166

J. Milk Food Techno!. Vol. 39, No. 3, Pages 166·169 (.March, 1976) Copyright © 1976, International Association of Milk, Food, and Environmental Sanitarians

Tyrosine and Activities of Pediococcus cerevisiae and Lactobacillus Species and the Production of in Fermented Sausages

S. L. RICE and P. E. KOEHLER Department ofFood Science

University ofGeorgia, Athens, Georgia 30602 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/39/3/166/2397373/0022-2747-39_3_166.pdf by guest on 28 September 2021

(Received for publication September 26, 1975)

ABSTRACT free is produced, it is decarboxylated to form In investigating formation of tyramine and histamine in a model sys· tyramine. Microorganisms commonly found in sausage tern, it was found that four commercial sausage starter cultures did not fermentations include Pediococcus, Lactobacillus, exhibit appreciable tyrosine or histidine decarboxylase activity. In Streptococcus, and Micrococcus (3). Bacteria belonging addition. other species of Pediococcus cerevisiae and Lactobacillus did to the group D streptococci are well known producers of not display appreciable decarboxylase activity. Mixtures of P. cerevisiae and Lactobacillus plantarum were also unable to produce significant tyrosine decarboxylase. The purpose of this research was levels of these amines. One species of Streptococcus tested was able to to investigate the role of the fermenting bacteria in the produce 34.5 llg of tyramine/hour under the assay conditions. When P. production of tyramine, emphasizing bacteria normally cerevisiae and L. plantarum were used as starter cultures to prepare used in sausage starter cultures. Histidine decarboxylase sausages, it was found that this treatment resulted in lower tyramine was assayed in these bacteria because of reports levels (approximately 200 f'g/g) than when the Streptococcus sp. was used as a starter culture (approximately 300 f'g/g). However, the use of implicating Pediococcus cerevisiae as the source of P. cerevisiae and L. plantarum did not result in a significantly lower histamine in sauerkraut (I I) and reports of high levels of tyramine level than when no starter culture was used. histamine in fermented sausages (5).

Presence of tyramine in fermented sausages has recent­ MATERIALS AND METHODS ly been reported (2, 5, 13). Tyramine is the major cause of Cultures hypertensive crises in patients treated with monoamine A list of the microorganisms used in the tyrosine and histidine oxidase inhibitors (1). Symptoms of hypertensive crisis decarboxylase assays and their sources is shown in Table 1. In addition include high blood pressure, headache, fever, and TABLE 1. Cultures examined for decarboxylase activity sometimes perspiration and vomiting (1, 8). A number of Microorganism Source deaths have been reported due to hypertensive attacks Pediococcus cerevisiaea Merck & Co., Inc. (1). Presence of tyramine in foods may also give rise to P. cerevisiaea Chr. Hansen's Lab., Inc. migraine headaches in susceptible individuals (9). P. cerevisiae ATCC 10791 P. cerevisiae ATCC 8042 Although foods normally contain small amounts of Lactobacillus biogenic amines, formation of large amounts has been plantaruma Merck & Co .. Inc. reported only in aged or fermented products. The factors L. plantaruma Chr. Hansen's Lab., Inc. L. plantarum ATCC 8014 that govern the formation of amines includes: (a) the L. acidophilus Food Science Dept., Univ. of Georgia availability of free amino acids, (b) presence of L. casei Food Science Dept., Univ. of Georgia microorganisms that can decarboxylate the amino acids, Streptococcus sp. Authors' isolate and (c) favorable conditions for the growth of aused as commercial starter cultures. the microorganisms and for the production of to testing pure cultures, mixtures of P. cerevisiae and Lactobacillus It decarboxylase . is well known that, during the plantarum from both Chr. Hansen's Lab., Inc. (Milwaukee, Wisconsin) ripening of cheese, liberation of amino acids occur. and Merck & Co., Inc. (Rahway, New Jersey) were assayed, since these Dierick et a!. (5) reported an increase in free amino acids starter cultures may be mixed in sausage production. All cultures were during the ripening of dry sausages, however, little carried on Bacto Micro Assay Culture Agar (Difco Laboratories, Inc., change in the concentration of free tyrosine was Detroit, Michigan), buffered with 1% CaC03• The Streptococcus sp. (probably Streptococcus jaecium) identified by the authors, was observed. Cantoni et a! (2) showed that free amino acids included in this study for comparative purposes. This Streptococcus sp. increased from 0.622% to 1.949% in the aging of a is a good producer of tyrosine decarboxylase and was isolated as a Compagnolo salami and a complete disappearance of contaminating bacterium in a preliminary decarboxylase study. free tyrosine was also observed. Both of these workers Assay of tyrosine and histidine decarboxylase reported an increase in the presence of tyramine during Bacteria for decarboxylase assay, grown in decarboxylase assay sausage ripening. These reports indicate that although media for 24 h at 30 C, were prepared for inoculation by washing the AMINO ACID DECARBOXYLASE$ OF LACTIC ACID BACTERIA 167

