EFFECT OF HERBAL PLANTS ON QUALITY AND MICROBIAL SAFETY OF MILK IN EASTERN ZONE OF TIGRAY, ETHIOPIA By HaftomYemane A Thesis Submitted to College of Veterinary Medicine, Mekelle University, in Partial Fulfillment of the requirements for the Degree of Master of science in Zoonosis and food safety June, 2015 Mekelle, Ethiopia Declaration This is to certify that this thesis entitled “effect of herbal plants in quality and microbial safety of milk in eastern zone of Tigray, Ethiopia” submitted in partial fulfillment of the requirements for the award of MSc. degree in Zoonosis and food safety to the College of veterinary medicine, department of veterinary medicine of Mekelle University done by Haftom Yemane ID. No. CVM/PG06/06 is an authentic work carried out by him under my guidance. The matter embodied in this project work has not been submitted earlier for award of any degree or diploma to the best of my knowledge and belief. Name of student: Haftom Yemane Signature __________________Date_____________ Name of Advisor: Dr. Habtamu Taddele Signature ______________ Date___________ Name of co-advisor: Dr. Yayneshet Tesfay Signature ____________ Date___________ EFFECT OF HERBAL PLANTS ON QUALITY AND MICROBIAL SAFETY OF MILK IN EASTERN ZONE OF TIGRAY, ETHIOPIA By Haftom Yemane BOARD OF EXTERNAL AND INTERNAL EXAMINERS 1. Dr. Desalegn Woldeyohanis (Assoc. professor of tropical infectious disease) Signature ________________________ 2. Dr. Endale Balcha (Assoc. professor of veterinary public health) Signature _______________________ Advisors 1. Dr. HabtamuTaddele (Assoc. Prof., Mekelle University, CVM) Signature ___________________ 2. Dr. YayneshetTesfay (PhD, International Livestock Research Institute ILRI) Signature ______________________ TABLE OF CONTENTS ACKNOWLEDGEMENTS ................................................................................................. I LIST OF TABLES .............................................................................................................. II LIST OF FIGURES .......................................................................................................... III LIST OF ABBREVIATION ............................................................................................. IV ABSTRACT ....................................................................................................................... V CHAPTER I: INTRODUCTION ........................................................................................ 1 1.1 Background and Justification ............................................................................... 1 1.2 Statement of the Problem ..................................................................................... 2 1.3 Significance and limitation of the study ............................................................... 3 1.4 General and Specific Objectives .......................................................................... 3 1.5 Hypothesis ............................................................................................................ 3 CHAPTER TWO: LITERATURE REVIEW ..................................................................... 4 2.1 Medicinal Plants in Ethiopia ..................................................................................... 4 2.2 Herbal Plants Used for fumigation and their Medicinal Value ................................. 4 2.2.1 Olea europaea L ................................................................................................. 4 2.2.2 Vernonia amygdalina ......................................................................................... 5 2.2.3 Solanum schimperianum hochst ......................................................................... 6 2.2.4 Acacia etbaica schweinf ..................................................................................... 6 2.2.5 Aloe elegans tod / aloe vera ............................................................................... 6 2.3 Importance of Milk in Human Health ....................................................................... 7 2.4 Compositional Quality and Characteristics of Milk .................................................. 7 2.5 Nutritional Value of Milk .......................................................................................... 8 2.6 Hygienic Quality of Milk .......................................................................................... 9 2.7 Bacteria in milk ......................................................................................................... 9 CHAPTER THREE: MATERIALS AND METHODS ................................................... 11 3.1. Study Area .............................................................................................................. 11 3.2 Survey on herbal Plants used for milk container fumigation ............................. 13 3.3 Study Plants ........................................................................................................ 13 3.4 Herbal Plant Collection ...................................................................................... 13 3.5 Plant Species Identification ..................................................................................... 14 3.6 Study Sample and Experimental Design ................................................................. 14 3.7 Milk Quality Parameters ......................................................................................... 14 3.8 Fumigation method ................................................................................................. 15 3.9 Bacterial Load Determination ................................................................................. 15 3.10 Bacterial Isolation ................................................................................................. 15 3.11 Statistical analysis ................................................................................................. 16 CHAPTER FOUR: RESULT ........................................................................................... 17 4.1 Questionnaire Survey Result ................................................................................... 17 4.2 Bacterial Load Determination Result ...................................................................... 19 The mean difference is x104 a reference to the best herbal plant and b the next and e the least................................................................................................................................ 25 4.3 Bacterial Isolation ................................................................................................... 25 CHAPTER FIVE: DISCUSSION ..................................................................................... 26 CHAPTER SIX: CONCLUSION AND RECOMMENDATIONS .................................. 31 CHAPTER SEVEN: REFERENCES ............................................................................... 32 CHAPTER EIGHT: ANNEXES....................................................................................... 38 I ACKNOWLEDGEMENTS First and for most, I would like to thank Almighty God for giving me full health, peace, strength and patience to complet of my study. I am thankful to ILRI, LIVES project for their financial support for my research work. I also want to forward my deep thanks to my advisors Dr. Habtamu Taddele and Dr.Yayneshet Tesfay for their constructive advice and encouragement in every step of my research work. My appreciation also goes to TARI staff members Dr. Zelalem Tesfay and Awet Estifanos for their technical support. It is my pleasure to thank Dr. Yohannes Hagos for his support in data analysis. I am also extremely thankful to Dr. Tadesse Kidusan for his unlimited commitment on identification of the herbal plants for their scientific names. I like to thank also the laboratory technicians of collage of veterinary medicine. I would be very glad to thank my beloved mam Abrehet Gebremicheal and my lovely wife Eliza Ayele and my little daughter Bersabeh Haftom, and my spiritual son Mual Asfawesen my friends Asfawesen Tekleab, Shishay Desta, Kiros Tsegay, Atakility Hailesilase, Dawit Tesfay Asefu Abadi, Ruta Tewelde, and Kidanemihret Kerbo for their comprehensive moral support and encouragement that enabled me to succeed my thesis. II LIST OF TABLES Table 1: Composition of milk from different animals ........................................................ 8 Table 2: Type of herbal plants used for fumigation and their mode of preparation ......... 17 Table 3: Respondents idea on milk spoilage..................................................................... 18 Table 4: Use of herbal plants and the level of importance by the interviewee ................. 19 Table 5: Treatments Mean of milk samples stored in plastic containers .......................... 20 Table 6: Treatments Mean of milk samples stored in calabash ....................................... 20 Table 7: Mean comparison of milk from plastic containers on the basis days ................. 22 Table 8: Mean comparison of milk from calabash containers on the basis days .............. 22 Table 9: Mean comparison of milk samples in calabash and plastic containers .............. 23 Table 10: Organoleptic taste result ................................................................................... 23 Table 11: Mean comparison of treatments........................................................................ 