PH12017000213A1

PH12017000213A1 - Preparation of pectin from mango peels

Info

Publication number: PH12017000213A1
Application number: PH12017000213A
Authority: PH
Inventors: Evelyn B Taboada; Francis Dave Siacor
Original assignee: Taboada Evelyn
Priority date: 2012-03-22
Filing date: 2017-07-21
Publication date: 2019-02-11
Also published as: WO2013141723A1; PH12017000214A1

Abstract

This invention relates to an improved process in the recovery and preparation of pectin from mango peels. In this improved process, the pectin-containing material is subjected to acid hydrolysis to extract the pectin, then separation of the filtrate containing the pectin material by a simple settling-decantation-filtration step, and recovery of the pectin material by alcoholic precipitation-mixing-and-settling step. The pectin precipitate is further dried and milled to obtain the pectin powder. The improved process is characterized by its pectin yield of 30 pcnt or greater. The pectin obtained herein is further characterized by its galacturonic acid content of 65 pcnt or higher and degree of esterification of 65 pcnt or greater, as it employs the defatted mango peel powder.

Description

mango peels indicates a good correlation to its antioxidative capacity and said observations were confirmed by other studies, although, interestingly, different varieties of Mangifera 7 indica L. Anacardiaceae, exhibited different characteristics and properties as well [Berardini - et al., 2005a; 2005b; Kim ef al., 2010; Larrauri ef al., 2007; Schieber et al., 2003].

In a related invention by Taboada and Siacor (2012), in which a patent application is filed Lo simultaneously with this invention, the mango peels were treated in a simple or integrated i" process to obtain a composition, referred to as mango peel powder (MPP), and characterized ) by its proximate analysis as shown in Table 1, and its bioactive components, more 5 specifically, its total polyphenolic and anthocyanin contents, as shown in Table 2 below: =

Rn

Table 1. Proximate analysis of the MPP composition.

Parameter Mango Peel Powder (MPP, %)

Moisture 4-7

Ash 4-7

Protein 3-5

Fat/Oil 18-23

Crude Fiber 15-18

Carbohydrate 40-56

Table 2. Total polyphenol and anthocyanin estimates of MPP.

Bioactive compounds MPP

Total Polyphenols (TP) content 50-120 (mg/g dry powder)

Total Monomeric Anthocyanins (TMA) 180-340 content (mg/100 g dry powder)

The said composition shows a considerable amount of fats/oils, which is an important composition, derived thereof and also crude fiber and carbohydrates. The latter two components are the sources of dietary fibers, sugars, and polysaccharides which have reportedly good health benefits. On the other hand, mango peel powder also contains high total polyphenolic and anthocyanin contents; the former is attributed mainly for its good antioxidant and antimicrobial activities.

Sources of Pectin

A fibre-containing pectin product was obtained from a plant material such as potato, tobacco and cacao, as described in a patent WO 2009/026936 or US 2010/0247582 by Sorensen et al.

Page 4 of 25

(2007). The method comprises the step, amongst others, of providing a plant material . comprising pectin having a degree of esterification of 55% or less. -

Beet pulp was tapped as source of pectin in a patent US 5,071,970 by Le Grand and Stevens =” (1990). In said patent, beet pulp was reduced to small particle size and hydrolyzed in HCI at = a low temperature for a long time and then peptized in hot water at 60-70°C for about one LE hours to produce an aqueous pulp slurry that is screened to recover the liquid which is filtered - and mixed with aluminum chloride to lower the pH about 0.1 pH unit to the range of 1.8-2.25 ~ pH. Ammonium hydroxide is then added to raise the pH to about 4.0-4.5 to form an ® aluminum pectinate precipitate, which is washed with an acidified alcoholic liquid to produce - high-to-medium methoxyl pectin which is separated and dried to a stable powder. Waste - streams are converted to animal feed or are recovered and recycled.

A EU patent 0664300 or US 6,143,346 by Glahn (1994) relates to pectin compositions and processes for preparing the same, obtained from lime, lemon, grapefruit, and orange. Said pectin composition has a high degree of esterification greater than 60% and a ratio of calcium sensitive pectin to the sum of calcium sensitive pectin and non-calcium sensitive pectin greater than 0.65.

A patent WO 00/24921 by Banister et al. (1998) relates to methods and reagents for preparing pectin from tomato, banana, and orange, especially the high molecular weight pectin having a high methoxyl content which can form a gel or a viscous solution in the presence of polyvalent metal ions such as calcium ions.

A patent US 6,787,177 by Crandall and McCain (2004) also revealed a process for extracting pectin from waste hull/hypocotyl streams during soybean processing. In said process, a soy pectin material was obtained comprising about 40% wt anhydrous galacturonic acid and about 8%wt protein. Chemical extraction agents in addition to or in conjunction with heat and agitation are used to extract pectin from the waste streams. The pectin is then precipitated in alcohol, washed and dried. Yield reported was 5.6% of pectin material.

