ICUMSA News n°8 – 1990

Message from the President

In this issue we have two letters commenting on the change of direction of the last four years. As your President I welcome this. Indeed, it was one of the reasons for setting up ICUMSA News. The dialogue should not stop here. Rather than run the risk of stopping the discussion by responding to these letters at this time, I propose that the issues raised be discussed further in the next issue and indeed at Colorado Springs if that is necessary.

It is with great regret that I advise the resignation due to ill-health of our General Secretary, Ted Whayman. He has just had a by-pass operation to rectify a heart problem. This was wholly successful, and he is now recuperating at home. In seeking to reduce his future workload he has asked to be relieved of his duties as General Secretary.

This, of course, I have accepted. in the remaining period of the 20th Session, Mr Bob McCowage, General Referee for Subject 1, will assist me as Ted’s replacement. On your behalf, I thank Ted for his contribution in the three years he has_ served our Commission and wish him a complete and speedy recovery.

To those people who have not yet decided whether to attend the Colorado Springs meeting, I would say “Don’t miss it or you will be sorry”. I met with the organising committee of the U.S. National Committee and also visited the Broadmoor venue in October. You can be assured that this is an outstanding conference venue in a beautiful city nestled at the foot of the Rockies. This will be the meeting at which we prepare the Commission for its second century. Not to be there will mean missing an opportunity to shape history. The Conference programme and extracurricular activities available to delegates will make this a great Session.

Subject 14 – Microbiology

Rapid microbiological tests

ICUMSA microbiological methods are available to check and compare the microbiological quality of sugars. Conventional methods are based on the visible growth of microorganisms. After cultivation, the organisms have grown to an extent that it is possible to count them without a microscope. In order to determine the contamination by a particular organism, special media are employed. Furthermore, the specification of pH, oxygen content and temperature help increase the selectivity for particular microorganisms. The aim is to facilitate the adaptation of a microorganism to a substrate and to shorten the lag-phase by improving the composition of the nutrient media. Generally, when using ICUMSA media with membrane filters we have incubation times of 48-72 hours.

Microbiological tests are carried out to give assurance of product quality for downstream applications, assurance that the product was manufactured in a·hygienic environment and that no pathogens are present. At the end of the production process sugar normally has a very low microorganism content. Contan1ination can occur on conveyor belts or during inadequate storage. Normally there is no multiplication of the microorganisms in the sugar. Our experience is that the major contaminants of sugar are spore-forming bacteria. Any vegetative microorganisms present are likely to be inactive or damaged. There are two important characteristics of microbiological control in sugar as far as the use of rapid methods are concerned:

1. low microorganism counts,

2. the prevailing occurrence of spores and inactive vegetative microorganisms.

In order to determine low microbial counts in sugar the microorganisms have to be concentrated by membrane filtration, so typically a 10 g sample is filtered. The use of membrane filtration for the enrichment of microorganisms from sugars with low microorganism counts simplifies the sample preparation and enhances the accuracy of detection. The determination of spores and inactive vegetative cells requires some time for reactivation which has to be taken into consideration in the specification of the cultivation time.

Rapid methods are nevertheless time saving because they establish microorganisms in a food sample directly, and they also shorten the incubation period for the cultivation of microorganisms. Rapid methods are however not readily applicable to the microbiological control of sugar because of low microorganism content and the presence of spores and damaged cells. Such samples have to be concentrated and their incubation time has to be prolonged. First and foremost, rapid methods are important for foods with a short shelf life. In order to market such products it is important to check as quickly as possible that they do not contain spoilage organisms or pathogens.

Impedance measurement and microfluorescence techniques are rapid methods permitting a reduction in incubation time.

The impedance method is based on the fact that metabolic products are concentrated in the nutrient solution during the growth of the microorganisms causing a change in the conductance of the nutrient medium. However, a detectable change in the conductance occurs only when the cell population exceeds a threshold level of about 106 microorganisms per cubic centimeter. Reproducible results obtained within a few hours are only found where higher microbiological counts exist in the form of vegetative cells. To measure low microbiological counts, e.g. yeasts in beet sugar, the sample preparation time and incubation time have to be increased. With the impedance measurement we have an automatic method permitting the checking of a large number of food samples with high microbiological counts. It is however important that the nature of the microflora is known as the nutrient media have to be chosen accordingly.

