In the dairy, the persistence of bacterial contaminants on surfaces is a critical problem due to the formation of spores and biofilms.
The presence of biofilms and spores in dairy plants poses a multifactorial threat that affects the economic stability, food safety, and operational efficiency of companies.
The main risks can be grouped into the following categories:
1. Financial and Quality Risks
Biofilm contamination directly affects a brand’s profitability and reputation:
- Reduced shelf life: Biofilms are responsible for the deterioration of organoleptic quality and the reduction in the shelf life of finished products. For example, in the United States, spore-forming bacteria are responsible for the loss of approximately one-third of the milk produced for consumption.
- Organoleptic changes: Bacteria that detach from biofilms can cause defects in taste, odor, or texture, leading to consumer complaints and dissatisfaction.
- Direct financial losses: The entire sector is being challenged by “serious economic problems” stemming from the need to manage persistent contamination that standard processes cannot resolve.
2. Health Risks and Food Safety
Consumer safety is the most critical risk:
- Foodborne illnesses: Strains such as Bacillus cereus can cause diarrhea or vomiting, posing a serious risk to public health.
- Failure of sterilization: Some spores are capable of surviving commercial sterilization processes, compromising the sterility of shelf-stable products and putting the company at risk of potential product recalls.
- Post-processing cross-contamination: Biofilms act as reservoirs from which microorganisms are released into the milk after pasteurization (post-pasteurization contamination), rendering the heat treatment ineffective.
3. Operational and Persistence Risks
Biofilms create a protected environment that renders standard management procedures ineffective:
- Chronic contamination: Once established, biofilms become a permanent source of contamination in the systems.
- Process resistance CIP: Cells within the biofilm are much more resistant to standard chemical washes (soda and acid) than free-floating (planktonic) cells, tolerating pH and temperature fluctuations that would normally kill them.
- Selection of super-resistant strains: The use of ineffective cleaning cycles can lead to the selection of spores with even greater resistance to alkalis or superior adhesion properties, making each subsequent production cycle more at risk than the previous one.
Microbial Ecology: The Key Players in Milk Powder Biofilms
The primary sources of contamination in powdered milk and reconstituted milk production lines are spore-forming bacteria, mainly belonging to the genera Bacillus and Geobacillus.
- Mesophilic bacilli: The Bacillus cereus group is particularly problematic due to its pathogenic potential and its ability to adhere tenaciously to stainless steel via appendages and the exospore.
- Thermophilic bacteria: Species such as Geobacillus stearothermophilus and Anoxybacillus flavithermus thrive in plant sections where temperatures range from 40 to 65 °C, such as evaporators and heat exchangers.
Often, the powdered milk itself is the initial source of these spores, which are then concentrated and accumulated during the thermal processing stages.
The main accumulation points identified include:
1 – Structural niches and “dead spots”
Biofilms tend to form in areas where the cleaning-in-place (CIP) system is less effective or where the surface is uneven:
- Hard-to-clean areas: Dead ends, joints, valves, cavities, and crevices.
- Rubber components and gaskets: Valve gaskets (such as mix-proof valves) and other non-metallic components are common bonding sites.
- Damaged surfaces: Cracks in corroded stainless steel can serve as hiding places for spores and biofilms, protecting them from the mechanical and chemical effects of washing.
2 – Storage and mixing tanks
These tanks are critical sources of contamination:
- Milk tanks: Biofilms were identified in the tanks for raw milk, pasteurized milk, and homogenized milk.
- Internal tank components: The internal complexity of cooling tanks—which includes agitators, thermostats, and temperature probes—hinders thorough cleaning and promotes bacterial growth.
- Effect of flow: In tanks, turbulent flow can paradoxically promote bacterial attachment by bringing microorganisms closer to the surfaces.
3 – Heat treatment systems
The varying temperatures along the production line promote the growth of specific types of biofilm:
- Pasteurizers and heat exchangers: Biofilms accumulate in feed pipes and plate heat exchangers.
- High-temperature sections (40–65 °C): In these areas, as in evaporators, thermophilic bacteria (e.g., Geobacillus) thrive, forming what are known as “process biofilms.”
- Probes and sensors: The sensor probes in direct steam injection (DSI) systems are potential accumulation points.
4 – Filtration and packaging lines
- Filters: Duplex filters located upstream of homogenizers and DSI systems can trap microorganisms that go on to form biofilms.
- Packaging machines: These represent a critical point for post-pasteurization contamination.
- Spray nozzles: In powder production facilities, even the spray nozzles at the end of the process can harbor bacteria.
Adhesion mechanisms
The adhesion of Bacillus cereus spores to stainless steel surfaces is a complex phenomenon resulting from a combination of structural characteristics, physicochemical properties, and environmental factors.
The main reasons for this steadfast commitment are:
- Surface hydrophobicity: Spores possess high surface hydrophobicity, which facilitates their binding to inert materials such as stainless steel. This physicochemical property is one of the key factors that allows the spore to “cling” to the surface.
- External cellular structures: Adhesion capacity is closely linked to the presence of specific structures on the spore’s outer surface, particularly the exosporium (an outer protective membrane) and the appendages (or filaments). These structures act as anchoring organs that facilitate initial contact and subsequent stable bonding with steel.
- Genetic and proteomic potential: Adhesion and subsequent growth within the biofilm are supported by the bacterium’s genetic potential. Proteomic studies have shown that, as early as the first two hours of biofilm formation, B. cereus expresses specific proteins that facilitate the colonization process.
