Production and Assessment of Sensory Properties of Probioticated Tigernut and Soybean Milk Stored Under Ambient and Refrigerated Temperatures

Background and Objective: The health benefits of probiotics have increased consumer’s demand for probiotic foods. Several food products suggested as delivery vehicles for probiotics to consumer. The production and sensory properties of probioticated tigernut and soybean milk stored under ambient and refrigerated temperatures were investigated. Materials and Methods: Lactic Acid Bacteria (LAB) isolated from tigernut and soybean was assessed for probiotics properties. LAB 2 and 3 were identified as Lactobacillus fermentum and L. plantarum , respectively and found to possess 100% Cumulative Probiotic Potential (CPP) was used to prepare probioticated drinks. Results: Probiotication of the drinks with the two probiotics resulted in the reduction of pH of the drinks and an increase in Total Titratable Acidity (TTA). The probioticated drinks had a higher score for all sensory attributes assessed which made them compare favourably with dairy yoghurt as against un-probioticated drinks. The LAB count of the stored probioticated drink increased during the period of storage while coliforms were not detected before and during the period of storage. The probioticated tigernut and soybean drinks were also generally acceptable compared to the un-probioticated counterparts. Conclusion: Findings from this study revealed that probiotic milk from soybean and tigernut was accepted based on its sensory attributes and may have promising commercial potential for vegetarians and individuals suffering from lactose intolerance.

foods containing the probiotic bacteria must maintain the characteristic sensory attributes of the traditional food, with this in mind, the most popular food delivery systems for probiotic have been fermented milk and yoghurt 1,6 .
Although milk and milk products have been generally accepted as the best probiotic food carrier, some of the limitations associated with this product are, the inability to digest lactose in milk due to the absence of β-galactosidase, an enzyme needed for digestion of lactose in some individuals, the presence of high level of unsaturated fat and cholesterol, allergic reaction to the milk protein in some individual, unsuitability of the food for vegetarians and relatively high cost as compared to other products 1,9 .
Plant-based products have been suggested as an alternative probiotic food carrier to dairy milk due to their low cost, availability, suitability for vegetarians and health benefits [10][11][12] .Moreover, there is an increasing demand for new flavours and tastes among consumers since the majority of the probiotic products in the market do not meet the needs of all consumer groups as they are mostly produced as yoghurt (a milk product) 1,11 .Considering the limitations associated with dairy-based probiotics, this research was therefore designed to produce and assess the sensory properties of non-dairy probiotic milk using soybean and tigernut milk as carriers for the delivery of locally isolated probiotics and lactic acid bacteria.

Study area:
The study was carried out in Akure City, Ondo State from January, 2019 to June, 2019.Akure City is the capital of Ondo State which is one of the 36 states in Nigeria, it lies about 70E15' North of the equator and 50E15' East of the Meridian.

