GB1595054A

GB1595054A - Capsules containing micro-organisms

Info

Publication number: GB1595054A
Application number: GB34261/76A
Authority: GB
Original assignee: Metal Box PLC
Current assignee: Crown Packaging UK Ltd
Priority date: 1977-11-14
Filing date: 1977-11-14
Publication date: 1981-08-05

Description

(54) IMPROVEMENTS RELATING TO CAPSULES CONTAINING MICRO ORGANISMS (71) We, METAL BOX LIMITED of Queens House, Forbury Road, Reading RG1 3JH, a British company, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: This invention relates to capsules containing micro-organisms, and to methods of treating a substance including use of such capsules. The term "capsule" as used herein means a body in which the micro-organisms are immobilized by encapsulation in a mass of a suitable material.

There has in the past been proposed by one worker a method of determining sterilizing or lethal values (F values) of micro-organisms, by putting small quantities of bacterial spores in a glass tube and embedding the glass tube in a foodstuff to be sterilized by heat treatment. In that method the spores are recovered after the sterilizing heat treatment and their survival rate measured; from this the lethal rate can be calculated. Such a method, which involves introducing the test organisms in their glass tube, into a food container, is both cumbersome and difficult, and is susceptible of inaccuracy. The use of encapsulated micro-organisms for measuring heating rates of meats indirectly has also been suggested, the vehicle for the micro-organisms being in this case a synthetic particle consisting of natural food products.

Other workers have proposed the use of bacterial spores for the indirect monitoring of thermal conditions for unspecified purposes, using a lethal micro-organism, viz. Bacillus anthracis, encapsulated in methyl methacrylate, the methacrylate capsule being made by a somewhat lengthy and inconvenient process. Yet another proposal that has been described involved studying thermal inactivation of Bacillus anthracis in so-called ultra-high temperature (UHT) systems for foodstuff sterilization, the "capsules" in this case being sphere of "Perspex" (trade mark) resin, in which the spores of the bacillus were encapsulated by a complex and time-consuming process. It was also necessary to use acetone in the treatment of the spheres during the process of recovering the spores; this would be likely to to be deleterious to the recovery of heat-injured spores and therefore harmful to the accuracy of the process.

Micro-organisms of various kinds are used, or are potentially useful, in many different kinds of process, falling basically into two categories, viz. (1) processes in which the micro-organisms act directly on a subject in order to effect changes by microbiological action, and (2) processes involving the application of heat to a substance, in which the micro-organisms are employed for the purpose of monitoring the process, the effectiveness of which is established by measuring the survival rate of the micro-organisms subjected, with the said substance, to the process itself.

An object of the present invention is to provide a form of capsule (as hereinbefore defined), which can be made easily, quickly and reliably; which can be made to a wide variety of shapes and sizes; which can retain micro-organisms in an immobilized state reliably without impairing their heat resistance, and for long periods; from which the micro-organisms are readily recoverable by simple, standard methods; and which can be employed in a wide variety of processes in both of the above-described categories.

According to the invention, in a first aspect, in a capsule (as hereinbefore defined) at least one variety of micro-organism is encapsulated in a gel of a non-acidic alginate insoluble in water. The alginate should be non-acidic to preserve the heat resistance of the mirco-organisms.

The or a said variety of encapsulated micro-organism may (by way of non-limiting example) be bacterial spores or bacterial vegetative cells, or yeast cells. The choice of micro-organism depends, of course, on the application to which the capsules are to be put.

Capsules according to the invention may be made by a simple process, an example of which will hereinafter be described, and which can include moulding the capsules to a required shape and size. Preferably for ease of manufacture, capsules according to the invention are spherical and preferably of a diameter of between 1 and 3 mm. The capsules will be difficult to handle if smaller than 1 mm in diameter, but it may for some applications be convenient to make them larger than 3 mm., and/or of a shape other than spherical.

A few examples of possible uses for capsules according to the invention, among the first of the two above-mentioned categories of process, are: as a means for preserving and transporting stock cultures, especially those which are difficult to preserve by any other means; as a means for preserving and transporting starter yeasts for use in processes such as brewing; as a means for immobilizing enzymes or cells to carry out microbiological reactions or fermentations in a wide variety of industrial or laboratory applications; as a simple means for adding active organisms to a silo, for control of the latter where the environment is such that the alginate is slowly broken down to release the micro-organisms; and use as a carrier for nitrogen-fixing bacteria in plant propagation.

As to process-monitoring applications (the second of the two process catgories aforementioned), many different kinds of substance require thermal threatment which needs to be monitored to ensure either that the correct thermal conditions exist, or that such thermal conditions exist for the required time, or both; but it is often inconvenient or impossible to achieve this by direct measurement, for example where a temperaturemeasuring device such as a thermocouple cannot be located in a substance which is in motion. One example of such a situation is the sterilization or pasteurization of foodstuffs in closed containers by application of heat in a cooker at a predetermined temperature for a period of time.

