**6. Lactic acid fermentation of tomatoes: effects on** *cis***/***trans* **lycopene isomers, β-carotene concentration and the formation of L(+) and D(-)-lactic acid**

The production of L-lactic acid and D-lactic acid isomers during the fermentation of different tomato varieties (var. Ronaldo and var. Cunero) by the bacteriocin-producing LAB *Lactobacil‐ lus* and *Pediococcus* spp. have been investigated. The influence of lacto fermentation on the lycopene and β-carotene contents and their relation to the colour characteristics of fermented tomato products were also investigated [75]. Tomato var. Cunero and Ronaldo, the LAB strains were used in this investigation. Tomato var. Cunero and Ronaldo were obtained from the Lithuanian Institute of Horticulture (Babtai, Lithuania) harvested in 2011. Pure cultures of *Lactobaccilus sakei KTU05-6, Pediococcus acidilactici KTU05-7* and *Pediococcus pentosaceus KTU05-8*, characterized as a bacteriocin producing strains [76] are from collection of Kaunas University of Technology (Kaunas, Lithuania) [75].

The LAB strains were propagated in nutrition media (moisture content 72 %), prepared by mixing extruded rice flour (100 g) and tap water. After addition of pure LAB cell suspension (5 g, 10.2 log10 colony-forming units (CFU) g-1) the mixture was incubated at optimal temper‐ atures (30 °C for *L. sakei*, 32 °C for *P. acidilactici* and 35 °C for *P. pentosaceus*) for 24 h. For comparison purpose control product was prepared using spontaneous fermentation of rice flour without bacterial inoculum at 30 °C for 48 h. Enumeration of LAB was carried out by plating the diluted samples onto MRS agar at 30 °C for 48 hours. Products obtained after propagation of individual LAB in rice media were used for fermentation of tomato pulp [75].

A rapid and specific Megazyme assay kit for simultaneous determination of L- and D-lactic acid (Megazyme Int., Bray, Ireland) in foods was used as reported by De Lima et al. [77] in this investigation. Extraction of carotenoids and carotenoid analysis by Reverse Phase Liquid Chromatography (RP-HPLC) were used [75] and the colour characteristics of fermented and untreated tomato pulp were evaluated of the surface using CIEL\*a\*b\* [78].

## **6.1. The effect of selected fermentation media on LAB viability**

substances produced by the tested LAB possess a proteinaceous nature. They might be bacteriocins because protease sensitivity is a key criterion in the classification of antimicrobial substances as BLIS [66]. In our previous studies, BLIS produced by *Lactobacillus sakei* KTU05-6 and *P. pentosaceus* KTU05-9 were designated as sakacin 05-6 and pediocin 05-9 [67]. We proposed that due to their broad inhibition spectrum, the presence of BLIS and organic acids in tested LAB is an indication that these bacteria can be used widely in the food industry as

Consumer interest for diverse fermented foods has increased in recent years because of the positive perception of their beneficial impact on health. Hence, there is an evident need to find novel methods and new food preservation agents from natural origins. Biopreservation refers to extending the shelf-life and enhancing the safety of foods using microorganisms or their

The food matrices in vegetables offer promising potential as sources and carriers of probiotic strains [70]. Vegetables are fundamental sources of water-soluble vitamins (vitamin C and group B vitamins), provitamin A, phytosterols, dietary fibres, minerals and phytochemicals [71] in the human diet. LAB are a small part (2.0–4.0 log10 CFU g−1) of the autochthonous microbiota of raw vegetables [37]. Under favourable conditions of anaerobiosis, water activity, salt concentration and temperature, raw fruits and vegetables may be subject to spontaneous lactic acid fermentation. In some cases, alcoholic fermentation takes place concomitantly [72]. Tomatoes are a rich source of a variety of nutritional compounds, especially key antioxidant components, such as the carotenoid lycopene, vitamin C, and a range of polyphenols. The possible protective characteristics of these antioxidants are of great interest, and consumers have already become aware of their potential importance. A survey of the literature revealed that a great deal of research has been conducted on the biochemical composition of tomatoes and their products [73]. Lycopene, a natural carotenoid found in tomatoes, has been reported to possess various health benefits, such as preventive properties against cardiovascular disease

**6. Lactic acid fermentation of tomatoes: effects on** *cis***/***trans* **lycopene**

University of Technology (Kaunas, Lithuania) [75].

