PH12017000078A1 - A process of extracting dietary fiber from cacao pod husk - Google Patents

Abstract

The present invention relates to the process of extracting dietary fiber from cacao, particularly from pod husks. The process includes enzymatic treatment to produce dietary fiber. Its application in food, feeds, beverage ant nutraceuticals is also disclosed herein.

Description

In embodiments of the invention, one or more selected enzymes are added to the CPHs particles or slurry to hydrolyze or degrade at least one type of fiber extracted from the CPHs cell walls. Suitable enzymes that target specific fibers and thereby result in end product with unique properties are used.

In accordance with another aspect, specific materials with varying properties may be derived from processed CPHs, depending on the particular methods, singly or combinations thereof, employed during processing of CPHs.

In a particular embodiment, the raw material is treated with different enzymes for the same period of time, with the resultant enzymatically-modified product exhibiting different functional properties from each other and from the raw material.

Varying the time period of treatment, from hours to days, may vary the functional, structural and nutritional properties of the resultant product.

In another embodiment, varying the suspending medium for enzymes brings about change in the resultant modified product, imparting different properties from the raw material.

In another embodiment, varying the enzyme loading brings about corresponding change in the resultant modified product, imparting different properties from the raw material.

In a particular embodiment, CPHs treated with different pectinase loading resulted to end products (fiber) with unique functional properties with varying values for water-holding capacity, swelling capacity, oil holding capacity and total phenolic content.

In a particular embodiment, varying the solids loading (amount of initial CPHs to buffer ratio) for pectinase treatment also resulted to end products (fiber) with varying functional properties such as water-holding capacity, swelling capacity, oil holding capacity and total phenolic content.

In a related embodiment, varying the reaction time for pectinase-treatment also led to production of modified fiber that is unique for the set of conditions employed. The functional properties such as water-holding capacity, swelling capacity, oil holding capacity and total phenolic content differ for each reaction time employed.

In summary of the earlier cited embodiments, the main factors (pectinase loading, solids loading and reaction time) generally had significant effects on all or most of the response variables. Furthermore, some interactions between the factors are also observed.

The reaction system maybe any appropriate vessel a person skilled in the art would know. The working volume may differ, depending on the desired properties of the end product. A typical run is to prepare a target amount of prepared CPHs powder, mix in the appropriate enzyme dose and allow the reaction to proceed at the target reaction time. A designated amount of enzyme powder is then added to an appropriate buffer to produce an enzyme solution at the desired appropriate for the 1S enzyme. The appropriate amount of CPHs powder is then added to the solution.

After which, the mixture is agitated well in a mechanical shaker throughout the reaction period. After the prescribed time is reached, the solution is heated, centrifuged and decanted. The supernatant is analyzed for total phenolic content (TPC) whereas the residue is dried in a cabinet type hot air oven. Afterwards, the dried residues are milled, sieved to obtain 60 mesh particles or finer particle sizes, depending on the intended use. These fibers may then be used for various applications.

In another embodiment, the mixture maybe dried, milled into smaller particle sizes to reduce the fiber size and improve texture and dispersion capability of the

CPHs fiber in a formulated product. Size reduction makes the texture of the particles feels smoother.

The added enzymes, singly or combinations thereof, hydrolyze or degrade the fibers of the raw material that target specific fibers and thereby result to an end product with specific properties in terms of nutritional and functional values that have distinct health benefits.

Once the desired endpoint is reached, the enzymatic reaction maybe terminated, by heating or acidification. The mixture maybe used as is, or the pH adjusted to desired level depending on the intended use.

The enzymatically-tailored product maybe incorporated as ingredient or additive in formulations and further process using methods known to people skilled in the art of producing functional /designer foods and other products.

In another embodiment, any form of the modified fiber may be used to obtain the desired product. Wet, frozen or dried modified fibers maybe used. Wet form may be taken directly as the press cake after enzymatic modification, and does not require rehydration. Frozen form is obtained after storing the fiber in the freezer, and this form also does not require rehydration. Thawing is necessary before it is incorporated in product formulations. Dry pomace is lighter and easier to store.

This form can be achieved through various methods known to persons skilled in the art.

In another embodiment, other polysaccharides and nutrients maybe obtained during the downstream processing for enzymatically-modified dietary fiber.

Subsequent washings and adjustment of pH to obtain a resultant fiber with specific properties suited for intended application generate polysaccharides and nutrients- rich materials which may find application in other industries, such as, but not limiting to, bio fertilizer, food additive, animal feed additive, etc.

If pH adjustment had not been made prior in the extraction process then sufficient amounts of organic acids such as citric acid, lactic acid, acetic acid, malic acid, succinic acid, or juices such as lemon juice and/or acidic juice concentrate may be added to the milled fiber to reduce the pH as desired. Acidification to under pH 4.5 is necessary for microbial stability.

In another embodiment, the modified CPHs fiber is shown to be safe when tested in mice. Oral toxicity testing done using 20 male and 20 female adult ICR mice that were equally divided into 4 treatment groups (n=5 male and 5 female per group): control group given distilled water, low dose, medium dose and high dose revealed no significant differences between the control and treatment groups regardless of the amount of dose given, on the parameters tested that included mean daily feed intake, mean daily water intake, mean body weight, mean blood creatinine, and alanine aminotransferase (ALT). The data indicated the safety of the modified CPHs fiber as an oral supplement.

In another embodiment, the efficacy of the modified CPHs fiber to lower glucose level in diabetic mice was proven. The trial involved 44 adult male ICR mice, 34 of which were intraperitoneally administered with streptozotocin to induce diabetes mellitus. The animals were divided into 5 treatment groups, namely, non- diabetic mice given distilled water (n=10), diabetic mice given distilled water (n=10), diabetic mice given glibenclamide (n=8) at 10 mg/kg BW, diabetic mice given psyllium (n=8) and diabetic mice given modified CPHs fiber (n=8) at 38g/60 kg BW. All treatments were administered via gavage once a day for 28 days, and mean fasting blood glucose level (mg/dL) was obtained and computed at days 1, 7, 14, 21 and 28. Results showed that the diabetic mice given modified CPHs had comparable mean fasting blood glucose levels with those administered with glibenclamide (positive control; anti-diabetic drug) and psyllium (commercial dietary fiber), thus confirming the glucose lowering activity of the CPHs fiber.

In accordance with another aspect, a comestible is provided comprising fiber- rich materials extracted from CPHs, wherein the fiber is extracted using at least one physical process or a combination of a physical process and enzymatic hydrolysis of

CPHs.

