Effect of probiotic fermentation on physico-chemical and nutritional parameters of milk-cereal based composite substrate

Abstract

Abstract

A dairy-cereal based composite substrate was prepared from whey-skim milk (60:40 v/v), germinated pearl millet flour (4.73% w/v) and liquid barley malt extract (3.27% w/v) and fermented using probiotic strain Lactobacillus acidophilus NCDC-13. Probiotic fermentation increased whiteness index, viscosity and water holding capacity of unfermented substrate. Fermentation caused a reduction in total solids, fat, ash and total dietary fibre content and increment in protein content. Fermentation brought a highly significant reduction in phytic acid (78%) and polyphenol (46%) content. The protein and starch digestibility increased significantly. The HCl- extractability of Ca, Fe, Mg and Zn of unfermented substrate was 32%, 25%, 64% and 17% respectively, which increased to 73%, 50%, 83% and 65% respectively after fermentation. Fermentation resulted in 77% decrease in phytate P as % of total P and significant increase in free P. The current investigation revealed that probiotic fermentation improved nutritional attributes of the composite substrate substantially. The low cost nutritionally enriched probiotic substrate can be utilized for preparation of a wide range of low- cost probiotic foods to address malnutrition and enhance immunity of common population.

Graphical Abstract

Introduction

Cereals are the cost-effective contributor of energy and nutrients to wide segments of population in developing country (Stefano et al. 2017). However, the sensory attributes of cereals and cereal products are inferior, due to the coarse nature of grains and darker colour. Presence of limiting amino acids (threonine, lysine, and tryptophan etc.), antinutrients (phytic acid, tannins and polyphenols), reduced the nutrients digestibility and low bio-availability of minerals designate cereals as inferior in terms of nutritional quality (Stefano et al. 2017). Several processing treatments have reported to improve nutritional quality of cereal and cereal products. Among them, fermentation is one of the best methods for improvement of nutritional quality of cereals (Arora et al. 2010; Sindhu and Khetarpaul 2003). Fermentation is associated with reduction in pH, creating a favourable environment for enzymatic degradation of phytate leading to release of minerals and protein and enhanced availability of these nutrients (Sindhu and Khetarpaul 2003; Stefano et al. 2017).

Pearl millet with scientific name Pennisetum typhoides is one of the most important food crop of India, which is capable of withstanding drought conditions (Serba et al. 2020). India is the largest producer of pearl millet (FAO 2020) with a productivity of 1239 kg/ha during 2015–2019 (Yadav et al. 2021). Although the grain is a superior foodstuff having ‘high-energy’ and a good amino acids balance, with major limitation being reduced palatability and presence of anti-nutrients. Antinutritional factors like phytic acid and polyphenols restricts the mineral bioavailability and digestibility of proteins and carbohydrates (Gull et al. 2016). Several processing technologies like cooking, milling, roasting and fermentation, can increase the nutritional qualities of pearl millet, and the best one is fermentation (Budhwar et al. 2020). Fermentation of pearl millet leads to a decline in the level of anti-nutrients with concomitant enhancement in nutritional attributes and palatability (Blandino et al. 2003).

Barley (Hordeum vulgare L.) is a widely consumed cereal grain having significant amounts of dietary fibres and bioactive components (Mitsou et al. 2010). Recently barley is gaining popularity as a human food, primarily due to its functional dietary component, β-glucan (Mitsou et al. 2010). Barley β-glucans demonstrates prebiotic properties due to their ability to reach the colon in undigested condition and can be selectively utilized by beneficial microbiota (Mitsou et al. 2010).

Probiotics are known for their multiple health beneficial properties. Probiotics have been used therapeutically to modulate immunity and treat several diseases (Kisan et al. 2019) when they are taken in a required suggestive concentration (Ganguly et al. 2019). Milk is the most common substrate for probiotic organisms. Limited attempts have been made to use other fermentation substrates such as cereals and combination of milk and cereals (Ganguly et al. 2013). The mixture of cereals and milk may offer better nutrition ultimately leading to value added functional product. The inclusion of cereal in dairy components would compensate for the lack of fiber in milk and the low digestibility and deficiency of certain amino acids in cereals. Probiotic fermentation of composite substrate can one way support the growth of probiotic organism and serve as suitable delivery medium and base material for new probiotic food products developments. Cereal–milk based composite fermented foods such as Kishk, Tarhanas, etc. play an important role as a good resources of nutrients in several countries (Georgala 2013). Probiotic fermentation of composite substrate has been found to improve nutritional quality by reducing antinutritional elements, improving mineral availability and enhancing protein and carbohydrate digestability (Ahuja et al. 2017; Di Stefano et al. 2017; Ganguly et al. 2013). However consistent, and systematic research work is required in this field.

