Quantification of high molecular weight (1→3)(1→4)-& 03B2 ... · and linear correlation...

Carlsberg Res. Commun. Vol. 53, p. 287-296, 1988

QUANTIFICATION OF HIGH MOLECULAR WEIGHT (1-,3)(1-,4)-p-D-GLUCAN USING CALCOFLUOR COMPLEX

FORMATION AND FLOW INJECTION ANALYSIS. II. DETERMINATION

OF TOTAL p-GLUCAN CONTENT OF BARLEY AND MALT by

K I M G. J O R G E N S E N l) and STEN A A S T R U P 2)

~)Department of Biotechnology, 2)Department of Brewing Chemistry and Technology, Carlsberg Research Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby

Keywords: Fluorescence, wheat, rye, sorghum, triticale, maize, high performance size exclusion chromatography

Complete solubilisation of barley or malt 13-glucan was obtained by extraction with dilute acid. Investigation of the extraction procedure by high performance size exclusion chromatography indicates that originally water insoluble high molecular weight 13-glucan is solubilized by degradation to smaller fragments. The fluorescence emitted by the specific complex formed between Calcofluor and D-glucan in solution was used to measure the total [3-glucan content of barley and malt samples. Examination of the residues after the extraction with dilute acid by degradation with (1--,3)(1--,4)-~-I~-glucanase and by fluorescence microscopy of Calcofluor stained residues confirmed the complete extraction of the [~-glucan. Comparison between the Calcofluor-FIA method and the enzymatic method of MCCLEARY and GLENNIE-HOLMES (20) revealed a high correlation between the values obtained by the two methods. 39 barleys and 57 malts were analysed for ~-glucan content with the two methods and linear correlation coefficients of r = 0.977 and r = 0.986, respectively, were found. The system enables a large number of barley or malt 13-glucan determinations to be performed within a short period of time with a minimum of manpower. The method is suitable for standard routine analysis in quality control laboratories.

1. INTRODUCTION In recent years a wide variety of assays for

quantification of the high molecular weight barley cell wall polysaccharide 13-glucan has been developed (6, 14). The fl-glucan content of barley has been estimated indirectly by measurement of extract viscosities (1, 7) and near infrared reflectance techniques (10), but problems were encountered in establishing an accurate calibration. Selective precipitations of fl-glucan

from extracts before carbohydrate determination have employed ammonium sulphate (21), CuSO4 (17) or Calcofluor (24). A major limita- tion with these procedures is the difficulty to render all fl-glucan soluble by the extraction and therefore frequently significant fractions are excluded from analysis. In the enzymatic methods the 13-glucan is solubilized by degradation with purified ( 1--,3)( 1 ~4)-[3-D-glucanases, and the products are measured either directly by a

Abbreviations: 13-glucan = mixed linkage (l~3)(1--,4)-[3-D-glucan; D.M. = dry matter; DMSO = dimenthyl sulfoxide; FIA = flow injection analysis; HPSEC = high performance size exclusion chromatography; MW = molecular weight.

Springer-Verlag 0105-1938/88/0053/0287/$02.00

K G JORGENSEN & S. AASTRUP: Quantification of total I]-glucan

Table I. Barley varieties and their l~-glucan content measured by the Caicofluor-FIA and Enzymatic methods. (a) - Proanthocyanidin-free type, (b) - Malting type, (c) - High Lysine type, (d) - winter barley. (I, II, III) - Grown at different locations

Variety Type Year and 13-glucan (% D.M.) location Calcofluor-FIA Enzymatic

method method

1. Ant-13-13 a 1981 DK-1 3.38 3.29 2. Keti 1981 DK-I 4.08 4. l 1 3. Ant- 13xLami a 1981 DK-I 3.85 3.74 4. Lami 1981 DK-I 4.72 4.59 5. Ant-13xNordal a 1981 DK-I 4.74 4.77 6. Nordal b 1981 DK-I 3.56 3.74 7. Ant-192 a 1981 DK-I 4.28 4.55 8. Georgie b 1981 DK-I 4.41 4.46 9. Ant-180 a 1981 DK-I 2.76 3.10

