Accepted Article
Article Type: Original Article
Antimicrobial activity of hop extracts against foodborne pathogens for meat applications
B. Kramer, J. Thielmann, P. Muranyi, J. Wunderlich, C. Hauser Fraunhofer Institute for Process Engineering and Packaging IVV, Freising, Germany
Correspondence to: Bernd Kramer, Department of Food Quality, Fraunhofer Institute for Process Engineering and Packaging (IVV), Giggenhauser Str. 35, D-85354 Freising, Germany. Email: [email protected], Phone: +49(0)8161-491-471, Fax: +49(0)8161-491666
Running Title: Antimicrobial hop extracts
ABSTRACT Aims: The objective of this study was the fundamental investigation of the antimicrobial efficiency of various hop extracts against selected foodborne pathogens in vitro, as well as their activity against L. monocytogenes in a model meat marinade and on marinated pork tenderloins. Methods and results: In a first step, the minimum inhibitory concentrations (MIC) of three hop extracts containing either α- or β-acids or xanthohumol were determined against test bacteria including Listeria monocytogenes, Staphylococcus aureus, Salmonella enterica and Escherichia coli by a colorimetric method based on the measurement of bacterial metabolic activity. Moreover, the
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/jam.12717 This article is protected by copyright. All rights reserved.
Accepted Article
influence of either lactic or citric acid on the antimicrobial activity of the hop extracts was evaluated. The efficiency of hop extracts as a natural food preservative was then tested in a model meat marinade at 2 °C and 8 °C respectively and finally on marinated pork. The experiments showed that Grampositive bacteria were strongly inhibited by hop extracts containing β-acids and xanthohumol (MIC values of 6.3 ppm and 12.5 ppm, respectively) whereas the antimicrobial activity of the investigated αacid extract was significantly lower (MIC values of 200 ppm). Gram-negative bacteria were highly resistant against all tested hop extracts. Acidification of the test media led to a decrease of the MIC values. The inhibitory activity of the hop extracts against L. monocytogenes was strongly reduced in a fat-containing model meat marinade, but the efficiency of β-acids in this matrix could be increased by lowering pH and storage temperatures. By applying 0.5 % β-acids at pH=5 in a model marinade, the total aerobic count of pork tenderloins was reduced up to 0,9 log10 compared to marinated pork without hop extract after two weeks of storage at 5 °C. Conclusions: β-acid containing hop extracts have proven to possess a high antimicrobial activity against Gram-positive bacteria in vitro and in a practice related application for food preservation. Significance and impact of the study: Antimicrobial hop extracts could be used as natural preservatives in food applications in order to extend the shelf-life and to increase the safety of fresh products.
Keywords: hop extracts, β-acids, pathogens, natural preservative, food safety, meat
INTRODUCTION Current consumer trends regarding the demand for fresh and minimally processed food products without additional chemical preservatives have promoted the research for new food preservation strategies. Naturally occurring compounds that show antimicrobial activity against spoilage and pathogenic microorganisms could potentially be applied for food preservation in order to extend the shelf-life and to improve the safety of foods and beverages at the same time. Many compounds from plant, animal or microbial sources like essential oils from thyme, basil, and oregano as well as specific proteins like lysozyme, lactoferrin or bacteriocins have been screened for their antimicrobial
This article is protected by copyright. All rights reserved.
Accepted Article
properties so far (Tiwari et al. 2009; Lucera et al. 2012). However, their application in food preservation remains scarce up to now, mostly due to a loss of efficacy in complex food matrices or alteration of organoleptic properties of foods when using effective concentrations (Burt 2004; Rasooli 2007; Tiwari et al. 2009). Fresh meat and meat products are susceptible to microbial deterioration, which is accompanied by changes in texture, color and taste. Contaminations of such products with pathogens like Listeria monocytogenes, Salmonella spp. or Escherichia coli can lead to foodborne diseases. For example, in 2012 a total number of 1533 foodborne outbreaks with 91034 confirmed salmonellosis cases in humans (61 reported deaths occurred in the European Union caused by Salmonella spp.), whereas over 20% of the strong-evidence outbreaks can be attributed to meat and meat products. In case of Listeria monocytogenes, the fatality rate (198 deaths) was very high in relation to the amount of confirmed listeriosis cases (1642 cases) (EFSA, 2014). A promising approach to reduce microbial hazards of raw meat products and to prolong their microbial stability may lie in the incorporation of natural antimicrobial substances in marinades. Among others, natural compounds like essential oils, chitosan, nisin and lysozyme have been investigated to replace chemical preservatives and to obtain “green label” products (Zhou, Xu and Liu, 2010).
