Microbiological Evaluation of Bio-Margarine from Three Different Vegetable Oils Using Probiotic Cultures of Lactic Acid Bacteria

Margarine was initially developed as a butter substitute. The product was designed to meet butter shortages caused by increasing urban populations during the industrial revolution as well as to produce a table spread with satisfactory keeping qualities for the armed forces [1]. However, over the years, margarine has established its own image and is used by consumers for a variety of purposes. Margarine is a water-in-oil emulsion produced from a non-dairy product created as a substitute for butter. While originally made from animal fat in 1800s, today the primary ingredient is vegetable oil. While butter is derived from animal fat, margarine is made with vegetable oil(s). This difference has an impact on taste, texture and nutrition. Since margarine primary component is vegetable oil, it lacks the cholesterol and saturated fat found in butter [2]. 

Bio-margarine is a margarine that is inoculated with probiotics (Lactic acid bacteria). Probiotics are live bacteria and yeasts that are good for the health, especially digestive system. Probiotics are often called “good” or “helpful” bacteria because they help keep the gut healthy. In recent years, different investigations support the importance of probiotics as part of healthy diet for humans and animals and as a way to provide a natural, safe and effective barrier against microbial infections [3]. According to the definition by the World Health Organization (WHO), Probiotics are “live microbial food supplements which, when administered in adequate amounts confer a health benefit on the host” [4]. Among the usually used microorganisms, Lactic Acid Bacteria (LAB) are regarded as a major group of probiotic bacteria. They are non-pathogenic, technologically suitable for industrial processes, acid tolerance and bile tolerance and produce antimicrobial substances [5]. They are classified as “Generally Recognized As Safe” (GRAS) microorganisms because of their long and safe use as starter cultures in fermented products [4]. 

Coconuts are known for their great versatility as evidenced by many traditional uses, ranging from food to cosmetics. They form a regular part of the diets of many people in the tropics and subtropics. Nutritionally, per 100gram serving of raw coconut meal supplies a high amount of total fat (33g), especially saturated fat (89% of total fat) and carbohydrates (24g). Coconut is being used by confectionaries, bakeries, biscuits, ice cream industries worldwide to enhance flavor and taste of various products. Coconut juice was found to be rich in calcium, protein and fat [6]. 

Melon seeds are globally popular and are valued for their sensory, nutritional and health attributes [7]. Apart from the nutritional components of melon, it also possesses other health benefits such as anti-cancer properties, heart health, cures kidney disease, maintains healthy skin, helps in weight loss, has anti-aging properties, promotes hair growth etc. [8]. 

Palm oil is a common cooking ingredient in the tropical belt of Africa, Southeast, Asia and parts of Brazil. It is used in the commercial food industry in other parts of the world and is widespread because of its lower cost and the high oxidative stability (saturation) of the refined product when used for frying [9]. 

There is a great increase in health risks associated with the consumption of animal fat which increases cholesterol level in human. In line with this, this research work was carried out in other to produce a bio-margarine which is enriched with probiotics using available local plant materials, since margarine primary component is vegetable oil and it lacks the cholesterol and saturated fats found in commercial butter. The objective of the study is to produce and evaluate the quality of bio-margarine from three different vegetable oils: palm, coconut and melon oils using probiotic cultures of lactic acid bacteria.

Sample Collection and Preparation 

Palm fruits (Elaeisguineensis), coconut (Cocosnucifera) and melon seeds (Citrullus colocynthia) from where the oils to be used for margarine production were obtained from Aba, New Market, Abia State, Nigeria. Commercial yoghurt from which Lactic Acid Bacteria (LAB) was isolated were obtained from a dairy product hawker in MOUAU Campus (Figure 1).

Figure 1: Coconut, palm fruits and melon seeds from where the oils to be used for margarine production.

Isolation and Characterization of Lactic Acid Bacteria

• Culture Media Preparation

The media MRS (de man, Rogosa and Sharpe) broth, MRS agar, nutrient agar, MacConkey agar and Peptone water were homogenized and sterilized at 121°C for 15minutes and was cooled to the temperature of 45°C before use. 

