Ind. Eng. Chem. Res. 2009, 48, 4681–4687
4681
Assessment of the Production of Oligomeric Compounds from Sugar Beet Pulp Martina Martı´nez,† Beatriz Gullo´n,† Henk A. Schols,‡ Jose´ L. Alonso,*,† and Juan C. Parajo´† Department of Chemical Engineering, Faculty of Science, UniVersity of Vigo (Campus Ourense), As Lagoas, 32004 Ourense, Spain, and Laboratory of Food Chemistry, Department of Agrotechnology and Food Sciences, Wageningen UniVersity, Bomenweg 2, 6703 HD, Wageningen, The Netherlands
In order to obtain pectin-derived oligosaccharides (mainly arabinooligosaccharides and oligogalacturonides), samples of sugar beet pulp (SBP) were subjected to hydrothermal processing under nonisothermal conditions. Experiments carried out to reach temperatures in the range 160-175 °C (corresponding to values of the severity factor R0 in the range 287-835 min) led to comparatively high concentrations of both oligogalacturonides and arabinooligosaccharides. When SBP was treated to achieve a maximum temperature of 160 °C (R0 ) 287 min) or 163 °C (R0 ) 357 min), the overall amount of oligomers present in the reaction liquors recovered by pressing accounted for 31.2 and 29.9 g/100 g of oven-dried SBP, respectively, with a limited amount of nonvolatile impurities (about 0.15 g/g of oven-dried matter) and a mass ratio of arabinooligosaccharides/oligogalacturonides of about 1:1. Spent solids were washed with water, and the washing liquors were assayed for oligomers. Recovery of washing liquors would increase the overall yield process by 10%, to reach near 33 g/100 g of oven-dried SBP. Washed spent solids (with increased cellulose content) were obtained as a process byproduct. 1. Introduction 1
According to Roberfroid, a prebiotic is “a selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microflora, that confer benefits upon host well-being and health”. The positive health effects achieved by prebiotics include (i) modification of the intestinal flora, leading to increased proportions and/or activity of healthy microorganisms; (ii) reinforcement of the immune system; (iii) decreased binding of patogenic microorganisms to the intestines; (iv) regulation of the bowel habit, with decreased constipation or duration of diarrhea periods; and (v) reduction of the cardiovascular risk, osteoporosis, and colorectal cancer.2-5 Nondigestible oligosaccharides (NDO) are the most known prebiotics.6 Oligosaccharides with prebiotic properties [including inulin, fructooligosaccharides (FOS), galactooligosaccharides (GaOS), and lactulose] are commercially available, and many others are under study. The interest of consumers in healthy foods fosters the search for new raw materials, production technologies, and products with new or more intense properties.7-9 Pectin-derived oligosaccharides (POS) are promising candidates for prebiotic properties. Sugar beet pulp (SBP) is a byproduct of the sugar industry abundant in Europe, Japan, and the United States.10 In Spain, 1 million of metric tons of SBP were produced in 2003, which were mainly employed for feed formulation. However, SBP has a high content of pectin, a complex polysaccharide mainly made up of three structural polymers: homogalacturonan (HG) and rhamnogalacturonans I and II (RGI and RGII). HG, the most abundant pectic polysaccharide, is made up of a backbone of galacturonic acid units linked R-(1,4), which can be partially esterified with methyl groups on C-6 and acetylated on O-2 and/ or O-3. The second most abundant polymer in pectins is RGI, made up of chains with alternating units of galacturonic acid and rhamnose, having branched arabinan, galactan, or even arabinogalactan chains. In this polymer, the galacturonic acid * To whom correspondence should be addressed. Fax: +34 988 38 70 01. E-mail:
[email protected]. † University of Vigo (Campus Ourense). ‡ Wageningen University.
