Enhancing Qotcho fermentation process utilizing lactic acid bacteria inoculum: microbial kinetics and metabolism

ABSTRACT

Various studies revealed that quality of Qotcho relies on fermentation time, microbial activities, and varieties of Enset. Qotcho fermentation time is the most dominant factor. Several months to years of fermentation time is a challenging task for food security in Ethiopia. Shortening this long fermentation process was the primary aim of the study. Qotcho samples that have been previously fermented over various months were collected for lactic acid bacteria (LAB) analysis. While collecting the samples, temperature and pH of each sample were measured and recorded as 38 ± 2.3°C, 29 ± 1.5°C, and 25 ± 1.3°C, and 5.88 ± 0.8, 5.43 ± 0.6, and 4.69 ± 0.4, respectively. Using a serial dilution, selective media plates were innoculated and incubated at different temperatures for 5 days. On the second day of incubation, microbial growth was observed on each plate. A colony forming units (CFU) of 30–35 colonies of LAB were utilized to check fermenting performance of the microbes on different fresh Enset varieties at various temperatures for 36 days. Over 20 days of incubation, the pH of the media declined for all Enset varieties. From the 24^th to the 28^th fermentation time, the pH value for all varieties became constant (4.50) and sharply increased to 5.30 after the 28^th. The quality of the Qotcho was determined to have similar characteristics and texture to the Qotcho that had been fermented for six months. The overall results showed that using LAB colony as a starter culture, about 26 days were enough to make Qotcho with good texture, odor, and color for consumption.

KEYWORDS:

• colony forming units
• enset
• lactic acid bacteria
• fermentation
• Qotcho
• microbes

Introduction

Ethiopia, which is food insecure and in a long-term crisis, would benefit from increasing use of Enset (false banana).^[1,2] Enset is a monocarpic short-lived perennial plant that has been farmed in Ethiopia since ancient times due to its drought tolerance, particularly in the southern region.^[3] It is a food source for around 20% of Ethiopia’s population, particularly in southern nations of Ethiopia.^[4,5] Enset can increase food security in drought-prone locations where the climate is warm but not excessively hot, thereby covering a significantly bigger area than is now used in Ethiopia.^[5–7] Enset cultivation is a simple way to help individuals achieve self-sufficiency in their livelihood.^[7] Moreover, pseudostem, corm, and stalk of Enset are rich in starch food.^[8] In a pit, a mixture of scraped pseudostem pulp, powdered corm, and essence stalk is fermented for different traditional food varieties, i.e., locally, known as Qotcho or Kocho. Qotcho can be baked into a thin bread that is high in Ca and Fe but low in protein contents.^[9,10] Chemical composition of Enset and its essential mineral concentration make it an excellent feed for ruminants.^[9] Many procedures are involved in traditional Qotcho making process, including leaf sheath separation, decorticating, squeezing, fermenting, and filtration.

Due to safe for ingestion, Lactic acid bacteria (LAB) are one of the oldest known food preservation strategies.^[11] Largely, LAB are employed in food fermentation,^[12] production of leather,^[13] textiles,^[14] and cosmetics.^[15] According to Dekeba Tafa and Abera Asfaw,^[4] LAB were the dominant microflora found in fermented Qotcho. LAB were employed not only for food fermentation, but also to destabilize the harmful microbes growing in the fermented food by creating an acidic medium.^[1] Understanding the fermentation behavior of distinct Enset varieties could enhance optimize the fermentation time and standardize Qotcho production scale up. The amount of time and energy required for Enset fermentation considering, improving quality and quantity of Qotcho product, reduce waste and identifying health issues are the main factors that should be considered. Qotcho processing is a tedious, labor-intensive old-age and unhygienic practice which requires efforts of food microbiologists and processing technologists to avoid spoilage during fermentation process. Various researches have been reported on Qotcho fermentation and microbial dynamics during fermentation period.^[1,2,4] The fermentation time of Qotcho varies from several months to years.^[1,4] However, as Qotcho fermentation time becomes higher, the quality of Qotcho (taste, color, texture, or aroma) getting better. The quality of Qotcho depends on the action of microbes especially LAB.^[2,16]

