Category Archives: Bacteriology

Bacteriology, Culture Media

Martin Lewis Agar: Principle, Composition, and Uses

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Martin Lewis Agar is a specialized microbiological culture medium widely used in clinical and research laboratories to isolate and identify pathogenic Neisseria species, such as Neisseria gonorrhoeae (causing gonorrhea) and Neisseria meningitidis (causing meningitis). This selective medium is critical for accurate diagnosis and effective management of these serious bacterial infections, making it an essential tool for microbiology students and professionals.

Historical Context of Martin Lewis Agar

Martin Lewis Agar is an advanced version of Thayer-Martin Agar, developed to improve the isolation of Neisseria gonorrhoeae from clinical specimens with mixed microbial flora. By refining the antibiotic composition and concentrations, Martin Lewis Agar enhances selectivity and recovery rates, making it more effective for isolating both Neisseria gonorrhoeae and Neisseria meningitidis. Understanding its development helps microbiology students appreciate the evolution of selective media in clinical diagnostics.

Principle of Martin Lewis Agar

Martin Lewis Agar operates as a selective medium designed to promote the growth of pathogenic Neisseria species while suppressing unwanted bacteria and fungi. It builds on the foundation of Thayer-Martin Agar with improved antibiotic formulations for better specificity.

How It Works:

  • Selective Inhibition: The medium contains antibiotics such as:
    • Vancomycin: Inhibits Gram-positive bacteria.
    • Colistin: Suppresses Gram-negative bacteria, except Neisseria species.
    • Anisomycin: Prevents fungal growth.
    • Trimethoprim: Inhibits Proteus species.
  • Nutrient-Rich Base: The medium uses chocolate agar enriched with hemoglobin to provide essential nutrients for Neisseria growth.
  • Growth Supplements: IsoVitaleX (or equivalent VX supplement) supplies vital growth factors like glucose, L-cysteine, and NAD to support Neisseria species.

Composition of Martin Lewis Agar

Martin Lewis Agar is a modified Thayer-Martin Agar with increased vancomycin levels to enhance selectivity. Its base is chocolate agar, with a final pH of 7.2 ± 0.2 at 25°C. Below is the detailed composition:

Composition per liter: 

ComponentsAmount
Pancreatic digest of casein7.5 g
Hemoglobin (Hb)10.0 g
Selected meat peptone7.5 g
NaCl (sodium chloride)5 g
K2HPO4 (Dipotassium phosphate)4 g
Corn starch1 g
KH2PO4 (Monopotassium phosphate)1 g
Supplement solution10 ml
VCAT inhibitor10 ml
Agar12 g
Final pH 7.2  ± 0.22 at 25°C

Source: Martin-Lewis agar is available as a prepared medium from BD Diagnostic Systems. 

Composition of Supplement Solution per liter: 

ComponentsAmount
Glucose100 g
L-cysteine HCI25.9 g
L-glutamine10 g
L-cystine1.1 g
Adenine1 g
Nicotinamide adenine dinucleotide0.25 g
Vitamin B120.1 g
Thiamine pyrophosphate0.1 g
Guanine HCl0.03 g
Fe(NO3).6H2O 0.02 g
p-Aminobenzoic acid0.013 g
Thiamine HCl3.0 mg

Source: The supplement solution IsoVitaleX® enrichment is available from BD Diagnostic Systems. This enrichment may be replaced by supplement VX from BD Diagnostic Systems. 

Composition of VCAT inhibitor per 10.0mL 

ComponentsAmount
Colistin7.5 mg
Trimethoprim lactate5 mg
Vancomycin 4 mg
Anisomycin0.02 g

Preparation of Martin Lewis Agar

  1. Add all components (except supplement solution and VCAT inhibitor) to 980 mL of distilled/deionized water.
  2. Gently heat and stir until boiling.
  3. Autoclave at 121°C (15 psi) for 15 minutes.
  4. Cool to 45–50°C.
  5. Aseptically add sterile supplement solution and VCAT inhibitor, mixing thoroughly.
  6. Pour into sterile Petri dishes.

Storage and Shelf Life

Dehydrated Media: Store in a sealed container in the dark at temperatures below 30°C.

Prepared Media: Refrigerate at 2–8°C and use within 60 days.

Caution: Avoid using media showing signs of deterioration (e.g., shrinking, cracking, or discoloration) or contamination, as this may lead to inaccurate results.

Uses of Martin Lewis Agar

Martin Lewis Agar is primarily used for:

  • Isolating Neisseria gonorrhoeae and Neisseria meningitidis from clinical specimens (e.g., urethral, cervical, or cerebrospinal fluid samples) with mixed microbial flora.
  • Supporting the growth of penicillinase-producing Neisseria strains when enriched with Penicillin G.
  • Facilitating accurate diagnosis of gonorrhea and meningitis in clinical settings.

Its selective antibiotics (vancomycin, colistin, anisomycin, and trimethoprim) effectively suppress Gram-positive bacteria, non-Neisseria Gram-negative bacteria, fungi, and Proteus species, ensuring reliable isolation of target pathogens.

Limitation of Martin Lewis Agar

  1. Nutritional Variability: Some Neisseria strains may exhibit poor growth due to nutritional differences.
  2. Antibiotic Sensitivity: Certain Neisseria gonorrhoeae strains may be inhibited by the antibiotics in the medium.
  3. Need for Non-Selective Media: A parallel non-selective medium (e.g., chocolate agar) is recommended to ensure comprehensive isolation.
  4. Confirmation Required: Additional biochemical or serological tests are necessary to confirm Neisseria species identity.

References

  1. Martin, J. E., Armstrong, J. H., & Smith, P. B. (1974). New system for cultivation of Neisseria gonorrhoeae. Applied microbiology, 27(4), 802–805. https://doi.org/10.1128/am.27.4.802-805.1974 
  2. Granato, P. A., Paepke, J. L., & Weiner, L. B. (1980). Comparison of modified New York City medium with Martin-Lewis Medium for recovery of Neisseria gonorrhoeae from clinical specimens. Journal of clinical microbiology, 12(6), 748–752. https://doi.org/10.1128/jcm.12.6.748-752.1980 
  3. Atlas, R.M., & Snyder, J.W. (2013). Handbook of Media for Clinical and Public Health Microbiology (1st ed.). CRC Press. https://doi.org/10.1201/b15973 
  4. Thayer J. and Martin J.E. Jr., 1966, Public Health Rep., 81:559
Biochemical Tests, Biochemistry

Mixed Acid Fermentation: Types and Products

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Fermentation is an anaerobic process of breaking down molecules like glucose and other carbohydrates. The fermentation process is usually helpful in alcohol production. Bacteria follow different fermentation pathways.

Among them, mixed acid fermentation is a characteristic feature of the family Enterobacteriaceae, especially the genera Citrobacter, Proteus, Shigella, Salmonella, Escherichia, Aeromonas, Yersinia, Vibrio, and some species of Aeromonas. Some anaerobic fungi also follow this pathway.

These microorganisms ferment monosaccharides, disaccharides, polyalcohol, and frequently polysaccharides. The glycolytic pathway of this type of fermentation produces lactic acid, succinic acid, formic acids, acetic acids, and ethanol.