units can be acetylated or methyl esterified. RGII is a polysaccharide made up of galacturonic acid, rhamnose, galactose, and unusual neutral sugars.11 According to the SBP composition, this feedstock could be a suitable starting material for obtaining a variety of NDO with biological activity, such as oligogalacturonides (OGaU), arabinooligosaccharides (AOS), and GaOS. Literature on the manufacture and evaluation of prebiotic effects of pectin-derived oligomers has been reported. Al-Tamimi et al.10 considered the in vitro fermentation of arabinan and AOS obtained by enzymatic hydrolysis of arabinan isolated from SBP by chemical processing and concluded that AOS have prebiotic potential. Mandalari et al.12 assessed the prebiotic effects of oligosaccharide-rich extracts obtained by enzymatic hydrolysis of bergamot peel, which exhibited potential as prebiotics. Hotchkiss et al.13 studied the in vitro fermentation of pectin oligosaccharides from Valencia oranges (mainly made up of arabinogalactan pectic side chains and xyloglucan) and concluded that they showed bifidogenic effects, as revealed by the increases in acetate, butyrate, and propionate concentrations upon fermentation. The above-mentioned studies employed POS obtained by twostage processing: pectin extraction (aided by externally added chemicals) and enzymatic hydrolysis with pectinases. Alternatively, the breakdown of pectic polymers to give soluble hydrolysis products can be achieved by treatments with hot, compressed water (hydrothermal or autohydrolysis treatments), a technology showing favorable features, such as (i) environmentally friendly character (water and feedstock are the only reagents), (ii) the ability to generate oligosaccharides in a single stage at favorable yields, (iii) faster reaction than enzymatic hydrolysis, and (iv) the spent solids from the process are enriched in cellulose, showing enhanced properties for their further benefit. On the other hand, a variety of processes (including carbohydrate decomposition, extractive removal, and lignin solubilization) result in the presence of undesired compounds in liquors,14 whose amount can be minimized by optimizing the operational conditions. This work deals with the experimental assessment of sugar beet pectin conversion into oligomers in aqueous media. The effects of treatment severity on both pulp solubilization and
10.1021/ie8017753 CCC: $40.75 2009 American Chemical Society Published on Web 03/26/2009
4682
Ind. Eng. Chem. Res., Vol. 48, No. 10, 2009
liquor composition (incluing oligomers, monomers, and nonvolatile impurities) were established. 2. Materials and Methods 2.1. Raw Material. SBP was provided by a local pulp factory (Azucarera Ebro), homogenized in a single lot to avoid compositional differences among aliquots, and stored in polyethylene bags at -18 °C until use. 2.2. Analysis of the Raw Material. Aliquots from the homogenized SBP lot were dried at 50 °C, milled to a particle size lower than 0.5 mm, and subjected to quantitative acid hydrolysis with 72% sulfuric acid followed by a quantitative posthydrolysis step (following the TAPPI T13m method). A sample of the liquid phase was assayed by HPAEC-PAD using a Dionex instrument (Dionex, Sunnyvale, CA). Separation of carbohydrates was carried out with a CarboPac PA-1 (4 mm × 250 mm) in combination with a CarboPac PA-1 guard column (4 mm × 50 mm) maintained at 30 °C and pulsed amperometric detection. The mobile phases were degassed with helium. Analyses were performed using a gradient of deionized water (eluent A), 200 mM sodium hydroxide (eluent B), and 2 M sodium acetate in 200 mM sodium hydroxide (eluent C). The total analysis time was 45 min. This method allowed the determination of glucose, galactose, xylose, rhamnose, and arabinose. For simplicity, the results are reported as glucan, galactan, xylan, rhamnosyl substituents, mannan and arabinan. Another sample of liquors was assayed for acetic acid using an Agilent HPLC fitted with a refractive index detector and an Aminex HPX-87H column (supplied by BioRad). The mobile phase (0.005 N H2SO4) was eluted at a flow rate of 0.6 mL/ min at 60 °C.15 The oven-dried weight of the solid phase from quantitative acid hydrolysis measured the content of Klason lignin. Uronic acids were determined by the method of Blumenkrantz and Asboe-Hansen,16 using galacturonic acid as a standard for quantification. Neutral-detergent fiber (NDF), acid-detergent fiber (ADF), and acid-detergent lignin (ADL) were determined according to the methods of Goering and Van Soest.17 Elemental nitrogen was determined with a Thermo Finnegan Flash EATM 1112 analyzer, using 130 and 100 mL/min of He and O2 and an oven temperature of 50 °C. Protein content was obtained by multiplying the elemental N content by 6.25. All determinations were made in triplicate. Moisture and ash were determined according to the methods ISO 638 and ISO 776, respectively. 2.3. Autohydrolysis Processing of Sugar Beet Pulp. SBP samples and water were mixed at the desired proportions, reacted in a 3.75 L stainless steel Parr reactor under nonisothermal conditions at a liquor-to-solid ratio of 12 kg/kg (oven-dried basis), and heated to the desired temperature (140, 150, 160, 163, 165, 167, 170, 180, 190, or 200 °C). The temperature profile followed during the heating and cooling process of the reactor is shown in Figure 1. In order to facilitate the comparisons between different reactors, the combined effects of time and temperature caused by the nonisothermal treatments were measured in terms of the severity factor (R0), defined as R0 )
- 100 ∫ exp[ T(t)14.75 ] dt t
0
(1)
where t is the time needed for achieving the desired temperature. At the end of treatments, liquors were separated by pressing (using a Enerpac P142 press operating at a presure of 10 kPa). Both spent solids and autohydrolysis liquors were quantified and analyzed as described below.
Figure 1. Temperature profile of the hydrothermal processing.
