Spices and their effect on the acceptability and microbial activity of milk and milk products

TABLE OF CONTENTS

DEDICATION

STATEMENT OF THE AUTHOR

ACKNOWLEDGEMENT

ABBREVIATIONS AND ACRONYMS

LIST OF TABLES

LIST OF FIGURES

LIST OF TABLES IN THE APPENDIX

ABSTRACT

1. INTRODUCTION
1.1. Background and Justification
1.2. Statement of the Problem
1.3. Research Objectives
1.4. Significance and Scope of the Study

2. LITERATURE REVIEW
2.1 Dairy Production in Ethiopia
2.2. Handling Practices of Milk and Milk Products in Ethiopia
2.3. Factors that Affect Storage Quality of Dairy Products
2.4. Functional Properties of Spices and Herbs
2.5. Spoilage Microorganisms of Dairy Products
2.6. Microbial Quality of Ayib and Ghee
2.7. Physico - chemical Properties of Ayib and Ghee
2.8. Consumer Acceptability of Dairy Product
2.9. Spices Used in Ayib and Ghee Production Process in Ethiopia
2.10. Consumption of Milk and Milk Products in Ethiopia

3. MATERIALS AND METHODS
3.1. Description of the Study Area
3.2. Study Design
3.2.1. Survey
3.3. Laboratory experiment
3.3.1. Preparation of Spice powder
3.3.2. Preparation of Ayib
3.3.3. Preparation of Ghee Sample
3.5. Treatment arrangement
3.7. Experimental design
3.8. Microbial analysis
3.8.1. Total Bacterial count
3.8.2. Yeast and Mold Count
3.8.3. Coliform count
3.82. Physio-Chemical Analysis
Total solids
Moisture content
Ash
Fat
PH
3.8.3. Consumer acceptability
3.84. Data and Statistical analysis

4. RESULTS AND DISCUSSIONS
4.1. Socio -Demographic and Economic Characteristics of Study Households
4.2. Dairy Cattle Production System in the Study areas,
4.2.1.Cattle Breeds
4.2.2. Reproductive Performance of Dairy Cattle
4.3. Milking and Storage Practices
4.4. Milk Production and Processing
4.5. Milk Processing
4.6. Use of Spices in Dairy Products
4.6.1. Fermented milk (Ergo)
4.6.2. Butter Milk (Arrera)
4.6.3. Traditional Butter (Kibe) and Ghee (Nitir Kibe)
4.6.4. Cottage cheese (Ayib)
4.7. Consumption and Utilization of Dairy Products
4.8. Constraints for Milk Production and Processing
4.9. Physico-chemical Properties and pH of Ayib
4.10. Antimicrobial activity of spices on Ayib
4.11. Consumer Acceptability of Ayib
4.12. Proximate Composition of Ghee Sample
4.13. Microbial quality of Ghee Samples
4.14. Consumer acceptability of Ghee samples

5. CONCLUSIONS AND RECOMMENDATIONS
5.1. Conclusions
5.2. Recommendations

6. REFERANCES
APPENDICES I
APPENDICES II
APPENDICES III
APPENDICES IV
APPENDICES V
APPENDICES VI
APPENDICES VII

BIOGRAPHICAL SKETCH

DEDICATION

I dedicated this thesis manuscript to memory of my father Yohannes Abula and my mother Adanach G/Wold for their unforeseen to my success.

STATEMENT OF THE AUTHOR

I, the undersigned, declare that this thesis is my original work and has not been presented for a Degree in any other University, and that all sources of materials used for the thesis have been properly acknowledged. First of all, I declare that this thesis is my work and that all sources of materials used for this Thesis have been properly acknowledged. This Thesis has been submitted in partial fulfillment of the requirements for Masters (MSc.) degree at Hawassa University and deposited at the University Library to be made available to borrowers under rules of the Library. I seriously declare that this thesis is not submitted to any other institution anywhere for the award of any academic degree and diploma.

Brief quotations from this thesis are allowable without special permission provided that accurate acknowledgment of source was made. Request for permission for extended quotation from or any part may be granted by head of school of Animal and range science and head of school of graduate studies.

ACKNOWLEDGEMENT

First of all I thank the Almighty God for his mercy and love, also giving me strength and to breathe during difficult time spent up on completion of this thesis work. I would like to sincerely acknowledge the following people and institutions. First and foremost, I would like to thank my supervisor Sintayehu Yigrem (PhD) for his advice, guidance, support and encouragement throughout my thesis work. Again for his follow up and keen interest in my work, constant supervision, and insightful critiques of drafts. I would like to express my deepest gratitude to my co-supervisor Mestawot Taye (PhD) for the timely advice on the way of study. I am greatly acknowledging the Hadero Tunito Zurie Woreda Agricultural office for the time they allot from starting up to the end of my study. I am grateful of thank developmental worker Tasawu Bulado and Muligeta and their other colleagues supporting and giving detailed information before and during data collection about Keble’s. I also would like to thank sanago my friend who helped me at experiment and indispensable friends who gladly shared my responsibilities when I was in need of it, finally I would also like to express my appreciation and heartfelt thanks to my family.

