Lactobacillus acidophilus: Classification, Benefits, Uses, and Side Effects of this Probiotic Bacterium

Table of Contents

• Introduction
• Classification
• Habitat
• Morphology
• Cultural Characteristics
• Biochemical Characteristics
• Role of Lactobacillus acidophilus as Biopreservation resource
• Role of Lactobacillus acidophilus in food and dairy industries
• Role of Lactobacillus acidophilus in human health
• Benefits of L. acidophilus probiotics
• Side effects of Lactobacillus acidophilus probiotics
• References

What is Lactobacillus acidophilus?

• Lactobacillus is a taxonomically diverse group of Gram-positive, non-sporing rods that are characterized by producing lactic acid as the sole end product of carbohydrate metabolism.
• Lactobacillus acidophilus is one of the most important species of this group, widely recognized for its extensive use in the production of probiotics with multiple health benefits.
• This bacterium is a natural part of the normal microbiota in humans, colonizing various regions of the body, including the oral cavity, gastrointestinal tract, and female genitourinary tract.
• Apart from the human body, L. acidophilus is also found in plant materials such as silage and in different foodstuffs and agricultural products.
• The species is frequently detected in fermented milk products such as cheese and yogurt, as well as in fermented beverages like wine and cider.
• Its occurrence in food may either contribute to the enhancement of flavors or be associated with spoilage.
• Most members of the Lactobacillus genus are regarded as non-pathogenic, and several, including L. acidophilus, are consumed as probiotics to prevent certain infections.
• Nevertheless, there have been some observed cases of bacteremia and other infections caused by Lactobacillus species, including L. acidophilus.
• A distinctive feature of L. acidophilus is its ability to grow under relatively low pH conditions, with optimal growth occurring at pH 5.5.
• The species name ‘acidophilus’ is derived from the words ‘acidum’ (acid) and ‘philus’ (loving), signifying its ability to thrive in acidic environments.
• The bacterium was first isolated from infant feces in 1900 by Moro, who named it Bacillus acidophilus. Later, in 1970, Hansen and Mocquot reclassified it as Lactobacillus acidophilus.
• Due to the large number of these bacteria commonly found in food, L. acidophilus has traditionally been regarded as a safe organism.
• However, more recently, some cases of lactobacillemia have been documented.
• Infections caused by L. acidophilus are sporadic and represent only about 0.05 to 0.48% of all reported cases of infective endocarditis and bacteremia.
• The species holds significant industrial applications, ranging from its use as probiotics for treating mild conditions to its utilization in the food and beverage industries.

Classification of Lactobacillus acidophilus

• Lactobacillus acidophilus belongs to the family Lactobacillaceae, as determined by phylogenetic analysis of 16S rRNA sequences.
• This family consists of three distinct genera: Lactobacillus, Paralactobacillus, and Pediococcus.
• At present, the genus includes about 96 species and 16 subspecies, and this number is expanding at a rate of approximately six new species per year.
• The initial classification of Lactobacillus species relied on phenotypic and metabolic characteristics, but has since shifted toward molecular methods such as DNA-DNA hybridization, 16S rRNA sequencing, and analysis of genomic GC content.
• Lactobacillus species can be categorized into groups according to carbohydrate metabolism, including:
• Obligately homofermentative species
• Heterofermentative species
• Facultatively heterofermentative species
• The widespread Lactobacillus species are further classified into 14 subgroups, with the L. acidophilus group being one of the most notable.
• L. acidophilus specifically belongs to the L. acidophilus subgroup, which is recognized as one of the most well-defined and deeply branched phylogenetic subgroups of Lactobacillus.
• The classification of members in this subgroup is based on DNA-DNA homology, and their genomic GC content ranges between 32% and 50%.

