PH12016000457A1 - Integrated processes for the treatment of mango wastes of fruit processing and the preparation of mango oil compositions from mango seed husks - Google Patents

Abstract

This invention pertains to simple and integrated processes which are appropriate and economically attractive methods for the instantaneous and efficient treatment of mango Mangifera indica L. Anacardiaceae) peels and seeds from fruit-processing, characterized in that the said processes, lead to the preparation of a plurality of compositions, which have shelf lives of at least six months and are commercially ready to use. Said processes also result in the most efficient treatment of the mango peels and seeds, which are oftentimes regarded as worthless, troublesome and potentially hazardous wastes. The said processes also allow the efficient utilization of energy and process equipment in the said process.

Description

Integrated processes for the treatment of mango wastes of fruit o processing and the preparation of © mango oil compositions from mango seed husks Ny oy

DESCRIPTION =

This invention relates to simple and integrated processes for the treatment of mango wastes " consisting of peels and seeds from fruit processing and the subsequent recovery and = preparation of compositions derived from such wastes. The methods allow for an efficient - and instantaneous processing of the mango wastes, resulting in its immediate treatment which © is highly imperative for such kind of wastes. R

Mango peels and seeds, which are commonly regarded as waste by-products in fruit processing, are becoming a potential hazard to public health and the environment, if not immediately handled and treated appropriately. At present, especially in many Asian countries like the Philippines, where mangoes are abundantly processed, the collected mango waste peels and seeds are simply dumped in landfills and open dumpsites. Such wastes tend to feed on many organisms, which cause its immediate rotting and degradation and resulting in the release of foul odor and generation of potentially hazardous leachates; many of said organisms are pathogenic in nature. Thus, there is an urgent need to establish the immediate i disposal and proper treatment of said wastes. On the other hand, studies revealed that mango peels and seeds are good sources of many commercially viable products such as pectin from peels and starch or flour and kernel oil from seeds, amongst others.

In this invention, the compositions immediately resulting from the simple and integrated processes developed herein are interestingly varied and said compositions, like the mango peel powder (MPP) and mango seed kernel powder (MSKP), have shelf lives of at least six months and can readily be used, for example, as ingredients in food and functional food preparations, in nutraceuticals, and as animal feeds or fodder. Further treatment of these compositions allows for the preparation of a plurality of semi-refined compositions with high nutritional and commercial value. The simple and integrated processes, which are the object of this invention, also result in the most efficient use of process equipment involved such as ;

Page 1 of 37 the dryer and mill, leading to more attractive and economical process routes in the o preparation of a plurality of compositions, most of which can readily be used as ingredients in ~ food, feed, and functional food preparations, in nutraceuticals, as antioxidants, as o antimicrobial agents, and so on. nt

Furthermore, the simple and integrated processes developed in this invention result in the most efficient treatment and utilization of mango wastes, thereby giving good value to - 40 seemingly worthless fruit by-products and eliminating its potential hazard to public health : and the environment. In addition, the mango seed husks, which is another important 7 composition resulting from this invention can be a good source of fuel or energy, which can eo be instantaneously utilized in the processes herein for its energy requirements, amongst other uses. 5 45

Background Information

Mango is a fleshy stone fruit belonging to the genus Mangifera, consisting of numerous tropical fruiting trees in the flowering plant family Anacardiaceae. The mango is native to

India from where it spread all over the world. While other Mangifera species (e.g. horse 50 mango, M. foetida) are also grown on a more localized basis, Mangifera indica — the common mango or Indian mango — is the only mango tree commonly cultivated in many tropical and subtropical regions, and its fruit is distributed essentially worldwide. Such that Mangifera indica is the scientific name of mangoes around the world, however, it has many varieties depending on the actual source it is cultivated, e.g. Brazilian variety, Egyptian variety, Indian 55 variety, Thai variety, Mexican variety, Philippine variety; notwithstanding the varieties made by tissue culture and other genetic manipulation techniques. It is commonly known that each of these mango varieties have distinct properties and characteristics, which lead as well to a variety of fruit compositions, properties, uses, and applications. 60 Among major fruit crops, mango (Mangifera indica L. Anacardiaceae) is one of the five most important fruits in the world with a total production of 39 million metric tons per year i (FAOSTAT, 2010). About 77% of this world production is coming from Asian countries such as India, China, Thailand, Indonesia, Philippines, Pakistan, and Bangladesh. As an important, nutritious and delicious tropical fruit, it is processed into various products such as 65 dried fruits, fruit bars, candies, flakes, juices, nectars, concentrates, jams, jelly, juice powders,

Page 2 of 37 and others. Mango processing industries utilize up to 25% of the mangoes produced, o equivalent to about 10 million metric tons per year worldwide and during its processing, huge ~ amounts of peels and seeds are generated as by-products, which are oftentimes regarded as 0 wastes. Its disposal is a major problem among many fruit processing industries because such - 70 wastes are easily spoiled, degraded, and is a potential hazard to both people and the = environment. The waste peels and seeds of mango amount to 35-60% of the total fruit > weight, of which mango peels form about 15-20% of whole mango fruit and the remaining - 20-40% may be composed of the mango seeds. On the other hand, the kernel inside the : mango seed represents from 45-75% of the seed and about 20% of the whole fruit. This LA 75 invention attempts to exploit such fruit wastes in order to recover more valuable = compositions or products from such seemingly worthless by-products in fruit processing. o

Studies on the proximate analysis and the various valuable bioactive components of different - mango varieties (e.g. Indian variety, Brazilian variety, Egyptian variety, Malaysian variety,

Nigerian variety, etc) reveal that depending on the variety, mango peels and seeds may have 80 varying carbohydrate, crude fiber, fat/oil, protein, moisture and ash contents and may also have varying levels of bioactive components, which may result in its different potential uses and applications in food and functional foods, in nutraceuticals and cosmetics, and pharmaceuticals. 85 Characterization and uses of mango peels

It is known that the mango peel contains a considerable amount of pectin and various bioactive compounds. In addition, the peel extracts exhibited potential antioxidant and anti- microbial properties; thus, is a potential ingredient in nutraceuticals and functional food products. For instance, processed mango peel was incorporated up to a 5% level into the 90 formulation of macaroni, which yielded an acceptable product with improved nutraceutical properties [Ajila CM, Aalami M, Leelavathi K, Prasada Rao UJS, Mango peel powder: A potential source of antioxidant and dietary fiber in macaroni preparations, Innovative Food

Science and Emerging Technologies 2010, 11, 219-224]. 95 Studies have also been conducted to characterize the mango peels. One work pertains to the characterization of mango peel (Indian variety) and results showed that this contains 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 CM, Bhat SG,

Page 3 of 37 :

: Prasada Rao UJS, Valuable components of raw and ripe peels from two Indian mango - 100 varieties, Food Chemistry 2007a, 102, 1006-1011]. =

The characteristic antioxidant and antimicrobial activities of mango peels are attributed to the ol bioactive components found therein. Results from studies showed that mango peels could be = a good source of bioactive compounds such as polyphenols (33.31-73.88 mg/g powder by wr 105 alcohol extraction and 54.67-109.7 mg/g powder by acetone extraction), carotenoids (365- o 3,945 ng/g powder), vitamin C (188-392 ng/g powder), and vitamin E (205-509 ug/g peel . powder). Further, mango peels also contain soluble dietary fiber (SDF = 15.70-28.05%) and wf 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 & 110 food and functional food applications [Ajila CM, Naidu KA, Bhat SG, Prasada Rao UJS. 5 2007b. Bioactive compounds and antioxidant potential of mango peel extract. Food

Chemistry 105, 982-988; 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]. In addition, the antioxidative capacity of the extracts 115 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 mango peels indicates a good correlation to its antioxidative capacity and said observations were confirmed by other studies, although, interestingly, different varieties of Mangifera indica L. Anacardiaceae, 120 exhibited different component characteristics and properties as well [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; Berardini N, Carle R, and Schieber A, Characterization of gallotannins and 125 benzophenone derivatives from mango (Mangifera indica L. cv. F Tommy Atkins) peels, pulp and kernels by high-performance liquid chromatography/electrospray ionization mass spectrometry. Rapid Communications in Mass Spectrometry 2004,18, 2208-2216; 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 : 130 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