ABBREVIATIONS AND ACRONYMS

Abbildung in dieser Leseprobe nicht enthalten

LIST OF TABLES

Table 1 .Dairy products and typical types of spoilage microorganisms or microbial activity

Table 2.Treatment arrangement for ayib sample

Table 3. Treatment arrangement for ghee

Table 4: Socio-demographic and Economic status of study households (n=60)

Table 5: Dairy Cattle breed type in the study areas (% within breed)

Table 6: The reproductive performance of the breeds in study site

Table 7: Milk hygienic practices

Table 8: Plants or herbs used by respondents for washing and smoking of utensils

Table 9: Milk production per day per house-hold(N=60)

Table 10: Souring time for Churning of the households (N=60)

Table 11: Volume of milk churned by respondents (N=60)

Table 12: Common herbs and spices used during ghee production in study sites

Table 13: Acceptable time of consumption for ghee by the consumers (n=60)

Table 14: Shelf stability for cottage cheese consumption (n=60)

Table 15: Physico-chemical properties of cheese from different treatments. (n=7)

Table 16: The effect of treating deferent levels of spice mix on microbial content of ayib (in log10 cfu/g)

Table 17. Sensory acceptability of ayib samples with the inclusion of spices powder mixture at different levels based on hedonic scale (n=25).

Table 18: Effect of spices treatment on ghee composition and pH (n=7)

Table 19: Microbial activity.(n=7) of Ghee samples treated with deferent levels of spice

Table 20: Sensory acceptability of based of stored ghee samples (n=33)

LIST OF FIGURES

Figure 1 Effect of the microbial initial contamination on shelf-life in a food

Figure. 2. Ayib and Ghee making flow charts

Figure 3. Trend in pH development of stored ayib from different treatments

LIST OF TABLES IN THE APPENDIX

Table 1.Pearson’s Correlations among Proximate Analysis and Microbial Activity Measure Parameters of Ghee Sample

Table 2.Pearson’s Correlations among Proximate Analysis and Microbial Activity Measure Parameters of cottage cheese sample

Table 3.ANOVA table for quality attributes of ghee sample versus weeks

Table 4.ANOVA table for quality attributes of cottage cheese sample versus days

The Effect of Spices on Microbial Activity and Acceptability of Milk and Milk Products

By: Natineal Yohannes

Advisor: Sintayehu Yigrem (PhD) and Co-advisor: Mestawot Taye (PhD)

ABSTRACT

Spices have been used by human being for thousands of years having multipurpose functional roles such as medicinal use, preservation of food, improving the flover and aroma of foods- including dairy products. This is also widely used in Ethiopian dairy products. This study was conducted in Tambaro woreda and Hosanna destrict to assess the traditional practices, types of spices and herbs used in preserving various dairy products in the study area, and to identify the effect of spices on cottage cheese (ayib) and ghee microbial activities and acceptability under controlled laboratory experiments. The study also explored the potential implications of spice uses towards the sensory properties of these dairy products. The experimental study was conducted as a follow-up to diagnostic survey. Ayib and Niter kibe (which are analogues to cottage cheese and ghee, respectively), were identified as main dairy products which farmers add spices and herbs as common practices in the study area. The cottage cheese were randomly assigned to either of three spice treatments Kororima and Tikur azimud, or Kororima and Netch azimud mixtures (equal ratio) at 0, 1, 3 and 5% levels by weight concentration, respectively. The ghee samples were randomly assigned to either of four spice treatments Kororima and Abish, or Tikur azimud and Netch azimud mixtures (equal ratio) at 0, 1, 3 and 5% levels by weight concentration, respectively. Cottage cheese samples were stored for 6 days and ghee samples for 30 days at ambient temperature before parameter analysis. Cottage cheese samples were analysed every 24 hrs, while the ghee samples were analysed once in a week for compositional, microbial and sensory properties, which included total solids, ash, fat content, pH, total bacterial count, coliform, and yeast and mould counts. The sensory analysis included flaver, aroma, taste and overall acceptability of ghee and cottage cheese samples. Data was analyzed using SPSS version.16. The most widely used spices and herbs applied to add flavour and/or extend the shelf of ghee in the study area were Nigella Sativa(Tikur azimud), Trachyspermum Copticum(Netch azimud), Aframomum Corrorima (Kororrima), Zingiber Offcinale (Ginger), Ocimum hardiense(Kosert) and Trigonella Foenum Graecum(Abish). The most widely used spices added onto cottage cheese included Aframomum Corrorima(Kororima), Nigella Sativa(Tikur Azimud), Trachyspermum Copticum (Netch azimud), Allium sativum(Netch shinkuret) and Capsicum spp(chilly). The experimental study highlighted that cottage cheese samples treated with 3% of Aframomum Corrorima and Trachyspermum Copticum mixtures received the highest acceptability and lower YAM count(5.746cfu/g ) and Coliform counts (1.792 cfu/g ). The mean microbial load is higher on untreated ayib sample ( 6.40 cfu/g YAM and 1.95cfu/g Coliform counts ). Coli forms were absent in all ghee samples. Ghee sample treated with 5% of Kororrima and Abish mixtures revealed relatively higher overall acceptability. Usage of natural extract of spices and herbs is safe and promising alternative for consumer preference, preservation and shelf life extension of the dairy products.

