ASSESSMENT OF THE FUNCTIONAL QUALITY AND SAFETY OF YOGHURTS PRODUCED WITH STARTER CULTURES OBTAINED FROM SELECTED COMMERCIALLY SOLD YOGHURTS

The study focused on the examination of laboratory prepared yoghurts which were fermented with selected starter cultures from commercially sold yoghurt. The starter cultures were molecularly identified (16s rRNA) as Enterococcus lactis, Lactobacillus plantarum, Lactobacillus pentosus, Pediococcuspentosaceus and Enterococcus durans. The isolates were examined for bile tolerance as an indicator of their ability to survive in the gut. The starter cultures were used to produce different yoghurts in the following order: Enterococcus lactis produced yoghurt, L. plantarum and L. pentosus produced yoghurt, Pediococcuspentosaceus produced yoghurt, E. durans produced yoghurt and yoghurt produced with all starter cultures. All yoghurts were examined for nutritional quality (vitamin A, B12 and C content, soluble and casein bound magnesium and calcium and proximate nutrient composition). At p ≤0.05, there was statistical significant difference in the nutritional content with P. pentosaceus contained yoghurt, E. durans contained yoghurt and yoghurt produced with a combination of all isolates recording the highest nutritional values and the lowest was observed with the control. Safety tests such as haematology and histology were carried out on wistar rats. After 7 days of feeding the rats in groups with the different yoghurts and a control without yoghurt, there were marked improvements in the red blood cell counts, white blood cell counts but no significant difference in the differentials at p ≤0.05. The isolates were also observed to have no disruptive effect on the morphology and structure of the small intestine. Overall, the use of these lactic acid bacteria strains showed immense benefits in their use as starter cultures and the study demonstrated safety of the final products for


INTRODUCTION
Yoghurt is a fermented milk product obtained from the milk or the milk products by the lactic acid fermentation through the action of Streptococcus salivarius subsp.Thermophilus, Lactobacillus delbrueckii subsp.Bulgaricus (FAO/ WHO, 1977).When a sufficient quantity of lactic acid is produced then the milk coagulates and this coagulated milk is called yoghurt.The probiotic yoghurt, having probiotic effect is a fermented milk product with adjuvant microorganisms.There are numerous advantages of consuming fermented dairy products containing probiotic bacteria.A high population of probiotic organisms in the colon contributes to good intestinal health.Consequently, consumption of products such as yoghurt containing viable probiotic organisms adds benefit to human gut health.Moreover, yoghurt supplies good quality proteins, also an excellent source of calcium, phosphorus, potassium and contains significant quantities of general vitamins.Yoghurt could be used for feeding, owing to its higher Ca/Na ratio (Demott, 1985).
Yoghurts vary in appearance, flavor and ingredients.The quality and composition of yoghurt of applied bacterial cultures affects the quality of the yoghurt obtained as the result of the milk fermentation processes.There is a symbiotic relationship between the two species of bacteria i.e Lactobacillus bulgaricus and Streptococcus thermophilus that is why there is more rapid acid development than in the single strain culture (Rasic et al., 1978;Tamime et al., 1980).
Various combinations of starter cultures are selected during manufacturing of yoghurt to achieve desirable characteristics of product and also to provide the consumers with a wide choice of therapeutic benefits.Depending on its activity, manufacturer usually adds 2 -4% yoghurt starter culture.These days, there has been increasing trends to fortify the dairy product with fruits (natural fruit juice, pulp, dry fruits) (Desai et al., 1994; Ghadge et al., 2008).Aesthetic value of new product can be increased by using fruit juice as a functional pigment in fermented milks with array of colors and flavor properties.Coissonet al., (2005) used Euterpeoleracea juice as functional pigment for yoghurt, which is dark purple in color having high anthocynin and phenolic content.
Yoghurt is a functional food.The functional food includes probiotics, prebiotics and synbiotics.Probiotics can be defined as "live microbial feed supplements that beneficially affect the host animal by improving its intestinal microbial balance" (Champagne et al., 2005).
Prebiotics is defined as "non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon".Symbiotic is a combination of probiotics and prebiotics that "beneficially affects the host by improving the survival and the implantation of live microbial dietary supplements in the gastro-intestinal tract by selectively stimulating the growth and/or by activating the metabolism of one or a limited number of health promoting bacteria" (Di Rienzo et al., 2000).
Yoghurt is one of the oldest fermented milk products known.Fermentation of milk involves the action of microorganisms, principally the lactic acid bacteria.These microorganisms sour the milk by converting the milk sugar lactose to lactic acid (Kagan, 1985).Yogurt gels are built of clusters of aggregated casein particles formed as a result of gradual fermentation of lactose by lactic acid bacteria (Horne, 1993).The