The hop plant Humulus Lupulus Linneus belongs to the family of the Cannabinaceae and has been used for beer brewing since ancient times. Besides giving beer its typical bitter taste, hop compounds possess distinctive antimicrobial and antioxidative properties. The antimicrobial activity of hop bitter acids namely, the α-acids (humulones) and β-acids (lupulones) has been demonstrated mainly against Gram-positive bacteria, whereas Gram-negative bacteria are either resistant or only affected by high concentrations of the hop resins (Teuber and Schmalreck, 1973, Haas and Barsoumian 1994). Prenylated flavonoids like xanthohumol also exhibit antimicrobial activity against Gram-positive bacteria but not against Gram-negative strains (Mizobuchi and Sato, 1985; Bhattacharya et al., 2003). Some fungi are also inhibited by hop acids and prenylchalcones (Mizobuchi and Sato 1984, Mizobuchi and Sato 1985) as well as protozoa (Srinivasan et al. 2004). The applicability of hop compounds as possible food preservatives has not been studied extensively so far. Larson et al. (1996)
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Accepted Article
Effect of hop extracts in a model meat marinade against L. monocytogenes In this study, the applicability of both most efficient hop extracts (Beta Bio 40, Xantho-Flav) for the preservation of raw meat was investigated in first instance by testing the inhibitory effect against L. monocytogenes in a model meat marinade. Figure 1 shows the survival and growth kinetics of L. monocytogenes in a marinade stored at 2 °C. The colony count of the reference samples increased from 3.5 to 7.0 log10 cfu ml-1 within 35 days of storage at pH 7. Both, β-acid extract and xanthohumol, did not have any bacteriostatic or bactericidal effects at 500 ppm and pH 7. When 1000 ppm was applied, the β-acid extract led to a reduction of the initial count of 2.9 log10 cfu ml-1 within 35 days. xanthohumol had only limited inhibitory effect at a concentration of 1000 ppm and caused an extended lag phase for about five days. At pH 5 (Figure 2) and a storage temperature of 2 °C, no growth of L. monocytogenes could be observed within the reference samples. A concentration of 100 ppm of the β-acid extract did not have any significant antibacterial effect, the viable cell count decreased by 0.5 log10 cfu ml-1, similar to the references. An accelerated reduction of the colony count could be observed at concentrations of 250 ppm (2.1 log10 cfu ml-1) and 500 ppm (> 3 log10 cfu ml-1) of the β-acid extract within 30 days of storage. When the samples were stored at 8 °C, the colony count of the references increased from 2*103 cfu ml1
to 2*107 cfu ml-1 within 14 days at pH 7.0 (Figure 3). A concentration of 1000 ppm of the β-acid
extract did not inhibit the growth of L. monocytogenes but caused an extended lag-phase of about three days. Almost no inhibitory effect could be observed with 500 ppm. When the pH of the model marinade was adjusted to 5.0 with citric acid, the colony count of the references increased from 2*103 cfu ml-1 to 106 cfu ml-1 within 14 days (Figure 4). β-acid extract (100 ppm) was sufficient to inhibit the growth of L. monocytogenes within 15 days. Higher concentrations (250 and 500 ppm) led to a reduction of the colony count by 2.0 and 2.5 log10 cfu ml-1 compared to the initial load respectively.
Application of hop extracts in a model meat marinade on pork The incorporation of 5000 ppm of Beta Bio 40 proved to have a positive effect on the microbial quality with regard to the total aerobic mesophilic cell count and the number of L. monocytogenes
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Figure 5 Legends to figures and tables Figure 1 Influence of Beta Bio 40 (β-acid extract) and Xantho-Flav (xanthohumol) on the growth of L. monocytogenes in the model meat marinade at a storage temperature of 2°C and a pH of 7.0. (●) reference, (▲) 1000 ppm Beta Bio 40, () 1000 ppm Xantho-Flav, (x) 500 ppm Beta Bio 40, (+) 500 ppm Xantho-Flav.
Figure 2 Influence of Beta Bio 40 (β-acid extract) on the growth of L. monocytogenes in the model meat marinade at a storage temperature of 2° and a pH of 5.0 (citric acid). (●) reference, (▲) 100 ppm Beta Bio 40, () 250 ppm Beta Bio 40, (x) 500 ppm Beta Bio 40.
Figure 3 Influence of Beta Bio 40 (β-acid extract) on the growth of L. monocytogenes in the model meat marinade at a storage temperature of 8°C and a pH of 7.0. (●) reference, (▲) 500 ppm Beta Bio 40, () 1000 ppm Beta Bio 40.
Figure 4 Influence of Beta Bio 40 (β-acid extract) on the growth of L. monocytogenes in the model meat marinade at a storage temperature of 8°C and a pH of 5.0 (citric acid). (●) reference, (▲) 100 ppm Beta Bio 40, () 250 ppm Beta Bio 40, (x) 500 ppm Beta Bio 40.
Figure 5 Influence of Beta Bio 40 (β-acid extract) on the total viable count and L. monocytogenes on pork loin slices marinated in a model meat marinade at a storage temperature of 2°C and a pH of 5.0 (citric acid). ). (●) reference, total aerobic, mesophillic cell count, (▲) hop containing marinade (5000 ppm), total aerobic, mesophillic cell count, (○) L. monocytogenes, reference, (Δ) L. monocytogenes, hop containing marinade (5000 ppm).
Table 1 MICs of the tested hop extracts against selected bacteria at pH values of 7.2 or 5.0. Acidification of the test media was done with citric acid (Cit) or lactic acid (Lac).
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for each concentration. Negative controls (water instead of hop compound) as well as blanks (sterile media instead of cell suspension) were included in each assay. 10 µl of a 1:10 (Gram-negative bacteria) or 1:80 (Gram-positive bacteria) mixture of the tetrazolium salt WST-8 and the electron mediator 2-methyl-1 4-naphthoquinone was added after an incubation period of 18 h. The plates were incubated at 37 °C between 0.5 and 2 h depending on the particular test strain. WST-8 is cleaved to a water soluble formazan dye by active microbial dehydrogenases, indicating metabolic activity of microbial cells. This reaction leads to the formation of a yellow color at which the intensity is proportional to the viable cell number (Tsukatani et al. 2008). The absorption was measured at 450 nm in a microplate reader (Spectramax 190, Molecular Devices, Sunnyvale, USA). The lowest hop concentrations where the absorption was not significantly (P < 0.05) higher than in the blanks were considered as MICs.