In processes for the packing of foodstuffs, there has in recent years been a substantial increase in the use of so-called ultra-high temperature (U.H.T) sterilization methods whereby flowable foodstuff, before being hermetically sealed in a container offering very high resistance to penetration by air and bacterial spores, for example a metal can, to form a sterile pack, is subjected for a compartively short period of time- a few seconds - to a temperature substantially in excess of 100"C. U.H.T. methods have been successfully utilized for liquid products such as milk and ice cream mixes and, recently, for certain viscous liquid products, though these methods of sterilizing are also applicable to particulate or other flowable solid products or to products consisting partly of solid particles and partly of liquid.

No sterilizing process is safe to use in production unless there is a means of ascertaining that it will kill all the undesired micro-organisms in the substance being treated. In so-called aseptic packing systems using U.H.T. processes, the flowable substance is treated whilst in continuous flow; this presents many problems, on this account and on account of the relatively short exposure time and high temperature in obtaining reliable evaluation of the micro-biological safety of the system.

There is a need for a method of evaluating micro-biological safety in U.H.T. processes which will be simple and reliable, and which can be applied not only to liquid products but also to semi-solid products which are flowable but which are in a heterogeneous and/or particulate form.

In addition, in more conventional packing processes where the product, such as a foodstuff, requires to be pasteurized or sterilized by application of heat for a predetermined time after the product has been sealed into the container, it is highly desirable to ensure that all bacteria such as may cause spoilage of the product, or food poisoning, are destroyed.

The method of ensuring this that is commonly used is to apply the heat for a period very substantially longer than that known to be sufficient for destroying any micro-organisms that might be present. Thus for example in foodstuffs, where for instance all traces of live spores of the most heat-resistant strain of Clostridium botulinum must be destroyed, the heating time is usually several times that needed to attain a lethal value Fo of 3.0, which is the number of minutes required at 250"F (121"C) to reduce the population of this strain of C. botulinum by factor of 1012.

Extending the heating time in this way is found in practice to be reliable, but it can have undesirable effects on the flavour or other characteristics of the products. It would be preferable to heat. for a time only just long enough to ensure destruction of all undesired micro-organisms, with perhaps a relatively small additional heating time to allow some reasonable margin of safety. To achieve this with safety, however, a reliable method of monitoring the process is essential. This calls either for measurement of the temperature in the product throughout the heating process or for some other method of ensuring that the heat has had the required effect. Direct temperature measurement is impracticable as mentioned above, where continuous cookers are used and the product is sealed into the container.

Preferably, for process-monitoring applications, the micro-organism (for at least one of said micro-organisms) is in the form of bacterial spores. Suitable spores include those of Bacillus stearothermophilus, Clostridium sporogenes and Bacillus subtilis; it should however be pointed out that some species or strains may be found by experiment to be more suitable than others for different applications, whilst a micro-organism eminently suitable in one application may be found to be entirely unsuitable for another.

According to the invention, in a second aspect, a method of treating a substance at elevated temperature, by heating the substance for a predetermined time, includes monitoring the treatment by the steps of: heating at least one capsule, according to the invention, in said first aspect thereof, under substantially identical temperature conditions and for the same time as, said substance, the capsule or capsules being disposed, during said heating process within an environment such that heat transfer to the capsule or capsules is no greater than that to any point in said substance; recovering the capsule or capsules; recovering the micro-organisms therefrom; and assessing the survival rate of the micro-organisms so recovered.

The assessment of survival rate may be performed in any suitable known manner, typically by incubation under strictly controlled conditions followed by counting the survivors, if any, by known techniques.

Where the substance is flowable, methods according to the invention may be employed whilst the substance is in continuous flow, before being introduced into the container, and is therefore suitable for use in continuous-flow U.H.T. sterilizing processes as well as for continuous-flow processes in chemical manufacture. To this end, the capsule is of an alginate capable of withstanding without deterioration, immersion in a medium at a temperature of up to 1500C. However, given this capability such a capsule may also be employed with processes in which the medium or substance in which it is immersed is contained in a container during heating thereof, not being in continuous flow.

Since the capsules according to the invention may in many applications be numerous and/or relatively small, it is convenient to provide a suitable means for holding them, either for transport or when the capsules are in actual use. Accordingly, in a third aspect the invention provides, in combination, at least one capsule according to the invention and a holder containing said at least one capsule. One example of such a capsule holder is a light frame of chemically-inert material, such as a plastics material, which can be slipped into a can (for use in a pasteurisation-monitoring process) before the can is filled with product, and is easily removed again for recovery of the capsule or capsules. Since its is desirable for the test micro-organisms to be at the point in the container least likely to be fully heat-treated, for example at the centre, such a frame provides a ready means for ensuring that they are always in the desired position. In another example, the holder is a solid block of material adapted to substitute for a foodstuff or other product in a test container. This block is made of material having heat-transfer charcteristics no better than that of the product which it replaces, so that survival of no test micro-organisms after the heating process will reliably indicate effectiveness of the latter to destroy indigenous microorganisms in the product in other similar containers subjected to the same process. Use of a solid block has the advantage that its recovery from an otherwise empty container is cleaner and easier than removal of either loose capsules, or capsules carried in a frame, from a container which also contains the product.