Finely-milled CPHs fibers may then be used to prepare nutraceuticals, beverage or food product, either as main ingredient or maybe included in various food products to provide enhanced nutrition and other characteristics, such as color, flavor, and mouthfeel. Suitable food products include, but are not limited to bread, cookies, and other bakery products, beverages, soups, spreads, puddings, smoothies, snack foods, yogurts, cereals, and fiber-rich supplements. The different forms of CPHs fiber may be added to products to impart cocoa flavor, improve natural fiber content of products, improve phytochemical contents and at the same time increase viscosity, smoothness and mouth filling.

The process typically comprises washing of freshly-collected CPHs, reducing the particle size of the CPHs by any method known to a person skilled in the art to increase the surface area, drying, milling and sieving. Further modification of dried

CPHs is done by combining the CPHs particles with a liquid, for example and without limitation, water, or appropriate buffer to form a slurry and adding one or more selected enzymes to the slurry. After the desired reaction period, the mixture is heated to inactivate the enzymes. Drying follows, then milling and sieving to remove large solids and isolating the modified fiber. The isolated fiber, and optionally one or more other extracted nutrients from the process may then be included in a formulation such as a comestible to increase the nutrition of the comestible.

An alternate process typically comprises washing of freshly-collected CPHs, reducing the particle size of the CPHs to increase the surface area to be extracted and breaking the cell walls of the CPHs by employing at least one physical process to increase the surface area on the particles, and adding one or more selected enzymes either suspended in small amounts of water or buffer, or just pure enzymes without the need of suspending them in water or buffer. In this process, drying step is omitted before enzymatic treatment. Moreover, when the CPHs byproducts are subjected to a physical process to break cell walls of the particles, additional liquid is released thereby forming a slurry. At the end of enzymatic reaction period, the slurry is heated to inactivate the enzymes. Separation of permeate and retentate follows, and then drying of the end products. Next, further reduction in particle size is employed, followed by sieving to remove large solids and isolating the modified fiber. The isolated fiber, and optionally one or more other extracted nutrients from the permeate and retentate, are then included in a formulation such as a comestible, to improve the nutritional and functional properties of the comestible.

In these particular embodiments of this method, it is not necessary to add a liquid to form slurry as the fresh CPHs contains sufficient liquid which is extracted after subjecting to physical process such as comminution and blending. The amount of water extracted is also enough for enzyme processes to be performed.

Other embodiments of this invention will become apparent in the examples of preferred embodiments of the invention.

The following examples serve to illustrate the present invention without, however, limiting the same thereto.

Example 1

The proximate analysis of the untreated and pectinase-treated fiber showed increased crude protein and crude fiber contents after enzymatic treatment. The proportion of carbohydrates and crude fat decreased (Table 1). The decrease in the carbohydrate content can be considered reasonable, since the pectin content in the treated fiber might have decreased due to the action of pectinase. The “lost pectin” may appear as the water-soluble hydrolysis products (galacturonic acids), which are separated from the fiber, and went with the liquid phase after enzyme treatment. The decrease in the proportion of the crude fat may also be attributed to the release of some lipids from the fiber matrix after pectinase treatment (and also went into the liquid phase). The ash might have also separated from the fiber and went to the liquid phase. The proportion of crude fiber may be attributed to the higher proportion of insoluble fiber, as a result of the decrease in pectin content (soluble fiber) due to pectinase treatment.

Table 1. Proximate analysis of CPHs and pectinase-treated CPHs.

Component ot irs ae) hari cated (% dry mass)

Ash 10.72 10.05

Crude fat 0.46 0

Crude protein 7.96 10.55

Crude fiber 25.38 32.11

Oy aS eee 0 eT

Example 2

The presence of lignins, hemicelluloses, celluloses as well as pectins in the cacao pod husk is a good indicator for its use as dietary fiber. Hemicelluloses and pectins share a remarkable ability to bind heavy metal compounds, which is promising and gives good hope for the promotion of health via fiber-containing food. Also, cellulose and lignin are able to bind heavy metals (though to a smaller extent than hemicellulose and pectins), and their binding ability varies with the source of origin of relevant fractions (Casterline and Yuoh, 1993; Borycka, Boryck and Zuchowski, 1996; Davidson and McDdonald, 1998; Nawirska and Oszmianski, 2001; Sangnark and Noomhorm, 2003).

The crude fiber profile analysis of the untreated and pectinase-treated fiber is presented in Table 2. Acid detergent fiber (ADF), which is composed of cellulose and lignin, is inversely related to the digestibility of a certain food material. On the other hand, neutral detergent fiber (NDF), which is mainly composed of cellulose, lignin and hemicellulose, reflects the amount of food material which can be consumed by an organism (i.e., dry matter intake) (Agri Analysis, Inc., 2009, cited by Sapin et al., 2013). There was an increase in the portion of the acid detergent fiber (ADF) and neutral detergent fiber (NDF) after subjecting the fiber to enzymatic treatment. This increase may be due to the decrease in the portion of lignin which may have been partially released as phenolic compounds from the fiber upon enzyme treatment (Faustino et al., 2010; Palonen, 2004 and Vanholme et al,, 2012). Increase ADF and NDF content could imply that the pectinase-treated fiber at optimum conditions would take longer time to be eaten due to its decreased digestibility and dry matter intake. With this, most of its essential nutrient content, such as protein, carbohydrates and ash, may be absorbed in the small intestine.

These may add to potential health benefit for health-conscious individuals since when high-fiber foods are consumed, it is expected that next food intake would be significantly reduced (FAO, 1998).

Table 2. Crude fiber profile of CPHs and pectinase-treated CPHs.

RRR

(% dry mass)

Acid detergent fiber (ADF) 35.36 44.20

Neutral detergent fiber (NDF) 48.08 66.70

Acid detergent lignin 24.09 18.73

Hemicellulose (HMC) 12.72 22.50

Example 3

To be used as dietary fiber source, it is desirable that the fiber materials should have the desired nutritional properties as well as functional and technological properties. Functional properties of dietary fiber material determine its suitability for use as functional food. With respect to hydration properties, dietary fiber with strong hydration property means that it can retain more water within its matrix. Incorporation in food will enhance viscosity as well as increase stool weight and potentially slow the rate of nutrient absorption from the intestine (Gallaher and Schneeman, 2001).

