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Antimicrobial Activity of Microgard™ Against Food Spoilage and Pathogenic Microorganisms1
Antimicrobial Activity of MicrogarcP Against Food Spoilage and Pathogenic Microorganisms' N. AL-ZOREKY
Department of Food Science and Technology J. W. AYRES College of Pharmacy
W. E. SANDINE Department of Microbiology Oregon State University Corvallis 973313804
Increased research attention is being given to the use in foods of naturally occurring metaboMicrogardm, a commercially available lites produced by selected bacteria to inhibit the fermented milk product containing antigrowth of undesirable microorganisms (3, 9). microbial metabolites, was a potent inhibSuch natural inhibitors could replace the use of itor for Gram-negative bacteria such as chemical preservatives. Pseudomonas, Salmonella, and Yersinia Microgardm. grade A skim milk fermented when 1% concentration was incorporated by specific dairy organisms and then pasteurinto agar media. Gram-positive Bacillus ized, was inhibitory to Pseudomonas and cercereus, Staphylococcus aureus, and tain fungi (25, 32). The product is added at 1% Listeria monocytogenes were insensitive to dairy products such as cottage cheese, yoto Microgardm. Kluyveromyces marxiagurt, and salad dressing. Microgardm is a p nus, an unidentified black yeast, and proved by the FDA and is used extensively in Penicillium expansum were partially supcottage cheese produced in the US (3). Also, pressed, whereas Aspergillus niger and a nondairy Microgardm is available for use in yogurt spoilage yeast were tolerant to 5% products such as sausage and bakery goods. Microgardm. Optimum activity of One million annual cases of enteric disease, Microgardm was at pH 5.3 and below; attributed to foodborne bacteria including Salthe concentration that gave complete inhimonella, Campylobacter, Yersinia, and Escherbition depended upon the number of bacichia coli, occur in the US. These infections teria present as well as the genus tested. cause loss of productivity and medical expenses Blood agar base reversed the antagonistic (14). Adding natural preservatives such as activity of Microgardm against PseudoMicrogardm may restrain the growth of some monas putida compared with plate count spoilage and pathogenic microorganisms, beagar. cause refrigeration alone (2 to 5'C) is known to (Key words: Microgardm, pathogens, anbe insufficient in preventing the growth of psytimicrobial activity) chrotrophic bacteria such as Clostridium botulinum type E,Yersinia enterocolitica, Aeromonas Abbreviation key: BHI = brain-heart infusion, hya'rophiliu, and Listeria monocytogenes (5,23, MIC = minimum inhibitory concentration, 24). Different approaches have been used to SPC = standard plate count. extend the shelf-life of food, including the use of buffered acids, low salt soy protein hydrolyINTRODUCTION sates, nisin, fermented whey, and organic acids (4, 21, 31). The present study reports on the effectiveness of Microgardm in inhibiting selected food Received Apnl 16, 1990. spoilage organisms as well as pathogenic miAccepted September 28, 1990. 'Technical Paper Number 9217, Oregon Agricultural croorganisms known to cause foodbome illnesses. Experiment station. ABSTRACT
1 9 1 J Dairy Sci 74:758-763
75 8
ANTIMICROBIAL. ACTIVITY OF MICROGARDm
MATERIALS AND METHODS
Microbial Strains and Culture Conditlons
759
tration of Microgardm that gave 100% inhibition of a 1:100 dilution of the test organism incubated 48 h at the assigned temperature (Table 1). This dilution provided lo6 to 107 cfu/d of the test organisms. Percentage inhibition was calculated as described by Litopoulou-Tzanetaki (15) and Freese et al. (6):
Table 1 shows the microorganisms used and the growth media employed. Foodborne pathogens were activated in brain-heart infusion (BHI) broth (Diico Laboratories, Detroit, MI) at 30°C for 24 h while spoilage Gram-negative standad plate count (SPC) of control psychrotrophs (Pseudomonas spp. and - SPC of sample Achromobacter) were propagated in lactose x loo. SPC control broth (Difco Laboratories, Detroit, MI). Lactobacillus spp. and fungi were propagated in MRS and malt extract (Difco Laboratories, influence of pH Detroit, MI), respectively. Between active Different pH conditions (5.0, 5.3, 5.6, and growth, cultures were maintained on BHI agar at 2 to 5'C. Each active organism was serially 5.8) were evaluated for effects on the inhibition of Pseudomonas putida by Microgardm using diluted with sterile .l% peptone water. the agar incorporation method. Sterile tartaric acid (10% solution) was used to lower the pH Preparatlon of the Growth Media of plate count agar (Difco Laboratories, Detroit, A selective or recommended medium (Table MI) and Microgardm. Incubation was at 30'C 1) was used to grow each bacterium. The pH of for 48 h. each was adjusted to 5.3 (Coming 125 pH meter, Medfield, MA) with 10% tartaric acid Effect of Type of Medium before autoclaving at 121'C for 15 min. Media on inhibition employed for fungal growth were acidified to The agar incorporation method (at pH 5.3) pH 4.0 prior to sterilization. These low pH was used to test the effectiveness of adjustments did not inhibit agar gelation because boiling of agar media was not employed Microgardm against Pseudomonas putida by before sterilization in order to avoid merheat- employing two agar media, plate count agar, and blood agar base (Difco Laboratories, ing. Detroit, MI); plates were incubated at 30'C for 48 h. MicrogaW Preparation and SusceptibilityTesting RESULTS AND DISCUSSION Liquid Microgardm (pH 6.2) was obtained from Wesman Foods, Inc., (Beaverton, OR) and kept refrigerated until use; it was acidified Screening to pH 5.30 (for bacterial screening) or 4.0 (for Among microorganisms tested, all but one fungi) with 10% sterile tartaric acid. Different concentrations of Microgardm (S, 1,3, and 5% of the Gram-negative bacteria were strongly vol/vol) were incorporated in the sterilized, inhibited by Miirogardm (100% inhibition at cool media and assayed against microorganisms 1% concentration). The exception was E. coli for their sensitivity using the agar incorporation V517 Fable 2). In this regard, it is of interest that diacetyl was found by Jay (11) to be method of Hogg et al. (8). inhibitory to all Gram-negative bacteria except E. coli. Similarly, some spheroplasts 6.om E. Minimum inhibitory Concentration coli were not inhibited by metabolites of LacMinimum inhibitory concentration (MIC) tobacillus plantarum (1). Also, Moustafa and was determined using the conditions for sus- Collins (22) stated that the rigidity of the cell ceptibility testing as described in the preceding wall of E. coli may contribute to its resistance section and was defined as the lowest concen- to inhibitors. Journal of Dairy Science Vol. 74, No. 3, 1991
760
AGZOREKY ET AL.
