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Processing of Apple Pomace for Bioactive MoleculesShashi Bhushan a; Kalpana Kalia a; Madhu Sharma a; Bikram Singh a; P. S. Ahuja a

a Institute of Himalayan Bioresource Technology (CSIR), Palampur, Himachal Pradesh, India

Online Publication Date: 01 December 2008

To cite this Article Bhushan, Shashi, Kalia, Kalpana, Sharma, Madhu, Singh, Bikram and Ahuja, P. S.(2008)'Processing of ApplePomace for Bioactive Molecules',Critical Reviews in Biotechnology,28:4,285 — 296

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Critical Reviews in Biotechnology, 28:285–296, 2008Copyright c© Informa UK Ltd.ISSN: 0738-8551 print / 1549-7801 onlineDOI: 10.1080/07388550802368895

Processing of Apple Pomace for Bioactive Molecules

Shashi Bhushan, Kalpana Kalia, Madhu Sharma, Bikram Singh,and P. S. AhujaInstitute of Himalayan Bioresource Technology (CSIR), Palampur, Himachal Pradesh, India

The growth of horticulture industries worldwide has generated huge quantities of fruit wastes(25%–40% of the total fruits processed). These residues are generally a good source of carbohy-drates, especially cell wall polysaccharides and other functionally important bioactive moleculessuch as proteins, vitamins, minerals and natural antioxidants. “Apple pomace” is a left-oversolid biomass with a high moisture content, obtained as a by-product during the processing ofapple fruits for juice, cider or wine preparation. Owing to the high carbohydrate content, applepomace is used as a substrate in a number of microbial processes for the production of organicacids, enzymes, single cell protein, ethanol, low alcoholic drinks and pigments. Recent researchtrends reveal that there is an increase in the utilization of apple pomace as a food processingresidue for the extraction of value added products such as dietary fibre, protein, natural an-tioxidants, biopolymers, pigments and compounds with unique properties. However, the centraldogma is still the stability, safety and economic feasibility of the process(s)/product(s) developed.This review is mainly focused on assessing recent research developments in extraction, isolationand characterization of bioactive molecules from apple pomace, along with their commercialutilization, in food fortification.

Keywords apple pomace, biocatalyst, dietary fibre, natural antioxidant, pectin, pigment

INTRODUCTIONThe research and development efforts on value addition and

efficient utilization of nutritionally rich agro-industrial residuessuch as whey, sugar beet pulp, cassava bagasse, apple po-mace, citrus waste, coffee pulp/husk, etc are gaining momen-tum around the world. The overall strategy for the utilizationof such food processing residues is generally built around fer-mentative or non-fermentative product development. In the caseof fermentative utilization, the residues are used as microbialsubstrates for the development of a variety of value added prod-ucts (Kennedy, 1994; Joshi and Pandey, 1999; Pandey et al.,2000; Lynd et al., 2005; Venus and Richter, 2007; Vendrus-colo et al., 2008). In non-fermentative concepts, agro-industrialresidues are processed for the extraction of bioactive molecules(Larrauri, 1999; Laufenberg et al., 2003; Schieber et al., 2003;Nawirska and Kwaúniewska, 2005).

“Apple pomace” is a left-over solid residue (25%–30% ofthe total processed fruits) obtained after the extraction of ap-ple juice. Several million tonnes of apple pomace are generated

Address correspondence to Shashi Bhushan, Institute of HimalayanBioresource Technology (CSIR), Post Bag-6, Palampur-176 061,Himachal Pradesh, India. Tel.: +911 894233339387; Fax: +911894230433; E-mail: sbhushan@ihbt.res.in

worldwide (Table 1). It has high moisture content (70%–75%)and biodegradable organic load (high BOD and COD values).High moisture content makes apple pomace bulky and suscep-tible to microbial decomposition. Huge piles of apple pomace,outside or near the industrial unit, not only violate the pollutioncontrol norms and industrial safety issues, but also lead to publichealth hazards. Owing to high transportation costs (being bulky)and the generation of foul smells (fast biodegradation by naturalflora), direct dumping is also a problem. Earlier, sun energy wasused for drying apple pomace in the open to reduce the bulk.This method makes apple pomace darker (enzymatic or oxida-tive browning) and unfit for value addition, especially for humanfood fortification. In addition to sun drying, a number of meth-ods have been reported for reducing the bulk of apple pomaceand to preserve it for further utilization (Fenton and Kennedy,1998; Constenla et al., 2002; Sun et al., 2007). The selection ofa particular method for pomace drying depends upon its energycost, change in nutritional profile and intended purpose.

Intrinsic to apple fruit nutrients, pomace also contains numer-ous phytochemicals in the form of simple sugars, pectin, dietaryfibres and natural antioxidants. In the past, apple pomace driedfor use as animal feed, fuel for boilers or added to soil as a con-ditioner (Singh and Narang, 1992; Takahashi and Mori, 2006).Since 1980, it has been extensively studied as a substrate formicrobial growth and used in the production of value added

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286 S. BHUSHAN ET AL.

