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Antioxidant and antimicrobial activity of Kitaibelia vitifolia extract as alterna-tive to the added nitrite in fermented dry sausage

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Vladimir S. Kurcubic, Pavle Z. Maskovic, Jelena M. Vujic, Danijela V.Vranic, Slavica M. Veskovic-Moracanin, e G.Okanovic, Slobodan V. Lilic

PII: S0309-1740(14)00075-8DOI: doi: 10.1016/j.meatsci.2014.03.012Reference: MESC 6389

To appear in: Meat Science

Received date: 4 March 2013Revised date: 9 March 2014Accepted date: 19 March 2014

Please cite this article as: Kurcubic, V.S., Maskovic, P.Z., Vujic, J.M., Vranic, D.V.,Veskovic-Moracanin, S.M., Okanovic, .G. & Lilic, S.V.,Antioxidant and antimicrobialactivity of Kitaibelia vitifolia extract as alternative to the added nitrite in fermented drysausage, Meat Science (2014), doi: 10.1016/j.meatsci.2014.03.012

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http://dx.doi.org/10.1016/j.meatsci.2014.03.012

http://dx.doi.org/10.1016/j.meatsci.2014.03.012

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Research paper

Antioxidant and antimicrobial activity of Kitaibelia vitifolia extract as alternative to the added

nitrite in fermented dry sausage

Vladimir S. Kurćubića*

, Pavle Z. Maškovića, Jelena M. Vujić

b, Danijela V. Vranić

c, Slavica M.

Vesković-Moračaninc, Đorđe G. Okanović

d, Slobodan V. Lilić

c

Vladimir S. Kurćubića*

aDepartment of Food Technology, Faculty of Agronomy, University of Kragujevac, Cara Dušana

34, 32000 Čačak, Republic of Serbia. Phone: +381 32 30 34 00; Fax: +381 32 30 34 01;

Mobile: +381 64 125 94 52; E-mail address: [email protected]

Pavle Z. Maškovića

aDepartment of Food Technology, Faculty of Agronomy, University of Kragujevac, Cara Dušana

34, 32000 Čačak, Republic of Serbia. Phone: +381 32 30 34 00; Fax: +381 32 30 34 01;

E-mail address: [email protected]

Jelena M. Vujićb

bDepartment of Chemistry and Chemical Engineering, Faculty of Agronomy, University of

Kragujevac, Cara Dušana 34, 32000 Čačak, Republic of Serbia. Phone: +381 32 30 34 00; Fax:

+381 32 30 34 01;


Danijela V. Vranićc

cInstitute for Meat Hygiene and Technology, Kaćanskog 13, 11000 Belgrade, Republic of Serbia.

Phone: +381 11 265 06 55; Fax: +381 11 265 18 25; E-mail address: [email protected]

Slavica M. Vesković-Moračaninc



Đorđe G. Okanovićd

dInstitute of Food Technology, Bulevar Cara Lazara 1, 21000 Novi Sad, Republic of Serbia.

Phone: +381 21 48 53 707; Fax: +381 21 45 07 30;

E-mail adress: [email protected]

Slobodan V. Lilićc



*Coresponding author: Vladimir S. Kurćubić, Department of Food Technology, Faculty of

Agronomy, University of Kragujevac, Cara Dušana 34, 32000 Čačak, Republic of Serbia.

Phone: +381 32 30 34 00; Fax: +381 32 30 34 01; Mobile: +381 64 125 94 52;


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Abstract

Fermented dry sausages (FDS) without nitrite added, fortified with bioactive phenol and

flavonoid compounds originating from the ethanol extract of Kitaibelia vitifolia were food matrix

for investigation of its antioxidant and antimicrobial potency. These activities were researched in

order to improve the sausages’ shelf-life, safety, and provide health benefits to consumers as

well. The oxidative stability of the FDS, containing two different levels of natural preservative,

was evaluated using five different contemporary methods for antioxidative activity. The activity

was tested on the 20th day of the refrigerated storage. Minimum inhibitory concentrations of the

sausage extract were determined against six microorganisms, using a micro dilution method.

Determined optimal effective concentration of dissolved K. vitifolia extract (12.5 g/kg of meat

dough) revealed strong antioxidant activity, and moderate antimicrobial activity against E. coli

(minimum inhibitory concentrations = 15.625 μg/mL). The modified sausages had typical

chemical-physical characteristics of FDS, controlled on 0, 13, 26 d of ripening and 20, 40 and 60

d of storage.

Key words: Kitaibelia vitifolia, ethanol extract, fermented dry sausage, antioxidant activity,

antimicrobial activity, proximate composition

1. Introduction

Fermented sausages are meat products made without heat treatment during the production

process, which enables the biological values of nutritionally essential elements (proteins,

vitamins, minerals) to stay unchanged. Despite the widespread production, Europe is still a major

producer and consumer of fermented dry sausages (FDS), with highest figures in Germany, Italy,

Spain, and France (Lücke, 1998; FICT 2002; Di Cagno et al., 2008).

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Modification of the conventional composition of fermented sausages in order to make them

healthier is technologically possible thanks to the addition of plant extracts, fibers and

vegetables, elimination/partial replacement of fats, and reduction of different additives (Mendoza

et al., 2001; Muguerza et al., 2004; Fernández-Ginés et al., 2005; Müller, 2006; Vasilev et al.,

2010). Nitrite reactions result in change in the color of cured meat, microbial inhibition,

antioxidant effects and flavor (Schrader, 2010). Reduction in the use of nitrites has become a one

of the most important aims for the meat processors. Nitrite is recognized as a potentially toxic

compound of cured meat, including chemical toxicity, formation of carcinogens (reactions with

some biogenic amines and formation of N-nitrosamines) in food or after ingestion, and

reproductive and developmental toxicity (Coughlin, 2006). Some experiments on nitrites

reduction were sucessfully applied (Sebranek et al. 2007; Yilmaz and Zorba, 2010; Kurt and

Zorba 2011; Hospital et al., 2012). Schrader (2010) reports that the two types of “uncured, no-

nitrate-or-nitrite-added” meat products are available on the marketplace, as natural or organic,

with a higher level of safety than the traditional products. The first type is a truly uncured

product, with no replacement for nitrate or nitrite, whose typical properties showed to be more

variable than that observed in conventionally cured meats. The second type is produced using

alternative methods which utilize naturally occurring nitrates and nitrites found in vegetables and

sea salts and demonstrate traditional color and flavor characteristics.

The above mentioned results suggest that active principles of plant species Kitaibelia vitifolia

have potential for being used in preservation of meat products, without available literature data

on its traditional use.

