Nutritional evaluation of genetically modified rice expressing human lactoferrin gene

Journal of Cereal Science 52 (2010) 350e355

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Nutritional evaluation of genetically modified rice expressing human lactoferrin gene Yichun Hu, Min Li, Jianhua Piao, Xiaoguang Yang* National Institute for Nutrition and Food Safety, Chinese Center for Disease Control and Prevention, No. 29, Nanwei Road, Xuanwu District, Beijing 100050, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 January 2010 Received in revised form 24 April 2010 Accepted 19 May 2010

The nutritional quality of a new strain of genetically modified rice (Oryza sativa L.) expressing human lactoferrin gene (hLF rice) was evaluated on the basis of components, nutrient digestibility in pigs, protein availability in rats and protein digestibility corrected amino acid scores (PDCAAS), and compared to its parental rice variety (PR rice). Although exogenous human lactoferrin gene was introduced, it did not interfere with the digestibility of protein, carbohydrates, fat and crude fiber. The revised protein efficiency ratio of hLF rice was increased to 2.50, which was significantly higher than that of PR rice. The PDCAAS of PR rice was 52.66 and its first limiting amino acid was lysine, while the PDCAAS of hLF rice was improved to 54.06 and its first limiting amino acid was tryptophan. Thus, it can be concluded that the nutritional quality of hLF rice is superior to PR rice according to the results of availability experiments and PDCAAS, and the hLF rice would be a superior strain of rice based on protein composition of the grain. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Genetically modified rice Human lactoferrin Nutritional evaluation Protein

1. Introduction Rice (Oryza sativa L.) is the second largest crop in the world, and feeds nearly 1/2 of the entire population. However, a large number of people subsisting on rice diets suffer from nutrition deficiency in terms of micronutrients such as iron, vitamin A, vitamin C and zinc. The World Health Organization (WHO) has recognized that anemia has catastrophic effects on the health and quality of life of at least 2 billion people, 90% of which are caused by iron deficiency (Chen and Zeng, 2007; WHO, 2009).

Abbreviations: AAS, amino acid scores; AD, apparent digestibility; ALB, albumin; ALP, alkaline phosphatase; ALT, alanine amiotransferase; AOAC, American Organization of Analytical Chemists; AST, aspartate aminotransferase; BUN, blood urea nitrogen; CHO, cholesterol; CRE, creatinine; EAL, endogenous amino acids losses; FAO, Food and Agriculture Organization; GLU, glucose; GRN, granulocyte; HCT, hematocrit; HDLC, high-density lipoprotein cholesterol; HGB, hemoglobin; hLF rice, genetically modified rice expressing human lactoferrin gene; LDLC, low-density lipoprotein cholesterol; LYM, lymphocyte; MCH, mean corpusular hemoglobin; MCV, mean corpusular volume; MO, mononuclear cell ratio; NRC, National Research Council of America; PDCAAS, protein digestibility corrected amino acid scores; PER, protein efficiency ratio; PLT, blood platelet count; PR rice, parental rice of genetically modified rice expressing human lactoferrin gene; RBC, red blood cell; TD, true digestibility; TG, triglyceride; TP, total protein; UNU, United Nations University; WBC, white blood cell count; WZSP, Wuzhishan miniature pig; WHO, World Health Organization. * Corresponding author. Tel.: þ86 10 8313 2798; fax: þ86 10 8313 2808. E-mail address: [email protected] (X. Yang). 0733-5210/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2010.05.008