Category	Classification
Domain	Bacteria
Phylum	Firmicutes
Class	Bacilli
Order	Lactobacillales
Family	Lactobacillaceae
Genus	Lactobacillus
Species	Lactobacillus acidophilus

Habitat of Lactobacillus acidophilus

• Lactobacillus acidophilus is frequently found in habitats that are rich in carbohydrates, which allows it to colonize a variety of natural environments such as plants, the mucosal surfaces of animals, and carbohydrate-rich food products.
• Within the human body, this bacterium is present in high numbers particularly in the mouth, gastrointestinal tract, and vaginal cavity
• In these regions, L. acidophilus plays an important role in maintaining the pH levels, which in turn provides protection against pathogenic organisms that are unable to survive in such acidic conditions.
• Beyond humans, L. acidophilus also colonizes the intestines of other mammals, including pigs, cattle, mice, and rats.
• Its colonization of the vaginal cavity has been shown to reduce colonization by the common vaginal pathogen Candida albicans, which is thought to result from L. acidophilus occupying the surface of the membrane and thereby preventing C. albicans from attaching.
• Another common habitat of this species is food products, especially fermented milk products and fermented beverages.
• In food, the presence of L. acidophilus may be beneficial by contributing to desirable flavors or harmful when it causes spoilage.
• Its frequent detection in milk products is attributed to the availability of lactose, which serves as an important carbohydrate source for its growth.
• L. acidophilus also occurs in silage or hay used as feed for domestic animals, where the bacterium is actively involved in fermenting sugars present in grass to produce silage.
• Additionally, the species has been isolated from manure, where under favorable substrate and temperature conditions, it is capable of doubling its population approximately every 20 minutes.

Morphology of Lactobacillus acidophilus

• The cells of Lactobacillus acidophilus are large, non-sporing rods that stain Gram-positive, although cultures older than 48 hours may appear Gram-negative.
• The length and curvature of the rods vary depending on the age of the culture, the composition of the medium, and oxygen tension, with dimensions ranging between 0.6–0.9 × 1.5–6 µm.
• The cells divide along a single plane, and their tendency to form chains differs among strains, being influenced by factors such as the growth phase and pH of the medium.
• Morphologically, the rods have rounded ends and can occur singly, in pairs, or in short chains.
• Asymmetrical cell division may result in the formation of wrinkled chains, and in rare cases, even ring structures.
• The bacteria are capable of developing a peritrichous flagellum depending on the culture medium and age. This flagellum may be observed during isolation but is often lost after transfer to artificial media.
• In L. acidophilus and other homofermentative lactobacilli, internal granulation is visible when stained with Gram stain or methylene blue.
• The cell wall of L. acidophilus is a typical Gram-positive structure, composed of peptidoglycan of the Lys-D-Asp type.
• Its cell membrane consists of a lipid bilayer with integrated proteins, and the fluidity of the membrane may change depending on environmental conditions.
• The cells contain membrane-bound teichoic acids, although cell wall-bound teichoic acids may be absent in some strains.
• The cytoplasm includes bacterial ribosomes, nucleoids, and large mesosomes.
• Mesosomes are formed by invaginations of the cytoplasmic membrane and are often filled with tubulin.

Cultural Characteristics of Lactobacillus acidophilus

• The nutrient requirement of Lactobacillus species is complex, which makes their isolation media also complex.
• An example of a non-selective medium for Lactobacillus is MRS medium, with a pH of 6.2 to 6.4.
• MRS medium is considered the medium of choice for the luxuriant growth of Lactobacillus from clinical samples.
• Acetate medium (SL) serves as a selective medium for the selective isolation of Lactobacillus.
• The presence of Tween 80 enhances the growth of L. acidophilus, particularly in media with a high content of acetate at pH 5.4.
• On culture media, L. acidophilus does not produce a characteristic odor, but in food it produces numerous volatile compounds that may either cause food spoilage or generate the pleasant aroma of fermented food.
• The optimum temperature range for growth is 30°C to 42°C, with the best growth at 35°C.
• On artificial media, L. acidophilus can grow within a pH range of 5 to 7, with optimum growth at pH 5.5.
• Luxuriant growth occurs under high oxygen tension, as most strains are aerobic. However, some strains, especially those isolated from food, are facultatively anaerobic, showing better growth at reduced oxygen tension and increased CO₂.
• The nutritional requirements of L. acidophilus include calcium pantothenate, folic acid, niacin, and riboflavin.
• In liquid media such as MRS broth, growth occurs throughout the medium, but the cells settle at the bottom once growth ceases.