centrifuged cells once with saline (0.85 %) solution and resuspending in by comparing the fluorescence of the samples to the fluorescence of a saline solution. Decarboxylase assay media was prepared by adding 10 standard containing 1 flglml histamine. g tryptone, 10 g yeast extract, 10 g glucose. 0.1 g tyrosine, and 0.1 g Tyramine ana(vsis of decarboxylase assay media histidine to 1 liter of distilled water. One milliliter (0.5 ml for each culture for mixed cultues) of the saline cell suspension was inoculated Tyramine was quantitated in the decarboxylase assay media in a into 100 ml of the above medium in a 250-ml Erlenmeyer flask. similar manner to that described for tyramine extraction and Cultures were incubated at 30 C and assays were done on duplicate quantitation in sausages (13). Five milliliters of the media were adjusted flasks. At time intervals of 0 (immediately after inoculation), 12, 24, to pH 10.0 with solid Na2C03• The liquid was saturated with NaCl and 48 h, 10.0 ml of medium was withdrawn with a sterile pipet. In (about 1.5 g), 3.75 ml of n-butanol were added, and the mixture was addition. at each time interval, 1.0 ml was withdrawn to determine shaken for 10 min using a wrist action shaker. After centrifugation the viable cell counts and 5.0 ml were withdrawn and frozen for a later tyramine in the butanol layer was determined as previously reported determination of tyramine or histamine. Plate counts were done by (13). using a medium containing 1 o/o tryptone, 1% yeast extract, 1 o/o glucose, Preparation of sausages and 2% agar. To further test the ability of starter cultures to produce tyramine, The pH of each 10-ml aliquot was measured using a pH meter sausages were made under identical conditions using the following Downloaded from http://meridian.allenpress.com/jfp/article-pdf/39/3/166/2397373/0022-2747-39_3_166.pdf by guest on 28 September 2021 equipped with a single probe electrode. Each aliquot was centrifuged variation with respect to the addition of cultures to sausage mixtures: for 20 min, the cells washed with 5 ml of saline solution, centrifuged (a) no culture added, (b) L. plantarum (Merck), (c) P. cere!'isiae again, and resuspended in 1.0 ml of saline solution. An aliquot of 0.5 (Merck), (d) Streptococcus sp., and (e) L. plamarum (:'vierck plus ml was used for each assay of tyrosine and histidine decarboxylase. Streptococcus sp. For L. plantarum and P. cerevisiae, the 6 oz cans of Tyrosine and histidine decarboxylase were assayed by a modified frozen concentrated cultures were thawed by placing in 21-24 C water. procedure of Levine and Watts (JO). The source (0.5 ml of cells Three millimeters of the culture were then added to 6.0 ml of sterile in saline solution) was added