Probiotic science and applications

Holobiont = single ecological unit consisting in eukaryote host organism (animal or plant) and microbiota. According to the

holobiontic theory all the animals and plants host lots of symbiotics microorganisms that influence the performance of the

entire holobiont. HOST ORGANISM (EUKARYOTE) AND A MICROBIOTA CONSIDER UNDER THE SAME ECOLOGICAL UNIT.

Phylosymbiosis is the evolution of relationship and interaction between the microbiota and host, in fact every host has a

perfectly adapted microbiota.

MICROBIOTA ≡ is the set of all microbial cells that colonize a specific ecosystem or habitat.

MICROBIOME ≡ is an entire habitat in which microorganisms reside and where they carry out their activity (included what they

produced, genomes and other molecules). [genetic materials + environment].

INTERACTIONS BETWEEN LIVING ORGANISMS IN AN ECOSYSTEM

Most of microorganism establish beneficial relationships with the host, instead pathogens are much less.

There are different form of interactions (/symbiosis):

• Neutralism: the coexistence of different species without any harm (damage) or benefit to them

• Commensalism: non-obligatory interaction between two organism. One takes advantage of the other's nourishment

[=nutrimento] or waste without causing harm or benefit

• Synergism/protocooperation: non-obligatory interaction in which both populations benefit. Both populations are able to

survive independently [bee and flowers].

• Mutualism/symbiosis: obligatory interrelation between two living organisms that determines benefit for both.

• Antagonism: interaction in which one or both living organisms in relation are damaged. The main antagonistic relationships

are:

o Antibiosis: one organism inhibits the growth and reproduction of

one other organism.

o Parasitism: one organism is a parasite that exploits [=sfuttare]

another (the host) for trophic [=nutrimento] reasons creating

biological damage. For example tick [=zecca].

o Predation: interaction in which an organism (predator) uses

another organism usually of a different species (prey) as a food

source. For example lion and zebra.

o Competition: interaction during which two living organisms are

looking for the same nutrient resource or habitat. (-) = damage/harm; (+) = benefit; (0) = no harm no benefit

SITE SPECIFIC DISTRIBUTIONS OF BACTERIAL PHYLA IN HEALTHY HUMANS

1-3% of the human mass is represented by microorganism (fungi and viruses), most of all bacteria. Firmicutes are most present,

followed by Bacteroides. The majority are collocated in colon, followed by mouth, skin, esophagus and stomach.

The human intestinal microbiota

99% of the human microbiota is collocated in the gut [=intestino]. They are important for human health.

From stomach to colon, the pH goes to neutrality. Consequently the UFC/mL increases and the types of microorganisms

changes. For example in the stomach there are Lactobacillus and Streptococcus that are not present in the colon, where we can

find Bacteroides, Clostridium, Enterobacteriaceae and Bifidobacterium.

THE MAIN FUNCTION OF THE INTESTINAL MICROBIOTA

The intestinal microbiota could be seen as a metabolically active organ of the human body and the main function are:

1. Barrier against the proliferation of (potential) pathogens that comes from ambient. They can do barrier effect or

competitive exclusion or colonization resistance.

2. Regulator of the immune system maturation and activity.

When we born we have any microorganism, then eating food our microbiota develops. It’s important to have a rich

biodiversity of microbiota because microbiota “teach” to immune system how to work.

! Immune system is based on recognize structural elements of bacterial cells that have specific ligand. For example

TLR-2 receptors recognize pili and murein.

3. Regulator of intestinal motility.

Microbiota regulate the secretion of serotonin inside the gut, modulating the intestinal motility.

4. Vitamin producer (vitamin K, B9, group B vitamins)

5. Modulator of the effect of food on host’s health

Without knowing the characteristic of microbiota, we can’t know if a food is healthy or not for specific human.

Microbiota use undigested carbohydrates, fermenting them and producing short chain fatty acids (acetate, butyrate

and propionate *). SCFA are absorbed and utilized by the host. For example they fermented fiber, in particular

undigested starch and non-starch polysaccharides.

* acetate, butyrate and propionate: the are important for host’s health and they recover energy from undigested food.

The microbiota can be transplanted.

There is a interaction among diet, microbiota and host: on the basis of what we eat, the composition of our intestinal microbiota

changes, and so human health condition. For this reason, a lot of diseases can be treated introducing probiotics in diet.

The origin of probiotics approach started with the use of bifidobacterial to treat childhood diarrhea [1906]. Even if

microorganisms with positive effect on health have been consumed for thousands of years through traditional fermented

products, without knowing the positive mechanism.

There is a correlation between microbiota and premature aging, because lactic bacterial can fight against intestinal putrefaction.

So we need to introduce useful microorganism with diet, for example with yogurt. The premature aging is caused by chronic

poisoning due to the presence of putrefactive microorganisms in gut.

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 to say that a food has a probiotic effect, there are some guidelines that must be followed:

1. First step is the phenotypic and genotypic identification for genus and species

It is important to understand his possible risk of it use in food and the relationship with human bodies.

In Italy is not admitted anymore the use of fantasy name ore obsolete name for probiotic in the product label. To understand

if a name can be used, there are some list of approved name lists of microorganisms: LPSN, NCBI and DSMZ. There is also the

scientific journal IJSEM, that public constant updating of changes in microbial taxonomy.

