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Research ArticleReceived: 13 May 2010 Revised: 11 September 2010 Accepted: 24 October 2010 Published online in Wiley Online Library: 23 December 2010

(wileyonlinelibrary.com) DOI 10.1002/jsfa.4237

Effects of rice dreg protein and its hydrolysateon growth performance and small intestinemorphology of early-weaned ratsXiang Li,a,b Hua Xiong,a∗ Kaiwu Yang,c Diwei Peng,a,b Wei Li,a,b

Yulong Yina,d and Jinhui Liue

Abstract

BACKGROUND: Protein hydrolysate has more efficient peptide absorption and less hypertonic protein absorption. This studyaims to investigate the influences of rice dreg (RD) protein and its hydrolysate on growth and somatropic axis in early-weanedSprague-Dawley rats in comparison with soybean (SB) protein.

RESULTS: Compared with a SB diet control, a rice dregs protein hydrolysate (RD-H) diet decreased average daily feed intake andfeed/gain in weeks 1 (P < 0.01) and 2 (P < 0.05), while an RD diet decreased average daily feed intake and feed/gain in week1 (P < 0.01). Average daily body gain exhibited no significant differences between either treatment during the 28-day period.After 28 d, villus height and lamina propria depth of jejunum and duodenum were greater in the RD-H treatment than in RDand control treatments (P < 0.01), while serum urea nitrogen concentration was lower in RD and RD-H treatments than in thecontrol (P < 0.05). There were no differences in serum growth hormone concentration, hepatic and muscle insulin-like growthfactor-1 expression, and muscle growth hormone receptor expression at 7 and 28 d (P > 0.05), but hepatic growth hormonereceptor expression was higher in RD-H treatment than in RD and control treatments after 7 d (P < 0.05).

CONCLUSION: Results showed that RD and RD-H diets improved feed utilisation efficiency in the first 2 weeks post-weaning andsmall intestine morphology at 28 d post-weaning for an early-weaned mammal compared with a SB diet.c© 2010 Society of Chemical Industry

Keywords: RD protein hydrolysate; intestine morphology; somatropic axis; early-weaned rats

INTRODUCTIONDietary protein can be degraded into free amino acids andoligopeptides by digestive enzymes in the gastrointestinal tract,whose hydrolytic capacities can be affected by the molecular formof the nitrogen supplied.1,2 Especially for early-weaned mammals,the source, concentration and quality of dietary protein haveimportant roles in the development of gastrointestinal disorders,intestinal damage, malabsorption and diarrhoea during the first1–2 weeks after weaning.3 – 6 Protein absorption in the formof oligopeptides, mainly di- and tripeptides, is more efficientcompared with an equivalent amount of free amino acid or wholeprotein, and its decreasing effect on the tonicity of the smallintestine could improve the absorption of dietary componentsand eliminate intestinal osmotic problems.7 – 10

Rice dreg (RD) is a by-product of rice syrup production andretains nearly all of the rice protein. Rice protein is an excellentsource of high-quality vegetable protein with hypoallergenicproperties.11 It is extracted from rice dreg, flour and bran, andis commercially available for use in the creep or starter feed ofearly-weaned pigs as a much cheaper source of dietary protein.12,13

However, the nutritional effects of RD protein and its hydrolysate(RD-H) are less well described. Thus, this study aims to investigatethe influences of RD and RD-H on intestinal morphology, growthand somatropic axis of early-weaned Sprague–Dawley rats and

develop a new high-quality vegetable protein source for early-weaned mammals.

EXPERIMENTALMaterialsRD (57.0% protein, dry matter basis) was provided by JiangxiHengtian Co. Ltd. (Jiangxi, Fengcheng, China). RD-H (62.5%

∗ Correspondence to: Hua Xiong, State Key Laboratory of Food Science andTechnology of Nanchang University, Nanchang, Jiangxi 330047, P.R. China.E-mail: huaxiong100@yahoo.com.cn

a State Key Laboratory of Food Science and Technology of Nanchang University,Jiangxi 330047, P.R. China

b Graduate School of Nanchang University, Jiangxi 330047, P.R. China

c Rhodia Rare Earths New Materials Co., Ltd, Liyang, Jiangsu 213000, P.R. China

d Laboratory of Animal Nutrition and Health and Key Laboratory of SubtropicalAgro-Ecology, Institute of Subtropical Agriculture, Chinese Academy of Sciences,Changsha, Hunan 410125, P.R. China

e Hunan Agriculture University Veterinary Faculty, Changsha, Hunan 410128,P.R. China

