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Use of bacteriophage-derived peptides to delay phage infections
Food Research International 37 (2004) 115–122 www.elsevier.com/locate/foodres
Use of bacteriophage-derived peptides to delay phage infections q,qq C.L. Hicks a
a,*
, P.A. Clark-Safko b, I. Surjawan a, J. OÕLeary
a
Department of Animal Sciences, University of Kentucky, 410 W. P. Garrigus Bldg., Lexington, KY 40546, USA b b1326 Glenwich Ave. Windermere FL 34786, USA Received 8 July 2003; accepted 15 September 2003
Abstract Peptides were prepared from structural proteins of Lactococcus lactis ssp. lactis c2 bacteriophage and used to inhibited c2 phage proliferation in L. lactis ssp. lactis C2 host. Culture media that contained phage peptides inhibited C2 growth slightly. However, when C2 culture was grown in media that contained c2 phage peptides and infected with c2 bacteriophage, host cell populations and time to growth apex increased over culture grown in medium without peptides. Growth time prior to lysis was extended 54 min when infected with 1 107 pfu/ml of c2 bacteriophage. Lysis slope was steeper for culture grown in medium with phage peptides than medium without phage peptide. Milk inoculated with bulk starters prepared with c2 phage peptides were more resistant to c2 phage proliferation than milk inoculated with bulk starter containing no c2 peptides or milks having c2 phage peptides added to the milk. Ó 2003 Elsevier Ltd. All rights reserved. Keywords: Bacteriophage; Proliferation; Peptides; Inhibition; Lactococcus lactis ssp. lactis C2
1. Introduction Bacteriophage (phage) infections in milk fermentations have been a major problem for many years and still cause most culture failures (Moineau et al., 1996). Phage may be introduced into cheese plants from many sources. Phage can be carried on clothing as well as in cheese whey and mist (Kosikowski, 1982; Neve, 1995). An infection occurs when phage are specific for the bacteria in use. Phage specificity may be visualized as a lock and key fit (phage receptor to bacteria receptor site). The host receptor site for phage adsorption (reversible step) generally consists of carbohydrates (usually rhamnose and/or glucose) (Valyasevi, Sandine, & Geller, 1991, 1994). Often several lactic phage are found absorbed onto a common spot on the host cell (Neve, 1995).
q
Published with the approval of the director of the Kentucky Agriculture Experiment Station as Journal Article Number 01-07-164. qq Research was conducted at the University of Kentucky. * Corresponding author. Tel.: +859-257-7537; fax: +859-257-7537. E-mail address: [email protected] (C.L. Hicks). 0963-9969/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2003.09.009
Presumably, phage spots occur where the polysaccharide coating on the cell membrane thins and nutrient transport occurs (Neve, 1995). Initial adsorption and movement of the phage on the cell surface appears to be facilitated by Brownian movement (Bao & Scott, 2000; Popescu, 1996) and a calcium binding domain such as the one located on the F protein of c2 phage (Lubbers, Waterfield, Beresford, Le Page, & Jarvis, 1995). After the phage has absorbed onto the surface of the host cell an irreversible attachment occurs between the phage adsorption protein (Lubbers et al., 1995) and a membrane component. Lactococcus lactis ssp. lactis C2 membrane contains a protein (99 kDa, pip gene) that binds with the c2 phage adsorption protein (Garbutt, Kraus, & Geller, 1997; Kraus & Geller, 1998; Monteville, Ardestrani, & Geller, 1994) to form the irreversible attachment step where the phage secures its tail onto the bacterial membrane by hooking spikes into the cell membrane (Neve, 1995; Valyasevi et al., 1991). After the tail is secured, the c2 phage ejects its DNA after being triggered by the pip gene protein (Gravey, van Sinderen, Twomey, Hill, & Fitzgerald, 1995). Once the phage DNA is in the cell it takes command over cell processes to perform transcription,
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translation, and replication (Powell, Tulloch, Hillier, & Davidson, 1992). Various technologies have been developed over the years to prevent phage attack in cheese plants. These technologies include the use of phosphate containing media for bulk starter production and culture rotation schemes (Jarvis, 1981; Ledford & Speck, 1979). Most industrial cultures are phage resistant mutants that are grown in phosphate media. Culture strains are rotated daily or when specific phage infections are detected (Cogan & Accoulas, 1990; Heap & Lawrence, 1976; Sing & Klaenhammer, 1993). Techniques using gene transfer for generating resistant starter strains have also been reported (Boucher, Emond, Parrot, & Moineau, 2001; Jarvis, 1981, 1978; Jarvis & Klaenhammer, 1986; Klaenhammer, 1987; Klaenhammer & Sanozky, 1985; McLandsborough, Sechaud, & McKay, 1998; Sing & Klaenhammer, 1993). Culture rotation is not without its problems. Rotations of cultures bring about strain to strain variations (Champagne & Lange, 1992; Heap & Lawrence, 1976; Lawrence, Heap, Limsowtin, & Jarvis, 1978) which can include differentiation of growth time and acid production. Culture variations may speed up or slow down plant processing time. If production is sped up, an excess of product may occur in one stage of processing while slower culture development delays production. The ideal concept would be to use one strain of a bacterium continuously which would eliminate culture rotation and strain to strain variation. One culture strain could be used, if cells were protected against all infecting bacteriophage. A technology using whey peptides as an additive to M17 medium was shown to prolong the time from phage inoculation to time of culture lysis (Onuorah, Hicks, & OÕLeary, 1995; Russell-Campbell Hicks & O’Leary, 1995). However, bacterial growth was inhibited slightly and the protection provided by this peptide was deemed insufficient to impede bacteriophage growth in an industrial cheese processing environment. It was theorized, that if peptides, that were highly specific to the cell receptor, could be isolated from bacteriophage coat proteins by enzymatic hydrolysis, they may compete with viable phage for binding sites on the host cell, thus inhibiting or preventing viable phage attachment (Hicks, 2001). If this technology reduces the number of phage attaching to cells a decrease in the rate of phage proliferation should be noted. However, as bacteria divide and reproduce, fewer peptides would be available to cover new bacterial receptor sites and phage proliferation would eventually occur after the peptide concentration became limited. This system, nonetheless, may prevent phage infection for a sufficient time to allow cheese fermentation. Therefore the objective of this study was to develop a phage peptide blocker to inhibit phage attachment and proliferation.
