THE EFFECT OF SUSPENDED SOLIDS ON THE FERMENTATION OF DISTILLER'S MALT WORT

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1 THE EFFECT OF SUSPENDED SOLIDS ON THE FERMENTATION OF DISTILLER'S MALT WORT By N. R. Merritt, B.Sc. {Scottish Grain Distillers, Lid., GlenochilResearch Station, Menstrie, Clacktnannanshire, Scotland) Received 26fA January Yeast growth is increased and the initial rate of fermentation is accelerated by the residue of insoluble material which is normally present in distiller's malt wort. The increase in yeast concentration is accompanied by a high glycerol content in the fermented liquor and the formation of higher alcohols, particularly Iso and 2-methyl, is also increased. The stimulation of yeast growth is independent of the state of aeration of the wort, suggesting that the effect is not associated with entrainment of oxygen by the solids, and the chemical nature and particle size of the suspended material do not appear to be important. Insoluble solids were without effect when yeast growth was limited by carbohydrate rather than by amino-nitrogen. The results suggest that a combination of electrostatic and absorption forces toncentrate yeast cells and amino adds at the solid-liquid Interface, increasing the rate of uptake of nutrients by the cell and thus diverting carbohydrate to processes associated with yeast growth. Introduction profound effect upon bacterial growth,*-1 During the preparation of distillery malt probably because of a concentration of wort a small quantity of insoluble spent grain essential nutrients at the solid-liquid interusually passes with the wort into the ferment- face,6 and Crowell & Guyrnon* have reported ing vessels. The concentration of this that the formation of higher alcohols during suspended material can vary widely and is ««fermentation of grape must is stimulated dependent upon many factors among which by grape skins or inert sobds. Accordingly, are? presumably, the depth and degree of Jhe effect of insoluble grain solids on the stirring of the grain bed, the condition of the fementation of distiller s malt wort by yeast mash-toin plates, the wort drainage rate, the has been studied and the results are presented nature of the malt and the type of grind used.»n *n»s paper. A typical wort might be expected to contain _ approximately 6% of dry solids and since Experimental this small amount of insoluble material in Laboratory mashing and fermentation prono way interferes with normal operations in cedure. Malt wort was prepared and ferthe distillery no attempt is usually made to rriented as described earlier5 using a constant remove it. However,,the presence of solid fermentation temperature of 3 C. In order surfaces in liquid media is known to have a to produce worts containing insoluble solids,

