The Hidden Side of Cedar

By Mélanie Trudel, Researcher, Innofibre

 

In Quebec, cedar is a conifer commonly used in landscaping. This tree, which can reach several meters in height, requires annual trimming. These cedar residues generate tons of biomass each year. Given the amount of residues available, it is interesting to explore ways to valorize this material to promote a circular economy. Among the many valorization possibilities, extracting molecules from this biomass for the development of bioactive ingredients for the cosmeceutical and nutraceutical markets presents a promising opportunity. This article will focus on the work carried out on developing an antimicrobial extract from cedar residues as part of a project funded from 2023 to 2024 by the Research and Transfer Assistance Program (PART) of the Ministry of Higher Education of Quebec (MES).

Why investigate the antimicrobial properties of cedar?

For nearly 10 years, Innofibre has developed expertise in extracting antimicrobial and antifungal molecules from forest biomasses such as spruce, poplar, and birch barks. With its specialized equipment, Innofibre can produce extracts at both the laboratory and pilot scales. Access to a level 2 microbiology laboratory also allows for testing the effectiveness of extracts on a variety of bacterial and fungal strains. Screening methods for handling a large number of extracts have also been developed at Innofibre to enhance the efficiency of quantitative tests.

How are antimicrobial molecules extracted?

Cedar residues must first be prepared through drying, sieving, and grinding. Drying the biomass prevents the growth of microorganisms on the residues. Grinding reduces the diameter of the residues to facilitate extraction. The finer the material, the greater the contact surface between the biomass and the liquid during extraction. Sieving removes contaminants such as sand and inorganic matter that could interfere with the extraction process. After these conditioning steps, extraction can be performed. Several extraction techniques exist. The method favored by Innofibre is based on a principle similar to that of a coffee machine, where a volume of water passes through a solid material (Figure 1).

 

                                                                                   Figure 1: Water extraction of conditioned cedar

Specifically, a determined mass of conditioned residues is placed in a paper-making washer as illustrated in Figure 1, and a volume of water circulates through the residues at a specific temperature. After several sequences of liquid circulation through the residues, the water is collected. This water contains certain molecules from the cedar that have dissolved in the water, similar to how coffee contains the molecules from ground coffee beans after percolation. This water rich in molecules now constitutes the extract. Cedar contains several water-soluble molecules such as sugars (polysaccharides), tannins, and terpenes (e.g., thujone). Some of these molecules are known to have antimicrobial properties. A liquid-liquid extraction process was subsequently performed to determine if it could concentrate the antimicrobial molecules further. Two extracts were produced as part of the project (Figure 2). The dry extracts were then formulated into different forms.

 

                                                                 Figure 2: Dry cedar extract with water (A) and organic solvent (B)

Extracts to the Rescue of Mastitis!

Mastitis is an infectious disease affecting the mammary glands of cattle. It can be caused by bacteria, fungi, yeasts, and algae. It is the disease causing the greatest economic loss for dairy producers, as milk from affected cows is unsuitable for consumption. In Canada, losses total more than 400 million dollars annually, which represents about 15% of the total net income of the dairy industry. Furthermore, only two antibiotics (Cefa-lak and Spectramast LC) are approved for the treatment of bacterial mastitis in Canada. Antibiotic resistance is becoming more frequent, making it more difficult to eradicate the disease. Additionally, as antibiotics are effective only against bacteria, an alternative treatment for this condition is necessary. Some forest extracts can be effective against various microorganisms such as bacteria and fungi. The project aimed to develop an extract from cedar residues that could be used to treat affected cows or prevent the spread of the disease through equipment cleaning.

What is antimicrobial efficacy?

The effectiveness of the two types of cedar extract produced was tested on two bacterial strains isolated from cows with mastitis, namely E. coli and S. aureus. The extracts were incorporated into four different formulations. These aimed to reproduce possible on-farm applications, such as an ointment applied to a cow’s teats, a teat dipping solution, a surface disinfectant, and a bedding additive. The antimicrobial efficacy of these formulations was measured by counting the number of residual bacteria following their contact with the formulations. Bacteria that were in contact were diluted in series, and these dilutions were spread on solid media containing nutrients for microorganisms. The bacteria that survived the treatment will feed on the nutrients in the medium and reproduce. The reproduction of a bacterium creates a colony on the solid medium (Figure 3), formed by the reproduction of that single cell. The number of colonies counted corresponds to the number of bacteria present per milliliter following contact with the formulation. This number is compared to a control that was not in contact with the formulation containing the extract. By subtracting the number of bacteria that were in contact from the number that was not in contact, the reduction in the number of active bacteria per milliliter can be calculated, expressed as log reduction of cells, due to the effect of the formulation. The log reductions represent the number of bacteria killed by the formulation. The extract used for the test shown in Figure 3 is the organic cedar extract. This test concluded that this extract is effective in reducing the number of Staphylococcus aureus bacteria. It could therefore be effective in controlling their proliferation and reducing the risk of mastitis development.

 

Figure 3: Solid media containing Staphylococcus aureus colonies. (A) Control – contact with formulation without antimicrobial extract and (B) contact with formulation containing antimicrobial extract

In conclusion, these results are promising for the valorization of cedar residues. Collaborations with research partners in the agri-food sector would help deepen knowledge in this area and combine efforts to validate these results under real use conditions. Further testing to evaluate other microorganisms will be required to validate the potential of these extracts.