Amino Acid Levels in Wort and Their Significance in Developing Malting Barley Varieties

Introduction

High levels of grain protein are the greatest restriction to increasing the selected malting barley pool in western Canada. International markets traditionally aim for less than 11.5 % protein in malting barley, but barley exported from Canada averaged greater than 12 % over the past 10 years (Fig 1). Higher protein barley is undesirable because of reduced potential fermentable extract which restricts the amount of beer that can be made from a barley’s malt. Canadian barley breeders, therefore, are often encouraged to develop varieties with potential for low protein. However, a certain level of barley protein is required to make a quality malt and protein is actually limiting in barley from some parts of the world. Canadian malting barley has a reputation for excellent quality, specifically high fermentability, which is indirectly a result of higher protein barley. Soluble protein in malt, resulting from hydrolysis of barley protein during malting and mashing, contributes to foam retention in the final beer and the amino acids and small peptides resulting from further degradation of the soluble protein, are essential for yeast nutrition during fermentation. Barley protein also provides greater potential to produce adequate levels of starch-degrading enzymes (Fig 2) which are essential for trouble-free fermentations (Evans et al 2003). The higher levels of protein in Canadian malting barley, therefore, contribute to excellent fermentation potential and adequate levels of foam positive proteins but some reduction in protein could still increase the amount of barley selected in western Canada.


Figure 1. Average protein content of 2-rowed malting barley exported from Canada, 1995-2004.

Any initiative to genetically reduce barley protein levels must proceed with caution, especially if extreme changes are expected. Altered barley must still have the potential to provide adequate levels of soluble malt protein for efficient fermentation and adequate beer quality. To ensure adequate protein degradation, breeding programs presently rely on percentage of soluble protein in Congress extract and Kolbach index. These values give some indication of adequate levels of degraded protein for beer foam as well as of nitrogenous nutrients for yeast. Free amino nitrogen (FAN), a measurement of amino acids and peptides, provides more specific information on nitrogenous nutrient status, but is seldom monitored. Fermentability and foam potential are not considered directly in breeding programs.


Data source: Langrell & Edney 1995– 2004
Figure 2. Diastatic power versus on protein levels in Canadian-grown Harrington barley.


The importance of monitoring free amino acid levels in malts of breeder lines has received limited attention. Levels are seldom even monitored in commercial malts because they are considered to be relatively constant in extracts made from malts of different varieties (Jones & Pierce 1964). These levels are also not considered to be limiting to fermentability in all-malt worts. However, they can be limiting in high-gravity and high-adjunct brewing (O’Connor-Cox & Ingledew 1989) and a recent study found significant relationships between several individual amino acids and fermentability in commercial malts (Yin et al 2004). The present study investigated the importance of monitoring free amino acids, versus other malt quality parameters, for determining fermentation potential in malting barley breeding programs.

Materials and Methods

A doubled haploid population (54 covered and 54 hulless lines) was used to compare levels of free amino acids and indeces of protein degradation to fermentability. The population was produced by anther culture techniques at Agriculture and Agri-Food Canada Brandon from the cross, TR251/HB345. TR251 is a covered breeding line with good malting potential while HB345 is a hulless breeding line with good agronomic traits and the allele for heat stable beta-amylase (sd2H). The 108 lines along with two replicates of each parent, were grown at Hamiota, Manitoba in 2002. Samples of the lines and parents (500 grams) were micromalted in a Phoenix Automated Micromalting machine (Adelaide, SA, Australia) according to the following schedule: Wet steep 6h, Air rest 2h, Wet steep 4h, Air rest 12h, Wet steep 4h, Air rest 4h, Wet steep 4h, Air rest 4h, Wet steep 4h (steeping at 13°C); Germination 100h (15°C), Kiln 12h @ 55°C, 6h @ 65°C, 2h @ 75°C, 4h @ 85°.

Standard methods of the ASBC (American Society of Brewing Chemists 1992) were used to prepare and analyse fine grind Congress malt extracts. Analysis of free amino acids was based on the method of Garza-Ulloa et al (1986) and was performed on a Beckman 7300 High Performance Amino Acid Analyzer (Beckman Coulter, Inc., Fullerton, CA 92834-3100).

A small scale method for measuring apparent attenuation limit (AAL) was used to determine the fermentation properties of the samples. The method incubated 40 ml of EBC wort (European Brewery Convention 1998) with 160 mg dried yeast (Mauribrew lager yeast, Toowoomba, Australia) at 25°C for 24 hours (Logue 1997).

Results and Discussion

The 108 samples showed a range in quality from very poor to excellent (Table 1) which is similar to what breeders would experience when screening lines for quality in their programs. As a result of the range in quality, fermentability was affected by a number of different malt quality factors, thus, masking, to some extent, direct effects of protein degradation and amino acids on fermentability.

