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Journal of Food Protection, Vol. 58, No.3, Pages 284-288Copyrighl©, International Association of Milk, Food and Environmental Sanitarians

Biogenic Amine Formation in Fresh Vacuum-Packaged BeefStored at -2°C and 2°C for 100 Days

ANGELIA R. KRIZEK, J. SCOTT SMITH* and RANDALL K. PHEBUS

Department of Animal Sciences and Industry, Call Hall, Kansas State University, Manhattan, Kansas 66506-1600

(MS # 94-156, Received June 30, 1994/Accepted November 9, 1994)

ABSTRACT

When fresh, vacuum-packaged, meat products are storedfor extended periods of time, undesirable changes, due tonaturally occurring microbial flora present during packag-ing occur. Lactobacillus spp. are known to form aminesthrough the decarboxylation of free amino acids. Tyramineand histamine can cause intoxication in individuals takingmonoamine oxidase-inhibiting drugs. This study determined1) the effect of storage temperature on bacterial growth andbiogenic amine production in vacuum-packaged beefsubprimals, 2) the effect of washing subprimals with waterto remove tyramine contamination, and 3) the penetrationof tyramine from the surface of the subprima1.

Inside rounds were vacuum packaged and stored at-2°C or 2°e. Samples were evaluated over 100 days foramine concentrations, total psychrotrophic· counts andlactic acid bacteria. Tyramine, putrescine and cadaver-ine were detected in this study. Significant levels (15Ilg/g) of tyramine were detected at 20 days of storage at2°C and 40 days of storage at -2°C. Putrescine andcadaverine were detected first at 40 days of storage at2°C and 60 days of storage at _2°e. Both treatmentgroups contained about 130 Ilg/g of tyramine at 100days of storage. Psychrotrophic plate counts and lacticacid bacteria counts were initially 103 colony formingunits (CFU)/cm2 and ranged from 106-107 CFUlcm2 at100 days of storage-. Even though tyramine was evidentat a depth of 6 mm from the surface of the cut, one-thirdof the amine was removed by washing the subprimalwith tap water.

Key words: Tyramine, putrescine, cadaverine, biogenicamines, vacuum-packaged, beef

Food products with extended shelf lives are rapidlygaining popularity because they are easier to market anddistribute. Advantages of vacuum-packaged fresh beef in-clude less weight loss from evaporation, lower transporta-tion costs, less surface trimming and longer product shelflife (13). Current vacuum packaging technology enablesbeef products to remain acceptable for consumption forabout 45 days (4). This broadens marketing potentials for

both the processor and consumer and allows more time forproducts to be in transit.

However, undesirable changes, which can cause lifethreatening conditions, can occur in vacuum-packaged beefwith extended shelf life. Because vacuum packaging cre-ates an anaerobic environment, Lactobacillus and Strepto-coccus spp. grow. Such proteolytic and decarboxylatingbacteria can produce amines known as biogenic or pressoramines, which are normal constituents of many foods weeat (19). Several species of lactobacilli and streptococcidemonstrate the ability to decarboxylate amino acids yield-ing biogenic amines. These species include Lactobacillusbuchneri, Lactobacillus 30a, Lactobacillus plantarum, Lac-tobacillus buchneri, Streptococcus faecium, Streptococcusmitis, Streptococcus lactis and several others (17).

Excessive ingestion of histamine and tyramine hasdetrimental effects on human physiological functions, mostnotable headaches, flushing and acute hypertension (10). Inaddition, individuals taking tranylcypromine sulfate andmonoamine oxidase-inhibiting (MAOI) drugs can sufferfrom biogenic amine intoxication (9,15,18). These drugscommonly are used as antidepressants. Tyramine toxicityoccurs more frequently than toxicity to any of the otherpressor amines in those taking MAOIs (23) and can resultin hypertensive attacks, strokes and even death (9). Al-though little information is available for the normal popu-lation, McCabe (9) has reported that only 6 mg of tyraminecan produce a reaction in individuals taking MAOI drugs,and 10-25 mg can cause severe headaches to intracranialhemorrhaging.