10. Galant a 1982 DK-I 4.79 4.82 11. Ant-13-13xRupal a 1982 DK-I 4.11 4.16 12. Catrin b 1982 DK-I 4.34 4.55 13. (Ant- 13xKristina)x Kristina a 1982 DK-I 3.94 4.11 14. Ant-! 3xCa-28348 a 1982 DK-I 3.85 4.01 15. Variety mix b 1982 DK 4.69 4.69 16, Triumph b 1982 DK-I 3.62 3.60 17. Nordal b 1982 DK-I 4.68 4.76 18. M-737 (Minerva mutant) b 1982 DK-I 3.85 3.77 19. M-892 (Minerva mutant) b 1982 DK-I 4.73 4.36 20. N-5247 (Nordal mutant) b 1982 DK-I 4.29 4.20 21. Ant-214 a 1982 DK-I 4.69 4.65 22. Galant a 1983 DK-II 4.55 4.50 23. Galant a 1983 DK-II 4.76 4.66 24. Gula b 1983 DK-II 5.08 4.89 25. Galant a 1983 DK-I 4.64 4.64 26. Ca-700202 c 1983 DK-I 2.37 2.36 27. Ant-13-13xRupal a 1983 DK-I 4.02 3.90 28. Galant a 1983 UK-I 5.24 5.40 29. Galant a 1983 UK-II 3.74 3.72 30. Galant a 1983 DK-11 4.22 4.24 31. Galant a 1984 DK-III 3.81 3.88 32. Ca-11734xGalant a 1984 DK-I 3.72 3.64 33. Ant-5022 b 1984 DK-I 3.05 3.30 34. Sonja d 1984 DK-I 4.25 4.18 35. Kaskade d 1984 DK-I 3.48 3.57 36. Ca-22344• a 1984 DK-I 3.78 3.78 37. Ca-26313• a 1984 DK-1 3.55 3.61 38. (Ant- 13-13xRupal)xAtem a 1984 DK-I 4.56 4.49 39. Triumph b 1984 DK-I 3.46 3.52

ch romophore reaction (11) or by glucose deter-

mina t ion after complete acid hydrolysis (4).

Other techniques (3, 5, 19, 22) employ a pre-ex-

trat ion step prior to enzymat ic degradat ion o f

the ~l-glucan to glucose. A commerc ia l ly avail-

able method (19, 20) uses two purified enzymes

sequentially: l ichenase f rom Bacillus subtilis

degrades [3-glucan to soluble fragments and

13-D-glucosidase complete ly hydrolyses the frag-

ments to glucose. Al though it is difficult to

288 Carlsberg Res. Commun. Vol. 53, p. 287-296, 1988

K.G. JORGENSEN & S. AASTRUP: Quantification of total 13-glucan

evaluate the specificity of the various techniques, it seems that some of the earlier enzyme based methods over-estimated the IB-glucan content, due to impurities such as starch-degrading enzymes. In an alternative procedure (26) the starch is first degraded by commercially avail- able crude preparations of thermostable a-amy- lase followed by a treatment with amylo-glucosidase. The 13-glucan is then precipitated and degraded using a crude cellulase preparation.

Complications arise when measuring 13-glucan content of malt because low molecular weight carbohydrates must be removed prior to analysis. In methods using IB-glucan specific enzymes, this is done by washing with aqueous ethanol (19), by dialysis (3) or indirectly by pretreatment with borohydride (9).

An automatic analyser measuring Calcofluor- I5-glucan complex formation by flow injection analysis (FIA) has been presented for the determination of IB-glucan in solutions, e.g. wort and beer (12, 15). This method measures the high molecular weight IB-glucan fraction without any pretreatment of the samples, making it very suitable for routine IB-glucan determination in quality control laboratories. However, to use this technique for measurement of IB-glucan in barley or malt, the IB-glucan must first be rendered soluble.

The present paper describes the extraction procedure which has been adapted for the use with the Calcofluor-FIA IB-glucan analyser to determine IB-glucan content of barley, malt and other cereals. The method is based on the extraction procedure described by AHLUWALIA and ELLIS (3).

The values of total I3-glucan content of barley and malt samples determined with the analyser is compared with the values obtained by em- ploying the enzymatic method of MCCLEARY, NURTHEN and GLENNIE-HOLMES (19, 20).

