The purpose of developing a functional snack product using Spirulina and Bilberry is to outline the applicable use of these two understudied and underutilized ingredients in the food industry space to fill the market gap in the development of functional food products for children. From chemical analysis conducted in previous research, both Spirulina and Bilberry have the potential to have functional benefits that can aid against oxidative stress and chronic diseases. Therefore, with the growing epidemic of childhood obesity, the aim of the development of the snack product was to target children with an on-the-go snack mini muffin to aid in the reduction of obesity in children.

After many trials, three mini muffin formulations were developed, one control (chocolate) and two “Spilberry” (combination name between Spirulina and Bilberry) muffins: 1% Spirulina + 4% Bilberry (1% S + 4% B) and 2% Spirulina + 8% Bilberry (2% S + 8% B). The formulations can be seen in Tables 1-3 . Staying within food industry trends, the functionality of each ingredient is found in Table 4 , utilizing alternative flours: almond and gluten-free all-purpose flours, as consumers are pushing for more alternative flours and gluten-free products in the light of celiac disease and gluten sensitivity/gluten intolerance.

Product quality and safety is one of the top priorities when developing a food product in an industry to sell. Therefore, to ensure maximum safety and quality of the control and “Spilberry” mini muffin products, shelf-life studies were conducted on all three product formulations for 9 days, in 3-day increments, with the muffins left out in a room temperature environment in a clam-shell packaging to mimic a grocery store bakery muffin. The following physiochemical properties were tested: pH, water activity (aw), and color based on Hunter’s LAB color scale where L* measures darkness to lightness (0 to 100) (L*), a* measures green (-) to red (+), and b* measures blue (-) to yellow (+).

To ensure consumer acceptability of the “Spilberry” mini muffins, a sensory panel was conducted on Alabama A&M University’s campus in the Department of Food and Animal Sciences’ Sensory Laboratory. Accessing consumer acceptability among minority adolescents, the sensory panel consisted of Black/African American (100%) males (26%) and females (74%) among the following ages: 17 (5%), 18 (47%), 19 (24%), 20 (13%), 21 (11%). The sensory participants were given a questionnaire to evaluate the three muffin formu-lations (control (with cocoa powder to mask color biasness), 1S% + 4%B, and 2%S + 8%B) for their acceptability, beginning with demographics and a pre-screen of questions as-sessing their consumption of muffins and awareness of Spirulina and Bilberry. To access consumer acceptability, the panelists were asked to rate the products using the 5-point Hedonic scale (1—Dislike very much, 2—Dislike a little, 3—Neither like nor dislike, 4—Like a little, 5—Like very much) based upon their acceptability of color, appearance, tactileness (touch), aroma, texture (mouthfeel), taste, and overall product acceptability.

Scanning Electron Microscopy (SEM) was used to examine the surface of the mini muffins to compare between subjective (consumer testing) and objective (texture analyzer) texture measurements. Proper sample preparation is important for SEM analysis. Volatile compounds such as water and oil must be removed from samples if any are present. Therefore, the mini muffin products were freeze-dried for 2 days to then be used for SEM analysis, ensuring all moisture was lost from the product, utilizing a VirTis Genesis Freeze Dryer. Reducing the particle size of each, the samples were placed on the SEM sample disk with Colloidal Silver Liquid. Afterward, the samples were coated with gold utilizing an Electron Microscopy Sciences Sputter Coating machine (Model: RV5; Code No.: A653-01-906; Serial Number: 037632871), to then be used for SEM analysis to be analyzed via micrographs of various specifications/magnifications.

The antioxidant activity of the muffin products (control (without cocoa powder), 1%S + 4%B, and 2%S + 8%B) was tested using the following assays: 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Ferric Reducing Antioxidant Potential (FRAP). Sample extractions were performed by mixing 100ml of 80% ethanol per 5g of the sample for 2 hours, to sonicate for 1 hour in a Bransonic M5800H Ultrasound Sonic Bath and centrifuged at 1107 g force at 4ºC for 20 minutes. Following, the product supernatants were filtered using Whatman #4 filter paper, then evaporated using a rotary evaporator and reconstituted using 10ml of the 80% ethanol solution. Afterward, the samples were stored in a 4ºC cooler for analysis.

