The Use of Tomato Powder Fermented with Pediococcus pentosaceus and Lactobacillus sakei for the Ready-to-Cook Minced Meat Quality Improvement

There is an increasing demand by consumers for high quality and nutritious meat products. The diverse nutrient composition of meat makes it an ideal environment for the growth and propagation of meat spoilage microorganisms and common foodborne pathogens. Therefore, it is essential that adequate preservation technologies are applied to maintain meat safety and quality (1). The processes used in meat preservation are principally concerned with inhibiting microbial spoilage, although other methods of preservation are sought to minimise deterioration such as colour and oxidative changes (2). The most investigated new preservation technologies for fresh meat ISSN 1330-9862 original scientifi c paper


Introduction
There is an increasing demand by consumers for high quality and nutritious meat products.The diverse nutrient composition of meat makes it an ideal environment for the growth and propagation of meat spoilage microorganisms and common foodborne pathogens.Therefore, it are nonthermal inactivation technologies such as high hydrostatic pressure (HHP), new packaging systems such as modifi ed atmosphere packaging (MAP) and active packaging (AP), natural antimicrobial compounds and biopreservation.Storage life is extended by using natural or controlled microorganisms, such as lactic acid bacteria (LAB) and their metabolites (e.g. as lactic acid and bacteriocins), which have been studied extensively (3,4).In our previous study, bacteriocin-like inhibitory substances (BLIS) produced by Lactobacillus sakei KTU05-6 and Pediococcus pentosaceus KTU05-9 were designated as sakacin 05-6 and pediocin 05-9, respectively (5).We proposed that these bacteria can be used widely in the food industry as biopreservatives due to their broad inhibition spectrum.
Consumer interest in various fermented foods has increased in recent years due to the positive perception of their impacts on health.Thus, there is a growing need for novel methods of preservation and natural food preservatives.Biopreservation by microorganisms or their metabolites can extend shelf life and enhance safety of foods (6,7).
Lactic acid fermentation represents the easiest and most suitable way to increase daily consumption of 'nearly' fresh fruits and vegetables.The safety of these products can be aided by the development of starter cultures.There has been progress in the development of antimicrobial LAB strains, which can be engineered to signifi cantly improve the quality, safety and acceptability of plant--based foods.Food matrices, such as vegetables, off er a promising performance as sources and carriers of probiotic strains (8).Vegetables are fundamental sources of water--soluble vitamins (vitamin C and group B vitamins), provitamin A, phytosterols, dietary fi bres, minerals and phytochemicals (9) for the human diet.LAB present a small part (2-4 log CFU/g) of the autochthonous microbiota of raw vegetables (10).When favourable conditions of anaerobiosis, water activity, salt concentration and temperature occur, raw vegetables and fruits may be subjected to spontaneous lactic acid fermentation.In some cases, alcoholic fermentation takes place concomitantly (11).
Tomatoes are rich sources of a variety of nutritional compounds and some key antioxidant components such as the carotenoid lycopene, vitamin C, and a range of polyphenols.Consumers have already become aware of the potential importance of the protective properties of these antioxidants.The biochemical composition of tomatoes has been reported extensively in the literature (12).Lycopene, a natural carotenoid, has also been reported to possess various health benefi ts, such as cardiovascular and cancer-preventive properties (13).
The aim of this study is to evaluate the infl uence of lactic acid fermentation of tomato pulp powder with bacteriocin-like inhibitory substances (BLIS) produced by Pediococcus pentosaceus KTU05-9 and Lactobacillus sakei KTU05-6 on the quality of fermented tomato products and their eff ect on the colour, carotenoid content and sensory characteristics of ready-to-cook minced pork meat.

Tomato powder and microorganisms
Tomato pulp powder was obtained from Obipektin AG (Bischofszell, Switzerland).The lactic acid bacteria (LAB), Pediococcus pentosaceus KTU05-9 and Lactobacillus sakei KTU05-6, used for the fermentation of tomato powder were obtained from the collection of the Kaunas University of Technology (Kaunas, Lithuania).The LAB were previously isolated from spontaneous rye sourdough (5) and selected due to their inhibition of undesirable bacteria (14,15).The LAB were stored at -80 °C and cultured at 35 °C (strain KTU05-9) or 30 °C (strain KTU05-6) for 48 h in MRS broth (CM0359; Oxoid Ltd, Hampshire, UK) prior to use.

