Production of low-protein cocoa powder with enzyme-assisted hydrolysis

doi:10.21203/rs.3.rs-2084395/v1

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Production of low-protein cocoa powder with enzyme-assisted hydrolysis

https://doi.org/10.21203/rs.3.rs-2084395/v1

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published 29 Jan, 2024

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Amino acid-related disorders are caused by a defect in the metabolic pathways of amino acid groups. These patients must follow a lifelong protein diet. The objective of this study was to produce a low-protein cocoa powder with enzymatic hydrolysis and precipitation method. Firstly, the solubility of cocoa powder was increased by heat and enzyme treatments (Amylase, Viscozyme and Alcalase). Then, protein level was decreased by isoelectric precipitation. According to obtained results, the solubility of cocoa powder rose from 28.61% to 50.69%. Protein content decreased by almost 40.00% and significant reductions in the amino acid profile were also provided; the highest ones were detected in methionine (100%), lysine (73.65%), leucine (53.64%), alanine (46.17%), and isoleucine (44.73%) levels. Low-protein cocoa powder had high phenolic content (25.10 mg/g GAE) and the changes in the antioxidant activities were not significant (p>0.05). Chocolate production has also been carried out successfully with the obtained cocoa powder.

enzyme-assisted hydrolysis

cocoa powder

low-protein product

amino acid disorder

Inherited metabolic diseases consist of 900 rare disorders, and about 30 of them can be treated with low protein diets (LPD). Phenylketonuria (PKU) is the most frequent one, on the other hand, maple syrup urine disease (MSUD), homocystinuria (HCU), type I and II tyrosinemia (TYR I and TYR II), organic acidemias (OAs) and urea cycle disorders (UCDs) are other inborn errors of amino acids [1-2]. The common aspect of these diseases is a deficiency or absence of a single enzyme causing a block in the metabolic pathway. Amino acid deficiency patients must follow a lifelong diet; otherwise, mental retardation, autism, motor development disorders and other health complications are inevitable [3].

Phenylketonuria is an inherited metabolic disorder that occurs with a deficiency or absence of phenylalanine hydroxylase enzyme synthesized in the liver. Because phenylalanine hydroxylase is responsible for converting phenylalanine to tyrosine, its lack causes the accumulation of phenylalanine and its metabolites in the blood [1, 4]. MSUD is another inborn error of metabolism caused by elevations of the branched-chain amino acids in the urine. MSUD patients should restrict branched-chain amino acids, including leucine, isoleucine and valine in their diets [5]. HCU is an autosomal recessive disease characterized by increased homocysteine and methionine levels [6]. TYR I and II are caused by deficiency of the enzymes fumarylacetoacetase and tyrosine aminotransferase, respectively and patients should apply lifelong diet therapy with low phenylalanine and tyrosine content [7]. OAs also occur by accumulation of organic acids in the blood due to various enzyme deficiencies [8]. UCDs result from inherited defects in one of the enzymes or transporter molecules of the urea cycle pathway, which provides removal of ammonia from the bloodstream [9].

Although there are studies and applications related to gene therapy, enzyme therapy or liver transplantation for treating amino acid disorder patients, a low protein diet has been the accepted method worldwide. Enzyme therapy is a high-cost treatment and can cause significant side effects. On the other hand, gene therapy has been effective in mice, but it is still far from clinical trials. Therefore, dietary management remains the key therapy to keep blood amino acid concentrations within limits [10, 11].

A lifelong low protein diet policy has been accepted and implemented in recent years. Consumption of low-protein food, a precursor-free protein substitute with all other amino acids and restriction of protein intake are three main aspects of dietary treatment [11, 12]. Meat and meat products, seafood, milk and dairy products, bread, nuts and eggs are examples of protein-rich foods that patients should avoid. Although some foods and beverages contain all nutritional supplements except precursor protein, such as bars and powders, some products can also be modified to decrease their protein content. Modified foods are produced by hydrolyzing proteins or using protein substitutes [13]. These products can satisfactorily replace conventional ones that patients should not consume or are allowed to consume only a minimal amount in their daily diet. Studies have shown that these hydrolysates are produced from milk, whey protein, rice and wheat flour. In the production of hydrolysates, the solubility has been increased by enzyme application, and protein has been removed by using different technologies, such as precipitation, adsorption or ultrafiltration [13-16].