2. Materials and methods 2.1. Culture and phage Lactococcus lactis ssp. lactis C2 and c2 bacteriophages were obtained from Klaenhammer (North Carolina State University, Raleigh, NC). 2.2. Propagation medium M17 medium, described by Terzaghi and Sandine (1975), was used as a growth medium for Lactococcus cultures. The medium was prepared from individual components or from a pre-formulated M17 medium (Fisher Scientific, Pittsburgh, PA). Pre-formulated medium was prepared by combining 37.5 g of M17 powder and 950 ml distilled water. All ingredients were mixed and sterilized as described by Terzaghi and Sandine (1975). A cold sterilized lactose solution was added (50 ml) to the sterilized M17 medium and was termed L-M17 (final lactose concentration 0.1%). 2.3. Preparation of phage enumeration plates Bottom agar (modified procedure of Douglas, Qanber-Agha, & Phillips, 1974) was prepared by adding 100 ml of 1 M CaCl2 6H2 0 containing 15 g of bactoagar to 37 g of M17 medium which was made up to 950 ml. The pH was adjusted to 6.9. The medium was sterilized at 121 °C for 15 min. After the medium was sterilized, cold sterilized lactose (50 ml) was added (final concentration 0.1%) at 45 °C. Approximately 10 ml of agar was dispensed into each Petri dish. Agar was allowed to solidify and plates were stored at 5 °C. Top agar was prepared using the same method except that 4.5 g agar was added to the M17 medium. To enumerate c2 phage, serial dilutions of bacteriophage were made in phosphate buffer (Martley, 1972). Top agar was steamed for 20 min and tempered to 45 °C. Active lactic C2 culture (1 109 cfu/ ml) was added (0.1 ml) to 2.5 ml top agar. The appropriate dilution of phage was added (0.1 ml), vortexed, and allowed to stand for 10 s. The mixture was then overlaid on the pre-prepared bottom agar plate. Plates were incubated at 26 °C for 6 h uninverted. Plaques were counted after 6 h of incubation. 2.4. Preparation of phage peptides using papain hydrolysis Ustunol and Hicks (1994) used 2% crude papain or a dried papaya extract to hydrolyze whey and then harvested the peptides using ultrafiltration techniques. A similar procedure was used to prepare phage peptides. Filtered (cell debris removed using a 0.45 lm membrane) phage (1 109 pfu/ml) was hydrolyzed with crude papain (2%) in L-M17 broth for 4 h at 26 °C. Digest was filtered through a 3000 molecular weight cut
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off (mwco) membrane (Amicon, Inc., Beverly MA) to remove phage debris and papain. Filtrate containing the phage peptides was tested for infectivity by assaying the filtrate as previously described (Douglas et al., 1974). Some filtrates were frozen ()20 °C) and freeze dried. 2.5. Evaluation of papain, bromelain and ficin to prepare phage peptides Five tubes of L-M17 broth (10 ml) with 0.1% CaCl2 were inoculated with 0.4 ml L. lactis ssp. lactis C2 (4 107 cfu/ml) and incubated (26 °C). After 1 h, one enzyme (papain, bromelain, or ficin [0.1%]) (Sigma Inc., St. Louis, MO) and 0.001 ml c2 phage (1 109 pfu) was added to three of the five tubes. The fourth tube served as a growth control. The fifth tube had 0.001 ml of c2 phage added after 1 h and served as a culture lysis control. Growth and cell lysis were observed (k600 nm , Milton Roy spectrophotometer) for 5 h by monitoring changes in turbidity. Digests were assayed for c2 phage titers as previously described. 2.6. Determination of ficin in the permeate Freeze dried permeate from ficin treated L-M17 medium containing c2 peptide was added to low heat reconstituted non-fat dry milk (Armour, Springfield, KY) (10 ml, 10% solids) at 1% (0.1 g) to determine the amount of residual ficin in the permeate. Crude ficin (0.01% and 0.025%) was added to 10 ml of non-fat dry milk to serve as coagulation controls. A tube of reconstituted non-fat dry milk served as a non-coagulating control. Coagulation of the milk was observed using the Berridge Clot Timing Method (Berridge, 1952). Bottles (125 ml) were rotated (3.0 rpm, 30° angle) and held at 31 °C. Milk clotting was visualized as a floc-containing film that fractured on the inside of the bottle. Bottles were observed repeatedly over a 5 h period for the presence of flocs. 2.7. Determination of activity of medium derived peptides Media with and without culture cell debris and with and without c2 phage were hydrolyzed with ficin (0.1%) to confirm that the peptide was derived from c2 phage. Hydrolyzed fractions were ultra-filtered (3000 mwco filter) under nitrogen pressure (4.06 atm. at 5 °C) and checked for viable phage using enumeration techniques. Permeates containing hydrolyzed peptides were freeze dried and stored at )20 °C until being used to prepare media formulations. 2.8. Preparation of phage peptides for additional research Propagated c2 phage in L-M17 broth was filtered (0.45 lm) to remove cellular debris into a sterile glass
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bottle. Bacteriophage in L-M17 broth was hydrolyzed with crude ficin (0.1%) for 4 h at 22–24 °C. The hydrolyzed mixture was ultra-filtered (3000 mwco filter) under nitrogen pressure (4.06 atm. at 5 °C) to remove ficin and phage debris from the permeate. Permeate was checked for viable phage using enumeration techniques and was found to be free of plaque forming units. Permeate containing hydrolyzed peptides was dialyzed (500 mwco, Spectra Por, Fisher Scientific, Pittsburgh, PA ) in 0.10 ionic strength sodium potassium phosphate buffer (pH of 6.4) for 6 h at 5 °C. Fresh phosphate buffer was replaced and dialysis continued for another 10 h. Dialyzed peptides were freeze dried and stored at )20 °C. 2.9. Media preparation and procedure for phage peptide blocking experiments Four test tubes of sterilized L-M17 broth (10 or 25 ml) containing CaCl2 (0.01%) with (two tubes) and without (two tubes) c2 phage peptide (2%) were inoculated (4% of 1 109 cfu/ml L. lactis ssp. lactis C2) and incubated at ambient temperature. Another tube containing L-M17 medium with only CaC12 added was used to zero the spectrophotometer (k600 nm ). Phage (0.001 ml of c2 phage, 1 107 pfu) was inoculated into two tubes (one with c2 phage peptides and one without peptides) after the tubes had been incubated for 1 h. Tubes with and without c2 peptides with added phage were used to determine the effect of the peptides on phage proliferation. Growth and lysis curves were determined spectrophotometrically (k600 nm ). Spectrophotometric readings were taken every 20 min for as long as 380 min. 2.10. Testing C2 culture in milk coated with and without c2 phage peptides C2 culture was grown in L-M17 medium with (1% and 2%) and without c2 phage peptides (3 starter cultures). Standardized (3.25% fat), pasteurized (72 °C for 17 s) milk (96 ml in 6 beakers at 31 °C) was prepared. Two milks were fortified with c2 phage peptides (1% and 2%, respectively). All milks were inoculated (4%) with culture as follows: two milks were inoculated with culture that was grown in medium containing 2% c2 peptide; one milk (containing 1% c2 peptide) was inoculated with culture that was grown in medium containing 1% c2 phage peptide; and three milks were inoculated with culture grown in medium containing 0% c2 phage peptide (This included the milk that contained 2% c2 phage peptide.). All milks were infected with 103 pfu/ml c2 phage, except for one milk that was inoculated with culture without peptide and one milk that was inoculated with culture containing 2% peptide. All milks were allowed to ripen for 1 h, prior to renneting (Chymosin EC 3.4.23.4, isozyme A, Pfizer, Milwaukee, WI). Curd
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was cut after approximately 25 min, heated from 31 to 37 °C in a 30 min period, and held at that temperature until the control pH declined to 5.6 (ca 1.25 h after cutting). Acid development (pH) was monitored at 20 min intervals throughout the period. Culture activity (pH) versus incubation time was plotted and analyzed. 2.11. Experimental procedure, data and statistical analysis All experiments were conducted using randomized block designs that were replicated three times. Growth and lysis curves were plotted for each replication. Measurements were taken from each plot to determine slope of the log growth and lysis rates, and for time from phage addition to growth apex (dependant variables). Effect of media with and without c2 phage peptides (independent variables) on these slopes and time to apex were statistically analyzed (SAS, 1994) using general linear models procedures, SS4 sums of squares, and orthogonal contrasts. These same procedures were used to test differences between end pH values of milk subjected to different culture treatments, except replication and time were used as co-variants.
3. Results and discussion L. lactis ssp. lactis C2 and its bacteriophage (c2) were utilized for this research because they comprise one of the best characterized and understood phage/host systems (Lubbers et al., 1995), even though C2 culture is less active than most cheese cultures. An L-M17 growth medium was selected for this research because of its transparency and because it is an excellent growth medium for lactic acid bacteria. Although this model system was not representative of a commercial culturemedium system it was selected because of the ease in which cell growth and lysis could be monitored. Since Hicks, Onuorah, and Surjawan (2000) observed that peptides derived from whey had an inhibitory effect on phage proliferation, it was theorized (Hicks, 2001) that peptides derived from specific phage structural proteins might have a greater inhibitory effect on phage proliferation than whey peptides. Since c2 phage coat protein (F protein) is thought to make the attachment between c2 phage and L. lactis ssp. lactis C2, the F protein would be the most likely target for protein hydrolysis. Also, since the c2 phage F protein is thought to contain one calcium binding site and one attachment site only one peptide may be active. Therefore, this research was undertaken to develop a phage peptide that would inhibit phage proliferation through competitive inhibition or the competitive binding of the phage peptide to the receptor on the host cell thus limiting the number of receptors available to bind native phage.