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Table 1. Amino acid composition (g·kg−1) of rice dregs and rice dregsprotein hydrolysate (based on dry matter)

Amino acid Rice dregsRice dregs protein

hydrolysate

Asparagine 57.86 60.66

Threonine∗ 18.11 18.66

Serine 35.75 33.68

Glutamine 119.06 123.52

Glycine 28.72 30.27

Alanine 32.99 35.14

Cystine 15.32 11.92

Valine∗ 37.51 37.70

Methionine∗ 12.10 8.98

Tyrosine 22.06 22.85

Phenylalanine∗ 24.06 48.14

Lysine∗ 30.02 31.49

Histidine∗ 30.45 36.02

Arginine∗ 20.13 22.09

Proline∗ 12.39 14.67

Tryptophan∗ 50.05 54.87

Isoleucine∗ 20.96 23.14

Leucine∗ 3.02 3.08

∗ Essential amino acid.

protein, dry matter basis) was prepared by limited hydrolysisof RD with trypsin (1 g kg−1) in pH 7.6 at 52.83 ◦C for 2.43 hwith a RD/water ratio of 170 g L−1. This was centrifuged at3000 × g for 10 min. The supernatant was then spray-dried foranalysis and used in experimental diets. The protein content wasdetermined according to the Kjeldahl method.14 The peptidemolecular weight distribution of dried RD-H was determined bygel filtration according to the method described by You et al.;15

70.9% of the peptides were between 1 and 5 kDa and 29.1%were <1 kDa. The amino acid composition of RD and RD-H wasmeasured in triplicate as described by Wu and Knabe16 and theresults are expressed as their mean values and are shown inTable 1.

DietsThree diets, including the RD protein diet, RD-H protein diet, andsoybean meal (SB) protein diet as control, were prepared. All dietswere isonitrogenous and isocaloric, and were formulated to meetor exceed the NRC17 recommendation. The composition and somenutrient levels of the diets are shown in Table 2.

Animals and housingSixty 21-d weaning female Sprague–Dawley rats (specific-pathogen-free), normally weaned at 4 weeks of age, wererandomly assigned to one of the three dietary treatments (n = 20per treatment). The rats were kept individually in cages withwire mesh floors in a room maintained at 24 ± 2 ◦C with a 12-hlight/dark cycle, and were offered food ad libitum and had freeaccess to water. During the 28-d experimental period, the ratswere weighed weekly after overnight fasting (12 h) and their feedintake recorded daily for the determination of average daily bodygain, average daily feed intake, and feed efficiency (feed/gain). Sixrats from each treatment were randomly selected for terminationat 8 am on day 7 and day 28. This study was legally approved by

Table 2. Composition and nutrient levels of experimental diets

Composition/nutrientlevel Soybean

Ricedregs

Rice dregsprotein

hydrolysate

IngredientCorn 530.0 632.6 652.2

Wheat bran 55.8 29.1 37.2

Soybean meal 262.5 0.0 0.0

Fish meal 50.0 50.0 50.0

Rice dregs 0.0 189.0 0.0

Rice dregs protein hydrolysate 0.0 0.0 167.5

L-Lysine 0.0 4.4 4.4

Coco oil 37.1 37.1 37.1

Soybean oil 20.0 20.0 20.0

Dicalcium phosphate 8.0 15.2 15.0

Calcium carbonate 20.0 6.0 0.0

Mineral premix 5.0 5.0 5.0

Vitamins 11.5 11.5 11.5

Citric acid 10.0 10.0 10.0

Choline chloride 1.0 1.0 1.0

Nutrient levelCrude protein 180.0 179.0 178.0

Metabolisable Energy (MJ kg−1) 13.5 13.5 13.5

Crude fibre 20.8 20.7 20.6

Calcium 12.0 12.0 12.1

Phosphorus 7.2 7.2 7.2

Lysine 11.9 11.9 11.9

Arginine 14.0 14.0 13.9

Oligopeptide 0.0 0.0 75.0

Results are given as g·kg−1, based on dry matter.The mineral premix provided the following per kilogram of diet: NaCl,0.5 g; K, 0.5 g; Mg, 0.2 mg; Fe, 120 mg; Zn, 30 mg; Mn, 75 mg; Cu, 10 mg;I, 0.5 mg; Se, 0.15 mg.The vitamins provided the following per kilogram of diet: vitamin A,14 000 IU; vitamin D3, 1500 IU; vitamin E, 120 IU; menadione, 5 mg;riboflavin, 12 mg; niacin, 60 mg; D-pantothenic acid, 24 mg; vitaminB12, 22 µg; thiamine, 13 mg; vitamin B6, 12 mg; biotin, 0.2 mg; folacin,6 mg.The nutrient levels are based on calculation.

the Animal Use and Care Committee of the Chinese Academy ofSciences.

