Accepted Manuscript

The effect of essential oil and extract from sage (Salvia officinalis L.) herbal dust
(food industry by-product) on the oxidative and microbiological stability of fresh pork
sausages

Branislav Šojić, Branimir Pavlić, Zoran Zeković, Vladimir Tomović, Predrag Ikonić,
Sunčica Kocić-Tanackov, Natalija Džinić
PII: S0023-6438(17)30874-5
DOI: 10.1016/j.lwt.2017.11.055
Reference: YFSTL 6684

To appear in: LWT - Food Science and Technology

Received Date: 4 September 2017

Revised Date: 27 October 2017
Accepted Date: 28 November 2017

Please cite this article as: Šojić, B., Pavlić, B., Zeković, Z., Tomović, V., Ikonić, P., Kocić-Tanackov,
Sunč., Džinić, N., The effect of essential oil and extract from sage (Salvia officinalis L.) herbal dust (food
industry by-product) on the oxidative and microbiological stability of fresh pork sausages, LWT - Food
Science and Technology (2017), doi: 10.1016/j.lwt.2017.11.055.

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 ACCEPTED MANUSCRIPT
1 The effect of essential oil and extract from sage (Salvia officinalis L.) herbal dust (food

2 industry by-product) on the oxidative and microbiological stability of fresh pork

3 sausages

5 Branislav Šojića,*, Branimir Pavlića, Zoran Zekovića, Vladimir Tomovića, Predrag Ikonićb,

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6 Sunčica Kocić-Tanackova, Natalija Džinića

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7
a
8 Faculty of Technology, University of Novi Sad, Bulevar Cara Lazara 1, 21000 Novi Sad,

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9 Serbia
b
10 Institute for Food Technology, University of Novi Sad, Bulevar Cara Lazara 1, 21000 Novi

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Sad, Serbia
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13 *Corresponding author: Tel.: +381214853714

14 E-mail address: sojic@tf.uns.ac.rs

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15
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16 Abstract

17 The effect of sage essential oil (SEO) and sage extract (SE) obtained from sage (Salvia
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18 officinalis L.) herbal dust (food industry by-product) on microbiological and oxidative

stability of fresh pork sausages was investigated. pH, microbiological analysis, TBARS value
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20 and sensory panel scores were assessed. Oxygenated monoterpenes, oxygenated

21 sesquiterpenes and diterpene polyphenols were the most abundant compounds present in SEO

22 and SE samples. SEO and SE addition resulted in significant (p<0.05) inhibition of microbial

23 growth. The inclusion of SEO and SE significantly (p<0.05) reduced the TBARS values.

24 Moreover, SE had a positive effect on sensory properties of fresh pork sausages. Hence, the

25 results of this study showed significant antioxidative and antimicrobial activities of SEO and

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26 SE obtained from sage filter tea processing by-products and potential of its utilization in

27 production of fresh pork sausages in order to enhance their stability and safety.

28 Keywords: sage by-product; supercritical fluid extraction; fresh pork sausage; oxidative

29 stability; microbiological stability

30

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31 Chemical compounds studied in this article

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32 1,1-diphenyl-2-picryl-hydrazyl-hydrate (Pubchem CID: 74358); α-Thujone (PubChem CID:

33 261491); Eucalyptol (PubChem CID: 2758); Borneol (Pubchem: CID: 64685); Camphor

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34 (Pubchem CID: 2537); 2-Thiobarbituric acid (PubChem CID: 2723628); Trichloroacetic acid

35 (PubChem CID: 6421); Butylhydroxytoluene (PubChem CID: 31404); n-Hexane (PubChem

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CID: 8058); 1,1,3,3-Tetraethoxypropane (PubChem CID: 67147);
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37
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38 Introduction

39 Fresh pork sausages are among the most common and the most popular processed meat
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40 products all around the world (Georgantelis, Ambrosiadis, Katikou, Blekas, & Georgakis,
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41 2007). Due to very high water activity, relatively high fat content, comminuted structure of

42 raw materials, high total number of microorganisms and lack of thermal processing these
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43 products are characterized with a short shelf life. The spoilage can be caused by microbial

contamination and lipid oxidation, leading to development of unacceptable sensory

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45 characteristics and even foodborne diseases. Hence, the inhibition of microbial growth and

46 delay of lipid oxidation are primary goals that can affect significantly the extension of shelf

47 life (Georgantelis et al. 2007; da Silveira, Luciano, Fronza, Cunha, Scheuermann, & Vieira,

48 2014).

