Tag Archives: species

March 29, 2022

Beyond Compliance: Understanding and Combating Contamination

By Jill Ellsworth MS, RDN, Tess Eidem, Ph.D.

As an emerging field in cannabis, contaminant testing remains a gray area for many businesses. The vast differences in state-by-state regulations, along with the frequent changes of previously established rules make testing a difficult, time-consuming process. But at its core, the science and reasoning behind why we test cannabis is very clear – consumer safety and quality assurance are key factors in any legal, consumer market. The implications of federal legalization make cannabis testing even more important to the future of the cannabis supply chain. Understanding the types of contaminants, their sources and how to prevent them is essential to avoiding failures, recalls and risking consumer safety.

When talking about cannabis contaminant testing there are four groups of contaminants: pesticides, heavy metals, foreign materials and microbes. The microbes found on cannabis include plant pathogens, post-harvest spoiling microbes, allergens, toxin release and human pathogens. While all of these can be lurking on the surface of cannabis, the specific types that are tested for in each state vary widely. Understanding the full scope of contaminants and looking beyond state-specific compliance requirements, cultivators will be able to prevent these detrimental risks and prepare their business for the future.

Environmental controls are essential to monitor and regulate temperature and humidity

Beyond just the health of the plant, both medical patients and adult use consumers can be adversely affected by microbial contaminants. To immunocompromised patients, Aspergillus can be life-threatening and both adult use and medical consumers are susceptible to allergic reactions to moldy flower. But Aspergillus is just one of the many contaminants that are invisible to the human eye and can live on the plant’s surface. Several states have intensive testing regulations when it comes to the full breadth of possible harmful contaminants. Nevada, for example, has strict microbial testing requirements and, in addition to Aspergillus, the state tests for Salmonella, STEC, Enterobacteriaceae, coliforms and total yeast and mold. Over 15 states test for total yeast and mold and the thresholds vary from allowing less than 100,000 colony forming units to allowing less than 1,000 colony forming units. These microbes are not uncommon appearances on cannabis – in fact, they are ever-present – so understanding them as a whole, beyond regulatory standards is a certain way to future-proof a business. With such vast differences in accepted levels of contamination per state, the best preparation for the future and regulations coming down the pipeline is understanding contamination, addressing it at its source and harvesting disease-free cannabis.

The risk of contamination is present at every stage of the cultivation process and encompasses agricultural practices, manufacturing processes and their intersection. From cultivation to manufacturing, there are factors that can introduce contamination throughout the supply chain. A quality control infrastructure should be employed in a facility and checkpoints within the process to ensure aseptic operations.

Microbial monitoring methods can include frequent/consistent testing

Cultivators should test their raw materials, including growing substrates and nutrient water to ensure it is free of microbial contamination. Air quality plays an important role in the cultivation and post-harvest processes, especially with mold contamination. Environmental controls are essential to monitor and regulate temperature and humidity and ensure unwanted microbes cannot thrive and decrease the value of the product or make it unsafe for worker handling or consumers. Developing SOPs to validate contact surfaces are clean, using proper PPE and optimizing worker flow can all help to prevent cross-contamination and are part of larger quality assurance measures to prevent microbes from spreading across cultivars and harvests.

Methods of microbial examination include air quality surveillance, ATP surface and water monitoring, raw materials testing, and species identification. Keeping control of the environment that product is coming into contact with and employing best practices throughout will minimize the amount of contamination that is present before testing. The solution to avoiding worst case scenarios following an aseptic, quality controlled process is utilizing a safe, post-harvest kill-step, much like the methods used in the food and beverage industries with the oversight of the FDA.

The goal of the grower should be to grow clean and stay clean throughout the shelf life of the product. In order to do this, it is essential to understand the critical control points within the cultivation and post-harvest processes and implement proper kill-steps. However, if a product is heavily bio-burdened, there are methods to recover contaminated product including decontamination, remediation and destroying the product. These measures come with their own strengths and weaknesses and cannot replace the quality assurance programs developed by the manufacturer.