Incorporation of hop extracts into a model meat marinade A basis oil-in-water emulsion for meat marinades was prepared from 20 % (w/w) of a rich cream (AVO-Werke, Belm, Germany), 80 % (w/w) deionized water and 0.5 % (w/w) of a compound (AVOWerke, Belm, Germany) consisting of Xanthan and whey protein. A stable emulsion was obtained by dispersing the lipid phase in the aqueous phase with an Ultra-Turrax Miccra D-8 (ART-Prozess und Labortechnik, Müllheim, Germany) for 5 min. After pasteurization at 85 °C for 15 min, the pH of the emulsion was at 7.0 ± 0.1. In order to study the impact of pH value on the antimicrobial activity of hop extracts in the emulsion, the pH was adjusted to 5.0 ± 0.1 with citric acid. The concentration of Beta Bio 40 was adjusted to 100, 250, 500 and 1000 ppm, the concentration of Xantho-Flav was adjusted to 500 and 1000 ppm. The hop extracts did not alter the initial pH of the emulsion. The inoculum of L. monocytogenes was prepared as described above and an initial cell density of approximately 103 cfu ml-1 was adjusted in the emulsion. Four replicates (each 10 mL) of each sample were stored under controlled temperature conditions of 2 °C and 8 °C in 15 ml tubes (Sarstedt, Nümbrecht, Germany) in order to simulate optimal (2 °C) and common (8 °C) storage conditions of fresh meat products. The samples were stored for up to 15 and 35 days respectively, depending on the temperature. Aliquots of 100 µl were taken every 2-4 days and the number of cfu per ml emulsion was determined by the pour
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plating method: appropriate dilutions of the emulsions were made with sterile ringer solution and 1 ml of each sample was mixed homogeneously with 12-15 mL of TSA (Oxoid, Hampshire, UK). The plates were incubated for 48 h at 37 °C. The data was analyzed using MS Excel 2010. Mean values and standard deviations of four replicates were determined.
Sensory analysis – Conventional profiling Fresh pork loins were cut into slices of 1 cm thickness, transferred into sterile PA/PE-bags (Interscience, Saint Nom, France) and marinated with 10 % (w/w) of the seasoned marinade “Piroschka” (AVO-Werke, Belm, Germany) containing either no or 5000 ppm Beta Bio 40. Bags were vacuum sealed. On the same day the slices were fried for 3 minutes on each side with 10 ml rapeseed oil in an aluminum pan. Sensory properties were assessed by a panel of 6 persons according to DIN 10967-1 (DIN, 1999). The intensity of the attribute “bitter” was evaluated in a 6 step interval scale (not detectable: 0; very strong: 5).
Application of hop extracts in a model meat marinade on pork Fresh pork loins were quartered lengthwise and cut into slices of approximately 15 g, with a thickness of 1 cm and a surface of about 10 cm². Collectively, 4 kg of pork slices were inoculated with 40 ml of a L. monocytogenes DSM 15675 cell suspension with a viable count of approximately 106 cfu ml-1. The cell suspension was homogenously dispersed before two slices were weighed individually into sterile PA/PE bags (Interscience, Saint Nom, France). According to the weight of the pork slices, 20 % (w/w) of the model marinade was added, containing either 500 or 5000 ppm at pH=5 ± 0.1 Beta Bio 40. The bags were vacuum-packed. For reference samples, pork loins were packaged the same way with pure marinade (without hop extract). All samples were stored at 5 °C up to 14 days. Aerobic, mesophilic cell count, Enterobacteria, Listeria and Lactobacilli were monitored as a function of time after 0, 1, 3, 6, 10, 14 days, using TSA (Merck, Darmstadt, Germany), Violet Red Bile Dextrose Agar (VRBD) (Merck, Darmstadt, Germany), Chromocult Listeria selective agar (CLSA) (Merck, Darmstadt, Germany) and de Man, Rogosa and Sharpe agar (MRS) (Merck, Darmstadt, Germany), respectively. Five individual samples were investigated at each point of measurement. Therefore, each
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sample was diluted 1:3 (w/w) with sterile ringer solution and the samples were homogenized in a Lab Blender for 1 min at 260 rpm (AES Chemunex, Bruchsal, Germany). The number of cfu g-1 was determined by the pour-plate method using appropriate decimal dilutions of the sample suspension in ringer solution. Incubation conditions were as follows: 24 h, 37 °C, aerobic for VRBD and CLSA; 48 h, 30 °C aerobic for TSA; 96 h, 30 °C, anaerobic for MRS. Data are presented as mean values ± standard deviations.
RESULTS Antimicrobial activity of hop extracts The determined MIC values of three hop extracts against four different test microorganisms are shown in Table 1. In comparison to the Gram-positive bacteria, the two Gram-negative bacteria S. enterica and E. coli were more resistant against all tested hop compounds. Xantho-Flav and Beta Bio 40 showed strong antibacterial effects against Staph. aureus and L. monocytogenes, with MIC values of 6.3 ppm (Xantho-Flav) and 12.5 ppm (Beta Bio 40) at a pH of 7.2 respectively. In contrast, the effect of Alpha Bio was lower. Both Gram-positive bacteria were inhibited at concentrations above 200 ppm at pH 7.2. Taking into account the particular amount of α-acids and β-acids prevalent in the extracts, β-acids (Beta Bio 40) showed an 8-fold and 16-fold stronger antimicrobial activity than α-acids (Alpha Bio) against L. monocytogenes and Staph. aureus respectively. With regard to E. coli, the MIC of the β-acid extract and the α-acid extract exceeded 5000 ppm at pH 7.2, which corresponds to the highest tested concentration. For the test strain S. enterica, the MIC value of both extracts matched with the highest tested concentration of 5000 ppm. The MIC value of Xantho-Flav against both Gram-negative bacteria exceeded the highest tested concentration of 200 ppm. Higher concentrations were not applied due to the intense yellow coloration and the low solubility of Xantho-Flav in the test media. Acidification of the test media with citric acid or lactic acid to a pH of 5.0 ± 0.1 led to a decrease of the MIC in almost all cases compared to the values determined at pH 7.2 ± 0.1, except for Staph. aureus when acidified with lactic acid (Table 1). The reduction of the MIC by lowering the pH value was in general more pronounced with the α-acid extract than with the β-acid extract or xanthohumol.
