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Effect of thyme essential oil and Lactococcus lactis CBM21 on the microbiota composition and quality of minimally processed lamb’s lettuce
Accepted Manuscript Effect of thyme essential oil and lactococcus lactis cbm21 on the microbiota composition and quality of minimally processed lamb’s lettuce Lorenzo Siroli, Francesca Patrignani, Diana I. Serrazanetti, Pamela Vernocchi, Federica Del Chierico, Alessandra Russo, Sandra Torriani, Lorenza Putignani, Fausto Gardini, Rosalba Lanciotti PII:
S0740-0020(16)30974-1
DOI:
10.1016/j.fm.2017.06.017
Reference:
YFMIC 2812
To appear in:
Food Microbiology
Please cite this article as: Lorenzo Siroli, Francesca Patrignani, Diana I. Serrazanetti, Pamela Vernocchi, Federica Del Chierico, Alessandra Russo, Sandra Torriani, Lorenza Putignani, Fausto Gardini, Rosalba Lanciotti, Effect of thyme essential oil and lactococcus lactis cbm21 on the microbiota composition and quality of minimally processed lamb’s lettuce, Food Microbiology (2017), doi: 10.1016/ j.fm.2017.06.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT EFFECT OF THYME ESSENTIAL OIL AND LACTOCOCCUS LACTIS CBM21 ON
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THE MICROBIOTA COMPOSITION AND QUALITY OF MINIMALLY
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PROCESSED LAMB’S LETTUCE
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Lorenzo Siroli(1), Francesca Patrignani(1), Diana I. Serrazanetti(2), Pamela Vernocchi(1,3),
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Federica Del Chierico(3), Alessandra Russo(3), Sandra Torriani(4), Lorenza Putignani(3,5),
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Fausto Gardini(1,2), Rosalba Lanciotti(1,2) *
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(1)
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Bologna, Campus of Food Science, Piazza Goidanich 60, 47521 Cesena, Italy
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Department of Agricultural and Food Sciences, Alma Mater Studiorum, University of
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(2)
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Goidanich 60, 47521 Cesena (FC), Italy
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(3)
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15, 00146 Rome, Italy
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(4)
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Italy
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(5)
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00165 Rome Italy
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Human Microbiome Unit, Bambino Gesù Children Hospital, IRCCS, Viale di San Paolo
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Department of Biotechnology, University of Verona, Strada le Grazie 15, 37134 Verona,
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Unit of Parasitology, Bambino Gesù Children Hospital, IRCCS, Piazza Sant’Onofrio 4,
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Interdepartmental Center for Industrial Agri-food Research, University of Bologna, Piazza
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*Corresponding Author: Rosalba Lanciotti, Department of Agricultural and Food Sciences,
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Alma Mater Studiorum, University of Bologna, Piazza Goidanich 60, 47521 Cesena, Italy.
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e-mail: [email protected]
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Phone +39 0547 338132
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ACCEPTED MANUSCRIPT Abstract
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The main aim of this work was to evaluate, at pilot scale in an industrial environment, the
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effects of the biocontrol agent Lactococcus lactis CBM21 and thyme essential oil compared
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to chlorine, used in the washing step of fresh-cut lamb’s lettuce, on the microbiota and its
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changes in relation to the time of storage. The modification of the microbial population was
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studied through pyrosequencing in addition to the traditional plate counts. In addition, the
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volatile molecule and sensory profiles were evaluated during the storage. The results showed
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no significant differences in terms of total aerobic mesophilic cell loads in relation to the
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washing solution adopted. However, the pyrosequencing data permitted to identify the genera
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and species able to dominate the spoilage associations over storage in relation to the treatment
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applied. Also, the analyses of the volatile molecule profiles of the samples during storage
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allowed the identification of specific molecules as markers of the spoilage for each different
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treatment. The sensory analyses after 3 and 5 days of storage showed the preference of the
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panelists for samples washed with the combination thyme EO and the biocontrol agent. These
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samples were preferred for attributes such as flavor, acceptability and overall quality. These
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results highlighted the effect of the innovative washing solutions on the quality of lettuce
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through the shift of microbiota towards genera and species with lower potential in decreasing
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the sensory properties of the product.
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Keywords: Minimally processed vegetables, Innovative washing procedures, Shelf-life,
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Pyrosequencing,
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ACCEPTED MANUSCRIPT 1. Introduction
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The consumption of minimally processed vegetables has significantly increased in recent
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years mainly because this category of products meet consumer needs for convenience and
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health Patrignani et al., 2015; Sillani and Nassivera, 2015). However, microbial
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contamination, spoilage and pathogenic species can grow during processing due to tissue
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damage and release of nutrients (Jeddi et al., 2014; Siroli et al., 2014). Currently the safety
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and shelf-life of this kind of products is based mainly on the maintenance of the cold chain
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and the sanitization of the raw materials (Alegre et al., 2010; Siddiqui et al., 2011).
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Nevertheless, in case of minimally processed vegetables, these steps are not able to
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significantly reduce the number of pathogenic and spoilage microorganisms (Brilhante São
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José and Dantas Vanetti, 2012; Petri et al., 2015). Nowadays, chlorine represents the most
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common decontaminant used at industrial level (Goodburn and Wallace, 2013; Ma et al.,
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2017), although chlorine-based compounds are corrosive, cause skin and respiratory tract
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irritation and react with the organic matter in the water leading to the formation of potentially
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harmful trihalomethanes (Brilhante São José and Dantas Vanetti, 2012; López-Gálvez et al.,
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2009). These limitations have stimulated the search for alternative technologies or sanitization
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solutions. Among the alternatives, the use of essential oils (EOs) and lactic acid bacteria
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(LAB) as biocontrol agents seems very promising. In fact, EOs or their constituents have
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shown excellent potential applications even in fresh-cut produce. These are substances with
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antifungal, antibacterial, antiviral and antioxidant activity and with a wide spectrum of action
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(Calo et al., 2015; Patrignani et al., 2015). Moreover, these natural antimicrobial compounds
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and some of their most active components are generally recognized as safe (GRAS) by the
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Food and Drug Administration (FDA) and are permitted as food additives by the European
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Food Safety Authority (EFSA) as well as by Japanese legislation (Patrignani et al., 2015). The
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use of these molecules in the washing of minimally processed vegetables has been widely
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studied with excellent results in recent years (Patrignani et al., 2015; Siroli et al., 2015a).
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ACCEPTED MANUSCRIPT Moreover, Siroli et al. (2015a) showed that the use of oregano and thyme EOs in the washing
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solution of lamb’s lettuce could replace chlorine solution showing the same or better
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performances in terms of safety aspects, shelf-life and sensory characteristics.
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Bioprotective microorganisms have also already been proposed for practical application in
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plant derived products (Trias et al., 2008). In particular, LAB represent indubitably the most
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important bioprotective cultures for non-fermented foods including minimally processed
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vegetables (Siroli et al., 2015b, 2015c).
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Despite the encouraging results obtained, the majority of the studies dealing with the use of
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alternative sanitizers were performed on laboratory scale and consequently were more
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difficult to transfer to industry (Wulfkuehler et al., 2013). Moreover, most of the studies have
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reported the effect of innovative washing solutions only on the total amount of microbial
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population, but only a few of them have investigated the effects of the treatments on the
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overall diversity and composition of the bacterial community of the product during the
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storage and their consequences on product quality (Leff and Fierer, 2013).
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In this context, the main aim of this work was to evaluate the effects of innovative washing
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solutions, containing the biocontrol agent Lactococcus lactis CBM21 and thyme essential oil,
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on the composition of the microbiota, the volatile molecule profiles and the sensory
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characteristics of fresh-cut lamb’s lettuce produced at pilot scale in a small Italian company.
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Moreover, the present research project aimed at identifying, through pyrosequencing, the
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main genera and species dominating the spoilage association during the storage and their
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relationship with the product volatile molecule and sensory profiles in relation to the washing
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applied. The innovative solutions were selected on the basis of the results obtained at
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laboratory level by Siroli et al. (2016), and compared to the traditional approach based on the
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use of chlorine solutions.
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2. Material and Methods 4
ACCEPTED MANUSCRIPT 2.1 Natural antimicrobials and microbial strain
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Thyme EO (Thymus vulgaris) was obtained from Flora s.r.l. (Pisa, Italy). The potential
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biocontrol agent L. lactis CBM21, nisin Z producer, belongs to the collection of the
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Department of Biotechnology of Verona University. This strain, before each trial was
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preliminarily grown in M17 broth (Oxoid Ltd., UK) and incubated aerobically at 30 °C for 24
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h.
