Table of Contents Page

Content Table of Contents

i-iii

Acknowledgements

iv-v

Abstract

vi

List of Abbreviations

vii

List of Figures

viii

List of Tables

ix

Chapter 1. Introduction I.

Introduction

1

II.

Research Objectives

2

III.

Hypothesis

3

Chapter 2. Literature Review I.

Probiotic

4-7

II.

Probiotic for Ruminants

7-9

III.

Lactic Acid Bacteria

IV.

9-10

A.

Lactobacillus brevis

10-11

B.

Lactobacillus buchneri

11-12

C.

Entercoccus faecium

12-13

Propionic Acid Bacteria

14

Propionibacterium jensenii

14-16

V.

Rumen Protozoa

16-18

VI.

Study of Rumen Fermentation A.

Rumen Ecosystem

B.

Rumen Fermentation

18 18-19 19

1.

Carbohydrates

2.

Proteins

21

3.

Lipids

21

4.

Gas Production

21

5.

Vitamin Synthesis

22

VII. Rumen Bacteria

19-20

22-23

Content

Page

Chapter 3. Material and Method I.

Diets and Diets Nutrient Analysis

24

II.

Probiotic Strains

24

III.

Probiotics Preparations

IV.

HFT (Hohenheimer Futterwert Test)

V.

23-24

A.

Diet Preaparation

25-26

B.

HFT Preaparation

26

C.

HFT Process

D.

Sample Collection

26-27 27

Probiotic Enumeration

27

Pour Plate Method VI.

25

27-28

Protozoa Enumeration

28

A.

Sample Preparation

28

B.


29

VII.

Autoclaved and Living Probiotics Experiment

29

VIII.

(NH4+) Ammonium Analysis

30

A.

Sample Preparation

30

B.

Distillations and Titrations

30

IX.

(VFA) Volatile Fatty Acids and Organic Acids Analysis

31

A.

Reagents

31

1.

Mobile phase

31

2.

Calibration substances

3.

Carrez reagent

32

Devices and Glass Material

32

HPLC-plant and material

32

Experiment

33

1.

Working procedures

33

a

Production of carrez reagent

33

b.

Production of mobile phase

33

c.

Calibration of the HPLC

33

d.

HPLC conditions

34

e.

Sample preparation

34

e.1.

Fermentation samples

34

e.2.

Carrez precipitation

35

B. C.

D.

Documentation and Evaluation

31-32

36

Content

Page

Chapter 4. Result and Discussion I.

II.

Result

37

A.

Diet and Diets Nutrient Analysis

37

B.

Gas Production

37-39

C.

(NH4+)

40-42

D.

Volatile Fatty Acids and Organic Acids Productions

43-51

E.

Probiotics Enumeration

52-53

F.


Ammonium Production

Discussion

54 55

A.

Gas Production

55-56

B.

(NH4+)

56-57

C.

Volatile Fatty Acids and Organic Acids Productions

57

D.

Probiotics Enumeration

57

E.


58

Ammonium Production

Chapter 5. Conclusion and Recommendation I.

Conclusion

II.

Recommendation

References Curriculum Vitae

59 59-60 61-67

Acknowledgements Praise be to Allah, the Cherisher and Sustainer of the worlds.

My sincere gratitude and deepest thanks go to Prof. Dr. Karl-Heinz Südekum, director of Animal Nutrition Group, Institute of Animal Sciences, University of Bonn. He had given me a great opportunity to conduct my MSc thesis under his supervision. I would like to thank him for his rounded and patient assistance during the study period.

I am also indebted to Dr. Saskia Kehraus, who introduced me basic knowledge of laboratory works in animal nutrition field, helped me with calculations and experiment arrangement.

My special thanks to Dr. Jürgen Hummel, who introduced me the world of HFT as well as his careful guidance, thoughtful comments and valuable advice during the period of my study.

I am also very much grateful to Prof. Dr. Helga Sauerwein, director of the Animal Physiology and Hygiene Group, Institute of Animal Science, University of Bonn and all group members, especially Mrs. Hannelore Brüssel for considerable and favours supports.

My deepest thanks to Dr. Edmund Mathis from Schaumann Company and Dr. Elisabeth Mayrhuber (Dr. Michaela Holzer) from Lactosan Company for their very numerous support.

I would like to acknowledge the supports provided by Mrs. Petra Jacquemien, Mrs. Nadja Wahl and Mrs. Evelyn Oettershagen. Their supports in the laboratory works were essential for the completion of this work.

I would like to use this opportunity to thank German Academic Exchange Service (DAAD) for inviting me to this study and financing through scholarship program.

I would like to thank to ARTS program, especially the program director Prof. Dr. Mathias Becker, program secretaries Mrs. Susanne Hermes and Mr. Jürgen Simons and all ARTS lecturers. I will never forget my beloved ARTS 2005 batch colleagues, especially Mr. Faith Chan and Mr. Ahmad Awais, for true and lovely friendship.

I convey my heart gratitude to my parents and wife, Mrs. Andri Pramesyanti Pramono to give me continuous moral support, encouragement, love and praying that gave me strength through my life.

Appreciations and thanks are also extended to my friends and colleagues in Bonn, especially in Institute of Animal Science, University of Bonn.

Abstract

Bacteria strains: Lactobacillus buchneri, Lactobacillus brevis, Enterococcus faecium and Propionibacterium jensenii in two different inoculums concentration (10 7 and 109 cfu/ml), were investigated for their probiotic properties in ruminal metabolism. The “Hohenheimer Futterwertest” (HFT, Hohenheimer gas test) was employed to determine the in vitro gas production of 8 and 24 hours incubation time, in 4 different ratios of grass silage and mixed concentrate (100:0; 75:25; 50:50; 25:75). The NH4+ (ammonium), VFA (volatile fatty acid) and lactic acid contents as well as probiotics and ruminal protozoa enumeration, were completed to estimate probiotic activity from the tested bacteria. Gas production, NH4+, organic acids (VFA and lactic acid) formation were affected by different probiotics colony concentration as well as diets type. Probiotics produced greater volume of gas at higher cell concentration in all diets type. The higher probiotics inoculums (10 increased

NH4+

9

cfu/ml) also

production and VFA and organic acids formation. Probiotics also produced similar

VFA and organic acids proportion. Cell concentrations of probiotics were also found to decrease after 24 hours incubation in HFT in vitro system. The P. jensenii was found as the most adaptable strain, when it cell density decline was observed at minimum level. Protozoa cell of probiotics treated experiments were found more dense as compared to controls (diet + HFT solutions + buffer+ filtered rumen liquor) and the before experiment sample (HFT solutions + buffer+ filtered rumen liquor). The autoclaved probiotics treatment showed the similar level of gas and NH 4+ production with its counterpart of living probiotic. The exception was indicated only by living P. jensenii which produced more gas and NH4+ production than in autoclaved treatment, at its high cell concentration (109 cfu/ml). Key words: Probiotic, gas production, in vitro, rumen

List of Abbreviation ADF

:

acid detergent fibre

ADL

:

acid detergent lignin

ATP

:

adenosine triphosphate

CA

:

crude ash

cfu

:

colony forming unit

CP

:

crude protein

CL

:

crude lipid

DFM

:

direct fed microbial

DNA

:

Deoxyribonucleic acid

FDA

:

food and drug administration

GRAS

:

generally regarded as safe

HFT

:

Hohenheimer futterwert test

HPLC

:

high-performance/pressure liquid chromatography

LAB

:

lactic acid bacteria

NDF

:

neutral detergent fibre

OMD

:

organic matter digestibility

RAC

:

readily available carbohydrates

rRNA

:

ribosomal ribonucleic acid

VDLUFA :

Verband Deutscher Landwirtschaftlicher Untersuchungs-und Forschungsantalten

VFA

volatile fatty acid

:

List of Figure Figure Figure 1. A schematic showing the major pathways of carbohydrate fermentation by ruminal becteria

Page 20

List of Tables Table

Page

Table 1.

Microorganisms considered as probiotics

Table 2.

Rates of production of acetic, propionic and n-butyric acid in the rumen

20

Table 3.

The characteristics of predominant ruminal bacteria

23

Table 4.

Probiotics colony density in powder and powder weight of each probiotic

25

Table 5.

Dilution pattern of 5 different concentration starting from the 1000 ppm standard

34

Table 6.

Sample dilution pattern

36

Table 7.

Diets and diet nutrient analysis

37

Table 8.

Gas production

39

Table 9.

Gas production of autoclaved and living probiotics

39

Table 10.

Gas production of diet component

39

Table 11.

Ammonium (NH4+) production

42

Table 12.

Ammonium

(NH4+)

42

Table 13-16.

VFA and organic acids production

44-47

Table 17-20.

Proportion of VFA and organic acids production

48-51

Table 21.

Probiotics enumeration

53

Table 22.

Protozoa enumeration

54

production of autoclaved and living probiotics

6

Chapter 1. Introduction I.

Introduction

Probiotics, in different ways of preparation and form, are used worldwide as animal feed supplement, with applications either for monogastric animals or ruminants. In ruminants, researchers have carried out their scientific works and published reports on probiotics of, for example, their mode of action, effectiveness and usefulness, as well as its function as an alternative agent replacing the banned antibiotics and other chemical-based growth promoter. Early use of probiotics as feed supplement dates back to the 1970´s, when probiotics were originally incorporated into feed to increase the growth of animal and to improve their health by increasing their resistance to disease (Martin and Nisbet 1992; Doyle 2001; Sharif 2003; Draksler et al. 2004; Anadón 2006) as well as their beneficial effect on the host by improving its microbial balance (Zhao et al. 1998).

An in vivo application of probiotics on non lactating Holstein cows showed the digestibility improvement of dry matter (DM) and acid detergent fibre (ADF), although it maintained the milk production and feed intake remained at the same level with control (Martin and Nisbet 1992). Abe et al. (1995) and Bomba et al. (2005) reported the influence of probiotics application on improving the mean daily body weight gain of growing calves and the animal health status by decreasing the diarrhoea incident.

Studies and investigations of single strain probiotics used as ruminant feed supplement have been conducted, while still only few and limited information can be obtained from comparison of the probiotic efficacy of different strains (Veiga-da-Cunha and Foster 1992; Yildirim and Yildirim 1999).

In this study, research was conducted to compare the effectiveness of probiotic strains in in vitro system to obtain data of probiotic effectiveness of different strains. Four different probiotics (Lactobacillus buchneri, L. brevis, Enterococcus faecium, Propionibacterium jensenii), were prepared in two different concentrations

(107 colony forming unit (cfu)/ml

and 109 cfu/ml) inoculated in four different diets (ratios of grass silage and mixed

concentrate (soybean meal and maize grain): 100:0; 75:25; 50:50, 25:75) and incubated in HFT (Hohenheimer Futterwert Test) in vitro system. The gas production was recorded after 8 and 24 hours incubation time. Other measurements and tests including: ammonium, volatile fatty acids and other organic acids (lactic acid) production, as well as protozoa and probiotics enumeration were performed to acquire data, of each probiotics ability to enhance feed digestibility and also the probiotics survival in a rumen like system. Propionibacterium jensenii was also included in the research as it has capacity to utilize lactate to produce propionate (as well as acetate and CO 2) an agent that acts as a precursor for hepatic glucose production. Theoretically, the efficiency of propionate as a source of energy in the form of ATP is 109% compared with glucose (Francisco et al. 2002).

In order to detect the influence of probiotics carrier media, experiment employing autoclaved and living probiotics cells also has been performed. II. Research objectives

1. Comparison of each strain to act as a probiotic and their survival during in vitro rumen-like fermentation. 2. Probiotics influence on ruminal feed degrdation. 3. Lactic to propionic acid conversion by Propionibacterium jensenii

III. Hypothesis

1. Probiotics survive during the feed digestion process and influence the normal microfauna (protozoa) population of ruminants (qualitative) 2. Probiotics enhance the feed digestibility rate, at certain colony concentration of application, at different rumen environment (quantitative) 3. Higher concentration of probiotics (cfu/ml) stimulates higher efficiency of digestion process.

Chapter 2. Literature Review I.

Probiotic

The word probiotic is derived from the Greek meaning for life and had several different meanings over the years. In 1974, Parker defined probiotic as organisms and substances which contribute to intestinal microbial balance. In an attempt to improve the definition, Fuller redefined probiotic as a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance (Fuller 1992). Schrezenmeir and de Vrese (2001) broadened the definition of probiotic with respect to host and habitat of the microflora as follows: a viable mono or mixed culture of microorganisms which applied to animal or man, beneficially affects the host by improving the properties of indigenous microflora. Nousiainen and Setälä (1988) have synthesised a description of probiotic as live indigenous microorganisms or nonantibiotic substances which decrease the number of intestinal infections and/or increase production and/or improve food hygiene by contributing to a better gastrointestinal environment.

