**3.1 Metabolic activity as a factor of organoleptic properties of fermented dairy products**

In the manufacturing process, starters are added for lactose fermentation, lipolysis (fat degradation), and proteolysis [29]. LAB that are naturally found in milk have a major role in the fermentation processes alongside the intentionally added starter cultures. The fermentation improves the general characteristics of the end product including texture and consistency, aroma, flavor, and the development of color. The intricate biochemical processes during fermentation involve many different enzymatic reactions. With an understanding of the strain/species specificity, a large screening for metabolic activity of newly isolated Bulgarian strains was carried out. Results with identified *L. plantarum* strains from cheese (unpublished data) and [20, 23, 24, 30] are summarized (**Figure 4**).

The produced LAB enzymes responsible for lipolysis and proteolysis in milk are among the key factors for the sensory qualities of taste and texture of cheese [31]. LAB possess different degrees of lipolysis, which is important for the selection of strains that can be used for starter cultures. During lipolysis, triglycerides hydrolyze into mono- and diglycerides, free fatty acids, and glycerol. The reduced glycerides participate in coagulation processes with different components of dairy foods that lead to the texture development of the final product [32] (Esteban-Torres et al., 2014). This characteristic is related to the flavor development of fermented dairy products [33].

During proteolysis, hydrolysis of the protein peptide bonds occurs and transforms them into peptides and free amino acids. Although many LAB are considered to have weak proteolytic activity, they possess complex proteinase/peptidase systems comprising peptidases on the cell wall initiating the degradation processes, peptide transporters, and intracellular peptides that break down peptides into shorter molecules and free amino acids [34]. Thus, essential amino acids may have accumulated in fermented products, due to the high peptidase activity (**Figure 4**) estimated for *L. plantarum* strains. For 8 of tested L. plantarum strains from white-brined cheese, accumulation of free amino acids from 0.170 to 0.609 mMGly/L was shown. The accumulated free amino acids are involved in reactions of deamination, transamination, decarboxylation, and desulfurization with an impact on the flavor profile of the end product [34]. At the same time, these 8 strains were characterized by low proteolytic activity. With a sample screening protocol [35], using milk agar and calcium caseinate agar (Fluka), we differentiated with low proteolytic activity all strains generating clear zone 1–8 mm, moderate zone 8-13 mm, and high zone >13 mm.

*From Traditional Bulgarian Dairy Products to Functional Foods DOI: http://dx.doi.org/10.5772/intechopen.108998*

#### **Figure 4.**

*Enzymatic activity of 31 L. plantarum strains isolated from artisanal Bulgarian dairy products (semiquantitative analysis with the API ZYM test strips, bioMérieux, France).*

Despite high peptidase activity (**Figure 4**), only 2 out of the 31 tested exponential *L. plantarum* cultures possessed high activity. The best results were obtained with 72 h cell-free supernatants (CFS) of *Lactobacillus* cultures. *L. bulgaricus* with yoghurt origin showed fast milk coagulation in combination with moderate to high proteolytic activity. Peptidase as well as β-galactosidase activity is promising for candidate probiotics, while other enzyme activities may contribute to organoleptic characteristics and stability of the products.

#### **3.2 Antagonistic LAB activity and food preservation**

It is well known that contamination of different types of foods with filamentous fungal microorganisms, yeast, and bacteria is the main reason for food spoilage. The presence of unwanted species in dairy products can cause different types of deterioration of organoleptic properties [36]. Also, molds such as *Aspergillus* and *Penicillium* can produce mycotoxins [37]. More than 60 yeast species have been identified as spoilage agents of dairy products, represented most frequently by a high diversity of *Candida* spp., as well as genera *Kluyveromyces*, *Geotrichum*, and *Yarrowia lipolytica* and phylum *Basidiomycota*, mainly *Cryptococcus* and *Rhodotorula* species [38]. Dairy product spoilage by yeasts results in visible alteration due to growth on the surface of the product, unpleasant changes in odor, flavor, or texture and production of different metabolites such as CO2, alcohols, aldehydes and esters, proteolytic and lipolytic enzymes [36], and biogenic amines [39].

