241 Journal of Food Protection Vol. 40, No. 4, Pages 241·245 fA.pril, 1977) Copyright © 1977, International Association of Milk, Food, and Environmental Sanitarians Production of Tyrosine and Histidine Decarboxylase by Dairy-Related Bacteria M. N. VOIGT and R. R. EITENMILLER Department ofFood Science University ofGeorgia, Athens, Georgia 30602 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/40/4/241/1649644/0362-028x-40_4_241.pdf by guest on 27 September 2021 (Received for publication August 2, 1976) ABSTRACT be harvested at either two-thirds maximal growth or at Manometric and radiometric procedures were used to determine the the end of active cell division (3). Acetone drying of ability of 38 dairy-related bacteria and four commercial starter bacteria cells is the standard method used to prepare preparations to produce tyrosine and histidine decarboxylase. All of the bacterial decarboxylases for biochemical studies, and 14 cultures had slight ability to cause release of C02 from carboxyl with the exception of ornithine decarboxylase, activities 14C-tyrosine and most released 14C0 from labeled histidine; however, 2 are not affected by the procedure (12, 33). because of inherent errors of the assay in detecting low levels of specific decarboxylase activity, the C02 release was not considered positive for A survey for tyrosine decarboxylase activity in bacteria specific decarboxylase activity unless the results were verified by the by Gale (12) included 800 streptococci; of these, manometric technique. One strain of Streptococcus lactis, a approximately 500 synthesized the enzyme. Most of the .Micrococcus luteus strain, and two Leuconostoc cremoris strains had active t}Tosine decarboxylase systems. Only Clostridium perfringens and 500 belonged to Lancefield's Group D and one culture an Escherichia coli strain were found to produce histidine was Streptococcus lactis. Koessler et al. (21) found five decarboxyiase. None of the commercial starter cultures produced the streptococci that produced tyrosine decarboxylase which enzymes. It was not determined whether tyramine or histamine was included S. lactis from milk with the activity. Various produced by the nonspecific decarboxylase activity. workers have reported the enzyme to be present in Eschen'chia coli (4, 11, 16). Gale found tyrosine Undesirable physiological reactions have been report­ decarboxylase activity in nine of 100 cultures of this ed following ingestion of foods containing biologically species. Lagerborg and Clapper {22) surveyed 33 active amines (1, 7, 19, 27). Rice et al. (30) discussed the lactobacilli and found one strain related to Lactobacillus roles of drug inducement and genetic tendencies in casei or Lactobacillus arabinosus that released carbon triggering these reactions. dioxide from tyrosine. They found an unknown Amines in food products are produced by specific Lactobacillus species with the same activity, and another amino acid decarboxylases, and amine build-up is the Lactobacillus species that decarboxylated both tyrosine result of the growth of decarboxylase-positive bacteria and histidine, a most unusual find, since this is the only under conditions favorable to enzyme synthesis and culture reported in the literature to possess both activity (10, 25, 26, 29, 33). Koessler et al. (21) postulated activities. Umezu (34) identified a tyrosine decarboxylase that decarboxylation may be a protective mechanism for in a strain of Leuconostoc mesenteroides var. Sake. bacteria against an acid environment. That is, when the Blackwell and Mabbitt (4), after noting the presence of hydrogen ion concentration becomes sufficient to threat­ Group D streptococci (10,000 colony forming units/g of en the life of the microorganism, a decarboxylase may be cheese) and E. coli in ripened cheese. concluded that synthesized. In 1946, Gale (12) summarized several these bacteria were the most likely source of the conditions that must be considered to optimize the decarboxylase enzymes responsible for formation of synthesis of bacterial decarboxylases. These conditions t}Tamine in cheese. Recently, Rice and Koehler (31) included most of the following: (a) the organism must surveyed several pediococci and lactobacilli common to have the genetic potential to synthesize the enzyme; (b) sausage starters and found the bacteria lacking in the particular substrate amino acid needs to be supplied tyrosine and histidine decarboxylase. A Streptococcus in the growth medium, as well as pyridoxine and species isolated by these authors was an active tyrosine­ nicotinic acid, except for histidine and glutamate decarboxylating organism. decarboxylase (2, 3, 14. 28); (c) the gro'"1h medium must Histidine decarboxylase is widely distributed in the be acid, usually obtained by supplying a fermentable genera Escherichia. Salmonella. Clostridium, Bacillus. carbohydrate to the medium (9, 17, 22. 25); (d) the and Lactobacillus (20. 