Physicochemical, Functional, Antioxidant, Pasting and FT-IR Spectroscopic Properties of Fermented Acorns and Sorghum Using Traditional Algerian Processes

Abstract

The fermentation of acorns and sorghum is an ancient practice among the inhabitants of northeastern Algeria. This study aimed to establish the traditional fermentation processes of acorns and sorghum through a regional survey conducted in Algeria. Additionally, it investigated the impact of fermentation on the physicochemical, functional, antioxidant, and pasting properties, as well as the FT-IR spectroscopic profiles of the flours derived from these fermented materials. Characteristics of fermented sorghum and acorn flours were compared with those of non-fermented flours. The study included a survey that was carried out in Algeria at the regional level to establish the traditional processes for fermented acorns and sorghum. The key findings reveal the existence of two production methods: the first, the oldest, involves fermentation in underground pits called Matmor, while the second, more recent, is conducted outside the Matmor. Most manufacturers employed the new process outside of the Matmor, usually in various sized and shaped containers to meet market demand. Acorns and sorghum flour, obtained by drying and grinding fermented acorns and fermented sorghum grains according to the process carried out outside the Matmor, are characterized by a unique biochemical, functional, and structural composition. Detailed analysis of the flours showed a significant decrease in their physicochemical properties after fermentation, with a simultaneous overall increase in antioxidant activity. Moreover, FT-IR spectroscopy suggests that fermentation differentially affects protein secondary structure and starch crystallinity.

1. Introduction

Fermentation is one of the oldest bioprocesses for producing fermented foods with desirable attributes such as long shelf life and good organoleptic qualities. Microorganism enzymes, particularly amylases, proteases, and lipases, hydrolyze polysaccharides, proteins, and lipids that naturally exist in food [1,2,3]. Microorganisms are also responsible for reducing or eliminating toxic ingredients and anti-nutritional factors present in raw materials [4]. Furthermore, fermentation is the most important traditional food processing technique in the world, especially in Africa, and is used for a variety of processed food products, including cereals, roots, and non-timber forest products. Therefore, a significant part of the diet of the inhabitants of this continent consists of fermented foods. The primary source of these foods is spontaneous fermentation [5,6,7].

Fermentation has multiple effects on food’s nutritional value. Proteins, carbohydrates (starch and fibers), and lipids influence techno-functional properties. Proteins, beyond their nutritional role, exhibit essential functional properties for the processing and final structure of food products. The structural characteristics of starch directly influence its thickening capacity, stability, and gelatinization. Extensive research has demonstrated that the internal molecular structure of starch plays a crucial role in its physicochemical properties. Specifically, relative crystallinity has been correlated with paste viscosity. The structure and composition of foods influence their properties, behavior, and interaction with the environment [1,3,8,9,10].

Research suggests that fermenting sorghum and acorn could be a promising method to modify their functional properties [11,12,13]. There is a growing interest among researchers in comprehending the various effects of fermentation on the functional properties of fermented products [1,7]. Two types of fermentation are commonly used: directed fermentation, which employs starter cultures, and spontaneous fermentation, which occurs naturally from microorganisms in the environment. The fermentation of sorghum and acorn is generally carried out over a short duration, not exceeding 72 h [14,15,16].

In Algeria, the only fermented food that has been scientifically documented is wheat in underground silos, or Matmor. According to a new procedure that takes several months, this process has been replaced by a fermentation that occurs outside of the Matmor [17,18].

In the absence of studies about traditional fermentation processes used for acorns and sorghum in Algeria, along with their impact on different characteristics of final products, it seems interesting to conduct this work.

The primary objective of this study is to contribute to knowledge preservation by highlighting the various traditional fermentation processes for acorns and sorghum through a survey conducted in several Northeastern Algerian localities. Additionally, this research aims to evaluate the impact of the fermentation process on the physicochemical, functional, antioxidant, and structural properties of the fermented products according to the traditional processes outlined in the survey results.

2. Materials and Methods

2.1. Raw Materials

Flours derived from fermented acorns (Quercus ilex) and non-fermented acorns were purchased from artisans (Benyahia K., Ziama Mansouriah, Jijel, Algeria). Similarly, flours derived from fermented sorghum (Sorghum bicolor) and non-fermented sorghum were obtained from artisans in the same region (Roula N., Texenna, Jijel, Algeria). The fermented flours used for measurable characteristics were produced using the traditional process conducted outside the Matmor detailed below, as the Matmor process was not employed by producers during the study period. Both fermented and non-fermented flours were sieved to achieve a homogeneous particle size of 200 μm.

2.2. Reagents

1,1-Diphenyl-2-picrylhydrazyl (DPPH),2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS), ammonium molybdate, Folin–Ciocalteu reagent, ascorbic acid, gallic acid, quercetin, ferric chloride (FeCl₃), potassium ferricyanide (K₃Fe(CN)₆), and trichloroacetic acid (TCA) were purchased from Sigma-Aldrich, Steinheim, Germany. All the other standards used, including the solvents, were of analytical grade.

2.3. Survey Area and Data Collection

To establish the traditional fermentation processes of acorns and sorghum, a survey was conducted in northeastern Algeria, encompassing 24 municipalities across the provinces of Jijel, Sétif, Skikda, and Mila, as shown in Figure 1. These areas were selected based on a pre-survey that identified regions with a high concentration of artisans specializing in fermenting acorns and sorghum. Information about producers was provided by these artisans of fermented and traditional foods in the studied area. Subsequently, interviews were conducted face-to-face with 80 producers, including 60 acorn producers and 20 sorghum producers, between April and December 2022.

Two questionnaires were used. One was designed for the fermented acorn producers, and the second was designed for the sorghum producers. Each questionnaire included 28 multiple-choice and open-ended questions divided into four categories.

First category: Sociodemographic information (gender, age, and education level).

Second category: Raw materials and equipment used for fermentation.

Third category: Various phases and conditions of traditional fermentation processes,

Fourth category: Treatment of products obtained after fermentation and consumption of fermented flours. A camera and a questionnaire were used for data collection and analysis.

2.4. Characterization of Fermented and Non-Fermented Acorns and Sorghum Flours

2.4.1. Physicochemical Analyzes

The analysis of protein and fat content was performed according to ISO Genéve, Switzerland [19,20]. Proteins were analyzed using the Kjeldahl method (ISO 1871:2009). The fat content was determined using the Soxhlet method (ISO 659:1998) (ASN 310 applications). The moisture content was determined according to the AACC 44-15A method, which is based on the principle of weight loss. The ash content was determined according to the following method: AACC 08-03.01. The insoluble, soluble, and total dietary fiber content were quantified following the AACC 991.43 method [21].

A pH meter (Jenway 3505, Chelmsford Essex, England) was used to measure the pH value according to the 02-52-01 method [21].