49 In order to reduce oxidative changes and to prevent bacterial growth several synthetic food

50 additives are regularly used by meat processors. However, in recent years due to increasing

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51 consumer awareness about potentially toxic effects and health issues the use of nitrites and

52 synthetic antioxidants (BHT, BHA, TBHQ) decreased, while demand for natural additives

53 rapidly increase (Georgantelis et al. 2007; Šojić et al., 2015; Zeng, Bai, Lu, & Dong, 2016;

54 Šojić et al., 2017). Many of natural antioxidants also exhibit antimicrobial activity and thus

55 have the advantage of being readily accepted by both consumers and meat processors (Sallam,

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56 Ishioroshi, & Samejima 2004; Georgantelis et al. 2007; Jajawardana et al., 2015). However,

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57 natural agents are often more expensive and less effective than synthetic ones. Consequently,

58 increasing attention is recently paid to the extraction of antioxidants from agro-food industry

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59 by-products (Mandic, Dilas, Cetkovic, Canadanovic Brunet, & Tumbas 2008; Lorenzo,

60 Sineiro, Amado, & Franco, 2014).

61
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Sage (Salvia officinalis L.) represents medicinal plant from Lamiaceae family, which has
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62 been recognized for various biological activities due to its interesting chemical profile.
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63 Besides widely used application in pharmaceutical industry, sage has found application as

64 flavouring agent in food products (Gali-Muhtasib, Hilan, & Khater, 2000) and fragrance in
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65 perfumes and cosmetics. Essential oil has been commonly isolated from sage by
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66 hydrodistillation and the most dominant terpene compounds in sage essential oil (SEO) are α-

67 and β-thujone, camphor and eucalyptol (Aleksovski & Sovova, 2007). Furthermore,
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68 supercritical fluid extraction (SFE) has been utilized for extraction of SEO due to its

advantages comparing to hydrodistillation (Aleksovski & Sovova, 2007; Glisic, Ivanovic,

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70 Ristic & Skala, 2010). Recently, sage herbal dust, which is generated in filter tea factory, has

71 been used as raw material for extraction of bioactive compounds (Pavlić et al., 2016; Zeković

72 et al., 2017). However, these extracts have not yet found application in either pharmaceutical

73 or food products.

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74 Therefore, the aim of this study was to determine antioxidant and antimicrobial effect of sage

75 essential oil and extract, obtained from sage tea processing by-products, during refrigerated

76 storage of fresh pork sausage.

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78 Materials and methods

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79

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80 Plant material

81 Sage (Salvia officinalis L.) originated from Montenegro was kindly donated by local filter tea

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82 factory, Fructus DOO (Bačka Palanka, Serbia). Sage herbal dust was obtained as by-product

83 in filter tea factory while all processing steps and applied unit operations were described

84
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elsewhere (Pavlić et al., 2016). Plant material had mean particle size <0.315 mm and moisture
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85 content of 7.24 ± 0.05%.
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86

87 Chemicals
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88 Commercial carbon dioxide (Messer, Novi Sad, Serbia) with >99.98% (m/m) purity was used
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89 for laboratory scale supercritical fluid extraction. The standard compounds for GC analysis:

90 α-pinene, β-pinene, camphor, methyl chavicol and eucalyptol were purchased from Dr
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91 Ehrenstorfer (Germany) and γ-terpinene, (+)-limonene, linalool, geraniol, carvacrol, eugenol

and α-terpineol were purchased from Carl Roth (Germany). α-Thujone, 1,1-diphenyl-2-picryl-
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hydrazyl-hydrate (DPPH·) and 2-thiobarbituric acid were purchased from Sigma-Aldrich

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94 GmbH (Germany). All other chemicals used were of analytical reagent grade.

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96 Essential oil extraction

97 Conventional (hydrodistillation) and novel (supercritical fluid extraction – SFE) extraction

98 techniques were used for recovery of sage essential oil. The official procedure from Ph.Jug.

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99 IV was applied for hydrodistillation. Briefly, 20 g of sage herbal dust and 400 mL of water

100 were mixed in 1 L round flask connected with Clevenger-type apparatus. Hydrodistillation

101 was performed for 2 h and essential oil was separated from aqueous phase after determination

102 of its yield (%; v/w).

103 The supercritical fluid extraction (SFE) was performed on laboratory scale high pressure

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104 extraction plant (HPEP, NOVA, Swiss, Efferikon, Switzerland) described in detail by Pekić et

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105 al. (1995). The main plant parts and properties, by manufacturer specification were: gas

106 cylinder with CO2, the diaphragm type compressor with pressure range up to 1000 bar,

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107 extractor with heating jacket for heating medium with internal volume 200 mL, maximum

108 operating pressure of 700 bar, separator with heating jacket for heating medium (with internal

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volume 200 mL, maximum operating pressure of 250 bar), pressure control valve,
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110 temperature regulation system and regulation valves. Sage herbal dust (35.0 g) was placed in
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111 an extractor vessel and extraction process was carried out for 4h at following conditions: 100

112 bar pressure, 40 ˚C temperature and 0.2 kg/h CO2 flow rate, while separator conditions were
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113 15 bar and 25 °C. Total extraction yield was measured and result was expressed as grams of
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114 total extractable compounds per 100 grams of plant material (g/100 g), i.e. percentage (%).

