Development of Gluten-Free Functional Bread Adapted to the Nutritional Requirements of Celiac Patients

: Celiac patients suffer from nutritional deﬁciencies before and during the maintenance of a gluten-free diet; this is due to non-fortiﬁed, mostly processed foods that are high in saturated fat and deﬁcient in minerals typically present in wheat. A literature search was carried out to determine the deﬁciencies of these patients in calcium, iron, ﬁber, folic acid, omega-3, vitamin B12 and vitamin D. Different formulations of gluten-free bread enriched with olive extract (hydroxytyrosol of natural (HXT O ) and synthetic (HXT S ) origin), acerola extract, citrus extract, spinach extract, calcium, iron and linseed were used. Antioxidant capacity, nutritional composition, folates, minerals, color and pH were studied, and a microbiological study and sensory analysis were conducted to assess organoleptic quality. These studies were carried out on days 0, 4, 7 and 11 to study their evolution. The results of the HXT S bread showed a higher antioxidant capacity, higher antimicrobial capacity and higher ﬁber content, as well as higher amounts of minerals. It also showed higher consumer acceptability, even relative to commercial gluten-free bread. The HXT O bread showed higher antimicrobial capacity than the control (C), higher ﬁber content and higher mineral content, but had lower antimicrobial capacity than HTX S bread. It also had better sensory acceptability than C but was worse than HXT S bread. Taking into account the physicochemical and organoleptic characteristics, the HXT S sample is the most suitable for enriching the diet of celiac patients.


Introduction
Celiac disease is an autoimmune disease in which patients' antibodies react against an enzyme after consuming gluten as part of the diet. This reaction mainly causes intestinal malabsorption problems that affect other areas of patients' health due to nutritional deficits. It is a disease highly influenced by both genetic and environmental factors, in addition to a wide range of clinical presentations. Its prevalence is approximately 1%, but more cases of celiac disease are diagnosed each year [1,2]. It increases morbidity and mortality in the people affected, but a strict gluten-free diet improves this, in addition to reducing the risk of complications such as gastrointestinal cancer [3].
Gluten is found mainly in wheat, rye and barley. Wheat is the largest cultivated cereal and the basis of many diets, including the Mediterranean diet. The average consumption rate of gluten is 5 to 20 g per day, and it is present in many of the fundamental foods of the diet, such as bread and pasta [4]. The current single treatment for this disease is following a gluten-free diet throughout life. According to a review by Gobbetti et al. (2018) [5], gluten-free products show a higher glycemic index, lower protein content and a higher percentage of fats, especially saturated, and a gluten-free diet results in a decreased intake of folic acid, vitamin B12, vitamin D, calcium, iron and other minerals. In addition, the gluten-free diet contains many processed products in order to replace gluten-based foods. All of these lead to an increased risk of metabolic syndrome and overweight and obesity. For the experiments, samples C, HXT O , HXT S and a commercial bread were used, the ingredients of which included water, corn starch, rice flour, vegetable fibers (psyllium, citrus), hydroxypropyl methylcellulose (thickener), sunflower oil, soya protein, yeast, salt, sugar, citric acid (acidifier) and lactose-free (lactose < 0.007 g/100 g), in order to compare the functional breads with a commercial supermarket bread.
For the first sample (HXT OES ), 5 g of spinach was added per 100 g of flour based on the article by [15]. The spinach flavor was very intense, which made it intolerable to taste and completely different from the normal taste of bread. From this sample, it was decided to reduce the amount of lyophilized spinach to 1 g/100 g flour. The remaining samples contain the quoted quantity.
In the next two, the addition of flaxseed and iron sulfate (FeSO 4 ) served as an experiment to create an iron-enriched product [17]. It started with a concentration of 8 g of ground flax and 220 mg of FeSO 4 per 100 g of flour and the same concentration of CBC, acerola, HXT and CaCO 3 as in HXT OES (HXT OFE1 ), and after a bibliographic search, the measure was adjusted, and 3.2 mg of FeSO4 was added per 100 g flour with the same amount of flax and natural extracts (HXT OFE2 ) [18,19]. In both cases, the taste of iron was intense, making it unacceptable organoleptically, so it was concluded that it would not be enriched with iron sulfate.
All 4 samples under study were run. Therefore, they were all subjected to the same production and storage conditions in order to reduce these external factors. The three samples and the commercial one were stored for 11 days in the refrigerator at 4 • C covered by a film; the representation of these conditions and ingredients of the different breads can be seen in Figure 1. Tests were carried out during this period (on days 0, 4, 7 and 11) to check the variations undergone by the time.   The sample drying method (AOAC, 1995) was used to determine the humidity. About 3 g of sample were weighed and allowed to dry at 110 • C in a forced air oven for 24 h.

Determination of Inorganic Matter
The technique and procedure of the AOAC were used [20], based on the complete incineration of the organic matter of the sample in a muffle furnace at 525 • C, leaving only the residue of inorganic matter. The empty porcelain crucible and 1 g of fresh sample are weighed and placed on a heating plate that reaches 525 • C. Leave approximately 24 h.

Determination of Total Nitrogen and Protein
The Kjeldahl procedure was followed for the determination [20]. The basis of the method is the destruction of organic matter with sulfuric acid that results in ammonium sulfate; adding an excess of sodium hydroxide releases ammonia that is distilled over boric acid, forming ammonium borate. After this, it is titrated with 0.1 N hydrochloric acid. About 2 g of fresh sample were weighed, and 7 g of catalytic mixture and 15 mL of concentrated sulfuric acid were added. The tubes were placed in a heating block reaching 450 • C and maintained for 1 h at that temperature. Once the time had elapsed, they were allowed to cool, and the distillation was carried out with the addition of 32% NaOH until it underwent a color change (light blue to brown) and was distilled for 4 min. It was collected in a flask with 25 mL of 4% boric acid and a protein indicator. The distillation product was titrated until the indicator was turned with 0.1 N hydrochloric acid.

