Effect,of,the,degree,of,milling,on,the,microstructure,and,composition,of,japonica,rice

Jie Liu,Yani Wu,Huihui Chen,Hongzhou An,*,Yu Liu,Jie Xu

a College of Food Science and Engineering,Henan University of Technology,Zhengzhou 450001,China

b Henan International Joint Laboratory of Grain Quality Analyzing and Processing,Zhengzhou 450001,China

c COFCO Engineering&Technology(Zhengzhou)Co.,Ltd.,Zhengzhou 451150,China

d Wilmar(Shanghai)Biotechnology Research&Development Center Co.,Ltd.,Shanghai 200137,China

Keywords:Degree of milling Loss of grain weight Residual bran Residual constituents

A B S T R A C T The effect of the degree of milling(DM)on the distribution of the residual bran layer on the surface of rice grains and subsequent rice quality is essential for rice milling technology.This study used scanning electron microscopy(SEM)and energy dispersive spectroscopy(EDS)to examine the microstructure of japonica rice grains,the microstructural changes in different bran portions after milling,and the content and distribution changes of the elements and nutrients in rice after milling to identify the components that can quantify bran residue according to DM.The SEM results revealed that the thickness of the bran layer and the depth of the grooves in different parts of a rice grain varied,while the depth of the grooves differed between varieties.When DM was 12%,aleurone layer(Al)cells embedded in endosperm(En)remained in the dorsal area.It was speculated that this was the reason why there was no significant difference in bran degree when the DM estimated via grain weight loss increased from 10%to 14%(P＞0.05).The EDS elemental mapping image showed the qualitative distribution of the elements,which was condensed in the bran and uniform in the En.The compositional changes revealed that milling caused the loss of rice nutrients,and the loss rate of DM at 0-10%was more serious than that of DM at 10%-14%.Higher DM(＞10%)could remove marginal bran from the dorsal groove.Therefore,the crude protein,ash,crude fat,dietary fiber,vitamin B1(VB1),and vitamin B2(VB2)content did not change significantly(P＞0.05),while the magnesium(Mg),kalium(K),and phosphorus(P)levels decreased substantially(P＜0.05).The microstructural and compositional changes indicated that the DM estimated via the grain weight loss increased from 2%to 10%,with the bran decreasing progressively.The DM estimated via the grain weight loss increased from 10%to 14%,with minimal changes in the residual bran in the dorsal grooves of the rice grains,while the Mg,P,and K levels presented precise DM indices during the rice milling process.This result will provide a theoretical reference for the accurate evaluations of DM.

Rice is an important food source,with the second largest yield in the world.It is a staple food for more than half of the global population[1,2].Postharvest loss reduction is essential for ensuring food security and long-term sustainable development.The consumption of overmilled rice is now considered one of the causes of“diseases of affluence”.Therefore,the effect of the degree of milling(DM)on the rice quality is essential for developing rice milling technology.

The quality of rice depends on its structure and composition.Brown rice differs significantly from milled rice in terms of appearance,composition,functionality,and nutritional content[3].The aleurone layer(Al)in brown rice is responsible for absorption during caryopsis development,as well as for transferring and accumulating nutrients,such as lipids,proteins,and mineral elements[4,5].The fundamental principle of DM testing methods is to determine how much bran is removed during milling.The primary technique is based on the extent of weight loss.Other methods used to estimate the DM can also be based on the loss of some constituents,colour changes in the rice grains,or bran layer staining[6-8].The weight reduction may include the loss of broken endosperm(En),especially for a high DM(＞12%,15%,or even 20%)[9].Therefore,the weight loss method cannot accurately reflect the removal of bran with a high DM.Staining is a convenient way to visualize how much of the bran remains or is removed.However,in the case of overmilling,the branvisualization methods are not sensitive since the residual bran in the dorsal grooves does not change during milling,while the weight loss continues to increase.The selection of representative components under high DM is critical for quantitatively estimating a residual chemical constituent.The cultivars,environment,and fertilizer influence the rice grain shape,density,bran,and composition[10-13].

The use of various methods assumes that the changes during progressive milling occur in more or less an identical or parallel manner.Especially if the loss in grain weight during milling is considered the reference standard,then any proposed method should have a reasonable proportionality with that of loss in grain weight.While there are challenges in the calculation of DM when the methods identify overmilling.It is hypothesized that the microstructure changes of bran layer can reveal the reason of the difference among approaches,and the change of nutrient composition would explore the compatibility of different approaches.