**isomers, β-carotene concentration and the formation of L(+) and D(-)-lactic**

The production of L-lactic acid and D-lactic acid isomers during the fermentation of different tomato varieties (var. Ronaldo and var. Cunero) by the bacteriocin-producing LAB *Lactobacil‐ lus* and *Pediococcus* spp. have been investigated. The influence of lacto fermentation on the lycopene and β-carotene contents and their relation to the colour characteristics of fermented tomato products were also investigated [75]. Tomato var. Cunero and Ronaldo, the LAB strains were used in this investigation. Tomato var. Cunero and Ronaldo were obtained from the Lithuanian Institute of Horticulture (Babtai, Lithuania) harvested in 2011. Pure cultures of *Lactobaccilus sakei KTU05-6, Pediococcus acidilactici KTU05-7* and *Pediococcus pentosaceus KTU05-8*, characterized as a bacteriocin producing strains [76] are from collection of Kaunas

metabolites [68]. In this aspect, LAB are very good candidates [69].

bio-preservatives.

140 Biotechnology

and cancer [74].

**acid**

As reported in the literature, the behaviour of different LAB depends on substrate composition, where bacteria in different substrates are able to produce different metabolites or increased biomass [79]. For maximum health benefits, it is important to have a significant number of viable LAB present in the probiotic product [80].

Extruded rice flour, a current product of the cereal processing industry, was found to show good fermentability. Counts of viable bacteria cells were measured between 6.62 and 8.50 log10 CFU g-1 after 48 h of analysed LAB cultivation in selected media (Table 1) [75]. The lowest biomass of bacteria was found in the spontaneously fermented rice media (5.57 log10 CFU g-1). According to the obtained results, rice flour is a suitable medium for LAB cultivation to produce a functional food while most likely maintaining the other functional properties of rice. These results are in agreement with Trachoo et al. [81], who showed a biomass increase of lactobacilli over 2.5 log10 CFU mL-1 during 24 h in a germinated rice broth [75].


The numbers are means followed by standard deviations (n = 3).

Means within a column with different superscript letters are significantly different (*p* < 0.05).

Samples: tomato products fermented with: P.p. – *P. pentosaceus,* P.a. – *P*. *acidilactici,* L. s. – *L. sakei;* SF – spontaneous fermented.

**Table 1.** The influence of fermentation media on LAB cell counts (log10 CFU g-1), pH and TTA values.

*L. sakei*, *P. acidilactici* and *P. pentosaceus* were found to be capable of sufficient rapid utilisation of tomato pulp for cell synthesis and organic acid production. They reduced the pH to 3.5–3.7 and increased the TTA to as high as 6.4. The viable cell counts reached 6.61 log10 CFU g-1 after 48 h of fermentation. In either case, tomato products treated with spontaneous fermentation had pH values that were higher by 7.2% and TTA values that were lower by 17.3 % than products treated with lactofermentation (Table 1) [75].

Acid production depends on the concentration of viable bacteria able to utilise the available carbohydrate sources in the substrate [82]. The viable LAB cells in the fermented tomato products were found to be lower on average by 30% (lactofermentation) or 49.2% (spontaneous fermentation) compared to the rice media (Table 1); however, three LAB counts measured after 48 h of fermentation varied between 4.54 and 6.61 log10 CFU g-1. To achieve health benefits, probiotic bacteria must be viable and available at a high concentration, typically approximately 6 log10 CFU g-1 of product [80]. According to Sindhu and Khetarpaul [83], probiotic fermenta‐ tion of indigenous food mixtures containing tomato pulp increases the acidity and improves the digestibility of starch and protein. Our results support the hypothesis that rice media contain the essential nutrients to support the growth of lactobacilli and can be directly used as a fermentation substrate of LAB. The obtained biomass levels are above the minimum required for a probiotic formulation.

Classic lactic acid vegetable fermentation is a microbial process that involves heterofermen‐ tative and homofermentative LAB, generally *Lactobacillus* and *Pediococcus* [82]. At a pH between 3.5 and 3.8, vegetables will be preserved for a long period of time [83]. Tomatoes treated by lactofermentation could be recommended as useful and safe products for human nutrition. Furthermore, fermented tomatoes could serve as a healthy product for vegetarians and consumers who are allergic to dairy products [75].

#### **6.2. The production of L- and D-lactic acid during lactofermentation of tomato pulp**

Our results showed that all the analysed LAB produced a mixture of L- and D-lactic acid (Figure 1), and the highest amounts of each form were determined in tomato products treated by spontaneous fermentation (7.18±0.03 and 7.67±0.11 mg/100 g, respectively). As reported by Hartman [85] and Li and Cui [86], *Lactobacilli amylophilus, L. bavaricus L. casei*, *L. maltaromicus*, and *L. salivarius* predominantly yield the L-isomer. Strains such as *L. delbrueckii*, *L. jensenii*, and *L. acidophilus* yield D-lactic acid or mixtures of both forms. LAB such as *L. pentosus*, *L. brevis* and *L*. *lactis* can ferment glucose into lactic acid through homolactic fermentation. The fermentation of rice with two strains of *L. delbrueckii* yielded 3.23 and 5.04 mg/100 g of D-lactic acid [87].

