Dormancy in malting barley: Studies on drying, storage, biochemistry and physiology

Abstract

The project report describes an investigation into the problem of dormancy in malting barley through a combination of three approaches. The influence of thermal processing, microbes on grain and biochemical changes in the grain are each examined.

Thermal processing

Samples of a batch of deeply dormant Triumph barley were dried to 12% moisture content at various rates, by varying the temperature of the drying air (27°, 38°, 48°) and its relative humidity. Drying times were in the range 4 - 24 h. Batches were then stored in sealed containers at 15°, 25° and 38°C. At intervals sub-samples were taken and their germinabilities were assessed. It was apparent that two independent processes were operating: (1) post-harvest maturation, causing a decline in dormancy, and (2) progressive loss of viability, preceded by a loss in vigour. Dormancy declined faster at higher storage temperatures, but the rate of decline was independent of the drying regime that had been used. At 15°C the rate of recovery from dormancy for one of the barleys was so slow that even after a years storage the grain was not fit to malt. Loss of viability was more rapid at higher temperatures.

Other samples of Triumph barley were dried to various moisture contents, between 9.4 and 14.5 % and were stored at 27° and 38°C. The initial rate of recovery from dormancy as still temperature dependent, but was independent of the moisture content of the grain. Grain samples dried to 9.4 and 10.3% moisture and stored at 38°C achieved germinations close to the viability value in both the 1 ml (agar) and 3 ml (agar) tests and showed no loss of viability in 30 weeks storage. On the other hand, grain held at 11% moisture deteriorated after 12 weeks and that stored at 13% and 14.5% moisture deteriorated after 3 weeks. The germination of samples dried to 9.4% moisture and stored at 38°C for 15 weeks or more (1 ml and 3 ml agar tests) was so good that it exceeded any value likely to be achieved with ambient storage. Since low moisture contents protect against loss of viability but do not impair break of dormancy, they offer the opportunity for higher temperature treatments. Further work is needed in this area.

Experiments carried out on other grain samples, including other samples of Triumph (12% moisture content), confirmed the initial conclusions based on experiments using the first sample of Triumph. However, in probit germination/storage time curves the results of the first trials tended to give two-stage, 'dog-leg' curves, which were much less apparent in the other data. Consequently, the initial data were not used in developing the probit description of dormancy. Data was stored on a computer data base, and used to develop a model of dormancy, based on probit analysis of the results combined with the data of other workers on the viability of barley. The model allows a computer simulation of the 'life history' of a batch of stored barley and permits the prediction of the rate of recovery from dormancy of samples of Triumph barley, stored under different conditions. At higher storage temperatures, the rate of emergence from dormancy fell below that predicted by probit analysis for germination values over about 80%. This may be associated with a loss of grain vigour, when warm storing grain at 12% moisture content.

The development of the probit model of dormancy employed 9 x 100 seed counts which reduced the standard deviation of the data. This enabled a better quantitative comparison of the rate of break of dormancy for Triumph barleys from two sites and two seasons. In the temperature range, 15-38°C, the effect of site and season was not distinguishable.

In the course of the experiments, grain was cooled and frozen after harvest and subsequent to warm storage for transportation. No effect on germinability or rate of break of dormancy was observed.

Microbes on grain

Various studies have shown that microbes depress the germination and vigour of barley. It was demonstrated that radioactive glucose was metabolised by microbes in the surface layers of the grains, and that the sugar did not reach the living tissues of the grain itself. Microbes on the grain can reduce triphenyl tetrazolium chloride to a red formazan, which can be quantified. This observation was used to develop a technique for quantifying the microbial populations. The technique lacked sensitivity, but showed that the microbes increased, especially during warm and aerated steeping and that growth was checked by dilute acid.

Measurements of the oxygen uptake rates of strips of husk and the water in which the husks are suspended confirmed the French reports that husk 'respiration' is initially lower in samples taken from mature grains than in samples taken from dormant grains. It was shown that the respiration was due to microbes which proliferated on the husk and in the surrounding water, supported by nutrients leached from the husk. Anaerobic and acidic steeping conditions check microbial multiplication.

Sulphur containing substances

Total thiols were determined in extracts of barley embryos using DTNB, (5,5'-dithiobis(2- nitrobenzoic acid)). Thiols were separated from crude barley extracts using binding to agarose substituted with p-hydroxymercuribenzoate (p-HMB agarose). Disulphides were recovered, as thiols, by trapping them in the effluent from the first p-HMB agarose column, on an ion exchange resin. After elution they were reduced with DTT and derivatised with DTNB and the derivatives were separated by h.p.l.c., being quantified by their absorbance at 330 nm. The major thiols in barley embryos were cysteine and glutathione, but traces of y-glutamylcysteine and cysteinylglycine were detected. The major disulphides were cystine and oxidised glutathione.

The changes in cysteine, cystine, glutathione and oxidised glutathione were determined in the embryos of mature and dormant samples of barley grown under malting conditions. The most striking changes were the declines in glutathione and oxidised glutathione early in the malting process and the rise in the latter as visible germination took place. No significant differences were found between the levels of the thiols and disulphides in dormant and mature grains.

When grains were steeped in hydrogen peroxide, as in the germinative capacity test, glutathione levels were slightly lower in the dormant than in the mature grain, but the cysteine and disulphide levels were essentially the same. When dormant and mature grains were steeped in a solution of 2,3-dimercaptopropanol (BAL) the germination of the mature grain was retarded, but that of the dormant grain was enhanced. The thiol and disulphide levels were the same in the dormant and mature grains. However, in both cases, the apparent levels of cysteine were greatly enhanced. This could not be confirmed by analysis of the amino-acids in extracts of the barley grains, and may have been due to the DTNB derivative of a metabolite of BAL co-chromatographing with DTNB-cysteine on h.p.l.c. Subsequent experiments showed that BAL appeared to be metabolised by barley grains, but attempts to characterise the substance, by mass spectrometry, have not been successful. The DTNB derivative of BAL does not interfere with the h.p.l.c. assay of the other thiols.

The fall in glutathione and oxidised glutathione, which occurs in the early stages of germination, is due - at least in part - to the transfer of these substances to the starchy endosperm. The thiols initially present in the 'non-embryo' part of the grain appear to be confined to the aleurone layer.

Dormant and mature samples of barley were steeped, with or without BAL, and were germinated for 3 or 5 days before kilning. The malt samples so obtained were kindly analysed by Pauls Malt Ltd. The BAL steep retarded malting in the mature grain, as reflected in the hot water extract, the diastatic power, the soluble nitrogen ratio and the wort viscosity. The, dormant barley, steeped with BAL, was less advanced than the water-steeped control after 3 days germination but after 5 days germination the BAL-treated samples were more advanced. However, the quality of the malts prepared from dormant barley, steeped in BAL. did not approach those prepared from the water-steeped, mature barley.