Table II. [~-glucan content of oats, sorghum, rye, wheat and maize. [~-glucan was measured after perchloric acid extraction with the Calcofluor-FIA method.

Sample Variety 13-glucan (% D.M.)

Oats - a: Stil (SF) 3.78 b: Titus (SF) 2.87 c: Puhti (SF) 3.15

Sorghum - a: Dabar 0.27 b: Safran 0.24 c: Felitia 0.27

Rye: Petkus (DK) 1.48

Wheat - a: Kalmar (S) 0.46 b: Orebro (DK) 0.48 c: Kraka (DK) 0.49

Triticale: 0.70

Maize - a: (DK) < 0.00 b: US3 0.10 c: (F) < 0.00

(Table II) were included in the test. Calcofluor TM was from Polysciences Ltd.,

Northampton, UK. All other chemicals used were of analytical grade.

2.2. Measurements of II-glucan The automatic method based on Calcofluor

complex formation and FIA as described in (12) was used for measurement of I~-glucan content in extracts made from barley, other cereals and malt.

For comparison, 13-glucan was also measured enzymatically as described by MCCLEARY and GLENNIE-HOLMES (20), with the commercial kit of Biocon Ltd., Worcestershire, UK.

2. MATERIALS and M E T H O D S 2.1. Materials

Barley varieties used included 39 samples as listed in Table I. From these barleys 57 malts were produced by pilot malting (2). For comparison of the [B-glucan contents: 3 oats, 3 maize, I rye, 1 triticale, 3 wheat and 3 sorghum varieties

2.3. Extraction of total l~-glucan For complete extraction of the total IB-glucan

content of cereals the following procedure was adopted (c.f. section 3.1).

Flour is produced by grinding the seeds (50- 100 g) in a Tecator Udy Cyclon grinder fitted with a 0.5 mm screen. The flour is carefully

Carlsberg Res. Commun. Vol. 53, p. 287-296, 1988 289

K.G. JORGENSEN & S. AASTRUe: Quantification of total [3-glucan

mixed before samples for analysis are taken. About 50 mg (barley, oats) or about 150 mg

(malt, wheat, rye, triticale, maize, sorghum) flour is poured into a 26x100 mm test tube having a teflon lined screw cap which ensures a tight closure of the tube during the boiling step. The exact weight is noted and 10 ml of deionized water is added. The flour suspension is mixed and placed in a boiling water bath for 1 h. The sample is cooled and 10 ml of acid (0.1 M- perchloric acid, 0.1 M-HCI or 0.075 M-H2SO4) added, the content mixed and boiled for 10 rain for complete solubilisation of all the [3-glucan. After cooling, the suspension is carefully mixed, centrifuged (l,000xg; 10 min) and the 13-glucan in the supernatant measured using the Calcoflu- or-FIA method. The results are expressed as % 13-glucan in dry matter (D.M.), calculated as described by JORGENSEN (12). All samples are made in duplicate.

2.4. Investigation of the efficiency of acid extraction

The efficiency of the acid extraction procedure was studied in the following way: 5 mg purified [~-glucan (from Biocon Ltd., Worcester- shire, UK, batch no. 515052) was boiled with 3.75 ml water for 1 h in pyrex test tubes closed with teflon lined screw caps. 3.75 ml of 0.1 M-perchloric acid was added and the samples placed in boiling water. After 0, 1, 2, 3, 5, 8, 10, 15, 20, 30 and 60 min of incubation, samples were removed and cooled. One set of samples was analyzed for 13-glucan content using the Calcofluor-FIA method and for reducing sugars by the method of McCleary and Glennie- Holmes (20). Another set of samples was neu- tralized with 1 M-NaOH and 2.6 ml of DMSO were added and the sample characterized by HPSEC using the procedure described by JORGENSEN (12).

2.5. Microscopy of flour samples Lyophilized pellets from acid treated flour,

and residual material from enzyme treated flour as well as untreated flour were stained with Calcofluor and Fast Green (2) and inspected using fluorescence microscopy.

" t .

c~

v

t -

C)

(5

3.0

6 . 0 i t i

5.0 e/~176 ~ o ~

zx~zx/A ~ A

4.0 / / ~ ~

/ .O

/ O~o~~ ~""- o ~ o

0 ~ ~ /

~o

2 . 0 i ~ i i l

5 10 15 20 25

Extract ion Time (min)

30

Figure 1. Time courses of 13-glucan extraction with perchloric acid for flour from 5 barley varieties.

