Review

Consumer demands for higher product quality and higher

product safety drive the development of non-invasive sterility tests that allow the assessment of product safety, yet leave

both the product and its package intact, and at fell market

value. In addition to being non-invasive, the ideal method

should be: rapid (permitting extensive testing), non-specific

(reacting to all types of microbial growth) and sensitive (mini-

mizing the necessary incubation time). This review presents some new methods for non-invasive sterility control in asepti-

cally packaged foods, which are still undergoing develop-

ment, but seem promising in that they fulfill some of the

above criteria.

Aseptically packaged food is processed with the aim of creating a so-called commercial sterile product. An important safety aspect of this concept has been defined as 'the absence of microorganisms capable of reproduc- ing in the food under normal non-refrigerated conditions of storage and distribution'L As consumer demand for higher product quality increases, so does the search for better production methods. Furthermore, an improved production output does, in turn, imply that the quality of testing methods has to he improved, and the proportion of samples tested increased. This consumer-driven circle of constantly improved quality may result in demands for the entire production lot of certain products (e.g. baby food and other special dietetic products) to he safety checked before they can be sold on the market, provided this is both technically and economically feas- ible. It is obvious that this demand for product safety drives the development of non-invasive testing methods that leave both products and packages intact, and at full (or increased) market value.

Some important criteria for method evaluation Non-spec i f i c i t y

Traditionally, microbial quality control methods have focused on assessing specific foodborne pathogens. A wide range of kits and instruments are now com- mercially available for the detection of well-known pathogens such as Sabnouella and Listeria. A great deal of research effort is also bei,ag put into the detection of Escherichia coli O157:H7 in foods-'. However, in or- der to check the safety of commercial sterile products, methods that detect the growth of any microorganism are needed. To date, there are only two commercial non-invasive methods on the market. The "Electester" (from Tuomo Halonen Oy, Toijaia, Finland) assesses viscosity changes ;n the product by first oscillating the product and then measuring the pattern of the

Harriet Gestrelius is at Tetra Pak Processing Systems AB. Ruben Rausings gala, S-221 86 Lund, Sweden. Tiina Matlila-Sandholm and Raija Ahvenainen are at VII Biotechnology and Food Research, Biologinkuja 1. PO Box 1500. FIN-02044 VIi', [spoo, Finland.

Methods for non-invasive sterility control in aseptically packaged foods

Harriet Gestrelius, Tiina Mattila-Sandholm and Raija Ahvenainen

subsequent damping of the induced motion in the fluid. "Tap Tone' (from Benthos Inc., North Falmouth, MA, USA) uses an electric field to create a tone, by inducing vibrations of the aluminum foil in the package: the amplitude of the tone changes if. for example, gas is pro- duced in a package without a headspaee. These indirect methods are often appropriate for detecting a number of different microorganisms, but as microorganisms pro- duce different effects, these methods must be checked against a wide variety of microorganisms in order to establish the extent of their applicability. It would, of course, be better if measurements could he made on fac- tors that are fundamentally and directly attributable to organic growth in the product. One such example, based on the assessment of the production of heat by growing microorganisms, will he presented below.

Sensi t iv i ty A high degree of sensitivity is important for a number

of reasons. A highly sensitive test method means that the required incubation time can he shortened: a short incubation time directly improves quality control by reducing the feedback time of the outcome of the micro- biological test on the sterilization and packaging processes. A shorter incubation time will also have a direct, favorable economic effect, by reducing the time the produced lot must he stored for before it can be dis- patched. A high degree of sensitivity also increases the applicability of indirect methods, as microorganisms that change the assessed property only slightly can be detected.

Rapid i ty As safety testing, for the reasons given alx~ve, must

be applied to a large proportion of the units produced, it is evident that a rapid test method is much more useful than a slow one. The output of modem sterilization and packaging equipment is of a magnitude that demands that the test methods used are rapid, if large numbers of samples are to he assessed. In those instances where the

Trends in Food Science & Technology December 1994 IVol. 51 ,~ qq4 El . . . . So;coLe Ltd t)~24 -2244 ~14"$07 tto 379

entire production lot is to be checked, the checking itself will directly increase the value of each package. Under such circumstances there will be a direct relationship between the speed of the assessment method and the product value.