2,2-diphenyl-1-picrylhydrazyl (DPPH) is a deep purple free radical scavenging assay used to evaluate the antioxidant potential of mini muffin products [10] . The principle of this assay is based upon the reduction of the deep, purple-colored DPPH radical to a pale yellow or colorless 2,2-diphenyl-1-hydrazine (DPPH-H) when it is in the presence of antioxidants [11] . For analysis, diluted 80% ethanol extracts of the mini muffin were prepared and combined with the DPPH radical. The two were then incubated and read at an absorbance of 517nm at 30-minute intervals for 90 minutes [12] .

Ferric Reducing Antioxidant Potential (FRAP) is an antioxidant test used to assess the reducing power of antioxidants present in various samples, including the mini muffins in the present study. Through a colorimetric reaction, the principle of the test is that in the presence of an antioxidant, the sample will reduce from ferric ions (FE³⁺) to ferrous ions (Fe²⁺). The FRAP assay involves the use of a FRAP reagent, which consists of a ferric chloride solution, a tripyridyltriazine (TPTZ) solution, and a buffer solution, typically an acetate buffer. The FRAP reagent reacts with the ferrous ions produced from the reduction of ferric ions, resulting in a blue-colored complex that absorbs light at 593 nm. The intensity of this blue color is directly proportional to the reducing power of the antioxidants in the sample. Therefore, in the procedure, the diluted mini muffin samples, extracted with 80% ethanol, were mixed with deionized water and the FRAP reagent to be incubated at 37˚C, and read at an absorbance of 593 nm [13] .

The shelf life of the control and “Spilberry” mini muffins was conducted over a 9-day period, with data taken in 3-day increments. Figure 1 shows the pH and water activity, and Figure 2 shows the color analysis of the top, side, and bottom of each product.

Over the 9-day period, the pH and water activity remained constant, with the pH ranging from 6.67 - 6.95, and water activity ranging from 0.84-0.89Aw. Color analysis of the top, side, and inside of the muffin product was conducted by using L*a*b* values to determine its color with L* measuring darkness to lightness (0 to 100) (L*), a* green (-) to red (+), and b* blue (-) to yellow (+). Overall, the L* seemed to decrease in lightness over the 9-day period, whereas the a* and b*value increased indicating a more of a red and yellow hue. The reason for the muffin’s darkening in color is oxidation during the shelf-life period, when left out in the room temperature environment within the clam shell packaging. The exposure to oxygen, light, and moisture could have contributed to the color change as well.

The pH of the 1% Spirulina + 4% Bilberry muffin increased from 6.62 (Day 1) to 6.90 (Day 9), whereas the water activity fluctuated between 0.82 - 0.86Aw ( Figure 3 ). The top, side, and inside of color of the muffin had slight changes during the shelf-life ( Figure 4 ). Low values of L* indicate the darkness of the product caused by the mixture between the dark green Spirulina powder and the dark purple-red Bilberry powder. Low a* values were present within the muffin product, which indicates the “redness” of the product which is produced by the higher amount of Bilberry to Spirulina in the formulation, and low levels of b* indicating the “bluer” hue of the muffins. Overall, the colors slightly changed over time with the a* and b* values increasing over time.

The pH of the 2% Spirulina + 8% Bilberry varied between 6.63 - 6.84, whereas the water activity decreased from 0.84 to 0.81, which could have been due to its exposure to oxygen, causing a loss in moisture. Increasing the Spirulina and Bilberry powder percentages in the 2% Spirulina + 8% Bilberry muffin formulations resulted in a rich dark purple hue, indicated by the low L*a*b* levels compared to the others. The reason for this could be the increased concentration of Spirulina and Bilberry interacting with each other to develop a rich dark purple/brown color. ( Figure 5 , Figure 6 )

One of the most important attributes of food products is its texture, which can be measured subjectively (human subjects) or objectively (instrumentation) [14] . Using objective measurements, Table 5 shows the results of the texture analysis of the three mini muffin formulations measuring post peak (N) hardness, integral (J), and slope (N/mm).

