency change [Hz]
Figure 2
Spectra obtained with the ultrasound streaming method from milk inoculated with S. epidermidis. Thin line: non-infected reference package; thick line: 4 days after inoculation; dotted line: 5 days after inoculation (the peaks are 5OHz electrical disturbances from the power supply)
Ultrasonic Doppler for sterility control: H. Gestrelius
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Frequency change [Hz] Figure 3 Spectra obtained with the ultrasound streaming method from milk inoculated with B. subtilis A. Thin line: non-infected reference package; dotted line: 6 days after inoculation; thick lines: 14 days after inoculation. The spectrum from day 6 indicates a slightly lower streaming velocity than from non-infected packages. The three different spectra from day I4 show the inconsistency between inoculated packages (the peaks are 50Hz electrical disturbances from the power supply)
Bacillus subtilis A Results
of a similar series of experiments
with B.
subtilis A is shown in Figure 3. As can be seen from the
spectra of day 6 and onwards, the maximum induced streaming velocity decreases in infected packages. However, as can be deduced from the spectral differences of day 14, there is a considerable variation between the individual packages. It seems that B. subtilis A changes some parameters in the milk, but not consistently in all packages. The results are compared with the observations (by destructive methods) that the bacterial content reached its maximum level of around 5 x 106c.f.u. ml-‘ml after 1.5 days, but no visible viscosity or other major changes were found in the packages over the 14 day period of the experiment.
DISCUSSION The above results indicate that an ultrasound Doppler method can be used for non-destructive detection of microorganisms that induce changes in the physical structure of milk. It seems that the method is fairly sensitive, as in both cases (S. epidermidis and B. subtilis A) it produced a reaction before any visible changes in viscosity or other major changes in physical properties could be detected. The Doppler measurement is also rapid enough (around 10 s) to be practically useful in an extensive testing programme. As the experiment can be characterized as a ‘test of concept’, all the practical arrangements can be greatly improved. Furthermore, as the spectra were used only for determining the maximum streaming velocity, most information concerning the amplitude and general shape were discarded. Hence, there is more information hidden in the spectra that might prove useful when a better understanding of the reflection mechanisms is obtained. For example, there is some preliminary evidence that gas-filled bubbles are the major ultrasound reflectors. If this is true, changes in amplitude
could be attributed to parameters that in different ways change the number of bubbles (gas production) or the properties of the bubbles (surface tension, oxygen content, pH etc.). If this type of more complex pattern recognition is to be applied, one possible method is to use artificial neural network techniques that look promising for dealing with pattern recognition problems. The successful ‘test of concept’ of the ultrasound Doppler method illustrates that there are new hightech methods that can be used for improving quality control in the food industry. The test also illustrates that the biological reality is very complex (S. epidermidis behaved quite differently from B. subtilis A) and it is therefore unlikely that there will be one method for checking the production and packaging of aseptic milk. There is clearly a need for starting up interdisciplinary projects and to do some lateral thinking and technology transfer. A good example of this lateral thinking is the attempt to transfer the ultrasound imaging techniques used within the medical sciences to food technology (Ahvenainen et al., 1989a, 1989b, 1989~). In addition to the physical properties discussed so far, chemical reagents or biosensors could be used for measuring chemical properties non-destructively. It can be concluded that it is likely that the best way of checking product safety in aseptic packaging will prove to be a combined method that takes into account physical as well as chemical changes within the product and uses modern information technology to analyse the patterns.
ACKNOWLEDGEMENTS The author would like to thank Dr Thomas Hertz, Dept. of Electrical Measurements, Lund Institute of Technology, Sweden for valuable technical assistance.