Biogenic amines are known to accumulate with time inextended shelf life, vacuum-packaged beef products (16).Studies have shown that vacuum-packaged beef stored for7 weeks at 1°C contained measurable amounts of tyramine,putrescine and cadaverine (3,4). In every case where tyra-mine was detected, Lactobacillus spp. also were identified.Edwards et a1. (3) also found that tyramine can accumulateto detectable concentrations after extended storage at nor-mal refrigeration temperatures, although sensory accept-ability was prolonged.

Because vacuum packaging has played and will con-tinue to play an integral role in the production and market-ing of beef products, there is a need to learn how to reduce

JOURNAL OF FOOD PROTECTiON, VOL. 58, MARCH 1995

BIOGENIC AMINES IN VACUUM-PACKAGED FRESH BEEF 285

biogenic amine presence and production. Therefore, theobjectives of this study were determining the effects ofstorage temperature on biogenic amine formation and cor-relating tyramine production to lactic acid bacteria growth,washing vacuum-packaged subprimals to reduce aminelevels, and penetration of amines into the muscle interior ofextended shelf life vacuum-packaged beef.

MATERIALS AND METHODS

SamplingBeef inside rounds were purchased from a local supplier

in the Manhattan, KS, area. Each subprimal was assignedrandomly as a replicate and cut into approximately 2 in.-thick roasts. Each roast was vacuum packaged (Model No.A300/l6, Multivac, Inc., Kansas City, MO) in a laminatedpouch (Koch, Kansas City, MO) with an average vacuum of599 ± 62 torr. Pouches were made of 3 mil nylon/polyethyl-ene with an oxygen transmission rate of 4.0 cc/lOO in.2(645.16 cm2)/h at O°C and water vapor transmission rates of0.6 cc/IOO in.2 (645.16 cm2)/24 h at 37°C. Packaged samplesthen were placed in storage at -2°C or 2°e.

Samples were taken before storage (day 0) and on days10, 20, 40, 60, 80 and 100 of storage. For penetrationstudies, additional samples were taken on days 60, 80 and100. For washing studies, samples were obtained on day100. Meat samples were obtained with 0.5 in. (1.3 cm) or1.5 in. (3.8 cm) coring tools. The larger coring tool wasused for penetration, washing and bacteriological studies,whereas the smaller tool was used for the amine study.Samples were taken from an area that had no fat cover oneither side of the roast. For amine, bacteriological, andwashing studies, cores were trimmed to 10.0 g by horizon-tally excising the middle section of the plug. For thepenetration study, the outermost ends of the core wereremoved to a specified depth (3, 6 and 9 mm) from thesurface of the meat. The outermost section weights wererecorded, and amine concentrations determined. To deter-mine the effects of removing amine contamination withwater, roasts were rinsed thoroughly with tap water undera faucet for approximately 30 s (3.0 L/min @ 16°C). Coresthen were removed and analyzed for amine concentration.

Amines were extracted immediately from the meatsamples. Sampling of each roast was done in duplicate, andcare was taken to avoid areas containing fat cover andconnective tissue. Each treatment group (storage tempera-ture: _2°C and 2°C; penetration: 3 mm, 6 mm and 9 mm;and washing) contained four replicates.

Amine extractionAmine extraction and analysis were conducted using a

modified method of Smith et al. (16). A 10.0 g sample wasobtained and placed in a Waring™ blender with 25.0 ml ofa 5% (wt/vol) solution of trichloroacetic acid and blendedat high speed for 15 s and then at medium speed for 45 s.The sample then was filtered through a Whatman™ No. 40fHter paper into a 50 ml volumetric flask. The flask wasbrought to volume with high performance liquid chroma-tography (HPLC) grade water. The dilutant was filteredthrough a 0.22 11mnylon 66 syringe filter (Alltech Associ-ates, Inc., Deerfield, IL) and placed in a glass vial. Sampleswere frozen and later analyzed by HPLC.

