Isolation, identification and characterization of predominant microorganisms in Swazi traditional fermented porridge (incwancwa) and optimization of fermentation conditions

ABSTRACT The objective of the study was to isolate, identify and characterize the predominant microorganisms during the fermentation of incwancwa and optimize fermentation conditions. The lactic acid bacteria (LAB) load from fermented uncooked samples ranged from 4.6 ± 1.46 to 6.56 ± 0.2 log CFU/g whereas the load for yeast and coliforms ranged from 1.5 ± 0.39 to 2.12 ± 0.43 log CFU/g and 2.14 ± 0.22 to 4.09 ± 0.57 log CFU/g, respectively. LAB were the dominant microorganisms in incwancwa where Lueconostoc lactis was the main. Fermentation time and temperature had significant (p < .05) effect on the sensory perception of the product. The best temperature and time was found to be 32°C to 35°C for 8 to 24 hours. Numerical optimization predicted that the preferred temperature and time combination was 35°C for 15 hours. Thus, it is feasible to produce an acceptable incwancwa product under temperature/time conditions of 35°C for 15 hours using starter cultures derived from the original indegonuos flora of traditional incwancwa.


Introduction
Fermented foods often have numerous advantages over the raw materials they are made from. Benefits of fermented foods include improvement in palatability and acceptability (by developing improved flavors, taste and textures), increased shelf life, enrichment of nutritive content, improved digestibility of protein and carbohydrates, removal of anti-nutrients, improved bio-accessibility of micronutrients and decreased cooking times (Bationo et al., 2020;Guyot et al., 2012;Kaur et al., 2017;Vilela et al., 2020). Studies have shown the positive impact of fermented food on gut microbiota and on health (Tsafrakidou et al., 2020;Vilela et al., 2020). The impact of lactic acid bacteria (LAB) fermentation on bioactive components of cereal-based products has also been reviewed and the ability of LAB to synthesize vitamins and other metabolites with health benefits has been reported (Capozzi et al., 2012).
Cereal-based fermented foods are increasingly being considered as sources and vehicles for probiotic microorganisms in the diet (Achi & Asamudo, 2015;Muyanja et al., 2012;Szydłowska et al., 2021). Currently, fermented milk is the main vehicle for providing probiotic microorganisms through commercial products. However, many consumers cannot digest dairy products or experience some allergies. Traditional fermented foods of plant origin have been found to contain probiotic lactic acid bacteria such as Lactobacillus plantarum (Szydłowska et al., 2021), indicating that they are a good substrate for growth of probiotic strains. Growth of LAB results in production of various metabolites including organic acids, amino acids, carotenoids, vitamins, and minerals that induce desirable responses in the human body (Kandylis et al., 2021;Sharma et al., 2020).
Incwancwa is a traditional fermented cereal food used as a breakfast meal in Eswatini. It is produced by spontaneously fermenting maize or sorghum flour at ambient temperatures followed by cooking. It is consumed while hot and is not stored for long periods. The estimated shelf life is about 24 hrs after cooking. The effect of processing method (Abegaz et al., 2004;Bationo et al., 2019Bationo et al., , 2020, composition of microbiota (Pswarayi & Gänzle, 2019), changes in microbial load and pH (Simango, 1997), effect of fortification (Afolabi et al., 2018;Maakelo et al., 2021) during fermentation of different products has been studied and reported. Detailed fermentation steps were previously documented for some Swazi fermented foods like umcombotsi, and emahewu (Simatende et al., 2015). There, however, lies a gap on the study of incwancwa since details of the preparation steps, biochemical changes during its preparation, and predominant microflora during its fermentation have not been systematically studied and recorded.
Recent developments in microbial classification have adopted the use of molecular techniques for identification of different strains of lactic acid bacteria (LAB) and other microbes associated with fermented foods down to species and strain level. These techniques include Polymerase Chain Reaction (PCR) and Amplified Fragment Length Polymorphism (AFLP). The use of molecular techniques allows for accurate identification of different species of microorganisms in food products (Beneduce et al., 2007). These technologies could be of benefit in accurately identifying and characterizing predominant microorganisms in incwancwa. According to available information, no systematic study on the microbial and physcio-chemical aspects of incwancwa have yet been done. Although the product is popular in Eswatini, there are no records documenting the production process. The aim of the study was to isolate, identify and characterize predominant microorganisms during fermentation of incwancwa and to optimize the fermentation conditions for an acceptable product. This will provide data necessary for further standardisation and potential commercial production.

