Chinese Journal of Animal Nutrition 2008 Vol. 20 (1) Open Access Study on Effect of Plant Small Peptides on Blood Biochemical Indexes and Gut Flora of Suckling Piglets ZHOU Shao 1 WANG Guan-bao 1,2 YANG Zai-bin 2* (1 College of Agricultural Husbandry, Qinghai University, Xining 810013, China; 2 College of Animal Science and Technology, Shandong Agricultural University, Taian 271018, China) Abstract: To study the effect of plant small peptides on blood biochemical indexes and gut flora of suckling piglets, four hundred PIC suckling piglets (initially 7±2 days of age) were randomly allotted into 4 treatments, with ten pigs per pen and ten replicate pens per treatment. From 7 to 21 days of age, each treatment was randomly fed with one of four diets that were formulated to contain 2% fish meal, 2% spray-dried porcine plasma (SDPP), 2% plant small peptides and 4% plant small peptides respectively. At the end of the trial, three piglets with moderate body weight in every treatment were killed after being bled from the anterior vena cava. The result indicated that: 1) diets supplemented with 4% plant small peptides could significantly improve the concentration of blood sugar and serum total protein and decrease the concentration of serum urea nitrogen (P<0.05); 2) the level of IgG of pigs fed 4% plant small peptides or 2% SDPP was higher than that of the control group (P<0.05).3) Diets supplemented with 4% plant small peptides could significantly decrease the amount of Escherichia coli (E. coli) and salmonella (P<0.05) while remarkably increase the amount of lactobacillus in cecum. Key words: Plant small peptides; Suckling piglets; Blood biochemical indexes; Gut flora In the feed industry of pigs, the breeding period of piglets takes an important role in the whole production. The health condition and production level of piglets are affected by piglets diarrhea and weaning stress syndrome all long. The strategies that relieving antigenicity stimulus by improving the nutrient level of the starter pig diets and adding additives to the diets, which make the suckling piglets get used to the starter diets more earlier, is studied frequently in the research area about suckling piglets earlier weaning technology, which will improve sow s generate rate and decrease the probability of disease s transmission from sows to piglets. Spray-dried porcine plasma (SDPP) is considered to be a high quality protein source to deal with these problems [1][2]. However, the use of SDPP is limited due to its expensive price, the short sources and the feed safety. Recently, the nutrition of small peptide received much attention because its transport system has many advantages, such as being absorbed rapidly, no competing, consuming little energy and not easily saturated and so on [3][4]. Thus, it is an efficient way in theory and in feed safety that providing good protein sources and relieving weaning stress syndrome Date received: 2007-05-21 Author information: ZHOU Shao, male, professor. Corresponding author, E-mail: yangzb@sdau.edu.cn of piglets by producing bioactive small peptides using various plant protein sources. The objective of this experiment is to study the effect of small peptide derived from plant on the blood biochemical indexes and gut flora by adding the plant small peptide into the starter pig diets, and provide theory for piglets early feeding and weaning. 1 Materials and methods 1.1 Trial Animals Four hundred crossbred PIC suckling piglets (initially (7±2) days of age, weighing an average of 2.61 kg) were randomly allotted into four groups with ten pigs per pen and ten replicate pens per group. The piglets were housed in an environment-regulated delivery room with lactating swine. The room temperature was maintained (20-22) and the temperature in boxes in which piglets sleep was maintained (30-35). Each pen had a nipple water to allow the piglets to consume water ad libitum. The piglets were induced to intake feed two times one day at the beginning of the trial. The trial started at 7 days of age and ended at 21 days of age when piglets weaned. 1.2 Diets