Amine analysisAmines were separated according to Van Boekel and

Arentsen-Stasse (20) as modified by Smith et al. (16) usinga Hewlett-Packard 1090A-Series II HPLC (Hewlett-Packard,Palo Alto, CA) with a 250 mm x 4.6 mm Bio-Sil Cl8 HL-90 reversed-phase analytical column (Bio-Rad Laboratories,Richmond, CA). The 10 mm x 4.6 mm guard column waspacked with Bio-Sil C 18 (5 11m)material (Alltech) and fittedwith OA5-llm column frits. The system and data processingwere controlled by a Hewlett-Packard ChemStation (Pascalseries) using software HP79988A Rev. 5.22 and HP79997 ARev. 5.20. All HPLC solvents were "Optima" pesticide gradeor better (Fisher Scientific Co., Pittsburgh, PA).

Monoamines (tyramine, tryptamine, phenylethylamine andhistamine). These amines were separated using an isocraticmobile phase of O.OlM I-heptane sulfonic acid and O.OIMpotassium phosphate (adjusted to pH 4.0 with IN H3P04) andmethanol (65:35 voVvol) at a flow rate of 1.0 mVmin. Themobile phase was sparged continuously with helium. Thecolumn temperature was maintained at 40°C. Amines weredetected at different wavelengths by an ultraviolet (UV)/visible diode-array detector at 206 nm (phenylethylamine),210 nm (histamine), and 220 nm (tyramine and tryptamine).Identification of the amine-containing peaks were confirmedby comparing UV sample spectra a against a spectral librarygenerated from pure amine standards.

Diamines (putrescine and cadaverine). Diamines wereanalyzed by the method described by Jones and Gilligan (7).Fifty IIIof amine extract solution and 50 III of Fluoraldehyde ™reagent solution (Pierce, Rockford, IL) of o-phthalaldehyde(OPA) were reacted for no longer than 45 s. Derivatizeddiamines were eluted with methanol and water (70:30, voVvol)at a 1.0 mVmin flow rate and detected by a HP l460Aprogrammable fluorescence detector using 231 nm excita-tion and 425 nm emission wavelengths. Identification ofHPLC peaks containing amines were confirmed by com-paring spectra against a spectral library generated frompure diamine standards.

Standard solutions of tyramine, tryptamine,phenylethylamine, histamine, putrescine and cadaverine wereprepared by dissolving an appropriate amount of the aminehydrochloride salt (Aldrich Chemical Co., Milwaukee, WI)in 20% methanol. All concentrations were expressed as thefree amine. Serial dilutions of the monoamines were be-tween 1.0 Ilg/ml and 100.0 Ilg/ml, and those of the di-amines were between 1.0 Ilg/ml and 50.0 Ilglml. A standardcurve for each amine was generated by plotting integratedpeak areas versus amine concentration. The coefficients ofdetermination for all standard curves were 0.994 or greater.These generated standard curves were used to determinethe quantity of amine contamination in meat samples.Analysis of amine accumulation consisted of duplicateextractions that were evaluated twice to obtain an averagepeak area.

Recovery of amines from beef samples was determinedby spiking 10.0 g of sirloin roast with an amount of theamine to produce a sample concentration of 100 Ilglml.Samples were analyzed as previously described. The mini-mal detectable level of amines was determined to be threetimes the background noise level.

JOURNAL OF FOOD PROTECTION, VOL. 58, MARCH 1995

286 KRIZEK, SMITH AND PHEBUS

Bacteriological analysisPsychrotrophic and lactic acid bacterial populations

were determined by excising a 1.5 in. (3.8 cm) diametercore from each replicate. Between samples, tools used toopen the packages as well as the coring devices werewashed, dried, submerged in 95% ethanol for 5 min, andflamed. The core and 90.0 ml of 0.1 % peptone water bufferwere placed together into a Stomacher bag (Spiral Biotech,Bethesda, MD) and stomached for 2 min.

Serial dilutions of core samples were analyzed for totalpsychrotrophic bacteria using plate count agar (Difco Labo-ratories, Inc., Detroit, MI) and for lactic acid bacteria usingoverlayed mithicillin-resistant Staphylococcus (MRS) agar(Fisher). Psychrotrophic bacterial enumeration was obtainedafter plates were incubated at 7°C for 10 days (5). For lacticacid bacterial enumeration, plates were incubated at 35°Cfor 48 h, and gram-positive, catalase-negative cocci or rodswere counted (6).

