天然防腐剂

ELSEVIER International Biodeterioration & Biodegradation (1995) 333-345 Copyright 0 1996 Elsevier Science Limited Printed in Great Britain. All rights reserved 0964-8305/95 $9.50 + 0.00 0964-8305(95)00074-7 The Quest for Natural Antimicrobials as Novel Means of Food Preservation: Status Report on a European Research Project S. Roller South Bank University, 103 Borough Road, London SE1 OAA, UK ABSTRACT At a time when consumers are demanding the partial or complete removal of chemically synthesized preservatives from foods, there is also an increased demand for convenience foods with long shelf-lives. These consu- mer-led trends have fuelled a renewed interest in the development of ‘more natural’ preservatives for extending the shelf-l&e and maintaining the safety of foods. Although the antimicrobial properties of many compounds from plant, animal and microbial sources have been reported, their potential for use as natural food preservatives has not been fully exploited. In this paper, the possible uses of natural antimicrobial compounds as food preservatives, used either singly or in combination, are explored. Specific examples are given from a current transnational research project on Natural Anti- microbial Systems sponsored jointly by the European Commission and a consortium of eight food companies. The results of trials with a range of potential natural preservatives including lyric enzymes, bacteriocins from lactic acid bacteria and plant antimicrobials in laboratory media and in a variety of foods and beverages including apple juice, milk, hard-cooked cheese (Emmental) andfresh fruit slices are discussed. Copyright 02 1996 Elsevier Science Ltd INTRODUCTION Consumer concern over the possible adverse health effects of certain food preservatives coupled with increasing demand for convenience foods with 333 334 S. Roller long shelf-lives has resulted in increasing pressure on manufacturers to remove chemically-synthesized additives from processed foods and to provide more ‘natural’ alternatives for the maintenance of food safety and shelf-life. These consumer-led trends have fuelled a renewed search for ‘more natural’ preservatives derived from plant, animal and microbial sources. In the last few decades, numerous reports have appeared in the scientific literature about the antimicrobial properties of various compounds from herbs and spices, fruits and vegetables, leaves and bark, animal tissues and microorganisms. For a comprehensive review of the subject, the reader is referred to a recent book by Dillon and Board (1994). Similarly, several chapters in the book by Gould (1995) are also relevant (Board, 1995; Hill, 1995; Nychas, 1995; Smulders, 1995). However, many natural compounds have remained scientific curiosities and very few have been exploited by application in foods. In 1991, a major transnational research project on Natural Anti- microbial Systems was initiated with the objective of identifying and assessing natural antimicrobial systems as novel means of extending the safety and quality of food. The content and activity of natural anti- microbial agents in plant, animal and microbial starting materials was examined and assessed for ease of extraction, purification and anti- microbial effectiveness. Inhibitory activity of the new antimicrobials was tested individually, as well as in combination with traditional preserva- tion techniques, against food spoilage and poisoning organisms in laboratory media and in foods. This work is still in progress and is being sponsored jointly by the European Commission’s Food Linked Agro- Industrial Research (FLAIR) Programme and a consortium of eight food companies including Aplin & Barrett Ltd (UK), BSN (France), CPC (UK) Ltd, Gist-brocades (Netherlands), Meat and Livestock Commission (UK), Nestle (Switzerland), Pepsi-Cola International (UK) and Unilever (UK). In the following paper, some of the findings from the above project are described. Since it is not the intention of this author to present a detailed research treatise of all the results obtained during the course of this project, the reader is advised to contact the individual partners directly for further details on specific aspects of the work. Table 1 shows the names, contact addresses and numbers, subject areas and publications for all the partners in this project. It should be noted that some aspects of the work have not been completed or may not be currently available for dissemi- nation because the partners may be seeking industrial exploitation. This paper is divided into three sections covering lytic enzymes, bacteriocins and plant antimicrobials. The quest for natural antimicrobials TABLE 1 335 Research Partners and their Subject Areas in the European Project on Natural Anti- microbial Systems Name of partner/s Institution Subject of research Publications to date Professor S. Roller (Coordinator) Professor R Board and Dr J. Lock Dr Cl. Nychas and Dr S. Tassou Dr G. Fitzgerald, Dr H. Coveney, E. Vaughan, E. Caplice and Professor C. Daly Professor C. Bourgeois and Ing D. Thuault Ing J.