This article goes deeper into spoilage at refrigeration temperature, a particular case of what is explained in spoilage microorganisms, and complements biofilms in food plants.
Table of contents
What a psychrotrophic microorganism is
A psychrotroph is a microorganism able to multiply at refrigeration temperature, even though its optimum growth temperature is higher. ISO 17410:2019 defines it operationally: the bacteria, yeasts and molds that form colonies on a solid medium after aerobic incubation at 6.5 °C. It is not a taxonomic group but an ability shared by very different genera, which is why the term covers everything from spoilage bacteria to a pathogen such as Listeria monocytogenes.
The same standard sums up in its introduction why they matter: they can spoil chilled foods through changes in odor and flavor, and some of those present in raw milk produce heat-stable enzymes that are not fully inactivated by pasteurization or sterilization and later break down the fat or protein of the already treated product. This article focuses on that group and on the cold chain. The general picture of yeasts, molds and acidophilic bacteria is in spoilage microorganisms.
Why refrigeration only slows things down
Cold reduces the growth rate, but in a psychrotroph it does not stop it. That is why the shelf life of a chilled food depends as much on how many cells reach the package as on the actual temperature at which it is kept. Small temperature differences change the outcome a great deal. The EFSA BIOHAZ Panel, citing Sumner and Jenson (2011), notes that in vacuum-packed lamb shoulders raising the temperature from −1.5 °C to 0, 2 or 5 °C cuts the time to spoilage by approximately 30%, 50% and 70%, respectively.
The same happens with Listeria monocytogenes. A study with five strains in BHI broth (Pennone et al., 2021) measured mean maximum growth rates of 0.043 to 0.048 h⁻¹ at 4.5 °C, 0.068 to 0.071 h⁻¹ at 7.8 °C and 0.145 to 0.187 h⁻¹ at 12 °C: between 4.5 and 12 °C the rate increased somewhat more than threefold, and the lag phase shortened as temperature rose. The FDA sums it up in its guidance for ready-to-eat foods: temperatures below freezing prevent the growth of L. monocytogenes, but at refrigeration temperature it multiplies slowly, so refrigeration is a less effective control measure for this pathogen than for others such as Salmonella.
The initial load is decisive, because cold only stretches the time it takes a population to reach spoilage levels. And cold chain breaks (loading and unloading, overloaded display cabinets, household refrigerators) add up. That quantitative reasoning is the basis of predictive microbiology.
Who spoils what: the environment decides
Of all the microbiota that reaches a food, only a fraction, the so-called specific spoilage organisms, produces the changes the consumer rejects. Which one dominates depends on the matrix, the packaging and the temperature.
Pseudomonas in fresh meat stored in air
In meat chilled in the presence of oxygen, pseudomonads dominate. According to EFSA, three species, Pseudomonas fragi, P. fluorescens and P. lundensis, are mainly responsible for slime formation and off-odors. Sensory spoilage usually becomes evident when the population reaches 10⁷–10⁸ CFU/cm², and EFSA used 10⁷ CFU/cm² as the end of shelf life in its models. In its 2016 opinion on meat transport, the panel concluded that chilling bovine or ovine carcasses to surface temperatures of up to 10 °C gave pseudomonad growth similar to or lower than chilling to the 7 °C core temperature required by Regulation (EC) 853/2004, whereas in pig carcasses the outcome depended on the target temperature and the chilling curve applied.
Lactic acid bacteria and Brochothrix under vacuum and modified atmosphere
When oxygen is removed, the picture changes. EFSA indicates that lactic acid bacteria and Brochothrix thermosphacta, oxygen-tolerant anaerobes, are usually the predominant spoilage organisms in meat stored under anaerobic conditions, together with psychrophilic clostridia in some cases. Cold-tolerant Enterobacteriaceae rarely cause spoilage unless there is temperature abuse. An example of how variable this group is: Lactococcus piscium, a psychrotrophic lactic acid bacterium that, according to a review by Saraoui et al. (2016), has been isolated from meat, seafood and vegetables packed under vacuum or modified atmosphere. Most studies indicate that it spoils meat, whereas in seafood it does not degrade sensory properties and some strains even inhibit L. monocytogenes.