It is essential to increase the variability of low-protein foods and to buy these products from the market shelves and to reach them easily. These can make the patients’ lives easier, just like diabetes and celiac patients. Therefore, this study aimed to investigate whether cocoa, a popular food with high protein content, can be turned into one of the products that amino acid disorder patients can consume. The novelty of this study was thought to be the production of low protein food instead of obtaining hydrolysate. For this purpose, cocoa proteins were hydrolyzed and then decreased in protein level by isoelectric precipitation. After spray drying the cocoa solution, the prepared low-protein cocoa powder was used for chocolate production. Solubility, sugar content, protein content, total phenolic content (TPC), antioxidant activities and amino acid profile were also investigated.

Materials

Methanol, iron (II) sulfate heptahydrate, sodium carbonate, Folin–Ciocalteu phenol reagent, gallic acid (≥98.0%) were supplied from Merck (Darmstadt. Germany). (±)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, 97%). 2,2-diphenyl-1-picrylhydrazyl (DPPH), TPTZ (2,4,6-tripyridyl-s-triazine), iron (III) chloride hexahydrate were purchased from Sigma (St. Louis, MO). Cocoa powder was supplied from Barry Callebaut (Eskişehir, Turkey). Enzymes used in this study, Amylase AG 300 L (300 AGU/mL), Viscozyme L (≥100 FBGU/g) and Alcalase (2.4 AU/g) were provided from Novozymes Enzim Dis Tic. Ltd. Sti (Istanbul, Turkey).

Production of low protein cocoa powder

Production steps of low protein content cocoa powder are shown in Figure 1. Cocoa powder was first defatted three times using hexane and dried in an oven at 30^oC for 24 h. Defatted cocoa powder was suspended in distilled water at 10 g/100 mL and heated on a magnetic stirrer from room temperature to 55^oC for 1 h. Amylase was added at a ratio of 1:10 (w/w, enzyme: substrate ratio), and the reaction was allowed for 3 h under recommended conditions (at pH 6 and 55 ^oC). Amylase AG 300 L enzyme is a fungal glucoamylase produced from a selected strain of Aspergillus Niger. It is a starch degrading enzyme that hydrolysis of 1.4-alpha bonds in starch and also 1.6-alpha bonds. The obtained suspension was centrifuged at 13.130 x g for 15 min to separate the soluble part and kept at 4 ^oC. The insoluble part of the suspension was cooled to -18 ^oC and followed by shock heating with distilled water at a temperature of between 95 and 100 ^oC. It was allowed to continue until the temperature had stabilized (approximately 30 min) to obtain a further treated suspension. Then, it was boiled under the condenser for 3 h to loosen the tight structure of the cell walls. After heat treatment, Viscozyme, which provided tissue hydrolysis and releasing proteins from plant material, was applied at a ratio of 1:10 for 3 h (at pH 6 and 60 ^oC). It is a cell wall degrading enzyme complex consisting of beta-glucanases, pectinases, hemicellulases and xylanases [16] To further break down the proteins, pH and temperature were adjusted to 7 and 50^oC, and Alcalase was added at for more 3 h. The enzymatic treatments were terminated by raising the temperature to 95 °C for 5 min, and the hydrolysates were centrifuged at 13.130×g for 15 min to discard the precipitants. The soluble parts were combined, and isoelectric precipitation was applied at pH 3.5 using 2M H₃PO₄ solution. After centrifugation, the pH was increased to 7.0 using 2M Ca(OH)₂ solution. The pH was adjusted using Ca(OH)₂ and H₃PO₄ instead of NaOH and HCl to avoid taste change by means of salt formation. Ca(OH)₂ and H₃PO₄ formed calcium phosphate, a highly insoluble salt, and was easily separated from the soluble part [16, 17]. The precipitants were again discarded with centrifugation, and finally, the solution was spray-dried.

