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INTRODUCTION
Eating patterns have undergone worldwide changes throughout the 20th century, characterized by a sedentary lifestyle and diets with high fat, sugar, and sodium contents, and low in fiber. This modification has been associated to the emergence of chronic, non-communicable diseases such as type II diabetes, obesity, heart conditions, cancer, and respiratory illnesses, representing a strong impact in population morbidity and their quality of life1-3.
Nutritional science studies on the issue have been increasingly associating feeding as a modulator in the processes related with degenerative diseases, aging, oxidative stress (which generates reactive oxygen species that also lead to damage and cellular death)1,4. In this context, nutritional science and the food industry play a key role in the development of healthy and functional foods with the purpose of preventing illnesses5,6.
The growth of the functional food market is currently on the rise since consumers nowadays look for not only safe or nutritious products, but they also demand natural, organic, or healthy food made from natural, innovative and non-traditional ingredients7,8.
Fruit and vegetable demand has increased considerably, this also entails an increase in their losses and waste because of inadequate handling methods and infrastructure. However, those discarded products are a rich source of bioactive compounds like natural pigments, phenolic compounds, fiber, minerals, and other components with health benefits9.
In this context, Salta province in northwestern Argentina, produces export-only blueberries (Vaccinium corymbosum L.). Only a low proportion is industrially used in the production of juices and derivatives, in which around 20-30% of solid waste is produced. Therefore, the agricultural wastes from blueberry juice processing and discarded export fruit could be incorporated into products using entire fruit, minimizing waste generation, and contributing to the development of new foods10. Blueberries are rich in phenolic compounds, such as anthocyanins, which can play an active role in the body against reactive oxygen species. This is attributed to their potential antioxidant properties9.
Currently, different types of legume flours have been incorporated into bakery products not only for their nutritional properties but also for the sensory characteristics they contribute to the final product. Prosopis nigra flour would be an interesting ingredient to give an added value to bakery products, since it stands out for its dietary fiber and phenolic compound contents, as well as its purple color11.
Snacks have become an important part of the daily diet in Western cultures. However, people generally opt for unhealthy products high in calories, fats, sugars, and sodium13. Based on this, the nutritional profile of snacks could be improved with the addition of healthy ingredients such as blueberries and alternative flours like Prosopis nigra, thus granting an added value to them.
Considering that, the food industry seeks to incorporate food waste into food matrices, to grant their functional properties, reduce agro-industrial losses and offer consumers healthy products based on non-traditional ingredients. The aim of this work was to develop a functional dietetic snack using black carob flour and discarded blueberries, and to evaluate its chemical and functional composition.
METHODOLOGY
Raw Materials
Dietetics snacks (DS) were prepared with the following ingredients: black carob flour (Prosopis nigra Grisebach Hieronymus) obtained in Sabores Andinos S. A., Buenos Aires, Argentina; wheat flour; fresh egg white; high oleic sunflower oil; chocolate essence and flavoring from a local Salta, Argentina market. The filling was made with discarded for-export Emerald blueberries (Vaccinium corymbosum L.). Sucralose was provided by Saporiti S. A., potassium sorbate and low methoxy pectin obtained from Gelfix S. A. and calcium lactate was obtained from Todo Droga, Córdoba, Argentina.
All the analytical reagents used for chemical determinations were obtained in MERCK S. A. and were analytical grade.
Snack formulation
DS were performed following a traditional recipe. Three formulations were prepared using different proportions of wheat flour (WF) and black carob flour (BCF): 60:40; 50:50; and 40:60, respectively. Ingredients and proportions used are shown in Table 1.
Table 1. Dough ingredients and proportions.
| Ingredients (g/100 g) | DS1 60:40 | DS2 50:50 | DS3 40:60 |
|---|---|---|---|
| WF/BCF | 29.3 | 24.2 | 19.5 |
| WF | 19.5 | 24.2 | 29.3 |
| Egg white | 24.4 | 24.4 | 24.4 |
| Sunflower oil | 26.8 | 26.8 | 26.8 |
| Sucralose | 0.015 | 0.015 | 0.015 |
| Potassium sorbate | 0.05 | 0.05 | 0.05 |
WF: Wheat flour; BCF: Black carob flour.