Sample collection:
Soybean and tigernuts were purchased from Adedeji Market, Akure Ondo State Nigeria.They were sorted and graded to remove dirt and debris.Other samples which include, industrially produced soymilk, local/street vended soymilk, FUTA (Food Science and Technology Department)produced tigernut milk, dairy milk and dairy yoghurt were also purchased in triplicates from street vendors, market places and beverage stores in different locations in Akure, Ondo State.After purchase, they were then refrigerated before the commencement of the analysis.
Preparation of soymilk: Soymilk was prepared according to the method of Afroz et al. 12 , with little modification.Soybean was sorted and cleaned to remove stones and damaged deformed seeds.Then the dry soybean was washed and soaked in water (500 g in 1 L) for 12 hrs.It was then rinsed and blanched at 60EC in 1.25% NaHCO 3 for 30 min to remove the bitterness and anti-nutritional factors (trypsin inhibitor).The soybeans were washed, manually dehulled and rinsed.The soybean seeds were blended using a blender in a ratio of 3:1 (water to beans on a weight basis).The mixture was filtered using a cheesecloth, the filtrate is obtained as the milk while the residue was discarded.The milk obtained was then brought to a boil.Fifty grams of white granulated sugar was added to taste and the drinks were subsequently bottled and stored at ambient and refrigeration temperature.
Preparation of tigernut milk: Tigernut milk was prepared 13 , with little modification tigernut extract was prepared by sorting out all unwanted objects and other rotten nuts, washed and blanched at 60EC in distilled water and soaked overnight in water containing 0.5% sodium bicarbonate to soften the fiber, the water was changed 2-3 times to avoid bad smell.The soaked tigernuts with ginger were milled in a blender at the ratio of 3:1 (water to nuts on a weight basis).The mash obtained was then sieved twice with a neat cloth to separate the extract.It was further strained to obtain a fine consistency.The filtered extract was heated at 90EC for 15 min, sweetened, cooled to 4EC and refrigerated for further processes 14  Isolation of microorganisms: Before the isolation, the raw materials which were tigernuts and soybean were each macerated using a sterile mortar and pestle.The different drinks samples purchased and the ones prepared were left to get accustomed to room temperature and shaken vigorously to suspend microbial content.Nine millilitres of distilled water was dispensed each into 10 clear test tubes and sterilized by autoclaving.After sterilization, each sample was serially diluted using sterile distilled water as diluents by weighing 1 g or 1 mL of the sample into the sterilized water, after which 1 mL of both 10G 8 and 10G 10 diluents were plated into the six different mediums (NA, PDA, EMB, MSA, SSA and MRS) in triplicate using the pour plate method.The plates were incubated at an appropriate temperature and time suitable for the organisms to be isolated 15,16 .
After incubation, the colonies that developed on the nutrient agar plates were counted and used to determine the total viable count of the samples (CFU mLG 1 ).This was done by multiplying the counted numbers of colonies by the dilution used.The representative colonies or the distinct colonies on the different medium plates were then purified by sub-culturing on fresh nutrient agar using the streaking method to obtain pure cultures of the isolates.The pure cultures obtained were observed for their morphological characteristics and were then transferred into nutrient agar slants for further biochemical identification.
Isolation of lactic acid bacteria: Lactic Acid Bacteria (LAB) strains were isolated from the samples and prepared drinks were stored at ambient and refrigerated temperature.Here, 1 mL of each of the samples was diluted to appropriate 8-fold dilutions, isolation of LAB was carried out by plating 1 mL of the 5th and 8th diluents using the pour plate method on MRS agar and incubated at 30EC for 48 hrs.The number of the colony was counted for the total LAB count after incubation.The cultures were purified by repeated streaking.The process was repeated at the 1 week interval for 4 weeks.The isolates were maintained on an MRS agar slant and kept at 4EC.

Assessment of probiotic properties of Lactobacilli isolates:
The probiotic potential of the isolated Lactobacilli was determined according to the method described by Prabhurajeshuwar and Chandrankanth 17 and Jung et al. 18 .Some of the parameters checked for were, tolerance to sodium chloride, bile salt and low-pH, growth at different incubation temperatures and in vitro antagonistic activity against selected pathogens.

Safety assessment of Lactobacilli isolates:
The safety proWle of the isolates of Lactobacillus was determined in vitro by their hemolytic activity, coagulase activity, DNase activity, gelatin liquefaction and antibiotic sensitivity 19,20 Molecular identification of Lactobacilli with the highest probiotic potential: The Lactobacilli isolates with the highest probiotic potential were further characterized and identified using the DNA sequencing technique.The identification procedure is based on 16S rRNA gene sequence similarity 21 .
Preparation of probioticated drinks: Probiotic soymilk and tigernut were prepared by filling 20 sterile bottles each with 500 mL soymilk and another 20 sterile bottles were filled with tigernut milk.Each of the 20 bottles was divided into 4 groups, each group containing 4 bottles of the drinks.These four groups For the inoculation, 1% standardized culture each of L. plantarum, L. fermentum and L. plantarum+L.fermentum under aseptic conditions was introduced into their respective bottles as indicated above.However, the control samples were left un-inoculated and all the jars were then incubated at 37EC for 8 hrs for fermentation 1 .

