Influence of Chemically-Modified Potato Starch (RS Type 4) on the Nutritional and Physiological Indices of Rats

Abstract

A biological study was undertaken to analyse the metabolic effect of feeding rats with an experimental diet in which cellulose was substituted with 20% contribution of chemically-modified potato starches (subjected to oxidation, esterification, cross-linking and dual modification). Caecum digesta mass was significantly higher in rats fed the experimental potato starch preparations compared to control group. Luminal ammonia concentration and pH of caecal or colonic content were lower as an effect of diets with all the investigated preparations. Compared to the cellulose-containing diet (control), all modified potato starch preparations raised the content of SCFA in caecum digesta when fed to rats. Significant lowering of the levels of triacylglycerols and total cholesterol was noticed for all chemically-modified starch preparations. The activity of β-glucuronidase determined upon the administration of potato starch preparations into rat diets was significantly lower as compared to the control diet. The results indicate that the chemically-modified potato starch preparations are a good substrate for the intestinal microecosystem and may promote the beneficial status of the gastrointestinal tract of rats. © Copyright by Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences.

Full text

© Copyright by Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences
Pol. J. Food Nutr. Sci., 2011, Vol. 61, No. 2, pp. 143-151
http://journal.pan.olsztyn.pl
INTRODUCTION
Themost common method for modelling physicochemical
andfunctional properties ofstarch is chemical modification,
which for food purposes is strictly limited interms ofthetype
ofchemical reactions, thekind ofmodifying agents, thede-
gree ofsubstitution ofstarch as well as thecontent ofim-
purities. Common types ofchemical reactions for starch
modification, for food purposes, include only three types,
i.e. oxidation, esterification andetherification. Thechemical
modification ofstarch may affect therate andextent ofits
digestion inthesmall intestine [Wolf et al., 1999]. It has been
stated that theoxidation or dextrinisation ofstarch, its substi-
tution with hydroxypropyl, acetyl or octenylsuccinate groups
as well as crosslinking diminish its digestibility [Wolf et al.,
1999; 2001]. From thenutritional point ofview, starches sub-
jected to chemical modification are acknowledged as resistant
starches type 4 (RS 4). Though resistant starch is not digest-
ed andnot absorbed inthegastrointestinal tract ofman, it
may be fermented by themicroflora inhabiting thecolon. By
these means, it may affect avariety offactors responsible for
theproper functioning oftheintestine, including: accelerating
transit andexcretion offaeces, increasing faecal bulk, modu-
lating pH ofintestinal digesta, reducing contents ofammonia
* Corresponding author: Tel. + 4889 523 4618; Fax: + 4889 524 0124
E-mail: m.wronkowska@pan.olsztyn.pl (Dr. M.Wronkowska)
andbile acids infaecal waters, andincreasing concentrations
ofshort-chain fatty acids [Silvester et al., 1995; Lopez et al.,
2001]. All these factors may positively affect theintestinal
ecosystem andtheactivity ofintestinal microflora. Silvi et
al. [1999] demonstrates that resistant starch could modify
thehuman gut microflora, particularly stimulating lactic acid
bacteria anddecreasing potentially pathogenic types such as
enterobacteria.
Thephysicochemical, morphological, thermal andrheo-
logical properties ofchemically-modified starch preparations
have been studied, but there is still alack ofwell-done nu-
tritional studies which discuss physiological consequences
oftheir ingestion. Thepurpose ofthis invivo study was to
investigate theresponse ofthegastrointestinal tract environ-
ment andserum lipids ofrats to diets containing some types
ofchemically-modified potato starch (subjected to oxidation,
esterification, cross-linking anddual modification) which are
commercially permissible for food uses.
MATERIALS ANDMETHODS
Materials
Native potato starch andits chemically-modified prepa-
rations were provided by Luboń SA Co. (WPZZ Luboń,
Poland). Thefollowing preparations were used inthenutri-
tional experiment: oxidised starch (OS, type ofmodification:
oxidation, International Numbering System (INS) provided
Influence ofChemically-Modified Potato Starch (RS Type 4) ontheNutritional andPhysiological
Indices ofRats
Małgorzata Wronkowska1*, Jerzy Juśkiewicz2, Zenon Zduńczyk2, Maria Soral-Śmietana1, Urszula Krupa-Kozak1
1Department ofChemistry andBiodynamic ofFood; 2Department ofBiological Properties ofFood;
Division ofFood Science, Institute ofAnimal Reproduction andFood Research ofthePolish Academy ofSciences,
Tuwima 10 str., 10–747 Olsztyn, Poland
Key words: chemically-modified potato starch, resistant starch, rats, cholesterol, SCFA, bacterial enzymes
Abiological study was undertaken to analyse themetabolic effect offeeding rats with an experimental diet inwhich cellulose was substituted with
20% contribution ofchemically-modified potato starches (subjected to oxidation, esterification, cross-linking anddual modification). Caecum digesta
mass was significantly higher inrats fed theexperimental potato starch preparations compared to control group. Luminal ammonia concentration
andpH ofcaecal or colonic content were lower as an effect ofdiets with all theinvestigated preparations. Compared to thecellulose-containing diet
(control), all modified potato starch preparations raised thecontent ofSCFA incaecum digesta when fed to rats. Significant lowering ofthelev-
els oftriacylglycerols andtotal cholesterol was noticed for all chemically-modified starch preparations. Theactivity ofβ-glucuronidase determined
upon theadministration ofpotato starch preparations into rat diets was significantly lower as compared to thecontrol diet. Theresults indicate that
thechemically-modified potato starch preparations are agood substrate for theintestinal microecosystem andmay promote thebeneficial status
ofthegastrointestinal tract ofrats.
Original paper
Section: Nutritional Research

144 M.Wronkowska et al.
by Codex Committee on Food Additives andContaminants,
No. 1404), acetylated starch (AS, type ofmodification: esteri-
fication, INS No. 1420), acetylated distarch adipate (ADA,
type ofmodification: dual treatment including esterification
with cross-linking, INS No. 1422), distarch phosphate (DP,
type ofmodification: cross-linking, INS No. 1412), andacet-
ylated distarch phosphate (ADP, type ofmodification: dual
treatment including esterification with cross-linking, INS No.
