Our scientific studies

The microbiota defines a community of microorganisms colonizing the human body. At all times of life, humans are associated with this population of microorganisms and their products1. Humans have co-evolved with microbes in the environment, and each body habitat has a unique set of microorganisms in its microbiota. The entire microbiota present in the human body is made up of more than 38,000 billion cells, or 0.3% of the total body weight2. The greatest concentrations of microbes occupy the intestine, skin and oral cavity and among the different microbiota, that of the digestive tract contains the most microorganisms in terms of number and species 2–5. Indeed, the intestinal microbiota is composed of more than a thousand species of microorganisms, such as viruses, bacteria or fungi 6–9.

First identified as vectors of disease during the 20th century 1011, it is now established that microorganisms are not only commensal, that is to say they colonize the body without causing harm, but above all symbiotic, that is to say they are beneficial to the proper functioning of the body12.

In the intestine, the microbiota is involved in a large number of functions essential for the proper functioning of the body. It is thus involved in major essential functions such as nutrition, development, immunity and well-being 13–15. The intestinal microbiota is therefore essential for the breakdown of food, the synthesis of vitamins and biomolecules useful to the body, and the absorption of fatty acids, calcium and magnesium 9,16.

Exposure to microorganisms from our birth and the appropriate assembly of the microbiota during childhood are essential processes for establishing an active immune system, necessary to prevent disease. Each individual has a distinct and unique population of microorganisms in their environment 17. Furthermore, this microbiota is also not constant throughout life, but changes significantly with age and in response to changes in diet. The intestine is constantly exposed to different elements, whether it is diet, ingested medications such as antibiotics or disease-carrying pathogens. A change in our lifestyle itself can lead to changes in the intestinal microbiota, as has been established in the case of physical activity 18.

The influence of lifestyle on the intestinal microbiota is really important. Comparing the microbiota of populations from industrialized countries with that of contemporary populations still living in a traditional way (for example, hunter-gatherers) highlights a different functional role of the microbiota between these two types of population. For example, the microbiota will no longer degrade the same elements ingested, the functional modification of the activity of the microbiota appearing to be linked to a loss of diversity of bacterial species 19,20.

The composition of the microbiota changes naturally over time, however the modifications undergone can have repercussions on health. It is now recognized that modifications of the microbiota can be involved in the development of a large number of non-infectious diseases21,22.

The relative balance between different microbial groups is therefore of paramount importance for the health of the host. We speak of dysbiosis when this balance is altered and this results in a modification of the normal functioning of the body 23. Dysbiosis can be associated with various diseases, such as inflammatory bowel diseases, type 1 diabetes, rheumatoid arthritis, asthma and obesity 24. It has also been shown that the intestinal microbiota could be involved in the development of cancers 25–27 and even influence mental health by disrupting the gut-brain dialogue 28– 30 (Figure 1).

Probiotics are live microorganisms which, when administered in adequate quantities, confer health benefits to the host 32. Currently, recognized probiotics are represented by the microbial species of lactobacilli, bifidobacteria, streptococci, saccharomyces, bacilli and enterococci. As explained previously, they are generally provided by fermented food products, non-fermented food products or food supplements 33.

Probiotics act on the host's intestinal microbiota in different ways:

• they improve the protective barrier function played by the intestine by regulating inflammation;
• they improve the selectivity of the protective barrier played by the intestine by regulating immunity;
• they increase the production of vitamins, minerals, short-chain fatty acids (SCFA) and growth regulators, thus strengthening the protective barrier played by the intestine;
• they increase acidity in the intestine, thus promoting its activity;
• they modify the composition of the intestinal microbiota.

The modifications induced by probiotics make it possible to prevent and/or treat diseases as varied as gastrointestinal diseases, hyperlipidemia and hypercholesterolemia, cancer, lactose intolerance, autoimmune diseases, bone disorders, neurodegenerative disorders. and metabolic33.

