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`March-April 1979
`Volume 44 : Number 2
`‘,
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`OURNAL of
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`Table 1~Percentage inhibition of three strains of C. perfringens by
`butylated hydroxyanisole®
`Percentage inhibition
`
`
`
`ppm BHA NCTC 8239—_—NCTC 10239 NCTC 8798
`
`
`
`
`
`0.0
`0.0
`0.0
`0
`8.3
`9.3
`0.0
`50
`29.5
`34.5
`2.9
`100
`100.0
`100.0
`100.0
`150
`100.0
`100.0
`100.0
`200
`
`
`
`100.0 100.0400 100.0
`
`2 Tested in Fluid Thioglycollate Medium at 37°C, inhibition deter-
`mined by Klett colorimeter readings after 12 hr growth. Percent-
`age inhibition was calculated by:
`% Inhibition =
`
`{Control — Control blank) — (Treatment — Treatment blank)
`(Contro! — Control blank)
`
`100
`
`Solutions were made containing 0, 50, 100 and 200 ppm BHA and
`were autoclaved. One milliliter of a seed culture of NCTC 10239
`was added to eachflask and, while being held at 37°C, a sample was
`withdrawn at 0.25, 5, 10, 20 and 30 min. The samples wereserially
`diluted in 0.1% peptone dilution blanks, plated on TSN agar, and
`incubated anaerobically at 45°C for 24 hr.
`Butylated hydroxyanisole and other
`food antimicrobials
`Sodium nitrite, sorbic acid, and the esters of para-hydroxyben-
`zoic acid (parabens) were tested to evaluate the possiblity of an
`inhibition of antimicrobial activity or a synergistic effect when these
`compoundsare used in conjuntion with BHA.Initially, several con-
`centrations of each of these additives were tested for antimicrobial
`activity against C. perfringens NCTC 10239. The two greatest con-
`centrations in which growth occurred ‘were used to test for antimi-
`crobial interactions with BHA. In each case the antimicrobial solu-
`tions with BHA were autoclaved, inoculated and growth monitored
`using the Klett colorimeter as previously described.
`Preliminary work with sodium nitrite showed 150 ppm to be
`completely inhibitory to C perfringens when added to FTM and
`autoclaved. Concentrations of 50 and 100 ppm sodiumnitrite were
`used with 0, 50, 100 and 150 ppm BHA.Each sample was prepared
`by dissolving an appropriate amountof solid sodium nitrite in 100
`ml FTM, adding stock BHA solutions and autoclaving. Filter-steri-
`lized nitrite was also tested in the initial study. However, the con-
`centrations that permitted growth of C. perfringens were so high
`that it was considered impractical to pursue testing since these levels
`(2000 ppm) greatly exceed the maximum allowedin foods.
`Total inhibition of C. perfringens by sorbic acid alone occurred
`at a concentration of 0.1%. Concentrations of 0.05% and 0.075%
`Sorbistat® (Pfizer and Co. Inc., NY) were dissolved in 100 mi of
`FIM, and the pH was adjusted to 6.5 + 0.02 with IN NaOH and IN
`HCl prior to the addition of BHA. Concentrations of BHA used
`were 0, 50, 100 and 150 ppm.
`In initial work involving the parabens, methyl and propylesters
`of parahydroxybenzoic acid in 2:1 and 3:1 ratios (Chichester and
`Tanner, 1968) were used. Solutions were made bydissolving appro-
`priate quantities of the methyl ester (Pfaltz and Bauer, Inc., Stam-
`ford, CT) and the propyl ester (Sigma Chemical Co., St. Louis, MO)
`in 95% ethanol to give 5%, 7.5% and 10% w/v solutions. Both mix-
`tures were tested at concentrations of 0.05%, 0.075% and 0.1% for
`their effect on C. perfringens, All concentrations were inhibitory
`except the 3:1 ratio at 0.05%. For work with BHA, 0.025% and
`0.05% solutions of the 3:1 ratio were used. The pH was adjusted to
`7,0 + 0.02, and BHAat 0, 50, 100 and 150 ppm was added,
`Effect of lipids on the antimicrobial
`activity of butylated hydroxyanisole
`Trypticase Sulfite Neomycin (TSN) Agar served as the basal
`medium for this study. Corn oil (Mazola) was used to obtain lipid
`concentrations of 1, 2, 3, 4 and 5% (v/v). Polyoxyethylene sorbitan
`monooleate (Tween 80®, Atlas Powder Co., Wilmington, DE) was
`
`200
`
`INHIBITION OF C. PERFRINGENS BY BHA...
