Moisture Requirements for Growth and Metabolite Production by Lactic Acid Bacteria

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1981, p Vol. 42, No /81/ $02.00/0 Moisture Requirements for Growth and Metabolite Production by Lactic Acid Bacteria J. A. TROLLER* AND J. V. STINSON The Procter & Gamble Company, Winton Hill Technical Center, Cincinnati, Ohio Received 21 November 1980/Accepted 22 June 1981 The effect of water activity (aw) reduction on growth and acid and diacetyl production by three lactic streptococci was studied. In addition, the influence of low moisture conditions on several bacteria of significance in the fermentation of sauerkraut was examined. The minimal aw supporting growth of dairy lactics was 0.93 in a medium adjusted with glycerol. Media adjusted with sucrose generally were more inhibitory than those in which glycerol was the humectant. Titratable acidity, although not related to the type of humectant, did depend on the aw of the medium and was directly related to the extent of growth. Diacetyl concentration increased in cultures of reduced a, when the media were adjusted with both humectants; however, the effect was greatest with glycerol. A lactic strain associated with sauerkraut fermentation appeared to grow at a lower miniimal aw in a glycerol-adjusted medium than in a system adjusted with NaCl; however, none of the sauerkraut organisms grew at a, levels of <0.95 when NaCl was the solute. Acid production appeared to be related to the presence and extent of growth at all of the aw levels studied. The moisture requirements of most lactic acid-producing bacteria of commercial significance have not been accurately defined. Practical experience with foods fermented by these organisms has been largely responsible for the empirical establishment of water activity (a,) minimal limits for growth in the 0.91 to 0.94 aw range. As a practical matter, it is known that insufficient salt in curing brines produces poor quality and even potentially pathogenic consequences in some lactic-fermented foods. Undersalting of cucumbers or sauerkraut permits aerogenic coliforms to grow with the result that cucumbers become bloated from excessive C02 production. Defects caused by coliforms in sauerkraut most commonly consist of foam and off-flavors. Lactic acid bacteria also suppress the growth of the food-borne pathogen Staphylococcus aureus in milk cultures (5) and in some types of fermented sausages (4). The association of lactic acid bacteria and staphylococci in products such as these may be influenced by the aw level of the particular food during processing or in the final product. In addition to influencing the type and progression of bacterial populations in fermentations, the inclusion of NaCl in bacteriological media may serve as a useful determinant of lactic species (14). Usually 4% NaCl is used for this purpose, a level that roughly corresponds to a,. Based on the extent of their growth in salted and sugared brines and foods, lactics could be characterized as moderately osmotolerant. However, the influence of reduced a, on the production of metabolites could, in addition, significantly alter the lactic acid content of brines and foods even though large numbers of the organism were present. We have shown elsewhere (19) that enterotoxin and protease activities in spent culture media obtained from two strains of S. aureus are inhibited by reduced a, to a greater degree than commensurate inhibition of growth would indicate. The production of propionic acid by Propionibacterium shermanii (13), on the other hand, appears to be related quite closely to the numbers of the organism present. Because lactic acid bacteria are important in the preservation and flavor development of many foods that are cured or salted or both, an attempt was made to ascertain the effects of a, on growth and production of acid or diacetyl or both. In this study, lactics significant in the dairy and sauerkraut industry were employed as test organisms. MATERIALS AND METHODS Organisms. Streptococcal dairy strains S. cremoris, S. diacetilactis, and S. lactis were obtained from R. W. Berg, Procter & Gamble Co., Cincinnati. The original source of these cultures was the culture collection of the Department of Microbiology at Ore- 682