Collection of incwancwa samples
Samples were obtained from three different homesteads where the cereal was produced on daily basis for private consumption, and also from three different care points. Households were selected using convenience sampling and samples of incwancwa were collected three times on three different days from each sampling point. The samples were collected at two stages: after fermentation before cooking; and after cooking in sterile 250 ml glass bottles and were transported in a cooler box to the laboratory for analysis.

Enumeration of LAB, coliforms and yeasts
Samples of fermented uncooked and cooked incwancwa were serially diluted in quarter strength Ringers solution to highest dilution of 10 −6 . Appropriate dilutions (those giving between 100 and 300 cfu) were plated on de Man Rogosa and Sharpe (MRS) agar and Violet Red Bile Glucose (VRBG) agar and incubated at 30°C for 48 hrs. To enumerate yeasts, portions of serially diluted samples were plated on Rose-Bengal Chloramphenicol agar (RBCA) and incubated (Lab Incubator model IN-665, Germany Industrial Corp, Taiwan) at 25°C for 3 days (Oxiod Limited, 2018).

Isolation of lactic acid bacteria
MRS plates inoculated with fermented uncooked incwancwa were used for isolation of LAB strains. Colonies with different morphologies were picked and purified by further streaking on MRS agar. The streaking was repeated until pure colonies were obtained. The colonies were taken through preliminary identification by conducting Gram staining and catalase tests. The identification involved microscopic observations and biochemical reactions of the strains. Pure colonies were picked from each of the plates using an inoculating loop and were suspended in MRS broth and incubated for 24 hours at 30°C (European Monoclonal Antibodies Network, 2018; Karen, 2010). Gram positive and catalase negative isolates were taken as presumptive LAB.

Preparation of concentrated cultures
The presumptive LAB strains were cultured in MRS broth at 30°C for 24 hours. The culture was then centrifuged in sterile centrifuge tubes to produce a pellet of pure LAB culture. The supernatant was discarded and the pellet re-suspended in MRS broth containing 20% (v/v) glycerol as described by Angshumanjana et al. (2016). The concentrated cultures were stored frozen until required for further tests.

Growth trials
Growth trials were performed to determine the ability of the isolates to grow individually in sorghum gruel. Fourteen presumptive strains were used for the trials, which had been previously stored in MRS broth containing 20% (v/v) glycerol. The culture was thawed and a loopful of each strain was revived in sterile Brain Heart Infusion (BHI) broth (Mast Group Ltd DM106D, Merseyside, UK), streaked on fresh MRS agar and incubated at 30°C for 48 hrs. A pure colony was picked from each of the plates using an inoculating loop and was suspended in MRS broth and incubated for 24 hours at 30°C. A concentrated culture was obtained by centrifuging the broth culture as previously described. The pellet obtained was washed with quarter strength Ringer's solution to remove excess broth and recentrifuged. The final pellet was re-suspended in fresh Ringers' solution (5 ml) and a portion (1 ml) of this culture was used to inoculate sterile sorghum gruel. The mixture was incubated at 30°C for 48 hours, whereby portions of the fermenting mixture were aseptically withdrawn at the following time intervals: 0, 6, 24 and 48 hours. LAB counts were determined by pour plating on MRS agar (Oxiod Limited, 2018). The pH was determined using a pH meter (pH50, XS, Instruments, PRC) calibrated using buffer solutions pH 4 and pH 7.

Identification of LAB by 16S rDNA sequencing
Out of the fourteen isolates that were selected for trial, seven that showed the best signs of rapid growth and acid production were selected for further identification using Polymerase Chain Reaction (PCR). In this technique, genomic DNA was extracted from the cultures using the Quick-DNA Fungal/ Bacterial Miniprep Kit (Zymo Research, Catalogue No. D6005). The 16S target region was amplified using OneTaq Quick-Load 2X Master Mix (NEB, Catalogue No. M0486) using the primers shown in Table 1. The PCR products were run on gel electrophoresis, which separates DNA, RNA and protein macromolecules and their fragments based on their size and charge. These were extracted with the Zymoclean Gel DNA Recovery Kit (Zymo Research, Catalogue No. D4001). The extracted fragments were sequenced in the forward and reverse direction (Nimagen, Brilliant DyeTM Terminator Cycle Sequencing Kit V3.1, and BRD3-100/1000) and purified using Zymo Research, ZR-96 DNA Sequencing Clean-up Kit (Catalogue No. D4050). The purified fragments were analyzed on the ABI 3500×l Genetic Analyzer (Applied Bio systems, Thermo Fisher Scientific) per sample, CLC Bio Main Workbench v7.6 was used to analyze the ab1 files generated by the ABI 3500×L Genetic Analyzer and results were obtained through Basic Local Area Sequencing Template (BLAST) search (NCBI) (Altschul et al., 1997;Xiao-Qin et al., 2013).

