Antimicrobial
`
`A Research Note
`Activity
`of Sodium Bicarbonate
`
`LAURA G. CORRAL,
`
`LAURIE S. POST, and THOMAS
`
`J. MONTVILLE
`
`I-Influence
`Table
`conditions
`(aerobic
`bicarbonate
`reouired
`
`of plating medium,
`versus
`anaerobic)
`to inhibit bacterial
`
`incubation
`of sodium
`
`level, and
`inoculum
`on
`the concentration
`orowth
`(%)
`cone
`Sodium bicarbonate
`reauired
`to inhibit arowth
`Anaerobic’
`Aerobic
`lnoculum
`(CFUlmL)
`100
`10,000
`4
`ndc
`2
`nd
`1
`nd
`1
`nd
`
`level
`
`10,000
`4
`4
`1
`1
`
`100
`nd
`nd
`nd
`nd
`
`nd nd
`>6
`>6
`>6
`>6
`z-6
`>6
`6
`6
`1
`1
`
`Plating
`medium’
`PCA
`MH
`PCA
`NH
`PCA
`:
`MH
`nd nd
`:
`nd
`>w
`TSC
`nd
`nd
`nd
`>6
`MH
`nd
`z-6
`BHI
`nd
`MH
`nd
`nd
`>6
`4
`4
`>6
`BHI
`4
`2
`>6
`MH
`1
`1
`>6
`BHI
`1
`1
`z-6
`MH
`1
`1
`2
`MRS
`1
`1
`1
`MH
`’ lnocula were prepared in Brain Heart Infusion (BHI) broth,
`for L. plantarum
`except
`and C. pedringens which ware grown
`(MRS.) and Fluid Thiog-
`in Lactobacillus
`MRS
`lycolata Medium,
`respectively.
`Other agars used were Plate Count Agar
`(PCA), Muel-
`ler Hinton
`(MH), end Trqptose
`Sulfite Cycloserine
`(Xx2) without
`cycloserine
`(Harmon,
`1984).
`b GasPak Systems
`C Not done
`d No
`inhibition
`
`Soecies
`P. fluorescens
`
`P. aeruginosa
`
`E. coli
`
`C. perfringens
`
`S. mutans
`
`S. feecalis
`
`S. aureus
`
`L. plantarum
`
`ABSTRACT
`Sodium bicarbonate (SB) inhibited the growth of bacteria and yeasts
`in agar media model systems under certain conditions. Escherichiu
`coli, Lactobacillus plantanrm, Staphylococcus aureus, and Pseudo-
`monas aeruginosu aerobic plate counts were reduced lO,OOO-fold by
`0.12M (1% wk) SB. Saccharomyces cerevisiae and Hansenula win-
`gei were more sensitive; counts were reduced lOO,OOO-fold by 0.06M
`SB. Potassium bicarbonate was equally inhibitory, but equimolar so-
`dium chloride had no effect, ruling out osmotic- and sodium-mediated
`mechanisms of inhibition. The bicarbonate ion was identified as the
`probable cause of SB-mediated inhibition although, in some cases,
`pH elevation played a significant role.
`
`INTRODUCTION
`SODIUM BICARBONATE
`(SB) is widely used in foods at
`levels up to 2% for leavening, pH-control, taste, and texture
`development (Lindsay, 1985). Data on the antimicrobial prop-
`erties of SB are limited. It is inhibitory to periodontal patho-
`gens (Newbrun et al., 1984; Gxra and Killoy, 1982; Miyasaki
`et al., 1986) and is used in dental preparations. SB is lethal to
`Aspergillus parasiticus and alters aflatoxin distribution in sur-
`viving cells (Montville and Goldstein, 1987). If SB inhibits
`other organisms, its GRAS (generally recognized as safe) status,
`low cost, and lack of toxicity would favor its use as a pre-
`servative. The objective of this study was to determine if SB
`has antimicrobial
`activity against several aerobic and anaerobic
`bacteria and against two common yeasts.