Color was measured using a 4Wave CR30-16 colorimeter (Planeta, Tychy, Poland), according to Bourekoua et al. [22], under the following settings: D65; observer angle: 10°; space: LAB; diam: 16 mm; style: 8/day. The CIE-L*a*b* system, in which L* stands for lightness, was used to identify the color. The chromatic coordinates a* and b* represent the redness+/greenness- and the yellowness+/blueness-, respectively. Additionally, the overall color difference (ΔE) was calculated as follows:

$$∆E=\sqrt{{\left(∆L\right)}^2+{(∆a)}^2+{\left(∆b\right)}^2}$$

(1)

2.4.2. Functional Properties

Water Retention Capacity

The water retention capacity (WRC) of the flours was determined using a procedure based on the method described by Badia-Olmos et al. [8]. An amount of 1 g of each sample was mixed with 10 mL of distilled water. The resulting suspensions were vortexed for 30 s (Vortex VELP Scientifica, model ZX3, Via Stazione, Italy) and then allowed to stand for 2 h at room temperature (22 ± 2 °C). The suspensions were then centrifuged for 10 min at 2100 rpm (using a Sigma 3–30 K centrifuge, Osterode am Harz, Germany). The supernatant was dried at 100 °C and the WRC was calculated as follows:

$$WRC\left({\displaystyle \frac{\mathrm{g}}{\mathrm{g}}}\right)={\displaystyle \frac{WWP-WDP}{WS}}$$

(2)

where WWP is the weight of wet precipitate (g), WDP is the weight of dried precipitate (g), and WS is the weight of sample (g).

Oil Retention Capacity

The oil retention capacity (ORC) of the flours was determined using the method described by Badia-Olmos et al. [8]. For this, 1 g of each flour sample was mixed with 10 mL of oil using a vortex mixer. The samples were then left to stand at room temperature (22 ± 2 °C) for 1 h before being centrifuged at 3709 rpm for 10 min. Excess oil was decanted by inverting the tubes onto absorbent paper, and the samples were left to drain. The ORC was calculated as follows:

$$ORC\left({\displaystyle \frac{\mathrm{g}}{\mathrm{g}}}\right)={\displaystyle \frac{SWIRO-WS}{WS}}$$

(3)

where SWIRO is the sample weight, including retained oil (g), and WS is the weight of the sample (g).

Swelling Capacity

The swelling capacity (SC) was determined following the method of Alam et al. [23], with slight modifications. An amount of 20 g of flour was weighed and added to a 100 mL graduated cylinder. Distilled water was then added to reach a total volume of 50 mL, and the mixture was stirred carefully. After 20 h, the final volume of the sample was measured, and SC was calculated using the following formula:

$$SC\left(\%\right)={\displaystyle \frac{\left(Vf-Vi\right)}{Vi}}\times 100$$

(4)

where Vi is the volume of the sample (mL) at 0 min, and Vf is the volume of the sample after 20 h (mL).

Emulsifying Capacity

The emulsification capacity (EC) of the flours was determined using a method described by Badia-Olmos et al. [8]. A 1 g sample of flour was mixed with 20 mL of distilled water and 20 mL of sunflower oil. This mixture was then emulsified using a homogenizer (Ultra Turrax IKA T18 digital, Burladingen, Germany) operating at 9500 rpm for 1 min. Subsequently, it was subjected to centrifugation at 4000 rpm for 10 min at room temperature to evaluate its emulsification capacity with the following formula:

$$EC \left(\%\right)={\displaystyle \frac{HEML}{HENL}}\times 100$$

(5)

where HEML is height of emulsion layer (mm) and HENL is height of entire layer (mm).

2.4.3. Pasting Properties

The pasting properties of fermented and non-fermented flours were evaluated using a Micro Visco-Amylo-Graph Brabender (Brabender, Duisburg, Germany) according to the method described by Kupryaniuk et al. [24]. An amount of 10 g of each flour sample were dispersed in 100 mL of distilled water. Pasting properties were evaluated at a constant rotation speed of 250 rpm with a sensitivity of 235 cmg. The temperature profile consisted of heating from 30 to 93 °C at a rate of 7.5 °C/min, holding at 93 °C for 5 min, cooling from 93 to 50 °C at a rate of 7.5 °C/min, and holding at 50 °C for 1 min. Brabender Viscograph software (version 4.1.1) was used to determine pasting properties. The following characteristics were evaluated: onset gelatinization temperature (OGT), peak viscosity temperature (PVT), onset gelatinization viscosities (OGV), peak viscosity (PV), and final viscosity (FV).

2.4.4. Antioxidant Properties

Ultrasound-assisted extraction was conducted following Ayad et al. [25] by mixing 1 g of flour with 10 mL of 80% Ethanol and sonicating using an ultrasonic cleaning bath (ultrasons-H, 50/60 hz, 720 W, Ctra. Nll Km: 585.1 Abrera (Barcelona) Spain) at 40 °C for 1 h. After extraction and cooling, the samples were filtered for analysis.

Total phenolic content (TPC) was measured using the Folin–Ciocalteu method described by Singleton and Rossi [26]. An extract sample was mixed with Folin–Ciocalteu reagent and Na₂CO₃ solution, incubated for 2 h, and absorbance was recorded at 765 nm (using a UV/visible spectrophotometer, Thermo Electron Corporation Evolution 100). Results, calculated using a gallic acid calibration curve, were expressed as mg of gallic acid equivalents per gram of dry weight (mg GAE/g dw).

The total flavonoid content (TFC) was determined following the method described by Djeridane et al. [27]. The procedure involved mixing the extract with a 2% AlCl₃ solution and measuring the absorbance at 430 nm after 10 min. The results were expressed as mg of quercetin equivalents per gram of dry weight (mg QE/g dw).

Antioxidant activity was assessed using several methods. The total antioxidant capacity of the extract was determined using the phosphomolybdate method described by Prieto et al. [28]. The DPPH assay was used to measure free radical scavenging activity following the protocol of Ismail et al. [29]. The ABTS radical scavenging activity was evaluated according to the method by Re et al. [30]. Additionally, the reducing power (RED) of the extract was determined using the method outlined by Oyaizu [31].

2.4.5. Fourier Transformed Infrared (FT-IR) Spectra Collection and Analysis

The flours from non-fermented and fermented acorns and sorghum were analyzed by Fourier-transform infrared (FT-IR) spectroscopy in order to investigate the changes in protein secondary structure and starch conformation induced by fermentation.