Key words: Antimicrobial, Cottage cheese, Ghee, Microbial activity, Sensory acceptability, Spices

1. INTRODUCTION

1.1. Background and Justification

Strong consumer demand for safe and high-quality foods can be attributed in part to the widespread availability and accessibility of quality health data and information. There are also new concerns about food safety due to increasing occurrence of new food-borne disease outbreaks caused by pathogenic micro-organisms. This raises considerable challenges, particularly since there is increasing unease regarding the use of chemical preservatives and artificial antimicrobials to inactivate or inhibit growth of spoilage and pathogenic micro-organisms (Tajkarimi et al., 2010).

Spices and herbs have been used for thousands of centuries by many cultures to enhance the flavor and aroma of foods. Early cultures also recognized the value of using spices and herbs in preserving foods and for their medicinal value. Studies in the past decade confirm that the growth of both gram-positive and gram-negative food borne bacteria, yeast and molds can be inhibited by garlic, onion, cinnamon, cloves, thyme, sage, and other spices. Essential oils extracted from spices and herbs are generally recognized as containing the active antimicrobial compounds. Studies conducted in Germany (Grohs et al., 2000 and Grohs & Kunz, 2000) with regard to possibilities of using spice mixtures with an objective of extending meat shelf-life have given positive effects. Some spices not only provide flavor and aroma to food and retard microbial growth, but are also beneficial in prevention of some off-flavor development. These attributes are useful in the development of snack foods and meat products (Peter, 1997).

Milk is one of the most nutritious foods due to its rich nutrient content. It is a good source of proteins, minerals (especially calcium and phosphorus) and vitamins. The nutritional value of milk as a whole is greater than the value of its individual nutrients because of its unique nutritional balance (Michel, 2006). In addition to being a nutritious food for humans, milk provides a favorable environment for the growth of microorganisms (O’Mahoney, 1988).

Undesirable microbes that cause the spoilage of dairy products are Gram-negative psychrotrophs, coli form bacteria, lactic acid bacteria, yeasts and moulds. In addition, various bacteria of public health concern such as Salmonella spp, Listeria monocytogenes, Campylobacter Jejuni, Yersinia enterocolitica, pathogenic strains of Escherichia coli and enterotoxigenic strains of Staphylococcus aureus may also be found in milk and dairy products (Asresie et al., 2013). The primary spoilage factors in butter are moulds, the majority of species being Thamnidium, Cladosporium and Aspergillus. However, proper heat treatment can destroy moulds in the cream, meaning that any mould contamination thereafter indicates contamination of water and air after production, and there is a protective effect of salt added to butter against moulds (Ahmed et al., 2016).

In different communities of Ethiopia, producers use various traditional preservatives and preservation techniques to increase shelf life of butter and ghee (Alganesh, 2002; Eyassu and Asaminew, 2014). Different plant herbs and spices are commonly used as additives in Ayib processing (Alemayehu et al., 2009; Binyam, 2008; Regu et al., 2016).

Preservatives in cheese processing may help to retard alterations caused by growth of microorganisms or enable physical properties, chemical composition, and original nutritional value to remain unaffected (Ismail et al., 1972). It is the widely used practices spicing dairy products like cheese, butter (ghee), ergo, etc, to extend to acceptable time period. This study was designed to explore the traditional practices of spice and herb usage on dairy products and its effect on consumer acceptability and microbial activities.

1.2. Statement of the Problem

Food poisoning is still a concern for both consumers and the food industry despite the use of various preservation methods. Through microbial activity alone, approximately one-fourth of the world’s food supply is lost. Milk is a highly nutritious food that serves as an excellent growth medium for a wide range of microorganisms. The microbiological quality of milk and dairy products is influenced by the initial flora of raw milk, the processing conditions, and post-heat treatment contamination. Consumers are also concerned about the safety of foods containing synthetic preservatives. There is a growing interest in using effective and nontoxic natural antibacterial compounds, such as extracts of herbs and spices especially for food preservation methods. Traditionally people in Kambata and Hadiya areas have been widely and extensively using herbs, spices and mixture of spices for preserving milk and milk products, to improve flavor and extend acceptability of the product by the consumers. The doses of spices or spices mixtures applied on dairy products such as cottage cheese, ghee, ergo (fermented milk) vary with consumers, as in traditional practices there is no such recipes knowing which mixtures and what level of inclusion could maximize the sensory qualities of products while minimizing the activities of undesirables microbial groups. As spices contain essential oil in it and this essential oil has antimicrobial compounds which in turn have the ability to inhibit spoilage microorganisms, it is necessary to set acceptable levels (amounts) of spices that optimize these two important quality traits. Therefore, this work is designed to assess production or practices of milk and effect of different level and composition of common spice on laboratory made Cottage cheese and ghee

