ABSTRACT

The aim of this study was to identify the adverse effect of fermented milk consumed by nigeria by examining the antimicrobial properties of Lactic Acid Bacteria (LAB) isolated from fermented milk collected from Fagge, Kumbotso, Dala, Gwale, Tarauni and Ungogo LGA in Kano state. Traditional fermented milk in Nigeria are produced by spontaneous fermentation using traditional utensils. In this study, thirty homesteads from six LGA that produce and process fermented milk were selected and interviewed using semi- structured questionnaires. Kindirmo and Maishanu have similar processing method whereby fermentation is achieved by accumulation of milk; mean while Wara is produced by allowing the milk to ferment naturally. The pH decreased logarithmically, nonlinearly over the fermentation period from 6.5 ± 0002 from first day of fermentation to 3.92±0.001 over 4 days.There was no significant difference (p<0.05) in the pH values between the three types of fermented milk preparations. Cell free supernatants (CFS) of 180 LAB isolated from traditional fermented milk were evaluated for antimicrobial activities against selected food borne pathogens; Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Candida albicans ATCC 14053, Bacillus cereus ATTC 10876 Geotrichum klebahnii (IKST F. Lab. isolate) Escherichia coli ATCC 25922 using the well diffusion method. Twenty LAB isolates that shown the highest inhibitory effects were selected for biochemical identification using API 50 CHL were identified as; Lactobacillus plantarum (53%), Lactobacillus rhamnosus (29%), Pediococcus pentosaceus (6 %), Lactobacillus paracasei ssp. paracasei (6%) and Lactococcus lactic ssp. lactis (6%), of which Pediococus pentosaceus showed the most inhibitory effect on all the indicator strains and they have potential to produce bacteriocin Pediocin and the most inhibited indicator strain belonged to yeast Candida famata. In addition to antimicrobial activities, the major organic acids in all three types of milk were found to be lactic acid 0.265±0.056 mg/L. However, the levels of volatile organic compounds in the naturally fermented  samples varied from one sample to another but,  butyric, acetic and propionic acid were found in trace amounts. Some of the volatile flavor  compounds found in Kindirmo, Wara and Maishanu  were  acetic acid, 2,3 Butanediol and Lactic acid.

 

 

 

CHAPTER 1: INTRODUCTION

Fermented milk is one of the foods which are highly respected and form part of daily intake in Nigeria. There are different types of fermented milk in Nigeria which have different preparation methods. The diversity of such fermented milk products is derived from the heterogeneity of traditions found in the Nigeria, cultural preference, different geographical areas where they are produced, the staple and/or by-products used for fermentation. These types of milk are mostly prepared using natural ingredients without any addition of preservatives.

Fermentation is a relatively efficient, low energy preservation process which increases the shelf life and decreases the need for refrigeration or other form of food preservation technology (Marshall, & Mejia, 2011). It is therefore a highly appropriate technique for use in developing countries and remote areas where access to sophisticated equipment is limited.

Traditional fermentation is a form of food processing where microbes for example, lactic acid bacteria (LAB) are utilized (Chelule et al., 2010). The technology of fermentation has been used for centuries as a form of food preservation technique for perishable food products (Hansen, 2002). Over the years, it became part of the cultural and traditional norm among the indigenous communities in most developing countries, especially in Africa. On this note, the people living in remote areas prefer fermented over unfermented milk due to improved flavor and texture that accompanies fermentation. In addition, these products are an important supplement to the local diet and provide vital elements for growth and good health.  This popularity has made fermented foods one of the main dietary components of the developing world.

 

Fermenting milk assist in the preservation of milk by generating  lactic acid and other antimicrobials,   production of flavor compounds (e.g. acetaldehyde and diacetyl) and other metabolites (e.g. extracellular polysaccharides) that confer consumer desired organoleptic properties to the product (Chelule, et al., 2010). Fermentation also improves the nutritional value of milk; it releases free amino acids and promotes synthesis of vitamins (Kailasapathy, 2008). Fermented milk contains special therapeutic or prophylactic properties against cancer and lactose intolerance (Mercenier, et al., 2002). Fermentation also, improves food safety through inhibition of pathogens (Adams & Mitchell, 2002) as well as removal of toxic compounds in food. These significant changes cause desirable biochemical effects resulting into development of new aroma, flavor, taste and texture thereby increasing the sensory quality, palatability and acceptability of the product.