2 VOL. 42, 1981 gon State University. The identity of the dairy cultures was confirmed with the medium of Reddy et al. (12). The sauerkraut-fermenting strains were obtained from J. R. Stamer, New York State Agricultural Experiment Station at Geneva, N.Y. Culture and growth conditions. Dairy lactics were stored on APT agar slants (Difco Laboratories, Detroit, Mich.) at 5 C and subcultured for 24 h at 32 C in APT broth. The basal milk medium, consisting of homogenized pasteurized milk containing 2% butterfat (nominal), was supplemented with 0.50% sodium citrate. Growth was at 32 C for the indicated periods of time in 300 ml of medium. The sauerkraut cultures were stored and subcultured similarly. Growth of these organisms was in APT broth incubated at 30 C. The a, level of the basal milk medium was adjusted with sucrose, glycerol, or NaCl. The concentrations of each solute required to produce stated a,, levels are shown in Table 1. aw determinations were made at 25 C with a Sina hygrometer operated and calibrated as described by Troller (17). The numbers of lactics were estimated by total count determinations on APT agar. Incubation was at 320C. Metabolite analyses. Diacetyl was analyzed in triplicate by the procedure of Owades and Jakovac as modified by Pack et al. (9). These methods were altered slightly by the addition of a condensor system to permit better recovery of dimethylglyoxime. Standard curves were established for various concentrations of glyoxime. The efficacy of the recovery procedure was estimated by spiking fresh samples of the milk medium with diacetyl. Recovery was >90% in all cases. Only the dairy lactic cultures were analyzed for diacetyl. Diacetyl levels are expressed in terms of total production and production relative to numbers of organisms. Acid production was measured in dairy and sauerkraut cultures. Titratable acidity, expressed as lactic acid, was measured by titrating with N NaOH to a ph 8.0 endpoint. The ph of media samples before titration also was measured. RESULTS AND DISCUSSION Dairy lactics. The effect of aw reduction on the growth of organisms used in this study was extensive. Growth suppression, in terms of rates and maximal numbers, occurred as a- levels were progressively reduced from 0.99 through TABLE 1. Solute content of media Amt (g/100 ml) of: aw Sucrosea Glycerol' NaClb (0.998) control a Homogenized milk containing 2% butterfat. b APT broth. WATER ACTIVITY AND LACTIC ACID BACTERIA or The lowest aw, that supported growth (0.93) was attained when glycerol was used to adjust a milk medium inoculated with S. lactis (Table 2). The miniimal a, at which growth of either S. cremoris or S. diacetilactis occurred was In media in which sucrose was used to adjust aw, the minimal a. for growth was 0.95 in media inoculated with S. diacetilactis (Table 3). Growth was not observed at 0.93 a. after 120 h TABLE 2. Effect of a. adjustment with glycerol on the growth of dairy lactic organisms in basal milk medium Growth Log Organism a,w Humn rate (divi- xisions/h) ma no./ml' S. diacetilactis Control Glycerol Glycerol Glycerol Glycerol No growth 4.34b 0.91 Glycerol No growth 4.34b S. lactis Control Glycerol Glycerol Glycerol Glycerol Glycerol No growth 5.84b S. cremoris Control Glycerol Glycerol Glycerol Glycerol No growth 4.41b 0.91 Glycerol No growth 4.41b a Total incubation time was 120 h. b Initial inoculum count. TABLE 3. Effect of a. adjustment with sucrose on the growth of dairy lactic organisms in basal milk medium Log Humec- Growth rate a- Organism aw tant (divisions/ man h) no./mla S. diacetilactis Control Sucrose Sucrose Sucrose Sucrose No growth 4.76b 0.91 Sucrose No growth 4.76b S. lactis Control Sucrose Sucrose Sucrose No growth 487b 0.93 Sucrose No growth 487b 0.91 Sucrose No growth 4.87b S. cremoris Control Sucrose Sucrose Sucrose No growth 438b 0.93 Sucrose No growth 438b 0.91 Sucrose No growth 4.38b a Total incubation time was 120 h. binitial inoculum count.