Shewanella in fish stored on ice
In temperate-water marine fish stored aerobically on ice, FAO identifies Shewanella putrefaciens as the specific spoilage bacterium. It uses the trimethylamine oxide (TMAO) in the muscle as an electron acceptor and reduces it to trimethylamine (TMA), responsible for the typical stale fish odor, and it also produces hydrogen sulfide (H₂S) and other sulfur compounds. According to the same document, under aerobic storage levels of 10⁸–10⁹ CFU/g of specific spoilage bacteria are needed for the fish to spoil. Pseudomonads, by contrast, spoil fish without producing TMA or H₂S, with fruity and putrid odors, and in fish packed with CO₂ the role passes to Photobacterium phosphoreum.
The taxonomy was refined later. Fonnesbech Vogel et al. (2005) isolated more than 500 H₂S-producing strains from Baltic cod, plaice and flounder stored on ice, and after storage most of them corresponded to Shewanella baltica, which grew well in cod juice at 0 °C. The spoilage reactions (TMAO reduction and H₂S production) do not change; what changed was the name of the main culprit.
Raw milk: the spoilage that survives heat treatment
In chilled raw milk the problem does not end with the bacterium. Regulation (EC) 853/2004 requires it to be cooled immediately on the holding to no more than 8 °C if collected daily, or no more than 6 °C if not collected daily. During that time in the cold, psychrotrophs grow, and according to a review by Sinha and Kelly (2026) pseudomonads can make up between 70% and 90% of the psychrotrophic population of chilled raw milk.
Many of those pseudomonads secrete AprX, a highly heat-stable extracellular metalloprotease. The review reports that it can retain between 25% and 88% of its activity after UHT treatment (138 °C for 18 to 20 s), with a D-value at 140 °C of 124 s, compared with 13 s for plasmin, milk's own protease. Pasteurization or UHT kills the bacterium, but the enzyme it has already produced keeps acting during storage of the finished product and causes bitterness, gelation, sedimentation and protein destabilization. That is why a microbiologically compliant product can fail on quality weeks later, and why effective control lies before heat treatment: low initial load, sustained cold and short holding times. How risks change with the dairy process is developed in microbiology in the dairy industry, and the role of tank and line surfaces as a source of recontamination, in biofilms in food plants.
Listeria monocytogenes: the pathogenic psychrotroph
Almost all psychrotrophs are a quality problem. L. monocytogenes is the relevant exception: a pathogen that grows in the refrigerator. The fundamentals of the pathogen are in Listeria monocytogenes: risks and detection methods.
That is why regulatory criteria are defined over the shelf life and not only when the product leaves the plant. In Regulation (EC) 2073/2005, ready-to-eat foods able to support the growth of L. monocytogenes (row 1.2) have a limit of 100 CFU/g during the shelf life and of not detected in 25 g before the food has left the immediate control of the producer. Products with pH ≤ 4.4 or aw ≤ 0.92, with pH ≤ 5.0 and aw ≤ 0.94, or with a shelf life of less than five days are automatically considered unable to support growth. Commission Regulation (EU) 2024/2895, applicable from 1 July 2026, extends not detected in 25 g to the whole period the product is placed on the market, unless the operator demonstrates that it will not exceed 100 CFU/g throughout the shelf life.
That demonstration relies on shelf-life studies. The technical guidance of the EU reference laboratory for Listeria (EURL Lm) classifies a food as able to support growth when the growth potential in a challenge test exceeds 0.5 log CFU/g. If there are no own temperature data, it proposes simulating the cold chain with 7 °C at the plant, 7 °C at retail and 10 °C at the consumer's home.
How they are counted: ISO 17410
ISO 17410:2019 is the horizontal method for enumerating psychrotrophs in food, feed, environmental samples from the production area and primary production samples. It replaced its 2001 edition and the dairy standards ISO 6730 and ISO 8552. Its key points:
- Surface plating on plate count agar (PCA), instead of the pour plating of the earlier dairy standards, because psychrotrophs are sensitive to the heat of molten agar.
- Aerobic incubation at 6.5 °C ± 1 °C for 10 days. It is a slow analysis: the result arrives when a good part of the shelf life of a fresh product has already gone by, so it is more useful for verifying the process and raw materials than for releasing batches.
- Counting from plates with fewer than 150 colonies, expressed according to ISO 7218, and performance testing of the medium according to ISO 11133.
- Rapid method for raw and pasteurized milk (Annex B, informative): incubation at 21 °C for 25 h. The standard warns that not all microorganisms that form colonies at 6.5 °C do so at 21 °C, so it is not an equivalent substitute.
