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Effect of addition of Opuntia ficus-indica mucilage on the biological leavening, physical, nutritional, antioxidant and sensory aspects of bread
Journal of Bioscience and Bioengineering VOL. xxx No. xxx, xxx, xxxx www.elsevier.com/locate/jbiosc
Effect of addition of Opuntia ficus-indica mucilage on the biological leavening, physical, nutritional, antioxidant and sensory aspects of bread Giorgia Liguori,1 Carla Gentile,2 Raimondo Gaglio,1 Anna Perrone,2 Rosa Guarcello,1 Nicola Francesca,1 Silvia Fretto,1 Paolo Inglese,1 and Luca Settanni1, * Department of Agricultural, Food and Forest Sciences, University of Palermo, Viale delle Scienze, 90128 Palermo, Italy1 and Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, University of Palermo, Viale delle Scienze, 90128 Palermo, Italy2 Received 3 June 2019; accepted 20 August 2019 Available online xxx
The addition of active compounds to enhance the functional properties of foods is a quite common practice. Recently, bread became one of the target foods to incorporate functional ingredients such as those deriving from Opuntia spp. So far, only Opuntia ficus-indica cladodes in powder has been tested. The addition of fresh O. ficus-indica mucilage (in substitution to water) did not influence the biological leavening of the doughs. The resulting breads showed a biological role of the cactus mucilage, because their antioxidant activity was higher than that of control wheat bread. The sensory analysis indicated a general appreciation of the breads enriched with O. ficus-indica mucilage by the judges. The inclusion of fresh cactus mucilage in bread production might increase the dietary antioxidant intake due to its daily worldwide consumption. Ó 2019, The Society for Biotechnology, Japan. All rights reserved. [Key words: Antimicrobial activity; Antioxidant activity; Biological leavening; Enriched bread; Cactus mucilage; Yeasts]
Functional foods provide health benefits. They represent one of the food sector that is growing more and more rapidly due to consumers’ increasing demand for foods that promote health and well-being (1). One of the first definition of functional foods, coined in Japan, was: “Food products fortified with special constituents that possess advantageous physiological effects” (2). Although most bioactive compounds are derived from plants (1), only a few species have been used to improve food healthy properties (3). Indeed, several factors affect the suitability of bioactive compounds pure or in mixture to be used as ingredients of functional foods. The in vivo efficacy of functionalizing agents depends on the doses added to the foods and the food matrices might affect the stability and/or bioavailability of the bioactive compounds. On the other hand, plant extracts could affect food sensory characteristics producing undesirable odors or tastes as well as negatively impacting the final texture (1). Opuntia ficus-indica (L.) Mill. is a drought-tolerant cactus, widely cultivated in semi-arid and arid regions worldwide. Several research showed that cactus pear fruits and cladodes could be used as sources of nutrients and phytochemicals for applications in food, pharmaceutical and nutraceutical industries (4). The major components of cladodes are carbohydratecontaining polymers, including a mixture of mucilage and pectin. Cladodes represent also a source of phytochemicals, in particular phenolic compounds (5). Mucilage is part of dietary fiber and generally refers to some part of fruits, vegetables, grains,
* Corresponding author. Tel.: þ39 091 23896043; fax: þ39 091 6515531. E-mail address: [email protected] (L. Settanni).
nuts and legumes (4). O. ficus-indica cladodes mucilage, due to its viscosity and chemical composition, is an interesting ingredient for food industry (6) that can be found in specialized storage cells or free within cells or intracellular spaces of the chlorenchymatic and parenchymatic tissue of the cladodes (4). Mucilage is a polysaccharide and based on its chemical composition, it is considered a polymer (similar to pectin), composed of arabinose, galactose, xylose and rhamnose as neutral sugars, and of a small amount of galacturonic acid (5e8). Opuntia cladodes are traditionally used as healthy nutrient and in folk medicine in South and Central America and North Africa but are scarcely used by the modern food and pharmaceutical industries. Bread from refined wheat flour is regularly eaten in significant quantities, as it is one of the main components of the human diet worldwide. Despite its nutritional value, with a high content of complex sugars and a very low lipid content, the nutraceutical value of this widely consumed product is scarce. On the other hand, bread, due to its wide consumption could be a good carrier for functional molecules. Fortified bread offer the possibility to introduce substances with health-enhancing properties through diet. Several studies evaluated the potential use of bread for delivering antioxidant molecules in particular by addition of natural raw materials rich in phenolic compounds (9,10). However, some studies were conducted on bread enrichment with Opuntia cladodes (11) but no studies focused on the effect of O. ficus-indica mucilage addition on bread and on its potential use as a new functional ingredient. The aim of this study was to evaluate the effect of addition of O. ficus-indica mucilage on the biological leavening, physical, nutritional, antioxidant and sensory aspects of bread.
1389-1723/$ e see front matter Ó 2019, The Society for Biotechnology, Japan. All rights reserved. https://doi.org/10.1016/j.jbiosc.2019.08.009
Please cite this article as: Liguori, G et al., Effect of addition of Opuntia ficus-indica mucilage on the biological leavening, physical, nutritional, antioxidant and sensory aspects of bread, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.08.009
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LIGUORI ET AL. MATERIALS AND METHODS
Fresh mucilage extraction and evaluation of antifungal activity Oneyear-old cladodes were collected from 4-year-old O. ficus-indica potted plants of the cultivar “Gialla”, located at Department of Agricultural, Food and Forest Sciences, University of Palermo (38 70 4.080000 N 13 220 11.280000 E, 29 m a.s.l). Three cladodes (one-year-old cladodes) were harvested from the same plant for mucilage extraction. Harvested cladodes were marked, packaged and transported to the laboratory where they were measured, weighed and processed for mucilage extraction, using a modified patented method of Du Toit et al. (12) developed in South Africa. Cladodes were washed with chlorinated water to improve mucilage shelf life and to remove impurities and spines. Cladodes chlorenchyma was removed with a peeler to obtain high pure quality mucilage. Cladodes were then sliced into squares and cooked in a microwave oven (900 W) for 3e5 min, until soft. The cooked, soft cladode pieces were then mixed using an Omni Mixer Homogenizer (mod. OmniMixer. 17,107, Dupont Instruments Sorvall, USA) to aid the mucilage extraction. The obtained pulp was then centrifuged using a Sigma centrifuge (mod. 6K15, Sigma Laborzentrifugen GmbH, Germany) at 8800 g for 15 min at 4 C, to separate the liquid mucilage from the solids. The mucilage was then decanted and weighed while the solid material left in the falcon tubes were discarded. No chemicals have been used during this extraction process and as such, the extracted mucilage obtained is natural and unadulterated by chemicals. The suitability of mucilage for the production of breads subjected to biological leavening depends on its harmlessness towards yeasts and lactic acid bacteria (LAB) which are the main agents of fermentation in yeasted and sourdough breads, respectively. For this reason, O. ficus-indica mucilage was tested for the potential inhibitory activity on several yeasts, mainly of cereal origin, in order to enlarge the knowledge on the antagonistic properties against unicellular fungi and versus the most relevant LAB species used for sourdough production (13,14). To this purpose, the yeast indicators were Aureobasidium pullulans AD14, Candida parapsilosis AD29, Cryptococcus aerius AD62, Cryptococcus carnesciens AD56, Cryptococcus fonsecae AD245, Cryptococcus flavescens AD17, Cryptococcus magnus AD122, Cryptococcus tephrensis AD157, Cryptococcus victoriae AD85; Filobasidum chernovii AD172; Filobasidum oeirense AD82; Holtermanniella festucosa AD413, Rhodosporidium babjevae AD224, Rhodosporidium toruloides AD258, Rhodotorula glutinis AD407, Rhodotorula mucilaginosa AD284, Saccharomyces cerevisiae Commercial (isolated from commercial baker’s yeast La Parisienne, AB Mauri Italy S.p.A., Casteggio, Italy), Sporidiobolus metaroseus AD244, and Sporobolomyces roseus AD397, while sourdough LAB were represented by Lactobacillus brevis SD46, Lactobacillus plantarum SD130, L. plantarum SD96, Lactobacillus sanfranciscensis SD22, Leuconostoc citreum SD142 and Weissella cibaria SD123. All strains except S. cerevisiae commercial belonged to the culture collection of the Agricultural Microbiology laboratory of the Department of Agricultural, Food and Forest Sciences, University of Palermo (Palermo, Italy). All yeast strains were grown overnight at 28 C in yeast peptone dextrose (YPD) broth (Oxoid, Milan, Italy). LAB were propagated overnight at 30 C in different media: lactobacilli in sourdough bacteria (SDB) broth prepared as