The mineral water and soft drink industry currently operates in an environment of intense competitive and regulatory pressure, where microbiological safety and sensory stability are not merely legal requirements but cornerstones of a brand’s reputation.
In this context, the issue of biofilms represents one of the most complex and insidious technical challenges for manufacturers.
A biofilm is not simply a collection of bacteria, but an organized and protected form of life that can be a source of chronic contamination.
Understanding the biochemistry that governs these communities is essential for overcoming the limitations of conventional detergents and adopting precision biotechnology solutions.
Water, the primary ingredient and essential medium, is treated and monitored at the source but travels through miles of pipes and tanks before reaching the bottling plant.
During this process, chemical, physical, and microbiological dynamics lead to a change in water quality compared to its initial state. The biological processes that occur within drinking water and mineral water distribution systems include the formation of biofilms on pipe walls, biocorrosion, and the degradation of network materials—phenomena that alter the taste and odor of the water and create conditions favorable for the proliferation of opportunistic microbes.
The Architecture of Biofilms: Biochemical Foundations and Developmental Dynamics
To address this issue, it is necessary to analyze the molecular structure of the biofilm. It is defined as a multicellular community held together by a matrix of extracellular polymeric substances (EPS) produced by the microorganisms themselves.
This matrix forms the functional and structural framework of the biofilm, determining its chemical and physical properties and providing protection against hostile environments, dehydration, UV radiation, and, above all, antimicrobial agents and disinfectants.
The biochemical composition of EPS varies depending on the microbial species involved and environmental conditions, but the fundamental components remain constant.
| EPS Component | Approximate Percentage | Primary Function |
| Protein | 40–60% | Structural integrity, enzymatic activity, adhesion to surfaces. |
| Polysaccharides | 20–30% | Water retention, 3D scaffold, physical protection. |
| eDNA (extracellular DNA) | < 10% | Structural cohesion, genetic exchange, matrix stabilization. |
| Lipids | Variable | Biodegradation, hydrophobic interactions, membrane stability. |
Proteins often make up the majority of the composition and are responsible for mechanical stability.
eDNA acts as a molecular glue; studies on Staphylococcus aureus have shown that the enzymatic removal of eDNA using DNase drastically increases the bacteria’s susceptibility to biocides.
In the beverage industry, this matrix protects microorganisms from the turbulent flows of the CIP system and chemically neutralizes disinfectants such as chlorine and peracetic acid, preventing them from reaching the living cells inside.
Biofilms and Mineral Waters: Life Cycle and Surface Colonization
The development of a biofilm follows specific kinetic stages that must be understood in order to determine the appropriate timing for intervention.
Biofouling begins within 1–10 seconds of water coming into contact with a clean surface through the formation of a “conditioning layer.” This layer of adsorbed organic molecules facilitates the initial adhesion of bacterial cells, which generally occurs within 6–8 hours.
Once irreversible adhesion has occurred, the cells begin to produce EPS, leading to the stabilization of the biofilm and the formation of protected microcolonies.
As it matures, the biofilm becomes a dynamic structure that periodically releases fragments or individual cells into the beverage stream, a phenomenon known as a “planktonic shower.”
This release is often unpredictable, which explains why routine microbiological samples may yield negative results one day and show spikes in contamination the next, making quality control a constant challenge.
The material of the piping is a critical factor. Although stainless steel is the industry standard, surface roughness caused by wear or corrosion (“rouging”) on the steel provides ideal niches for the initial attachment of bacteria, making maintenance of metal surfaces essential.
Plastics such as PVC and HDPE may exhibit different colonization patterns.
The microbiological context: key players and major defects
The soft drink and mineral water industry is susceptible to contamination by specific microorganisms, which thrive due to the products’ inherent characteristics, such as acidic pH,CO2, the presence of sugars, and sometimes organic preservatives.