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2.2 Preparation of lamb’s lettuce products
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Lamb’s lettuce (Valerianella locusta sp.) was supplied by the SIPO s.r.l (Santarcangelo di
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Romagna, Rimini, Italy). and has been stored and prepared at pilot scale in the same company
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and with the same procedures of industrial production. The lamb’s lettuce process is reported
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in Figure 1.
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Four different washing solutions were prepared with running water:
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a) Chlorine 120 mg/L (Control)
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b) Thyme EO 250 mg/L
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c) Biocontrol agent L. lactis CBM21 (Inoculated at a level of 6 log CFU/ml)
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d) Thyme EO 125 mg/L + L. lactis CBM21 (Inoculated at a level of 6 log CFU/ml)
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The inoculation level of L. lactis and the concentrations of thyme EO were selected on the
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basis of previous results obtained at laboratory scale (Siroli et al., 2016, 2015a, 2015b).
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Thyme EO was conveyed to the washing solution through 1% ethanol, while the biocontrol
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agent was grown in M17 broth (Oxoid Ltd., UK) incubated aerobically at 30 °C for 24 h then
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the cells were harvested by centrifugation at 8,000/g for 15 min in a J-25 Beckman Coulter
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centrifuge at room temperature, re-suspended in saline solution and inoculated in the washing
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solution. The pH of the washing solutions, before the treatments, resulted 7.04 in case of
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addition of chlorine 120 mg/L, 6.85 and 6.76 in case of addition of thyme EO 250 mg/L and
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biocontrol agent.
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Prepared lettuce was immersed in an industrial washing tank and agitated in the appropriate
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washing solution at 8 °C for 1 min (10 kg of lettuce in 180 L of washing solution). After the
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treatment, the lettuce was spin dried and packaged into PP trays and closed by a plastic PP
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bags. Each package was filled with 80 g of lettuce. Samples were stored at 6 °C until the end
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of shelf-life (12 days). Finally, we obtained around 85 lettuce bags for each condition.
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2.3 Microbiological analyses
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During storage, the cell loads of mesophylic aerobic bacteria, lactic acid bacteria and yeasts,
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were evaluated by plate counting on Plate Count Agar (PCA, Oxoid Ltd., UK), Man Rogosa
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and Sharpe Agar (MRS, Oxoid Ltd., UK) supplemented with cycloheximide (0.05%) (Sigma-
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Aldrich) and Sabouraud Dextrose Agar (SAB, Oxoid Ltd., UK), respectively. The inoculated
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L. lactis strain was evaluated by plate counting on M17 agar (Oxoid Ltd., UK). To confirm
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the presence of L. lactis, bacterial colonies from M17 plates were isolated and subsequently
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identified by sequencing the 16S rRNA region according to the protocol described by De
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Angelis et al. (2006). After homogenization, samples were serially diluted in physiological
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solution (10 g of sample diluted into 90 ml of physiological water (0.9% (w/v) NaCl). Plates
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were incubated at 30 °C for 48h in case of yeasts and mesophilic aerobic bacteria, at 37 °C for
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24-48h for all the other species. For the detection of the natural occurring Listeria
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monocytogenes, Salmonella spp. and Escherichia coli the methods reported by Siroli et al.
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(2015b) were used. The microbiological analyses were performed immediately after
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treatments and after 3, 5, 7, 10, and 12 days of storage. All the microbiological analyses were
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performed in triplicate on three independent samples.
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2.4 Color, volatile molecule profiles analyses and panel test 6
ACCEPTED MANUSCRIPT Lamb’s lettuce color was determined using a color-spectrophotometer mod. Colorflex
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(Hunterlab, USA). Color was measured using the CIELab scale and Illuminant D65. The
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instrument was calibrated with a white tile (L*98.03, a* - 0.23, b* 2,05). Results were
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expressed as L* (luminosity), a* (red-green index) and b* (yellow-blue index). The color
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analysis was performed immediately after treatments and after 3, 5, 7, 10 and 12 days of
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storage. Forty-five readings were obtained from three independent samples for each lamb’s
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lettuce condition.
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Lettuce bags, containing 80 g of product were used for headspace volatile compound analysis
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by GC/MS-SPME technique. Samples were analyzed immediately after treatments and after 5
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and 10 days of storage. The samples were conditioned for 30 min at 37 °C and each sample
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was analyzed by following the method reported by Siroli et al. (2014). Compounds were
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identified using the Agilent Hewlett–Packard NIST 98 mass spectral database. All the
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analyses were performed in triplicate from three independent samples.
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A panel test was performed immediately after treatments and after 3 and 5 days of storage.
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Thirty untrained consumers made up the panel, and the quality parameters evaluated were
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odor, color, browning, texture, flavor, acceptability and overall quality. Each of the qualitative
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parameters considered was evaluated by consumers on a scale from 1.0 (very poor) to 5.0
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(very good). Consumers evaluated four different conditions: control lettuce, lettuce washed
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with thyme EO (250 mg/L), lettuce washed with the biocontrol agent and lettuce washed with
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both thyme EO (125 mg/L) and the biocontrol agent.
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2.5 Genomic DNA extraction pyrosequencing and quantitative analysis of the microbiome
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composition
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DNA was adjusted to 10 ng/µL concentration and used as template for 16S Metagenomic 454
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Sequencing Analyses. 7
ACCEPTED MANUSCRIPT Total genomic DNA was extracted directly from lamb’s lettuce kit Mo-bio (Cambrex
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Biosciences, Bergamo, Italy). DNA from the entire set of 12 samples, was employed for the
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V1-V3 region of 16S ribosomal RNA (rRNA) locus amplification for the pyrosequencing step
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on a 454-Junior Genome Sequencer (Roche 454 Life Sciences, Branford, USA) according to
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the pipeline described by Ercolini et al. (2012).
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2.6 Statistics and bioinformatics
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For each sample, the microbiological and volatile data were the mean of three different
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measurements. Regarding microbiological, color and panel test data, statistical analysis was
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performed using Statistica software (version 8.0; StatSoft, Tulsa, Oklahoma, USA). Means
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were compared using ANOVA followed by LSD test at p<0.05 level to mark differences
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between treatments with same storage times. The volatile molecule profiles were analyzed
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using ANOVA followed by a principal component analysis (PCA) performed using Statistica
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software (version 8.0; StatSoft, Tulsa, Oklahoma, USA) to obtain a visual overview of
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volatile molecule profile analyses.
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The bioinformatic workflow applied in this study derives from the pipeline elaborated by
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Kuczynsky et al. (2012).
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At first result filtering was performed with 454 Amplicon signal processing; and sequences
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were analyzed by using Quantitative Insights into Microbial Ecology (QIIME 1.8.0) software.
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To detect Operational Taxonomic Units (OTUs), row reads were demultiplexed and reads
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with an average quality score lower than 25, shorter than 300 bp and with an ambiguous base
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calling were excluded from the analysis to reduce the overall error rates. Sequences that
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overcame the quality filter were denoised (Reeder and Knight, 2010) and singletons were 72
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excluded. The OTUs defined by a 97% of similarity were collected by using the uclust
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method (Edgar, 2010) and the representative sequences were submitted to PyNAST, for the
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sequence alignment the employed method was UCLUST and the database for OTUs matching
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was greengenes (v 13.8). Finally, an OTU table was created with the absolute abundance of
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each OTU across the samples.
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The α and β diversity and the Kruskal wallis test were performed using QIIME software,
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using “alpha_rarefaction.py, beta_diversity_through_py, group_significance.py” scripts.
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3. Results
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3.1 Effect of the Lactococcus lactis CBM21 and thyme essential oil on the microbiological
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quality of lamb’s lettuce
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In table 1 the cell loads are reported of Lactococcus lactis CBM21 in relation to the washing
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solution used. Lamb’s lettuce cell loads immediately after the treatment were 5.1 and 5.4 log
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CFU/g in the samples treated with the biocontrol agent alone and in combination with thyme
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EO, respectively. The biocontrol agent cell load decreased during storage by about 0.7-1.3 log
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CFU/g after 5 days of storage at 6 °C irrespective of the presence of thyme EO in the washing
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solution (Table 1). In the control samples and in those treated only with thyme EO, LAB cell
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loads were lower than 3.0 log CFU/g also after 12 days of storage at 6 °C.
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As shown in Table 2, no significant differences were evidenced in the total aerobic
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mesophilic cell loads among the samples for the whole period of storage at 6 °C. In fact, after
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12 days of storage the load of the total aerobic mesophilic in the samples ranged between 7.2
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and 7.6 log CFU/g, irrespective of the washing solution adopted. Moreover, no significant
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differences were found for yeast and total coliform cell loads in relation to the washing
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solution adopted, whose loads resulted below 3.0 and 2.5 log CFU/g respectively, during the
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whole period of storage (data not shown). In any case, these microbial groups, due to their
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low cell loads were unable to play a key role in the spoilage of the product irrespective of the
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washing procedures applied. L. monocytogenes and Salmonella spp. were absent in 25 g of
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products, while E. coli was always under the detection limits (1.0 log CFU/g) after 12 days of
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storage irrespective of the washing solution used (data not shown).
