Int. J. Food Eng. 2021; 17(1): 75–82 Elif Aykın-Dinçer*, Keziban Kübra Güngör, Emine Çağlar and Mustafa Erbaş The use of beetroot extract and extract powder in sausages as natural food colorant https://doi.org/10.1515/ijfe-2019-0052 products of betalamic acid and amino acids or amines, Received February 12, 2019; accepted August 6, 2020; respectively. Approximately 60 betacyanins and 33 published online August 24, 2020 betaxanthins have been found in nature [4, 6]. According to Food Additives Regulation (EC) No 1333/ Abstract: Four colorants (control, carmine, beetroot 2008 of the Council of Europe, beet juice is not considered extract and beetroot extract powder) and two methods as a food additive, whereas betalains extracted from (fermentation and heat treatment) were used in the pro- beetroot have been accepted as an additive and are labeled duction of sausages. The betalain content, total phenolic as E-162. E-162 gives a pink color when applied to food [2]. It substance content and ORAC and TEAC values of concen- is commercially used in the production of wine, jams, trated beetroot extract were 562.08 mg/L, 27.72 mg GAE/ marmalade and strawberry-flavored dairy products such as mL, 33.96 µmol TE/mL and 35.70 mmol TE/L, respectively. yogurt and ice cream. Betalains are not toxic when The moisture content, pH value, lightness (L*), yellowness compared to synthetic colorants and do not cause allergic (b*) and odor values of heat-processed sausages were reactions [7]. While acid-resistant anthocyanins are mostly higher than those of fermented sausages. 2-thiobarbituric used as natural pigments, the color of betalains is relatively acid reactive substances (TBARS) values were lower in stable over a wide pH range (3–7), and they are a suitable sausages with beetroot extract (20.51 μmol·MDA/kg) and colorant for low-acid foods [8]. Their coloring power is very powder (19.03 μmol MDA/kg) than for control and carmine high because betalains have a higher molar extinction treatments. The use of beetroot extract and powder posi- coefficient than synthetic colorants [7]. On the other hand, tively affected the sensory appearance, color, flavor and betalains have been proven to be beneficial to human overall acceptance of sausages. Thus, beetroot extract and health due to their potential to possess anti-inflammatory powder could be used as alternatives to carmine in sausage activity, inhibit lipid oxidation and peroxidation, increase production. resistance to the oxidation of low-density lipoproteins and provide chemo-preventive effects [9]. Keywords: beetroot; betalain; carmine; colorant; sausage. Sodium nitrate and nitrite are used in meat products for various purposes such as providing an antimicrobial effect, typical taste and aroma, and desired curing color, 1 Introduction and preventing autooxidation [10]. However, nitrate and nitrite cause the formation of carcinogenic nitroso com- Beetroot (Beta vulgaris L. ssp. vulgaris) is a two-year her- pounds such as N-nitrosodimethylamine in cured meat baceous plant from the Amaranthaceae family, a good products [11]. It is also stated that nitrate is a potentially antioxidant source for phenolic compounds and betalains toxic food additive. Because of these, the use of synthetic [1, 2]. Betalain is a water-soluble nitrogenous pigment, and additives has been reduced because of their toxicity, which its basic structure consists of betalamic acid [3]. Betalamic in turn has gradually increased the demand for natural acid, which forms the structure of the betalains, evolves colorants such as annatto, curcumin, betalains, cochineal into betacyanins (red) and betaxanthin (yellow) as a result carmine, tomato, pepper and Monascus fungi, which pro- of biosynthesis with different molecules [4, 5]. Betacyanins vide a stable red color [12–14]. are betanidine glycosides; betaxanthins are condensation Carmine colorant is a colorful red extract obtained from female cochineal insects living on Opuntia cacti in tropical regions such as South Africa, Mexico and Arizona *Corresponding author: Elif Aykın-Dinçer, Department of Food [15, 16]. The color of heat-resistant carmine is very similar to Engineering, Faculty of Engineering, Akdeniz University, Antalya, the color of cured meat products [17]. In the European 07058, Turkey, E-mail: [email protected]. https://orcid.org/ Union, the use of carmine is restricted due to its allergen 0000-0003-4427-9819 Keziban Kübra Güngör, Emine Çağlar and Mustafa Erbaş, Department effect, and the amount permitted for use as a food additive of Food Engineering, Faculty of Engineering, Akdeniz University, in meat products is between 100 and 250 ppm [14]. Antalya, 07058, Turkey Consumer concerns about the use of carmine in food Open Access. © 2020 Elif Aykın-Dinçer et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International License. 