RUMINAL FERMENTATION, PRODUCTION AND FATTY ACID QUALITY OF MILK OF LATE LACTATION DAIRY GOAT FED PUFA-DIET
SUPPLEMENTED WITH YEAST AND Curcuma xanthorrhiza Roxb
ENDANG SULISTYOWATI
THE GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY BOGOR
PERNYATAAN MENGENAI DISERTASI DAN
SUMBER INFORMASI SERTA PELIMPAHAN HAK CIPTA*
Dengan ini saya menyatakan bahwa disertasi berjudul Ruminal Fermentation, Production and Fatty Acid Quality of Milk of Late Lactation Dairy Goat Fed PUFA-Diet Supplemented with Yeast and C. xanthorrhiza Roxb adalah benar karya saya dengan arahan dari komisi pembimbing dan belum diajukan dalam bentuk apa pun kepada perguruan tinggi mana pun. Sumber informasi yang berasal atau dikutip dari karya yang diterbitkan maupun tidak diterbitkan dari penulis lain telah disebutkan dalam teks dan dicantumkan dalam Daftar Pustaka di bagian akhir disertasi ini.
Dengan ini saya melimpahkan hak cipta dari karya tulis saya kepada Institut Pertanian Bogor.
Bogor, Februari 2014
RINGKASAN
ENDANG SULISTYOWATI. Fermentasi Rumen, Produksi dan Kualitas
Asam Lemak Susu pada Kambing Perah dengan Fase Laktasi Akhir yang laktasi seperti kambing perah, diperlukan perbaikan nutrisional dalam ransum. Hasil- hasil penelitian sebelumnya telah dilakukan pada kambing atau sapi perah yang diberi suplemen ragi, pasta tape- temulawak atau curcuma (C. xanthorrhiza Roxb), Tabut blok, dan konsentrat laktasi yang mengandung ragi dan temulawak dapat meningkatkan produksi susu dan memperbaiki kualitas susu. Oleh karena itu, tiga penelitian yang meliputi evaluasi nutrisi konsentrat-PUFA (polyunsaturated fatty acid) selama penyimpanan, analisis ransum-PUFA secara fermentasi in vitro, dan aplikasi ransum- PUFA secara in vivo telah dilakukan. Ransum yang mengandung sumber PUFA, yaitu jagung giling sangrai, tepung kedelai sangrai, dan minyak jagung yang disuplementasi ragi Saccharomyces cereviseae dan curcuma diaplikasikan pada kambing perah dengan fase laktasi akhir.
kecernaan bahan kering (KCBK) dan kecernaan bahan organik (KCBO), N-NH3, VFA total dan parsial, TPC, dan CH4. Hasil menunjukkan bahwa, walaupun KCBK- KCBO dan N- NH3 terendah; namun, didukung dengan populasi protozoa yang rendah dan produksi VFA yang tinggi, ransum-PUFA dengan ragi dan curcuma bisa dipertimbangkan sebagai ransum yang paling potential dalam meningkatkan metabolism nutrien dalam rumen.
Penelitian ketiga, in vivo, dilakukan untuk mengevaluasi performan ransum-PUFA dengan suplementasi ragi dan curcuma terhadap produksi dan komposisi nutrisi serta asam lemak susu, mastitis status, kecernaan nutrien, dan metabolit darah pada 20 ekor Peranakan Ettawa (PE) pada fase laktasi akhir (sekitar 4 bulan laktasi). Rancangan perlakuan berdasarkan pengelompokan tingkat produksi susu dengan menggunakan Rancangan Kelompok dengan 5 perlakuan dan 4 ulangan. Perlakuan terdiri atas tanpa aditif (PD0), 3 tablet laktasi Asifit (PDA), 0.5% yeast (PDY), 2% curcuma (PDC), dan kombinasi 0.5% yeast dan 2% curcuma (PDM) yang ditambahkan sebagai topping menjelang pemberian makan pagi (06.00) dan sore (15.00); Asifit diberikan per oral pagi hari. Sumber PUFA selain konsentrat- PUFA dari penelitian sebelumnya, juga dicampurkan kulit kedele dari limbah produksi tahu. Produksi susu dicatat sebelum, selama, dan setelah perlakuan. Perlakuan PDC mengandung lemak lebih rendah dibanding PDM, dan lebih tinggi serat kasar tetapi rendah ADF dan Phosphor (P). Pada ransum- PUFA dengan ragi (PDY) menunjukkan rendah protein. Produksi susu selama perlakuan secara kuantitatif tinggi pada PDA dan PDY. Pada pasca perlakuan, produksi ini lebih tinggi (P<0.05) dibanding perlakuan PDY dan PDC; sedangkan PDM berada diantaranya. Komposisi susu pada PDM secara kuantitas lebih rendah untuk lemak, 4% FCM, % dan berat protein, rasio lemak: protein, bahan kering, dan bahan kering tanpa lemak (BKTL), namun lebih tinggi untuk laktosa dan Ca. Indikator mastitis dan metabolit darah tidak terpengaruh (P>0.05) oleh perlakuan. Namun, secara kuantitas PDM menujukkan angka terendah untuk SCC, terendah Hb, PCV,dan glukosa, prolaktin dan trigliserida. Pada perlakuan PDM, konsumsi nutrien lemak antarperlakuan tertinggi (P<0.05), sementara, kecernaan nutrisi sebagian besar juga dipengaruhi oleh perlakuan (P<0.05 atau P<0.01). Perlakuan ransum-PUFA dengan curcuma (PDC) menunjukkan paling rendah untuk BK, BO, PK, LK, dan GE (gross energy); sementara, PDY dan PDM tinggi dalam kecernaan nutrisi. Disimpulkan bahwa 0.5% ragi dan 2% curcuma bubuk yang ditambahkan pada ransum-PUFA (PDM) adalah yang terbaik dalam hal nutrisi dan metabolit serta produksi susu pasca perlakuan (dengan semakin berlanjutnya fase laktasi) pada kambing perah.
Hasil penelitian yang berkaitan dengan asam lemak menunjukkan bahwa PDM mengandung tinggi asam lemak total, asam lemak rantai sedang, rantai panjang, dan asam lemak tak jenuh; namun, rendah dalam SCFA, n6/n3 rasio, dan indeks atherogenik. Kualitas susu kambing perah yang diberi pakan PUFA- diet dengan ragi dan curcuma ini dapat dipertimbangkan sebagai produk yang lebih sehat. Oleh karena itu, ransum PUFA dengan 0.5% ragi dan 2% curcuma merupakan ransum yang dipilih untuk diaplikasikan pada ternak kambing perah.
ENDANG SULISTYOWATI. Ruminal Fermentation, Production and Fatty Acid Quality of Milk of Late Lactation Dairy Goat Fed PUFA-Diet Supplemented with Yeast and C. xanthorrhiza Roxb. Supervised by TOTO
TOHARMAT, ASEP SUDARMAN and KOMANG G. WIRYAWAN.
population and high VFA production in the goat rumen fluid, the PUFA- diet with a mixture of yeast and curcuma additives was considered the most potential diet to improve nutrient metabolism in rumen.
The third experiment, in vivo, was to evaluate milk production and health of dairy goat fed PUFA-diet supplemented with yeast of curcuma. The were 20 crossbred Ettawa goats in the late lactation selected based on their production levels and grouped them in Randomized Block design to receive 5 treatments of no supplement (PD0), 3 tablets of Asifit (PDA), 0.5%/d yeast (PDY), 2%/d curcuma (PDC), and a mix of 0.5%/d yeast and 2%/d curcuma (PDM) in diets containing concentrate with PUFA sources (roasted ground corn, roasted soy bean meal, and corn oil), soybean by-product, and King Grass. The variables evaluated were milk yield, milk composition, mastitis status, nutrient digestibility, and blood metabolites. Milk yield was recorded daily pre, during, and post treatment. The PDC had lower ether extract than that of mix diet and showed higher crude fiber but lower ADF; diet with yeast had lower crude protein; and mix diet showed lower P content. Milk yield during treatment was higher in PDA and PDY. Post treatment, these milk yields were higher (P<0.05) than those in PDY and PDC; while the PDM was in between. There were tendencies that mix diet (PDM) had lower milk fat, 4% FCM, % protein and weight, fat: protein ratio, dry matter, and solid non fat, but it showed higher lactose and Ca percentage. Mastitis indicators and blood metabolites were not affected by treatments. However, PDM showed lowest SCC, higher Hb, PCV, and glucose but had lower prolactin and triglyceride. In PDM, nutrient intake of ether extract was the highest (P<0.05), while nutrient digestibilities were mostly affected significantly (P<0.05 or P<0.05). The PDC had the lowest DM, OM, CP, EE, and GE; whereas PDY and PDM showed higher digestibilities. As conclusion, a mix supplement of 0.5%/d dried yeast and 2%/d curcuma powder was considered reasonable since it showed a better recovery in milk yield after treatment with progressing lactation in dairy goat.
