Advancing analytical microbiology in the dairy industry

Innovation is a key contributor for product superiority. Among innovations in the dairy industry, the study of fermenting microorganisms takes an important part as they are essential ingredients in product manufacturing. Fermenting microorganisms are often used as a mix of species composing a beneficial microflora in the final dairy product for (i) texture and organoleptic properties; (ii) for product preservation against pathogens; (iii) for health benefit properties. All these beneficial aspects are mostly driven by fermenting microorganisms bringing added value to the products. Among beneficial microbes, probiotic used in dairy products brings health benefits through its consumption. Hence, an increasing interest in the commercial exploitation of selected lactic acid bacteria (LAB) and probiotics in the food industry gives rise to many new products launched each year (Figure 1).

Sales of yoghurt and yoghurt-related products including Greek yoghurt, fermented milk product associated to functionality, or traditional products like kefir have increased 40 per cent since 2008 and are projected to increase between five to seven per cent per year between now and 2017 (source: Mintel). Growth of sales is also associated with a necessity to characterize more and more complex dairy products; e.g. fermented milk associated with probiotics often contains two more species than usually used in yoghurt, and traditional products like kefir or cheese contain a genuine microflora composed of several species of bacteria, yeasts and moulds, whose complete diversity is not well known. In parallel, to guarantee quality of some dairy food categories or dairy ingredients, CODEX and WHO/FAO have provided definitions of yoghurt and probiotic categories, respectively^2,3. Both definitions state that microorganisms have to be alive and in a sufficient number in final products. Therefore, dairy microbiology needs methods with high discriminatory power to quantify probiotics in more and more complex dairy products, with information related to their ability to survive along the shelf life.

Some standardised methods based on culture media are available to qualify these beneficial bacteria and are widely used to monitor enumeration of fermenting microorganisms or probiotics in the final product as recommended by standardisation committees. Selective culture methods rely on cultivability of microorganism but it’s actually a narrow way to represent only a part of the bacterial population present in the product. Indeed, viable but not cultivated (VBNC) bacteria are not taken into account by these methods as well as their metabolic activities. Additionally, these culture-dependent methods are time-consuming, labour-intensive and show poor discriminatory power. Therefore, the dairy industry requires new, alternative methods to perform qualitative and quantitative measurements of fermented milk products and this represents the stakes of analytical microbiology today. We would like to shed light here that this challenge can be faced with alternative methods based on molecular biology or flow cytometry, which could offer new analytical solutions to dairy microbiology.

Fuel product superiority with bioanalytic management: From conception to standardisation

Before going deeper into technologies, we would like to introduce our analytical management system (Figure 2). For us, innovation in bioanalytics is a key priority to leverage performance of methods. The performance is represented by the combination of basic analytical criteria (specificity, sensitivity, repeatability, reproducibility, linearity, robustness with the uncertainty value, accuracy, precision), as well as the cost and speed of analysis to meet the industrial demands.

• Firstly, bioanalytics requires to be connected to cutting-edge technologies and should select the best one in this ‘Analytical Cloud’ to translate it into a performing method that could be applied in food industry. Selection of technologies that will produce the methods of tomorrow could be fostered with pre-competitive research through cooperative ways of working with academic and private partners.
• The second phase is the ‘Proof of Concept’ that corresponds to the first results that we can get from testing a new idea with the selected technology into our domain of application. Here we expect to demonstrate the idea feasibility and to verify its potential to be used in R&D or quality control.
• The third phase, called ‘Proof of Performance’, aims to optimise the analytical criteria cited above to reach the highest performance. The performance of methods is the key driver, as it warrants the delivery of reliable analytical results at an industrial scale later.
• The last phase is to work on standardisation and it requires qualifying the performance of the methods that were already optimised. It generally implies contribution of an analytical network of laboratories for intra- and/or inter-laboratories testing in order to know result variability using the same method. This step is necessary to industrialise method use in multiple laboratories. The International Organization for Standardization (ISO) and International Dairy Federation (IDF) are particularly key partners in delivering standards in the dairy industry.
• For the final industry application of the method, a constructive way to perform analysis for long-term needs is to work with partners that can provide commercialised instruments and ready-to-use kits produced in a standard way.

The system should be managed by a balanced portfolio to deliver a continuum of methods from conception to standardisation. Overall, these different inter-connected phases are thus integrated in an analytical management process that continually feeds innovation, quality and superiority of dairy products.