Probiotic definition also has been released by an Expert Consultation at a meeting convened by the FAO/WHO in 2001: probiotic are live microorganisms which when administrated in adequate amounts conferring health benefit on the host (Julliand 2006).

The U.S. Food and Drug Administration (FDA) use the term of direct-fed microbial (DFM) instead of probiotics and has narrowed the definition to a source of live (viable), naturallyoccurring microorganisms (including viable cultures of fungi and bacteria). The most commonly used probiotics organisms are the lactic acid bacteria, which are found in large number in the gut of healthy animals and do not appear to effect them adversely, defined as GRAS, Generally Regarded as Safe by FDA (Martin and Nisbet 1992; Krehbiel et al. 2003).

In terms of ruminant production system, the efficacy of bacterial DFM has been studied most extensively in the neonatal dairy calf. Feeding calves viable culture of the species Lactobacillus and Streptococcus has been reported to decrease the incidence of

diarrhoea. The decreased incidence of diarrhoea might be associated with a consistently increased shedding of Lactobacillus and an inconsistent decreased shedding of coliforms in faeces in response to supplement of Lactobacillus (Krehbiel et al. 2003).

An effective probiotic is required to operate under a variety of different environmental conditions and to survive in many different forms. It should therefore have the following characteristics: a. It should be capable of being prepared as a viable product on an industrial scale. b. It should remain stable and viable for long periods under storage and field conditions. c. It should have the ability to survive (not necessarily grow) in the intestine. d. It must produce a beneficial effect in the host animal. (Fuller 1992).

Tuomola et al. (2001) described one of the more important selection criteria for probiotics: is the ability to adhere to the intestinal mucosa, as the adhesion to the intestinal mucosa is considered to be a prerequisite for colonization, as well as important for stimulation of the immune system.

The beneficial claims made for probiotic supplementation are numerous and include: a. Improved growth rate of farm animals; due to suppression of a sub clinical infection with a growth-depressing microorganism. b. Improved utilization of food; by increased efficiency of existing digestive process or by promoting the digestion of previously indigestible substances, by manipulating and regulating rumen metabolism. c. Improved milk production by dairy cows; obtained particularly with fungal supplement such as Saccharomyces cerevisiae or Aspergillus oryzae. d. Increased egg production. e. Improved health; either by direct antagonism or by stimulation of immunity. (Fuller, R 1992; Salminen et al. 1993; Noceck et al. 2002).

One of the selection criteria for probiotics is the production of antimicrobial substances, for example bacteriocins (Tuomola et al. 2001).

Today a large range of defined strains of probiotics belong to the group of lactic acid bacteria (Bifidobacterium, Lactobacillus, Streptococcus, Enterococcus and Lactococcus species),

Bacillus sp.,

fungi (Aspergillus

sp.) and yeast (Saccharomyces sp.,

Kluyveromyces sp.) (Julliand 2006).

Table 1. Microorganisms considered as probiotics Lactobacillus species -

L. acidophilus L. amylovorus L. casei L. crispatus L. delbrueckii subsp. bulgaricus L. gallinarum L. gasseri L. johnsonii L. paracasei L. plantarum L. reuteri L. rhamnosus

Bifidobacterium species

Other lactic acid bacteria

Nonlactic acid bacteria

-

-

- Bacillus cereus var. toyoi - Escherichia coli strain nissle - Propionibacterium freudenreichii - Saccharomyces cerevisiae - S. boulardii

B. adolescentis B. animalis B. bifidum B. breve B. infantis B. lactis B. longum

-

Enterococcus faecalis E. faecium Lactococcus lactis Leconostoc mesenteroides Pediococcus acidilactici Sporolactobacillus inulinus Streptococcus thermophilus

Source: Holzapfel et al. (2001) In the European Union, probiotics are under the control of Regulation (EC) 1831/2003 of 22 September 2003 on additives for use in animal nutrition (OJEC L268 of 18.10.2003). Probiotics are classified as Zootechnical Additives which is one of the five categories and functional groups, stated as Digestibility enhancers, Gut flora stabilizers, substances which favourably affect the environment and others (Julliand 2006).

In recognition of the importance of assuring safety, even among a group of bacteria that is “Generally Regarded As Safe” (GRAS), FAO/WHO working group recommends that probiotic strains should be characterized at a minimum with the following tests: 1. Determination of antibiotic resistance patterns 2. Assessment of certain metabolic activities 3. Assessment of side-effects during human studies 4. Epidemiology surveillance of adverse incidents in consumers

5. If the strain under evaluation belongs to a species that is a known mammalian toxin producer, it must be tested for toxin production. 6. If the strain under evaluation belongs to a species with known haemolytic potential, determination of haemolytic activity is required

Standard criteria for Probiotic clinical evaluation are: safety, efficacy, effectiveness and surveillance (FAO/WHO 2002). II. Probiotics for Ruminants

Digestion of food in the rumen occurs by a combination of microbial fermentation and physical breakdown during regurgitation of the food by rumination. No host animal enzymes are thought to be involved.

A major advantage of foregut fermentation compared with hindgut fermentation is that the microbial cells formed as a result of the fermentation are available to the host as they pass down the tract to the gastric stomach of the ruminant, the abomasum. Indeed, microbial protein is the most important source of amino acids for absorption.

During early life, when milk is the main ingredient, food tends to bypass the rumen. The distance between the end of the oesophagus and the reticulo-omasal orifice, through which food leaves the rumen, is small. Young animals have a reflex which closes the vestigial oesophagus between these two orifices, the so-called oesophageal groove, so that food passes directly from the oesophagus to the omasum and then to the abomasums. Some food does enter the rumen, however, and the rumen becomes inoculated with the bacteria, protozoa and fungi in preparation for weaning, when the adult, fibre-digesting population takes over.

The potential benefits of probiotics to ruminants are therefore perhaps even greater than with monogastric animals. The prevention of scouring in calves is essentially the same problem as in other species. In ruminants, however, benefits may also be obtained by enhancing the rate at which the rumen flora and fauna develop or, once the fermentation

has fully established in the adult ruminants by stimulating fermentation (Wallace and Newbold 1992).

1. Development of digestive function

The development of a functional microbial population in the gut of the newborn ruminant facilitates not only the digestion of fibre by the host but also helps protect the gut from infection by pathogenic organisms. Ruminants are born with a sterile gastrointestinal tract. As the animal begins to consume solid feed, the microbial population in the rumen increases and begins to resemble that of the adult ruminant. The end-products of microbial fermentation encourage the development and enlargement of the rumen (Wallace and Newbold 1992).

2. Prevention of diarrhoea

Diarrhoea can be caused by enterotoxigenic bacteria colonizing the gut. To induce diarrhoea by enterotoxin production, E. coli must first colonize the gut. It has been suggested that probiotics might be used either to displace enterotoxigenic E. coli from the gut wall or to promote a healthy bacterial population which excludes coliforms from the gut (Wallace and Newbold 1992).

3. Development of rumen fermentation

In addition to the prevention of diarrhoea, bacterial and fungal probiotics have also been used to enhance the development and maintenance of stable rumen fermentation (Wallace and Newbold 1992).

III.

Lactic Acid Bacteria

Lactic acid bacteria (LAB) is a group of Gram-positive bacteria united by a constellation of morphological, metabolic and physiological characteristics. The general description of the bacteria included in the group is Gram-positive, nonsporing cocci or rods, which produce

lactic acid as the major end product during the fermentation of carbohydrates. Recent taxonomic revision of these genera suggests that the lactic acid bacteria comprise the following: Aerococcus, Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Streptococcus, Tetragenococcus and Vagococcus (Axelsson 1993).

An important characteristic used in the differentiation of the LAB genera is the mode of glucose fermentation under standard conditions. Under these conditions, LAB can be divided into two groups: heterofermentative, which ferment glucose to lactic acid, ethanol/acetic acid and CO2 and homofermentative, which convert glucose almost quantitatively to lactic acid (Axelson 1993).

Certain heterofermentative lactic acid bacteria often encountered in food and beverage fermentations are able to utilize the uronic acids as a sole carbon source for energy. Essentially equimolar quantities of CO2, acetic acid and lactic acid are produced from the glucuronic acid fermentation by L. brevis. Colorimetric determination of residual glucuronic acid by carbazole, indicate that almost 94% of the original glucuronate was converted to the final acidic end products (Stamer and Stoyola 1967).

LAB colonize various habitats in the bodies of humans and animals, such as mucous membranes of the oral cavity, intestine and vagina. The following LAB have been found in animal hosts: Lactobacillus. acidophilus, L. murinus, L. intestinalis, L. salivarius, L. agilis, L. ruminis, L. vitulinus, L. hamsteri, L. aviaries, L. casei, L. reuteri and L. brevis (Bernardeau et al. 2006).

One expanding application of LAB is the addition of probiotic strains to various products to enhance the health of humans and animals through their favorable effects on the microflora of the gastrointestinal tract (Bernardeau et al. 2006). Data form various studies on probiotics strains of lactic acid bacteria have produced information on the probable mechanism of action. Proposed mechanisms include the suppression of harmful bacteria and viruses and stimulation of local and systemic immunity. The ability of probiotic bacteria to suppress the growth of pathogens has been attributed to the production of antibacterial

substances such as lactic acid, peroxide, bacteriocins, and bacteriocin-like inhibitory substances (Salminen et al. 1993).

Bacteriocins are proteneinaceous compounds produced by bacteria that exhibit a bactericidal or bacteriostatic mode of action against sensitive bacterial species due to their application in food preservation (Yildirim and Yildirim 1999; Faye et al. 2000).

A. Lactobacillus brevis

Super kingdom

:

Bacteria

Phylum

:

Firmicutes

Class

:

Actinobacteria

Order

:

Lactobacillales

Family

:

Lactobacillaceae

Genus

:

Lactobacillus

Species

:


Lactobacillus brevis is a heterofermentative bacterium that utilises hexoses by the 6phosphogluconate pathway, producing lactic acid, CO 2 and ethanol and/or acetic acid in equimolar amounts. It can be isolated from many different environments and it is frequently used as starter culture in silage fermentation, sourdough and lactic-acidfermented types of beer (Ogunbanwo et al. 2003; Vancanneyt et al. 2006).

In beverages obtained by alcoholic fermentation, lactobacilli may contribute to the quality of the product but may also cause spoilage (Joint Genome Institute). L. brevis is a major contaminant of spoiled beer. The organism can grow in beer in spite of the presence of antibacterial hop compounds that give the beer a bitter taste (Sakamoto et al. 2001; Thelen et al. 2006).

B. Lactobacillus buchneri

Super kingdom

:

Bacteria

Phylum

:

Firmicutes

Class

:

Actinobacteria

Order

:

Lactobacillales

Family

:

Lactobacillaceae

Genus

:

Lactobacillus

Species

:


Some strains of Lactobacillus brevis, L. buchneri and L. reuteri can grow on glycerol by cofermenting it with glucose or fructose. The first two organisms can also coferment glycerol with ribose or lactate. All these bacteria have a coenzyme B12-dependent dehydratase that convert glycerol to 3-hydroxypropionaldehyde (3-HPA), which is then reduced to 1,3-propanediol (1,3-PDL) (Veiga-Da-Cuncha and Foster 1992).

Manca de Nadra et al. (1986) reported that L. buchneri metabolized arginine via the arginine dihydrolase pathway, producing ornithine, ammonia, carbon dioxide and ATP, involving following enzymes: arginine deiminase, ornithine transcarbamylase and carbamate kinase.

Yildirim and Yildirim (1999) confirmed the active compound produced by L. buchneri isolated from commercially fermented vegetable product. This active compound identified as Buchnerin LB, which has inhibitory activity against several food borne pathogens and food

spoilage

bacteria

including

Listeria,

Bacillus,

Enterococcus,

Micrococcus,

Lactobacillus, Leuconostoc, Pediococcus and Streptococcus. Buchnerin LB is heat-stable (90-121 °C for 15 minutes), resistant to catalase, peroxidase, lipase, ribonuclease A, amylases and organic solvent, but will loose its activity completely after treatment with certain proteases, α-chymotrypsin, trypsin, proteinase K, pepsins and pronase E.