Scientific hypothesis
The present study investigates the functional quality and safety of yoghurts produced from starter cultures obtained from randomly sampled commercial yoghurts and conducted with the following objectives: 1. Molecular identification of starter cultures Yoghurt production with identified starter cultures 2. Evaluation of physicochemical and nutritional properties that contribute to final product quality such as pH, titratable acidity, reducing sugar concentration, vitamin content, mineral composition and proximate nutrient composition 3. Assessment of probiotic potential of the yoghurts with antibacterial assay and safety to the intestine

MATERIAL AND METHODS
The media that were used in this work include nutrient agar, Macconkey agar, Eosinemethyleneblue agar (EMB), peptone water.The composition of the media, their method of production and the list of other equipment and reagents used in this work are presented in the appendix.

Sample Collection
Six different brands of bottle packaged yoghurt were brought from hawkers and beverage stores in Ikeji Arakeji, Osun State.Isolates from each brand was used and the brands were designated A, B, C, D and E. The samples were brought to the laboratory, stored in the refrigerator and analyzed within 6 hours of collection.

Analysis of Sample
Each sample was serially diluted using sterile distilled water as diluents according to (Prescott et al., 2002) and 1 milliliter of 10 -3 sample was plated in duplicate using the pour plate method on nutrient agar media.The plates were incubated at 37 °C for 24hrs.After incubation the colonies developedon the nutrient agar plates were counted and used to determine the total bacterial count of the yoghurt samples (CFU.ml - ).The representative colonies on the plates were sub-cultured on fresh nutrient agar to obtain pure cultures of the isolates.The pure cultures were then transferred into nutrient agar slants for molecular characterization.

Identification of Isolates
Molecular identification of the lactic acid bacteria isolate involved Denaturing Gradient Gel Electerophoresis, Polymerase Chain reaction and pure sequencing of 16s rRNA (bacteria) genes as described by Akabanda et al.

Inoculum Preparation
Pure cultures of the bacterial isolates were inoculated in sterilized lactose broth and incubated at 37 o C for 18 hours

Preparation of Yoghurt
One hundred milliliteroutof whole milk collected from lactating cow was poured into a 500 mL beaker; was brought to boiling point without being allowed to boil and immediately cooled to 60 °C.3.5 grams of non fat dry milk was added to the milk and stirred vigourously with a glass rod to dissolve the powder.The mixture was allowed to cool to 45 °C. 10 milliliter of 18 hour culture of the lactic acid bacteria was added in single, double and allied fermentation trials.This was done separately for the purpose of comparison.The beakers were covered with aluminium foil and incubated for 6 hours at 45 °C until it becomes firm (Fassara, 2010).

Determination of Physicochemical parameter
The determination of physic-chemical parameters such as pH, total acidity, reducing sugar and optical density were determined according to the Association of Analytical Chemists, (2000).

Proximate Analysis and Analysis of Nutrient Composition
The proximate analysis and determination of nutrient composition of the prepared yoghurt were determined according to A.O.A.C (2000) and ASEAN (2011).

