3.2.1. Effect of Flour Composition and Fermentation Time on pH and Titratable Acidity (TA)
The interaction effect of both factors on pH and TA are indicated in
Table 2. The pH of control samples (unfermented porridge) was in the range of 5.22 to 5.26. However, with increased fermentation time, the pH decreased to values below four pH units for fava bean flour-rich samples (MBF
2 and MBF
3).
Shameta samples collected from households of lactating mothers made from similar ingredients composition to MBF
1 resulted in a pH value of 3.9 [
13]. Furthermore, the pH values of MBF
1 and MBF3 fermented for 12 days were in line with some Ethiopian traditional fermented foods such as
Azo and
Cheka, with values of 3.81 and 3.74, respectively, while the pH of MBF
1 fermented for the same days was slightly lower than
Borde with a value of 4.2 [
60,
61,
62]. The result of this study shows that the pH of the controlled study was more or less the same as the pH value of traditionally processed
Shameta made at the household level.
The decrease in pH value with an increase in fermentation time is expected for fermented products due to the growth and dominance of LAB and yeast to produce lactic acid and other organic acids during the fermentation [
63,
64,
65]. A significant change in pH values for all formulations was observed in the first week of fermentation. The pH value decreased from an average of 5.24 units to 4.29 units after eight days of fermentation. From a food safety point of view, the required pH reduction below 4.5 could be achieved within the first week of fermentation. However, the products were fermented up to the 12th day to attain the required nutritional values and sensory properties.
Change in titratable acidy (TA) negatively correlated with change in pH of the fermenting foods. As indicated in
Table 2, the TA increased with a decrease in pH value with fermentation time. However, the TA values observed in this study were lower than the TA value (0.82%) of the
Shameta sample collected from households of lactating mothers made using the same ingredient composition (MBF
1) as reported in a previous study [
13]. The higher TA value associated with over-fermentation of the product could affect the sensorial property of the same product, resulting in low acceptability of the product by the product consumers. Both the pH and TA values recorded in this study and the previous work carried out on microbial dynamics during the fermentation of
Shameta [
14] indicate that a maximum of 12 days of fermentation of
Shameta could be sufficient enough to attain the desired product maturity and safety.
3.2.2. Effect of Flour Composition and Fermentation Time on Proximate Composition
The highest moisture content value was observed in MBF
1 fermented for 12 days (66.32 g/100g), while the lowest was in the MBF
2 control sample (60.58 g/100g). Accordingly, all control samples (cooked porridge not fermented for the second round) have lower moisture content than porridge fermented for the second-round ‘
Shameta’ (
Table 3). This might be associated with the microbial breakdown of polysaccharides to simple sugar during fermentation, while there could be free water in the control samples as the large polymer polysaccharides do not bind water molecules [
66,
67]. Food’s moisture content levels significantly impact the aesthetic values: taste, texture, appearance, shape, and even safety of products. Accordingly, an excessive amount of water could facilitate the spoilage of food products as its excessive losses could also change the sensorial properties of foods, making the product unacceptable [
68]. Regarding the moisture content, almost all of the formulated products assessed in the study were near the category of intermediate moisture foods [
69] in which some of the water bound that makes the products safe for some more weeks in combination with the inhibitory effects of low pH of the products (
Table 2) and essential oils in spices and herbs added during preparation (
Figure 1). The moisture contents of all
Shameta formulations (MBF
1-3) were lower than the co-fermented
Ogi made of maize soybean (67.35 g/100g) and millet soybean (67.86 g/100g). However, the values align with co-fermented
Ogi made of sorghum soybean with a moisture content of 64.52 g/100g [
70].
