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Effects of gluconodeltalactone and Lactobacillus plantarum on the production of histamine and tyramine in fermented sausages
baternuthmalJounud ~fF~nal Micn~hioh~gy. 17 (1~3) 31|3-31~ U~Iq~3 Elsevier Science Publishers B.V. All rights reserved 016S-1605/93/S1~6,110
3113
FOOD I;0546
Effects of gluconodeltalactone and Lactobacillus plantarum on the production of histamine and tyramine in fermented sausages S. Bun~:i~ ,t, Lj. Paunovi~ b, V. Tcodorovi~ ", D. Radi~i~ ~', G. Vojinovi~ c, D. Smiljani~ " a n d M. Balti~ " " Unirersily of Belgrade. |'eterinao' Factdty. I)el~trlment .for Meat Ilygicm" am/Techmdo, k~'. Ih'lgrmh; Yug~ahtt'ia. t, "BEK' ,ih'at hulu.~try. Micnd~iohJ.¢y Laborator)'. Zrt'nittntno Yugoshwhl and ' Ytegoshar ht~titute .for 3h'at Technology. Belgnuh', )'ugoshtt'h,
(Received I June 1992: revision received 20 July IH¢}2:accepted 2h July 1~2)
Fermented sausages were made experimentally with addition of 0.3'; (w/w) glucon~.t¢ltalactone (GDL) or a starter culture of I.actohacilh,s phmtarutn, unable to produce histamine or tyramine. Controls were produced without GDL and starter culture. During 15 days preparation and storage pcriegls, number of bacteria and lactobacilli, pll, and levels of histamine, tyrosin¢ and tyramine were monitored, and organolepti¢ evaluations were carried out. Maximal histamine levels were ItL41 p,g/g of dry matter in the GDl.-added sausages. 18.64 p.g/g in the control, and 17.21| ,ug/g in the starter sausages. Maximal tyraminr levels were 1249.16 p,g/g in lh¢ (;DE sausages, 1101.16 #g/g in the control, and ~)6.35 p.g/g in the st:trier s;.lus;~tges.S~tus;.lges produced with GDL pr{wcd less acceptable ill organoleplic evaluation due to Jill unpleasant s~mr flax'our.
Key words: Fermented sausage: ilistaminu; Tyramine; I.a¢tohacilhes phmtarton; (;luconodel~ak|cton¢
Introduction
Fermented sausage is a product that offers favourablc conditions for the formation of biogcnic amines. The microbial counts and activities arc substantial, the p r o d u c t i o n process is lengthy, r e q u i r i n g higher t e m p e r a t u r e s , a n d a c e r t a i n a m o u n t of protcolysis takes place. It is, therefore, u n d e r s t a n d a b l e why m o r e or less h i s t a m i n e (Rice ct al., 1975; T a y l o r et al., 1978: R a m a n t a n i s et al., 1985; T s c h a b r u n et al., 1990), t y r a m i n e (Rice a n d Koehler, 1976: V a n d e k e r c k h o v c , 1977; E i t e n miller et al., 1978; R a m a n t a n i s et :d., 1985) a n d t r y p t a m i n c ( R a m a n t a n i s et al., 1985) can be f o u n d in this type of sausage. T h e r e are n o r e p o r t e d d a t a in literature o n the a m i n e c o n t e n t s in Yugoslav types of f e r m e n t e d sausages.
(,brrespondence mhiress: Prof Dr Sava Bun~i~,Veterinary. Faculty, Department fi~r Meat ilygicnc and Technology, Bul. JNA 18. Belgrade, Yugoslavia.
304 A classical mode of production of fermented sausages implies lengthy fermentation and drying processes with the help of natural micropopulation. To shorten the process and speed it up, starter cultures of lactic acid bacteria can be added to the sausage mixture, instead of a starter culture, .some add gluconodcltalactonc (GDL) to lower thc pH more rapidly, to stabilize the colour, suppress the microorganisms which cause spoilage, and to simplify the production. It has been reported that the use of lactic acid starter cultures could hinder the formation of biogenic amines in fermented sausages (Rice and Koehler, 1976: Eitenmiller et al., 1978: Taylor et al., 1978). There are no reports of effects of GDL on the formation of biogenic amines. Howcver, rccent investigations (Teodorovi~ et al., 1991) suggested that GDL was likely to stimulate substantially the histamine production by Morganella morganii in a histidine-containing broth. Thus, the objective of the present study was to examine the effect of GDL ea the formation of the most important amines, histamine and tyramine, in fermented sausages, and to evaluate its effect in relation to use of a starter culture.