2.3.1. Analysis of Spent Solids from Treatments. Spent solids were washed with distilled water at a liquid to solid mass ratio of 2 kg/kg (oven-dried, native sugar beet pulp basis), pressed to recover the washing liquors, and air-dried. Washing liquors and solid residues were quantified and assayed for composition. Samples of dried spent solids were milled to a particle size below 0.5 mm and analyzed using the same methodology described for the raw material. Liquors from the quantitative acid hydrolysis were analyzed for sugars and acetic acid by HPLC by the method described above.15 In HPLC chromatograms, galactose, xylose, mannose, and rhamnose were eluted together. For simplicity, as the concentration of galactose is higher than that of the coeluted sugars (see compositional data of the raw material), the overall peak was quantified as galactose. Consequently, HPLC data are reported in terms of glucan, galactan, arabinan, and acetyl groups. Uronic acids and nitrogen were determined using the methods described in section 2.2. 2.3.2. Analysis of Authohydrolysis Liquors. Samples of reaction liquors were filtered through 0.45 µm membranes and analyzed by HPLC for monosaccharides, furfural, hydroxymethylfurfural (denoted HMF), formic acid, and acetic acid. Another sample was subjected to quantitative posthydrolysis and analyzed by HPLC for acetyl groups. Nonvolatile compounds (NVC) were measured by oven drying at 105 °C until constant weight. Determination of oligogalacturonides (OGaU) by the m-hydroxydiphenyl method16 was hindered by interferences with reaction byproducts generated in the strongly acidic medium required by the reagent. Because of this, OGaU in autohydrolysis liquors were treated with an enzymatic concentrate rich in endopolygalacturonase (“Viscozyme 1.5L” from Aspergillus aculeatus, kindly supplied by Novozymes, Madrid, Spain), to yield monogalacturonic acid, which was determined by HPLC using the same method employed for monosaccharide and acetic acid quantification. Polygalacturonase (PGase) activity was determined by measuring the rate of D-galacturonic acid formation from 0.5% w/v polygalacturonic acid (Sigma Chemical). One unit of enzymatic activity (U) is defined as the amount of enzyme catalyzing the formation of 1 µmol of D-galacturonic acid per min at 37 °C and pH 5. Enzymatic assays were carried out at 37 °C for 40 h in Erlenmeyer flasks with orbital agitation (150 rpm) at an enzyme loading of 45 U/g of liquor. Sodium acetate buffer (50 mM) was employed to keep the pH at 5. In enzymatic assays, AOS, GaOS and glucooligosaccharides (GOS) present in liquors were also quantitatively hydrolyzed, as confirmed by chemical posthydrolysis in media catalyzed with sulfuric acid. The contents of oligomers (AOS, GaOS, OGaU,
Ind. Eng. Chem. Res., Vol. 48, No. 10, 2009 Table 1. Composition of the Raw Material fraction
wt %, oven-dried basis
glucan galactan xylan rhamnosyl moieties mannan arabinan acetyl groups galacturonan Klason lignin neutral-detergent fiber (NDF) acid-detergent fiber (ADF) acid-detergent lignin (ADL) ashes protein (from nitrogen determination)
20.1 5.3 1.0 1.4 1.1 17.5 2.6 21.0 4.8 60.0 24.5 3.4 4.5 10.8
and GOS) were calculated on the basis of the increase in each monomer concentration (arabinose, galactose, galacturonic acid, and glucose, respectively) obtained upon quantitative hydrolysis of liquors. The composition of washing liquors was assayed by the same methods employed for autohydrolysis liquors. 3. Results and Discussion 3.1. Composition of the Raw Material. Table 1 lists compositional data of the SBP lot employed in this study. Galacturonan, glucan, and arabinan were the major components of the raw material, followed by galactan, rhamosyl moieties, mannan, and xylan. These results are in good agreement with the ones reported by Bertin et al.18 Additional characterization of the raw material was carried out by fiber analysis, which led to contents of neutral-detergent fiber, acid-detergent fiber and acid-detergent lignin of 60, 24.5, and 3.4 wt % (oven-dried basis), respectively. From these data, the estimated cellulose content was 21.1%. These results are in the range reported by other studies.19 A comparison between the results of Table 1 and the fiber analysis data confirms that the glucan present in the raw material corresponded to cellulose. The results obtained for the contents of protein and ashes (10.8 and 4.5 wt %) are also in agreement with the results reported by FEDNA.19 3.2. Effect of the Hydrothermal Processing on Raw Material Solubilization. Samples from the homogeneized feedstock lot were subjected to hydrothermal processing under nonisothermal conditions (following the heating profile shown in Figure 1) to reach maximal temperatures in the range 140200 °C. Once the target temperatures were reached, the media were cooled, and the solids were recovered, washed, and ovendried to measure the weight loss caused by treatments. Table 2 lists data on the temperature dependence of SBP weight loss. The degree of solubilization increased with temperature, particularly in the range 140-167 °C (where the solubilization percentage increased from 24.4 to 57.6 wt %). The rate of weight loss became slower above this latter temperature. Under the severest conditions assayed, 63.9% of the initial mass was solubilized. The spent solids obtained under these conditions were mainly made up of cellulose and lignin, which are less susceptible to the hydrothermal processing than other polysaccharides.20,21 3.3. Effects of the Hydrothermal Processing on the Composition of Spent Solids. Hydrothermal processing causes a variety of effects, including removal of extractives and acidsoluble lignin, as well as hydrolytic breakdown of hemicelluloses22 and polygalacturonic acid.23 Depending on the operational conditions, the predominant reaction products from hemicelluloses and polygalacturonic acid can be high-molecular weight compounds, oligomers, monomers, or decomposition products.
