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M:FoodMicrobiology&
Safety
Rice Hull Smoke Extract Inactivates SalmonellaTyphimurium in Laboratory Media and ProtectsInfected Mice against MortalitySung Phil Kim, Mi Young Kang, Jun Cheol Park, Seok Hyun Nam, and Mendel Friedman
Abstract: A previously characterized rice hull smoke extract (RHSE) was tested for bactericidal activity against SalmonellaTyphimurium using the disc-diffusion method. The minimum inhibitory concentration (MIC) value of RHSE was0.822% (v/v). The in vivo antibacterial activity of RHSE (1.0%, v/v) was also examined in a Salmonella-infected Balb/cmouse model. Mice infected with a sublethal dose of the pathogens were administered intraperitoneally a 1.0% solutionof RHSE at four 12-h intervals during the 48-h experimental period. The results showed that RHSE inhibited bacterialgrowth by 59.4%, 51.4%, 39.6%, and 28.3% compared to 78.7%, 64.6%, 59.2%, and 43.2% inhibition with the medicinalantibiotic vancomycin (20 mg/mL). By contrast, 4 consecutive administrations at 12-h intervals elicited the most effectiveantibacterial effect of 75.0% and 85.5% growth reduction of the bacteria by RHSE and vancomycin, respectively. Thecombination of RHSE and vancomycin acted synergistically against the pathogen. The inclusion of RHSE (1.0% v/w)as part of a standard mouse diet fed for 2 wk decreased mortality of 10 mice infected with lethal doses of the Salmonella.Photomicrographs of histological changes in liver tissues show that RHSE also protected the liver against Salmonella-induced pathological necrosis lesions. These beneficial results suggest that the RHSE has the potential to complementwood-derived smokes as antimicrobial flavor formulations for application to human foods and animal feeds.
Keywords: antimicrobial effects, liquid smoke, mice, rice hull smoke extract, Salmonella Typhimurium, vancomycin
Practical Application: The new antimicrobial and anti-inflammatory rice hull derived liquid smoke has the potential tocomplement widely used wood-derived liquid smokes as an antimicrobial flavor and health-promoting formulation forapplication to foods.
IntroductionRice (Oryza sativa L.) is a major source of nourishment for
the world’s population. World production of rice is estimated ataround 685 million tons, equivalent to that of wheat (FAOSTAT2010). The United States produces about 10 million tons andSouth Korea about 7 million tons. Rice hulls accounting for 20%of the rice crop are a byproduct of postharvest rice processing(Foo and Hameed 2009). Rice hulls consist mainly of lignin,hemicellulose, cellulose, and hydrated silica (Salanti and others2010). A byproduct of the combustion of rice hulls is the smokethat is generated.
Smoke has been used to preserve food naturally, as well as to givedistinctive flavors to food. Liquid smoke has gained widespreadacceptance in the food industry, replacing traditional smoking
MS 20110883 Submitted 7/21/2011, Accepted 9/29/2011. Author Kim is withthe Dept. of Molecular Science & Technology and author Nam is with the Dept. ofBiological Science, Ajou Univ., Suwon, 443–749, Republic of Korea. Author Kangis with the Dept. of Food Science and Nutrition, Kyoungpook Natl. Univ., Daegu702–701, Republic of Korea. Author Park is with the Natl. Inst. of Animal Science,Rural Development Administration, Suwon 441–706, Republic of Korea. AuthorFriedman is with U.S. Dept. of Agriculture, Western Regional Research Center, Agri-cultural Research Service, Albany, CA 94710, U.S.A. Direct inquires to author Nam(E-mail: [email protected]) or Friedman (E-mail: [email protected]).
practices. Liquid smoke preparations have advantages over tradi-tional smoking because they are easier to apply and provide moreconsistent results. Wood-derived liquid smoke is now widely usedin foods including cheese (Garabal and others 2010), meat (Olsenand others 2005; Gedela and others 2007a, 2007b; Martin andothers 2010), and seafood (Niedziela and others 1998; Thuretteand others 1998; Sunen and others 2003; Martinez and others2010). Additional antimicrobial effects of wood-based smoke aredescribed in the literature (Asita and Campbell 1990; Faith andothers 1992; Estrada-Munoz and others 1998; Sunen 1998; Sunenand others 2001; Holley and Patel 2005). In the United States, liq-uid smoke has been granted generally-recognized-as-safe (GRAS)status as a natural flavoring (Cadwallader 2007).