1.3. Research Objectives

General Objective

ü To assess the traditional practices, types of spices and herbs used in preserving various dairy products in Tambaro and Hosanna districts and determine the effects of selected spices on the microbial activity and consumer acceptance of selected dairy products (controlled lab experiment).

Specific objectives:

- To assess the traditional processing practices and type of spices and herbs those are widely applied in milk and milk products in the study area.
- To test and identify the acceptable levels of spice combinations that optimize consumers acceptability while minimizing undesirable microbes in cottage cheese and ghee

1.4. Significance and Scope of the Study

This study adds to the knowledge base to the little published information regarding the effect of natural preservatives such as spices and herbs on microbial activities by specifically addressing common Ethiopian dairy products, common spices and most prevalent microbes in dairy foods. Moreover the study based to exploit the indigenous knowledge of the areas regarding the use of natural preservatives and processing methods of foods especially dairy product usage, processing, consumption and preservation techniques.

2. LITERATURE REVIEW

2.1 Dairy Production in Ethiopia

Ethiopia has great potential for dairy development due to its large livestock population, the Favorable climate and the relatively disease free environment for livestock. Given the considerable potential for small holder income and employment generation from high value dairy products development of the dairy sector can contribute significantly to poverty alleviation and nutrition in the country (Mohammed et al., 2004). Cattle has the largest contribution (81.2%) of the total national annual milk output, followed by goats (7.9%), camels (6.3%) and sheep (4.6%) (CSA, 2009). A recent report by CSA (2010/11) indicated that the total production of cow milk is about 4.06 billion liters, and this translates to an average daily milk production/cow of 1.86 liters/day.

2.2. Handling Practices of Milk and Milk Products in Ethiopia

Milk from healthy animals is sterile, however; postharvest handling practices are the major sources of microbial contamination of raw milk (Coorevits et al., 2008). Contamination of milk at farm level to processing centers could be attributed to handling of milk with unclean equipment, use of poor-quality water for cleaning, and the use of ineffective and inappropriate sanitation agents (Yilma, 2012).

Microbial growth induces changes in the taste and odor of milk such as sour, putrid, bitter, malty, fruity, rancid, and unclean. During milking, microorganisms can enter the milk from the skin of the teats, which are often contaminated by dung, soil, or dust. Appropriate housing and care of the cows is an essential measure to promote clean udders. All milk-handling equipment (utensils) must be effectively cleaned and disinfected so as to reduce the levels of microbial contamination. Cleaning is the most important part of the sanitation process, and if milking equipment/utensils are not kept physically clean, chemicals and other disinfectants are likely to be ineffective (Saran, 1995). The micro flora in butter may be affected by the chemical composition of milk, differences in traditional processing methods, packaging material, storage conditions and post processing handling (Kacem and Karam, 2006). The safety of cheese with respect to food born diseases is a great concern around the world and in developing countries (Ashenafi et al., 2006). This is especially true in Ethiopia where the consumption of Ethiopian cottage cheese is typically manufactured in small dairy farms under poor hygienic conditions is common practices (Alehilign, 2004).

2.3.Factors that Affect Storage Quality of Dairy Products

Shelf life defined as the stage when a food maintains the expected quality desired by the consumer. Foods outside of the shelf life will experience a loss in the desired qualities and an increased chance of microbial (bacteria/fungi) action. The quality of the product and its ability to stay at good condition for a longer time without any change is important to consumer (Caplan and Barbano, 2013). The ultimate shelflife of a product will be determined by its deterioration on storage. The main mechanisms of deterioration are: microbiological, structural, biochemical and chemical (Wilbey, 1997). Food spoilage is greatly influenced by environmental conditions concerning the food matrix, microbial characteristics, temperature, pH, water activity (aw), processing time, etc. The main objective of studying the influence of environmental factors in food preservation is to inhibit spoilage due to microbial survival and growth and/or occurrence of chemical reactions.

Regarding microbial growth, environmental conditions can affect largely the microbial load along the food chain. They can be classified as:

-Physical factors, such as temperature, food matrix.
- Chemical factors, such as pH, preservatives, etc.
-Biological factors, such as competitive flora, production of metabolites or inhibiting compounds etc.
-Processing conditions such as slicing, mixing, removing, washing, shredding etc. as well as influencing transfer of microorganisms (cross-contamination events)( Antonio et al., 2012 ).

