Screening and Optimization of <i>α</i>-Glucosidase Inhibitor Production by Potent Strain of <i>Bacillus subtilis</i> Isolated from Peruyaan, Fermented Soy-Food of Northeast India

Kabir, Mir Ekbal; Borah, Anupriya; Barman, Hiranmoy; Sharmah, Bhaben; Afzal, Nazim Uddin; Phukan, Tridip; Kalita, Jatin; Manna, Prasenjit

doi:https://doi.org/10.1155/2024/3199103

Journal of Food Biochemistry

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Abstract Introduction Materials and Methods Results Discussion Conclusion Abbreviations Data Availability Ethical Approval Conflicts of Interest Authors’ Contributions Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2024 | Article ID 3199103 | https://doi.org/10.1155/2024/3199103

Screening and Optimization of α-Glucosidase Inhibitor Production by Potent Strain of Bacillus subtilis Isolated from Peruyaan, Fermented Soy-Food of Northeast India

Mir Ekbal Kabir,^1,2Anupriya Borah,¹Hiranmoy Barman,^1,2Bhaben Sharmah,^1,2Nazim Uddin Afzal,^1,2Tridip Phukan,^2,3Jatin Kalita,^1,2and Prasenjit Manna^1,2

Academic Editor: Abd El-Latif Hesham

Received22 Dec 2023

Revised28 Feb 2024

Accepted05 Mar 2024

Published15 Mar 2024

Abstract

Background. Fermented soy foods exhibit the capability to inhibit the α-glucosidase enzyme. The bacteria isolated from these fermented soy foods may contribute to the production of a higher quantity of α-glucosidase inhibitor (α-GI) in optimized condition. Aim. The present study aims to isolate α-glucosidase inhibitor producing bacterial strain from peruyaan, a traditional fermented soy food of Arunachal Pradesh, optimize the production of α-GI for maximum yield, and assess the compounds. Result. Bacillus subtilis PM NEIST_4 (identified by 16S rRNA gene sequencing) was isolated as a potential strain that exhibit maximum α-glucosidase inhibition. This strain showed maximum α-GI productivity in Mueller Hinton Broth (MHB). The cultivation parameters for fermentation of MHB were optimized in order to maximize the yield of α-GIs. The α-GI productivity of 1616.613 ± 84.54 U/mL was observed before optimization, which increased by almost three times after optimization (4808.324 ± 13 U/mL). In vitro assays against α-glucosidase enzyme revealed a significant IC₅₀ value of 596.532 ± 44.80 μg/mL after the optimized study compared to that before optimization (IC₅₀ 1705.617 ± 43.95 ug/mL). Besides, α-glucosidase enzyme inhibitory property of fermented MHB (FMHB) was found to be unaffected at varied range of pH and temperature. Oral gavaging of FMHB in sucrose-loaded Wistar rat exhibited maximum reduction in postprandial blood glucose at 400 mg/kg body weight. Furthermore, FMHB was fractionated using solvents of increasing polarity including n-hexane, ethyl acetate, and n-butanol. Ethyl acetate fraction that exhibited the strongest inhibitory action against the α-glucosidase enzyme (IC₅₀ value of 251.55 ± 19.65 μg/mL), showed the presence of pyrrolopyrazine derivatives through GC-MS and LC-MS analyses. Conclusion. The present study demonstrated the isolation of a potent α-GI producing bacterial strain from traditional fermented soy food, and the optimization of the bacterial fermentation process was achieved to maximize the yield of α-GI. These findings underscore promising industrial potential of B. subtilis PM NEIST_4, advocating the use of indigenous microbial resources for the production of α-GI.

1. Introduction

Diabetes is a serious metabolic disorder that is highly ranked as a global pandemic on the international health agenda and is characterized by hyperglycemia [1, 2]. Diabetes can be most commonly classified into Type 1 (insulin-dependent diabetes mellitus), Type 2 (noninsulin-dependent diabetes mellitus), and gestational diabetes. Type 2 diabetes mellitus is primarily responsible for the current global epidemic of the disease, accounting for more than 90% of the diagnosed cases of diabetes mellitus [3]. However, studies indicate relatively lower epidemiological prevalence of type 2 diabetes within the Asian population, where a higher consumption of fermented soy products is observed [4]. Over a long period of time, soybean (Glycine max) has evolved as a dietary staple in a majority of Asian countries [5]. It is indeed a nutritious source of high-quality protein for human consumption [6]. However, soybeans typically contain certain anti-nutritional factors in addition to some toxic compounds like agglutinin, soyatoxin, proteinase inhibitors, and urease [7]. Interestingly, the fermentation of soybeans facilitates the degradation of some of these antinutrients while also producing small-sized proteins, bioactive peptides, and active isoflavones that exert a favourable effect on a variety of physiological conditions, such as diabetes [8, 9]. It has been scientifically proven that fermented soybeans help to combat cancer and tumors as well as protect against cardiovascular diseases, neurodegenerative diseases, inflammation, obesity, and diabetes [10]. Most of the Asian countries, including India, have a wide practice of fermenting foods with Bacillus species [11]. The type of food might differ depending on the country; however, the use of Bacillus for fermentation is quite common. Fermentation of soybeans with Bacillus produces viscous substances to finally yield Natto (Japan), Chongkukjang (Korea), Pepok (Myanmar), Sieng (Cambodia, Laos), and Thua nao (Thailand) [12]. In North-Eastern region of India, consumption of fermented soybeans is an age-old tradition followed by various ethnic groups since the origins in Mongolia [8]. Aakhone (Nagaland), Bekang (Mizoram), Hawaijar (Manipur), Kinema (Sikkim), Tungrymbai (Meghalaya), and Peruyaan (Arunachal Pradesh) are some of the naturally fermented soybean foods prevalent in different parts of North-East (NE) India. It is noteworthy that in all these fermented soybean foods; B. subtilis is the most predominant [13]. B. subtilis has been the subject of extensive research for a very long time, and hence, it is currently one of the well characterized low-GC Gram positive bacteria [14]. As a multifunctional probiotic organism, B. subtilis has been an ideal choice for numerous applications ranging from nanoparticle biosynthesis [15, 16] to the production of enzymes [17–20], vitamins [21, 22], antibiofilm, and antimicrobial materials [23]. At the same time, a few studies on probiotic B. subtilis unraveled a new dimension revealing its antimicrobial, antiviral, and anticancer properties [24]. Food and Drug Administration (FDA) has acknowledged B. subtilis as a GRAS (Generally Recognized as Safe) organism [25]. In addition, B. subtilis is widely known for its superior fermentation properties, substantial product yields (20–25 g/L), and the complete absence of harmful by-products [26]. However, the microbial system produces a very low amount of products in fermentation which poses a challenge to separate them for industrial preparations. Consequently, in order to achieve the highest possible yield of the desired product, the fermentation conditions must be improved [27]. To find the ideal relationship between the fermentation rate and the independent variables, statistical optimization is a helpful tool for analyzing the interactions between various elements. It is a set of statistical and mathematical methods that have been applied to the development, enhancement, and optimization of different processes in order to maximize biomass output or bacterial harvests [28]. Recent studies have revealed that bacterial strains, such as Bacillus amyloliquefaciens SY07 [29], Bacillus amyloliquefaciens AS385 [30], Bacillus methylotrophicus K26 [31], Bacillus subtilis B2 [32], and Paenibacillus sp. TKU042 [33] have the capability of producing α-Glucosidase inhibitors (α-GIs) directly into the fermentation broth. Inhibition of α-glucosidase activity is considered to be a well-established strategy for managing postprandial hyperglycemic pathophysiology linked with type 2 diabetes, delaying the digestion and absorption of oligosaccharides by competitively blocking α-glucosidase enzyme at the brush border of small intestine [34, 35]. Several commercially available α-GIs, like acarbose, voglibose, and miglitol, have been clinically used to treat diabetes mellitus because of their ability to efficiently control postprandial blood glucose level [36]. However, these treatments accompany some undesirable side effects, such as diarrhea, nausea, vomiting, and may cause potential harm to the kidneys and liver due to which safer and natural α-GIs need to be explored and studied further [37]. The present study aims to isolate α-GI producing bacterial strain from peruyaan, a traditional fermented soy food, optimize the production of α-GI for maximum yield and assess the compounds via GC-MS and LC-MS analyses.