C. Enterococcus faecium

Super kingdom

:

Bacteria

Phylum

:

Firmicutes

Class

:

Bacilli

Order

:

Lactobacillales

Family

:

Enterococacceae

Genus

:

Enterococcus

Species

:

Enterococcus faecium

Enterococci were described for the first time by Thiercelin in 1899. They were thought to be a new Gram-positive diplococcus and were later included in the new genus Enterococcus, proposed by Thiercelin and Jouhaud in 1903, with the type species Enterococcus proteiformis. The genus Enterococcus was finally described by Schleifer and Kilpper-Bälz (1984), who used DNA:DNA hybridisation to demonstrate that Streptococcus faecalis and Streptococcus faecium were sufficiently distinct from other streptococci to warrant their transfer to a separate genus. Based on 16S rRNA cataloguing, DNA:DNA and DNA:rRNA hybridisation and serological studies with superoxide dismutase antisera, the streptococci sensu lato were subdivided into three genera: Streptococcus sensu stricto, Enterococcus and Lactococcus (Wijaya 2003).

Enterococci strain constitutes a major part of the environment and intestinal microflora in humans and animals (Viteniene et al. 2004). E. faecium is commonly used in animal probiotics to modulate immunity by decreasing the adhesion molecule sICAM-1 in addition to CD54 (on monocytes) and CD11b (on lymphocytes) (Reid 2006). Marcinakova et al. (2004) reported that E. faecium strain EF 9296 was sensitive to ampicillin, vancomycin, rifampicin, tetracycline, chloramphenicol and erythromycin, but resistant against kanamycin.

E. faecium also produce a huge variety of antimicrobial

substances including organic acids, hydrogen peroxide and bacteriocins (Viteniene et al. 2004).

Zeyner (2006) stated

that daily oral supplementation of Enterococcus faecium (DSM

10633 NCIMB 10415) early from birth until weaning, reduces the portion of piglets suffering from diarrhoea. E. faecium also has been reported produced pH and heat stable proteases sensitive bacteriocins which active against Listeria monocytogenes, synthesized and excreted formic, acetic and isobutyric acid (Arihara et al. 1993; Viteniene et al. 2004). Other researchers reported the inhibitory action of E. faecium isolated from chicken intestine, which was active against Salmonella in vitro and in vivo, and was clinically effective in the treatment and prevention of chicken diarrhoea (Viteniene et al. 2004). Noceck et al. (2003) described that feeding a direct-fed microbial product (DFM) containing E. faecium and yeast increased DMI, milk production and milk-protein percentage, when applied 21 days prepartum through 70 days of lactation. IV. Propionic Acid Bacteria Propionibacterium jensenii Super kingdom

:

Bacteria

Phylum

:

Firmicutes

Class

:

Actinobacteria

Order

:

Actinomycetales

Family

:

Propionibacteriaceae

Genus

:

Propionibacterium

Species

:


Source: Stackebrandt et al. (2006).

Propionibacteria are Gram-positive, non-acid-fast and non-motile rods which do not produce endospores. Although there is tendency towards anaerobiosis and growth is improved in an atmosphere of 5% CO 2, both aerobically and anaerobically, cultures are catalase-positive. The morphological appearance of propionibacteria depends very much on cultural conditions, in particular oxygen tension and pH of the medium. Under anaerobic conditions and in neutral media, the cells appear as very short rods with

rounded end sometimes resembling streptococci in appearance. Under aerobic conditions with adequate aeration and in similar medium, long rod-shape cells of irregular or even branched appearance may be formed (Harrigan 1998).

The genus Propionibacterium is divided into the cutaneous and the dairy or classical species. Classical species include Propionibacterium jensenii, P. acidipropinici, P. freudenreichii subsp. freudenreichii and shermanii and P. thoenii. The industrial significance of this group lies in their contribution of the characteristic flavour, texture and eyes of Swiss cheese, their prolific production of propionic acid and their ability to synthesize vitamin B12 (Grant and Salminen 1988; Grinstead and Barefoot 1991, Herve et al. 2001).

Propionibacteria are natural inhabitants of the rumen microflora and comprise 1.4% to 4.3% of the total microbial population. In dairy cows, the population of propionibacteria range from 103 to 104 cfu/ml in the rumen (Alemán et al. 2005).

Together with Propionibacterium freudenreichii, P. acidopropionici and P. theonii, P. jensenii shown potential probiotic effects, such as the production of propionic acid (also an important mould inhibitor, used in food and feed preservations) bacteriocins (natural food preservatives), vitamin B12, synthesis of β-galactosidase enzyme, growth stimulation of bifidobacteria, reduction of intestinal enzyme involved in the production of carcinogenic compounds and favourable affects on lipid metabolism and the immune system of the host (Hammond et. al 1993; Adams 2002; Hugenholtz et al. 2002; Huang and Adams 2003; Schweninnger and Meile 2004).

Used as DFM, Propionibacterium prevented lactate accumulation, because these bacteria convert lactate and glucose to acetate and propionate. Supplementation with lactateutilizing Propionibacterium increased butyrate concentration, acetate concentration, protozoan numbers, VFA accumulation and ruminal NH 3 concentration in ruminal fluid as well as it reduced metabolic acidosis risk (Ghorbani et al. 2002).

The Propionibacteria are well known for their production of inhibitory metabolites. During fermentative metabolism, they convert glucose and lactate to propionate, acetate and carbon dioxide. The inhibitory effects of propionate and acetate are potentiated by the low pH encountered in Swiss cheese and other fermented products; the undissociated forms are effective against gram-negative bacteria (Grinstead and Barefoot 1991).

Among the dairy propionibacteria, two bacteriocins have been described; propionicin PLG1 from P. thoenii P127 and jenseniin G from P. thoenii (former P. jensenii) P126. Propionicin PLG -1 is active against a variety of microorganisms such as propionibacteria, as well as many other gram-positive and gram-negative bacteria and even fungi (Faye et al. 2000).

Jenseniin G is a heat-stable protein with bactericidal action against closely related classical propionibacteria and displays extended activity against lactococci and lactobacilli. Production of a jenseniin G or other compounds that inhibit faster-growing lactic acid bacteria would provide a competitive advantage for propionibacteria. This bacteriocin therefore has a potential role in preventing over acidification of yoghurt (Grinstead and Barefoot 1991; Faye et al. 2000). V.

Rumen Protozoa

Rumen protozoa were discovered in 1843 and an excellent taxonomy was compiled in 1927 by Russian protozoologist, Dogiel. It represents a substantial part of the rumen microbial population contributing up to 50% of the total microbial biomass.

Protozoa in the rumen are ciliate species, although flagellate species do occur. In general, the flagellate protozoa have been observed to occur in relatively low numbers in adult ruminants and are quite small size as compared to the ciliates. Rumen ciliates need specific environmental conditions to survive: oxygen must be absent and the temperature must be maintained at 39 oC (Prins 1990; Nsabimana et al. 2003; Dehority 2004; Regensbogenova et al. 2004).

The rumen ciliates are the most abundant protozoa in the rumen (105 -106 per ml rumen contents in healthy animal) and are involved in host metabolism and digestion of plant material. By classical morphological criteria more than 250 species of ciliates have been described which live in the rumen of various feral and domesticated ruminants. They have evolved into highly specialized group fitted to survive only in the rumen or a closely related habitat. They are anaerobic, can ferment plant materials for energy, and can grow at rumen temperatures in the presence of billions of accompanying bacteria. The rumen protozoa vary considerably in size, ranging from approximately 15 to 250 μm in length and 10 to 200 μm in width for the different species. The ciliate protozoa which occur in the rumen are all classified in the phylum Ciliophora. Ciliate protozoa in the rumen are classified on the basis of the micro- and macronucleus and the presence and morphology of exterior spines and lobes or internal skeletal plates as well as the shape and size of cells (Ogimoto and Imai 1981; Dehority 2004; Regensbogenova et al. 2004).

The ciliates found in the rumen are divided into three orders, Prostomatida, Trichostomatidfa and Entodiniomorphida, which composes most of rumen ciliates (Ogimoto and Imai 1981).

In vitro studies have suggested that 19-28% of total cellulose activity can be attributed to protozoa. The concentration of protozoa in ruminal contents generally increases with the addition of concentrates to roughage diets. Diets containing between 40 and 60% concentrate will support maximal protozoan numbers with a diverse fauna containing species in most of the genera. Concentrates provide a source of rapidly fermentable carbohydrate for ruminal microorganisms, which produce VFA, thereby reducing ruminal pH. Protozoa concentrations usually begin to decrease when the level of concentration exceeds 60%. One possibility would be the effect of rate of passage of high-concentrate diets. Both level and physical forms of the diet can increase passage rate, which in turn reduces protozoal concentrations or defaunates the rumen. The most important aspect of protozoa is their ability to engulf large molecules, protein, degrade fibrous and nonfibrous CHO or even ruminal bacteria, as their main supply of protein. In addition, protozoa play a role in regulating bacterial N turnover in the rumen, and they supply soluble protein to sustain microbial growth. Because protozoa are not able to use ammonia N, a fraction of

previously engulfed insoluble protein is later returned to the rumen fluid in the form of soluble protein (Franzolin and Dehority 1996; Varga and Kolver 1997; Bach et al. 2005).

Chagan et al. (1999) suggested the significant relation of ciliate protozoa to the rumen methanogenesis. The methanogens associated with protozoa may be divided into two categories, which colonize extracellularly or intracellularly. Indeed, the elimination of ciliate protozoa from the rumen may prevent 30 to 45% of ruminal methane emissions.

Onodera (1986) has shown that the rumen protozoa could produce lysine from 2,6diaminopimelate (DAP), though they could barely synthesize DAP from acetate or aspartate. Then the protozoa were found to produce lysine by utilizing the bound form of DAP contained in the peptidoglycan of rumen bacterial cell walls.

Protozoa play an important role in the biodegradation of plant toxins and mycotoxins, the regulation of ruminal conditions such as pH and redox potential, eliminate certain pathogens from the digestive tract of ruminants and protecting them from disease and so improving food safety of edible animal products (Nsabimana et al. 2003).

VI. Study of Rumen Fermentation

Ruminant nutrition studies are often accompanied by estimates of some easily measured parameters in rumen fluid, including pH, volatile fatty acids (VFA) and ammonia concentrations. These often help to explain the effects of different dietary manipulations on host animal nutrition. A. Rumen Ecosystem

Certain features of the rumen are common to almost all ruminants and feeding situation. It is an anaerobic environment with a very low redox potential and a temperature of 39 – 41 °

C. The pH is maintained fairly constant (pH 6 – 7) by the buffering capacity of bicarbonate

and the absorption of fermentation endproducts through the rumen wall. It is densely populated by a wide variety of bacteria, protozoa and fungi.

The fermentation pattern of carbohydrate is also similar. Pentoses enter the pathway as fructose-6-phospate or glyceraldehydes-3-phospate via the hexose monophospate way, which is the major pathway, or via phospoketolase. Proteins are degraded to peptides and amino acids, with the rate of protein degradation being correlated to feed protein solubility. The main fermentation products are CO 2, volatile fatty acids (VFA) and NH3. Branchedchain VFA, formed from the branched-chain amino acids, are essential growth factor for many cellulolytic organisms. Another feature is the similarity of the endproducts, acetate, propionate and butyrate, irrespective of the type of feed (Murphy 1990).

B. Rumen Fermentation

1. Carbohydrates

In the rumen of animal fed at a low level, essentially 100% of the readily available carbohydrates (RAC) will be fermented by the rumen microorganisms. The principal end products of fermentation are the volatile fatty acids (primarily acetic, propionic, and butyric), CO2, CH4 and heat. The animal, in turn, uses the volatile fatty acids as a source of energy for its life processes. The fibrous carbohydrates are also fermented by rumen microorganisms and the end products are the same, although less propionic acid is produced normally than from RAC. Because animal tissues do not produce cellulase or hemicellulase, microbial fermentation is the only means by which animal can indirectly use these complex carbohydrates. Domestic ruminants in developing countries are still fed forage-based diets, when the diets of cattle in developed countries are often supplemented with 50 to 90% grain. Feeding unadapted animals large amounts of cereal grains such as wheat may result in abnormal rumen fermentation, which is associated with high production of lactic acid and a very acid rumen. This condition may cause death if severe enough (Russell and Rychlik 2001; Pond et. al 2005).

Figure 1. A schematic showing the major pathways of carbohydrate fermentation by ruminal bacteria (Russell and Rychlik 2001).