Even though many studies were conducted on Qotcho fermentation and microbial dynamics, no one has tried to find a solution to minimize fermentation time of Qotcho using LAB as a starter culture yet. To enhance food security in the country, the longtime of Qotcho fermentation should be minimized. In this study, long time of traditional Qotcho fermentation was addressed using LAB as starter culture by keeping the quality parameters of Qotcho. The starter culture of LAB was prepared through successive identification, characterization, and isolation procedures. All the biochemical changes that occurred during Qotcho fermentation under different treatment conditions, concentration of microbial growth, and their growth rate kinetics were investigated intensively. The LAB were purified in separate and sterile MRS media and employed to check their fermentation performance on decorticated and chopped Enset. The long tradition Qotcho fermentation period was substantially shortened by this system for further food security enhancement.

Materials and methods

Chemicals and materials

Enset varieties (Agade, Disho, and Gimbo) were collected from Hadiya Zone (Morsito-Wereda) of SNNP Regional State. De Man Rogosa Sharpe (MRS) agar media with chemical composition of beef extract (10.0 g), yeast extract (5.0 g), sodium acetate (5.0 g), tween 80 (1.0 g), disodium phosphate (2.0 g), potassium phosphate (2.0 g), ammonium citrate (2.0 g), magnesium sulfate (0.2 g), manganese sulfate (0.05 g) was purchased. All the chemical reagents and solutions used in this investigation were of analytical grade and provided by Sigma-Aldrich (Addis Ababa, Ethiopia). A high-purity water (resistivity higher than 18 MΩ cm at 25 ℃) was used to prepare solutions and chemicals. The pH values were measured with a pH meter (Eutech Instruments Pte, Ltd, 6plus pH meter, Singapore).

Sample collection and preparation

Preliminary survey was performed in Enset growing area of Hadiya Zone (Morsito-Wereda) of SNNP Regional State, Ethiopia. Morsito is a small town which located 30 km to the Northern-West of Hosanna city, Hadiya zone. Household traditional experimental pits were prepared on Enset farms at the selected site as described in Figure 1a. The area was chosen primarily because of the availability of numerous Enset varieties that have been grown at different seasons. Community-based Qotcho fermentation process was performed from late October to early December, and on occasion from May to mid-June every year. Enset may also be processed all year as long as fresh Enset leaves are available for usage at initial stages of processing.^[17] However, Qotcho processing was not recommended during the wet season since the land becomes muddy and occasionally floods would happen.^[18]

Figure 1. Geographical location of study site (a) and schematic representation of the study process flow diagram (b).

Thus, on three Months intervals (October, January and February 2022), three types of most common Enset varieties (Agade, Disho, and Gimbo) were chopped together and allowed to ferment naturally in a prepared ground pit through household based traditional processing method for six months (Month-6), three months (Month-3), and one month (Month-1) respectively. The pit sizes, the geographical area, amount of the chopped Enset varieties, and quantity of samples (~5 kg each) were the same for each experimental operation. The only difference was on the experiment fermentation period (6, 3, and 1 Months). The process was supported by local experienced female farmers who were well known for traditional Qotcho making process in the area. The samples (Month-6, Month-3, and Month-1 fermented Qotcho) were collected randomly from different portions of the fermenting mass aseptically using sampling tongs. The samples were wrapped with Enset leaf (Coba) and stored into a sterile polyethylene bag independently by assigning as 1 M, 3 M, and 6 M corresponding to Month-1, Month-3 and Month-6, respectively. Then, the samples were taken to laboratory and stored at 4°C. While collecting the samples, temperature and pH of the fermented masses were recorded using temperature sensor and pH meter respectively. At the same time, three fresh Enset varieties (Agade, Gimbo and Disho) were collected from the same area and chopped into small pieces using traditional Qotcho processing method and prepared for fermentation in laboratory.

