Psychrotrophic Microbiota Research Articles

Development of microbiological criteria for evaluating frozen fish based on the amount of psychrotrophic microflora

Unrefrigerated fish and seafood are dominated by mesophilic microflora. At the same time, during their storage in the conditions of refrigerating chambers, a cold-loving – psychrotrophic microbiota dominates, which, according to many scientists, causes organoleptic and chemical changes in fish and affects sanitary and hygienic indicators. Therefore, the microbiological assessment of frozen fish by the content of psychrotrophic microbiota, which participates in its reduced safety and quality, will make it possible to propose preventive measures against spoilage. The aim of the work was to develop a microbiological criterion for the hygiene of the technological process of frozen fish based on the assessment of the content of psychrotrophic microbiota. In the work, standard methods were used, in particular, the microbial number was determined in the samples at a temperature of (30 ± 1) ºС incubation of crops for 72 hours and at a temperature of (6.5 ± 0.5) ºС incubation for 10 days (psychrotrophic microbiota). It was established that during the storage of frozen fish at a temperature of -18 °С, the mesophilic microflora is inactivated, as a result of which its number gradually decreases. However, the intensity of inactivation of mesophilic bacteria in samples with insignificant microbial insemination is, on average, 1.4 times faster, compared to samples with a larger number of microorganisms. In our opinion, this is due to the fact that the composition of mysophilous microflora is largely represented by genera of bacteria that show tolerance to cold. At the same time, the psychrotrophic microflora of frozen fish does not become inactivated during the 8-month storage period at a temperature of -18 °C, and is more stable compared to the mesophilic one. That is, no significant increase or decrease in the number of microorganisms was noted. As a result, by the amount of psychrotrophic microflora of frozen fish, determined at any stage of storage, it is possible to judge its amount at the time of freezing. The amount of psychrotrophic microflora more objectively characterizes the hygienic conditions of production, compliance with the freezing temperature. The microbiological hygiene criteria of the technological process of frozen fish production were substantiated and developed based on the estimation of the amount of psychrotrophic microflora (n = 5; c = 3; m = 10000 CFU/g; M = 50000 CFU/g). These criteria indicate compliance with a set of hygienic measures from freezing to circulation of fish. Therefore, the determined microbiological criterion for the quantity of psychrotrophic microflora in frozen fish complements the existing methods of evaluating the hygiene of the technological process and aims to broadly assess the microbiological safety of frozen fish.

Short communication: Effect of bactofugation of raw milk on counts and microbial diversity of psychrotrophs

Bactofugation is a centrifugal process for removing spores of microorganisms from milk, especially when it is destined for cheese making. Other microorganisms may be removed in bactofugation. This study aimed to verify the effect of milk bactofugation on the counts and microbial diversity of psychrotrophs. The raw milk was preheated (≈55°C) before being bactofuged, and samples were collected from 3 batches of milk: refrigerated raw, preheated, and bactofuged, representing the immediate conditions before and after bactofugation. The mean psychrotrophic counts of the 3 batches were 3.08 (±1.69) × 106, 193 (±232), and 20 (±26) cfu/mL, respectively. Preheating was sufficient to eliminate 99.99% of the raw milk psychrotrophs, but bactofugation further reduced 89.66% of psychrotrophs from preheated milk. Lysinibacillus fusiformis was the most frequently isolated species (45.7%) among the psychrotrophs of raw milk and, proportionally, were more frequent in preheated (37.5%) and bactofuged (60%) milk. Bacillus invictae (20%), Enterococcus faecalis (10%), and Kurthia gibsonii (10%) were also isolated from bactofuged milk. Albeit in small numbers, psychrotrophic, thermoduric, and spore-forming bacteria with known proteolytic and lipolytic activity remained in the milk after bactofugation, which apparently had no effect on a specific population of microorganisms but proportionally reduced the entire psychrotrophic microbiota of raw milk.