described by Kline and Sugihara (15), while Leuconostoc citreum and W. cibaria in modified-de Man-RogosaSharpe (mMRS) agar, prepared as reported by Corona et al. (16). Freeze-dried O. ficus-indica mucilage powder, obtained after 48 h in a ScanVac CoolSafe 4e15 L freeze-dryer at 100 C, was re-suspended in water at 200 mg mL1 as commonly performed for extracts of botanical origin (17) and tested by well diffusion assay. A water agar base medium (1.5% agar) was overlaid with soft agar YPD containing the yeast strains at a cell density of approximately 105 CFU mL1. Circular wells were obtained by means of a sterile cork borer and filled in with 100 mL of O. ficus-indica mucilage solution. Production of doughs The doughs were processed from wheat flour type “0” SISA Manitoba (Molino Rossetto, Pontelongo, Italy) and O. ficus-indica (90% of water) mucilage. Two dough trials were obtained by yeast leavening: CTR, control dough prepared exclusively from wheat flour; MUC, dough prepared from wheat flour and O. ficus-indica mucilage in place of water. Doughs of 406 g were obtained from 246 g of wheat flour, 150 mL of tap water (CTR trial) or 150 mL O. ficus-indica mucilage (MUC trial), 4 g of baker’s yeast (Lesaffre Italia S.p.A., Trecasali, Italy) and 6 g of NaCl. The fermentation was performed at 28 C for 2 h. All productions were carried out in duplicate and repeated in two consecutive weeks for a total of eight replicates (breads) for each trial. Monitoring of acidification and biological leavening The acidification process and the levels of the main fermenting microorganisms were analyzed at beginning (T0) and end of fermentation (T2). The monitoring of the acidification was performed by measuring pH using the pH meter Russell RL060P (Thermo Fisher Scientific, Beverly, MA, USA) and total titratable acidity (TTA), determined by titratation with 0.1 N NaOH (mL of NaOH 10 g1 of dough). The microbiological analyses were performed on 10 g of each dough. The samples were diluted (1:10) in Ringer’s solution (Oxoid) through homogenization carried out by stomacher (BagMixer 400, Interscience, Saint Nom, France) and then serially diluted. The following media and growth conditions were used to determine the different microbial groups from the cell suspensions: plate count agar (PCA), incubated aerobically for 72 h at 30 C for total mesophilic microorganisms (TMM); mMRS agar and SDB agar, incubated anaerobically and aerobically, respectively, were used to detect LAB, after 48 h at
J. BIOSCI. BIOENG., 30 C; Wallerstein laboratory (WL) nutrient agar, added with chloramphenicol (0.05 g mL1) to avoid bacterial growth, spread-plated and incubated aerobically for 72 h at 28 C for yeasts. All media and chemical components were purchased from Oxoid. Plate counts were performed in triplicate. Baking and analyses of bread attributes The ingredients were mixed by means of the mechanical mixer SilverCrest Bread Maker SBB 850 A1 (Kompernass GMBH, Bochum, Germany) applying the dough mode for 15 min and the resulting doughs (100 g) were placed into rectangular stainless steel baking pans (143 79 mm, top inside; 129 64 mm, bottom outside; depth inside 57 mm) as indicated by the Method 10-10B of the American Association of Cereal Chemists (18). At the end of fermentation, the doughs were baked in the oven Compact Combi (Electrolux, Pordenone, Italy) applying a 2-step program, including 5 min at 200 C with the combi cooking function, followed by 15 min at the same temperature with the convection heat function. After cooling at room temperature for 30 min, all breads were evaluated for their quality attributes. Firstly, the weight loss was determined and the breads were divided into two portions. The height of the two halves was evaluated as reported by Schober et al. (19) by caliper. The two central slices of each bread were then scanned (Epson Perfection 4180 Photo, Seiko Epson Corp., Japan) to perform the image analysis with the ImageJ software (National Institutes Health, Bethesda, MD, USA). Scansions were performed at 350 dpi of resolution and the resulting images were saved in TIFF format, cropped to a square of 207 207 pixels (representing 15 15 mm of the slice area) and converted to grey-level (8 bit). The Otsu’s threshold algorithm was applied and the following parameters were determined: void fraction, representing the fraction of the total area corresponding to the bread pores; cell density expressing the number of cells/cm2; and mean cell area in mm2. Crust color was determined on four points, while crumb color was evaluated on three points of the central slices with a Chroma Meter (CR-300; Minolta, Osaka, Japan). Lightness (L*), redness (a*), and yellowness (b*) of the Hunter’s scale were expressed according to the International Commission on Illumination (CIE) L*a*b* system. Bread firmness was determined by means of the Instron-5564 (Instron Corp., Canton, MA, USA). The breads were subjected to a load cell force of 50 kg and the results were expressed as maximum resistance to compression (compressive stress, N mm2). Sensory evaluation A panel of 14 judges, consisting of seven women and seven men aged between 22 and 65 years old, were specifically trained for bread attribute evaluation. The sensory attributes of the final breads were analyzed following the guidelines of the ISO 6658 (20). The judges were asked to evaluate 20 descriptors considering those suggested by Comendador et al. (21), Rodrigues et al. (22) and Martins et al. (23), including color and thickness of crust, color, porosity, alveolation, and alveolation uniformity for crumb, intensity, bread and unpleasant odor, intensity, bread and unpleasant aroma, salty, acid, astringent, bitter, taste persistency, adhesiveness in mouth, crispness and the overall assessment. The scores were given using a line scale anchored on the left (visual analogue scale) with dislike/low quality and on the right with like/high quality. The hedonic scale results were converted as distance (cm) of mark from the left end of the line. Preparation of extracts from O. ficus-indica mucilage, doughs, and breads Samples of mucilage as well as of both series (CTR without any addition, and MUC with added mucilage) of doughs and final breads were submitted to analysis. Doughs were analyzed at beginning (T0) and end of fermentation (T2). Two different 5 g aliquots of each sample were analyzed. Each aliquot was extracted for three times with 15 mL 70% ethanol and was subjected to stirring at retention time for 5 min in vortex. After a cleanup step via centrifugation (5000 g at 4 C for 10 min) and filtration (Millex HV 0.45 mm filter) the supernatants of each extraction cycle were collected and combined. Aliquots of each extracts were stored at 20 C until analysis. Total phenolic content analysis Total phenolic content (TPC) of extracts from O. ficus-indica mucilage and of both series (CTR and MUC) of doughs and breads was determined by the reduction of phosphotungstic-phosphomolybdic acid (FolinCiocalteu’s reagent) to blue pigments, in alkaline solution according to FolinCiocalteau’s method (24). Total phenolic content was expressed as mg gallic acid (GA) equivalents (GAE) per 100 g, dried weight (DW) for breads, and fresh weight (FW) for mucilage and doughs. All measurements were done in three replicates. Evaluation of antioxidant activities Antioxidant properties of extracts from mucilage, and control and fortified bread were evaluated using two antioxidant 0 capacity assays, the [2,2 -azinobis (3-ethylbenzothiazoline- 6-sulfonic acid)], (ABTS) radical cation decolorization assay (25) and the ferric reducing antioxidant potential (FRAP). ABTS assay ABTS assay was performed according to Re et al. (26). ABTSþ was prepared by reaction of ABTS with potassium persulfate. Samples were analyzed at five different dilutions, within the linearity range of the assay, as previous described by Gentile et al. (27). The calibration curve was constructed using 6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid (Trolox), an hydrophilic analog of vitamin E. The total antioxidant activity (TAA) was expressed as mmol Trolox equivalents (TE)/ 100 g1 DW or FW. All measurements were repeated twice. Ferric reducing antioxidant power Ferric reducing antioxidant power (FRAP) assay was performed according to Benzie and Strain (28). The FRAP reagent was prepared daily by mixing (8:1:1, v/v) 0.3 M acetate buffer pH 3.6, 10 mM 2,4,6-
Please cite this article as: Liguori, G et al., Effect of addition of Opuntia ficus-indica mucilage on the biological leavening, physical, nutritional, antioxidant and sensory aspects of bread, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.08.009
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tripyridyl-S-triazine (TPTZ) and 20 mM FeCl3. FRAP reagent (160 mL) were mixed with 30 mL of water and 10 mL of sample or standard. The absorbance at 595 nm was measured after 30-min incubation at 37 C. The calibration curve was constructed using Trolox. Ferric reducing power was expressed as mmol TE 100 g1 DW or FW. All measurements were repeated twice. Statistical analyses All data were tested for differences using the one-way analysis of variance (ANOVA; general linear model) followed by Tukey’s multiple range test for P 0.05 whereas Student’s t-test was used to determine the significance and the difference between control and O. ficus-indica mucilage enriched breads using XLStat add-in ver. 2014.5.03 (Addinsoft) for Microsoft Excel.
