RESEARCH ARTICLEImpact of acid and heat –moisture treatment combination on physicochemical characteristics and resistant starch contents of sweet potato and yam starches Pham Van Hung1,
Trang 1RESEARCH ARTICLE
Impact of acid and heat –moisture treatment combination on
physicochemical characteristics and resistant starch contents
of sweet potato and yam starches
Pham Van Hung1, Nguyen Thi Huyen My1and Nguyen Thi Lan Phi2
1
School of Biotechnology, International University, Vietnam National University in HoChiMinh City, HoChiMinh City, Vietnam 2
Faculty of Chemical Engineering, HoChiMinh City University of Technology, HoChiMinh City, Vietnam
The objective of this study is to investigate formation of slowly digestible starch (SDS) and
resistant starch (RS) and change in physicochemical properties of sweet potato and yam
starches under a combination of acid and heat–moisture treatments using three mild
organic acids including acetic acid, lactic acid and citric acid and heating temperature at
110°C for 8 h The results show that the SDS and RS in sweet potato starch significantly
increased from 6.6 and 14.7% in native starch to 8.7–13.2% and 37.5–42.1% in acid and
heat–moisture treated starches, respectively Likewise, the SDS and RS in yam starch
increased from 4.7 and 21.6% in native starch to 10.0–11.3% and 39.0–46.4% in the treated
starches, respectively The RS content in the acid and heat–moisture treated starches was
also significantly higher than that of the heat–moisture treated starches without acid
hydrolysis Yam starch produced higher amount of RS under acid and heat–moisture
treatment as compared to sweet potato starch at the same condition Swelling power and
viscosity of starches significantly decreased, whereas the solubility significantly increased
after treatments The citric acid had the most impact on RS formation and starch properties,
followed by lactic acid and acetic acid As a result, the combination of acid and heat–moisture
treatment is a useful method to produce higher amount of resistant starch, which can be
applied for functional foods
Received: May 29, 2014 Revised: July 8, 2014 Accepted: July 14, 2014
Keywords:
Acid hydrolysis / Heat–moisture treatment / Resistant starch / Sweet potato / Yam
1 Introduction
Starch, the most abundant reserve carbohydrate of many
plants, is also a major component of many food products
Starch in foods after consumption can be classified into
three types based on their digestibility: a rapidly digestible
starch (RDS), slowly digestible starch (SDS) and resistant
starch (RS) [1] RDS and SDS are completely digested and absorbed in the human small intestine that cause the increase in blood glucose level, whereas RS escapes digestion in small intestine and does not contribute to the blood glucose level of healthy individuals Recently, the SDS and RS have received much attention for both its potential health benefits and functional properties The SDS are known to have potential health benefits in a stable glucose metabolism, diabetes management, mental perfor-mance, and satiety [2] The SDS may be used by athletes to provide a longer-lasting source of systemic glucose [3, 4] Although RS is not digested, it is fermented in the large intestine by human microflora to produce short-chain fatty acids The potential health benefits of RS have been reported
as prevention of colon cancer, hypoglycemic effects, substrate for growth of the probiotic microorganisms,
Correspondence: Dr Pham Van Hung, School of Biotechnology,
International University, Vietnam National University, Quarter 6,
Linh Trung Ward, Thu Duc District, HoChiMinh City, Vietnam
E-mail: pvhung@hcmiu.edu.vn
Fax: þ84-8-37244271
Abbreviations: HMT, heat –moisture treatment; RDS, rapidly
digestible starch; RS, resistant starch; SDS, slowly digestible
starch; SPS, sweet potato starch; YS, yam starch
Trang 2reduction of gall stone formation, hypocholesterolemic
effects, inhibition of fat accumulation, and increased
absorption of minerals [5] Therefore, the recent studies
focus on production of high amounts of RS from various
starch sources and application of RS as a“low-carbohydrate”
ingredient in food formulations [3]
SDS and RS can be produced by various modification
techniques including physical, chemical, and enzymatic
modifications Heat–moisture treatment (MHT) is a
well-known hydrothermal method to increase the levels of SDS
and RS in starches without destroying their granular
structure [6] In HMT, starch granules are treated at low
moisture levels (<35% moisture, w/w) for a certain time
period (15 min–16 h) and at high temperatures (84–
120°C) [7] Chung et al [8] reported that the RDS decreased