Materials and Methods
Sausage preparation The composition of sausage mix was the following: 40 kg frozen beef trimmings with 20% of fatty tissue, 40 kg frozen pork leg, 20 kg frozen pork fat, 2.2 kg salt. 30 g ascorbic acid, and 20 g sodium nitrite. All ingredients were ground in a cutter (Kramer Gr';.ibe 173/136-325, Germany) at 14(}0 rpm for 2 min and then at 280{) rpm for another 2 rain. The mix was divided into three portions for three different fermentations. The type A fermentation was prepared with the addition of 3 g of GDL in 10 ml of saline (0.9% NaCI) per kg of sausage mix. To type B was added i0 ml of Lactobacillus plantanlm culture suspended in saline (concentration about 10S/ml) per kg. The suspension was prepared from a 24 h L. plamarum culture in MRS (Merck 10660) broth incubated at 30"C. The strain was first isolated from a fermented sausage and screened for production of histamine and tyramine in broths added histidine and tyrosine, where it was found not to produce these compounds (unpublished results). To type C (control) was added l0 ml of saline per kg of sausage mix. Each sausage mix portion was hand-mixed in plastic containers for 3 rain and stuffed into 50-mm diameter collagenous casings (Koteksproduct, Yugoslavia), which has oxygen permeability of 9415.3 NTP cm3/24 h/bar, and was left to ripen and dry for 15 days, as previously described (Bun~,i~ et al., 1991). Finished sausages were held at room temperature {18-20°C) for an additional 15-day period. Sausage preparations were carried out on two different occasions.
Sampling Each type (A, B, C) was sampled immediately upon stuffing (zero time) and then after 1, 2, 3, 6, 15, and 30 days. Each sample was analyzed in duplicate.
3115
pH
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The pH was measured using a pH meter MA 57115 (lskra, Slovenia) with a combined electrode inserted into the middle of a sausage.
Bacteriological analysis Ten to 15 g samples were taken aseptically from the middle of the sausages, placed in sterile stomacher bags and homogenized lbr 2 min in sterile saline (ratio 1 : 9), using a Stomacher (G-4IM), Iskra, Slovenia). Series of decimal dilutions were prepared and aerobic mesophilic bacteria were determined by plating appropriate dilutions onto plate count agar {Torlak 2295, Yugoslavia) incubated for 48-72 h at 30°C. Lactobacilli were determined on MRS agar plates (Merck 111661})incubated for 48-72 h. at 30°C.
Tyrosbu'. histamhze and tyramhae analysis Tyrosine was determined quantitatively by colorimctry in a sausage extract according to Wt~t)d ct al. (19421. The level of histamine was determined fluorometrically according to Shore 11971) by use of a Spckol-10 spectrophotometer (Karl Zeiss, Germany) equipped with a FK fluorescence ~¢cessory, Tyramine was determined by a thin-layer chromatographic procedure developed by Langner and Kellinghusen (1979) on 0.25 mm silicagel G plates (Merck) and densitometric measurements (Camag, USA).
Organoleptic assessment The sausages were assessed in a ranking test according to ISO-8587 standards {Anonymous, 1988). Each of 7 trained assessors ranked the 3 samples of sausages (A, B, and C), which had been marked by code letters, during the total of 3 repeated tasting sessions. Ranking of the samples was done according to the acceptability of their flavour.
Expression of the results Results arc means of two individual experiments with each sample processed in duplicate.
Results
The number of aerobic mesophilic bacteria for all types of sausages was about 104/g initially, increasing to numbers as high as 10S/g during the ripening period. At the end of storage period, the bacterial counts in all sausages were reduced to about 5 X 105/g (results not included). The initial concentration of lactobacilli was highest in type B sausage with added starter culture (4.2 × 10~'/g). Types A and C were almost equal with significantly lower initial number (6 x 103/g). Finished sausages (day 15) showed
6.1 5.9
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,¢ n ~ - - J - - . , to,-~,.--~ Fig. I. pli and concentrations of histamine in experimental sausages. (,',) Control (('): (~) GDL
sausage(A);(e) starter culture sausage(B).
equal number of lactobacilli for the A and C sausages (6 × 107/g), whereas the number was higher in the B sausages (8 x 10'J/g). Over the subsequent storage period the concentrations of lactobacilli dropped significantly, reaching a similar level in all sausages - about 10~/g (results not included). During the first days of the ripened period, pH tended to drop significantly (Fig. 1). The drop was most pronounced (l{~west level pH 5.32) and most rapid in GDL sausages. The pH in sausages prepared with starter culture dropped to 5.43, and in the control to 5.62. In the second half of the ripening period and during the storage period, the pH rose in all sausages. Formation of histamine, in all three fermentations, was observed throughout the fermentation and storage periods (Fig. 1). in the end of the ripc,ning period (day 15), the histamine contents of the GDL and control sausages appeared to be similar (15.02 and 14.81 /,tg/g, respectively), and slightly lower in starter sausages (12.24 p,g/g). During the storage period, histamine concentration rose to 19.41 /.tg/g (A), 18.64/,tg/g (B) and 17.20/~g/g (C). Increasing levels of tyrosine were observed in the beginning of the ripening period (Fig. 2). After an apparent peak level the content of tyrosine decreased during the storage period with no significant difference between the three types of sausages. The concentration of tyramine kept rising throughout the period of ripening and storage. Only minor differences were observed with final levels after 30 days of 1249.16/.tg/g (A), 1101.71 p.g/g (C), and 906.35/zg/g (B).