4683
Table 2 shows compositional data of the various spent solids obtained in this work. As expected, harsher operational conditions resulted in spent solids having increased cellulose content. From the experimental data, the solubilization percentage (SP) of a given fraction (F-fraction) was expressed in terms of the F-fraction solubilization percentage (FSP), defined as FSP ) 100 -
FC × SY FCRM
(2)
where the F-fraction refers to glucan, arabinan, galactan, galacturonan, acetyl groups, or protein; FC and FCRM are the F-fraction contents of the spent solids and raw material, respectively; and SY is the solid yield (defined as the percentage of recovered solid respect the raw material weight, oven-dried basis). The FSP of glucan varied in a narrow range, with an average value of 25%. The percentage of glucan retained in spent solids (about 75%) compares favorably with the results reported for other substrates such as corncobs (for which 54.2-60.5% of glucan recovery has been reported).24 The contents of arabinan (An), galactan (Gan), galacturonan (GaUn), and acetyl groups (AcO) decreased continuously with the severity of treatments, owing to the susceptibility of these components to hydrolytic degradation. This behavior was more pronounced in the temperature range 140-170 °C. Operating at 170 °C (R0 ) 588 min), An was almost completely removed from the solid phase, whereas FSP values of 88.1, 84.5, and 70.3% were determined for GaUn, AcO, and Gan, respectively. In the treatment at 200 °C (R0 ) 4460 min), FSP values of 95.4, 88.3, and 80% were obtained for GaUn, AcO, and Gan, respectively. According to these results, An showed the highest susceptibility toward hydrolytic degradation, whereas Gan showed the highest resistance, particularly under harsh operational conditions. Even though protein was solubilized in treatments (with FSP in the range 20.6-54.4%, based on nitrogen determinations), the concomitant solid solubilization results in the production of spent solids whose nitrogen content increased with temperature (up to 16 wt % or protein equivalent, in comparison with 10.8 wt % for the raw material). The protein solubilization values determined in this work are in the range reported for the hydrothermal processing of rice husks (43-51%).25 According to the above data, hydrothermal processing allows the hydrolytic breakdown of polysaccharides different from cellulose, leaving spent solids enriched in both cellulose and protein (which could be used for specific purposes). 3.4. Effect of the Operational Conditions on the Liquor Composition. SBP shows the ability to retain remarkable amounts of water (up to 26.5 mL/g).18 Because of this, liquors were separated from spent solids by pressing instead of filtration. Compositional results concerning the composition of liquors are listed in the following sections. 3.4.1. Content of Nonvolatile Compounds. Autohydrolysis of SBP caused a variety of effects, including solubilization of pectin, hemicelluloses, and protein. Both soluble products and decomposition products are produced from these substrates, most of them of nonvolatile character (which can be quantified by oven-drying of liquors). The data in Figure 2, which shows the content of nonvolatile components (NVC) as a function of temperature, present a maximum (0.0498 g NVC/g liquor) at 170 °C (R0 ) 588 min). At lower temperatures, the major contribution to NVC corresponded to the solubilization of the above-mentioned fractions, whereas the decomposition reactions (for example, monosaccharides to aldehydes) were predominant
4684
Ind. Eng. Chem. Res., Vol. 48, No. 10, 2009
Table 2. Pulp Solubilization (expressed as percentage of dry raw material) and Chemical Composition of Spent Solids (expressed as percentage of the oven-dried, treated solid) at Various Maximal Temperatures
pulp solubilization glucan galactana arabinan acetyl groups galacturonan protein others a
140 °C
150 °C
160 °C
163 °C
165 °C
167 °C
170 °C
180 °C
190 °C
200 °C
24.4 21.2 7.5 16.1 2.1 17.7 11.4 24.1
40.3 25.5 8.1 12.8 1.7 14.1 13.1 24.9
49.8 29.6 7.3 8.2 1.3 8.4 15.7 29.6
53.7 31.6 7.4 8.3 1.1 8.6 15.8 27.3
55.2 33.7 8.1 7.1 1.3 8.6 15.7 25.5
57.6 33.9 7.0 4.7 1.0 8.1 16.7 28.7
58.4 33.2 6.2 2.7 1.0 6.0 16.6 34.3
61.3 37.9 7.1 3.3 1.1 5.7 16.4 28.6
62.9 45.1 5.7 0.6 1.1 2.9 14.9 29.7
63.9 44.2 4.8 0.3 0.8 2.7 13.7 33.5
Includes minor amounts of mannose, rhamnose, and xylose.
Figure 2. Effect of temperature on the NVC and ONVC contents of reaction liquors.