Solid-state fermentation of tomato powder
Samples were prepared by mixing the tomato powder (300 g) with appropriate amounts of water and LAB culture suspension (10 g), containing an average of 10.1 log of colony-forming units (CFU) per g (moisture content of 45 %, by mass) and incubated at 35 °C (P.pentosaceus) or 30 °C (L.sakei) for 48 h.Spontaneous fermentation of tomato powder was carried out at 30 °C for 48 h without LAB starters.

Analysis of fermented tomato products
The pH value was measured and recorded using a pH electrode (PP-15; Sartorius, Goett ingen, Germany).For microbiological analysis, 10 g of sample were homo genised with 90 mL of sterile saline (0.9 %).Serial dilutions (10 -4 -10 -8 ) of this suspension were made, spread onto MRS agar and incubated under anaerobic conditions at 30 °C (for L. sakei and spontaneous fermentation) and 35 °C (for P. pentosaceus) for 72 h.The LAB cell number was calculated and expressed as log CFU/g.

Preparation of ready-to-cook minced meat
Fresh pork loin was obtained from a local market and used for the preparation of the ready-to-cook minced meat.Meat was minced with a meat grinder (hole diameter of 5.0 mm; Meissner AG, Biedenkopf-Wallau, Germany) and mixed with the tomato product (fermented with LAB or spontaneously) at selected mass fractions (10 and 30 %).Control samples were prepared using minced meat with and without the addition of nonfermented tomato powder.Minced meat samples were covered with plastic fi lm and stored at 4 °C for 24 h until analysis.Nontreated and thermally treated (cooked in water at 100 °C for 10 min) meat samples were subjected to measurements of colour and carotenoid content, and sensory analysis.

Extraction and analysis of carotenoids in ready-to-cook minced meat products
Prior to analysis, the samples of ready-to-cook minced meat were pureed using a blender (LBC15 Laboratory Blender, Lab Depot Inc., Dawsonville, GA, USA), followed by a few minutes of homogenisation with a Polytron ® PT 1200E homogeniser (Kinematica AG, Luzern, Switzerland) at room temperature.A mass of 2 g of homogenate was extracted with 100 mL of hexane with the addition of 0.1 % butylated hydroxytoluene (BHT) at room temperature.Extracts were stored at −20 °C and equilibrated to room temperature before analysis by HPLC.
The working solutions (1 mg/mL) of β-carotene and lycopene standards (Sigma-Aldrich, Taufk irchen, Germany) were prepared daily.The mobile phase consisted of solution A (methanol 100 %) and solution B (methyl tert--butyl ether 67 % and ethyl acetate 33 %).The solvent composition for the isocratic elution was as follows: 40 % solvent A and 60 % solvent B. The elution fl ow rate was 1.5 mL/min and the injection volume was 10 mL.Detection was registered at the wavelength of 473 nm.Carotenoids were identifi ed based on the elution times in comparison with standard reference samples and concurrence with wavelengths for standard compounds.

Sensory evaluation of ready-to-cook minced meat
Sensory analysis of the ready-to-cook minced meat was performed according to the ISO 8586-1:1993 ( 17) method by fi ft een judges using a 10-score hedonic scale (from 10=extremely like, to 0=extremely dislike).Coded samples were served and water was provided for rinsing between the sensory evaluations of the samples.

Colour measurements
The colour characteristics of samples were evaluated at three diff erent positions of the surface using CIELab system (CromaMeter CR-400, Konica Minolta, Kyoto, Japan).L* is a measure of lightness, from completely opaque (0) to completely white (100), a* is a measure of redness (or -a* of greenness), and b* of yellowness (or -b* of blueness).The hue angle (h°) value of the samples indicates the colour tone and was calculated as follows (18):

Statistical analysis
All analytical determinations were performed at least in triplicate.The obtained data were analysed using statistical package SPSS for Windows XP v. 15.0 (SPSS Inc., Chicago, IL, USA).Signifi cance of diff erences among the treated samples was evaluated by using Duncan's multiple range tests at a 95 % level of confi dence.