Spray drying

Maltodextrin was used for spray drying of cocoa powder. The feed solution was prepared in a ratio of 1:1 w/w (maltodextrin: the dry matter of cocoa solution). The spray drying was performed on a Mini Spray Dryer Büchi B-290 (Büchi Labortechnik AG, Flawil, Switzerland). A spray nozzle 0.7 mm internal diameter and compressed air at 6 bar were employed. The volumetric feed flow and air flow for atomization were programmed to 8.0 mL/min and 480 L/h. respectively. The set temperature was 160 ^oC. The proportions of maltodextrin to cocoa solution and other conditions were determined by preliminary experiments.

Determination of solubility

To determine the solubility of cocoa powders, 10 g of sample was mixed with 90 g of distilled and agitated at 20 ^oC for 1 hour. Then, the mixture was poured a tube and centrifuged at 13.130×g for 5 min. The upper soluble layer was used to measure the dry matter content [17].

Determination of sugar content

Glucose concentration of samples were determined according to the method of Cerit and Demirkol, (2021) [18]. Obtained cocoa solution before spray drying process was filtered with the 0.45 μm nylon filters. Ten microliters of the sample was injected to the HPLC system (Hitachi, Tokyo, Japan), which consists of L-2130 pump, L-2200 autosampler, L-2490 refractive index detector, L-2300 oven. Isocratic elution was performed with an acetonitrile: water (85:15) solution at a flow rate of 0.8 mL/min. Column (GL sciences, Inertsil NH₂ column, 250 mm × 4.6 mm i.d.) operating temperature and injection volume were 40 °C and 10 μL, respectively. Sugar concentrations were calculated with calibration curves prepared in the range of 0–5000 ppm. The R² values of glucose and fructose were 0.998 and 0.999, respectively.

Determination of protein content

The protein content of samples were determined using the Kjeldahl method. Protein nitrogen was converted to % protein using the coefficient 6.25 [19]. Protein content was determined for each pH case (pH: 3.0. 3.5. 4.0. 4.5. 5.0).

Determination of amino acid profiles

Amino acid profile of samples was performed with the LC-MS/MS system. The procedure was performed by the JASEM’s protocol (Table 1). For the quantification of amino acids (L-taurine, L-phenylalanine, L-tyrosine, L-leucine, L-isoleucine, L-methionine, , L-valine, L-glutamic acid, L-aspartic acid, L-threonine, L-serine, L-alanine, L-glycine, L-proline, L-cystine, L-arginine, L-ornithine, L-histidine, L-lysine), first, 0.5 g of the sample was taken into a screw-capped glass tube and 4 mL of the JASEM Amino Acid Reagent (JASEM JSM-CL-508, Istanbul, Turkey) were added for hydrolyses reaction at 110^oC for 24 h. Then, the hydrolysate was centrifuged at 5000 x g for 5 min, and the supernatant was diluted to 1 mL with distilled water. This procedure was repeated to obtain 800-fold dilution. Diluted hydrolysate (50 µL) was transferred to a vial, and 50 µL of stable isotope labeled internal standard and JASEM acidic hydrolysis reagent were added. The JASEM quantitative amino acids kit protocol (Sem Laboratuvar Cihazları A. Ş, Istanbul, Turkey) were applied for LC-MS/MS analysis. The analysis was carried out on an Agilent 1260 Infinity HPLC system (Agilent Technologies, Santa Clara, USA) connected to an Agilent 6460 tandem mass spectrometer equipped with electro spray ionization (ESI) probe. Three milliliters of aliquot were injected into Jasem amino acid column (JASEM JSM-CL-575) with a flow rate of 0.7 mL/min. The results were expressed as mg amino acid/100 g sample [20, 21].