Means ± standard error (n = 3). Significant differences (p ≤ 0.05) of muffin texture properties, shown in columns, indicated by letters “abc”.

Post peak (N) is the amount of force involved in compressing the muffin sample. Comparing the three mini muffins, there were no significant differences among the three. However, the highest post peak value was seen in the 2 S% + 8 B% (170.00 ± 5.13 N), whereas the lowest was found in the control (152.33 ± 3.67 N). The reasoning behind this can be due to high amounts of Spirulina and Bilberry incorporated into the product, specifically incorporating higher amounts of the Spirulina microalgae. Integral (J) calculates the work per unit fracture surface area of the product [15] . The integral range of the muffin formulations ranged from 1.49 ± 0.15 to 2.48 ± 0.24 (control - 2S%+8B%) with 2 S% + 8 B% (2.48 ± 0.24 N) being significantly higher than control (1.49 ± 0.15 N) and 1 S% + 2 B% (1.52 ± 0.18 N). Opposite results were found in the slope (N/mm), where the control (11.36 ± 0.48 N) was higher compared to 1 S% + 2 B% (10.40 ± 1.69 N) and 2 S% + 8 B% (8.15 ± 1.23 N) muffins.

Snacking is very common among college students. With their busy schedules, it is important for them to consume snacks that are not only tasty but filling and provide them with satiety and energy throughout their productive days. Therefore, accessing college students was important for this study, as well as with childhood obesity and other chronic diseases being the more prominent in minority adolescents, accessing consumer acceptability among these individuals was crucial. Therefore, consumer acceptability of the product was accessed using 38 minority panelists, whom were asked to give their acceptability of color, appearance, tactileness (touch), aroma, texture (mouthfeel), taste, and overall product acceptability based upon a 5-Point Hedonic scale (1—Dislike very much, 2—Dislike a little, 3—Neither like nor dislike, 4—Like a little, 5—Like very much). The results for each muffin formulation can be seen in Figures 7-9 .

Figure 7 represents the product attribute scores of the control mini muffin based upon consumer acceptability. Out of the 38 participants, the majority of the individuals liked the color, appearance, and touch, but disliked the texture, taste, and overall acceptability of the chocolate mini muffin. Whereas the two “Spilberry” mini muffins showed opposite results. Panelists disliked the color and aroma, but liked the overall touch, texture, taste, and overall acceptability of the 1% S + 4% B mini muffin. Consumers did not respond as well to the 2% S + 8% B compared to the 1% S + 4% B in regard to color, appearance, and taste. However, some of the individuals liked the color and appearance better than the 1% S + 4% B but disliked the taste and overall acceptability more. Overall, consumers accepted each formulation in terms of different product attributes, mainly taste, texture, and touch. A few consumers were not pleased with the color of the two “Spilberry” formulations, due to the mixture of the extremely dark green and dark red colors that caused a dark green-blue hue (1% S + 4% B) and a dark red-purple hue (2% S + 8% B). Both are rather acceptable on their own; however, in combination, the off-green and dark purple colors are not acceptable. The reason for this is due to consumer perception and what they previously were accustomed to. Consumers are not accustomed to seeing green-blue and red-purple cake, bread-like products, unless it is artificial colored icing. Therefore, perception is very key when it comes to sensorial results.

Overall, the results of the product development suggested that Spirulina and Bilberry, are acceptable to be utilized in food products to increase their use in the food industry for functional food health benefits among adolescent nutrition product development.