Physicochemical characteristics of the probioticated drinks:
The pH and Titratable Acidity (TTA) of the probioticated milk were determined 22 .

Determination of proximate composition of the probioticated non-dairy milk:
The moisture content, ash content, crude protein, crude fat and crude fibre were determined using the methods of Wu and Wu 23 .Carbohydrate (CHO), a soluble carbohydrate was calculated by subtracting the sum of the percentage contents of moisture, crude protein, crude lipid, ash and crude fibre from 100.

Sensory evaluation probioticated non-dairy milk:
The previous methods 13,24 were used to evaluate the organoleptic characteristics (colour, taste, flavour and texture) and overall acceptability of the probioticated non-dairy milk.Fifteen panellists comprised of microbiology undergraduate students, students from other Departments and some teaching and non-teaching staff members of Federal University of Technology Akure, Ondo State, Nigeria who are conversant with the products were used.A nine-point hedonic scale ranging from excellent or extremely like (score = 9) to very poor or extremely dislike (score = 0) was adopted 24,25 .The samples were presented in random order and the panellists were asked to pour a little of the product in their mouth for some seconds and grade it based on what they thought suitable to qualify it and water was used for rinsing the mouth in between samples.

Microbiological quality of the probioticated drinks:
The microbiological quality of the product was determined by checking for the presence of coliforms in the drink and monitoring its microbial load during the storage period using the pour plate method according to the method of Adebayo-Oyetoro et al. 13 , Yoon et al. 26 and Awaisheh et al. 27 .

Statistical analysis:
The data obtained were statistically analysed using SPSS version 20, the results obtained were compared using Analysis of Variance (ANOVA) and the tests of significance were evaluated using Duncan's Multiple Range Test at p<0.05.The results obtained were computed as the mean of triplicate±standard deviation.

Total viable, total lactic acid bacteria, coliform and fungal counts of the samples:
The total viable counts of both soybean and tigernut under different treatments, as well as those of t he drinks produced and purchased, are presented in Fig. 1.The unwashed tigernut had the highest bacterial load of 2.9×10 8 CFU gG 1 and the least was seen in the blanched soybean 1.0×10 7 CFU gG 1 .Unwashed tigernut (UT) had the highest count of 8.5×10 7 CFU mLG 1 of LAB while the blanched soybean and tigernut (BB, BT) had no LAB growth.Moreover, the unwashed tigernut had the highest coliform count (1.2×10 7 CFU gG 1 ) and the blanched raw material had no coliform bacteria.However, among the drinks analysed, the locally produced/street vended soymilk (LSM) had the highest bacterial count of 2.0×10 8 CFU mLG 1 while the one produced by the well-established manufacturer had no growth in Fig. 2.However, as shown in Fig. 2, Dairy Yoghurt (DY) had the highest LAB count (4.0×10 7 CFU mLG 1 ).On the other hand, Dairy Milk (DM) and Industrially Produced Soymilk (ISM) had no LAB growth.The LSM has the highest coliform count (2.0×10 7 ) CFU mLG 1 , while there were no coliforms in the other sample.The fungal counts of soybean and tigernut and the milk obtained from them are also presented in Fig. 1

Probiotic property of isolated lactic acid bacteria:
The probiotic properties of the isolated lactic acid bacteria were determined according to their tolerance to bile, acid, salt, growth at various temperatures and their antagonistic effect on pathogenic bacteria.The salt tolerance of isolates is presented in Table 2.It was observed that all the isolates tolerated 2, 3.5 and 6% salt concentration for 24 hrs of incubation.The acid tolerance assessment revealed that only LAB 3 survived for 2 hrs at pH 2, others did not grow at this pH but they all grew well at pH 3-8 in Table 3.
The influence of incubation temperature on the growth of LAB isolates also revealed that all grew at an incubation temperature of 15 and 37EC, however, only LAB 1 grew evidently at 45EC incubation temperature as shown in Table 4. Table 5 shows the bile salt tolerance of isolates.All the isolates tolerated bile salt concentration of 0.1-0.4% for 24 hrs while none of the isolates survived in 0.5% bile salt concentration.
Inhibitory property of the isolated LAB on selected pathogens: The inhibitory effect of LAB isolates on selected pathogens is shown in Fig. 3.The LAB isolate designated LAB 3 had a good inhibitory effect on all the isolates with the highest effect on Escherichia coli (18 mm) and the least effect was seen against Pseudomonas aeruginosa (12 mm).