Page 4 of 37 polyphenolics. Innovative Food Science and Emerging Technologies 2005b, 6, 442-452; 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. 0s 135 Food Chemistry 2010, 121, 429-436; Larrauri JA, Ruperez P, and Saura-Calixto F, Mango . peels fibres with antioxidant activity. Zeitschrift fur Lebensmittel-Untersuchung und- it

Forschung A 2007, 205, 39-42; Schieber A, Berardini N, and Carle R, Identification of > flavonol and xanthone glycosides from mango (Mangifera indica L. cv. Tommy Atkins) peels oO by high-performance liquid chromatography-electrospray ionization mass spectrometry. o 140 Journal of Agricultural and Food Chemistry 2003, 51, 5006-5011]. ur

Recovery of pectin from mango peels -

Mango peels were also found to be a rich source of pectin, with a high degree of esterification o [Srirangarajan AN and Shrikhande AJ, Mango peel waste as a source of pectin. Current 145 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,

Tandon DK, Kalrat SK, Singh BP, and Garg N, Characterization of pectin from mango fruit waste. Indian Food Packer 1991, 45, 9— 12; Beerh OP, Raghuramaiah B, and Krishnamurthy

GV, Utilization of mango waste: Peel as a source of pectin. Journal of Food Science and 150 Technology 1976, 13, 96-97; Berardini ef al., 2005a and 2005b cited above].

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, 155 used in fillings, medicines, and sweets, and as a stabilizer in fruit juices and milk drinks. 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 ef al., 2005a and 2005b cited above]. 160 Characterization and uses of mango seed kernel

Depending on the fruit variety, mango seed kernels (MSK) contain on a dry weight basis an average crude protein content of 6.7%, total lipid of 12.3%, crude fiber of 2.7%, moisture content of 8.5%, ash content of 2.5%, and the remaining amount is carbohydrate [Abdalla

AEM, Darwish SM, Ayad EHE, El-Hamahmy RM, Egyptian mango by-product I.

Page 5 of 37

165 Compositional quality of mango seed kernel. Food Chemistry 2007a, 103, 1134-1140]. -

Although a low amount of crude protein was obtained, the quality of protein was good = because it was rich in all essential amino acids. Total essential amino acid content was 32.1 g on per 100 g protein, with most of the essential amino acids equal to or exceeding the "

FAO/WHO reference proteins, while the total non-essential amino acids was about 52.2 g per - 170 100 g protein. The lipids in MSK contain high fractions of oleic acid (46.1%) and stearic ot acid (38.3%). -

Further, mango seed kernels of two cultivars of Indian variety (Chausa and Dusheri) were LA found to constitute about 18% of the total fruit and had 5% protein, 6-7% crude fat, 0.19- a 175 0.44% tannins, iodine value of 34-44 and saponification number of 202-213 [Dhingra S and -

Kapoor AC, 1985. Nutritive value of mango seed kernel. Journal of Science of Food and o

Agriculture 36, 752-756]. The principal fatty acids in the oil were oleic acid (42%) and stearic acid (39%), which are similar with the Egyptian variety. Although the protein content was rather low in these mango varieties, the essential amino acid index and protein quality 180 index were high, thus indicating good quality of the protein in mango seed kernel. This finding is similar to the mangoes of Indian variety. Other studies also explored the oils which are extracted from mango seed kernel, which is known as mango seed kernel oil, obtained from the mango (Mangifera indica). The oil is semi-solid at room temperatures, but melts on contact with the skin, making it appealing for baby creams, sun care balms, hair products, and 185 other moisturizing products. The oil is a soft yellow color with a melting point of 23-27°C [Moharram YG and Moustafa AM, Utilization of mango seed kernel (Mangifera indica) as a source of oil, Food Chemistry 1982, 8, 269-276; Hemavathy J, Prabhakar JV, and Sen DP,

Composition of polar lipids of Alphonso mango (Mangifera indica) kernel. Journal of Food

Science 1987, 52, 833-834; Hemavathy J, Prabhakar JV, and Sen DP, Drying and storage 190 behaviour of mango (Mangifera indica) and composition of kernel fat. Asian Food Journal 1988, 4, 59-63; Mahale SM and Goswami-Giri AS, Composition and Characterization of

Refined Oil Compared with Its Crude Oil from Waste Obtained from Mangifera indica,

Asian Journal of Research Chemistry 2011, 4(9), 1415-1419]. 195 Antioxidant and antibacterial activities of mango seed kernel

In addition, mango seed kernels also contained about 112 mg total phenolic compounds per 100 g dry kernel powder, and among the various phenolic compounds identified, tannin and :

Page 6 of 37

‘ vanillin were in highest amounts found [Abdalla AEM, Darwish SM, Ayad EHE, El- -

Hamahmy RM, Egyptian mango by product 2: Antioxidant and antimicrobial activities of wr 200 extract and oil from mango seed kernel. Food Chemistry 2007, 103, 1141-1152]. In this o work, the antioxidant and antimicrobial activities of mango seed kernel extract and oil iy indicated very positive results suggesting that the Egyptian mango seed kernel can be utilized = as a natural antioxidant and antimicrobial agent in foods. a = 205 Other studies also dealt with the potential of mango seed kernel to exhibit antioxidant and . antimicrobial activities [Kaur J, Rathinam X, Kasi M, Leng KM, Ayyalu R, Kathiresan S, LIT

Subramaniam S, Preliminary investigation on the antibacterial activity of mango (Mangifera cy indica L. Anacardiaceae) seed kernel, Asian Pacific Journal of Tropical Medicine 2010, 707- - 710; Soong YY and Barlow PJ, Antioxidant activity and phenolic content of selected fruit o 210 seeds, Food Chemistry 2004, 88, 411-417; Soong YY and Barlow PJ, Quantification of gallic acid and ellagic acid from longan (Dimocarpus longan Lour.) seed and mango (Mangifera indica L.) kernel and their effects on antioxidant activity, Food Chemistry 2006, 97, 524- 530]. 215 Effect of drying of mango peels and seeds

Studies also showed that the drying temperature of the mango peels and seed kernel during the preparation has an influence on the polyphenolic content of such materials. The phenol content of both is largely responsible for their antioxidant activity and such phenol contents are affected by the drying temperatures as well as drying methods [Dorta E, Lobo MG, 220 Gonzalez M, Using drying treatments to stabilize mango peel and seed: Effect on antioxidant

Activity, LWT-Food Science and Technology 2012, 45, 261-268].

Production of mango seed kernel starch or flour, use as supplement in baking, and as feed for animals 225 Mango seed kernel starch is a carbohydrate obtained from the mango seed kernel. This consists of a large number of glucose units joined together by glycosidic bonds. This polysaccharide is produced by all green plants as an energy source. Pure starch is a white, tasteless and odorless powder that is insoluble in cold water or alcohol. Depending on the plant, starch generally contains 20 to 25% amylose and 75 to 80% amylopectin. Starch is

Page 7 of 37

‘ 230 processed to produce many of the sugars in processed foods. When starch is stripped of oO protein, this becomes softer and is used as flour and is preferred in baking. =

The production of flour from mango seed kernel was also explored [Arogba SS, Physical, » chemical and functional properties of Nigerian mango (Mangifera indica) kernel and its ol 235 processed flour. Journal of the Science of Food and Agriculture 1997, 73, 321-328; Arogba >

SS, The performance of processed mango (Mangifera indica) kernel flour in a model food ow system. Bioresource Technology 1999, 70, 277-281]. In these works, wheat flour was . substituted with mango seed kernel flour at various ratios in the baking of biscuits. Results wi showed that an increase in the carbohydrate, protein, and energy content were notable in = 240 biscuits with mango kernel flour incorporated into it. In terms of color, texture, flavor and - over-all acceptability, the 50:50 biscuit grade was preferred; although other grades were also - favored but mostly for its brown coloration, which was attributed to the presence of residual tannin in the mango kernel flour. The findings on this work is claimed to have encouraged commercial manufacture of biscuits blended with mango kernel flour as mango is abundant 245 in Nigeria.