Psychrotrophic Microbiota in Milk and Fermented Milk Products

Psychrotrophic Microbiota in Milk and Fermented Milk Products

Identification and metagenetic characterisation of Listeria monocytogenes-harbouring communities present in food-related industrial environments

The main aim of this study was to localise, identify and characterise the Listeria monocytogenes-harbouring bacterial communities present in food related premises via 16S rRNA gene metagenetic analysis. With this scope, 319 environmental samples coming from a wide variety of surfaces of fish (n = 120), meat (n = 80) and dairy industries (n = 119), were firstly analysed following ISO 11290–1 and ISO 11290–2 norms. Direct L. monocytogenes quantification was possible in 9 samples (2.8%) with values between 2.00 and 3.97 log CFU/cm2. After enrichment, an overall L. monocytogenes incidence of 12.54% (n = 40) was obtained, being samples from meat industry the most contaminated. Molecular serotyping assays showed that most of the isolates belonged to 1/2b-3b-7 subgroup, followed by 1/2a-3a and 1/2c-3c. These results combined with AscI and ApaI PFGE macrorestriction patterns, yielded 7 different L. monocytogenes clusters. Nevertheless, no clear ecological relationships could be stablished. High amounts of L. monocytogenes-associated psychrotrophic microbiota were obtained in all cases with values above 9 log CFU/cm2 in some cases. Metagenetic analysis of one representative sample per each food industry type (fish, meat, dairy) demonstrated that Actinobacteria (53.16%) was mostly present in the meat sample whereas Proteobacteria was the most representative phylum in dairy (69.58%) and fish (97.11%) samples. Subsequent operational taxonomic units (OTUs) analysis, showed a wide variety of taxa associated with L. monocytogenes such as spoilage-associated genera (e.g. Psyschromonas or Shewanella), lactic acid bacteria genera (e.g. Lactococcus or Lactobacillus) or pathogenic species such as Yersinia enterocolitica. It was thus demonstrated, that L. monocytogenes is capable to both survive with different bacteria in different ecological niches, highlighting once more the need for proper surveillance schedules so as to guarantee the safety of the food products.

Monitoring of spoilage-associated microbiota on modified atmosphere packaged beef and differentiation of psychrophilic and psychrotrophic strains.

This study aimed to explore the discriminatory power of MALDI-TOF MS as a high-throughput method to monitor growth dynamics of meat-spoiling bacteria on modified atmosphere packaged (MAP) beef and to differentiate psychrophilic and psychrotrophic spoilage-associated strains. MAP beef steaks were incubated for 21days at constant 4 and 10°C. Development of headspace gas composition, pH, CFU and spoilage-associated microbial composition were monitored. MALDI-TOF MS exhibited high discriminatory power for reliable, high-throughput identification of spoilage-associated, psychrotrophic microbiota. Microbiota development was highly dependent on initial abundance of specific species. Organoleptic onset of spoilage was concomitant with an alteration of headspace atmosphere and pH. Screening for psychrophiles at 4°C on beef revealed the abundance of Leuconostoc gelidum subsp. gelidum TMW2·1998 with characteristic psychrophilic growth behaviour. We suggest that control of initial contaminants is crucial to predict the onset of spoilage and that headspace atmosphere and pH are important parameters with spoilage-indicative potential. MALDI-TOF MS proved suitable for high-resolution monitoring of psychrotrophic and psychrophilic spoilage-associated microbiota and enables improved insights in the spoilage progress. The presence of psychrophilic strains on beef is the likely causative for unexplained spoilage events.

The main spoilage-related psychrotrophic bacteria in refrigerated raw milk

Refrigerated raw milk may contain psychrotrophic microorganisms that produce thermoresistant exoproteases and lipases, which may compromise the quality of processed fluid milk and dairy products during storage. The aim of this work was to quantify and identify the deteriorating psychrotrophic microbiota in Brazilian refrigerated raw milk using genetic diversity analysis. The mean psychrotrophic count was 1.1 × 104 cfu/mL. Of the total isolates, 47.8 and 29.8% showed deteriorating activity at 35°C within 48 h and 7°C within 10 d, respectively. Among the proteolytic species, more isolated by this study were Lactococcus lactis (27.3%), Enterobacter kobei (14.8%), Serratia ureilytica (8%), Aerococcus urinaeequi (6.8%), and Bacillus licheniformis (6.8%). Observed among lipolytics were E. kobei (17.7%), L. lactis (15.6%), A. urinaeequi (12.5%), and Acinetobacter lwoffii (9.4%). The isolates S. ureilytica, E. kobei, Pseudomonas spp., and Yersinia enterocolitica potentially produced alkaline metalloprotease (aprX). Despite the low counts, a considerable portion of the psychrotrophic microbiota presented spoilage potential, which reaffirms the need for rigor in the control of contamination and the importance of rapid processing as factors that maintain the quality of milk and dairy products.