Reactions Involved in Mixed Acid Fermentation

In mixed acid fermentation reactions, two stages are present. The first stage of mixed acid fermentation is glycolysis, converting glucose to pyruvate. Here two NADH molecules are produced.

The second stage of mixed acid fermentation follows the following reactions the conversion of the pyruvate produced after glycolysis to one or more end products. The two NADH molecules produced in the first stage are reoxidized to NAD+. The reactions in the second stage are discussed below.

Mixed acid fermentation in E coli

Lactate Production

The enzyme lactate dehydrogenase catalyzes the formation of lactate/lactic acid. Here, glycolysis generates two molecules of pyruvate. Each molecule converts to lactate in the presence of a NADH+H+ molecule.

The overall reaction of lactate production is as follows:

Pyruvate → Lactate; in presence of lactate dehydrogenase and NADH+H+, which converts to NAD+

Acetate Production

In this reaction, pyruvate converts to acetyl CoA with the enzyme pyruvate dehydrogenase and NADH catalysts. The acetyl CoA now converts to acetate, which produces ATP by substrate-level phosphorylation. The conversion of acetyl CoA to acetate is a two-step process requiring two separate enzymes; phosphate acetyltransferase and acetate kinase.

The overall reaction of this reaction is as follows;

  1. Pyruvate → Acetyl CoA in the presence of pyruvate dehydrogenase
  2. Acetyl CoA + Phosphate → Acetyl phosphate + CoA in the presence of phosphate acetyltransferase.
  3. Acetyl phosphate + ADP → Acetate + ATP. 

Ethanol Production

The reduction of Acetyl CoA with the help of NADH forms the third end product of mixed acid fermentation; ethanol. This ethanol production is a two-step reaction and requires the enzyme alcohol dehydrogenase.

The overall reaction of this step of fermentation is;

  1. Acetyl CoA + NADH +H+ → Acetaldehyde + NAD+ + CoA
  2. Acetaldehyde + NADH + H+ →  Ethanol + NAD+ 

Formate Production

The cleavage of pyruvate helps in the production of Formate. The enzyme pyruvate-formate-lyase catalyzes this production reaction. The enzyme pyruvate-formate-lyase also plays an essential role in regulating anaerobic fermentation in Enterobacteriaceae.

The overall reaction of the formate production is as follows;

Pyruvate + CoA  → Acetyl CoA + Formate; catalyzed by pyruvate-formate-lyase. 

Succinate Production

The production of succinate is a multi-step process. The glycolytic pathway intermediate, phosphoenol pyruvate, is the first substrate for carboxylation to form oxaloacetate, with the enzyme phosphoenol pyruvate carboxylase catalyzing this step.

In the second step, the oxaloacetate converts to malate in the presence of malate dehydrogenase. The third step is the formation of fumarate from the dehydration of malate in the fact of fumarate hydratase.

Phosphoenol pyruvate + HCO3 → Oxaloacetate +Phosphate

Oxaloacetate + NADH + H+ → Malate + NAD+ 

Malate → Fumarate + H2O

The final step is succinate production by reducing formate catalyzed by the enzyme fumarate reductase.

Fumarate + NADH + NAD+ →  Succinate + NAD+

The reduction is the anaerobic respiration reaction that uses electrons in NADH dehydrogenase and the electron transport chain. ATP is produced using electrochemical balance and ATP synthetase—this step of fermentation produces ATP, not through substrate-level phosphorylation.

Hydrogen and Carbon Dioxide Production

The enzyme formate hydrogen lyase catalyzes the conversion of formate to hydrogen carbon dioxide gas. The production of these gases helps in preventing acidic condition inside the cells.

End Products of Mixed Acid Fermentation

The end products of mixed acid fermentation are as follows:

  1. Lactate
  2. Acetate
  3. Formate
  4. Succinate
  5. Ethanol

The concentration of these end products of fermentation vary depending on the environment and microorganism but it may be at the 4:1 ratio of neutral to acid products. Hydrogen and carbon dioxide is formed in bacteria with formate hydrogen lyase complex.

Application of Mixed Acid Fermentation

Mixed Acid fermentation is applicable in various fields of science, especially for producing many helpful end products. The applications of mixed acid fermentation are as follows:

  1. The use of single bacteria can help produce various products in biotechnology and the food industry.
  2. Likewise, many different strains of bacteria have been metabolically engineered in the laboratory to increase the yield of the specific end product.
  3. This fermentation method is applied to identify bacteria in the laboratory. Methyl red test is standard for detecting the bacteria following the mixed acid fermentation reaction. Here, the test solution turns red if the pH drops below 4.4 in the presence of those microorganisms that follows the mixed acid fermentation pathway.

References

  1. Thakker, C., Martínez, I., San, K. Y., & Bennett, G. N. (2012). Succinate production in Escherichia coli. Biotechnology journal, 7(2), 213–224. https://doi.org/10.1002/biot.201100061
  2. Vuoristo, K. S., Mars, A. E., Sangra, J. V., Springer, J., Eggink, G., Sanders, J. P., & Weusthuis, R. A. (2015). Metabolic engineering of the mixed-acid fermentation pathway of Escherichia coli for anaerobic production of glutamate and itaconate. AMB Express, 5(1), 61. https://doi.org/10.1186/s13568-015-0147-y
  3. Böck, A. (2009). Fermentation. Encyclopedia of Microbiology, 132–144. https://doi.org/10.1016/b978-012373944-5.00074-2
Bacteriology, General Microbiology, Mycology

Extremophiles: Their Types and Applications

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Prokaryotic life has existed since the beginning of evolution. Their ability to adapt and mutate as per the environmental need is commendable. Some can tolerate the extreme environmental conditions whereas most need these conditions for growth. Thermophiles, acidophiles, and barophiles are some of the terms that denote organisms that endure the harsh environment, which are collectively termed extremophiles.   

Extreme conditions found in the Earth

So, extremophiles thrive in extreme environments typically considered hostile to life. These environments include extreme temperatures, high or low pH levels, high salinity, high pressure, and even high radiation levels. The discovery of extremophiles has expanded our understanding of the limits of life and the conditions under which life can exist.

Extremophiles have high use in different fields because of their unique ability. From enzyme production to astrobiological studies, their applications to benefit human life is increasing significantly.   

Different Types of Extremophiles

As per their abilities to tolerate and thrive on different extreme environmental conditions, extremophiles are of various types. Acidophiles, thermophiles, barophiles, alkaliphiles, psychrophiles, halophiles, and radio-resistant organisms are some of the extremophiles. 

Thermophiles

Thermophiles in Yellowstone national park

Thermophiles are the organisms that thrive in extremely high temperatures, i.e., above 60°C (140°F) and up to around 122°C (252°F). These are present in hydrothermal vents, hot springs, and geothermal areas. Gases, minerals, and metals in the regions are the only sources of nourishment for the organism. 

Thermoplasma acidophilus, Bacillus stearothermophilus, and Thermus aquaticus are examples of thermophiles. However, the thermophile that can thrive in temperatures as high as 121.11°C or 250°F is Methanopyrus kandleri.