TABLE 1World apple pomace generation

QuantityCountry (thousands of tonnes) Reference

Spain 20 Gullon et al., 2007Germany 250 Endreß, 2000New Zealand 20 Lu and Foo, 1998India 3–5 HPMC, 2007Brazil 13.75 Villas-Boas et al., 2003Iran 97 Pirmohammadi et al., 2006Japan 160 Takahashi and Mori, 2006United States 27 Roberts et al., 2004

products such as organic acids, enzymes, single cell proteins,low alcoholic drinks, ethanol, biogas, pigment and baker’s yeast(Hang et al., 1982; Sharma, 1983; Kalia et al., 1992; Wiacek-Zychlinska, et al. 1994; Hang and Woodams, 1995; Shojaosadatiand Babaeipour, 2002; Seyis and Aksoz, 2005; Bhushan and

Joshi, 2006; Attri and Joshi, 2006; Gullon et al., 2007). Ven-druscolo et al. (2008) recently reviewed in detail the researchand development efforts currently underway on apple pomacethroughout the world for fermentative product development.

Since 1985, a continuous increase was observed in the num-ber of publications in peer reviewed journals per year (Figure 1a)dealing with apple pomace utilization. In this period, R & D ini-tiatives were more focused, on apple pomace conversion intovalue added product development (Joshi et al., 1996; Masoodiet al., 2002; Sudha et al., 2007), or on extracting the bioactiveconstituents for food fortification or food enrichment (Fig. 1b).Efforts were also made to understand the addition of apple po-mace on the nutritional profile of prepared food products andon regulatory mechanisms, as evident from the percentage ofpublications in the area of nutrition and dietetics as well as bio-chemistry and molecular biology (Masoodi et al., 2002; Zhanget al., 2003; Sembries et al., 2003; Devarajan et al., 2004;Lantto et al., 2006; Sehm et al., 2006; Fridrich et al., 2007;Gutzwiller et al., 2007). The fact is also supported by the utiliza-tion and disposal hierarchy of food processing residuals (Brandtand Martin, 1994), according to which recovery of human

FIG. 1. R & D publications on apple pomace: in relation to (a) year-wise and (b) subject-wise percentage (Source: Web of Sciences,2008).

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PROCESSING OF APPLE POMACE FOR BIOACTIVE MOLECULES 287

consumption products from agricultural residues has been pri-oritized at the second position of the management strategies.Besides fermentative product development, since the 1920s, ef-forts have continuously focused on the isolation of bioactivemolecules from apple pomace, with the execution of a pectinextraction industrial unit in the USA and Europe. In additionto pectin, apple pomace is also used for extraction of dietaryfibre, xyloglucan, natural antioxidant and aromatic compounds(Carson et al., 1994; Almosnino et al., 1996; Barwal and Kalia,1997; Watt et al., 1999; Lu and Foo, 1997; Schieber et al., 2003;Canteri-Schemin et al., 2005; Figuerola et al., 2005; Royer et al.,2006; Guyot et al., 2007). Apple pomace has also been consid-ered for new applications such as textile dye removal (Robinsonet al., 2002), press aid (Roberts et al., 2004), as a heavy metalabsorbent (Nawirska, 2005) and as a protein stabilizer (Lanttoet al., 2006). Thus, value addition of such residues will not onlyprovide the means to industrial economic stability but will alsohelp in reduction of environmental pollution, judicious man-agement of natural bioresources and availability of nutrition-ally enriched food products, as well as industrially importantbiomolecules.

APPLE PROCESSING AND ITS BY-PRODUCTSThe world production of apples in 2006–2007 was 46.1 mil-

lion tonnes, with more than a 50% (24.5 million tonnes) con-tribution from China, followed by the USA. China is slowlycapturing the world apple market in fresh, as well as pro-cessed, products. India is far behind and holds ninth posi-tion but is among the top 25 apple producing countries in theworld with a total production of 1.3 million tonnes, (MFPI,2006). Out of the total production, about 71% of the fruit ismarketed as being fresh, while 25%–30% of the fruit is pro-cessed into juice, cider, and frozen and dried processed products(Figure 2).

Apple juice concentrate (AJC) is the major processed productthroughout the entire world (64% of the total of all processed

apple products) and is prepared either from specific processingvarieties or low grade and culled fruits that are otherwise un-suitable for the fresh market. Global apple juice production wasestimated at 1.4 million metric tonnes during 2004–2005, withChina’s contribution alone being 600,000 tonnes. India produces4500 tonnes per annum of AJC and this is equivalent to about0.64% of the total world production. The total recovery of thejuice in industrial production process (Figure 3) is about 70%–75%, generating 25%–30% apple pomace and 5%–11% sludge(liquid waste obtained after clarification and sedimentation withbentonite). Apple pomace consists mainly of apple skin/flesh(95%), seeds (2%–4%) and stems (1%). In India, around 3000–5000 tonnes of apple pomace are generated annually, with 2000–3000 tonnes in Himachal Pradesh alone. Sludge contains applejuice (5–6◦Brix TSS) and bentonite particles (added during clar-ification of the juice), and there seems to be a direct loss of about10% juice, which can be recovered easily by adoption of efficientprocessing techniques.

NUTRITIONAL PROFILE OF APPLE POMACEThe apple fruit is highly nutritious and contains carbohy-

drates, proteins, minerals and natural antioxidants. These fruitsare invaluable in terms of:

• Elimination of certain harmful substances from thebody.

• Prevention of decomposition of protein matter in thealimentary canal.

• The malic acid being beneficial to the bowels, liver andbrain.

• Help in prevention of anemia, constipation, stomachdisorders, hypertension, rheumatic afflictions, etc.

