Control of halobacterial damage on brine cured hides with halocin20 November 2006
Introduction Halophilic Archaea of the family Halobacteriaceae are dominant microorganisms in hypersaline environments such as salt lakes, crystalliser ponds of solar salterns, salt mines, hypersaline soda lakes (Grant et al, 1998; Oren 2000). Halophilic Archaea can also grow in artificially salted environments such as salted fish, salted meat, salted hides and certain fermented food products (Thai fish sauce) (Thongthai et al, 1991; Bailey and Birbir 1993; Oren 2000). They are easily detected in such habitats since they produce a red to orange pigmentation. These pigments are generally carotenoids which are used to stimulate an active photorepair system to repair thymine dimers formed by ultraviolet radiation (DasSarma and Arora 2001). Extremely halophilic Archaea require at least 1.5-2 M NaCl for growth and optimally most species require 2-4 M NaCl (Grant et al, 2001). Although halophilic Archaea produce a wide variety of biotechnological products such as bacteriorhodopsins, halorhodopsins, compatible solutes, biopolymers, biosurfactans, exopolysaccharides, polyhydroxyalkanoates, flavouring agents, isomerases, hydrolases, nucleases, amylases, proteases, lipases, anti-tumour drugs and liposomes (Grant et al,1998; Oren 2000; Eichler 2001; Rodriguez-Valera et al, 1991), these organisms may also cause significant damage on brine cured hides (Bailey and Birbir 1993; Birbir and Ilgaz 1996; Birbir et al, 1996; Bitlisli et al, 2004) and salted foods such as fish (Graihoski 1973), meat, cheese, olive, tomato paste, grape leaves and pickles with their hydrolytic enzymes (Birbir et al, 2004a). Haloarchaeal damage on hides or skins Our earlier studies on halophiles showed that brine cured hides processed in different countries had extremely halophilic Archaea (Bailey and Birbir 1993; Birbir 1997). In the US, 131 brine cured hides were tested for extremely halophilic Archaea and 98% of them contained these microorganisms. A total of 332 extremely halophilic Archaeal strains were isolated and 94% of these strains were protease positive (Bailey and Birbir 1993). Moreover, 35 salt-cured French and Russian hides were tested for extremely halophilic microorganisms and 91% of them contained these microorganisms (Birbir 1997). From these hides, 85 extremely halophilic strains were isolated and 67% of the strains were protease positive. Furthermore, researchers found that most salted sheepskins contained extremely halophilic Archaea and 53-74% of extremely halophilic Archaea showed proteolytic activity (Bitlisli et al, 2004). Presence of proteolytic haloarchaeal strains in salt affect hide quality adversely. Halophilic Archaea can cause discoloration of the flesh side of the skins or hides. This condition is referred to as 'red heat' in the hide industry. This discolouration is due to massive growth of halophilic Archaea (Didato et al, 1999). In addition, hair slip or pin prick can be seen on hides that have been inadequately salt packed or brine cured. Proteolytic halophilic Archaea in salt may grow in the hair follicles of hides and cause the degradation of the entire follicle leaving a hole in the grain (Didato et al, 1999). Furthermore, microorganisms grown on hides may cause uneven dyeing in leathers (Bitlisli et al, 2004). It was also demonstrated that extremely halophilic Archaea damaged the grain of brine cured hides within seven weeks at a temperature of 41°C. This damage was easily observed by the naked eye and scanning electron microscopy clearly showed that the damage done by halophilic microorganisms resembled sueded grain (Bailey and Birbir 1993). In addition, it was also mentioned that halophilic Archaea caused a complete disruption of collagen fibres and production of sponge-like vesicles within hides (Vreeland et al, 1998). Natural antibacterial substances (Bacteriocins) Considerable attempts have been made to use bactericides during brine curing of hides (Vivian 1969; Hendry et al, 1971; Birbir and Bailey 2000). Effective bactericides (Birbir and Bailey 2000; Weiss and Thornton 1984; Lollar and Kallenberger 1986; Mitchell 1987) and bile salts (Vreeland and Bailey, 1999) have been recommended to prevent halobacterial damage during brine curing of hides but the use of bactericides have been questioned due to environmental pollution risk, mutation of bacteria and bacterial resistance on repeated uses. In recent years, the control of the bacterial population by natural antimicrobial substances is an important alternative to chemicals. It is known that thirty genera of the domain Bacteria produce bacteriocins to inhibit closely related species or even different strains of the same species (Pelczar et al, 1993; Shand 1999). Many of human bacterial flora synthesise and release bacteriocins (Prescott et al, 1993). There are many different bacteriocins including those produced by bacteria normally found in the intestine. Bacteriocins may give their producers an adaptive advantage against other bacteria. Sometimes, they may increase bacterial virulence by damaging host cells such as mononuclear phagocytes. Bacteriocin Nisin A produced by the lactic acid bacteria strongly inhibits the growth of a wide range of Gram-positive bacteria and is used as a preservative in food industry (Madigan and Martinko 2006). Kwaadsteniet et al (2005) explained that 3944 Da bacteriocin (ST15) produced by Enterococcus mundtii which was isolated from soy beans inhibited the growth of Gram-positive and Gram-negative bacteria such as Lactobacillus sakei, Enterococcus faecalis, Bacillus cereus, Propionibacterium sp, Clostridium tyrobutyricum, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae and Streptococcus caprinus. Ghrairi et al, (2005) mentioned that Lactococcin MMT24, which is a novel bacteriocin produced by Lactococcus lactis MMT24, was isolated from a Tunisian traditional cheese. The bacteriocin showed a narrow antimicrobial activity against closely related lactic acid bacteria. Oh et al (2006) found that bacteriocin produced by Lactococcus sp HY 449 inhibited the growth of Staphylococcus epidermidis ATCC 12228, Staphylococcus aureus ATCC 65389, Streptococcus pyogenes ATCC 21059 and Propionibacterium acnes ATCC 6919. Researchers explained that the bacteriocin produced by Lactococcus sp. HY 449 might be a useful antimicrobial substance to control the growth of Propioni-bacterium acnes and to prevent skin-inflammation and acne. Ammor et al (2006) used 87 lactic acid bacteria (LAB) (36 Lactobacillus sakei, 22 Enterococcus faecium, 16 Lactococcus garvieae, eleven Vagococcus carniphilus and two Enterococcus sp isolated from a small-scale facility producing traditional dry sausages to screen for antagonistic activity against other LAB and some spoilage and pathogenic microorganisms, also isolated from the same processing facility. The main goal of their research was to investigate LAB antibacterial activity within the facility microbial ecosystem and to select interesting strains for the role of bio-preservatives. Twenty-one Enterococcus faecium, six Vagococcus carniphilus, four Lactococcus garvieae, three Lactobacillus sakei and two Enterococcus sp were shown to inhibit the growth of some indicator microorganisms in an agar well diffusion assay. Except two Lactobacillu sakei and an Enterococcus sp, all these isolates exhibited antibacterial activity against Listeria innocua but only three Enterococcus faecium, five Vagococcus carniphilus and three Lactococcus garvieae displayed also antagonistic activity against Staphylococcus aureus. The five Vagococcus carniphilus isolates were also found to be inhibitory for the Gram-negative bacterium Hafnia alvei. Sarkar (2006) stated that application of more than one bacteriocin may be advantageous to minimise the possibility of survival of microflora resistant to a particular bacteriocin. Natural antibacterial substances (halocins) As in domain Bacteria, extremely halophilic Archaeal strains in domain Archaea produce antimicrobial substances called halocins to kill or inhibit other halophilic Archaea in the same or different environmental niche. Researchers have explained that halocin production was a near-universal feature of haloarchaeal rods and, based on antagonism studies, hundreds of different types have been found to exist (Price and Shand 