at temperatures higher than 170 °C. Under the severest conditions assayed, the NVC content of liquors reached 0.0369 g/g of liquor. 3.4.2. Content of Oligomers and Acetyl Groups. Operating under suitable autohydrolysis conditions, polysaccharides can be principally converted into high-molecular weight material and oligomers. In this work, as a consequence of the methodology employed, the oligomeric and polymeric components present in the autohydrolysis liquors have been quantified as monomers, and their concentrations are expressed in terms of “oligomers” independently from both the molecular weight and the backbone they are linked to. Figure 3 shows the temperature dependence of the composition of polysaccharide-derived products present in autohydrolysis liquors. As it can be seen in Figure 3a, the major components of liquors corresponded to An and GaUn hydrolysis products. At low temperatures, the concentrations of OGaU were higher than those of AOS, probably due to the presence of small molecules in the raw material. However, the slope of the curve corresponding to AOS was sharper, revealing a faster generation in the autohydrolysis media, which makes them the most abundant components at temperatures higher than 155 °C. Temperatures in the range 150-175 °C (R0 ) 138-835 min) led to comparatively high concentrations of both OGaU and AOS, suggesting that both fractions could be solubilized at high yields simultaneously. The maximum concentration of AOS (15.1 g/L, corresponding to 88% conversion of the initial An into soluble products) was achieved at 167 °C (R0 ) 475 min). These data compare favorably with the results reported for other substrates such as
Eucalytus globulus, for which 13.4 g/L of xilooligosaccharides were obtained at 196 °C (R0 ) 3190 min).26 Harsh conditions resulted in decreased concentrations of both fractions, with a more marked drop in the case of AOS, which confirmed the easier decomposition of arabinose-containing oligomers. A comparison of data in Figure 3a,b confirms that arabinose was the major product from AOS decomposition, even if experiments carried out under harsh conditions resulted in arabinose decomposition: up to 38% of the potential arabinose present in the raw material in the experiment carried out at 200 °C (R0 ) 4460 min). On the other hand, material balances also confirmed the generation of decomposition products from OGaU and their hydrolysis products, a finding in agreement with literature data.23,28 In addition, the OST to monosaccharide ratio (which measures the process selectivity) reached 7 g/g at 160 °C, a result higher than the ones reported for Eucalyptus wood (OST/monosaccharides ) 5)14 and barley wastes from malting industries (OST/ monosaccharides ) 6).20 Table 3 shows data on the concentrations of volatile products (acetic acid, formic acid, furfural, and HMF) present in liquors from the various treatments. Acetic acid, generated from acetyl groups of hemicelluloses and pectin, was the most abundant volatile reaction product, followed by formic acid and saccharide-decomposition products (HMF and furfural). Oligomers made up of structural units different from galacturonic acid and arabinose (denoted GaOS, owing to their higher proportion of galactose) were also present in liquors, at concentrations remakable lower than those of OGaU and AOS.
Ind. Eng. Chem. Res., Vol. 48, No. 10, 2009
4685
Figure 3. Effect of temperature on the composition of reaction liquors: (a) oligomers and (b) monosaccharides. Table 3. Other Products Generated in the Reaction Media (expressed as g of product/L of reaction liquors) at Various Maximal Temperatures fraction
140 °C
150 °C
160 °C
163 °C
165 °C
167 °C
170 °C
180 °C
190 °C
200 °C
acetic acid HMF furfural formic acid
0.1 0.0 0.0 0.0
0.2 0.0 0.0 0.0
0.4 0.0 0.0 0.0
0.4 0.0 0.0 0.0
0.5 0.0 0.0 0.4
0.6 0.0 0.0 0.4
0.8 0.1 0.0 0.5
1.4 0.1 0.0 0.8
1.9 0.3 0.1 0.7
2.3 0.5 0.5 0.8
The data in Figure 3a confirmed that GaOS behaved as reaction intermediates, reaching a maximum concentration (3 g/L) at an intermediate temperature (160 °C or R0 ) 287 min), where generation and decomposition are balanced. Harsher temperatures resulted in GaOS decomposition and in flat galactose profiles, confirming the similar contribution of galactose generation from oligomers and galactose decomposition into HMF. Under the severest operational conditions assayed, the decreased concentration of galactose was due to the higher incidence of decomposition reactions. Many of the properties and biological functions of pectins are believed to be determined by ionic interactions between homogalacturonans.29 In comparison with other pectins, SBP