As a fresh fruit, apple pomace contains significant amountsof phytochemicals such as carbohydrates, proteins, vitamins andminerals. The nutritional profile (Table 2) of dried apple pomacereveals a high content of carbohydrates with a fermentable sugar

FIG. 2. World apple production and processing status.

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288 S. BHUSHAN ET AL.

FIG. 3. Industrial processing of apple juice concentrate (AJC).

content of up to 50% (Hang and Walter, 1989; Bhushan, 2002;Gullon et al., 2007). The apple pomace carbohydrates mainlyconsist of galacturonic acid (49%–64%), arabinose (14%–23%)and galactose (6%–15%), with minor amounts of rhamnose, xy-lose and glucose (Mehrlander et al., 2002). Besides carbohy-drates, apple pomace is rich in proteins, vitamins (A and C)and minerals (P, K, Mn, Ca, Mg and Fe) (Hang and Walter,1989; Johr et al., 1960; Kennedy, 1994). It is a good source ofnatural antioxidants (catechins, procyanidins, caffeic acid, phlo-ridzin, phloretin glycosides, quercetin glycosides, chlorogenicacid, etc). Apple pomace, including seeds, contains polypheno-lics (Table 3) with the strong antioxidant activity of quercetinglycosides, phloridzin and its oxidative products (Lu and Foo,2000; Schieber et al., 2002; Guyot et al., 2007). The seed con-tains about 80% of fatty acids, the majority being linoleic acidand oleic acid.

PROCESSING OF APPLE POMACE FOR BIOACTIVEMOLECULESDietary Fibre

“Dietary fibres” are the plant cell wall compounds (non-starchpolysaccharides), which are resistant to hydrolysis by digestiveenzymes in humans. They are intrinsic and intact in plants andconsist of mainly cellulose, hemicelluloses, pectic substancesand lignins (Trowell, 1974). Diets with inadequate dietary fibre

content are generally associated with constipation, diverticu-losis, cardiovascular disease and cancer (Trowell, 1985). Di-etary fibres are known to exert a number of protective effectson cardiovascular diseases, colorectal cancer, obesity and di-abetes (Salmeron et al., 1997; Ellegard et al., 2000). Fibresbind excess hydrochloric acid, cholesterol and gastric juices,increase the faecal bulk and stimulate intestinal peristalsis (Jenk-ins et al., 1998; Nawirska and Kwasniewúka, 2005). The fruit-and vegetable-based fibres are known to have better nutritionalquality than cereal formulations, because of the presence of sig-nificant amounts of associated active compounds (flavonoids,carotenoids, higher fibre content) in balanced composition (sol-uble/insoluble dietary fibre ratio, water- and fat-holding capac-ities, lower energy value and phytic acid content).

Apple pomace contains a significant amount of non-starchpolysaccharides (35%–60% dietary fibre), with a high amountof insoluble fibre (36.5%) as well as soluble fibre (14.6%) (Chenet al., 1988; Gallaher and Schneeman, 2001; Villas-Boas et al.,2003; Sudha et al., 2007). The main constituents of these dietaryfibres are pectins (5.50%–11.70%), cellulose (7.20%–43.60%),hemicelluloses (4.26%–24.40%), lignins (15.30%–23.50%) andgums. Cellulose, pectin and lignin are water insoluble, whilegalacturonic acid and hemicelluloses are water soluble. A num-ber of fibre enriched bakery products were prepared by addingdried apple pomace powder on a wheat flour replacement basis.

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TABLE 2Proximate composition of apple pomace

Composition CompositionConstituents (dry weight basis) Constituents (dry weight basis)

Moisture (%) 3.90–10.80 Alcohol-soluble fraction of carbohydrateProtein (%) 2.94–5.67 Saccharose (%) 3.80–5.80Total carbohydrate (%) 48.0–62.0 Glucose (%) 19.50–19.70Fibre (%) 4.70–51.10 Fructose (%) 48.30Insoluble fibre 36.50 Xylose, mannose and galactose (%) 1.20–4.40Soluble fibre 14.60 L-malic acid (%) 2.60–3.20Fat (ether extract, %) 1.20–3.90 Arabinose and rhamnose (%) 7.90–6.0Pectin (%) 3.50–14.32 Glucooligosaccharides (%) 3.40–3.80Ash (%) 0.50–6.10 Xylooligosaccharides (%) 3.0–3.70

Minerals Arabinooligosaccharides (%) 0.20–0.40Phosphorus (%) 0.07–0.076 Uronic acid (%) 2.70–3.40Potassium (%) 0.43–0.95 Alcohol-insoluble fraction of carbohydrateCalcium (%) 0.06–0.10 Glucan (%) 41.90–42.90Sodium (%) 0.20 Starch (%) 14.40–17.10Magnesium (%) 0.02–0.36 Cellulose (%) 7.20–43.60Copper (mg/kg) 1.10 Polysaccharides of xylose, mannose and galactose (%) 13.0–13.90Zinc (mg/kg) 15.00 Polysaccharide of arabinose and rhamnose (%) 8.10–9.0Manganese (mg/kg) 3.96–9.00 Acid detergent lignin (%) 15.20–20.40Iron (mg/kg) 31.80–38.30 Uronic acid (%) 15.3