24 Table 12: Bacterial isolates from the different treatment groups ..................................... 25 Table 13: Bacteriological taste format .............................................................................. 40 Table 14: Milk quality taste format .................................................................................. 41 Table 15: Organoleptic taste format ................................................................................. 42 III LIST OF FIGURES Figure 1: Map of the study districts .................................................................................. 12 Figure 2: Olea europaea L (left) and Vernonia amygdalina (right) ................................. 43 Figure 3: (S. schimperianum hochst (left) and Acacia etbaica schweinf (right) ............... 43 Figure 4: Aoe elegans........................................................................................................ 44 Figure 5. Labelled Calabash and plastic milk containers ................................................. 44 Figure 6. During fumigation ............................................................................................. 45 Figure 7. Plant collection for species identification.......................................................... 45 IV LIST OF ABBREVIATION ASL Above Sea Level BoARD Bureua of Agricultural Rural Development Cfu: Colony Forming Units CNCS: College of Natural and Computational Science CVM: Collage of Veterinary Medicin FAO: Food and Agricultural Organization ILRI: International Livestock Research Institute LIVES: Livestock Irrigation Value chain for Ethiopian Smallholders SPC: Standard Plat Count WHO: World Health Organization WOARD: Wereda Office of Agricultural Rural Development V ABSTRACT The study was conducted to understand the effect of herbal plants on quality and microbial safety of milk and to identify the best plant used for fumigation of milk container in Eastern zone of Tigray, Ethiopia. A total of 84 respondents, 52.4% (n = 44) from Ganta-afeshum and 47.6% (n = 40) from Kilteawilaelo were interviewed. From these, 81% (n = 68) of them encountered milk spoilage. 70.2% (n = 59) respondents, used herbal plants as fumigators of milk containers to prevent milk spoilage. The milk samples collected from the selected farms were poured in to 6 calabash and 6 plastic containers each with 200 ml of milk. The milk poured in to the untreated plastic and calabash containers were used as control group. Organoleptic tastes like odor and taste as well as physical attributes like temperature and pH were carried out. Standard plate count and coliform count were used for bacterial load determination. Herbal plants namely Olea europaea L, Vernonia amygdalina, Solanum schimperianum hochst, Acacia etbaica schweinf, Aoe elegans were used for milk fumigation. The data obtained were analyzed using SPSS Statistical software version 17.0 (descriptive analysis (frequency), mean comparison T independent test, General leaner model multifactorial repeated two way ANOVA) analysis method were applied. The lowest bacterial count was obtained from milk samples fumigated by V. amygdalina for both standard plate count (7.8x105) and coliform count (4.5x105). The highest bacterial count was recorded from samples of the untreated or control groups. The bacterial count between treated and untreated milk container had statistically significant variation (p = 0.000). Milks samples stored in calabash containers had excellent taste and odor in day one and day two compared to plastic containers. Milk container fumigated with Vernonia amygdalina improve the quality and microbial safety of milk. Key words: Calabash, Coliform, Herbal plant, Plastic, Fumigation, Standard plat count 1 CHAPTER I: INTRODUCTION 1.1 Background and Justification In Ethiopia, medicinal plants have been used as traditional medicine to treat different human ailments by the local people from time immemorial. These medicinal plants are estimated to be over 700 species (Kebebew and Addis, 1996). Being a land of diverse climatic and edaphic potentials, several of such indigenous and exotic species and essential plants could luxuriously grow in Ethiopia and provide remarkable benefits to the national economy (Endashaw, 2007). 80% of the human population and 90% of livestock rely on traditional medicines (Getachew et al., 2001). Traditional medicine is an integral part of the culture, belief and lifestyle of Ethiopian peoples (Tesfaye et al., 2009). The issue of medicinal plant conservation in Ethiopia today calls for aggressive studies and documentation before accelerated ecological and cultural transformation distorts the habitats of these plants and culturally held knowledge bases (Endashaw, 2007). Since most traditional knowledge in Africa is transmitted orally, from generation to generation, knowledge of wild plants is in danger of being lost as habitats, value systems and natural environments change (Haile and Delenasaw, 2007). Thus, valuable indigenous knowledge associated with medicinal plants permits proper documentation (Awas et al., 2012). The use of milk and milk products as human food has got a very long history. Milk is one of the most important foods of human beings. It is universally recognized as a complete diet due to its essential components (Javaid et al., 2009). However, health risk to consumers can be associated with milk, due to the presence of zoonotic pathogens and antimicrobial drug residues (Bradley, 2002). The quality of milk may be lowered by a numbers of factors such as adulteration, contamination during and after milking and the presence of udder infections (Esron et al., 2005). Milk plays a vital role in building a healthy society and can be used as vehicle for rural development as well. Fresh or raw milk as diet contributes a great role for survival to infants in human and animals in all over the world (Buyser et al., 2001). Milk could be naturally a good medium for microbial growth. Due to such nature of milk, there is no 2 complete prevention mechanism of microbial contamination, that's why the microbial contamination is major factor in determining quality of milk (Buyser et al., 2001). Microbial contamination can originate from different sources including from the milking cow and air, milking equipment’s, feed, soil, faces and milking place (Coorevits et al., 2008). Moreover, the utensils, animal skin, environment, water used for adulteration can cause great contamination Transportation of milk at ambient temperature results in enhanced and multiple number of microbes that deteriorate the overall milk quality (Oliver et al., 2005). Quality and safety improves the shelf life of the milk and also help to introduce new and different products which can be convenience for the consumer (Goff and Griffiths, 2006). The storage life of a dairy product has the better time during which the product can remains wholesome and having no physical or sensory defects (Casewell, 2003). 1.2 Statement of the Problem Consumption of raw or highly contaminated milk has commonly been associated with food borne illness. The world still faces great problems of food borne diseases associated with contamination of the milk supply. About 1.3 million cases of active diarrhea in children less than five years in the developing world due to contaminated milk (FAO, 2004). It is possible to milk animals in such a clean way that the raw milk contains only 500 to 1,000 bacteria per ml. usually the total bacteria count after milking is up to 50,000 per ml (FAO, 2004). Milk contains a natural inhibitory system which prevents a significant rise in the bacteria count during the first 2 - 3 hours and if it is cooled within this period to 4 ˚C, it maintains nearly its original quality. Timely cooling ensures that the quality of the milk remains good for processing and consumption (O’Connor, 1994). However, in rural places where there is no refrigerator facility people use traditional approaches to maintain the quality and safety of milk. In Tigray use of herbal plants as milk preservation/milk container fumigation is a common practice for long period of time. However, there is no documented study on the role of these herbal plants and all herbal plants are considered as equally effective in improving the quality and microbial safety of milk. 3 1.3 Significance and limitation of the study The main purpose of this research is to identify the herbal plants used by the farmers, test them under laboratory condition and recommend the best plant that can be widely used in the area. Thus, consumer demand for safe and high quality milk in areas where there is lack of refrigeration facility can be achieved. Limitation of the study I can’t address all over the region in which more herbal plants can be available rather than the five herbal plant I had use. Plant active ingredient extraction and use as additive on the milk as fumigation might have a better result but due to financial limitation I can’t do that. 1.4 General and Specific Objectives The general objective of the present research was to understand the effect of herbal plants on quality and microbial safety of milk in Eastern zone of Tigray. Specific objectives were:  To assess and select herbal plants used as milk container fumigators in eastern zone, Tigray  To evaluate the quality of milk preserved using these herbal plants as milk container fumigators.  