Antibiotic sensitivity pattern of the isolated LAB:
The sensitivity pattern of isolated LAB to different antibiotics is shown in Table 6.LAB isolate designated LAB 1 was found to be resistant to Streptomycin, Gentamycin and Chloramphenicol while LAB 2 and LAB 3 were found to be susceptible to all the tested antibiotics.

Cumulative Probiotic Potential (CPP) of the isolated LAB:
The results of the cumulative probiotic potential of the LAB isolates are presented in Table 8 isolate LAB 2 and 3 had the highest score which was 100% among the three isolated LAB.While LAB 1 had 75%.

DISCUSSION
This study evaluated the suitability of tigernut and soymilk in the production of non-dairy probiotic drinks and also monitored the effect of probiotication on microbial and sensory properties of the non-dairy milk.
From the microbiological evaluation of the raw materials, it was observed that the total bacteria, lactic acid bacteria, coliform and fungi count of the unwashed tigernuts and soybean sample is higher than their washed and blanched counterpart.This high microbial load could be a result of pre-harvest contamination of both soybean and tigernut from the soil where they were harvested or due to postharvest contamination from the market or the handlers 28 .
Locally vended soymilk had the highest microbial count and this high viable microbial load in these drinks could be attributed to inadequate hygienic measures in production or inadequate processing recontamination or non-aseptic handling and inadequate heat treatment 29 .Although the high viable bacterial counts obtained do not usually constitute a health risk they can serve as an indication of an overall lack of hygiene 28 .The presence of coliform in a particular sample can be used as an indicator to determine its quality, safety and integrity 30 .However, the presence of coliforms in the unwashed raw materials and the street-vended soymilk constitute a public health concern as it could indicate the presence of other pathogenic organisms.
Three Lactic Acid Bacteria (LAB), Lactobacillus casei, L. fermentum and L. plantarum, were isolated from the raw soybean and tigernut and their milk.Sebastia et al. 28 had earlier isolated similar organisms from tigernut milk and other dairy products collected from Akure.
Microorganisms with potential probiotics application must survive in the bile salts environment of the intestine to play beneficial roles in the gut 31,32 .The three LAB isolates were able to tolerate and survived in growth medium supplemented with 0.1-0.4% concentration of bile salt, however, none survived in a higher concentration of bile salt.The result obtained is similar to the observation of Fijad 31 .This is expected and essential for probiotics since the physiological bile salts concentration of 0.3% is a level achieved normally in the human intestine.
The LAB isolates in this study were able to tolerate acidic pH of 3-5 and a basis pH of 8, however, only Lactobacillus plantarum was found to tolerate pH 2 for two hours of incubation.However, Lactobacillus casei and Lactobacillus fermentum present no visible growth at this pH.This is observation is contrary to the result of Halder et al. 33 , who reported that Lactobacillus plantarum was unable to tolerate this low level of pH 2. The three LAB isolates also grew well at the lower temperature of 15 and 30EC, while Lactobacillus casei and Lactobacillus plantarum showed no growth at 45EC.
All the LAB isolates show a good inhibitory effect against spoilage and pathogenic microorganisms.Lactobacillus plantarum was found to antagonize all the test bacteria with the highest inhibition against E. coli (18 mm).This inhibition could be a result of the high amount of lactic acid produced which leads to a reduction in the pH of the growth environment, thus making it hostile to other microorganisms 34 .In addition to this, it has been well documented that the un-dissociated forms of these lactic acids also diffuse through the pathogenic bacteria cell membrane and dissociate inside the cell and releases H + which resultantly acidifies the cytoplasm to cause the collapse of the electrochemical proton gradient, leading to bacteriostasis and the eventual death of the susceptible bacteria 35 .
All LAB isolates except Lactobacillus casei were susceptible to all the antibiotics used, this complies with the safety guidelines stated by Marchwińska et al. 36 .However, the resistance of Lactobacillus casei to some of the antibiotics agrees with the previous results 37 .The probiotic microorganisms must be safe and a safety test using haemolysis, DNA hydrolysis, coagulation of blood and gelatin liquefaction revealed that the Lactobacillus spp., with probiotic potential were safe.This is similar to the result obtained by Halder et al. 33 whose isolates were unable to liquefy gelatin and cause haemolysis.
The demonstration of Cumulative Probiotic Potential (CPP) of the Lactobacilli strains has been considered an improved criterion for probiotic validation 36 .The CPP obtained for the Lactobacilli strains used in this study was found to vary from one organism to the other.Lactobacillus fermentum and Lactobacillus plantarum had a CPP of 100% which made them fulfil the criteria of FAO/WHO 37 .Similarly, Halder et al. 33 also reported that Lactobacillus plantarum and some other isolates from curd had a CPP of 100%.
The overall acceptability of the probioticated drinks prepared was found to be more than average and this agrees with the reports of Adebayo-Tayo et al. 38 who indicated that the acceptability of milk blends from plant sources (tigernut, soy and groundnut milk) compared favourably with dairy milk.Also, El-Shenawy et al. 14  The microbial quality analysis revealed the absence of coliforms in non-dairy milk products.Yoghurt should contain a minimum of 4×10 6 CFU mLG 1 LAB 36 .According to Terpou et al. 39 , a high viable count (>10 7 CFU mLG 1 ) after 4 weeks of storage is important for maximum health beneWts.The result from this study agrees with the guidelines of Terpou et al. 39 as the refrigerated probioticated drinks support and maintained the growth and survival of the probiotics microorganisms throughout the storage period with the minimal probiotic count of 3.39×10 7 CFU mLG 1 .The Lactic Acid Bacteria (LAB) viability during cold storage is fundamental as it leads to the production of organic acids (mainly lactic acid) which consequentially lead to the reduction in pH value, it also ensures microbiological stability and prevents food-borne pathogens or spoilage microorganisms.