The concentration of D-lactic acid in fermented tomato products was measured between 4.05±0.05 and 6.34±0.04 mg/100 g, and the concentration of L-lactic acid ranged from 4.26±0.04 to 7.19±0.08 mg/100 g (Figure 1). The results of our study indicate that compared to spontane‐ ous fermentation, the use of *P. pentosaceus* allowed a reduction in the content of D-lactic acid in tomato products by 11.8% (Figure 1). Fermentation with *P. acidilactici* and *L. sakei* reduced the content of the latter isomer at a higher level (on average by 40.6%).

**6.2. The production of L- and D-lactic acid during lactofermentation of tomato pulp**

tomato pulp increases the acidity and improves the digestibility of starch and protein. Our results support the hypothesis that rice media contain the essential nutrients to support the growth of lactobacilli and can be directly used as a fermentation substrate of LAB. The obtained biomass levels are above the minimum required for a probiotic

Classic lactic acid vegetable fermentation is a microbial process that involves heterofermentative and homofermentative LAB, generally *Lactobacillus* and *Pediococcus* [82]. At a pH between 3.5 and 3.8, vegetables will be preserved for a long period of time [83]. Tomatoes treated by lactofermentation could be recommended as useful and safe products for human nutrition. Furthermore, fermented tomatoes could serve as a healthy product for vegetarians and consumers who

Our results showed that all the analysed LAB produced a mixture of L- and D-lactic acid (Figure 1), and the highest amounts of each form were determined in tomato products treated by spontaneous fermentation (7.18±0.03 and 7.67±0.11 mg/100 g, respectively). As reported by Hartman [85] and Li and Cui [86], *Lactobacilli amylophilus, L. bavaricus L. casei*, *L. maltaromicus*, and *L. salivarius* predominantly yield the L-isomer. Strains such as *L. delbrueckii*, *L. jensenii*, and *L. acidophilus* yield D-lactic acid or mixtures of both forms. LAB such as *L. pentosus*, *L. brevis* and *L*. *lactis* can ferment glucose into lactic acid through homolactic fermentation. The fermentation of rice with two strains of *L. delbrueckii* 

Figure 1. Concentrations of L- and D-lactic acid in fermented tomato products. Samples: fermented with LAB: P.p. – *P. pentosaceus*, P.a. –

formulation.

are allergic to dairy productsb [75].

*L. sakei*, *P. acidilactici* and *P. pentosaceus* were found to be capable of sufficient rapid utilisation of tomato pulp for cell synthesis and organic acid production. They reduced the pH to 3.5–3.7 and increased the TTA to as high as 6.4. The viable cell counts reached 6.61 log10 CFU g-1 after 48 h of fermentation. In either case, tomato products treated with spontaneous fermentation had pH values that were higher by 7.2% and TTA values that were lower by 17.3 % than

Acid production depends on the concentration of viable bacteria able to utilise the available carbohydrate sources in the substrate [82]. The viable LAB cells in the fermented tomato products were found to be lower on average by 30% (lactofermentation) or 49.2% (spontaneous fermentation) compared to the rice media (Table 1); however, three LAB counts measured after 48 h of fermentation varied between 4.54 and 6.61 log10 CFU g-1. To achieve health benefits, probiotic bacteria must be viable and available at a high concentration, typically approximately 6 log10 CFU g-1 of product [80]. According to Sindhu and Khetarpaul [83], probiotic fermenta‐ tion of indigenous food mixtures containing tomato pulp increases the acidity and improves the digestibility of starch and protein. Our results support the hypothesis that rice media contain the essential nutrients to support the growth of lactobacilli and can be directly used as a fermentation substrate of LAB. The obtained biomass levels are above the minimum

Classic lactic acid vegetable fermentation is a microbial process that involves heterofermen‐ tative and homofermentative LAB, generally *Lactobacillus* and *Pediococcus* [82]. At a pH between 3.5 and 3.8, vegetables will be preserved for a long period of time [83]. Tomatoes treated by lactofermentation could be recommended as useful and safe products for human nutrition. Furthermore, fermented tomatoes could serve as a healthy product for vegetarians