3.1. Papain hydrolysis of phage proteins Ustunol and Hicks (1994) reported that whey peptides could be prepared by hydrolyzing whey with a general protease such as papain. Therefore, papain was added to c2 phage filtrates in hopes that peptides could be cleaved from phage structural proteins. After the papain had reacted for 4 h, debris from the digests was removed using a 3000 mwco membrane. However, these 3000 mwco filtrates were not completely free of viable c2 phage. Phage titers remaining in the 3000 mwco filtrates averaged 1 101 pfu/ml. Although this titer was low, it was still enough to infect, and eventually, destroy C2 culture that was incubated in medium containing the filtrate. Thus the papain treatment appeared insufficient to hydrolyze all c2 phage structural proteins that are necessary for attachment and the 3000 mwco membrane did not fully exclude all viable phage that was in the digest or post contamination occurred. When the 3000 mwco filtrate was heat treated at 80 °C for 1 h no viable c2 phage remained in the filtrate. When L-M 17 medium with and without heat treated filtrate was inoculated with L. lactis ssp. lactis C2 and incubated for 1 h at 26 °C before being infected with or without c2 phage, no inhibition of phage proliferation was observed when the media contained papain c2 phage filtrate. However, growth of L. lactis ssp. lactis C2 in media that contained the heat treated papain filtrate was somewhat inhibited at the added filtrate concentration. Since culture growth was inhibited and phage proliferation was only slightly affected at the filtrate concentrations used, experimentation with other proteases was initiated. 3.2. Bromelain and ficin hydrolysis of phage proteins Two other general food proteases were then compared to papain. Although papain, bromelain, and ficin are all cysteine-proteinases and used as meat tenderizers, the latter two are used less often because they soften meat too quickly rendering too much proteolytic activity (Romans, Costello, Jones, Carlson, & Ziegler, 1985). Bromelain is noted to have less cleavage activity than ficin (Romans et al., 1985). Since these two enzymes have broader specificity than papain, it was thought that they might produce smaller peptides and may have the ability to reduce phage titers in the filtrate to zero. To test this hypothesis, lactic C2 culture was grown in L-M17 medium which contained one of the three proteases and infected with c2 bacteriophage 1 h after inoculation. Two controls were also run, both without enzyme but with and without c2 phage added. Media inoculated with C2 culture were observed for turbidity development. Both the control and the papain containing media that were infected with c2 phage cleared within 3 h suggesting that viable c2 phage were present. Inoculated L-M17 media containing the bromelain and
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ficin had no observed clearing and were very turbid after 5 h (optical density > 1.0) suggesting that the C2 culture reached its stationary phase without lysing even though they had been infected at the same concentration as the papain medium. These data suggested that ficin and bromelain were proteolytic enough to destroy the phage structural proteins or remove their adsorption proteins during hydrolysis such that the c2 phage proliferation was dramatically inhibited. Since little culture growth inhibition was observed when these two enzymes were present in the growth medium, it was hypothesized that the resulting peptides were not blocking metabolic receptors (Joklik, Wilett, Amos, & Wilfert, 1992), that little or no cell lysis occurred, and that the peptidoglycan layer of the cell surface membrane was protecting the cells from enzyme degradation. When ficin and bromelain filtrates (3000 mwco) were prepared from the digests and assayed for viable phage using enumeration techniques, no phage plaques were observed on the plates, suggesting that these filtrates were free of viable phage. Both ficin and bromelain filtrates were presumed to be equally acceptable for additional research to determine if the peptides in these filtrates would competitively inhibit adsorption of viable c2 phage. However, since ficin costs less than bromelain, ficin was deemed to be the commercially viable enzyme and was used for all further research on competitive inhibition of peptides hydrolyzed from c2 phage. 3.3. Determination of residual ficin in the filtrate L-M17 medium containing c2 phage (1 109 pfu) was hydrolyzed with ficin as described. Cell debris and ficin were removed from the filtrate containing the peptides by using a 3000 mwco ultrafiltration membrane. Ficin activity in filtrate (permeate) was assayed using the Berridge Clot Timing Method (Berridge, 1952) by adding 0.1 g of freeze dried permeate to 10 ml of reconstituted skim milk. Three controls were also prepared, one with reconstituted skim milk only and two with ficin added at concentrations of 0.025% and 0.01%, respectively. Skim milk containing 0.025% ficin coagulated after 3.69 min (average). This percentage of ficin showed that small amounts of crude enzyme coagulate the milk very quickly. When the ficin concentration was reduced to 0.01%, the milk coagulated after 45.23 min (average). Even at a lower enzyme concentration the casein in milk first formed small flocs that came together to form larger, visible curd particles. This concentration of ficin took 41.54 min longer to coagulate milk than the milk containing 0.025% ficin. The bottles containing only reconstituted skim milk or 1% freeze dried permeate in the reconstituted skim milk never coagulated within the 5 h period that the samples were observed. Even trace amounts of ficin would have caused the reconstituted skim milk to coagulate within 5 h. If any
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ficin was present in the freeze dried permeate, the concentration would be so low that it would not affect any yield or quality aspect of cheese production. Since ficin has a molecular weight of approximately 29,000, it would not be expected to go through a 3000 mwco filter. 3.4. Determination of activity of medium derived peptides To determine if the active peptides were derived from ficin hydrolyzed L-M17 medium, filtrates (3000 mwco) of ficin hydrolyzed L-M17 media (filtrate contained hydrolyzed peptides and other low molecular weight compounds from the L-M17 medium) were prepared and freeze dried. Paired tubes of L-M17 medium (two sets of tubes) with (2%) and without freeze dried filtrates were assayed for activity by inoculating the media with C2 culture and infecting one of the paired tubes with c2 phage after 1 h. No differences (p > :52) were observed between the slope of the log growth, time to lysis and lysis slope of C2 culture that was grown on media containing filtrate from ficin hydrolyzed L-M17 medium and the non-filtrate containing L-M17 medium (Fig. 1). Filtrates from ficin hydrolyzed L-M17 medium inhibited culture growth slightly, thus time to lysis (233.5 vs. 244 min) shifted 10.5 min (average) prior to the lysis of nonfiltrate media. Therefore, any peptides derived from L-M17 or ficin were deemed to only impede L. lactis ssp. lactis C2 growth without inhibiting phage proliferation. In fact, the L-M17 medium hydrolysate may have actually enhanced phage proliferation rate. When these data were compared with data from medium containing c2 peptide, a dramatic difference in phage proliferation was observed, thus additional research using 2% peptide was initiated.
Fig. 1. Growth of Lactococcus lactis ssp. lactis C2 culture in M17 medium with ðjÞ and with out ðÞ M17 medium ficin digest added (2% peptide, growth controls). Two other growth tubes inoculated with L. lactis ssp. lactis C2 were infected with L.lacits ssp. lactis c2 bacteriophage with ðrÞ and without ðNÞ Ml7 medium ficin digest peptides added.
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3.5. Addition of 2% phage peptide to the growth medium When L-M17 medium with and without 2% c2 phage peptide (mixture of phage peptides plus M17 peptides with other low molecular weight compounds from the medium) was inoculated with 4 107 cfu/ml C2 culture, the culture grown in medium without peptide grew faster (p < :07Þ than the culture in medium containing the peptide (Fig. 2). The stationary phase of the culture grown in medium containing the peptide was reached approximately 40 min after the culture grown in medium without peptide. Culture growth inhibition was similar to that observed for C2 culture grown in medium containing peptides from the L-M17 ficin digest (Fig. 1) suggesting that a peptide derived from the L-M17 medium ficin digest may be impeding the growth of the C2 culture independent of the peptides derived from c2 phage. Presumably, a large portion of the peptides added to this medium were derived from the bacteriophage since the viable phage concentration in the medium was 109 pfu/ml before hydrolyzation and less than one pfu/ ml after hydrolyzation. Although the ficin hydrolyzation step did not impede the C2 culture growth, one could imagine that a small amount of hydrolyzed peptide
Fig. 2. Growth of Lactococcus lactis ssp. lactis C2 in M17 medium with ðjÞ and without ðÞ ficin digested L. lactis ssp. lactis c2 bacteriophage c2 peptides added (2% c2 peptide). Two other growth tubes inoculated with L. lactis ssp. lactis C2 were infected with L.lactis ssp.lactis c2 bacteriophage with ðrÞ and without ðNÞ c2 phage peptides added.
could be derived from host cell proteins. Also, note that growth slopes (Fig. 2) did not decrease until the stationary phase was reached suggesting that culture agglutination was not a problem (Ustunol & Hicks, 1994) in this model system within the 380 min fermentation period. No differences in the initial log slope was observed for culture grown in media with and without added peptides (Table 1). Growth time of C2 culture in L-M17 medium with added peptide and phage was extended (p < :01) from 182 to 236 min before lysis occurred (Table 1), an increase of 54 min compared to culture grown in medium without peptide. Culture growth in the medium containing peptide also increased (p < :01) during this time period (approximately 0.26 increase in optical density). These data suggested that the addition of phage peptide to a growth medium protected the C2 culture from c2 phage and allowed the culture to increase in cell numbers before lysis occurred. Culture grown in medium with peptide had a slightly steeper lysis slope (p < :07) than the culture grown in medium without added peptide (Table 1). Since cell numbers were much higher in the medium with peptide, total cell surface area would have been much greater thus saturation of the cells membrane with peptide may have become limiting by the time c2 phage numbers exceeded C2 cell numbers, thus resulting in a sharper growth apex and a steeper lysis slope. When these data were compared to that of Onuorah (1996), it appears that phage peptides may be a better inhibitor of phage proliferation than whey peptides. OnuorahÕs (1996) results show a significant reduction in cell growth when whey peptides were added to the growth medium. The c2 phage peptide does not appear to depress cell growth as much as the whey peptides, suggesting that the whey peptides may bind to the receptors that are important to metabolite transport, whereas the c2 phage peptide may be binding to a different receptor on the cell membrane. Also, Onuorah (1996) reported using 0.5 g of whey peptide/ml for optimum growth time to the growth apex. Phage peptides in these experiments were added at 0.02 g peptide/ml
Table 1 Differences in slope of lag phase, slope of log phase, time to apex, and lysis slope of C2 culture grown in M17 medium with and without (w/wo) phage peptides and with and with out c2 phage
Culture grown in Ml7 medium Culture grown in M17 medium with phage peptides Absolute differences a
b;c
Slope of lag phase (dx=dy) w/wo phage
Slope of log phase (dx=dy) w/wo phage
Time to growth apex (min) with phage
Slope of lysis (dx=dy) with phage
0.0012a
0.0047a
182b
)0.0091b
0.0011a
0.00433a
236c
)0.014c
0.0001
0.0004
54
)0.0049
Nonsignificant. Differences of significance within columns at p < :01, n ¼ 3.