RESULTS AND DISCUSSION Antagonism test of O. ficus-indica mucilage Dhaouadi et al. (29) reported data on the antimicrobial properties of O. ficus-indica polyphenolic extract showing mainly antibacterial properties against some human pathogens, including Staphylococcus aureus, Staphylococcus epidermidis, Bacillus cereus, Pseudomonas aeruginosa, Escherichia coli and Salmonella spp. In our work, we tested O. ficus-indica mucilage against some representative sourdough LAB in view of its application during sourdough fermentation. Dhaouadi et al. (29) also tested O. ficus-indica polyphenolic extract against the yeast species Candida albicans, for this reason, in the present work, we better investigated on the ability of several yeasts, mainly of cereal origin, to grow in presence of O. ficus-indica mucilage for its addition in yeasted bread production. Of course, also a commercial starter S. cerevisiae strain was included in the assay. O. ficus-indica mucilage did not inhibit the growth of any of the yeast and LAB species used as target microorganisms for the inhibitory test (results not shown). These results confirmed the suitability of O. ficus-indica mucilage in both bread production typologies, yeasted and sourdough bread. Leavening process The fermentation process of the doughs prepared with and without O. ficus-indica mucilage was followed by acidification kinetics and microbiological evolution. As reported in Fig. 1, the initial pHs of CTR and MUC trials were quite distant with values of 5.9 and 5.3, respectively. Thus, the addition of the mucilage determined a relevant drop of pH. After 2 h of fermentation, the pH value decrease more consistently in CTR rather than MUC trial. Fig. 1 shows how TTA data were correlated linearly with those of pH. Also for this parameter, the highest differences during fermentation were shown by CTR trial with 0.8 mL NaOH 1 N of increase from 0 to 2 h, while barely 0.4 mL
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NaOH 1 N were registered in MUC trial. Other works reported a similar trend of TTA in dough samples supplemented with other ingredients that exert a buffering activity (30). The results of the cell counts for the microbial groups investigated are reported graphically in Fig. 2. The levels of TMM were almost superimposable to those of yeasts indicating the dominance of bakers’ yeasts from the beginning until the end of the fermentation process. In particular, yeasts were counted at 108 CFU g1 in both trials. These results clearly showed that O. ficus-indica mucilage supplementation did not negatively influence the development of the leavening agents. Regarding LAB, which are generally present in wheat flour (31), were found at similar levels of about 105 CFU g1 both on mMRS, used for the detection of generic food LAB, and on SDB, applied for the enumeration of sourdough LAB (13) at T0 as well as after 2 h, indicating that LAB did not interfere with the yeast fermentation. The contribution of indigenous yeasts in this work can be considered negligible, since the presence of yeasts in wheat and cactus cladodes composite flours are not reported to exceed 103 CFU g1 (32). Physical characteristics of breads The quality characteristics of the breads obtained from CTR and MUC trials are reported in Table 1. The height of the breads registered on the central slices were not statistically different for CTR and MUC trials. The height of the breads is linearly and directly proportional to volume increase (16). Thus, the substitution of water with O. ficus-indica mucilage did not affect the development of bread volume. This is different from what reported when O. ficus-indica powder was used; Shin and Lee (33) registered a decrease of the specific loaf volume of bread added with O. ficus-indica powder compared to the control bread. On the contrary, the weight baking loss was influenced by this operation, because MUC bread weight was significantly lower than CTR one. This result has to be related to the lower water content of the doughs of MUC trial, since O. ficus-indica mucilage contained 90% of water and, when added to the flour (246 g) in substitution to water (150 mL in CTR trial) the real amount of water was 135 mL. This also affected the firmness of the breads that was positively correlated with the mucilage supplementation and the resulting MUC bread was characterized by a higher firmness than CTR bread. Similar results were also showed by Ayadi et al. (34) who found that the firmness of doughs produced from mixtures of wheat flour and O. ficus-indica cladode powder was higher than that displayed by the control dough.
FIG. 1. Kinetics of acidification during fermentation. Closed symbols, pH. Open symbols, TTA. CTR, control, wheat flour; MUC, wheat flour and O. ficus-indica mucilage. Bars represent standard deviation of the mean.
Please cite this article as: Liguori, G et al., Effect of addition of Opuntia ficus-indica mucilage on the biological leavening, physical, nutritional, antioxidant and sensory aspects of bread, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.08.009
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FIG. 2. Microbiological counts during fermentation. (A) CTR, control wheat flour; (B) MUC, wheat flour and O. ficus-indica mucilage. PCA, plate count agar; mMRS, modified-de ManRogosa-Sharpe agar; SDB, sour dough bacteria agar; WL, Wallerstein laboratory nutrient agar. Results indicate mean values and standard deviation of three plate counts.
Crumb colour was more influenced than crust colour by the presence of the mucilage in the doughs. Regarding crust, only yellowness resulted different between the two trials, with the highest levels registered for CTR breads. In case of addition of cladode powder to produce cakes, Ayadi et al. (34) showed a decrease of L* and a* for crust and crumb color in comparison to the control trial. Shin and Lee (33) reported an increase in redness of crust and crumb of bread in presence of O. ficus-indica powder. The image analysis indicated that O. ficus-indica mucilage influenced only cell density with the lowest value showed by MUC. To our knowledge, no previous work focussed on these aspects when O. ficus-indica cladode powder was used to produce breads. However, Hrusková et al. (35) reported that cereal fortification causes the progressive change of bread volume and crumb morphology, including cell density.
Functional properties of O. ficus-indica mucilage and breads O. ficus-indica cladodes contain several potentially active ingredients. The content of fibers, mineral and phenolic compounds is particularly interesting (36). Cladodes powder was proposed as ingredient for fortified milk-based drinks, cereals, and bakery products (11,37). In particular, it was reported that a bread enriched with O. ficus-indica cladodes, by substitution of wheat flour with whole cladodes powder at 5% level, had acceptable sensorial attribute, and it also showed increased total phenolics content and antioxidant potential with respect to control bread (11). In the present work we evaluated mucilage from O. ficus indica cladodes as a new functional ingredient for enhancing nutraceutical properties of wheat bread. TPC and antioxidant activity of the O. ficus-indica mucilage used in our study are reported in Table 2. In the studied sample, TPC was
Please cite this article as: Liguori, G et al., Effect of addition of Opuntia ficus-indica mucilage on the biological leavening, physical, nutritional, antioxidant and sensory aspects of bread, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.08.009
Mean cell area (mm2)
13.06 0.49a 12.00 0.55a NS
37.33 0.78a 30.52 0.42b ***
Cell density (n.cm2)
1.01 0.13a 1.64 0.14b **
b* a* L*
69.49 2.48b 76.15 3.18a * 30.91 2.37a 23.34 2.94b *
b* a*
17.31 2.04a 15.17 1.68a NS 47.36 4.14a 41.71 3.49a NS 11.98 0.66b 13.80 0.85a * 43.33 1.07a 42.01 1.23a NS CTR MUC Statistical significance
Weight loss (g) Strain
Height (mm)
L*
16.19 0.67b 19.17 1.31a *
6.60 0.47b 9.06 0.31a **
Void fraction (%) Firmness value (N) Crumb color Crust color
TABLE 1. Characteristics of breads supplemented with Opuntia ficus-indica mucilage.
CTR, control wheat flour; MUC, wheat flour and O. ficus-indica mucilage. Results indicate mean values SD of four determinations (carried out in duplicate for two independent productions). Data within a column followed by the same letter are not significantly different according to Tukey’s test. P value: * P 0.05; ** P 0.01; *** P 0.001; NS, not significant. Data within a line followed by the same letter (a, b) are not significantly different according to Tukey’s test. L*, lightness; a*, redness; b*, yellowness.