Microbial growth analysis and experimental set up

Microbial growth analysis is the quantification of the growth of microorganisms under controlled conditions, such as bacteria, yeast, and fungi. It is critical for a wide range of applications, including food and beverage production, pharmaceuticals, biotechnology, and environmental monitoring. Thus, number microbial growth was quantified from each sample (Month-1, Month-3, and Month-6) at different dilution concentration on petri-plates. Serial dilution ranges were prepared 1 to 5 (10⁻¹ to 10⁻⁵ g L⁻¹) dilution factor and 0.1 mL volumes of aliquots from each sample were plate-spread in triplicates on pre-dried plates. However, 10⁻¹ g L⁻¹ to 10⁻³ g L⁻¹ factorial dilutions were rejected due to high number of microbial growths on the medium. The composition of each plate was an average of 22.5 mL of MRS agar media for each to count aerobic mesophilic microbial colonies at incubation temperatures of 25, 30 to 35°C for 5 days. Microbial analysis and colonies of mesophilic microbes were counted for each agar plate with colony counter and recorded as colony forming units (CFU) within 6 h intervals. LAB were identified and quantified from each sample. Sample Month-6 was selected for further study. The identified LAB were purified and re-cultured repeatedly through the identification of morphological structure of the microbial colonies (structure, size, and color). Re-culturing of LAB was conducted by inoculating 4–5 colonies of LAB from sample Month-6 that have been fermented at the temperature of 30°C. The plates were then incubated at a temperature of 30°C for 5 days to obtain pure and numerous LAB colonies. The overall process was described in Figure 1b. Eventually, pure colonies of LAB were obtained and stored at 4°C for further experiment.

Fermentation performance trial on fresh Enset varieties

A fresh Enset fermentation performance trial entails evaluating the fermentation characteristics and quality attributes of various Enset varieties systematically. Flasks of 500 mL were utilized to prepare broth media for each fresh Enset variety with approximately 342 mL working volume and thoroughly mixed with shaker. The broth media were prepared according to the chemical nutrient composition of the MRS Media including the fresh Enset samples as carbon source. An average of 30 pure colonies of LAB were picked with pre-sterilized ring rod and inoculated to the fresh Enset containing flasks. Then, the flasks along with control flask were incubated anaerobically at temperatures of 25, 30, and 35°C for 36 days in shaker incubator. The pH of the fermentation process for each Enset trial samples including the control flasks were measured over 36 days at 4 days interval. Moreover, odor, texture, and color of the fermented samples were compared with previously traditional household fermented Qotcho.

Results and discussion

Study site and fermentation

The overall traditional household Qotcho fermentation process was presented in Figure 2. Mature Enset plants (such as Agade, Disho, and Gimbo) were decorated and processed traditionally at the study site. The fermentation pit was prepared, and the floor of the pits as well as the walls of the pits were lined with layers of fresh Enset leaves, as presented in Figure 2a. More new leaves were added to the pit surface to avoid the entrance of air. Moreover, Figure 2b,c demonstrated the aseptic techniques of fermented Qotcho sampling from the study site. The samples were assigned as 6 M, 3 M, and 1 M corresponding to fermented Qotcho over six months, three months, and one month respectively (Figure 2c). Besides, Figure 2d showed the community-based traditional Qotcho fermentation techniques that have been practiced for centuries.

Figure 2. Sample collection and traditional Qotcho fermentation: Pit of fermentation for samples (Month-1, Month-3 and Month-6) (a), fermented mass wrapping with Enset leaves (koba) (b) and Samples of Month-1, Month-3 and Month-6 in polyethylene bag (c).

