Accepted Manuscript

Combination of garlic essential oil, allyl isothiocyanate, and nisin

Z as bio-preservatives in fresh sausage

Monyque Kais Araújo, Aline Marzaleck Gumiela, Keliani Bordin,

Fernando Bittencourt Luciano, Renata Ernlund Freitas de Macedo

PII: S0309-1740(18)30031-7
DOI: doi:10.1016/j.meatsci.2018.05.002
Reference: MESC 7552
To appear in: Meat Science
Received date: 15 January 2018
Revised date: 7 March 2018
Accepted date: 2 May 2018

Please cite this article as: Monyque Kais Araújo, Aline Marzaleck Gumiela, Keliani
Bordin, Fernando Bittencourt Luciano, Renata Ernlund Freitas de Macedo , Combination
of garlic essential oil, allyl isothiocyanate, and nisin Z as bio-preservatives in fresh
sausage. The address for the corresponding author was captured as affiliation for all
authors. Please check if appropriate. Mesc(2017), doi:10.1016/j.meatsci.2018.05.002

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 ACCEPTED MANUSCRIPT

Combination of garlic essential oil, allyl isothiocyanate, and nisin Z as bio-

preservatives in fresh sausage

Monyque Kais Araújoa , Aline Marzaleck Gumielaa , Keliani Bordina , Fernando

Bittencourt Luciano a , Renata Ernlund Freitas de Macedoa,*

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Graduate Program in Animal Science, School of Life Sciences, Pontifícia Universidade

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Católica do Paraná, Rua Imaculada Conceição-1155, 80215-901 Curitiba, Brazil

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*Corresponding author: Tel: +55 41-3299-2690; fax +55 41-3299-2615. E-mail:

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renata.macedo@pucpr.br
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Abstract

The effects of natural antimicrobial compounds (garlic essential oil [GO], allyl isothiocyanate

[AITC], and nisin Z [NI]) on microbiological, physicochemical and sensory characteristics of

fresh sausage were assessed. The minimum inhibitory concentrations (MICs) and the

minimum bactericidal concentrations (MBCs) towards Escherichia coli O157:H7 and

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Lactobacillus plantarum were determined in vitro. Sausages inoculated with E. coli O157:H7,

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were treated with different combinations of antimicrobials and assessed for microbiological

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and physicochemical parameters during storage (6 C for 20 d). Treatments that presented the

greatest antimicrobial effects were subjected to sensory evaluation. Combinations of 20

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mg/kg NI + 125 µL/kg GO + 62.5 µL/kg AITC or 20 mg/kg NI + 62.5 µL/kg GO +125 µL/kg
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AITC were effective in reducing E. coli O157H7 and spoilage lactic acid bacteria, and

maintained the physicochemical characteristics of fresh sausage. Combinations of NI, GO and

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AITC were effective to improve the safety and the shelf life of fresh sausage, with no impact
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on its sensory acceptance.

Keywords: pathogens; essential oil; natural compounds; meat products

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1. Introduction

Brazil plays an important role in the global meat market, being the second largest

producer in the world after the USA (Brasil, 2016). Among the meat products, fresh sausage

is one of the most consumed; however, because of high water activity and the absence of heat

treatment, it is susceptible to microbial spoilage and have short shelf life (Terra, 1998).

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Pathogenic bacteria, such as E. coli O157:H7 can also be found in meat-related raw

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materials. The presence of this serotype has been reported in foodborne illness outbreaks

associated with undercooked meat products. According to Centers for Disease Control and

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Prevention (CDC, 2016) in the USA, in 2013, 2014, 2015, and 2016 years, there were

respectively 33, 12, 19, and 9 reported cases of foodborne illness associated with the
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consumption of ready-made salads, ground meat, chicken salad, and alfalfa sprouts. In
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addition, the control of spoilage bacteria is commercially important to ensure product stability

and extended shelf life. Lactic acid bacteria are among the most important spoilage bacteria in
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sausages and other meat products stored under refrigeration (Niven, 1986; Nychas,
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Skandamis, Tassou, & Koutsoumanis, 2008).

