The Effect of Autochthonous Starter Culture , Sugars and Temperature on the Fermentation of Slavonian Kulen

Slavonian kulen is the most representative traditional Croatian fermented pork sausage produced in rural settings using a traditional technology and industrial settings according to a modifi ed recipe (which includes the addition of commercial bacterial starter cultures, nitrate and nitrite salts and isoascorbate). This sausage is traditionally made from the fi rst and second class pork previously cleaned of connective tissue, damaged parts, blood vessels and fatback, spiked with salt, red hot and sweet paprika powder and garlic, the stuffi ng ultimately being fi lled into the pork appendix (intestinum caecum). Once the stuff ing is conditioned, the sausage is smoked, fermented, dried and ripened for several months (1,2). Due to the various technological processes used for the sausage production, including hurdle technology, the activity of technological ISSN 1330-9862 original scientifi c paper


Introduction
Slavonian kulen is the most representative traditional Croatian fermented pork sausage produced in rural settings using a traditional technology and industrial settings according to a modifi ed recipe (which includes the addition of commercial bacterial starter cultures, nitrate and nitrite salts and isoascorbate).This sausage is traditionally made from the fi rst and second class pork previ-microfl ora (especially vivid during the fermentation process) and long-term maturation, complex microbiological, physicochemical and biochemical processes take place resulting in the change of fundamental building materials (fats, proteins and carbohydrates), water loss and increase in dry mass (3).
Nowadays, bacterial starter cultures are widely used in the industrial production of fermented sausages to overcome the fermentation problems, reduce the variability of the product quality, limit the growth of spoilage bacteria by accelerating fermentation, and improve the sensory properties of fermented meat products (4).Fermentation is the crucial phase of dry sausage curing, since in this stage major physical, biochemical and microbiological transformations occur (5,6).Meat conservation by fermentation is characterised by several factors: pH decrease, changes in the initial count of microfl ora, reduction of nitrates fi rst to nitrites and later on to nitric oxide, formation of nitrosomyoglobin, solubilisation and gelifi cation of myofi brillar and sarcoplasmic proteins, proteolytic, lipolytic and oxidative changes, and dehydration (7).The process is also characterised by the increase in the number of lactic acid bacteria (LAB) from 10 3 -10 5 to 10 6 -10 9 CFU/g, as well as by glycolytic sugar degradation and the increase in lactic acid concentration.The increase in lactic acid concentration causes a pH decrease from the initial 5.7 to 5.5 in slow-fermented sausages and to 4.6 (sometimes even 4.2) in fast-fermented sausages (8,9).Bacterial starter cultures used for meat fermentation are the preparations of viable bacteria that exhibit a desired metabolic activity, primarily acidifi cation, and are responsible for the development of aroma during meat fermentation (10).They are composed of the strains of lactic acid bacteria primarily belonging to the Lactobacillus or Pediococcus genus, as well as of coagulase-negative staphylococci (CNS) and the members of the Micrococcaceae genus (4).
Fermentation is most intense during the fi rst few hours, when the temperature rises up to the values optimal for LAB growth.This can take from 12 h to 7 days or even longer, depending on the product type, additives, production technology and production environment, i.e. temperature and relative air humidity.Higher temperatures and higher relative air humidity (RH) speed up the fermentation process and decrease the pH value.Depending on the bacterial strains, fermentation can be carried out at temperatures ranging from 18-24 °C or higher for 1-2 days, or at lower temperatures (10-12 °C) for over a week.However, in some cases fermentation can last for over a week even if taking place at higher fermentation temperatures (e.g. in the production of Greek and some Italian sausages) (11,12).
Since the glucose content in meat is too low or much too variable, and in order to provide suffi cient quantities diff erent carbohydrates, like glucose, sucrose, lactose, maltodextrin, corn syrup, starch and sorbitol are added to the fermented sausage stuffi ng.These carbohydrates enhance the growth of technological microfl ora, primarily LAB (13).The most common substrate used in this type of fermentation is glucose, consumed during the LAB exponential growth phase.
Fermentation temperature, mass fraction and the type of added sugar directly aff ect the pH decrease rate.
Low pH inhibits the growth of pathogens and spoilage bacteria, while glucose and salt increase the osmotic pressure, which favours the growth of autochthonous technological bacteria.Sugars, mostly glucose, facilitate dry sausage fermentation, since they serve as a substrate for the lactic acid production and contribute to the specifi c aroma development.Up to 2 % (on average 0.3-0.8%) of sugars are added into the fermented sausage stuffi ng to ensure the pH decrease from the initial 5.8-6.0 to 4.8-5.4(14).
Inoculation of the sausage stuffi ng with a starter culture composed of the selected LAB (i.e.homofermentative Lactobacilli and/or Pediococci) and non-pathogenic CNS and/or Kocuria improves the quality and safety of the fi nal product and contributes to the standardisation of the production process (13)(14)(15)(16).
The goal of this study is to evaluate the impact of an isolated and well-characterised autochthonous starter culture, glucose and maltodextrin (w=0.8 %) and two fermentation temperatures (12 and 20 °C) on the fermentation process and quality of the Slavonian kulen.