2. Materials and Methods

2.1. Materials

2.1.1. Chemicals/Reagents and Solutions

Nutrient Broth (NB), Nutrient Agar (NA), Brain Heart Infusion Broth (BHI), Mueller Hinton Broth (MHB), Tryptone Soya Broth (TSB), Luria Bertani Broth (LB), Sodium chloride, Sodium carbonate, Sodium bicarbonate were purchased from Himedia, India. Gram staining kit (77730-1KT-F), 10X Phosphate-Buffered Saline (PBS), Saccharomyces cerevisiae α-glucosidase (G5006), 4-Nitrophenyl α-D-glucopyranoside (N1377), acarbose (A8980), RPMI media (R1383), and FBS were purchased from Sigma, USA. Streptomycin and penicillin were purchased from GIBCO. Analytical grade solvents including n-hexane, ethyl acetate, and n-butanol were purchased from Fisher Chemical. Biochemical analysis kits (Robonik) are sourced from India.

2.1.2. Equipments

Weighing Balance (UniBiocAP, Shimadzu), High-Pressure Steam Autoclave (EQUITRON, Medica instrument), pH Meter (LAQUA, Horiba), Laminar air flow (Optics Technology), Shaking incubator (Kuhner Lab-Therm LT-XC), Compound microscope (Leica MD750, ICC50), Field Emission Scanning Electron Microscope (STEM, JEOL, JEM 2100 Plus), Freeze dryer (FreeZone, Labconco), Microplate Reader ((Epoch 2, BioTek)). Thermal Cycler (Applied Bio Systems, ThermoFisher Scientific), Nucleic Acid Electrophoresis Gel System (Bio-Rad), Molecular Imager (ChemiDoc Imaging System, Bio-Rad, US), Biosafety level −2 laminar air flow cabinet (1300 SEREIES A2, Thermo Scientific), CO₂ incubator (CellXpert, eppendorf), Liquid chromatography-mass spectrometry (Agilent), Gas chromatography-mass spectrometry (Agilent), Biochemical Analyzer (AutoLab VerSA, Euro Diagnostic System).

2.2. Sample Collection

The fermented soybean, peruyaan, was obtained from a local market in Arunachal Pradesh, India. To ensure a completely sterile environment, the sample was delicately enclosed within a pristine zip-lock plastic pouch and then kept at a temperature of −20°C until it underwent analysis for microbiological evaluations.

2.3. Isolation of Bacteria from Peruyaan

A quantity of 10 g of peruyaan from the stock was delicately ground with a previously sterilized mortar and pestle, then transformed into a harmonious mixture by suspending it in 90 mL of sterile 0.9% NaCl solution followed by vigorous agitation for a duration of 60 min, which was serially diluted 10-fold over a range of 10⁻¹ to 10⁻⁶ in sterilized test tubes. A 100 μL from each diluted suspension were individually plated onto distinct nutrient agar plates, each plate meticulously designated for its corresponding dilution level. The plates that were introduced with the microbial specimens underwent an incubation period of 24 h at a temperature of 37°C, facilitating the emergence of well-defined colonies. These colonies, characterized by their unique morphological attributes including shape and texture, were subsequently isolated and transferred onto fresh nutrient agar plates through subculturing, thereby ensuring the cultivation of pure and unadulterated clusters.

2.4. Screening of α-GI Producing Bacteria

Based on α -glucosidase inhibition activity, the potent bacteria was selected. Therefore, for the screening procedure, isolated bacteria were separately incubated (final OD₆₀₀ = 10⁻³), in 250 mL conical flask having 100 mL NB medium (1.3%) as the only source of carbon/nitrogen and allowed to undergo fermentation at a cultivation temperature of 37°C using a shaker incubator at 125 rpm for 72 h [28].

2.5. α-Glucosidase Inhibition Assay

The evaluation of the potential of the experimental samples to inhibit α -glucosidase was conducted using the previous outlined methodology with some modification [38]. In a concise manner, different volumes of supernatant of the fermented culture media sample were used for the assay, making a total of 50 μL with the vehicle. This 50 μL sample mixture was preincubated with 25 μL of α-Glucosidase enzyme (at a concentration of 0.5 U/mL) for a duration of 15 min. As a positive control, samples were replaced with 50 μL of acarbose at varying concentrations. Subsequently, 50 μL of the substrate 4-Nitrophenyl α-D-glucopyranoside (at a concentration of 5 mM) in phosphate buffer (pH 6.8) was introduced to initiate the reaction. The amalgamation was then subjected to an incubation period of 30 min at 37°C. In the end, a halt to the reaction was achieved by introducing 50 μL of a solution containing sodium carbonate (Na₂CO₃) at a concentration of 100 mM. The absorbance was subsequently measured at 405 nm using microplate reader. The α-glucosidase inhibition was calculated by using the formula: percent (%) inhibition.

Where A is the optical density (OD) of reaction mixture containing vehicle and B is the OD of reaction mixture containing test sample with vehicle. Furthermore, the inhibition was expressed in terms of unit activity/mL (U/mL) and IC₅₀ value. One U was calculated with the volume of the test sample required for 50% inhibition of the enzyme and IC₅₀ value was calculated with the concentration of the test sample required for 50% inhibition of the enzyme. The experimental samples were freeze dried and later dissolved in respective vehicle to make different concentration of the samples to determine its IC₅₀. The measurement was carried out in triplicate.

2.6. Molecular Identification of Bacterial Strain

The 16S ribosomal RNA gene sequence analysis was chosen as the method of choice for identifying the potent bacteria. In order to accomplish this, genomic DNA was isolated employing the phenol-chloroform method with certain modification [39]. Agarose gel electrophoresis and spectrophotometric analysis were used to qualitatively and quantitatively validate extracted genomic DNA. Following that, the genomic DNA underwent targeted sequence amplification using primers 27F (GTTTGATCCTGGCTCAC) and 1492R (GGTTACCTTGTTACGACTT) in an automated thermal cycler (Applied Bio Systems, ThermoFisher Scientific). The amplicons were visualized on a 1% agarose gel having EtBr, and the sequencing was conducted by Eurofins Genomics India Pvt. Ltd. In the end, the sequence acquired was subjected to a comparison using BLAST (basic local alignment search tool) and subsequently submitted to the GenBank sequence database at NCBI to obtain an accession number. Besides, a phylogenetic tree was also constructed using Maximum Likelihood method.