The natural mineral water industry is unique due to regulatory requirements that mandate bottling the water exactly as it flows from the source, thereby limiting disinfection treatments.
In this oligotrophic environment (where nutrients are scarce), selective pressure favors species capable of making the most of the limited organic resources available.
Conventional microbiology often fails to detect the true extent of such contaminants due to the presence of viable but non-cultivable (VBNC) cells. Furthermore, testing small volumes (250 ml) can be misleading.
Below is a list of the microorganisms most commonly involved in biofilm formation in these industrial settings and responsible for the main organoleptic defects.
Pseudomonas spp.
They are among the most common colonizers. Species such as P. aeruginosa are considered model organisms for the study of biofilms and are ubiquitous in water and soil.
Pseudomonas aeruginosa is a primary colonizer of pipes and fill heads.
Under nutrient-deprived conditions, P. aeruginosa produces alginate, a highly hydrated, polyanionic EPS that protects cells from environmental stress and oxidizing agents.
Pseudomonas can break down proteins and lipids found in more complex beverages, releasing amines and esters that impart putrid or rancid flavors.
Acetic acid bacteria (AAB)
Genus such as Acetobacter and Gluconobacter (e.g., G. liquefaciens) are commonly found in flavored waters and non-carbonated soft drinks. They metabolize sugars and residual ethanol, producing acetic acid, which alters the flavor.
Lactic acid bacteria (LAB)
Lactobacillus and Leuconostoc are particularly problematic in beverages containing fruit juices. Many strains are resistant to organic acids such as benzoic and sorbic acids. They are responsible for the “ropiness” (stringiness) of the product due to the production of dextrans, exopolysaccharides that form gelatinous masses resembling “slime.”
Thermoacidophilic bacteria (TAB)
Species such as Alicyclobacillus acidoterrestris pose a threat to fruit juices and pasteurized acidic beverages, as their spores survive heat treatment and can germinate, producing guaiacol, which gives the product a “medicinal” or “earthy” taste.
Contaminants in Purified Water
In pharmaceutical settings or systems handling special-purpose water, bacteria such as Ralstonia pickettii and Burkholderia cepacia are known to proliferate in ultrapure water systems, where nutrient concentrations are minimal.
Sulfate-Reducing Bacteria (SRB)
A specific hazard in mineral water lines is posed by sulfate-reducing bacteria (SRB), obligate anaerobic microorganisms that thrive even in systems that appear to be oxygenated, thanks to the protection provided by the biofilm.
The most significant genus in industrial settings is Desulfovibrio (e.g., D. desulfuricans, D. vulgaris), which is ubiquitous in aquatic environments and capable of colonizing drinking water and mineral water distribution systems.
Although mineral water may contain oxygen, the thickness of the biofilm creates chemical gradients: the basal layers of the biofilm become anoxic, providing the ideal habitat for these bacteria. In these protected niches, SRBs reduce the sulfates naturally present in the water to produce hydrogen sulfide, a highly volatile and toxic gas.
For example, bottled mineral water may contain dimethyl sulfide (DMS) and, to a lesser extent, dimethyl selenide (DMSe), with concentrations in the range of a few ng/L—well below toxicological levels but sufficient to trigger the sense of smell.
The buildup of these bacteria usually indicates a long-term microbiological problem and poor hygiene in the pipes or storage tanks.
Yeasts and Molds
Yeasts are the primary cause of spoilage in soft drinks due to their tolerance for low pH levels and carbonation.
Yeasts such as Z. bisporus are particularly insidious because they can survive under conditions of highCO2 pressure and very low water activity.