Cultural Characteristics on Different Media

a. Nutrient Agar (NA)

• Colonies on NA plates are small, convex, smooth, and glistening, with an entire margin.
• Colony size ranges between 2–5 mm, and they appear opaque without pigmentation.
• Some strains, particularly those isolated from food, may produce mucoid colonies due to slime production.

b. MRS Agar

• Colonies on MRS agar are slightly opalescent, appearing light to medium in color.
• Most strains form opaque, convex, smooth, and glistening colonies with an entire margin, while some may produce rough colonies.
• Slight proteolytic activity can occur, visible as media clearing, due to cell wall-bound or cell wall-released proteases.

c. Blood Agar (BA)

• Colonies on blood agar are small to medium, grey-colored colonies that show very weak β-hemolysis.
• The hemolysis caused by L. acidophilus is referred to as bleaching, because while it produces changes resembling β-hemolysis, the stromata of red blood cells remain intact.

Biochemical Characteristics of Lactobacillus acidophilus

S.N.	Biochemical Characteristic	L. acidophilus
1	Capsule	Non-capsulated
2	Shape	Rod
3	Gram Staining	Gram-positive
4	Catalase	Negative (-)
5	Oxidase	Negative (-)
6	Citrate	Negative (-)
7	Methyl Red (MR)	Negative (-)
8	Voges Proskauer (VP)	Positive (+)
9	Oxidative-Fermentative (OF)	Oxidative
10	Coagulase	Negative (-)
11	DNase	Negative (-)
12	Urease	Negative (-)
13	Gas	Negative (-)
14	H2S	Negative (-)
15	Hemolysis	β-hemolytic
16	Motility	Some strains are motile with single flagella
17	Nitrate Reduction	Negative (-)
18	Gelatin Hydrolysis	Negative (-)
19	Pigment Production	Negative (-)
20	Indole	Negative (-)
21	TSIA (Triple Sugar Iron Agar)	Alkali/Alkali (Red/Red)
22	Spore	Non-sporing

S.N.	Substrate	L. acidophilus
1	Amygdalin	Positive (+)
2	Arabinose	Positive (+)
3	Cellobiose	Positive (+)
4	Dulcitol	Negative (-)
5	Fructose	Positive (+)
6	Galactose	Positive (+)
7	Glucose	Positive (+), obligately homofermentative
8	Glycerol	Positive (+)
9	Glycogen	Positive (+)
10	Hippurate	Negative (-)
11	Inulin	Negative (-)
12	Inositol	Negative (-)
13	Lactose	Positive (+)
14	Malonate	Positive (+)
15	Maltose	Positive (+)
16	Mannitol	Negative (-)
17	Mannose	Positive (+)
18	Pyruvate	Negative (-)
19	Raffinose	Positive (+)
20	Rhamnose	Positive (+)
21	Ribose	Negative (-)
22	Salicin	Positive (+)
23	Sorbitol	Negative (-)
24	Starch	Positive (+)
25	Sucrose	Positive (+)
26	Trehalose	Negative (-)
27	Xylose	Positive (+)

S.N.	Enzyme	L. acidophilus
1	Acetoin	Negative (-)
2	Acetate Utilization	Positive (+)
3	β-galactosidase	Positive (+)
4	Esculin Hydrolysis	Positive (+)
5	Casein Hydrolysis	Negative (-)
6	Lactase	Positive (+)
7	Lysine	Negative (-)
8	Ornithine Decarboxylase	Positive (+)
9	Phenylalanine Deaminase	Negative (-)

Role of Lactobacillus acidophilus as Biopreservation resource

• Lactobacillus acidophilus has several mechanisms that help it compete against other microorganisms, including pathogenic strains.
• It exerts antimicrobial activity against food-borne microorganisms.
• This is achieved by producing inhibitory substances such as:
• Organic acids
• Hydrogen peroxide
• Ammonia
• Bacteriocins
• Diacetyl
• Antimicrobial activity is also attributed to the production of lactic acid and hydrogen peroxide.