pectin is rich in acetyl groups.30 Owing to this, autohydrolysis results in acetylated oligomers, whose properties are affected by the substitution pattern. Figure 3a shows the temperature dependence of the concentration of acetyl groups linked to oligomers (AcO) in the reaction media. The variation pattern is similar to the one observed for OGaU and can be interpreted on the basis of the simultaneous effects caused by (i) generation of acetylated oligosaccharides from pectin and (ii) acetyl group hydrolysis to give acetic acid. 3.4.3. Content of Non-Saccharide, Nonvolatile Products. The difference between the concentrations of nonvolatile components and the total saccharide concentrations (including
4686
Ind. Eng. Chem. Res., Vol. 48, No. 10, 2009
Table 4. Material Balances and Conversion Yields (a) Reaction Liquors (data based on 100 kg of oven-dried sugar beet pulp) T (°C) 140 150 160 163 165 167 170 180 190 200
recovered liquors (kg)
liquor recovery after pressing (%)
Glc
Gal
Ara
GOS
GaOS
AOS
AcO
OGaU
677.0 763.6 935.6 907.8 949.0 965.2 983.0 1002.0 1015.0 1025.0
55.3 61.6 74.9 72.4 75.6 76.8 78.1 79.4 80.4 81.1
0.15 0.10 2.11 2.14 2.41 2.66 2.70 2.87 2.66 2.09
0.45 0.15 2.13 2.21 2.58 2.87 3.06 3.20 3.30 2.93
0.06 0.20 0.29 0.36 0.57 0.77 0.99 2.72 6.36 9.28
0.50 0.52 1.47 1.47 1.38 1.39 1.41 1.29 1.12 0.40
0.43 1.18 2.83 2.69 2.73 2.73 2.80 2.33 2.46 1.40
3.48 7.88 12.95 13.11 14.22 14.60 14.67 13.23 9.01 1.78
0.93 1.41 1.89 1.87 1.93 1.96 1.93 1.49 0.94 0.41
7.46 10.01 12.06 10.77 10.08 9.58 8.84 4.37 1.45 0.71
(b) Washing Liquors (data based on 100 kg of oven-dried sugar beet pulp) T (°C) 140 150 160 163 165 167 170 180 190 200
recovered liquors (kg)
Glc
Gal
Ara
GOS
GaOS
AOS
AcO
OGaU
188.5 196.0 195.0 238.4 196.4 227.0 191.0 182.0 196.0 201.0
0.03 0.02 0.22 0.32 0.27 0.30 0.25 0.22 0.24 0.21
0.08 0.03 0.29 0.32 0.26 0.33 0.27 0.24 0.29 0.27
0.01 0.03 0.03 0.05 0.07 0.09 0.09 0.20 0.57 0.89
0.00 0.02 0.24 0.22 0.18 0.20 0.14 0.12 0.12 0.14
0.00 0.11 0.38 0.43 0.36 0.40 0.33 0.30 0.41 0.45
0.29 0.88 1.02 1.45 1.32 1.40 1.13 0.95 0.84 0.28
0.04 0.15 0.14 0.21 0.10 0.19 0.12 0.08 0.08 0.06
0.50 0.77 0.97 1.14 0.90 0.95 0.75 0.32 0.13 0.07
(c) Conversion Yields (expressed as g of monomer equivalents/100 g of monomer equivalents in raw material)
glucan conversion into Glc galactan conversion into Gal arabinan conversion into Ara glucan conversion into GOS galactan conversion into GaOS arabinan conversion into AOS acetyl groups conversion into AcO galacturonan conversion into OGaU
140 °C
150 °C
160 °C
163 °C
165 °C
167 °C
170 °C
180 °C
190 °C
200 °C
0.77 5.31 0.32 2.23 4.37 18.94 26.95 34.71
0.53 1.77 1.17 2.42 13.24 44.07 43.05 46.97
10.46 24.07 1.62 7.67 32.87 70.21 56.01 56.77
11.00 25.94 2.05 7.56 31.88 73.21 57.43 51.91
12.00 29.00 3.19 7.00 31.64 78.14 55.82 47.85
13.25 32.75 4.33 7.14 31.95 80.48 59.32 45.92
13.21 34.04 5.46 6.96 31.97 79.41 56.59 41.80
13.83 35.22 14.69 6.31 26.94 71.30 43.24 20.45
12.95 36.71 34.87 5.56 29.33 49.50 27.97 6.88
10.26 32.72 51.14 2.40 18.90 10.33 12.86 3.38
oligo- and monosaccharides) gives an estimate of the nonsaccharide, nonvolatile products (here denoted as “other nonvolatile compounds”, ONVC) present in liquors. This fraction includes extractives, extracted protein and protein-derived compounds (particularly, Maillard reaction products), acidsoluble lignin, soluble mineral components, etc. Figure 2 shows the dependence of ONVC profiles, expressed as mass fractions with respect to NVC. At temperatures below 160 °C (R0 ) 287 min), ONVC accounted for about 0.15 g/g of NVC and increased markedly under harsher conditions. This increase was partially due to the presence of nitrogen-containing compounds in liquors, since the “protein equivalent” contents (based on elemental nitrogen determination) increased from 0.63 g/L (at 140 °C) up to 2.92 g/L (at 180 °C). The ONVC content of liquors obtained at temperatures below 160 °C compares favorably with those reported by Vegas et al. using barley industrial wastes (0.29 g/g of NVC)20 or rice husks (0.32 g/g of NVC).27 Finally, an oligosaccharide to nonvolatile impurities ratio of 5 g/g was observed for liquors obtained at 160 °C, which compares well with the values of 2.08 and 1.98 that were reported by Vegas et al. for other substrates.20,27 3.5. Material Balances. Table 4 shows data concerning the amount of autohydrolysis liquors recovered in the pressing stage, and the amounts of valuable products present in them (expressed as g/100 g of oven-dried SBP). The amount of liquors recovered increased with the severity of treatments (from 55% of the overall liquid phase in the experiment carried out at 140 °C (R0 ) 53 min) up to about 80% in treatments at temperatures in the range 170-200 °C (R0 in the range 588-4460 min). This behavior is directly related to the compositional changes
