Biogenic amine formation

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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 -2C and 2C for 100 Days


    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)


    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-2C or 2e. 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 at2C and 40 days of storage at -2C. Putrescine andcadaverine were detected first at 40 days of storage at2C and 60 days of storage at _2e. 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 1C 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



    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.


    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 OC and water vapor transmission rates of0.6 cc/IOO in.2 (645.16 cm2)/24 h at 37C. Packaged samplesthen were placed in storage at -2C or 2e.

    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 @ 16C). 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: _2C and 2C; 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 40C. 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 l460