A capsule according to the invention may, for protection from hostile environments, be encased or enveloped in, or coated by, a casing, envelope or coating (as the case may be) having protective qualities against such environments, for example, highly acid environments.

Suitable dyes may be added to the alginate during manufacture of the capsules, to give a simple colour code by which individual capsules or batches of capsules can be readily identified.

Any process involving the handling of micro-organisms can be potentially dangerous or harmful if any micro-organisms go astray. For this reason, capsules according to the invention should, in general, only be used where suitable safeguards are provided. This is particularly true in applications in which the capsules are used for monitoring a process involving any kind of foodstuff, beverage or medicine. Preferably such monitoring should, for this reason, not ttike place as a matter of routine during actual production runs, unless fully and closely supervised at all times by properly qualified staff trained in the use of microbiological techniques. Where, however, personnel having such training are available to use the capsules according to the invention (in whatever application) the latter may be used with advantage.

Various embodiments of the invention will now be described, by way of example only, with reference to the accompanying diagrammatic drawings, in which: Figure 1 illustrates a preferred method of making capsules according to the invention; Figure 2 shows an alternative method of making capsules according to the invention; Figure 3 is a section through a capsule having a protective coating; Figure 4 is a diagram illustrating methods according to the invention applied to a continuous-flow heating process; Figure 5 is a graph illustrating survival (plotted on a logarithmic scale) of Bacillus stearothermophilus in capsules according to the invention subjected under test conditions to temperatures such as would obtain in U.H.T. heating processes; Figure 6 is a diagram illustrating the passage of filled food cans and test cans through a sterilising cooker during the setting-up of a food canning process; Figure 7 is a graph illustrating survival (plotted on a logarithmic scale) of Bacillus stearothermophilus in capsules according to the invention, subjected to heat treatment with meat paste; Figure 8 is a transverse sectional elevation of a test can, in one form suitable for use in the process illustrated in Figure 6, and having a holder therein containing capsules according to the invention, taken on the line VIII-VIII in Figure 9; Figure 9 is a sectional plan view taken on the line IX-IX in Figure 8; and Figure 10 is a transverse sectional elevation of a test can, in another form suitable for use in the process illustrated in Figure 6, and containing a holder in the form of a solid block, having therein capsules according to the invention.

Referring to Figure 1, a capsule 10 comprises at least one variety of micro-organism encapsulated in a solid alginate, so as to immobilize the spores, In the example to be described, the capsule 10 is one of a plurality of spherical capsules, each consisting of calcium alginate in which the micro-organisms comprise bacterial spores of a single variety or species. It should however be understood that capsules such as those described herein may be used for the immobilization by encapsulation of micro-organisms other than bacterial spores.

The species may for example be one of the following: (a) Bacillus stearothermophilus, Strain TH24, held under acquisition number NCD01096 in the culture collection of the National Collection of Dairy Organisms at the National Institute for Research in Dairying, Shinfield, Reading, Berkshire.

(b) Clostridium sporogenes, Strain PA3679, held under acquisition number 8053 in the culture collection of the National Collection of Industrial Bacteria at the Torry Research Station of the Department of Scientific and Industrial Research, P.O. Box 31, 135 Abbey Road, Aberdeen, Scotland.

(c) Bacillus subtilis, Strain 786, held in the same culture collection as C. sporogenes above at the Torry Research Station.

The capsules 10 have been made by the following method: The two bacillus species and the clostridium species were grown by known methods in suitable nutrients, each species being grown separately. The thermophile, B. stearothermophilus was incubated at 37"C. On completion of sporulation, spores were harvested -and washed thoroughly before being suspended in distilled water.

Capsules 10 were then made in an atmosphere made sterile by laminar air flow, one batch of capsules being made for each of the above-mentioned species. A measured quantity of the live spore suspension was added to, and thoroughly mixed with, a sterile aqueous solution of sodium alginate of the required strength (typically 2-4% of a buffered aqueous dispersion), and of carefully standardised viscosity. The alginate solution was contained, in a known quantity, in a bottle containing also glass beads. The resulting mixture of spores and sodium alginate dispersion was then carefully added, drop by drop, to a sterile 2% aqueous solution of calcium chloride (CaC12), 11, Figure 1, by means of a sterile syringe having a stainless steel needle 12 the tip 13 of which was cut in a plane transverse to the needle axis. The needle 12 was placed vertically over the CaCl2 solution 11 to disperse the drops 14 of the mixture into the solution. Each drop 14 gelled immediately upon falling into the CaC12, to form a spherical capsule 10 containing a random distribution of spores. The capsules were left in the CaC12 solution for at least 18 hours and up to 48 hours, depending on capsule size. During this time the sodium alginate was converted to calcium alginate (which is insoluble in water and non-acidic) and became hardened. In addition it is thought that the spores absorb from the calcium ions from the gel; a high calcium content like a non-acidic environment, is associated with high reproducible heat resistance, so that upon being subjected to heat the spores will not be destroyed too quickly.

