Effect of Okra Pectin on the Sensory, Physicochemical and Microbial Quality of Yoghurt

The objective of this work was to investigate the effect of okra pectin from two genotypes (asha and agbagoma) on the physicochemical, sensory and microbial quality of yoghurt. Okra pectin concentrations (w/w pectin to milk powder ratios) of 0.2%, 0.4% and 0.6% were used in the yoghurt preparation and its water holding capacity, titratable acidity and pH were analyzed against a control (0.0% pectin) weekly for a month. Consumer acceptability tests for the yoghurt samples were carried out using 50 untrained panellists on a 7-point hedonic scale. Total aerobic microorganisms present in the most preferred samples were enumerated over a four-week period. Results indicated that samples containing 0.2% asha pectin were most preferred by panelists. Water holding capacity varied significantly with those containing asha pectin higher than agbagoma counterparts and the control. There was a decline in pH with increasing pectin concentration and over the storage period. Samples containing agbagoma pectin had lower pH (3.60 - 4.32) compared to samples containing asha pectin (4.22 - 4.45). Titratable acidity increased during the storage period and with increasing pectin concentration. After four weeks of storage at 4˚C sample containing 0.2% agbagoma pectin had the least microbial count (7.6 × 10 5 ± 4.51 cfu/g), followed by the sample containing 0.2% asha pectin (2.4 × 10 7 ± 11.14 cfu/g) and the control (8.6 × 10 7 ± 5.57 cfu/g). The study revealed that addition of okra pectin at 0.2% improved the consumer acceptability of yoghurt and 0.2% agbagoma pectin inhibits the proliferation of aerobic microbes. Addition of okra pectin also improved the water holding capacity and reduced whey exudation.


Introduction
Okra (Abelmoschus esculentus) is an important vegetable crop situated in the Malvaceae family and is usually cultivated for its immature pods [1] [2]. The immature pod is characterized by a thick and slimy mucilage which often finds use as thickening agent for soups and stews in most African cuisines and rarely as industrial raw material [3]. It is used in traditional medicine as a dietary meal in the treatment of gastric irritations and dental diseases [3]. Okra is used as a brightening agent in electro deposition of metals. It is also used to reduce friction in pipe in flow as well as preventing flocculation in the fabric and paper industry [4]. Okra mucilage has been found to have potential uses as an extender for serum albumin and as an additive to dried egg white as well as a fat substitute in chocolate, frozen desserts and cookies [5]. The texture of fried yam snack has been improved using okra polysaccharides [6]. Okra polysaccharides have also been used as additives for flour and as a clarifying agent in the sugar cane [3].
The mucilage is used as a thickener to impart characteristic viscosity and smoothness called the okra-effect. This is because it forms a stringiness when dispersed in water. Okra polysaccharides have good emulsion properties in acidic environment and can be used in fruit drinks and acidified dairy products [7]. Okra polysaccharides are predominantly pectins [8]. Pectins are complex heteropolysaccharides containing mainly partially methoxylated galacturonic acid residues [9]. It is extensively distributed in a majority of the fruits and vegetables as the structural unit of fresh cells and the junction between the cells. The pectin molecule is linked by 1,4-D-galacturonic acid which has side chains containing L-rhamnose and other neutrals sugars such as D-galactose and L-arabinose [10].
The viscosity of pectin solutions depends on chemical and physical characteristic of the pectins, on the ionic strength of the solution and on the presence of sugar.
According to previous studies, the molecular weight of the pectin affects the gel properties strongly [11]. Other factors that affect the gelation properties of pectin is the length of the pectin side chain as well as the distribution to the methyl group as well as the degree of acetylation. Pectin with shorter side chains gives better gelation properties than pectin with longer side chains [12] [13].
Pectin in the okra pods has functional properties that can be employed in the food industry [8] [14] [15]. These functional properties include; gelling agents, emulsifying agents, water holding capacity and fat substitutes. Pectin can be used in yoghurt to improve the water holding capacity and reduce syneresis in yoghurt. Yoghurt is an acidified milk product that contains calcium that is bioavailable and is also convenient for lactose intolerant individuals [16]. They may also contain probiotics that are live microorganisms which confer health benefits to  [17]. The common yoghurt types found on the market are the Greek yoghurt and the set style yoghurt. The consumption of yoghurt over the years has been on the ascendency [18]. However, syneresis, an undesired occurrence which affects the quality and textual attributes in yoghurt, especially Greek type yoghurt is observed in many instances.
Stabilizers are commonly used to combat syneresis and improve the textual properties of yoghurt [19]. Most stabilizers used are hydrocolloids and they include; pectin, alginates, xanthan gum, carrageenan, locust bean gum, gum Arabic, tragacanth and karaya gum [20]. Okra pectin is a novel functional ingredient that has been shown as effective emulsifying candidates in acidic environments with a potential application for acidified food processing [14]. However, the quality of yoghurt as influenced by okra pectin from different genotypes has not been studied.
The objective of this work, therefore, was to study the effect of okra pectin from two genotypes on the physicochemical, sensory and microbial quality of yoghurt.

