Effectiveness of Lactiplantibacillus plantarum in enhancing the folate content of injera made with different cereals

Abstract Fermentation can contribute to the supply of essential vitamins like folate, but studies exploring this potential are scarce. Injera is an Ethiopian fermented pancake‐like flatbread made from different cereals. The study aimed to investigate the effect of injera‐making process using different cereals (tef, sorghum, wheat, and barley) on folate content and to evaluate the effectiveness of Lactiplantibacillus plantarum in enhancing folate of injera made with different cereals. Cereals were used alone or in combination (tef and sorghum (1:1), wheat and sorghum (3:1), sorghum (100%), and barley (100%)). L. plantarum previously isolated from tef dough and ersho (fermentation batch) collected from the households were used as starters. Folate content of the flour, dough, and injera was determined by microbiological assay. Contribution of consumption of injera made with different cereals to the folate requirement of children and women of reproductive age was evaluated. Among the studied cereals, the highest average folate content (49.9 μg/100 g) was observed in 100% sorghum flour and the least (32.2 μg/100 g) in 100% barley flour, on dry weight basis. After fermentation, the highest average folate content (60.1 μg/100 g) was observed in 100% sorghum dough fermented with L. plantarum. Highest average folate content (15.45 μg/100 g) per fresh weight was observed in wheat and sorghum (3:1)‐blend injera fermented with L. plantarum. Consumption of L. plantarum–fermented injera made with different cereals contributed up to 8% of the recommended folate intake of women of reproductive age.

is mainly related to poor dietary diversity and reduced food intake (Dewey & Brown, 2003).The deficiency of folate has been implicated in a wide variety of disorders such as Alzheimer's disease, coronary heart diseases, osteoporosis, increased risk of breast and colorectal cancer, poor cognitive performance, hearing loss, and NTDs (LeBlanc et al., 2007(LeBlanc et al., , 2011)).Inadequate maternal folate status has also been associated with low infant birth weight, preterm delivery, and fetal growth retardation (Scholl & Johnson, 2000).Megaloblastic anemia and elevated blood concentrations of homocysteine have also been linked to folate deficiencies (Aslinia et al., 2006;Ho et al., 2011).
Insufficient folate intake is also reported to be associated with high blood homocysteine concentration, a potential risk factor for coronary heart disease and stroke (Djuric et al., 2008).Consequently, mandatory folic acid fortification has been implemented in more than 60 countries around the world (Crider et al., 2011).
On the other hand, very high folic acid intake has been associated with high concentrations of unmetabolized folic acid in the blood, which has been associated with the growth of preneoplastic lesions, cancer, and cognitive impairments (Herrmann & Obeid, 2011b;Hoekstra et al., 2008;Hooijberg et al., 2004Hooijberg et al., , 2006;;Morris et al., 2010).Besides, high daily folic acid consumption could mask and delay the diagnosis of an underlying vitamin B12 deficiency and thereby allow vitamin B12 deficiency-associated neuropathies to progress (Institute of Medicine, 2000).Although the natural form (folate) is safer relative to the synthetic form (folic acid), animal-source foods and selected dark-green leafy vegetables are dietary sources; regular consumption of these foods is limited in low-and middle-income countries (LMIC) (Zerfu et al., 2016).This along with the low availability of folic acid-fortified foods leaves behind a significant proportion of the population living in LMIC to be at a greater risk of folate deficiency and its adverse effects (Haidar, 2010;McLean et al., 2008;Taye et al., 2015).
In recent years, the natural fortification of folate in fermented foods by the introduction of microbes that produce folate has attracted attention.Lactic acid bacteria (LAB) and yeasts, which are the main actors of fermentation, could produce up to 100 μg/L and 145 μg/g cell mass, respectively (Hjortmo et al., 2005;Iyer & Tomar, 2009;Sybesma et al., 2003).Up to 256 μg /L total folate production to the growing medium by genetically engineered L. lactis ssp.lactis was also reported by Sybesma et al. (2003).
Cereal-based foods are widely consumed as the primary staple foods in many countries of the world, especially in Africa (Tamene, Kariluoto, et al., 2019).Many cereal-based staples typically undergo a fermentation step before consumption (Humblot & Guyot, 2008).
For example, injera is a leavened, flat, and round cereal-based fermented bread made from different cereals (Mengesha et al., 2022).It is a staple food that is widely consumed in Ethiopia (Baye et al., 2013).
Injera in Addis Ababa (Ethiopia's capital) is preferably made with tef (Eragrostis tef)-an ancient pseudo-cereal that has its center of origin and diversity in Ethiopia (Baye, 2014).A recent study investigated the effect of injera processing (fermentation and baking) on tef injera samples collected from Addis Ababa (Tamene, Kariluoto, et al., 2019).In a subsequent study, a folate-producing Lactiplantibacillus plantarum P2R3FA was isolated from tef fermentation and it was successful in increasing the folate content of injera made with tef (Tamene, Baye, et al., 2019;Tamene et al., 2022).However, whether similar folate-producing effects can be expected for injera made from the fermentation of other cereals remain unknown.
According to Baye et al. (2013), while injera is preferably prepared from tef in major cities, tef-and sorghum-based injera are commonly consumed in the lowlands, whereas barley and wheat injera are common in the highlands of Ethiopia.Therefore, this study aimed to investigate the effect of injera preparation using different cereals on folate concentration.In addition, it investigated the possibility of enhancing the folate content of injera made with different cereals using L. plantarum P2R3FA as a starter isolated from prior tef fermentation.

