Impact of particle size of pulverized citrus peel tissue on changes in antioxidant properties of digested fluids during simulated in vitro digestion


食品科学与人类健康(英文) 2020年1期

Yidi Cai,Wei Qin,Sunantha Ketnawa,Yukiharu Ogawa

Graduate School of Horticulture,Chiba University,648 Matsudo,Matsudo 271-8510,Japan




Recent epidemiological studies have shown that larger amount of consumption of fruits and vegetables can reduce the risk of some chronic diseases[1-4].The fruits and vegetables process could influence their nutritional attributes including biological active compounds[5-9]and antioxidant activities[10],therefore,it is generally considered to be a cause of the degradation of natural food products.This indicates processed fruits and vegetables are regarded as lower health-promoting capacity than fresh ones[11].Thus,assessing the impact of food processing on the natural attribute of foodstuffs is one of the contemporary critical issues connecting to human health[12,13].

Vegetables and fruits are frequently processed by cut,stewed,baked,fried and etc.,to enhance eating quality[14]and/or to improve nutritional quality[15-18].The nutritional compounds of plant-based foods are mostly encapsulated by its cell-matrix,which is compartmentalized by cell walls and cell membranes.A cell wall consists of several soluble and insoluble compounds such as pectin and cellulose,the main constituents of dietary fiber,which cannot be decomposed by human digestive organs[19,20].Thus,the intracellular substances are needed to be eluted as bioavailable nutrients from the mechanical or chemical damaged cell-matrix by the microstructural changes or heat processing[21].

Recently,it was reported that the effect of microstructural changes in plant tissue on the elution of intracellular pigmented substances to several types of solvent using pulverized citrus peel powders classified into the various particle size distribution[22].This report also suggested the impact of microstructural changes of plant tissue on the bioavailability of intracellular pigmented substances that could strongly connect to digestive properties of plant tissue.Moreover,health-promoting compounds like antioxidative substances in foods are chemically unstable than the main nutrients such as carbohydrates because they can easily be oxidized and degraded[23].As a consequence,changes in the antioxidant properties can be used as a measurable parameter to assess such digestive properties of fruits and vegetables.

When applied to food analysis,the antioxidant activity measurements may be different depending on the assay used.Since a single method,however,may not accurately evaluate the antioxidant property,several assays are usually employed for example,1,1-dipheny1-2-picrylhydrazyl(DPPH)and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulphonic acid)(ABTS)free radical scavenging assay are used to detect free-radical scavenging ability based on an electron transfer and involves reduction of a colored oxidant[24].Transition metals,such as Fe2+,are well-known stimulants of lipid peroxidation and their chelation may help to retard the peroxidation and subsequently prevent food rancidity thus metal(ferrous)chelating ability(MIC)could retard those reaction[25].Ferric reducing antioxidant power(FRAP)assay is different from the others as there are no free radicals involved but the reduction of ferric iron(Fe3+)to ferrous iron(Fe2+)is monitored[26].Besides,total phenolic content(TPC)also measured as common antioxidant substances[27].

This study aimed to examine the effect of structural changes of plant tissue on the release of intracellular bioactive compounds during digestion.A flavedo tissue of citrus peel,which is rich in antioxidants such as phenolic compounds in the group of carotenoids and flavonoids[28],was employed as a model of plant tissue.Various size powders of pulverized flavedo tissue were classified into four fractions by its particle size distributions and assumed as the sample materials for different microstructures.Several antioxidant activities of the digestion fluid of the powders during simulated in vitro gastrointestinal digestion,including changes in TPC,were investigated in the current study.

2.Materials and methods

2.1.Materials and sample preparation

Citrus fruits(Citrus unshiu,cv.unknown)were harvested in Kumamoto,Japan,and purchased from the local store in Matsudo,Japan in January 2019,used as a fresh material in this study.The fresh samples were thoroughly washed by tap water and grated from the surface of peel using a grater(Microplane,Ikesyo,Tokyo,Japan)to collect crude tissue of the flavedo layer[22].The collected crude tissue was frozen and preserved at-30°C using a freezer(NW 101953,Haier Japan,Osaka,Japan),and then freezedried using a freeze dryer(FDU-1100,Eyela,Tokyo,Japan).The freeze-dried tissue sample was roughly pulverized using a mortar,and finely pulverized using an electric grinder(EU6820 P,Panasonic,Osaka,Japan).The pulverized tissue powder was sieved using 125,180,355,500 and 710 μm mesh sieves and separated to four groups of various particle size distributions,which were 125-180,180-355,355-500 and 500-710 μm.