Determination of Total Fat
For the determination of total fat, a Soxhlet-type Tecator extractor was used [20]. The dried empty aluminum cups and about 1 g of dried sample are weighed in a cellulose cartridge. After introducing the samples into the extractor, 50 mL of ether is added to each sample and the extraction process that lasts about 3 h with different steps is started. The ether heated to 80 • C extracts the fat from the sample, which fell on the aluminum vessel that was previously weighed. At the end of the process, they are put in the stove to remove the excess ether and the glass is reweighed with the residue of the fat removed.

Determination of Dietary Fiber
For the determination of the fiber, procedure 985.29 of the AOAC, described by Prosky et al. (1985) [21], was used. Then, 1 g of the sample was weighed, and 50 mL of phosphate buffer 0.05 M pH 6 was added. After this, the sample was taken to a bath at 95 • C, and the α-amylase was added. After 30 min, adjust pH 4.5, and add amyloglucosidase at 60 • C for 30 min. After finishing the digestion, add ethanol to precipitate the fiber; once precipitated, filter it. At the end of the filtration, the residue is left in the oven, and once dried, the proteins and ashes of this residue are determined.

Determination of Carbohydrates
The determination of carbohydrates is carried out by difference according to the recommendations of the FAO and WHO [22] (1982); based on the results, the determinations of fat (F), ash (A), protein (P), moisture (M) and dietary fiber (DF) determinations are obtained so that Carbohydrates (%) = 100 − (F + A + P + M + FD).

Determination of the Energy Value
The energy value of the samples is obtained by summing the energy value of the protein, carbohydrate and fat of each sample using the energy conversion factors of Atwater [23]. Extraction and deconjugation of folates from samples: Folates were extracted from the sample following the procedure described by Konings et al. (1999) and Pfeiffer et al. (1997) [24,25]. One gram of sample was mixed with 25 mL of extraction buffer (50 mmol/L, 50 mmol/L HEPES, containing 2 g sodium ascorbate/100 mL and 10 mmol/L 2-mercaptoethanol, pH 7.85) under a nitrogen atmosphere. The extraction mixtures, in screw-capped tubes, were placed in a boiling water bath for 10 min, cooled on ice and homogenized. Then, the pH was adjusted to 4.9 with 60 mmol/L HCl. Enzymatic deconjugation and purification of samples were carried out following the methodology described by Vahteristo et al. (1996) [26]. An aliquot of 5 mL was incubated for 3 h at 37 • C under a nitrogen atmosphere with 1 mL of hog kidney conjugase prepared from fresh pig kidneys, as described by Gregory, Sartain and Day (1984) [27], and 1 mL of α-amylase preparation (20 mg/mL in 1 g Na ascorbate/100 mL). To inactivate the enzymes, the samples were boiled at 100 • C for 5 min and then cooled on ice. The samples were filtered through 0.45 µm pore size and purified and strong anion-exchange (SAX) cartridges connected to a Supelco 12-port vacuum manifold. First, the cartridges were conditioned with 3 mL of n-hexane, methanol and Mili-Q water and then equilibrated with 3 mL of purification buffer (10 mmol/L dipotassium hydrogen phosphate, 2-mercaptoethanol (v/v), pH 7.0). Second, the sample was slowly loaded and eluted with 2 mL of elution buffer (10 g sodium chloride/100 mL, 10 mmol/L sodium acetate, 1 g ascorbic acid/100 mL) at a flow rate of <0.5 mL/min. The eluted sample was weighed.
HPLC analysis of folates: The separation and analysis of samples were performed with an HPLC/MS system consisting of an Agilent 1290 Infinity II Series HPLC (Agilent Technologies, Santa Clara, CA, USA) equipped with an Automated Multisampler module and a High-Speed Binary Pump and connected to an Agilent 6550 Q-TOF Mass Spectrometer (Agilent Technologies, Santa Clara, CA, USA) using an Agilent Jet Stream Dual electrospray (AJS-Dual ESI) interface. Experimental parameters for HPLC and Q-TOF were set in MassHunter Workstation Data Acquisition software (Agilent Technologies, Rev. B.08.00). Standards or samples (20 µL) were thermostatted at 4 • C and injected onto a Zorbax Eclipse Plus C18 (2.1 × 100 mm, 1.8 µm) HPLC column at a flow rate of 0.4 mL/min. Column was equilibrated at 30 • C. Solvents A (MilliQ water with 0.1% formic acid) and B (acetonitrile) were used for the compound separation.
The mass spectrometer was operated in the positive mode. The nebulizer gas pressure was set to 40 psi, whereas the drying gas flow was set to 16 L/min at a temperature of 150 • C, and the sheath gas flow was set to 12 L/min at a temperature of 300 • C. The capillary spray, nozzle, fragmentor and octopole 1 RF Vpp voltages were 4000 V, 1000 V, 350 V and 750 V, respectively. Profile data in the 100-600 m/z range were acquired for MS scans in 2 GHz extended dynamic range mode with 3 spectra/s, 333.3 ms/spectrum and 1999 transients/spectrum. Reference mass at 121.0509 was used for mass correction during the analysis. Data analysis was performed with MassHunter Qualitative Analysis Navigator software (Agilent Technologies, Rev. B.08.00).

Mineral Determination
Prior to mineral analysis, digestion was performed using a microwave laboratory station (Ethos D) type Ethos plus 1 purchased from Milestone Inc. (USA). For each sample, 5 g of dry matter and 10 mL of soluble and dialyzable fractions resulting from digestion were weighted in a Teflon digestion vessel with 7 mL of concentrated (65%) nitric acid (HNO 3 ) and 1 mL of 30% hydrogen peroxide (H 2 O 2 ). Then, the sample was subjected to a microwave program as follows: step 1, 25-200 • C for 10 min at 1000 W; step 2, 200 • C for 10 min at 1000 W. Digests were finally made up with deionized water to 25 mL in acid-washed standard flasks. The following elements were measured using inductively coupled plasma mass spectrometry (ICP-MS) (Thermo electron X7 inductively coupled plasma mass spectrometry, model X series, UK): sodium (Na), potassium (K), calcium (Ca), magnesium (Mg), manganese (Mn), phosphorus (P), iron (Fe), zinc (Zn), selenium (Se), and copper (Cu). ICP-MS operating conditions were the following: nebulizer gas flow, 0.91 L min −1 ; radio frequency (RF), 1200 W; lens voltage, 1.6 V; cool gas, 13.0 L min −1 ; auxiliary gas, 0.70 L min −1 .

