Search form


Contact details
Traineeship proposition
Abstract Bachelor Project FBT 2018-2019: Preparation of a quantitative method for determination of the enzyme activity of amylase by fluorescence
Metagenics is responsible for the production of food supplements. It produces, among other things, Enzygest and Similase® Total. The first food supplement is a digestive supplement that works in two phases. The first phase optimizes the functioning of the stomach, the second phase optimizes the digestion and absorption in the small intestine. The second food supplement, Similase® Total, supports the digestion in a natural way; for the digestion of carbohydrates, fats and proteins. This is due to the presence of several digestive enzymes including amylase, protease, maltase, lactase, etc.
The aim of this bachelor project is to set up an analysis method to determine the enzyme activity of α-amylase in these food supplements. When purchasing these raw materials, each time a certificate is supplied with the enzyme activity. Quality control and quality assurance is extremely important, so that is why Metagenics wants to check whether the enzyme activity of amylase matches the certificate; in other words, what the producer guarantees. This analysis method is also drawn up because the samples should no longer be sent externally, because this is a big cost for the company. It is therefore the intention to be able to carry out this analysis internally. If it is possible to determine the enzyme activity in the raw materials, an attempt will be made to determine the enzyme activity in the food supplements.
The Invitrogen EnzChek® Ultra Amylase Assay kit is used to determine the enzyme activity of α- amylase. The level of enzyme activity is determined based on fluorescence measurements. The Tecan Spark® 20M Multimode Microplate Reader is used for this. The aim is to be able to determine the activity of the raw materials based on a standard series of alpha-amylase. A standard series is set up between 0 and 20 mU/mL. The fluorescence measurements of this standard series are done at a temperature of 37 ° C; what the human body temperature should represent, and incubation for 45 minutes to determine where the optimum incubation period is.
A graph can be drawn up from the fluorescence measurements in which fluorescence is displayed versus time. From this it could be deduced that an incubation period of 30 minutes is enough. However, it was not yet possible to determine the enzyme activity of amylase in the raw materials because the fluorescence values did not end up in the range of the standard series.
This could mean that the enzyme activity of amylase is much lower than claimed by the producer. Because the standard and raw material samples have not yet been externally analysed for the mutual enzyme activity, it cannot yet be concluded whether the (provisionally) drawn up analysis method for the determination of the enzyme activity using fluorescence works correctly. This should be done in the future whether or not to confirm the internal results. From the internal analysis results and those external analysis results, it can then be decided whether the method can be further optimized or whether the cause of the low fluorescence intensity of the analysed raw material samples should be further compared to that of the corresponding standard. Because of this reason, it is not yet possible to say with certainty whether the enzyme activity of amylase present in the food supplements Similase® Total and Enzygest meets the requirements.
Abstract bachelorproef 2016-2017Determination of hygroscopicity in food supplements by measuring water activity

The main purpose of this study is to determine the hygroscopicity of the food supplement products which contain probiotics and yeast. Measurement is performed by using the water activity meter in the laboratory of Metagenics Belgium. The measured data will be used to keep the Aw-value of the powder mixtures lower than 0,2 during the processing and storage.

A humid environment is a breeding ground for all sorts of microscopic organisms and can also cause physical and chemical changes which will harm the product. The lowest water activity value needed for the growth of mold is 0,6, but bacteria need an Aw-value of 0,8 to be able to grow. High humidity can also promote vitamin inactivation, lipid oxidation and enzymatic/non-enzymatic reactions in a product. Each of the named factors has a specific min/max Aw-value and promoting any of them can reduce the shelf life and the quality.

Before measuring the hygroscopicity or sorption behaviour of a product in function of time, the product is dried. The drying method can be chosen depending on the product. The temperature sensitive powder mixtures are dried by reducing the environment humidity using standard salt with 6% Relative Humidity (RH). The non-temperature sensitive powder mixtures were dried in the oven for two hours at 75°C. Thereafter, the powder gets moistened with 58%/75% RH standard salt.

As the moisture moves from the standard salt to dried product, the water activity of the product will rise and the time will be recorded by the computer.

The tested products, Probactiol Junior Chewable, Probactiol Plus and Probactiol DUO-V2, each showed two deflections on the graph, Nutrimonium-3-0 and Probactiol DUO had only one. The time (in minutes) that powders needed to reach the water activity 0,2 was respectively 18 from Aw 0,096, 31 from Aw 0,104, 127 from Aw 0,036, 24 from Aw 0,084 and 81 from Aw-value of 0,068.

Some products showed deflections on the graph after reaching a certain Aw-value. This is a specific characteristic of each product. The hygroscopicity of a powder mixture is dependent on his composition and presence of polar/non-polar substances. Nutrimonium is the most hygroscopic product due to its composition and probactiol DUO is the least hygroscopic product.

By taking into account the time a product needs to reach a certain water activity, the product can be prevented from exceeding 0,2 Aw-value. This can limit the growth of microorganisms and deactivate components in a product.

Samenvatting eindwerk 2011-2012: Ontwikkelen van een methode voor het bepalen van magnesiumglycerofosfaat met HPLC-ELSD
Magnesium is een mineraal dat voorkomt in de vorm van magnesiumglycerofosfaat in één van de belangrijkste producten van Metagenics, namelijk Metarelax. Momenteel wordt de concentratie magnesium bepaald door middel van titraties. Om een preciezere bepaling te hebben wil men nu gebruik maken van de HPLC. Hiervoor is een ELSD nodig als detector. De bedoeling is om een methode te ontwikkelen met deze nieuwe detector om de concentratie magnesium te bepalen.
Bij de ELSD wordt, met behulp van een vernevelingsgas, de mobiele fase verneveld in microdruppeltjes. De vloeistof die aanwezig is in deze druppeltjes zal verdampen en de oorspronkelijk opgeloste componenten blijven achter in het vernevelgas (perslucht of een inert gas zoals stikstofgas). De gesuspendeerde partikels komen daarna in een lichtstraal terecht. Het signaal van het verstrooide licht wordt gemeten met een fotomultiplicator
Om de methode te ontwikkelen worden telkens parameters gewijzigd tot men een methode bekomt die een goed resultaat geeft. Deze parameters zijn onder andere de kolom, het oplosmiddel, de mobiele fase en het debiet van de mobiele fase. Er is een methode gevonden die een goed resultaat geeft en die gebruik maakt van volgende instellingen:
  • Kolom: Primsep 100 5µm 250mmx4,6mm
  • Kolomtemperatuur: 35°C
  • Debiet mobiele fase: 0,5mL/minuut
  • Injectievolume staal: 10µl
  • Temperatuur ELSD: 40°C
  • Gasflow: 1,5L/minuut
  • Gain: 1
  • Looptijd: 25 minuten
Als mobiele fase wordt gebruik gemaakt van water/acetonitril/TFA (30/70/0,15) en als oplosmiddel 1mL 3N HCl dat aangelengd wordt tot 100mL met water.
In de toekomst moet de methode nog gevalideerd worden. Hiervoor zal men verschillende parameters moeten testen zoals herhaalbaarheid, nauwkeurigheid, precisie, robuustheid, …


Edward Vlietinckstraat 20
8400 Oostende


Traineeship supervisor
Stefan Daenekynt
Via Map