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3.2 Microbiome characterization of lamb’s lettuce microbiota
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A pyrosequencing analysis was performed on the samples of lamb’s lettuce in relation to
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washing solutions and time of storage (0, 5, 10 days). A total of 34821 sequencing reads were
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obtained from all the analyzed samples. To characterize the ecological species diversity in
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lamb’s lettuce products, Shannon index was calculated (Li et al., 2012). and performed by
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grouping samples based on storage time. An increase of Shannon index was observed in T5
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set, respect T0 and T10 groups (Figure 2A), while assembling samples by washing solution,
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the alpha diversity index was higher respect to controls (Figure 2B). The β-diversity data
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obtained, representing the comparison of microbial communities based on their dissimilar
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composition, analyzed by Principal Coordinate Analysis (PCoA), provided a clear separation
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among the samples based on storage time (Figure 3), while a significant clustering of the
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samples based on washing solution was not highlighted (data not shown).
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The analyses of the significance of the Operational Taxonomic Units (OTU) identified
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highlighted that the main represented phyla in the complete dataset were Proteobacteria,
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Bacteroidetes, Actinobacteria and Firmicutes. In particular, Proteobacteria, and Firmicutes
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showed a decrease during storage time in all samples, while Bacteroidetes showed an opposite
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trend (p<0.005) (Figure 4A). Those washed with L. lactis+Thyme EO showed a more
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different profile with higher level of Proteobacteria and Actinobacteria and lower level of
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Bacteroidetes compared to the other samples, although the differences on OTU distribution
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were not statistically significant (Figure 4B). The analysis at genus level evidenced a different
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profile among the samples in relation to storage time (Figure 4C) and to the washing solution
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adopted (Figure 4D). During the storage period an increase of Flavobacterium spp.,
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Oxalobacteriaceae, Janthinobacterium spp. and Pedobacter spp. was observed. By contrast a
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decrease of Sphingomonas spp., Comamonadaceae, Methylophilaceae and Caulobacteraceae
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was detected. The analysis of data by clustering the samples based on the washing solution
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time, samples washed with alternative solutions were characterized by a higher presence of
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Pedobacter spp., Chriseobacterium spp., Rhodococcus spp. and Comamonadaceae compared
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to control samples. By contrast, samples washed with chlorine showed a higher presence of
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Oxalobacteraceae, Sphingomonas spp., and Methylophilaceae compared to samples washed
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with the alternative solutions.
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As expected the pyrosequencing analysis evidenced the presence of Lactococcus lactis only in
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the samples washed with the solution containing the selected biocontrol agent. However, in
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the samples washed only with the biocontrol agent the average relative abundance of
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Lactococcus lactis was around twice than in the samples washed with the solution also
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containing thyme (Figure 4D).
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3.3 Effects of natural antimicrobials on volatile molecule profiles of lamb’s lettuce
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The biocontrol agent and/or thyme EO affected the lamb’s lettuce volatile molecule profiles
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leading to the identification of 98 molecules belonging to different chemical classes such as
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alcohols, ethers, esters, aldehydes, ketones and hydrocarbons (data not shown). Specific
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volatile fingerprintings, in relation to the washing solution, were obtained. The washing with
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thyme EO was reflected in the presence of γ-terpinene, p-cymene, β-phellandrene, limonene,
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terpinolene, camphene, caryophyllene, thymol and carvacrol, that represent the characteristic
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molecules of thyme EO. During the storage period, the increase in molecules obtained by the
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transformation of the main component of thyme EO (acetic acid methyl ester, ethyl acetate
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and thymol methyl ether) was evident. The addition of the biocontrol agent determined
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significant changes in molecules classes, in particular an increase of aldehydes (hexanal, 2-
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hexanal and benzaldehyde) and alcohols (1-butanol, 3-methyl, phenyl ethyl alcohol).
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In order to better show the effects of the washing solution on the volatile molecule profiles,
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the GC-MS-SPME data were subjected to a Principal Component Analysis (PCA) excluding
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transformation by lettuce tissues or microorganisms according to literature data (Siroli et al.,
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2015a). The results are shown in Figures 5A and B. The PCA analysis showed that the control
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samples were well separated from the other samples, although the clustering was influenced
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by the storage time. In fact, the control samples immediately after packaging and those after 5
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days of storage constituted a cluster (cluster 1) well separated along the main component 1,
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which explains about 28.53% of the variance, from control samples stored for 10 days at 6 ° C
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(Cluster 2). Cluster 1 was characterized mainly by hexanal, 2-hexanal, benzene acetaldehyde,
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α-pinene, β-pinene and methylene chloride while cluster 2 by molecules such as 6-Methyl-5-
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hepten-2-ol, 6-Methyl-1-heptene, dimethyl sulphide and 1-hexanol. Cluster 2 was separated
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from the samples subjected to the alternative washing solution and stored for 10 days along
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the main component 2 explaining the 20.23% of the total variance (cluster 3). These differed
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from the other samples washed with the biocontrol agent and/or oil thyme EO, which grouped
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in two other clusters separated along the main component 1 in relation to the storage time
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(cluster 4 and 5). Cluster 3 was characterized by the molecules acetic acid methyl ester,
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benzyl alcohol and ethyl acetate while the grouping of cluster 4 was mainly affected by
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butanol, 1-propanol, hexanal and 2-hexanal.
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Moreover, a sensory evaluation through panel tests was performed immediately and after 3
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and 5 days of storage (Figure 6). The panel test immediately after packaging showed no
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significant differences regarding color and texture parameters. As expected, the odor of
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lettuce resulted significantly higher for controls and samples washed with the biocontrol
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agent. Contrarily, with regard to flavor intensity, acceptability and overall quality samples
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washed with the combination of innovative treatments resulted significantly higher than the
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controls. After 3 days of storage, the samples washed with thyme EO alone or in combination
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with the biocontrol agent showed a significantly higher overall acceptability and color
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intensity compared to the others. The samples washed with the combination of the innovative
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the samples washed with thyme EO showed the highest scores of browning. Sensory analysis
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carried out on the fifth day of storage confirmed the positive effects on flavor, acceptability,
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and odor intensity of the alternative washing solutions. The combination thyme EO and
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biocontrol culture showed the highest scores regarding acceptability, flavor, texture, and the
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overall quality. However, the color analysis performed immediately after packaging and after
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3, 5, 7, 10 and 12 days of storage at 6 °C did not show significant differences among the
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samples (data not shown).
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4. Discussion
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The microbiological data and the main quality parameters showed no significant difference
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regarding the shelf life and safety of the product in relation to the washing solution adopted.
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The composition of the microbial population was similar in terms of species recorded to that
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reported by literature independently on the washing solution adopted. In fact, the prevalence
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of gram- negative microorganisms was demonstrated in all the samples analyzed over storage
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with Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria as predominant phyla.
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These phyla are also reported as the dominant ones in minimally processed vegetables by
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Rastogi et al. (2012). Moreover, these results are in agreement with those obtained in
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laboratory trials by Siroli et al. (2015a), that showed the same reduction of the naturally
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occurring microbial population when lamb’s lettuce was washed either with chlorine (120
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mg/L) or thyme EO.
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However, significant differences in relation to the washing solution adopted were pointed out
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by volatile molecule profiles and sensory analyses performed during storage. In fact, the
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GC/MS SPME showed that the components of thyme EOs were transformed in alcohols by
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microorganisms and plant tissues. The transformation of hexanal, 2-hexanal, neral, geranial
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and other six atom aliphatic aldehydes by the plant tissues and yeasts to alcohols (mainly 1-
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ACCEPTED MANUSCRIPT hexanol, 3-hexen-1-ol, nerol and geraniol) endowed with lower antimicrobial properties
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compared to their precursors is well documented (Belletti et al., 2004; Patrignani et al., 2013;
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Siroli et al., 2014). The PCA analyses of the volatile molecule data showed the grouping of
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the samples mainly based on the time of storage and washing solution adopted. In fact, the
340
samples treated with the innovative washing solutions resulted significantly different over
341
time compared to the control samples, demonstrating completely different spoilage patterns.
