Tài liệu Luận văn làm giàu protein củ sắn bằng cách lên men với nấm men làm thức ăn cho lợn địa phương ở lào

HUE UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY NOUPHONE MANIVANH NUTRITIVE IMPROVEMENT OF CASSAVA ROOT AND ITS UTILISATION IN TARO FOLIAGE AND BANANA STEMS BASAL DIETS FOR LOCAL PIG PRODUCTION IN SMALLHOLDERS IN LAO PDR DOCTOR OF PHILOSOPHY IN ANIMAL SCIENCES HUE, 2019 HUE UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY NOUPHONE MANIVANH NUTRITIVE IMPROVEMENT OF CASSAVA ROOT AND ITS UTILISATION IN TARO FOLIAGE AND BANANA STEMS BASAL DIETS FOR LOCAL PIG PRODUCTION IN SMALLHOLDERS IN LAO PDR SPECIALIZATION: ANIMAL SCIENCES CODE: 9620105 DOCTOR OF PHILOSOPHY IN ANIMAL SCIENCES SUPERVISORS 1: ASSOCIATE PROFESSOR DR. LE VAN AN 2: ASSOCIATE PROFESSOR DR. TRAN THI THU HONG HUE, 2019 GUARANTEE I hereby guarantee that scientific work in this thesis is mine. All results described in this thesis are righteous and objective. They have been published in Journal of Livestock Research for Rural Development (LRRD) http://www.lrrd.org Hue University, 2019 Nouphone MANIVANH, PhD student i DEDICATION To my parents, my husband (Phoneouthai Thiphavanh), my daughter (Southida Thiphavanh) and my son (Kanlaya Thiphavanh) ii ACKNOWLEDGEMENTS The research in this PhD thesis was conducted four experiments with supported from Mekong Basin Animal Research Network (MEKARN II) project for funding this thesis research and the scholarship for the PhD study. I am grateful for the support from all of those people and institutions: I would like to express my sincere gratitude to the Mekong Basin Animal Research Network (MEKARN II) project for funding this theses research and the scholarship for the PhD study. I would like to thanks the Faculty of Agriculture and Forestry, Souphanouvong University, Luagprabang province, Laos, for allowing me study leave and helping me to carry out the experiments. I would like to express my cordial and faithful gratitude to my main supervisors, Associate Professor Dr. Le Van An and co-supervisor, Associate Professor Dr. Tran Thi Thu Hong for their support, guidance, and valuable advice for writing paper. I would like to express deeply gratitude to Professor Dr. Thomas Reg Preston Director of the University of Tropical Agriculture (UTA) for his good discussion, valuable advice and useful guidance during my studies and research project. My sincere thanks to Professor Dr. Ewadle, International Coordinator MEKARN II project; Dr. Vanthong Phengvichith, National Agriculture and Forestry Researcher Institute (NAFRI), Lao PDR; Dr. Kieu Borin, MEKARN II regional coordinator for their facilitation, help and support to the whole course. Professors, lecturers and assistant lectures in Hue University of Agriculture and Forestry and MEKARN II program, for giving me care and useful knowledge; Dr. Vongpasith Chanthakhoun, Dean of Faculty of Agriculture and Forestry, Souphanouvong University for his help and encouragement. I am also grateful to my friends on the PhD course from Cambodia, Laos and Vietnam for their good friendship and sharing Lastly I would like to express special thanks to my husband (Phone outhai Tiphavanh), my parents and all my brothers and sisters for their support, encouragements and patience. iii ABSTRACTS The study was aimed at improving protein content of cassava root (Manihot esculenta Crantz) by solid-state fermentation with yeast (Saccharomyces cerevisiae), urea and di-ammonium phosphate (DAP) additive and its utilization as protein source in the diets of Moo Lath Pig in Laos. Four experiments were carried out with “two in cassava root fermentation experiments, two experiments were conducted with Moo Lath pig using taro silage (TS) replaced by protein-enriched cassava root (PECR) as protein sources on growing trial and digestibility. In chapter 2, experiment 1. Cassava root was fermented with yeast, urea and DAP in a solid-state fermentation to determine the degree of conversion of crude to true protein; and experiment 2 the limiting factor to the synthesis of true protein from crude protein in the fermentation of cassava root could be the decrease in pH in the fermentation substrate preventing the hydrolysis of urea to ammonia and thus decreasing the availability of nitrogen for growth of the yeast. The following experiment to determine the degree of conversion of crude to true protein, pH and ammonia. In experiment 1. The experiment was arranged as a 2*3*4 factorial in a completely randomized design (CRD). The treatments were: root processing (steamed and not steamed); DAP: 0, 1 and 2% of the substrate DM. The fermentation was over 14 days with samples taken for determination of true and crude protein (CP) at 0, 3, 7 and 14 days. In experiment 2. A CRD was used with 2 treatments arranged as a 2*9 factorial. The treatments were anaerobic and aerobic fermentation. The substrate was cassava root 93.6% + DAP 2% + urea 1.4% + yeast 3% (DM basis). True, crude protein, ammonia and pH were measured at 0 and 3h after preparing the substrates and every 24h until end of day 7 (0, 3h, 1, 2, 3, 4, 5, 6 and 7 day). Experiment 1 (chapter 2). The true protein (TP) in cassava root increased with a curvilinear trend (R2 = 0.98) from 2.30 to 6.87% in DM as the fermentation time increased from zero to 14 days; the ratio of true to crude protein increased from 24.6 to 63.7 over the same period. Increasing the proportion of DAP from zero to 2% of the substrate DM increased the TP from 5.6 to 7.3% in DM after 14 days of fermentation. Steaming