The influence of some starter cultures and GDL on the formation of biogenic amines in dry sausages

The influence of five common starter cultures and glucono-delta-lactone (GDL) on the formation of histamine, tyramine, putrescine, cadaverine, spermine and spermidine in dry sausages was studied. Sausages were manufactured in a pilot plant from two different batches of raw material. No major differences were observed between the startercultures studied in the biogenic amine levels detected duringripening. The lowest levels of histamine were detected in sausages fermented by GDL and Staphylococcae with or without lactic acid bacteria as a starterculture. In pure culture studies performed with a turbidometric method in MRS broth, non-starter lactic acid bacteria isolated from sausages were found to be more sensitive to acidic conditions than the starter strains used in the study. The addition of 2% histidine to MRS broth resulted in a tremendous increase in histamine production (from 1-2 to 6000 ppm). However, in histidine-fortified MRS broth with GDL addition, only 54 ppm of histamine was formed. According to these results, the pH decrease caused by GDL addition decreases histamine formation in dry sausages and in MRS broth. The differences in pH decrease may be one reason for the very varying concentrations of histamine detected in retail dry sausages.


Introduction
Over 30 years ago the beneficial influence ofadded starter cultures was shown to improve and to stabil- ize the quality of dry sausages.NIINIVAARA (1955)  recommended the use of micrococci as a starter culture (resulting in the development of Baktofer- ment 61) and Nurmi (1966) used a combination of Lactobacillus plantarum with micrococcae (resulting in the development of Duploferment 66).Since then, many new starter cultures have been developed.However, lactic acid bacteria are still used in most combinations of dry sausage manufacture.
There are several reports of high levels of bio- genic amines in fermented sausages obtained from retail markets (Rice el al. 1975, Vandekerckhove 1977, Ramantanis 1982, Wortberg and  WOLLER 1982, PECHANEK et al. 1983, Pfannhauser and Pechanek 1984, Bauer et al. 1989,  Tschabrun et al. 1990).Factors such as starter cultures, raw material and pH decrease, which af- fect the formation of biogenic amines during saus- age fermentation, are not yet fully understood.It is therefore difficult to make improvements in saus- age fermentation as long as the critical points still remain largely unascertained.Biogenic amines are basic nitrogenous com- pounds mainly formed by decarboxylation of amino acids or by amination and transamination of aldehydes and ketones (Maga 1978, Pfann- hauser  andPechanek 1984, Askar andTreetow 1986).Some aromatic and heterocyclic amines, such as tyramine, histamine and phenylethylamine, have vasoactive properties making their presence in food a potential public health concern (lenistea 1971, Luthy and Schlatter 1983,   Taylor   1983, Askar and Treptow 1986,  Moneret-Vautrin 1985).The aliphatic diamines, putrescine and cadaverine, as well as the polyamines, spermine and spermidine, occur univer- sally in animals and plants, and at least putrescine and spermidine are found in most bacteria.They are important in the regulation of nucleic acid function and protein synthesis, and probably also in the sta- bilization of membranes (Smith 1981).They also potentiate the effect of histamine (Bjeldanes et al.   1978).
In an earlier study the pH decrease caused by addition of GDL decreased significantly the forma- tion of histamine in minced meat.The lower was the pH on the first day of incubation, the less amines were formed (Maijala et al. 1993).GDL hydrolyzes spontaneously in water to gluconic acid and causes a decrease in pH.It is used in the meat industry for dry sausage manufacture, especially with starter cultures which do not contain lactic acid bacteria (LAB).However, the addition of GDL can sometimes be detrimental to flavour and accelerate the development of rancidity (LOCKE and  Hechelman 1986).The purpose of this work was to study biogenic amine formation in sausages fer- mented by five common starter cultures in Finland.The effect of accelerated pH decrease in dry saus- ages was also studied in the second trial by adding different GDL and glucose combinations to the sausages.

Sausage manufacture
The sausages were prepared from frozen meat originating from one cutter of a commercial meat plant (82 kg pork, 55 pork fat, 69 kg beef and 3% NaCl).The mass was divided into five samples and processed in a Seydelmann cutter (K4l) with addi- tion of sodium nitrite (NaNO2) as a 10% aqueous solution (60 ppm), KNO3 120 ppm, ascorbic acid (0.025%) and spices.
This trial was repeated twice.In the second trial, different levels of addition of GDL (Finnsugar Bio- products, Finland) together with glucose were stud- ied in conjunction with the starter cultures 1-4.Each sample was divided into three subsamples designated a, b and c.No GDL but 0.6% glucose was added to the samples (a) as in the first trial, 0.25% GDL and 0.4% glucose were added to samples (b), and 0.5% GDL and 0.20% glucose to samples (c).The sausages (weight 300 g, diameter 60 mm) were stuffed into fibrous casings and ripened for 2 days at 23°C (93 %rh), 1 day at 21°C (85 %rh) and then for 4 days at 20°C (85 %rh).The remainder of the ripening and storage for up to 7 weeks was at 10°C (70 %rh).The sausages were lightly smoked during days 1-5.