3. RESULTS 3.1. An optimal procedure for extracting total

II-glucan The Calcofluor-FIA method (12), was devel-

oped to measure [3-glucan in solution. In order to measure the total ~-glucan content of barley or malt, the [3-glucan must be completely solubilized prior to analysis. For this purpose, a two- step procedure was applied. 1) Boiling of the flour with water for 1 hour releases water soluble 13-glucan and 2) boiling with acid extracts the non water soluble high molecular weight ~-glucan fraction. Step 1) ensured highly repro- ducible results. The boiling time was not found critical, it could often be reduced to 30 minutes. For finding the optimal time of acid extraction, 5 barley samples with contrasting levels of [3-glucan content were investigated. As seen in Figure l all samples showed the same kinetics of solubilization irrespective of J3-glucan content. Up to about 8 rain of incubation with perchloric acid increasing amounts of 13-glucan were solubilized, between 8 and 15 min the 13-glucan level was constant while upon further extraction a


K.G. JORGENSEN & S. AASTRUP: Quantification of total [~-glucan

6.0 i i i i i i i

~a 5.5 ,/. 5.0

e-

_~ 4.5 O

4.0 , r N= 39

E < 3.52.53.0 / � 9 r== 0.977 * ~ .

2.0 I I i i i i i

2.0 2.5 3.0 3.5 4.0 4.5 5,0 5.5 6.0

Enzymatic fl-Glucan (% D.M.)

Figure 2. The relationship between total barley I~-glucan measured enzymatically and by the Calcofiuor- FIA method. Regression line: "FIA 13-glucan (% D.M.)" = 1.03 x "Enzymatic ~-glucan'- 0.15 (% D.M.).

2.5

~. 2.0 13

~- 1.5 t~ o

( .9

< t J_

0.5

i i i i

�9 � 9 1 4 9 �9

�9 r = 0.986***

I I I I

0.5 1.0 1.5 2.0 2.5 Enzymatic fl-Glucan (% D.M.)

Figure 3. The relationship between total malt [~-glucan measured enzymatically and by the Calcofluor-FIA method. Regression line: "FIA 13-glucan (% D.M.)" = 1.02 x "Enzymatic ~-glucan"- 0.06 (% D.M.).

decrease in the amount of 13-glucan occurred. After 30 rain extraction time, the 13-glucan content was about 90% of that observed at 10 min extraction.

As a consequent of these results, the 1 hour preboil with water and a 10 min 0.05 M-perchloric acid extraction was chosen for complete solubilization of barley and malt 13-glucan. The reproducibility of this extraction technique, and measurement with the Calcofluor-FIA method was tested. The 5 barleys representing different levels of fl-glucan were measured 8 times, and coefficients of variation (C.V.) between 1.0 and 1.7% were found.

The thirty-nine barley samples and 57 malt samples were extracted using the procedure outlined above. The results were compared with the 13-glucan contents measured by the enzymatic kit using the extraction method of MCCLEARu and GLENNIE-HOLMES (20) (Table I, Figures 2 and 3). The linear correlation coefficients between the values determined by the two methods were r = 0.977 and r = 0.986 for barley and malt, respectively. The regression line for barley is described by the equation: "FIA I]-glucan (% D.M.)" = 1.03 • "Enzymatic [~-glucan"- 0.15 (%

D.M.), and that for malt by the equation: "FIA 13-glucan (% D.M.)" = 1.02 x "Enzymatic I~-glucan" - 0.06 (% D.M.).

The close relationship between the values obtained by the two extraction methods was further confirmed by analysing the residue from the extracts. Residual material from the acid extractions of barley flour (Figure 2) was analyzed for I~-glucan by the enzymatic method. Very low 13-glucan contents of 0.05-0.12% D.M. were obtained. Likewise, the enzymatically treated flour was extracted with acid and solubilized material analyzed with the Calcofluor-FIA method. Between 0.09 and 0.16% D.M. of 13-glucan was found. Finally, the effectiveness of the acid extraction procedure was examined by microscopy (Figure 4). Comparing untreated flour and acid extracted flour by white light inspection (Figure 4.A. 1 and 4.A.2), it was found that the extraction procedure solubilized the endosperm, whereas husk material was left. In UV light a bright fluorescence from Calcofluor stained cell wall material was observed in the preparation of untreated flour (Figure 4.B.1), but few fluorescent particles could be found in residues after the acid extraction (Figure 4.B.2).