Statistical analysisStatistical analysis of the data was· performed using

Least Squares Analysis of Variance (14). Dependent vari-ables were bacterial numbers and tyramine concentrations;independent variables were treatment and time.

RESULTS

The lowest detectable concentration for all amines frommeat samples was 1.0 Ilglg. Average recoveries from spikedmeat samples for observed amines were histamine (87.2%,C.v. 3.3%), phenethylamine (43.1%, C.v. 6.7%), tryptamine(23.95%, C.v. 8.1%), and tyramine (49.4%, C.v. 4.8%). Nocorrections for recoveries were made on the amine levelsreported here.

Histamine, phenethylamine and tryptamine were notdetected in any of the samples. Figures I and 2 show thelevels of tyramine, putrescine and cadaverine detected invacuum-packaged meat samples during 100 days of stor-age at -2 and 2°C. Tyramine was found to accumulate tovery high levels (140 Ilg/g) regardless of storage tempera-ture. The highest measured level of tyramine was181.1llg/g in a sample that had been stored at 2°C for 100days. Both temperature treatments showed detectable in-creases in tyramine concentrations starting at 20 days. Therate of tyramine accumulation decreased at day 80.Tyramine intoxication would be possible after 20 days ofstorage at 2°C and 40 days of storage at _2°C in oursamples. It should be noted that samples stored for 100days would be considered organoleptically inedible. Pu-trescine was detected after 100 days of storage at average

Figure I. Biogenic amine formation in vacuum-packaged beefstored for 100 days at -2°C (0 tyramine; 0 putrescine; L1cadaverine). Figure 3. Lactic acid bacteria plate counts in vacuum-packaged

beef stored for lOO days at -2°C and 2°C (0 _2°C, 0 2°C).

1008040 60Days

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160Ci 140....~120-c: 100oiii 80•...."E 60Q)

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oo

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8.... 7~120 N- E6c: 100 00 -5:.;::; 80ctl :::::l•... LL.4-c: 60 0Q) (930 40c: 320() 20

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0 20 40 600

80 100 0 20 40 60 80Days

100Days

Figure 2. Biogenic amine formation in vacuum-packaged beefstored for lOO days at 2°C (0 tyramine; 0 putrescine; L1cadave rine).

Figure 4. Psychrotrophic bacteria plate counts in vacuum-pack-aged beef stored for 100 days at _2°C and 2°C (0 -2°C, 0 rc).

JOURNAL OF FOOD PROTECTION, VOL. 58, MARCH 1995

BIOGENIC AMINES IN VACUUM-PACKAGED FRESH BEEF 287

TABLE I. Measurement of tyramine in penetration and washingstudies of vacuum-packaged subprimals stored at 2°C.

Days of Average J.1g1gstorage Treatment tyramine detected S.D.

60 total 10 g sample 81.9 12.460 3 mm penetration 103.1 42.560 6 mm penetration 73.0 33.260 9 mm penetration N.D.

80 total 10 g sample 119.5 37.280 3 mm penetration 124.7 64.880 6 mm penetration 69.6 36.280 9 mm penetration N.D.

100 total 10 g sample 141.4 26.7100 3 mm penetration ]56.0 37.7]00 6 mm penetration 131.0 5.9100 9 mm penetration N.D.]00 water wash* 103.2 23.0

S.D.: Standard deviation.N.D.: Not detected.* Subprimals were rinsed approximately 30 s on each side

with tap water.

concentrations of 99 Ilg/g at _2°C and 44 Ilg/g at 2°e.Cadaverine was detected after 100 days of storage ataverage concentrations of 27 Ilg/g at _2°C and 54 Ilg/g at2°e.