TABLE 1. Mimrmnisms and assay media used in this study. -~~ ~
Organism
source
Assay medium and incuketion temperature
wesmaul ATCC ATCC
Plate count agar (PCA), 30'C PCA, 30'c PCA, 30'c Violet red bile agar, 30'C
ATCC
Salmonella-ShigeUa (SS) agar, 37'C SS agar, 37'C
Gram-negative
Pseudomonas putida w Pseudomonas aeruginosa 419
Achromobacter dclicatulus 19103 Escherichia coli V 517 Salmonella paratyphi 9281 Salmonella tphimurium OSU Shigella dysenteriae E19b Campylobacter jejuni 29428 Yersinia enterocolitica 23715 Aeromonas hydrophila 7965 Gram-positive Lactobacillus P Lactobacillus 79A Lactobacillus 79B Lactobacillus brevis Lactobacillus plantarm MT Bacillus cereus var. mycoides Staphylococcus aureus Listeria monocytogenes 7644 Propionibacterium shennanii 9616
pungi Yeast4 Y Kluyveromyces marxianus 8554 Black yeast5 Aspergillus niger OSU PeniciIium erpanswn OSU'
osu
OSUZ
ss agar, 37'c B N W agar, ~ 42'C Macconlrey agar, 30'c Nutrient agar, 30'C
osu
ATCC ATCC ATCC
Pickles Wesmanl wesman'
osu ~icrolire3 os+ osu2
ATCC ATCC yogurt
ATCC wesman' Sweet condensed milk
osu
MRS MRS MRS MRS
agar, 37'C (Gas Pak, BBL, Baltimore, agar, 37'c (Gas Pak) agar, 37'C (Gas Pak) agar, 37'C (Gas Pak)
MD)
MRS agar, 37'C (Gas Pak) Nuhiat agar, W C Manit01 salt agar, 37'C Brain-heart infusion, trypticase soy agar, blood agar base, 30'C Thioglycollate agar, 30'C (Gas Pak)
Potato dexeose agar (PDA), 30'C PDA, 30'c Mycophile agar (MA), 30'C MA, 25'C
MA,
2Sc
'Weman POO~S, w., ~eaverton,OR. kulture collection, Oregon State University, Department of Microbiology. JM~CIOW~ ~ e ~ h n i c sara~ota, s. PL. 4lsolated from yogurt. 'Isolated from cottage cheese.