The capsules were subsequently washed free of CaCl2 solution with sterile distilled water, and stored at 4"C in a moist condition in sealed containers.

Capsule size, when the capsules are made by a method such as the above, using drop-by-drop dispersing of a mixture of spores and alginate into a reagent such as to cause the drop to gel, is determined by the external diameter of the needle 12. For example, needles of 26, 30 and 31 gauge produce drops which gel into capsules of approximately 2.2, 2.0 and 1.6mm respectively. The diameter of the spherical capsule 10 is in the range 1 to 3 mm.

When recovery of spores (alive or dead) from three capsules 10 is required, the latter are first dissolved in a solution of citrate, preferably a 1% aqueous solution of sodium potassium citrate. The spores are recovered from the suspension of spores in the citrate solution, using a suitable known nutrient for recovery of each of the micro-organism species. The nutrient is then, in known manner, kept for a measured period of time under controlled conditions, and the number of surviving spores is then counted by any suitable known method. An alternative to citrate solution is a solution of sodium hexametaphosphate in water, containing glass beads to assist agitation.

The best concentration of soluble alginate for the initial aqueous dispersion is found for most purposes to be 3%. Sodium alginate sold under the trade mark "Manucol DM" is found to give capsules having satisfactory mechanical properties for use with processes involving heat, and for moulding to suitable shapes.

It has been found that viable spores can, in suitable nutrient substrates, grow out from the capsules. An alternative method of assessing survival rates is therefore to heat the capsules under controlled conditions such as to allow survival of a small (and known) proportion of the surviving spore population, and then to allow these few remaining survivors to grow out from the capsule, thus avoiding prior dissolution of the alginate.

The number of spores that can be recovered is exemplified in Table 1, which shows the spore recovery rate for two of the above-mentioned micro-organism species, with those for a third species included by way of comparison, in a typical set of experiments carried out on capsules made by the process described above. The spores were not subjected to heating before the spores were recovered but were maintained at constant temperature such that no germination to the vegetative state could take place, and therefore no increase in cell numbers.

TABLE 1 Organism Estimated original Measured number of number of spores spores recovered per capsule per capsule When After prepared 1 year at 4"C C. sporogenes (PA 3679) 105 - 106 1 x 105 3 x 105 B. stearothermophilus ) Strain TH 24) ) 105 - 106 106 106 Strain "X" 105 2 x 105 2 x 105 The figures given for C. sporogenes reflect inaccuracy in measuring method, but we infer from them that no decrease in its population took place.

The third micro-organism in Table 1, strain "X", is an unidentified facultative thermophilic anaerobe which was found to grow from spores after prolonged incubation at 30"C but rapidly on incubation at 45-550C.

It should be noted that repeated tests show Bacillus stearothermophilus to have a particularly consistent recovery pattern from capsules made as described above with reference to Figure 1.

Capsules according to the invention can be of any desired shape, for example the cylindrical shape shown in Figure 2. However, it will be appreciated that the spherical shape of the capsule 10 is particularly easy to achieve using a method such as has already been described hereinbefore with reference to Figure 1. Cylindrical capsules could be made by, for example, reacting the spore-bearing sodium alginate with the calcium chloride in a vertical (e.g. a cylindrical tubular) reaction vessel of suitable cross-sectional size, extruding the resulting gel from such vessel through an orifice of any desired shape, and cutting the capsule from the extrudate.

Alternatively, the capsules may be formed to any desired moulded shape by moulding the gel in a mould, before the hardening process (mentioned in the description earlier herein with reference to Figure 1) is completed. The moulded capsules will then be returned to the calcium chloride solution to complete the hardening of the capsules.

Some environments to which the capsules of the present invention may be required to be subjected can be hostile to the alginate of which they are made, since alginates are not chemically inert. They may for example be attacked by any metal chelators present in a product in which they are immersed, or by some acid environments. In addition, some additives in food products, for example polyphosphates, affect the heat resistance of the alginate capsules. To protect the capsules from hostile environments, the alginate may be modified, for example by addition of magnesium carbonate. The capsules may alternatively be encased or enveloped in a shell, or coated with a coating of a compatible material having suitable protective qualities. Figure 3 shows a calcium alginate capsule 10 having a vitreous outer shell or coating 60 formed by dipping in an aqueous slip of glazing silica. Such a shell is very thin and can readily be cracked by, for example, agitating the capsule with glass beads in the vessel containing the citrate solution in which the calcium alginate is to be dissolved for recovery of the spores therefrom.

Referring now to Figure 4, a flowable substance passes through a conduit 50, in continuous flow, and is subjected to heat applied by a suitable medium such as steam in a heating jacket or oven 51 through which the conduit 50 extends. The rate of flow of the substance is controlled so as to control the rate at which it is heated. Before the substance reaches the heating zone, a capsule 10, of the kind described with reference to Figure 1, is introduced into the flowing substance (as indicated at 52). The capsule is recovered after the heating process, and to this end Figure 4 shows, purely diagrammatically, a sump 53 for collection of the capsules, the sump having a spillway 54 to permit the substance to continue its flow without interruption. The flowable substance may be liquid or a mixture of liquid and flowable solid, or just flowable solid (for example a solid in granular or other particulate form, the particles of which are substantially lighter than the capsules 10).