Milled CPHs were subjected to enzymatic treatment, with control that incorporated water only. The resulting fiber products differ in water- and oil- absorption capacities (Table 3). The reduction in WAC and OAC after enzyme treatment affected the viscosity of sample when suspended in water or oil. Treated material can be used as functional ingredients to control texture through their abilities to bind water and to stabilize emulsions.

Table 3. Water absorption capacity and oil absorption capacity of enzymatically- treated CPHs.

Water Absorption Oil Absorption

Treatment Capacity Capacity (WAC) (OAC)

Starting CPHs material 15.60 2.75

Enzymatically-treated fiber:

Water 3.16 1.39

Cellulase (1.0 %, BIOTECH 4.49 2.43 enzyme)

Xylanase (0.5%, Sigma) 4.61 1.64

Cellulase (0.5%, Sigma) 4.17 2.41

SS)

Example 4

The reducing sugar (RS) in the supernate was monitored to determine immediate changes in the composition of cacao pod husk fiber during enzymatic treatment. Results presented in Table 4 show that enzyme- treated samples released more RS than the untreated sample. Higher values were also observed with pectinase- treated sample than BIOTECH cellulase-treated cacao fiber.

Table 4. Changes in reducing sugars (RS) in the supernate during enzymatic treatment of dietary fiber from cacao.

Treatments

A B Cc

Time Untreated Pectinase Cellulase (hour) (0.5%, Sigma) (0.5%, BIOTECH)

RS RS % difference over RS % increase over (mg/mL) (mg/mL) Treatment A (mg/mL) Treatment A 0 088 ~ 0.88 0 CED 0

Lo 0% vis 8229 147 8312 xoorer 1% 9406 tel 94L 312s 232 8560 174 3920

Example$

Plant non-starch polysaccharides possess antioxidant properties and may be exploited as potential novel antioxidants. This suggests possibilities for the use of fibers with high antioxidant activities as ingredients that allow the stabilization of fatty foodstuffs, thereby improving their oxidative stability and prolonging their shelf life. Table S shows that lower amount of polyphenols are observed in the permeate in samples treated with xylanase (Sigma'™) and cellulase (Sigma™) as compared to untreated and cellulase (BIOTECH)-treated samples, after 4 hours of treatment.

Table 5. Amount of polyphenols and reducing sugars in the supernate after 4 hours of enzymatic treatment.

Supemnate

Treatment Reducing Sugar, Total Polyphenols, mg/mL pg/mL 1% Cellulase¢/BIOTECH ~~ 219 ~~ 60080 _0.5% Xylanase/Sigma 307 4683

Example 6

There is big potential of cacao pod husk fiber as natural source of antioxidants, as can be seen in Table 6. This has relatively higher total phenolic content and DPPH scavenging activity as compared to other waste/by-products analyzed. It also shows better DPPH activity than the synthetic antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT).

Table 6. Phenolic content and DPPH scavenging activity of different agricultural wastes and by-products. ~~ Total Phenolic DPPH Scavenging

Content Activity per 20 pg

Sample (mg/g dry GAE/assay matter)” (% DPPH inhibition)

Waste/by- Calamansipeel 251 A134 product Coffeehusk ~~ 35% 29 ~ Mango seed powder 108.3 90.09 -

Butylated hydroxyanisole } 57.50

Synthetic ~~ (BHA) antioxidants Butylated hydroxytoluene } 34.19 (BHT)

OU — ? Extractant used: 50% acetone

Example 7

Milled CPHs were separated into two-those that passed through the 60 mesh sieve and those that did not which have bigger particle size (designated as <60 mesh). The functional properties of CPH with varying particle size is shown in

Table 7. Functional properties of dietary fiber material determine its suitability for use as functional food. With respect to hydration properties, dietary fiber with strong hydration property means that it can retain more water within its matrix.

Incorporation in food will enhance viscosity as well as increase stool weight and potentially slow the rate of nutrient absorption from the intestine (Gallaher and

Schneeman, 2001). The fiber from cacao pulp and cacao pulp with skin, of larger particle size (<60 mesh) also exhibited less than 10 ml/gram WAC. What is noticeable is that cacao husk fiber with smaller particle size (60 mesh) exhibited 15.60 to 20.00 ml/g, especially the fiber from cacao skin. The observed WAC is comparable in most of the previously reported fibers: 9.77 g water/ g grapefruit fiber (Fernandez et al., 1993) and similar to the value reported by Grigelmo- Miguel and Martiin-Belloso (1999), more than 11 g water/g orange fiber and about 10.66 g water/g citrus fruits fiber (Robertson et al., 2000). Differences in the oil absorption capacity of the different fibers might also be attributed to the variation in the physical and chemical structures of these fibers. According to Femenia et al. (1997)

and Lopez et al. (1996), fat adsorption capacity (FAC) depends on surface properties, overall charge density, thickness, and hydrophobic nature of the fiber particle. The swelling capacity of fiber concentrates was also comparable with values obtained from other agricultural wastes such as Valencia orange (6.11 ml/g dry matter) and Fino 49 lemon (9.19 ml/g dry matter).

In separate trials where enzyme concentration was varied, it was found out that gelation capacity is affected by enzymatic treatment (Tables 8 and 9). Along with water and oil-absorption capacities, gelation capacity is also critical in the development of products that will include CPHs fiber.

Table 7. Functional properties of CPH fibers with varying particle size.

Lo]

Dried Cacao Husk Fiber

Property Cacao peel/skin Cacao pulp Cacao pulp with skin 60 mesh <60 mesh 60 mesh <60mesh 60 mesh <60 mesh

Water absorption 2000 1580 16.40 4.65 15.60 3.85 capacity, mL/gram

Oil absorption 2.55 1.60 2.10 1.80 2.75 1.10 capacity, mL/gram

Bulk density, g/cm’ 0.74 0.71 0.70 0.70 0.61 0.71

Swelling power, 18.14 16.66 17.56 5.53 17.66 5.32 mL/gram

Starch solubility, % 39.76 16.86 30.38 8.51 30.02 10.42

Foam capacity, % 0.00 1.92 0.00 3.85 0.00 5.77

Foam stability, % - 98.11 - 99.07 - 96.36

Gelation capacity 8%/soft 8%/soft 8%/soft . 8%/soft 8%/soft

Percentage Nd incorporation/ gel gel gel gel gel gel i 16%/firm 16%/firm 16%/firm 16%/firm 16%/firm description Nd gel gel gel gel gel

Not detected

Table 8. Gelation capacity of 60 mesh enzymatically-modified fiber from cacao pod husk after 4 hours of treatment (Enzyme level: 0.5% w/w). ~~ Description of Gel Formed

Sample . Liquid, Curdy. Soft Firm

Sample Size ’ ’ Gel, Gel,

Type Max Max .