Thermophiles are of different types; simple, extreme, and hyperthermophiles. Simple thermophiles tolerate temperatures from 50-64°C or 122-147.2°F. Extreme thermophiles tolerate or need high temperatures for survival, i.e., 65-79°C or 149-174.2°F. Hyperthermophiles can tolerate temperatures as high as 80°C or 176°F but not below 50°C.

Psychrophiles

Psychrophiles or Cryophiles are the organisms that love or require freezing temperatures, i.e., -20°C to +20°C. These organisms have 15°C or lower optimal growth temperature, with maximum growth temperature at about 20°C and minimum temperature at 0°C or lower. These are found in polar regions, deep-sea environments, and glaciers. Vibrio marinus and V psychroerythrus are the first true psychrophiles.

Here, Colwellia psychrerythraea isolated from Artic marine sediments was the first sequenced genome of psychrophile. The Artic permafrost bacteria, Planococcus halocryophilus, grows at the lowest growth temperature, i.e., -15°C with 50 days generation time. True psychrophiles that grow under sub-freezing temperatures have a longer generation time; Psychromonas ingraham grows after ten days at -12°C, and Psychrobacter arcticus grows at -10°C after 39 days of generation time. The only and first archaeon isolated is Methanogenium frigidum from Ace Lake Antarctic.

Acidophiles

The organisms can thrive in highly acidic conditions, i.e., pH levels below 3. These grow in acid mine drainage, volcanic springs as well as acidic soils. Acidobacterium, Picrophilus, Ferroplasma, and Leptospirillum also grow in these acidic sites.

Acidithiobacillus thiooxidans isolated from soil-rock-sulfur composts was the first reported acidophile. Many acidophiles are metal resistant and can generate ATP or energy from metals like ferrous iron.   

Most extreme acidophiles belong to the archeal group Acidianus, Metallosphaera, Pyrococcus, Desulfurococcus, Sulfurisphaera, Sulfolobus, Picrophilus, Stygiolobus, and Thermoplasma.

Alkaliphiles

The organisms that adapt to highly alkaline environments, with pH above 9, are called alkaliphiles. These are found in soda lakes, alkaline lakes, and alkaline solids. 

The aerobic alkaliphilic microorganisms include Bacillus, Micrococcus, Pseudomonas, Streptomyces, Bogoriella, Halomonas, Alkalibacillus, yeasts, and filamentous fungi isolated from various environments. 

Vagococcus, Marinobacter, Alkalimonas, Paenibacillus, Rhodobaca, Dietzia, and Reseinatrobacter are isolated from the alkaline Lonar Lake of India. Whereas Alkaliphilic actinomycete, Bogoriella caseilytica was reported from a Soda lake in Africa. Likewise, Dietzia natronolimnaios was reported from an East African Soda Lake.

Other organisms include cyanobacteria like Spirulina platensis, Spirulina maxima, and Chorococcus species and anoxygenic phototrophic bacteria like Halorhodospira and Ectothiorhodospira.   

Halophiles

The organisms which require high saline (salty) environments like salt lakes, salt flats, and pans are called halophiles. Haloarchaea class and Nanohaloarchaeota subphylum are the only archaea that show halophilism. According to their requirements, halophiles have three categories; slight (0.34-0.85 M salt), moderate (0.85-3.4M), and extreme halophiles (3.4-5.1M salt).  

Halophiles dominate the most hypersaline environments on Earth, with many surviving salt concentrations close to saturation levels. Eubacteria are mostly halotolerant in nature, which means these bacteria do not rely on salt to thrive but can tolerate some salt concentrations. 

Barophiles

The organisms that can adapt and thrive in high-pressure environments are barophiles or piezophiles. These are found in areas like the deep sea where the pressure is several hundred times higher than Earth’s surface, i.e., above 380 atm. Halophiles cannot survive without high pressure, also called obligate barophiles. Halomonas Salaria, Gram-negative proteobacteria, is an example of obligate halophiles that requires 1000 atm pressure. 

The barophiles are sensitive to ultraviolet rays and susceptible to UV radiation, because of which many of the barophiles grow in the dark. Other examples of barophiles include xenophyophores, found in the deepest ocean trench. 

Radio-resistant Organisms

The organisms capable of thriving in high radiation levels, like ionizing radiation, are called radio-resistant organisms. These grow near nuclear reactors and waste storage sites in radioactive environments. 

Deinococcus radiodurans is a bacterial species highly resistant to radiation levels up to 1.5✕105 rad (rad=radiation absorbed dose) of acute ionizing radiation but only 6000 rad/hour of chronic radiation. The bacteria grow in radiation-contaminated areas like deserts, oceans, and seas. This bacteria is highly resistant to hypertonic stress, oxidation as well as desiccation. 

In addition to the Deinococcus species nitrogen-fixing cyanobacterium Anabaena species, Micrococcus, Bacillus, and Actinobacteria are known radio-resistant bacteria. 

Polyextremophiles

Those organisms that thrive in more than one extreme condition are called polyextremophiles. For example, most barophiles are psychrophilic,i.e., requiring low temperature for growth as well as high pressure. 

Likewise, bacteria like Clostridium paradoxum are haloalkaliphilic moderately thermophilic organisms; Deinococcus radiodurans are radio-resistant, acidophiles, and psychrophiles. 

Role of Extremophiles in Human Well-being

The study of extremophiles is significant in different fields like biotechnology, environmental science, and astrobiology. Their study has surely helped us understand the potential for life in various extreme environments on Earth. The roles of extremophiles in human well-being are as follows:

  1. Drug discovery: The pharmaceutical application of extremophiles depends on the unique compounds these organisms produce for living in extreme environments. The screening and study of the medicinal values of these compounds help in the discovery of new drugs in humans and also in veterinary treatment.
  2. Environmental applications: The demand for cleaning the Earth without any ramifications to its natural sources has exponentially risen in the past few years. The enzymes produced by extremophiles are significantly useful in bioremediation, which cleans up contaminated environments using living organisms. Some extremophiles can also degrade toxic pollutants like metal.
  3. Enzyme production: Extremophiles also produce some enzymes that can function in an extreme environment. The other term for these enzymes are extremoenzymes. These are highly stable and active even in high temperatures, high and low pH levels and are helpful in industries.
  4. Astrobiology: The study of extremophiles has given insights into the limit of life on Earth and the existence of life anywhere else in the universe. Research on extremophiles will surely guide the search for extraterrestrial life by designing an instrument that detects energy. The study will also help develop technologies and strategies for space exploration and habitation.   
  5. Industrial process: The enzymes produced by extremophiles are useful in biofuel like ethanol. The extremophiles also have applications in producing textiles, chemicals, and other materials. These help in obtaining more sustainable and efficient alternatives to conventional methods.    
  6. Agricultural and food production: Another sector that can benefit from extremophiles is agriculture and food production. The issues of decreased agricultural productivity due to various environmental and human factors and risk food security is at rise. The extremophiles can contribute to developing crop varieties resistant to extreme environmental conditions like drought or salinity. 
  7. Biotechnological application: Biotechnological application of extremophiles includes methods like PCR, where amplification of DNA using Taq polymerase from thermophiles occurs, organisms used in mining under extreme conditions, and the production of carotenoids for cosmetic and food industries. 