The purpose of the present study was to develop no-nitrite-added fermented dry sausages,

modified by using ethanol extract from the overground part (stems, leaves and flowers) of

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Kitaibelia vitifolia as a functional ingredient. The primary aim of study was to select the most

suitable effective concentration of extract to be added, on the basis of the oxidative and

microbiological stability of FDS during storage under different conditions (aerobic or anaerobic

packs). The impact of the nitrites replacement on the quality aspects of FDS was yet another aim.

2. Material and methods

2.1. Plant material

Kitaibelia vitifolia is a member of the Malvaceae family, an imposing and undemanding

Mallow from ex-Yugoslavia. The above-ground part of the test plant was collected in Central

Serbia (latitude 40.1965, longitude 20.28352, and 322 m above sea) in May 2009, at its

flowering stage. This robust, perennial, shade-loving plant has bold, maple-like leaves (silvery

when young) and a stalk-full of small white-to-pink flowers. Plant hardiness of K. vitifolia is in

the zone 6 (-23.33 °C to -17.78 °C). The species was identified and the voucher specimen was

deposited at the Department of Botany, Faculty of Biology, University of Belgrade (16350

BEOU, Lakušić Dmitar).

2.2. Preparation of herb extract

Samples prepared from the over ground part of the plant Kitaibelia vitifolia (10.0 g) were

extracted by 96 % ethanol (100.0 mL) as a solvent. The extraction process was carried out using

an ultrasonic bath (Brason and Smith-Kline Company, B-220) at room temperature for 1 hour.

The goal was the highest extraction yield of phenol acids. After filtration, 5 mL of the liquid

extract was used for extraction yield determination. The solvent was removed by a rotary

evaporator (Devarot, Elektromedicina, Ljubljana) under vacuum, and was dried at 60 °C to

constant weight. The dried extracts were stored in glass bottles at 4 °C to prevent oxidative

damage until analysis. Extract of the Kitaibelia vitifolia was dissolved in sterile distilled water at

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concentrations of 3 % (w/v) and 10 % (w/v), before adding in fortified production batches PB II

and PB III of FDS. Value of pH of the prepared extract was 4.5. Spectrophotometric

measurements were performed using a UV-VIS spectrophotometer MA9523-SPEKOL 211

(ISKRA, Horjul, Slovenia). The chemical composition of ethanol herb extract of Kitaibelia

vitifolia used in present study was previously determined by Mašković et al. (2011).

2.3. Sausage formulation and processing

Three production batches (PB) of fermented dry sausages (FDS), about 20 kg each, were

prepared accordingly to the procedure described bellow. Three formulations of FDS were made

with frozen pork shoulder (40 %), beef meat II category (loin, back, shoulder – 30 %) and frozen

pork back fat (30 %). The following additives were added in gram per kilogram quantities to the

meat mixture of PB I: spice for Kulen (Lay Gewürze) 11 g/kg, Paprika extract - Oleorezin

30.000 FE (Lay Gewürze) 1 g/kg, nitrite salt 27 g/kg, TARI®

S77 [GdL (E575), sugars, salt,

sodium-isoascorbat (E 316)] 9 g/kg. This original mixture was used as control sample. To assess

the influence of the various concentrations of herb extract, nitrite was replaced by dissolved herb

extracts of the K. vitifolia in effective concentration of 30.0 g/kg of meat dough in PB II and 12.5

g/kg of meat dough in PB III. To avoid the influence of its own antioxidant potential of hot

pepper and garlic to the accuracy of the results of determining antioxidant effects, these

traditional spices were not added to the experimental sausages.

The FDS were prepared on the same day and in an identical manner in a small-scale plant

“Kotlenik-promet” Ltd (Lađevci, Central Serbia), in accordance with industrial processing.

Partially defrosted meat and pork fat were first cut into small pieces using a guillotine (Sind

Šabac, Serbia). The meat was minced using a meat grinder (REX Technologie GmbH & Co. KG)

down to the size of about 8 mm, and then transferred to the cutter. All of the other ingredients

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were added and mixed with minced meat in a cutter for 4 min at the temperature of -1 to -3 °C.

Herb extract of Kitaibelia vitifolia or nitrite salt were added to the prepared meat dough in

accordance with the following recipe: PB I was prepared as a control group with nitrites; PB II

was manufactured with K. vitifolia extract prepared at concentrations of 3 % (w/v), and added to

meat dough in quantity of 30.0 g/kg. PB III was produced with K. vitifolia extract prepared at

concentrations of 10 % (w/v), and added to meat dough in quantity of 12.5 g/kg. As a following

process, each stuffing was filled into natural pork casings of 36-38 mm diameter (country of

origin: Spain), using a filling machine (VEMAG, model ROBBY-2, 1998) at 2 °C. Filled

sausages were hand-paired, hung on metal rods, set on the cart and transported to an air-

conditioned chamber for ripening, smoking and drying.

Temperature and relative humidity (RH) in the air-conditioned chamber during the process of

ripening, smoking and drying was changed: 22 °C/92% on 1

st day, 20

°C/88% on 2

nd day, 19

°C/86% on 3

rd day, 18

°C/82% for 4

th day, 17

°C/78% for 5

th day and 15

°C/72% from the 6

th to

26th

days. Sausages were smoked 5 hours daily (from the 3rd

to the 5th

d) with filtrated smoke

from beech wood, smoked temperature 18 °C. At the end of the ripening process, which lasted

for 26 days, sausages were stored at 4 °C. Each PB of sausages was divided in two groups: first

was aerobically packed and the second one was vacuum packed (Inauen Machinen, AG VC 999)

using polyethylene (PE) bags thickness 0.07 mm (Blik-produkt Kikinda, Serbia). Chemical-

physical analysis was carried out during processing (ripening: 0th

, 13th

and 26th

day) and storage

at 4 °C (after 20, 40 and 60 days). Antioxidant experiments were conducted on 20

th day of

storage (46 days from the 0 d).

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2.4. Preparation of sausage extract

Wrappers were removed from FDS samples, stuffed, chopped, and then homogenized in the

blender. Ten grams of homogenized sausages sample were taken and dissolved in 10 mL of

distilled water to obtain a concentration of 1 mg per 1 mL of distilled water. The aqueous extract

of FDS was prepared for antioxidant and antimicrobial activity testing.

2.5. Determination of total phenol content in FDS

Total phenols in experimental FDS were estimated according to Singleton et al. (1999). The

aqueous extract of sausages was diluted to the concentration of 1 mg/mL, and aliquots of 0.5 mL

were mixed with 2.5 mL of Folin-Ciocalteu reagent (previously diluted 10-fold with distilled

water) and 2 mL of NaHCO3 (7.5 %). After 15 min at 45 °C, the absorbance was measured at 765

nm using a spectrophotometer against a blank sample. Total phenols were determined as gallic

acid equivalents (mg GA/g extract), and the values are presented as mean of three

determinations.