Results dealing with fatty acid demonstrated some consistencies in diet containing polyunsaturated fatty acid (PUFA) supplemented with 0.5% yeast and 2% curcuma (C. xanthorrhiza Roxb) that were high in total fatty acid, MCFA, LCFA, and PUFA. Milk fatty acid of goat fed with this diet showed high in LCFA and MUFA; while it was low in SCFA, n6/n3 ratio, and atherogenicity index. These milk qualities were optimally considered good in terms of healthier product. Therefore, the PUFA- diet with 0.5% yeast and 2% curcuma was a reasonable choice to be applied for dairy goat.
© Hak Cipta Milik IPB, Tahun 2014
Hak Cipta Dilindungi Undang-Undang
Dilarang mengutip sebagian atau seluruh karya tulis ini tanpa mencantumkan atau menyebutkan sumbernya. Pengutipan hanya untuk kepentingan pendidikan, penelitian, penulisan karya ilmiah, penyusunan laporan, penulisan kritik, atau tinjauan suatu masalah; dan pengutipan tersebut tidak merugikan kepentingan IPB
Dissertation
as part of requirement to fulfill PhD degree at
Nutrition and Feed Science Study Program
RUMINAL FERMENTATION, PRODUCTION AND FATTY ACID QUALITY OF MILK OF LATE LACTATION DAIRY GOAT FED PUFA-DIET
SUPPLEMENTED WITH YEAST AND C. xanthorrhiza Roxb
THE GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY BOGOR
2014
Close exam examiner: Prof Dr Ir Dewi Apri Astuti, MS Dr Despal
Approved by Advisory Committee
Prof Dr Ir Toto Toharmat, MAgrSc Main Supervisor
Acknowledged by
Head of Study Program Nutrition and Feed Science
Dr Ir Dwierra Evvyernie A, MS MSc
Dean of Graduate School
Dr Ir Dahrul Syah, MScAgr Prof Dr Ir Komang G. Wiryawan
Co- Supervisor
Dr Ir Asep Sudarman, MRurSc Co- Supervisor
Name : Endang Sulistyowati
SID : D162110011
Dissertation title : Ruminal Fermentation, Production and Fatty Acid Quality of Milk of Late Lactation Dairy Goat Fed
PUFA – Diet Supplemented with Yeast and Curcuma xanthorrhiza Roxb
Date of Examination: 16 January 2014
Date of Graduation:
ACKNOWLEDGEMENT
Above all, I would humbly distinguish the Most Merciful Allah swt who led me to take this opportunity, pursuing my dream, my ultimate study. Secondly, I would like to acknowledge Directorate General of Higher Education (DGHE) of the Republic of Indonesia through BPPS for granting scholarship during the enrollment in Nutrition and Feed Science at the Graduate School of Bogor Agricultural University.
I would like to sincerely deliver my greatest gratitude to my Advisors: Prof Dr Ir Toto Toharmat, MAgrSc, Dr Ir Asep Sudarman, MRurSc, and Prof Dr Komang G Wiryawan for their advice, expertness, encouragement, and support. I would also express my appreciation to Prof Dr Dewi Apri Astuti, MS, Dr Anuraga Jayanegara, and Dr Ir Sumiati, MS MSc who had guided me for the preliminar y exams; also to Dr Ir Dwierra Evvyernie A, MSc as head of study program and Mr. Supriadi as her Assistant who have been very kindly taking care of the paper work concerning the process of study at this department. Highly recognitions were addressed to Prof Dr Ir Dewi Apri Astuti, MS and Dr Despal who had been my examiners in close exam (December 3, 2013). I would also recognize Prof Dr Ir Ali Khomsan, MS and Prof Dr Ir Lily Warly, MAgrSc who had dedicated their valuable time to serve as examiners in open exam; as well as to Prof Dr Ir Luki Abdullah MAgrSc and Prof Dr Ir Panca Dewi Manuhara Karti MS in January 16, 2014. My special gratitude was also extended to Prof Dr Ir Toto Toharmat, Nutrition Laboratory; Fapet- IPB Microbiology Laboratory; Fateta- IPB Microbiology Laboratory; Ms. Endang at PAU-IPB Proxymate Laboratory; IPB- Laboratory Terpadu; Kesmavet- FKH IPB Laboratory; IPB- Biofarmaka Laboratory; Balai Penelitian Tanaman Obat dan Rempah- Deptan; Balai Besar Pascapanen Laboratory- Deptan; Physiological Nutrition Laboratory- Deptan; and Faperta-UNIB Microbiology Laboratory; and Kimia Farma Laboratory. Special thanks also went to Mr. Syauqi who allowed me to use his facility at Cordero Dairy Goat Farm in Ciapus, Bogor during the in vivo experiment; Mr. Eko and co- workers as well as IPB Polytechnic students who had worked together effortlessly during the research at the farm.
Sincere appreciations were also addressed to all my lectures, staffs and colleagues for their sharing experience, knowledge, and friendship at Bogor Agricultural University. Special thanks were extended to Drh Ria P Santoso, MS, Dr Ir Nyoto Santoso, MS, Ir Fiastri, IMBR and FWB students, Sistanto, and University of Dehasen Bengkulu as part of my big family.
Kusuma Wardani, SSos, and Bambang Satrio Nugroho, SPi who have been praying and loving me as always.
This Dissertation is dedicated for you all and my students at Animal Science Department, University of Bengkulu.
