Assessment of Nunu Produced Using Pure Starter Culture
Chapter One
AIM OF THE STUDY
The present study was undertaken with the aim to investigate and assess microorganism present in ‘Nunu’ produced using pure starter culture.
OBJECTIVES OF THE STUDY
- To inoculate pure starter culture into freshly prepared ‘Nunu’.
- To isolate and enumerate Lactic Acid Bacteria present in the produced ‘Nunu’.
- To isolate, characterize and enumerate other microorganisms present in the produced ‘Nunu’.
- To make useful recommendations based on my findings.
CHAPTER TWO
LITERATURE REVIEW
LACTOBACILLALES
Lactobacillales or lactic acid bacteria (LAB) are a clade of Gram-positive, low-GC, acid-tolerant, generally non-sporulating, non-respiring, either rod- or cocci-shaped bacteria that share common metabolic and physiological characteristics. These bacteria, usually found in decomposing plants and milk products, produce lactic acid as the major metabolic end product of carbohydrate fermentation. This trait has, throughout history, linked LAB with food fermentations, as acidification inhibits the growth of spoilage agents. Proteinaceous bacteriocins are produced by several LAB strains and provide an additional hurdle for spoilage and pathogenic microorganisms. Furthermore, lactic acid and other metabolic products contribute to the organoleptic and textural profile of a food item. The industrial importance of the LAB is further evinced by their generally recognized as safe (GRAS) status, due to their ubiquitous appearance in food and their contribution to the healthy microflora of human mucosal surfaces. The genera that comprise the LAB are at its core Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, and Streptococcus, as well as the more peripheral Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Sporolactobacillus, Tetragenococcus, Vagococcus, and Weisella; these belong to the order Lactobacillales.
CHARACTERISTICS OF LAB
The lactic acid bacteria (LAB) are either rod-shaped (bacillus), or spherical (coccus), and are characterized by an increased tolerance to acidity (low pH range). This aspect helps LAB to outcompete other bacteria in a natural fermentation, as they can withstand the increased acidity from organic acid production (e.g., lactic acid). Laboratory media used for LAB typically include a carbohydrate source, as most species are incapable of respiration. LAB are catalase negative. They consist of the organelles of a simple bacterial structure. LAB are amongst the most important groups of microorganisms used in the food industry (Sonomoto and Yokota, 2011).
METABOLISM OF LAB
Two main hexose fermentation pathways are used to classify LAB genera. Under conditions of excess glucose and limited oxygen, homolactic LAB catabolize one mole of glucose in the Embden-Meyerhof-Parnas pathway to yield two moles of pyruvate. Intracellular redox balance is maintained through the oxidation of NADH, concomitant with pyruvate reduction to lactic acid. This process yields two moles of ATP per mole of glucose consumed. Representative homolactic LAB genera include Lactococcus, Enterococcus, Streptococcus, Pediococcus, and group I lactobacilli.
Hetero-fermentative LAB use the pentose phosphate pathway, alternatively referred to as the pentose phosphoketolase pathway. One mole of glucose-6-phosphate is initially dehydrogenated to 6-phosphogluconate and subsequently decarboxylated to yield one mole of CO2. The resulting pentose-5-phosphate is cleaved into one mole glyceraldehyde phosphate (GAP) and one mole acetyl phosphate. GAP is further metabolized to lactate as in homofermentation, with the acetyl phosphate reduced to ethanol via acetyl-CoA and acetaldehyde intermediates. In theory, end products (including ATP) are produced in equimolar quantities from the catabolism of one mole of glucose. Obligate heterofermentative LAB include Leuconostoc, Oenococcus, Weissella, and group III lactobacilli.
SCIENTIFIC REVIEW OF LAB
The importance of Lactic Acid Bacteria in fermented non-dairy foods and beverages was reviewed previously in the early 1990 in an overview of the role of lactic acid bacteria in fermented fish products, vegetables and yoghurt (Lee, 1994). The role of Lactic Bacteria in rice wine, grape wine and beer fermentation has also been reported, this have shown the versatile application of LAB in use to initiate or support fermentation (Rhee et al., 2003).
