Probiotic science and applications

DEFINITION OF PROBIOTICS

The word probiotics is used for the first time in 1956, but it was used to indicate the capability of promote and improve the living organism survive. Nowadays the definition accepted by the scientific community is proposed by FAO and WHO in 2002: live microorganisms that, when administrated in adequate amounts, confer a health benefit on the host. In the legal definition, with the word "microorganism" means not only Lactoacillus and Bifidobacteria but also other microorganism that don't need to be bacteria. Probiotics need to be alive in sufficient amounts when consumed, but it's not required in the gastrointestinal tract. The amount of alive microbes must be the same used in clinical study because it has demonstrate positive effect on health, shown in the target host population. Probiotics need to be "administrated" because it's not necessary to be eaten [but they can enter in the organism in other way]. In order to be able todire che un alimento ha un effetto probiotico, ci sono alcune linee guida che devono essere seguite: 1. Il primo passo è l'identificazione fenotipica e genotipica per genere e specie. È importante comprendere il possibile rischio del suo utilizzo nel cibo e la relazione con il corpo umano. In Italia non è più ammesso l'uso di nomi fantasiosi o obsoleti per i probiotici sull'etichetta del prodotto. Per capire se un nome può essere utilizzato, ci sono alcune liste di nomi approvati di microorganismi: LPSN, NCBI e DSMZ. C'è anche la rivista scientifica IJSEM, che pubblica costantemente aggiornamenti sulle modifiche nella tassonomia microbica. Il metodo ufficiale per specificare che un ceppo appartiene a una specie è l'ibridazione del DNA-DNA; ma può essere utilizzata anche la sequenza del DNA che codifica per il 16S rRNA (più veloce). [Per i lieviti possiamo fare l'analisi della sequenza dei domini D1 e D2 del 26S rRNA]. La sequenziatura del gene 16S rRNA viene effettuata in alcune fasi specifiche: 1. Ottenimento del microorganismo in coltura pura 2. Estrazione del DNADNA from the cells of the strain <strong>1. PCR amplification of the 16s rRNA gene with universal primers</strong> 2. Sequencing of the amplicon, it takes therefore 3 reactions to sequence the gene entirely 3. Preparation of the nucleotide sequence 4. Search for homologies in genomic databases. If using BLAST we have a similarity > 98% in order to know the species we need to use DNA/DNA hybridization to have a definitive confirmation. The DNA-DNA hybridization must be conducted between the total DNA isolated from the strain under study and the total DNA extracted from a reference stain. We can't use directly DNA-DNA hybridization to identify a completely unknown bacterial strain, but we use first the 16S rRNA. <strong>2. Second step is the functional characterization with vitro test and animal studies</strong> <strong>3. Third step is a safety assessment in vitro (or in animal) and than in human</strong> <strong>4. Last step is another trial in human in order to verify the effectiveness of the microorganism. If the effectiveness is positive</strong>wehave to do another study in human to compare probiotics with standard treatment of a specific condition. In 2005 also Italian Ministero della Salute has given a probiotic definition:

Viable and active microorganisms that confer health benefits of the host when consumed, in adequate quantities, as part of a food or supplement.

The difference with the FAO and WHO definition is that in the Italian one probiotics has to be used exclusively in the food context (not medicine). Probiotics need also to be active when they arrive in the gut, so they must reach intestine, multiply and exert a balancing action on the intestinal microflora by direct colonization. The microorganisms currently used as probiotics can be divided into three categories:

• Lactic acid bacteria (LAB). Phylum Firmicutes, Gram + and low GC [=Guanina e Citosina]. Some of them are pathogens. They are immobile and have a rod [=bastoncini] (Lactobacillus) or cocci (Streptococcus, Enterococcus, Lactococcus) shape. They are all

anaerobes they can use both glucose and pentoses as substrates. They produce lactic acid, ethanol, CO and sometimes a small amount of acetic acid.

heterofermentative they can degrade hexose producing lactic acid. They can degrade pentose producing lactic acid and ethanol (not CO₂).

The acid is produced in D, L and DL form according to the genus. The D form can’t be metabolized by human cells.

The most common probiotics are Lactobacilli that can be divided into different groups (genus), because lactobacillus genus is too much heterogenic for both phenotypic and genotypic aspect.

