University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Faculty Publications in Food Science and Technology Food Science and Technology Department September 1993 pH Homeostasis in Lactic Acid Bacteria Robert W. Hutkins University of Nebraska-Lincoln, [email protected] Nancy L. Nannen University of Nebraska-Lincoln Follow this and additional works at: https://digitalcommons.unl.edu/foodsciefacpub Part of the Food Science Commons Hutkins, Robert W. and Nannen, Nancy L., "pH Homeostasis in Lactic Acid Bacteria" (1993). Faculty Publications in Food Science and Technology. 28. https://digitalcommons.unl.edu/foodsciefacpub/28 This Article is brought to you for free and open access by the Food Science and Technology Department at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Faculty Publications in Food Science and Technology by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. pH Homeostasis in Lactlc Acld Bacterial ROBERT W. HUTKINS and NANCY L. NANNEN Department of Food Science and Technology University of Nebraska-Lincoln Lincoln 68583.0919 ABSTRACT especially important concern for lactic acid The ability of lactic acid bacteria to bacteria used as dairy starter cultures because regulate their cytoplasmic or intracellular these obligate acid-producers must cope with pH is one of the most important physio- low pH, high acid environments during ordi- logical requirements of the cells. Cells nary growth in milk. This paper reviews the unable to maintain a near neutral in- effects of low pH, and in particular, low in- tracellular pH during growth or storage tracellular pH (pHin), on growth and metabo- at low extracellular pH may lose viabil- lism of lactic acid bacteria and discusses how ity and cellular activity. Despite the im- cytoplasmic pH regulates cell metabolism and portance of pH homeostasis in the lactic how cytoplasmic pH is regulated in the lactic acid bacteria, however, an understanding acid bacteria. of cytoplasmic pH regulation has only recently begun to emerge. This review GROWTH OF LACTIC ACID describes the specific effects of low pH BACTERIA IN MILK on lactic acid bacteria, reports recent re- In general, growth of lactic acid bacteria search on the physiological role of in- continues as long as 1) carbohydrates, amino tracellular pH as a regulator of various acids, and other nutrients are available; 2) metabolic activities in lactic acid bacte- toxic or inhibiting compounds, such as hydro- ria, and presents the means by which gen peroxide, are removed, degraded, or lactic acid bacteria defend against low diluted; or 3) the hydrogen ion concentration is intracellular pH. Particular attention is maintained above the level that a specific devoted to the proton-translocating strain can tolerate. For example, growth of ATPase, an enzyme that is largely Lactococcus lactis ssp. lactis, Lactococcus lac- responsible for pH homeostasis in fer- tis ssp. cremoris, and Streptococcus ther- mentative lactic acid bacteria. mophilus in milk occurs until the pH reaches (Key words: pH, pH homeostasis, lactic approximately 4.5, despite nonlimiting concen- acid bacteria) trations of nutrients and the relative absence of inhibitory compounds (21, 61. 98). Low pH Abbreviation key: pHout = extracellular pH, (or high lactic acid) is frequently growth- pHi, = intracellular pH, ApH = pH gradient, limiting for lactic acid bacteria that are grown H+-ATPase = proton-translocating ATPase. in milk or in weakly buffered bacteriological media (12, 61). INTRODUCTION Although lactic acid bacteria may fre- quently be isolated from acid habitats (69), The means by which microorganisms toler- such sour and are commonly thought ate variations of environmental pH been an as milk, has to favor low pH environments, except for cer- active area of investigation [for reviews, see tain Lactobacillus species, lactic acid bacteria (16, 41, 46, 51, 76)]. pH homeostasis is an are probably best characterized as neutrophiles. For example, optimal growth rates for the mesophilic dairy starter cultures, L. lactis ssp. Received February 10, 1992. kactis and L. lactis ssp. cremoris have been Accepted April 28, 1992. reported to occur within an external or ex- Published as Paper Number 9795, Journal Series Nebraska Agricultural Experiment Station, Lincoln tracellular pH (pHout) range of 6.3 to 6.9 (12, 685834919, 13,37). The optimal pHout for the thermophilic 1993 J Dairy Sci 76:2354-2365 2354 SYMPOSIUM: DAIRY STARTER CULTURES 2355 lactic acid bacterium S. themphilus was be- ied the damage to L.. fuctis ssp. fuctis ML3 tween 6.5 and 7.5 (9, 72). Among the lactic resulting from growth at low pH. Maximum acid bacteria used as dairy starters, only the specific growth rates occurred at pHout 6.3. lactobacilli (Lactobacillus helveticus and Lac- Cell damage, determined as the ratio of the tobacillus delbrueckii ssp. bulgaricus) appear transient growth rate to the normal growth rate, to grow optimally at acid pH; maximal