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Effect of hop extracts in a model meat marinade against L. monocytogenes In this study, the applicability of both most efficient hop extracts (Beta Bio 40, Xantho-Flav) for the preservation of raw meat was investigated in first instance by testing the inhibitory effect against L. monocytogenes in a model meat marinade. Figure 1 shows the survival and growth kinetics of L. monocytogenes in a marinade stored at 2 °C. The colony count of the reference samples increased from 3.5 to 7.0 log10 cfu ml-1 within 35 days of storage at pH 7. Both, β-acid extract and xanthohumol, did not have any bacteriostatic or bactericidal effects at 500 ppm and pH 7. When 1000 ppm was applied, the β-acid extract led to a reduction of the initial count of 2.9 log10 cfu ml-1 within 35 days. xanthohumol had only limited inhibitory effect at a concentration of 1000 ppm and caused an extended lag phase for about five days. At pH 5 (Figure 2) and a storage temperature of 2 °C, no growth of L. monocytogenes could be observed within the reference samples. A concentration of 100 ppm of the β-acid extract did not have any significant antibacterial effect, the viable cell count decreased by 0.5 log10 cfu ml-1, similar to the references. An accelerated reduction of the colony count could be observed at concentrations of 250 ppm (2.1 log10 cfu ml-1) and 500 ppm (> 3 log10 cfu ml-1) of the β-acid extract within 30 days of storage. When the samples were stored at 8 °C, the colony count of the references increased from 2*103 cfu ml1
to 2*107 cfu ml-1 within 14 days at pH 7.0 (Figure 3). A concentration of 1000 ppm of the β-acid
extract did not inhibit the growth of L. monocytogenes but caused an extended lag-phase of about three days. Almost no inhibitory effect could be observed with 500 ppm. When the pH of the model marinade was adjusted to 5.0 with citric acid, the colony count of the references increased from 2*103 cfu ml-1 to 106 cfu ml-1 within 14 days (Figure 4). β-acid extract (100 ppm) was sufficient to inhibit the growth of L. monocytogenes within 15 days. Higher concentrations (250 and 500 ppm) led to a reduction of the colony count by 2.0 and 2.5 log10 cfu ml-1 compared to the initial load respectively.
Application of hop extracts in a model meat marinade on pork The incorporation of 5000 ppm of Beta Bio 40 proved to have a positive effect on the microbial quality with regard to the total aerobic mesophilic cell count and the number of L. monocytogenes
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during storage at 5 °C (Figure 5). This 10-fold higher concentration was chosen because no antibacterial effect could be observed at a concentration of 500 ppm (data not shown). Furthermore, previous trials have shown that applications in food matrices need much higher concentrations than the determined MIC in vitro. The sensory analysis using conventional profiling (DIN, 1999) on pork with the seasoned marinade “Piroschka” containing 5000 ppm Beta Bio 40 evidenced that the attribute “bitter” was not observable (5000
>5000
>200
pH 5.0 ± 0,1 (Cit)
1250
2500
>200
pH 5.0 ± 0,1 (Lac)
625
1250
>200
pH 7.2 ± 0.1
5000
5000
>200
pH 5.0 ± 0.1 (Cit)
1250
2500
>200
pH 5.0 ± 0.1 (Lac)
625
1250
>200
DSM 11822
L. monocytogenes DSM 15675
E. coli DSM 498
S. enterica DSM 11320
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Figure 5 Legends to figures and tables Figure 1 Influence of Beta Bio 40 (β-acid extract) and Xantho-Flav (xanthohumol) on the growth of L. monocytogenes in the model meat marinade at a storage temperature of 2°C and a pH of 7.0. (●) reference, (▲) 1000 ppm Beta Bio 40, () 1000 ppm Xantho-Flav, (x) 500 ppm Beta Bio 40, (+) 500 ppm Xantho-Flav.
Figure 2 Influence of Beta Bio 40 (β-acid extract) on the growth of L. monocytogenes in the model meat marinade at a storage temperature of 2° and a pH of 5.0 (citric acid). (●) reference, (▲) 100 ppm Beta Bio 40, () 250 ppm Beta Bio 40, (x) 500 ppm Beta Bio 40.
Figure 3 Influence of Beta Bio 40 (β-acid extract) on the growth of L. monocytogenes in the model meat marinade at a storage temperature of 8°C and a pH of 7.0. (●) reference, (▲) 500 ppm Beta Bio 40, () 1000 ppm Beta Bio 40.
Figure 4 Influence of Beta Bio 40 (β-acid extract) on the growth of L. monocytogenes in the model meat marinade at a storage temperature of 8°C and a pH of 5.0 (citric acid). (●) reference, (▲) 100 ppm Beta Bio 40, () 250 ppm Beta Bio 40, (x) 500 ppm Beta Bio 40.
Figure 5 Influence of Beta Bio 40 (β-acid extract) on the total viable count and L. monocytogenes on pork loin slices marinated in a model meat marinade at a storage temperature of 2°C and a pH of 5.0 (citric acid). ). (●) reference, total aerobic, mesophillic cell count, (▲) hop containing marinade (5000 ppm), total aerobic, mesophillic cell count, (○) L. monocytogenes, reference, (Δ) L. monocytogenes, hop containing marinade (5000 ppm).
Table 1 MICs of the tested hop extracts against selected bacteria at pH values of 7.2 or 5.0. Acidification of the test media was done with citric acid (Cit) or lactic acid (Lac).