It is possible to partially control probiotic intake by consuming certain cheeses, yogurts or other fermented dairy products containing probiotics 34,35. These natural sources of probiotics nevertheless have significant limitations, as the heterogeneous composition of a healthy gut microbiota requires an adequate quantity and diversity of probiotics. Our modern lifestyle is often accompanied by very intense activity rhythms and with little time to devote to the selection and preparation of a varied and appropriate diet, which limits the maintenance of a bacterial flora. diversified 36. Another limitation of the intake of probiotics through food lies in the quantity of probiotics naturally present in foods, which is very variable and often very low. From this perspective, food supplements are becoming increasingly important because they provide a reliable, controlled and substantial source of essential probiotics.

The viability of probiotics is essential to obtain the benefits associated with their consumption. According to FAO/WHO guidelines, probiotic products must contain at least 107 colony-forming units (CFU) of vital microorganisms per gram at the expiration date to be considered effective 37. However, most clinical studies that have validated the benefits of probiotics in humans used very high doses of probiotics, ranging from 108 to 1011 CFU 38. Depending on the quantity ingested and taking into account the effect of storage on the viability of probiotics, a daily intake of 108-109 probiotic microorganisms is essential to obtain a beneficial action in the human organism, while maintaining an optimal balance between effectiveness and safety 39,40. Considering these parameters established by the scientific community, the formulation proposed by DIJO presents a very high concentration of probiotics, providing 149 live bacteria per capsule. In addition, the dosage recommended by DIJO (2 capsules per day) provides a total of 289 microorganisms per day, which places it among the richest probiotic products on the market (Table 1).

To achieve a high final concentration of probiotics, a crucial step is the final isolation of the bacteria from their culture medium. Direct freezing of concentrated probiotics may be considered sufficient when the probiotics are intended to be added to food products. However, if probiotics are to be included in a dry pharmacological preparation (capsules, tablets, sachets) and possibly mixed with other probiotic strains/species, the concentrated bacteria must be dried. The most economical drying method is spray drying, widely used in the industry thanks to its speed and inexpensive procedures. After concentration, the bacteria are sprayed as an aerosol in a hot chamber which dries the remaining water. Despite its economic advantages, this technique can produce harmful stress on bacteria, significantly reducing their availability. In particular, heat stress caused by high temperature and dehydration are the two main mechanisms leading to the inactivation and loss of viability of probiotics 41.

Instead, DIJO opted for the very low temperature freeze-drying technique, known as freeze drying, which is a more effective procedure for concentrating probiotics, although more expensive (Figure 2)42. The bacteria are first frozen below a critical temperature and then dried by vacuum sublimation in two phases: primary drying, during which unbound water is removed and secondary drying, during which bound water is removed. eliminated. The main limitation of this procedure is due to the crystallization of water and solutes during the freezing process: the solid structure of the crystals can damage the bacterial membrane and reduce the viability of the probiotics. This disadvantage is overcome during the first freezing stage by the use of cryo-preservatives which significantly improve the resistance of bacteria and reduce crystallization 42. After drying, the product can be ground to a desired particle size, which allows quantify the quantity of bacteria in the final formulation (capsules, tablets or sachets) (Figure 2)43.

Several methods have been developed to deliver probiotics to the gastrointestinal tract, such as pharmaceutical formulations and food products (Table 2).

Commercially available probiotic-enriched foods offer a preferential system for reaching customers, but it has several limitations. First, food fortification requires many steps to introduce probiotics into food. In order to ensure the quality and therapeutic effectiveness of probiotic foods, it is imperative that probiotics maintain their viability and integrity throughout the process of food fermentation, storage and consumption, when in fact, multiple environmental factors can undermine microbial survival and functionality 45. Environmental variables specific to each different food carrier (e.g. fruit juices rather than dairy products), can significantly influence the survival and effectiveness of bacteria which are often linked to short expiration date of food. All these variables prevent a clear evaluation of probiotic effects, even more so in the case of combining different probiotics.

From this perspective, food supplements are considered more effective compared to enriched food base systems 46: freeze-dried probiotic preparations (capsules, tablets or liquids) have the advantages of providing a concentrated amount of probiotics that can be maintained at the resting state in a controlled environment. This type of preparation also has greater success when combining different species/strains, as environmental factors and stress are reduced to a minimum 47.

Pure probiotic preparations, without the addition of preservatives, have the advantage of avoiding possible collateral effects such as intolerance or allergic reactions.