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which resulted in a longer growth time before cell lysis occurred, suggesting that phage peptides are more effective for inhibiting phage proliferation than whey peptides. 3.6. Testing C2 culture in milk coated with and without c2 phage peptides Bulk starters of C2 culture grown in L-M17 medium with and without phage peptides were used to inoculate milk (with and without c2 phage peptide) that had been infected with and without c2 phage. The pH decreased faster (end pHs were lower, p < :05) in milk inoculated with C2 culture grown in medium without added c2 phage peptide than in milk inoculated with culture grown in medium containing c2 peptide (Fig. 3) suggesting that the c2 peptides were slightly inhibiting culture growth in milk similar to that observed for cultures in M17 medium. These two milks had better (p < :0001) acid production (pH 5.63 and 5.71, respectively after 3.7 h of fermentation) than the other four milks. However, milks that were inoculated with culture grown in c2 peptide (both the 2% peptide medium and 1% peptide medium with1% peptide added to the milk) and infected with c2 phage did continue to produce acid (to pH 6.01) throughout the fermentation period. The cultures in these two milks produced more acid (p < :05) than the cultures that were in infected milk with and without added peptide in the milk. No differences were observed
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in acid production in milks with or without added peptides. When 2% c2 peptide was added to the milk and inoculated with culture grown in medium without peptide, acid development stalled (pH 6.28) after 100 min (curd was coagulated) and the pH decline at a much slower rate. These data suggest that lysis of the culture was nearly complete by the time the chymosin formed a soft coagulum. Acid production in milk inoculated with culture grown in medium without peptide and infected with c2 phage stopped (pH 6.25) after 100 min of fermentation suggesting that lysis was complete. Note that acid production started again (Fig. 3) after 180 min as C2 mutants gained in numbers. These results suggest the culture grown in media containing c2 peptide was partially protected from c2 phage proliferation and lysis during the fermentation period. Culture grown in peptide containing media was protected more than when the same amount of peptide was added to the milk, or when no peptide was present. Acid production generally continues if lysis of the culture has not occurred by the time the coagulant forms a curd and prevents the movement of newly released phage. This phenomenon was observed with cultures that were grown in all media that contained c2 phage peptides. Thus cultures grown in media containing phage peptides may be more resistant to phage infections and this technology may provide an additional barrier to phage infection under manufacturing conditions.
4. Conclusions
Fig. 3. Effect of c2 phage peptide on the fermentation activity (pH) of C2 lactic culture with and with out c2 phage present. All standardized, pasteurized milk was inoculated with 4% culture. Cultured milks were ripened (31 °C for 1 h), rennetted (ca. 25 min cutting time), and cook to 37 °C and held at the temperature. M, C2 lactic culture grown in LM17 medium without c2 phage peptides and inoculated into milk; , C2 lactic culture grown in L-M17 medium containing 2% c2 phage peptide and inoculated into milk; N, C2 lactic culture grown in L-M17 medium containing 2% c2 phage peptide and inoculated into milk infected with 103 pfu/ml c2 phage; }, C2 lactic culture grown in LM17 medium containing 1% c2 phage peptide and inoculated into milk containing 1% c2 phage peptide and infected with 103 pfti/ml c2 phage; r, C2 lactic culture grown in L-M17 medium and inoculated into milk containing 2% c2 phage peptide and infected with 103 pfu.ml c2 phage; , C2 culture grown in L-M17 medium and inoculated into milk infected with 103 pfu/ml.
Phage peptides were successfully hydrolyzed from c2 bacteriophage using ficin or bromelain. Ficin and bromelain hydrolysates destroyed the ability of c2 bacteriophage to infect a C2 culture. Ficin was chosen as the most likely commercial enzyme because of its lower cost. Residual ficin was not found in the c2 phage peptide permeate (3000 mwco). Peptides derived from hydrolyzed L-M17 medium (ficin digest) were found to be inhibitory to C2 culture growth and were not inhibitory to c2 phage proliferation. However, ficin hydrolyzed c2 phage peptides were effective in prolonging the growth time of C2 lactic culture in L-M17 medium and milk when infected with c2 bacteriophage. C2 culture was protected from c2 phage through the ripening and renneting periods when C2 bulk starter was prepared in a medium containing the c2 phage peptides.
References Bao, G., & Scott, S. (2000). Langevin dynamics of ing-motion proteins. Annuals of Biomedical Engineering, 28(Suppl. 1), S32. Berridge, N. J. (1952). An improved methods of observing the clotting of milk containing rennin. Journal of Dairy Research, 19, 328–329.
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Parrot, M., & Moineau, S. (2001). DNA Boucher, I., Emond, E, sequence analysis of three Lactococcus lactis plasmids encoding phage resistance mechanisms. Journal of Dairy Science, 84, 1610– 1620. Champagne, C. P., & Lange, M. (1992). Preparation of starters and their subsequent sensitivity to bacteriophage. Cultured Dairy Products Journal, 27(2), 16–21. Cogan, T. M., & Accoulas, J. P. (1990). Starter cultures: Types, metabolism, and bacteriophage. In R. K. Robinson (Ed.), The microbiology of milk: Vol. 1. Dairy microbiology (2nd ed., pp. 77– 114). London: Elsevier. Douglas, F., Qanber-Agha, A., & Phillips, V. (1974). Medium for the propagation and assay of lactic and other phages. Laboratory Practical, 23, 3–5. Garbutt, K. C., Kraus, J., & Geller, B. L. (1997). Bacteriophage resistance in Lactococcus lactis engineered by replacement of a gene for a bacteriophage receptor. Journal of Dairy Science, 80, 1512– 1519. Gravey, P., van Sinderen, D., Twomey, D. P., Hill, C., & Fitzgerald, G. F. (1995). Molecular genetics of bacteriophages and natural defense systems in the genus Lactococcus. International Dairy Journal, 5, 905–947. Heap, H. A., & Lawrence, R. C. (1976). The selection of starter strains for cheese making. New Zealand Journal of Dairy Science and Technology, 11, 16–20. Hicks, C .L., inventor (2001). Cheese making with bacteriophage resistant bacteria. University of Kentucky, assignee. Pat. No. 6,297,042. October 2. Hicks, C. L., Onuorah, C. E., & Surjawan, I. (2000). Use of hydrolyzed whey peptide to inhibit culture agglutination. Journal of Dairy Science, 83, 1196–1202. Jarvis, A. W. (1981). The use of whey-derived phage resistant starter strains in New Zealand cheese plants. New Zealand Journal of Dairy Science and Technology, 16, 25–31. Jarvis, A. W. (1978). Serological studies of a host range mutant of a lactic streptococcal bacteriophage. Applied and Environmental Microbiology, 36, 785–789. Jarvis, A. W., & Klaenhammer, T. R. (1986). Bacteriophage resistance conferred of lactic Streptococci by the conjugative plasmid pTR2030: Effects on small isometric-, large isometric-, and prolate-headed phages. Applied and Environmental Microbiology, 51(6), 1272–1277. Joklik, W. K., Wilett, H. P., Amos, D. B., & Wilfert, C. M. (1992). Bacteriophage. In Zinsser (Ed.), Microbiology (pp. 906–928). Norwalk, CT: Appleton and Lange. Klaenhammer, T. R. (1987). Plasmid-directed mechanisms for bacteriophage defense in lactic streptococci. FEMS Microbiology Review, 46, 313–325. Klaenhammer, T. R., & Sanozky, R. B. (1985). Conjugal transfer of plasmid from Streptococcus lactis ME2 encoding phage resistance, nisin resistance, and lactose fermenting ability: Evidence for high frequency conjugal plasmid responsible for abortive infection of virulent bacteriophage. Journal of General Microbiology, 131, 1531–1541. Kosikowski, F. V. (1982). Cheese and fermented milk foods (2nd ed., pp. 18, 27–29). Brooktondale, NY: Kosikowski FV and Associates. Kraus, J., & Geller, B. L. (1998). Membrane receptor for prolate phage is not required for infection of Lactococcus lactis by small or large isometric phage. Journal of Dairy Science, 81, 2329– 2335. Lawrence, R. C., Heap, H. A., Limsowtin, G., & Jarvis, A. W. (1978). Cheddar cheese starters: Current knowledge and practices of phage characteristics and strain selection. Journal of Dairy Science, 61,1181–1191.