The spectra were collected using a SHIMADZU IRAffinity-1S FT-IR spectrometer (SHIMADZU, Burladingen, Germany) equipped with a diamond attenuated total reflectance (ATR) accessory. They were obtained between 4000 and 400 cm⁻¹, with a resolution of 4 cm⁻¹. Each measurement was performed by conducting five separate analyses. To ensure an optimal signal-to-noise ratio, 128 scans were accumulated. Subsequently, each spectrum was baseline corrected using Origin software (Version 8.0724 PRO, Origin Lab Corporation, Northampton, MA, USA). The amide I bands (1600–1700 cm⁻¹) of all spectra were baseline corrected and area normalized. To visualize the changes in the secondary structure of proteins induced by fermentation, the spectra were smoothed by 7 points to reduce noise, and a secondary derivative of the spectrum was obtained using Origin software (Version 8.0724 PRO, Origin Lab Corporation, Northampton, MA, USA). In order to study the effect of fermentation on starch conformation, the proportions of crystalline and amorphous fractions of starch were evaluated by the intensity ratio I (1047 cm⁻¹)/I (1022 cm⁻¹) [32].

2.5. Data Analysis

The response rate was calculated as the percentage of respondents divided by the total survey population, rounded to the nearest whole number. Statistical analyses of the survey were performed using Epi Info statistical software (version 7.2, CDC, Atlanta, GA, US). Study results were expressed as the mean of three replicates, with the standard deviation (SD) calculated in Excel 2007. Means were compared using one-way analysis of variance (ANOVA), followed by a post hoc Fisher’s Least Significant Difference (LSD) test performed with Minitab 19 software (Minitab Inc., State College, PA, USA). The significance of differences between groups was tested at the p < 0.05 level.

3. Results and Discussion

3.1. Results of the Survey

The characteristics and sociodemographic structure of respondents depending on the two questionnaires (for acorn and sorghum processes) are presented in

Table 1. Characteristics and sociodemographic structure of 80 respondents.

	Acorns	Sorghum	Total
Number of respondents	60	20	80
Type of process
Outside the Matmor	58	13	71
Inside the Matmor	2	7	9
Gender
Women	59	0	59
Men	1	20	21
Age
28–39	16	05	21
40–59	34	08	42
60–86	10	07	17
Study level
Illiterate	13	09	22
Primary, secondary	39	11	50
University	08	00	8

.

The oldest method of fermenting acorns and sorghum involved placing them in underground pits, called the Matmor process. However, this traditional practice is tending to disappear, replaced by various utensils of varying shapes and sizes. Actually, only nine respondents (Table 1) still use the Matmor process, which is threatened by rural exodus and the difficulty of emptying the Matmor. Among these nine respondents, only two are the producers of fermented acorns inside the Matmor. The rest (n = 7) are producers of fermented sorghum. The survey showed that there were more producers of fermented acorns (n = 60) than those making fermented sorghum (n = 20) for both processes. Among the respondents, there were 59 women who only fermented acorn. The age of the people interviewed ranged from 28 to 86 years old, with the highest number of respondents (n = 42) falling between 40 and 59 years old (Table 1). The most prevalent category was primary and secondary education (n = 50).

3.1.1. Main Traditional Steps in the Preparation of Fermented Acorns and Sorghum

Based on the survey results concerning the traditional fermentation steps of the selected materials, two diagrams illustrating the fermentation process with the derived flours were established and are shown in Figure 2 and Figure 3 for acorns and sorghum, respectively. These Figures depict the fermentation process as described by the interviewed producers, based on the calculated percentages. The images in each Figure serve to visually represent the two processes.

Dry Cleaning

Dry cleaning is the first step defined by all interviewed producers (100%) for both the outside and inside Matmor processes, and for both types of materials (acorns and sorghum). This step preserves the natural flora initially present on the surface of the grains, which plays a crucial role in the fermentation process [33,34].

Wet Cleaning

The water cleaning process, also known as washing, is not used in the traditional Matmor process as reported by producers. However, it constitutes an essential step in the new fermentation process (outside the Matmor) adopted by 56.89% of producers working with fermented acorns and 76.92% of those working with fermented sorghum. This approach avoids using disinfectants to preserve the grain’s initial natural flora.

Fermentation

• Inside the Matmor

For Balout el-Matmor, which means buried acorns, there are two main types of pits used for acorn fermentation. The first type consists of pits naturally filled with ground water. The acorns are placed in mesh bags, then stacked in the pit, which is then sealed with wood and soil. The second type of pit used is the Matmor storage pit, where the pit is filled with acorns, then clean water is added until the acorns are completely submerged, and the pit is sealed. Fermentation lasts between 7 and 8 months, as reported by 100% of producers.

Bechna el-Matmor refers to sorghum buried in storage pits throughout the cold season. The sorghum is placed in the Matmor storage pit; then clean water is added until the seeds are completely submerged. The Matmor is then sealed with a zinc sheet and soil to achieve complete sealing. Fermentation duration varies among producers, with 71.42% fermenting for one and a half years, and 28.57% fermenting for up to two years. Producers relate this variation to soil characteristics such as microbial composition, pH, texture, organic matter content, and temperature.

The biochemical composition of raw materials, moisture content and absence of air inside the Matmor generate fermentation phenomena. These conditions create a favorable environment for the development of certain bacteria, such as lactic bacteria present in the raw materials [17,35].

• Outside the Matmor

Acorn and sorghum production in the Matmor has been enriched by a new process external to the Matmor to meet local market demands, similar to the modernization of other traditional practices like fermented wheat production in Algeria [18,36].

Fermentation of acorns and sorghum now often occurs in specific containers like plastic buckets (63.79% of producers of acorns) and plastic barrels (93.10% of producers of sorghum). The process involves immersing the ingredients in water using a specific water/ingredient ratio of 425 L (575 kg) for the acorn and 410 L (590 kg) for the sorghum. The water used comes directly from the tap at room temperature and is not pretreated. In the fermentation process, the humidity and temperature promote enzymatic reactions that alter the physicochemical and pasting properties of substances, change their chemical structure, induce the production of phenolic compounds, and increase antioxidant activity.

The majority of acorn producers (63.79%) use additives. Most commonly (83.78%), these additives are added only at the beginning of fermentation, while some producers (16.22%) add them both initially and during water changes. Notably, 62.07% of acorn producers change the water at varying intervals: 27.7% change it every 15 days for the first month, 22.22% change it every two months, and 13.88% change it every month and a half.

The most frequently used additive is salt, as reported by 56.75% of producers, added at a rate of 1 kg per 1000 L of water. Vinegar is another popular additive, used at a 1 L per 50 L of water ratio, as reported by 21.62% of producers. Some producers (13.51%) incorporate unconventional additives like pistachio leaves. Others use various plant materials, including pistachio oil, lemon tree leaves, apple tree leaves, olive tree leaves, basil leaves, and orange tree leaves.

Spontaneous fruit fermentations involve a wide variety of microbes, and if lactic acid fermentation is delayed or the pH remains high, spoilage bacteria can thrive. Back-sloping, along with the addition of salt and vinegar, helps to reduce this risk. These additives promote the growth of beneficial lactic acid bacteria over spoilage bacteria [18,37,38].