`
`© CONTROL
`o 100 PPM BHA
`4 200 PPM BHA
`
`
`
`2345 67 8
`
`§ Wit 2
`
`
`
`KLETTUNITS
`
`100
`
`HOURS
`
`Fig. 1—Inhibition of C, perfringens NCTC 10239 by butylated
`hydroxyanisole in Fluid Thioglycollate Medium at 37° C.
`
`utilized at 0.5% to insure dispersion of the oil. This compoundis an
`emulsifier with a high hydrophile-lipophile balance (HLB), suitable
`for oil-in-water emulsions (Griffin and Lynch, 1968). Each lipid
`concentration was tested with and without BHA at 200 ppm. The
`control (0% lipid) was TSN without any other ingredients. In addi-
`tion,
`the effects of the other ingredients were tested by_use of
`treatments with TSN and_BHA only; TSN and Tween 80® only;
`and TSN with Tween 80© and BHA. The TSN agar was prepared
`according to the manufacturer’s directions and quantities were dis-
`pensed into 200 ml prescription bottles. The quantitits added were
`such that when the oil was addedlater, the final volume was 150 ml
`excluding the volume due to the emulsifier and BHA. The BHA was
`then added to the appropriate bottles and all the bottles were auto-
`claved for 15 min at 121°C. After autoclaving, the bottles were held
`at 52°C and the emulsifier and corn oil were added aseptically. Pour
`plates were prepared with this media and a 14 hr culture of C
`perfringens NCYC 10239. Serial dilution of the culture were made
`in 0.1% peptone. Immediately prior to pouring the plates, the bot-
`tles were vigorously shaken for 20 sec. Pouring was carried out ona
`refrigerated surface to speed hardening of the medium. The plates
`were incubated anaerobically at 45°C for 24-36 hr. Percentage in-
`hibition was calculated from the plate counts using the formula:
`
`% Inhibition =
`
`Control plate count - Treatment plate count x 100
`Control plate count
`°
`
`RESULTS & DISCUSSION
`Growth inhibition studies
`In the inital study, all three strain of C. perfringens were
`completely inhibited by 150 ppm BHA in Fluid Thioglyco-
`llate Medium (FTM) when grown at 37°C. Growth at 50
`and 100 ppm BHA was delayed or inhibited to a lesser
`degree (Table 1). The average plate count of the control
`cultures after 15 hr of incubation at 37°C was 2 x 10°
`cells/ml, whereas the cultures containing 200 and 400 ppm
`BHA had no viable organisms. Growth curves for the NCTC
`10239 strain tested in media containing 0, 100 and 200
`ppm BHAare given in Figure 1.
`Subsequent experiments showed that 150 ppm was not
`always completely inhibitory. In several studies at 45°C,
`growth occurred in the solutions with 150 ppm BHA. The
`reasons for this variability are not known. The resistance
`(or susceptibility) of a microorganism is highly dependent
`on the age and health of the culture used (von Schelhorn,
`1953). Other factors that cannot be totally accounted for
`
`Vol. 44, No. 2 (1979)-JOURNAL OF FOOD SCIENCE—565
`
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`

`300
`250
`
`
`
`KLETTUNITS
`
`55 60 65
`
`70
`pH
`
`75
`
`80
`
`85
`
`
`
`
`
`LOG\9C.perfringens/imi
`
`
`
`o CONTROL
`
`a 50 PPM BHA
`
`4100 PPM BHA
`
`* 150 PPM BHA
`
`
`
`
`Q
`
`5
`
`10
`
`IS
`
`20
`
`«25
`
`30
`
`MINUTES
`
`Fig. 2—The effect of pH on the inhibition of C. perfringens NCTC
`10239 by 100 ppm BHA in Fluid Thiaglycollate Medium at 45°C
`after 9 hours incubation,
`
`Fig. 3—Lethality rate of butylated hydroxyanisole on C. perfringens
`NCTC 10239 in a buffer system at 37°C as determined on Trypti-
`case Sulfite Neomycin Agar.
