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Pollach G.: Enzymatic determination of the total fermentable sugars in sugar factory products (German). Zuckerindustrie 107 (1982) pp. 603-606.

Abstract: An enzymatic method for determination of the total fermentable sugars in the absence of melibiase is described. The results of the method are relevant to utilization of sugar factory products by Saccharomyces cerevisiae. In principle, the sum of glucose and fructose, after hydrolysis of the oligosaccharides, is determined by means of β-fructosidase. The aim of this work is to obtain a reproducibility that is adequate for the determination of major sample components, by modifying the well-known hexokinase method.

Paper presented to the Scientific Committee of CITS in Norwich, June 1-3, 1981. Introduction: For trade relations between the sugar industry on the one hand and the fermentation- and yeast industry on the other hand, the sum of fermentable sugars in products rather than the sucrose content is relevant. The use of Saccharomyces cerevisiae for production of ethanol and baker’s yeast is dominant in the fermentation industry, compared to other variants of fermentation. This micro-organism is able to utilize sucrose, glucose, fructose, kestoses, galactose and one third of raffinose. With neglect of galactose, which is only present in raw juice in low amounts [1], the sum of all other relevant sugars can be determined with one single enzymatic determination. A method like this, based on the well-known enzymatic technique [2], is described in the following. In principle this method is already known and represented by an intermediate result of the sucrose determination according to Schoenrock and Costesso [3]. The present paper aims at an improved reproducibility of this enzymatic method. There is some demand of improvement, according to the ICUMSA report 1978 [4]: The precision of an enzymatic sucrose determination using the hexokinase method was poor and studies on improvement were recommended. In contrast to other fields of application, the variation of enzymatic results is high, compared to the variation of sucrose as a main component of samples. The same is true for "fermentable sugars (FS)".

The principle of the method is summarized here:

	Principle:
							Glucose
							Fructose
	Hydrolysis of oligosaccharides by β-fructosidase:
	Sucrose	+	H2O			→	Glucose	+	Fructose
	Raffinose	+	H2O			→	Melibiose	+	Fructose
	Kestoses	+	2 H2O			→	Glucose	+	Fructose	+	Fructose
	Phosphorylation by hexokinase:
	Glucose	+	ATP			→	G-6-P	+	ADP
	Fructose	+	ATP			→	F-6-P	+	ADP
	Isomerisation by phosphoglucose-isomerase:
	F-6-P					↔	G-6-P
	Oxidation by G-6-P-dehydrogenase (G6P-DH):
	G-6-P	+	H2O	+	NADP+	→	Gluconate-6-P  +  NADPH  +  H+