Ultrasonic imaging The application o f non-invasive methods developed

by medical science to solve quality problems encoun- tered in food production is an interesting idea. Studies carried out using medical equipment that creates images of reflected ultrasound have shown that ultrasound imaging at a frequency of 5 MHz can be used for the non-invasive testing of the microbiological quality of non-viscous and viscous milk-based aseptic products, such as milk, vanilla and chocolate sauces, and the UHT base material for soft ice cream, packaged in plas- tic or aluminum foil laminated containers x-'~. Ultrasound imaging detects structural changes and gas bubbles caused by microbial enzymes, and can detect the

Fig. 1 Ultrasound images of ultrahigh-temperature vanilla sauce

taken through the package wall ISaranex container), after incubation for 5.5 d at +25"C, using a Toshiba SAL-77B imager designed for medical use.

lat, Non-inoculated reference sample ~no bacterial growth); drl, sample inoculated with fscherichia colilbacterial count of tO ~ cfu/gL

ISource: R. Ahvenainen, uc~published.)

spoilage of milk products when the bacterial count has reached a level of 10-~-10 x colony-forming units (cfu) per gram (see Fig. l). As gas bubbles are detected by the ultrasound, the product itself cannot contain air (which, for example, excludes certain puddings from being tested using this method).

Ultrasound images of milk products can be digitized and the numerical values obtained (which quantify, for example, the number and size of the air bubbles) corre- late well with bacterial counts". However, different milk products need different incubation times in order to obtain bacterial counts that are high enough to produce clear changes in ultrasound images. Studies carried out on ultrasound imaging confirm the necessity of optimiz- ing incubation times and other conditions before taking the actual measurement.

A problem with ultrasound imaging at 5 MHz is that the acoustic impedance of cardboard-based materials at this frequency is too high to permit any measurements to be taken through these types of packages. Decreasing the frequency to 0.5-2 MHz allows penetration of the cardboard x, but at this lower frequency only larger par- ticles reflect the ultrasound waves. This reduces the sensi- tivity of the method, such that only heavily spoiled milk can be detected at these lower frequencies. Increasing the ultrasound frequency should, in theory, improve the sensitivity of the method. However, in practice ultra- sound waves with a frequency of 7.5 MHz also detect the interfaces within the milk product itself and its par- ticles (probably fat particles), which gives a slightly turbid image even for steri!e milk. So the net result of increasing the frequency is a decreased sensitivity in the detection of spoilage'L

Ultrasound imaging is a fairly rapid and sensitive method for detecting spoiled milk. Thus, ultrasound imaging could be developed into a method suitable for testing large numbers of samples after incubation for only 3 - 7 d. However, in order to become commercially viable, it is necessary to be able to detect spoilage through cardboard. This might be achieved by deter- mining an optimal combination of frequency, dynamic range, echo enhancement and gamma compensation".

Ultrasonic Doppler Another non-invasive method based on ultrasound

technology has been proposed by Gestrelius et aL "ul

An ultrasonic beam that is transmitted into a liquid will create a controlled motion called ultrasonic streaming nz. The velocity of this streaming depends on various physi- cal factors such as viscosity, sound absorption, speed of sound and possibly other factors that might be affected by microbial activity within a sample of milk. The velocity spectrum of such ultrasonic streaming can be determined by measuring the Doppler shift (frequency drop) of sound waves reflected from particles or gas bubbles in the milk. The faster the particles move away from the ultrasound transducer, the greater is the Doppler shift.

The experimental setup includes the following com- ponents:

380 trends in Food Science & Technology December 1994 lVol. 5l

• a transducer that transmits ultrasonic beams into the package;

• a receiver that measures the frequency change caused by the induced acoustic streaming in the product;

• a spectrum analyzer and a personal computer that processes the reflected sound into a spectrum of Doppler shifts (see Fig. 2).

Tests performed on packaged infected ultrahigh- temperature (UHT) milk demonstrate that microorgan- isms that induce major structural changes in the milk, like coagulation, can be clearly detected after an incu- bation time of 4 -5 d, whereas microorganisms that only induce minor structural changes need a longer preinou- bation time.

The potential of this method lies in the fact that a measurement can be taken within 10s, and that it seems

to be sensitive enough to detect non-sterile packages after an incubation period of only a few days. The fact that the method is rapid opens up the possibility of extensive testing of the production lot. whereas the high degree of sensitivity minimizes the required incubation time before the product can be sold on the market.

Limiting factors of this method are that it only works on liquid foodstuffs, and that it is an indirect method. the specificity of which is unknown. Hence. there is a possibility that microbial growth that induces very minor textural changes to the product may not be de- tected. Thus there appears to he a lot of work to be done before this method can be applied more generally.