2 Vol. 73, 1967] merritt: effect of suspended solids on fermentation 485 spent grains were taken from a pilot mashtun, washed exhaustively in order to remove traces of fermentable material and added to clear worts after the dry matter of the material had been determined. In certain cases the spent grains were fractionated by wet-sieving. The solids were washed with water through a series of sieves of 1-, 18-, 3- and 1 mesh (B.S. 41, Endecotts Ltd.) and the fine material passing the 1-mesh sieve was collected and recovered by centrifugation. The five separate fractions so obtained were then added individually to worts after the determination of moisture content. Worts containing a natural dis tribution of insoluble solids were obtained by drawing distiuery samples and in these cases control worts free from spent grains were obtained by nitration. Yeast growth under carbohydrate limitation. Malt wort prepared in the usual way and containing 13-5 g. carbohydrate and 2*5 mg. a-amino nitrogen per 1 ml. was diluted 1-fold with water. Sufficient casein hydrolysate was added to the medium to restore the a-amino nitrogen to its original level, and the carbohydrate content of the wort was adjusted to 2-5% with glucose. Portions of this dilute wort were seeded with -1% (w/v) distiller's pressed yeast and fermented in the presence or absence of 25 mg. dry solids per 1 ml. Yeast growth was estimated daily by centrifugation of a 25-ml. sample. The yeast so obtained was washed and dried at 15 C. for 4 hr. Corrections for the amounts of spent grains present were made where necessary. The state of glucose-limitation of wort was verified in a subsidiary experiment in which yeast growth was found to be directly related to the carbohydrate content up to a concentration of at least 5% in the dilute medium. Aeration. Three conditions of aeration were used. In normal fermentations, wort was contained in bottles closed with water traps and the only air available was that dissolved in the wort and that of the headspace of the bottle {2-75 vol. per vol. of wort). For anaerobic conditions, fermentations were fitted with glass spargers and were de-aerated by means of a vigorous stream of high purity nitrogen for 2 hr. before the addition of the yeast and for a further 1 hr. after pitching. In aerobic fermentations the wort was aerated as described earlier8 throughout the whole fermentation period at a rate of 5 ml. air per litre of wort per hr. Analyses. Glycerol was estimated as de scribed earlier.6 Higher alcohols were ob tained by distillation8 and after 1-fold concentration were determined by gas chromatography using a -ft in. x 15 ft. copper column packed with 5% polyethylene glycol 555M and with Embacel 6/8 mesh as solid support. The temperature of the column was programmed from 35 to 85 C. Results and Discussion When solids derived from spent grain were added to clear malt wort a striking effect on yeast growth was evident. As little as -5% dry solids in the wort caused an increase of more than 1% in the amount of yeast produced during the first 18 hr. of fermentation (Table I), the period in which TABLE Ykast Growth in Wort Containing Insoluble Malt Solids Insoluble solids content of wort (g. per 1 ml.) I New yeast growth (g. per 1 ml. at 25% dry matter) Fermentation time (hr.) about 9% of the total carbohydrate of the wort is metabolized. During the second phase of the fermentation, autolytic processes cause a steady decline in yeast dry matter content which tends to obscure the differences between bright and solids-containing worts. However, it is clear that an asymptotic TABLE II Effect of Suspended Solids on the Rate op Fermentation of Wort Time (hr.) Ethanol production (g. per 1 ml.) Insoluble solids in wort (mg. per 1 ml.)

3 48G merkitt: effect of sisit.npf.i) solids on fermentation fj. Inst. Brew. relationship exists between yeast growth and the concentration of insoluble materials (Table I), which is reflected in the initial rate of fermentation. As the results in Table II show, the rate of ethanol production in wort containing -5% suspended solids is in creased by about 1%, although the final alcohol concentration is actually higher in worts which are free from solids (Tables II and III). TABU- III Glycerol and Ethascol Production in thk Presbnce of Insoluble Solids Wort 1 o Insoluble solids content (nig. per 1 ml.) "i 5 Glycurol production (nir. per 1 ml.) Ethanol production (g. per 1 ml.) :i In anaerobic fermentation processes a relationship between the production of new yeast and the formation of glycerol is to be expected5 and in the present study it has been found that the increase in yeast growth caused by material suspended in the wort is accompanied by a high glycerol content in the fully fermented liquor (Table III). NordstrSm7 has recently deduced a quanti tative correlation which suggests that the formation of 1 g. of yeast dry matter in a glucose-ammonium salts medium should be accompanied by the production of 5- or 7-5 m. moles of glycerol according to the synthetic mechanisms involved. This amount would presumably be less in complex media such as wort in which the yeast is supplied with pre-formed amino acids so that the present findings, in which approximately 4-5 m. moles of glycerol were produced per g. of yeast dry matter, agree well with the theoretical calculations. Fusel oil production was also influenced by the presence of insoluble solids as shown in Table IV. With the possible exception of n-propanol, the formation of all the major higher alcohols was affected by the solids but the increased concentration of iso and 2-methyl was particularly pronounced. Thus the total effect of insoluble material in wort appears to be a stimulation of yeast growth associated with increases in the formation of glycerol and higher alcohols and accordingly leading to a small reduction in ethanol production. Increases in higher alcohol production due to insoluble material have been reported before2 but have usually been ascribed to an adsorption of oxygen by the solids.2-8 In the present instance, the influence of suspended solids is unlikely to be associated with entrainment of oxygen into the wort since similar results were obtained after worts containing insoluble material had been de-aerated by vigorous sparging with nitrogen for 2 hr. before fermentation. In any case, it may be relevant to note that, in agreement with Cowland & Maule1 but in contrast to Crowell & Guymon,2 we have found that moderate levels of aeration cause no increase in higher alcohol production in clear or in turbid worts (Table V). Insoluble material stimulates yeast growth in both static and stirred fermentations so that its activity is presumably unrelated to TABLE Effect ov Insoluble Solids on Higher Alcohol Production IV Wort Insoluble solids content (mg. per 1 ml.) n-propanol Production of higher alcohols (mg. per 1 ml.) Iso 2-Methyl butanot 3-Methyl Total o 3S no fl 'fl G