In a previous study (Kim and others 2011b), we described theproduction and composition of a new liquid rice hull smoke ex-tract (RHSE) with a smoky aroma and sugar-like odor preparedby pyrolysis of rice hulls followed by liquefaction of the result-ing smoke. RHSE contained 161 compounds characterized bygas chromatography and mass spectrometry. In vitro and in vivocell and mouse assays showed that the extract exhibited strongantioxidative, antiallergic, and anti-inflammatory activities, similarto those we previously reported for black rice bran (Choi andothers 2010). Related studies by other investigators showed thata rice hull extract inhibited the growth of the toxic cyanobac-terium Microcystis aeruginosa and that far-infrared radiated rice hull
Journal of Food Science C© 2011 Institute of Food Technologists R©No claim to US original government works
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extracts possessed significant reactive-oxygen-scavenging and pro-tective effects against oxidative DNA damage (Lee and others2003; Jeon and others 2006). Based on the cited beneficial bioac-tivities, we suggested that RHSE derived from a major agriculturalbyproduct could serve as a new biomaterial for the improvementof food quality and safety and the environment.
To promote the application of the extract to food, the mainobjective of this study was to evaluate the antimicrobial activity ofRHSE against the foodborne pathogen Salmonella Typhimuriumin laboratory media and in infected mice and to compare the resultswith those obtained with the medicinal antibiotic vancomycinused as a positive control. To our knowledge, this is the firstreport on antimicrobial properties of RHSE against a foodbornepathogen.
Materials and Methods
MaterialsMueller–Hinton agar, Nutrient broth, and NBGP (Nutrient
broth containing 0.05% phenol red and 10% glucose) were pur-chased from Becton Dickinson (San Jose, Calif., U.S.A.). Van-comycin was obtained from Sigma (St. Louis, Mo., U.S.A.). TheRPMI 1640 medium and fetal bovine serum (FBS) and othercell culture reagents were obtained from Hyclone Laboratories(Logan, Utah, U.S.A.). Murine interferon-γ (IFN-γ ) was pur-chased from PharMingen (San Diego, Calif., U.S.A.). All reagentsof analytical grade were purchased from Sigma and used withoutfurther purification.
Bacterial strain and culture conditionsSalmonella enterica serovar Typhimurium (ATCC #14028)
(Salmonella Typhimurium) cells were obtained from the AmericanType Tissue Culture Collection (ATCC, Manassas, Va., U.S.A.)and kept as frozen glycerol stock at –70 ◦C. Cells in frozen stockwere streaked onto agar medium to produce cell colonies, andthen a single colony was transferred to liquid medium. For prepa-ration of inocula, cells were grown for 20 h at 37 ◦C in Nutrientbroth. For infection, cultured bacterial cells were recovered bycentrifugation (13,000 rpm for 30 s), then washed once withphosphate-buffered saline (PBS, pH 7.4) and resuspended in PBS.The turbidity of the cell suspensions was measured. The cell sus-pensions were diluted with PBS to the desired concentration ofbacteria using a standard curve of optical density versus bacterialnumber determined as CFU.
Rice hull smoke extract (RHSE)The industrial production of the RHSE, its content of 161
compounds characterized by gas chromatography and mass spec-trometry, and beneficial bioactivities in chemical and cell assays aredescribed in our previous publication (Kim and others 2011b).
In vitro bactericidal assayThe inactivation of the Salmonella by RHSE was determined
using the disc inhibition zone assay described by Rojas-Grau andothers (2006) with some modification. RHSE stock was dilutedto 0.1%, 0.5%, and 1.0% (v/v) in PBS. An aliquot (20 μL) ofeach sample was then spotted onto 6-mm disc and allowed todry for 15 min. The discs were then placed on plates containingMueller–Hinton agar, which had been previously spread with 0.1mL of inoculum containing 1 × 108 CFU/mL of bacteria previ-ously standardized as mentioned above. The plates were incubatedat 37 ◦C for 24 h. The diameter of the growth inhibition zones
around the discs was measured using a ruler. The tests were carriedout in triplicate for each formulation.