the cassava root prior to fermentation improved slightly (p=0.67) the conversion of crude to TP. Experiment 2 (chapter 2). The pH decreased with iv fermentation time, according to an almost linear trend, from 5.8 immediately after mixing the substrate, to 5.47in 3h and to 3.43 after 7 days. The level of CP after mixing the substrate and additives was 10.35% in DM and did not change over the 7 days of fermentation. TP in the substrate increased from 2.37 to 6.97% in DM as the fermentation time increased from zero to 7 days. There were no differences in all these criteria as between the aerobic and anaerobic condition, other than a tendency for the pH to fall slightly more quickly in the first 4 days in the anaerobic condition followed by a slower rate of fall to reach almost the same final value after 7 days, as for the aerobic condition. Experiment 3 (chapter 3). A growth trial was conducted with 12 Moo Lath pigs with average 14.8 ±1.89 kg initial live weight in a CRD, with three replications of four treatments. The aim of the study was to determine the effect of replacing TS with PECR in a basal diet of ensiled banana stem (BS). There were positive responses in dry matter (DM) intake, live weight gain, feed conversion ratio, as the percentage of PECR in the diet was increased (zero to 15% in DM ). It was concluded that the replacing of taro foliage silage with PECR improved the quality of the overall diet, which resulted in higher intake, growth rate, better feed conversion ratio and economical efficiency. Experiment 4 (chapter 4). Four castrated male Moo Lath pig, weighing on average 15 kg were allotted at random to 4 diets within a 4*4 Latin square design, to study effects on DM intake, digestibility and N retention of levels of protein-enriched cassava root (PECR) as 0, 25, 50 and 75% in combination with TS as 80, 55, 30 and 5% with constant levels of ensiled banana stem 20% (all on DM basis). PECR at 25% in a diet led to increases in feed intake, diet digestibility and N retention in native Moo Lath pigs and PECR could be the result of its superior biological value compared with the protein in the taro foliage. These criteria declined linearly when the proportions of PECR were increased to 50 and 75% of the diet DM. Key words: banana pseudo-stem, di-ammonium phosphate, probiotic, solidstate fermentation, urea, yeast, crude protein, true protein, ammonia, pH, Moo Lath pig v TABLE OF CONTENTS GUARANTEE ................................................................................................................. i DEDICATION ....................................................................................................... ii ACKNOWLEDGEMENTS ................................................................................... iii ABSTRACTS ....................................................................................................... iv TABLE OF CONTENTS ....................................................................................... vi LIST OF FIGURES ............................................................................................... xi LIST OF PHOTO ................................................................................................ xiii LIST OF TABLES .............................................................................................. xiii LIST OF ABBREVIATIONS ............................................................................... xv INTRODUCTION .................................................................................................. 1 1. PROBLEM STATEMENT.................................................................................. 1 2. OBJECTIVES ............................................................................................................. 1 3. HYPOTHESES ........................................................................................................... 3 4. SIGNIFICANCE/INNOVATION OF THE DISSERTATION.................................. 1 CHAPTER 1: LITERATURE REVIEW ........................................................................ 5 1. PIG PRODUCTION IN LAOS .................................................................................. 5 1.1. ROLE OF PIG PRODUCTION ........................................................................... 5 1.2. PIG POPULATION ............................................................................................. 6 1.3. PIG PRODUCTION SYSTEM IN LAOS ............................................................ 8 1.3.1. Commercial pig production .......................................................................... 8 1.3.2. Semi-commercial pig production ................................................................. 9 1.3.3. Smallholder pig production .......................................................................... 9 1.3.4. Main problems in smallholder pig production systems ............................ 12 1.3.5. Important points to improve smallholder pig production system ............. 12 1.4. LOCAL PIG BREEDS RAISED BY SMALLHOLDERS ................................ 13 1.4.1. Moo Lath .................................................................................................... 13 1.4.2. Moo Chid, Moo Markadon or Moo Boua ................................................... 14 1.4.3. Moo Nonghad or Moo Hmong ................................................................... 15 vi 1.4.4. Moo Deng or Moo Berk ............................................................................. 