In the present study, both Cumpylobucrer jejuni ATCC 29428 and Shigella dysenteriae E19b did not grow in control plates (pH 5.3) not containing Microgardm (Table 2). Previous reports stated that Cumpylobucrer jejuni was not able to grow in nutrient broth at pH 5.3 or in yogurt at pH 4.2 to 5.3 (2, 12). Therefore, the pH condition (5.3) used to assay Microgardm likely was below the minimum pH of growth for these bacteria. Concerning Shigella dysenteriae, some strains, such as strain 1, were inhibited by Salmonella-Shigella agar (29), and this also may explain the absence of growth of this particular pathogen in this medium. Interestingly, when C. jejuni ATCC 29428 grew in Brucella agar @ifco Laboratories, Journal of Dairy Science Vol. 74, No. 3, 1991
Detroit, MI) at 42'C (CampyPak, Microerophilic System, BBL, Baltimore, MD) and pH slightly above 5.3, it undment conversion from rods to cocci as observed when examined microscopically. Moran and Upton (19, 20) reported similar results. Microgardm was not able to retard the growth of the Gram-positive bacteria tested, although Lisreria monocyrogenes ATCC 7644 gave variable results (Table 2). Hence, Microgardm is apparently rather specific for inhibiting Gram-negative bacteria, whereas nisin is inhibitory for Gram-positive bacteria (7). Some fungi screened were suppressed by MicrogarP, although no complete inhibition was achieved (Table 2). The insensitivity of some fungi may be related to proteolytic en-
761
ANTIMICROBIAL ACTMTY OF MICROGARDm
TABLE 2. Antagonistic activity of MicrogaxP at pH 5.3 against different microorganisms as revealed during the agar incorporation assay. Test organism
Sensitivity'
Gram-negative Pseudomonas putida W Pseudomonas aeruginosa 419 Achromobacter delicatulus 19103 Escherichia coli V517 Salmonella paratyphi 9281 Salmonella typhimurium OSU Shigella dysenterioe E19b Campylobacter jejuni 29428 Yersinia enterocolitica 23715 Aeromonas hydrophila 2965 Gram-positive Lactobacillus isolate P Lactobacillus 79A Loctobacillus 79B Latobacillus brevis OSU LaCiobaciNus plantarum MT Bacillus cereus OSU Staphylococcus aureus OSU Propionibacterium shermanii %16 Liszmb monocytogenes 7644
tt tt
++
-
tt tt
NG NG U ii-
w Yeast Y
s
+ + +
Kluyveromyces marxianus 8554 Black yeast Aspergillus niger OSU Penicillwn exvansum OSU
'NG = No growth in control plates @H 5.30). -H = complete inhiiition (no growth), stimulation effect, f = variable results, - = no inhibition.
+ = partial inhibition, S
=
zymes produced (5, 16, 18, 30), which degrade some inhibitors or raise the pH. However, yeasts were inhibited when Microgardm was prepared under certain fermentation conditions; in fact, yeast or mold inhibitory activity can be independently potentiated by varying fermentation conditions in preparing Microgardm (Weber, 1989, personal communication).
centration of Microgardm (1%) after being exposed for less than 42 h. Thus, a higher Microgardm concentration was required to achieve complete inhibition for more than 42 h of incubation at 37'C compared with Salmonella paratyphi (Table 3). Staphylococcus aureus is reportedly able to initiate growth after being inhibited by metabolites of LuctobacilZus plantarum (1). Total inhibition by 3% Microgardm was achieved against high microMinimum inhibitory Concentration Determination bial loads (lo6 to lo7 cfu/ml), and this inhibition lasted for more than 7 d at 30'C against The MIC values against Gram-negative bacsame sensitive Gram-negative psychrotrophic teria tested are shown in Table 3. Pseudomonas organism (Figure 1). Furthermore, 1% putida was dramatically inhibited by low con- Microgardm gave the same inhibition for more centrations of Microgardm, whereas Pseudo- than a week when tested against Achromobacmonas aeruginosa ATCC 419 required a higher fer (data not shown). MIC value (3%) to achieve the same inhibition. The latter bacterium is known for its resistance Effectiveness of Microgard" to some antibiotics (17). Yersinia and Aeromo- as a Function of pH nas organisms were highly vulnerable to Microgardm, as evident from their low MIC Figure 2 illustrates the activity of Microvalues (Table 3). However, Salmonella gardm as a function of pH and shows that typhimunum was able to overcome a low con- Microgard" functioned optimally below Journal of Dairy Science Vol. 74, No. 3, 1991
762
ALZOREKY ET AL.
TABLE 3. Minimum inhibitory concentration (MIC) of Microgdw to came 1UO% inhibition of various Gram-negative bacteria as revealed by the agar incorporation assay. The middle column shows the numbers of organisms present in the assay medium. Strain
Numbea of organisms
Pseudomonas purida W Pseudomonas aeruginosa 419 Achromobacter delicatulus 19103 Salmonella paratyphi 9281 Salmonella typhimuriwn OSU Yersinia enterocolitica 23715 Aeromonns hydrophila 7965
(cfo/ml) 6 X lo7 6.8 x io7 5 x 106 2.5 x io7 4 x io7 1.5 x io7 6.6 X IO6
pH 5.6. Although 3% Microgardm gave complete inhibition against the test bacterium at pH 5.3, 1% concentration gave the same inhibition when cells were diluted further to give about 104 cfu/ml (data not shown). This property renders it useful as a shelf-life extender of acid foods. Compared with other food preservatives, diacetyl and propionate also are much more inhibitory at pH values below 7.0 (6, 11). However, ascorbic acid was bacteriocidal for Campylubucrer jejuni even at pH 7.0 (12). Antagonistlc Effect of Some Medla Components on Mlcrogard"
The results shown in Figure 3 revealed that a low concentration of Microgardm (1%) was not able to retard the growth of Pseudomoms putida when a rich medium such as blood agar base was used for growth compared with plate count agar. Jay (11) reported similar results
(96 vol/vol) 1 3 1
1 3 1 1
whereby diacetyl was most effective in plate count but not in BHI agar or cooked meat medium. The antimicrobial activity of specific compounds can be reversed by some substances such as albumin, glucose, ash, serum, and phosphate (10, 11, 13, 22, 26, 27, 28). These observations indicate that food composition may be a factor influencing the inhibitory activity of Microgardm. This does not appear to be a detriment with dairy products, however, because Microgardm inhibitory activity is readily demonstrated in both cottage cheese and yogurt (25). Therefore, higher concentrations of Microgardm may be required in certain foods, and for this purpose both spray-dried and freezedried preparations as well as a liquid concentrate have recently been developed.