Figures 10-13 show the micrograph images from the SEM of the chocolate control, 1% S + 4% B, and 2% S + 8% B muffin products. The purpose of using SEM was to analyze the texture and air pocket formations within the product to compare and understand why some consumers accepted the texture of one muffin (1% S + 4% B) over the other (2% S + 8% B). Figure 10 shows major air pocket formation within the chocolate muffin, which could be caused by the leavening agent used, overmixing, aeration, etc. These air pockets are often associated with a lighter, more open crumb structure. Figure 11 shows very nice structural formations of the 1% S + 4% B muffin product, which shows a well-defined and consistent microstructure with evenly distributed air pockets and a uniform crumb texture. This suggests that the lower concentration of Spirulina and Bilberry may have contributed to a more desirable texture by maintaining better batter consistency and air incorporation. In contrast, Figure 12 shows some differences in texture across the surface of the 2% S + 8% B muffin. It can be observed that the product has a crumbly and compact texture in comparison to the other two muffins in Figure 13 . This uneven texture is indicative of a denser and more compact structure. The reasoning behind this discovery could be the increased concentration of Spirulina. Spirulina is a microalga and can be used as a thickening agent in the product. During preparation of the raw muffin batter, the texture of the 2% S+ 8% B muffin batter was thicker compared to the other two products, which resulted in a thicker more compact texture after baking. This increased viscosity led to a denser batter that did not rise as much during baking, resulting in a more compact and crumblier final product. Thus, the higher Spirulina content appears to have significantly impacted the texture of the muffins, influencing consumer preference to prefer the 1% S + 4% B muffin over the 2% S + 8% B muffin.

The results for DPPH and FRAP of the control (without cocoa powder) and “Spilberry” muffin samples can be seen in Table 6 and Table 7 . The purpose of the control without cocoa powder was to have a pure control to measure the antioxidant activity of the Spirulina and Bilberry without the interference of the bio-active compounds present in cocoa powder.

The DPPH radical exhibits a deep purple color due to the delocalization within its aromatic rings. When the DPPH radical accepts an electron from an antioxidant, it becomes neutralized, causing the color to change from deep purple to pale yellow. The radical scavenging assay was conducted using an absorbance of 517 nm [11] . Table 6 presents the DPPH antioxidant activity of control and “Spilberry” muffins extracted with 80% ethanol.

*Data was averaged in triplicates (n = 3). Data based on serving size of 2 muffins (30 g). Significant differences (p ≤ 0.05) of muffin extracts indicated by letters “abc”.

DPPH ranged from 47% to 90% % inhibition with the 2%S + 8%B muffin being 1.35 - 1.91 higher compared to the 1% S + 4% B and control muffins. In order for the muffin products to be considered inhibiting the DPPH radical as an antioxidant, the % inhibition must be above 50%. Fortunately, both muffins containing Spirulina and Bilberry showed an overall inhibition greater than 50%, suggesting that Spirulina and Bilberry are two powerful antioxidants that can be used in a functional food product.

The Ferric Reducing Antioxidant Potential (FRAP) assay measures the reduction of ferric-tripyridyltriazine [Fe^III(TPTZ)]³⁺ to the ferrous complex [Fe^II(TPTZ)]²⁺ by antioxidants, resulting in a dark blue color [16] . Figure 6 illustrates the FRAP values for the control and “Spilberry” muffins.

*Fe (II): Ferric iron; Data was averaged in triplicates (n = 3). Data based on serving size of 2 muffins (30 g). Significant differences (p ≤ 0.05) of muffin extracts indicated by letters “abc”.

Following similar trends from the DPPH antioxidant assay, FRAP was significantly higher in the 2% S + 8% B (177.08 ± 0.26) formulation compared to 1% S + 4% B (125.42 ± 0.97) and the control (73.76 ± 3.00) muffin which was a 2.4 fold increase. Suggesting that as concentrations of both Spirulina and Bilberry increased, antioxidant potential/power increased as well.

Overall, the DPPH and FRAP increased as Spirulina and Bilberry concentrations increased from 1% Spirulina + 4% Bilberry to 2% Spirulina + 8% Bilberry.