Lactic bacteria in clinical samples, dairy derivatives and probiotic preparations: qualitative and quantitative culture, molecular identification (PCR and sequencing).
Probiotics are live microorganisms that, when administered in the appropriate amount, promote health benefits for the person who ingests them as long as they meet the definition and requirements established by the World Health Organization (WHO) and the Organization of Nations. United (FAO).
Lactic acid bacteria (LAB) are grouped into the Lactobacillaceae family that comprises about 20 different genera, among which the following stand out: Aerococcus, Alloiococcus, Carnobacterium, Dolosigranulum, Enterococcus, Globicatella, Lactobacillus, Lactococcus, Lactosphaera Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus, and Weisella. Of them, Lactobacillus is the most extensive of all.
In 1919 the classification of BAL began as a group of physiologically uniform and morphologically heterogeneous organisms including cocci and bacilli of variable length (0.5-0.8 µm) and cell wall with gram-positive structure. They are non-sporulated microorganisms (except Sporolactobacillus inulinus), non-mobile, anaerobic, microaerophilic or aerotolerant; neither oxidase nor catalase producers, they lack cytochromes, they do not reduce nitrate to nitrite and they produce lactic acid as the only or main product of carbohydrate fermentation. In addition, LABs are acid-tolerant and can develop in a pH range between 3.2 and 9.6, although most develop better at a pH between 4.0 and 4.5.
LABs are also classified, according to the final metabolites of carbohydrate fermentation, into homofermentative (they only produce lactic acid as the final metabolite) and heterofermentative (they produce lactic acid and other final metabolites), and according to the growth temperature in mesophiles and thermophiles.
These bacteria are widely used because they produce metabolites that fulfill specific functions, among which the production of propionic, citric and lactic acid stands out; they also produce antimicrobial substances, hydrogen peroxide, bacteriocins, exopolysaccharides (EPS); compounds that contribute to the taste, smell, texture, and nutritional value of fermented foods and products; production of lactic drinks, silage (preservation of forage by acidification), vitamins and probiotics.
The consumption of LABs as probiotics has been associated with a series of beneficial effects for the digestive, immune, endocrine, cardiovascular and nervous systems, among which the following stand out:
- Maintenance of intestinal biota, intestinal movement, improvement in intestinal permeability, reduction of diarrhea and treatment of digestive problems and reduction of symptoms due to lactose malabsorption.
- Modulation and stimulation of the immune system.
- Possible mechanisms of action with potential for the prevention and treatment of colon and other organ cancer.
- Effects on the cardiovascular system: as a possible antithrombrombotic by releasing peptides with the ability to inhibit the formation of thrombus (clot) in blood vessels, antihypertensive by moderately lowering blood pressure and as hypocholesterolemic by lowering serum cholesterol preventing cardiovascular diseases.
- Benefits attributed to probiotics in general.
To achieve the beneficial effect on the health of the host, a probiotic culture must be ingested in adequate quantity. The recommended amount of BAL is in the range of 106-107 CFU/g of product. LABs generally used as a probiotic are commercially available in these amounts in fermented products, food preparations, and supplements. The genera of BAL most used as probiotics are: Bifidobacterium animalis subsp. animalis and B. animalis subsp. lactis, B. brevis, B. bifidum, B. infantis, B. longum; Lactobacillus acidophilus, L. casei, L. reuteri, L. plantarum, L. casei GG; Streptococcus salivaris subsp. thermophilus.
However, there are autochthonous lactic strains in meats, vegetables, milk, etc., with proteolytic and lipolytic activity, acidifying capacity and gas production that allow to improve or deteriorate the useful life and the microbiological quality of the final product. Among the typically isolated genera, Lactobacillus, Lactococcus, Leuconostoc and Pediococcus stand out.
LABs have specific nutritional requirements, they are sensitive to changes in pH, the presence of salt, carbohydrates, vitamins, etc. In addition to nutritional requirements, temperature is one of the most important factors influencing the proper growth of LABs and their differentiation. For this reason, they must be grown at the optimum temperature and in media with nutrients that favor their growth.
The isolation and quantitative culture of lactic acid bacteria is carried out in a solid, selective and differential medium. The recommended culture media are: MRS (De Man, Rogosa and Sharpe agar) that favors the isolation of the Lactobacillus genus; M17 supplemented with sugars for the isolation of the Lactococcus and Streptococcus genera; MSE medium (Mayeux, Sandine and Ellike); and the KAA medium (Kanamycin, Aesculin, Acid) to promote the growth of the genus Leuconostoc and Enterococcus. However, molecular methods make it easier to detect and identify these bacteria in order to know exactly which strain is being analyzed. Currently, for taxonomic identification, the genomic sequence encoding 16S rRNA is used as a target, but sometimes there are not enough differences in this sequence between some species and it is necessary to carry out a prior isolation in culture using selective culture media.
Tests carried out at IVAMI:
- Isolation and qualitative or quantitative culture of lactic acid bacteria in selective and differential media according to the genus to be isolated.
- Qualitative molecular detection by specific PCR.
- Identification of isolated colonies by molecular methods (PCR and sequencing).