`
`may also liave contributed to these variable results, such as
`. differences in handling of samples and small differences in
`oxidation-reduction potential, pH, or temperature.
`The ethanol used as a carrier for BHA was shownto have
`no deleterious effects on C. perfringens growth. Similarly,
`there was little difference in inhibition between cultures
`grown‘in media containing autoclaved versus filter-sterilized
`BHA: This ifidicates that BHA wasnot being volatilized and
`lost during the autoclaving process, nor changed in some
`way as to enhanceor destroy its antimicrobialactivity.
`The activity and potential effectiveness of several food
`antimicrobials is dependent on the pH at which they are
`used, At all of the pH values tested the growth of C. per-
`fringens. was partially inhibited by 100 ppm BHA, butin-
`hibition was greatest at the pH extremes where the organ-
`ism was already under considerable stress (Figure 2). This
`increased inhibition does not necessarily indicate that BHA
`has optimal activity at
`these pH values, but rather, that
`there is an increase in the susceptibility of the organism to
`BHAat the pH extremes that were used.
`Experiments using cultures of different ages indicated
`that the susceptibility of C. perfringens to BHA is age de-
`pendent, Fourteen to eighteen hour cultures grown in FTM
`at 45°C were more -resistant
`to inhibition by BHA than
`cultures less than 14 or over 18 hr old. The 14 to 18 hr
`cultures also gave more consistent results for percentage
`inhibition by BHA.Plate counts of all the cultures were in
`the range of 2 x 108 to 3 x 108 cells/ml. Apparently the
`variability in inhibition by BHA that occurred with cultures
`of different ages was due to differences in metabolic rates
`(i.e., a fast metabolism in a young culture rendering it more
`susceptible to BHA inhibition) or to the presence of toxic
`metabolic end products in the medium. Further study on
`the actual mechanisms of BHAaction on theindividual cell
`may explain this finding.
`Lethality rate of BHA
`The lethality rate study was conducted to determine
`whether inhibitory concentrations of BHA were actually
`killing C. perfringens or simply preventing growth. There-
`sults of this study are shown in Figure 3. In the dilution
`buffer used for this study, the number of viable cells re-
`mained constant in the control throughout the test period,
`
`as indicated by plating on TSN agar. Upon addition of 50
`ppm BHA to the medium, this number was immediately
`reduced by approximately one log cycle and by two and
`one-half log cycles after 30 min incubation. At 100 and 200
`ppm BHA, the numberof viable cells decreased even more
`dramatically, and by 10 min incubation, the plate counts
`showed that almost no viable cells were present. Although
`recovery of possibly injured cells may not have occured on
`the TSN agar, these results indicate that BHA was bacteri-
`cidal rather than bacteriostatic.
`
`BHAandother food antimicrobials
`Foods are seldom,
`if ever, contaminated by only one
`type of microorganism. Forthis reason, more than one anti-
`microbial is often employed to insure complete inhibition
`of all the organisms present. This practice may also allow
`the use of smaller quantities of the individual antimicro-
`bials. Occasionally, preservatives that are used together will
`enhance each other (von Schelhorn, 1953), but thereis also
`the chance that one compound might reduce the other’s
`activity. The tests using BHA with other antimicrobials
`were conducted for this reason, The results are shown in
`Table 2. It appears that none of the antimicrobials tested
`reduced the activity of BHA against C. perfringens. Rather,
`the data indicate that adding BHA may makeit possible to
`reduce the concentrations of the other antimicrobials need-
`ed to achieve inhibition via a synergistic effect, This may be
`of interest
`to the food industry as a possible means of
`reducing the amounts of these antimicrobials used in food
`products. For instance, if testing of Clostridium botulinum
`with BHA and nitrite showed a response similar to that of
`C. perfringens, the amount of residual nitrite necessary for
`protection against botulism might be reduced, although
`BHA’s usefulness in high fat products may be limited as
`discussed below. Likewise,
`the amount of sorbic acid or
`parabens might be reduced in other food products if BHA
`were added to these. Additional work using food systemsis
`needed to determine whether the synergistic effects would
`exist in these processed foods as they do in a broth test
`system.