307
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'
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.~_
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Fig. 2. Contents of tymsinc and tyraminc in experimental ~ausagcs. ( A ) Control (C): ( ~ ) G D L ~ausagc ( A k (e) starter culture ~ausagc (B}.
in the organoleptic evaluations (results no~ shown) the flavour of sausages with starter (B) was ranked significantly better than tl;c GDL (A) and control sausages (C), when evaluated after 15 days (end of ripening) and after 30 days (end of storage). The sausage prepared with GDL had an unpleasant sour flavour.
Discussion The maximal histamine detected in three experimental fermentations ( < 19 /zg/g) was far below the limits considered toxic. Low histamine levels ( < 25 p,g/g) in a number of fermented sausages were reported by Taylor et al. (1978), Ramantanis et al. (1985), Rice et al. (1975), and Tschabrun et al. (1991)). However, in few occasions high and potentially toxic histamine contents have been recorded, e.g. Vandekerckhove (1977) who found 285.9 ~g of histamine/g of dry matter in 'pure pork' sausage, while Taylor et al. (1978) reported 245/.tg/g in an Italian dry salami and 550 ~ g / g in a pepperoni sausage. The highest histamine level (654 ~ g / g ) was found in an Austrian dry sausage (Tschabrun et al., 1990). Tyramine concentrations in our sausages were much higher than the levels of histamine, and the maximal concentrations observed in all our fermentations, i.e. 1249.16 # g / g (A), 1101.71 p g / g (C) and 906.35 # g / g (B), should be considered very high and potentially toxic. Very high tyramine level was also reported by Vandekerckhove (1977) who found as high as 1506.3 t.tg/g of dry matter in one sample of fermented sausage. Other authors have reported concentrations of
31IS tyramine much lower than ours, e.g. Ramantanis et al. (1985) reported up to 93.24 /zg/g, Ricc and Koehlcr 11976) up to 3110 p,g/g, and Eitenmiller et al. (1978) up to 326 p,g/g in fermented sausages. Formation of very high tyramine concentrations in all three types of our sausages is difficult to explain. This may be due to an extensive proteolysis (i.e. liberating amino acids), which possibly t{x~k place in meat before freezing a n d / o r during the fermentation. A strong tyrosine decarboxylase activity of the natural mieropopulation could also bc involved. However, it is unclear why the concentration of histamine remained much lower than tyramine in the same sausage. It seems unlikely that this phenomenon may be a consequence of higher concentration of tyrosine, which encourage the tyraminc formation (Eitenmiller and Koehler, 1978), than hislidine in the sausage mix because most proteins of muscle and connective tissues contain equal levels of these two amino acids (Partmann and Schlaszus, 1978; Penet et al., 1983; Krilova and Ljaskovskaja, 1968); the single exception is elastin containing 1.5% tyrosine and no histidinc (Krilova and Ljaskovskaja, 1968). Reports of lcnistea (1971) and Yamada 11967) indicated that the final concentrations of histamine and tyramine in foods also depend upon the degradation by microbial diamine oxidase (DAO) and monoamine oxidase (MAO), but we have no information whether histamine degradation took place more extensively than tyramine degradation in our sausages. On the other hand, our results may indicate that the addition of GDL favours fo~,~aiion of tyramine (Fig. 2). This may be duc to GDL hydrolysis which liberates acid which then, in its turn, lowers the pH of the sausages rapidly and to a considerable degree. The lowest pH was thus observed in GDL fermentation. Amino acid dccarboxylase activity is stronger in acidic environment (Rice et ai., 1976; Sinell, 1978). Furthermore, in such an environment, bacteria are encouraged more strongly to produce these enzymes, as a part of their defence mechanisms (Voigt and Eitcnmillcr, 1977). Generally, our investigation revealed that fermented sausages produced in manners which have been often used in local meat industries may be unsafe with respect to tyramine content. Histamine seems to be a minor problem in this type of sausage. Relatively small differences in the content of tyramine between sausages with added GDL or starter and fermented spontaneously indicate that the formation of this compound is influenced also by other factors. Further investigations should be carried out to identify and control them.
