Lipolytic Starter Culture Effects on Production of Free Fatty Acids in Fermented Sausages

Fermented sausages, inoculated with lipolytic and non lipolytic starter cultures and a control containing glucono-δ-lactone (GDL) and an antibiotic mixture were prepared. The sausages were analyzed on the basis of color, pH, water activity, weight
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  538  JOURNAL OF FOOD SCIENCE —Volume 63, No. 3, 1998 MICROBIOLOGY  Lipolytic Starter Culture Effects onProduction of Free Fatty Acidsin Fermented Sausages P.M. KENNEALLY, G. SCHWARZ, N.G. FRANSEN, and E.K. ARENDT  All authors are affiliated with the Dept. of Food Technology, National Food Biotech-nology Centre, Univ. College Cork, Cork, Ireland. Address inquiries to Dr E.K. Arendt.  ABSTRACT Fermented sausages, inoculated with lipolytic and non lipoly-tic starter cultures and a control containing glucono-  -lac-tone (GDL) and an antibiotic mixture were prepared. The sau-sages were analyzed on the basis of color, pH, water activity,weight loss, moisture and fat contents. Microbiological analy-ses were performed to measure total viable counts, lactoba-cilli and, staphylococci and micrococci. The increase in totaland individual free fatty acids, and lipid oxidation was moni-tored. Results showed no difference (p  0.05) in the level offree fatty acids between treatments, indicating that lipolysiswas most likely due to endogenous meat enzymes.Key Words: fermented meats, lipolysis, endogenous en-zymes INTRODUCTION S AUSAGE   MANUFACTURE   IS   CONSIDERED   TO   OCCUR   IN   THREE phases: formulation (mixing of ingredients), fermentation, and ripen-ing (Garcia et al., 1992; Fernandez et al., 1995a). Lipid forms animportant fraction in fermented sausages (Demeyer et al., 1974; Actonand Dick, 1976; Chasco et al., 1993), and the distinctive flavor wasfound to be related to hydrolytic changes in the lipid fraction duringripening (Roca and Incze, 1990). Other flavor components are derivedfrom carbohydrates, lipid oxidation, proteolysis, seasonings and curingsalts (Verplaetse, 1994). Among flavor compounds reported in dryfermented sausages are aldehydes, ketones and esters (Johansson et al.,1994). Traditionally lipolysis has been mainly attributed to bacteriallipase activity (Demeyer et al., 1974; Palumbo and Smith, 1977; Rocaand Incze, 1990) although meat enzymes may also have an effect (Wallach,1968; Dobbertin et al., 1975; Garcia et al., 1992). Oxidative changes of unsaturated fatty acids resulting in the production of lipid peroxides andcarbonyl compounds have been related to both chemical reactions andbacterial metabolism (Cerise et al., 1973, Demeyer et al., 1974).Most dry sausages are produced by adding starter cultures mainlycomposed of lactic acid bacteria (LAB) and either staphylococci ormicrococci (Hammes and Knauf, 1994). The LAB are the essentialagents of fermentation, as the inherent stability of the product is pri-marily dependent on controlled conversion of sugars into acid (Bacus,1984). They are responsible for the pH drop, which in turn can inhibitgrowth of spoilage and pathogenic microorganisms (Vignolo et al.,1989). This pH drop is also responsible for gelation of proteins whichcauses the product to become sliceable, and gives the product itscharacteristic texture (Buckenhüskes, 1990). However the physiolog-ical properties of lactic starters may not ensure a sensory quality of traditionally produced dry fermented sausages. It is generally accept-ed that this is because LAB have very limited lipolytic ability (Stad-houders and Veringa, 1973; El Soda et al., 1986) and this pertainsmainly to low molecular weight fatty acid triglycerides such as tribu-tyrin and tricaproin. Thus, staphylococci and micrococci are used incombination with LAB due to their ability to contribute to flavormainly through lipolytic activities, and also due to their abilities toreduce nitrate and produce catalase (Lücke, 1986; Nychas and Ark-oudelos, 1990). Previous works have shown that tissue lipases wereimportant in lipolysis (Talon et al., 1992; Montel et al., 1993), and thatstaphylococci have little lipolytic activities under conditions normallyfound in sausages (Sorensen and Jakobsen, 1996). Thus, endogenousmeat enzymes probably have a major effect in the lipolytic process infermented sausages as one study