Statement of Problem

Kindirmo, Masanza or Mabasi and Maishanu are common fermented milk products in the rural areas of the northern Nigeria. Like  any other traditional fermented milk in the developed countries, these types of milk serve as  substitutes for traditional vegetables, beans or meat supplementing nutrition for low income households and for women and children in rural area (Bille, et al., 2002). In addition, the fermentation is based on spontaneous process due to the growth of the micro flora naturally present in the raw material. Therefore, the quality of the fermentation end product dependents on the microbial load and spectrum present in the raw material. Since Kindirmo, Masanza or Mabasi and Maishanu are not prepared uniformly by the rural processors, they have fluctuating qualities due to natural fermentation, uncontrolled fermentation temperature and time.  Failure of fermented milk to cause food poisoning in spite of uncontrolled fermentation environments suggests unique chemical properties in these products. Therefore, there is a need to examine the diversity of LAB, the antimicrobial content of the milk and to purify the most predominant LAB in traditional fermented milk from the northern parts of Nigeria. Previous antibacterial activity studies have been performed by isolating LAB from fermented products. However, LAB isolated from Nigeria traditionally fermented milk has not been intensively studied.

Objectives of the Study

The overall aim of the study was to identify the adverse effect of fermented milk consumed by nigeria by examining the antimicrobial properties of Lactic Acid Bacteria (LAB) isolated from fermented milk.

The specific objectives of this project.com.ng/list-of-project-and-materials/">research project were:

1. To analyze the physicochemical properties (pH, viscosity and titratable acidity) for the three types traditional fermented milk in Nigeria.
2. To isolate different types of lactic acid bacteria using selective media such as Rogosa for

Lactobacillus spp. and M17 for Lactococcus spp. e.t.c.

3. To identify starter culture strains to the species and sub-species level using biochemical and genetic identification methods (API 50 CHL and 16S rDNA).
4. To screen the isolated strains for antimicrobial compounds with the ability to act as a natural competitive substance inhibiting other microorganisms that share the same niche; these include: hydrogen peroxide, organic acids, diacetyl and bacteriocins.
5. To determine the concentration of different organic acids (lactic and acetic acids) using conversional method (titration) and chromatographic method (HPLC).

1.5 Significance of the Study

Fermented milk plays an important role in the diet of low income and the majority of people living in the rural areas in Nigeria. This research work provides a baseline data on the antimicrobial activities of Nigerian fermented milk products. It focuses on antimicrobial compounds profiling of different starter cultures from fermented milks produced in Nigeria which has not been reported previously. In addition, this research identified the types of micro flora in traditional fermented milk to the genus level. Once the identity of the milk fermenting micro flora is known it would be possible to optimize fermentation process through alteration and modification of starter cultures so as to improve the quality of fermented milk products for possible commercialization purpose. Furthermore, knowledge of the starter cultures metallic products would facilitate their ease of extraction and subsequent use in food industries as preservatives. Alternatively, the isolated LAB could be used as probiotics. Micro flora profiling is significant as it may unravel species’ diversity and concentrations in the fermented milk; it may shed light on the organisms’ ecological characteristics as they interact with the host and the environment at large.

1.6 Limitation of the Study

Preferably, the study was to include all LGA in Kano state in Nigeria, but Nigeria is a very big country and there are many different cultural groups with different traditional foods and fermented milks. It would be an expensive, time consuming and labor intensive to screen all fermented milk products in Nigeria. DNA sequencing facilities are not available in Nigeria; genetic identification of the starter cultures was therefore done at Inqaba Biotech Laboratory in South Africa. During the screening and strains identification phases, a large number of isolates was obtained. Therefore, only isolates which showed outstanding results on different tests, in particular, the  bacteriocengenic strains which showed protective characteristics were considered for identification  on molecular diagnosis’ and other antimicrobial tests such as diacetyl, hydrogen peroxide and organic acids using conversional analytical techniques, since screening all isolates was going to be a very expensive and time consuming process.

 

 

 

 

CHAPTER 2 LITERATURE REVIEW

2.1. The Origin of Fermented Milk

Traditional Fermented milk is defined by Abdelbasset & Djamila, (2008) ; as a dairy product obtained by the spontaneous fermentation of milk, which may have been made from products obtained from milk with or without any modification of their composition, through appropriate process of microorganisms which results in a lowering of the pH with or without coagulation. The traditional fermentations are taking place as a result of the activities of natural flora present in the food or added from the environment. Fermentation of milk is a very ancient practice of man which has been passed down from generation to generation (FAO, 1990).

The definite origin of fermented milks is indefinite but there is no doubt that their consumption dates back to prehistoric times and its  believed that fermented milks originated in the Near-East, perhaps before Phoenician era, and spread through central and Eastern Europe (Lefoka,  2009) .  Though the composition and microbiology of fermented milks were not known, they were more preferred comparing to raw milk because of their beneficial effects over fresh milk. With these advantages, fermented milk preserved the high quality nutrients present in milks in a relatively stable form (Panesar, 2011). In addition, the fermented milk could be stored at warm temperatures and be safely consumed for several days. Apart from nutritional benefits, fermented milk was also known to contain pharmaceutical benefits. It was prescribed for curing disorders of the stomach, intestines and other illnesses (Gandhi, 2006) . The history of fermented milks is now often described in terms of a sequence of several generations of products (Steinkraus, 2002): the first generation (800 BC until 1900 BC), when the micro flora was not defined and differed from villages to villages.  In the early years of milk fermentation, milk was simply allowed to be fermented by its normal micro flora, but the actual process was not completely understood.  Cultures could be maintained by inoculating fresh milk with fermented milk (Kerr & McHale, 2001). This first generation was followed from about 1910 by a second generation of fermented milks in which the micro flora was defined and the fermentation process was controlled (Chandan, & Shah, 2013).

