Isolation and Characterization of Probiotic Lactic acid Producing Bacteria in Kenyan Traditionally Fermented Milk
Wachira Susan, Kirui Stella, Bakari Chaka*
1Department of Biological Sciences, Maasai mara university, Kenya
2Department of Mathematics and physical sciences, Maasai mara university, Kenya
Received Date: 09/12/2020; Published Date: 21/12/2020
*Corresponding author: Bakari chaka, Department of Mathematics and physical sciences, Maasai mara university, Kenya
Cite this article: Wachira Susan, Kirui Stella, Bakari Chaka*. Isolation and Characterization of Probiotic Lactic acid Producing Bacteria in Kenyan Traditionally Fermented Milk. Op Acc J Bio Sci & Res 6(3)-2020.
Artificial intelligence is a new phenomenon that has occupied a prominent place in our present lives. Its presence in almost any industry that deals with any huge sheer volume of data are taking advantage of AI by integrating it into its day-to-day operation. AI has predictive power based on its data analytic functionality and some levels of autonomous learning, which its raw ingredient is just the massive sheer volume of data. Artificial intelligence is about extracting value from data, which has become the core business value when insight can be extracted. AI has various fundamental applications. This technology can be applied to many different sectors and industries. There has been a tremendous use of artificial intelligence in Nanotechnology research during the last decades. Convergence between artificial intelligence and Nanotechnology can shape the path for various technological developments and a large variety of disciplines. In this short communication, we present such innovative and dynamic sites utilizing artificial intelligence and its sub-sets of machine learning driven by deep learning in Nanotechnology.
Keywords: Artificial Intelligence; Machine Learning; Deep Learning; Nanoscience; Nanotechnology; Atomic Force Microscopy; Simulations, Nano computing
Abbreviations: AI: Artificial Intelligence; AFM: Atomic Force Microscope; STM: Scanning Tunneling Microscope; ML: Machine Learning; DL: Deep Learning; BMI: Brain Machine Interfaces; EEG: Electroencephalograph; HPC: High-Power Computing; PSPD: Position-Sensitive Photodiode; IoT: Internet of Things; TCO: Total Cost of Ownership; ROI: Return on Investment
Fermented foods have been used as a source of lactic acid producing bacteria (LAB) by different communities all over the world. LAB is of great importance in the human body. They have been classified into various families which include; Lactobacillaceae, Aerococcaceae, Carnobacteriaceae, Enterococcaceae, Leuconostocaceae, and Streptococcaceae. These bacteria have several importance in the human body like acting as probiotics. Probiotics are live micro-organisms, when administered in adequate amounts confer a health benefit on the host [1,2]. The word probiotic comes from the Greek word ‘pro’ meaning promoting and ‘biotic’ meaning life. Probiotics were discovered in the early 20th century, when Ellie Metchnikoff, known as the ‘father of probiotics’ had observed that rural dwellers of Bulgaria lived to very old ages despite extreme poverty and harsh climate . His theory was that health could be improved by manipulating host friendly bacteria in sour milk. The International association for probiotics and prebiotics (ISAPP) in conjunction with other medical and scientific experts were also able to differentiate between foods which contained live cultures and those which contained probiotics . Live cultures are any foods with fermentation microbes which need no specific research is required to prove their existence. Probiotics are supported by convincing evidence on their benefit to man and have clearly defined strains.
The human body naturally contains normal flora and harmful bacteria. A balance between the two is necessary and when it is disrupted (dysbiosis) can cause diseases like ulcerative colitis, irritable bowel syndrome amongst others . For probiotics to take effect it requires a prebiotic. This is a non-digestible carbohydrate that acts as food for probiotics and other bacteria. A prebiotic helps a probiotic by combining to form a synergetic effect known as symbiotic. Most probiotics identified are oligosaccharides and are resistant to human digestive enzymes. They go through the gastral-intestinal system without being digested and get fermented in the lower colon to form short chained fatty acids that nourish the microbes in the colon. Probiotics can be identified in a variety of natural sources including bananas, barley, garlic, honey, milk, Mustard, soybean, sugarcane, tomato and wheat amongst others. In Kenya, fermented milk (mala) is a common supplement for fermented milk lactic acid producing bacteria strains. Two samples will be used for the study; from the Maasai community, and the Kalenjin community to individual diets. With the cultural diversity of the various tribes, fermented milk has developed distinct characteristics because of the different additions.