References

  • Moyer, C.L., Eric Collins, R. and Morita, R.Y. (2017) ‘Psychrophiles and Psychrotrophs’, in Reference module in Life Sciences. Elsevier, pp. 298–303. 
  • Merino, N. et al. (2019) ‘Living at the extremes: Extremophiles and the limits of life in a planetary context’, Frontiers in Microbiology, 10. doi:10.3389/fmicb.2019.00780.  
  • Scoma, A. et al. (2019) ‘The polyextremophilic bacterium clostridium paradoxum attains piezophilic traits by modulating its energy metabolism and cell membrane composition’, Applied and Environmental Microbiology, 85(15). doi:10.1128/aem.00802-19. 
  • Kanekar, P.P., Kanekar, S.P. (2022). Radiophilic, Radioresistant, and Radiotolerant Microorganisms. In: Diversity and Biotechnology of Extremophilic Microorganisms from India. Microorganisms for Sustainability. Springer, Singapore. https://doi.org/10.1007/978-981-19-1573-4_8
Biochemical Tests, General Microbiology

Biuret Test: Principle, Procedure, and Uses

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Protein analysis is done for various reasons; for example, in clinical laboratories, it is used for determining disease by analyzing serum proteins. The biuret test is one of the methods of protein analysis.

The biuret test is a colorimetric test that helps detect specific proteins or peptide bonds in given analytes. It is followed by spectrophotometry for quantification. 

The test requires the use of a biuret reagent. This reagent is a solution that consists of hydrated copper (II) sulfate, sodium hydroxide, and potassium sodium tartrate.

The use of copper (II) ions present in the biuret reagent results in the formation of purple coloration if peptides are present. The intensity of the purple color is measured using a spectrophotometer.

Principle of Biuret Test

Biuret test requires testing the analytes with biuret reagents. The reagent is a mixture of potassium sodium tartrate (KNaC4H4O6 or C4H4KNaO6), copper (II) sulfate or cupric sulfate (CuSO4), and sodium hydroxide (NaOH).

Sodium hydroxide makes the solution alkaline, and potassium sodium tartrate is the chelating agent. The potassium sodium tartrate helps stabilize the cupric ions in the mixture and maintains the alkaline solution’s solubility.

The four nitrogen atoms present in the protein peptides bind to the reagent’s copper (II), resulting in a change of cupric ions to cuprous ions and displacement of peptide hydrogen under alkaline conditions.

Nitrogen binding to cupric ions also results in donating the lone pairs of an electron from nitrogen to the copper ions to form coordinated covalent bonds. The coordinate covalent bond with cupric ions forms a chelate complex that absorbs light with a wavelength of 540 nm, which imparts purple color. Hence, the formation of purple color indicates the presence of proteins in the analyte.

The test depends on the peptide bonds instead of the presence of amino acids in the sample, so it can help measure the protein concentration in whole tissue samples. Proteins purified using ammonium sulfate ((NH4)2SO4) may give false positive results due to nitrogen in ammonia.

Reagents and Materials Required

The reagents and materials required for the biuret test are as follows:

  1. The reagent required for performing a the test is biuret reagent. The biuret reagent is prepared by mixing 1% solution of CuSO4 (1 gm CuSO4 in 100 ml water) and 1.2 grams of potassium sodium tartrate. 10 ml 10% solution of NaOH (10 gm NaOH in 100 ml water) is added to the above mixture, known as a biuret reagent.  The solution is blue in color due to the presence of the CuSO4.
  2. Other equipment required for the test are test tubes, a dropper or a pipette, a test tube holder, and a stand.

Procedure of Biuret Test

The following steps are followed to perform the biuret test:

  1. Take three clean and dry test tubes.
  2. In the first tube, add 1-2 ml test sample. Likewise, add 1-2 ml of egg albumin in the second one; in the third tube, add 1-2 ml of distilled water. The egg albumin is a positive control, whereas distilled water is a negative control for this test.
  3. Then, add 1-2 ml biuret reagent in all three tubes.
  4. After that, properly shake all the tubes to mix the reagent and samples/analytes. Then, let the mixture in the tubes stand for at least 5 minutes.
  5. Finally, observe the color change.

Precautionary measures 

  1. Use test tube holders when holding the tubes with a solution.
  2. When preparing biuret reagent, handle NaOH carefully as it is a strong base that might cause corrosion when exposed to the skin.

Result Interpretation

The results of the biuret test are interpreted as follows:

Observation Interpretation
1. The color of the first solution changes to purple.
2. The color of the first solution changes to pink .
3. No change in the color of the first solution.		 1. Presence of proteins. (Positive biuret test)
2. Presence of peptides (Positive biuret test)
3. Absence of proteins or peptides (Negative biuret test)
The color of the second tube changed to purple. Postive biuret test (Positive control)
No change was seen in the color of the solution in the third tube.. Negative biuret test ( Negative control)

Uses of Biuret Test

The biuret test is used mainly for diagnostic purposes, like determining serum proteins. Other applications of this test are as follows:

  1. The test helps in determining the type of proteins in unknown samples.
  2. It is used for quantification of protein by using a spectrophotometer alongside.
  3. It can help determine proteins in the urine, CSF, and other body fluids.
  4. The test helps determine the presence of specific proteins during food analysis.

Advantages of Biuret Test

The advantages of the biuret test are as follows:

  1. The test is simple and inexpensive.
  2. It can detect nitrogen from only peptide bonds.
  3. Very few components interfere with the test.
  4. The color is stable, so it causes less deviation.
  5. It is also a rapid test.
  6. It can detect proteins with at least four peptide bonds.

Disadvantages of Biuret Test

  1. The presence of amino acid histidine can give false positive results because there is a presence of nitrogen.
  2. If the buffer used to purify proteins has ammonium and magnesium salts, it can hinder the test.
  3. The presence of carbohydrates and fats can also hinder the test.
  4. This test alone cannot help in quantifying the protein in the sample; spectrophotometric analysis is required for quantification.
  5. Its sensitivity is lower than the Folin Lowry test.
  6. Only soluble proteins are helpful in this test, and different proteins give different colors, so standardization of colors is required for known proteins.

References

  1. A. Bianchi-Bosisio, PROTEINS | Physiological Samples, Editor(s): Paul Worsfold, Alan Townshend, Colin Poole, Encyclopedia of Analytical Science (Second Edition), Elsevier, 2005, Pages 357-375, ISBN 9780123693976, https://doi.org/10.1016/B0-12-369397-7/00494-5. (https://www.sciencedirect.com/science/article/pii/B0123693977004945)
  2. Mohanlal Sukhadia University. (n.d.). Biuret test – Mohanlal Sukhadia University. https://www.mlsu.ac.in/econtents/2207_Biuret%20test.pdf.
Biochemical Tests, General Microbiology

Lipid Hydrolysis Test: Principle, Procedure, and Result

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Lipid hydrolysis or lipase test helps determine the ability of bacteria to produce lipase and hydrolyze lipids. Lipids are fatty, waxy, and oily bio-compounds. These include fats and triglycerides.  