Capsules 10 may be passed at known intervals through the apparatus, to provide what amounts to a form of batch testing. After removal, they are dissolved in citrate solution and the spores recovered, incubated and counted in the manner already described herein.

Continuous-flow heating processes such as that exemplified in Figure 4 may be employed, in various chemical processes under suitably-qualified supervision, in the monitoring of or during setting-up procedures, by suitably-trained staff, preparatory to a process for food or drug manufacture, for example an ultra-high temperature sterilization process for milk or other foodstuffs. In such a process, after being sterilized, the foodstuff is aseptically packed in containers by known techniques.

In another type of process (not illustrated) capsules 10, of suitable shapes and sizes, are made by the process described with reference to Figure 1, or by any other suitable process modified therefrom, so as to contain micro-organisms for the purpose of transporting the latter so that upon subsequent release of the micro-organisms from the capsules, the micro-organisms can perform a positive function. This is in contrast to the monitoring of a process such as that described with reference to Figure 4, in which the function of the micro-organisms is to be destroyed during transport and to be subsequently counted.

In one example, nitrogen-fixing bacteria are encapsulated in the manner described with reference to Figure 1 and the resulting capsules are placed in soil adjacent to plant seeds for the purpose of inoculating the soil by slow release of the bacteria into the soil upon degeneration of the alginate by its chemical environment.

In another example, starter cultures (yeasts) for fermented products such as beer are encapsulated in the alginate for the purpose of transporting them, the yeast cultures being released by dissolving the alginate at the brewery so as to enable fermentation to take place.

In a further example, micro-organisms of any type capable of surviving the encapsulation process (and particularly heat-resistant organisms) are made into the alginate capsules as a general and convenient means for enabling them to be stored for long periods and transported from place to place.

Whilst capsules as described herein can be used in process evaluation, there are substantial risks in using them for any such purpose where the micro-organisms concerned do not constitute an integer of the actual process being evaluated but are, as in the example described with respect to Figure 4, provided only for that part of the process which is the evaluating part thereof. Where suitably tranined or qualified staff are supervising the process with proper safeguards, the risk of capsules and/or the micro-organisms therefrom going astray can be controlled. However, if for example in a food, beverage or pharmaceutical process such safeguards and supervision are lacking, there is a danger of the product becoming contaminated or spoilt. For this reason, in practice the use of the capsules for process evaluation is of interest at the present time principally in connection with the setting up of a process. Examples include food canning, U.H.T. milk sterilization, aseptic packing of liquid or solid foodstuffs and the processes for manufacture of containers for foodstuffs (for instance the heat-curing of protective lacquers on the interior of a beer can). The description which follows with reference to Figures 5 to 10 should be read bearing in mind that proper supervision and safeguards, and any relevant statutes and official regulations must be observed at all times without exception during the performance of the process, or use of the capsules and associated equipment, to be described.

Referring therefore to Figure 6, a method of treating a substance in the form of a solid foodstuff, at elevated temperatures, includes introducing a capsule 10, made as described with reference to Figure 1 and preferably by a suitable carrying member, into a conventional metal can 21, after the can has been filled with the foodstuff but before the can is closed. The can is closed conventionally by seaming an end closure member over the open end of the can to effect hermetic sealing of the latter. The sealed can is one of several marked on their exterior with special markings (for instance by being painted in a bright colour) to identify them as being the test cans. In other respects these test cans and their contents are identical with cans 23 containing food, Figure 6, which are filled and closed in the usual way by the canner and passed, by a conveyor indicated diagrammatically at 24, through a cooker in the form of an autoclave 25 in which they are subjected to heating, by means not shown, at a temperature of just under 100"C to pasteurize the foodstuff. The time for which the cans of food are subjected to this heating is predetermined by appropriate control of the speed of the conveyor 24. The sealed test cans 21 are interposed in the line of cans 23 at appropriate intervals. After being passed through the autoclave 25, each test can 21 is removed and opened, the foodstuff therein being discarded and the capsule 10 recovered before being dissolved in a citrate solution and the spores recovered from the latter as already described. The surviving spores are counted. Absence of any survivors indicates that all the test spores originally in the capsule 10 were destroyed in the heating process; but, as discussed hereinafter, the presence of some survivors may depending on their number - indicate that, whilst not all of the test spores were destroyed, the food has still been adequately processed.

In a modified form of the method described with reference to Figure 6, the temperature of the autoclave is substantially greater than 100"C, and the time for which the cans of food are subjected to heating is reduced accordingly. This is then a "high-temperature" process.

Figure 7 illustrates a typical effect of heat treatment on cans of mixed meat paste, such as may be expected from a typical process as just described using the capsules 10, the spores used being those of Bacillus stearothermophilus, with heating at 1210C (250"F). At this temperature the F0 or horizontal axis is a direct measure of the process time in minutes.