Sample collectionRats were anaesthetised with sodium pentobarbital (60 mg kg−1)intraperitoneally before decapitation. Blood samples were col-lected in cloth tubes and allowed to clot for 60 min on ice beforecentrifugation at 3000 × g for 10 min to obtain serum. Serum sam-ples were immediately stored at −20 ◦C until analysis for growthhormone (GH) and urea nitrogen (UN) concentration.

After blood sampling, rats were paunched for tissue samples.The liver and right hind leg muscle samples were immediatelycollected and frozen in liquid nitrogen, and then stored at −70 ◦Cfor subsequent assays of growth hormone receptor (GHR) andinsulin-like growth factor-1 (IGF-1) mRNA levels.

The small intestine was dissected free of its mesentery anddivided approximately into duodenum, jejunum and ileum.Lengths of segments (1 cm) were removed from the pylorus endof the duodenum, the jejunum and the ileum just anterior to theileo-caecal valve. Intestinal segments were opened longitudinally,

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rinsed thoroughly with ice-cold physiological saline solution,spread out on glass plates, and then submerged immediatelyin 10% Bouinn’s fixative solution (TFA: trinitrophenol, formalin andglacial acetic acid solution, 75 : 25:5, v/v/v) for 24 h.

Analytical proceduresMeasurement of serum urea nitrogen and growth hormoneconcentrationSerum urea nitrogen was measured spectrophotometrically usingan automatic biochemistry analyser, while serum concentrationsof GH were measured with a rat radioimmunoassay kit (BeijingChemclin Biotech Co., Ltd., China). Samples were analysed induplicate with an intra-assay coefficient of variation of 6.8–9.2%at GH concentrations of 2.2–7.0 g L−1.

Gut morphologySmall intestine segments were processed, embedded and stainedas described by Sagher et al.18 and Kang et al.19 with minormodifications. After fixation in TFA for 24 h, each segment wasdehydrated over a 3-d period with increasing concentrations ofethanol (70, 80, 95 and 100%) at 25 ◦C. They were embedded inparaffin wax and then refrigerated at 4 ◦C to sufficiently hardenthe paraffin before sectioning. Four 5-µm cross-sections of eachsegment were deparaffinised with xylene for 7 min, and thenstained with haematoxylin and eosin. Villous height, crypt depthand lamina propria depth were measured with computer-assistedmicroscopy (Micrometrics; Nikon ECLIPSE E200, Japan). Only villiattached to the lamina propria and with well-defined tips weremeasured. Ten measurements of villous height, 10 of crypt depthand 10 of lamina propria depth were taken from each of the foursections, so that a total of 40 measurements from each area and 40villous height/crypt depth ratios were used as experimental units.

Reverse transcribed polymerase chain reactionTotal RNA was extracted from liver and muscle samples with TriZolReagent (Invitrogen, Guangzhou, China) according to the manu-facturer’s protocol, and then dissolved in diethyl-pyrocarbonate(DEPC)-treated water. The concentration and purity of extractedRNA were determined by spectroscopy at 260 and 280 nm. A5 µg sample of total RNA was then reverse transcribed into cDNAusing the Revert Aid First Strand Synthesis kit (Promega, Shang-hai, China). Subsequently, the reverse-transcribed product (2.5 µL)was amplified in a total volume of 25 µL containing 0.5 µmol L−1

each of the primer (final concentration) and 12.5 µL of SYBRgreen I. Glyceraldehyde-3-phosphate dehydrogenase (3-GAPDH)was used as a housekeeping gene. The primers used were asfollows: GHR (upstream) 3′-TCA CCG ACA CCA AAC AG-5′; GHR(downstream) 3′-TTA CTC GTC AGG GCA CA-5′; IGF-1 (upstream)3′-CAC ATA GAA ATA ACC TCC ACG-5′; IGF-1 (downstream) 3′-CCACCT GCG AGA AGT CAA-5′; 3-GAPDH (upstream) 3′-TCC CTA CTACAA GAC CCG-5′, and 3-GAPDH (downstream) 3′-GAA CCC GATGTA CTC CTG-5′. PCR products of GHR and IGF-1 were 137 and 79bp, respectively. Real-time RT-PCR assays were performed with ABIPRISM 7900HT system using the SYBR Premix Ex Taq kit (TaKaRa,Dalian, China). Thermocycler reaction conditions were as follows:95 ◦C for 30 s, followed by 45 cycles of 95 ◦C for 5 s, 53 ◦C for 30 s,and 72 ◦C for 20 s. Each PCR run included a no-template control andduplicates of control and samples. A dissociation curve of RT-PCRproducts was performed to inspect the specificity of amplification.No amplification of unspecific products was observed. Relative