The limitation of these formulations is that freeze-dried bacteria are very sensitive to storage and exhibit a rapid decline in microorganism viability over time. An overall reduction of 10-15% in live cells is generally recognized for all bacterial strains freeze-dried using freeze-drying technology, but different studies have identified some strain-specific differences. For example, a preparation of Lactobacillusacidophilus without any preservative showed a drastic decrease in bacterial viability over four months of observation at any storage temperature (room temperature, refrigerated, frozen, Figure 3)48. Increased sensitivity to storage conditions also results in a reduction in the efficiency of bacteria to survive in gastrointestinal transit. Another study recently carried out on freeze-dried lactobacilli strains (L. Plantarum and L. Rhamnosus ) evaluated the survival of these strains between 2 and 6 months (Figure 4). To do this, their effectiveness/viability was measured after exposure to simulated saliva (SS), simulated gastric fluid (SGF), and simulated intestinal fluid (SIF). These different media were used to simulate oro-gastro-intestinal transit. The probiotics analyzed showed for both strains a significant and constant reduction in their ability to survive in the gastrointestinal environment. Lactobacillus strains stored for 2 months retained viability ≥ 88, 66 and 45% after consecutive exposure to SS, SS --> SGF and S-->SGF --> SIF. Lyophilized cells after 4 months of storage exhibited ≥70, 45 and 30% viability during SS, SS --> SGF and SS --> SGF --> SIF transit respectively. The survival of lyophilized cells after 6 months of storage was ≥60, 44 and 28% in SS, SS --> SGF and SS --> SGF --> SIF 49 respectively.

According to these results, a pure preparation of probiotics, like that offered by DIJO, must be consumed within a very short time after production. Indeed, local production and the reduction of steps in the distribution chain is essential for this type of product.

The composition of the microbiota changes naturally over time, however the modifications undergone can have repercussions on health. It is now recognized that modifications of the microbiota can be involved in the development of a large number of non-infectious diseases21,22.

The relative balance between different microbial groups is therefore of paramount importance for the health of the host. We speak of dysbiosis when this balance is altered and this results in a modification of the normal functioning of the body 23. Dysbiosis can be associated with various diseases, such as inflammatory bowel diseases, type 1 diabetes, rheumatoid arthritis, asthma and obesity 24. It has also been shown that the intestinal microbiota could be involved in the development of cancers 25–27 and even influence mental health by disrupting the gut-brain dialogue 28– 30 (Figure 1).

The first observations of beneficial bacteria were made by Elie Metchnikoff in 1905, proposing for the first time in history, the theory that microorganisms present in fermented yogurt could be directly linked to increased life expectancy of the Bulgarian population 50. Interest in foods enriched with probiotics grew over time, when more and more scientific publications began to support their beneficial effect on human health (Figure 5) 51. It became evident that the selection of probiotics was based on biological (food safety and functional aspects) and technical characteristics. Crucial aspects for the selection and production of probiotics were quickly identified and included: possible pathogenicity, antibiotic resistance, viability and stability during transit through the gastrointestinal tract, benefits in terms of system modulation immune system, possible antagonistic characteristics
and genetic and physiological stability 52. ​​Furthermore, the ability to adhere to the intestinal mucosa is one of the most important selection criteria for probiotics, because adhesion is considered a necessity for colonization and is a common and fundamental characteristic of all probiotics.
The global definition of probiotics was finally established during a series of joint FAO and WHO expert consultations (2001, 2002 and 2006) and it specifies that probiotics are "live micro-organisms which, when administered in adequate amounts, confer a health benefit to the host"32. Numerous scientific studies on probiotics had already been carried out at the end of the 1990s on animal models and gave encouraging results 53–55. However, clinical trials with probiotics only developed in the first decades of the 200s and grew exponentially, after the WHO recognized the potential effects of probiotic treatment on their ability to:
- restore the number of bacteria at the base naturally present in a niche, a number which may have decreased and caused disease
- counteract pathogens and disease(s), allowing the recovery of the patient and their microbial flora

Today, the effects of probiotics on the intestinal microbiota and general health are well known. Probiotics exhibit a wide range of beneficial effects both local, on the intestinal biome, and systemic, on the immunosystem and metabolism (Figure 5). Due to the heterogeneity of the organisms used in probiotic treatments, these effects may vary from one strain or species to another or be more specific for certain strains or species: the combination of different sources of probiotics becomes increasingly the best approach to achieve synergistic results 56.