3 684 TROLLER AND STINSON of incubation at 32 C. Neither S. lactis nor S. cremoris was able to grow at 0.95 a,. Although S. lactis was not particularly tolerant of low a, when this water limitation was achieved with sucrose, maximal counts were obtained by this organism at the lowest aw at which it would grow, It also is interesting that, of the three dairy lactics tested, S. lactis was the most tolerant to low a, levels when glycerol was the humectant and one of the least tolerant, in terms of reduced growth rate, when the a, was poised with sucrose. In general, sucrose was somewhat more restrictive of growth than glycerol. A similar situation exists with regard to the growth of many types of bacteria, i.e., they tolerate lower aw levels when glycerol is the solute as compared to other solutes. An example is the work of Baird-Parker and Freame (1) in which three types of Clostridium botulinum were found to be more sensitive to reduced aw levels when this reduction was obtained with NaCl than with glycerol. A similar result was reported for Bacillus cereus by Raevuori and Genigeorgis (11). Marshall et al. (7), on the other hand, reported that NaCl was less inhibitory to S. aureus than glycerol. As expected, the production of the two metabolites, lactic acid and diacetyl, did not occur at aw levels at which growth was absent. Titratable APPL. ENVIRON. MICROBIOL. acidity in both glycerol and sucrose-containing systems was generally unrelated to the type of humectant but was related to the a, level. Diacetyl content was greater (Tables 4 and 5) in media in which the a, content had been adjusted by the addition of humectants than in the unadjusted control media. The amount of diacetyl present in most of the a, series tested was inversely proportional to a, except, in some cases, at the very lowest a, at which growth occurred. This relationship also held for differential levels of diacetyl, which were directly related to maximal numbers of lactics. The highest concentrations of diacetyl were detected in S. diacetilactis cultures of reduced aw. The remarkable increase in diacetyl production at reduced a, was unexpected. Both of the humectants employed in these studies are utilizable by many organisms; little is known, however, about their catabolism and subsequent conversion to 4-carbon molecules (such as diacetyl) by lactics. Sucrose is rarely fermented by S. cremoris, and glycerol is not fermented. For this reason and because both humectants produced similar results with regard to growth and metabolite production, we conclude that the data obtained are a response to a, reduction and are not an intrinsic, solute-related phenomenon. Although diacetyl is not listed in Bergey's Manual of Determinative Bacteriology (2) as Downloaded from TABLE 4. Effect of a. adjustment with glycerol on the production of acid and diacetyl in basal milk medium at 32 C. Production of: Acid Diacetyl Organism aw Humectant Maximum % titrata- ph ug/20 ml' pg/108b blea S. diacetilactis Control Glycerol Glycerol Glycerol Glycerol None Glycerol None S. lactis Control Glycerol Glycerol Glycerol Glycerol None Glycerol None S. cremoris Control Glycerol Glycerol Glycerol Glycerol None Glycerol None a Calculated as lactic acid. b Samples taken after 120-h incubation. on August 20, 2018 by guest

4 VOL. 42, 1981 TABLE 5. WATER ACTIVITY AND LACTIC ACID BACTERIA 685 Effect of a. adjustment with sucrose on the production of acid and diacetyl in basal milk medium at320c Production of: Organism aw Humectant Acid Diacetyl Maximum % ph pig/20 Mlb /LgI108b titratable S. diacetilactis Control Sucrose Sucrose Sucrose C Sucrose Sucrose S. lactis Control Sucrose Sucrose Sucrose Sucrose Sucrose S. cremoris Control Sucrose Sucrose Sucrose Sucrose Sucrose a Calculated as lactic acid. b Samples taken after 120-h incubation. c-, Not done. one of the metabolites of either S. cremoris or S. lactis, our work showed that detectable levels of this compound were produced, in the presence of citrate, by both of these organisms. As expected, S. diacetilactis produced the highest levels of diacetyl. With few exceptions, wherever growth occurred and even at, or close to, minimal a, levels, diacetyl production also was observed. Maximal titratable acidity, calculated as lactic acid, generally followed growth levels and rates. This was