To reduce the microbiological risk in foods and to decrease the use of synthetic
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additives, essential oils (EO) and antimicrobial peptides have been studied extensively.
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(Holley & Patel, 2005). EO have a wide range of antimicrobial activities against various

species of bacteria such as Escherichia coli O157:H7, Bacillus cereus, Staphylococcus

aureus, Staph. epidermidis, Listeria monocytogenes, Salmonella Typhimurium, Yersinia

enterocolitica, Proteus vulgaris. (Burt, 2004; Nuryastuti, Van der Mei, Iravati, Aman, &

Krom, 2009; Tiwari, Valdramidis, O’Donnel, Muthukumarappan, & Cullen, 2009; Silveira,

Luciano, Fronza, Junior, Scheuermann, & Vieira, 2012; Andrade, Barbosa, Probst, & Junior,
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2014). Among the chemical antimicrobial components present in these oils are terpenes (e.g.,

p-cymene), terpenoids (e.g., thymol and carvacrol), phenylpropenes (e.g., eugenol and

cinnamaldehyde), and other compounds containing sulfur and nitrogen (e.g., allyl

isothiocyanate and allicin) (Hyldgaard, Mygind, & Meyer, 2012).

Due to their high volatility, the addition of EO to foods may cause undesirable

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sensorial changes (Lis-Balchin, Buchbauer, & Hirtenlehner, 1998); additionally, in food,

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higher concentrations of EO are required than in experimental media to obtain the same

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inhibitory effect, due to the interaction of EO with food compounds, such as meat fat. (Burt,

2004). Therefore, the use of essential oils extracted from plants usually used as seasoning in

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foods is a way of reducing the negative sensory consequences. This way, the use of garlic
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may increase the shelf life of foods because of the antibacterial and antioxidant action

(Abubakar, 2009; Fujisawa, Watanabe, Suma, Origuchi, Matsufuji, Seki, & Ariga, 2009;
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Alorainy, 2011; Wu, Santos, & Fink-Gremmels, 2015; Horita, Farías-Campomanes, Barbosa,
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Esmerino, Cruz, Bolini, Meireles, & Pollonio, 2016), whereas mustard oil can reduce pH

inside the bacterial cells such as E. coli O157:H7 and Salmonella (Nadarajah, Han, & Holley,
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2005; Turgis, Borsa, Millette, Salmieri, & Lacroix, 2008).

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The combined use of EO and other natural preservatives, such as peptides as nisin (NI)

in fresh sausages, can reduce their concentration while maintaining the antimicrobial
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efficiency. Furthermore, nisin exerts preferential action on gram-positive bacteria (Breukink,

Wiedemann, Van Kraaij, Kuipers, Sahl, & Kruijff, 1999; Brotz & Sahl, 2000), which could

favor the control of spoilage lactic acid bacteria without affecting sensory characteristics of

the food (Kuwano, Tanaka, Shimizu, Nagatoshi, Nou, & Sonomoto, 2005). Therefore, this

could be a natural strategy for inhibiting the growth of both gram-positive and gram-negative
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bacteria in sausages, thus increasing the safety and prolonging the shelf life of this product

(Trajano, Lima, Souza, & Travassos, 2009; Mathenjwa, Hugo, Bothma, & Hugo, 2012).

The antimicrobial effect of combination of EO and nisin (NI) has been previously

reported in experimental media (Solomakos, Govaris, Koidis, & Botsoglou, 2008; Turgis,

Borsa, Millette, Salmieri, & Lacroix, 2012; Yoon, Bajpai, & Kang, 2011); however there is a

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lack of studies evaluating their combination applied in more complex food matrices such as

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fresh sausage and their impact on its physicochemical and sensory quality.

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The objectives of this study were 1) to evaluate the in vitro inhibitory effect of a

combination of allyl isothiocyanate, garlic oil, and nisin Z on a pool of E. coli O157:H7 and a

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pool of Lactobacillus plantarum strains and 2) to evaluate the effects of the combination of
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these natural compounds on microbiological stability, physicochemical characteristics and

sensory properties of fresh sausage.

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2. Materials and methods

2.2. Materials
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Allyl isothiocyanate (AITC, 95%) was purchased from Sigma-Aldrich Inc. (St. Louis,
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MO, USA). Garlic oil (GO) was purchased from Zhengzhou Sigma Chemical Co., Ltd.

(Henan, China) and, according to the manufacturer’s specification, GO was prepared by steam
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distillation and had the following composition: 51.8% allicin, 0.3% moisture, and 47.9% other

thioester compounds (allyl sulfite and allyl disulfite). Nisin Z (NI, 2.5%), was purchased from

Handary (Brussels, Belgium). E. coli strains O157:H7 (02-0627, 02-0628, 00-0381, 02-0304,

and NM 02-1840) were donated by Dr. Richard A. Holley of the Department of Food Science

at the University of Manitoba, Winnipeg, Canada. All the strains were non-carriers of the stx1

and stx2 genes. An L. plantarum pool, composed of indigenous strains (194, 201, 202, 212,
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and 227), isolated from artisan fermented meat sausages and belonging to the culture

collection of the Laboratory of Agri-Food Technology, served as a model for the in vitro

assays for antimicrobial activity of the natural compounds.

