The Culture Condition For Maximum Lipase Production By Isolated Micrococcus

Project and Seminar Material for Microbiology

Chapter One

1.0 Introduction

Ugba also called ukpaka is a popular food delicacy in Nigeria especially among Ibo ethnic group. It is rich in protein and is obtained by a solid state fermentation of the seed of African oil bean tree (Pentaclethra macrophyllaBenth). It is essential food item from various traditional ceremonies where it is mixed with slices of boiled stock fish (ugba and okpoloko). The natural fermentation of the seed which at present is still done at the house-hold level, renders the production nutritious, palatable and non-toxic (Enujiugha, 2002).

Its production, like many African fermented foods depends, entirely on mixed fermentation by microorganism from diverse source.

Some of these seeds have been exploited as soup bases such as Gbegiri from Vigna unguicuata (Akanbi, 1992). Others are fermented and used as condiments and seasonings such as okpei from Prosopis africana (Achi, 1992) and ogiri from Ricinus cummunis (Odunka, 1989). Another of such seeds is the African oil bean seed (Pentaclethra macrophylla Benth), a highly nutritious leguminous crop seed abundant in the rain forest areas of west and central Africa.

Pentaclethra macrophylla Benth is a large woody plant abundant in the rain forest areas of west and central Africa. It’s origin in Nigeria is believed to be around 1937 (Ladipo, 1984); where it is found in the South Nigeria, (Mbajunwa et al, 1998).

“Ugba” Pentaclethra macrophylla Benth belongs to the Family Leguminosae and sub-family microsoideae (Keay, 1989 and NFTA, 1995).

Ugba seeds are irregular and oval, they are flat, black and hard pods. It is composed of oil, protein and small amounts of carbohydrate (Obeta, 1982).

Production of Ugba is still on age old traditional family in the rural area. The fermentation depends on random inoculation of boiled slices of the oil bean seed by microorganism within he immediate environment. Earlier report attribute the sources of the micro-organism to the leaves used in packaging, human handling, container and utensils used in processing (Obeta, 1993, Odunfa & Oyeyiola, 1985).

A number of researches have been carried out to know what causes this fermentation, and was attributed to micro-organisms that are probably introduced through the air, water, and banana leaves. Organisms such asMicrococus roseus, Micrococcus luteus, Leuconostoc mesenteroides,Staphylococcus aureus, Staphylococcus epidermidis, Bacillus substilis,Bacillus megaterium and Bacillus circclans, E-Coli

Fermented African oil bean seed (ugba), has a high rate of susceptibility to microbial spoilage and therefore has a very short self life of 1-2 weeks (Enujiugha & Olajundoye, 2001).

Manifestation of food spoilage are many and vary typically resulting in an off smell, colour, taste and texture.

Reports by Mbajunwa (1998) Obeta, (1983) and Nwagu et al. (2010) indicates that micrococcus species do not play an active role during microbial fermentation of Ugba.

However, further work on spoilage association of ugba by Nwagu et al(2010) showed that the population of Micrococcus sp increased with increase in keeping time of ugba. This indicates the ability of micrococcus to thrive in the alkalophilic environment while constituting as a spoilage organism of ugba. This may be attributed to the ability of microorganism to produce lipase, lipase, or protease able to utilize protein, carbohydrate or lipid content of ugba as source of nutrition (Njoku et al., 1990).

Lipase are defined as glycerol ester hydrolases (EC3.1.1.3) hydrolyzing tri-di and mono-glycerides present at oil-water interface (Saxena et al, 1999). Some lipases are also able to catalyze esterification, trans-esterification and enantioselective hydrolysis reaction (Nine et al, 2001, Shintre et al, 2002). The interest in microbial lipase production has increased in the last decades, because of its large potential in a wide range of industrial applications and additives in food processing (flavour modification).

1.1 Aims and Objectives

This work is aimed at the following;

Isolating micrococcus from fermented ugba
Producing lipase from the micrococcus from ugba

Chapter Five

5.0 Discussion, Conclusion and Recommendation

5.1 Discussion

The microorgnism isolated from the fermented African Oil bean seed (Ugba) in this project tally with already known organism isolated from previous studies on the microbiology study of Ugba like those of Orji and Mbata (2008). Interestingly, Table 2 shows the biochemical test used for identification of microorganism. Micrococcus was identified and it was positive Gram stain i.e. (Gram +ve), also it was catalase positive and positive to methyl red test, it was negative in other biochemical test like oxidase test, coagulase test and citrate test.

This result shows that Micrococcus was able to produce lipase and therefore could utilize the oil content of ugba. This also explains the ability to survive the highly alkalophilic environment encountered during post fermentation of ugba (Njoku et al., 1990).

Bacteria involved in the fermentation are probably introduced through the air, water, utensil, banana leaves or by handling during preparatory stages. This is because the initial step of boiling for 16 – 18 hours would kill most of the natural microbial floral of the seeds (Mbata and Orji, 2008). The ability to produce this enzyme will enable the successful colonization of this organisms when introduced in the food, and will enable it to multiply and establish itself as a spoilage association. Micrococcus species have been reported to produce enzymes like lipases, this is in agreement with the work.

African oil bean are largely composed of oil, protein and relatively small amount of carbohydrate (Enujiugha et al., 2005); the ability to breakdown these components of the seed is an important characteristics of organism able to ferment and persist till the seed spoils. This explains why Micrococcus spp and Bacillus spps, which are known to breakdown oil, were prominent throughout the experiment.

This result also shows that Micrococcus can produce lipase in different kinds of medium.

5.2 Conclusion

This study was carried out to determine the culture condition for maximum lipase production by isolated micrococcus from ugba.

Micrococcus isolated from ugba was confirmed as a very good lipase producer of extracellular enzyme. Basal medium gave the most condition for lipase production and therefore the highest lipase yield.

5.3 Recommendation

Proper handling of ugba during production, or product pasteurization after production may be necessary to kill Micrococcus sp in ugba and halt the enzyme production which participates in spoilage. More work is required to further characterize the lipase produced and to determine its usability in biotechnological processes.
Therefore, researchers should take not that in production of lipase enzyme from micrococcus isolated from ugba, Basal medium is best recommended for a higher yield than using other medium like olive oil and Palm oil medium.