Food Microbiology (FM) | Bioactive Compounds or Metabolites: Function and Application
Microbiol. Biotechnol. Lett. 2024; 52(3): 288-297
https://doi.org/10.48022/mbl.2407.07007
Mst. Sarmin Sultana1, Maya Khatun1,2, and Md. Ajijur Rahman1*
1Department of Pharmacy, University of Rajshahi, Rajshahi-6205, Bangladesh
2Pharmaceutical Sciences Research Division, Bangladesh Council of Scientific and Industrial Research (BCSIR), Dhaka - 1205, Bangladesh
Correspondence to :
Md. Ajijur Rahman, ajijurrahman.pharm@ru.ac.bd
Fermented cooked rice is known to have remedial properties and good for health, but there is a lack of scientific knowledge to prove their beneficial effects. In this study, we investigated the presence of antibiotic-producing bacteria in fermented cooked rice. The probiotic properties of the bioactive isolates were also investigated. A total of seven pure isolates were isolated from fermented cooked rice prepared from parboiled cooked Aus rice in the lab following traditional methods. All the isolates were gram-positive, can grow at thermophilic range of temperatures, and all but CRS9 were able to coagulate milk. Three strains exhibited moderate to high broad-spectrum antibacterial activity against the test bacteria including Shigella brodie, Bacillus cereus, Escherichia coli, Pseudomonas sp., Staphylococcus aureus, and Proteus sp. Analysis of 16S rDNA sequences of the strain CRS8 and CRS11 showed that they belong to the genus Bacillus as they exhibited >99% identity to several strains of Bacillus. Both strains could survive the highly acidic conditions and can tolerate bile acid indicating their potential to be the candidate probiotic strains. The strain CRS8 was resistant to penicillins and cephalosporins including amoxicillin, penicillin, cephalothin, however, the CRS11 was sensitive to all the antibiotics tested. This is the first report that fermented cooked rice is a source of antibiotic-producing Bacillus sp. The probiotic properties of the Bacillus isolates from fermented cooked rice were also investigated for the first time.
Keywords: Fermented cooked rice, Bacillus sp., antimicrobial activity, probiotic properties, antibiotic resistance
From ancient times people from around the world consume fermented foods, however, in the recent years, there has been a surge in popularity of fermented foods in the Western countries due to their health benefits [1, 2]. Historically, fermentation was used to preserve foods such as fish, milk, rice, vegetables, cereals, fruits etc. as well as to increase their taste and aroma. The production of organic acids, alcohol and bacteriocins during fermentation helps to reduce growth of pathogenic as well as food-spoiling microorganisms thus, increase their shelf-life [2]. Fermentation also helps to reduce the amount of antinutrients such as nondigestible carbohydrates and thus increase the pool of vitamins, minerals and essential amino acids [3]. Fermented cooked rice (also called panta bhat, poitabhat, watered rice, sour rice etc.) is a form of fermented rice prepared generally by soaking cooked parboiled rice overnight in water. It is consumed since ancient times in Bangladesh and in many parts of India mainly during the hot seasons [4]. It is generally eaten as a breakfast with salt, onion, chilli and sometimes with vegetables, potato mash, fish, or meat etc. It is popular among the low- and middle-income rural people as a regular breakfast, however, it is widely consumed on the day of Pahela Baishakh or Bengali new year [5]. In Ayurvedic medicine, fermented cooked rice is considered as cold food and is used as a food for children with fever. It also lowers the blood pressure, has diuretic and sedative effect [6]. It contains a small amount of alcohol produced during the overnight soaking of the cooked rice.
