walls were broken, the active ingredients would contact thefoodandthereforebecomemorebio-available.However,tra-ditional solvent extraction of essential oil requires a largequantityof toxicorganicsolvent.Atthesametime,itislabor-intensive, requires long extraction time, and has low extrac-tion yield. Thus the direct use of ultra-ﬁne powders of foodspices to replace essential oil should be explored.Ball mills have been successfully used for grinding raw materials into ultra-ﬁne powders because of their ease of operation, higher grinding rate, lower energy consumptionand considerably less plant space compared with other ﬁne-grinding machines (Jimbo 1992; Gao and Forssberg 1995).This can reduce the particle sizes of plants down to 10
m orlower (Lowrison 1974). However, to the best of our knowl-edgethereisnoliteratureinvestigatingthefeasibilityof ultra-ﬁne powders of spices as potential antimicrobial agents. Inthis work, we grinded cinnamon and clove using a high-frequency oscillatory type ball mill, and examined the anti-microbialactivitiesobtainedtheultra-ﬁnepowdersof cinna-mon and clove against ﬁve spoilage organisms
frommeats.Theeffectof ball-milledtimeonthepowdergranular-ity and the particle sizes on the antimicrobial effectivenesswere evaluated.
MATERIALS AND METHODS
Preparation of Bacterial Strains
(strain AS1.823)were obtained from Microbial Culture Collection Center of Guangdong Institute of Microbiology (Guangdong, China).
(1A01916) was provided by MarineCultureCollectionofChina(Fujian,China).
(CICC 6141) was obtained from ChinaCenter of Industrial Culture Collection (Beijing, China).Man–Rogosa–Sharpe (MRS) medium and streptomycinthalliumacetate actidion (STA) medium were purchasedfrom Qingdao Hope Bio-Technology Co., Ltd. (Qingdao,China).
were suspended in asterile nutrient broth (NB) medium, and incubated at37
1C and 30
1C for 24 h, respectively.
was transferred into a sterile MRS medium andincubated at 37
1C for 48 h,whereas
wastransferred to a sterile STA medium and incubated at30
1C for 48 h. One milliliter stock culture was standard-ized through two successive 24 h growth cycles in an appro-priate broth (NB, MRS or STA). The cell from thestandardized culture was inoculated in a sterile medium andincubated to obtain a cell suspension at exponential phasecontaining approximately 2
Preparations and Characterizations ofUltra-Fine Powders
) and clove (
Eugenia caryo- phyllus
) were purchased from Jinyaotang Drugstore inWuhan, Hubei Province, China. The samples were dried at50C for 24 h, and then milled for 50, 60, 70, 80 and 90 minusing a high-frequency oscillatory type ball mill at 20
1C.The cinnamon powders were coded as CI-50, CI-60, CI-70,CI-80 and CI-90, the clove powders as CL-50, CL-60, CL-70,CL-80 and CL-90,respectively.The particle size of a sample was analyzed using a particlesize analyzer (LS 13320, Deckman Coulter Inc., Fullerton,CA)andthemeanparticlesize(d
)ofasamplewasestimatedas a median diameter from the count value based on weightpercent cumulative curve.
Determination of Antibacterial Activities
The antibacterial assays of cinnamon and clove powdersagainst selected
were determined by broth dilutionmethod.Eachtestedbacteriumwasculturedinacorrespond-ing medium described above. A series of broths containingtested powders were prepared, the concentrations of eachtested powder incorporated in broth were 0,50,100,150,200and 250 mg/mL. Each bacterium was suspended in sterilewater and diluted to approximately 2
cfu/mL. Then1 mL bacterial suspension was added to 9 mL broth contain-ing a sample, and the mixture was cultured at 37C for 48 hwith stirring to prevent the sample from depositing.The sur-vivingbacteriawereconﬁrmedbyplatecount.Theinhibitory effect (I %) was calculated using the following formula:
I A B A
isthenumbersof bacteriawithoutanysampleinthetest tube;
is the numbers of bacteria with sample in the testtube.Three independent measurements were prepared in eachcase. The MIC of a sample was deﬁned as the lowest concen-tration of the sample in which more than 90% bacteria couldnot grow.
Data were subjected to analysis of variance and Duncan’smultiple-range tests using analysis of variance procedure.Differencesbetweenmeansof bacterialcounts(cfu/mL)wereregarded to be statistically signiﬁcant when
0.05. Experi-ments were replicated twice for each powder sample at eachconcentration.
Journal of Food Safety
(2011) 291–296 © 2011 Wiley Periodicals, Inc.