Ledford, R. A., & Speck, M. L. (1979). Injury of lactic streptococci by culturing in media containing high phosphates. Journal of Dairy Science, 62, 781–784. Lubbers, M. W., Waterfield, N. R., Beresford, T. P. J., Le Page, R. W. F., & Jarvis, A. W. (1995). Sequencing and analysis of the prolateheaded lactococcal bacteriophage c2 genome and identification of the structural genes. Applied and Environmental Microbiology, 61, 4348–4356. Martley, F. G. (1972). The effect of cell numbers in streptococcal chains on plate counting. New Zealand Journal of Dairy Science and Technology, 7, 7–11. McLandsborough, L. A., Sechaud, L., & McKay, L. L. (1998). Synergistic effects of abiE or abiF from pNP40 when cloned in combination with abiD from pBF61. Journal of Dairy Science, 81, 362–368. Moineau, S., Borkaev, M., Holler, B. T., Walker, S. A., Kondo, J. K., Vedamutha, E. R., & vandenbergh, P. A. (1996). Isolation and characterization of lactococcal bacteriophage from cultured buttermilk plants in the United States. Journal of Dairy Science, 79, 2104–2111. Monteville, M. R., Ardestrani, B., & Geller, B. L. (1994). Lactococcal bacteriophages require a host cell wall carbohydrate and a plasma membrane protein for adsorption and ejection of DNA. Applied and Environmental Microbiology, 60(9), 3204–3211. Neve, H. (1995). Bacteriophage. In T. M. Cogan & J. P. Accoulas (Eds.), Dairy starter cultures (pp. 157–186). New York: VCH Publishers. Onuorah, C. E. (1996). Use of hydrolyzed whey peptide blockers to inhibit culture agglutination and phage proliferation in lactic bulk starter culture. Ph.D. dissertation, University of Kentucky, Lexington, KY. Onuorah, C. E., Hicks, C. L., & OÕLeary, J. (1995). Use of peptide blockers to inhibit phage proliferation in lactic bulk starter. Journal of Dairy Science, 78(Suppl. 1), 110, D48. Popescu, A. I. (1996). Cell–cell interactions. A physical approach. Bioelectrochemcal Bioengineering, 40(2). Powell, I. B., Tulloch, D. L., Hillier, A. J., & Davidson, B. E. (1992). Phage DNA synthesis and host DNA degradation in the life cycle of Lactococcus lactis bacteriophage c6A. Journal of General Microbiology, 138, 945–950. Russell-Campbell, E., Hicks, C. L., & OÕLeary, J. (1995). Inhibition of lactic bacteriophage proliferation in bulk starters. Journal of Dairy Science, 78(Suppl. 1), 111, D50. Romans, J. R., Costello, W. J., Jones, K. W., Carlson, C. W., & Ziegler, P. T. (1985). The meat we eat (12th ed., pp. 542–543). Danville, IL: The Interstate Printers and Publishers. SAS (1994). SAS Systems for Windows (Release 6.10 ). Cary, NC: SAS Inst. Inc. Sing, W. D., & Klaenhammer, T. R. (1993). A strategy for rotation of different bacteriophage defenses in a lactococcal single-strain starter culture system. Applied and Environmental Microbiology, 59(2), 365–372. Terzaghi, B. E., & Sandine, W. E. (1975). Improved medium for lactic streptococci and their bacteriophages. Applied and Environmental Microbiology, 29(6), 807–813. Ustunol, Z., & Hicks, C. L. (1994). Use of an enzyme-treated, wheybased medium to reduce culture agglutination. Journal of Dairy Science, 77, 1479–1486. Valyasevi, R., Sandine, W. E., & Geller, B. L. (1991). A membrane protein is required for bacteriophage c2 infection of Lactococcus lactis ssp. lactis C2. Journal of Bacteriology, 173, 6095–6100. Valyasevi, R., Sandine, W. E., & Geller, B. L. (1994). Lactococcus lactis ssp. lactis C2 bacteriophage sk1 involves rhamnose and glucose moieties in the cell wall. Journal of Dairy Science, 77, 1–6.
Use of bacteriophage-derived peptides to delay phage infections q,qq C.L. Hicks a
a,*
, P.A. Clark-Safko b, I. Surjawan a, J. OÕLeary
a
Department of Animal Sciences, University of Kentucky, 410 W. P. Garrigus Bldg., Lexington, KY 40546, USA b b1326 Glenwich Ave. Windermere FL 34786, USA Received 8 July 2003; accepted 15 September 2003
Abstract Peptides were prepared from structural proteins of Lactococcus lactis ssp. lactis c2 bacteriophage and used to inhibited c2 phage proliferation in L. lactis ssp. lactis C2 host. Culture media that contained phage peptides inhibited C2 growth slightly. However, when C2 culture was grown in media that contained c2 phage peptides and infected with c2 bacteriophage, host cell populations and time to growth apex increased over culture grown in medium without peptides. Growth time prior to lysis was extended 54 min when infected with 1 107 pfu/ml of c2 bacteriophage. Lysis slope was steeper for culture grown in medium with phage peptides than medium without phage peptide. Milk inoculated with bulk starters prepared with c2 phage peptides were more resistant to c2 phage proliferation than milk inoculated with bulk starter containing no c2 peptides or milks having c2 phage peptides added to the milk. Ó 2003 Elsevier Ltd. All rights reserved. Keywords: Bacteriophage; Proliferation; Peptides; Inhibition; Lactococcus lactis ssp. lactis C2
1. Introduction Bacteriophage (phage) infections in milk fermentations have been a major problem for many years and still cause most culture failures (Moineau et al., 1996). Phage may be introduced into cheese plants from many sources. Phage can be carried on clothing as well as in cheese whey and mist (Kosikowski, 1982; Neve, 1995). An infection occurs when phage are specific for the bacteria in use. Phage specificity may be visualized as a lock and key fit (phage receptor to bacteria receptor site). The host receptor site for phage adsorption (reversible step) generally consists of carbohydrates (usually rhamnose and/or glucose) (Valyasevi, Sandine, & Geller, 1991, 1994). Often several lactic phage are found absorbed onto a common spot on the host cell (Neve, 1995).
q
Published with the approval of the director of the Kentucky Agriculture Experiment Station as Journal Article Number 01-07-164. qq Research was conducted at the University of Kentucky. * Corresponding author. Tel.: +859-257-7537; fax: +859-257-7537. E-mail address: [email protected] (C.L. Hicks). 0963-9969/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2003.09.009
Presumably, phage spots occur where the polysaccharide coating on the cell membrane thins and nutrient transport occurs (Neve, 1995). Initial adsorption and movement of the phage on the cell surface appears to be facilitated by Brownian movement (Bao & Scott, 2000; Popescu, 1996) and a calcium binding domain such as the one located on the F protein of c2 phage (Lubbers, Waterfield, Beresford, Le Page, & Jarvis, 1995). After the phage has absorbed onto the surface of the host cell an irreversible attachment occurs between the phage adsorption protein (Lubbers et al., 1995) and a membrane component. Lactococcus lactis ssp. lactis C2 membrane contains a protein (99 kDa, pip gene) that binds with the c2 phage adsorption protein (Garbutt, Kraus, & Geller, 1997; Kraus & Geller, 1998; Monteville, Ardestrani, & Geller, 1994) to form the irreversible attachment step where the phage secures its tail onto the bacterial membrane by hooking spikes into the cell membrane (Neve, 1995; Valyasevi et al., 1991). After the tail is secured, the c2 phage ejects its DNA after being triggered by the pip gene protein (Gravey, van Sinderen, Twomey, Hill, & Fitzgerald, 1995). Once the phage DNA is in the cell it takes command over cell processes to perform transcription,
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translation, and replication (Powell, Tulloch, Hillier, & Davidson, 1992). Various technologies have been developed over the years to prevent phage attack in cheese plants. These technologies include the use of phosphate containing media for bulk starter production and culture rotation schemes (Jarvis, 1981; Ledford & Speck, 1979). Most industrial cultures are phage resistant mutants that are grown in phosphate media. Culture strains are rotated daily or when specific phage infections are detected (Cogan & Accoulas, 1990; Heap & Lawrence, 1976; Sing & Klaenhammer, 1993). Techniques using gene transfer for generating resistant starter strains have also been reported (Boucher, Emond, Parrot, & Moineau, 2001; Jarvis, 1981, 1978; Jarvis & Klaenhammer, 1986; Klaenhammer, 1987; Klaenhammer & Sanozky, 1985; McLandsborough, Sechaud, & McKay, 1998; Sing & Klaenhammer, 1993). Culture rotation is not without its problems. Rotations of cultures bring about strain to strain variations (Champagne & Lange, 1992; Heap & Lawrence, 1976; Lawrence, Heap, Limsowtin, & Jarvis, 1978) which can include differentiation of growth time and acid production. Culture variations may speed up or slow down plant processing time. If production is sped up, an excess of product may occur in one stage of processing while slower culture development delays production. The ideal concept would be to use one strain of a bacterium continuously which would eliminate culture rotation and strain to strain variation. One culture strain could be used, if cells were protected against all infecting bacteriophage. A technology using whey peptides as an additive to M17 medium was shown to prolong the time from phage inoculation to time of culture lysis (Onuorah, Hicks, & OÕLeary, 1995; Russell-Campbell Hicks & O’Leary, 1995). However, bacterial growth was inhibited slightly and the protection provided by this peptide was deemed insufficient to impede bacteriophage growth in an industrial cheese processing environment. It was theorized, that if peptides, that were highly specific to the cell receptor, could be isolated from bacteriophage coat proteins by enzymatic hydrolysis, they may compete with viable phage for binding sites on the host cell, thus inhibiting or preventing viable phage attachment (Hicks, 2001). If this technology reduces the number of phage attaching to cells a decrease in the rate of phage proliferation should be noted. However, as bacteria divide and reproduce, fewer peptides would be available to cover new bacterial receptor sites and phage proliferation would eventually occur after the peptide concentration became limited. This system, nonetheless, may prevent phage infection for a sufficient time to allow cheese fermentation. Therefore the objective of this study was to develop a phage peptide blocker to inhibit phage attachment and proliferation.