When the lethal value Fro is 3, there are approximately 103 5 survivors among the test spores.

On this basis, a count of less than 103 5 survivors, where the initial number in the capsule was 106, indicates a lethal rate more than sufficient to destroy Clostridium botulinum. Thus as far as destruction of this latter toxic, indigenous micro-organism is concerned, the heating process will have been satisfactory if terminated immediately thereafter and without all the test spores having been destroyed.

However, it is found that the heat resistance of spores of particular species of micro-organism is reduced by inhibitors of various kinds, such as polyphosphates, which are present in many preserved foods and were present in the meat paste used in experimental work on which Figure 7 is based. By way of illustration, Table II below shows the number of survivors per capsule at 2500F (121"C) when capsules of Bacillus stearothermophilis were heated in capillary tubes in samples of the same meat paste diluted with one part of water.

TABLE II Time at 250"F Survivors per (Fo) (Minutes) capsule 0 105 to 106 10 2.0 to 2.3 x 103 20 None From Table II it will be seen that the time taken to reduce the live spores to zero is substantially longer than in the undiluted meat paste. This is but one illustration of the fact, emphasised earlier herein, that before a particular micro-organism is used in a method according to the invention, its suitability for the particular desired application must be determined by experiment. For example, we have shown that the number of Bacillus stearothermophilis spores surviving, when used in calcium alginate capsules in cans of processed peas, remains constant for a period longer than the total time for conventional heating processes for this product. Although this micro-organism if thus clearly unsuitable for use with canned processed peas, a less resistant type of micro-organism, such as Bacillus subtilis, will be suitable. Processed peas do not contain the inhibitors mentioned above, so where these latter are present, a test micro-organism having relatively high resistance to heat is chosen.

Still with reference to Figure 6, in an alternative method the test cans 21, after being removed from the conveyor 24, are not opened but are allowed to incubate so as to allow any surviving spores to multiply throughout the contents of the can. If they eventually burst, the blown can indicates that not all the spores were destroyed by heating. If at the same time one of the ordinary cans 23 is incubated as a control under the same conditions and its contents show no signs of spoilage by the time that the test can 21 bursts, there is a very high possibility that the control can 23 is sterile, and thus that the heating process is sufficiently effective. This method can be carried out at the production line, or in the laboratory to establish the parameters for a heating process prior to commencing production. Conventionally a large number of cans of food, incubated for several weeks, are used for establishing the parameters before a heat pasteurization or sterilization process is undertaken in commercial production. Even then the results can be relatively unreliable.

The method just described, using encapsulated micro-organisms, not only shortens the time required for the test but also enables the process parameters to be established with greater certainty. Table III, below, illustrates the results of two such tests with heating at 250"F (121"C), on cans of two different food products, using in each case a test can inoculated with Clostridium sporogenes and an uninoculated control can. In both tests, each can was incubated for the period indicated at 370C.

TABLE III Product Process time Inoculated can Control can (min.) Time Result Time Result Processed peas 1.3 3-4 days Blown 11 days Sound Stewed Steak in gravy 4.0 14 days Sound 14 days Sound From Table III it will be seen that a process time of 1.3 minutes at 1210C is too short for safety in processed peas, whilst 4 minutes at the same temperature for the stewed steak is safe. The normal process time at this temperature to give protection against Clostridium botulinum is, as already indicated, 3 minutes; but it will be understood that this is not the only potentially toxic or spoilage-inducing micro-organism that must be protected against, and therefore the need has existed for simple methods of testing such as that now exemplified in Table III.

The following remarks apply to a continuous-flow process as exemplified by Figure 4, or to a sterilization process for products already packed in containers, as already mentioned with reference to Figures 6 and 7.

Referring now to Figure 5, this Figure represents actual test results and shows the survival pattern for spores of Bacillus stearothermophilus which were heated in the laboratory, in a buffer solution in capillary tubes at 127.50C and 135"C. The spores were in encapsulated form as described with reference to Figure 1, and there were initially about 1.5 x 106 spores per capsule for the test at the lower temperature and about 1 x 106 for that at the higher temperature. Further results, this time in terms of survivors per capsule after an exposure time of 14 seconds, are given in Table IV below. Each result is averaged from the survivors counted on five capsules carrying an initial 0.5 x 106 to 1 x 106 spores of Bacillus stearothermophilus per capsule.

TABLE IV Process temperature Exposure time (sec.) Survivors per capsule 138"C 14 3.4 x 104 " 9.4 x 104 it 11 1.3 x 105 139"C 14 None It will at once be seen from the foregoing that a relatively small temperature change produces a very substantial change in the spore survival pattern at U.H.T. process temperatures for Bacillus stearothermophilus. It has been found that survival patterns for this bacillus are quite consistently repeatable; and hence at U.H.T. process temperatures these spores provide a remarkably accurate means for indirectly measuring temperature, when they are encapsulated and used according to the methods already described.