November 14, 2019

Beyond THC: Encouraging Cannabinoid and Terpene Production with LEDs

By Andrew Myers

No Comments

For years, tetrahydrocannabinol (THC) got all the attention. While THC certainly delivers its own benefits (such as relaxation and pain relief), there’s a whole host of other – and often overlooked – compounds found in cannabis with important benefits as well. THC is truly only the tip of the iceberg when it comes to cannabis’s potential.

As the cannabis industry evolves with changing consumer tastes and developing medical research, growers may employ techniques to boost cannabinoid and terpene profiles in their harvests – beyond merely focusing on THC. Advanced LEDs allow growers to elicit specific biological responses in cannabis crops, including increased concentrations of these naturally occurring chemical compounds.

The Foundation of Cannabis’s Effects
Whether used medicinally or otherwise, cannabis has changed our society and many of our lives – and there’s a collection of naturally occurring chemical compounds, known as cannabinoids and terpenes, to thank.

The cannabinoids THC and CBD are the most common and well-researched, however they are accompanied by more than 200 additional compounds, including cannabinol (CBN), cannabigerol (CBG) and tetrahydrocannabivarin (THCV), among others.
The cannabis plant also contains terpenes. These structures are responsible for giving flowers (including cannabis), fruits and spices their distinctive flavors and aromas. Common terpenes include limonene, linalool, pinene and myrcene.

Both cannabinoids and terpenes are found in the cannabis plant’s glandular structures known as trichomes. Look closely, and you’ll notice trichomes coating the cannabis flowers and leaves, giving the plant an almost frosty appearance.

macropistil/trichome — A macro view of the trichomes and pistils on the plant

Trichomes – which are found across several plant species – are a key aspect of a cannabis plant’s survival. The specific combination of metabolites produced by trichomes may attract certain pollinators and repel plant-eating animals. Moreover, trichomes (and specifically THC) may act as the plant’s form of sunscreen and shield the plant from harmful ultraviolet rays.

While they play an essential part in the cannabis plant’s lifecycle, trichomes are volatile and easily influenced by a range of environmental factors, including light, heat, physical agitation and time. Therefore, environment is a defining variable in the development of these important structures.

How LEDs Support Cannabinoid and Terpene Development in Crops
Spectrally tunable LEDs give indoor cannabis growers unparalleled control over their crops. As research has expanded about plants’ responses to the light spectrum, growers have discovered they are able to elicit certain physiological responses in the plant. This phenomenon is called photomorphogenesis. At its root, photomorphogenesis is a survival tactic – it’s how the plant responds to miniscule changes in its environment to increase the chances of reaching full maturity and, eventually, reproducing. While cultivated cannabis plants won’t reproduce at an indoor setting, growers can still use the light spectrum to encourage strong root and stem development, hasten the flowering process and the development of bigger, brightly colored flowers.

It makes sense that using the proper light spectrums may also have an impact on the production of specific cannabinoids and terpenes – an important factor when responding to highly specific consumer needs and desires, both within medical and adult-use markets.

Here are a few more reasons why utilizing full-spectrum LEDs can lead to higher quality cannabis:

Lower Heat, but the Same Intensity.
When compared to HPS, fluorescent and other conventional lighting technologies, LEDs have a much lower heat output, but provide the same level of intensity (and often improved uniformity). This represents an enormous advantage for cannabis cultivators, as the lights can be hung much closer to the plant canopy without burning trichomes than they would be able to with other lighting technologies.
UV Light. Cannabinoids and terpenes are part of the cannabis plant’s natural defense mechanism, so it makes sense that lightly stressing plants can boost cannabinoid and terpene numbers. Some studies illustrate an increase in UV-B and UV-A light can lead to richer cannabinoid and terpene profiles.¹ It’s a fine line to walk, though – too much UV can result in burned plants, which leads to a noticeable drop in cannabinoids.
Full-Spectrum Capabilities. The cannabis plant evolved over millions of years under the steady and reliable light of the sun. Full-spectrum is the closest thing to natural sunlight that growers will be able to find for indoor growing – and they’ve been shown to perform better in terms of cannabinoid development. A 2018 study titled “The Effect of Light Spectrum on the Morphology and Cannabinoid Content for Cannabis Sativa L.,” explored how an optimized light spectrum resulted in increased expression of cannabinoids CBG and THCV.²