Molds, on the other hand, usually grow only in the presence of dissolved oxygen, often settling in the headspace of bottles or in non-carbonated products where the seal is not airtight.
| Microbial Species | Type of Beverage | Defect Found |
| Zygosaccharomyces bisporus | Carbonated and flavored beverages | Package swelling (stuffing), gas production. |
| Zygosaccharomyces bailii | Beverages with preservatives | Extreme resistance to sorbates and benzoates, sediment. |
| Saccharomyces spp. | Sugary beverages | Unwanted fermentation, alcoholic off-flavor. |
| Aspergillus and Penicillium | Non-carbonated soft drinks | Formation of soft white masses (“cottony masses”), visual changes. |
The Limits of Traditional Chemistry: Why Conventional CIP Fails
Traditional Cleaning-In-Place (CIP) involves a series of washes using acidic and alkaline solutions (typically caustic soda and nitric or citric acid) at high temperatures (70–85°C).
Although this approach is effective at removing organic and mineral residues, its effectiveness against mature biofilms is limited for several biochemical reasons.
Physical and chemical resistance
Caustic soda solutions often fail to penetrate the entire thickness of the biofilm. The reaction between the alkali and the outer polymers can create an even more compact outer layer that protects the cells beneath.
Similarly, oxidizing disinfectants such as chlorine or peracetic acid are consumed by the organic matrix of the EPS before they can act on the bacteria.
It has been shown that sessile bacteria (in biofilms) are 100 to 1,000 times more resistant to disinfectants than their planktonic counterparts.
Material degradation and ecological niches
Excessive exposure to high temperatures and harsh chemicals damages the system’s components.
Rubber gaskets begin to degrade at pH levels above 9, and prolonged exposure to strong acids can cause “rouging” (corrosion) of stainless steel surfaces.
These damaged areas have microscopic pores where biofilm can accumulate, protected from the mechanical action of the CIP flow.
In essence, overly aggressive chemical cleaning can, paradoxically, create the conditions for faster and more extensive microbial recolonization.
The Enzymatic Revolution: Precision Mechanisms for Eradication
Enzymatic cleaning represents the paradigm shift needed for modern biofilm management.
Rather than a blanket chemical attack, enzymes act as specific biological catalysts that break down the molecular bonds of the EPS matrix, disrupting the biofilm’s supporting structure without damaging the surfaces.
Types of enzymes and molecular targets
The most recent scientific studies highlight the effectiveness of enzymatic cleaning against the EPS matrix, which is proportional to the variety and type of enzymes used.
Biorem® 3G, a third-generation enzymatic formulation patented by Realco-Piramide, is a unique blend on the market consisting of 8 families of different enzymes specifically designed to address the complex heterogeneity of the matrix. The 8 families include:
- Proteases: Essential for breaking the peptide bonds in proteins, which make up to 50% of the EPS. They are crucial for the eradication of Pseudomonas and Bacillus biofilms.
- Polysaccharidases (amylase, cellulase, pectinase), dispersin: Target complex sugars. Cellulase is vital against acetic acid bacteria biofilms, while pectinase and amylase remove fruit residues and starches that serve as nutrients for recolonization. Dispersin specifically targets PNAG, a specific polysaccharide in the EPS matrix, which is essential for strengthening the integrity of the organic matrix.
- Lipases: They act on fatty residues and the lipid components of the matrix, increasing the surface wettability and allowing other enzymes to penetrate more deeply.
- DNases: They degrade eDNA, which recent studies indicate is a key structural component for the integrity of mature biofilms.
Proven efficacy and synergy with biocides
Enzymatic action is not usually biocidal (it does not directly kill bacteria), but rather “disintegrating.”
Once the matrix has been hydrolyzed by enzymes, the bacteria revert to their planktonic form, making them vulnerable to common disinfectants such as peracetic acid.
Comparative studies have shown that incorporating enzymes into the cleaning protocol can result in a reduction in microbial load of more than 5 log, whereas traditional CIP often achieves no more than a 2- to 3-log reduction on mature biofilms.
In addition, enzymatic cleaning allows operations to be carried out at much lower temperatures, drastically reducing energy costs and equipment wear and tear.