**6.2. The production of L- and D-lactic acid during lactofermentation of tomato pulp**

Our results showed that all the analysed LAB produced a mixture of L- and D-lactic acid (Figure 1), and the highest amounts of each form were determined in tomato products treated by spontaneous fermentation (7.18±0.03 and 7.67±0.11 mg/100 g, respectively). As reported by Hartman [85] and Li and Cui [86], *Lactobacilli amylophilus, L. bavaricus L. casei*, *L. maltaromicus*, and *L. salivarius* predominantly yield the L-isomer. Strains such as *L. delbrueckii*, *L. jensenii*, and *L. acidophilus* yield D-lactic acid or mixtures of both forms. LAB such as *L. pentosus*, *L. brevis* and *L*. *lactis* can ferment glucose into lactic acid through homolactic fermentation. The fermentation of rice with two strains of *L. delbrueckii* yielded 3.23 and 5.04 mg/100 g of D-lactic

The concentration of D-lactic acid in fermented tomato products was measured between 4.05±0.05 and 6.34±0.04 mg/100 g, and the concentration of L-lactic acid ranged from 4.26±0.04 to 7.19±0.08 mg/100 g (Figure 1). The results of our study indicate that compared to spontane‐ ous fermentation, the use of *P. pentosaceus* allowed a reduction in the content of D-lactic acid in tomato products by 11.8% (Figure 1). Fermentation with *P. acidilactici* and *L. sakei* reduced

the content of the latter isomer at a higher level (on average by 40.6%).

products treated with lactofermentation (Table 1) [75].

and consumers who are allergic to dairy products [75].

required for a probiotic formulation.

acid [87].

142 Biotechnology

**Figure 1.** Concentrations of L- and D-lactic acid in fermented tomato products. Samples: fermented with LAB: P.p. – *P. pentosaceus*, P.a. – *P. acidilactici*, L.s. – *L. sakei*; SF – spontaneous fermented

In summary, *P. pentosaceus* can produce D-rich lactic acid (L/D ratio 0.64), while the other strain, *L. sakei*, produces L-rich lactic acid (L/D ratio 1.61). Fermentation with *P. acidilactici* and spontaneous fermentation gave almost equal amounts of both lactic acid isomers (L/D ratio 1.17 and 1.07, respectively). *P. acidilactici*, L.s. – *L. sakei*; SF – spontaneous fermented The concentration of D-lactic acid in fermented tomato products was measured between 4.05±0.05 and 6.34±0.04 mg/100 g, and the concentration of L-lactic acid ranged from 4.26±0.04 to 7.19±0.08 mg/100 g (Figure 1). The results of our study

By evaluating our knowledge of the potential toxicity of D-lactic acid in terms of nutrition, we can report that tomato products prepared using a pure culture of LAB were found in all cases to be safer than those treated with spontaneous fermentation. The level of D-lactic acid in pure LAB-fermented tomato products was significantly lower (*p*<0.05) than that in those spontane‐ ously fermented (Figure 1). Based on these results, *L. sakei KTU05-6* could be selected as the Llactic acid bacteria and is recommended for the fermentation of tomatoes [75]. indicate that compared to spontaneous fermentation, the use of *P. pentosaceus* allowed a reduction in the content of Dlactic acid in tomato products by 11.8% (Figure 1). Fermentation with *P. acidilactici* and *L. sakei* reduced the content of the latter isomer at a higher level (on average by 40.6%). In summary, *P. pentosaceus* can produce D-rich lactic acid (L/D ratio 0.64), while the other strain, *L. sakei*, produces L-rich

#### **6.3. Trans/cis lycopene and β-carotene contents in fermented tomato products** lactic acid (L/D ratio 1.61). Fermentation with *P. acidilactici* and spontaneous fermentation gave almost equal amounts of

The results from our analysis of lycopene and β-carotene contents in fermented tomato products are presented in Figure 2. The highest concentration of total carotenoids (on average 6.83 mg/100 g) were measured in a var. Cunero sample fermented with *P. pentosaceus* and in a var. Ronaldo sample fermented with *L. sakei*. However, fermentation with the latter bacteria increased the total level of carotenoids by 41.1 and 33.6%, respectively, compared to untreated samples. Compared to untreated tomatoes, fermentation with *P. acidilactici* reduced the concentration of total carotenoids by 3.6% in the samples of var. Cunero and var. Ronaldo (3.96 and 4.61 mg/100 g, respectively), which was accompanied by a reduction in β-carotene content (Figure 2) [75]. both lactic acid isomers (L/D ratio 1.17 and 1.07, respectively). By evaluating our knowledge of the potential toxicity of D-lactic acid in terms of nutrition, we can report that tomato products prepared using a pure culture of LAB were found in all cases to be safer than those treated with spontaneous fermentation. The level of D-lactic acid in pure LAB-fermented tomato products was significantly lower (*p*<0.05) than that in those spontaneously fermented (Figure 1). Based on these results, *L. sakei* KTU05-6 could be selected as the Llactic acid bacteria and is recommended for the fermentation of tomatoes [75].

