Developing cookies containing Chlorella : proximate composition, carotenoid content, and sensory acceptance

. Chlorella is a unicellular alga that contains high nutritional and bioactive compounds, especially protein and carotenoids. Therefore, it is potential as the ingredient for functional food. This study aimed to develop a cookie product with the addition of Chlorella . Besides, we also investigate the effect of Chlorella addition at the various concentration on cookies on the chemical properties and consumer acceptance. Analysis of chemical properties included proximate and total carotenoid content. A 9-point hedonic test determined consumer acceptance. The results were statistically analyzed by one-way analysis of variance (ANOVA). The results showed that the addition of Chlorella significantly increased the protein content but decreased the carbohydrate content (by difference). The addition of Chlorella also significantly increased the total carotenoid content. The highest carotenoid was reached by cookies containing 3% Chlorella with the content of 51.97±0.49 μ g/g. The cookies containing a 1% concentration of Chlorella had the highest overall acceptance (7.19 “Like moderately”), which were influenced by the acceptance of aroma, taste, and after taste.


Introduction
Chlorella is a unicellular green alga belonging to Chlorophyceae that lived in the freshwater and marine aquatic system [1].Chlorella is known for its high protein content that ranges from 55-60% dry weight.The protein is mainly composed of simple and essential amino acids, such as leucine, isoleucine, valine, lysine, methionine, histidine, threonine, phenylalanine, and tryptophan.The simple amino acids are more accessible to be absorbed by the human body [2].In addition, Chlorella also contains bioactive compounds such as omega-3 poly-unsaturated fatty acids (PUFAs), bioactive polysaccharides, pigments, vitamins, and phenolic compounds beneficial for human health.The well-known bioactive polysaccharide that presents in Chlorella -1,3-glucan soluble fiber.Other than that, Chlorella also contained rhamnose and mannose [3].The pigments present in Chlorella are chlorophyll and carotenoids.Lutein dominates the carotenoid in Chlorella.The rest of the carotenoids found in Chlorella -carotene, and ß-carotene [4].Vitamins present in Chlorella biomass are vitamin A, B3, B6, B9, and B12 [5].
Chlorella has been identified as "GRAS" or "Generally Recognized as Safe" to consume.For centuries, Chlorella has been consumed as a nutritional supplement in tablets or powder and consumed by mixing the dried biomass with water.Recommended daily intake for Chlorella biomass to show a health effect ranges from 3-10 grams per day [6].Consumption of Chlorella biomass could promote good health.PUFAs found in Chlorella can inhibit the synthesis of the proinflammatory mediator associated with diabetes [7].The -1,3-glucan soluble fiber also plays an antidiabetic effect and lower blood cholesterol levels [8].Carotenoids and polyphenols in Chlorella could act as an antioxidant.Carotenoids present in Chlorella have been used for application in human health related to cardiovascular diseases and macular degeneration [9].Vitamin A, B3, B6, B9, and B12 are beneficial for the biochemical function that happens in the body [10].
Due to its high nutrition and bioactive compounds, Chlorella could be utilized as functional food ingredients to increase its functionality.Nowadays, Chlorella is widely advertised as a modern healthy food that leads to the development of Chlorella-supplemented, -fortified, or -substituted food products, such as biscuits, bread, extruded snacks, pasta, and yogurt [11].
Though, the utilization of microalgae biomass in food products is familiar in several countries in Asia, such as Japan and Taiwan [10].In Indonesia, the utilization of microalgae in commercial food products is still limited.The utilization of Chlorella in commercial food products could increase the value of the food itself and minimize malnutrition in Indonesia.This study aimed to develop a cookie product with Chlorella powder to produce a functional food that might provide health benefits and increase Chlorella accessibility in Indonesia.This study analyzed the cookie chemical characteristics, including the nutritional content (proximate analysis) and carotenoid content on three different levels of Chlorella powder concentrations: 1%, 2%, and 3% of the total weight.The acceptance of cookies containing Chlorella was investigated by using the hedonic test.

Cookies production
Cookies without containing Chlorella powder were used as a control.The cookies containing Chlorella were made using Chlorella powder with a ratio of 1%, 2%, and 3% from the total weight, which was then mentioned as Cookies Containing Chlorella (CCC) 1, CCC 2, and CCC 3 (Table 1).The Chlorella powder (BioFinest®) used in this experiment was commercially available.In the making of CCC, the wheat flour content was reduced according to the concentration of Chlorella powder since it was considered a dry ingredient.The other ingredient, such as eggs, vanillin, butter, sugar, and baking powder, remained the same in each sample.The cookies were made using the creaming method by high-speed mixing (Oxone OX-855) of the wet ingredients, such as butter, sugar, eggs, baking powder, and vanillin until the mixture formed a soft and foamy texture.The wet ingredients were mixed for seven minutes.Next, the dry ingredients, such as flour and Chlorella powder, were mixed until combined, then sifted and gradually added to the wet ingredient's mixture.Finally, the mixtures were slowly folded until it formed a dough.The dough was sheeted to reach the thickness of 1 cm, then moulded and baked at 150°C for 30 minutes.The cookies diameter was 4 cm.The cookies were then cooled and packed in enclosed containers for further examination.