2. Materials and methods 2.1. Culture and phage Lactococcus lactis ssp. lactis C2 and c2 bacteriophages were obtained from Klaenhammer (North Carolina State University, Raleigh, NC). 2.2. Propagation medium M17 medium, described by Terzaghi and Sandine (1975), was used as a growth medium for Lactococcus cultures. The medium was prepared from individual components or from a pre-formulated M17 medium (Fisher Scientific, Pittsburgh, PA). Pre-formulated medium was prepared by combining 37.5 g of M17 powder and 950 ml distilled water. All ingredients were mixed and sterilized as described by Terzaghi and Sandine (1975). A cold sterilized lactose solution was added (50 ml) to the sterilized M17 medium and was termed L-M17 (final lactose concentration 0.1%). 2.3. Preparation of phage enumeration plates Bottom agar (modified procedure of Douglas, Qanber-Agha, & Phillips, 1974) was prepared by adding 100 ml of 1 M CaCl2 6H2 0 containing 15 g of bactoagar to 37 g of M17 medium which was made up to 950 ml. The pH was adjusted to 6.9. The medium was sterilized at 121 °C for 15 min. After the medium was sterilized, cold sterilized lactose (50 ml) was added (final concentration 0.1%) at 45 °C. Approximately 10 ml of agar was dispensed into each Petri dish. Agar was allowed to solidify and plates were stored at 5 °C. Top agar was prepared using the same method except that 4.5 g agar was added to the M17 medium. To enumerate c2 phage, serial dilutions of bacteriophage were made in phosphate buffer (Martley, 1972). Top agar was steamed for 20 min and tempered to 45 °C. Active lactic C2 culture (1 109 cfu/ ml) was added (0.1 ml) to 2.5 ml top agar. The appropriate dilution of phage was added (0.1 ml), vortexed, and allowed to stand for 10 s. The mixture was then overlaid on the pre-prepared bottom agar plate. Plates were incubated at 26 °C for 6 h uninverted. Plaques were counted after 6 h of incubation. 2.4. Preparation of phage peptides using papain hydrolysis Ustunol and Hicks (1994) used 2% crude papain or a dried papaya extract to hydrolyze whey and then harvested the peptides using ultrafiltration techniques. A similar procedure was used to prepare phage peptides. Filtered (cell debris removed using a 0.45 lm membrane) phage (1 109 pfu/ml) was hydrolyzed with crude papain (2%) in L-M17 broth for 4 h at 26 °C. Digest was filtered through a 3000 molecular weight cut
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off (mwco) membrane (Amicon, Inc., Beverly MA) to remove phage debris and papain. Filtrate containing the phage peptides was tested for infectivity by assaying the filtrate as previously described (Douglas et al., 1974). Some filtrates were frozen ()20 °C) and freeze dried. 2.5. Evaluation of papain, bromelain and ficin to prepare phage peptides Five tubes of L-M17 broth (10 ml) with 0.1% CaCl2 were inoculated with 0.4 ml L. lactis ssp. lactis C2 (4 107 cfu/ml) and incubated (26 °C). After 1 h, one enzyme (papain, bromelain, or ficin [0.1%]) (Sigma Inc., St. Louis, MO) and 0.001 ml c2 phage (1 109 pfu) was added to three of the five tubes. The fourth tube served as a growth control. The fifth tube had 0.001 ml of c2 phage added after 1 h and served as a culture lysis control. Growth and cell lysis were observed (k600 nm , Milton Roy spectrophotometer) for 5 h by monitoring changes in turbidity. Digests were assayed for c2 phage titers as previously described. 2.6. Determination of ficin in the permeate Freeze dried permeate from ficin treated L-M17 medium containing c2 peptide was added to low heat reconstituted non-fat dry milk (Armour, Springfield, KY) (10 ml, 10% solids) at 1% (0.1 g) to determine the amount of residual ficin in the permeate. Crude ficin (0.01% and 0.025%) was added to 10 ml of non-fat dry milk to serve as coagulation controls. A tube of reconstituted non-fat dry milk served as a non-coagulating control. Coagulation of the milk was observed using the Berridge Clot Timing Method (Berridge, 1952). Bottles (125 ml) were rotated (3.0 rpm, 30° angle) and held at 31 °C. Milk clotting was visualized as a floc-containing film that fractured on the inside of the bottle. Bottles were observed repeatedly over a 5 h period for the presence of flocs. 2.7. Determination of activity of medium derived peptides Media with and without culture cell debris and with and without c2 phage were hydrolyzed with ficin (0.1%) to confirm that the peptide was derived from c2 phage. Hydrolyzed fractions were ultra-filtered (3000 mwco filter) under nitrogen pressure (4.06 atm. at 5 °C) and checked for viable phage using enumeration techniques. Permeates containing hydrolyzed peptides were freeze dried and stored at )20 °C until being used to prepare media formulations. 2.8. Preparation of phage peptides for additional research Propagated c2 phage in L-M17 broth was filtered (0.45 lm) to remove cellular debris into a sterile glass
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bottle. Bacteriophage in L-M17 broth was hydrolyzed with crude ficin (0.1%) for 4 h at 22–24 °C. The hydrolyzed mixture was ultra-filtered (3000 mwco filter) under nitrogen pressure (4.06 atm. at 5 °C) to remove ficin and phage debris from the permeate. Permeate was checked for viable phage using enumeration techniques and was found to be free of plaque forming units. Permeate containing hydrolyzed peptides was dialyzed (500 mwco, Spectra Por, Fisher Scientific, Pittsburgh, PA ) in 0.10 ionic strength sodium potassium phosphate buffer (pH of 6.4) for 6 h at 5 °C. Fresh phosphate buffer was replaced and dialysis continued for another 10 h. Dialyzed peptides were freeze dried and stored at )20 °C. 2.9. Media preparation and procedure for phage peptide blocking experiments Four test tubes of sterilized L-M17 broth (10 or 25 ml) containing CaCl2 (0.01%) with (two tubes) and without (two tubes) c2 phage peptide (2%) were inoculated (4% of 1 109 cfu/ml L. lactis ssp. lactis C2) and incubated at ambient temperature. Another tube containing L-M17 medium with only CaC12 added was used to zero the spectrophotometer (k600 nm ). Phage (0.001 ml of c2 phage, 1 107 pfu) was inoculated into two tubes (one with c2 phage peptides and one without peptides) after the tubes had been incubated for 1 h. Tubes with and without c2 peptides with added phage were used to determine the effect of the peptides on phage proliferation. Growth and lysis curves were determined spectrophotometrically (k600 nm ). Spectrophotometric readings were taken every 20 min for as long as 380 min. 2.10. Testing C2 culture in milk coated with and without c2 phage peptides C2 culture was grown in L-M17 medium with (1% and 2%) and without c2 phage peptides (3 starter cultures). Standardized (3.25% fat), pasteurized (72 °C for 17 s) milk (96 ml in 6 beakers at 31 °C) was prepared. Two milks were fortified with c2 phage peptides (1% and 2%, respectively). All milks were inoculated (4%) with culture as follows: two milks were inoculated with culture that was grown in medium containing 2% c2 peptide; one milk (containing 1% c2 peptide) was inoculated with culture that was grown in medium containing 1% c2 phage peptide; and three milks were inoculated with culture grown in medium containing 0% c2 phage peptide (This included the milk that contained 2% c2 phage peptide.). All milks were infected with 103 pfu/ml c2 phage, except for one milk that was inoculated with culture without peptide and one milk that was inoculated with culture containing 2% peptide. All milks were allowed to ripen for 1 h, prior to renneting (Chymosin EC 3.4.23.4, isozyme A, Pfizer, Milwaukee, WI). Curd
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was cut after approximately 25 min, heated from 31 to 37 °C in a 30 min period, and held at that temperature until the control pH declined to 5.6 (ca 1.25 h after cutting). Acid development (pH) was monitored at 20 min intervals throughout the period. Culture activity (pH) versus incubation time was plotted and analyzed. 2.11. Experimental procedure, data and statistical analysis All experiments were conducted using randomized block designs that were replicated three times. Growth and lysis curves were plotted for each replication. Measurements were taken from each plot to determine slope of the log growth and lysis rates, and for time from phage addition to growth apex (dependant variables). Effect of media with and without c2 phage peptides (independent variables) on these slopes and time to apex were statistically analyzed (SAS, 1994) using general linear models procedures, SS4 sums of squares, and orthogonal contrasts. These same procedures were used to test differences between end pH values of milk subjected to different culture treatments, except replication and time were used as co-variants.