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On the other hand, the results of pyrosequencing showed significant differences of microbiota
343
composition in relation to the washing solution. In fact, the analysis of the microbiota of the
344
different lettuce samples highlighted the ability of both the biocontrol culture as well as thyme
345
EO to significantly change the relative proportions between the different spoilage species. In
346
the products washed with the Lactococcus lactis CBM21, the incidence of high potential
347
spoilage microorganisms, such as those belonging to the family of Pseudomonadaceae, was
348
reduced. Even thyme EO, including when used alone, had a significant effect on product
349
microbiota. In fact, this natural antimicrobial has a broad spectrum of action, the effectiveness
350
of which however varies considerably in relation to the species, composition and chemical
351
and physical composition of the system conditions (Bagamboula et al., 2004; Gutierrez et al.,
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2008; Ivanovic et al., 2012).
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Samples washed with the innovative washing solutions after 10 days of storage showed a
354
significantly higher presence of esters such as ethyl acetate and acetic acid methyl ester,
355
generally associated with a positive fruity smell, and some alcohols such as benzyl alcohol
356
and phenyl ethyl alcohol due to the reduction of spoilage microbial groups such as
357
Pseudomonadaceae and Methylophilaceae that are more associated with the production of
358
sulphur compounds and volatile amines.
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The differences found in the microbial population composition of the products in relation to
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the washing conditions adopted can explain, in addition to the differences of the volatile
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molecule profiles, the different sensory evaluation of the samples. The sensory analysis
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ACCEPTED MANUSCRIPT showed the preference by the panelists for lamb’s lettuce obtained with alternative washing
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solutions immediately after packaging. By the fifth day of storage the samples obtained by
364
washing with thyme EO in combination with the biocontrol agent showed a major overall
365
quality compared to all the other treatments. In fact, it is well documented that differences in
366
microbial population of fresh vegetables can result in different organoleptic features (Di
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Cagno et al., 2009; Gutierrez et al., 2009; Siroli et al., 2015b). In fact, Gutierrez et al. (2009)
368
identified volatile spoilage markers in minimally processed lettuce and carrots washed with
369
chlorine or oregano EO. In particular, they showed that an increase in Enterobacteria and
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Pseudomonas was associated with a loss of aroma, texture and color. Furthermore, the
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competition of the biocontrol agents for the space and the nutrients and their production of
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antimicrobials including nisin, are reported as the main mechanisms through which the
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biocontrol agents inhibit natural occurring microorganisms of products modifying their
374
spoilage patterns in relation to the sensitiveness of the microorganisms present (Siroli et al.,
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2016). In addition, it is widely reported that natural antimicrobials, such as thyme EO, have a
376
strong influence on the microbial metabolism and volatile molecule profile, and consequently
377
on the organoleptic characteristics of the product (Gutsche et al., 2012; Picone et al., 2013;
378
Siroli et al., 2015d). No negative effects on color were observed within 12 days of storage for
379
samples washed in the presence of the selected biocontrol agent. These results, in
380
disagreement with those of other authors (Siroli et al., 2015b; Trias et al., 2008), highlighted
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the importance of the selection of the suitable biocontrol agents in relation to the product and
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process considered and the expected shelf-life.
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4. Conclusions
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This work highlighted a marked effect of different washing solutions on the microbial
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community of lettuce resulting in differences in the volatile profiles and the sensory 15
ACCEPTED MANUSCRIPT characteristics of minimally processed lettuce. In fact, the innovative washing solutions
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affected the quality of lettuce through the shift of microbiota towards genera and species with
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lower potential in decreasing the sensory properties of the product. Moreover, this work,
390
conducted in an industrial environment, indicated that the protocol adopted can be easily
391
upscaled to answer innovation needs of small to medium-sized industries. However, the
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definitive industrialization of the proposed biotechnological solutions will only happen after
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further studies have been performed on the effects on microbial interactions in the product
394
and on the mechanisms of action of the EO chosen against spoilage and pathogenic
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microorganisms.
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Figure 1. Working protocol used to prepare lamb’s lettuce; the addition of lactic acid bacteria
399
and/or thyme EO was performed during the washing phase; samples washed with chlorine
400
(120 mg/L) represented the controls.
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Figure 2A,B. Shannon-Weaver index box plot. α-diversity analysis for sample grouped by
402
time of storage (a) and treatment (b). The table showed interquartile (25-75%) (qi) range,
403
median values, and minimum (min) maximum (max) Shannon index value.
404
Figure 3. Metagenomics β-diversity plots. PCs plots of the diversity in the microbial
405
communities from all samples stratified by time of storage.
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Figure 4 A-D. Graphical representation of OTUs distribution at phylum (a,b) and genus (c,d)
407
level. In panel a,c the samples were grouped by time of storage, and in panel b,d samples
408
were grouped by treatment. The OTUs highlighted in the legend were statistically significant
409
(p≤0.05).
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411
different times of storage) and loadings (7B molecules detected by GC/MS/SPME) on the
412
factor-plane (1x2). PC1 and PC2 explained 28.75% and 20.20% of the total variance
413
respectively.
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Figure 6. Sensory data of lamb’s lettuce, in relation to the washing conditions used,
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immediately after treatments (A) and after 3 days (B) and 5 days (C) of storage at 6 °C.
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Acknowledgment
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This experimental research was supported by the national project AGER-STAY FRESH 2010
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ACCEPTED MANUSCRIPT Table 1. L. lactis CBM21 count on lamb’s lettuce in the presence or not of thyme EO day 0
day 3
day 5
day 7
day 10
day 12
L. lactis CBM211
5.1±0.23a
5.0±0.24a
4.4±0.60a
4.5±0.21a
4.1±0.25a
3.7±0.33a
Thyme eo + L. lactis CBM212
5.4±0.22a
5.1±0.16a
4.1±0.29a
4.1±0.21a
3.7±0.13a
3.5±.28a
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Counts are expressed in Log cfu/g (+/− standard deviation). At the same time of storage, means followed by different letters are significantly different (p<0.05) LAB counts in samples washed with chlorine and thyme EO ranged between 2.4 and 3.0 log cfu/g during the whole period of storage Samples washed with the biocontrol agent L. lactis CBM21 (Inoculated at a level of 6 log cfu/mL in the washing solution)
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Samples washed with thyme essential oil 125 mg/L + L. lactis CBM21 (Inoculated at a level of 6 log cfu/mL in the washing solution)
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1
6.33±0.33
day 3 a
6.89±0.35
day 5 a
7.06±0.37
day 7 a
7.27±0.33
day 10 a
7.45±0.31
day 12 a
7.32±0.29a
6.83±0.23a 7.29±0.13a 7.45±0.13a 7.70±0.25a 7.86±0.32a 7.20±0.23a
Thyme eo3
6.46±0.31a 7.17±0.37a 7.16±0.35a 7.50±0.24a 7.72±0.18a 7.46±0.21a
Thyme eo + L. lactis CBM214
6.43±0.28a 7.19±0.23a 7.26±0.20a 7.61±0.16a 7.76±0.19a 7.58±0.22a
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Counts are expressed in Log cfu/g (+/− standard deviation). At the same time of storage, means followed by different letters are significantly different (p<0.05) Samples washed with chlorine 120 mg/L (Control)
2
Samples washed with the biocontrol agent L. lactis CBM21 (Inoculated at a level of 6 log cfu/mL in the washing solution)
3
Samples washed with thyme essential oil 250 mg/L
4
Samples washed with thyme essential oil 125 mg/L + L. lactis CBM21 (Inoculated at a level of 6 log cfu/mL in the washing solution)
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Washing 1 min with running water in automatic industrial washing tank; Water T 8 °C; lamb’s lettuce/water 5 Kg/180 L Different washing condition: a) 120 mg/L chlorine (Control) b) Thyme E 250 mg/L c) Biocontrol agent L. lactis CBM21 (6 log CFU/mL) d) Thyme essential oil 125 mg/L + L. lactis CBM21 (6 log CFU/mL)
Figure 1
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Draining centrifuging lamb’s lettuce 2 times for 30 seconds in an industrial centrifuge
Packing in PP trays and closed by PP bags: 80 g of product for each bag
Storage, 6 °C
ACCEPTED MANUSCRIPT 7 Shannon-Weaver index
6 5 4 3
1 0
T0
T5
T10
qi
4.860942216
5.446432836
4.926446819
min
3.959366506
5.298256693
4.295143764
mediana
5.594608979
5.146433337
5.49546136
5.981543668
5.398179036
q3
5.304873591
5.788076324
5.216533887
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7 Shannon-Weaver index
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LACT L. lactis CBM21
LACT_TIM CBM21+Thyme
TIM eo Thyme
4.043310821
5.189112752
5.229862073
5.325723432
min
3.959366506
5.13688117
5.161467453
5.155985504
mediana 4.127255135
5.241344334
5.298256693
5.49546136
max
4.295143764
5.981543668
5.398179036
5.594608979
q3
4.211199449
5.611444001
5.348217864
5.545035169
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ACCEPTED MANUSCRIPT Innovative solutions as an alternative to chlorine to guarantee lamb’s lettuce shelf-life
-
Samples washed with alternative solutions showed a different volatilome than controls
-
Washing solution affect the quality of lamb’s lettuce microbiota
-
The use of innovative washing solutions affect the composition of spoilage microflora
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The sensory analyses highlight the preference by the panelists for innovative products
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S0740-0020(16)30974-1
DOI:
10.1016/j.fm.2017.06.017
Reference:
YFMIC 2812
To appear in:
Food Microbiology
Please cite this article as: Lorenzo Siroli, Francesca Patrignani, Diana I. Serrazanetti, Pamela Vernocchi, Federica Del Chierico, Alessandra Russo, Sandra Torriani, Lorenza Putignani, Fausto Gardini, Rosalba Lanciotti, Effect of thyme essential oil and lactococcus lactis cbm21 on the microbiota composition and quality of minimally processed lamb’s lettuce, Food Microbiology (2017), doi: 10.1016/ j.fm.2017.06.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT EFFECT OF THYME ESSENTIAL OIL AND LACTOCOCCUS LACTIS CBM21 ON
2
THE MICROBIOTA COMPOSITION AND QUALITY OF MINIMALLY
3
PROCESSED LAMB’S LETTUCE
4
Lorenzo Siroli(1), Francesca Patrignani(1), Diana I. Serrazanetti(2), Pamela Vernocchi(1,3),
5
Federica Del Chierico(3), Alessandra Russo(3), Sandra Torriani(4), Lorenza Putignani(3,5),