Effects of temperature and pH on microbial dynamics

During sampling of fermented Qotcho, temperature and pH were measured in situ in the study site. As shown in Figure 3(a,b), the temperatures and pH of fermented Qotcho over Month-1, Month-3, and Month-6 were 38 ± 2.3°C, 29 ± 1.5°C, and 25 ± 1.3°C, and 5.88 ± 0.8, 5.43 ± 0.6, and 4.69 ± 0.4, respectively. Comparing the amount of temperature, samples from fermented Qotcho over Month-1 showed higher temperature records over the other samples. This result indicated that high metabolic reactions of microbials activities were taking place on the initial fermentation stages of Qotcho fermentation. On contrast, the acidity of the fermented Qotcho Month-6 was more acidic than others. This also gives a clue to what kinds of microbes could resist and live in acidic environment. Consequently, LAB were the dominant microbes observed and identified from the fermented Qotcho Month-6. Low number of yeasts and fungus in fermented Qotcho could be attributed to a lack of oxygen in the tightly packed and sealed fermenting mass leading to acidic environment.^[19] Molds were responsible for the black coloration of loosely wrapped Qotcho fermenting pits. When fermented Qotcho was removed from fermenting pits, it got quickly contaminated with bacteria, and the predominant spoiling microbes including fungi, penicillium, and trichoderma species.^[20] Because of the warmer temperature and fermentable sugars produced by amylolytic microbes, coliforms, and Enterobacteriaceae species, the early-stage Qotcho fermentation (Month-1) was more susceptible for higher microbial growth kinetics as shown in Figure 3c. The result revealed that fermented Qotcho below 1 a month composed of numerous active microflorae comparing to fermented Qotcho over three months (Month-3 and Month-6). This could be due to the production of secondary metabolites (antimicrobial compounds) which led to few microbial species that can persist for the secondary metabolites. Consequently, microbial growth becomes significantly lowered^[19] with more similar strains. At the incubation temperature of 30°C, the growth of microbes started to decline over five days and became like each other. The graph was correlated to well-known Monod rate growth kinetics of microorganisms. This graph did not correspond to lactic acid bacteria development, but rather to the growth of all mesophilic microorganisms in the samples. Lactic acid bacteria growth was accelerated at the end of Qotcho fermentation (Month-6). Higher microbial colony counts were obtained at 30°C and at three days of incubation period.

Figure 3. Dynamic nature of microbial fermentation: Temperature record (a), pH measurement (b) colony forming units of LAB in laboratory at 30°C (c), and pH of fermentation in laboratory at 30°C (d) of Qotcho samples.

During microbial growth, pH was also recorded as a function of incubation time. Lower pH values were observed on fermented Qotcho over six months (Month-6) than other samples. The lower pH value in fermented Qotcho in Month-6 might be due to the action of a single strain of LAB.^[17] At the end of Qotcho fermentation, it was predicted that LAB biochemical changes would increase at a faster pace as the temperature rose as shown in Figure 3d. The biochemical and kinetic behavior of a group of microbes and temperature increased as the number of counts of fermenting microorganisms increased while the pH value of the fermenting mass decreased as demonstrated.^[20] Increasing LAB counts resulted in dropping in pH value, corresponding to low quantities of other harmful microbes. Because most of these species lack amylase, products containing polysaccharides but no large quantities of simple sugars are typically stable against the activities of yeasts and LAB.^[13]

The growth of mesophilic microorganisms reduced as Qotcho fermentation duration rose (from Month-1 to Month-6). The decrease in mesophilic microbial count was caused by other amylolytic bacteria rather than LAB development. The bacteria that could survive at lower pH could be LAB, which could thrive as the pH of the fermenting media fell. As fermentation duration (Months) rose, only one strain was able to resist and dominate the process. Bacillus spp., in the form of their spores, appeared at a larger frequency as the count of different groups of microorganisms fell toward the end of fermentation. Otherwise, at the pH values reported at the end of fermentation, the active vegetative forms were frequently suppressed. During cassava fermentation, Bacillus species are found to break down starch and release fermentable sugars.^[21] Lactobacillus species, for example, prevailed in fermented Qotcho in Month-6. This was because the microorganisms secreted secondary metabolites and antimicrobial compounds that might kill other microbes and only microbes that could resist the toxic and acidic environment could grow. Amylolytic lactobacilli have been observed to grow during the fermentation of plant components.