Sutton et al. (2003) have reported the differences VFA rate production of lactating dairy cows received normal and low-roughage diets. The complete figure of different VFA production were shown in the table as follow

Table 2. Rates of Productions of acetic, propionic and n-butyric acids in the rumen (Sutton et al. 2003) Normal

Low roughage

De novo synthesis, mol/d Acetic Propionic Butyric

60.1 15.3 5.8

55.4 34.0 3.3

Net production, mol/d Acetic Propionic Butyric

56.5 16.8 6.5

49.0 36.2 4.8

Molar proportion of net production, mol/100 mol total VFA Acetic Propionic Butyric

71.0 21.2 7.9

54.5 40.1 5.4

Concentration, mM Acetic Propionic Butyric

58.7 16.9 11.0

48.6 36.6 8.8

Molar proportions of concentrations, mol/100 mol total VFA Acetic Propionic Butyric

68.0 19.4 12.6

51.6 39.0 9.4

2. Proteins

Rumen microorganisms also are capable of utilizing simple N sources such as urea (a normal mammalian excretory product), amino acids, nitrates, biuret, and amines. Thus, urea or other economically competitive nonprotein nitrogen sources may be substituted for protein in ruminant diets in many instances. The net effect of normal rumen metabolism of protein is that it allows the animal to exist on a wide variety of diets, but overall efficiency of protein utilization is low because it is biologically inefficient to degrade and resythesize complex molecules such as protein (Pond et al. 2005).

3. Lipids

In the rumen, the microbes do not greatly alter the fat fraction, although some lipids may be synthesized. Rumen microbes modify the unsaturated fatty acids by either saturating them or causing changes in the location of the double bonds or altering the normal cis bond to a trans double bond. In addition, some of the microbial fat synthesis results in the

production of fatty acids with an odd number of carbon atoms in the chain and with branched chains in the molecule (Pond et al. 2005).

4. Gas Production

Anaerobic fermentation such as those that occur in the rumen result in the production of copious amounts of gases. A fairly typical composition of the gases would be: 65% CO 2, 25 to 27% CH4, 7% N and trace amounts of O2, H2 and H2S. Methane, which has a high heat equivalent, represents a direct loss of energy to the animal (Pond et al. 2005).

5. Vitamin Synthesis

Rumen microorganisms have the capability of synthesizing essentially all of the B-complex vitamins required by the host animal. Although some synthesis may occur in the large intestine or cecum of other species, the amount synthesized in the rumen is probably greater than that in the lower GI tract (Pond et al. 2005).

VII.

Rumen Bacteria

The bacteria number 109 – 1010 per ml of rumen contents. Over 200 species have been identified, most are non-spore forming anaerobes. Bacteria play a dominant role in all facets of ruminal fermentation. Interactions between microorganisms are an important feature of rumen fermentation. The activities of a given species of bacteria may vary from one strain of that species to another. The total number of bacteria and the relative population of individual species vary with animal’s diet; for example, diet rich in concentrate foods promote high total counts and encourage the proliferation of lactobacilli (Russell and Rychlik 2001; Mc Donald et al. 2002).

A variety of ruminal bacteria produced end products that could not be detected in ruminal fluid, and these intermediate (e.g. succinate and lactate) are subjected to secondary fermentations by other species (e.g. Selenomonas ruminantium and Megashpera elsdenii). The most active cellulolytic ruminal bacteria (Ruminicoccus albus, Ruminococcus

flavefaciens and Fibrobacter succinogenes) require ruminal fluid, due to branched-chain volatile fatty acids, derivate from a distinctly different population of amino acid fermenting bacteria (Megashpera elsdenii and Peptostreptococcus anaerobius) (Russell and Rychlik 2001). The rumen bacteria and its fermentation products were given in table 2.

Table 3. The characteristics of predominant ruminal bacteria. Species

Fermentation products

Fibrobacter succinogenes Ruminococcus albus Ruminococcus flavefaciens Eubacterium ruminantium Ruminobacter amylophilus Streptococcus bovis Succinomonas amylolytica Prevotella ruminocola, albensis, brevis, and bryantii Butyrivibrio fibrisolvens Selenomonas ruminantium Megasphaera elsdenii Lachnospira multiparus Succinivibrio dextrinosolvens Anaerovibrio lipolytica Peptostreptococcus anaerobius Clostridium aminophilum Clostridium sticklandii Wolinella succinogenes Methanobrevibacter ruminantium Abbreviations are as follows: L, lactate; S, succinate; H2, hydrogen; F, formate; A, acetate; E, ethanol; B, butyrate; P, propionate; Br, branched-chain volatile fatty acids; and CH4, methane.

S, F, A A, F, E, H2 S, F, A, H2 A, F, B, L S, F, A, E L, A, F, E S, A, P S, A, F, P B, F, A, H2 L, A, P, B, F, H2 P, A, B, Br, H2 L, A, F, H2 S, A, F, L A, S, P Br, A A, B A, Br, B, P S CH4

Chapter 3. Material and Method

I.

Diets and Nutrient Analyses of Diets

Diets (100%, 75%, 50% and 25% grass silage content) and diet components (grass silage, soybean meal and maize grain) were analysed to obtain data of their compositions, including: crude ash, crude protein, crude fibre and lipid as well as ADF (acid detergent fibre), ADL (acid detergent lignin) and NDF (neutral detergent fibre). All sample examination was conducted in Animal Nutrition Laboratory, Institute of Animal Science. Van Soest method was applied to examine ADF, NDF and ADL contents, while VDLUFA

(Verband

Deutscher

Landwirtschaflicher

Untersuchungs-und

Forschungsantalten) methods were employed to determine crude ash (according to method no. 8.1), crude protein (method 4.1.1 and 4.1.2), crude lipid (method 5.1.1) and crude fibre (method 6.1.1) contents. II.

Probiotic Strains

Probiotic strains used in the experiment were provided by Lactosan (Lactosan, Starterkulturen Gesellschaft m.b.H., Kapfenberg, Austria). Probiotics were three lactic acid bacteria strains: Lactobacillus buchneri,

L. brevis

and Enterococcus faecium and one propionic acid bacteria: Propionibacterium jensenii, obtained in powder form, with different colony density per gram for each probiotic sample. III. Probiotic preparation Serial dilutions are a series of dilutions. In bacterial work, dilutions are usually performed in series of 1/10 or 1/100 dilutions. A series is used because it allows us to take samples and analyze at different concentrations (McLandsborough 2005). Four probiotic strains, in powder form with certain initial colony densities (provided by Lactosan) were weighed and diluted in a dilution series in the sterile physiological solution (0.9% NaCl), in order to obtain a distinct final colony density: 109 cfu (colony forming units)

per ml. The 107 cfu/ml probiotics were prepared by diluting 0.1 ml (100 μl) of 109 cfu/ml solution into 9.9 ml 0.9% NaCl. Table 4. Probiotics colony density in powder and powder weight of each probiotic Probiotic

Colony density in powder (cfu/ml)

Powder weighed (g)

1012

0.3


11

0.75

11

0.6

11

1.5


4 x 10

Enterococcus faecium

5 x 10


2 x 10

Note: Data of colony density in probiotic powder were provided by Lactosan

IV.

HFT (Hohenheimer Futterwerttest)

The HFT or gas test method is based on the accumulated 24 h gas production by a substrate, incubated in a syringe with rumen liquor and a nutritive solution. Gas production is proportional to the amount of digestible carbohydrates, and thus highly correlated to the energy value of feedstuffs or to the in vivo organic matter digestibility (OMD) (Menke and Steingass 1988). A.

Diet Preparation

Diets used in the experiment were 4 different mixed ratios of grass silage and concentrate (Soybean meal and maize grain; with proportion approximately 11:10). The ratios of silage concentrate were: 100:0; 75:25; 50:50 and 25:75. Concentrates and silage were obtained from Animal Nutrition Laboratory feed stock. Grass silage was collected, chopped, deep frozen (Liebherr) and freeze dried (Martin Christ® Alpha 1-4 LSC with rotary vane vacuum pump Vacuubrand® Vacuubrand GmbH + Co. KG.). All feed samples (silage and concentrates) are ground in 1 mm sieve grinding machine (Retsch KG type ZM 1).

The crude protein content (g/kg DM) of 50:50 diet was calculated first, in order to have value of 180 g/ kg DM crude protein content. The other diets were obtained by mixing homogenously silage and concentrate according to the ratio of each diet, to have at least 200 g diet stock of each ratio. B.

HFT Preparation

HFT incubation media was prepared in Woulff flask mixed with magnetic agitator and heated to 39 °C in a water bath in the following order: 580 aquadest, 0.5 ml trace element solution, 290 ml buffer solution, 290 ml macro element solution and 1.5 ml resazurin solutions. Buffer was prepared freshly, by mixing element as follow: 35 g NaHCO 3, 4 g (NH4)HCO3 in 1 l aquadest. Macro element solution consist elements as follow: 5.7 g Na 2HPO4, 6.2 g KHPO4, 0.6 g MgSO4 x 7H2O and 1 l aquadest. Reduction solution was freshly prepared by mixing 58 distillated water, 2.5 ml 1 N NaOH and 380 mg 35% Na2S (Steingass and Menke 1988). C.

HFT Process

Approximately 200 mg (± 2 mg) of mixed-feed samples from each feed ratio were weighed for each HFT syringe. Feed samples were weighed and placed on a weighing boat with a removable stem so that the material can be put on the bottom of the syringe without leaving sample remains stuck to the side of the syringe. A 30 ml incubation media (rumen fluid + all HFT solutions) was added into each HFT syringe followed by inoculation of 1 ml diluted probiotic strain. Then Vaseline-greased HFT piston was adjusted at 30 ml syringe volume. Any air bubbles which get in are brought to the surface by gentle shaking and removed through the capillary attachment by a careful upward movement of the piston. After shutting the clamp on the tube, the exact volume (V 0) is read off on the calibration and the syringe is placed in the rotor which is inside the pre-heated (39 °C) incubator (Menke and Steingas 1988). Second gas production measurement was at 8 hours incubation (V8). After the gas at 8 hours read off, the piston was returned to the 30 ml mark, if the volume measured after 8

hours exceeds the 50 ml mark. The final gas measurement was at 24 hours incubation (V24). D.

Sample collection

After 24 hours incubation, syringes were removed from the incubator and placed on a container filled with ice, to decrease the temperature and stop activity of microbial metabolisms. As much as 2.5 ml of each syringe was dropped into a 20 ml centrifuge tube as sample, for probiotics enumeration. The rest of the sample from each treatment’s repetition was placed in a 30 ml plastic bottle. For VFA test, 1.5 ml sample was taken from the plastic bottle and placed in two Eppendorf tubes, while 5 ml sample was collected for organic acid test and the (approximately 20 ml) for Ammonium (NH4+) test. V.

Probiotic Enumeration

Pour plate method The pour plate method involves adding a small volume of sample (0.1-2.0 ml) to melted agar (44-46 °C) and then pouring the mixture into plates. This method usually used to grow organisms that need lower oxygen tension (such as microaerophilic organisms). The plates are then incubated for the required time. Plates can be stored at 35 °C for 48 hours or at 20-28 °C for 5-7 days. Using the pour plate method, colonies are generally small and compact and therefore easier to count. To obtain accurate quantitative analyses of cell numbers, petri dishes should have relatively diluted bacterial sample (25 to 250 cfu/plate) (Harrigan 1998; McLandsborough 2005). Probiotic enumerations were conducted in Animal Hygiene and Physiology Laboratory and included steps as follow: 2.5 ml sample obtained from before and after 24 hours HFT incubation. Samples were stored in Falcon centrifuge tube (20 ml) and prepared in the dilution series. A dilution series were applied in order to have the countable colony formed. As much as 1 ml from each of the 5 highest dilutions series was poured into a sterile Petri dish. Approximately 10 ml melting agar media [MRS agar (Lactobacillus-Agar de Man Rogosa and Sharpe, Merck) for Lactobacilli and Enterococci, RCM agar (Clostridium-Agar,

Oxoid) for Propionibacteria] is added into each Petri dish. Petri dishes were then placed inside anaerobic jar and set in 30 °C incubator for 5 days incubation. After 5 days incubation, colonies formed were observed and counted. Numbers of bacteria per ml were obtained by formula as follow: Bacteria/ml

VI.

A.