QUALITY OF BREADS SUPPLEMENTED WITH CACTUS MUCILAGE 0.34 0.06a 0.39 0.07a NS
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lower than the value reported by Lanuzza et al. (38) for methanolic extract from whole cladodes. Differences can be due to the microwave cooking for mucilage preparation and then separation of the liquid mucilage from the solid part. In order to evaluate the intrinsic reducing capability of O. ficusindica mucilage, we employed in solution antioxidant assays. Since it has been reported that a single assay is not sufficient to predict the antioxidant potential of plant extracts and that the results from different assays can help to elucidate the mechanism involved in the observed activities (39), redox active properties were investigated through ABTS and FRAP assay. Antioxidant activity was higher in FRAP assay than in ABTS assay. However, the differences found between the two techniques might be imputable to the variability in the hydrophilicity of the reaction mixtures, as well as to the different ability of the antioxidant compounds to reduce ABTS free radicals and ferric ions (40). To evaluate the effects of mucilage inclusion into dough, we measured TPC in both dough and bread samples of both CTR and MUC series. Although we found polyphenols also in CTR dough, MUC dough showed a higher value. In particular, mucilage addition increased dough TPC about 2.6 times (Fig. 3). After mucilage incorporation in dough, contribution to TPC in MUC dough was close (76.86 mg GAE 100 g1 FW) to the value (74.89 mg GAE 100 g1 FW) predicted considering the TPC of mucilage and the amount of added mucilage (37.5 g) into 100 g of dough. Moreover, after two hours of fermentation the TPC did not change significantly in both CTR and MUC dough (Fig. 3). These results suggest that the dough matrix and the fermentation process do not affect the solubility and stability of antioxidant components of mucilage. In our results, TPC of both CTR and MUC dough was strongly affected by baking (Table 3). TPC had a 75% decrease in CTR bread and 35% decrease in MUC bread. In any case, after baking MUC bread showed a TPC seven times higher than CTR bread while the TPC in MUC bread was very close to the value (53.74 mg GAE 100 g1 FW) predicted considering the TPC of mucilage (Table 2) and the amount of added mucilage (37.5 g) to 100 g of dough. These results suggest that after backing the mucilage polyphenols are more resistant than those naturally present in wheat flour. Polyphenols possess a large variety of bioactive effects and are strong antioxidative molecules (41,42). On the other hand, bioactivity of plant chemicals, including polyphenols, has been especially related to their antioxidant properties, which are useful to prevent oxidative stress and also influence redox-dependent cell functions (43). In this study, the antioxidant activity of control and fortified bread was measured using both ABTS and FRAP assays. Similarly to mucilage extracts, bread extracts antioxidant activity was higher when obtained with FRAP assay than with ABTS (Table 3). Although CTR bread itself showed an antioxidant activity, a significant effect of the added mucilage was found. In particular, the inclusion of mucilage into dough produced in final bread an increase of antioxidant activity of about 1.6 and 2.3 times by ABTS and FRAP assay, respectively. These values were higher than the predicted ones (62.21 mmol TE 100 g1 DW and 989.12 mmol TE 100 g1 DW for ABTS and FRAP assay respectively) considering the antioxidant activity of mucilage and the amount added to dough. This discrepancy can be the result of a synergistic action between antioxidants from mucilage and those naturally present in dough.
TABLE 2. Total phenolic content (TPC), antioxidant activity by ABTS and FRAP assays of O. ficus-indica mucilage. TPC (mg GAE 100 g1 FW) 122.23 3.95
ABTS (mmol TE 100 g1 FW)
FRAP (mmol TE 100 g1 FW)
31.52 0.33
1.92 0.28
GAE, gallic acid equivalent; TE, Trolox equivalent. Results indicate mean values SD of three determinations.
Please cite this article as: Liguori, G et al., Effect of addition of Opuntia ficus-indica mucilage on the biological leavening, physical, nutritional, antioxidant and sensory aspects of bread, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.08.009
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FIG. 3. Total polyphenolic content (TPC) of extracts from CTR and MUC doughs at T0 and T2. TPC was determined by Folin-Ciocalteau method as reported in Materials and methods. Results indicate mean values and standard deviation of three determinations. Different lowercase letters indicate significant differences at P 0.05 as measured by Tukey’s multiple range test (P 0.0001).
TABLE 3. Total phenolic content (TPC), antioxidant activity by ABTS and FRAP assays of CTR and MUC baked breads. TPC (mg GAE 100 g1 DW)
Sample CTR MUC Statistical significance
b
7.92 1.51 54.72 4.92a ***
ABTS (mmol TE 100 g1 DW) b
50.40 1.70 83.63 0.68a ***
FRAP (mmol TE 100 g1 DW) 269.12 s4.07b 626.12 8.01a ***
CTR, control baked bread; MUC, baked bread with O. ficus-indica mucilage; GAE, gallic acid equivalent; TE, Trolox equivalent. Results indicate mean values SD of three determinations. Within the same series, TP, ABTS or FRAP; three asterisks indicate significant differences with respect control bread (CTR) as measured by Student’s t-test (P 0.001). Data within a line followed by the same letter (a, b) are not significantly different according to Tukey’s test.
FIG. 4. Spider diagrams of descriptive sensory analysis of breads. CTR, control wheat flour; MUC, wheat flour and O. ficus-indica mucilage.
Please cite this article as: Liguori, G et al., Effect of addition of Opuntia ficus-indica mucilage on the biological leavening, physical, nutritional, antioxidant and sensory aspects of bread, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.08.009
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QUALITY OF BREADS SUPPLEMENTED WITH CACTUS MUCILAGE
On the other hand, obtained results can be influenced by the formation of Maillard reaction products typical of the baking process. Sensory attributes of breads The spider plot reported in Fig. 4 shows the sensory evaluation of the final breads by the judges. Except taste persistence, salty and astringent parameters, all other attributes were evaluated as particularly different between the bread supplemented with O. ficus-indica mucilage and that processed from wheat flour only. The highest scores were registered for crust color and odor intensity of MUC trial. Also aroma intensity of MUC trial was higher than that measured in the control dough. Porosity, alveolation and bread odor were registered at levels much higher in CTR trial. Although the addition of the mucilage determined a quite different sensory profile of the resulting bread, the overall assessment, intended as an overall rating of the breads expressed considering all parameters with their levels of evaluation, of MUC bread was only slightly lower than that reached by CTR bread indicating a certain appreciation of the novel bread. Moreno-Álvarez et al. (32) who evaluated the sensory impact of breads produced from wheat and O. ficus-indica cladodes composite flours reported a general appreciation by the judges. Our results revealed that the substitution of water with liquid O. ficus-indica mucilage did not affect the fermentation process and the food quality attributes of bread while also attaining an acceptable quality evaluation from the sensory panel. Moreover, mucilage addition enriched bread with bioactive components, strongly increasing its health properties. In conclusion, considering the popularity of bread in human diet worldwide, the use of mucilage in this bakery product could really benefit human nutrition contributing to increase the dietary antioxidant intake. ACKNOWLEDGMENTS This research did not receive any specific grant from funding agencies, but it was partially financed with the personal research funds of Luca Settanni provided from University of Palermo (FFR_D13_2018/2021). The authors declare no conflict of interest. References 1. Sanguansri, L. and Augustin, M. A.: Microencapsulation in functional food product development, pp. 295e317, in: Smith, J. and Charter, E. (Eds.), Functional food product development. Wiley-Blackwell, Hoboken (2010). 2. Martirosyan, D. M. and Singh, J.: A new definition of functional food by FFC: what makes a new definition unique? Funct. Food Health Dis., 5, 209e223 (2015). 3. Gaglio, R., Gentile, C., Bonanno, A., Vintaloro, L., Perrone, A., Mazza, F., Barbaccia, P., Settanni, L., and Di Grigoli, A.: Effect of saffron addition on the microbiological, physicochemical, antioxidant and sensory characteristics of yoghurt, Int. J. Dairy Technol., 72, 208e217 (2019). 4. Sáenz, C., Sepúlveda, E., and Matsuhiro, B.: Opuntia spp. mucilage’s: a functional component with industrial perspectives, J. Arid Environ., 57, 275e290 (2004). 5. Nazareno, M. A.: Nutritional properties and medicinal derivatives of fruits and cladodes, pp. 152e158, in: Inglese, P., Mondragon, C., Nefzaoui, A. and Saen, C. (Eds.), Crop ecology, cultivation and uses of cactus pear. Food and Agriculture Organization of the United Nations and the International Center for Agricultural Research in the Dry Areas, Rome (2017). 6. Ginestra, G., Parker, M. L., Bennett, R. N., Robertson, J., Mandalari, G., and Narbad, A.: Anatomical, chemical, and biochemical characterization of cladodes from prickly pear [Opuntia ficus-indica (L.) Mill.], J. Agric. Food Chem., 57, 10323e10330 (2009). 7. Sepúlveda, E., Sáenz, C., Aliaga, E., and Aceituno, C.: Extraction and characterization of mucilage in Opuntia spp. J. Arid Environ., 68, 534e545 (2007). 8. Madjdoub, H., Rousdeli, S., and Dertani, A.: Polysaccharides from prickly pear and nopals of Opuntia ficus-indica: extraction, characterization and polyelectrolyte behavior, Polym. Int., 50, 552e560 (2001). 9. Altunkaya, A., Hedegaard, R. V., Brimer, L., Gökmen, V., and Skibsted, L. H.: Antioxidant capacity versus chemical safety of wheat bread enriched with pomegranate peel powder, Food Funct., 30, 722e727 (2013).