Figures 3 and 4 illustrate lactic acid bacterial (LAB)growth and total psychrotrophic bacterial growth with stor-age. Initial LAB and total psychrotrophic populations werelQ3 CFU/cm2• Lactic acid bacteria and psychrotrophic bac-terial growth at 2°C reached the stationary growth phase by20 and 40 days of storage at log 6.4 and 7.2 CFU/cm2,

respectively. Bacterial populations at the -2°C storage tem-perature appeared to reach a stationary growth phase byapproximately 60 days. Lactic acid bacteria growing at_2°C steadily increased to log 7.5 CFU/cm2 at 80 days anddecreased to log 6.9 CFU/cm2 at 100 days of storage. Inaddition, LAB and total psychrotrophic counts for samplesstored at -2°C exceeded counts for samples stored at 2°Cshortly during 40 days of storage. Statistical analysis indi-cated that bacterial numbers were unaffected by storagetemperature over the course of the study (P<0.05). Inaddition, tyramine levels were not significantly different(P<0.05) between storage temperatures after 100 days ofstorage. However, significant differences (P<0.05) in tyra-mine levels at different storage temperatures were evidentat 20 and 40 days of storage.

Table 1 indicates the results of the washing and penetra-tion studies. Samples stored for 100 days contained averagesof 141.41lg/g of tyramine before and 103.2 Ilg/g of tyramineafter a water rinse. Thus, rinsing caused an average reductionof 38.21lg/g for these samples (P<0.07). Evidence of tyraminepresence at various depths from the subprimal surface ex-plains the difficulty of using a water wash to remove tyramine.Penetration of tyramine was evaluated after 60, 80 and 100days of storage at depths of 0 to 3 mm, 3 to 6 mm and 6 to

9 mm. Tyramine was found in highest concentrations at theo to 3 mm level ranging from 103 Ilg/g at 60 days to 1561lg/g at 100 days of storage. At the second measured level ofpenetration (3 to 6 mm), slightly less tyramine was found,ranging from 73 Ilg/g at 60 days to 131 Ilg/g at 100 days ofstorage. No tyramine was detected 6 to 9 mm from thesurface of the cut.

DISCUSSION

Information on biogenic amine formation in fresh,vacuum-packaged beef stored for more than 50 days islimited. Dainty et a1. (3) investigated the formation ofputrescine and cadaverine in vacuum-packaged beef in-oculated with Hafnia alvei and Serratia liquefaciens for51 days of storage at 1°e. Their inoculated study indi-cated that putrescine reached 10 Ilg/g and cadaverineaccumulated to 200 Ilg/g. Our uninoculated study showsthe formation of these diamines but not at the samemagnitude. In addition, our study indicates that cadaver-ine concentrations exceeded putrescine concentrations at2°C throughout the experiment, but were reversed at _2°C.The explanation for this interaction is unknown but prob-ably relates to differences in predominating bacterial spe-cies. Smith et a1. (16) and Dainty et a1. (3) showed thatbiogenic amine production becomes evident when bacte-rial loads approach log 6 CFU/cm2• In our study, biogenicamine formation also became apparent in all samples atlog 6 CFU/cm2 microbial load. Smith et a1. (16) measuredLAB on vacuum-packaged beef stored for 120 days at1°e. Initial LAB counts in their study were less than 10CFU/cm2 in comparison to log 3 CFU/cm2 for the presentstudy. However, after 20 days of storage at 1°C, Smith eta1. (16) detected approximately log 4.5 CFUlcm2 LAB,which was the first point of biogenic amine detection insome samples. These results parallel those in our study.When bacterial counts reached log 6 to log 7 CFU/cm2 inboth studies, bacterial growth tended to shift to the sta-tionary phase, and amine production continued. Smith eta1. (16) showed approximately 130 Ilg/g of tyramine after90 days of storage at 1°C in comparison to 141 Ilg/g oftyramine after 100 days of storage at 2°C in our study.Edwards et a1. (3) showed a similar trend in production oftyramine for preinoculated, vacuum-packaged, beef samplesheld at 1°C for 7 weeks. They also found that tyraminewas not evident in samples until bacterial numbers ap-proached log 6 CFU/cm2• Edwards et a1. (4) and Rozbehet a1. (13) associated biogenic amine formation withLactococcus spp., Lactobacillus spp., Leuconostoc spp.,Brochothrix thermosphacta, and Pseudomonas spp. inrefrigerated, fresh, vacuum-packaged beef.