-R. Kerjean and Dr F. Girard South Bank Lytic enzymes, University, 103 chitosan, barrier- Borough Road, forming London SE1 OAA, compounds, UK (in bacteriocins collaboration with the Leatherhead Food Research Association, Surrey, UK) University of Bath, Claverton Down, Bath BA2 IAY, UK Institute of Technology of Agricultural Products, S Venizelou 1, Athens, Greece University College Cork, Western Road, Cork, Ireland ADRIA, 6 Rue de l’Universite, 29 19 1 Quimper, France Institut Technique Salmonellae in egg- based products, acidulants and oils, cinnamic acid, combinations of antimicrobials, microbial ecology of foods Olive phenolics; mint; other plant phenolics and essential oils Bacteriocins from lactic acid bacteria Bacteriocins from lactic acid bacteria for the prevention of cheese spoilage by Clostridum tyrobutyricum: screening and biochemistry Bacteriocins from du Gruyere, 73 Rue lactic acid bacteria de Saint Brieuc, for the prevention 35062 Rennes, of cheese spoilage France by Clostridum tyrobutyricum: cheese trials Samelis et al., 1994; others not released due to commercial sensitivity Lock and Board (1994. 1995a, b) Tranter et al. (1993) Tassou and Nychas (1994) Tassou et a/. (1995a,b) Tassou and Nychas (1995a,b) Vaughan et al. (1994) others not released due to commercial sensitivity None due to commercial sensitivity None due to commercial sensitivity 336 S. Roller LYTIC ENZYMES AGAINST YEAST SPOILAGE Many lytic enzymes now used in the food industry to degrade unwanted polysaccharides have potential for use as novel and ‘natural’ food preser- vatives. One such enzyme, lysozyme from hen egg whites, has been known for many years and is used against clostridial spoilage in hard-cooked cheeses in France (Fox & Grufferty, 1991). However, lytic enzymes against yeasts have not been used to date. The objective of this part of the work, carried out by the author of this paper, has been to identify readily available lytic enzymes and evaluate their potential for use as novel anti- microbial agents against spoilage yeasts in laboratory media and, when appropriate, in foods and beverages. The lytic enzymes were selected on the basis of their reported substrate specificities. For example, mannanases were chosen to attack the mannan component of yeasts and glucanases were studied for their ability to degrade the glucan component of yeasts. Although the aim of this study was to identify food-grade enzymes with antimicrobial activity, non-food grade preparations were not deliberately excluded and were also tested. Thirteen commercially-available lytic enzymes were tested against eight yeasts at a range of pH values and temperatures. All yeasts used in this study, except Kluyveromyces lactis, had been isolated from spoiled beverages and donated to the project by a co-sponsoring company (Pepsi- Cola International). The yeasts were grown in Malt Extract Broth, Agar and/or UHT-treated, clear apple juice (pH 3.5). The yeasts were: Kluyver- omyces lactis, Saccharomyces cerevisiae 28 (from juice-based beverage, preservative-resistant), S. cerevisiae 3085 (from juice), S. cerevisiae SD (from Pepsi premix), S. exiguis 391 (from tea, low preservative resistance), Schizosaccharomyces pombe (from apple juice), Zygosaccharomyces bailii 906 (from carbonated beverage, gas producer, benzoate-resistant, low pH- resistant) and Z. bailii H.P. (from Hawaiian Punch containing 40s 500 ppm benzoate, very preservative resistant). All enzyme stock solutions were prepared in phosphate buffer adjusted to the optimum pH for each enzyme. The well assay was used for testing the inhibitory activity of enzymes against yeasts. A 10% agar solution (malt extract agar) was seeded with 1% of an overnight culture of yeast and the mixture was poured into Petri dishes. Once solid, wells of 1 cm in diameter were bored in each plate and approximately 170 ,ul of the enzyme solutions were added to each well. The plates were incubated at 4°C for 2 h to allow diffusion of the solution into the agar, then incubated at the appropriate temperature for the experiment. Zones of inhibition (in mm) showing a complete absence of growth were measured periodically until the plates had overgrown. Zones of growth reduction were also recorded. The quest for natural antimicrobials 331 In addition, yeast growth in the presence of selected enzymes in apple juice was assessed by monitoring absorbance at 540nm using microtitre plate assays at room temperature. Of all the 13 enzymes tested, Lyticase, SP299 and Novozym 234 showed the greatest antimicrobial activity. Lyticase was a b-glucanase from Arthrobacter luteus supplied