WO1980002458A1

WO1980002458A1 - Process for prophylactic registration of putrefaction in food

Info

Publication number: WO1980002458A1
Application number: PCT/DK1979/000025
Authority: WO
Inventors: A Bentsen
Original assignee: Bioteknisk Inst; A Bentsen
Priority date: 1979-05-01
Filing date: 1979-09-05
Publication date: 1980-11-13
Also published as: DK178879A; EP0035013A1

Abstract

Rot in food products or raw materials for food products is detected prophylactically by determining spectral data on a sample and comparing the spectral data thus recorded with corresponding data being statistically correlated to the probability of development of rot. To detect Phoma and/or Fusarium infections in potatoes, a thin layer is peeled off the potatoes, and they are then irradiated with ultraviolet excitation light. Any resulting fluorescence with a wave length of about 475-500 nm is isolated and fluorescence data are recorded which are compared with predetermined data for the potato variety examined. This method may be carried out by an apparatus which comprises means (23) for emitting UV light, means (13) for detecting the intensity of UV excited fluorescence and read-out means (25), a magazine (21) for potatoes, as well as means (10, 11, 12) between the magazine (21) and detecting means (13) for isolating fluorescence with a wave length of about 475-500 nm.

Description

PROCESS FOR PROPHYLACTIC REGISTRATION OF PUTREFACTION IN FOOD

The present invention relates to a method of direct, prophylactic determination of rot in food products or raw materials for food products, in particular of detection of infections in potatoes caused by the fungi Phoma and Fusarium.

The invention will be explained more fully below in relation to said fungus infections in potatoes, but may similarly be used for prophylactic determination of other conditions of rot, e.g. the presence of ochratoxins or aflatoxins caused by Aspergillus infections in grain varieties.

The Phoma species which is particularly harmful to potatoes is Phoma exigua, especially the variety foveata and to a less degree the variety exigua. The most harmful Fusarium species is Fusarium coerulum.

These fungi cause diseases that develop during storage, in particular, and which entail that even very considerable quantities of potatoes must be rejected.

The storage temperature, in particular, determines which fungus will develop during storing, Phoma having a growth optimum temperature of about 2-8ºC, which is unfortunately also the most appropriate temperature for storing potatoes over an extended period of time, Fusarium having a somewhat higher growth optimum of 12-15°C. Further, a high air humidity (95-100%) seems to promote the growth of both fungi during storing. Fungus infections may of course be detected by visual inspection when the infection has reached a sufficiently advanced stage, but then it is usually too late to avoidrejection of the entire, stored quantity or at any rate a significant loss in the usual sorting of potatoes before they are sold. Accordingly, it is of great practical and economic importance to provide a method which is capable of disclosing Phoma and/or Fusarium infections at a very early stage, preferably before the potatoes are stored.

A chose study of the changes in the potato caused by the growth of the fungi has revealed that a large number of components which fluoresce when irradiated by ultraviolet light will be formed in the infected tissue and in the surrounding tissue by metabolizing the chlorogenic acid present in all varieties of potatoes. These components include caffeic acid, scopolin, scopoletin, aesculin, aesculetin and umbelliferone, cf. Lashin, Meddelelser fra Kartoffelafdelingen, Bioteknisk Institut, (Reports from the Potato Division, Biotechnical Institute 14, 1976, p. 9-14, which also reports previous works.

The difficulty of utilizing this fluorescence for determination, of Phoma and Fusarium infections consists in isolating a fluorescence specific of these infections, the reason being that also chlorogenic acid and thus uninfected potatoes, too, exhibit fluorescence under UVirradiation. Fluorescent compounds may also be formed in other diseases. Finally, susceptibility to the fungi and the development of the infections vary from variety to variety.

A conventional solution to this problem would comprise extracting the fluorescent chemical compounds, including the compounds specific of the fungus infections, separating them, e.g. by thin layer chromatography and then analyzing the individual components separately, and in this connection examining the intensity of their fluorescence by conventional fluorometry on a solution in relation to standard.

This method has in fact been tried, Mosch and Mooi, 1975, but it is cumbersome and time-consuming, and it also requires much special-purpose instrumentation and specially trained staff. Moreover, it is totally unsuitable for determinating infections "in the field" before the potatoes are transported from the location of cultivation to the storage location.

Lashin op.cit. has proceeded one step further. He has proposed two visual test methods based on the UV-fluorescence.