In the present study, the highest crude protein was observed in MBF
3 fermented for 12 days (16.56 g/100g), followed by the same sample fermented for ten days (16.32 g/100g). However, the lowest was observed in the MBF
1 control sample (12.22 g/100g), followed by the same sample fermented for eight days (12.82 g/100g). The difference might be associated with a higher proportion of fava beans than the MBF
1 sample. In addition, the prolonged fermentation time could contribute to the release of bound proteins and other macromolecules. It is possible to blend cereals’ relatively poor protein quality with protein-rich food crops to achieve a nutritionally balanced diet [
71]. Studies showed that the fermentation of cereals with lactic acid bacteria and yeast cultures increased the protein content of the fermented foods [
72,
73]. With high counts of LAB and yeast in the cereal-based fermented products, the microbial cell mass protein (commonly called single-cell protein) could also contribute to the total crude protein of the fermented product. Omemu and others [
70] also indicated the incremental effect of fermentation on the crude protein content of
Ogi. However, crude protein contents in the present study are different than the values in co-fermented
Ogi made of maize–soybean (6.53 g/100g), millet–soybean (6.64 g/100g), and sorghum–soybean (8.44 g/100g) at a ratio of 66.67:33.33 and fermented for 48 h. Contrastively, the crude protein content (18.28 g/100g) of complimentary food made of a formulation of maize, haricot bean, and cooked banana flour at a ratio of 30:60:10 and fermented for 36 h is greater than the values in the present finding [
74].
The proteins in human milk come from the diet and maternal body stores. The potential to supply the extra protein required by lactating mothers (20 g/day) among the formulations assessed in the present study is the highest in MBF
3 fermented for 12 days (82.8%), followed by the same sample fermented for 10 days (81.6%). The extra protein requirement yield in the present finding is greater than the yield in
Borde (48%), made of 100% maize fermented for four days, and average values of co-fermented
Ogi (36.02%) made of maize–soybean, millet–soybean, and sorghum–soybean fermented for two days [
54,
55]. However, it was lower than complimentary food (91.4%) made of maize, haricot bean, and cooked banana flour (30:60:10) fermented for 36 h [
74]. The finding revealed that to improve the extra protein requirement yield of
Shameta, supplementing primary ingredients (maize and barley) with legumes such as fava bean is a necessity.
As indicated in
Table 3, the flour compositions and fermentation time showed significant differences in crude fat contents. The prolonged fermentation time from the 8th to the 10th and 12th day has provided better crude fat contents. The better crude fat content in MBF
1 could be due to the relatively high proportion of maize flour rich in fat content and rapeseed oil added during the preparation of
Shameta (
Figure 1). Studies have shown that maize is richer in fat content than fava beans [
26,
75]. Maize and rapeseed oil are rich in mono- and polyunsaturated fatty acids with good oxidative stability [
76,
77,
78]. Fatty acids are considered to be a fundamental building material for the structural components of cells, tissues, organs, and synthesis of specific biologically active substances, and facilitate the absorption and transport of fat-soluble vitamins [
79].
In general, the crude fat content in the present finding is far greater than what was reported from other cereal-based fermented foods in Ethiopia, including
Borde (6.9 g/100g) made of maize [
60] and
Cheka (1.3 g/100g) [
62] made of maize and taro leaves (approximately in the ratio of 70:30) and fermented for four days. Therefore,
Shameta is an excellent candidate to promote supplementary fermented cereal-based food rich in fat to support the strength and health of lactating mothers. In addition, the better fat content, likely rich in mono- and polyunsaturated fatty acids, could contribute to better breastfeeding of newborns [
80].
One of the ingredients of
Shameta, barley, contains significant amounts of soluble fiber (beta-glucans), which microorganisms can ferment to produce short-chain fatty acids, an important energy source for the brain, muscles, and tissues [
81,
82]. In addition, the fatty acids contribute to lowering the pH to prevent the growth of pathogenic microorganisms and reduce peptide breakdown and toxin formation [
83,
84]. Fava bean is also a rich source of oligosaccharides, the third most abundant nutrient in breast milk behind lactose and fat, and serves as prebiotic soluble fibers for the infant’s gut, ensuring proper immune responses [
85,
86].