2.7. Optimization of Cultivation Parameters for Production of α-GIs

Five different media, including NB, BHI, MHB, LB, and TSB were used to culture potent bacteria. Considering this, a temperature of 37°C with an agitation speed of 125 rpm and a 100/250 mL medium/flask volume ratio were set as the cultivation parameters for 9 days. Since MHB resulted to be the most favorable for the production of α-GIs, therefore it was selected for optimization of the fermentation parameters, that includes cultivation temperature (25, 30, and 37°C), media volume (50, 100, 150, and 200 mL), and the amount of seed culture (OD₆₀₀ = 10⁻⁵, 10⁻⁴, 10⁻³, 10⁻², 10⁻¹). The culture supernatant of each of the fermented media was allowed to go under centrifugation at 4000 rpm for 20 min, followed by filtration, and then utilized to assess the inhibitory activity of yeast α-glucosidase [33].

2.8. Measurement of Inhibitor Stability

The pH stability of the inhibitor were determined by mixing FMHB with HCl to decrease the pH or mixed with NaOH for increasing it. In particular, 0.5 mL of FMHB (2 mg/mL) were treated with a pH range from 1 to 13 at 37°C for 30 min and subsequently, its α-Glucosidase inhibition efficacy was evaluated at pH 6.8 [40]. To evaluate the thermal stability of the inhibitors, the samples were subjected to a temperature range of 30°C to 120°C for a duration of 30 min. Following this, the inhibitory activity of the sample against yeast α-Glucosidase was assessed [40].

2.9. Cell Culture

HCT-8, human ileocecal adenocarcinoma cells were provided by ATCC, USA and thereafter cultured in RPMI (Sigma) media, supplemented with solutions, including 10% FBS, 1% streptomycin, and 1% penicillin. The cells were maintained under incubation at 37°C and 5% CO₂ [41]. The cells were allowed to grow in a standard T25 flask until confluency reached to 80–90%. The cells were trypsinized prior to confluency and the culture medium was changed at every alternate day. At 1 × 10⁵/mL cell density, the cells were further seeded onto experimental plates.

2.9.1. Cytotoxicity Assay

After 90% confluency was achieved, HCT-8 cells were treated with different concentrations of FMHB for determining its cytotoxic effect. In particular, the cells were pretreated with 0, 1, 2.5, 5, and 10 mg/mL concentration of FMHB (as treatment groups) and PBS (as a control) for 20 h. After the treatment, cell culture media was discarded and Alamar blue reduction bioassay was performed to evaluate cell cytotoxicity [42].

2.10. Animal Study

Adult male Wistar rats were obtained from the Department of Veterinary Parasitology, College of Veterinary Science, Guwahati, Assam. The animals were given a two-week acclimatization period under laboratory conditions before the commencement of the experiments. The rats were kept in polycarbonate cages, housed in a clean room maintained at a temperature of 22−24°C with a 12 h light/dark cycle and relative humidity at 55 ± 5%. The animals were provided with a standard pellet diet and regular drinking water. The animal study was approved by the Institutional Ethical Committee and CPCSEA (Committee for the Purpose of Control and Supervision on Experiments on Animals), Ministry of Environment and Forests, New Delhi, India [2028/G)ReBi/S/18/CPCSEA].

2.10.1. Acute Toxicity Study

The oral acute toxicity study was conducted following the guidelines of Organization for Economic Co-operation and Development (OECD) [43]. A total of two groups of Wistar rats, five in each, weighing 75 ± 5 g were selected for this study. The FMHB extract was orally administered to each of the five animals with a single dose of 2000 mg/kg body weight (BW), and control group was administered with normal water. The animals were observed individually for the first 5 min to avoid any clinical sign of concern. Later, each of them were checked upon after every 30 min for the first 6 h after dosing, followed by periodic observation for 24 h and, daily thereafter, for a total of 2 weeks to detect any mortality and behavioral changes that include food intake, body weight, drinking water, urination, faeces, abnormal respiration, salivation, lacrimation, skin texture and color, eye color, abnormal jumping, head twitches, scratching, Straub tail, loss of balance, drowsiness, and aggressiveness. The body mass of the animals were also measured on day 1, day 7, and day 14.

2.10.2. Biochemical Analysis for Liver and Kidney Function Tests

After two-weeks of observation, the rats were sacrificed, and blood samples were obtained via a heart puncture procedure. The collected blood was then processed to extract plasma, which was subsequently utilized for liver and kidney function tests. The levels of alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were quantified to assess liver function. Additionally, creatinine levels were measured to evaluate kidney function.

2.10.3. Effect of FMHB on Blood Glucose in Sucrose-Loaded Rat

The effect of FMHB on the reduction of postprandial plasma glucose in sucrose-gavaged rat was performed according to the experimental animal protocol described by Wresdiyati et al., with some modification [44]. Adult male rats were allocated into six groups, with six animals in each group. All rat had similar average plasma glucose level and BW. The groups were divided as follows: normal group receiving only water, control group receiving normal water and sucrose, a drug control group receiving acarbose and sucrose, and three treatment groups receiving different doses of FMHB (100 mg/kg BW, 200 mg/kg BW, and 400 mg/kg BW) and sucrose. Prior to the experiment, all animal groups underwent an overnight fasting period of 16 h. Subsequently, they were orally administered water, acarbose, and the extract according to their respective experimental groups. After 20 min, a sucrose solution (3 g/kg BW) was orally administered to control, drug control, and treatment groups. Blood glucose level was measured at intervals of 0, 30, 60, 90, and 120 min by glucometer. The changes in blood glucose from the basal level after treatment were analyzed and represented as delta blood glucose level [45].

2.11. α-Glucosidase Inhibition Efficacy of the Solvent Fractions from FMHB

About 600 mL of FMHB was fractionated with different solvents by employing the solvent-solvent partitioning method. The partitioning was performed by using solvents of increasing polarity, that include n-hexane, ethyl acetate, and n-butanol with equal volume of these three solvents (thrice) and then concentrated under reduced pressure at 40°C using a rotary evaporator [46]. Thereafter, the activity of these three concentrated fractions along with residual aqueous fraction was assessed through α-glucosidase inhibition assay and their respective IC₅₀ values were calculated.

2.12. Assessment of Compounds in Potent Fraction of FMHB

The fraction showing the highest activity against α-glucosidase inhibition was further analyzed by performing GC-MS and LC-MS analyses to assess the potential compounds.

The GC-MS analysis was performed in Agilent 8890 GC system coupled with Agilent 7010 B GC/TQ mass spectrophotometer and HP-5MS ultra inert column (30 m × 250 μm × 0.25 μm) (Agilent). Helium gas was used as carrier gas with a flow rate of 1.0 ml/min. The injection temperature was 250°C, and 0.3 μL sample was injected in split mode with a split ratio of 1 : 50. The ion-source temperature was 230°C. The column temperature program was as follows: initially hold for 1 min at 50°C and then raised up to 200°C at the rate of 8°C/min and held for 2 min and finally increased up to 260°C at the rate of 15°C/min and hold for 3 min. The total run time was 28.75 min. Agilent Mass Hunter Qualitative Analysis 10.0 software was used for the data analyses and the compound identification was achieved by using NIST Mass Spectral Database (NIST 17): NIST MS Search v.2.3.

The LC-MS analysis was conducted in a UHPLC system connected with mass spectrometer. The chromatographic separation was carried out in a C18 column (Waters, 2.1 × 50 mm, 1.7 μm) operated at 45°C. The flow rate was maintained at 0.3 mL/min, and 0.1% formic acid in water (solvent A), and acetonitrile (solvent B) were used as mobile phase solvents. The total run time was 5 min, and with the elution conditions: 0.5–2 min, 90% A; 2-3 min, 10–90% B, returned to the initial condition (10% B) for 3–5 min. PDA detector was used to acquire the complete spectrum at each time point in the chromatogram. The generation of ions was achieved by using electron spray ionization (ESI) mode and the mass spectra were acquired in positive mode. The data acquisition and processing were performed in MassLynx software.