Sources: Chen et al., 1988; Hang and Walter, 1989; Kennedy, 1994; Joshi and Sandhu, 1994; Masoodi and Chauhan, 1998; Bhushan, 2002;Constenla et al., 2002; Schieber et al., 2003; Marcon et al., 2005; Virk and Sogi, 2004; Nawirska and Kwaúniewska, 2005; Sudha et al.,2007; Gullon et al., 2007)

Chen et al. (1988) investigated the effect of fibre source andfibre concentration on physical and baking properties of bread,cookies and muffins. They used commercially available fibresof apple (spray dried), wheat bran and oat bran at 0%, 4%, 8%and 12% concentration with uniform particle size (130 µm).The results showed that apple fibres had a higher content ofdietary fibre (61.90%), lipids (2.45%), and lower amounts ofproteins (7.25%) and ash (1.27%), than other fibre sources. Irre-spective of fibre source, a significant increase in water absorp-tion, product weight and dough development time was observedwith the increase in fibre concentration. A chemical analysis ofthe finished product showed that the bakery products preparedby adding apple fibres had a higher dietary fibre content thanother sources. Hence, the authors have advocated the additionof apple fibres (up to 4%) for food enrichment without compro-mising product quality and acceptance by consumers. In anotherstudy, cakes were prepared with direct incorporation of apple po-mace in the wheat flour and the baking properties were evaluated(Masoodi et al., 2002). They used dried apple pomace powderof different particle sizes (30 µm, 50 µm and 60 µm) with awheat flour replacement basis at 0%, 5%, 10% and 15% con-centration. A significant increase in batter viscosity (82.6 poise,113.6 poise and 307.6 poise) was observed with an increase inpomace concentration (5%, 10% and 15% pomace) comparedto the control (64.6 poise) at uniform particle size (30 mesh).

The results revealed a significant increase in cake weight withthe increase in pomace concentration and particle size, whichcaused the batter properties to deteriorate to an unacceptablelevel. Cakes were also prepared by direct incorporation of finelyground apple pomace (150 µm) in wheat flour at 0%, 10%, 20%and 30% on a flour replacement basis (Sudha et al., 2007). Therheological properties of the dough revealed an increase in wa-ter absorption, resistance to extension and pasting temperature,with the increase of apple pomace content in the dough. Theyobserved a significant increase in cake weight and texture, anda decrease in cake volume with the increase in concentration ofapple pomace. Nutritional characteristics showed that productsprepared with apple pomace incorporation (25% apple pomaceblends) had significantly higher amounts of dietary fibre (14.2%)as compared to the control (0.47%). It was inferred that applepomace not only improved the dietary fibre content of the bakedproduct, but also increased the phenolic content (3.15 mg g−1) ata 25% addition over the control (2.07 mg g−1). However, thesefindings still require validation on a product safety and stabilityscale before recommendations for commercial food fortificationor nutritionally enriched product development can be made. Itis worth mentioning that there is still no accessible report onapple pomace derived food products for human consumption ona commercial scale. Hence there seems to be a wide scope inthis segment of food processing.

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290 S. BHUSHAN ET AL.

TABLE 3Total phenolic compounds identified in apple pomace fractions

and seeds

Apple pomace

Catechin Quercetin 3-arabinofuranosideEpicatechin Quercetin 3-rhamnosidep-Coumaric acid Quercetin-O-pentosidep-Coumaroylquinic acid Sinapic acid-O-glucosideCaffeic acid-O-glucoside Apigenin*Chlorogenic acid Chrysoeriol*Caffeoylquinic acid Eriodictyol-hexoside*Cyanidin 3-glucoside Eriodictyol*Dicaffeoylquinic acid Hesperidin-O-pentoside*Ferulic acid Luteolin*3-Hydroxyphloridzin Luteolin-7-O-glucoside*Kaempferol-O-glucoside Luteolin-7-O-galactoside*Procyanidin B2 Naringenin*Phloridzin Naringenin-7-O-rutinoside*Phloretin Naringenin-O-hexoside*Phloretin xyloglucoside Naringenin-O-hexoside*Quercetin Naringenin-O-glucuronide*Quercetin 3-diglucoside Naringenin-7-O-neohesperidoside*Cyanidin 3-glucoside Naringenin-7-O-glucoside*Quercetin 3-rutinoside Protocatechuic acid*Quercetin 3-galactoside Quercetin-O-pento-hexoside*Quercetin 3-glucoside Rhamnetin*Quercetin 3-xylanoside Rhamnetin 3-glucoside*Quercetin-O-hexoside Salicylic acid*Quercetin

3-arabinopyranosideApple seed

Catechin PhloridzinEpicatechin Phloretinp-Coumaric acid Phloretin xyloglucosidep-Coumaroylquinic acid Quercetin 3-galactosideChlorogenic acid Quercetin-3-O-glucosideProcyanidin B2 Quercetin-3-O-rhamnoside

*Reported for first time in apple or apple pomace fraction.Sources: Lu and Foo, 1997, 2000; Schieber et al., 2002, 2003; Sanchez-Rabaneda et al., 2004; Guyot et al., 2007, Cetkovic et al., 2008.