Microorganisms
Starter cultures, bacterial strains Lactobacillus plan tarum 1K and Staphylococcus carnosus 4K1, characterised as functional (1,(7)(8)(9)(10)(11)(12), are originally isolated from the traditional Croatian fermented sausages.These strains are utilised in an industrial sausage production.Bacterial strains were acquired from the collection of microorganisms stored in the Laboratory for General Microbiology and Food Microbiology, Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb, Croatia.Lactobacillus plantarum 1K and Staphylococcus carnosus 4K1 were kept at -70 °C in de Man-Rogosa-Sharpe (MRS) broth (Difco™, Detroit, MI, USA) and nutrition broth (Biolife, Milano, Italy) with 30 % (by volume) glycerol.The strains were activated in the above mentioned broths and maintained at 4 °C until propagation.

Preparation of wet biomass
The LAB were grown in the MRS broth at 30 °C for 48 h, while the Staphylococcus was grown in the nutrition broth at 37 °C for 48 h.Bacterial cells were harvested under aseptic conditions as follows: fi rst they were centrifuged (at 6000×g for 10 min) at room temperature, then washed three times in salt water (0.5 %) and fi nally resuspended in a sterile salt water.A standard dilution method aft er a 48-hour incubation at 37 °C in MRS and nutrition agar was used to assess the total viable count (TVC).The fi nal counts of bacterial cells were: 10 11 L. plantarum 1K viable bacterial cells per g of wet biomass, and 10 7 S. carnosus 4K1 viable bacterial cells per g of wet biomass (17).

Sample preparation
Slavonian kulen was produced in a pilot plant (Faculty of Food Technology, Osĳ ek, Croatia) intended for dry sausage production, equipped with a programmable au-tomated ripening chamber.The Slavonian kulen stuffi ng was prepared according to the traditional recipe: pork meat (of the fi rst and second class, 91.8 %), pork fatback (5 %), garlic (0.2 %), red hot paprika powder (0.4 %), sweet red paprika powder (0.6 %) and salt (2 %).The meat was ground through a grinding plate with 8-mm diameter holes, while the pork fatback was ground through a grinding plate with 6-mm holes.The batches with added autochthonous starter culture were inoculated with L. plantarum 1K in the fi nal concentration of about 10 6 cells per g and S. carnosus 4K1 in the fi nal concentration of about 10 3 cells per g.To these samples, 0.8 % of glucose or maltodextrin (both from Sigma-Aldrich, Taufk irchen, Germany) were added, as well.
The Slavonian kulen stuffi ng was stuff ed into the pig appendix.Aft er stuffi ng, the raw samples were cold--smoked using a dry hardwood (hornbeam, beech and its sawdust) every few days (for 3-4 h) for two weeks.A 20--day fermentation process was carried out at two diff erent temperatures (Table 1) and relative humidity (RH) of 80 to 90 %.Aft er smoking and fermentation, all samples were left to rest in the ripening chamber at the temperature from 14 to 17 °C and RH from 70 to 80 %.The entire technological process running under the processing conditions detailed above lasted for 90 days.A total of 15 samples of raw Slavonian kulen were divided into fi ve batches (Table 1).