A patent application US 2009/0110798 by Gusek et al. (2006) pertains to a process for obtaining pectin from a group of pectin-containing materials such as citrus fruit, apple, sugar

Page 5 of 25 beet, sunflower soybean, and mixtures thereof, comprising the treatment of said materials in = aqueous medium with cellulase and hemicellulase enzymes, to release pectin from said pectin-containing materials, thereby forming a pectin solution. = ~

High-quality pectins may be extracted from pectin-containing plant materials with microwave ~ energy under pressure as described in a patent WO 00/65929 by Fishman and Chau (1999). ©

The pectin obtained is characterized by increased molecular weight, size and intrinsic a) viscosity when compared to pectin extracted by conventional heating techniques. -

A patent US 4,686,187 by Sakai and Katsuragi (1981) revealed a different process for = preparing pectin by subjecting a plant tissue containing pectic substances, such as Citrus ik unshiu and Citrus natsudaidai to the action of microorganism, which possesses an activity liberating pectin from a plant tissue, or of a culture broth or processed material thereof to liberate pectin from said plant tissue and subsequently recovering the pectin. Microorganisms used in said process belongs to the genus Endomyces, Endomycopsis, Saccharomyces,

Shizosaccharomyces, Pichia, Hansenula, Debaryomyces, Hanseniaspora, Torulopsis,

Candisa or Kluyveromyces. Although such a process is novel, it results in very low yield (up to 9% only) of the pectic material.

Recovery methods of pectin

The processes for preparing pectin are well-known, as are the many uses for this product composition. In general, the typical pectin processes include the following steps: (1) Acid extraction at low pH of the plant starting material containing pectin, (2) Purification of the liquid extract, and (3) Isolation of the extracted pectin from the liquid.

In the first step, the pectin-containing material, which is usually a plant material, is treated with dilute acid solutions such as nitric, sulfuric, hydrochloric, or other inorganic or organic acids to remove the pectin from the cellulose components of the material. The commonly used plant starting materials are citrus peels from juice production and apple pomace from apple juice and cider production. Other plant sources such as orange peels, sugar beet (before or after sugar extraction), beet pulp, sunflower heads (after the removal of seeds), mango peels, and other vegetables or waste products from plants, are also considered in the prior art.

Page 6 of 25

Extraction conditions are selected such that a majority of the pectin molecules contained in — the plant material is extracted and transferred to the extracting medium. In many cases as o reported in the prior art, the quantity and quality of the extracted pectin depends on the raw = material source and the selection and control of the extraction conditions such as pH, 0 temperature and extraction time.

After the acid extraction step, a mixture containing the pectin-containing liquid and the spent = plant material is formed. This mixture is then subjected to further purification, which is the - second step herein, in which the solid plant material is removed by filtration, centrifugation, = or other conventional separation steps. The extract can, optionally, be further purified by ion- = exchange, adsorption, and concentrated by evaporation of part of the water. Alternatively, ol the purification step can be carried out by reverse osmosis, concentrating and purifying the extract in the same step.

The pectin in the acid extract can also be isolated by reacting with aluminum salts after adjusting the pH. The aluminum pectinate gel thus formed is treated with alcohol/acid mixture to wash out the aluminum salt and transformed the pectin into pectic acid. The pectic acid can then be neutralized and a substantial amount of the water is removed by washing with slightly alkaline alcohol.

More commonly, the pectin is isolated by treating the pectin solution with appropriate alcohol to render the pectin insoluble in the ensuing blend of alcohol and water. Any alcohol or other organic solvent miscible with water can be used, most often ethanol, methanol, or isopropyl alcohol. The insolubilized pectin is separated from the alcohol/water mixture by appropriate means such as filtration, centrifugation, etc. The resulting pectin is dried and milled to the desired particle size.

Pectins produced industrially are made up primarily of polygalacturonic acid chains in which rhamnose may be found. Natural sugars may be attached to the rhamnose units. The anhydrogalacturonic acid makes up at least 65% of the dry matter in commercial type pectins.

The galacturonic acids are partly esterified with methyl alcohol. According to convention, pectins with more than 50% of the carboxylic acid groups esterified with methyl alcohol are referred to as high methoxyl pectins, having a corresponding degree of esterification (% DE)

Page 7 of 25 of greater than 50%; whereas pectins with less than 50% of the carboxylic groups esterified i, with methyl alcohol are called low methoxyl pectins, with corresponding degree of i. esterification (% DE) of less than 50%. =

The extract as obtained by the commercial production is composed of those molecules that - are soluble under the conditions of pH, temperature, and incubation time used during the © extraction. The extract is composed of a mixture of molecules which differ according to fo molecular weight, distribution of molecular weight, and degree of esterification. -

Recovery of polyphenolic compositions from plant sources =

Two alternative processes for the combined recovery of pectin and polyphenols, were also developed, which can easily be integrated in an existing pectin production process [see

Berardini et al., 2005b cited above]. In said alternative process, the alcoholic precipitation of pectin is integrated in the recovery of polyphenols by using an adsorber resin, combined with polyphenolic elution with methanol and its subsequent evaporation and lyophilization.

Furthermore, a patent US 2002/0187207 by Pascale et al. (2002), which is similar to the process by Berardini ef al. (2005b) revealed a method for extracting, fractionating, and purifying polyphenolic compounds originating from fresh plant sorting deviations using a high adsorption and elution performance resin.

In another invention described by a patent US 5,932,623 by Tanabe et al. (1995), a process was developed for the production of fruit polyphenols from unripe Rosaceae fruit. Said process provides fruit polyphenols obtained by subjecting the unripe fruits of Rosaceae such as apples to pressing and/or extraction and then purifying the resulting juice or extract. The polyphenolic compositions derived herein are claimed to be an antioxidant, a hypotensive agent, an antimutagenic agent, an antiallergic agent and an anticarcinogenic agent.