The fiber contents of different formulations assessed in the current study were significantly different (
p < 0.05) from each other (
Table 3). The possible reduction in crude fiber content during the fermentation process could be attributed to the partial solubilization of cellulose and hemicellulosic materials in fermentation by the activities of microbial enzymes [
87]. Although the reduction in fiber content during fermentation is sound, as a high fiber content increases the viscosity of food, which reduces food intake, it plays an essential role in increasing the utilization of nitrogen and absorption of some other micronutrients [
88,
89]. The average fiber content of the current formulations is lower than complementary food made of maize, haricot bean, and cooking banana flour (4.21 g/100g) but significantly different than values in
Cheka (1.1 g/100g),
Injera (pancake-like bread made of teff) (2.8 g/100g) made of 100% teff (
Eragrostis tef) fermented for 24 h and co-fermented
Ogi (0.30 g/100g) [
62,
70,
74,
90].
The ash content of the present finding ranged between 3.75 and 2.45 g/100g for MBF
3 and MBF
1 control samples, respectively, with an average value of 3.05 g/100g. The initial ash content for the fresh porridges showed significant differences due to differences in the proportion of ingredients used to make the porridges. Porridge using a relatively higher fava bean composition (10 or 15%) showed better ash content than porridge with 5% fava bean. Variations in ash content might be associated with a higher mineral concentration in fava bean than in maize and barley flours [
57,
91]. However, the difference has little association with fermentation time compared to the effect of variation in ingredient composition.
The average ash content of all the different formulations is significantly different than values in a complementary food made from maize, haricot bean and cooking banana flour (2.23 g/100g), co-fermented
Ogi (3.01 g/100g),
Cheka (0.75 g/100g) and wheat-based
Borde (0.78 g/100g), while it was lower than the maize-based
Borde (3.7 g/100g) [
60,
62,
70,
74,
92]. The variation could be attributed to differences in composition, preparation steps, and fermentation time.
Shameta could contribute to total ash intake, translating to a better mineral supply than other commonly consumed cereal-based fermented foods like
Cheka and
Borde.
Most of the values of carbohydrates in the present findings were not significantly different (
p > 0.05) from each other (
Table 3). The highest value was observed in MBF
1 fermented for eight days (69.23 g/100g) and the lowest in MBF
3 fermented for 12 days (66.19 g/100g). The difference might be associated with the higher accumulation of carbohydrates in maize than in fava bean [
26]. The recorded carbohydrate content is significantly different than value observed in
Azo (16.6 g/100g) made of sorghum and endod (Phytolaca dodecandra) leaves (50:50) as significant ingredients fermented for 30 days,
Cheka (9.6 g/100g), and co-fermented
Ogi (22.26 g/100g), but lower than complementary food made of maize, haricot bean and cooking banana flour (71.16 g/100g) [
61,
62,
70,
74].
Carbohydrates are primary energy sources that comprise 55% of the total caloric intake [
93]. Therefore, lactating mothers should consume at least 100g/kg/day of carbohydrates from locally available food crops [
92]. The Recommended Dietary Allowance (RDA) of carbohydrates for lactating women is 160 g/kg/day [
93]. Accordingly, MBF
3 fermented for 12 days contributed 41.37% of carbohydrates. The current selected
Shameta formulation (MBF
3) contributes the highest carbohydrate for lactating mothers when compared to other cereal-based fermented condiments, beverages, and porridges such as
Azo (10.38%),
Cheka (6%), and
Ogi (13.91%), while this was slightly lower than complementary food made of maize, haricot bean and cooking banana flour (44.48%) [
61,
62,
70,
74].
Most of the values of gross energy in the present finding were not significantly different (
p > 0.05) from each other, with the highest value in MBF
1 fermented for 12 days (446.87 Kcal/100g) and the lowest in MBF
3 fermented for 8 days (425.49 Kcal/100g) (
Table 3). The gross energy in the present finding is significantly different from values in a complementary food made of maize, haricot bean, and cooked banana flour (397.11 Kcal/100g) and co-fermented
Ogi (218.77 Kcal/100g) [
70,
74]. Although the increase in energy requirements during lactation is maximal compared to protein requirements, if the energy intake is low, protein will be used for energy production rather than its primary role [
94]. According to the extra energy demand for exclusive breastfeeding from birth to six months postpartum (500 kcal/day), the MBF
3 fermented for 12 days provides 85.38% for lactating mothers. All gross energy values recorded in the current study are significantly different than values in
Injera (76.8),
Azo (18.3), and
Cheka (18.8%) of the extra energy required for lactating mothers, respectively [
61,
62,
95].