Physicochemical analysis
The water content was determined gravimetrically (18) at 103 °C (model Epsa 2000; Bari, Velika Gorica, Croatia).The total protein content was determined by the Kjeldahl method (19) using a Unit 8 Basic digestion block (Foss, Hillerød, Denmark) and a Kjeltec 8400 automated distillation and titration device (Foss).The total fat content was determined by the Soxhlet method (20).The sample was submitt ed to digestion in an acidic environment followed by petroleum ether-induced fat extraction using a Soxtherm 2000 automated device (C.Gerhardt Gmbh & CO.KG, Königswinter, Germany).Collagen content was determined by the spectrophotometric analysis of hydroxyproline ( 21) using a spectrophotometer model DR/4000U (Hach, Düsseldorf, Germany).Titration method was applied in order to determine the salt content (22) using 2 g of each sample.Samples were homogenised with sand and 3 mL of water and the content was transferred into a 100-mL volumetric fl ask, stirred and placed into a water bath at 100 °C for 15 min.Aft er cooling, the fl ask was fi lled with water up to the mark and fi ltered.An aliquot (25 mL) of the fi ltrate was transferred into an Erlenmeyer fl ask containing a few drops of K 2 CrO 4 as an indicator (62 g per 100 mL of water) and titrated with 0.1 M AgNO 3 until a persistent reddish colour was obtained.The volume and concentration of the titration reagent were used to calculate the salt content.A homogenate diluted with distilled water (1:10) was used for pH determination with pH/Ion 510 Bench pH/Ion/mV Meter (Eutech Instruments Pte Ltd/ Oakton Instruments, Vernon Hills, IL, USA).The water activity (a w ) was determined at room temperature ((20±2) °C) using a Rotronic Hygrolab 3 (Rotronic AG, Bassers dorf, Switzerland), according to the manufacturer's instruc tions.Analytical grade chemicals were used for all analyses of physicochemical parameters.For each batch, analyses were done in triplicate.

Texture profi le analysis
Texture profi le analysis was performed at room temperature with a Universal TA.XT2i SMS Stable Micro Systems Texture Analyser (Stable Microsystems Ltd., Go dalming, UK) equipped with a P/75 cylindrical probe.This involved cutt ing of samples into 1.5-cm thick slices, subsequently compressed twice to 60 % of their original thickness.Force-time curves were obtained at the crosshead speed of 5 mm/s and the equal recording speed.Determined parameters involved (23): hardness, i.e. the maximum force required to compress the sample, springiness, i.e. the ability of a sample to recover its ori ginal form once the deforming force has ceased, cohe siveness, i.e. the extent to which a sample can be deformed prior to rupture, and chewiness, i.e. the eff ort to masticate the sample before swallowing, calculated by multiplying hardness by cohesiveness and springiness.Seven measurements were taken for each sample of the fi ve batches.

Microbiological analysis
The determination of microbial population in Slavonian kulen samples employed classical microbiological analyses (Table 2; 24-29).The isolation of microbial populations was carried out according to the microbiological criteria for food (30) and included the isolation of Enterobacteriaceae, Staphylococcus aureus and sulphite-reducing Clostridia, Salmonella sp. and Listeria monocytogenes.Aft er the casing was aseptically removed and discarded, 10 g of a sample were homogenised in 90 mL of sterile 0.5 % saline solution and serially diluted before plating on selective nutrient media (Biolife) (Table 2; 24-29).The incubation took place under aerobic conditions at 37 °C for 48 h.Microbial growth (CFU/g) was determined using the traditional plate counting.

Isolation and identifi cation of microbial population
The identity and viability of the applied autochthonous starter cultures and natural microbial population in Slavonian kulen samples were determined using API 50 CH and API Staph identifi cation kits (bioMérieux, Marcy l'Etoile, France).Colonies randomly taken from the MRS (150 colonies) and the Baird Parker agar (100 colonies) (26), descriptive of fi ve samples, were determined using identifi cation kits and then authenticated using a Microfl ex LT™ matrix-assisted laser desorption-ionisation time-of-fl ight mass spectrometer (MALDI-TOF MS; Bruker Daltonik, Bremen, Germany) (31).For identifi cation purposes, the peaks from the generated mass spectra were compared to the reference spectra of the integrated database using the MALDI Biotyper soft ware (Bruker Daltonik).