Bogor, February 2014
TABLE OF CONTENT
LIST OF TABLE LIST OF FIGURE LIST OF APPENDIX
1. INTRODUCTION 1
2. THE NUTRITIVE PERFORMANCES OF PUFA- CONCENTRATE
SUPPLEMENTED WITH YEAST AND Curcuma xanthorrhiza Roxb
STORED IN 2-6 WEEKS
SUPPLEMENTED WITH YEAST AND Curcuma xanthorrhiza Roxb
Introduction 21
Material and Methods 22
Results and Discussion 24
Conclusion 29
4. MILK YIELD, MILK COMPOSITION, AND BLOOD
PARAMETERS OF LATE LACTATION DAIRY GOAT FED POLYUNSATURATED FATTY ACID DIET SUPPLEMENTED WITH YEAST AND Curcuma xanthorrhiza Roxb
Introduction 30
Material and Methods 31
Results and Discussion 35
Conclusion 43
LIST OF TABLE
2.1 Ingredients of PUFA-concentrate supplemented with yeast and C. xanthorrhiza Roxb concentrate supplemented with yeast and C. xanthorrhiza Roxb stored for 2-6 weeks
11
2.4 Ether extract, crude protein, crude fiber, N-free extract, and gross energy in PUFA-concentrate supplemented with yeast and C. xanthorrhiza Roxb stored for 2-6 weeks
15
2.5 Neutral detergent fiber, ADF, Ca, and P in PUFA-concentrate supplemented with yeast and C. xanthorrhiza Roxb stored for 2-6 weeks
13
2.6 Tannin and curcumin in PUFA-concentrate containing yeast and C. xanthorrhiza Roxb during 2-6 weeks storage
14
2.7 Microbes population in PUFA-concentrate supplemented with yeast and C. xanthorrhiza Roxb stored for 2-6 weeks
15
2.8 Correlation between moisture and other nutrient in PUFA-concentrate supplemented with yeast and C. xanthorrhiza
Roxb(PCM)
16
2.9 Fatty acid contents in PUFA-concentrate supplemented with yeast and C. xanthorrhiza Roxb stored for 2 weeks
17
2.10 Fatty acid contents in PUFA-concentrate supplemented with yeast and C. xanthorrhiza Roxb stored for 6 weeks
19
3.1 Composition of ingredients in PUFA-diet supplemented with yeast and C. xanthorrhiza Roxb used in in vitro goat ruminal fermentation
22
3.2 Nutrient contents of PUFA-diet supplemented with yeast and C. xanthorrhiza Roxb fermented in vitro in goat rumen liquor
24
3.3 Fatty acid contents in PUFA-diet supplemented with yeast and C. xanthorrhiza Roxb
25
3.4 In vitro dry matter and organic matter digestibility (IVDMD and IVOMD) and fermentation characteristics of PUFA-diet supplemented with yeast and C. xanthorrhiza Roxb in goat rumen liquor
27
3.5 Protozoa, TPC, and methane gas production of PUFA-diet supplemented with yeast and C. xanthorrhiza Roxb in goat rumen liquor fermentation in vitro
28
4.1 Ingredients and nutrient composition of PUFA-diet containing Asifit, yeast and C. xanthorrhiza Roxb for dairy goat
32
4.2 Effects of feedingPUFA-diets supplemented with Asifit, yeast
and C. xanthorrhiza Roxb on nutrient intakes of dairy goats
4.3 Effects of feeding PUFA-diets supplemented with Asifit, yeast and C.xanthorrhiza Roxb on nutrient composition of feces of dairy goats
36
4.4 Effects of feding PUFA-diets supplemented with Asifit, yeast and C.xanthorrhiza Roxb on nutrient digestibility of dairy goats
36
4.5 Effects of feeding PUFA-diet supplemented with Asifit, yeast and C.xanthorrhiza Roxb on milk yield and composition of dairy Goats
38
4.6 Effects of feeding PUFA-diets supplemented with Asifit, yeast and C.xanthorrhiza Roxb on microbial composition and mastitis indicator ofdairy goats
41
4.7 Effects of feeding PUFA-diets supplemented with Asifit, yeast and C.xanthorrhiza Roxb on blood parameters of dairy goats
42
5.1 Ingredients and nutrient composition of PUFA-diet containing Asifit, yeast and C. xanthorrhiza Roxb for dairy goats
46
5.2 Nutrient composition of PUFA-diet containing Asifit, yeast and C. xanthorrhiza Roxb for dairy goats
48
5.3 Total fatty acid contents in PUFA-diets supplemented with Asifit, yeast and C. xanthorrhiza Roxb for dairy goat
50
5.4 Fatty acid contents in milk of dairy goat fed PUFA-diet supplemented with Asifit, yeast and C. xanthorrhiza Roxb
51
5.5 Fatty acid contents in feces of dairy goat fed PUFA-diet supplemented with Asifit, yeast and C. xanthorrhiza Roxb
53
5.6 Fatty acid and PUFA correlation among intake, milk, feces of dairy goat fed PUFA-diet supplemented with Asifit, yeast and C. xanthorrhiza Roxb
54
LIST OF FIGURE
2.1 Ingredients and steps in preparing the yeast: (a) yeast ingredients; (b) mixing; (c) dough; (d) mouting-drying
7
2.2 Steps in preparing the curcuma powder: (a) shredding Curcuma xanthorrhiza Roxb; (b) drying; (c) oven; (d) grinding
7
3.1 Some activities conducted in the in vitro experiment: (a) PUFA-diet; (b) in vitro fermentation; (c) TPC test; (d) methane test; (e) tannin test; (f) NH3 test
23
4.1 Some activities done during the experiment: (a) PE goat; (b) additives; (c) diets; (d) blood; (e) milk; (f) feces samples
34
4.2 Milk production along the research (pre, during, and post treatment)
LIST OF APPENDIX
1 Breed and lactation state of each dairy goat used in the research
64
2 Body measurements of dairy goat used in the research 65
3 Goat and treatment arrangement during in vivo experiment 66 4 Environment condition around housing at Cordero Farm in
Ciapus, Bogor
67
5 Anova of PUFA-concentrate moisture 70
6 DMRT of PUFA-concentrate moisture 70
7 Anova of PUFA-concentrate dry matter 70
8 DRMT of PUFA-concentrate dry matter 70
9 Anova of PUFA-concentrate ash 71
10 Anova of PUFA-concentrate organic matter 71
11 DMRT-week of PUFA-concentrate organic matter 71
12 DMRT-treatment of PUFA-concentrate organic matter 71
13 ANOVA of PUFA-concentrate ether extract 72
14 ANOVA of PUFA-concentrate crude protein 72
15 ANOVA of PUFA-concentrate crude fiber 72
16 DMRT of PUFA-concentrate crude fiber 72
17 ANOVA of PUFA-concentrate NFE 73
18 DMRT-week of PUFA-concentrate NFE 73
19 DMRT-treatment of PUFA-concentrate NFE 73
20 ANOVA of PUFA-concentrate ADF 73
21 ANOVA of PUFA-concentrate ADF 74
22 DMRT-week of PUFA-concentrate ADF 74
23 DMRT-treatment of PUFA-concentrate ADF 74
24 ANOVA of PUFA-concentrate saccharomyces 74
25 DMRT of PUFA-concentrate saccharomyces 75
26 ANOVA of PUFA-concentrate bascillus 75
27 DMRT of PUFA-concentrate bascillus 75
28 ANOVA of dry matter consumption 75
29 ANOVA of organic matter consumption 75
30 ANOVA of crude protein consumption 76
31 NOVA of crude fiber consumption 76
32 ANOVA of ether extract consumption 76
33 DMRT of ether extract consumption 76
34 ANOVA of NFE consumption 76
35 ANOVA of gross energy consumption 77
36 ANOVA of NDF consumption 77
44 DMRT of dry matter digestibility 78
45 ANOVA of organic matter digestibility 79
46 DMRT of organic matter digestibility 79
47 ANOVA of crude protein digestibility 79
48 DMRT of crude protein digestibility 79
49 ANOVA of crude fiber digestibility 79
50 DMRT of crude fiber digestibility 80
51 ANOVA of ether extract digestibility 80
52 DMRT of ether extract digestibility 80
53 ANOVA of NFE digestibility 80
54 DMRT of NFE digestibility 80
55 ANOVA of gross energy digestibility 81
56 DMRT of gross energy digestibility 81
57 ANOVA of NDF digestibility 81
58 DMRT of NDF digestibility 81
59 ANOVA of ADF digestibility 81
60 DMRT of ADF digestibility 82
74 ANOVA of tridecanoid acid C13:0 in milk 84
75 DMRT of tridecanoic acid C13:0 in milk 84
76 Milk production of goats fed PUFA- diets along the research
85
1. INTRODUCTION Background
Milk production in Indonesia is increasing yearly, however, it has not fulfilled milk demand. In 2011, milk production was 926 103 tons, while the total demand of milk and milk product was 3,903 103 tons (DGLAH 2011). The lack of this milk demand would be imported for about 76.27% a year; with an international milk price of $400/ton, it would cost around $1,190,800,000/yr. On the other hand, with 17,483 103 population of goat in 2011, supposed 0.5% out of them was dairy goat and 30% of them was in lactation with 0.5 kg/d milk production, in one lactation period would be producing milk about 1,967 tons. There might be a potential alternative of milk production as much as 0.05%/yr to supply national milk demand. This assumption might be too low compared to data released by FAO that was about 1.56% goat milk contributed by Indonesia to the world goat milk (Thiruvenkadan 2012). This huge gap of milk consumption and milk production should be narrowed. At the same time, the quality of the milk needs to be improved as well. For these purposes there has to be nutrition manipulation that could be applied to the dairy ruminants, such as dairy goat.
In terms of fatty acid (short, medium- chain, saturated branched, mono- polyunsaturated, cis- trans conjugated) contents in animal product, including milk, have been receiving a lot of attention concerning of human health (Chilliard et al. 2003). Therefore, it needs efforts in improving rumen kinetics relating to nutrients of the feed provided, especially in improving the polyunsaturated fat, PUFA. This could be achieved by applying diet rich in PUFA (such as roasted corn grain, roasted soybean meal, and corn oil) supplemented with temulawak (Curcuma xanthorrhiza Roxb) and yeast.