ZHOU Shao et al.: Study on Effect of Plant Small Peptides on Blood Biochemical Indexes and Gut Flora The plant small peptides was a mixture of small peptides derived from many plant proteins which were firstly enzymolysised by compound enzyme and then processed via solid-fluid separation, grading depuration, vacuum concentration and spray drying. It contains 80% protein and 70% protein in form of small peptide. The average molecular weight of small peptide is around 1 000 Daltons. All diets in this experiment were formulated according to NRC (1998) nutrient requirements of pigs (Table 1).Each group was randomly fed one of four diets containing 2% fish meal, 2% spray-dried porcine plasma (SDPP), 2% plant active peptides and 4% plant active peptides respectively. Table 1 Composition and nutrient level of the test diets (dry-matter basis, %) Ingredients Corn 48.8 49 49.4 49.6 Dehulled soybean meal 13 13 13 13 Compressed protein 2.5 2.5 2.5 2.5 Whey meal 10 10 10 10 Fish meal 1.9 Spray-dried porcine plasma 2 Full cream milk powder 10 10 10 10 Globulinemia 2.9 2.4 2.1 Plant small peptides 2 4 Soybean oil 2.4 2.1 2.2 1.9 Glucose 4 4 4 4 Premix * 4.5 5 4.8 5 Total 100 100 100 100 Nutrient levels DE MJ/kg 14.02 14.02 14.02 14.02 CP 21.0 21.0 21.0 21.0 lysine 1.42 1.42 1.42 1.42 Met+Cys 0.81 0.81 0.81 0.81 Ca 0.88 0.88 0.88 0.88 P 0.74 0.74 0.74 0.74 * Premix provided per kilogram of complete diet: Mn, 40 mg; Fe, 130 mg; Zn, 130 mg; Cu, 15 mg; I, 35 mg; Se, 3 mg; vitamin A, 11,025IU; vitamin D 3, 2,203 IU; vitamin E, 80 IU; menadione (menadione sodium bisulfate complex), 4.4 mg; thiamine, 4.4 mg;riboflavin, 11 mg; d-pantothenic acid, 35mg; niacin, 59.5 mg; choline, 330 mg; Folic Acid,0.9 mg;biotin,0.5 mg; vitamin B12, 55 µg. 1.3 Index determination and method 1.3.1 Blood biochemical indexes At 21 days of age, these piglets were used to collect the blood sample 10~15 ml from the anterior vena cava using vacuum tubes. The blood was immediately centrifuged at 3000r/min for 30 minutes and supernatants were frozen at -20 until used for analysis of blood sugar (BS), serum total protein (STP), serum urea nitrogen (SUN)and antibody level. The BS, STP and SUN was determined by glucose oxidase method, biuret method and urase enzyme colorimetric method respectively. The antibody level in serum was determined by an automatic biochemical analyzer (Hitachi 7150, Japan). 1.3.2 Gut flora Twelve piglets were sacrificed after the blood sample was collected, and then 5cm posterior segment of cecum and midpiece of colon were immediately ligated at both terminal to prevent the contamination and then stored at -80 for analysis of gut flora. The amount of microbe was determined with ATB Expression expert system (BioMerieux, France). The amount of bacteria was expressed with logarithm of bacteria's numbers in 1g content of intestine tract [lg (cfu)/g] 1.4 Statistical analysis The data were analyzed using SPSS 10.0 and expressed as mean±sem. One-way analysis of variance (factor=group) was done for all piglets. Duncan s multiple range test was used to compare the significance of differences among group
2008 Vol. 20 (1) http://www.chinajan.com/oa/darticle.aspx?type=view&id=080207 means. A difference was considered significant at p<0.05. 2.2 Antibody level 2 Results 2.1 Blood biochemical indexes The concentration of BS and STP were highest in group III and IV(Table 2). They were significantly higher than the other two groups (P<0.05).The concentration of SUN of pigs fed 4% plant small peptides was notably lower than the other three groups. As shown in Table 3, the level of IgA and IgM in serum had not significant difference (p>0.05) (Table 3), especially IgA, whose content in four groups was on the same level. The IgG level in Group II and IV was not significantly different with each other (p>0.05) but higher compared to the other groups (p<0.05). Table 2 Biochemical indexes in serum of the tested piglets (mmol/l) Blood sugar 6.79±0.13 b 6.49±0.18 b 7.62±0.19 a 7.82±0.11 a Serum total protein 56.60±0.21 b 55.33±0.19 c 58.13±0.39 a 58.70±0.42 a Serum urea nitrogen 6.10±0.02 c 8.40±0.06 a 7.20±0.10 b 5.24±0.21 d Table 3 Antibody level in serum of suckling piglets IgG (g/l) 1.51±0.06 b 2.12±0.04 a 1.40±0.08 b 1.98±0.05 a IgA (g/l) 0.02±0.003 0.02±0.003 0.02±0.003 0.02±0.003 IgM (g/l) 0.34±0.04 0.39±0.03 0.32±0.04 0.35±0.01 2.3 Effect of different treatments on flora in large intestine peptide were significantly higher than that of pigs fed fish meal (p<0.05). In cecum, the amount of lactobacillus of pigs fed 4% plant active peptide were significantly higher than In this trial, the amount of E. coli and salmonella in large pigs fed SDPP (p<0.05), but it was not significantly different intestine of piglets fed 4% plant active peptides were in colon (p>0.05). significantly lower than that of pigs fed fish meal or 2% SDPP (p<0.05) (Table 4). On the contrary, the amount of lactobacillus in cecum and colon of pigs fed plant active Table 4. Amount of microbe in cecum and colon of suckling piglets Cecum E. coli 6.76±0.10 a 6.92±0.04 a 6.43±0.06 b 6.25±0.05 b Salmonella 4.70±0.10 b 4.99±0.06 a 4.36±0.03 c 4.37±0.03 c Lactobacillus 5.86±0.06 bc 5.70±0.05 d 5.96±0.08 ab 6.16±0.06 a Colon E. coli 6.06±0.02 a 6.06±0.03 a 5.96±0.04 ab 5.88±0.03 b Salmonella 4.63±0.03 a 4.69±0.02 a 4.45±0.03 b 4.30±0.06 c Lactobacillus 5.76±0.07 b 5.96±0.06 a 5.98±0.06 a 6.05±0.04 a * The amount of bacteria was represented with the logarithm of bacteria's numbers in 1g content of intestine tract [lg (CFU)/g].
