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Šojić, B.; Putnik, P.; Danilović, B.; Teslić, N.; Bursać Kovačević, D.; Pavlić, B. Lipid Extracts Obtained by Supercritical Fluid Extraction. Encyclopedia. Available online: https://encyclopedia.pub/entry/56643 (accessed on 25 May 2024).
Šojić B, Putnik P, Danilović B, Teslić N, Bursać Kovačević D, Pavlić B. Lipid Extracts Obtained by Supercritical Fluid Extraction. Encyclopedia. Available at: https://encyclopedia.pub/entry/56643. Accessed May 25, 2024.
Šojić, Branislav, Predrag Putnik, Bojana Danilović, Nemanja Teslić, Danijela Bursać Kovačević, Branimir Pavlić. "Lipid Extracts Obtained by Supercritical Fluid Extraction" Encyclopedia, https://encyclopedia.pub/entry/56643 (accessed May 25, 2024).
Šojić, B., Putnik, P., Danilović, B., Teslić, N., Bursać Kovačević, D., & Pavlić, B. (2024, May 12). Lipid Extracts Obtained by Supercritical Fluid Extraction. In Encyclopedia. https://encyclopedia.pub/entry/56643
Šojić, Branislav, et al. "Lipid Extracts Obtained by Supercritical Fluid Extraction." Encyclopedia. Web. 12 May, 2024.
Lipid Extracts Obtained by Supercritical Fluid Extraction
Edit

Supercritical fluid extraction (SFE) has been recognized as the green and clean technique without any negative impact on the environment. Although this technique has shown high selectivity towards lipophilic bioactive compounds, very few case studies on the application of these extracts in final products and different food matrices were observed.

meat products supercritical fluid extraction lipid extracts natural antioxidants antimicrobials