Materials
The milk powder and sugar were obtained from the local market, Ayigya-Kumasi and the starter culture was obtained from the Beef and Dairy Research Station, Boadi-Kumasi. Okra pectin samples (asha and agbagoma) used were isolated and characterized as previously reported [8].

Yoghurt Preparation
Different concentrations of okra pectin thus 0, 0.2%, 0.4% and 0.6% w/w (pectin to milk powder ratio) were added to reconstituted powdered milk and stirred thoroughly with a wooden spatula till there were no lumps. The milk was then pasteurized at 85˚C for 30 min. While pasteurizing, the milk was stirred to ensure effective homogenization. The pasteurized milk was cooled to 43˚C and starter culture (Streptococcus thermophilus and Lactobacillus bulgaricus) was inoculated. It was then fermented in an air tight container at 43˚C for 4 h. The yoghurt was stored at 4˚C after fermentation [21] pending analysis.

Sensory Evaluation
Consumer acceptability test was carried out using a 7-point hedonic scale with 1-dislike very much and 7-like very much. The attributes evaluated include

Determination of pH, Titratable Acidity (TA) and Water Holding Capacity (WHC)
The pH of yoghurt was measured on 7 days' interval with the electrode of a pH meter standardized with the appropriate buffer. Measurements were made in triplicates and readings were performed at 25˚C [22].
Titratable acidity was determined using [22] method. 2.5 ml of the yoghurt was placed in a beaker and 25 ml of distilled water added. NaOH of concentration 0.1 N was then titrated against the yoghurt. The volume of NaOH used was recorded as ml of 0.1 N NaOH used. The titratable acidity (TA) was calculated using Formula (1) [22]: The TA was determined in duplicates, every week for a month.
The method used for water holding capacity was reported by [23] with slight modification. Yoghurt samples (5 g) were centrifuged at 5000 rpm for 15 min at 4˚C and the extent of trapped serum phase quantified and expressed as a percentage of yoghurt volume by measuring the volume of supernatant. Syneresis measurements of yoghurt were made in triplicates. Percentage water holding capacity was calculated using Formula (2): where, W 1 = Weight of whey after centrifugation, W 2 = Yogurt weight.

Determination of Total Aerobic Count
The total aerobic count (TAC) was carried out to determine the presence or

Statistical Analysis
Results for sensory evaluation were analysed statistically using SPSS version 20 (IBM SPSS Statistics, US). Water holding capacity, pH and titratable acidity were analysed using multivariate test, followed by Tukey LSD post hoc multiple com-