In the case of microfluorescence techniques, one involves counting microcolonies while the other involves counting organisms directly. With the microcolony fluorescence method a reduction of incubation time can also be achieved. Before the colonies have grown into macroscopic size, they are stained with a fluorescent dye on the membrane filter so that it is possible to count colonies more easily with a fluorescence microscope. However, this reduction of the incubation time is accompanied with time consun1ing microscopic work. With low microbiological counts the method becomes relatively inaccurate. Moreover, scanning a whole membrane filter for a single microorganism is very tedious. It is also possible to determine the microbiological count of a sample directly by counting under a microscope.

The direct epifluorescence filter technique is a rapid method involving filtering the sample on a membrane with the adhering bacteria and yeasts being stained with fluorescent dye and counted microscopically. The disadvantage of this method is that it is not possible to distinguish clearly between living and dead cells. This method requires accurate preparation and experience in the use of a microscope. The counting of bacteria is exceptionally tiresome and inaccurate. This method is more suitable for yeasts, but again, the expenditure of work is too great for the search for only one yeast cell. Moreover, this method cannot be used to count spores.

Another method for the direct inspection of microorganisms in sample relies upon the properties of cell contents. The adenosine triphosphate (ATP) content of a sample is determined by measuring bioluminescence. ATP exists in all living microbiological cells in a nearly constant amount. The measurement is based on a luciferine-luciferase reaction with ATP, thereby emitting light proportional to the mole content of ATP. Because of the low microbial counts, this method is not suitable for measuring the extent of sugar contamination. Moreover, it is impossible to differentiate between species or to detect spores.

Endotoxins are produced by gram-negative bacteria. It is possible to determine a low endotoxin concentration directly with the limulus-test. This method is however restricted to the detection of gram-negative bacteria. Even then it cannot be applied to actual cell counts as the endotoxins of dead cells interfere with the measurement. Since the bacteria occurring in sugar are mainly gram-positive, this test is not suitable for microbiological control in sugar factories.

There are some rapid tests which can differentiate between a gram-positive or gram-negative reaction. They have been developed mainly for the determination of pathogenic organisms.

It is possible to detect E. coli in a sample by adding 4-methylumbelliferyl-P-D-glucuronide (MUG) to the culture medium. If E. coli grows in the medium the enzyme P-D-glucuronidase produces splits the MUG. The positive reaction can be detected with fluorescence (UV-light, 366 nm). Most rapid tests for determination of pathogenic organisms have been developed to shorten the time of identification, e.g. of salmonella, listeria and coliforms. These tests are mainly suitable for process control in order to find the causes of contamination and to eliminate them. The emphasis in microbiological control of the sugar end product is on determination of product specific microorganisms. At present conventional methods are still being mainly used because rapid methods do not give comparable results for the tests required.

Letters to the Editor

I. Letter by Leon A. Anhaiser, Vice President ICUMSA

The twentieth Session of ICUMSA, scheduled for 1990 in Colorado Springs, Colorado, USA, presents both a challenge and an opportunity for the organization in its efforts to adjust to the most prolific technological period known to man. It will also be a time when ICUMSA will be” faced with important decisions about what type of organization it wants to be.

At this critical juncture in ICUMSA’s history, it is worthwhile to pause and consider some of the issues facing ICUMSA and the questions they raise:

There has been a trend toward the concept of only one official test method. At the same time, ICUMSA has indicated a willingness to implement IUPAC criteria for the evaluation and validation of methods. If more than one method meets these criteria, on what basis, then, can one method be accepted, and one be denied?

Allowing only one method implies a regulatory function. Obviously, ICUMSA is not in a position to dictate what methods a country or company can use. The exclusion of methods which qualify on their merits could result in the reduction of influence by our organization. On the other hand, having no limit on the number of methods implies a methods validation organization.

ICUMSA methods have always been accepted on the basis of the need they fulfil for the sugar industry. If ICUMSA is perceived as a legitimate and prestigious organization, then its methods are accepted by the industry. If it is not perceived this way, then its methods are not accepted, no matter what ICUMSA says.

ICUMSA already has more than one method in some instances: Two HPLC methods and one GLC method for sucrose in molasses; HPLC and enzymatic methods for raffinose; two methods for copper in white sugar; two methods for lactic acid; several tests for loss on drying; three tests for particle size.

What about too many methods? The expense and trouble of a collaborative test is not lightly undertaken. One of the functions of a Methods Review Committee could be to ascertain that a need exists for the method before the collaborative test is undertaken.