• Isolation of Microorganisms 

Serial dilutions were made from the samples using a sterile pipette. This was done by mixing 1ml of the homogenized sample to 9ml of sterile peptone water to give 1:10 dilution, the dilution was continued up to 10^-4. Using sterile pipette, 0.1ml of appropriate dilutions was inoculated onto the MRS agar, MacConkey agar and nutrient agar plates. The plates were incubated at 37°C for 48hours. At the end of the incubation, representative colonies of the various isolates were selected at random and sub cultured repeatedly to obtain a pure culture [10]. 

• Culture Preservation 

Cultures of LAB isolated were sub cultured into nutrient medium. The stock cultures were stored on agar slants at 4⁰C for subsequent use.

Characterization of Isolates 

• Gram’s Staining 

The pure isolate was stained as described by Beveridge [11]. A thin smear of the isolate was made on a clean glass slide and heat-fixed by flaming. Two drops of crystal violet were added to the smear for 1 minute. The drops of crystal were washed with water and stained with Gram's iodine solution for 1 minute. The stain was decolorized by flooding the slide with alcohol until no more violet colouration was observed. Two drops of Safranin reagent were added for 10 seconds rinsed again with tap water and blotted dry using a filter paper. Observation was made using oil immersion objective microscope. Gram-positive bacteria were characterized by purple coloration while gram-negative cells were pinkish in colour. This staining technique also shows the different shapes and arrangement of the bacteria cells.

Biochemical Test 

• Catalase Test 

Two to three (2-3) drops of 3% hydrogen peroxide was placed on a clean grease-free slide using a pipette and a loopful of pure culture of the isolates were added and emulsified. Formation of visible bubbles was taken as a positive test while absence of bubbles was regarded as a negative test [10]. 

• Methyl Red Test 

The isolates were inoculated into various MR-VP (Methyl Red Voges- Proskauer) broth bottles and appropriately labeled. The bottles were incubated for five days. After incubation, five drops of methyl red were added to the bottles for methyl red test. A positive test is indicated by appearance of red color. Negative test is indicated by a yellow coloration of the media [10].

• Voges-Proskauer (VP) Test 

The isolates were inoculated into various MR-VP broth bottles and appropriately labeled. The bottles were incubated for five days. After incubation, 1ml of the MR-VP broth with test organisms was transferred to sterile test tubes. Fifteen (15) drops of VP reagent A were added to the test tubes followed by 5 drops of VP reagent B. A positive test was indicated by appearance of pink-red color. Negative test was indicated by a light yellow coloration of the media [10]. 

• Indole Test 

The isolates were inoculated into a bijou bottle containing 3ml of sterile tryptone broth and incubated at 35-37°C for 48 hours. Indole production was tested by adding 7 drops of Kovac’s reagent and examined for formation of a ring of red colour in the surface layer within 5 minutes. A ring of red layer indicates a positive test. Absence of a ring of red layer indicates a negative test [10]. 

• Citrate Utilization Test 

This was used to detect the ability of each isolate to use citrate as its sole source of carbon and energy. Slopes of Simmons citrate agar were prepared in bijou bottles following the manufacturer’s directives. Using a sterile straight wire, the slope was first streaked with a saline suspension of the isolates and then the butt stabbed and incubated at 35°C for 48 hours. A bright blue color was recorded as a positive test [10].

Technological Properties of Lab Isolates (for Selection of Suitable Isolates) 

• Hydrogen Sulphide (H₂S) Production 

Hydrogen sulphide (H₂S) production test is used for the detection of hydrogen sulphide (H₂S) gas produced by an organism. An inoculum from each isolate was transferred aseptically to a sterile Triple Sugar Iron Agar (TSIA) slant. The inoculated slants were incubated at 35^oC for 24hours and the results observed. Hydrogen sulfide combines with the iron in the media to produce iron sulfide (FeS). The presence of H₂S producing organism is detected by the turning of the agar slants to black color. 