On average, the fermented tomato samples of var. Cunero had 24.7 % lower β-carotene and 11.5% higher lycopene content compared to untreated tomatoes. In contrast, the β-carotene

**6.3. Trans/cis lycopene and β-carotene contents in fermented tomato products**

The results from our analysis of lycopene and β-carotene contents in fermented tomato products are presented in Figure 2. The highest concentration of total carotenoids (on average 6.83 mg/100 g) were measured in a var. Cunero sample fermented with *P. pentosaceus* and in a var. Ronaldo sample fermented with *L. sakei*. However, fermentation with the latter bacteria increased the total level of carotenoids by 41.1 and 33.6%, respectively, compared to untreated samples. Compared to untreated tomatoes, fermentation with *P. acidilactici* reduced the concentration of total carotenoids by 3.6%

Figure 2. Carotenoid contents in untreated and fermented with different LAB tomato products. Samples: Control – untreated tomato pulp; tomato pulp fermented with: P.p. – *P. pentosaceus*; P.a. – *P. acidilactici* MI807; L.s. – *L. sakei*, SF – spontaneous fermented. **Figure 2.** Carotenoid contents in untreated and fermented with different LAB tomato products. Samples: Control – un‐ treated tomato pulp; tomato pulp fermented with: P.p. – *P. pentosaceus*; P.a. – *P. acidilactici* MI807; L.s. – *L. sakei*, SF – spontaneous fermented.

concentrations in all the fermented tomato products of var. Ronaldo were generally higher, with an average increase of 69.4% compared to untreated tomatoes (Figure 2) [75]. On average, the fermented tomato samples of var. Cunero had 24.7 % lower β-carotene and 11.5% higher lycopene

A 24.8% increase in lycopene content was reached in the var. Ronaldo samples after fermen‐ tation with *L. sakei.* Spontaneous fermentation or treatment by *P. pentosaceus* reduced the concentration of lycopene by 11.0 and 4.4%, respectively, compared to the control sample (Figure 2). content compared to untreated tomatoes. In contrast, the β-carotene concentrations in all the fermented tomato products of var. Ronaldo were generally higher, with an average increase of 69.4% compared to untreated tomatoes (Figure 2) [75].

According to these results, lactic acid fermentation generally had a positive effect on the lycopene and total carotenoid contents of the fermented tomato products. The β-carotene contents were influenced not only by which LAB was used but also by the variety of tomato. As reported in the literature, compositional variation of lycopene in tomatoes occurs as a consequence of varietal differences, climate conditions, agricultural variables, stage of maturity, harvesting and post-harvest handling and conditions during storage [75]. Other researchers reported lycopene values within the range of 3.1–7.7 mg/100 g for different tomato cultivars [88]. However, Camara et al. [89] reported a lycopene concentration of 6–15 mg/100 g for whole fresh tomato fruit [89], which is higher than the results of this investigation. Lycopene content may be directly affected by the pH of the fruit, as the low pH of red tomatoes accumulates more lycopene [90]. A 24.8% increase in lycopene content was reached in the var. Ronaldo samples after fermentation with *L. sakei.* Spontaneous fermentation or treatment by *P. pentosaceus* reduced the concentration of lycopene by 11.0 and 4.4%, respectively, compared to the control sample (Figure 2). According to these results, lactic acid fermentation generally had a positive effect on the lycopene and total carotenoid contents of the fermented tomato products. The β-carotene contents were influenced not only by which LAB was used but also by the variety of tomato. As reported in the literature (*36,37*), compositional variation of lycopene in tomatoes occurs as a consequence of varietal differences, climate conditions, agricultural variables, stage of maturity, harvesting and post-harvest handling and conditions during storage[75]. Other researchers reported lycopene values within the range of 3.1–7.7 mg/100 g for different tomato cultivars [88]. However, Camara et al. [89] reported a lycopene

Our analysis of all-*trans* and *cis-*lycopene showed that the amounts of both isomers depended significantly on the tomato variety and were slightly affected by the LAB strain used for fermentation (Figure 3). The fermented tomato products of var. Ronaldo had all-*trans-* and *cis*concentration of 6–15 mg/100 g for whole fresh tomato fruit [89], which is higher than the results of this investigation. Lycopene content may be directly affected by the pH of the fruit, as the low pH of red tomatoes accumulates more lycopene [90].

compared to the tomato products of var. Cunero [75].