Brief description of drawings

Figure 1 shows the diagram of a simple process for the treatment of mango peels from fruit- processing, described fully in a related invention, wherein mango peel powder (MPP) and the corresponding defatted or oil-extracted composition (MPP*) and its fat/oil extract are obtained.

Page 8 of 25

Figure 2 shows the diagram of the integrated process in the treatment of mango wastes of i. fruit processing described fully in a related invention, whereby the immediately resulting compositions in this process are indicated, namely: mango peel powder (MPP), mango seed ht kernel powder (MSKP), mango seed husks (MSH) or mango seed husk powder (MSHP), and - the corresponding defatted or oil-extracted compositions from said compositions above. -

Figure 3 shows the diagram of an improved process, which is a continuation of the simple process in Figure 1 or integrated process in Figure 2 for the treatment of mango wastes, » wherein process steps are added to treat further the defatted mango peel powder (MPP*) and obtain the mango pectin and the polyphenolic compositions. o -

In a related invention mentioned earlier, simple and integrated processes are developed as “ illustrated in Figures 1-2, wherein mango (Mangifera indica L. Anacardiaceae) peels are treated instantaneously and efficiently. Among the resulting compositions from the said invention, are the compositions derived from mango peels, which are relevant to the invention herein as follows: 1. A composition, which is the mango peel powder (MPP), derived from the treatment of treating mango (Mangifera indica L. Anacardiaceae var. Philippines) peels, characterized by the following proximate analysis: moisture 4-7%, ash 4-7%, protein 3-5%, fat/oil 18- 23%, crude fiber 15-18%, and carbohydrate 40-56%. 2. A composition, which is the defatted mango peel powder (MPP*), characterized in that said composition is obtained from the mango peel powder (MPP) derived from the simple or integrated processes of the said invention described earlier. 3. A composition which is the polyphenolic concentrate, characterized in that said composition contains relatively high concentrations of polyphenols derived from the mango peel powder (MPP) obtained in the simple or integrated process of the invention described earlier.

The Invention

The object of the invention is the improved process in the recovery and preparation of pectin and polyphenolic compositions, which can be combined with the simple or integrated processes described earlier and illustrated in Figures 1-2. The said improved process is comprised of the following steps, as shown in Figure 3:

Page 9 of 25

(a) Acid hydrolysis of the pectin-containing material, which is the defatted mango peel i, powder (MPP*), at a pH range of 1.5-3.5 and temperature of 60-90°C for about 30- - 120 minutes, ” (b) Separation of the filtrate or liquid extract resulting in step (a) from the spent mango - peel powder, by simple decantation after settling of the spent MPP* or by simple ~ filtration using cheesecloth, or a combination of these two steps, © (c) Alcoholic precipitation of pectin from the filtrate or liquid extract obtained in step (b), combined with mixing-settling processes, whereby ethanol or methanol is employed, wv preferably ethanol at a volumetric ratio of 1:5 (ethanol-to-filtrate), o (d) Purification of the alcoholic solution obtained in step (c) after separation of the wet - pectin precipitate, preferably combined with the recovery of the alcohol from the = alcoholic solution obtained during the said process step (c), and recycling said alcohol back to process step (c), and (e) Drying and milling of the wet pectin precipitate obtained in step (c).

The improved process in the recovery and preparation of pectin and polyphenolic compositions, which is the object of this invention, is characterized in that the said improved process: i. employs the defatted mango peel powder derived from the mango peel powder (MPP), obtained in either the simple or the integrated process of a related invention, ii. allows the recovery of pectin with a galacturonic acid content of 70% or higher and high methoxyl content corresponding to a degree of esterification of 65% or greater, iii. results in pectin yield of 30% or higher, iv. results in a “zero-waste” process, wherein no wastes are generated, v. results in the efficient use of alcohol as precipitating agent for pectin, vi. allows the use of the combined method of purification and alcohol recovery in the preparation of polyphenolic composition.

In this invention, pectin yield is defined as the amount of pectin obtained based on the amount of defatted mango peel powder used as starting material for the process. The degree of esterification (% DE) of pectin is defined as the number of methyl-esterified galacturonic

Page 10 of 25 acid units expressed as a percentage of the total galacturonic acid in the pectin molecule obtained. Further, pectin content is also expressed in terms of galacturonic acid content, - using said galacturonic acid as standard. =

In this invention, one relevant resulting composition obtained by either the simple or the Th integrated process discussed in the related invention and illustrated in Figures 1-2, that is the + defatted mango peel powder (MPP*), is treated accordingly to obtain a plurality of 0 compositions derived thereof. As illustrated in Figure 3, the defatted mango peel powder - (MPP*) undergoes acid hydrolysis in process step 8 to extract the pectin material. Two dilute - acid solutions were considered in this process step, namely: sulfuric acid or hydrochloric = acid, which are effective in giving good pectin recovery. Acid hydrolysis of the defatted mango peel powder (MPP*) at a low pH range, preferably at pH 1.5-3.5, more preferably at pH 1.5-2.5, and most preferably at 1.5, using either dilute sulfuric acid or hydrochloric acid solution, but preferably hydrochloric acid solution is employed. Furthermore, a weight ratio of defatted mango peel powder (MPP*), which is the pectin-containing material, to water of 1:10 to 1:40 is employed during said process, preferably at a ratio of 1:10-1:30, more preferably at a ratio of 1:10-1:20, and most preferably at a ratio of 1:15. The hydrolysis step is carried out by heating the mixture at 60-90°C, preferably at 70-90°C, more preferably at 80-90°C and most preferably at 80°C to facilitate faster and more efficient pectin extraction : 20 from the defatted mango peel powder for about 30 to 120 minutes. Acid hydrolysis was preferably done at an incubation time of 60-120 minutes, more preferably at 90-120 minutes, and most preferably at 120 minutes.