• Determination of Lactic Acid, Hydrogen Peroxide and Diacetyl Production by LAB Isolates 

For accurate measurements, the test organisms were grown in MRS broth. The broths were inoculated with 0.1ml of a suspension of LAB specie and incubated. Incubation was for 72h at 37°C. Cultures were centrifuged at 3000rpm for 15min. Known volume of the supernatant fluid was used for all the titrations at specified time interval [12]. 

• Quantitative Estimation of Lactic Acid 

The quantity of lactic acid produced by antimicrobial producing isolates at 24hr, 48hr and 72hr were determined by transferring 25ml of broth cultures of test organisms into 100ml flasks. The production of lactic acid was determined by titration with 0.25mol/L NaOH and 1ml of phenolphthalein indicator. The titratable acidity was calculated as lactic acid (% v/v). Each millilitre of IN NaOH is equivalent to 90.08mg of lactic acid. The titratable acid was then calculated according to AOAC [10].

Titratable acidity (% lactic acid) :  (ml NaOH x N NaOH x M.E. x 100) / Volume of sample used (ml)

Volume of sample used (ml)

• Determination of Diacetyl Formation 

Diacetyl production was determined by transferring 25ml of broth cultures of test organisms into 100ml flasks. Seven and half millilitres of 1M Hydroxylamine solution was added to the flask and to a similar flask for residual titration. Both flasks were titrated with 0.IN HCL to a greenish yellow end point using bromophenol blue as indicator. The equivalence factor of HCl to diacetyl is 21.52mg. The concentration of diacetyl produced was calculated according to the method of Food Chemicals Codex.

AK = (b – s) (100E) / W

AK=Percentage of diacetyl; b=No of ml of 0.IN HCl consumed in titration of sample; E=Equivalence factor; W=Volume of sample; s=No of ml of 0.IN HCl consumed in titration of sample [10].

• Quantitative Estimation of Hydrogen Peroxide Formation 

Hydrogen peroxide production was determined by transferring 25ml of broth cultures of test organisms into 100ml flasks. Twenty millilitres of dilute sulphuric acid was added to 25ml of the supernatant and titration was carried out with 0.1M potassium permanganate, which is equivalent to 1.7mg of hydrogen peroxide. A decolourization of the sample was regarded as the end point [10].

H₂O₂+2KMnO₄+3H₂S0₄→K₂SO₄+4H₂O+O₂

H₂O₂ = (ml KMnO₄ x NKMNO₄ x M.E x 100) / (ml H₂S0₄ x Vol of sample)

ml KMnO₄ =Volume of Sample used

NKMnO₄ =Normality of KMnO₄

ml H₂SO₄ =Volume of H₂S0₄ used

M.E: Equivalent factor.

Acidification Activity

MRS broth was inoculated with a 24h old culture of each isolate and incubated at 37ºC for 72h. At intervals of 24, 48 and 72h, the culture was centrifuged at 3000 × g for 5min and the supernatant recovered was used for pH measurement using a pH meter. Change in pH was calculated as the change in pH from an initial 6.05, which was the pH of the MRS broth at the time of inoculation (0h).

Extraction of Oils 

Melon seed and coconut oils were produced using method of extraction described by AOAC [10]. Palm fruit oil was produced using the method described by Enyi and Ojimelukwe [13]. The oils were rationalized in discrete portions of ratios to obtain 9 samples of composite oil: all containing coconut, melon and palm oils in part (Table 1 & Figure 2).

Table 1: Blends formulation.

Figure 2: Samples of palm fruit oil, melon seed oil and coconut oil.

In line with design of the experiment the last three oils ratios were inoculated with LAB isolates before conversion to margarine. These were so because their melon and palm oil content were relatively higher. These experimental adjustment produced samples MAG 007, MAG 008 and MAG 009. Test sample 010 was a commercially available margarine used as a control.

