Faster Clostridium Detection in Milk with Raman Spectroscopy

Milk analysis: cow with milk bottle © 2rogan - stock.adobe.com

Clostridium contamination poses a major challenge for the dairy industry, especially in the production of hard and semi-hard cheeses. The bacteria’s gas production during fermentation causes defects such as cracks and slits in cheese, leading to economic losses and quality issues. Traditional detection methods are slow, labor-intensive, and often lack specificity. In a groundbreaking study published in Applied Spectroscopy, a team of researchers led by Daniele Barbiero, Fabio Melison, Luca Poletto, and colleagues from the National Research Council’s Institute for Photonics and Nanotechnologies and Veneto Agricultura in Italy has revealed a Raman spectroscopy-based solution to this longstanding problem (1). There is a history of analysis of milk using Raman spectroscopy (1–3).

The Need for a Better Detection Method

Clostridium spores are remarkably resilient, surviving pasteurization and reactivating during cheese aging. Their metabolic activity leads to the production of hydrogen and carbon dioxide gases, which compromise the texture and flavor of cheeses such as Grana Padano. Current detection techniques, including microbiological and molecular methods, have limitations: they are time-consuming, require specialized personnel, or are cost-prohibitive for routine use in the dairy industry (1).

Microbiological methods, such as the most probable number (MPN) approach, lack specificity and take several days to yield results. Molecular techniques like polymerase chain reaction (PCR) can be faster but face challenges with complex food matrices and high costs. Similarly, immunological assays struggle with sensitivity, often missing low concentrations of spores. Recognizing these challenges, the research team sought to develop an efficient, cost-effective, and user-friendly alternative (1).

Raman Spectroscopy as a Solution

The new detection method leverages Raman spectroscopy, a non-invasive technique that analyzes the unique vibrational properties of molecules (1-3). By targeting the hydrogen gas produced exclusively by Clostridium metabolism, the Raman-based system distinguishes Clostridium from other bacteria, such as Bacilli, which produce only carbon dioxide (1).

The researchers designed a compact and cost-effective Raman gas analyzer tailored to meet the needs of the dairy industry. Key components include a solid-state laser for excitation, a custom spectrometer, and a borosilicate glass vial optimized for gas sampling. The system’s automated routine simplifies the detection process, eliminating the need for highly trained personnel (1).

Experimental Design and Validation

In the study, milk samples inoculated with Clostridium spores were incubated in vials and analyzed using both the Raman spectroscopy method and a reference MPN-based method. The Raman setup utilized a 532 nm laser to excite gas molecules in the vial’s headspace, and a complementary metal-oxide-semiconductor (CMOS) detector captured the scattered light. The system’s sensitivity allowed for the detection of hydrogen signals as low as 29 spores per liter (1).

The Raman spectroscopy method identified the first infected vial within 27 hours of incubation, compared to 44 hours required by the standard method. The entire detection campaign was completed in just 54 hours using Raman spectroscopy, nearly halving the 96-hour timeframe of the traditional approach. Importantly, the Raman method demonstrated perfect agreement with expected infection rates, confirming its accuracy and reliability (1).

Advantages Over Traditional Methods

The Raman spectroscopy-based system offers numerous advantages (1):

Speed: Detection times are significantly reduced, enabling faster decision-making in dairy processing.

Sensitivity: The system reliably detects Clostridium at low spore concentrations, preventing late-blowing defects in cheese.

Cost-Effectiveness: By using inexpensive materials and requiring minimal training, the system is accessible to a wide range of dairy operations.

Specificity: Unlike traditional methods, Raman spectroscopy distinguishes Clostridium from competing bacteria based on unique hydrogen gas signatures.

Implications for the Dairy Industry

The adoption of this technology could revolutionize quality control in the dairy sector. Rapid identification of Clostridium allows producers to take preventive measures, reducing economic losses and maintaining product quality. Additionally, the system’s automated and non-invasive design aligns with the industry’s push for efficiency and sustainability.

This study represents a significant advancement in food safety and quality assurance. By harnessing the power of Raman spectroscopy, the researchers have addressed a critical challenge in dairy production, offering a faster, more reliable, and cost-effective method for detecting Clostridium in milk. Future research could further optimize the system and expand its applications to other areas of food safety.

References

(1) Barbiero, D.; Melison, F.; Cocola, L.; Fedel, M.; Andrighetto, C.; Dea, P. D.; Poletto, L. Raman Spectroscopy Applied to Early Detection of Clostridium Infection in Milk. Appl. Spectrosc.2024, 78 (12), 1256–1262. DOI: 10.1177/00037028241252693

(2) He, H.; Sun, D. W.; Pu, H.; Chen, L.; Lin, L. Applications of Raman Spectroscopic Techniques for Quality and Safety Evaluation of Milk: A Review of Recent Developments. Crit. Rev. Food Sci. 2019, 59 (5), 770–793. DOI: 10.1080/10408398.2018.1528436

(3) Mazurek, S.; Szostak, R.; Czaja, T.; Zachwieja, A. Analysis of Milk by FT-Raman Spectroscopy. Talanta 2015, 138, 285–289. DOI: 10.1016/j.talanta.2015.03.024