Table 3. Growth performance of rats weaned on the experimentaldiets for 28 days

Item Soybean Rice dregsRice dregs protein

hydrolysate

Body weight (g)

Initial 65.4 ± 1.92 65.3 ± 1.70 65.4 ± 1.65

Final 202.5 ± 18.57 195.4 ± 20.75 205.4 ± 19.73

ADG (g·day−1)

7 d 3.6 ± 1.09 3.8 ± 0.81 3.8 ± 0.87

14 d 6.2 ± 1.06 6.8 ± 1.10 6.6 ± 1.21

21 d 6.2 ± 1.06 6.8 ± 1.10 6.6 ± 1.21

28 d 3.8 ± 0.95 3.8 ± 1.43 3.9 ± 1.65

1–28 d 4.9 ± 1.35 4.7 ± 1.29 5.0 ± 1.52

ADFI (g·day−1)

7 d 11.2 ± 1.20A 10.1 ± 1.03B 10.0 ± 1.01B

14 d 23.8 ± 1.97A 24.6 ± 2.24A 20.9 ± 2.91B

21 d 23.9 ± 3.72 23.0 ± 2.51 21.1 ± 2.63

28 d 23.5 ± 4.31 22.7 ± 3.20 19.9 ± 3.74

1–28d 20.2 ± 2.61 19.4 ± 2.31 19.1 ± 2.81

Feed/Gain

7 d 3.2 ± 0.41a 2.8 ± 0.61b 2.7 ± 0.60b

14 d 3.7 ± 0.60a 3.5 ± 0.59ab 3.1 ± 0.76b

21 d 3.4 ± 0.82 3.0 ± 0.62 2.9 ± 0.43

28 d 6.6 ± 1.20 5.4 ± 1.15 5.3 ± 1.03

1–28 d 4.2 ± 0.95 3.9 ± 0.73 3.6 ± 0.79

Data are expressed as mean ± SE.Small letter superscripts in the same row refer to different significancelevels (P < 0.05).Capital letter superscripts in the same row refer to significant differences(P < 0.01).ADFI, average daily feed intake; ADG, average daily body gain.

quantification of mRNA was based on amplification cycle thresh-old (Ct) values of each sample. Relative to 3-GAPDH, the resultantvalue (fold changes) of genes were expressed as 2−��C(t)s.20

Statistical analysisData were analysed by one-way ANOVA using the GLM procedureof SAS (SAS Institute Inc., Cary, NC, USA). When treatment effectswere significant (P < 0.05), means among treatments werecompared with Duncan’s multiple range test. The experimentalunit for all statistical procedures was the individual rat.

RESULTSGrowth performance of ratsTable 3 presents the growth performances of rats weaned onthe three different dietary proteins. There were no significantdifferences in average daily body gain among the three treatmentsthroughout the 28-d experiment period. Compared with controls,rats weaned on RD-H diets consumed significantly less feed in thefirst (P < 0.01) and second weeks (P < 0.05), and rats weaned onRD diets consumed less feed in the first week (P < 0.01). Trends infeed efficiency were similar to those of average daily feed intake.Feed/gain was the lowest in the RD-H group, while that in thecontrol was the highest in the first 2 weeks (P < 0.05). This alsodomenstrated that the rice protein diets had higher efficiencies offeed use in rats in the first week post-weaning when comparingwith the soybean protein diet. During the last 2 weeks, neither

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Figure 1. Serum urea nitrogen concentrations of the early-weaned rats on7 and 28 d. Values are means (±SE) of six rats. Serum nitrogen concentrationwas significantly lower in RD and RD-H treatments than in SB treatment on28 d (P < 0.05), but not significantly different among the three treatmentson 7 d (P > 0.05).

average daily feed intake nor feed efficiency exhibited significantdifferences among the three treatments.

Serum urea nitrogen and growth hormone concentrationsCompared with controls, rats fed RD and RD-H diets hadlower serum urea nitrogen concentrations on 28-d post-weaning(P < 0.05) (Fig. 1). However, serum GH concentration was notsignificantly influenced by the dietary treatment during the 28-dexperimental period (P > 0.05) (Fig. 2).