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Fausto Gardini(1,2), Rosalba Lanciotti(1,2) *
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7 8
(1)
9
Bologna, Campus of Food Science, Piazza Goidanich 60, 47521 Cesena, Italy
SC
Department of Agricultural and Food Sciences, Alma Mater Studiorum, University of
10
(2)
11
Goidanich 60, 47521 Cesena (FC), Italy
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(3)
13
15, 00146 Rome, Italy
14
(4)
15
Italy
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(5)
17
00165 Rome Italy
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Human Microbiome Unit, Bambino Gesù Children Hospital, IRCCS, Viale di San Paolo
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Department of Biotechnology, University of Verona, Strada le Grazie 15, 37134 Verona,
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Unit of Parasitology, Bambino Gesù Children Hospital, IRCCS, Piazza Sant’Onofrio 4,
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Interdepartmental Center for Industrial Agri-food Research, University of Bologna, Piazza
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*Corresponding Author: Rosalba Lanciotti, Department of Agricultural and Food Sciences,
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Alma Mater Studiorum, University of Bologna, Piazza Goidanich 60, 47521 Cesena, Italy.
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e-mail: [email protected]
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Phone +39 0547 338132
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ACCEPTED MANUSCRIPT Abstract
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The main aim of this work was to evaluate, at pilot scale in an industrial environment, the
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effects of the biocontrol agent Lactococcus lactis CBM21 and thyme essential oil compared
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to chlorine, used in the washing step of fresh-cut lamb’s lettuce, on the microbiota and its
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changes in relation to the time of storage. The modification of the microbial population was
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studied through pyrosequencing in addition to the traditional plate counts. In addition, the
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volatile molecule and sensory profiles were evaluated during the storage. The results showed
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no significant differences in terms of total aerobic mesophilic cell loads in relation to the
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washing solution adopted. However, the pyrosequencing data permitted to identify the genera
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and species able to dominate the spoilage associations over storage in relation to the treatment
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applied. Also, the analyses of the volatile molecule profiles of the samples during storage
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allowed the identification of specific molecules as markers of the spoilage for each different
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treatment. The sensory analyses after 3 and 5 days of storage showed the preference of the
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panelists for samples washed with the combination thyme EO and the biocontrol agent. These
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samples were preferred for attributes such as flavor, acceptability and overall quality. These
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results highlighted the effect of the innovative washing solutions on the quality of lettuce
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through the shift of microbiota towards genera and species with lower potential in decreasing
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the sensory properties of the product.
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Keywords: Minimally processed vegetables, Innovative washing procedures, Shelf-life,
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Pyrosequencing,
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ACCEPTED MANUSCRIPT 1. Introduction
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The consumption of minimally processed vegetables has significantly increased in recent
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years mainly because this category of products meet consumer needs for convenience and
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health Patrignani et al., 2015; Sillani and Nassivera, 2015). However, microbial
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contamination, spoilage and pathogenic species can grow during processing due to tissue
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damage and release of nutrients (Jeddi et al., 2014; Siroli et al., 2014). Currently the safety
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and shelf-life of this kind of products is based mainly on the maintenance of the cold chain
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and the sanitization of the raw materials (Alegre et al., 2010; Siddiqui et al., 2011).
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Nevertheless, in case of minimally processed vegetables, these steps are not able to
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significantly reduce the number of pathogenic and spoilage microorganisms (Brilhante São
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José and Dantas Vanetti, 2012; Petri et al., 2015). Nowadays, chlorine represents the most
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common decontaminant used at industrial level (Goodburn and Wallace, 2013; Ma et al.,
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2017), although chlorine-based compounds are corrosive, cause skin and respiratory tract
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irritation and react with the organic matter in the water leading to the formation of potentially
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harmful trihalomethanes (Brilhante São José and Dantas Vanetti, 2012; López-Gálvez et al.,
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2009). These limitations have stimulated the search for alternative technologies or sanitization
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solutions. Among the alternatives, the use of essential oils (EOs) and lactic acid bacteria
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(LAB) as biocontrol agents seems very promising. In fact, EOs or their constituents have
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shown excellent potential applications even in fresh-cut produce. These are substances with
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antifungal, antibacterial, antiviral and antioxidant activity and with a wide spectrum of action
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(Calo et al., 2015; Patrignani et al., 2015). Moreover, these natural antimicrobial compounds
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and some of their most active components are generally recognized as safe (GRAS) by the
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Food and Drug Administration (FDA) and are permitted as food additives by the European
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Food Safety Authority (EFSA) as well as by Japanese legislation (Patrignani et al., 2015). The
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use of these molecules in the washing of minimally processed vegetables has been widely
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studied with excellent results in recent years (Patrignani et al., 2015; Siroli et al., 2015a).
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ACCEPTED MANUSCRIPT Moreover, Siroli et al. (2015a) showed that the use of oregano and thyme EOs in the washing
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solution of lamb’s lettuce could replace chlorine solution showing the same or better
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performances in terms of safety aspects, shelf-life and sensory characteristics.
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Bioprotective microorganisms have also already been proposed for practical application in
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plant derived products (Trias et al., 2008). In particular, LAB represent indubitably the most
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important bioprotective cultures for non-fermented foods including minimally processed
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vegetables (Siroli et al., 2015b, 2015c).
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Despite the encouraging results obtained, the majority of the studies dealing with the use of
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alternative sanitizers were performed on laboratory scale and consequently were more
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difficult to transfer to industry (Wulfkuehler et al., 2013). Moreover, most of the studies have
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reported the effect of innovative washing solutions only on the total amount of microbial
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population, but only a few of them have investigated the effects of the treatments on the
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overall diversity and composition of the bacterial community of the product during the
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storage and their consequences on product quality (Leff and Fierer, 2013).
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In this context, the main aim of this work was to evaluate the effects of innovative washing
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solutions, containing the biocontrol agent Lactococcus lactis CBM21 and thyme essential oil,
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on the composition of the microbiota, the volatile molecule profiles and the sensory
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characteristics of fresh-cut lamb’s lettuce produced at pilot scale in a small Italian company.
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Moreover, the present research project aimed at identifying, through pyrosequencing, the
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main genera and species dominating the spoilage association during the storage and their
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relationship with the product volatile molecule and sensory profiles in relation to the washing
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applied. The innovative solutions were selected on the basis of the results obtained at
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laboratory level by Siroli et al. (2016), and compared to the traditional approach based on the
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use of chlorine solutions.
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2. Material and Methods 4
ACCEPTED MANUSCRIPT 2.1 Natural antimicrobials and microbial strain
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Thyme EO (Thymus vulgaris) was obtained from Flora s.r.l. (Pisa, Italy). The potential
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biocontrol agent L. lactis CBM21, nisin Z producer, belongs to the collection of the
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Department of Biotechnology of Verona University. This strain, before each trial was
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preliminarily grown in M17 broth (Oxoid Ltd., UK) and incubated aerobically at 30 °C for 24
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h.
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2.2 Preparation of lamb’s lettuce products
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Lamb’s lettuce (Valerianella locusta sp.) was supplied by the SIPO s.r.l (Santarcangelo di
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Romagna, Rimini, Italy). and has been stored and prepared at pilot scale in the same company
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and with the same procedures of industrial production. The lamb’s lettuce process is reported
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in Figure 1.