Similar studies on the use of mango seed kernel as flour supplement in bread and biscuit baking were also reported [Ashoush IS and Gadallah MGE, Utilization of Mango Peels and

Seed Kernels Powders as Sources of Phytochemicals in Biscuit, World Journal of Dairy & 250 Food Sciences 2011, 6 (1), 35-42; El-Soukkary FAH, El-Sahn MA, and Mohamed HMA,

Physico-chemical and nutritional evaluation of mango seed kernel and its utilization for pan bread supplementation. Zagazig Journal of Agriculture and Research 2000, 27, 1319-1342;

Mohamed EM and Girgis AY, Utilization of mango seed kernels for improving stability of some oils and biscuit production. Journal of Agricultural and Science 2005, 30(8), 4625— 255 4636; Kaura M, Singha N, Sandhua KS, Gurayab HS, Physicochemical, morphological, thermal and rheological properties of starches separated from kernels of some Indian mango cultivars (Mangifera indica L.), Food Chemistry 2004, 85, 131-140]. Another study also pertains to the use of mango seed kernels as chicken feed [Joseph JK and Abolaji J, Effects of replacing maize with graded levels of cooked Nigerian mango seed kernels (Mangifera 260 indica) on the performance, carcass yield and meat quality of broiler chickens, Bioresource

Technology 1997, 61, 99-102].

Page 8 of 37

‘ Potential use of mango seed husks for briquetting -

Briquettes are blocks of flammable matter used as fuel. Common types of briquettes are = 265 charcoal briquettes and biomass briquettes. Biomass briquettes are made from agricultural waste. Briquettes made of rice husks, straw, sunflower husks, buckwheat, and others are wo classified as biomass briquettes, which are a replacement for fossil fuels such as oil or coal, cl and can be used to heat boilers in manufacturing plants. This can also replace charcoal or briquettes. These are a renewable source of energy and avoid adding fossil carbon to the - 270 atmosphere. ;

LE

There are two types of briquettes, namely: the charcoal briquettes and the biomass briquettes. 5

The charcoal briquettes are sold commercially for cooking food, which can include: wood charcoal, mineral char, limestone, sawdust and others. Some briquettes are compressed and oO 275 dried and extruded into hard blocks. This is a common technique for low rank coals. They are typically dried to 12-18% moisture, and are primarily used in household and industry.

The biomass briquettes are usually made of agricultural by-products and wastes, which are milled or pulverized [Amaya A, Medero N, Tancredi N, Silva H, Deiana C, Activated carbon 280 briquettes from biomass materials. Bioresource Technology 2007, 98, 1635-1641]. The extrusion production technology of briquettes is the process of extruding milled agricultural wastes or finely shredded wood waste (sawdust) under high pressure. The quality of such briquettes, especially the heat content is much higher compared with other methods like using piston presses. Some typical physical and proximate analysis of briquettes are as follows: 285 briquette specific density - 1,225 kg/m’, bulk density - 618 kg/m>, moisture (wet basis) - 9.02%, ash (dry basis) - 7.92%, volatile matter (dry basis) - 80.5%, fixed carbon (dry basis) - 11.58%, and heating value of 18.6 MJ/kg [Singh RN, Bhoi PR, Patel SR, Modification of commercial briquetting machine to produce 35mm diameter briquettes suitable for gasification and combustion. Renewable Energy 2007, 32, 474-479). 290 : Sawdust briquettes have developed over time with two distinct types: those with holes through the center, and those that are solid. Both types are classified as briquettes but are formed using different techniques. A solid briquette is manufactured using a piston press that compresses sandwiched layers of sawdust together. Briquettes with a hole are produced with : 295 a screw press. The hole is from the screw thread passing through the center, but it also

Page 9 of 37 increases the surface area of the log and aids efficient combustion. In another embodiment of - this invention, the mango seed husks are dried and milled, according to the said integrated ~ process developed herein, molded and pressed into briquettes. Pressing pressures, sizes, - shapes, and addition of binders at different ratios are considered in the production of good oy 300 quality briquettes from such material. =

Potential use of seed husks as adsorbents -

In some studies, seed husks were also found to be useful adsorbent of dyes in wastewater : [Davila-Jimenez MM, Elizalde-Gonzalez MP, Hernandez-Montoya V, Performance of 1 305 mango seed adsorbents in the adsorption of anthraquinone and azo acid dyes in single and aE binary aqueous solutions, Bioresource Technology 209, 100, 6199-6206; Franca AS, Oliveira

LS, Pedro I.A. Santos PIA, Silvanio A. Saldanha SA, Samantha A. Salum SA, Mango seed 0 husks as biosorbents for basic dyes, Journal of Biotechnology 2008, 136S, S647-S677;

Vasanth Kumar K and Kumaran A, Removal of methylene blue by mango seed kernel 310 powder, Biochemical Engineering Journal 2005, 27, 83-93].

Challenges in the prior art

A patent WO 01/78859 by Carle et al. (2000) showed a method for obtaining useful materials from the by-products of fruit and vegetable processing involving apple or citrus marc. 315 Although the method is efficient, it is unsuitable for mango waste peels and seeds. In other prior art, particularly studies in literature, the mango peels are usually processed separately from the mango seeds, in spite of the fact that these two mango wastes are simultaneously produced during fruit processing and thus are also simultaneously disposed of. In the case of treating mango peels, the focus is also limited to either pectin production only, or looking at 320 the bioactive compounds derived from it only, except in one case in which both aspects of mango peel processing were tackled [see Berardini ef al., 2005a and 2005b cited earlier].

Approaches in the processing of mango peels and seeds were oftentimes lab-scale and are difficult to translate into industrial scale operation, as these either involve expensive chemicals and/or highly-intricate process equipment. 325

From the cited studies, it is very clear that efforts were directed to the uses and applications of such mango wastes when treated and tapped as sources of valuable compounds like pectin, oil, butter, starch, flour, proteins, and bioactive compounds exhibiting antioxidant,

Page 10 of 37 :

antimicrobial, and enzymatic activities, and others. Up to now, the processes in treating - 330 appropriately such mango by-products are sporadic and scarce, which do not address = sufficiently the many challenges which impeded the full and commercial exploitation of said on mango peels and seeds. An important aspect in said undertaking is the fact that mango peels os and seeds are easily degraded, i.e. in less than 24 hours, when left untended in a tropical = environment, these are already attacked by pests, insects and degrading microorganisms. o 335 Such that a suitably instantaneous process that treats such wastes and convert them into stable o and commercially-viable compositions would be necessary. Since mango by-products are : seemingly useless to many fruit processing industries, the treatment process has to be simple 1 and cost-effective in order to render it economically attractive for industrial implementation.

Such aspects are believed to be sufficiently and satisfactorily addressed in the object of this 5 340 invention. .

Brief description of drawings

Figure 1 shows the diagram of a simple process for the treatment of mango seeds, which is the object of this invention, wherein mango seed kernel powder (MSKP) and mango seed 345 husk (MSH) or mango seed husk powder (MSHP), and the corresponding defatted or oil- extracted compositions (MSKP*, MSHP*) and its fat/oil extracts are obtained.

Figure 2 shows the diagram of a simple process for the treatment of waste mango peels, which is the object of this invention, wherein mango peel powder (MPP) and the corresponding defatted or oil-extracted composition (MPP*) and its fat/oil extract are 350 obtained.

Figure 3 shows the diagram of an integrated process for the treatment of mango wastes of fruit processing, which is the object of this invention. The immediately resulting compositions in this process are indicated, namely: mango peel powder (MPP), mango seed kernel powder (MSKP), mango seed husks (MSH) or mango seed husk powder (MSHP), and 355 the corresponding defatted or oil-extracted compositions. In addition, the corresponding fat/oil extracts from the plurality of compositions can be in pure or combined form. In this integrated process, when washing and blanching is done in process step 1, the wash water is collected and treated via an alcoholic fermentation to obtain another set of compositions such as the alcoholic products, single cell proteins or biomass and carbon dioxide gas, all of which 360 are collected for further use.

