Asian Journal of Dairy and Food Research

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Full Research Article

Diversification of Cocoa Drinks with Chlorella vulgaris Hydrolysate as a Functional Innovation for Health Benefits

Dian Iriani1,*, Harifa Syah Putra1, Bustari Hasan1, Trisla Warningsih2, Zahtamal3, Hannan Khairu Anami4, Ronita Uli Simatupang1
  • https://orcid.org/0000-0002-4930-1651, https://orcid.org/0009-0009-7114-4646, https://orcid.org/0000-0002-6587-0651, https://orcid.org/0000-0001-6654-5845, https://orcid.org/0000-0003-1989-9258, https://orcid.org/0009-0003-8438-3634, https://orcid.org/0009-0002-1010-8333
1Department of Fisheries Products Technology, Faculty of Fisheries and Marine Science Universitas Riau, Indonesia.
2Department of Social Economy of Fisheries, Faculty of Fisheries and Marine Science, Universitas Riau, Indonesia.
3Department of Public Health, Faculty of Medicine, Universitas Riau, Indonesia.
4Hospital of Universitas Riau, Universitas Riau, Indonesia.

ABSTRACT

Background: Cocoa has long been known as the main ingredient in making chocolate, which is applied in food such as very popular chocolate drinks. Thus, cocoa is believed to have a promising approach in terms of diversification and innovation of health products. One of them is the fortification of Chlorella protein hydrolysate which is considered a sustainable protein source for the future in increasing the protein value of health drinks. The purpose of this study was to evaluate the sensory and physicochemical properties   of cocoa drinks fortified with Chlorella hydrolysate.

Methods: The experimental design used was a non-factorial completely randomized design (CRD) with 4 treatment levels, in the fortification of Chlorella protein hydrolysate into cocoa drinks 0% (HPC0), 10% (HPC1), 15% (HPC2), 20% (HPC3).

Result: Chlorella protein hydrolysate had a protein content of 90.47%. Overall, the sensory value of cocoa drink was highly favored by panelists (p<0.001) and the addition 20% of Chlorella hydrolysate (HPC3) obtained the highest taste value. Moreover, the chemical composition for all treatments increased and HPC3 had the highest protein (11.15%) with 72.97% of solubility and 2.27 ppm of phenol (p<0.001). Cocoa drinks can be widely applied worldwide for diversification and innovation of valuable products.

INTRODUCTION

Indonesia is one of the world’s third largest cocoa producing countries after Gading Coast and Ghana, with a production of 13% (Priyono, 2021; FAO, 2024). Cocoa contains polyphenols and flavonoids that have the potential to act as antioxidants. Around 10% of the dry weight of cocoa beans also contains bioactive compounds such as catechins, anthocyanins and proanthocyanidins (Zugravu and Otelea, 2019). However, cocoa beans also contain theobromine, polyphenols and flavonoids which can cause a bitter taste after being processed into chocolate so that sugar is usually added to beverage products to provide a sweet taste (Goya et al., 2022; Cempaka et al., 2021; Nidhilangelo and Antony, 2024). Along with the development of health and sustainability trends, the diversification and innovation of cocoa-based products are growing. Cocoa can be developed and applied as a health drink.
       
One interesting breakthrough is the combination of cocoa with nutrient fortification such as Chlorella protein hydrolysate. Chlorella has attracted the interest of scientists globally because it has essential bioactive compounds such as pigments, proteins, carbohydrates, vitamins and polyunsaturated fatty acids (PUFA) (Lorenzo et al., 2023; Levasseur et al., 2020; Je and Yamaoka, 2022; Balasubramaniam et al., 2021). Chlorella has been exploited commercially and the most widely used genera are Chlorella for functional foods (Beheshtipour et al., 2012). The potential of Chlorella sp. can be used as a nutrient for a food product because it has a high protein nutritional content of 51-58% and antioxidant content such as phenolics (Iriani et al., 2011; Iriani et al., 2017; Iriani et al., 2023., Iriani et al., 2025).
       
Various studies have revealed that the quality of products such as cheddar cheese, bread and cosmetics after the addition of Chlorella has no negative impact on the physicochemical characteristics (Gouveia et al., 2007; Iriani et al., 2023; Diprat et al., 2020; Batista et al., 2017; Bhatnagar et al., 2024). For example, chocolate showed low oxidative stability and peroxide value and maintained its quality after fortification with lyophilized and encapsulated Scenedesmus obliquus (Hlaing  et al., 2020). Yogurt fortified with Chlorella (0.25%) improved water retention and maintained whey quality for 28 days of storage (Barkallah et al., 2017). Chocolate milk fortified with Spirulina-LEB-18 improved solubility and decreased hygroscopicity (<10%) (Oliveira et al., 2021). Nutritional properties evaluated by the addition of Spirulina (10%) in Saudi Arabian date drinks resulted in good quality for the body’s immunity (Aljobair et al., 2021).
       