1) In the so-called cut test 50 tubers are halved and mixed separately in a plastic bag. The bags are incubated separately for 7 days at 8ºC. Then the cut face is visually examined under UV-light to estimate the extent of the fluorescent area. The tubers are divided into 6 groups according to area percentage, and a contamination index may then be calculated on the basis of this.

This method suffers from the drawback that even though in principle it can be carried out immediately after the potatoes have been lifted, it will be a good while before the result is reliable because the sensitivity of the method is limited since the Phoma and Fusarium infections must have advanced so much that the fluorescence produced will be vigorous enough to dominate other fluorescence at visual observation.

2) In the so-called quality test, which it is proposed to carry out during the period of storage to detect changes in quality, if any, caused by Phoma or Fusarium infections, 50 tubers are carefully peeled and their surfaces are visually examined under UV-light, the index of the surface being evaluated on the basis of the area analogously with the cut test. Lashin reports that at least 10 days should lapse before the evaluation is made, and that the method is much less sensitive than the cut test.

Though these methods represent an improvement over the previous methods, they have, however, the serious drawback that they are time-consuming, and that their sensitivity is insufficient for detecting Phoma and Fusarium infections at early stages.

Thus, these methods are not very suitable for a rapid detection allowing the permissible storage period for a given quantity of potatoes to be fixed reliably, said storage period being i.a. a function of the developmental stage of the fungi.

It should be added that Lashin's examinations were all conducted on potatoes of the variety Bintje which has turned out to be particularly susceptible to Phoma and Fusarium infections. The infection develops relatively rapidly over a large face, which of course suggests using the proposed area method. As for a number of other varieties, e.g. Hansa, which to an increasing degree substitutes Bintje, the infections develop, point by point, more in the depth, and accordingly measurements based on the area method are very unreliable.

The object of the present invention is generally to provide a method of prophylactic determination of rot in food products or raw materials, and more particularly to provide a method and an apparatus which (a) permit direct detection of Phoma and/or Fusarium infections in potatoes without any other pretreatments than peeling optionally preceded by washing, (b) are sufficiently sensitive to compensate for variations from variety to variety in the development of the infections and for the infections to be detected at a very early stage in the fungal development so that the permissible period of storage can be fixed with a reasonable amount of certainty, (c) do not make special demands on apparatus and staff, and (d) are suitable 'for examinations in the field.

This general object is achieved by the method of the invention which is characterized by the features defined in the characterizing portion of claim 1.

The invention is based on the recognition that development of rot is reflected in recordable spectral data which may be determined for various food products so that once a series of standard data, has been determined a comparison with the recorded spectral data of a given sample will show the developmental stage of rot and, particularly, a prophylactic determination can be made. The most appropriate measures for the food product concerned can then be taken on the basis of such a prophylactic determination, be it rejection, omission of storage or combatting or removing organisms causing rot. It applies in particular to potatoes that in respect of Phoma and Fusarium infections a specific and vigorous fluorescence with the wave length of about 475500 nm stated in claim 4 will be obtained if infected potatoes are subjected to excitation with ultraviolet light, and that fluorescence produced by other causes will be safely screened off by isolating this narrow range. Experiments with potatoes have shown that the optimum wave length of the excitation light is about 365 nm, producing the most pronounced fluorescence at 475-500 nm.

This characteristic wave length has been found after visual comparison of healthy potatoes, potatoes infected by Phoma and Fusarium in various developmental stages, and potatoes infected by Phytophthora, common scab and other diseases. All these potatoes exhibit a more or less wide spectre fluorescence when irradiated with ultraviolet light having a wave length of 365 nm.

However, when the potatoes were observed through a FabryPerot interference wedge at various wave lengths a rough determination revealed that in Phoma and Fusarium infected potatoes the fluorescence blazes vigorously at about 500 nm, and that this blaze does not occur in potatoes which are not infected by Phoma or Fusarium.

An analysis of the potatoes has shown that this fluorescence, which as shown by the wave length has a strong light-blue colour, is primarily caused by aesculin:

which is the 6-glucopyranoside of aesculetin (6,7dihydroxycumarin) :

as well as scopoletin which is aesculetin-6-methylether or 7-hydroxy-6-methoxycumarin:

In serious Phoma and Furasium infections the fluorescence is so predominant that it can be observed visually, while in less serious infections it will often be dominated by the natural fluorescence of the potato, which explains the insufficient sensitivity in the previous methods.

In order to utilize this fluorescence the method of the invention departs from the conventional fluorometric principle which would suggest extraction for isolating and identifying these compounds in that according to the invention the characteristic fluorescence is isolated by purely optical means from other fluorescence which is not specific of Phoma and Fusarium.

