**2. LAB as probiotic agents**

### **2.1. Overview of probiotics**

The most tried and tested manner in which the gut microbiota composition may be influenced is through the use of live microbial dietary additions, as probiotics. In fact, the concept dates back as far as prebiblical ages. The first records of ingestion of live bacteria by humans are over 2,000 years old. However, at the beginning of this century probiotics were first put onto a scientific basis by the work of Metchnikoff (1908). He hypothesised that the normal gut microflora could exert adverse effects on the host and that consumption of 'soured milks' reversed this effect. The word ''probiotics'' was initially used as an anonym of the word ''antibiotic''. It is derived from Greek words pro and biotos and translated as ''for life''. The origin of the first use can be traced back to Kollath (1953), who used it to describe the restoration of the health of malnourished patients by different organic and inorganic supplements. Later, Vergin (1954) proposed that the microbial imbalance in the body caused by antibiotic treatment could have been restored by a probiotic rich diet; a suggestion cited by many as the first reference to probiotics as they are defined nowadays. Similarly, Kolb recognized detrimental effects of antibiotic therapy and proposed the prevention by probiotics (Vasiljevic and Shah, 2008) Later on, Lilly and Stillwell (1965) defined probiotics as "…microorganisms promoting the growth of other microorganisms". Following recommendations of a FAO/WHO (2002) working group on the evaluation of probiotics in food, probiotics, are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host (Sanders, 2008; Schrezenmeir and De Vrese, **2001)**. The idea of health-promoting effects of LAB is by no means new, as Metchnikoff proposed that lactobacilli may fight against intestinal putrefaction and contribute to long life. Such microorganisms may not necessarily be constant inhabitants of the gut, but they should have a "…beneficial effect on the general and health status of man and animal"

species, but are mostly lactic acid bacteria (Mercenier et al, 2003).

**2. LAB as probiotic agents** 

**2.1. Overview of probiotics** 

health. Since their discovery, LAB has been gained mush interest in various applications, as starter cultures in food and feed fermentations, pharmaceuticals, probiotics and as biological control agents. In food industry, LAB are widely used as starters to achieve favorable changes in texture, aroma, flavor and acidity (Leory and De Vuyst, 2004). However, there has been an important interest in using bacteriocin and/or other inhibitory substance producing LAB for non-fermentative biopreservation applications. Du to their antimicrobial and antioxidant activities some LAB strains are used in food biopreservation. However, LAB are generally regarded as safe (GRAS) to the consumer and during storage, they naturally dominate the microflora of many foods (Osmanağaoğlu and Beyatli, 1999; Parada et al., 2007). Many of the indications for probiotic activity have been obtained from effects observed in various clinical situations. Even, there are few strains that have officially gained the status of pharmaceutical preparation; each of these effects is gradually being supported by a number of clinical studies or human intervention trials, performed in a way that resembles the traditional pharmacological approach (placebo-controlled, double blind, randomized trials) and the strains used in these studies belong to different microbial

The most tried and tested manner in which the gut microbiota composition may be influenced is through the use of live microbial dietary additions, as probiotics. In fact, the concept dates back as far as prebiblical ages. The first records of ingestion of live bacteria by humans are over 2,000 years old. However, at the beginning of this century probiotics were first put onto a scientific basis by the work of Metchnikoff (1908). He hypothesised that the normal gut microflora could exert adverse effects on the host and that consumption of 'soured milks' reversed this effect. The word ''probiotics'' was initially used as an anonym of the word ''antibiotic''. It is derived from Greek words pro and biotos and translated as ''for life''. The origin of the first use can be traced back to Kollath (1953), who used it to describe the restoration of the health of malnourished patients by different organic and inorganic supplements. Later, Vergin (1954) proposed that the microbial imbalance in the body caused by antibiotic treatment could have been restored by a probiotic rich diet; a suggestion cited by many as the first reference to probiotics as they are defined nowadays. Similarly, Kolb recognized detrimental effects of antibiotic therapy and proposed the prevention by probiotics (Vasiljevic and Shah, 2008) Later on, Lilly and Stillwell (1965) defined probiotics as "…microorganisms promoting the growth of other microorganisms". Following recommendations of a FAO/WHO (2002) working group on the evaluation of probiotics in food, probiotics, are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host (Sanders, 2008; Schrezenmeir and De Vrese, **2001)**. The idea of health-promoting effects of LAB is by no means new, as Metchnikoff proposed that lactobacilli may fight against intestinal putrefaction and contribute to long life. Such microorganisms may not necessarily be constant inhabitants of the gut, but they should have a "…beneficial effect on the general and health status of man and animal"