115
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116 GC-MS and GC-FID analysis

GC-MS analysis was performed on Agilent GC890N system coupled to mass spectrometer
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118 model Agilent MS 5759. The HP-5MS column (30 m length, 0.25 mm inner diameter and

119 0.25 µm film thickness) was used. Helium flow rate was 2 mL/min. Obtained extracts were

120 dissolved in methylene chloride and injected volume of solution was 5 µL with split ratio

121 30:1. Temperature conditions were as follows: injector temperature 250 ˚C, detector

122 temperature 300 ˚C; initial 60 ˚C with linear increase of 4 ˚C/minute to 150 ˚C. Compounds

123 were identified using the NIST 05 and Wiley 7n data base by comparing their spectral data to

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124 those from mass spectral libraries and with spectral data of standard compounds. GC-FID

125 analysis was used for quantitative determination of dominant monoterpene compounds in

126 sage essential oil (γ-terpinene, (+)-limonene, eucalyptol, α-terpineol, methyl chavicole,

127 carvacrol, α-pinene, β-pinene, α-thujone, eugenol, linalool, camphor and geraniol). Standard

128 compounds dissolved in methylene chloride at different concentrations (1 – 500 µg/mL) were

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129 used for creation of calibration curves which are describing dependence of peak area at

different concentrations (R2>0.99). All experiments were performed in triplicates and results

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130

131 are expressed as milligrams per gram of extract (mg/g).

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132

133 In vitro antioxidant activity

134
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Free radical scavenging activity of samples was determined using DPPH assay, previously
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135 described by Espín et al. (2000). A certain volume of sample diluted in ethyl acetate was

mixed with 95% methanol and 90 µM 1,1-diphenyl-2-picryl-hydrazyl (DPPH·) in order to

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137 obtain different final concentrations. After incubation at room temperature for 60 min, the
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138 absorbance was measured at 515 nm and result was expressed as radical scavenging capacity
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139 (RSC, %) which was calculated using following equation:

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A × 100
%RSC = 100 − (1)
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140 where Asample is the absorbance of sample solution and Ablank is the absorbance of blank probe.
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141 Antioxidant activity was further expressed as inhibition concentration at 50% of RSC value

142 (IC50). IC50 represents the concentration of test solution required to obtain 50% of radical

143 scavenging capacity, expressed as μg per mL. All experiments were performed in triplicate,

144 and results are expressed as mean values.

145

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146 Preparation of fresh pork sausage

147 Fresh pork sausages (type of Petrovská klobása) were produced in meat processing pilot plant

148 within Institute of Food Technology Novi Sad (FINS). Fresh boneless chilled pork shoulder

149 and back fat were purchased from a local industrial plant. Pork shoulder (74.48% moisture,

150 19.34% protein, 5.05% fat, 0.98% ash) and back fat were minced using an electric meat

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151 grinder with 8 mm grinding plate. The sausage batter was obtained by mixing minced pork

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152 (80%) and back fat (20%) with salt (1.80%), sweet paprika powder (1.00%), red hot paprika

153 powder (0.70%), caraway (0.20%) and garlic paste (0.07%), in meat mixer. The amount of

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154 seasonings was calculated in relation to minced meat and back fat weight. The grounded

155 meat, back fat and other ingredients were blended for approximately 5 min, until the

156
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homogenous batter was obtained, and the temperature of approximately 7°C was recorded.
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157 The resulting mixture (55.81% moisture, 15.21% protein, 23.19% fat, 3.01% ash) was divided
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158 into seven batches. Sage essential oil (SEO) and sage extract (SE) were added separately in

159 six of them, at concentrations of 0.05 µL/g (SEO1; SE1), 0.075 µL/g (SEO2; SE2) and 0.1
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160 µL/g (SEO3; SE3), representing different treatments. SEO and SE were mixed with salt
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161 before being mixed with other ingredients. The remaining batch was used as control sample

162 (C). All batches were stuffed in natural casings (pig small intestines; Ø ≈ 32 mm). Sausages
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163 were packed in polyethylene plastic bags (without vacuum) and stored at 3 ± 1 ˚C, under dark

conditions, for 8 days.

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165

166 Samples

167 Samples taken at distinct periods of storage included three randomly selected sausages from

168 each batch after 0, 2, 4, 6 and 8 days. Analyses were carried out at the day of sampling, and

169 were done in duplicate for each sample.

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171 pH determination

172 The pH of samples was measured using the portable pH meter Testo 205 (Testo AG, USA)

173 equipped with a combined penetration tip with temperature probe. The pH meter was

174 calibrated before the readings using 2 buffer solutions (pH=4.00 ± 0.05 and pH = 7.00 ± 0.01

175 at 20 ± 2 ˚C).