by 10.2, 14.0, and 15.1%, the SDS content increased by 2.5,
2.8, and 4.7%, and the RS content increased by 7.7, 11.2, and
10.4% for corn, pea, and lentil starches, respectively, when
these starches were heat–moisture treated at 120°C as
compared to native starches The SDS contents of maize,
potato, cocoyam, yam, plantain, rice, and sweet potato under
hydrothermal treatments also increased as compared to the
native starches [9, 10] The increase in thermo-stable SDS
and RS suggests that some interactions formed during
hydrothermal treatments may have survived after
gelatini-zation, thereby partly restricting accessibility of starch
chains to the hydrolysing enzymes [8] The different
chain-length distribution and crystallinity of starch were key
factors for improvement of SDS and RS of treated starch
Hung et al [11] reported that the debranched potato starch
with significantly higher chain length (35.4 glucose units)
formed double helices with more dense crystalline structure
resulting in more resistance to enzyme digestion as
compared to the debranched cassava starch having the
chain length about 32.4 glucose units Partial acid hydrolysis
of starches prior heat–moisture treatments improved RS
yield over the heat–moisture treatments without acid
hydrolysis [12, 13] Rice starch treated with citric acid
followed by heat treatments reduced its RDS content, but
increased its SDS content as compared to control and native
starches [14] The internal structure and physicochemical
properties of the acid and heat treated starches were also
changed such as producing more various short chains,
forming different crystallites that have different melting
temperatures, increasing in apparent amylose content and
cold-water solubility, and decreasing in viscosity and
gel-forming ability [14, 15] Thus, the acid and heat–moisture
treatment not only improves the SDS and RS contents of
starch, but also changes its physicochemical properties In
this study, the effects of a combination of acid and heat–
moisture treatments using various organic acids including
acetic, lactic and citric acids on the formation of SDS and RS,
and physicochemical properties of sweet potato and yam
starches are investigated
2 Materials and methods
2.1 Materials Two kinds of fresh tubers, purple sweet potato (Ipomoea batatas) and yams (Dioscoreaceae atatas) grown in Dong Thap and Long An provinces of Vietnam, respectively, were used in this study and their starches were isolated The purple sweet potato and yam tubers were harvested after planting approximately 3–4 months, depending on each cultivar, soil and growing conditions
Alpha-amylase from A oryzae (30 U/mg) and amylo-glucosidase from A niger (300 U/mL) used in this study were purchased from Sigma–Aldrich Co (St Louis, MO, USA) Other chemicals were purchased from Merck Co (Darmstadt, Germany)
2.2 Starch isolation The starch was isolated according to the method of Lawal [16] with minor modification This traditional method has been widely used to produce the commercial starch products in the traditional craft villages in Vietnam without using chemicals or enzymes to purify starch The purple sweet potato and yam tubers were washed with tap water to remove any type of contamination The cleaned tubers were peeled, sliced and ground with small volumes of distilled water using a Waring blender (7015N, Waring1 Commercial, USA) Then the homogenate was passed through a sieve of 0.232 mm in aperture size The extraction was repeated three times and then the resultant starch slurry wasfiltered through the sieves (0.232 and 0.105 mm in aperture size) and centrifuged at 1500g for 20 min After removing the supernatant, the sediment was washed thoroughly
in distilled water for three times Finally, the starch sediment was recovered and dried in an oven at 40°C for
24 h to 10–11% moisture
After isolation, the starches were analyzed for their chemical composition to evaluate their purity Amylose content of starch was determined according to the method previously descried by Hung et al [17] Protein content was determined using a Kjeldahl digestion system (KI 26, Gerhardt, Germany) based on the standard AACC Approved Method 46-10 [18] Lipid contents were determined by extraction with hexane for 6 h using a Soxhlet apparatus (EV6, Gerhardt, Germany) and ash content was determined by burning in a muffle furnace at 550°C for 3 h [18] Total starch was calculated as follows: total starch (%, db)¼ 100% protein content (%, db) lipid content (%, db) ash (%, db) 2.3 Acid and heat–moisture treatments of starches The isolated sweet potato and yam starches (100 g) were directly weighed into screw capped bottles and then
