Bioprotection in Vegetable Foods Antonio Gálvez Prof. of Microbiology Dept.of Health Sciences University of Jaen, Spain agalvez@ujaen.es
Microorganisms in vegetable foods Cross Raw materials Processing contamination Pathogens Toxin FOOD Spoilage producers Beneficial effects Antimicrobial resistance
ANTIMICROBIAL SUBSTANCES FROM LAB ANTIBACTERIAL ANTIFUNGAL LAB Organic acids Organic acids Carboxylic acids Tetramic acid Hydroxy-fatty acids Aldehydes Aldehydes Miscellaneous Cyclic dipeptides Bacteriocins Synergistic action Antifungal peptides
ANTIFUNGAL LAB FROM VEGETABLE MATERIALS AND FOODS Lactococcus Lactobacillus L. lactis Lb. acidophilus Lb. amylovorus Leuconostoc Lb. brevis Ln. citreum Lb. coryniformis Ln. mesenteroides Lb. hammesii Lb. paracollinoides Pediococcus Lb. pentosus P. acidilactici Lb. plantarum P. pentosaceous Lb. reuteri Lb. sakei Weissella Lb. sanfranciscensis W. cibaria W. confusa W. paramesenteroides
ANTIFUNGAL LAB AS BIOPROTECTIVE CULTURES Inhibition of spoilage and mycotoxigenic fungi Fresh fruits and vegetables Treated Fruit juices Sourdoughs and breads Control Rice cakes Fermented beverages Prevention of Korean rice wine spoilage (Ryu et al. http://dx.doi.org/10.1016/j.fm.2014.01.011)
POTENTIAL OF BACTERIOCINOGENIC LAB IN THE PRESERVATION OF VEGETABLES AND FRUITS Bioprotective cultures for fruits and vegetables • Increased safety of minimally processed vegetables due to the inhibition of A. hydrophila , Staph. aureus , E. coli and L. monocytogenes • Growth inhibition of E. coli and S. typhimurium and inactivation of L. monocytogenes on wounded Golden Delicious apples and Iceberg lettuce cut leaves • Increased safety and shelf life of lamb´s lettuce and apple cubes • Improving the safety and shelf life of fruits and vegetables by nisin- producing strains Starter cultures in vegetable fermentations • Avoiding stuck fermentations • Acceleration of fermentation • To improve the consistency and quality of the final products • Protection from undesirable spoilage and pathogenic microorganisms
Fermented table olives as sources of strains producing antimicrobial substances Plantaricin genes detected in L. pentosus strains plnA plnD plnJ plnNC8 plnW Antimicrobial activity of LAB from table olives (Abriouel et al., 2012 (doi: 10.1016/j.fm.2012.07.006)
POTENTIAL OF LAB BACTERIOCINS IN PRESERVATION OF FRESH VEGETABLES AND FRUITS Nisin •Reducing the surface bacterial load on whole melon and transfer of pathogens from the surface of melons to freshly cut pieces •Reducing the load of pathogens on the surface of minimally processed mangoes (nisin film) Pediocin •Preserving minimally processed papaya (alginate coatings) Pentocin MQ1 •Extension of the shelf life of bananas Enterocin 416K1 •Growth inhibition of L. monocytogenes on apples and grapes Prevention of banana spoilage (Wayah & Philip, https://doi.org/10.3389/fmicb.2018.00564)
Enterocin AS-48 Wide Circular bactericidal bacteriocin spectrum • Bacillus • Alicyclobacillus • Clostridium • Listeria monocytogenes • Staphylococcus aureus • Lactobacillus • Pediococcus • Escherichia coli* • Salmonella*
ENTEROCIN AS-48 FOR BIOPROTECTION OF VEGETABLES Fresh vegetables Fruits AS-48 Soups, purees Canned vegetables and sauces Synergy with other Fermented Cereals hurdles vegetables Chemical preservatives Beverages Bakery products Essential oils Tª, HIPEF, HHP
• Applications in fermented foods •Manzanilla Aloreña table olives •Short fermentation during cold storage (ca. one week) •Packed in brine with condiments •Do not withstand pasteurization •Short shelf life due to refermentation Bacteriocins No refermentation at (nisin, AS-48) room temperature HHP Reduced salt content Essential oils
• Fermented beverages Inactivation of spoilage bacteria in Bacillus licheniformis beer (except hop-resistant strains) 6 Log CFU/ml 4ºC Inactivation of spoilage bacteria in 4 white wine (but not in red wine) 2 Inactivation of spoilage lactic acid 0 0 5 10 15 bacteria and endospore formers in Time (days) apple ciders Final bacteriocin concentrations were 2 ( ▲ ), 4 ( ● ), and 10 µg/ml ( □ ). Controls ( Ο ). Endospores of B. licheniformis were more resistant to AS-48. Endospore inactivation improved in combination with mild heat treatment