to the reaction vessel containing 2.9 ml saline. This suspension was added to the sausage preparation at the acetate buffer (1.0 M. pH 5.5) containing 3.7 x w-s M pyridoxal appropriate time. For studies involving the inoculation of the phosphate. The reaction was initiated by introduction of 0.1 ml of the Streptococcus sp., the culture was grown for 24 h at 30 C in Bacto 4 4 , r CJ carboxyl-L-histidine or r CJ carboxyl-L-tyrosine (Cala­ Micro Inoculum Broth (Difco). Cells were harvested by centrifugation, 3 tomic, Inc., Los Angeles, Ca.), 5 x 10· M and approximately 0.1 flCL washed with saline solution, and resuspended in approximately 3.5 ml The substrate was kept frozen until ready for use. All reactions were of saline solution. Three milliliters of the saline suspension of carried out at room temperature in a Dubnoff Metabolic Shaker Streptococcus sp. were used for inoculation into the sausage (Precision Scientific Co.. Chicago, Ill.). The reaction vessel was preparation. For the mixed culture, i.e. L. plantarum plus stoppered and incubated for I h. At the end of the incubation period, Streptococcus sp., only 0.1 ml of the saline-washed Streptococcus 2.0 ml of 1.2 N perchloric acid was injected through the side of the vial suspension was used. and the resulting hole covered with tape. The solution was allowed to Sausages were prepared in a manner similar to that described by incubate for additional 30 min to allow liberation of the C02 and DeKetelaere et al. (4). Composition of the sausage mixture is given in absorption onto a rolled filter paper (1 x 3 em, 3 MM Whatman) Table 2. The frozen meats were first cut with a band saw into containing phenylethylamine (Packard Instrument Co., Downers Grove. Ill). The filter paper rolls were attached to a No. 2 rubber TABLE 2. Composition mixture stopper via a wire clip and were dipped into the phenylethylamine just [ngredients Amount before initiation of the reaction. 6.8 kg After completion of the incubation period, the wire clips containing Beef trimmings Pork trimmings 4.1 kg the filter papers were added to polyethylene scintillation vials Pork hearts 2.15 kg containing 10.0 ml of toluene Omnifluor (0.4%, New England Nuclear, Lean pork 1.25 kg Boston, Mass.). The 14C was monitored for 5 min in a Beckman Model Salt (NaCl) 425 g LS-100 C liquid scintillation counter (Fullerton, Ca.). Controls Glucose lOS g consisted of adding 0.5 ml of saline solution to the reaction mixture in Cervelat seasoninga 91 g place of the cell suspension. The counting efficiency was determined 1.05 g 4 using standard r CJ benzene (Radiation Safety Officer, Safety Services aA. C. Legg Packing Co., Birmingham, Alabama Dept., University of Georgia} with 4.150 disintergrations/min (dpm).