Since the beginning of the 1990s, the scientific community has become increasingly aware that
combinations of different strains and species of probiotics are more effective than administering a
alone. During this period, Pesce de Ruiz Holgado's group conducted a series of experiments on
different in vivo models, during which he and his collaborator evaluated the effect of a treatment
simple and multi-strain probiotic on bacterial infections 55. In particular, they were able to show that
Lactobacillus acidophilus and Lactobacillus casei could better protect mice against Salmonella Typhimurium infection when combined, rather than individually. Mice received one of the fermented products (alone or in combination, 20% suspension in drinking water, giving a total of 2.4×109 viable organisms) for 8 days, followed by infection. oral with S. Typhimurium. Mice fed with the combination of the two probiotics showed a 100% survival rate 21 days after infection, compared to controls (fed with skimmed milk) or the single probiotic treatment (20% survival rate). The survival rate was characterized by a better immune response to the infection and better elimination of salmonella particles from the liver. These results were quickly confirmed by the publication of (Paubert-Braquet et al, 1995) 54 who observed increased resistance to S. Typhimurium in mice fed with fermented milks containing a mixture of different species of
Lactobacillus (1.1×108 of St. thermophilus, 8.2×108 of Lb. bulgaricus, 0.8×108 of Lb. casei). The observed survival was 87.5% after 14 days compared to 0% of the control group, the survival rate with treatment
Lactobacillus casei alone being significantly lower.
Today it has become clear that multi-species supplements with characteristics
different have a better chance of colonization and present synergistic effects and better
chance of survival and adhesion. Furthermore, positive relationships between strains can increase their
biological activity 57. Many studies were carried out last year to evaluate the possible effect of combinations of probiotics not only in the treatment of pathological symptoms, but
also from the perspective of preserving a healthy lifestyle. Studies on probiotic modulation of physiological parameters (body weight, blood pressure, blood sugar), sports performance and
cognitive processes are particularly interesting.

i) Combination of probiotics in the control of physiological parameters
(body weight, blood pressure and blood sugar)

Changes in dietary habits and the increased availability of foods high in
calories have made overweight and obesity one of the most serious health problems of our time.
The World Health Organization (WHO) estimated that 39% of people over the age of 18 were in
overweight, and the global prevalence of obesity has almost tripled since 1975. Nearly 2.8 million deaths
per year are the consequence of being overweight and conditions associated with obesity, such as
dyslipidemia, high blood pressure and insulin resistance, which lead to disease risk
coronary heart disease, ischemic stroke, type 2 diabetes mellitus and cancer (statistics
of WHO, 2020). All these characteristics may be linked to an imbalance in intestinal microbiota
which are the consequence of obesity and overweight 58.
Many studies have been carried out in recent years on large cohorts, but
main meta-analysis studies crossing all the results obtained agree that mixtures
Multi-strain and multi-species probiotics perform better than single strains in controlling
of body weight. (Zhang et al., 2016) analyzed a total of 25 studies involving 1931 people and found
showed that the consumption of probiotic mixtures could significantly reduce the index
body mass (-0.65 in BMI with p-value <0.01) compared to less reduction
and not statistically significant for single strains (-0.12 BMI with p-value = 0.17) (see Table
3) 59. These effects on outcomes were more evident in the obese population and over a longer period of time
administration (>8 weeks) (see Table 3). A similar conclusion was reached by the meta-analysis of
(Koutnikova et al., 2019) on larger cohorts of publications (105) involving more than 6800
patients: improvements in BMI and blood glucose were mainly observed with the combination
several strains of bifidobacteria (Bifidobacterium breve, B. longum), St. thermophilus and L. acidophilus,
L. casei, L. delbrueckii.
60