Page 11 of 37

Figure 4 shows the diagram of a process which is a continuation of the simple or the - integrated processes for the treatment of mango wastes of fruit processing, wherein process = steps are added to refine the compositions obtained from the simple or integrated processes > described earlier and acquire herein additional valuable compositions. . 365 or

The Invention “

In this invention, a simple process is developed in which mango (Mangifera indica L. o

Anacardiaceae) seeds, regarded as wastes of fruit processing, are treated instantaneously and . efficiently, comprising the following steps as illustrated in Figure 1: MH 370 (a) washing of mango seeds in process step 1, @ (b) drying of the mango seeds at a temperature range of 50-80°C in process step 2, pt (a) cracking of the dried mango seeds to obtain the kernel and separate the outer layer yi of the seed, which is the seed husk, in process step 3, (b) milling of the dried mango seed husks to obtain the composition referred to as 375 mango seed husk powder (MSHP) in process step 4, (c) further drying of the seed kernel obtained in process step 3 using the same dryer in step (b), in process step 2, (d) milling of the dried mango seed kernel using the same mill in step (d) to obtain the composition referred to as mango seed kernel powder (MSKP), in process step 4, 380 (e) subjecting any or all of the resulting compositions obtained above to a defatting step or oil extraction step in process step 5 for the recovery and preparation of a plurality of compositions, which are extracts mainly composed of mango fat/oil, referred to as mango oil or mango oil extract, one of which is mango seed kernel oil (MSKO) extract and the other is the mango seed husk oil (MSHO) extract, 385 (f) recovery of the solvent such as ethanol or methanol in process step 7, to obtain the compositions which are fat/oil extracts obtained in process step 5, in concentrated mixtures and recycling the recovered solvent into process step 5, where it is used for defatting, and (g) treatment of the wash water obtained from process step 1 by employing 390 fermentation in process step 6, to obtain a plurality of compositions such as ethanol, single-cell proteins or biomass, and carbon dioxide (CO,) gas.

Page 12 of 37

The object of the invention herein which is the simple process described above and illustrated o in Figure 1 is characterized in that the said simple process: o 395 i. leads to the recovery and preparation of a plurality of compositions, which have > shelf lives of at least six months, without undergoing degradation, when said 7 compositions are packed tightly and stored in a clean and dry place, ~ ii. allows for the preparation of a plurality of compositions, which are commercially - ready to use, o 400 iii. allows for the recovery and preparation of a plurality of compositions using the . same set of process equipment units, thereby, simplifying the over-all process itself, = iv. results in at least the dramatic minimization and at most the elimination or full = treatment of the mango seeds, which are oftentimes regarded as worthless, 2 troublesome and potentially hazardous wastes, a 405 v. allows the use of one of the resulting compositions, which is the mango seed husks, as powder and/or as is, as solid fuel and alternative source of energy as needed in the said process itself; which consequently further results in substantial energy savings, and finally vi. allows the efficient utilization of energy and process equipment in the said simple 410 process.

In another embodiment of the invention, another similar simple process is developed, in which mango (Mangifera indica L. Anacardiaceae) peels, regarded as wastes of fruit processing, are treated instantaneously and efficiently, comprising the following steps as 415 illustrated in Figure 2: (a) washing and blanching of mango peels in process step 1, (b) drying of the mango peels at a temperature range of 50-80°C in process step 2, (c) milling of the dried mango peels to obtain the composition referred to as mango peel powder (MPP) in process step 4, 420 (d) subjecting the resulting composition obtained above to a defatting step or oil extraction step in process step 5 for the recovery and preparation of another composition, which is the extract mainly composed of mango fat/oil, referred to as mango peel oil (MPO) extract, (e) recovery of the solvent such as ethanol or methanol in process step 7, to obtain the 425 composition which is the mango peel oil (MPO) extract obtained in process step 5, in

Page 13 of 37 concentrated mixtures and recycling the recovered solvent into process step 5, where o ; it is used for defatting, and - (f) treatment of the wash water obtained from process step 1 by employing fermentation os in process step 6, to obtain a plurality of compositions such as ethanol, single-cell ? 430 proteins or biomass, and carbon dioxide (CO) gas. rs [RR

The object of the invention herein which is the simple process described above and illustrated o in Figure 2 is characterized in that the said simple process: - : i. leads to the recovery and preparation of a plurality of compositions, which have shelf + 435 lives of at least six months, without undergoing degradation, when said compositions = are packed tightly and stored in a clean and dry place, ® ii. allows for the preparation of a plurality of compositions, which are commercially ready to use, iii. allows for the recovery and preparation of a plurality of compositions using the same 440 set of process equipment units, thereby, simplifying the over-all process itself, iv. results in at least the dramatic minimization and at most the elimination or full treatment of the mango peels, which are oftentimes regarded as worthless, troublesome and potentially hazardous wastes, and finally v. allows the efficient utilization of energy and process equipment in the said simple 445 process.

In another embodiment of this invention, a process is developed which is an integration of appropriate and economically attractive methods described earlier and illustrated in Figures 1 and 2, for the treatment of mango (Mangifera indica L. Anacardiaceae) wastes such as peels 450 and seeds from fruit-processing, comprising the following steps, as further illustrated in

Figure 3: (a) segregation and washing of mango peels and seeds in process step 1, (b) drying of the mango peels, seeds and seed kernel at a temperature range of 50-80°C in process step 2, 455 (c) cracking of the dried mango seeds to obtain the kernel and separate the outer layer of the seed, which is the seed husk, in process step 3, (d) milling of the dried mango peels to obtain the composition referred to as mango peel powder (MPP) in process step 4, !

Page 14 of 37

(e) further drying of the seed kernel obtained in process step 3 using the same dryer in - 460 step (b), in process step 2, x (f) milling of the dried mango seed kernel using the same mill in step (d) to obtain the 0 composition referred to as mango seed kernel powder (MSKP), in process step 4, » (g) milling of the dried mango seed husk obtained in process step 3 using the same mill in a. process step 4, to obtain the composition referred to as mango seed husk powder w 465 (MSHP), . (h) subjecting any or all of the resulting compositions obtained above to a defatting step . or oil extraction step in process step Sfor the recovery and preparation of a plurality of bk compositions, which are extracts mainly composed of mango fat/oil, © (i) recovery of the solvent such as ethanol or methanol in process step 7, to obtain the o 470 compositions which are fat/oil extracts obtained in process step §, in concentrated o mixtures and recycling the recovered solvent into process step 5, where it is used for defatting, and (j) treatment of the wash water obtained from process step 1 by employing fermentation in process step 6, to obtain a plurality of compositions such as ethanol, single-cell 475 proteins or biomass, and carbon dioxide (CO) gas.

The object of the invention, which is the integrated process described herein and illustrated in

Figure 3, is characterized in that the said integrated process: i. leads to the recovery and preparation of a plurality of compositions, which have shelf 480 lives of at least six months, without undergoing degradation, when said compositions are packed tightly and stored in a clean and dry place, ii. allows for the preparation of a plurality of compositions, which are commercially ready to use, iii. allows for the recovery and preparation of a plurality of compositions using the same 485 set of process equipment units, thereby, simplifying the over-all process itself, iv. results in at least the dramatic minimization and at most the elimination or full treatment of the mango peels and seeds, which are oftentimes regarded as worthless, troublesome and potentially hazardous wastes, v. allows the use of one of the resulting compositions, which is the mango seed husks, as 490 powder and/or as is, as solid fuel and alternative source of energy as needed in the

Page 15 of 37 said process itself, which consequently further results in substantial energy savings, o and finally - vi. allows the efficient utilization of energy and process equipment in the said integrated process. . 495 =

In many fruit processing industry, the peels are specifically separated from the fruit during or peeling and consequently, these are collected in one vessel; which is conveniently separated o from the seeds - another by-product after the removal of mango pulp. The washing of peels . and seeds is also incorporated into the processing itself to maintain sanitary and clean +1 500 workplace. Thus, when mango peels and seeds are obtained from such fruit processing = factory, the segregation and washing step in process step 1 of the simple processes in Figures & 1-2 or the integrated process in Figure 3, is skipped. In such an event, there is no wash water “ required, thus, no wash water to be treated as well. However, occasionally, these peels and seeds need to be washed to remove unwanted particulate matter. Consequently, the collected 505 wash water contains some considerable fruit pulp, rendering it rich in reducing sugars, in which the reducing sugar content reaches a range of 10-40%, depending on the amount of wash water used per kilogram of fresh mango wastes washed. The wash water is collected and treated by a fermentation process to obtain a plurality of compositions, e.g. alcoholic products such as ethanol, simultaneously with single cell proteins such as biomass, and 510 carbon dioxide (CO,), all of which can be collected for further use.