This is the most important tip for indoor growers: your plants’ environment is everything. It can make or break a successful harvest. That means cultivators are responsible for ensuring the plants are kept in ideal conditions. Lights are certainly important at an indoor facility, but there are several other factors to consider that can affect your lights’ performance and the potency of your final product. This includes your temperature regulation, humidity, the density of plants within the space, CO2 concentration and many other variables. For the best results, your lights should be fully aligned with other environmental controls in your space. Nothing sabotages a once-promising crop like recurrent issues in the indoor environment.

solsticegrowop_feb — Indoor cultivation facilities often use high powered lights that can give off heat

Cannabinoids and terpenes take time to develop – so cultivators will want to avoid harvesting their plants too early. On the other hand, these compounds begin to degrade over time, so growers can’t wait too long either.

Cultivators seeking potent cannabinoid and terpene profiles must find a happy medium for the best results – and the best place to look is where cannabinoids and terpenes develop: the trichomes. With a microscope, cultivators can get up close and personal with these sparkly structures. Younger plants begin with clear trichomes, which eventually become opaque and change to amber. Once your plants show amber-hued trichomes, they’re ready for harvest.

The truth here is that there’s no perfect formula to elicit show-stopping cannabinoids and dizzying terpenes with every harvest. A lot of cannabis cultivation is based around trial-and-error, finding what works for your space, your business and your team. But understanding the basics around indoor environmental controls like lighting and temperature – and how they can affect the development of cannabinoids and terpenes – is an excellent place to start. Using high quality equipment, such as full-spectrum LED lighting can boost both cannabinoid and terpene production, resulting in richer, more potent and higher quality strains.

References:

Lyndon, John, Teramura, Alan H., Coffman, Benjamin C. “UV-B Radiation Effects on Photosynthesis, Growth and Cannabinoid Production of Two Cannabis Sativa Chemotypes.” August 1987. Photochemistry and photobiology. Web. https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1751-1097.1987.tb04757.x?&sid=nlm%3Apubmed
Magagnini G., Grassi G., Kotiranta, S. “The Effect of Light Spectrum on the Morphology and Cannabinoid Content of Cannabis sativa L.” 2018. Medical Cannabis and Cannabinoids. Web: https://www.karger.com/Article/FullText/489030

October 21, 2019

3 Essential Components of Microbial Safety Testing

By Heather Ebling

1 Comment

Microbial contamination on cannabis products represents one of the most significant threats to cannabis consumers, particularly immunocompromised patients who are at risk of developing harmful and potentially fatal infections.

As a result, regulatory bodies in the United States and Canada mandate testing cannabis products for certain microbes. The two most popular methods for microbial safety testing in the cannabis industry are culture-based testing and quantitative polymerase chain reaction (qPCR).

When considering patient safety, labs should choose a method that provides an accurate account of what is living on the sample and can specifically target the most harmful microbes, regardless of the matrix.

1. The Method’s Results Must Accurately Reflect the Microbial Population on the Sample

The main objective of any microbial safety test is to give the operator an indication of the microbial population present on the sample.

Figure 1: MA data collected directly from plant material before and after culture on 3M petrifilm and culture-based platforms.

Culture-based methods measure contamination by observing how many organisms grow in a given medium. However, not all microbial organisms grow at the same rate. In some cases, certain organisms will out-compete others and as a result, the population in a post-culture environment is radically different than what was on the original sample.

One study analyzed fifteen medicinal cannabis samples using two commercially available culture-based methods. To enumerate and differentiate bacteria and fungi present before and after growth on culture-based media, all samples were further subjected to next-generation sequencing (NGS) and metagenomic analyses (MA). Figure 1 illustrates MA data collected directly from plant material before and after culture on 3M petrifilm and culture-based platforms.

The results demonstrate substantial shifts in bacterial and fungal growth after culturing on the 3M petrifilm and culture-based platforms. Thus, the final composition of microbes after culturing is markedly different from the starting sample. Most concerning is the frequent identification of bacterial species in systems designed for the exclusive quantification of yeast and mold, as quantified by elevated total aerobic count (TAC) Cq values after culture in the total yeast and mold (TYM) medium. The presence of bacterial colonies on TYM growth plates or cartridges may falsely increase the rejection rate of cannabis samples for fungal contamination. These observations call into question the specificity claims of these platforms.