a. Organic Acids

• Produced as end products of homofermentation of carbohydrates (lactic acid, acetic acid, propionic acid).
• These acids lower the pH of the medium and suppress the growth of pathogens and spoilage organisms.
• Mechanisms of antimicrobial action:
• Interference with cell membrane integrity
• Inhibition of active transport
• Reduction of intracellular pH
• Effective against both Gram-positive and Gram-negative bacteria.

b. Diacetyl and Aldehydes

• L. acidophilus produces flavor compounds such as diacetyl and acetaldehyde with antimicrobial activity.
• Diacetyl is formed during citrate metabolism and contributes aroma and flavor to butter and fermented milk products.
• Gram-negative bacteria and some yeasts are more sensitive to diacetyl and acetaldehyde compared to Gram-positive bacteria.

c. Hydrogen Peroxide

• Produced by L. acidophilus through various mechanisms in the presence of oxygen.
• Antimicrobial effects include:
• Oxidation of sulfhydryl groups, leading to enzyme denaturation
• Peroxidation of membrane lipids, which increases membrane permeability
• Effective in inhibiting the growth of psychrotrophic and pathogenic microorganisms, even under refrigeration conditions.

d. Bacteriocins

• Proteinaceous substances with bactericidal activity against different microbes.
• Provide a competitive advantage in ecological niches shared with other microorganisms.
• When consumed, help L. acidophilus compete with gastrointestinal pathogens.
• Classified into four groups:
• Class I: Lantibiotics, produced in response to microbial attack or colonization.
• Class II: Small, heat-stable proteins; non-lanthionine peptides.
• Class III: Larger proteins; heat-labile.
• Class IV: Complex molecules with lipid and carbohydrate moieties; some hydrophobic and heat-stable.
• Examples of bacteriocins:
• Lactobacin B: Active against L. bulgaricus, L. helveticus, L. lactis, and L. leichmannii.
• Lactacin F: Active against Enterococcus faecalis and L. fermentum.
• Acidocin A and B: Active against Listeria monocytogenes, Clostridium sporogenes, and Brochothrix thermosphacta.

Role of Lactobacillus acidophilus in food and dairy industries

• Lactobacillus acidophilus is widely used in the dairy industry for producing acidophilus milk, yogurt, miru-miru, and kefir.
• Acidophilus milk is distinct from non-fermented milk as it is suitable for people who lack the lactase enzyme, since the lactose is broken down by the β-galactosidase enzyme of L. acidophilus.
• Some strains of L. acidophilus act as starter cultures during milk fermentation, where they acidify milk and contribute to flavor and texture through proteolytic enzyme activity.
• As a starter culture, L. acidophilus aids in converting lactose into lactic acid, which lowers the pH of milk and causes its coagulation.
• The development of different flavors in fermented milk products is due to volatile metabolites and the incorporation of carbon dioxide during metabolism.
• In cheese, flavor formation occurs during the ripening process, which involves both starter and non-starter Lactobacillus species.
• L. acidophilus supports faster ripening of cheeses like cheddar and reduces the occurrence of bitterness.
• In yogurt, flavor is enhanced by the combined activity of Streptococcus thermophilus and Lactobacillus species.
• Common flavor compounds produced by L. acidophilus during milk fermentation include organic acids such as acetic acid and propanoic acid, as well as diacetyl, acetaldehyde, and acetoin.
• The texture of yogurt is improved by exopolysaccharides produced by L. acidophilus, which act as thickening agents.
• Coagulation, induced by L. acidophilus, further enhances yogurt texture by neutralizing negative charges in milk.
• L. acidophilus is also available in milk powder form, providing extended shelf life and natural biopreservation.
• Beyond milk and dairy, L. acidophilus contributes to the production of other fermented foods such as soymilk, soy-based yogurt, kombucha, fermented vegetable juices, kimchi, sausages, and salami.