Curcuma xanthorrhiza Roxb, within its root, contains some bioactives such as curcuminoids (3% of dry matter) consisting of curcumin (C), demethoxycurcumin, and bisdemethoxycurcumin (Rukayadi et al. 2008); and xanthorrizol (33.2% of rhizome oil) reported by Sirat et al. (2008). Curcumin has molecular weight of C21H20O6 that would decrease due to the changes of
poliphenol weight during radiation process of curcuma powder and curcuma simplicia. This condition resulted in degradation of covalent bond that changed to free phenol becoming highly active in antioxidant compared to that of in curcumin tablet and fresh curcuma (Nurlidar and Chosdu 2008). Bioactive of curcumin also functions as antibiofilm (Rukayadi et al. 2008), antimicrobial, anti- inflammation, anticancer (apoptosis, antiangiogenesis), detoxification, neuro protection, and antiaging (Hwang 2008); elevates bile production, lessens tissue inflammation and plasma LDL (low density lipoprotein) in rabbit (Wientarsih and Meulen 2008). It was also reported that in early identification, curcumin and xanthorrhizol worked very strong as antibacterial and effectively slow down the growth of Staphylococcus aureus, Salmonella paratyphi, Trichophyton gypseum, and Mycobacterium tuberculosis (Benson 2012).
are decreasing SCC (somatic cell count), Staph. aureus, and E. coli contents in milk.
In organic (less use of antibiotic synthetic) dairy farm, it was found decreasing CNS (coagulase negative staphylococci) and environment Streptococcus sp in milk sample (Suriyasataporn 2010). Normal SCC level of dairy cow milk was less than 1 x 105 cfu/ml; while the infected cow was 1 x 106 cfu/ml (BytyQi et al. 2010). It has been reported also that SCC (consisting of 75% leucocytes: neutrophils, macrophages, lymphocytes, erythrocytes, monocytes; and 25%epithelial cells) in infected dairy cows were facing potential loss of milk production for up to1200 kg/lactation with SCC of 2.2 - 4.5 x 106 cells/ml (Sharma et al. 2011). There was correlation between high level of SCC and mastitis associated with decreasing lactose, α-lactalbumin, and milk fat (Harmon 1994).
Combination of polyherbal in low level (125 mg/kg body weight) produced milk optimally in dairy goat (Mirzaei and Prasad 2011). Curcuma, including C. xanthorrhiza Roxb, also contains bioactive steroid that served as lactagogum, conserving the continuity of cell differentiation of epithel cells in normal tissue, mucose secretion, alveolus proliferation, and ductul growth in mammary gland. Hormones relating to this process such as prolactin is the most secreted hormon right before partus (Larson 1985). Concentration of prolactin in dairy goat blood decreased from 18.55 ng/ml in 15 days of lactation to 5.88 ng/ml in 150 days of lactation (Singh and Ludri 2002). Therefore, curcuma besides maintaining lactation, also containing other secondary compound, such as tannin and saponin that function in microbial rumen activity.
Ruminal kinetics are expressed by microbial rumen activity in digesting fiber that its dynamics were affected by protozoa functioning as predator for bacteria (Gutierez 2007). Saponin (triterpenoid and steroid saponin) generated from any materials were identified detrimental toward protozoa (antiprotozoa) and as defaunating agent in rumen with the effect of detergent foaming on the surface of cell membrane (Francis et al. 2002). Saponin generated from lerak (Sapindus rarak) extract of 0.8 mg/ml rumen liquor decreased protozoa population and affected composition and total bacteria population after 24 hours in vitro fermentation. Population of F. succinogenes bacteria was dropped with increasing consentrate; whereas P. ruminicola increased markedly with increasing Sapindus. Decreasing protozoa was associated with decreasing activity of methanogen; modifying H2 to be propionate supported by bacteria with certain hydrolyzed (easily accumulated, toxic) or condensed (bound, safe). Tannin from chestnut was the most reasonable in reducing CH4 gas per digested organic matter
with the lowest C2/C3 ratio (Jayanegara et al. 2008). Methane gas is affected during fermentation process of fat source of diet and metabolized it through lipid hydrolysis process with end results of triglyceride, free fatty acid, glycerol, then
continued in β- oxidation to yield acetate and H2, as the precursors ofmethane
Lipid supplementation produced fatty acid, including CLA (conjugated linoleic acid) based on its source; high in cooking oil and fresh grass, but, low in unprotected grain oil (Chilliard et al. 2003). Free fatty acid and CLA in the rumen will be biohydrogenized (hydrolyzed by bacteria with addition of 2 ion H+ to be saturated; in conversely, it should be bypassed then biosynthesized as unsaturated fat in milk). Eventually, it is transported into mammary gland (as in precursor), changed from linolenic acid (C18:3 cis 9, cis 12, cis 15) in several steps to become stearate (C18:0), then ultimately converted into oleate in mammary gland. This process is optimazed by SCD (steoroyl- CoA desaturase) enzyme. Fatty acid content of milk of goat fed sunflower and soy bean oil was lowered in saturated fat and atherogenicity index (1.21- 1.71), but increased in CLA compared to control. This suggested that unsaturated fatty acid in blood vessel was going up that made this index down as expected (Chilliard et al. 2003).
Yeast, mostly mentioned as Saccharomyces cereviseae in any forms, dried or liquid, is widely used as rumen enhancer or fermenting feed supplement with variable effects on dry matter and organic matter digestibility, ruminal microbes, and ruminal fermentation activity (pH rumen, VFA, lactate, milk production, and fatty acid) as described by Desnoyers et al. (2009). Lynch and Martin (2002) reported that yeast, both in culture and cells showed no different activities; however with high dose (0.73 g/l) produced lower CH4 and C2/C3 in in vitro fermentation of diet containing Bermuda grass. Viability of yeast cell in dry form was decreasing (from 9.88 x 1010 cfu/g to 5.43 x 1010 cfu/g) after 3 months of storage in 400C (Sullivan and Bradford 2011). Supplementation of RumiSacc, commercial yeast, was not significantly different from control in affecting BUN (blood urea nitrogen), cholesterol, triglyceride, and glucose of dairy cow (Yalcin et al. 2011).
could increase milk production for 0.1 kg/d with significant reduction in milk fat to 2.09% (Sulistyowati 2009).
However, along with these researches, there has not been studied on ruminal activity, blood metabolites, milk fatty acid quality and milk production, especially in dairy goat, moreover during late lactation. Therefore, three experiments had been conducted (nutrient quality of stored PUFA-concentrate, in vitro ruminal fermentation, and in vivo application of PUFA-diet supplemented with yeast and C. xanthorrhiza Roxb) in late lactating dairy goat.
Problems
Based on above description, it has been analyzed several problems that need to be improved:
1. Milk production of dairy goat was very low compared to its actual capacity.
2. Milk quality, especially milk fatty acid was high in saturated fat.
3. Ruminal fermentation needs to be manipulated with nutrient originated from PUFA –diet supplemented with curcuma and yeast.
Research Purposes
Based on the identified problems, three experiments were designed to meet those three purposes:
1. To evaluate nutritional quality of PUFA- concentrate containing roasted ground corn, roasted soybean meal, corn oil supplemented with curcuma and yeast stored in 2, 4, and 6 weeks.
2. To analyze performance of PUFA- diet supplemented with curcuma and yeast in in vitro ruminal fermentation.
3. To evaluate PUFA- diet added with Asifit tablet (a commercial tablet for women in lactation), curcuma and yeast on nutrient digestibility, blood metabolites, mammary health, production and fatty acid quality of milk of late lactating dairy goat.
Research Outcomes
Out of each experiment, there would be a result that could be applied, those are: 1. One PUFA – concentrate formula that is durable in nutritional quality
during the 2- 6 weeks of storage.
2. One PUFA- diet formula that is optimal in in vitro ruminal fermentation that is optimal metabolically.
3. One formula of PUFA- diet that is optimal for mammary health, production and fatty acid quality of milk of late lactating dairy goat.
Research Hypothesis
2. Curcumin, tannin, yeast (Saccharomyces cereviseae), and PUFA sources (roasted ground corn, roasted soybean meal, and corn oil) in PUFA- diet were able to reduce unwanted microbes, such as protozoa in such a way that would improve metabolic rumen in biohydrogenation process.