1. Introduction

In 2020, modern meat market was USD 838 billion, and it is expected to further increase up to USD 1 trillion by 2025. During the same year, 328 million tons of meat were produced in the world, which is equal to 35 kg of meat/capita. Hence, meat market has large share in international food industry [1]. One of the largest challenges in meat processing is oxidation processes that are dangerous for human health and the market value of meat products.
Oxidative reactions (lipid and protein oxidation) and the growth and activity of microbial populations are the two main causes of deterioration in the quality of meat and meat products [2]. Due to the relatively high content of unsaturated lipids and the presence of a variety of oxidants in muscles, meat and meat products are highly susceptible to lipid and protein oxidation. Hydroperoxides as primary and volatile compounds (e.g., aldehydes, ketones, organic acids) as secondary products of lipid oxidation led to a reduction in nutritional content and shelf-life, as well as market value of the final products [3][4][5]. Moreover, oxidative reactions in muscle protein have a strong influence on the overall quality of meat and meat products. Muscle peptides in interacting with reactive oxygen species lead to the formation of covalent intermolecular cross-linked proteins, resulting in protein aggregation and fragmentation. The changes in proteins in muscles promote the reduction in water-holding capacity, loss of biological functionality of proteins, and reduction in sensory quality (colour, flavour and texture) of meat and meat products [6][7][8][9]. Generally, the prevention of oxidative reactions (e.g., lipid and protein oxidation) in the meat products is a key challenge for meat processing technology. Lipid oxidation in meat and meat products is usually evaluate by measuring the amount of peroxide value (PV), thiobarbituric acid-reactive substances (TBARS), whereas the sulphydryl and carbonyl group generated during the meat processing and storage show the level of protein oxidation [8].
Depending on the initial hygiene and the preservation method used, the growth of microbial population (spoilage and pathogenic) can cause spoilage and accordingly various foodborne poisonings in meat and meat products [9]. The growth of spoilage bacteria, yeasts and molds can decompose the main components of meat (e.g., lipids and proteins, and vitamins) and led to slime, unpleasant flavour and abnormal discolouration that reduced the shelf-life of the final products [2].
The pathogenic bacteria (e.g., Salmonella spp., Escherichia coli O157:H7, Listeria monocytogenes) are primarily responsible for numerous foodborne diseases and food poisoning, which is why they are considered the most important limiting factor in the meat industry [9].
To prevent negative changes (chemical and microbial) during processing, meat processors usually use numerous additives (e.g., antioxidants and antimicrobials) [1]. However, most of these additives are synthetic and may have adverse effects on human health. For example, some meat products are classified as Group 1 carcinogens by the World Health Organisation (WHO) due to the use of nitrites and nitrates, which are very common in meat processing [8][10]. They are added to meat as an important antioxidant that prevents the growth of bacteria and off-flavours while preserving the appealing colour of the products. Namely, the use of nitrites and nitrates contributes to the of development of the typical reddish-pink colour and flavour of cured meat products. On the other hand, nitrites and nitrates promote the formation of N-nitrosamines, which are associated with cancer risks. The current Serbian legislation has restricted the maximum amount of nitrate or nitrite that may be added in processed meat expressed as NaNO2 or NaNO3 to 100 and 150 mg/kg, depending on the type of product. Besides nitrites and nitrates, there are other synthetic additives such as butylated hydroxytoluene, butylated hydroxyanisole, tertiary butyl hydroquinone and propyl gallate, which are used to ensure microbial safety and prevent oxidation of products, but also have carcinogenic effects on humans [11][12]. Therefore, in the last decade, the use of natural antioxidants and antimicrobial agents, isolated from various plant materials has received considerable attention in meat processing [2][13]. The natural compounds used as antioxidants and antimicrobial agents in meat products are mainly various essential oils (EOs) or plant extracts [2][4][8]. The ability of these compounds to provide oxidative stability and protection against pathogenic and spoilage microorganisms in meat products has been extensively studied.
Most research has focused on the use of medicinal and aromatic plants with proven antioxidant and antimicrobial potential [14][15][16], but in recent years there has been an increasing use of fruit extracts [17][18][19]. In addition to conventional extraction methods, supercritical fluid extraction (SFE) has proven to be a useful method to obtain extracts of high quality and good biological activity, rich in phenolic and flavonoid compounds. Moreover, supercritical fluid extracts do not contain solvent residues and preserve the aroma of the plant [13].
Nutraceuticals and bioactive compounds can be isolated from natural resources by various extraction techniques before further use as natural additives in food matrices. Conventional extraction processes (Soxhlet extraction, hydrodistillation, etc.) can be effectively used for the recovery of lipophilic bioactive compounds. However, the use of these methods is decreasing, which is related to their limitations. The major problems with traditional extraction techniques are the use of expensive and toxic organic solvents (hexane, methylene chloride, etc.), poor yield and quality of the final product, and huge consumption of time, energy and resources.
The recent trend and challenges posed by green chemistry place high demands on chemical engineering technologies. The most important aspects have been defined by Chemat et al. [20] and are related to the use of alternative solvents that are non-toxic, non-flammable and without toxic residues in the obtained herbal extracts, the use of renewable resources or plant cultivation instead of uncontrolled harvesting of natural resources and the discovery and development of new extraction processes with non-hazardous solvents, low energy requirements, low cost, renewable natural products and high-quality extracts with bioactive molecules. Classical extraction methods are overwhelmed with the above requirements, whereas emerging extraction techniques are increasingly capable of meeting these requirements. Within the concept of green extraction, novel technologies are being developed that include various extraction techniques such as high-pressure extraction (supercritical or subcritical fluid extraction), microwave-assisted extraction, ultrasound-assisted extraction, extraction accelerated by pulsed electric fields, enzyme-assisted processes and extraction with natural deep eutectic solvents.
SFE has been particularly recognized as the green and clean technique with no negative impact on the environment. Moreover, this technique has shown high selectivity towards lipophilic bioactive compounds and the ability to obtain extracts with concentrated target compounds without traces of potentially harmful solvents. De Melo et al. [21] provided an extensive literature review on the application of SFE to isolate a wide range of bioactive compounds from natural sources, whereas Essien et al. [22] reviewed in detail the recent advances in the field of SFE application. An enormous number of studies have been carried out by the scientific community focusing on the SFE of different plant matrices with the aim of determining the extraction kinetics and understanding the phenomena occurring in this process, maximizing the yield of the target compound(s), determining the chemical profile and/or evaluating the bioactivity of the extracts, and finally optimizing the process and adapting it for industrial production. However, there are very few case studies on the application of these extracts in final products and different food matrices. A good example is the case study on sage (Salvia officinalis L.) herbal dust, where the SFE study started with the evaluation of the process kinetics [23], determination of chemical profile and in vitro bioactivity [24], which was followed by the application of these extracts obtained by SFE as natural additives in fresh pork sausages [25], minced pork meat [26] and fresh kombucha cheese [27].