2.3. Antimicrobial activity: in vitro essay

2.3.1. Inoculum preparation

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E. coli O157:H7 strains were kept frozen in TSB broth solution (Tryptone Soya Broth;

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Himedia Laboratories Ltd, Mumbai, India) and glycerol (ratio 75:25) at 80 C. The pool of

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E. coli O157:H7 strains was obtained by the addition of 100 μL of each isolated strain (grown

in TSB at 37 C for 24h) to 9.5 mL of TSB broth with pH adjusted to 6.0. The pool was

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incubated at 37 C for ~5 h until a mid-exponential growth was reached (7 log to 8 log
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CFU/mL; 0.5 McFarland scale). The L. plantarum pool was prepared by adding 20 μL of each

strain [grown in MRS broth at 37 C for 48h (KASVI, Paraná, Brazil)] into 9.5 mL of MRS
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broth. The pool was incubated at 37 °C for ~5 h until a population count ranging from 7 log to
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8 log CFU/mL; 0.5 McFarland scale.

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2.3.2 Determination of minimum inhibitory concentration (MIC) and minimum bactericidal

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concentration (MBC)
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A stock solution of each EO containing 20 μL of GO or AITC + 980 μL of dimethyl

sulfoxide (DMSO) was prepared to facilitate solubilization (maximum of 2% in the final

solution). Treatments were performed in screw-capped tubes by adding 7.9 mL of BHI broth,

80 μL of the pool of E. coli O157:H7 or the pool of L. plantarum and antimicrobials at

concentrations described in Table 1 (final volume of 8 mL). Control groups contained 7.760
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mL BHI broth, 160 μL dimethyl sulfoxide and 80 μL bacterial inoculum. Then, tubes were

incubated at 37 ° C for 24 h.

After incubation and serial dilutions, the number of viable cells of E. coli O157:H7

was determined in Sorbitol MacConkey agar plates (HIMEDIA, Mumbai, India), incubated at

37 °C for 24 h (log CFU/mL), and L. plantarum counts were performed in MRS agar plates

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(KASVI, Paraná, Brazil), incubated at 37 °C for 48 (log CFU/mL).

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MIC was considered as the lowest antimicrobial concentration causing a reduction of

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the target bacteria count by >90% (~1 log), and MBC was defined as the lowest antimicrobial

dose causing a reduction of the target bacteria count by >99.9% (~ 3 log) (Perricone, Arace,

Corbo, Sinigalia, & Bevilacqua, 2015).

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2.4. Preparation of fresh sausage
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The sausages were prepared in accordance with a basic commercial formulation

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presented in Table 2.

The pork leg and bovine boneless chuck were ground in an electric meat-grinder (PC-
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10, Eccel Metalúrgica, Brusque, SC, Brazil) with an 8-mm disc. The dry ingredients were
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added to and manually mixed with the batter for 2 min. Subsequently, the pool of E. coli

O157:H7 was added to the mixture (final concentration of 6 log CFU/g), and mixed manually
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for 2 min. The antimicrobial compounds were added at the end of the batter preparation, with

subsequent stuffing using a mechanical stuffer (Jamar model EFI, Tupã, SP, Brazil) into

natural sheep casing (Brascase Alimentos, Fazenda Rio Grande, PR, Brazil), previously

sanitized with 10% lactic acid solution for 30 min. The sausages were stored in sterile plastic

bags (Laborclin, model 570671, Vargem Grande, Pinhais, Paraná, Brazil) and kept at 6°C for

20 d. At 5-day intervals, samples were withdrawn and subjected to microbiological (counts of

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E. coli O157:H7, indigenous E. coli, lactic acid bacteria, and psychrotrophic aerobic bacteria)

and physical chemical analyses (determination of pH and instrumental color).