Consumer Preference and Acceptability
Variation in the concentration of okra pectin as well as okra variety resulted in varying degree of preference by the sensory panellist in the current study (Table   1; Figure 1; Figure 2). It was observed that, yoghurt sample containing 0.2% pectin was preferred most for both okra genotypes. However, agbagoma recorded a higher degree of preference (56%) as opposed to 44% recorded by asha for the same okra pectin concentration ( Figure 1; Figure 2). This result is in agreement with that of [24] who also observed that 0.6% pectin addition had the least liked sensory properties. Conversely, yoghurt sample containing 0.6% okra pectin was least preferred for both okra varieties. The preference of samples containing okra pectin over the control gives an indication of the inclination of consumers to purchase these samples over the control.
The current study revealed that okra genotype from which the pectin was extracted and the concentration of incorporation into yoghurt had significant effect on sensory outcomes as perceived by the panellists (   well as the control sample.
With regards to taste of the yoghurt, the mean values ranged between 4.40 ± agbagoma pectin and 0.6% asha and agbagoma pectin were least liked as compared to the control. This finding is not consistent with the findings of [25] who stated that the improvement of the taste of yoghurt was proportional to the pectin addition rate from 0% -0.6%. The sour taste of yoghurt is as a result of the fermentation of lactose into lactic acid by two bacteria strains namely Lactobacillus bulgaricus and Streptococcus thermophiles. Specific yoghurt flavour is contributed by the acetaldehyde during the lactic acid fermentation [26]. Flavour is the overall impression of food and is the combination of aromatics, taste and mouthfeel. The milk proteins and pectin interact in a way that causes the unfolding of the milk proteins making hydrophilic groups accessible. These hydrophobic groups serve as extra sites for the binding of volatile compounds [27].
Samples containing the 0.6% had the least liked aftertaste. This finding corresponds with [25] who found that yoghurt with 0.6% pectin had the least liked aftertaste. Studies by [24] found that the addition of stabilizers above 3.5% resulted in undesired flavours.
The thickness of samples with 0.4% asha pectin was most liked but there was no significant difference between that and the control while the sample containing 0.6% pectin was least liked. For samples containing agbagoma pectin, samples containing 0.2% pectin was liked most as compared to the control. There was no significant difference in the overall acceptability of samples containing agbagoma pectin. However, sample containing 0.6% asha pectin had the least overall acceptability. This result is in agreement with that obtained by [24] who stated that 0.6% pectin addition has the least liked sensory properties on the overall. The results make it evident that pectin from different varieties of okra have significantly different impacts on the consumer acceptability of yoghurt.

Physicochemical Properties
Water holding capacity of protein gels in yoghurt is important because it is directly related to syneresis which is an undesirable occurrence. Syneresis is due to the intrinsic instability of gels. The lower the water holding capacity, the higher the occurrence of syneresis [23]. Sample containing 0.4% agbagoma pectin had the highest water holding capacity on the first day of analysis, however, the difference was not significant with the other samples. There was a gradual decline in the water holding capacity of the samples over the week ( There were significant differences in water holding capacity observed between the samples containing asha pectin. There were no significant differences between the water holding capacity between 0.2% and 0.0% on the first day of storage.   Table 2). This sample exhibited lesser water holding capacity than the control. Studies by [28] found that increasing pectin content increased the water holding capacity and the increase was directly proportional to the pectin content. This is as a result of the interaction between proteins and water. The intrinsic factors that could affect the water holding capacity include the protein conformation and hydrophobicity of protein. The presence of pectin further helps in binding of water and improving the texture. The water is trapped in the matrix of the pectin as it forms a three dimensional structure. Pectin also interacts with proteins hence, increasing hydration properties and gel stability [28].  [30]. The production of lactic acid (predominant acid in yoghurt) during the fermentation of yoghurt is responsible for the formation of gel network which gives yoghurt its characteristic texture [31]. The lactic acid produced also confers the characteristic flavour and taste to the yoghurt. Reduction in pH after fermentation is however not desired since it leads to excess sourness, wearing off and textural defects. Hence, it is very important to closely monitor the pH and titratable acidity to assure the consumer of consistency in product characteristics. The usual acceptable pH of yoghurt is 4.5. Several reports indicate that the rapid decrease in pH at the beginning of fermentation is of importance for the quality of the end product [29].
There was a decline in pH in the samples containing agbagoma pectin. Increasing concentration of the okra pectin leads to a resultant decrease in the pH of the samples. The pH decreased slightly for all samples with the sample containing 0.4% having the lowest pH after the storage period (Table 3).
For samples containing asha pectin, the change in pH was directly proportional to the rate of pectin addition. The pH increased with increasing pectin concentration. There were no significant differences in pH of all the samples on the 14 th day of storage. During the time of storage, there was slight reduction in the pH in all the samples ( Table 3).
The reduction in pH on storage can be attributed to the utilization of residual carbohydrate by viable microorganisms. The decline in pH values may also be due to continued fermentation by lactic acid bacteria and the contribution of acidity of the added stabilizer [28]. [32] recorded a pH decreased from 6.70 to 4.34 from the time of inoculation of the bacterial culture to the completion of the yoghurt manufacturing processing. Streptococcus thermophilus and Lactobacillus bulgaricus are the bacteria strains responsible for converting lactose into lactic acid in yoghurt. They live in symbiosis and there exists a synergy between the two bacteria which relates to a mutual stimulation. This stimulation relates mainly to the growth, acidification, and the production of aromatic compounds. Streptococcus thermophilus is stimulated by the amino acids and small peptides coming from the proteolytic activity of Lactobacillus bulgaricus. The stimulation of L. bulgaricus is allotted to formic acid, pyruvic acid, and carbon dioxide produced by S. thermophilus. Both microbial species are homofermentative bacteria, which produce lactic acid starting from lactose milk. The production of lactic acid results in a lowering pH. Some reports [33] attributed the reduction in pH to microbial activity while others [34] attributed the post acidification during storage as due to the presence of β-galactosidase which is active at temperatures of 0˚C -5˚C. Furthermore, [35] suggested that the drop in pH during storage was as a result of the activities of residual enzymes produced by starter culture during fermentation.
The pH range of samples containing asha pectin was 4.45 -4.22. The samples containing agbagoma pectin recorded pH of 4.32 -3.60 during the storage period. Yoghurt samples containing agbagoma pectin exhibited lower pH (Table 3).
There were significant differences between the titratable acidity of the samples (p < 0.05; Table 4). Titratable acidity refers to the measure of the total acid contained in a food. Titratable acid is sometimes referred to as total acid. This is different from pH of food which is the logarithm of the concentration of free hydrogen ions. The titratable acidity is a better predictor of the acid impact on flavour. The ability of microorganisms to grow in a particular food is more dependent on the pH than titratable acidity. The titratable acidity of the samples increased with increasing pectin concentration. A previous study [25] also observed an increase in titratable acidity with increase in pectin concentration.
According to [24], addition of stabilizer slightly increased the rate of acid development. There was an increase in the titratable acid content during the storage period which could be due to the accumulation of organic acids. This was also observed by [26].
Samples containing asha pectin exhibited higher titratable acidity as compared to the sample containing agbagoma pectin. Differences observed is as a result of