References Anonymous11988) Sensoryanalysis- MethodologyRanking. InternationalOrganizationof Standardization ISO, Ref. No. ISO 8587-1988 (E). Bun?:i~. S., Paunovi~. Lj. and Radi~i~, D. 11991) The fate of Listeria momJcytogt,nes in fermented sausages and in vacuum-packagedfrankfurters.J. Food. Prof. 54, 413-417. Eitenmiller, R.R., Koehler, P.E. and Reagan, J.O. 11978) Tyramine in fermented sausage: factors affectingformationof tyramineand tyrosinedecarboxylase.J. Food Set. 43, 689-693.
lenistea, ('. (1~)71) Bacterii, I production and destrvction of hisl;m)im: in fi~ds and l't~l I~)i~)ning c~,uscd by histamine. Die Nahrung 15, IliL}- 113. Krih)v~l. N.N. and Ljasko~,,skaja. I.N. (I~)l')S) Biohimija Mjasa. Pis~:vaja Promislem)st, Mo.sk~..a 513. l.angncr, I l.J. and Kcllinghu.sen, M. ( IL)7~)JDunn~,chichtchromatotzraphischc Trenmmg biogener Amine in Fischkonscrven. spc~,i~:ll Ilislamin. Dic Flcisehcr~:i, 4, 312-314. P:lrtmann. W. and Schlaszus. II. (1~)7~) Freic Aminosauren in Kalb- und Schweinefleiseh naeh Lagerung in koh!endioxidreichen At.n,b,,,pharcn. Fleisch~,.'irlsch. 5g (,~), $43-846. Pcnel. I-.S.. Worthington. R.I-., Philips. R.I). and M~lon, J.N. (1~)83) Free amino acids of raw and cl~ked ground beef alld pork. J. F ' ~ d Sci. 48. 2~,~8-2~)t). Ramantani.s, S., Fashcnder. ('.P. and Wenzel, S, (It)~5) UnlernuchL, ngen zur Biklung wm Ilistamin, Tyramin und "]'D'plamin in Rohwurslem Arch. Lehensmittelhyg. 36, *~- I I. Rice, S.. |:.ilenmiller, R.R. and Koehler, P.l:,. (1'~75) Ilistamine and lyramme ¢onlcnl of meal pmduc!s. ,]. Milk Food Tcchnol. 38, 251%258. Rice, S.I.. and K~,chlcr, P.I-. (1~}70)T.~rosine and histidinc dccarboxylase aclivilics of I'edirw~'('us c'en'ri.siae and I.a('t,,ha~'ill,,s species and tile production ~f t~.,'ramhle in Iermeltted ,'.iaus;.|ges. ,I. Milk Shor~. P.A. (I~)71) Fh.oromelri~' as~a~ of hi,~lamine. Melhod,~ l~nzvmol. 17, ,~42-845. Sitlell, l lJ. (197S) liiogene Amine al.~ Risikofakloren in dcr l:i,~chh.~giene. A[ch, l,el~nsmillelh.~'~. 29, 2lh~-21l). Taylor, S.l... l,cather~o~d, M. and l,iehcr, ILR. (I~)7S) A ~ur~'c~' ~f hi~lami.~e levels in sausatze.~. J, I : ~ I Prol. 41. (,34-~,37.
'l'eodor~wi~, V., Bun~:i~, S. a.)d Smiljani~. D, Slud.,. of some fach~r~, influencing the histamine pr~luclion in meal. I:h:isch~,,'irt.,,ch. (iq press). "rschal~rul), R., Sick, K.. Bauer, l:. a.)d K.'anncr, P. (l~)~|) Bildung ~.on Ilistamin in schniltl'csten Rohwurslen. Fleisehwirlsch. 7ll, 448-451. Vandekerckhove. P. (1~)77) Amines ill d~' fernlellled sausage, J. F~od Sci. 42, 2S3-285. Voigl, M,N. aqd l-itenn'dllcr, R.R. (1~}77) Production of tyrosine a.ld histidine decarhoxylase I~y dairy-rclah:d hacleria. J. Food Prol. 4(l, 241-245. W ~ d . A..I., Sigvrdss~m, (;.J. and Dyer, W,J. (1~}42) J. Fish. Res. Bd. ('an. 6, 53= Ref. D. Pearson, I~"l. "Applic:llion of chemical melh~l.,, fl)r Ihc assessment ()1 heel qualily. II Methods related to prolein breakdown'. J. Sci. I:~u~d Atzric. 19. 3(~fl-3(~). Yamada, I1., Uwajima, T., Kumagai, I1., Willilllilhc, M, ilnl.I Ogal;i, K. ( 1 ~ 7 ) BJ~:Ie[iill monoamin¢ oxydase.s. Part I. Pulificalio.~ and cO'slallizalioq of lyramine oxyda.,,e of Sarcma hHl'~l. Ag,ric. Biol. ('hem. 31. ~I)-F,~)(~.