2.2.Simulated in vitro gastrointestinal digestion

A simulated in vitro gastrointestinal digestion technique following Donlao’s[29]method was applied for this study with some modifications.The mixture of 19 mL of simulated gastric fluid and 165 mL of distilled water was put into a thermostatic grass beaker(Double tubes reactor,Sansyo,Tokyo,Japan)and the temperature was maintained at 37°C.One gram of sample powder was put into the mixture with stirring using magnetic stirrer and the pH of the powder mixture was adjusted to 1.2 by adding an appropriate concentration of HCl solution.This treatment was finished within 1 min after sample powder adding.The mixture was stirred for 1 h as a simulated gastric digestion stage.A small amount of digested fluid(0.5 mL)was collected at 1,5,10,15,30 and 60 min after the gastric stage was started.After the gastric digestion stage,the pH of the mixture was adjusted to 6.8 by adding an appropriate concentration of NaOH solution.Then,23 mL of simulated small intestinal fluid was put into the mixture,which was stirred for 2 h as a simulated small intestinal digestion stage.The digested fluid was continuously collected at 5,10,15,30,60,90 and 120 min after the small intestinal stage was started.The collected liquid was placed into a centrifuged tube and immediately mixed with 4.5 mL of 70%(V/V)methanol to terminate the enzyme reactions.The mixed liquid sample was centrifuged at 4000×g for 10 min,and then the supernatant was collected and used to determine the total phenolic content(TPC)and antioxidant activities.The sediments after centrifuge were used for microscopic observation.


To observe changes in the apparent properties of sample powders during simulated digestion,the digested powder samples were collected at 60 min(end of the gastric digestion stage),120 min(after 1 h for the small intestinal digestion began)and 180 min(end of the small intestinal stage).The collected powder samples were mixed with a tiny amount of distilled water and placed on a glass slide and covered by a cover slip,then observed with a simple observation mode of inverted microscope built-in laser scanning microscope system(LSM510,Carl Zeiss,Oberkochen,Germany).

Particle diameter was measured as a circumscribed circle equivalent diameter using graphic software(Photoshop CC2019,Adobe,San Jose,CA,USA).Approximately 100 particle images were randomly selected,and the diameter was averaged in this study.To evaluate changes in the color of powder surface during simulated digestion,approximately 100 particles were randomly selected and captured using a digital camera system(Axiovision 4.8,Carl Zeiss,Oberkochen,Germany).The photographic conditions such as exposure time were calibrated and standardized.The RGB represents the color composition in the microscope images,namely red(R),green(G),and blue(B).RGB values of individual powders in the captured images were measured and approximately 100 particle images were randomly selected and averaged using the graphic software(Photoshop CC2019,Adobe).

2.4.Total phenolic content(TPC)

Total phenolic content(TPC)was determined following Folin-Ciocalteu[30].10%(V/V)of Folin-Ciocalteu solution and 7.5%(m/V)of Na2CO3solution were prepared.Twenty five microliters(μL)of collected sample solution during simulated digestion was mixed with 125 μL of 10%(V/V)Folin-Ciocalteu solution in a microplate,and then 100 μL of 7.5%(m/V)Na2CO3solution was poured into the mixture.The mixture was allowed to stand at room temperature for 1 h and the absorbance at 740 nm was measured using microplate reader(Multiskan FC,Thermo Fisher Scientific,Waltham,MA).Distilled water was used as a blank.The TPC was presented as a mg gallic acid equivalent(GAE)per one gram of dried sample powder.

2.5.DPPH radical scavenging activity

A DPPH assay was conducted applying the Brand-William’s method[31]with some modifications,50 μL of sample solution was mixed with 1.95 mL of 60 μmol/L DPPH solution in test tube.The mixture was allowed to stand for 30 min in dark at room temperature.Then,200 μL of the mixture was transferred to microplate and the absorbance at 520 nm was measured using microplate reader(Multiskan FC,Thermo Fisher Scientific).Methanol was used as a blank.The DPPH value was presented as a μmoL Trolox equivalent per one gram of dried sample powder.

Fig.1.Appearance of sample powders for the various size fractions for freeze-dried and several digestion stages.a-d:freeze-dried,e-h:digested for 60 min(60 min at gastric stage),i-l:digested for 120 min(60 min at smallintestinal stage),m-p:digested for 180 min(120 min at smallintestinal stage);a,e,i,m:156 μm,b,f,j,m:211 μm,c,g,k,o:390 μm,d,h,l,p:633 μm.Scale bar shows 300 μm.