Some bacteria catabolizes lipid, especially in high-fat dairy product, to generate energy (ATP). These bacteria may also use lipids for synthesizing various biomolecules by entering different pathways.

Since lipids are complex fatty acids, they require further degradation before bacteria metabolize them. The extracellular enzyme lipase produced by bacteria helps in the hydrolysis of lipids.  

The bacteria that can produce lipase belong to Enterobacteriaceae, Clostridium, Staphylococcus, and Neisseria. Many fungal species also demonstrate lipolytic ability. 

Principle of Lipid Hydrolysis Test

Lipid is complex biological compounds that include fats and oils. The hydrolysis of fats occurs in the presence of the enzyme lipases. Some bacteria can produce this enzyme, whereas others cannot. 

Although many oils can be used for lipid hydrolysis tests, tributyrin oil is the most commonly used lipid for determining the lipolytic effects of the bacteria. The oil is simple fat or triglycerides or triacylglycerols.   

Triglycerides comprise glycerol and three long-chain fatty acids. Although tributyrin is simple fat, it is large and cannot enter the cell. Some cells can secrete the enzyme lipase to break down tributyrin before cellular uptake.

The breaking down of tributyrin releases glycerol converted into dihydroxyacetone phosphate, an intermediate product of glycolysis. Beta-oxidation catabolizes the fatty acids; two carbons combine with Coenzyme A to form acetyl Coenzyme A.

The acetyl CoA enters into the Krebs cycle to produce energy. Glycerol and fatty acids are useful in both anabolic and catabolic pathways alternatively. Tributyrin agar is opaque, and when the plate is inoculated with lipase-positive organisms, clear zones appear around the growth. The absence of the clear indicates the organism does not produce lipase. 

The testing lipid hydrolysis can also occur in spirit blue agar. The composition of the agar is an emulsion with tributyrin oil and spirit blue dye as a color indicator. The media is opaque and light blue. Lipase-positive bacteria produce clear halos around the growth. However, the lightening of the medium is not considered positive growth.          

Materials Required for Lipid Hydrolysis Test

A lipid hydrolysis test requires some laboratory equipment and materials. The equipment and materials needed for the test are as follows: 

  1. Tributyrin agar base: It has peptone, yeast extract, and agar. The pH of the base is 7.5 ± 2 at 25℃. 
  2. Tributyrin: It is also known as glycerol tributyrate or propane-1,2,3-triyl tributyrate. Its molecular formula is C15H26O6.   
  3. Petri plates: The use of Petri plates is for preparing culture media or tributyrin agar.  
  4. Test tubes: The test tubes are used for preparing bacterial inoculum.    
  5. Autoclave: This equipment is used for sterilizing tributyrin agar during preparation.  
  6. Incubator: Tributyrin plate inoculated with bacterial isolates is incubated during the test.  
  7. Beaker: The glassware is used to transfer liquid during the tributyrin agar preparation. 
  8. Inoculating loop: The inoculating loop of standard size is used for streaking bacterial inoculum in the tributyrin agar.  
  9. Bunsen burner: It is used to sterilize inoculating loops and preparing tributyrin agar. 
  10. Analytical balance: It is used for weighing powder for preparing tributyrin agar. 
  11. Conical flask: It is used for preparing tributyrin agar. 

Composition of Tributyrin Agar Base

CompositionAmount
Peptone5.00 g
Yeast Extract3.00 g
Agar15.00 g
Final pH7.5±2 at 25°C

Procedure of Lipid Hydrolysis Test

The lipid hydrolysis test involves streaking bacterial inoculum in tributyrin agar. The clear zone around the growth indicates a positive for lipase production.  

Preparation of Tributyrin Agar

Before conducting a lipid hydrolysis test, preparing tributyrin agar is an important step. The steps for preparing tributyrin agar depend on the manufacturing company. The steps for the preparation of tributyrin agar manufactured by Himedia are as follows:

  1. In 990 ml of distilled water, mix 23 grams of tributyrin agar base. 
  2. After that, add 10 ml of tributyrin to the mixture above.
  3. Then, boil the solution to mix the base and tributyrin thoroughly.
  4. Sterilize the solution by autoclaving at 121℃ or 15 lbs pressure for 15 minutes. 
  5. Cool the solution to 40-45℃.
  6. After that, pour the solution into sterile Petri plates.  

Steps of Lipid Hydrolysis Test

Above: Positive for lipase test (presence of clear zone around the growth).
Below: Negative lipase test (absence of clear zone around the growth).

Following are the steps for performing a lipid hydrolysis test:

  1. In a tributyrin agar, inoculate a loopful of freshly isolated bacterial samples in a straight streak line or circle the size of a dime using a sterile inoculation loop.
  2. Incubate the streaked plate at 37℃ for 24-48 hours for aerobic bacteria and 72 hours for anaerobic bacteria.
  3. After incubation, observe the plate for a clear zone and interpret the result.

Precautions

There are some precautionary steps to follow during performing lipid hydrolysis tests; they are as follows:

  1. While sterilizing the inoculation loop, heating should be done until the loop turns red hot. 
  2. Cooling down to the right temperature is necessary before picking up the bacterial colony.
  3. Maintaining proper incubation time is required. At least incubate for four days for anaerobic bacteria. 

Result Interpretation

The lipid hydrolysis test’s result interpretation is based on the presence or absence of the clear zone around the bacterial growth.

  1. Positive result: There is a clear zone around the inoculation area. Staphylococcus aureus, S saprophyticus, Clostridium botulinum, Bacillus subtilis, Pseudomonas aeruginosa, etc., are bacteria that show positive results. 
  2. Negative result: There is no clear zone around the inoculation area. Clostridium perfringens, C difficile, Escherichia coli, Klebsiella pneumoniae, K oxytoca, etc, are bacteria that shows negative result.   

Quality Control

The quality control bacteria used for this test are as follows:

  1. Positive control: Staphylococcus aureus ATCC 12600 is used as the positive control.
  2. Negative control: Clostridium perfringens ATCC 1292 is used as the negative control.

Uses of Lipid Hydrolysis Test

The lipid hydrolysis test is a biochemical test to determine the lipolytic properties of some bacteria. It helps identify the bacteria. Its use is as follows:

  1. Differentiating lipolytic bacteria from non-lipolytic bacteria, especially in dairy industries. 
  2. Identification of bacteria of the genus Clostridium, Corynebacterium, Bacillus, and Moraxella. 

Limitation of Lipid Hydrolysis Test

The lipid hydrolysis test is a practical test for identifying lipolytic bacteria. However, it also has some limitations, which are as follows:

  1. The time for incubation is longer compared to another biochemical test.
  2. The lipid hydrolysis test is not confirmatory. Further biochemical testing is required to confirm the bacteria.
  3. The growth of fastidious bacteria is not possible. 