Probiotics are live microorganisms which when administered in adequate amounts, confer a health benefit on the host [7]. Dairy and dairy-related products are main source of probiotic microorganisms. Along with the dairy products, probiotic bacteria including the species of the genus
Although fermented cooked rice is traditionally known to be healthy and claimed to exert remedial properties, there is a lack of sufficient information on the presence of probiotic bacteria in it. In this study, we isolated and identified
500 gm of parboiled Aus rice was collected from a local market in Rajshahi and brought into the lab for cooking and fermentation. The rice was washed twice with 1 L of distilled water and any excess water was drained. 1.5 L of distilled water was taken in an aluminium pot and the drained rice was poured into the water and cooked until soft consistency was achieved. The cooled rice was transferred to an autoclaved earthen pot and 1.5 L of sterile distilled water was added. The pots were covered with aluminium foil and incubated overnight at room temperature or 37℃ to ferment and allow the growth of bacteria present in the cooked rice.
To determine the CFUs per millilitre of fermented cooked rice, the samples were taken in a sterile mortar and triturated until a smooth suspension is formed. 1 ml of the suspension was then serially diluted in sterile saline water (0.85% NaCl) and 100 μl of each dilution was plated onto MRS agar plates. The plates were incubated for 16 h aerobically and the CFUs per millilitre of the fermented cooked rice were determined. Pure colonies with distinct morphological characteristics were picked using sterile toothpicks and transferred to fresh MRS agar or LB agar plates. Each isolate was preserved in 20% glycerol at -20℃.
The colony morphology of the pure isolates was determined visually. The gram-staining and catalase tests were performed using the standard procedures. The ability of the strains to grow at different temperatures (10℃ and 45℃) and salt concentration (1.5%−7.5%), the ability to use different carbohydrate sources (glucose, fructose, sucrose, maltose, lactose, xylose, and starch), the ability to coagulate milk and change in pH of the milk after milk coagulation were determined for each pure isolate.
A single pure colony of the isolates was grown at 37℃for 16 h in 5 ml LB broth with shaking. Genomic DNA was extracted from 1.5 ml culture using the Wizard Genomic DNA Purification Kit (Promega, USA) following the manufacturer’s instructions for Gram-positive bacteria. The purified genomic DNAs were checked on 1% agarose gel to check the purity.
The 16S rDNA sequence was amplified by PCR using Hot Start Green Master MixM7432 (Promega). A 20 µl PCR reaction mix contained 10 µl Master Mix (10X), 1 µl of genomic DNA, 1 µl of both primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-CGGTTACCTTGTTACGAC TT-3′) and 7 µl molecular grade H2O. The PCR conditions were as follows: initial denaturation for 5 min at 95℃ followed by 35 cycles of denaturation at 95℃ for 30 s, annealing at 48℃ for 30 s, an extension at 72℃ for 1.5 min and final extension at 72℃ for 5 min. The amplified PCR products were checked on a 1% (w/v) agarose gel and visualized in a gel documentation system. The PCR products were purified using the PCR Clean-Up System (A9281, Promega) and sequenced using Sanger Sequencing.
To obtain the homologous sequences of the partial 16S rDNA sequences of the isolates, the EZBiocloud.net(4) was used. The phylogenetic tree was constructed using the MEGA11 (5). The neighbour-Joining method (6) was used to construct the phylogenetic tree.
The ability of the isolates to produce antimicrobial substances was determined according to the method described previously with minor modifications [12]. Briefly, the isolates were streaked in a single line on MH agar plates and incubated for 20−22 h at 37℃. On the following day, the freshly grown test bacterial strains (
The antibiotic resistance phenotype of the isolates was determined using the Kirby-Bauer disk diffusion susceptibility test protocol (ASM, 2009). Briefly, the freshly grown broth culture of the isolates was adjusted to 0.5 McFarland Standard using sterile saline. The isolates were inoculated on MH agar plates using a sterile cotton swab. The plates were allowed to dry for 5 min after inoculation and antibiotic disks (Amoxicillin 30 µg, Ciprofloxacin 5 µg, Kanamycin 30 µg, Cephalothin 30 µg and Penicillin G 10 µg) were placed using forceps. The plates were incubated for 16−18 h and the zone of inhibition around the antibiotic disks was measured in millimetres in triplicates.
