3.1. Effect of Fermentation Time on the Viability of Lactic Acid Bacteria in Pearl Millet Extract during Fermentation
The effect of fermentation time on the survival of Leuconostoc mesenteroides
and Pediococcus pentosaceus
as lactic acid bacteria (LAB) is shown in Figure 1
ranging from 3.32 to 7.97 log CFU·mL−1
in plain non-alcoholic pearl millet beverage (PNAPMB) and 3.58 to 8.38 log CFU/mL in moringa supplemented pearl millet beverage. Initial LAB in plain non-alcoholic pearl millet beverage (PNAPMB) was 3.32 log CFU·mL−1
and significantly increased to 7.96 log CFU·mL−1
in 12 h. The lag phase was not visible as the cells immediately grew exponentially. The cells did not take long to adapt to the new environment. Thereafter, the growth of LAB was not significant between 12 and 15 h. At this point, the available nutrients were being depleted and bacteria started to compete for remaining nutrients. Leuconostoc mesentoroides
which stops growing at a pH of 4.0–4.5 could also have been halted as the pH was between 4.71 and 4.13. The cells decreased from 8.16 (15 h) to 7.97 CFU·mL−1
In moringa-supplemented non-alcoholic pearl millet beverage (MSNAPMB), the LAB started at 3.58 log CFU·mL−1
and remained stationary for 6 h followed by exponential growth (p
< 0.05) to 8.38 log CFU·mL−1
. The cells could have been in the lag phase adapting to the new environment during the first 6 h. Thereafter, the cells multiplied at a maximal rate utilizing the available nutrients in the beverage. This is contrary to Simango [23
] who reported a sharp increase in LAB in the first 6 h and thereafter remained the same during the fermentation of Mahewu,
a non-alcoholic fermented cereal beverage. The total LAB cells after 18 h were 108
in MSNAPMB and PNAPMB, respectively, which is ideal for organisms to confer health benefits to hosts [24
]. Since no death phase was visible in MSNAPMB, this could mean that Moringa oleifera
leaf extract supported the growth of LAB, which is in agreement with Hekmart et al. [24
] who reported positive effects of Moringa oleifera
seed on the survival of Lactobacillus rhamnosus
GR-1 in yoghurt with added sugar. The report suggested that added sugar could have acted as a food source to the LAB. In addition, this could mean that MSNAPMB can be fermented beyond 18 h should a sour beverage be desired.
3.2. Effect of Fermentation Time on the pH and Total Titratable Acidity of Pearl Millet Extract during Fermentation
The effect of fermentation time on the pH and total titratable acidity (TTA) of pearl millet extract for the production of plain and moringa supplemented non-alcoholic pearl millet beverages is shown in Figure 2
. There was a significant decrease in the pH for plain non-alcoholic pearl millet beverage (PNAPMB) during the 18 h fermentation period from 5.05 to 4.14. The pH did not decrease significantly from the onset of fermentation until after 12 h due to an increase in the activity of the lactic acid bacteria (LAB) growth breaking down the starch in pearl millet extract into simple sugars. The released monomeric sugars were utilised in the production of lactic acid which depressed the pH. Similarly, there was a significant decrease in pH of MSNAPMB after 12 h to 3.65. However, the pH was lower in MSNAPB compared to PNAPMB, which could be because the moringa leaf extract powder supported the growth of LAB.
The total titratable acidity (expressed as % lactic acid) during the 18 h fermentation period ranged from 0.14 to 0.22% for PNAPMB and 0.17 to 0.38% for MSNAPMB.
The total titratable acidity (TTA) did not significantly change from the onset of fermentation until 12 h has elapsed. At this point, the lower amount of lactic acid was produced by LAB. After 12 h, the TTA of PNAPMB and MSNAPMB increased significantly to 0.22% (18 h) and 0.38% (18 h), respectively. This was caused by the decrease in pH which increased the acid content of the beverages. The decrease in pH and increase in TTA agrees with [25
] who reported a decrease in pH range 5.04–5.17 to 3.74–4.35 and an increase in TTA from 1.28 to 2.59 g.L−1
during the fermentation of rice flour with various lactic acid bacteria. In summary, there was an inverse relationship between the pH and TTA with the pH decreasing from 5.05 to 4.14 while the TTA increased from 0.14 to 0.22%.