2.5. Microbiological analysis

Sausage samples (25g) were aseptically transferred to sterile plastic bags containing

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225 mL of 0.1% peptone water (HIMEDIA, Mumbai, India) and homogenized in a stomacher

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(IUL Instruments, Barcelona, Spain). From the first dilution (1:10), subsequent dilutions were

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performed and plated in Sorbitol MacConkey agar plates (HIMEDIA, Mumbai, India),

incubated at 37 C for 24 h to determine counts of E. coli O157:H7 (colorless colonies) and

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indigenous E. coli (pink purple colonies) (Thompson et al., 1990). The lactic acid bacteria
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count was determined on MRS agar plates (KASVI, Paraná, Brazil) incubated at 37 C for 48

h. The aerobic psychrotrophic bacteria were counted on PCA agar (HIMEDIA, Mumbai,
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India) after incubation of the plates at 6 °C for 7 days. All results were expressed in log
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CFU/g.
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2.6. Physicochemical characterization

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The pH levels were measured using a digital pHmeter (HI253 Hanna Instruments,

Dallas, Texas, USA) and the electrode inserted at three random sites in the sausage, in
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accordance with Adolfo Lutz Institute Analysis Manual (Ial, 2008).

The instrumental color was evaluated by the ASTM International (2001) method using

a portable colorimeter (Konica Minolta CR 410, Tokyo, Japan) and L*, a*, and b* color

coordinates. Color was measure on three different points of surface of sausages using light

source D65, opening diameter of 50 to 53 mm, and angle of observation of 2. The hue values
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[h = ARCTAN (b/a)], which indicate intensity of discoloration, and C* (chroma; c = √a2xb2),

the latter indicates color saturation, were calculated.

2.7. Sensory analysis

Three formulations of fresh sausage were prepared: NC) a control, formulated with

beef, pork, and condiments, without the addition of the natural antimicrobials and two other

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formulations, F2 and F3, formulated with the same ingredients, but with addition of EO and

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NI at different concentrations (Table 3). The preparation of the sausage followed the same

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procedure described above, but without the addition of E. coli O157:H7. The sausages were

cooked on an electric grid (Oster, model 4777-33, Chicago, IL, USA) until reaching an

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internal temperature of 71 C. Sausages were sliced (~1 cm thickness) and samples (2 slices
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for each panelist) were coded with its own 3-digit random number for the evaluation. An

affective test was used to analyze the sensory acceptance of sausages, using a nine-point
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structured hedonic scale ranging from 1 (disliked extremely) to 9 (liked extremely). The test
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was performed by a panel of 100 untrained volunteers (32 men and 68 women, aged 18–50

years). The panelists were asked to evaluate the appearance, odor, flavor, texture and overall
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liking of the samples (Dutcosky, 2014). The sensory analysis was performed with the
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approval by the Ethics Committee (Process number 1.675.506) at the School of Life Sciences,

Pontifícia Universidade Católica do Paraná.

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2.8. Statistical analysis

Statgraphics® Centurion XVI version 16.1.11 (Statpoint Technologies, Warrenton,

Virginia, USA) was used for statistical analysis of the data.

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The in vitro antimicrobial activity results were analyzed by one-way ANOVA and

means were compared by Tukey test (P < 0.05).

The microbiological and physicochemical data were analyzed using a random block

design, considering a mixed linear model including formulation and storage time as fixed

effects and replication as a random effect. Means were compared by Tukey test (P < 0.05).

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The microbiological and physicochemical assays were performed two times independently

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and differences between replicates were not significant (P < 0.05). The microbiological assays

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were conducted in duplicate (n = 4) and physicochemical analysis in triplicate (n = 6).

Sensory assay was conducted in one section and data were analyzed using a random

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block design, considering a mixed linear model including the fixed effect of formulation and
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random block effect (panelist). Means were compared by Tukey test (P < 0.05).
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Results and Discussion

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3.1. In vitro antimicrobial activity

The concentrations of AITC and GO used in this study were based on MIC values
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determined in our previous unpublished studies on the combination of AITC and GO against
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E. coli O157:H7. AITC and GO showed an in vitro synergistic inhibitory effect on E. coli

O157:H7 at concentration of 0.0125 μL/mL for both EO. Thus, the concentrations of EO used
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in combination with NI in MIC assays were 0.0125 μL/mL, 0.00625 μL/ mL and 0.00312 μL/

mL. NI was added at concentrations of 125 IU/mL (0.12 mg/mL) and 250 IU/mL (0.24

mg/mL).

NI concentration at 0.24 mg/mL combined with EO concentrations from 0.00625

μL/mL to 0.0125 μL/mL caused a large reduction (>3 log CFU/mL) (Burt, 2004) of both E.

coli O157:H7 and L. plantarum populations compared to control (Table 1).