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Article Type: Original Article
Antimicrobial activity of hop extracts against foodborne pathogens for meat applications
B. Kramer, J. Thielmann, P. Muranyi, J. Wunderlich, C. Hauser Fraunhofer Institute for Process Engineering and Packaging IVV, Freising, Germany
Correspondence to: Bernd Kramer, Department of Food Quality, Fraunhofer Institute for Process Engineering and Packaging (IVV), Giggenhauser Str. 35, D-85354 Freising, Germany. Email: [email protected], Phone: +49(0)8161-491-471, Fax: +49(0)8161-491666
Running Title: Antimicrobial hop extracts
ABSTRACT Aims: The objective of this study was the fundamental investigation of the antimicrobial efficiency of various hop extracts against selected foodborne pathogens in vitro, as well as their activity against L. monocytogenes in a model meat marinade and on marinated pork tenderloins. Methods and results: In a first step, the minimum inhibitory concentrations (MIC) of three hop extracts containing either α- or β-acids or xanthohumol were determined against test bacteria including Listeria monocytogenes, Staphylococcus aureus, Salmonella enterica and Escherichia coli by a colorimetric method based on the measurement of bacterial metabolic activity. Moreover, the
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/jam.12717 This article is protected by copyright. All rights reserved.
Accepted Article
influence of either lactic or citric acid on the antimicrobial activity of the hop extracts was evaluated. The efficiency of hop extracts as a natural food preservative was then tested in a model meat marinade at 2 °C and 8 °C respectively and finally on marinated pork. The experiments showed that Grampositive bacteria were strongly inhibited by hop extracts containing β-acids and xanthohumol (MIC values of 6.3 ppm and 12.5 ppm, respectively) whereas the antimicrobial activity of the investigated αacid extract was significantly lower (MIC values of 200 ppm). Gram-negative bacteria were highly resistant against all tested hop extracts. Acidification of the test media led to a decrease of the MIC values. The inhibitory activity of the hop extracts against L. monocytogenes was strongly reduced in a fat-containing model meat marinade, but the efficiency of β-acids in this matrix could be increased by lowering pH and storage temperatures. By applying 0.5 % β-acids at pH=5 in a model marinade, the total aerobic count of pork tenderloins was reduced up to 0,9 log10 compared to marinated pork without hop extract after two weeks of storage at 5 °C. Conclusions: β-acid containing hop extracts have proven to possess a high antimicrobial activity against Gram-positive bacteria in vitro and in a practice related application for food preservation. Significance and impact of the study: Antimicrobial hop extracts could be used as natural preservatives in food applications in order to extend the shelf-life and to increase the safety of fresh products.
Keywords: hop extracts, β-acids, pathogens, natural preservative, food safety, meat
INTRODUCTION Current consumer trends regarding the demand for fresh and minimally processed food products without additional chemical preservatives have promoted the research for new food preservation strategies. Naturally occurring compounds that show antimicrobial activity against spoilage and pathogenic microorganisms could potentially be applied for food preservation in order to extend the shelf-life and to improve the safety of foods and beverages at the same time. Many compounds from plant, animal or microbial sources like essential oils from thyme, basil, and oregano as well as specific proteins like lysozyme, lactoferrin or bacteriocins have been screened for their antimicrobial
This article is protected by copyright. All rights reserved.
Accepted Article
properties so far (Tiwari et al. 2009; Lucera et al. 2012). However, their application in food preservation remains scarce up to now, mostly due to a loss of efficacy in complex food matrices or alteration of organoleptic properties of foods when using effective concentrations (Burt 2004; Rasooli 2007; Tiwari et al. 2009). Fresh meat and meat products are susceptible to microbial deterioration, which is accompanied by changes in texture, color and taste. Contaminations of such products with pathogens like Listeria monocytogenes, Salmonella spp. or Escherichia coli can lead to foodborne diseases. For example, in 2012 a total number of 1533 foodborne outbreaks with 91034 confirmed salmonellosis cases in humans (61 reported deaths occurred in the European Union caused by Salmonella spp.), whereas over 20% of the strong-evidence outbreaks can be attributed to meat and meat products. In case of Listeria monocytogenes, the fatality rate (198 deaths) was very high in relation to the amount of confirmed listeriosis cases (1642 cases) (EFSA, 2014). A promising approach to reduce microbial hazards of raw meat products and to prolong their microbial stability may lie in the incorporation of natural antimicrobial substances in marinades. Among others, natural compounds like essential oils, chitosan, nisin and lysozyme have been investigated to replace chemical preservatives and to obtain “green label” products (Zhou, Xu and Liu, 2010).
The hop plant Humulus Lupulus Linneus belongs to the family of the Cannabinaceae and has been used for beer brewing since ancient times. Besides giving beer its typical bitter taste, hop compounds possess distinctive antimicrobial and antioxidative properties. The antimicrobial activity of hop bitter acids namely, the α-acids (humulones) and β-acids (lupulones) has been demonstrated mainly against Gram-positive bacteria, whereas Gram-negative bacteria are either resistant or only affected by high concentrations of the hop resins (Teuber and Schmalreck, 1973, Haas and Barsoumian 1994). Prenylated flavonoids like xanthohumol also exhibit antimicrobial activity against Gram-positive bacteria but not against Gram-negative strains (Mizobuchi and Sato, 1985; Bhattacharya et al., 2003). Some fungi are also inhibited by hop acids and prenylchalcones (Mizobuchi and Sato 1984, Mizobuchi and Sato 1985) as well as protozoa (Srinivasan et al. 2004). The applicability of hop compounds as possible food preservatives has not been studied extensively so far. Larson et al. (1996)
This article is protected by copyright. All rights reserved.