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176

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177 Microbiological analysis

178 Microbiological analyses were performed on three samples from each group of the fresh pork

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179 sausages in duplicate. Twenty grams of samples were homogenized for 10 minutes at 200 rpm

180 (Unimax 1010, Heidolph, Germany) in 180 mL 1 g/L buffered peptone water (Merk,

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Darmstadt) and then serial of decimal dilution were prepared (up to 10-3). One milliliter of
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182 each dilution was placed in a sterile Petri plate and poured with appropriate media depending
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183 on the type of tested microorganism. The following microbial analyses were performed: total

184 number of aerobic mesophilic bacteria (ISO 4833:2003), Salmonella spp. (ISO 6579:2008),
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185 Escherichia coli (ISO 7251:2005), Listeria monocytogenes (ISO 11290-2:1998). Results were
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186 expressed as a log cfu/g.

187
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188 TBARS determination

TBARS (2-thiobarbituric acid reactive substances) test was performed according to the
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189
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190 method of Botsoglou, Fletouris, Papageorgiou, Vassilopoulos, Mantis & Trakatellis (1994)

191 with modifications described in Šojić et al. (2015). TBARS values were expressed as

192 milligrams of malondialdehyde per kilogram of sample (mg MDA/kg).

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194 Sensory analysis

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195 Sensory evaluation of the investigated sausages was performed by ranking tests (ISO 8587,

196 2006; ISO 8589, 2007; Lawless & Heymann, 2010). Sausages were taken from refrigerated

197 storage (3°C) and baked in oven at 163 °C for 1 h. The sausages were served warm (50-60°C)

198 and analyzed by a panel of 70 consumers. Consumers were asked the following: “Please rank

199 the samples in numerical order according to your preference“ (from like extremely - 1 to

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200 dislike extremely - 7) or „ Please rank the intensity of sage odour and flavour“ (from none - 1

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201 to very strong - 7). Consumers between 19 and 65 years old were students and staff members

202 of the Faculty of Technology Novi Sad. The sensory analyses were performed immediately

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203 after stuffing (0), and after 4 and 8 days of refrigerated storage.

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205 Statistical analysis

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206 Statistical analysis was carried out using STATISTICA 12.0 (StatSoft, Inc., Tulsa, OK, USA).
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207 All data were presented as mean value with their standard deviation indicated (mean ± SD).

208 Variance analysis (ANOVA) was performed, with a confidence interval of 95% (p<0.05).
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209 Means were compared by Fisher's LSD test.

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211 Results and discussion

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212

Essential oil and extract characterization

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214 Chemical profile of sage herbal dust essential oil (SEO) and extract obtained by SFE (SE)

215 was determined by GC-MS and GC-FID analysis and results are presented in Table 1.

216 According to results, it could be observed that oxygenated monoterpenes (α-thujone, camphor

217 and eucalyptol), oxygenated sesquiterpenes (viridiflorol) and diterpene polyphenols

218 (epirosmanol) were the most abundant compounds present in SEO and SE samples. Besides

219 that, certain oxygenated monoterpenes (β-thujone, borneol and bornyl acetate) and

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220 sesquiterpene hydrocarbons (trans-cariophyllene and α-humulene) were observed in SE with

221 relative percentage >1% (Table 1). Similar chemical profile was observed in sage extract

222 obtained by SFE at 100 bar and 50 ˚C (Glisic et al., 2010). Even though, less terpenoid

223 compounds were detected in SEO comparing to SE, similar compounds had the highest

224 relative percentage in both samples. According to literature data, oxygenated monoterpenes

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225 (eucalyptol, α-thujone, β-thujone and camphor) were the most dominant compounds (≈70%)

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226 in sage essential oil obtained by hydrodistillation (Aleksovski & Sovova, 2007; Glisic et al.,

227 2010; Occhipinti, Capuzzo, Arceusz & Maffei, 2014). In both SEO and SE, the most

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228 abundant subgroups of terpenoid compounds were oxygenated monoterpenes and diterpenes.