100
C
MIC
100
75
a
1 % Microgard
c 75
0 .c
.-0
2c
50
8
25
25
0
0
c .-
0 50
L
-
c -
3
1
% Microgard Figure 1. Influence of incubation time of PseudomoMs purida on the minimum inhibitory concentration of M i c r o g a r P deteamined by the agar incorporation method. Assay plates were seeded with 106 to 107 cfu/ml.
Journal of Dairy Science Vol. 74, No. 3, 1991
5.0
5.3
5.6
5.8
PH Pigare 2. Correlation between inhibition of Pseudomonas pwido by MicrogarP and pH of the agar medium assayed by the agar incorporation method. Assay plates were seeded with lo6 to io7 du/ml.
ANTIMICROBIAL ACTIVlTY OF MICROGARDm
'0°1
.-
a c 25
1
Ot
3
1 o/o
Microgard
Figure 3. The antagonistic effect of growth medium on inhibition by Microgardm assayed against Pseudomonas pur& by the agar incorpomtion method. Assay medium was seeded with io6 to lo7 a d . REFERENCES 1 Anderson, R 1986. Inhibition of Stuphylococcus uureus and spheroplasts of Gram-negative bacteria by an antagonistic compound produced by a strain of Loctobacilluc plantarum. Int. J. Food Microbiol. 3:149. 2 % Z., A. Annan-prah. M Janc, and J. Zajc-Satler. 1987. Yoghurt: an unlikely source of Campylobacrer jejunilcoli. J. Appl. Bactcriol. 63202. 3 Daeschel, M A. 1989. Antimicrobial substances from lactic acid bacteria for use as food preservatives. Fbod Technol. 43(1):164. 4Debevere. J. M 1987. The use of buffered acidulant systems to improve the microbiological stability of acid foods. Food Microbial. (Lomi.) 4:lM. 5 meet, G.H., and M. A. Mise 1987. The occurrence and growth of yeasts in dairy products. Int. J. Food Microbiol. 4 145. 6 Freese, E.,C. W. She% and E.Galliers. 1973. Function of lipophilic acids as antimicrobial food additives. Nature (Loud.) 241:321. 7Henning. S., R M Hammes, and W. P. Hammes. 1986. Studies on the mode of action of nisin. Int. J. Food Microbiol. 3:121. 8Hogg, G. M., M. F. Patterson. and J. G. Barr. 1987. Correlation between antibiotic sensitivity testing by conventional and conductivity measnrements. J. Appl. Bacteriol. 62189. 9 Hoover, D. G., K.J. Dishart and M A. Hermes. 1989. Antagonistic effect of Pediococcus spp. against Listeria monocyfogenes. Food Biotechnol. (NY) 3183. lOIsmaeel, N.. J. R. Furr, and A. D. Russell. 1986. Reversal of the surface effects of chlorhexidine. diacetate on cells of Providencia staurfii. J. Appl. Bacteriol. 61:373. 11 Jay, James M. 1982. Antimicrobial properties of diacetyl. Appl. Environ. Microbiol. 44525. 12 Juven, B. J., and J. Katmer. 1986. Effects of ascorbic, isoascorbic and dehydroascorbic acids on the growth and survival of Campylobacter jejuni. J. Appl. Bacteri01. 61539. UKabara, Jon J. 1978. Fatty acids and derivatives as antimicrobial agents: a review. Ch. 1. The pharmacological effects of lipids. Jon J. Kabara, ed. Am.
763
oil chem. Soc., champaign, IL. 14Kvenberg, J. E., and D. L. Archer. 1987. Economic impact of colonization control of food-born disease. Food Techeol. 47(7):77. 1 5 L i t o p o u l o ~ - T ~ t a kE. i , 1987. Interactions of PediOCOCCUF penrosuceus and some food-borne pathogens. Food Microbiol. (Lond.) 4293. 16Magan, N., and J. Lacey. 1986. Water relations and metabolism of propionate in two yeasts from hay. J. Appl. Bacteriol. a 1 6 9 . 17Mehta, A. M,K. A. Patel, and P. J. Dave. 1983. Isolation and purification of an inhibitory protein from Lactobacillus ackbphilus AC1. Microbios 37:43. 18 Miyakoshi, S., H. Uchiyama, T. Someya, T. Satoh, and T. Tabuchi. 1987. Dismiution of the methylcitric acid cycle and B-oxidation pathway for propionate c a t a b h in fungi. Agric. Biol. Chem. 51:2381. 19 Moran, A. P., and M.E.Upton. 1987. Factors affecting production of coccoid forms by Campylobacrer jejuni m solid media during incubation. J. Appl. Bacteriol. 62:527. POMoran, A. P., and M. E. Upton. 1987. Effect of medium supplements, illumination and superoxide dismutase on the production of the coccoid forms of Campylokter jejuni ATCC 29428.1. Appl. Bacteriol. 62:43. 21 Morris, C. E. 1985. Derived from whey, it's a milk replacer. Food Eng. (NY) 5763. 22MousIafa, H. H., and E. B. Collins. 1969. Effect of selected food additives on growth of Pseudomonas frugi. J. Dairy Sci. 52335. 23 Palumbo, Samuel A. 1986. Is refrigeration enough to restmin foodborne pathogens? J. Food Prot. 491003. 24 Phillips, J. D., and M. W. Griffiths. 1987. The relations between temperature and growth of bacteria in dairy products. Food Microbiol. (Loud.) 4172. 25 Salih, M. A., W. E. Sandine, and J. W. Ayres. 1989. Inhibitory effects of Microgardm on yogurt and cottage cheese spoilage organisms. J. Dairy Sci. 73:887. 26 Schales, 0. 1951. Effect of albumin on the antibacterial activity of &tones. Arch Biochem. Biophys. 