Triple Extraction: What Comes Out of the Mushroom at Each Stage, vs Hot Water Only and Double Extraction

In brief5 points · 1-minute read

• Three solvents act one after the other on a single mushroom: first cold water, then alcohol, and finally hot water under pressure, and each solvent pulls out its own family of compounds.
• Cold water captures proteins and polyphenols before any heating, alcohol dissolves triterpenes and hericenones, and hot water releases β-glucan from the chitin-glucan wall.
• Measured so far: β-glucan of 23.21 to 28.16 percent on the finished extracts at TÜV Austria, and all four mushrooms tested free of lead, arsenic and mercury.
• The alcohol-stage compounds, such as ganoderic acids and hericenones, and microbiology have not been measured yet, and the next batch is headed for those tests.
• Triple extraction is a coverage method, not a quality score: what proves the extract is a laboratory certificate on the finished product, not the number of stages.

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Triple extraction is three solvents, one after the other, on the same mushroom: cold water, alcohol, then hot water under pressure. Each stage pulls out a different family of compounds, because each family dissolves in something different. This page shows the mechanism, stage by stage, and says exactly which part of it we have already measured on our extracts, and which part we have not yet.

• The four extracts that go through all three stages, 50 ml:
• Reishi
• Lion’s Mane
• Cordyceps
• Turkey Tail & Reishi

What comes out of the mushroom at each stage? The mechanism, in motion

The fresh mushroom goes into the first vessel, and its solid residue travels from vessel to vessel. In each vessel the solvent pulls out what dissolves in it and leaves the rest for the next stage. The colored dots in the animation are families of compounds, not single molecules and not quantities: this is a mechanism diagram, not a measurement chart.

If you want the long version, with the history and the full protocol, it is in the triple extraction article. Here we stay with the mechanism: why three solvents, what each of them does, and where the line runs between what the literature says and what we measured ourselves.

Why can't you just soak a mushroom in water?

Because the compounds we care about are locked behind a cell wall built of chitin and glucans, a fibrous matrix that room-temperature water barely penetrates. Without heat or a suitable solvent, most of the β-glucan stays in the mushroom, and the triterpenes, which do not dissolve in water, stay in it at any temperature.

A mushroom's cell wall is different from a plant's. Instead of cellulose it has chitin, the same rigid polysaccharide that builds the exoskeleton of insects, and it is chemically bonded to a network of β-glucans to form a chitin-glucan complex. The review by Gow, Latgé and Munro describes the wall as a scaffold of load-bearing fibrous polysaccharides with proteins and further sugars embedded in it. That network gives the mushroom its strength, and for exactly that reason it makes extraction hard: part of the β-glucan is itself structural material of the wall, and releasing it means breaking the bonds that hold it in place.

On the other side, the mushroom's small compounds, triterpenes, sterols and phenols, do not dissolve well in water at all. Hot water can break a wall, but it will not dissolve what needs alcohol. That is where the idea of sequential solvents comes from: not because "more stages is better", but because each family of compounds has a solvent it dissolves in, and no single solvent suits all of them.

Stage 1: what does cold water pull out, and why does it go first?

Room-temperature water pulls out of the mushroom whatever dissolves in water without the help of heat: proteins, polyphenols and small sugars. In a study that split three edible mushrooms into sequential fractions, the room-temperature water fraction was the largest by weight and consisted mainly of proteins, sugars and polyphenols; most of the β-glucan was left for the stages after it.

Why first? Because everything that comes out here comes out without heat ever touching it. Once this stage ends, the mushroom meets alcohol and then boiling water, and whatever is left in it by then goes through harsher conditions. The order of the stages is a rule of protection: the gentle step first, the strong one after.

Among the proteins that interest mushroom researchers is a family called fungal immunomodulatory proteins (FIP). In a study on the enoki mushroom, cold water pulled the protein FIP-fve out of fresh mushroom with no chemical reagent at all. And here comes the honesty we owe you: not every protein of this kind is heat-sensitive. The same research group showed that reishi's LZ-8 protein keeps its activity after heating to 100 degrees Celsius for 30 minutes and even after pressure sterilization. So we do not write "the heat would have destroyed them". We write what is true: the cold-water stage captures them before heat touches them at all, and whatever in this group is heat-sensitive, and some of it is, comes out here intact.

Stage 2: what does alcohol pull out, and why seven weeks?