2.THE NUTRITIVE PERFORMANCES OF PUFA- CONCENTRATE SUPPLEMENTED WITH YEAST AND Curcuma xanthorrhiza Roxb
STORED IN 2-6 WEEKS
ABSTRACT
The objective of this experiment was to evaluate the nutritive performances of PUFA- concentrate supplemented with yeast and Curcuma xanthorrhiza Roxb stored in 2-6 weeks. There were four PUFA- concentrates, namely, no supplement (PC0), 0.5%yeast (PCY), 2%curcuma powder (PCC), and 0.5%yeast with 2%curcuma powder (PCM).
Yeast (containing 3.6 x 107 cfu/g) and curcuma powder (containing 0.8% curcumin) were
self made. These concentrates were evaluated for nutrition and fatty acid contents during
2 and 6 weeks of storage. Results showed that moisture and Saccharomyces cereviseae
(20.68x 106 cfu/g) increased significantly (p<0.05) in 6 weeks of storage; whereas, dry matter (DM) and organic matter(OM), crude fiber (CF), nitrogen free extract (NFE) decreased significantly (p<0.05). The total PUFA (P), P/S, Monounsaturated fatty acid (MUFA), and long chain fatty acid (LCFA) contents were found higher in PUFA- concentrate with 2%curcuma powder. Whereas, the PUFA- concentrate with 0.5%yeast and 2%curcuma powder was higher in unsaturated (U) fat and the ratio of U/S. Combining all nutrient performances during the storage of 2- 6 weeks, the PUFA- concentrate with 0.5%yeast and 2% curcuma powder was considered nutritionally healthy.
Key words: curcuma, nutrient, PUFA-concentrate, yeast, storage
INTRODUCTION
Concentrate is the main nutrient source for livestock, including dairy goats. Its nutritional values are importantly taken into account in order to fulfill the requirement for improved production and good quality of milk produced. Several feed supplements such as yeast have been applied extensively. It was reported as optimal level in a rate of 3g/dairy goat (Sulistyowati and Mega 2002), 20g/dairy cow (Sulistyowati et al. 2010b), 56g/dairy cow (Hristov et al. 2010), and 50g Rumisacc / dairy cow (Yalçın 2011). Yeast contain active viable cells, especially Saccharomyces cereviseae that would be beneficial for nutrient digestion through fermentation within the concentrate all the way to rumen system.
(a) (b) (c) (d)
Figure 2.2 Steps in preparing C. xanthorrhiza Roxb powder: (a) shredding; (b) drying; (c) oven; (d) grinding
Schmidely et al. 2005). Concentrate supplemented with roasted ground corn, yeast, and curcuma powder showed optimal levels in PUFA, ratio of PUFA/saturated (P/S), and n6/n3 designated for dairy cows (Sulistyowati et al. 2010b).
Dairy goats and their products are getting popular in terms of preference and nutrition for human health. Therefore, a part of this research was designed to evaluate concentrate containing roasted corn grain, roasted soy bean meal, and corn oil as PUFA sources supplemented with yeast and Curcuma xanthorrhiza Roxb, stored for 2, 4, and 6 weeks
MATERIALS AND METHODS
Feed Supplements Preparation
Yeast supplement was prepared by modification on a procedure of Pusbangtepa (1981). The ingredients were 500g of rice flour, 50g of cassava tuber,10.5%of sugar, 10g of garlic, 20.5%of Alpinia galanga Sw, 10g of lemon juice, 10g of local (Bengkulu, Indonesia) yeast, and 500g of water; mixed, mounted in 10g each, then sun dried. This yeast contained 3.6 107 cfu/g. These are ingredients and steps in preparing the yeast; shown in Figure 2.1.
(a) (b) (c) (d)
Figure 2.1. Ingredients and steps in preparing the yeast: (a) yeast ingredients; (b) mixing; (c) dough; (d) mouting-drying
PUFA- Concentrate Preparation
The concentratewas designed for lactating dairy goat with 30 kg of body weight and 1kg of milk production (NRC 1981). There were several ingredients combined in this concentrate as can be seen in Table 2.1.
Table 2.1 The ingredients of PUFA-concentrate supplemented with yeast and C. xanthorrhiza Roxb
PC0: PUFA concentrate with no supplementation; PCY: PUFA concentrate with 0.5%yeast; PCC: PUFA
-concentrate with no yeast and 2%curcuma; PCM:PUFA --concentrate with 0.5%yeast and 2%curcuma; 1half
roasted; 2 all roasted; 3 for 30kg dairy goat with 1kg of milk production (NRC 1981). NA: not available.
The ground corn was half roasted, while the soy bean meal was all. Both were roasted in 80° C for about 25 minutes until it was becoming light brown. Together with corn oil, these roasted soy bean meal, and roasted ground corn were intended as PUFA sources in concentrate, containing 46.46%, 50.35%, and 34.69% total fatty acid, respectively. Fatty acid contents of corn oil, roasted soybean meal, and roasted ground corn are presented in Table 2.2.
Cassava meal was prepared from the fresh tubers, sliced about 1 mm- thick, sun dried, then grinded as powder. Mixing of the ingredients were started by the smallest portion, manually as homogenized as possible. Each supplement was then added accordingly for each treatment.
Treatments and Experimental Design
Randomized Design with repeated measurement and split plot statistical analysis. Data were tabulated and analyzed for variance with any differences were detected by Duncan Multiple Range Test in significances of P<0.05 or P<0.01 according to Lentner and Bishop (1986).
Table 2.2 Fatty acid contents of corn oil, roasted soybean meal, and roasted ground corn
Fatty acid (% of total fat) Corn oil Roasted SBM Roasted ground
corn
Cis-11,14-Eicosedienoic Acid, C20:2 0.07 0.05 nd
Heneicosanoic Acid, C21:0 nd nd 0.03
Nutrient analyses of dry matter, crude protein (CP), crude fiber (CF), and ether extract (EE) were determined according to AOAC (2000). While, NFE was calculated as 100% - (moisture + ash + EE + CP + CF). The content of ADF (Acid Detergent Fiber) were analyzed by the method of Goering and Van Soest (1970). Minerals of Ca and P were analyzed using Atomic Absorbance Spectrophotometer (AAS) according to the procedure of AOAC (2000).
was then extracted by soxhlet method in 96% ethanol, continued with evaporation using rotavapor. Residue from this process was washed with aquadest then centrifuged; the solid was crystallized by adding MeOH. This crystal with orange- brown color is curcuminoid, in which curcumin was part of it (Sutrisno et al. 2008). Tannin was analyzed by extracting 3g of sample in boiling water, then strained and added with 1% FeCl3. Whenever green, blue, and black colors were
detected in Spectrophotometer , this means positive indicator for tannin.
Microbial analyses for bacteria population was quantified by weighing 1g of fineground sample, diluted in 10 ml of aquadest. This solution was serially diluted in 6 folds (106), then 0.1 ml was taken appropriately and inoculated onto 10 ml NA (Nutrient Agar) plate, made in double. Plates were incubated unaerobic at 400C for 2 days. Fungi analysis was conducted with the same procedure, except it was inoculated onto 10 ml PDA (Potato Dextrose Agar). Population of colony forming unit (cfu) found in each plate divided by sample weight timed dilution frequencies. The mean value from the double plate counts was used for statistical analysis.
Fatty acid methyl esters (FAME) were determined by using gas chromatography (GC) Shimadzu 2010 series. There are several calculations of total short chain fatty acid (C4- C10), medium chain fatty acid (C12-C16), long chain fatty acid (C>16), mono unsaturated, poly unsaturated, saturated, unsaturated, ratio of PUFA/saturated, and unsaturated/saturated fatty acid. Fatty acid ratio of n-6/n-3 was calculated using this formula (Schmidely et al. 2005 ): n6/n3 = (linoleic acid + arachidonic acid)/linolenic acid
While, atherogenicity score was calculated with this formula (Ulbrict and Southgate 1991):
Atherogenicity index : (C12 + 4C14 + C16)
(total unsaturated fat)
RESULTS AND DISCUSSION
Moisture, Dry and Organic Matter
PUFA- concentrate supplemented with yeast and C. xanthorrhiza Roxb stored for 2- 6 weeks are presented in several tables. In Table 2.3 most of the treatments were higher in moisture (16.38%), lower in dry matter (-2.05%), and lower in organic matter (-2.81%) significantly (P<0.05) during 6 weeks of storage, compared to those in week 2 and week 4 in three treatments, except PUFA- concentrate supplemented with yeast and curcuma (PCM).