3.2.3. Effect of Flour Composition and Fermentation Time on Minerals Contents
Calcium (Ca) is one of the essential mineral elements for better recovery and strength of lactating mothers. Calcium deficiency in maternal nutrition could lead to hypertensive conditions, pregnancy disorders, lower blood pressure, and osteoporosis [
96]. Results in
Table 4 showed that flour formulations had a significant effect on the Ca content than the effects of fermentation time. Porridge prepared from flour composition rich in fava beans results in better Ca content (15%) than others. A relatively high Ca content was observed in MBF
3 porridge fermented for 12 days (61.3 mg/100g), followed by the same formulation fermented for 10 days (60.7 mg/100g). However, the lowest Ca content was observed in the MBF
1 formulation (25.8 mg/100g) because of its small proportion of fava bean flour (5%). Even if the fermentation time appears to have some effect on the Ca content, overall, the effect was not as significantly high as that of the composition of ingredients.
The results of this study are in agreement with complementary foods made of maize, haricot bean, and cooking banana flours (30:60:10) with a value of 61.43 mg/100g; however, this value is lower than the average value in co-fermented
Ogi (2073.54 mg/100g) made of maize–soybean, millet–soybean, and sorghum–soybean with a cereal–soybean ratio of 66.67:33.33 and fermented for 48 h [
70,
74]. According to the present analysis, the highest recorded Ca content could meet only close to 6.1% of DRA of Ca for lactating mothers. However, ‘
Injera’ made from 100% teff (Eragrostis tef) and fermented for 24 h, could meet 16.77% of DRA for lactating mothers [
90].
Cheka, the other form of cereal-based fermented food made of maize and taro leaves (approximately 70:30) and fermented for four days, could meet only 1.47% DRA [
62]. Even if there is an improvement in the Ca content by more than double as compared to the control, there is a need to have additional Ca from other sources, including the consumption of
Shameta with
Injera, to improve Ca contents for rapid recovery and strength of lactating mothers.
The iron (Fe) and Zinc (Zn) contents of the
Shameta formulations were not significantly (
p > 0.05) affected by the fermentation time (
Table 4). In this study, the Fe and Zn contents improved with an increment in the proportion of fava bean flour in the mix from 5 to 15%. The highest Fe content (8.83 mg/100g) and better Zn contents were observed in MBF
3 porridge fermented for 12 days (
Table 4).
The Fe contents in most of the analyzed samples were different than values in complementary foods made of maize, haricot bean, and cooking banana flour (5.69 mg/100g) and
Ogwo (0.34 mg 100 g
−1) made of malted sorghum, un-malted sorghum and potato (54.55:27.27:18.18) fermented for 48 h; however, the contents were lower than the Fe contents of
Cheka (18.3 mg 100 g
−1) and
Injera (15.4 mg 100 g
−1) [
62,
90,
97,
98]. Iron deficiency revealed during lactation may not only be because of a lack of access to iron-rich foods. However, it could also be associated with complications related to iron status before pregnancy, hemorrhage after delivery, low-vitamin C diet, excessive consumption of tannin-rich foods, frequent pregnancies, and early pregnancy. According to this study, the Fe content in MBF
3 porridge fermented for 12 days could meet close to 98% of the recommended dietary allowance of Fe for lactating mothers.
Although the demand for Zn increases during lactation is required for many biological activities, it has been reported that the amount of zinc in breast milk is independent of Zn in diet [
99]. Fermented porridge produced from MBF
3 samples fermented for different days could provide close to 74% of RDA of Zn for lactating mothers. These values are better than those reported from cereal-based fermented foods in Ethiopia, such as
Cheka and
Injera, which could provide only 7.67 and 20% of RDA, respectively [
62,
90]. Thus, the mineral composition of fermented products could be improved by considering the ingredients’ compositions more than monitoring the fermentation time. Therefore, further improvement in mineral content can be achieved through home or industry-based formulations for better health and recovery of the mothers and their infants.