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Please cite this article as: Liguori, G et al., Effect of addition of Opuntia ficus-indica mucilage on the biological leavening, physical, nutritional, antioxidant and sensory aspects of bread, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.08.009
Effect of addition of Opuntia ficus-indica mucilage on the biological leavening, physical, nutritional, antioxidant and sensory aspects of bread Giorgia Liguori,1 Carla Gentile,2 Raimondo Gaglio,1 Anna Perrone,2 Rosa Guarcello,1 Nicola Francesca,1 Silvia Fretto,1 Paolo Inglese,1 and Luca Settanni1, * Department of Agricultural, Food and Forest Sciences, University of Palermo, Viale delle Scienze, 90128 Palermo, Italy1 and Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, University of Palermo, Viale delle Scienze, 90128 Palermo, Italy2 Received 3 June 2019; accepted 20 August 2019 Available online xxx
The addition of active compounds to enhance the functional properties of foods is a quite common practice. Recently, bread became one of the target foods to incorporate functional ingredients such as those deriving from Opuntia spp. So far, only Opuntia ficus-indica cladodes in powder has been tested. The addition of fresh O. ficus-indica mucilage (in substitution to water) did not influence the biological leavening of the doughs. The resulting breads showed a biological role of the cactus mucilage, because their antioxidant activity was higher than that of control wheat bread. The sensory analysis indicated a general appreciation of the breads enriched with O. ficus-indica mucilage by the judges. The inclusion of fresh cactus mucilage in bread production might increase the dietary antioxidant intake due to its daily worldwide consumption. Ó 2019, The Society for Biotechnology, Japan. All rights reserved. [Key words: Antimicrobial activity; Antioxidant activity; Biological leavening; Enriched bread; Cactus mucilage; Yeasts]
Functional foods provide health benefits. They represent one of the food sector that is growing more and more rapidly due to consumers’ increasing demand for foods that promote health and well-being (1). One of the first definition of functional foods, coined in Japan, was: “Food products fortified with special constituents that possess advantageous physiological effects” (2). Although most bioactive compounds are derived from plants (1), only a few species have been used to improve food healthy properties (3). Indeed, several factors affect the suitability of bioactive compounds pure or in mixture to be used as ingredients of functional foods. The in vivo efficacy of functionalizing agents depends on the doses added to the foods and the food matrices might affect the stability and/or bioavailability of the bioactive compounds. On the other hand, plant extracts could affect food sensory characteristics producing undesirable odors or tastes as well as negatively impacting the final texture (1). Opuntia ficus-indica (L.) Mill. is a drought-tolerant cactus, widely cultivated in semi-arid and arid regions worldwide. Several research showed that cactus pear fruits and cladodes could be used as sources of nutrients and phytochemicals for applications in food, pharmaceutical and nutraceutical industries (4). The major components of cladodes are carbohydratecontaining polymers, including a mixture of mucilage and pectin. Cladodes represent also a source of phytochemicals, in particular phenolic compounds (5). Mucilage is part of dietary fiber and generally refers to some part of fruits, vegetables, grains,
* Corresponding author. Tel.: þ39 091 23896043; fax: þ39 091 6515531. E-mail address: [email protected] (L. Settanni).
nuts and legumes (4). O. ficus-indica cladodes mucilage, due to its viscosity and chemical composition, is an interesting ingredient for food industry (6) that can be found in specialized storage cells or free within cells or intracellular spaces of the chlorenchymatic and parenchymatic tissue of the cladodes (4). Mucilage is a polysaccharide and based on its chemical composition, it is considered a polymer (similar to pectin), composed of arabinose, galactose, xylose and rhamnose as neutral sugars, and of a small amount of galacturonic acid (5e8). Opuntia cladodes are traditionally used as healthy nutrient and in folk medicine in South and Central America and North Africa but are scarcely used by the modern food and pharmaceutical industries. Bread from refined wheat flour is regularly eaten in significant quantities, as it is one of the main components of the human diet worldwide. Despite its nutritional value, with a high content of complex sugars and a very low lipid content, the nutraceutical value of this widely consumed product is scarce. On the other hand, bread, due to its wide consumption could be a good carrier for functional molecules. Fortified bread offer the possibility to introduce substances with health-enhancing properties through diet. Several studies evaluated the potential use of bread for delivering antioxidant molecules in particular by addition of natural raw materials rich in phenolic compounds (9,10). However, some studies were conducted on bread enrichment with Opuntia cladodes (11) but no studies focused on the effect of O. ficus-indica mucilage addition on bread and on its potential use as a new functional ingredient. The aim of this study was to evaluate the effect of addition of O. ficus-indica mucilage on the biological leavening, physical, nutritional, antioxidant and sensory aspects of bread.
1389-1723/$ e see front matter Ó 2019, The Society for Biotechnology, Japan. All rights reserved. https://doi.org/10.1016/j.jbiosc.2019.08.009
Please cite this article as: Liguori, G et al., Effect of addition of Opuntia ficus-indica mucilage on the biological leavening, physical, nutritional, antioxidant and sensory aspects of bread, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.08.009
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LIGUORI ET AL. MATERIALS AND METHODS
Fresh mucilage extraction and evaluation of antifungal activity Oneyear-old cladodes were collected from 4-year-old O. ficus-indica potted plants of the cultivar “Gialla”, located at Department of Agricultural, Food and Forest Sciences, University of Palermo (38 70 4.080000 N 13 220 11.280000 E, 29 m a.s.l). Three cladodes (one-year-old cladodes) were harvested from the same plant for mucilage extraction. Harvested cladodes were marked, packaged and transported to the laboratory where they were measured, weighed and processed for mucilage extraction, using a modified patented method of Du Toit et al. (12) developed in South Africa. Cladodes were washed with chlorinated water to improve mucilage shelf life and to remove impurities and spines. Cladodes chlorenchyma was removed with a peeler to obtain high pure quality mucilage. Cladodes were then sliced into squares and cooked in a microwave oven (900 W) for 3e5 min, until soft. The cooked, soft cladode pieces were then mixed using an Omni Mixer Homogenizer (mod. OmniMixer. 17,107, Dupont Instruments Sorvall, USA) to aid the mucilage extraction. The obtained pulp was then centrifuged using a Sigma centrifuge (mod. 6K15, Sigma Laborzentrifugen GmbH, Germany) at 8800 g for 15 min at 4 C, to separate the liquid mucilage from the solids. The mucilage was then decanted and weighed while the solid material left in the falcon tubes were discarded. No chemicals have been used during this extraction process and as such, the extracted mucilage obtained is natural and unadulterated by chemicals. The suitability of mucilage for the production of breads subjected to biological leavening depends on its harmlessness towards yeasts and lactic acid bacteria (LAB) which are the main agents of fermentation in yeasted and sourdough breads, respectively. For this reason, O. ficus-indica mucilage was tested for the potential inhibitory activity on several yeasts, mainly of cereal origin, in order to enlarge the knowledge on the antagonistic properties against unicellular fungi and versus the most relevant LAB species used for sourdough production (13,14). To this purpose, the yeast indicators were Aureobasidium pullulans AD14, Candida parapsilosis AD29, Cryptococcus aerius AD62, Cryptococcus carnesciens AD56, Cryptococcus fonsecae AD245, Cryptococcus flavescens AD17, Cryptococcus magnus AD122, Cryptococcus tephrensis AD157, Cryptococcus victoriae AD85; Filobasidum chernovii AD172; Filobasidum oeirense AD82; Holtermanniella festucosa AD413, Rhodosporidium babjevae AD224, Rhodosporidium toruloides AD258, Rhodotorula glutinis AD407, Rhodotorula mucilaginosa AD284, Saccharomyces cerevisiae Commercial (isolated from commercial baker’s yeast La Parisienne, AB Mauri Italy S.p.A., Casteggio, Italy), Sporidiobolus metaroseus AD244, and Sporobolomyces roseus AD397, while sourdough LAB were represented by Lactobacillus brevis SD46, Lactobacillus plantarum SD130, L. plantarum SD96, Lactobacillus sanfranciscensis SD22, Leuconostoc citreum SD142 and Weissella cibaria SD123. All strains except S. cerevisiae commercial belonged to the culture collection of the Agricultural Microbiology laboratory of the Department of Agricultural, Food and Forest Sciences, University of Palermo (Palermo, Italy). All yeast strains were grown overnight at 28 C in yeast peptone dextrose (YPD) broth (Oxoid, Milan, Italy). LAB were propagated overnight at 30 C in different media: lactobacilli in sourdough bacteria (SDB) broth prepared