Poverty and malnutrition are serious trouble in developing country and should be main criteria for new product development (OECD/FAO 2011). Malnutrition is associated with reduced immunity and occurrence of infectious diseases. Although, probiotic and fermented foods are most popular food item in functional food sector, they are eventually non-existent within poorer population as they are costly and less available. Availability of low-cost probiotic foods to society will strengthen the immunity to wider section of population. The aim of current study is to investigate the effect of probiotic fermentation on probiotic fermentation on physico-chemical and nutritional parameters of milk-cereal based composite substrate.

Materials and methods

Probiotic strain Lactobacillus acidophilus NCDC-13 was collected from National Collection of Dairy Cultures (NCDC) at National Dairy Research Institute, Karnal, India. Probiotic organism grown for sixteen hour in skim milk (~ 8 × 10⁸ cfu ml⁻¹) used as inoculums. Acid (paneer) whey (TS 6%, fat 0.25%, pH 5.5) was filtered and mixed with skim milk (total solids, TS 9%, fat 0.15%, pH 6.7) in the ratio 60:40 to obtain whey-skim milk (WSM), whose pH was adjusted to 6.8 with 25% KOH. Pearl millet grains were cleaned, washed and soaked in water in the ratio 1:3 for 24 h at ambient temperature (changing of soak water at 4 h). The grains were germinated at 25 °C for 48 h. The germinated grains were dried at suitable temperature (55–60 °C) for 8 h untill a final moisture content of 5–6% subjected to grinding and sieving (52 mesh) to obtain germinated pearl millet flour (GPMF). Liquid barley malt extract (LBME) was obtained from Malt Company (India) Limited, Gurgaon, India having solids by refractometer 80.5%, protein on dry basis 5.17%, ash 1.453%, pH 5.72 and specific gravity 1.398.

Preparation of composite substrate

A composite dairy-cereal substrate consisting WSM (60:40 v/v), GPMF (4.73% w/v) and LBME (3.27% w/v) was heat treated at 95 °C for 5 min and cooled to 37 °C after heat-treatment prior to inoculation using L. acidophilus NCDC-13 at 4%. The sample was incubated at 37 °C for 8 h. At the end of the fermentation process probiotic count reached to 10¹⁰–10^11 CFU/ml with pH value of 5.2–5.1. The unfermented (UF) and fermented (F) samples were subjected to various analysis as given below.

Physical analysis

Colour measurement

The colour of unfermented and fermented substrate was measured by reflectance spectroscopy technique employing reflectance meter, colour flex (Hunter lab, Reston, Virginia, USA) and the Universal Software (Version 4.10) after calibration of instrument. Data were received in terms of L*, a*, and b* values of the international colour system. Whiteness index (WI), chroma (C*), hue angle (h*) and colour differences (ΔE*) can be obtained from following equations (Sanz et al. 2008):

$$Whitenessindex\left(\mathit{WI}\right)=100-\sqrt{{\left(100-L^{\ast }\right)}^2+a^{\ast 2}+b^{\ast 2}}$$

$$Croma\left(C^{\ast }\right)=\sqrt{a^{\ast 2}+b^{\ast 2}}$$

$$Hueangle\left(h^{\ast }\right)=arctan(b^{\ast }/a^{\ast })$$

$$Colourdifferences\left(\mathrm{\Delta }E\right)=\sqrt{{\left(L_0-L^{\ast }\right)}^2+{(a_0-a^{\ast })}^2+{\left(b_0-b^{\ast }\right)}^2}$$

where subscript ‘o’ refers to the colour reading of initial or unfermented sample used as the reference and ΔE indicates colour change of fermented sample from reference sample.

Viscosity and flow properties

Viscosity and flow properties of unfermented and fermented substrate were determined at 20 ± 0.1 °C using Viscostar plus Viscometer (Fungilab, Spain) using low viscosity accessories (AMP assembly) of coaxial cylinders (Spindle TL-5) as attachment. The sample volume required was 8 ml and the shear rate (s⁻¹) was obtained by multiplying R.P.M by 1.32. The relationship between apparent apparent viscosity μ_a (Pa s) and shear rate γ (s-1) can be expressed as:

$${\mu }_a=K{\gamma }^{n-1}$$

where μ_a is the apparent viscosity (Pa s), γ is the shear rate (s⁻¹), K is the consistency index (Pa sⁿ), and n is the flow behavior index. The higher K value indicates a more viscous the fluid. In this model the parameter n indicates a physical property characterizing a non-Newtonian behaviour.

Water holding capacity

Water-holding capacity of unfermented and fermented samples was determined using procedure by Guzman-Gonzalez et al. (1999). About 20 ml of sample was centrifuged in 30 ml centrifuge tubes for at REMI laboratory centrifuge for 30 min at 1250 × g at 20 °C. The whey expelled was removed and weighed. The water holding capacity (WHC) was determined by:

$$WHC=\frac{100\times \left(Y-WE\right)}{Y}$$

where WHC = water holding capacity; Y = weight of sample and WE = whey expelled.