Production of Margarine from Blends of Palm Fruit, Melon and Coconut Oil 

Margarine was produced according to the method described by Sayed et al. [14]. A basic recipe that included 81.7% oil blend, 0.3% emulsifier, 16% water, 0.8% salt, 0.9% skim milk powder, 0.2% flavour, 0.01% antioxidant and 0.003% colour was used for margarine production. Emulsifier, antioxidant, flavor and color were dissolved in the heated oil phase. Salt and skim milk powder were dissolved in the water phase. The water phase was added gradually to the oil phase while agitating it to form a nice emulsion. For the solidification of margarine, the emulsion was stirred for 10 minutes and then cooled in ice bath containing 10% sodium chloride (NaCl). The emulsion was then mixed and solidified at a temperature of 11°C. All samples were packaged carefully in plastic containers and analyzed for microbial growth. Samples MAG 007, MAG 008 and MAG 009 were set apart for inoculation with the chosen lactic acid bacteria isolates (Figure 3). 

Figure 3: Samples of Biomargarine 001-008. 

Inoculation of Margarine with Chosen Lactic Acid Bacteria (Starter Culture) 

Inoculation of the isolate was done by pour plate method as described by Ezeama [15]. LAB isolate with desirable Technological properties were selected and grown on nutrient agar plates and incubated for 24hrs. After incubation, the resulting colonies were incubated for 24h on MRS broth. The overnight grown cultures were then inoculated into the various sterile margarine samples in 5ml proportions using a sterile needle and syringe. The inoculated margarine samples were appropriately stored at room temperature for 72hr and examined microbiologically. 

Microbiological Analysis 

The microbiological examination of the margarine samples was carried out using the pour plate method as described by Ezeama [15]. The samples were examined for total viable count, total coliform count, and total fungal count during storage on two day interval for twelve (12) days.

Morphological and Biochemical Characteristics of Isolates 

Three (3) probiotic starter cultures were isolated in this study from commercial yoghurt. The results of the phenotypic and biochemical characterization of the LAB isolates are shown in table 2. The results reveal that all isolates were Gram-positive, catalase-negative and non-endospore forming. Based on their phenotypic and biochemical characteristics, the isolates were presumptively identified to belong to the genus Lactobacillus; this was done with particular reference to Bergey’s Manual of Systematic Bacteriology, based on their phenotypic profiles. The isolation of the isolates is in agreement with the work of Buchanan and Gibbons [16], who isolated six (6) lactic acid bacterial species based on their colonial morphology and biochemical characteristics. One isolate was cocci. The cocci were presumptively identified to belong to genus Streptococcus with reference to Bergey’s Manuel of Systematic Bacteriology (2000), this is supported with the work of Ifeanyi et al. [17], in which only two LAB which comprise the starter cultures (Streptococcus thermophilus and Lactobacillus bulgaricus) were isolated from yoghurt samples. 

Table 2: Morphological and biochemical characteristics of the lactic acid bacteria isolates. 

Note: MR = Methyl red, VP: Voges prokauer, SH: Starch hydrolysis

+ Gram-positive

– Gram-negative

Technological Properties of the Isolates 

• Metabolites of the Isolated Microbes 

The technological properties of the isolates are shown in table 3. Technological properties were investigated with a view of selecting appropriate starter cultures for use in the bio-margarine production. 

Table 3: Metabolites of the Microbial Isolates. 

Hydrogen peroxide produced by the LAB strains ranged from 0.006 to 0.09mg/l, the highest was produced by Enterococcus sp lactobacillus at 72 hours of incubation. The quantity of hydrogen peroxide produced by the test isolates varies among the species of the organisms. Hence, it was observed that as the incubation period increased, the quantity of hydrogen peroxide (H₂O₂) produced by three of the isolates (L. acidophilus, S. thermophiles and L. bulgaricus) increased from 24 to 48 hours while the quantity produced by the rest of the isolates increased from 24 to 72 hours. Among the microbial isolates, Enterococcus sp lactobacillus produced the highest concentration of hydrogen peroxide (0.09mg/l) and which was lower than the value reported in the study of Damisa-Okorhi and Ataikiru [18], who obtained a value of 1.91mg/l of hydrogen peroxide production by Lactobacillus. This difference may be attributed to possible difference in lactose-hydrolyzing/galactosidase enzyme activity in metabolic activities of the strains and media used. 