REFERENCES Ahvenainen,
R., Mattila, T. and Wirtanen, G. (1989a) Ultrasound penetration through different packaging materials - a nondestructive method for quality control of packaged UHT milk. Lebensm. Wissensch. Technol. 22, 268-272
Ahvenainen, R., Wirtanen, G. and Manninen, M.(1989b) Ultrasound
imaging - a non-destructive method for control of the microbiologica! quality of aseptically packaged foodstuffs. Lebensm. Wissensch. Technol. 22, 273-278 Ahvenainen,
R., Wirtanen, G. and Man&en, M. (1989~) Ultrasound imaging - a non-destructive method for monitoring the microbiological quality of aseptically packaged milk products. Lebensm. Wissensch. Technol. 22, 382-386
Ishikawa, K. (1985) Whar is Total Quaky
Way Prentice-Hall,
Control? The Japanese Englewood Cliffs, NJ, p. 81
FDA (1993) Code of Federal Regulations 21 part 113. April 1. Office of the Federal Register National Archives and Records Administration Gestrelius, H., Hertz, T.G., Naamu, M., Persson, H.W. and Lindstriim, K. (1993) A non-destructive ultrasound method for microbial quality control of aseptically packaged milk. Lebensm. Wissensch. Technol. 26, 334-339 Lighthill, J. (1978) Waves in Fluids Cambridge University Press, Cambridge, UK, pp. 337-351 Meijer, G.C.M., Kerkvliet, H.M.M. and Toth, F.N. (1994) Non-invasive detection of micro-organisms using smart temperature sensors. Sensors Actuators B Chemical 18 (l-3). 276-281
Food Control 1994 Volume 5 Number 2
105
testing
of aseptic
milk;
ultrasonic
Doppler;
INTRODUCTION Total quality control (TQC) is one of the most important issues in the production and packaging of food today. Quality cannot be attained by simply checking the end-product, on the contrary it must be worked into the company in a number of different ways ranging from management policies and reorganization of responsibilities to the introduction of improvement teams. Most quality programmes aimed at applying good manufacturing practice (GMP) or hazard analysis critical control points (HACCP) procedures try to readjust the quality control focus from inspection of the end product made by QC specialists to in-line selfinspection of the various processes. Although the introduction of such a scheme will reduce the need for a comprehensive inspection of end products, testing of critical parameters such as product safety will remain. This is in keeping with a growing trend to let product safety responsibility remain with the producer. Such a notion re-evaluates the importance of inspection, as the issue of product liability and responsibility requires assembling of data for evidence that will hold in court,
Tetra Pak Processing Systems AB, Lund, Sweden. Presented at the third EFFoST conference ‘Food Control - On-Line Control for Improved Quality’, 26-29 September 1993. Porto, Portugal 0956-7135/94/02/0103-03
@ 1994 Butterworth-Heinemann
Ltd
acoustic
streaming,
in-line
quality
and not only for the quality assurance itself (Ishikawa, 1985). A decrease in product defect rate per se does imply larger samples in order to control quality and in some cases, e.g. baby food and other special dietetic products (SDPs), the entire production may need to be checked, if it is technically possible. Thus, the value of non-destructive testing of safety parameters, in which the product and packages are left intact and at full market value, will increase as customer demand for safety increases. In the case of aseptic packaging, one important aspect of product safety can be defined as ‘the absence of microorganisms capable of reproducing in the food under normal non-refrigerated conditions of storage and distribution’ (FDA, 1993). In order to be able to continuously improve the processes of sterilization and aseptic packaging while maintaining a high quality (close to zero-defect) product to the consumers, it is important to have a non-destructive testing method that detects the presence or growth of microorganisms as part of the in-line self-inspection quality scheme. Preferably, the method shall be rapid enough to permit testing of many packages and sensitive enough to provide results after a short period of incubation. The only non-destructive commercial test today is the Electester (Tuomo Halonen Oy, Finland), which is used for milk products in carton packages and is based on the detection of viscosity changes caused by spoilage of the product. There is a need to develop other Food Control 1994 Volume 5 Number 2
103
Ultrasonic Doppler for sterility control: H. Gestrelius
methods, Some ideas on such non-destructive methods for spotting growth of microorganisms are given below. A non-destructive method for detecting microorganisms in aseptic packages should preferably have a high sensitivity (in order to detect low concentrations of microorganisms early), but low selectivity (it should preferably detect any microorganism). Basically, the ideal test would be one that directly detected any living microorganism but as this is still not possible, the next best thing is to measure chemical and/or physical properties that are secondary to growth of microorganisms. Chemical changes may range from alterations in pH and oxygen content to the presence of specific molecules. An example of a new possibility based on chemical properties is the development of a ‘smart package’ with selective sensors incorporated into the package material itself. Traditionally, non-destructive tests have been based on the physical changes in the texture of the product (viscosity, presence of bubbles and coagula, etc.) that are secondary to growth of microorganisms. Although this approach is predominant in methods based on the physical properties, there are some interesting new experiments being done to measure the small amounts of heat that microorganisms produce while growing (Meijer et al., 1993). In an attempt to find better ways to non-destructively detect bacterial growth, it was decided to test whether a method based on ultrasound Doppler could be employed to spot infected packages. This test was carried out as a joint project between Tetra Pak and Lund Institute of Technology (Gestrelius et al., 1993).