Characterisation of fermented tomato pulp powder
Results of pH changes and bacterial growth during tomato pulp powder fermentation are presented in Fig. 1.The study showed that tomato powder fermentation time had a signifi cant (p≤0.05)infl uence on bacterial growth rate and changes in pH.Medium with tomato powder in-duced the growth of the LAB (Fig. 1).The cell growth of L. sakei strain (7.53 log CFU/g) was more intense than of P. pentosaceus (6.35 log CFU/g) during 24 h of fermentation.Spontaneous fermentation of tomato powder reduced the cell growth by 38 %, compared to the fermentation with pure LAB.The tomato powder medium seems to be more suitable for L. sakei; the viable cell count of the bacteria reached 8.15 log CFU/g aft er 48 h of fermentation.A signifi cant (p≤0.05)reduction in viable cells was found in the tomato powder samples fermented with P. pentosaceus (26.3 %) aft er 48 h of fermentation.In contrast, the spontaneous fermentation of tomato powder showed an increase of bacterial cell counts of 36.2 % during longer fermentation (Fig. 1).
In our previous study (19), L. sakei and P. pentosaceus strains during tomato pulp fermentation reduced the pH up to 3.5-3.7,and the viable cell counts of the tested strains reached 6.61 log CFU/g aft er 48 h of tomato pulp fermentation.Spontaneously fermented tomato pulp had pH values higher by 7.2 % than the lactofermented one.
Both L. sakei and P. pentosaceus were found to be capable of rapid utilisation of tomato pulp powder for cell synthesis and organic acid production.The most intensive formation of organic acids was noticed in the tomato powder samples fermented with P. pentosaceus (pH=4.1).The pH of the medium was lower (4.1-4.15)than the critical 4.5 value (Fig. 2); in either case, spontaneously fermented tomato products had pH values slightly higher than the lactofermented products (Fig. 2).
Many authors consider the number of probiotic cells in a food item required to benefi t health to be between 10 6  and 10 8 CFU/g (20,21).In selecting probiotic bacteria for industrial manufacturing processes, safety, functional and technological characteristics should be considered.Functional properties include the viability of cells, stability of the cells within a food matrix, strain characteristics and fermentation technology (21,22).Our results support the hypothesis that the tomato medium contains the essential nutrients to support the growth of lactobacilli and can be directly used as a fermentation substrate for probiotic lactic acid bacteria.Therefore, the LAB counts were compared to the value of 6.35-8.15log CFU/mL, which is considered to be the minimum probiotic count in functional products.

Colour characteristics and carotenoid content of fermented tomato products
The results of carotenoid analysis of tomato powder samples and their colour values are presented in Fig. 3.According to the results, fermentation signifi cantly (p≤ 0.05) increased the carotenoid content and infl uenced the colour characteristics of the tomato powder products (Fig. 3).Total carotenoid mass fractions up to 95.56 and 98.96 mg per 100 g were measured in tomato samples fermented with P. pentosaceus and L. sakei, respectively.In addition, spontaneous fermentation increased the content of total carotenoids, but not as eff ectively; the content of carotenoids in the spontaneously fermented samples was 43.7 % lower compared with the lactofermented samples (Fig. 3).
This study confi rmed previous results (19) about the increase of lycopene and total carotenoid contents during lactofermentation of tomatoes.The use of P. pentosaceus and L. sakei strains for fermentation of tomato powder increased the mass fractions of total carotenoids on average by 41.1 and 33.6 %, respectively, compared to the untreated sample.Lycopene content was found to be lower by 11.0 % in spontaneously fermented tomatoes, compared to the control.
Fermentaton with LAB leads to a disruption of lycopene-protein complexes, breaking down the tomato cell matrix and yielding higher levels of total carotenoids due to increased concentration of cis-lycopene (19).Lycopene content may be aff ected directly by the pH, as at low pH tomatoes accumulate more lycopene (23).
Colour is an important quality att ribute of the food and bioprocess industries that infl uences consumer choice and preference.The measurement of food product colour has been used as an indirect evaluation of other quality att ributes, such as fl avour and contents of pigments, due to the simple and fast measurements and the good correlation with other physicochemical properties (24).
A signifi cant (p≤0.05)decrease in yellowness (b*) and redness (Fig. 3) with increased contents of β-carotene and lycopene in the tomato powder aft er 48 h of fermentation was detected.In all tomato powder samples, the colour tone (h°) showed mean values ranging from 29.3 to 34.1, indicating an orange colour.Thus, the samples with a lighter colour (higher L* values) tended to be more yellow-orange than the samples with a darker colour (lower L* values).However, the lactofermented samples showed a colour tone (h°) 7.3 and 12.9 % lower compared with the spontaneously fermented and tomato powder samples, respectively (Fig. 3).A strong negative linear correlation was observed between β-carotene and lycopene contents and lightness (L*) (R 2 =0.835 and R 2 =0.846, respectively), and also between the colour tone (h°) and lycopene content (R 2 =0.904, p≤0.001).A strong positive linear relationship was observed between L* and h° (R 2 =0.830, p≤0.001).The colour change of a tomato product is an indication of the fermentation procedure and is related to its pigment concentration.
Our previous study showed a weak correlation between the yellow colour (b*) values and β-carotene content and a strong correlation between b* and lycopene content in tomato samples (19).As was reported by Fernán dez-Ruiz et al. (25), the measurement of the yellow coordinate (b*) could be used for predicting lycopene content in tomato products.