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The concentration of EO (0.00625 μL/mL) in combination with a 50% reduced initial

concentration of NI (0.12 mg/mL) (T6) maintained a large reduction in E. coli O157:H7 count

(>3 log CFU/mL), but showed a medium reduction in L. plantarum count, 1 to 3 log

CFU/mL. NI at 0.24 mg/mL combined with ¼ initial concentration of EO (0.00312 μL/mL)

promoted a large reduction in E. coli O157:H7 population (> 3 CFU/ mL), but a slight

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reduction in L. plantarum count (~1 log CFU/mL).

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Conversely, Olasupo, Fitzgerald, Gasson, & Narbad (2003) observed no antimicrobial

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effect of nisin A on E. coli and S. Typhimurium in vitro in combination with other natural

compounds (thymol, carvacrol, eugenol, cinnamic acid, or diacetyl). In vitro studies

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conducted by Kruger (2006) and Rohani, Moradi, Mehdizadeh, Saei-Dehkordi, & Griffiths
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(2011) yielded MIC values of 5 μL/mL (oregano oil) + 6 mg/mL (nisin Z) and 0.1 μL/mL

(garlic oil) + 12.5 IU/mL (nisin Z), respectively, against L. monocytogenes. The Kruger study
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(2006) revealed an in vitro MIC of NI (6 mg /mL) to be 25-fold higher than the concentration
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in the present study. Rohani et al. (2011) found a MIC of GO against L. monocytogenes 8-fold

higher than the highest dose used in the present study against L. plantarum. Nevertheless, the
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MIC for NI reported by these authors was 20-fold lower than the concentration used in the
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present work (250 IU/mL NI). This variation in the antimicrobial action of nisin may be

attributed to the different culture media used in the in vitro assays. In addition, the high
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concentrations of divalent cations such as Fe2+, Mn2+, Zn2+, Cu2+, and some salts (Bellamy,

Takase, Wakabayashi, Kawase, & Tomita, 1992; Ellison, 1994; Branen & Davidson, 2004) in

the medium may hinder the inhibitory action of nisin on microorganisms. Kuwano, Tanaka,

Shimizu, Nagatoshi, Nou, & Sonomoto (2005) stated that the high concentration of NaCl (100

mM) in the medium might reduce the antimicrobial activity of nisin Z against E. coli via

several cationic amino acids (Buchman, Banerjee, & Hansen, 1988). In the present study,
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TSB medium contained 75 mM NaCl, which may also have contributed to the higher

inhibitory dose.

Considering that the greatest reductions in the populations of these microorganisms

were obtained with treatments T1, T3, and T4 (Table 1), the last two treatments were selected

to be tested in sausage, because in these treatments, the concentrations of the essential oils

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were lower than those used in T1.

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3.2. Effects on sausage stability

Concentrations of antimicrobial compounds applied to sausages were 10 fold higher

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than the used on in vitro study because the characteristics of the meat matrix, which is
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composed of lipids and proteins, may interact with the essential oils and affect their action.

The microbial counts of E. coli O157:H7 throughout the storage period of fresh sausage under
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refrigeration are shown in Figure 1.

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E. coli O157:H7 counts were reduced starting on the 5th day (P < 0.05) by the

combination of NI and essential oils compared to controls (negative and positive: NC and PC,
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respectively, in the figures) or NI alone (F1). The reductions in E. coli O157:H7 counts after
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20 d were 1.29, 2.06, and 2.12 log CFU/g in treatments F1, F2, and F3, respectively. Chacon

(2006), Solomakos, Govaris, Koidis, & Botsoglou, (2008), and Djenane, Socorrista,
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Yanguela, Idir, Gómez, & Roncalés (2012) evaluated the effects of lavender essential oil (2

μL/g) and mint (1 μL/g), thyme essential oil (6 μL/g) in combination with nisin Z (500 or

1000 IU/g) and microencapsulated AITC (4.98 μL/g) against E. coli O157:H7 in ground beef,

respectively. The treatments reduced the pathogen counts between 3.10 to 5.07 log CFU/g.

The reductions were greater, but the concentrations of the essential oils and nisin Z were
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respectively 16- to 96-fold and 25- to 50-fold higher than the concentrations of the present

study.

Indigenous E. coli counts were reduced to undetectable levels (<1 log CFU/g) after 10

d by F2 and F3, whereas with the treatment with NI alone, this reduction was achieved on the

15th day. After 20 d, the values of E. coli counts in all treatments were lower than those

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established by the Brazilian legislation (Brazil, 2001) for coliforms at 45 C/g. Lemay,

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Choquette, Delaquis, Gáriepy, Rodrigue, & Saucier (2002) also observed a gradual reduction

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in E. coli counts in chicken meat under all the conditions tested (control, sodium lactate, and

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mustard essential oil). After 14 days, E. coli was near or below the detection level (<1 log

CFU/g) for all treatments, including control.