The official method to specify that a strain belongs to a species is the DNA-DNA hybridization; but it can be used also the DNA

sequences encoding of 16S rRNA (faster). [For yeast we can do the analysis of the sequence of domains D1 and D2 of 26S rRNA].

The sequencing of the 16S rRNA gene is done in some specific phase:

1. Obtaining the microorganism in pure culture

2. Extraction of the DNA from the cells of the strain

3. PCR amplification of the 16s rRNA gene with universal primers

4. Sequencing of the amplicon, it takes therefore 3 reactions to sequence the gene entirely

5. Preparation of the nucleotide sequence

6. 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.

2. Second step is the functional characterization with vitro test and animal studies

3. Third step is a safety assessment in vitro (or in animal) and than in human

4. Last step is another trial in human in order to verify the effectiveness of the microorganism. If the effectiveness is positive we

have 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 with fermentative metabolism (so they can’t produce ATP), but there are few exceptions like Lacto-

coccus lactis that can do cellular respiration if a source of heme group is provided. They are catalase negative, so they can’t

carry out the decomposition of H O into H O + O .

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They are acid resistance (pH<5).

Some of them live in association with plants, taking nutrients released after death and decomposition of plant tissues. They

are also part of animals microbiota. In food they can be found in sauerkraut, wine, beer and milk.

They can be divided into three metabolic groups:

o →

Homofermentative they use just glucose producing lactic acid. They can’t use pentoses.

o →

Obligate heterofermentative they can’t degrade glucose because they don’t have fructose 1,6 diphosphate al-

dolase so they use pentose phosphate pathway. The final products are lactic acid, ethanol and CO and sometime a

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small amount of acetic acid.

o →

Facultative heterofermentative they can degrade hexose producing lactic acid. They can degrade pentose pro-

ducing lactic acid and ethanol (not CO ).

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

The most common probiotics are Lactobacilli that can be divided into different group (=genus), because lactobacillus genus

is too much heterogenic for both phenotypic and genotypic aspect.

o →

Lactobacillus delbrueckii group: L. acidophilus, L. helveticus homofermentative

o →

Lacticaseibacillus casei group: L. casei, L. paracasei facultative heterofermentative

o →

Lactiplantibacillus plantarum: L. plantarum facultative heterofermentative

o →

Limosilactobacillus reuteri group: L. reuteri obligate heterofermentative

o →

Ligilactobacillus salivarius homofermentative

Occasionally used as probiotics are: Enterococcus faecium; Leuconostoc mesenteroides, Lactococcus lactis; Streptococ-

cus 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 bifidobacte-

rial 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 tolerate

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

We can found them in supplements or medicine. If they are in medicine, they are not recognize as probiotics for Italian law

[for example the Enterogermina that contain Bacillus clausii].

Instead Bacillus coagulant in supplement is a probiotics. In supplement can be also added formulation of different 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 lipopolysaccharides

surface 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 of milk/sugar-rich solutions with kefir grains, which made of polysaccharide en-

trapping yeast, lactic acid bacteria and acetic acid bacteria.

The final products of fermentation are lactic acid, acetic acid, ethanol and CO .

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KOMBUCHA

is a traditional black/green tea beverage “fermented” by a symbiotic community of acetic acid bacteria (family: Acetobacter-

aceae) and osmophilic yeast (resistant at high sugar concentration).

The microorganisms derived from a previous batch that are Komagataeibacter (genus).

The presence of a carbon source (generally sucrose added previously) allows the activation of microbic metabolic processes

(cellular respiration) generating a carbonated and acid drink.

After 10-14 days of incubation the acetic microorganisms form a mature bacterial cellulose biofilm that appears on the top (the

“cellulose” is the exopolysaccharide produced by acetic).

YOGURT

Is the product of the acid fermentation of milk with Lactobacillus delbruekii subsp. bulgaricus 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 spe-

cies 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 pro-

ducer (so I don't fully know its microbiological composition).

It is demonstrate that the evidence of health benefit is strain specific and products like yogurt contains live microorganism

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 in the UE before 1997.

EFSA has approved one health claims related to live yogurt culture that improved lactose digestion. In these yogurt there are

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Lactobacillus delbruekii and Streptococcus thermophilus at least 10 CFU/g and in the individuals with lactose maldigestion the

presence of live yogurt culture (=claim) can improve lactose digestion (=claimed effect).

STRAIN and TAXONOMY CHARACTERIZATION – step 1

The probiotic effects are strain dependent, and probiotic strains are deposited in an internationally recognized culture collec-

tion. It is therefore generally accepted that the effects of one probiotic strain cannot be extrapolated from another strain, alt-

hough phylogenetically related. In other words, clinical studies must be conducted on each specific strain so that it can be at-

tributed a probiotic property.

FAO and WHO guidelines suggest that probiotics should be typing (=characterized) by genetic methods; the two proposed tech-

niques are RAPD and PFGE because they have high discriminating power. Nowadays the sequency of the entire genome is used.

Identification of the identity of the strain is not a critical aspect only in cases where there is sufficient scientific evidence

to support the fact that the health effect is not strain-specific (in yogurt, where S. thermophilus and L. delbrueckii subsp.

bulgaricus can increase the digestion of lactose in lactose intolerant individuals).

Strain ≡ is made-up [costituita] of the descendants of a single isolation in pure culture and usually is made up of a succession of

cultures ultimately derived from an initial single colony.

In the same strain there still be genetic difference, but they are accepted.

Genetic drift is a result of progres