To evaluate the plants for their effectiveness in minimizing the bacterial count by replicating the traditional processing methods. 1.5 Hypothesis Fumigating milk containers does not improve the organoleptic and microbial quality of milk, and between herbal plants traditionally used for fumigation have no major difference in maintaining the desired quality of milk. 4 CHAPTER TWO: LITERATURE REVIEW 2.1 Medicinal Plants in Ethiopia The cultivation and use of spices, herbs, medicinal and other essential oil bearing plants is not new to Ethiopia. It is as old as the crop themselves, and its history can be traced back to the reign (Endashaw, 2007). Ethiopia is the origin and/or center of diversity for many of these plant species. The various literature available show the significant role of medicinal plants in primary health care delivery in Ethiopia where 70% of human and 90% of livestock population depend on traditional medicine similar to many developing countries particularly that of Sub-Saharan African countries. The traditional health care is culturally deep rooted with oral and written pharmacopoeia (O’Connor, 1994). Thousands identified medicinal plant species are reported in the Ethiopian Flora, however, many others are not yet identified. About 300 of these species are frequently mentioned in many sources. The greater concentration of medicinal plants are found in the south and south western Ethiopian parts of the country following the concentration of biological and cultural diversity (Edwards, 2001). The various citations made from various written records of medicinal plants from central, north and northwestern part of Ethiopia are thus small fractions of medicinal plants present in Ethiopia. Some study on the Bale Mountains National Park in the South East Ethiopia revealed that the area, as much as it is a biodiversity hotspot, also turned out to be a medicinal plant hotspot with 337 identified medicinal species of which 24 are endemic (National Herbarium, 2004; Ermias Lulekal, 2005; Haile Yineger, 2005). The species comprised of 283 used as human medicine, 47 used as livestock medicine and 76 species used for both human and livestock by the community healers, harvesters, traders and users. 2.2 Herbal Plants Used for fumigation and their Medicinal Value 2.2.1 Olea europaea L Olive tree (Olea europaea L.) is one of the most important trees in Mediterranean countries, where they cover 8 million ha, and accounting for almost 98% of the world crop. This demonstrates the great economic and social importance of this crop and the possible benefits to be derived from utilization of any of the byproducts derived from it (Guinda, 5 Albi, Camino, & Lanzo´ n, 2004). Olive leaves are one of the byproducts of farming of the olive grove; they can be found in high amounts in the olive oil industries (10% of the total weight of the olives) and they accumulate during pruning of the olive trees (Tabera et al., 2004). Popular medicine and phytotherapy use olive leaves to treat and prevent hypertension, and for their hypoglycemic, antiseptic and diuretic properties (Bruneton & Iridoides, 1993). They were formerly used as a folk remedy for combatting fevers and other diseases, such as malaria, but this use was dropped. Several reports have shown that olive leaf extract has the capacity to lower blood pressure in animals and increase blood flow in the coronary arteries (Zarzuelo, 1991), relieve arrhythmia and prevent intasteinal muscle spasms (Garcia, Castillo, 2000). There is an increasing interest in the phenolic compounds in olive by-products, due to their biological properties. Olive oil polyphenols possess good antioxidant activities (Gordon, & Gameiro, 2003). Also olive leaves are a source of several antioxidants (Bouaziz & Sayadi, 2005) 2.2.2 Vernonia amygdalina Vernonia amygdalina, a member of the leaf of about 6 mm diameter size and elliptic shape plants is widely used as a medicinal plant. The leaves are green with characteristic odour and bitter taste. There are about 200 species of Vernonia (Iwalokun et al., 2006). The leaves are used for human consumption; washed before eating to get rid of the bitter taste. They are used as vegetable and stimulate the digestive system, as well as they reduce fever. Furthermore, they are used as local medicine against leech, which are transmitting bilharziose. (Igboasoiyia et al., 2008). It is very unlikely that a single molecule is responsible for such varied activities; instead multiple molecules, working alone or in combination with others, are much more likely to be these biological activities (Mohammed, 2008). Pharmacologically the leaf of Vernonia amygdalina extract is used in medicine as an antimalarial, antibacterial, laxative, antihelmitic, antithrombotic, and both hypoglycemic and hypolipidaemic effect in diabetic patients. Phytochemical screening of the plant has revealed the presence of saponins, glycosides and tannins, which are known to be bioactive purgative principles. Flavonoids are also present in bitter leaf and its antioxidant effect may play a beneficial role in cancer prevention, and offer some protection against diabetes and arterosclerosis (Georgewill, et al., 2009). 6 2.2.3 Solanum schimperianum hochst The genus Solanum is the largest genera of the family Solanaceae consisting of more than 1700 species distributed all over the world. The genus is represented by about 16 species (Antonio et al., 2004). Several species of genus Solanum are used in the folk medicine of different countries, specially Brazil, India, Taiwan, Germany, South Africa and Kenya, as remedy for various ailments such as hypoglycemic, hepatoprotective, laxative, appetizer, cardiotonic, antispasmodic, renal pain, epilepsy, gastric, liver disorder, treatment of bronchitis, itches, body aches and cancer (Ferro et al., 2005). Genus solanum is a rich source for many classes of compounds such as alkaloids, steroids and phenolic compounds. Solanum schimperianum Hochst grows throughout Southern region of Saudi Arabia and widely distributed in the tropical Africa (Yoshimitsu et al., 2003).the plant has no folkloric usage. The methanol extract of Solanum schimperianum was reported to have significant antitrypanosomal activity (Al-Rehaily et al., 2011). 2.2.4 Acacia etbaica schweinf .Acacia etbaica Schweinf subspecies etbaica within the family Leguminosae is a one of the most widespread plant in east Africa (Wood, 1997). Acacia etbaica has a wide occurrence, from semi-desert scrubto wooded grassland at altitudes that extend from 1200 to 2000 m. The tree is widely used to make the pillars and beams of earthen houses in Ethiopi (Phillips, 1995). The leaves of Acacia etbaica subspecies etbaica, are generally used as in folk medicine in different counties. Microscopic examination of acacia etbaica showed that the paracytic stomata and non-glandular, unicellular, curve trichomes are characterized for Acacia etbaica subspecies etbaica. Phytoconstituets like carbohydrates, glycosides, saponins, flavonoids, coumarins, tannins, triterpenes, sterols, amino acid and protein were presented; these compounds are known to have curative activity against several pathogens and therefore can be suggested for the treatment of different diseases. (Alferi et al 2014). 2.2.5 Aloe elegans tod / aloe vera Aloe vera (A. vera) L. is a cactus-like perennial plant belonging to family liliaceae, widely distribution in the tropical and subtropical regions of the world have been chosen in the present study (Lanjhiyana et al., 2011). Most of Aloe species are indigenous to Africa, but now have wide distribution in the tropical and subtropical regions of the world. They are 7 grown in warm climates, both as wild and cultivated plants, in countries in southern, eastern and northern Africa [Rai S. et al., 2011). The genus Aloe contains over 400different species with Aloe barbadensis Miller (A. vera), Aloe aborescens and, Aloe chinensis being the most popular. Aloe barbadensis Miller is considered to be the most biologically active (Bozzi A. et al., 2007). Traditionally, A. vera gel is used both, topically (treatment of wounds, minor burns, and skin irritations) and internally to treat constipation, coughs, ulcers, diab, headaches, arthritis, immune-system deficiencies (Bozzi et al., 2007). A. vera has been used throughout history in folk medicine as valuable ingredient for the food, pharmaceutical and cosmetic industries (Lanjhiyana et al., 2011). Fresh aloe juice from the inner leaf parenchyma contains 96% water, polysaccharides consisting mainly of D- glucose and D-mannose, tannins, steroid, enzymes, plant hormones, amino acids, vitamins and minerals (Mohamed, 2011). Leaf exudates and mucilaginous gel of A. vera possesses anti-inflammatory, antifungal, antibacterial, anticancer, antioxidant, cytoprotective, cardiac stimulatory and immune modulatory activities. 2.3 Importance of Milk in Human Health The use of milk and milk products as human food has got a very long history. The milk - as it is meant to be the first and sole food for offspring of mammals it is an almost complete food. It contains in a balanced form all the necessary and digestible elements for building and maintaining the human and animal body (Fekadu Beyene and Abrahamsen, 1994). In addition it contains immunoglobulin which protect the newly born against a number of diseases. Milk and milk products have an immune enhancing property as well, particularly for the benefit of immune suppressed people. In addition, milk contains various properties, which make it easy to convert into different milk products or to use it as an ingredient for other food items. Various human cultures have their own traditional ways of using milk and preparing different milk products (Feleke, 2003). 