A minority of sorghum producers (23.07%) use additives, most commonly pistachio leaf (66.66%) or vinegar (33.33%), at a 1 l/50 L ratio, primarily to accelerate the refinement process as reported by Yao et al. [35].

All producers (100%) reported that the utensils used for fermentation are tightly sealed. A total of 81.03% of acorn producers and 61.53% of sorghum producers stored them in darkness. The fermentation of acorns has led to many reflections on the duration of fermentation, ranging from one and a half months to eight months.

The most common durations are 5 months for 10.34% of producers, 6 months for 48.27% of producers, and 7 months (10.34%). Sorghum fermentation lasts 8 months for 53.84% of producers, between 6 and 8 months for 15.84%, and 6 months for 30.76% of producers.

Description and Treatment of Products Obtained after Fermentation

At the end of the fermentation period outside the Matmor process, the volume of water in the vessels significantly decreases. However, respondents practicing the inside Matmor fermentation process of acorns and sorghum report the complete absence of water upon opening the storage pits. The final materials from both methods are very moist. The fruits and seeds are characterized by a strong odor and an increased volume, and their taste becomes acidic, while their texture becomes soft. The acorn changes from a light brown hue to a dark brown hue after fermentation, whereas the sorghum changes from a dark brown hue to a light gray. After fermentation, the processing of fermented acorns continues with washing, peeling, and sun drying, spanning several days.

Producers prefer direct exposure of the seeds to the sun, which accelerates the drying process and facilitates the subsequent grinding step. Grinding is an essential operation in the treatment of solid materials, aiming to reduce and control particle size. This step is closely linked to the previous drying phase, as the texture and water content of the raw materials directly influence particle size [39,40]. Fermented sorghum also undergoes a sun drying phase, resulting in finely ground dry products. The obtained flours are primarily used for making black couscous, as reported by 80% of respondents, and to a lesser extent for making breads, as reported by 20% of respondents.

3.2. Physico-Chemical Properties of Fermented and Non-Fermented Flours

Based on survey results indicating that a majority of producers ferment acorns and sorghum outside of the Matmor, fermented acorn and sorghum flours undergo different measurements compared to non-fermented flours. The results are displayed below.

3.2.1. pH-Values and Color of Flours

Results of pH values and color of non-fermented and fermented flours are presented in

Table 2. pH value and color of acorn and sorghum flours before and after fermentation.

Flours	NAF	FAF	NSF	FSF
pH	5.60 ± 0.01 b	4.37 ± 0.01 d	6.48 ± 0.02 a	4.99 ± 0.01 c
Color
L*	83.92 ± 0.22 a	62.25 ± 0.29 d	64.44 ± 0.37 c	71.54 ± 0.37 b
a*	4.07 ± 0.12 c	6.53 ± 0.30 a	5.55 ± 0.41 b	5.41 ± 0.17 b
b*	13.93 ± 0.08 a	12.87 ± 0.78 a	7.16 ± 0.33 c	11.37 ± 0.41 b
ΔE	-	21.55	-	8.25

Data are expressed as mean values ± standard deviation; ^a–d letters indicate statistically significant differences between groups in rows at the p < 0.05 level. NAF—non-fermented acorn flour, FAF—fermented acorn flour, NSF—non-fermented sorghum flour, FSF—fermented sorghum flour.

.

pH-Value

Table 2 displays the pH values of the flours before and after fermentation. Non-fermented sorghum flour (NSF) had the highest pH (6.48), followed by non-fermented acorn flour (NAF), which had a pH value of 5.60. After fermentation, the pH of sorghum flour (FSF) decreased to 4.99, whereas fermented acorn flour (FAF) had the lowest pH (4.37). Both samples showed a significant decrease (p ≤ 0.05) in the value of pH after the fermentation process. These results are supported by those found by Amina et al. [13] and Hashemi et al. [14] for fermented acorn dough, and those conducted by Correia et al. [41] for fermented sorghum dough. These authors have reported similar pH reductions. It is important to note that this reduction in pH is a common phenomenon during the fermentation process, and is generally related to carbohydrate degradation, which causes food acidification. Several previous studies have shown that this acidification contributes to food preservation during storage by creating pH levels at which many microorganisms cannot survive [13,14,42,43].

Color

Evaluation of fermented flour quality primarily relies on analysis of smell and color, according to 96% of surveyed individuals. Therefore, color was measured using L*, a*, b* values and ΔE (Table 2). Research by Sánchez-García et al. [44] suggests a ΔE > 5 indicates significant color changes visible to the human eye. Acorn flours exhibit the highest ΔE (21.55), while sorghum flours have the lowest (8.25). While color changes occur in both types of fermented flour, they are more pronounced in those derived from acorns.

Non-fermented acorn flour exhibited the highest lightness value (83.92), closely followed by fermented sorghum flour (71.54). Non-fermented sorghum flour reached L* value of 64.44, while fermented acorn flour had the lowest value (62.25) of lightness. Interestingly, acorn flour showed a significant decrease (p ≤ 0.05) in lightness after fermentation, whereas sorghum flour exhibited a significant increase (p ≤ 0.05) following fermentation. Similar studies showing an increase in brightness after the fermentation of pearl millet were observed by Akinola et al. [45], and also by Olamiti et al. [46] after sorghum fermentation. Furthermore, all respondents have noted that the brightness decrease in acorn flour could be due to the oxidation of the fermented grains after peeling. They observed that fermented grain appears light in color after peeling, but its exposition to air during drying makes the color darker.

Fermented acorn flour exhibited the highest a* value (6.53), followed by non-fermented (5.55) and fermented (5.41) sorghum flours. Notably, non-fermented acorn flour has the lowest value (4.07). The values of a* significantly increased in acorn flour, unlike sorghum flour, which showed no significant change. This indicates that FAF contained higher red pigmentation than NAF, unlike sorghum flour, which underwent no significant change in its red pigmentation after fermentation.

Both non-fermented (13.93) and fermented (12.87) acorn flour exhibited the highest b* values, followed by fermented sorghum flour (11.37). Non-fermented sorghum flour had the lowest value (7.16). Interestingly, fermentation caused no significant change (p ≥ 0.05) in the b* value of acorn flour. Conversely, fermented sorghum flour showed a significant increase (p ≤ 0.05) in this value, indicating enhanced yellow pigmentation after fermentation.

3.2.2. Proximate Composition

Table 3. The proximate composition of the flours before and after fermentation.