PectinThe utilization of apple pomace for pectin production is con-

sidered to be one of the most practical approaches. Fruit pectinshave been employed as gelling agents, thickener texturizers,emulsifiers and stabilizers in food processing, and the cosmeticsand pharmaceutical industries (Thakur et al., 1997). In the foodindustry, pectin is used in products such as jams and jellies; fruitpreparation; bakery jellies; confectionery; yogurt and acidifiedmilk drinks; beverages; and frozen foods. The non-toxicity andbiocompatibility of fruit pectin render its application as a car-rier for drugs, e.g. for oral administration of colon-specific drug

delivery (Holst et al., 2006). The gelation properties of pectinare a major factor in terms of its suitability for a particular appli-cation and gelation these properties are known to be influencedby the length of side-chain branches, the degree of acetylationand the pattern of esterification (Constenla et al., 2002).

Apple pomace consists of approximately 10%–15% pectinon a dry weight basis (Endreß, 2000; Wang et al., 2007). Pectinfrom dried apple pomace is usually extracted with diluted min-eral acids at elevated temperatures to solubilize the protopectin,followed by alcohol precipitation (May, 1990). The dried applepomace is ground to powder (particle size 80 mesh) and extru-sion is performed (by twin-screw extruder) at different speeds,feed rates and moisture content (Hwang et al., 1998). Extrusionhas been found to exert a more solubilizing effect than the com-mercial acid extraction process. However, it is effective only inincreasing the pectin yield with a lower degree of esterification,which has limited industrial application. The authors have ob-served a cohesive effect of pH of the buffer, extraction time andsolid:liquid ratio on pectin yield. The pomace obtained fromGranny Smith apples, on a laboratory scale, was dried at differ-ent temperatures (60◦C, 70◦C, 80◦C and 105◦C) to evaluate theeffect on pectin characteristics, composition, colour and gelpointtemperature (Constenla et al., 2002). Galacturonic acid contentseemed to be unaffected by the drying temperature; however, sig-nificant effects were noticed on pectin colour and the gelpointtemperature (maximum at 80◦C). Extremes of drying temper-ature resulted in more brownish pectin colour, either owing toenzymatic browning (at a lower drying temperature, 60◦C) orcaramelization (at a higher drying temperature, 105◦C). Canteri-Schemin et al. (2005) used nine apple varieties to study the effectof variety, particle size and kind of acid used for extraction onpectin yield. The apple pomace flour with a particle size higherthan 600 µm showed a lower pectin yield (maximum between106 µm and 250 µm) and a statistical difference was noticedamong the varieties with respect to pectin yield. However, thishas little relevance to industrial apple pomace processing, asthere is always a combination of number of varieties. Amongthe acid used for pectin extraction, citric acid showed the highestaverage yield (13.75%) and was reported to be more econom-ical and safe. Wang et al. (2007) used a microwave-assistedextraction system, at a frequency of 2450 MHz, for extractionof pectin from dried apple pomace powder (particle size 0.6 ×10−3 to 1.5 × 10−3) and the analytical data was verified usingresponse surface methodology. Microwave-assisted extractionreduced the pectin extraction time considerably. The authorsconcluded from the experimental results that “extraction timeand buffer pH” and “pH and solid:liquid ratio” had significanteffects on pectin yield and on the degree of methylation. The op-timum predicted yield (0.158 g g−1 dried apple pomace powder)was recorded with pH 1.01 and HCl buffer, 499.4 W microwavepower, 20.8 min extraction time and 0.069 solid:liquid ratio. Re-cently, the effect of extraction conditions on yield and purity ofapple pomace pectin by alcoholic precipitation was investigated(Garna et al., 2007). The extraction was carried out using two

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different conditions of pH and process time by heating 50 g ofapple pomace in 1 L of acidic water, with continuous stirringat 250 rev min−1 in a jacketed stainless steel reactor at a tem-perature of 90◦C. The low pH (1.5) with the longer extractionperiod (3 h) resulted in a high yield of pectin (8.9%), while aslight increment in pH (2.0) with a decrease in extraction time(2 h) produced pectin with a high content of galacturonic acid.The researchers were able to obtain pectin with a high degree ofmethylation from both extraction conditions, but with a brownhue that developed owing to the oxidation of phenolic com-pounds. Efforts were made to improve the colour of the applepomace pectin by bleaching it with an alkaline peroxide (Re-nard et al., 1996). The process helped in improving the extractedpectin colour, but led to a loss of polyphenolics and to a degra-dation of the major part of the pectins, resulting in a lower pectinyield. Later, Schieber et al. (2003) developed a method for thecombined recovery of pectin and polyphenolics. This processhelped the production of a light coloured pectin with the simul-taneous recovery of phenolic compounds, which can be used asa source of antioxidants. At present, fruit pectins with varyingproperties are in great demand and hence there is a need forprocesses for the development of products with tailored proper-ties at an industrial level, and this is a great challenge for R&Dpersonnel.

XyloglucanIn addition to pectin, apple pomace is also a good source

of cellulose and hemicelluloses – xyloglucan mostly fuco-galactoxyloglucans, representing up to 18% of apple cell wallpolysaccharides (Renard et al., 1995; Watt et al., 1999; Cailiet al., 2006). Cellulose is used for the production of variousindustrial products (methylcellulose, hydroxypropylcellulose,carboxymethylcellulose) having wide commercial applications.However, the exploitation of xyloglucan extraction from applepomace is at an infant stage. Xyloglucans are not solubilized dur-ing the hot acidic treatment employed for pectin extraction. Re-nard et al. (1995) suspended depectinized apple pomace (1 g) in100 mL alkali solution (pH 7.0) and centrifuged after incubationfor 15 min. They found that the increased alkali concentrationand extraction time resulted in high xyloglucan production.