What about comparability of test methods? The General Referee and Methods Review Committee could recommend that comparative collaborative studies be made whenever this is felt to be necessary.

ICUMSA in the past has done this for a number of tests, including methods for loss on drying and chromatographic and other methods for sucrose in molasses, and so it is not a new concept.

In the case of fundamental values and reference tables, it is appropriate, on the other hand, to have only one accepted and official value.

ICUMSA meets only once every four years. At this rate, new methods of analysis require, at best, eight years before becoming official and usually twelve years is considered normal. Under this schedule, a method has a good chance of becoming obsolete before it is officially adopted.

Should ICUMSA conduct yearly evaluations of methods and evolve into an organization able to cope with the growing need for more rapid dissemination of collaborative testing results and acceptance of new methods? What is the rule of the National Committees in this scheme of methods evaluation?

The reorganisation of the numerous committees was designed to enhance and consolidate subjects in what would seem to be a more effective manner of achieving the goals of the organization. Will all of those changes result in a more responsive ICUMSA? Does ICUMSA mean one method or does it enhance all scientific methods? Colorado Springs may hold the answer.

II. Thoughts on the Work of ICUMSA

by A. Emmerich and E. Reinefeld (Braunschweig, F.R.G.)

In a few months time, the ICUMSA members will meet for the 20th Session in Colorado Springs. Behind us we have a time with many changes within our organisation. The next Session will probably be governed by discussions about the rearrangement of ICUMSA’s activities, about its advantages, but also about its disadvantages. We should not overlook, however, the activities of many referees during the recent years, which will surely form an approach to our main object to provide well-founded methods of analysis for the industry, for the trade and for other parties interested in sugar. The authors intend to contribute some thoughts to this discussion based on their knowledge of the development of ICUMSA during the last decades. The most important item will surely be the rearrangement of the Subjects and in this connection the subdivision in Product Subjects and so-called Scientific Subjects. This system will only work satisfactorily if the distribution of tasks between these two kinds of Subjects will be clearly defined and if there is a close cooperation between the Referees involved. For instance, investigations or collaborative tests about invert sugar determinations in raw sugar should be planned by the General Referee l (Raw Sugar) together with the Referee 15 (reducing sugars) consulting also the Referees 8 (GLC), 9 (HPLC) and 11 (Enzymatic methods). Only in this way superfluous duplication of work and diverging developments within ICUMSA can be avoided. We should not, as has already been done – question the re-arrangement but should fill it with life.

In order to achieve this to our opinion some sort of guidance of the working groups and their referees by a coordinating board will be necessary. Such a board already exists since the 17th Session 1978 in Montreal: the “Steering Committee”. It is, however, inactive since that time. The members of the Steering Committee – one of them should be the referee of Subject 3 (Methods Format, Collaborative Tests and Statistical Treatment of data) – should meet soon after a Session in order to review the results and to elaborate a catalogue of those tasks, which are to be regarded as urgent. In the same period the referees should be found and appointed by the President. The referees responsible for the most important tasks could be informed accordingly already within a short time after a Session. So, there is time enough to plan necessary activities in close cooperation between the Steering Committee, the referees and their associates. In this way it should be possible to start the investigations at the latest during the year following a session.

What was common practice up to now? In most cases one year and more time passed before the report of the Proceedings was available. During this time nothing happened. The activities for collaborative tests and for the discussion of the contents of the Reports to be presented were, with only a few exceptions, limited to the year immediately before the next Session. Even then, the referees were practically left alone. And – after all – they chose from the Recommendations of the preceding Session those items which corresponded to their preference and to their own ideas.

We think that tightening and guidance according to the proposals given above will lead to a more effective function of our Commission. Substantial preconditions have already been established. Since the 18th Session in Dublin (1982) criteria are available for the format of methods and for the adoption- of ICUMSA methods. From that time there are instructions about the execution and the statistical evaluation of collaborative tests. They had only to be adapted to the international agreements within IUPAC. In addition, a further problem should be thoroughly discussed once more: Should ICUMSA recommend only one method for every analytical purpose, or should more methods be admitted? There are good arguments for both alternatives. There is still some time left for the National Committees to discuss this matter. In Colorado Springs we should have a decision.

Editor: R. Piek, Klein Spanuit 9, B-3300 Tienen, December 1989 – Tel. +32 16/8 1 24 36 – Telex 222 51 – Telefax +31 1 6/82 03 17