Characteristics showing the survival patterns for particular micro-organisms at different temperatures, such as those shown in Figure 5, can be used in the performance of heating processes at U.H.T. process temperatures as for processes at lower temperatures. Thus the survival figure determined after recovery of the capsules 10, Figure 4, shows the thermal conditions to which these capsules were subjected by being heated while passing along the conduit 50.

To prevent contamination by the test micro-organisms of the milk or other flowable substance under test and intended for packing, apparatus as described above with reference to Figure 4 may be modified so that the conduit 50 is parallel with a main conduit 55 and constitutes a test branch into which the flowable substance can be diverted by means of a valve 56 when required. Both branches 50 and 55 are of identical length and cross-section in the heating zone, so that flow and thermal conditions in the branch 50 are substantially identical with those in the conduit 55. With this arrangement the spillway 54 is not necessary, and the sump or container 53 can be removable with the heated capsules 10.

When capsules carrying micro-organisms are immersed in a substance, then, unless the purpose for which they are to be used requies that they be left in place, e.g. to incubate any surviving micro-organisms in order to affect the substance in some desired way, they need not be in direct contact with the substance. With some products, indeed, a capsule buried in a mass of a substance may be difficult to find, particularly if the product is a dense solid.

Removal of the capsule may with some products by unpleasant for the operator and will almost certainly be messy. This is undesirable under laboratory conditions, especially since it can lead to the twin dangers of (a) a substance contaminated with micro-organisms being spread around, and (b) the operators inadvertently or deliberately licking their fingers, thus taking in micro-organisms. If the substance is a solid requiring cutting, there is also a danger of damage to the capsule by the cutting implement. Figures 8 to 10 illustrate two embodiments in which these disadvantages are reduced or avoided.

Referring to Figures 8 and 9, a capsule holder in one form consists of a frame in the form of a spider 70, moulded in one piece from a plastics material which is chemically inert both to the capsules and to the product 20 contained within the metal can 21. The spider 70 comprises a central core 73 having a pocket 74 and radial fins 75 adapted to fit easily in the can 21 so that the holder 70 can readily be removed from the latter.

The capsules 10 are inserted in the pocket 74, so that when the spider 70 is fitted in the can, the capsules lie in the centre of the can. A stopper, not shown, may be fitted in the top of the pocket over the capsules. The spider 70 is inserted in the can 21 before the latter is filled with the product, 20, after which a can end 76 is seamed to the can in the usual way and the resulting pack is subjected, as a test pack in a manner as hereinbefore described, to heating. When the pack is opened after the heating process, the spider 70 is easily removed for recovery of the capsules 10 therefrom.

In the further embodiment shown in Figure 10, the test can 21 contains no product at all.

The can 21 is similar to the cans which do contain product. Before the can end 76 is seamed on to the test can 21, a capsule holder in the form of a solid cylindrical block 77 is inserted into the can. The block 77 is of a material having a known heat transfer coefficient no greater than that of the product in the other cans, for example a suitable plastics material, and is of such dimensions as to fit snugly but removably in the can. The material of the block 77 is such as to be chemically inert to the capsules 10 when heated, and is formed for example by moulding. To improve its heat transfer characteristics, the block 77 may have cavities (not shown) therein; such cavities may comprise holes drilled through its outer surface, or for example the block 77 may be formed of a foamed plastics material. The cavities, if present, may contain air, water or any other suitable medium such as to give the required heat transfer characteristics with respect to the product in the other cans.

In both of the embodiments shown in Figures 8-10, a basic criterion for location of capsules and design and choice of material of the capsule holder, is that during the heating process the heat transfer (i.e. the rite of heat transfer and the total amount of heat transferred) to the capsule or capsules l() is no greater than that to any point in the product 20 (if any) in the test can and no greater than that to the product in any "normal" cans being tested at the same time.

The block 77 has a pocket 78 for containing the capsules 10, and a stopper 79 to entrap the capsules in the pocket in the centre of the can 21. The end 76 is seamed on and the test can is then subjected to heating; upon subsequent opening of the can, the block 77 is removed and the capsules 10 extracted from it.

Both the spider 70, Figures 8 and 9, and the block 77, Figure 10, may be so made that, after being sterilized, they can be used over and over again.

Whilst the capsule holders 70 and 77 are shown as containing loose capsules, it will be understood that capsules may be moulded into the holder or otherwise suitably united with it. For example one or more capsules may be adhesively bonded to any internal or external surface of the holder.

Where a plurality of capsules are placed in different positions in a container for any purpose, or attached to or inserted into different parts of a capsule holder, they may advantageously be colour-coded so as to be readily distinguishable from each other after removal. Such colour coding may, in process-monitoring applications, also be used to identify capsules from two or more different test packs, or capsules used at different times in a continuous-flow process. Colour coding can be achieved by introducing a suitable chemically and biological compatible dye or pigment into the alginate suspension or calcium chloride solution used in the manufacture of the calcium alginate capsules, made in the manner described above.

It will be understood that in any one capsule as described herein, there may be more than one micro-organism species. This is useful where, for example, it is required to establish the parameters beforehand for a particular process. On recovery, some capsules are incubated in a nutrient suitable for one species and unsuitable for the others, other capsules in a nutrient suitable for another of the species, and so on. In this way the different species can be isolated from each other for subsequent counting of survivors.