Villus height, crypt depth, lamina propria depth, and villusheight/crypt depth ratioThe influence of dietary treatment on small intestine morphologyof early-weaned rats is illustrated in Figs 3, 4 and 5. Compared withthe control diet, the RD-H diet, but not the RD diet, significantlypromoted the growth of villi in jejunum of rats on 7 d post-weaningand in small intestine of rats on 28 d post-weaning. Rats fed onthe RD-H diet had higher lamina propria depths in duodenum

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7d 28d

GH

, ng·

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SBRDRD-H

Figure 2. Serum GH concentrations of the early-weaned rats on 7 and28 d. Values are means (±SE) of six rats. No significant differences wereobserved in serum GH concentration among the three treatments on 7 dor 28 d (P > 0.05).

and jejunum on 28 d post-weaning, accompanied by significantlylarger villous heights and insignificantly larger crypt depths in smallintestine, but dietary protein had no significant influence on thevillous height/crypt depth ratio during the 28-d period (P > 0.05).These results implied that the RD-H diet could promote enterocytegrowth in small intestine as well as the cellular growth of laminapropria in duodenum and jejunum on 28 d post-weaning.

Growth hormone receptor and insulin-like growth factor-1mRNA levelsThe influences of dietary protein on IGF-1 and GHR mRNAexpression in the liver and muscle were illustrated in Fig. 6.Compared with controls, hepatic expression of IGF-1 mRNA wasimproved by the RD-H diet but suppressed by the RD diet on 7 d,and suppressed by both the RD-H and RD diets on 28 d (P > 0.05).In contrast, muscle expression of IGF-1 mRNA was improved by theRD-H diet on 7 d and suppressed by the RD diet on 28 d (P > 0.05)(Fig. 6A). Hepatic expression of GHR in rats weaned on the RD-Hdiet was the highest among the treatments on 7 d, but decreasedto equal that in SB diet and to be slightly higher than that in RDdiet at the end of the experiment (Fig. 6B).

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SB RD RD-H SB RD RD-H SB RD RD-H

Villus height Crypt depth Lamina propria depth

(um

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duodenumjejunumileum

Figure 3. Micro-measure results of small intestine for the early-weaned rats on 7 d. Values are means (±SE) of six rats. Lamino propria depth (P < 0.01) andvillus height (P < 0.05) of jejunum were significantly higher in RD-H treatment than in RD and control treatments. Lamino propria depth of duodenumwas higher in RD-H treatment than in RD (P > 0.05) and control (P < 0.05) treatments and there were no significant differences between RD treatmentand control treatment. Dietary treatment had no significant influence on villus heights of duodenum and ileum, crypt depth of small intestine, or laminopropria depth of ileum (P > 0.05).

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Figure 4. Micro-measure results of small intestine for the early-weaned rats on 28 d. Values are means (±SE) of six rats. Means without a common capitalletter are significantly different, P < 0.01. Villus height of small intestine and lamina propria depths of duodenum and jejunum were significantly higherin RD-H treatment than in RD and control treatments (P < 0.01). Crypt depth of duodenum was significantly lower in RD treatment than in RD-H andcontrol treatments (P < 0.01), but there were no significant differences in crypt depths of jejunum and ileum or lamina propria depth of ileum amongthe three treatments on 28 d (P > 0.05).

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DISCUSSIONDietary protein is generally agreed to be the most satiatingmacronutrient, mainly by its stimulation of the release ofcholecystokinin,21,22 which varies with dietary protein source andconcentration and has a short-term regulatory effect on daily en-ergy intake.23 – 25 This is demonstrated again in the present study.Rice protein from either RD or RD-H could lead to a significantlydecreased average daily feed intake than SB protein during thefirst week post-weaning. Moreover, RD-H diets contributed tolower feed consumption among the three treatments throughoutthe experiment, which was also reported by Cudennec et al.26

They found that protein hydrolysate from fish could stimulatethe release of cholecystokinin from intestinal endocrine cells anddemonstrated that this stimulation was due mainly to peptideswith molecular weight from 1000 to 1500 Da. Additionally, thepalatability of RD and RD-H diets may also contribute to the lowerfeed consumption in the first week post-weaning. Thus, excessiveaddition of protein hydrolysate in the diet may not be beneficial forhigh growth performance in terms of daily limited nutrient intake.

Serum urea nitrogen is derived from the deamination of aminoacids in systemic protein metabolism, and depends mostly onthe amount and balance of amino acid in the system.27 Previousstudies have demonstrated that a lower serum urea nitrogen levelis tightly correlated with higher nitrogen utilisation efficiency andless urea synthesis.28 In the present study, rats weaned on theRD and RD-H diets had similar serum urea nitrogen levels thatwere lower than those on the SB diet. The lower feed/gain ratiosin the RD and RD-H dietary treatments than in the SB treatmentimply that dietary nitrogen from RD and RD-H diets are utilisedmore efficiently and have higher feed conversion efficiencies thansoybean protein. Thus, rice protein is more favourable for weaningrats than soybean protein.