1414).
Chemical analysis ofthematerial
TheAOAC [1990] method was employed to determine
contents ofash andproteins. Theresistant starch, considered
as thestarch fraction not hydrolysed invitro by pancreatic
α-amylase (from porcine pancreas, SIGMA, A-3176), was
determined according to themethod by Champ et al. [1999].
Theproducts ofhydrolysis were extracted with 80% (v/v) eth-
anol andthenon-digested material was solubilised in2 mol/L
KOH, then hydrolysed with amyloglucosidase (Novozymes,
AMG 300L) into glucose. Theglucose was quantified with
aglucose oxidase/peroxidase analysis kit (Liquick Cor-GLU-
COSE 120, Cormay, Poland) andmeasured spectrophoto-
metrically at 500 nm.
Theinvitro hydrolysis
Susceptibility ofthestudied potato starch preparations
to pancreatic α-amylase was determined invitro according to
Soral-Śmietana & Wronkowska [2000] using 200 Uofpor-
cine pancreatic α-amylase (from porcine pancreas, SIGMA,
A-3176) per 1 gram ofasample. Thesample (500 mg) was
suspended in20 mL ofan enzyme solution andthehydrolysis
was carried out for 24 h at 37°C.Prior to hydrolysis, isopro-
panol (100 µL) was added to thesample to inhibit thegrowth
ofmicrobes during incubation. Theenzyme was inactivated
with 95% (v/v) ethanol andafter centrifugation at 3,000 ×g/10
min, thesamples were dried at atemperature below 30ºC.
Scanning electron microscope (SEM)
Changes inthemicrostructure oftheinvestigated starches
after invitro hydrolysis were analysed by covering dry samples
with agold layer andvisualizing at an acceleration of10 KeV
on ascanning electron microscope (JSM 5200, Japan).
Feeding experiment
Theprotocol used inthis animal study was approved by
theInstitutional Animal Care andUse Committee, at theUni-
versity ofWarmia andMazury, Olsztyn, Poland. Theexperi-
ment was conducted on 56 male Wistar rats, aged 4 weeks,
divided into experimental groups of8 rats each. Theanimals
were housed individually under standard conditions: temper-
ature 21–22°C, relative air humidity 50–70%, 12-h light:dark
cycle, intensive ventilation (air turnover 10/h), andad libitum
access to water andfeed. Thenutritional experiment lasted
4 weeks. Thecomposition ofexperimental diets is presented
inTable 1. Cellulose (200 g/kg) was added as asource ofdi-
etary fibre. Intheexperimental treatments, thewhole dietary
cellulose was substituted with native or chemically-modified
potato starch preparations. After theexperiment, therats
were anaesthetized with sodium pentobarbitone according to
therecommendations for euthanasia ofexperimental animals
followed by the12 h offasting [Close et al., 1997].
Indices ofdiets intake andutilization
Body weight gain ofrats andfeed intake were determined
individually. Coefficients ofapparent nitrogen digestibil-
ity andutilisation were calculated inthefirst 5-day period
ofexperimental feeding, from daily N intake andN excretion
infaeces or infaeces andurine, respectively. Faeces andurine
were collected from rats ineach group for 5 days, preceded
by a10-day preliminary period. Nitrogen inthesamples was
determined for each rat according to theKjeldahl method
[AOAC, 1990].
Physiological parameters ofthecaecum
After laparotomy, blood samples were taken from thetail
vein, andthen thecaecum andcolon with contents were re-
moved andweighed. Samples offresh digesta were used
for immediate analysis ofdry matter, ammonia andshort
chain fatty acids (SCFA), andtheremainder was transferred
to tubes andstored at -70°C.Thecaecal andcolonic walls
were flushed clean with ice-cold saline, blotted on filter pa-
per andweighed for tissue mass. Thecaecal andcolonic pH
was measured using amicroelectrode andapH/ION meter
(model 301, Hanna Instruments, Vila do Conde, Portugal).
Dry mass ofcaecal contents was determined after primary
drying at 50–60oC for 24 h, with asecondary drying at 105oC
to determine theconstant mass. Infresh caecal digesta, am-
monia was extracted andtrapped inasolution ofboric acid
inConway’s dishes, anddetermined by direct titration with
sulfuric acid [Hofirek & Haas 2001].
TABLE 1. Composition ofexperimental diets (g/100 g diet).
Diets*
C NS OS AS ADA DP ADP
Cellulose (C) 20.0
Native potato starch (NS) – 20.0 – ––––
Oxidised starch (OS) – – 20.0 ––––
Acetylated starch (AS) – – – 20.0 – –
Acetylated distarch adipate
(ADA) – – – – 20.0 – –
Distarch phosphate (DP) – – – – 20.0 –
Acetylated distarch
phosphate (ADP) – – – – – – 20.0
Casein 14.0 14.0 14.0 14.0 14.0 14.0 14.0
DL-methionine 0.2 0.2 0.2 0.2 0.2 0.2 0.2
Soy oil 8.0 8.0 8.0 8.0 8.0 8.0 8.0
Cholesterol 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Mineral mixa 3.5 3.5 3.5 3.5 3.5 3.5 3.5
Vitamin mixb 1.0 1.0 1.0 1.0 1.0 1.0 1.0
Corn starch 52.8 52.8 52.8 52.8 52.8 52.8 52.8
aAIN-93G-MX [Reeves, 1997]; bAIN-93G-VM [Reeves, 1997]; *Diets: C
– control with cellulose; NS – with native potato starch; OS – with oxi-
dised starch; AS – with acetylated starch; ADA – with acetylated distarch
adipate; DP – with distarch phosphate; ADP – with acetylated distarch
phosphate. Thediets were isoenergetic andisonitrogenous.