Glucose and fructose are split off from oligosaccharides by β-fructosidase and thus added to free glucose and fructose. In presence of ATP, the monosaccharides are phosphorylized by hexokinase (HK). With the aid of phosphoglucose-isomerase (PGI) an equilibrium is established between both phosphates. By oxidation, Glucose-6-phosphate (G6P) is withdrawn from this equilibrium and NADPH is formed. Its characteristic absorbance is measured at or near 340 nm. The sample preparation in advance of the enzymatic analysis comprises a dilution and eventually a clarification according to Carrez [5]. A high dilution with high precision is necessary for samples with high content of “fermentable sugars (FS)”. Beside precision of weighing and measurement, special care is necessary to avoid contamination of diluted solutions. A method of sample preparation, which proved well effective, is explained in detail in the appendix. Since the range of concentration is quite well known for main components, test solutions with approx. constant sugar content (around 100 mg FS/L) can be used. The solutions are made from a sample weight which is dependent on the expected result. Thus the absorbance scale can be used to a great extent and a linearity error of the photometer can be almost eliminated. The random error of sample preparation results from the sum of 2 gravimetric and 3 volumetric measures. Sample preparation, based on analytical balances and classical volumetric flaks, will not limit the overall precision of the enzymatic method. Lowering of random errors: Within the enzymatic assay, random errors are lowered by preparation of pre-mixtures of enzymes immediately prior to filling the cuvettes. More precisely, the following advantages are derived from this:
1.	The number of pipetting steps into a cuvette is reduced to a minimum of four.
2.	Random errors by individual contamination of a cuvette or by pipetting suspensions are eliminated.
3.	For those pipetting steps, which are relevant for the overall precision, Hamilton syringes with high repeatability are used. But these syringes must be rinsed with the solution before dosing.
The following mixtures are pipetted into the cuvettes:
a)	0.5 mL of redistilled water, standard solution or sample solution.
b)	0.1 mL of solution of β-fructosidase, dissolved in citrate buffer, pH = 4.6.
c)	2.0 mL of solution of NADP+, ATP and PGI in buffer, pH = 7.6.
d)	0.1 mL of solution of HK and G6P-DH-suspension in buffer, pH = 7.6.
In order to keep systematic errors as low as possible, results are evaluated by use of a sucrose standard. Therefore, the absolute dosing is of secondary importance (for control purposes a calibration of the dosing equipment via analytical balance is recommended). After addition of b) a break of 15 min is kept, which can be used to prepare the mixtures c) and d). Absorbance E1 is measured 3 min after addition of c), absorbance E2 is measured 20 and 25 minutes after addition of d). The difference in absorbance E2-E1 is evaluated. Pipetting technique: Hamilton syringes (types 1002 TLLCH and 1750 TLLCH, both equipped with a device for precise repetition), which show better repeatability than the wide-spread type of automatic pipettes with one-way tips, are used for dosing of the main volumes of 0.5 and 2.0 mL. Sufficient solution is available from sample- and standard solution to rinse the 0.5 mL syringe several times. The second syringe (2 mL) is rinsed repeatedly too, but the solution is put back to the mixing vessel. Syringes and mixing vessels are always used for the same purpose, in order to avoid contamination with traces of enzymes or sucrose. The lower volumes of 0.1 mL are dosed with usual pipettes, but contaminations are washed from the one-way tips by return of solution to the mixing vessel, charged with a magnetic stirrer, at the beginning. Thus any contamination of the tip will be distributed to all cuvettes, whereas the blank cuvette, which is usually placed at the first position, is mostly endangered in the conventional method. For reasons of standardisation of all cuvettes the enlargement of a small error through addition of 0.1 mL hexokinase solution after measurement of E1 - instead of 0.02 mL in case of the classical method [2] - is consciously taken into account. The results are too low, but only by 4 % of the difference E1Sample - E1Blank. This error would have to be considered in case of extremely coloured samples, but normally it is negligible. Potassium-dichromate test: In order to determine - in a simple way - the total error, caused by the two main dosing instruments, the cuvette path length tolerances, the intermixing procedure and the photometer, a proposal of Walter [6] was used in a modified form. As a first step, 2 mL of redistilled water were dosed with the buffer/enzyme syringe into a series of cuvettes and E1 values were measured. Subsequently 0.5 mL of potassium dichromate solution (300 mg/L for 334 nm) was added with the aid of the sample syringe and E2 was measured, after intermixing of the dichromate solution. The absorption differences E2-E1 were evaluated. Table 1 shows the results of an experiment with a photometer Vitatron MPS, one-way cuvettes (Sarstedt 67.741) and Hamilton syringes 0.5 and 2.0 mL.