The quantity of lactic acid produced by the LAB strains ranged from0.38-1.38mg/l, LAB 2 (L. fermentum) isolate was the highest producer at 72 hours of incubation. Reasonable quantity of lactic acid was produced by the isolates, and this was in agreement with the report of Pinthong et al. [19], who reported that lactic acid bacteria could lead to products with sufficient acidity (low pH) for good keeping properties through production of organic acids. Lactic acid is a major product of fermentation of carbohydrate by lactic acid bacteria, and this was evident in the isolates under investigation. This is an important technological property of LAB which may impart positively on the quality attributes of thebio-margarine, especially shelf-stability, aroma and flavors characteristics [20]. 

The Diacetyl (DA) produced by the isolates ranged from 0.38-0.78mg/l. The highest diacetyl was produced after 72 hours of incubation by Streptococcus thermophilus (LAB 3) isolate. Reasonable quantity of dicaetyl was produced by the presumptive LAB isolates. Diacetyl has a strong, buttery flavor and is essential at low concentrations in many dairy products, and other functional food products. However, based on the performance of the presumptive Lactobacillus stains of the presumptivestrains in terms of diacetyl production, it seems they could make a good bio-preservative culture. This is as a result of the strains’ production of comparatively higher concentrations of the antimicrobial than the Enterococci strains. Production of relatively high DA concentration has been observed to contribute significantly to exertion of antagonism by LAB species against most unwanted organisms [21]. 

The acetic acid produced by the isolates ranged from 0.34 to 1.26mg/l. The highest acetic acid was produced at 72 hours of incubation by LAB 4 (Lactobacillus plantarum) isolate. There was an increase in the acetic acid produced by the isolates with increase in fermentation time, leading to proportional decrease in pH. This was in line with the work of Harper and Collins [22], who reported an increase production of acids (lactic acid and acetic acid) with a decrease in pH during production of furundu (a traditional fermented Sudanese roselle from Hibiscussabdariffa L.seed). The production of reasonable level of acid by LAB will also help improve the flavor of the product. Lactobacillus fermentum had the highest rate of acidification with a pH of 3.7 in 24 hours of incubation while LAB 3 and 8 (Streptococcus thermophilus and Enterococcus sp lactobacillus) had the least of acidification with equal pH of 4.8 at 24 hours of incubation which reduced to pH values of 3.8 and 3.7 respectively at 72 hours of incubation. Enterococci spp. was however, poor producers of lactic acid and diacetyl compared to their Lactobacilli counterparts. Hence, the potential antimicrobial activities of the latter strains could make them better potential starter cultures in food production and in preventing the growth of undesirable spoilage and pathogenic bacteria during food preservation processes [23]. 

The early production of comparatively higher concentrations of lactic acid by lactic acid bacteria could serve as an important factor in competitive elimination of unwanted organisms [24]. From the result of this study, Latobacillus fermentum produced a high amount of lactic acid (0.84mg/l) at 24h of incubation and this may be very significance in food bio-preservation processes [25]. 

The fast acidifying strains of LAB could be good candidates for fermentation process as primary starter culture in comparison with poor acidification strains [26]. Similarly, it has been reported that the faster the decrease in pH to <4, the faster the growth inhibition of the fermenting medium against pathogens such as Salmonella spp. [21]. 

Reduction in pH during the fermentation is usually due to the fermentative transformation of carbohydrates to lactic acid and acetic acid by LAB. This could account for the results obtained in this study; LAB 1 (Lactobacillus acidophilus) showed a reduction in pH from 4.5 to 3.8, LAB 2 (L. fermentum) from 3.7 to 3.2 and LAB 3 (Streptococcusthermophilus). From 3.8 to 4.8 during fermentation. In addition to lowering the pH and acid production (acetic, lactic and carbonic), LAB contribute to food preservation by the production of a vast array of antimicrobial compounds and proteins [27]. 

Lactic acid bacteria are present in fermented foods because they are able to survive under high acidic conditions and also have the ability to produce a high level of lactic acid. Reasonable amount of lactic acid was produced as a major end product of fermentation of carbohydrate by the screened isolates. This gives the product more stable shelf quality with characteristic aroma and flavors. 