2. Supercritical Fluid Extraction of Lipophilic Bioactives

2.1. Basic Principles of SFE

Solid–liquid extraction (SLE) using organic solvents such as toluene, hexane, heptane, petroleum ether, dichloromethane, chloroform, ethanol, methanol, etc., is a traditionally accepted procedure for the recovery of various classes of lipophilic compounds. This process is usually carried out at lower temperatures than conventional hydrodistillation (HD), thus reducing the risk of possible chemical changes and/or degradation of target compounds due to elevated temperatures. Although non-polar organic solvents have good affinity and selectivity for lipophilic bioactives (essential oils, carotenoids, non-polar polyohenols), concomitant lipophilic compounds such as waxes may be co-extracted, reducing the content of target compounds in the extracts. The use of organic solvents in SLE is associated with other major problems such as particularly high costs due to the consumption of expensive resources, poor environmental impact due to the discharge of the chemicals used, and the major risk of human toxicity that may occur during production or due to the traces of the solvents in extracts and final products.
On the other hand, HD is the most commonly used method for isolating non-water-soluble compounds with high-boiling points such as Eos, which consists only of volatile compounds. This technique is the most widely used in the world due to its simplicity, it is considered the most cost-effective technique for the production of most Eos at a reasonable price in less developed countries [28]. Although HD is already an established and widely used procedure for isolating pure Eos, this technique has some disadvantages in terms of chemical changes such as hydrolysis and thermal degradation that occur during the process and can affect the quality and bioactivity of the product. Moreover, this process consumes a lot of energy for steam generation, heating and cooling. Since the process can take several hours, it is very time-consuming, and prolonged contact between the fresh or dry plant matrix and the heated water increases the risk of undesirable chemical changes and thermal degradation of Eos [28].
SFE has been developed as an environmentally friendly and clean technique that can overcome the above challenges associated with SLE and is an excellent alternative and a current method of choice for the isolation of lipophilic compounds. Over the last 40 years, this technique has become increasingly important in the food, pharmaceutical and cosmetics industries. The most important aspects of these improvements over SLE and HD are reduced solvent consumption, preservation and reuse of energy, reduced time, improved selectivity and total yield, and avoidance of degradation of bioactive compounds by high temperatures.
The supercritical state of matter is associated with a tremendous effect on its physico-chemical properties, which is the main principle of SFE. For example, the increase in pressure leads to an increase in the density of supercritical fluid, whereas some of its transport properties (diffusivity, surface tension and viscosity) are more similar to those gasses [22]. The low viscosity and enormous diffusivity of the supercritical fluid led to an improvement in the heat and mass transfer coefficients of pressurized fluids and consequently to better penetration into the pores of the plant material and rapid dissolution of the target compounds. Among the liquids considered for this purpose, carbon dioxide is recognized as the most commonly used solvent in SFE processes, mainly because its relatively mild critical conditions (31.3 °C and 73.8 bar). In addition, CO2 has other desirable properties as it is non-flammable, non-explosive, available in high purity, relatively cheap, non-toxic (generally recognized as safe-GRAS) and inert [29], which makes it very suitable for the SFE processes.
Extraction and separation are the two main steps of the SFE process. The solvent is first compressed to the working pressure and temperature, which allows its diffusion into the plant matrix and mass transfer from the solid to the liquid phase [30]. The separation of supercritical fluid and lipid extracts can be easily achieved by lowering the pressure and/or temperature in the separator, releasing the gaseous fluid and recovering the solvent-free extracts. Some SFE plants are equipped with two or more separators when it is necessary to separate the extracts into different fractions immediately by choosing the appropriate combination of pressure and temperature [31]. Moreover, the recirculation of the solvent in industrial scale SFE processes could be achieved to improve their efficiency.