229 Higher relative percentage of oxygenated monoterpenes (50%) was observed in SE, while

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SEO exhibited higher content of diterpenes (33%) comparing to its content in SE sample
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231 (24%) (Figure 1). It should be pointed out that these compounds were responsible for
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232 biological activity of S. officinalis essential oil, particularly diterpenes for high antioxidant

233 potential (Babovic et al., 2010) and oxygenated monoterpenes for antimicrobial effects
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234 (Bozin, Mimica-Dukic, Samojlik & Jovin, 2007). Even though, GC-MS represents commonly
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235 used procedure for chemical characterization of EOs, relative percentage of determined

236 compounds does not represent suitable quantitative indicator in extracts obtained by SFE due
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237 to coextraction of various non-volatile lipids. Therefore, GC-FID analysis was utilized to

determine content of certain monoterpene compounds (γ-terpinene, (+)-limonene, eucalyptol,

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238
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239 α-terpineol, methyl chavicole, carvacrol, α-pinene, β-pinene, α-thujone, eugenol, linalool,

240 camphor and geraniol) in SEO and SE. It could be observed that eucalyptol (103.82 and 32.75

241 mg/g), α-thujone (170.48 and 91.54 mg/g) and camphor (177.74 and 107.48 mg/g) were the

242 most abundant monoterpenes in SEO and SE, respectively. Higher content of oxygenated

243 monoterpenes in SEO was rather expected since hydrodistillation provides “pure” EO, while

244 various lipids were commonly coextracted with volatile compounds by SFE.

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245 Antioxidant activity of SEO and SE samples towards DPPH radicals expressed as IC50 value

246 was 0.4823 and 0.0242 mg/mL, respectively. Even though, SE sample had lower content of

247 oxygenated monoterpenes, antioxidant activity of this sample was higher comparing to SEO,

248 due to high antioxidant potential of coextracted compounds, particularly diterpene

249 polyphenols. Antioxidant activity observed in SEO sample was higher comparing to S.

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250 officinalis EO from Spain (IC50 = 4.20 mg/mL) (Viuda‐Martos, Ruiz Navajas, Sánchez

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251 Zapata, Fernández‐López & Pérez‐Álvarez, 2010) and Algeria (IC50 = 2.00 mg/mL) (Adrar,

252 Oukil & Bedjou, 2016). Furthermore, sage extract obtained by SFE had lower antioxidant

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253 activity (IC50 = 0.23 mg/mL) comparing to previously analyzed SE sample (Babovic et al.,

254 2010). This could be explained by variation of SFE conditions which were set for extraction

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of diterpene polyphenols (350 bar and 100 ˚C) after previous extraction of essential oil
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256 fraction (115 bar and 40 ˚C).
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258 pH changes in fresh pork sausages

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259 The effect of SEO and SE on the pH values of fresh pork sausages is shown in Table 2. At the
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260 beginning of storage pH values ranged from 5.60 to 5.66. Obtained results showed very good

261 corresponding with literature data for this type of sausage (Crist, Williams, Schillinga, Hood,
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262 Smith, & Campano, 2014; Fasolato et al., 2016; Sharma, Mendiratta, Agrawal, Gurunathan,
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263 Kumar, & Singh, 2017). Significant drop of pH was registered in each sausage group during 8
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264 days of refrigerated storage. Most probably, this was result of growth and metabolic activity

265 of lactic acid bacteria, as it was previously reported by a number of authors (Wenjiao,

266 Yunchuan, Junxiu, & Yongkui, 2014; Jayawardana, Liyanage, Lalantha, Iddamalgoda, &

267 Weththasinghe, 2015; Sharma et al., 2017). However, the lowest numerical values were

268 registered in sausages C and SEO1, being in accordance with the highest microbial growth

269 (Table 3).

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270

271 Microbiological analysis

272 The microbiological profile of fresh pork sausage during 8 days of storage under refrigeration

273 is shown in Table 3. The addition of SEO and SE significantly (p<0.05) reduced the total

274 number of aerobic mesophilic bacteria (TBC). Probably, it was the consequence of sage

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275 antimicrobial properties which could be attributed to oxygenated monoterpenes (Burt, 2004),

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276 which consisted approximately 50% of SEO. In case of S. officinalis EO, eucalyptol (1,8-

277 cineole), α-thujone and camphor were related to antimicrobial effects (Delamare, Moschen-

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278 Pistorello, Artico, Atti-Serafini & Echeverrigaray, 2007). However, it should be also pointed

279 out that other EO constituents possess antimicrobial effects, as well as that their synergistic

280
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effect could increase antimicrobial potential in food preservation (Hyldgaard, Mygind &
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281 Meyer, 2012). Even though, content of oxygenated monoterpenes was approximately twice
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282 lower in SE sample (Table 1), there was no significant difference in antimicrobial effects of

283 SEO and SE samples (Table 3).

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284 The initial TBC ranged from 5.56 log cfu/g (SE2) to 6.90 log cfu/g (C). As expected, for all
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285 treatments the TBC significantly (p<0.05) increased during 8 days of storage. At the end of

286 storage, TBC was significantly (p<0.05) different between the samples, being arranged as
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287 follows: C>SE1≥SEO1>SEO2>SEO3>SE2>SE3. After 8 days, TBC was lower than 7 log

cfu/g just in samples SE2 and SE3, showing considerable effect of sage extract on reduction
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289 of microbial growth.

290 Three analyzed foodborne pathogenic bacteria (Salmonella spp., E. coli, L. monocytogenes)

291 were not detected both in control and treated samples.