Accepted Article
Effect of hop extracts in a model meat marinade against L. monocytogenes In this study, the applicability of both most efficient hop extracts (Beta Bio 40, Xantho-Flav) for the preservation of raw meat was investigated in first instance by testing the inhibitory effect against L. monocytogenes in a model meat marinade. Figure 1 shows the survival and growth kinetics of L. monocytogenes in a marinade stored at 2 °C. The colony count of the reference samples increased from 3.5 to 7.0 log10 cfu ml-1 within 35 days of storage at pH 7. Both, β-acid extract and xanthohumol, did not have any bacteriostatic or bactericidal effects at 500 ppm and pH 7. When 1000 ppm was applied, the β-acid extract led to a reduction of the initial count of 2.9 log10 cfu ml-1 within 35 days. xanthohumol had only limited inhibitory effect at a concentration of 1000 ppm and caused an extended lag phase for about five days. At pH 5 (Figure 2) and a storage temperature of 2 °C, no growth of L. monocytogenes could be observed within the reference samples. A concentration of 100 ppm of the β-acid extract did not have any significant antibacterial effect, the viable cell count decreased by 0.5 log10 cfu ml-1, similar to the references. An accelerated reduction of the colony count could be observed at concentrations of 250 ppm (2.1 log10 cfu ml-1) and 500 ppm (> 3 log10 cfu ml-1) of the β-acid extract within 30 days of storage. When the samples were stored at 8 °C, the colony count of the references increased from 2*103 cfu ml1
to 2*107 cfu ml-1 within 14 days at pH 7.0 (Figure 3). A concentration of 1000 ppm of the β-acid
extract did not inhibit the growth of L. monocytogenes but caused an extended lag-phase of about three days. Almost no inhibitory effect could be observed with 500 ppm. When the pH of the model marinade was adjusted to 5.0 with citric acid, the colony count of the references increased from 2*103 cfu ml-1 to 106 cfu ml-1 within 14 days (Figure 4). β-acid extract (100 ppm) was sufficient to inhibit the growth of L. monocytogenes within 15 days. Higher concentrations (250 and 500 ppm) led to a reduction of the colony count by 2.0 and 2.5 log10 cfu ml-1 compared to the initial load respectively.
Application of hop extracts in a model meat marinade on pork The incorporation of 5000 ppm of Beta Bio 40 proved to have a positive effect on the microbial quality with regard to the total aerobic mesophilic cell count and the number of L. monocytogenes
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Figure 5 Legends to figures and tables Figure 1 Influence of Beta Bio 40 (β-acid extract) and Xantho-Flav (xanthohumol) on the growth of L. monocytogenes in the model meat marinade at a storage temperature of 2°C and a pH of 7.0. (●) reference, (▲) 1000 ppm Beta Bio 40, () 1000 ppm Xantho-Flav, (x) 500 ppm Beta Bio 40, (+) 500 ppm Xantho-Flav.
Figure 2 Influence of Beta Bio 40 (β-acid extract) on the growth of L. monocytogenes in the model meat marinade at a storage temperature of 2° and a pH of 5.0 (citric acid). (●) reference, (▲) 100 ppm Beta Bio 40, () 250 ppm Beta Bio 40, (x) 500 ppm Beta Bio 40.
Figure 3 Influence of Beta Bio 40 (β-acid extract) on the growth of L. monocytogenes in the model meat marinade at a storage temperature of 8°C and a pH of 7.0. (●) reference, (▲) 500 ppm Beta Bio 40, () 1000 ppm Beta Bio 40.
Figure 4 Influence of Beta Bio 40 (β-acid extract) on the growth of L. monocytogenes in the model meat marinade at a storage temperature of 8°C and a pH of 5.0 (citric acid). (●) reference, (▲) 100 ppm Beta Bio 40, () 250 ppm Beta Bio 40, (x) 500 ppm Beta Bio 40.
Figure 5 Influence of Beta Bio 40 (β-acid extract) on the total viable count and L. monocytogenes on pork loin slices marinated in a model meat marinade at a storage temperature of 2°C and a pH of 5.0 (citric acid). ). (●) reference, total aerobic, mesophillic cell count, (▲) hop containing marinade (5000 ppm), total aerobic, mesophillic cell count, (○) L. monocytogenes, reference, (Δ) L. monocytogenes, hop containing marinade (5000 ppm).
Table 1 MICs of the tested hop extracts against selected bacteria at pH values of 7.2 or 5.0. Acidification of the test media was done with citric acid (Cit) or lactic acid (Lac).
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for each concentration. Negative controls (water instead of hop compound) as well as blanks (sterile media instead of cell suspension) were included in each assay. 10 µl of a 1:10 (Gram-negative bacteria) or 1:80 (Gram-positive bacteria) mixture of the tetrazolium salt WST-8 and the electron mediator 2-methyl-1 4-naphthoquinone was added after an incubation period of 18 h. The plates were incubated at 37 °C between 0.5 and 2 h depending on the particular test strain. WST-8 is cleaved to a water soluble formazan dye by active microbial dehydrogenases, indicating metabolic activity of microbial cells. This reaction leads to the formation of a yellow color at which the intensity is proportional to the viable cell number (Tsukatani et al. 2008). The absorption was measured at 450 nm in a microplate reader (Spectramax 190, Molecular Devices, Sunnyvale, USA). The lowest hop concentrations where the absorption was not significantly (P < 0.05) higher than in the blanks were considered as MICs.
