probiotic strain lactobacillus plantarum nbimcc 2415 with ... · biotechnol. & biotechnol. eq....

1624 Biotechnol. & Biotechnol. eq. 24/2010/1

Article DOi: 10.2478/v10133-010-0016-4 FB

Keywords: probiotics, antioxidant activity, antimicrobial activity, Lactobacillus plantarum, meat starter cultures, cell disintegration

Biotechnol. & Biotechnol. eq. 2010, 24(1), 1624-1630

Introductionthe lactic acid bacteria fermentation ability has been used by people for production of fermented goods (raw-dried meat products, yoghurt, cheese, fermented fruits and vegetables, bread) since the antiquity. nowadays, selected lactic acid bacteria and bifidobacteria strains are applied in the production of such foods. these strains are added as concentrates, which contain high number of viable cells. they are called “starter cultures” (1).

the probiotic strains for fermented meat products should be able to survive under the conditions in the stomach and the intestines – low values of ph and high concentrations of bile salts (4, 5, 9). they should also show high antimicrobial activity against pathogenic and toxigenic microorganisms in the raw meat at temperatures suitable for fermentation and ripening (2). the inclusion of lactobacilli strains with antioxidant activity in starter cultures provides another source of antioxidants or antioxidant synthesis during fermentation and ripening of meat products (6, 8, 11).

A new approach in their production is the application of lactic acid bacteria and bifidobacteria strains with probiotic properties, which provides a purposeful fermentation process, meat products’ safety and high content of microorganisms with beneficial influence on the microflora of the gastro-intestinal

tract. in such a way, meat products could turn into healthy foods.

the aim of this study is to investigate the antioxidant activity of Lactobacillus plantarum as well as its inhibition of pathogenic and toxigenic microorganisms at 15-18ºc.

Materials and Methodsthe antimicrobial activity of Lactobacillus plantarum nBiMcc 2415 (National Bank for Industrial Microorganisms and cell cultures) was determined against the following pathogenic microorganisms: Escherichia coli Atcc 25922, Escherichia coli Atcc 8739, Proteus vulgaris G, Salmonella sp., Salmonella abony ntcc 6017, Staphylococcus aureus Atcc 25093, Staphylococcus aureus Atcc 6538 P, Listeria monocytogenes and Listeria monocytogenes i which were granted by hMi-Plovdiv.

in the present work the following nutrient media were used for determination of the antimicrobial activity: MRS-agar (MeRcK, Germany); lAPtg10 (Peral de Portillo); lBG (luria-Bertani with 1% glucose).

the materials for disintegration of cells included:1. TRIS with рН 8 (g/dm3, cm3/dm3): tRiS base– 121.0;

concentrated hcl – 42.0;2. 0.5М EDTA with рН 8 (g/dm3): EDTA – 186.0; NaOH

– 20.0;3. PeB-buffers:

3.1. PeB i (cm3/dm3): TRIS with рН 8 – 25.0; EDTA with рН 8 – 50.0;

3.2. PeB ii (g/dm3): SDS – 20.0; NaOH – 12.0;

ProbIotIc straIn LactobaciLLus pLantarum nbIMcc 2415 wIth antIoxIdant actIvIty as a starter culture In the ProductIon of drIed ferMented Meat Products

P. nedelcheva1, Z. Denkova1, P. Denev2, A. Slavchev1, A. Krastanov3

1University of Food Technologies, Department of Microbiology, Plovdiv, Bulgaria2institute of organic chemistry with centre of Phytochemistry-BAS, laboratory of Biologicaly Active Substances, Plovdiv, Bulgaria3University of Food Technologies, Department of Biotechnology, Plovdiv, Bulgariacorrespondence to: Albert Krastanove-mail: [email protected]