| Chemicals and raw materials
Unless otherwise specified, all the necessary chemicals and reagents used in this work were purchased from Sigma-Aldrich Chemie GmbH (Buchs, Switzerland).The raw materials (tef, wheat, sorghum, and barley) were obtained from the local market of Addis, Ethiopia.

| Sampling and sample preparation
Information on the different types of cereals used for injera making was obtained from the literature.The most commonly used grain types and mixing ratios for injera preparation were taken from Baye et al. (2013) and actual observations of selected households.

| Preparation of injera made with different cereals and inoculums
The traditional injera-making process was developed after detailed observations from 10 selected households.Households were selected deliberately, where blended cereals are used for injera making, and this resulted in a flow diagram presented in Figure 1.Briefly, the dough was prepared by using blended and/or whole flour of 0.5 kg, 0.75 L sterile tap water, and 125 mL starter culture (0.5:0.75:0.125)(w/v/v).The 125 mL starter culture was prepared by mixing 62.5 mL saline containing the L. plantarum strain and 62.5 g flour (1:1) (v/g); similar proportion was also used for ersho starter.Thereafter, 200 mL sterile tap water was carefully added to cover the surface of the batter and incubated for 4 days at room temperature (firstphase fermentation).After 4 days of fermentation, liquid present on top of the batter was decanted and replaced by a similar amount of fresh tap water except for barley fermentation where there was no supernatant.Before baking injera from the fermented batter, 1/11th of the fermented batter was mixed with sterile tap water (1:3) (v/v), boiled for about 10 min, and cooled to a temperature of around 46°C.The resulting product (absit) was added back to the remaining fermented batter to enhance proper fermentation.In case of barley fermentation, sterile cold water was used instead of absit.The batter was allowed to ferment for 2 h at 25°C until gas production was observed (second-phase fermentation).Finally, the fermented liquid batter (450 mL) was poured onto a hot clay with cover and was baked for 2 min to make the final product, injera.Three baking were performed for each inoculum mentioned under Section 2.3.1.

| Sampling
During injera preparation, pH of the batter was measured initially before fermentation and after second-stage fermentation.Fermented batter was sampled to measure dry matter (DM) and folate contents before the addition of absit, and injera was also sampled to measure DM and folate contents and subjected to sensory analysis.DM was analyzed by oven drying at 105°C.pH was measured using an aliquot of dough immediately after diluting it with deionized water (1:1) (v/v).