2.6. Determination of flavonoid content in FDS

Total flavonoids in experimental FDS were determined according to Brighente et al. (2007).

A total of 0.5 mL of 2 % aluminum chloride (AlCl3) in methanol was mixed with the same

volume of methanol solution of plant extract. After 1 hour at room temperature, the absorbance

was measured at 415 nm in a spectrophotometer, compared to a blank sample. Total flavonoids

were determined as rutin equivalents (mg RU/g dry extract), and the values are presented as

mean of three determinations.

2.7. Determination of total antioxidant (AOX) capacity

The total antioxidant activity was evaluated by the phosphor-molybdenum method (Prieto et

al., 1999). The assay is based on the reduction of Mo (VI) - Mo (V) by antioxidant compounds

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and subsequent formation of a green phosphate/Mo (V) complex at acid pH. A total of 0.3 mL of

sausage sample extract was combined with 3 mL of reagent solution (0.6 M sulfuric acid, 28 mM

sodium phosphate and 4 mM ammonium molybdate). The tubes containing the reaction solution

were incubated at 95 °C for 90 min. Then, the absorbance of the solution was measured at 695

nm using spectrophotometer against the blank after cooling to room temperature. Methanol (0.3

mL) as a sausage extract was used in its blank form. Ascorbic acid (AA) was used as a standard

and the total antioxidant capacity was expressed in milligrams of ascorbic acid per gram of dry

sausage extract.

2.8. Determination of DPPH free radical scavenging activity

The method used by Takao et al. (1994) was adopted with suitable modifications from

Kumarasamy et al. (2007). DPPH (2, 2-dephenyl-1-picrylhydrazyl) (8 mg) was dissolved in

MeOH (100 mL) to obtain a concentration of 80 µg/mL. Serial dilutions were carried out with

the stock solution (1 mg/mL) of the sausage extract. Solutions (2 mL each) were then mixed with

DPPH (2 mL) and left to stand for 30 min for any reaction to occur, and the absorbance was

measured at 517 nm. Ascorbic acid (AA), gallic acid (GA) and butylated hydroxytoluene (BHT)

were used as reference standards and dissolved in methanol to make the stock solution with the

same concentration (1 mg/mL). Control sample was prepared containing the same volume

without test compounds or reference antioxidants. Ninety-five percent a methanol sausage

extract was used as a blank. The DPPH free radical scavenging activity (%) was calculated using

the following equation:

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The percentage inhibition values were calculated from the absorbance of the control (Ac) and

of the sample (As), where the controls contained all the reaction reagents except the extract or

positive control substance.

The IC50 value, defined as the concentration of the test material that leads to 50 % reduction

in the free radical concentration, was calculated as µg/mL through a sigmoid dose-response

curve.

The relation between the inhibition percentage and the concentration of the researched

extracts of sausages was determined and it was inserted into statistical software OriginPro 8.

Dislinear algorithm was used for the results of IC50 because the inhibition depends on the

relation of nonlinear curve fit Growth and the sigmoidal dose-response function. The function

which is then received as a result of this is as following:

In which Y stands for the absorbance of the measured solutions, X stands for the

concentration of the tested extracts of sausages, while Xо, А1, А2 and p are statistical

parameters. After solving the formula following the X parameter we get the following formula:

If we take that Y = 50 in this formula, we get the value of IC50.

2.9. Determination of inhibitory activity against lipid peroxidation

Antioxidant activity was determined by the thiocyanate method (Hsu et al., 2008). Serial

dilutions were carried out with the stock solution (1 mg/mL) of the sausage extracts, and 0.5 mL

of each solution was added to linoleic acid emulsion (2.5 mL, 40 mM, pH 7.0). The linoleic acid

emulsion was prepared by mixing 0.2804 g linoleic acid, 0.2804 g Tween-20 as emulsifier in 50

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mL 40 mM phosphate buffer and the mixture was then homogenized. The final volume was

adjusted to 5 mL with 40 mM phosphate buffer, pH 7.0. After incubation at 37 °C in the dark for

72h, a 0.1 mL aliquot of the reaction solution was mixed with 4.7 mL of ethanol (75 %), 0.1 mL

FeCl2 (20 mM) and 0.1 mL ammonium thiocyanate (30 %). The absorbance of the mixture was

measured at 500 nm and the mixture was stirred for 3 min. Ascorbic acid, gallic acid, α-

tocopherol and BHT were used as reference compounds. To eliminate the solvent effect, the

control sample, which contained the same amount of solvent added to the linoleic acid emulsion

in the test sample and reference compound, was used. Inhibition percent of linoleic acid

peroxidation was calculated using the following formula:

2.10. Measurement of ferrous ion chelating ability

The ferrous ion chelating ability was measured by the decrease in absorbance at 562 nm of

the iron (II)-ferrozine complex (Yan et al., 2006; Carter, 1971). One milliliter of 0.125 mM

FeSO4 was added to 1.0 mL sample (with different dilutions), followed by 1.0 mL of 0.3125 mM

ferrozine. The mixture was let to equilibrate for 10 min before the absorbance was measured.

The ability of the sample to chelate ferrous ion was calculated relative to the control (consisting

of iron and ferrozine only) using the formula:

2.11. Determination of hydroxyl radical scavenging activity

The ability of K. vitifolia to inhibit non site-specific hydroxyl radical-mediated peroxidation

was carried out according to the method described by Hinneburg et al. (2006). The reaction

mixture contained 100 µL of sausage extract dissolved in water, 500 µL of 5.6 mM 2-deoxy-D-

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ribose in KH2PO4-NaOH buffer (50 mM, pH 7.4), 200 µL of premixed 100 µM FeCl3 and 104

mM EDTA (1:1 v/v) solution, 100 µL of 1.0 mM H2O2 and 100 µL of 1.0 mM aqueous ascorbic

acid. Tubes were vortexed and incubated at 50 °C for 30 min.1 mL of 2.8 % TCA and 1 mL of

1.0 % TBA were added to each tube afterwards. The samples were vortexed and heated in a

water bath at 50 °C for 30 min. The extent of oxidation of 2-deoxyribose was estimated from the

absorbance of the solution at 532 nm. The values are presented as a mean of three

determinations.