15 2. REQUIREMENT OF PROTEIN AND AMINO ACID FOR GROWING PIGS .... 17 3. FEED STUFFS FOR PIG IN LAOS ........................................................................ 19 3.1. LOCAL FEED AVAILABLE FOR PIG ............................................................ 19 3.1.1. Taro (Colocasia esculenta) ......................................................................... 20 3.1.2. Cassava (Manihot esculenta Crantz) .......................................................... 23 3.1.3. Banana pseudo (Musa sapientum Linn) stems ........................................... 25 4. METHOD TO IMPROVE NUTRITIVE VALUE FOR FEED STUFF WITH LOW PROTEIN CONTENT .................................................................................................. 27 4.1. SACCHAROMYCES CEREVISIAE (S. CEREVISIAE) .......................................... 27 4.2. SOLID-STATE FERMENTATION (SSF) ........................................................ 29 4.2.1. Application of solid-state fermentation ...................................................... 29 4.2.2. Advantage solid-state fermentation ............................................................ 29 4.2.3. Factors influencing solid-state fermentation .............................................. 29 4.2.4. Problem of solid-state fermentation............................................................ 30 4.2.5. Conditions necessary for fermentation ....................................................... 30 4.3. PROTEIN ENRICHMENT ................................................................................ 31 5. UTILIZATION OF FORAGE-BASED DIET FOR PIGS ....................................... 32 5.1. EFFECT OF TARO FOLIAGE AS PROTEIN SOURCE ON FEED INTAKE AND DIGESTIBILITY OF PIGS ............................................................................. 32 5.2. EFFECT OF TARO FOLIAGE AS PROTEIN SOURCE ON GROWING PERFORMANCE OF PIGS ..................................................................................... 33 5.3. EFFECT OF TARO FOLIAGE REPLACING BANANA PSEUDO STEM ON GROWTH AND DIGESTIBILITY OF PIGS .......................................................... 34 5.4. EFFECT OF ENSILED TARO FOLIAGE REPLACED BY PROTEINENRICHED CASSAVA ROOT ON GROWTH PERFORMANCE AND DIGESTIBILITY ...................................................................................................... 35 REFERENCES ............................................................................................................. 36 CHAPTER 2 ................................................................................................................. 51 IMPROVING NUTRITIVE VALUE OF CASSAVA ROOTS ................................... 51 (Manihot esculenta Crantz) .......................................................................................... 51 vii ABSTRACT ............................................................................................................. 51 INTRODUCTION .................................................................................................... 53 EXPERIMENT 1: ......................................................................................................... 54 MATERIALS AND METHODS .............................................................................. 54 Experimental design ............................................................................................. 54 Measurements ....................................................................................................... 55 Chemical analysis ................................................................................................. 56 Statistical analysis ................................................................................................. 56 RESULT AND DISCUSSION ................................................................................. 56 Changes in the mass of substrate during fermentation ......................................... 59 EXPERIMENT 2. ......................................................................................................... 61 MATERIALS AND METHODS .............................................................................. 61 Experimental design ............................................................................................. 61 Measurements ....................................................................................................... 62 Chemical analysis ................................................................................................. 62 Statistical analysis ................................................................................................. 62 RESULT AND DISCUSSION ................................................................................. 62 DISCUSSION ........................................................................................................... 65 CONCLUSIONS ...................................................................................................... 66 REFERENCES ......................................................................................................... 67 CHAPTER 3 ................................................................................................................. 70 REPLACING TARO (Colocasia esculenta) SILAGE BY PROTEIN-ENRICHED CASSAVA ROOT IMPROVED THE NUTRITIVE VALUE OF A BANANA STEM (Musa sapientum Linn) BASED DIET AND SUPPORTED BETTER GROWTH IN MOO LATH PIG .......................................................................................................... 