Sensory analysis
The fi nal Slavonian kulen samples from all fi ve batches (obtained aft er 90 days) were subjected to a quantitative descriptive analysis.The analysis was performed by a panel of seven (three male and four female) trained experts according to the ISO standard (32).The panellists had completed three preliminary training sessions in order to familiarise themselves with the involved samples.Twelve att ributes were examined and rated on a 5-point scale: 1 standing for poorly perceived or absent and 5 standing for intensely perceived.During these three training sessions, an agreement on the descriptors to be targeted by the analysis was made.This agreement included: two external att ributes (appearance and hardness), two att ributes describing the slice (colour uniformity and sliceability), fi ve att ributes describing the experience during mastication (fl avour intensity, juiciness, smokiness, acidic taste and saltiness) and three att ributes describing the product smell (spicy odour, lactic acid odour and mouldy odour).The appearance of Slavonian kulen samples was assessed visually, while the hardness was assessed by palpation.The sliceability as a textural property was assessed visually (to see whether any crumbling is present or whether the pieces of the sausage are falling out).A three-digit code was used for coding of sausage samples.Samples were served cut into approx.0.4 cm thick slices.In order to cleanse the palate water was offered between the analyses.

Statistical analysis
The results are shown as mean value±standard deviation.Experimental output was processed using the analysis of variance (ANOVA) and the Fisher's least signifi cant diff erence (LSD), with the signifi cance defi ned at the level of probability of 95 % (p<0.05).Statistical analysis was carried out using the Statistica v. 12.7 soft ware (StatSoft Inc.Tulsa, OK, USA).