experimented by the solid phase along the autohydrolysis reaction: at 140 °C, most of the initial pectin (a fraction with high ability for retaining water) remained in the solid phase, but it was removed in the experiment at 200 °C. Table 4c lists the conversion yields achieved for each reaction product and polymer. The results determined for the arabinan conversion into arabinooligosaccharides (70.2%) and galacturonan conversion into oligogalacturonides (56.7%) were in the range of reported data.14,20 A comparative analysis of the operational conditions employed in this work leads to the conclusion that the most favorable operation corresponded to nonisothermal processing up to 160-163 °C (R0 in the range 287-357 min), conditions under which the overall amount of oligomers present in the raw liquors recovered by pressing accounted for 31.2 and 29.9 g/100 g of oven-dried SBP, respectively, with a limited amount of ONVC (about 0.15 g/g of NVC) and a mass ratio AOS/OGaU close to 1:1. In order to increase the process yield, spent solids were washed with water at a liquor to solid ratio of 2:1 g/g of raw material and separated by pressing. Table 4b lists the corresponding results, which show that the product recovery can be increased by about 10% for treatments carried out at intermediate severities, with an overall product yield of about 33 g/100 g of oven-dried SBP. 4. Conclusions In order to obtain pectin-derived oligosaccharides (mainly AOS and OGaU) suitable to be used as prebiotic food ingredients, samples of sugar beet pulp were subjected to
Ind. Eng. Chem. Res., Vol. 48, No. 10, 2009
hydrothermal processing under nonisothermal conditions up to maximal temperatures in the range 140-200 °C (R0 in the range 53-4460 min). Treatments with maximal temperatures in the range 160-175 °C (R0 ) 287-835 min) led to comparatively high concentrations of both OGaU and AOS, suggesting that both fractions could be solubilized at high yields simultaneously. When SBP was treated at 160 °C (R0 ) 287 min) or 163 °C (R0 ) 357 min), the overall amount of oligomers present in the reaction liquors recovered by pressing accounted for 31.2 and 29.9 g/100 g of oven-dried SBP, respectively, with a limited amount of undesired nonsaccharide products (about 0.15 g/g of NVC) and a mass ratio AOS/OGaU close to 1:1. The recovery of liquors from solid washing allowed us to increase the product yield by about 10%. The experimental data show that pectic oligomers (for which prebiotic properties have been reported) can be obtained from SBP at high yield (about 33 g/100 g of oven-dried SBP). In addition, spent solids from hydrothermal processing and washing show increased contents of both cellulose and protein and could be used for specific applications in other fields. Nomenclature AcO ) acetyl groups AOS ) arabinooligosaccharides Ara ) arabinose An ) arabinan FOS ) fructooligosaccharides FSP ) F-fraction solubilization percentage Gal ) galactose Gan ) galactan GaUn ) galacturonan GaOS ) galactooligosaccharides Glc ) glucose GOS ) glucooligosaccharides HMF ) hydroxymethylfurfural NVC ) nonvolatile compounds OGaU ) oligogalacturonides ONVC ) other nonvolatile compounds R0 ) severity factor SBP ) sugar beet pulp
Acknowledgment Authors are grateful to the Ministry of Science and Innovation of Spain (Project ref: CTQ2008-05322/PPQ, partially funded by the FEDER funds of the European Union) for the financial support of this work. Literature Cited (1) Roberfroid, M. Prebiotics: The Concept Revisited. J. Nutr. 2007, 137, 830S. (2) Swennen, K.; Courtin, C. M.; Delcour, J. A. Non-Digestible Oligosaccharides with Prebiotic Properties. Crit. ReV. Food Sci. Nutr. 2006, 46, 459. (3) Tuohy, K. M.; Rouzaud, G. C. M.; Bru¨ck, W. M.; Gibson, G. R. Modulation of the Human Gut Microflora towards Improved Health Using Prebiotics. Assessment of Efficacy. Curr. Pharm. Des. 2005, 11, 75. (4) Roberfroid, M. B. Prebiotics and Probiotics: Are they Functional Foods? Am. J. Clin. Nutr. 2000, 71, 1682S. (5) Bamba, T.; Kanauchi, O.; Andoh, A.; Fujiyama, Y. A New Prebiotic from Germinated Barley for Nutraceutical Treatment of Ulcerative Colitis. J. Gastroenterol. Hepatol. 2002, 17, 818. (6) Gibson, G. R.; Ottaway, P. B.; Rastall, R. A. In Prebiotics: New DeVelopments in Functional Foods; Chandos Publishing Limited: Oxford, UK, 2000. (7) Menne, E.; Guggenbuhl, N.; Roberfroid, M. Fn-type Chicory Inulin Hydrolysate Has a Prebiotic Effect in Humans. J. Nutr. 2000, 130, 1197.