- Mechanical factors (Flow): Processing conditions affect adhesion. For example, turbulent flow within the equipment can promote adhesion by physically bringing the spores closer to the equipment walls than under static conditions.
- Resistance to cleaning: Spores not only adhere easily, but are also capable of surviving standard cleaning cycles (CIP). In some cases, chemical treatments can even select for spores with specific surface properties that make them even more effective at adhering.
Once attached, these spores can germinate, multiply, and form mature biofilms that become chronic reservoirs of contamination, protecting the bacteria from disinfectants and making it difficult to eradicate them from production lines.
Why can the standard CIP system fail?
The failure of traditional Cleaning-in-Place (CIP) systems to combat biofilms is due to a combination of structural, chemical, and biological factors that make these microbial communities extremely resilient.
The main reasons why CIP is unable to completely remove these milk powder biofilms are:
1 – The protective barrier of the EPS matrix
Biofilms are not simply accumulations of bacteria, but communities embedded in a matrix of extracellular polymeric substances (EPS) produced by the bacteria themselves. This matrix acts as a physical and chemical shield that:
- It protects bacteria from the action of biocides, rendering them inaccessible.
- It enables the microorganisms within the biofilm to withstand changes in temperature and pH much better than their planktonic counterparts (those floating freely in the milk).
- It requires very harsh treatments using powerful oxidizing agents or specific enzymes to be dissolved, which is beyond the capabilities of standard detergents.
2 – Chemical tolerance and adaptation
CIP procedures typically use caustic soda (NaOH) and nitric acid (HNO₃), but many bacterial strains have developed specific resistances:
- Alkali resistance: Strains such as Bacillus licheniformis have demonstrated high resistance to hot caustic soda (80 °C), surviving washing cycles.
- Survival difference: While planktonic cells are reduced by more than 6 log units after 10 minutes of contact with NaOH or HNO₃ at 80 °C, cells in biofilms show a smaller reduction despite twice the exposure time (20 minutes).
- Selection of resistant strains: Paradoxically, chemical cleaning can select spores with specific surface properties (such as hydrophilicity) or induce alkali tolerance, making the residual biofilm even more difficult to remove in the future.
3 – Persistence and reattachment of spores
Bacillus and Geobacillus spores are a critical factor in CIP failure:
- Survival cycle: Spores survive washing, can reattach to surfaces during the CIP process itself, and quickly form a new biofilm.
- Anchoring structures: The presence of appendages and the exosporium on the spores facilitates their tenacious adhesion to stainless steel, preventing their mechanical removal.
4 – Physical constraints and structural limitations
The CIP is a closed-loop system based on the flow of cleaning agents, but it has physical limitations:
- Hard-to-reach areas: Biofilms thrive in places where the mechanical and chemical action of washing is weak, such as dead ends, crevices, joints, valves, and gaskets.
- Damaged surfaces: Irregularities or damage in stainless steel provide a haven for biofilms, shielding them from the cleaning solution.
- 3D Architecture: Mature biofilms develop complex, compact three-dimensional (multilayered) structures that are inherently resistant to conventional industrial cleaning methods.
Innovative Strategies: Enzymatic Cleaning
To effectively combat biofilm formation, it is necessary to go beyond traditional cleaning.
Enzymatic cleaners represent one of the most promising strategies for overcoming the ineffectiveness of traditional CIP systems:
- Breakdown of the matrix: Enzymes act by hydrolyzing the organic matrix of the biofilm (composed of extracellular polymeric substances, or EPS). This action “opens up” the protective structure, allowing disinfectants to more easily reach and kill the bacteria nestled inside.
- Incorporation into the CIP system: The addition of enzymes to the CIP system’s wash solutions has been shown to significantly improve biofilm removal efficiency compared to chemical treatments alone.
- Optimization of alkaline washing: Sources suggest enhancing the alkaline washing step with enzymatic treatments to promote more thorough removal of biofilm from surfaces. The effectiveness of these enzymes can be further enhanced if they are solubilized in alkaline buffers containing surfactants.
For this reason Piramide, thanks to the ongoing work of Realco’s R&D department, is able to provide Biorem®, an enzymatic solution specifically designed to:
- Contain enzymes belonging to multiple enzyme classes, and thus targeting different molecular targets
- Containing enzymes with high substrate specificity
- Contain enzymes with high activity
These factors are extremely important to consider whenever evaluating an enzymatic approach for the removal of biofilm—or, more precisely, its amorphous matrix. Products with a limited range of enzyme classes, low specificity, and low activity can lead to mediocre results.
The Biorem® 3G Solution
Biorem® 3G is the only octavalent enzyme cocktail on the market capable of hydrolyzing the maximum number of components in the organic matrix of the biofilm, making it the most effective solution, especially for entrenched and resistant biofilms.
Thanks to the broad-spectrum and targeted action of Biorem® 3G in hydrolyzing the various components of the extrapolymere matrix of biofilms, it is possible to eliminate and reduce the risk of biofilm formation by these microorganisms. Furthermore, the enzymatic approach does not promote the development of resistance and is able to bypass the tolerance mechanisms provided by the matrix itself, ensuring greater effectiveness in removing biofilms from work environments.
The Piramide team specializes in applying customized enzymatic protocols tailored to specific production environments, and in developing pre- and post-treatment diagnostic strategies using state-of-the-art analytical methods.
Contact us for advice or a treatment plan:
Email: info@piramide-ambiente.it
Phone: 0332-826017