The mango peels inherently have tannins, which cause the brown color of the peel after exposure to the atmosphere due to some enzymatic reactions; such a component leads to the dark brownish color of the mango peel powder (MPP) composition obtained down the 515 process line. Such tannins can be removed easily by blanching of the fresh mango peels, if so desired. To address this option, the simple process (Figure 2) and integrated process (Figure 3) of this invention can further employ a washing step of the mango peels in process step 1 in combination with a blanching step, in which washing of mango peels uses hot water at a temperature of about 70-90°C, preferably at 75-85°C, more preferably at 80°C for about 5 to 520 15 minutes and preferably for 10 minutes, leading to the removal of the tannins or the brown coloring matter of the mango peels, which actually caused its bitter taste. Said process step 1 is further characterized in that the water vaporized from the dryer in process step 2 is utilized as the agent for heating or in combination with the wash water for blanching; thereby

Page 16 of 37 minimizing the energy requirement of the said process step. Only mango peels need to be - 525 subjected to blanching step. The mango seeds or seed husks do not need to be blanched o- because the tannin content of the said materials does not affect its further use or application.

Pod

In drying, which is process step 2 of the simple and integrated processes of this invention as - illustrated in Figures 1-3, the peels and/or seeds are separately spread (in trays or in conveyor > 530 belts) and dried at a temperature range of 50-80°C using an oven dryer with hot air until o moisture content is less than 10%, preferably at a moisture content of 4-7%, and more . preferably at 5-6%. In this drying step, the peels and the seed kernels are preferably dried at Li a temperature of 50-70°C, more preferably at 60°C, while the seed and seed husks are dried = preferably at higher temperature of 70-80°C and most preferably at 80°C. The peels and seed o 535 kernel need to be dried more preferably at 60°C to avoid high temperature effects on the o polyphenol content of such materials. It is commonly known in the prior art that the polyphenol content of the peels or kernels decreased and are degraded at high drying temperatures. The drying step may be done batch-wise, intermittently, or semi-continuously for each of the said material, thereby rendering the drying unit efficiently utilized. 540

Further, the drying of such materials are scheduled in such a way that efficient utilization of energy is implemented by heating the seeds or seed husks in the dryer at any employed temperature, as they are not temperature-sensitive, and during times when temperatures are adjusted or ramped to its desired levels. In this way, no heat energy is wasted. In addition, 545 the water evaporated from the dryer, which is in large quantities, can be collected and utilized in other steps of the simple or integrated processes where hot air or water vapor stream is needed such as during blanching of mango peels or in any other process step, i.e. during acid hydrolysis at 80°C in the recovery of pectin as illustrated further in Figure 4, and so on. 550 The dried peel is milled and powdered as indicated in process step 4 in either the simple process (see Figure 2) or integrated process (see Figure 3), weighed, and is passed through a 50-200 um sieve screen. The resulting composition is referred to herein as mango peel powder (MPP). It is packed in desired amounts, sealed, and stored in a dry and clean place for further use. The finely powdered peel with particle sizes of 50-200 um, preferably at 50- 555 150 pm, more preferably at 60-140 um, more preferably at 70-120 pm and best at 80-100

Page 17 of 37

. pum, can readily be used for food, functional food, and nutraceutical applications, and for o further treatment and refining. Occasionally, the powdered composition with bigger particle or sizes of about 150-200 pm or higher are either re-milled to reduce its size ranges, or utilized @ as ingredients for animal feeds. rs 560 ~

In another embodiment of the invention, as shown in Figure 1 or Figure 3, the dried mango seeds are cracked manually or using a decorticator or cracker, in order to obtain the seed o kernel which are found inside the seed and separated from the seed husks, which are the outer - covering of the seeds. The seed husks are collected and weighed, and further dried if needed, + 565 and milled to powder. The powdered husks are passed through a 50-200 um sieve screen, and = this important composition is referred to herein as mango seed husk powder (MSHP).

Furthermore, the obtained mango seed kernel is sent back to the drier for further drying at a o. temperature range of 50-70°C, more preferably at 60°C, until such time that it has a moisture content of 10% or less, preferably at a moisture content of 4-7%, and more preferably at 5- 570 6%. The dried seed kernel is then milled, weighed and passed through a 50-200 um sieve screen. The finely powdered seed kernel with particle sizes of 50-200 um, preferably at 50- 150 pm, more preferably at 60-140 um, more preferably at 70-120 um and best at 80-100 um, can readily be used for food, functional food, and nutraceutical applications, and for further treatment and refining. The resulting composition is referred to herein as mango seed 575 kernel powder (MSKP).

In another embodiment of the invention, the seed kernel after cracking may be soaked for 1-4 hours in a solution containing a preservative such as sodium bisulfite, citric acid or others. In this example, a solution containing 0.16% sodium bisulfite was used to soak the seed kernel 580 for 1-4 hours, filtered, collected and dried in the same dryer in process step 2of the simple process (see Figure 1) and of the integrated process (see Figure 3). Drying was done until the moisture content is less than 10%, preferably at a moisture content of 4-7%, and more preferably at 5-6%. The soaking step of the mango seed kernel was skipped in another set of examples in order to see its effect on the shelf life of the resulting composition. The dried 585 seed kernel was then milled, weighed, and sieved through a 50-200 um sieve screen. Results showed that soaking the kernel with a solution containing a preservative did not have an effect in lengthening the shelf life of the mango seed kernel powder. However, this process

Page 18 of 37 step is important in helping keep the pathogenic micro-organisms out of the seed kernels o especially during unavoidable storage of the said material prior to drying and when the drying = 590 step takes longer than necessary. 0: >

Resulting from the embodiments of the said invention, are the following compositions po derived from the simple and integrated treatment processes of mango (Mangifera indica L. &

Anacardiaceae var. Philippines) peels and seeds: o 595 1. A composition referred to herein as mango peel powder (MPP), which is on characterized by its proximate analysis: moisture 4-7%, ash 4-7%, protein 3-5%, LF fat/oil 18-23%, crude fiber 15-18%, and carbohydrate 40-56%. @ 2. A composition referred to herein as mango seed kernel powder (MSKP), which is characterized by its proximate analysis: moisture 4-7%, ash 1-3%, protein 4-6%, z 600 fat/oil 20-28%, crude fiber 5-7%, and carbohydrate 48-66%. 3. A composition specifically referred to herein as mango seed husks, either in powder form or as is, which is characterized by its proximate analysis: moisture 4-8%, ash 1- 3%, nitrogen 0.3-1%, fixed carbon 88-95% (including fat/oil 9-13%), and heating value of 15-23 MJ/kg. 605 4. A composition which is any combination in any proportion of two or more of the mango powder compositions described above.

The shelf lives of the mango peel powder (MPP) and mango seed kernel powder (MSKP), which have potential uses in food, functional food, and nutraceutical applications, reach at 610 least six months when these are packed tightly and stored in a dry and clean place, without undergoing degradation. Such long shelf lives of the compositions is supposedly attributed to its very low moisture content, which is unfavorable for molds or other degrading microorganisms to thrive, and the presence in relatively high quantities of polyphenolic compounds which act as antimicrobial agents themselves in the said compositions. 615

In this invention, mango (Mangifera indica L. Anacardiaceae var. Philippines) peels and seeds were used and separately collected from a local mango fruit processing factory. The peels and seeds were already segregated, washed, and cleaned accordingly prior to its collection, thus, these steps were skipped in one series of examples. 620

Page 19 of 37

Another aspect of the object of this invention is the treatment and re-use of wash water for - alcoholic fermentation, as shown in process step 6 of the simple and integrated processes in ~

Figures 1-3. In this part of the process, the wash water is characterized, pretreated and used as an sugar source in alcoholic fermentation by yeasts. Established fermentation conditions such as 625 pH, temperature, and aeration are considered when dealing with yeast growth, fermentation = time, and volatile composition in order to produce an alcoholic beverage of good quality. wr

Sugar (as reducing sugar) and ethanol concentrations were analyzed using the standard - methods. Yeast growth is measured by gravimetric method. The alcoholic beverage produced : is also subjected to sensory evaluation. Lr 630 i

In another embodiment of the invention, the MPP, MSKP, and MSHP compositions undergo ao subsequent defatting or oil extraction step in process step 5 to recover and obtain their ° corresponding fat/oil extracts, which are additional valuable compositions resulting from the simple or integrated processes. On the other hand, the defatted MPP, MSKP, and MSHP, 635 referred to herein as MPP*, MSKP*, and MSHP*, respectively, are also obtained which are treated further to obtain other valuable compositions thereof.