In these current study, Lactobacillus acidophilus, Streptococcus sp. and L. bulgaricusstrains produced considerable concentrations of organic acids and other technological properties, and were therefore selected as potential starter cultures for the production of bio-margarine. The selection was in agreement with the reports of various previous studies [26].

Qualitative Determination of Bile Salts Hydrolases Activity 

Bile salts at a concentration of 0.3% had different degrees of inhibition on the 3 tested strains of lactic acid bacteria used in this study. The results were analyzed using the standards: resistant strains (d≤15min), tolerant strains (15<d≤40min), weakly tolerant strains (40<d<60min), and tolerant strains (d≥60min). Twenty-one (72%) of the tested strains resisted 0.3% bile; their tolerances to bile are showed in table 3. Two strains (Lactobacillus acidophilus and Lactobacillus bulgaricus) were considered to be tolerant strains. Kalui et al. [28], reported that 18 of the 19 L. plantarum tested were able to grow in broth supplemented with 0.3% bile salts following exposure to pH 2.5. [29]. Showed that, among several strains of L. acidophilus and Bifido bacterium studied, only a few strains survived under the acidic conditions and bile concentrations normally encountered in fermented products and in the gastrointestinal tract, respectively. Their findings indicated that tolerance to acid and bile salt is strain specific. Lactobacillus fermentum RS-2 strain was reported that were able to tolerate upto1.0% bile concentrations efficiently. Succi et al. [30], reported that L. rhamnosus RSI3 strain was able to tolerate up to 1.0% bile concentrations with less than 2 log cycle reduction in their cell counts. Inthe present study 3 isolates that survived at the pH 2.0 and 3.0 were further screened for their ability to grow at 3% of bile concentration-a qualitative assay to address resistance to bile acid. The effect of bile on the viability of lactobacilli isolates is presented in table 4. 

Table 4: Qualitative determination of bile salts hydrolases activity (Min). 

Note: D = Degree of inhibition to bile salts hydrolases activity

+ = Positive resistant to bile salts hydrolases activity 

Antimicrobial Activity 

Table 5 shows the antibacterial activity of Lactobacilli as a zone of inhibition against these pathogens. The extracellular overnight-spent supernatant of Lactobacilli exhibited varying zones of inhibition from 19mm to 31mm depending upon the tested pathogen and Lactobacilli isolates. 

Table 5: Antimicrobial activities of lactic acid bacteria strain. 

Note: + = Low inhibition (1mm)

++ = Moderate inhibition (3mm)

+++ = Very high inhibition (5mm) 

In this study, growth inhibition as observed on agar-well indicates that the assayed lactobacilli produced antimicrobial products that were able to inhibit growth of Streptococcus pyogenes, E. coli, S. aureus, Candida albicansall of which are pathogens. Abhijit et al. [31], reported that antimicrobial activities of the tested Lactobacillus spp. had broad inhibitory spectrum, against yeast and bacteria both of gram-negative and gram-positive. Antimicrobial activity is thought to be an important means for probiotic bacteria to competitively exclude or inhibit activities of harmful or pathogenic intestinal microbes. It is generally believed that the resident gastrointestinal microflora in vivo provides protection for the host against possible colonization by pathogenic bacteria. Several reports have been documented on the ability of probiotic lactobacilli and bifidobacteria to inhibit the cell association and invasion by pathogenic bacteria. L. acidophilus has been reported to inhibit gram negative and gram-positive pathogens, such as Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium, and Enterobacter cloacae [32].

Microbial Evaluation 

• Microbial Counts of Margarine Samples Stored for Various Days 

The load of various microbes in the margarine samples stored on day 1 (zero day) were below 10cfu/g with the exception of the samples MAG 007, MAG 008 and MAG 009 which had total viable counts, total Lactobacillus count and total Staphylococcus ranges from of 4.8x10⁶–5.4x10⁶cfu/g, 4.6x10⁶-5.2x10⁶cfu/g and 2.0x10⁵–2.0x10⁵cfu/g respectively (Table 6). 

Table 6: Microbial counts of the bio-margarine samples. 