Trang 3moisture content of sample was adjusted to 30% by
adding a measured volume of each acid solution (0.2 M
lactic acid, 0.2 M acetic acid, or 0.2 M citric acid) After
dispersion, the bottles were equilibrated at room temperature
for 24 h before heating at 110°C for 8 h After heat–
moisture treatment, the starch samples were neutralized
with 1 M sodium hydroxide and then washed thoroughly
with distilled water The treated starches were recovered
by centrifuging at 10,000g for 30 min and then dried at 40°C
for 24 h
2.4 Determination of starch fractions (RDS, SDS, RS)
Rapid digestible starch (%RDS), slowly digestible starch (%
SDS) and resistant starch (%RS) of the native and treated
sweet potato and yam starches were measured based on the
method of Englyst et al [1] with a moderate modification as
follows Starch (0.3 g, db) was mixed with 20 mL of sodium
acetate buffer (pH 6.0) and then boiled for 30 min in a water
bath The sample was equilibrated at 37°C for 15 min and
then an enzyme solution (5 mL) ofa-amylase (1400 U/mL)
and amyloglucosidase (13 AGU/mL) was added The starch
solution was incubated with shaking at 37°C for 120 min
The total glucose concentrations of the 20 min-digested
and 120 min-digested hydrolysates (G20 and G120,
respec-tively) were determined using the phenol–sulfuric acid
method The remained residue was intensively hydrolyzed
with 7 M KOH and then with amyloglucosidase (50 AGU/
mL) Thefinal hydrolysate was then determined for total
glucose concentration (TG) The values obtained for G20,
G120, and TG were used to calculate for RDS, SDS, and RS
as follows
RDS¼ G20 0:9
SDS¼ ðG120 G20Þ 0:9
RS¼ ðTG G120Þ 0:9
2.5 Determination of degree of polymerization of
starch
The number-average degrees of polymerization (DPn) of
native and treated starches were determined by the method of
Hizukuri et al [19] DPn was calculated as the difference
between reducing residues and total glucose concentration of
the starches [20]
2.6 Determination of X-ray crystallization of starch
X-ray diffraction analysis was performed using an X-ray
diffractometer (Rigaku Co., Ltd, Rint-2000 type, Tokyo, Japan)
operated at 40 kV and 80 mA Diffractograms were obtained
from 2° 2u to 35° 2u with a scanning speed of 8o/min and
scanning step of 0.02o[20]
2.7 Determination of swelling power of starch Swelling powers of native and treated sweet potato and yam starches were determined according to the method of Sasaki
et al [21] with a slight modification The starch sample (0.16 g, db) was dispersed with distilled water (5 mL) and then heated from 50 to 90°C at 10°C intervals After keeping at those temperatures for 30 min, the heated sample was quickly cooled to room temperature in cold water and centrifuged at 3000g for 15 min The supernatant was carefully removed and swelling power was calculated as described by Hung
et al [17]
2.8 Determination of solubility of starch Solubility (%) of starch was determined according to the methods described by Leach et al [22] with a slight modification The native and treated sweet potato and yam starches (0.5 g) was suspended in 30 mL of distilled water in
50 mL-falcon tubes and heated in a thermostatically controlled water bath for 30 min at different temperature range from 50 to 90°C at 10°C intervals Then the tubes were rapidly cooled to room temperature before centrifuging at 1500g for 30 min Then the supernatant (soluble starch) was poured into an aluminum dish and dried at 120°C for
4 h The solubility of starch was calculated following the formula:
Solubilityð%Þ ¼ m2 m1ðgÞ
mass of starchðgÞ 100 where: m1, the aluminum dish weight; m2, the weight of aluminum dish containing the soluble starch
2.9 Determination of viscosity of starch solution Viscosity of native and treated starches was measured using
a Brookfield Viscometer LVDV-E (Brookfield Engineering Laboratories, USA) according to the official method described by the International Starch Institute with slight modification [11] A starch slurry (2% starch) was cooked in
a boiling water bath for 15 min with continuously stirring and additional 15 min without stirring The paste was cooled down to 50°C and measured the viscosity in centipoises (cP) at 50°C with spindle SC4-18 at a speed
of 50 rpm
2.10 Statistical analysis All tests were performed at least in duplicate Analysis of variance (ANOVA) was performed using Duncan’s multiple-range test to compare treatment means at P< 0.05 using SPSS software (SPSS Inc., USA)
Trang 43 Results and discussion
3.1 Chemical composition of isolated starches
Chemical compositions of starches isolated from sweet
potato and yam tubers are shown in Table 1 The amylose