Determination of Calcium and Magnesium
The analytical method used for the analysis of heavy metal concentration was the Atomic Absorption Spectroscopy (AAS) using the calibration plot method.For each element, the instrument was auto-zeroed using the blank (distilled water) after which the standard was aspirated into the flame from the lowest to the highest concentration.The corresponding absorbance was obtained by the instrument and the graph of absorbance against concentration was plotted.The samples were analyzed in duplicates with the concentration of the metals present being displayed in parts per million (ppm) after extrapolation from the standard curve (Greenberg et al., 1985).

In-vitro antibacterial activity
The in-vitro antibacterial activity of the yoghurts was carried out by agar well diffusing assaywith Mueller-Hinton agar (Lab M) (Clinical and Laboratory Standards 2013).The yoghurts were centrifuged and the supernatant was tested against Salmonella typhimurium ATCC 14028, Escherichia coli ATCC 29929 and Staphylococcus aureus ATCC 29293.

Biological material
This was done by modifying the methodology of Hounkpatin et al. ( 2013).The animal material was composed of 14 male albino Wistar rats weighing about 250 grams.These rats were purchased from a local rat farmer in Ado-Ekiti, Nigeria and were acclimated for 3 days before the experiments.They wereplaced in designed sterile polypropylene cagesat room temperature (25 to 30 °C).Thecages were illuminated with a sequence of 12 h light and 12h darkness.The rats 63 had free access to water and food.

Treatment of Wistar rats with the yoghurts
The wistar rats in duplicates were fed differently with the different yoghurts twice daily and supplemented with grains while the control was fed only with grains and water.The animals were fed for a period of 7 days after which they were sacrificed for haematological and histological assays.

Blood collection and haematological analysis
This was done by modifying the methodology of

Hounkpatin et al. (2013).
After 7 days of treatment, rats were fastedovernight.They were weighed before the collection of blood and sacrifice.All samples were taken between 7 and 9 am to avoid variations due to circadian rhythm.Whole blood was obtained from a puncture of the retro-orbital sinus by the conventional method (Van Hercket al., 1992).Blood samples collected in ethylene diamine tetra-acetic acid (EDTA)anticoagulant tubes (8.5%) wasquickly returned bymixing with anticoagulant in the tube.All blood samples were labeled and immediately conveyed to the laboratory for analysis.Hematological parameters were analyzed: Packed Cell Volume (PCV), white blood cell count (WBC) and its differentials such as: Leukocyte, Eosinophil, Neutrophil, Basophils and Monocyte counts.All hematological parameters were analyzed in the "Haematology Unit, Federal Polytechnic Ado-Ekiti, Ekiti State, Nigeria Medical Laboratory using the automated method withthe automatic analyzer "Haematology auto analyzer Sysmex KX-21N".

Histology
The tissue were dehydrated in an ascending grade of alcohol (ethanol), cleared in xylene and embedded in paraffin wax.Serial sections of 7 microns thick were obtained using a rotatory microtome.The deparaffinised sections were stained routinely with haematoxylin and eosin.Photomicrographs of the desired results were obtained using digital research photographic microscope in the laboratory (Eweka and Om'Iniabohs, 2011).

Analysis of data
The results were presented as the mean standard values of three replicates.A one-way analysis of variance (ANOVA) was carried out using SPSS 16.0.Significance was accepted at p ≤0.05.