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293 Thiobarbituric acid reactive substances (TBARS)

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294 Lipid oxidation was evaluated by determining the levels of TBARS

295 (mg malondialdehyde/kg). At the beginning of storage TBARS values varied in very narrow

296 interval from 0.03 (SE2; SEO3) to 0.06 mg malondialdehyde/kg (control) (Table 2). In all

297 treatments TBARS values significantly increased (p<0.05) during 8 days of storage,

298 indicating the occurrence of lipid oxidation (Zhang, Lin, Leng, Huang, & Zhou, 2013; Šojić et

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299 al., 2015).

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300 After 6 day of storage, sausages produced with the addition of SEO and SE had significantly

301 (p<0.05) lower TBARS values compared to C samples. Additionally, at the end of storage, the

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302 TBARS test showed differences between the samples in the following order:

303 C>SEO1>SEO2≥SE1≥SEO3≥SE2≥SE3. These results demonstrate a good antioxidative

304
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potential of SEO and especially SE, which showed the highest inhibitory potential against
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305 lipid oxidation in concentration of 0.1 µL/g. Antioxidant effects of EOs could be achieved
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306 through scavenging free radicals, the inhibition of lipid peroxidation, the chelating of

307 transition metal ions (Bozin et al., 2007). Besides monoterpene hydrocarbons, diterpene
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308 polyphenols were designated as the major subgroup of sage bioactive compounds responsible
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309 for high antioxidant potential (Babovic et al., 2010). Higher antioxidant activity of SE sample

310 could be potentially explained by the synergistic effects of terpenoids and other lipids which
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311 are simultaneously extracted by SFE. Similarly, the addition of ground sage in concentrations

from 0.05 to 0.15% decreased lipid oxidation in Chinese-style sausage (Zhang et al., 2013).
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312
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313 Also, Estévez, Ventanas & Cava (2006) reported that sage extract oil had a good antioxidative

314 potential in liver pâté during 90 days of storage (0.1%). According to Tisserand & Balacs

315 (1995), permitted content of thujones in food and beverages is <0.5 mg/kg due to their acute

316 toxicity in increased concentrations. Since concentration of SEO and SE in pork sausages was

317 0.05 – 0.1 mL/kg and α-thujone concentration in these samples was 170.48 and 91.54 mg/g

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318 (Table 1), respectively, thujones content in final product was certainly bellow maximal

319 permitted content.

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321 Sensory evaluation of fresh pork sausages

322 The sensory attributes (preference, odour and flavour) of C, SEO and SE fresh pork sausages

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323 are showed in Table 4. Intensity of odour and flavour in sausages produced with 0.05 µL/g of

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324 sage extract (SE1) was not significantly different (p>0.05) compared to C samples. Generally,

325 sausages produced with the addition of SE had a significantly (p<0.05) lower intensity of

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326 odour and flavour compared to sausages treated with the SEO. Effects of SEO and SE on

327 sensory properties of sausages were undoubtedly connected with their chemical profile. It has

328
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been noted that hydrodistillation provides “pure” essential oil and monoterpene compounds
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329 with characteristic sensory properties are concentrated in this sample. On the other hand, SFE
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330 allows coextraction of concomitant lipids such as waxes and resins, thus diluting terpene

331 concentration in extracts and reducing sensory effects. Besides that, waxes could detain
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332 evaporation of the terpenes due to hydrophobic interaction, which could also lead to milder
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333 sensory effects comparing to EO.

334 Immediately after stuffing, 0.05 µL/g of sage essential oil and sage extract (SEO1; SE1) and
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335 0.075 µL/g of sage extract (SE2) had no significant (p>0.05) negative effect on sensory

properties of sausages. On the other hand, at the end of storage, all treated sausages had
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336
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337 significantly (p<0.05) lower scores for preference compared to control and SEO3 samples.

338 Deterioration of preference in C samples may be a consequence of microbial growth.

339 Except SEO3, all other concentrations of SEO and SE have a positive effect on sensory

340 properties of fresh pork sausages during storage period. Similarly, Zhang et al. (2013)

341 determined that ground sage in concentrations from 0.10 to 0.15 µg/g had a no negative effect

342 on overall sensory acceptability of Chinese-style sausage.

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343

344 Conclusion

345 The results of this study showed that the application of sage essential oil (SEO) and sage

346 extract (SE) retarded the lipid oxidation in fresh pork sausages. SE at the concentrations of

347 0.075 µL/g and 0.1 µL/g was the most effective against microbial growth. Moreover, SE

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348 obtained by SFE provided better sensory properties of fresh pork sausages, suggesting

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349 advantage of novel extraction technique. Another aspect which should be considered is

350 utilization of sage herbal dust as raw material for essential oil recovery and utilization in food

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351 products. Valorization of these by-products could lead towards sustainable and economically

352 efficient production of natural extracts. The overall results show that SEO and especially SE,

353
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as natural antioxidant and antimicrobial agents, could be successfully applied in formulation
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354 of fresh pork sausages.
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355

356 Acknowledgements
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357 These results are also part of the project TR31032, which is financially supported by the
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358 Ministry of Education, Science and Technological Development, Republic of Serbia.

