**3. Research initiatives on LAB from Philippine fermented foods**

### **3.1. Bacteriocin research**

Bacteriocins are antimicrobial proteins or peptides produced by certain bacterial strains. Unlike the peptide antibiotics they usually have a narrow spectrum of antimicrobial activity, usually inhibiting growth of closely related bacterial species or strains and lacking lethality to the producer strain (Riley and Wertz, 2002).

The bacteriocins of LAB are small, cationic, hydrophobic, or amphiphilic peptides or small proteins, composed of 20 to 60 amino acid residues (Chen & Hoover, 2003). The bactericidal mode of action and biochemical properties depend on the protein moiety that could be specific to a particular LAB strain, *i.e.* the N-terminal amino acids as determinant of receptors in the cell wall of the susceptible strains/species and C-terminal amino acids for the biochemical properties. LAB bacteriocin must have the following desirable properties: "(1) not active and nontoxic to eukaryotic cells, (2) become inactivated by digestive proteases, having little influence on the gut microbiota, (3) low pH and heat-tolerant, (4) have a relatively broad antimicrobial spectrum, against many food-borne pathogenic and spoilage bacteria, (5) show a bactericidal mode of action, usually acting on the bacterial cytoplasmic membrane: no cross resistance with antibiotics, and (6) have genetic determinants that are usually plasmid-encoded, facilitating genetic manipulation" (Apaga, 2012 as cited from Abriouel et al., 2007).

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

Landang Visayas,

Suka Whole Phil

Sinamak Luzon

Pinakurat Visayas,

to the producer strain (Riley and Wertz, 2002).

Puto

**3.1. Bacteriocin research** 

Tapuy Luzon Rice, glutinous rice

Mindanao

Mindanao

Toyo Whole Phil Soybeans

**Table 1.** Regional Lactic Acid-Fermented Specialties in the Philippines

Puto Whole Phil Rice, sugar *L. mesenteroides, E. faecalis, P.* 

Cassava, or buli palm

Sugar cane juice (for sukang Iloco), palm inflorescence sap (for sukang tuba)

> Sugar cane juice, spices (chilies, onions, garlic)

Coconut sap, chilies,

(Sources: Banaay et al., 2004; Besas and Dizon, 2012; Lee, 1999; Olympia et al., 1995; Sanchez, 2008; Tan et al., 2007)

Bacteriocins are antimicrobial proteins or peptides produced by certain bacterial strains. Unlike the peptide antibiotics they usually have a narrow spectrum of antimicrobial activity, usually inhibiting growth of closely related bacterial species or strains and lacking lethality

The bacteriocins of LAB are small, cationic, hydrophobic, or amphiphilic peptides or small proteins, composed of 20 to 60 amino acid residues (Chen & Hoover, 2003). The bactericidal mode of action and biochemical properties depend on the protein moiety that could be specific to a particular LAB strain, *i.e.* the N-terminal amino acids as determinant of receptors in the cell wall of the susceptible strains/species and C-terminal amino acids for the biochemical properties. LAB bacteriocin must have the following desirable properties: "(1) not active and nontoxic to eukaryotic cells, (2) become inactivated by digestive proteases, having little influence on the gut microbiota, (3) low pH and heat-tolerant, (4)

**3. Research initiatives on LAB from Philippine fermented foods** 

Bibingka Whole Phil Rice, sugar Baked rice cake

Pangasi Mindanao Rice Unknown Wine

balanghoy Mindanao Cassava Unknown Steamed cake Basi Luzon Sugar cane Unknown Wine

salt, various spices Unknown

Tuba Whole Phil Coconut sap Unknown Wine Lambanog Whole Phil Coconut sap Unknown Wine

flour Unknown

*cerevisiae* (in mixed fermentation with *Saccharomyces cerevisiae*)

*Leuconostoc, L. plantarum* (in mixed fermentation with molds and yeasts)

*Leuconostoc, Lactobacillus, Streptococcus* in the initial fermentation phase only

Unknown

*P. halophilus, E. faecalis, L. delbrueckii* (in mixed fermentation with *Aspergillus sojae* and *Saccharomyces rouxii*)