Sensory evaluation
Samples were prepared by mixing sorghum flour with distilled water and added in to 50 ml glass bottles, then sterilized using an autoclave (Tuttnauer, model 2540 ML USA) for 30 minutes at 121°C. Thereafter, a loopful of the concentrated culture was then aseptically added to the sterile sorghum gruel by first reviving them in sterile Brain Heart Infusion (BHI) broth (Mast Group Ltd DM106D, Merseyside, UK). Samples were made in replications of four for each run and incubated. Samples were then added into boiling water and left to boil with stirring for 10 minutes. Water was added to the mixture and cooked further without stirring for approximately 30 minutes. The samples were cooled and used for sensory evaluation. The sensory evaluation was conducted using 52 untrained panellists to determine consumer acceptability of the incwancwa. The panellists were simultaneously presented with coded incwancwa samples and tasted the samples in sensory evaluation booths. The degree of liking was recorded on a 9-point hedonic scale labelled as follows: 1 = dislike extremely, 2 = dislike very much, 3 = dislike moderately, 4 = dislike slightly, 5 = neither like nor dislike, 6 = like slightly, 7 = like moderately, 8 = like very much, 9 = like extremely.

Optimization of fermentation conditions
A face centred central composite design (CCD) was used to investigate the effect of fermentation time and temperature on the sensory attributes of incwancwa (Montgomery, 2001). The coded and actual values of time and temperature are presented in Table 2. A total of 13 runs were generated using Stat -Ease Design-Expert version 7.0. This involved the making of 13 different samples with different temperature, time combination as generated by the software and were presented to the panellists. Graphical optimization was used to determine region of optimal combination of the fermentation time and temperature of incwancwa based on minimum of "like" intensity. The contour plots of different attributes were super-imposed to come up with an overlay plot (Montgomery, 2001). The overlay plot was used to determine the optimal region based on maximum like intensity optimization criteria. The desirability function (Montgomery, 2001) was used to numerically optimize the blend proportions. For n attributes, the overall desirability D based on the desirability value (d i ) of each attribute is given by:

Data analysis
Data from enumeration of microorganisms was analysed using Statistical Package for Social Science (SPSS) version 20. The least significant difference (LSD) was used to compare treatment means for the uncooked and cooked samples, between sampling days and between sampling points as shown in Tables 2, 3 and 4. Significant difference was accepted at p < .05. Optimization was done using Design Expert version 7.