Max Min

Conc Conc.

Conc. Conc.

Water/ Milledsample ~~ 4% 8% 12% 14%

Control =60mesh 6% 10% 14% 16% op 260mesh 4% 8% 14% 16%

Pectinase- Milledsample 6% 12% 20% -

es 2 60mesh 0 4% 12% 20% -

Cellulase. ~~ Milledsample ~~ 6% 10% 16% 18% treate d _= 60 mesh emt rt rte 6% en 8 ronnie 14% rei 16% me ~~ ...>8mesh 4% 10% 16% 18%

Xylanase. ~~ Milledsample 4% 8% 14% 16% oe =60mesh 4% 8% 14% 16% > 60 mesh 4% 8% 14% 16% 0

Table 9. Gelation capacity of milled and 60 mesh enzymatically-modified fiber from cacao pod husk after 4 hours of treatment (Enzyme level: 0.05% w/w).

Samole Description of Gel Formed ample . — :

Type Sample Size Liquid, Curdy, Soft Gel, Firm Gel,

YP Max Conc Max Conc. Max Conc. Min Conc.

Water/ Milledsample ~~ 2% 8% 14% 16% eo > 60mesh 4% 0 8% 18% 20%

Cellulase Milled sample 2% 10% 16% 18% treated =60mesh 4% 8 16% 18% ee >60mesh 4% 8% 16% 18%

Xvian Milled sample 6% 10% 14% 16% > 60 mesh 4% 8% 14% 16%

Alia

Example 8

In a specific experiment on pectinase modification of CPHs, the results from

Response Surface Methodology (RSM) - based experimental design on water absorption capacity is presented in Table 10. The values of WAC changes with varying combinations of pectinase loading, solids loading and reaction time.

Table 10. RSM-based (Box Behnken) design experimental results of water absorption capacity (WAC). 0 ~~ Factor Response

Pectinase Solids Reaction WAC

Std .

Run Loading Loading Time (mL water/g (U/g pectin) (Yow/v) (hour) cacao) 1 10 25 2.00 4.25 3.51 2 6 500 2.00 4.25 3.03 3 3 25 6.00 4.25 3.79 4 4 500 6.00 4.25 2.79 5 15 25 4.00 0.50 3.15 6 2 500 4.00 0.50 2.95

8 12 500 4.00 8.00 1.83 9 13 263 2.00 0.50 3.48 10 5 263 6.00 0.50 3.27 11 9 263 2.00 8.00 3.20 12 8 263 6.00 8.00 2.57 13 14 263 4.00 4.25 1.98 14 16 263 4.00 4.25 1.92 15 17 263 4.00 4.25 2.04 16 1 263 4.00 4.25 2.18 17 11 263 4.00 4.25 2.13 be _________________________________________________________________________________________]

As compared to the lowest value of WAC in the factorial experiment (WAC = 3.92 mL/g, achieved at pectinase loading of 300 U/g pectin, solids loading of 4.00 %w/v and reaction time of 6.00 hours), a lower value of WAC (1.83 mL/g) was obtained during RSM-designed experiments at pectinase loading of 500 U/g pectin, solids loading of 4.00 %w/v and reaction time of 8.00 hours. This decrease in WAC could be due to the increased amount of pectinase in the suspension causing more pectin molecules to be hydrolyzed; thus, reducing the capacity of the fiber to hold water. Furthermore, longer reaction time could have also contributed to the decrease in WAC since this also resulted to the hydrolysis of more pectin in the fiber. On the other hand, the highest WAC was observed at low pectinase loading (25 U/g pectin), high solids loading (6.00 %w/v) and moderate reaction time (6.00 hours).

This was expected since at high solids loading, pectin was also high, thus, with less pectinase loading, less pectinase was available to hydrolyze the pectin in the fiber; hence, the WAC increased.

Example 9

In a specific experiment on pectinase modification of CPHs, the results from

Response Surface Methodology (RSM) - based experimental design on oil- absorption capacity is presented in Table 11. The values of OAC, another hydration property considered, changes with varying combinations of pectinase loading, solids loading and reaction time.

Table 11. RSM-based (Box Behnken) design experimental results of oil holding capacity (OHC).

EE

Factor Response

Std Run Pectinase Solids Reaction OHC

Loading Loading Time (mL oil/g (U/g pectin) (Yow/v) (hour) cacao) 1 10 25 2.00 4.25 2.87 2 6 500 2.00 4.25 2.78 3 3 25 6.00 4.25 2.98 4 4 500 6.00 4.25 2.55 15 25 4.00 0.50 2.21 6 2 500 4.00 0.50 2.05 7 7 25 4.00 8.00 2.33 8 12 500 4.00 8.00 1.97 9 13 263 2.00 0.50 2.88 5 263 6.00 0.50 2.79 11 9 263 2.00 8.00 2.70 12 8 263 6.00 8.00 2.46 13 14 263 4.00 4.25 2.12 14 16 263 4.00 4.25 2.18 17 263 4.00 4.25 2.19 16 1 263 4.00 4.25 2.16 17 B 263 4.00 4.25 2.00

From Table 11, the lowest value of OAC (1.97 mL/g) was obtained at high 5 pectinase loading (500 U/g pectin), moderate solids loading (6.00 %w/v) and longer reaction time (8.00 hours) while the highest OAC (2.98 mL/g) was observed with low pectinase loading (25 U/g pectin), high amount of solids (6.00 %w/v) and moderate reaction time (4.25 hours). The decrease in OAC upon the addition of pectinase in the mixture could be attributed to the liberation of fat-binding 10 components from the fiber.

Example 10

The results of the swelling capacity (SC), another important hydration 15 property, are shown in Table 12. The values of SC ranged from 1.49 mL/g to 4.47 mL/g. The lowest value was achieved at moderate values of pectinase loading (263

U/g pectin), solids loading (4.00 %w/v) and reaction time (4.25 hours). On the other hand, the highest value was attained from the combinations of low pectinase loading (25.00 U/g pectin), high solids loading (6.00 %w/v) and moderate reaction time (4.25 hours). This difference SC could be due to less amount of pectinase in the mixture which could have caused a decrease in hydrolysis of pectin in the fiber; hence, high SC was obtained.

Table 12. RSM-based (Box Behnken) design experimental results of swelling capacity (SC).

