**3.5. Applications**

578 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

(1492R) 97% *P. acidilactici* UL5; 99% *P. acidilactici* DSM20284 (27F) 99% *P. acidilactici* LAB 001; 99% *P. acidilactici* DSM20284

(1492R) 97% *P. acidilactici* UL5; 99% *P. acidilactici* DSM20284 (27F) 99% *P. acidilactici* LAB 001; 99% *P. acidilactici* DSM20284

(1492R) 99% *P. acidilactici* L94; 99% *P. acidilactici* DSM20284 (27F) 98% *P.acidilactici* JS-9-4; 99% *P. acidilactici* DSM20284

(1492R) 98% *P. acidilactici* UL5; 99% *P. acidilactici* DSM20284 (27F) 97% *P. acidilactici* LAB 001; 99% *P. acidilactici* DSM20284

**Table 2.** Identification and bacteriocin gene determination of putative *Pediococcus acidilactici* through

Purification of bacteriocin peptides or small proteins into homogeneity is necessary in order to fully characterize them, particularly the determination of molecular mass, the primary structure or amino acid sequence and secondary structure. For pediocin, it was found that a simple and rapid method is effective for its purification. This method involves adsorption of pediocin onto the cell wall of the producer cell at pH 6 and 0.05 M NaCl and then subsequent desorption at pH 2.0 and 1 M NaCl (Elegado et al., 1997; Yang et al., 1992). This method seemed more applicable to pediocin but not with the lactococcin, nisin or plantaricin. The reason is not clear but it could be related to variation in cell wall properties. The pH-adsorption/desorption method was able to provide materials for pH and temperature tolerance assays, estimation of molecular mass through SDS-PAGE, residual activity determination after protease, amylase and other enzyme actions (Laxamana et al., 2011). Enough amount of semi-purified bacteriocin from pediococci using this method was obtained for further purification through preparative reverse phase HPLC for various characterization studies, including the determination of secondary structures by circular dichroism and confirmation of double bonds through trypsin digestion and electrospray mass spectrometry (Elegado and Kwon, 1998). Other preparative purification methods prior to reverse phase HPLC and spectrometry included ion exchange chromatography and gel

(doubtful) - Abuel (2007) ped+ ; plan+ ped+

100% *P. acidilactici* UL5 Elegado and

Villarante (2011); Elegado and Perez (2012)

Apaga (2012)


Apaga (2012)

Apaga (2012) ped+

Perez (2012) ped+

ped+ ; plan+ ped+ ; REP

ped+ [99% *P. acidilactici* genomic scaffold]; plan+

ped+ [99% pediocin operon; pSMB72];

API: *Pediococcus pentosaceus*(good) partial 16S rRNA gene ID: *P. acidilactici*

API: *P. acidilactici* (doubtful); partial 16S rRNA gene ID: *P. acidilactici*

partial 16S rRNA gene ID: *P. acidilactici*

API: *Lactococcus lactis* (good) partial 16S rRNA gene ID: *P. pentosaceus*

partial 16S rRNA gene ID: *P. acidilactici*

partial 16S rRNA gene ID: *P. acidilactici*

16S rRNA and pediocin gene PCR amplification and sequencing.

**3.3. Purification and characterization of bacteriocins** 

K2A2-1 API: *P. acidilactici*

K2A2-3

K2A2-5

K2A1-1

K3A2-2

K3A2-3

S3

Pediocins and plantaricins are the commonly found bacteriocins in Philippine fermented foods so far studied. Their antimicrobial properties have been investigated in several studies (Banaay et al., 2004; Elegado et al., 2003, 2004, 2007; Marilao et al., 2007). Although pediocins

and plantaricins show promise, their applications are limited at present because it is a wellknown fact that other bacteriocins aside from nisin are not yet approved for food use. For pediocins and plantaricins, the most practical use for now would be dermatological and animal health care use. But since the bacteriocin-producing LAB are of GRAS status, those with probiotic properties such as tolerance to acidic pH (2.0 -3.0) and bile (0.3%) and adhesion properties to intestinal mucosa would be an advantage when used as adjunct inocula in fermented food products (Gervasio and Lim, 2007).