According to survey reports (Alganesh and Fekadu ,2012; Eyassu and Asaminew, 2014; Debela, 2016), traditional Ethiopian ghee can be kept at ambient temperature of between 20 to 30°C for 11.10, 19.20 and 7.3 to 7.7 months, respectively in East Wollega, North west and West Shewa, without significant change on quality.

2.4. Functional Properties of Spices and Herbs

Spices, herbs and their constituents are generally recognized to be safe (GRAS), either because of their traditional use without any documented detrimental impact or because of dedicated toxicological studies (Smid and Gorris, 1999). According to the U.S. Food and Drug Administration (FDA), spice is an “aromatic vegetable substance in the whole, broken, or ground form, the significant function of which in food is seasoning rather than nutrition” and from which “no portion of any volatile oil or other flavoring principle has been removed” (Sung et al., 2012).

Being natural foodstuffs, spices and herbs appeal to many consumers who question the safety of synthetic food additives. Some spices and herbs used today are valued for their antimicrobial activities and medicinal effects in addition to their flavor and fragrance qualities. The extracts of many plant species have become popular in recent years and attempts to characterize their bioactive principles have gained momentum for varied pharmaceutical and food processing applications. The antimicrobial activities of plant extracts form the basis for many applications, including raw and processed food preservation, pharmaceuticals, alternative medicines and natural therapies (Lis-Balchin and Deans, 1997).

Most essential oils have been classified as general recognize as safe(GRAS) and the antimicrobial activity of essential oils and spices are considered to result in extending the shelf life of a wide variety of foods (Burt, 2004).

Many studies have reported that phenolic compounds in spices and herbs significantly contributed to their antioxidant and pharmaceutical properties (Cai et al., 2004; Shan et al., 2005 and Wu et al., 2006). Some studies claim that the phenolic compounds present in spices and herbs might also play a major role in their antimicrobial effects (Hara-Kudo et al., 2004). There has been no large scale systematic investigation of the relationship between bacterial inhibition and total phenolic content of spices and herbs. In addition to imparting characteristic flavors, certain spices and herbs prolong the storage life of foods by preventing rancidity through their antioxidant activity or through bacteriostatic or bactericidal activity, also to food borne pathogenic bacteria (Beuchat and Golden, 1989; Shelef et al., 1980). Previous study reports (Cai et al., 2004 and Shan et al., 2005) showed that a highly positive linear relationship exists between antioxidant activity and total phenolic content in some spices and herbs.

Herbs have found many uses in treating number of diseases and their herbal extracts can be used in pharmaceuticals, ayurvedic formulation, confectionery, nutritional foods, ready-to-drink mixes, instant foods, seasonings, dairy products, seasoning blends, etc. Therefore, fortification of herbs in dairy products could provide value added, functional dairy product. Moreover, use of some specific herbs will help in shelf life extension of otherwise perishable dairy products (Lalita et al., 2017).

2.5. Spoilage Microorganisms of Dairy Products

Troublesome spoilage microorganisms include aerobic psychrotrophic Gram-negative bacteria, yeasts, molds, heterofermentative lactobacilli, and spore-forming bacteria. Psychrotrophic bacteria can produce large amounts of extracellular hydrolytic enzymes, and the extent of recontamination of pasteurized fluid milk products with these bacteria is a major determinant of their shelf life. Fungal spoilage of dairy foods is manifested by the presence of a wide variety of metabolic by-products, causing off-odors and flavors, in addition to visible changes in color or texture. Coliforms, yeasts, heterofermentative lactic acid bacteria, and spore-forming bacteria can all cause gassing defects in cheeses (Ledenbach and Marshall, 2009).

Table 1 .Dairy products and typical types of spoilage microorganisms or microbial activity

Abbildung in dieser Leseprobe nicht enthalten

Source (Ledenbach and Marshall, 2009)

So methods to extend shelf life based on altering factors leads to food spoilage and addition of treatments. Traditional methods used to extend shelf life of milk and milk products include using smoking with plants leaf or stems on milk equipments, fermentation of milk, and application of spices on milk products. Modern methods used to extend shelf life of milk and milk products involves Pasteurization of milk, storing milk products in a refrigerator, Ultra Heat Treating (UHT) of milk etc.The end of shelf-life can be defined as the time needed for Specific Spoilage Organisms (SSO) to multiply from an initial contamination level to a spoilage level, or the time invested by SSO to produce a certain metabolite causing sensorial rejection (Koutsoumanis and Nychas, 2000). At high contamination levels, less time would be needed by SSO to reach the minimum spoilage level, thus, shelf-life will be reduced. The following graph depicts the bacterial growth curve (Fig.1)

Abbildung in dieser Leseprobe nicht enthalten

Source: (Antonio et al., 2012). Figure 1 Effect of the microbial initial contamination on shelf-life in a food.