With the rapid development of transgenic technology, the nutritional quality of traditional rice could be optimized to compensate for natural deficiencies. Recently, several rice cultivars were developed with the aim of alleviating iron-deficiency anemia (Goto et al., 1999; Suzuki et al., 2003). However, systematic nutritional evaluation for these strains is rarely reported. Nutritional quality of a new strain of genetically modified rice expressing human lactoferrin gene (hLF rice), developed by Zhejiang University, China, was evaluated in this paper. The iron content of hLF rice was increased to 24.7 mg/kg, which was twice that of its parental rice (PR rice) “Xiushui 110” (Oryza sativa L. ssp. japonica). The principle of substantial equivalence for GM food assessment (OECD, 1996) was no longer applicable to the hLF rice, in which the content of iron was intentionally increased. Therefore an extra nutritional evaluation was conducted to determine whether the protein expressed by exogenous human lactoferrin gene had affected digestion or utilization of total protein in hLF rice and utilization of other nutrients. Two animal experiments were conducted to evaluate the nutritional quality of hLF rice compared with PR rice in terms of digestibility of protein, carbohydrates, fat and fiber as well as availability of protein in diets of inbred Wuzhishan miniature pigs (WZSP) and growing Wistar rats. Moreover, the PDCAAS method was also applied in this study to make a comprehensive nutritional evaluation for hLF rice.

Y. Hu et al. / Journal of Cereal Science 52 (2010) 350e355

2. Experimental 2.1. Materials The hLF rice and the PR rice were harvested in 2009 and obtained from Changxing transgenic experimental farm, Zhejiang University, Hangzhou, China. By use of Agrobacterium-mediated method, the codon-optimized human lactoferrin gene (Gt1 gene as promoter and PEP carboxylase gene from corn as terminator) was transformed into “Xiushui 110”. The hLF protein was specifically expressed in the seeds rather than other tissues to a concentration of 0.5 g/100 g seeds. The whole grain from PR rice and hLF rice were ground to a 0.5-mm mesh screen before analysis or inclusion into the diets. 2.2. Animals and diets 2.2.1. Digestibility experiment The ileal analysis method was used to determine digestibility of main nutrients in both PR rice and hLF rice. Eight healthy castrated male WZSPs, weighing 30e35 kg, were individually housed in stainless steel cages. All WZSP were surgically fitted with postvalve T-intestinal cannulas after 16-h fasting control. Six WZSPs, which were well recovered from the surgery and with good appetite, were selected. A replicated 2  2 Latin square design was applied for PR rice and hLF rice comparison. The formulation of diets has been described by Han et al. (2006). All WZSPs were fed twice per day with equal amounts of each meal at 12-h intervals (8:00 and 20:00). The casein diet, which was served to determine the endogenous amino acid losses (EAL), was supplied to all WZSPs finally to avoid affecting the nutritional condition of WZSPs. Each diet was supplied for 7 days. During each of the last 3 days, ileal digesta was continuously collected by self-designed honeycomb ducts (from 8:00 to 20:00) and frozen immediately at 20  C. Afterwards, all pigs were sacrificed at the end of the experiment and dissected to inspect whether cannulation had caused intestinal abnormalities. Routine pathological examination for intestinal tracts was carried out to observe if there were any pathological changes. 2.2.2. Availability experiment The availability experiment was designed to evaluate in vivo absorption and utilization of protein. The protein efficiency ratio (PER) method (Ge, 2004) was used to determine protein availability of PR rice and hLF rice by use of growing Wistar rats. Sixty weanling Wistar rats, weighing 60e80 g, were randomly divided into three groups, ten male rats and ten female rats for each group. By taking maximum protein addition as the principle, protein content of each diet was formulated up to 10%, while the insufficient part was supplemented by casein. All the other ingredients were added according to the formulation recommended by AIN-93G (Reeves et al., 1993). Rats in each group were given PR rice diet, hLF rice diet and casein diet respectively, and with the casein diet serving as a control. At the end of the experiment, blood of each rat was taken from eye plexus to determine the blood routines and blood biochemistry. All the animals in this study had free access to feed and water, and the care and treatment protocols were approved by the Experimental Animal Welfare and Ethics Committee of the Chinese Center for Diseases Control and Prevention (China CDC).

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et al., 1998). Therefore the digestibility of protein/amino acids was calculated by true digestibility (TD) which includes EAL, while the digestibility of other nutrients was calculated by apparent digestibility (AD). Parameters of digestibility were calculated using the following Eqs. (1), (2) and (3) (Ge, 2004; Han et al., 2006).