Proximate analysis
Proximate analysis was based on the AOAC procedure, and the result obtained was in the percentage of dry basis.
The ash content was analyzed by the dry-ashing method using a furnace at 550°C.The analysis of protein content using the Kjeldahl method based on AOAC 2001.11.2005 using KjelDigester for sample digestion.The protein content was analyzed using the nitrogen conversion factor of 6.25.The lipid was measured by Weibull hydrolysis and extracted using hexane.The carbohydrate content was calculated by difference.

Total carotenoid analysis
The analysis of total carotenoid content was based on AOAC 938.04.The analysis was using a spectrophotometer at 436nm.The standard curve was -carotene standard solution in seven different concentrations 0.05 mg/L, 0.75 mg/L, 1.50 mg/L, 2.25 mg/L, 3.00 mg/L, 3.75 mg/L, and 4.50 mg/L.

9-point hedonic test
The acceptance of control cookies and cookies containing Chlorella (CCC) was measured using a 9-point hedonic test.The panelists were expected to give ratings using nine preference scales on the sensory attributes, such as color, aroma, texture, taste, after-taste, and overall.Scale 1 represents "dislike extremely," 2 represents "dislike very much", 3 represents "dislikes moderately", 4 represents "dislike slightly", 5 represents "neither like nor dislike", 6 represents "like slightly", 7 represents "like moderately", 8 represents "like very much" and 9 represents "like extremely".
The hedonic test was carried out in BINUS University, Jakarta, Indonesia, by 52 untrained panelists aged over 18 years old and able to understand the hedonic testing procedure.The panelists mainly were university students, lecturers, and staff aged 19-55 years old.

Statistical analysis
The data obtained from proximate analysis and total carotenoid analysis were analyzed in triplicate.Data from all parameters in proximate analysis, total carotenoid content, and hedonic were subjected to one-way analysis of variance (ANOVA) with Duncan post hoc test (p<0.05).The hedonic test result of these sensory attributes: color, aroma, taste, and texture were correlated with overall acceptance using Pearson's correlation test (p<0.05).

Penalty analysis
JAR test results are the intensity of sensory attributes: green color, Chlorella aroma, sweetness, milk flavor, and smoothness.Penalty Analysis was conducted to determine which attribute that need to be improved in each formulation.Penalty analysis was done manually, which consisted of 3 main steps.The first step was calculating the respondent percentage for each category (too little, JAR, and too much).The second step was calculating the mean drop, which can be done by subtracting the acceptance level average of too little/too many groups from the acceptance level average of the JAR group.The last step was to make a plot with the xaxis representing respondent percentage and the y-axis representing a mean drop.