76 E. Aykın-Dinçer et al.: The use of beetroot extract and extract powder in sausages products have increased over the past years, due to the fact together in a mincing machine. Then, other additives (salt, seasoning that this colorant is obtained by crushing insects [18]. mix and sodium tripolyphosphate) were added, and the mixture was Therefore, carmine is not preferred by most manufacturers, homogenized for about 5 min. The batter was divided into four groups for different colorant sources (control, carmine, beetroot extract and the search for alternative food colorants is ongoing. concentrate and beetroot extract powder). The amount of colorant to In this study, the effect of different colorants (control, be used in the sausage production was determined according to its beetroot extract, beetroot powder and carmine) and treat- coloring power and the literature [12, 13]. Carmine and beetroot ments in sausage production (fermentation and heat- extract powder were diluted in the water used in the formulation and processing) on lipid oxidation, color stability and sensory mixed into the batter. The formulations for all sausage groups are given in Table 1. The mixtures were stuffed into artificial sausage characteristics of sausage was investigated. For this pur- casings with 28 mm diameter, and these casings were knotted at pose, the functional properties of beetroot extract were also 15-cm intervals. Each colorant group included two different treat- analyzed. ments (fermentation and heat-processing). Some of the sausages were ripened in a climate room for 15 days at 10 ± 1 °C and 78% relative humidity (RH) for fermentation treatment. The rest of the sausages were cooked until the core temperature reached 68 °C and 2 Materials and methods were stored in refrigerator conditions until analyzed. A total of 8 kg was used for one replication, and two replicates were carried out for Beetroot used in the production of colorant, and lean-boneless beef, analyses. beef fat and spices for use in the production of sausages were obtained from well-known grocery store in Antalya. 2.4 Total betalain analysis 2.1 Preparation of beetroot extract The betaxanthin and betacyanin content of concentrated beetroot extract was determined spectrophotometrically at 480 and 538 nm, The extraction of beetroot was performed according to the method of respectively, according to the method of Stintzing et al. [7] Total Wiley and Lee, with modifications [19]. Once beetroots were washed, betalains were the sum of both betacyanins and betaxanthins. The their shells were peeled off and their size was reduced. In order to betalain content of the extract was calculated from the following increase mass transfer to solvent, the beetroots were chopped into equation. cubes (0.5 × 0.5 × 0.5 cm) before the extraction process. The beetroot pieces were added to water containing 0.5% citric acid at a ratio of 1:3 mg A × DF × MW × 1000 Betalain content (beetroot:water). Extraction was carried out in a shaking water bath L e × l (WiseBath WSB-30, Seoul, South Korea) at 80 °C for 1 h and a shaking rate of 150 rpm. The beetroot extract obtained by this method was where A is absorbance, DF is the dilution factor, and l is the optical cooled to room temperature and then filtered with Whatman filter path length of the tube (1 cm). The molecular weight (MW) and molar paper (no. 4). extinction coefficient (e) of betacyanin and betaxanthin are given as The beetroot extract was concentrated in a rotary evaporator MW = 550 g/mol; e = 60.000 L/mol · cm and MW = 308 g/mol; (Laborota 4000, Heidolph Instruments GmbH & Co. KG, Schwabach, e = 48.000 L/mol cm, respectively. Germany) in a 200-mm Hg vacuum and a temperature of 50 °C. Some of the concentrated extracts were adjusted to 25°Brix for analysis, and the remainder were dried by the freeze-drying technique (Operon fdu & Table : The formulations of sausages. fdb type, Gyeonggi-do, South Korea). Ingredients Quantity (%) 2.2 Freeze drying of beetroot extract Control Sausage Sausage Sausage sausage with with with The concentrated extract was spread in a thin layer over trays and carmine beetroot beetroot frozen at −80 °C for 24 h. After the freezing process, the trays were put extract extract powder into a freeze-dryer (Operon FDU&FDB type, Gyeonggi-do, South Ko- Beef . rea). Freeze drying was started at −40 °C (shelf temperature) at an Beef fat absolute chamber pressure of 5333 Pa (40 mm Hg), and the process Water temperature reached room temperature at the end of the freeze-drying Salt . process in 48 h. The powder obtained was stored in a hermetically Seasoning mix sealed glass jar until