Minimal inhibitory concentration (MIC)The MIC of RHSE was determined following the method of
Mbaveng and others (2008) with some modifications. RHSE wasdiluted in Nutrient broth with glucose and phenol red (NBGP) tovarious concentrations as shown in Figure 2. Freshly prepared bac-terial culture (90 μL) at a final concentration of 5 × 105 CFU/mLafter 20-h incubation was mixed with RHSE samples (10 μL) ina well of a 96-well plate. The plates were covered with a sterileplate sealer and agitated to mix the contents using a plate shaker.The plates were incubated at 37 ◦C for 24 h. Bacterial growth wasdetermined by measuring the change in color of the NBGP inthe wells from red to yellow by monitoring the optical density at595 nm in a microplate reader (Model 550, Bio-Rad, Hercules,Calif., U.S.A.). MIC is defined as the lowest concentration of ricehull liquid smoke that inhibited visible growth and prevented achange in color of the NBGP. Specific bactericidal activity wascalculated according to the following formula:
% bactericidal activity
= (ODRHSE − ODNBGP)/(ODvehicle − ODNBGP) × 100.
MicePathogen-free female Balb/c mice, aged from 6 to 8 wk, were
obtained from Orient Inc. (Seoul, Korea). After acclimation for 1wk, the mice were housed under a 12-h light/dark cycle with atemperature range of 20 to 22 ◦C and relative humidity of 50 ±10◦C. The mice were fed freely a pelletized commercial chow dietand sterile tap water during the entire experimental period. Foodwas withheld for a period of 12 to 15 h before the experiments.
In vivo bactericidal assayThree groups of 10 mice each (PBS-treated negative control,
RHSE-treated experimental groups, and vancomycin-treated pos-itive control) were used for bacterial infection studies. The lethaldose of Salmonella (killing mice at 5 to 7 d post-infection) was de-termined to be 1 × 105 CFU. Mice were infected intraperitoneallywith a sublethal dose (1 × 104 CFU) of Salmonella. A solution ofthe RHSE (1.0%, 200 μL) was injected into the peritoneal cavityat 0, 12, 24, or 36 h after bacterial infection. An additional 3 injec-tions of the same dose were administered at 12-h intervals. Then,mice were sacrificed by cervical dislocation at 48-h post-infection.The abdominal cavities were opened and repeatedly washed with atotal of 10 mL of PBS as described by Kodama and others (2001).Bacterial counts in the pooled PBS wash were determined byplating technique to measure CFU levels as described above.
Mice salmonellosis studyThe salmonellosis assay was carried out following the method
of Kim and others (2008) with modification. Briefly, 2 groupsof 10 mice each (PBS-treated control and RHSE-treated experi-mental group) were used for bacterial infection. After acclimationfor 1 wk, mice were fed a pelletized commercial chow diet with-out and with RHSE (1.0%, v/w) during the 16-d experimentalperiod. After feeding each diet for 14 d, mice were infected in-traperitoneally with a lethal dose of Salmonella (1 × 105 CFU). Todetermine the survival rate, mice were observed for an additional14 d after bacterial infections.
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Nitrogen oxide (NO) productionNO was measured by determining the concentration of its sta-
ble oxidative metabolite nitrite using the microplate assay methoddescribed by Xie and others (1992). Mice fed a pelletized commer-cial chow diet without and with an RHSE (1.0%) for 14 d wereinfected intraperitoneally with 1 × 104 CFU of Salmonella, andthen housed for a further 2 d. Thereafter, mice were sacrificedby cervical dislocation. Isolation and purification of mice peri-toneal macrophages were performed as described by Narumi andothers (1990). Peritoneal macrophages were cultured in a 96-wellplate (1 × 105 cells/well) with IFN-γ (10 U/mL) for 6 h. Then,the cultures were left standing for 48 h. To measure the nitriteconcentration, the culture medium (100 μL) was mixed with anequal volume of Griess reagent (1.0% sulfanilamide and 0.1.0% N-(1-naphthyl)-ethylenediamine dihydrochloride in 5% phosphoricacid) at room temperature for 15 min. The absorbance at 570 nmwas determined with a microplate reader (Model 550, Bio-Rad)using a standard calibration curve for sodium nitrite.