2.12. Determination of nitrogen content

The nitrogen content (N x 6.25) was determined by the Kjeldahl method using a semi

automatic distillation unit Analyse Unit, Kjeltec TM 8400 (FOSS, Sweden), in a Basic 250,

Tecator TM digester (Foss, Sweden) according to the manufacturer’s instructions: User Manual

TM digester, 1001 3846/Rev.4, Foss, Sweden; User Manual book - Analyse Unit, Kjeltec TM

8400, 6002 3655/Rev.3, Foss, Sweden (SRPS ISO 937/1992).

2.13. Determination of water content, total fat content and total ash content

Water content was determined by drying at 103 °C ± 2

0C (SRPS ISO 1442/1998). Total fat

was determined by Soxhlet extraction with petroleum ether after acid hydrolysis of the sample

(SRPS ISO 1443/1992). Ash content was determined by measuring the mass of residue after

annealing at 550 °C ± 25

°C (SRPS ISO 936/1999).

2.14. Determination of Na content

After cooling, samples were transferred into a 50 mL volumetric flask with de-ionized water.

Analyses were carried out on atomic absorption spectrometer “SpectrAA 220” (Varian, Palo

Alto, California, USA) according to Varian AAS Analytical methods (Flame Atomic Absorption

Spectrometry Analytical Methods). All reagents used were of analytical grade and equipment

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was pre-calibrated appropriately with standardized solutions prior to measurement. Samples

were prepared by microwave digestion (ETHOS TC, Milestone S.r.l., Sorisole, Italy) according

to manufacturer’s recommendations (Tips and Techniques for ETHOS Series Microwave Lab

Stations, an Operations Overview). 0.5 g of the sausage sample was treated with 8 mL of nitric

acid (HNO3) and 2 mL of hydrogen peroxide (30 % H2O2); temperature program was as follows:

5 min from room temperature to 180 °C then 10 min hold at 180

°C.

2.15. Determination of pH values

The pH value of the samples was measured by laboratory pH-meter (Cyber Scan 510 5 pH

Meter, EUTECH Instruments, The Netherlands) according to SRPS ISO 2917/2003). 10 g of

minced sample was homogenized with 100 mL of 0.1M KCl solution in CombiMax 600 blender

(Brown GmbH, Kronberg, Germany). The pH value of the resulting slurry is measured directly

with digital pH meter (accuracy ±0.01 pH units) when achieves a constant value. Calibration of

the pH meter at 20±2 °C before use is conducted using 2 buffer solutions (pH=4.00±0.05 and

pH=7.00±0.01). pH value is expressed as mean of three determinations.

2.16. Activity of water (aw) determination

The activity of water value was determined using a hygrometer (FA-st/1 aw meter, GBX

Scientific Instruments, France) at 25±1 °C according to the manufacturer’s instructions: FA-sr/1

User Manuel GBX Scientific Instruments, France. Before carrying out of measurements, the

equipment recalibrates using the salt standards given in Annex A, ISO 21807:2004 (E).

Examined samples were homogenized in CombiMax 600 blender (Brown GmbH, Kronberg,

Germany). Approximately 5 g of homogenous sample is put in disposable cup, completely

covering the bottom of the cup and filling not more than half of it. The activity of water value

was directly measured by a hygrometer with accuracy of ±0.003.

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2.17. Antimicrobial activity

The antibacterial activity was tested against the Staphylococcus aureus ATCC 25923,

Escherichia coli ATCC 25922, Proteus vulgaris ATCC 13315, Proteus mirabilis ATCC 14153

and Bacillus subtilis ATCC 6633. The antifungal activity was tested against the mold Aspergillus

niger ATCC 16404. The mold was cultured on potato-glucose agar for 7 days at a room

temperature of 20 °C. Then they were cultured on a new potato-glucose substrate for 7 more

days. The culturing procedure was performed four times, after which pure cultures requiring

determination were obtained. Samples for examination were aqueous extract of sausages from all

three PB and both storage manners. Minimum inhibitory concentrations (MIC) against the test

microorganisms were determined using a micro dilution method in 96 multi-well micro titer

plates (Satyajit et al., 2007). All tests were performed in Muller-Hinton broth (MHB) with the

exception of mold, in which case Sabouraud dextrose broth was used. A total of 100 µL stock

solution of sausage extract (200 µL/mL) was pipetted into the first row of the plate. Fifty µL of

Mueller-Hinton or Sabouraud dextrose broth (supplemented with Tween 80 at a final

concentration of 0.5 % (v/v) for analysis of sausage extract) was added to the other wells. Fifty

µL from the first test wells was pipetted into the second well of each micro titer line, and then 50

µL of scalar dilution was transferred from the second to the twelfth well. Ten µL of resazurin

indicator solution (prepared by dissolution of a 270 mg tablet in 40 mL of sterile distilled water)

and 30 µL of nutrient broth were added to each well. Finally, 10 µL of bacterial suspension (106

CFU/mL) and mold suspension (3 × 104 CFU/mL) was added to each well. The growth

conditions and the sterility of the medium were checked for each strain. Standard antibiotic

amracin was used to control the sensitivity of the tested bacteria, whereas ketoconazole was used

as control against the tested mold. Plates were wrapped loosely with cling film to ensure that

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bacteria did not become dehydrated and prepared in triplicate, and then they were placed in an

incubator at 37 °C for 24

h for the bacteria and at 28

°C for 48

h for the molds. Color change was

then assessed visually. Any color change from purple to pink or colorless was recorded as

positive. The lowest concentration at which color change occurred was taken as the MIC value.

The average of 3 values was calculated and represented the MIC for the tested compounds and

standard drug.

2.18. Sensory evaluation

At the end of fermentation (26 d), FDS were tested by a panel of 5 trained assessors. Panelists

were recruited from the staff of the Institute for Meat Hygiene and Technology, Belgrade,

Republic of Serbia, chosen on the basis of previous experience in consuming FDS. Assessor’s

senses were previously tested using a test to determine the sense of taste (SRPS ISO 3972/2002)

and test for training of assessors in the detection and recognition of odors (SRPS ISO

5496/2002). Using quantitative - descriptive test (SRPS ISO 6658/2002), with grading scale from

one to five (1-unacceptable, 5-optimum), sensory properties of FDS were assessed (cut color,

consistency, smell, taste and overall impression).

2.19. Statistical analysis

All measurements in chemical and physic-chemical analyses were carried out repeatedly for

three times and results were subject to analysis of variance (ANOVA) using SPSS software,

version 15.0 (SPSS, Chicago, Illinois, USA). Differences between means were determined by the

least significant difference (LSD) test, and significance was defined at P < 0.05. Differences

between means calculated for aerobically and anaerobically packaged sausages were determined

by non parametric Mann-Whitney Test.