Table 1 LC-MS/MS analytical parameters.

Initial pressure	110 bar
Column temperature	30^oC
Polarity	Positive
Gas temperature	150^oC
Gas flow	10 L/min
Sheath gas temperature	400^oC
Sheath gas flow	10 L/min
Nebulizer pressure	40 psi
Capillary voltage	2000 V (positive)

Determination of total phenolic content and antioxidant activities

The samples were defatted at the beginning of the process. Therefore, the extraction was directly applied according to Capanoglu et al. (2008) method with some modifications [22]. A hundred milligram of cocoa powder was weighed and 3 mL of methanol: water (75:25, v/v) solution was added. The solutions were vortexed and they were incubated in the ultrasonic bath (Bandelin Sonorex, RK 100H, Germany) for 15 min to increase the extraction efficiency. After centrifugation (13.130× g, 4°C, 10 min), supernatants were separated and the above procedure was repeated twice. All supernatants were collected, and the final volume was adjusted to 10 mL.

The prepared extracts were diluted one-fifth ratio for TPC analysis. Then, 2 mL of distilled water and 0.2 mL of Folin–Ciocalteu reagent were added, and kept for 3 min. After addition of 1 mL of sodium carbonate (20% w/v) solution, the mixture was incubated for one hour at room temperature. The absorbance was recorded at 765 nm wavelength by using a UV-VIS spectrophotometer (Shimadzu UV mini 1240. Japan), and the standard curve was created by 0-500 ppm gallic acid solutions (R² = 0.998) [23].

The antioxidant activities of cocoa powders were performed using two spectrophotometric methods: DPPH scavenging activity and FRAP assay. DPPH scavenging activity was determined according to Brand-Williams et al. (1995) with some modifications [24]. Briefly, 200 µL of prepared extracts were diluted in a ratio of 1:10 and mixed with 3 mL of 0.051 mmol/L DPPH solution, followed by incubation at room temperature for 30 min. The absorbance was read at 517 nm wavelength, and different concentrations of standard Trolox solution (0-50 ppm) were prepared to construct the standard curve (R² = 0.996).

FRAP assay was performed using the method developed by Benzie and Strain (1996) [25]. In a test tube, 0.1 mL of extract (dilution factor. 1:10). 8 mL of FRAP reagent prepared by mixing 300 mmol/L acetate buffer (pH 3.6), 10 mmol/L TPTZ, and 20 mmol/L FeCl₃.6H₂O at the ratio of 10:1:1 (v/v) and 1.2 mL of distilled water and were added. The mixtures were incubated in a 37 ^oC water bath for 15 min. The absorbance was determined at 593 nm, and the calibration curve was constructed with standard FeSO₄ solutions (0-1000 ppm. R²= 0.999).

Chocolate production

The ingredients used for chocolate production were cocoa, sugar and cocoa butter in a ratio of 14%, 45% and 40%, respectively. Other ingredients such as milk fat or milk powder were not added because this product was prepared for PKU patients. Codex Alimentarius (2003) indicates that the chocolate shall not contain less than 14% fat-free cocoa solid; therefore, the recipe of chocolate was adjusted according to it [26]. First, cocoa butter was liquefied by the bain-marie method and then the cocoa powder was added and mixed with a mixer (Fakir-Mezza plus. Germany). The mixture was ground in melanger (Santha. USA). Lecithin was added after 6 hours of thinning process and mixed for more 30 min. Finally, the chocolate mass was cooled to 28°C, then heated to 32 °C and tempering was completed [27]. Chocolate production was repeated for unprocessed cocoa powder and low-protein cocoa powder.

Statistical analysis

The results are expressed as mean ± standard deviation. The statistical analyses were performed using SPSS (version 11.5. SPSS Inc.. USA). T-test (α = 0.05) was applied to show the difference between control and low-protein cocoa powder.