145
Chemically-Modifi ed Potato Starch
Bacterial enzyme activity inthecaecal digesta was mea-
sured by therate ofp- or o- nitrophenol release from ni-
trophenylglucosides according to themethod ofDjouzi &
Andrieux [1997], modified by Juśkiewicz & Zduńczyk [2004].
Thefollowing substrates were used: for β-glucuronidase,
p-nitrophenyl-β-D-glucuronide; for α-galactosidase, p-ni-
tro phenyl-α -D-galactopyranoside; for β-galactosidase, o-ni-
trophenyl-β-D galactopyranoside; for α-glucosidase, p-ni-
trophenyl-α-D glucopyranoside; andfor β-glucosidase,
p-nitrophenyl-β-D glucopyranoside. Thereaction mixture con-
tained 0.3 mL ofasubstrate solution (5 mmol/L) and0.2mL
ofa1:10 (v/v) dilution ofthecaecal sample in100 mmol/L
phosphate buffer (pH 7.0) after centrifugation at 10,000 × g
for 15 min. Incubation was carried out at 37°C andp- or o-
nitrophenol was quantified at λ=400 nm andat λ=420 nm, re-
spectively, after addition of2.5 mL of0.25 mol/L cold sodium
carbonate. Theenzymatic activity ofα - andβ-glucosidases,
α- andβ-galactosidases, andβ-glucuronidase, was expressed
as micromoles ofproduct formed per minute (unit) per 1 g
ofdigesta inthefresh caecal sample. Caecal digesta samples
were subjected to an SCFA analysis using gas chromatography
(Shimadzu GC-2010; Shimadzu, Kyoto, Japan). Thesamples
(0.2 g) were mixed with 0.2 mL offormic acid, diluted with
deionised water andcentrifuged at 10,000 × g for 10min. Su-
pernatant was loaded onto acapillary column (SGE BP21,
30m x 0.53 mm) using an on-column injector. Theinitial oven
temperature was 85°C, andwas raised to 180°C by 8°C/min
andheld for 3 min. Thetemperatures offlame ionization de-
tector andtheinjection port were 180°C and85°C, respective-
ly. Thesample volume for agas chromatography analysis was
1 µL.Thecaecal SCFA pool was calculated as theconcentra-
tion ofSCFA inthecaecum (µmol/g) multiplied by themass
ofthecaecal contents (g) andwas expressed inmicromoles
per 100 g ofbody weight.
Blood serum analysis
Blood samples collected from tail veins were left for 1 h
at aroom temperature to aggregate red blood cells. Blood
serum was purified by centrifugation at 2,500 × g for 15 min
at 4°C, andstored at –70°C after freezing with liquid nitro-
gen. Concentrations ofthefollowing indices were determined
intheblood serum: triacylglycerols (TG) (ChF Reagent,
catalog No. 220–200), glucose (ChF Reagent, catalog No.
210–200), total cholesterol (TC) (ChF Reagent, catalog No.
150–200), andhigh-density lipoprotein (HDL-C) cholester-
ol fraction (ChF Reagent, catalog No. 160–100). Log (TG/
HDL-C) was calculated as an atherogenic index ofserum.
Statistical analysis
Results ofthephysiological response ofthetreated
animals are expressed as means andpooled standard er-
ror (SEM). Statistical comparisons were done transversely
among different dietary groups. Data were analysed by one-
-way ANOVA, with one factor (diet). If significance was ob-
served (p<0.05), theDuncan’s multiple range test was used
to identify differences intheeffect ofindividual diets. Calcu-
lations were made with STATISTICA 6.0 software (StatSoft
Corporation, Kraków, Poland).
RESULTS ANDDISCUSSION
Thecontent ofresistant starch innative andchemically-
modified potato starch preparations was: native potato starch
– 77.2% dry matter; ADA – 76.5% d.m.; AS – 75.6% d.m.;
OS – 74.5% d.m.; DP – 14.5% d.m. andADP – 13.5% d.m.
Themodification ofpotato starch, namely esterification,
changed slightly theRS content inacetylated starch (AS)
andacetylated distarch adipate (ADA) preparations. Also
inoxidised starch (OS) thecontent ofresistant starch did
not change significantly compared to native potato starch.
Thenext type ofmodification, i.e. cross-linking, which was
applied to obtain distarch phosphate (DP), caused asignifi-
cant reduction inRS content. Inturn, dual modification, i.e.
acombination ofsubstitution andcross-linking which was
used to produce acetylated distarch phosphate (ADP), re-
sulted inthelowest RS content among thestarch prepara-
tions applied. Protein andash contents inall potato starch
preparations were almost similar andreached from 0.20 to
0.37% d.m. for proteins andfrom 0.23 to 0.39% d.m. for ash.
Theaddition ofstarch preparations applied inthestudy
to therat diets was stipulated based on our previous in-
vestigations andsome literature data according Wurzburg
[1986] andBird et al. [2006]. According to recommenda-
tion oftheWorld Health Organization [FAO Report, 1998],
thedaily intake ofthedietary fibre should be at therange
of20–40 g/day. Therefore inour study thecontrol diet con-
tained 20% ofcellulose and20% ofinvestigated starches
intheexperimental diet.
Body weight gains oftherats anddiets intake inexperi-
mental groups are presented inTable 2. Both, diet intake
andfinal body weight gain oftherats from theADA group
turned out to be thelowest as compared to thecontrol
andNS groups (p<0.05). Hodgkinson et al. [1982] demon-
strated that feeding animals with theaddition ofacetylated
waxy maize starches evoked an increase intheir body weight
TABLE 2. Diet intake, body weight gain andnitrogen apparent digestibility ofrats fed experimental diets.
Diets* SEM
C NS OS AS ADA DP ADP
Diet intake (g/28 days) 562.1a531.5ab 523.4abc 514.5bc 487.0c531.9ab 529.5abc 5.7
Body weight gain (g/28 days) 134.1ab 132.4ab 136.7ab 131.0ab 117.4b143.8a130.7ab 2.5
Nitrogen apparent digestibility (%) 82.1a 80.4ab 81.7ab 81.4ab 79.9b 80.1b80.9ab 0.2
*Diets: C – control with cellulose; NS – with native potato starch; OS – with oxidised starch; AS – with acetylated starch; ADA – with acetylated distarch
adipate; DP – with distarch phosphate; ADP – with acetylated distarch phosphate; a,b,c – values inthesame row with different letters are significantly
different (p<0.05); SEM – pooled standard error ofthemeans (standard deviation for all rats divided by square root ofrat number, n=56).