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The results of antioxidant activity examinations were presented as mean ± standard

deviations of three determinations. Statistical analyses were performed using Student´s t-test and

one way analysis of variance. Multiple comparisons of means were done by LSD (least

significant difference) test. A probability value of P < 0.05 was considered significant. All

computations were made by employing the statistical software (SPSS, version 15.0). IC50 values

were calculated by nonlinear regression analysis from the sigmoid dose-response inhibition

curve.

3. Results and discussion

3.1. Antioxidant activity

Mašković et al. (2011) showed that the major phenol component of Kitaibelia vitifolia

ethanol extract is rosmarinic acid (2.937 mg/g of plant extract) by HPLC/DAD analysis. Next to

it, lower concentrations of the following phenol acid were determined in milligram per gram

quantities of the K. vitifolia extracts: p-hydroxybenzoic acid 0.182, caffeic acid 0.103,

chlorogenic acid 0.044, syringic acid 0.042, p-coumaric acid 0.031, ferulic acid 0.093 and

quercetin 0.004. The presence of phenolic hydroxyl groups of these acids is partly responsible

for the expressed antioxidant power of plant extract of K. vitifolia and modified sausage to which

a plant extract is added. There are reports in the literature stating that there is a highly positive

relation between total phenolic content and antioxidant activity in many plant species (Kırca and

Arslan, 2008).

The results on total phenol, flavonoid, and total antioxidant capacity are given in Table 1.

IC50 values were determined for each measurement: DPPH free radical scavenging activity,

inhibitory activity against lipid peroxidation, metal chelating activity, hydroxyl radical

scavenging activity, and presented in Table 2.

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Table 1

Table 2

FDS containing herb extracts of K. vitifolia have slower rates of oxidation than those with

nitrite salts. The antioxidant capacity related to the DPPH radical revealed that the values in the

samples of FDS fortified with herb extract (PB II and PB III) were lower than the values of the

control sausages, indicating a stronger antioxidant activity of herb extracts in relation to the

nitrite salt, traditionally used as a preservative.

Using five different methods for antioxidative activity testing (total antioxidant capacity,

DPPH scavenging activity, inhibitory activity against lipid peroxidation, metal chelating activity

and hydroxyl radical scavenging activity), the strongest effect was confirmed in samples of FDS

from PB III. Dissolved herb extract of K. vitifolia (10 %, w/v) applied in effective concentration

of 12.5 g/kg of meat dough in FDS PB III revealed stronger action then dissolved extract (3 %,

w/v) in concentration of 30 g/kg of meat dough in FDS PB II (Table 2).

K. vitifolia herbal extract exerted a powerful antioxidant effect. The results are more

significant in light of the well-known fact that fat from frozen meat and solid fat (used in the

production of FDS in present study) easily oxidized and the oxidation of fats is prominent in the

fermented sausages smaller diameter (Müller, 2006). Another reason demonstrating the

significance of our results was that the antioxidant effectiveness of all flavonoids was reduced in

products with higher fat content (Finotti and Di Majo, 2003). Our study confirmed the benefit of

anaerobic packaging of meat products through slower increase in fat oxidation during storage,

similar like reported by Ansorena and Astiasaran (2004).

The antioxidant capacity in the fortified sausages was the result of their composition,

containing phenol acids and flavonoids from K. vitifolia extract. Antioxidant activity is thus

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sequestering free radical as a result of their hydrogen-donating capacity. Total phenol and

flavonoid content was highest in fortified fermented dry sausage belonging production batches

III, followed by the sausages from PB II and PB I, with a much lower content of these

ingredients. This finding is consistent with results obtained in the study, and suggests that the

level of antioxidant activity depends on the amount of these ingredients. Extensive and diverse

research indicated that the phenol compounds, especially flavonoids, have proved to have

positive effects on human health and protect against cancer (Harris et al., 2007), cardiovascular

diseases (Mazza, 2007) and inflammatory, allergic and ulcerous disorders (Jung et al., 2007;

Lien and Chuong, 2008). Antioxidant and anti-hypertensive effects (Hwang and Yen, 2008) are

also of great importance. This fact suggests that the processing and storage conditions that would

help preserve these compounds in the FDS should be sought in order to make the benefits until

the last moment consumers buy the products.

3.2. Antimicrobial activity

The results on antimicrobial activity obtained by the dilution method are given in Tables 3

and 4. Minimum inhibitory concentrations (MIC) were determined for 6 microbial strains.

Inhibitory activity against bacteria and mold was higher in samples that belong to PB modified

with K. vitifolia extracts. In samples prepared from anaerobically packed FDS, level of

antimicrobial activity was different between control and nitrites-free PB against all tested

microbes. Generally, in samples prepared from aerobically stored FDS, values for MIC were

same in PB I and PB II samples, against P. vulgaris and B. subtilis. Dissolved herb extract

applied in effective concentration of 12.5 g/kg of meat dough added in FDS of PB III have

positive influence on antimicrobial effects. Stronger activity revealed in PB III against P.

vulgaris and A. niger, in anaerobically packed FDS. In aerobically packed FDS, stronger activity

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observed in PB III samples, against P. vulgaris, B. subtilis and A. niger. The highest

susceptibility to the extract of fortified sausages among the bacteria tested was exhibited by E.

coli (MIC = 15.625 μg/mL), in both PB II and PB III, regardless of the method of packaging.

Results of our previous examination of antimicrobial activity and determining MIC values of

ethanol extract of the Kitaibelia vitifolia in vitro (Mašković et al., 2011) were basis to continue

with the research of antibacterial and antifungal activities in food matrix of fortified FDS.

Application of plant extracts for control of meat spoilage bacteria and food-borne pathogens

requires the evaluation of efficacy within meat products or in model systems that closely

simulate meat composition. In general, the efficacy of many added antimicrobials may be

reduced by certain food components (Glass and Johnson, 2004). Our study was comparable to

bellow mentioned studies done using herb extracts on food-borne microorganisms.

Experimentally results of Fullerton et al. (2011) showed that the phenol compounds of extract of

sorrel (H. sabdariffa, fam. Malvaceae) exhibited antibacterial activity against the E. coli bacterial

strains. With the number of hydroxyl groups present on the phenol ring there is increased

hydroxylation, and with increased hydroxylation there will be an increased antimicrobial

activity. Our results compared to those of phytochemical studies of Saravanakumar et al. (2009)

indicated a much stronger activity of K. vitifolia, despite the facts that Thespesia populnea (L.)