Preparation of samples for chemical and nutritional analysis

The samples for chemical and nutritional analysis were prepared by procedure described by Ganguly et al. (2013) with modification. The fermented as well as unfermented substrates were oven dried at 60 °C for 24 h to a constant weight and finally ground in cyclone mill using 0.5 mm sieve.

Chemical analysis and calculation of total energy

The TS, fat, protein, ash, carbohydrate, starch was analysed as per AOAC (1995). Total energy was calculated using following formulae:

$$\text{Energy value}=\%\text{Protein}\times 4+\%\text{Carbohydrate}\times 4+\%\text{Fat}\times 9.$$

Total dietary fibre analysis

The total dietary fibre content of the dry samples were estimated by Megazyme Dietary Fibre Kit (K-ACHDF 11/08) using the modified method of AOAC (1995).

Nutritional analysis

Estimation of phytic acid, phytate phosphorus and total phosphorus

The phytic acid and phosphorus content of samples was estimated using the phytic acid estimation kit (Megazyme International Ireland Limited). Total polyphenols were determined according to the Prussian blue spectrophotometric method (Ganguly et al. 2013). A standard curve was prepared, using tannic acid.

Estimation of in vitro protein and starch digestibility

In vitro protein digestibility (IVPD), was measured by taking about 200 mg of sample into a 50 ml centrifuge tube followed by addition of 15 ml of 0.1 M HCl containing 1.5 mg pepsin (Ganguly et al. 2013). The tube was incubated at 37 °C for 3 h. After incubation, the sample was treated 10% (w/v) trichloroacetic acid (TCA) 10 ml and centrifuged (50,000 g) for 2 min at room temperature. Nitrogen in the supernatant was estimated by micro-kjeldahl method (AOAC 1995). Digestibility was calculated using the following formula:

$$ProteinDigestibility\left(\%\right)=\frac{NinSupernatant-NinEnzyme}{Ninsample}\times 100$$

Starch digestibility (in vitro) was estimated by the spectrophotometric method as described by Ganguly et al. (2013). Defatted sample (50 mg) were dispered in 1.0 ml of 0.2 M phosphate buffer at pH 6.9. About 20 mg pancreatic α-amylase was dissolved in 50 ml of same buffer and 0.5 ml of it were added to the sample suspension followed by incubation at 37 °C for 2 h. After incubation about 2 ml of 3–5 dinitrosalicylic acid (10% aqueous solution) was added and the mixture was heated for 5 min in water bath. After cooling, the solution was made up to 25 ml with distilled water and filtered before measurement of the absorbance at 550 nm. A blank was run simultaneously. Maltose was used for the standard curve preparation the in vitro starch digestibility values were stated as percent maltose released from 100% starch present in the sample.

Bioavailability of minerals

Minerals were determined by the dry-ashing method using Atomic Adsorption Spectroscopy (Hitachi 2-5000 Series) (Ganguly et al. 2013). Total and phytate phosphorus was estimated by Megazyme kit. Bio-availability of minerals were estimated after the samples were treated by 0.03 N HCl as described by El Maki et al. (2007). Extractability of each element was calculated as percentage of the total amount of the mineral.

$$\text{Mineral}\text{extractability}\%=\frac{\text{Mineral}\text{extractable}\text{in}0.03\text{N HCl}\left(\text{mg}/100\text{g}\right)}{\text{Total}\text{Mineral}\left(\text{mg}/100\text{g}\right)}\times 100$$

Statistical analysis

The obtained experimental data was subjected to suitable statistical analysis using MS-Excel 2010 and SPSS 16.0 Software. Student’s t-test and analysis of variance (ANOVA) was applied for testing the significance of difference between two or more treatments respectively.

Results and discussions

Effect of fermentation on physical parameters

Effect of fermentation on colour parameter

Colour is an important parameter for acceptability of product. The colour profile changed as a result of probiotic fermentation. “L” value represents lightness (100) and blackness (0); “a” represents red (+ ve) to green (− ve) hues, whereas “b” represents yellow (+ ve) to blue (− ve) hues (Sanz et al. 2008). Fermentation resulted in a significant (P < 0.05) increment in L* value (UF-53.03; F-58.77), leading to an increased whiteness. The increase in L* as a result of fermentation may be due to increase in protein content and decrease in ash content in fermented substrate as compared to unfermented substrate. Protein particles are capable of scattering light in visible spectrum, leading to whitening effect. Whiteness index, which represents the overall whiteness of food products was 50.17 in UF which increased significantly (P < 0.05) to 54.83 in fermented substrate. It is generally considered that the ash content is a critical factor affecting the whiteness of flours (Lu et al. 2005). Fermentation enhanced the whiteness of rice flour (Lu et al. 2005) as a result of starch purification and the decreased level of ash content. Increase in luminosity(whiteness) during the course of fermentation was observed for fermentation of pearl millet based ‘Degue’ (Hama et al. 2009).