Lactic Acid Bacteria in Philippine Traditional Fermented Foods 581

technologies and cultures used for the production of some traditional as well as developed technologies that have arisen from the culture-based studies conducted in earlier years.

In recent years culture-based approaches in LAB isolation have become more targeted for detection of bacteriocin-producers and those that have potential as probiotics. In one initiative, LAB isolates from fermented foods were screened for bacteriocin production and a PCR-based assay was used to detect specific bacteriocin-encoding genes. Acid and bile tolerance were also determined. Among all the isolates tested, *Lactobacillus fermentum* 4B1 and *Lactobacillus pentosus* 3G3 (later identified as *Pediococcus acidilactici*) have been identified as most promising for the development of new probiotic food products, hence they were chosen for subsequent biomedical application assays (Lim and Gervacio, 2007). In another study, LAB from traditionally fermented wine and vinegar from Visayas and Mindanao were isolated, identified, and tested for inhibitory activity against *Enterococcus faecium*, *Listeria innocua*, and *Staphylococcus aureus*. Five *Lactobacillus paracasei* and one *Lactobacillus brevis* showed antimicrobial properties against the tester strains (Licaros and Bautista, 2009). With the advent of molecular techniques, the existence of non-culturable microorganisms has been acknowledged especially since the occurrence of culture-bias is already wellaccepted. Culture-independent approaches, therefore, have been gaining popularity in microbial diversity studies and this includes researches on microorganisms found in fermented foods. The microbial populations in selected Philippine fermented foods were assessed through Polymerase Chain Reaction followed by Denaturing Gradient Gel Electrophoresis (PCR-DGGE) in two recent studies (Dalmacio et al., 2011; Larcia, 2010). Food samples tested include *burong mustasa* (fermented mustard), *alamang* (fermented shrimp paste), *burong isda* (fermented rice-fish mixture), *balao-balao/burong hipon* (fermented riceshrimp mixture), *tuba* (sugar cane wine), and *sinamak* (spiced vinegar). Analysis of the 16S rRNA gene sequences revealed the presence of several LAB that have not been reported in these food products before. *Weissella cibaria*, *Lactobacillus plantarum*, *Lactobacillus pontis*, *Lactobacillus panis*, and *Lactobacilus fermentum* were detected in *burong mustasa* (Larcia, 2010). *L. panis* and *L. fermentum* were present in *alamang*; *L. pontis* and *L. plantarum* in *burong isda*; *L. panis*, *L. pontis*, and *L. fermentum* in burong hipon; and *W. cibaria*, *L. pontis*, *L. panis*, *L.* 

*fermentum* and *L. plantarum* in *burong mustasa* (Dalmacio et al., 2011).

gene library for Philippine fermented foods (Dalmacio et al., 2011).

**4.2. Biomedical applications** 

1. Anti-Obesity

The results of the two studies using molecular approaches in defining diversity of LAB in Philippine fermented foods show that culture-independent approaches are efficient tools for the analysis of microbial populations in fermented foods. Majority of the identified bacteria (LAB and other bacterial groups) have not been reported in culture-dependent studies. As such, the isolated bacterial 16S rRNA genes were cloned to have an initial partial 16S rRNA

Obesity is defined as an abnormal or excessive fat accumulation that presents risks to health. Probiotics can help in fighting obesity by reducing lipid absorption through its action on bile

Perhaps another importance of bacteriocin-producing LAB is their effectiveness in biomedical applications. In one study, for example, partially-purified pediocin K2a2-3, through pHmediated bacteriocin extraction method, was found cytotoxic against human colon adenocarcinoma (HT29) and human cervical carcinoma (HeLa) cells *in vitro* as determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (Villarante et al., 2011). Other potential biomedical applications will be discussed in the succeeding section.