Table 2.3 Moisture, dry matter, organic matter, and ash in PUFA-concentrate supplemented with yeast and C. xanthorrhiza Roxb stored for 2-6 weeks
Moisture content of the concentrate would be another important reason for the reduction in colony forming unit later. This is in conjunction with data on the viability of the cells as it is displayed in Table 2.7; that it showed a positive relationship when the lowest moisture happened in the 6 weeks of storage resulted in the highest concentration of Saccharomyces cereviseae as shown in PUFA- concentrate supplemented with yeast and curcuma (PCM). Increasing water availability and high temperature will activate yeast cells that eventually will be ceased because of inadequate available nutrients (Sullivan and Bradford 2011). Results in this experiment approved that dry matter and organic matter were decreasing with higher moisture content in the PUFA- concentrate with no supplements, yeast, or curcuma only, resulting in lower concentrations of S. cereviseae.
Ether Extract, Crude Protein, and Gross Energy
Other nutrients, such as crude protein, N-free extract (NFE), and gross energy in PUFA- concentrate supplemented with yeast and curcuma were not significantly changed within 2- 6 weeks of storage (Table 2.4).
Table 2.4 Ether extract, crude protein, crude fiber, N-free extract, and gross energy in PUFA-concentrate supplemented with yeast and C. xanthorrhiza Roxbstored for 2-6 weeks
However, ether extract contents in week-2 of PC0 (8.96%), PCY (8.41%), and PCC (8.98%) were significantly higher (P<0.05) than those in week- 6. While, in PCM concentrate, its ether extract was remained relatively the same during 2- 6 weeks storage. However, this concentrate showed the lowest ether extract in the 2 weeks storage. The crude fiber and NFE of 2 and 4 weeks storage were higher significantly (p<0.05) than that of in 6 weeks. Reductions were quantitatively found in crude protein (-8.01%) and gross energy (-6.65%) within 6 weeks of storage of all PUFA- concentrates. These data suggested that the longer the PUFA- concentrates (with or without yeast and curcuma supplementation) stored, the less nutritional values shown.
In conjunction with these facts (Table 2.3) that the average moisture content was increasing while the organic and dry matters were decreasing; consequently, other nutritional contents within the organic matter such as ether extract, crude fiber, crude protein, and gross energy were found diminishing as well. This suggested that during storage, fermentation process was happening where nutrients were broken down significantly. These carbon contents in the nutrients were being taken up for microbial growth, such as it happened in coincidence with higher population of Saccharomyces sp (2.35 107 cfu/g ) and Bacillus sp (11.43 107 cfu/g) as it can be seen in the next table .
Acid Detergent Fiber, Ca, and P
Within 2 weeks of storage, ADF contents of all PUFA- concentrates were not significantly different as can be seen in Table 2.5. Only ADF was affected, in week 2 and 4 were higher (P<0.05) than that of during 6 weeks of storage.
Table 2.5 Neutral detergent fiber, ADF, Ca, and P in PUFA-concentrate supplemented with yeast and C. xanthorrhiza Roxb stored for
2-6 weeks
In this week, PUFA- concentrate without any supplement (PC0) showed the highest ADF (33.95%). While, the lowest ADF (22.54%) carried by PUFA- concentrate with both supplements (PCM). In all treatments, the differences between week-2 and week-6 of storages were found in ADF (4.35%), Ca (32.84), and P (7.23%).
In overall, all three treated PUFA- concentrates were lower (P<0.05) in ADF compared to that of in control diet. Suggesting that these supplements (yeast, curcuma, and their combination) worked well in reducing fiber. The PCM was considered the most nutritional since it showed significantly (P<0.05) the lowest ADF with the highest Ca and P. Other study showed that basal diet for dairy cows in which 56g XP yeast incorporated, was having 31% NDF, 20.4% ADF, 0.96% Ca, and 0.39% P (Hristov et al. 2010). Compared to our data for, the NDF and P were more than doubled, while Ca was one third of those in XP diet.
Tannin and Curcumin
Bio actives detected in the C. xanthorrhiza Roxb used in this experiment were 0.8% curcumin and 1.58% tannin. Tannin levels in four treatments of PUFA- concentrate quantitatively were decreasing as much as 32.78% in 6 weeks storage compared to those in 2 weeks storage (Table 2. 6). Among the treatments, PUFA- concentrate with no supplements (PC0) quantitatively contained the lowest tannin; while PUFA- concentrate with yeast and curcuma supplements (PCM) had the highest one during 2- 6 weeks of storage. This suggested that curcuma powder contributed some amount of tannin in the concentrate. Tannins in all type of concentrate found in this study were not as high as it was in polyherbal combination (3.69%) as reported by Mirzaei and Prasad (2011).
Curcumin levels in both treatments, PCC and PCM were found the same in week -2, yet decreasing in week-6; however, it was slightly higher in PCM. These results showed that tannin and curcumin concentrations were found not as much high as than those in their original sources when other feedstuffs were combined together and depended upon its level added as well. Curcuminoids in
Table 2. 6 Tannin and curcumin in PUFA-concentrate containing yeast and C. xanthorrhiza, Roxb during 2-6 weeks storage
Bioactive PC0 PCY PCC PCM Average
PC0: PUFA -concentrate with no supplementation; PCY: PUFA -concentrate with 0.5% yeast; PCC: PUFA -concentrate with no yeast and 2% curcuma; PCM:PUFA -concentrate with 0.5% yeast and 2%
C. longa contained curcumin (1.06- 5.65%), demethoxycurcumin (0.83- 3.36%), and bisdemethoxycurcumin (0.42 – 2.16%) as observed by Jayaprakasha et al. (2002).
Microbial Viability
This self made yeast contained 3.6 107 cfu/g. The viable cell was within the range of other commercial yeast products (2.93 107 – 1.43 1013 cfu/g) that had been surveyed by Sullivan and Bradford (2011). They reported that there was a decrease in the viability of the cell approximately 90% per month when the active dry yeast was kept under high temperature of 400C; however, there was no significant difference of viable cells stored during spring and summer as the variability of the samples were so wide due to different distribution.
In previous research, the yeast was prepared in the ambient temperature of around 310C then added into PUFA- concentrate and kept in another ambient temperature of around 26.940C with average moisture of 78.83. These places of yeast preparation were certainly lower in the ambient temperature compared to the previous temperature.
Results in Table 2.7 showed that Saccharomyces sp was not significantly different within 2 weeks and 6 weeks; however, they were significantly (P<0.05) higher in 6 weeks compared to those in 2 weeks of storage.
Being the highest one was PUFA- concentrate with yeast and curcuma or PCM (23.5 106 cfu/g) or 2.35 107 cfu/g in 6 weeks of storage. This amount of viable cell seemed to be well protected by the addition of curcumin in the concentrate, while it was stored under suitable ambient temperature. This kind of concentrate would be beneficial for ruminants.
Bacillus sp were found significantly different (P<0.05) within 2 and 4 toward 6 weeks, while population in week 4 was about the same as in week 2. However, diet with combination of yeast and curcuma (PCM) in 4 weeks showed the highest (P<0.05) Bacillus sp. This might happen as there was yeast that could
Table 2.7 Microbes population in PUFA-concentrate supplemented with yeast and C. xanthorrhiza Roxb stored for 2-6 weeks
Microbe PC0 PCY PCC PCM Average
increase the availability of nutrient; together with nutrient available from curcuma then made this diet as a good source of Bacillus sp as well as Saccharomyces sp to grow more than that in separated supplement diets.
These S. cereviseae and Bacillus sp in PUFA- concentrate with curcuma powder in this experiment were higher compared to those in Tabut block (300g) containing 20% curcuma diluents and 35% yeast-fermented cassava stored in 12 weeks, were 9.5 104 cfu/g and 1.5 107 cfu/g, respectively (Sulistyowati et al. 2008b). Further study showed that Saccharomyces cereviseae supplementation was reported to increase organic matter digestibility generating a higher energy availability for milk yield in buffalos (Campanile et al. 2008).