as described by Kline and Sugihara (15), while Leuconostoc citreum and W. cibaria in modified-de Man-RogosaSharpe (mMRS) agar, prepared as reported by Corona et al. (16). Freeze-dried O. ficus-indica mucilage powder, obtained after 48 h in a ScanVac CoolSafe 4e15 L freeze-dryer at 100 C, was re-suspended in water at 200 mg mL1 as commonly performed for extracts of botanical origin (17) and tested by well diffusion assay. A water agar base medium (1.5% agar) was overlaid with soft agar YPD containing the yeast strains at a cell density of approximately 105 CFU mL1. Circular wells were obtained by means of a sterile cork borer and filled in with 100 mL of O. ficus-indica mucilage solution. Production of doughs The doughs were processed from wheat flour type “0” SISA Manitoba (Molino Rossetto, Pontelongo, Italy) and O. ficus-indica (90% of water) mucilage. Two dough trials were obtained by yeast leavening: CTR, control dough prepared exclusively from wheat flour; MUC, dough prepared from wheat flour and O. ficus-indica mucilage in place of water. Doughs of 406 g were obtained from 246 g of wheat flour, 150 mL of tap water (CTR trial) or 150 mL O. ficus-indica mucilage (MUC trial), 4 g of baker’s yeast (Lesaffre Italia S.p.A., Trecasali, Italy) and 6 g of NaCl. The fermentation was performed at 28 C for 2 h. All productions were carried out in duplicate and repeated in two consecutive weeks for a total of eight replicates (breads) for each trial. Monitoring of acidification and biological leavening The acidification process and the levels of the main fermenting microorganisms were analyzed at beginning (T0) and end of fermentation (T2). The monitoring of the acidification was performed by measuring pH using the pH meter Russell RL060P (Thermo Fisher Scientific, Beverly, MA, USA) and total titratable acidity (TTA), determined by titratation with 0.1 N NaOH (mL of NaOH 10 g1 of dough). The microbiological analyses were performed on 10 g of each dough. The samples were diluted (1:10) in Ringer’s solution (Oxoid) through homogenization carried out by stomacher (BagMixer 400, Interscience, Saint Nom, France) and then serially diluted. The following media and growth conditions were used to determine the different microbial groups from the cell suspensions: plate count agar (PCA), incubated aerobically for 72 h at 30 C for total mesophilic microorganisms (TMM); mMRS agar and SDB agar, incubated anaerobically and aerobically, respectively, were used to detect LAB, after 48 h at
J. BIOSCI. BIOENG., 30 C; Wallerstein laboratory (WL) nutrient agar, added with chloramphenicol (0.05 g mL1) to avoid bacterial growth, spread-plated and incubated aerobically for 72 h at 28 C for yeasts. All media and chemical components were purchased from Oxoid. Plate counts were performed in triplicate. Baking and analyses of bread attributes The ingredients were mixed by means of the mechanical mixer SilverCrest Bread Maker SBB 850 A1 (Kompernass GMBH, Bochum, Germany) applying the dough mode for 15 min and the resulting doughs (100 g) were placed into rectangular stainless steel baking pans (143 79 mm, top inside; 129 64 mm, bottom outside; depth inside 57 mm) as indicated by the Method 10-10B of the American Association of Cereal Chemists (18). At the end of fermentation, the doughs were baked in the oven Compact Combi (Electrolux, Pordenone, Italy) applying a 2-step program, including 5 min at 200 C with the combi cooking function, followed by 15 min at the same temperature with the convection heat function. After cooling at room temperature for 30 min, all breads were evaluated for their quality attributes. Firstly, the weight loss was determined and the breads were divided into two portions. The height of the two halves was evaluated as reported by Schober et al. (19) by caliper. The two central slices of each bread were then scanned (Epson Perfection 4180 Photo, Seiko Epson Corp., Japan) to perform the image analysis with the ImageJ software (National Institutes Health, Bethesda, MD, USA). Scansions were performed at 350 dpi of resolution and the resulting images were saved in TIFF format, cropped to a square of 207 207 pixels (representing 15 15 mm of the slice area) and converted to grey-level (8 bit). The Otsu’s threshold algorithm was applied and the following parameters were determined: void fraction, representing the fraction of the total area corresponding to the bread pores; cell density expressing the number of cells/cm2; and mean cell area in mm2. Crust color was determined on four points, while crumb color was evaluated on three points of the central slices with a Chroma Meter (CR-300; Minolta, Osaka, Japan). Lightness (L*), redness (a*), and yellowness (b*) of the Hunter’s scale were expressed according to the International Commission on Illumination (CIE) L*a*b* system. Bread firmness was determined by means of the Instron-5564 (Instron Corp., Canton, MA, USA). The breads were subjected to a load cell force of 50 kg and the results were expressed as maximum resistance to compression (compressive stress, N mm2). Sensory evaluation A panel of 14 judges, consisting of seven women and seven men aged between 22 and 65 years old, were specifically trained for bread attribute evaluation. The sensory attributes of the final breads were analyzed following the guidelines of the ISO 6658 (20). The judges were asked to evaluate 20 descriptors considering those suggested by Comendador et al. (21), Rodrigues et al. (22) and Martins et al. (23), including color and thickness of crust, color, porosity, alveolation, and alveolation uniformity for crumb, intensity, bread and unpleasant odor, intensity, bread and unpleasant aroma, salty, acid, astringent, bitter, taste persistency, adhesiveness in mouth, crispness and the overall assessment. The scores were given using a line scale anchored on the left (visual analogue scale) with dislike/low quality and on the right with like/high quality. The hedonic scale results were converted as distance (cm) of mark from the left end of the line. Preparation of extracts from O. ficus-indica mucilage, doughs, and breads Samples of mucilage as well as of both series (CTR without any addition, and MUC with added mucilage) of doughs and final breads were submitted to analysis. Doughs were analyzed at beginning (T0) and end of fermentation (T2). Two different 5 g aliquots of each sample were analyzed. Each aliquot was extracted for three times with 15 mL 70% ethanol and was subjected to stirring at retention time for 5 min in vortex. After a cleanup step via centrifugation (5000 g at 4 C for 10 min) and filtration (Millex HV 0.45 mm filter) the supernatants of each extraction cycle were collected and combined. Aliquots of each extracts were stored at 20 C until analysis. Total phenolic content analysis Total phenolic content (TPC) of extracts from O. ficus-indica mucilage and of both series (CTR and MUC) of doughs and breads was determined by the reduction of phosphotungstic-phosphomolybdic acid (FolinCiocalteu’s reagent) to blue pigments, in alkaline solution according to FolinCiocalteau’s method (24). Total phenolic content was expressed as mg gallic acid (GA) equivalents (GAE) per 100 g, dried weight (DW) for breads, and fresh weight (FW) for mucilage and doughs. All measurements were done in three replicates. Evaluation of antioxidant activities Antioxidant properties of extracts from mucilage, and control and fortified bread were evaluated using two antioxidant 0 capacity assays, the [2,2 -azinobis (3-ethylbenzothiazoline- 6-sulfonic acid)], (ABTS) radical cation decolorization assay (25) and the ferric reducing antioxidant potential (FRAP). ABTS assay ABTS assay was performed according to Re et al. (26). ABTSþ was prepared by reaction of ABTS with potassium persulfate. Samples were analyzed at five different dilutions, within the linearity range of the assay, as previous described by Gentile et al. (27). The calibration curve was constructed using 6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid (Trolox), an hydrophilic analog of vitamin E. The total antioxidant activity (TAA) was expressed as mmol Trolox equivalents (TE)/ 100 g1 DW or FW. All measurements were repeated twice. Ferric reducing antioxidant power Ferric reducing antioxidant power (FRAP) assay was performed according to Benzie and Strain (28). The FRAP reagent was prepared daily by mixing (8:1:1, v/v) 0.3 M acetate buffer pH 3.6, 10 mM 2,4,6-
Please cite this article as: Liguori, G et al., Effect of addition of Opuntia ficus-indica mucilage on the biological leavening, physical, nutritional, antioxidant and sensory aspects of bread, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.08.009
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tripyridyl-S-triazine (TPTZ) and 20 mM FeCl3. FRAP reagent (160 mL) were mixed with 30 mL of water and 10 mL of sample or standard. The absorbance at 595 nm was measured after 30-min incubation at 37 C. The calibration curve was constructed using Trolox. Ferric reducing power was expressed as mmol TE 100 g1 DW or FW. All measurements were repeated twice. Statistical analyses All data were tested for differences using the one-way analysis of variance (ANOVA; general linear model) followed by Tukey’s multiple range test for P 0.05 whereas Student’s t-test was used to determine the significance and the difference between control and O. ficus-indica mucilage enriched breads using XLStat add-in ver. 2014.5.03 (Addinsoft) for Microsoft Excel.