= number of colonies dilution x amount plated

Protozoa enumeration

Sample preparation

Protozoa number was calculated with chamber method. Samples were preserved with formaldehyde, as an agent which causes death of protozoa and allows preservation of the sample which then might be stored for a long time. A 4% formalin (formaldehyde) solution was prepared from 37% formaldehyde stock solution. Volume of 9 ml of the solution was stored in a plastic tube with lid (volume 20 ml). Numbers of tube contain 9 ml formalin were prepared regarding treatment number. One ml sample of before incubation (filtered rumen fluid + HFT solutions) and after (filtered rumen fluid + HFT solutions+ diet + probiotic) was added to each tube to have 1/10 dilution of sample. Sample then stored in room temperature. B. Protozoa enumeration As much as 0.1 ml (100 μl) sample was poured into the Fuchs-Rosenthal chamber under the cover glass through side rift of the chamber. The protozoa were counted in 80 small rectangular prisms at 100 x magnification. Total count was conducted in respectively, 80 small rectangular squares of Fuchs-Rosenthal chamber, included the cells which touch the upper and left borders and excluded the one which touch the lower and right borders of big square. The sub sample on the other side/rafter of the chamber was counted in the same way.

The cell calculation use the formula as follow: Number off cells ml-1 = (n1 + n2)/2 x 103 x d With: n1

= number of cells counted in upper rafter

n2

= number of cells counted in lower rafter

D

= dilution factor

Source: Couteau (1996). VII.

Autoclaved and Living Probiotics Experiment

In order to obtain data of probiotics carrier media influences, the experiment employing autoclaved and living cells of probiotics were conducted. The same amount of probiotics for each, autoclaved and living cell experiment was prepared by weighing certain amount of probiotics powder follow information given in Table 3. Probiotics powder than diluted into two different dilution of 0.9% NaCl to have two colony concentrations (107 and 109 cfu/ml). Two series, each has two different colony concentrations were obtained. One series than undergone autoclaving steps (conducted in animal hygiene and physiology laboratory), to obtain autoclaved probiotic cells. VIII.

(NH4+) Ammonium Analysis

Ammonium (NH4+) tests were conducted in Animal Nutrition Department laboratory, using Kjeldahl method (without digestion step) of ammonium analysis. A. Sample Preparation After defreezing samples, pH of each sample was measured (Knick digital-pH-meter 646). Each sample was weighed in Kjeldahl tube (Sartorius® Precisa 404 M SCS). During weighing process, the samples temperature was maintained low. B. Distillations and Titrations

Distillation process was conducted followed procedure described in Kjeldahl method. A trapped solution was prepared by applied 25 ml 0.05 M H 2SO4 and indicator H3BO3 in a small flask. As much as 5 ml 1 N NaOH was added into each sample before distillation. Distillation was carried out using distillation equipment (Gerhardt Vapodest 2), run approximately 3 minutes for each sample. The titration was conducted using 0.05 M NaOH. Point of titration acquired was indicated by the change of indicators colour changed from purple to light green (greenish). The quantity of NaOH used for titration of each sample was recorded and calculated to gain the NH4+ contents. Prior to the sample titration, blanks titrations were taken place, as well as the titration of standard 0.05 M H2SO4 (10 ml 0.05 M H 2SO4 standard volumetric solution AppliChem) to get the NaOH titter.

IX. VFA (Volatile Fatty Acids) and Organic Acids Analysis Organic acids analysis including: acetate, propionic, iso-butyric, n-butyric, iso-valerinat, nvalerinat and lactic and formic acid were conducted using HPLC method by Lactosan. The method followed the Proceedings suggested by Merck for the use of column (ICSep ION300). The column is suitable for the separation of organic acids, coal hydrates and alcohols. The samples were prepared before over the Carrez precipitation, in order to precipitate available proteins and fats. The samples were centrifuged in Auto sampler vials. Quantification was made via a RI Detector by a calibration with external standard. A. Reagents

1. Mobile phase Sulphuric acid 0.005 M (MERCK, art. no. 1.09981.0001, Titrisol, 0.5 mol/l; 1 N)

2. Calibration substances: a. Maltose (MERCK, art. no. 1.05912.0025, 99%)

b. Lactose (MERCK, art. no. 1.07660.0250, mono hydrate, 95%) c. Citrate (SIGMA ALDRICH, art. no. 24 062-1, mono hydrate, 99 %) d. D(+)Glucose (MERCK, art. no. 1.08337.0250, 99 %) e. D(-)Xylose (MERCK, art. no. 1.08689.0025, 99 %) f. D(-)Fructose (MERCK, art. no. 1.04007.0250, 99%) g. D Mannitol (SIGMA ALDRICH, art. no. M235-7, 98%) h. L(+)Arabinose (SIGMA ALDRICH, art. no. A9 190-6, 98%) i. Succinic (MERCK, art. no. 8.22260.2500, 99%) j.

L(+) Lactic acid, Litiumsalz (MERCK, art. no. 8.22084.0100)

k. Glycerol (MERCK, art. no. 8.18709.1000, 99%) l. Formic acid (MERCK, art. no.. 1.00264.0100, 98%) m. Acetic acid (MERCK, art. no. n. Diacetyl (MERCK, art. no. 8.03528.0100, 98%) o. 1,2-Propandiol (SIGMA ALDRICH, art. no. 39,803-9, 99.5%) p. 1,3-Propandiol (SIGMA ALDRICH, art No. P5,040-4, 98%) q. Acetaldehyde (MERCK, art. no. 1.00005.0100, 99.5%) r. Propionic acid (MERCK, art. no.. 8.00605.0100, 99%) s. Ethanol (LACTAN, art. no. t. Iso-butyric acid (MERCK, art. no. 8.00472.0100, 99%) u. 2-Propanol (MERCK, art. no. 1.09634.1000, 99%) v. n-Butyric acid (MERCK, art. no. 8.00457.0100, 99%) w. 1-Propanol (SIGMA ALDRICH, art. no. 29,328-8, 99.5%) x. Iso-Valerian acid (MERCK, art. no. 8.00820.0005, 99%) y. Valerian acid (MERCK, art. no. 8.00821.0100, 98%)

3. Carrez reagents: a. K4[Fe(CN)6 ] (MERCK, art. no. 1.04984.0100). b. ZnSO4. 7 H2O (MERCK, art. no. 1.08883.0100).

B. Devices and Glass Materials

HPLC - plant and material a. Hewlett Packard HPLC system 1100: Auto sampler, Isocrates pumps, column furnace, interface 35900, RI-Detektor1047 A. b. A column (TRANSGENOMIC, ICSep ION-300, art. no. ICE-99-9850, and/or WAGNER of LOEFFLER, ICSep ION-300, art. no. CH0-0800). c. Auto sampler vials 2 ml (WAGNER & MUNZ, art. NR. 1109 0356). d. Vial caps (WAGNER & MUNZ, art. NR. 1103 0209)

C.

Experiment

1.

Working procedures

a.

Production of the Carrez reagents For precipitating proteins and fats. -

Precipitation reagent C1: 1.06 g K4[Fe(CN)6 ]. 3 H2O in 10 ml Distillate H2O (note: Potasiumhexacyanoferrat (II))

-

Precipitation reagent C2: 2.88 g ZnSO4 x 7 H2O were kept in 10 ml distillate H2O solutions in the refrigerator.

b.

Production of the mobile phase From Titrisol and Milli-Q water (conductivity 16 µΩ/cm), 0.5 M sulphuric acid were prepared and stored in low temperature. For the mobile phase refill, 2 L of 0.005 M H2SO4 was prepared: Dilution: 1:100, 20 ml concentrated acid (0.5mol/l) in the 2 L measuring flask filled up with Milli-Q water.

c.

Calibration of the HPLC The calibration substances are summarized in 5 different external standard solutions. To have 1000 ppm standard, as much as 0.1 g of each substance

was weighed in 100 ml clean and dry flask. Flask filled up with Milli-Q water. The samples weights were noted. Flask filled up with Milli-Q water. Points of calibration: The standards 2 were injected, and produced calibration 2 levels. The list for calculating the calibration table was present as Excel paper. In this data sheet the actual weighted-in quantities and the purities of the chemicals were considered. The file for each calibration was stored under the date of the new calibration. For examination in regular intervals standards with 100 ppm were injected and the evaluated data were saved in separated sheet.

Table 5.

Dilution pattern of 5 different concentrations starting from the 1000 ppm

standard Stages

d.

e.

ppm

Dilution factor

Flaks (ml)

Std. 1000 ppm

Vial levels

Level 1/2

10

1 : 100

10

0.1

5

Level 3/4

40

1 : 25

25

1

5

Level 5/6

100

1 : 10

100

10

15

Level 7/8

500

1:2

10

5

5

Level 9/10

1000

-

100

-

3

HPLC conditions -

HPLC conditions

-

Injection volume 40 µl

-

Temperature 42 °C

-

Stop time 55 min

-

Post time 5 min

-

Maximum pressure 100 bar

-

Minimum pressure 7 bar of

-

Mobile phase 0.005 molecular H2SO4

Sample preparation

e.1.

Fermentation Samples

For determination of the fermented acid samples from fermentation projections, 1 ml the cell suspension was centrifuged (Eppifuge, 12.500 U/min, 10 min). The cell projection was diluted (1:40 ) at the same time in Milli-Q water and the Carrez precipitation was prepared (1715 µl Milli-Q water, 45 µl sample, 20 µl precipitation reagent c1, 20 µl precipitation reagent C2). The diluted sample was again centrifuged (Eppifuge, 12.500 U/min, 30 min). The projection was transferred into a Vial and injected over the automatic sample injector into the HPLC.

e.2.

Carrez - precipitation

The Carrez - precipitation to the clean up of the samples one accomplishes. Proteins and fats (could load the HPLC column) were removed by the complex formation of the 2 precipitation reagents from the sample. It is important that both complex agents in the same concentration are added is achieved and the precipitation conducted at pH values between 4-7. The quantity of the reagents C1 and C2 can be amended depending upon volumes and degree of pollution of the samples. Down aforementioned were at present the by routine used sample processing.

Sample dilutions: Sample were diluted in distilled H2O in Eppendorf (with dilution 1:5 780 µl, with dilution 1:40 - 1715 µl); without H2SO4 or mobile phase (otherwise too sour; pH value has to lie between 4 and 7) 200 µl (1:5 diluted) and/or 45 µl (1:40 diluted) using Eppendorf pipette sample was pipetted into the water. Addition solution C1: 10 µl and/or 20 µl also step by, well mixed (vortex) leave for approximately 1 minute.

Addition solution C2: 10 µl and/or. 20 µl also step by, well mixed (vortex) and leave for 5 10 min.

Table 6. Sample dilution pattern: Type of Sample

Dilutions

Dest. H20 (µl)

Sample (µl)

C1 (µl)

C2 (µl)

Silage extract

1:5

780

200

10

10

Fermentation sample

1:40

1715

45

20

20

Centrifugation: 30 min 12500/min approx. 500 - 700 µl clear supernatants directly in Vials transferred.

D.

Documentation and evaluation

The samples over the automatic sample injector into the HPLC were injected and analyzed. The evaluation of the Chromatogram was made by the software (HP Chemstation). Using an Excel-macro the calculations of the dilutions were accomplished. Subsequently, the data in the respective data sheets were documented.

Chapter 4. Result and Discussion I.

Result

Experiments of 100% and 75% grass silage ratio of each repetition were carried out at the same run by employing the same rumen fluid, when 50% and 25% grass silage diet, as well as its repetition, were performed at the same run applying the same rumen liquor. A. Diets and Nutrient Analysis of Diets

Result of diets and diet nutrient analysis were given in table 7 as follows: Table 7. Diet and diet nutrient analysis. Nutrient content (g/kg DM) Diet/component CA

CP

CL

CF

NDF

ADF

ADL

Grass silage/diet 100%

73.6

129

25.9

254

531

277

19

Diet 75 % grass silage

64.0

167

33.3

221

481

229

13

Diet 50% grass silage

54.3

194

32.9

165

371

166

12

Diet 25% grass silage

49.1

216

34.7

109

294

109

8

Soybean meal

66.6

488

30.1

71.0

280

103

25

Maize grain

15.3

83.6

33.4

22.1

159

25

2

Abbreviations are follows: ADF: acid detergent fibre; ADL: acid detergent lignin; CA.: crude ash; CF: crude fibre; CL: crude lipid; CP: crude protein; DM: dry matter; NDF: neutral detergent fibre

B. Gas Production

The gas productions mentioned are gas productions recorded after 24 hours of incubation in HFT. Treatments applied were different probiotics concentrations (10 7 and 109 cfu/ml), different grass silage to mixed-concentrate ratios (100:0; 75:25; 50:50; 25:75) and different probiotics strains.