K.G. JORGENSEN & S. AASTRUP: Quantification of total I~-glucan

Figure 4. Low magnification microscopy (x 4) of barley flour stained with Calcofluor and Fast Green: 4.A. 1 & 4.B. 1 = untreated barley flour, 4.A.2 & 4.B.2 = residual material from the acid extraction. A = white light inspection, B = inspection in UV light.

This verifies that the extraction very effectively solubilize the barley endosperm cell walls and hence the [3-glucan polysaccharide.

3.2. Examination of the extraction process It was found that the amount of 13-glucan

resulting from extraction was time dependent with a maximum at about 10 rain (Figure 1). In order to determine the cause for the reduction of the ~-glucan content upon prolonged acid extraction, a solution of 13-glucan made of the Biocon preparation batch no. 515052 wasboiled with perchloric acid for various length of time and analyzed with the Calcofluor-FIA method.

As observed in Figure 5, the response of the Calcofluor fluorescence was unaffected up to 15 min, whereafter the amount of fl-glucan dimin- ished. After 60 min of incubation the amount detected with Calcofluor had decreased from 250 mg. L" to about 100 mg. L -~. This indicated a degradation of the 13-glucan into fragments which do not complex with Calcofluor. The degradation of 13-glucan was further studied by HPSEC (Figure 6). The MW profile of the untreated preparation consisted of a very high MW fraction which occurred within the exclusion volume of the column (Vo), giving a broad shoulder on a sharp peak, followed by a very distinct peak near the total volume of the column (Vt). After 1 min incubation with


K.G. JORGENSEN & S. AASTRUP" Quantification of total 13-glucan

300 , , ,

250 '

,~ 200

E "-" 150

e-

--~ 100 &

50

~ 0 0 0

I I I I I

10 20 30 40 50 60

Ext ract ion Time (min)

Figure 5. The effect of perchloric acid incubation on ~-glucan preparation, Biocon batch no. 515052.13-glucan was measured by the Calcofluor-FIA method.

perchloric acid, the high MW fraction decreased in size and after 10 min of incubation, the high MW fraction disappeared completely. By reducing sugar analysis it was shown that these changes in molecular weight distribution corre- sponded only to an increase of the degree of hydrolysis of 1% and 2% after 20 and 30 min of acid extraction, respectively. Furthermore, determination of 13-glucan content in extracts made of barley and malt before and after the 10 min acid treatment gave identical results indi- cating that no degradation of [I-glucan to MW below the detection limit had taken place.

Based on these findings which suggest a mild acid hydrolysis during extraction, tests were performed substituting the perchloric acid with dilute concentrations of nESO 4 or HC1 for extracting the 13-glucan of barley fluor. The relationship between measured 13-glucan and incubation time is shown in Figure 7 for one flour sample and perchloric, H2SO4 and HC1 as extraction acids. No significant difference in effectiveness of extraction was found among the acids used, when the 13-glucan contents of the 5 barley samples were measured by the Calcoflu- or-FIA method, the use of the less expensive and less hazardous H2SO4 instead of perchloric acid is therefore recommended.

I I I

20

10

Vo vt I I I

0 10 2 0 3 0

E l u t i o n T i m e ( m i n )

Figure 6. HPSEC of perchloric acid extracted 13-glucan preparation, Biocon batch no. 515052. The values at the individual profiles indicate incubation time in 0 - 20 minutes with 0.05 M-perchloric acid in a bath with boiling water.