Alcohol dissolves what water does not: triterpenes, sterols and phenols. In reishi these are the ganoderic acids, the family most reishi research is interested in; in lion's mane they are the hericenones of the fruiting body. In our process this stage runs for seven weeks, because time, not temperature, does the work here.

The literature on reishi is unambiguous about the solvent. In an optimization study that tested heat, time and ethanol concentration, the conditions that yielded the most triterpenes and phenols were high ethanol concentrations, around 62 to 90 percent, in two different methods. And a phytochemical study that went looking for the compounds themselves isolated 29 different triterpenes from an ethanol extract of reishi fruiting bodies, 11 of them described there for the first time. These are not compounds that water pulls out.

In lion's mane it matters to separate two families that get confused all the time. Hericenones are in the fruiting body, and in the study that examined them they were isolated from an ethanol extract of the fruiting body. Erinacines, by contrast, are mycelium compounds, and a 2025 systematic review deals with them explicitly as mycelium compounds. Our extracts are from fruiting bodies only, so when we talk about the alcohol stage in our process, we are talking about hericenones. Anyone promising you erinacines from a fruiting-body extract probably has not checked.

On the seven weeks: this is a maceration in a sealed, dark vessel, with agitation, without heating. Sonication (ultrasound) is a complementary step in our process, not a replacement; in our view it has not yet bypassed what a long time in alcohol does. That is a producer's position, not a laboratory finding, and we mark it as such.

Stage 3: what does hot water under pressure pull out?

Hot water is the classic solvent for β-glucan and the other polysaccharides of the cell wall. Heat and pressure break the chitin-glucan bonds and release the long chains into the water. This is the stage responsible for the one number we have already measured on our extracts: β-glucan of 23.21 to 28.16 percent.

The professional literature works almost entirely this way: mushroom polysaccharides are extracted in hot water, and sometimes precipitated afterwards in ethanol to concentrate them. A study on an edible mushroom showed extraction in water at 60, 80 and 100 degrees Celsius, and a comparison with a high-pressure pre-treatment, which recommended precisely the combination of pressure and moderate hot water. Another study, on reishi, arrived at optimal conditions of 121 degrees Celsius for two hours followed by ethanol precipitation, and obtained a fraction rich in β-glucan. And the quantification method our laboratory uses, the Megazyme kit for mushroom and yeast β-glucan, is the same kit that appears in these studies when the aim is to tell real β-glucan apart from "total polysaccharides", a number that inflates itself easily with starch or added fiber.

Note why this stage comes after the alcohol and not before it. If the process started with boiling, the proteins that stage 1 captured would meet heat before they came out, and the triterpenes of stage 2 would stay behind in a cooked mushroom. The order is part of the method.

What is the difference between hot water only, two solvents and triple extraction?

Hot water only pulls out β-glucan and misses everything that is alcohol-soluble. Two solvents (double extraction: hot water and alcohol) cover the two big families. Triple extraction adds a cold stage before everything, and with it an order: the heat comes last. The table says exactly what each method covers, without exaggerating.

This is a coverage table, not a quantity table. It says which family can be present in an extract at all, given the solvent that was used, not how much of it there is. Only a laboratory certificate answers the question of quantity, and that comes further down.

Why does the order matter: cold, alcohol, and only then hot?

Because the three stages are not parallel, they are sequential on the same mushroom. What the first stage did not pull out is left for the next one, under harsher conditions. Arranging the stages from gentle to aggressive ensures that each family meets its solvent under the mildest conditions that are enough for it.

There is a practical side too: after the alcohol stage, the solid residue has already been "cleaned" of fats and triterpenes, and hot water meets a more exposed cell wall. In sequential separation studies, like the one that fractionated three edible mushrooms, this picture is clear: each fraction contains something different, and the structural β-glucan concentrates precisely in the late fractions, the ones that need heat or alkali to come out. Our method aims at the same principle without a chemical base: water, alcohol and pressure only.

What does each of our extracts carry, and which stage does it come from?

All four extracts go through the same three stages, but each mushroom brings a different family to the alcohol stage, and each extract has its own mushroom-to-liquid ratio. The table separates what has been measured from what the literature says should be there.