Materials and Methods
The milk was obtained from the Maasai community and Kalenjin community, Sample bottles, Sodium acetate, Sodium hydroxide, Yeast extract, Glucose broth, Tween 80.
1.2.1. Study Size: The study size of this experiment covered two Kenyan communities; the Maasai and the Kalenjin. These two tribes are well known for the unique ways in which they locally ferment their milk with different additives to give the milk a distinct taste and texture.
1.2.2. Sampling Design: The method of sampling that was used in this design is simple random for the communities chosen. The milk samples came from random cow sources from each area chosen respectively. Two samples were required from each community to enable the comparison between the lactobacillus.
1.2.3. Isolation: Isolation involved creation of a media that was able to effectively sustain sample LAB cultures. An enriched nutrient agar media was used. It was enriched using; Sodium acetate, Sodium hydroxide, Yeast extract, Glucose broth, and Tween 80. This media was weighed out and the optimum quantities were obtained which supported the growth of the cultures. After preparation of the media each was inoculated from the three different samples. Growth of the colonies was then observed at intervals of 24 and 48 hours. When the colonies were formed, isolates were obtained for pure culture so as to obtain pure isolates for morphology observation and further analysis.
1.2.4. Gram Staining: This test was done to authenticate the presence of LAB in the culture. Gram positive bacteria turns violet blue due to the presence of a thick peptidoglycan in their cell wall. Gram negative bacteria stains red due to a thinner peptidoglycan in their cell . The procedure involved; preparing a heat fixed sample, adding the primary stain (crystal violet blue) followed by Gram’s iodine after washing of the unbound primary stain. The slide was then rinsed in acetone or alcohol followed by use of running distilled water. The secondary stain safranin was then added before incubating for one minute. Observations were done to determine the presence of LAB.
1.2.5. Sodium Chloride Tolerance: Isolated samples of lactobacilli were grown in enriched media in several test tubes each containing different concentrations of sodium chloride from 1-9%. After autoclaving the media for 15 minutes in 15lbs pressure at 121 °C, the test tubes were then inoculated with 10 microliters of overnight culture of Lactobacillus and then incubated anaerobically at 37°C for 24 hours. The bacterial growth was measured afterwards .
1.2.6. Catalase Test: Catalase test was done by aseptically mixing 1ml of hydrogen peroxide in a test tube and adding 1ml of culture. For positive results, there was a formation of bubbles to indicate evolution of gas. .
1.3. Morphological Analysis
After culturing, various qualities of the colonies including; the shape, the color, and the size of the colony was observed. Cell morphology was also observed upon further investigation to confirm the shape, on whether it was rod shaped or cocci of individual bacterial cells. Likewise, it was observed to indicate on whether they occurred in pairs or individually, with clear understanding on the spore formation and size.
1.4. Data Analysis
Data collection was done through experimentation and observation methods. The experiments done provided a data base for analysis and comparison of the results obtained from the different sources
2.1. Isolation of Lab: (Table 1)
2.2.1. Colony Morphology: (Table 2) (Figure 1)
2.2.2. Cell Morphology: (Table 3) (Figure 1)
Table 1: MRS media vs enriched media.
Table 2: Colony morphology of cultured lactic acid producing bacteria.
Table 3: Cell morphology of cultured lactic acid producing bacteria.
Isolation of LAB requires specific conditions which are provided by the ideal media; De Man Ragosa and Sharpe (MRS). In the absence of this media a substitute was established using nutrient agar media which was enriched using various compounds to make it an enriched media and a selective media. This media had to provide conditions which optimize growth of LAB.
The media created was able to be selective and only allow the growth of the LAB that was inoculated. The beef extract found in the nutrient agar, glucose and the added yeast extract provided nutritional value to the bacteria. Sodium acetate made the media selective and allowed only the growth of LAB colonies. Sodium hydroxide stabilized the pH of the media to make it conducive for culture. The pH of the media ranged between 6.5-7.2. In the Maasai samples, growth was observed with the optimum growth being between 36-48 hours. The inoculates were then isolated for a pure culture. The Kalenjin sample also yielded results in the selective media and growth was observed between 36-48 hours. The control yielded growth of the colonies of LAB found in the sample.