suggested that lipolysis in a ferment-ed Belgian sausage was almost exclusively brought about by the en-zymes present in muscle and fat tissue (Molly et al., 1996).Our objective was to investigate the influence of lipolytic startercultures and endogenous meat enzymes on the production of free fattyacids in fermented sausages. MATERIALS & METHODS Materials Two trials were performed, in which 5 batches of fermented sau-sages with different starter strains were prepared (Table 1). The initialsausage mixture contained (%w/w) beef (45), pork (25), pork back fat(30). Other ingredients were added (g/kg) as follows, NaCl (28.0),NaNO 2  (0.14), sodium ascorbate (0.5), sodium glutamate (1.0), whitepepper (2.5), pepper coarse/shredded (1.0), cardamom (0.5), clove(0.2), caraway seed (0.2), glucose (3.0), and lactose (5.0). Ingredientswere mixed in a bowl chopper and appropriate starter cultures wereadded during mixing. Staphylococcus xylosus  (7) (STX7), and  Micro-coccus varians  (13) (MCV13) were lipolytic strains, and Staphylo-coccus carnosus  M17 and  Micrococcus varians  (4) (MCV4) werenon lipolytic strains, determined by Kenneally et al., (1997). Themixture was then stuffed into 50 mm diameter R2 casings (Naturin,Germany) using a piston filler. Sausages were placed in a ripeningchamber (Ness & Co GmbH, D 73626 Remshalden, Germany)equipped with a NESS digitronic rf 3 d-n process control system,under the following conditions of temperature and relative humidity(R.H.): day 0 to 3; 24  C and 88–92% RH, day 3 to 8; 18  C and 86–92% RH, day 8 to 10; 18  C and 84–90% RH, day 10 to 15, 18  C and82–88% RH, and day 15 to 18; 18  C and 78–84% R.H. The sausageswere smoked after 3 days, 5 days, and again after 8 days in a Sümannsmoking chamber (15–20  C) for 30 min. After reaching 30% weightloss, the sausages were removed from the ripening chamber, vacuumpackaged and stored at 13  C. Table1—Experimental design of salami trials-Inoculation details ofthe different sausages SampleStarter AddedGDL a Ab a STX7 c Lb. sake LS-25/  S. xylosus   (7) b 0   MCV13Lb. sake LS-25/  M. varians (13)0   MCV14Lb. sake LS-25/  M. varians (4)0   M17Lb. sake LS-25/  S. carnosus M17  0   ContNone1.1%   a GDL  % Glucono-  -Lactone added; AB  Antibiotic mixture added (  ) or not(  )(20,000 U.IPenicillin, 20 mg Streptomycin and 50 mg Amphotericin/kg of sausage). b LS-25 has now been renamed LAD. c Strains S. xylosus   (7) and M. varians   (13) are lipolytic starters. Strains S. carnosus    M17 and M. varians   (4) are non lipolytic starters.  Volume 63, No. 3, 1998 —   JOURNAL OF FOOD SCIENCE 539 Sampling Five sausages from each batch were kept separate and used tomeasure the average weight loss for each batch during fermentationand ripening. On sampling days, a whole sausage from each batchwas transferred to the laboratory and two 20g slices were takenaseptically for microbiological analyses from opposite ends of thesausage. For color, pH and water activity measurement a 40g slicewas taken, along with two more 15g slices for analyzing lipid oxida-tion. From the remainder the casing was removed and it was homog-enized for 2 min in a blender (Krups Rotary 500, Solingen, Germa-ny) at full speed, to give a homogenous mass. A sample (10g) wasthen taken to measure the level of total free fatty acids by the coppersoaps method. The remainder was then vacuum packaged and storedfrozen at  18  C for further use. For analysis of free fatty acids(FFA) by Gas Chromatography (GC), and for moisture and fat anal-ysis, the appropriate weight of sample was cut from the frozen ho-mogenous mass and thereafter vacuum packaged and allowed tothaw at 4 ° C overnight. A whole sausage from each batch was usedfor sensory evaluation. Microbiological analyses Sausage samples (10g) were weighed into a stomacher bag underaseptic conditions and were diluted in 90 mL of 2% sterile bufferedpeptone water (Oxoid, Basingstoke, Hampshire, England). The sam-ples were homogenized in a Laboratory Blender, Stomacher 400(Seward, London, UK) for 2 min. Ten fold dilution series were pre-pared using sterile ˘ strength ringers (Merck, Darmstadt, Germany).Appropriate dilutions were spread plated on PM medium, consistingof 1% tryptone (Difco Laboratories, Detroit ,USA), 0.5% yeast ex-tract (Difco), 0.5% NaCl (Merck), 0.1% glucose (BDH Laboratorysupplies, Poole, England) and 1.5% agar (Difco), to determine sta-phylococci and micrococci counts. They were differentiated from oth-er strains on the basis of colony morphology. Appropriate dilutionswere also spread plated on MRS agar (Oxoid) to determine lactobacil-li counts. To determine total viable counts appropriate dilutions werepour plated with Plate Count Agar (PCA) (Oxoid). All plates wereincubated at 30  C, with PM agar and MRS agar being incubated for48h and PCA for 72h. Tests were carried out in duplicate on twodifferent samples.  