Deciphering the complexity of dairy products with molecular tools

A growing scientific literature showed that enumerating fermenting microbes or probiotics in dairy products could be successfully performed with molecular methods. Recently, we published a review showing the new analytical opportunities of using PCR-based methods in the dairy industry⁴. The recent advances in quantitative PCR (qPCR) applications offer a faster and reliable analytical tool to perform experiments with high throughput/automation analyses and thus could subsequently reduce costs per assay, increase reliability of results and meet industrial demands. The strength of the molecular technics is its high discriminatory power to target multiple microorganisms in samples. Moreover, the boom of genome sequencing provides a rich support of genetic information for designing a specific molecular biomarker. We emphasise that the dairy industry cannot settle for basic qPCR, one of whose major drawbacks is that qPCR counts could not be directly associated with cell viability as samples are often composed of dead/viable cell mixtures. Fortunately, viability PCR (v-PCR), a new kind of PCR, has been developed for several years to overcome this limitation using impermeable nucleic binding dye like ethidium monoaside bromide (EMA) or propidium monoaside (PMA) prior DNA extraction and qPCR.

In this technique, the membrane integrity is used as a viability marker of the microorganism so that a compromised membrane can be permeable to the dye which can penetrate only into dead cells, intercalate with DNA and subsequently inhibit DNA amplification, and thus allow discrimination between viable cells, including VBNC, and dead cells. Proof of concept of this new method and its reliability was attested on dairy products to quantify viable probiotic strains of L. acidophilus, L. casei and B. lactis in fermented milk and B. animalis, L. rhamnosus and L. helveticus species in Cheddar cheese⁵. V-PCR method was also applied to quantify viable probiotics in faecal samples from people having ingested fermented milk products⁶. This method could therefore be used for evaluating probiotic’s ability to resist stressful conditions during their transit through the gastrointestinal tract. The increasing popularity of v-PCR produces many promising results but more and more different protocols were developed without proper evaluation of the robustness.

Standardisation of real-time PCR methods is growing with sets of guidelines like MIQE that describe the minimum information necessary to evaluate qPCR experiments⁷, or ISO22119:2011⁸ guidelines that define requirements to detect food-borne pathogens in foodstuffs by PCR and qPCR. The recent recommendations for better use of qPCR in the food industry should be used to develop alternative methods based on qPCR for quantification of dairy microorganisms^9,10. Digital PCR or droplet digital PCR that are based on the principle of the most probable number for target quantification appears to be a new generation of PCR that could facilitate standardisation of PCR. Indeed, it provides absolute quantification without the need of setting up standard curves and also produces data with better accuracy than qPCR. Furthermore, this system opens great opportunities for developing multiple testing in a single analysis, which increases throughput of analysis. No application has yet been described for microbe’s quantification in food. However, good performances of dPCR from proof of concept studies in clinical application and the recent availability of guidelines for warrant delivery of dPCR data of quality, i.e. dMIQE¹¹, suggest that dPCR or even v-dPCR could bring new analytical benefits in food microbiology in the future.

Complementary to these targeted molecular approaches, new opportunities for bioanalytic have appeared with the great development of untargeted approaches. They are classically referred to as the ‘omics’ approaches and are able to provide a more complete picture of product biological composition. Metagenomic, thanks to the boom of high throughput DNA sequencing, is a very helpful tool to draw the taxonomic description of microorganisms present in a sample. Usually performed to study microbial diversity in complex environments like gut microbiota or environmental samples, this technology has been recently applied to complex dairy foodstuffs, such as raw milk¹², cheese¹³ and kefir¹⁴. Most of the time, metagenomic reveals presence of taxa not traditionally identified by culture-based approaches. Moreover, viability dye can be associated with the metagenomic to provide information related to the viable community present in sample¹². Thus, a food v-metagenomic approach could be very powerful to optimise and control manufacturing of complex dairy products like cheese or kefir that contains diverse bacteria, yeasts and moulds. However, metagenomic is associated with sillico analyses and therefore requires a development of expertise in bioinformatic to treat the high quantity of data. This untargeted screening shows a great potential to be used in the future in routine product testing to check product compliance in a real time single analysis, both for safety control consideration and for conformity regarding specification of beneficial bacteria count. But this interesting perspective would be reached only if the performance of the untargeted screening with food metagenomic is confirmed, hence moving from a proof of concept phase to a well-standardised method.

Flow cytometry: Technology without border

Flow cytometry (FCM) is not a new technology. FCM in combination with fluorescent techniques have been widely used in clinical diagnosis, pharmaceutical application and fundamental research for the multiparameter analysis of cell populations. This technique was introduced 10 years ago in dairy industry for the evaluation of somatic cell load and total bacteria count in raw milk, which has revolutionised the grading of raw milk, producing rapid and accurate results¹⁵.