Identification and re-culturing of LAB

Even though the MRS agar media were selective for lactobacilli, the growth medium was composed of many microbes such as molds, yeast, and other non-deductible microbes. The colonies of microbial growth in all petri-plates were recorded in 162 experimental runs over 5 days of incubation time at temperature of 25, 30, and 35°C. Microbes that have been grown at a temperature of 25 and 35°C were rejected due to the observation of small microbial colonies. Identification, isolation, and re-culturing of LAB was conducted though visual morphological analysis (structure, size, and color) at a temperature of 30°C from fermented Qotcho over Month-6 only. The methods of re-culturing and purifying of the LAB were conducted repeatedly by inoculating 4 to 5 colony of LAB on petri-plate and incubating at temperature of 30°C according to Weldemichael et al.^[2] The growth of the microbes was counted and recorded as colony forming units (CFU) over 5 days. After several re-culturing process, pure colonies were obtained and stored in refrigerator at temperature of 4°C for further analysis for trial fermentation. The microbial growth identification, culturing, and re-culturing on media plates were represented in Fig. S1a. The availability of biochemical and kinetic behavior of microorganisms in fermenting mass was significantly connected to the pH of fermenting mass.

It was observed that samples that were taken from fermented Qotcho Month-1 exhibited an exponential microbial rate growth. While samples that have taken from fermented Qotcho over Month-3 and 6 displayed a relatively stationary growth kinetics. This was due to fell of nutritional content responsible for amylolytic microbial development, as well as the buildup of secondary metabolites. The microbial nutritional composition altered the concentration of the microbes in the sample. Even if the media were selective for lactobacilli growth, many microbial colonies, such as molds, lactic acid bacteria, yeast, and other nondeductible microbes were observed (Fig. S1a). Analyzing and understanding the morphological appearance (structure, size, color, etc.) of lactobacilli (LAB), LAB were easily separated. Since LAB have semi-round structure, small size and white color, the colonies were identified, isolated, and re-cultured in a new media at a temperature of 30°C over the optimal period of incubation. After several re-culturing processes, a white colony of LAB was isolated and purified. Eventually, approximately 6.2 × 10⁶ colonies of lactobacilli were quantified within a single petri-plate. This pure LAB were stored at a temperature of 4°C in the refrigerator for further analysis.

Fermentation capability evaluation of LAB on fresh enset varieties

The fermenting capability of LAB for fresh Enset varieties entails evaluating the ability of LAB to ferment Enset in its raw form. Enset is a crop known for its starchy pseudostems and corms, and it is commonly used for food and fermentation in certain parts of Ethiopia. Lactobacillus species, for example, play an important role in the fermentation process by converting carbohydrates into organic acids and other metabolites that contribute to the final flavor, texture, and preservation of the product.^[11,12,15] Three fresh Enset varieties (Agade, Gimbo and Disho) were selected as trial Enset samples on which LAB applied as shown in Fig. S1b. An average of 50 pure colonies of LAB were picked with pre-sterilized ring rod and inoculated to the fresh Enset containing flasks and allowed to ferment over 36 days at temperatures of 25, 30, and 35°C. The pH of the fermentation process for each Enset trial samples including the control flasks was measured over 36 days at 4 days interval. Moreover, odor, texture, and color of the fermented samples were compared with previously traditional household fermented Qotcho.