Table 1	Results of a dichromate test: Add 2 mL of redistilled water into empty cuvettes and measure E1. Add 0.5 mL of potassium-dichromate-solution (about 300 mg/L for 334 nm), mix and measure E2.
(E2 - E1) * 1000
670	670	670	671	670.5	668
671.5	670.5	671.5	670	670.5	671
671.5	670	671	672	669	670.5
Mean	670.47
n	18
sr	0.96
C.V.r	0.14

The results of table 2 show the repeatability which was achieved with the aid of the specified device in case of analyses of sugar samples. Eight independent repetitions were examined, covering a) a blank, b) a sucrose standard solution and c) a syrup sample with approx. 60% fermentable sugars, inclusive sample preparation. In table 2, the results are stated for all repetitions of standard solutions, calculated via molar absorption coefficient and specified in % of theory. Taking into account the purity of refined sugar, an unexplained error of 1% was found for all steps of analysis, but a higher accuracy cannot be expected due to the photometer specification. The sample results were calculated according to a relative procedure, based on the individual standard for every sample. The repeatability of the standard values would allow one standard for a group of samples instead of 8 individual standards. If repeatability figures like those shown in table 2 can be achieved again and again for this enzymatic determination - after optimizing of the analysis instruments with the aid of a potassium dichromate test - then double determinations would be sufficient for a routine analysis. Thus a method with acceptable analysis expenditure would be available.

Table 2	Repeatability figures for the enzymatic method: Calculation based on individual sucrose standards. Concentrated raw juice was used as a model sample at that time.
Overall repetitions: New standard and new sampling	Sucrose standard (% of theory)	Sample % Fermentable sugars (as monosaccharides)
1	99.1	62.88
2	99.1	62.57
3	99.0	62.41
4	99.0	62.34
5	99.3	62.30
6	99.3	62.07
7	99.0	62.39
8	98.8	62.34
Mean	99.08	62.41
sr	0.17	0.23
C.V.r	0.17	0.38

The applicability of the specified methodology to other variants with rather identical demand of repeatability, such as enzymatic sucrose determination (exact: sucrose + kestoses * 0.68) or the determination of the sum of fermentable sugars in presence of melibiase and/or alpha-glucosidase, was not examined within the framework of this paper.