3456. 27 Shibasaki, I., and N. Kato. 1978. Combined effects on antibacterial activity of fatly acids and their esters agaiust Gram-negative bacteria. Ch.2. The phaunacological effects of lipids. Jon J. Kabara, ed. Am. Oil chem. soc., Champaigs IL. 28sR. P. 1984. Effect of buffering media with phosphates on antibiotic resistance of lactic streptococci. Appl. Environ. Microbiol. 47:1175. 29 Speck, M. L., ed. 1984. Compendium of methods for the microbiological examination of foods. Am. Public Health Assoc., Washington. DC. 3OSnihL0, M L., and V. Makinen. 1984. Tolerance to acetate, propionate and sorbate by Saccharomyces cerevisiae and Torulopsis holmii. Food Microbiol. (Loud.) 1:lM. 31 Vallejdordoba, B., S. Ndai, W. D. Powrie, and T. Bevddge. 1987. Extended shelf life of franwlrters and fish franldurter-analogs with added soy protein hydrolysates. J. Food Sci. 521133. 32 Weber, G. H.,and W. A. Broich 1986. Shelf-life extension of cultured dairy foods. Cult. Dairy Prod. J. 21(4):19.
Journal of Dairy Science Vol. 74, No. 3, 1991
Department of Food Science and Technology J. W. AYRES College of Pharmacy
W. E. SANDINE Department of Microbiology Oregon State University Corvallis 973313804
Increased research attention is being given to the use in foods of naturally occurring metaboMicrogardm, a commercially available lites produced by selected bacteria to inhibit the fermented milk product containing antigrowth of undesirable microorganisms (3, 9). microbial metabolites, was a potent inhibSuch natural inhibitors could replace the use of itor for Gram-negative bacteria such as chemical preservatives. Pseudomonas, Salmonella, and Yersinia Microgardm. grade A skim milk fermented when 1% concentration was incorporated by specific dairy organisms and then pasteurinto agar media. Gram-positive Bacillus ized, was inhibitory to Pseudomonas and cercereus, Staphylococcus aureus, and tain fungi (25, 32). The product is added at 1% Listeria monocytogenes were insensitive to dairy products such as cottage cheese, yoto Microgardm. Kluyveromyces marxiagurt, and salad dressing. Microgardm is a p nus, an unidentified black yeast, and proved by the FDA and is used extensively in Penicillium expansum were partially supcottage cheese produced in the US (3). Also, pressed, whereas Aspergillus niger and a nondairy Microgardm is available for use in yogurt spoilage yeast were tolerant to 5% products such as sausage and bakery goods. Microgardm. Optimum activity of One million annual cases of enteric disease, Microgardm was at pH 5.3 and below; attributed to foodborne bacteria including Salthe concentration that gave complete inhimonella, Campylobacter, Yersinia, and Escherbition depended upon the number of bacichia coli, occur in the US. These infections teria present as well as the genus tested. cause loss of productivity and medical expenses Blood agar base reversed the antagonistic (14). Adding natural preservatives such as activity of Microgardm against PseudoMicrogardm may restrain the growth of some monas putida compared with plate count spoilage and pathogenic microorganisms, beagar. cause refrigeration alone (2 to 5'C) is known to (Key words: Microgardm, pathogens, anbe insufficient in preventing the growth of psytimicrobial activity) chrotrophic bacteria such as Clostridium botulinum type E,Yersinia enterocolitica, Aeromonas Abbreviation key: BHI = brain-heart infusion, hya'rophiliu, and Listeria monocytogenes (5,23, MIC = minimum inhibitory concentration, 24). Different approaches have been used to SPC = standard plate count. extend the shelf-life of food, including the use of buffered acids, low salt soy protein hydrolyINTRODUCTION sates, nisin, fermented whey, and organic acids (4, 21, 31). The present study reports on the effectiveness of Microgardm in inhibiting selected food Received Apnl 16, 1990. spoilage organisms as well as pathogenic miAccepted September 28, 1990. 'Technical Paper Number 9217, Oregon Agricultural croorganisms known to cause foodbome illnesses. Experiment station. ABSTRACT
1 9 1 J Dairy Sci 74:758-763
75 8
ANTIMICROBIAL. ACTIVITY OF MICROGARDm
MATERIALS AND METHODS
Microbial Strains and Culture Conditlons
759
tration of Microgardm that gave 100% inhibition of a 1:100 dilution of the test organism incubated 48 h at the assigned temperature (Table 1). This dilution provided lo6 to 107 cfu/d of the test organisms. Percentage inhibition was calculated as described by Litopoulou-Tzanetaki (15) and Freese et al. (6):