`
`MATERIALS 8z METHODS
`BACIERIA were tested for sensitivity to SB in Mueller Hinton Agar
`(MI-I, Difco), a universal medium for testing anti-microbial com-
`pounds (Matson and Barry, 1974), and media appropriate for specific
`microorganisms, as indicated in Table 1. These were prepared at SB
`concentrations of 0 to 6%. Also prepared were media which contained
`potassium bicarbonate (KB) and NaCl in molar amounts equivalent
`to the Na+ and HCO? provided by inhibitory SB concentrations.
`Media to serve as pH controls were adjusted to pH 10 using 5N NaOH.
`Details of stock culture preservation and media preparation appear
`elsewhere (Corral, 1987). The bacteria were grown to mid-log or
`stationary phase, diluted to lo4 or lo2 CFU/mL, 1 mL added to pour
`plates of media tempered to 45” C, and incubated aerobically or an-
`aerobically as indicated in Table 1. All
`incubations were at 37” C,
`except for the pseudomonads, which were at 26” C. Conditions pre-
`venting any colony formation were considered inhibitory.
`To examine the inhibition of bacteria in a liquid system, Brain Heart
`Infusion broth (BHI, Difco Laboratories, Detroit, MI) was prepared
`in citrate phosphate buffer at pH 5.6, 6.0, 7.0, and Tris-HCI buffer
`at pH 8.6 (Costilow, 1981). SB was added to each medium at 0 to
`10% and the pH readjusted with HCl. Control media, which did not
`contain SB, were adjusted to pH 9.4 with, 5N NaOH. All media were
`filter-sterilized
`(0.45 pm) and used immediately. Cells were grown
`
`the Dept. of
`are affiliated with
`Authors Corral and Montville
`Food Science, New Jersey Agricultural Experiment Station, Cook
`College, Rutgers-the
`State Univ., New Brunswick, NJ 08903.
`Author Post,
`formerly
`affiliated with Rutgers Univ.,
`is now with
`M&M/Mars,
`Hackettstown,
`NJ. Inquiries
`should be addressed
`to
`Dr. Montville.
`
`I
`
`(BBL, Cockeysville,
`
`MD)
`
`at any sodium
`
`bicarbonate
`
`concentration
`
`tested.
`
`to mid-log phase, harvested, washed, and adjusted to lo6 ClWmL.
`The broths were inoculated to a final concentration of l(Y CFU/mL,
`incubated aerobically without agitation, and examined for turbidity at
`48 hr. Conditions completely preventing turbidity were scored as in-
`hibitory.
`Yeast Peptone Dextrose (YPD) agars containing 0 to 1.00% (w/v)
`SB were prepared to examine inhibition of the yeasts. Additional
`media were prepared to serve as controls for KB, NaCl, and pH.
`Twenty-four hour yeast cultures were serially diluted to inocula levels
`of 102 through 10s CPU/mL, plated and incubated. All experiments
`were conducted in duplicate and repeated once; the data represent the
`results of four trials.
`
`RESULTS & DISCUSSION
`PHASE OF GROWTH was not a factor in SB sensitivity; only
`data from stationary phase inocula are presented in Table 1.
`Aerobic growth of E. coli, S. aureus, P. aerughosa, and L.
`plantanrm was prevented by 1% SB in BHI or MH agars. Of
`the obligate and facultative anaerobes, only L. plantantm was
`sensitive to low SB concentrations under anaerobic conditions.
`However, twice as much SB was required to inhibit anaerobic
`L. plantancm compared with aerobic conditions when high
`inocula were used. Inoculum size and medium used were not
`major determinants of SB sensitivity, although in some cases
`there was increased sensitivity when low inocula were plated
`on MH agar. This suggests that the cell’s physicochemical
`environment may play some role in its SB sensitivity. KB
`
`Volume 53, Nd. 3, 1988-JOURNAL
`
`OF FOOD SCIENCE-981
`
`This article is protected by copyright and is provided by the University of Wisconsin-
`Madison under license from John Wiley & Sons. All rights reserved.
`
`Petitioner Dr. Squatch
` Ex. 1022
`
`

`

`SODIUM BICARBONATE INHIBITION. . .
`
`to the bacteria (data not shown).
`(0.12M) was also inhibitory
`Both SB and KB elevated the pH of agar media to between
`9.00 and 9.87. Agar media adjusted with NaOH to a target
`pH value of 10 had final pH values of 9.00 to 9.76 and was
`also inhibitory. Growth occurred at pH 19.0.