Correlation between Moisture Content and Other Nutrient in PUFA-Concentrate
The correlations between moisture content and other nutrient in PUFA- concentrate supplemented with yeast and C. xanthorrhiza Roxb (PCM) were in negative manner. Higher correlations were found in DM, OM, ether extract, ADF, and bacillus; while the lower correlation was found in crude fiber; as shown in Table 2.8.
Fatty Acid Profile
Fatty acid methyl esters (FAME) contents in these concentrates (Table 2.9 and Table 2.10) were showing specific amount even though they contained the same level and sources of PUFA (roasted ground corn, roasted soy bean meal, and corn oil). This might represent the presence of yeast and curcuma or their combination in reaction with fatty acid content in concentrate during the bio hydrogenation reaction. Total fatty acid of PUFA- concentrate with 2% curcuma (PCC) seemed to be the highest (79.25%), while the one without supplement (PC0) was the lowest (33.74%), in 2 weeks of storage. In the 6 weeks of storage, the same treatment (PCC) showed the highest total fatty acid (62.64%), while the PUFA- concentrate with 0.5% yeast and 2% curcuma (PCM) had the lowest total fatty acid (46.54%). This described that curcumin only, with the amount of 0.07- 0.10% in PUFA- concentrate would be optimal in hydrogenation process such that it would yield the highest total fatty acid.
Table 2.8 Correlation between moisture and other nutrient in PUFA-concentrate supplemented with yeast and C. xanthorrhiza Roxb (Y2C20)
Moisture - nutrient Equation (Y) Correlation (r)
Organic matter Y= -0.485x + 88.74 0.87
Ether extract Y= -1.266x +19.37 0.99
Crude protein Y= -0.192x + 13.15 0.46
Crude fiber Y= -0.101x + 13.70 0.10
ADF Y = -8.151x + 95.59 0.89
Saccharomyces cereviseae Y = 121.4x- 930.2 0.41
As shown in the highest total fatty acid, polyunsaturated fatty acid (PUFA) was also found the highest in concentrate with 2% curcuma, both in 2 weeks (41.77%) and 6 weeks (24.07%) of storage. Obviously, it was decreasing with the longer time of storage. The total PUFA of PUFA-concentrate with yeast was almost twice (29.25%) as it was in PUFA-concentrate with no supplements in 2 weeks of storage. This, in part, could be described that yeast in RumiSacc contained PUFA (26.42%) that might contribute to the total PUFA of concentrate or diet as reported by Yalçın et al. (2011). Previous study (Sulistyowati et al. 2010c) of concentrate containing 0.5%yeast, 10.5%curcuma powder, with palm oil, corn oil, and roasted ground corn showed that total PUFA was ranging from 77.23% (in 1.5% corn oil and 3% roasted ground corn) to 79.19% (in 4.5% palm oil). These are about 2- 3 times higher than the results in the present experiment.
Table 2.9 Fatty acid contents in PUFA-concentrate supplemented with yeast and C. xanthorrhiza Roxb stored for 2 weeks
Fatty acid (% total fat) PC0 PCY PCC PCM SED
Cis-11,14-Eicosedienoic Acid, C20:2 0.07 0.04 0.06 0.10 0.03
Heneicosanoic Acid, C12, C21:0 0.02 nd 0.03 0.02 0.01
The fatty acid profile of PCC was considered the highest among other treatments; C18:2 (38.47% of total FA), C18:1 (27.02%), and lower levels of C18:3 (3.24%) and C18:0 (0.05%) in 2 weeks. In 6 weeks, PCC was still showing the highest fatty acid profile; C18:1 (21.94%), C18:2 (21.88%), C18:3 and C18:0 were lower. Curcumin in this concentrate containing corn oil, roasted soy bean meal, and roasted corn, seemed to be able to protect unsaturated fat from further hydrogenation. This is more in positive way since there is no fermentation effect from yeast in this treatment, so that it would not cause a certain level of rancidity that might lead to hydrogenation of much more unsaturated fat. Fatty acids were also detected in soy bean oil that the highest contents were found in C18:2 (54%) and C18:1(22%), as reported by (Bouattour et al. 2008).
It means that, only the P/S ratio in 2 week that was close to this; whereas, the n6/n3 was 8 – 9 times higher in this current research. The ratio of n6/n3 in the 2 weeks (12.71) and 6 weeks (14.59) were much higher than those in soy bean oil (4.69- 6.12), reported by Bouattour et al. (2008). These ratios in PUFA- concentrate were far above the recommended range of n6/n3 for human wise is between 5:1 and 10:1. However, it is necessary to check this ratio in the milk of goat fed with this concentrate.
Table 2.10 Fatty acid contents in PUFA-concentrate supplemented with yeast and C. xanthorrhiza Roxb stored for 6 weeks
Fatty acid (% total fat) PC0 PCY PCC PCM SED
Cis-11,14-Eicosedienoic Acid, C20:2 0.08 0.12 0.08 0.04 0.03
Cis-5,8,11,14,17-Eicosapentaenoic
PC0: PUFA -concentrate with no supplementation; PCY: PUFA -concentrate with 0.5% yeast; PCC: PUFA
–conc.with no yeast and 2% curcuma; PCM:PUFA –conc. with 0.5% yeast and 2% curcuma. Nd: not
For a comparison, the ratio of P/S were 0.88 and 1.69, respectively in saturated fat (prilled hydrogenated fat) containing diets and unsaturated fat (Ca soaps of long chain fatty acid) as reported by Harvatine and Allen (2006). These suggested that the type of fat supplemented in concentrate would determine its type of fatty acid. Different PUFA are not secreted freely from each other, therefore, any other vegetable oil as supplement would respond differently on its fatty acid content (Chilliard et al. 2006).
The atherogeneicity index increased in PUFA- concentrate with the more complete supplement in 2 weeks; on the other hand, it decreased in PUFA- concentrate with the same treatments in 6 weeks of storage. This index supposedly dealing with unhealthy saturated fat that might lead to some kind of blood vessel disease (Ulbricht and Southgate, 1991). However, other researchers (Mensink et al. 2003; Knopp and Retzlalaff 2004, Chilliard et al. 2006; Bouattour et al. 2008) declared that there is little fact that the medium chain fatty acid (C12:0, C14:0, and C16:0) caused in atherogenic effect. This saturated fat is even better compared to high carbohydrate with low fat; and it would be of concern only if fat consumption is excessive. The average index in this experiment was 0.10 and 0.27 during the 2 and 6 weeks of storage, respectively. These could be considered as low levels in the feed that will be healthy for ruminants and eventually into their product that will be beneficial for human health.
CONCLUSION
3. IN VITRO GOAT FERMENTATION OF PUFA- DIET SUPPLEMENTED WITH YEAST AND C. xanthorrhiza Roxb
ABSTRACT
This experiment, in vitro, was conducted to evaluate the ruminal performances of
PUFA-diet (containing PUFA-concentrate with soybean byproduct of tofu industry and King grass) supplemented with yeast and C. xanthorrhiza Roxb.. Experimental design being applied was Randomized Complete Block of 4 ruminal liquor derived from four slaughtered goats and 4 treatments (PD0-no supplement, PDY- 0.5% yeast, PDC-2% curcuma, and PDM- 0.5% yeast + 2% curcuma), in 3 replications. Variables being
analyzed were pH, N-NH3, total and partial VFA, TPC, and CH4. Results showed that in
spite of having the lowest organic and dry matter digestibilities as well as N- NH3; however, supported by low protozoa population and high VFA production in the goat rumen fluid, the PUFA- diet with a mixture of yeast and curcuma additives was considered the most potential diet to improve nutrient metabolism in rumen
Key words : curcuma, in vitro fermentation, PUFA- diet, yeast.
INTRODUCTION
Ruminal fermentation performance was affected mostly by diet being ingested. Diet containing high concentrate with polyunsaturated fatty acid (PUFA) may increase production, improve milk fat and milk fatty acid in ruminants, including goat. However, to make such improvements, it needs any bioaditives, such as yeast and curcuma to be supplemented into diet that would determine kinetics of rumen fermentation.
