A Lyophilized Water Extract of Melissa officinalis L. as an Effective Natural Antioxidant during the Storage of Dry Fermented Sausages High in α-Linolenic Acid and DHA

Abstract: A pre-emulsified mixture of linseed and algae oils (15/10) and stabilized with 686 ppm of a lyophilized water extract of Melissa officinalis， was successfully applied in dry fermented sausages to increase the ω-3 PUFA content. The objective of this work was to evaluate the stability of this modified formulation during the storage and to compare it to that of a traditional formulation. Traditional and modified products were stored during 90 days at 4 oC in aerobic conditions. Fatty acid profiles， TBARS and volatile compounds derived from oxidation were analyzed at 0， 30 and 90 days of storage. The fatty acid profiles did not significantly change along the storage period. The stabilizing effect of the natural antioxidants of M. officinalis could contribute to detect no losses of ω-3 PUFA in Modified (30 days: 2.13 g/100 g of product， 90 days: 2.33 g/100 g of product)， whereas in Control products a slightly significant reduction was detected (30 days: 0.34 g/100 g of product， 90 days: 0.29 g/100 g of product). After 90 days， the increases of TBARS and hexanal content were much higher in Control than in Modified (Control: 1.41 mg MDA/kg 17，915 ng dodecane/kg of dry matter; Modified: 0.48 mg MDA/kg 2，496 ng dodecane/kg of dry matter). In conclusion， the lyophilized water extract of M. officinalis protected high ω-3 PUFA of dry fermented sausages from oxidation along the storage time， guaranteeing the nutritional improvements achieved with the modified formulation.

Key words: Natural antioxidants， oxidation， sausages， omega-3， linseed oil， algae oil.

1. Introduction

Recently many authors have developed strategies to obtain new formulations of healthier meat products trying to improve their lipid fraction [1-10]. The lipid profile modification implies an increase of the poly-unsaturated (PUFA) fraction， specially of the ω-3 PUFA content， leading to a lower ω-6/ω-3 ratio， in order to achieve the current recommendations [11]， and consequently， obtaining some beneficial effects as protective role against cardiovascular risk factors， inflammatory effects， sudden death， obesity， insulin-resistance and mental illness [12-18]. However， a described drawback for this strategy points out that the inclusion of a higher PUFA content involves a higher lipid oxidation susceptibility， developing off-flavour volatile compounds deteriorating the quality， color and even the nutritional value of the products [19， 20]. Consequently， the acceptability of the meat and meat products could be compromised [21].

With the aim of preventing the oxidation of meat products， compounds with antioxidant activity should be included in the formulation， being an effective mean for delaying lipid oxidation [22]. Ansorena [2]

used BHA + BHT as inhibitors of the lipid oxidation in dry fermented sausages enriched in ω-3 PUFA. In the last years， the use of natural sources of antioxidants against the development of rancidity in food and also， in the protection on the human health， are being studied as an interesting approach in food research [23-25]. Food industry has shown great interest on vegetable extracts from medicinal plants， spices， fruits and edible plants due to their technological and healthy roles. These products， in some cases， had shown a greater antioxidant efficacy than synthetic antioxidants， which is attributed to their content in antioxidant compounds such as polyphenols， lycopene and α-Tocopherol [8， 26-30]. In particular， Melissa officinalis (Lemon balm) is an edible plant that belongs to the Lamiaceae family， and it is considered an interesting source of antioxidant components [31]. In fact， the antioxidant properties of methanolic and ethanolic extracts of Melissa officinalis have been already pointed out [32].

The objective of this work was to evaluate the stability during storage of dry fermented sausages enriched in ω-3 PUFA by means of a pre-emulsified mixture of linseed and algae oils and stabilized by a lyophilized water extract of Melissa officinalis L.

2. Material and Methods

2.1 Experimental Design

Water extracts of Melissa officinalis were prepared as follows: 50 g of dried leaves were weighted and added to 500 mL of distilled water， preheated at 100 °C. The mixture was subjected to reflux during 30 minutes at the temperature above. Extraction process was repeated with another 500 mL of distilled water， and both extracts were joined and completed with distilled water to a final volume of 1 L. Extracts were filtered using filter to remove insoluble particles. Water extraction was performed in triplicate.