Biodiversity of culturable psychrotrophic microbiota in raw milk attributable to refrigeration conditions, seasonality and their spoilage potential

Refrigerated storage of raw milk promotes the growth of psychrotrophic bacteria, some of which produce heat-stable exoenzymes causing dairy product spoilage. The effects of storage conditions and season on the biodiversity of psychrotrophs in raw milk were examined using matrix-assisted laser desorption time of flight mass spectrometry and 16S rRNA analysis. The ability of psychrotrophs to produce protease, lipase and phospholipase C was determined. The predominant genera found were Pseudomonas (19.9%), Bacillus (13.3%), Microbacterium (5.3%), Lactococcus (8.6%), Acinetobacter (4.9%) and Hafnia (2.8%); a considerable number of isolates were hitherto unknown species and genera. Diversity varied significantly (P < 0.05), depending on the storage temperature, time, initial microbiota and season. The predominant isolates showed significantly higher heat-stable exoenzyme activities after heating at 142 °C for 4 s. Improving the quality of milk products may require differential processing of raw milk depending on the type of microbiota present, storage temperature and seasonality.

Atividade proteolítica de bactérias psicrotróficas em leites estocados em diferentes temperaturas

A difusão do uso da tecnologia do resfriamento, como estratégia para manutenção das características microbiológicas e organolépticas do leite, não associada aos cuidados básicos de higiene na obtenção do produto, fez com que os micro-organismos psicrotróficos emergissem como as principais bactérias deteriorantes da indústria láctea. Este estudo teve por objetivo avaliar como o metabolismo psicrotrófico proteolítico responde a diferentes temperaturas de incubação do leite, delineando um paralelo entre multiplicação de psicrotróficos, quantidade de micro-organismos com capacidade de hidrolisar proteínas e quantidade de GMP (glicomacropeptídeo) liberada. As amostras de leite, coletadas diretamente do tanque de resfriamento, foram submetidas à contagem de micro-organismos psicrotróficos, contagem de micro-organismos proteolíticos e determinação da concentração de GMP, em diferentes tempos (12 h; 24 h; 48 h) e temperaturas (4°C; 8°C; 12°C) de armazenamento. Não houve linearidade entre os parâmetros microbiológicos e o GMP aferido, segundo o binômio tempo/temperatura. A maior concentração de GMP (5,07 µg/mL) foi aferida no binômio 8°C/24 h (T8/M24). Esses dados indicam a necessidade de estudos sobre o metabolismo da microbiota psicrotrófica, de forma a elucidar questões básicas e de profunda relevância sobre seu metabolismo.

Changes in psychrotrophic microbial populations during milk creaming to produce Grana Trentino cheese

The aim of this study was to study the psychrotrophic microbiota developing during milk creaming of Grana Trentino cheese-making. 138 isolates from raw whole milk, cream and skim milk samples were screened by Randomly amplified polymorphic DNA PCR biotyping and representative strains of each biotype were characterised by partial 16S rRNA gene sequencing and enzymatic activity. Pseudomonadaceae were commonly isolated in cream samples while Streptococcaceae and Enterobacteriaceae in milk samples. Moraxellaceae and Flavobacteriaceae were found in both cream and milk samples. More than 80% of psychrotrophic isolates could grow at 37 °C. All Flavobacteriaceae and half of Pseudomonadaceae biotypes displayed proteolytic activity on milk agar even at low temperatures such as 10 °C. All Streptococcaceae and some of Enterobacteriaceae displayed acidifying activity and almost all Acinetobacter spp. ( Moraxellaceae) displayed lipolytic activity towards tributyrin. Even if psychrotrophic bacteria is not the dominant microbial group in raw milk, their total number increases during creaming and becomes one of the most present group together with Lactic Acid Bacteria. Their enzymatic activities may be key players in determining milk quality for cheese making.

Application of high-pressure processing and nisin to mechanically recovered poultry meat for microbial decontamination

The combined effect of high-pressure, nisin and acidification on the mesophilic and psychrotrophic microbiota of mechanically recovered poultry meat (MRPM) was evaluated. Nisin (0, 12.5, 100 and 200 ppm) and 1% of glucono-delta-lactone were added to MRPM. Samples were treated by combinations of pressure (350 and 450 MPa), time (5 and 15 min) and temperature (−20 and 20 °C). Both alone or in combination with nisin, pressurization at 20 °C was more lethal than that at −20 °C. The significantly highest decreases in counts occurred in samples with 200 ppm of nisin treated at 450 MPa: 5.3 log CFU/g for mesophiles and above 7.5 log CFU/g (nondetected growth) for psychrotrophs. So, this combined treatment can improve the safety and shelf-life of MRPM.