Flours	NAF	FAF	NSF	FSF
Moisture (%)	9.277 ± 0.0001 c	10.474 ± 0.001 a	9.481 ± 0.001 b	9.172 ± 0.003 d
Protein (%)	7.22 ± 0.04 c	5.40 ± 0.02 d	10.37 ± 0.04 a	9.91 ± 0.12 b
Fat (%)	8.28 ± 0.002 b	13.59 ± 0.001 a	4.02 ± 0.02 c	3.65 ± 0.01 d
TF (%)	18.07 ± 0.001 d	26.69 ± 0.01 c	36.35 ± 0.03 a	30.06 ± 0.02 b
SF (%)	2.98 ± 0.01 a	2.27 ± 0.01 b	1.96 ± 0.001 d	2.04 ± 0.02 c
IF (%)	15.09 ± 0.02 d	24.42 ± 0.02 b	28.01 ± 0.01 a	24.26 ± 0.04 c
Ash (%)	2.25 ± 0.001 c	1.29 ± 0.001 d	2.44 ± 0.001 b	2.50 ± 0.001 a

Data are expressed as mean values ± standard deviation; ^a–d letters indicate statistically significant differences between groups in rows at the p < 0.05 level. NAF—non-fermented acorn flour, FAF—fermented acorn flour, NSF—non-fermented sorghum flour, FSF—fermented sorghum flour, IF—insoluble fiber, SF—soluble fiber, TF—total fiber.

shows the proximate composition of non-fermented and fermented flours.

Moisture Content

The moisture content of flour plays a crucial role in its storage and preservation [47]. Data from Table 3 shows that FAF had the highest moisture content with 10.47%, followed by NSF with 9.48%, NAF with 9.27%, and finally FSF with a value of 9.17%. Overall, the moisture analysis results of the flours used in this study comply with the standards established by Codex Alimentarius, who establishes 15% as the maximum value that allows for their long-term storage [48].

Protein Content

As shown in Table 3, non-fermented sorghum flour contained the highest protein content with a value of 10.37%, followed by fermented sorghum flour with 9.91%. Next comes non-fermented acorn flour with 7.22%, while fermented acorn flour had the lowest protein content at just 5.40%. Protein content in acorn and sorghum decreased significantly after fermentation (p < 0.05). Similar results have been observed in previous studies, despite variations in fermentation duration and methods. Correia et al. [41] reported a decrease in sorghum protein content, while Abdelseed et al. [49] and Makawi et al. [11] reported an increase in protein content for sorghum after fermentation. The decrease in protein content may be due to the breakdown of proteins into smaller components (polypeptides, peptides, and amino acids) resulting from microbial activity. These components are then utilized by microorganisms for energy and nitrogen during fermentation [18,50]. The increased protein content of fermented sorghum reported in other studies may be attributed to the microbial synthesis of proteins from metabolic intermediates during fermentation [51].

Fat Content

The highest fat content was depicted in fermented acorns, with 13.59%, as indicated in Table 3, followed by non-fermented acorn flour (8.28%) and non-fermented sorghum flour (4.02%). Fermented sorghum flour presented the lowest values with 3.65% fat content. The fat content of acorn flour significantly increased (p ≤ 0.05) after fermentation. A similar result was reported by Sara et al. [17] in the fermentation of durum wheat. In contrast, the fat content of sorghum significantly decreased after fermentation. Makawi et al. [11] also reported a similar observation. This decrease is likely caused by increased lipolysis, a process that breaks down fats into fatty acids and glycerol during fermentation [11,18].

Total, Soluble and Insoluble Fiber Content

Table 3 shows the content of total fiber (TF), soluble fiber (SF), and insoluble fiber (IF) in the four types of flour. Non-fermented sorghum flour has the highest TF content with 36.35%, followed by fermented sorghum flour (30.06%), fermented acorn flour (26.69%), and finally non-fermented acorn flour (18.07%). In terms of insoluble fiber, non-fermented sorghum flour also has the highest value at 28.01%, followed by fermented acorn flour (24.42%) and fermented sorghum flour (24.26%). Non-fermented acorn flour had the lowest value at 15.09%. Regarding soluble fiber, non-fermented acorn flour had the highest content at 2.98%, followed by fermented acorn flour (2.27%). Fermented sorghum flour had a content of 2.04%, while non-fermented sorghum flour had the lowest value at 1.96%.

Our observations indicate an increase in TF and IF and a decrease in soluble fiber after acorn fermentation. This is in agreement with the results of Sánchez-García et al. [44] on the fermentation of white quinoa. The high biomass production can be attributed to the increase in insoluble fiber, as suggested by Sánchez-García et al. [44]. In contrast, sorghum fermentation seems to decrease TF and IF while increasing soluble fiber. This decrease in total and insoluble dietary fiber content during fermentation could be due to the activation of hydrolytic enzymes. These enzymes break down dietary fibers, and convert insoluble fibers into soluble fiber fractions [17,52].

Ash Content

Minerals trapped and associated within complex structures are largely responsible for their low bioavailability. Fermentation stands out as one of the transformation methods used to release these complex minerals and make them more easily assimilable [53]. The fermented sorghum flour had the highest ash content, reaching 2.50%, as presented in Table 3. It is closely followed by non-fermented sorghum flour, which showed an ash content of 2.44%. Next, non-fermented acorn flour had an ash content of 2.25%, while the lowest content (p < 0.05) was observed in fermented acorn flour, with only 1.29%. In this study on sorghum samples, a significant increase (p < 0.05) in ash content was observed after fermentation. Similar results have been noted in other studies, such as that of Makawi et al. [11], where an increase of ash content was observed for sorghum after fermentation. Likewise, Amina et al. [13] observed an increase in bioavailability in acorns samples after fermentation. This increase can be attributed to the activity of the microbial enzyme phytase, which releases minerals and thus makes them more accessible [11,18,53].

Interestingly, in the case of acorn samples, these results were an exception. A significant decrease in ash content was observed. Previous studies have also reported such decreases after fermentation, such as in the work of Sara et al. [17] on wheat samples. The impact of fermentation on mineral bioavailability is influenced by several environmental factors, including humidity, optimal pH, adequate temperature promoting enzymatic reactions of microorganisms, and the transformation of tannins into phenols. Phenols can complex minerals and limit their absorption, which could explain why, in some cases, the degradation of phytates (which bind minerals) during fermentation does not result in an increase in mineral bioavailability [53,54].

3.3. Functional Properties of Fermented and Non-Fermented Flours

Functional properties of non-fermented and fermented acorns and sorghum flours are summarized in

Table 4. Functional properties of flours before and after fermentation.

Flours	NAF	FAF	NSF	FSF
WRC (g/g)	1.88 ± 0.01 a	1.79 ± 0.001 b	1.65 ± 0.001 c	1.30 ± 0.01 d
ORC (g/g)	0.58 ± 0.02 c	0.40 ± 0.001 d	1.51 ± 0.14 a	1.16 ± 0.02 b
SC (%)	47.73 ± 0.02 a	40.88 ± 0.02 b	24.18 ± 0.02 c	19.33 ± 0.02 d
EC (%)	10.72 ± 0.01 c	10.32 ± 0.001 d	16.66 ± 0.01 a	16.59 ± 0.01 b

Data are expressed as mean values ± standard deviation; ^a–d letters indicate statistically significant differences between groups in rows at the p < 0.05 level. NAF—non-fermented acorn flour, FAF—fermented acorn flour, NSF—non-fermented sorghum flour, FSF—fermented sorghum flour, WRC—water retention capacity, ORC—oil retention capacity, SC—swelling capacity, EC—emulsifying capacity.