Resulting further from the embodiments of this invention is a plurality of additional compositions as follows: 640 5. A composition referred to herein as defatted mango peel powder (MPP*), characterized in that said composition is derived from the mango peel powder (MPP) obtained earlier in the simple or integrated processes of the said invention. 6. A composition referred to herein as defatted mango seed kernel powder (MSKP¥), characterized in that said composition is derived from the mango seed kernel powder 645 (MSKP) obtained earlier in the simple or integrated processes of the said invention. 7. A composition referred to herein as defatted mango seed husk powder (MSHP*), characterized in that said composition is derived from mango seed husk powder (MSHP) obtained earlier in the simple or integrated processes of the said invention. 8. A composition which is any combination in any proportion of two or more of the 650 defatted mango powder compositions described above. 9. A composition referred to herein as mango peel oil extract, characterized in that said composition is composed mainly of fats/oils derived from the mango peel powder : (MPP) obtained earlier in the simple or integrated processes of the said invention.

Page 20 of 37

10. A composition referred to herein as mango seed kernel oil extract, characterized in o 655 that said composition is composed mainly of fats/oils derived from the mango seed = kernel powder (MSKP) obtained earlier in the simple or integrated processes of the > said invention. ~ 11. A composition referred to herein as mango seed husk oil extract, characterized in that - said composition is composed mainly of fats/oils derived from mango seed husk o 660 powder (MSHP) obtained earlier in the simple or integrated processes of the said o invention. . 12. A composition which is any combination in any proportion of two or more of the 1 mango oil extracts described above, which is characteristically composed of fats/oils, = derived from the MPP, MSKP, and MSHP compositions obtained from the simple or @ 665 integrated processes described herein. I.

In another embodiment of this invention, the immediately resulting compositions obtained in the simple and integrated processes discussed above and illustrated in Figures 1-3, are further treated and refined to obtain a plurality of additional compositions derived thereof. For 670 instance, the defatted mango peel powder (MPP*) obtained in the simple or integrated process, is further treated via acid hydrolysis in process step 8, to extract pectin, as illustrated in Figure 4. Then, the resulting mixture is filtered in process step 9 to separate the filtrate and the spent defatted mango peel powder (spent MPP*). While the latter is dried and milled to obtain the dried spent defatted mango peel powder for further use, e.g. as ingredient in animal 675 feeds, the filtrate undergoes alcoholic precipitation, mixing and settling in process step 10; afterwhich the obtained wet pectin precipitate is further dried and milled in process step 11, to obtain an important composition which is the mango pectin powder. On the other hand, the alcoholic solution obtained in process step 10 is another important composition, which is an extract characterized by its high polyphenol content. This composition is subjected to 680 purification steps in process step 12, which include alcohol recovery, to obtain a more concentrated composition of polyphenolics. The alcohol recovered in this process step is collected and recycled back to process step 10 where it is used for the alcoholic precipitation- mixing-settling process. An appropriate solvent such as ethanol or methanol is used and a solvent make-up is needed in process step 10, to ensure the favorable conditions for the said : 685 process step. The concentrate obtained after process step 12 is again another important composition, referred to herein as polyphenolic concentrate A (see Figure 4) obtained after

Page 21 of 37 the purification of the alcoholic solution and characterized by its relatively very high - concentrations of polyphenolics. - 690 In another embodiment of the said invention, the treatment of the defatted mango seed kernel . powder (MSKP*), which is a composition obtained earlier in the simple or integrated process (see Figure lor 3), is done to recover the starch that it largely contained in process step 13, as vr illustrated in Figure 4. The obtained kernel starch is then dried and milled in process step 11 o and collected for further use, while the spent defatted mango kernel powder (spent MSKP*) . 695 is also separately dried and milled in the same drier and mill of process step 11, and collected +1 for further use, e.g. as ingredient in animal feeds. An aqueous and/or organic extract may be = obtained in process step 13 during starch recovery, and said extract undergoes further o purification methods in process step 14 to obtain a composition, characterized by its 0 relatively high concentrations of polyphenolic compounds derived from mango seed kernel ] 700 powder, referred to herein as polyphenolic concentrate B (see Figure 4). This composition may be combined with the other composition, referred to as polyphenolic concentrate A, which is also characterized by its high polyphenolic content, obtained from process step 12.

In another embodiment of the said invention, the defatted mango seed husk powder 705 (MSHP*), which is a composition obtained earlier in the simple or integrated process illustrated in Figure 1 or 3, is further treated by mixing with appropriate amounts of binding agent, such as cassava starch, sawdust or others, in process step 15 (see Figure 4). The : obtained mixture is further molded and pressed in process step 16 to obtain briquettes, which is an important composition of the said invention herein, which can be used as alternative 710 solid fuel and source of energy. The mango seed husk or the defatted mango seed husk powder can be used directly as well, for instance, as fuel in boilers, for energy generation in the simple or integrated process itself in the said invention; thereby, minimizing if not eliminating the external energy requirements of the over-all process. 715 Resulting further from the embodiments of this invention is a plurality of additional compositions as follows: 13. A composition referred to herein as polyphenolic concentrate, characterized in that said composition contains relatively high concentrations of polyphenolic compounds

Page 22 of 37 derived from the mango peel powder (MPP), which is obtained earlier in the simple or - 720 integrated process of the said invention. = 14. A composition referred to herein as polyphenolic concentrate, characterized in that on said composition contains relatively high concentrations of polyphenolic compounds " derived from the mango seed kernel powder (MSKP), which is obtained earlier in the = simple or integrated process of the said invention. o 725 15. A composition which is any combination at any proportion of the two polyphenolic - concentrates derived from the mango peel powder and mango seed kernel powder. . 16. A composition which is the dried and defatted spent mango peel powder. LA 17. A composition which is the dried and defatted spent mango seed kernel powder. CE 18. A composition which is any combination at any proportion of the two dried and - 730 defatted spent compositions of mango peel powder and mango seed kernel powder. 5 19. A composition which is a briquette derived from the defatted mango seed husk powder obtained by the simple or integrated processes of the said invention. 20. A composition which consists of mango pectin, in liquid, gel, or powder form, characterized in that the said composition is derived from the defatted mango peel 735 powder obtained by the simple or integrated process of the said invention. 21. A composition which consists of mango kernel starch, characterized in that the said composition is derived from defatted mango seed kernel powder obtained by the simple or integrated processes of the said invention. 740

Page 23 of 37 :

EXAMPLES ~

Example 1. Preparation of mango peel powder (MPP), mango seed kernel powder ® 745 (MSKP), and mango seed husk powder (MSHP) ns

About 5 kgs each of mango (Mangifera indica L. Anacardiaceae var. Philippines) peels and : seeds were separately collected from a local mango fruit processing factory. The peels and or seeds were already segregated, washed, and cleaned accordingly prior to its collection, thus, o these steps were skipped in one series of experiments. The peels and seeds were separately o 750 spread in trays and dried at a temperature range of 50-80°C using an oven dryer until vy moisture content is less than 10%, preferably at a moisture content of 4-7%, and more = preferably at 5-6%. The dried peel was milled and powdered using a Wiley mill, weighed, > and was passed through a 50-200 pm sieve screen. Occasionally, when the obtained powder 2 compositions have bigger particle sizes, these are re-milled until the preferably small particle 755 sizes were obtained. The resulting composition, in said example, is referred to herein as mango peel powder (MPP). This composition can readily be used for food and nutraceutical applications, especially the composition with suitably smaller particle sizes, while the powdered peels with bigger particle sizes of about 150-200 pum can alternatively be utilized as ingredients for animal feeds. 760