Mitogen-induced lymphocyte proliferation and IFN-γ levelsThe spleen and liver were aseptically excised from the sacrificed
mice from the above NO production study. Spleen cell suspen-sions were prepared by gently pressing the spleen through a stain-less steel mesh according to the method described by Teixeira andothers (2006). Thereafter, the cells were suspended in RPMI1649medium supplemented with 5% FBS and seeded into 24-wellculture plates in the presence of Concanavalin A (2.5 μg/mL)to stimulate T cells. After stimulation for 72 h at 37 ◦C in 5%CO2, cell proliferation levels were determined by measuring ata wavelength of 490 nm in a microplate. The cell numberswere determined using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyohenyl)-2-(4-sulfophenyl)-2H-tetrazolium)) as-say in the form of the CellTiter 96 aqueous nonradioactive cellproliferation assay kit (Promega, Madison, Wis., U.S.A.) followingthe manufacturer’s instructions. The fold-increase of cell growthwas determined by setting as 1 the growth of spleen cells frompathogen-free mice in FBS-supplemented RPMI1640 mediumwithout Con A. IFN-γ in the culture supernatant of spleen cellsfrom infected or mock-treated mice without Con A was deter-mined by an ELISA kit (Biosource Intl., Camarillo, Calif., U.S.A.)following the manufacturer’s instruction. After the reaction, the
absorbance of the final solution at 420 nm was read in a microplatereader (Model 550, Bio-Rad laboratories).
Histology of liver tissueThe liver was excised from Salmonella-infected mice fed diets
with or without RHSE (1.0%), and then fixed with paraformalde-hyde (4%) in phosphate buffer (PBS, 0.5 M, pH 7.4). The lobeswere isolated, embedded in paraffin, and sectioned at 6 μm. Thesections were mounted on glass slides and dewaxed using xyleneand ethanol. Finally, the sections were stained with hematoxylinand eosin (H&E). Histological changes were observed under alight microscope at a magnification × 100.
Statistical analysisResults are expressed as the mean ± SD of 3 independent exper-
iments. Significant differences between means were determinedby ANOVA using the Statistical Analysis Software Package (SAS,
0.2 0.4 0.6 0.8 1.00
20
40
60
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100
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Figure 2–Minimum inhibitory concentrations (MIC, %) value of RHSEagainst Salmonella Typhimurium determined from a dilution series. Eachdiluted RHSE sample in NBGP was mixed with bacteria (5 × 105 CFU) ina 96-well microplate reader. After incubation at 37 ◦C for 24 h, bacterialgrowth was determined by measuring the change of color in the wells at595 nm. Plotted values are means ± SD (n = 3).
Figure 1–Quantitation of inhibition zones on agarplate following exposure of SalmonellaTyphimurium to 0.1, 0.5, or 1.0% RHSE. Valuesnot sharing common superscripts are significantlydifferent at P < 0.05 (n = 3).
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Cary, N.C., U.S.A.), with P < 0.05 being regarded as statisticallysignificant.
Results and Discussion
Antimicrobial effects of the RHSE and vancomycinFigure 1 shows a photograph of the 3 increasing inhibition
zones on an agar plate of Salmonella induced by 3 concentrationsof RHSE. Figure 1 shows that these significantly different zonesdiameters (including the 6-mm disk size) range from 10 to 19 mm.These results demonstrate for the first time that RHSE has antibi-otic properties.
Figure 2 depicts a plot of the effect of 10 concentrations ofextract in the range of 0.2% to 1.0% on the inactivation of thebacteria. The plot increases steeply and then levels off at 0.822%,the MIC value that represents complete inhibition of growth.