3.4. Proximate Composition of Plain, Moringa-Supplemented, and Traditional Non-Alcoholic Pearl Millet Beverages
The proximate composition of plain non-alcoholic pearl millet beverage (PNAPMB), moringa supplemented non-alcoholic pearl millet beverage (MSNAPMB), and traditional non-alcoholic pearl millet beverage (TNAPMB) is shown in Table 1
. The moisture content differed significantly (p
< 0.05) between the beverages and was 91.74, 91.03, and 87.59% in PNAPMB, MSNAPMB, and TNAPMB, respectively. This is due to the volume of water added during the production of the beverages. The ash content was 2.00, 1.56, and 1.18% in PNAPMB, MSNAPMB, and TNAPMB, respectively. The lower ash content of MSNAPMB could be due to the utilization of mineral elements by L. mesenteroides
and P. pentosaceus
during fermentation. The lower ash content could also be due to the lactic acid bacteria whose enzymatic activity resulted in the breakdown of the beverage components into absorbable forms caused by enrichment of the beverage with Moringa oleifera
leaf extract powder. It was reported by [27
] that the decrease in ash content was caused by the supplementation of beverage with moringa leaf powder.
The protein content differed significantly (p
< 0.05) between PNAPMB (1.62%) and MSNAPMB (2.17%). TNAPMB was comparatively significantly lower in protein (1.50%). The higher proteins in MSNAPMB could be attributed to moringa leaf extract powder which contains about 19.95% protein as reported by [28
]. However, the protein content of 2.17% in MSNAPMB was lower than that reported by [29
] (4.63%) at 5% moringa seed flour. This could be due to the level of moringa used and/or moringa seed flour instead of moringa leaf powdered extract as in this study. This is also an indication that the increase in moringa levels caused an increase in protein. This is supported by [27
] and [30
] who reported that the addition of moringa seed flour and moringa leaf flour increased the protein content of pearl millet flour, and maize-ogi
In addition, the increase in protein content could be related to the solubilisation of insoluble proteins of raw pearl millet, rice flour, and the synthesis of protein by lactic acid bacteria during fermentation (Nour and Ibrahim, 2014; Nour et al., 2016). Non-soluble proteins aggregate and settle depending on the pH of the beverage. If these proteins become soluble in the beverage during fermentation the protein content could increase considerably.
The total fat content was 0.92, 0.65, and 1.54% in PNAPMB, MSNAPMB, and TNAPMB, respectively. Saturated fats were high in TNAPMB (0.48%) in comparison to PNAPMB (0.23%) and MSNAPMB (0.16%). The polyunsaturated fats in PNAPMB (0.45) and TNAPMB (0.60%) differed significantly from that of MSNAPMB (0.32%). The fatty acids identified in the beverages were palmitic acid and stearic acid (saturated fats (SFA)), oleic acid (monounsaturated fats (MUFA)), and linolelaidic acid (polyunsaturated fats (PUFA)). Oleic acid and palmitic acid were the highest in TNAPMB followed by PNAPMB. MSNAPMB had the highest amount of stearic acid followed by TNAPMB. These fatty acids (SFA, MUFA, and PUFA) are prime in pearl millet and occur naturally [31
]. The presence of palmitic acid was low in PNAPMB (0.19%) and MSNAPB (0.14%) in comparison to the TNAPMB (0.41 ± 0.03%) and stearic acid was also low in PNAPMB (0.05 ± 0.00%) and MSNAPMB (0.02 ± 0.03%) when compared to TNAPMB (0.07 ± 0.02%). Palmitic acid is associated with an increased risk of coronary heart diseases and tumours while stearic acid is associated with a neutral effect on blood total and low-density lipoprotein (LDL) cholesterol levels. However, these saturated fatty acids are lower in cereal beverages in comparison to yoghurt with palmitic and stearic acid accounting for 16.54% and 11.73%, respectively [32
]. Oleic acid (omega-9) was 0.45% in TNAPMB, 0.24% in PNAPMB, and 0.17% in MSNAPMB. Linolelaidic acid is an essential omega-6 fatty acid important in the prevention of diseases related to cardiovascular and cancer [33