The general observation of the gas production showed that at 10 9 cfu/ml concentrations probiotic strains produced higher gas production compared to 10 7 cfu/ml densities. The highest gas production was produced by Propionibacterium jensenii; 102.2 ml at 109 cfu/ml in 75% grass silage diet. When the lowest was 48.4 ml produced by Lactobacillus brevis at 107 cfu/ml in 100% grass silage diet. At 109 cfu/ml P. jensenii also had highest level of gas production when compared to another three probiotic strains and control

(Table 8).

Probiotics also have been observed to produce higher amount of gas in 50% and 25% of grass silage ratio than in higher proportion of grass silage; 100% and 75%. The exception results were performed by P. jensenii at 109 cfu/ml in 100% and 75% grass silage. P. jensenii strain produced higher gas production then in 50% and 25% grass silage ratio (Table 8).

Similar pattern of gas productions between different probiotic cell concentration and probiotic strain have also been recorded from experiment employing autoclaved and living probiotic cultures. Gas productions of 109 cfu/ml probiotics concentration in both treatments were higher than at 10 7 cfu/ml. The single exception was the gas production of autoclaved L. buchneri cell. when the gas has been higher produced at lower colony density. The gas productions of autoclaved probiotics experiment, including its control have been observed to have higher values when compared with gas production of living cells probiotics. Excluding gas production of L. brevis and living cell, which is higher than its counterpart (Table 9).

P. jensenii at 109 cfu/ml of

Table 8. Gas production Grass Silage (%)

Exp. cfu/ml 1

100 2 1 75 2 1 50 2 1 25 2

L.buchneri

Gas Production (ml/200 mg DM) L. brevis E. faecium P. jensenii

E+07 E+09 E+07 E+09

49.6 56.9 51.4 63.1

48.4 69.6 58.1 70.5

49.0 61.8 49.8 60.2

49.7 100.4 49.9 83.8

E+07 E+09 E+07 E+09

50.0 59.3 53.5 62.5

51.4 69.5 52.0 74.0

51.5 65.3 57.3 69.6

50.6 102.2 54.7 90.8

E+07 E+09 E+07 E+09

57.9 65.0 53.4 62.5

58.9 70.0 54.7 70.4

56.9 66.5 49.1 67.8

56.2 87.1 52.8 86.5

E+07 E+09 E+07 E+09

58.8 66.1 59.9 67.0

59.6 76.9 59.6 71.0

58.0 72.6 57.8 72.8

59.6 88.4 59.0 91.3

Control 49.2 51.9 51.4 53.2 58.1 53.7 59.3 59.6

Abbreviations are follow: cfu: colony forming unit; DM: dry matter; E+07: 107; E+09: 109; Exp. : experiment; L. buchneri/brevis; Lactobacillus buchneri/brevis; E. faecium: Enterococcus faecium; P. jensenii: Propionibacterium jensenii;

Table 9. Gas Production of autoclaved and living probiotics (Note: experiment were conducted in 50% grass silage diet)

Gas Production (ml/200 mg DM) L. brevis E. faecium P. jensenii

Experiment

cfu/ml

Autoclaved

E+07 E+09

57.8 64.1

57.2 73.8

57.3 69.6

57.2 78.8

57.8

Living

E+07 E+09

56.6 63.0

54.3 75.2

55.8 68.1

55.8 89.7

56.0

L.buchneri

Control

Abbreviations are follow: cfu: colony forming unit; E+07: 107; E+09: 109; L. buchneri/brevis; Lactobacillusbuchneri/brevis; E. faecium: Enterococcus faecium; P. jensenii: Propionibacterium jensenii;

Table 10. Gas production of diets component Diets component

Gas Production (ml/200 mg)

Silage

49.9

Soybean meal

52.5

Maize grain

73.1

Soybean meal + maize grain

65.8

C. Ammonium (NH4+) Production Data of ammonium (NH 4+) production of 100% and 75% silage diet has shown significant different result between replicates. The first experiment has shown lower result (maximal value 8.87 mg/30ml) then its replicates (15.2 mg/30ml) (Table 11). The first replicate displayed a similar pattern of NH 4+ production between 2 different diets (100% and 75%) between probiotic strains and between 2 colony concentrations. All ammonium produced from less colony density (10 7 cfu/ml) were lower than control, when at 109 cfu/ml L. brevis and P. jensenii represented probiotics strains which produced higher NH4+ than control values.

The E. faecium also the only probiotics strain that

produced lower NH4+ in 100% silage diet and slightly higher (75% silage diet), at 109 cfu/ml when compared to 107 cfu/ml (Table 11).

The second replicates of the same treatment produced different pattern of data between 100% and 75% silage concentration diet. At 100% silage, the NH 4+ production of 107 cfu/ml probiotics were higher than at 109 cfu/ml. in all strains except P. jensenii, when at 75% silage diet the more dense probiotics colony (10 9 cfu/ml) produced higher NH4+ than at 107 cfu/ml. The NH4+ productions at 107 cfu/ml of treatment with 75% silage were lower than control, except

L. buchneri which produced slightly different NH4+ when

compared with control (Table 11). NH4+ productions of 50% and 25% have been conducted in 2 replications, which also displayed different pattern between replicates. All NH 4+ production of probiotics from the first replicate in 50% silage was higher than control except NH 4+ production from L. buchneri at 109 cfu/ml. While all probiotic strains in 25% silage were producing higher NH 4+ than control, except E. faecium. Both in 50% and 25% silage of first replicate. The P. jensenii produced highest NH4+ at 109 cfu/ml, when E. faecium was the highest for 107 cfu/ml (Table 11). From the second replication of 50% and 25% silage, the data figured the higher NH 4+ production at 109 cfu/ml than 107 cfu/ml and controls for all probiotic strains. At colony density 107 cfu/ml and grass silage concentration 50%. NH4+ production from all probiotics

strains were lower than control, when L. buchneri and P. jensenii produced higher NH4+ than control at 25% grass silage (Table 11). The NH4+ production analysis of first experiment of lower grass silage rations were higher than at higher grass silage rations for all probiotics strains in two different colony concentrations. The second experiment figured similar results of NH4+ produced in different grass silage concentrations and in different probiotic strains (Table 11). The NH4+ productions of autoclaved probiotics including its control have been measured to have higher values than on living probiotics. The result of NH4+ production of living P. jensenii at 109 cfu/ml was the only value which was higher than its counterpart of autoclaved cells (Table 12).

Table 11. Ammonium (NH4+) Production Grass Silage (%)

Exp.

cfu/ml

1

NH4+ Production (mmol/L) L.buchneri

L. brevis

E. faecium

P. jensenii

Control

E+07 E+09

2.59 2.95

2.90 3.82

3.00 1.88

2.81 4.70

3.50

2

E+07 E+09

6.11 4.69

6.51 5.45

5.80 5.51

5.55 9.12

5.54

1

E+07 E+09

2.81 3.10

2.99 3.65

2.44 2.58

3.05 5.32

3.55

2

E+07 E+09

5.50 5.92

5.33 6.50

5.12 6.09

5.14 7.31

5.49

1

E+07 E+09

5.97 6.39

5.36 6.26

5.54 6.33

5.62 7.35

6.10

2

E+07 E+09

6.31 5.63

6.48 7.58

7.79 6.79

7.15 9.45

6.25

1

E+07 E+09

6.23 6.92

5.97 7.06

6.12 6.89

6.05 8.08

6.02

2

E+07 E+09

6.92 7.51

6.35 8.39

7.67 5.96

7.29 9.31

6.03

100

75

50

25

Abbreviations are follow: cfu: colony forming unit; E+07: 107; E+09: 109; Exp. : experiment; L. buchneri/brevis; Lactobacillus buchneri/brevis; E. faecium: Enterococcus faecium; P. jensenii: Propionibacterium jensenii;

Table 12. Ammonium (NH4+) production of autoclaved and living probiotics Experiment

cfu/ml

Autoclaved

Living

NH4+ Production (mmol/L) L.buchneri

L. brevis

E. faecium

P. jensenii

Control

E+07 E+09

4.76 4.42

4.58 5.20

5.00 5.17

5.01 5.92

5.06

E+07 E+09

4.55 5.23

4.27 5.21

4.34 4.91

4.69 6.29

4.52

7

9

Abbreviations are follow: cfu: colony forming unit; E+07: 10 ; E+09: 10 ; Exp. : experiment; L. buchneri/brevis; Lactobacillus buchneri/brevis; E. faecium: Enterococcus faecium; P. jensenii: Propionibacterium jensenii;

D. Volatile Fatty Acids and Other Organic Acids Production

There were no significant differences between probiotics in their performance producing volatile fatty acids (VFA) and other organic acids (lactic and formic acid).

The higher probiotics concentration the higher VFA and organic acids were produced. The VFA and organic acids production value from treatments was also higher than blanks (experiment sample without diet and probiotics addition) in all diet ratios. The results from lower probiotics inoculums (107 cfu/ml) were slightly different with controls (without probiotics inoculation). At colony concentration 10 9 cfu/ml, probiotic strain P. jensenii synthesized higher VFA and organic acids than the other 3 strains.

Detection limit in HPLC system used by Lactosan was 50 mg/l. Lactic and valeric acids result mostly lower than 50 mg/l. Therefore the values of both organic acids can not be considered. Butyric, iso-butyric and formic acids values in all experiment were 0, thus result were not shown on the table.

The complete figures of results (including each diet replications) are given in table 13 to 20.

Table 13. VFA and organic acids production Grass Silage (%)

Exp.

Microbes

cfu/ml

Acetic acid

Propionic acid

Butyrate

Isovaleric acid

Valeric Acid

Lactic acid

mmol/L Blank

2.3

0.7

0.4

0.1

0.0

0.0

1E+07

4.3

1.4

0.6

0.1

0.1

0.0

1E+09

4.7

1.5

0.7

0.1

0.1

0.0

1E+07

4.3

1.4

0.6

0.1

0.1

0.0

1E+09

5.3

1.7

0.8

0.2

0.1

0.0

1E+07

4.4

1.4

0.6

0.1

0.1

0.0

1E+09

4.9

1.6

0.7

0.1

0.1

0.0

1E+07

4.5

1.5

0.6

0.1

0.1

0.0

1E+09

6.6

2.3

1.2

0.3

0.2

0.0

Control

4.4

1.4

0.6

0.1

0.1

0.0

Blank

2.6

0.7

0.4

0.1

0.0

0.0

1E+07

4.7

1.4

0.6

0.1

0.1

0.0

1E+09

5.0

1.5

0.7

0.1

0.1

0.0

1E+07

4.7

1.4

0.6

0.1

0.1

0.0

1E+09

5.6

1.7

0.8

0.1

0.1

0.0

1E+07

4.7

1.4

0.6

0.1

0.1

0.0

1E+09

5.6

1.7

0.8

0.1

0.1

0.0

1E+07

4.8

1.5

0.6

0.1

0.1

0.0

1E+09

6.4

2.3

1.0

0.2

0.1

0.0

5.0

1.5

0.6

0.1

0.1

0.0

L. buchneri

L. brevis 1 E. faecium

Propioni

100

L. buchneri


Propioni

Control

7

9

Abbreviations are follow: cfu: colony forming unit; E+07: 10 ; E+09: 10 ; Exp.: experiment L. buchneri/brevis; Lactobacillus buchneri/brevis; E. faecium: Enterococcus faecium; P. jensenii: Propionibacterium jensenii;

Table 14. VFA and organic acids production Grass Silage Exp. (%)

Microbes

cfu/ml

Acetic acid

Propionic acid

Butyrate

Isovaleric acid

Valeric Acid

Lactic acid

mmol/L Blank

2.3

0.7

0.4

0.1

0.0

0.0

1E+07

4.2

1.3

0.7

0.1

0.0

0.0

1E+09

4.7

1.5

0.8

0.1

0.1

0.0

1E+07

4.3

1.4

0.7

0.1

0.1

0.0

1E+09

5.3

1.6

0.9

0.2

0.1

0.0

1E+07

4.3

1.4

0.7

0.1

0.1

0.0

1E+09

5.2

1.6

0.9

0.1

0.1

0.0

1E+07

4.5

1.4

0.7

0.1

0.1

0.0

1E+09

6.9

2.2

1.3

0.3

0.2

0.0

Control

4.6

1.4

0.7

0.1

0.1

0.0

Blank

2.6

0.7

0.4

0.1

0.0

0.0

1E+07

4.7

1.4

0.7

0.1

0.1

0.0

1E+09

5.1

1.5

0.8

0.1

0.1

0.0

1E+07

4.7

1.4

0.7

0.1

0.1

0.0

1E+09

5.8

1.7

0.9

0.2

0.1

0.0

1E+07

4.8

1.4

0.7

0.1

0.1

0.0

1E+09

5.5

1.7

0.9

0.1

0.1

0.0

1E+07

4.7

1.4

0.7

0.1

0.1

0.0

1E+09

6.7

2.4

1.1

0.2

0.2

0.0

5.0

1.5

0.8

0.1

0.1

0.0

L. buchneri


Propioni

75

L. buchneri


Propioni

Control

Abbreviations are follow: cfu: colony forming unit; E+07: 107; E+09: 109; Exp.: experiment L. buchneri/brevis; Lactobacillus buchneri/brevis; E. faecium: Enterococcus faecium; P. jensenii: Propionibacterium jensenii;


Exp.

