Biochemical Test of Lactobacillus spp.

Lactobacillus spp. is a group of gram-positive, rod-shaped bacteria that belong to the lactic acid bacteria (LAB) family. They are widely distributed in nature and can be found in various habitats, such as the human and animal gastrointestinal tract, oral cavity, vagina, fermented foods, and plant materials. They are also used as probiotics, starter cultures, and bio-preservatives in the food industry.

Lactobacillus spp. have some common characteristics that distinguish them from other bacteria. Some of these are:

• They are facultative anaerobes, meaning they can grow in the presence or absence of oxygen.

• They produce lactic acid as the main end product of carbohydrate fermentation, which lowers the pH of their environment and inhibits the growth of pathogens and spoilage microorganisms.

• They have a complex nutritional requirement and need various vitamins, amino acids, peptides, and minerals for growth.

• They are catalase-negative, meaning they do not produce the enzyme catalase that breaks down hydrogen peroxide into water and oxygen.

• They are non-spore-forming, meaning they do not form resistant structures that can survive harsh conditions.

• They are generally non-motile, meaning they do not have flagella or other structures that enable movement.

Lactobacillus spp. can be classified into different species based on their phenotypic and genotypic characteristics. Some of the commonly used methods for the identification and differentiation of Lactobacillus spp. Are:

• Morphological observation: The shape, size, arrangement, and staining properties of the bacterial cells can be observed under a microscope.

• Biochemical tests: The ability of the bacteria to ferment different carbohydrates, produce gas, hydrolyze certain substrates, and utilize specific compounds can be tested using various media and reagents.

• Molecular methods: The genetic material of the bacteria can be analyzed using techniques such as polymerase chain reaction (PCR), DNA sequencing, DNA hybridization, and ribotyping.

In this article, we will focus on some of the biochemical tests that can be used to identify and characterize Lactobacillus spp. These tests include:

• Fermentation of L. acidophilus

• Enzymatic reactions of Lactobacillus spp.

These tests will help us understand the metabolic capabilities and physiological functions of Lactobacillus spp., as well as their potential applications in food and health.

Lactobacillus spp. are Gram-positive, non-spore-forming, rod-shaped bacteria that belong to the lactic acid bacteria (LAB) group. They are widely distributed in nature and can be found in various habitats, such as dairy products, fermented foods, plants, animal intestines, and mucosal surfaces. They are also part of the normal microbiota of the human gastrointestinal tract, vagina, and oral cavity.

Lactobacillus spp. has several properties that make them beneficial for human health and food production. Some of these properties are:

• Lactic acid production: Lactobacillus spp. can ferment different types of carbohydrates and produce lactic acid as the main end product. Lactic acid lowers the pH of the environment and inhibits the growth of pathogenic and spoilage microorganisms. It also contributes to the flavor, texture, and shelf-life of fermented foods, such as yogurt, cheese, sauerkraut, and kimchi.

• Probiotic effects: Lactobacillus spp. can modulate the immune system and enhance the intestinal barrier function by competing with pathogens for adhesion sites, producing antimicrobial substances, stimulating the production of mucin and immunoglobulins, and influencing the cytokine profile. They can also improve the digestion and absorption of nutrients, prevent diarrhea and constipation, and alleviate lactose intolerance and irritable bowel syndrome.

• Bacteriocin production: Lactobacillus spp. can produce bacteriocins, which are ribosomally synthesized peptides with antimicrobial activity against closely related or unrelated bacteria. Bacteriocins can be used as natural preservatives in food or as alternative therapies for infections caused by antibiotic-resistant bacteria.

• Vitamin synthesis: Lactobacillus spp. can synthesize various vitamins, such as B1, B2, B6, B12, folic acid and K2. These vitamins are essential for human metabolism and can prevent or treat deficiencies.

• Biofilm formation: Lactobacillus spp. can form biofilms on different surfaces, such as teeth, implants, and catheters. Biofilms are complex communities of microorganisms embedded in a matrix of extracellular polymeric substances. Biofilms can protect bacteria from environmental stresses, such as pH changes, dehydration, antibiotics, and host defenses. However, biofilms can also cause dental caries, infections, and biofouling.

These are some of the properties of Lactobacillus spp. that make them important for human health and the food industry. In the next section, we will discuss the fermentation of L. acidophilus, one of the most studied species of Lactobacillus.

L. acidophilus is a homofermentative anaerobic microorganism, meaning it only produces lactic acid as an end product of fermentation; and that it can only ferment hexoses (not pentoses) by way of the EMP pathway (glycolysis). For every glucose molecule that undergoes fermentation in L. acidophilus, the energy yield is two ATPs.

Lactic acid production by L. acidophilus has several benefits for food production and human health. Lactic acid lowers the pH of the food, inhibiting the growth of spoilage and pathogenic bacteria. It also contributes to the taste, flavor, and texture of fermented foods such as yogurt, cheese, and sourdough bread. Moreover, lactic acid can modulate the immune system, improve intestinal health, and prevent infections by competing with harmful microbes for adhesion sites and nutrients.

L. acidophilus can ferment various types of sugars, such as glucose, fructose, galactose, lactose, and sucrose. However, some strains of L. acidophilus may have different preferences and abilities to utilize these sugars. For example, L. acidophilus NCFM can grow faster on fructose than on glucose or lactose, while L. acidophilus LA-5 can grow equally well on all three sugars. Therefore, the choice of substrate may affect the growth rate and lactic acid production of different strains of L. acidophilus.

Lactobacillus spp. are able to produce various enzymes that are involved in different metabolic pathways. Some of these enzymes are:

• Lactase: This enzyme hydrolyzes lactose, the main sugar in milk, into glucose and galactose. Lactase is essential for the growth of Lactobacillus spp. on milk-based media and for the production of lactic acid from lactose. Lactase activity can be detected by using a pH indicator such as phenol red, which turns yellow in acidic conditions. Lactobacillus spp. are usually lactase-positive, meaning that they can ferment lactose and lower the pH of the medium.

• Catalase: This enzyme decomposes hydrogen peroxide into water and oxygen. Hydrogen peroxide is a toxic by-product of aerobic respiration that can damage cellular components. Catalase activity can be detected by adding a drop of hydrogen peroxide to a bacterial colony and observing the formation of bubbles. Lactobacillus spp. are usually catalase-negative, meaning that they do not produce this enzyme and are sensitive to hydrogen peroxide.

• Arginine deiminase: This enzyme converts arginine, an amino acid, into citrulline and ammonia. Arginine deiminase is involved in the production of energy and the regulation of pH in some bacteria. Arginine deiminase activity can be detected by using a colorimetric indicator such as bromocresol purple, which turns yellow in alkaline conditions. Lactobacillus spp. are usually arginine deiminase-positive, meaning that they can hydrolyze arginine and raise the pH of the medium.

• β-galactosidase: This enzyme hydrolyzes β-galactosides, such as lactose and ONPG (o-nitrophenyl-β-D-galactopyranoside), into galactose and another sugar or compound. β-galactosidase is involved in the catabolism of β-galactosides and the regulation of gene expression in some bacteria. β-galactosidase activity can be detected by using a chromogenic substrate such as ONPG, which turns yellow when cleaved by the enzyme. Lactobacillus spp. Vary in their β-galactosidase activity, depending on the species and strain.

These are some of the enzymatic reactions of Lactobacillus spp. that can be used for their identification and characterization. By performing these tests, one can determine the metabolic capabilities and physiological properties of these bacteria.

We are Compiling this Section. Thanks for your understanding.