Assessment of the Spoilage Microbiota during Refrigerated (4 °C) Vacuum-Packed Storage of Fresh Greek Anthotyros Whey Cheese without or with a Crude Enterocin A-B-P-Containing Extract

Abstract

Although fresh whey cheeses are prone to rapid deterioration, mainly by psychrotrophic Gram-negative bacteria and lactic acid bacteria (LAB), data on the specific spoilage species in traditional Greek whey cheeses are scarce. Therefore, this study quantified growth and characterized the primary spoilage bacteria in fresh Anthotyros whey cheeses stored at 4 °C in a vacuum for 40 days, without or with an added 5% (v/w) of an enterocin A-B-P crude extract (CEntE). Psychrotrophic Pseudomonas spp., Aeromonas spp., Hafnia spp. and Serratia spp. grew faster than LAB during early storage. However, LAB outgrew the Gram-negative bacteria and prevailed by mid to late storage in all cheese batches, causing a strong or milder batch-dependent natural acidification. Two major non-slime-producing and two minor biotypes of Leuconostoc-like bacteria, all identified as Leuconostoc mesenteroides by 16S rRNA sequencing, dominated the LAB association (76.7%), which also included four subdominant Carnobacterium maltaromaticum biotypes (10.9%), one Leuconostoc lactis biotype (3.3%) and few Lactococcus (1.6%), mesophilic Lactobacillus (0.8%) and Enterococcus (0.8%). Growth and distribution of LAB and Gram-negative species were strongly batch-dependent and plant-dependent. The CEntE neither retarded growth nor altered the whey cheese spoilage association but enhanced LAB growth and the declines of Gram-negative bacteria by late storage.

Growth and Biocontrol of Listeria monocytogenes in Greek Anthotyros Whey Cheese without or with a Crude Enterocin A-B-P Extract: Interactive Effects of the Native Spoilage Microbiota during Vacuum-Packed Storage at 4 °C

“…The preparation of the crude enterocin A-B-P extract (CEntE) and most methods followed in this study were according to the procedures described by Sameli et al [37]. Additional experimental methods, such as the whey cheese inoculation with L. monocytogenes and additional analyses relating to the interactive growth of the pathogen with the whey cheese spoilage microbiota, are described in later paragraphs.…”

“…No MW644963), the multiple Ent+ (entA-entB-entP) E. faecium KE82 (MW644969) and the multiple Ent+ (entA-entB-entP) E. faecium KE118 (not yet deposited in GenBank) [38]. The same CEntE (150 mL) used by Sameli et al [37] was also used in the challenge experiments of this follow-up study for direct data comparison. The CEntE containing enterocin A (secreted by all three strains) plus enterocins B and P (secreted by KE82 and KE118) was prepared without preceding pH adjustment and stored at -30 • C until use [37].…”

“…The same CEntE (150 mL) used by Sameli et al [37] was also used in the challenge experiments of this follow-up study for direct data comparison. The CEntE containing enterocin A (secreted by all three strains) plus enterocins B and P (secreted by KE82 and KE118) was prepared without preceding pH adjustment and stored at -30 • C until use [37]. The Ent+ antilisterial activity, the enterocin titer (400 AU/mL) and the stability of the enterocin titer of the thawed CEntE after every use [37] were determined by the well diffusion assay procedures previously described by Vandera et al [39].…”

“…Eight retail VP samples (500 g each) of fresh Anthotyros cheese, representing four batches previously coded A, B, C and D, were obtained from two semi-industrial dairy plants located in Epirus, Greece [37]. In specific, fresh cheese batches A and B were products of the traditional dairy Pappas Bros. (Skarfi E.P.E., Filippiada, Greece), our collaborator SME in the BIO TRUST project, while batches C and D were fresh retail Anthotyros cheese products of another traditional dairy located near Ioannina.…”

“…In a previous study, we assessed the effects of a crude enterocin A-B-P extract on the evolution and species composition of the specific spoilage microbiota prevailing in fresh vacuum-packaged Anthotyros whey cheeses stored at 4 • C [37]. This research is a follow-up challenge study to evaluate growth interactions of L. monocytogenes with the specific spoilage bacterial species during refrigerated (4 • C) storage of fresh vacuumpackaged Anthotyros whey cheeses without or with enterocin A-B-P supplementation.…”