2.13. Statistical Analysis

Each experiment was conducted in triplicate, at a minimum, and the findings were depicted as the mean ± standard deviation (SD). The data were statistically analyzed with the application of Sigma Stat software (Jandel Scientific, SanRafael, CA), subjecting them to one-way ANOVA and comparing all the groups using Student–Newman–Keuls post hoc method. A was considered statistically significant.

3. Results

3.1. Isolation and Screening of the Promising Strain

During isolation and screening procedure, 23 of the bacterial colonies were isolated from peruyaan. Among the 23 isolates, 4 isolates showed inhibitory activity against α-glucosidase enzyme and were named as P2 MEK, P4 MEK, P6 MEK, and P8 MEK. Analysis of the α-glucosidase inhibitory activity of these 4 isolates revealed that isolate P6 MEK exhibited the strongest inhibitory activity against α-glucosidase (95.42 ± 0.08%) compared to others (Figure 1). This potent strain gave an IC₅₀ value of 1705.617 ± 43.95 μg/mL with α-GI productivity of 1616.61 U/mL after 72 h fermentation (Table 1).

3.2. Identification of the Potent Isolate, P6 MEK

The colony displays a distinctive milky-white, round morphology, characterized by a dry surface (Figure 2(a)). Microscopic examination unveiled the bacterial strain to be Gram-positive (Figure 2(b)). Furthermore, scanning electron microscopy (SEM) analysis revealed the presence of rod-shaped bacteria measuring approximately 2-3 μm in length (Figure 2(c)). For molecular identification, the 16S rRNA gene was amplified via PCR and subsequently visualized on a 1% agarose gel (Figure 2(b)). By partial sequencing of 16S rRNA gene amplicons, the isolate was identified as Bacillus subtilis PM_NEIST_4 (accession no. OP810678.1) and its consanguinity were analyzed via phylogenetic tree contraction (Figure 2(e)).

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3.3. Optimization of Cultivation Parameters for Maximal α-GI Productivity

3.3.1. Effect of the C/N (Carbon/Nitrogen) Source on α-GI Production

To investigate the potency of α-GI production by B. subtilis PM_NEIST_4, five sources of C/N, namely 1.3% NB, 3.7% Brain Heart Infusion broth (BHI), 2.1% Mueller Hinton Broth (MHB), 3% Tryptone Soya Broth (TSB), and 2.5% Luria Bertani Broth (LB) were investigated. In order to determine α-GI productivity, the culture supernatants were appropriately diluted to obtain inhibitory activity and then expressed as U/mL. Considering α-GI productivity, significant differences were observed among the five culture supernatants as depicted in Figure 3(a). MHB exhibited the highest α-GI productivity with 1892.34 U/mL on day 6, followed by NB with 1616.61 U/mL on day 3, TSB with 1374.72 U/mL on day 5, LB with 1037.59 U/mL on day 6, and BHI with 624.93 U/mL on day 3, respectively. Considering α-GI productivity, MHB was found as the best source of C/N, and therefore, it was selected for subsequent experiments. Additionally, the cell growth of B. subtilis PM_NEIST_4 was also monitored during the process of fermentation by measuring the absorbance of the cell suspension at 600 nm after every 24 h for 9 days. As illustrated in Figure 3(b), no correlation was found between bacterial growth and α-GI productivity.

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Figure 3

The effect of C/N sources on α-GI production through fermentation with Bacillus subtilis PM_NEIST_4 using 1.3% NB, 3.7% BHI, 2.1% MHB, 3.1% TSB, and 2.5% LB as the sole sources of C/N. Fermentation was carried out at 37°C in a shaker incubator set at 125 rpm and medium/flask volume of 100/250 mL at 0.001 OD₆₀₀. The inhibition against α-glucosidase enzyme was calculated and expressed in U/mL (a); growth of bacterium expressed as OD₆₀₀ (b). Data are expressed as mean ± SD (n = 3).

3.3.2. Effect of Specific Parameters on α-GI Production

In order to achieve maximal α-GI productivity, MHB was selected as the best C/N source to investigate the effect of certain fermentation parameters that include cultivation temperature, culture volume, and concentration of bacteria in terms of ODs.

As shown in Figure 4(a), B. subtilis PM_NEIST_4 exhibited the highest α-GI productivity (1941.20 U/mL on day 7) at 37°C, approximately 2- and 3.3-fold higher than those at 30°C (884.32 U/mL on day 6) and 25°C (575.18 U/mL on day 2), respectively. Thus, temperature at 37°C was selected as the suitable cultivation temperature to further determine the optimal culture volume for maximum α-GI productivity. Figure 4(b) depicts that α-GI productivity was best achieved in 50 mL medium (4682.46 U/mL) on day 6 of cultivation, followed by 100 mL (1801.63 U/mL), 150 mL (1652.80 U/mL), and 200 mL medium (766.39 U/mL) on 7^th day of cultivation, respectively. Consequently, a culture volume of 50 mL medium in 250 mL conical flask (1 : 5 ratio) at a temperature of 37°C was selected to investigate the effect of different OD₆₀₀ of bacteria on α-GI productivity on 6^th day of cultivation. The results showed that an OD₆₀₀ of 10⁻⁴ gave the highest α-GI productivity of 4808.32 U/mL, followed by 10⁻³, 10⁻², 10⁻⁵, and 10⁻¹ OD₆₀₀ with 3822.88, 3636.49, 3298.148, and 3106.704 U/mL α-GI productivity, respectively (Figure 4(c)). Furthermore, as shown in Figure 5, the IC₅₀ value of FMHB after the optimized study was evaluated as 596.532 ± 44.80 μg/mL, which was almost 3 times less than that of FMHB before optimization.

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In summary, B. subtilis PM_NEIST_4 effectively produced α-GIs in 2.1% MHB-containing media, maintaining an OD₆₀₀ of 10⁻⁴ at 37°C at a culture volume of 50 mL in 250 mL Erlenmeyer flask, using a shaker incubator at 125 rpm for 6 days. Table 1 represents the fermentation parameters before and after the optimized study.

3.4. Thermal and pH Stabilities of FMHB α-GIs

A temperature range of 30°C to 120°C resulted in FMHB α-GIs being strongly thermostable. The activity was still greater than 95% even when treated at a high temperature of 120°C (Figure 6(a)). At a pH range from 1 to 13, no major differences in α-glucosidase inhibitory activity of FMHB were observed. As shown in Figure 6(b), the stability of α-GI was 91% to 97% at a varied pH range.

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3.5. Toxicity Test of FMHB on Cells and Animal Model

To examine the cytotoxicity of FMHB, the Alamar blue bioreduction assay was performed by using intestinal epithelial cells, HCT-8 treated with different concentrations of FMHB (1, 2.5, 5 and 10 mg/mL). The cells treated with PBS (pH 7.4) served as the control. As observed in Figure 7, treatments with FMHB did not show any significant alteration on cell viability even at high concentrations. The findings from acute toxicity studies revealed no unusual behavior, with all subjects demonstrating a 100% survival rate throughout the two-week treatment period (Table 2). Moreover, there were no significant differences in the liver (ALT and ALP) and kidney (creatinine) function tests between normal and those gavaged with FMHB (Table 3).