2.2. Influence of SFE Parameters

Efficiency, yield and selectivity of the SFE process depend largely on the choice of the main process parameters (pressure, temperature, solvent flow rate, particle size, etc.). Therefore, the application of SFE requires the use of a very specific high pressure process equipment with integrated control of the factors affecting the process. SFE parameters must be selected according to the physicochemical properties of the target compounds and the process should be frequently optimized for each case study to achieve maximum yield of the target compound(s). Solvent solubility is directly affected by pressure and temperature; therefore, they are often considered the most important SFE parameters. Increasing the solvent density and thus improving the solubility is associated with increasing the pressure. This leads directly to a higher total extraction yield, which in certain cases can represent a significant improvement in process efficiency. However, the increased solubility leads to the co-extraction of other lipophilic compounds. For example, an SFE extract of coriander obtained at 100 bar and 40°C would be rich in volatile terpenoids (mainly linalool), whereas SFE at 300 bar and 40°C would tremendously improve the total yield due to co-extraction of fatty oil, resulting in low selectivity towards terpenoids [32]. This leads to dilution of active compounds with concomitants and requires further complications in purification of the extract. Although increasing the process pressure could decrease the selectivity towards the target compounds, in certain cases the co-extracted compounds could have a positive effect on the bioactivity of the extracts obtained [24].
Contrarily to pressure, an increase in temperature has a negatively effect on solvent density, often resulting in poor extraction yields. However, temperature affects both solvent properties and vapor pressure of solutes, which could improve their solubility and lead to higher yield [33]. Since the effects of temperature on solvent density and vapor pressure of solutes are contradictory, the final effect on total extraction yield cannot be easily predicted and must be observed experimentally. This suggests that a suitable selection of solvent selectivity could be made by adjusting the pressure and temperature. According to the literature data, most SFE experiments are carried out at a pressure of 100–400 bar and a temperature of 40–60 °C range [21], which allows a density of 200 to 900 kg m−3 of supercritical carbon dioxide, making it a suitable solvent for the isolation of lipid compounds. Understanding the thermodynamic and kinetic nature of the SFE process is critical to ensure high selectivity of extraction and reduce the possibility of co-extraction of non-target compounds [22].
For example, EO terpenoid-rich extracts may often be contaminated with other non-volatile lipids that could affect the bioactivity of the extract and degrade its quality. It should be emphasized that the selectivity of supercritical carbon dioxide is limited to different classes of lipid compounds and is very limited for the recovery of polar and moderately polar bioactives. The selectivity and solubility of supercritical carbon dioxide can be modified by adding various co-solvents, which can be either polar (ethanol and methanol) or non-polar (hexane and methylene chloride). Since non-polar organic solvents have low selectivity, their use as co-solvents could have a positive effect on the total extraction yield. However, their use is not often recommended because it affects the green character of the SFE. On the other hand, ethanol is most commonly used as a co-solvent for SFE due to its low miscibility with carbon dioxide, lower toxicity and easy removal from the extract [22]. This approach directly improves the spectrum of target compounds to moderately polar bioactives that can be successfully isolated using SFE. One might assume that SFE has very limited applicability for the recovery of polar plant bioactives such as anthocyanins. However, there are numerous applications of supercritical CO2 with the addition of ethanol as a co-solvent for the preparation of anthocyanin-rich extracts, which have been discussed elsewhere [34].
In addition to pressure and temperature, solvent flow rate and extraction time are also studied as important parameters of SFE. Mass transfer parameters such as axial dispersion, convective mass transfer coefficient and accumulation of extracted compounds in the supercritical phase are highly influenced by the flow rate of solvent [16]. Increasing the flow rate of the solvent directly improves mass transfer by increasing the concentration gradient created by the constant supply of fresh solvent. However, in some cases, this factor could lead to a decrease in the total extraction yield. Excessive solvent flow may reduce the contact time between solvent and matrix and prevent mass transfer caused by internal diffusion. Therefore, solvent flow and its consumption must be evaluated from both technological and economic points of view, as increased solvent flow could cause unnecessarily high solvent consumption.
Time is also an essential attribute of any technological process. Increasing the extraction time would usually lead to an asymptotic increase in the total extraction yield, and time is closely related to solvent consumption. Therefore, optimization of SFE can be performed by selecting the total extraction yield or the yield of target compounds as the responses [24]. Most studies on the optimization of SFE are based on this approach, which is efficient for laboratory scale studies but has severe limitations in industrial processes. The process optimized by this approach could not often be scaled up to industrial scale because it is not economically feasible to perform extractions up to a diffusion-controlled time period due to the excessive time required. Therefore, recent studies have proposed a combined approach in which the extraction kinetics are modeled in a first step and then the initial gradient is optimized either by artificial neural networks (ANN) or by response surface methodology (RSM) [23][35][36][37][38]. In addition to the above process parameters, the SFE process is highly dependent on the properties of the plant matrix, particle size and distribution and post-harvest treatment of the material, which must be considered and evaluated for each case study.