The method of the invention also represents a departure from Lashin's method, which would suggest automatized determination of area, e.g. by scanning. This principle has been rejected partly because of the apparatus it requires as a scanning apparatus would be extremely expensive to construct, but primarily because of the variations from variety to variety in the development of the infection, which, as mentioned, makes area determination unreliable in respect of a plurality of widely used varieties of potatoes. In respect of potatoes it has in the method of the invention been elected to integrate the total emission of light, i.e. the intensity of the fluorescent light, to achieve the best possible correlation to the actual contamination load, rather than to determine the extent of the infected, i.e. fluorescent, surface area.

According to the invention this may be dome by means of a filter arrangement adapted to the wave length range:

1) To prevent natural fluorescence of the subsequent filters and to withhold excitation light a UV barrier absorption filter is disposed in the front of the ray path. A filter of the type Wratten no. 2A is suitable for this purpose.

2) To withhold light with wave lengths below about 475 nm an LWP absorption filter (low-pass filter) is then mounted. A filter of the type Wratten no. 4 is suitable as it absorbs all light with wave lengths below about 460 nm.

3) To withhold light with wave lengths above about 500 nm an SWP interference filter (high-pass filter) is then mounted. A filter of the type SWP 500 Ferroperm is suitable as it reflects light with wave lengths above about 500 nm.

In principle, the last two filters might be replaced by a band pass filter corresponding to the desired wave length range. Conventional band pass filters permit, however, also side bands with other wave lengths to pass, which might disturb measurements. This is obviated by the preferred combination of a low-pass filter and a high-pass filter.

The invention also provides an apparatus for carrying out the method of the invention in connection with potatoes. The apparatus is characterized by the features defined in the characterizing portion of claim 6.

The invention is illustrated in greater detail in the drawings, in which fig..1 schematically shows an embodiment of an apparatus for carrying out the method of the invention in connection with potatoes,

fig. 2 shows schematically a section of the detector and the filter system disposed in front of it, and

fig. 3 shows a block diagram of the electric circuit between the detector and the read-out means.

Fig. 1 shows an apparatus comprising a housing 20 whose bottom is provided with a removable magazine 21 in which the peeled potato tubers 22 to be examined are placed. One or more tubes 23 capable of emitting ultraviolet light of the desired wave length, preferably about 365 nm, are disposed in the upper part of the housing, which may e.g. be shaped as a removable cover. Said upper part also includes a detector 24 with filters located in front of it which are connected (not shown) to the screening box 25 which contains an analog/digital converter connected to a digital display.

Fig. 2 shows a section of the filter arrangement described above where 10 is the UV absorption filter, 11 is the LWP filter and 12 is the SWP filter.

A light sensitive detector 13, whose sensitivity range includes at least said wave range, is disposed behind the filters (10, 11, 12) to produce a measurement value for the extent of the Phoma and/or Fusarium infection. In front of the detector, and preferably in front of the filter arrangement mentioned above, there are provided light deflecting means arranged to focus as much of the fluorescent light from the potatoes towards the detector. This may e.g. be achieved by a suitably shaped convex lens or other curved lens 14.

The detector is a light sensitive means for producing an output signal that represents the intensity of the light received. In the preferred embodiment of the apparatus of the invention the detector is a photo diode, a blue silicon diode; in particular, being expedient. A suitable light sensitive photo resistor, such as a CdS-LDR sensor may be used as well.

Fig. 3 shows the electric circuit between the photo diode and the read-out means. The photo diode 1 emits, depending upon the light intensity, a signal to a calibration circuit 2 which applies a calibrated, analog signal to an analog/digital converter 3 arranged to produce, on the output, a digital representation of the light intensity received. The digital information is decoded by a drive circuit 4 for display on suitable read-out means, e.g. as a three-figure decimal number by means of digit displays 5, 6 and 7.

The intensity of the fluorescent light within the wave range already mentioned may vary very much from measurement to measurement, depending upon the extent of the infection, and the calibration unit 2 can therefore be adjusted, e.g. by means of the potentiometer 8 so that the final measurement result may be produced as the difference between the number shown on the digit display in a calibration position where the photo diode 1 is not irradiated and the figure resulting from irradiation of the photo diode. The analog/digital converter 3 is preferably of the type that produces a digital output signal depending upon the period of charge of a capacitor connected to the analog input signal. As it is desirable to demonstrate any fungal infection as early as possible, the low intensity of light may cause inconvenient electric noise in the circuit, but this may be offset by a suitably large time constant in connection with said capacitor. A measurement period as short as possible is, however, also of interest, and the time constant may therefore be varied, e.g. by means of the variable capacitor 9 shown in the figure or by a corresponding modification of the charge circuit of the capacitor for changing the time of integration.