Most of the food for amino acid metabolic disorder patients is in the form of mixtures of specific and limited nutritional components. These patients can not generally consume basic food sources in their diets [2]. As far as we know, there is no research about the production of low-protein cocoa powder. Cocoa is a popular food with a characteristic taste and unique properties such as health-promoting effects. It is a primary raw material of chocolate and is also used as an ingredient in many preparations, such as cookies, drinks, bread and cakes [28, 29]. For the reasons mentioned above, in this study, low protein-cocoa powder was produced to provide an important alternative and diversity in patients’ diets and improve their life quality.

Cocoa powder has low solubility due to the high fat content and a large number of polysaccharide-based carbonyl groups [30]. Therefore, the first step of low-protein cocoa production was increasing the solubility of powder with heat and enzymatic treatments. Amylase and Viscozyme were used for starch and cell wall degradation, respectively. Alcalase was also added for further break down the proteins in cocoa powder. The reducing sugar contents of cocoa powder before and after treatment are given in Table 2. Although reducing sugar were not detected with the HPLC-RID detector in the control powder, (sugar content is specified as 0.4 g/100 g on the product label), the amount of glucose and fructose were found to be 21.34 and 1.14 g per 100 g of low-protein cocoa powder. It has been reported that free sugar content of cocoa bean is not more than 2-4% (dry weight) and predominant sugars are sucrose, fructose and glucose. While reducing sugar increases during fermentation due to enzymatic reactions, it decreases after drying and roasting processes. Therefore, there was no reducing sugar in control sample, but, it was determined after enzymatic treatments. Moreover, chemical conversion of sucrose into glucose and fructose due to pH drop during processing may also increase the reducing sugar concentration [31, 32]. The solubility of cocoa also increased from 28.61% to 50.69% (Table 2). Blondeel et al. (2009) stated that their invention provided at least 50% solubility of cocoa powder. In addition, it was reported that 14.5% of starch and 22.5% of cellulose converted to glucose, dextrin and glucose oligomers which affected the solubility [17].

Table 2 Sugar content and solubility of samples.

Samples

Glucose

(g/100 g)

Fructose

(g/100 g)

Solubility

(%)

Control cocoa powder

28.61±0.08

Low-protein cocoa powder

21.34±1.21*

1.14±0.30*

50.69±0.07*

_{* shows the significant difference between control and low-protein cocoa powders (p<0.05). nd: not detected}

The protein content of cocoa powder is reported as approximately 18-19 g/100 g in the literature, and it was determined as 17.8 g/100 g in this study [29]. Preliminary studies showed that precipitation of proteins was highest when the pH of the solution was adjusted to 3.5. Therefore, proteins were precipitated with H₃PO₄solution at pH 3.5 (Figure 2). Then, the cocoa solution was spray-dried with the help of maltodextrin, and the amino acid profile of both control and low-protein cocoa powder were quantified with the LC-MS/MS system (Table 3). Glutamic acid was the most relevant amino acid amongst the samples: 42.78 mg/kg. Serine occupied the second position in control samples with a value of 21.27 g/kg. Cystine, glycine and taurine were not detected. Adeyeye et al. (2010) reported that glutamic acid and cystine were the most and least abundant amino acids in cocoa nibs, similar to our findings [33].

Table 3. Content and type of amino acid as quantified by LC–MS/MS.