Later on, FCM was recognised as an ideal tool to evaluate the metabolic activity of the LAB and probiotic starter cultures. It has been successfully used to predict LAB physiology during strain propagation for ferment manufacturing. FCM has also been applied to assess the viability of probiotic starter cultures through storage and to monitor cell damage after stress treatments¹⁶. There is a range of fluorescent probes associated with various cellular functions (e.g. membrane integrity, membrane potential, intracellular pH, enzymatic activity, intracellular ions), making the fine characterisation of the cell physiology possible. Meanwhile, the viable population could be enumerated in a near real-time analysis (<1h). Quantification result by FCM are generally more than that found on the plate, which reveals that there is a considerable amount of VBNC population in the dairy starter culture during processing, storage and stress treatment¹⁰.

FCM has been proposed as a means to enumerate viable probiotic populations in dairy products. However, while a whole population can be characterised, distinction between near genera or species remains difficult. Recently in Danone, we developed a specific FCM method to enumerate viable Bifidobacteria in commercial products by using double staining of antibody and viability probe¹⁷. With the development of custom antibodies, specific FCM enumeration of probiotics will be accessible to characterise more and more complex dairy products. Beyond the simple enumeration, FCM provide higher knowledge about microbial fitness in the products from production until the end of shelf life.

FCM appears as a very promising tool for analyses of raw milk, starter culture and final products in the dairy industry. The implantation of this technology as routine analysis requires an automated system and standardised method. Today, some automated FCM are accessible in the market, while the commercialized kits are rather dedicated to the detection of microbiology contaminants. Since 2012, an ISO standardised FCM method is being set-up under coordination of the IDF, for enumeration of LAB in starter cultures and their applications¹⁸.With the development of appropriate kits and validation of the ISO method, FCM will be a prospective microbiology analytical solution for tomorrow’s challenge.

Conclusion

Analytical methods to detect food-borne pathogens have been well developed and standardised for safety control. Meanwhile, methods with high performance to qualify beneficial bacteria are still missing in spite of the exponential growth of probiotic products in the dairy market (Figure 3).

Today, culture-based methods are validated as a reference in food microbiology but only three ISO/IDF culture-based methods for the enumeration of fermenting bacteria and probiotics are available. Moreover, these methods are facing great challenges to analyse more and more complex dairy products. Innovation on culture medium as chromogenic media might overcome some limitations of these widely routine used methods. The promising flow cytometry methods offer more rapid and accurate enumeration, with fine metabolic characterisations of each bacterium in the products. With the publication of IDF/ISO method and development of appropriate kits for the automation system that already exist in the dairy industry, it will again revolutionise analysis food. Alternative methods based on molecular biology like v-PCR have shown high discrimination power on complex products and rapid time-to-result in many proof of concept studies. Evaluation of the performance through method standardisation and commercialised automation kits are still needed to enhance further application. Fast developments of untargeted approaches as metagenomic also bring new opportunities to analyse very complex dairy products.

The future of analytical methods for the dairy industry will be ‘on-line’, ‘real-time’ and ‘all-in-one’, with advancing analytical technologies that should show a total picture of every microorganism in the products; it will provide information from pathogen identification to probiotics fitness in one analysis. The targeted analyses will be replaced by untargeted screening, which meets all the needs from safety to superiority aspects.

The key points for the management of the analytical system are the stimulation of continued innovation and the promotion of win-win collaborations with different partners in each phase of the system.

References

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About the authors

Mickaël Boyer, PhD, is an analytical microbiology and molecular biology Team Leader for Danone Nutricia Research. From 2003 to 2008, he worked in academia as a microbial ecologist to study plant probiotics and their application as a natural fertiliser. He developed expertise in molecular biology and microbiology. From 2008 to 2011, he held a scientific position in an academic infectious disease centre and has experience in virology and metagenomic for application in health. From 2011, he has developed analytical expertise in dairy microbiology to speed up innovation at Danone

Jing Geng is an analytical microbiology scientist in Danone Nutricia Research. In 2008, she obtained her PhD in Microbiology from Wuhan University, China. From 2008 to 2010, she worked in the Institute Curie in Paris to study the biofilm formation by flow cytometry. Since 2010, the focus of her work contributes to the development and application of advanced microbiology methods for scientific research, products development and quality control in dairy industry.