146 M.Wronkowska et al.
gains by ca. 5% as compared to theanimals not administered
modified starches. Inturn, inamodel study conducted on
rats Wronkowska et al. [2002] showed that, as compared to
thecontrol diet containing 10% ofcellulose, an experimental
diet inwhich cellulose was substituted with 10% ofphysically-
modified potato starch preparation elicited astatistically sig-
nificant increase inbody weight gain oftheanimals.
For thecontrol group (C), nitrogen apparent digest-
ibility was thehighest (not statistically significant) incom-
parison with groups receiving theinvestigated potato starch
preparations (Table 2). Thelowest values ofthat indicator
were noticed when thediets contained acetylated distarch
adipate (ADA) anddistarch phosphate (DP). Results ofthis
study proved areduction innitrogen concentration infaeces
andurine ofanimals (data not shown). Thecharacteristics
ofnitrogen metabolism is strictly linked with thetype ofdi-
etary carbohydrates [Pastuszewska et al., 2000]. Ferment-
able carbohydrates enhance faecal N excretion by promoting
bacterial proliferation andalso by accelerating thedigestive
transit time [Younes et al., 1995]. Krupa-Kozak et al. [2010]
found astatistically significant decrease innitrogen apparent
digestibility ofrats fed diets inwhich 30% ofcorn starch was
substituted with bean or pea starches.
Results oftheanalysis ofcaecum andcolon parameters
were provided inTable 3. As compared to thecontrol diet,
inwhich thenon-digestible constituent was cellulose, all diets
with theexamined starches caused astatistically significant
(p<0.05) increase incaecal tissue mass andcaecal digesta
bulk. Thehighest mass ofcaecal digesta was found for
thediet containing distarch phosphate (DP). Inturn, thedi-
ets were not observed to evoke analogous changes incolonic
parameters (Table 3). Literature data presents that prepara-
tions containing different types ofRS significantly influence
themetabolism andmorphology ofthegastrointestinal tract,
including hypertrophy oflarge intestinal tissue andbulking
effect [Ebihara et al., 1998; Wronkowska et al., 2002; Annison
et al., 2003]. All types ofRS are fermentable, so their bulk-
ing action is rather variable andmuch less than that ofwheat
bran which is thought as amost effective faecal bulking agent
[Topping & Clifton, 2001].
Ingroups C andOS, asignificantly higher dry mat-
ter content inthecaecal digesta was found as compared to
theother groups (Table 3). Both, pH values andtheconcen-
tration ofammonia inthecaecal digesta ofthecontrol group
were significantly higher than inall experimental groups
(C vs. theother treatments, p<0.05). Thelowest pH value
ofthecaecal digesta was determined inOS group. ThepH
ofcolonic content was lower for all experimental groups
compared with thecontrol group. Microflora metabolizes ni-
trogenous compounds that enter thelarge intestine into putre-
factive catabolites, such as ammonia andphenols, which may
negatively affect gastrointestinal tract health. Fermentable
carbohydrates may decrease theconcentration ofputrefactive
compounds by providing gut microflora with an additional
energy [Younes et al., 1995]. Cermak et al. [2002] demon-
strated that luminal ammonia inhibited sodium andchloride
absorption inthedistal colon ofrats. Til et al. [1986] showed
that feeding hydroxypropyl distarch phosphate from potato
starch led to an increase intissue mass ofthecaecum andco-
lon ofrats. Inturn, Kishida et al. [2000] found an increase
inthecaecal wall andcontents after theaddition ofgelati-
nized hydroxypropyl distarch phosphate from tapioca starch
to therats diet.
Astatistically significant (p<0.05) increase inthepool
ofSCFA inthecaecal content was observed inall groups
fed diets with theaddition ofthenative potato starch andits
modified preparations (Table 4). Inrespect ofthecontrol
group, all experimental groups were characterised by asig-
nificant increase inconcentrations ofacetic, propionic, bu-
tyric andvaleric acids as well as by considerably diminished
concentrations ofiso-butyric andiso-valeric acids. Thesig-
nificant increase inthetotal content ofSCFA inthecaecum
was due to significant acidification ofthecaecal andcolonic
contents (Table 3) observed for all types oftheinvestigated
potato starch. Acetylated starches could be used to raise
theSCFA level inthelarge bowel ofrats as presented by An-
TABLE 3. Caecal andcolonic parameters ofrats fed experimental diets.
Diets* SEM
C NS OS AS ADA DP ADP
Caecum
Mass oftissue (g/100 g BW) 0.29c0.69a0.73a0.64ab 0.57b0.68a0.65ab 0.02
Mass ofdigesta (g/100 g BW) 0.96d3.23ab 3.59ab 3.12ab 2.33c3.87a2.87bc 0.15
Dry matter ofdigesta (%) 28.2a24.0b28.1a23.0b24.4b24.5b22.8b0.40
Ammonia (mg/g digesta) 0.26a0.19b0.20b0.20b0.21b0.19b0.19b0.01
pH ofcaecal contents 6.88a5.21bc 5.12c5.32bc 5.38b5.16bc 5.34bc 0.08
Colon
Mass oftissue (g/100 g BW) 0.65 0.64 0.65 0.64 0.61 0.63 0.61 0.01
Mass ofdigesta (g/100 g BW) 0.67 0.72 0.76 0.62 0.62 0.70 0.62 0.02
pH ofcolonic contents 6.64a5.08b5.07b5.07b5.06b5.04b4.95b0.08
*Diets: C – control with cellulose; NS – with native potato starch; OS – with oxidised starch; AS – with acetylated starch; ADA – with acetylated distarch
adipate; DP – with distarch phosphate; ADP – with acetylated distarch phosphate; a,b,c – values inthesame row with different letters are significantly
different (p<0.05); BW – body weight; SEM – pooled standard error ofthemeans (standard deviation for all rats divided by square root ofrat number,
n=56).