To prevent regrowth, regular (weekly or monthly) use of Biorem® prevents microorganisms from secreting an EPS matrix thick enough to provide protection.
Sustainability and long-term economic benefits
In addition to its technical effectiveness, enzymatic cleaning addresses the beverage industry’s growing demand for sustainability (ESG).
- Water Conservation: Enzymes require less intensive rinsing cycles than caustic soda, as they do not leave behind aggressive chemical residues that need to be diluted extensively.
- Biodegradability: Enzymatic solutions are naturally biodegradable, reducing the environmental impact of wastewater and making the work of industrial wastewater treatment plants easier.
- Extending Equipment Lifespan: By reducing the frequency of cleaning at extreme temperatures and with strong acids, wear on rotary valves, nozzles, and elastomer seals is minimized.
- Operational Efficiency: Although the cost of the enzymatic formulation may be higher, the savings resulting from reduced product rework, plant downtime, and energy consumption make the ROI (Return on Investment) extremely attractive.
Conclusions and Implications for Industry
The issue of biofilms in mineral water and soft drinks is not merely a microbiological challenge, but a management risk that can compromise the entire value chain.
Science has unequivocally demonstrated that traditional sanitization methods are no longer sufficient to combat the resilience of evolved microbial communities protected by complex EPS matrices.
Adopting a biotechnology strategy based on enzymatic cleaning allows companies to shift from a “reactive” approach (addressing problems only after the product has already been contaminated) to a “proactive” approach that ensures complete control.
Piramide’s Biofilm Expert approach, which combines the identification of biofilm-forming strains with their removal using the patented Biorem® enzyme cocktail, ensures sustainable and long-lasting hygiene.
Biorem® 3G is the only octavalent enzyme blend on the market capable of hydrolyzing the maximum number of components in the organic matrix of biofilms, making it the most effective solution, particularly for deeply rooted 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 inherent in 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
Bibliography
- Biofilm Formation in Water Distribution Systems – MDPI
- Persistent Threats: A Comprehensive Review of Biofilm Formation, Control, and Economic Implications in Food Processing Environments –
- Evaluation of Enzymatic Cleaning on Food Processing Equipment and the Bacterial Microflora of Food Products
- Isolation of Extracellular Polymeric Substances from Biofilms of the Thermoacidophilic Archaea Sulfolobus acidocaldarius
- Pseudomonas aeruginosa: A typical biofilm-forming pathogen and an emerging but underestimated pathogen in food processing
- Biofilms and Their Impact on the Food Industry
- Microbiological contamination profile in soft drinks
- Microbial attachment and biofilm formation in brewery bottling plants
- Enzymes Boost the Biofilm-Removal Efficiency of Cleaners –
- Extracellular matrix-degrading enzymes as a strategy for controlling biofilms formed by foodborne microorganisms
Biofilm and Mineral Water: FAQ
The primary legal reference in Italy is Legislative Decree No. 176 of October 8, 2011, No. 176, which transposes European Directive 2009/54/EC on the exploitation and marketing of natural mineral waters, supplemented by the Ministerial Decree of February 10, 2015, on the criteria for evaluating the characteristics of natural mineral waters.
From a microbiological standpoint, the standard stipulates that, at the source, the total microorganism content must be consistent with the normal microbial flora of the water and demonstrate effective protection of the source against any contamination; the determination of the total microbial count must be carried out both at 20–22 °C after 72 hours and at 37 °C after 24 hours. Furthermore, pathogenic microorganisms and indicators of contamination (such as Escherichia coli and other coliforms, fecal streptococci, Pseudomonas aeruginosa, and spore-forming sulfite-reducing anaerobes) must be absent in the specified sample volumes.
