BS ISO 13082:2011 pdf free download Milk and milk products — Determination of the lipase activity of pregastric lipase preparation
1 Scope
This International Standard specifies a method for the determination of the lipase activity. lt is intended for thepreparation of pregastric lipase and rennet paste, both of animal origin.
NOTE No reference method was used to check this method as no stable standard can be found. On the other hand.a reference method can be omitted as the substrate is reproducible and well defined.
2Terms and definitions
For the purposes of this document, the following terms and definitions apply.2.1
international lipase unitILU
amount of lipase activity that releases butanoic acid, also known as butyric acid, at a rate of 1,25 umol/minunder specified conditions
NOTE1 Lipase activity is expressed either in international lipase units (ILU) per gram of product or IlLu per militre ofproduct.
NOTE2 The definition is based on the direct consumption of titrant while not considering that a small molar fraction ofthe butyric acid (4 %) is not dissociated and thus cannot be titrated.As such, that creates a small error in the definition.
3Principle
Triglyceride esters are hydrolysed by lipase. The free fatty acids (as butyric acid) released from the substratetributyrin are titrated in a pH-stat with sodium hydroxide. The amount of sodium hydroxide consumed within adefined period is used to calculate the activity in lLU per millilitre or lLU per gram.
Due to the non-existence of a reference standard, it is recommended that a control (known) sample beincluded in the test.
4Reagents
During the analysis,unless otherwise stated, use only reagents of recognized analytical grade and distilledwater or demineralized water or water of equivalent purity.
The brand of chemicals can affect the result.Therefore, before using a brand other than the one mentioned,verify whether it gives the same result.
4.1 Tributyrin (glycerintributyrate or glyceryl tributyrate), e.g.Merck No.1.01958.01001) or similar.4.2Sodium caseinate, e.g. Sigma C86541) or similar.
4.3Lecithin,from soya bean, e.g.BDH Prod. 298631) or similar.
4.4 Liquid paraffin. Use paraffin which is highly liquid (or similar light mineral oil), e.g. MerckNo.7174.10001), or similar.
4.5Soda lime granules [Carbosorb1′)],e.g,BDH no 3311041) or similar.
4.6 Sodium hydroxide solution, c(NaOH) 0,025 mol, which can either be purchased or be prepared asfollows.
Using a pipette (5.1), add 25,00 ml of 1 moll sodium hydroxide with an accurately known titre into a container.Dilute with water to 1 000 ml.
The 0,025 moll NaOH solution can be kept in a closed container, protected against carbon dioxide in the airby use of a CO2 trap with soda lime (4.5) at room temperature for at least 1 month. f necessary, seek advicefrom the supplier of the equipment or reagent. Change the soda lime at least once a year.
When changing the sodium hydroxide batch, check the actual stability of the titre by comparing the old andnew titrant, e.g. using a control sample.
For samples with low activity and manual titrations, use a 0,010 mol/l NaOH instead of a 0,025 moMl NaOHsolution.As such, the 0,010 moll NaOH solution gives a higher and more useful consumption of tirant.Prepare the 0,010 moll NaOH solution freshly before use (unless the titre has been checked) as it is unstable.lf using the 0,010 mol/ NaOH solution, correct the calculation according to the formulae in 8.1.
4.7 Lecithin solution, with a mass per volume fraction of 10 %. Weigh 10,0 g of lecithin in a suitable bottle.Use magnetic stirring to dissolve it in approx. 95 ml of liquid paraffin,which may take between 1 day and2 days of mixing. When the lecithin is completely dissolved, make it up to a total volume of 100 ml with theliquid paraffin.
when stored in a refrigerator, the lecithin solution is stable for 1 year.
4.8 Control sample. Include a control sample of known activity in each series of test for lipase samples.Collect the results and use them for the evaluation of the variation of the test.
The control sample can be the last sample analysed or another well-known sample.
When carrying out the method for the very first time, use a control sample obtained from another laboratory orthe first sample analysed being kept as control sample for the next series of analyses. If needed, store thecontrol sample(s) in a freezer.
NOTE lt can be difficult to get a suitable control sample for rennet paste.
5 Apparatus
Usual laboratory equipment and, in particular, the following.
The laboratory equipment can be substituted by other equipment verified as giving similar results.
5.1 Micropipette or any other pipette , of capacities 1 ml and 10 ml with a repeatability of 0,5 % or higher.