21, 27). Evidence for the enz}me medium should be unbuffered (3); (e) the gro\\'ih in E. coli include the finding by Gale (12) that 14 of 155 temperature should be Q.7 C; and (j) the culture should cultures possessed the enzyme and the report by Hanke 242 VOIGT AND EITENMILLER and Koessler (16) that six of 20 eolifonn cultures Tyrosine and histidine decarboxylase activities were determined by synthesized the enzyme. Havelka (1 TJ noted that 71.4 o/o of an isotopic assay method adapted from the procedure of Levine and all Enterobacteriacae isolated from the imported marine Watts (23) and by a manometric method. The isotopic procedure consisted of incubating LO ml of cultured skim milk with L 9 ml of 1 M fish produced histamine. Eggerth (8) found 40 strains of sodium acetate buffer containing 2.5 x 10-4 M tyrosine or histidine, pH bacteria of intestinal origin capable of decarboxylating 5.5, and 100 111 (0.125 f'Ci) of 14C-carboxyl tyrosine or histidine histidine. Histidine decarboxylase activity was found by (Ca!Atomic, Los Angeles) at 27 C for 60 min in a reaction vessel (a Rodwell (32) in strains of Lactobacillus pentoaceticus and polyethylene scintillation vial). All buffers contained 1.05 x to-• M Lactobacillus bifidus as well and in Lactobacillus 30a and ~ireptomycin sulfate to inhibit bacterial gro.,.,'th. All reactions were carried out in duplicate. Following incubation, 2 ml of 1.2 N perchloric Lactobacillus 60. However, the number of dairy-type acid was injected into the flasks to stop the reactions and to liberate 14 14 lactobacilli found to contain the enzyme has been small. dissolved C02• The liberated C02 was trapped on filter paper (3 MM Horakova et al. (18) isolated a Lactobacillus thought to Whatman, 1 x 3 em rolled cylinders) containing hyamine hydroxide to related to Lactobacillus acidophilus that decarboxy­ (New England Nuclear, Boston). The filter paper trap was suspended over the reaction solution by a paper clip inserted into a No. 2 rubber lated histidine. In 1968, DeKoning (TJ reported that a Downloaded from http://meridian.allenpress.com/jfp/article-pdf/40/4/241/1649644/0362-028x-40_4_241.pdf by guest on 27 September 2021 stopper. After shaking for 30 min in a Dubnoff Metabolic Shaker Lactobacillus was responsible for formation of toxic (Burrell Corp., Pittsburgh) to insure complete liberation and 14 amounts of histamine in a Gouda Cheese. The author entrapment of dissolved C02 , the filter paper was transferred to a indicated that the lactobacilli were an unusual contami­ scintillation vial containing 10 ml of a toluene scintillation fluor (4 g of nant originating in the rennet. 2,5-diphenyloxazola (PPO), 100 mg of P-bis-[2-(5-phenyloxazoyl)] ben­ The objective of the present study was to survey dairy zene (PO POP), and 1000 ml toluene, all Beckman, Fullerton, California) and then counted in a Beckman Model LS-100 C Liquid Scintillation starter cultures and dairy-related microflora for tyrosine Counter. Controls for background correction consisted of reaction and histidine decarobxylase activity. The study was mixtures containing heat inacitvated culture media or acetone-dried conducted because bacterial decarboxylase studies have cells. Reaction rates were reported as nMoles C02 released/min/rug of not included a variety of dairy-type bacteria. It was acetone dried cells, nMoles C02 released/min/ml of skim milk culture, intended that the information would indicate the or nMoles C02 released/minim! of sonicated extract. Manometric assays were carried out on a Gilson Differential potential of dairy starters to contribute to tyramine and Respirometer (Gilson Medical Electronics, Inc., Middleton, Wis.). histamine build-up. Enzyme preparation for the manometric assay consisted of acetone­ dried cells grown in TYEB and resuspended in 0.5 M sodium acetate, MATERIALS AND METHODS pH 5.5. One milliliter of the acetate buffer and 1 ml of the cell suspension were added to the main compartment of the Gilson flask. The 38 dairy-type cultures and the four starter cultures were obtained One side arm of the flask contained 0.5 ml of substrate (0.5 M tyrosine from Miles Laboratories, Chr. Hansens's Laboratory, Kraft Foods, and or histidine in 0.5 M sodium acetate buffer, pH 5.5). Reaction com­ the Universities of Nebraska, Wisconsin, North Carolina State, and ponents were allowed to equilibrate for 10 min at 27 C before the Georgia. Cultures were maintained in broth containing 1% of the substrate was tipped in. Release of C0 were monitored for 60 min and following in distilled water: tryptone, yeast extract, and dextrose 2 re5ults were reported as nMoles C0 released/min/rug of cells. ITYEB). In addition, the TYEB contained 100 mg of tyrosine and 2 histidine/liter. Cultures were activated at 27 C by repeated transfer in RESULTS AND DISCUSSION skim milk or TYEB allowing 24 h between each transfer. Cells grown in TYEB were harvested after 24 h of growth at 27 C, The decarboxylase Data in Tables 1-3 show the ability of the various assays were carried out on the preparations from the cultures grown in bacteria to decarboxylate tyrosine and histidine. The 10% skim milk and in the TYEB medium.