This study investigates the influence of DM on the microstructural changes and elemental distribution of the bran layer via scanning electron microscopy(SEM)and energy dispersive spectroscopy(EDS)while examining the constituent changes in the rice by analyzing the crude protein,ash,crude fat,dietary fiber,vitamin B1(VB1),vitamin B2(VB2),and mineral content.This provides clues to a precise index for uniform milling and intelligent processing.

2.1.Rice milling

Three classic Chinese rice varieties,i.e.,japonica cultivar Xinfeng 2(X2),Liaojing Rice(LR),and Super Rice(SR),were obtained from Yuanyang(Henan province),Benxi(Liaoning province),and Changchun(Jilin province),respectively.The brown rice grains of each cultivar(200 g,moisture content of 15.5%,15.0%,14.6%for X2,LR,and SR)were milled for 20 s at 1060 r/min using an abrasive emery mill(Grit size,36#)(Satake,TM05C,Suzhou,China).The samples were then milled to different DMs of 2%,4%,6%,8%,10%,12%,and 14%using a laboratory metal mill(Puyun,LTJM-9099,Shanghai,China).The DM was calculated using the following formula[14]:

wherem1is the mass of milled rice,g;m0is the mass of brown rice,g.

The prepared samples were screened using a 10-mesh screen to remove the bran,separately packed in sealed polyethylene bags,and stored in a refrigerator below 4°C for further use.

2.2.SEM

The X2 sample was selected to examine the microstructures of different parts of the rice grain.The X2 rice with DMs of 6%and 12%were used to evaluate the effect of milling on the dorsal and ventral microstructures.The X2,LR,SR,and Koshihikari(Ko)(Shinjuku-ku,Japan)brown rice samples were used to assess the grooves of different cultivars.First,a slight gap was cut along the periphery near the middle of the rice grain using a thin blade.Second,the rice kernel was broken by hand to expose the transverse section.Next,a 2 mm-thick lamina with a complete surface was cut from the fractured surface of the rice kernel.Finally,the lamina was directly pasted onto a stage using a conductive adhesive.Under an acceleration voltage of 15 kV,the whole and part of the transverse section of the sample were observed with a scanning electron microscope(FEI,model Quanta 250 FEG,USA)at varying magnifications(130×,150×,and 2000×).

2.3.Determination of the bran degree

The bran degree refers to the percentage of the residual bran on the surfaces of the rice grains to the total area of rice grains.The bran degree was determined according to the following steps delineated in the instruction manual of a rice appearance detector(JMWT-12,China):12 g of head rice was weighed and immersed in deionized water for 1 min,and then exposed to eosinY-methylene blue dye(Dongfujiuheng Instrument Technology Co.,Ltd.,China)for 2 min and 80%ethanol for 1 min in sequence.The sample was rinsed three times with 80%ethanol and dried,after which the bran degree was measured using a rice appearance detector(JMWT-12,China).

2.4.EDS

The transverse sections of the X2 brown rice grains with DMs of 6%and 12%were selected for the elemental analysis to examine the effect of milling on the distribution of the elements.EDS(EDAX,model Apollo XLT SDD,USA)was used to determine the relative content of magnesium(Mg),kalium(K),phosphorus(P),silicon(Si),carbon(C),and oxygen(O)at an accelerating voltage of 20 kV.

2.5.Chemical analysis

Brown rice grains with different DMs(2%,4%,6%,8%,10%,12%,and 14%)were ground into powder using a hammer-type whirlwind mill(JXFM110,Hangzhou Big Material Photoelectric Technology Co.,Ltd.,China)for further analysis.The moisture content of the rice was determined by weighing the grains before and after drying in a natural air circulation oven at(105±3)°C for 24 h.The moisture content was calculated using the following formula:

whereW0is the weight of rice grains before drying,g;W1is the weight of rice grains after drying,g.

The ash,crude protein,crude fat,dietary fiber,mineral,VB1,and VB2 content was determined according to the AACC Method 08-01,46-11A,30-20,32-41,40-75,86-80,and 86-70,respectively[15].