The resulting activities were expressed as nmoles of C02 evolved/h. approximately 2-4 em cubes. The remaining ingredients were added to the meats and mixed by hand. After the meat had softened, the mixture Histamine analysis of decarboxylase assay media was passed through a 3/16-inch plate. After remixing by hand, the To verify histidine decarboxylase activity by bacterial cultures, the mixture was divided into five equal lots. At this point the starter decarboxylase assay media were assayed for histamine by a cultures, as described above, were added. Sausage mixtures were modification of the method of Shore (14). One milliliter of the medium stuffed into 688-mm diameter smoke permeable fibrous casings (Tee-Pak, Inc., Danville. Ill.) using an E-Z Pak Automatic Sausage was mixed with 4.0 ml of0.4 N HCI04 • A 2.0-ml aliquote of the mixture was transferred to a centrifuge tube containing 5.0 ml of Stutler (E.F. Zuber Engineering & Sales, Co., Minneapolis, Minn.). n-butanol:chloroform (3:2. vollvol), 0.25 ml 5 N NaOH, and O.iS g Sausages were placed in a 22 C incubator (approximately 65 o/o relative NaCJ. The tube was shaken for 5 min and centrifuged to remove any humidity) and the temperature was lowered at 18 C over a period of 5 free histidine. The butanol:chloroform layer was transferred to a days. On the sixth day sausages were smoked for 4 h with a heavy second tube containing 2.5 mi of NaCl-saturated 0.1 N NaOH, shaken smoke at 43 C and 80%relative humidity. Sausages were then placed in for 1 min, and centrifuged. A 4-ml aliquot of the washed butanol a drying chamber at 16 C and 70% relative humidity for 30 days. extract was transferred to a third tube containing 2.5 ml of 0.1 N HCI Tyramine content of the sausages was determined as described and 7.5 ml of n-heptane. The tube was shaken for 1 min, centrifuged, previously ([3). Duplicate samples were extracted and the butanol and the organic phase was removed by aspiration. To 2.0 ml of this acid extracts were spotted twice and compared to standard amounts of phase containing histamine was added 0.4 ml of 1 NNaOH, followed by tyTamine spotted on the same plate. 0.1 ml of o-phthalaldehyde (OPT) (10 mg/ml in methanol). After 4 min at room temperature, 0.2 ml of 3 N HCI was added. The contents of the RESULTS AND DISCUSSION reaction tubes were mixed using a vortex mixer following each addition. No tyrosine decarboxylase activity for the various P. The tluorescence at 450 nm. resulting from activation at 360 nm, was measured on a Turner Model 430 Spectrofluormeter (G. K. Turner cerevisiae test stains was detected throughout the 48-h Associates, Palo Alto, Ca.). Histamine concentration was determined culturing period. Viable cell counts showed that the 168 RICE AND KOEHLER bacteria had achieved the stationary phase within this TABLE 4. Activity of tyrosine decarboxylase in cultures (nmoles C02 time. This is important, since Gale (7) reported that evolved/h) bacteria primarily produce amino acid decarboxylases towards the end of the growth cycle. All the microorganisms had produced low pH values (4.41-4.95) Streptococcus sp. P. cerevisiae (Merck) and within the assay period. Again, this is important since L. plantarum (Merck) ND 1.527 the production of amino acid decarboxylases is highest at P. cerevisiae (Hansen) and low pH (7). No tyramine was detected in the media at any I. (Hansen) ND ND ND 0.905 time. It appears from these data that P. cerevisiae strains are not able to decarboxylate tyrosine when grown under lower than that obtained by the other microorganisms. these conditions. Tyramine levels of up to 300 11g/ml were detected in the