The extracts were lyophilized with a freeze-dryer-cryodo (Telstar， Barcelona， Spain)， previous freezing at -80 °C in a MDF-V5386S Ultra-Low-Temperature Freezer (Sanyo Electric Co.， Ltd.， Japan). 21.8 g of lyophilized material were obtained from 100 g of Melissa dried leaves. The lyophilized material was subsequently used for the evaluation of the antioxidant capacity and for application as ingredient in the dry fermented sausage formulation.

686 ppm of this lyophilized water extract of Melissa officinalis L. were used for controlling the lipid oxidation of a pre-emulsified mixture of linseed and algae oils (15/10). According to the procedure described by García-í?iguez de Ciriano [9] this emulsion was used as a partial substitute of 25% of the pork back fat in a particular type of dry fermented sausages (Chorizo) (Modified). A batch prepared according to a traditional formulation was used as Control (Control). Processing conditions are those described by Muguerza [33].

Control and Modified products were stored during 90 days at 4 oC in aerobic conditions. Total fatty acid profiles， TBARS and volatile compounds derived from oxidation were analyzed at 0 (Final product)， 30 and 90 days of storage.

2.2 Determination of the Total Fatty Acid Profile

The method of Folch [34] was used for the extraction of lipids. Fatty acids were determined in the lipid extract by gas chromatography， previous methylation with boron trifluoride/methanol [35]. The quantification of fatty acids was based on the internal standard method， using heptadecanoic acid methyl ester (Sigma， St. Louis， MO， USA). 2.3 Determination of TBARS Value

TBARS value was determined according to Tarladgis [36] with modifications by Tarladgis [37]. Results are shown in mg malonaldehyde (MDA)/kg sample.

2.4 Volatile Compounds from Lipid Oxidation

A Likens-Nickerson extraction followed by a

3. Results and Discussion

The enrichment of dry fermented sausages with linseed and algae oils increased the content onα-linolenic acid (ALA) and docosahexaenoic acid(DHA) in the products [9]. The changes in the lipid profile of these products were analyzed during their storage period (Table 1). After 30 days of storage， no significant differences were found for ALA compared to Final product in Modified batches， whereas a slight reduction was noticed after 90 days (1.6%). Valencia[39] did not find significant differences of ALA in dry fermented sausages enriched in linseed oil and stabilized with synthetic antioxidants (BHA + BHT) when they were stored from 2 to 5 months in aerobic condition. Besides， in the present work， DHA remained constant in the Modified batches (1.99 ±0.01 g/100 g of FAME) along the storage. Also， the total ω-6 content was not modified during the storage. In consequence， the interesting ω-6/ω-3 ratio observed in the final product (1.96) was kept below 2 until the end of storage， and was significantly lower than that observed for Control products (mean = 16). The different

natural susceptibility of SFA (saturated fatty acids) and PUFA to oxidation might induce changes of the ratio PUFA/SFA along storage. However， this ratio remained constant as well， pointing to the high stability of the new formulation.

Results expressed on basis on 100 g product confirmed again the stability of the PUFA fraction in Modified products (Fig. 1). Even slight increases ofω-3， ALA and DHA were detected after 90 days compared to 30 days of storage， which could be attributed both to the maintenance of the fatty acids un-oxidized and to a slight loss of moisture during the aerobic conditions of storage.

In summary， the nutritional improvement of the new formulation reported by García-í?iguez de Ciriano [9] could be guaranteed along the self-life of the products， due to the adequate preservation of these compounds by the protective role of M. officinalis.

Although the modified formulation resulted in a healthier product along the time， it is greatly important to evaluate the stability problems related to the potential lipid oxidation that might overcome along the storage time. Results obtained from TBARS analysis did not show relevant oxidation problems in the tested experimental conditions (Table 2). During the first 30 days， Modified batches showed higher values than Control batches. After 90 days of storage， the TBARS was only 0.48 ppm in Modified batches

4. Conclusion

In conclusion， the lyophilized water extract of M. officinalis protected ω-3 PUFA and long chain ω-3 PUFA of dry fermented sausages from oxidation along the storage time， guaranteeing the nutritional improvements achieved with the new formulation.

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