2.6.Statistical analysis

Origin software(Version 95C,Massachusetts,USA)was used for data analysis,while Duncan"s multiple range test in the SPSS software(Version 16.0,Chicago,USA)was used to analyze the significance of the data at level ofP＜0.05.

3.1.Microstructure of the brown rice

The SEM of the X2 brown rice showed microstructural differences in the dorsal,centrilateral,and ventral parts of the rice grain(Fig.1).The pericarp(Pe),seed coat(Se),Al,subaleurone layer(sAl),and En were arranged successively from the outside to the inside.The backscattered electron images showed that the Al cells looked like luminous spheres[16].The larger the average atomic number of atoms in the sample,the brighter the area.Therefore,it is helpful to observe and distinguish the Al according to the contrast of brightness.The Al was brighter than other parts since the enrichment of minerals leads to a higher average atomic number.

Fig.1.The SEM images of different parts of the transverse section of the X2 brown rice.

As shown in Fig.1,the Pe consisted of fiber walls,while the grain was wrapped in Pe of different thicknesses.The thickness of the Pe on the dorsal portion was thicker than that in the other parts.The Se was transversely elongated next to the Pe.Aleurone cells of different shapes and sizes were distributed in different areas of the brown rice.The thickness of one layer of rectangular aleurone cells was about 12-23 μm in the ventral area(Fig.1a)and 5-18 μm in the ventrilateral area(Fig.1b).The thickness of four to five layers of irregular aleurone cells was about 50-63 μm in the dorsal area(Fig.1e),while that of one to two layers of polygonal aleurone cells was about 10-20 μm in the dorsolateral area(Fig.1d),and one layer of irregular aleurone cells was about 15-25 μm in the centrilateral area(Fig.1c).The dorsal area contained a higher number of Al than the ventral area,which was consistent with the conclusions of Rosario et al.[17]and Wang et al.[18].This is mainly because the Al develops earlier in the dorsal area and presents more cell layers during the development of the rice kernel.The Al thickness in different parts of the brown rice was different,which was consistent with the conclusion of Wu et al.[19].The En was distributed in the innermost part and was wrapped by an Al.Due to the arrangement of different tissues,the Pe and Se were removed first during milling,followed by the Al.Therefore,the continuous removal of bran exposed the En and improved the whiteness of the rice.

The central cross sections of the X2,LR,SR,and Ko rice samples showed longitudinal furrows and grooves on the kernel surfaces,displaying different groove depths in different locations(Fig.2).The groove depth near the dorsal area was deeper than that in the ventral region.The groove depth is considered a vital characteristic of rice grains.The rice kernel characteristics were affected by rice growth,environmental conditions,and rice genetics[20,21].The groove depth of the four rice cultivars also varied,requiring different degrees of polishing to attain a smooth,white surface[21].Specific rice varieties require suitable milling techniques,especially cultivars with deep dorsal grooves[22-24].When rice is overmilled,the bran in the grooves is ultimately removed.Therefore,the depth of the grooves affects the milling characteristics of rice and also presents a significant challenge when measuring the degree of bran removal under high DM.

3.2.Effect of the DM on the microstructure of the bran layer

The rice was categorized into four classes based on the DM,namely under-milled(DM 3%-4%),medium-milled(DM 5%-6%),fully-milled(DM 7%-8%),and overmilled(DM＞8%)[25].The effect of milling on the microstructure of the Al of the rice grains was explored by observing the microstructure of the rice kernel during mediummilling and overmilling.The microstructures of the ventral and dorsal areas of X2 at DMs of 6% and 12% were observed via SEM.The complete histological structure in the dorsal and ventral areas of the brown rice was shown in Fig.3(1)and Fig.3(2).When the DM reached 6%,the Pe and Se on the surface of the brown rice were removed,exposing the uneven surface of the Al(Fig.3(3)and Fig.3(4)).A significant Al remained in the dorsal area(Fig.3(3)),while it was minimal in the ventral region(Fig.3(4)).When the DM reached 12%,the aleurone cells embedded in the En remained in the dorsal area(Fig.3(5)),while the En in the ventral region was exposed completely,with no Al cells on the surface(Fig.3(6)).Therefore,the ventral Al was removed before the dorsal Al,and polishing smoothed the surfaces of the rice grains.

Fig.2.SEM images of the transverse section of the brown rice.