However, the application of algae biomass in large quantities can cause green color and fishy taste which are usually unacceptable in the product (Iriani et al., 2017). In addition, nutritional quality depends on the type of microalgae and processing conditions which may have low digestibility and lower nutrient availability (Qazi et al., 2021; Muys et al., 2019). One way to overcome this may be to improve the functional properties of Chlorella in the form of hydrolysate. Chlorella protein hydrolysate shows interesting properties such as antioxidant, antihypertensive, anti-inflammatory, anticancer and antimicrobial (Cunha et al., 2022). Through the hydrolysis process, the rigid cell wall of Chlorella becomes weak, making it easier for protease reactions to access intracellular proteins (Cunha et al., 2022). In addition, hydrolyzed microalgae peptides also have physical properties such as solubility, emulsifying and foaming which can be useful for industrial applications that have an impact on improving the nutritional value of food products. Therefore, fortification of Chlorella protein hydrolysate in cocoa beverage products is one of the best ways to improve overall healthy food intake with low side effects compared to intact protein and free amino acids from Chlorella biomass (Iriani et al., 2017; Morgan et al., 2021; Ornella et al., 2024). This reason is also supported by Pushpa et al., (2018) that beverages formulated through the protein hydrolysis process can enhance nutritional quality, making them a health drink recommended by the WHO.
       
Thus, cocoa processed and consumed in the form of delicious beverages is believed to have a promising approach for Chlorella hydrolysate fortification. The utilization of algae proteins to improve texture and nutritional value has been carried out for more than 20 years (Ferreira et al., 2021; Bhatnagar et al., 2024). Previous studies have revealed that the beneficial properties of Chlorella can be further enhanced in the form of protein hydrolysates. This is also because Chlorella biomass has a green color and a fishy taste that interferes with consumer preferences and product sensory. Therefore, instead of adding algae biomass as a whole, isolating proteins derived from algae and adding them to food products can be a promising solution to overcome this problem.
       
To the author’s knowledge, there has been no comprehensive study on the application and fortification of Chlorella proteins in cocoa. Considering that the global algae protein market has been growing and continues to increase to expand. The purpose of this study was to provide a comprehensive interpretation of cocoa formulations fortified with Chlorella protein hydrolysate as functional drink. This study also summarized the effects of Chlorella hydrolysate fortification on the sensory, nutritional, solubility and phenolic properties of cocoa. This latest study related to cocoa product development helps in-depth insight into the current trends related to algae protein extraction which is very important for the successful commercialization of algae protein in the cocoa industry.

MATERIALS AND METHODS

Time and place
 
This research was conducted from August to December 2024. Samples were processed and analyzed at the Fishery Products Processing Laboratory, Fishery Products Biotechnology Laboratory, Fishery Products Chemistry Laboratory, Faculty of Fisheries and Marine Sciences, Universitas Riau.
 
Materials and equipment
 
The main materials used in this study consisted of cocoa powder and Chlorella vulgaris was cultivated in the Fishery Products Biotechnology Laboratory, Faculty of Fisheries and Marine Science Universitas Riau, Indonesia. Other ingredients were distilled water, H2SO4 (Merck, Germany), ethanol 96% (Merck, Germany), ethanol 70% (Merck, Germany), ethyl acetate (Merck, Germany), hexane (Merck, Germany), PP indicator (phenolpthalein) (Merck, Germany), kjeldhal tab catalyst (Merck, Germany), 45% NaOH, 20%, boric acid (H3BO3) (Merck, Germany), methyl blue and methyl red indicators (Merck, Germany), HNO3 (Merck, Germany). The equipment used in this study were digital scales (Kova), incubator (binder), measuring cylinder (Pyrex), beaker (Pyrex), spatula, Erlenmeyer flask (Pyrex), hot plate (Thermo Scientific), porcelain cup (Oem), filter paper (Hellma), desiccator (Duran) and UV-Vis spectrophotometer (Optima).
 
