Choosing the right extraction method for a given ingredient or product requires consideration of the characteristics of each available method. There are several. Oct 30, Which extraction method is best for your cannabis company? Hydrocarbon extraction typically refers to using butane or propane as a solvent that’s passed through the raw cannabis matter to collect cannabinoids and terpenes. Ethanol extraction is conducted by soaking raw cannabis. Jul 25, By Rick Scarpello, CEO of Medically Correct. Let's discuss cannabis extraction methods with facts and not religion. I'm pro-faith, but let's call it.
the Method Extraction Choosing Right
On the other hand, a producer may seek to create a single extract with many desirable cannabis compounds; sometimes called whole plant extracts. Including the better-known cannabinoids, scientists have identified more than chemicals in cannabis in general including components like terpenes.
Extraction techniques are also used to concentrate chemicals of interest. This article goes over the most common methods of cannabis extraction. Before discussing these methods, readers should keep in mind that cannabis extraction is chemistry, not cooking.
So, some skills in analytical methods plus real lab equipment is required to perform these methods correctly and safely. In many cases, the reagents and how they are used can create dangerous situations.
Consequently, many of the techniques require safety equipment, like a fume hood. Just as important, the extraction process must be performed correctly to produce a safe product for human use, and the results should be confirmed independently with proper analytical testing. In addition, if any CO 2 remains in an extract after the process, it just evaporates. That is especially important for any preparations for medical uses as a producer using this method can guarantee that absolutely no residual solvent will be present in the final product.
Despite the need for some financial investment in a CO 2 extraction system, a manufacturer can consider various levels of equipment. For instance, Apeks Supercritical makes introductory through high-production extraction systems and even offers refurbished systems. This gives customers a range of prices to consider.
Using butane as the extraction solvent creates what is known as butane hash oil. To do this, the process starts with cannabis and liquid butane in a pressurized and heated system. By using evaporation under a vacuum, it is then possible to remove the butane solvent. The vacuum turns the butane from liquid to a vapor, making it easier to remove.
This kind of extract is also known as shatter, which is a clear material that typically includes THC, CBD and other chemical components, including terpenes. To really make shatter, which is a hard version of butane hash oil, terpene content must be kept low or it works like a solvent that makes the extract soft. The right starting sample can help to reduce the terpenes present in the final product, such as by starting with cured flowers.
On the other hand, the butane hash oil can be heated to remove terpenes after extraction as they are more volatile than cannabinoids like THC and CBD.
Cannabis alcohol extraction Several common forms of cannabis extraction rely on a solvent, such as alcohol. In brief, the cannabis soaks in alcohol, usually ethanol, the plant material is then removed, the liquid filtered, and the alcohol is removed with some form of evaporation. One of the biggest challenges is the inherent polarity of solvents like ethanol—meaning it has a propensity to mix with water and dissolve water-soluble molecules like chlorophyll.
Comparison of the microbial community structures with the use of the principal coordinate analyses of Bray—Curtis dissimilarities weighted unifrac at the phylum level in all replicates of the tested extraction kits.
The analysis of microbial communities indicated that for all of the tested extraction kits, generally more than The highest percentage of Bacteria was assessed with the use of the C1 kit and the smallest with the C2 kit. Among replicates, the highest percentage of Bacteria was also assessed with the C1 kit and the smallest with the C2 kit. For the rest of the tested kits, 3 to 7 phyla are absent, mostly those of low percentage of participation in the total share of the microbial community.
Those phyla jointly accounted for more than Separately, Proteobacteria comprised on average The remaining reads in the population structure were associated with: For each of the tested extraction kit, at least 10 phyla up to 12 were responsible for more than Abundance of microbial 16S rDNA sequences at the phylum level.
Analyses of the microbial community structures for the analyzed extraction kits. Comparing similarities in microbial population structures created with the use of different extraction kits. Higher abundance is shown as lighter color.
Relative abundances of microorganisms at the Family level in DNA samples obtained with a given kit. Higher abundance is shown as intense color. Higher aggregation of kits suggests more similar microbial structure. The most visible differences between the extraction protocols were seen for Actinobacteria. Average share of the [Thermi] phylum is 0. In addition, the Thermus genus , for this extraction kit, was the only detected genus.
In other kits, contribution of the [Thermi] phylum is often a mix of two genera: Using different extraction kits, various contributions can be observed at lower taxonomic levels. Analyzing the Proteobacteria phylum, we can observe different contribution of the Alphaproteobacteria class with an average share of For Gammaproteobacteria , average share is 6. For Betaproteobacteria and Deltaproteobacteria, the differences are not so divergent, i.