The prominent roles of particular Lactic Acid Bacteria belonging to the genera Lactobacillus and Pediococcus also the role of Leuconostocmesenteriodes, its rapid growth, producing carbondioxide and acids leading to the modification of the environment (another important ability besides fermentation) as well as conditions that favour the growth of other lactic acid bacteria has been reviewed (Lee et al., 1983).
Interest in microorganisms as a component of biological diversity has been renewed in recent years (Alsoppet al., 1995). The interest in microorganisms occurring in food is primarily due to the biotechnological potential of new bacterial species and strains (Leisneret al., 1999). Several published reports have indicated the presence of Lactobacillus strains in sheep and cow milk (Mobarezet al., 2008). Additionally, several studies have shown the inhibitory activities of numbers of Lactic acid Bacteria such as Lactobacillus brevisisolated from Turkish dairy products (Aslimet al.,2005) and Lactobacillus acidophilus isolated from Iranian yoghurt against Staphylococcus aureus(Mobarezet al., 2008). Numerous reviews have suggested that some Lactic Acid Bacteria were able to control the growth of some pathogen microorganisms such as L. monocytogenesin food products (Calleweartet al., 2000; Mataragas et al., 2003; Tantilloet al., 2002).
In history, milk played a major role as nutritional source and since 1900’s, the start of golden era of industrial microbiology. It was also economically significant because larger quantity of milk was being processed daily in factories for the demand of fermented food products (Singh et al., 2009). The tenet, “let food be thy medicine and medicine be thy food” was embraced 2500 years by Hippocrates, the father of medicine, However, this “food as medicine” philosophy fell in relative obscurity in the 19th century with the advent of modern drug therapy. In 1908, Elie Metchnikoff, proposed that the acid producing organisms (Lactic acid Bacteria) in fermented dairy products could prevent fouling in the large intestine and this leads to a prolongation of the life span of consumers. Later on the concept of probiotics was introduced and defined, further highlighting the therapeutic uses of LAB (Fuller, 1989).
A program aimed at extensive sequencing of the genome of Lactic acid Bacteria was announced in 2002 by the Lactic acid Bacteria Genome Sequencing Consortium (Klaenhammeret al., 2002). The actual breakthrough occurred only in the last 7 years. This would improve the use of LAB in Biotechnology.
CHAPTER THREE
MATERIAL AND METHODS
The project was carried out in microbiology laboratory of department of biological sciences, University of Abuja, Main Campus, along Airport road Abuja.
SAMPLE COLLECTION
Nunu sample was bought from local producers at the Main Campus University of Abuja, Abuja. The sample was transported to the Microbiology Laboratory of University of Abuja for isolation and identification of LAB. The sample was prepared in the Laboratory.
STERILIZATION AND DISINFECTION OF MATERIAL
Work bench was disinfected with cotton wool soaked in 70% ethanol. The glass wares used in the investigation were all wrapped in foil aluminum paper and sterilization in hot air oven at 121oC for 15 minutes.
Sterilization of media and distilled water was done by autoclaving at 121oC and 15psi for 15 minutes.
Sterilization of the inoculating and bent glass rod was done by flaming to redness before and after use.
MEDIA USED
The media in this investigation is de Man Rogosa and sharpe Agar(MRSA).
Preparation Of Media
10grams of de Man Rogosa and sharpe Agar powder was weighed and dissolved in 250ml of distilled water in a conical flask. The solution was boiled to dissolve completely and then autoclaved at 121oC and 15psi for 15 minutes for sterilization, it was allowed to cool before dispensing into the petri dishes.
CHAPTER FOUR
RESULT AND ANALYSIS
ISOLATION AND IDENTIFICATION OF LAB
One sample of “nunu” was used for isolation of Lactic Acid Bacteria labeled 1-4. This isolates involving Gram staining, visual microscopy, catalase test and motility test.
All the isolates were gram positive, rods or cocci, appeared singly, in pairs, in tetrads and chains. Cell were nom-motile, and negative to catalase.
These strains were then classified into genus level using profile matching method, 4 isolates were separate into groups.