4 Vol. 73, 1967] mkrritt: effect ok suspended solids on fermentation 487 TABLE V Alcohol Frouuction Under Varvixi; Conditions of Akration Higher alcohol production (nig. per 1 ml,) Fermentation conditions* n-propanol lso 2-Methyl 3-Methyl Total Anaerobic Normal Aerobic ie 1-D I-H B c-r> * For details sec text. any change in the pattern of yeast sedimenta tion, and when spent solids taken from a mash-tun were fractionated by wet-sieving and the individual fractions added separately to clear worts, all fractions were active to the same degree. It appears, therefore, that particle size is of no significance and the nature of the suspended material also seems to be unimportant since cellulose powder, celite or filter paper all cause effects similar to those obtained with spent grains. In the standard malt wort used in this study the growth of the yeast was limited by the availability of amino acids. However, it was found that when an experimental wort was diluted with casein hydrolysate so as to Fermentation time (days) Fig. 1. The effect o( insoluble solids on yeast growth under conditions of amino acid or carbohydrate limitation. AH wort contained 2-6 tog. a-atnino nitrogen per 1 ml., and 2-6% and 13-5% carbohydrate for conditions of carbohydrate and amino acid limitation, respectively. # Clear wort; O O Wort containing -25% insoluble solids. produce a medium in which the carbohydrate concentration was the limiting factor, sus pended solids caused no increase in the growth of the yeast (Fig. 1). According to Mitchell6 the interface between most naturally-occur ring substances and a growth medium at physiological ph is negatively charged, in which case a concentration of amino acids and other positively charged material should take place at the surface of the solids suspended in the wort. Yeast itself also canies a negative charge during fermentation but the yeast cell wall has strongly adsorptive properties and Jansen & Mendlik* have shown that these adsorption forces can be stronger than electrokinetic repulsion, so that yeast cells might also be expected to collect at the surface of the insoluble material. Thus the situation in wort containing suspended solids appears to be that a concentration of yeast cells and growth-promoting nitrogenous com pounds occurs at the solid surface, so resulting in an increase in the rate of uptake of nutrients by the yeast, and hence leading to an acceleration in the rate of cell growth. This has the effect of diverting more carbo hydrate into biochemical processes associated with yeast growth and in consequence the amount of carbohydrate metabolized by the pathways of fermentation is slightly de creased. Acknowledgements. The author wishes to express his thanks to the Directors of the Distillers Company Limited for permission to publish this paper, and to Dr. M. Pykc, F.R.S.E., for his interest during the course of the investigation. References 1. Cowland, T. \\\, & Maule, D. U., this Journal, 195, Crowcll, E. A., & Giiymon, J. F., Anitr. J. Enol. <S- Vilirult., 193, 14, 214.

5 488 merritt: effect of suspended solids on fermentation [J. Inst. Brew 3. Heukelckian. M.. & Heller, A., /. Bad., 14, 4, Janscn, H. E., & Mendlik, F.. Proc. Eur. Brew. Com. Nice fl. Merritt, N. R.. this Journal, 16, Mitchell, P., in Bacterial Physiology. New York: Academic Press, Nordstrdm, IC, Ada Client. Scand., , 11. S. Webb, A. D., & Ingraham, J. L., in Advances iii Applied Mierobiol., Vol. 6. New York: Academic Press, Wiles, A. E., Proc. Eur. Brew. Conv. Brighton, ZoBcli, C. H.. J. Butt , 39.