Microbiological and chemical analyses
One sausage from each sausage type (8,1-4) was taken as a sample 6 times during ripening.The cover was cleaned with 70% ethanol and removed aseptically.The whole sausage was cut into small pieces and homogenized mechanically.Thereafter the mass was mixed well and samples were taken for analysis of biogenic amines and microbiological content and for measurement of pH and a w - pH values were measured directly from the samples using an Orion Research Incorporated SA 520 pH/mV meter equipped with a Ross 1M pH electrode no.8163 (Switzerland).After a 2-h adaptation period aw values were obtained at 25°C from a sample of 25-30 g using a Rotronic Hygroskop (Fattore Vitale & Co, Italy).A 10 g sample of mixed sausage was serially diluted with a diluent containing 0.1% peptone and 0.85% NaCl in sterile deionized water.Coliforms were counted on Violet Red Bile Agar (VRB, Orion; ISO 4832), fecal streptococci on Slanetz-Bartley agar (SB, Oxoid, NCFA 68), moulds and yeasts on Malt extract agar (Oxoid) with added chlortetracycline (100 mg/1) and chloramphenicol (100 mg/1), Staphylococcae on blood agar base (BBL) containing 5% defibrinated blood and LAB on de Man, Rogosa and Sharpe agar with sorbic acid (MRS-S, MRS of LAB M with added sorbic acid of Fluka, incubated at 20-22°C for 5 days anaerobically).Biogenic amines were determined using the HPLC method of Eerola et al. (1993).The detection limits were 1 mg/kg for tyramine, histamine, spermine, sper- midine and cadaverine and 2 mg/kg for putrescine.

Histamine production by pure cultures
The influence of low pH achieved with GDL or by the initial addition of lactic acid was studied by a turbidometric method.At the same time the pH values were monitored in similar cultures main- taned in small tubes at 30°C.Histamine was analyzed from tubes by HPLC after 5 days of incuba- tion.Because no major differences were observed between the growth patterns of the four starter cul- tures, one strain (GS-11) isolated from Duploferment 66 (L.plantarum) was selected to represent the starter cultures.Two non-starter lactic acid bacteria (NSLAB) isolated from the sausages during ripening were also used.G-106 produced 930 ppm histamine and 920 ppm tyramine and G-261 produced < 1 ppm histamine and 400 ppm tyramine in fortified MRS broth (Maijala 1993).
LAB strains (preserved at -75°C) were incubated in MRS broth (Oxoid) at 30°C for 24 h. 100 pi of this culture was inoculated into a fresh MRS broth and incubated at 30°C for exactly 24 h.These cultures were used for turbidometric inoculations.The cell densities (cfu/ml) of the cultures were deter- mined by culturing serial dilutions on MRS-S plates.10 pi of a 10" 1 dilution of MRS-broth cul- tures (logio 8 cfu/ml) was added to the wells of Honeycomb plates, each containing 390 pi ofbasic broth.As a basic broth either pure MRS broth, MRS broth with lactic acid (pH 4.7) or MRS broth with GDL (1% or 2%), all with or without L-histidinemonohydrochloride (Merck 4350), were used.Three replicate wells were used both for the samples and for the negative controls.The cuvettes were incubated in a data logging turbidometer incu- D bator (Bioscreen , Labsystems Oy, Finland) at 30°C for 120 h.The filter giving the lowest pos- sible background absorbance values was chosen (600 nm) and the optical density was measured at 30 min intervals.