Our results showed no histamine, phenethylamine andtryptamine formation. These results agree with Edwards eta1. (4) who reported that vacuum-packaged beef productsshowed no more than 3 Ilg/g accumulation of histamine.Vanderkerckhove (19) reported less than 7 Ilg/g ofphenethylamine in dry fermented meat products, whichcaused intoxication in patients taking a monoamine oxi-dase inhibiting drug. Tryptamine has been found in fruits,vegetables and cheeses but generally at lower levels than

288 KRIZEK, SMITH AND PHEBUS

histamine and tyramine (12). Even though tryptamine andtyramine show similar pharmacological actions, tryptamineintoxication has not been reported.

Rice, Eitenmiller and Koehler (1 J) stated that 3.7 Ilglgof tyramine caused intoxication in patients undergoing MAOIdrug treatment. Blackwell and Mabbit (1) determined that5-10 mg of tyramine could cause a moderate food-druginteraction in MAOI therapy patients. Sullivan (18) re-ported that consumption of 6 mg of tyramine produced arise in blood pressure and that 10-25 mg intake inducedsevere hypertension.

No reports exist for putrescine or cadaverine intoxica-tion. However, these diamines may take an active role inhistamine intoxication and cancer promotion (2,22). Pu-trescine and cadaverine do not directly cause intoxicationproblems, but may exhibit indirect effects. Chu andBjeldanes (2) showed that putrescine and cadaverine po-tentiate toxicity of histamine by increasing the rate ofhistamine transport across the gut wall. Warthesen et al.(22) reported that putrescine and cadaverine treated withnitrite in a high temperature-low moisture system formednitrosopyrrolidine and nitrosopiperidine, two nitrosaminesassociated with carcinogenicity. In general, these dia-mines are used as spoilage indicators in meat products (8).

Results from this study indicate that fresh beef storedfor extended periods of time in vacuum packages canaccumulate potentially toxic levels of tyramine (>15 mg)under normal refrigerated storage. Enough tyramine wasproduced by 40 days of storage at -2°e and 20 days ofstorage at 2°C to cause interactions in individuals takingMAOI drugs. However, Taylor (19) reported that 80 mg oftyramine can be consumed without elevation in bloodpressure by individuals not using MAOI drugs.

This research has addressed the penetration of aminesfrom the surface of the subprimal or the effects of washingsubprimals with water on amine concentration. Our resultsindicate that amines penetrate no more than 9 mm from thesurface of the cut over 100 days of storage. The resultsfrom our washing study also support the penetration results.Because amines were found at various levels from thesurface of the subprimal, only 38.2 Ilg/g of tyramine wasremoved by rinsing the sub primal with tap water.

CONCLUSIONS

This study indicates that biogenic amine productionshould be considered when storing vacuum-packaged beefbeyond 40 days. At -2°C storage temperature, the onset ofbiogenic amine formation is delayed by 20 days. However,tyramine and putrescine concentrations are virtually thesame after 80 days of storage regardless of storage tem-perature. Because biogenic amines do not penetrate morethan 9 mm from the surface of the subprimal, rinsingvacuum-packaged beef with water is effective in decreasingthe amine contamination by about 30%. However, therewould still be enough tyramine present after 60 days ofstorage (60 Ilg/g) so that an eight ounce portion wouldcontain a potentially toxic level (13.6 mg/227 g) for indi-viduals using MAOI drugs.

ACKNOWLEDGMENTS

Contribution No. 94-537-J from the Kansas Agricultural ExperimentStation. This material is based upon work supported by The CooperativeState Research Services, U.S. Department of Agriculture, under agreementNo. 89-34187-4511.

REFERENCES

I. Blackwell, B. L. and A. Mabbit. 1965. Tyramine in cheese related tohypertensive crisis after monoamine oxidase inhibitions. Lancet1:938-940.

2. Chu, C. H. and L. F. Bjeldanes. 1981. Effect of diamines, poly-amines and tuna fish extracts on the binding of histamine to mucinin vitro. J. Food Sci. 47:79-80, 88.