Weight loss, color and pH The decrease in weight of 5 designated sausages in each batch wasmeasured (Mettler Toledo 16000, Greifensee-Zurich, Switzerland)and weight loss was calculated on a percentage basis. Color measure-ments were carried out using a Chroma-meter CR 300 (Minolta, Os-aka, Japan) to measure CIE L*a*b* values, (L*   whiteness, a*  redness, b*   yellowness). Color was analyzed at 5 points on thecentral part of the cut surface of the sausage sample. pH measure-ments were carried out using a pH 320 meter, (WTW, 82362 Weil-heim, Germany) fitted with an Ingold LoT 406-M6-DXK-S7/25 pHprobe and a WTW TFK 15/E temperature probe. pH was measured at3 different points within the sausage sample.  Water activity, lipid oxidation and free fatty acids Water activity measurements were carried out using an Aqua LabCX-2 water activity meter (Decagon Devices Inc., Pullman, WA).Water activity was measured in 3 slices (5g each) of the sausagesample. Lipid oxidation (TBA reactive substances) was measured ontwo slices (10g each) of the sausage by the 2-thiobarbaturic acidmethod of Ke et al.(1977) and expressed as mg malondialdehyde/kgfat.   For free fatty acids, samples (10g) were homogenized with 90 mLof distilled water for 3 min using an Ultra Turrax T 25 homogenizer(Janke and Kunkel, Staufen, Germany) at 24000 rpm. This mixturewas used to determine the total free fatty acids by the copper soapsmethod (Leuschner et al., 1997). Total free fatty acids (FFA) wereexpressed as mg FFA/g fat. Total and individual free fatty acids Free fatty acids were measured by Gas Liquid Chromatography(GLC) according to a modified method of that employed by Martinez-Castro et al. (1986). Homogenized sausage samples (5g) were groundwith a mortar and pestle along with 5 mL of 11N H 2 SO 4  and 0.8 mLof distilled water until a paste resulted. The paste was transferred to acentrifuge tube along with 10 mL of diethyl ether (BDH). The mixturewas shaken on a reciprocal shaker (IKA - Schüttler MTS 4, Janke andKunkel, Staufen, Germany) at 400 rpm for 10 min. The tubes wereallowed to settle another 10 min. Subsequently 3 mL of the ether layerwas transferred to a glass screw cap test tube and to this, 0.2 mL of 20% tetramethyl ammonium hydroxide (TMAH), (Sigma ChemicalCo., Missouri, USA), in methanol (Alkem Chemicals Ltd, Dublin,Ireland), 0.5 mL of absolute alcohol (B.P Chemicals Ltd., Dublin,Ireland) and 1 mL of distilled water, were added. The test tubes wereshaken on a reciprocal shaker for 1–2 min at 400 rpm, and the contentsallowed to settle for 10 min. The top layer contained the fatty acidmethyl esters derived from esterified fatty acids (glycerides), whilethe bottom layer contained the TMAH soaps of free fatty acids. Thelayers were separated, and to 0.5 mL of the TMAH soap layer, 0.5 mLof a solution of 1 mg/mL pentadecanoic acid methyl ester (Sigma) inabsolute alcohol (B.P Chemicals, Ltd.) was added as internal stan-dard. The free fatty acids were separated and quantified by GLC usinga Shimadzu GC-14A GLC equipped with an SGE BP21 column. Theoven temperature was set at 50  C and raised at a rate of 10  C/ min to200  C and held at that temperature for 17 min. The temperature wasthen raised at a rate of 9  C/min to a final temperature of 227  C and heldat that temperature for 15 min. Injector and detector temperatures wereset at 250  C. FFA’s were identified by comparison of retention timeswith those of authentic standards. Peak areas were calculated using aWaters “millenium” integration software package and the concentra-tion of the FFA’s were calculated as mg/g of fat. Moisture and fat Samples were prepared as described. Moisture and fat were mea-sured according to AOAC methods (AOAC, 1995a, b). Two rectan-gular pads and one circular pad were tared in the CEM AVC 80machine (CEM Corporation, NC) and 