Table 1. Average Malt Quality of the 108 Malt Samples Studied

	Barley Protein %	Fine Extract %	Soluble Protein %	FAN mg/L	ß-Glucan ppm	DP °L	α-Amylase DU	AAL %	Alcohol v/v%
Average	13.8	82.5	6.33	262	137	185	59.2	80.8	3.5
Maximum	15.8	87.6	7.58	335	466	306	81.1	85.7	3.9
Minimum	12.0	76.1	5.53	202	29	107	21.7	71.9	2.9

Soluble protein and FAN were found to have insignificant correlations with fermentability (r²=0.014 and 0.039 respectively). The sum of all individual free amino acid did show a higher correlation coefficient but still insignificant (r²=0.112). Individual levels of both serine and cysteine were found to correlate highly, significantly with fermentability for the samples tested (Fig 3 & 4). Serine levels also correlated well (r²=0.441***) with α-amylase levels and levels of both serine and α-amylase were significantly lower in hulless versus covered samples (Edney et al 2004). This suggested that the serine/fermentability correlation was related to α-amylase levels, which are known to affect fermentability, and not serine directly. However, serine has been shown by others (Yin et al 2004) to be related to fermentation which, in combination with the significant cysteine correlation, and to a lesser extent, tryptophan and phenylalanine (r²=0.186* and 0.174*, respectively), still supported the monitoring of amino acids as an indication of fermentation potential.


Figure 3. Effect of levels of serine on fermentability of the 108 malt samples studied.


Figure 4. Effect of levels of cysteine on fermentability of the 108 malt samples studied.

Conclusions

Percentage of soluble protein in a wort, Kolbach index and FAN levels provided no information on the nitrogenous nutrient status of the worts with respect to fermentability for the breeding population studied. Individual amino acids did explain some of the variability in fermentation, despite masking by other parameters such as levels of enzymes and ß-glucan, suggesting that monitoring of free amino acid could be of benefit. However, the cost of such testing with early generation lines would be prohibitive, although, monitoring at final stages, just prior to commercialization, might be warranted. This would be especially important in altered lines with low levels of barley protein.

References

American Society Of Brewing Chemists (1992). Methods of Analysis, 8th ed. The Society, St. Paul, MN.
European Brewery Convention (1998). Analytica-EBC. Verlag Hans Carl Getränke-Fachverlag, Nürnberg, Germany.

Edney, M.J., Legge, W.G., Rossnagel, B.G., Collins, H.M. (2004). Malting quality of a hulless/covered doubled haploid barley population. Proceedings International Barley Genetics Symposium, Brno, Czech Republic, June 20-26 (eds. J. Spunar, J. Janikova) CD-ROM pp 418-424.

Evans, S.E., Van Wegen, B., Ma, Y., Eglinton, J. (2003). The impact of the thermostability of a-amylase, ß-amylase, and limit dextrinase on potential wort fermentability. J. Am. Soc. Brew. Chem. 61:210-218.

Garza-Ulloa H., Cantú R.G., Gajá A.M.C. (1986). Determination of amino acids in wort and beer by reverse-phase high-performance liquid chromatography. J. Am. Soc. Brew. Chem. 44:47-51.

Jones, M., Pierce, J.S. (1964). Absorption of amino acids from wort by yeasts. J. Inst. of Brew. 70:307-315.

Langrell, D.E., Edney, M.J. (1995–2004). Quality of western Canadian malting barley. Canadian Grain Commission, Winnipeg, MB.

Logue S.J. (1997). The Waite Barley Quality Evaluation Laboratory Barley Quality Report, University of Adelaide.
O’Connor-Cox, W.S.C., Ingledew, W.M. (1989). Wort nitrogenous sources – Their use by brewing yeasts: A review. J. Am. Soc. Brew. Chem. 47:102-108.

Yin, X.S., Strasser, G.H., Ladish, W.J. (2004). Wort amino acid composition of different barley varieties and effect on nitrogen assimilation, Proceedings World Brewing Congress, San Diego, CA, July 25-28 CD ROM.

Edney, M.J.(1)*, Legge, W.G. (2) and Rossnagel, B.G. (3)
* Corresponding author: medney@grainscanada.gc.ca
(1) Grain Research Laboratory, Canadian Grain Commission, Winnipeg, R3C 3G8 Canada
(2) Brandon Research Station, Agriculture and Agri-Foods Canada, Brandon, R7A 5Y3 Canada
(3) Crop Development Centre, University of Saskatchewan, Saskatoon, S7N 5A8 Canada

Presented at the 18th North American Barley Researchers Workshop, July 17-20, 2005