3. Dainty, R. H., R. A. Edwards, C. M. Hibbard and S. V. Ramantanis.1986. Bacterial sources of putrescine and cadaverine in chill storedvacuum-packaged beef. J. Appl. Bacteriol. 61: 117-123.

4. Edwards, R. A., R. H. Dainty, C. M. Hibbard and S. V. Ramantanis.1987. Amines in fresh beef of normal pH and the role of bacteria inchanges in concentration observed during storage in vacuum packsat chill temperatures. J. Appl. Bacteriol. 63:427-434.

5. Food and Drug Administration. 1992. Bacteriological AnalyticalManual. 7th ed. Association of Official Chemists International,Arlington, VA.

6. Frank, J. F., G. L. Christen and L. B. Bullerman. 1992. Tests forgroups of microorganisms. Ch. 8. In R. R. Marshall (ed.). StandardMethods for the Microbiological Examination of Dairy Products.16th ed. American Public Health Association, Washington, DC.

7. Jones, B. N. and J. P. Gilligan. 1983. O-Phthaldialdehyde precolumnderivatization and reversed-phase high-performance liquid chroma-tography of polypeptide hydrolysates and physiological fluids.J. Chromatogr. 266:471-482.

8. Legarreta, I. G. and A. M. C. Gallardo. 1991. Detection of biogenicamines as meat spoilage indicators. J. Muscle Foods 2:263-278.

9. McCabe, B. J. 1986. Dietary tyramine and other pressor amines inMAOI regimens: a review. J. Am. Diet Assoc. 86:1059-1064.

10. Morrow, J. D., G. R. Margolies, J. Rowland and L. J. Roberts. 1991.Evidence that histamine is the causative agent in scromboid-fishpoisoning. N. Engl. J. Med. 324:716-720.

II. Rice, S. L., R. R. Eitenmiller and P. E. Koehler. 1975. Histamine andtyramine contents of meat products. J. Milk Food Techno\. 38:256-258.

12. Rice, S. L., R. R. Eitenmiller and P. E. Koehler. 1976. Biologicallyactive amines in food: A review. J. Milk Food Technol. 39:353-358.

13. Rozbeh, M., N. Kalchayanand, R. A. Field and M. C. Johnson. 1993.The influence of biopreservatives on the bacterial level of refriger-ated vacuum packaged beef. J. Food Safety 13:99-111.

14. Statistical Analysis System Institute Inc. 1989. SAS Users Guide:Basics, Version 6.06 Ed. SAS Institute Inc., Cary, NC.

15. Sen, N. P. 1969. Analysis and significance of tyramine in foods.J. Food Sci. 34:22-26.

16. Smith, J. S., P. B. Kenney, C. L. Kastner and M. M. Moore. 1993.Biogenic amines in vacuum packaged fresh beef. J. Food Prot.56:497 -500,532.

17. Stratton, J. E., R. W. Hutkins and S. L. Taylor. Biogenic amines incheese and other fermented foods: A review. J. Food Prot. 54:460-470.

18. Sullivan, E. A. and K. I. Shulman. 1984. Diet and monoamine oxidaseinhibitors: A re-examination. Can. J. Psychiatry. 29:707-711.

19. Taylor, S. L. 1990. Other Microbial Intoxications. Ch. 9. pp. 160-168. In D. O. Cliver (ed.). Foodborne Diseases. Academic Press,Inc., San Diego, CA.

20. Van Boekel, M. A. J. S. and A. P. Arentsen-Stasse. 1987. Determi-nation of aromatic amines and their precursors in cheese by high-performance liquid chromatography. J. Chromatogr. 389:267-272.

21. Vanderkerckhove, P. 1977. Amines in dry fermented sausage.J. Food Sci. 42:283-285.

22. Warthesen, J. J., R. A. Scanian, D. D Bills and L. M. Libbey. 1975.Formation of heterocyclic N-nitrosamines from the reaction of nitriteand selected primary diamines and amino acids. J. Agric. FoodChern. 23:898-902.

23. Wheatley, A. M. and K. F. Tipton. 1987. Determination of tyraminein alcoholic and nonalcoholic beers by high performance liquidchromatography with electrochemical detection. J. Food Biochem.11:133-142.