| Folate measurement
The total folate content of tef flour, dough, and injera was determined in triplicates using the reference microbiological method after trienzyme extraction (Kariluoto et al., 2004;Tamene, Baye, et al., 2019).The total folate content was determined based on the growth of folate-dependent strain, Lactobacillus rhamnosus ATCC 7469 as the test organism, and (6S)-5-formyltetrahydrofolate (5-HCO-H4 folate) as the calibrant.Performance of the method was checked by testing a blank sample and certified reference material (BCR-121 whole meal flour).Only folate contents in the range of certified value (500 ± 70 ng/g dry matter) were accepted.In addition, for triplicate samples, folate content variations of >10% were not accepted.Analytical procedures were carried out under yellow or subdued light.Alternatively, samples and calibrants were covered with aluminum foil.Sample extracts were kept under nitrogen atmosphere whenever necessary.

| Effect of processing
The effect of cooking (baking) and fermentation on the total folate content was evaluated and results were expressed as percentage retention.

| Contribution of injera consumption to the recommended nutrient intake (RNI) of folate
Based on the existing data on injera consumption from the Ethiopian National Food Consumption Survey as indicated in Tamene, Kariluoto, et al. (2019) and daily folate requirements (required or recommended nutrient intakes [RNI]) from World Health Organization (WHO, 2004), the contribution of consumption of injera made with different cereals to the RNI of folate was estimated for children and women of reproductive age.These population groups were selected because they represent a higher risk for folate deficiency.

| Sensory evaluation
The aim of this experiment was to determine whether the folateenriched injera made with different cereals is acceptable or not by potential consumers.The acceptability and sensory profile for the prepared injera were rated by 30 adult healthy volunteers (women of reproductive age), using a 9-point hedonic scale.The 30 judges of adult healthy volunteers were randomly selected and were provided with the randomly sequenced samples for testing.They were asked to evaluate the products for color, taste, odor, texture (mouth feel), and overall acceptability.As described in Tamene et al. (2022), the panelist used a 9-point hedonic scale to evaluate the acceptability of injera, where 1 = liked extremely and 9 = disliked extremely.They were instructed to cleanse their mouth with water before testing the next sample.

| Statistical analysis
Statistical analysis of folate and sensory acceptability was computed using SPSS version 25 software.The folate analyses were carried out in triplicate and the average values and standard deviations were calculated.The differences between means of folate values in different cereal flour, dough, and injera as well as differences in acceptability ratings were evaluated using analysis of variance (ANOVA) and

| pH of the dough
The pH of the initial dough made with four different blended cereals recorded at 25°C ranged from 5.5 to 6.1 with an average of 5.8 ± 0.3.
After fermentation, the pH value of doughs made with different cereals and fermented with L. plantarum ranged from 3.38 to 3.43 with an average value of 3.36 ± 0.07, whereas the pH value of ershofermented doughs made with different cereals ranged from 3.30 to 3.42 with an average value of 3.41 ± 0.09.There was no significant difference in pH value of all doughs regardless of the cereal types and starters used.

| DM content of flour, dough, and injera
The DM content of flour, dough, and injera made with different cereals and starters were analyzed and results are shown in Table 2.
The average DM of different flour ranged from 91.7 ± 0.57% to 92.7 ± 0.57% by mass.There was no significant difference in DM content of all the flours.
The average DM contents of ersho-fermented dough ranged from 35.6 ± 0.31% to 40.8 ± 0.25% by mass.For L. plantarum-fermented dough, the DM was in the range of 37.9 ± 0.94% and 41.5 ± 0.35% by mass.Significantly higher DM (41.5 ± 0.35% by mass) was observed for dough made with sorghum, whereas there was no significant difference in DM content between the doughs made from the other cereals.
The average DM content of injera made with ersho ranged from 37.2 ± 0.53% to 45.3 ± 0.26% by mass.With the exception of injera made with tef and sorghum blend, which had significantly higher DM (45.3 ± 0.26), there was no significant difference in DM contents for all the other injeras fermented with ersho.Regarding injera fermented with L. Plantarum, the average DM content ranged from 39.9 ± 0.4% to 44.7 ± 0.5% by mass.The DM of injera made from barley (39.9 ± 0.4% by mass) was significantly lower than the injera made from other cereals.
The average folate contents of injera made with different cereals and fermented using ersho ranged from 17.5 ± 0.7 to 23.6 ± 0.7 μg/100 g.Among the different blends, significantly lower folate contents (17.5 ± 0.7 μg/100 g) were observed in tef and sorghum-blended injera.However, there was no significant difference in folate contents among the other injera samples (p ≥ .05).