LAB can be used not only as starter cultures but also as protective cultures to improve the safety and/or shelf life of the product [40]. Their preservative action is due to the combined action of a number of antimicrobial metabolites produced during the fermentation process. LAB produce a large range of antimicrobial substances, including organic acids (lactic, acetic, etc.), fatty acids, antifungal peptides, reuterin, and bacteriocins [41–44]. Formally, the metabolites produced by LAB can be divided into two main groups: substances with a low molecular mass < 1000 Da and substances with a high molecular mass > 1000 Da, such as bacteriocins/bacteriocin-like inhibitory substances (BLIS). To be defined as food-grade bioprotective cultures, LAB strains are selected according to their antimicrobial properties.

As bioprotective cultures, LAB are expected to possess antibacterial/antifungal activity that is exhibited and maintained throughout the manufacturing process and storage time, to have no impact over the functions of the starter cultures, not to modify the organoleptic properties of the final product, to be used with the lowest possible inoculum that maintains the same activity to reduce the cost value, and to have easy propagation and resistance to technological processes [38]. In the later stages of ripening, *lactobacilli* are well adapted to the environment inside the cheese, withstanding the low pH, high salt concentration, absence of sugars, and anaerobic conditions, and they may produce BLIS.

*Lactobacillus*, as well as *Leuconostoc, Lactococcus, Pediococcus,* and *Weissella*, are the most frequently cited genera to possess antifungal properties and have been the most evaluated *in situ* in recent years as well [45]. Many *Lactobacillus* species, including *L. plantarum, Levilactobacillus brevis, L. casei,* and *Lacticaseibacillus paracasei*, were shown to exhibit antifungal activity against a large spectrum of fungal representatives, including *Penicillium, Kluyveromyces, Candida, Rhodotorula*, *Aspergillus,* etc., which are among the most common spoilage microorganisms in dairy products [46–52]. Other lactobacilli, such as *Limosilactobacillus fermentum, Lacticaseibacillus rhamnosus, Schleiferilactobacillus harbinensis, L. helveticus,* and *Lactobacillus amylovorus*, were shown to be able to extend the shelf life when added as adjunct cultures in yoghurt and cheese [49, 53–55].

The well-expressed profile of antagonistic activity of the autochthonous LAB microbiota of traditional fermented foods against a number of pathogens and foodassociated contaminants is established. Our strains, isolated from yoghurt, whitebrined cheese, and "Kashkaval," show а strain-specific broad spectrum of activity against Gram (+) and Gram (−) food spoilage microorganisms and clinical pathogens (**Figure 5**).

#### **Figure 5.**

*Antagonistic activity of strains isolated from artisanal Bulgarian dairy products against food-associated pathogens and contaminants.*

*From Traditional Bulgarian Dairy Products to Functional Foods DOI: http://dx.doi.org/10.5772/intechopen.108998*

Initial results with *L. plantarum* strains from white-brined cheese were highly promising [23]. In the presence of 5% (v/v) CFS from 12 newly isolated strains from cheese and yoghurt cultures, the growth of *Pseudomonas aeruginosa* PA01 (from a patient with cystic fibrosis) was significantly inhibited up to 50–80% (unpublished data). A group of active strains belonging to the species *L. plantarum* and *L. rhamnosus* from dairy products inhibited *Staphylococcus aureus* MRSA as well as out-patient antibiotic-resistant strains [56]. In situ results of antibacterial/antifungal activity of whey fractions, skimmed milk, cow milk, and soya milk are also important [24]. For the strains of LAB, which form the microbiota of traditional fermented foods, a well-expressed profile of antagonistic activity against a number of pathogens and food-associated contaminants is established. Our isolated strains from yoghurt, white-brined cheese, and "Kashkaval" show antibacterial activity against Gram (+) and Gram (−) bacterial representatives, but their antifungal activity is also well expressed (**Figure 5**).