Table 1 shows the microorganisms used and the growth media employed. Foodborne pathogens were activated in brain-heart infusion (BHI) broth (Diico Laboratories, Detroit, MI) at 30°C for 24 h while spoilage Gram-negative standad plate count (SPC) of control psychrotrophs (Pseudomonas spp. and - SPC of sample Achromobacter) were propagated in lactose x loo. SPC control broth (Difco Laboratories, Detroit, MI). Lactobacillus spp. and fungi were propagated in MRS and malt extract (Difco Laboratories, influence of pH Detroit, MI), respectively. Between active Different pH conditions (5.0, 5.3, 5.6, and growth, cultures were maintained on BHI agar at 2 to 5'C. Each active organism was serially 5.8) were evaluated for effects on the inhibition of Pseudomonas putida by Microgardm using diluted with sterile .l% peptone water. the agar incorporation method. Sterile tartaric acid (10% solution) was used to lower the pH Preparatlon of the Growth Media of plate count agar (Difco Laboratories, Detroit, A selective or recommended medium (Table MI) and Microgardm. Incubation was at 30'C 1) was used to grow each bacterium. The pH of for 48 h. each was adjusted to 5.3 (Coming 125 pH meter, Medfield, MA) with 10% tartaric acid Effect of Type of Medium before autoclaving at 121'C for 15 min. Media on inhibition employed for fungal growth were acidified to The agar incorporation method (at pH 5.3) pH 4.0 prior to sterilization. These low pH was used to test the effectiveness of adjustments did not inhibit agar gelation because boiling of agar media was not employed Microgardm against Pseudomonas putida by before sterilization in order to avoid merheat- employing two agar media, plate count agar, and blood agar base (Difco Laboratories, ing. Detroit, MI); plates were incubated at 30'C for 48 h. MicrogaW Preparation and SusceptibilityTesting RESULTS AND DISCUSSION Liquid Microgardm (pH 6.2) was obtained from Wesman Foods, Inc., (Beaverton, OR) and kept refrigerated until use; it was acidified Screening to pH 5.30 (for bacterial screening) or 4.0 (for Among microorganisms tested, all but one fungi) with 10% sterile tartaric acid. Different concentrations of Microgardm (S, 1,3, and 5% of the Gram-negative bacteria were strongly vol/vol) were incorporated in the sterilized, inhibited by Miirogardm (100% inhibition at cool media and assayed against microorganisms 1% concentration). The exception was E. coli for their sensitivity using the agar incorporation V517 Fable 2). In this regard, it is of interest that diacetyl was found by Jay (11) to be method of Hogg et al. (8). inhibitory to all Gram-negative bacteria except E. coli. Similarly, some spheroplasts 6.om E. Minimum inhibitory Concentration coli were not inhibited by metabolites of LacMinimum inhibitory concentration (MIC) tobacillus plantarum (1). Also, Moustafa and was determined using the conditions for sus- Collins (22) stated that the rigidity of the cell ceptibility testing as described in the preceding wall of E. coli may contribute to its resistance section and was defined as the lowest concen- to inhibitors. Journal of Dairy Science Vol. 74, No. 3, 1991
760
AGZOREKY ET AL.
TABLE 1. Mimrmnisms and assay media used in this study. -~~ ~
Organism
source
Assay medium and incuketion temperature
wesmaul ATCC ATCC
Plate count agar (PCA), 30'C PCA, 30'c PCA, 30'c Violet red bile agar, 30'C
ATCC
Salmonella-ShigeUa (SS) agar, 37'C SS agar, 37'C
Gram-negative
Pseudomonas putida w Pseudomonas aeruginosa 419
Achromobacter dclicatulus 19103 Escherichia coli V 517 Salmonella paratyphi 9281 Salmonella tphimurium OSU Shigella dysenteriae E19b Campylobacter jejuni 29428 Yersinia enterocolitica 23715 Aeromonas hydrophila 7965 Gram-positive Lactobacillus P Lactobacillus 79A Lactobacillus 79B Lactobacillus brevis Lactobacillus plantarm MT Bacillus cereus var. mycoides Staphylococcus aureus Listeria monocytogenes 7644 Propionibacterium shennanii 9616
pungi Yeast4 Y Kluyveromyces marxianus 8554 Black yeast5 Aspergillus niger OSU PeniciIium erpanswn OSU'
osu
OSUZ
ss agar, 37'c B N W agar, ~ 42'C Macconlrey agar, 30'c Nutrient agar, 30'C
osu
ATCC ATCC ATCC
Pickles Wesmanl wesman'
osu ~icrolire3 os+ osu2
ATCC ATCC yogurt
ATCC wesman' Sweet condensed milk
osu
MRS MRS MRS MRS
agar, 37'C (Gas Pak, BBL, Baltimore, agar, 37'c (Gas Pak) agar, 37'C (Gas Pak) agar, 37'C (Gas Pak)
MD)
MRS agar, 37'C (Gas Pak) Nuhiat agar, W C Manit01 salt agar, 37'C Brain-heart infusion, trypticase soy agar, blood agar base, 30'C Thioglycollate agar, 30'C (Gas Pak)
Potato dexeose agar (PDA), 30'C PDA, 30'c Mycophile agar (MA), 30'C MA, 25'C
MA,
2Sc
'Weman POO~S, w., ~eaverton,OR. kulture collection, Oregon State University, Department of Microbiology. JM~CIOW~ ~ e ~ h n i c sara~ota, s. PL. 4lsolated from yogurt. 'Isolated from cottage cheese.