3.6. Effect of FMHB on Postprandial Blood Glucose Level in Animal Model

To determine the effect of FMHB on postprandial plasma glucose level, three doses of FMHB, 100, 200, and 400 mg/kg BW were administered to male Wistar rats. As illustrated in Figure 8, significant reduction in plasma glucose level were observed for all the three doses administered after sucrose loading. Although, treatment with FMHB at a dose of 100 mg/kg BW demonstrated remarkable effect on suppressing plasma glucose level (Figure 8(a)); however, it was less effective as compared to the higher doses. Treatment with FMHB at a dose of 200 mg/kg BW showed the best reducing effect on blood glucose after 30 and 60 min (Figure 8(b)). At the same time, FMHB at a dose of 400 mg/kg BW reduced blood glucose level maximum after 90 and 120 min effectively (Figure 8(c)). The test groups showed significant reduction in blood glucose level when compared to the animal treated with sucrose along with standard drug, acarbose at a dose of 50 mg/kg BW (Figure 8(d)). In comparison to animals administered with sucrose alone, the comprehensive glycemic response exhibited a reduction of 34.9%, 67.4%, 69.9%, and 58.9% upon treatment with 100, 200, and 400 mg/kg BW of FMHB, and 50 mg/kg BW of acarbose, respectively (Figure 8(e)).

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Figure 8

The effect of FMHB (a) 100 mg/kg⋅BW; (b) 200 mg/kg⋅BW; (c) 400 mg/kg⋅BW); and acarbose (d) on postprandial plasma glucose level in sucrose fed male Wistar rats. Blood glucose level was measured at a regular interval of 30 min consecutively for 2 h. The areas under the curve (AUC) of the glucose response was analysed (e). Data are expressed as mean ± SD (n = 6). “∗” denotes the significant difference from normal (received water) (p < 0.05); “#” denotes the significant difference from sucrose-fed group (p < 0.05); @ denotes the significant difference from FMHB treated group at a dose of 100 mg/kg BW (p < 0.05).

3.7. Assessment of α-Glucosidase Inhibitory Activity of Solvent Fractions from FMHB

Based on the polarity, four fractions, namely, n-hexane, ethyl acetate, n-butanol, and aqueous fractions were collected from crude FMHB. Among them, the ethyl acetate fraction of FMHB showed the highest α-glucosidase inhibition activity followed by n-butanol, aqueous fraction, and n-hexane. The IC₅₀ values of each respective fraction were found to be 251.55 ± 19.65 μg/mL, 985.75 + 51.99 g/mL, 2197.63 + 58.04 g/mL, and 4129.63 + 156.47 g/mL (Figure 9).

3.8. Identification of Compounds in the Solvent Fraction of Bacterial Culture Broth (FMHB) with Promising α-Glucosidase Inhibitory Activity

To identify the compounds responsible for the activity of the ethyl acetate fraction, both GC-MS and LC-MS analyses were performed (Figure 10). The GC-MS analysis of the ethyl acetate fraction demonstrated five major peaks at the acquisition time of 20.629, 22.226, 22.496, 22.677, and 27.145 min (Figure 10(a)). The corresponding GC-MS library revealed the presence of 3-propylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione (compound 1; C₁₀H₁₆N₂O₂), 3-isobutylhexahydropyrrolo [1,2-a]pyrazine-1,4-dione at 22.226, 22.496, and 22.677 min (compound 2, C₁₁H₁₈N₂O₂), and 3-benzylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione (compound 3; C₁₄H₁₆N₂O₂) (Figure 10(c)). Furthermore, LC-MS analysis of ethyl acetate fraction also revealed the presence of compound 1 (calculated exact mass for C₁₀H₁₆N₂O₂ 196.1212, found [M + H]⁺ 197.1310), compound 2 (calculated exact mass for C₁₁H₁₈N₂O₂ 210.1368, found [M + H]⁺ 211.1494), and compound 3 (calculated exact mass for C₁₄H₁₆N₂O₂ 244.1212, found [M + H]⁺ 245.1331) in the ethyl acetate fraction of FMHB (Figures 10(b) and 10(c)).

(a)

(b)

(c)

4. Discussion

Persistent elevation of blood glucose concentration in diabetic conditions can lead to severe complications such as diabetic nephropathy, neuropathy, retinopathy, hepatic steatosis, cardiovascular disease, and pancreatic disorders [48]. Consumption of fermented soy-based foods has been well known to minimize high blood glucose concentration in diabetic individuals [49]. Daily intake of these fermented food products can help to manage numerous chronic disorders and gain multiple health benefits [49]. In multiple in vitro, in vivo, and clinical settings, the antidiabetic potential of fermented soybeans has been demonstrated, revealing a wide spectrum of therapeutic targets [49]. Inhibiting key digestive enzymes like α-glucosidase can help to reduce postprandial hyperglycemia associated with diabetes by delaying the digestion of carbohydrates [50]. Studies have revealed that certain glucosidase inhibitors are also found to be naturally present in fermented soybean foods such as tempeh, douchi, doenjang, and miso [51]. However, the knowledge of any such enzyme inhibitor present in fermented soy foods of North-East India is very limited. Previous studies have revealed that a few species of Streptomyces, Actinoplanes, and Flavobacterium have the capability of producing α-GIs that form the basis of antidiabetic drugs available commercially [29]. In addition, some bacterial strains like Bacillus amyloliquefaciens SY07 [29], Bacillus amyloliquefaciens AS385 [30], Bacillus methylotrophicus K26 [31], Bacillus subtilis B2 [32], and Paenibacillus sp. TKU042 [33] also possess the α-GI-producing ability to a large extent. Taken together, it can be stated that Bacillus species is a predominant microorganism that can be potentially used to yield α-GIs and help to regulate postprandial hyperglycemic pathophysiology associated with diabetes. Bacillus species stand out for their dual functionality, combining robust probiotic attributes with exceptional fermentation characteristics [24]. Notably, strains such as B. subtilis exhibit resilience in gastrointestinal conditions, making them effective probiotics that contribute to gut health and immune modulation. Simultaneously, their impressive fermentation capabilities enable the efficient conversion of various substrates into valuable products like enzymes and bioactive compounds that have tremendous health and therapeutic benefits.

In this investigation, a total of 23 bacteria were isolated from peruyaan, a traditional fermented soy food of Arunachal Pradesh. Among these diverse bacterial colonies, four remarkable strains (P2 MEK, P4 MEK, P6 MEK, and P8 MEK) were recognized as α-GI producers (Figure 1). Moreover, from these four strains, the most potent strain has been characterized, identified, and designated as Bacillus subtilis PM_NEIST_4 (Figure 2). Thereafter, the study focuses on optimizing the fermentation process using B. subtilis PM_NEIST_4 by exploring cultivation parameters such as culture media, temperature, culture volume, and OD₆₀₀ of bacteria in media [28] (Figures 3 and 4). Optimization of these culture parameters led to a significant increase in α-GI productivity, reaching 4808.324 ± 137.26 U/mL. In comparison, before optimization, the productivity was 1616.613 ± 84.54 U/mL (Table 1). Furthermore, an initial IC₅₀ value of 1705.617 ± 43.95 μg/mL was improved to a value of 596.532 ± 44.80 μg/mL (Figure 5). Both these α-GI productivity and IC₅₀ resulted after optimization strongly supported that the cultivation parameters considered to yield maximum α-GI productivity were successfully able to improvise the FMHB by approximately three times.