Headspace indicators The application of headspace indicators represents a

consumer-friendly appro~ch for detecting the spoilage of aseptic packages. Tnz concept of beadspace indi-

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . cators differs from that of ~ther non-invasive methods in

~ ~ small transparent window to allow inspection of the

Fig. 2

that the indicators are ad~ r d to the package itself. Thus the packages need to be ,auai,la-efi;. or at least have a

indicator. The indicators react, producing color changes. which can then be compared with a reference color by machine and/or by visual inspection. This means that spoilage checks can be carried out both at the factory line and by the consumers themselves.

Oxygen indicators Most of the indicators used so far have been de-

veloped for the detection of the leakage of oxygen into packages normally containing a low oxygen level (0.1-0.2%). The Japanese are the market leaders in this technology, as is evident from their R&D efforts and the number of patents concerned with oxygen detection indicators~-~'L According to Krumhal and KareF °, an oxygen-~nsit ive dye component should fulfill the fol- lowing criteria:

• the color change should be distinct, lasting and rapid in contaminated packages:

• the dye should not react to reducing components within the food itself;

• the dye should be sensitive enough to oxygen such that change occurs within 1-8 h of leakage;

• the color of the indicator should be easily di~ernible on visual inspection of the package.

Usually, these oxygen detectors are contained within separate pouches that are transparent, and most of them have not been destgned for aseptic packages.

Principle of the ultrasound streaming method. Ultrasound transmitted The two common problems encountered with many into a liquid induces a streaming motion. Sound reflected by particles dyes are that the3,' are too sensitive to oxygen, and that

the color reaction they undergo is often reversible. It travelling with the streaming is detected and demodulated. The should be remembered that headspaoe indicato~ should demodulated signal is Fourier transformed into a s0ectmm not react to residual oxygen levels that are allowed in representing the velocity distribution of the particles in the streaming, the packages. The resistance to heat treatments and the T, transmitter; R, receiver; M, mixer used to demodulate the signal, stability of the dyes at various stages of the packaging (Source: H. Gestrelius, unpublished.) process are other problem areas. For safety reasons the

Trends in Food Science & Technology December 1994 [Vol. 5] 381

indicator should consist of components that are allowed to make direct contact with the food.

Applications in aseptic packaging Although several patenls describing oxygen detection

indicators exist (18 from Japan, four from the USA and one from the UK), none of these have been applied to aseptic thud packaging. The most likely reason for this is that defective seals, which result in increased oxygen levels, are only one cause of defective packages in an aseptic production line, and an indicator should be able to detect many other contamination sources. One sol- ution could be the use of several indicators, one for detecting the leakage of oxygen, and another for detect- ing chemical or microbial spoilage. Experiments. car- ried out by Mattila et al.~9 in which an oxygen indicator was used in conjunction with a pH indicator showed that the packages needed to be preincubated at two different temperatures; the oxygen indicator detected leakers at +4°C, whereas the pH indicator indicated microbial spoilage at +30°C.

The detection of microbial or chemical spoilage in aseptic packages could be achieved by using indicators designed to react with the by-products of microbial metabolism in the package headspace. Carbon dioxide production has been used as an indicator of microbial fond spoilage, and in fact some of the pH and redox indi- cators already tested have been shown to react to carbon dioxide levels in the headspace ~2~--'4. Volatile compounds are other components of importance in the headspace, and many of them are rather specific with regard to the microorganisms causing the spoilage, the food type and the components in the food. The production of certain volatile compounds has been shown to indicate spoilage earlier than any other microbial or sensory change 25. Most volatile compounds are produced as a result of the breakdown of amino acids and fatty acids, and are often species specific. However, metabolites that are common to many microorganisms, like dimethylsulphide, ethanol, propanol and acetaldehyde, also exist.

Headspace indicators are both sensitive and easy to check. In fact, it is even feasible that the end consumers could check the product themselves. This opens up the possibility of greatly reducing the incubation time before the product is dispatched. The present limitations lie in the specificity of the indicators, which, at present, are not adequate for the detection of microbial growth in aseptic packaging. However, it should be possible to develop indicators that are sensitive to metabolites that are more relevant. Such developmental work should be preceded by studies on products that are contaminated with the most relevant contaminants and pathogens, and those volatile substances that are commonly found in such products should be chosen for indication purposes. Hence, the commercial use of headspace indicators for aseptic products lies a couple of years away.

Smart temperature sensors Metabolically active and growing microorganisms

consume energy and, in turn. generate small amounts of

heat. Meijer et al . 26 have developed a method for detect- ing the small temperature increase of a product caused by growing microorganisms. Basically, the method uses a setup that consists of: a specially designed calorimeter, smart temperature sensors and data processing equipment.