]. The increase in fat during fermentation could be due to the transformation of carbohydrates to fat; meanwhile, the decrease could be caused by the utilization of fat by lactic acid bacteria present in the beverage during fermentation [27
]. In contrast, [29
] reported an increase in fat content from 1.67 to 2.20% when 5% moringa seed flour was added to the beverage. However, [27
] reported a decrease in the oil content in fermented sorghum with 5% moringa seed flour. Fermentation decreases the long-chain fatty acid content in finger millet [31
The beverages differed significantly (p
< 0.05) in sucrose content 5.06, 5.31, and 3.78% in PNAPMB, MSNAPMB, and TNAPMB, respectively. Glucose (2.05%) and fructose (0.28%) were also present in TNAPMB. The sucrose could be from the added sugar during the production and available free sucrose found in millet [34
]. The fibre in the beverages could have been utilised by fermenting LAB [27
The carbohydrate content differed significantly (p < 0.05) between PNAPMB (4.31%), MSNAPMB (5.02%), and TNAPMB (9.4%). The energy content in PNAPMB, MSNAPMB, and TNAPMB was 113.23, 130.23, and 197.48 kJ·100 mL−1, respectively, and differed significantly (p < 0.05). There was no significant increase in carbohydrate and energy as a result of supplementation with 4% moringa leaf extract. The lack of an increase in carbohydrate and energy was expected as moringa leaf extract is not a source of carbohydrate and also due to complete utilisation of the carbohydrate by the LAB. The higher energy in TNAPMB could be due to the presence of carbohydrates in the beverage due to insufficient breakdown of carbohydrates by the spontaneous LAB fermentation.
3.5. Colour Characteristics of Pearl Millet Non-Alcoholic Beverages
The colour of plain non-alcoholic pearl millet beverage (PNAPMB), moringa supplemented non-alcoholic pearl millet beverage (MSNAPMB), and traditional non-alcoholic pearl millet beverage (TNAPMB) is shown in Table 2
. The beverages differed significantly (p
< 0.05) in lightness, redness, yellowness, chroma, and hue. Pearl millet non-alcoholic beverage with moringa leaf extract (MSNAPMB) was darker, less red, and yellower than the TNAPMB while the one without the extract (PNAPMB) was lighter, greener, and yellower than the traditional beverage (TNAPMB). The added moringa leaf extract powder made the beverage darker, redder, and yellower than the one without the extract (PNAPMB). The colour of the beverages in terms of hue can be described as light yellowish-green, unsaturated for PNAPMB; grey yellowish-red, saturated for MSNAPMB; and light yellowish-red, saturated for TNAPMB (Figure 4
The total colour difference (∆E)
between the TNAPMB and MSNAPMB (10.60) and PNAPMB (5.91) indicate a perceivable difference in colour between the novel beverages and the traditional since the colour difference is greater than one, the just-noticeable difference. However, the PNAPMB could be acceptable by the consumers since the ∆E
falls between 4 and 8. The MSNAPMB may not be accepted due to the dominant greenish colour from the moringa leaf extract. A similar decrease in consumer acceptability with an increase in Moringa oleifera
leaf powder (4% and 6%) in mahewu (a fermented maize beverage) compared to 2% was reported [13
3.7. Volatile Compounds in the Non-Alcoholic Pearl Millet Beverages
The 42 compounds identified in the non-alcoholic pearl millet beverages included sugars, alcohols, alkanes, ketones, esters, fatty acids, carbonyl compounds, and organic acids are outlined in Table 3
. The distribution of the metabolites among the beverages is outlined in Figure 6
. The node sizes are ranked according to betweenness centrality. More metabolites were identified from the TNAPMB compared to the other beverages. The important metabolites with high betweenness centrality were melezitose (129.71), 3-deoxy-d-mannoic lactone (129.71), D-mannopyranose (64.86), 5-Hydroxymethylfurfural (49.73), Methyltris(trimethylsiloxy)silane (49.73), 2 Ethyl-oxetane (15.12), and lactose (15.12). All the beverages contained melezitose, 3-deoxy-d-mannoic lactone, and D-mannopyranose. Melezitose is a non-reducing trisaccharide produced by many sap eating insects like Cinara pilicornis