ADð%Þ ¼ 100 

Ndigesta Mdiet   100 Mdigesta Ndiet

(1)

Mdiet Mdigesta

(2)

EALðg=kgÞ ¼ Ndigesta 

TDð%Þ ¼ AD þ

EAL  100 Ndiet

(3)

where Mdiet is the content of chromium in diet (mg/kg), Mdigesta is the content of chromium in digesta (mg/kg), Ndiet is the content of nutrients in diet (g/kg), and Ndigesta is the content of nutrients in digesta (g/kg). PER and revised PER were calculated with Eqs. (4) and (5) (Ge, 2004),

PER ¼

Body weight increment of rats in experimental period Protein intake in experimental period (4)

Revised PER ¼

PER of experimental group  2:5 PER of control group

(5)

where 2.5 is the correction factor. Protein digestibility corrected amino acid scores (PDCAAS) of hLF rice and PR rice were calculated according to Eqs. (6) and (7) (Ge, 2004). The scoring model of reference protein was the scoring model for preschool children revised by the FAO/WHO/UNU expert panel in 1985 (Ge, 2004).

AASð%Þ ¼

Content of amino acid in measured protein ðmg=gÞ Content of amino acid in reference protein ðmg=gÞ (6)

PDCAASð%Þ ¼ AAS  TD

(7)

where AAS is the amino acid score. Protein, amino acids, fat, water, ash content, carbohydrates, chromium in rice diets and digesta were all determined in compliance with the National Standards of China (Standards Press of China, 2005). Blood routines were measured by automatic hematology analyzer (ACT-5diff, Beckman), and blood chemistry was measured by an automatic biochemical analyzer (7080, HITACHI). Statistical analysis was performed using SAS 9.1 for Windows statistical software. Differences among comparisons of digestibility values, PER values, blood routines and blood biochemistry obtained from pigs and rats were statistically analyzed by the TTEST procedure, and P < 0.05 was considered as significance limit.

3. Results 3.1. Digestibility of main nutrients

2.3. Analytical methods Ileal digesta was freeze-dried and ground through a 0.250-mm mesh screen. The animals excrete EAL because of digestive secretions, mucoprotein, desquamated epithelial cells etc. (Moughan

All pigs were normal in diet intake and water drinking, and were in good condition during the whole experiment. Although there was no statistical significant difference in total amino acid concentration (P ¼ 0.96), the composition of amino

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acids in hLF rice protein was different from that of PR rice (Table 1). There were no statistically significant differences in digestibility of protein, carbohydrates, fat and crude fiber between hLF rice and PR rice (Table 1). Except for digestibility of methionine and tyrosine in PR rice, digestibilities of all amino acids were above 90% for both hLF rice and PR rice. With the exception of tryptophan, glycine, proline and cysteine, the mean digestibility of amino acids in hLF rice was slightly higher than that in PR rice, but there was no statistically significant difference in digestibility of all amino acids between hLF rice and PR rice. The results of pathological examination showed that there was dropsy in layers of intestinal wall (see Fig. 1A), necrosis ablated in epithelial cells of mucosa, vascular dilatation and hyperemia in submucosa in two cases of intestinal wall where T-cannula was settled. In four other cases of T-cannula-settled intestinal walls, serous layer and muscle was found incrassated, numerous fibrous connective tissues were proliferated, and a large number of inflammatory cells were infiltrated. However, the upper and lower sides of T-cannulasettled intestinal wall were observed as normal in all pigs (see Fig. 1B).

3.2. Protein availability All rats were normal in diet intake and water drinking, and were in good condition during the whole experiment. Protein intakes, body weight increment and PER of each group are shown in Table 2. The protein intakes of each group were similar, but the increment of body weight was obviously different. In terms of PER, the hLF group displayed no statistical difference from the casein group (P > 0.5), but was significantly higher than the PR group (P < 0.01). The revised PER of the hLF group was also significantly higher than that of the PR group (P < 0.01).