2.4 Compositional Quality and Characteristics of Milk The composition of milk is not constant, but shows a wide variation. In the first place the composition depends on the species of animal. But also within a species there is a big differences between breeds and individual animals within a breed. The composition might even change from day to day, depending on feeding and climate. But also during one 8 milking the first milk drop differs from the last milk drops (Firew et al., 2008). Average figures of the composition of milk from cows, sheep and goats are given in Table 1. Table 1: Composition of milk from different animals Nutrients Cow’s milk Goats milk Sheep milk Water 87.2 % 85.8 % 81.6 % Total Solids 12.8 % 14.2 % 18.4 % Fat 4.0 % 4.9 % 6.5 % Protein 3.4 % 4.3 % 6.7 % Lactose 4.5 % 4.1 % 4.3 % Ash (minerals) 0.9 % 0.9 % 0.9 % Source: Firew et al., 2008 Milk is a yellowish white non-transparent liquid. Fresh milk has a pleasant soft and sweet taste and carries hardly any smell. Consumer acceptance of milk is greatly affected by its flavour. There are several factors which may produce off flavours and/or odours in milk. Some of the more common causes of flavour and odour problems are feed and weed flavours, strong smelling plants, like wild onion or garlic, strong flavored feedstuffs such as poor quality silage, cow barn flavors from dung, etc. Milk obtained from a dirty or poorly ventilated environment or from improperly cleaned milking equipment can have bad odour/flavor. Rancid flavours are caused by excessive agitation of milk during collection and/or transport (Mogesse, 1990; Javaid et al., 2009). 2.5 Nutritional Value of Milk Milk is very tasteful and is an excellent source of high quality protein that can be digested easily. Milk also contains lots of important vitamins and minerals. In many countries milk and milk products provide 5 – 10 % of the total calories of the daily human diet. It represents one of the best natural sources of essential amino acids for human nutrition. (Javaid et al., 2009). Moreover, milk is an outstanding source of calcium and a good source of phosphorus. As these 2 elements play an essential role in building the bones and teeth in the body, it is clear that milk was included in the diet of humans in all their stages of life. In fact milk is the most important source of calcium in the diet of almost all people. These nutritional attributes have made milk a mainstay in the diet, particularly of growing 9 children. It is recommended to drink 3-4 glasses of milk per person per day (Caswell, 2003). 2.6 Hygienic Quality of Milk Milk when it emerges from a healthy udder contains only a very few bacteria. However, milk is a perishable product. It is an ideal medium for micro-organisms and as it is a liquid, it is very easily contaminated and invaded by bacteria. Almost all bacteria in milk originate from the air, dirt, dung, hairs and other extraneous substances. In other words, milk is mainly contaminated with bacteria during milking. It is possible to milk animals in such a clean way that the raw milk contains only 500 to 1,000 bacteria per ml. usually the total bacteria count after milking is up to 50,000 per ml. However, counts may reach several millions bacteria per ml. That indicates a very poor hygienic standard during milking and the handling of the milk or milk of a diseased animal with mastitis (O’Connor, 1994). Raw milk is one of the most suitable media for the growth of a wide variety of bacteria. Especially immediately after milking when it is almost at body temperature. However, milk contains a natural inhibitory system which prevents a significant rise in the bacteria count during the first 2 - 3 hours. If milk is cooled within this period to 4 ˚C, it maintains nearly its original quality. Timely cooling ensures that the quality of the milk remains good for processing and consumption. The bacterial load in fresh raw milk was less than 50,000 per ml when it reaches the collection point or processing plant. To prevent a too high multiplication of bacteria, the milk has to be produced as hygienic as possible and was cooled or heated at the earliest. Hygienic milk only originates from mastitis free and healthy animals. Cows suffering from a disease may secrete the pathogenic bacteria, which cause their disease, in the milk they produce. Consumption of raw milk therefore might be dangerous to the consumer. Some of these diseases, like tuberculosis, brucellosis and anthrax, can be transmitted to the consumer (FAO, 2003). 2.7 Bacteria in milk The major group of bacteria in milk is the group of lactic acid bacteria. These are able to use the lactose in the milk and convert it into lactic acid. The most important family in this group is the Streptococcus lactis. These multiply and grow very fast when the milk is kept at ambient temperatures after milking. The produced lactic acid causes the natural souring 10 of milk. The primary source of these bacteria is the environment: air, dust, dirty equipment and operators, etc. How soon the milk turns sour depends on the degree of contamination and on the temperature of the milk (Javaid et al., 2009). Therefore, proper cleaning and sanitizing procedures are essential to control the quality of milk. Cooling to a temperature of 4 ºC makes the bacteria inactive and prevents them to grow and produce the lactic acid (FAO, 2003). There are also types of micro-organisms which make use of other milk components, like the proteins and the milk fat. All this microbial activity deteriorates the milk quality. Therefore only fresh milk of tasteed quality was used as raw material to enable processing into high quality milk products. For this reason the dairy industry strictly controls the quality of the incoming milk from the dairy farmers. If the milk quality does not fulfill the set minimum quality standards, it is rejected. This means an economical loss to the farmer. Most countries have implemented special laws and regulations concerning the composition and hygienic quality of milk and milk products to protect both the consumers and the public health. When high counts become a problem it is generally due to one or more of the following reasons: improper cleaning of milking equipment (the most common cause, high bacteria counts in milk, improper cooling of milk and occasionally, a herd experiencing a high prevalence of bacterial infection (Feleke, 2003). 11 CHAPTER THREE: MATERIALS AND METHODS 3.1. Study Area The study was conducted in eastern zone of Tigray region, Northern Ethiopia. The eastern zone of Tigray covers about 6050 km2 with a total population of 830,503 (52% female and 48% male). It is divided into seven districts. It consists of about 414,408 cattle and 13,977 ha of irrigated area largely used for vegetable and fruit production. The elevation of districts in the zone ranges from 946 to 3,298masl. Annual rainfall is variable within a range of 420-689mm. Temperature ranges from 8 to 26°C (BoARD, 2014). Two districts, Kilte-Awlaelo and Ganta-Afeshum, were selected based on the history of application of herbal plants as fumigators of milk containers. The human population of the area is 830503. The cultivated area by rain are covers 94859.4 ha, and by irrigation 24256.2 grazing land 140994.2. Livestock population cattle 414408 small ruminant 1297203 and poultry 546472 bee colony 83624 (Zonal BoARD, 2012) Ganta-afeshum district/Adigrat: is about 117 km north of Mekelle, capital of Tigray National Regional state and located geographically at 14o24' and 14o21'N Latitude and 39o13' and 39o37'E Longitude. The study area is classified in to two agro-ecological zones: highland (3 Tabias) and middle land (17 Tabias). The altitude of the district ranges from 1800 to 3200 m.a.s.l. Agriculture is the main economic source of the district. Farmers practice mixed farming system comprising crop, livestock, and agro-forestry subsystems. Livestock husbandry is the main integral part of the farming system of the district (WoARD, 2014). According to the national agro-ecological zonation, the study area falls under the Central Cereal Production Zone, classified as wheat and barley production area with uni-modal rainfall pattern (USAID, 2000). The annual rainfall of the districts varies from 350 to 650 mm while the rainfall pattern is erratic and unpredictable. Out of the total annual rainfall greater than half falls between July and August. The mean minimum and maximum temperature ranges from 8 to 25 0C (WoARD, 2014). 12 Figure 1: Map of the study districts Kilte-awlaelo district/Wikro: is found at distance of 45 km north of Mekelle and geographically located between 13046' - 13059' N latitude and 390 36'-390 42' E longitude (WOARD, 2014). The district has a total area of 101,758 ha and the arable land accounts 19,809.5 ha. The altitude ranges from 1900 – 2460 m.a.s.l. The livelihood of inhabitants of the district fully depends on subsistent agriculture. The dominant types of agriculture in the district is mixed crop livestock farming system, but small scale rain fed crop farming is the main pillar of livelihood. Besides to the major rain fed farming, irrigation is also practiced in some areas of the district especially since 2005 due to the high attention given by the government to water harvesting and utilization. Annual temperature varies from 17- 23oC. Weina-Dega is the agro-climatic zone that prevails in the district which experiences frequent shortage of rain fall and hence moisture stress is the main problem for the agricultural production. The area exhibits uni-modal type of erratic and unreliable rainfall 13 distribution which occurs between June and August ranging from 350-450 mm (WOARD, 2014). 