2.6.ABTS radical scavenging activity

An ABTS assay was conducted following Floegel method[32]with some modifications.In a microplate,100 μL of sample solution was mixed and stirred with 0.32 mL of a prepared ABTS solution of which adjusted absorbance to between 0.7 and 0.8 at 740 nm was measured using spectrophotometer(V-630bio,Jasco,Tokyo,Japan).The mixture was placed in dark at 36°C for 10 min and then measured its absorbance at 740 nm using microplate reader(Multiskan FC,Thermo Fisher Scientific).Distill water was used as a blank.The ABTS value was presented as a mg ascorbic acid equivalent per one gram of dried sample.

2.7.Metal ion(ferrous)chelating ability(MIC)

An MIC assay was conducted following Gülc¸in method[33]with some modifications.Three milliliters of sample solution was mixed and stirred with 50 μL of 2 mmol/L FeCl2solution and 100 μL of 5 mmol/L ferrozine solution in test tube.The mixture was allowed to stand for 10 min in dark at room temperature.Then,transferred 315 μL of the mixture to microplate and the absorbance at 560 nm was measured using microplate reader(Multiskan FC,Thermo Fisher Scientific).Distilled water was used as a blank.The MIC value was presented as a μmol ethylenediaminetetraacetic acid(EDTA)equivalent per one gram of dried sample powder.

2.8.Ferric reducing antioxidant power(FRAP)

A FRAP assay was conducted following Benzie’s method[34]with some modifications.Three hundred mmol/L acetate buffer(pH 3.6),10 mmol/L TPTZ solution and 20 mmol/L FeCl3solution were mixed at a ratio of 10:1:1(V/V)as a FRAP reagent.Twenty microliters of sample solution was mixed and stirred with 130 μL of FRAP reagent in a microplate.The mixture was incubated at 37°C for 30 min,and then the absorbance at 595 nm of the mixture was measured using microplate reader(Multiskan FC,Thermo Fisher Scientific).Distilled water was used as a blank.The FRAP value was presented as a μmol FeSO4equivalent per one gram of dried sample powder.

2.9.Statistical analysis

All data are reported as means±standard deviations over three replications.t-test was applied for the results of particle diameters and RGB values to determine significant differences between freeze-dried and digested sample powders at a significance level of P<0.05 using R software(R for windows x64,version 3.4.2).

3.Results and discussion

3.1.Appearance of particles

Fig.1 depicts the appearance of sample powders and cells for the various particle fractions at each gastrointestinal digestion stage.A diameter of the circumscribed circle of a particle image was regarded as particle diameter and cell diameter in this study.Approximately 100 particle images were randomly selected and calculated the averaged diameter.The calculated result is shown in Table 1.According to the significant differences(P>0.05)between freeze-dried and digested sample powders,there were no obvious changes in the particle size and cell size due to the simulated digestion by which powders could absorb the digestive liquids.The shape of powders also tended to be stable even it was digested.

Table 1 Averaged particle diameters and cell diameters for the various particle size fractions at before and after the simulated in vitro gastrointestinal digestion.

Table 2 RGB values of particle surface for the various particle size fractions at before and after the simulated in vitro gastrointestinal digestion.

In contrast,RGB values of sample powders,particularly,B values of all particle size ranges significantly changed between the freezedried sample,which is considered as a sample before digestion,and the digested sample as shown in Table 2.This could be considered that the pigmented compounds connecting to B values were eluted from the cell-matrix of the citrus peel tissue during simulated digestion.However,the powders for larger particle fractions(390 μm and 633 μm)showed no significant differences in R and G values between before and after digestion,although the smaller powders(156 μm and 211 μm)showed significant changes.These results indicated that the particle size,which was micron scale but larger than individual cell size,could influence the degree of elution of selective intracellular substances from the tissue during digestion.

3.2.Total phenolic content(TPC)

Fig.2 depicts changes in the TPC values of digested fluid from each particle size fraction during simulated gastrointestinal digestion.The phenolic compounds could be eluted from sample powders at 1 min of digestion,and it tended to be continuously stable during digestion.Particularly at 1 min of digestion,which can be considered that sample powders were just after soaking in the gastric fluid,the larger particle size fraction tended to show higher TPC value than the smaller fraction.According to the results for the appearance of powders shown in Table 2,the RGB values for the smaller sample powders were mostly decreased.This could indicate that the most pigmented substances of smaller powders should be eluted.In contrast,the larger sample powders could maintain more pigmented substances than the smaller powders.These results regarding the effect of particle size were not correspondent with the result for the TPC values shown in Fig.2.It might suggest that the color of digested citrus peel powders had not so strongly relations with the eluted TPC values.