Acid tolerance is an important property of probiotic microorganisms. To determine the acid tolerance ability of the isolates, they were grown in pH adjusted MRS broth. A volume of overnight broth culture was adjusted to 0.5 McFarland standard and 1 ml of the adjusted suspension was added to 99 ml MRS broth of different pH range (pH 1 to pH 4) and incubated with shaking at 200 rpm at 37℃. The CFUs/ml were determined at 0, 1, 2, and 4 h by removing 100 μl of the culture at each time point followed by plating on MRS agar plate in different dilution. The number of colonies was counted after incubation at 37℃ for 16−18 h.
Fresh cow bile was collected aseptically from an abattoir in Rajshahi city. The water portion was evaporated in a hot air oven and stored in the refrigerator. To make MRS broth with 0.3% bile, 0.3 gm of the dried cow bile was added to 100 ml MRS broth and autoclaved. A volume of overnight culture of the isolates was adjusted to 0.5 McFarland standard and 1 ml of the adjusted suspension was added to 99 ml MRS broth with 0.3% bile. The flasks were then incubated with shaking at 200 rpm at 37℃. The CFUs/ml were determined at 0, 1, 2, and 4 h by removing 100 µl culture at each time point followed by plating on MRS agar. The number of colonies appeared on the plates were counted after incubation at 37℃ for 16−18 h.
The partial 16S rDNA sequences of CRS8 and CRS11 were submitted to GenBank, and the accession numbers ON630269 and ON630270 were given, respectively for CRS-8 and CRS-11.
After overnight aerobic fermentation of the cooked parboiled Aus rice in an earthen pot, a diluted aliquot of the triturated fermented rice samples was plated on MRS agar and colonies on the plates were counted. The CFUs/ml of the fermented rice suspension was 18.4×107. Seven colonies of distinct morphology were picked from the plates and purified for further investigation (Fig. 1).
Among the seven pure isolates of fermented rice, six were white or creamy white and one was yellow. All of them were gram-positive and all but CRS9 were rod-shaped. Four isolates were catalase-positive and the remaining three were catalase-negative (Table 1). None but CRS8 could grow at 10℃. All the isolates were thermophilic and could grow at 45℃ (Table 1). Four isolates including CRS8, CRS10, CRS11, CRS12 and CRS14 coagulated cow milk and resulted in decline of pH (Supplementary Table S1). All the isolates were able to tolerate up to 4.5% of salt (NaCl), whereas three isolates including CRS11, CRS13 and CRS14 were able to grow with 7.5% of salt (Supplementary Table S2). All the isolates were able to utilize glucose and fructose. CRS9 and CRS13 failed to utilize sucrose and maltose. None of them could utilize lactose and maltose and only two isolates, CRS8 and CRS11 could utilize starch as a carbon source (Supplementary Table S3).
Table 1 . Morphological and phenotypic properties of fermented rice isolates.
Isolates | Colony colour | Gram staining | Cell shape | Catalase activity | Growth at different temperature | |
---|---|---|---|---|---|---|
10℃ | 45℃ | |||||
CRS8 | White/Creamy white | + | Rod | + | + | + |
CRS9 | Creamy white | + | Cocci | - | - | + |
CRS10 | Creamy white | + | Rod | + | - | + |
CRS11 | Creamy white | + | Rod | + | - | + |
CRS12 | Yellow | + | Rod | + | - | + |
CRS13 | Creamy white | + | Rod | - | - | + |
CRS14 | Creamy white | + | Rod | - | - | + |
The antimicrobial activity of the isolates was carried out using the cross-streaking method. Two gram-positive and four gram-negative bacteria were used to indicate the inhibitory activity of the isolates against them. Among the seven pure isolates, three isolates were found to exhibit broad-spectrum antibacterial activity. The distance of inhibition (DOI) of CRS14 was higher than CRS8 and CRS11. The active isolates showed highest activity against
Table 2 . Distance of inhibition (DOI) of the pure isolates from fermented cooked rice against the test bacteria.