RESULTS AND DISCUSSION
The commercial yoghurts used in the study are presented as a randomsampling of five different brands.All of five samples were used as starter cultures for the production of yoghurts in the laboratory.An assessment of the physical properties of the commercial yoghurt showed that sample A and B were thick and sour, sample C was very thick and very sour while sample D and E were slightly thick and sour.Samples A, B and E showed smooth consistency while samples C and D were coarsely smooth.The isolates were identified through the characterization of the 16S rRNA sequence and were analysed with the Basic Local Alignment Search Tool for sequence alignment (Table 1) while figure 1.0 shows the phylogenetic relationship between the organisms.The lactic acid bacterial isolates used in the study were isolated from the commercial yoghurts and their cell morphology and Gram stain reaction were observed.All isolates were observed to be Gram positive short rods and coccobacilli.The bacterial isolates were tested for their ability to survive and grow on bile salts and tested positive.The isolates used for the laboratory production of yoghurt in this study are lactic acid bacteria which have been reported as probiotic in functional dairy.Enterococci are lactic acid bacteria in large numbers and are naturally present in vegetables, plant material and food stuff; especially those of animal origin such as dairy products Pediococcuspentosus and Pediococcuspentosaceus to be of high probiotic quality and were isolated from Indonesian fermented foods in a bid to review health promoting lactic acid bacteria.
After 24 hours of incubation at 37 o C on MacConkey agar with bile salt, all isolates were observed to survive the growth condition.After 18 hours of incubation of the laboratory prepared yoghurts at 37 o C, physical parameters such as texture, taste, consistency and colour were determined while taste was determined as the only organoleptic property.Yoghurt produced with Enterococcus lactis (L.A) and yoghurt produced with Lactobacillus plantarum and L. pentosus (L.B) as starter cultures were thick and sour; yoghurt produced with Pediococcuspentosaceus (L.C) as starter culture was slightly thick and very sour while yoghurt produced with Pediococcuspentosaceus and E. durans as starter cultures (L.D) and yoghurt produced with E. durans as starter culture (L.E) were slightly thick and sour.They all showed a smooth consistency with a creamy colouration.The pH of the yoghurts (Table 2) after 8 hours of fermentation was found to be within the range of 5.51 in Pediococcuspentosaceus produced yoghurt and 5.76 in Enterococcus durans produced yoghurt.This finding corresponds with the report of Obi et al. (2016) who reported pH 4 -5.5 and 5.5 as optimum.The study is also in agreement with the report of Charles et al. (2016), who reported titratable acidity between 0.02 -0.06%.The control revealed the progress of fermentation and decrease in pH of the samples inoculated with the lactic acid bacteria as starter cultures.The study also revealed increasing optical density in all samples and decreasing trend in the reducing sugars as a result of the fermentative activities of the starter cultures and this is supported by the report of Shao-Chi et al. (2007).
The vitamin content (A, B12 and C) as determined in all the yoghurts had varying differences (p ≤0.05) which was as a result of different starter cultures and combinations of the starter cultures (Table 3).The vitamin A and B12 content of yoghurt produced with Pediococcuspentosaceus, Enterococcusduransand a combination of all the isolates resulted in higher vitamin A content when compared with others.The vitamin A and B12 content recorded in this study is higher than the record found in the report of Ihemeje et al. (2015).The combination of all the starter cultures resulted in higher vitamin C contentwhich is lower when compared to the report of Ihemeje et al. (2015).This increase may be as a result of more efficient oxidation of sugar by the consortium of lactic acid bacteria to ascorbic acid in a fermentative path way of L-galactose to L-ascorbic acid (vitamin C) as reported by Berry (2002).Pediococcus pentosaceus used as starter culture also produced yoghurt at a close range with the combined isolates, vitamins are essential organic compounds required in very small amounts to maintain the fundamental functions of the body (Hassen et al., 2010).
The soluble and casein bound calcium and magnesium of the yoghurts showed varying concentrations of magnesium and calcium which showed statistically significant differences (p ≤0.05) (Table 4.0).The soluble magnesium recorded lied between 4.140 ±0.007 -5.713 ±0.002 while the raw milk before fermentation was 5.650 ±0.007.Therefore, there was depletion in the soluble magnesium content as it is a mineral requirement of lactic acid bacteria (Boyaval, 1989) but yoghurt produced with Enterococcus durans as starter culture recorded improved magnesium content and was found to have the highest magnesium content.The highest soluble calcium in the study was observed with yoghurt produced with all the isolates followed by Enterococcus durans and Pediococcus pentosaceus with relatively high values.The casein calcium in this study were relatively higher than the concentrations found in the soluble calcium content in yoghurt which had starter cultures of Lactobacillus plantarium and L. pentosus as dual starter cultures, Pediococcus pentosaceus and Enterococcus durans as dual starter culture and Enterococcus durans as starter culture.However, Enterococcus lactis and the combination of the isolates gave improved magnesium content in the casein bound magnesium.The result of the study in terms of calcium and magnesium content is in contrast with the work of Gad et al. (2010) who reported higher calcium and magnesium values.
The report of this study agrees with the work of Miguel et al. ( 2003) who reported such variations between the soluble and casein bound magnesium and calcium.
The proximate composition of the yoghurts as determined in comparison with the raw, recorded a decrease.However, the carbohydrate content increased in all the yoghurts produced.The highest protein content was found in the yoghurt produced with Enterococcus lactis while the highest carbohydrate content was observed with the yoghurt produced with Pediococcuspentosaceus as starter culture.There was significant difference in all tested parameters of the different yoghurts (p ≤0.05) (Table 5).The fat content of the yoghurts were relatively low.Fat plays an important role in improving the consistency of yoghurt and also provides twice the energy of carbohydrate and protein   6).The yoghurts were observed to express antibacterial activity although at minimal levels while the highest inhibitory activity was found in an assay performed using the synergistic potential of all the laboratory prepared yoghurts.This is in agreement with the report of Hami ( 2011 The examination of the yoghurts influence on haematological parameters using Wistar ratsshowed improvements in the tested parameterswhich was demonstrated in the difference between the control and the treated samples (Table 7).The study revealed that combined use of the isolates always yielded better outcomes in terms of parameters examined.In a previous study, Shu et al. (1999) reported that 4 weeks of consumption of Lactobacillus rhamnosus, Lactobacillus acidophilus, and Bfidobacterium lactis had no adverse effect on mice general health status including haematology.In this study there was general increase in total white blood cell, packed cell volume but there was no significant effect on differentials such as the neutrophils, leukocytes, eosinophils, monocytes and basophil count.The report of this study differs from Hamid et al. (2008) as the study recorded higher blood cell counts as a result of longer exposure to treatment.A high level of packed cell volume is an indication that the rats are not anemic while a lower level is an indicationof anemia (Aboderin and Oyetayo, 2006).The white blood cell is important in defending our body against infection (Aboderin and Oyetayo, 2006).However, the leukocyte counts cannot give specific information and this necessitated the differential counts.The lymphocytes did not change significantly and this is in agreement with the report Hamid et al. (2008).
Figure 2 shows a section of small intestinal tissue, the submucosa (SM), muscularis mucosa (MM) and serosa arrow.The projection of the intestinal villi into the lumen together with the intestinal glands are lined by columnar epithelium, numerous goblets cells are seen.No sign of intestinal inflammation.Figure 3 shows a section of intestinal tissue with marked disruption of the mucosa layer (line), other layers of the intestine appear normal.Figure 4 shows a section of small intestinal tissue, the submucosa (SM), muscularis mucosa (MM) and serosa.The projection of the intestinal villi into the lumen together with the intestinal glands are lined by columnar epithelium, numerous goblets cells are seen.Arrow shows vascular leucocytes Margination.Figure 5 shows a section of the small intestinal tissue, the submucosa (SM), muscularis mucosa (MM) and serosa arrow.The projection of the intestinal villi into the lumen together with the intestinal glands which are lined by columnar epithelium, numerous goblets cells are seen.No sign of intestinal inflammation.Figure 6 shows a section of intestinal tissue with intestinal villi projection into the lumen, the intestinal crypt and adjoining layers (line) appear essentially normal.Figure 7 shows a section of unremarkable intestinal layers, mucosa, sub-mucosa (SM), muscularis and mucosa appear essentially normal.Figure 8 shows a section of intestinal tissue with some disruption of the mucosa layer (line), other layers of the intestine appeared normal.The projection of the intestinal villi into the lumen together with the intestinal glands is lined by columnar epithelium.The lactic acid bacteria strains were reported to cause no morphological abnormalities to the small intestine.The report of this study is in the affirmative to the reports of Ramiahet al., (2009) who reported the safety examination of Enterococcus mundtii and Lactobacillus plantarum and the organisms were found to have caused no histological abnormalities or inflammation.Cheng-Chihet al., (2014) also examined Lactobacillus acidophilus, L. pentosus, L. plantarum, L. reuteri and Enterococcus faecium and their toxicity.The small intestines of the Wistar rats fed with the different yoghurts which contained starter cultures; Enterococcus lactis, L. pentosaceus, L. pentosus, L. plantarum and E. duransshowed no abnormality, inflammation or damage.