contents of sweet potato and yam starches were 18.7 and
22.3%, respectively The higher amylose content of yam
starch than that of sweet potato starch was also reported by
Hoover [23] The difference in amylose contents of sweet
potato starch and yam starch might affect RS formation
during acid and heat–moisture treatments and the
physico-chemical properties of the treated starches The native
starches of sweet potato and yam starches isolated by repeated
dispersion and sedimentation in distilled water contained
high levels of proteins (1.1 and 0.8%, respectively) and lipids
(0.9 and 1.3%, respectively), but low amount of ash content
(0.1%) The purity of the isolated sweet potato and yam
starches was not significantly different (97.9 and 97.8%,
respectively)
3.2 Starch fractions (RDS, SDS, RS) of native and
treated starches
Amounts of rapid digestible starch (RDS), SDS, and RS of
native and treated sweet potato and yam starches are given in
Table 2 In native form, sweet potato starch contained higher
amount of RDS and SDS, but lower amount of RS as
compared to those of yam starch The amount of SDS of sweet
potato and yam starches did not change after heat–moisture
treatment, whereas the amount of RS of these starches
significantly increased The higher amount of RS in the heat–
moisture treated yam starch than the heat–moisture treated
sweet potato starch was also observed when these starches
were treated at the same condition A combination of acid and
heat–moisture treatments significantly increased amounts of
SDS and RS as compared to the native or heat–moisture
treatment only For sweet potato starch, the amount of SDS
increased from 6.6% in native form to 13.2, 8.7, and 11.5%
after heat–moisture treatment combined with lactic acid,
acetic acid and citric acid, respectively, while the amount of
RS raised from 14.7 to 40.1, 37.5, and 42.1% at the same treatment condition For yam starch, the amount of SDS increased from 4.7 to 10.0, 11.6, and 11.3% and the amount of
RS increased from 21.6 to 41.0, 39.0, and 46.4% after lactic acid, acetic acid and citric acid and heat–moisture treatments, respectively The results in this study also indicate that treatment with citric acid produced the highest amounts of
RS in sweet potato starch and yam starch (42.1 and 46.4%, respectively), followed by treatment with lactic acid (40.1 and 41.0%, respectively) and with acetic acid (37.5 and 39.0%, respectively) The previous study also reported that high amount of RS (57.5%) in acid-treated lentil starch was obtained by spraying with 2.2 M HCl and incubated at 140°C for 3 h [24] Shin et al [14] also reported that treatment of rice starch with citric acid followed by heat treatments reduced its RDS content, but increased its SDS content as compared to control and native starches The citric acid-treated starch was found to have more various short chains, which are composed of linear or branched chains than did native rice starch [14, 15] The internal structure of the acid-treated rice starch was more or less spherical and composed of a number of double helices [15] Thus, the formation of RS after heat–moisture treatment is due to either improve the order of the crystalline fraction or enhance the proportion of this fraction in starch granules [6] Moreover, the partial acid hydrolysis of starches prior heat–moisture treatment can enhance the mobility of the molecules and allow more
efficient rearrangement, therefore, improved RS yield over the heat–moisture treatments without acid hydrolysis [12, 13] The results show that amount of RS in the treated yam starch was higher than that in the treated sweet potato starch
at the same treatment condition Thus, the differences in internal structures and amylose contents of sweet potato and yam starches affected the formation of RS in these starches during acid and heat–moisture treatments
Table 1 Chemical composition (%, w/w, db) of sweet potato
and yam starches
Amylose content (%) 18.7 0.5 22.3 0.3
SPS, sweet potato starch; YS, yam starch.
All data presented as mean of duplicate experiments SD.
Table 2 RDS, SDS, and RS contents (%, w/w, db) of native and treated sweet potato and yam starches
Treatment
Native 78.7d 6.6a 14.7a 73.8d 4.7a 21.6a HMT 66.1c 6.7a 27.2b 64.1c 4.9a 31.0b LA–HMT 46.6 a 13.2 d 40.1 d 49.0 b 10.0 b 41.0 d AA–HMT 53.8b 8.7b 37.5c 49.4b 11.6c 39.0c CA–HMT 46.4a 11.5c 42.1e 42.3a 11.3c 46.4e SPS, sweet potato starch; YS, yam starch; LA –HMT, lactic acid and heat –moisture treatment; AA–HMT, acetic acid and heat–moisture treatment; CA –HMT, citric acid and heat–moisture treatment; RDS, rapid digestible starch; SDS, slowly digestible starch; RS, resistant starch.