Steamed rice cake

Wine; beer

Dried jelly pellets pellets, rice substitute

> Vinegar, condiment, flavoring

Spiced vinegar, condiment, flavoring

Spiced vinegar, condiment, flavoring

Condiment, flavoring agent, seasoning

Fermented rice, cassava, sugar cane, coconut, soya

LAB bacteriocins have attracted attention in recent years because of their generally regarded as safe (GRAS) status and good value as natural biopreservatives which can find applications in the food and cosmetic industries (Cleveland et al., 2001; Daeschel, 1993; Riley and Wertz, 2002). Nisin, produced by strains of *Lactococcus lactis*, has been used in over 50 countries as anti-listerial and anti-clostridium substance. LAB bacteriocins with selective inhibition on food pathogens such as *Listeria monocytogenes*, but no inhibition on important lactic acid bacterial inocula such as the noted probiotic *Lactobacillus paracasei* or *Lactobacillus rhamnosus*; and yogurt-producing *Lactobacillus delbrueckii* subsp. *bulgaricus* and *Lactococcus thermophilus*, may provide advantage over those that have a wider spectrum of antimicrobial activity and would kill these beneficial organisms, including nisin (De Vos, 1993; Jack and Ray, 1995; Nielsen et al., 1990). Hence, efforts on the search for LAB bacteriocins and elucidation of their properties are actively being pursued by several research laboratories. The future holds a wide array of LAB bacteriocins available for various specific applications.

### **3.2. Isolation and identification of bacteriocin-producing LAB**

Some efforts on the isolation of bacteriocin-producing LAB had been started for more than a decade now in two major research institutions in the country namely: University of the Philippines Los Banos (specifically, the National Institutes of Molecular Biology and Biotechnology or BIOTECH-UPLB and the Institute of Biological Sciences or IBS-UPLB) and the Philippine Root Crop Research and Training Center, Visayas State University (VSU). These two institutions branched out knowledge on bacteriocin research through affiliate tutorship, as thesis advisers and as trainors to students and staff from a few other academic institutions which also did bacteriocin researches like University of Santo Tomas (UST), University of the Philippines Manila (UPM), De La Salle University (DLSU) and Ateneo de Manila University (ADMU). BIOTECH-UPLB and IBS-UPLB jointly worked on bacteriocins of *Lactobacillus plantarum* or plantaricins and those of *Pediococcus acidilactici* or pediocins. On the other hand, VSU devoted some efforts on the enterocins of *Enterococcus* spp. (Tan et al., 2001). DLSU also tried isolation of bacteriocin-producing LAB for food applications. UST was able to isolate bacteriocinlike inhibitory substances against medically important pathogens like *K. pneumoniae*  (Dedeles et al., 2011). UPM and ADMU worked on human and animal health applications of bacteriocins.

Various fermented food products with proteinaceous components were the major sources of isolated LAB for bacteriocin screening. Such fermented food products are home-grown or produced by small enterprises and are still commercially available from

public markets in Luzon, Philippines and some parts of the Visayas like Leyte island. Examples of Philippine indigenous fermented foods that were good sources of bacteriocin-producing LAB are fermented rice and shrimp (*balao-balao*), fermented rice and fish mixture (*burong kanin at isda*), fermented pork (*burong babi*) in Central Luzon (Elegado et al., 2003; Gervasio and Lim, 2007) and fermented pork and sweet potato (*agos-os*) in Eastern Visayan region (Samar and Leyte). On the other hand, pickled vegetables like mustard leaf (*burong mustasa*) and green papaya (*achara*), fermenting fruits like pickled green mango, *bignay* or mango wine (Samnang 2010), fermented salted fish (*bagoong*), spicy sausages (*longganisa*) may contain some LAB but often times they are not bacteriocinogenic (Gervasio and Lim, 2007). The obvious reasons are the presence of inhibitory substances like salt, spices, alcohol or acid and of course the dearth of proteinaceous materials in the food material.

Lactic Acid Bacteria in Philippine Traditional Fermented Foods 577

Elegado et al. 2003

Laxamana et al.

Apaga (2012)

Elegado and

Elegado and

Apaga (2012)

Apaga (2012)

Apaga (2012) ped+

Apaga (2012) ped+

Apaga (2012) ped-

Apaga (2012) ped+

Perez (2012) ped+ ; REP; ped+

Perez (2012) ped+

Apaga (2012) ped+

Apaga (2012) ped+

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

ped+ [100% pediocin operon;PSMB74];

(2011) ped+ ; REP

**Bacteriocin gene by PCR; fingerprinting; HOMOLOGY** 

ped + ; REP and RAPD

ped+;[ 99% *P. acidilactici* bacteriocin genes ; pSMB74 ]

*P. acidilactici* **type strain REFERENCE** 

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

(1492R) 98% *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) 98% *P. acidilactici* 8D2CCH01MX; 99% *P. acidilactici* DSM20284

(1492R) 99% *P. acidilactici* DSM20284 (27F) 99% *P. acidilactici* DSM20284

(1492R) 98% *P. acidilactici* UL5; 99% *P. acidilactici* DSM20284; (27F) 99% *P. acidilactici* 8D2CCH01MX ; 99% *P. acidilactici* DSM20284

(1492R) 96% *P. acidilactici* UL5; 99% *P. lolii to* NGRI0510Q (27F) 99% *P. acidilactici* LAB 001 ; 99% *P. lolii* NGRI 0510Q