Total Aerobic Count
The outcome of the aerobic plate assay of the yoghurts indicated the presence of some aerobic microorganisms in the yoghurt samples. The colonies upon enumeration exhibited an exponential increase in population over the study period producing a positive gradient slope with increasing microbial load with increasing time; thus a directly proportional relationship between time and microbial load for all the samples (Figure 3). This trend was however not observed by [36] who stated that there was a decrease in the total plate count during the storage period and attributed it to the reduction in the viability of the lactic acid bacteria.
The result indicates the agbagoma genotype to have recorded the least microbial proliferation considering the initial and final counts of 1.3 × 10 2 ± 5.77 cfu/g and 7.6 × 10 5 ± 4.51 cfu/g, respectively over the control and asha genotype.
Statistical analysis of the results however reveals no significant difference in the microbial loads of the samples (p > 0.05). This notwithstanding, lower microbial load of the agbagoma variety is clearly seen in Figure 4. After four weeks of storage at 4˚C sample containing 0.2% agbagoma pectin had the least microbial count, 7.6 × 10 5 ± 4.51 cfu/g, followed by the sample  containing 0.2% asha pectin (2.4 × 10 7 ± 11.14 cfu/g) and the control (8.6 × 10 7 ± 5.57 cfu/g). Table 3 indicates that yoghurt with 0.2% agbagoma pectin had the lowest pH of 3.61 on the fourth week of storage. This pH was lower than that observed by the control and the sample containing 0.2% asha pectin which were 4.30 and 4.32, respectively. The acidic nature of the sample containing agbagoma pectin could have slowed/inhibited the growth of the aerobic microbes.

Conclusion
Different okra genotypes had varying effects on the physicochemical, sensory and microbial quality of yoghurt. Altering the concentration at which okra pectin is incorporated into yoghurt also had significant effect on the properties evaluated. Addition of okra pectin at 0.2% improves the consumer acceptability of yoghurt and 0.2% agbagoma pectin inhibits the proliferation of aerobic microbes. Addition of okra pectin also improves the water holding capacity and reduces whey exudation.

Conflicts of Interest
The authors declare that there is no conflict of interest.