Effect of temperature on growth and fermentation

Temperature is an important factor influencing the rate of fresh Enset fermentation, the composition of the microbial community, and the final Qotcho product quality. The fermentation process for Enset varieties is dependent on the activity of microorganisms in the Enset plant that are responsible for breaking down of starches. Temperature influences the rate of enzymatic and microbial activity, and thus has a significant impact on the overall fermentation process and the final Qotcho quality. Figure 4 demonstrated how microbial growth and fermentation of Enset trail vary over the temperature of the fermenting medium. Higher incubation temperature (35 ℃) aided in the rapid growth of microorganisms and the rapid fermentation process, resulting in a shorter fermentation period for all Enset trial varieties as shown in Figure 4 (b,c) respectively. The Enset varieties (type) did not have a significant impact on fermentation process. Comparing to the control fermentation, three of the fresh Enset varieties (Agade, Gimbo and Disho) followed similar pattern of microbial growth and fermentation at fermentation temperature of 30°C over 36 days. In fact, higher microbial colonies were observed on fermenting media containing Agade as a source of carbon. The result suggested, Disho was more easily fermentable following Agade and Gimbo was least fermentable (Figure 4d). After 27 days of fermentation, the number of colonies of LAB started to decline on the reacting medium, indicating the Enset sample was fermented.^[22] Understanding the temperature-Enset fermentation or temperature-microbial growth kinetics relationship is critical for optimizing the process, ensuring food safety, and producing Enset-based products with desirable sensory characteristics. Traditional knowledge and local practices are frequently used to adapt fermentation processes to specific temperature conditions.

Figure 4. Microbial growth kinetics in the form of CFU at different temperatures: LAB fermentation on Agade (a), LAB fermentation on Disho (b) LAB fermentation on Gimbo (c) and LAB fermentation on all samples with control at 30°C (d) over 36 days of fermentation.

Effect of temperature on pH of Qotcho product

Depending on several factors such as fermentation stage, microbial community, and local processing practices, pH of Qotcho products is also affected by temperature. Mostly, pH was dropped if Enset fermented by LAB. LAB are responsible for excreting lactic acid as secondary metabolite and then the pH of the medium could drop. Thus, the lactic acid concentration was measured through pH meter for 36 days with 4 days interval at different temperature ranges (25, 30, and 35 °C) as shown in Table 1. Comparing the ranges of temperature and types of Enset varieties for pH variation to control, no significant change was observed (Figure 5a-c). However, higher temperature facilitated faster fermentation, the pH was maintained constant over 27 days, indicating that the type of Enset variety could not contribute a significant difference in pH. Comparing each experimental Enset trial varieties pH value for Disho fermentation dropped faster within eight days than the other varieties. This faster fermentation showed that carbohydrates that found in Disho variety were more suitable for the growth of LAB. In general, all experimental Enset trial varieties attained almost the same pH value at 24–28^th and then started to increase as shown in Figure 5a.

Figure 5. pH measurement at different temperatures: pH record on Agade (a), pH record on Disho (b) pH record on Gimbo (c), and pH record on all samples with control at 30°C (d) over 36 days of fermentation.

Table 1. Colony forming units of LAB in different fresh Enset varieties at different temperature ranges.

Samples	Fermentation time (days)	pH record	CFU (10 ×10^5)
Agade	0	6.91 ± 0.35	0.00
	8	5.69 ± 0.29	0.76 ± 0.04
	16	5.09 ± 0.26	0.98 ± 0.05
	24	4.49 ± 0.23	1.13 ± 0.06
	32	5.14 ± 0.26	1.46 ± 0.07
Disho	0	6.95 ± 0.35	1.63 ± 0.08
	8	5.27 ± 0.26	1.79 ± 0.11
	16	5.31 ± 0.27	1.81 ± 0.09
	24	4.54 ± 0.23	1.54 ± 0.08
	32	5.25 ± 0.26	1.57 ± 0.07
Gimbo	0	6.94 ± 0.35	1.33 ± 0.06
	8	5.61 ± 0.28	1.24 ± 0.06
	16	5.27 ± 0.26	1.18 ± 0.05
	24	4.55 ± 0.23	1.07 ± 0.05
	32	5.39 ± 0.27	0.92 ± 0.06