The resulting mixture is then filtered in process step 9 to separate the filtrate and the spent defatted mango peel powder (spent MPP*). In this process step, simple decantation of the filtrate after a few minutes of settling of the spent MPP* works most efficiently, especially that the filtrate has an increased viscosity with increasing amount of pectin extracted; however, the use of a simple filter cloth is also equally efficient and quick as the spent MPP* is easily removed by then. Thus, a combination of a simple settling-decantation-filtration process step to separate the filtrate (liquid extract) from the spent solids is the most preferable step as this allows the full recovery of the said filtrate containing pectin and said spent solid (spent mango peel powder) for recycling or further use. While the collected spent MPP* is dried and milled in process step 11 to obtain the dried and milled spent MPP* for further use,

Page 11 of 25 e.g. as ingredient in animal feeds, the filtrate undergoes alcoholic precipitation-mixing-and- » settling in process step 10; after which the obtained wet pectin precipitate is dried and milled - in process step 11, to obtain an important composition herein which is the mango pectin fi powder. During alcoholic precipitation, ethanol or methanol can be used. In this invention, =~ ethanol is preferred as it is acceptably food-grade. After addition of appropriate quantities of . alcohol, employing a volumetric ratio of 1:5 (ethanol-to-filtrate), that is, for every 1 liter of © filtrate, about 200 mL of ethanol (95% pure) is used, the mixture is then stirred thoroughly wt for a few minutes, during which time the pectin gel is visibly formed in the mixture. -

Afterwhich, the resulting pectin precipitate (which is semi-solid, having a gel-like form) is = allowed to settle for a few minutes. This pectin gel-like material is then withdrawn as wet pectin precipitate. This is preferably dried and milled to the desired particle size in process step 11 to obtain the dry mango pectin powder.

On the other hand, the alcoholic solution obtained in process step 10 is another important composition, which is an extract with high polyphenol concentrations. This solution is subjected to a purification step in process step 12, which includes alcohol recovery, to obtain a more concentrated composition of polyphenolics. The purification step may involve ion- exchange, adsorption or simple evaporation of the alcohol at reduced pressure. The alcohol recovered in this process step is collected and recycled back to process step 10 where it is used again for the alcoholic precipitation-mixing-settling process. An appropriate alcohol such as ethanol or methanol is used and an alcohol make-up is needed in process step 10, to ensure the favorable conditions for the said process step. The concentrate obtained after process step 12 is another important composition, referred to herein as polyphenolic concentrate (see Figure 4), derived after the purification of the alcoholic solution. The said composition is characterized by its relatively high concentration of polyphenolics.

EXAMPLES

Example 1. Recovery of pectin from mango peel powder (MPP)

Pectin was extracted from the defatted mango peel powder (MPP*) obtained by the simple or integrated process described in a related invention and illustrated in Figures 1-2 herein. The pectin extraction process employs the acid hydrolysis and alcoholic precipitation methods.

Page 12 of 25

A known amount of defatted mango peel powder (MPP*) is mixed with water at different . weight ratios (1:10-1:40) and the pH of the mixture was adjusted to a certain pH point (1.5- - 3.5) by adding a dilute solution of acid (using 0.IN sulfuric acid or 0.1N hydrochloric acid). o

The resulting mixture was stirred and heated at a certain temperature (60-90°C) for different - time periods (30-120 minutes). After which, the mixture was allowed to cool down, during - which time, the spent solids were also settled down. When the mixture has cooled down to © be conveniently handled, the filtrate is decanted to another vessel. Then, the spent solids » together with the remaining adhering filtrate is filtered using an ordinary filter or alternatively v using cheesecloth, to efficiently separate the filtrate from the spent solids (mango peel = powder). The filtrate collected in another vessel was further mixed with ethanol (95% pure) ~ at a certain volumetric ratio of 1:5 (ethanol-to-filtrate), and mixed thoroughly to precipitate = the pectin. The mixture was allowed to settle the pectin precipitate, then, filtered through an ordinary filter to efficiently separate the pectin precipitate and the alcoholic filtrate. Washing of the wet pectin precipitate is done using known amounts of ethanol. The wet pectin precipitate was then dried, and milled to produce the powdered mango pectin. Some quantities of pectin is set aside for further analysis in terms of its galacturonic acid content employing the standard methods and yield on the basis of dried defatted mango peel powder sample used. The obtained pectin composition is also analyzed for its degree of esterification (% DE) according to the procedures described herein [Singthong et al., 2004]. On the other hand, the alcoholic solution after alcoholic precipitation was collected and analyzed for its total polyphenol (TP) and total monomeric anthocyanin (TMA) contents as described herein [Wolfe ef al., 2003; Ajila ef al., 2007a; Vieira et al., 2011].