Xyloglucan was extracted with 4 M KOH from a commercialapple pomace source with and without pectinase pretreatment(Watt et al., 1999). Different oligosaccharides were isolated fromthe 1,4-β-D-glucanase hydrolysate and characterized using 13CNMR spectroscopy. Enzymatic treatment of the apple pomaceresulted in the production of lower molecular weight xyloglucan,with a lower viscosity compared to non-enzymatic treatment. Analmost similar composition of neutral sugars was found in allthe purified xyloglucan fractions, except for a slight variationowing to apple variety and type of pomace used for extraction.The intrinsic viscosity of the purified xyloglucan was reportedto be 244 mL g−1. The results were comparable to earlier re-ports of Renard et al. (1995) (240 mL g−1), despite differentextraction procedures. In both studies, the yield of xyloglucan

was reported to be influenced by the liquid:solid ratio, time ofextraction, concentration of alkali, type of pomace and pro-cess temperature. Furthermore, ultrasound-assisted extraction(50 kHz frequency with an input power of 160 W) was observedto reduce the time by a factor of three over the alkali extractionmethod (Caili et al., 2006). The three independent variables,i.e. liquid (34.4):solid (1) ratio, KOH concentration (3.3 M) andextraction time (2.5 h) were optimized using response surfacemethodology. The conversion of xyloglucan into compoundssuch as thickening agents and texture modifiers or as a sourceof biologically active oligosaccharides having medicinal prop-erties, is possible. Therefore the extraction of xyloglucan fromapple pomace can provide a new means for the development ofhigh-value biomolecules.

Natural AntioxidantsA range of polyphenolics compounds have been isolated

from apple pomace, such as cinnamic acid and its deriva-tives, epicatechin, epicatechin dimer (procyanidin B2), trimer,tetramer and oligomer, quercetin-3-glycosides phloridzin and3-hydroxyphloridzin (Table 3). The nature of procyanidinpresent in apple pomace has been established by 13C-NMRspectroscopy, by the acid-catalyzed degradation reaction withphloroglucinol and by electro-spray mass spectrometry (Foo andLu, 1999). Earlier, these authors had investigated Gala apple po-mace as a potential source of polyphenols (Lu and Foo, 1997).The fraction was obtained by extracting apple pomace powderwith 70% aqueous acetone. The extract was concentrated andleft-over residue defatted with hexane, concentrated and freezedried before separation on Sephadex LH-20 using methanol assolvent. Out of the total polyphenols present (7.24 g kg−1 DM),quercetin glycosides accounted for more than half (4.24 g kg−1

DM), followed by phloridzin and its oxidative products. The elu-tion order of the seven quercetin glycosides from apple pomacewas established by Schieber et al. (2002) using HPLC stationaryphase with hydrophilic endcapping system. The separation wasachieved within 8 min for all compounds and the elution orderwas found to be Q-3-O-rutinoside, Q-3-O-galactoside, Q-3-O-glucoside, Q-3-O-xyloside, Q-3-O-arabino-pyranoside, Q-3-O-arabino-furanoside and Q-3-O-rhamnoside. The predom-inant quercetin glycosides detected were Q-3-O-galactoside,Q-3-O-arabinosoide, Q-3-O-glucoside and Q-3-O-xyloside.

In the subsequent year, Schieber et al. (2003) reported a pro-cess for the simultaneous recovery of pectin and polyphenolsfrom apple pomace. The acidic extract of apple pomace (pH 2.8,TSS 3.1◦Brix) preheated to 60◦C was passed through an Amber-lite XAD 16 HP resin- (1.77 L) containing glass column at a flowrate of approximately 10 bed volumes per hour. Pectins present inthe extract easily pass through the resin and pectin-containing ef-fluent was collected at the bottom. Pectin residues were removedwith distilled water until no pectin could be recovered by alco-hol precipitation. The process resulted in a light coloured pectinwith a slight increase in the degree of esterification and galactur-onic acid units. The adsorbed phenolic compounds were eluted

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292 S. BHUSHAN ET AL.

TABLE 4

Apple pomace (mg kg−1 DM)

Schieber et al., 2003Apple seed (mg kg−1 DM)

Phenolic compounds Lu and Foo, 1997 Drum dried Lyophilisate Schieber et al., 2003

Catechin nd* nd 2,400 ndEpicatechin 640 nd 9,300 9.6Caffeic acid 280 nd nd nd3-Hydroxyphloridzin 270 nd nd ndChlorogenic acid nd nd 14,300 119.8Phloretin-xyloglucoside 170 nd nd 47.4Phloretin nd nd nd 6.3Phloridzin 1,420 nd 11,400 1,915.0Quercetin 3-galactoside 1,610 224.2 nd 12.9Procyanidin B2 nd nd 9,300 17.0Quercetin 3-glucoside 870 27.6 3,900 5.9Quercetin 3-xyloside 530 114.4 1,800 ndQuercetin 3-arabinoside 980 223.0 1,100 ndQuercetin 3-rhamnoside 470 297.5 4,700 25.0p-Coumaroylquinic acid nd nd 1,800 9.4