Body weight modulation is often associated with improvement of other parameters
systemic factors such as blood pressure and blood sugar. A recent meta-analysis of 9 studies
differences in the effects of probiotics on blood pressure revealed that the consumption of
probiotics significantly reduced overall systolic blood pressure by 3.56 mmHg and blood pressure
diastolic blood pressure of 2.38 mmHg compared to the control groups. This meta-analysis suggests that
the best results were obtained in reducing pre-existing hypertension conditions
(hypertensive patients), when several species of probiotics were consumed and the duration of
the intervention was ≥8 weeks61. These same composition parameters of the probiotic mixture
(multi-strain probiotics containing L. acidophilus, S. thermophilus, L. bulgaricus, and/or B. lactis) and
duration of administration also reduced blood glucose concentration in patients
with type II diabetes, since Lactobacillus, Bifidobacterium, and Streptococcus species are
known to regulate glycemic control via satiety signaling, intestinal integrity, and
antioxidant protection of pancreatic cells 62.

ii) Advantage of the combination of probiotics in the practice of sport

Gastrointestinal (GI) and upper respiratory tract (RSV) symptoms are discomfort
common among endurance athletes. Several studies have shown different results from
probiotics on these symptoms, probably linked to the probiotic strain, the dose, the period of
consumption or even the form of administration (capsules, sachets or fermented milk). However there is
a common agreement on the fact that the strains ingested in combination over a period of consumption
longer may show better results in minimizing RSV and GI symptoms 63.
Multi-strain probiotic supplements containing L. acidophilus, B. bifidum and B.
animalis ssp lactis have been tested in numerous trials, treatments lasting at least one month. These studies have
reports positive results, characterized by better preservation of the intestinal barrier in cases
prolonged efforts (Lamprecht and Frauwallner, 2012) and plasma concentrations of endotoxins higher
low 64.65. (Strasser et al., 2016), reported that multi-strain probiotics were able to reduce
the occurrence of symptoms in RSV by 2.2 times after 12 weeks in the treated group compared to the
placebo. This improvement was correlated with a significant reduction in tryptophan, phenylalanine
and their primary catabolites in the blood66.

iii) Effect of probiotics on cognitive processes and mood
The gut microbiota has been shown to actively influence proper development and

functioning of the immune system and the brain in what is today called the “microbiota-
gut-brain" (MIC). An unfavorable change in the gut microbiota disrupts the symbiosis

maintained by the MIC axis and has been associated with serious illnesses such as schizophrenia, Alzheimer's disease
Parkinson's and major depression 67. Patients suffering from this disorder often had low levels
reduced from the two main genera of probiotics, Lactobacillus and Bifidobacterium 68. Even if a
treatment with single strains allows us to better understand the function and contribution of each
probiotic, multi-strain probiotics may be more powerful in humans. In fact, different
combinations of L. acidophilus, B. bifidum, St. thermophilus, L. casei and B. bifidum have shown several
resumption of positive antidepressant effects 69,70. Positive results have also been obtained on
young students (average 20 years old) who showed improvement in panic and anxiety
neurophysiological, negative affect, worry and increased negative regulation of
mood after regularly receiving multi-strain probiotics 71. The same beneficial results
were obtained by (Akkasheh et al., 2016) where a 12-week treatment with L. acidophilus, L. casei, B.
bifidum, and Lactobacillus fermentum induced a significant improvement in cognitive functions and status
metabolism of elderly patients with Alzheimer's69.

The most popular sources of probiotics selected for their organic properties are
made up of the genera Lactobacillus, Bifidobacterium, Streptococcus, Saccharomyces, Bacillus,
Enterococcus. These species are widely used in fermented food products, products
non-fermented foods, as well as in functional food supplements and nutraceuticals 72.
Bifidobacteria and lactobacilli are the most represented in the intestinal tract of
humans and animals and, therefore, the most used in the production of fortified foods. These
two species can easily be confused, but they have metabolic differences that
make them complementary in terms of survival and functions. For example, compared to lactobacilli,
bifidobacteria are not as tolerant of acids and their growth cannot be described as
“facultative anaerobic”. Indeed, bifidobacteria produce lactic acid like lactobacilli
from the fermentation of carbohydrates, but in addition, they produce acetic acid which
requires specific catabolic pathways not shared with lactic acid bacteria.