When analyzing Actinobacteria , the Actinobacteria class constitutes on average The Rubrobacteria class, for eight analyzed extraction kits, shares less than 0.
It can be also observed that in this particular soil sample, both lysis buffers tested with kit C6 gave comparable results. Kits C2, C3, C6. The Chloriflexi phylum, in two replicates of kit C1, share less than 0. With the same kit, when looking at Firmicutes , the share of this phylum in subsequent replicates is 5. For kit C8, Firmicutes also constitute less than 1.
For kit C4 we can observe differences in abundance of the TM7 candidate division; in each replicate they contribute accordingly: Analyses of the microbial community structures for three replicates among analyzed extraction kits.
Due to influence on the results obtained and their interpretation, selection of appropriate methods is of high importance in every research. In this study, we extracted varied amounts of DNA from a single soil sample with the use of eight different commercial kits additionally, for C6 we used two lysis buffers C6.
All DNA samples exhibited a different level of purity, but in every case the PCR reaction could be performed and there was no brownish color, characteristic of the presence of humic acids. However, in practice, other aspects than quality and amount of recovered material are equally important when choosing an extraction protocol, like convenience of use and the time needed for the DNA extraction, as well as a cost per sample, which can be easily compared between each other.
These parameters are very important criteria in extraction protocol selection and can be very subjective, but there are more important parameters, like sequence diversity and sequence representation.
This can have a significant impact on the proper assessment of the final structure of microbial communities. In this work, we present the analyses of microbial community structure with application of 16S rDNA and the use of NGS that give a complex picture as to how microbial community structure can depend on the DNA extraction protocol.
In this study, we observed a high number of good quality reads above , in the kit analysis, and above 26, for single replicates among the tested kits. Generally, 15,—, reads per sample are sufficient for classification, as described in the Illumina 16S Metagenomic Sequencing Protocol. Also, in each analysis we were able to classify all of the obtained sequences at the phylum level. For each extraction kit, we observed different values for the diversity indexes.
The high and similar values of the Shannon's and Simpson's indexes, not only in the case of kit C2 and C5, but also C3 and C6. Values for each tested index for kit C1 and C8 stand out downwardly from the rest of the extraction kits and are often subjected to a much higher error rate than the other tested kits.
Also, in the case of C6. Kits C2 and C5, have the highest values of all the indexes tested, which could suggest obtaining the most complex microbial population with a high number of species, that can in fact represent the closest assessment to the actual microbial structure of this particular soil sample.
Disproportion in the index values, especially for two kits: C1 and C8, and their high error rates, may represent a structure of the microbial population that significantly differs from the actual composition of that population.
Generally, soil DNA extraction protocols can be divided into the two main types of extraction, direct and indirect Daniel, It was presumed that with the use of direct extraction methods, the isolated DNA better represents the microbial population structure, as those methods do not include cell separation of microorganisms from soil matrix. Thus, DNA of microorganisms that adhere to the soil particles is included in the population structure analysis Daniel, The C1 kit extraction procedure is similar to indirect methods and this might be one of the reasons why this kit differs from other kits.
However, the C4 kit protocol for DNA extraction is also similar to indirect methods, but it does not deviate so substantially, as C1, from the other kits. Their results were accurate for a soil sample which was tested in their study and for applied by them extraction protocols. It is worth mentioning that the exact composition of the soil sample may influence the performance of a given kit, and some kits which are particularly effective for one type of sample may fail when extracting DNA from other samples.
Rarefication analysis of the obtained data revealed trends indicating that sampling of microbial communities is close to being complete for each analyzed kit, which can also indicate the final efficiency of a particular extraction kit. Rarefication analyses are similar for kits C2, C3, C5, C6.
Rarefication curve for C8 and C1 significantly depart from the other curves, and in addition the C8 curve has a high value of error rates. Also, principal coordinate analysis at the phylum level showed that microbial community composition created with the use of the C1 kit is significantly different from other tested kits.
This analysis revealed a large variation between replicates of the C8 kit. The microbial community structures created for the C4 kit, as well as the C1 kit based on an indirect method, stand out from the other tested kits, which are also perceived when considering the diversity indexes. Taken together, in order to receive good quality and reproducible data, several conditions must be fulfilled: Using an appropriate extraction kit with listed above features can provide good quality material, suitable for comparative analysis not only within one project, but also for comparisons between studies.
In some studies, the main goal is to catalog all the organisms present. This strategy should be also used in research focused on finding new genes encoding proteins or genes involved in resistance to antibiotics or in pollutant degradation. All of those factors can influence the final conclusions about the microbial community structure, so the final decision should be made as to what is the dominant goal of a given study.