Data followed by the same superscript letter in the same column are not significantly different (P 0.05).
Trang 53.3 Degree of polymerization of native and treated
starches
Table 3 shows the degrees of polymerization (DPs) of native
and acid-treated sweet potato and yam starches The results
indicate that the sweet potato starch contained longer average
chain length than did the yam starch The longer average
chain length of sweet potato starch as compared to the yam
starch was due to the lower amylose content and the higher
branched chain length distribution of amylopectin existed in
sweet potato starch than those in yam starch [25]
The treatment of the starches with organic acids under
heat–moisture treatment significantly reduced their DPs as
compared to the native starches The DPs of sweet potato
starch reduced from 1059.2 glucose units to 123.3, 370.1, and
49.2 glucose units after treating with lactic acid, acetic acid,
and citric acid, respectively Likewise, the DPs of yam starch
also reduced from 683.5 glucose units to 56.3, 234.7, and 30.6
glucose units after treating with lactic acid, acetic acid and
citric acid, respectively Shin et al [15] reported that the citric
acid-treated starch contained the short chain-length
mole-cules derived from both linear and branched chains from
amylopectin and linear chains from amylose Thus, the
amylose and amylopectin in sweet potato and yam starches
were partially hydrolyzed at different degrees depending on
different kinds of organic acids The results in this study
agreed with other previous reports [15, 26, 27] Among three
organic acids used, the citric acid had the highest impact on
hydrolyzing of hydrocarbon chains in both sweet potato and
yam starches resulting in the lowest DPs, followed by lactic
acid and acetic acid Thus, the increase in RS contents of the
acid and heat–moisture treated starches was due to the
presence of the low-molecular-weight hydrolysates (both
branched and linear structures of amylose and amylopectin)
produced by acid hydrolysis The formation of double helices
and compartmentalization of amylose–amylose,
amylopec-tin–amylopectin, and amylose–amylopectin chains during
heat–moisture treatment were considered to resist to enzyme
hydrolysis, therefore, increase the amounts of SDS and RS in
the treated starches [8, 28]
3.4 X-ray crystallization of starches X-ray diffraction patterns of native and treated sweet potato and yam starches are shown in Fig 1 The native sweet potato starch exhibited the A-type crystal with the major peaks at around d-spacings 5.8 Å (line 3b), 5.2 and 4.8 Å (line 4a, 4b) and 3.8 Å (line 6a), whereas the native yam starch showed the B-type crystal with a peak at 15.8 Å (line 1), a broad medium intensity lines at about 5.9 Å (line 3a, 3b), a strong line at 5.2 Å (line 4a) and a medium intensity double at 4.0 and 3.7 Å (lines 6a, 6b) as classified by Zobel [29] These results are consistent with the previously reported [27] After acid and heat–moisture treatment, the crystalline structure of sweet potato starch, having the A-type crystal, did not change the X-ray diffraction pattern However, the B-type crystalline structure of yam starch after treatment with acid and heat was changed into the C-type structure, which is close to the A-type crystal structure The similar results were obtained for all treated starches The change in crystalline structure of starch from B-type
to A-type after heat–moisture treatment was also observed for potato and yam starches, while other types of starches such as taro, cassava and cereal starches did not have an altered X-ray diffraction pattern after heat–moisture treatment [7, 30] In this study, the partial hydrolysis by organic acids did not affect the crystalline structure of starches even though more short chain-length molecules were produced
3.5 Swelling powers of native and treated starch Figure 2 shows the results of swelling powers of the native and treated sweet potato and yam starches In the native form, the swelling power significantly increased when the heating temperatures increased from 60 to 90°C After acid treatment, the swelling power of the treated starches significantly reduced as compared to that
of the native starch The results indicate that swelling power of the acetic acid-treated slightly increased when heating temperature was higher than 70°C, whereas the starches treated with lactic acid or citric acid hardly swelled even though heating at 90°C The previous studies also reported that the reduction of pasting properties of the acid-treated starches was due to partial acid hydrolysis of amylose and amylopectin in both crystalline and amorphous regions [14, 31] Thus, the less water-holding capacity of the acid and heat–moisture treated starches was due to the presence of the short chain-length molecules produced by acid hydrolysis In addition, the heat–moisture treatment also enhanced interactions between amylose and amylopectin molecules, strength-ened intramolecular bonds and formation of amylose–lipid complexes resulting in reduction in swelling power of the treated starched [32]
Table 3 Degree of polymerization (DPn) of native and treated
sweet potato and yam starches
SPS, sweet potato starch; YS, yam starch; LA –HMT, lactic acid and
heat –moisture treatment; AA–HMT, acetic acid and heat–moisture
treatment; CA –HMT, citric acid and heat–moisture treatment.