The values of a* and b* were respectively 2.52 and 16.46 before fermentation and 3.13 and 18.17 after fermentation. Fermentation resulted in more intense redness and yellowness of the substrate. The statistical analysis revealed that fermentation had non-significant effect on redness (a*) and significant (P < 0.05) effect on yellowness. Fermentation resulted in release of pigments of pearl millet and barley malt that caused more intense red and yellow colour of fermented samples. Figure 1 depicts the effect of fermentation on colour profile of composite substrate. Garcia-Perez et al. (2005) observed an increment in a* and b* values in orange fibre fortified yoghurt fermentation. In an another study the L*, a* and b* values reduced as the fermentation proceed resulted in a darker, more green-less reddish and more blue- less yellow samples in tarhana prepared with different wheat germ/ bran content (Bilgicli and Ibanoglu 2007). Similar trend of reduction in a* and b* values was observed during Degue fermentation (Hama et al. 2009). Increase in C* value is an indication of enhancement of colourfulness (Sanz et al. 2008). Fermentation caused a highly significant (P < 0.01) increase in C* value. The h* values indicated that all samples were red in colour and there was non- significant (P > 0.05) change in hue as a result of fermentation.

Fig. 1.

Effect of fermentation on colour profile of composite substrate. a L* value, b a* value, c b* value, d Whiteness index, e C* value, f h* value. UF—unfermented substrate, F—fermented substrate. Values with different superscripted letter in a graph differs significantly (P < 0.05)

With the aim of determining whether the differences in colour in between UF and F samples recorded by instruments can be identified by human eye, parameter ΔE* was calculated, with the colour parameters of unfermented samples being taken as reference point. It was observed that ΔE* values greater than 3 indicate differences evident to the human eye (Sanz et al. 2008). The average ΔE* in this study was 6.022, so it can be expected that colour difference between UF and F samples could be differentiated by human eye.

Effect of fermentation on viscosity of composite substrate

The unfermented substrate was fluid-like before fermentation. Fermentation caused a significant (P < 0.05) increment in viscosity of composite substrate at all speeds (rpm) at 20 °C. This agrees with the report by Amankwah et al. (2009). The increase in viscosity may be due to network or gel formation as a result of the biochemical and physicochemical changes during substrate fermentation.

The apparent viscosity of UF and F substrates, decreased with an increase in the shear rate, indicating a non-Newtonian fluid behaviour. The decrease in the apparent viscosity of both substrates with the increase in shear rate at the specific temperature indicates that the samples had shear thinning characteristics. The result is in agreement with results obtained for inulin fortified yogurt (Donkor et al. 2007); symbiotic lactic beverage (Castro et al. 2008). The reduction in the apparent viscosity with the increasing shear rate in fermented beverage products, may be linked to multiple factors. This might be resulted from the destruction of weak bonds and a reduction in the sum of the electrostatic repulsion and hydrophobic interaction between the gel molecules. Karazhiyan et al. (2009) reported that the increase in shear rate and reduction in viscosity may occur due to a breaking of the macromolecular structures in solution, due to the shear force. Alparslan and Hayta (2002) reported that the hydrodynamic forces, at the beginning of shearing, are more severe, causing greater rupture. Over time, these forces generate system stretching allowing the alignment with the flow and, consequently, a reduction in the viscosity values.

The relationship between flow behavior index, consistency index, shear rate and apparent viscosity described by the power law model is given below:

$$\mu a=K{\gamma }^{n-1}$$

where, μ_a is the apparent viscosity (Pa s), γ is the shear rate (s⁻¹), K is the consistency index (m Pa sⁿ), and n is the flow behavior index. The larger the value of K, the thicker would be the product and therefore, more viscous the fluid. In this model the parameter n constitutes a physical property that characterizes a non-Newtonian behaviour. The r² values for curve fitting ranged from 0.991 to 0.999 for all cases.

The flow behavior index (n) of UF and F substrates were 0.83 and 0.49 respectively at 20 °C. The consistency index, K, of UF and F substrates were 22.38 and 185.91 m Pa sⁿ respectively. Figure 2c and d represent the effect of fermentation on n and K values of composite substrate. The increase in K values of unfermented substrate indicated an increase in viscosity as a result of fermentation. A lower K value was reported for fermented boza samples indicating a decrease in viscosity. Some authors reported a decrease, whereas some researchers observed first a decrease then an in viscosity during fermentation. Fermentation brought about lowering of the n values, causing the greater departure from Newtonian behavior (n = 1), which indicated a greater resistance to flow and hence the existence of a more entangled structure (Sanz et al. 2008).