The Live Dead Problem

Figure 2: The enzyme is instantaneously inactivated when lysis buffer is added

One of the common objections to using qPCR for microbial safety testing is the fact that the method does not distinguish between live and dead DNA. PCR primers and probes will amplify any DNA in the sample that matches the target sequence, regardless of viability. Critics claim that this can lead to false positives because DNA from non-viable organisms can inflate results. This is often called the Live-Dead problem. However, scientists have developed multiple solutions to this problem. Most recently, Medicinal Genomics developed the Grim Reefer Free DNA Removal Kit, which eliminates free DNA contained in a sample by simply adding an enzyme and buffer and incubating for 10 minutes. The enzyme is instantaneously inactivated when lysis buffer is added, which prevents the Grim Reefer Enzyme from eliminating DNA when the viable cells are lysed (see Figure 2).

2. Method Must Be Able to Detect Specific Harmful Species

Toxic Aspergillus spp., which is responsible for at least one confirmed death of a cannabis patient, grows poorly in culture mediums and is severely underreported by current culture-based platforms. And even when Aspergillus does grow in culture, there is a certain non-pathogenic Aspergillus species that look remarkably similar to their pathogenic cousins, making it difficult to speciate using visual identification alone.

Conversely, qPCR assays, such as the PathoSEEK, are designed to target DNA sequences that are unique to pathogenic Aspergillus species, and they can be run using standard qPCR instruments such as the Agilent AriaMx. The primers are so specific that a single DNA base difference in the sequence can determine whether binding occurs. This specificity reduces the frequency of false positives in pathogen detection, a frequent problem with culture-based cannabis testing methods.

Additionally, Medicinal Genomics has developed a multiplex assay that can detect the four pathogenic species of Aspergillus (A. flavus, A. fumigatus, A. niger, and A. terreus) in a single reaction.

3. The Method Must Work on Multiple Matrices

Figure 4: The team also placed TSB without any E. coli onto a petrifilm to serve as a control.

Marijuana infused products (MIPs) are a very diverse class of matrices that behave very differently than cannabis flowers. Gummy bears, chocolates, oils and tinctures all present different challenges to culture-based techniques as the sugars and carbohydrates can radically alter the carbon sources available for growth. To assess the impact of MIPs on colony-forming units per gram of sample (CFU/g) enumeration, The Medicinal Genomics team spiked a known amount of live E. coli into three different environments: tryptic soy broth (TSB), hemp oil and hard candy. The team then homogenized the samples, pipetted amounts from each onto 3M™ Petrifilm E. coli / Coliform Count (EC) Plates, and incubated for 96 hours. The team also placed TSB without any E. coli onto a petrifilm to serve as a control. Figures 3 and 4 show the results in 24-hour intervals.

Table 1: DNA was spiked into various MIPs

This implies the MIPs are interfering with the reporter assay on the films or that the MIPs are antiseptic in nature.

Many MIPs use citric acid as a flavoring ingredient which may interfere with 3M reporter chemistry. In contrast, the qPCR signal from the Agilent AriaMx was constant, implying there is microbial contamination present on the films, but the colony formation or reporting is inhibited.

Table 3: SenSATIVAx DNA extraction can successfully lyse the cells of the microbes

Table 2: Different numbers of DNA copies spiked into chocolate

This is not an issue with DNA-based methods, so long as the DNA extraction method has been validated on these matrices. For example, the SenSATIVAx DNA extraction method is efficient in different matrices, DNA was spiked into various MIPs as shown in Table 1, and at different numbers of DNA copies into chocolate (Table 2). The SenSATIVAx DNA extraction kit successfully captures the varying levels of DNA, and the PathoSEEK detection assay can successfully detect that range of DNA. Table 3 demonstrates that SenSATIVAx DNA extraction can successfully lyse the cells of the microbes that may be present on cannabis for a variety of organisms spiked onto cannabis flower samples.