3113
FOOD I;0546
Effects of gluconodeltalactone and Lactobacillus plantarum on the production of histamine and tyramine in fermented sausages S. Bun~:i~ ,t, Lj. Paunovi~ b, V. Tcodorovi~ ", D. Radi~i~ ~', G. Vojinovi~ c, D. Smiljani~ " a n d M. Balti~ " " Unirersily of Belgrade. |'eterinao' Factdty. I)el~trlment .for Meat Ilygicm" am/Techmdo, k~'. Ih'lgrmh; Yug~ahtt'ia. t, "BEK' ,ih'at hulu.~try. Micnd~iohJ.¢y Laborator)'. Zrt'nittntno Yugoshwhl and ' Ytegoshar ht~titute .for 3h'at Technology. Belgnuh', )'ugoshtt'h,
(Received I June 1992: revision received 20 July IH¢}2:accepted 2h July 1~2)
Fermented sausages were made experimentally with addition of 0.3'; (w/w) glucon~.t¢ltalactone (GDL) or a starter culture of I.actohacilh,s phmtarutn, unable to produce histamine or tyramine. Controls were produced without GDL and starter culture. During 15 days preparation and storage pcriegls, number of bacteria and lactobacilli, pll, and levels of histamine, tyrosin¢ and tyramine were monitored, and organolepti¢ evaluations were carried out. Maximal histamine levels were ItL41 p,g/g of dry matter in the GDl.-added sausages. 18.64 p.g/g in the control, and 17.21| ,ug/g in the starter sausages. Maximal tyraminr levels were 1249.16 p,g/g in lh¢ (;DE sausages, 1101.16 #g/g in the control, and ~)6.35 p.g/g in the st:trier s;.lus;~tges.S~tus;.lges produced with GDL pr{wcd less acceptable ill organoleplic evaluation due to Jill unpleasant s~mr flax'our.
Key words: Fermented sausage: ilistaminu; Tyramine; I.a¢tohacilhes phmtarton; (;luconodel~ak|cton¢
Introduction
Fermented sausage is a product that offers favourablc conditions for the formation of biogcnic amines. The microbial counts and activities arc substantial, the p r o d u c t i o n process is lengthy, r e q u i r i n g higher t e m p e r a t u r e s , a n d a c e r t a i n a m o u n t of protcolysis takes place. It is, therefore, u n d e r s t a n d a b l e why m o r e or less h i s t a m i n e (Rice ct al., 1975; T a y l o r et al., 1978: R a m a n t a n i s et al., 1985; T s c h a b r u n et al., 1990), t y r a m i n e (Rice a n d Koehler, 1976: V a n d e k e r c k h o v c , 1977; E i t e n miller et al., 1978; R a m a n t a n i s et :d., 1985) a n d t r y p t a m i n c ( R a m a n t a n i s et al., 1985) can be f o u n d in this type of sausage. T h e r e are n o r e p o r t e d d a t a in literature o n the a m i n e c o n t e n t s in Yugoslav types of f e r m e n t e d sausages.
(,brrespondence mhiress: Prof Dr Sava Bun~i~,Veterinary. Faculty, Department fi~r Meat ilygicnc and Technology, Bul. JNA 18. Belgrade, Yugoslavia.
304 A classical mode of production of fermented sausages implies lengthy fermentation and drying processes with the help of natural micropopulation. To shorten the process and speed it up, starter cultures of lactic acid bacteria can be added to the sausage mixture, instead of a starter culture, .some add gluconodcltalactonc (GDL) to lower thc pH more rapidly, to stabilize the colour, suppress the microorganisms which cause spoilage, and to simplify the production. It has been reported that the use of lactic acid starter cultures could hinder the formation of biogenic amines in fermented sausages (Rice and Koehler, 1976: Eitenmiller et al., 1978: Taylor et al., 1978). There are no reports of effects of GDL on the formation of biogenic amines. Howcver, rccent investigations (Teodorovi~ et al., 1991) suggested that GDL was likely to stimulate substantially the histamine production by Morganella morganii in a histidine-containing broth. Thus, the objective of the present study was to examine the effect of GDL ea the formation of the most important amines, histamine and tyramine, in fermented sausages, and to evaluate its effect in relation to use of a starter culture.