abstractLactobacillus plantarum NBIMCC 2415 resistant to low and high values of pH and pepsin, and to high concentrations of bile salts was selected. It was demonstrated that Lactobacillus plantarum NBIMCC 2415 inhibited the growth of pathogenic microorganisms such as Escherichia coli ATCC 25922, Escherichia coli ATCC 8739, Proteus vulgaris G, Salmonella sp., Salmonella abony NTCC 6017, Staphylococcus aureus ATCC 25093, Staphylococcus aureus ATCC 6538 P, Listeria monocytogenes and Listeria monocytogenes I at temperature 15-180C in the meat products. The high antioxidant activity of this strain against peroxide radicals (ORAC results) was determined. Experimental series of raw-dried meat products were produced. Reduction of the microflora of the meat raw-dried sausages and high concentration of viable cells of L. plantarum NBIMCC 2415 were observed, which makes them functional foods.

1625Biotechnol. & Biotechnol. eq. 24/2010/1

3.3. lysozyme solution: PeB i + lysozyme (5 g/dm3).the materials for antioxidant activity measurement were:4. 2,2’-azobis(2-amidinopropane) dihydrochloride

(AAPh)- 0.5151M: dissolve 0.717g AAPh (Mw=271.2, purity-97%) in 5ml phosphate buffer (75mM, ph 7.4). Prepared prior every experiment;

5. Solutions of fluorescein (FL):5.1. concentrated Fl: 8.37x10-4M: dissolve 11.25mg

Fl (Mw=376.28, 70% active substance) in 25ml phosphate buffer (75mM, pH 7.4). Durability 4-5 months on dark,-18°c;

5.2. Stock Fl: 4.185x10-6M: 0.050ml of the concentrated Fl to 10ml with phosphate buffer (75mM, pH 7.4). Durability 7 days in dark place, 4-6°c;

5.3. Working solution: 6.3x10-8M: 0.48ml of Stock solution to 32 ml with phosphate buffer (75mM, ph 7.4). Prepared daily and kept in a dark bottle during experiments.

6. trolox;7. Phosphate buffer: 75mM, ph 7.4 (mixture of nah2P04

and na2hPo4);8. Fluorimeter: excitation wavelength = 485nm, emission

filter = 520nm.

analytical methodsFor determination of the viable counts of L. plantarum nBiMcc 2415 the strains were cultivated in skimmed cow’s milk for a fixed time at the optimal temperature for their growth. the cultures were diluted tenfold according to the method of serial dilutions in saline solution (7). After that they were spread on Petri dishes with MRS media (10), which were incubated for 3 days at 37°c until single colonies were formed.

the method for determination of resistance of L. plantarum nBiMcc 2415 in model gastric juice conditions (12) was performed as follows. the cultures were incubated for 16 h in MRS-broth. the culture medium was then centrifuged at 5000 min-1 for 10 min. this was followed by two-act washing of the pellet with 0.1M phosphate buffer, ph 7.0. the cells were resuspended to the original volume with the buffer. Buffer with ph 2.0, containing 2 g/dm3 nacl and 3.2 g pepsin with activity 0.7 FiP U/mg and buffer with ph 7.0 and the same content were incubated with 2% of the bacterial suspension. they were thermostated at 37°c and samples were taken on the 0, 2, 4 and 24 h of incubation. Proper dilutions were made, according to the method of the serial tenfold dilutions in 0.1% peptone water and plated in MRS-agar. the plates were incubated at 37°c until single colonies were formed. the colonies were counted and the results were expressed as colony-forming units per milliliter (cFU/cm3).

the method for determination of the bile tolerance of L. plantarum nBiMcc 2415 (2, 12) comprised several steps. the first steps, until bacterial suspension was obtained, were the same as those in the method for determination of resistance of

L. plantarum nBiMcc 2415 in model gastric juice conditions. MRS-broth (50 cm3), containing different concentrations of bile salts (0, 0.05, 0.1, 0.15, 0.3, 1%) was incubated with 0.5 cm3 of the cell suspension. the lactic acid bacteria were cultivated at 37°c and the viable cell counts were determined on the 0, 2, 4, 6, 8 and 24 h according to the described method.