3. Natural Extracts Obtained by SFE and Their Application in Meat Products

The main studies conducted on the application of natural extracts obtained by SFE in meat and meat products are summarized in Table 1 and Table 2. The extracts were isolated from medicinal and aromatic plants (e.g., Echinacea spp., oregano, rosemary, sage, ginger, winter savory, wild thyme) and from fruit berries (e.g., tamarillo, acerola, pomegranate, chokeberry).
Table 1. Antioxidant effects of extracts obtained by SFE in meat and meat products.
Plant Extract Dose Meat/Meat Product Storage Effect Reference
Echinacea
angustifolia
2 mL/kg Frozen chicken meat −20 °C, 10 days Reduced lipid and protein
oxidation
[14]
Tamarillo
epicarp
200 mg/kg Cooked beef 4 °C, 9 days Reduced lipid oxidation [39]
Oregano 1 and 3 g/kg Fish patties 4 °C,
9 days
Reduced cholesterol oxidation [40]
Raspberry pomace 0.5 and 1% Beef burger 4 °C,
26 days
Increased lipid oxidation; no effect in preserving colour [17]
Sage herbal dust 0.05, 0.075 and 0.100 µL/g Fresh pork sausages 3 °C, 8 days Reduced lipid oxidation;
preserved sensory characteristics
[25]
Ginger 0.2% Fish burger 4 °C, 8 days Reduced lipid oxidation; Negative effect on sensory characteristics [41]
Winter savory 0.075 and 0.150 µL/g Fresh pork sausages 3 °C, 8 days Reduced lipid and protein oxidation;
preserved sensory characteristics
[42]
0.2 µL/g Precooked pork chops 4 °C, 6 days Reduced lipid and protein oxidation;
preserved colour, texture and sensory characteristics
[15]
Wild thyme
by-product
0.075 and 0.150 µL/g Ground pork patties 4 °C,
6 days
Reduced lipid and protein oxidation;
preserved colour
[43]
Pomegranate peel 100 ppm Bluefish patties 4 °C,
9 days
Reduced lipid and protein oxidation;
no effect in preserving colour
[44]
Chokeberry
extract
2% Raw pork burgers and cooked ham 4 °C, 7 days (burger) and 13 days (ham) Reduced lipid oxidation (burger);
no negative effect on sensory
characteristics (burger and ham)
[18]
Table 2. Antimicrobial effect of supercritical fluid extracts in meat and meat products.
Plant Extract Dose Meat/Meat Product Storage Effect Ref.
Acerola 0.0063, 0.0125, 0.025 and 0.05% (w/v) Water buffalo steaks 4 °C,
21 days
B. thermosphacta and
Pseudomonas spp.
[41]
Raspberry pomace 0.5 and 1% Beef burger 4 °C,
26 days
No significant effect on the number of B. thermosphacta, Pseudomonas sp., LAB 1 and Enterobacteriaceae [17]
Sage herbal dust 0.05, 0.075 and 0.1 μL/g Fresh pork sausages 3 °C, 8 days Reduction in AMB 2 count [25]
Winter savory 0.075 and 0.150 µL/g Fresh pork sausages 3 °C, 8 days Reduction in AMB count and Enterobacteriacea [42]
Wild thyme
by-product
0.075 and 0.150 µL/g Ground pork patties 4 °C, 6 days Reduction in total plate count,
Enterobacteriaceae and LAB
[43]
Chokeberry pomace extract 2% Pork slurry 4 °C, 16 days bacteria (LAB), and aerobic mesophilic bacteria (AMB)
Reduction in the growth of L. monocytogenes,
B. thermosphacta, P. putida, and AMB
[45]
1 LAB—lactic acid bacteria; 2 AMB—aerobic mesophilic bacteria.
Echinacea spp. is a well-known medicinal plant from North America with strong antioxidant potential [14]. Gallo et al. [14] investigated the use of Echinacea angustifolia extracts as a potential natural antioxidant in chicken burgers. The plant extracts were obtained by conventional (water/ethanol) and emerging (SFE) extraction techniques. Both types of plant extracts were added to chicken meat at a concentration of 2 mL/kg in. The obtained meat product was cooked and stored at −20 °C for 10 days. In terms of strong antioxidant potential, both kinds of extracts were effective in reducing lipid and protein oxidation in chicken burger during storage, but the extract obtained by SFE showed higher efficiency and better selectivity than the conventional one. Additionally, the aroma and texture of the chicken burgers with Echinacea extracts were acceptable to consumers.
Oregano (Origanum vulgare L.) is used in food industry as a flavouring agent and natural additive because of its flavouring properties, which are due to its essential oil. Oregano essential oil is mainly rich in terpenoid compounds (e.g., carvacrol, thymol, p-cymene and γ-terpinene), which have obvious preservative potential in various foods [40].
Rosemary (Rosemarinus officinalis L.) is known as a medicinal and aromatic plant with high content of bioactive compounds such as rosmarinic acid, carnosic acid and carnosol, which have significant antioxidant, antiviral, antibacterial and anti-inflammatory potential [40].