ZHOU Shao et al.: Study on Effect of Plant Small Peptides on Blood Biochemical Indexes and Gut Flora 3 Discussion 3.1 Effect of plant small peptides on blood biochemical indexes Shen et al. [5] found that diets supplemented with small peptides could increase the concentration of BS and BTP and decrease the concentration of SUN in serum of hog. Blood sugar directly provides energy to animal s vital movement. Within the normal limits(4.2~8.3mmol/l), high concentration of blood sugar could enhance the immunity and resist the harmful invasion. In this trial, plant small peptides supplemented to diets significantly increase the concentration of BS, maybe it was because that the plant small peptides participate the neuroregulation and glyconeogenesis of BS. The serum total protein include albumin and globulin. They could maintain plasmatic osmotic pressure, participate the transportation of nutrient, retain dynamic balance of histone, maintain PH value of blood and provide energy in stringency. It has been indicated that, compared to the same level amino acid, small peptide was absorpted more higher, which could increase the concentration of protein in serum within a period of time [6]. Serum total protein (STP) has a important relationship with the metabolism of protein in vivo. It was used to measure the utilization rate of amino acid and protein [7]. The decreasing of SUN concentration means that increasing of nitrogen deposition and protein utilization. While the increasing of SUN concentration means that protein catabolism reinforced [8]. Boza et al. [9] found that compared to the same level amino acid, diets containing small peptide could significantly increase protein transformation efficiency and nitrogen deposition. The result in this trial indicated that plant small peptide was a nitrogen source whose transportation was very high and it could directly participate protein synthesis in vivo and so decreased the deposition of urine nitrogen. Effect of plant small peptide on antibody level in serum From their birth to 21 days of age, suckling piglets obtain the antibody from the colostrum to maintain their immunity. After 21 days of age, piglets can form their own antibody [10]. The level of immune globulin is the lowest at 21 days of age, once piglets intake solid diets in this period, their ability of resisting pathogenic bacteria that enter gastrointestinal tract from feed and water is weak, which will result in piglets sickness. Among four immune globulins of pigs, the concentration of IgG was the highest. It could activate the complement system and some leucocytes, such as macrophage, phagocytize and demolish virus and bacteria when combining with invading virus and bacteria [11]. In this trial, the concentration of IgG in Group II and Group IV was the highest, which indicated that diets supplemented with opportune SDPP and plant active peptides could elevate the concentration of IgG in serum, and then induce the activation of some immune function, therefore plant active peptides have the function as an immunoenhancer. These results confirmed the data of Wang et al. [12]. 3.2 Effect of plant small peptides on gut flora in large intestine Yue [13] observed that diets supplemented with 1000mg/kg, 3000mg/kg or 5000mg/kg soybean bioactive peptide could significantly decrease the amount of E. coli and salmonella while significantly promote the growth of lactobacillus. Cordoba et al. [14] observed that piglets fed soy oligopeptides under enterotoxigenic Escherichia coli (ETEC) colibacillosis showed better recovery after antibiotic treatment, which reflected that oligopeptide allows better recovery of the gut epithelium. E. coli was a kind of Gram-negative bacteria, both aerobe and anaerobe. It can make young animals diarrhea and get hydropsy. Salmonella is a kind of pathogenic bacterium. It often invades young animals body and lead to acute septicemia, gastroenteritis and other region inflammation. Lactobacillus, a kind of beneficial bacteria in intestine tract, can product some antibiosis compounds, such as bacteriocin, which can inhibit some harmful bacteria. Otherwise, it can cohere on the epithelial cell and occupy the adhere sites of epithelial cell which forms a protective biomembrane preventing pathogenic bacteria s allocation and diversion. He [15] found that salmonella, E. coli and bacillus were normal flora in piglet s gastrointestine. At the normal condition, normal gastrointestinal flora was benefit to parasitifer [16]. After piglets weaned, the gastrointestinal flora changed. The mount of pathogenic bacterium (E. coli. E.g.) increased and beneficial bacterium (lactobacillus, e.g.) was decreased [17]. It was thought that plant active peptides themselves had the sterilizing function or they could be used to ferment by some beneficial bacterium such as lactobacillus. This function showed by plant active peptides was similar to