Appendix
Detailed working procedure. Reagents and solutions, partly derived from [7]. Apparatus:
-	Spectrophotometer for measurement at 340 nm, or spectral-line photometer with filter for measurement at 334 or 365 nm.
-	Cuvettes, working volume: 2-3 mL, path length: 1 cm.
-	Hamilton syringes for repeatable dosing near 2 mL (1002 TLLCH) and 0.5 mL (1750 TLLCH), C.V. max. 0.1%.
-	Pipettes with disposable tips, 0.1 mL, C.V. max. 1%.
-	Pipettes with disposable tips, 0.01 and 0.02 mL, C.V. max. 5%.
-	Analytical balance, laboratory balance, magnetic stirrer, teflon-coated magnetic rods.
-	Plastic syringes: 1 mL, 5 mL, 10 mL.
-	Volumetric flasks: 500 mL, 200 mL, 100 mL.
-	Pipettes: 20 mL.
-	Filters, funnels.
-	Small flasks with screw caps and sealing, 15 mL, 5 mL (from enzymatic kits).
Reagents:
-	Triethanolamine hydrochloride, analytical grade.
-	Sodium hydroxide, analytical grade.
-	Magnesium sulphate – MgSO4 * 7 H2O, analytical grade.
-	Nicotinamide-adenine-dinucleotide phosphate di-sodium salt (NADP-Na2), Boehringer Mannheim (B.M.) no. 128031, no.128040.
-	Sodium hydrogen carbonate – NaHCO3, analytical grade.
-	Adenosine-5'-triphosphate di-sodium di-hydrogen salt (ATP-Na2H2), B.M. no. 126888, no. 127523, no. 127531.
-	Hexokinase/Glucose-6-P-Dehydrogenase (HK/ G6P-DH), B.M. no. 127183, no. 127825.
-	Phosphoglucose isomerase (PGI), B.M. no. 127396.
-	β-fructosidase, B.M. no. 104914, no.104922.
-	Pure sucrose (refined quality).
-	Citric acid.1 H2O, analytical grade.
-	Trisodium citrate.2 H2O, analytical grade.
-	Zinc sulphate, ZnSO4.7 H2O, analytical grade.
-	Potassium ferrocyanide - K4[Fe(CN)6].3H2O.
Preparation of solutions:
-	Sodium hydroxide solution: 5 mol/L, 0.1 mol/L.
-	Buffer pH 4.6 (0.32 mol/L): Dissolve 3.45 g of citric acid plus 4.55 g of trisodium citrate in approx. 75 mL of redistilled water in a 100 mL volumetric flask. Adjust the pH to 4.6 by addition of sodium hydroxide solution (5 mol/L). Fill up to the mark with redistilled water.
-	β-fructosidase solution: Dissolve 10 mg of substance in 2 mL of buffer 4.6 for 20 analyses. Prepare fresh solution daily.
-	Buffer pH 7.6 (0.375 mol/L triethanolamine 5 mmol/L Mg2+): Dissolve 7 g of triethanolamine hydrochloride and 0.125 g of magnesium sulphate in approx. 75 mL of redistilled water in a 100 mL volumetric flask. Adjust the pH to 7.6 by addition of sodium hydroxide solution (5 mol/L). Fill up to the mark with redistilled water.
-	NADP+ solution (approx. 11.5 mmol/L): Dissolve 50 mg NADP-Na2 in 5 mL of redistilled water. The solution is stable at least for 4 weeks at +4 °C.
-	ATP solution (approx. 81 mmol/L): Dissolve 250 mg of ATP-Na2H2 and 250 mg of NaHCO3 in 5 mL of redistilled water. The solution is stable at least for 4 weeks at +4 °C.
-	PGI suspension: Ready to use.
-	HK/G6P-DH suspension: Ready to use.
-	Potassium ferrocyanide solution = CARREZ I (85 mmol/L): 35.9 g/L.
-	Zinc sulphate solution = CARREZ II (250 mmol/L): 71.9 g/L.
Sample preparation:
1.	Very viscous samples are diluted by adding 10 g of redistilled water per 100 g of sample (results to be multiplied by 1.1). The amount of sample, dosed into a volumetric flask (500 mL), depends on the expectable result E (in % FS). The amount of sample is 50/E g, e.g. 1 g of sample with 50 % FS. Dosing is done with plastic syringes (1 mL, 5 mL or 10 mL). The syringe is rinsed with sample several times, filled and finally weighed. The graduation on the syringe is used as an orientation guide for dosing of the right amount of sample. The syringe is re-weighed after dosing of the sample into the volumetric flask and the difference between the two weightings is used as sample weight.
2.	For samples which give a turbid solution, a Carrez clarification is used: redistilled water is added to the volumetric flask on a laboratory balance, up to approx. 10 g of diluted sample. Subsequently 1.25 mL of Carrez I, 1.25 mL of Carrez II and 2.5 mL of 0.1 m NaOH are added, with the liquid in the volumetric flask mixed after every addition. Finally the volumetric flask is filled up to the mark and the solution is mixed and filtered through filter paper.
3.	For samples which give a clear solution, the volumetric flask is filled up with redistilled water and mixed without Carrez clarification.
4.	A sucrose solution with 1000 mg sucrose per litre is prepared.