METHODS
AND MATERIALS
The material used was UHT treated milk with 3% fat packaged in Tetra Brik Aseptic (375 ml) packages with headspace and pulltab. The metal pulltab opener, which measures 15 x lOmm, was used as a window for transmitting ultrasound waves into (and receiving reflected ultrasound waves from) the sealed packages. The packages were inoculated with either Stuphylococcus epidermidis, which is known to induce coagulation fast, or Bacillus subtilis A, which only induces slow and small changes in the texture. The ultrasound beam transmitted into the liquid induces a streaming motion known as acoustic streaming (Lighthill, 1978). In addition to characteristics of the beam, the velocity of this streaming is dependent of the viscosity, speed of sound and ultrasound absorption of the liquid. When moving particles (or gas bubbles) reflect sound, the frequency of the sound is changed due to the Doppler effect. Hence, the velocity of the induced acoustic streaming in the liquid is proportional to the frequency change Af of the reflected ultrasonic waves, while the amplitude of the reflected waves depends on the acoustic properties and the amount of particles (or bubbles) in the liquid. By measuring the amplitude of the different frequency changes, a spectrum diagram like Figure I could be constructed. The obtained spectrum can be analysed in various ways to obtain information about the liquid (see Discussion), but in this work the analysis was limited to finding the maximum frequency change, which is proportional to the maximum streaming velocity. A maximum 104
Food Control 1994 Volume 5 Number 2
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Frequency change [Hz]
Figure 1 Spectrum obtained with the ultrasound streaming method from milk. The frequency change of the reflected ultrasonic waves is proportional to the velocity of the induced acoustic streaming in the liquid, while the amplitude of the reflected waves depends on the acoustic properties and the amount of particles in the liquid
frequency change of 200 Hz corresponds to a maximum streaming velocity of approximately 30 mm s-l. The measurement time was around lOs, during which 13 spectra were acquired and averaged. RESULTS Staphybcoccns
epidemidis
As can be seen from Figure 2, a clear decrease in streaming velocity (decrease in maximum frequency change) could be seen 4-5 days after inoculation. This development over time is compared with the observations (by destructive methods) that the bacterial content flattened out at around 108c.f.u. ml-’ after 2 days and that the milk had coagulated around day 5. However, no packages opened on day 4 had coagulated. These observations suggested that, in the case of S. the ultrasound method does detect epidermidis, infected packages. The method also spots the infected packages before any visible coagulation of the milk could be observed. -20 7
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Frequency change [Hz]
Figure 2
Spectra obtained with the ultrasound streaming method from milk inoculated with S. epidermidis. Thin line: non-infected reference package; thick line: 4 days after inoculation; dotted line: 5 days after inoculation (the peaks are 5OHz electrical disturbances from the power supply)
Ultrasonic Doppler for sterility control: H. Gestrelius
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Frequency change [Hz] Figure 3 Spectra obtained with the ultrasound streaming method from milk inoculated with B. subtilis A. Thin line: non-infected reference package; dotted line: 6 days after inoculation; thick lines: 14 days after inoculation. The spectrum from day 6 indicates a slightly lower streaming velocity than from non-infected packages. The three different spectra from day I4 show the inconsistency between inoculated packages (the peaks are 50Hz electrical disturbances from the power supply)
Bacillus subtilis A Results
of a similar series of experiments
with B.
subtilis A is shown in Figure 3. As can be seen from the
spectra of day 6 and onwards, the maximum induced streaming velocity decreases in infected packages. However, as can be deduced from the spectral differences of day 14, there is a considerable variation between the individual packages. It seems that B. subtilis A changes some parameters in the milk, but not consistently in all packages. The results are compared with the observations (by destructive methods) that the bacterial content reached its maximum level of around 5 x 106c.f.u. ml-‘ml after 1.5 days, but no visible viscosity or other major changes were found in the packages over the 14 day period of the experiment.