`
`Effect of lipids on BHAactivity
`Unsaturated lipids are extremely reactive compounds
`
`EaanenotinaSMRDta
`
`566—JOURNAL OF FOOD SCIENCE—Vol, 44, No. 2 (1979)
`eli
`
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`

`INHIBITION OF C. PERFRINGENS BY BHA...
`
`Table 2—Inhibition of C. perfringens NCTC 10239 by several combi-
`nations of butylated hydroxyanisole, nitrite, sorbic acid and
`
`parabens® 4
`% Inhibition®
`
`0.0
`0.0
`74
`
`5.6
`0.0
`6.5
`
`3.9
`20.4
`38.3
`
`16.1
`39.9
`71.7
`
`17.5
`37.9
`59.7
`
`79.1
`100.0
`100.0
`
`32.7
`76.3
`93.2
`
`Additive Oppm BHA 50 ppm BHA 100 ppm BHA 150 ppm BHA *
`Nitrite:
`Opom
`50 ppm
`100 ppm
`Sorbic acid:
`0.0
`0%
`0.05% . 17.8
`0.075% 22.8
`Parabens®:
`41.4
`12.0
`a
`0.0
`0%
`93.6
`38.8
`18.2
`8.6
`0.025%
`
`
`
`
`31.8 38.9 90.60.05% 100.0i
`@ Tested in Fluid Thioglycollate Medium at 45°C,inhibition deter-
`mined by Klett colorimeter readings after 12 hr growth. Percent-
`age inhibition was calculated by:
`% Inhibition =
`
`‘
`(Control — Control blank)— (Treatment — Treatment blank) x 100
`(Control -- Control! blank)
`
`P Each value was determined from the mean of three readings.
`© A 3:1 mixture of the methyl and propyl esters of para-hydroxy-
`benzoic acid
`d Not determined
`
`Chang, H.C. and Branen, A.L, 1975. Antimicrobial effects of buty- .
`lated hydroxyanisole (BHA). J., Food Sci. 40: 349.
`Chichester, D.F. and Tanner, F.W. Jy, 1968, Antimicrobial food -
`additives; In “(Handbook of Food*Additivés,’ Ed. Furia, T.E., p.
`137. Chemical Rubber Co., Cleveland, OH.
`Dework, F.M. Jr. 1972, Sporulation of Clostridium perfringens
`Type A in vacuum-sealed meats, Appl. Microbiol. 24: 834.
`Dugan, L. Jr. 1976. Lipids. In “Principles of Food Science, Part 1;
`Pood Chemistry,” Ed. Fennena, O.R., p. 139. Marcel Dekker,
`ne, NY,
`Forrest, J.C., Aberle, E.D., Hedrick, H.B., Judge, M.D. and Merkel,
`R.A. 1975. “Principles of Meat Science, ” W.H. Freeman and”
`Co,, San Francisco,
`Griffin, W.C. and Lynch, M.J. 1968. Surface active agents. In
`“Handbook of Food Additives,” Ed. Furia, T.E., p. 413. Chemi-
`cal Rubber Co., Cleveland, OH.
`Robach, M.C., Smoot, L.A. and Pierson, M.D. 1977. Inhibition of _
`Vibrio parahaemolyticus 04:Ki1 by butylated hydroxyanisole,
`J. Food Prot, 40: 549.