Note: TVC, total viable count, TFC, total fungal count, TLC, total lactobacillus count, TSC, total staphylococcus count, TCC, total coliform count 

The microbial load of bio-margarine samples wereseen to be dominantly Lactobacillus with mild Streptococcus strains in culture plates (or culture). The day one product of MAG 007 gave 95.8% Lactic acid bacteria and 4.20% of Streptococcus. Sample MAG 008 yielded 96.1% Lactobacillus and 4.90% Streptococcus. MAG 009 gave 96.3% Lactobaccillus and 4.7% Streptococcus for O hour. 

After day 3 (72 hours) storage of the products, it was observed that there were progressive rise in TVC, TLC and TSC of samples MAG 007, MAG 008 and MAG 009 while the other samples showed no significant growth. For instance, the Total Viable Count (TVC) of the samples MAG 007, MAG 008 and MAG 009 increased from 1^st to 3^rd day of storage by 1.8x10⁶cfu/g respectively. This state could be likened to the stationary phase of growth of the starter flora inoculated into the margarine sample. On the six day of storage, the number of microorganisms in the margarine samples uninoculated with LAB remain below 10cfu/g while the TVC, TLC and TSC in the margarine samples inoculated with the three strains of LAB reached their peak values which never changed after the 10^th and 12^th days of storage of the samples. 

The microbial load remained at this level throughout the period of experimentation. The deduction here indicated that the starters were able to make use of the nutrient matrix of the margarine to undergo growth and multiplication.

Since they were majorly protein concentrate the protein proximate of MAG 007 and MAG 008 increased progressively along storage line. Those of the uninoculated margarine products remain unchanged as there were no microbial growth dictated in them. From table 2, the data concerning microbial colonies were recorded using a colony counter while the morphological characteristics and the cultural properties were carried out using a compound binocular microbial microscope. In this table 2, that showed the morphological and cultural characteristics considered. The identifying microbes were cell shape, cell structure, cellular arrangement, presence of spores, cell mortality, colony color, colony appearance, colony edge, cell appendage, presence of pigment and talli. It was observed that Lactobacillus bulgaricus, L. acidophillus and streptococcus thermophilus were culturally and morphologically seen dominating samples MAG 007, MAG 008 and MAG 009 respectively. Other microbes that would have occurred in the uninoculated margarine samples were viewed to be destroyed by processing treatment.

MAG 001, uninoculated margarine with 90% coconut, 2% melon and 8% palm oils; MAG 002, uninoculated margarine with 80% coconut, 4% melon and 16% palm oils; MAG 003, uninoculated margarine with 70% coconut, 8% melon and 22% palm oils; MAG 004,uninoculated margarine with 60% coconut, 10% melon and 30% palm oils; MAG005, uninoculated margarine with 50% coconut, 20% melon and 30% palm oils; MAG006, uninoculated margarine with 40%, 30% and 30% palm oils; MAG 007, margarine with 60% coconut, 10% melon and 30% palm oils inoculated with L. acidophilus MAG 008, margarine with 50% coconut, 20% melon and 30% palm oils inoculated with S. thermophilus; MAG 009, margarine with 40% coconut,30% melon and 30% palm oils inoculated L. bulgaricus; MAG 010 (Table 7).

Table 7: Microbial counts of the bio- margarine samples. 

Note: TVC, total viable count, TFC, total fungal count, TLC, total lactobacillus count, TSC, total staphylococcus count, TCC, total coliform count

MAG 001, uninoculated margarine with 90% coconut, 2% melon and 8% palm oils; MAG 002, uninoculated margarine with 80% coconut, 4% melon and 16% palm oils; MAG 003, uninoculated margarine with 70% coconut, 8% melon and 22% palm oils; MAG 004, uninoculated margarine with 60% coconut, 10% melon and 30% palm oils; MAG005, uninoculated margarine with 50% coconut, 20% melon and 30% palm oils; MAG006, uninoculated margarine with 40%, 30% and 30% palm oils; MAG 007, margarine with 60% coconut, 10% melon and 30% palm oils inoculated with L. acidophilus MAG 008, margarine with 50% coconut, 20% melon and 30% palm oils inoculated with S. thermophilus; MAG 009, margarine with 40% coconut, 30% melon and 30% palm oils inoculated L. bulgaricus; MAG 010 (Table 8).