EE "" Response

Std Run Pectinase Solids Reaction SC

Loading Loading Time (mL liquid/g (U/g pectin) (Yow/v) (hour) cacao) 1 10 25 2.00 4.25 3.32 2 6 500 2.00 4.25 3.44 3 3 25 6.00 4.25 4.47 4 4 500 6.00 4.25 3.43 5 15 25 4.00 0.50 2.49 6 2 500 4.00 0.50 2.48 7 7 25 4.00 8.00 2.78 8 12 500 4.00 8.00 2.24 9 13 263 2.00 0.50 2.83 5 263 6.00 0.50 3.35 1 9 263 2.00 8.00 3.29 12 8 263 6.00 8.00 2.65 13 14 263 4.00 4.25 2.19 14 16 263 4.00 4.25 1.49 17 263 4.00 4.25 2.04 16 1 263 4.00 4.25 1.95 _ 17 11 263 4.00 4.25 1.75

Example 11 10 Table 13 summarizes the favored conditions of each factor presented in

Examples 7, 8 and 9 where each response is dependent on the conditions employed during the modification process. The desired outcome can be predicted based on the conditions employed during the modification process. 15 Table 13. Summary of the favored conditions for each factor to obtain the desired outcome of the target responses.

Responses

Factors wae OAC = SC TPC

Desired Outcome

Low Low Low High

Pectinase loading . . (Ulg pectin) high moderate high low

Solids loading moderate moderate moderate high ews

Renton yme high moderate low high

Le

As observed from Table 13, favored conditions for each factor varied, and sometimes “opposing”. For example, a high pectinase loading is favored for a desired low value of WHC, but a low pectinase loading is needed for a desirable high value of TPC content of the cacao fiber. Thus, in order to satisfy these multiple responses, numerical optimization was conducted with the goals of minimizing the

WAC, OAC, SC and maximizing TPC,,;s From these goals, the optimum conditions were obtained by the numerical optimization algorithm of the Design

Expert ® software, and these are summarized in Table 14.

Table 14. Operating range and optimum conditions for pectinase modification. “Pectinase loading, U/g pectin 25 500 344

Solids loading, Y%ew/v 2 6 4.35

Reaction time, hour 0.5 8 8.00

Example 12

Untreated and pectinase-treated powdered CPHs were also subjected to

Glucose Dialysis Retardation Index (GDRI) Test. GDRI is a useful in vitro index to predict the effect that fiber has on the delay of glucose absorption in the gastrointestinal tract (Lopez et al., 1996). GDRI was determined as described by

Lecumberri et al. (2007). The resulting fiber products differ in GDRI values.

The modified fiber product showed lower GDRI value from the starting material (untreated fiber), as shown in Figure. 1. Since GDRI is related to the soluble dietary fiber (SDF), uronic acids, internal structure and surface properties of the fiber, it was expected that a decrease in the said parameter would be observed due to the reduction of pectin in the fiber (Edwards et al., 1987 & Wolever, 1990, cited by Lecumberri et al., 2006). Nevertheless, the GDRI value of the pectinase- treated fiber at the optimum conditions (15.06 %) was found to be comparable with other commercial fibers. It was relatively higher than Carob pod fiber (8.23 %) and wheat bran (5.3%) (Adiotomre et al., 1990, cited by Sapin et al., 2013) but lower than guar gum (25.80), apple pectins (25.79 %) and citrus pectins (27.01%) (Bravo et al., 1994, cited by Lecumberri, 2006). Furthermore, it was also higher than the obtained value (4.40 %) of Lecumberri et al., (2006) for untreated cacao husk obtained from Barcelona, Spain.

Example 13

Using 20 male and 20 female adult ICR mice, acute oral toxicity testing of

CPHs fiber was done. The mice were equally divided into 4 treatment groups (n=>5 male and 5 female per group): control group given distilled water, low dose CPHs fiber, medium dose CPHs fiber and high dose CPHs fiber. All treatments were given via gavage once a day for 14 days. Doses administered to male mice were 19g/60kg BW for the low CPHs fiber, 38g/60 kg BW for the medium CPHs fiber

I5 and 57g/60kg BW for the high CPHs fiber; while the female mice was given the following doses: 12.5g/50 kg BW for the low CPHs fiber, 25g/50kg BW for the medium CPHs fiber and 37g/50kg BW for the high CPHs fiber group.

Mean daily feed intake, mean daily water intake, mean body weight, mean blood creatinine, and alanine aminotransferase (ALT) were computed and compared per treatment. Results showed no significant differences between the control and treatment groups regardless of the amount of dose given (Tables 15a to 15f). These data indicate the safety of the CPHs fiber as an oral supplement

Table 15a. Mean daily feed intake (g) of male and female mice given water and different concentrations of CPHs fiber.

Treatment Groups n Mean Daily Feed Intake Mean Daily Feed Intake (g) of Male Mice (g) of Female Mice

Control 5 3.18 2.27

Low CPHs fiber 5 3.29 2.45

Medium CPHs fiber 5 2.68 2.71

Table 15b. Mean daily water intake (mL) of male and female mice given water and different concentrations of CPHs fiber.

SE

Treatment Groups n Mean Daily Water Intake Mean Water Intake p (mL) of Male Mice (mL) of Female Mice

Control 5 7.20 7.77

Low CPHs fiber 5 7.97 7.00

Medium CPHs fiber 5 8.20 6.94

High CPHs fiber 5 6.42 7.38

A

Table 15¢c. Mean body weight (g)/(mL) of male mice given water and different concentrations of CPHs fiber at Day 1 (before treatment) vs. Day 14.

SS

Mean Body Weight Mean Body Weight (g)

Treatment Groups n (2) at Day 1 at Day 14

Control 5 35.96 36.22

Low CPHs fiber 5 35.60 35.78

Medium CPHs fiber 5 33.84 33.46

JHghCPHs fiber S$ 3438 30s

Table 15d. Mean body weight (g)/(mL) of female mice given water and different concentrations of CPHs fiber at Day 1 (before treatment) vs. Day 14. © MeanBody Mean Body

Treatment Groups n Weight (g) Weight (g) at Day 1 at Day 14

Control 5 26.46 26.76

Low CPHs fiber 5 28.18 27.60

Medium CPHs fiber 5 30.22 30.26

Table 15e. Mean blood creatinine (mg/dL) and alanine aminotransferase (ALT, U/L) of male mice given water and different concentrations of CPHs fiber at

Day 1 (before treatment) vs. Day 14.