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The functional properties of flours result from the complex interaction between the various factors and components. Notably, proteins and starch play a crucial role in food product manufacturing, enabling the adjustment of formulations to meet the specific needs of each food [9,23]. As shown in Table 4, non-fermented acorn flour exhibited the highest water retention capacity (WRC) with 1.88 g/g, followed by fermented acorn flour (1.79 g/g), and then sorghum flour (1.65 g/g). Fermented sorghum flour had the lowest WRC (1.3 g/g). Fermentation significantly reduces water retention capacity (p ≤ 0.05), mirroring the findings of Makawi et al. [11] for fermented sorghum flour. A high WRC in flours indicates their exceptional quality, which supports the formation of a rigid gel structure that enhances the texture and volume of high-quality bakery products [23]. Similarly, the swelling capacity (SC) followed significantly (p < 0.05) in the following order: non-fermented acorn flour (47.73%), fermented acorn flour (40.88%), sorghum flour (24.18%), and fermented sorghum flour (19.33%). Fermentation significantly reduced the SC of the flours (p ≤ 0.05), aligning with the results of Mudau et al. [3] for fermented millet flours and Amina et al. [13] for fermented acorn flour. The high SC of flours could potentially facilitate the preparation of aqueous food formulations [23]. Conversely, their low SC makes them ideal for producing nutritionally dense foods for infants [3]. As suggested by Alam et al. [23] and Badia-Olmos et al. [8], the observed reduction in WRC and SC after fermentation appears to be primarily explained by modifications in the physical state of starch and a decrease in protein content. Regarding oil retention capacity (ORC), sorghum flour showed the highest value (1.51 g/g), followed by fermented sorghum flour (1.16 g/g), non-fermented acorn flour (0.58 g/g), and fermented acorn flour (0.40 g/g). Both sorghum samples showed a significant decrease (p ≤ 0.05) in oil retention capacity after fermentation. Makawi et al. [11] also reported a decrease in oil retention capacity in sorghum flour after fermentation. Sorghum flour, whether fermented or not, exhibits the highest values in terms of ORC. According to Mudau et al. [3] and Oloyede et al. [55], it can also act as a flavor preservative, suggesting that it can be incorporated into foods to enhance their taste. There were noted variations in emulsifying capacity across flour types. Non-fermented sorghum flour exhibited the highest rates (16.66%), closely followed by fermented sorghum flour (16.59%). Non-fermented acorn flour showed a higher emulsifying capacity (10.72%) compared to fermented acorn flour (10.32%). This decline can be attributed to the reduction in protein content after fermentation. Protein emulsifying capacity is closely linked to its hydrophobic nature, which reduces tension at the oil–water interface and forms a shell, preventing oil droplet coalescence. Higher protein content leads to a denser and stronger interfacial film, contributing to emulsion stability [8,9].

3.4. Pasting Properties of Fermented and Non-Fermented Flours

Table 5. Pasting properties of flours before and after fermentation.

Flours	NAF	FAF	NSF	FSF
OGT (°C)	37.5 ± 0.8 c	65.6 ± 1.1 b	32.1 ± 1.5 d	84.7 ± 0.2 a
PVT (°C)	92.9 ± 1.2 b	92.9 ± 0.5 b	35.0 ± 0.7 c	93.5 ± 0.7 a
OGV (mPas)	17.0 ± 2.1 c	21.0 ± 5.6 b	40.0 ± 2.8 a	17.0 ± 1.4 c
PV (mPas)	179.0 ± 4.2 b	187.0 ± 12.7 a	120.0 ± 7.7 d	166.0 ± 7.0 c
FV (mPas)	206.0 ± 7.0 d	266.0 ± 8.4 a	186.0 ± 2.8 c	255.0 ± 11.3 b

Data are expressed as mean values ± standard deviation; ^a–d letters indicate statistically significant differences between groups at the p < 0.05 level. NAF—non-fermented acorn flour, FAF—fermented acorn flour, NSF—non-fermented sorghum flour, FSF—fermented sorghum flour, OGT—onset gelatinization temperature, PVT—peak viscosity temperature, OGV—onset gelatinization viscosities, PV—peak viscosity, FV—final viscosity.

summarizes the onset gelatinization temperature (OGT), peak viscosity temperature (PVT), onset gelatinization viscosities (OGV), peak viscosity (PV), and final viscosity (FV) of non-fermented and fermented sorghum and acorn flours.

The highest onset gelatinization temperature (OGT) was observed in fermented sorghum flour (84.7 °C), followed by fermented acorn flour (65.6 °C), non-fermented acorn flour (37.5 °C), and finally non-fermented sorghum flour (32.1 °C). This progression indicates a significant increase (p ≤ 0.05) in OGT after fermentation for both flours, suggesting a significant delay in the starch gelatinization process following fermentation.

Regarding peak viscosity temperature, it was highest for fermented sorghum flour (93.5 °C), closely followed by both fermented and non-fermented acorn flour (92.9 °C), and finally non-fermented sorghum flour (35 °C). Interestingly, the peak viscosity temperature remains constant before and after fermentation for acorn flour, while it significantly increases (p ≤ 0.05) after fermentation for sorghum flour.

This increase in temperatures after fermentation is generally related to the effect of fermentation processes on the starch structure and its repercussions on its thermal properties. Specifically, fermentation induces changes in starch granule size, morphology, amylose and amylopectin distribution, as well as the proportion of crystalline structure. These structural adjustments notably influence the gelatinization and viscosity temperatures of starch after fermentation [3,56,57,58].

Non-fermented sorghum had the highest onset gelatinization viscosity (40 mPas), followed by fermented acorns (21 mPas), and then fermented sorghum and non-fermented acorns, both with 17 mPas. Fermentation significantly (p ≤ 0.05) affected onset gelatinization viscosity, with acorn viscosity increasing and sorghum viscosity decreasing.

The highest peak viscosity was observed in fermented acorn flour (187 mPas), followed by non-fermented acorn flour with 179 mPas, then fermented sorghum with 166 mPas, and finally non-fermented sorghum with 120 mPas. After fermentation, a significant increase (p ≤ 0.05) in flour peak viscosity was observed.

Peak viscosity is influenced by the reduction in starch granule rigidity in flours after fermentation, resulting from acidic and enzymatic actions on starch. During the fermentation process, these actions increase granule erosion, and enzymatic invasion can lead to significant branching and shortening of starch chain length, consequently altering the pasting properties of fermented flour [58,59,60].

