Beet red food colourant can be produced more sustainably with engineered Yarrowia lipolytica.

Synthetic food colourants are widely used in the food industry, but consumer concerns about safety and sustainability are driving a need for natural food-colour alternatives. Betanin, which is extracted from red beetroots, is a commonly used natural red food colour. However, the betanin content of beetroot is very low (~0.2% wet weight), which means that the extraction of betanin is incredibly wasteful in terms of land use, processing costs and vegetable waste. Here we developed a sustainability-driven biotechnological process for producing red beet betalains, namely, betanin and its isomer isobetanin, by engineering the oleaginous yeast Yarrowia lipolytica. Metabolic engineering and fermentation optimization enabled production of 1,271 ± 141 mg l-1 betanin and 55 ± 7 mg l-1 isobetanin in 51 h using glucose as carbon source in controlled fed-batch fermentations. According to a life cycle assessment, at industrial scale (550 t yr-1), our fermentation process would require significantly less land, energy and resources compared with the traditional extraction of betanin from beetroot crops. Finally, we apply techno-economic assessment to show that betanin production by fermentation could be economically feasible in the existing market conditions.

Metabolic engineering of betacyanin in vegetables for anti-inflammatory therapy.

Betalains, which consist of the subgroups betaxanthins and betacyanins, are hydrophilic pigments that have classically been used for food colorants. Owing to their strong antioxidant property, their usefulness for application for therapeutic use is also expected. In addition, as betalains are mainly naturally available from plants of the order Caryophyllales, including beet (Beta vulgaris), metabolic engineering for betalain production in crops such as vegetables, fruits and cereals may provide new food resources useful for healthcare. Here we conducted metabolic engineering of betacyanins in tomato fruits and potato tubers. The transgenic tomato fruits and potato tubers with coexpression of betacyanin biosynthesis genes, CYP76AD1 from B. vulgaris, DOD (DOPA 4,5-dioxygenase) and 5GT (cyclo-DOPA 5-O-glucosyltransferase) from Mirabilis jalapa, under control of suitable specific promoters, possessed dark red tissues with enriched accumulation of betacyanins (betanin and isobetanin). The anti-inflammatory activity of transgenic tomato fruit extract was superior to that of wild-type fruit extract on macrophage RAW264.7 cells stimulated with lipopolysaccharide (LPS), as a result of decreased LPS-stimulated transcript levels of proinflammatory genes. These findings were in accord with the observation that administration of the transgenic tomato fruits ameliorated dextran sulfate sodium (DSS)-induced colitis as well as body weight loss and disease activity index in mice, via suppression of DSS-stimulated transcript levels of pro-inflammatory genes, including Tnf (encoding TNF-alpha), Il6, and Ptgs2 (encoding cyclooxygenae 2). Intriguingly, given the fact that the transgenic potato tuber extract failed to enrich the anti-inflammatory activity of macrophage cells, it is likely that metabolic engineering of betacyanins will be a powerful way of increasing the anti-inflammatory property of ordinary foods such as tomato.

Dehydrogenation of Betacyanins in Heated Betalain-Rich Extracts of Red Beet (Beta vulgaris L.).

Betacyanins are a group of water-soluble red-violet compounds containing nitrogen in their structure. These are biosynthesized in red beetroot (Beta vulgaris L.), a widely consumed vegetable that contains significant amounts of nutritious and bioactive compounds which are also found in dietary supplements. This contribution presents results of betacyanin thermal oxidation (resulting in dehydrogenation) interrelated with decarboxylation in selected acetate/phosphate buffers at pH 3-8 and at 85 °C, which may be of particular significance for formulation and performance of foods. Most of the reaction products were detected at the highest concentrations in the acidic solutions (pH 3-4). The main dehydrogenation reaction pathways were monitored by LC-DAD-MS/MS and were associated with decarboxylation of the principal extract pigments, betanin/isobetanin and neobetanin, at carbon positions C-2 and C-17. Additional reactions are accompanied by the 2,15-decarboxylation processes at different dehydrogenation levels with 15-decarboxy-betanin and 2,15-bidecarboxy-betanin, structurally elucidated by NMR analysis, as the distinct indicators of this route type. For other novel pigments detected, 2,15-bidecarboxy-xanbetanin, 2,15-bidecarboxy-xanneobetanin and 2,15,17-tridecarboxy-neobetanin, additional high resolution mass spectrometric analyses were performed and confirmed their molecular formulas.

Thermoprotective properties of Opuntia ficus-indica f. inermis cladodes and mesocarps on sheep lymphocytes.