A particularly important point regarding biofilm management: the regulations prohibit water treatment processes, the addition of bactericidal or bacteriostatic substances, and any treatment capable of altering the microbial composition of the water. The law also requires that water collection systems, pipelines, reservoirs, and, in particular, washing and bottling facilities be constructed and operated in a manner that meets hygiene requirements and prevents any alteration of the water’s characteristics. This framework is complemented by the general food hygiene requirements set forth in Regulation (EC) No. 852/2004, which requires the implementation of an HACCP system. Consequently, since it is not possible to treat the water itself, microbiological safety is ensured primarily through proper design and strict hygiene practices in the systems—precisely the area where biofilm control becomes critical.
It is worth considering a few key points. First and foremost, accreditation (ISO/IEC 17025) and specific experience in the mineral water and food industries, because the parameters and sampling methods are highly specialized. Then there is the need for methods suitable for biofilm analysis: it is not enough to analyze the water; surface sampling techniques (swabs, probes) and targeted research on indicator microorganisms and the contaminants most relevant to the sector are required.
Also useful are the ability to provide interpretation and technical support (not just the report), response times compatible with production schedules, and the ability to integrate the analyses into a continuous monitoring plan. A partner that combines laboratory data with process hygiene expertise—as is the case with Biorem® audits—helps translate the results into concrete actions on the production line.
Traditional chemical detergents (alkaline and acidic) act on the surface of the biofilm but struggle to penetrate the EPS matrix, often leaving behind a residual layer from which contamination can reform. Enzymatic products, on the other hand, are formulated to “digest” the structural components of the biofilm (proteins, polysaccharides), detaching it from surfaces and making its removal more effective.
For mineral water bottling lines, where it is not possible to treat the water with bactericidal agents, the most appropriate approach combines a preliminary analysis to identify critical points, a targeted enzymatic cleaner, and an application protocol (CIP/COP) tailored to the specific system. This is the approach behind Piramide’s Biorem® program, which combines contamination detection, elimination, and prevention with a dedicated audit service.
Prevention involves three complementary approaches. In terms of plant design: smooth surfaces suitable for food contact, geometries that prevent stagnation and dead spots, and fittings and gaskets in good condition. From a management perspective: regular, validated CIP/COP cleaning protocols, incorporating the proper combination of time, temperature, mechanical action, and cleaning agent, as well as scheduled microbiological monitoring. In terms of corporate culture: training of staff and traceability of hygiene procedures.
A crucial aspect for mineral water is that regulations prohibit the “disinfection” of the water itself: hygiene measures must therefore focus on the facility. With this in mind, enzymatic technologies—such as those offered by Piramide through its Biorem® program—act on the biofilm matrix, breaking it down at a deep level to remove the deposit and reduce its recurrence, while operating at a neutral pH and moderate temperatures.
Biofilm acts as a reservoir of microorganisms that can be released into the finished product by water flow or mechanical processes, compromising its microbiological compliance. The main risk is the presence of indicator or potentially pathogenic microorganisms (such as Pseudomonas aeruginosa, which is frequently associated with humid environments) and, more generally, an increase in the microbial load beyond expected levels.
Unlike free-floating (planktonic) microorganisms, those organized into biofilms are much more resistant to sanitization treatments: the EPS matrix reduces the penetration of cleaning and disinfecting agents. For this reason, biofilm contamination tends to be recurrent and difficult to eradicate, with potential consequences for food safety, product recalls, and brand reputation.
Biofilm forms when microorganisms in the water adhere to a surface (pipes, valves, tanks, fillers) and begin to produce a matrix of extracellular polymeric substances (EPS): a viscous film that protects them and keeps them anchored. The process proceeds through successive stages: initial reversible adhesion, stable adhesion, maturation of the three-dimensional structure, and, finally, detachment of fragments that can contaminate other areas of the system.
Factors that promote its growth include, above all, the presence of residual nutrients, rough, worn, or poorly welded surfaces, water stagnation, and dead spots in the circuit (areas with little flow), as well as inadequate cleaning routines. In mineral water production systems, even moderate temperatures and extended downtime between production runs can create ideal conditions for microbial growth.