Fig. 2.

Effect of probiotic fermentation on rheological properties of composite substrate (at 20 °C). a Viscosity, b Water holding capacity, c Flow behavior index (n), d Consistency coefficient (K). UF—unfermented substrate, F—fermented substrate. Values with different superscripted letter in a graph differs significantly (P < 0.05)

Effect of fermentation on water holding capacity of composite substrate

Water holding capacity (WHC) is an important parameter which indicates network or gel formation. WHC increased significantly (P < 0.05) as a result of fermentation (Fig. 2b). Lower WHC is related to an unstable gel network and excessive rearrangement of a weak gel network (Donkor et al. 2007) in which water cannot be entrapped within the holes three- dimensional network (Donkor et al. 2007). Higher WHC in fermented substrate could be related with a network formation as a result of fermentation, which can entrap more water. Similar increase in WHC was reported by Pyo and Song (2009) during soygurt fermentation. Increase in water holding capacity was noticed after 8 h fermentation in rice as well as in rice and barley- based vegetable yoghurt-like beverages (Coda et al. 2012).

Effect of fermentation on proximate composition of composite substrate

Effect of fermentation on total solids of composite substrate

The total solids content reduced significantly (P < 0.05) as a result of probiotic fermentation. Loss in dry matter as a result of fermentation had been reported by several workers. The loss in dry matter is might to be due to physiological activities of fermentation process of organisms that utilized part of the substrate nutrients, causing a reduction in dry matter (Abdelhaleem et al. 2008). Table 1 represents the effect of fermentation on composition of composite substrate.

Table 1.

(a and b) Effect of fermentation on nutrient composition (% DM) of composite substrate

	Unfermented	Fermented
a. Nutrients
Total Solids	14.73 ± 0.05a	14.37 ± 0.006b
Fat	2.83 ± 0.17a	2.5 ± 0.29a
Protein	16.33 ± 0.29a	17.79 ± 0.51b
Ash	5.50 ± 0.05a	5.43 ± 0.03a
Fibre	13.10 ± 0.21a	12.5 ± 0.06b
Starch	8.93 ± 0.09a	8.90 ± 0.06a
Carbohydrate	53.31 ± 0.16a	52.82 ± 0.48a
Energy (Kcal)	339.5 ± 1.60a	340.5 ± 2.23a
b. Minerals content (mg/100 g DM) and bioavailability of minerals *
Ca	303.6 ± 1.58a (32.06 ± 1.36a)	302.6 ± 3.0a (72.82 ± 0.26b)
Fe	1.36 ± 0.196a (25.13 ± 1.36a)	1.49 ± 0.198b (50.36 ± 1.58b)
Mg	8.63 ± 0.086a (64.09 ± 0.63a)	8.17 ± 0.091b (83.19 ± 0.89b)
Zn	3.23 ± 0.082a (17.35 ± 0.55a)	2.54 ± 0.028b (64.94 ± 1.17b)

Data represent mean and standard error from three replicates. Different superscript in the same column differ significantly (P < 0.05). Values in parenthesis in 1 (b) represent (%) bioavailability of respective minerals

Effect of fermentation on fat content of composite substrate

The average fat content of UF and F substrate was 2.83 g and 2.50 g per 100 g dry matter respectively (Table 1a). There was non-significant (P < 0.05) decrease in fat content as a result of fermentation. Significant decrease in fat content due to fermentation also reported in pearl millet (Ahmed et al. 2009), soy-rabadi (Gupta and Nagar 2008), maize (Gernah et al. 2011). On the contrary, an increase in fat content due to fermentation was reported in pearl millet based Fura (Inyang and Zakari 2008) and maize-soyabean weaning blends (Amankwah et al. 2009). No change in fat content was observed by some workers (Sindhu and Khetarpaul 2005; Hama et al. 2009; Arora et al. 2010). Reduction in fat content may be due to depletion and utilization of fat during fermentation, which is a catabolic process to obtain energy for microbial activity (Gupta and Nagar 2008).

Effect of fermentation on ash content of composite substrate

There was non-significant (P > 0.05) decrease in total ash content due to fermentation (Table 1a). The observed reduction in ash content might be attributed to possible losses of dry matter and volatiles which normally took place during fermentation. Several workers reported either decrease (Inyang and Zakari 2008; Temitope and Taiyese 2012) or increase (Gupta and Nagar 2008; Amankwah et al. 2009; Gernah et al. 2011) or no change (Sindhu and Khetarpaul 2005; Hama et al. 2009; Arora et al. 2010) in ash content due to fermentation.