Amino acid	Control cocoa powder (g/kg)	Low protein cocoa powder (g/kg)	Reduction (%)
Alanine	3.69±0.01	1.98±0.02*	46.17
Arginine	11.58±0.01	7.08±0.03*	38.85
Aspartic Acid	19.00±0.02	11.34±0.04*	40.34
Cystine	nd	nd	nd
Glutamic Acid	42.78±0.02	28.64±0.08*	33.06
Glycine	nd	nd	nd
Histidine	5.46±0.01	5.71±0.01	0.00
Isoleucine	1.53±0.17	0.85±0.01*	44.73
Leucine	10.54±0.92	4.89±0.05*	53.64
Lysine	2.36±0.09	0.62±0.02*	73.65
Methionine	1.50±0.01	nd*	100.00
Ornitine	12.68±0.01	11.35±0.02*	10.49
Phenylalanine	11.63±0.07	9.87±0.15*	15.12
Proline	18.91±0.29	15.47±0.04*	18.18
Serine	21.27±0.02	15.07±0.05*	29.16
Threonine	17.04±0.04	15.09±0.01*	11.45
Tyrosine	5.30±0.47	5.19±0.35	2.05
Valine	14.28±0.02	13.07±0.01*	8.48
Taurine	nd	nd	nd

_{* shows the significant difference between control and low-protein cocoa powders (p<0.05). nd: not detected}

When the amino acid profile of low-protein cocoa was examined, significant reductions in amino acid concentrations were obtained except for histidine and tyrosine (p<0.05). The most remarkable reduction rates in amino acid concentrations were 100.00%, 73.65%, 53.64%, 46.17% and 44.73% for methionine, lysine, leucine, alanine and isoleucine, respectively. Among these amino acids, reducing leucine and isoleucine level is important for MSUD patients who must follow lifelong dietary restrictions in terms of branched-chain amino acids [5]. In addition, phenylalanine removal was 15.12% which is significant for PKU patients [10]. As a result of these findings, it can be said that low-protein cocoa can be used as an alternative in the diets of protein metabolic disorder patients.

Epidemiological studies have shown that cocoa contains high concentration of polyphenols that is associated with positive effects on health, such as decreasing the risk of cardiovascular disease and amelioration of insulin sensitivity [34, 35]. Therefore, changes in TPC and antioxidant activity of low-protein cocoa were examined and the results are shown in Table 4. While TPC of the control sample was determined as 15.82 mg/g GAE, it was 25.10 mg/g GAE in low-protein cocoa powder. Gültekin-Özgüven et al. (2016) were reported the TPC of cocoa powder as 18.0 mg catechin equivalent/g in dry defatted base [36]. In another study, TPC ranged from 41.55 mg GAE/g in natural cocoa powder to 10.67 mg GAE/g in alkalized cocoa powder, which is in parallel with our findings [37]. The TPC value of low-protein cocoa was significantly higher compared to the control sample. It is thought that the reasons for this difference may be the heat treatment and enzymes during the process. Because the cellular structure degradation of cocoa occurred, and bound phenolic compounds were released in consequence of both heat and enzymatic treatments. In the control sample, phenolic compounds may be bound to proteins or cell wall polysaccharides, which block the extractability of phenolic compounds from the cocoa matrix [38]. In addition, the phenolic content was measured with the Folin-Ciocalteu reagent, which reacts with other reducing compounds such as pigments, carbohydrates and Maillard reaction products. Because the reducing sugar content increased in low-protein cocoa powder, the formation of melanoidins could provide the increase in the TPC [39-41].

Table 4. Total phenolic content and antioxidant activities of samples

Samples

TPC

(mg GAE/g)

DPPH

(mg Trolox/ g)

FRAP

(mg FeSO₄/ g)

Control cocoa powder

15.82±0.76

22.37±0.25

222.14±1.88

Low-protein cocoa powder

25.10±1.44*

22.70±0.17

224.12±0.77

_{* shows the significant difference between control and low-protein cocoa powders (p<0.05).}

There is a growing interest in the content of antioxidant compounds in food, and many studies have investigated cocoa's antioxidant properties that affect the consumption rate of the products. In this study, the antioxidant activities of cocoa samples were determined with two spectrophotometric methods. The results of DPPH scavenging activity and FRAP are given in Table 4. According to the results, DPPH values of control and low protein cocoa samples were 22.37 mg/g Trolox and 22.70 mg/g Trolox, respectively. FRAP values were also found to be 222.14 mg/g FeSO₄and 224.12 mg/g FeSO₄, respectively. There were no significant differences between samples when antioxidant results were compared (p>0.05). The fact that there was no change in the antioxidant activity as a result of the process is a positive outcome for this study.

