`
`OURNAL of
`
`March-April 1979
`Volume 44 2 Number 2
`..
`.f
`
`FOOD SCIENCE
`
`
`Board of Editors
`5
`J. Augustin (‘81)
`L. Beuchat (81)
`F. Clydesdale (81)
`H. Herring (81)
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`H. Swaisgood (81)
`R. Toledo (81)
`
`Scientific Editor
`Bernard J. Liske
`Asst. Scientific Editor
`Anna May Schenck
`Managing Editor
`Berna rd Schulo'aft
`
`Director of Publications
`John B. Klis
`_
`Publisher
`Calvert L. Willey
`
`Z. Carpenter (79)
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`G. Flick (79)
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`J. Litchfield (79)
`D. Lund (79)
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`
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`Table 1—Percentage inhibition of three strains of C, perfringens by
`butylated hydroxyanisolaI
`Percentage inhibition
`
`
`
`
`
`
`
`NCTC 10239ppm BHA_____.__.—.____—NCTC 8798 NCTC 8239
`
`
`
`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
`
`3Tested 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 blankl— (Treatment — Treatment blank)
`(Control — Control blank)
`
`X 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 each flask and, while being held at 37° C, a sample was
`withdrawn at 0.25, 5, 10, 20 and 30 min. The samples were serially
`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
`compounds are 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 calorimeter 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 sodium nitrite were
`used with 0. 50, 100 and 150 ppm BHA. Each sample was prepared
`by dissolving an appropriate amount of 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 allowed in 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 ml of
`FTM, and the pH was adjusted to 6.5 t 0.02 with 1N NaOH and 1N
`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 propyl esters
`of parahydroxybenzoic acid in 2:1 and 3:1 ratios (Chichester and
`Tanner, 1968) were used. Solutions were made by dissolving 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
`1
`7.0 t 0.02, and BHA at 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
`
`iNHiB/TION OF C. PERFR/NGENS BY BHA .
`
`.
`
`.
`
`
`
`KLETTUNITS
`
`200
`
`IOO
`
`A 200 PPM BHA
`
`0 CONTROL
`0 IOO PPM BHA
`
`
`
`23456789l01ll2
`
`Houhs
`
`Fig. 1—lnhibition of C. perfringens NCTC 70239 by butylated
`hydraxyanisole in Fluid Thioglyco/lare Medium at 37° C.
`
`utilized at 0.5% to insure dispersion of the oil. This compound is 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 Twéen 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 quantities added were
`such that when the oil was added later, 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
`perfrmgens 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 on a
`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
`Control plate count
`
`X100
`
`RESULTS & DISCUSSION
`Growth inhibition studies
`In the inital study, all three strain of C. perfringens were
`completely inhibited by 150 ppm BHA1n 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 108
`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 BHA are given in Figure 1.
`Subsequent experiments showed that 150 ppm was not
`always completely inhibitory. In several studies at 450 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, N0. 2 (1979i—JOURNAL OF FOOD SCIENCE—565
`
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`
`
`300
`250
`
`I50
`
`IOO
`
`
`
`KLETTUNITS
`
`5.5 ’6.0
`
`6.5
`
`7.0
`pH
`
`7.5
`
`8.0
`
`8.5
`
`
`
`LOGIoC.perfringensnnl
`
`
`
`o CONTROL
`
`
`
`
`:1 50 PPM BHA
`
`A IOO PPM BHA
`
`‘ l50 PPM BHA
`
`
`
`O
`
`5
`
`IO
`
`IS
`
`20
`
`25
`
`30
`
`MINUTES
`
`Fig. 2— The effect of pH on the inhibition of C. perfringens NCTC
`10239 by 700 ppm BHA in Fluid Thiog/ycal/ete Medium at 45°C
`after 9 hours incubation.
`
`Fig. 3—Letha/ity rate of buty/ated hydroxyaniso/e on C, perfringens
`NCTC 10239 in a buffer system at 37° C as determined on Trypti-
`case Sulfite Neomycin Agar.