• Lactobacillus delbrueckii group: L. acidophilus, L. helveticus (homofermentative)
• Lacticaseibacillus casei group: L. casei, L. paracasei (facultative heterofermentative)
• Lactiplantibacillus plantarum: L. plantarum (facultative heterofermentative)
• Limosilactobacillus reuteri group: L. reuteri (obligate heterofermentative)
• Ligilactobacillus salivarius (homofermentative)

Occasionally used as probiotics are: Enterococcus faecium; Leuconostoc mesenteroides, Lactococcus lactis; Streptococcus thermophilus

Bifidobacteria

(BFB)Phylum Acinobacteria, Gram+ and high GC. They are not consider pathogenic.

From their energy metabolism they produce acetic acid and lactic acid in proportion 3:2, even if the proportion depends by the species of the microorganism. The enzyme fructose-6-phosphate phosphoketolase F6PPK convert fructose-6P into erythrose-4P and acetyl-P. Than, with the action of transaldolase and transketolase, we have the production of acetic acid and lactic acid. [F6PPK substitute the aldolase in LAB].

They’re optimal growth temperature is 35-39°C.

They are immobile and have a pleomorphic rod shape (pleomorphic= possono avere forme a bastoncino diverse), aspori-genous. They are strict anaerobes and they are negative for the catalase test. They do anaerobic fermentation, producing acids but not gas.

The only original habitat is gut (of different animals) and they are transmitted directly from mother. The one that we find in dairy products comes from cross contamination between animal gut and milk.

B.

breve and B. bifidum are found exclusively in the human intestine and they represent a perfect example of co-evolution between humans and microorganisms [because human body has produced itself probiotics microorganism]. All bifidobacterial of human intestinal origin are considered potential probiotics. The most used are: B. breve, B. bifidum, B. longum subsp. infantis, B. longum subsp. longum, B. animalis subsp. lactis. The last one is the most common in food because it tolerates oxygen and acidity much better than other bifidobacteria, but it isn't from human origin.

Unlike lactobacillus, bifidobacteria are not commonly associated with food. Non-LAB and non-BFB bacteria and yeast can be found in supplements or medicine. If they are in medicine, they are not recognized as probiotics for Italian law [for example the Enterogermina that contains Bacillus clausii]. Instead, Bacillus coagulant in supplement is a probiotic. In supplements, formulations of different bacteria and yeast can also be added.

probiot-ics microorganism. Like food supplement we can find also Escherichia coli strain Nissle 1917, one of the very few gram-negative probiotics. Generally gram-negative are not good like probiotics because of the presence of lipopolysaccharides in the outer surface, that are not recognize by immune system. But in the strain Nissle 1917 there is a mutation that make lipopolysaccharidessurface different.

IS IT POSSIBLE CONSIDERING TRADITIONAL FERMENTED FOOD PRODUCTS POTENTIAL PROBIOTICS?

These types of products are associated with health benefits but they are not probiotics because they haven’t a well-defined strain. There are lots of fermented food that contain microorganisms, for example:

• Fontina: Enterococcus faecium, Streptococcus thermophilus, Enterococcus faecalis
• Olives: Lactobacillus plantarum (to eliminate oleuropein), Pediococcus spp., Yeasts
• Kimchi: Leuconostoc mesenteroides and many different lactobacilli

KEFIR is a drink produced through the fermentation

htmlbulgaricus and Streptococcus thermophilus. These thermophilic lactic bacteria must be viable in a total quantity of no less than 10 million per gram and each of the two species must be present in no less than 1 million per gram until the end of shelf-life (until the expiry date indicated on the package). To affirm that yogurt is a probiotic microorganisms must arrive in the gut alive, and it has been demonstrated. BUT there are some reasons why yogurt should not be considered probiotic: • Microbiological reason: yogurt is a food that is produced with different bacterial strains [=ceppo], depending on the producer (so I don't fully know its microbiological composition). It is demonstrated that the evidence of health benefit is strain specific and products like yogurt contain live microorganisms but the strain composition (and stability) is not well defined. • Legal reason: in Europe, yogurt is a conventional food not a novel food, instead probiotics are novel food. Novel food.

≡ food that had not been consumed to significant degree by humans