2.2 Traditional Fermented Milk

A wide variety of fermented milk products have been produced in different parts of Africa as a method of milk preservation. The major source of fermented milk is from cow but, but sheep, goat, buffalo, camel and horse milk are also used in some cultures. The fermentation process occurs spontaneously by back-sloping; i.e. inoculation of the raw material with a small quantity of the previously performed successful fermentation. Therefore, back-slopping results in dominance of the best adapted strains (Steinkraus, 2002). It represents a way, be it unconsciously, of using a selected starter culture to shorten the fermentation process and to reduce the risk of fermentation failure. This traditional method of preparing fermented milk is still used in some less sophisticated societies. Modern techniques of milk fermentation, on the other hand, use starter cultures with known characteristics (Chandan, & Shah, 2013). The advantage of modern techniques over the traditional methods is the production of consistent products that are less likely to spoil and are relatively safe.

2.3 African Traditional Fermented Milk

Fermented Milk is a major component of the traditional diet in many LGA in Africa. Most of the milk produced is consumed in the house mostly by children and excess is infrequently sold in open markets. Due to limitation of cold storage facilities such as refrigerators in many African countries, milk is stored at ambient temperatures which usually fermented rapidly by the natural flora.

Fermented milk produced in South Africa (Amasi) was described by (Beukes et al., 2001). The product is consumed in different LGA of Southern African, including Zimbabwe, South Africa and Lesotho. Amasi is an unsweetened curd which is traditionally prepared from unpasteurized bovine (cow’s) milk which is allowed to ferment spontaneously in a pottery (clay pot) or gourd

(“calabash”) for two to three days at ambient temperature. Beukes, et al. (2001) also reported that clay pots have a better flavor of Amasi than calabashes because a calabash needs to be seeded with microbial inoculums before it could be used for the production of fermented milk.  The microbial flora responsible for the fermentation is derived from the air, raw milk and walls of the containers. After coagulation, the whey is drained through a plugged hole at the bottom of the container.  According to a study which was done by Feresu and Muzondo (1990), the predominant LAB isolated from Amasi   belongs to Lactobacillus helveticus, Lb. plantarum, Lb. delbrueckii subsp. lactis, Lb. paracasei subsp. paracasei and Lb. paracasei subsp. pseudo– plantarum.

Amasi is reported to have consistency slightly thicker than yoghurt and with a pH between 3.6 and 4.2 (Todorov, et al., 2007). Although normally consumed with thick corn-meal porridge, Amasi is also consumed between meals with ground sorghum which is similar to muesli.

2.4 Fermentation Process

The term “fermentation” comes from Latin word fermentum (to ferment) (Gogineni et al., 2013). The historical definition describes fermentation as the process in which chemical changes in an organic substrate occur as the result of action of microbial enzymes (Bamforth, 2005). The fermentation processes can be classified as spontaneous and induced (Bamforth, 2005). A wide variety of microbes can be responsible for the fermentation process of milk including lactic acid bacteria (LAB), acetic acid bacteria (AAB), yeasts and mycelia fungi (Ross et al., 2002).  The fermentation process of traditional fermented milk depends on the end products of lactic acid bacteria formed during the fermentation of lactose. LAB may be classified as homofermentative or heterofermentative based on their by-products of sugar (e.g., lactose) fermentation (Rattanachaikunsopon & Phumkhachorn, 2010).

Homo-fermentative LAB; Use the Embden-Meyerhof-Parna’s pathway to generate two moles of lactate per mole of glucose and derive approximately twice as energy per mole of glucose as hetero fermentative lactic acid bacteria (Bassyouni et al., 2012). They include Lactococcus spp. that is used in dairy starter culture applications, while, other homo-fermentative LAB include yoghurt strains consisting of rods (Lactobacillus delbruckii subspecies bulgaricus, Lactobacillus (Lb.) acidophilus) and cocci (Streptococcus salivarius subsp. thermophilus) and thermophilic strains that might be used in cheese (e.g., Lb. helveticus) (Gandhi, 2006).

Hetero-fermentative LAB; produce equal molar amounts of lactate, carbon dioxide and ethanol from lactose via the hexose monophosphate or pentose pathway (figure 1.1). These type of LAB are rarely used as dairy starter cultures, Although , they are not commonly used  in milk and dairy products , if they are  allowed to grow  significantly in numbers, they can cause defects related to their acid and CO₂ production, such as slits in hard cheeses or bloated packaging in other dairy products. They include Leuconostoc spp. (Gram-positive cocci) and Gram-positive rods such as Lactobacillus brevis, Lb. fermentum and Lb. reuteri.