Our analysis of all-*trans* and *cis-*lycopene showed that the amounts of both isomers depended significantly on the tomato variety and were slightly affected by the LAB strain used for fermentation (Figure 3). The fermented tomato products of var. Ronaldo had all-*trans-* and *cis-*lycopene contents that were higher on average by 25.9 and 62.6%, respectively, results of this investigation. Lycopene content may be directly affected by the pH of the fruit, as the

(Figure 3). The fermented tomato products of var. Ronaldo had all-*trans-* and *cis-*lycopene contents that were higher on average by 25.9 and 62.6%, respectively, compared to the tomato products of var.

lycopene contents that were higher on average by 25.9 and 62.6%, respectively, compared to the tomato products of var. Cunero [75]. low pH of red tomatoes accumulates more lycopene [90]. Our analysis of all-*trans* and *cis-*lycopene showed that the amounts of both isomers depended significantly on the tomato variety and were slightly affected by the LAB strain used for fermentation

Cunero [75].

concentrations in all the fermented tomato products of var. Ronaldo were generally higher,

**Figure 2.** Carotenoid contents in untreated and fermented with different LAB tomato products. Samples: Control – un‐ treated tomato pulp; tomato pulp fermented with: P.p. – *P. pentosaceus*; P.a. – *P. acidilactici* MI807; L.s. – *L. sakei*, SF –

**β-carotene lycopene β-carotene lycopene**

**Control P.p. P.a. L. s. SF**

**var. Cunero var. Ronaldo**

**6.3. Trans/cis lycopene and β-carotene contents in fermented tomato products**

The results from our analysis of lycopene and β-carotene contents in fermented tomato products are presented in Figure 2. The highest concentration of total carotenoids (on average 6.83 mg/100 g) were measured in a var. Cunero sample fermented with *P. pentosaceus* and in a var. Ronaldo sample fermented with *L. sakei*. However, fermentation with the latter bacteria increased the total level of carotenoids by 41.1 and 33.6%, respectively, compared to untreated samples. Compared to untreated tomatoes, fermentation with *P. acidilactici* reduced the concentration of total carotenoids by 3.6% in the samples of var. Cunero and var. Ronaldo (3.96 and 4.61 mg/100 g, respectively), which was accompanied by a

A 24.8% increase in lycopene content was reached in the var. Ronaldo samples after fermen‐ tation with *L. sakei.* Spontaneous fermentation or treatment by *P. pentosaceus* reduced the concentration of lycopene by 11.0 and 4.4%, respectively, compared to the control sample

According to these results, lactic acid fermentation generally had a positive effect on the lycopene and total carotenoid contents of the fermented tomato products. The β-carotene contents were influenced not only by which LAB was used but also by the variety of tomato. As reported in the literature, compositional variation of lycopene in tomatoes occurs as a consequence of varietal differences, climate conditions, agricultural variables, stage of maturity, harvesting and post-harvest handling and conditions during storage [75]. Other researchers reported lycopene values within the range of 3.1–7.7 mg/100 g for different tomato cultivars [88]. However, Camara et al. [89] reported a lycopene concentration of 6–15 mg/100 g for whole fresh tomato fruit [89], which is higher than the results of this investigation. Lycopene content may be directly affected by the pH of the fruit, as the low pH of red tomatoes

Our analysis of all-*trans* and *cis-*lycopene showed that the amounts of both isomers depended significantly on the tomato variety and were slightly affected by the LAB strain used for fermentation (Figure 3). The fermented tomato products of var. Ronaldo had all-*trans-* and *cis-*

range of 3.1–7.7 mg/100 g for different tomato cultivars [88]. However, Camara et al. [89] reported a lycopene

Our analysis of all-*trans* and *cis-*lycopene showed that the amounts of both isomers depended significantly on the tomato variety and were slightly affected by the LAB strain used for fermentation (Figure 3). The fermented tomato products of var. Ronaldo had all-*trans-* and *cis-*lycopene contents that were higher on average by 25.9 and 62.6%, respectively,

with an average increase of 69.4% compared to untreated tomatoes (Figure 2) [75].

respectively, compared to the control sample (Figure 2).

compared to the tomato products of var. Cunero [75].

reduction in β-carotene content (Figure 2) [75].

(Figure 2).

[75].

spontaneous fermented.