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Lactic acid bacteria count under the influence of all treatments involving NI showed a

significant reduction on the 5th day of storage (P < 0.05). Lactic acid bacteria counts were
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reduced to >6 log CFU/g, starting on the 10th day, reached a plateau, and stayed constant
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until 20 d. The above value is important because it represents a maximal count of lactic acid

bacteria as an indicator of spoilage of meat products. In another study, the use of bay leaf
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essential oil (0.5 or 1.0 μL/g) did not have a significant effect on lactic acid bacteria, which
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remained in fresh sausage at >7 log CFU/g (Silveira, Junior, Scheuermann, Secchi, & Vieira,

2014).
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Psychrotrophic bacteria were presented at approximately 6 log CFU/g, and treatments

had no effect on this population compared to negative control (Figure 1). The psychrotrophic

microbiota in meat is mainly Aeromonas and Pseudomonas, which multiply rapidly at high

water activity, in a neutral environment, and in the presence of oxygen (Jeppesen, 1995).

Aeromonas and Pseudomonas can actively multiply in aerobically packaged products and can
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be a part of the dominant microbiota at the end of a storage period (Mano, Pereda, &

Fernando, 2002). The fact that the sausage was not vacuum-packed may have contributed to

the growth of this group of microorganisms in the product because the antimicrobial activity

of essential oils is related to the amount of available oxygen. This situation can occur because

in an atmosphere with low oxygen diffusion, oxidative changes in oils are smaller or because

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cells that obtain energy through anaerobic metabolism are more sensitive to the toxic action of

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essential oils (Paster, Juven, Shaaya, Menasherov, Nitzan, Weisslowics, & Ravid, 1990).

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3.2 Effects on sausage stability

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Figure 2 shows the pH values of fresh sausage stored for 20 d. There was a slight
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increase in pH in all treatments during the storage and at the end of the storage the pH of the

sausage samples treated with the combination of the natural compounds (F2 or F3) was lower
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than pH in the controls, 6.05 and 6.18, respectively. The increase in pH may be related to the
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reduction in lactic acid bacteria counts, which have the ability to increase the acidity of meat

through metabolism (Delazari, Leitão, & Hsu, 1977), whereas increased counts of
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psychrotrophic bacteria, which can use proteinaceous compounds as a source of energy, lead
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to the production of ammonia and, consequently, to higher pH (Jay, 2000). In the studies by

Kitakawa (2002) and Figueiró (2013), both authors observed a decline of pH in fresh sausage
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and fresh pork sausage, respectively, associated with an increase in lactic acid bacteria counts.

In contrast, in the study by Georgantelis (2007), there was an increase in pH from 5.8 to 6.2 in

fresh pork sausage, and this increase was associated with the presence of enterobacteria and

Pseudomonas.

With regards to the color coordinates, the L* coordinate indicated a difference

between treated samples and control on the 20th day, and all samples showed a reduction in
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luminosity during the storage. These data are in agreement with the results of Chiavaro,

Zanardi, Bottari, & Ianieri (2008), who confirmed a reduction in luminosity of fresh pork

sausage during 15 days of storage. Here, at the end of the storage period, the sausage samples

subjected to F2 or F3 showed similar L* but higher than L* of the samples that were treated

with pure NI only. These differences may be due to the color heterogeneity of the samples

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(the presence of lighter and darker spots); this heterogeneity is an inherent characteristic of

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sausage.

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Color intensity (saturation; C*) and discoloration (hue; Figure 3) showed significant

differences among treatments (P < 0.05). Nevertheless, despite the lower C*, the sausage

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samples with an added antimicrobial agent (F1, F2, and F3) showed less discoloration (more
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hue) than the control groups did (NC and PC). Because the chroma (C*) of food represents

the sum of its colors, both red and yellow contribute to its values (AMSA, 2012). As
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yellowness was higher in controls, this could explain higher C* in controls sausages.
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However, treated samples yielded higher values for the red color (a*) as compared to controls.