147
Chemically-Modifi ed Potato Starch
nison et al. [2003]. Inahuman study Clarke et al. [2007]
found that theapplication ofacetylated starches was apo-
tentially effective method for delivering significant quantities
ofspecific SCFAs to thecolon. However, not all RS oftype 4
appear to be equal intheir effects on large bowel SCFA.Ebi-
hara et al. [1998] reported that theingestion ofhydroxy-
propylated starches increased faecal bulk inrats but did not
affect changes inSCFA.Inour study, theresults indicated
strongly that thetype ofindustrial processing itself, rather
than theRS content affected theeffects ofthepreparations
applied on thecaecal SCFA yield. Intheinvestigated potato
starch preparations, dextrins ofdifferent molecular mass can
be formed during starch degradation andthese degradation
products are supposed to have effect on thephysiological
processes intheanimals examined. It is inline with sugges-
tions provided by several authors that thephysiological ef-
fects ofchemically-modified starches are affected by thetype
andextent ofmodification [Ebihara et al., 1998; Ferguson &
Jones, 2000; Nugent, 2005].
Asignificant indicator ofthephysiological effect is theac-
tivity ofbacterial glycolytic enzymes analysed incaecal diges-
ta (Table 5). Incomparison to thecontrol diet, astatistically
significant (p<0.05) increase was observed intheactivities
ofα-glucosidase andβ-galactosidase upon theadministra-
tion ofall experimental diets under study. Distinguishing
activities ofβ-glucosidase as well as α- andβ-galactosidase
were noted upon theapplication ofthediet with acetylated
starch (AS). Yet, worthy ofspecial attention is adiminished
activity ofβ-glucuronidase as affected by theaddition ofna-
tive potato starch andchemically-modified starches to diets,
except for ADP (Table 5). Theincrease inβ-galactosidase
andα-glucosidase activities andthedecrease inthat
ofβ-glucuronidase could be considered beneficial for
thehost. β-Galactosidase andα-glucosidase activities may
improve thefermentation oflactose andresistant starch,
leading to theproduction ofSCFA andlactic acid which are
asource ofenergy for colonic tissues [Cummings & Macfar-
lane, 1991]. Theincrease inβ-glucosidase activity is more am-
biguous because its hydrolytic activity is responsible both for
thegeneration oftoxins [Mallet & Rowland, 1988], andfor
theproduction ofbacterial glucoside derivatives which are
assumed to be responsible for protection against chemically-
induced cancer [Rowland & Tanaka, 1993]. β-Glucuronidase
is involved inthegeneration oftoxic andcarcinogenic me-
tabolites inthehindgut [Reddy et al., 1992]. Wronkowska et
al. [2002] reported thedecreasing ofβ-glucuronidase activ-
ity incaecal digesta ofrats fed diets with 10% ofphysically-
modified starch preparations obtained from wheat, potato or
pea starch, as compared with diets containing native starches.
Theprevious invivo study [Krupa-Kozak et al., 2010] con-
TABLE 5. Caecal bacterial enzyme activity ofrats fed experimental diets.
Diets* SEM
C NS OS AS ADA DP ADP
α-Glucosidase (U/g c.c.) 0.13b1.29a1.33a1.55a1.42a1.33a0.13b0.07
β-Glucosidase (U/g c.c.) 0.04c0.09abc 0.07bc 0.14a0.12ab 0.07bc 0.04c0.01
α-Galactosidase (U/g c.c.) 0.06b0.05b0.06b0.09a0.07ab 0.04b0.06b0.00
β-Galactosidase (U/g c.c.) 0.35d0.99ab 0.66c1.21a0.99ab 0.88bc 0.35d0.05
β-Glucuronidase (U/g c.c.) 0.16a0.07b0.08b0.10b0.10b0.10b0.16a0.01
c.c. – caecum contents; *Diets: C – control with cellulose; NS – with native potato starch; OS – with oxidised starch; AS – with acetylated starch;
ADA – with acetylated distarch adipate; DP – with distarch phosphate; ADP – with acetylated distarch phosphate; a,b,c – values inthesame row with
different letters are significantly different (p<0.05); SEM – pooled standard error ofthemeans (standard deviation for all rats divided by square root
ofrat number, n=56).
TABLE 4. Caecal digesta total short chain fatty acids (SCFA) pool ofrats fed experimental diets.
SCFA pool (µmol/100 g BW) Diets* SEM
C NS OS AS ADA DP ADP
Acetic 63.2d339.0ab 324.4ab 307.2ab 221.0c384.8a282.1bc 16.1
Propionic 12.10b73.20a71.91a96.72a74.437a79.90a77.00a4.61
Iso-butyric 0.61a0.05b0.04b0.14b0.08b0.09b0.04b0.03
Butyric 7.85c51.41a40.58a25.61b25.38b47.59a40.43a2.58
Iso-valeric 0.74a0.12b0.14b0.54a0.13b0.14b0.08b0.05
Valeric 0.92c10.28a7.93b6.09b5.97b10.35a6.01b0.55
Total 85.4d474.9ab 444.1ab 436.0ab 327.5c522.2a406.0bc 22.1
*Diets: C – control with cellulose; NS – with native potato starch; OS – with oxidised starch; AS – with acetylated starch; ADA – with acetylated distarch
adipate; DP – with distarch phosphate; ADP – with acetylated distarch phosphate; a,b,c,d – values inthesame row with different letters are significantly
different (P<0.05); BW – body weight; SEM – pooled standard error ofthemeans (standard deviation for all rats divided by square root ofrat number,
n=56).

148 M.Wronkowska et al.
firmed thesignificant decrease ofβ-glucuronidase activity
incaecal digesta when rats were fed adiet containing dietary
legume starches, bean andpea or their microwaved starch
preparations.