3. Results and discussion L. lactis ssp. lactis C2 and its bacteriophage (c2) were utilized for this research because they comprise one of the best characterized and understood phage/host systems (Lubbers et al., 1995), even though C2 culture is less active than most cheese cultures. An L-M17 growth medium was selected for this research because of its transparency and because it is an excellent growth medium for lactic acid bacteria. Although this model system was not representative of a commercial culturemedium system it was selected because of the ease in which cell growth and lysis could be monitored. Since Hicks, Onuorah, and Surjawan (2000) observed that peptides derived from whey had an inhibitory effect on phage proliferation, it was theorized (Hicks, 2001) that peptides derived from specific phage structural proteins might have a greater inhibitory effect on phage proliferation than whey peptides. Since c2 phage coat protein (F protein) is thought to make the attachment between c2 phage and L. lactis ssp. lactis C2, the F protein would be the most likely target for protein hydrolysis. Also, since the c2 phage F protein is thought to contain one calcium binding site and one attachment site only one peptide may be active. Therefore, this research was undertaken to develop a phage peptide that would inhibit phage proliferation through competitive inhibition or the competitive binding of the phage peptide to the receptor on the host cell thus limiting the number of receptors available to bind native phage.
3.1. Papain hydrolysis of phage proteins Ustunol and Hicks (1994) reported that whey peptides could be prepared by hydrolyzing whey with a general protease such as papain. Therefore, papain was added to c2 phage filtrates in hopes that peptides could be cleaved from phage structural proteins. After the papain had reacted for 4 h, debris from the digests was removed using a 3000 mwco membrane. However, these 3000 mwco filtrates were not completely free of viable c2 phage. Phage titers remaining in the 3000 mwco filtrates averaged 1 101 pfu/ml. Although this titer was low, it was still enough to infect, and eventually, destroy C2 culture that was incubated in medium containing the filtrate. Thus the papain treatment appeared insufficient to hydrolyze all c2 phage structural proteins that are necessary for attachment and the 3000 mwco membrane did not fully exclude all viable phage that was in the digest or post contamination occurred. When the 3000 mwco filtrate was heat treated at 80 °C for 1 h no viable c2 phage remained in the filtrate. When L-M 17 medium with and without heat treated filtrate was inoculated with L. lactis ssp. lactis C2 and incubated for 1 h at 26 °C before being infected with or without c2 phage, no inhibition of phage proliferation was observed when the media contained papain c2 phage filtrate. However, growth of L. lactis ssp. lactis C2 in media that contained the heat treated papain filtrate was somewhat inhibited at the added filtrate concentration. Since culture growth was inhibited and phage proliferation was only slightly affected at the filtrate concentrations used, experimentation with other proteases was initiated. 3.2. Bromelain and ficin hydrolysis of phage proteins Two other general food proteases were then compared to papain. Although papain, bromelain, and ficin are all cysteine-proteinases and used as meat tenderizers, the latter two are used less often because they soften meat too quickly rendering too much proteolytic activity (Romans, Costello, Jones, Carlson, & Ziegler, 1985). Bromelain is noted to have less cleavage activity than ficin (Romans et al., 1985). Since these two enzymes have broader specificity than papain, it was thought that they might produce smaller peptides and may have the ability to reduce phage titers in the filtrate to zero. To test this hypothesis, lactic C2 culture was grown in L-M17 medium which contained one of the three proteases and infected with c2 bacteriophage 1 h after inoculation. Two controls were also run, both without enzyme but with and without c2 phage added. Media inoculated with C2 culture were observed for turbidity development. Both the control and the papain containing media that were infected with c2 phage cleared within 3 h suggesting that viable c2 phage were present. Inoculated L-M17 media containing the bromelain and
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ficin had no observed clearing and were very turbid after 5 h (optical density > 1.0) suggesting that the C2 culture reached its stationary phase without lysing even though they had been infected at the same concentration as the papain medium. These data suggested that ficin and bromelain were proteolytic enough to destroy the phage structural proteins or remove their adsorption proteins during hydrolysis such that the c2 phage proliferation was dramatically inhibited. Since little culture growth inhibition was observed when these two enzymes were present in the growth medium, it was hypothesized that the resulting peptides were not blocking metabolic receptors (Joklik, Wilett, Amos, & Wilfert, 1992), that little or no cell lysis occurred, and that the peptidoglycan layer of the cell surface membrane was protecting the cells from enzyme degradation. When ficin and bromelain filtrates (3000 mwco) were prepared from the digests and assayed for viable phage using enumeration techniques, no phage plaques were observed on the plates, suggesting that these filtrates were free of viable phage. Both ficin and bromelain filtrates were presumed to be equally acceptable for additional research to determine if the peptides in these filtrates would competitively inhibit adsorption of viable c2 phage. However, since ficin costs less than bromelain, ficin was deemed to be the commercially viable enzyme and was used for all further research on competitive inhibition of peptides hydrolyzed from c2 phage. 3.3. Determination of residual ficin in the filtrate L-M17 medium containing c2 phage (1 109 pfu) was hydrolyzed with ficin as described. Cell debris and ficin were removed from the filtrate containing the peptides by using a 3000 mwco ultrafiltration membrane. Ficin activity in filtrate (permeate) was assayed using the Berridge Clot Timing Method (Berridge, 1952) by adding 0.1 g of freeze dried permeate to 10 ml of reconstituted skim milk. Three controls were also prepared, one with reconstituted skim milk only and two with ficin added at concentrations of 0.025% and 0.01%, respectively. Skim milk containing 0.025% ficin coagulated after 3.69 min (average). This percentage of ficin showed that small amounts of crude enzyme coagulate the milk very quickly. When the ficin concentration was reduced to 0.01%, the milk coagulated after 45.23 min (average). Even at a lower enzyme concentration the casein in milk first formed small flocs that came together to form larger, visible curd particles. This concentration of ficin took 41.54 min longer to coagulate milk than the milk containing 0.025% ficin. The bottles containing only reconstituted skim milk or 1% freeze dried permeate in the reconstituted skim milk never coagulated within the 5 h period that the samples were observed. Even trace amounts of ficin would have caused the reconstituted skim milk to coagulate within 5 h. If any
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ficin was present in the freeze dried permeate, the concentration would be so low that it would not affect any yield or quality aspect of cheese production. Since ficin has a molecular weight of approximately 29,000, it would not be expected to go through a 3000 mwco filter. 3.4. Determination of activity of medium derived peptides To determine if the active peptides were derived from ficin hydrolyzed L-M17 medium, filtrates (3000 mwco) of ficin hydrolyzed L-M17 media (filtrate contained hydrolyzed peptides and other low molecular weight compounds from the L-M17 medium) were prepared and freeze dried. Paired tubes of L-M17 medium (two sets of tubes) with (2%) and without freeze dried filtrates were assayed for activity by inoculating the media with C2 culture and infecting one of the paired tubes with c2 phage after 1 h. No differences (p > :52) were observed between the slope of the log growth, time to lysis and lysis slope of C2 culture that was grown on media containing filtrate from ficin hydrolyzed L-M17 medium and the non-filtrate containing L-M17 medium (Fig. 1). Filtrates from ficin hydrolyzed L-M17 medium inhibited culture growth slightly, thus time to lysis (233.5 vs. 244 min) shifted 10.5 min (average) prior to the lysis of nonfiltrate media. Therefore, any peptides derived from L-M17 or ficin were deemed to only impede L. lactis ssp. lactis C2 growth without inhibiting phage proliferation. In fact, the L-M17 medium hydrolysate may have actually enhanced phage proliferation rate. When these data were compared with data from medium containing c2 peptide, a dramatic difference in phage proliferation was observed, thus additional research using 2% peptide was initiated.