Preparation of incwancwa
The steps for the preparation of incwancwa based on the description by the respondents are presented in Figure 1. Two major steps identified were fermentation followed by cooking. Fresh sorghum flour is mixed with warm water (32 ± 3°C) and the slurry is inoculated with a small portion (approximately 20% of the total mass) of slurry from a previous fermentation and fermented for 12 to 48 hours. The slurry is added into boiling water and boiled with stirring for 5 to 10 minutes. More water is added to the mixture and cooked further without stirring for 15 to 30 minutes. The product is then cooled and ready to serve. Understanding the traditional process will pave the way to refine and come up with an improved processing method for better quality product in terms of safety, nutritional and sensory attributes (Bationo et al., 2019). Table 3 shows the microbial load in cooked and uncooked incwancwa across all locations and all sampling days; whereas Table 4 shows the microbial load of cooked and uncooked incwancwa over sampling days, and sampling locations, respectively. LAB had the highest counts (Tables 3 and 4) compared to yeasts and coliforms, which is similar to previous studies with different cereal-based fermented foods and beverages (Guyot et al., 2012;Miguel et al., 2012). The high number of LAB in the uncooked mixture was also in agreement with what had been reported in other studies on other types of cereal fermented products (Blandino et al., 2003;Kostinek et al., 2005). Lactobacillus species were reported to be predominant in kenkey, gari, agbelima, with L. plantarum being identified most often (Amoa-Awua et al., 1996; Kostinek et al., 2005). Species of the genera Leuconostoc, Lactobacillus, Streptococcus, Pediococcus and Micrococcus are the most common in the fermentation of food and beverages produced using cereals such as sorghum, wheat, maize and rice (Capozzi et al., 2012;Guyot et al., 2012;Miguel et al., 2012). LAB frequently predominate in fermented foods due to their ability to tolerate low pH. LAB also produce a variety of antimicrobial substances that create conditions that are unfavorable for the growth of pathogens and spoilage microorganisms (MacDonald et al., 1990). The co-occurrence and interdependent association between LAB and yeast in African traditional fermented foods have been reported by several authors (Akinleye et al., 2014;Gadaga et al., 1999;Miguel et al., 2012;Steinkraus, 1996). LAB create an acidic environment which is conducive for yeast proliferation, while yeasts produce vitamins and other essential growth factors such as amino acids for the lactic acid bacteria (Steinkraus, 1996). Yeasts contribute to the typical flavor of the fermented product and some show enzymatic ability that may contribute to breaking down of starch and also allow improved absorption of essential minerals (Amoa-Awua et al., 1996;Omemu et al., 2007). Although coliforms are commonly found in different cereal-based fermented foods and beverages (Almeida et al., 2007;Gadaga et al., 1999), the population found in Las medias en las columnas seguidas de letras distintas son significativamente diferentes a p < .05. Table 3. Average number of microorganisms (log CFU/g) in the samples from different sampling days before cooking and after cooking.

Diversity of microbes in incwancwa
Tabla 3. Número medio de microorganismos (log CFU/g) en las muestras de diferentes días de muestreo antes de la cocción y después de la cocción.  incwancwa was lower than what was reported for other sorghum-based cereal products. Generally, coliforms had low counts in both the fermented cooked and uncooked samples for all the sampling points, compared to the LAB (Table 3). This is in agreement with other studies of LAB fermentation by Oyarekua (2011), who reported low counts of enterobacteria and other gram-negative bacteria, which indicates that their growth may have been inhibited by the presence of lactic acid and hence low pH, resulting in a decrease in the population. The presence of coliforms generally suggests feacal contamination and/or contamination from microbes that are naturally found in the soil. This in turn suggests possible exposure to pathogens that cause gastrointestinal diseases such as diarrhoea and typhoid fever. Poor environmental sanitation and/or personal hygiene can be responsible for much of the contamination, while improper storage leads to multiplication of pathogens in food.

Effects of cooking on microbial quantity
Generally, a decrease in microbial load was observed after cooking and a significant difference (p < .05) was observed between the cooked and uncooked samples. The average initial LAB count in the uncooked sample was 5.64 Log CFU/g, which was significantly reduced to an average of 1.82 Log CFU/g. The yeast count was also reduced from an average of 1.83 log CFU/g before cooking to 0.82 log CFU/ g after cooking. Similarly, the population of coliforms showed a significant reduction from 2.80 log CFU/g to 1.05 log CFU/g due to cooking. These observations indicate that a significant number of the microorganisms were susceptible to heat treatment. Microbial cell reduction occurs slowly just above maximal growth temperatures. However, the rate of death increases markedly as the temperature is raised. Some survivors that could be thermoduric were found even after cooking. This suggests that there is a need to optimize the cooking conditions to ensure product safety and shelf stability