The highest final viscosity value was observed for fermented acorn flour (266 mPas), followed by fermented sorghum flour (255 mPas), followed by non-fermented acorn flour (206 mPa) and finally non-fermented sorghum flour (186 mPas). After fermentation, the final viscosity significantly increased (p ≤ 0.05). A similar phenomenon of increased final viscosity following fermentation has been observed in previous studies. It is well established that final viscosity is closely correlated with amylose content. Indeed, an increase in amylose leads to a concomitant increase in final viscosity, as highlighted in the works of Oloyede et al. [55] and Mudau et al. [3].

3.5. Antioxidant Properties of Fermented and Non-Fermented Flours

The antioxidant properties of fermented and non-fermented flours translated by total phenolic content, total flavonoid content, and antioxidant activities are presented in

Table 6. Total phenolic content, total flavonoid content, and antioxidant activities of acorn and sorghum flours before and after fermentation.

Flours	NAF	FAF	NSF	FSF
TPC (mg GAE/g dw)	10.16 ± 0.17 a	9.17 ± 0.03 b	4.89 ± 0.01 d	7.80 ± 0.05 c
TFC (mg QE/g dw)	0.30 ± 0.001 c	0.26 ± 0.04 d	0.34 ± 0.01 b	0.44 ± 0.01 a
TAC (mg AAE/g dw)	5.74 ± 0.04 a	5.03 ± 0.05 b	1.38 ± 0.01 d	2.92 ± 0.02 c
ABTS IC50 (mg dw/mL)	0.0035 ± 0.0002 c	0.0007 ± 0.0002 c	0.4500 ± 0.0173 a	0.0296 ± 0.0005 b
DPPH IC50 (mg dw/mL)	0.19 ± 0.001 c	0.34 ± 0.001 c	4.09 ± 0.01 b	9.57 ± 0.24 a
RED A0.5 (mg dw/mL)	0.19 ± 0.02 d	2.72 ± 0.17 c	21.31 ± 0.46 a	5.18 ± 0.06 b

TPC—total phenolic content, TFC—total flavonoid content, TAC—total antioxidant capacity, ABTS—2,2′-azino-bis(3-éthylbenzothiazolin-6-sulfonate), DPPH—2,2-diphényl-1-picrylhydrazyl, RED—reducing power, NAF—non-fermented acorn flour, FAF—fermented acorn flour, NSF—non-fermented sorghum flour, FSF—fermented sorghum flour. ^a–d letters indicate statistically significant differences between groups in rows at the p < 0.05 level.

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The total phenolic content (TPC) and total flavonoid content (TFC) of flours before and after fermentation are presented in Table 6. Overall, the results showed that non-fermented acorn flour had the highest values of TPC and the best antioxidant activities with TAC, DPPH, and RED, followed by fermented acorn flour, fermented sorghum flour, and finally non-fermented sorghum flour. The highest values for TPC were 10.16 mg GAE/g dw or NAF, against 4.89 mg GAE/g dw, for NSF. For TFC, the FSF had the highest value with 0.44 mg QE/g dw, against 0.26 mg QE/g dw for FAF. The TAC results ranged from 1.38 mg AAE/g dw to 5.74 mg AAE/g dw. The ABTS assay findings also revealed that non-fermented sorghum had the highest IC₅₀ value, indicating relatively weak antioxidant activity, with an IC₅₀ of 0.4500 mg dw/mL. In comparison, fermented sorghum exhibited enhanced antioxidant activity with an IC₅₀ of 0.0296 mg dw/mL. The highest antioxidant activities were observed for non-fermented acorn and fermented acorn, with IC₅₀ values of 0.0035 mg dw/mL and 0.0007 mg dw./mL, respectively. DPPH assay values showed that fermented sorghum had the highest IC₅₀ value, at 9.57 mg dw/mL, indicating its low antioxidant activity. This was followed by non-fermented sorghum, with an IC₅₀ of 4.09 mg dw/mL. Acorn flour, both fermented and non-fermented, displayed the highest antioxidant activity, with IC₅₀ values of 0.34 and 0.19 mg dw/mL, respectively. Regarding reducing power (RED) values, non-fermented sorghum exhibited the highest value with 21.31 mg dw/mL, against non-fermented acorn with 0.19 mg dw/mL. After fermentation, reducing power significantly increased (p < 0.05) in acorns, leading to an increase in antioxidant activity. In contrast, fermentation significantly decreased (p < 0.05) reducing power in sorghum, thus reducing antioxidant activity.

Our results revealed that fermentation significantly (p < 0.05) reduced TPC and TFC in acorns, unlike sorghum, where fermentation significantly (p < 0.05) increased these values. The decrease in TPC and TFC in fermented acorn, might be related to the production of compounds with fewer free hydroxyl groups. Wiczkowski et al. [61] reported a decrease in anthocyanin content, an important class of phenolics in fermented red cabbage.

Jiménez-López et al. [62] also noticed a decrease in epicatechin concentration during the fermentation process of caper berries. On the other hand, the increase in TPC and TFC in fermented sorghum, could be due to an increase in free and soluble conjugated phenolic compounds and the bioavailability of free hydroxyl groups.

A similar increase in TPC was reported by Bei et al. [63] for oat fermentation. Similarly, an increase in TPC and TFC was observed by Zhang et al. [64] after buckwheat fermentation.

The increase in antioxidant activities in fermented sorghum indicated that compounds produced by fermentation are more active antiradicals. In addition, the higher reducing power might be related to the hydrogen-donating ability of the contained reductions [65].

Studies like that of Zhu et al. [66] on lactic acid fermentation of sweet potato residue reported that fermented samples produced higher DPPH scavenging activity after fermentation, while ABTS scavenging capacity showed no significant difference after fermentation.

Yue et al. [67] found no significant change in RP after the fermentation of Laminaria japonica. Fermentation can enhance the antioxidant capacity of plant materials by increasing the content of compounds capable of neutralizing free radicals. Therefore, fermentation can be considered a useful method for developing functional foods [67,68].

3.6. Structural Properties of Flours and Analysis of the Secondary Structure of Proteins

To better understand the molecular characteristics of fermented and non-fermented flours, an analysis was conducted on the chemical groups, the secondary structure of proteins, and the starch conformation. A fitting curve was created for this purpose. As illustrated in Figure 4, the FT-IR spectra of fermented and non-fermented acorn and sorghum flours generally exhibited marked differences in terms of frequencies and peaks. These differences were observed both between sorghum and acorn and within samples of the same flour, whether fermented or not. The region from 4000 to 3200 cm⁻¹ represents the stretching vibration of OH (hydroxyl) groups [69,70]. In both fermented and non-fermented sorghum flour, the frequency of 3838 cm⁻¹ was present, but the peak intensity decreased in fermented sorghum flour. Peaks at 3738 and 3626 cm⁻¹ shifted to 3730 and 3614 cm⁻¹ in fermented sorghum flour. In non-fermented acorn flour, peaks at 3849, 3722, 3595, and 3232 cm⁻¹ were shifted to 3838, 3730, 3603, and 3224 cm⁻¹ with the same intensity. Peaks at 3371 and 3263 cm⁻¹ in non-fermented sorghum flour disappeared in fermented sorghum flour, related to the OH stretching vibrations of polysaccharides and polyphenols [71]. In the region from 3000 to 2000 cm⁻¹ attributed to the CH stretching group [70]: NSF 2912 cm⁻¹ shifted to 2927 cm⁻¹ in FSF. In NAF, 2908 cm⁻¹ is shifted to 2916 cm⁻¹ in FAF.