Fig.2.Changes in the total phenolic content(TPC)in digested fluids from the various size fractions of sample powders during simulated digestion.

3.3.Antioxidant activities

Fig.3 depicts changes in the DPPH,ABTS,MIC and FRAP values of the digested fluid for each particle size fraction during simulated gastrointestinal digestion from 1 min of the gastric stage to the end of the digestion.The simulated gastric digestion stage was the first 60 min in which the larger particle size fractions tended to show not significantly but comparatively higher values than the smaller fractions.This result could follow the result of TPC as shown in Fig.2.However,the values of the antioxidant activities for the digestive fluid of various powder size fractions,particularly for the DPPH and MIC,showed drastically changes after 60 min of gastric digestion.Because the concentration of the dissolved TPC in the digestive fluid would not decrease[35]and the pH at the start of simulated small intestinal digestion was adjusted to 6.8,it could be due to the pH-dependency of the assays[36].At this moment on the pH change,the values for the DPPH and FRAP assays were decreased,but the MIC assay showed an increasing trend.Despite the value of the ABTS in the gastric step was unstable,it also showed a slight decrease at the moment for the pH change.This trend could be due to the comparatively lower dependence on the pH of the ABTS[37].In the meanwhile,the values for those were mostly stable during the small intestinal digestion stage and there were not obviously shown the dependency of powder size fractions.

Most substances in plant tissue are enveloped in cell-matrix which consists of the plant cell wall.The plant cell wall is mainly contained cellulose,which is an indigestible substance for human digestive system.Although the plant cell wall could be supposed to allow penetrating most materials,it can be realistically classified two types of individual cell conditions for the cell-matrix of the sample powders;one is a disrupted cell damaged by pulverizing at the fringe portion of the particles(Damaged)and the another is an intact cell at the inner part of the particles(Intact)as shown in Fig.4.A previous study[22]on the relationship between the particle size of pulverized plant tissue and the elution property of intracellular substances reported that the smaller particles had the higher eluted percentage of intracellular pigmented substances than the larger particles,which had lower cell disruption percentages.Therefore,it was assumed that the eluted TPC in the digested fluid which could be considered as pigmented substances derived from the inside of the cell-matrix of sample peel powders would show the dependency on the particle size.It was also considered that the eluted TPC could be related to the changes in the antioxidant activities of digested fluids during digestion.However,the result in this experiment indicated the TPC and several antioxidant activitiies had no dependency on the particle size fractions.Furthermore,the antioxidant activities of the digested fluid of larger particles during gastric digestion tended to be higher.In spite of these results,the degree of pulverization for plant tissue might relate to the sustain of antioxidant activities,particularly at the gastric digestion stage for the intracellular substances of intact cells,which would have less oxidative.This also suggested that the finer powders of plant-based foods might have lower antioxidant activity during digestion.

Fig.3.Changes in the values of DPPH,ABTS,MIC and FRAP in digested fluids from the various size fractions of sample powders during simulated digestion(A:DPPH,B:ABTS,C:MIC,D:FRAP).

Fig.4.Schematic diagram of mechanical damages for the citrus peel tissue.


The various particle size samples employed in this study can be regarded as one of the structural attributes for the plant-based foods that strongly connects to the processing conditions.Although the result of changes in RGB values for the digested powders during digestion was not correspondent to,the particle sizes would influence on the changes in antioxidant activities during digestion,especially at the gastric digestion stage.Our result showed the larger particles,which involves larger numbers of the intact cell,tended to show the higher antioxidant activities of their digested fluid.This suggested the lower percentages of damaged cells in the plant tissue could be sustained its antioxidant activities.In other words,the intracellular substances of the intact cell might be protected from the oxidative reactions even if it was processed before intake.

Ethics statements

Our research did not include any human subjects and animal experiments.

Declarations of Competing Interest

The authors declare no conflict of interest.The Watanuki International Scholarships Foundation had no role in study design,data collection,or analysis.The authors alone are responsible for the content and writing of the paper.


The authors are grateful to the Watanuki International Scholarships Foundation for providing a scholarship to the first author.