3.2 Survey on herbal Plants used for milk container fumigation A semi-open structured questionnaire was prepared, pre-tasted and translated into local language before administration. A total of 84 respondents, 52.4% (n=44) from Ganta- Afeshum and 47.6% (n=40) from Kilte-Awilaelo were involved in this interview. A systematic method of sampling were used to select dairy farm owners for the interview. The questionnaire survey focused on assessing the type of plants used for milk container fumigator, rank of the plants based on interviewee’s perception, reason why they categorize as grade one and others were main focus of the questioner (Annex I). 3.3 Study Plants Herbal plants used by smallholder dairy farmers as milk container fumigators to maintain the quality of milk were considered as study samples. The plants were chosen based on interviews and discussions with the local farmers. So that, the plants which were used frequently by the farmers to minimize spoilage of milk and for better aroma to the milk were used for the experiment. From a the herbal plants used in both districts, five herbal plants most commonly used by smallholder dairy farm owners were selected The plants used were Olea europaea L, Vernonia amygdalina, Solanum schimperianum hochst, Acacia etbaica schweinf, Aoe elegans (figure, 2, 3, and 4). These plants were used by the farmers for fumigation of milk containers and believed to serve as an extender of shelf life of milk and provide aroma to the milk. For all herbal plants the stem or branch parts are used for smokeing except Awilie (Olea europaea L) where the stem and leaf parts are used together for fumigation of the milk containers. 3.4 Herbal Plant Collection The collection of herbal plants from the study areas was done in consultation with the traditional milk preservative and processing practitioners. Collection practices were done in a way that ensures the long term survival of wild populations and their associated habitats. Only ecologically nondestructive systems of collection were applied which means 14 I have taken the plant sample without harming the future survival of the plant. So the stem, branch and leaf part of the selected plants were taken for experiment. 3.5 Plant Species Identification The collected plant specimens were coded by their local names and were transported by plant presser and plastic bag to avoid drying as well as for species identification purpose (Figure 7). After collection and drying, voucher specimens were identified by botany specialists in Mekelle University College of Natural and Computational Science, Biology Department for their scientific names. 3.6 Study Sample and Experimental Design In this study only milk taken from bovine was considered as study sample. The milk sample was taken from five different farms proven that the milk container is not fumigated. Smallholder dairy farms with more than three milking cows were considered as study farm and the milk was taken from the pulled milk container immediately after milking. The milk was collected from non-clinical mastitis cows. Milk sample collection was accomplished under hygienic condition to minimize an excessive contamination of the milk. The milk samples collected from the selected farms were poured in to 6 calabash and 6 plastic containers each with 200 ml of milk. Milk with untreated plastic and calabash containers was used as control group (Figure, 5). Five plastic and five calabash containers fumigated with each of the medicinal plants were used as the experimental groups. These herbal plants used for milk container smoker were taken as a treatment and then each plant was tasted in 5 batch of milk samples as replication for its effectiveness in maintaining the quality of milk according to the traditional use. 3.7 Milk Quality Parameters Organoleptic tastes (odor and taste), and physical tastes (temperature and pH) were carried out within 24 hours difference between the first and second days examination. The organoleptic test was done by three person for each batch or five times. The persons involving in the organoleptic test are those who have an experience of milking and milk fumigation. The scoring method was done as 1. Excellent 2. Very good 3. Good 4. Bad odor 5. Normal milk odor (Annex 4) 15 3.8 Fumigation method A fire from charcoal was prepared in which the fire checked for any smoke out from it. After proven that the fire doesn’t have a smoke out and then the dried herbal plant was inserted to the fire and then the fire was covered with aluminum foil with a small opening which can be used as an opening for the smoke outside. The dried milk containers were hold/suspend upside down to the smoke for 3-5 minutes. After I saw a smoke is come out from the milk container immediately putting the container on the ground and pour with a milk and then tightly clothed (Figure, 6) 3.9 Bacterial Load Determination Bacterial load determination was conducted for the control and experimental milk samples. Standard plate count (SPC) and coliform count methods were used for determining the number of total viable bacteria and coliforms in milk, respectively. Serial tenfold dilution up to 106 dilutions was prepared for each sample using 0.85% sterile saline solution. Pour on plate method was used to make viable count. After incubation of 24 and 48 hours, plates from the different dilutions having bacterial colonies ranging from 30 – 300 were counted using the illuminated colony counter. The counts for each plate were expressed as colony forming unit of the suspension (cfu/mL) (Quinn et al., 2002). All milk samples were kept at room temperature to replicate the traditional application of the plants. Milk from every batch was equally distributed to all treatments. 3.10 Bacterial Isolation Bacterial isolation from treated and control milk samples was done according Quinn et al. (2002) with some modification. A loopful of milk sample was streaked on tryptose blood agar base enriched with 7 % defibrinated sheep blood (Oxoid, UK) and MacConkey agar (Oxoid, UK) plates using the quadrant streaking method. Both agar plates were incubated aerobically at 37 °C for 24–48 h and examined for characteristic bacterial colonies. Pure culture colonies were selected and sub-cultured on general purpose medium, nutrient agar (Oxoid, UK), and incubated aerobically at 37 °C for 24–48 h for further biochemical identification. Bacterial organisms were identified according to their Gram reaction and morphology, catalase taste, triple sugar iron agar taste and urease taste. In addition, 16 mannitol salt agar was used to differentiate Staphylococcus aureus from other Staphylococcus spp. 3.11 Statistical analysis The Data collected was inserted to computer using SPSS Statistical software version 17.0. The data from questioner survey was analyzed using descriptive data analysis and the data from laboratory examination was analyzed using mean comparison T independent test, general leaner model multifactorial repeated two way ANOVA (post hock) analyzing method. Statistical model was (Intercept), treatment, location, container, days. 17 CHAPTER FOUR: RESULT 4.1 Questionnaire Survey Result From the 84 respondents interviewed, 81% (n=68) of them encounter milk spoilage in their farm. Regarding the frequency of milk spoilage, 63.2% (n=43) and 36.8% (n=25) of the respondents indicated a weekly and a daily milk spoilage, respectively. Only 11.8% (n=8) of the farms undergoes further processing or fermentation of the remaining spoilage (after raw milk consumption/sale) to avoid spoiled and 23.5% (n=16) respondents of the spoiled milk were given to pet animals the rest of the farms, 64.7% (n=44), the milk which is not used immediately could be spoiled and dumped. In 70.2% (n=59) of the farms were used herbal plants as fumigators of milk containers to prevent milk spoilage and increase its shelf life (Table 3). The local and scientific name as well as mode of preparation is described in (Table 2) Table 2: Type of herbal plants used for fumigation and their mode of preparation S/N Local name Scientific name Part of plant used Mode of preparation1 1. Awlie Olea europaea L. Leaf and stem A leaf and stem part of the plant is taken and washed with clean water and dried, after that put on a fire and the smoke will fumigate the milk container till it became warm. Finally, the milk will be poured in to the container immediately and then tightly clothed. 2. Grawa Vernonia amygdalina Stem A stem part of the plant is taken and washed with clean water and dried, after that put on a fire and the smoke will fumigate the milk container till it became warm. Finally, the milk will be poured in to the container immediately and then tightly clothed. 3. Qorenet Solanum schimperianum hochst Branch A branch part of the plant is taken and washed with clean water and dried, after that put on a fire and the smoke will fumigate the milk container till it became warm. Finally, the milk will be poured in to the container immediately and then tightly clothed. 