Sol. Ex Correa belongs to the same family - Malvaceae, and contained flavonoids, alkaloids,

tannins and anthroquinone glycosides. Moreno et al. (2006) determined the antimicrobial activity

of rosmarinic acid which showed inhibitory activity for neither E. coli nor B. subtilis. Caffeic,

ferulic and p-coumaric acids inhibit E. coli (Herald and Davidson, 1983). Shan et al. (2007)

confirmed that among the five bacteria tested, S. aureus was the most sensitive to the 46 extracts,

while E. coli was the most resistant. A possible explanation for these observations may lay in the

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facts that Gram-negative bacteria have an outer membrane and a unique periplasmic space not

found in Gram-positive bacteria (Duffy and Power, 2001). There is some evidence that minor

components have a critical part in antibacterial activity, probably by producing a synergistic

effect between the other components (Burt, 2004).

The mechanisms of action of each phenol compound against various bacteria are also very

complicated (Kalemba and Kunicka, 2003; Burt, 2004). Therefore, it is necessary to conduct a

more profound research on the relationship between antimicrobial activity and chemical structure

of each phenol compound in the K. vitifolia extract.

Table 3

Table 4

3.3 Sensory evaluation

There were not differences in cut color, taste and overall acceptability due to dissolved herb

extract applied in effective concentration of 12.5 g/kg of meat dough added in FDS of PB III,

compared to the control with added nitrites. Between controls PB I and PB III there are

significant differences (P < 0.01) in the consistency and smell of fermented sausages.

Fig. 1.

3.4. Proximate composition of FDS

Both control (PB I) and fortified, no nitrites added, formulas of sausages (PB II and III) had

final values within or under considered borderlines defining FDS (Toldrá, 2010): pH ranking

between 5.20 and 5.80 (5.33; 5.66 and 5.81), a moisture significantly lower than 30 % (17.25;

17.32 and 18.87), final values for aw under 0.9 - 0.91 (0.718; 0.712 and 0.733) and M : P ratio

lower than 2.3 : 1 (0.74 : 1; 0.71 : 1 and 0.79 : 1).

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Table 5 and 6 show the results obtained in the chemical and physic-chemical analyses of

different sausage formulations. The protein content increased significantly (P < 0.05) during

whole ripening period, and showed highest values on 20 d and 40 d of storage, in all production

batches of FDS. At the end of the ripening period (26 d), the protein content of FDS was 23.33 %

to 24.42 %, very similar like results (21.46 % to 23.55 %) reported by Živković et al. (2012). The

moisture content declined significantly (P < 0.05) during whole ripening period in all

experimental groups, and continued until the 60 d of storage in aerobically packed sausages from

all PB. In anaerobically packed sausages of all PB, values for moisture content increased slightly

to 20 d, and showed decrease of 40 and 60 d of storage. Anaerobically packaging showed better

performance in preventing loss on drying of the FDS during storage. At the end of ripening,

experimental sausages showed a very low level of moisture content (17.25 % to 18.87 %), lower

than in Greek traditional sausages (28.22 % to 34.70 %), which are very similar in composition.

The moisture content level was also lower than in Serbian fermented sausages (23.50 % to 36.70

%) prepared exclusively of pork meat and back fat (Kozačinski et al., 2008). In his investigation

of chemical composition of Serbian Sremska fermented sausage, at the end of ripening (14 d),

moisture content decreased to the level of 25.11% to 27.89% (Živković et al., 2012). The fat

content increased significantly (P < 0.05) during whole ripening period, in all experimental

groups. All production batches of experimental sausages contain at the end of the drying period

(26 d) more than 50 % fat, which makes the texture of experimental sausages gently. Sodium

content in sausages slightly increased during ripening and storage (PB I and PB III), but 13 d in

PB II have higher value than 26 d, and after that gradually increased, because of dehydration

process.

3.5. Water activity (aw)

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Fermented products will obtain a longer shelf life due to the reduction of water activity value

(drying, salting) or a combined effect of aw decrease and pH decrease (Vandendriessche, 2008).

Water activity (aw) levels of FDS decreased from 0.937 to 0.712 during ripening, typical for

similar products in Greece (from 0.84 to 0.87 on 0 day to 0.77 to 0.8 on 28 d), reported by

Kozačinski et al. (2008), and for a similar product of a narrow diameter (Ø approximately 30

mm) resulting from slow maturation (Vesković Moračanin, 2007). During the ripening process

aw-value of fermented sausages constantly decreased, representing a factor of safety in

production. This decrease happened due to the decreased water content, salt diffusion and the

dehydration that the pieces undergo during the drying–ripening stage (Lorenzo, 2014). With a

number of other obstacles, "hurdle effect" acting one after the other in a specific order (nitrite,

pH, aw and antimicrobial properties of lactic acid bacteria - LAB), a factor in the development of

microbiologically stable product (Leistner, 2000). Statistical analysis revealed a statistically

significant difference (p <0.05) between aw-values of samples of the control group sausages

(PBI) and sausage with added herbal extract, during all 60 days of storage. Also, sausages

packed in vacuum had higher values of aw (PB I - 0.619; PB II - 0.635; PB III - 0.693) compared

to sausage packed under aerobic conditions (PB I 0.595; PB II 0.621; PB III 0.633). Differences

were statistically significant (p <0.05). As can be seen in Tables 5 and 6, values of water activity

dropped continually to 60 d of storage. The obtained values of aw, in all groups indicate products

that are from the point of failure microbiologically stable, thus their storage at ambient

temperatures possible (Incze, 1987).

Table 5

Table 6

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3.6. pH

The initial pH value in PB I, PB II and PB III samples of FDS varies in a broad interval,

possibly caused by very low pH value of K. vitifolia extract added (pH 4.5). The decline in the

pH value during the first days of ripening (Tables 5 and 6) is very important due to the inhibition

of undesired bacteria, rate of conversion of color, and formation of desired flavor in fermented

dry sausages (Lücke, 1998). Results of our research confirmed the usual trend of microbial

growth in fermented sausages, where the number of LAB increases at the very beginning of the

fermentation and leads to rapid decrease of pH value of FDS. The similar dynamics and pH

values determined in the moment when the ripening completed in Greek, Serbian and Croatian

FDS, reported by Kozačinski et al. (2008). Salgado et al. (2005) reported an increase of pH value

in the latter stages of the ripening process appears to be more related to the decrease in lactic acid

content than to the formation of low molecular weight nitrogen compounds.