CONCLUSION
As determined in this study, the final product quality of yoghurt or functional dairy is dependent on the selected lactic acid bacteria (LAB) cultures; therefore the choice of organism(s) to drive the fermentation is as important as the product itself.The study also demonstrated the importance of using animal models to determine the safety of the LAB cultures to be employed during yoghurt production.The use of more than one starter culture results in enhanced product quality as use of combined isolates in this study gave better outcomes in terms of nutritional quality.Finally, yoghurt is an important source of nutrients required for the body and also a remedy for gastrointestinal disorders caused by pathogens.
Food and Drug Administration (FDA, 2008) standard of identity for yogurt drinks specifies >8.25% milk solids-not-fat and fat levels to satisfy nonfat yogurt (<0.5%), low-fat yogurt (2%), or yogurt (>3.25%) before the addition of other ingredients (Chandan et al.,2006).Yogurt is among the most common dairy products consumed around the world (Saint-Eve et al., 2006).As the popularity of yogurt products continues to grow, manufacturers are continuously investigating value-added ingredients to entice health-conscious consumers (Allgeyer et al., 2010).
(Girraffa, 2003); Katie and Carol, 2009).Because they produce bacteriocins, Enterococcus species have been used widely over the last decade in the food industry as probiotics or as starter cultures (Foulquie, et al., 2006).Studies on the microbiota of traditional cheeses of Mediterranean countries produced mainly from raw milk of sheep, goats and cows indicate that Enterococci are a relevant component of the natural cultures involved in fermentation and play important role in the final quality of the product.They are also used to extend product shelf life and improve the hygienic safety of foodstuffs because they produce antimicrobial substances such as lactic acid, hydrogen peroxide, and bacteriocins (enteroxins) (Franz et al., 2007).According to Franz et al. (2007) and Naoualet al. (2010), Enterococcus durans and Enterococcus lactis are reported as non pathogenic Fabianova, (2010) reported the presence of members of the enterobacteriaceae in raw cow milk and their presence may be as a result of their presence in the raw milk used for the production of the yoghurts.Kumar et al. (2016) reported the use of lactobacillus plantarium in yoghurt fermentation properties and was said to give excellent antimicrobial and final product quality.Swain et al. (2014) and Lilis (2015) described