Abbreviations: DNBS, dinitrobenzene sulfonic acid; DSS, dextran sodium sulfate; IL-10, interleukin 10; NA, not available; TNBS, trinitrobenzene sulfonic acid.

**Table 1.** Selected organisms that are used as probiotic agents (Gareau et al., 2010).

**(**Holzapfel et al., 2001; Belhadj et al., 2010). Other definitions advanced through the years have been restrictive by specification of mechanisms, site of action, delivery format, method, or host. Probiotics have been shown to exert a wide range of effects. The mechanism of action of probiotics (e.g, having an impact on the intestinal microbiota or enhancing immune function) was dropped from the definition to encompass health effects due to novel mechanisms and to allow application of the term before the mechanism is confirmed. Physiologic benefits have been attributed to dead microorganisms. Furthermore, certain mechanisms of action (such as delivery of certain enzymes to the intestine) may not require live cells. However, regardless of functionality, dead microbes are not probiotics (Sanders, 2008). In relation to food, probiotics are considered as "viable preparations in foods or dietary supplements to improve the health of humans and animals". According to these definitions, an impressive number of microbial species are considered as probiotics. (Holzapfel et al., 2001). For gastrointestinal ecosysteme, however, the most important microbial species that are used as probiotics are lactic acid bacteria (LAB) (Table 1).

Lactic Acid Bacteria as Probiotics:

Characteristics, Selection Criteria and Role in Immunomodulation of Human GI Muccosal Barrier 201

proliferate and colonise at this specific location. Probably only host-specific microbial strains are able to compete with the indigenous microflora and to colonise the niches. Besides, the probiotic strain must be tolerated by the immune system and not provoke the formation of antibodies against the probiotic strain. So, the host must be immuno-tolerant to the probiotic. On the other hand, the probiotic strain can act as an adjuvant and stimulate the immune system against pathogenic microorganisms. It goes without saying that a probiotic has to be harmless to the host: there must be no local or general pathogenic, allergic or mutagenic/carcinogenic reactions provoked by the microorganism itself, its fermentation

Pathogenicity and infectivity

Good sensory properties

Adhesion to mucosal surface

Validated and documented health effects

Antagonistic activity towards gastrointestinal

Antimutagenic and anticarcinogenic properties

pathogens, i.e., Helicobacter pylori, Candida albicans

Phage resistance Large-scale production

Bile tolerance

Immunomodulation

**Table 2.** Key and desirable criteria for the selection of probiotics in commercial applications (Vasiljevic

When probiotic strains are selected, attributes important for efcacy and technological function must be assessed and a list of characteristics required for all probiotic functions is required. Basic initial characterization of strain identity and taxonomy should be conducted, followed by evaluation with validated assays both in studies of animal models and in controlled studies in the target host. In vitro assays are frequently conducted that have not been proved to be predictive of in vivo function. Technological robustness must also be determined, such as the strain's ability to be grown to high numbers, concentrated, stabilized, and incorporated into a nal product with good sensory properties, if applicable, and to be stable, both physiologically and genetically, through the end of the shelf life of the product and at the active site in the host. Assessment of stability can also be a challenge, since factors such as chain length and injury may challenge the typical assessment of colony-

Cholesterol metabolism Lactose metabolism

Virulence factors—toxicity, metabolic activity and

intrinsic properties, i.e., antibiotic resistance

Desired viability during processing and storage

products or its cell components after decrease of the bacteria (Desai, 2008).

Technological criteria Genetically stable strains

Functional criteria Tolerance to gastric acid and juices

**General Property** Safety criteria Origin

Desirable physiological

criteria

and Shah, 2008).