The initial pH of each Enset varieties (Agade, Disho, and Gimbo) was approximately 6.80. Although the final pH values of the experimental trial were comperably similar, the longer duration of fermentation at 30°C temperature could result in the production of more acid. The dominance of LAB during the active stages of Enset fermentation was in agreement with the report of Birmeta et.al. and Hunduma et.al..^[1,20] As all LABs were homofermentative, more acid would be produced per mole of fermentable sugar and the rate of pH fall would be faster.^[23] The lowest pH achieved during the fermentation was approximately 4.50 for all varieties as shown in Figure 5a. The lower pH value showed high growth of LAB in the media beyond that LAB could not grow i.e. the maximum growth was attained at that pH value. After a minimum pH value was attained for all Enset variaties, the pH value started to increase as fermentation time was running.[²⁴] This pH value increment showed the fermentable nutrient decreased and there was high competition of microbes in the fermenting media.

Similar pH value was reached only toward the end of fermentation at day of 28^th of fermentation. The coliforms and other Enterobacteriaceae members helped to reduce the pH of the fermenting mass at first. Later on, homofermentative lactobacilli took over the process and dominated the flora until fermentation was completed.^[22] The pH dropped and the titratable acidity increased as the lactobacilli growth increased. Throughout the fermentation process, the yeast count remained low. The ideal temperature for the growth of mesophilic microorganisms was around 30°C. The evaluation of fermentation capability was critical to understand how LAB can contribute to the development of fermented Enset products with improved quality, flavor, and nutritional properties. Overall temperature can affect the pH of Qotcho during fermentation by affecting the rate of acid production by LAB. Higher temperatures have the tendency to accelerate acidification. It is important to note, however, that the specific pH levels achieved during Qotcho fermentation can vary depending on several factors, including traditional practices, microbial communities, and regional preferences. Monitoring and controlling pH during fermentation can be critical for achieving the desired Qotcho product quality and safety.

Statistical analysis

That pH data obtained during the Enset trial variaties fermentation was analyzed using minitab. General factorial design was employed to determine the optimal operating conditions. The model was analyzed to correlate pH to Enset sample varieties, temperature, and fermentation time. Using the stepwise mechanism, analysis of variance (ANOVA) was generated as described in the Table 2. From Table 2, the P-value of the samples was higher than 0.05 (0.326), indicating that the Enset variaties could not contribute significant impact on pH variation. However, temperature, fermentation time, and the interaction of temperature and fermentaion time were significant as shown Fig. S2. As a result the the optimal model was determined as shown in Equn. 1 and 2.

(1) $\mathit{pH}=\mathit{k}+\mathit{q}\sum _{\mathit{i}=1}^n{T}_i+\mathit{p}\sum _{\mathit{i}=1}^f{F}_i\pm \mathit{s}\sum _{\mathit{i}=1}^{\mathit{n}\times \mathit{f}}{T}_i\times F_i$(1)

(2) $\mathit{pH}=5.33\pm \mathit{q}\sum _{\mathit{i}=1}^n{T}_i\pm \mathit{p}\sum _{\mathit{i}=1}^f{F}_i\pm \mathit{s}\sum _{\mathit{i}=1}^{\mathit{n}\times \mathit{f}}{T}_i\times F_i$(2)

Table 2. Analysis of Variance (ANOVA), General factorial regression: pH versus samples, temperature, fermentation time.