A note on Cordyceps: the compounds it is identified with, cordycepin and adenosine, are nucleosides that dissolve in water. So with Cordyceps the water stages do most of the work, and the alcohol stage contributes mainly sterols. That is not a drawback, it is chemistry. On the ratio, and on why 1:2 fresh is not 1:2 dried, there is a separate page.

What has already been measured on our extracts, and at which laboratory?

β-glucan 1,3/1,6 was measured on the finished extract, not on the mushroom, at TÜV Austria in July 2025, by the Megazyme method. Four certificates, one for each extract that was sent, are open to read. Alpha-glucan, meaning starch, was not detected in any of them.

• 28.16Cordyceps · β-glucan, percent
• 25.65Reishi · β-glucan, percent
• 23.93Lion's Mane · β-glucan, percent
• 23.21Turkey Tail & Reishi · β-glucan, percent
• Next testGanoderic acids, hericenones, cordycepin

These numbers are the proof that stage 3 works: β-glucan at this level on a finished liquid extract means the cell wall was opened and the chains moved into the liquid. For comparison, extracts based on mycelium grown on grain tend to show less than 7 percent, while a good fruiting body sits in the 25 to 40 range. All the certificates, with report numbers you can verify with the laboratory, are on the lab testing page.

And it has to be said what this number does not say. β-glucan is a measure of stage 3. It says nothing about stage 2, so you cannot infer from it how many ganoderic acids there are in our reishi. Anyone who wants to know that needs a separate certificate, and that comes further down.

Is the source clean? Heavy metals, pesticides and PAH

Yes, and it was tested. No extraction method cleans a contaminated mushroom: whatever goes into the first vessel reaches the bottle. So our four mushrooms were tested at an external laboratory for heavy metals, for 610 pesticides and for combustion contaminants (PAH), and the certificates are open next to the β-glucan certificates.

• Not detectedLead · arsenic · mercury, in all four mushrooms
• 0 of 610Pesticides quantified; compliant with the European standard
• Below the limitPAH, the four markers and their sum, in every mushroom

The numbers in words: heavy metals were tested by the ICP method, and lead, arsenic and mercury were not detected in any of the four mushrooms; cadmium and aluminium appear on the certificates with a specific value for each mushroom, and we do not merge them into a single number. The pesticide screen covered 610 active substances by the EN 15662 method, and none of them was quantified, in line with the European residue regulation. The four measured PAH, and their sum, were below the method's limit of quantification in each of the mushrooms. A mushroom that grows on our farm in the Galilee, on a substrate we prepare ourselves, arrives at extraction clean, and that is not a claim but a certificate.

What has not been measured yet, and what will the next batch be tested for?

The alcohol-stage compounds, microbiology on the extract, and genetic species identity of the mushrooms. The four existing certificates are from our first and so far only batch; the next batch is entering production now, and each of its extracts will be sent for a full panel of tests. The moment a certificate comes back, its number will appear here in place of the words "next test".

We write this list here, and do not hide it, for a simple reason: a test that was not done is information. "All our extracts are lab tested" would have been a more convenient sentence, and it is not true. What is true is that all our tests are open, and that on the lab page you can see exactly which extract was tested.

Is triple extraction "better"? The honest answer

Triple extraction is not a quality metric and proves nothing by itself. It is a coverage method: three solvents cover three families of compounds, in an order that protects the delicate ones. What proves the method worked is a number on the finished extract, and right now we have such a number for one stage out of three.

There are producers on the market who write "double extraction" or "full spectrum" and attach no certificate at all; there are those who attach "total polysaccharides", a number that can be inflated with starch or inulin; and there are international brands that measure everything and publish everything, and that is the bar we measure ourselves against. This page does not claim to decide who is "the best". It shows a mechanism the literature supports, one number an external laboratory confirmed, and an open list of what has not been measured yet. Anyone comparing brands can now do it on the basis of what each one actually published; ours is here, and how to read such a certificate is here too.

Fresh, not dried: what does it change for the three stages?

Almost all mushroom extracts in the world are made from dried mushroom, because drying is a logistical solution for material that travels from far away. In our case the mushroom grows on the farm in the Galilee and goes from harvest to freezer to extraction with no drying step. As far as we know, we are among the very few in the world who work this way.