In the present study, both Cumpylobucrer jejuni ATCC 29428 and Shigella dysenteriae E19b did not grow in control plates (pH 5.3) not containing Microgardm (Table 2). Previous reports stated that Cumpylobucrer jejuni was not able to grow in nutrient broth at pH 5.3 or in yogurt at pH 4.2 to 5.3 (2, 12). Therefore, the pH condition (5.3) used to assay Microgardm likely was below the minimum pH of growth for these bacteria. Concerning Shigella dysenteriae, some strains, such as strain 1, were inhibited by Salmonella-Shigella agar (29), and this also may explain the absence of growth of this particular pathogen in this medium. Interestingly, when C. jejuni ATCC 29428 grew in Brucella agar @ifco Laboratories, Journal of Dairy Science Vol. 74, No. 3, 1991
Detroit, MI) at 42'C (CampyPak, Microerophilic System, BBL, Baltimore, MD) and pH slightly above 5.3, it undment conversion from rods to cocci as observed when examined microscopically. Moran and Upton (19, 20) reported similar results. Microgardm was not able to retard the growth of the Gram-positive bacteria tested, although Lisreria monocyrogenes ATCC 7644 gave variable results (Table 2). Hence, Microgardm is apparently rather specific for inhibiting Gram-negative bacteria, whereas nisin is inhibitory for Gram-positive bacteria (7). Some fungi screened were suppressed by MicrogarP, although no complete inhibition was achieved (Table 2). The insensitivity of some fungi may be related to proteolytic en-
761
ANTIMICROBIAL ACTMTY OF MICROGARDm
TABLE 2. Antagonistic activity of MicrogaxP at pH 5.3 against different microorganisms as revealed during the agar incorporation assay. Test organism
Sensitivity'
Gram-negative Pseudomonas putida W Pseudomonas aeruginosa 419 Achromobacter delicatulus 19103 Escherichia coli V517 Salmonella paratyphi 9281 Salmonella typhimurium OSU Shigella dysenterioe E19b Campylobacter jejuni 29428 Yersinia enterocolitica 23715 Aeromonas hydrophila 2965 Gram-positive Lactobacillus isolate P Lactobacillus 79A Loctobacillus 79B Latobacillus brevis OSU LaCiobaciNus plantarum MT Bacillus cereus OSU Staphylococcus aureus OSU Propionibacterium shermanii %16 Liszmb monocytogenes 7644
tt tt
++
-
tt tt
NG NG U ii-
w Yeast Y
s
+ + +
Kluyveromyces marxianus 8554 Black yeast Aspergillus niger OSU Penicillwn exvansum OSU
'NG = No growth in control plates @H 5.30). -H = complete inhiiition (no growth), stimulation effect, f = variable results, - = no inhibition.
+ = partial inhibition, S
=
zymes produced (5, 16, 18, 30), which degrade some inhibitors or raise the pH. However, yeasts were inhibited when Microgardm was prepared under certain fermentation conditions; in fact, yeast or mold inhibitory activity can be independently potentiated by varying fermentation conditions in preparing Microgardm (Weber, 1989, personal communication).
centration of Microgardm (1%) after being exposed for less than 42 h. Thus, a higher Microgardm concentration was required to achieve complete inhibition for more than 42 h of incubation at 37'C compared with Salmonella paratyphi (Table 3). Staphylococcus aureus is reportedly able to initiate growth after being inhibited by metabolites of LuctobacilZus plantarum (1). Total inhibition by 3% Microgardm was achieved against high microMinimum inhibitory Concentration Determination bial loads (lo6 to lo7 cfu/ml), and this inhibition lasted for more than 7 d at 30'C against The MIC values against Gram-negative bacsame sensitive Gram-negative psychrotrophic teria tested are shown in Table 3. Pseudomonas organism (Figure 1). Furthermore, 1% putida was dramatically inhibited by low con- Microgardm gave the same inhibition for more centrations of Microgardm, whereas Pseudo- than a week when tested against Achromobacmonas aeruginosa ATCC 419 required a higher fer (data not shown). MIC value (3%) to achieve the same inhibition. The latter bacterium is known for its resistance Effectiveness of Microgard" to some antibiotics (17). Yersinia and Aeromo- as a Function of pH nas organisms were highly vulnerable to Microgardm, as evident from their low MIC Figure 2 illustrates the activity of Microvalues (Table 3). However, Salmonella gardm as a function of pH and shows that typhimunum was able to overcome a low con- Microgard" functioned optimally below Journal of Dairy Science Vol. 74, No. 3, 1991
762
ALZOREKY ET AL.