Shelf-Life Study
pH was measured at days 0, 4, 7 and 11 in triplicate and was determined with the help of a pH meter (Crison GLP21) after mixing 5 g of fresh sample with the same amount of distilled water at room temperature [20].
Color was measured using a Konica Minolta CR-410 chromameter (Minolta Camera Co., Osaka, Japan), and the DP-400 data processor of "AQ instruments" was used to measure crust color (CIE Lab* values). CIE L* values (lightness), CIE C* values (saturation), a* values (red-green) and b* values (yellow-blue) were measured. The color coordinates were analyzed at 0, 4, 7 and 11 in triplicate.

Determination of Antioxidant Capacity
Antioxidants are compounds that inhibit the formation or spread of free radicals. They achieve this by donating a hydrogen atom or by transferring electrons [28,29]. In this study, four methods were used for the measurement of the antioxidant activity of breads, namely, oxygen radical scavenging capacity, DPPH radial scavenging activity, ABTS radial scavenging activity and antioxidant capacity to reduce ferric ions [30].
Absobality of Oxygen Radicals (ORAC) AAPH (2,2 -azobis (2-amidinopropane) dihydrochloride) is used, which, upon decomposition, produces peroxyl radicals used and oxidized to fluorescein protein that reduces its emission light at 528 nm. If the radicals are reduced, the light emission will remain high, and, therefore, the antioxidant power is high [29,31]. A straight Trolox standard (vitamin E analogue, antioxidant) is used, which is used as a reference [32].
It was performed following the protocol previously described by Ehlenfeldt and Prior (2001) [33]. A 96-well black plate was used, the edges of which were filled with 200 µL of distilled water; the blanks consisted of 20 µL of phosphate buffer (used for dilution of samples) and 20 µL of samples and Trolox standards. After the preparation of the plate, the process is started in the Synergy HT plate reader after a purge with water and the corresponding reagents. The GEN 5 program was used in which the protocol was stipulated (200 µL of fluorescein in each well; after 15 min, 20 µL of AAPH (0.216 g in 10 mL of phosphate buffer) was added in each well and then measured with an excitation of 485 nm and an emission of 528 nm every minute for an hour and a half). The whole process is carried out at 37 • C. With the support software, the area under the curve was obtained for all measures, and the data were extrapolated by Trolox standard curves (having known concentration).

Iron Reducing Antioxidant Power (FRAP)
This technique is based on the reduction of the complex of TPTZ (2,4,6-tripyridyl-striazine) and ferric iron (Fe +3 ) by antioxidants, reaction after which iron converts to ferrous iron (Fe +2 ), which produces a blue color. Variations are detected by spectrophotometry that is measured at 593 nm wavelength [34].
The technique was performed based on the test by Benzie and Strain (1999) [34]. The reagents are TPTZ (10 mM), hydrochloric acid (40 mM HCl), sexahhydrate ferric chloride (FeCl 3 •6H 2 O 20 mM), as well as 300 mM acetate buffer with pH 3.6). FRAP solution with 20 mL of acetate buffer, 2 mL TPTZ and 2 mL of FeCl was performed 3•6H 2 O. In the cuvette, 1 mL of reagent FRAP and 100 µL samples or standards were added, and absorbance at 593 nm at 4 min was measured. Blank is the FRAP solution. To compare the results, a Trolox standard curve was performed.

DPPH Technique
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) is a free radical with a high absorbance at 515 nm; when interaction with an antioxidant compound, DPPH no longer has an unpaired electron and thereby loses the absorbance in direct proportion to the amount of antioxidant [35].
The technique was performed based on the methodology described by Brand- Williams et al. (1995) and Sánchez-Moreno et al. (1998) [36,37]. The DPPH reagent was made with 0.0063 g of DPPH and 250 mL of ethanol, keeping it protected from light. 3.9 mL of DPPH reagent were mixed and 100 µL samples and standards by measuring absorbance at 515 nm after 30 min mixing (kept protected from light). Zero was measured with methanol, and the blank was DPPH reagent without sample.

ABTS Technique
The ability of antioxidant compounds to capture the radical cation that is generated after the reaction of 2,2 -azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) with MnO 2 is evaluated. The radical generated absorbs at a wavelength of 734 nm, but after being neutralized by the antioxidants, it decreases its absorbance directly proportionally [29,38].
The method described by Re et al. (1999) [39]. The necessary reagents were a phosphate-buffered saline at pH 7.4; ABTS (7 mM) activated by passing through a filter with manganese oxide II (MnO 2 ). Then, 1 mL ABTS solution was used and 100 µL of sample or standard. Water was used for zeroing and for blank the ABTS solution with an absorbance of approximately 0.700 (adjustment with water and MnO 2 ). It was stirred for 30 s, and the absorbance was measured after 2 min at 734 nm.

Microbiological Analysis
Total microbiological growth of total coliform count (TCC), Escherichia coli, total vial count (TVC) and molds and yeasts (TMY) were determined at days 0, 4, 7 and 11 from elaboration. Mass seeding was performed on PCA (to determine mesophilic aerobes), rapid E. coli (to determine coliforms and E. coli) and oxytetracycline-glucose yeast extract (OGYE) agar medium. Peptone water was used to make dilutions. Analyses were performed in a laminar flow hood (Telstar, BIO-II-A, Madrid, Spain). After seeding, plates were incubated for 24 h at 37 • C for E. coli and TCC, 48 h at 37 • C for TVC and 96 h at room temperature for TMY. Analyses were performed in triplicate and expressed in cfu/g.