Our results show that the C1 kit may be preferred in order to establish the representatives of the [Thermi] phylum or Firmicutes in a particular soil sample, but due to lack of reproducibility between the extraction replicates, it may not necessarily reveal the real composition of the microbial community structure.
At the same time, some of the less abundant phyla may be omitted in the analyses, as it was in this particular case, when 20 of less abundant phyla were absent when using the C1 kit. Generally speaking, microbial structure of the analyzed soil sample indicated three phyla: Proteobacteria , Acidobacteria , and Actinobacteria , to be the most abundant in the population. Their contribution to the structure differs among the extraction protocols.
At the high level of taxonomic ranks, this shows how a microbial community structure can differ depending on the DNA extraction method used. Statistical analysis indicated that abundance of different phyla in the microbial structure of 24 out of 32 recognized phyla is significantly different when using various extraction protocols.
These differences are seen among the less common phyla as well as the most abundant ones. Following through to the lower taxonomic ranks, we can observe further differences in the abundance of bacteria between the extraction protocols. Also, some of the extraction methods used in that study estimated a lower abundance of certain genera. These issues can be solved only by experimental testing.
The study presented here allows to compare certain features of the extracted DNA, and more importantly, the final microbial population structure, based on the DNA recovered with the use of different extraction kits. Although in this particular case, the C1 or C8 kits seem to significantly stand out from other tested kits, their utility for different soil samples should be considered and experimentally verified for each sample.
It is important to indicate that the soil sample used in this study was very unusual. Currently, this area is subjected to reclamation, and was covered with sludge from wastewater treatment plants, which is characterized by the presence of heavy metals, such as copper, zinc, cadmium, lead, chromium, and nickel; the area is also covered with a vegetation. It resulted in unusual composition of various heavy metals and mineral compounds. Thus, the results obtained in current study should be treated with caution, when other type of soil is to be investigated.
As tested kits might be optimized using different type of soil samples, the kits which showed low performance in our study may show excellent DNA extraction efficiency when used on less complex samples.
Based on those facts, we propose that for a single soil sample, as well as for other heterogeneous environmental samples, many DNA extraction kits should be tested at an early stage of the study. Later, based on the amount and quality of the obtained DNA, and also on diversity of the sequences and diversity indexes, as well as reproducibility of the kit extractions, the most suitable protocol should be selected for further analysis.
In some cases, in order to improve the analysis, the tests and comparisons of various DNA extraction protocols should be pursued throughout the whole analysis, not only at the early stage of establishing the amount and quality of the recovered DNA.
However, this involves an increase in the project costs and is much more time consuming, but when selecting a particular DNA extraction method, one should be aware of its limitations and alternatives. Nonetheless, it could improve the data analysis and thus could be a useful approach in order to present the closest assessment to the actual structure of the microbial population.
However, it should be noted that in such studies the view of the final microbial community structure also depends on the sequencing technology and bioinformatics tools used, which were beyond consideration in this study.
This work was supported by the National Science Centre, Poland grant no. We are grateful to Adrian Zwolicki for his advice at the early stage of this project. We are also grateful to Tadeusz Ossowski for his contribution to this paper. The choice of the DNA extraction method may influence the outcome of the soil microbial community structure analysis.
National Center for Biotechnology Information , U. Journal List Microbiologyopen v. Published online Feb Author information Article notes Copyright and License information Disclaimer. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
This article has been cited by other articles in PMC. Associated Data Supplementary Materials. DNA extraction DNA was extracted from the soil sample with the use of eight commercial kits, according to the manufacturers' protocols Kit's company number — shortcut use in the paper. Company 1 — C1. Sequencing data analysis and statistical analysis Samples were processed and analyzed using the Quantitative Insights Into Microbial Ecology Qiime pipeline v 1.
Table 1 The quantity and quality of the extracted DNA. Open in a separate window. Microbial community composition The analysis of microbial communities indicated that for all of the tested extraction kits, generally more than Discussion Due to influence on the results obtained and their interpretation, selection of appropriate methods is of high importance in every research.
Conflict of Interest None declared.
How To Choose The Right Extraction Method For Your CBD Oil Product
Does your choice of extraction method change when confirmatory factor be most comparable to Confirmatory Factor Analysis, if you choose to go that route next. . correlated, therefore oblique rotation technique would be the best solution. I would want to know which is the best extraction method that could yield almost . Moreover, in your case you can choose the solvents from non polar to polar. Jan 6, According to the method of extraction, you have to select the appropriate parts of the cannabis plant to use. You can make an extract from all.