has. Bifidobacterium

newborns. Bifidobacteria are gram-positive, curved, bifurcated (split, shaped) cells.
of X or Y) in the shape of a stick. They are mainly found in the human intestinal environment and
animal: the human intestine, the animal intestine (murine, rabbit, bovine, chicken and insect) and the oral cavity. THE
bifidobacteria are widely distributed among living organisms that provide parental care
to their offspring, such as mammals, birds and social insects, but have never been found
in other animals such as fish and reptiles. Given this exclusive presence, the
direct transmission of bifidobacterial cells from parents/caregivers to offspring could be a
important reason for their ecological distribution 73.
The genus includes 32 described species. In the manufacture of fermented milk products derived from
milk, Bifidobacterium bifidum is the most used species, while Bifidobacterium longum and
Bifidobacterium infantis are the predominant species in the stools of breastfed infants 73. B.
bifidum and B. infantis are appreciated for their ability to synthesize appreciable quantities of
certain vitamins, such as thiamine, folic acids, biotin and nicotinic acids 74.

i) Bifidobacterium bifidum

Bifidobacterium bifidum is a probiotic strain that has been used as a major ingredient for the manufacturing of nutraceutical products and as a starter dairy product since the 2000s. The various bio-functional effects and industrial application potential of B. bifidum have been characterized and proven by in vitro and in vivo studies and clinical studies (Table 4). The B. bifidum bacteria is very useful in improving metabolic processes in the intestine (such as carbohydrate metabolism, vitamin production and the breakdown of food-derived catabolites). It is also useful in strengthening the host's immune system by controlling the composition of the gut biome and directly interfering with intestinal cells. Several studies have suggested the important role of B. bifidum as an anticancer 77 and as an immune system enhancer 78,79. These observations were confirmed by clinical trials where B. bifidum showed beneficial effects in reducing symptoms of allergic/autoimmune diseases 80,81, irritable bowel syndrome 82 and alleviating cognitive degeneration in diseases linked to aging such as Alzheimer's disease 83.

ii) Bifidobacterium infantis (32624)

Bifidobacterium longum subsp infantis 35624 (B. infantis) was originally isolated from healthy human gastrointestinal tissues resected approximately 15 years ago. It is the strain most represented in the intestine of infants. In the gastrointestinal mucosa, epithelial cells are the first to encounter microbes. B. infantis has been shown to adhere to gastrointestinal epithelial cell lines, without inducing NFKB activation or chemokine secretion93, suggesting that this microbe exerts immunomodulatory effects on intestinal cells that mediate pro-inflammatory responses. -host inflammation to enteric pathogens 94.

The most studied effects of B. infantis concern its ability
to modulate the host immune response by directly differentiating
the immature lymphocytic population (T cell; resident cells of the thymocyte), via a dendritic cell interaction, towards the T-REG cells, involved in the tolerance mechanism. As a result, differentiation towards immunoreactive lymphocytes (TH, T helper cells) is significantly reduced under the effect of B. infantis. This observation was validated by the administration of B. infantis to healthy volunteers: in fact, the treated group showed a significant increase in T-REG cells in the blood compared to the control group as well as a consistent reduction in cytokines. circulating inflammatory diseases 95. The clinical application of these results in human patients has shown a positive effect on pathologies linked to increased activation of the immune system and inflammation. In particular, administration of B. infantis improved symptoms during
allergic reactions 96.97, in patients suffering from irritable bowel syndrome in adults 97, diarrhea and intestinal infection in newborns 98.99 (Figure 7).