SFEs extracted from oregano and rosemary were added to fish patties (Atlantic salmon) to reduce cholesterol oxidation. The results of this study indicate that the addition of oregano and rosemary extracts (1 and 3 g/kg) effectively reduced cholesterol oxidation products (7α-hydroxycholesterol, 7β-hydroxycholesterol and 7-ketocholesterol) in fish patties during 14 days of cold storage [40].
Sage (Salvia officinalis L.) has long been used as a medicinal and aromatic plant, for its specific flavour and its strong antioxidant and antimicrobial effects. The predominant compounds in sage essential oil are monoterpene ketones, although its strong antioxidant potential is mainly associated with the presence of diterpene polyphenols [24][25]. Šojić et al. [25] evaluated the effect of extracts (Soxhlet and SFE) isolated from sage herbal dust on the quality and safety of fresh pork sausages during 8 days of cold storage. It was found that the extracts obtained by Soxhlet extraction and SFE added at 0.050, 0.075 and 0.100 µL/g decreased lipid oxidation and total plate count in fresh pork sausages. Moreover, the extract obtained with SFE was more effective against microbial growth and provided better sensory quality of fresh pork sausages, indicating the advantages of novel extraction technique. It should also be mentioned that the sage herbal dust extract obtained by SFE effectively reduced the growth of some pathogenic bacteria (Listeria monocytogenes and Escherichia coli) in ground pork [16][26]. Danilović et al. [26] observed that sage extract obtained by SFE at a concentration of 0.4, 0.6 and 1 μL/g can reduce the growth of E. coli in ground meat during 8 days of cold storage for. In another study, Danilović et al. [16] showed that the same extract at a concentration of 0.300 µL/g, can effectively reduce the number of L. monocytogenes and, consequently extend the shelf-life of ground meat by up to 6 days.
Ginger (Zingiber officinale) has a high content of bioactive compounds, including gingerol and its derivatives, with a strong antioxidant potential [41]. The essential oil and extracts obtained from ginger have great potential as an alternative to synthetic antioxidants in foods. Mattje et al. [6] examined the effect of application of ginger extracts (0.2%) on the quality of fish burgers (Oreochromis niloticus). The fish burgers were cold stored for 8 days. The extracts were obtained by conventional hydrodistillation and by the emerging SFE technique. Both extracts reduced lipid oxidation in the beef burgers. It is also worth highlighting that the extract obtained by SFE showed better antioxidant potential than the conventional one, probably due to as the higher content of gingerol compounds. In terms of sensory properties, Mattje et al. (2019) found that the use of ginger extract at 0.2% had a negative impact on the aroma of the burger, especially in the batches prepared with the extract obtained by SFE. The use of lower ginger extract content is important to maintain the overall quality of the meat products.
Winter savory (Satureja montana L.) is known as a medicinal plant with diverse pharmacological activities due to its chemical form. The most abundant compounds in the essential oil of winter savory are phenolic terpenoids, thymol, and carvacrol, which have potent antioxidant and antimicrobial potential [42]. Šojić et al. [42] studied the effect of winter savory essential oil and extract (0.075 and 0.150 µL/g) on lipid oxidation and microbial growth of fresh pork sausages stored at 3 ± 1 °C for 8 days. The essential oil was obtained by hydrodistillation, whereas the extract was obtained by SFE. In the above-mentioned study, Šojić et al. [42] found that the application of both types of extracts delayed lipid oxidation and microbial growth and prolonged the shelf-life of fresh pork sausages. It should be emphasized that the extract obtained by SFE had a stronger antioxidant and antimicrobial potential than the essential oil, most probably due to the higher concentration of co-extracted non-volatile lipids. It was found that terpenoid-rich extracts of winter savory could be used as a natural food additive in cured meat products. In another study, Jokanović et al. [15] investigated the effect of winter savory extracts on the shelf-life of marinated meat. Winter savory extract (0.200 µL/g) obtained by SFE showed a strong protective effect against lipid and protein oxidation and preserved the sensory quality of precooked pork chops during 6 days of cold storage for [15].

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