The α-GI produced by B. subtilis PM_NEIST_4 was also proved to have high thermal and pH stabilities up to 120°C and pH 13, respectively, with more than 90% activity observed in both the cases (Figure 6). This signifies that the α-glucosidase inhibitory property of the optimized FMHB would remain unaffected even when exposed to high temperature. Thus, it may be concluded that the FMHB can persist and maintain its stability for a longer duration. At the same time, as the pH of the gastrointestinal tract varies greatly, therefore it is one of the essential factors that must be considered for determining the pH stability of α-GI produced in the culture supernatant of FMHB. In this regard, the experimental results indicate that the FMHB can withstand various pH levels in the digestive tract without compromising its α-glucosidase inhibitory activity.

Treatment with FMHB demonstrated no toxic effects in both cellular and animal models, even at higher concentrations (Figure 7, Tables 2, and 3). For elucidating the effect of FMHB on animal model, three different doses of FMHB were administered to the sucrose-fed rat model. Results demonstrated that two doses, namely, 200 mg/kg and 400 mg/kg showed remarkable reduction in postprandial plasma glucose level at different time intervals, almost comparable to that of positive control (acarbose) (Figure 8). The results indicate that FMHB has the potential to contribute to lowering postprandial blood glucose by inhibiting the breakdown of sucrose into glucose in the intestinal tract. Thereafter, FMHB was fractionated using solvents of increasing polarity that include n-hexane, ethyl acetate, and n-butanol. The ethyl acetate fraction of FMHB showed the highest inhibitory activity against α-glucosidase enzyme (Figure 9). Based on this result, the ethyl acetate fraction was allowed to undergo GC-MS and LC-MS analyses for compound assessment. In this regard, GC-MS analysis of the ethyl acetate fraction demonstrated five major peaks at different acquisition times and LC-MS analysis of the same also revealed the presence of different compounds based on calculated mass. Both the analyses of ethyl acetate fraction suggested the presence of three pyrrolopyrazine derivatives- 3-propylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione, 3-isobutylhexahydropyrrolo [1,2-a]pyrazine-1,4-dione, and 3-benzylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione (Figure 10). Pyrrolopyrazine derivatives have been reported to exhibit a wide range of biological activities including antioxidant, anti-inflammatory, kinase inhibitory, antimicrobial, antiviral, antifungal, as well as antitumor [52]. However, there is no report so far that advocates the α-glucosidase inhibitory efficacy of pyrrolopyrazine and its derivatives.

The present study demonstrated the isolation of a potent α-GI producing bacterial strain, B. subtilis PM_NEIST_4 from traditional fermented soy food (peruyaan), and thereafter, the optimization of bacterial fermentation process was achieved to maximize the yield of α-GI (Figure 11). These findings underscore promising industrial potential of B. subtilis PM_NEIST_4, advocating the use of indigenous microbial resources for the production of α-GI.

5. Conclusion

Numerous commercially available α-GIs have been used clinically to manage hyperglycemia in diabetic conditions. Despite their effectiveness, these drugs are often associated with undesirable side effects, including diarrhea, nausea, vomiting, kidney problems, and liver damage. In addition, a significant disparity exists between the substantial production of α-GIs in the industry and their extensive consumption in the market. To address this challenge, there is a need to explore safer natural α-GIs and maximize its productivity in order to meet the ends. In this study, Bacillus subtilis PM_NEIST_4, isolated from peruyaan, showed remarkable inhibition of α-glucosidase enzyme activity. In order to enhance the productivity of α-GI, fermentation conditions have been optimized. This imperative optimization process aims to fix various parameters within the fermentation environment, including C/N source, cultivation temperature, medium/system volume ratio, OD₆₀₀ of B. subtilis PM_NEIST_4 in culture media, and cultivation duration, with the overarching goal of maximizing the yield of α-GIs. This finding not only emphasizes the significant role of this bacterium for production of α-GI but also suggests its promising application as a therapeutic tool for managing hyperglycemia. While the results showed promising evidence of its antidiabetic potential, it is however necessary to perform further studies in order to fully understand the underlying mechanism of its action. Hence, clinical trials are encouraged for evaluating the antihyperglycemic efficacy of α-GI produced by this bacterium. In conclusion, the outcome of this study will be helpful for the development of a novel therapeutic approach to prevent the attainment of high blood glucose concentrations in diabetic conditions. This has significant implications for the effective management of hyperglycemic pathophysiology in individuals with diabetes.

Abbreviations

α-GI:	α-Glucosidase inhibitor
BHI:	Brain Heart Infusion
FBS:	Fetal bovine serum
FMHB:	Fermented Mueller Hinton Broth
GC-MS:	Gas chromatography-mass spectrometry
LC-MS:	Liquid chromatography-mass spectrometry
LB:	Luria Bertani Broth
NA:	Nutrient Agar
NB:	Nutrient Broth
OD:	Optical density
PBS:	Phosphate-Buffered Saline
SEM:	Scanning electron microscopy
TSB:	Tryptone Soya Broth.

Data Availability

The data used to support the findings of this study are available from the corresponding author upon reasonable request.

Ethical Approval

The animal study was approved by the Institutional Ethical Committee and CPCSEA (Committee for the Purpose of Control and Supervision on Experiments on Animals), Ministry of Environment and Forests, New Delhi, India (2028/G)ReBi/S/18/CPCSEA).

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors’ Contributions

Mir Ekbal Kabir conceptualized the study, performed investigation, proposed the methodology, contributed to data curation, perform formal analysis, wrote the original draft, wrote, reviewed, and edited the article, and contributed to visualization. Anupriya Borah performed investigation, proposed the methodology, contributed to data curation, performed formal analysis, wrote the original draft, and reviewed and edited the article. Hiranmoy Barman, Nazim Uddin Afzal, and Bhaben Sharmah performed investigation, contributed to data curation, and reviewed and edited the article. Tridip Phukan performed formal analysis, supervision, and validation. Jatin Kalita performed supervision and validation. Prasenjit Manna conceptualized the study, performed investigation, proposed the methodology, contributed to data curation, performed formal analysis, provided resources, performed supervision, prepared the original draft, reviewed and edited the article, and performed validation. Mir Ekbal Kabir and Anupriya Borah contributed equally to this manuscript.

Acknowledgments

The authors are thankful to the Director, CSIR-NEIST for his support, and CSIR-HRDG, India, for providing the Senior Research Fellowship to Mr. Mir Ekbal Kabir (16/06/2019 (i) EU-V dtd 20-11-2019 and Sr. No. 1061931174) and Hiranmoy Barman (Nov/06/2020(i)) EU-V and Cert. No. JUN20C03006), UGC, Government of India, for providing Senior Research Fellowship (SRF) to Nazim Uddin Afzal (UGC Ref. No. 191620230693), DST, India, for providing the Junior Research Fellowship to Bhaben Sharmah (DST/INSPIRE Fellowship/IF200419), and also DBT, India, for providing fellowship to Ms. Anupriya Borah and fund for conducting the study (BT/PR45260/NER/95/1929/2022).