observed for the three lactic cultures and both humectants. Sauerkraut lactics. Lactic acid-producing organisms originally isolated from sauerkraut were included in this study because, unlike the dairy lactics, these organisms are more commonly associated with salt-containing environments (10). For this reason, growth under restrictive osmotic conditions might be expected and, as is the case with S. aureus (7), glycerol might be more poorly tolerated by these organisms than NaCl. To test the forner hypothesis, four sauerkraut organisms were grown in APT broth adjusted to a number of a, levels with NaCl (Table 6). In all cases, a, levels <0.99 produced significant decreases in total numbers of bacteria and in growth rates. Titratable acidity did not respond appreciably to reduced a. levels until the aw was reduced to <0.96. Hetero- and homofermentative species of lactobacilli, as represented by L. brevis and L. plantarum, respectively, appeared to respond similarly to osmotic stress. Of the four species tested, Leuconostoc mesenteroides was slightly less osmotolerant than Pediococcus pentosaceus or either of the Lactobacillus species, which agrees with the statements of Stamer (16). In the sauerkraut fermentation process, salt (5 to 8% NaCl, 0.98 to 0.95 aw) has the vital function of restricting the growth of undesirable organisms, such as yeasts or gram-negative bacteria, in addition to performing functions unrelated to microbiology. The growth of many gram-negative bacteria, such as Enterobacter or Flavobacterium, ceases at levels <0.97 (18); however, the data in Table 6 show that most of the organisms examined in these studies grew, albeit slowly, at 0.95 a,. The influence of solute type of growth and acid production by one of these organisms, L. plantarum, can be determined by comparing the appropriate portion of Tables 6 and 7. In the former, the solute was NaCl, whereas in the latter it was glycerol. Growth and acid production occurred at lower aw levels when glycerol was used to bind water in the medium than when NaCl was the solute. This result was similar to that found by Baird-Parker and Freame

5 686 TROLLER AND STINSON (1) for C. botulinum and by Jakobsen et al. (6) for B. cereus. On the other hand, a survey by Marshall et al. (7) indicated that bacteria that were considered to be relatively more salt tolerant were substantially more sensitive, at a given a, level, to glycerol than to NaCl. In our work, the sauerkraut lactics that we studied would be considered to be salt tolerant based on the data in Table 6, yet at least one of the species studied, L. plantarum, was more tolerant to glycerol than to NaCl. These studies show that the lactic acid-producing organisms examined in our experiments tolerated fairly high solute levels in growth media. Growth of either dairy or sauerkraut lactics was not observed at a, levels <0.93 and, in most cases, 0.95 a, was the minimal level permitting growth. The sauerkraut-related cultures were TABLE 6. Growth and acid production by sauerkraut organisms where medium aw was adjusted with NaCI Growth Acid production Titrata- Organism aw Log ble Divi- maxi- acidity ph sions/h mum maxino./ml mum %a L. mesenteroides _b 0.30c P. pentosaccus c L. plantarum c L. brevis c a Calculated as lactic acid. -, Inoculum level or lower. Residual titratable acidity. APPL. ENVIRON. MICROBIOL. TABLE 7. Growth and acid production by L. plantarum where medium aw was adjusted with glycerol Growth Acid production Log Titratable Divi- maxi- acidity H sions/h mum maximum P no./mil %a c b a Calculated as lactic acid. b, Inoculum level or lower. Residual titratable acidity. somewhat more osmotolerant than the dairyrelated strains, although this increased osmotolerance could be attributed to solute-related differences. Although aw reductions resulted in slower growth and reduced production of lactic acid, this was not the case with diacetyl production by the dairy lactic strains. In general, diacetyl production was enhanced by aw reduction and, in some cases, the highest concentrations of diacetyl were found at miniimal aw levels for growth. The reason that the production of one specific metabolite increases, whereas the production of at least one other by-product, lactic acid, is unaffected is not known. Christian and Waltho (3) have shown that the