Conclusions

In our study, we were able to produce healthier meat product with no nitrites added and

contain in addition health-promoting bioactive phenol and flavonoid compounds. The present

study is the first report on the evaluation of the antioxidant and antimicrobial activities of K.

vitifolia extract, which requires assessment of efficacy within meat products, because of the well-

known problems of incompatibilities of natural preservative with food matrix properties. This

study has demonstrated a great potential of Kitaibelia vitifolia ethanol extract in preserving of

fermented dry sausage during production and refrigerated storage. Therefore, determined optimal

concentration of dissolved K. vitifolia extract (10 % w/v, applied in effective concentration of

12.5 g/kg of meat dough) revealed the strongest activity of five different contemporary methods

for antioxidative effects testing. The highest susceptibility to the extract of fortified sausages

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among the bacteria tested was shown by E. coli (MIC = 15.625 μg/mL). Improvement of shelf-

life and microbiological safety for no nitrites added sausages fortified with herb extract provide

safer products for the consumers. Herb extract did not interfere in the sensory acceptance of the

product. Obtained results of this study showed proximate composition similar with other well-

known fermented dry sausages produced in region, confirmed their good quality and safety.

Further research should be done in order to determine the relationship between antimicrobial

activity and chemical structure of each phenol compound in the K. vitifolia extract. Recognizing

the fact that nitrites have a broad spectrum of effects (especially antibacterial activity against

Clostridium botulinum), we believe that it is necessary to improve the investigation of the use of

herb extracts as nitrite replacement in the future, and promote new approaches, such as using a

low dose of synergistic antimicrobial combination of plant extracts.

Acknowledgements

The work is part of the research project record number 046009 III project, funded by the

Ministry of Education, Science and Technological Development of the Republic of Serbia.

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Fig. 1. Results of the sensory evaluation (quantitative - descriptive analysis) carried out in the

samples of FDS formulated with different K. vitifolia extract concentrations (PB III and PB II) and in

the samples of FDS belonging control group (PB I)

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Table 1 Total phenol and flavonoid content and total antioxidant capacity of experimental

fermented dry sausage

Packaging/PB number Total phenolics

(mg GA/g)

Flavonoids

(mg RU/g)

Total antioxidant

capacity

(μg AA/g)

Aerobically packs PB I 60.16±0.34 31.82±0.34 55.35±0.78

Aerobically packs PB II 73.56±0.45 28.09±0.98 65.13±0.25

Aerobically packs PB III 87.45±0.65 24.99±0.75 93.45±0.62

Anaerobically packs PB I 56.56±0.51 35.93±0.56 60.28±0.55

Anaerobically packs PB II 74.54±0.35 28.75±0.78 80.76±0.35

Anaerobically packs PB III 88.97±0.55 25.85±1.05 98.69±0.69

AA, Ascorbic acid; GA, Gallic acid; RU, Rutin.

Table 2 Antioxidant activities in experimental fermented dry sausage and standards

Packaging/PB number

aIC50 (µg/ml)

DPPH

scavenging

activity

Inhibitory activity

against lipid

peroxidation

Metal chelating

activity

Hydroxyl radical

scavenging

activity

Aerobically packs PB I 43.49±0.34 54.37±0.11 32.79±0.18 46.56±0.19

Aerobically packs PB II 27.45±0.55 34.67±0.49 22.55±0.35 33.35±0.67

Aerobically packs PB III 19.67±0.81 21.53±0.83 21.08±0.35 23.68±0.91

Anaerobically packs PB I 39.67±0.47 34.87±1.23 25.75±0.92 39.05±0.36

Anaerobically packs PB II 23.87±0.15 29.45±1.31 23.99±0.57 20.67±0.87

Anaerobically packs PB III 15.57±0.95 22.20±0.91 20.57±0.77 16.95±0.54

Gallic acid 3.79±0.69 255.43±11.68 - 59.14±1.10

Ascorbic acid 6.05±0.34 > 1000 - 160.55±2.31

BHT 15.61±1.26 1.00±0.23 - 33.92±0.79

α-Tocopherol - 0.48±0.05 - -

Results are mean ± SD values from three experiments. The 50 % inhibitory concentration (IC50) values were determined by

nonlinear regression analysis.

BHT, butylated hydroxytoluene; DPPH, 2, 2-diphenyl-1-pycrilhydrazyl.

Table 3 Minimum inhibitory concentrations (MIC) in the anaerobically packed fermented dry

sausage

MIC μg/mL

Microbial strains ATCC

number PB I PB II PB III Amracin Ketoconazole

Bacteria’s

Staphylococcus aureus 25923 62.50 31.25 31.25 0.97 /

Escherichia coli 25922 31.25 15.625 15.625 0.97 /

Proteus vulgaris 13315 62.50 62.50 31.25 0.49 /

Proteus mirabilis 14153 62.50 31.25 31.25 0.49 /

Bacillus subtilis 6633 62.50 31.25 31.25 0.24 /

Mold

Aspergillus niger 16404 125.00 62.50 31.25 / 0.97

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Table 4 Minimum inhibitory concentrations (MIC) in the aerobically packed fermented dry

sausage

MIC μg/mL

Microbial strains ATCC

number PB I PB II PB III Amracin Ketoconazole

Bacteria’s

Staphylococcus aureus 25923 62.50 31.25 31.25 0.97 /

Escherichia coli 25922 31.25 15.625 15.625 0.97 /

Proteus vulgaris 13315 62.50 62.50 31.25 0.49 /

Proteus mirabilis 14153 31.25 31.25 31.25 0.49 /

Bacillus subtilis 6633 125.00 125.00 62.50 0.24 /

Mold

Aspergillus niger 16404 125.00 62.50 31.25 / 0.97

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Table 5 Comparison between chemical-physical characterization data of various production

batches (PB) of fermented dry sausages during fermentation and ripening

VARIABLE DAYS/

package

PB I PB II PB III

MEAN±SD1 MEAN±SD MEAN±SD

PROTEINS

(g/100g)

0 15.15a*

±0.29 13.18b±0.10 13.67

c±0.15

13 20.52a±0.19 20.24

b±0.08 20.78

a±0.11

26 Aer 23.33a±0.01 24.42

b±0.11 23.77

c±0.06

26 A-Aer 23.19a±0.07 24.56

b±0.04 24.00

c±0.14

MOISTURE

(g/100g)

0 49.86a±0.09 51.30

b±0.14 52.83

c±0.12

13 29.93a±0.14 30.48

b±0.19 29.87

a±0.08

26 Aer 17.25a±0.04 17.32

a±0.08 18.87

b±0.23

26 A-Aer 17.58a±0.16 18.47

b±0.04 18.88

c±0.19

FATS

(g/100g)

0 31.97a±0.06 33.20

b±0.10 30.34

c±0.04

13 44.33a±0.03 46.11

b±0.11 45.05

c±0.02

26 Aer 54.70a±0.42 53.10

b±0.55 51.88

b±0.00

26 A-Aer 54.85a±0.20 51.83

b±0.20 51.68

b±0.18

ASH (g/100g)