by Sigma Chemical Co. The enzyme cocktails Novozym 234 and SP299 were supplied by Novo Nordisk. Novozym 234 was a multicomponent lytic enzyme preparation from Trichoderma harzianum reportedly suitable for the production of protoplasts from yeasts and fungi. The main enzyme activities reported for Novozym 234 were 1,3-a-glucanase, 1,3+glucanase, laminarinase, xylanase, chitinase and protease. Similarly, SP299 was also an enzyme cocktail from T. harzianum that was reportedly suitable for use in yeast hydrolysis, special cleaning of yeast soils and processing of plant tissues. This was an experi- mental preparation intended for laboratory trials only. The enzyme complex consisted of 1,3-a-glucanase with side activities from cellulase, laminarinase, xylanase, chitinase and proteinase. None of the three enzyme preparations were food-grade. Four of the eight yeasts tested in this study were resistant to the anti- microbial action of lyticase up to a concentration of 2000U/ml at 25°C pH 7.5 (the reported temperature and pH optima for the enzyme). For a definition of a unit of activity, please see Table 2. However, four of the target yeasts were sensitive, as shown in Table 2. Of these, clear zones of inhibition were shown around one of the strains only (S. cerevisiue 28), whilst the other three strains showed zones of reduced growth around the well in the agar plate. Generally, a dose response was observed. Zones of inhibition/growth reduction were reduced with time possibly due to poor diffusion of the enzyme through the agar, instability of the enzyme with time and/or degradation of Lyticase by proteolytic enzymes released from the target cells. The inhibitory effect was very strain-specific. Studies with Lyticase were expanded to include a wider range of pH values (7.5, 6.8 and 5.8) and temperatures (18 and 25°C) as shown in Table 3. Since the reported pH optimum for Lyticase was 7.5, it was not unexpected that antimicrobial activity declined as the pH of the medium was reduced. By contrast, although the temperature optimum for Lyticase was 25°C inhibition was greater at the lower temperatures tested. It has recently been reported that antimicrobial activity of lysozyme against Listeriu monocytogenes increased (mainly by extending the lag phase) at reduced temperatures (reduced from 25 to 5°C) and pH (reduced from 7.2 to 5.5) suggesting that conditions of optimal pH and temperature may not be necessary for antimicrobial activity of enzymes (pH 6.2 and 25°C for lysozyme) (Johansen et al., 1994). 338 S. Roller TABLE 2 Inhibition of Growth of Four Spoilage Yeasts in Malt Extract Agar at 25°C pH7.5, by Lyticase (a fl-Glucanase). Inhibition Zones were Measured in mm. Clear Inhibition Zones are Indicated by an Asterisk; all other Zones Indicate Reduced Growth Organism Time (days) Enzyme dose Enzyme dose Enzyme dose (100 U”jml) (1000 U “/ml) (2000 U “/ml) S. cerevisiae 28 2 2.0 6.0 * 6.7 * 4 0 3.7 3.7 * 5 0 4.0 4.0 * 6 0 3.7 4.0 * S. cerevisiae 3085 1 0 3.3 4.7 2 0 5.0 4.7 Z. bailii 906 4 0 1.0 4.7 5 0 1.3 3.7 6 0 0 2.7 Z. bailii H.P. 2 0 8.3 8.0 4 0 3.7 4.7 5 0 4.0 4.7 6 0 4.0 4.0 ‘One unit of Lyticase was defined by the supplier, Sigma Chemical Co., as producing a change in absorbance at 800 nm of 0.001 per min at pH 7.5 and 25°C in a 3-ml suspension of brewer’s yeast. TABLE 3 Inhibition of Growth of Four Spoilage Yeasts in Malt Extract Agar at Two Temperatures and Three pH Values by 2000 U a/ml of Lyticase (a /I-Glucanase) after 48 h of Incubation. Inhibition Zones were Measured in mm. Clear Inhibition Zones are Indicated by an Asterisk; all other Zones Indicate Reduced Growth Organism pH5.8 (25°C) pH5.8 (18”CO pH6.8 (25°C) pH6.8 (18OC) pH 7.5 (2YC) S. cerevisiae 28 2 2* 3 3* 7* S. cerevisiae 3085 2 2 3 5 5 Z. bailii 906 2 3 2 5 5 Z. bailii H.P. 3 2 2 2 8 “Units of activity for Lyticase are defined in Table 2. It has been reported that a combination of /I-glucanase and chitinase was necessary to produce protoplasts from fungi (Yabuki et al., 1984). Therefore, the enzyme cocktail SP299, containing both of these activities, was tested for antimicrobial action against the target yeasts. None of the organisms were inhibited by 0.4 and 0.8 U/ml of the enzyme preparation The quest for natural antimicrobials 339 TABLE 4 The Effect of SP299 (33.6Ua/ml) on Yeasts in Malt Extract Agar at Two Temperatures and Two pH Values. Inhibition Zones were Measured in mm. Clear Inhibition Zones are Indicated by an Asterisk; all other Zones Indicate Reduced Growth Organism pH5.5 (25°C) pH5.8 pH5.8 pH6.8 pH6.8 (25°C) (18°C) (25°C) (18°C) K. lactis N.D. 7* S. cerevisiae 28 0 6 S. cerevisiae 3085 0 3 S. cerevisiae SD 0 0 S. exiguis 0 2 Sch. pombe 8 3* Z. bailii 906 0 2 Z. bailii H.P. 0 3 10 7* 5* 5 2 0 0 0 2 2 2 1 3 0 3 4 IO 5* 3 0 2 1* 5 3 aOne unit of activity is defined by the supplier, Novo Nordisk, as the