2.6. Microbial Quality of Ayib and Ghee

Coliforms, which are associated with fecal contamination, are killed by pasteurization. When present in milk, they are regarded as "indicators" of post-pasteurization contamination as a result of poor sanitation (Jayarao et al., 2004). According to Council Directive 92/46(EEC ,1992) the maximum limit of coliforms for soft cheese made from heat-treated milk is 10[5]cfu/g. The yeast and mould counts in butter samples collected from southern Ethiopia (Mekdes, 2008) ranged from 5.52 to 5.74 cfu/g. Moulds are the primary spoilage factors in butter and their presence in butter indicates post production contamination from air or water(Alganesh and Yetenayet, 2016). Microbiological limit specification for butter settled by Ethiopian standard for total plate count is 10[6] cfu/ml (ES, 2009).

2.7. Physico - chemical Properties of Ayib and Ghee

Requirements of ghee set by Ethiopian standard(2009) indicates that the level of milk fat min, 99.6%, Moisture -, Acidity 0.4 max, % by mass as oleic acid , Peroxide value 0.6 , max, milliequivalent of oxygen/ 1kg fat. O'mahony (1988) reported that ayib comprises 79% water, 14.7% protein, 1.8% fat, 0.9% ash and 3.1% soluble milk constituents and the yield should be at least 1 kg of Ayib from 8 liters of milk (12.5%). Appropriate temperature of curd-cooking coupled with the low pH of the product should make Ayib a safe and nutritious product with an improved keeping quality (Berhanu and Tsehayneh, 2014).

2.8. Consumer Acceptability of Dairy Product

Sensory testing is designed to validate the length of time that a product will remain with the same “acceptable quality” level or presents “no changes in desired sensory characteristics” over the storage time of a product (IFTS, 1993; Kilcast and Subramanian, 2000).

The sensory characteristics of a product such as color, flavor, and aroma effects consumer

satisfaction (Zellner et al., 2014; Van Loo et al., 2015). For the food industry, the detection of oxidative off-flavors by taste or smell is the main method of deciding when a lipid-containing food is no longer fit for consumption (Shahidi and Zhong, 2005).The sensory quality characteristics of cultured dairy products are not as clearly defined as for other dairy products. Specific geographical differences may exist in consumer preferences for flavor intensity, body and texture characteristics, and /or color and appearance features of many cultured dairy products (Body felt et al., 1988). The flavor of soft cheeses can either be natural or acquired. The natural or primary cheese flavor is regarded as that which originates from the enzymatic activity of the starter microbes, whereas the acquired or secondary flavor is due to the addition of other ingredients in the finished products (Scott, 1998).

Flavor of ghee is greatly influenced by the fermentation of the cream or butter and the heating process. Carbonyls, lactones and free fatty acids are reported to be the key Ghee flavoring compounds (Sserunjogi et al., 1998). Much of the typical flavor, in ghee, comes from the burned milk solid non-fat remaining in the product (O'Mahony, 1988). The most important factor controlling the intensity of flavor in ghee is the temperature of clarification. The acidity of the cream or butter also affects the flavor of ghee and shelf life of ghee is also dependent on the method of preparation (Ganguli and Jain, 1972).

2.9. Spices Used in Ayib and Ghee Production Process in Ethiopia

Nigella sativa (Tikur azimud)

The genus name Nigella is a diminutive of the Latin niger (black), referring to the seeds.. Nigella sativa (black caraway, also known as black cumin, nigella, and kalonji) is an annual flowering plant in the family Ranunculaceae , native to south and southwest Asia. The dry-roasted seeds flavor curries, vegetables, and pulses. They can be used as a "pepper" in recipes with pod fruit, vegetables, salads, and poultry. In some cultures, the black seeds are used to flavor bread products, and are used as part of the spice mixture panch phoron (meaning a mixture of five spices) and alone in many recipes in Bengali cuisine and most recognizably in naan (Bramen, 2011). Nigella is also used in Armenian string cheese, a braided string cheese called majdouleh or majdouli in the Middle East. N. sativa oil contains linoleic acid, oleic acid, palmitic acid, and trans-anethole, among other minor constituents (Bharat B. A., 2015.)

Aframomum Corrorima (Kororrima)

Aframomum corrorima is a species in the ginger family, Zingiberaceae and the plant native to Tanzania, western Ethiopia (in vicinity of Lake Tana and Gelemso), south western Sudan, and western Uganda. The spice, known as kororima, Ethiopian cardamom, or false cardamom and cultivated in both Ethiopia and Eritrea (NGRP, 2011).It is obtained from the plant's seeds (usually dried), and is extensively used in Ethiopian and Eritrean cuisine. It is an ingredient in berbere, mitmita, awaze, and other spice mixtures, and is also used to flavor coffee. In Ethiopian herbal medicine, the seeds are used as a tonic, carminative, and laxative (Jansen, 2002).