Test bacterial strains | Distance of inhibition (DOI) of the isolates in millimetre | ||||||
---|---|---|---|---|---|---|---|
CRS8 | CRS9 | CRS10 | CRS11 | CRS12 | CRS13 | CRS14 | |
14.3 ± 1.5 | 0 | 0 | 3 ± 0 | 0 | 0 | 21 ± 1.7 | |
15 ± 0.5 | 0 | 0 | 8.5 ± 0.5 | 0 | 0 | 18.8 ± 1.8 | |
0 | 0 | 0 | 0 | 0 | 0 | 19.6 ± 0.5 | |
0 | 0 | 0 | 0 | 0 | 0 | 9 ± 0 | |
20 ± 1.5 | 0 | 0 | 14 ± 0.2 | 0 | 0 | 30.3 ± 0.5 | |
10 ± 0.5 | 0 | 0 | 6.1 ± 0.2 | 0 | 0 | 14.3 ± 0.5 |
The nearly complete 16S rDNA sequences of CRS8 and CRS11 were analyzed, and it was found that both belong to the genus
Among the three active isolates, CRS8 and CRS11 were selected to analyse their probiotic properties. The results of acid tolerance ability of CRS8 and CRS11 is presented in Fig. 3. Both isolates could tolerate pH as low as 1. At pH1, the average number of viable cells for CRS8 was 600 per ml at 1 h of incubation. The number of CFU/ml decreased to 100 at 3 h. However, at pH 2 and 3, the number of CFU increased over time. At pH2, the average number of CFU/ml for CRS8 at 1 h was 1900 which increased to 2500 at 3 h. The cell viability increased significantly at pH 3. The similar trend for was found for CRS11, where the number of viable cells at pH 1 decreased significantly with time, however, at pH 2 and 3, the number of viable cells increased significantly with the increase of incubation time (Fig. 3). This indicates that the strains can survive the strong acidic conditions of stomach.
The bile tolerance ability of CRS8 and CRS11 was studied using 0.3% cow bile in MRS broth. The results showed that both for CRS8 and CRS11, the number of viable cells per millilitre of broth reduced over time (Fig. 4). For CRS8, the number of viable cells per millilitre was 18,300 after one hour which reduced to 1,200 at 3 h. For CRS11, the average number of viable cells per millilitre of MRS was 13,300. The number declined to 720 after 3 h of incubation with 0.3% bile (Fig. 4). This indicates that the isolates are bile tolerant for until 2 h and when they are incubated at same bile concentration for longer time the number of viable cells declined exponentially.
The antibiotic resistance phenotype of CRS8 and CRS11 was studied against five antibiotics (amoxicillin, ciprofloxacin, kanamycin, cephalothin and penicillin G). It was found that CRS8 was sensitive to all antibiotics tested, however, CRS11 was resistant to all antibiotics except ciprofloxacin (Table 3).
Table 3 . Antibiotic resistance phonotype of CRS8 and CRS11 against antibiotics of different classes. R and S in the parenthesis indicates resistant and sensitive phenotypes, respectively.
Antibiotics | Zone of inhibition (mm) | |
---|---|---|
CRS8 | CRS11 | |
Amoxicillin (30 µg) | 35.6 ± 1.15 (S) | 9 ± 1 (R) |
Ciprofloxacin (5 µg) | 30.6 ± 1.15 (S) | 27 ± 1.5 (S) |
Kanamycin (30 µg) | 34.7 ± 2.51 (S) | 17 ± 1.5 (R) |
Cephalothin (30 µg) | 16.5 ± 1.15 (S) | 0 (R) |
Penicillin G (10 µg) | 29.8 ± 1.6 (S) | 0 (R) |
Although fermented cooked rice (poitabhat, fermented rice or sour rice) is traditionally consumed from ancient times by millions of people of Bangladesh and India, it failed to gain universal recognition as a healthy food due to insufficient scientific information. It received huge media attention when it became the winning dish of MasterChef Australia in 2021. Following that, numerous newspaper articles, TV and other electronic media including BBC Bangla, NDTV, Times of India, The Daily Star, UNB and so on published news on the benefits of fermented cooked rice, however, none of these articles cited any published journal articles or books. Although, the presence of micronutrients in fermented cooked rice has been investigated before [13], according to our knowledge there was no scientific study published on the presence of bacteria producing antibacterial metabolites and exhibiting probiotic properties which could relate the claimed benefits of fermented cooked rice.