3.3. Total Ii-glucan content of various cereals In addition to the 13-glucan determination of

barley and malt, the extraction procedure and the Calcofluor-FIA method were used to quan- tify the 13-glucan content of some cereal samples (Table II). The oat samples were found to have a I~-glucan content very similar to the levels


K.G. JORGENSEN & S. AASTRUP" Quantification of total 13-glucan

5.0

4.5 [

c5

C t~ O

I

4.0

3.5

3.0

2.5

2.0

/o

I I I

5 10 15 20

Extract ion Time (min)

Figure 7.13-glucan in samples taken at different times during extraction, using different acids for extraction and with final concentrations as noted. [] = 38 mM- H2SO4, ~7 = 50 mM-HC1, �9 = 50 mM-perchloric acid.

found in barley, i.e. in the range from 2.5 to 5.5% D.M. (Figure 2), while the rye sample had half of that amount. The triticale sample was found to contain less fl-glucan than the rye sample, but more than the wheat samples. Small amounts of 13-glucan could be found in sorghum but no detectable amounts in maize. The fl-glucan values of these samples were not confirmed by the enzymatic method, however, the contents were found to be in good agreement with previously published results using an enzymatic method (20).

4. DISCUSSION 4.1. Specificity of the Calcofluor-FIA method

Referring to Table I, Figures 2, 3 and 4, it is evident that the method is highly specific, measuring exclusively 13-glucan. Other sources of carbohydrate polymers which might be present in the extract do not seem to complex with

Calcofluor. Possible interfering carbohydrate polymers could be: starch, cellulose, polymers containing only (l~3)-13-D-glycosidic linkages, or pentosans. WOOD (23) has reported that spectral changes in Calcofluor are caused by glucose polymers containing (1--,3)-15-D- or

(1-,4)-13-o-glycosidic linkages, and that starch does not affect the fluorescence intensity. WOOD et al. (25) have further shown that wheat pentosans do not affect Calcofluor fluorescence, and it has recently been shown by JORGENSEN et al. (15), that treatment of wort or beer samples with purified endo(1 ~3)(l--,4)-13-D-glucanase from bacillus subtilis removed all substances causing increase in Calcofluor fuorescence intensity. This suggests that barley pentosans do not react significantly with Calcofluor. Cellulose which binds to Calcofluor must be solubilized in order to react with Calcofluor in the FIA system. Therefore, previous studies (23) on the reaction between Calcofluor and soluble (1--,4)-13-D-glucans have been carried out using derivatised cellulose (e.g. carboxy-methyl cellulose etc.). The results obtained by fluorescence microscopy (Figure 4), make it unlikely that the mild acid extraction conditions would solubilize cellulose. Interference from (1--,3)-13-D-glucan is not a significant problem, either because (i) the cereals investigated contain a low level, or (ii) the material is not solubilized. However, a detailed survey of other possible interfering components must be carried out before generalizations on the specificity of Calcofluor for measurement of 13-glucan in components other than endosperm cells are considered.

13-glucan contents of barley and malt were measured with a low coefficient of variation. Small amounts of flour were analyzed (about 50 mg for barley), so a proper mixing of the flour must be done before a sample is taken for analysis to avoid a sampling error. However, a procedure using a larger amount of flour could easily be made e.g. by dilution of the sample before analysis or by injection of smaller vol- umes in the FIA system.

4.2. The extraction process Examination of the extraction process used to

solubilize 13-glucan quantitatively by acid hydrolysis showed that a minimum time and concentration of acid is required and that too long extraction time has to be avoided. HPSEC data indicate that the MW distribution is altered during acid hydrolysis. It was shown by FOLOAGER and JORGENSEN (8) that an increase


K.G JORGENSEN & S. AASTRUP: Quantification of total 13-glucan

in Calcofluor fluorescence intensity only takes place with 13-glucan molecules having a size which is larger than 104 dalton. It is therefore essential that the 13-glucan during extraction is not degraded to fragments smaller than 104 dalton, a condition which can be achieved by adjustment of both the extraction time and the concentration of the acid.

The results of the proposed extraction procedure are to some extent in conflict with the results from AHLUWAL1A and ELLIS (3). In the latter investigation perchloric acid extraction (50 mM and boiling for 3 min) was used to solubilize 13-glucan prior to determination with specific enzymes. It was postulated that no significant acid hydrolysis or degradation of the 13-glucan occurred. However, the hydrolysis was judged by measurement of free glucose, which is not a very sensitive method for evaluating a small degree of hydrolysis. Increases in the glucose level are observed only after a high degree of hydrolysis (16). Furthermore, AHLUWALIA and ELLIS (3) determined the effect on MW distribution using gel permeation chromatography with a relatively small column. All the 13-glucan was found to elute in the void volume of the column and a degradation of the 13-glucan would not have been observed.