The low temperature of storage and the antioxidant activity of the essential oils may have
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influenced the stability of the coloring of the product because the antioxidant action of
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essential oils can be attributed to their capacity for sequestering free radicals and for

inhibition of hydrolytic and oxidative enzymes (Burt, 2004; Bakkali, Averbeck, Averbeck, &
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Idaomar, 2008; Tajkarimi, Ibrahim, & Cliver, 2010). Considering that fresh sausage has a

characteristic red color, high yellow color values are not desirable in this type of product, and

the samples treated with the essential oils and NI combinations showed desirable color

characteristics.

3.2. Sensory analysis

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The results of the sensory analysis are presented in Table 3. The analysis was

performed on samples of three treatments: 1) NC- sausage formulation only, 2) F2-

formulation + 125 μL/kg GO + 62.5 μL/kg AITC and 3) F3- formulation + 62.5 μL/kg GO +

125 μL/kg AITC. A significant difference (P < 0.05) was observed between the control and

the treated samples for odor, flavor, texture and overall liking.

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We found that the characteristics of color, flavor, texture, and overall liking showed an

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acceptability index above 70%, indicating acceptance by the panelists. Only for the odor

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attribute, were the batches of sausage with antimicrobial agents acceptable at less than 70%,

probably because of the volatility of the essential oils that interfered with the aroma of the

sausage samples.
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As in the present experiment, in the study by Silveira, Luciano, Fronza, Junior,

Scheuermann, & Vieira (2014), the addition of bay leaf oil (from Laurus nobilis; 0.05 g per
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100 g or 0.1 g per 100 g) to Tuscan sausage was acceptable for the panelists, but the control
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sample showed better acceptance. In the present study, the strongest effect of the addition of

essential oil was observed for taste and the overall liking, which yielded scores between
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“neither liked nor disliked” and “liked slightly.” On the other hand, in the study by Govaris,
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Solomakos, Pexara, & Chatzopoulou (2010) on the use of oregano essential oil (0.6 g per 100

g or 0.9 g per 100 g) in sheep meat, both doses of the oil were sensorially acceptable, and the
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taste and overall rating of the sample containing essential oil at 0.6 g per 100 g received a

significantly higher (P < 0.05) score than the control did. In the study by Horita, Farías-

Campomanes, Barbosa, Esmerino, Cruz, Bolini, Meireles, & Pollonio (2016), the addition of

garlic and its derivatives as garlic oil to sausage samples did not make a significant difference

(P < 0.05) in relation to flavor and general rating.

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For all the evaluated attributes, the scores received by the samples treated with the

different doses of the antimicrobials were above 6.0, which denotes “liked slightly,” meaning

that the samples were enjoyed by the panelists. Although the control group of samples yielded

greater acceptance by the panelists relative to the treated samples, we found that both the

samples containing the essential oils and NI showed acceptance by the consumers and

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therefore could have potential to be commercialized.

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3. Conclusions

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The tested combinations of essential oils and NI caused a significant reduction in the

E. coli O157:H7 population in fresh sausage as compared to the control groups (no treatment).

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The combinations of these antimicrobials were also effective in maintaining the stability of
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the fresh sausage by controlling the growth of spoilage lactic acid bacteria and by maintaining

the red color and pH in the sausage samples. Sensory evaluation revealed that both
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formulations containing the natural antimicrobial compounds were acceptable to panellists,

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thus indicating a commercial potential of such foods. This study provides data for the

development of natural compounds for inhibition of pathogens and for increasing the shelf life
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of fresh sausage, thereby helping the food industry to offer safer and more sustainable foods
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to the consumer.
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Table 1 In vitro evaluation of bactericidal effects of nisin Z in combination with allyl

isothiocyanate and garlic oil on E. coli O157:H7 and L. plantarum
Bacterial population* (log
Antimicrobial Treatment
CFU/g)
Treatments
NI AITC GO E. coli
L. plantarum
(mg/mL) (µL/mL) (µL/mL) O157:H7
C - - - 10.40 ± 0.01a 8.11 ± 0.02a

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T1 0.24 0.0125 0.0125 2.00 ± 0,00e 2.51 ±0.03f

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T2 0.24 0.0062 0.0062 4.62 ±0.01d 5.00 ± 0.02d

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T3 0.24 0.0125 0.0062 2.00 ± 0.00e 2.60 ± 0.00ef
T4 0.24 0.0062 0.0125 2.00 ± 0.00e 2.67 ± 0.01e

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T5 0.24 0.0031 0.0031 6.68 ± 0.01b 7.06 ± 0.02b
T6 0.12 0.0062 0.0062 4.93 ± 0.00c 5.44 ± 0.03c
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*
Bacterial counts after 24 h of incubation
NI, nisin Z; AITC, allyl isothiocyanate; GO, garlic oil
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Table 2 Formulations of samples of fresh mixed sausage