Table 3 below shows some selected results at different operating conditions during acid hydrolysis and alcoholic precipitation of pectin from the defatted mango peel powder.

Experimental trials and analysis were done in triplicates, preferably in two sets of triplicates.

Table 3. Yields and % DE of pectin at different conditions during recovery process.

Sample Acid pH Sample:Water Pectin Yield Degree of Esterification

Trial used Ratio (w.v) (%) (%) 1 H,S0, 2.5 1:40 1-6 37-61 2 H,SO, 2.5 1:30 18-32 38-60 3 H,SO, 2.5 1:20 24-34 47-67 4 HCI 2.5 1:20 27-42 68-75 5 HCI 1.5 1:15 32-40 67-84 6 HCI 1.5 1:10 14-32 60-80

Page 13 of 25

Results showed that a notably high yield of 32-40% pectin was obtained when a pectin- - containing material, that is the defatted mango peel powder (MPP)-to-water volumetric ratio fi of 1:15 and hydrochloric acid at pH 1.5 were employed during acid hydrolysis at 80°C for 120 minutes. Good yields in the range of 27-42% of pectin were also obtained when a ~ defatted MPP-to-water volumetric ratio of 1:20 was employed at the same conditions of pH, 0 temperature, and incubation period. The distinctly high yield of the process is attributed to " the small particle sizes of the mango peel powder used as pectin-containing material, which v maximizes the surface areas of the particles exposed for pectin extraction during acid = hydrolysis. In addition, the defatting of the pectin-containing material prior to hydrolysis o could have also contributed to a much simpler extraction process, in which the starting be material is free of other unnecessary components, especially fats and oils, prior to pectin extraction.

Example 2. Analysis of the Collected Alcoholic Solution after Pectin Extraction

The alcoholic solution (see Figure 3) obtained after separation of pectin was analyzed for its total polyphenol (TP) and total monomeric anthocyanin (TMA) contents according to the procedures described earlier. Results in Table 4 showed that the solution contains notably high contents of total polyphenols (TP) ranging from 700-1,710 mg per liter of alcoholic solution. An average total polyphenol content of 850-1,350 mg per liter of alcoholic solution was noted. However, there was also a negligible amount of total monomeric anthocyanins (TMA). The results indicate that the alcoholic solution obtainable after pectin precipitation is a good source of polyphenolic substances, even prior to its subsequent purification, rendering it a good ingredient already in nutraceuticals, functional foods, pharmaceuticals, and cosmetic applications, and therefore, is a valuable composition resulting from this invention.

Table 4. Total polyphenol and monomeric anthocyanin estimates of alcoholic solution.

Sample Trial Total Polyphenolics (TP) Total Monomeric Anthocyanin (TMA) (mg/L solution) (mg/L solution) 1 1,150-1,650 negligible 2 750-1,100 negligible 3 700-870 negligible 4 1,000-1,710 negligible average 850-1,350 negligible

Page 14 of 25 hy

Example 3. Analysis of the Spent Mango Peel Powder after Pectin Extraction .

The spent mango peel powder collected after pectin extraction was collected and further ol analyzed for its proximate analysis by standard methods [AOAC, 2000] and its remaining - total polyphenolic and anthocyanin contents by the methods described earlier. Results in -

Table 5 shows that the said spent mango peel composition still comprises of good quantities ~ of carbohydrates, crude fiber, and protein. In addition, the spent mango peel powder also Lo contains a considerable amount of polyphenolics, about 45-70 mg per gram of spent material, ia such that said composition is a good source of such bioactive compounds. Further drying of - the said composition reduced its moisture content, and after re-milling to powder, it is 5 rendered a good material for food, feed, and functional food applications. In short, the spent = mango peel is also a very useful composition obtainable by the improved process; thereby, Hi illustrating clearly that the process is generating zero-wastes.

Table 5. Proximate analysis of spent mango peel powder after recovery of pectin.

Sample Proximate Analysis (%)

Trial Moisture Ash Protein Fat/Oil Crude Fiber ~~ Carbohydrates

A 87-90 0.25-0.35 0.3-1.0 negligible 4.3-8.0 0.65-8.2

B 89-91 0.20-0.25 0.2-0.8 negligible 6.6-6.7 1.25-4.0

C 89-91 0.20-0.25 0.2-0.9 negligible 3.1-7.0 0.85-7.5

Table 6. Total polyphenol estimates of spent mango peel powder.

Sample Trial Total Polyphenolics (TP) Total Monomeric Anthocyanin (TMA) (mg/g spent MPP) (mg/g spent MPP)

A 45-50 negligible

B 45-50 negligible

C 45-70 negligible

Page 15 of 25

DOCUMENTS CITED .

Patent literature: i.

Banister NE, Sime JT, Cheetham PSJ. Pectins and the preparation thereof. Patent WO 00/ ol 24921, October 24, 1998. 0

Crandall PG and McCain AM. Production of pectin from soybeans. Patent US 6,787,177, o

September 7, 2004. i

Fishman M and Chau HK. Extraction of pectin by microwave heating under pressure. Patent r-

WO 00/65929, April 30, 1999. -

Glahn PE. Pectin process and composition. EU Patent 0664300 or US 6,143,346, February 12, 1993.

