**6.** *Lactobacillus bulgaricus* **– The contribution to modern healthy nutrition**

gut microbiota can also enhance host immunity through MyD88-independent mechanisms (MyD88 – Myeloid differentiation primary response gene 88). Notably, colonization of GF mice by commensal bacteria induces development of Th-17 cells in the intestine, which is important

**Figure 1.** Probiotic modulation of the gastrointestinal mucosal immune system. While intestinal epithelial cells (IECs) exposed to pathogenic microbes or related stimuli produce proinflammatory mediators such as interleukin 8 (IL-8) and tumour necrosis factor a (TNF-a), probiotics suppress the production of these cytokines and instead induce anti-in‐ flammatory mediators such as transforming growth factor b (TGF-b) and thymic stromal lymphopoietin (TSLP), which can promote the differentiation of immature dendritic cells (iDCs) to regulatory dendritic cells (DCregs). Macrophages in the inflamed mucosa produce high amounts of IL-6, and probiotics can decrease their IL-6 production and increase

Probiotics regulate host innate and adaptive immune responses by modulating the functions of dendritic cells, macrophages, and T and B lymphocytes. Probiotics regulate immunomo‐ dulalory functions through the activation of toll-like receptors, which is one of the mechanisms of regulation. Recent studies indicate that probiotics activate innate immunity by enhancing adaptive immune response [20, 53]. One of the proposed mechanisms is by activation of toll-

Regulatory dendritic cells are the primary professional antigen presenting cells (APCs) modulating adaptive immune responses. Probiotics containing *L. acidophilus, L. casei, L. reuteri, E. bifidium,* and *Streptococcus thermophilus*, stimulate dendritic cells to produce IL-10, TGF-β, COX-2, and indoleamine 2,3-dioxygenase, which in turn increase the formation of CD4 Foxp3 regulatory T cells (Tregs) and the suppressor activity of naturally occurring CD4 CD25 Tregs. They also decrease responsiveness of T and B lymphocytes and the number of T helper (Th) 1, Th2, and Th17 cytokines without inducing apoptosis. This mixture suppressed 2,4,6-

for protection against *Citrobacter rodentium* infection [52].

**5. Immune cells**

IL-10 production.

202 Immunopathology and Immunomodulation

like receptors.

*Lactobacillus bulgaricus* is the only probiotic microorganism named after a certain territory and nation. It only multiplies in the region of modern Bulgaria, coinciding with ancient Thrace. It mutates and stops its multiplication after 1-2 fermentations in other regions of the world. Bulgarian traditional food comprises *Lactobacillus bulgaricus. Lactobacillus bulgaricus* is included in the production of Bulgarian food products based on lactic acid, such as yogurt, feta yogurt, white brine cheese, other cheeses and cream, humanized baby food, probiotic functional foods, and whole food supplements [56-58].

*Lactobacillus bulgaricus* was known to the Thracians – the ancient population that lived in what is at present Bulgarian land, more than 7–8 thousand years ago. The word yogurt is Thracian and means hard, solid milk. During his tour in Thrace, the Greek scientist Herodotus (484–425 BC) wrote that the Thracians prepare special fermented dairy food, which is a gift from their Gods [59].

The father of probiotics and Nobelist – Ilya Metchnikoff (the Russian scientist) attributes the long and healthy life of Bulgarians largely to the yogurt consumption and in particular to the local bacterium in yogurt in his work "Prolongation of Life" [Figure 2].

In 1905 in Geneva, the Bulgarian student Stamen Grigorov isolated *Lactobacillus bulgaricus*from yogurt, brought from Bulgaria [Figure 3].

BSS (Bulgarian State Standard) for yogurt was established in Bulgaria in compliance with European standards of origin. Responding to this standard, the lactic acid fermentation should be accomplished using only *Lactobacillus bulgaricus* and *Streptococcus thermophilus*. Yogurt is a

**Figure 2.** Prof. I. Metchnikoff

**Figure 3.** Dr Stamen Grigorov

fermented milk product with the typical bacterial cultures *Lactobacillus bulgaricus* and *Streptococcus thermophilus* (the standards of identity, published in the US Code of Federal Regulations).

In Commission Regulation (Elf) No 432/2012 in the list of the permitted health claims made on foods, the only probiotic microorganisms included are *Lactobacillus bulgaricus* and *Streptococcus thermophilus*. According to the claim, in doses over 108 colony forming units, they improve lactose digestion [60].

In the 1990s, *Lactobacillus bulgaricus* was used in the production of probiotic functional food for astronauts, which was tested during the flight of the second Bulgarian astronaut in Space [61],

Until recently it was considered that *Lactobacillus bulgaricus* can be isolated from plants – dogwood, rose cup, etc. Bulgarian scientists, N. Alexandrov and D. Petrova have isolated *Lactobacillus bulgaricus* and other fermented milk probiotic microorganisms from spring water in Bulgaria [62, 63]. There is experimental and clinical scientific evidence from around the world of the beneficial effects of *Lactobacillus bulgaricus* in stimulating the immune system, regulating functions and microbial flora of the gastrointestinal tract, including diarrhea and dysbacteriosis, reducing the risk of cancer, radiotherapy protecting effect, regulating choles‐ terol levels, competitive inhibition of pathogenic strains causing infections, alleviating lactose intolerance, and anti-mutagenic effect. *Lactobacillus bulgaricus* has a beneficial effect on the health of women because of its similarity to the Doederlein flora in the vagina of the woman [64]. *Lactobacillus bulgaricus*, unlike other probiotic microorganisms, which secrete only L (+) lactic acid, secretes L (+) and D (-) lactic acid during fermentation. This determines its powerful antioxidant and anticancer effect. *Lactobacillus bulgaricus* produces antimicrobial substance, known as Bulgariacan, which is resistant to heat and active against highly virulent (pathogenic) strains of microorganisms [56, 57]. It has been shown that *Lactobacillus bulgaricus* adheres best on the colonic mucosa, followed by a rapid reproduction in the body. This is crucial for its more powerful treatment and detoxic effect than other lactic acid bacteria probiotics.

*Lactobacillus bulgaricus* is reproduced best with *Streptococcus thermophilus*. To gain rapid biomass and biologically active substances and to potentiate their effect, joint cultivation of *Lactobacillus bulgaricus* and *Streptococcus thermophilus* is needed. Lots of scientific studies of yeasts, containing *Lactobacillus bulgaricus* and *Streptococcus thermophilus*, used in yogurt production, were carried out in Bulgaria. The results show that these specific bacteria of yogurt are stable and survive after passage through the human gastrointestinal tract.

Certain Bulgarian companies are using modern biotechnology, and wrap strains of *Lactobacil‐ lus bulgaricus* and other probiotic microorganisms with a natural coating, consisting of components of the growing medium, during fermentation. This helps the production of probiotics and the isolation of the new probiotic strains from spring water, which normally survives under changing climatic conditions. Theses probiotic microorganisms should retain their stability and vitality when passing through the gastrointestinal tract, as well as when stored in shops at temperatures of up to 24o C for the entire shelf life.

*Lactobacillus bulgaricus* is the ancient contribution of mankind to modern science and to the creation of the first healthy foods in the world.