Fig.3.SEM images of X2 with different DMs estimated via grain weight loss.

When the DM＞10%,no significant changes were evident in the bran degree of the three rice cultivars(P＞0.05)(Table 1).Based on the microstructural changes,it can be inferred that progressive milling did not change the residual bran in the grooves at high DM levels.

Table 1Bran degree of the rice with different DMs estimated via grain weight loss.

3.3.Effect of the DM on the rice composition

3.3.1.Effect of the DM on the elemental distribution

Fig.4.The ventral EDS of X2 at different DMs estimated via grain weight loss.

The elemental mapping image for X2 showed the distribution of Mg,P,K,Si,C,and O in the bran in the ventral area(Fig.4(1)).The Mg,P,K,Si,and C were present as bright bands in the bran layers,revealing that the levels of these elements were higher in the outer layer of the brown rice than in the En.C and O were evenly distributed in the En and bran.At a DM of 6%,the bright Si bands disappeared,while those of Mg,P,and K became narrow and vague(Fig.4(2)).At a DM of 12%,no bright bands were evident,and only shadows characterized the elements in the image(Fig.4(3)).No significant changes were apparent in the elemental distribution in the En.This showed the richness of these mineral elements in the Al and the varied elemental distribution patterns.Furthermore,the disappearance of the bright bands from the elemental map indicated the complete removal of the ventral Al of the rice kernel,while even part of the outer layer of the En might be milled away.

3.3.2.Effect of the DM on the content of main compositions

Fig.5.Changes in the content composition in the rice with different DMs estimated via grain weight loss.

Since rice represents the main staple food in the world,its nutritional value is vital.Milling affects not only the whiteness of rice but also its quality and nutritional value[22,26-28].The ash,crude fat,crude protein,dietary fiber,VB1,and VB2 levels showed a declining trend in the milled samples(Fig.5).

The ash content decreased from 1.11%-1.25% to 0.27%-0.34%as the DM increased.When the DM exceeded 10%,the ash content displayed no significant changes(P＞0.05).At a DM of 8%,the ash content accounted for about 38%-43%of that in brown rice.It indicated that the ash content in the bran was higher than that in the En,which was consistent with the mineral results in Section 3.3.1.

The crude fat content in the rice decreased from 2.19%-2.65%to 0.25%-0.61%,with an improvement in the DM.Significant changes(P＜0.05)were evident in the crude fat content at a DM below 10%.At a DM exceeding 10%,the crude fat content changes were minimal(P＞0.05).At a DM of 14%,the crude fat content accounted for 10%-23%of the fat content in the brown rice.This was consistent with the fact that most lipid components in the brown rice were located in the lipid bodies in the Al and other tissues,while the En contained negligible lipid quantities[29,30].

The protein content is a crucial physicochemical characteristic affecting the cooking quality and taste of rice[8,31,32].The crude protein content decreased significantly from 10.18%to 9.47%when the DM increased from 4%to 8%in the LR cultivar.No significant changes were evident in the crude protein content when the DM exceeded 8%in X2 and 10%in SR,respectively.At a DM of 14%,the crude protein content accounted for 85%-92% of the protein content in the brown rice.This confirmed that rice En protein,consisting mostly of glutelin and prolamin,accounted for 80%or more of the total protein,which was consistent with the conclusions of Lamberts and Resurrecion et al.[8,33].Rice bran protein with average compositions of 34% albumin,15%-26% globulin,4% prolamin,and 11%-27%glutelin were removed with progressive milling[34].

At a DM below 10%,the dietary fiber content in the three cultivars decreased significantly(P＜0.05).When the DM exceeded 10%,the dietary fiber content changed little(P＞0.05)as the rice was milled.This was consistent with the conclusions of Kim et al.[35].At a DM of 8%,the dietary fiber content accounted for about 14%-20% of the total.Therefore,rice bran is a concentrated source of dietary fiber.The decrease in dietary fiber at a DM of 0-2%was more significant than at any subsequent milling stage(Fig.5).The major carbohydrates in the bran were represented by cellulose and hemicellulose(or pentosan),while no plant starch was present in the Pe layer[36].The distinct bright band in the outer layer of the brown rice was visible in Fig.4(1-C)but is absent from Fig.4(2-C).This highlighted the richness of dietary fiber in the outer layer of the brown rice bran.