Theaddition ofdistarch phosphate (DP) andacetylated
distarch adipate (ADA) to thediets was found to decrease
glucose concentration (p<0.05) intherat blood serum (Ta-
ble6). All chemically-processed potato starch preparations
led to asignificant drop intheserum level oftriacylglycerols
(TG). On theother hand, all starch treatments caused asig-
nificant decrease inthetotal cholesterol andalso asignificant
increase intheratio ofHDL-C/total cholesterol intheserum.
However, thecalculated log (TG/HDL-C) index ofserum
atherogenicity was thehighest inthecontrol group andits sig-
nificant reduction was found inall experimental treatments.
It should also be emphasized that thelowest log (TG/HDL-
C) index was observed intheDP group (p<0.05 vs. all other
groups).
De Deckere et al. [1993] try to explain theeffect ofRS on
serum total cholesterol andtriacylglycerols concentrations:
(1) serum cholesterol might have been decreased by RS as
aresult ofan increased faecal excretion ofsterols andinhib-
ited via SCFA ofcholesterol synthesis intheliver; (2) serum
TAG concentration may be lowered due to theeffects on lipid
absorption andhepatic fatty acid synthesis. It has been re-
ported inan invivo test that acetylated potato starch inameal
improved theglycemic, insulinemic andsatiating properties,
because theacetylation decreased thesusceptibility ofstarch
to α-amylase [Raben et al., 1997]. Some researchers have
observed that thecross-linking modification may reduce
therate ofstarch digestion, however inthis regard thetype
andextent ofcross-linking seem to be ofparamount impor-
tance [Woo & Seib, 2002]. Several studies have shown that
high amylose corn starch (RS type 2) reduces serum choles-
terol andtriacylglycerols concentration inrats [Sacquet et al.,
1983] andhamsters [Ranghotra et al., 1997]. Inan invitro
study, acetylated potato starch was characterised by thehigh-
est cholesterol binding capacity incomparison with other in-
vestigated chemically-modified potato starches [Wronkowska
et al., 2008]. Kishida et al. [2002] found that high amylose
corn starch exerted ahypocholesterolemic effect incaecoto-
mized rats fed acholesterol-free diet, andthis effect was most
likely mediated through theenlargement ofthebile acids pool
intheintestine andincreased faecal excretion ofbile acids,
proving that various ways leading to thehypocholesterolemic
effect are attributable to RS preparations.
For in-depth recognition ofthecourse ofhydrolysis
ofthechemically-modified preparations ofpotato starch in-
vestigated inthis study, an analysis was conducted ofthemi-
crostructure ofthese starch preparations subjected to 24-h
hydrolysis with pancreatic α-amylase (Figure 1). After 24-h
activity ofthat enzyme, asurface-damaging effect could be
observed, being typical ofgranules ofthenative potato starch
(NS) [Gallant et al., 1992]. Incontrast, themodified prepara-
tions revealed asignificant effect ofthedestruction ofgranule
structure, especially inthecase oftheoxidised starch (OS),
distarch phosphate (DP) andacetylated distarch phosphate
(ADP). Thestudy proved that upon theprocess ofmodifi-
cation, those preparations were hydrolysed by pancreatic
α-amylase by theformation ofpin-like channels from thesur-
face to theinterior ofthegranules, which is typical ofce-
real starches with Atype crystalline structure. Incontrast,
intheacetylated preparation (AS), observations have shown
theradial mode ofamylolysis only inmore damaged frag-
ments ofthegranules andcollapse oftheexternal structure
intheinter-crystalline space. Generally, more or less intensive
superficial erosion was observed intheAS andADA prepa-
rations, which is typical course oftheamylolysis ofpotato
starch.
Results ofthemicrostructural study seem to be espe-
cially interesting inrespect oftheDP andADP preparations,
inwhich RS content was determined to be low. Micropho-
tographs ofthese preparations proved thesignificance
ofthephysical status ofpotato starch preparations andalso
thetype ofmodification used, provided as asubstrate for in-
testinal microflora. Thedigestion ofstarch granules is acom-
plex process, consisting from afew stages, i.e.: adsorption
oftheenzyme by starches; penetration oftheenzyme through
asubstrate, which is linked with porosity ofgranules; andat
theend – theprocess ofhydrolysis. Potato starch andother
starches with type B crystalline structure are digested super-
ficially, whereas cereal starches are hydrolysed through pores
andchannels that may be used by theenzyme [Lehmann &
Robin, 2007]. Our research proved that theprocess ofhy-
TABLE 6. Serum indices ofrats fed experimental diets.
Diets* SEM
C NS OS AS ADA DP ADP
Glucose (mmol/L) 12.19a11.58a12.27a11.27ab 10.30b10.45b11.69a0.15
TG (mmol/L) 1.54a1.39ab 1.26bcd 1.33bc 1.14de 1.02e1.18cde 0.03
TC (mmol/L) 2.81a2.24b2.18b2.32b2.21b2.15b2.26b0.04
HDL-C (mmol/L) 1.01b1.24a1.04b1.18ab 1.07ab 1.15ab 1.15ab 0.02
HDL-C/TC (%) 36.02b55.23a47.58a51.01a48.44a53.23a51.08a1.24
Log (TG/HDL-C) 0.18a0.05b0.09b0.05b0.03bc -0.05d0.01c0.00
*Diets: C – control with cellulose; NS – with native potato starch; OS – with oxidised starch; AS – with acetylated starch; ADA – with acetylated distarch
adipate; DP – with distarch phosphate; ADP – with acetylated distarch phosphate; a,b,c,d – values inthesame row with different letters are significantly
different (p<0.05); SEM – pooled standard error ofthemeans (standard deviation for all rats divided by square root ofrat number, n=56); TC – total
cholesterol; TG – triacylglycerol; HDL-C: HDL fraction ofcholesterol.

149
Chemically-Modifi ed Potato Starch
FIGURE 1. Microphotographs (SEM) ofthepotato starch preparations subjected to 24-h hydrolysis with pancreatic α-amylase. NS – native potato
starch; OS – oxidised starch; AS – acetylated starch; ADA – acetylated distarch adipate; DP – distarch phosphate; ADP – acetylated distarch phosphate.