Gusek TW, Zullo L, Eyal AM. Process for obtaining pectin. Patent application WO 2006/096884 or US 2009/0110798, March 9, 2005.

Le Grand F and Stevens NC. Process for producing pectin with a high-to-medium methoxyl content from beet pulp. Patent US 5,071,970, February 12, 1990.

Pascale G, Amiot-Carlin MJ, Escudler JL, Mikolajczak M, Martin M. Method for extracting fractionating and purifying polyphenolic compounds originating from fresh plant sorting deviations using a high adsorption and elution performance resin. Patent US 2002/0187207,

February 15, 2002.

Sakai T and Katsuragi T. Process for preparing pectin from plant tissues. Patent US 4,686,187, August 11, 1987.

Sorensen OB and Larsen PF. Method of preparing fibre-containing pectin product and pectin product hereof. Patent WO 2009/026936 or US 2010/0247582, August 29, 2007.

Page 22 of 25

Taboada EB and Siacor FDC. Integrated processes in the treatment of mango wastes of fruit = processing and the preparation of compositions derived thereof. Patent PH applied on March - 22,2012. ro

Tanabe M, Kanda T, Yanagida A. Process for the production of fruit polyphenols from unripe -

Rosaceae fruit. Patent US 5,932,623, November 9, 1995.

Non-patent literature: -

Ajila CM, Bhat SG, Prasada Rao UJS. Valuable components of raw and ripe peels from two —

Indian mango varieties. Food Chemistry 2007a, 102, 1006-1011. fal

Ajila CM, Naidu KA, Bhat SG, Prasada Rao UJS. Bioactive compounds and antioxidant potential of mango peel extract. Food Chemistry 2007b, 105, 982-988.

Barreto JC, Trevisan MTS, Hull WE, Erben G, De Brito ES, Pfundstein B, Wurtele G,

Spiegelhalder B, Owen RW. Characterization and Quantitation of Polyphenolic Compounds in Bark, Kernel, Leaves, and Peel of Mango (Mangifera indica L.). Journal of Agricultural

Food Chemistry 2008, 56, 5599-5610.

Beerh OP, Raghuramaiah B, and Krishnamurthy GV. Utilization of mango waste: Peel as a source of pectin. Journal of Food Science and Technology 1976, 13, 96-97.

Berardini N, Fezer R, Conrad J, Beifuss U, Carle R, and Schieber A. Screening of mango (Mangifera indica L.) cultivars for their contents of flavonol O- and xanthone C-glycosides, anthocyanins and pectin. Journal of Agricultural and Food Chemistry 2005a, 53, 1563-1570.

Berardini N, Knodler M, Schieber A, Carle R. Utilization of mango peels as a source of pectin and polyphenolics. Innovative Food Science and Emerging Technologies 2005b, 6, 442-452,

Page 23 of 25

Hassan FA, Ismail A, Hamid AA, Azlan A, Al-sheraji SH. Characterization of fibre-rich ” i. powder and antioxidant activity of Mangifera pajang K. fruit peels. Food Chemistry 2011, . 126, 283-288 = iy

Kim H, Moon JY, Kim H, Lee DS, Cho MJ, Choi HK, Kim YS, Mosaddik A, Cho SK. .

Antioxidant and antiproliferative activities of mango (Mangifera indica L.) flesh and peel. wo

Food Chemistry 2010, 121, 429-436. ws

Kratchanova M, Benemou C, Kratchanov C. On the Pectic Substances of Mango fruits. =

Carbohydrate Polymers 1991, 15, 271-282.

Larrauri JA, Ruperez P, Borroto B, and Saura-Calixto F. Mango peels as a new tropical fibre:

Preparation and characterization. Lebensmittel-Wissenschaft und-Technologie 1996, 29, 729— 733.

Larrauri JA, Ruperez P, and Saura-Calixto F. Mango peels fibres with antioxidant activity.

Zeitschrift fur Lebensmittel-Untersuchung und-Forschung A 2007, 205, 39-42.

Rehman ZU, Salariya AM, Habib F, Shah WH. Utilization of Mango Peels as a Source of

Pectin. Journal Chemical Society of Pakistan 2004, 26(1), 73-76.

Schieber A, Berardini N, and Carle R. Identification of flavonol and xanthone glycosides from mango (Mangifera indica L. cv. Tommy Atkins) peels by high-performance liquid chromatography-electrospray ionization mass spectrometry. Journal of Agricultural and

Food Chemistry 2003, 51, 5006-5011.

Srirangarajan AN and Shrikhande AJ. Mango peel waste as a source of pectin. Current

Science 1976, 45, 620-621.

Tandon DK and Garg N. Mango waste: A potential source of pectin, fiber, and starch. Indian

Journal of Environmental Protection 1999, 19, 924-927.

Page 24 of 25

Tandon DK, Kalrat SK, Singh BP, and Garg N. Characterization of pectin from mango fruit oe waste. Indian Food Packer 1991, 45, 9— 12. . oo —

Singthong J, Cui SW, Ningsanond S, Goff HD. Structural characterization, degree of _ esterification and some gelling properties of Krueo Ma Noy (Cissampelos pareira) pectin. =

Carbohydrate Polymers 2004, 58, 391-400. ww

Vieira FGK, Borges GDS, Copetti C, Di Pietro PF, Nunes EDC, Fett R. Phenolic compounds = and antioxidant activity of the apple flesh and peel of eleven cultivars grown in Brazil. <r

Scientia Horticulturae 2011, 128, 261-266. po 011

Wolfe K, Wu X, Liu RH. Antioxidant activity of apple peels. Journal of Agricultural and

Food Chemistry 2003, 51, 609-614.