5.	20 mL of solutions resulting from steps 2 - 4 are diluted with redistilled water up to 200 mL. This step of dilution can be eliminated for samples with a content of FS lower than 10 %, if the weight of sample is 5/E instead of 50/E. Be careful to avoid a contamination of the very diluted final solutions with approx. 100 mg/L of FS.
Enzymatic analysis:
For a group manipulation with up to 6 cuvettes, 3 small flasks with teflon magnetic stirring rods are prepared as follows: A) capacity 5 mL, filled with 2 mL of β-fructosidase-solution; B) capacity 15 mL (empty); C) 5 mL capacity (empty). Flasks B) and C) must be clean, but not dry.
1.	A Hamilton syringe (0.5 mL) is rinsed three times before dosing, and afterwards 0.5 mL of redistilled water (=blank), calibration standard (105.3 mg/L FS) or sample solution are dosed into dry cuvettes.
2.	A pipette with disposable tips is used to add 0.1 mL β-fructosidase. The solution is sucked and put back to the flask five times, with the magnetic stirrer running. Afterwards 0.1 mL of solution is dosed into every cuvette. After intermixing by gentle shaking of the cuvette holder a waiting time of 15 min is kept.
3.	During this break the solutions in flasks B) and C) are prepared (volumes given per one cuvette): 2 mL of buffer 7.6 are dosed to flask B) and 0.1 mL to flask C). Then 0.1 mL NADP+-solution, 0.1 mL of ATP-solution and 0.01 mL PGI-suspension are dosed to flask B) and 0.02 mL HK/G6P-DH-suspension to flask C). The solutions in flask B) and flask C) are mixed with magnetic stirrers.
4.	After a reaction time of 15 min in the cuvettes, according to step 2, the enzyme mixture in flask B) is placed on the magnetic stirrer and a Hamilton syringe (2 mL) is rinsed several times with the solution, which is always put back to flask B). After rinsing the syringe, the stirrer is switched off and 2 mL of solution are dosed to every cuvette.
5.	The solution in the cuvettes are mixed with plastic stirrers, and after 3 min the absorption E1 is measured.
6.	A pipette with disposable tips is used to add 0.1 mL of the solution in flask C). With the magnetic stirrer running, the solution is sucked and put back to flask C) five times. Afterwards 0.1 mL of solution is dosed into every cuvette.
7.	The solution in the cuvettes is mixed again and after 20 min and 25 min the absorption E2 is measured. If the two measurements vary by more than 0.001 absorption, the analysis is disturbed and one of the stock enzymes (PGI) has to be replaced. The reaction must be completed after 20 min (25 min is a check), although evaluation is done via sucrose standard solution.
8.	Hamilton syringes and flasks B) and C) are immediately rinsed with redistilled water, flask A) can be used for further determinations.
Calculation of results:
1.	Calculate E2 - E1 for every cuvette.
2.	The difference E2 - E1 of the blank is subtracted from the other differences. The residual net difference is designated ED. A drop of the dilution 1:10 during the sample preparation has to be considered in the following formula: FS (%) = 50 * 1.053 * EDsample / EDstandard / EW FS = mass content of "Fermentable Sugars", expressed as monosaccharides EW = weight of sample in g per 500 mL volumetric flask ED = net absorption difference (water corrected)

References:
[1]	Hollaus, F.; Wieninger, L.; Braunsteiner, W.: Experiences with enzymatic determination of raffinose by means of galactose-dehydrogenase in beet and sugar factory products (German). Zucker 30 (1977) pp.653-658.
[2]	Bergmeyer, U.: Methods of enzymatic analysis (German). 2nd ed., Verlag Chemie, Weinheim/Bergstrasse (1970).
[3]	Schoenrock, K.W.R.; Costesso, D.: The Spectral Photometric Determination of Sucrose in Sugar Beets and Sugar Beet Products via Specific Enzyme Systems. J. Amer.Soc.Sugar Beet Technol. 18 (1975) pp. 349-359.
[4]	ICUMSA: Proc. of the 17thSess. in Montreal (1978), Subject 8, p.120.
[5]	Schormüller, J.: Handbook of food chemistry (German). Vol. 2/2, Springer Verlag, Berlin-Heidelberg-New York (1967).
[6]	Walter, E.: Check of laboratory equipment for enzymatic determinations (German). Alimenta 19 (1980) pp. 159-164.
[7]	BOEHRINGER MANNHEIM GmbH.: Methoden der enzymatischen Lebensmittelanalytik / Methods of enzymatic food analysis (German). Mannheim (1980).