In this study, we aimed to isolate bacteria from the fermented cooked rice having antimicrobial activity and to investigate their probiotic properties. The fermented cooked rice was prepared in the lab using the traditional method - cooking of rice followed by soaking the cooked rice in water overnight. After overnight fermentation, an aliquot of the rice samples was plated on MRS agar plate in different dilution. Seven pure colonies from the dilution plates were transferred to fresh MRS agar plates and their phenotypic and cultural characteristics were tested. Two catalase-positive, rod shaped, gram-positive isolate namely CRS8 and CRS11 were selected for identification up to species level using 16S rDNA sequencing and their probiotic characteristics such as tolerance to low pH and bile acids as well as resistance to antibiotics etc. were studied [14].
The analysis of 16S rDNA sequences using BLAST and EzBioCloud database as well as the phylogenetic tree showed that both CRS8 and CRS11 strains belong to the genus
Both CRS8 and CRS11 exhibited broad-spectrum anti-bacterial activities. Twenty-seven
Low pH and bile salt resistance properties are considered very important selection criteria of a probiotic strain. The potential probiotic strains must be resistant to low pH and bile acid as they need to pass through the highly acidic conditions of stomach before they reach the small intestine [17]. The mechanisms of acid tolerance in gram-positive bacteria include use of proton pumps, the protection of macromolecules, changes of the cell membrane and cell envelope and production of alkaline substances [18]. Four stress-related genes have been identified in
Tolerance to bile salts is an important property of probiotic isolates. Bile is produced in the liver, stored in gall bladder, and secreted into the duodenum to aid digestion of dietary fats. Bile salts, a main ingredient in bile, is produced by conjugation of bile acids with amino acid glycine, taurine or sulphate [21]. Bile salts and the remaining unconjugated bile acids present in the bile are toxic to bacteria, thus, to survive the bile attack, bacteria entering the gut through oral route must exert bile acid tolerance [22]. Disruption of plasma membrane is the main mechanism by which bile salts cause bacterial cell death [23]. Bile tolerance in
Resistance of potential probiotics to commercial anti-biotics can be a beneficial property of probiotics when they need be administered during the antibiotic therapy to avoid side effects of antibiotics incurred due to alteration gut microbiota [24]. However, this approach has the risks of transfer of transferrable antibiotic resistance genes residing on mobile genetic elements from the probiotic strain to the pathogens. The strain CRS8 were sensitive to all antibiotics tested, however, CRS11 was moderate to highly resistant to all the antibiotics except ciprofloxacin. This indicates the presence of different set of resistance genes among the two related strains.
Several species of the genus
Several strains of
The results of this study provide evidence that fermented cooked rice is a source of
Our study is limited by the number of rice samples used to isolate bacterial strains to screen their antibacterial activities and probiotic characteristics. Further studies with different kinds of rice may yield different kinds of bacteria having interesting biological properties.
We gratefully acknowledge the contribution of Professor Dr. Anwar Ul Islam for his critical thoughts during performing the experiments. The study was partially funded by the internal funding of the Department of Pharmacy, University of Rajshahi.
MSS and MK performed the investigations and analyzed the data, MAR conceptualized the experiment, supervised the work and wrote the manuscript. I certify that the above information is true and correct. All the authors contributed to the study and the manuscript. If the manuscript is accepted for publication, we agree to transfer all copyright ownership of the manuscript to the Journal of Microbiology and Biotechnology, which covers the rights to use, reproduce, or distribute the article.
The authors have no financial conflicts of interest to declare.
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