359
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360 References

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480 officinalis) on the oxidative stability of Chinese-style sausage during refrigerated storage.
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Table 1. GC-MS and GC-FID profile of volatile essential oil compounds determined in sage
essential oil and sage extract

Compound tRa [min] Relative percentage [%] Content [mg/g]

GC-MS GC-FID
SEO SE SEO SE
Monoterpenes
β-Myrcene 4.460 n.d.b 0.06 n/ac n/a
α-Terpinene 5.067 n.d. 0.09 n/a n/a

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p-Cymene 5.283 n.d. 0.21 n/a n/a
Limonene 5.341 0.36 0.39 13.64 3.89
Eucalyptol 5.420 3.30 5.24 103.82 32.75
γ-Terpinene

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6.106 n.d. 0.05 <1 <1
cis-Linalool oxide 6.556 n.d. 0.04 n/a n/a
Dehydro-p-cymene 6.997 n.d. 0.08 n/a n/a

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α-Thujone 7.487 5.26 10.14 170.48 91.54
β-Thujone 7.781 4.09 2.47 n/a n/a
Camphor 8.643 12.74 13.39 177.74 107.48
Borneol 9.480 3.28 4.55 n/a n/a

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Menthol 9.653 n.d 0.19 n/a n/a
4-Terpineol 9.689 n.d. 0.23 n/a n/a
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Carvotanaceton 11.826 n.d. 0.03 n/a n/a
trans-Geraniol 12.191 n.d. 0.07 <1 <1
Bornyl acetate 12.861 1.26 1.14 n/a n/a
Camphene 13.123 n.d. 0.12 n/a n/a
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Thymol 14.111 1.61 0.58 n/a n/a

Carvacrol 14.400 0.95 0.29 2.32 4.14
Sesquiterpenes
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γ-Cariophyllene 16.531 n.d. 0.09 n/a n/a

trans-Cariophyllene 16.942 1.62 1.87 n/a n/a
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Aromadendren 17.530 n.d. 0.07 n/a n/a

α-Humulene 18.009 n.d. 2.24 n/a n/a
Ledene 19.240 n.d. 0.35 n/a n/a
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Cariophyllene oxide 21.927 0.42 0.67 n/a n/a

Viridiflorol 22.386 18.42 18.24 n/a n/a
Ledole 22.586 n.d. 0.09 n/a n/a
Humulene oxide 22.720 n.d. 0.32 n/a n/a
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Cariophyllene 24.690 n.d. 0.77 n/a n/a

Diterpene
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Epirosmanol 34.120 26.25 20.14 n/a n/a

Phytol 35.324 n.d. 0.39 n/a n/a
Ferruginol 40.274 1.81 0.67 n/a n/a
Total 85.01 85.27 468.00 239.80
a
retention time [min]
b
not detected
c
not applicable
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Table 2. Effect of different concentrations of sage essential oil (SEO) and extract (SE) on pH values and TBARS values
(mg malondialdehyde/kg) in fresh pork sausage during storage

pH value

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Storage Treatments
day C SEO1 SE1 SEO2 SE2 SEO3 SE3
0 5.62±0.04Aba 5.60±0.05Ba 5.61±0.02Bb 5.60±0.01Ba 5.61±0.01ABa 5.66±0.02Aa 5.61±0.01Aba

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2 5.54±0.03Db 5.58±0.03BCa 5.63±0.03Aa 5.62±0.01Ab 5.64±0.01Aa 5.61±0.02ABa 5.56±0.03CDb
4 5.47±0.02Ec 5.52±0.01BCb 5.57±0.02Ac 5.55±0.03ABbc 5.56±0.02ABb 5.57±0.04Ab 5.50±0.01CDb

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Cc Cc Abd Acd ABCc BCc
6 5.46±0.01 5.45±0.01 5.51±0.02 5.53±0.03 5.50±0.02 5.47±0.01 5.47±0.06BCc
8 5.40±0.02Cd 5.40±0.01Cd 5.46±0.01ABe 5.46±0.06ABd 5.49±0.01Ac 5.44±0.03ABCc 5.42±0.02BCc
TBARS value (mg malondialdehyde/kg)

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Storage Treatments

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day C SEO1 SE1 SEO2 SE2 SEO3 SE3
0 0.06±0.01Ac 0.04±0.01ABd 0.04±0.01ABd 0.04±0.01ABc 0.03±0.02Bc 0.03±0.01Bc 0.04±0.01ABc
2 0.10±0.08Ab 0.13±0.01Ac 0.13±0.01Ac 0.12±0.02Ab 0.11±0.01Ab 0.12±0.01Ab 0.10±0.01Ab