On the other hand, the dried mango seeds were cracked manually or using a decorticator or cracker, in order to obtain the seed kernel which are found inside the seed and separated from the seed husks, which are the outer covering of the seeds. The seed husks were collected and weighed, and further dried if needed, and milled using the Wiley mill. The powdered husks 765 were passed through a 50-200 um sieve screen. The resulting composition is referred to herein as mango seed husk powder (MSHP). Further in another set of experiments in this ; example, the mango seed kernel was soaked for 1-4 hours in 0.16% sodium bisulfite solution, filtered, collected and dried in the same dryer. Drying was done until the moisture content : was less than 10%, preferably at 4-7%, and more preferably at 5-6%. The soaking process of 770 the mango seed kernel was skipped in another set of experiments in order to see its effect on the shelf life of the resulting composition. The dried seed kernel was then milled, weighed, and sieved through a 50-200 pm sieve screen. The said composition is referred to as mango seed kernel powder (MSKP). The said composition were packed tightly and set aside for

Page 24 of 37 further analysis. Some sample compositions were packed separately and stored in a clean - 775 and dry place for shelf-life analysis. =

For at least three trials, the average masses of the resulting plurality of compositions obtained - and their corresponding percentages relative to the wet fresh samples of mango peels or seeds = are shown in Table 1. Results show that dried peels composed only 18-24% of the wet fresh o 780 mango peel sample, while dried seed kernel and seed husk composed 22-32% and 24-30%, = respectively, of the fresh wet mango seed sample. These results indicate that a large quantity - of water or moisture (about 60-70% of the wet weight of sample) has to be evaporated during LF drying to obtain the preferably dry compositions at moisture contents less than 10%. be] 785 Table 1.Mass of compositions expressed as % in wet fresh mango waste samples. o

Sample compositions Mass collected per 5- % of wet fresh sample kg fresh sample (kg)

Mango peel powder (MPP) 09-12 18-24% of wet peel

Mango seed kernel powder (MSKP) 1.1-1.6 22-32% of wet seed

Mango seed husk powder (MSHP) 12-15 24-30% of wet seed

Example 2.Proximate analysis of MPP and MSKP

The obtained sample compositions in Example 1 were subjected to proximate analysis by 790 determining its moisture, ash, protein, and fat content according to the standard methods. . Nitrogen content is estimated by Micro-Kjeldhal method and is converted to protein content by using a factor of 6.25. The fat content is determined by Soxhlet method as described by the standard methods. The carbohydrate content may be obtained by difference in the proximate analysis and/or determined by the colorimetric analysis using phenol-sulfuric acid 795 method after hydrolyzing the samples with 6N HCI at 100°C and 6h [Dubois M, Gilles KA,

Hamilton JK, Roberts PA and Smith F, Colorimetric method for the determination of sugars and related substances. Analytical Chemistry 1956, 28, 350-356]. D-galacturonic acid is used as standard for carbohydrate analysis. Crude fiber content is measured using the gravimetric method described by the standard methods. Analysis of samples was done in triplicate. 800

Results in Table 2 show that the mango peel powder (MPP) composition contain high quantities of carbohydrates, fat/oil, and crude fiber, while the mango seed kernel powder (MSKP) contains high quantities of carbohydrates and fat/oil. The analysis indicates that said :

Page 25 of 37 compositions have distinctly higher quantities of the important components such as - 805 carbohydrates and fat/oil, when compared with the compositions of other varieties reported = so far. This further indicates that said compositions are good sources of such components os especially important in food, functional food, and nutraceutical applications. ) -

Table 2. Proximate analysis of the compositions. MPP and MSKP.

Parameter Mango Peel Powder Mango Seed Kernel Powder — (MPP, %) (MSKP, %) fod

Moisture 4-7 - 4-8 *

Ash 4-7 1-3 na

Protein 3-5 4-6 LA

Fat/Oil 18-23 20-28 ©

Crude Fiber 15-18 5-7 = :

Carbohydrates 40-56 48-66 = 810 ae ou

Example 3. Determination of bioactive compounds in MPP and MSKP

The bioactive compounds in mango peel powder (MPP) and mango seed kernel powder : (MSKP) prepared in Example 1 were analyzed and grouped into two big classes: the total ; 815 polyphenols and the total monomeric anthocyanins. Estimation of each of these bioactive compound groups were done following the methods described in these studies [Wolfe K, W

X, Liu RH, 2003. Antioxidant activity of apple peels. Journal of Agricultural and Food :

Chemistry 2003, 51, 609-614; Ajila CM, Naidu KA, Bhat SG, Prasada Rao UJS, Bioactive compounds and antioxidant potential of mango peel extract. Food Chemistry 2007, 105, 982- : 820 988; 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. Scientia Horticulturae 2011, 128, 261-266]. For total polyphenol estimation, gallic acid is used as standard. The total monomeric anthocyanin content is expressed as mg cyanidin 3-galactoside equivalent per 100 g dried sample (mg cy-3-gal/100 g sample). 825 Results in Table 3 show that the MPP and MSKP compositions have considerable quantities of polyphenolic compounds. In addition, while the MPP composition contains high contents of total monomeric anthocyanins (TMA), this is negligible in MSKP compositions. Thus, the i

MPP composition is also a good source not only of polyphenolic compounds but also of : anthocyanins. 830

Page 26 of 37

Table 3. Total polyphenol and anthocyanin estimates of MPP and MSKP. -

Bioactive compounds MPP MSKP “

Total Polyphenols (TP) content 50-120 70-100 on (mg/g dry powder) x

Total Monomeric Anthocyanins (TMA) 180-340 negligible content (mg/100 g dry powder) = > 835 It is known that the total polyphenol content in mango peel and seed kernels are attributed to its characteristically high antioxidative and antimicrobial activities. The total polyphenols in o mango peel powder and mango seed kernel powder contained herein, coupled with very low . moisture contents in the said compositions, have helped in lengthening the shelf lives of the 1 said compositions. The MSKP soaked in bisulfite solution has no different observed effect = 840 with regards to its shelf life when compared with sample compositions not soaked in bisulfite @ solution. pe

Example 4. Extraction of fats and oils from mango peel powder (MPP), mango seed kernel powder (MSKP), and mango seed husk powder (MSHP) 845 Different solvents may be used in the extraction of fats and oils from the said compositions obtained in Example 1. Solvents such as hexane, ethanol, acetone, mixture of chloroform— methanol (2:1) may be used for the extraction of lipids from known amounts of dried powder by shaking the mixture in an incubator shaker at room temperature. In some cases, the extraction temperature may be raised to see the extent of its influence on the extraction 850 efficiency. The meal or spent powder is then filtered off under vacuum with washings of the residue. The process may be repeated with half quantity of solvent for maximum extraction of lipids and extracts are pooled together in a round-bottomed flask or any vessel with cap.

The solvent is removed and recovered under vacuum by rotary evaporator and the lipids are collected, purified, and analyzed. In this example, ethanol is used as solvent and the 855 extraction process is done at 80°C, which is roundabout the boiling point temperature of ethanol, in an incubator-shaker rotated at 100-150 rpm for about 2-3 hours. Results in Table : 4 show that MPP, MSKP, and MSHP have considerable quantities of fat/oil that can be extracted from said compositions using ethanol as solvent. The said quantities are distinctly higher than in other mango varieties reported so far. The corresponding oil extracts recovered

Page 27 of 37 ]

860 from said compositions are collected for further use. Some samples are set aside for further o analysis. =

Table 4. Percentage of Fat/Oil extracted from MPP, MSKP, and MSHP compositions. re

Sample Composition Fat/Oil extracted using Ethanol in a ht given amount of powder sample (%)

Mango Peel Powder (MPP) 18-23 o

Mango Seed Kernel Powder (MSKP) 20-28 pi

Mango Seed Husk Powder (MSHP) 9-13 foo} 865 LH

Example S. Determination of bioactive compounds in the oil extracts obtained from the = compositions: MPP, MSKP, and MSHP ©

The total polyphenolic (TP) and total monomeric anthocyanin (TMA) contents of the fat/oil extracts obtained from the defatting of the MPP, MSKP, and MSHP compositions in 870 Example 4, namely: mango peel oil (MPO) extract, mango seed kernel oil (MSKO) extract, and mango seed husk oil (MSHO) extract were analyzed according to the methods mentioned earlier in Example 3. The total polyphenols and the total monomeric anthocyanins of the ; extracts are shown in Table 5 below. Results showed that the mango peel oil extract contains high concentrations of total polyphenols (TP) at a range of 300-350 mg/g extract and total 875 monomeric anthocyanins (TMA) at a range of 100-300 mg/100 g dried sample. More preferably, the oil with total polyphenol(TP) content at 320-340 mg/g extract and total monomeric anthocyanin (TMA) content of 150-260 mg/100 g dried sample is obtained. On ] the other hand, the mango seed kernel oil has total polyphenol (TP) content of 100-150 mg/g extract and more preferably at a range of 130-140 mg/g extract. Total monomeric 880 anthocyanin content was negligible in mango seed kernel oil. For mango seed husk oil extract, the total polyphenol content is just about 1-2 mg/g extract with negligible TMA content. 885 Table 5. Total polyphenol and anthocyanin estimates of the oil extracts from various ] compositions. Values in parenthesis are the content range obtained more preferably. .