The results of blood routine examination (Table 3) showed that apart from significant difference in MCH between the hLF group and the PR group (P < 0.01 for male, P < 0.05 for female), there was no significant difference between the two groups. Except for a significant difference in HGB of female rats (P < 0.01) between the hLF group and the casein group, there was no significant difference between the hLF group and the casein group. The blood chemistry examination showed that AST of female rats in the hLF group was significantly lower than that of the casein group (P < 0.05), serum TP of the hLF group was significantly higher than that of the PR group (male and female, P < 0.05) and of the casein group (female, P < 0.05), which indicated that the nutritional condition of rats in hLF group was superior to the other two groups. BUN of the hLF group was significantly higher than the casein group (male, P < 0.01) while there was no significant difference with the PR group (P > 0.05). The blood CRE, which reflected the nutritional condition of internal organs, had no statistical difference in all groups. HDLC (male and female, P < 0.01) and CHO (male, P < 0.01) of the hLF group was higher than the casein group, and LDLC (P < 0.01) of the hLF group was lower than the casein group, while CHO, LDLC and HDLC of the hLF group had no statistical difference with the PR group. The casein diet was mainly made from corn starch and casein which was different from the components in rice, and this might be the reason for the difference between hLF and casein groups in CHO, LDLC and HDLC. Also there was no difference among the three groups in ALT, ALB, ALP, GLU and TG. 3.3. PDCAAS The results of PDCAAS for PR rice and hLF rice are shown in Table 4. The PDCAAS of PR rice and hLF rice were 52.66 and 54.06, respectively. The PDCAAS of hLF rice was slightly higher than that of PR rice. The limiting amino acids for both PR rice and hLF rice were

Table 1 Content and digestibility of main nutrients in hLF rice and PR rice. Digestibilitya,b (%)

Main nutrients/brown rice (g/kg)

Crude protein Carbohydrates Fat Fiber Amino acids Threonine Valine Methionine Isoleucine Leucine Phenylalanine Lysine Tryptophan Aspartate Serine Glutamate Glycine Alanine Tyrosine Histidine Arginine Proline Cysteine a b c d

c

PR rice

hLF rice

P value

PR rice

hLF rice

Variation at protein base (%)

82.0 756.00 15.00 7.78

81.4 764.00 20.00 8.88

e e e e

92.03 95.86 82.02 42.58

   

4.95 0.24 1.79 3.05

93.36 95.64 81.71 46.20

   

3.51 0.20 1.81 5.45

0.739 0.147 0.508 0.213

2.7 4.7 1.8 3.0 7.1 4.6 2.7 0.5 7.0 4.1 15.0 3.6 4.6 1.6 2.0 6.2 3.3 1.0

2.7 4.5 1.6 3.0 7.1 4.3 3.2 0.5 7.2 3.8 13.3 3.6 4.8 1.8 1.9 6.4 3.7 1.1

0.74 3.55 10.46 0.74 0.74 5.83 19.39 0.74 3.62 6.63 10.68 0.74 5.12 13.33 4.30 3.99 12.95 10.81

92.03 93.94 88.68 92.47 92.35 95.94 95.27 100d 93.41 93.06 90.68 100d 93.62 88.61 96.12 97.86 100d 92.14

      

6.83 4.57 5.08 5.21 4.14 3.84 6.96

93.43 94.76 91.10 93.94 93.25 96.21 98.83 100d 94.63 93.55 91.88 100d 95.85 92.25 96.46 98.68 100d 91.79

      

5.76 4.05 3.77 4.10 2.98 4.08 6.64

0.709 0.748 0.110 0.599 0.675 0.908 0.385 0.238 0.678 0.856 0.608 0.242 0.471 0.051 0.868 0.479 0.864 0.792

 4.94  5.14  4.34    

5.33 4.29 3.55 1.94

 2.36

 4.89  3.85  3.49    

4.96 2.83 3.48 1.94

 2.04

Values are means  SEM, n ¼ 6. The digestibility of protein and amino acids was calculated by true digestibility and the digestibility of other nutrients was calculated by apparent digestibility. “e” Stands for decreased value comparing to the PR rice, while the increased value recorded without any mark. Digestibility  100% was regarded as 100%.