References

A. Chaudhury, C. Duvoor, V. S. Reddy Dendi et al., “Clinical review of antidiabetic drugs: implications for type 2 diabetes mellitus management,” Frontiers in Endocrinology, vol. 8, p. 6, 2017.
View at: Google Scholar
P. Z. Zimmet, D. J. Magliano, W. H. Herman, and J. E. Shaw, “Diabetes: a 21st century challenge,” Lancet Diabetes & Endocrinology, vol. 2, no. 1, pp. 56–64, 2014.
View at: Publisher Site | Google Scholar
P. Zimmet, K. G. Alberti, D. J. Magliano, and P. H. Bennett, “Diabetes mellitus statistics on prevalence and mortality: facts and fallacies,” Nature Reviews Endocrinology, vol. 12, no. 10, pp. 616–622, 2016.
View at: Publisher Site | Google Scholar
D. Y. Kwon, J. W. Daily, H. J. Kim, and S. Park, “Antidiabetic effects of fermented soybean products on type 2 diabetes,” Nutrition Research, vol. 30, pp. 1–13, 2010.
View at: Publisher Site | Google Scholar
S. Badole and S. Bodhankar, “Glycine max (soybean) treatment for diabetes,” Bioact Food Diet Interv Diabetes, vol. 77, pp. 77–82, 2012.
View at: Google Scholar
D. Das, M. E. Kabir, S. Sarkar, S. B. Wann, J. Kalita, and P. Manna, “Antidiabetic potential of soy protein/peptide: a therapeutic insight,” International Journal of Biological Macromolecules, vol. 194, pp. 276–288, 2022.
View at: Publisher Site | Google Scholar
S. Aslam, A. Zuberi, M. W. H. Chan, and J. Mustaquim, “Effect of lemna minor and glycine max on haematological parameters, glucose level, total protein content and anti-oxidant enzyme activities in ctenopharyngodon idella and Hypophthalmichthys molitrix,” Aquaculture Reports, vol. 19, Article ID 100616, 2021.
View at: Publisher Site | Google Scholar
D. Das, S. Sarkar, A. Dihingia et al., “A popular fermented soybean food of northeast india exerted promising antihyperglycemic potential via stimulating PI3K/AKT/AMPK/GLUT4 signaling pathways and regulating muscle glucose metabolism in type 2 diabetes,” Journal of Food Biochemistry, vol. 46, no. 12, Article ID e14385, 2022.
View at: Publisher Site | Google Scholar
R. Mukherjee, R. Chakraborty, and A. Dutta, “Role of fermentation in improving nutritional quality of soybean meal—a review,” Asian-Australasian Journal of Animal Sciences, vol. 29, no. 11, pp. 1523–1529, 2015.
View at: Publisher Site | Google Scholar
F. G. do Prado, M. G. B. Pagnoncelli, G. V. de Melo Pereira, S. G. Karp, and C. R. Soccol, “Fermented soy products and their potential health benefits: a review,” Microorganisms, vol. 10, no. 8, p. 1606, 2022.
View at: Publisher Site | Google Scholar
R. Loying, J. Kalita, and P. Manna, “Rice-based alcoholic fermented beverages of north north-east india: insight into ethnic preparation, microbial intervention, ethnobotany, and health benefits,” Journal of Food Biochemistry, vol. 2024, Article ID 7769743, 23 pages, 2024.
View at: Publisher Site | Google Scholar
D. Shin and D. Jeong, “Korean traditional fermented soybean products: jang,” Journal of Ethnic Foods, vol. 2, no. 1, pp. 2–7, 2015.
View at: Publisher Site | Google Scholar
J. P. Tamang, “Naturally fermented ethnic soybean foods of India,” Journal of Ethnic Foods, vol. 2, no. 1, pp. 8–17, 2015.
View at: Publisher Site | Google Scholar
C. R. Harwood, S. Pohl, W. Smith, and A. Wipat, “Bacillus subtilis: model Gram-positive synthetic biology chassis,” Methods in Microbiology, vol. 40, pp. 87–117, 2013.
View at: Google Scholar
A. Ullah, X. Yin, F. Wang et al., “Biosynthesis of selenium nanoparticles (via Bacillus subtilis BSN313), and their isolation, characterization, and bioactivities,” Molecules, vol. 26, no. 18, p. 5559, 2021.
View at: Publisher Site | Google Scholar
A. Ullah, J. Mu, F. Wang et al., “Biogenic selenium nanoparticles and their anticancer effects pertaining to probiotic bacteria—a Review,” Antioxidants, vol. 11, no. 10, p. 1916, 2022.
View at: Publisher Site | Google Scholar
M. Naveed, Y. Wang, X. Yin et al., “Purification, characterization and bactericidal action of lysozyme, isolated from Bacillus subtillis BSN314: a disintegrating effect of lysozyme on gram-positive and gram-negative bacteria,” Molecules, vol. 28, no. 3, p. 1058, 2023.
View at: Publisher Site | Google Scholar
M. Naveed, S. Wen, M. W. H. Chan et al., “Expression of BSN314 lysozyme genes in Escherichia coli BL21: a study to demonstrate microbicidal and disintegarting potential of the cloned lysozyme,” Brazilian Journal of Microbiology, vol. 55, pp. 215–233, 2023.
View at: Publisher Site | Google Scholar
R. Singh, S. Langyan, S. Sangwan et al., “Optimization and production of alpha-amylase using bacillus subtilis from apple peel: comparison with alternate feedstock,” Food Bioscience, vol. 49, Article ID 101978, 2022.
View at: Publisher Site | Google Scholar
S. Saravanakumar, N. N. Prabakaran, R. Ashokkumar, and S. Jamuna, “Unlocking the gut’s treasure: lipase-producing bacillus subtilis probiotic from the intestine of microstomus kitt (lemon sole),” Applied Biochemistry and Biotechnology, vol. 52, pp. 1–14, 2023.
View at: Publisher Site | Google Scholar
A. Berenjian, R. Mahanama, A. Talbot et al., “Efficient media for high menaquinone-7 production: response surface methodology approach,” New Biotech, vol. 28, no. 6, pp. 665–672, 2011.
View at: Publisher Site | Google Scholar
J. Song, H. Liu, L. Wang et al., “Enhanced production of vitamin K 2 from bacillus subtilis (natto) by mutation and optimization of the fermentation medium,” Brazilian Archives of Biology and Technology, vol. 57, no. 4, pp. 606–612, 2014.
View at: Publisher Site | Google Scholar
A. Ullah, Z. A. Mirani, S. Binbin et al., “An elucidative study of the anti-biofilm effect of selenium nanoparticles (SeNPs) on selected biofilm producing pathogenic bacteria: a disintegrating effect of SeNPs on bacteria,” Process Biochemistry, vol. 126, pp. 98–107, 2023.
View at: Publisher Site | Google Scholar
N.-K. Lee, W.-S. Kim, and H.-D. Paik, “Bacillus strains as human probiotics: characterization, safety, microbiome, and probiotic carrier,” Food Science and Biotechnology, vol. 28, no. 5, pp. 1297–1305, 2019.
View at: Publisher Site | Google Scholar
R. M. Martinez, “Bacillus subtilis,” Brenner’s Encyclopedia of Genetics (Second Edition), Academic Press, Cambridge, MA, USA, 2013.
View at: Google Scholar
J. van Dijl and M. Hecker, “Bacillus subtilis: from soil bacterium to super-secreting cell factory,” BioMed Central, vol. 23, 2013.
View at: Google Scholar
Z. Yuan, Z. Lanhong, S. Jun, J. Xiaofeng, and S. Mi, “Effects of culture conditions and surfactants on marine lysozyme S-12 production by Bacillus sp isolated from East China sea,” African Journal of Microbiology Research, vol. 7, no. 26, pp. 3341–3350, 2013.