3 to 5g of sample were spreadbetween the two rectangular pads. The sample and three pads were putin the microwave oven section and the moisture was measured bydrying at 90% power for 4 min. The pads and sample after dryingwere transferred to the solvent extraction section of the CEM AVC 80system and the fat extracted. The pads were redried in the microwaveoven section at 100% power for 1 min. The pads minus the fat andmoisture were reweighed automatically, and the fat % was displayed.Fat and moisture measurements were carried out on two samples fromeach sausage. Sensory evaluation The aim of the sensory evaluation was to determine whether addi-tion of a lipolytic starter culture would affect the sensorial quality,according to the method of Kramer et al. (1974).Sensory measurements determined were, taste, appearance, over-all acceptability and presence of rancidity. Measurements were carriedout on four different samples, each containing a different starter cul-ture (lipolytic staphylococci (STX7), lipolytic micrococci (MCV13),non lipolytic staphylococci (M17) and non lipolytic micrococci(MCV4)). Control sausages were not evaluated due to the presence of antibiotics. Each sample was tested on one occasion (after 25 days)for each batch, once per subject for each treatment.The method employed was a preference test, using a rank prefer-ence design. Nine untrained subjects recruited from the laboratorywere used for the experiment, which was carried out in a sensoryevaluation room with separate compartments for each subject, undernormal environmental conditions using 100w artificial lighting. Sam-ples were sliced to 4 mm thickness (diameter 50 mm) and held in a  540  JOURNAL OF FOOD SCIENCE —Volume 63, No. 3, 1998  Free Fatty Acids in Fermented Sausages . . . plastic container covered with cling film, at room temperature. All 4samples were then placed on a paper plate, with code numbers as-signed to each sample (STX7  34, MCV13  35, MCV4  36,M17  37), and held at room temperature. Subjects were allowed toevaluate samples in the order of their choice. Panelists were asked torank the samples, on the basis of taste, appearance and overall accept-ability, in order of preference on a scale of 1–4 (1  best, 4  worst).Panelists were also asked to determine if rancidity was present in anysamples, on a scale of 0–4 (0  no rancidity, 4  extreme rancidity).Water was provided for subjects to rinse their mouths between samples.Data were collated by hand and input into an Excel 5.0 worksheetand thereafter examined for significance using Friedmans Two-WayAnalysis of Variance (in the case of taste, appearance and overall accept-ability) or Repeated measures ANOVA ( in the case of rancidity). Statistical methods All results, except for results of sensory evaluation, measured on anumber of days were analyzed by Analysis of Variance, using a splitplot design with two trials (SPSS 7.5.1s, Norussis, 1993). In thisdesign each trial was divided into 5 main plots, with the 5 treatmentsrandomly assigned to these main plots. Subsequently, each main plotwas divided into a number of smaller plots (i.e split plots), dependingon the number of sampling times (days) for a specific parameter. Thedifferent days were then randomly assigned to these split plots (Meadet al., 1993). Results of sensory evaluation were analyzed by Fried-mans Two-Way Analysis of Variance in the case of taste, appearanceand overall acceptability. The occurrence of rancidity was analyzed byrepeated measures ANOVA. RESULTS & DISCUSSION Microbiological analyses All results shown are the averages of counts of the two trials.Staphylococci and micrococci counts on PM agar (Fig. 1) indicatedthat bacterial counts started at an initial level of 10 7 CFU/g sausage inall samples except the control where the initial level was ca. 10 4  CFU/ g. In the case of  Micrococcus varians  (13) and (4) and Staphylococ-cus carnosus  M17 a slight decrease in bacterial numbers over 35 dayswas observed, which corresponds with previous reports (Garcia etal., 1992). The numbers of staphylococci/micrococci in the controlsausage remained almost constant over 35 days   due to the added   anti-biotic mixture. Staphylococcus xylosus (7) counts had reached a levelof  10 6  CFU/g after 2 days, and after 7 days it decreased to  5  10 4 . A final level of 5   10 3  CFU/g