In recent years, with the increasing public concern about the safety of animal-derived food, the research and development of feed additives that are green, safe, and effective substitutes for antibiotics and antimicrobial drugs have become a major focus. Probiotic preparations have emerged in response to this demand. In 2002, the European Food and Feed Cultures Association (EFFCA), a leading European authority, defined probiotics as live microorganisms that, when ingested in adequate amounts, confer one or more specific, scientifically validated health benefits to the host. Microecological preparations refer to live bacterial preparations made from beneficial microorganisms naturally found in animals. Among these, lactic acid bacteria (LAB) microecological preparations have become a key area of research in recent years. Among various microbial additives, LAB preparations are widely used and considered highly effective as feed microbial additives. They are predominant symbiotic bacteria in the digestive tracts of many aquatic animals, capable of forming normal flora. LAB is also one of the first microbial strains in China to be approved for direct use as feed-grade microbial additives. LAB can colonize the intestinal tract of aquatic animals, effectively regulating intestinal flora balance, secreting lactic acid, and producing various digestive enzymes that help with food digestion and metabolism, promoting the absorption and utilization of nutrients. Additionally, they can compete with pathogenic bacteria for nutrients and adhesion sites, secrete bacteriocins, and inhibit the growth of harmful bacteria, thereby improving the intestinal environment, balancing the gastrointestinal microbiota, and enhancing animal health. LAB provides an efficient, harmless, and pollution-free new pathway for the healthy development of the feed and aquaculture industries.

1. Physiological Characteristics of Lactic Acid Bacteria

Lactic acid bacteria refer to a group of Gram-positive bacteria that do not produce spores and primarily ferment carbohydrates to produce lactic acid. Their morphological, metabolic, and physiological characteristics vary. Most LAB cells are rod-shaped or spherical, non-sporulating, non-motile or slightly motile, heat-sensitive but acid-tolerant, capable of growing in environments with pH levels between 3.0 and 4.5, and have strict nutritional requirements, including the need for various amino acids, vitamins, and peptides in addition to carbohydrates. Within the animal body, LAB lowers the pH through biological antagonism, preventing and inhibiting the invasion and colonization of pathogenic bacteria and degrading harmful substances like nitrosamines, ammonia, indoles, and skatole. The LABs discovered in nature, which fall into at least 23 genera in bacterial taxonomy, include Lactobacillus, Bifidobacterium, Carnobacterium, Streptococcus, Leuconostoc, Pediococcus, and Sporolactobacillus. Most LAB thrive in anaerobic or microaerobic, acidic environments rich in minerals and organic nutrients, and are widely distributed in fermented products (e.g., silage, pickles, yogurt) and animal digestive tracts.

2. Mechanisms of Action of Lactic Acid Bacteria

2.1 Antibacterial Mechanisms

The survival and colonization of bacteria in the animal intestine depend on several factors, including gastric acid, bile salts, digestive enzymes, immune responses, endogenous microorganisms, and their production of antibiotics. Any change in these factors may affect bacterial survival. LAB's antibacterial mechanisms are rooted in their biochemical properties, which alter the living environment and growth mechanisms of pathogenic bacteria, achieving antibacterial effects.

2.1.1 Inhibiting Harmful Bacteria by Acid Production:
LAB ferment lactose in the body to produce large amounts of acetic and lactic acids, which lower the environmental pH, making the intestinal environment acidic. Pathogenic bacteria in the intestine prefer a pH of 7.0-7.4 for optimal growth. The acid production by LAB and the resulting cell lysis decrease intestinal pH, inhibiting the colonization and growth of pathogenic bacteria on the intestinal wall, leading to their reduced viability, aging, and death, thereby maintaining intestinal ecological balance and normal physiological function.

2.1.2 Inhibiting Harmful Bacteria by Producing Antibacterial Substances:
In addition to organic acids, LAB produce other antibacterial substances, such as hydrogen peroxide and benzoic acid, as well as lysozymes and other antibacterial agents like nisin, lacticin, and acidophilin. These products inhibit pathogenic bacteria in the intestine. Hydrogen peroxide and bacteriocins inhibit pathogens by a mechanism known as adhesion resistance. Adhesion to the intestinal mucosa is a prerequisite for pathogenic bacteria to colonize and cause clinical symptoms. There is a competition for attachment sites on the small intestine epithelium between beneficial and pathogenic microorganisms, known as competitive exclusion. If beneficial strains occupy more of these sites, pathogenic bacteria are excluded. LAB inhibit pathogenic bacteria by competing for space, time, adhesion sites, and nutrients, thereby preventing their adhesion to intestinal mucosal epithelial cells.

2.2 Nutritional Mechanisms

LAB, when metabolically active within the animal body, directly provide the host with essential amino acids, vitamins, and digestive enzymes (e.g., amylase, protease, cellulase), and improve the digestion and absorption rates of minerals (e.g., calcium, phosphorus, iron, magnesium), thereby enhancing nutritional metabolism and promoting growth and productivity. Additionally, the acidic metabolic products of LAB acidify the intestinal environment, which is optimal for the activity of various digestive enzymes (e.g., amylase at pH 6.5, glucoamylase at pH 4.4), aiding nutrient digestion and absorption. The production of organic acids also enhances intestinal peristalsis and secretion, further promoting nutrient digestion and absorption.