This study aims to investigate the thermoprotective properties of Opuntia ficus-indica f. inermis. Extracts were prepared from cladodes (CE) and mesocarps (ME), then subjected to a spectrophotometric and LC-MS analyses. Lymphocytes were isolated from peripheral blood of non-stressed sheep, supplemented with CE, ME, betanin or α-tocopherol, and subjected to two thermal treatments: 40 and 41 °C, for 6 h. Viable lymphocytes and H2O2 production were evaluated. The antioxidant activity of ME was 3.43 folds higher than CE. The LC-MS analysis of CE and ME allowed identifying 11 phenolic acids, 2 flavanones, 6 flavones, 3 flavonols and 1 betanin type betacyanin. Lymphocytes mortality increased linearly as function of the severity and the duration of heat stress. This mortality was correlated with H2O2 production. At 41 °C, only ME allowed maintaining lymphocytes viability. Moreover, ME was more efficient than CE in reducing H2O2 production. This thermoprotection was ensured by betaxanthin and betacyanin pigments. Interestingly, betanin was more efficient than α-tocopherol in preventing hyperthermia-induced lymphocytes' mortality. We report here for the first time the thermoprotective properties of cladodes and mesocarps of Opuntia ficus-indica f. inermis. Betanin was able to maintain lymphocyte viability through reducing H2O2 production, and therefore the oxidative-induced heat stress.

Trans-epithelial transport of the betalain pigments indicaxanthin and betanin across Caco-2 cell monolayers and influence of food matrix.

PURPOSE: This study investigated the absorption mechanism of the phytochemicals indicaxanthin and betanin and the influence of their food matrix (cactus pear and red beet) on the intestinal transport. METHODS: Trans-epithelial transport of dietary-consistent amounts of indicaxanthin and betanin in Caco-2 cell monolayers seeded on Transwell(R) inserts was measured in apical to basolateral (AP-BL) and basolateral to apical (BL-AP) direction, under an inwardly directed pH gradient (pH 6.0/7.4, AP/BL) mimicking luminal and serosal sides of human intestinal epithelium. The effect of inhibitors of membrane transporters on the absorption was also evaluated. Contribution of the paracellular route was investigated after EDTA treatment of the cell monolayer. In vitro digestion of betalainic food was performed to provide a post-intestinal fraction containing bioaccessible pigments. RESULTS: Apparent permeability coefficients (P(app)) in the absorptive direction were (4.4 ± 0.4) × 10⁻6 and (3.2 ± 0.3) × 10⁻6 cm s⁻¹ for indicaxanthin and betanin, respectively. Transport of indicaxanthin was non-polarized, linear as a function of time and concentration, and unaffected by inhibitors of membrane transporters. Betanin exhibited significantly different bidirectional P(app) values and non-linear efflux kinetics. The concentration-dependent betanin efflux was described by a kinetic model including one non-saturable (K(d) = 0.042 µL cm⁻² min⁻¹) and one saturable component identified as the apical multidrug resistance-associated protein 2 (MRP2; K(m) = 275 µM; J(max) = 42 pmol min⁻¹ cm⁻²). Permeation of both betalains increased remarkably after EDTA treatment of the cell monolayer. Neither indicaxanthin nor betanin underwent metabolic transformation. Food matrix did not affect trans-epithelial transfer of indicaxanthin, but reduced the absorption rate of betanin, red beet more than cactus pear. CONCLUSIONS: Dietary indicaxanthin and betanin can substantially be absorbed through paracellular junctions of intestinal epithelial cells. Additional trans-membrane permeation can be considered for betanin, whose absorption is limited by a MRP2-mediated efflux and negatively affected by its food matrix. Present findings are consistent with the quite higher bioavailability of indicaxanthin over betanin established in humans.

Betacyanin and other antioxidants production during growth of Opuntia stricta (Haw.) fruits.

Mature cactus pears from Opuntia stricta have a dark purple color due to high betacyanin concentration, whose biosynthesis is initiated with the amino acid L-tyrosine as a primary precursor. This study followed the maturation and ripening processes of Opuntia stricta fruits to harvest them at high betacyanin and other antioxidant concentrations. Fruits lasted 9 months for final ripening. Physical and compositional changes at different maturation and ripening stages have been determined. Thus, ripe fruits were around 4.72 ± 0.10 cm length, 2.94 ± 0.05 cm diameter and 22.71 ± 0.20 g weight; moisture and pH were maintained at 87.05 ± 0.19 % and 3.37 ± 0.12, respectively. Purple pigment production started in the ovary of immature fruits four months after anthesis (MAA). Concentration of all analyzed metabolites increased from immature (4 MAA) until ripe (9 MAA) stage. In ripe fruits, reducing sugars were 4.72 ± 0.54 g/100 g ff and total phenols 135.17 ± 0.68 mg gallic acid/100 g ff. Metabolites identified by HPLC were the betacyanins: betanin (60.17 ± 1.08 mg/100 g ff), isobetanin (7.58 ± 0.94 mg/100 g ff) and betanidin (13.48 ± 0.87 mg/100 g ff). Also, L-ascorbic acid (35.03 ± 1.06 mg/100 g ff) and L-tyrosine (4.43 ± 0.73 mg/100 g ff) were determined. Furthermore, the addition of L-tyrosine or L-dopa to fruit pulp of moderately ripe fruits, increased betacyanin concentrations 17 (103.3 ± 3.8 mg/100 g) and 32 % (114.3 ± 4.1 mg/100 g), respectively.