Extraction and hydrolysis of Chlorella protein
 
The method introduced by Winarni et al., (2022) and Gao et al., (2020) with modified. Chlorella was cultured for 12 days under room temperature. Chlorella was harvested by centrifugation for 4 minutes, at 4000 rpm to obtain a paste. Furthermore, it was lyophilized with a freeze dryer at a temperature of -63.5oC for 3 days to obtain algae powder. Furthermore, the algae powder was extracted with 0.3 M NaCl (1:10 w/v) at pH 10 and incubated for 3 hours at 5oC. Then, it was centrifuged at 4000 rpm for 26 minutes. The supernatant was precipitated for 10 minutes using 0.5 M HCl until pH 4.2. It was centrifuged again and the precipitate was mixed with distilled water (2:1 v/v) at pH 7.2 using 1 N NaOH and it was lyophilized using a freeze dryer. Furthermore, the algae protein was dissolved with 5% phosphate buffer and 2% pancreatin enzyme was added in the substrate (Gharehbeglou et al., 2024). Then, it was incubated at 50oC for 3 hours and inactivated at 90oC for 15 minutes. The solution was centrifuged at 4000 rpm for 20 minutes and the supernatant was lyophilized using a freeze dryer at -63.5oC for 6 hours.
 
Procedure for making cocoa drinks with Chlorella hydrolysate
 
The cocoa drink formulation refers to Cempaka et al., (2021) which was modified as shown in Table 1. The concentrations of Chlorella hydrolysate were 0% (HPC0), 10% (HPC1), 15% (HPC2) and 20% (HPC3), this percentage was obtained from the total amount of cocoa powder used.

Table 1: Formulation of Chlorella hydrolysate in cocoa drinks.


 
Sensory evaluation
 
Organoleptic analysis refers to SNI (2006). Chlorella protein hydrolysate was dissolved using 200 mL of hot water for 2 minutes and added 2 spoons of cocoa. As many as 80 untrained panellists for this evaluation. This analysis provides a preference value for the quality of product characteristics according to the numerical scale that had been given. The scores given range from 1 to 9 (1 = very much dislike; 6 = like; 9 = very much like). The higher of value, the more preferred of the product.
 
Proximate analysis
 
Chlorella protein hydrolysate was measured for protein, fat, carbohydrate, water, ash and fiber analysis according to AOAC (2005).
 
Solubility test
 
Solubility testing based according to Mosii et al., (2023)  was carried out to measure the solubility level of the cocoa-Chlorella powder produced. A total of 1 g of powder (a) was dissolved in 20 mL of distilled water then filtered it with Whatman No. 42 filter paper. Before use, the filter paper was dried in an oven at 105oC for 30 minutes and weighed (b). After filtering, the filter paper was dried again in an oven for 1 hour at a temperature of 105oC. After that, the filter paper was cooled in a desiccator and then weighed until a constant weight was achieved (c).
 
  

Analysis of phenol content
 
Total phenol analysis refers to Hazra et al., (2008). A total of 5 mg was dissolved in 5 mL of demineralized water. The sample dilution results were taken as much as 1 mL, 1 mL of 96% ethanol, 5 mL of distilled water and 0.5 mL of 50% Folin Ciocalteau reagent were added. The mixture was left for 5 minutes and 1 mL of 5% Na2C3 was added. The mixture was homogenized and then incubated in the dark for one hour. The standard used was gallic acid with concentrations  of 20, 40, 60, 80 and 100 ppm. The standard solution and sample were measured using a UV-Vis spectrophotometer at a wavelength of 725 nm. The concentration values   of the standard solution and the absorbance of the blank were plotted on the x and y axes respectively in the linear regression equation:
 
y = ax + b
 
The equation formed is used to obtain the total compound value (mg GAE/G). The calculation of total phenol uses the following formula:
 
F = CxFP/a
 
Description:
F = Phytochemical compound (mg GAE/g extract).
C = Standard concentration (mg/L).
a = Linear regression.
FP = Dilution factor.
 
Data analysis
 
The experimental design used in the study of Chlorella hydrolysate fortification in cocoa drink was a completely randomized design (CRD) non-factorial with 4 levels of treatment: 0% (HPC0), 0.5% (HPC1), 0.75% (HPC2), 1% (HPC3) of cocoa weight and tri replicate. Chemical composition data were analyzed and tabulated in table form. Significant differences in treatment (p<0.001) using Duncan multiple range test (DMRT), Statistical for Social Science (IBM SPSS 26).