by enzyme action. It is part of honeydew, which acts as an attractant for ants and also as a food for bees. 3-deoxy-d-mannoic lactone has been reported for antimicrobial activity. D-mannopyranose is d-mannose in its six-membered ring form, a sugar monomer of the aldohexose carbohydrates. It is important in the glycosylation of certain proteins in human metabolism. TNAPMB and PNAPMB contained 5-Hydroxymethylfurfural and Methyltris(trimethylsiloxy)silane. 2 Ethyl-oxetane and lactose were found in PNAPMB and MSNAPMB. 5-hydroxymethylfurfural is a flavouring substance in a wide variety of heat-processed products. The identified compounds contribute to the taste, aroma, and biological and medicinal potential of the beverage. For instance, the ester (3-deoxy-d-mannoic lactone) contributes to the flavour of the beverage during fermentation and has an antibacterial effect which results in a safer product [35
]. The N, N’-dibutyl-N, N’-dimethyl- have immune-modulating properties while 5-Hydroxymethylfurfural have antioxidant and antiproliferative properties [35
]. n-Hexadecanoic acid (palmitic acid) is a fatty acid with anti-inflammatory activities [36
]. Lactose that is present in PNAPMB and MSNAPMB could be from the skim milk used during the freeze-drying of isolated lactic acid bacteria. This is supported by its absence in TNAPMB, which was not inoculated with isolated probiotics.
The organic acids produced may preserve the beverage through the inhibition of pathogenic microorganisms. The nutritional content of the beverage is also improved. The identified compounds with their biological and medical uses prove that the beverages are not meant for refreshing only but have many benefits to consumers.
3.8. Sensory Characteristics of the Non-Alcoholic Pearl Millet Beverages
The demography of the panellists who evaluated the non-alcoholic pearl millet beverages (NAPMBs) is shown in Table 4
. There were 50 panellists made-up of 24 and 71% of males and females, respectively, of which 52% were black, 10% coloured, and 2% white; 16% were staff members and 82% were students; 52% were South African citizens, and 24% were international students; and 77% less or equal to 29 years, 10% between the age of 30–39, and 10% were 40 years old or above.
shows the sensory parameters of NAPMBs. The mean rating for appearance for the plain non-alcoholic pearl millet beverage (PNAPMB), moringa-supplemented non-alcoholic pearl millet beverage (MSNAPMB), and traditional non-alcoholic pearl millet beverage (TNAPMB) were 5.9, 5.5, and 4.5, respectively. The beverages differed significantly (p
= 0.037) in appearance with PNAPMB being the most preferred. The mean ratings for the colour of PNAPMB, MSNAPMB, and TNAPMB were 5.8 (liked slightly), 5.6 (liked slightly), and 4.8 (neither liked nor disliked), respectively, and differed significantly (p
= 0.007). The PNAPMB was golden brown, MSNAPMB was greenish-and-golden in colour while TMNAPMB was milky in colour and appearance. PNAPMB and MSNAPMB made using pearl millet extract appeared similar to commercial soft drinks; hence, they were preferred. In contrast, TNAPMB contained particles of starch, proteins, and minerals, which could have affected the colour. TNAPMB beverage was made with no stabilisers and the sedimentation of particles could be attributed to the lower scores.
The mean score for the aroma of PNAPMB was 5.3 (neither liked nor disliked), 4.7 (neither liked nor disliked) for MSNAPMB, and 4.2 (disliked slightly) for TNAPMB; hence, the beverages differed significantly (p = 0.020). The organic acids and metabolites produced during fermentation by Leuconostoc mesentoroides and Pediococcus pentosaceus could be responsible for the aroma of the beverages. Indigenous cereal slurries lack flavour but develops flavour during fermentation when volatile substances (diacetyl, acetic acid, butyric acid, amino acids, aldehydes, etc.) are developed.