Fig. 1. Growth of Lactococcus lactis ssp. lactis C2 culture in M17 medium with ðjÞ and with out ðÞ M17 medium ficin digest added (2% peptide, growth controls). Two other growth tubes inoculated with L. lactis ssp. lactis C2 were infected with L.lacits ssp. lactis c2 bacteriophage with ðrÞ and without ðNÞ Ml7 medium ficin digest peptides added.
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3.5. Addition of 2% phage peptide to the growth medium When L-M17 medium with and without 2% c2 phage peptide (mixture of phage peptides plus M17 peptides with other low molecular weight compounds from the medium) was inoculated with 4 107 cfu/ml C2 culture, the culture grown in medium without peptide grew faster (p < :07Þ than the culture in medium containing the peptide (Fig. 2). The stationary phase of the culture grown in medium containing the peptide was reached approximately 40 min after the culture grown in medium without peptide. Culture growth inhibition was similar to that observed for C2 culture grown in medium containing peptides from the L-M17 ficin digest (Fig. 1) suggesting that a peptide derived from the L-M17 medium ficin digest may be impeding the growth of the C2 culture independent of the peptides derived from c2 phage. Presumably, a large portion of the peptides added to this medium were derived from the bacteriophage since the viable phage concentration in the medium was 109 pfu/ml before hydrolyzation and less than one pfu/ ml after hydrolyzation. Although the ficin hydrolyzation step did not impede the C2 culture growth, one could imagine that a small amount of hydrolyzed peptide
Fig. 2. Growth of Lactococcus lactis ssp. lactis C2 in M17 medium with ðjÞ and without ðÞ ficin digested L. lactis ssp. lactis c2 bacteriophage c2 peptides added (2% c2 peptide). Two other growth tubes inoculated with L. lactis ssp. lactis C2 were infected with L.lactis ssp.lactis c2 bacteriophage with ðrÞ and without ðNÞ c2 phage peptides added.
could be derived from host cell proteins. Also, note that growth slopes (Fig. 2) did not decrease until the stationary phase was reached suggesting that culture agglutination was not a problem (Ustunol & Hicks, 1994) in this model system within the 380 min fermentation period. No differences in the initial log slope was observed for culture grown in media with and without added peptides (Table 1). Growth time of C2 culture in L-M17 medium with added peptide and phage was extended (p < :01) from 182 to 236 min before lysis occurred (Table 1), an increase of 54 min compared to culture grown in medium without peptide. Culture growth in the medium containing peptide also increased (p < :01) during this time period (approximately 0.26 increase in optical density). These data suggested that the addition of phage peptide to a growth medium protected the C2 culture from c2 phage and allowed the culture to increase in cell numbers before lysis occurred. Culture grown in medium with peptide had a slightly steeper lysis slope (p < :07) than the culture grown in medium without added peptide (Table 1). Since cell numbers were much higher in the medium with peptide, total cell surface area would have been much greater thus saturation of the cells membrane with peptide may have become limiting by the time c2 phage numbers exceeded C2 cell numbers, thus resulting in a sharper growth apex and a steeper lysis slope. When these data were compared to that of Onuorah (1996), it appears that phage peptides may be a better inhibitor of phage proliferation than whey peptides. OnuorahÕs (1996) results show a significant reduction in cell growth when whey peptides were added to the growth medium. The c2 phage peptide does not appear to depress cell growth as much as the whey peptides, suggesting that the whey peptides may bind to the receptors that are important to metabolite transport, whereas the c2 phage peptide may be binding to a different receptor on the cell membrane. Also, Onuorah (1996) reported using 0.5 g of whey peptide/ml for optimum growth time to the growth apex. Phage peptides in these experiments were added at 0.02 g peptide/ml
Table 1 Differences in slope of lag phase, slope of log phase, time to apex, and lysis slope of C2 culture grown in M17 medium with and without (w/wo) phage peptides and with and with out c2 phage
Culture grown in Ml7 medium Culture grown in M17 medium with phage peptides Absolute differences a
b;c
Slope of lag phase (dx=dy) w/wo phage
Slope of log phase (dx=dy) w/wo phage
Time to growth apex (min) with phage
Slope of lysis (dx=dy) with phage
0.0012a
0.0047a
182b
)0.0091b
0.0011a
0.00433a
236c
)0.014c
0.0001
0.0004
54
)0.0049
Nonsignificant. Differences of significance within columns at p < :01, n ¼ 3.
C.L. Hicks et al. / Food Research International 37 (2004) 115–122
which resulted in a longer growth time before cell lysis occurred, suggesting that phage peptides are more effective for inhibiting phage proliferation than whey peptides. 3.6. Testing C2 culture in milk coated with and without c2 phage peptides Bulk starters of C2 culture grown in L-M17 medium with and without phage peptides were used to inoculate milk (with and without c2 phage peptide) that had been infected with and without c2 phage. The pH decreased faster (end pHs were lower, p < :05) in milk inoculated with C2 culture grown in medium without added c2 phage peptide than in milk inoculated with culture grown in medium containing c2 peptide (Fig. 3) suggesting that the c2 peptides were slightly inhibiting culture growth in milk similar to that observed for cultures in M17 medium. These two milks had better (p < :0001) acid production (pH 5.63 and 5.71, respectively after 3.7 h of fermentation) than the other four milks. However, milks that were inoculated with culture grown in c2 peptide (both the 2% peptide medium and 1% peptide medium with1% peptide added to the milk) and infected with c2 phage did continue to produce acid (to pH 6.01) throughout the fermentation period. The cultures in these two milks produced more acid (p < :05) than the cultures that were in infected milk with and without added peptide in the milk. No differences were observed
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in acid production in milks with or without added peptides. When 2% c2 peptide was added to the milk and inoculated with culture grown in medium without peptide, acid development stalled (pH 6.28) after 100 min (curd was coagulated) and the pH decline at a much slower rate. These data suggest that lysis of the culture was nearly complete by the time the chymosin formed a soft coagulum. Acid production in milk inoculated with culture grown in medium without peptide and infected with c2 phage stopped (pH 6.25) after 100 min of fermentation suggesting that lysis was complete. Note that acid production started again (Fig. 3) after 180 min as C2 mutants gained in numbers. These results suggest the culture grown in media containing c2 peptide was partially protected from c2 phage proliferation and lysis during the fermentation period. Culture grown in peptide containing media was protected more than when the same amount of peptide was added to the milk, or when no peptide was present. Acid production generally continues if lysis of the culture has not occurred by the time the coagulant forms a curd and prevents the movement of newly released phage. This phenomenon was observed with cultures that were grown in all media that contained c2 phage peptides. Thus cultures grown in media containing phage peptides may be more resistant to phage infections and this technology may provide an additional barrier to phage infection under manufacturing conditions.
4. Conclusions
Fig. 3. Effect of c2 phage peptide on the fermentation activity (pH) of C2 lactic culture with and with out c2 phage present. All standardized, pasteurized milk was inoculated with 4% culture. Cultured milks were ripened (31 °C for 1 h), rennetted (ca. 25 min cutting time), and cook to 37 °C and held at the temperature. M, C2 lactic culture grown in LM17 medium without c2 phage peptides and inoculated into milk; , C2 lactic culture grown in L-M17 medium containing 2% c2 phage peptide and inoculated into milk; N, C2 lactic culture grown in L-M17 medium containing 2% c2 phage peptide and inoculated into milk infected with 103 pfu/ml c2 phage; }, C2 lactic culture grown in LM17 medium containing 1% c2 phage peptide and inoculated into milk containing 1% c2 phage peptide and infected with 103 pfti/ml c2 phage; r, C2 lactic culture grown in L-M17 medium and inoculated into milk containing 2% c2 phage peptide and infected with 103 pfu.ml c2 phage; , C2 culture grown in L-M17 medium and inoculated into milk infected with 103 pfu/ml.
Phage peptides were successfully hydrolyzed from c2 bacteriophage using ficin or bromelain. Ficin and bromelain hydrolysates destroyed the ability of c2 bacteriophage to infect a C2 culture. Ficin was chosen as the most likely commercial enzyme because of its lower cost. Residual ficin was not found in the c2 phage peptide permeate (3000 mwco). Peptides derived from hydrolyzed L-M17 medium (ficin digest) were found to be inhibitory to C2 culture growth and were not inhibitory to c2 phage proliferation. However, ficin hydrolyzed c2 phage peptides were effective in prolonging the growth time of C2 lactic culture in L-M17 medium and milk when infected with c2 bacteriophage. C2 culture was protected from c2 phage through the ripening and renneting periods when C2 bulk starter was prepared in a medium containing the c2 phage peptides.
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