Trigonelle Foenum Graecum (Abish)

An annual heavily scented Mediterranean leguminous plant, Trigonella foenum-graecum, with hairy stems and white flowers: cultivated for forage and for its medicinal seeds. It is an aromatic Eurasian plant, Trigonella foenumgraecum, of the legume family and its seed used as seasoning especially in curry and medical (http:www.The freedictionary.com/ Trigonella+foenum-graecum).

Trachyspermum Copticum (Netch azimud)

Trachyspermum copticum contain the oil called ajowan oil which contain compounds thymol, gamma-terpinene, p-cymene, and beta-pinene. Alpha-pinene, alpha-thujene, beta-myrcene, carvacrol, limonene, and terpinene-4-ol.the oil has antioxidant, anti-inflammatory, antibacterial, antifungal activity (AOS product, 2016).

2.10. Consumption of Milk and Milk Products in Ethiopia

Milk products like curd and cheese are widely used and consumed in many part of the world and available in market for many generations. There is still an increase demand of consumer due to free from artificial preservatives, high quality natural food and free of contaminating micro-organisms (Berhanu and Tsehayneh.,2014).

Dairy products form part of the diet of many Ethiopians. They consume dairy products either as fresh milk or in sour form. According to Central Agricultural Census Commission (CSA, 2003) at national level 48.2% of the milk is consumed as whole fresh milk or in fermented form whereas 46.6% is used for butter making and only 5.2% marketed.

The consumption pattern of milk and milk products produced at home varies depending upon the amount of milk produced per household, dairy production system and market access, season of the year, and fasting period (particularly for the followers of Orthodox Christian) (Azage et al., 2013). Fresh milk, Ergo, whey(Aguat), Ethiopian cottage cheese (Ayib) and traditional butter (Kibe) are the most common milk products produced and consumed by different part of the country. Butter is a traditional food which is widely consumed all over the world, directly or as ingredient in processed food such as pastries and convenience dishes. Its nutritional value (due to high content of fats, vitamins and minerals), and unique and pleasant flavor make butter practically appreciated by consumers. Butter can be made directly from milk or by separation of milk and subsequent churning of the cream (Kacem and Karam, 2006). Ayib is an important source of nutrients and serves as a staple diet. It may be consumed fresh as side dish, or it may be spiced with hot spices, salt and other herbs (Gonfa et al., 2001). Ayib mixed with cooked and minced cabbage leaves, fresh and melted butter and spiced chilli-powder and served along with minced raw meat (kitfo) to be eaten with spoon. This preparation is frequently eaten with thick flat bread made from false banana flour (kocho) (FAO, 1990).

Ghee is the most widely used milk product in the Indian sub-continent. Ghee is also gaining popularity in Australia, Arabian countries, the United States, the United Kingdom, Belgium, New Zealand, Netherlands and many other African and Asian countries (Illingworth, et al., 2009). Ghee can be defined as a pure clarified fat exclusively obtained from milk, cream or butter, by means of processes involving application of heat at atmospheric pressure, which results in the almost total removal of moisture and solid nonfat and which gives the product a characteristic flavor and physical structure and texture (Illingworth et al. , 2009). According to Codex Alimentarius (FAO/WHO, 1997, 2006).Traditional ghee manufacture is based on individual experience and taste. Addition of combinations of spice powders, chopped tubers and or dry herbs, leaves and stems of green spices are used in traditional ghee making (Alganesh, 2002; Hailemariam and Lemma, 2011). Traditional Ghee “Neter kibe” is a more convenient product than “kibe” in the tropics, because of its better shelf life under warm conditions. “Neter kibe” has an attractive appearance, a grainy texture and a light yellow color. At room temperature it is semi-solid. It has a pleasant odor and good taste (Gonfa, et al., 2001). Ghee made as such is either added into a traditional sauce (wot) to impart good flavor, used as a sauce during raw meat consumption, or drank with coffee. It is also consumed by mixing it with Metata Ayib (Eyasu and Assaminew, 2014).

3. MATERIALS AND METHODS

3.1. Description of the Study Area

The study was conducted in Kambata Tambaro zone (Tambaro woreda) and Hadiya zone (Hosanna areas) in Southern Nation Nationalities and Peoples Regional State of Ethiopia (SNNPR). The geographical location of the Kambata zone is between latitude 7[0].10' –7.5[0] 0' N and 37[0].34'-38[0].07' E longitude. Topographically, the zone lies between an elevation ranges of 501-3000 meters above sea level. Latitudinal location of Hadiya zone is 7°30′N 37°45′E to  / 7.5°N 37.75°E and elevation range of 1500 to 3000 meters above sea level.7.5; 37.75 Durame (main town of Tambaro district) and Hosanna city are situated 350 km and 200 km far from Addis Abeba, respectively. The temperature of the study areas ranges between 12oC and 30oC. The average annual rain fall of the areas ranges from 1167 mm to 1583mm. The main agro-ecologies of Tambaro district is mid-highlands and low lands, while Hosanna areas are typically cold highlands (SNNPR, 2008). Predominant agricultural system of the areas is mixed crop livestock production, where perennial crops such as Enset and coffee, as well as root crops and other food crops are cultivated as part of the mixed crop-livestock production. Cattle are the most abundant and important livestock species in the areas and dairy products such as cottage cheese and butter/ghee are widely produced as home food and cash commodities.