TABLE 3. Activity of histidine decarboxylase in cultures (nmoles decarboxylase assay media. A decarboxylase activity of Downloaded from http://meridian.allenpress.com/jfp/article-pdf/39/3/166/2397373/0022-2747-39_3_166.pdf by guest on 28 September 2021 C02 evolved/h) 250 nmoles C02 evolved/h is equivalent to 34.5 11g of tyramine/h. Given ample substrate and sufficient Culture numbers of these organisms (10 8 cells/g), tyramine levels P. cerevisiae (Merck) of 300-900 11glg could be formed within 9-26 h at the P. cerevisiae (Hansen) 0.397 ND above rate of amine formation. Pederson (12) has reported P. cerevisiae (A TCC 10791) ND ND P. cerevisiae (A TCC 8042) 0.139 ND the presence of a Streptococcu:sfaecalis-like bacteria that Streptococcus sp. 0.105 ND reached a level exceeding 10 8 cells/g after 16 hat 41 C in P. cerevisiae (Merck) and a Chorizo-type sausage. It is possible that the I. plantanun \Merck) ND ND ND 0.175 P. cerevisiae (Hansen) and Streptococcus sp. was a strong tyramine producer and L planrarum (Hansen) ND ND ND 0.030 that S. faecalis-like bacteria are common contaminates aND·Not detected. in sausages. The tyrosine content of sausage is reported to be 4.54 mglg (6). Assuming a 10o/o release of tyrosine Table 3 shows the histidine decarboxylase activities of during proteolysis and 100% conversion to tyramine, it is the Pediococcu:s strains. Low, variable activity was possible that a tyramine level of 344 !Ag/g may arise. observed. The activities detected under these conditions Tyrosine levels of sausages probably vary widely due to are too low to give rise to any appreciable quantities of differences in moisture and fat content. Sausages with histamine. The highest activities (0.637 nmoles low fat contents may be able to attain much higher C02/evolved/hr) could give rise to only 3.5 X w- 3 j-Ig tyramine levels. histamine/106 cells/h. Assay of the media for histamine Table 3 also shows the histidine decarboxylase showed that less than 3~-tg/ml of histamine was formed. It activities for the Streptococcus sp. A very slight is apparent that under these conditions strains of P. decarboxylase activity was detected at 12 h. The fact that cerevisiae are unable to produce substantial amounts of no activities were detected at any ether times would tend histamine. to decrease the significance of the histidine As with P. cerevisiae, no tyrosine decarboxylase decarboxylase activity detected at 12 h. It seems that the activity was detectable for any of the Lactobacillus spp. Streptococcus sp. would not be capable of producing tested curing the 48-h incubation period. It is apparent appreciable quantities of histamine. from the viable cell counts that all the bacteria had The tyrosine decarboxylase activities for the mixed achieved stationary phase after 48 h of incubation. The cultures (P. cerevisiae and L. plantarum) (Table 4) final pH of the Lactobacillus media was, in all instances, indicate the absence of this enzyme except at 48 h. Both quite low (pH 4.46-4.58). In addition, negligible levels of mixed cultures showed very slight activity at this time. tyramine were found in the assay media. It appears that Assay of culture media for tyramine showed no these Lactobacillus spp. are unable to produce tyramine appreciable build-up of this amine. The viable cell under these conditions. counts of the mixed cultures indicated that the No histidine decarboxylase activities were detectable microorganisms had attained stationary phase between for any of the iactobacilli during the 48-h culturing 12-24 h. The pH of the culture media had dropped to period. When the media was assayed for histamine, less 4.51-4.67 at the end of the 48-h time period. It seems than 3 1-1g/ml of histamine was detected. It is apparent that. although slight tyrosine decarboxylase activity was that, under the assay conditions, the Lactobacillus spp. detected, no appreciable levels of tyramine could cannot produce histidine decarboxylase. accumulate under these conditions. Table 4 shows the results of the tyrosine decarboxylase Table 3 indicates the histidine decarboxylase activities assays for the Streptococcus sp. This microorganism of the mixed cultures. Again, no activity was detected showed high decarboxylase activity at all assay times. except at 48 h. Both mixed cultures produced slight From the viable cell counts, it can be seen that the histidine decarboxylase activity at this time. Analysis of bacteria had reached stationary phase in 12 h. Unlike the assay media for histamine showed the presence of less the other organisms tested, the pH of the media was 4.80 than 3~-tg/ml of histamine. It seems unlikely that, under after only 12 h. The ultimate pH obtained (4.36) was also these conditions, the mixed culture of P. cerevisiae and AMINO ACID DECARBOXYLASES OF LACTIC ACID BACTERIA 169