The calorimeter is made of polystyrene foam and contains 100 cavities for one-liter packages. Each cavity is equipped with a smart sensor. The packages are insu- lated from the environment so that heat generated by microbial metabolism in infected packages causes a small but detectable increase in the temperature of infected packages relative to non-infected packages. As the ambient temperature, and thus the temperature of all the packages, may change by several degrees Celsius during the microbial growth phase, this small tempera- ture increase can only be measured relative to the other packages. Hence, the individual insulation of the pack- ages makes it easier to discern infected packages from non-infected packages.

The smart temperature sensors are novel devices 27, and the term 'smart" indicates that some information- processing circuits are integrated with the sensor el- ements on the same chip. These sensors are capable of reliably measuring temperature changes in the product of 0.005°C. In order to obtain this high sensitivity, some technical measures were taken. A metal plate was inserted between the sensor and the package in order to imoruve the physical contact with the package, while the self-heating and noise problems of the sensors were circumvented by sequentially powering the individual sensors for a measurement time of only 30 ms, and then averaging the results over 100 measurements.

In order to reduce the effects of the ambient tempera- ture, inaccmate temperature sensors and initial tempera- ture differences between the packages, the temperature of an individual package is always compared with the temperature of its neighbors by means of a computer program. This type of signal 'filtering' markedly im- proves the sensitivity of the method and temperature differences of 0.005°C are easily detected, although the ambient temperature may change by several degrees. Experiments have shown that infected packages can be detected by measuring a relative temperature increase of ~0.8°C alter an incubation period of some 30 h.

The great potential of the sm,-'Lrt temperature sensor method lies in the fact that by measuring energy dissi- pation, which is directly coupled to all organic growth, it actually succeeds in combining high setlsitivity with low specifi~,ity. This makes the smart temperature sen- sor method unique among the others discussed in this review.

One of the limitations of this method is the fact that measurements have to be taken before and during the microbial exponential growth phase. As the exact timin~ of this is uncertain, each package must be assessed over a 10-20 h period. The requirement for calorimeters also puts a practical limit on the number of packages that can be assessed simultaneously. These two limitations result in a throughput that is slow compared with the high out- put of modem packaging equipment. Hence, the method

382 Trends in Food Science & Technology December 1994 [Vol. 51

i Table 1. P . . . . . ;slat-us of . . . . . . . a "d d e v ~ e l o p i n g m e i h ~ fo r n~o n : inv-asives te rilit y coni;e/-in asepficaU, p a c k a g ~ l f o ~ . . . . . . . . . .

Method Type of changes registered Non-specificity Sensitivity Rapld~

Ultrasonic imaging Physical structures, such as bubbles ++ ++ ++

Ultrasonic Doppler Viscosily, physical structures ++ +÷ ++

Headspace indicators Chemical + ++ +++

Smart temperature sensors Tem0erature +++ +++ +

% low; +% medium; ++% high

~s probably more suitable for smaller batches and also References for the sample testing of larger batches, t Code of federal Regulations (1993) [21 C.F.R. §I 131, US Government

Conclusions There has been an increasing interest in the develop-

ment of new non-invasive methods for detecting the growth of microorganisms and the spoilage of products during recent years. New methods have been suggested and compared with existing ones, but so far none of these methods have succeeded in meeting the three 'ideal' criteria of non-specificity, high sensitivity and rapidity (see Table I). Changes in physical structure are measured in both ultrasonic imaging and the newer ultrasonic Doppler methods, as well as in the two avail- able commercial methods, Electester and Tap Tone. How- ever, there are a number of microbes that do not produce major changes in physical structure, so detecting this type of indirect change is not a very reliable way to check for non-sterility. The only method that directly measures microbial growth is the smart temperature sensor method, which measures the minute temperature increases that ,arc produced by all growing microbes. However, this method requires that measurements are carried out before aod during the exponential growth phase of the microbes, which makes the assessment time rather long and uncertain, limiting the applicability of the method.

Headspace indicators differ from the other methods in that reactive dyes are added to the package itself. This has the advantage of reducing the equipment necessary to register the changes, and in many cases the final con- trol step can be carried out by the consumers them- selves. Similar methods have been very successful in specific analyses of blood, urine, etc. So, if an indicator that reacts with a chemical substance that is commonly produced as a result of most microbial growth can be produced at a price that is not too high, this would be close to an ideal solution. Another possibility is, of course, to combine the different methods in a way that would minimize their individual shortcomings.

However, the problems of non-invasively controlling aseptically packaged foods are not yet solved. Research is in progress and new methods are being suggested. Although some possibilities have been presented in this review, there are likely to be more lo come in the next couple of years.

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Trends in Food Science 8, Technology December 1994 Wol. 51 383