References

  1. Leboffe, M.J. and Pierce, B.E. (2011) “Differential media,” in A Photographic Atlas for the 4th edition microbiology laboratory. 4th edn. Englewood, CO: Morton Pub. Co., pp. 77–78. 
  2. Madigan, M.T., Brock, T.D. and Stahl, D.A. (2011) Brock Biology of Microorganisms. 13th edn. Boston: Pearson/Benjamin Cummings. 
  3. Tributyrin Agar Base W/O Tributyrin (no date) HiMedia Leading BioSciences Company. HiMedia. Available at: https://www.himedialabs.com/us/m157-tributyrin-agar-base-wo-tributyrin.html
Bacteriology

Fermented Foods: Types, Examples, and Health Benefits

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Fermentation is derived from the Latin verb fevere, which means to boil. It is an ancient method to preserve food and transform food materials by utilizing the metabolic activity of microorganisms. (1)

Fermented foods may be defined as the foods or beverages produced through the activities of microorganisms that influence the component of food in terms of organoleptic properties (flavor, texture, and color). Fermented food products are produced worldwide as they have health benefits and can be preserved for an extended period.

Advantages of Fermented foods

Traditionally, the foods were fermented to prevent spoilage and preserve for an extended period. Some of the few advantages of fermenting foods are given below.

  1. It preserves the foods and is economical; for example, cheese prepared from milk can be preserved for longer than the source.
  2. It also removes the unwanted or harmful properties of raw materials.
  3. The presence of microorganisms enhances the nutritional content of food.
  4. In comparison to raw materials, the organoleptic properties of fermented foods are improved due to fermentation.

Traditional Fermented Foods

Fermented foods can be produced from raw materials such as milk, soybean, cabbage, fruits, etc. Other organisms, such as Lactobacillus, ferment these raw materials. Some of the foods produced from these raw materials are mentioned below:

Fermented Milk Products

Milk is used as raw materials for producing foods such as yogurt, curd, kefir, cheese, probiotic drinks, etc.

Yogurt: Yogurt is a popular dairy product fermented by two bacterial species, Streptococcus thermophilus, and Lactobacillus bulgaricus, from milk. In addition, a few other organisms act as starter cultures: Bifidobacterium spp., Lactobacillus delbrueckii, Lactobacillus casei, and Lactococcus lactis, among others. These bacteria ferment milk and produce lactic acid, which drops the milk pH from 7 to 4-5, which coagulates it. Produced lactic acid causes the sour taste of yogurt and inhibits the growth of spoilage bacteria.

Yogurt

Fermented Food from Soybeans

Soybean is a nutritional food rich in protein (42%), oil (18%), and low carbohydrates (17%). The bean consists of phospholipids, nucleic acids, and vitamins such as thiamine, riboflavin, and niacin. (2) Even though it is nutritious, some compounds cannot be digested, which, after fermentation, are reduced to a lower molecular weight and are made digestible. Some fermented soybean products include Soy sauce, Miso, Tempeh, Natto, etc.

Soy sauce

It is famous in Japan and is known as shoyu, a deep red-brown liquid having a salty taste and distinct aroma. Its primary raw material is soybean, and other ingredients are wheat, salt, and a mixture of molds, bacteria, and yeasts. (3)

Natto

It is originated in Japan more than 1,000 years ago. It is a fermented whole soybean of two types: Hama-natto, fermented by the action of Aspergillus, and Itohiki-natto, fermented by Bacillus natto (3).

Natto

Likewise, Tempeh is popular in Indonesia and is eaten as a critical protein source. Several species of Rhizopus are used to ferment soybean, mainly Rhizopus oligosporous Saito. (3)

Tempeh

Miso

Miso is a fermented soybean paste that is popular in Japan. Soybean is mainly used as a substrate for miso production; other legumes such as chickpeas or novel bases like nuts may also be used. Traditionally, it used to be prepared by fermentation soybeans with Koji (produced from Aspergillus oryzae). (4) Organisms such as Bacillus subtilis, Bacillus amyloliquefaciens, Staphylococcus gallinarum, and Staphylococcus kloosii are used as a starter culture for the fermentation of soybeans to produce Miso.(5) For Miso production, two steps of fermentation are carried out. In the first fermentation, barley or rice is used as a raw material, and fermentation is carried out by Aspergillus oryzae forming a product known as Koji. Thus, the Koji is mixed with cooked soybean, salt, pure yeasts, and lactic acid bacteria, and the second fermentation is carried out. After this process, it is aged for years and packaged as Miso.

Miso

Fermented Food from Vegetables

Vegetables such as cabbages, olives, cucumber, onions, peppers, green tomatoes, carrots, okra, celery, and cauliflower can be fermented and traditionally have been used as a means to preserve. Some fermented vegetables are kimchi, sauerkraut, olives, pickles, and mustard pickles.

Kimchi

Kimchi is a fermented vegetable dish that originated in Korea. The main ingredients are Chinese cabbage or radishes and flavoring ingredients such as chili, pepper, garlic, onion, ginger, and seasonings such as salt, soybean sauce, sesame seed, and other additional foods such as carrot, apple, pear, and shrimp. All these ingredients are mixed, and the fermentation occurs naturally by microorganisms in the cabbage or other elements. Although fermentation occurs naturally, for the commercial production of kimchi, different bacteria are added as a starter culture, including Leuconostoc, Lactobacillus, Pseudomonas, Pantoea, and Weissella genera. But the concentration of bacteria differs in different stages of fermentation; bacteria belonging to Leuconostoc genera dominate the first three days of fermentation. Since various ingredients are added to kimchi, the composition of microbes may vary accordingly. For example, the concentration of Weissella is high, and Leuconostoc and Lactobacillus are lower after adding red pepper powder. (5)

Kimchi

Sauerkraut

It originated in the 4th century and is a common form of preserving cabbage. It is popular in Germany and is now common in other European, Asian countries, and the United States. The raw material is cabbage and salt (2.3-3.0) % and is fermented spontaneously. Organisms that contribute to fermentation are Leuconostoc spp., Lactobacillus spp., and Pediococcus spp. (5)

Sauerkraut

Cucumber (pickling) is eaten raw after fermentation or pickling. Dry salting and brine salting are widely used. During brine salting, salt is added in 5-8% amounts. The unwanted bacteria are removed, allowing lactic and yeast to increase during the primary fermentation, lasting 2 to 3 days. The significant organisms for fermentation include Lactobacillus plantarum and L.brevis, followed by Pediococcus.

Fermented Beverages

Fermented beverages are in either alcoholic form or stimulant form. Beer and wines are alcoholic drinks, while tea, coffee, and cocoa are stimulant beverages.

Alcoholic beverages

Alcoholic beverages are famous worldwide and are being produced in different societies. It is mainly based on the conversion of sugar to alcohol by yeasts. The primary organism involved in the fermentation of alcohol is yeasts, and the raw material used in most distilled liquors are honey, sugarcane, palm sap, beetroot, maize, or corn. The yeasts used in mainly brewing belong to Saccharomyces cerevisiae, with each strain having its characteristics, while some yeasts belong to Saccharomyces ellipsoids.

Wine

In the case of wine, historically, the fermentation of grape species called Vitis vinifera produces wine. When a grape is ripe, its moderate acidity is favorable to make wine. Its natural sugar content acts as a raw ferment material and can produce wine with 10% or higher alcohol. It contains tannins that provide flavor and protect wines from spoilage bacteria. Other fruits such as guava, mangoes, pineapple, tangerine, cashew fruit, and many more contain 10-20 % fermentable sugar, which can be used to produce wine. Still, the names of fruits included papaya wine, guava wine, and so on.