Variability of microbial loads over sampling days
The microbial counts of cooked and uncooked samples at different days of sampling are presented in Table 4. The results indicated that there were significant differences (p < .05) in the yeast counts in both cooked and uncooked samples taken on different days at the different locations. The yeast counts ranged from 2.01 log CFU/g to 1.6 log CFU/ g and 0.95 log CFU/g to 0.62 log CFU/g for uncooked and cooked samples, respectively. A significant difference (p < .05) was observed between sampling day 3 and sampling day 1 and 2 while sampling day 1 and 2 had no significant difference (p > .05) indicating inconsistencies in day-to-day operations. The counts ranged from 5.35 log CFU/g to 5.80 log CFU/g for the fermented uncooked samples whereas the range for cooked samples was from 1.7 log CFU/g to 1.82 log CFU/g (Table 3). Significant differences (p < .05) in coliforms among sampled days were observed for uncooked samples where the count ranged from 2.65 log CFU/g to 2.88 log CFU/g. A significant difference was observed between sampling day 3 and sampling day 1 and 2. The differences in the microbial load between the sampling days confirm the variability of microbial concentration owing to the spontaneous nature of the fermentation. Other factors that can lead to variability in microbe concentration between sampling days can be the time of day, relative humidity, relative concentration of particles or organisms, and the performance of the air. Microbial counts also varied according to sampling locations (Table 4, with the counts ranging from 1.5 log CFU/g to 2.1 log CFU/g in uncooked fermented incwancwa and 0.41 Log CFU/g to 1.2 log CFU/g for cooked incwancwa). The variabilities observed in microbial load over sampling days and sampling locations are indicative of the need to standardize the process parameters in order to obtain products of consistent quality and possibly commercialize the process and the product.

Preliminary tests
Several isolates, which had different colony shapes and sizes were picked and subjected to microscopic observations and biochemical tests. Fourteen isolates were rod shaped, gram positive, and catalase negative and were therefore taken as presumptive Lactobacillus strains (Rhaiem et al., 2016). The presumptive lactic acid bacteria isolates were then selected for growth trials and PCR identification.

Growth trials
Growth trials were done to select LAB strains that grew well in sorghum gruel. The strains were inoculated into a sterile sorghum gruel and left to ferment for 48 hours. Figure 2 shows changes in pH and LAB counts in the gruel over time. The pH dropped gradually for some of the isolates and rapidly for others by the end of the 48 hours. The lowest pH attained after 48 h was pH 4.02, obtained with Isolate 4 from an initial pH of 5.87. Isolates that showed rapid decline in pH within the first 6 hours of fermentation, in the order of decline were isolates 4(CX4), 2 (CX2), 1(CX1), 12(CX12), 7(CX7), 9(CX9), and 3(CX3), respectively. The low pH provides protection of the product from pathogenic microorganisms (Gadaga et al., 2004). Seven isolates that had the lowest final pH at the end of 48 h of fermentation were further identified to species level using PCR.
The growth patterns as a function of fermentation time of the LAB isolates ( Figure 3) were generally similar to typical trends in LAB fermentation. The highest LAB counts of 6.1 log CFU/g were obtained for isolate 1, while the lowest of 4.6 log CFU/g were recorded for isolate 9. The fourteen isolates displayed in Figure 2 showed an increase in LAB counts as time increased. Based on the observations in Figures 1 and 2, final pH after fermentation ranged from 4.02 to 6.02. In a separate study, MacDonald et al. (1990) reported final pH of 4.6 to 4.8 that when Lactobacillus plantarum was used to ferment millet and sorghum. The current observations indicated that the chosen isolates were potential starter cultures for sorghum fermentation. LAB have consistently been associated with traditional fermentation of sorghum into beer (Steinkraus, 1996;Tsafrakidou et al., 2020;Vilela et al., 2020).