was detected after 35 daysshowing that the rate of decline of this starter culture under sausageconditions was greater than any of the other strains used.Lactobacilli counts on MRS agar clearly showed that all inoculatedsausages started at5   10 7  CFU/g and increased rapidly after 24h up to 10 8 –10 9 CFU/g and counts remained almost constant up to day 35 (Fig. 2). Theinitial lactobacilli counts found in the uninoculated control were  10 4 CFU/g and within 24h had decreased to a  10 3  CFU/g after whichnumbers increased slightly, giving a level of  10 4  CFU/g after 2days. Thereafter numbers increased further up to a final level of  10 5 CFU/g after 35 days. Overall however, there was very little increasein numbers over the duration of the trial, due to the presence of antibi-otics. Total aerobic counts on PCA seemed to follow the same trend aslactobacilli counts on MRS agar (Results not shown) pH determination Results of pH measurement are averages of two independent trials.The pH of all inoculated sausages started at an initial pH of 5.75,thereafter it decreased rapidly to 5.1 after 24h. After 2 days pH 5.0was reached and after 7 days a further decrease to pH 4.9 was ob-served. From day 7 a slight increase in pH was observed, which couldbe a result of the production of ammonia and other basic compoundsarising from proteolytic activity. Due to the addition of GDL in thecontrol sausage the pH decreased rapidly, reaching 4.8 after 24h,thereafter increasing slightly up to 4.9. The pH increase in controlsausages could also be due to increased proteolysis resulting fromlysis of microbial cells, which in turn could lead to increased ammoniaformation. Statistical evaluation showed significant treatment   dayeffects (p  0.001), and day effects (p   0.001). Moisture content Results of moisture content are averages of two independent trialsand showed that the initial moisture content of all sausages was be-tween 50 and 54%. The four sausages inoculated with starter culturesfollowed similar patterns of moisture loss and all reached a final levelof ca. 31–32% moisture after 21 days. The control sausage showedthe lowest level of moisture % on almost every sampling day andreached a final level of 30% moisture. This different pattern could beexplained by the more rapid pH drop in the control which resulted ina faster gel formation, followed by syneresis, and therefore faster Fig. 1—Staphylococci/micrococci counts in salamis over time.—  — STX7; —  — MCV13; —  — MCV4; —  — M17; —  — Control.Fig. 2—Lactobacilli counts in salamis over time. Symbols, same asFig. 1.  Volume 63, No. 3, 1998 —   JOURNAL OF FOOD SCIENCE 541 weight loss. No significant difference was found between treatments(p  0.05), nor did treatment alter the day effect. Fat content, weight loss, water activity and color  Fat content increased over the period of the trials, and followed asimilar pattern for all samples, with no differences between treatments[(p  0.05)(results not shown)]. Weight loss followed a pattern similarto fat content (results not shown). Water activity of all samples de-creased from an initial 0.960 to 0.900 in all samples over the ripeningperiod, with no differences found between treatments [(p  0.05)(resultsnot shown)]. No differences were observed for CIE a* and b* [(p  0.05)(results not shown)]. CIE L* showed a treatment   day effect(p  0.01), but on further examination using multiple comparison t-testno difference was in evidence between treatments (p  0.05). There-fore, added staphylococci or micrococcci did not have any influenceon weight loss, water activity or color formation. Total free fatty acids The results of total free fatty acids (FFA) from trial 1 (Fig. 3)clearly showed that the level of FFA started at   zero. The levels of FFA increased slowly up to 7 days, reaching a level of between 4 and16 mg FFA/g fat, with the control sausage giving the highest FFA.From day 15 the level of FFA seemed to increase rapidly up to >50mg/g fat in all cases at day 21, with little difference between sausagesinoculated with lipolytic starters and those inoculated with non lipoly-tic starters and the control. This suggested that lipolysis in the sausag-es was independent of bacterial enzymes and therefore more likely tobe due to endogenous enzymes, as has been suggested (Molly et al.,1996). From day 21 onwards