"Natural antimicrobials to increase the shelf-life and safety of. minimally processed vegetables"

Transcription

1 "Natural antimicrobials to increase the shelf-life and safety of minimally processed vegetables" Francesca Patrignani, Lorenzo Siroli, Rosalba Lanciotti University of Bologna, Department of Agricultural and Food Sciences

2 Project coordinator: Prof. M. Cristina Nicoli

3 Project coordinator for UNIBO: Prof. Rosalba Lanciotti

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5 UNIBO OBJECTIVES WITHIN STAY FRESH PROJECT. Evaluate the potential of some essential oils and their components to improve the safety and the shelf life of lamb s lettuce (Valerianella locusta) 2. Isolate, identify, characterize for their technological features LAB isolated from fresh-cut lettuce in order to select potential bio-control agents 3. Evaluate the effect of the selected essential oils, in combination with selected LAB, on the shelf-life and safety as well as on the organoleptic properties of minimally processed Lamb s lettuce (Valerianella locusta) 4. Evaluate the scaling up of the optimized Laboratory conditions

6 MINIMALLY PROCESSED VEGETABLES Products that contain live tissues or those that have had slight modifications to their freshness condition but have kept their quality and character similar to those of fresh produce Increasing interest is due to: Remarkable convenience of use Wide range of choice

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8 Main issues related to minimally processed fruit and vegetables: Vegetable tissue metabolism Growth of spoilage microorganisms Ineffectiveness and safety issues of traditional sanitizers Presence of pathogenic microorganisms Short shelf-life Food safety Refrigeration Modified atmosphere Searching for natural alternatives to ensure safety and quality : Essential oils Biocontrol agents UV radiation Vacuum impregnation

9 ESSENTIAL OILS Essential oils are complex mixtures of volatile compounds, characterized by a strong sensory impact and produced by several plants as secondary metabolites. Functions in nature Defense from microorganisms and insects Activities related to pollination and seed dispersal

10 Essential oils and their costituents Antimicrobial activity Wide activity spectrum GRAS according to the FDA Antioxidant activity Low cost Green consumerism In food sector: flavouring agents, antioxidant, antimicrobials Their use is dependent of : Organoleptic compatibility Standardization of composition in terms of active compounds Comprehension of the action mecchanisms Evaluation of the potential of application in food in relation with the product composition and storage

11 Location and action mecchanisms of essential oils and their components in bacterial cells

12 Use of Biocontrol agents Bioprotective cultures Lactic acid bacteria Competition Production of Antimicrobial Compounds Foods GRAS, Low impact on product features Organic Acids Bacteriocins Diacetyl Acetoin Reuterin

13 1. Natural antimicrobials to prolong the shelf-life of minimally processed lamb s lettuce

14 Essential oils and their mixture used on lamb s lettuce: Oregano oil (250ppm) Thyme oil (250ppm) Oregano oil/thyme oil (125/125ppm) Oregano oil/carvacrol (125/100ppm) Characterization of the composition and the antimicrobial activity of the essential oils employed Evaluation of the effects of the added antimicrobials on lamb s lettuce shelf life: Microbiological analyses Texture and colour analyses Volatile molecule sample profiles

15 Lamb s lettuce working protocol Valerianella locusta Laterr Purchase at a local retailer Washing 2 min with running water with 120 ppm chlorine Manual agitation, 8 C (Valerian:water, 1:10 w/v) Chlorine or EO Draining Taking valerian with the basket of a manual centrifugal Centrifugation (30 s) Manual handling of valerian placed in the drum of the manual centrifuge Centrifugation (30 s) Packaging in 30 µm-thick BOPP bags (valerian: headspace, 1:1) storage, 4-6 C

16 p-cymene 35% Carvacrol 28% Abundance TIC: ORIGANO4.D\data.ms GC-MS-SPME of Oregano oil limonene linalol Thymol 5% Time--> p-cymene 28% Carvacrol 16% Abundance ϒ-terpinene 15% GC-MS-SPME of Thyme oil limonene TIC: TIMOROSSO.D\data.ms Linalol 2% Thymol 11% Time-->

17 No significant differences of cell loads of mesophylic aerobic bacteria with respect to the controls were detected independently on the washing solution used The GC-MS-SPME data of the volatile molecule profiles analysed through a PCA showed the clustering of the samples mainly on the basis of the time of storage Fresh Withered No significantly differences of the colour parameters were detected during 8 days of storage. Contrarily a loss of turgidity was observed after 3 days in samples dipped in thyme-oregano and after 5 days in samples dipped in oregano and oregano-carvacrol;

18 So considering all the parameters, Thyme EO was the best candidates for the following trials

19 Optimization of lamb s lettuce production process 13 C

20 No significant differences of cell loads of mesophylic aerobic bacteria, yeats and LAB The products washed with thyme and oregano, similarly to chlorine, were able to with respect to the controls were detected independently on the washing solution maintain good color and turgidity attributes during over 10 days of storage at 6 C used; PCA analysis the electronic nose data grouped the samples on the basis of storage time showing that the volatile profiles of the products were quite unaffected by the added antimicrobials; C T0 C T3 C T8 C T10 Oregano T0 Oregano T3 Oregano T8 Oregano T10 Thyme T0 Thyme T3 Thyme T8 Thyme T10

