since it is relevant to the propriety of assessing the relative nutrient values of

Transcription

1 18 thorne: bios requirements of brewery yeasts [Jan.-Feb., 1949 of the correlation coefficient which is classed as not significant means that there is at least one chance in ten that such a result could be obtained fortuitously from a sample of experimental results; a significant value is such that there is less than one chance in twenty that it could arise fortuitously. Values intermediate between these are regarded as doubtfully significant, while values which could arise by chance less than once in a hundred times are considered very significant. THE BIOS REQUIREMENTS OF SOME TOP-FERMENTATION BREER EASTS IN RELATION TO THE NITROEN SOURCE By R. S.. Thorne, Ph.D. (Received by the Chairman: 10/12/47) It has been suggested that the bios requirements of yeast may vary to some extent according to the nature of the nitrogen source. In view of the undoubtedly complex nature of the synthetic reactions involved in the production of proteins by yeast from simple nitrogen compounds, such a suggestion is inherently likely. Consequently it was felt that this question should be examined more closely since it is relevant to the propriety of assessing the relative nutrient values of different nitrogen sources under constant conditions of bios dosage. The experiments reported in the following paper show that certain differences in the bios requirements of yeasts are apparent when a comparison is made between ammonia and amino acids as nutrients. These differences are, however, quite small and do not affect to any appreciable extent the assessment of their relative nutrient values. differences in the bios requirements of amino acids between themselves have been established. It has further been shown that the bios require ments of different top-fermentation brewery yeasts may vary quite markedly; in fact, out of four strains studied three different kinds of bios requirement were found. ildiers (La Cellule, 1901, 18, 313) dis covered that yeast was unable to grow on a synthetic nutrient medium containing only sugar, ammonia and mineral salts, but that it would do so as soon as a small quantity of yeast-water was added to the medium. The unknown growth-promoting factor or factors evidently present in yeast-water was named "bios" by ildiers. After four decades of research in the vitamin field it has become clear that ildiers' bios is a complex of growth factors of which the most significant, from the point of view of yeast nutrition, are inositol, pantothenic acid, biotin, aneurin and pyridoxin. All these, and many other, growth factors are known or suspected to be essential to the metabolic activities of living cells in general; but in the study of any par ticular organism, e.g. yeast, a growth factor only becomes nutritionally important when the cell is either incapable of synthesizing it, necessitating an external supply, or when the rate of synthesis is slow enough to act as a brake on the rate of growth. The role of these growth factors in the economy of the cell is imperfectly under stood at present. The function of inositol (I) is unknown; it differs from the'other yeast growth factors in that its effect is only apparent in relatively large dosage. Panto thenic add (II) and biotin (III) are nutrition ally the most important of these factors.