Effect of fermentation on carbohydrate, starch and fibre content of composite substrate

Total dietary fibre reduced significantly (P < 0.05) as a result of fermentation (Table 1a). The reduction in fibre content could be attributed due to solubilisation of fibre by microbial enzyme (Sindhu and Khetarpaul 2005). Similar findings had been reported by Arora et al. (2010) in barley based food mixture, Gupta and Nagar (2008) in soya rabadi, Gernah et al. (2011) in maize, Temitope and Taiyese (2012) in ‘oti-oka’ beverage. Contradictory results had been reported by Amankwah et al. (2009) in weaning blends, whereas non significant change in fibre was observed in ‘Fura’ (Inyang and Zakari 2008) due to fermentation.

Starch content did not vary significantly due to fermentation, probably because the organism was starch hydrolysis negative. Reduction in starch as a result of fermentation had been reported in soy rabadi (Gupta and Nagar 2008), Degue (Hama et al. 2009), barley based food mixtures (Arora et al. 2010), whereas starch level did not vary in pearl millet based ‘oti-oka’ beverage fermented by combination of organisms (Temitope and Taiyese 2012).

Carbohydrate content reduced (Table 1a) non-significantly (P > 0.05) due to fermentation of composite substrate. Reduction in carbohydrate might be credited to increased activity of amylolytic enzymes resulting in breakdown of complex carbohydrates to simpler sugars. The simple sugars are responsible for providing energy for the fermenting microorganism for possible synthesis of other compounds, which would also associated with increased availability of nutrients in fermented samples. Similar trend was observed after fermentation of ‘Fura’ (Inyang and Zakari 2008), maize-soybean weaning blends (Amankwah et al. 2009) and maize (Gernah et al. 2011). The opposite trend in fermentation was reported by Gupta and Nagar (2008) in soya rabadi, Hama et al. (2009) in ‘Degue’ and Temitope and Taiyese (2012) in ‘oti-oka’ beverage.

Effect of fermentation on energy content of composite substrate

Energy value of unfermented substrate was 339.5 kcal/100 g dry matter which increased non-significantly (P < 0.05) to 340.5 kcal/100 g as a result of fermentation. Fermentation is generally associated with reduction in energy value due to utilization of the nutrients by microorganisms for growth. Gupta and Nagar (2008) in soya- rabadi, Sade (2009) in pearl millet and Gernah et al. (2011) in maize had reported reduction in energy value due to fermentation. However, Onweluzo and Nwabugwu (2009) observed significant (P < 0.05) increase in metabolizable energy after fermentation. Conversion on nutrients as a result of fermentation resulted in energy improvement (Onweluzo and Nwabugwu 2009) in fermented substrate.

Effect of fermentation on nutritional parameters of composite substrate

Effect of fermentation on phytic acid content

Phytic acid is the major antrinutritional factor present in pearl millet. In cereals phytic acid can interact with several different food components. Phytic acid can bind cations within a phosphate group, between two phosphate groups of the same molecule or between phosphate groups of different phytic acid molecules which render it a strong chelating agent. This chelating property helps phytic acid to bind with minerals and makes them unavailable. At the same time phytic acid form complexes with protein and carbohydrate and reduced their digestion. Phytic acid content of unfermented and fermented substrate was 637.3 mg and 138.4 mg per 100 g dry matter respectively (Fig. 3a). Fermentation brought about highly significant (P < 0.01) reduction in phytic acid content (~ 78%). Presence of microbial phytase in fermenting organism might be one of the reasons of phytic acid reduction. The optimal temperature for phytase activity is known to range between 35 to 45 °C and fermentation was carried out at 37 °C, which was very favourable for microbial phytase activity. Phytase dephosphorylates phytate in successive steps that terminates with the formation of inositol and phosphoric acid. Reduction in phytic acid content during fermentation had been reported by various researchers due to fermentation of various cereal based foods (Onweluzo and Nwabugwu 2009; Gernah et al. 2011; Temitope and Taiyese 2012; Stefano et al. 2017).

Fig. 3.

Effect of fermentation on anti-nutrients content and digestibility in composite substrate; a phytic acid content, b polyphenol content, c protein digestibility, d starch digestibility. UF—unfermented substrate, F—fermented substrate. Values with different superscripted letter in a graph differs significantly (P < 0.05)

Effect of fermentation on polyphenols content

Polyphenols content of UF substrate was 137.8 mg per 100 g dry matter which reduced to 74.37 mg per 100 g dry matter as a result of fermentation (Fig. 3b). There was 46% reduction in polyphenols content due to fermentation and the reduction was highly significant (P < 0.01). The reduction in polyphenolic content was due to presence of polyphenol oxidase present in microflora. Similar reduction in polyphenols during fermentation had been reported earlier in various fermented foods (Sindhu and Khetarpaul 2003; Temitope and Taiyese 2012).