**Concentration / (mg/100 g)**

144 Biotechnology

accumulates more lycopene [90].

lycopene [90].

concentration of 6–15 mg/100 g for whole fresh tomato fruit [89], which is higher than the results of this investigation. Lycopene content may be directly affected by the pH of the fruit, as the low pH of red tomatoes accumulates more Ronaldo (b) products. Samples: Control – untreated tomato pulp; tomato products fermented with: P.p. – *P. pentosaceus*, P.a. – *P. acidilactici*, L. s. – *L. sakei*; SF – spontaneous fermented). **Figure 3.** Content of all-*trans*- and *cis*-lycopene in fermented tomato of var. Cunero (a) and var. Ronaldo (b) products. Samples: Control – untreated tomato pulp; tomato products fermented with: P.p. – *P. pentosaceus*, P.a. – *P. acidilactici*, L. s. – *L. sakei*; SF – spontaneous fermented.

9

Figure 3. Content of all-*trans*- and *cis*-lycopene in fermented tomato of var. Cunero (a) and var.

The control samples of var. Ronaldo had 3.3-fold higher *cis*-lycopene (3.4 mg/kg) compared to var. Cunero (11.3 mg/kg) (Fig. 3). Fermentation by *P. pentosaceus* or *L. sakei* increased the *cis*lycopene contents on average by 30.6 and 8.5%, respectively in the products of var. Cunero and var. Ronaldo. A lower increase in *cis*-lycopene was noticed during fermentation of the var. Cunero tomatoes with *P. acidilactici* as well as during spontaneous fermentation (an average increase of 9%). Similarly, lactofermentation using *P. acidilactici* and *L. sakei* increased the *cis*lycopene contents by 5.8% on average in the tomato products [75].

The fermentation of var. Cunero and var. Ronaldo tomatoes by *P. pentosaceus* and *L. sakei* produced an average of 22.2% more all-*trans*-lycopene compared to the controls (204.6 and 296.0 mg/kg, respectively) (Figure 3) [75].

The *cis/trans* ratio of var. Cunero and var. Ronaldo tomatoes were 1.67 and 3.81, respectively. The highest *cis/trans* ratio was found in the var. Cunero samples fermented by *L. sakei* (2.08), following that of var. Ronaldo samples fermented by *P. acidilactici* (4.90) and spontaneous fermentation (4.09) [75].

It is known from the literature that in human subjects, lycopene from *cis*-isomer-rich tomato sauce is more bioavailable than that from all-*trans*-rich tomato sauce [91]. Because of the positive effect of lactofermentation on the *cis/trans* lycopene ratio, fermented products of the var. Ronaldo tomato, fermented with *P. acidilactici* or *L. sakei*, could be recommended as more biologically accessible products with greater functional value.

#### **6.4. Colour characteristics of fermented tomato products**

The results from our analysis of the red (a\*) and yellow (b\*) colour coordinates of fermented tomato products are presented in Table 2. No relation was found in the var. Cunero samples between the yellow colour coordinate (b\*) and total carotenoid, lycopene or β-carotene contents (*p* > 0.05) (Table 2). However, the red colour coordinate (a\*) slightly correlated (R2 = 0.672) with β-carotene content.

In contrast, a weak relation was noticed between colour coordinate b\* of var. Ronaldo and total carotenoid or β-carotene contents (R<sup>2</sup> = 0.581 or R2 = 0.596, respectively) (Table 2). In addition, samples of this variety showed a strong relation between colour coordinate b\* and lycopene content (R2 = 0.825, *p* = 0.03). No significant relations were observed between a\* and β-carotene or lycopene contents (*p* > 0.05) (Table 2) or between total carotenoids and the colour tone (ho ) or colour purity (C) values of the var. Cunero and var. Ronaldo samples (Table 3) [75].

The best estimation for β-carotene content was obtained using the b\* chromaticity value from the whole fruit measurements or the transformed a\*2 value from the pure measurements [91]. Neither model, however, could explain more than 55% of the variation in β-carotene levels, suggesting that chromaticity values may not be appropriate for estimating tomato β-carotene content. It has been stated that the inspection of different chromaticity values and regression models suggest that colorimeter readings may not be highly useful for estimating β-carotene content in the tomato fruit [92].