The premise for good feed conversion efficiency is theabsorption of dietary nutrient in the small intestine, which dependsgreatly on intact morphology and sound physiological function,and is enhanced by villus growth.29,30 Destruction of villus in thesmall intestine brought about by weaning with a solid diet isusually associated with its dysfunction, which could contributeto low nutrient absorption, diarrhoea, and even enteritis. In thepresent study, there was no diarrhoea among the three treatmentgroups during the 28-d experiment period. Villous height of thesmall intestine in rats weaned on the RD-H diet was the highestthroughout the experiment and significantly higher than othersat the end of the experiment (P < 0.01). This indicate that a dietsupplemented with RD-H efficiently prevents weaning-inducedvillous atrophy and/or promotes the recovery and growth ofdestroyed villi, subsequently mitigating physiological stresses andgrowth depression resulting from early weaning. This could belargely due to its decreased hypertonicity10 and more efficientutilisation3,31,32 of peptides in RD-H than that of whole protein inRD and SB materials. Another factor that could have contributedto this effect is the relatively higher content of glutamic acid of riceprotein than soybean protein because glutamine is a primary fuelfor growth and renewal of intestinal mucosa33 and is obligatoryfor intestinal mucosal mass and integrity,34,35 and defence ofmucosa against toxic and peroxidative damage.36 Meanwhile, ascompared with the RD diet, it should not be ignored that there

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Figure 6. Expressions of IGF-1 (A) and GHR (B) mRNA for the early-weanedrats on 7 and 28 d. Values are means (±SE) of six rats. (A): No differenceswere observed among the three treatments on 7 or 28 d in liver or muscleIGF-1 expression (P > 0.05). (B) Hepatic expression of GHR was significantlyenhanced by RD-H diet on 7 d (P < 0.05) but no significant differenceswere observed on 28 d (P > 0.05). Dietary treatment did not significantlyaffect GHR expression in muscle on 7 d or 28 d (P > 0.05).

are anti-nutrition factors present in the SB diet, specifically thetrypsin inhibitor, which was correlated inversely with the growthof villous.37

Although nutritional and hormonal factors have complexinfluences on body growth, the current somatomedin hypothesissuggests that the GH–IGF-1 axis plays a major role in growthregulation.38,39 The anabolic effects of GH are mediated by IGF-1,and IGF-1 levels are dependent on the expression of IGF-1 mRNAphosphorylated by the binding of GH to GHR in liver.38 – 40 SerumGH levels were not significantly influenced by dietary treatment,and muscle expression of GHR mRNA was found to be unaffectedby dietary protein, which concurs with recent research.41,42 On theother hand, an RD-H diet could significantly enhance hepatic GHRexpression in rats in the first week post-weaning (Fig. 6B), whichmay be responsible for the slightly higher IGF-1 expressions inliver at 7 d and in muscle at 7 and 28 d post-weaning (Fig. 6A). Thisobservation is consistent with the report that the increase in liverGHR expression can be attributed to an increase in serum IGF-1level,43 which is dependent on the expression of IGF-1 mRNA inliver and non-hepatic tissues.44

CONCLUSIONCompared to SB protein, ingestion of rice protein could helpameliorate small intestine morphology of weaned rats on 28 dpost-weaning, as well as improve the feed conversion efficiencyin the first 2 weeks, and this was better for the RD-H diet thanfor the RD diet, though there was no difference in average dailybody gain among treatments throughout the experiment periodor even RD-H diet was not beneficial for a greater average dailyfeed intake in the first 2 weeks post-weaning. Additionally, a dietwith addition of RD-H had no adverse effect on the expression ofGH–IGF-1 axis in liver and muscle of rats during the 28-d post-weaning period, but enhanced the hepatic GHR mRNA expressionon 7 d post-weaning. The present form in peptide of protein fromRD-H may be responsible for its special effect in growth. RD-Hhas the potential to be an effective protein source in balancedearly-weaned diet and food.

ACKNOWLEDGEMENTSThis study was jointly supported by grants from the Programsof State Key Laboratory of Food Science and Technology,Nanchang University (Programs No. SKLF-TS-200818 and SKLF-MB-200809), the 863 High-tech Development Program of China(No. 2008AA10Z332), and the Major High-tech IndustrializationProject of Jiangxi Province (No. Gan2008212).