Beer

It is a beverage, is obtained by the fermentation of malted cereals. Malted cereal is extracted with water and other carbohydrates. This extract is boiled with hops and later cooled and fermented with yeast. Fermentation of cereal extracts by Saccharomyces is a crucial step involved during brewing. Even though all Saccharomyces strains produce ethanol, the type of strain used determines the production of beers and is classified as lager or ale beers. (6) Lager beer: It is a bottom-fermented beer, where barley is fermented by Saccharomyces carlsbergensis (also called bottom-fermenting yeast). Ale beer: It is a top-fermented beer produced by Saccharomyces cerevisiae (Top-fermenting yeast).

Beer

In addition, there are distilled beverages such as whiskey, rum, brandy, etc. Whiskey, rum, and Brandy are obtained by distillation of aqueous extract of infusion of malted barley or other cereals, the fermented juice of cane syrup or sugarcane derivates, and fermented grape, apple, pear, wine, and so on, respectively.

Stimulant beverages

Stimulant beverages such as tea, coffee, and cocoa are mainly produced in the rainforest zones of the Indian sub-continent, South America, and West Africa, respectively. A few stimulant beverages are discussed below.

In the case of coffee fermentation, it originated in Ethiopia. The microorganisms used in fermentation include spore-forming and non-spore-forming bacteria. In addition, other lactic acid bacteria (Leuconostoc spp and Lactobacillys spp) and yeasts (Saccharomyces spp and Schizosaccharomyces spp.) are also used. Processing of coffee involves two methods: the wet method and the dry method.

While using the wet method, fruits are passed through a pulping machine to remove the pulp, and pectinolytic enzymes produced by microorganisms remove the mucilage. On the other hand, while using the dry method, fruits are dehulled to remove dry outer portions. The coffee may also be dried by exposing it to the sunlight.

Coffee beans

The tea was originated in South-East China and is now popular worldwide. Young tea leaves are handpicked and withered. Leaves are squeezed to extract juices by rolling, which is further spread on the surface of the leaves. Juice consists of polyphenols exposed to oxidation, changing the green color to brown. This is followed by chemical reaction fermentation. The tea is then subjected to hot air between 80 – 90⁰C, sorted, and graded. (3)

Dried tea leaves

Cocoa is native to South America but is now produced in Ghana, Ivory Coast, Cameroon, Malaysia, and Nigeria. The tree grows a pod consisting of 40-60 seeds. The mucilaginous outer covers of sources are broken. They are allowed to ferment by microbial action changing the seed color from pinkish to black, and the aroma is due to the lactic acid bacteria.

Cocoa pods

Health Benefits of Fermented Foods

Fermented foods have a long history of safe use and are accepted widely by the public owing to their favorable health effects. The positive impacts of these have attracted consumers in large numbers.

  1. Fermented food consumption positivity impact mood, brain activity, and gut microbes. Yogurt consumption reduces the risk of cardiovascular disease, type-2 diabetes, and mortality. Intake of kimchi is linked to anti-diabetic and anti-obesity effects. (7)
  2. Fermentation transforms the raw materials allowing the food to be tolerated by consumers. This process improves the digestibility of starch and polypeptides by breaking them down into oligosaccharides and amino acids. For instance, the casein micelle of milk is destabilized by the fermentation of bacteria present in the milk, enhancing the digestibility of milk protein. In addition, the fermentation process causes the catabolism of protein, lipid, and carbohydrate-producing bioactive compounds, which lowers blood pressure and cholesterol, has anti-cancer effects and improves metabolic syndromes and overall immune functions. The dairy products fermented by Lactobacillaceae also produce Vitamins B7, B11, and B12 beneficial for health.
  3. Microorganisms such as Zymomonas, Leuconostoc, Pediococcus, and Streptococcus species and other members of the Lactobacillaceae family produce exopolysaccharides. These organisms have high molecular weight exopolysaccharides from simple sugars found in raw materials. EPS-producing LAB helps in immunomodulation which depending on factors, may be suppressive or stimulatory.
  4. Fermented food modulates the gut microbiome. For example, wine is rich in polyphenols, alters gut microbiota, and lowers total cholesterol and blood pressure. It also inhibits pathogenic bacteria and benefits the beneficial bacteria. (7)

References

  1. Terefe NS. Food Fermentation [Internet]. Reference Module in Food Science. Elsevier; 2016. 1–3 p. Available from: http://dx.doi.org/10.1016/B978-0-08-100596-5.03420-X
  2. Sharma R, Garg P, Kumar P, Bhatia SK, Kulshrestha S. Microbial fermentation and its role in quality improvement of fermented foods. Fermentation. 2020;6(4):1–20.
  3. Okafor N. Modern Industrial Microbiology and Biotechnology [Internet]. Vol. 3, Science Publishers. 2007. 954–955 p. Available from: http://www.socgenmicrobiol.org.uk/pubs/micro_today/book_reviews/MTNOV07/MTN07_12.cfm
  4. Allwood JG, Wakeling LT, Bean DC. Fermentation and the microbial community of Japanese koji and miso: A review. J Food Sci. 2021;86(6):2194–207.
  5. Dimidi E, Cox SR, Rossi M, Whelan K. Fermented foods: Definitions and characteristics, impact on the gut microbiota and effects on gastrointestinal health and disease. Nutrients. 2019;11(8).
  6. Bokulich NA, Bamforth CW. The Microbiology of Malting and Brewing. Microbiol Mol Biol Rev. 2013;77(2):157–72.
  7. Heinen E, Ahnen RT, Slavin J. Fermented Foods and the Gut Microbiome. Nutr Today. 2020;55(4):163–7.
Bacteriology, General Microbiology

Prebiotics: Mechanisms, Sources, and Examples

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Glenn Gibson and Marcel Roberfroid first introduced the concept of prebiotics in 1995. They stated that a “Prebiotic is a nondigestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improves host health.” This was later redefined in 2008 as “a selectively fermented ingredient that results in specific changes in the composition and/or activity of the gastrointestinal microbiota, thus conferring benefits upon host health.”

Prebiotics are included in a human diet containing different types of fruits, vegetables, and legumes, which are a source of plant polysaccharides and are fermented by microbes. The prebiotics food includes broccoli, carrots, Jerusalem, Psyllium seed husk, garlic, onions, asparagus, wheat, oat, rye, barley, tomatoes, berries, root tubers, and root vegetables(sweet potato, potato), and so on. Nowadays, prebiotics are produced commercially as a supplementary diet.

Mechanism of Action of Prebiotics

Human enzymes such as pancreatic and salivary amylase digest few carbohydrates (present in our diet) by breaking glycosidic linkages present in them, while those not digested act as a substrate for bacterial fermentation. These non-digestible substances include oligosaccharides and polysaccharides which are usually called prebiotics and can withstand digestion and absorption in the small intestine. However, these get fermented by the intestinal flora especially Bifidobacteria and Lactobacillus in the large intestine which positively impacts the gut. 