The standard includes no performance data because no interlaboratory study was organized, and it clarifies that psychrotrophs are mainly used for process monitoring. A psychrotrophic count tells you how many cells able to grow in the cold are present, not which species they are or whether they are pathogenic. To interpret the number, it is worth reviewing what CFU are and how they are interpreted.
Which TAAG kits relate to this topic
No TAAG kit replaces the ISO 17410 count: the kits in the catalog are for qualitative detection, not enumeration. Where they do contribute is in the part of the problem that is answered with presence or absence.
- Listeria in chilled foods and surfaces. Ampliora 2.3 Listeria spp. and L. monocytogenes (V-SF46, real-time PCR with probes, Mila design) and Specio 2.3 Listeria spp. and L. monocytogenes (V-SF04, real-time PCR with melting curve) detect and identify Listeria spp. and L. monocytogenes in food, beverages and living and inert surfaces, with declared industries of beverages, dairy, meat and poultry and ready-to-eat. Specio 2.3 declares a sensitivity of 1 CFU/25 g in food. Specio 1.4 L. monocytogenes (V-SF28) is limited to the pathogenic species.
- Spoilage bacteria in dairy. Specio 00.1 Bacteria (V-SF14, real-time PCR with melting curve) detects and identifies more than 80 spoilage bacteria, with validated matrices that include natural milk, skimmed milk, fortified milk, chocolate milk and cream, as well as surfaces. The catalog does not publish the list of species, so before using it to look for Pseudomonas it is advisable to confirm with TAAG that the genus is in the panel. It detects the bacterium, not the AprX protease already formed.
The catalog has no kits for Shewanella, Photobacterium or lactic acid bacteria in packaged meat; its lactobacilli kits are validated in beer. In those cases, the plate count remains the tool. To choose by matrix and industry, see how to choose a TAAG kit.
Conclusion
The cold chain is a tool for buying time, not for stopping psychrotrophs: every extra degree shortens shelf life and every break adds up. Which organism dominates depends on the matrix and the packaging, from pseudomonads in meat stored in air to lactic acid bacteria under vacuum, Shewanella in fish on ice and the heat-stable proteases that pseudomonads leave in milk. Listeria monocytogenes adds a health risk to that same mechanism, which is why regulation now looks at the whole shelf life. Counting psychrotrophs with ISO 17410, controlling the initial load and detecting Listeria in time are parts of the same program.
About TAAG
Consult TAAG on the scope of Ampliora 2.3 Listeria spp. and L. monocytogenes and Specio 00.1 Bacteria for your chilled products and your process surfaces.
Frequently asked questions
What is the difference between a psychrotrophic and a mesophilic microorganism?
A psychrotroph can multiply at refrigeration temperature even though it grows faster at higher temperatures; a typical mesophile barely grows in the cold. ISO 17410 defines psychrotrophs operationally as those that form colonies after aerobic incubation at 6.5 °C. That is why a good mesophile count does not guarantee a long refrigerated shelf life.
Why does a chilled food spoil anyway?
Because cold reduces the growth rate of psychrotrophs but does not stop it. EFSA, citing Sumner and Jenson (2011), indicates that in vacuum-packed lamb raising the temperature from −1.5 °C to 5 °C cuts the time to spoilage by approximately 70%. The initial load and the actual temperature along the whole chain determine how long the product lasts.
Why can UHT milk gel or turn bitter if it has no live bacteria?
Because the pseudomonads that grew in the chilled raw milk secrete the AprX protease, which withstands heat treatment. A 2026 review reports that it retains between 25% and 88% of its activity after UHT. The enzyme keeps breaking down the protein during storage even though the bacterium is already dead.
How long does a psychrotroph count according to ISO 17410 take?
The standard incubates the plates at 6.5 °C for 10 days, with surface plating on plate count agar. For raw and pasteurized milk it includes an informative rapid method at 21 °C for 25 h, although it warns that not all microorganisms that grow at 6.5 °C form colonies at 21 °C.
Why is Listeria monocytogenes different from other psychrotrophs?
Because it is a pathogen, not just a spoilage agent, and it multiplies slowly under refrigeration, so cold controls it less well than other pathogens. That is why in the EU the criteria apply throughout the shelf life and, from 1 July 2026, not detected in 25 g applies on the market unless the operator demonstrates that it will not exceed 100 CFU/g.