NS
OS
AS
ADA
DP
ADP

150 M.Wronkowska et al.
drolysis ofpotato starch subjected to various treatments
ofchemical modification might proceed inadiversified mode,
untypical oftype B tuber starches, which was additionally re-
flected inthepresent invivo study.
SUMMARY
Chemically-modified potato starches containing RS type
4 are widely used inthefood industry rather for technologi-
cal than nutritional reasons. Insummary, thepresent study
provides data regarding theeffect ofdiets supplemented by
different kinds ofchemically-modified potato starch on small
mammals (rats). Partial substitution ofrats diets by theex-
perimental starches caused asignificant increase inbody
weight gain compared with thecontrol animals. For all inves-
tigated starch preparations, astatistically significant increase
ofthetotal SCFA (especially butyric acid) inthecaecal di-
gesta was found. As an effect ofdiets with all theinvestigated
preparations, thesignificant lowering ofluminal ammonia
concentration, pH ofcaecal or colonic content, triacylglyc-
erols, total cholesterol andactivity ofβ-glucuronidase incae-
cum content were noticed compared to thecontrol rats.
Thepositive physiological results obtained invivo inamodel
experiment with rats should, however, be verified inahuman
model research.
ACKNOWLEDGEMENTS
This study was financed by theMinistry ofScience
andHigher Education project No. N312016 32/1279 (2007–
–2009). Theauthors wish to thank thePolish company WPPZ
S.A.Luboń for all industrial potato starch preparations.
Theauthors declare that there are no conflicts ofinterest to
disclose.
REFERENCES
1. Annison G., Illman R.J., Topping D.L., Acetylated, propionylated
or butyrylated starches raise large bowel short-chain fatty acids
preferentially when fed to rats. J.Nutr., 2003, 133, 3523–3528.
2. AOAC, Official Methods ofAnalysis. (15th ed.). 1990, Arlington,
Virginia, USA.
3. Bird A.R., Brown I.L., Topping D.L., Low andhigh amylase
maize starches acetylated by acommercial or alaboratory pro-
cess both deliver acetate to thelarge bowel ofrats. Food Hydro-
coll., 2006, 20, 1135–1140.
4. Cermak R., Minck K., Lawnitzak C., Scharrer E., Ammonia in-
hibits sodium andchloride absorption inrat distal colon. Exp.
Physiol., 2002, 87, 311–319.
5. Champ M., Martin L., Noah L., Gratas M., Analytical methods
for resistant starch. 1999, in: Complex Carbohydrates inFoods
(eds. S.Sungsoo Cho, L.Prosky, M.Dreher). Marcel Dekker
Inc., New York, pp. 184–187.
6. Clarke J.M., Bird A.R., Topping D.L., Cobiac L., Excretion
ofstarch andesterified short-chain fatty acids by ileostomy sub-
jects after theingestion ofacetylated starches. Am. J.Clin. Nutr.,
2007, 86, 1146–1151.
7. Close B., Banister K., Baumans V., Bernoth E.-M., Bromage N.,
Bunyan J., Erhardt W., Flecknell P., Gregory N., Hackbarth H.,
Morton D., Warwick C., Recommendation for euthanasis ofex-
perimental animals: Part 2. Lab. Animals, 1997, 31, 1–32.
8. Cummings J.H., Macfarlane G.T., Thecontrol andconsequences
ofbacterial fermentation inthehuman colon – areview. J.Appl.
Bact., 1991, 70, 443–459.
9. De Deckere E.A.M., Kloots W.J., van Amelsvoort J.M.M., Resis-
tant starch decreases serum total cholesterol andtriacylglycerol
concentrations inrats. J.Nutr., 1993, 123, 2142–2151.
10. Djouzi Z., Andrieux C., Compared effect ofthethree oligosac-
charides on metabolism ofintestinal microflora inrats with ahu-
man faecal flora. Brit. J.Nutr., 1997, 78, 313–324.
11. Ebihara K., Shiraishi R., Okuma K., Hydroxypropyl-modified
potato starch increases fecal bile acid excretion inrats. J.Nutr.,
1998, 128, 848–854.
12. Ferguson M.J., Jones G.P., Production ofshort-chain fatty acids
following invitro fermentation ofsaccharides, saccharide esters,
fructo-oligosaccharides, starches, modified starches andnon-
starch polysaccharides. J.Sci. Food Agr., 2000, 80, 166–170.
13. FAO/WHO, 1998, Carbohydrates inHuman Nutrition: Report
ofaJoint FAO/WHO Expert Consultation, 14–18 April 1997,
97–104.
14. Gallant D.J., Bouchet B., Buléon A., Pérez S., Physical character-
istics ofstarch granules andsusceptibility to enzymatic degrada-
tion. Eur. J.Clin. Nutr., 1992, 46, suppl. 2, 3–16.
15. Hodgkinson A., Davis D., Fourman J., Robertson W.G., Roe
F.J.C., Acomparison oftheeffects oflactose andoftwo chemi-
cally modified waxy maize starches on mineral metabolism
intherat. Food Chem. Toxicol., 1982, 20, 371–382.
16. Hofirek B., Haas D., Comparative studies ofruminal fluid col-
lected by stomach tube or by puncture ofthecaudoventral rumi-
nal sac. Acta Vet. Brno, 2001, 70, 27–33.
17. Juśkiewicz J., Zduńczyk Z., Effects ofcellulose, carboxymethyl-
cellulose andinulin fed to rats as single supplements or incom-
binations on their caecal parameters. Comp. Biochem. Phys. A,
2004, 139, 513–519.
18. Kishida T., Nakai Y., Ebihara K., Hydroxypropyl-distarch
phophate from tapioca starch reduces zinc andiron absorption,
but not calcium andmagnesium absorption, inrats. J.Nutr.,
2001, 131, 294–300.