Materials and Methods
Sausage preparation The composition of sausage mix was the following: 40 kg frozen beef trimmings with 20% of fatty tissue, 40 kg frozen pork leg, 20 kg frozen pork fat, 2.2 kg salt. 30 g ascorbic acid, and 20 g sodium nitrite. All ingredients were ground in a cutter (Kramer Gr';.ibe 173/136-325, Germany) at 14(}0 rpm for 2 min and then at 280{) rpm for another 2 rain. The mix was divided into three portions for three different fermentations. The type A fermentation was prepared with the addition of 3 g of GDL in 10 ml of saline (0.9% NaCI) per kg of sausage mix. To type B was added i0 ml of Lactobacillus plantanlm culture suspended in saline (concentration about 10S/ml) per kg. The suspension was prepared from a 24 h L. plamarum culture in MRS (Merck 10660) broth incubated at 30"C. The strain was first isolated from a fermented sausage and screened for production of histamine and tyramine in broths added histidine and tyrosine, where it was found not to produce these compounds (unpublished results). To type C (control) was added l0 ml of saline per kg of sausage mix. Each sausage mix portion was hand-mixed in plastic containers for 3 rain and stuffed into 50-mm diameter collagenous casings (Koteksproduct, Yugoslavia), which has oxygen permeability of 9415.3 NTP cm3/24 h/bar, and was left to ripen and dry for 15 days, as previously described (Bun~,i~ et al., 1991). Finished sausages were held at room temperature {18-20°C) for an additional 15-day period. Sausage preparations were carried out on two different occasions.
Sampling Each type (A, B, C) was sampled immediately upon stuffing (zero time) and then after 1, 2, 3, 6, 15, and 30 days. Each sample was analyzed in duplicate.
3115
pH
#tletlsllrettlOllS
The pH was measured using a pH meter MA 57115 (lskra, Slovenia) with a combined electrode inserted into the middle of a sausage.
Bacteriological analysis Ten to 15 g samples were taken aseptically from the middle of the sausages, placed in sterile stomacher bags and homogenized lbr 2 min in sterile saline (ratio 1 : 9), using a Stomacher (G-4IM), Iskra, Slovenia). Series of decimal dilutions were prepared and aerobic mesophilic bacteria were determined by plating appropriate dilutions onto plate count agar {Torlak 2295, Yugoslavia) incubated for 48-72 h at 30°C. Lactobacilli were determined on MRS agar plates (Merck 111661})incubated for 48-72 h. at 30°C.
Tyrosbu'. histamhze and tyramhae analysis Tyrosine was determined quantitatively by colorimctry in a sausage extract according to Wt~t)d ct al. (19421. The level of histamine was determined fluorometrically according to Shore 11971) by use of a Spckol-10 spectrophotometer (Karl Zeiss, Germany) equipped with a FK fluorescence ~¢cessory, Tyramine was determined by a thin-layer chromatographic procedure developed by Langner and Kellinghusen (1979) on 0.25 mm silicagel G plates (Merck) and densitometric measurements (Camag, USA).
Organoleptic assessment The sausages were assessed in a ranking test according to ISO-8587 standards {Anonymous, 1988). Each of 7 trained assessors ranked the 3 samples of sausages (A, B, and C), which had been marked by code letters, during the total of 3 repeated tasting sessions. Ranking of the samples was done according to the acceptability of their flavour.
Expression of the results Results arc means of two individual experiments with each sample processed in duplicate.
Results
The number of aerobic mesophilic bacteria for all types of sausages was about 104/g initially, increasing to numbers as high as 10S/g during the ripening period. At the end of storage period, the bacterial counts in all sausages were reduced to about 5 X 105/g (results not included). The initial concentration of lactobacilli was highest in type B sausage with added starter culture (4.2 × 10~'/g). Types A and C were almost equal with significantly lower initial number (6 x 103/g). Finished sausages (day 15) showed
6.1 5.9
:g
5.
2O
.~_
0
5
L r .1£pen
10
i
1,5
i
20
i
2,5
30"
DoY-el
,¢ n ~ - - J - - . , to,-~,.--~ Fig. I. pli and concentrations of histamine in experimental sausages. (,',) Control (('): (~) GDL
sausage(A);(e) starter culture sausage(B).
equal number of lactobacilli for the A and C sausages (6 × 107/g), whereas the number was higher in the B sausages (8 x 10'J/g). Over the subsequent storage period the concentrations of lactobacilli dropped significantly, reaching a similar level in all sausages - about 10~/g (results not included). During the first days of the ripened period, pH tended to drop significantly (Fig. 1). The drop was most pronounced (l{~west level pH 5.32) and most rapid in GDL sausages. The pH in sausages prepared with starter culture dropped to 5.43, and in the control to 5.62. In the second half of the ripening period and during the storage period, the pH rose in all sausages. Formation of histamine, in all three fermentations, was observed throughout the fermentation and storage periods (Fig. 1). in the end of the ripc,ning period (day 15), the histamine contents of the GDL and control sausages appeared to be similar (15.02 and 14.81 /,tg/g, respectively), and slightly lower in starter sausages (12.24 p,g/g). During the storage period, histamine concentration rose to 19.41 /.tg/g (A), 18.64/,tg/g (B) and 17.20/~g/g (C). Increasing levels of tyrosine were observed in the beginning of the ripening period (Fig. 2). After an apparent peak level the content of tyrosine decreased during the storage period with no significant difference between the three types of sausages. The concentration of tyramine kept rising throughout the period of ripening and storage. Only minor differences were observed with final levels after 30 days of 1249.16/.tg/g (A), 1101.71 p.g/g (C), and 906.35/zg/g (B).