The snacks' filling was prepared using discarded Emerald blueberries, sucralose (0,01%), calcium lactate (0,055%), potassium sorbate (0,05%) and low methoxy pectin (1%).
Blueberries (BB) were previously washed by immersion and crushed in a blender for thirty seconds, sweetener was then added and mixed with a spoon. The mixture was cooked during 10 minutes with constant agitation. The blueberry filling was allowed to cool for 1 hour at room temperature. The complete process of the snack's development is indicated in the Figure 1.
Chemical composition analysis
Proximal chemical composition (moisture, proteins, lipids, carbohydrates, ashes, and sodium) was determined by the procedures of AOAC methods. All analyses were performed in triplicate12.
The total caloric value (TCV) was determined considering all the ingredients used in the formulation. Nutritional information was calculated using 4, 9 and 4 calories per gram of protein, fat, and carbohydrate, respectively.
For the comparison of the proximate composition, a commercial product with similar characteristics was taken as a reference, whose chemical composition per 100 g is detailed below: caloric value 476kcal, carbohydrates 77 g, proteins 8 g, total fats 15 g, dietary fiber 5 g, sodium 100 mg.
Functional compound determination
We studied total polyphenols, anthocyanins, proanthocyanidins and antioxidant activity. All bioactive compounds were analyzed in the BCF, the BB and DS to evaluate changes produced by the processes applied (crushing, kneading, cooking). Dietary fiber was quantified in the product with the highest antioxidant activity.
The fractions of bioactive compounds were quantified using colorimetric methods measured in extracts prepared according to Sciammaro (2015) with some modifications13. A solid-liquid extraction was performed using 50% diluted acetone in a 1:2 ratio (sample/solvent). The sample was stirred at 800 rpm for 40±1 minutes, filtered with Whatman paper N° 4 and kept in refrigerator temperature (2-8 ºC) in amber bottles until used.
Total polyphenols (TPF): were identified according to Singleton and Rossi's (1965) technique with some modifications14. Extracts (50 µl) were oxidized with Folin-Ciocalteu reagent (100 µl) and mixed with distilled water (2350 µl). Finally, the neutralization was made with sodium carbonate concentrate to 20% (50 µl). The absorbance was measured at 765 nm. Results were expressed on calibration curve as milligram of gallic acid equivalents per 100g (mg GAE/100g).
Total anthocyanins (TA): were determined using pH-differential method Giusty-Wrolstald (2000)15. An aliquot was diluted (1:2 v/v) with two buffers, pH 1.0 (0.025 M potassium chloride) and pH 4.5 (0.4 M sodium acetate). After 15 minutes of incubation at room temperature, the absorbance was measured at 510 and 700 nm. TA was expressed as Cyanidin-3-glucoside/100 g according to the following equations:
Equation 1:
Equation 2:
Where:
A is 
; Mw is the molecular weight (449.20 g/mol); DF is the dilution factor; ε is the molar extinction coefficient (26,900 L/M/cm for cyanidin-3-glucoside); and l is the pathlength (1 cm).
Proanthocyanidins (PAT): were quantified by vanillin-HCl method described by Price et al., (1978)16. First, 20 µL of extract were reacted with 180 µL of methanol, secondly 1.2 mL of vanillin (at 4% w/v in methanol) was incorporated and stirred. Finally, 600 µL of concentrated HCl was added and protected from light for 30 minutes. Samples were measured at 500 nm and values obtained were calculated in catechin calibration curve. PAT were expressed in milligram catechin equivalent per 100 g (mg CE /100 g).
Antioxidant activity (AA): free radical scavenging ability of black carob flour was measured using radical cation ABTS method described by Re et al., (1999)17. Radical cation was prepared by incubating the ABTS solution with a 2.45 mM potassium persulfate for 16 hours in darkness at room temperature. Subsequently was diluted with methanol to a final absorbance of 0.7 at 734 nm. For AA determination, 30 µl of sample were added to a cuvette containing 3 mL of the ABTS solution. The absorbance was measured at 734 nm, and results were expressed as µmol Trolox equivalent per 100 g (µmol TE/100 g) using Trolox curve as standard.