2008 Vol. 20 (1) http://www.chinajan.com/oa/darticle.aspx?type=view&id=080207 that of antibiotics to a certain extent, which could provide a theory evidence for decreasing the application of antibiotics in diets of piglets. [6] Coma J, Carrion D, Zimmeman D R. Use of plasma urea nitrogen as a rapid response criterion to determine the lysine requirement of pigs. Journal of Animal Science, 1995, 73:472-481. 4 Conclusion In this trial, diets supplemented with 4% plant small peptide could significantly increase the concentration of [7] Gao Feng, Jiang Yun, Zhou Guang-hong, Han Zheng-kang, Zhu Xu-dong. Effect of fructooligosaccharides (FOS) on growth, metabolism and immune in weaning piglets. Animal Husbandry & Veterinary Medicine,2001,33(6):8-9. blood sugar, serum total protein and serum antibody level (P<0.05), and significantly decrease the concentration of serum urine nitrogen (P<0.05). In this trial, diets supplemented with 4% plant small [8] Boza, J. J., D. Moennoz, J. Vuichoud, et al. 2000. Protein hydrolysate vs free amino acid-based diets on the nutritional recovery of the starved rat. European Journal of Nutrition. 39: 237-243. peptide could significantly decrease the amount of E. coli. and salmonella (P<0.05) in large intestine and significantly increase amount of lactobacillus in cecum (P<0.05). [9] Ao Chang-jin, Hou Xian-zhi, Zhao Zhi-gong, Yang Yu-fen, Yibigeletu, Yu Zhi-hong. 1998. Changes in serum IgG content in suckling piglet. Journal of Inner Mongolia Institute of Agriculture References: and Animal Husbandry. 19(2) : 18-21. [10] WANG Jing-yan, ZHU Sheng-geng, XU Chang-fa. 2002. [1] Hu Qi-wei, Wang Chun-wei, Guo Shi-dong, Song Hua-jun. Effect of spray dried porcine plasma on grown performance and blood biochemical indexes of postweaning piglets. Biochemistry. 3rd edn. High education Press, Beijing. [11] WANG Tian, FU Yong-ming, LV Jun-long, et al. 2003. Effects of mini-peptide on the growth performance and the development of [1] Kats L. J., Nelssen J. L, Tokach M. D., et al. 1994. The effect of spray-dried porcine plasma on growth performance in the early-weaned Pig. J. Anim. Sci. 72: 2075-2081. small intestine in weaning piglets. Animal Husbandry & Veterinary Medicine. 35 (6):4-8. [12] YUE Hong-yuan. 2004. Study on effects of soybean bioactive [2] Webb, K. E. 1990. Intestine absorption of protein hydrolysis products: A review. J. Anim. Sci. 68: 3011-3022. peptides on growth performance and mechanism in weaning pigs. M. Thesis, Chinese agriculture university, Beijing. [3] Webb, K. E., Jr., J. C. Matthews, D. B. Dirienzo. 1992. Peptide absorption: A review of current concepts and future perspectives. J. Anim. Sci. 70: 3248-3257. [13] Cordoba X, Dvm, Borda E, et al. Soy oligopeptides in weaning pig nutrition. Feed international. 200, 3: 14-18. [14] He Ming-qing. Animal microecology. Bei Jing: China agriculture [4] Shen Feng, Wang Tian, Zhang Li-li, Huang Wei-dong. Effects of the mini-peptide preparation on the growth performance, slaughter performance and the serum biochemical indexes in finishing pigs. China Feed, 2006(2):30-32. Press,1994. [15] WANG Xu-ming, CHEN Zong-ze, YUAN Yi. Study on mechanism of probiotic. JiLin Agriculture Science. 2002, 27 (1): 50-53. [5] Li, De-fa, X. H. Zhao, T. B. Yang, E. W. Johnson, P. A. Thacker. 1999. A comparison of the intestinal absorption of amino acids in piglets when provided in free form or as a dipeptide. Asian-Aus. J. Anim. Sci. 12 (6) 939-943. [16] Mathew, A.G., Robbins, C.M. Influence of galactosyl lactose on energy and protein digestibility, enteric microflora and performance of weaning pigs. Journal of Animal Science. 1997, 75: 1009-1016.