Results
The levels of biogenic amines increased during ripening, as was reported e.g. by Ramantanis  (1982) (Table 1).In both trials the final levels of tyramine were lowest in the sausages fermented by Baktoferment 61 + GDL.However, the addition of GDL also decreased to some extent the tyramine levels detected in sausages fermented by Duploferment 66 and Flora-Carn SL (Fig. 1).
The influence of the raw material was especially clear on the formation of histamine.In the first trial hardly any histamine was detected but in the second trial the amounts increased up to 108 ppm.The lowest levels of histamine in the second trial were detected in the sausages fermented by Baktofer- ment 61 + GDL and Flora-Cam SL (Table 1).Histamine levels also clearly decreased when GDL was added (Fig. 2).There were no major differ- ences between the starter cultures 1 -4 regarding the levies of other amines during ripening.
In both trials the most rapid pH decrease was in the sausages fermented by Baktoferment 61 + GDL, as expected.The addition of GDL with a smaller amount of glucose also lowered the pH values in the beginning of the fermentation (Table 2).There were no great differences between the a w values of different sausages during ripening.The a w values decreased from 0.98 to 0.90-0.93 (21days) and to 0.78-0.84(49 days) in these trials.
The results of LAB and Staphylococcus in saus- ages fermented with starter cultures 1-4 without GDL addition are presented in Table 3.The levels of coliforms were < log 1.0 except in the raw mater- ial of the second trial (log 1.1).The levels of fecal streptococci (< log 3.1), yeasts (< log. 4.7) and Bacillus spp. (< log 3.6) also remained at the nor- mal level for this type of product.No moulds were detected in any of the samples.The GDL addition had no influence on the bacterial counts measured.
In pure culture studies the NSLAB strains (G-106 and G-261) decreased the pH slowly as compared with the starter strain GS-11.However, the final pH values with all these strains were al- most equal after 5 days of incubation in pure MRS broth (Table 4).GS-11 also grew much better than the NSLAB strains in pure MRS broth (Fig. 3-5).Acidification of MRS broth below pH 5.0 with lactic acid or 1 % GDL increased the lag time and decreased the growth rate of the NSLAB strains but not of GS-11.Furthermore, the addition of 2% GDL with or without histidine to MRS broth prevented the growth of NSLAB during five days of Fig. 1.Tyramine levels detected in sausages fermented with different GDL and glucose combinations.For starter cultures, see Table 2.The results of sausages fermented by B, I and 4 have been presented previously (Maijala and Eerola 1993).Fig. 2. Histamine levels detected in sausages fermented with different GDL and glucose combinations.For starter cultures, see Table 2.The results of sausages fermented by B, I and 4 have been presented previously (Mauala and Eerola 1993).
Table 1.Amounts of biogenic amines during the ripening of dry sausages in two trials (mg/kg).B and the numbers 1-4 refer to the different starter cultures used, see Table 2. (a = 0.6% glucose + no glucono-delta-lactone, n.d.= not detected).

First trial:
Second trial: incubation, whereas GSII showed only a slightly decreased growth curve as compared with the 1% GDL addition.Histamine was not produced by the strains GS-II and G-261 in any of the broth com- positions studied.G-106 produced 1 ppm histamine in pure MRS broth, 2 ppm in pure MRS broth at pH 4.7 and 11 ppm in MRS + GDL.Addition of his- tidine resulted in a great increase of histamine in MRS+H (5964 ppm) and MRS+H (pH 4.7) (6120 ppm).However, in MRS+H broth with GDL addi- tion only 55 ppm of histamine was detected.

Discussion
In this study, histamine was only detected in the second trial, in which the number of LAB was higher than in the first trial.This conforms with the results of Kranner et al. (1991).These authors reported that the formation of undesirably high amounts ofhistamine is dependent on a sufficiently high number of histamine-producing microorganisms and on the presence of free histidine as substrate.Furthermore it is influenced by the storage temperature and by the freshness of the meat used for production.Tschabrun et al. (1990) also found more LAB and total bacteria in dry sausages with  high histamine content than in those with low hista- mine.These authors could reduce the histamine content also by using very fresh meat.Eitenmiller et al. (1978) reported that the use of Pediococcus cerevisiae decreased the formation of tyramine in dry sausages.On the basis of brand- specific studies of retail sausages, Taylor et al.  (1978) also proposed that proper control of natural fermentation could largely prevent histamine for- mation.In the present study there were no great differences between the starter cultures studied concerning amines formed during ripening.How- ever, it was interesting that even if the starter cul- tures used were histamine-and tyramine-negative in vitro, the lowest levels of histamine were found in sausages fermented by Baktoferment 61 + GDL or by the starter cultures containing LAB with GDL addition.
This phenomenon may be explained by the fact that the amine-positive NSLAB strains did not tol- erate acidic conditions as well as the aminenegative starter culture in pure culture studies.This is rea- sonable because one of the most important criteria for the selection of starter strains has been their good lactic acid production capacity coupled with acid tolerance.Because the accelerated pH de- crease caused by GDL addition increased the lag time and decreased the growth rate of NSLAB strains, it is probable that their number will remain lower for a longer time in sausages with GDL addi- tion.This gives the starter culture the opportunity to outgrow the histamine-positive NSLAB strains.Because the main increase in histamine is detected during the first two weeks (Tschabrun et al. 1990)  it is probable that the decrease in the number of e.g.histamine-positive strains during this period de- creases the histamine levels detected in the final product.

Table 3 .
Results of microbiological studies during the ripening of dry sausages (login cfu/g) in two trials.B and the numbers 1-4 refer to the different starter cultures used, see Table2.* = not studied.