Sensory Analysis
The tasting room for the sensory evaluation was air-conditioned and free of influencing factors.
It was conducted with 30 untrained panelists aged between 20 and 58 years. Samples were coded with four random digits and presented individually to the panelists on a plate, as shown in Scheme 1. Mineral water was provided for mouthwash between samples. The attributes measured were appearance, aroma, texture, taste, flavor, color, juiciness, purchase intention and overall acceptability. A five-point hedonic scale was used to evaluate the attributes of the breads; panelists scored on a scale of 1 (a little) to 5 (a lot).

Statistical Analysis
Data were analyzed with the statistical package SPSS 23.0 (Statistical Package for the Social Science for Window (IBM, Armonk, New York, NY, USA). A descriptive statistical analysis of the results was performed, as well as ANOVA test using pairs of factors and Pearson's correlation of the data over time. A ratio of p < 0.05 was considered significant, while p < 0.01 was considered highly significant. It was conducted with 30 untrained panelists aged between 20 and 58 years. Samples were coded with four random digits and presented individually to the panelists on a plate, as shown in Scheme 1. Mineral water was provided for mouthwash between samples. The attributes measured were appearance, aroma, texture, taste, flavor, color, juiciness, purchase intention and overall acceptability. A five-point hedonic scale was used to evaluate the attributes of the breads; panelists scored on a scale of 1 (a little) to 5 (a lot).

Statistical Analysis
Data were analyzed with the statistical package SPSS 23.0 (Statistical Package for the Social Science for Window (IBM, Armonk, New York, NY, USA). A descriptive statistical analysis of the results was performed, as well as ANOVA test using pairs of factors and Pearson's correlation of the data over time. A ratio of p < 0.05 was considered significant, while p < 0.01 was considered highly significant. Table 2 shows the approximate composition of the different extracts that provide macronutrients to the gluten-free bread in the study. High values of all macronutrients were observed in both samples, highlighting the quantity of all macronutrients analyzed in spinach compared to flaxseed.  Table 2 shows the approximate composition of the different extracts that provide macronutrients to the gluten-free bread in the study. High values of all macronutrients were observed in both samples, highlighting the quantity of all macronutrients analyzed in spinach compared to flaxseed. The high protein content of spinach (29.36 g/100 g) observed in this study is consistent with previous studies, representing 28.05 g/100 g [40] and 27.8 g/100 g [41]. On the other hand, a slightly higher carbohydrate content was observed for spinach (32.36 g/100 g) than in a previous study which obtained 30.28 g/100 g [41]. However, with respect to fiber, a slightly lower content of 32.01 g/100 g was found than in a previous study where 35.21 g/100 g [41] was obtained and contrary to what Junejo et al. (2021) [40] found where 8.82 g/100 g was obtained, a lower value of fiber compared to that obtained in the current study.

Macronutrients of Flaxseed and Spinach Powder
Flaxseed, on the other hand, was found to have a higher proportion of fat (40.89 g/100 g), followed by carbohydrates (28.89 g/100 g) and protein (21.99 g/100 g), which was comparable to some recent studies [42][43][44].

Macronutrients of the Samples
Nutritional differences were obtained between the different breads, as can be seen in the following table (Table 3). The proteins in Com, HXT O and HXT S samples had similar values and showed no significant differences, while C is the one with the least amount of protein and is highly significant (p < 0.01). These findings were also observed by Galla et al. (2017) [41] in their study, where they found that the addition of thorn in the biscuits increases the amount of protein compared to the control biscuit. Other studies, such as Khemiri et al. (2020) [45] and Krupa-Kozak et al. (2021) [46], observed that the addition of green leafy vegetables or seaweed increases the percentage of protein in gluten-free bread compared to the control. Also, the fat in HXT O and HXT S is significantly higher than Com and C (p < 0.01) due to the addition of flaxseed, which provides them with a higher percentage of fat, providing omega-3 [47]. Flaxseeds are rich in α-linolenic acid [48], which is especially important for good cardiovascular health. The amount of protein in this study is lower than those obtained by Dur et al. (2019) [49], who developed a gluten bread enriched with spinach powder and ferrous sulphate (FeSO 4 ). The results for fat may be due to the fact that, compared to other studies, spinach contains a significant level of fat between 3-6 g/100 g [40,41]. It may also be due to the addition of hydroxytyrosol, as Martínez-Zamora et al. (2020) [50] obtained a higher amount of fat in a fish stick compared to the control.
Carbohydrates depend on the amount of moisture, fat, fiber and protein. The highest carbohydrate content was obtained in the Com, and the bread with the lowest level was HTX S , with significant differences between all samples (p < 0.01). Carbohydrates were slightly lower in HXT O and HXT S because of the amount of flaxseed added, which lowered the proportion of carbohydrates, as well as the addition of spinach, as observed by Galla, who found that, with the addition of spinach, the biscuits decreased their carbohydrate content. Filip and Vidrih (2015) [51] produced protein-rich pasta using 40% pea protein isolate and 3% dry spinach powder and obtained a carbohydrate reduction (44.6, 40.4%) of 77% in wheat pasta.
The sample with the highest energy value was Com, and the one with the lowest energy value was HTX S ; in all of them, significant differences were obtained (p < 0.01), except in the Control sample compared with HXT O -their energy value is very similar. There were no significant differences in moisture between the samples; the only sample where a lower value than the others was observed was the Com. Very similar results were obtained as was found in the study of Dur et al. (2019) [49], where their bread contained about 50% of carbohydrates.
The ashes were higher in HXT O and HXT S , being highly significant in the case of HXT O (p < 0.01) and less significant in the case of HXT S (p < 0.05). This is due to the addition of spinach and flaxseeds, which provide a large number of minerals that are very important in celiac patients when they are recovering from the disease. Very similar results were obtained to the study of Dur et al., 2019 [49], where their bread contained about 2% of ashes. In addition, Galla et al. (2017) [41] found the same finding in biscuits, where incorporating spinach increases the ash content compared to the control.
The highest fiber content was obtained in HTXs and the lowest in Com, and there were significant differences between all samples (p < 0.05). As shown in Table 2, it can be observed that the enriched breads have a higher fiber content; this could be due to the acerola since, according to the study of Carmo et al. (2018) [52], the fiber content of acerola is approximately 70-80% on a dry basis. Also, flax and spinach because, according to Bekhit et al. (2018) and El-Sayed (2020) [53,54], flax contains about 20-35% and linseed 20%. In addition, the flour used has a high content of corn starch and rice flour, which contain vegetable fibers that, apart from helping the cohesion of the bread, as well as the agglutination of the dough, which is essential for achieving the correct texture, also provides fiber [55]. The results obtained for fiber, where a higher content was observed in the enriched breads, were also obtained by Galla et al. (2017) [41] in the spinach-enriched biscuits compared to the control. The increase in fiber, along with the increase in the amount of healthy fat, helps the enriched samples have a lower glycemic index, according to the literature [56][57][58]; this reduces the risk of metabolic syndrome and associated diseases. Table 4 shows the concentration of each of the four folate monoglutamates, as well as the total folate monoglutamates and the total folate concentration in the three developed breads and commercial bread. Total folate concentration was highest in the HXT O bread (2879.31 µg folic acid equivalents/100 g FW) followed by bread HXT S (2112.12 µg folic acid equivalents/100 g FW), C (1330.13 µg folic acid equivalents/100 g FW), and COM (839.55 µg folic acid equivalents/100 g FW), with significant differences of p < 0.01. In the folic acid (FA) content, significant differences of p < 0.01 were observed between the different breads, except for the HXT S bread with the Com bread and the control. Regarding the tetrahydrofolate (THF) content, significant differences (p < 0.05) were found between the different breads, except for the HXT O bread with Com and HXT S with Control. In the 5M-THF (5-methyltetrahydrofolate) composite, significant differences of p < 0.05 were observed in all breads. No significant differences were observed between samples regarding 5F-THF (5-formyltetrahydrofolate) content, but it could be found that the one with the highest 5F THF content was HXT O , and the one with the lowest 5F-THF content was COM. The main form of natural folate found in all our samples was folic acid, which is consistent with the results of studies where FA was found to be the most predominant natural form of folate in legumes [59]. However, López-Nicolás et al. (2014) [60] observed that the main abundant natural form of folate was 5M-THF in their gluten breads enriched with spinach and chard, but this is perhaps because they only analyzed two natural forms of folate, 5M-THF and THF.