Incorporation of hop extracts into a model meat marinade A basis oil-in-water emulsion for meat marinades was prepared from 20 % (w/w) of a rich cream (AVO-Werke, Belm, Germany), 80 % (w/w) deionized water and 0.5 % (w/w) of a compound (AVOWerke, Belm, Germany) consisting of Xanthan and whey protein. A stable emulsion was obtained by dispersing the lipid phase in the aqueous phase with an Ultra-Turrax Miccra D-8 (ART-Prozess und Labortechnik, Müllheim, Germany) for 5 min. After pasteurization at 85 °C for 15 min, the pH of the emulsion was at 7.0 ± 0.1. In order to study the impact of pH value on the antimicrobial activity of hop extracts in the emulsion, the pH was adjusted to 5.0 ± 0.1 with citric acid. The concentration of Beta Bio 40 was adjusted to 100, 250, 500 and 1000 ppm, the concentration of Xantho-Flav was adjusted to 500 and 1000 ppm. The hop extracts did not alter the initial pH of the emulsion. The inoculum of L. monocytogenes was prepared as described above and an initial cell density of approximately 103 cfu ml-1 was adjusted in the emulsion. Four replicates (each 10 mL) of each sample were stored under controlled temperature conditions of 2 °C and 8 °C in 15 ml tubes (Sarstedt, Nümbrecht, Germany) in order to simulate optimal (2 °C) and common (8 °C) storage conditions of fresh meat products. The samples were stored for up to 15 and 35 days respectively, depending on the temperature. Aliquots of 100 µl were taken every 2-4 days and the number of cfu per ml emulsion was determined by the pour
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plating method: appropriate dilutions of the emulsions were made with sterile ringer solution and 1 ml of each sample was mixed homogeneously with 12-15 mL of TSA (Oxoid, Hampshire, UK). The plates were incubated for 48 h at 37 °C. The data was analyzed using MS Excel 2010. Mean values and standard deviations of four replicates were determined.
Sensory analysis – Conventional profiling Fresh pork loins were cut into slices of 1 cm thickness, transferred into sterile PA/PE-bags (Interscience, Saint Nom, France) and marinated with 10 % (w/w) of the seasoned marinade “Piroschka” (AVO-Werke, Belm, Germany) containing either no or 5000 ppm Beta Bio 40. Bags were vacuum sealed. On the same day the slices were fried for 3 minutes on each side with 10 ml rapeseed oil in an aluminum pan. Sensory properties were assessed by a panel of 6 persons according to DIN 10967-1 (DIN, 1999). The intensity of the attribute “bitter” was evaluated in a 6 step interval scale (not detectable: 0; very strong: 5).
Application of hop extracts in a model meat marinade on pork Fresh pork loins were quartered lengthwise and cut into slices of approximately 15 g, with a thickness of 1 cm and a surface of about 10 cm². Collectively, 4 kg of pork slices were inoculated with 40 ml of a L. monocytogenes DSM 15675 cell suspension with a viable count of approximately 106 cfu ml-1. The cell suspension was homogenously dispersed before two slices were weighed individually into sterile PA/PE bags (Interscience, Saint Nom, France). According to the weight of the pork slices, 20 % (w/w) of the model marinade was added, containing either 500 or 5000 ppm at pH=5 ± 0.1 Beta Bio 40. The bags were vacuum-packed. For reference samples, pork loins were packaged the same way with pure marinade (without hop extract). All samples were stored at 5 °C up to 14 days. Aerobic, mesophilic cell count, Enterobacteria, Listeria and Lactobacilli were monitored as a function of time after 0, 1, 3, 6, 10, 14 days, using TSA (Merck, Darmstadt, Germany), Violet Red Bile Dextrose Agar (VRBD) (Merck, Darmstadt, Germany), Chromocult Listeria selective agar (CLSA) (Merck, Darmstadt, Germany) and de Man, Rogosa and Sharpe agar (MRS) (Merck, Darmstadt, Germany), respectively. Five individual samples were investigated at each point of measurement. Therefore, each
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sample was diluted 1:3 (w/w) with sterile ringer solution and the samples were homogenized in a Lab Blender for 1 min at 260 rpm (AES Chemunex, Bruchsal, Germany). The number of cfu g-1 was determined by the pour-plate method using appropriate decimal dilutions of the sample suspension in ringer solution. Incubation conditions were as follows: 24 h, 37 °C, aerobic for VRBD and CLSA; 48 h, 30 °C aerobic for TSA; 96 h, 30 °C, anaerobic for MRS. Data are presented as mean values ± standard deviations.
RESULTS Antimicrobial activity of hop extracts The determined MIC values of three hop extracts against four different test microorganisms are shown in Table 1. In comparison to the Gram-positive bacteria, the two Gram-negative bacteria S. enterica and E. coli were more resistant against all tested hop compounds. Xantho-Flav and Beta Bio 40 showed strong antibacterial effects against Staph. aureus and L. monocytogenes, with MIC values of 6.3 ppm (Xantho-Flav) and 12.5 ppm (Beta Bio 40) at a pH of 7.2 respectively. In contrast, the effect of Alpha Bio was lower. Both Gram-positive bacteria were inhibited at concentrations above 200 ppm at pH 7.2. Taking into account the particular amount of α-acids and β-acids prevalent in the extracts, β-acids (Beta Bio 40) showed an 8-fold and 16-fold stronger antimicrobial activity than α-acids (Alpha Bio) against L. monocytogenes and Staph. aureus respectively. With regard to E. coli, the MIC of the β-acid extract and the α-acid extract exceeded 5000 ppm at pH 7.2, which corresponds to the highest tested concentration. For the test strain S. enterica, the MIC value of both extracts matched with the highest tested concentration of 5000 ppm. The MIC value of Xantho-Flav against both Gram-negative bacteria exceeded the highest tested concentration of 200 ppm. Higher concentrations were not applied due to the intense yellow coloration and the low solubility of Xantho-Flav in the test media. Acidification of the test media with citric acid or lactic acid to a pH of 5.0 ± 0.1 led to a decrease of the MIC in almost all cases compared to the values determined at pH 7.2 ± 0.1, except for Staph. aureus when acidified with lactic acid (Table 1). The reduction of the MIC by lowering the pH value was in general more pronounced with the α-acid extract than with the β-acid extract or xanthohumol.