307
.¢.,
'
'
/
[1500
A
.~_
Lr l p ~ n
O~/S
Fig. 2. Contents of tymsinc and tyraminc in experimental ~ausagcs. ( A ) Control (C): ( ~ ) G D L ~ausagc ( A k (e) starter culture ~ausagc (B}.
in the organoleptic evaluations (results no~ shown) the flavour of sausages with starter (B) was ranked significantly better than tl;c GDL (A) and control sausages (C), when evaluated after 15 days (end of ripening) and after 30 days (end of storage). The sausage prepared with GDL had an unpleasant sour flavour.
Discussion The maximal histamine detected in three experimental fermentations ( < 19 /zg/g) was far below the limits considered toxic. Low histamine levels ( < 25 p,g/g) in a number of fermented sausages were reported by Taylor et al. (1978), Ramantanis et al. (1985), Rice et al. (1975), and Tschabrun et al. (1991)). However, in few occasions high and potentially toxic histamine contents have been recorded, e.g. Vandekerckhove (1977) who found 285.9 ~g of histamine/g of dry matter in 'pure pork' sausage, while Taylor et al. (1978) reported 245/.tg/g in an Italian dry salami and 550 ~ g / g in a pepperoni sausage. The highest histamine level (654 ~ g / g ) was found in an Austrian dry sausage (Tschabrun et al., 1990). Tyramine concentrations in our sausages were much higher than the levels of histamine, and the maximal concentrations observed in all our fermentations, i.e. 1249.16 # g / g (A), 1101.71 p g / g (C) and 906.35 # g / g (B), should be considered very high and potentially toxic. Very high tyramine level was also reported by Vandekerckhove (1977) who found as high as 1506.3 t.tg/g of dry matter in one sample of fermented sausage. Other authors have reported concentrations of
31IS tyramine much lower than ours, e.g. Ramantanis et al. (1985) reported up to 93.24 /zg/g, Ricc and Koehlcr 11976) up to 3110 p,g/g, and Eitenmiller et al. (1978) up to 326 p,g/g in fermented sausages. Formation of very high tyramine concentrations in all three types of our sausages is difficult to explain. This may be due to an extensive proteolysis (i.e. liberating amino acids), which possibly t{x~k place in meat before freezing a n d / o r during the fermentation. A strong tyrosine decarboxylase activity of the natural mieropopulation could also bc involved. However, it is unclear why the concentration of histamine remained much lower than tyramine in the same sausage. It seems unlikely that this phenomenon may be a consequence of higher concentration of tyrosine, which encourage the tyraminc formation (Eitenmiller and Koehler, 1978), than hislidine in the sausage mix because most proteins of muscle and connective tissues contain equal levels of these two amino acids (Partmann and Schlaszus, 1978; Penet et al., 1983; Krilova and Ljaskovskaja, 1968); the single exception is elastin containing 1.5% tyrosine and no histidinc (Krilova and Ljaskovskaja, 1968). Reports of lcnistea (1971) and Yamada 11967) indicated that the final concentrations of histamine and tyramine in foods also depend upon the degradation by microbial diamine oxidase (DAO) and monoamine oxidase (MAO), but we have no information whether histamine degradation took place more extensively than tyramine degradation in our sausages. On the other hand, our results may indicate that the addition of GDL favours fo~,~aiion of tyramine (Fig. 2). This may be duc to GDL hydrolysis which liberates acid which then, in its turn, lowers the pH of the sausages rapidly and to a considerable degree. The lowest pH was thus observed in GDL fermentation. Amino acid dccarboxylase activity is stronger in acidic environment (Rice et ai., 1976; Sinell, 1978). Furthermore, in such an environment, bacteria are encouraged more strongly to produce these enzymes, as a part of their defence mechanisms (Voigt and Eitcnmillcr, 1977). Generally, our investigation revealed that fermented sausages produced in manners which have been often used in local meat industries may be unsafe with respect to tyramine content. Histamine seems to be a minor problem in this type of sausage. Relatively small differences in the content of tyramine between sausages with added GDL or starter and fermented spontaneously indicate that the formation of this compound is influenced also by other factors. Further investigations should be carried out to identify and control them.