RESULTS AND DISCUSSION Antagonism test of O. ficus-indica mucilage Dhaouadi et al. (29) reported data on the antimicrobial properties of O. ficus-indica polyphenolic extract showing mainly antibacterial properties against some human pathogens, including Staphylococcus aureus, Staphylococcus epidermidis, Bacillus cereus, Pseudomonas aeruginosa, Escherichia coli and Salmonella spp. In our work, we tested O. ficus-indica mucilage against some representative sourdough LAB in view of its application during sourdough fermentation. Dhaouadi et al. (29) also tested O. ficus-indica polyphenolic extract against the yeast species Candida albicans, for this reason, in the present work, we better investigated on the ability of several yeasts, mainly of cereal origin, to grow in presence of O. ficus-indica mucilage for its addition in yeasted bread production. Of course, also a commercial starter S. cerevisiae strain was included in the assay. O. ficus-indica mucilage did not inhibit the growth of any of the yeast and LAB species used as target microorganisms for the inhibitory test (results not shown). These results confirmed the suitability of O. ficus-indica mucilage in both bread production typologies, yeasted and sourdough bread. Leavening process The fermentation process of the doughs prepared with and without O. ficus-indica mucilage was followed by acidification kinetics and microbiological evolution. As reported in Fig. 1, the initial pHs of CTR and MUC trials were quite distant with values of 5.9 and 5.3, respectively. Thus, the addition of the mucilage determined a relevant drop of pH. After 2 h of fermentation, the pH value decrease more consistently in CTR rather than MUC trial. Fig. 1 shows how TTA data were correlated linearly with those of pH. Also for this parameter, the highest differences during fermentation were shown by CTR trial with 0.8 mL NaOH 1 N of increase from 0 to 2 h, while barely 0.4 mL
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NaOH 1 N were registered in MUC trial. Other works reported a similar trend of TTA in dough samples supplemented with other ingredients that exert a buffering activity (30). The results of the cell counts for the microbial groups investigated are reported graphically in Fig. 2. The levels of TMM were almost superimposable to those of yeasts indicating the dominance of bakers’ yeasts from the beginning until the end of the fermentation process. In particular, yeasts were counted at 108 CFU g1 in both trials. These results clearly showed that O. ficus-indica mucilage supplementation did not negatively influence the development of the leavening agents. Regarding LAB, which are generally present in wheat flour (31), were found at similar levels of about 105 CFU g1 both on mMRS, used for the detection of generic food LAB, and on SDB, applied for the enumeration of sourdough LAB (13) at T0 as well as after 2 h, indicating that LAB did not interfere with the yeast fermentation. The contribution of indigenous yeasts in this work can be considered negligible, since the presence of yeasts in wheat and cactus cladodes composite flours are not reported to exceed 103 CFU g1 (32). Physical characteristics of breads The quality characteristics of the breads obtained from CTR and MUC trials are reported in Table 1. The height of the breads registered on the central slices were not statistically different for CTR and MUC trials. The height of the breads is linearly and directly proportional to volume increase (16). Thus, the substitution of water with O. ficus-indica mucilage did not affect the development of bread volume. This is different from what reported when O. ficus-indica powder was used; Shin and Lee (33) registered a decrease of the specific loaf volume of bread added with O. ficus-indica powder compared to the control bread. On the contrary, the weight baking loss was influenced by this operation, because MUC bread weight was significantly lower than CTR one. This result has to be related to the lower water content of the doughs of MUC trial, since O. ficus-indica mucilage contained 90% of water and, when added to the flour (246 g) in substitution to water (150 mL in CTR trial) the real amount of water was 135 mL. This also affected the firmness of the breads that was positively correlated with the mucilage supplementation and the resulting MUC bread was characterized by a higher firmness than CTR bread. Similar results were also showed by Ayadi et al. (34) who found that the firmness of doughs produced from mixtures of wheat flour and O. ficus-indica cladode powder was higher than that displayed by the control dough.
FIG. 1. Kinetics of acidification during fermentation. Closed symbols, pH. Open symbols, TTA. CTR, control, wheat flour; MUC, wheat flour and O. ficus-indica mucilage. Bars represent standard deviation of the mean.
Please cite this article as: Liguori, G et al., Effect of addition of Opuntia ficus-indica mucilage on the biological leavening, physical, nutritional, antioxidant and sensory aspects of bread, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.08.009
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FIG. 2. Microbiological counts during fermentation. (A) CTR, control wheat flour; (B) MUC, wheat flour and O. ficus-indica mucilage. PCA, plate count agar; mMRS, modified-de ManRogosa-Sharpe agar; SDB, sour dough bacteria agar; WL, Wallerstein laboratory nutrient agar. Results indicate mean values and standard deviation of three plate counts.
Crumb colour was more influenced than crust colour by the presence of the mucilage in the doughs. Regarding crust, only yellowness resulted different between the two trials, with the highest levels registered for CTR breads. In case of addition of cladode powder to produce cakes, Ayadi et al. (34) showed a decrease of L* and a* for crust and crumb color in comparison to the control trial. Shin and Lee (33) reported an increase in redness of crust and crumb of bread in presence of O. ficus-indica powder. The image analysis indicated that O. ficus-indica mucilage influenced only cell density with the lowest value showed by MUC. To our knowledge, no previous work focussed on these aspects when O. ficus-indica cladode powder was used to produce breads. However, Hrusková et al. (35) reported that cereal fortification causes the progressive change of bread volume and crumb morphology, including cell density.
Functional properties of O. ficus-indica mucilage and breads O. ficus-indica cladodes contain several potentially active ingredients. The content of fibers, mineral and phenolic compounds is particularly interesting (36). Cladodes powder was proposed as ingredient for fortified milk-based drinks, cereals, and bakery products (11,37). In particular, it was reported that a bread enriched with O. ficus-indica cladodes, by substitution of wheat flour with whole cladodes powder at 5% level, had acceptable sensorial attribute, and it also showed increased total phenolics content and antioxidant potential with respect to control bread (11). In the present work we evaluated mucilage from O. ficus indica cladodes as a new functional ingredient for enhancing nutraceutical properties of wheat bread. TPC and antioxidant activity of the O. ficus-indica mucilage used in our study are reported in Table 2. In the studied sample, TPC was
Please cite this article as: Liguori, G et al., Effect of addition of Opuntia ficus-indica mucilage on the biological leavening, physical, nutritional, antioxidant and sensory aspects of bread, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.08.009
Mean cell area (mm2)
13.06 0.49a 12.00 0.55a NS
37.33 0.78a 30.52 0.42b ***
Cell density (n.cm2)
1.01 0.13a 1.64 0.14b **
b* a* L*
69.49 2.48b 76.15 3.18a * 30.91 2.37a 23.34 2.94b *
b* a*
17.31 2.04a 15.17 1.68a NS 47.36 4.14a 41.71 3.49a NS 11.98 0.66b 13.80 0.85a * 43.33 1.07a 42.01 1.23a NS CTR MUC Statistical significance
Weight loss (g) Strain
Height (mm)
L*
16.19 0.67b 19.17 1.31a *
6.60 0.47b 9.06 0.31a **
Void fraction (%) Firmness value (N) Crumb color Crust color
TABLE 1. Characteristics of breads supplemented with Opuntia ficus-indica mucilage.
CTR, control wheat flour; MUC, wheat flour and O. ficus-indica mucilage. Results indicate mean values SD of four determinations (carried out in duplicate for two independent productions). Data within a column followed by the same letter are not significantly different according to Tukey’s test. P value: * P 0.05; ** P 0.01; *** P 0.001; NS, not significant. Data within a line followed by the same letter (a, b) are not significantly different according to Tukey’s test. L*, lightness; a*, redness; b*, yellowness.