iii) Bifidobacterium longum

Bifidobacterium longum is the most common bacteria present in newborns and represents, along with other Bifidobacterium species, up to 90% of the bacteria in an infant's gastrointestinal tract which drops to 3% in the gastrointestinal tract. -intestinal of an adult 101. B. longum is considered a versatile bacterium because it is capable of metabolizing several substrates. This ability is linked to the high number of genes associated with oligosaccharide metabolism acquired as a result of gene duplications and horizontal gene transfers, indicating that B. longum is under selective pressure to increase its ability to compete for various substrates in the gastrointestinal tract102.
The carbohydrate metabolic capabilities of the longum subspecies appear to be more oriented toward the metabolism of complex plant carbohydrates and do not have the same ability as the infantis subspecies to degrade human breast milk oligosaccharides. 103. Some strains of B. longum have been found to have a high tolerance to stomach acid and bile, suggesting that these strains could
survive in the gastrointestinal tract to colonize the small and large intestines. In fact, longum is also rich in bile salt hydrolases allowing the hydrolysis of bile salts into amino acids and
in bile acids. This function is not yet completely clear, although B. longum may use amino acid products to better tolerate bile salts.104
Recent clinical trials of B. longum have shown beneficial effects in the treatment of patients with irritable bowel syndrome 105,106. Different in vivo studies suggest a possible role of this bacteria in the modulation of the autoimmune response in models of type II diabetes and immunological pathologies due to gluten intolerance107,108.

b. Lactobacillus

The genus Lactobacillus includes more than 200 species characterized by phylogenetic and metabolic diversity that exceeds that of a typical bacterial family. These bacteria are very dependent on nutrients, such that they are preferentially found in the food processing organs of humans, rodents, farm animals, but also insects and birds 109 (Figure 8). Lactobacilli can be subdivided into subfamilies based on their dependence on the host: free-living bacteria are found in the environment (grass, soil) and have a relatively large set of genes ; nomadic bacteria are not autonomous and colonize different organs of the host and they can migrate into different niches; finally, host-specific bacteria that are highly adapted to function in specific hosts 110. These metabolic behaviors reflect the size of their genome: the large genome of free-living and nomadic groups gives them more flexibility and flexibility. adaptability, while the limited genome of host-specific genes gives them higher metabolic efficiency, but limited to a specific environment. In addition to the common characteristics of lactobacilli metabolism, certain characteristics are specific to each subtype. In the group of nomads, we find certain species of Lactobacillus, such as L. plantarum, L. casei, L. paracasei and L. rhamnosus, which, although not autochthonous in the strict sense, have adaptations to intestinal ecosystems and the cavity oral, allowing them to persist at least for a limited time.

The most commonly used probiotic bacteria belong to lactic acid bacteria, and more particularly to those of the ancient genera Lactobacillus and Enterococcus111. The production of bioactive peptides from lactic acid bacteria is often linked to health benefits. Bioactive peptides are released from proteins by microbial or non-microbial enzymatic hydrolysis 112. Bioactive peptides can act as immune modulators 113, and are antihypertensive by inhibiting angiotensin converting enzyme (ACE) 114, such as in yogurts115, and antioxidants as in sourdough 116 and yogurt too117. Antioxidant peptides have been found in soy milk fermented by lactic acid bacteria and can effectively scavenge free radicals118. Antioxidant properties can also be the consequence of the production of extracellular polysaccharides by bacteria which secrete them into the lumen of the digestive tract 119.
Other health benefits are linked to consuming products fermented using probiotic strains of lactic acid bacteria. Indeed, the consumption of yogurt gave interesting results in relation to type 2 diabetes in a meta-analysis of the consumption of dairy products 120. Lactic acid bacteria also produce antimicrobial compounds whose properties may also contribute to the establishment of probiotic strains in the host 121 and thus counter pathogenic bacteria in the gastrointestinal tract.

i) Lactobacillus casei group (LCG): Lactobacillus casei, paracasei and rhamnosus

The Lactobacillus casei group (LCG), consisting mainly of the closely related species L. casei, L. paracasei and L. rhamnosus, is one of the most studied species due to its commercial, industrial and health potential. Commercially, they are used to ferment dairy products, often improving the flavor and texture of foods. LCG bacteria present several intrinsic advantages which make them one of the most exploited probiotics: in fact, LCG have a particular metabolic characteristic which gives them great resistance to the acidic environment of the intestine 1, to the oxidative stress derived metabolism 123 and osmotic and temperature variations during production processes 124,125.
The mechanisms by which these bacteria have, directly or indirectly, a beneficial effect on human health are not yet fully understood and require further study. Potential mechanisms include production of antimicrobial substances such as bacteriocins, strengthening of the epithelial barrier through attachment, competition for pathogen binding sites, or modulation of the immune system [ 126 ].
LCG bacteria have been widely studied for their potential properties in improving human health in different areas, such as allergies, obesity cancer and dysregulation.
of the intestinal microbiota (Table 5) 127.