CHAPTER FIVE
DISCUSSION, CONCLUSION AND RECOMMENDATION
INTRODUCTION
Many traditional fermented milk products were made in Asia, Africa, the middle East and Northern and Eastern Europe (Savadogo et al.,2004). Studies on the microbiological characteristics of several fermented milk products have been carried out in countries such as Indonesia (Yodoamijoyo et al., 1983; Hosono et al., 1989); Zimbabwe ( Mutukumira,1995;Gadaga et al.,1999); South Africa (Keller and Jordan,1990; Beukes et al.,2001); Morocco (Hamama,1992); Tanzania( Isono et al.,1994); Burkinafaso (Savadogo et al.,2004); Sudan (Abdelgadin et al.,1998 and 1994); Nigeria (Eka and Ohaba, 1977, Atanda and Ikenebom,1989) and Ethopia (O’Connor et al.,1993: O’Mahony,1988).
The nature of fermented products depends on the local indigenous microflora which in turn reflects the climatic conditions of the area (Savadogo et al.,2004). Thus traditional fermented milk in regions with a cold climate contained Mesophilic bacteria such as Lactococcus and Leuconostoc Spp. While Thermophilic bacteria, which include mostly Lactobacillus and Streptococcus prevailed in regions with a hot, subtropical or tropical climate (Thomas, 1985; Tamine and Robinson, 1988; Kurmann, 1984).
This fermented milk product, nunu, are consumed as food and the market value and storage life are improved over that of raw milk Lactic acid bacteria are mostly associated with the production of fermented milk products. They play key role in producing desirable flavour, aroma and good physical appearance in fermented milk product (Tannock, 2004).
LAB starter culture for the production of nunu was isolated from purchased nunu samples which confers desirable flavor characteristics in certain products (Marshall,1987).
Generally, result shown that the pH of all the nono samples decreased as the milk fermented. This agreed with the work of Oyewole (1990). Who stated that the acidity of fermenting milk is normally only noticeable when the pH falls to about 5.5. This showed that the LAB inoculated into the pasteurized milk were able to carry out acid fermentation which resulted in the production of lactic acid. AchiAkobor (2000) and Nout (1991) also reported the decrease in pH during fermentation process of traditional fermented food products.
Fenwema (1985) also established in his earlier report that pasteurization and fermentation with starter culture improve the flavor and taste of milk products.
CONCLUSION
Various LAB do exist in the nunu sample that was collected from the Fulani local producers in the permanent site, University of Abuja, Abuja. The result obtained showed four (4) isolates of Lactic Acid Bacteria which can be grouped into four (4) genera of Lactobacillus, Leuconostoc, Lactococcus and Pediococcus.
The bacteria colonies of LAB counted from the petri dishes concurring with the work of Giraffa (1998).
Lactic Acid Bacteria contributes to the enormous demands on fermented food and has been proven to be used as starter culture in the improvement of nutritional properties, better flavor or aroma, upgrading of substances to higher value products and improved health aspect. Milk fermented with mixed starter culture of LAB had the highest quality and acceptability as was evident in this project work (Fenwema,1985). The result also showed that the pH dropped from 6.29- 5.58 and this is because LAB inoculated into the pasteurized milk were able to carry out acid fermentation which resulted in the production of lactic acid. Also nunu produced with starter culture possess antimicrobial properties and when stored at refrigerated temperature not only improved the flavor but also extended its shelf-life.
RECOMMENDATION
- Lactic acid bacteria contribute to enormous nutritional and health benefits. Extensive work should be performed to discover its secrets and importance in the production of
- The research fund and expertise should be extended and harnessed to achieve this purpose.
- The micro-organisms isolated from nunushould be characterized using modern molecular methods and the technological properties of the dominant types determined to facilitate selection and development of starter cultures from them for the production of nunu, this way the fermentation process will be controlled thereby increasing the quality of the product.
- LAB fermented foods in the diet should be encouraged.
- A standard processing method that will ensure nunuof the highest microbial and nutritional quality should be developed and the technology be transferred to the local producers.
- To avoid self-contamination of product, critical hazard points should be guided jealously during production coupled with maintenance of environmental and personal hygiene.
- Education of producers on good manufacturing practices including basic hygienic principles will equally be crucial in achieving a quality standard product.
REFERENCES
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- Akabanda, F., Owusu-Kwarteng, J., Glover, R. K. L.and Tano-Debrah, K. (2010). Microbiological characteristics of Ghanaian traditional fermented milk product, Nunu. Nature and Science,8: 178–187.
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