the determination of the antibiotic resistance of L. plantarum nBiMcc 2415 was performed according to the agar diffusion method, but with paper disks with a definite antibiotic concentration. L. plantarum nBiMcc 2415 was cultivated in its growth medium and centrifuged at 5000 min-1 for 10 min. the biomass was resuspended in saline solution to the original volume and the growth media were inoculated with 0.1 cm3 of this suspension. the plates were left for 1-2 h at room temperature and after that the disks with d=6 mm were placed. the Petri dishes were incubated at 37°c for 24-48 h and the zones of clearance were determined.

the used culture - Lactobacillus plantarum nBiMcc 2415, was cultivated in de Man, Rogosa, Sharpe (MRS)-broth with 1% inoculum for 48h at 30°С in 400 cm3 jars. the total viable cell count of the lactic acid bacteria was determined thorough the spread plate method on MRS-agar. the culture was poured in 50 cm3 test tubes and was centrifuged at 5000 rpm for 10 min. 50 cm3 of the culture medium was added to the biomass. Second centrifugation came after, at 5000 rpm for 10 min. the obtained sediments from both centrifugations were resuspended with saline solution and were centrifuged one more time at 5000 rpm for 10 min. the rinsing was realized twice. Several methods for disintegration of cells were performed as different kinds of lyses were applied on the obtained sediments.

• enzymatic disintegration:twice rinsed microbial cell sediment was resuspended with

25 cm3 from reagent 2.3.3. it was homogenized and incubated at 37°С for 1h with periodical mixing in every 5 min. It was centrifuged at 3500 rpm for 10 min. the supernatant was separated from the sediment for antioxidant activity measurement.

• Alkaline disintegration:twice rinsed microbial cell sediment was resuspended with

2 cm3 from reagent 2.3.3. it was homogenized and incubated at 37°С for 30 min with periodical mixing in every 5 min; after that reagent 2.3.3. was added. the well homogenized sample was placed at 37°С for 20 min. It was centrifuged at 3500 rpm for 10 min. the supernatant was separated from the sediment for antioxidant activity measurement.

• temperature disintegration:twice rinsed microbial cell sediment was resuspended

with 2 cm3 distilled water and was placed in high temperature amplitude conditions (from -30°С to 100°С). The sample was held for 10-20 s in the respective conditions. the test tube was poured to 50cm3 and was centrifuged at 5000 rpm for 10 min. the supernatant was separated for antioxidant activity measurement.


• Ultrasonic disintegration:twice rinsed microbial cell sediment was resuspended

with 25 cm3 distilled water. the well homogenized sample was subjected to sonication in ice bath. the disintegration was performed by 40 cycles of sonication for 30 s with 1 min pause between each cycle. the sample was poured to 50cm3 with distilled water and was centrifuged at 5000 rpm for 10 min. the supernatant was separated from the sediment for antioxidant activity measurement.

the antioxidant activity measurement was performed according to the following experimental conditions. For each 200 μl well the reaction mixture contained:

1. FL – 170 μl, 5.36x10-8M in final concentration;2. AAPH – 20 μl, 51.51mM in final concentration;3. Sample – 10 μl.The 170 μl FL working solution and 10 μl sample were

pipetted in the wells and incubated at 37° for 10 min. the gain of the fluorimeter was adjusted and 20 μl AAPH was added. the AAPh should always be added in the row with the blank and standard samples last. the gain should be adjusted on every run. The initial fluorescence was measured. After that the fluorescence readings were taken on every cycle. For blank sample, phosphate buffer solution was used. trolox solutions with concentrations of 12.5, 25, 50, 100 and 200 μM were used for building the standard curve. The final ORAC values were calculated using a regression equation between the trolox concentration and the net area under the curve and are expressed as trolox equivalents as micromole per liter or gram (μmol TE/dm3; μmol TE/g).

results and discussionthe lactic acid bacteria inhabit animal and human gastro-intestinal tract. they transform substrates with the formation of short-chain fatty acids, bacteriocins, etc. through which they inhibit pathogenic and toxigenic microorganisms’ growth and push them out of the biological niche. this is the way they support the equilibrium of the gastro-intestinal microflora.