The second derivative of the spectra of non-fermented and fermented sorghum and acorn flours is shown in Figure 5. The second derivative of the FT-IR spectrum in the amide I region (1600–1700 cm⁻¹) displays that both non-fermented flours were characterized by the presence of three zones. The region between 1620 and 1640 cm⁻¹ is attributed to the β-sheet structure [72,73,74]. The spectral region between 1649 and 1659 cm⁻¹ is attributed to the α-helix structure [75], while the region ranging from 1660 to 1688 cm⁻¹ represents the β-turn structure [38,72,74]. For the non-fermented sorghum flour, two peaks at 1624 and 1639 cm⁻¹ in the β-sheet structure spectral region were observed. Another peak at 1651 cm⁻¹ represents the α-helix structure. A final peak at 1674 cm⁻¹ represents the β-turn structure. After the fermentation of the sorghum flour, we observed that the positions of some peaks shifted, some peaks disappeared, and some new peaks appeared. The second derivative of the fermented sorghum flour shows peaks at 1632 cm⁻¹ corresponding to the β-sheet structure, the peak at 1646 cm⁻¹ corresponding to the disordered structure (region between 1640 and 1648 cm⁻¹) according to Emmambux and Taylor [54], and peaks at 1662 and 1674 cm⁻¹ corresponding to the β-turn structure. The overlay of the second derivatives of the spectra of fermented and non-fermented sorghum flours showed a large peak attributed to the β-turn structure compared to that of the non-fermented sorghum flour. Additionally, the α-helix structure disappeared, and the disordered structure represented by the peak at 1646 cm⁻¹ appeared. These changes indicate the reorganization (including degradation and aggregation) of sorghum proteins during the fermentation process. The α-helix structure was completely degraded by fermentation. Such degradation led to the appearance of disordered structures and an increase in the β-turn structure, indicating protein aggregation. Our findings are entirely consistent with those of Yasar et al. [76] and Alrosan et al. [77], who recorded a total disappearance of the α-helix structure, the appearance of random coils, and an increase in β-turns in soybean meal and lentil proteins subjected to fungal and water kefir seed fermentation, respectively. According to Wang et al. [78], there is a clear indication of enhanced protein digestibility in the reduced α-helix component. The primary mechanisms of action by which the fermentation process changed the secondary protein component are attributed to the microbial and endogenous enzymes present in the food [79,80]. According to Alrosan et al. [77], these changes in secondary structure have a strong positive correlation with improved protein quality.

The second derivative of the amide I region (1600–1700 cm⁻¹) of the fermented and non-fermented acorn flour spectrum was analyzed, revealing clear peaks at 1624 and 1637 cm⁻¹ (β-sheet), 1655 cm⁻¹ (α-helix), and 1674 cm⁻¹ (β-turn structure). We observed that there were slight modifications in the secondary structure of the proteins in acorn flour induced by fermentation. It was observed that the β-sheet, α-helix, and β-turn structures decreased slightly after fermentation. Lactic acid fermentation modifies the content and composition of proteins due to microbial enzymes and other components, such as acids. These acids can affect proteins in two different ways [2]. On the one hand, as seen in acorn flour, they can upset the ionic connections between the side chains of proteins that maintain the structure. Nevertheless, as we have seen with sorghum flour, it can also lead to the loss of secondary and tertiary structures. The FTIR results confirm the previous findings regarding the decrease in protein content in sorghum flour due to the action of enzymes from microorganisms involved in the fermentation process, resulting in the loss of secondary structure. As for acorn flour, we assume that the intensity of fermentation is more pronounced, as accelerated protein degradation has led to the formation of amino acids. Therefore, despite the decrease in protein content in acorn flour, the percentages of secondary structures appear to remain stable.

Analysis of Starch Conformation

The FT-IR band at 1047 cm⁻¹ is associated with the crystalline fractions of starch, while the band at 1022 cm⁻¹ is thought to be linked to the amorphous regions. The absorbance ratio (R_1047/1022) can be used to describe the amount of ordered structure in starch [32,81]. The R_1047/1022 of both acorn and sorghum flours decreased after the fermentation processes. However, the decrease was more pronounced in sorghum flour than in acorn flour. Non-fermented sorghum flour had the highest R_1047/1022 (0.67), followed by non-fermented acorn flour (0.66), fermented acorn flour (0.60), and finally fermented sorghum flour (0.06). The reduction in R ratio observed in fermented sorghum and acorn starch was primarily a result of alterations in the ordered structure of certain starch molecules after fermentation. Fermentation induces modifications in the distribution of branched starch chains, consequently reducing their short-range order [82]. These findings are corroborates with those of Yang et al. [83], who reported the breakdown of branched starch during the fermentation of maize starch by lactic acid bacteria. Higher R values in non-fermented flours indicate a dominance of crystalline conformation over amorphous conformation. This, in turn, suggests a stronger tendency for starch retrogradation [32,84].

4. Conclusions

This study reported traditional processes for fermenting sorghum and acorn. Two main processes were described for each material: the oldest, which is now abandoned, involved fermentation in underground pits called Matmor, while the newer process was carried out outside the Matmor with various utensils. The biochemical composition of the raw materials, the moisture content, and the absence of air generate fermentation phenomena. The flours from outside the Matmor process were analyzed. The physicochemical, pasting, functional, antioxidant, and structural properties revealed significant modifications induced by fermentation. Functional properties decreased notably, in oil retention capacity and emulsifying capacity, while pasting properties increased markedly. The increase in final viscosity of fermented flours makes them desirable ingredients for enhancing the quality of food products. Additionally, the results show that fermentation can improve the antioxidant capacity of plant materials by increasing the content of compounds capable of neutralizing free radicals. Therefore, fermentation can be considered a useful method for developing functional foods. Fourier-transform infrared spectroscopy (FT-IR) analysis highlighted clear differences between flours before and after fermentation, which shows an improvement in the digestibility of sorghum proteins in the reduced α-helix component. The reduction in the R ratio after fermentation is mainly the result of alterations in the ordered structure of certain starch molecules after fermentation. Fermentation induces changes in the distribution of branched starch chains, thus reducing their short-range order.

Overall, the findings deepen our knowledge of this organic resource by emphasizing the advantages of fermentation and its potential for a range of applications in the food industry, particularly for leavened gluten-free products.