(1492R) 98% *P. acidilactici* UL5; 99% *P. acidilactici* DSM20284 (27F) 99% *P. acidilactici* 8D2CCH01MX; 99% *P. acidilactici* DSM20284

(1492R) 99% *P. acidilactici* IMAU20090 (27F) 98% *P. acidilactici* DSM20284

(1492R) 99% *P. acidilactici* UL5 (27F) 98% *P. acidilactici* DSM20284

(1492R) 95% *P. acidilactici* UL5 (27F) 98% *P. acidilactici* UL5; 99% *P. acidilactici* DSM20284

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

(1492R) 97% *P.acidilactici* UL5 99% *P. acidilactici* 8D2CCH01MX; (27F) 98% *P. acidilactici* DSM20284

**ISOLATE/** 

AA-5a

4E2

4E4

4E5

4E6

4E10

4BL7

3G3

3G8

3F3

3F8

3F10

IG7

**STRAIN No. IDENTIFICATION (primer) HOMOLOGY to** 

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

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

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

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

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

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

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

API CHL50 ID: *Lactobacillus pentosus*(doubtful) partial 16S rRNA gene ID: *P. acidilactici*

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

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

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

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

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

In one of the first isolation studies for bacteriocinogenic LAB, various proteinaceous fermented foods native to Central and Southern, Philippines were screened for bacteriocin-producing bacterial isolates. Seventy one out of several hundreds of colonyforming unit isolated by agar plate streaking were found antagonistic to the indicator microorganism, *Lactobacillus plantarum* ATCC 14917, through direct assay. By "spot-onlawn" assay by pH-neutralized culture supernatant, nine (9) isolates were confirmed to be bacteriocin producers (Elegado et al., 2003). Banaay et al. in 2004 also reported on the isolation of 1,100 putative LAB from indigenous fermented foods in Luzon, Philippines. A strain of *Lactobacillus plantarum* was selected as the best bacteriocin producer. In another study, out of the 160 putative LAB obtained from 19 fermented food products from public markets in Central Luzon, 32 LAB isolates were found to be bacteriocinogenic (Gervasio and Lim, 2007). Santiago et al. (2008) were also able to find two LAB isolates, *Lactobacillus fermentum* LBA-19 and *Lactobacillus casei* LTI-21, screened from among several LAB isolates from various fermented food products from different regions in the Philippines.

Being pleomorphic, identification of LAB is quite challenging. A combination of various microbiological and molecular biology tools would help in finding the real identity. Banaay et al. (2004) did a thorough identification of the bacteriocinogenic LAB isolate using conventional morphological, biochemical and physiological methods, chemotaxonomic methods, as well as molecular methods. This is especially relevant to the identification of *Lactobacillus plantarum* which is a known pleomorphic bacteria. Most other Philippine LAB researchers often times directly apply 16S rRNA gene sequencing and homology search for LAB purified through repeated agar streaking and putatively identified as LAB just after determining its acid–forming, Gram positive and catalase negative properties. (Elegado et al., 2003; Gervasio and Lim, 2007; Santiago et al., 2008). Aside from 16S rRNA genes, other conserved genes were used for identification such as phenylalanyl-tRNA synthase (*pheS*) gene (Dedeles et al., 2011). Detection of bacteriocin genes through PCR may also be helpful in confirming the identity of the bacteriocinogenic LAB as well as the probability of producing the bacteriocin (Table 2).


proteinaceous materials in the food material.

regions in the Philippines.

bacteriocin (Table 2).

public markets in Luzon, Philippines and some parts of the Visayas like Leyte island. Examples of Philippine indigenous fermented foods that were good sources of bacteriocin-producing LAB are fermented rice and shrimp (*balao-balao*), fermented rice and fish mixture (*burong kanin at isda*), fermented pork (*burong babi*) in Central Luzon (Elegado et al., 2003; Gervasio and Lim, 2007) and fermented pork and sweet potato (*agos-os*) in Eastern Visayan region (Samar and Leyte). On the other hand, pickled vegetables like mustard leaf (*burong mustasa*) and green papaya (*achara*), fermenting fruits like pickled green mango, *bignay* or mango wine (Samnang 2010), fermented salted fish (*bagoong*), spicy sausages (*longganisa*) may contain some LAB but often times they are not bacteriocinogenic (Gervasio and Lim, 2007). The obvious reasons are the presence of inhibitory substances like salt, spices, alcohol or acid and of course the dearth of