2000). Halocins are natural proteinaceous antimicrobials which were first discovered by Francisco Rodriguez-Valera et al, in 1982. The main reason for the existence of halocins has always been that they reduce competition by lysing competitors and enrich the environment for the producer strains (Rodriguez-Valera et al, 1982). In this research, forty extreme halophiles were screened against each other for production of halocins; seven of forty were found to produce effective halocin against other 39 halobacterial strains. Five of the seven producers inhibited a large number (19 to 35) of the 40 strains, while the remaining two inhibited only a few (1-3) (Rodriguez-Valera et al, 1982). In other halocin studies, 147 extremely halophilic strains were screened against each other for production of halocins; 144 of the 147 were found to produce halocins and twenty of the 144 were sensitive to their own halocin and none of the isolates was completely insensitive to all halocin. The results of this study showed that some of the strains inhibited nearly all the strains and the others inhibited only a few. Both studies indicated that there were numerous classes or groups of halocins and the halocin production was a practically universal feature of haloarchaea (Rodriguez-Valera et al, 1982; Meseguer et al, 1986). Halocins always reduce competition among haloarchaeal strains (Rodriguez-Valera et al, 1982). Liu et al (2003) stated that many species of family Halobacteriaceae produce halocin. They found that halocin C8, excreted by the Halobacterium strain AS7092, had a very wide activity spectrum, including most haloarchaea and even some haloalkaliphilic rods. When a sensitive strain of Halorubrum saccharovorum was exposed to halocin C8, the treated cells swelled at the initial stage. The cell wall appeared to be nicked and the cytoplasm was then extruded out, and the whole cell was eventually completely lysed. They explained that halocin C8 was a novel microhalocin and its primary target might be located in the cell wall of the sensitive cells. Haloarchaeal growth on hides may be prevented with natural antimicrobial compounds such as halocins produced by halophilic Archaea. In our halocin study, 56 extremely halophilic archaeal strains isolated from Tuz Lake, Kaldirim saltern, Kayacik saltern and Tuzköy salt mine were screened for antagonistic activity against each other. Twelve of the 19 Tuz Lake strains, twelve of 18 Kaldirim Saltern's strains, five of seven Kayacik Saltern's strains and eight of the twelve Tuzkoy Salt Mine strain were gelatinase positive (Birbir et al, 2004b). It was found that seven of the 19 Tuz Lake's strains, sixteen of 18 Kaldirim saltern's strains, two of seven Kayacik saltern's and ten of twelve Tuzköy salt mine's strains produced halocin against each other. Five of the seven Tuz Lake producers inhibited a large number (12-16) of the Tuz Lake's strains, while the remaining two inhibited only a few (1-2). The results showed that most of the gelatinase positive strains in Tuz Lake might be inhibited by gelatinase negative strains of Tuz Lake and Kayacik saltern. Three of the sixteen Kaldirim saltern's producers inhibited a large number (9-13) of this saltern's strains, while the remaining thirteen inhibited only a few (1-7). Two of Kayacik saltern's producers inhibited five Kayacik saltern's strains. The gelatinase negative strain of Kayacik saltern inhibited all the gelatinase positive strains of this saltern. Five out of ten of the Tuzköy salt mine's products inhibited a large number (6-10) of the Tuzköy salt mine's strains, while the remaining five inhibited only a few (1-4). The results showed that most of the gelatinase positive strains in Tuzköy salt mine might be inhibited by gelatinase negative strains of Tuzköy salt mine and Kayacik saltern strains (Birbir et al, 2004b). In conclusion, it was found that gelatinase negative halocin producers inhibited gelatinase positive strains. Therefore, it was suggested that these gelatinase negative halocin producers or their isolated halocins may be used in preventing the proteolytic haloarchaeal damage that can occur during brine curing of hides (Birbir et al, 2004).