Role of Lactobacillus acidophilus in human health

• Many Lactobacillus species are recognized as human probiotics, and several strains of L. acidophilus are known to provide multiple health benefits.
• Reported benefits include reducing gastrointestinal symptoms in lactose-intolerant individuals, relieving constipation, treating infantile diarrhea, and showing activity against Helicobacter pylori.
• Studies have shown that L. acidophilus helps prevent gastric inflammation associated with H. pylori infections.
• The bacterium supports individuals with lactose intolerance by degrading lactose through the action of β-galactosidase.

Negative impacts

• Although generally beneficial, L. acidophilus has occasionally been linked to infections, particularly in immunocompromised individuals where it can act as an opportunistic pathogen.
• Reported infections include lactobacillemia, which in severe cases may progress to endocarditis and bacteremia.
• The mechanisms of infection are not fully understood, but possible factors include enzymatic activity, bacterial translocation, mucin degradation, and platelet aggregation.
• Colonization of the intestinal tract is essential for probiotic benefits but also contributes to bacterial adherence in infection cases.
• Some strains exhibit amino acid decarboxylase activity, producing biogenic amines that may be harmful to the host.
• Protease production, which supports colonization and tissue invasion, has been linked to endocarditis in rare cases.
• Despite these risks, infections caused by L. acidophilus are extremely rare and generally occur only in individuals with pre-existing conditions.

Positive impacts

• The most significant health benefit of L. acidophilus is its role as a probiotic.
• Probiotics are live microorganisms that, when consumed in appropriate amounts, provide health benefits to the host.
• While the importance of gut microbiota in preventing gastrointestinal infections has long been recognized, the use of probiotics for prevention and therapy has gained prominence more recently.
• Selection of L. acidophilus as a probiotic is based on criteria such as its ability to survive passage through the upper gastrointestinal tract, tolerance to gastric juices, and antagonistic activity against intestinal pathogens.
• L. acidophilus can stabilize and modulate intestinal microbiota and demonstrates strong adherence to epithelial cells.
• Dairy products are common carriers of L. acidophilus, including pasteurized milk, ice cream, cheeses, and fermented milk.
• Yogurt is a classic probiotic product containing L. acidophilus that has been consumed for many years.
• Cheese also serves as an effective probiotic carrier due to its low oxygen content and high lipid concentration, which support bacterial survival.

Benefits of L. acidophilus probiotics

Digestive benefits

• Supports the breakdown and metabolism of lactose, helping to reduce the effects of lactose intolerance.
• Minimizes common lactose-intolerance symptoms such as intestinal pain, diarrhea, gas, and bloating.
• Promotes the growth of beneficial gut microbiota by releasing essential growth factors that aid digestion.
• Reduces symptoms of irritable bowel syndrome (IBS), including diarrhea, constipation, and bloating.
• Produces bacteriocins and other metabolic compounds that inhibit the growth of harmful intestinal pathogens.

Immunity

• Strengthens the immune system by maintaining a healthy balance of gut microbiota and producing antimicrobial compounds.
• Helps lower inflammation, especially in the digestive tract.
• Plays a role in preventing infections caused by Helicobacter pylori, a common pathogen of the digestive system.
• Produces metabolic products that discourage colonization by pathogenic bacteria, contributing to its preservative action.

Other benefits

• Assists in preventing and treating vaginal yeast infections; when combined with antibiotics, it helps clear infections more quickly.
• Improves the gut microbiota of non-breast-fed infants, supporting early digestive health.
• Shows potential anti-tumor properties, partly due to its association with higher levels of beneficial lipoproteins.
• Helps reduce symptoms of atopic dermatitis and may contribute to better skin health.

Side effects of Lactobacillus acidophilus probiotics

• The use of Lactobacillus acidophilus probiotics is generally considered safe; however, some minimal side effects can occur with their intake.
• A common side effect associated with L. acidophilus and other similar probiotics includes gas, bloating, and other mild digestive issues.
• In certain cases, rashes and acne may develop, which can depend on the immune condition of the individual, as probiotics have the potential to induce inflammation.
• These side effects are usually minor, short-lived, and typically resolve on their own within 12–14 days.
• If severe or persistent effects are experienced, the use of probiotics should be discontinued immediately, and medical assistance should be sought without delay.

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