19. Kishida T., Nogami H., Ogawa H., Ebihara K., Thehypocho-
lesterolemic effect ofhigh amylose cornstarch inrats is medi-
ated by an enlarged bile acid pool andincreased faecal bile acid
excretion, not by caecal fermented products. J.Nutr., 2002, 132,
2519–2524.
20. Krupa-Kozak U., Juśkiewicz J., Wronkowska M., Soral-Śmietana
M., Zduńczyk Z., Native andmicrowaved bean andpea starch
preparations – physiological effects on theintestinal ecosystem,
caecal tissue andserum lipids inrats. Brit. J.Nutr., 2010, 103,
1118–1126.
21. Lehmann U., Robin F., Slowly digestible starch – its structure
andhealth implications: areview. Trends Food Sci. Tech., 2007,
18, 346–355.
22. Lopez H.W., Levrat-Verny M.-A., Coudray C., Besson C., Kres-
pine V., Messager A., Demigné C., Rémésy C., Class 2 resistant
starches lower plasma andliver lipids andimprove mineral reten-
tion inrats. J.Nutr., 2001, 131, 1283–1289.
23. Mallet A.K., Rowland I.R., Factors affecting thegut microflora.
1988, in: Role oftheGut Flora inToxicity andCancer. Academic
Press, London, 347–382

151
Chemically-Modifi ed Potato Starch
24. Nugent A.P., Health properties ofresistant starch. Brit. Nutr.
Found. Bull., 2005, 30, 27–54.
25. Pastuszewska B., Kowalczyk J., Ochtabińska A., Dietary carbo-
hydrates affect caecal fermentation andmodify nitrogen excre-
tion patterns inrats. II.Studies with diets differing inprotein
quality. Arch. Anim. Nutr., 2000, 53, 335–352.
26. Raben A., Andersen K., Karberg M.A., Holst J.J., Astrup A.,
Acetylation ofor beta-cyclodextrin addition to potato: benefi-
cial effect on glucose metabolism andappetite sensations. Am.
J.Clin. Nutr., 1997, 66, 304–314.
27. Ranghotra G.S., Gelroth J.A., Leinen B.S., Hypolipidemic effects
ofresistant starch inhamster is not dose dependent. Nutr. Res.,
1997, 17, 317–323.
28. Reddy B.S., Engle A., Simi B., Goldman M., Effect ofdietary
fiber on colonic bacterial enzymes andbile acids inrelation to
colon cancer. Gastroenterology, 1992, 102, 1475–1482.
29. Reeves P.G., Components oftheAIN-93 diets as improvements
intheAIN-76A diet. J.Nutr., 1997, 127, 838–841.
30. Rowland I., Tanaka R., Theeffects oftransgalactosylated oligo-
saccharides on gut flora metabolism inrats associated with ahu-
man fecal microflora. J.Appl. Bacter., 1993, 74, 667–674.
31. Sacquet E., Leprince C., Riottot M., Effect ofamylomaize starch
on cholesterol andbile acid metabolisms ingermfree (axenic)
andconventional (holoxenic) rats. Repr. Nutr. Devel., 1983, 23,
783–792.
32. Silvester K.R., Englyst H.N., Cummings J.H., Ileal recovery
ofstarch from whole diets containing resistant starch measured
invitro andfermentation ofileal effluent. Am. J.Clin. Nutr.,
1995, 62, 403–411.
33. Silvi S., Rumney C.J., Cresci A., Rowland I.R., Resistant starch
modifies gut microflora andmicrobial metabolism inhuman
flora-associated rats inoculated with faeces from Italian andUK
donors. J.Appl. Microbiol., 1999, 86, 521–530.
34. Soral-Śmietana M., Wronkowska M., Resistant starch ofpea ori-
gin. Żywność. Nauka. Technologia. Jakość, 2000, 23, 204–212.
35. Til H.P., Feron V.J., Immel H.R., Vogel W.F., Chronic (89-week)
feeding study with hydroxypropyl distarch phosphate, starch ac-
etate, lactose andsodium alginate inmice. Food Chem. Toxicol.,
1986, 24, 825–834.
36. Topping D.L., Clifton P.M., Short chain fatty acids andhuman
colonic function: roles ofresistant starch andnon-starch poly-
saccharides. Phys. Rev., 2001, 81, 1031–1064.
37. Wolf B.W., Bauer L.L., Fahey G.C., Effects ofchemical modi-
fication on invitro rate andextent offood starch digestion: an
attempt to discover aslowly digested starch. J.Agr. Food Chem.,
1999, 47, 4178–4183.
38. Wolf B.W., Woelver T.M.S., Bolognesi C., Zinker B.A., Garleb
K.A., Firkins J.F., Glycemic response to afood starch estrified
by 1-octenyl succinic anhydride inhumans. J.Agr. Food Chem.,
2001, 49, 2674–2678.
39. Woo K.S., Seib P.A., Cross-linked resistant starch: preparation
andproperties. Cereal Chem., 2002, 79, 819–825.
40. Wronkowska M., Juśkiewicz J., Soral-Śmietana M., Nutritional
andphysiological effects ofnative andphysically – modified
starches ofdifferent origin on rats. Pol. J.Food Nutr. Sci., 2002,
11/52, SI 1, 62–67.
41. Wronkowska M., Soral-Śmietana M., Krupa U., Chapter 9:
Invitro hydrolysed chemically modified potato starch – binding
ofcholesterol andbile acids. 2008, in: Starch: Recent Progress
inBiopolymer andEnzyme Technology (eds. P.Tomasik, E.Ber-
toft, A.Blennow). Polish Society ofFood Technologists. Cracow,
pp. 123–130.
42. Wurzburg O.B., Nutritional aspects andsafety ofmodified food
starches. Nutr. Rev., 1986, 44, 74–79.
43. Younes H., Demigne C., Behr S., Remesy C., Resistant starch
exerts alowering effect on plasma urea by enhancing urea N
transfer into thelarge intestine. Nutr. Res., 1995, 15, 1199–1210.
Received November 2010. Revision received andaccepted Janu-
ary 2011.