Effect of fermentation on protein digestibility

The in vitro protein digestibility of unfermented substrate was 47.77% which improved significantly (P < 0.05) to 73.38% upon fermentation (Fig. 3c). Protein digestibility is influenced by the presence of various antinutrients and protein structure. Improvement in protein digestibility of fermented products is mainly linked with enhanced proteolytic activity of fermenting organism. An increment in amino nitrogen by fermentation indicates partial breakdown of protein to peptide and amino acid subsequently causing an improved protein digestibility. In addition, phytic acid decreased considerably during fermentation which is a well-known inhibitor of proteolytic enzymes. This might contribute partly towards improvement in protein digestibility of the composite substrate in the current study. Various workers had reported increased protein digestibility upon fermentation in different foods (Sindhu and Khetarpaul 2003).

Effect of fermentation on starch digestibility

The in vitro starch digestibility (expressed as mg maltose released per g sample) of unfermented substrate was 32.52, which improved significantly (P < 0.05) to 73.72 as a result of fermentation (Fig. 3d). The low digestion of starch might be credited to presence of amylase inhibitors, phytate and polyphenols in foods as well as chain length of starch. Amylase present in cereals and microbes cleaves amylose and amylo-pectin to maltose and glucose. Decrease in amylase inhibitor activity might be associated with an improved starch digestibility. Sindhu and Khetarpaul (2003) observed maximum increase in starch digestibility (96%) after sequential fermentation with S. boulardii and L. casei in food mixture containing rice flour, milk coprecipitate, sprouted green gram paste and tomato pulp.

Effect of fermentation on mineral content and minerals bio- availability

The levels of Ca, Fe, Mg and Zn in unfermented samples were 303.6, 1.36, 8.63 and 3.23 mg/100 g dry matter respectively (Table 1b). The Ca level (302.6 mg/100 g) did not change after fermentation, whereas Fe level increased significantly to 1.488 mg/100 g dry matter, where as Mg and Zn content decreased slightly to 8.17 and 2.54 mg/100 g respectively. Several workers had reported no change in minerals during fermentation in fermented Indian bread (Bhatia and Ketarpaul 2012). Increase in mineral contents due to fermentation was noticed by Inyang and Zakari (2008) in fura fermentation and Temitope and Taiyese (2012) in ‘oti-oka’ beverage. Eltayeb et al. (2008), Ahmed et al. (2009) and Sade (2009) observed reduction mineral contents as a result of fermentation in pearl millet. The reduction in Mg and Zn content might be due to the fact that they were used up as the source of energy during fermentation, while, Fe might have been synthesized by fermentation process. Table 1b represents effect of fermentation on Ca, Fe, Mg and Zn contents respectively.

The HCl extractability is an index for bioavailability of minerals in human system. Fermentation resulted in highly significant (P < 0.01) increment in HCl extractability of Ca, Fe, Mg and Zn. The HCl extractability of Ca, Fe, Mg and Zn of unfermented substrate was 32%, 25%, 64% and 17% respectively, which increased to 73%, 50%, 83% and 65% respectively due to fermentation. The enhancement of minerals availability due to fermentation might be attributed to the decrease in phytic acid content. Most of the minerals (Ca, Fe etc.) are bound with phytic acid and formation of a protein-phytate-mineral complex is taken. Phytic acid reduction during fermentation may cause releasing of these metallic ions in free form and therefore, may account for increment in minerals bio-availability in fermented products (Stefano et al. 2017).

Similarly during probiotic fermentation of food blends consisting of germinated pearl millet flour, whey powder and tomato pulp caused a significant (P < 0.05) improvement in availability of Ca, Fe and Zn (Arora et al. 2010).

Effect of fermentation on free and phytate phosphorus content

Total phosphorus decreased significantly (P < 0.05) as a result of probiotic fermentation. Free phosphorus content increased significantly (P < 0.05), whereas phytate phosphorus content reduced highly significantly (P < 0.01) as a result of fermentation (Table 2). Phytate phosphorus content of unfermented sample was 21.44% of total phosphorus which decreased to 4.92% of total phosphorus as a result of probiotic fermentation. Fermentation resulted in 77% decrease in phytate P as % of total P. Reduction in phytic acid during fermentation released phytate phosphorus resulting an increment in free phosphorus depicting better availability of phosphorus in fermented substrate. Reduction in phytate P content had been observed earlier in pearl millet and wheat flour rabadi (Sindhu and Khetarpaul 2003). A highly significant negative relationship (Table 3) was obtained for free phosphorus with phytic acid and polyphenols, whereas a highly significant positive relation was obtained for phytate phosphorus with phytic acid and polyphenols. This indicates reduction in antinutrients resulted in increase in free phosphorus and decrease in phytate phosphorus.

Table 2.