not all of the lactic acid bacteria strains show these properties. that is the reason why selection of strains with probiotic activities is needed. lactic acid bacteria, which are involved in the probiotics and probiotic foods, must be able to survive in the conditions of gastro-intestinal tract – in low and alkaline ph values, presence of pepsin and bile salts and

to be resistant to antibiotics. in order to be applied as starter cultures in the raw-dried meat goods production, lactic acid bacteria have to survive in the conditions of foods’ ripening (15-18°c), to have low proteolytic and lipolytic activities, to show high level of antimicrobial activity and to take part in the formation of sensorial complex. the addition of cultures with high antioxidant activity influences the sensorial properties of meat foods and increases their safety.

survival of L. plantarum nbIMcc 2415 in model conditions of gastro-intestinal tractlactic acid bacteria, which could be included as components of probiotics and probiotic foods, must be resistant to the acidic conditions of the stomach, as well as to the bile acids in the small intestines. the bacteria, which enter the digestive tract, pass through an active acidity range from ph 2.0 to ph 8.0. Different microorganisms vary in their ability to survive in such extreme conditions. Some of them perish entering the stomach, and others survive long enough and preserve their viability and reproductive capacity. in the concentrated form of probiotics or probiotic foods, the lactobacilli reach the stomach in the conditions of low ph values and the presence of pepsin. After that, together with the digested food, they pass through the small intestines, where the concentration of bile acids is up to 0.3% and reach the large intestine, which has a neutral ph value.

We investigated the ability of Lactobacillus plantarum nBiMcc 2415 to develop in model conditions of digestion – enzymes, low ph values, and the presence of bile salts.

the growth of Lactobacillus plantarum nBiMcc 2415 in a medium with low and neutral ph values and the addition of pepsin was studied (table 1). the concentration of the viable Lactobacillus plantarum nBiMcc 2415 cells decreased one fold in a low ph medium for 4 h.

Pepsin has a ph-optimum at ph 1.0-2.0. it can attack the peptide components of the cell wall. This influences the cells in the logarithmic phase of growth. the cells in the stationary phase are resistant to its action, they preserve their viability and when the stress factor is removed, they form colonies on the surface of the lAPtg10-agar. At neutral ph values, both in the buffer and the nutrient medium, the action of the enzyme is suspended and in the presence of nutrients the microbial cells grow and reproduce.

table 1Reduction of the viable cell counts of Lactobacillus plantarum nBiMcc 2415 at acid (ph 2.0) and neutral (ph 7.0) ph values and in the presence of pepsin

strain

viable cell counts, cfu/cm3

0 h 2 h 4 h 24 hph 2.0

+ pepsinph 7.0

+ pepsinph 2.0

+ pepsinph 7.0

+ pepsinph 2.0

+ pepsinph 7.0

+ pepsinph 2.0

+ pepsinph 7.0

+ pepsinLactobacillus plantarum nBiMcc 2415 2.104 2.105 2.104 2.1.105 1.104 1.104 10 10


the cells of Lactobacillus plantarum nBiMcc 2415 are the most resistant to the addition of bile salts in the nutrient medium. Moreover, 0.3% of bile salts in medium suspended the development of the strain, while maintaining a high concentration of viable cells (over 105 cfu/cm3) (fig. 1). For the period of 8-24 h an increase of the total viable cells count was observed in a medium with 1% bile salts (fig. 1). the high concentration of antimicrobials substances is a premise for advent of resistant cells, which on their turn grow and proliferate. this was expressed by the increase of the viable cell counts after 8 h.