4. Seraw Acacia etbaica schweinf Stem A stem part of the plant is taken and washed with clean water and dried, after that put on a fire and the smoke will fumigate the milk container till it became warm. Finally, the milk will be poured in to the container immediately and then tightly clothed. 5. Hatsat Aoe elegans Branch A branch part of the plant is taken and washed with clean water and dried, after that put on a fire and the smoke will fumigate the milk container till it became warm. Finally, the milk will be poured in to the container immediately and then tightly clothed. 1 The mode of preparation is according to the dairy farm owners experience 18 Table 3: Respondents idea on milk spoilage Variables Number Percentage Method of spoilage control (n = 84) Putting in cooler 25 29.8% Fumigation 59 70.2% Problem of milk spoilage (n = 84) Yes 68 81% No 16 19% Frequency of milk spoilage (n = 68) Daily 25 36.8% Weekly 43 63.2% Fate of spoiled milk (n = 68) Dumped 44 64.7% Given to pet 16 23.5% Fermented 8 11.8% In the study area smallholder dairy farm owners use the above mentioned herbal plants unselectively. However, the respondents were requested to rank these medicinal plants for their effectiveness and 41.2% (n=28) of them ranked Solanum schimperianum hochst as their first choice followed by Aoe elegans, Vernonia amygdalina, Olea europaea L, Acacia etbaica schweinf from 2nd – 5th, respectively. 38 (55.9%) respondents were believe that, fumigation of the milk container with the above herbal plants as a solution to prevent milk spoilage for two days. 51.5% (n=35) respondents had confirm that, the herbal plants has a better improvement both in odor and taste. In relation the impact 82.4% (n=56) interviewee confidentially witnessed for the positive impact of the herbal plants on the milk quality (Table 4). 19 Table 4: Use of herbal plants and the level of importance by the interviewee Variables Number Percentage Herbal plant type as first choice (n = 68) Olea europaea L 4th (n=8) 11.7% Vernonia amygdalina 3rd (n=10) 14.7% Solanum schimperianum hochst 1st (n=28) 41.2% Acacia etbaica schweinf 5th (n=6) 8.8% Aoe elegans 2nd (n=16) 23.5% Why the plants are best (n = 68) Good in odor 16 23.5% Good in taste 17 25% Good in odor and taste 35 51.5% Fumigation prevent spoilage (n = 68) One day 21 30.9% Two day 38 55.9% Three days 9 13.2% Fumigation has positive impact (n = 68) Yes 56 82.4% No 12 17.6% 4.2 Bacterial Load Determination Result Each of the plant materials were tested for the effectiveness in minimizing the bacterial count and for each treatment group five replica were made. In analyzing the result the mean of day one and day two were summed together. For the whole milk at day 0, temperature, pH, SPC and coliform counts were 25.4oC, 5.3, 20.4 x105/cfu/ml and 15.6 x105/cfu/ml, respectively. Milk samples from fumigated plastic containers showed statistically significant variation for both standard plate count and coliform counts (p = 0.000). The lowest bacterial count was obtained from milk samples fumigated by V. amygdalina for both standard plate count (7.8x105) and coliform count (4.5x105). The highest bacterial count was seen from the sample taken untreated or control groups (Table 5). 20 Table 5: Treatments Mean of milk samples stored in plastic containers Treatment groups Mean comparison with Standard Plate Count Mean comparison with Coliform Count Mean comparison with To Mean comparison with pH Mean ± SD P Value Mean ± SD P Value Mean ± SD P Value Mean ± SD P Value Control 16.4x105±4.95 0.0000 13.2x105±9.19 0.0000 22.22±1.21 0.6060 3.07±0.27 0.0976 O. europaea L 9.1x105±5.65 7.3x105±7.07 21.60±0.85 2.80±0.28 V. amygdalina 7.8x105±7.07 4.5x105±7.78 21.64±0.25 2.84±0.02 S. schimperianum hochst 11.4x105±1.41 9.8x105±2.12 21.87±0.18 2.61±0.09 A. etbaica schweinf 10.2x105±11.31 9.2x105±4.24 21.34±0.48 2.78±0.03 A. elegans 10.6x105±7.78 10x105±4.42 21.56±.0.74 2.61±0.18 Table 6: Treatments Mean of milk samples stored in calabash Treatment groups Mean comparison with Standard Plate Count Mean comparison with Coliform Count Mean comparison with To Mean comparison with pH Mean ± SD P Value Mean ± SD P Value Mean ± SD P Value Mean ± SD P Value Control 16.8x105±2.83 0.0000 12.7x105±0.78 0.0000 21.50±0.98 0.5719 3.26±0.22 0.0658 O. europaea L 9.4x105±5.66 7.3x105±9.19 20.75±0.49 3.16±0.01 V. amygdalina 8.4x105±3.54 4.7x105±7.07 20.90±0.70 2.94±0.09 S. schimperianum hochst 11.9x105±7.07 9.9x105±9.19 21.09±0.01 2.92±0.01 A. etbaica schweinf 10.8x105±2.83 10.4x105±2.82 21.10±0.14 3.02±0.03 A. elegans 11.3x105±5.66 10x105±5.66 21.34±.0.84 2.70±0.03 21 Similarly, milk samples from fumigated calabash containers showed statistically significant variation for both standard plate count and coliform counts (p = 0.000). The lowest bacterial count was obtained from milk samples fumigated by V. amygdalina for both standard plate count (8.4x105) and coliform count (4.7x105). The highest bacterial count was seen from samples of the untreated or control groups (Table 6). Considering temperature and pH as factors for the treatments, no statistically significant variation was observed between the different treatment groups for both milk samples stored in plastic and calabash containers (Table 5 and 6). Mean comparisons of bacterial counts were also conducted in standard plate count and coliform count at different storage days taking all treatments as one. The effects of temperature and pH in relation to storage time were also analyzed. Similar to the above analysis, for the whole milk at day 0 temperature, pH, SPC and coliform counts were 25.4oC, 5.3, 20.4x105/cfu/ml and 15.6 x105/cfu/ml, respectively. Mean comparison of day one and day two of standard plate count (P = 0.6094) and coliform count (P = 0.6384) from milk sample stored in plastic containers didn’t show statistically significant association (Table 7). Moreover, mean comparison of day one and day two of standard plate count (P = 0.7096) and coliform count (P = 0.6365) from milk sample stored in calabash containers didn’t show statistically significant association (Table 8). However, temperature showed a statistically significant relation with storage times both in plastic (P = 0.0141) and calabash (P = 0.0466) stored milk samples (Table 7 and 8). 22 Table 7: Mean comparison of milk from plastic containers on the basis days Days of storage Mean comparison days in Standard Plate Count Mean comparison days in Coliform Count Mean comparison days in To Mean comparison days in pH Mean ± SD P Value Mean ± SD P Value Mean ± SD P Value Mean ± SD P Value Day one 11.4x105±28.9 0.6094 9.4x105±29.10 0.6384 22.09±0.52 0.0141 2.88±0.22 0.1212 Day two 10.5x105±20.27 8.6x10±29.27 21.31±0.39 2.69±0.17 Mean Total 10.9x105±28.57 9x10±28.15 21.70±0.60 2.78±0.21 Table 8: Mean comparison of milk from calabash containers on the basis days Days of storage Mean comparison days in Standard Plate Count Mean comparison days in Coliform Count Mean comparison days in To Mean comparison days in pH Mean ± SD P Value Mean ± SD P Value Mean ± SD P Value Mean ± SD P Value Day one 11.8x105±29.08 0.7096 9.5x105±26.13 0.6365 21.38±0.42 0.0466 3.04±0.25 0.5438 Day two 11.1x105±29.68 8.8x105±29.45 21.85±0.40 2.10±0.16 Mean Total 11.4x105 ±28.21 9.1x105±26.86 21.11±0.48 2.99±0.20 23 In addition, mean comparisons of bacterial counts were made in standard plate count and coliform count considering the storage containers. However, the association in both cases was insignificant. Temperature (P = 0.0147) and pH (P = 0.0167) showed statistically significant association with the type of container (Table 9). In this case also all treatments were considered as one. The mean temperature in calabash and plastic containers was 21.11±0.48 and 21.10±0.60, respectively. The mean pH in calabash and plastic containers was 2.99±0.20 and 2.78±0.21, respectively. Table 9: Mean comparison of milk samples in calabash and plastic containers Container type Mean comparison on Standard Plate Count Mean comparison on Coliform Count Mean comparison in To Mean comparison in pH Mean ± SD P Value Mean ± SD P Value Mean ± SD P Value Mean ± SD P Value Calabash 11.4x105±28.21 0.6551 9.1x105±26.86 0.8834 21.11±0.48 0.0147 2.99±0.20 0.0167 Plastic 10.9x105±28.57 9x105±28.15 21.10±0.60 2.78±0.21 Total 11.1x105±27.90 9.1x105±26.92 21.41±0.61 2.89±0.21 Milks samples stored in calabash containers had excellent taste and odor in both day’s (day one and day two) compared to milk samples stored in plastic containers (Table 10). But all plants had very nice aroma which has their own nature. In relation to the days, at day one the smell and taste was very strong than day two. Table 10: Organoleptic taste result Container type Days Organoleptic Day one Day two Control x x Odor Control x x Test Calabash xxxx xxx Odor Calabash xxxx xxx Taste Plastic xxx xx Odor Plastic xxx xx Taste N.B. xxxx- excellent, xxx- very good, xx-good, x- normal milk odor 24 The comparative analysis of the control group with the treatment group was revealed for the statistically significant difference with the control group in their bacterial load (Table 11). The highest mean difference was encountered from milk samples treated by Vernonia amygdalina and the lowest mean difference was seen in milk samples treated Solanum schimperianum hochst. According