*nd = not detected

with a two bed volume of methanol, and after solvent removal invacuo, residues were lyophilized at 0.075 mbar for 80 h. The to-tal lyophilisate contained about 12% of phenolic compounds anda fraction was characterized by HPLC using a stationary phasewith hydrophilic endcapping. The main phenolic compoundsdetected were phloridzin, chlorogenic acid and quercetin-3-O-glycosides, with appreciable amounts of catechin and procyani-din. The authors also investigated the phenolic profile of wetand three-stage drum-dried apple pomace. Industrial drying ofapple pomace has been reported to have a non-significant ef-fect on the total yield or phenolic profile except for flavanols,which were adversely affected. Sanchez-Rabaneda et al. (2004)identified about 60 phenolic compounds in different apple po-mace fractions (peel, core, seed, calyx, stem and soft tissues)using LC/MS/MS and also verified the structure of the differentcompounds. Twenty-two new phenolic compounds were char-acterized from apple or apple pomace, especially naringenin,luteolin and their derivatives. Factors such as pressure, temper-ature, ethanol concentration and extraction time were found toinfluence the subcritical extraction of polyphenols from applepomace (Adil et al., 2007). The phenolic profile of apple seedsseems to be limited (Table 4) in comparison to apple skin (Luand Foo, 1998; Schieber et al., 2003). The effect of enzymatictreatment on the yield of phenolic compounds was also investi-gated under different extraction conditions (Zheng et al., 2008).The pectinase treatment of apple pomace improved the extrac-tion of polyphenolics compounds under optimized conditions of

enzyme reaction, i.e. pH 3.6, enzyme to pomace ratio of 12%,37◦C and an 11 h reaction period. This resulted in higher yields oftotal phenolics (about 28%) and flavonoid content (about 50%)in comparison to the control.

Polyphenols from apple extracts have been shown to inhibittumour-cell proliferation in vitro (Eberhardt et al., 2000). Theantioxidant properties of apple pomace polyphenols were eval-uated using a β-carotene/linoleic acid system, free radical scav-enging activity using DPPH (1,1-diphenyl-2-picryl-hydrazyl)and superoxide anion radical scavenging activities by a cellularxanthine/xanthine oxidase system as a superoxide source (Luand Foo, 2000). Epicatechin and quercetin 3-glycosides frompomace showed the highest activity, while phloridzin exhibitedmoderate activities in comparison to Vitamins C and E. Ex-cept for phloridzin (0.60 EC50), all of the apple polyphenolsexhibited good DPPH-scavenging properties, two to three timeshigher than vitamins C (0.35 EC50) or E (0.30 EC50). EC50 is theamount of antioxidant necessary to decrease the initial DPPHconcentration by 50%. Apple pomace polyphenols were foundto be effective superoxide scavengers, in comparison to vita-mins C and E; however, among themselves, procyanidins wereobserved to be superior to quercetin 3-glycosides, chlorogenicacid, 3-hydroxyphloridzin and phloridzin.

These studies revealed that the polyphenols responsible forthe antioxidant activity in apple are still present in the po-mace, and can easily be extracted for food fortification or nu-traceutical product development. Apple pomace can therefore

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PROCESSING OF APPLE POMACE FOR BIOACTIVE MOLECULES 293

become an inexpensive and readily available source of dietaryantioxidants.

Source of BiocatalystThe intrinsic enzymes system of apple pomace had been em-

ployed for biocatalysis. Biocatalysis can be defined as the uti-lization of natural catalysts, such as enzymes (either isolatedor intrinsic to the living cells) to perform chemical transforma-tions on organic compounds. Since the 1980s there have beena few research groups working on the production of volatilealdehydes and alcohols by apple peripheral tissues from in situor added linoleic acid or linolenic acid (Paillard, 1979; Ambidand Fallot, 1980; Drawert et al., 1986). Almosnino and Belin(1991) explored an apple pomace intrinsic enzymes system forthe bioconversion of polyunsaturated fatty acids in the produc-tion of aromatic compounds. Fresh, oxidized (exposed to air)and frozen Golden Delicious apple pomace (10 g each) wasused as an enzyme source for the chemical transformation oftechnical linoleic acid (75% linoleic acid) in 150 mL of McIl-vaine buffer reaction volume using Tween 80 as an emulsifier(37.5 µl). The reaction was carried out at 18◦C for 4 h. Sulphurdioxide and ascorbic acid were used to preserve and reducethe browning of the apple pomace (caused from oxidation bypolyphenol oxidase). Micronized fresh apple pomace preservedwith 60 p.p.m. of SO2 , and 500 p.p.m. of ascorbic acid was foundto increase the yield of hexanal and 2,4-decadienal by 56% and95%, respectively. The endogenous enzymes system contained alipoxygenase that converted linoleic acid to 9,13-hydroperoxide(Kim and Grosch, 1979) and these intermediates were furthercatalyzed by hydroperoxide lyase, resulting in the formation ofvolatile compounds, i.e. hexanal and 2,4-decadienal (Schreierand Lorenz, 1982; Hatanaka et al., 1986). Hexanal formationrequired both the lipoxygenase and hydroperoxide lyase en-zymes, while formation of 2,4-decadienal only depended onthe presence of lipoxygenase for conversion into hydroperox-ides (Almosnino and Belin, 1991). Later, the process was scaledup in 5 L and 10 L Trimix reactors, in a 2 L buffered mediumcontaining 33% or 50% apple pomace and 0.5% or 2% linoleicacid (Almosnino et al., 1996). The biocatalysis, occurring dur-ing biochemical transformation, was monitored by the quantifi-cation of hexenal (reflecting the expression of the double en-zyme system, i.e. lipoxygenase and hydroperoxide lyase) and2,4-decadienal production (reflecting the expression of lipoxy-genase and β-scission decomposition). The authors found thatthe reaction at 25◦C, with continuous oxygen flow to vesselheadspace, resulted in high productivities of both aromatic com-pounds. The best hexenal yield (160 mg kg−1) was obtained inan alkaline pH (9.0) after 48 h, exhibiting the pH optima ofthe enzyme complex. However, both the enzymes appeared tobe unaffected by acidic conditions in reaction buffers pH 1.4–4.8.

