340 [J. Inst. Brew. BREWING INDUSTRY RESEARCH FOUNDATION SIGNIFICANCE OF THE USE OF HOPS IN REGARD TO THE BIOLOGICAL STABILITY OF BEER I. REVIEW AND PRELIMINARY STUDIES By R. M. Macrae, B.Sc, Ph.D., M.I.Biol. (Brewing Industry Research Foundation, Nutfield, Surrey) Received 2] si March, 1964 The extent of survival of brewery bacteria, i.e,, two strains of Acetobocter spp. and a Lactobacillus sp., and one wild yeast, Pichia membranaefaciens, in beer brewed from wort hopped at different rates has been studied. The gram-negative species and the wild yeast were apparently unaffected by the presence of hop materials and the Laetobacillus sp., although initially inhibited, recovered despite the presence of hop antiseptics. The enhancement of the biological stability of beer attributed to the use of hops has therefore perhaps been overemphasized. Introduction Much of the past work on the subject of the preservative value of hops has been con cerned with hop substances and bacterial infections which lack definition. This communication, it is emphasized, is concerned with antiseptic potency as assessed by biological methods. This definition is essential in view of the loose manner in which the term preservative value (P.V.) is often employed. Thus, because of much previous work suggesting a connection between the hop resins and the biological preservative value,2-4!19'24'25 it has frequently been the practice to describe the potential P.V. of a hop sample in terms of its resin analysis. Reference to the work of Chapman,2 McHugo9 and Williams & Todd,27 however, suggests that there is only a rough correlation between the biological P.V. and the resin content. Many earlier studies on biological P.V. employed uncharacterized or poorly charac terized organisms which sometimes had little significance as causative agents of brewery spoilage. Several were used simply as "biological reagents," being specially selected because of their apparent sensitivity to the antimicrobial materials present in hops. Moreover, the procedures employed for testing biologically active substances from hops have varied. Chapman2 made dilutions of a boiling water extract of hops with molten nutrient agar. After sterilization, three drops of a culture of a spore-forming aerobic bacillus were added and the effect of the hop extract was noted by counting the number of colonies developing in the agar. On the other hand Walker19 examined the antiseptic potency of the a and /3 resin fractions and subfractions by measuring the suppression of growth of four different organisms for fixed periods of time. Sediments from "forced" beers were employed to test water extracts of hops by Hopkins & Fraser,6 who estimated the antiseptic action by the inhi bition of the development of acidity. In 1932, Walker, Hastings, Farrar & Day21 concluded that the grading of antiseptic strengths of beers was similar to that of the worts from which they had been brewed. These workers stated further that hops could be graded by chemical methods to give an estimate of their biological preservative value, and that gravimetric analysis could provide a satisfactory basis for estimating the relative resistance of the derived beers to infection. In a series of studies on the biological stability of beer and the effect of hop anti septics on lactobacilli, Shimwell12-18 firstly13 concluded that possibly no bacterium exhi bited such a resistance towards hops as did Saccharobacillus, now named Ladobacillus.11 However, in later publications16'17 he showed that other organisms notably gram-negative bacteria were more hop resistant. These experiments were carried out using hopped
Vol. 70, 1964] MACRAE: HOPS IN REGARD TO BIOLOGICAL STABILITY OF BEER 341 brewer's wort rather than beer. In beer, Shimwell using the accumulation of acid as the criterion of biological instability found that the preservative effect of hops could be overlaid by the presence of residual sugar. In a review of previous studies,18 Shimwell concluded that the best routine precautions against bacterial infection were to employ as high a hop rate and as low an initial ph value as practicable, whilst keeping residual carbohydrates to a minimum. were shown to vary markedly in their relative sensitivities to different antiseptic agents. A mathematical treatment was given in a later paper by Walker & Parker,84 and the loss of preservative value which takes place during boiling and fermentation was also reported.8 >B Finally, mention may be made of the work of Williams & Todd" who carried out a series of brewings with hops having Iowa acid contents; they showed that these hops conferred upon beers produced 12 I 2 3 45 6 Fig. 1. Acelobacler strain A grown in beer: Incubated aerobicatly (full lines) in beer from unhopped wort (%); in beer from wort hopped at A lb. per brl. (A) ' aru' 'n beer from wort hopped at 3 1b. per brl. ( ). Incubated anaerobically (broken lines) in beer from unhopped wort (O); in beer from wort hopped at A 1b. per brl. (A) ' and in beer from wort hopped at 3 