2 Jan.-Feb., 1049] thorne: bios requirements of brewery yeasts 19 Biotin is fully active in the exceedingly low concentration of 10* gnns. per ml. It appears (inzler, Burk and du Vigneaud, Arch. Biochem., 1945, 5, 25) to take part in the synthesis of some nitrogenous substance involved in the fermentation cyde. The pyrophosphate of aneurin (IV) is the cocarboxylase of yeast which is involved in the pyruvic acid -* acetaldehyde stage of the HOH (I) CH,OHC(CHi),CONHCH,CH,CO0H CH,,-C (II) /CH,CH,CH,CH,COOH.NH-CH-C v ^NH CH CH,/ NH, (III) N = C C CH, I C CH, N N CH^^ / CH fe~ HO # (IV) CH.OH \ CH.OH C-CH,CH,OH fermentation cycle. Pyridoxal phosphate, a substance closely related to pyridoxm (V) functions as the coenzyme of amino add decarboxylases (Baddiley and ale, Nature, 1945, 155, 727) and also as the coenzyme of transaminase (Lichstein, unsalus and Umbreit, /. Biol. Chem., 1945, 161, 311), so that it must clearly play an important part in protein metabolism. Schultz, Atkin and Frey (ibid., 1940, 135, 267) in a study of the bios requirements of yeast, found that when urea was used as a nitrogen source, more bios IIB (biotin) and less bios IIA (pantothenic acid) were needed than when the nitrogen source was ammonia. They therefore suggested that the bios requirements of yeast might be considerably affected by the form of nitrogen supplied and consequently that such work as that of the present writer on the nutrient efficiency of different amino acids (this Journ., 1941, 255) might profitably be re-examined with this consideration in mind. The present paper reports the results of experiments on the bios requirements of four top-fermentation yeasts designated {ibid., 1944, 222),, and in the presence of a number of different nitrogen sources, mostly amino acids. The yeasts are single cell cultures; was originally isolated from a Burton yeast, from a London yeast and from a orkshire yeast; the origin of is unknown but it closely resembles a Midland yeast. Experimental The basal nutriment medium used was as follows: 1,000 mis. distilled water/' 2 gnns. potassium dihydrogen phosphate 1 grm. magnesium sulphate (cryst.) 0-2 grm. calcium sulphate 0-01 grm. ferrous sulphate (cryst.) 60 gnns. sucrose 3*5 mis. lactic acid 12 mis. 50 per cent, potassium lactate soln. To this medium was added 035 grm. assimil able nitrogen in the form of ammonium phosphate or an amino add, and the pa of the medium adjusted to 6-0. Finally, appro priate amounts of the desired growth factors were added. The media were put out in 100 ml. portions in special fermentation vessels (ibid., 1939, 13), inoculated with washed suspensions of the yeasts and grown for 4 days with constant shaking at 21 C. Periodic measurements of yeast growth and fermentation were made in order to evaluate (a) the final yeast growth, (6) the initial relative growth rate and (c) the fermentation factor (ibid., 1941, 255). The individual figures quoted in the subsequent tables are mean values obtained usually from

3 20 thorne: bios requirements of brewery yeasts [Jan.-Feb., s TABLE I Tbe Effects of Various Combinations of Inositol (I), Pantotbbnic Acid (P) and Biotin (B) Using easts,, and, with Ammonium Phosphate as the Nitroobn Source addition. I. P. B. IP. IB. PB. IPB. Final yeast growth in grrns. per litre ± M Initial relative growth rate ± Fermentation factor ± C (100)» * This is an arbitrarily chosen standard value of the fermentation factor. TABLE II The Effects of Various Combinations of Inositol (I), Pantotbbnic Acid (P) and Biotin (B) Using easts 6470,, and, with lutamic Acid as the Nitrogen Source addition. I. P. B. IP. IB. PB. IPB. Final yeast growth in grms. per litre ± Initial relative growth rate : t l-eo Fermentation factor ± (1-00)

4 Jan.-Feb., 1949] thorne: bios requirements of brewery yeasts 21 measurements made on eight separate cul tures. In all cases the standard deviations of these mean values are specified. The first series of experiments was designed to ascertain the effects of inositol, pantothenic acid and biotin, used singly and in combina tion, upon the four yeasts when ammonium phosphate was the source of nitrogen. A considerable experience of the growth of yeast in synthetic media has shown that satisfactory growth is obtained if these growth factors are used in the following concentrations: Inositol: 20 mgrms. per litre; Pantothenic add (as calcium salt): 2 mgrms. per litre; Biotin: 1 /xgrm. per litre (1 /xgrm. = 1/1000 mgrm.). Accordingly, these concentrations were used in the present series of measurements. Eight