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发表于 2007-1-17 20:57
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|发表于：上海
今天就再发1/3好了，明天我再把后面的discussion部分传上来
Materials and methods
■ Starch sources
Lentil (Lens esculenta Medic.) seeds and cocoyam (Xanthosoma sagittifolium(L.) Schott.) roots were purchased from a local market in Caracas (Venezuela). Commercially isolated potato starch was purchased from Lyckeby Starkelsen (Kristianstad, Sweden).
■ Starch isolation
Starch was isolated as described elsewhere [14]. Briefly,decorticated lentil grains or peeled and diced cocoyam roots were homogenised in a liquidiser using one volume of water.The suspension was filtered through a 280-mesh cloth several times (usually 7 times), each time adding one volume of water, until no material passed through the cloth. The filtered material (essentially starch) was washed out with an equal volume of water,three or four times by centrifugation (1,500 g for 15 min), removing the lipid layer out of the surface in each centrifugation step until none appeared.Before the last washing,the pH was adjusted to neutrality.Then,the starch was dried in an oven at 45 °C for 24 h, sieved out(250 μm pore), and stored at room temperature. Additionally,lentil grains were steeped in 62.5mmol/l NaOH solution for 24 h at room temperature and washed out extensively afterwards, as a pretreatment to facilitate the peeling of the grains. Yield was 24 % and 45 % (d. b.) for lentil and cocoyam starches, respectively. It was low as the method used [14] was developed for the isolation of amaranth starch and no attempt to improve the yield for other sources was made because the emphasis was put on a high starch purity. The estimated starch content of the preparations used in this study was 94.7 % and 96.8 % for lentil and cocoyam, respectively [6].Additionally, the protein content was 0.24% for lentil [15] and 0.56% for cocoyam[16] starches. Therefore, the starting materials for the pyrodextrinisation step were mainly starch and the impact of other components, such as antinutritional factors,was most likely minor.
■ Starch pyrodextrinisation
Isolated starches (22 g) were sprayed with 0.5ml HCl(1.82 g acid/kg starch), mixed thoroughly and left overnight at room temperature. Then, they were heated at 140 °C for 3 h, milled and sieved through a 250 μm pore size mesh [17].
■ Starch pre-digestion
To remove the digestible starch, both native and pyrodextrinisedstarches were pre-digested using an Englyst starch kit (Englyst Carbohydrate Services Ltd,Cambridge,UK) with some protocol modifications. Briefly,0.8 g starch and 50 mg guar gum were weighed in a centrifuge tube, then 10 ml of 5 g/l pepsin suspension in 50 mmol/l HCl were added, vortex mixed and incubated for 30 min at 37 °C.To simulate small intestinal digestion,five glass balls and 10 ml 0.25 mol/l sodium acetate solution were added to each tube and they were shaken well by hand.After equilibrating at 37 °C, an enzyme mixture(pancreatin from porcine pancreas, amyloglucosidase from Aspergillus niger and invertase from yeast; 5ml)was added and incubated for 2 h in a shaking water bath(70 strokes/min).Then,the glass balls were taken out and the tubes were spun at 1,500 g for 5min. The non-digestiblefraction from the pre-digested native starches was recovered from the pellets, freeze-dried, pooled,milled in a mortar, and used for the fermentation experimentsThis fraction contained the resistant starch, as defined by Englyst et al. [18], and guar gum.In contrast, the non-digestible fraction from the predigested pyrodextrinised starches remained in the supernatant after centrifugation, due to their solubility in water.However, as the products of the pre-digestion step were also present in the supernatant, these were removed by dialysis (overnight dialysis against water at 4 °C, followed by five 1-hour repeat dialysis steps with stirring) using dialysis bags with small Mw cut off(2,000).Retentates were pooled, freeze-dried, milled in a mortar, and used for fermentation. This fraction contained the non-digestible component from the pre-digested pyrodextrin and the enzymes from the starch kit.As the SCFA profile was one of the variables studied, it was important to consider the effect of other fermentable substrates present in the system. Therefore,potato native and pyroconverted starches were fermented as described below, but without the pre-digestion step to look at the impact of the presence of either guar gum or the enzymes (from the starch kit) in the non-digestible fractions obtained from native or pyrodextrinised starches, respectively. There were no discernible effects of either component.
■ Fermentation of non-digestible fractions
Non-digestible fractions (and glucose, as a readily fermented control) were fermented according to Edwards et al. [19] in four to six independent determinations (actual sample size is given in Table 1). Faecal slurry was made using freshly voided faeces (within 45 min from evacuation),donated by healthy adults (34–36 years,one female), homogenised in 0.1mol/l Na,K-phosphate buffer, pH6.5 (pre-boiled, cooled and kept in an oxygen free nitrogen (OFN) atmosphere until used) using a liquidiser.Starch samples (100 mg for test cultures or none in case of control cultures) were suspended in 10 ml 160 g/l faecal slurry (previously filtered through a nylon stocking) in McCartney bottles (28 ml capacity). Each bottle was fitted with a holed, screw cap with a rubber lining to allow flushing of the culture with OFN before incubation.The bottles were incubated horizontally in a shaking water bath (50 strokes/min) at 37 °C for 24 h. After incubation, produced gas was released and measured using a calibrated syringe. A culture aliquot (2 ml) was used to measure pH and frozen at –20 °C for later SCFA analysis.The remaining slurry was boiled for 30 min and frozen to assess residual starch and carbohydrate.Time zero cultures were immediately boiled for 30 min and frozen to estimate total starch and carbohydrate.