The infl uence of fermented tomato powder on sensory characteristics of ready-to-cook minced meat
Signifi cant diff erences were found in the intensity of acidity, colour and acceptability of ready-to-cook minced meat with diff erent amounts of tomato powder fermented with pure LAB or spontaneously fermented (Fig. 4).The intensity scores for acid taste of ready-to-cook minced The ready-to-cook minced meat samples with 30 % tomato powder had the most intensive colour (5.5-6.5) and acidity (5.2-6.7) (Fig. 4).
The ready-to-cook minced meat with 10 % tomato powder fermented with L. sakei and P. pentosaceus had the highest acceptability scores (9.4 and 8.8, respectively) (Fig. 4).Acceptability scores of the control sample (without additives) were found to be lower by 34.2 and 5.6 %, respectively, than those with fermented tomato powder.The ready-to-cook minced meat samples with 30 % fermented tomato powder had a lower acceptability (by 30.3 %) than those with 10 % fermented tomato powder, due to an undesirable acidic taste.However, the ready-to-cook minced meat samples with 30 % nonfermented tomato powder were rated as highly acceptable (an average of 7.7) (Fig. 4).

The infl uence of tomato additives on colour and carotenoid content of ready-to-cook minced meat
Considering the colour properties of tomato products and the benefi cial eff ects on human health (26)(27)(28), their addition to meat products could reduce the necessity of adding synthetic colourants and yield products with a bett er nutritional profi le due to the higher content of plant-derived bioactive compounds (lycopene and phenolic compounds) (29).The Joint FAO/WHO Expert Committ ee on Food Additives (JECFA) agreed that lycopene (both natural and synthetic) is acceptable as a food colourant and established a group acceptable daily intake (ADI) of 0-0.5 mg of both preparations per kg of body mass per day (30).According to Descalzo et al. (31), mass fractions of β-carotene in fresh meat could range between 5 and 50 μg per 100 g depending on food intake.Few studies have reported the use of tomato products or lycopene in meat products.Candogan (32) and Deda et al. (33) reported on the impact of the addition of tomato paste or tomato peel on the quality and sensorial properties of beef patt ies and frankfurters.Calvo et al. (34) used tomato peel for the enrichment of dry fermented sausages with lycopene.However, data on the eff ect of fermented tomato products on ready-to-cook minced meat quality parameters have not been found.
Our study showed that the addition of fermented tomato powder significantly aff ected (p≤0.05) the colour characteristics of the ready-to-cook minced meat (Table 1).The controls (without tomato powder) had signifi cantly higher (p≤0.05)values of lightness (L*) and the lowest (p≤0.05)values of redness (a*) and yellowness (b*).The addition of 10 and 30 % of fermented tomato powder in-Table 1. Eff ect of the addition of tomato powder on the colour characteristics of nontreated and thermally treated (10 min  creased the redness by 70.2 and 77.8 % and yellowness by 1.8 and 1.9-fold, respectively, of the ready-to-cook minced meat, which were more att ractive to consumers than the control samples.Spontaneous fermentation improved the yellowness of ready-to-cook minced meat at a lower level (28.5 %) (Table 1).These tendencies were detected for both nontreated and thermally treated ready-to-cook minced meat samples.
Large variation in the colour parameters of meat products due to the diff erent measurement conditions, the composition of the meat products and the size of the meat particles has been reported (35,36).According to our results, the meat colour parameters were signifi cantly affected by the amount of the added tomato powder and the treatment of ready-to-cook minced meat.In general, the addition of lycopene to meat products, through the addition of tomato pasta or peel, led to changes in the typical red colour of the meat products by increasing the yellow colour and providing an orange tone (37,38).