| Effect of fermentation on folate content of dough
Percentage retention of folate after fermentation was calculated by comparing the amount of folate content in tef flour and dough on DM basis (Figure 2a,b).A value above 100% indicated that fermentation led to increases in folate, whereas retention values less than 100% indicated consumption/losses of folate.
Folate retention after fermentation of different cereals with L.
plantarum ranged from 120 to 141% (Figure 2a).This showed more than 100% folate retention for all of the doughs fermented with the strain regardless of the blend used.The highest retention (141%) was recorded in dough made with wheat and sorghum blend (3:1).In contrast, fermentation with ersho yielded retention ranging from 68% to 107% (Figure 2b).This showed less than 100% folate retention for all doughs with the exception of barley, which had folate retention of 107%.The higher retention for barley dough may be due to the absence of decantation process which may prevent the losses of folate and/or due to the presence of naturally occurring folate-producing microorganisms involved in the fermentation process.

| Effect of baking on folate content of injera made using ersho and L. plantarum
Regardless of the type of cereal blends and the type of inoculants or starters used for fermentation, retention of folate after baking was less than 100%, with an average of 66% (56% to 76%) for L. plantarum (Figure 2c) and 68% (60% to 76%) for ersho (Figure 2d).

| Folate content and calculated contribution of injera made with different cereals and starters to folate requirements (RNI)
The average folate content of injera made with different cereals and fermented with ersho was in the range of 7.82 ± 0.61 and 9.79 ± 0.28 μg/100 g per fresh weight (FW).The lowest average folate content (7.82 ± 0.61 μg/100 g FW) was recorded in barley injera and the highest (9.79 ± 0.28 μg/100 g FW) was observed in wheat and sorghum blend (3:1) injera (Table 4).
However, the average total folate content of injera made with different cereals and fermented with L. plantarum ranged from 13.13 ± 0.59 to 15.45 ± 0.58 μg/100 g FW.The least folate content (13.13 ± 0.59 μg/100 g FW) was observed in barley and the highest (15.45 ± 0.58 μg/100 g FW) was noted in wheat and sorghum blend (3:1) injera.
Contribution of injera consumption to folate requirements was calculated by estimating folate supplied by the average portion size of injera consumed by children (66 g/day) and women of reproductive age (202 g/day) (Tamene, Kariluoto, et al., 2019), relative to the RNI of folate (150 μg/day) for children and (400 μg/day) for women of reproductive age (WHO, 2004).The least folate contribution to RNI was observed in injera fermented by traditional process with ersho, which contributed 3.4% to 4.3% of the RNI for children and 3.9% to 4.9% of the RNI for women of reproductive age.The highest contributions to the folate RNI to both population groups were observed by wheat and sorghum injera, whereas the least contribution was observed for barley injera.
Folate content of L. plantarum-fermented injera was much higher than the ersho-fermented injera and contributed to 5.7% to 6.8% of the RNI for children and 6.6% to 7.8% of the RNI for women of reproductive age.
TA B L E 3 Folate content (μg/100 g) of flour, dough, and injera fermented with L. plantarum and ersho, DM basis.

| Sensory acceptability of different injera made using L. plantarum and ersho
All of the samples made using different cereals and selected inoculums were found to be in the acceptable range with the exception of sorghum injera, which was rated as neither liked nor disliked by the panelists (Table 5).The overall acceptability test result showed that sorghum injera fermented with both inoculums (ersho and L. plantarum P2R3FA) was the least acceptable product, whereas tef and sorghum blended and 100% barley injeras fermented with both inoculums were the most acceptable injera.
Looking at each specific parameter, with the exception of color, for all other sensory parameters (taste, odor, texture, and appearance), wheat and sorghum blended and 100% sorghum injeras fermented with both inoculums were found to be the least liked product.(Tamene, Kariluoto, et al., 2019).