REFERENCES1 Klein S, Kinney J, Jeejeebhoy K, Alpers D, Hellerstein M, Murray M, et al,

Nutrition support in clinical practice : review of published dataand recommendations for future research directions. Clin Nutr16:193–218 (1997).

2 McClave SA, Do peptide-based enteral formulas provide any benefitover intact protein diets? Nutrition 11:395–397 (1995).

3 Boza JJ, Moennoz D, Vuichoud J, Jarret AR, Gaudard-de-Weck D andBallevre O, Protein hydrolysate vs free amino acid-based diets onthe nutritional recovery of the starved rat. Eur J Clin Nutr 39:237–243(2000).

4 Yue LY and Qiao SY, Effects of low-protein diets supplementedwith crystalline amino acids on performance and intestinaldevelopment in piglets over the first 2 weeks after weaning. LivestSci 115:144–152 (2008).

5 Vente-Spreeuwenberg MAM, Verdonk JM, Bakker GC, Beynen AC andVerstegen MWA, Effect of dietary protein source on feed intake andsmall intestinal morphology in newly weaned piglets. Livest ProdSci 86:169–177 (2004).

6 Opapeju FO, Rademacher M, Payne RL, Krause DO and Nyachoti CM,Inflammation-associated responses in piglets induced with post-weaning colibacillosis are influenced by dietary protein level. LivestSci 131:58–64 (2010).

7 Siemensma AD, Weijer WJ and Bak HJ, The importance of peptidelengths in hypoallergenic infant formulae. Trends Food Sci Technol4:16–21 (1993).

8 Adibi SA, Glycyl dipeptides: new substrates for protein nutrition. J LabClin Med 113:665–673 (1989).

9 Parrado J, Bautista J and Machado A, Production of soluble enzymaticprotein hydrolysate from industrially defatted nondehulledsunflower meal. J Agric Food Chem 39:447–450 (1991).

10 Clemente A, Enzymatic protein hydrolysates in human nutrition.Trends Food Sci Technol 11:254–262 (2000).

11 Van Hooser B and Crawford LV, Allergy diets for infants and children,Compr Ther 15:38–47 (1989).

12 Yun JH, Yong JS and Chae BJ, Effects of feeding rice proteinconcentrate on growth performance and ileal digestibility in early-weaned pigs. Asian Australas J Anim 18:384–389 (2005).

13 Hou ZP, Yin YL, Huang RL, Li TJ, Hou R, Liu Y, et al, Rice proteinconcentrate partially replaces dried whey in the diet for early-weaned piglets and improves their growth performance. J Sci FoodAgric 88:1187–1193 (2008).

wileyonlinelibrary.com/jsfa c© 2010 Society of Chemical Industry J Sci Food Agric 2011; 91: 687–693

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Effects of rice protein on growth in rats www.soci.org

14 Cunniff PA, Official Methods of Analysis, 16th edition. Association ofOfficial Analytical Chemists, Washington, DC, on CD ROM (1998).

15 You L, Zhao M, Cui C, Zhao H and Yang B, Effect of degree of hydrolysison the antioxidant activity of loach (Misgurnus anguillicaudatus)protein hydrolysates. Innov Food Sci Emerg 10:235–240 (2009).

16 Wu G and Knabe DA, Free and protein-bound amino acids in sow’scolostrum and milk. J Nutr 124:415–424 (1994).

17 National Research Council (NRC), Nutrient requirements of thelaboratory rat, in Nutrient Requirements of Laboratory Animals, 4thedition. National Academy Press, Washington DC, pp. 10–79 (1995).

18 Sagher FA, Dodge JA, Johnston CF, Shaw C, Buchanan KD and Carr KE,Rat small intestinal morphology and tissue regulatory peptides:effects of high dietary fat. Br J Nutr 65:21–28 (1991).

19 Kang P, Yin YL, Ruan Z, Pan J, Hu Q, Deng ZY, et al, Effect ofreplacement of lactose with partially hydrolysed rice syrup onsmall intestine development in weaned pigs from 7 to 21 days. J SciFood Agric 88:1932–1938 (2008).

20 Fu WJ, Hu J, Spencer T, Carroll R and Wu G, Statistical models inassessing fold change of gene expression in real-time RT-PCRexperiments. Comput Biol Chem 30:21–26 (2006).

21 Woods SC and D’Alessio DA, Central control of body weight andappetite. J Clin Endocrinol Metab 93:S37–S50 (2008).

22 Karhunen LJ, Juvonen KR, Huotari A, Purhonen AK and Herzig KH,Effect of protein, fat, carbohydrate and fibre on gastrointestinalpeptide release in humans. Regul Pept 149:70–78 (2008).