RESULTS AND DISCUSSION

Sensory evaluation
 
The sensory values   of cocoa drink fortitified with Chlorella hydrolysate were shown in Table 2. The aroma value of cocoa fortified with Chlorella protein hydrolysate as a functional beverage in HPC0 and HPC1 was significantly different from HPC2 and HPC3 (p<0.001). For the taste value, HPC0 was significantly different from HPC1, HPC2 and HPC3 (p<0.001). For the appearance value, HPC0 was significantly different from HPC2 and HPC3, but not significantly different from HPC1 and HPC1 was not significantly different from HPC2 (p<0.001). The appearance value also had the same pattern as the texture value (p<0.001).

Table 2: Sensory value of cocoa drink fortified with Chlorella hydrolysate.


       
Overall, panellists preferred HPC3, due to the taste value increased with the addition of Chlorella protein hydrolysate, however, the aroma, appearance and texture values tended to decrease. The decrease in value was still in the category preferred by consumers (>7). In this study, panellists tended to detect a slight fishy aroma from cocoa products but the aroma was not as strong as the addition of microalgae biomass in chocolate reported by Hlaing et al., (2020). Chlorella hydrolysate and microenca psulated algae have better appeal in terms of taste and other sensory aspects aspects (Fradique et al., 2010). This encourages the potential for the development of new microalgae-based products. Therefore, the development of functional foods enriched with microalgae can be a new era for new food products with increased consumer acceptance.

Cocoa nutritional content
 
Chlorella protein hydrolysate contained 90.47% protein. In this study, the addition of Chlorella protein hydrolysate to cocoa can increase protein levels which are good for nutritional needs. Chlorella protein hydrolysate showed higher protein content results, but fat content, carbohydrate content and moisture content tended to decrease compared to HPC0 (p<0.001). The nutritional content of cocoa drink fortified Chlorella protein hydrolysate was shown in Table 3.
       
In this study, the low water content of cocoa can inhibit microbial growth so that it can extend the shelf life of the product (Quek et al., 2007). This reason is supported by GEA Niro Research Laboratory (2010), that the product standard of less than 10% is considered non-hygroscopic. Thus, all treatments showed low hygroscopicity and the desired characteristics for dehydration. In this study, the formulation of adding protein hydrolysate to cocoa can increase protein levels which are good for nutritional needs. HPC3 has a high protein content (12.12%) from other treatments, even higher than the addition of Chlorella microencapsulation to cocoa drinks reported by (Oliveira et al., 2021) which was 12%. This increase in protein levels also increases physical stability, which can be seen from the characteristics of cocoa. HPC3 also has higher solubility (72.97%) and phenol content (2.27 ppm) (Table 3). The higher solubility was due to more peptide bonds in the Chlorella protein hydrolysate, which were enzymatically hydrolyzed into the product (Sinaga et al., 2025).

Table 3: Nutritional content of cocoa fortified with Chlorella protein hydrolysate.



Table 3 also shown that the cocoa drink with 20% of Chlorella protein hydrolysate obtained higher value of protein content than cocoa drink fortified Chlorella powder  (9.52%) (Vattaah et al., 2024a) and it also shown that the cocoa drink had higher protein content than probiotic drink fortified Chlorella (3.77%) (Iriani et al., 2024). So, Chlorella protein hydrolysate give the new development in a drink innovation.
       
The increase in phenolic compound content indicates biological activity in reducing free radicals (Tejano et al., 2019). Secondary metabolites such as phenol can stabilize metabolic disorders (Savitri et al., 2019). This reason is also supported by Lakshmi et al., (2024) and Balamurugan et al., (2022). Therefore, HPC3 with addition 20% of Chlorella protein hydrolysate in cocoa drink is the best treatment that is promising as a functional drink.

CONCLUSION

In this study, high-quality Chlorella protein hydrolysate, with a protein content of 90.47%. Furthermore, fortification of Chlorella protein hydrolysate in cocoa had a significant effect on sensory values (taste, aroma, texture, appearance). The chemical composition with the addition of 20% Chlorella protein hydrolysate showed an increase in protein content of 12.12%, exhibiting solubility characteristics of 72.97% and phenol content of 2.27 ppm. The results of this study indicate that the 20% of Chlorella protein hydrolysate in cocoa drink was the best treatment and demonstrates antioxidant potential as a health drink.

ACKNOWLEDGEMENT

The present study was supported by Ministry of Education and Culture, Research and Technology of Indonesia, the Directorate of Research, Technology and Community Service (DRTPM) and the Institution of Research and Community Service (LPPM) Universitas Riau for funding this research in the year 2024.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article.

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