there seemed to be a decrease in totalFFA with all sausages reaching a final level of 33–42 mg/g fat after 49days which could be due to lipid oxidation, although results of TBARsdid not confirm this.Results of total FFA from trial 2 (Fig. 4) showed that as in trial 1,the level of FFA started at an initial level of zero. The level of FFA thenincreased gradually, reaching a level of 6-14 mg/g fat after 7 days withsausages inoculated with  Micrococcus varians (4)producing the low-est amount of FFA and those inoculated with  Micrococcus varians (13) producing the greatest amount. The different pattern of FFAproduction between trial 1 and trial 2 could possibly be due to differ-ences in raw material which was further emphasized by results of initial malondialdehyde concentration which varied between the twotrials. After 21 days all samples showed FFA within the range 21–31mg/g fat. From day 21 to 28 all samples showed a slight increaseexcept those inoculated with MCV13 which actually showed a slightdecrease, with the control showing the highest FFA. From day 28 today 35 however, the control, and sausages inoculated with MCV13and STX7 all showed a rapid increase in FFA, reaching a final 45–53mg FFA/g fat in all cases, with little difference between samples. Thisagain emphasized the possibility that lipolysis was most likely due toendogenous enzymes. However, in the two remaining samples, fromday 28 to day 35 there was no increase in level of FFA. In the controlsausage and those inoculated with MCV13 and STX7 a decrease inlevel of FFA was evident from day 35 to day 49, which correspondedwith results from trial 1. In sausages inoculated with M17 and MCV4an increase in the level of FFA in the same timescale was observed. Inall cases a final level 38 to 45 mg FFA/g fat was reached by day 49.Statistical evaluation showed no differences between treatments(p  0.05). Therefore overall, results of both trials seemed to suggestthat lipolysis was most likely due to endogenous enzymes, with bac-terial lipases having only minor effects. This was suggested by Mollyet al. (1996) who showed that added micrococcaceae had no effect onthe level of FFA in a Belgian sausage. Total and individual FFA by GC Results shown for trial 1 and trial 2 (Table 2) are averages of foursamples   standard deviation. Results from trial 1 (Table 2) indicatedthat the levels of total FFA for each sample started at a different level,with the sausages inoculated with STX7 having the highest startinglevel of FFA at day zero and those inoculated with M17 having thelowest level. It would appear from results on day 15 that the sausageinoculated with M17, (a non lipolytic starter strain) had the highestlevel of FFA. This was closely followed by the control and sausageinoculated with STX7 (lipolytic starter), with the sausage inoculatedwith MCV13 giving the lowest level of FFA. This showed that asausage inoculated with a non lipolytic starter and an uninoculatedsausage both produced higher levels of FFA than those inoculatedwith lipolytic starter strains, suggesting that lipolysis was due in themajor part to endogenous meat enzymes. On day 35, the sausageinoculated with STX7 gave the highest level of FFA, followed byM17 and the control. Little difference was observed between controlsand sausages inoculated with lipolytic or non lipolytic strains, againindicating the possibility that lipolysis was mostly due to endogenous Fig. 3—Total free fatty acids in salamis over time, as measured by thecopper soaps method [(Mean   standard deviation(Trial 1)].Symbols, same as Fig. 1.Fig. 4—Total free fatty acids in salamis over time, as measured by thecopper soaps method [(Mean   standard deviation(Trial 2)]. Symbolssame as Fig. 1.  