DISCUSSION The above results indicate that an ultrasound Doppler method can be used for non-destructive detection of microorganisms that induce changes in the physical structure of milk. It seems that the method is fairly sensitive, as in both cases (S. epidermidis and B. subtilis A) it produced a reaction before any visible changes in viscosity or other major changes in physical properties could be detected. The Doppler measurement is also rapid enough (around 10 s) to be practically useful in an extensive testing programme. As the experiment can be characterized as a ‘test of concept’, all the practical arrangements can be greatly improved. Furthermore, as the spectra were used only for determining the maximum streaming velocity, most information concerning the amplitude and general shape were discarded. Hence, there is more information hidden in the spectra that might prove useful when a better understanding of the reflection mechanisms is obtained. For example, there is some preliminary evidence that gas-filled bubbles are the major ultrasound reflectors. If this is true, changes in amplitude
could be attributed to parameters that in different ways change the number of bubbles (gas production) or the properties of the bubbles (surface tension, oxygen content, pH etc.). If this type of more complex pattern recognition is to be applied, one possible method is to use artificial neural network techniques that look promising for dealing with pattern recognition problems. The successful ‘test of concept’ of the ultrasound Doppler method illustrates that there are new hightech methods that can be used for improving quality control in the food industry. The test also illustrates that the biological reality is very complex (S. epidermidis behaved quite differently from B. subtilis A) and it is therefore unlikely that there will be one method for checking the production and packaging of aseptic milk. There is clearly a need for starting up interdisciplinary projects and to do some lateral thinking and technology transfer. A good example of this lateral thinking is the attempt to transfer the ultrasound imaging techniques used within the medical sciences to food technology (Ahvenainen et al., 1989a, 1989b, 1989~). In addition to the physical properties discussed so far, chemical reagents or biosensors could be used for measuring chemical properties non-destructively. It can be concluded that it is likely that the best way of checking product safety in aseptic packaging will prove to be a combined method that takes into account physical as well as chemical changes within the product and uses modern information technology to analyse the patterns.
ACKNOWLEDGEMENTS The author would like to thank Dr Thomas Hertz, Dept. of Electrical Measurements, Lund Institute of Technology, Sweden for valuable technical assistance.
REFERENCES Ahvenainen,
R., Mattila, T. and Wirtanen, G. (1989a) Ultrasound penetration through different packaging materials - a nondestructive method for quality control of packaged UHT milk. Lebensm. Wissensch. Technol. 22, 268-272
Ahvenainen, R., Wirtanen, G. and Manninen, M.(1989b) Ultrasound
imaging - a non-destructive method for control of the microbiologica! quality of aseptically packaged foodstuffs. Lebensm. Wissensch. Technol. 22, 273-278 Ahvenainen,
R., Wirtanen, G. and Man&en, M. (1989~) Ultrasound imaging - a non-destructive method for monitoring the microbiological quality of aseptically packaged milk products. Lebensm. Wissensch. Technol. 22, 382-386
Ishikawa, K. (1985) Whar is Total Quaky
Way Prentice-Hall,
Control? The Japanese Englewood Cliffs, NJ, p. 81
FDA (1993) Code of Federal Regulations 21 part 113. April 1. Office of the Federal Register National Archives and Records Administration Gestrelius, H., Hertz, T.G., Naamu, M., Persson, H.W. and Lindstriim, K. (1993) A non-destructive ultrasound method for microbial quality control of aseptically packaged milk. Lebensm. Wissensch. Technol. 26, 334-339 Lighthill, J. (1978) Waves in Fluids Cambridge University Press, Cambridge, UK, pp. 337-351 Meijer, G.C.M., Kerkvliet, H.M.M. and Toth, F.N. (1994) Non-invasive detection of micro-organisms using smart temperature sensors. Sensors Actuators B Chemical 18 (l-3). 276-281
Food Control 1994 Volume 5 Number 2
105