3

4

5

6

7

8

9

10

0 2 4 6 8Time, h

log

N

0%0,05%0,10%0,15%0,30%1%

fig. 1. Variation in the Lactobacillus plantarum nBiMcc 2415 viable cells concentration under the influence of different amounts of bile salts in the medium

antimicrobial properties of L. plantarum nbIMcc 2415the strain Lactobacillus plantarum nBiMcc 2415 is included in starter cultures for meat products with higher ph and lower processing temperature. this was the reason we studied the antimicrobial activity of Lactobacillus plantarum nBiMcc 2415 against pathogenic, toxigenic and saprophytic microorganisms at drying temperature (15-18oС) for raw-dried meat products.

a

0

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0 3 6 9 12 15Time, d

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N

L.plantarum 2415k*

L.plantarum 2415m**

S.aureus 6538 Pk

S.aureus 6538 Pm

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L.plantarum 2415m

S. aureus 25093k

S. aureus 25093m

*- cultivated as pure cultures**-cultivated as mixed culturesfig. 2. Growth dynamics of the Staphylococcus aureus strains and Lactobacillus plantarum nBiMcc 2415 as pure and mixed cultures at 15-180Сa) Staphylococcus aureus Atcc 6538 Pb) Staphylococcus aureus Atcc 25093

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L.plantarum 2415m

L.monocytogenesIk

L.monocytogenesIm

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L.plantarum 2415m

L.monocytogenesk

L.monocytogenesm

fig. 3. Growth dynamics of the Listeria monocytogenes strains and Lactobacillus plantarum nBiMcc 2415 as pure and mixed cultures at 15-180Сa) Listeria monocytogenes ib) Listeria monocytogenes


high total viable cell counts for the pure culture of Lactobacillus plantarum nBiMcc 2415 were achieved on day 6 (1012 cfu/cm3) at 15-18oС and lasted during 15 days of cultivation (fig. 2). During the cultivation of Lactobacillus plantarum nBiMcc 2415 the medium acidity raised (table 2) and metabolites were synthesized, which inhibited the growth of Staphylococcus aureus (fig. 2). one of the two representatives lost its viability and on 15th day there were none living cells, while the other had viable cells at the end of the experiment which exceeded 102 cfu/cm3.

table 2Alteration in titratable acidity of the culture medium of Lactobacillus plantarum nBiMcc 2415

Pathogen sample15-18°Сbeginning end

E. coli atcc 8739lABcontrol sample 28 54MixlAB+Pathogen 25 61.5Pathogencontrol 19 45

E. coli atcc 25922lABcontrol sample 28 54MixlAB+Pathogen 21 56Pathogencontrol 18 50

L. monocytogeneslABcontrol sample 28 54MixlAB+Pathogen 24 56Pathogencontrol 17.5 41.5

L. monocytogenes IlABcontrol sample 28 54MixlAB+Pathogen 22 55Pathogencontrol 19 51.5

s. abony ntcc 6017lABcontrol sample 28 54MixlAB+Pathogen 22.5 61Pathogencontrol 18 60

salmonella sp.lABcontrol sample 28 54MixlAB+Pathogen 23 66Pathogencontrol 19 47

s. aureus atcc 25093lABcontrol sample 28 54MixlAB+Pathogen 21 60Pathogencontrol 18 63

s. aureus atcc 6538 PlABcontrol sample 28 54MixlAB+Pathogen 24 53.5Pathogencontrol 18 55.5

p. vulgaris GlABcontrol sample 28 54MixlAB+Pathogen 24 57Pathogencontrol 18 51

Similar relation was observed for the mixed cultures of the listeria and Lactobacillus plantarum nBiMcc 2415. For Listeria monocytogenes i (fig. 3a) - the viable cell count reduction started at day 3, after which it suddenly decreased. on the 15th day, there were no living cells (fig. 3a), while Listeria monocytogenes kept its viability to 102 cfu/cm3 cells (fig. 3b). During the growth of Lactobacillus

plantarum nBiMcc 2415, the medium’s ph changed (table 2) and metabolites with antimicrobial activity were produced (bacteriocins), which inhibited listeria’s growth. the reduction degree of viable cells was strain specific (fig. 3).