Yeast has been known as rumen enhancer. In vitro fermentation of diet added with Diamond- V XP yeast showed higher in dry matter digestibility (DMD), organic matter digestibility (OMD), and total volatile fatty acid (VFA) than those of diet with A- Max yeast with the same dose (57g/d) as reported by Miller- Webster et al. (2002); while supplementation of Saccharomyces cereviseae culture of 0.35g/l produced higher concentration of acetate (C2) than that of higher dose of 0.73g/l (Lynch and Martin 2002).
Based on these data, a study on in vitro fermentation of PUFA- diet supplemented with yeast and curcuma PUFA- diet had been conducted to observe in vitro goat rumen fermentation performance.
MATERIALS AND METHODS
Yeast, Curcuma Powder, and PUFA- Diet Preparation
Yeast was prepared from the previous (first) experiment according to Pusbangtepa (1981). Curcuma powder was also provided from experiment I through the processes of slicing, drying, grinding, and powdering). The PUFA- diet was reformulated as it was in the PUFA- concentrate then combined with soybean by-product from local tofu industry. Ground corn was half roasted; while soy bean meal was all roasted in 800C for about 20 minutes. These ingredients together with corn oil were designated as PUFA sources in the diets. The PUFA- diets were formulated accordingly to their treatments as can be seen in Table 3.1.
Nutrient contents were assessed based on proxymate analysis (AOAC, 1990); ADF was analysed according to Van Soest (1990). Curcumin was analysed using maseration method (Sutrisno et al. 2008), tannin was analysed by modification of Folin- Ciocalteu method (Harborne 1987). Calcium was detected using Atomic Absorbance AA7000 Shimadzu Co. Serial no A 306647-00345. rumen liquor of slaughtered goats. Tubes were soaked and flowed with CO2 for 30
seconds, checked for pH ( 6.5 – 6.9) using pH meter; tubes were then probed with ventilated rubber caps, fermented for 48 hours. When fermentation done, the
Table 3.1 Composition of ingredients in PUFA-diet supplemented with yeast and C. xanthorrhiza Roxb used in in vitro goat ruminal fermentation
Ingredients (%) PD0 PDY PDC PDM
caps were taken of from the tubes, add with 2 -3 drops of HgCl2 to stop microbial
growth (at this point, analyses were conducted for protozoa population, total plate count, TPC, and methane gas, CH4). Fermentor tubes were then centrifuged in 4.000 rpm for 10 minutes. Substrates were separated as solid in the bottom and supernatant above. The supernatant was used for volatile fatty acid, VFA, and N- ammonia, N-NH3analyses; while the solid was then added with 50ml solution of pepsin-HCl 0.2%, centrifuged in 4.000 rpm for 15 minutes. This substrate was then reincubated for 48 hours without probes; then strained through whatman paper no 41 (that has been known for its weight) using vacuum pump. The solid left in the paper was transferred into baker glass, ovened in 105⁰C for 24 hours. The glass, paper, and residue were taken out from the oven, put them in excicator then weighed them to get its dry matter content. Afterward, these samples were ashed in electrical tanur for 6 hours in 450 – 600oC. Then weighing them to get their dry and organic matters to calculate the in vitro dry matter digestibility (IVDMD) and in vitro organic matter digestibility (IVOMD). Blanco was prepared from the residue of the fermentation without samples. These digestibilities were calculated as if that of in hidrolytic post ruminal fermentation after two 24 hour period (Metode Tilley and Terry 1963).
Analysis on N- NH3 was conducted using micro diffusion Conway (General Laboratory Procedure 1966). Analysis of VFA in partial containing of acetate (C2), propionate (C3), isopropionat, butirate (C4), isobutirat, valerate, and isovalerate were detected using gas chromatography (GC) Chromopack 9002 with centrifuge IEC micromac RF type 3593, column capillary WCOT fused silica ID coating FFAP-CB. Methane (CH4) was measured using GC Shimadzu 8-APT detector TCD and C- R6A Chromatopack.
Analysis of total plate count (TPC) was conducted by weighing 50 g sample, added with 450 ml butterfield phosphate solution. Out of this solution, take 10 ml sample, added with 90 ml of butterfield phosphate solution as 101 dilution, then any dilution (101,102,103 or more) was done by pippetting 1 ml sample into 9 ml of butterfield phosphate solution, soaked using vortex. Putting 1 ml out of this sample onto PCA agar media, incubated in 35oC for 48 hours, placed the glass in upside down positition. These are (Figure 3.1) some of the activities conducted in this in vitro experiment.
(a) (b) (c) (d) (e) (f)
Experimental Design and Data Analysis This level fulfilled the protein requirement for 30 kg dairy goat with 1 kg milk as stated in NRC (1981), that was 12%. Other result showed that protein content in diet with extruded soybean in dairy goat was much higher 18.5 ± 0.38% or 42.75% higher (Schmidely et al. 2005). In quantity, PDM, diet with a mixture of yeast and curcuma, contained higher organic matter, lower ADF, higher Ca, and higher fat than other diets. This result approved that these two additives could improve nutrient content.
Diet with high concentrate in this experiment was about 80% could decrease ether extraxt of the diet that eventually decreased milk fat. However, since in this research used fat sources (roasted ground corn, roasted soy bean meal, and corn oil), the ether extraxt of these diets were relatively high (4.0- 4.43%), except in control diet (3.75%). This result was in accordance with others that diet without extruded soybean contained very low fat (1.38%), while the one with that ingredient showed high fat content (5.19%) as reported by Schmidely et al. (2005). Diet with corn oil was reported to have higher fat, 5.62% (Bouattour et al.
Table 3.2 Nutrient contents of PUFA-diet supplemented with yeast and C. xanthorrhiza Roxb fermented in vitro in goat rumen liquor
2008). It seemed that bioactives, curcumin and tannin did not affect the fat content of the diets.
Fatty Acid Content in PUFA- Diets
Among other diets (Table 3.3), the one with a mixed additives (PDM) quantitatively had the lowest saturated fatty acid (SFA), highest unsaturated fatty acid (UFA), highest UFA/SFA ratio, lowest n6/n3 ratio and atherogenicity.
Table 3.3 Fatty acid contents in PUFA-diet supplemented with yeast and C. xanthorrhiza Roxb
Cis-11,14-Eicosedienoic Acid, C20:2 0 0.07 0.09 0.1
Cis-5,8,11,14,17-Eicosapentaenoic acid,C20:5n3 0 0.02 0 .04
Heneicosanoic Acid, C12, C21:0 0.02 0.05 0.03 0.06
However, curcuma supplementation in diet (PDC) numerically decreased monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid (PUFA) compared to that of in yeast diet (PDY).
It has been known that curcumin played a role in disturbing lipid peroxidation, when the process of demethylation finished, consequently its function as antioxidant was over as well. It means that, curcumin will be functioning depends on the availability of the substrate that is going to be catabolysed. It was reported that curcuminoid as much as 0.0133mg/ml could slow down peroxidation of linoleic acid (Tonnesen 1988). The implication of the result in this research was that curcumin in C. xanthorrhiza, Roxb functioned much stronger in fatty acid reduction when it was singly; in contrast, its effect would be weaker when it was combined with yeast. This can be seen in the level of linoleic acid in PDC that was lower (10.8%) compared to that of PDM (14.85%).
Short chain fatty acids (C10 and C11) were not detected in control, yeast, and curcuma diets, very little level showed in the mixed PUFA- diet. Long chain fatty acids (C18:0 to C18:3n3) were higher in both yeast (PDY) and yeast + curcuma diet (PDM. These fatty acids were reported high in diet with supplementation of forage tannin, extract tannin, or extract saponin, was about 83.720% (Khiaosa- Ard et al. 2008).
Atherogenicity index being numerically the lowest was in PDC (0.87%), that was accordingly due to the ability of curcumin in reducing medium fatty acid (MCFA) C12, C14, C16 making the unsaturated fatty acid increased. However, when curcuma was combined with yeast (PDM), this index would increase, suggesting that there was fermentation process going on as the effect of the yeast. The fermentation effect with yeast (PDY) was also increased the index higher than that of in PDC.
Indexes in this research were much lower (the highest was 1.44% in PD0) compared to control diet (2.20%) and 20% extruded soybean (3.48%) as reported by Schmidely et al. (2005). Implication of the lower index in diet containing