Besides enzyme systems, apple pomace also contains min-eral elements such as iron, zinc, copper, calcium and magnesium(Table 2). As these micro-elements act as cofactors in many

biosynthetic processes, they can be investigated for new appli-cations either in biotransformations or bioconversions.

Pigment MoleculesOxidation of polyphenols by polyphenoloxidase leads to the

development of yellow–orange coloured juices or ciders duringprocessing in the presence of oxygen, which has a direct correla-tion with the concentrations of hydroxycinnamic acid, flavanolsand dihydrochalcones, i.e. phloridzin and phloretin xylogluco-side (Lea, 1984; Nicolas et al., 1994). Phloridzin is a phenoliccompound present in apple pomace and its enzymatic oxida-tion has been shown to produce yellow products (Lu and Foo,1997; Ridgway et al., 1997; Schieber et al., 2003). Phloridzin,using apple leaf enzyme systems, was first converted into 3-hydroxyphloridzin and later into the corresponding o-quinones(Raa and Overeem, 1968). A brown reddish colour was ob-tained, and it was proposed that these products resulted fromthe phloroglucinol nucleus and quinones in head-to-tail oxida-tive coupling reactions. Recent development in instrumentationand analytical techniques has led to the identification and gener-ation of structural information on compounds such as phloridzinoxidation products (POP). With the help of mass spectrometryand NMR, monomeric structures resulting from oxidation andintramolecular oxidative coupling with simultaneous ring rearo-matisations of POP to a colourless compound (POPi), whichwere then converted to yellow pigment products (POPj), wereelucidated (Le Guerneve et al., 2004). The oxidation of phlo-ridzin by mushroom polyphenol oxidase in POP production ofyellow pigment, as an alternative to tartrazine for application asa food dye, was evaluated (Guyot et al., 2007). Mushroom PPOwas added to pre-incubated phloridzin (dissolved in phosphatebuffer 100 mM, pH 6.5) at 30◦C. The reaction time was 0–52 h,and the solution was immediately stabilized by the addition of200 µL of o-phosphoric buffer and 100 µL of sodium fluoridesolution. They studied oxidative product formation kinetics, theeffect of enzyme concentration, the colouring power of POPj,stability as influenced by pH and temperature, and radical scav-enging activities. The POPj molecule exhibited strong colouringpower, and 30 mg L−1 was found enough to reach half saturationof the colour at pH 3. Researchers observed that an acidic pH(2.2–5.0) had no pronounced effect on the colour characteris-tics; however, an alkaline pH (≥ 6.0) resulted in the formationof orange pigment from the yellow. the free radical scavengingactivity of phloridzin and its oxidative products was also exam-ined. The studies revealed that phloridzin and POPj had a veryweak antioxidant activity, while POPi had similar activity to thatof epicatechin, ascorbic acid and Trolox. A similar free radicalscavenging activity of phloridzin was observed by Lu and Foo(2000). The food and cosmetic industries are always looking fornew highly water-soluble yellow pigments and the phloridzinpigment could be an alternative food dye. However, significantresearch efforts would still be required to make this a viableprocess on an industrial scale.

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294 S. BHUSHAN ET AL.

CONCLUSIONSThe processing of apple pomace and other industrial food

residues for extraction of bioactive molecules can play an im-portant role in efficient bioresource utilization and can provide amethod for the nutritional enrichment of foods at a low cost. Asper the reviewed literature, target molecules from apple pomaceare dietary fibre, pectin (especially tailored polysaccharides withunique functional properties) and, more importantly, natural an-tioxidants. Dietary fibres from fruit residues have more benefi-cial effects on human health than cereals owing to the presenceof associated bioactive molecules. Polysaccharides tailored forunique functional properties can be employed for the develop-ment of industrial molecules such as bioabsorbents, biopolymersor compounds for effluent treatment.

A lot of interest has been generated regarding natural antiox-idants, owing to their role as a free radical scavenger, as theseare associated with a number of degenerative diseases such ascancer and atherosclerosis. The recovered antioxidants from ap-ple pomace showed good free radical scavenging activities, andhence could be used as a nutraceutical and a food supplement.In addition to these bioactive molecules, the biocatalytic prop-erties of apple pomace can be explored for the conversion oflow value substrates to high value product formation. However,further R&D efforts are required for product development andfor the assessment of their safety and stability with respect toassociated human health benefits. It is also worth mentioningthat apple crops are heavily sprayed with chemicals owing totheir susceptibility to insect and pathogen attacks, and thereforea toxicity analysis of apple pomace will be required prior toprocessing.

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