lb. per brl. { ) Walker, Hastings & Farrar20 introduced what they termed the "log phase method" for the evaluation of the bacteriostatic potency of substances derived from hops, and later commented upon its theoretical basis.23 Phenol was used as an antiseptic standard, and a species of Laclobacillus isolated from cheese as the test organism. This bacterium was later replaced with another from a similar source, as Walker found that the original strain of the organism had weakened after two years of cultivation in the labora tory. Walker acknowledged the difficulties in interpretation which might arise from variability in the behaviour of the organism. Thus, the two strains of lactobacilli used from them a significant preservative value measured by the restriction in the develop ment of acidity after inoculation with a strain of Lactobacillus pastoriamts. Howard8 reviewed chemical and biological methods for the estimation of hop P.V. It was concluded that, although microbiological tests presented the only direct means of determining the bacteriostatic potency of hop derivatives in beer, they left much to be desired. Lupulone, humulone, tsohumulone and humulinone were tested for their bacterio static action by Cook & Harris,8 who measured the suppression of growth by dilution of these compounds in laboratory
342 MACRAE: HOPS IN REGARD TO BIOLOGICAL STABILITY OF BEER [J. Inst. Brew. media. Two strains of Lactobacillus spp. were employed amongst other organisms and active dilutions of all compounds lay between 1:20,000 and 1:100,000, with the exception of lupulone which was inactive against one of the strains. Further work on substances from hops was carried out by Hough, Howard & Slater7 who compared total counts of organisms growing in media with and without the addition of the test compounds. It was shown that humulone and the related com ponents cohumulone, adhumulone and isohumulone had similar antiseptic potencies for 1 hr. under reflux (a) without hops and (6) with minced hops at the equivalent rates of J lb. per brl. and 3 Ib. per brl. The hops were Kent Fuggles from the 1961 crop, and had an initial a acid content of 5-1%. The worts were dispensed in duplicate 2-litre volumes in tall glass tubes (2 in. x 00 in.) and pitched with 5 g. (moist weight) of brewing yeast strain N.C.Y.C. 1040 {Sacch. cerevisiae), grown in Roux bottles containing 200 ml. of unhopped wort at 25 C. on a reciprocating shaker. Fermentations were continued until the sp. gr. had fallen to 1-010, which normally 4 S 6 Fig. 2. Acelobacler strain B grown in beer. Key as in Fig. 1. against one strain of Lactobacillus plantarum, but a slight variation in activity was shown with a strain of Laclobacilltis pastorianus. Humulinone and the related compounds /sohumulinone and a humulinone boiling product were not markedly bacteriostatic. The purpose of the work reported in this paper is to re-examine what relevance the use of hops bears to the biological stability of beer. Experimental and Discussion Wort was produced by mashing an all- Proctor malt grist for 90 min. at 65-6 C. (150 F.) with distilled water containing 200p.p.m. gypsum. After adjusting the sp. gr. to 1-040, 4-litre quantities were boiled took 87-90 hr. at 18 C. After chilling to 3 C, 500-ml. quantities of the beers were filtered through Oxoid membrane filters and were checked for sterility. This method was adopted to avoid possible artefacts introduced by pasteurization. The ph value of each was determined, and all were found to lie between 3-80 and 3*95. Values of 13 and 42 mg. per litre bitterness were given by the Brenner method1 for the beers hopped at i and 3 lb. per brl. respectively. It was noted incidentally, that the degree of yeast head formation was correlated with the hopping rate, the largest head being formed in the wort hopped at 3 lb. per brl. The sterile beers were dispensed in 10-ml. volumes into sterile test tubes closed by
Vol. 70, 1964] macrae: hops in regard to biological stability of beer 343 cotton wool plugs. Six tubes of each beer were inoculated singly with each of the four organisms enumerated below. These were Pichia membranaefaciens, N.C.Y.C. 326, and a Lactobacillus sp., isolated from spoiled beer; and two strains of Acelobacter spp. from brewery pitching yeast. The tubes were incubated at 25 C. in such an arrangement that triplicate samples of each beer and organism were forced both aerobically and anaerobically. Viable counts were then undertaken using the Miles & Misra10 tech- I 2 Fig. 3. Pichia membranaefaciens grown in beer. Key as in Fig. I. nique on samples taken initially and after 1, 2, 3 and 7 days of incubation. Three suitable dilutions were made, and plate counts were carried out in duplicate on the requisite medium for each organism, viz., Lactobacillus sp., medium "L",*8 Acetobader sp., medium "A",28 and Pichia membranaefaciens, M.Y.G.P.28 The above procedure was specifically designed with regard to the growth of brewery organisms in beer thus giving, it is hoped, some practical relevance to