3.2.4. Effect of Flour Composition and Fermentation Time on Anti-Nutritional Factors and Antioxidant Capacity
The adverse health effect of phytate in the diet reduces the absorption of minerals such as Zn
2+, Fe
2+/3+, Ca
2+, Mg
2+, Mn
2+, and Cu
2+; Zn and Fe deficiencies in particular have been reported as a consequence of high phytate intake [
100]. A report indicated that a high level of dietary tannin (120 mg/kg) reduces protein absorption and damages the intestinal walls [
101]. This study aimed to minimize the negative impact of phytate and tannin in traditionally produced fermented porridge. Results of the study showed that both the formulation of flour to make the porridge and fermentation time have significantly different (
p < 0.05) effects on the phytate and tannin contents. The concentrations decreased with a decrease in maize flour from 90% to 75% due to an increase in fava bean proportion.
Similarly, an increased fermentation time significantly reduced the phytate and tannin contents. For instance, after 12 days of fermentation, the phytate content decreased by 84, 70, and 71% compared to fresh
Shameta for MBF
1, MBF
2, and MBF
3, respectively (
Table 5). Similarly, the tannin concentration decreased by 88, 78.5, and 78.5% after 12 days of fermentation for the same formulations. As expected for fermented food products, the fermentation time’s impact was significant compared to flour formulations.
The phytate content recorded in the current study is significantly lower than its content in complementary food made of maize, haricot bean, and cooking banana flour (30:60:10) (36.99 mg/100g) fermented for 36 h and
Kutukutu (12.4 mg/100g) made of 100% corn fermented for 24 h, while it is greater than co-fermented
Ogi (0.2 mg/100g) made of maize–soybean, millet–soybean, and sorghum–soybean with a cereal/soybean ratio of 66.67:33.33 and fermented for 48 h [
70,
74,
102]. Consumption of dietary phytate up to 500 mg/day leads to a 0.04 mg/day reduction in zinc absorption [
103], while Ndie and Okaka [
104] reported that the levels of phytate between 23.5 and 130.65 mg/kg are high enough to be associated with health risk. However, the phytate contents in this study are below 1% after one week of fermentation of the product.
The tannin content recorded in this study is also lower than the content in complementary food made of maize, haricot bean, and cooking banana flour (31.32 mg/100g) but significantly different than value in co-fermented
Ogi (0.13 mg/100g) [
70,
74]. According to Ndie and Okaka, [
104], levels of tannins up to 108.3 mg/kg are high enough to be associated with health risks beyond reducing the bioavailability of nutrients. However, the tannin level for fermented porridge is significantly lower than 1% for all durations of fermentation. The observed lower value could be associated with the combined effects of first-stage fermentation, intermediate cooking, and second-stage fermentation of the product. The absence or lower value of tannin will increase the prevalence of high bioavailability of minerals and proteins, which is necessary for lactating mothers in support of their rapid recovery, strength, and health.
Consuming food products rich in phytochemicals during pregnancy and lactation is a critical component of dietary guidelines to protect mothers and infants from oxidative damage and related diseases [
105]. The result showed that the MBF
3 sample fermented for 12 days had a better DPPH scavenging ability with a lower IC50 value than other formulations fermented at different fermentation times. However, in all formulations, the potential for scavenging activities increased as fermentation time increased. Adebo and Gabriela Medina-Meza [
106] also reported that an increase in fermentation time increased the total phenolic contents and antioxidant activities of whole cereal grains. The ability of fermentation to improve the antioxidant activity is primarily due to an increase in the number of phenolic compounds and flavonoids as a result of the structural breakdown of plant cell walls by microbial hydrolysis reaction [
106,
107]. Meanwhile, the variation in antioxidant activities in control samples might be due to the variation during the roasting of spices and intermediate cooking to make porridge [
108,
109,
110]. Generally, in addition to providing protein and some minerals, the consumption of
Shameta improves the health of lactating mothers and newborns by preventing oxidative stress.