TA B L E 4
Contribution of injera made with different cereals and starters to folate intake requirements (RNI).
In Ethiopia, injera is mainly produced from tef, but it is also prepared from other different cereals like barley, sorghum, and wheat either alone or in combination.Few studies focused on investigating the effect of tef injera preparation on folate contents and the extent to which folate-producing strains (L.plantarum) can be used to enhance the folate content of injera.The current study investigated the effect of injera preparation made with different cereals fermented with ersho or a folate-producing L. plantarum strain priorly isolated from tef fermentation.We the sensory acceptability of the resulting injera and estimated the contribution to folate requirement.
The current study shows that all of the cereals' flour used in our study are relatively good sources of folate.The impact of fermentation (one of the steps in injera preparation) was highly variable.In the case of doughs fermented with L. plantarum, although highly variable, fermentation leads to net folate production.However, doughs fermented with the traditional process using ersho led to reduction of folate with the exception of barley dough which may be due to the presence of other naturally occurring folate-producing microorganisms and/or the lack of decantation process that may lead to folate loses.In contrast with fermentation, baking invariably led to folate losses, which is in line with findings from Tamene, Kariluoto, et al. (2019).
Recent studies showed that fermented foods such as cereals and dairy products have the potential to increase folate intake and potentially prevent adverse effects related to folate deficiencies (Rollán et al., 2019;Saubade et al., 2017).Our study has also revealed the possibility of increasing folate through fermentation as illustrated, for example, by the greater than 100% folate retention observed in barley fermentation.This result suggests that folateproducing microorganisms may dominate those that do not produce or consume folate as previous studies confirmed the co-occurrence of both folate-producing and consuming microorganisms during fermentation process (Okoroafor et al., 2019).The type of microorganisms and the overall conditions that led to folate production in barley fermentation need further detailed examination.Fermentation of all cereals using L. plantarum always led to net folate production, suggesting that the strain is as effective in increasing folate during injera fermentations of other cereals as in tef fermentation.
As baking is the second major step in injera preparation, any folate that is originally present in a food or produced as a result of fermentation needs to survive the baking temperature to have meaningful health impact.Folate is known to be heat sensitive and cooking/baking conditions can cause varying levels of losses (Lešková et al., 2006).Folate losses due to baking are quite common and have been reported by different scholars (Kariluoto et al., 2004;Lešková et al., 2006;Tamene, Kariluoto, et al., 2019).However, some examples of baked injera (ersho-fermented 100% sorghum injera and L. plantarum-fermented 100% barley injera) retained more than 75% of the folates.This finding suggests that folate losses can be minimized if injera baking process is optimized.
Based on portion size estimates obtained from the national food consumption survey, the average folate content in our ershofermented injera samples made from different cereals contributes to less than 5% of the folate RNI for children (1-3 years) and woman of reproductive age.Use of the same cereals with equivalent proportions but changing the inoculum (L.plantarum instead of ersho) yields injera with better folate contents, which can contribute to more than 5% of the daily folate requirement of both women and young children.
Assuming that two servings (∼350 g/serving) of injera are consumed per day by women of reproductive age, injera made from cereals other than tef could contribute to more than 10% of the RNI if L. plantarum is used as inoculum.The present study clearly showed that all injera prepared using the traditional process had the lowest folate content as compared to its counterpart process which used L. plantarum.
The contribution of consumption of these injeras made using L. plantarum to folate requirement is a bit lower than tef injera fermented with the same strain (Tamene et al., 2022).This could be obviously due to differences in the initial total folate content where significantly higher initial total folate is observed in tef flour (Tamene, Kariluoto, et al., 2019) as compared to flours of any cereals used in this study.Otherwise, in both cases, the strain L. plantarum is found to be effective in enhancing the folate content by doubling the contribution of injera relative to the traditional process which uses ersho as inoculum.

Preparation of injera made with L. plantarum and ersho as inoculums
Blending ratio used for the preparation of injera.
Values are means and standard deviations of triplicate samples.Different lower-case superscript letters along the column indicate a statistically significant difference at p < .05 and different upper case superscript letters across the row indicate a statistically significant difference at p < .05.
TA B L E 5 Sensory acceptability of different injera fermented with ersho and L. plantarum starters.