the study. The averages of the viable counts obtained from the plates were employed to construct Figs. 1-4. The 24-hr, samples of the beers infected with one strain of Acelobacter sp. were con taminated and could not be counted, but it is clear from Figs. 1 and 2 that the presence of hop materials makes little difference to the growth of the strains tested. As would be expected with an aerobic organism, rather less growth took place during incubation in an anaerobic environment, such growth as occurred presumably being facilitated by oxygen previously dissolved in the beers. Pellicle formation after two days of incu bation of the beers infected with Pichia membranaefaciens precluded accurate viable counts being carried out, though by that time the early growth trend had been established (Fig. 3), showing the picture to be similar, the organism clearly being insensitive to hops. Since it is an aerobic species, growth is relatively poor under anaerobic conditions. The results with the bacteria confirm for beer the findings of Shimwell10 who used wort. He reported that gram-negative species were unaffected or even stimulated by hops and in the present work both Acetobacler strains, which are gram-negative, were insensitive to the hop substances in the beers. This finding has a greater relevance to the bio logical stability of beer than the behaviour of organisms in wort, in view of losses and changes in hop materials during fermentation. In view of the spoilage difficulties associated with wild yeasts it might be anticipated that hop substances have little inhibitory effect on their growth in beer, and this has been confirmed using one common beer-contami nating species. When the species of Lactobacillus was inoculated into beers hopped at different rates, the effect on its survival was in marked contrast to that on the other organisms (Fig. 4). Thus, in unhopped beer the bac terium can multiply both anaerobically and aerobically. At an equivalent hopping rate of lb. per brl., however, the growth of the organism is inhibited, the effect being more pronounced under anaerobic conditions. It should be noted that some recovery of growth takes place, and the effect is bacteriostatic rather than bactericidal. This would suggest that the organisms would continue to metabolize and go on producing acid under these conditions. At the higher hopping rate equivalent to 31b. per brl. a killing action was demonstrated when the incubation was carried out anaerobically. In the aerobic
344 MACRAE: HOPS IN REGARD TO BIOLOGICAL STABILITY OF BEER [J. Inst. Brew. 3 4 Fig. 4. Lactobacillus sp. grown in beer. Key as in Fig. 1. samples, however, although growth was markedly inhibited, a recovery of the organism was evident after the initial effect. Even with the gram-positive Lactobacillus sp., therefore, it seems that under the conditions of the experiments survival of the organism in presence of relatively high concentrations of hop materials is possible. This ability of lactobacilli to recover from the inhibitory action of hops, suggesting the development of resistance to hop materials, is now under further investigation. These results imply that the significance of hops in relation to the enhancement of the biological stability of beer may in some respects have been overemphasized. Acknowledgement. The author acknow ledges the encouragement of Dr. A. H. Cook, F.R.S., Director of the Foundation. References 1. Brenner, M., Vigilante, C, & Owades, J. L., Proc. A. M. Amer. Soc. Brew. Chem., 1956, 48. 2. Chapman, A. C, this Journal, 1925, 13. 3. Cook, A. H., & Harris, G., /. chem. Soc, 1950, 1873. 4. Hastings, J. J. H., & Walker, T. K., this Journal, 1928, 550. 5. Heron, H., this Journal, 1941, 178. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. Hopkins, R. H., & Fraser, J. E., this Journal, 1928, 387. Hough, J. S., Howard. G. A., & Slater, C. A., this Journal, 1957, 331. Howard, G. A., this Journal, 1953, 36. McHugo, C. W., this Journal, 1938, 294. Miles, A. A., & Misra, S. S., /. Hyg., Lond., 1938, 38, 732. Pederson, C. S., in Bergey's Mannual of Deter minative Bacteriology. London: BaiUiere, Tindall & Cox Ltd., 1957. Shimwell,. L., this Journal, 1935, 245. Shimwell,. L., this Journal, 1935, 481. Shimwcll,. L., this Journal, 1936, 127. Shimwcll,. L., this Journal, 1936, 452. Shimwell,. L., this Journal. 1937. 111. Shimwcll,. L., this Journal, 1937, 191. Shimwell,. L., this Journal, 1937, 450. Walker, T. K., this Journal, 1925, 562. Walker, T. K., Hastings, J. J. H., & Farrar, E. J., this Journal, 1931, 512. Walker. T. K., Hastings, J. J. H., Farrar, E. J.. & Day, F. E., this Journal, 1932, 303. Walker, T. K., Hastings, J. J. H., & Parker, A., this Journal. 1940, 304. Walker. T. K., & Parker. A., this Journal. 1937. 17. Walker, T. K.. & Parker, A., this Journal, 1940, 235. Walker, T. K., & Parker, A., this Journal. 1940, 337. Wickerham, L. J., Tech. Bull. No. 1029. U.S. Dept. Agric, 1951. Williams, D. O., & Todd, J., this Journal, 1951, 450. Williamson, D. H., this Journal, 1959, 154.