Mean Blood Mean Blood Mean Blood Mean Blood

Treatment Groups ALT Levels ALT Levels Creatine Levels Creatine Levels at Day 1 at Day 14 at Day 1 at Day 14

Control 18.38 14.62 <0.05 <0.05

Low CPHs fiber 22.22 17.63 <0.05 <0.05

Medium CPHs fiber 25.18 16.06 <0.05 <0.05 *Normal values: ALT: 26 - 77 IU/L; Creatinine: 0.3 — 1.0 mg/dl (Harkness and Wagner, The

Biology and Medicine of Rabbits and Rodents, 5" Ed.) *Normal values: ALT: 27-60 IU/L; Creatinine: 0.3 — 0.43 mg/dl (Julius Santos Thesis, CVM,

UPLB, 2010)

Table 15f. Mean blood creatinine (mg/dL) and alanine aminotransferase (ALT,

U/L) of female mice given water and different concentrations of CPHs fiber at Day 1 (before treatment) vs. Day 14.

Mean Blood ~~ Mean Blood ~~ Mean Blood Mean Blood

Treatment Groups ALT Levels ALT Levels Creatine Creatine

P at Day | at Day 14 Levels at Day Levels at Day 1 14

Control 14.13 19.26 <0.05 <0.05

Low CPHs fiber 12.75 19.21 <0.05 <0.05

Medium CPHs fiber 33.52 19.49 <0.05 <0.05 *Normal values: ALT: 26 - 77 IU/L; Creatinine: 0.3 — 1.0 mg/dl (Harkness and Wagner, The

Biology and Medicine of Rabbits and Rodents, 5" Ed.) *Normal values: ALT: 27-60 IU/L; Creatinine: 0.3 — 0.43 mg/dl (Julius Santos Thesis, CVM,

UPLB, 2010)

Example 14

The glucose lowering activity of CPHs fiber was evaluated using 44 adult male

ICR mice, 34 of which were intraperitoneally administered with streptozotocin to induce diabetes mellitus. All animals were then divided into 5 treatment groups: Non- diabetic mice given distilled water (n=10), diabetic mice given distilled water (n=10), diabetic mice given glibenclamide (n=8) at 10 mg/kg BW, diabetic mice given psyllium (n=8) and diabetic mice given CPHs fiber (n=8) at 38g/60 kg BW. All treatments were administered via gavage once a day for 28 days. Mean fasting blood glucose level (mg/dL) was obtained and computed at days 1, 7, 14, 21 and 28. Results showed that the diabetic mice given CPHs fiber had comparable mean fasting blood glucose levels with those administered with glibenclamide (positive control; anti- diabetic drug) and psyllium (commercial dietary fiber), thus confirming the glucose lowering activity of the cacao by-product. (Table 16; Figure 2).

Table 16. Mean fasting blood glucose level (mg/dL) of the different treatment groups.

Treatment Groups on Dayl Day? Day 14 Day 21 Day 28

Non-diabetic + distilled water 10 53.50 53.40 50.60 49.70 51.70

Diabetic + distilled water 10 189.00 196.50 197.50 202.30 205.30

Diabetic + glibenclamide 8 159.00 124.88 115.25 116.38 139.25

Diabetic + Psyllium 8 153.88 108.63 106.50 111.00 124.13

CPHs fiber given to male mice for this experiment is 38g/60kg BW.

Mice subjected to 12 hours fasting.

Example 15. Applications as bakery ingredient

Baked products were formulated such as pandesal, cookies and cupcakes in order to take advantage of the dietary and functional properties of modified fiber from CPHs. Results are presented in Table 17.

Table 17. Mean’ sensory scores’ and other properties of baked products with

Sonor Properties Pandesa nsory Trop 10% 20% | 0% 25% 375%] 25% 10%

Color 7.13a 6.80b 8.60a 8.33a 8.07a 6.80a 6.20b

Sweetness 7.24a 6.76b - - -

Saltiness 6.76a 6.84a - - -

Aroma 7.60a 6.84b 8.20a 7.87a 7.80a 6.80a 6.25b

Flavor 6.58a 7.04a 8.27a 8.00a 7.67a 7.25a 6.05b

Texture/mouth feel |7.02a 6.82a 8.33a 8.13a 7.27a 7.25a 5.95b

Aftertaste - - - - - 6.73a 5.73b

Overall acceptability |7.18a 6.89a | 845a 8.07a 7.87a 8.07a 6.25b

Phenolic content, ug 289.50 454.87 | 286.77 669.55 905.82 | 388.69 626.29

GAE equiv/g

EE

’% Increase 57.12 133.48 215.87 61.13 (Decrease) oT TT ! N=15. Means followed by the same letter within a given sensory attribute are 10 . not significantly different from each other at P< 0.05. “ Range of scores:

Color: 1, Dislike extremely; 5, Neither like nor dislike; 9, Like extremely

Sweetness: 1, Dislike extremely; 5, Neither like nor dislike; 9, Like extremely

Saltiness: 1, Dislike extremely; 5, Neither like nor dislike; 9, Like extremely

Aroma: 1, Dislike extremely; 5, Neither like nor dislike; 9, Like extremely

Cacao flavor: Extremely bland; 5, Neither bland nor creamy; 9, Extremely creamy

Texture/Mouthfeel: 1, Extremely rough; 5, Neither rough nor smooth, 9, Extremely smooth

General acceptability: 1, Extremely unacceptable; 5, Neither unacceptable nor dislike; 9, Extremely acceptable

Given the benefit of the above disclosure and description of exemplary embodiments, it will be apparent to those skilled in the art that numerous alternate and different embodiments are possible in keeping with the general principles of the invention disclosed here. Those skilled in the art will appreciate that there are numerous variations and permutations of the above described systems and techniques that fall within the spirit and scope of the invention as set forth in the appended claims.

A PROCESS OF EXTRACTING DIETARY FIBER FROM

CACAO POD HUSK

TECHNICAL FIELD OF THE INVENTION

The present invention relates to processes for production and modification of cacao pod husks (CPHs) by enzymatic action to vary its functionalities. The processes have resultant products that include tailored dietary fibers and flours with improved manufacturing qualities and versatile uses and applications for various industries. More particularly, the dietary fiber and flour of the present invention have decreased water and oil absorption capacities, swelling properties and gel hardness compared to the starting material.

The present invention also relates to food and nutraceutical products containing the modified CPHs.