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Four different washing solutions were prepared with running water:
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a) Chlorine 120 mg/L (Control)
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b) Thyme EO 250 mg/L
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c) Biocontrol agent L. lactis CBM21 (Inoculated at a level of 6 log CFU/ml)
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d) Thyme EO 125 mg/L + L. lactis CBM21 (Inoculated at a level of 6 log CFU/ml)
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The inoculation level of L. lactis and the concentrations of thyme EO were selected on the
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basis of previous results obtained at laboratory scale (Siroli et al., 2016, 2015a, 2015b).
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Thyme EO was conveyed to the washing solution through 1% ethanol, while the biocontrol
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agent was grown in M17 broth (Oxoid Ltd., UK) incubated aerobically at 30 °C for 24 h then
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the cells were harvested by centrifugation at 8,000/g for 15 min in a J-25 Beckman Coulter
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centrifuge at room temperature, re-suspended in saline solution and inoculated in the washing
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solution. The pH of the washing solutions, before the treatments, resulted 7.04 in case of
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addition of chlorine 120 mg/L, 6.85 and 6.76 in case of addition of thyme EO 250 mg/L and
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biocontrol agent.
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Prepared lettuce was immersed in an industrial washing tank and agitated in the appropriate
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washing solution at 8 °C for 1 min (10 kg of lettuce in 180 L of washing solution). After the
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treatment, the lettuce was spin dried and packaged into PP trays and closed by a plastic PP
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bags. Each package was filled with 80 g of lettuce. Samples were stored at 6 °C until the end
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of shelf-life (12 days). Finally, we obtained around 85 lettuce bags for each condition.
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2.3 Microbiological analyses
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During storage, the cell loads of mesophylic aerobic bacteria, lactic acid bacteria and yeasts,
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were evaluated by plate counting on Plate Count Agar (PCA, Oxoid Ltd., UK), Man Rogosa
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and Sharpe Agar (MRS, Oxoid Ltd., UK) supplemented with cycloheximide (0.05%) (Sigma-
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Aldrich) and Sabouraud Dextrose Agar (SAB, Oxoid Ltd., UK), respectively. The inoculated
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L. lactis strain was evaluated by plate counting on M17 agar (Oxoid Ltd., UK). To confirm
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the presence of L. lactis, bacterial colonies from M17 plates were isolated and subsequently
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identified by sequencing the 16S rRNA region according to the protocol described by De
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Angelis et al. (2006). After homogenization, samples were serially diluted in physiological
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solution (10 g of sample diluted into 90 ml of physiological water (0.9% (w/v) NaCl). Plates
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were incubated at 30 °C for 48h in case of yeasts and mesophilic aerobic bacteria, at 37 °C for
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24-48h for all the other species. For the detection of the natural occurring Listeria
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monocytogenes, Salmonella spp. and Escherichia coli the methods reported by Siroli et al.
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(2015b) were used. The microbiological analyses were performed immediately after
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treatments and after 3, 5, 7, 10, and 12 days of storage. All the microbiological analyses were
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performed in triplicate on three independent samples.
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2.4 Color, volatile molecule profiles analyses and panel test 6
ACCEPTED MANUSCRIPT Lamb’s lettuce color was determined using a color-spectrophotometer mod. Colorflex
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(Hunterlab, USA). Color was measured using the CIELab scale and Illuminant D65. The
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instrument was calibrated with a white tile (L*98.03, a* - 0.23, b* 2,05). Results were
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expressed as L* (luminosity), a* (red-green index) and b* (yellow-blue index). The color
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analysis was performed immediately after treatments and after 3, 5, 7, 10 and 12 days of
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storage. Forty-five readings were obtained from three independent samples for each lamb’s
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lettuce condition.
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Lettuce bags, containing 80 g of product were used for headspace volatile compound analysis
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by GC/MS-SPME technique. Samples were analyzed immediately after treatments and after 5
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and 10 days of storage. The samples were conditioned for 30 min at 37 °C and each sample
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was analyzed by following the method reported by Siroli et al. (2014). Compounds were
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identified using the Agilent Hewlett–Packard NIST 98 mass spectral database. All the
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analyses were performed in triplicate from three independent samples.
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A panel test was performed immediately after treatments and after 3 and 5 days of storage.
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Thirty untrained consumers made up the panel, and the quality parameters evaluated were
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odor, color, browning, texture, flavor, acceptability and overall quality. Each of the qualitative
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parameters considered was evaluated by consumers on a scale from 1.0 (very poor) to 5.0
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(very good). Consumers evaluated four different conditions: control lettuce, lettuce washed
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with thyme EO (250 mg/L), lettuce washed with the biocontrol agent and lettuce washed with
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both thyme EO (125 mg/L) and the biocontrol agent.
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2.5 Genomic DNA extraction pyrosequencing and quantitative analysis of the microbiome
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composition
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DNA was adjusted to 10 ng/µL concentration and used as template for 16S Metagenomic 454
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Sequencing Analyses. 7
ACCEPTED MANUSCRIPT Total genomic DNA was extracted directly from lamb’s lettuce kit Mo-bio (Cambrex
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Biosciences, Bergamo, Italy). DNA from the entire set of 12 samples, was employed for the
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V1-V3 region of 16S ribosomal RNA (rRNA) locus amplification for the pyrosequencing step
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on a 454-Junior Genome Sequencer (Roche 454 Life Sciences, Branford, USA) according to
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the pipeline described by Ercolini et al. (2012).
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2.6 Statistics and bioinformatics
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For each sample, the microbiological and volatile data were the mean of three different
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measurements. Regarding microbiological, color and panel test data, statistical analysis was
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performed using Statistica software (version 8.0; StatSoft, Tulsa, Oklahoma, USA). Means
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were compared using ANOVA followed by LSD test at p<0.05 level to mark differences
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between treatments with same storage times. The volatile molecule profiles were analyzed
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using ANOVA followed by a principal component analysis (PCA) performed using Statistica
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software (version 8.0; StatSoft, Tulsa, Oklahoma, USA) to obtain a visual overview of
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volatile molecule profile analyses.
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The bioinformatic workflow applied in this study derives from the pipeline elaborated by
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Kuczynsky et al. (2012).
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At first result filtering was performed with 454 Amplicon signal processing; and sequences
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were analyzed by using Quantitative Insights into Microbial Ecology (QIIME 1.8.0) software.
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To detect Operational Taxonomic Units (OTUs), row reads were demultiplexed and reads
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with an average quality score lower than 25, shorter than 300 bp and with an ambiguous base
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calling were excluded from the analysis to reduce the overall error rates. Sequences that
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overcame the quality filter were denoised (Reeder and Knight, 2010) and singletons were 72
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excluded. The OTUs defined by a 97% of similarity were collected by using the uclust
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method (Edgar, 2010) and the representative sequences were submitted to PyNAST, for the
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sequence alignment the employed method was UCLUST and the database for OTUs matching
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was greengenes (v 13.8). Finally, an OTU table was created with the absolute abundance of
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each OTU across the samples.
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The α and β diversity and the Kruskal wallis test were performed using QIIME software,
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using “alpha_rarefaction.py, beta_diversity_through_py, group_significance.py” scripts.
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3. Results
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3.1 Effect of the Lactococcus lactis CBM21 and thyme essential oil on the microbiological
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quality of lamb’s lettuce
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In table 1 the cell loads are reported of Lactococcus lactis CBM21 in relation to the washing
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solution used. Lamb’s lettuce cell loads immediately after the treatment were 5.1 and 5.4 log
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CFU/g in the samples treated with the biocontrol agent alone and in combination with thyme
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EO, respectively. The biocontrol agent cell load decreased during storage by about 0.7-1.3 log
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CFU/g after 5 days of storage at 6 °C irrespective of the presence of thyme EO in the washing
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solution (Table 1). In the control samples and in those treated only with thyme EO, LAB cell
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loads were lower than 3.0 log CFU/g also after 12 days of storage at 6 °C.
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As shown in Table 2, no significant differences were evidenced in the total aerobic
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mesophilic cell loads among the samples for the whole period of storage at 6 °C. In fact, after
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12 days of storage the load of the total aerobic mesophilic in the samples ranged between 7.2
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and 7.6 log CFU/g, irrespective of the washing solution adopted. Moreover, no significant
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differences were found for yeast and total coliform cell loads in relation to the washing
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solution adopted, whose loads resulted below 3.0 and 2.5 log CFU/g respectively, during the
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whole period of storage (data not shown). In any case, these microbial groups, due to their
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low cell loads were unable to play a key role in the spoilage of the product irrespective of the
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washing procedures applied. L. monocytogenes and Salmonella spp. were absent in 25 g of
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products, while E. coli was always under the detection limits (1.0 log CFU/g) after 12 days of
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storage irrespective of the washing solution used (data not shown).