The unique development of aromas and/or flavour depends on the chemical composition of the substrate (type of cereal, sprouted, etc.), an environmental condition during fermentation (pH, temperature, and anaerobic/aerobic) and starter culture [37
]. TNAPMB was carried out by chance fermentation made up of a diversity of lactic acid bacteria and other bacteria which could have resulted in the unacceptable aromas, and thus lower rating, unlike PNAPMB and MSNAPMB, which were fermented using known purified cultures. The slight differences between PNAPMB and MSNAPMB could be due to moringa extract powder which could have released other volatile compounds during fermentation. The beverage was fermented in a closed vessel, unlike traditional beverages, which are simply covered with a cloth to exclude foreign matters. During closed fermentation CO2
is not allowed to escape and is dissolved in the beverage, this may be ideal for anaerobic lactic acid bacteria but it may lead to spoilage and the creation of unwanted flavours or aromas. The closed system could also have caused all the released metabolites to be contained within the beverage. The level of diacetyl compound (2,3-butandione) could be high due to the lack of aeration. Hence, when citric acid was used in the beverage the ‘off-like’ flavour became intense because diacetyl is synthesised during utilization of citric acid. Pediococcus pentosaceus
could also be responsible for the production of diacetyl at high levels [38
]. Some of the panellists related TNAPMB to mahewu and porridge since the beverage is fermented as a whole and the flour was cooked through gelatinization.
The beverages differed significantly (p
= 0.001) in taste. The mean rating for taste was 5.3 for PNAPMB, 4.9 for MSNAPMB, and 5.4 for TNAPMB, meaning the beverages were neither liked nor disliked in taste. TNAPMB was rated high followed by PNAPMB then MSNAPMB. The cocktail of bacteria could be responsible for the sweet–sour taste profile of TNAPMB, whereas only selected lactic acid bacteria (L. mesentoroides
and P. pentosaceaus
) were used in PNAPMB and MSNAPMB. The preference for TNAPMB could have been due to cultural preferences by people who are familiar with the natural fermented ethnic beverages. The lower rating of MSNAPMB could be due to the fresh leaf earthy flavour of moringa leaf extract in the beverage. The majority (77%) of the panellists were youth (≤29 years old) and are loyal supporters of carbonated drinks in South Africa. According to StatsSA [39
], the South African population reported an increase in the growth rates of the elderly people meaning the beverages have the potential for growth among the older generation who are familiar with non-alcoholic cereal beverages such as ‘magewu
The mean score for the mouthfeel of the beverages did not differ significantly (p
= 0.094). The similarity could be because the beverages were all fermented. Phytates, phenols, and tannins found in pearl millet could be responsible for the mouthfeel of the beverages. Murevanhema and Jideani [40
] also reported the influence of tannin on the mouthfeel of fermented Bambara groundnut milk. However, lactic acid bacteria during fermentation resulted in low pH (3.65–4.14) and built-up of lactic acid (0.22–0.42%) and pasteurisation of the beverages at high temperature could have resulted in the reduction in these antinutrients.
PNAPMB, MSNAPMB, and TNAPMB had a mean score for overall acceptability of 5.8, 4.9, and 5.4, respectively. The beverages differed significantly (p < 0.05) in overall acceptability. PNAPMB was rated high followed by TNAPMB then MSNAPMB. The overall acceptability was influenced by all the other attributes of appearance, colour, aroma, and taste. PNAPMB had a bright golden-brown appearance resembling most grape flavoured beverages; hence, it was preferred. TNAPMB had a creamy-milk appearance the panellist could have related to ‘umgqomothi’ (African beverage) with which they are familiar. MSNAPMB was scored lower, which could be explained by the greenish colour and fresh earthy leaf aroma from moringa leaf powder. The taste of the beverages could be improved in future work. Possible approaches will be flavoured, carbonated, and blended with other cereals.