media, differing in their bios content, were used, viz. no growth factors (control), inositol only, pantothenic acid only, biotin only, inositol + pantothenic acid, inositol -f- biotin, pantothenic add + biotin, and inositol 4- pantothenic add + biotin. The experimental results are given in Table I. The results of an exactly similar series of experiments using glutamic add in place of ammonium phosphate as the nitrogen source are given in Table II, and the two sets of data may conveniently be considered to gether. In the absence of all three growth factors none of the yeasts grows or ferments satis factorily with either nitrogen source. The addition of inositol only or of biotin only produces as a rule only a small improvement if any; the addition of pantothenic acid alone tends to give a somewhat greater improve ment. It is more informative to take the growth and fermentation in the presence of all three factors as a standard of comparison and to contrast with this the figures obtained when one or other of the factors is absent (the last four columns of Tables I and II). Since the figures for final growth, initial relative growth rate, and fermentation tend to run parallel with each other there is some justification in taking an average value derived from all three of them. This is done in Table IIA which shows the percentage reduction of this averaged growth character istic caused by the omission of growth factors from the medium. The absence of biotin reduces the response of all the yeasts, though the reduction is rather less marked with and. /It also seems that the amino add is a little less sensitive to absence of biotin than ammonium phosphate. Absence of inositol causes only a slight reduction of response with yeasts, and, but a distinctly greater one with yeast ; the reductions are perhaps a little greater with ammonium phosphate than with glutamic add..absence of pantothenic add produces a marked reduction in the growth of yeasts, and, but little or none in ; the need for pantothenic acid seems appreciably TABLE IIA Percentage Reduction in rowth and Fer mentation of easts,, and in thb Absence of Different rowth Factors east. Nitrogen source. Ammonium phosphate lutamic acid inositol panto thenic acid biotin greater when glutamic add is the nitrogen source than when this is ammonia. Appar ently the differences among the yeasts in respect of bios requirements are more con spicuous than the differences between the nitrogen sources. Roughly, it can be said that all the yeasts require biotin, all but require pantothenic add and none but requires inositol. ith glutamic add the requirement for pantothenic add is slightly greater, and that for inositol slightly less, than with ammonia. The response of the four yeasts to varia tions in the concentrations of pantothenic add and biotin was next examined, using ammonium phosphate as the nitrogen source. Inositol in a concentration of 20 mgrms. per litre was present in all the media. Then, with a fixed concentration of 1 ftgrm. of biotin per litre, the concentration of panto thenic acid in different media was adjusted to 0005, 0026, 0-128,, 3-2 and 16

5 22 thorne: bios requirements of brewery yeasts [Jan.-Feb., 1949 TABLE The Effects of Varying trb Concentrations of Pantothbnic Acid and Biotin Using basts,, and, with Ammonium Phosphate as the Nitrogbn Source III 1 figrm. biotin per litre. 2-0 mgrms. pantothenic per litre. acid Pantothenic acid. mgrms. per litre: Biotin, jigrms. per litre: Final yeast growth in grins, per litre ± Initial relative growth rate! ± Fermentation factor ± (1-00) TABLE IV The Effects of Varying the Concentrations of Pantothbnic and biotin. Using east, with Ammonium Phosphate, Alaninb, Lbucine and lutamic Acid as the Nitrogbn Sources 1 ftgrm. biotin per litre. Pantothenic acid, mgrms. per litre: mgrms. pantothenic acid per litre Biotin, pgrms. per litre: Final yeast growth in grins, per litre ± 0-6. Arom. phos. Alanine.... Lcucinc lut add ' Initial relative growth rate ± Amm. phos. Alanine.. Leucine lut, acid Fermentation factor ± Amm. phos. Alanine Leucine lut acid (100)