Drying has a cost that is hard to quantify without measurement, so we make no chemical claims here about the advantage of fresh. What can be said: the proteins and compounds that stage 1 is interested in are exactly the group that heat drying touches first, and our cold-water stage meets them in their original state. Freezing in our process is for preservation only, not for improving extraction; anyone telling you that freezing "bursts cells" in mushrooms has not read the chitin studies. On fresh versus dried in depth, and on fruiting body versus mycelium, we have separate pages.

What is the connection between a "1:3 ratio" and "triple extraction"? None

These are two separate facts that get confused all the time. The mushroom-to-liquid ratio says how many grams of mushroom went into each milliliter of extract, and it varies from extract to extract in our range: 1:2.5 for Cordyceps, 1:2 for Lion's Mane, 1:3 for Reishi and for Turkey Tail & Reishi. Triple extraction says which solvents, and in what order. The number 3 in both is a coincidence.

The two facts together, plus one more, appear on every bottle of ours: the ratio of that extract, triple extraction by the GTC 3X™ method (Glucan-Terpenoid Complex, three stages), and local cultivation in the Galilee. None of them replaces the certificate.

Which extract is right for me?

The four extracts go through the same triple extraction; the difference between them is the mushroom, and so the stage 2 family of compounds and the goal. Reishi for sleep and calm, Lion's Mane for focus and memory, Cordyceps for energy and breath, and Turkey Tail & Reishi for immune support. If you are not sure, there is a short quiz.

The bottom line

Triple extraction is a mechanism, not a slogan: cold water for proteins and polyphenols before heat touches them, alcohol for seven weeks for triterpenes and hericenones, and hot water under pressure for the β-glucan locked in the chitin-glucan wall. The literature supports every stage, an external laboratory confirmed the third on our extracts, 23.21 to 28.16 percent, and the source was tested and found clean of heavy metals, pesticides and PAH. We have not yet measured the alcohol stage, and the next batch will answer that. Until then, the most honest thing you can do with missing information is write it down.

Frequently asked questions

What is the difference between triple extraction and two-solvent extraction?

Two-solvent extraction uses hot water and alcohol and covers the two big families: β-glucan and triterpenes. Triple extraction adds a cold-water stage before them, which captures proteins, polyphenols and small sugars before the mushroom meets heat, and sets an order: cold, alcohol, and only then hot. The difference is the cold stage and the order, not "more of everything".

Does triple extraction prove the extract is high quality?

No. Triple extraction is a coverage method, not a metric. What proves quality is a laboratory certificate on the finished extract. We have such a certificate for β-glucan (TÜV Austria, 23.21 to 28.16 percent) and not yet for the alcohol-stage compounds. We say that in the open.

Why don't most producers do triple extraction?

Mostly time and cost. Our alcohol stage alone runs seven weeks, with the vessel occupied the whole time, and the hot-water stage needs pressure equipment; a single hot-water extraction is finished in hours. On top of that, no label regulation requires a producer to state the extraction method, so a product can say "mushroom extract" and nobody checks how it was made. Our answer is not the word "triple" but the certificate on the finished extract.

What comes out in the cold-water stage that would not come out in hot water?

In hot water most proteins and polyphenols come out as well, but after heating. The difference in the cold stage is that they come out before any heat, so even the sensitive ones among them are captured intact. Not every fungal protein is heat-sensitive: reishi's immunomodulatory protein LZ-8, for example, survives 100 degrees Celsius. The cold stage is insurance, not a claim that heat destroys everything.

Why does the alcohol stage take seven weeks?

Because triterpenes, sterols and hericenones are not water-soluble and move into alcohol slowly, without heating. In optimization studies on reishi, high ethanol concentrations yielded the most triterpenes. In our process time, not temperature, does the work: maceration in a sealed, dark vessel, with agitation, for seven weeks. Sonication is a complementary step, not a replacement.

Are there erinacines in the extract?

Not expected, and that is deliberate. Erinacines are compounds of the lion's mane mycelium, and our extracts are from fruiting bodies only. The fruiting body's distinctive compounds are the hericenones, and they come out in the alcohol stage. A hericenone test on the extract is on the next-batch list.

What exactly was measured, and on what?

β-glucan 1,3/1,6 was measured on the finished extract, at TÜV Austria, in July 2025, with the Megazyme kit. Four certificates: Cordyceps 28.16, Reishi 25.65, Lion's Mane 23.93, Turkey Tail & Reishi 23.21 percent. Alpha-glucan, meaning starch, was not detected. The source was tested as well: lead, arsenic and mercury were not detected in the four mushrooms, none of 610 pesticides was quantified, and PAH was below the limit of quantification. All certificates are open on the lab testing page.