`
`may also lfave 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 shown to 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 indicates that BHA was not being volatilized and
`lost during the autoclaving process, nor changed in some
`way as to enhance or destro’yits antimicrobial activity.
`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»
`fringene was partially inhibited by 100 ppm BHA, but in-
`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
`BHA at 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 sresistant
`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 BHA action on the individual 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. The re-
`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 number of viable cells decreasedeven more
`dramatically, and by 1b 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.
`
`BHA and other food antimicrobials
`Foods are seldom,
`if ever, contaminated by only one
`type of microorganism. For this 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 there is 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 make it 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 Closrridium 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 systems is
`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 BHA activity
`Unsaturated lipids are extremely reactive compounds
`
`"hum-inning.).La
`
`566—JOURNAL OF FOOD St‘lENCE—Voi. 44, No. 2 (1979)
`,, ,. ,74‘ ,i,
`
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`
`1 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 process
`that occurs (Dugan, 1976). The reaction between BHA and
`a lipid molecule in effect “uses up” the BHA molecule,
`é making it no longer effective as an antioxidant. The same
`lg: reaction could also result in destruction of the antimicro-
`«i-
`bial activity of BHA. Similarly, the hydrophobic nature of
` Jr
`, BHA and its solubility in lipophilic compounds might result
`in it being localized within the lipid portion of the medium,
`
`Jams{29%
`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-
`frz'ngens growth. This inhibitory action of Tween 80®
`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 for lipid samples containing
`BHA than 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 known if these observations can be
`applied directly to food products. The decreased antimicro-
`bial activity of BHA due to Tween 80® and lipid indicates
`that a partitioning of BHA into the lipid 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 reduce its antimicrobial activity.
`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
`
`
`
`
`
`.\v:«uup,inn/‘3‘...
`
`
`
`
`
`
`
`
`
`
`
`15$».tu“EK‘JW,1.\xfifiafifjk‘wa
`
`
`
`..wfi»-.
`
`INHIBITION OF C. PEHFRINGENS BY BHA .
`
`.
`
`.
`
`Tab/e 2—lnhibition of C. perfringens NCTC 7 023.9 by several combi-
`nations of butylated hydroxyanisole, nitrite, sorbic acid and
`
`parabens’L r
`% Inhibitionb
`
`Additive 0 ppm BHA 50 ppm BHA 100 ppm BHA 150 ppm BHA w
`Nitrite:
`0 ppm
`50 ppm
`100 ppm
`Sorbic acid:
`0.0
`0%
`0.05% . 17.8
`0.075% 22.8
`Parabensc:
`0%
`0.025%
`0.05%
`
`0.0
`0.0
`7.4
`
`0.0
`8.6
`31.8
`
`5.6
`0.0
`6.5
`
`3.9
`20.4
`38.3
`
`—d
`18.2
`38.9
`
`16.1
`39.9
`71.7
`
`17.5
`37.9
`59.7
`
`12.0
`38.8
`90.6
`
`79.1
`100.0
`100.0
`
`32.7
`76.3
`93.2
`
`41.4
`93.6
`100.0
`
`aTested in Fluid Thioglycollare 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)
`
`b Each value was determined from the mean of three readings.
`‘3 A 3:1 mixture of the methyl and propyl esters of paravhydroxy-
`benzoic acid
`6 Not determined
`
`Chang, H.C. and Branen, AL. 1975. Antimicrobial effects of buty- -
`lated hydroxyanisole (BHA). J.. Food Sci. 40: 349.
`Chichester, D.F. and Tanner; F.W. Jr. 1968.‘Antimicrobial food‘
`additives: In “Heindbook of Food‘Additivés?’ Ed. Furia. ’I‘.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;
`nc., NY.
`food Chemistry,” Ed. Fennena, O.R.. p. 139. Marcel Dekker.
`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'
`C0,, 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 parabeemolyticus 04:K11 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. 1953. 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.
`
`#
`
`Vol. 44, No. 2 (1979i—JOURNAL OF FOOD SGIENCE—567
`
`UCB Biopharma SPRL (IPR2019-00400)
`Exhibit 2033 Page 5
`
`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.
`
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