The results of the carotenoid analysis of the ready-to--cook minced meat are presented in Table 2.The lycopene and β-carotene contents of the ready-to-cook minced meat increased from 0.48 to 10.32 mg per 100 g and from 0.19 to 1.95 mg per 100 g, respectively, depending on the percentage of tomato powder in the formulation (10 or 30 %) (Table 2).The addition of lactofermented tomato powder provided β-carotene and lycopene contents of on average 1.12 and 3.63 mg per 100 g (10 % of tomato powder), respectively, and 1.86 and 9.99 mg per 100 g (30 % of tomato powder), respectively.The ready-to-cook minced meat with spontaneously fermented tomato powder had β-caro tene and lycopene contents that were 43 and 75 % lower, respectively, compared to the ready-to-cook minced meat with lactofermented tomato powder.The lowest mass fractions of β-carotene and lycopene measured in the ready-to-cook minced meat samples were 0.9 and 0.48 mg per 100 g with 10 % and 0.25 and 1.46 mg per 100 g with 30 % of nonfermented tomato powder, respectively.
The content of lycopene in ready-to-cook minced meat with lactofermented tomato powder was higher than that reported by Doménech-Asensi et al. (37) in mortadella aft er the addition of tomato paste.The lycopene in our ready-to-cook minced meat samples is expected to be more available, since it originates from tomato powder in which both heat treatment and fermentaton with LAB lead to higher levels of total carotenoids due to an increased concentration of cis-lycopene (19).
Cooking the ready-to-cook minced meat in water lowered the β-carotene and lycopene contents as well as the total carotenoid content (Fig. 5).Mean carotenoid losses of up to 22-28 % and up to 30-41 % were detected in ready-to-cook minced meat samples with 10 and 30 % fermented tomato powder, respectively.As a consequence, the highest loss of total carotenoids was found in samples with 10 and 30 % of nonfermented tomato powder (38 and 49 %, respectively) (Table 2).
Heat and diff erent food matrices are factors that have an eff ect on lycopene isomerisation and autoxidation.Lycopene may isomerise to mono-or poly-cis forms in the presence of heat or fat, or during dehydration.Heat treatment of oil-rich meat products supplemented with tomatoes could increase lycopene losses due to the heat treatment in the fat-water emulsion (38,39).
The addition of fermented tomato powder did not aff ect negatively technological processing, so the minced meat with 10 % fermented tomato powder was a suitable product with the added benefi t of the presence of lycopene and a positive eff ect on overall consumer acceptance.

Conclusions
The direct use of Pediococcus pentosaceus KTU05-9 and Lactobacillus sakei KTU05-6 in fermentation of tomato powder increases its carotenoid content, and makes it a benefi cial additive that improves the colour and nutritional value of minced meat.L. sakei showed a 43.8 % more intensive cell growth during the solid-state fermentation of tomato powder compared to P. pentosaceus.Moreover, according to our previous research (19), the P. pentosaceus strain produces mainly d-lactic acid (l/d ratio of 0.64), while L. sakei produces mainly l-lactic acid (l/d ratio of 1.61) during the fermentation of tomatoes.Because of the potential toxicity of d-lactic acid in food, the tomato products prepared using selected LAB cultures are safer than those that are spontaneously fermented.
The addition of tomato products to minced pork meat allowed to change the typical colour of the meat products by increasing the yellow colour and providing an orange tone.The Lactobacillus sakei KTU05-6 could be recommended for the fermentation of tomato powder to improve the nutritional value and colour of the minced meat without eff ects on overall consumer acceptance.This is the fi rst report of tomato powder fermented with selected lactobacilli used as a source of lycopene and β--carotene for meat products, and more research is needed to explain the mechanism of carotenoid increase during fermentation.

Table 2 .
Eff ect of the addition of tomato powder on carotenoid content in nontreated and thermally treated (10 min in water at 100 °C) ready-to-cook minced meat bMean values in a column with diff erent lett ers are signifi cantly diff erent (p≤0.05).TP=tomato powder, SPF=spontaneously fermented tomato powder, Pp=tomato powder fermented with P. pentosaceus, Ls=tomato powder fermented with L. sakei