Bioactive compounds MP oil MSK oil MSH oil extract extract extract

Total Polyphenols (TP) content 300-350 100-150 1-2 . (mg/g extract) (320-340) (130-140) :

Total Monomeric Anthocyanins (TMA) 100-300 negligible negligible : content (mg/g extract) (150-260) q

Example 6. Production and characterization of briquettes from MSHP :

Page 28 of 37 :

The mango seed husk powder (MSHP) obtained in Example 1 was mixed with a minimal o : amount (e.g. 1-10%) of binding agent such as cassava starch. The mixture is placed in or 890 appropriate molds and pressed employing an appropriate pressure to produce the briquettes. eo

The briquette is characterized by its proximate analysis and its heating value is determined ro using the oxygen bomb calorimeter. Standard methods in proximate analysis are followed as - described earlier in Example 2. Results showed that the briquettes have a heating value of + 15-23 MJ per kg and since its proximate analysis shown in Table 6 below indicates very low o 895 ash and moisture contents and very high fixed carbon content, this indicates that utilizing - mango seed husks for the preparation of briquettes is a good alternative use of such an MSHP . composition derived from the treated mango seeds from fruit processing. =

Table 6. Proximate analysis of Mango Seed Husk Powder. .

Parameter Mango Seed Husk Powder (MSHP) Fu?

Moisture, % 4-8 :

Ash, % 1-3

Nitrogen, % 0.3-1.0

Fixed Carbon, % (by diff) 88-95

Heating value (MJ/kg) 15-23 900

Example 7. Recovery of pectin from mango peel powder (MPP)

Pectin was extracted from mango peel powder (MPP) by employing the acid hydrolysis and alcoholic precipitation methods. A known amount of MPP is mixed with water at different 905 weight ratios (1:10-1:40) and the pH of the mixture was adjusted at a certain pH point (1.5- 3.5) by adding a dilute solution of acid (using 0.1N sulfuric acid or 0.1N hydrochloric acid).

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 is filtered through an ordinary filter : or alternatively using a cheesecloth. The filtrate was collected, further mixed with ethanol at 910 a certain volumetric ratio of 1:5, and mixed thoroughly to precipitate the pectin. The mixture was further filtered through an ordinary filter to separate the pectin precipitate and the alcoholic filtrate. The wet pectin precipitate was weighed, dried, and milled to produce the powdered mango pectin. The said composition is analyzed for its yield based on the defatted mango peel powder sample used, galacturonic acid content, and the degree of esterification 915 (% DE). 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. Pectin content is also

Page 29 of 37 i expressed in terms of galacturonic acid content using galacturonic acid as standard. The - degree of esterification (% DE) of pectin is defined as the number of methyl-esterified - galacturonic acid units expressed as a percentage of the total galacturonic acid in the pectin o 920 molecule obtained. Standard methods of analysis were done based on reported literature . : presented earlier. On the other hand, the alcoholic filtrate was analyzed for its total ot polyphenol (TP) and total monomeric anthocyanin (TMA) contents. 7 "

Results showed that a notably high yield of 27-42% pectin with galacturonic acid content . 925 greater than 70% was obtained when an MPP to water volumetric ratio of 1:20 and pH 1.5 LM using 0.1N hydrochloric acid solution were employed during acid hydrolysis at 80°C for 120 er minutes. Ethanol was used to precipitate the pectin at a volumetric ratio of 1:5 (ethanol to = filtrate). The corresponding degree of esterification (% DE) of the pectin compositions 0 ; obtained was in a range of 68-75%. 930

On the other hand, the alcoholic filtrate obtained after filtration of pectin was analyzed for its total polyphenol (TP) and total monomeric anthocyanin (TMA) contents. Results showed that the filtrate contains notably high contents of total polyphenol (TP) ranging from 700-1,710 mg per liter of alcoholic filtrate, but has negligible amounts of total monomeric anthocyanins 935 (TMA). The results suggest that during pectin extraction, the anthocyanins initially present in the mango peel powder may have gone with the pectin precipitate. Thus, the mango pectin may also be rich in anthocyanins. Nevertheless, the alcoholic filtrate is a valuable composition resulting from this invention, which is a good source of polyphenolic substances, supposedly exhibiting high antioxidative and antimicrobial activities, thus, a good ingredient ] 940 in nutraceuticals, functional foods, pharmaceuticals, and cosmetic applications.

Page 30 of 37 1

Claims (12)

1. pa othe {- 2011 - | Ar wy danse Pf g bag ft B22 tr 4 + CLAIMS |-70l6 ET

   I. A process for the treatment of mango (Mangifera indica L. Anacardiaceae) seeds, = 945 comprising the following steps: hn hi (a) washing of seeds, - (b) drying, > (c) cracking of the dried seeds to separate the kernels from the husks, i. (d) milling to powder forn{_ of the dried husks and/or kernels, p Po? 950 (e) defatting or oil extraction of the powdered compositions using a solvent, 5 (f) recovery and recycling of the solvent in step (e), and (g) fermentation of wash water obtained from step (a).
2. 2. A process for the treatment of mango (Mangifera indica L. Anacardiaceae) peels and © seeds, comprising the following steps: + 955 (a) segregation and washing of peels and seeds, (b) drying, (c) cracking of the dried seeds to separate the kernels from the husks, : (d) milling to powder of the dried peels, kernels, and/or-htisks;—> (e) defatting or oil extraction of the powdered compositions using a solvent, 960 (f) recovery and recycling of the solvent in step (e), and (g) fermentation of the wash water obtained from step (a).
3. 3. A mango ssf 10) extract obtainable from the defatting of mango seed husks by the proces according to Claim 1.
4. 4. A mango seed husk oil (MSHO) extract obtainable from the defatting of mango seed 965 husks by the process according i Claim 2.
5. 5. A mango seed husk oil (MSHO) extract obtainable from the defatting of mango seed husks by the process according to Claim 1, characterized in that the composition is comprising of the following: a plurality of fatty acids and a plurality of polyphenols.
6. 6. A mango seed husk oil (MSHO) extract obtainable from the defatting of mango seed 970 husks by the 1 process according to Claim 2, characterized in that the composition is comprising of the following: a plurality of fatty acids and a plurality of polyphenols.
7. 7. The composition according to Claims 3-6, wherein the total polyphenol content is at ] least 1 mg per gram extract. : Page 31 of 37 ras
8. 8. The composition according to Claims 3-7, which is any combination of the mango - 975 seed husk oil (MSHO), mango peel oil (MPO), and mango seed kernel oil (MSKO) = extract compositions. Go

   /
9. 9. The composition according to Claim 8, which is any combination in any proportion of ~ the mango seed husk oil (MSHO), mango peel oil (MPO), and mango seed kernel oil (MSKO) extract compositions. = 980
10. 10. The composition according to Claims 29, where manky (Memes indica L. o Anacardiaceae var. Philippines)peels and/or seeds are used. ) . :
11. 11. The use of composition according to Claims 3-10 as antioxidant agent. LF
12. 12. The use of composition according to Claims 3-10 as antimicrobial agent. Tr 985 LI i : Page 32 of 37