TABLE 3. Minimum inhibitory concentration (MIC) of Microgdw to came 1UO% inhibition of various Gram-negative bacteria as revealed by the agar incorporation assay. The middle column shows the numbers of organisms present in the assay medium. Strain
Numbea of organisms
Pseudomonas purida W Pseudomonas aeruginosa 419 Achromobacter delicatulus 19103 Salmonella paratyphi 9281 Salmonella typhimuriwn OSU Yersinia enterocolitica 23715 Aeromonns hydrophila 7965
(cfo/ml) 6 X lo7 6.8 x io7 5 x 106 2.5 x io7 4 x io7 1.5 x io7 6.6 X IO6
pH 5.6. Although 3% Microgardm gave complete inhibition against the test bacterium at pH 5.3, 1% concentration gave the same inhibition when cells were diluted further to give about 104 cfu/ml (data not shown). This property renders it useful as a shelf-life extender of acid foods. Compared with other food preservatives, diacetyl and propionate also are much more inhibitory at pH values below 7.0 (6, 11). However, ascorbic acid was bacteriocidal for Campylubucrer jejuni even at pH 7.0 (12). Antagonistlc Effect of Some Medla Components on Mlcrogard"
The results shown in Figure 3 revealed that a low concentration of Microgardm (1%) was not able to retard the growth of Pseudomoms putida when a rich medium such as blood agar base was used for growth compared with plate count agar. Jay (11) reported similar results
(96 vol/vol) 1 3 1
1 3 1 1
whereby diacetyl was most effective in plate count but not in BHI agar or cooked meat medium. The antimicrobial activity of specific compounds can be reversed by some substances such as albumin, glucose, ash, serum, and phosphate (10, 11, 13, 22, 26, 27, 28). These observations indicate that food composition may be a factor influencing the inhibitory activity of Microgardm. This does not appear to be a detriment with dairy products, however, because Microgardm inhibitory activity is readily demonstrated in both cottage cheese and yogurt (25). Therefore, higher concentrations of Microgardm may be required in certain foods, and for this purpose both spray-dried and freezedried preparations as well as a liquid concentrate have recently been developed.
100
C
MIC
100
75
a
1 % Microgard
c 75
0 .c
.-0
2c
50
8
25
25
0
0
c .-
0 50
L
-
c -
3
1
% Microgard Figure 1. Influence of incubation time of PseudomoMs purida on the minimum inhibitory concentration of M i c r o g a r P deteamined by the agar incorporation method. Assay plates were seeded with 106 to 107 cfu/ml.
Journal of Dairy Science Vol. 74, No. 3, 1991
5.0
5.3
5.6
5.8
PH Pigare 2. Correlation between inhibition of Pseudomonas pwido by MicrogarP and pH of the agar medium assayed by the agar incorporation method. Assay plates were seeded with lo6 to io7 du/ml.
ANTIMICROBIAL ACTIVlTY OF MICROGARDm
'0°1
.-
a c 25
1
Ot
3
1 o/o
Microgard
Figure 3. The antagonistic effect of growth medium on inhibition by Microgardm assayed against Pseudomonas pur& by the agar incorpomtion method. Assay medium was seeded with io6 to lo7 a d . REFERENCES 1 Anderson, R 1986. Inhibition of Stuphylococcus uureus and spheroplasts of Gram-negative bacteria by an antagonistic compound produced by a strain of Loctobacilluc plantarum. Int. J. Food Microbiol. 3:149. 2 % Z., A. Annan-prah. M Janc, and J. Zajc-Satler. 1987. Yoghurt: an unlikely source of Campylobacrer jejunilcoli. J. Appl. Bactcriol. 63202. 3 Daeschel, M A. 1989. Antimicrobial substances from lactic acid bacteria for use as food preservatives. Fbod Technol. 43(1):164. 4Debevere. J. M 1987. The use of buffered acidulant systems to improve the microbiological stability of acid foods. Food Microbial. (Lomi.) 4:lM. 5 meet, G.H., and M. A. Mise 1987. The occurrence and growth of yeasts in dairy products. Int. J. Food Microbiol. 4 145. 6 Freese, E.,C. W. She% and E.Galliers. 1973. Function of lipophilic acids as antimicrobial food additives. Nature (Loud.) 241:321. 7Henning. S., R M Hammes, and W. P. Hammes. 1986. Studies on the mode of action of nisin. Int. J. Food Microbiol. 3:121. 8Hogg, G. M., M. F. Patterson. and J. G. Barr. 1987. Correlation between antibiotic sensitivity testing by conventional and conductivity measnrements. J. Appl. Bacteriol. 62189. 9 Hoover, D. G., K.J. Dishart and M A. Hermes. 1989. Antagonistic effect of Pediococcus spp. against Listeria monocyfogenes. Food Biotechnol. (NY) 3183. lOIsmaeel, N.. J. R. Furr, and A. D. Russell. 1986. Reversal of the surface effects of chlorhexidine. diacetate on cells of Providencia staurfii. J. Appl. Bacteriol. 61:373. 11 Jay, James M. 1982. Antimicrobial properties of diacetyl. Appl. Environ. Microbiol. 44525. 12 Juven, B. J., and J. Katmer. 1986. Effects of ascorbic, isoascorbic and dehydroascorbic acids on the growth and survival of Campylobacter jejuni. J. Appl. Bacteri01. 61539. UKabara, Jon J. 1978. Fatty acids and derivatives as antimicrobial agents: a review. Ch. 1. The pharmacological effects of lipids. Jon J. Kabara, ed. Am.
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Journal of Dairy Science Vol. 74, No. 3, 1991