23 Shi G, Leray V, Scarpignato C, Bentouimou N, Bruley Des Varannes S,Cherbut C, et al, Specific adaptation of gastric emptying to diets withdiffering protein content in the rat : is endogenous CCK implicated?Gut 41:612–618 (1997).

24 Leray V, Segain J-P, Cherbut C and Galmiche J-P, Adaptation to low-protein diet increases inhibition of gastric emptying by CCK. Peptides24:1929–1934 (2003).

25 Hall WL, Millward DJ, Long SJ and Moragan LM, Casein and whey exertdifferent effects on plasma amino acid profiles, gastrointestinalhormone secretion and appetite. Br J Nutr 89:239–248 (2003).

26 Cudennec B, Rozenn RP, Elisa C and Martine FP, Peptides from fishand crustacean by-products hydrolysates stimulate cholecystokininrelease in STC-1 cells. Food Chem 111:970–975 (2008).

27 Shriver JA, Carter SD, Sutton AL, Richert BT, Senne BW and Pettey LA,Effects of adding fiber sources to reduced-crude protein, aminoacid-supplemented diets on nitrogen excretion, growthperformance, and carcass traits of finishing pigs. J Anim Sci81:492–502 (2003).

28 Rosell VL and Zimmerman DR, Threonine requirement of pigsweighing 5 to 15 kg and the effect of excess methionine in dietsmarginal in threonine. J Anim Sci 60:480–486 (1985).

29 Patience JF, Thacker PA and De Lange CFM, Swine Nutrition Guide, 2ndedition. Prairie Swine Center, Saskatoon, pp. 10–11 (1995).

30 Li DF, Swine Nutrition, 2nd edition. China Agricultural Press, Beijing,pp. 18–21 (2003).

31 Webb KEJ, Intestinal absorption of protein hydrolysis products: Areview. J Anim Sci 68:3011–3022 (1990).

32 Walton MJ, Cowey CB, Coloso RM and Adron JW, Dietary requirementsof rainbow trout for tryptophan, lysine and arginine determinedby growth and biochemical measurements. Fish Physiol Biochem2:161–169 (1986).

33 Souba WW, Herskowitz K, Salloum RM, Chen MK and Austgen TR, Gutglutamine metabolism. J Parenter Enter Nutr 14:45–50 (1990).

34 Wu G, Intestinal mucosal amino acid catabolism. JNutr 128:1249–1252(1998).

35 Reeds PJ and Burrin DG, The gut and amino acid homeostasis. Nutrition16:666–668 (2000).

36 Reeds PJ, Burrin DG, Stoll B, Jahoor F, Wykes L, Henry J, et al, Enteralglutamate is the preferential source for mucosal glutathionesynthesis in fed piglets. Am J Physiol 273:E408–E415 (1997).

37 Zarkadas LN and Wiseman J, Inclusion of different processed full fatsoy bean in diets for piglets, in The Weaner Pig, ed. by Varley Mand Wiseman J. British Society of Animal Science, Penicuik, UK,pp. 45–55 (2000).

38 LeRoith D, Bondy C, Yakar S, Liu JL and Butler A, The somatomedinhypothesis. Endocr Rev 22:53–74 (2001).

39 Frank SJ, Growth hormone signaling and its regulation: Preventiontoo much of a good thing. Growth Horm JGF Res 11:201–212 (2001).

40 Florini JR, Ewton DZ and Coolican SA, Growth hormone and theinsulin-like growth factor system in myogenesis. Endocr Rev17:481–517 (1996).

41 Straus DS and Takemoto CD, Effect of dietary protein deprivationon insulin-like growth factor (IGF)-I and-II, IGF-binding protein-2; and serum albumin gene expression in rat. Endocrinology127:1849–1860 (1990).

42 Brameld JM, Gilmour RS and Bitteru PJ, Glucose and amino acidsinteract with hormones to control expression of insulin-like growthfactor-I and growth hormone receptor mRNA in cultured pighepatocytes. J Nutr 129:1298–1306 (1999).

43 Peng M, Pelletier G, Palin MF, Veronneau S, LeBel D and Abribat T,Ontogeny of IGFs and IGFBPs mRNA leves and tissue concentrationsin liver, kidney, and skeletal muscle of pig. Growth Dev Aging60:171–187 (1996).

44 Sara V and Hall K, Insulin-like growth factors and their binding proteins.Physiol Rev 70:591–614 (1990).

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