• Fruit juices Alicyclobacillus acidoterrestris Salmonella enterica A HiPEF 40ºC B 1000 4.5 4.25 775 Treatment time (µs) 4 3.75 550 3.5 3.25 325 Vegetative cells as well as endospores of A. 3 acidoterrestris are highly sensitive to AS-48 (2.5 2.75 µ g/ml at 4 and 15ºC) in commercial and fresh- 100 30 37.5 45 52.5 60 made juices AS-48 (µg/ml) Inactivation of S. enterica improved in AS-48 (30-60 µ g/ml) combination with HiPEF
Control of pathogenic and spoilage bacteria in fresh produce Control AS-48 AS-48 + Acetic acid (0.5%) L. monocytogenes AS-48 + Citric acid (0.5%) AS-48 + Propionate (0.1%) AS-48 + Propionate (0.5%) B. cereus AS-48 + Sorbate (0.1%) AS-48 + Lactic acid (0.1%) AS-48 + Lactic acid (0.5%) B. weinhestephanensis AS-48 + Lactate (0.1%) AS-48 + Lactate (0.5%) AS-48 + Nitrite (50 ppm) Enterocin AS-48 reduced AS-48 + Nitrite (100 ppm) Treatment AS-48 + Nitrate (50 ppm) viable cell counts in fresh AS-48 + Nitrate (100 ppm) AS-48 + TSP (1.5%) produce ( sprouts, sliced AS-48 + TSTMP (0.1%) AS-48 + TSTMP (0.5%) fruits ). AS-48 + Thiosulfate (0.01 N) AS-48 + Permanganate (25 ppm) AS-48 + Propy-p-HB (0.1%) AS-48 + Propyl-p-HB (0.5%) Strong synergistic activity with AS-48 + PHBME (0.1%) AS-48 + PHBME (0.5%) several chemical preservatives, AS-48 + Peracetic acid (80 ppm) AS-48 + HDP (0.1%) essential oils, and phenolic AS-48 + Hypochlorite (25 ppm) AS-48 + Hypochlorite (50 ppm) compounds. AS-48 + Hypochlorite (100 ppm) 0 1 2 3 4 5 6 Log CFU/ml
Inactivation of Gram-negative bacteria in sprouts combination with other antimicrobials Control • Salmonella enterica AS-48 • Escherichia coli Lactic acid (1.5%) Lactic acid + AS-48 • Aeromonas hydrophila Trisodium P (1.5%) • Shigella sonnei Trisodium P + AS-48 • Enterobacter aerogenes PolyP (0.1%) PolyP + AS-48 • Yersinia enterocolitica Peracetic acid (80 ppm) • Pseudomonas fluorescens Peracetic acid + AS-48 HDP (0.5%) HDP + AS-48 0 1 2 3 4 5 6
Inactivation of Listeria monocytogenes in apple cubes Activated coatings 6 •Chitosan Pectin Log10 CFU/g •Caseinate 4 •Alginate 2 •K-carrageenate 0 0 1 3 7 •Xanthan gum Time (days) Pectin 6 •Pectin + EDTA •Starch Log10 CFU/g 4 •Carboxymethyl cellulose 2 •Methyl cellulose 0 0 1 2 3 4 5 6 7 Time (days)
Effect of pectin-bacteriocin coating in chopped parsley 100% Relative abundance (%) 100% Relative abundance (%) 90% 90% 80% 80% 70% 70% 60% 60% 50% 50% 40% 40% 30% 30% 20% 10% 20% 0% 10% C0 C3 C5 C10 PB0 PB3 PB5 PB10 0% Pseudomonas Gamma Proteobacterium Rheinheimera Flavobacterium Acinetobacter Shigella Proteobacteria Bacteroidetes Stenotrophomonas Salmonella Firmicutes Actinobacteria Unc. low G+C G+ Paenibacillus Sphingobacterium Erwinia Pantoea Rest Viable counts (Log 10 CFU/g ± SD) at different storage times (days) Treatment 0 3 7 10 Control 6.3 ± 0.05 6.8 ± 0.09 7.8 ± 0.09 9.4 ± 0.06 PB 2.6 ± 0.07 2.3 ± 0.42 3.4 ± 0.24 4.0 ± 0.52 Grande et al., 2017. doi: 10.1016/j.foodres.2017.05.011.
Effect on bacterial diversity of cherimoya pulp 100 90 80 Relative abundance (%) 70 60 50 40 30 20 10 0 Pantoea vagans Pantoea agglomerans Pantoea ananatis Enterobacter aerogenes Enterobacter asburiae Enterobacter kobei Escherichia fergusonii Leclercia adecarboxylata Raoultella terrigena Serratia fonticola Serratia liquefaciens Serratia plymuthica Serratia proteamaculans Erwinia aphidicola Erwinia billingiae Erwinia persicina Yersinia ruckeri Cronobacter turicensis Acinetobacter johnsonii Pseudomonas psychrophila Pseudomonas putida Pseudomonas fragi Stenotrophomonas rhizophila Enterococcus casseliflavus Enterococcus gallinarum Leuconostoc kimchii Leuconostoc mesenteroides Leuconostoc sp. C2 Bacillus firmus Bacillus stratosphericus Bacillus plakortidis Bacillus nealsonii Bacillus pumilus Bacillus thuringiensis Micrococcus luteus Perez Pulido et al.2015. doi: 10.1016/j.ijfoodmicro.2014.11.033.
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