In one of the first isolation studies for bacteriocinogenic LAB, various proteinaceous fermented foods native to Central and Southern, Philippines were screened for bacteriocin-producing bacterial isolates. Seventy one out of several hundreds of colonyforming unit isolated by agar plate streaking were found antagonistic to the indicator microorganism, *Lactobacillus plantarum* ATCC 14917, through direct assay. By "spot-onlawn" assay by pH-neutralized culture supernatant, nine (9) isolates were confirmed to be bacteriocin producers (Elegado et al., 2003). Banaay et al. in 2004 also reported on the isolation of 1,100 putative LAB from indigenous fermented foods in Luzon, Philippines. A strain of *Lactobacillus plantarum* was selected as the best bacteriocin producer. In another study, out of the 160 putative LAB obtained from 19 fermented food products from public markets in Central Luzon, 32 LAB isolates were found to be bacteriocinogenic (Gervasio and Lim, 2007). Santiago et al. (2008) were also able to find two LAB isolates, *Lactobacillus fermentum* LBA-19 and *Lactobacillus casei* LTI-21, screened from among several LAB isolates from various fermented food products from different

Being pleomorphic, identification of LAB is quite challenging. A combination of various microbiological and molecular biology tools would help in finding the real identity. Banaay et al. (2004) did a thorough identification of the bacteriocinogenic LAB isolate using conventional morphological, biochemical and physiological methods, chemotaxonomic methods, as well as molecular methods. This is especially relevant to the identification of *Lactobacillus plantarum* which is a known pleomorphic bacteria. Most other Philippine LAB researchers often times directly apply 16S rRNA gene sequencing and homology search for LAB purified through repeated agar streaking and putatively identified as LAB just after determining its acid–forming, Gram positive and catalase negative properties. (Elegado et al., 2003; Gervasio and Lim, 2007; Santiago et al., 2008). Aside from 16S rRNA genes, other conserved genes were used for identification such as phenylalanyl-tRNA synthase (*pheS*) gene (Dedeles et al., 2011). Detection of bacteriocin genes through PCR may also be helpful in confirming the identity of the bacteriocinogenic LAB as well as the probability of producing the


Lactic Acid Bacteria in Philippine Traditional Fermented Foods 579

Tolerant to pH 2-9 and 121 °C

MW = 4,626 Da

Optimum pH = 5-7 Resistant to boiling but not to autoclaving

Tolerant to pH 2-9; slight loss of activity at 100 °C; loss of activity at 121 °C; tolerates high salt; est. MW = 6,500 Da by SDS-PAGE

filtration chromatography (Elegado et al., 2003), and hydrophobic interaction chromatography (Villarante et al., 2011). This method could also be applied with bacteriocins of pediococci and lactobacilli. The properties obtained from well characterized

Reversed-phase HPLC

Hydrophobic interaction and ion-exchange chromatographies Reversed-phase HPLC

**Table 3.** List of purified and characterized bacteriocins from LAB isolated from Philippine indigenous

Bacteriocin production is largely dependent on the nutrients and nitrogen content of the fermentation medium. For instance, increased yeast extract concentration and polypeptone amount increases bacteriocin production. Molasses, raw sugar and sago hydrolyzates of amylase digestion were found to be good carbon sources. Other possible substrate base and supplements are cheese whey, coconut water and rice bran extract. Initial sugar concentration of usually 2 to 3% and inoculation rate of 3% by volume of at least 108

Bacteriocin production is highly dependent on cell or biomass growth. LAB are microaerophilic and most are either mesophilic or slightly thermophilic. The following conditions are applicable to their production: pH= 5.5 to 6.0; temperature = 35 – 40 °C; agitation = 50 rpm; without aeration. Usually, bacteriocin is optimally produced or secreted in the culture broth during the early stationary phase of growth. For *Pediococcus acidilactici*, culturing at 40 °C promotes earlier optimum bacteriocin production of around 10-12 hours.

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

**3.4. Optimization of bacteriocin production through fermentation kinetics** 

Reversed-phase HPLC MW = 3,830 Da

**Isolate Identity Bacteriocin Purification mode Properties** 

*acidilactici* pediocin pH adsorption/desorption

*plantarum* plantaricin Gel filtration chromatography

*acidilactici* pediocin pH adsorption/desorption

*acidilactici* pediocin pH adsorption/desorption

cells/mL provides good bacteriocin production (Elegado et al., 2001).

At 37 °C, bacteriocin production is from 14-16 hours (Sagpao et al., 2007).

bacteriocinogenic LAB are shown in Table 3.

*acidilactici* pediocin

AA5a *Pediococcus* 

BS25 *Lactobacillus* 

K2a2-3 *Pediococcus* 

K2a2-1 *Pediococcus* 

4E5 *Pediococcus* 

fermented foods.

**3.5. Applications** 

**Table 2.** Identification and bacteriocin gene determination of putative *Pediococcus acidilactici* through 16S rRNA and pediocin gene PCR amplification and sequencing.