Results and Discussion
Physicochemical analysis of the meat and fatback (Table 3) used for Slavonian kulen stuffi ng showed the mass fraction of the basic building blocks (fat, moisture, proteins and collagen) to be in accordance with the literature data on fresh pork meat and fatback analysed 24 h post mortem.The water activity and pH values are also in agreement with the data obtained by the previous research on similar samples (2).A drop in pH values measured 24 h post mortem down to below 6 should be observed in meat used for dry sausages, but also for other meat products.This indicates the normal course of post mortal glycolysis, lactic acid production and pH value dropdown.
In Slavonian kulen, changes in mass fraction of individual building blocks and water activity (a w ) cease aft er 20 days (Table 4), mostly due to drying.The obtained a w of 0.91 was lower than 0.946 and 0.93 reported for a Spanish sausage fermented under similar conditions, but using a diff erent commercial starter culture (5,33).On the other hand, other authors reported the a w of 0.88 on day 21 of fermentation and drying of chorizo with added 1 % of glucose and Lactobacillus sakei K29 and whose fermentation and drying took place under similar conditions of temperature and relative humidity (34).Temperature and RH in the automated chamber during the production of all fi ve Slavonian kulen batches are presented in Figs. 1 and 2. The RH and temperature were within the limits specifi ed for Slavonian kulen production stages (Table 1).
Mass loss determined in all fi ve Slavonian kulen batches is presented in Fig. 3.It is far more substantial in the samples fermented at a higher temperature (20 °C) in the presence of sugars and an autochthonous starter culture, which explains lower moisture contents in these samples (33,34).
During the fermentation phase, the water content in batch B3 decreased from the initial 64.3 % to the lowest 52.6 %.The moisture content was signifi cantly aff ected by the treatments (p<0.05)(Table 4).Samples fermented at higher temperature had lower moisture contents regardless of the addition of sugars and autochthonous starter culture.Other studies (33,34) have reported a lower moisture content (47.53 %) in a Spanish dry sausage fermented using a commercial starter culture (Lactobacillus sakei, Staphylococcus carnosus and Staphylococcus xylosus) at 15 °C and relative humidity of 80 %.In another study (5), a higher moisture content was reported (56.63 %) in the Spanish salchichon fermented using a commercial starter culture (Pediococcus pentosaceus and Micrococcus varian) and supplemented with 0.45 % sugar (dextrose, lactose and maltodextrin).Moisture content and a w variations may be related to the diff erences in the used microorganisms, the mass fraction and the type of added sugar, and the larger diameter of the Slavonian kulen casing (around 100 mm) than that of the Spanish sausage (60 mm).The salt mass fraction increased from the initial 2 % in raw stuffi ng to the average value of 2.7 % aft er 20 days of processing, which complies with the results of similar research in the fi eld (35) (Table 4).Fermentation of dry sausages is usually monitored through changes in pH values (36)(37)(38).Changes in the pH values during 20 days of processing in all fi ve batches of the Slavonian kulen are presented in Fig. 4. The results show that in the samples of Slavonian kulen fermented at 20 °C, a substantial decrease in the pH value was seen after 5 days, while in the samples fermented at 12 °C such a decrease was noticed aft er 7 days.This drop was more intense in the samples fermented at 20 °C and supplemented with an autochthonous starter culture and 0.8 % glucose (batch B3), whose pH dropped from the initial 5.55 to 4.42 aft er 18 days of fermentation (39) (Fig. 4).The Slavonian kulen fermentation is signifi cantly diff erent from the fermentation of similar Spanish sausages (chorizo).This diff erence is characterised by a slower pH drop seen in all samples under this study.The lowest pH value of 4.42, registered in this study in batch B3, was seen on the fermentation day 17, while in the chorizo samples the lowest pH was reached on the ripening day 7 under similar fermentation conditions (34).
The decrease in the pH value comes as a consequence of the presence of lactic acid emerging due to the activity of technological microfl ora (LAB and CNS) and glycolysis, i.e. the transformation of sugar into lactic acid (40).  5.In fermented sausages, the optimal colour formation strongly depends on the fermentation and the resulting pH decrease and oxygen depletion.
The L* values were signifi cantly modifi ed aft er the addition of the autochthonous starter culture and sugar, as well as the change of fermentation temperature.The highest L* values were obtained of the batch B3 samples.Generally, samples fermented at higher temperature had higher L* values (Table 5).A higher L* value may be associated with lower pH values and consequently higher lactic acid concentrations established in these samples.In meat, higher lactic acid concentrations cause exudation once the meat proteins have reached their isoelectric point (41).The a* value was higher in the Slavonian kulen samples fermented at lower temperature.The decrease in a* value can only be associated with the eff ect of lactic acid on diff erent myoglobin forms (myoglobin, nitrosomyoglobin and oxymyoglobin).This acid may partially or totally denaturise this compound.Some authors have reported that this acid decreases redness (41).The yellowness was altered markedly (p<0.05)(Table 5) by diff erent fermentation temperatures and the addition of sugars and autochthonous starter culture.Higher fermentation temperature and the addition of autochthonous starter culture decreased yellowness b* value.This decrease could probably be att ributed to the salt content (due to the eff ect of salt on oxygen solubility in the meat batt er)   (41), which was higher in samples fermented at higher temperature in the presence of glucose and maltodextrin and autochthonous starter culture (Table 4).
In view of the above, a more striking downfall of oxymyoglobin content, which greatly contributes to the value of this colour coordinate, is to be expected.Other authors have also claimed that microorganisms produce metabolites that induce oxidation of meat and fat present in the sausage (42) and, by doing so, contribute to the decrease in this value.Colour parameters measured aft er 20 days were similar to those of kulenova seka (whose stuffi ng and production process are virtually the same as for Slavonian kulen, the diff erence being that the stuffi ng is stuff ed into the end part of the pig large intestine) and the Slavonian homemade sausage (1,2).
The infl uence of autochthonous starter culture, glucose, maltodextrin and fermentation temperature on the instrumental texture profi le (established aft er 20 days of fermentation) is shown in Table 6.The addition of autochthonous starter culture and sugars and variations in fermentation temperature signifi cantly aff ected (p<0.05)hardness and chewiness.As expected, hardness and chewiness reached the highest values in batch B3 samples, followed by batch B5 samples, and were the lowest in batch B4 samples (fermented at 12 o C).During fermentation, pH decreased and solubilised myofi brillar proteins aggregated to form a gel.
Therefore, the variability of texture across Slavonian kulen samples seen during fermentation could be explained by pH diff erences (Fig. 4).
The decrease in the pH gradually induces the aggregation of proteins, leading to the formation of an ordered protein network that contributes to fi rmness (43).
In addition, acid solubilisation of collagen may occur (44).It has been demonstrated that if the pH falls below the isoelectric point of the muscle protein, solubilisation of the protein is higher producing fi rmer sausages (45).Our results are in agreement with the above mentioned, since only Slavonian kulen samples supplemented with autochthonous starter culture and 0.8 % glucose and fermented at 20 °C had pH values below 4.5 during fermentation (Fig. 4).A major eff ect of autochthonous starter culture and sugar addition, and fermentation temperature on springiness and cohesiveness (Table 6) was detected aft er the end of fermentation.Generally, batches fermented at higher temperature had higher texture parameter values than samples fermented at lower temperature, irrespective of autochthonous start er culture and sugar addition.
In the Slavonian kulen samples, Salmonella sp., Enterobacteriaceae, sulphite-reducing Clostridia and L. monocytogenes were not found (Table 7).Bacterial counts reached at the end of the Slavonian kulen fermentation are shown in Table 5.The highest counts of 1.2•10 7 of LAB and 9.6•10 3 CFU/g of CNS were determined in batch B3 samples.In general, higher LAB and CNS counts were established in the samples fermented at higher temperature (Table 7).
The results of biochemical (API) testing of the Slavonian kulen samples (Table 8) showed that the dominant microfl ora in the samples supplemented with autochthonous starter culture was Lactobacillus plantarum, while in the control sample the predominant microfl ora was Lactobacillus sakei.In the samples without autochthonous starter culture, the most represented CNS strains were S. saprophyticus, S. equorum and S. warneri, while in the samples with the autochthonous starter culture the most represented CNS strain was proven to be S. curvatus (testing accuracy 99-99.9%).The dominance of the above strains in the latt er samples is to be expected since these strains are the components of the starter culture (17,31,46).Therefore, we can conclude that the applied autochthonous starter culture has bett er adapted to the meat environment and to the specifi c manufacturing process than the indigenous microbial population present in the control sample.In order to avoid possible misclassifi cation, in addition to physiological (API) tests, the identifi cation of the two applied bacterial species (an autochthonous starter culture) was confi rmed using MALDI-TOF MS, which corroborated the presence of L. plantarum 1K (data not shown) and S. carnosus 4K1 (data not shown).A complex interaction among physicochemical, biochemical and microbiological processes, playing a role in the formation of chemical compounds, and the modifi cation of molecules responsible for the texture and appearance of the fi nal product, also determine its sensory characteristics.The average scores given by the panellists for all fi ve batches at the end of the Slavonian kulen manufacturing process (90 days) are shown in Fig. 5.As for the external att ributes, the highest hardness score was given to batch B3 samples (4.4±0.7), while the appearance of the samples of all fi ve batches was so similar that the assessors could not detect any diff erences among them.The slicing ability and colour intensity of batch B3 and B5 samples also scored highly.
During the fermentation of dry sausages, LAB are known to produce lactic acid (47) responsible for the sour taste (48) and odour of the product, while a mouldy odour is ordinarily associated with 1-octen-3-ol, a typical component of mushroom odour (49).Batches fermented at higher temperature in the presence of glucose or maltodextrin and autochthonous starter culture (Fig. 5) had higher scores for all three att ributes.
As for the smell, that of lactic acid was dominant, especially of the batches fermented at higher temperature and supplemented with glucose or maltodextrin and autochthonous starter culture, while mouldy and spicy odour scored low in all fi ve batches.