CONCLUSION
Conclusively, the production of soymilk and tigernut milk and the probiotication process were sufficient to produce probiotic drinks that met the standard, thus enabling it to compete with the commercial probiotic yoghurt in the market.The probioticated tigernut and soymilk drinks were also generally acceptable compared to the un-probioticated counterparts.

SIGNIFICANCE STATEMENT
This study discovered that probiotic drink produced from non-dairy sources, soybean and tigernut milk has promising marketability potential as they compete favourably with probiotic drink produced from dairy milk.The results from this study revealed that the non-dairy probiotic milk will be an ideal drink for vegetarians and individuals suffering from lactose intolerance.

Fig. 1 :
Fig. 1: Microbial load of the raw materials BC: Bacterial count, LC: Lactic acid bacterial count, CC: Coliform count and FC: Fungal count

characteristics of Lactic Acid Bacteria (LAB) isolates:
The biochemical of Lactic Acid Bacteria (LAB) isolates is shown in Table1.The tentative identity of the isolates from their biochemical characteristics are Lactobacillus casei, L. fermentum and L. plantarum.

Table 1 :
Biochemical characteristics of Lactic Acid Bacteria (LAB) isolates

Table 2 :
Tolerance of the isolated LAB to different salt concentrations

Table 9 :
Molecular Identity of probiotic LAB isolates

Table 10 :
1 )iform count of the non-dairy milk and their probioticated variants before and after storage at both refrigerated and ambient temperature (CFU mLG1 )

Table 11 :
Total lactic acid bacteria count of the stored refrigerated drinks (10 8 CFU mLG 1 ) reported the acceptability of an equal proportion of blends of tigernut, soy and groundnut milk.