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3.2 Microbiome characterization of lamb’s lettuce microbiota
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A pyrosequencing analysis was performed on the samples of lamb’s lettuce in relation to
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washing solutions and time of storage (0, 5, 10 days). A total of 34821 sequencing reads were
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obtained from all the analyzed samples. To characterize the ecological species diversity in
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lamb’s lettuce products, Shannon index was calculated (Li et al., 2012). and performed by
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grouping samples based on storage time. An increase of Shannon index was observed in T5
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set, respect T0 and T10 groups (Figure 2A), while assembling samples by washing solution,
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the alpha diversity index was higher respect to controls (Figure 2B). The β-diversity data
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obtained, representing the comparison of microbial communities based on their dissimilar
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composition, analyzed by Principal Coordinate Analysis (PCoA), provided a clear separation
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among the samples based on storage time (Figure 3), while a significant clustering of the
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samples based on washing solution was not highlighted (data not shown).
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The analyses of the significance of the Operational Taxonomic Units (OTU) identified
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highlighted that the main represented phyla in the complete dataset were Proteobacteria,
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Bacteroidetes, Actinobacteria and Firmicutes. In particular, Proteobacteria, and Firmicutes
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showed a decrease during storage time in all samples, while Bacteroidetes showed an opposite
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trend (p<0.005) (Figure 4A). Those washed with L. lactis+Thyme EO showed a more
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different profile with higher level of Proteobacteria and Actinobacteria and lower level of
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Bacteroidetes compared to the other samples, although the differences on OTU distribution
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were not statistically significant (Figure 4B). The analysis at genus level evidenced a different
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profile among the samples in relation to storage time (Figure 4C) and to the washing solution
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adopted (Figure 4D). During the storage period an increase of Flavobacterium spp.,
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Oxalobacteriaceae, Janthinobacterium spp. and Pedobacter spp. was observed. By contrast a
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decrease of Sphingomonas spp., Comamonadaceae, Methylophilaceae and Caulobacteraceae
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was detected. The analysis of data by clustering the samples based on the washing solution
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time, samples washed with alternative solutions were characterized by a higher presence of
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Pedobacter spp., Chriseobacterium spp., Rhodococcus spp. and Comamonadaceae compared
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to control samples. By contrast, samples washed with chlorine showed a higher presence of
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Oxalobacteraceae, Sphingomonas spp., and Methylophilaceae compared to samples washed
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with the alternative solutions.
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As expected the pyrosequencing analysis evidenced the presence of Lactococcus lactis only in
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the samples washed with the solution containing the selected biocontrol agent. However, in
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the samples washed only with the biocontrol agent the average relative abundance of
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Lactococcus lactis was around twice than in the samples washed with the solution also
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containing thyme (Figure 4D).
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3.3 Effects of natural antimicrobials on volatile molecule profiles of lamb’s lettuce
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The biocontrol agent and/or thyme EO affected the lamb’s lettuce volatile molecule profiles
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leading to the identification of 98 molecules belonging to different chemical classes such as
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alcohols, ethers, esters, aldehydes, ketones and hydrocarbons (data not shown). Specific
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volatile fingerprintings, in relation to the washing solution, were obtained. The washing with
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thyme EO was reflected in the presence of γ-terpinene, p-cymene, β-phellandrene, limonene,
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terpinolene, camphene, caryophyllene, thymol and carvacrol, that represent the characteristic
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molecules of thyme EO. During the storage period, the increase in molecules obtained by the
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transformation of the main component of thyme EO (acetic acid methyl ester, ethyl acetate
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and thymol methyl ether) was evident. The addition of the biocontrol agent determined
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significant changes in molecules classes, in particular an increase of aldehydes (hexanal, 2-
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hexanal and benzaldehyde) and alcohols (1-butanol, 3-methyl, phenyl ethyl alcohol).
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In order to better show the effects of the washing solution on the volatile molecule profiles,
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the GC-MS-SPME data were subjected to a Principal Component Analysis (PCA) excluding
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ACCEPTED MANUSCRIPT from the analyses the molecules from thyme EO and those deriving from their further
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transformation by lettuce tissues or microorganisms according to literature data (Siroli et al.,
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2015a). The results are shown in Figures 5A and B. The PCA analysis showed that the control
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samples were well separated from the other samples, although the clustering was influenced
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by the storage time. In fact, the control samples immediately after packaging and those after 5
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days of storage constituted a cluster (cluster 1) well separated along the main component 1,
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which explains about 28.53% of the variance, from control samples stored for 10 days at 6 ° C
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(Cluster 2). Cluster 1 was characterized mainly by hexanal, 2-hexanal, benzene acetaldehyde,
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α-pinene, β-pinene and methylene chloride while cluster 2 by molecules such as 6-Methyl-5-
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hepten-2-ol, 6-Methyl-1-heptene, dimethyl sulphide and 1-hexanol. Cluster 2 was separated
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from the samples subjected to the alternative washing solution and stored for 10 days along
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the main component 2 explaining the 20.23% of the total variance (cluster 3). These differed
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from the other samples washed with the biocontrol agent and/or oil thyme EO, which grouped
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in two other clusters separated along the main component 1 in relation to the storage time
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(cluster 4 and 5). Cluster 3 was characterized by the molecules acetic acid methyl ester,
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benzyl alcohol and ethyl acetate while the grouping of cluster 4 was mainly affected by
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butanol, 1-propanol, hexanal and 2-hexanal.
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Moreover, a sensory evaluation through panel tests was performed immediately and after 3
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and 5 days of storage (Figure 6). The panel test immediately after packaging showed no
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significant differences regarding color and texture parameters. As expected, the odor of
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lettuce resulted significantly higher for controls and samples washed with the biocontrol
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agent. Contrarily, with regard to flavor intensity, acceptability and overall quality samples
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washed with the combination of innovative treatments resulted significantly higher than the
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controls. After 3 days of storage, the samples washed with thyme EO alone or in combination
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with the biocontrol agent showed a significantly higher overall acceptability and color
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intensity compared to the others. The samples washed with the combination of the innovative
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the samples washed with thyme EO showed the highest scores of browning. Sensory analysis
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carried out on the fifth day of storage confirmed the positive effects on flavor, acceptability,
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and odor intensity of the alternative washing solutions. The combination thyme EO and
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biocontrol culture showed the highest scores regarding acceptability, flavor, texture, and the
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overall quality. However, the color analysis performed immediately after packaging and after
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3, 5, 7, 10 and 12 days of storage at 6 °C did not show significant differences among the
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samples (data not shown).
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4. Discussion
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The microbiological data and the main quality parameters showed no significant difference
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regarding the shelf life and safety of the product in relation to the washing solution adopted.
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The composition of the microbial population was similar in terms of species recorded to that
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reported by literature independently on the washing solution adopted. In fact, the prevalence
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of gram- negative microorganisms was demonstrated in all the samples analyzed over storage
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with Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria as predominant phyla.
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These phyla are also reported as the dominant ones in minimally processed vegetables by
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Rastogi et al. (2012). Moreover, these results are in agreement with those obtained in
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laboratory trials by Siroli et al. (2015a), that showed the same reduction of the naturally
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occurring microbial population when lamb’s lettuce was washed either with chlorine (120
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mg/L) or thyme EO.
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However, significant differences in relation to the washing solution adopted were pointed out
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by volatile molecule profiles and sensory analyses performed during storage. In fact, the
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GC/MS SPME showed that the components of thyme EOs were transformed in alcohols by
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microorganisms and plant tissues. The transformation of hexanal, 2-hexanal, neral, geranial
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and other six atom aliphatic aldehydes by the plant tissues and yeasts to alcohols (mainly 1-
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ACCEPTED MANUSCRIPT hexanol, 3-hexen-1-ol, nerol and geraniol) endowed with lower antimicrobial properties
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compared to their precursors is well documented (Belletti et al., 2004; Patrignani et al., 2013;
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Siroli et al., 2014). The PCA analyses of the volatile molecule data showed the grouping of
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the samples mainly based on the time of storage and washing solution adopted. In fact, the
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samples treated with the innovative washing solutions resulted significantly different over
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time compared to the control samples, demonstrating completely different spoilage patterns.