Conclusion
The addition of glucose, maltodextrin and autoch thonous starter culture and using diff erent fermentation temperatures (12 and 20 °C) aff ected the Slavonian kulen fermentation.In conclusion, the batches fermented at higher temperature with added autochtho nous start er culture had higher values of all textural parameters and L* and a* values, higher counts of lactic acid bacteria and coagulase--negative staphylococci, and lower moisture, a w and b* values regardless of sugar addition.Sensory analysis, undertaken at the end of the production process, proved higher fermentation temperature and autochthonous starter culture addition to have the greatest infl uence on the Slavonian kulen fermentation.Finally, it is important to emphasise that all of the investigated instrumental colour and texture parameters could be useful in monitoring the product evolution and, moreover, in an easy-to-perform control over the total duration of the Slavonian kulen fermentation and ripening.
CIE L*a*b* values of the Slavonian kulen samples are shown in Table

Table 1 .
Samples of the Slavonian kulen prepared according to the traditional recipe with the addition of glucose or maltodextrin and fermented with autochthonous starter culture at diff er-

Table 2 .
Classical microbiological and biochemical (API) methods used for the isolation and identifi cation of microbial population in Slavonian kulen

Table 4 .
Basic chemical composition, salt content and water activity (a w ) of the Slavonian kulen samples, determined aft er 20 days of fermentation a Values are mean±S.D., N=3.Values in the same column marked with diff erent lett ers in superscript are signifi cantly diff erent (p<0.05)

Table 5 .
Colour measurements of the Slavonian kulen samples aft er 20 days of fermentation

Table 6 .
Texture profi le of the Slavonian kulen samples aft er 20 days of fermentation

Table 7 .
Microbiological analysis of the Slavonian kulen samples aft er 20 days of fermentation N.D.=not determined

Table 8 .
Biochemical (API) test results for the Slavonian kulen samples obtained aft er 20 days of fermentation