BACKGROUND OF THE INVENTION

The cacao industry is producing a lot of waste considering that only 10% of cacao fruit is the bean which is commercially- processed. The remaining 90% is often always discarded as waste. Considering that CPHs comprise majority of the fruit, solid waste residues are generated, thus serious solid waste management problems are likely to occur (Chun et al., 2013). Although these husks are used in low value applications such as animal feeds in some areas, they still remain underexploited and their disposal still remains a problem at present.

One sustainable strategy for the management of these solid waste residues would be via “resource recovery”, or processing CPHs wastes into products of added value. Enzymatic modification provides a reasonable utilization potential for CPHs, thus adding value to by-products of the cacao processing industry.

Studies have found that CPHs are rich sources of dietary fiber, pectin, protein and polyphenols. According to Bonvehi and Beneria (1998), CPH is composed of 43.8 % dietary fiber. Lecumberri et al. (2007) also conducted same analysis and found that 60.54% of CPHs is dietary fiber (DF), and is comparable to whole grains and cereal which are known to be rich in fiber. Also, its fat content is relatively low compared to cocoa bean and it has negligible sugar content as well. Several products made from CPH have already been commercialized. Usually, they are processed to produce air-dried CPHs pellet which becomes component of animal feeds for poultry, pigs and sheep due to the vast essential compounds. Also, once fermented with Pleurotus ostreatus, it can be used as broiler finisher diet (ICCO, 2013; Oddoye et al., 2013).

CPHs could be an inexpensive source of polyphenols and soluble fibers (Bruna et al., 2009). Polyphenols dictate the anti-oxidant capacity of a given substance; the higher the amount, the greater is the tendency of the substance to prevent oxidative damage by scavenging free radicals. Thus, CPHs provide valuable nutrients and fiber.

These properties indicate the potential of these residues as potential food ingredients or healthy food additives or nutraceuticals. However, no studies have been reported on its use as a food ingredient for human consumption. This may be hindered by the inherently high water absorbing capacities of its fiber, as manifested by the formation of an emulsion upon its addition in water. Some form of modification might render the water absorbing capacity and other physico-chemical properties of the cacao pod husks to be more suitable for (human) food applications.

The use of fibers from new origins like CPHs that are currently not fully exploited and the possibility of modifying the fibers by enzymatic treatments and combining them with other components will enhance their nutritional and sensory characteristics, and probably widen the fields of application for dietary fibers. With the consumers’ increasing consciousness on the consumption of fiber-rich food, this invention will benefit the field of food science and technology as it uncovers a new potential source of fiber- and antioxidant-rich powder that can be used in place of other expensive commercial fiber enhancers. Thus, cheap yet effective dietary fiber supplement will be accessible to most people so that they may be able to reach the required 20 to 25 grams dietary fiber intake per day as prescribed by the Food and

Agriculture Organization of the United Nations (FAO) and World Health

Organization (Barba and Cabrera, 2008).

It would be beneficial to process CPHs to arrive at a useful product in order to avoid such waste. Incorporating the enzymatically-modified CPHs to form innovative products like functional/ designer food will open new market segments.

Additionally, this approach of managing solid waste residues will contribute towards achieving clean/green productivity objectives of the cacao processing industry. Environmentally speaking, this invention is designed to alleviate the serious solid waste management problems and human health risks arising from the frequently discarded CPHs. Lastly, this will offer new opportunities for local cacao farmers and growers as they will yield extra income from their cacao plantations and production facilities, through the addition of value to the solid CPHs waste for use in various applications.

SUMMARY OF THE INVENTION

One advantage of embodiments of the present invention is the processing and modification of CPHs resulting to modified fiber with distinct functional properties such as, but not limiting to low viscosity, low gelling capacity and variable water and oil absorption capacities depending on the conditions employed. A further advantage of the embodiments of the present invention is the utilization of fibers from CPHs employing physical, enzymatic or combinations thereof, and without employing chemical modification processes. Another advantage of the embodiments is the overall effect to the cacao processing industry where tremendous amount of solid wastes is reduced by adding value to these processing wastes.

In accordance with one aspect, a method is provided comprising treatment of

CPHs to prevent browning prior to drying and particle reduction. The material obtained contains both soluble and insoluble fiber.

In accordance with another aspect, a method is provided comprising modification of CPHs fiber through enzymatic treatment where the CPHs are combined with one or more enzyme solutions to form a slurry, mixing or agitating the slurry, heating the slurry to deactivate enzymes and filtering or centrifuging the slurry to provide a retentate and a permeate. The retentate contains both soluble and insoluble fibers. The resulting products differ in nutritional, functional and structural properties from the raw material. The modified CPH can be subsequently used in the manufacture of foodstuffs, pharmaceuticals and nutraceuticals.

It will be appreciated by those skilled in the art that at least certain exemplary embodiments of the process and products described resulted to modified fiber with different structural and functional properties from the starting material.

In accordance with another aspect, the efficacy and safety of the modified

CPHs is established. Oral toxicity testing on mice given different levels of fiber showed no significant differences among treatments in terms of mean daily feed intake, mean daily water intake, mean body weight, mean blood creatinine, and alanine aminotransferase (ALT).

In another embodiment, the glucose-lowering activity of CPHs fiber is observed in diabetic-induced mice where comparable mean fasting blood glucose level (mg/dL) was obtained with those administered with glibenclamide (positive control; anti-diabetic drug) and psyllium (commercial dietary fiber (CDF)). These embodiments indicate the safety of the CDF as an oral supplement.

In accordance with another aspect, a comestible is provided comprising fiber- rich materials extracted from CPHs, wherein the fiber is extracted using at least one physical process or a combination of a physical process and enzymatic hydrolysis of

CPHs.

These and other aspects, features and advantages of the invention or of certain embodiments of the invention will be further understood by those skilled in the art from the following description of exemplary embodiments. This invention addresses waste management problems associated with waste cacao husks, in anticipation of a resurgence of the Philippine cacao industry. The “resource recovery approach” involving the conversion of waste into value-added products (such as anti-oxidant rich food ingredients) is seen to contribute to the economic benefits of a revitalized cacao industry.

DETAILED DESCRIPTION

For purposes of the invention, the term “raw material” as used herein refers to fresh CPHs (with beans removed) or CPHs that has undergone drying and milled into different particle sizes using any milling or particle reduction equipment, or any method known to a person skilled in the art. The term “enzymatically-modified” and “enzymatically-tailored” product refers to resultant product from the enzymatic treatment of the raw material.