One of the most popular cocoa products is chocolate which consists of cocoa butter, sugar and other flavoring agents such as vanillin, milk ingredients or spices. In this study, cocoa, cocoa butter and sugar were used to produce chocolate for patients with the amino acid disorder. The main aim was to see if the produced low-protein cocoa could be used to make chocolate. The same procedure was applied during the production, including mixing the ingredients, thinning and tempering processes, respectively [27]. Chocolates produced with control and low-protein cocoa under laboratory conditions are shown in Figure 3. The color of a food product is the primary quality parameter to attract the consumer. When the colors of chocolate samples were compared, significant difference was seen. Chocolate with low-protein cocoa seemed lighter and redder than the control sample. The main reason for this difference is the addition of maltodextrin during spray drying to increase efficiency. In preliminary studies, a maltodextrin concentration of 1:2 w/w (maltodextrin: the dry matter of cocoa solution) was tried. However, the high sugar amount present in the low-protein cocoa strongly interacted with water molecules which caused agglomeration. In the study of Ribeiro et al. (2020), maltodextrin addition on the flow properties of cocoa pulp powder obtained by spray and freeze drying were investigated. They used 15% and 30% maltodextrin DE20 in cocoa pulp samples and reported that the higher the maltodextrin concentration in cocoa pulp, the lower the product agglomerated by spray drying [42]. Therefore, the maltodextrin concentration of 1:1 w/w was used during spray drying, which resulted in a lighter color.

In this study, the solubility of cocoa was increased by heat and enzyme treatments, and then protein level was decreased by isoelectric precipitation. Then, the solution was dried with a spray dryer using maltodextrin as a carrier. Changes in solubility, sugar content, protein content, amino acid profile and antioxidant activities were examined. The results indicated that the solubility increased significantly with the treatments, and protein content decreased by almost 40% according to the control sample. Moreover, significant reductions were obtained in the amino acid profile except for histidine and tyrosine. While the total phenolic content of low-protein cocoa was higher, the antioxidant activities did not change. Finally, the obtained cocoa samples were successfully used for chocolate production. Thus, it is proposed to be applied in various food products such as ice cream, cookies, hazelnut cream. In summary, if this cocoa product takes place on the market shelves, it can increase the variability of low-protein foods for patients with the amino acid disorder. In future studies, different ways such as ultrafiltration or activated carbon can be investigated to reduce the protein content of cocoa, efficiently.

DPPH 2,2-diphenyl-1-picrylhydrazyl

FRAP ferric reducing antioxidant power

GAE Gallic acid equivalents

HCU homocystinuria

LPD Low protein diets

MSUD Maple syrup urine

OAs Organic acidemias

PKU Phenylketonuria

TPC Total phenolic content

TYR I Type I tyrosinemia

TYR II Type II tyrosinemia

UCDs Urea cycle disorders

Funding

This work was supported by the Commission of Scientific Research Projects of Sakarya University (Sakarya, Turkey) [Project Numbers: 2021-7-24-27].

Authorship contribution statement

İnci Cerit: Methodology, Formal analysis, Writing - original draft, Review & editing. Konul Mehdizade: Formal analysis. Ayşe Avcı: Formal analysis,Review & editing. Omca Demirkol: Conceptualization, Methodology, Review & editing, Supervision. All authors approved the final version of the manuscript.

Data Availability

All data generated or analyzed for this study are included in this published article.

Declarations

Competing interests The authors declare no competing interests.

Ethical Approval Ethical approval was not required for this research.

Consent to Participate Not applicable.

Consent for Publication Not applicable.

Conflicts of Interest The authors declare that they have no conflict of interest.

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Production of low-protein cocoa powder with enzyme-assisted hydrolysis

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