In practice, a Phoma or Fusarium infection is detected as follows:

The apparatus (digital display) is set at zero by a measurement with switched-on excitation light withoutany potatoes in the magazine. The apparatus is then calibrated to a suitably selected numerical value, e.g. 100, by means of an external fluorescent standard, e.g. a piece of paper impregnated with a substance, such as aesculin or umbelliferone, that fluoresces in the relevant wave length range. The integration period (time constant) is set at the selected, suitably low value, e.g. 1 second.

Then a suitable number of potato tubers are placed in themagazine so as to cover its bottom. The tubers are selected from a quantity to be examined, are washed, and a thin layer has been peeled off so that the outer skin is essentially removed.

Then the excitation light is switched on, and the intensity-of the fluorescence caused by Phoma and/or Fusarium is reflected in the numerical value appearing on the display.

It has been found that fluorescence values above a certain limit, which varies somewhat from potato variety to potato variety and depends of course also upon the selected calibration, are in practice a reliable indication of Phoma and/or Fusarium infections, which can be confirmed by a visual examination of potatoes for visible infections on the surface and subsequent peeling and determination of fluorescence by the apparatus.

At lower values which approach this limit, the risk of a visible or impending appearance of a visible Phoma and/or Fusarium infection in the quantity is imminent, and in practice measures must be taken for early sorting of the quantity in the usual manner and sale of the non-rejected potatoes.

The numerical value measured can, once the above variations from variety to variety have been determined, be used as a criterion for the permissible storage period of the potato quantity examined because, other things being equal, low numerical values allow longer storage periods.

Accordingly, great practical interest attaches to the possibility of obtaining exact measurements in the low range where the effect of thermal noise may be specially disturbing. Therefore, it is advisable to increase the time constant from e.g. 1 second by e.g. a factor of 10, thus reducing or eliminating the impact of noise. This is done after recalibration, e.g. to a numerical value of 1000 for the external standard used. Similarly, a series of standard data may be prepared for each variety of potato for the various developmental stages of the fungus infection, so that the permissible storage period may be determined with a great measure of certainty by comparison with a sample on the basis of these data.

Similarly, standard data may be prepared for other food products once the conditions, such as wave length of excitation light and spectral data detected, have been determined under which the determination can be carried out most expediently.

Claims

P a t e n t C l a i m s - - - - - - - - - - - - - - - - - - - - - - - -

1. A method of direct, prophylactic determination of rot in food products or raw materials for food products, ch a r a c t e r i z e d by determining spectral data on a sample and comparing the spectral data thus determined with corresponding data being statistically correlated to the probability of development of rot.

2. A method according to claim 1, c h a r a c t e ri z e d in that the spectral data determined on the sample are fluorescence data induced by ultraviolet light.

3. A method according to claim 1 or 2, c h a r a c t e r i z e d in that the food products are potatoes and the potential rot is essentially caused by Phoma or Fusarium infections.

4. A method according to claim 3, c ha r a c t e r i z e d by irradiating potatoes with ultraviolet excitation light once a thin layer has been peeled off them, isolating any resulting fluorescence with a wave length of about 475-500 nm and recording fluorescence data which are compared with predetermined data for the variety of potatoes examined.

5. A method according to claim 1, c h a r a c t e r i z e d by isolating the fluorescence with a wave length of 475-500 nm by means of a combination of a UV barrier absorption filter, a low-pass filter which absorbs light with wave lengths below about 460 nm, and a high-pass filter which reflects light with wave lengths above about 500 nm.

6. An apparatus for carrying out the method according to claim 4, comprising means for emitting UV light, means for detecting the intensity of UV excited fluorescence and read-out means, c h a r a c t e r i z e d in that it comprises a magazine for potatoes to be examined, and that means are provided between the magazine and the detecting means for isolating fluorescence with a wave length of about 475-500 nm.

7. An apparatus according to claim 6, c h a r a c t e ri z e d in that the isolating means comprise a combination of a UV barrier absorption filter, a lowpass filter which absorbs light with wave lengths below about 460 nm, and a high-pass filter which reflects light with wave lengths above about 500 nm.