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4 0.11±0.08Ab 0.15±0.01Ac 0.13±0.02Ac 0.12±0.01Ab 0.13±0.01Ab 0.13±0.01Ab 0.13±0.01Aa
Aa Bb BCa Ca CDa Db
6 0.33±0.01 0.20±0.01 0.19±0.01 0.17±0.01 0.16±0.01 0.14±0.02 0.12±0.01Eab

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8 0.36±0.02Aa 0.24±0.01Ba 0.17±0.01CDb 0.19±0.02Ca 0.15±0.01DEa 0.17±0.01CDa 0.14±0.02Ea
(A-C)
Values with different letters in the same row are significantly different (p<0.05); Values with different letters (a-c) in the same

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column are significantly different (p<0.05)
C EP
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Table 3. Effect of different concentrations of sage essential oil (SEO) and extract (SE) on microbiological profile (log cfu/g) in fresh pork
sausage during storage

Microorganisms Storage Treatments

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day C SEO1 SE1 SEO2 SE2 SEO3 SE3
Ac BCe BCd Ce Ce Bc
0 6.90±0.02 5.74±0.01 5.68±0.03 5.59±0.01 5.56±0.01 6.03±0.05 5.70±0.02BCe
2 6.10±0.01Ae 6.00±0.02Bd 5.70±0.05Ed 5.88±0.01Cd 5.60±0.02Fd 5.80±0.02Dd 6.00±0.01Bd

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Total mesophilic aerobic Ad Bc Cc Dc Bc Cb
4 6.63±0.03 6.38±0.02 6.26±0.02 6.01±0.01 6.38±0.04 6.23±0.01 6.39±0.01Bc
count
6 7.15±0.03Ab 6.84±0.02Cb 6.41±0.02Fb 6.73±0.02Db 6.50±0.02Eb 6.98±0.02Ba 6.85±0.05Ca

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8 7.66±0.04Aa 7.29±0.01Ba 7.30±0.02Ba 7.15±0.01Ca 6.72±0.02Ea 7.10±0.02Da 6.50±0.02Fb
0 ND ND ND ND ND ND ND
2 ND ND ND ND ND ND ND

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Salmonella spp. 4 ND ND ND ND ND ND ND

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6 ND ND ND ND ND ND ND
8 ND ND ND ND ND ND ND
0 ND ND ND ND ND ND ND

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2 ND ND ND ND ND ND ND
E. coli 4 ND ND ND ND ND ND ND

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6 ND ND ND ND ND ND ND
8 ND ND ND ND ND ND ND

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0 ND ND ND ND ND ND ND
2 ND ND ND ND ND ND ND
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L. monocytogenes 4 ND ND ND ND ND ND ND
6 ND ND ND ND ND ND ND
8 ND ND ND ND ND ND ND
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(A-E) (a-c)
Values with different letters within each parameter in the same row differ significantly (p<0.05); Values with different letters in the same
column differ significantly (p<0.05)
AC
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Table 4. Summary of rank sums for each sensory attribute

Storage Sensory Treatments

day attribute C SEO1 SE1 SEO2 SE2 SEO3 SE3

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Odour 188d 316bc 188d 350b 206d 443a 269c
e c de
0 Flavour 134 330 165 387b 201d 463a 280c
212d 258cd 212cd 338b 237cd 441a 264c

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Preference
Odour - - - - - - -
4 Flavour - - - - - - -

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b c c
Preference 335 235 223 268c 248c 433a 220c
Odour - - - - - - -

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8 Flavour - - - - - - -
a c c c
Preference 473 196 213 208 226c 413b 233c

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Number of products = 7; Number of assessors (consumers) = 70; LSD = 50.1 (at the 0.05 risk)

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Monoterpene
a) hydrocarbons
0%

Diterpenes
35% Oxygenated
monoterpenes
40%

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Oxygenated
Sesquiterpenes
sesquiterpenes
2%
23%

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Monoterpene
b) hydrocarbons
Diterpenes

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1%
25%
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Oxygenated
monoterpenes
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45%
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Oxygenated
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sesquiterpenes
23% Sesquiterpenes
6%
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Figure 1. GC-MS profile of main subgroups of terpenoid compounds detected in a) essential

oil and b) extract of sage herbal dust
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Highlights

• Sage essential oil and extract (SEO; SE) were obtained from sage tea processing
by-product.
• SE was obtained by environmentally friendly "green" extraction technique.
• Use of SEO and SE reduced lipid oxidation and microbial growth in fresh
sausages.

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• Good sensory properties of fresh pork sausages were preserved due to SE
addition.

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