Folate Content
According to market consultant Mintel, Spain ranks as the third country in the world that has launched the most gluten-free products, after Brazil and the United States (AECOC, 2016) [61]. Furthermore, according to the food consumption report, Spain increased its consumption of gluten-free products by 3.08% in 2020 [62]. Spanish citizens consumed 8.5 g of gluten-free bread per day. Taking these data into account, the results obtained in the present study showed that consumption of studio bread could reach 48-80% RDI of folate (400 µg), respectively, with the highest HXT O and the lowest HXT S . Brevik et al. (2005) [63] reported that bread is one of the foods that contribute the most to total folate intake, even more than other folate-containing foods, such as vegetables and fruits. Vegetables, as rich sources of folate and other vitamins, are widely recommended for balanced diets; therefore, breads enriched with spinach and chard would be a good choice to provide an additional source of folates [60]. In addition, besides celiac patients, it would also be a good option for pregnant women since they would supplement their diet and thus prevent neural tube defects in newborns; this would thus assist 40% of those who experience unplanned pregnancies through the inclusion of folic acid fortification in the bread as the base food of the Mediterranean diet [10]. It would also be a good alternative because celiac patients have deficiencies in the intake of micronutrients, including iron; folic acid; vitamins A, B6, B12, D, E and K; copper; and zinc. Due to malabsorption [64], gluten-free products also contain significantly lower levels of vitamins D, E and B 12 ; iron; folate; magnesium; potassium; and sodium than foods containing gluten [65]. It has also been shown that only 5% of gluten-free breads contain the four mandatory fortification nutrients (calcium, iron, niacin and thiamine), while 28% are fortified only with calcium and iron. However, it should be noted that not all countries require fortification of foods. Some countries only require wheat-based products to be fortified, and these rules do not apply to "dietetic or special foods", such as gluten-free products. This lack of fortification may increase the risk of micronutrient deficiency in subjects with celiac disease who are following what appears to be an adequate gluten-free diet [65].

Minerals
The results of the minerals are shown in Table 5. No arsenic, beryllium, bismuth, lanthanum, selenium or vanadium were found in any of the samples, and no differences were seen between the samples of the amount of cadmium, cobalt, potassium, lithium, molybdenum, sodium, nickel, rubidium, antimony, titanium and zinc. High aluminum content was observed in the C and in Com, being the lowest in the HXT S (p < 0.01). Arsenic was only detected in sample C (0.07 mg/Kg) and in the Com (0.03 mg/Kg). In boron, copper and iron, the highest content of these minerals was found in HXT S (1.69 mg/kg; 1.63 mg/kg; 10.16 mg/kg) (p < 0.05). However, higher contents of silicon and strontium were observed in the HXT O (31.99 mg/Kg; 3.46 mg/Kg) (p < 0.05). In the case of thallium and zinc, the content of C is greater than that of the other samples (p < 0.05). It was also observed that in the case of zinc, it was higher in the developed samples than in the Com; this may be due to the fact that flaxseeds (5.5 mg/100 g) provide a large amount of zinc [47]. Regarding the iron content, this is higher in the samples that had spinach added (p < 0.05). With respect to calcium, chromium, magnesium and manganese, significantly higher contents were observed in the fortified breads than in the control and commercial breads. For phosphorus, a small significant difference was found between the control (0.05 g/100 g) and the formulated breads. The same finding was obtained by [41], who observed an increase in phosphorus when spinach was added to biscuits.
The minerals which are increased in C can be distinguished between two main groups because the majority of them are found in cereals and minerals which increase in all samples enriched or some of them due to the various ingredients added.
According to authors such as Pennington and Schoen (1995) and Soni et al. (2001) [66,67], cereals are the main source of aluminum through food. The main ingredients in sample C were corn starch and rice flour. It was noted that enrichment in other extracts caused a lower concentration of aluminum since the percentage of cereals was reduced. Spinach is also a food rich in aluminum [68], and the higher spinach content in this sample compensates for the lower percentage of cereals. Arsenic is present in corn starch and rice, so something similar to aluminum occurs [69]. Thallium was mostly seen in sample C; this is because cereals are rich in thallium [70].
Boron increased proportionally to the amount of spinach added, being very low in sample C and quite high in the HXT S sample. In the rest of the samples, the value is similar and intermediate. Based on the literature, spinach is rich in boron and is the ingredient used with a higher concentration of this mineral [71,72]. Chromium was quite increased in the enriched samples; green leafy vegetables such as spinach are sources of chromium [73]. Copper was similarly increased in all enriched breads. The main source of copper is the flax and light contribution of spinach; in the same way, it occurs with magnesium (380 mg/100 g) and phosphorus (470 mg/100 g) [47], very abundant minerals in flaxseeds and somewhat less in spinach [47]. Manganese was especially increased in the enriched samples with respect to C and Com, reaching double the amount of this. Manganese is especially abundant in spinach and somewhat less in flaxseeds [47,74]. The increase in silicon, especially in fortified breads, suggests that the main source is spinach enrichment. Even more obviously, it occurs with the strontium mineral; in the fortified breads sample, an increase of more than 3 times compared to the rest of the bread samples was observed and more than 13 times compared to C [74].
The minerals that stand out the most are calcium and iron, as these are common deficiencies in celiac patients, and this fortification was specifically sought. In calcium, an increase of 10% was observed in the enriched breads with respect to C and 3% in Com compared to the enriched ones, which contain spinach and flaxseeds (170 mg/100 g) [47], ingredients in which this mineral is present. In iron, which is found naturally in spinach, flaxseed (7.06 mg/100 g) [47] and lentil flour [47,[74][75][76], an increase of 36-39% was observed for reformulated breads compared to Com and 3-5% compared to C. This finding was also found by Galla et al. (2017) [41], who observed an increase in calcium and iron by adding spinach to the biscuits, but the observed increase was much higher, with calcium increasing by 100-305.62% and iron by 31.23-176.84%.

pH and Color during Different Days of Storage
The pH results can be seen in Table 6; it was observed that HXT O has the highest. A significant increase (p < 0.05) in pH from D0 to D11 was observed in the HXT O , HXT S and control loaves, but a decrease in pH was observed in the control loaf. On days 0 and 4, the HXT O had the highest pH (5.88;5.90), and the control had the lowest pH (4.85;5.08), but on day 7, the HXT S had the highest pH (5.90), and the control had the lowest pH (5.11). On day 11, a higher pH was observed in HTX O (5.90) and a lower one in control (5.13).
In the study by Moore et al. (2008) [77], no significant differences were seen in the pH of the sample over time, but it was only studied at 24 and 48 h with sourdough bread. The pH value is affected by many conditions, such as the amount of CO 2 formed by fermentation and other ingredients, such as calcium carbonate and spinach. The absence of variation of the HXT O and HXT S samples can be associated with greater stability provided by the number of natural antioxidants. v-x Means in the same column that are not followed by a common letter in superscript are significantly different between days of analysis (p < 0.05).
The color measurements of the different breads can be seen in Table 7. A significantly higher difference (p < 0.05) in brightness (L*) was observed in the C and Com compared to the enriched breads (HTX O and HXT S ) on all days. The variable a* was observed to be significantly lower in the enriched breads representing the green shades compared to the C and Com breads, which is found on day 0, day 4, day 7 and day 11. This coincidence was also observed in the variable b* (yellow), where it was found to be significantly higher in the enriched breads than in the C and Com bread on all days. The hue angle (h) also follows the same pattern of measurements, being higher in the HXT S and HXT O and lowest in the C and Com. These findings of how the inclusion of spinach affects bread variables, increasing the b* variable and decreasing L* and a*, were also found by Junejoet al. (2017) [40] in gluten breads. All measurements (L*, a*, b*, C*, h) of the C and Com were found to be highly significant (p < 0.01) with respect to the HXT O and HXT S breads; however, there is no significant difference between the HXT O and HXT S breads. Regarding the color variables in the same samples during the storage time, no major changes were found, the brightness was not affected during storage; however, the a*, b*, C* showed a tendency to decrease from day 0 to day 11 in all the breads, finding a significant difference (p < 0.01) in the C* measure with respect to the days of storage, but only with a significant difference (p < 0.01) in the b* in the control, Hxts, commercial and control breads.
The differences in color are due to the addition of lyophilized spinach, which causes darker dough with a more greenish and yellowish tone. The color differences are evident to the naked eye, being imperceptible between the HXT O and HXT S (there is no significant difference). The C increased its luminosity with the passing of days, which is an unfavorable characteristic since gluten-free breads already have a lighter color due to their high content of starches and rice flour [78]. The absence of correlation of brightness change of HXT O and HXT S can also be associated with greater stability of the sample and, therefore, better maintenance of color and conditions during storage.  . v-y Means in the same column that are not followed by a common letter in superscript are significantly different between days of analysis (p < 0.05).

Characterisation of Preservative Extracts
The knowledge of the antioxidant activity of antioxidant ingredients allows a comparative evaluation between the preservative extracts analyzed. The results obtained from each method are shown in Table 8. Table 8. Antioxidant activity of natural extracts by measuring their ABTS and DPPH represented in the percentage of chelating activity (%) and antioxidant activity (µmol TE/g) of the samples (n = 4).

Samples
Chelating Activity Percent (%) Antioxidant Activity (µmol TE/g) Firstly, considering the results presented in Table 8, it can be observed that HXTs, HXTo and acerola showed the highest chelating capacity against the radical cations DPPH and ABTS. At the same time, the lowest values were presented by CBC with 9.55% and spinach with 44.06% of chelation in DPPH.

ABTS
On the other hand, the scavenging activity against the hydrophilic radical ABTS is generally lower than against DPPH. In both methods, the hydroxytyrosol stands out, especially the hydroxytyrosol from synthetic sources. As a result, the oxygen radical scavenging capacity of HXTs was 5% higher than that of HXTo, 10% higher than that of extract A, 45% higher than that of sp and 79% higher than that of CBC. For the ferricreducing extract, the antioxidant power of HXTs was 72% higher than that of HXTo. Furthermore, the percentage chelating activity of HXTs against ABTS radicals was only 11% compared to HXTo. This was due to the purity of the HXT (99.2%) compared to the 7.26% bioactive compound content in HXTo. HXT is known for its antioxidant potential, which is ten times higher than green tea and twice as high as coenzyme Q10 [79]. It also has a major role as a free radical scavenger and outstanding efficacy under stressful conditions. This antioxidant activity is based on the chemical structure of this phytochemical compound: a phenolic ring consisting of a catechol group and three hydroxyl groups [80].

Antioxidant Capacity during the Shelf-Life Study
The results of the antioxidant capacity of the different samples in different samples are shown in Table 9. The methods were chosen based on the bibliography [75,81,82]. It is important to highlight the importance of carrying out more than one study of antioxidant capacity in order to understand the behavior of antioxidants [83]. Table 9. Chelating activity percent (%) and antioxidant activity (µmol TE/g) of the samples of bread (n = 4). Significantly (p < 0.01) higher antioxidant capacity was found in all methods in the fortified samples compared to the control and commercial samples. This finding was also reported by Junejo et al. (2021) [40], who observed that the addition of spinach improved antioxidant versus control activities; this could be due to the presence of phytochemicals and bioactive compounds of spinach such as polyphenols, lutein, lycopene, α-carotene,-and α-tocopherol [84].

Control HXT O HXT S Com
FRAP and DPPH were significantly higher in HTX S on all days of the analysis. However, in ORAC, the highest antioxidant capacity was found in HXT O on days 0 (754.75 µmol TE/g) (p < 0.01). In ABTS, the highest concentration was obtained on days 0 (60.58%), 4 (45.37%) and 7 (39.38) in HXT S (p < 0.01). It was observed that in all methods, commercial bread was significantly lower (p < 0.05).
The difference in the averages between HXT O and HXT S is especially interesting since the only difference between them is the origin of hydroxytyrosol. This demonstrates that the chemically synthesized hydroxytyrosol has a greater antioxidant capacity than the hydroxytyrosol extracted from the olive leaf. In all methods and samples, a decrease was observed as the days progressed, where a decrease was found with a great difference in the control bread and commercial bread; however, a low decrease was observed in the enriched breads, especially from day 4 onwards. In the case of HXT S , there is not so much variation as the days go by. This may be because synthetic hydroxytyrosol is more stable, provides greater antioxidant protection and is more sustained over time ( Table 9).
The HXT O and HXT S samples showed a higher antioxidant capacity consistent with the studies of Branciari et al. (2017) [85] on hydroxytyrosol enriched with meat and [86], who analyzed the antioxidant capacity in vitro of hydroxytyrosol and tyrosol. In both, a high antioxidant capacity of hydroxytyrosol was determined by ORAC, DPPH and FRAP. Moreover, [87], in his gluten-free breads developed with chestnut flour at different concentrations, observed significant differences in the stability of the antioxidant capacity throughout the days. This also occurs in our results since HXT S presents much greater stability of the antioxidant capacity with respect to HXT O due to HTXs at antioxidant purity (99.2%) compared to 7.26% bioactive compound content in HXTo.
In the studies by Jensen, Oestdal, Clausen, Andersen and Skibsted (2011) and Jensen, Ostdal, Skibsted, and Thybo (2011) [88,89], it is revealed that the variation of antioxidant capacity over time depends on the bread formulation; this could explain the difference between control and commercial enriched breads. Paciulliet al. (2016) [87] observed that the antioxidant capacity decreased from day 1, results that coincide with those obtained. However, Jensen, Oestdal, Clausen, Andersen and Skibsted (2011) [88] observed a very low decrease in their samples of whole wheat bread compared to the results that we found, where a much higher decrease in antioxidant capacity is observed, but in bread with Hxts from day 4 onwards, the decrease is very low, and it could be said that it is almost stable.

Microbiological Analysis
The results of the microbiological study are shown in Table 10. C: control; HXT O : organic hydroxytyrosol; HXT S : synthetic hydroxytyrosol; TVC: total vial count; TCC: total coliform count; TMY: total molds and yeasts. a-c Different letters within the same row indicate significant differences between samples at different times of analysis (p < 0.05). w-z Different letters within the same column indicate significant differences between samples (p < 0.05).
HXT S on the initial day presented the lowest number of total microorganisms, which shows that the synthetic source of HXT (99%purity) inhibits the total growth of microorganisms with respect to the control and commercial supermarket bread, but with respect to the passage of storage time a change can be observed in the bread with HXTs, as it increases the growth of mesophilic aerobic microorganisms; however, in molds and yeasts, it is in the bread with HXTo. With these results, it can be extrapolated that hydroxytyrosol has a higher shelf life and microbiological safety compared to the control, including a possible higher antimicrobial effect. This finding coincides with the results obtained in the study by Martínez-Zamora et al. (2020) [80], who developed burgers with different types of hydroxytyrosol and observed that HXTs has a high antimicrobial capacity against mesophilic aerobic microorganisms.
In other studies using fish patties, the authors observed the same antimicrobial reaction due to hydroxytyrosol and natural extracts (citrus extracts) [50,90]. Shapira and Mimran (2007) [91] suggested that their antimicrobial effect could be due to phenolic compounds. This antimicrobial activity was also shown in pork sausages made with emmer wheat, almonds, hazelnuts and pomegranate and citrus extracts [92][93][94]. This behavior can also be compared with previous studies by Azaizeh et al. (2011) [95], which also demonstrated the antibacterial power of olive extracts (as sources of HXT) in lamb patties.
According to RM Law number 615-2003 SA/DM, (2003) [96], bakery and pastry products with or without filling that do not require refrigeration must include the following microorganisms: molds, E. coli, Staphylococcus aureus, Clostridium perfringens and Salmonella sp. In the case of bread without filling, only mold analysis is required, and the allowed limit is 100-1000 CFU/g. In the samples developed, it is fulfilled on day 0-less in the Com since it is above 103 CFU/G; from day 4, no bread falls within the rule.

Sensory Analysis
In the sensory analysis, with the samples prepared as in Scheme 1, parameters of the organoleptic quality of each piece of bread were measured, and the samples were ordered according to their taste, from highest to lowest. In Table 11, the difference between the averages received is shown. Com stood out, and more than 50% of the tasters gave it a score of 5, and 40% placed it in the first place. C is the one with the least first places (only 10%) and earned the most last-place ratings (36.7%). HXT O is the one with the most second places (33.3%), and the HXT S sample had a great acceptability, having the second most first-place ratings (30%) after Com. Differences in score regarding the texture of C with HXT O are highly significant (p < 0.01) and significant (p < 0.05) for C with the HXT S , with C's texture being the lowest valued. For the color, highly significant differences were observed in Com with HXT O and HXT S and significant differences with C, with Com being better valued. Regarding juiciness, the difference between C and the rest of the samples is highly significant, with C having a worse evaluation in all cases. If the average score of the samples is compared, C was the one that received the highest score, followed by the HXT S , HXT O and finally C. The order of choice by the testers was Com> HXT S > HXT O > C, as shown in Figure 2. However, Zhou et al. (2023) [97] obtained different results, as they found that by supplementing vegetables in breads, they provide greater satisfactory acceptability on the part of the consumer.
The texture and juiciness (related qualities) of C were poor compared to the other samples, so the ingredients added to HXT O and HXT S improved these two characteristics, making the bread more enjoyable. However, the differences in color are significant, with those of HXT O and HXT S being worse valued, but sample C also scored low. The best value was obtained by the Com sample since it showed a darker color, which reinforces the study of Wronkowska et al. (2013) [78], in which it was observed that a darker color is more satisfactory in gluten-free bread for the tester.
Taking into account these results, HXT S showed a good acceptability, despite having a very different appearance from "normal" bread. This finding was also obtained by Zamora- Martínez et al. (2020) [80], who found better near-commercial acceptability results in HXTs lamb burgers compared to HXTo. After analyzing the characteristics of the tasters, it is even more striking since the majority (63.3%) were people between 18 and 30 years who tend to be more reluctant to foods enriched for their health and to vegetables in general. The texture and juiciness (related qualities) of C were poor compared to the other samples, so the ingredients added to HXTO and HXTS improved these two characteristics, making the bread more enjoyable. However, the differences in color are significant, with those of HXTO and HXTS being worse valued, but sample C also scored low. The best value was obtained by the Com sample since it showed a darker color, which reinforces the study of Wronkowska et al. (2013) [78], in which it was observed that a darker color is more satisfactory in gluten-free bread for the tester.
Taking into account these results, HXTS showed a good acceptability, despite having a very different appearance from "normal" bread. This finding was also obtained by Zamora-Martínez et al. (2020) [80], who found better near-commercial acceptability results in HXTs lamb burgers compared to HXTo. After analyzing the characteristics of the tasters, it is even more striking since the majority (63.3%) were people between 18 and 30 years who tend to be more reluctant to foods enriched for their health and to vegetables in general.

Conclusions
The samples with HXT showed a greater antioxidant capacity, a more stable pH, a better nutritional value and a greater number of minerals, especially calcium, of folates (especially important due to the generalized deficiency in celiac patients), in addition to an antimicrobial activity compared with the control and less degradation and aging. In the sensory analysis, the samples with hydroxytyrosol showed a higher acceptability than the control; HXTS was the one with the highest acceptability, reaching levels similar to those obtained by commercial gluten-free bread. With these results, it can be concluded that the fortified samples meet the proposed objectives since they are healthier and have a greater contribution of specific nutrients, and the acceptability of the product compared to the control was higher.
After analyzing the deficiencies of celiac patients before and after establishing a gluten-free diet, it can be determined that the HXTO and HXTS samples are a good source of calcium, omega-3, fiber and other minerals such as boron, chromium, copper, magnesium, manganese, phosphorus, silicon and strontium-elements that are usually deficient in gluten-free products and that are beneficial for the health of celiac patients. Taking into account the physicochemical and organoleptic characteristics, the HXTS sample is the most appropriate to enrich the diet of celiac patients.

Conclusions
The samples with HXT showed a greater antioxidant capacity, a more stable pH, a better nutritional value and a greater number of minerals, especially calcium, of folates (especially important due to the generalized deficiency in celiac patients), in addition to an antimicrobial activity compared with the control and less degradation and aging. In the sensory analysis, the samples with hydroxytyrosol showed a higher acceptability than the control; HXT S was the one with the highest acceptability, reaching levels similar to those obtained by commercial gluten-free bread. With these results, it can be concluded that the fortified samples meet the proposed objectives since they are healthier and have a greater contribution of specific nutrients, and the acceptability of the product compared to the control was higher.
After analyzing the deficiencies of celiac patients before and after establishing a glutenfree diet, it can be determined that the HXT O and HXT S samples are a good source of calcium, omega-3, fiber and other minerals such as boron, chromium, copper, magnesium, manganese, phosphorus, silicon and strontium-elements that are usually deficient in gluten-free products and that are beneficial for the health of celiac patients. Taking into account the physicochemical and organoleptic characteristics, the HXT S sample is the most appropriate to enrich the diet of celiac patients.
Funding: This research received no external funding. Data Availability Statement: Not applicable.

Conflicts of Interest:
The authors declare no conflict of interest.