6 Jan.-Feb., 1049] thorne: bios requirements of brewery yeasts 23 mgrms. per litre, i.e. the concentrations were successively increased by a factor of 5. Similarly, with a fixed concentration of 2 mgrms. of pantothenic add per litre, the biotin concentrations were adjusted to 002, 0-2, 2 and 20 pgrms. per litre. The experi mental data obtained by growing the four yeasts in these media are given in Table III. Inspection of these figures shows that for pantothenic acid there is with yeasts, and a maximal concentration at mgrms. per litre; increasing the concentra tion beyond this value does not give any further improvement in the yeast response indeed, there tends to be a just perceptible decline. east, as already found, does not require pantothenic acid and hence altera tions in the concentration of this factor are without effect on its response. Over the range of concentrations of biotin examined there is a gradual improvement in the response of each of the yeasts which is, how ever, practically complete at 2 jugrms. per litre. To summarize, it can be said that varying the concentrations of pantothenic acid and biotin does not disclose any differ ences between the yeasts apart from the fact, already found, that yeast does not need pantothenic acid. An exactly similar series of experiments was carried out using only yeast, but four different nitrogen sources, namely, ammonium phosphate, alanine, leucine and glutamic acid, with the results* shown in Table IV. ith ammonium phosphate, alanine and leucine, maximal response to pantothenic acid is obtained, as before, at a concentration of mgrms. per litre; in the case of glutamic acid a slightly greater response is obtained when the concentration is increased to mgrms. per litre. The response to biotin Teaches a maximum value when its concentration is 2 /igrms. per litre. Thus the four nitrogen sources are alike in their quantitative requirements for panto thenic acid and biotin with the reservation that glutamic acid may possibly have a slightly greater requirement for the former. (This is not at variance with the previous finding that the yeasts are distinctly more sensitive to the absence of pantothenic acid when glutamic acid is the nitrogen source). Attention was next directed to the effects of some other substances upon yeast growth and fermentation, namely, aneurin, pyridoxin nicotinic acid and riboflavin. east was employed, with ammonium phosphate as the nitrogen source. Inositol, panto thenic acid and biotin in the concentrations already specified, were used in all the media. The concentrations of the remaining factors were: aneurin: 0-1 rngrm. per litre; pyri doxin: 0-1 mgrm. per litre; nicotinic acid: O'l mgrm. per litre; riboflavin: 0-2 mgrm. per litre. These were used singly and in all possible combinations, the results obtained for yeast growth and fermentation being shown in Table V. An analysis of these TABLE V The Effects of Aneurin (An), Pyridoxin (Pr), Nicotinic Acid (Nc) and Riboflavin (Rb) in all Possible Combinations upon east 6470, with Ammonium Phosphate as the Nitrogen Source addition An.. Pr.. Nc.. Rb.. An + Pi- Ann-Nc An+ Rb Pr +Nc Pr +Rb Nc + Rb Pr + Nc + Rb An + Nc + Rb An + Pr + Rb An + Pr + Nc An + Pr + Nc + Rb Standard deviations Final yeast growth in grois. per litre ±0-4 Initial relative growth rate ±008 Ferment* ation factor. (100) ±002 figures enables the net result to be expressed very briefly. Aneurin alone has a slightly depressive effect, to the extent of 6 per cent., on yeast response; pyridoxin alone has a slightly stimulative effect, to the extent of 4 per cent. Aneurin and pyridoxin in com bination cause quite a noticeable improve ment in response, the average value being 12 per cent. Nicotinic add and riboflavin, singly or together, exercise practically no modifying effect on these results.