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4. Discussion

Fig. 1. (A) Layers of cannula-settled intestinal wall of WZSP (dropsy). There were pathological changes in intestinal wall of cannula-settled, and this was an example of dropsy. (B). The upper side of T-cannula-settled intestinal wall of WZSP (normal). There were no pathological changes in lower and upper side of intestinal wall of WZSP.

lysine, tryptophan and threonine. The first limiting amino acid of PR rice was lysine, which was the same as in other traditional grains. However, the first limiting amino acid of hLF rice was tryptophan given the relatively lower PDCAAS. The PDCAAS of other essential amino acids for both PR rice and hLF rice were all above 100%.

Table 2 Protein intakes, body weight increment and PER for each group. Group

Gender

Protein intakesa (g)

Body weight incrementa (g)

PER

Revised PER

PR group

Male Female

39.67  5.66 41.07  5.15

69.80  1.76 73.07  1.78

1.76 1.78

1.94 2.00

hLF group

Male Female

37.86  4.13 40.20  4.84

85.44  2.25 89.74  2.23

2.25 2.23

2.49** 2.51**

Casein group

Male Female

42.53  4.76 44.21  4.93

96.40  2.26 98.14  2.22

2.26 2.22

e e

*Significantly different vs PR group (P < 0.05). **Significantly different vs PR group (P < 0.01). a Values are means  SEM, n ¼ 10.

The protein composition of hLF rice was intentionally altered by introduction of exogenous hLF gene, in an attempt to improve its overall nutritional condition. The principle of substantial equivalence was no longer applicable to hLF rice. Therefore simple analysis of composition was not sufficient to make a nutritional assessment. Animal experiments were needed to assess the digestibility of the changed nutrient or its influence on the digestibility/availability of other constituents and other effects on the physiological processes in the animals (Flachowsky et al., 2005). As the content of exogenous protein in the same genetically modified food may vary because of different processing conditions, in this study the addition of hLF rice in each diet was designed to reach maximum amount. The content of hLF rice reached 97.8% and 82.78% in protein digestibility and availability experiments respectively, and other nutrients aside from protein in diets met the requirements of NRC (National Research Council, 1998) and AIN93G (Reeves et al., 1993) respectively. Inbred WZSP pigs were used as experimental animals to study nutrient digestibility of hLF rice compared with PR rice. The pigs’ anatomy, physiological characteristics and mechanism of diseases are extremely similar to that of humans, and therefore they are an ideal experimental animal model for the study of nutritional metabolism, heterogenic transplantation, or cardiovascular disease (Zhang and Feng, 2007). The ileal rather than fecal analysis method was used to determine nutrient digestibility in WZSPs. Digesta was collected by T-cannulas at the distal ileum. The ileal analysis method directly reflects the actual digestion in the intestine. Because of continuously rubbing with T-cannulas, pathological changes such as dropsy in intestinal walls, necrosis ablated in epithelial cells of mucosa and proliferation in fibrous connective tissues were found in the intestinal walls where cannulas were settled. However, both the upper and the lower side of cannulasettled intestinal walls were normal. It could be concluded that all pigs were in normal physiological situation and the settlement of cannulas did not affect the digestion of experimental diet. There was no statistical difference in protein digestibility between the two cultivars, which indicated that the protein of hLF rice could be digested as well as that of traditional PR rice. The digestibility of proteins and amino acids in both hLF rice and PR rice was close to that of GM rice with cowpea trypsin inhibitor (hsien rice), which was also conducted in pigs (Han et al., 2006). However, the digestibility of protein in both cultivars was higher than true digestibility of cooked rice (88  4%) reported by FAO/WHO (FAO/ WHO, 1989; Juliano, 1993). The result was in accordance with the conclusion that the protein digestibility in cooked rice is lower than raw rice reported by Boisen et al. (2001). The digestibility of our studied rice was lower than that of brown rice (hsien rice) reported by Eggum et al. (1981), which was conducted in rats by using a nitrogen balance method. The difference may be due to different rice cultivars, methods applied and animal models. In addition to protein, there were no differences in digestibility between PR rice and hLF rice in terms of carbohydrates, fat and fiber. Therefore, the transferred exogenous gene does not influence the digestion of main nutrients, and main nutrients in hLF rice can be identically digested as the PR rice. Body weight was the most direct index reflecting nutritional status. The results of body weight increment of rats during the availability experiment showed that there was no statistical difference between hLF and casein (control) groups, but the body weight increment of rats in the PR group was much lower than hLF and casein (control) groups. The Association on of Official Analytical Chemists (2007) recommended that PER was an essential index for nutritional evaluation of protein. The quality of food protein could