growth began to occur at medium pH or pbut5.0, and occurs at pH 5.5 to 5.8 (9, 95). the specific growth rate approached zero at Not only do most lactic acid bacteria grow pHout 4.0. A reduction in the specific activity more slowly at low pH, but acid damage and of the enzymes hexokinase and acetate kinase, loss of cell viability may also occur in cells as well as the overall glycolytic activity, also held at low pH. In fermented dairy products, occurred at pH 4.0. Although pHin was not such as yogurt or cultured buttermilk, whether measured, Harvey (37) suggested that the cells the lactic acid bacteria are viable or injured by were able to maintain constant pHh as long as the lactic acid and low pH environment, once pbutwas >5.0. Many other workers (18, 55, the desired pH is reached frequently is techno- 72, 98) have reported that growth of lactic logically irrelevant. Moreover, inhibition of the streptococci and lactococci decreases sharply starter culture by lactic acid and low pH acts to when the medium pH reaches 5.0. Similarly, prevent, in part, overacidification of the fin- pH-induced damage also occurred in the fecal ished product. In the production of yogurt, for coccus, Enterococcus fuecaZis (formerly Strep- example, fermentation lowers the pH of the tococcus faeculis). Below pH 5.0, derangement milk from 6.5 to between 4.0 and 4.5, and the of membrane structures and solute leakage oc- milk coagulates. Unrestricted growth and fer- curred (60). Although the changes were revers- mentation in yogurt (or postacidification) by ible in both L Zuctis ssp. lactis and E. faeculis, the lactic culture may result in excessive acid a lag in fresh media occurred following ex- and flavor defects. In other cases, however, posure to these low pH (37, 60). maintenance of culture viability under acid Bender et al. (1 1) studied the effect of gross conditions is desired and necessary. Cultures membrane damage caused by acidification in and culture adjuncts (e.g., Lactobacillus various oral streptococci. Damage was as- acidophilus) that are added to yogurt for ther- sessed by measurement of the release of mag- apeutic value must remain viable during stor- nesium ions from cells that were incubated at age at low pH and, ultimately, must survive specific pH at room temperature. Magnesium the acid stomach environment. Mixed genus, was not lost from Streptococcus mutuns, Strep- thermophilic cultures used for Italian cheese tococcus sanguis, and Streptococcus salivarius manufacture (i.e., S. themphilus and Lb. hel- at pH near neutrality, but magnesium efflux veticus) are usually propagated or cultivated occurred after the pH was lowered to <4.0. At together; that the former is more acid-sensitive pH 2 or 3, release of magnesium was rapid and than the latter may result in variable strain extensive. In contrast, Lactobacillus casei ratios. In the industrial production of starter leaked magnesium only at pH <3. Those wor- cultures, sensitivity to acid is undesirable be- kers (1 1) suggested that differences in the sus- cause high cell densities may not be achieved, ceptibility to gross membrane damage caused and cell viability may be impaired (77). by acidification appeared to vary among organ- isms and was correlated with the degree of acid tolerance. ACID DAMAGE DURING GROWTH AT LOW pH Conventionally prepared, milk-based (with- out added buffers) starter cultures are espe- The basic knowledge that starter cultures cially prone to acid damage because the final grow best at neutral or near neutral pH, that medium pH usually is <5.0. Such cultures, if acid environments are damaging to cells, that they are not used soon, behave poorly in fer- starters tend to lose activity during prolonged mented milks, resulting in slow or sluggish storage at low pH, and that ovempened or culture performance (39, 84). To minimize overacidified cultures perform slowly or result these acid-damaging effects in dairy starter in inferior productions has long been recog- cultures, the starter culture industry has nized (54, 55, 77, 84). Harvey (37) fmt stud- devoted considerable effort to development of Journal of Dahy Science Vol. 76. No. 8, 1993 2356 HUTKINS AND NANNEN TABLE 1. Optimal pH for intracellular enzymes from lactic acid bacteria. ~~~~~ PH Enzyme Organism Optimum Reference B-D-Galactosidase Streptococcus thcmphilus 7.1 8-D-Galactosidase S. themphilus 8.0 Phosphofructokinase Lactobacillus bulgaricus 8.2 6-D-Phosphogalactoside Lactococcus lactis galactohydrolase ssp. cremris 7.0 Acylglyccrol Lactococcus lactis ssp. lactis acylhydrolase (lipase) L lactis ssp. cremris 7.0-8.5 Pyruvate kinase L loctis ssp. lactis 6.9-7.5 D-Tagatose 1.6-diphosphate L kactis ssp. lactis aldolase L &tis ssp. cremris 7.0-7.3 Glucose-6-phosphate dehydrogenase Lactobacillus casei 6.8 6-Phosphogluconate de hydrogenase Lb. casei 6.8 Aminopeptidase L lactis ssp. cremoris 7 .O Intracellular proteinase L lactis ssp. lactis 7.5 Aminopeptidase Lb.