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Effect of hop extracts in a model meat marinade against L. monocytogenes In this study, the applicability of both most efficient hop extracts (Beta Bio 40, Xantho-Flav) for the preservation of raw meat was investigated in first instance by testing the inhibitory effect against L. monocytogenes in a model meat marinade. Figure 1 shows the survival and growth kinetics of L. monocytogenes in a marinade stored at 2 °C. The colony count of the reference samples increased from 3.5 to 7.0 log10 cfu ml-1 within 35 days of storage at pH 7. Both, β-acid extract and xanthohumol, did not have any bacteriostatic or bactericidal effects at 500 ppm and pH 7. When 1000 ppm was applied, the β-acid extract led to a reduction of the initial count of 2.9 log10 cfu ml-1 within 35 days. xanthohumol had only limited inhibitory effect at a concentration of 1000 ppm and caused an extended lag phase for about five days. At pH 5 (Figure 2) and a storage temperature of 2 °C, no growth of L. monocytogenes could be observed within the reference samples. A concentration of 100 ppm of the β-acid extract did not have any significant antibacterial effect, the viable cell count decreased by 0.5 log10 cfu ml-1, similar to the references. An accelerated reduction of the colony count could be observed at concentrations of 250 ppm (2.1 log10 cfu ml-1) and 500 ppm (> 3 log10 cfu ml-1) of the β-acid extract within 30 days of storage. When the samples were stored at 8 °C, the colony count of the references increased from 2*103 cfu ml1
to 2*107 cfu ml-1 within 14 days at pH 7.0 (Figure 3). A concentration of 1000 ppm of the β-acid
extract did not inhibit the growth of L. monocytogenes but caused an extended lag-phase of about three days. Almost no inhibitory effect could be observed with 500 ppm. When the pH of the model marinade was adjusted to 5.0 with citric acid, the colony count of the references increased from 2*103 cfu ml-1 to 106 cfu ml-1 within 14 days (Figure 4). β-acid extract (100 ppm) was sufficient to inhibit the growth of L. monocytogenes within 15 days. Higher concentrations (250 and 500 ppm) led to a reduction of the colony count by 2.0 and 2.5 log10 cfu ml-1 compared to the initial load respectively.
Application of hop extracts in a model meat marinade on pork The incorporation of 5000 ppm of Beta Bio 40 proved to have a positive effect on the microbial quality with regard to the total aerobic mesophilic cell count and the number of L. monocytogenes
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during storage at 5 °C (Figure 5). This 10-fold higher concentration was chosen because no antibacterial effect could be observed at a concentration of 500 ppm (data not shown). Furthermore, previous trials have shown that applications in food matrices need much higher concentrations than the determined MIC in vitro. The sensory analysis using conventional profiling (DIN, 1999) on pork with the seasoned marinade “Piroschka” containing 5000 ppm Beta Bio 40 evidenced that the attribute “bitter” was not observable (5000
>5000
>200
pH 5.0 ± 0,1 (Cit)
1250
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625
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5000
5000
>200
pH 5.0 ± 0.1 (Cit)
1250
2500
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pH 5.0 ± 0.1 (Lac)
625
1250
>200
DSM 11822
L. monocytogenes DSM 15675
E. coli DSM 498
S. enterica DSM 11320
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Figure 5 Legends to figures and tables Figure 1 Influence of Beta Bio 40 (β-acid extract) and Xantho-Flav (xanthohumol) on the growth of L. monocytogenes in the model meat marinade at a storage temperature of 2°C and a pH of 7.0. (●) reference, (▲) 1000 ppm Beta Bio 40, () 1000 ppm Xantho-Flav, (x) 500 ppm Beta Bio 40, (+) 500 ppm Xantho-Flav.
Figure 2 Influence of Beta Bio 40 (β-acid extract) on the growth of L. monocytogenes in the model meat marinade at a storage temperature of 2° and a pH of 5.0 (citric acid). (●) reference, (▲) 100 ppm Beta Bio 40, () 250 ppm Beta Bio 40, (x) 500 ppm Beta Bio 40.
Figure 3 Influence of Beta Bio 40 (β-acid extract) on the growth of L. monocytogenes in the model meat marinade at a storage temperature of 8°C and a pH of 7.0. (●) reference, (▲) 500 ppm Beta Bio 40, () 1000 ppm Beta Bio 40.
Figure 4 Influence of Beta Bio 40 (β-acid extract) on the growth of L. monocytogenes in the model meat marinade at a storage temperature of 8°C and a pH of 5.0 (citric acid). (●) reference, (▲) 100 ppm Beta Bio 40, () 250 ppm Beta Bio 40, (x) 500 ppm Beta Bio 40.
Figure 5 Influence of Beta Bio 40 (β-acid extract) on the total viable count and L. monocytogenes on pork loin slices marinated in a model meat marinade at a storage temperature of 2°C and a pH of 5.0 (citric acid). ). (●) reference, total aerobic, mesophillic cell count, (▲) hop containing marinade (5000 ppm), total aerobic, mesophillic cell count, (○) L. monocytogenes, reference, (Δ) L. monocytogenes, hop containing marinade (5000 ppm).
Table 1 MICs of the tested hop extracts against selected bacteria at pH values of 7.2 or 5.0. Acidification of the test media was done with citric acid (Cit) or lactic acid (Lac).
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