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Table 3 Blood routines and blood chemistry examination of rats. Specification

PR group

hLF group

Male Blood routinesa WBC (109/L) LYM (%) MO (%) GRN (%) RBC (1012/L) HGB (g/L) HCT (%) MCV (fl) MCH (pg) PLT (109/L) Blood chemistrya ALT (U/L) AST (U/L) TP (g/L) ALB (g/L) ALP (U/L) GLU (mmol/L) BUN (mmol/L) CRE (mmol/L) CHO (mmol/L) TG (mmol/L) LDLC (mmol/L) HDLC (mmol/L)

9.49 78.86 8.55 13.29 6.34 126.89 0.36 55.93 19.85 1414.50 30.80 226.90 71.39 40.07 253.80 2.15 4.96 44.07 2.76 0.58 0.38 2.58

Female  3.06  5.07  3.10  3.37  0.55  9.39  0.05  1.76  0.85  390.39            

8.74 78.51 9.43 12.06 5.81 124.00 0.32 57.50 21.00 1386.60

7.69 34.99 3.83 3.40 49.37 0.28 0.81 2.53 0.39 0.23 0.10 0.30

25.90 192.60 73.50 38.74 188.70 2.65 4.92 48.49 2.18 0.65 0.30 2.18

Casein group

Male  1.18  4.56  3.48  2.57  0.25  5.53  0.04  1.28  0.63  114.63            

3.48 22.78 4.08 3.69 17.42 0.65 0.70 4.77 0.21 0.39 0.05 0.24

11.80 78.30 7.11 14.49 6.42 134.70 0.37 57.65 21.08 1262.10 39.33 240.00 77.04 39.37 294.50 2.46 5.01 48.11 2.89 0.70 0.40 2.70

Female  1.58  2.84  1.86  3.91  0.49  8.43  0.05  1.89  0.59**  388.17            

6.04 32.97 4.89* 2.61 62.66 0.55: 0.71:: 5.59 0.27:: 0.20 0.07 0.23::

Male

Female

9.63  81.40  7.60  11.00  5.59  120.70  0.33  58.26  21.74  1341.40 

1.09 5.59 2.55 4.66 0.26 3.74:: 0.05 1.14 0.57* 172.36

11.00 79.02 7.85 13.13 5.85 134.00 0.33 58.20 21.15 1071.20

           

5.52 54.14: 5.08*: 2.89 47.36 0.95 1.62 4.93 0.29 0.21 0.05: 0.25::

36.80 256.70 74.42 37.44 243.63 3.18 4.16 52.27 2.16 0.73 0.37 1.92

27.30 179.10 78.61 41.37 184.20 2.96 5.02 47.45 2.34 0.66 0.27 2.28

 2.61  5.17  2.78  6.85  1.53  7.39  0.09  1.99  0.58  383.21            

6.01 65.40 4.05 3.45 45.76 0.66 0.61 6.68 0.31 0.28 0.08 0.30

9.92 80.97 7.69 11.32 5.83 130.70 0.37 59.02 21.62 1381.40 30.20 249.10 71.93 39.15 185.10 2.50 4.16 53.67 1.87 0.92 0.34 1.69