Source	DF	Adj SS	Adj MS	F-Value	P-Value
Model	21	59.3432	2.82587	71.27	0.000
Linear	12	57.7299	4.81082	121.34	0.000
Samples	2	0.0900	0.04501	1.14	0.326
Temperature	2	0.4025	0.40252	10.15	0.002
Fermentation Time	9	57.2373	6.35970	160.40	0.000
2-Way Interactions	18	1.6133	0.17925	4.52	0.000
Temperature*Fermentation Time	18	1.6133	0.17925	4.52	0.000
Error	98	3.8856	0.03965

where k, q, p and s are constants, n is temperature level, T_i is temperature, f is level of fermentation time and F_i fermentation time. Following the optimized model in this study, normal probability plot was generated. The normal probability curve was ploted percent distribution versus residuals. Thus, 95% of the pH data were observed within the specific range of values as shown in Figure 6a. The individual plot in Figure 6b was used to assess outlier data points by visualizing how individual data points were distributed along the fitted or predicted value. The plot in this regression analysis demonstrated the actual observed values on the y-axis and a relevant independent variable on the x-axis. Each data point on the plot represented a single observation, which aids in comprehending the spread and distribution of data points. The difference between an observed or measured value and the predicted or expected value is commonly referred to as residual, and it is used in regression analysis to assess the goodness of fit of a model. A positive residual indicated that the observed value was greater than the predicted value, whereas a negative residual was the opposite. Residuals could help understand how well the model was approximated to the data. Since the assumptions of the model were met, the residuals were randomly distributed around zero in a well-fitting model as shown in histogram plot of Figure 6c. Observation order versus residuals was also an important aspect of regression analysis used for statistical model validation. As shown in Figure 6d, the data points were observed sequentially in time series. The average data points were also normally distributed along the zero values of y-axis. The statistical regression model analysis for colony forming units of LAB was presented in Fig. S3. The overall regression analysis obtained using the above statistical models was important aspects of statistical analysis. However, their relationship depends on the nature of the data and specific analysis being conducted. Thus, the predicted model could be a fundamental optimized parameter for the mentioned parameters in Qotcho fermentation studies. The statistical regression analysis of pH variation to temperature and fermentation time could provide important insights into how LAB affect the fermentation process and the resulting in quality Qotcho products. It could aid in validation of the hypothetical fermentation process model, guide process optimization, and contribute to scientific understanding of Qotcho quality parameters using LAB as a starter culture in Enset fermentation.

Figure 6. Statistical analysis of pH measurement at different temperatures for all samples: Normal probability plot (a), individual point plot (b) histogram of residual plot (c), and data point plot on time series (d) over 36 days of pH observation.

Conclusion

Qotcho fermentation process is a traditional food fermentation process used to make a staple food product in Southern and Southwestern regions of Ethiopia. Qotcho is made from the starchy core of the Enset plant (Ensete ventricosum), also known as the “false banana.” Based on the local respondents, Qotcho fermentation demands several months to years to get quality Qotcho product. As fermentation time increased to years, the quality of the product also increased. Much research has been done to understand the microbial dynamics during fermentation process. However, the long Qotcho fermentation time has not been addressed yet. In this research, lactic acid bacteria (LAB) were used as a starter culture to enhance Qotcho fermentation within the specified period. LAB were identified, isolated, and re-cultured from previously fermented Qotcho and applied on different decorticated and chopped fresh Enset varieties to facilitate the Qotcho fermentation process. Based on microbiological analysis, pH, color, texture, and odor, the Qotcho making process was minimized to approximately 27 days. Thus, using LAB as a starter culture, 27 days could be enough to Qotcho with good quality and the research could open a new door for Qotcho standardization process.

Authorship contribution statement

The contribution of authors for this paper is as follows: Redae Nuguse Berhe: Conceptualization; Original draft; Methodology, Review, and Investigation.

Acknowledgments

Our thankfulness has gone to Addis Ababa University, School of chemical and Bio-Engineering laboratory and Wachemo University for contribution to complete this research.

Disclosure Statement

No potential conflict of interest was reported by the author(s).