Figure 2
Figure 2 Histology of the small intestine of wistar rat fed without yoghurt (Control) .Note: Control intestine, X100, HE STAIN.HE (Haematoxylin and Eosin stain).

Figure 3 Figure 4
Figure 3 Histology of the small intestine of wistar rat fed with Enterococcus lactis containing yoghurt.Intestine, X100, HE STAIN.

Figure 5 Figure 6
Figure 5 Histology of the small intestine of wistar rat fed with Pediococcus pentosaceus containing yoghurt.Intestine, X100, HE STAIN.

Figure 7
Figure 7 Histology of the small intestine of wistar rat fed with Enterococcus durans containing yoghurt.Intestine, X100, HE STAIN.
(Ehirim and Onyeneke, 2013).The findings of this study in terms of proximate composition agree with the report of Igbabul et al. (2014) and Dairy Council (2013).
The yoghurts were observed to exhibit antibacterial activity against selected bacterial isolates representative of the Gram positives and Gram negatives as in Staphylococcus aureus and Escherichia coli and Salmonella typhi respectively (Table ) and Okiki et al. (2018) who established such antibacterial properties in nono fermented spontaneously by naturally occurring lactic acid bacteria.A number of studies have found probiotic consumption to be useful in the treatment of many types of diarrhea and intestinal disorders (Isolauri et al., 1991; Oksanen et al., 1990 and Siitonen et al., 1990).Antimicrobial effects of lactic acid bacteria are formed by producing some substances such as organic acids, carbondioxide, hydrogen peroxide, diacetyl, low molecular weight antimicrobial substances and bacteriocins (Ouwehand and Vesterlund, 2004; Romanova and Urminska 2017).