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On the other hand, the results of pyrosequencing showed significant differences of microbiota
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composition in relation to the washing solution. In fact, the analysis of the microbiota of the
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different lettuce samples highlighted the ability of both the biocontrol culture as well as thyme
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EO to significantly change the relative proportions between the different spoilage species. In
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the products washed with the Lactococcus lactis CBM21, the incidence of high potential
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spoilage microorganisms, such as those belonging to the family of Pseudomonadaceae, was
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reduced. Even thyme EO, including when used alone, had a significant effect on product
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microbiota. In fact, this natural antimicrobial has a broad spectrum of action, the effectiveness
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of which however varies considerably in relation to the species, composition and chemical
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and physical composition of the system conditions (Bagamboula et al., 2004; Gutierrez et al.,
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2008; Ivanovic et al., 2012).
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Samples washed with the innovative washing solutions after 10 days of storage showed a
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significantly higher presence of esters such as ethyl acetate and acetic acid methyl ester,
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generally associated with a positive fruity smell, and some alcohols such as benzyl alcohol
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and phenyl ethyl alcohol due to the reduction of spoilage microbial groups such as
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Pseudomonadaceae and Methylophilaceae that are more associated with the production of
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sulphur compounds and volatile amines.
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The differences found in the microbial population composition of the products in relation to
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the washing conditions adopted can explain, in addition to the differences of the volatile
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molecule profiles, the different sensory evaluation of the samples. The sensory analysis
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solutions immediately after packaging. By the fifth day of storage the samples obtained by
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washing with thyme EO in combination with the biocontrol agent showed a major overall
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quality compared to all the other treatments. In fact, it is well documented that differences in
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microbial population of fresh vegetables can result in different organoleptic features (Di
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Cagno et al., 2009; Gutierrez et al., 2009; Siroli et al., 2015b). In fact, Gutierrez et al. (2009)
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identified volatile spoilage markers in minimally processed lettuce and carrots washed with
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chlorine or oregano EO. In particular, they showed that an increase in Enterobacteria and
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Pseudomonas was associated with a loss of aroma, texture and color. Furthermore, the
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competition of the biocontrol agents for the space and the nutrients and their production of
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antimicrobials including nisin, are reported as the main mechanisms through which the
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biocontrol agents inhibit natural occurring microorganisms of products modifying their
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spoilage patterns in relation to the sensitiveness of the microorganisms present (Siroli et al.,
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2016). In addition, it is widely reported that natural antimicrobials, such as thyme EO, have a
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strong influence on the microbial metabolism and volatile molecule profile, and consequently
377
on the organoleptic characteristics of the product (Gutsche et al., 2012; Picone et al., 2013;
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Siroli et al., 2015d). No negative effects on color were observed within 12 days of storage for
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samples washed in the presence of the selected biocontrol agent. These results, in
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disagreement with those of other authors (Siroli et al., 2015b; Trias et al., 2008), highlighted
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the importance of the selection of the suitable biocontrol agents in relation to the product and
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process considered and the expected shelf-life.
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4. Conclusions
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This work highlighted a marked effect of different washing solutions on the microbial
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community of lettuce resulting in differences in the volatile profiles and the sensory 15
ACCEPTED MANUSCRIPT characteristics of minimally processed lettuce. In fact, the innovative washing solutions
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affected the quality of lettuce through the shift of microbiota towards genera and species with
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lower potential in decreasing the sensory properties of the product. Moreover, this work,
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conducted in an industrial environment, indicated that the protocol adopted can be easily
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upscaled to answer innovation needs of small to medium-sized industries. However, the
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definitive industrialization of the proposed biotechnological solutions will only happen after
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further studies have been performed on the effects on microbial interactions in the product
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and on the mechanisms of action of the EO chosen against spoilage and pathogenic
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microorganisms.
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Figure caption
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Figure 1. Working protocol used to prepare lamb’s lettuce; the addition of lactic acid bacteria
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and/or thyme EO was performed during the washing phase; samples washed with chlorine
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(120 mg/L) represented the controls.
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Figure 2A,B. Shannon-Weaver index box plot. α-diversity analysis for sample grouped by
402
time of storage (a) and treatment (b). The table showed interquartile (25-75%) (qi) range,
403
median values, and minimum (min) maximum (max) Shannon index value.
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Figure 3. Metagenomics β-diversity plots. PCs plots of the diversity in the microbial
405
communities from all samples stratified by time of storage.
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Figure 4 A-D. Graphical representation of OTUs distribution at phylum (a,b) and genus (c,d)
407
level. In panel a,c the samples were grouped by time of storage, and in panel b,d samples
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were grouped by treatment. The OTUs highlighted in the legend were statistically significant
409
(p≤0.05).
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different times of storage) and loadings (7B molecules detected by GC/MS/SPME) on the
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factor-plane (1x2). PC1 and PC2 explained 28.75% and 20.20% of the total variance
413
respectively.
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Figure 6. Sensory data of lamb’s lettuce, in relation to the washing conditions used,
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immediately after treatments (A) and after 3 days (B) and 5 days (C) of storage at 6 °C.
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Acknowledgment
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This experimental research was supported by the national project AGER-STAY FRESH 2010
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ACCEPTED MANUSCRIPT Table 1. L. lactis CBM21 count on lamb’s lettuce in the presence or not of thyme EO day 0
day 3
day 5
day 7
day 10
day 12
L. lactis CBM211
5.1±0.23a
5.0±0.24a
4.4±0.60a
4.5±0.21a
4.1±0.25a
3.7±0.33a
Thyme eo + L. lactis CBM212
5.4±0.22a
5.1±0.16a
4.1±0.29a
4.1±0.21a
3.7±0.13a
3.5±.28a
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Counts are expressed in Log cfu/g (+/− standard deviation). At the same time of storage, means followed by different letters are significantly different (p<0.05) LAB counts in samples washed with chlorine and thyme EO ranged between 2.4 and 3.0 log cfu/g during the whole period of storage Samples washed with the biocontrol agent L. lactis CBM21 (Inoculated at a level of 6 log cfu/mL in the washing solution)
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Samples washed with thyme essential oil 125 mg/L + L. lactis CBM21 (Inoculated at a level of 6 log cfu/mL in the washing solution)
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ACCEPTED MANUSCRIPT Table 2. Mesophilic aerobic bacteria count on lamb’s lettuce treated with thyme EO, the biocontrol agent, their combination or chlorine day 0 Chlorine
1
6.33±0.33
day 3 a
6.89±0.35
day 5 a
7.06±0.37
day 7 a
7.27±0.33
day 10 a
7.45±0.31
day 12 a
7.32±0.29a
6.83±0.23a 7.29±0.13a 7.45±0.13a 7.70±0.25a 7.86±0.32a 7.20±0.23a
Thyme eo3
6.46±0.31a 7.17±0.37a 7.16±0.35a 7.50±0.24a 7.72±0.18a 7.46±0.21a
Thyme eo + L. lactis CBM214
6.43±0.28a 7.19±0.23a 7.26±0.20a 7.61±0.16a 7.76±0.19a 7.58±0.22a
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Samples washed with the biocontrol agent L. lactis CBM21 (Inoculated at a level of 6 log cfu/mL in the washing solution)
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Samples washed with thyme essential oil 250 mg/L
4
Samples washed with thyme essential oil 125 mg/L + L. lactis CBM21 (Inoculated at a level of 6 log cfu/mL in the washing solution)
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Washing 1 min with running water in automatic industrial washing tank; Water T 8 °C; lamb’s lettuce/water 5 Kg/180 L Different washing condition: a) 120 mg/L chlorine (Control) b) Thyme E 250 mg/L c) Biocontrol agent L. lactis CBM21 (6 log CFU/mL) d) Thyme essential oil 125 mg/L + L. lactis CBM21 (6 log CFU/mL)
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Draining centrifuging lamb’s lettuce 2 times for 30 seconds in an industrial centrifuge
Packing in PP trays and closed by PP bags: 80 g of product for each bag
Storage, 6 °C
ACCEPTED MANUSCRIPT 7 Shannon-Weaver index
6 5 4 3
1 0
T0
T5
T10
qi
4.860942216
5.446432836
4.926446819
min
3.959366506
5.298256693
4.295143764
mediana
5.594608979
5.146433337
5.49546136
5.981543668
5.398179036
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5.304873591
5.788076324
5.216533887
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TIM eo Thyme
4.043310821
5.189112752
5.229862073
5.325723432
min
3.959366506
5.13688117
5.161467453
5.155985504
mediana 4.127255135
5.241344334
5.298256693
5.49546136
max
4.295143764
5.981543668
5.398179036
5.594608979
q3
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5.611444001
5.348217864
5.545035169
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ACCEPTED MANUSCRIPT Innovative solutions as an alternative to chlorine to guarantee lamb’s lettuce shelf-life
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Samples washed with alternative solutions showed a different volatilome than controls
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Washing solution affect the quality of lamb’s lettuce microbiota
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The use of innovative washing solutions affect the composition of spoilage microflora
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The sensory analyses highlight the preference by the panelists for innovative products
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