70 ABSTRACT ............................................................................................................. 70 INTRODUCTION .................................................................................................... 70 MATERIALS AND METHODS .............................................................................. 71 Experimental design ............................................................................................. 74 Data collection ...................................................................................................... 75 Chemical analysis ................................................................................................. 75 viii Statistical analysis ................................................................................................. 75 RESULTS AND DISCUSSION ............................................................................... 75 DISCUSSION ........................................................................................................... 79 CONCLUSIONS ...................................................................................................... 80 REFERENCES ......................................................................................................... 80 CHAPTER 4 ................................................................................................................. 83 APPARENT DIGESTIBILITY AND N RETENTION IN GROWING MOO LATH PIGS FED ENSILED TARO FOLIAGE (Colocasia esculenta) REPLACED BY PROTEIN-ENRICHED CASSAVA ROOT (Manihot esculenta Crantz) ................... 83 ABSTRACT ............................................................................................................. 83 MATERIAL AND METHODS ................................................................................ 85 Experimental design ............................................................................................. 85 Measurements and data collection ........................................................................ 86 Statistical analysis ................................................................................................. 87 RESULTS AND DISCUSSION ............................................................................... 87 Chemical composition .......................................................................................... 87 Feed intake ............................................................................................................ 87 Apparent digestibility ........................................................................................... 89 Nitrogen balance ................................................................................................... 90 DISCUSSION ........................................................................................................... 91 CONCLUSIONS ...................................................................................................... 93 REFERENCES ......................................................................................................... 93 GENERAL DISCUSSION AND CONCLUSIONS .................................................... 96 1. GENERAL DISCUSSION ....................................................................................... 96 1.1. IMPROVING NUTRITIVE VALUE OF CASSAVA ROOT (MANIHOT ESCULENTA CRANTZ) BY FERMENTATION WITH YEAST (SACCHAROMYCES CEREVISIAE) UREA AND DI-AMMONIUM PHOSPHATE (DAP) ......................... 96 1.2. EFFECT OF THE USE OF PROTEIN ENRICHED OF CASSAVA ROOT (MANIHOT ESCULENTA CRANTZ) ON INTAKE, DIGESTIBILITY, N BALANCE AND GROWTH PERFORMANCE OF LOCAL PIG. ...................................................... 99 2. GENERAL CONCLUSIONS ................................................................................. 103 ix 3. IMPLICATION AND FURTHER RESEARCH.................................................... 104 3.1 IMPLICATION................................................................................................. 104 3.2. FURTHER RESEARCH.................................................................................. 106 REFERENCES ............................................................................................................. 96 PUBLISHCATION LIST ........................................................................................... 112 x LIST OF FIGURES CHAPTER 1 .................................................................................................................... 5 Figure 1. Number of pigs in Laos from 2010-2016 ............................................. 7 Figure 2. Characteristic of pig in northern, central and southern in 2005-2015 .. 8 CHAPTER 2 .................................................................................................................. 51 EXPERIMENT 1: .......................................................................................................... 54 Figure 1. The level of crude and true protein after fermented 14 days ............. 58 Figure 2. Curvilinear response in the true and crude protein ratio with increasing length of fermentation........................................................................................ 58 Figure 3. Effect of level of DAP on concentration of true protein after 14 days of fermentation................................................................................................... 