4687
(8) Rao, V. A. The Prebiotic Properties of Oligofructose at Low Intake Levels. Nutr. Res. (N.Y.) 2001, 6, 843. (9) Tuohy, K. M.; Ziemer, C. J.; Klinder, A.; Kno¨bel, Y.; Pool-Zobel, B. L.; Gibson, G. R. A Human Volunteer Study To Determine the Prebiotic Effects of Lactulose Powder on Human Colonic Microbiota. Microb. Ecol. Health Dis. 2002, 14, 165. (10) Al-Tamimi, M. A. H. M.; Palframan, R. J.; Cooper, J. M.; Gibson, G. R.; Rastall, R. A. In vitro Fermentation of Sugar Beet Arabinan and Arabino-oligosaccharides by the Human Gut Microflora. J. Appl. Microbiol. 2006, 100, 407. (11) Ralet, M. C.; Cabrera, J. C.; Bonnin, E.; Que´me´ne´r, B.; Hellı´n, P.; Thibault, J. F. Mapping Sugar Beet Pectin Acetylation Pattern. Phytochemistry 2005, 66, 1832. (12) Mandalari, G.; Nueno Palop, C.; Tuohy, K.; Gibson, G. R.; Bennett, R. N.; Waldron, K. W.; Bisignano, G.; Narbad, A.; Faulds, C. B. In Vitro Evaluation of Prebiotic Activity of a Pectic Oligosacharide-Rich Extract Enzymatically Derived from Bergamot Peel. Appl. Microbiol. Biotechnol. 2007, 73, 1173. (13) Hotchkiss, A. T. J. R.; Manderson, K.; Tuohy, K. M.; Widmer, W. W.; Nunez, A.; Gibson, G. R.; Rastall, R. A. Bioactive Properties of Pectic Oligosaccharides from Sugar Beet and Valencia Oranges. Proceedings from the 233rd ACS National Meeting; Chicago, IL, March 25-29, 2007. (14) Va´zquez, M. J.; Garrote, G.; Alonso, J. L.; Domı´nguez, H.; Parajo´, J. C. Refining of Autohydrolysis Liquors for Manufacturing Xylooligosaccharides: Evaluation of Operational Strategies. Bioresour. Technol. 2005, 96, 889. (15) Gonza´lez-Mun˜oz, M. J.; Domı´nguez, H.; Parajo´, J. C. Depolymerization of Xylan-Derived Products in an Enzymatic Membrane Reactor. J. Membr. Sci. 2008, 320, 224. (16) Blumenkrantz, N.; Asboe-Hansen, G. New Method for Quantitative Determination of Uronic Acids. Anal. Biochem. 1973, 54, 484. (17) Goering, H. K.; Van Soest, P. J. Forage Fiber Analysis. In Agricultural Handbook 379; Agricultural Research Service, U.S. Dept. of Agriculture: Washington, DC, 1970; pp 5-11. (18) Bertin, C. H.; Rouau, X.; Thibault, J. F. Structure and Properties of Sugar Beet Fibres. J. Sci. Food Agric. 1988, 44, 15. (19) FEDNA: Spanish Federation for the Animal Nutrition Development, available at http://www.etsia.upm.es/fedna/subp_humedos/remolacha_pulpa.htm. (20) Vegas, R.; Alonso, J. L.; Domı´nguez, H.; Parajo´, J. C. Manufacture and Refining of Oligosaccharides from Industrial Solid Wastes. Ind. Eng. Chem. Res. 2005, 44, 614. (21) Garrote, G.; Kabel, M. A.; Schols, H. A.; Falque´, E.; Domı´nguez, H.; Parajo´, J. C. Effects of Eucalyptus globulus Wood Autohydrolysis Conditions on the Reaction Products. J. Agric. Food Chem. 2007, 55, 9006. (22) Garrote, G.; Domı´nguez, H.; Parajo´, J. C. Production of Substituted Oligosaccharides by Hydrolytic Processing of Barley Husk. Ind. Eng. Chem. Res. 2004, 43, 1608. (23) Miyazawa, T.; Funazukuri, T. Hydrothermal Production of Mono(galacturonic acid) and the Oligomers from Poly(galacturonic acid) with Water under Pressures. Ind. Eng. Chem. Res. 2004, 43, 2310. (24) Garrote, G.; Ya´n˜ez, R.; Alonso, J. L.; Parajo´, J. C. Coproduction of Oligosaccharides and Glucose from Corncobs by Hydrothermal Processing and Enzymatic Hydrolysis. Ind. Eng. Chem. Res. 2008, 47, 1336. (25) Vegas, R.; Kabel, M.; Schols, H. A.; Alonso, J. L.; Parajo´, J. C. Hydrothermal Processing of Rice Husks: Effects of Severity on Product Distribution. J. Chem. Technol. Biotechnol. 2008, 83, 965. (26) Gullo´n, P.; Gonza´lez-Mun˜oz, M. J.; Domı´nguez, H.; Parajo´, J. C. Membrane Processing of Liquors from Eucalyptus globulus Autohydrolysis. J. Food Eng. 2008, 87, 257. (27) Vegas, R.; Luque, S.; Alvarez, J. R.; Alonso, J. L.; Domı´nguez, H.; Parajo´, J. C. Membrane-Assisted Processing of XylooligosaccharideContaining Liquors. J. Agric. Food Chem. 2006, 54, 5430. (28) Miyazawa, T.; Funazukuri, T. Production of Mono- and OligoSaccharides from Hydrothermal Degradation of Polygalacturonic Acid. In Hydrothermal Reactions and Techniques. Proceedings of the 7th International Symposium on Hydrothermal Reactions, Changchun, China. 14-18 December 2003; 2003. (29) Ridley, B. L.; O’Neill, M. A.; Mohnen, D. Pectins: Structure, Biosynthesis, and Oligogalacturonide-Related Signalling. Phytochemistry 2001, 57, 929. (30) Voragen, A. G. J.; Pilnik, W.; Thibault, J. F.; Axelos, M. A. V.; Renard, C. M. G. C. Pectins. Food Sci. Technol. 1995, 67, 287.
ReceiVed for reView November 19, 2008 ReVised manuscript receiVed February 25, 2009 Accepted February 25, 2009 IE8017753