Total dietary fiber (TDF): soluble dietary fiber (FDS) and insoluble dietary fiber (FDI), were determined by the gravimetric enzymatic technique18.
Statistical analysis
An analysis of variance (one-way ANOVA) was performed to evaluate any significant differences between formulations in their chemical composition and possible variation in bioactive compounds. Multiple comparisons were also evaluated by Tukey's post-hoc test using the software INFOSTAT student Version 2018.
RESULTS
Chemical composition
The proximal composition of DS is presented in Table 2.
Table 2: Proximal composition of DS (100 g).
| DS1 | DS2 | DS3 | |
|---|---|---|---|
| TCV (kcal) | 383 (SD 2a) | 395 (SD 3a) | 367 (SD 2a) |
| Cho (g) | 45.36 (SD 1.17a) | 47.91 (SD 0.99a) | 48.48 (SD 2.40a) |
| Prot (g) | 13.63 (SD 0.28a) | 13.05 (SD 0.73a) | 13.13 (SD 0.25a) |
| TF (g) | 15.89 (SD 0.04ab) | 16.61 (SD 0.59b) | 14.87 (SD 0.46a) |
| MS (%) | 16.87 (SD 0.40a) | 18.56 (SD 0.11b) | 22.83 (SD 0.85c) |
| AS (g) | 1.98 (SD 0.02a) | 1.98 (SD 0.02a) | 1.97 (SD 0.02a) |
| SDM (mg) | 166.82 (SD 1.00a) | 161.58 (SD 7.41b) | 130.33 (SD 8.96b) |
SD mean standard deviation (n=3). TVC: Total caloric value; Cho: Carbohydrates; Prot: Proteins; TF: Total fat; MS: Moisture; AS: Ashes; SDM: Sodium. DS: Dietetic Snack. Values with different superscript characters within a row are significantly different (p<0.05).
TCV in snacks showed no significative differences. Calories were lower than commercial snack available (476 kcal/100 g). According to these results, we can confirm that snacks have a reduction of 20%, 17% and 23% of the total caloric value in DS1, DS2 and DS3 respectively.
No significative differences were found in carbohydrate content in the snacks formulated. However, a reduction of 41%, 38% and 37% of this nutrient was achieved in the snacks, respectively.
Protein content showed no significant difference throughout the different DS formulations. Besides, levels were also higher than commercial snacks available (8 g/100 g), that means an increase of 70.37%, 63.12% and 64.12% of total protein content for SD1, SD2 y SD3 respectively. Significant differences were found with respect to fat and moisture. No significant differences in ash content were observed.
Functional compounds
Values obtained for bioactive compounds fractions in DS are expressed in Table 3.
Table 3. Bioactive compounds in DS (100 g).
| Sample | TP - (mg GAE) | TA - (mg Cyanidin-3-glucoside) | PAT - (mg CE) | AA - (µmol TE) |
|---|---|---|---|---|
| DS1 | 266.27 - (SD 4.54a) | 110.90 - (SD 34a) | 11.77 - (SD 54a) | 202.55 - (SD 2.47a) |
| DS2 | 185.04 - (SD 1.00b) | 97.67 - (SD 12b) | 9.35 - (SD 43b) | 245.11 - (SD 4.66b) |
| DS3 | 150.81 - (SD 1.42c) | 127.08 - (SD 25c) | 23.29 - (SD 10c) | 194.39 - (SD 2.47a) |
TPF: Total Polyphenols; TA: Total anthocyanins; PAT: Proanthocyanidins; AA: Antioxidant activity. SD: Standard deviation (n=3). Values with different superscript characters within a row are significantly different (p<0.05).
PAT values obtained were lower than the original matrix (236.34 SD16.75 mg CE/100 g) presenting losses of 95.02%, 96.04% and 90.14%.
AA in this case showed a reduction compared with BCF+BB (284.63±3.78 µmol TE) approximately 28.83%, 13.88% and 31.70% for DS1, DS2 and DS3 respectively.
As we mentioned before, TDF was determined in the snack with higher antioxidant activity, DS2 was selected for this analysis. Values of TDF, IF and SF are indicated in Table 4.
DISCUSSION
The snacks developed had an improved nutritional profile compared to commercially available snacks and added value due to the ingredients used.