Trang 63.6 Solubility of native and treated starches
The solubility degrees of native and treated sweet potato and
yam starches are given in Fig 3 The results show that the
solubility of starch significantly increased after acid
treatment The solubility degrees of starches treated with
different organic acids increased in order: acetic acid< lactic
acid< citric acid These results are positively related to the
DPs of the treated starches Thus, the increase in the solubility of the treated starches was due to the high amount
of short chain amyloses produced by acid hydrolysis, which easily dissociate and diffuse out of granules during swelling [33] In addition, the results also indicate that the solubility degrees of the native and treated potato starches significantly increased at temperature more than 70°C, whereas the solubility degrees of the native and treated yam
Figure 1 X-ray diffraction pattern of native and treated sweet potato and yam starches SPS, sweet potato starch; YS, yam starch; LA-HMT, lactic acid and heat –moisture treatment; AA-HMT, acetic acid and heat–moisture treatment; CA-HMT, citric acid and heat–moisture treatment.
Trang 7starches significantly increased when starch solution was
heated at more than 80°C
3.7 Viscosity of starch solution
The viscosities of 2% starch solutions measured using a
Brookfield Viscometer LVDV-E are shown in Fig 4 In the
native form, the native sweet potato starch had significantly
lower viscosity than did the yam starch The results also show
that the viscosities of starch solutions significantly reduced
when starches were treated with different organic acids The
reduction in viscosity was also observed by Haros et al [31] for corn treated with lactic acid and by Shin et al [14] for rice treated with citric acid Thus, the presence of more short chain molecules produced by partial hydrolysis of starch using organic acids resulted in lower the viscosities of the treated starches
4 Conclusions
In this study, organic acids including acetic acid, lactic acid and citric acid were used to partially hydrolyze sweet potato
Figure 2 Swelling power (g/g) of native and treated sweet potato
and yam starches SPS, sweet potato starch; YS, yam starch; LA –
HMT, lactic acid and heat –moisture treatment; AA–HMT, acetic
acid and heat –moisture treatment; CA–HMT, citric acid and heat–
moisture treatment.
Figure 3 Solubility (%, w/w) of native and treated sweet potato and yam starches SPS, sweet potato starch; YS, yam starch; LA – HMT, lactic acid and heat –moisture treatment; AA–HMT, acetic acid and heat –moisture treatment; CA–HMT, citric acid and heat– moisture treatment.
0
5
10
15
20
25
30
Sample
LA-HMT AA-HMT CA-HMT
Figure 4 Viscosity (cP) of native treated sweet potato and yam starches SPS, sweet potato starch; YS, yam starch;
LA –HMT, lactic acid and heat–moisture treatment; AA –HMT, acetic acid and heat– moisture treatment; CA –HMT, citric acid and heat –moisture treatment.
Trang 8and yam starches under heat–moisture treatment By a
combination of acid and heat–moisture treatment, the SDS
and RS contents of the starches significantly increased as
compared to the native starch or heat–moisutre treated starch
(control) The physicochemical properties of the starches
were also changed after acid and heat–moisture treatments
The high amounts of SDS and RS in the treated starches are
desired to produce the low-carbohydrate foods, which have
many benefits for diabetes and overweight’s patients As a
result, the combination of acid and heat–moisture treatment
is a useful method to produce higher RS content applied in
functional food processing
This research is funded by the National Foundation for Science
and Technology Development (NAFOSTED) of Vietnam under
grant number 106.99-2012.26
The authors have declared no conflict of interest
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