7 _ 24 thorne: bios requirements of brewery yeasts Qan.-Feb., 1940 In order to ascertain the validity of this conclusion under a wider range of con ditions a series of experiments was designed in which yeast 6470 was grown with ammon ium phosphate, glycine, alanine, leucine, glutamic add, aspartic add, asparagine, arginine, histidine or tryptophane as the nitrogen source; inositol, pantothenic add and biotin in the standard concentrations were used in all the media, together with together cause a reduction of 3 per cent, in yeast response. Closer inspection of the data shows that the omission of aneurin and pyridoxin from the medium causes on the average a greater reduction in response when the nitrogen source is an amino add than when it is ammonia: the average reduction with ammonia (from this and the previous experiment) is 10 per cent., while the average reduction with amino adds is 18 per cent. TABLE VI The Effects of Aneurin (An), Pyridoxin (Pr), Nicotinic Acid (Nc) and Riboflavu* (Rb) upon east 8479, with Ammonium Phosphate, lycinb, Alanine, lutamic Acid, Aspartic Acid, Asparaginb, Arginine, Histidine and Tryptophane as the Nitrogen Sources a e IS3 O Final yeast growth in grms. per litre ± 0-4. addition An Pr An + Pr Nc + Rb An + Pr + Nc + Rb Initial relative growth rate ± 005. addition An Pr An + Pr Nc + Rb An + Pr + Nc + Rb »l F C * Fermentation factor ± addition An Pr An + Pr Nc + Rb An + Pr + Nc + Rb (100) optional additions of aneurin, and pyridoxin singly or together, and nicotinic add and riboflavin with and without aneurin and pyridoxin. The results of these experiments are summar ized in Table VI. The average trend disdosed by an examination of these figures is that aneurin alone reduces the yeast response by 2 per cent., pyridoxin increases it by 6 per cent. and aneunn and pyridoxin together by 16 per cent. Nicotinic add and riboflavin It should be observed that those amino adds, e.g. glycine and histidine, which, under the conditions of the writer's earlier experiments (ibid., 1941, 255), were practically useless as yeast nutrients are not materially improved under the present conditions with known amounts of inositol, pantothenic add, biotin, aneurin and riboflavin. It is instructive to compare the earlier results (1041) with these later ones (1947) using averaged growth

8 Jan.-Feb., 1949] thorne: bios requirements of brewery yeasts 26 characteristics and taking the values for ammonium phosphate (100) as standards of comparison. The relevant figures are given in Table VIA. It will be seen that the agree ment between the two sets of figures is TABLE VIA The Relative Values op Different Nitrogen Sources (Ammonium Phosphate = 100) Deter mined in 1041 with Inositol, Pantothbnic Acid and bios iib, and in 1047 with inositol, Pantothenic Acid, Biotin, Aneurin and Pyridoxin Ammonium phosphate. lycinc Alanine Leucine lutamic acid Aspartic acid. Asparagine Arginine Histidino Tryptophane no SI remarkably dose, considering the fluctua tions in the response of yeast to a defined chemical medium which are always observed even over much shorter periods of time than 6 years. The only differences which might possibly be significant are found with aspartic acid and tryptophane, but the writer would hesitate to ascribe these to varying responses of the amino acids to the different bios supplements employed in the two sets of experiments. Discussion and Summary The processes whereby a simple source of nitrogen such as an ammonium salt or an amino acid is converted by yeast into pro? teins must be of extreme complexity, involv ing the co-operation of many growth factors and the delicate balancing of many inter locking chemical reactions. The growth factors themselves will be the products of the yeast cells' own synthetic activity or, where this fails, will be assimilated directly from the nutrient medium. It seems a priori rather probable that, since different nitrogen sources will alter at any rate the initial step of the protein synthesis, they may also necessitate a somewhat different balance of the growth factors to achieve the maximum yeast response of which they are capable. On the other hand, it can hardly be doubted that this initial stage of the nitrogen meta bolism is but a small proportion of the whole series of transformations taking place, and that the requirement for those growth factors not implicated in it should be unaffected by the nature of the nitrogen source. The experimental results presented in this paper do indicate that there are differences, though not very great ones, between the sensitivities of yeasts to bios deficiencies when the source of nitrogen is, on the one hand, ammonia, and on the other, amino acids. Perhaps the most significant differ ence of this kind is that which is shown when aneurin and pyridoxin are absent from the media. The growth of yeast is lowered by about 10 per cent, as a result of this de ficiency when the nitrogen source is ammonia, but is lowered nearly twice as much if the nitrogen source is an amino acid. Since pyridoxin, as mentioned above, appears to be directly concerned in the decarboxylation and transamination of amino acids this result, as