59 Figure 4. Changes in the mass of substrate during the fermentation ................. 60 Figure 5. Proportion of the original substrate fermented during different stages of the fermentation ............................................................................................. 60 EXPERIMENT 2. .......................................................................................................... 61 Figure 1. Effect of fermentation time on pH of cassava root fermented with yeast, urea and DAP, under anaerobic and aerobic condition ........................... 64 Figure 2. Effect of fermentation on true and crude protein content of cassava root supplemented with urea, DAP and yeast .................................................... 64 Figure 3. Distribution of the nitrogen as urea, ammonia and true protein at the beginning and after 7 days of fermentation ....................................................... 65 CHAPTER 3 .................................................................................................................. 70 Figure 1. Effect of supplementation with PECR on DM intake of pigs by replacing taro silage and ensiled banana stem as a basal diet ........................... 76 Figure 2. Relationship between live weight gain and PECR content of the diet 77 Figure 3. Relationship between feed conversion ratio and PECR content of the diet ..................................................................................................................... 78 xi CHAPTER 4 .................................................................................................................. 83 Figure 1. Mean values for DM intake by pigs fed diets in which taro silage was replaced by PECR .............................................................................................. 88 Apparent digestibility............................................................................................. 89 Figure 2. Mean values for apparent digestibility of DM and crude protein in pigsfed diets in which taro silage was replaced by PECR ................................. 89 xii LIST OF PHOTO CHAPTER 1: LITERATURE REVIEW ........................................................................ 5 Photo 1. Local pigs are allowed to scavenge freely all year round .................. 10 Photo 2. Local pigs in pen ................................................................................ 11 Photo 3. Feed stuffs available in farm condition .............................................. 12 Photo 4. Moo Lath ............................................................................................ 14 Photo 5. Moo Chid, Moo Markadon or Moo Boua .......................................... 15 Photo 6. Moo Nonghad or Moo Hmong ........................................................... 15 Photo 7. Moo Deng or Moo Berk ..................................................................... 16 CHAPTER 2 ................................................................................................................. 51 EXPERIMENT 1: ......................................................................................................... 54 Photo 1. The steaming of the cassava root ....................................................... 55 Photo 2. Aerobic fermentation of the cassava root .......................................... 55 CHAPTER 3 ................................................................................................................. 70 Photo 1. Wooden boards 30cm above the base of the barrel ............................ 72 Photo 2. The bamboo strips placed above the boards ....................................... 72 Photo 3. The steaming of the ............................................................................ 72 cassava root ....................................................................................................... 72 Photo 4. Mixing cassava root with urea, di-ammonium phosphate (DAP) and yeast .................................................................................................................. 72 Photo 5. The mixed substrate was put in bamboo baskets covered with plastic netting ............................................................................................................... 73 Photo 6. The protein-enriched cassava root ...................................................... 73 Photo 7. Taro (Colocasia esculenta) were chopped by hand ........................... 73 Photo 8. Taro (Colocasia esculenta) were wilted for 24h to reduce the moisture .......................................................................................................................... 73 Photo 9. Taro silage in the plastic bag .............................................................. 73 Photo 10. Ensiled taro after 14 days ................................................................. 73 Photo 11. Banana stems were chopped by hand into small pieces ................... 74 xiii Photo 12. Ensiled banana stems in 200 liter PVC ............................................ 74 Photo 13. Housing made from local materials.................................................. 74 Photo 14. Moo Lath pig used in the experiment ............................................... 74 xiv LIST OF TABLES CHAPTER 1 .................................................................................................................... 5 Table 1. Number of meat consumption in 2017 of Lao PDR .............................. 