Microbes

cfu/ml

Acetic acid

Propionic acid

Butyrate

Iso-valeric acid

Valeric Acid

Lactic acid

mmol/L Blank

2.8

0.8

0.4

0.1

0.0

0.0

1E+07

4.8

1.4

0.9

0.1

0.1

0.0

1E+09

5.3

1.5

0.9

0.2

0.1

0.0

1E+07

4.9

1.4

0.9

0.1

0.1

0.0

1E+09

5.8

1.7

1.0

0.2

0.1

0.0

1E+07

5.1

1.5

0.9

0.2

0.1

0.0

1E+09

5.5

1.6

1.0

0.2

0.1

0.0

1E+07

4.9

1.4

0.9

0.2

0.1

0.0

1E+09

6.5

2.1

1.2

0.2

0.2

0.0

Control

4.9

1.4

0.9

0.1

0.1

0.0

Blank

2.8

1.0

0.7

0.1

0.1

0.0

1E+07

5.0

1.8

1.3

0.2

0.1

0.0

1E+09

5.4

1.9

1.5

0.1

0.1

0.0

1E+07

4.9

1.8

1.3

0.2

0.1

0.0

1E+09

5.7

2.1

1.5

0.2

0.1

0.0

1E+07

4.9

1.8

1.3

0.1

0.1

0.0

1E+09

5.6

2.1

1.5

0.2

0.1

0.0

1E+07

4.9

1.8

1.3

0.1

0.1

0.0

1E+09

6.4

2.7

1.8

0.2

0.1

0.0

5.0

1.8

1.3

0.1

0.0

0.0

L. buchneri


Propioni

50

L. buchneri


Propioni

Control

Abbreviations are follow: cfu: colony forming unit; E+07: 107; E+09: 109; Exp.: experiment L. buchneri/brevis; Lactobacillus buchneri/brevis; E. faecium: Enterococcus faecium; P. jensenii: Propionibacterium jensenii;


Exp.

Microbes

cfu/ml

Acetic acid

Propionic acid

Iso-valeric acid

Butyrate

Valeric Acid

Lactic acid

mmol/L Blank

2.8

1.0

0.7

0.1

0.1

0.0

1E+07

4.9

1.5

1.0

0.2

0.1

0.0

1E+09

5.2

1.5

1.0

0.2

0.1

0.0

1E+07

5.0

1.5

0.9

0.2

0.1

0.0

1E+09

5.9

1.8

1.1

0.2

0.1

0.0

1E+07

4.8

1.4

0.9

0.2

0.1

0.0

1E+09

5.7

1.6

1.1

0.2

0.1

0.0

1E+07

5.0

1.5

0.9

0.2

0.1

0.0

1E+09

6.6

2.1

1.3

0.3

0.2

0.0

Control

5.0

1.5

1.0

0.2

0.1

0.0

Blank

2.8

0.8

0.4

0.1

0.0

0.0

1E+07

4.8

1.8

1.4

0.2

0.1

0.0

1E+09

5.3

1.9

1.5

0.2

0.1

0.0

1E+07

4.9

1.8

1.3

0.2

0.1

0.0

1E+09

5.8

2.2

1.6

0.2

0.1

0.0

1E+07

4.9

1.8

1.4

0.1

0.1

0.0

1E+09

5.5

2.1

1.6

0.2

0.1

0.0

1E+07

4.9

0.9

1.4

0.1

0.1

0.0

1E+09

6.4

2.7

1.9

0.2

0.1

0.0

5.1

1.9

1.4

0.2

0.1

0.0

L. buchneri


Propioni

25

L. buchneri


Propioni

Control

7

9

Abbreviations are follow: cfu: colony forming unit; E+07: 10 ; E+09: 10 ; Exp.: experiment L. buchneri/brevis; Lactobacillus buchneri/brevis; E. faecium: Enterococcus faecium; P. jensenii: Propionibacterium jensenii;

Table 17. Proportion of VFA and organic acids production Grass Silage (%)

Exp.

Microbes

cfu/ml

Acetic acid

Propionic acid

Butyric acid

Isovaleric acid

Valeric Acid

Lactic acid

Total

% Blank

65.32

20.81

9.89

3.00

0.99

0.00

100.00

1E+07

66.45

21.48

9.15

1.80

0.94

0.20

100.00

1E+09

66.42

21.31

9.26

1.89

0.94

0.17

100.00

1E+07

66.64

21.46

8.94

1.82

0.93

0.21

100.00

1E+09

65.72

21.71

9.70

1.88

1.00

0.00

100.00

1E+07

66.71

21.54

8.73

1.83

0.94

0.25

100.00

1E+09

65.54

21.79

9.56

1.87

1.02

0.22

100.00

1E+07

66.26

22.07

8.94

1.83

0.90

0.00

100.00

1E+09

62.82

21.55

11.29

2.47

1.79

0.09

100.00

Control

66.72

21.50

8.93

1.85

0.89

0.12

100.00

Blank

67.0

18.76

11.23

2.29

0.28

0.43

100.0

1E+07

67.99

20.66

8.65

1.63

0.82

0.25

100.00

1E+09

67.99

20.37

9.04

1.75

0.85

0.00

100.00

1E+07

67.97

20.86

8.71

1.67

0.78

0.00

100.00

1E+09

67.33

20.62

9.44

1.70

0.91

0.00

100.00

1E+07

68.17

20.82

8.60

1.60

0.81

0.00

100.00

1E+09

67.46

20.51

9.28

1.71

0.91

0.12

100.00

1E+07

67.91

20.73

8.59

1.63

0.82

0.33

100.00

1E+09

64.18

22.90

9.54

1.92

1.34

0.12

100.00

67.98

20.75

8.66

1.60

0.73

0.28

100.00

L. buchneri

L. brevis

1 E. faecium

Propioni

100

L. buchneri

L. brevis

2 E. faecium

Propioni

Control

Abbreviation are follow: cfu: colony forming unit; Exp.: experiment; 1E+07: 107; 1E+09: 109. E. faecium: Enterococcus faecium; L. brevis/buchneri: Lactobacillus brevis/buchneri. P. jensenii: Propionibacterium jensenii


Exp.

Microbes

cfu/ml

Acetic acid

Propionic acid

Butyric acid

Isovaleric acid

Valeric Acid

Lactic acid

Total

% Blank

65.32

20.81

9.89

3.00

0.99

0.00

100.00

1E+07

66.05

20.59

10.59

1.91

0.74

0.12

100.00

1E+09

65.57

20.69

10.67

1.96

0.99

0.11

100.00

1E+07

66.01

20.92

10.29

1.82

0.87

0.09

100.00

1E+09

66.00

20.04

10.86

1.96

0.97

0.16

100.00

1E+07

66.01

20.98

10.15

1.80

0.88

0.19

100.00

1E+09

65.50

20.60

10.94

1.86

1.00

0.10

100.00

1E+07

66.18

20.64

10.45

1.82

0.90

0.00

100.00

1E+09

63.35

20.37

12.06

2.34

1.74

0.14

100.00

Control

66.19

20.42

10.30

1.83

1.00

0.26

100.00

Blank

67.0

18.76

11.23

2.29

0.28

0.43

100.0

1E+07

67.18

19.72

10.29

1.71

0.79

0.31

100.00

1E+09

67.08

19.67

10.47

1.79

0.88

0.11

100.00

1E+07

67.26

20.31

10.00

1.66

0.76

0.00

100.00

1E+09

66.54

19.88

10.73

1.76

0.91

0.18

100.00

1E+07

67.34

19.94

10.10

1.65

0.79

0.17

100.00

1E+09

66.53

20.07

10.57

1.74

0.86

0.23

100.00

1E+07

66.80

20.34

10.16

1.72

0.78

0.21

100.00

1E+09

63.43

22.25

10.83

2.00

1.43

0.05

100.00

67.06

19.97

10.30

1.72

0.08

100.00

L. buchneri


Propioni

75

L. buchneri


Propioni

Control

0.87 7

9

Abbreviation are follow: cfu: colony forming unit; Exp.: experiment; 1E+07: 10 ; 1E+09: 10 . E. faecium: Enterococcus faecium; L. brevis/buchneri: Lactobacillus brevis/buchneri. P. jensenii: Propionibacterium jensenii


Exp.

Microbes

cfu/ml

Acetic acid

Propionic acid

Butyric acid

Isovaleric acid

Valeric Acid

Lactic acid

Total

% Blank

66.37

19.57

10.28

2.81

0.72

0.26

100.00

1E+07

66.90

18.79

11.83

1.60

0.87

0.0

100.00

1E+09

66.15

18.46

11.66

1.98

1.49

0.25

100.00

1E+07

66.51

18.67

11.64

1.90

1.29

0.00

100.00

1E+09

66.02

19.10

11.75

2.16

0.84

0.12

100.00

1E+07

66.05

19.03

11.68

2.17

1.06

0.00

100.00

1E+09

65.54

18.86

11.94

1.91

1.46

0.29

100.00

1E+07

66.13

18.74

11.60

2.38

0.93

0.22

100.00

1E+09

63.24

20.96

11.76

2.25

1.54

0.27

100.00

Control

66.04

19.12

11.70

1.99

0.92

0.23

100.00

Blank

65.66

18.82

11.20

2.74

1.26

0.32

100.00

1E+07

65.42

19.17

11.83

2.18

1.31

0.10

100.00

1E+09

65.69

19.13

12.27

1.79

1.01

0.10

100.00

1E+07

65.71

19.51

11.75

2.00

1.03

0.00

100.00

1E+09

65.50

19.38

11.90

1.97

1.02

0.22

100.00

1E+07

65.72

19.56

11.78

1.80

1.03

0.11

100.00

1E+09

65.63

19.65

11.92

1.79

0.93

0.09

100.00

1E+07

66.03

19.40

11.63

1.88

0.83

0.24

100.00

1E+09

63.38

21.36

11.87

1.99

1.40

0.00

100.00

66.04

19.64

11.85

1.82

0.65

0.00

100.00

L. buchneri

L. brevis

1 E. faecium

Propioni

50

L. buchneri

L. brevis

2 E. faecium

Propioni

Control

Abbreviation are follow: cfu: colony forming unit; Exp.: experiment; 1E+07: 107; 1E+09: 109. E. faecium: Enterococcus faecium; L. brevis/buchneri: Lactobacillus brevis/buchneri. P. jensenii: Propionibacterium jensenii


Exp.

Microbes

cfu/ml

Acetic acid

Propionic acid

Butyric acid

Isovaleric acid

Valeric Acid

Lactic acid

Total

% Blank

66.37

19.57

10.28

2.81

0.72

0.26

100.00

1E+07

64.83

19.09

13.17

2.17

0.67

0.06

100.00

1E+09

65.09

18.96

12.95

2.13

0.82

0.04

100.00

1E+07

65.50

19.24

12.43

2.00

0.69

0.14

100.00

1E+09

64.25

19.27

12.31

2.23

1.64

0.30

100.00

1E+07

64.68

18.74

12.60

2.82

1.15

0.00

100.00

1E+09

64.79

18.62

12.61

2.51

1.30

0.18

100.00

1E+07

64.42

19.84

12.28

2.36

0.88

0.23

100.00

1E+09

62.87

20.48

12.30

2.52

1.69

0.13

100.00

Control

64.42

19.15

12.55

2.66

1.13

0.10

100.00

Blank

65.66

18.82

11.20

2.74

1.26

0.32

100.00

1E+07

64.65

19.73

12.52

2.05

0.93

0.13

100.00

1E+09

65.06

19.26

12.36

2.12

1.14

0.06

100.00

1E+07

64.91

19.91

12.14

2.03

0.85

0.17

100.00

1E+09

64.95

19.75

12.17

1.93

1.20

0.00

100.00

1E+07

64.92

19.92

12.30

1.91

0.89

0.05

100.00

1E+09

64.66

19.71

12.65

1.88

0.98

0.13

100.00

1E+07

72.06

10.84

14.01

2.10

0.84

0.15

100.00

1E+09

62.53

21.41

12.53

2.12

1.37

0.05

100.00

64.96

19.78

12.32

2.03

0.86

0.05

100.00

L. buchneri

L. brevis

1 E. faecium

Propioni

25

L. buchneri

L. brevis

2 E. faecium

Propioni

Control

7

9

Abbreviation are follow: cfu: colony forming unit; Exp.: experiment; 1E+07: 10 ; 1E+09: 10 . E. faecium: Enterococcus faecium; L. brevis/buchneri: Lactobacillus brevis/buchneri. P. jensenii: Propionibacterium jensenii

Probiotics enumeration

Probiotic enumerations were conducted in Laboratory of Animal Hygiene and Physiology. Probiotics growth and colony appearance were observed and enumerated before and after 24 hours incubation. Each experiment mentioned has two repetitions of before and after incubation.