The control samples of var. Ronaldo had 3.3-fold higher *cis*-lycopene (3.4 mg/kg) compared to var. Cunero (11.3 mg/kg) (Fig. 3). Fermentation by *P. pentosaceus* or *L. sakei* increased the *cis*lycopene contents on average by 30.6 and 8.5%, respectively in the products of var. Cunero and var. Ronaldo. A lower increase in *cis*-lycopene was noticed during fermentation of the var. Cunero tomatoes with *P. acidilactici* as well as during spontaneous fermentation (an average increase of 9%). Similarly, lactofermentation using *P. acidilactici* and *L. sakei* increased the *cis*-

The fermentation of var. Cunero and var. Ronaldo tomatoes by *P. pentosaceus* and *L. sakei* produced an average of 22.2% more all-*trans*-lycopene compared to the controls (204.6 and

The *cis/trans* ratio of var. Cunero and var. Ronaldo tomatoes were 1.67 and 3.81, respectively. The highest *cis/trans* ratio was found in the var. Cunero samples fermented by *L. sakei* (2.08), following that of var. Ronaldo samples fermented by *P. acidilactici* (4.90) and spontaneous

It is known from the literature that in human subjects, lycopene from *cis*-isomer-rich tomato sauce is more bioavailable than that from all-*trans*-rich tomato sauce [91]. Because of the positive effect of lactofermentation on the *cis/trans* lycopene ratio, fermented products of the var. Ronaldo tomato, fermented with *P. acidilactici* or *L. sakei*, could be recommended as more

The results from our analysis of the red (a\*) and yellow (b\*) colour coordinates of fermented tomato products are presented in Table 2. No relation was found in the var. Cunero samples between the yellow colour coordinate (b\*) and total carotenoid, lycopene or β-carotene contents (*p* > 0.05) (Table 2). However, the red colour coordinate (a\*) slightly correlated (R2

In contrast, a weak relation was noticed between colour coordinate b\* of var. Ronaldo and total carotenoid or β-carotene contents (R<sup>2</sup> = 0.581 or R2 = 0.596, respectively) (Table 2). In addition, samples of this variety showed a strong relation between colour coordinate b\* and lycopene

or lycopene contents (*p* > 0.05) (Table 2) or between total carotenoids and the colour tone (ho

The best estimation for β-carotene content was obtained using the b\* chromaticity value from the whole fruit measurements or the transformed a\*2 value from the pure measurements [91]. Neither model, however, could explain more than 55% of the variation in β-carotene levels, suggesting that chromaticity values may not be appropriate for estimating tomato β-carotene content. It has been stated that the inspection of different chromaticity values and regression models suggest that colorimeter readings may not be highly useful for estimating β-carotene

or colour purity (C) values of the var. Cunero and var. Ronaldo samples (Table 3) [75].

= 0.825, *p* = 0.03). No significant relations were observed between a\* and β-carotene

=

)

lycopene contents by 5.8% on average in the tomato products [75].

biologically accessible products with greater functional value.

**6.4. Colour characteristics of fermented tomato products**

296.0 mg/kg, respectively) (Figure 3) [75].

fermentation (4.09) [75].

146 Biotechnology

0.672) with β-carotene content.

content in the tomato fruit [92].

content (R2

The numbers are means followed by standard deviations (n = 3). Means within a column with different superscript letters are significantly different (*p* < 0.05).

Samples: control – untreated tomato pulp; tomato products fermented with: P.p. – *P. pentosaceus,* P.a. – *P*. *acidilactici L.,* s. – *L. sakei;* SF – spontaneous fermented; R2 – correlation coeficient.

**Table 2.** Colour coordinates (a\*, b\*) of tomato var. Cunero and var. Ronaldo samples and their correlations between total carotenoids, lycopene and β-carotene contents


The numbers are means followed by standard deviations (n = 3). Means within a column with different superscript letters are significantly different (*p* < 0.05).

Samples: control – untreated tomato pulp; tomato products fermented with: P.p. – *P. pentosaceus,* P.a. – *P*. *acidilactici L.,* s. – *L. sakei;* SF – spontaneous fermented; R2 – correlation coeficient.

**Table 3.** Colour tone (ho ) and purity (C) of tomato var. Cunero and var. Ronaldo samples and their correlation with total carotenoid contents

The overall results indicate that lycopene content could be measured simply and quite accurately across a wide range of tomato genotypes using chromaticity values taken from fruit puree [91]. In contrast, Liu et al. [93] reported that treating tomatoes with a daily light treatment enhances exocarp lycopene accumulation with minimal effect on the colour. Arias et al. [59] also observed that the b\* characteristic was not appropriate for predicting the lycopene content of tomatoes.

According to the obtained results, colour tone (ho ) and purity (C) are not suitable indicators of the total carotenoid content in the evaluation of tomato products. We postulate that measuring the yellow coordinate (b\*) could be a simple and non-destructive method for predicting lycopene concentration in tomato products [94].