Besides their pollution and hazardous aspects, in many cases, CPHs might have a potential for recycling raw materials or for conversion into useful products of higher value as a by-product, or even as raw material for other industries, or for the use as food or feed/fodder after biological treatment.

One advantage of embodiments of the present invention is the processing and modification of CPHs resulting to modified fiber with distinct functional properties such as, but not limiting to low viscosity, low gelling capacity and variable water and oil absorption capacities depending on the conditions employed. A further advantage of the embodiments of the present invention is the utilization of fibers from CPHs employing physical, enzymatic or combinations thereof, and without employing chemical modification processes.

It is an advantage of at least certain embodiments of the invention to extract nutrients from CPH to selectively produce various components, for example and without limitation, polysaccharides, fibers, sugars and combinations thereof. It is a further advantage of the invention to utilize the extracted nutrients in various uses, such as but not limited to, ingredients or additives in food, feeds, cosmetics and nutraceutical formulations, as known to persons skilled in the art. These components can provide comestible products such as food and beverage products with desirable appearance, taste and health properties. The formulated products can claim to have an advantage of having increased fiber content.

It will be clear from various embodiments of the invention that overall advantage of the invention is to make use of nutrients obtained from CPHs that might otherwise be discarded as wastes. These and other advantages and features of the invention or of certain embodiments of the invention will be apparent to those skilled in the art from the following disclosure and description of exemplary embodiments.

It will be necessary that CPHs be collected right away after processing that involves removal of seeds and the mucilaginous portion attached to the seeds.

This is to ensure high quality of the end products in terms of microbial and chemical properties. Decomposition rapidly progresses if CPHs will not be processed right away. Upon arrival at the processing area, CPHs are immediately washed, processed to reduce the size, dried and milled.

The dried CPHs fiber of 60 mesh size is used for enzymatic treatment. Water is used as the control. For enzymatically-treated samples, enzymes are dissolved in buffers with appropriate pH (pH 5.0 for cellulase, pH 4.0 for pectinase and pH 4.5 for xylanase). Fermentation proceeded for 4 h at room temperature in a laboratory shaker and after the prescribed time, the slurry is heated to inactivate the enzymes, dried in a cabinet type hot air oven and then milled to achieve desired mesh size for further evaluation.

In the preparation of the raw materials, CPHs are subjected to physical treatments to reduce the particle size. The byproduct particle size is reduced according to aspects of the invention using any suitable means, typically one or more physical means disclosed herein as suitable for breaking plant cell walls; namely for example and without limitation, cutting, shredding, slicing, grinding, shearing, extruding, homogenizing, pulverizing, comminuting and combinations thereof.

In accordance with one aspect, a method is provided comprising treatment of

CPHs to prevent browning prior to drying. In some cases, cut/sliced CPHs are soaked in not more than 0.1% food-grade metabisulfite solution to prevent further browning for at least 30 min. before putting in a drying. Drying maybe accomplished by sun-drying or in a mechanical dryer of any types that are familiar to persons skilled in the art. In cases where processing cannot be performed, the fresh CPHs are immediately stored in the freezers for subsequent processing to prevent microbial decomposition and deterioration of quality.

The process of milling dried CPHs chips can be done using any type of milling machine known to a person skilled in the art. Sieving using fine mesh sieves to obtain the desired particle size follows. The powdered forms can be packed in aluminum foil packs or plastic bags, or any appropriate containers that can withstand heat treatment. The packed product maybe subjected to heat sterilization at 15psi for at least 15 minutes to eliminate microorganisms that will affect the quality of the product and render the product safe for human use when incorporated in various formulations. The material obtained contains, but not limited to, both soluble and insoluble fiber, phenolic antioxidants, proteins, fat and carbohydrates.

In accordance with another aspect, a method is provided comprising modification of CPHs fiber through enzymatic treatment where the dried CPHs are combined with one or more enzymes in appropriate buffer or water to form a slurry, mixing or agitating the slurry, heating the slurry to deactivate enzymes, filtering or centrifuging the slurry to provide a retentate and a permeate. The retentate contains both soluble and insoluble fibers. The resulting products differ in nutritional, functional and structural properties from the raw material. The modified CPHs can be subsequently used in the manufacture of foodstuffs, pharmaceuticals and nutraceuticals. The permeate contains, but not limited to, soluble fibers such as pectin and gelling agents, phenolic antioxidants, sugars and bio-flavors and colorants.

In another embodiment, it is not necessary for CPHs to undergo drying before enzymatic modification. Slurry may be obtained by applying physical process to raw materials before enzymatic process is performed. When fresh raw materials, or

CPHs particles are subjected to a physical process such as blending or mixing at high speed to break cell walls of the particles, additional liquid is released thereby forming a slurry.

In a preferred embodiment, the raw material is treated with enzymes suspended in appropriate aqueous solution under conditions appropriate for the enzyme used. The temperature, pH, and time of treatment vary and affect the properties of the resultant product. The enzymes used are those that can modify the polysaccharides present in CPH.

Claims (5)

CLAIMS What is claimed is:

1. A process of producing dietary fiber from cacao pod husk comprising the following steps: ~~ = or a. reducing the size of cacao pod husk by drying in a hot-air cabine®type 3 - dryer, milled in a hammer mill and passed onto a 60 mesh sieve .

b. treating of cacao pod husk with anti-browning substances; = : 5 c. subjecting the cacao pod husks particles to a physical process tazbrea i cell walls of the by-product particles, namely for example and Wihou 3 limitation, cutting, shredding, slicing, grinding, shearing , extrudthg, i homogenizing, pulverizing, comminuting and combinations thereof;

d. adding an enzyme solution comprising of at least one or a mixture of xylanase, pectinase and cellulase;

€. mixing or agitating the cacao pod husk particles with the enzyme solution;

f. heating the mixture of cacao pod husk and enzyme solution;

g. cooling the mixture by introducing cold air (fan) or just allowing to stand at room temperature; and h. separating the retentate from the permeate to obtain the dietary fiber.

2. The process according to Claim 1, wherein the enzymes are selected from cellulose, pectinase and xylanase, or combinations thereof.

3. The process according to Claim 1 wherein the enzyme is preferably pectinase.

» 4. The process according to Claim 1 wherein the pectinase is added at the rate of 25 to 500 U/g pectin, for 0.5 to 8 hours reacting time and 2 to 6 % w/v solids loading.

5. The dietary fiber obtainable from the process according to Claim 1 wherein it can be used as additive in nutraceuticals, beverages, feeds and food formulations.