View at: Publisher Site | Google Scholar
M. Naveed, H. Tianying, F. Wang et al., “Isolation of lysozyme producing Bacillus subtilis Strains, identification of the new strain Bacillus subtilis BSN314 with the highest enzyme production capacity and optimization of culture conditions for maximum lysozyme production,” Current Research in Biotechnology, vol. 4, pp. 290–301, 2022.
View at: Publisher Site | Google Scholar
Y. Gao, W. Bian, Y. Fang et al., “α-Glucosidase inhibitory activity of fermented okara broth started with the strain Bacillus amyloliquefaciens SY07,” Molecules, vol. 27, no. 3, p. 1127, 2022.
View at: Publisher Site | Google Scholar
S. Onose, R. Ikeda, K. Nakagawa et al., “Production of the α-glycosidase inhibitor 1-deoxynojirimycin from Bacillus species,” Food Chemistry, vol. 138, no. 1, pp. 516–523, 2013.
View at: Publisher Site | Google Scholar
H. Lee, H.-H. Shin, H. R. Kim, Y.-D. Nam, D.-H. Seo, and M.-J. Seo, “Culture optimization strategy for 1-Deoxynojirimycin-producing bacillus methylotrophicus K26 isolated from korean fermented soybean paste, doenjang,” Biotechnology and Bioprocess Engineering, vol. 23, no. 4, pp. 424–431, 2018.
View at: Publisher Site | Google Scholar
Y. P. Zhu, K. Yamaki, T. Yoshihashi et al., “Purification and identification of 1-deoxynojirimycin (DNJ) in okara fermented by Bacillus subtilis B2 from Chinese traditional food (Meitaoza),” Journal of Agricultural and Food Chemistry, vol. 58, no. 7, pp. 4097–4103, 2010.
View at: Publisher Site | Google Scholar
V. B. Nguyen, A. D. Nguyen, Y.-H. Kuo, and S.-L. Wang, “Biosynthesis of α-glucosidase inhibitors by a newly isolated bacterium, paenibacillus sp. TKU042 and its effect on reducing plasma glucose in a mouse model,” International Journal of Molecular Sciences, vol. 18, no. 4, p. 700, 2017.
View at: Publisher Site | Google Scholar
U. Hossain, A. K. Das, S. Ghosh, and P. C. Sil, “An overview on the role of bioactive α-glucosidase inhibitors in ameliorating diabetic complications,” Food and Chemical Toxicology, vol. 145, Article ID 111738, 2020.
View at: Publisher Site | Google Scholar
S. R. Quah, International Encyclopedia of Public Health, Academic Press, Cambridge, UK, 2016.
A. M. Dirir, M. Daou, A. F. Yousef, and L. F. Yousef, “A review of alpha-glucosidase inhibitors from plants as potential candidates for the treatment of type-2 diabetes,” Phytochemistry Reviews, vol. 21, no. 4, pp. 1049–1079, 2022.
View at: Publisher Site | Google Scholar
F. Lu, J. Sun, X. Jiang et al., “Identification and isolation of α-glucosidase inhibitors from siraitia grosvenorii roots using bio-affinity ultrafiltration and comprehensive chromatography,” International Journal of Molecular Sciences, vol. 24, no. 12, Article ID 10178, 2023.
View at: Publisher Site | Google Scholar
M. I. Kazeem, J. O. Adamson, and I. A. Ogunwande, “Modes of inhibition of α-amylase and α-glucosidase by aqueous extract of Morinda lucida Benth leaf,” BioMed Research International, vol. 527570, p. 24, 2013.
View at: Google Scholar
L. Minnullina, D. Pudova, E. Shagimardanova, L. Shigapova, M. Sharipova, and A. Mardanova, “Comparative genome analysis of uropathogenic morganella morganii strains,” Frontiers in Cellular and Infection Microbiology, vol. 9, p. 167, 2019.
View at: Publisher Site | Google Scholar
V. B. Nguyen, A. D. Nguyen, and S. L. Wang, “Utilization of fishery processing by-product squid pens for α-glucosidase inhibitors production by paenibacillus sp,” Marine Drugs, vol. 15, no. 9, p. 274, 2017.
View at: Publisher Site | Google Scholar
S. Deng, W. He, A. Y. Gong et al., “Cryptosporidium uses CSpV1 to activate host type I interferon and attenuate antiparasitic defenses,” Nature Communications, vol. 14, no. 1, pp. 1456–37129, 2023.
View at: Publisher Site | Google Scholar
S. Sarkar, P. Sadhukhan, D. Das et al., “Glucose-sensitive delivery of vitamin K by using surface-functionalized, dextran-capped mesoporous silica nanoparticles to alleviate hyperglycemia,” ACS Applied Materials & Interfaces, vol. 14, no. 23, pp. 26489–26500, 2022.
View at: Publisher Site | Google Scholar
Oecd, “Test no 423: acute oral toxicity- acute toxic class method2002,” OECD Guidelines for the Testing of Chemicals, Section 4, vol. 14.
View at: Google Scholar
T. Wresdiyati, S. Sa'diah, A. Winarto, and V. Febriyani, “Alpha-glucosidase inhibition and hypoglycemic activities of Sweitenia mahagoni seed extract,” HAYATI Journal of Biosciences, vol. 22, no. 2, pp. 73–78, 2015.
View at: Publisher Site | Google Scholar
H. Mohamed Sham Shihabudeen, D. Hansi Priscilla, and K. Thirumurugan, “Cinnamon extract inhibits α-glucosidase activity and dampens postprandial glucose excursion in diabetic rats,” Nutrition & Metabolism, vol. 8, pp. 46–11, 2011.
View at: Publisher Site | Google Scholar
A. Bhatia, B. Singh, R. Arora, and S. Arora, “In vitro evaluation of the α-glucosidase inhibitory potential of methanolic extracts of traditionally used antidiabetic plants,” BMC Complementary and Alternative Medicine, vol. 19, no. 1, pp. 74–2482, 2019.
View at: Publisher Site | Google Scholar
K. M. M. Hasan, N. Tamanna, and M. A. Haque, “Biochemical and histopathological profiling of Wistar rat treated with Brassica napus as a supplementary feed,” Food Science and Human Wellness, vol. 7, no. 1, pp. 77–82, 2018.
View at: Publisher Site | Google Scholar
S. Sarkar, M. Ekbal Kabir, J. Kalita, and P. Manna, “Mesoporous silica nanoparticles: drug delivery vehicles for antidiabetic molecules,” ChemBioChem, vol. 24, no. 7, Article ID e202200672, 2023.
View at: Publisher Site | Google Scholar
D. Das, S. B. Wann, J. Kalita, and P. Manna, “Insight into the efficacy profile of fermented soy foods against diabetes,” Food Bioscience, vol. 53, Article ID 102665, 2023.
View at: Publisher Site | Google Scholar
L. Gong, D. Feng, T. Wang, Y. Ren, Y. Liu, and J. Wang, “Inhibitors of α‐amylase and α‐glucosidase: potential linkage for whole cereal foods on prevention of hyperglycemia,” Food Science and Nutrition, vol. 8, no. 12, pp. 6320–6337, 2020.
View at: Publisher Site | Google Scholar
S. Yu, W. Wang, S. Li et al., “Glucoregulatory properties of fermented soybean products,” Fermentation, vol. 9, no. 3, p. 254, 2023.
View at: Publisher Site | Google Scholar
F. Dehnavi, S. R. Alizadeh, and M. A. Ebrahimzadeh, “Pyrrolopyrazine derivatives: synthetic approaches and biological activities,” Medicinal Chemistry Research, vol. 12, pp. 1–26, 2021.
View at: Publisher Site | Google Scholar

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Copyright © 2024 Mir Ekbal Kabir et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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