`When lo5 CFU/mL of E. co&, S. mutans, S. faecalis, or C.
`pe$-ingens were inoculated into BHI broth, there was little or
`no inhibition at pH 5.6 and 6.0 (Table 2). All four organisms
`were inhibited at pH 7.0; SB was more effective at pH 8.6. It
`was difficult
`to maintain stable pH values in media which
`contained SB. Media initially adjusted to pH 5.6, 6.0, 7.0,
`and 8.6 reached final values of 7.0, 8.1, 8.8, and 9.4, re-
`spectively. All four species grew in BHI (0% SB) adjusted to
`pH 9.4 with NaOH. The pH of BHI without SB did not drift.
`Yeasts were more sensitive to SB than were the bacteria.
`At inoculum levels of lo* and lo3 CFU/mL, 0.25% SB pre-
`vented growth; H. wingei was inhibited at inoculum levels of
`105 CFU/mL. S. cerevtiiae required 0.50% SB to inhibit growth
`of high inocula. Growth was not inhibited by 0.06M NaCl,
`but was inhibited by 0.06M KE3 and at NaOH-generated al-
`kaline pH values.
`In broth experiments, growth was not inhibited by alkaline
`pH controls. Alkalinity has also been excluded as the agent of
`SB inhibition against A. parasiticus (Montville and Goldstein,
`1987) and oral anaerobes (Newburn et al., 1984). These stud-
`ies, and our findings, have found KB to be equally inhibitory
`to SB, suggesting that HCO,
`is the inhibitory agent. Bicar-
`bonate:potassium
`ratios regulate
`the morphology of some
`streptococci (Tao et al., 1987). Bicarbonate may alter mem-
`brane permeability
`(Sears and Eisenberg, 1961; Jones and
`Greenfield, 1982). SB also uncouples oxidative phosphoryla-
`tion (Daniels, et al., 1985; Newbrun, et al. 1984) by stimu-
`lating mitochondrial ATPases which are very similar to bacterial
`ATPases (Maloney, 1987). The increased sensitivity of facul-
`tative anaerobes when grown aerobically may be caused by
`the synergistic effect of H,Oa with SB (Miyasaki et al., 1986).
`Growth inhibition
`in solid agar media could, however, be ex-
`plained solely on the basis of pH elevation. Bacterial inhibition
`by SB occurred only at pH > 7.0. This agrees with previous
`findings that SB inhibits A. parasiticus at pH 7.5, but not at
`5.5 (Montville and Goldstein, 1987). While this would appear
`
`Table P-Minimum
`hibition
`of bacteria
`incubated
`aerobically
`
`in-
`for
`required
`bicarbonate
`of sodium
`concentration
`at 100,000 CFUImL
`into EHI broth
`and
`inoculated
`at 37°C for 48 hr
`bicarbonate
`Cone (%) of sodium
`for
`inhibition
`at pH
`6.0
`7.0
`10
`10
`>lO
`8
`>lO
`8
`>lO
`10
`
`required
`
`8.6
`4
`6
`6
`6
`
`5.6
`Soecies
`10
`Escherichia
`coli
`>lO”
`Streptococcus
`mutans
`>lO
`Streptococcus
`faecalis
`>lO
`Clostridium
`perfrngens
`a Denotes
`no
`inhibition
`at any concentration
`
`tested.
`
`the use of SB in foods, SB’s buffering capacity can
`to limit
`elevate the pH of hot dogs and soybeans without adversely
`affecting protein functionality
`(Bechtel et al., 1985; Lu and
`Jassen, 1986). Browning reactions and off flavors generated
`at high SB concentrations may place limits on its use. In ad-
`dition, pH elevation might cause some acid foods to move into
`the pH range where Clostridium botulinum becomes a prob-
`lem. Thus, while the applicability of SB inhibition
`to various
`foods might be possible, each application should be the subject
`of laboratory evaluations.
`
`REFERENCES
`Bechtel, P.J., McKeith, F.K., Martin, S.E., Basgall, E.J., and Novakofski,
`J.E. 1985. Properties of frankfurters processed with different
`levels of
`sodium bicarbonate. J. Food Protect. 48: 861.