Discrepancy in caloric values of each snack could be related to fill nature and dough proportions used in each one.
Significative differences were found in TPF. Results were lower than the initial quantity in BCF+BB (900.05 SD57.29 mg EAG/100 g). Losses represent the 70%, 79% and 83% of the compounds in DS1, DS2 and DS3 respectively.
TA content was lower in the final products in comparison with BCF+BB at initial quantity (151.54 SD12,54 mg Cyanidin-3 glucoside/100 g). Decreasing was 26.83%, 35.54% and 16.14% for DS1, DS2 and DS3, respectively.
Values obtained, with respect to carbohydrates, were lower to Macías et al. (2013) who studied cookie formulations with a partial substitution of white carob flour (80:20), reporting 66.67 g/100 g19. It was also lower than Zavala Chingay (2016), who investigated the development of cookies with the replacement of white carob flour in different proportions (4, 8 and 12%)20 reporting 72.52; 70.42 and 68.30 g/100 g, respectively. Also, the values obtained were lower than those compared with the commercial product (77 g/100 g). Differences found could be attributed to ingredient proportion and flour variety as well as the fact that no white sugar was used in the formulation.
All values of protein were higher than Escobar et al. (2009), who reported values of 10.7 and 13.3 mg/100 g in the study of cookie development with carob cotyledon flour and wheat flour mixtures (10:90 and 20:80)18. According to results obtained, we can also claim the snack as “high protein food”21.
Although the amount of fat was like that found in the commercial product, the DS would have a healthy fat profile due to the fat used. In addition to the various values observed could be associated to the different flour percentage added. Data found was like Macias et al. (2013), for cookies made with wheat flour and BCF, reporting fats values of 15.3, 15.9 g/100 g and 17.3, 16.7 g/100 g19.
In terms of moisture, this parameter increased proportionally to the quantity of BCF added, that could be attributed to flour sugars composition who can absorb the environmental humidity20,21. Protein content also has an influence in product moisture, mayor protein levels produced a more viscous dough with less expansion during the baking process due to the water absorption capacity of proteins when gluten structure formed22.
In reference to ashes, Macias et al., (2013) and Escobar et al. (2009) analyzed substitution of carob flour in cookies at different proportions (10-20%), shown quantities ranged up to 0.98 and 0.60 g/100 g; 1.2 and 1 g/100 g respectively18,19. The variations observed could be associated to variety of legume, portion used, and proportions applied in dough.
The decrease in TPF can be explained because of the heat treatment applied, producing ruptures and degradation of covalent bonds23-25. According to polyphenol intake studies, there is a relationship between the consumption of this compounds and the appearance of non-communicable diseases26-28. Besides, estimated polyphenol intake is around 500-900 mg28, according to these values, the snacks' formulations cover the 53.25%; 37.08% y 30.16% for DS1, DS2 and DS3 respectively.
Changes in TA can be attributed to anthocyanins' susceptibility to high temperature, light, and oxygen. These conditions provoke adverse effects over their structure26-27.
Losses in PAT as anthocyanins could be attributed to heat conditions applied. Besides, PAT determination could be conditioned for non-extractable pigments retained in the original matrix28.
Changes in AA can be explained for the chemical reactions produced during the cooking process like mailoindins from Maillard reaction25.
Dietary fiber values found were lower than Escobar et al. (2009) who studied the same fractions of fiber indicating 2.80; 2.18 and 0.62 g/100 g in cotyledon algarroba flour at 20% of substitution18. Differences found could be attributed to flour origin and variety, proportion used, and the fruit filling added.
In comparison with similar commercial products (5 g/100 g), TDF was higher with a percentage around 59%. Thus, a snack ration (30 g) covers the 16.5% of daily recommendations for dietary fiber. In addition, this product can be claimed as “high fiber content” according to stablished in Argentinian Food Code21.
CONCLUSIONS
The development of snacks with black carob flour and discarded blueberries was possible. The formulations performed had functional properties, were high in protein content and reduced in total caloric value in comparison with commercial products available. DS2 was the snack with the greatest antioxidant activity and, according to fiber values, it is a product “high in fiber content”. Despite the losses caused by the cooking process in the content of bioactive compounds, the antioxidant activity was greater than 50% in all the snacks produced.