WHAT WE CLAIM IS: 1. A capsule (as hereinbefore defined) wherein at least one variety of micro-organism is encapsulated in a gel of non-acidic alginate insoluble in water.

2. A capsule according to Claim 1, wherein the alginate is calcium alginate.

3. A capsule according to Claim 1 or Claim 2 having, as the or a said variety of encapsulated micro-organism, bacterial spores or bacterial vegetative cells.

4. A capsule according to any one of the preceding claims, having, as the or a said variety of encapsulated micro-organism, yeast cells.

5. A capsule (as hereinbefore defined) being a capsule substantially as hereinbefore described with reference to, and as illustrated in, any one of Figures 1 to 3 of the accompanying drawings.

6. A capsule according to Claim 5, being a capsule made by a method substantially as hereinbefore described with reference to, and as illustrated in, Figure 1 of the accompanying drawings.

7. A capsule according to any one of Claims 1 to 5, made by moulding the micro-organism-containing alginate to a required shape.

8. In combination, at least one capsule according to any one of the preceding claims and a holder containing said at least one capsule, the holder being constructed, arranged and adapted to be used substantially as hereinbefore described with reference to, and as illustrated in, Figures 8 and 9 or Figure 10 of the accompanying drawings.

9. A method of treating a substance at elevated temperature by heating the substance for a predetermined time, wherein the method includes monitoring the treatment by the steps of: heating at least one capsule, according to any one of Claims 1 to 7, under substantially identical temperature conditions, and for the same time as, said substance, the capsule or capsules being disposed, during said heating process within an environment such that heat transfer to the capsule, or capsules is no greater than that to any point in said substance; recovering the capsule or capsules; recovering the micro-organisms therefrom;

**WARNING** end of DESC field may overlap start of CLMS **.

Claims

**WARNING** start of CLMS field may overlap end of DESC **. and is of such dimensions as to fit snugly but removably in the can. The material of the block 77 is such as to be chemically inert to the capsules 10 when heated, and is formed for example by moulding. To improve its heat transfer characteristics, the block 77 may have cavities (not shown) therein; such cavities may comprise holes drilled through its outer surface, or for example the block 77 may be formed of a foamed plastics material. The cavities, if present, may contain air, water or any other suitable medium such as to give the required heat transfer characteristics with respect to the product in the other cans. In both of the embodiments shown in Figures 8-10, a basic criterion for location of capsules and design and choice of material of the capsule holder, is that during the heating process the heat transfer (i.e. the rite of heat transfer and the total amount of heat transferred) to the capsule or capsules l() is no greater than that to any point in the product 20 (if any) in the test can and no greater than that to the product in any "normal" cans being tested at the same time. The block 77 has a pocket 78 for containing the capsules 10, and a stopper 79 to entrap the capsules in the pocket in the centre of the can 21. The end 76 is seamed on and the test can is then subjected to heating; upon subsequent opening of the can, the block 77 is removed and the capsules 10 extracted from it. Both the spider 70, Figures 8 and 9, and the block 77, Figure 10, may be so made that, after being sterilized, they can be used over and over again. Whilst the capsule holders 70 and 77 are shown as containing loose capsules, it will be understood that capsules may be moulded into the holder or otherwise suitably united with it. For example one or more capsules may be adhesively bonded to any internal or external surface of the holder. Where a plurality of capsules are placed in different positions in a container for any purpose, or attached to or inserted into different parts of a capsule holder, they may advantageously be colour-coded so as to be readily distinguishable from each other after removal. Such colour coding may, in process-monitoring applications, also be used to identify capsules from two or more different test packs, or capsules used at different times in a continuous-flow process. Colour coding can be achieved by introducing a suitable chemically and biological compatible dye or pigment into the alginate suspension or calcium chloride solution used in the manufacture of the calcium alginate capsules, made in the manner described above. It will be understood that in any one capsule as described herein, there may be more than one micro-organism species. This is useful where, for example, it is required to establish the parameters beforehand for a particular process. On recovery, some capsules are incubated in a nutrient suitable for one species and unsuitable for the others, other capsules in a nutrient suitable for another of the species, and so on. In this way the different species can be isolated from each other for subsequent counting of survivors. WHAT WE CLAIM IS:

1. A capsule (as hereinbefore defined) wherein at least one variety of micro-organism is encapsulated in a gel of non-acidic alginate insoluble in water.

2. A capsule according to Claim 1, wherein the alginate is calcium alginate.

and assessing the survival rate of the micro-organisms so recovered.

10. A method according to Claim 9 for treating a flowable substance, the method being substantially as hereinbefore described with reference to, and as illustrated in, Figure 4 of the accompanying drawings.

11. A method according to Claim 9 for treating a substance contained in a succession of containers, the method being substantially as hereinbefore described with reference to, and as illustrated in, Figure 6 of the accompanying drawings.

12. A test container for use in a method according to Claim 9 or Claim 11, the test container including a combination according to Claim 8.