Results and discussion
Table 2 showed the proximate composition (w/w on a dry basis) of control and cookies containing Chlorella.The ash content of four samples ranged between 1.63 -1.69%, showing an insignificant difference between the control samples and the cookies containing Chlorella regarding Chlorella concentration.There was no clear increment trend of fat content in cookies containing Chlorella.The addition of 3% Chlorella resulted in a significant difference in fat content compared to control (p<0.05).CCC showed a significant increment of crude protein content (p<0.05), which CCC 3 reached the highest percentage of 9.18%.The higher Chlorella composition on cookies decreased carbohydrate content.The control sample contained 68.5% carbohydrate, while CCC contained carbohydrates that ranged between 66.71 -67.32%.Values are mean ± SD from triplicate determination.The numbers followed by different superscripts in the same row show significant differences (p <0.05).
The small addition of Chlorella powder in cookies might lead to insignificant ash content, regardless of 1-3% of Chlorella powder.The ash content from Chlorella biomass is around 5-10% of biomass on a dry basis.The Chlorella ash composition is smaller than other microalgae species such as Haematococcus lacustris (29% w/w on a dry basis) and Dunaliella salina (49% w/w on a dry basis) [12].Our result showed a similar outcome with a previous report [12], suggesting a negligible effect of Chlorella addition to ash fat content of cookies.Batista et al. [12] suggested that the addition of less than 5% of Chlorella biomass in cookies did not show any significant difference in ash content.The significant ash content resulted from the addition of 6% Chlorella sp. in cookies.Besides, the addition of Chlorella biomass at the concentrations 2% and 6% did not significantly alter cookies fat content [12].
Our study showed a small increment in lipid content of around 0.36 -0.57% due to adding Chlorella in cookies.A significant increment occurred by adding 2% and 3% of Chlorella.The lipid composition of Chlorella biomass is varied regarding species strain and cultivation condition, ranging from 1-12% of biomass on a dry basis.The result was contrary to the previous report.However, it was suggested that a certain amount of Chlorella addition may increase the total lipid in food products that contain Chlorella [12].
A significant difference showed in protein and carbohydrate composition between control and CCC.The result suggested a significant increment of protein composition, while the CCC showed a significantly lower carbohydrate composition.Chlorella contains high protein composition, ranging between 20-60% of the biomass, which might be attributed to the increment of protein composition in cookies.The lower composition of carbohydrates was possibly caused by a reduction of flour composition in the formula of CCC.Wheat flour has a higher carbohydrate content ranging from 64-71%, while Chlorella has a lower carbohydrate content ranging from 12-26% of the dry weight of biomass [13,14].Batista et al. [12] reported a similar result in which the addition of 2% Chlorella biomass increased around 1% of protein composition in cookies.The significant decreased in carbohydrates was shown in the addition of 6% Chlorella from 72.6% to 67.7%.
Analysis of total carotenoid content on control cookies and CCC samples to determine if there is a significant increase in the total carotenoid content in cookies.The results showed a significant difference between four samples: control cookies, CCC 1, CCC 2, and CCC 3 with an average total carotenoid value of 2.98±0.00Values are mean ± SD from triplicate determination.The numbers followed by different superscripts in the same row show significant differences (p <0.05).
Accumulation of carotenoid in Chlorella was affected by the species strain and growth condition [12].Chlorella fusca contained total carotenoid around 79.55 μg/g of dry weight, while Chlorella vulgaris composed total carotenoid around 81.81 μg/g [15].Chlorella genus primarily produced carotenoid pigments such as astaxanthin, canthaxanthin, and lutein [15,16].Previous study reported that consumption of 6 g dried Chlorella carotene, and zeaxanthin, indicating the potency of Chlorella as a carotenoid source with human health benefit [17].
The sensory acceptance of CCC was measured by using a 9-point hedonic scale.The panelists were expected to give ratings based on nine preference scales based on six sensory attributes: color, aroma, texture, taste, after-taste, and overall.The sensory evaluation of cookies was shown in Table 4.The results suggested that CCC 1 had an insignificant difference in sensory acceptance with control cookies.The color acceptance of CCC 1 was slight significantly lower to control that might be attributed to darker color due to green color from Chlorella.The cookies containing 1% Chlorella had a slightly higher sensorial score on attributes of aroma, texture, taste, and overall compared to control cookies, suggesting the preferable one was 1% CCC.The higher Chlorella concentrations, CCC 2 and CCC 3, decreased the sensory acceptability in the attributes of color, aroma, texture, taste, and after taste.CCC 3 had the lowest acceptance score in all sensory attribute.
Our results of the sensory acceptance of CCC were similar to the report of Batista et al. (2017).The 6% Chlorella biomass concentration resulted in unpleasant acceptance, especially in cookies taste and smell attributes.
Moreover, the tasters gave an unacknowledged perception for buying intention of 6% Chlorella-containing cookies, suggesting the rejection in the acceptance [12].Hexanal, the aldehyde, is the most volatile compound detected in Chlorella vulgaris that influenced fresh green grass odor formation [18].Therefore, hexanal and chlorophyll possibly led to the establishment of grassy taste, resulting in less favorable organoleptic acceptance in Chlorella-containing food products [19].In this study, Pearson's correlation test was performed to determine which attributes influenced the overall preferences of cookies, which was determined by correlation coefficient (Table 5).The result showed that all the attributes influenced the overall preferences, which were indicated by the significant difference of the correlation coefficient value based on a two-tailed test (<0.05).Although, aroma, taste, and after taste have higher correlation coefficients: 0.71, 0.78, 0.78, respectively.The result suggested that overall likings were influenced by aroma, taste, and after taste which was shown by the highest correlation coefficient.Moreover, the result supported by sensory acceptance where control cookies and 1% Chlorella-containing cookies had a higher acceptance score for the attributes of aroma, taste, and after-taste.Values are mean ± SD from triplicate determination.The numbers followed by different superscripts in the same row show significant Table 5.The correlation coefficient between sensory attributes to the overall preferences.

Conclusion
The addition of Chlorella in the cookie affected the proximate composition, which showed a significant increase in protein and fat while showed a decrease in carbohydrate content.Moreover, the addition of Chlorella in cookies resulted in a significant increment in carotenoid content, regardless of 1-3% of Chlorella powder.Cookies containing 1% Chlorella powder (CCC 1) showed the highest acceptance score.In contrast, the higher addition of Chlorella powder in cookies caused a reduction in sensory acceptance.The overall acceptance was strongly correlated to the aroma, taste, and after-taste attributes.For further research, reformulation might be essential to develop a cookie containing a higher concentration of Chlorella powder but well-acceptance to the consumer palate.

Table 1 .
Formulation of Cookies Samples.

Table 2 .
Proximate composition of control cookies and cookies containing Chlorella (CCC) in % (w/w) on a dry basis.

Table 3 .
Total carotenoid content of control cookies and cookies containing Chlorella (CCC).

Table 4 .
Total sensory acceptance of control cookies and cookies containing Chlorella (CCC).