3.2. Colony Morphology
3.2.1. Maasai Sample Culture
After incubation all samples indicated the presence of LAB. Upon further investigation, where pure culture samples of the colonies were obtained, the morphology of the different colonies was described. In sample one the colony appeared white in color. It was rounded and had a smooth margin. The bacteria grew horizontally across the media with no elevation. Sample two colony presented itself as white in color. The colony was round and had a smooth margin. There was a slight elevation in the colony showing aerial growth. The third isolate was white and the colony was round. It had a smooth margin and aerial growth was also observed. These characteristics are common for most LABs .
3.2.2. Kalenjin Sample Culture
There were three samples of traditionally fermented milk that were tested from cow milk from the Kalenjin community from Eldoret and Bomet. All the three samples incubated were able to culture LAB. A pure culture of an individual colony was obtained from each. The isolate from the first sample was creamy in color. It had a rounded shape and a smooth margin. There was slight aerial growth of the colony. The second sample isolate appeared white in color. It had a smooth margin and was rounded in shape. The isolate grew horizontal to the substrate with no elevation. The second colony isolate was creamy and rounded in shape. It had a smooth margin and is flattened along the substrate. The third sample isolate was creamy in color, was rounded in shape, had a smooth margin and is flattened along the media surface [10-12].
3.3. Cell Morphology
3.3.1. Maasai Sample
A pure culture from each sample was done and then various tests were carried out on the isolates. The first sample had isolates which were gram positive and upon further observation under the microscope, the cells appeared rod shaped and short. This is a characteristic of LAB under Family Lactobaocillaceae. The second isolate also had gram positive cells . They appeared rod shape and rounded in shape. These cells were in the Family Lactobaocillaceae, the third isolate also had gram positive bacteria. When observed under the light microscope they presented as rod shaped bacteria which were squared. The majority of isolates were LAB of Lactobacillaceae with the exception of a few unidentified species .
3.3.2. Kalenjin Sample
After the colony morphology was observed a pure culture was performed to identify and characterize the cells. In the first isolate gram-positive cells were identified. The cells were rod shaped and appeared in pairs. This is a characteristic of family Lactobaocillaceae. The second isolate presented itself as gram positive spherical cells which are common characteristics of LAB from the family Streptococcaceae. The third isolate had gram positive rod shaped which were rounded cells. These are common characteristics of Family Lactobaocillaceae.
3.4. Salt Tolerance
All the samples did well under low concentrations of sodium chloride ranging between 2% to 6% concentration. These are the optimum standards for LABs to survive in an environment .
3.5. Catalase Test
All the sample isolates tested negative in this test. This test showed that the isolates were in fact LABs as they do not.
3.5.1. Spore Formation
The isolates were all non- spore producing bacteria. This is also common for LABs. Researchers from different countries have been able to isolate lactic acid bacteria from different fermented foods. These foods are consumed by locals in their countries just as traditionally fermented milk is consumed here in Kenya. In areas such as Gurgaon and Lakshmangarh scientists were able to isolate lactobacillus from curd samples [16,17]. They used the conventional media De Man Ragosa and Sharpe (MRS) agar media and were able to characterize them by the phenotypic characteristics.
This experiment was able to isolate and characterize LAB using an enriched media. Unlike industrial starter cultures which offer only one variety of microflora, traditionally fermented milk offers a wider array of bacteria. However, these bacteria may not offer a health benefit and might instead cause infection in the body. Only a few strains of the bacteria are beneficial to the host and the remainder may either be infective or not affect the host in any way.
Deep Learning (DL) is the subset of machine learning that, on the other hand, is the subset of artificial intelligence. The structure of the human brain inspires deep learning. Deep learning algorithms attempt to draw similar conclusions as humans would by continually analyzing data with a given logical structure. To achieve this, deep learning uses a multi-layered structure of algorithms called neural networks. Just as humans use their brains to identify the patterns and classify the different types of information, neural networks can be taught to perform the same data tasks.
Whenever humans receive new information, the brain tries to compare it with known objects. Deep neural networks also use the same concept. By using the neural network, we can group or sort the unlabeled data based on similarities among the samples in the data. Artificial neural networks have unique capabilities that enable deep learning models to solve tasks that machine learning models can never solve.
One of the main advantages of deep learning lies in solving complex problems that require discovering hidden patterns in the data and/or a deep understanding of intricate relationships between a large number of interdependent variables. When there is a lack of domain understanding for feature introspection, Deep Learning techniques outshine others, as you have to worry less about feature engineering. Deep Learning shines when it comes to complex problems such as image classification, natural language processing, and speech recognition.
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