L. plantarum could produce appreciable levels of sausages are the presence of tyrosine decarboxylase-pro­ histamine. ducing microorganisms, such as the Group D Streptococcus and the availability of free tyrosine as a TABLE 5. Tyramine contents of experimental sausages substrate. The low acid conditions that occur in sausage Treatment Tyramine ( g/g) fermentations are favorable for the decarboxylation of Control 211a amino acids. The presence of decarboxylating bacteria Lactobacillus plantarum 212a Pediococcus cerevisiae 160a may be controlled, at least in part, by proper methods of Streptococcus sp. 298b sanitation and hygiene in production plants. Use of short L. plantarum and Streptococcus sp. 285b fermentation times and cooking of the to kill Values with the same superscript are not significantly different from decarboxylating bacteria may also help to control each other, while values with different superscripts are significantly different (p < 0.05). t}Tamine levels in sausages. REFERENCES The tyramine contents of experimental sausages is Downloaded from http://meridian.allenpress.com/jfp/article-pdf/39/3/166/2397373/0022-2747-39_3_166.pdf by guest on 28 September 2021 1. Blackwell, B .. E. Marley, J. Price. and D. Taylor. 1967. Hyperten­ presented in Table 5. These values were calculated sive interactions between monoamine oxidase inhibitors and food­ assuming a recovery of 73o/o (13). All of the sausages stuffs. Br. J. Psychiatry 113:349-365. contained tyramine, while none of the samples contained 2. Cantoni, C., M .. Bianchi, and G. Beretta. 1974. Amino acids his­ exceedingly large amounts of the amine (i.e greater than tamine. and tyramine variation during ripening of dry sausage. 400 1-lg/g). Both sausages prepared with the Ind. Aliment. (Bucharest) 13:75-78. [Chern. Abstr. 82:2803f (1974)]. Streptococcus sp. were significantly higher in tyramine 3. Deibel. R. H., C. F. Niven, and G. D. Wilson. 1961. Microbiology than the other sausages. It appears from these data, that of meat curing. Ill. Some microbiological and related technologi­ the Streptococcus sp. was able to produce greater cal aspects in the manufacture of fermented sausages. Appl. Mi­ amounts of tyramine than in the control and other crobiol. 9:156-161. treatments, regardless of whether it was initially present 4. DeKetelaere, A., D. Demeyer, P. Vandekerckhove, and I. Ver­ vaeke. 1974. Stoichiometry of carbohydrate fermentation during in high or low populations. It is possible that the dry sausage ripening. J. Food Sci. 39:297-300. Streptococcus sp. was more competitive than L. 5. Dierick, N., P. Vandekerckhove, and D. Demeyer. 1974. Changes plantarum for the available nutrients. Even in the in nonprotein compounds during dry sausage ripening. J. Food presence of a good tyrosine decarboxylase microorgan­ Sci. 39:301-304. ism {Streptococcus sp.), relatively low levels of tyramine 6. FAO. 1970. Amino-acid content of foods and biological data on proteins. Food and Agricultural Organization of the United Na­ were found.lt is possible that the production of tyramine tions: Nutrition Studies No. 24. p. 120. was partially inhibited by some environmental factors or 7. Gale, E. F. 1946. Bacterial amino acid decarboxylases. Adv. En­ that the amount of substrate was limiting. Some zymol. 6:1-32. ingredients, e.g. spices, may have inhibited the 8. Goodman, L. S., and A. Gilman.1965. The pharmacological basis It of therapeutics. Macmillan, London. p. 197. decarboxylase activity. is also possible that the level of 9. Hanington, E. 1967. Preliminary report on tyramine headache. tyramine may have been decreased by catabolic Br. Med. J. 2:550-551. pathways. 10. Levine, R. J., and D. E. Watts. 1966. A sensitive and specific assay The other sausages produced with only P. cerevisiae or for histidine decarboxylase activity. Biochem. Pharmacol. 15:841- L. plantarum did not show a significantly different 849. 11. Mayer, K .. G. Pause, and U. Vetsch. 1973. Formation of biogenic amount of tyTamine than that found in the control. The amines during sauerkraut termentation. Obst- Gemuese- Verwert. source of tyramine in these sausages and in the control is Ind. 58:307-309. probably the decarboxylase activity of contaminating 12. Pederson, C. S. 1971. Microbiology of food fermentations. Avi bacteria. It appears that use of starter cultures did not Publishing Co., Inc .. Westport, Conn. p. 153. serve to control their growth and the production of 13. Rice, S., R. R. Eitenmiller, and P. E. Koehler. 1975. Histamine and tyramine content of meat products. J. Milk Food Techno!. 38: It tyTamine. is also possible that tyramine may be 256-258. produced synergistically with the bacteria present in the 14. Shore. P. A. 1971. The chemical determination of histamine. pp. natural flora. 89-97. In D. Glick (ed.) Methods of biochemical analysis. Vol. The main factors that govern productionoftyraminein Supplemental. Interscience Publishers, New York.