4.3. Application of Calcofluor-FIA method The Calcofluor-FIA method as presented in

this investigation represents a rapid technique for the determination of 13-glucan at all stages of the brewing process. Its possible use in malt and wort production has previously been presented by JORGENSEN and AASTRUP ( 13). AS the method requires a minimum of manpower, and a large number of analyses can be performed within a day, it is a good candidate for a new routine method in quality control laboratories.

A C K N O W L E D G E M E N T S The authors wish to thank Fil.Dr. L. MUNCK

and Dr. B. AHRENST-LARSEN, heads of Dept. of Biotechnology and Dept. of Brewing Chemistry and Technology, respectively, for their support in this work. The advise of our colleagues Drs. K. ERDAL, B.S. ENEVOLDSEN, Prof. D. VON

WETTSTEIN and R.G. FULCHER and the skilful technical assistance by Mrs. L. FREDERIKSEN, Mrs. E. MEILING, Mr. M JACOBSEN and Mr. K.T. HANSEN is greatly appreciated.

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ty of an acid flour extract of barley and its 13-glucan content. Carlsberg Res. Commun. 44, 28%304 (1979)

2. AASTRUP, S. a K. ERDAL: Quantitative determination of endosperm modification and its relationship to the content of 1,3:l,4-13-glucan during malting of barley. Carlsberg Res. Commun. 45, 369-379 (1980)

3. AHLUWALIA, B. & E.E. ELLIS: A rapid and simple method for determination of starch and 13-glucan in barley and malt. J. Inst. Brew. 90, 254-259 (1984)

4. ANDERSON, M.A., J.A. COOK & B.A. STONE: Enzy- matic determination of 1,3:1,4-13-glucans in barley grain and other cereals. J. Inst. Brew. 84, 233-239 (1978)

5. BAMFORTH, CW.: Penicillum funiculosum as a source of 13-glucanase for the estimation of barley 13-glucan. J. Inst. Brew. 89, 391-392 (1983)

6. BAMVORTH, CW.: Barley 13-glucans, their role in malting and brewing. Brew. Dig. June, 22-35 (1982)

7. BENDELOW, V.M.: Determination of non-starch polysaccharides in barley breeding programmes. J. Inst. Brew. 81, 127-130 (1975)

8. FOLDAGER, L. 8, K.G. JORGENSEN: The molecular weight distribution of I]-glucan in wort from different barley varieties at different stages of malting. Carlsberg Res. Commun. 49, 525-534 (1984)

9. HENRY, R.J. & AB BLAKENEY: Determination of total fl-glucan in malt. J. Inst. Brew. 92, 354-356 (1986)

10. HENRY, R.J.: Near-infrared reflectance analysis of carbohydrates and its application to the determination of (l~3),(1--,4)-13-D-glucan in barley. Car- bohydr. Res. 141, 13-19 (1985)

11. HENRY, R.L.: A simplified enzymic method for the determination of (1-*3)(l-*4)@D-glucans in barley. J. Inst. Brew. 90, 178-180 (1984)

12. JORrENSEN, K.G: Quantification of high molecular weight (l-*3)(1-*4)-I~-D-glucan using Cal- cofluor complex formation and flow injection analysis. I. Analytical principle and its standard- ization. Carlsberg Res. Commun. 53, 277-285 (1988)

13. JORGENSEN, K.G. & S. AASTRUP: Analysis of [3-glu-


K.G. JORGENSEN & S. AASTRUP: Quantification of total 13-glucan

can in wort. Proc. Eur. Brew. Conv. Symp., Mono- graph XI, Maffiiers. 262-273 (1986)

14. JORGENSEN, K.G & S. AASTRUP" Determination of 13-glucan in barley, malt, wort and beer. In: Modern Methods of Plant Analysis. Beer Analysis. H.F. Linskens, J.F. Jackson, eds., Springer Verlag, Vol. VII, pp. 88-108 (1988)

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Accepeted by S.O. ANDERSEN


Quantification of high molecular weight (1→3)(1→4)-& 03B2 ... · and linear correlation...

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