3.2. Study Design

This study has two parts: survey and laboratory experiment. For the survey part, purposive sampling procedure was employed for select kebeles and households. Kebeles were stratified as rural and urban. Then one from the urban and one from rural areas of each study site were selected. A total of 30 households per study site based on the availability of at least one lactating dairy cow were selected. For the experimental work, cottage cheese and butter were made in the laboratory with the common spices used to preserve cottage cheese and butter in the study area based on the survey result.

3.2.1. Survey

Both secondary and primary data sources were used for the study. Primary data was collected from purposively selected households. Secondary data source was from the respective Kebeles and Woreda’s offices. Structured questionnaires were used to collect the primary data. The questionnaire was pre-tested and adjusted before actual interview. Informal discussions with key informants, kebele leaders and developmental agents of the selected kebeles and personal observations were also made to supplement the data.

3.3. Laboratory experiment

3.3.1. Preparation of Spice powder

Spice used in the experiment included Aframomum Corrorima (Kororima), Nigella sativa (Tikur azimud), Trigonelle Foenum Graecum (Abish) and Trachyspermum Copticum( Netch azimud). Dried spices were obtained from market and dry matter and moisture percentage of was determined. Spice toasted to ease grinding. The spice was grounded using electric kitchen grinder, packed and put in refrigerator until use.

3.3.2. Preparation of Ayib

Fresh milk was obtained from Hawassa dairy farm. The milk was adequately fermented, churned and the butter was collected. Butter milk was heated at a temperature of 50 [0] C for 55 min and cooled overnight. The whey was drained using sieve for about one hour to separate the cheese curd.

3.3.3. Preparation of Ghee Sample

Ghee was produced by melting the butter collected during ayib production by heating until foam was formed over the surface, and spices powder mixtures was added when the temperature approaches the boiling point by carefully removing the foam with spoon. When the color changed from light yellow to light brown, it was removed from the heating source and was allowed to cool down. The clear liquid, the sweetly aromatic ghee, floating on the heavier solids was strained through a fine-meshed strainer into the plastic beaker was allowed to cool.

Figure. 2. Ayib and Ghee making flow charts.

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3.5. Treatment arrangement

In the laboratory experiment ayib and ghee were produced and laid in to 7 different treatments each. There 7th treatment comprised either ayib or ghee as a control that did not receive any spice treatment. To set up the other 6 treatments for ayib and ghee three and four types of ground spices were mixed in 1:1 ratio respectively.

Table 2.Treatment arrangement for ayib sample.

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Where AC: Aframomum corrorima, TC: Trachyspermum copitcum, NS: Nigella sativa,

Ayib sample in each treatment weighing 900 gm was divided in to 6 equal parts making it 150 gm after receiving respective treatment. Each treatment was replicated and was stored for 6 days. And also sampled every 24 hr for analysis.

Table 3. Treatment arrangement for ghee

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Where AC: Aframomum corrorima, TC: Trachyspermum copitcum, NS: Nigella sativa, TF: Trigonella foenum

Ghee sample in each treatment weighing 400 gms was divided in to 4 equal parts making it 100 gm after receiving respective treatment. Each treatment was replicated and was stored for 4 week. Sampled every week for analysis.

3.7. Experimental design

The experiment laid out in a2x3 and 4x3 splitplot design for ayib and ghee respectively treatment by levels effect having three replications. Treatments were Aframomum corrorima and Trachyspermum copitcum powder mixtures, Aframomum corrorima and Nigella sativa powder mixtures at 1:1 ratio and no spice powder (control) added in fresh cottage cheese at three levels (1%, 3% and 5%). And for ghee sample Aframomum corrorima and Trigonella foenum powder mixtures, Nigella sativa and Trachyspermum copitcum mixtures and at three levels (1%, 3% and 5%) control no spice added to ghee during making process.

3.8. Microbial analysis

3.8.1. Total Bacterial count

For total bacterial count (TBC) was analyzed by taking 11gram of each sample mixing it with 99 ml of previously autoclaved buffered peptone water with stomacher bag. Further dilutions were prepared by pouring 1ml of the sample to 9 ml of buffered peptone water. After making the appropriate dilution (10[0] -10-[6]), 1ml of diluted sample was pour plated with plate count agar in duplicate. Count was made after incubating the plates for 48 hours at 32 [0]c. The total number of colonies between 30 and 250 were used for computing the cfu/g values (Richardson, 1985).

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