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L.plantarum 2415m

E.coli 8739k

E.coli 8739m

b

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Time, d

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L.plantarum 2415k

L.plantarum 2415m

E.coli 25922k

E.coli 25922m

fig. 4. Growth dynamics of the Escherichia coli strains and Lactobacillus plantarum nBiMcc 2415 as pure and mixed cultures at 15-180Сa) Escherichia coli Atcc 8739b) Escherichia coli Atcc 25922

Both Escherichia coli strains were resistant to the influence of Lactobacillus plantarum nBiMcc 2415. the concentration of the viable cells decreased in the mixed cultures with Lactobacillus plantarum nBiMcc 2415 at 15-18oС. This process started around day 3, diminished slowly and in the end of the cultivation more than 103 cfu/cm3 were determined (fig. 4).

Similar relation was observed for the other representatives of enterobacteriaceae, the Salmonella strains. the decrease in the Salmonella cells numbers in the mixed culture with Lactobacillus plantarum nBiMcc 2415 started after the 3rd day and it was also related to the increase of the medium titratable


acidity above 40oТ (fig. 5, table 2). During further cultivation at 15-18oС their viable cell counts decreased slowly and on day 15 varied from 102 to 104 cfu/cm3 (fig. 5). the reduction was 2-3 folds in comparison with the cell concentration in the beginning of the process. the number of the Salmonella viable cells dropped under the minimal infective dose (105 cfu/cm3).

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Salmonella sp.k

Salmonella sp.m

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L.plantarum 2415m

S. abony 6017k

S. abony 6017m

fig. 5. Growth dynamics of the Salmonella strains and Lactobacillus plantarum nBiMcc 2415 as pure and mixed cultures at 15-180С

a) Salmonella sp.

b) Salmonella abony ntcc 6017

Lactobacillus plantarum nBiMcc 2415 inhibited the growth of Proteus vulgaris G but the inhibition was not complete. in a mixed culture, the viable cell numbers of both strains were reduced and this process started on day 3 for Proteus vulgaris G and on day 6 for Lactobacillus plantarum nBiMcc 2415. At the end of the experiment, the number of living Lactobacillus plantarum nBiMcc 2415 cells diminished to 1010 cfu/cm3 and those of Proteus vulgaris G to

102 cfu/cm3, and this was significantly lower than the minimum infective dose for Proteus vulgaris G (105 cfu/cm3)(fig. 6).

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g N

L.plantarum 2415k

L.plantarum 2415m

P.vulgaris Gk

P.vulgaris Gm

fig. 6. Growth dynamics of the Proteus vulgaris G and Lactobacillus plantarum nBiMcc 2415 and medium titratable acidity as pure and mixed cultures at 15-180С

the titratable acidity of the medium increased in the pure Lactobacillus plantarum nBiMcc 2415 culture as well as in the mixed culture with Proteus vulgaris G and its range was 18-60oТ (table 2).

antioxidant properties of L. plantarum nbIMcc 2415oxidation is a needed reaction in an organism’s metabolism. When the metabolism is disturbed, products from incomplete oxidation of oxygen (free radicals) are produced. they damage human tissues and cause different diseases. the application of probiotic bacteria with antioxidant activity in foods increases their biological activity and quality, as well as their shelf-life. Four methods for cell disintegration of the selected strain Lactobacillus plantarum nBiMcc 2415 were investigated. it was determined that the most appropriate is the alkaline lysis method. With this method L. plantarum nBiMcc 2415 showed high antioxidant activity (5.57 μmol TE/logN) (table 3). it was demonstrated, that as a component of meat starter cultures L. plantarum nBiMcc 2415 not only realized the purposeful fermentation process, but preserved the colour, formed the flavour and increased the meat products’ shelf-life as well.

Raw-dried meat sausages were prepared with addition of Lactobacillus plantarum nBiMcc 2415 in the amount of 108 cfu/g, for the realization of the desired lactic acid fermentation. the product was microbiologically tested during the fermentation and drying process. the obtained data are shown in table 4.