2.3 Immune Mechanisms

LAB enhance animal immunity in two ways:

1. They influence non-specific immune responses by boosting monocyte and macrophage activity, stimulating the secretion of reactive oxygen species, lysozyme enzymes, and monocyte factors.
2. They stimulate specific immune responses, such as increasing IgA, IgM, and IgG levels on mucosal surfaces and in serum to enhance humoral immunity, promoting the proliferation of T and B lymphocytes to boost cellular immunity. Schiffrin et al. (1994) found that Lactobacillus johnsonii Lj1 and Bifidobacterium lactis Bb12 enhance phagocytic cell activity against Escherichia coli in vitro. When used with Salmonella, Streptococcus thermophilus acts as an immunoadjuvant, significantly increasing serum IgA levels. The mechanisms by which LAB stimulate the immune system are still being studied. Ouwehand et al. (1999) proposed possible pathways for immune stimulation by ingesting probiotics (including LAB). Antigenic substances pass through follicle-associated epithelium in lymph nodes via two routes:
3. Microbial metabolites or fragments, as small molecular antigens, pass directly through regular epithelial cells or through tight junctions between epithelial cells.
4. Microbial cells are delivered to macrophages encapsulated in M-cells through pinocytosis. After entering lymphoid tissue, antigens are either processed by antigen-presenting cells or directly handed over to lymphocytes to produce corresponding immune responses.

2.4 Detoxification and Prevention of Putrefactive Products

Certain bacteria can neutralize or reduce the toxic effects of endotoxins and other harmful substances. Bifidobacterium has been found to prevent ammonia production in intestinal contents, while Bacillus cereus reduces ammonia concentrations in intestinal contents and the hepatic portal vein, decreasing the levels of harmful substances like phenols and indoles.

3. Health Benefits and Therapeutic Effects of LAB on Animals

Numerous feeding and clinical trials at home and abroad have shown varying results, with both positive and negative effects, though positive effects are more common. The inconsistencies may be due to factors such as the animal species, age, feeding environment, strain quality, and stress conditions, but the beneficial effects of LAB on animal health are widely recognized.

3.1 Improving Gastrointestinal Function and Reducing the Incidence of Gastrointestinal Diseases

LAB is dominant in the gut flora. After ingestion, they ferment to produce acids, altering the gastrointestinal environment, inhibiting harmful bacterial growth, and balancing the gastrointestinal microbiota. They also produce adhesins that bind tightly to intestinal mucosal cells, colonizing the mucosal surface and forming a physiological barrier, thereby restoring host resistance, repairing the intestinal bacterial barrier, and treating gastrointestinal diseases.

3.2 Enhancing Immunity and Promoting Animal Health

LAB enhance animal immunity by stimulating both non-specific and specific immune responses, inducing interferon production, promoting cell division, producing antibodies, and enhancing cellular immunity, thus increasing the body's disease resistance.

3.3 Promoting Animal Production, Increasing Feed Efficiency, Reducing Feeding Costs, and Improving Economic Returns

LAB produce various nutrients within the animal body, such as vitamins, essential amino acids, and digestive enzymes, enhancing nutritional metabolism and promoting growth and productivity. LAB also lower intestinal pH, creating an acidic environment conducive to enzyme activity, improving nutrient digestion and absorption, and reducing feeding costs.

4. Current Application Status

In summary, LAB has preventive, therapeutic, nutritional, and health benefits. In recent years, the drawbacks of antibiotic feed additives have become increasingly recognized, prompting many researchers to focus on overcoming these drawbacks through microecological research, which has driven the development of microecological preparations. LAB preparations are now widely used in various fields, including pig, poultry, cattle, sheep, and aquaculture.

5. Issues and Prospects

5.1 Strain Selection

Currently, probiotic preparations for aquaculture sold on the market primarily include photosynthetic bacteria, Lactobacillus, Bifidobacterium, EM bacteria, Bacillus, and their cultures. The selection and activity determination of LAB strains, a key component of probiotic preparations, are crucial in determining their efficacy. Qualified LAB strains must possess the following characteristics:

1. Strong anti-pathogenic bacteria activity, effective colonization, and high production of digestive enzymes.
2. Able to maintain high vitality and fermentation activity during processing, storage, and transportation, with a survival rate over 90% and storage stability.
3. The final fermentation product should have low pH, strong antibacterial effects, and beneficial microbial species composition, with certain environmental adaptability (e.g., high temperature, acid and salt resistance).
4. Possess strong stress tolerance and resilience to stressors, contributing to the improvement of breeding environments.

5.2 Fermentation Process and Quality Control

The production process and quality control of LAB preparations require close attention to ensure stability, activity, and effectiveness. This includes optimizing fermentation conditions, using high-quality raw materials, and monitoring the final product for consistent quality.

5.3 Application Method and Dosage

Determining the appropriate dosage and application method for LAB preparations is critical for achieving optimal results. Overuse or underuse can lead to diminished effectiveness or negative side effects.

5.4 Regulatory and Safety Considerations

As the use of LAB preparations in aquaculture increases, regulatory frameworks must be established to ensure safety, efficacy, and environmental sustainability. This includes conducting thorough safety assessments, monitoring long-term effects, and establishing guidelines for proper use.

6. Conclusion

Lactic acid bacteria preparations represent a promising and eco-friendly alternative to antibiotics in aquaculture. Their ability to improve animal health, enhance immune function, and promote growth makes them an essential component of sustainable aquaculture practices. Continued research and development in this field will likely lead to even more effective and widely adopted LAB-based solutions.