Figure 3 compares the in vivo effect of a 1.0% RHSE in miceinfected with Salmonella to that of the widely used medicinal an-tibiotic vancomycin. In terms of CFU, the treatment with RHSEmirrored the treatment with vancomycin in reducing bacteria inthe peritoneal cavity, although in every case the vancomycin wassomewhat more effective. The last 2 bar graphs show the observedreduction in the in vivo Salmonella counts following consecutive in-jection of the RHSE into the intraperitoneal cavity after 0, 12, 24,and 36 h during the 48-h experimental period. The graphs indi-cate that under these conditions, both the extract and vancomycininduced comparative in vivo reductions of the Salmonella.
Additional studies depicted in Figure 4 show that the combina-tion of vancomycin (10 mg/kg mouse) and 1.0% RHSE exhibiteda synergistic effect that was similar to the 20 mg/kg vancomycintreatment. This is evident from a comparison of the reductionin the CFU count induced by vancomycin or RHSE alone (2ndand 4th bars, respectively) and the reduction induced by the com-bination (5th bar), which is greater than those induced by theindividual compounds.
Figure 3–In vivo bactericidal activity of RHSE. Balb/c mice infected in-traperitoneally with Salmonella Typhimurium (1 × 104 CFU) were admin-istrated 1.0% RHSE in PBS (200 μL, v/v) at the indicated time points afterinfection. At 48-h post-infection, surviving bacteria in peritoneal exudateswere counted by plating technique for measuring CFU. Analogous experi-ments were also carried out with the same volume of PBS and vancomycin(20 mg/kg) as negative and positive controls. The last 2 bar graphs showthe observed reduction in the in vivo Salmonella counts following consec-utive injection of the RHSE into the intraperitoneal cavity after 0, 12, 24,and 36 h during the 48-h experimental period. Plotted values are means ±SD (n = 3).
Effect of RHSE on histopathology of livers of infected miceFigure 5 shows photomicrographs of 4 histology slides derived
from tissues of normal, infected, and RHSE-treated mice. Notethere are pathological lesions (necrosis) in the livers of the infectedmouse and their amelioration in livers of infected mice treatedwith 0.5% or 1.0% RHSE.
Related previous studies reported that in a murine salmonel-losis model, Salmonella Typhimurium caused liver injury throughinvolvement of tumor necrosis factor-α (TNF-α) and lipid perox-idation (Lincopan and others 2005) and that probiotics protectedmice against Salmonella-induced liver damage (Rishi and others2008). Therefore, it is likely that the strong antioxidant activityof RHSE (Kim and others 2011b) has the potential to suppressa rapid increase in the level of cellular peroxidation that resultedfrom stimulation of TNF-α. This is a likely mechanism by whichthe 1.0% RHSE treatment ameliorated the liver damage observedin the present study.
Effect of RHSE on mortality of infected miceFigure 6 illustrates the survival of infected mice orally fed a 1.0%
RHSE for 2 wk. The infected (positive) group all died on day 7,whereas RHSE-treated group all died on day 14. Therefore, onaverage, the life span of the group orally fed RHSE was aboutdouble that of the untreated infected mice. As shown in Table 1,the beneficial effect of RHSE was accompanied by a reduction ofSalmonella counts from 5.7 × 102 CFU (untreated mice) to 3.6 ×102 CFU (treated mice).
Effect of RHSE on NO production, lymphocyte proliferation,and IFN-γ levels of infected mice
Table 1 shows the inhibitory effect of RHSE on the produc-tion of NO in mouse peritoneal macrophages. Compared to thecontrol value of 15.32 μM, NO produced by macrophage cellsinduced a reduction to 11.75 μM or a 23.4% decrease follow-ing treatment with RHSE. Suppression of NO production is at-tributed to antioxidant activity, and is considered to be associatedwith anti-inflammatory effects (Kim and others 2011a).