An organoleptic evaluation of the produced experimental lot of raw-dried meat sausages with the addition of Lactobacillus plantarum nBiMcc 2415 was made by specialists. the data from table 4 shows that during the fermentation process the


spoilage microflora was inhibited and the enterococci viable cell counts were reduced, which guaranteed the product safety as well as its quality. on the other hand, the product contained high concentration of viable cells (3.5.1010 cfu/g) of the probiotic strain Lactobacillus plantarum nBiMcc 2415, which turns the product into probiotic and healthy, and this increased its shelf-life

conclusionsthe strain L. plantarum nBiMcc 2415 preserved high total viable cell counts in the simulated low ph-values with the addition of pepsin, as well as in the presence of high bile salt concentrations. This confirms the results of Pennacchia et al. (9) on the requirements of probiotic cultures. in earlier investigations this probiotic culture was characterized with low proteolytic and lipolytic activity, low ability to form biogenic amines and stability of the growth in a medium with high concentrations of NaCl (15%), which fulfills the criteria for starter cultures (1).

L. plantarum nBiMcc 2415 grew in the raw sausage mass and produced lactic and other organic acids, bacteriocins and other biologically active compounds (1), which inhibit the development of pathogenic, toxigenic and saprophytic microorganisms to a large extent. this ensures the safety of both production and the end product. the inclusion of this culture provided the desired fermentation process in the raw sausage mass and reduced the pathogenic flora. The application of starter cultures with antioxidant activity preserved the colour of the meat products and delivered substances with antioxidant activity for the organism as well (6). L. plantarum nBiMcc 2415 showed comparatively high antioxidant activity. in the end product high concentration of viable cell counts (more than 109cfu/g) were determined, which was a necessary

requirement for a beneficial effect on the human organism, according to Donald & Brown, 1993 (3).

references1. ammor M.s. and Mayo b. (2007) Meat Science, 76, 138-

146.2. ammor s., dufour e., Zagores M., chaillou s.,

chevallier I. (2005) Food microbiology, 22, 529-538.3. donald J. and brown n.d. (1993) let’s live, november,

45-47.4. erkkila s. and Petaja e. (2000) Meat Science, 55, 297-

300.5. Klingberg t.d., axelsson l., naterstad K., elsser d.,

budde b.b. (2006) int. J. of Food Microbiology, 105, 419-431.

6. Kullisar t.M., Zilmer M., Mikelsaar M., vikalemm t., annuk h., Kairane c., Kilk a. (2002) international J. of Food Microbiology, 72, 215-224.

7. Macrae r., robinson r.K., sadier M. (1993) Food technology and nutrition, 5, 3082-3085.

8. ouwehand a.c., salminen s., Isolauri e. (2002) Antonie Van leeuwenhoek, 82, 279-289.

9. Pennacchia c., ercolini d., blaiotta G., Pepe o., Mauriello G., villani f. (2004) Meat Science, 67, 309-317.

10. Portillo P.M.c., amoroso M.J., oliver G. (1988) Milchwiessenschaft, 48(8), 490-491.

11. saidef J.a.o. and Gilliland s.e. (2005) J. Dairy Sci., 88, 1352-1357.

12. valdez G.f. and taranto M.P. (1990) Methods in Biotechnology, 14, 368-371.

table 3Antioxidant activity of the Lactobacillus plantarum nBiMcc 2415 determined through four different methods of cell disintegration

strain disintegration method

total viable cell count of the culture medium, cfu/cm3

antioxidant activity,μmol TE/log N

Lactobacillus plantarum nBiMcc 2415

temperature

3.84.1012

-Alkaline 5.57enzymatic 2.04Ultrasound -

table 4Microbiological analysis of the raw-dried meat sausage during the fermentation, drying and preservation process

day рН tvcc*,cfu/g

s. aureus,cfu/g

salmonella,cfu/g

E. coli,cfu/g

Enterococcus,cfu/g

lab**,cfu/g

4 5.2 Up to 10 - - - Up to 104 8.4.109

14 5.5 Up to 10 - - - Up to 103 5.4.1010

48 5.5 Up to 10 - - - Up to 10 3.5.1010

*tVcc - total viable cell count**lAB – lactic acid bacteria

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