6 Table 2. Statistic of livestock population in Laos (2010-2016)........................... 7 Table 3. Pig population in Laos (2005-2015) ...................................................... 7 Table 4. Classification of phenotype characteristics and reproductive performance of native pigs produced under smallholder farm (SHPF) conditions in Lao PDR....................................................................................... 16 Table 5. Dietary amino acid requirements of growing-finishing pigs (NRC 1998) .................................................................................................................. 17 Table 6. Chemical composition of taro (Colocasia esculenta) in DM basis ..... 21 Table 7. Planted area, yield and production of cassava root ............................. 23 Table 8. Proximate nutrient composition of Cassava root and leaves ............... 24 Table 9. Planted area, yield and production of banana ...................................... 26 CHAPTER 2 .................................................................................................................. 51 EXPERIMENT 1: .......................................................................................................... 54 Table 1. Composition of the substrates (DM basis) .......................................... 55 Table 2. Mean values for DM, OM, crude protein; true protein and ratio of TP/CP at different stages of the fermentations (% in DM) ............................ 57 Table 3. Effect of level of DAP on concentration of crude protein, true protein and ratio of TP/CP after 14 days of fermentation (% in DM) ....................... 59 Table 4. Changes in the mass of fresh (FM) and dry (DM) substrate during the fermentation ....................................................................................................... 59 Table 5. Chemical composition (g/kg of DM)................................................... 60 EXPERIMENT 2. .......................................................................................................... 61 Table 1. Changes in pH, crude protein (CP), true protein (TP) and ammonia in cassava root fermented with yeast, urea and DAP under aerobic or anaerobic conditions ........................................................................................................... 63 xv CHAPTER 3 .................................................................................................................. 70 Table 1. The chemical composition of feed ingredients (% in DM, except DM which is on fresh basis)...................................................................................... 75 Table 2. Mean values for DM intake (g/day) by pigs fed taro silage (TS) and ensiled banana stem (BT) supplemented with protein enriched cassava root (PECR)............................................................................................................... 76 Table 3. Mean values for live weight changes of growing pigs during the experiment ......................................................................................................... 77 Table 4. Feed ingredient costs (LAK) ............................................................... 78 Table 5. Economic analysis of experimental treatments (LAK) ....................... 79 CHAPTER 4 .................................................................................................................. 83 Table 1. The chemical composition of feed ingredients (% in DM, except DM which is on fresh basis)...................................................................................... 87 Table 2. Mean values of DM intake by pigs fed protein-enriched cassava root (PECR) replacing taro silage with constant levels of ensiled banana stem ....... 88 Table 3. Apparent digestibility (%) of diets with PECR replacing ensiled taro foliage with constant levels of ensiled banana stem .......................................... 89 Table 4. Mean values for N balance in pigs fed protein enriched cassava root replacing taro silage with constant levels of ensiled banana stem ..................... 91 xvi LIST OF ABBREVIATIONS AA Amono acids ADG Average daily gain ANOVA Analysis of variance ADF Acid detergent fibre AOAC Association of Official Analytical Chemists BS Banana stem ensilage BW Body weight Ca Calcium CF Crude fibre CSF Classical swine fever Cl Chloride CRD Completely randomized design Cm Centimetre CP Crude protein °C Degree Celsius DAP Di-ammonium phosphate DE Digestible energy DLF Department of Livestock and Fisheries DM Dry matter EAA Essential amino acids EE Ether extract EBS Ensiled banana stem FAO Food and Agriculture Organization of the United Nation FW Fresh weight FCR Feed conversion ratio g Gram GDP Gross domestic product h Hour xvii ha Hectare HCN Hydrocyanic acids Kg Kilogram Lao PDR Lao People’s democratic republic LWG Live weigh gain LW Live weigh L Liter m Meter ME Metabolisable energy MAF Ministry of Agriculture and Forestry Mekarn Mekong Basin Animal Research Network N Nitrogen NRC National Research Council NAFES National Agriculture and Forestry Extension Service NAFRI Institute National Agriculture and Forestry Research NE Net energy NPN None protein nitrogen NFE Nitrogen-free extract F Neutral detergent fibre NST Non steamed NP Non-protein OM Organic Matter P Phosphorus PECR Protein-enriched cassava root PECP Protein-enriched cassava pulp pH Power of/potential Hydrogen Prob/p Probability RCBD Randomised Complete Block Design RDB Rice distillers’ by-product xviii