From the comparison of before and after incubation colony density in all probiotic strains and diets a decrease of colony density was indicated. except P. jensenii in the first trial of 100% and 75% grass silage diet. The colony density of P. jensenii before incubation was lower than after incubation (Table 21). After incubation observation at colony concentration 10 9 cfu/ml. P. jensenii has shown the lowest colony density decrease. The E. faecium competed with

P. jensenii in

giving the lowest colony decreasing data of before and after incubation (first trial of 75% and 50% grass silage diet and second trial of 100% diet) (Table 21).

Table 21. Probiotics enumeration Grass Silage (%)

6

Exp.

cfu/ml

Incubation

L.buchneri

x10 cfu/ml L. brevis E. faecium

P. jensenii

E+07

Before After

150 0.17

330 0.0015

610 0.022

0.2 0.56

E+09

Before After

8200 0.33

31000 4.6

47000 1.5

1.7 0.98

E+07

Before After

490

2100

1000

700

0.46

0.65

3.3

12

E+09

Before After

5600 3.5

35000 12

61000 15

95000 53

E+07

Before After

150 0.15

330 0.005

610 1.9

0.2 0.98

E+09

Before After

8200 0.49

31000 3.2

47000 7.8

1.7 5.4

E+07

Before After

490

2100

1000

700

2.5

2.5

1.1

31

E+09

Before After

5600 6.2

35000 6.5

61000 21

95000 44

E+07

Before After

130

140

470

310

0.65

0.019

0.3

24

E+09

Before After

24000

18000

34000

58000

0.17

0.037

0.29

35

1 100 2

1 75 2

1 50 E+07

Before After

180

140

330

660

0.26

0.053

0.32

0.93

E+09

Before After

22000 0.093

18000 13

35000 0.32

61000 0.98

E+07

Before After

130

140

470

310

0.84

2.7

9.8

5.3

E+09

Before After

24000

18000

34000

58000

0.77

1.3

13

55 660

2

1 25 E+07

Before After

E+09

Before After

2

180

140

330

0.63

1.2

11

13

22000

18000

35000

61000

16

140

1.0

1.3 7

9

Abbreviations are follow: cfu: colony forming unit; E+07: 10 ; E+09: 10 ; L. buchneri/brevis; Lactobacillus buchneri/brevis; E. faecium: Enterococcus faecium; P. jensenii: Propionibacterium jensenii;

E. Protozoa enumeration

The protozoan cell density at all probiotics concentration different diet ratios and probiotics strains has shown similar results. The slight differences have been observed were between before (without inoculation of probiotics-rumen fluid + HFT solutions) and after incubation. The detail result of protozoa enumeration is presented in Table 22.

Table 22. Protozoa enumeration Grass Silage (%)

6

Protozoa Enumeration ( x 10 /ml)

Exp.

cfu/ml

1 100 2 1 75 2 1 50 2 1 25 2

L.buchneri

L. brevis

E. faecium

P. jensenii

Before

Control

E+07 E+09

1.33 4.13

1.60 1.90

1.20 1.88

0.90 1.78

2.85

1.10

E+07

0.75

0.77

0.95

0.55

E+09

0.78

1.20

0.85

0.73

0.40

0.48

E+07 E+09

1.83 1.70

1.68 1.60

1.48 2.28

1.55 2.38

2.85

1.43

E+07

1.00

0.98

1.80

1.63

E+09

1.15

1.10

1.45

0.85

0.40

0.85

E+07 E+09

1.35 0.68

1.33 2.18

1.20 1.98

1.45 1.43

0.05

0.90

E+07

1.53

1.70

1.50

1.41

E+09

1.88

1.55

1.88

1.65

0.85

1.65

E+07 E+09

1.38 1.78

1.00 1.73

6.25 2.53

1.83 2.75

0.05

2.08

E+07

1.53

1.60

1.70

1.45

E+09

1.95

1.28

2.55

2.03

0.85

1.63

Abbreviations are follow: cfu: colony forming unit; E+07: 107; E+09: 109; Exp.: experiment ; L. buchneri/brevis; Lactobacillus buchneri/brevis; E. faecium: Enterococcus faecium; P. jensenii: Propionibacterium jensenii;

II. Discussion

The variability of results within treatment (between replicates) was mainly caused by low numbers of run (experiment) which were performed. which produced less number of repetition for each treatment. The ideal setting of experiment is each treatment should contain more than two comparable run. which develop more data.

Each experiment has been arranged and conducted according to the laboratory equipments set up. as well as the time table of the laboratory staffs. A. Gas Production

The HFT method determined carbohydrate digestibility through gas production during in vitro fermentation. Thus gas production value is related to feed digestion rate. Gas productions of probiotics-treated experiments as well as their controls from diets with low grass silage proportion were higher when compared to diets with higher grass silage ratio (Table 7). Laboratory analysis of diet nutrient content pointed that CF, ADF as well as NDF values of diets with higher ratio of grass silage were greater than diet with low portion of grass silage (Table 6). It indicates that the grass silage dominant diet contains more indigestible part of carbohydrate rather than in the concentrate dominant diet.

Experiment applying the autoclaved and living cells of probiotics was conducted to measure possible influence of media used as a carrier of probiotics product. The data shown similar results between treatments attribute to the probiotics carrier influences. During probiotic inoculums preparation. the treatments with higher probiotics concentration were prepared by weighing more probiotics powder; in order to obtain colony number requested (Table 3).

Autoclaving process should be also considered as a factor that elevates the digestibility of the carrier media. The complex form of nutrients in the carrier media could have been de-

arranged. producing simple forms which serve as available nutrient sources for microbial fermentation process.

The single result was an exception. as living cell of P. jensenii at 10

9

cfu/ml produced

greater gas production than its counterpart. indicating the independent action of probiotics living cell and can be interpreted that P. jenseni is high potential probiotics strain. B. (NH4+) Ammonium Production

Bach et al. (2005) reported that the protein degradation is affected by pH and type of ration or substrate being fermented. Protein degradation is reduced as pH decreased. Protein degradation is lower with beef-type ration (10:90 forage –to-concentrate ratio) than in dairy-type ration (60:40 forage-to-concentrate ratio), when pH of both rations was maintained above 6.0. Results of NH4+ production of lower grass silage concentration were higher than experiment with higher grass silage rations. on account of different fermentation rate has occurred due to different type of substrate. The reduction of protein degradation is not only due to a pH effect. but is also related to type of substrate being fermented (Bach et al. 2005).

As the low ratio grass silage diets comprise higher proportion of concentrates, which have high protein contents (Table 3). The NH4+ production of lower ratio grass silage diet were higher than in grass silage dominant diet. The production of NH4+ also found greater in more dense probiotic inoculums (10 9 cfu/ml), as probiotic my served as bacterial protein source. Higher production of NH4+ in autoclaved probiotic treatment probably developed from the media carrier. The autoclaved treatment has been gone through autoclaving process which degraded complex compounds into simple and more available compounds.

C. Volatile Fatty Acids and Other Organic Acids Production

Hungate et al. (1961) described that the average production of VFA in lactating cows per day was 10.5 moles butyric acid, 12.8 moles propionic acid and 40 moles acetic acid. Detail investigation from Sutton et al. (2003) demonstrated the influence of diet type on the production rate of VFA (Table 2).

The VFA proportion of acetate, propionate and butyrate from the experiments is roughly 65, 21 and 8 % from total volume VFA which were produced (100%, including iso-valeric, valeric and lactic acid).

At the same diet treatment the production of propionic acid from P. jensenii is higher than the other probiotics strain (at their high colony concentration

(10 9 cfu/ml), whereas at

107 cfu/ml there were no significant different of proponic acid production between probiotic strains.

D. Probiotics Enumeration

The probiotics colonies of all strains after incubation sample in different treatments were lower than the starting colony number (before incubation), mainly due to the extreme rumen environment.

The P. jensenii acted as the most potent probiotic under the rumen condition, indicated that P. jensenii is more adaptable to the rumen (in vitro) environment than the other tested probiotics.

E. Protozoa Enumeration

Protozoa enumeration results showed that protozoa number in most of probiotics-treated experiments was higher when compared to controls (without probiotics inoculation) and to the before fermentation samples (contains only filtered rumen fluid + all HFT solutions). It indicated that protozoa population was positively influenced by the addition of probiotics (bacterial) population.

In rumen environment bacteria act as main protein source for

protozoa (Bach et al. 2005).

Result also indicated that different diet ratios did not influence the protozoa population, although Franzolin and Dehority (1996) mentioned in their study that the concentration of protozoa in ruminal contents generally increases with the addition of concentrates to roughage diets. Concentrates provide a source of rapidly fermentable carbohydrate for ruminal microorganisms, which produce VFA, thereby reducing ruminal pH. The fall in ruminal pH is generally accompanied by a decrease in protozoal concentrations and in some instance a complete disappearance of the protozoa. The application of buffer solutions that maintain the environment pH during fermentation process could be one of the reasons.

Conclusion and Recommendations I.

Conclusions

1.

From the gas production test and series of sample analysis, probiotic strains had more influence on the rumen fermentation process, when they were administered at high colony concentration (109 cfu/ml).

2.

The experiments indicated that P. jensenii has the most effective probiotic agent when applied at concentration higher than 10 7 cfu/ml.

3.

The

variation

of

diet

composition

has

also

influenced

probiotics

effectiveness.

II.

Recommendations

1.

Number of replications of each treatment should be greater than two, in order to provide more comparable value and to prevent ambiguous interpretation.

2.

An experiment, comparing probiotic activity of P. jensenii at colony concentration 108 and 109

cfu/ml is intriguing, as P. jensenii showed

outstanding performance as potential probiotic agent.

3.

The information of component used in the experiment should be completely collected, as contents of each component my give influence on the results.

4.

An in vivo experiment of potential probiotics candidate should be accomplished to obtain comprehensive data before their applications on the farm.

5.

A mixed culture of probiotic could is a promising research in the future, as in a composite form probiotic cultures could have better performance when compare to its single form.

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Curriculum Vitae Name

: Taufiq Wisnu Priambodo

Place/date of Birth

: Jakarta/23 July 1972

Sexes

: Male

Marital Status

: Married

Nationality

: Indonesia

Religion

: Islam

Education Background Year

Level

Field//Major

1979-1985

Elementary School

Jakarta

1985-1988

Junior High School

Jakarta

1988-1991

Senior High School

Biology

Institution/Place

Jakarta

1991-1996

First degree

Biology (thesis in microbiology)

University of Indonesia/Depok

2003

Non degree training

Advance Industrial Biotechnology

GBF (Germany Society for Biotechnology)/Braunschweig

2005-2007

Master degree

Animal Nutrition (thesis in probiotics)

University of Bonn/Bonn

Working/Research Experience Year

Institution

Field/Project

Place

1996

Laboratory of Microbiology, University of Indonesia

Screening of antimicrobial compound from Rhizopus actives against tested bacteria (First degree thesis)

Depok

1996

R&D Centre for Biotechnology, Indonesian Institute for Sciences

Genetic engineering of Ruminoccoccus albus

Cibinong

1997

Friesche Vlag Indonesia Dairy Industry

Production trainee

Jakarta

Since 1999

Biotech Centre, The Agency For Assessment and Application of Technology

Research and application of probiotic as feed supplement for ruminant

Serpong

2003

Laboratory of Food Microbiology and Hygiene, University of Hamburg

Vitamin B12 enhancement in tempe (Indonesian traditional fermented food) using Lactobacillus strains

Hamburg