Formulations
Ingredients
NC* PC† F1 F2 F3
Lean pork (g/kg) 595 595 595 595 595
Beef (g/kg) 275 275 275 275 275
Water (g/kg) 100 100 100 100 100
NaCl (g/kg) 24 24 24 24 24

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Sodium erythorbate (g/kg)*

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0.12 0.12 0.12 0.12 0.12
Parsley powder (g/kg) 1 1 1 1 1

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Onion powder (g/kg) 2 2 2 2 2
White pepper powder (g/kg) 2 2 2 2 2

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Nutmeg powder (g/kg) 0.26 0.26 0.26 0.26 0.26
Curing salt (91% NaCl + 9% 0.62 0.62 0.62 0.62 0.62
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**
NaNO 2 ) (g/kg)
E. coli O157:H7 (log UFC/g) 0 6 6 6 6
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Nisin Z (mg/kg) 0 0 20 20 20
Allyl isothiocyanate (μL/kg) 0 0 0 62.5 125.0
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Garlic oil (μL/kg) 0 0 0 125.0 62.6

*
NC: Negative control, i.e., without bacterial inoculation
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†
PC: Positive control, i.e., with bacterial inoculation and without treatment
*
Food Pryme, Sorocaba, SP, Brazil
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**
BRC Ingredientes, Rio Claro, SP, Brazil
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Table 3. Sensory characteristics of samples of fresh mixed sausage subjected to various

treatments (Mean ± Standard error)
Characteristics NC F2 F3
Color 7.22 ± 0.15ab 6.74 ± 0.17b 7.26 ± 0.13a
Odor 6.94 ± 0.17a 6.10 ± 0.20b 6.23 ± 0.19b
Tenderness 7.06 ± 0.15a 7.31 ± 0.14a 6.92 ± 0.16a
Flavor 7.52 ± 0.13a 6.34 ± 0.22b 6.76 ± 0.19b

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7.44 ± 0.10a 6.65 ± 0.16b 6.72 ± 0.15b

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Overall rating
NC = Control formulation without treatment; F2 = formulation + 125 µL/kg GO + 62.5 µL/kg

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AITC, F3 = formulation + 62.5 µL/kg GO + 125 µL/kg AITC
Tests were conducted by untrained panelists (n = 100), which used an affection hedonic scale

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(1-extremely poor and 9- extremely good)
Different lower case letters show significant differences (P < 0.05) in the same row
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Figure 1. Population of (A) E. coli O157:H7; (B) Native E. coli; (C) lactic acid bacteria; and
(D) psychrotrophic bacteria in fresh sausage during 20 d of storage (6°C). NC = negative
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control, PC = positive control, T1 = 20 mg/kg NI, T2 = 20 mg/kg NI + 125 µL/kg GO + 62.5
µL/kg AITC, T3 = 20 mg/kg NI + 62.5 µL/kg GO +125 µL/kg AITC
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Figure 2. Results of pH measurement in fresh sausage with addition of nisin Z, allyl

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isothiocyanate, and/or garlic oil. Samples were stored at 6 C for 20 days. NC = negative
control, PC = positive control, T1 = 20 mg/kg NI, T2 = 20 mg/kg NI + 125 µL/kg GO + 62.5
µL/kg AITC, T3 = 20 mg/kg NI + 62.5 µL/kg GO + 125 µL/kg AITC
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Figure 3. Color coordinates (A) a*; (B) b*; (C) hue, and L* (D) evaluated in samples of fresh
mixed sausage. Samples were stored at 6 C and evaluated for 20 days. NC = negative control,
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PC = positive control, T1 = 20 mg/kg NI, T2 = 20 mg/kg NI + 125 µL/kg GO + 62.5 µL/kg
AITC, T3 = 20 mg/kg NI + 62.5 µL/kg GO + 125 µL/kg AITC.
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Highlights

 Combinations of essential oils (EO) and nisin (NI) significantly

reduced E. coli O157:H7 count in sausage
 EO and NI were effective in controlling spoilage lactic acid bacteria in
sausage
 Natural antimicrobials maintained the red color and pH in the
sausage samples
 Fresh sausages containing combinations of EO and NI were sensory

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acceptable

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 This study showed the commercial potential of fresh sausages
containing EO and NI

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Figure 1
Figure 2
Figure 3