The VB1 in SR,X2,and LR decreased significantly from the brown rice to the milled rice at a DM of 10%(P＜0.05).No significant changes occurred in the VB1 content since the DM exceeded 10%.When the DM was 4%-10%,the VB1 level decreased rapidly,with a loss of about 50%-62%,indicating that VB1 was mainly distributed on the surface of the bran.The VB1 content at a DM of 14% accounted for about 14%-29%of the VB1 content of brown rice,confirming that the Al contained approximately 33%and the En 6.3%of the total VB1[37].The overmilling of rice drastically lowered the VB1 level,causing deficiency diseases like beriberi.Although the VB2 content was extremely low,even in the brown rice,and was further reduced during milling,the decline was not dramatic.At a DM of 10%,the VB2 content in the brown rice was about 56%-70%,indicating primary and even distribution in the En.The loss of VB1 and VB2 caused by milling was about 71%-86%and 35%-47%,respectively,showing a more significant VB1 decline than VB2,evidencing a greater milling effect.The nutritional quality of the rice deteriorated as the milling progressed,which was consistent with the conclusions of Wang et al.[11,12,38].The DM of 10% with a small portion of the bran(about 0.3%)remained on the surface of the rice grain was critical.When the DM fell within 10% to 14%,there was no further significant change in the measured values of both chemical and nutrient compositions(P＞0.05).Therefore,ash,crude fat,crude protein,dietary fiber,VB1,and VB2 are not suitable for quantifying the Al changes caused by milling at high DM values.

3.3.3.Effect of the DM on the mineral content

The minerals in rice grains are not evenly distributed,and most,such as P,Mg,K,and Mn in brown rice,are more abundant in the outer bran layers[39].Lamberts et al.[8]confirmed that about 61%of minerals are present in bran.Studies have shown that the distribution of minerals in rice affects its nutritional and sensory quality[40,41].Minerals are highly important to the nutritional content of rice.However,this study showed that the mineral content began to decline immediately as the DM increased.

The levels of minerals in the brown rice were,from high to low,P＞K＞Mg＞Ca(Fig.6).Although the P content was the highest,its decline was the most significant.This showed that these four minerals were unevenly distributed in the grains and declined in the three cultivars during progressive milling.No significant changes were evident in the Ca content when the DM increased from 10%to 14%.However,the P,Mg,and K levels changed significantly when the DM increased from 10% to 14%,therefore they could be used as an index of DM calibrated against the weight-loss scale for the overmilled rice.

Fig.6.The mineral content in the rice with different DMs estimated via grain weight loss.

This study investigated the influence of DM on the microstructural and constituent changes in rice.The traditional methods used for DM estimation present various challenges since the loss of En accounts for the majority of grain weight loss due to overmilling.A high DM adversely affects the bran degree results when using the staining method for determination.It is hypothesized that the microstructural changes can reveal the differences among the approaches,while the compositional changes may highlight the compatibility between these strategies.When the DM estimated via the loss of grain weight increases from 10%to 14%,the visual approximation of the DM in the residual bran is not significantly different(P＞0.05).A DM exceeding 10%facilitates the removal of marginal bran from the dorsal groove.Therefore,the crude protein,ash,crude fat,dietary fiber,VB1,and VB2 content display no significant changes(P＞0.05),while the Mg,K,and P levels decrease substantially(P＜0.05).The results of this study,combined with the microstructural and compositional changes,showed that:(1)The bran layer is progressively removed from under-milled rice,medium-milled rice,and fully-milled rice.(2)Minimal changes are evident in the residual bran in the dorsal grooves of the overmilled rice,while the Mg,P,and K in the residual bran tissue can serve as a precise index of DM.This result provides more accurate estimation of DM and theoretical guidance for milling.

Author contributions

Jie Liu:Writing-original draft,Conceptualization,Methodology,Writing-review&editing.Yani Wu:Conceptualization,Formal analysis,Writing-original draft.Huihui Chen:Conceptualization,Methodology,Data curation,Investigation.Hongzhou An:Validation,Supervision,Funding acquisition,Project administration,Writing-review &editing.Yu Liu:Investigation.Jie Xu:Resources.

Conflicts of Interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This study was supported financially by the National Key Research and Development Program of China(Grant Number 2017YFD0401101-01).