`Shih, A.L, and Harris, N.D, 1977, Antimicrobial activity of selected ~
`antioxidants, J, Food Prot, 40; 520.
`von Schelhorn, M. 1954. Efficacy and specificity of chemical food
`preservatives, Food Technol. 7: 97.
`Ms received 6/10/78; revised 8/17/78; accepted 8/24/78.
`
`Presented at the 37th Annual Meeting of the Institute of Food
`Technologists, Philadelphia, PA, June 5-8, 1977.
`Scientific Paper No. 5111, College of Agriculture Research Cen-
`ter, Washington State University, Pullman, WA.
`
`el
`
`Vol. 44, No. 2 (1979)—JOURNAL OF FOOD SCIENCE—567
`
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`
`E which undergo autoxidation by reaction with oxygen and
`* subsequent formation of free radicals and hydroperoxides.
`
`Phenolic antioxidants such as BHA are active in preventing
`€ autoxidation by donating a hydrogen atom to the unstable
`lipid free radical, interfering with the chain reaction procegs
`
`that occurs (Dugan, 1976). The reaction between BHA and
`a lipid molecule in effect ‘uses up” the BHA molecule,
`
`making it no longer effective as an antioxidant. The same
`- reaction could also result in destruction of the antimicro-
`; bial activity of BHA. Similarly, the hydrophobic nature of
`‘S BHAand its solubility in lipophilic compounds might result
`in it being localized within the lipid portion of the medium,
`rendering it unavailable to act against those microorganisms
`which grow in the aqueous phase of the medium. An inves-
`"tigation of whether the interaction of BHA with lipid in a
`microbiological medium would affect the antimicrobial ac-
`tivity of BHA was, therefore, conducted.
`The use of corn oil in TSN agar with Tween 80® as an
`emulsifier proved to be an effective method for production
`of an emulsion for this study. However, it was observed
`that Tween 80® caused considerable inhibition of C. per-
`fringens growth. This inhibitory action of Tween 30@
`masked the effects of BHA inhibition at different lipid con-
`centrations. It did appear that an increase in lipid concen-
`tration caused a reduction in the inhibition of C perfringens
`by BHA,indicating some interaction between BHA and the
`lipid. Antimicrobial activity still
`remained, however, as
`greater inhibition was observed forlipid samples containing
`BHAthan for those without BHA.
`Foods are extremely complex, varied systems, and sev-
`eral factors may control the effect of lipids on BHA inhibi-
`tion. Therefore, it is not knownif these observations can be
`applied directly to food products. The decreased antimicro-
`bial activity of BHA due to Tween 80©
`andlipid indicates
`that a partitioning of BHA into thelipid phase of the med-
`ium is taking place. This same type of partitioning may also
`occur in a food product. If so, the potential for use of BHA
`as an antimicrobial is limited since it is currently allowed
`only in foods containing lipids, and these compounds ap-
`pear to reduceits antimicrobialactivity.
`In conclusion, it has been shown that butylated hydrox-
`yanisole does have a marked antimicrobial activity against
`Clostridium perfringens and will consistently inhibit
`its
`growth at a concentration of 200 ppm. This antimicrobial
`activity was bactericidal under the conditions of this study
`and also heat stable, withstanding the stress of autoclaving.
`Several factors appear to be important in the extent of
`inhibition of C. perfringens by BHA. These include the age
`and physiological state of the culture, temperature, the pH
`of the medium, and the presence of lipids. This last factor
`may limit
`the usefulness of BHA as an antimicrobial in
`lipid-containing food products.
`REFERENCES
`
`i:
`
`Ayaz, M. 1975. Studies on the physiological characterization of
`staphylococci and the effects of butylated hydroxyanisole and
`butylated hydroxytoluene on growth and enterotoxin produc-
`tion of Staphylococcus aureus, Ph.D.
`thesis, Washington State
`University, Pullman, WA
`
`q&
`
`Laetih
`
`BRepEtroteininsheEAEhsTet
`
`AnASWDennywestae
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