 2.70  4.72  2.05  3.93  0.28  6.73  0.05  1.73  0.56  109.91            

8.07 47.93 2.87 3.44 48.65 0.60 0.84 4.96 0.21 0.34 0.10 0.21

*Significantly different vs PR group (P < 0.05); : significantly different vs casein group (P < 0.05) **Significantly different vs PR group (P < 0.01); :: significantly different vs casein group(P < 0.01). ALB, albumin; ALP, alkaline phosphatase; ALT, alanine amiotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; CHO, cholesterol; CRE, creatinine; GLU, glucose; GRN, granulocyte; HCT, hematocrit; HDLC, high-density lipoprotein cholesterol; HGB, hemoglobin; LDLC, low-density lipoprotein cholesterol; LYM, lymphocyte; MCH, mean corpuscular hemoglobin; MCV, mean corpuscular volume; MO, Mononuclear cell ratio; PLT, blood platelet count; RBC, red blood cell; TD, true digestibility; TG, triglyceride; TP, total protein; WBC, white blood cell count. a Values are means  SEM, n ¼ 10.

be evaluated by revised PER by means of describing the level of availability and absorption of food protein (Ge, 2004). The result of the availability experiment showed the revised PER of the hLF group was equal to standard casein (Ge, 2004); while revised PER of the PR group was only 78.8% of standard casein. After introduction of hLF gene into traditional PR rice, the mean revised PER calculated from female and male rats significantly increased from 1.97 to 2.50. It was concluded that, compared with PR rice, protein in hLF rice was more easily utilized and absorbed by animals, which could be considered as a better source of protein than PR rice. Although there were certain differences between each group in terms of blood routines and blood chemistry, all parameters tested were normal, which suggested that the growth and development of rats were all under normal physiological condition during the whole processing of the availability experiment. The method of PDCAAS is based on composition of essential amino acids in food protein, protein digestibility and the ability of

food protein to satisfy the requirement of essential amino acids for humans. It is, therefore, also a good method for protein quality evaluation. As indicated from the PDCAAS result, with a 27 percentage increase in lysine compared with PR rice, the first limiting amino acid of hLF rice is tryptophan rather than lysine, as in traditional grains. Although the tryptophan’s PDCAAS was slightly lower in hLF rice, the final PDCAAS of hLF rice was still higher than that of PR rice. The proportion of amino acids in hLF rice was optimized and the quality of protein in hLF rice is better than that in PR rice. In conclusion, the present results suggest that, instead of interfering with the digestion and metabolism of the main nutrients in hLF rice, introduction of the hLF gene in hLF rice promoted the growth and development of animals. Efficacy studies are needed to determine whether the hLF rice can alleviate iron-deficiency anemia.

Table 4 The results of PDCAAS for PR rice and hLF rice. Amino acids

Threonine Valine Methionine þ Cystine Isoleucine Ieucine Phenylalanine þ tyrosine Histidine Lysine Tryptophan a

PDCAASa(%)

AAS(%) Scoring model

PR rice

hLF rice

PR rice

34 35 25 28 66 63 19 58 11

98.28 162.89 136.72 130.01 131.93 119.63 128.05 56.56 53.22

97.56 157.07 131.72 132.50 132.25 118.76 120.91 67.67 50.26

88.24 150.58 133.39 120.75 122.05 119.37 123.08 52.66 55.07

Values are the mean weights  SEM, n ¼ 6.

hLF rice         

4.44 4.99 3.05 1.92 1.89 3.55 4.54 3.93 2.73

91.15 150.38 118.07 124.47 123.33 107.10 116.63 66.88 54.06

        

5.61 5.72 4.69 5.43 3.94 3.71 4.21 4.49 1.97

Y. Hu et al. / Journal of Cereal Science 52 (2010) 350e355

Acknowledgements We would like to thank Zhicheng Shen and his team for rice samples provided, to Baofan Wan and Jun Shen for cannulation surgery and to Tianjin Chen for English editing.

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