to the mean difference, ranking of the herbal plants on their ability to decrease the bacterial count in milk was performed and the plants are listed from first to the least as: Vernonia amygdalina, Olea europaea L, Acacia etbaica schweinf, Aoe elegans, Solanum schimperianum hochst. In this analysis, mean of day one and day two were summed together. Table 11: Mean comparison of treatments Group 1 Group 2 Mean difference P value S.E Control Olea europaea L b65.50 0.001 6.279 Vernonia amygdalina a86.15 0.000 Solanum schimperianum hochst e41.85 0.008 Acacia etbaica schweinf c50.65 0.003 Aoe elegans d44.80 0.006 Olea europaea L Control -65.50 0.001 6.279 Vernonia amygdalina 20.65 0.250 Solanum schimperianum hochst -23.65 0.140 Acacia etbaica schweinf -14.85 0.838 Aoe elegans -20.70 0.247 Vernonia amygdalina Control -86.15 0.000 6.279 Olea europaea L -20.65 0.250 Solanum schimperianum hochst -44.30 0.006 Acacia etbaica schweinf -35.50 0.020 Aoe elegans -41.35 0.009 S. schimperianum Control -41.85 0.008 6.279 Olea europaea L 23.65 0.140 Vernonia amygdalina 44.30 0.006 Acacia etbaica schweinf 8.80 1.000 Aoe elegans 2.95 1.000 Acacia etbaica Control -50.65 0.003 6.279 Olea europaea L 14.85 0.838 Vernonia amygdalina 35.50 0.020 Solanum schimperianum hochst -8.80 1.000 25 Aoe elegans -5.85 1.000 Aoe elegans Control -44.80 0.006 6.279 Olea europaea L 20.70 0.247 Vernonia amygdalina 41.35 0.009 Solanum schimperianum hochst -2.95 1.000 Acacia etbaica schweinf 5.85 1.000 The mean difference is x104 a reference to the best herbal plant and b the next and e the least The bacterial organisms isolated from the different treatment groups are summarized in Table 14. Shigella organism was the most commonly isolated organisms and Staphylococcus organisms were also isolated 4.3 Bacterial Isolation Table 12: Bacterial isolates from the different treatment groups Treatments MacConkey agar Blood agar Salmonella Shigella agar Manitol salt agar Control +++++ +++++ +++++Sh2 + Olea europaea L +++++ +++++ +++++Sh + Vernonia amygdalina +++++ +++++ +++++Sh + Solanum schimperianum hochst +++++ +++++ +++++Sh + Acacia etbaica schweinf +++++ +++++ +++++Sh + Aoe elegans +++++ +++++ +++++Sh + 1 +++++ means positive in the five samples; +++++Sh means shigella not salmonella; + means only in one sample was staphylococcus grown 26 CHAPTER FIVE: DISCUSSION 5.1. Perception of respondents From the herbal plants used in both districts, five herbal plants most commonly used by smallholder dairy farm owners were selected. These plants are Awilie (Olea europaea L), grawa (Vernonia amygdalina), Qorenet (Solanum schimperianum hochst), Seraw (Acacia etbaica schweinf), Hatset (Aoe elegans). These plants were used by the farmers for fumigation of milk containers and believed to serve as an extender of shelf life of milk and provide aroma to the milk. Similar finding that agrees with this from previous studies which showed milk can be preserved by fumigation of milk containers and fumigation is a common traditional practice carried out by pastoralists for the sake of improving milk quality and aroma in Kenya by (Wayua et al., 2009) and (Kipsang, 2011). From the 84 respondents interviewed, 81% (n=68) of them encounter milk spoilage in their farm. Regarding the frequency of milk spoilage, 63.2% (n=43) and 36.8% (n=25) of the respondents indicated a weekly and a daily milk spoilage, respectively. These figures are indicators of a serious milk spoilage problem to smallholder dairy farms as most of these farmers don’t have a refrigeration facility to maintain the quality of milk. Only 11.8% (n=8) of the farms undergoes further processing or fermentation of the remaining spoilage (after raw milk consumption/sale) to avoid spoiled and 23.5% (n=16) respondents of the spoiled milk were given to pet animals the rest of the farms, 64.7% (n=44), the milk which is not used immediately could be spoiled and dumped. This is clearly showed that there is a massive loss of milk due to spoilage as a result significant economic loss on dairy industry was happened this could contribute as a negative factor to expand dairy farm in the area. 70.2% (n=59) of the farms were used herbal plants as fumigators of milk containers to prevent milk spoilage and increase its shelf life. So majority of the farm owners those who use the herbal plant as fumigator has less spoilage if they use the herbal plant properly. In the study area smallholder dairy farm owners use the above mentioned herbal plants unselectively. However, the respondents were requested to rank these medicinal plants for their effectiveness and 41.2% (n=28) of them ranked Solanum schimperianum hochst as 27 their first choice followed by Aoe elegans, Vernonia amygdalina, Olea europaea L, Acacia etbaica schweinf from 2nd – 5th, respectively. The reason why the plants are ranked as best was based on odor and taste of the milk. In addition respondents explain that, the trend they have about the continuous use of the plants was receive from their ancestors and they need to keep that so Solanum schimperianum hochst was most common for a long period of time that’s why it is the most used plant in the area. Long time familiarity of the smoke can contribute to the first choice of the plant. 38 (55.9%) respondents were believe that, fumigation of the milk container with the above herbal plants can be used as a solution to prevent milk spoilage for two days. 51.5% (n=35). This was confirmed with bacterial count done in this study with concert evidence of decreasing bacterial count due to smoke. And also respondents had confirm that, the herbal plants has a better improvement both in odor and taste. This is due to the aromatic nature of the herbal plants. In relation to the impact 82.4% (n=56) interviewee confidentially witnessed for the positive impact of the herbal plants on the milk quality. This concept was supported by previous research done by (Ashenafi, 1996) shows that according to the local understanding of dairy farm owners, smoking of milk containers imparts special taste and odour to the milk, and disinfects the containers, thus reducing the loads of microorganisms, thereby extending the shelf life of milk. In addition a faster development of aerobic mesophilic microorganisms occurred in milk kept in non-smoked as compared to smoked containers. 5.2.Bacterial load determination The selected five plants were tested for the effectiveness in minimizing the bacterial count. And for each treatment group five replica were made. During analysis to obtain the mean each plant, day one and day two were summed together. For the whole sampled milk (day 0) temperature, pH, SPC and coliform counts were 25.4oC, 5.3, 20.4 x105/cfu/ml and 15.6 x105/cfu/ml, respectively. Milk samples from fumigated and control on plastic containers showed statistically significant variation for both standard plate count and coliform counts (p = 0.000). This indicates that there is a difference on bacterial load level because of the fumigation. The lowest bacterial count was obtained from milk samples fumigated by V. amygdalina for both standard plate count (7.8x105) and coliform count (4.5x105). The 28 highest bacterial count was seen from the sample taken untreated or control groups. Similarly, milk samples from fumigated calabash containers showed statistically significant variation for both standard plate count and coliform counts (p = 0.000). The lowest bacterial count was obtained from milk samples fumigated by V. amygdalina for both standard plate count (8.4x105) and coliform count (4.7x105). The highest bacterial count was seen from samples of the untreated or control groups. This result has similarity from a research result which is previously done by (Onyango, 2011) Total plate counts increased constantly in all the milk samples up to the 14th day. However, there was a significant difference between the fermented milk samples without any treatment with plate counts of 10.00 – 11.40 log cfu/ml and the three herbs Lippia javanica, Olkingiri and Olea european. The fermented milk treated with Olea europaea had the lowest total plate counts of 5.05 - 8.38 log cfu/ml at P<0.05 level of significance. The total plate counts for the fermented milk treated with plant extracts fell within the expected range in cultured milk, which is 106 – 109 (Mathara et al., 1995). Considering temperature and pH as factors for the treatments, no statistically significant variation was observed between the different treatment groups for both milk samples stored in plastic and calabash containers. So PH as well as temperature do not influenced due to the herbal plant fumigated and non-fumigated and in between the plants too. Comparisons of bacterial counts between the treatments were conducted in different storage days by taking all treatments as one. Mean comparison of day one and day two of standard plate count (P = 0.6094) and coliform count (P = 0.6384) from milk sample stored in plastic containers and standard plate count (P = 0.7096) and coliform count (P = 0.6365) from milk sample stored in calabash containers didn’t show weak statistically significant association. However, temperature showed a statistically significant relation with storage times both in plastic (P = 0.0141) and calabash (P = 0.0466) stored milk samples. So a milk which is treated with herbal plant stored for one day or for two day doesn’t have visible and meaningful change on bacterial load of the milk. In