presence of proline in a culture medium of low aw seems to counteract the osmotic effects of the solute. Measures (8) reported that specific amino acids were accumulated intracellularly to create isoosmotic conditions across the cell membrane. One of these amino acids, gamma-aminobutyric acid, may very well be accumulated by lactic acid bacteria when the aw of the medium reaches an inhibitory level. Once accumulated, the pool of excess gamma-aminobutyric acid could then serve as a convenient source of precursor for diacetyl. A simple deamination plus an oxidative step would then be required to produce diacetyl. Although this is, at present, highly hypothetical, we observed during these studies that citrate supplementation did not seem to be required for diacetyl production by S. diacetilactis at low aw levels, but was necessary at higher a,. Perhaps the more conventional pathway to diacetyl, which requires citrate as a precursor, was in some way circumvented. Certainly the fact that the synthesis of lactic acid, an intermediate in the normal synthesis of diacetyl, is suppressed

6 VOL. 42, 1981 with an a, increase would argue that the increased levels of diacetyl did not accrue as a result of the synthesis of this compound via conventional biosynthetic pathways. If so, the source of the increased diacetyl levels must be elsewhere. A logical candidate might be a partial conversion of the gamma-aminobutyric acid pool, termed by Measures (8) a "compatible solute," into diacetyl. LITERATURE CITED 1. Baird-Parker, A. C., and B. Freame Combined effect of water activity, ph and temperature on the growth of Clostridium botulinum from spore and vegetative cell inocula. J. Appl. Bacteriol. 30: Buchanan, R. E., and N. E. Gibbons (ed.) Bergey's manual of determinative bacteriology, 8th ed. The Williams & Wilkins Co., Baltimore. 3. Christian, J. H. B., and J. A. Waltho Water relations of Salmonella oranienburg stimulation of respiration by amino acids. J. Gen. Microbiol. 43: Daly, C., M. Lachance, W. E. Sandine, and P. R. Elliker Control of Staphylococcus aureus in sausage by starter cultures and chemical acidulation. J. Ed. Sci. 38: Gilliland, S. E., and M. L. Speck Antagonism of lactic streptococci toward Staphylococcus aureus in associated milk cultures. Appl. Microbiol. 28: Jakobsen, M., 0. Filtenborg, and F. Bramsnaes Germination and outgrowth of the bacterial spore in the presence of different solutes. Lebensm. Wiss. Technol. 5: Marshall, B. J., D. F. Ohye, and J. H. B. Christian Tolerance of bacteria to high concentrations of NaCl and glycerol in the growth medium. Appl. Microbiol. 21: WATER ACTIVITY AND LACTIC ACID BACTERIA Measures, J. C Role of amino acids in osmoregulation of nonhalophilic bacteria. Nature (London) 257: Pack, M. Y., W. E. Sandine, P. R. Elliker, E. A. Day, and R. C. Lindsay Owades and Jakovac method for diacetyl determination in mixed strain starters. J. Dairy Sci. 47: Pederson, C. S., and M. N. Albury The influence of salt and temperature on the microflora of sauerkraut fermentation. Food Technol. 8: Raevuori, M., and C. Genigeorgis Effect of ph and sodium chloride on growth of Bacillus cereus in laboratory media and certain foods. Appl. Microbiol. 29: Reddy, S. M., E. R. Vedamuthu, C. V. Washam, and G. W. Reinbold Differential agar medium for separating Streptococcus lactis and Streptococcus cremoris. Appl. Microbiol. 18: Ruegg, M., H. Glattli, and B. Blanc Einfluss der Wasseraktivitat auf Vermahrung und Stoffwechsel von Propionsauerbakterien. Schweiz. Milchwirtsch. Forsch. 5: Sandine, W. E., P. C. Radich, and P. R. Elliker Ecology of the lactic streptococci. J. Milk Food Technol. 36: Sherman, J. M., and W. R. Albus Some characters which differentiate the lactic-acid streptococcus from streptococci of the pyogenes type occurring in milk. J. Bacteriol. 3: Stamer, J. R Lactic acid bacteria in food microbiology. In Food microbiology: public health and spoilage aspects. AVI Press, Westport, Conn. 17. Troller, J. A Statistical analysis of a,, measurements obtained with the Sina Scope. J. Food Sci. 42: Troller, J. A., and J. H. B. Christian Water activity and food. Academic Press, Inc., New York. 19. Troller, J. A., and J. V. Stinson Influence of water activity on the production of extracellular enzymes by Staphylococcus aureus. Appl. Environ. Microbiol. 35: