Marta
Mesias
*ab,
Francisco J.
Morales
a,
Cristina
Caleja
cd,
Tânia C. S. P.
Pires
cd,
Ricardo C.
Calhelha
cd,
Lillian
Barros
cd and
Eliana
Pereira
cd
aInstitute of Food Science, Technology and Nutrition, ICTAN-CSIC, José Antonio Novais 6, 28040-Madrid, Spain. E-mail: mmesias@ictan.csic.es; Tel: +3491 549 2300
bDepartment of Nutrition and Food Science, Faculty of Pharmacy, Complutense University of Madrid, Plaza Ramón y Cajal s/n., 28040 Madrid, Spain
cCentro de Investigação de Montanha (CIMO), Instituto Politécnico de Bragança, Campus de Santa Apolónia, 5300-253 Bragança, Portugal
dLaboratório Associado para a Sustentabilidade e Tecnologia em Regiões de Montanha (SusTEC), Instituto Politécnico de Bragança, Campus de Santa Apolónia, 5300-253 Bragança, Portugal
First published on 27th March 2024
This study evaluated the nutritional profile and fiber content of innovative formulations of wheat-based biscuits enriched with chia seeds, carob flour and coconut sugar. The in vitro antioxidant, cytotoxic, anti-inflammatory and antimicrobial activities were also investigated to understand the potential health advantages of the incorporation of these new ingredients. The novel biscuits demonstrated significant improvements in protein and mineral content, with increases of 50% and 100% in chia biscuits, and up to 20% and 40% in carob biscuits, respectively. Fiber also notably increased, particularly in samples containing 10% carob flour, which increased four times as compared to wheat-based samples. The new ingredients exhibited antibacterial and antifungal activity, particularly against Yersinia enterocolitica (minimum inhibitory concentration 1.25 mg mL−1 in coconut sugar) and Aspergillus fumigatus (minimum inhibitory concentration/minimum fungicidal concentrations 2.5/5 mg mL−1 in chia seeds). However, the final biscuits only displayed antifungal properties. Carob flour and chia seeds had a remarkably high capacity to inhibit the formation of TBARS and promoted greater antioxidant activity in biscuit formulations, with EC50 values decreasing from 23.25 mg mL−1 (control) to 4.54 mg mL−1 (15% defatted ground chia seeds) and 1.19 mg mL−1 (10% carob flour). Only chia seeds exhibited cellular antioxidant, anti-inflammatory and cytotoxic activity, attributes that were lost when seeds were added into the biscuits. These findings highlight the potential health benefits of these ingredients, particularly when incorporated in new wheat-based formulations.
Chia seed (Salvia hispanica L.) is characterized by its high content of dietary fiber (18–40%) and polyunsaturated fatty acids, especially from the ω-3 group, with α-linolenic acid being the most representative (60% of the total fatty acids). Additionally, it is a source of proteins (15–24%), carbohydrates (26–41%), and minerals (4–6%).5,6 Compared to traditional crops such as wheat, corn, rice, and oats, chia has a higher content of protein, fiber, and lipids. Chia also boasts bioactive compounds with strong antioxidant activity, including gallic, caffeic, chlorogenic, cinnamic, or ferulic acid.6 It also shows a high water absorption capacity, resulting in a clear gel known as chia mucilage. These properties make chia a functional food with remarkable nutritional characteristics, ideal for use in the food industry as a foam-stabilizing agent, suspending agent, emulsifier, adhesive, or binder with water-holding capacity.5 As such, incorporating chia into certain food formulations is desirable from both a technological and nutritional perspective.7–9
Carob flour is obtained from the carob pod of the leguminous tree Ceratonia siliqua L., by dehulling and deseeding the pulp, which is then oven dried and mechanically milled.10 Despite carob pulp being mainly used for animal feed and the production of sugar syrup,11 it has recently been recognized as an important source of nutrients and essential elements for human metabolism, healthy growth and disease prevention, such as dietary fiber, polyphenols and minerals, while having low amounts of fat.12 Moreover, it is gluten-free, which makes carob flour an excellent ingredient for developing new food products with enhanced nutritional value and a suitable alternative for people with celiac disease or gluten intolerance.13–15 For this reason, carob flour is increasingly being used in bakeries,12 particularly in Mediterranean countries that are facing issues related to climate change.
Reducing the sugar content is another issue for new food formulations. In 2015, the World Health Organization (WHO) recommended that the consumption of added sugars should be reduced to less than 10% of total caloric intake.16 Cereals and their by-products are the main sources of added sugar, accounting for 19.1% of the total dietary sugar intake.17 Therefore, it is crucial to consider alternatives to sucrose, such as carob flour and other sweeteners (i.e. coconut sugar), which can be added to cereal-based foods and can provide healthier alternatives to combat obesity and other diet-related health issues.18,19
In recent years, several investigations have been focused on cereal-based foods formulated with chia and carob flour and there is no information for coconut sugar. The majority of these studies have assessed the psychochemical attributes and consumer acceptability of the final products, including textural and sensorial analysis.8,9,14,15,20,21 Nevertheless, the researches of the nutritional changes in novel formulations containing carob, chia and coconut sugar are limited or even nonexistent. In this context, the aim of this study was to evaluate the nutritional profile and the fiber content of new formulations of wheat-based biscuits enriched with novel ingredients such as chia seeds, carob flour, and coconut sugar. The goal was designing a cereal-based product that offers added nutritional and physiological values respect to the traditional wheat-based biscuit. Different biological activities in vitro, including antioxidant, cytotoxic and anti-inflammatory were also assessed to determine the health-enhancing effects of the reformulated food. Additionally, the antimicrobial activity of novel ingredients was further investigated to set more insight into their potential use as food preservatives and additives aimed at enhancing the safety and shelf life of biscuits. The findings from this research will provide valuable knowledge regarding the health properties of chia seeds, carob flour and coconut sugar, highlighting their potential applications in the food industry.
- Ground chia biscuits (GC): wheat flour was replaced by ground chia seed, with percentages in the final weight of 5% (GC5), 10% (GC10) and 15% (GC15).
- Defatted ground chia biscuits (DGC): wheat flour was replaced by defatted ground chia seed, with percentages in the final weight of 5% (DGC5), 10% (DGC10) and 15% (DGC15).
- Carob flour biscuits (CF): wheat flour was replaced by carob flour, with percentages in the final weight of 1% (CF1), 5% (CF5) and 10% (CF10).
- Coconut sugar biscuit (CS): 100% of white sugar was replaced by coconut sugar (CS100).
- Carob flour and coconut sugar biscuits: wheat flour was replaced by carob flour, with percentages in the final weight of 1% (CF1), 5% (CF5), and 10% (CF10), and 100% of white sugar was replaced by coconut sugar (CS100).
The total amount of solids in the dough remained the same. Ingredients were thoroughly mixed and the dough was rolled out to disks with the diameter of 6.5 cm and the thickness of 2 mm, and baked at 180 °C for 22 minutes in a convention oven (Memmert UFE 400, Germany).
FAMEs analysis was conducted using a gas chromatograph (GC) equipped with a flame ionization detector (FID), a split/splitless injector, and a capillary column (30 m × 0.32 mm ID × 0.25 μm, Macherey-Nagel). The identification of fatty acids was performed by comparing the relative retention times of FAME peaks obtained from the oil extracted from samples with a reference standard FAME mixture. The specific details of the chromatography separation and determinations were previously described by Reis et al.26 The results were expressed in relative percentage (%).
Gram-positive bacteria (B. cereus, L. monocytogenes, S. aureus) were incubated in fresh blood agar containing 7% sheep blood, while Gram-negative bacteria (E. cloacae, E. coli, P. aeruginosa, S. enterica, Y. enterocolitica) were incubated in Muller Hilton agar. Both types of bacteria were incubated at 37 °C for 24 hours to maintain the exponential growth phase. Bacterial suspensions were prepared at a concentration of 1.5 × 106 CFU mL−1. Malt agar plates were used for the micromycetes (fungal strains) and they were incubated at 25 °C for 72 hours. After that, fungal spores were recovered from the agar surface using sterile 0.85% saline solution containing 0.1% Tween 80 (v/v). Fungal spore suspensions were adjusted to a concentration of 1.0 × 105 UFC mL−1.
Results were presented as minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum fungicidal concentrations (MFC) and expressed in mg mL−1.
For the cellular antioxidant activity assessment, the method described by de la Fuente et al.30 was followed. Extracts at various concentrations (ranging from 500 to 2000 μg mL−1) were prepared in H2O. The extracts were incubated with a murine macrophage cell line (RAW 264.7); obtained from the European Collection of Authenticated Cell Cultures (ECACC) at 37 °C for 1 hour. After incubation, the medium was discarded, and the cells were washed twice with 100 μL of HBSS. Then, 100 μL of AAPH (600 μM) was added to the cells. The reaction was carried out using a plate reader Biotek FLX800 (Bio-Tek Instruments, Inc., Winooski, VT, USA) with fluorescence filters set at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. Fluorescence values were recorded every 5 minutes over a period of 1 hour, and the differences in the areas under the curve (AUC) were calculated. Quercetin standard was used as a positive control. The CAA values were determined using the equation , where
represents the integrated area under the sample fluorescence versus time curve, and
represents the integrated area from the control curve. The results were expressed as a percentage of inhibition of the oxidation reaction.
Control | Wheat flour replacers | Control | Sugar replacer | |||
---|---|---|---|---|---|---|
Wheat flour | Ground chia seed | Defatted ground chia seed | Carob flour | White sugar | Coconut sugar | |
Results are mean ± standard deviation (n = 3). nd: not detected. Different letters in the same line indicate significant differences between samples (p < 0.05). | ||||||
Proximate composition (g/100 g fresh sample) | ||||||
Protein | 9.47 ± 0.31c | 21.69 ± 0.25e | 18.06 ± 0.14d | 5.28 ± 0.07b | nd | 0.92 ± 0.44a |
Fat | 1.16 ± 0.01b | 32.03 ± 0.42d | 8.21 ± 0.06c | 0.42 ± 0.01ab | nd | 0.20 ± 0.06a |
Total saturated fat | 0.30 ± 0.01b | 8.36 ± 0.08d | 2.49 ± 0.06c | 0.13 ± 0.01a | nd | 0.11 ± 0.01a |
C6:0 (%) | nd | 1.10 ± 1.01b | 0.40 ± 0.01a | nd | nd | nd |
C8:0 (%) | nd | 0.50 ± 0.20a | 0.70 ± 0.10a | nd | nd | nd |
C14:0 (%) | 0.20 ± 0.10a | nd | nd | 0.40 ± 0.01b | nd | nd |
C16:0 (%) | 23.10 ± 0.50a | 17.60 ± 2.40a | 21.30 ± 1.20a | 25.30 ± 3.80a | nd | 46.70 ± 0.01b |
C18:0 (%) | 1.60 ± 0.01a | 6.90 ± 0.40bc | 8.01 ± 0.40c | 5.40 ± 0.80b | nd | 7.80 ± 0.10c |
Total unsaturated fat | 0.83 ± 0.03c | 22.93 ± 1.15e | 5.39 ± 0.07d | 0.26 ± 0.02b | nd | 0.09 ± 0.01a |
C18:1n9 (%) | 16.01 ± 0.80b | 11.70 ± 0.30a | 13.30 ± 0.60a | 42.80 ± 0.40d | nd | 34.01 ± 0.40c |
C18:2n6 (%) | 52.30 ± 0.10c | 18.60 ± 0.30b | 18.20 ± 0.50b | 16.20 ± 4.30ab | nd | 8.90 ± 0.60a |
C18:3n3 (%) | 3.40 ± 0.10a | 41.30 ± 3.80a | 34.20 ± 2.40b | 2.70 ± 1.30a | nd | nd |
Total carbohydrates | 79.41 ± 0.38c | 36.19 ± 0.04a | 61.33 ± 0.25b | 87.70 ± 0.03d | 99.97 ± 0.00f | 94.12 ± 0.36e |
Fiber | 3.41 ± 0.42b | 25.60 ± 0.31c | 31.23 ± 0.42d | 35.80 ± 0.51e | nd | 1.30 ± 0.06a |
Soluble sugars | 0.50 ± 0.06a | 0.93 ± 0.04a | 0.79 ± 0.06a | 30.86 ± 0.26b | 104.41 ± 0.69d | 95.46 ± 1.48c |
Fructose | nd | 0.14 ± 0.02a | 0.11 ± 0.03a | 9.04 ± 0.13c | nd | 2.42 ± 0.06b |
Glucose | nd | nd | nd | 2.80 ± 0.05b | nd | 4.12 ± 0.14c |
Sucrose | 0.50 ± 0.06a | 0.79 ± 0.01a | 0.68 ± 0.03a | 19.02 ± 0.09b | 104.41 ± 0.69d | 88.92 ± 1.29c |
Moisture | 9.44 ± 0.00f | 5.54 ± 0.00d | 7.31 ± 0.00e | 4.41 ± 0.00c | 0.03 ± 0.00a | 3.62 ± 0.00b |
Total mineral content | 0.52 ± 0.06a | 4.54 ± 0.12d | 4.99 ± 0.05e | 2.19 ± 0.03c | nd | 1.14 ± 0.05b |
Energy value (kcal/100 g) | 352.32 ± 0.16b | 417.44 ± 2.58c | 266.53 ± 0.06a | 232.48 ± 1.61a | 399.89 ± 0.01b | 376.76 ± 0.06b |
Novel formulations of biscuits were prepared with partial or total replacement of traditional ingredients (wheat flour and white sugar) by innovative ingredients (chia seeds, carob flour and coconut sugar) (ESI Table 1†). Chia biscuits were formulated with both defatted and non-defatted ground chia seeds in a range between 5 and 15% of the final weight. Despite chia seeds are allowed as an ingredient in baked goods at concentrations below 10%,34 experiments were conducted with percentages as high as 15% to gain a deeper understanding of the nutritional composition and in vitro bioactivities of the biscuits formulated with these seeds. Similarly, carob flour was added to the biscuits in a range between 1 and 10% of the final weight. As expected, the addition of chia led to an increase in protein, fat, fiber, and mineral content, while concurrently reducing total carbohydrate content without modifying the energy value of the final product (Table 2). This change resulted in an improved nutritional profile when compared to the control sample. The rise in fiber and mineral content was particularly pronounced when defatted seeds were incorporated, resulting in a slight uptick in fat content when they were added at percentages of 10 and 15%. In the case of chia biscuits, the increase in fat content resulted in a positive outcome since chia is abundant in polyunsaturated fatty acids, primarily omega-3 fatty acids (C18:3n3, linolenic acid). These fatty acids are considered essential because the human body cannot synthesize them and they must be obtained through dietary sources.7 At this point, it is important to mention that an increase in unsaturated fatty acids could promote lipid oxidation during storage. Therefore, although the chemical modifications of biscuit during storage are not within the scope of the present study, it is relevant to carefully assess the potential impact of lipid oxidations throughout the shelf life in a risk/benefit context. Previous studies conducted by our research group reported an acceleration of lipid oxidation in chia-enriched biscuits, particularly due to the increase in polymerization compounds, which suggests a decreased shelf life of the product by promoting rapid rancidity.35 Consistent with the results of this study, several researchers have also documented the increase of proteins, fiber, and fats when chia is included in formulations of bakery products.8,9
Control | GC5 | GC10 | GC15 | DGC5 | DGC10 | DGC15 | |
---|---|---|---|---|---|---|---|
Proximate composition (g/100 g fresh sample) | |||||||
Protein | 5.98 ± 0.32a | 7.50 ± 0.10cde | 8.30 ± 0.04ef | 9.16 ± 0.03f | 7.32 ± 0.08bcde | 7.98 ± 0.20de | 7.94 ± 0.24de |
Fat | 14.55 ± 0.02ab | 16.51 ± 0.08de | 18.15 ± 0.23f | 20.51 ± 0.39g | 14.75 ± 0.24ab | 15.67 ± 0.05cd | 16.61 ± 0.01e |
Total saturated fat | 1.82 ± 0.07a | 2.15 ± 0.06b | 3.86 ± 0.18c | 5.84 ± 0.05e | 1.93 ± 0.03a | 2.04 ± 0.04ab | 4.72 ± 0.05d |
C6:0 (%) | nd | nd | 0.50 ± 0.10a | 1.90 ± 0.80b | nd | nd | 4.10 ± 1.70c |
C8:0 (%) | nd | nd | 0.20 ± 0.10a | 0.70 ± 0.20b | nd | nd | 0.80 ± 0.10b |
C14:0 (%) | 0.10 ± 0.01a | 0.10 ± 0.01a | 0.10 ± 0.01 | 0.20 ± 0.01b | 0.10 ± 0.01a | 0.10 ± 0.01a | 0.20 ± 0.01b |
C16:0 (%) | 8.50 ± 0.20a | 8.70 ± 0.20a | 14.01 ± 0.30b | 17.60 ± 1.30c | 8.80 ± 0.40a | 8.60 ± 0.01a | 14.30 ± 0.80c |
C18:0 (%) | 4.10 ± 0.10a | 4.20 ± 0.01a | 6.50 ± 0.10c | 8.10 ± 0.40d | 4.20 ± 0.01a | 4.30 ± 0.20a | 9.01 ± 0.40d |
Total unsaturated fat | 12.41 ± 0.21bc | 14.03 ± 0.79d | 13.57 ± 0.21c | 11.83 ± 0.20b | 12.51 ± 0.23bc | 13.26 ± 0.68c | 10.45 ± 0.23a |
C18:1n9 (%) | 29.80 ± 0.10bc | 27.30 ± 0.01a | 37.01 ± 0.40d | 37.10 ± 0.50d | 28.60 ± 0.30abc | 28.60 ± 0.31abc | 24.40 ± 0.90e |
C18:2n6 (%) | 55.30 ± 0.20d | 52.90 ± 0.01d | 33.20 ± 1.20c | 24.10 ± 3.30b | 53.10 ± 0.01d | 51.01 ± 1.01d | 34.10 ± 2.20a |
C18:3n3 (%) | 0.20 ± 0.01a | 4.80 ± 0.10c | 4.60 ± 0.20c | 4.00 ± 0.70bc | 3.10 ± 0.01b | 5.01 ± 0.31c | 4.40 ± 0.10a |
Total carbohydrates | 76.25 ± 0.39g | 73.00 ± 0.37cde | 70.44 ± 0.39b | 67.26 ± 0.47a | 74.18 ± 0.02def | 72.94 ± 0.12cd | 71.99 ± 0.17bc |
Fiber | 2.63 ± 0.06a | 5.80 ± 0.31c | 8.13 ± 0.53f | 10.83 ± 0.66h | 7.47 ± 0.64e | 9.57 ± 0.95gh | 13.73 ± 1.35j |
Soluble sugars | 19.21 ± 0.01abcd | 18.90 ± 0.10abcd | 19.45 ± 0.15bcd | 18.61 ± 0.02abcd | 18.15 ± 0.78abc | 18.69 ± 0.89abcd | 19.03 ± 0.81abcd |
Fructose | nd | nd | nd | nd | nd | nd | 0.14 ± 0.01a |
Glucose | nd | nd | nd | nd | nd | nd | 0.20 ± 0.01a |
Sucrose | 19.21 + 0.01c | 18.90 ± 0.10abc | 19.45 ± 0.15c | 18.61 ± 0.02abc | 18.15 ± 0.78abc | 18.69 ± 0.89abc | 18.69 ± 0.80abc |
Moisture | 2.10 ± 0.00b | 1.29 ± 0.01a | 1.36 ± 0.00a | 1.05 ± 0.01a | 2.34 ± 0.01b | 1.68 ± 0.01ab | 1.24 ± 0.00a |
Total mineral content | 1.11 ± 0.05ab | 1.69 ± 0.19bcd | 1.75 ± 0.21bcd | 2.01 ± 0.05cd | 1.40 ± 0.14abc | 1.73 ± 0.27bcd | 2.22 ± 0.06d |
Energy value (kcal/100 g) | 449.40 ± 0.14c | 447.39 ± 0.37c | 445.78 ± 0.34c | 447.00 ± 1.71c | 428.92 ± 1.73b | 426.47 ± 0.84b | 414.33 ± 0.17a |
Control | CF1 | CF5 | CF10 | CS100 | CF1-CS100 | CF5-CS100 | CF10-CS100 | |
---|---|---|---|---|---|---|---|---|
Results are mean ± standard deviation (n = 3). nd: not detected. Different letters in the same line indicate significant differences between samples (p < 0.05). | ||||||||
Proximate composition (g/100 g fresh sample) | ||||||||
Protein | 5.98 ± 0.32a | 6.53 ± 0.21abc | 6.65 ± 0.09abc | 6.26 ± 0.34ab | 7.10 ± 0.04abcd | 6.92 ± 0.07abcd | 6.58 ± 0.00abc | 7.06 ± 0.45abcd |
Fat | 14.55 ± 0.02ab | 14.64 ± 0.08ab | 14.80 ± 0.12ab | 14.26 ± 0.01a | 14.20 ± 0.08a | 14.56 ± 0.14ab | 15.09 ± 0.12bc | 14.99 ± 0.00abc |
Total saturated fat | 1.82 ± 0.07a | 1.93 ± 0.08a | 1.95 ± 0.05a | 1.89 ± 0.01a | 1.92 ± 0.04a | 2.02 ± 0.03ab | 2.28 ± 0.05b | 2.01 ± 0.06ab |
C6:0 (%) | nd | nd | nd | nd | nd | nd | nd | nd |
C8:0 (%) | nd | nd | nd | nd | nd | nd | nd | nd |
C14:0 (%) | 0.10 ± 0.01a | 0.1 ± 0.0a | 0.1 ± 0.0a | 0.1 ± 0.0a | 0.1 ± 0.0a | 0.1 ± 0.0a | 0.1 ± 0.0a | 0.1 ± 0.0a |
C16:0 (%) | 8.50 ± 0.20a | 8.6 ± 0.3a | 8.4 ± 0.1a | 8.4 ± 0.2a | 8.8 ± 0.5a | 8.9 ± 0.2a | 9.2 ± 0.1a | 8.7 ± 0.1a |
C18:0 (%) | 4.10 ± 0.10a | 4.5 ± 0.0a | 4.7 ± 0.1ab | 4.8 ± 0.1ab | 4.6 ± 0.2ab | 4.9 ± 0.3ab | 5.8 ± 0.3bc | 4.6 ± 0.3ab |
Total unsaturated fat | 12.41 ± 0.21bc | 12.32 ± 0.31bc | 12.47 ± 0.27bc | 12.02 ± 0.15b | 11.94 ± 0.07b | 12.14 ± 1.01b | 12.42 ± 0.21bc | 12.59 ± 0.24bc |
C18:1n9 (%) | 29.80 ± 0.10bc | 30.6 ± 0.2c | 30.5 ± 0.0c | 30.5 ± 0.0c | 30.5 ± 0.4c | 30.5 ± 0.3c | 28.3 ± 0.1ab | 27.7 ± 0.2a |
C18:2n6 (%) | 55.30 ± 0.20d | 53.4 ± 0.2d | 53.6 ± 0.4d | 53.6 ± 0.5d | 53.3 ± 0.6d | 52.6 ± 0.1d | 53.8 ± 0.5d | 56.0 ± 0.2d |
C18:3n3 (%) | 0.20 ± 0.01a | 0.2 ± 0.0a | 0.2 ± 0.0a | 0.2 ± 0.0a | 0.3 ± 0.1a | 0.3 ± 0.1a | 0.2 ± 0.0a | 0.3 ± 0.0a |
Total carbohydrates | 76.25 ± 0.39g | 76.19 ± 0.36g | 75.98 ± 0.31fg | 75.02 ± 0.49fg | 74.98 ± 0.16fg | 75.09 ± 0.13fg | 74.79 ± 0.12efg | 75.18 ± 0.48fg |
Fiber | 2.63 ± 0.06a | 2.70 ± 0.06a | 5.09 ± 0.02b | 9.11 ± 0.14g | 2.86 ± 0.10a | 3.93 ± 0.34b | 6.12 ± 0.01d | 11.34 ± 0.05i |
Soluble sugars | 19.21 ± 0.01abcd | 19.37 ± 0.45abcd | 20.08 ± 0.26cd | 20.90 ± 0.69d | 16.74 ± 0.01a | 17.30 ± 0.31ab | 18.07 ± 0.22abc | 18.11 ± 0.09abc |
Fructose | nd | nd | 0.20 ± 0.00b | 0.66 ± 0.02d | 0.37 ± 0.01c | 0.47 ± 0.02c | 0.73 ± 0.00d | 1.15 ± 0.01e |
Glucose | nd | nd | 0.18 ± 0.02a | 0.25 ± 0.01b | 0.58 ± 0.01c | 0.58 ± 0.05c | 0.75 ± 0.04d | 1.04 ± 0.02e |
Sucrose | 19.21 + 0.01c | 19.37 ± 0.45a | 19.70 ± 0.25c | 19.98 ± 0.71c | 15.79 ± 0.00a | 16.26 ± 0.24ab | 16.59 ± 0.25ab | 15.91 ± 0.06a |
Moisture | 2.10 ± 0.00b | 1.80 ± 0.00ab | 1.43 ± 0.00ab | 3.24 ± 0.02c | 2.67 ± 0.01bc | 2.18 ± 0.01b | 2.27 ± 0.02b | 1.19 ± 0.00a |
Total mineral content | 1.11 ± 0.05ab | 0.84 ± 0.05a | 1.15 ± 0.11ab | 1.20 ± 0.014ab | 1.04 ± 0.04ab | 1.24 ± 0.08ab | 1.27 ± 0.00ab | 1.58 ± 0.03bcd |
Energy value (kcal/100 g) | 449.40 ± 0.14c | 449.50 ± 0.42c | 441.87 ± 0.70c | 416.06 ± 0.09a | 442.42 ± 1.31c | 441.45 ± 0.07c | 437.25 ± 0.70bc | 419.49 ± 0.01ab |
The addition of carob flour fundamentally resulted in a significant increment of the fiber content in biscuits, particularly at substitutions level of 5% and 10% (Table 2). Babiker et al.14 described that a complete replacement of wheat flour with carob flour in cookies led to a significant rise in protein content (from 8.94% to 13.49%) and a decrease in fat content (from 14.20% to 12.30%). However, a 50% substitution with carob flour only exerted residual changes in the composition. In the present study, the protein content reached 9.24%, and the fat content was 14.00%, which closely resembled the levels in the control sample. Despite the proximate composition of the biscuits with carob was very similar to the control biscuit, the notable increase in fiber, even with just 1% incorporation of carob, suggests an improvement for health in this new formulation. This is particularly significant given the well-documented health benefits associated with increased fiber intake. Research, such as the recent review by Ioniţă-Mîndrican et al.,36 has linked higher fiber consumption to various positive health outcomes, including reduced risk of diabetes, obesity, constipation, as well as a lowered risk of coronary heart diseases and stroke. The nutritional composition of the biscuits made with carob flour and coconut sugar was remarkably similar to those made with common sugar. However, there was a slight increase in protein, mineral and fiber content in the former, suggesting a mild additional improvement in the nutritional profile compared to the control sample. Given the low-fat content of the flours used, there was not a significant change in the lipid profile that warrants further comment (Table 2).
Fig. 1 depicts the proximal composition and fiber content of both the control biscuit and the new formulations containing chia, carob, and coconut sugar. The formulations with 10% chia and carob were specifically chosen for comparison under similar conditions, adhering to the maximum allowable concentration of chia in baked products.34 As mentioned before, the novel biscuits exhibited notable improvements in protein and mineral content (up to 50%), and substantially there was a rise in fiber when compared to traditional wheat-based biscuits, particularly in samples containing carob flour (up to four times). These findings underscore the potential health advantages associated with the incorporation of these new ingredients into the reformulated biscuits.
Wheat | GC | DGC | CF | WS | CS | Positive controls | ||
---|---|---|---|---|---|---|---|---|
nt: not tested. Results are mean ± standard deviation (n = 3). MIC: minimum inhibitory concentration; MBC: minimal bactericidal concentration. MBC: minimal fungicidal concentration. WF: Wheat flour. GC: Ground chia seed. DGC: defatted ground chia seed. CF: carob flour. WS: White sugar. CS: Coconut sugar. | ||||||||
Antibacterial activity | Streptomycin | Ampicillin | ||||||
Gram-negative bacteria | ||||||||
E. cloacae | 20/>20 | 20/>20 | 20/>20 | 20/>20 | >20/>20 | >20/>20 | 0.007/0.007 | 0.15/0.15 |
E. coli | >20/>20 | 20/>20 | >20/>20 | 20 />20 | 20/>20 | 20/>20 | 0.01/0.01 | 0.15/0.15 |
P. aeruginosa | 20/>20 | >20/>20 | >20>20 | 20/>20 | 20/>20 | 20/>20 | 0.06/0.06 | 0.63/0.63 |
S. enterica | >20/>20 | 20/>20 | 20/>20 | 10/>20 | 20/>20 | 20/>20 | 0.007/0.007 | 0.15/0.15 |
Y. enterocolitica | >20/>20 | 5/>20 | 5/>20 | 2.5/>20 | 10/>20 | 1.25/>20 | 0.007/0.007 | 0.15/0.15 |
Gram-positive bacteria | ||||||||
B. cereus | >20/>20 | >10/>20 | >10/>20 | 10/>20 | >20/>20 | >20/>20 | 0.007/0.007 | nt/nt |
L. monocytogenes | >20/>20 | >10/>20 | >10/>20 | 20/>20 | 20/>20 | 20/>20 | 0.007/0.007 | 0.15/0.15 |
S. aureus | >20/>20 | 20/>20 | 20/>20 | 10/>20 | 10/>20 | 20/>20 | 0.007/0.007 | 0.15/0.15 |
Antifungal activity | Ketoconazole | |||||||
A. brasiliensis | 10/>20 | 10/>20 | 10/>20 | 10/>20 | 10/>20 | 10/>20 | 0.06/0.125 | — |
A. fumigatus | 2.5/20 | 2.5/5 | 2.5/5 | 2.5/20 | 2.5/5 | 5/20 | 0.5/1 | — |
Ground chia seed | Defatted ground chia seed | Carob flour | CS 100% | Carob flour + coconut sugar 100% | Positive control | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Control | GC5 | GC10 | GC15 | DGC5 | DGC10 | DGC15 | CF1 | CF5 | CF10 | CS100 | CF1-CS100 | CF5-CS100 | CF10-CS100 | Ketoconazole | |
Results are mean ± standard deviation (n = 3). MIC: minimum inhibitory concentration; MBC: minimal bactericidal concentration. | |||||||||||||||
A. brasiliensis | 10/20 | 10/20 | 10/20 | 10/20 | 10/20 | 10/20 | 5/10 | 5/10 | 10/20 | 10/20 | 10/20 | 10/20 | 10/>20 | 2.5/20 | 0.06/0.125 |
A. fumigatus | 1.25/2.5 | 2.5/5 | 5/10 | 2.5/5 | 2.5/5 | 2.5/5 | 2.5/5 | 2.5/5 | 5/20 | 5/20 | 5/20 | 5/20 | 10/>20 | 2.5/20 | 0.5/1 |
In a recent revision concerning plants utilized as antimicrobials, it has been elucidated that Salvia species are notably abundant in various bioactive compounds, including terpene derivatives, essential oils, phenolic compounds, flavonoids, and tannins. These compounds have been closely associated with a range of bioactive properties, including antimicrobial activities, ascribed to Salvia species.42 In this sense, previous research by Kobus-Cisowska et al.43 reported the antimicrobial activity of ethanolic extracts from chia seeds. Consistent with our findings, ground chia seeds have shown antibacterial potential against L. monocytogenes, P. aeruginosa, and E. coli. This activity is primarily attributed to the high content of chlorogenic, ferulic, and protocatechuic acids, with greater extraction efficiency achieved when the seeds are ground. Similarly, antimicrobial properties have been previously described in carob samples.44 Recently, Djebari et al.45 evaluated the biological activities of extracts obtained from different edible parts of carob tree, including pulp and gum. Their study demonstrated that these extracts could inhibit the growth of pathogenic bacteria at concentrations of 20 mg mL−1, and completely halt bacterial growth at higher concentrations (>50 mg mL−1). The authors concluded that extracts from carob pulp and gum show promise as alternatives to synthetic additives in the medicinal industry. In this context, they may serve as potential antioxidant agents and preservatives that combat bacterial contamination. In the case of carob, the antimicrobial effect can be mainly attributed to the high content of phenolic acids, gallotannins, and flavonoids.46 While there is limited information in the literature regarding the antimicrobial activity of sugar samples, certain studies have demonstrated that other coconut-derived products, such as coconut water47 and virgin coconut oil,48 are capable of inhibiting the growth of microorganisms such as Salmonella typhimurium and Staphylococcus aureus.
None of the novel formulations exhibited antibacterial activity against the tested microorganisms as compared with wheat-based biscuits, with the exception of biscuits CF10-CS100, which inhibited the growth of B. cereus at concentration of 20 mg mL−1 (data not shown). However, all biscuits displayed antifungal activity (Table 4). Among them, CF10-CS100 highlighted for its inhibition against A. brasiliensis followed by the biscuit containing 15% defatted chia seeds (DGC15). In contrast, the control sample exhibited the highest inhibition against A. fumigatus, which proved to be the most sensitivity to all the extracts. Based on the literature, it is anticipated that the activity against microorganisms would diminish or even vanish with heat treatment. Xu et al.49 noted significant reductions in the fungistatic activity of flaxseed when exposed to high temperatures. Similar decreases in antimicrobial properties have been documented in other heat-treated products, including honey.50 At this point, it is worth considering the possible potential antimicrobial effect of Maillard reaction compounds generated during the heat treatment. In this regard, Diaz-Morales et al.51 reported the antimicrobial properties of melanoidins isolated from various bakery products, including bread and biscuits, suggesting their capacity to enhance food shelf-life and safety. These authors also noted that the antimicrobial properties of melanoidins can vary, influenced by both the microorganism species and the type of melanoidin. This variability could explain the differences observed in the present study for each biscuit, since the development of the products of the Maillard reaction and consequently its activity could depend on the composition of the ingredients and the amount incorporated to the formulations. Previous studies conducted by our research group has indicated that incorporating defatted flour into biscuit formulations led to a substantial rise in Maillard reaction products, including acrylamide and furan compounds.52 Furthermore, research by Turfani et al.13 highlighted an increase in the browning of wheat bread enriched with carob flour. This was attributed not only to the color of the added flour but also to a higher occurrence of the Maillard reaction during baking, a consequence of elevated lysine content in carob as compared with wheat.
Ground chia seed | Defatted ground chia seed | |
---|---|---|
Cellular antioxidant activity: quercetin: 95 ± 5% oxidation inhibition at 0.3 μg mL−1. Anti-inflammatory activity: IC50 values for dexamethasone: 6.3 ± 0.4 μg mL−1 (RAW 264.7). Cytotoxic activity: IC50 values for ellipticine: 1.23 ± 0.03 μg mL−1 (AGS), 1.21 ± 0.02 μg mL−1 (Caco-2), 1.02 ± 0.02 μg mL−1 (MCF-7), 1.01 ± 0.01 μg mL−1 (NCI-H460). Different letters in each row correspond to significant differences among extracts of chia seeds (p < 0.05). | ||
Cellular antioxidant activity (oxidation inhibition % at 2 mg mL−1) | ||
RAW 264.7 | 20% | 62% |
Anti-inflammatory activity (IC50 μg mL−1) | ||
RAW 264.7 | 18.9 ± 1.7 | >400 |
Cytotoxic activity (GI50 μg mL−1) | ||
AGS | 202.1 ± 4.2a | 191.8 ± 6.7a |
Caco-2 | 130.4 ± 11.9a | 382.4 ± 8.2b |
MCF-7 | 220.7 ± 6.9a | 323.4 ± 2.2b |
NCI-H460 | 298.3 ± 10.0b | 175.5 ± 14.3a |
The high antioxidant capacity of chia seeds has been reported by other authors, mainly associated with the high content of polyphenolic compounds and bioactive peptides.53,54 Recently, Mas et al.54 reported the ability of chia polyphenols to reduce lipid peroxidation. Coelho et al.53 described that protein hydrolysates from defatted chia seeds have potential in vitro and in vivo antioxidant capacity and can effectively inhibit lipid oxidation in food models. In agreement with affirmations of Mas et al.,8 the supplementation of biscuits with chia seeds could be recommended to improve antioxidant properties of foods even when heat treated. These authors reported that the addition of 10% defatted chia seeds in cookies increased the polyphenol content and the in vitro antioxidant capacity of the formulations. Carob flour has also been noted to enhance the antioxidant capacity of various bakery products. Several studies, have demonstrated that the antioxidant power, measured by FRAP (Ferric Reducing Antioxidant Power) was notably elevated in bread made from blends of wheat flour and carob flours, especially unrefined carob flours at substitutions of 10% or more. Similarly, assays like ABTS (2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid), DPPH (2,2-diphenyl-1-picrylhydrazyl) and HPS (Hydrogen peroxide scavenging) have indicated an increased antioxidant capacity in muffins supplemented with carob powder.13 This enhancement was linked to a greater contribution of total phenolic compounds and total flavonoids content in the baked goods.15 In addition to the composition of the innovative ingredients, it is essential to consider the contribution of Maillard reaction compounds generated during baking to the enhancement of the antioxidant capacity in the biscuit samples, as previously reported in other bakery products.18
In contrast to the antioxidant activity observed in the TBARS assay, for all the ingredients and biscuit formulations, only chia seeds exhibited cellular antioxidant, anti-inflammatory and cytotoxic activity (Table 5). The highest concentration tested (2 mg mL−1) inhibited the oxidation reaction generated in the RAW macrophages by 20% in the extracts from ground chia seed and by 62% in those prepared from defatted ground chia seeds. Regarding the anti-inflammatory activity, the concentration of extract required to inhibit 50% NO production was from 18.9 μg mL−1 in ground chia seeds whereas defatted samples did not present activity at the maximum concentration tested (> 0.4 mg mL−1). The anti-inflammatory effect of chia seed could be due to the presence of lipids in this matrix and particularly to the high content of polyunsaturated fatty acids, which have been previously associated with anti-inflammatory activity by acting as competitor substrates for the inhibition oxidation of arachidonic acid.55 High levels of polyunsaturated fatty acids in oils obtained from sea bass and bream head oils have been associated with the inhibition of NO in LPS-stimulated macrophage cells.30 Regarding the cytotoxic activity, the colon cancer cells (Caco-2) and the breast cancer cells (MFC-7) exhibited significantly higher susceptibility to the ground chia seeds (GI50 = 130.4 μg mL−1 and 220.7 μg mL−1, respectively) as compared with defatted ground chia seeds at the tested concentrations (GI50 = 382.4 μg mL−1 and 323.4 μg mL−1, respectively). In contrast, defatted samples inhibited to a significant greater extent the proliferation of lung cancer cells (NCI-H460) (GI50 = 175.5 μg mL−1vs. 298.3 μg mL−1 in ground chia seeds), whereas similar growth inhibition effects of stomach cancer cell lines (AGS) were observed for both samples (GI50 range: 191.8–202.1 μg mL−1). Again, the fat content of the seeds could be considered as responsible for this activity, since previous works have revealed the antiproliferative effect of omega-3 fatty acids against colon and lung cancer cells.56,57 Together with the fat content, other components of the seeds such as peptides could also be involved in cellular inhibition, which would explain the activity in the defatted ground chia seed.58
Footnote |
† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4fo00204k |
This journal is © The Royal Society of Chemistry 2024 |