Table 8: Microbial counts of the bio-margarine samples. 

Note: TVC, total viable count, TFC, total fungal count, TLC, total lactobacillus count, TSC, total staphylococcus count, TCC, total coliform count 

MAG 001, uninoculated margarine with 90% coconut, 2% melon and 8% palm oils; MAG 002,uninoculated margarine with 80% coconut ,4% melon and 16% palm oils; MAG 003,uninoculated margarine with 70% coconut,8% melon and 22% palm oils; MAG 004,uninoculated margarine with 60% coconut,10% melon and 30% palm oils; MAG005,uninoculated margarine with 50% coconut,20% melon and 30% palm oils; MAG006,uninoculated margarine with 40%,30% and 30% palm oils; MAG 007, margarine with 60% coconut,10% melon and 30% palm oils inoculated with L. acidophilus MAG 008,margarine with 50% coconut,20% melon and 30% palm oils inoculated with S. thermophilus; MAG 009, margarine with 40% coconut,30% melon and 30% palm oils inoculated L. bulgaricus; MAG 010 (Table 9).

Table 9: Microbial counts of the bio-margarine samples. 

TVC, total viable count, TFC, total fungal count, TLC, total lactobacillus count, TSC, total staphylococcus count, TCC, total coliform count 

MAG 001, uninoculated margarine with 90% coconut, 2% melon and 8% palm oils; MAG 002,uninoculated margarine with 80% coconut ,4% melon and 16% palm oils; MAG 003,uninoculated margarine with 70% coconut,8% melon and 22% palm oils; MAG 004,uninoculated margarine with 60% coconut,10% melon and 30% palm oils; MAG005,uninoculated margarine with 50% coconut,20% melon and 30% palm oils; MAG006,uninoculated margarine with 40%,30% and 30% palm oils; MAG 007, margarine with 60% coconut,10% melon and 30% palm oils inoculated with L. acidophilus MAG 008,margarine with 50% coconut,20% melon and 30% palm oils inoculated with S. thermophilus; MAG 009, margarine with 40% coconut,30% melon and 30% palm oils inoculated L. bulgaricus; MAG 010 (Table 10).

Table 10: Microbial counts of the bio-margarine samples. 

Note: TVC, total viable count, TFC, total fungal count, TLC, total lactobacillus count, TSC, total staphylococcus count, TCC, total coliform count 

MAG 001, uninoculated margarine with 90% coconut, 2% melon and 8% palm oils; MAG 002, uninoculated margarine with 80% coconut, 4% melon and 16% palm oils; MAG 003, uninoculated margarine with 70% coconut, 8% melon and 22% palm oils; MAG 004, uninoculated margarine with 60% coconut, 10% melon and 30% palm oils; MAG005, uninoculated margarine with 50% coconut, 20% melon and 30% palm oils; MAG006, uninoculated margarine with 40%, 30% and 30% palm oils; MAG 007, margarine with 60% coconut, 10% melon and 30% palm oils inoculated with L. acidophilus MAG 008, margarine with 50% coconut, 20% melon and 30% palm oils inoculated with S. thermophilus; MAG 009, margarine with 40% coconut, 30% melon and 30% palm oils inoculated L. bulgaricus; MAG 010.

The findings of this study established that margarine samples with total microbial counts comparable to a commercial margarine sample can be produced from blends of palm, melon seed and coconut oils especially when inoculated with species of lactic acid bacteria. Inoculation of lactic acid bacteria was able to reduce total viable, total fungi, total lactobacillus, total streptococcus and total coliform counts when compared to the non inoculated samples. The use of lactic acid bacteria in margarine production could be very beneficial especially as this has the potential of advantageously replacing the commercial margarine, since this bio-margarine (margarine enriched with probiotics) could have tremendous health benefits. From the results of this study, the use of L.acidophilus, L.bulgaricus and S.thermophilus enhanced quality attributes of the margarine.

The authors thank all those that played different roles in seeing that this research work was a success.

The authors declare no conflict of interest.

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