Page 25 of 25

DESCRIPTION .

This invention pertains to the improved processes in the recovery of pectin from mango peels - and the subsequent preparation of polyphenolic compositions derived thereof. Mango peels wo are commonly regarded as waste by-products in fruit processing; and in tropical countries, 0 these are oftentimes simply dumped in landfills and open dumpsites. Studies revealed that - mango peels, which are a seemingly useless by-product of fruit processing, contain high LH quantities of carbohydrates and crude fiber, and can be an excellent source of pectin and =o polyphenolic compositions. Said mango peels also exhibit good antioxidative and - antimicrobial activities which are attributed to its high polyphenolic content. © ©

In this invention, it is revealed that pectin and polyphenolic compositions can be recovered . more efficiently from mango peels by the implementation of an improved process. The said compositions resulting from this improved process can readily be used as ingredients in food and functional food preparations, in nutraceuticals, cosmetics, and pharmaceutical applications.

Background Information

Mango peel is a significant by-product or waste from mango processing industries. Studies showed that the mango (Mangifera indica L. Anacardiaceae, Indian variety) peels contain a considerable amount of carbohydrates (20.8 -28.2%), crude fibre (3.28-7.4%), protein (1.45- 2.05%), fat (2.16-2.66%), moisture (66.0-75.25%) and ash (1.30-3.0%) [Ajila et al., 2007a].

Further, mango peels also contain soluble dietary fiber (SDF = 15.70-28.05%) and insoluble dietary fiber (IDF = 28.99-50.33%), with an IDF/SDF ratio of 1.68-1.99, which indicates that such a composition has more health beneficial effects, when incorporated in food and functional food applications. The proximate analysis of mango peel of Indian variety is similar to the peels of mango (Mangifera indica L., Hayden Cuban variety), which has soluble dietary fiber (21.6-28.1%), insoluble dietary fiber (12.8-23.8%), ash (2.85%), protein (5.25%), oil (2.5%), total sugar (soluble fiber-21.6%), and total sugar (insoluble fiber-12.8%) [Larrauri ef al., 1996]. On the other hand, the bambangan fruit (Mangifera pajang K.), which is three times as large as commercial mango (Mangifera indica), found in Malaysia, Brunei, and Indonesia, contain about 27% peels, which has a proximate analysis of: moisture (3.9%), protein (4.6%), fat (2.9%), ash (2.7 %), carbohydrate (7.3%) and total dietary fiber (72.3%)

Page 2 of 25

[Hassan ef al., 2011]. Such examples show that mango peels contain indeed a considerable . amount of carbohydrates and crude fiber, which are sources of sugars, soluble and insoluble o dietary fiber and bioactive components, the latter of which is attributed to the antioxidant i. activity of the peels. ” oo

On the other hand, mango peels also contain a considerable amount of pectin, which is © usually expressed in terms of galacturonic acid composition. Pectin is a structural polysaccharide found in the primary cell walls of terrestrial plants. It is produced ~ commercially as a white to light brown powder, mainly extracted from citrus and apple peels = and is used in food as a gelling agent and as a source of beneficial dietary fiber, used in =o fillings, medicines, and sweets, and as a stabilizer in fruit juices and milk drinks. Peels of two -

Guinean mango varieties (Ceni and Springfield) has reportedly 24.5% and 22.3% of pectin for Ceni and Springfield varieties, respectively [Kratchanova ef al., 1991]. Mango peels of

Pakistan variety were also reported to have 21.0% pectin yield at optimum conditions investigated [Rehman et al., 2004]. Similar findings are also obtained in other studies [Srirangarajan and Shrikhande, 1976; Tandon and Garg, 1999; Tandon et al., 1991; Beerh et al., 1976; Berardini et al., 2005a; 2005b].

Furthermore, mango peels are proven to be a good source of bioactive compounds. Several studies showed that it contains bioactive compounds such as polyphenols (33.31-73.88 mg/g powder by alcohol extraction and 54.67-109.7 mg/g powder by acetone extraction), carotenoids (365-3,945 pg/g powder), vitamin C (188-392 pg/g powder), and vitamin E (205- 509 ng/g peel powder) [Ajila et al., 2007b]. Polyphenolics were reportedly extracted from mango (Mangifera indica L. cv.‘Tommy Atkins’) peels and characterized by high- performance liquid chromatography/electrospray ionization mass spectrometry [Barreto ef al., 2008; Berardini ef al., 2004]. Eighteen gallotannins compounds and five benzophenone derivatives were detected in said mango peels which were tentatively identified as gallolated maclurin and iriflophenone glucosides. Gallotannins quantified by the rhodanine assay amounted to 1.4 mg/g dm in the peels (expressed as gallic acid). In addition, the antioxidative capacity of the extracts from mango peels exceeded that of mangiferin and quercetin 3-O-glucoside, respectively, thus demonstrating mango peels to be a suitable source of health-beneficial compounds. The amount of polyphenolic compounds in the extracts of

Page 3 of 25

Claims

AMENDED CLAIMS -

1. A process for the preparation of pectin and polyphenolic compositions from mango - (Mangifera indica L. Anacardiaceae) peels, comprising the following steps: = (a) acid hydrolysis of the pectin-containing material, (b) separation of the filtrate or liquid extract from the spent pectin-containing ~ material, 0 (¢) alcoholic precipitation of pectin from the filtrate, (d) purification of the pectin-free solution, combined with alcohol recovery and - recycling, and o (e) drying and milling of the pectin precipitate, = characterized in that the said process employs the defatted mango peel powder as the wr pectin-containing material.