Effect of fermentation on free and phytate phosphorus content (mg/100 g DM)

Fermentation condition	Total phosphorus (TP)	Free phosphorus (FP)	Phytate phosphorus (PP)	PP, % of TP
Unfermented	865.3 ± 5.89a	679.20 ± 0.15a	186.1 ± 5.99a	21.44 ± 1.10a
Fermented	791.30 ± 5.13b	752.30 ± 3.42b	39.02 ± 3.0b	4.92 ± 0.35b

Data represent mean ± SE, n = 3. Within rows, values having different superscript are significantly different (P < 0.05)

Table 3.

Correlation of phytic acid and polyphenols with free and phytate phosphorus, in-vitro digestibility of protein and starch and bio- availability of minerals

Parameters	X1	X2
	Phytic acid	Polyphenols
FP	768.61–0.13X1 R2 = -0.93**	838.35–1.16X2 R2 = -0.97**
PP, % of TP	0.031X1 + 0.656 R2 =  + 0.99**	0.27X2-14.85 R2 =  + 0.97**
In vitro protein digestibility	Y = 82.064 − 0.054 * X1 R2 = − 0.959**	Y = 106.263 − 0.425 * X2 R2 = − 0.984**
Multiple linear regression	Y = 97.337 − 0.019 * X1 − 0.269X2 R2 = − 0.95**
In vitro starch digestibility	Y = 85.17 − 0.083 * X1 R2 = − 0.984**	Y = 121.685 − 0.647 * X2 R2 = − 0.987**
Multiple linear regression	Y = 107.695 − 0.032 * X1 − 0.398X2 R2 = − 0.95**
BA- Ca	Y = 84.129 − 0.082 * X1 R2 = − 0.996**	Y = 120.174 − 0.639 * X2 R2 = − 0.997**
Multiple linear regression	Y = 103.684 − 0.038 * X1 − 0.345X2 R2 = − 0.999**
BA- Fe	Y = 57.257 − 0.05 * X1 R2 =  − 0.990**	Y = 79.299 − 0.39 * X2 R2 = − 0.997**
Multiple linear regression	Y = 73.685 − 0.013 * X1 − 0.295X2 R2 = − 0.997**
BA- Mg	Y = 88.502 − 0.038 * X1 R2 = − .987**	Y = 105.388 − 0.299 * X2 R2 = − 0.987**
Multiple linear regression	Y = 96.816 − 0.019 * X1 − 0.147X2 R2= − 0.987**
BA- Zn	Y = 78.154 − 0.095 * X1 R2 = − 0.998**	Y = 120.081 − 0.745 * X2 R2 = − 0.994**
Multiple linear regression	Y = 91.69 − 0.065 * X1 − 0.239X2 R2 = − 0.999**

FP free phosphorus, PP phytate phosphorus, TP total phosphorus, BA bioavailability

Correlation of antinutrients with digestibility and minerals availability

The regression equation and correlation coefficient of phytic acid and polyphenols with in vitro protein and starch digestibility and in vitro bio-availability of minerals have been expressed in Table 3. In all cases a very highly significant (P < 0.01) negative correlation was observed. Negative correlation between antinutrients and digestibility was also observed by Sindhu and Khetarpaul (2003) in probiotic food mixture. The probable reason for increment in digestibility and mineral availability as a result of fermentation may be reduction in antinutrients content. As in all cases the level of phytic acid and polyphenols was found to have a highly significant (P < 0.01) negative correlation with HCl-extractability of minerals. Similar findings were reported by several workers.

Conclusion

Probiotic fermentation of composite substrate by L. acidophilus (NCDC-13) reduced total solids, fat, ash and total dietary fibre content, whereas protein content increased due to fermentation. Probiotic fermentation was effective in reducing the phytic acid, polyphenols and phytate phosphorus (as % total P) content with concomitant increment in in vitro protein and starch digestibility and bio-availability of minerals in the composite pearl millet-barley- dairy based substrate. A highly significant negative correlation was observed between phytic acid and polyphenols with in vitro protein and starch digestibility and in vitro minerals bio- availability. The substrate comprising of underutilized raw materials can be used as a base for a wide range of low- cost probiotic foods.

Abbreviations

%

Percent

°C

Degree Celsius

ANOVA

Analysis of variance

et al.

And co-workers

FAO

Food and Agricultural Organization

g

Gram

H

Hour

M

Molar

mg

Milligram

mL

Mililiter

AOAC

Association of Official Analytical Collaboration

MRS

De Man Rogosa Sharpe

N

Normality

Kg

Kilogram

L*

Lightness

a*

Redness

b*

Yellowness

R²

Coefficient of determination

RPM

Revolutions per minute

CFU

Colony forming units

S

Second

w/v

Weight by volume

Authors' contributions

SG: Project administration, data analysis, writing; LS: Supervision, Methodology, resources; AKS: Conceptualization, Funding.

Funding

The study was funded by National Agricultural Innovation Project (NAIP), C-1800.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Consent to participate

Corresponding author is providing consent to participate in reviewing process.

Consent for publication

Consent is being provided for publication.