addition, there is no difference on improving the quality and microbial safety of the milk due to container difference which is in calabash as well as in plastic milk containers. This is revealed by the mean comparisons of bacterial counts were made in standard plate count and coliform count 29 considering the storage containers. However, the association in both cases was insignificant. Were as Temperature (P = 0.0147) and pH (P = 0.0167) showed statistically significant association with the type of container. In this case also all treatments were considered as one. The mean temperature in calabash and plastic containers was 21.11±0.48 and 21.10±0.60, respectively. The mean pH in calabash and plastic containers was 2.99±0.20 and 2.78±0.21, respectively. In calabash due to its nature which can resist by being influenced by the external air condition the milk in calabash was slightly with low temperature and high PH. Milks samples stored in calabash containers had excellent taste and odor in both day’s (day one and day two) compared to milk samples stored in plastic containers (Table 10). But all plants had very nice aroma which has their own nature. In relation to the days, at day one the smell and taste was very strong than day two. This is because the smoke in calabash can intermingle with the calabash itself so this can help on maintain the aroma for a long time in comparative to plastic in which can’t hold the smoke for a long period. The comparative mean analysis of the treatments control group with the treatment group was revealed for the statistically significant difference with the control group in their bacterial load. The highest mean difference was encountered from milk samples treated by Vernonia amygdalina and the lowest mean difference was seen in milk samples treated Solanum schimperianum hochst. According to the mean difference, ranking of the herbal plants on their ability to decrease the bacterial count in milk was performed and the plants are listed from first to the least as: Vernonia amygdalina, Olea europaea L, Acacia etbaica schweinf, Aoe elegans, Solanum schimperianum hochst. In this analysis, mean of day one and day two were summed together. This is due to the nature of Vernonia a previous research result indicates that, Vernonia amygdalina is used as an antibacterial, laxative and acts as an antioxidant (Georgewill, et al., 2009). 30 5.3.Bacterial isolation The bacterial organisms isolated from the different treatment groups in total Shigella organism was the most commonly isolated organisms from the salmonella shigella agar culture This is due you to the reason that Shigella spp. grow in a pH range of 5–9 (ICMSF 1996). (Zaika, 2001) demonstrated that S. flexneri is tolerant to acid and can survive at pH 4 for 5 days in broth when incubated at 28°C. Shigella spp. are better able to survive lower pH conditions at reduced temperatures. 31 CHAPTER SIX: CONCLUSION AND RECOMMENDATIONS The present study five herbal plants commonly used as milk container fumigator in the study area these are Olea europaea L, Vernonia amygdalina, Solanum schimperianum hochst, Acacia etbaica schweinf, Aoe elegans. The lowest bacterial count was obtained from milk samples fumigated by Vernonia amygdalina on standard plate count as well as on coliform count. So Vernonia amygdalina was identified as the best in improving the quality and microbial safety of milk. Herbal plant used for fumigation of milk container used in plastic as well as in calabash doesn’t have a difference in improving the milk quality but milk in calabash has an excellent taste and odor in compared to plastic containers. In this study all herbal plants used for milk container fumigation has very nice aroma with their exclusive natural taste and they can significantly able to decrease the bacterial load of milk. The smoke in calabash can intermingle with the calabash itself so this can help on maintain the aroma for a long time in comparative to plastic in which can’t hold the smoke for a long period. Generally milk treated with herbal plant through fumigation has a better quality than the milk which is untreated. Based on the above conclusion the following recommendations are forwarded  The research result is obtained from two districts only herbal plants used for milk fumigation has to asses and tasteed in the whole region.  Vernonia amygdalina found to be the best herbal plant so further investigation has to be done other than the traditional method (extraction of the active ingredient and incorporate with milk for preservation).  Herbal plants are degraded from time to time due to deforestation and global warming so conservation of herbal plants should be done undertaken to be used for all kind of food preservation. 32 CHAPTER SEVEN: REFERENCES Alferi Saleh Kassem1*, Algifri A Naser2, Alshakka Mohammed Ahmed3, Taleb Muna1, Munaiem Ramzi Tariq, 2014 Anatomical and Phytochemical Studies of the Leaves of Acacia etbaica subspecies Etbaica. Al-Rehaily, J.A., Ahmad, M.S., Mustafa, J., Al-Oquil, M., Hassan, W.H., Khan, S.I., Lhan, I.A., 2011. 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The effect of temperature and low pH on survival of Shigella flexneri in broth. Journal of Food Protection 64(8):1162–1165 Zarzuelo, A., 1991. Vasodilator effect of olive leaf. Planta Medica, 57, 417–419. 38 CHAPTER EIGHT: ANNEXES Annex I. Questioner survey on herbal plant used for milk preservative I. General information CODE _________ 1. Name of the respondent ____________________ 2. Date of interview _____________ Name of enumerator_______________ 3. Town __________Zone _____________Sub city _________________ 4. Cell phone number (if any) ________________ II. Technical questions 1. Do you encounter problem of milk spoilage? 1= Yes 2= No 2. If yes, how frequent the spillage problem is in your household? 1. Every day 2. Every week 3. Every two weeks 4. Every three weeks 5. Every month 3. What was the fate of spoiled milk? 1. Dump 2. Given to pate animals 3. Fermentation and further processing 5 Other, specify ________________ 4. What methods do you use to minimize spoilage of milk and milk products? 1. Store at cooler place 2. Store in refrigeration 3. Use traditional milk fumigation 4. Others, specify_____________________ a. If you use traditional preservative method what are the plants used for preservation. A. Plant preservative specify____________________ S/N Type of plant used for milk preservative Part used (leaf, stem, root, fruit, seed, whole, etc ) 1. 2. 3. 5. Which one do you think the best? ___________________________ 6. Why do you think it is the best?_______________________ 7. For how long have you been used? 1. Less than 1 year 2. 1-3 years 3. More than 3 years 8. What is the procedure used for the herbal plant to be used as milk preservative? 39 Name of the plant 1. ________________________________ (001) 2. ________________________________ (002) 3. _________________________________ (003) Steps (For enumerator (put the plant code number above the steps) 1. _______________________________ 2. _______________________________ 3. _______________________________ 9. Does it have any positive or negative impact on the quality and safety of milk? Explain? ____________________________________________ 10. Do you think the herbal plants you use have a preservative effect? A. yes B. No if yes? How long can be preserve milk preservation method? You can explain about each and different herbal plants. __________________________________ 11. What is the status of most useful milk preserving herbs? a. Decreasing b. Increasing c. Stable 12. If decreasing, list three important apparent causes? ______________________ 40 Annex II Table 13: Bacteriological taste format S/N Bacteriological taste Plant type Whole sample milk a day zero Sample no ……. Day one Type milk container Day two Type milk container Clash plastic Clash plastic 1. Plat count Control 2. Coliform count 3. Plat count Olea europaea L 4. Coliform count 5. Plat count Vernonia amygdalina 6. Coliform count 7. Plat count Aoe elegans 8. Coliform count 9. Plat count Solanum schimperianum hochst 10. Coliform count 11. Plat count Acacia etbaica schweinf 12. Coliform count 41 Annex III:- Table 14: Milk quality taste format S/N Milk taste type Plant type Whole sample milk a day zero Sample no ……. Day one Type milk container Day two Type milk container Clash plastic Clash plastic 1. Temperature Control 2. PH 3. Temperature Olea europaea L 4. PH 5. Temperature Vernonia amygdalina 6. PH 7. Temperature Aoe elegans 8. PH 9. Temperature Solanum schimperianum hochst 10. PH 11. Temperature Acacia etbaica schweinf 12. PH N. B:- Grading system 1. Excellent 2. Very good 3. Good 4. Bad odor 5. Normal milk odor 42 Annex IV:- Table 15: Organoleptic taste format S/N Organoleptic taste Plant type Whole sample milk a day zero Sample no ……. Day one Type milk container Day two Type milk container Clash plastic Clash plastic 1. Odor Control 2. Taste 3. Odor Olea europaea L 4. Taste 5. Odor Vernonia amygdalina 6. Taste 7. Odor Aoe elegans 8. Taste 9. Odor Solanum schimperianum hochst 10. Taste 11. Odor Acacia etbaica schweinf 12. Taste 43 Figure 2: Olea europaea L (left) and Vernonia amygdalina (right) Figure 3: (S. schimperianum hochst (left) and Acacia etbaica schweinf (right) 44 Figure 4: Aoe elegans Figure 5. Labelled Calabash and plastic milk containers 45 Figure 6. During fumigation Figure 7. Plant collection for species identification