2. The process according to Claim 1, wherein sulfuric acid is used during acid hydrolysis.

3. The process according to Claim 1, wherein hydrochloric acid is used during acid hydrolysis. ~

4. The process according Claims 1-3, wherein a dilute acid solution is used.

5. The process according to Claim 1, wherein a pH range of 1.5-3.5 is employed during acid hydrolysis.

6. The process according to Claim 1, wherein a pH range of 1.5-2.5 is employed during acid hydrolysis.

7. The process according to Claim 1, wherein a pH of 1.5 is employed during acid hydrolysis.

8. The process according to Claim 1, wherein a temperature range of 60-90°C is employed during acid hydrolysis.

9. The process according to Claim 1, wherein a temperature range of 70-90°C is employed during acid hydrolysis.

10. The process according to Claim 1, wherein a temperature range of 80-90°C is employed during acid hydrolysis.

11. The process according to Claim 1, wherein a temperature of 80°C is employed during acid hydrolysis.

12. The process according to Claim 1, wherein the incubation time during acid hydrolysis is at 30-120 minutes. Page 16 of 25

13. The process according to Claim 1, wherein the incubation time during acid hydrolysis i» is at 60-120 minutes.

14. The process according to Claim 1, wherein the incubation time during acid hydrolysis = is at 90-120 minutes. -

15. The process according to Claim 1, wherein the incubation time during acid hydrolysis is at 120 minutes. 0

16. The process according to Claim 1, wherein a ratio of pectin-containing material to 3 water of 1:10 to 1:40 (wt:vol) is employed. -

17. The process according to Claim 1, wherein a ratio of pectin-containing material to : water of 1:10 to 1:30 (wt:vol) is employed.

18. The process according to Claim 1, wherein a ratio of pectin-containing material to ol water of 1:10 to 1:20 (wt:vol) is employed.

19. The process according to Claim 1, wherein a ratio of pectin-containing material to water of 1:15 (wt:vol) is employed.

20. The process according to Claim 1, wherein a combined settling-decantation-filtration step is employed in the separation of filtrate and spent solids.

21. The process according to Claim 1, wherein ethanol is used during alcoholic precipitation.

22. The process according to Claim 1, wherein methanol is used during alcoholic precipitation.

23. The process according to Claim 1, wherein an alcohol-to-filtrate volumetric ratio of 1:5 is employed during alcoholic precipitation.

24. The process according to Claim 1, wherein a combined alcoholic precipitation- mixing-settling process is employed.

25. The process according to Claim 1, wherein purification of alcoholic filtrate is combined with alcohol recovery.

26. The process according to Claim 1, wherein the alcohol recovered during the purification step is recycled.

27. The process according to Claim 1, wherein the pectin composition is in liquid form.

28. The process according to Claim 1, wherein the pectin composition is in powder form.

29. The process according to Claim 1, wherein mango (Mangifera indica L. Anacardiaceae var. Philippines) peels is used. Page 17 of 25

30. A pectin material derived from the defatted mango (Mangifera indica L. Anacardiaceae) peels, characterized in that the composition has a galacturonic acid content of 70% or higher.

31. A pectin material derived from the defatted mango (Mangifera indica L. - Anacardiaceae) peels, characterized in that the composition has a degree of ~ esterification of 65% or higher. pe

32. A pectin material obtainable from the process according to Claim 1, characterized in » that the composition is derived from the defatted mango (Mangifera indica L. - Anacardiaceae) peels. =

33. A pectin material obtainable from the process according to Claim 1, characterized in that the composition is derived from the defatted mango (Mangifera indica L. o : Anacardiaceae) peels and is having a galacturonic acid content of 70% or higher.

34. A pectin material obtainable from the process according to Claim 1, characterized in that the composition is derived from the defatted mango (Mangifera indica L. Anacardiaceae) peels and is having a degree of esterification of 65% or higher.

35. The composition according to Claims 30-34, which is in liquid form.

36. The composition according to Claims 30-34, which is in gel form.

37. The composition according to Claim 30-34, which is in powder form.

38. The composition according to Claim 30-37, which is in any combination with other pectin compositions. i

39. The composition according to Claim 38, which is in any combination in any proportion with other pectin compositions.

40. The composition according to Claim 30-39 wherein mango (Mangifera indica L. Anacardiaceae var. Philippines) peels are used.

41. The use of composition according to Claims 30-39 as ingredient for food, feed, and functional food applications.

42. The use of composition according to Claims 30-39 as ingredient in nutraceutical applications.

43. The use of composition according to Claims 30-39 as ingredient in cosmetic applications.

44. The use of composition according to Claims 30-39 as ingredient in pharmaceutical applications. Page 18 of 25