■ Short-chain fatty acid assay
SCFA were estimated by gas liquid chromatography using a TRACE™ 2000 gas chromatograph (ThermoQuest Ltd,Manchester, UK) equipped with a flame ionization detector (250 °C) and using a Zebron ZB-Wax capillary column (15 m x 0.53 mm id x 1 μm film thickness),made of polyethylene glycol (catalogue No.7EK-G007-22,Phenomenex,Cheshire, UK). Nitrogen (30 ml/min) was used as the carrier gas. Internal standard solution(86.1 mmol/l 3-methyl-n-valeric acid, 0.1ml) and concentrated orthophosphoric acid (0.1 ml) were added to 0.8ml culture aliquots. The mixture was extracted three times with 3ml diethyl ether each time, centrifuged and the ether layers pooled. One microlitre of ether extract was automatically injected (230 °C, splitless) into the column.Then, the column temperature was held at 80 °C for 1 min, increased at 15 °C/min until 210 °C and held for 1min. The peak integrals were analysed using Chrom-Card 32-bit software version 1.07β5 (2000) by ThermoQuest (Milan, Italy) using an averaged (n=5)response factor for each external standard(166.5mmol/l acetic, 135.0mmol/l propionic,113.5 mmol/l isobutyric, 113.5 mmol/l n-butyric,97.9mmol/l isovaleric, 97.9mmol/l n-valeric,86.1 mmol/l n-hexanoic, 76.8 mmol/l heptanoic and 69.3 mmol/l n-octanoic acid solutions, pH8) as calibration method.All the standards were from Sigma-Aldrich Company Ltd. (Dorset, UK), except acetic acid glacial,which was from Fisher Scientific (Loughborough, UK).
■ Starch and carbohydrate analyses
Starch and carbohydrate were estimated both before(total) and after (residual) 24 h anaerobic incubation to quantify the degree of fermentation. Total starch was measured using the enzymatic procedure described by Englyst et al. [18].Residual starch was estimated according to Edwards et al. [19] based upon Englyst et al. [18].These two methods are essentially the same and quantify the starch content as glucose released after an in vitro simulation of both gastric and small intestine enzymatic digestion. The specificity of the Englyst method,however,underestimates the “starch” content in modified starches such as pyrodextrins because of the presence of atypical bonds that cannot be hydrolysed by the enzymes used in the method. Therefore, measurement of carbohydrate by a more general, chemical assay was necessary to estimate the degree of fermentation of the pre-digested pyrodextrins.Total and residual carbohydrates were estimated, by sampling from the same preparations made for total and residual starch analyses, using the anthrone-sulphuric acid method,which is a suitable method for the estimation of glucose-based carbohydrates like starches [20,21]. A modification was made to perform the assay into 96-well microtitration plates. The reaction was carried out mixing 40 μl sample, standard or blank with 100 μl 10.3 mmol/l anthrone in concentrated sulphuric acid and incubated at 92 °C for 3min. It has been shown that these assay conditions are able to quantify high molecular weight polymers like commercial soluble starch [21].Absorbance at 630 nm was read using a Dynatech MR5000 microplate reader and analysed with Dynatec Reader software version 1.1 (Dynatech Laboratories Inc., Chantilly, VA, USA). An appropriate calibration curve was made with each plate using glucose as standard[21]. Standard solutions for both starch and carbohydrate determinations were prepared dissolving glucose in slurry supernatants (1,500 g for 5min) from control cultures at time zero.
■ Statistical analysis
Variables were described as mean ± standard deviation.Statistical analysis was made using Minitab for Windows software, release 10.51 Xtra (Minitab Inc., State College,PA,USA).One- or two-sided unpaired t-test was used to compare means of pre-digested pyrodextrinised against pre-digested native starches where appropriate.A probability level less than 0.05 was used to indicate a significant difference between means.
Results
■ Fermentation of native and pyrodextrinised starches
The fermentation properties of both pre-digested native and pyrodextrinised starches from several sources after 24 h in vitro anaerobic incubation with human faeces are shown in Table 1. In cultures containing the pre-digested native starches, 99, 98, and 95% of potato, lentil, and cocoyam starches, respectively, were fermented. Although a similar trend was found for the pre-digested pyrodextrinised samples, the modified starch was measured as carbohydrate content by a chemical method [21] to overcome the specificity of the enzymatic starch assay[18].With this approach, 77, 75, and 81% of the carbohydrate in potato, lentil, and cocoyam pre-digested pyrodextrins were fermented, respectively.
■ Total SCFA
The net total SCFA, estimated as mmol/l of fermentedculture, was similar for all starches (Table 1). Comparable amounts were produced using glucose as substrate (58.8±4.4 mmol/l, n=6).However,when net total SCFA were corrected for initial culture carbohydrate content,SCFA production remained similar for all the pre-digested native starch sources, but the total SCFA for the pre-digested pyrodextrinised starches were significantly higher (between 43% and 75%) than their corresponding predigested native starches (p<0.023,one-sided t-test). Control cultures (n=6), i. e. those fermented without starch samples, produced 32.4±9.1 mmol/l of total SCFA, achieved pH 6.5±0.1and produced 7±3ml gas.
■ Molar proportions of SCFA
All pre-digested pyrodextrins showed a significantly higher (p<0.017, one-sided t-test) molar ratio of propionate (around twofold) when fermented compared with their pre-digested native starch. In addition, the acetate molar ratio decreased (p<0.04, one-sided t-test) by about 24% (Table 1). There was no difference in the nbutyrate molar proportion (p>0.82).
■ Fall in pH and gas produced
The culture pH was 6.6±0.1 (n=6) at time zero. After fermentation, all the cultures showed a decrease in pH, but the fall in pH was significantly less for the pre-digested pyroconverted samples (p<0.03, one-sided ttest). The fall in pH observed for glucose (1.7±0.3,n=6)was similar to all the pre-digested native starches (Table 1). The volume of gas produced was the same for all samples, including glucose (17±5 ml, n=6). |
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