ii) Lactobacillus plantarum

Lactobacillus plantarum is a very diffuse Lactobacillus, present in a wide spectrum of products
dairy, vegetables, meat, silage, wine and in the gastrointestinal, vaginal and urogenital tracts 140. The ubiquitous presence of L. plantarum is supported by high adaptive capacities and metabolic pathway 141. L . plantarum was initially selected for its ability to maintain the integrity of dairy products against contaminating microorganisms 142. These antifungal and antimicrobial functions rely on the high production of a particular class of bacterial toxins, bacteriocins, which rendered L. plantarum is interesting for both the food industry and probiotic supplements. Indeed L. plantarum colonizes the gastric tube of the host and offers protection against gastrointestinal pathogens (Table 6) 143.
L. plantarum has shown an important effect in reducing the effect of certain human pathologies affecting the fatty-intestinal tract. Clinical studies have shown the beneficial effects of L. plantarum in antibiotic-derived diarrhea 144, diabetes and fat metabolism 145.
Other evidence links gut microbial population homeostasis to mental well-being. In this perspective, the use of L. plantarum on patients with major depression, in addition to standard chemical treatment, has been shown to be effective in reducing blood kynorunin concentration and improving cognitive functions 146,147.

iii) Lactobacillus acidophilus

Lactobacillus acidophilus belongs to the specific group of Lactobacilli and presents important differences compared to the group of L. casei and L. Plantarum. In fact, L. acidophilus presents a limited genome, compared to nomadic lactobacilli, which provides a set of genes conferring high survival efficiency in a limited number of niches. As its name suggests, it easily colonizes an acidic environment that can be found, in the human body, in the gastric tube (from the cavity
oral to the colon) and in the vaginal environment. This species of bacteria is one of the first to be extensively studied for applications in dairy products and health improvement,
presenting a large literature already before the 2000s 154. L. acidophilus present characteristics common to other lactobacilli, such as better adhesion to intestinal epithelial cells (Buck et al., 2005), extreme resistance to acids and salts bile 155 and
production of antibiotic bacteriocin 154.

L. acidophilus has been tested in clinical trials that show how it can influence systemic homeostasis and improve symptoms of several metabolic syndromes. Consumption of probiotics containing L. acidophilus in combination with Bifidobacterium species reduces bloating in adults with functional intestinal disorders 156. The antibiotic and anti-inflammatory properties of L. acidofilus have helped reduce the incidence and duration of symptoms, incidence of colds and flu with prescribed antibiotics in children aged 3 to 5 years 157. L. acidophilus supplemented with other lactobacilli has also been shown to be effective in reducing symptoms of bacterial pathogens in vaginosis 158 and Clostridium difficile infections159. In vitro experiments have also suggested an anticancer effect, correlated with the production of conjugated linoleic acid and the possible elimination of pro-carcinogenic metabolic byproducts 160.

vs. Lactococcus lactis

Lactococcus is a genus of lactic acid bacteria and is known as a homofermenter because it produces lactic acid almost exclusively as a result of glucose fermentation. Its homofermentative character can be modified by adjusting environmental conditions such as pH, glucose concentration and nutrient limitation. Recently, Lactococcus lactis have evolved from agents used in the food industry to qualified vehicles for mucosal drug delivery due to their evolved ability to bind mucosal surfaces and modify immune responses (Figure 9). 161. Many modified strains have been produced in recent years with the aim of directly delivering proteins and DNA to the mucosa of the respiratory and digestive tracts in order to improve mucosal tolerance. This process involves the ability of the antigen
administered through the mucosa to regulate local and systemic immune responses162.
Numerous experimental tests have been carried out in recent years with encouraging results. For example, L. lactis NCDO 2118 reduces symptoms of recurrent colitis in an in vivo model of induced colitis. Bacteria-enhanced early IL-6 production plays a role in enhancing mucosal repair and preserving colonic IL-10 production, which could be responsible for important effects anti-inflammatories 163. In addition, oral treatment with the same
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