QUALITY OF BREADS SUPPLEMENTED WITH CACTUS MUCILAGE 0.34 0.06a 0.39 0.07a NS
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lower than the value reported by Lanuzza et al. (38) for methanolic extract from whole cladodes. Differences can be due to the microwave cooking for mucilage preparation and then separation of the liquid mucilage from the solid part. In order to evaluate the intrinsic reducing capability of O. ficusindica mucilage, we employed in solution antioxidant assays. Since it has been reported that a single assay is not sufficient to predict the antioxidant potential of plant extracts and that the results from different assays can help to elucidate the mechanism involved in the observed activities (39), redox active properties were investigated through ABTS and FRAP assay. Antioxidant activity was higher in FRAP assay than in ABTS assay. However, the differences found between the two techniques might be imputable to the variability in the hydrophilicity of the reaction mixtures, as well as to the different ability of the antioxidant compounds to reduce ABTS free radicals and ferric ions (40). To evaluate the effects of mucilage inclusion into dough, we measured TPC in both dough and bread samples of both CTR and MUC series. Although we found polyphenols also in CTR dough, MUC dough showed a higher value. In particular, mucilage addition increased dough TPC about 2.6 times (Fig. 3). After mucilage incorporation in dough, contribution to TPC in MUC dough was close (76.86 mg GAE 100 g1 FW) to the value (74.89 mg GAE 100 g1 FW) predicted considering the TPC of mucilage and the amount of added mucilage (37.5 g) into 100 g of dough. Moreover, after two hours of fermentation the TPC did not change significantly in both CTR and MUC dough (Fig. 3). These results suggest that the dough matrix and the fermentation process do not affect the solubility and stability of antioxidant components of mucilage. In our results, TPC of both CTR and MUC dough was strongly affected by baking (Table 3). TPC had a 75% decrease in CTR bread and 35% decrease in MUC bread. In any case, after baking MUC bread showed a TPC seven times higher than CTR bread while the TPC in MUC bread was very close to the value (53.74 mg GAE 100 g1 FW) predicted considering the TPC of mucilage (Table 2) and the amount of added mucilage (37.5 g) to 100 g of dough. These results suggest that after backing the mucilage polyphenols are more resistant than those naturally present in wheat flour. Polyphenols possess a large variety of bioactive effects and are strong antioxidative molecules (41,42). On the other hand, bioactivity of plant chemicals, including polyphenols, has been especially related to their antioxidant properties, which are useful to prevent oxidative stress and also influence redox-dependent cell functions (43). In this study, the antioxidant activity of control and fortified bread was measured using both ABTS and FRAP assays. Similarly to mucilage extracts, bread extracts antioxidant activity was higher when obtained with FRAP assay than with ABTS (Table 3). Although CTR bread itself showed an antioxidant activity, a significant effect of the added mucilage was found. In particular, the inclusion of mucilage into dough produced in final bread an increase of antioxidant activity of about 1.6 and 2.3 times by ABTS and FRAP assay, respectively. These values were higher than the predicted ones (62.21 mmol TE 100 g1 DW and 989.12 mmol TE 100 g1 DW for ABTS and FRAP assay respectively) considering the antioxidant activity of mucilage and the amount added to dough. This discrepancy can be the result of a synergistic action between antioxidants from mucilage and those naturally present in dough.
TABLE 2. Total phenolic content (TPC), antioxidant activity by ABTS and FRAP assays of O. ficus-indica mucilage. TPC (mg GAE 100 g1 FW) 122.23 3.95
ABTS (mmol TE 100 g1 FW)
FRAP (mmol TE 100 g1 FW)
31.52 0.33
1.92 0.28
GAE, gallic acid equivalent; TE, Trolox equivalent. Results indicate mean values SD of three determinations.
Please cite this article as: Liguori, G et al., Effect of addition of Opuntia ficus-indica mucilage on the biological leavening, physical, nutritional, antioxidant and sensory aspects of bread, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.08.009
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FIG. 3. Total polyphenolic content (TPC) of extracts from CTR and MUC doughs at T0 and T2. TPC was determined by Folin-Ciocalteau method as reported in Materials and methods. Results indicate mean values and standard deviation of three determinations. Different lowercase letters indicate significant differences at P 0.05 as measured by Tukey’s multiple range test (P 0.0001).
TABLE 3. Total phenolic content (TPC), antioxidant activity by ABTS and FRAP assays of CTR and MUC baked breads. TPC (mg GAE 100 g1 DW)
Sample CTR MUC Statistical significance
b
7.92 1.51 54.72 4.92a ***
ABTS (mmol TE 100 g1 DW) b
50.40 1.70 83.63 0.68a ***
FRAP (mmol TE 100 g1 DW) 269.12 s4.07b 626.12 8.01a ***
CTR, control baked bread; MUC, baked bread with O. ficus-indica mucilage; GAE, gallic acid equivalent; TE, Trolox equivalent. Results indicate mean values SD of three determinations. Within the same series, TP, ABTS or FRAP; three asterisks indicate significant differences with respect control bread (CTR) as measured by Student’s t-test (P 0.001). Data within a line followed by the same letter (a, b) are not significantly different according to Tukey’s test.
FIG. 4. Spider diagrams of descriptive sensory analysis of breads. CTR, control wheat flour; MUC, wheat flour and O. ficus-indica mucilage.
Please cite this article as: Liguori, G et al., Effect of addition of Opuntia ficus-indica mucilage on the biological leavening, physical, nutritional, antioxidant and sensory aspects of bread, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.08.009
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On the other hand, obtained results can be influenced by the formation of Maillard reaction products typical of the baking process. Sensory attributes of breads The spider plot reported in Fig. 4 shows the sensory evaluation of the final breads by the judges. Except taste persistence, salty and astringent parameters, all other attributes were evaluated as particularly different between the bread supplemented with O. ficus-indica mucilage and that processed from wheat flour only. The highest scores were registered for crust color and odor intensity of MUC trial. Also aroma intensity of MUC trial was higher than that measured in the control dough. Porosity, alveolation and bread odor were registered at levels much higher in CTR trial. Although the addition of the mucilage determined a quite different sensory profile of the resulting bread, the overall assessment, intended as an overall rating of the breads expressed considering all parameters with their levels of evaluation, of MUC bread was only slightly lower than that reached by CTR bread indicating a certain appreciation of the novel bread. Moreno-Álvarez et al. (32) who evaluated the sensory impact of breads produced from wheat and O. ficus-indica cladodes composite flours reported a general appreciation by the judges. Our results revealed that the substitution of water with liquid O. ficus-indica mucilage did not affect the fermentation process and the food quality attributes of bread while also attaining an acceptable quality evaluation from the sensory panel. Moreover, mucilage addition enriched bread with bioactive components, strongly increasing its health properties. In conclusion, considering the popularity of bread in human diet worldwide, the use of mucilage in this bakery product could really benefit human nutrition contributing to increase the dietary antioxidant intake. ACKNOWLEDGMENTS This research did not receive any specific grant from funding agencies, but it was partially financed with the personal research funds of Luca Settanni provided from University of Palermo (FFR_D13_2018/2021). The authors declare no conflict of interest. References 1. Sanguansri, L. and Augustin, M. A.: Microencapsulation in functional food product development, pp. 295e317, in: Smith, J. and Charter, E. (Eds.), Functional food product development. Wiley-Blackwell, Hoboken (2010). 2. Martirosyan, D. M. and Singh, J.: A new definition of functional food by FFC: what makes a new definition unique? Funct. Food Health Dis., 5, 209e223 (2015). 3. Gaglio, R., Gentile, C., Bonanno, A., Vintaloro, L., Perrone, A., Mazza, F., Barbaccia, P., Settanni, L., and Di Grigoli, A.: Effect of saffron addition on the microbiological, physicochemical, antioxidant and sensory characteristics of yoghurt, Int. J. Dairy Technol., 72, 208e217 (2019). 4. Sáenz, C., Sepúlveda, E., and Matsuhiro, B.: Opuntia spp. mucilage’s: a functional component with industrial perspectives, J. Arid Environ., 57, 275e290 (2004). 5. Nazareno, M. A.: Nutritional properties and medicinal derivatives of fruits and cladodes, pp. 152e158, in: Inglese, P., Mondragon, C., Nefzaoui, A. and Saen, C. (Eds.), Crop ecology, cultivation and uses of cactus pear. Food and Agriculture Organization of the United Nations and the International Center for Agricultural Research in the Dry Areas, Rome (2017). 6. Ginestra, G., Parker, M. L., Bennett, R. N., Robertson, J., Mandalari, G., and Narbad, A.: Anatomical, chemical, and biochemical characterization of cladodes from prickly pear [Opuntia ficus-indica (L.) Mill.], J. Agric. Food Chem., 57, 10323e10330 (2009). 7. Sepúlveda, E., Sáenz, C., Aliaga, E., and Aceituno, C.: Extraction and characterization of mucilage in Opuntia spp. J. Arid Environ., 68, 534e545 (2007). 8. Madjdoub, H., Rousdeli, S., and Dertani, A.: Polysaccharides from prickly pear and nopals of Opuntia ficus-indica: extraction, characterization and polyelectrolyte behavior, Polym. Int., 50, 552e560 (2001). 9. Altunkaya, A., Hedegaard, R. V., Brimer, L., Gökmen, V., and Skibsted, L. H.: Antioxidant capacity versus chemical safety of wheat bread enriched with pomegranate peel powder, Food Funct., 30, 722e727 (2013).
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Please cite this article as: Liguori, G et al., Effect of addition of Opuntia ficus-indica mucilage on the biological leavening, physical, nutritional, antioxidant and sensory aspects of bread, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.08.009