PCR identification of lactic acid bacteria
Out of the fourteen isolates that were selected for trial, seven that showed the best signs of rapid growth, acid production, and were gram positive, catalase negative and rod shaped, were further identified using Polymerase Chain Reaction (PCR). The Basic Local Alignment Search Tool (BLAST) results indicate the similarity between the sequence queried and the biological sequences within the National Center for Biotechnology Information (NCBI) database that are used as standards. Table 5 shows the Blast results of all the presumptive isolates. All the isolates were identified as Leuconostoc lactis. Leuconostoc spp. are chemoorganotrophics with obligate requirement for a fermentable carbohydrate (Dellaglio et al., 1995). They require nutritionally rich media including complex growth factors and amino acids. Following the results of the identification, starter cultures derived from the original indigenous flora can be used of traditional incwancwa of consistent quality.     Leuconostoc strains are beneficial for numerous technological aspects linked to their capacity to produce organic acids, carbon dioxide, dextrans and, especially, aromatic compounds, such as diacetyl, acetaldehyde and acetoin (Flórez et al., 2016). Thus, well-characterized strains are intentionally added as starter or adjunct cultures in many production processes to control the fermentations and contribute to the organoleptic and rheological properties of the final product (Hemme & Foucaud-Schuman, 2003). Leuconostic are generally regarded as safe (GRAS) due to their long history of safe consumption through traditional fermented foods. The European Food Safety Authority (EFSA) considers Leuconostoc to be suitable for the qualified presumption of safety (QPS) in their safety assessment, which requires that technological strains intended to be introduced into the food chain should lack acquired or transferable resistance determinants to antimicrobials of clinical and veterinary importance to prevent lateral spread of strains with acquired transferrable resistance (Flórez et al., 2016).
Other studies evaluating the microbial communities of cereal-based fermentations expose a great diversity of microbial communities with lactic acid bacteria being the dominant (Schoustra et al., 2013). Various species of lactic acid bacteria have also been isolated from raw sorghum powder (Kunene et al., 2000). Hounhuigan et al. (1993) isolated L. brevis, L. curvatus, P. pentosaceus and Pediococcus acidilactici during spontaneous fermentation of mawe, a maize-based fermented product. Leuconostoc sp. have also be isolated from Boza, a sweet thick beverage made from wheat, millet maize, rice, barley and oats and consumed in Turkey, Albania, Romania, and Bulgaria (Hancioğlu & Karapinar, 1997). Also, a Zimbabwean maize porridge, mutwiwa is reported to be produced using lactic acid bacteria, other bacteria, and yeast as starter cultures,  and tobwa, which is another Zimbabwean origin cereal made from maize has lactic acid bacteria as starter cultures (Gadaga et al., 1999). Agarry et al. (2010) reported that Leuconostoc. lactis, together with L. plantarum, and L. fermentum, is used as a starter culture in the fermentation of a Nigerian non-alcoholic fermented cereal beverage made from millet/wheat/rice/malted rice known as Kunun-zaki. Lueconostic lactis is very common in the fermentation of cereal-based products (Efiuvwenwere & Akona, 1995).

Sensory evaluation and optimization
The sensory attributes including taste, texture, appearance, flavor, aroma and overall acceptability were significantly affected by fermentation time and temperature (p < .05). Response surface trends for taste, texture, flavor, aroma and overall acceptability as a function of fermentation time and temperature were similar. The sensory scores increased with increase in fermentation time from 6 h to 15 h followed by a decrease as the fermentation continued to 24 h ( Figure 4). The response surfaces also showed that the sensory panel gave increasing ratings for taste, texture and aroma as temperature increased from 25°C to 35°C. However, with respect to flavor and overall acceptability decreased as the temperature increased from 25°C to 35°C. The optimum region obtained by superimposing the six contour maps is shown in the yellow shaded region of the overlay plot ( Figure 5). This optimum region provides the coordinates of possible optimal levels of fermentation time and fermentation temperature. The criteria for the optimal region were sensory attributes greater or equal to the "like" intensity. The ranges of the fermentation time and temperature in the optimal region are 8.6-24 hours and 32.5-35°C for fermentation time and fermentation temperature, respectively. The numerical optimization using the criteria of maximizing all the sensory attributes revealed that the optimal time and temperature combinations which give the best result were 35°C and 15 h for fermentation time and temperature, respectively, with a desirability value of 0.89. The optimal time and temperature combinations which give the best result were 35°C and 14.56 hours for fermentation time and temperature respectively

Conclusions
The study showed that incwancwa is made by spontaneous fermentation of either sorghum or maize flour followed by cooking. The microorganisms involved in the fermentation included coliforms, yeasts, and lactic acid bacteria. The predominant LAB strain identified was Leuconostoc lactis, which is well known for production of flavour compounds. Some coliforms were detected after cooking, suggesting post cooking contamination and hence need for improved hygiene among food handlers. The best temperature and time range required to produce an acceptable product was found to be 32° to 35°C for 8 to 24 hours. Numerical optimization predicted that the preferred temperature and time combination was 35°C for 15 hours. Further research is needed to determine the characteristics of microorganisms that survive the heat treatment. It is likely that most of the mesophilic lactic acid bacteria are eliminated by cooking leaving thermoduric microorganisms that may include some that cause spoilage. This may negatively impact on the shelf life of the final product. It is also important to Figura 5.. Gráfico superpuesto que muestra la región oprimal del tiempo de fermentación y la temperatura.
determine the nutritional content of incwancwa to establish the benefits of consuming the product.

Disclosure statement
No potential conflict of interest was reported by the authors.