542  JOURNAL OF FOOD SCIENCE —Volume 63, No. 3, 1998  Free Fatty Acids in Fermented Sausages . . . enzymes. On day 49 the salami inoculated with STX7 gave the highestlevel of FFA but the level of FFA was lower than that observed forday 35, which was also evident for the other four samples. Results of trial 2 (Table 2) also showed that the starting levels of FFA on day zerovaried between the 5 sausages. Those inoculated with STX7 againgave the highest level of FFA, with the sausage inoculated with M17giving the lowest level. At day 15 however, the sausages inoculatedwith lipolytic starter cultures (STX7 & MCV13) had the highest lev-els, followed by the control, although there was little difference be-tween samples. The two salamis inoculated with non lipolytic starterstrains gave the lowest results. On day 35 the level of FFA in sausagesinoculated with lipolytic starters had decreased in relation to day 15,and gave results comparable to those inoculated with non lipolyticstarters. The control gave the highest level of FFA. On day 49 thehighest concentration of FFA was found in the control, which showedan increase from day 35. The only other sausage to show an increasefrom day 35 to 49 was that inoculated with MCV4. Excluding thecontrol salami at day 49 the sausages inoculated with lipolytic and nonlipolytic starters gave comparable results. Therefore lipolysis wasprobably due mostly to endogenous meat enzymes, as was suggested(Molly et al., 1996). The increase in major FFA (concentration at day35   concentration at day 0) (Table 3) showed that in both trials themain increase in FFA concentration was detected for C14:0, C16:0,C16:1, C18:0, C18:1, C18:2 and C18:3, which corresponded withprevious reports (Fernandez et al., 1995b; Stahnke, 1995; Molly et al.,1996). The calculated production of the various fatty acids variedgreatly between trial 1 and trial 2, which could also be a reflection of  Table 2—Total free fatty acids a  in salamis over time, as measuredby GC Trial 1STX7MCV13MCV4M17CONT Day 09.84  1.148.86  0.108.41  0.977.69  0.459.42  1.44Day 1546.09  4.4533.57  2.3137.42  1.2849.20  2.6149.06  3.32Day 3596.82  6.6069.94  3.5973.32  3.0792.11  2.7777.59  7.29Day 4987.47  4.6158.46  2.7562.89  6.3763.24  6.8641.55  4.03 Trial 2STX7MCV13MCV4M17CONT Day 014.74  1.3912.88  0.4813.78  0.8212.83  1.0714.73  0.53Day 1555.94  4.4552.38  2.2745.61  2.7336.33  2.2449.66  3.52Day 3543.32  1.5244.41  3.4937.72  1.8040.11  3.8461.57  5.10Day 4939.32  0.6640.52  2.0243.85  3.1637.96  3.6866.20  1.33 a Mean Total FFA mg/g fat   standard deviation. Table 3—Calculated production (mg/g) of the major individual freefatty acids detected in salamis between day 0 and day 35, as mea-sured by GC Trial 1C14:0C16:0C16:1C18:0C18:1C18:2C18:3 STX71.7615.642.847.2640.0816.101.52MCV131.2811.811.955.4428.6010.120.74MCV41.0811.942.725.4731.6010.690.73M170.7413.893.316.4637.7218.351.34CONT1.2212.342.445.9033.1911.840.78 Trial 2C14:0C16:0C16:1C18:0C18:1C18:2C18:3 STX70.283.780.591.889.5310.470.95MCV130.293.830.861.5011.5011.321.08MCV40.182.680.491.4810.079.981.16M170.233.500.631.7410.459.040.84CONT0.455.911.842.7921.3213.171.60 the difference in raw materials used. The difference in proportions of C18:1 to C18:2 in trial 2 as compared to trial 1 could be due toconsumption of C18:1 by the bacteria present. Another possible ex-planation of the shift in proportions is that C18:1 could have beenconverted to C18:2 by chemical or microbiological means. Note alsothat the concentrations of total FFA as measured by the copper soapsmethod did not match those values achieved by GC, which suggeststhat GC is more sensitive. Statistical analyses showed no differencesbetween treatments (p  0.05) regarding total and individual FFA, there-by suggesting that lipolysis was due mostly to endogenous enzymes. Lipid oxidation Lipid oxidation results (TBARs) as mg malondialdehyde/kg fatfrom trial 1 (Fig. 5) showed that all samples reached their highest levelof malondialdehyde at day 35 or day 49. For sausages inoculated withSTX7 and MCV13 the highest level was  1.2 mg malondialdehyde/ kg fat, whereas sausages inoculated with MCV4 and M17 gave opti-mum levels of 1.5–1.9 mg/kg. In the control the level of lipid oxida-tion was the highest, with levels after 49 days reaching  3.5 mgmalondialdehyde/kg fat.Results from trial 2 (Fig. 6) showed that the starting values of TBARs were well in excess of those in trial 1 showing differences inraw materials. In sausages inoculated with STX7 it was evident thatthere was a decrease in level of TBARs from day 0 to day 49 but theconcentration of malondialdehyde never reached a value of  1.5 mgmalondialdehyde/kg fat. In sausages inoculated with MCV13 andMCV4, the highest level of TBARs was at day 35 with MCV13 Fig. 6—TBARs of salamis over time [(Mean   standard deviation (Trial2)].Fig. 5—TBARs of salamis over time [(Mean   standard deviation (Trial1)].
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