Betalain production is possible in anthocyanin-producing plant species given the presence of DOPA-dioxygenase and L-DOPA.

BACKGROUND: Carotenoids and anthocyanins are the predominant non-chlorophyll pigments in plants. However, certain families within the order Caryophyllales produce another class of pigments, the betalains, instead of anthocyanins. The occurrence of betalains and anthocyanins is mutually exclusive. Betalains are divided into two classes, the betaxanthins and betacyanins, which produce yellow to orange or violet colours, respectively. In this article we show betalain production in species that normally produce anthocyanins, through a combination of genetic modification and substrate feeding. RESULTS: The biolistic introduction of DNA constructs for transient overexpression of two different dihydroxyphenylalanine (DOPA) dioxygenases (DODs), and feeding of DOD substrate (L-DOPA), was sufficient to induce betalain production in cell cultures of Solanum tuberosum (potato) and petals of Antirrhinum majus. HPLC analysis showed both betaxanthins and betacyanins were produced. Multi-cell foci with yellow, orange and/or red colours occurred, with either a fungal DOD (from Amanita muscaria) or a plant DOD (from Portulaca grandiflora), and the yellow/orange foci showed green autofluorescence characteristic of betaxanthins. Stably transformed Arabidopsis thaliana (arabidopsis) lines containing 35S: AmDOD produced yellow colouration in flowers and orange-red colouration in seedlings when fed L-DOPA. These tissues also showed green autofluorescence. HPLC analysis of the transgenic seedlings fed L-DOPA confirmed betaxanthin production. CONCLUSIONS: The fact that the introduction of DOD along with a supply of its substrate (L-DOPA) was sufficient to induce betacyanin production reveals the presence of a background enzyme, possibly a tyrosinase, that can convert L-DOPA to cyclo-DOPA (or dopaxanthin to betacyanin) in at least some anthocyanin-producing plants. The plants also demonstrate that betalains can accumulate in anthocyanin-producing species. Thus, introduction of a DOD and an enzyme capable of converting tyrosine to L-DOPA should be sufficient to confer both betaxanthin and betacyanin production to anthocyanin-producing species. The requirement for few novel biosynthetic steps may have assisted in the evolution of the betalain biosynthetic pathway in the Caryophyllales, and facilitated multiple origins of the pathway in this order and in fungi. The stably transformed 35S: AmDOD arabidopsis plants provide material to study, for the first time, the physiological effects of having both betalains and anthocyanins in the same plant tissues.

In vitro digestion of betalainic foods. Stability and bioaccessibility of betaxanthins and betacyanins and antioxidative potential of food digesta.

Betalains are considered to be bioactive dietary phytochemicals. The stability of betacyanins and betaxanthins from either fresh foods or manufactured products of cactus pear fruit ( Opuntia ficus indica L. Mill. cv. Gialla and Rossa) and red beet ( Beta vulgaris L. ssp. vulgaris) was assessed in a simulated oral, gastric, and small intestinal digestion and compared with the digestive stability of purified pigments. A minor loss of indicaxanthin, at the gastric-like environment only, and a decrease of vulgaxanthin I through all digestion steps were observed, which was not affected by food matrix. In contrast, food matrix prevented decay of betanin and isobetanin at the gastric-like environment. Loss of betacyanins, either purified or food-derived, was observed during the small intestinal phase of digestion. Betalamic acid accumulated after digestive degradation of purified pigments, but not of food betalains. Betaxanthins were wholly soluble in the aqueous (bioaccessible) fraction after ultracentrifugation of the postintestinal (PI) digesta, whereas release of betacyanins from the matrix was incomplete. PI digesta inhibited dose-dependently the oxidation of methyl linoleate in methanol, an effect not correlated with the betalain content. The data suggest that digestive stability controls bioaccessibility of dietary betaxanthins, whereas additional factors, relevant to the food matrix and style of processing, affect betacyanin bioaccessibility.

Beta vulgaris L. suspension cultures permeabilized with triton X-100 retain cell viability and betacyanines production ability: a digital image analysis study.

The influence of Triton X-100 on Beta vulgaris L. permeabilized cell culture viability, regrowth, and ability to produce betacyanines was evaluated in this study. A non-destructive method based on the analysis of images in the RGB (red, green, blue) system was developed to estimate betacyanines content. A treatment for 15 min with 0.7 mM Triton X-100 induced the release of 30% of betacyanines without loss of cell viability (>or=70%). After this permeabilization treatment, B. vulgaris cultures regrew normally, reaching a maximum biomass concentration of 48% higher than non-permeabilized cultures after 14 days of culture. Also, maximum betacyanines concentration was only 25% lower than that of non-permeabilized cultures.