Figure 8
Figure 8 Histology of the small intestine of wistar rat fed with Enterococcus lactis containing yoghurt.Intestine, X100, HE STAIN.

Table 2
Physicochemical assessment of the laboratory prepared yoghurts.
Note: SD: Standard deviation, L.A: Yoghurt produced with Enterococcus lactis as starter culture, L.B: Yoghurt produced with Lactobacillus plantarum and Lactobacillus pentosus as starter cultures, L.C: Yoghurt produced with Pediococcus pentosaceus as starter culture,L.D: Yoghurt produced with Pediococcuspentosaceus and Enterococcus durans as yoghurt starter cultures,L.E: Yoghurt produced with two isolates of Enterococcus durans as starter culturesand ABCDE: Yoghurt produced with all isolates as starter cultures.

Table 3
Vitamin content of the laboratory prepared yoghurts.
Note: ND: Not Determined, L.A: Yoghurt produced with Enterococcuslactis as starterculture, L.B: Yoghurtproduced with Lactobacillus plantarum and Lactobacillus pentosus as startercultures, L.C: Yoghurt produced with Pediococcus pentosaceus as starter culture, L.D: Yoghurt produced with Pediococcus pentosaceus and Enterococcus durans as yoghurt starter cultures, L.E: Yoghurt produced with two isolates of Enterococcus durans as starter cultures and ABCDE: Yoghurt produced with all isolates as starter cultures.

Table 4
Determination of soluble and casein bound magnesium and calcium.
Note: ND: Not Determined, Superscript a-g denotes statistical difference in mean at P≤0.01.L.A: Yoghurt produced with Enterococcus lactis as starter culture, L.B: Yoghurt produced with Lactobacillus plantarum and Lactobacillus pentosus as starter cultures, L.C: Yoghurt produced with Pediococcus pentosaceus as starter culture,L.D: Yoghurt produced with Pediococcuspentosaceus and Enterococcus duransas yoghurt starter cultures,L.E: Yoghurt produced with two isolates of Enterococcus durans as starter cultures and ABCDE: Yoghurt produced with all isolates as starter cultures.

Table 5
Proximate analysis of the laboratory prepared yoghurts.Standard Deviation, ND: Not Determined, Superscript a-g denotes statistical difference in mean at p ≤0.01.A: Enterococcus lactis as yoghurt starter culture.B: Lactobacillus plantarum and Lactobacillus pentosus as yoghurt starter culture.C: Pediococcuspentosaceusas yoghurt starter culture.D: Pediococcuspentosaceus and Enterococcus duransas yoghurt starter culture.E: Two isolates of Enterococcus durans as yoghurt starter culture.ABCDE: All isolates

Table 6
Antibacterial assay of the laboratory prepared yoghurts on selected clinical isolates using well agar diffusion.Enterococcuslactisas yoghurt starter culture, B: Lactobacillus plantarum and Lactobacillus pentosus as yoghurt starter culture.C: Pediococcus pentosaceus as yoghurt starter culture.D: Pediococcus pentosaceus and Enterococcus durans as yoghurt starter culture.E: Twoisolates of Enterococcus durans as yoghurt starter culture, ABCDE: All isolates.

Table 7
Hematologicalevaluation of Wistarratstreatedwiththepreparedyoghurts.Enterococcus lactis as yoghurt starter culture.B: Lactobacillus plantarum and Lactobacillus pentosus as yoghurt starter culture.C: Pediococcuspentosaceusas yoghurt starter culture.D: Pediococcuspentosaceus and Enterococcus duransas yoghurt starter culture.E: Two isolates of Enterococcus durans as yoghurt starter culture.ABCDE: All isolates.