amount of enzyme which releases one microequivalent of reducing sugar per min from insoluble 1.3-cc-glucan at 40°C pH 5.5 at a concentration of glucan of 1.5%. (units of activity are defined in Table 4). At 8.4 and 33.6 U/ml and culture conditions of 25°C and pH 5.5, only Sch. pombe was sensitive, as shown in Table 4. However, when the temperature of incubation was reduced from 25 to 18”C, and/or the pH was raised from 5.5 to 5.8 or 6.8, inhibition zones against all the target yeasts, except S. exiguis, were recorded (Table 4). It was not possible to increase the concentration of the enzyme above 33.6 U/ml as the preparation was available in liquid form only. As with Lyticase, inhibition was strain-specific and zones of reduced growth surrounding the clear zones of no growth were observed. The latter effect may have been due to poor diffusion of the enzyme preparation through the agar, instability of the enzyme with time and/or degradation of the enzyme preparation by proteolytic enzymes released from the target cells. Similarly, as with Lyticase, antimicrobial activity was favoured at pH values approaching neutral, although the manufacturer’s reported pH optimum, based on the c+glucanase component of the cocktail, was 4.5. This suggests that the antimicrobial properties of SP299 may have been due to an enzyme other than glucanase. Unlike Lyticase, a reduction in temperature from 25 to 18°C generally had little effect on the anti- microbial activity of SP299. K. lactis and Sch. pombe were most sensitive to SP299 whilst S. cerevisiae SD was resistant at all concentrations, pH values and temperatures investigated. Novozym 234, marketed as an enzyme cocktail for the protoplasting of fungi, was shown to inhibit two of the eight target yeasts used in this study at pH 5.5. S. exiguis was not inhibited by lOU/ml of this enzyme 340 S. Roller preparation, but at 100 U/ml an inhibition zone of 4.8 mm was observed. Sch. pombe was more sensitive, producing an inhibition zone of 3.5 mm in the presence of 10 U/ml and 4.8 mm in the presence of 100 U/ml. Neither of these two organisms had been identified as particularly sensitive to conventional chemical preservatives (benzoate) used in beverages. The remaining six yeast strains, three of which had been identified as benzoate- resistant, were not inhibited by Novozym 234. The effects of the lytic enzymes on growth of the eight target yeasts in apple juice at pH 3.5 were investigated by monitoring absorbance at 540nm at room temperature (results not shown). In general, the enzymes failed to inhibit growth of the yeasts in apple juice. As most of the enzymes had pH optima ranging from 4.5 to 7.5, it is probable that the low pH of the juice resulted in poor activity. However, a growth delay of up to 15 h was observed with some enzyme/yeast combinations and the effect appeared to be dose-related (results not shown). The extension of the lag phase in the presence of the enzyme at low pH is in agreement with a recent report on the antimicrobial effects of lysozyme at low pH on L. monocytogenes (Johansen et al., 1994). Although a growth lag of 15h would offer no commercial advantage, the results suggested that higher levels of addition of some of the enzymes may have improved their anti- microbial efficacy. It was concluded that some lytic enzymes currently available on the market, such as Lyticase, SP299 and Novozym 234, have potential for development as natural food preservatives against spoilage yeasts. Anti- microbial action by lytic enzymes was favoured at pH values approaching neutral with very little or no action occurring at pH values below 5. There- fore, applications would be limited to pH-neutral foods, e.g. processed cheese. Inhibition by lytic enzymes tended to be strain-specific, thereby limiting their usefulness. A lower incubation temperature (18 vs 25°C) sometimes enhanced the inhibitory action of the enzymes. Further work at temperatures below 18°C and in pH-neutral foods would need to be carried out to confirm the potential of lytic enzymes as natural preservatives for foods. Any further development of the use of the specific enzyme prepara- tions tested in this study would also involve regulatory clearances. ANTIMICROBIAL PEPTIDES FROM LACTIC ACID BACTERIA Bacteriocins for chilled foods Many lactic acid bacteria produce peptides known as bacteriocins with bactericidal action against a limited range of bacteria usually closely rela- The quest for natural antimicrobials 341 ted to the producer organism. The best known of these bacteriocins is the lanthionine-containing nisin from Lactococcus luctis. Nisin has been known for over 50 years and is used effectively in processed cheese, cheese spreads, dairy desserts and canned foods worldwide (Delves-Broughton, 1990). In the last 20 years, rising interest in natural antimicrobials has result