0 3.13a±0.06 2.99

b±0.01 3.13

a±0.03

13 4.44a±0.04 4.13

b±0.03 4.40

a±0.20

26 Aer 4.70aS#

±0.01 5.09b±0.00 5.52

c±0.01

26 A-Aer 4.10a±0.00 5.14

bS±0.01 5.50

c±0.01

Na

(g/100g)

0 1.01a±0.00 1.90

b±0.02 0.98

c±0.00

13 1.37a±0.00 2.00

b±0.02 1.34

a±0.02

26 Aer 1.40b±0.00 1.40

b±0.00 1.52

c±0.02

26 A-Aer 1.35aS

±0.01 1.44bS

±0.00 1.50c±0.00

pH

0 6.36a±0.02 5.32

b±0.03 5.00

c±0.03

13 5.21a±0.03 5.56

b±0.01 5.48

c±0.20

26 Aer 5.33a±0.01 5.66

b±0.00 5.81

cS±0.00

26 A-Aer 5.48aS

±0.01 5.72bS

±0.02 5.74b±0.00

aw

0 0.94±0.00 0.93±0.00 0.93±0.00

13 0.83a±0.00 0.85

b±0.00 0.85

b±0.01

26 Aer 0.72a±0.00 0.71

b±0.00 0.73

cS±0.00

26 A-Aer 0.73a±0.00 0.73

aS±0.00 0.72

b±0.00

*a-c

Means in the same rows on the same sampling days followed by different superscript letters

are significantly different (P < 0.05). 1Standard deviation.

#S

Letter S in superscript means significant difference between samples of FDS in anaerobically

(A-Aer) and aerobically (Aer) manner of storage.

Note: 0, 13, 26 - sampling days (start, middle and finishing of ripening and drying).

Aerobically packed FDS: (Aer); anaerobically packed FDS: (A-Aer).

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Table 6 Comparison between chemical-physical characterization data of various production

batches (PB) of fermented dry sausages during storage

VARIABLE DAYS/

package

PB I PB II PB III

MEAN±SD1 MEAN±SD MEAN±SD

PROTEINS

(g/100g)

20 Aer 22.22a±0.06 22.65b±0.16 25.99c±0.09

20 A-Aer 21.80a±0.21 23.90b±0.06 26.12c±0.16

40 Aer 24.60a±0.08 25.41b±0.08 24.19c±0.17

40 A-Aer 25.06a±0.47 23.15b±0.23 23.54b±0.14

60 Aer 23.12a±0.07 24.69b±0.18 24.18c±0.10

60 A-Aer 22.23a±0.03 20.55b±0.11 24.39c±0.07

MOISTURE

(g/100g)

20 Aer 16.72a±0.03 16.72a±0.03 16.30ab±0.20

20 A-Aer 16.83±0.00 16.49±0.27 16.92±0.11

40 Aer 15.59a±0.07 15.85±a0.24 15.85a±0.24

40 A-Aer 17.62a±0.20 16.53b±0.03 17.73a±0.10

60 Aer 13.34a±0.06 12.01b±0.10 14.45c±0.14

60 A-Aer 19.23a±0.09 17.74b±0.21 18.62c±0.14

FATS

(g/100g)

20 Aer 56.50a±0.23 55.20b±0.28 51.80c±0.20

20 A-Aer 56.15a±0.15 54.19b±0.05 51.28c±0.16

40 Aer 54.30a±0.22 55.30b±0.20 54.30aS±0.32

40 A-Aer 52.00a±0.22 55.10b±0.11 53.00c±0.00

60 Aer 57.92a±0.08 57.72a±0.11 55.81b±0.19

60 A-Aer 53.35a±0.05 55.50b±0.50 51.81c±0.19

ASH (g/100g)

20 Aer 5.21a±0.03 5.39b±0.01 5.95c±0.01

20 A-Aer 5.22a±0.06 5.43b±0.03 5.67c±0.03

40 Aer 5.32a±0.01 5.72b±0.01 5.62c±0.01

40 A-Aer 5.42a±0.01 5.20b±0.01 5.66c±0.01

60 Aer 5.51a±0.03 5.60b±0.02 5.53a±0.02

60 A-Aer 5.14a±0.03 5.55b±0.04 5.20a±0.04

Na

(g/100g)

20 Aer 1.40a±0.00 1.44b±0.00 1.59c±0.00

20 A-Aer 1.40a±0.00 1.47bS±0.01 1.59c±0.01

40 Aer 1.47a±0.00 1.50b±0.02 1.64cS±0.00

40 A-Aer 1.50aS±0.00 1.51a±0.01 1.61b±0.02

60 Aer 1.52a±0.00 1.53b±0.00 1.66cS±0.00

60 A-Aer 1.53aS±0.00 1.56a±0.03 1.64b±0.00

pH

20 Aer 5.42a±0.00 5.61b±0.01 6.04cS±0.00

20 A-Aer 5.41a±0.00 5.70bS±0.00 5.78c±0.00

40 Aer 5.92aS±0.00 5.86b±0.00 5.93a±0.01

40 A-Aer 5.75a±0.00 5.87b±0.01 5.98cS±0.00

60 Aer 5.80a±0.00 5.99bS±0.00 6.08c±0.00

60 A-Aer 5.77aS±0.00 5.79a±0.17 6.24b±0.31

aw

20 Aer 0.720a±0.00 0.707b±0.00 0.690c±0.00

20 A-Aer 0.725aS±0.00 0.700b±0.00 0.711cS±0.00

40 Aer 0.680a±0.00 0.689c±0.00 0.689c±0.00

40 A-Aer 0.708a±0.00 0.717cS±0.00 0.717cS±0.00

60 Aer 0.595a±0.00 0.621b±0.00 0.633c±0.00

60 A-Aer 0.619aS±0.00 0.635bS±0.00 0.693cS±0.00

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*a-cMeans in the same rows on the same sampling days followed by different superscript letters

are significantly different (P < 0.05). 1Standard deviation.

#S

Letter S in superscript means significant difference between samples of FDS in anaerobically

(A-Aer) and aerobically (Aer) manner of storage.

Note: 20, 40, 60 - sampling days (storage) of aerobically (Aer) and anaerobically packed FDS

(A-Aer).

Aerobically packed FDS: (Aer); anaerobically packed FDS: (A-Aer);

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Highlights

Produced healthier fermented dry sausage (FDS) that are no nitrites added.

FDS fortified by bioactive phenol and flavonoid compounds from Kitaibelia vitifolia.

Significantly increase of oxidative stability in fortified food matrix of FDS.

Great antimicrobial activity of fortified sausage, highest against E. coli.

Optimal effective concentration of herb extract was 12.5 g/kg of meat dough.

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