The enzymes produced by bacteria ferment complex polysaccharides and produce secondary metabolites, short-chain fatty acids (SCFAs) such as propionate, butyrate, acetate, and gases. These by-products encourage the activity and proliferation of good native bacteria in the gastrointestinal tract and reduce pathogenic bacteria. They also protect other body parts, such as the central nervous, cardiovascular, and immune systems.

Fermentation of Prebiotics

Criteria for Eligibility as Prebiotic

For the food ingredients to be prebiotic, they need to fulfill the following criteria:

  1. Resistant to hydrolysis and not absorbed in the upper part of the gastrointestinal tract
  2. Selective substrate for one or few beneficial bacteria that is naturally present in the colon.
  3. Beneficial to the host’s health and able to change the flora in order to make it healthier.
  4. Encouraged to thrive or metabolically trigger induce luminal or systemic effects that are beneficial to host health.
  5. For commercially produced, must have a good storage life at room temperatures such as heat, and dehydration.

Prebiotic Examples

In human diets, prebiotics exists in small concentration and includes oligosaccharide carbohydrates as well as non-carbohydrates that meet the above-mentioned criteria. The most widely known prebiotics includes inulin, galactooligosaccharides, and fructooligosaccharides while other include polyphenols, lactitol, xylooligosaccharides, isomaltooligosaccharides are emerging prebiotics. Some of the widely used prebiotics are:

Examples of prebiotics

Galacto-oligosaccharides (GOS)

GOS are composed of two to eight sugar units, two of which are galactose and two disaccharides (containing two galactose units), and one of the sugar units is terminal glucose. The chemical structures vary by branching, chain length, and glycosyl linkages. Food that contains galactooligosaccharides includes lentils, chickpeas, dairy products, beans, and so on. 

Biological activity: It enhances the production of short-chain fatty acids, reduces the count of potentially pathogenic bacteria, promotes normal function of the intestine, lipid and carbohydrate metabolism, and so on. It naturally occurs in breast milk and stimulates the intestinal Bifidobacteria and Lactobacilli in infants. Since it has a positive effect on immune function, it is commercially available and is added to dairy products such as yogurt and infant formulas.  

Inulin

It is a non-digestible water-soluble storage polysaccharide found in many plant-based foods. These are polymers consisting of a linear chain of β-2,1-linked d-fructofuranose molecules terminated at the reducing end by a sucrose-type linkage by a glucose residue. It is difficult for inulin to be absorbed and digested by the human small intestine due to the existence of β-()-D-frutosyl fructose bonds between the fructose unit and the isomeric carbon of inulin, but it can be fermented by the intestinal flora of the human large intestine. Some examples of food that contains inulin include Asparagus, Dandelion root, onions, chicory, burdock, leeks, and so on.  

Helianthus tuberosus  tubers

Biological activity: It increases the apparent calcium and magnesium absorption, relieves constipation, manages blood sugar and blood lipid with diabetes type II in elderly people, suppresses the appetite reduces weight, and enhances gut microbiome such as Bifidiobacteria.

Fructooligosaccharides (FOS)

It is inulin-type fructans, which have low calories and are non-digestible carbohydrates. These are composed of linear chains of fructose units, linked by β-2,1 bond. Food that contains fructooligosaccharides: tomatoes, garlic, onions, wheat, rye, chicory root, seaweed, sugar cane, rice bran, papaya, beetroot peels, and so on. 

Biological activity: It enhances the body’s intestinal flora, lowers the risk of heart disease and certain cancers, relieves constipation, improves lipids in hyperlipidemia, stops the production of putrefactive substances in the gut, and promotes healthier digestion. They have a number of advantageous physiological effects, including less carcinogenicity, improved mineral absorption in the intestine, and decreased levels of triacylglycerols, phospholipids, and serum cholesterol.

Advantages of Prebiotics

Prebiotics has more positive effects on human health. Some of these advantages are mentioned below:

  1. These affect mainly the gastrointestinal tract as well as distant organs such as the central nervous system, immune system, and cardiovascular system, as their degradation forms majorly SCFAs which enter the blood circulation by diffusing through gut enterocytes. 
  2. The fermented product of prebiotics is mostly acids which decreases the pH of the gut from 6.5 to 5.5 that alters the population of gut microbes. Lactobacilli and Bifidobacteria decrease the harmful bacteria. As an example, the colonization of Salmonella in the epithelium is prevented due to the adhesion of mannose to Salmonella.
  3. Inhibits the activities of spoilage bacteria like Clostridium spp., such as an increase in Bifidobacteria in the gut, harmful fermentation products are less likely to occur.
  4. In the intestinal epithelium, butyric acid reduces the growth of lesions such as adenomas and carcinomas in the gut.
  5. Gut microbiota affects the central nervous system activity through the “gut-brain axis”. Some prebiotics, like FOS and GOS, regulates synaptic proteins, neurotransmitters (such d-serine), and brain-derived neurotrophic factors. 

Limitations of Prebiotics

The intake of prebiotics also has a few limitations which are mentioned below:

  1. Prebiotics may negatively impact lipid profiles by generating certain SCFAs, namely acetate. Acetyl-CoA, which is a substrate for the synthesis of fatty acids in hepatocytes, can be produced from acetate. This may explain why blood levels of cholesterol and triglycerides rose following rectal acetate infusion.
  2. Prebiotics are fermented in the colon and have an osmotic effect in the intestinal lumen. They could cause bloating and flatulence.
  3. High doses cause diarrhea and abdominal pain.
  4. Large daily doses have lately been linked to a rise in gastroesophageal reflux.

References 

  1. Davani-Davari, D., Negahdaripour, M., Karimzadeh, I., Seifan, M., Mohkam, M., Masoumi, S. J., … Ghasemi, Y. (2019). Prebiotics: Definition, types, sources, mechanisms, and clinical applications. MDPI, 8(3), 1–27. https://doi.org/10.3390/foods8030092
  2. Gibson, G. R., & Roberfroid, M. B. (1995). Dietary modulation of the human colonic microbiota: Introducing the concept of prebiotics. Journal of Nutrition, 125(6), 1401–1412. https://doi.org/10.1093/jn/125.6.1401
  3. Rahim, M. A., Saeed, F., Khalid, W., Hussain, M., & Anjum, F. M. (2021). Functional and nutraceutical properties of fructo-oligosaccharides derivatives: a review. International Journal of Food Properties, 24(1), 1588–1602. https://doi.org/10.1080/10942912.2021.1986520
  4. You, S., Ma, Y., Yan, B., Pei, W., Wu, Q., Ding, C., & Huang, C. (2022). The promotion mechanism of prebiotics for probiotics: A review. Frontiers in Nutrition, 9(2), 1–22. https://doi.org/10.3389/fnut.2022.1000517                                                                                              
  5. https://atlasbiomed.com/blog/inulin-prebiotic-fiber/
  6. Holscher, H.D. (2017) Dietary fiber and prebiotics and the gastrointestinal microbiota. Gut Microb 8, 172–184                                               
  7. Marteau P, Seksik P. Tolerance of probiotics and prebiotics. J Clin Gastroenterol. 2004 Jul;38(6 Suppl):S67-9. doi: 10.1097/01.mcg.0000128929.37156.a7. PMID: 15220662.