References Anonymous11988) Sensoryanalysis- MethodologyRanking. InternationalOrganizationof Standardization ISO, Ref. No. ISO 8587-1988 (E). Bun?:i~. S., Paunovi~. Lj. and Radi~i~, D. 11991) The fate of Listeria momJcytogt,nes in fermented sausages and in vacuum-packagedfrankfurters.J. Food. Prof. 54, 413-417. Eitenmiller, R.R., Koehler, P.E. and Reagan, J.O. 11978) Tyramine in fermented sausage: factors affectingformationof tyramineand tyrosinedecarboxylase.J. Food Set. 43, 689-693.
lenistea, ('. (1~)71) Bacterii, I production and destrvction of hisl;m)im: in fi~ds and l't~l I~)i~)ning c~,uscd by histamine. Die Nahrung 15, IliL}- 113. Krih)v~l. N.N. and Ljasko~,,skaja. I.N. (I~)l')S) Biohimija Mjasa. Pis~:vaja Promislem)st, Mo.sk~..a 513. l.angncr, I l.J. and Kcllinghu.sen, M. ( IL)7~)JDunn~,chichtchromatotzraphischc Trenmmg biogener Amine in Fischkonscrven. spc~,i~:ll Ilislamin. Dic Flcisehcr~:i, 4, 312-314. P:lrtmann. W. and Schlaszus. II. (1~)7~) Freic Aminosauren in Kalb- und Schweinefleiseh naeh Lagerung in koh!endioxidreichen At.n,b,,,pharcn. Fleisch~,.'irlsch. 5g (,~), $43-846. Pcnel. I-.S.. Worthington. R.I-., Philips. R.I). and M~lon, J.N. (1~)83) Free amino acids of raw and cl~ked ground beef alld pork. J. F ' ~ d Sci. 48. 2~,~8-2~)t). Ramantani.s, S., Fashcnder. ('.P. and Wenzel, S, (It)~5) UnlernuchL, ngen zur Biklung wm Ilistamin, Tyramin und "]'D'plamin in Rohwurslem Arch. Lehensmittelhyg. 36, *~- I I. Rice, S.. |:.ilenmiller, R.R. and Koehler, P.l:,. (1'~75) Ilistamine and lyramme ¢onlcnl of meal pmduc!s. ,]. Milk Food Tcchnol. 38, 251%258. Rice, S.I.. and K~,chlcr, P.I-. (1~}70)T.~rosine and histidinc dccarboxylase aclivilics of I'edirw~'('us c'en'ri.siae and I.a('t,,ha~'ill,,s species and tile production ~f t~.,'ramhle in Iermeltted ,'.iaus;.|ges. ,I. Milk Shor~. P.A. (I~)71) Fh.oromelri~' as~a~ of hi,~lamine. Melhod,~ l~nzvmol. 17, ,~42-845. Sitlell, l lJ. (197S) liiogene Amine al.~ Risikofakloren in dcr l:i,~chh.~giene. A[ch, l,el~nsmillelh.~'~. 29, 2lh~-21l). Taylor, S.l... l,cather~o~d, M. and l,iehcr, ILR. (I~)7S) A ~ur~'c~' ~f hi~lami.~e levels in sausatze.~. J, I : ~ I Prol. 41. (,34-~,37.
'l'eodor~wi~, V., Bun~:i~, S. a.)d Smiljani~. D, Slud.,. of some fach~r~, influencing the histamine pr~luclion in meal. I:h:isch~,,'irt.,,ch. (iq press). "rschal~rul), R., Sick, K.. Bauer, l:. a.)d K.'anncr, P. (l~)~|) Bildung ~.on Ilistamin in schniltl'csten Rohwurslen. Fleisehwirlsch. 7ll, 448-451. Vandekerckhove. P. (1~)77) Amines ill d~' fernlellled sausage, J. F~od Sci. 42, 2S3-285. Voigl, M,N. aqd l-itenn'dllcr, R.R. (1~}77) Production of tyrosine a.ld histidine decarhoxylase I~y dairy-rclah:d hacleria. J. Food Prol. 4(l, 241-245. W ~ d . A..I., Sigvrdss~m, (;.J. and Dyer, W,J. (1~}42) J. Fish. Res. Bd. ('an. 6, 53= Ref. D. Pearson, I~"l. "Applic:llion of chemical melh~l.,, fl)r Ihc assessment ()1 heel qualily. II Methods related to prolein breakdown'. J. Sci. I:~u~d Atzric. 19. 3(~fl-3(~). Yamada, I1., Uwajima, T., Kumagai, I1., Willilllilhc, M, ilnl.I Ogal;i, K. ( 1 ~ 7 ) BJ~:Ie[iill monoamin¢ oxydase.s. Part I. Pulificalio.~ and cO'slallizalioq of lyramine oxyda.,,e of Sarcma hHl'~l. Ag,ric. Biol. ('hem. 31. ~I)-F,~)(~.