far as it goes, fits into the picture quite well. On the data at present available it is not possible to say whether there are any differences among the amino acids them selves in their requirements for aneurin and pyridoxin. A further conclusion from the results presented is that absence of biotin causes a differential response as between ammonia and amino acids; yeast seems more able to dispense with this factor when the nitrogen source is an amino add. The differences in bios requirements be tween the nitrogen sources examined are, however, much less conspicuous than the specific differences.between the four yeasts examined even though each of these is of the top-fermentation brewery type. To obtain adequate growth and fermentation under the experimental conditions adopted, irrespect ively of the nitrogen source, yeast requires biotin, yeasts 6179 and require pantothenic add and biotin, and yeast requires inositol, pantothenic add and biotin. As far as the quantitative requirements for pantothenic add and biotin are concerned, maximal yeast response is obtained when the concen trations of these factors are approximately 0-1 mgrm. per litre and 2 /tgrms. per litre respectively; these values appear scarcely to be affected by the nature of the nitrogen source. Finally, such differences as may exist between the bios requirements imposed by

9 26 shimwell: a study of ropiness in beer [Jan.-Feb., 1949 different nitrogen sources are evidently so small as not to invalidate comparative studies of the nutrient efficiencies of different amino acids made with constant dosages of the growth factors. The author's best thanks are due to Miss Doreen L. Birkett and Miss Myra. Hulse for assistance with the experimental work. Institute of Brewing Research Laboratories, University of Birmingham. 8th December, A STUD OF ROPINESS IN BEER By J. L. Shimwell, D.Sc, F.R.I.C. Part III. Ropiness due to Lactobacllll In Parts I and II of this Study (this Journ., 1947, 280; 1948, 237) the incidence of ropiness in beer due to Acetobacter species and tetrad-forming Streptococcus species respectively has been discussed. hich of the two types is the more prevalent it is difficult to estimate. Most brewers would probably say the latter, since cocci are widely believed to be the chief cause of ropiness. It has to be borne in mind, however, that both Acetobacter viscosutn and A. capsulatum often adopt an almost spherical form {loc. cit.), and several cases of ropiness submitted to the writer as being due to cocci have proved to be due to one or other of these Acetobacter species. As regards virulence, there is little doubt that ropiness due to Str. damnosus varieties is, of the two types, far more dangerous and difficult to combat, for exclusion of air cannot be used to suppress growth, as is the case with the acetic acid bacteria. hatever may be the relative prevalence of ropiness due to these two types of bacteria the writer's experience is that together they are responsible for most instances of ropiness occurring in British beers. Out of all the cases encountered in the last twelve years only two were due to other bacterial types, namely lactobacilli. It is felt, however, that this study would not be complete without reference to such rare cases*r In the first of these instances, previously referred to (Shimwell, ibid., 1936, 127), the organism was not actually isolated from ropy beer, but from a sample of pitching yeast. On inoculating unhopped beer with a pure culture of the organism only acidity and "silky" turbidity was produced, but when hopped beer was substituted bacterial growth took place but ropiness was produced. The hypothesis was advanced that possibly the hop antiseptic present might have stimulated the bacterial cell to surround itself with slime, a phenomenon somewhat analogous to that later observed in the case of Sir. damnosus var. viscosus, where COa saturation appeared to fulfil a similar function (Part II of this study, ibid., 1948, 237). Unfortunately, cultures of this organism were not preserved and it has therefore not been possible to make a proper study of it. Since, however, it was ram-positive, nonmotile, facultative and aciduric, it seems probable that it was a species of Ladobacillus. Compared with L. pastorianus it was a much smaller and shorter rod about 0-4/x x 1-5/x (although the variable morphology of the former species must be borne in mind). hen producing ropiness in hopped beer the organism grew chiefly as single rods with occasionally pairs (Fig. 1), whilst in unhopped beer chains were the predominating form (Fig. 2). (The high magnification (xl200) should be noted.) The cultural characters on solid media were uncharacteristic, similar to those of other lactobacilli. Lactobacillus pastorianus var. brownii. The other slime-producing lactobacillus was isolated by B. M. Brown from ropy bottled ale and was handed to the writer for study. On inoculating beer with this organism, the same phenomenon was encountered as with Str. damnosus var. viscosus (this study, Part II, loc. cit.), i.e. ropiness was not pro duced in "flat" beer, only acidity and turbidity ensuing, but if bottled beersaturated