21 2/3. Natural antimicrobials and lactic acid bacteria to improve safety and shelf-life of minimally processed lamb s lettuce

22 Isolation, identification and characterization in vitro of potential biocontrol agent ISOLATION OF LAB FROM COMMERCIAL PRODUCTS During and at the end of shelf-life 15 STRAINS ISOLATED FROM LAMB S LETTUCE

23 a Symbols: -No inhibition zone; +small inhibition zone (0.5 to 1 mm); ++medium inhibition zone (1 to 2 mm); +++large inhibition zone (>2mm)

24 Bio-control agent selected: Lb. plantarum V7B3 Essential oil selected: Thyme EO 250 ppm Same washing protocols employed in previous phases Inoculum of LAB (7 log cfu/g) and/or pathogenic microorganisms (L. monocytogenes and E. coli (3 log cfu/g)) and or natual antimicrobials In the washing or dipping step The Lactobacillus plantarum V7B3 was absolutely well adapted to the considered systems and it was not affected by the presence of the natural antimicrobials

25 Mesophilic counts A B log cfu/g Chlorine+pathogens V7B3+thyme EO+pathogens Thyme EO+pathogens pathogens log cfu/g Chlorine+pathogens V7B3+thyme EO+pathogens Thyme EO+pathogens pathogens time (d) time (d) E. coli L. monocytogenes

26 2/3. Use of natural antimicrobials combined with a nisin-producing Lactococcus lactis strain, to improve the safety and shelf-life of minimally processed lamb s lettuce

27 Screening for selection of a nisin Z-producing Lactococcus lactis strain Technological characterization and evaluation of the antimicrobial activity of L. lactis CBM21 Effects of L. lactis CBM21, in combination with natural antimicrobials, on microbiological quality of minimally processed lamb s lettuce

28 Bio-control agent selected: Lc. Lactis CBM 21 Thyme EO 250 ppm Same washing protocols employed in previous phases Inoculum of LAB (7 log cfu/g) and/or pathogenic microorganisms (L. monocytogenes and E. coli (3 log cfu/g)) and or natual antimicrobials In the washing step

29 Main Results The bio-control agent reduced its viability of 1 log cycle after 10 d of lamb s lettuce storage Major decrease of the mesophilic count with respect to the Lb. plantarum previously tested Same efficacy of chlorine against E.coli but increased against L. monocytogenes The combination of Thyme EO and Lc. Lactis CBM21 increased the shelf-life of lamb s lettuce (intotal 12 d) with respect to the control samples washed with chlorine (in total 7d) The presence of the bio-control agent and thyme EO did not affect sensorial quality of Lamb s lettuce

30 4. Scaling up

31 Results Mesophilic count Control L. Lactis Thyme Thyme+ L. lactis T0 log cfu/g Chlorine 120 ppm Lc. lactis CBM 21 Thyme 250 ppm Thyme+Lc. lactis CBM 21 time (d) T3 8.0 Yeasts 7.0 Chlorine 120 ppm T5 log cfu/g Lc. lactis CBM 21 Thyme 250 ppm 2.0 T time(d) T12

32 Chlorine 120 ppm T=0 Chlorine 120 ppm T=10 Thyme oil T=0 Thyme oil T=10 P R I N C I P A L Lactococcus lactis cbm 21 T=0 Lactococcus lactis cbm 21T=10 M I C R O B I O T A Lactococcus lactis cbm 21 + thyme oil T=0 Lactococcus lactis cbm 21 + thyme oil T=10

33 Odour 5 Final score 4 3 Colour T=0 d Control Lc lactis CBM 21 Thyme Acceptability Browing Theme+ Lc. lactis CBM 21 Flavour Texture Odour 5 4 T=5 d Final score 3 Colour Control Lc lactis CBM 21 Thyme Acceptability Browing Theme+ Lc. lactis CBM 21 Flavour Texture

34 Lab trials Conclusions The work of three years permitted to select Thyme essential oil as best candidate to prolong safety and shelf-life of lamb s lettuce Thyme oil had effects comparable to chlorine action without detrimental effects on colour and quality indexes Optimization of the process conditions and the inclusion of bio-control agents have increased the lamb s lettuce quality in terms of safety and shelf-life The combination of thyme oil and Lactococcus lactis CBM 21 determined the major decrease of L. monocytogenes when deliberately inoculated in lamb s lettuce Scaling up The trials performed confirmed the results obtained at lab level, confirming the chance of the passage of scale. Thyme oil and Lactococcus lactis CBM 21: same effects of chlorine in terms of safety but able to modify the microbiota with increased sensorial properties.

35 Food Microbiology Group Campus of Food Science, University of Bologna Thank you for your attention