`Cerra, M.B. and Killoy, W.J. 1982. The effect of sodium bicarbonate and
`hydrogen peroxide on the microbial
`flora of periodontal pockets. J. Per-
`iodont. 63: 599.
`Corral, L.G. 1987. Antimicrobial
`activity of sodium bicarbonate against
`food-relate bacteria and yeasts. M.S. thesis, Rutgers Univ., New Brims-
`wick, NJ.
`Costilow, R.N. 1981. Biophysical factors in growth. In “Manual of Methods
`for General Bacteriology,” p. 66. American Society for Microbiology,
`Washin
`n,DC.
`Daniels, P .A., Krishnamurthi, R., and Rizvi, S.S.H. 1985. A review of ef-
`fects of carbon dioxide on microbial growth and food quality. J. Food
`Protc ?ct. 48: 532.
`Harmc ~n, S.M. 1984. Clostridium
`ens: enumeration and identifi-
`perfrin
`n. Chl 17. In “Bacteriological An f. ytmal Manual,” p. 17.00-17.10.
`AOAC, Arlin
`on, VA.
`
`Jones, R.P. an tit Greenfield, P.F. 1982. Effect of carbon dioxide on yeast
`growth and fermentation. Enzyme Microb. Technol. 4: 210.
`Lindsay, R.C. 1985. Food Additives. Ch. 10. In “Food Chemistry,” O.R.
`Fennema (Ed.), p. 632. Marcel Decker, Inc., New York, NY.
`Lu, J.Y. and Al Jassen, MS. 1986. Characterization of a flour
`repared
`
`from sodium bicarbonate soaked and steamed sovbeans. J. Foo B Process
`Preserv. 10: 177.
`Maloney, P.C. 1987. Pumps and Carriers: Nutrient Trans
`ort in Bacteria.
`Ch. 2. In “Food Microbiolo
`4 Vol. I, Concepts in Physio ogy and Metab-
`
`olism.” T.J. MontviIle
`(Ed. P , p. 36. CRC Press, Inc., Boca Raton, FL.
`
`Matson, J.M. and Barry, A.L. 1974. Susce
`testing: diffusion
`tibility
`test
`
`
`procedures. In “Manual of Clinical Micro il. lology”, p. 418. American So-
`ciety for Microbiology, Washington, DC.
`Miyasaki, K.T., Genco, R.J., and Wilson, M.E. 1986. Antimicrobial
`prop-
`erties of hvdroeen neroxide and sodium bicarbonate individuallv and in
`combinati& a&n&
`selected oral Gram-negative
`facultative ba&eria. J.
`Dent. Res. 65:-1142.
`MontvilIe, T.J. and Goldstein, P.K. 1987. Sodium bicarbonate reduces vi-
`ability of As
`in Csapek’s agar and inhibits
`the for-
`ergillus
`paroszticus
`
`mation of a8 atoxin Bz and G1. Appl. Environ. Microbial. 53: 2303.
`Newbrun. E.. Hoover. CL. and Rvder. MI. 1984. Bactericidal action of
`bicarbonate
`ion on selected periodonto1 pathogenic microorganisms. J.
`Peridontol. 55: 658.
`Sears, D.F. and Eisenber R.M. 1961. A model representing a physiolog-
`
`ical role of carbon dioxl eat the cell membrane. J. Gen. Physiol. 44: 869. 3
`Tao, L., Taxer, J.M., and MacAlister, T.J. 1987. Bicarbonate and potas-
`sium rerrulation of the shane of Streutococcus mutans PllC 104498. J.
`Bacteri& 169: 2543.
`-
`-
`MS received B/19/87; revised l/14/88; accepted l/21/88.
`
`at the 47th Annual
`Presented
`17-19,
`1987, La8 Vegas, NV.
`Station.
`Exueriment
`of the New Jersev Aericultural
`Thin
`is oublication
`D-10540-1-87
`and wa8 supported
`by State and U.S. Hatch
`Fun&
`&d
`by The Chkch
`and Dwighi
`company,
`Inc.
`
`Meeting
`
`of the
`
`Institute
`
`of Food Technologists,
`
`June
`
`9824OlJRNAL OF FOOD SCIENCE-Volume
`
`53, No. 3, 1988
`
`