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Vancomycin - 10 mg/kg 20 mg/kg - 10 mg/kg
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Figure 4–Comparison of in vivo bactericidal effects of RHSE and the medic-inal antibiotic vancomycin. Balb/c mice infected intraperitoneally withSalmonella Typhimurium (1 × 104 CFU) were administrated an RHSE solu-tion (200 μL, 1.0%) and/or vancomycin at the indicated concentrations.At 48-h post-infection, living bacteria in peritoneal exudates were countedby plating technique for measuring CFU. Bars not sharing a common letterare significantly different between groups at P < 0.05.
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Rice hull smoke extract inhibits Salmonella . . .
Figure 5–Photomicrographs showing effect ofRHSE on formation of pathological lesions inlivers of mice infected with SalmonellaTyphimurium. Liver specimens obtained 2 dafter intraperitoneal injection of RHSE from themice infected with Salmonella Typhimurium(1 × 104 CFU) were fixed with 4%paraformaldehyde. The sections were thenstained with hematoxylin and eosin (H&E).Magnification × 100. Figures represent resultsfrom at least 3 individual experiments. Arrowsindicate representative hemorrhagic necrosis.
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Figure 6–Effects of the dietary administration of RHSE on Salmonella Ty-phimurium infection induced lethality. Balb/c mice (10 mice per group)were fed for 2 wk a standard mouse diet supplemented with RHSE (1.0%,v/w), followed by infection with a lethal dose of Salmonella (1 × 105 CFU)administered intraperitoneally. PBS was used as the vehicle in the controlgroup. Plotted values are means of triplicate determinations.
Table 1 also shows that RHSE treatment had no effect on lym-phocyte proliferation, suggesting a lack of components such aspolysaccharides that have mitogenic potential. The treatment also
somewhat increased production of IFN-γ by the spleen cells invivo, from 316.24 to 346.78 pg/mL. This result suggests that thetreatment stimulated the cellular immune system through activa-tion of Th1 cells or macrophages. However, the expressed effectseems to be in a highly limited range.
ConclusionsThe inhibitory effect of RHSE against Salmonella in laboratory
media and in infected mice was determined with the aid of astandard bacterial assay and an in vivo mouse model in which theextract was administered both intraperitoneally and orally as partof a standard diet. The in vitro results show that antimicrobial effec-tiveness of the extract approaches that of the widely used medicinalantibiotic vancomycin. In vivo, the observed reduction in pathogenlevels was accompanied by reduction in NO levels produced bythe peritoneal macrophages and an increase in the levels of IFN-γfrom splenocytes. The observed change in the latter biomarker im-plies that the extract also stimulated the cellular immune system.The extract also protected the mice against Salmonella-inducedliver necrosis and mortality. These beneficial effects suggest thatRHSE is a novel bioactive, antimicrobial formulation that meritsfurther study for its potential to improve microbial food safety.Possible applications in veterinary and human medicine also meritstudy. However, it is first necessary to demonstrate that RHSEis safe. The previous (Kim and others 2011b) and present studieswere designed to contribute to this assessment.
Table 1– Effects of dietary administration of a 1.0% solution of RHSE on reduction of intraperitoneally infected SalmonellaTyphimurium, NO production in peritoneal macrophages, lymphocyte proliferation in the spleen, and IFN-γ release by spleniclymphocytes.1
Nr of colonies Lymphocyte proliferationSample (×102 CFU/mL) Nitrite (μM) (fold-increase) IFN-γ (pg/mL)
Vehicle, PBS 5.7 ± 0.42a 15.32 ± 1.08a 1.60 ± 0.17a 316.24 ± 16.71b
RHSE, 1.0% 3.6 ± 0.28b 11.75 ± 0.91b 1.54 ± 0.13a 346.78 ± 11.90a
1Fold-increase was determined as the ratio of proliferation of Con A-stimulated splenocyte in Salmonella-infected mice subjected to RHSE or PBS to that of unstimulated splenocytesin normal mice.aValues in the column not sharing a common letter are not significantly different at P < 0.05.
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AcknowledgmentsThis research received grant support from the Agenda Program
(Nr. 200901OFT113068122), Rural Development Administra-tion, Republic of Korea. We thank Carol E. Levin for assistancewith the preparation of the manuscript. USDA is an equal oppor-tunity employer.
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Vol. 71, Nr. 1, 2012 � Journal of Food Science M85