Are there solvents in the extract?

Only water and food-grade ethanol. No other organic solvent is used at any stage. The ethanol stays in the final extract at the concentration declared on the label, and it is also what preserves the extract over time.

Why is the mushroom fresh and not dried, and what does it change?

Because we grow it ourselves in the Galilee and do not need to dry it in order to ship or store it. The mushroom goes from harvest to freezer to extraction, and the freezing is for preservation only. We do not claim this improves extraction without a measurement; what is certain is that the cold-water stage meets a mushroom that never went through an oven.

Scientific sources (peer-reviewed papers)

• The fungal cell wall: a scaffold of fibrous polysaccharides, chitin and glucans, with proteins embedded in it (review) — Gow NAR, Latgé JP, Munro CA. Microbiology Spectrum, 2017;5(3) — View on PubMed
• Sequential fractionation of three edible mushrooms: the room-temperature water fraction is the largest and consists of proteins, sugars and polyphenols; structural β-glucan concentrates in the late fractions — Pérez-Bassart Z, Fabra MJ, Martínez-Abad A, et al. Food Chemistry, 2022;402:134207 — View on PubMed
• Cold water extracts the immunomodulatory protein FIP-fve from fresh mushroom without chemical reagents — Tung CH, Lin CC, Wang HJ, et al. Journal of Food and Drug Analysis, 2018;26(3):1005-1014 — View on PubMed
• The immunomodulatory proteins FVE (enoki) and LZ-8 (reishi) keep their activity after 100 degrees Celsius for 30 minutes, pressure sterilization and freezing — Tong MH, Chien PJ, Chang HH, et al. Journal of Agricultural and Food Chemistry, 2008;56(9):3160-3166 — View on PubMed
• Optimization of triterpene and phenol extraction from reishi: ethanol concentrations of 62.5 to 89.5 percent yielded the most, with heat and with ultrasound — Oludemi T, Barros L, Prieto MA, et al. Food & Function, 2018;9(1):209-226 — View on PubMed
• 29 triterpenes isolated from an ethanol extract of reishi fruiting bodies, 11 of them described for the first time — Li DW, Liu M, Leng YQ, et al. Phytochemistry, 2022;203:113339 — View on PubMed
• Mushroom polysaccharide extraction in hot water at 60, 80 and 100 degrees Celsius, with and without high-pressure pre-treatment; the recommendation: pressure plus moderate hot water — Tepsongkroh B, Thaihuttakij C, Supawong S, et al. Food Chemistry: X, 2023;19:100864 — View on PubMed
• Reishi polysaccharides: optimal conditions of 121 degrees Celsius for two hours followed by ethanol precipitation, for a β-glucan-rich fraction — Dos Santos JF, Sauruk da Silva K, Iacomini M, et al. Pharmaceuticals, 2026;19(6):946 — View on PubMed
• Edible mushroom polysaccharides in hot water followed by alkali; β-glucan 1,3/1,6 quantified with the Megazyme kit — Petraglia T, Latronico T, Fanigliulo A, et al. Molecules, 2023;28(5):2176 — View on PubMed
• Hericenone C isolated from an ethanol extract of the lion's mane fruiting body; the derivative without the fatty acid is more active — Tamrakar S, Wang D, Hiraki E, et al. Molecules, 2023;28(11):4549 — View on PubMed
• Erinacines are compounds of the lion's mane mycelium: a systematic review of preclinical models — Spangenberg ET, Moneypenny A, Bozzo GG, Perreault ML. Frontiers in Pharmacology, 2025;16:1582081 — View on PubMed
• The active components of Cordyceps: cordycepin, adenosine, sterols and polysaccharides (review) — Chen L, Liu X, Zheng K, et al. Molecules, 2024;29(21):5107 — View on PubMed

This page describes an extraction method and what has been measured on it. It is not medical advice and does not replace a consultation with your physician, especially if you take medication, are pregnant or breastfeeding. The laboratory numbers refer to the extracts that were tested, as detailed on the lab testing page. *These statements have not been evaluated by the Food and Drug Administration. This product is not intended to diagnose, treat, cure, or prevent any disease.*

Read next

• Triple extraction: the full article, history and protocol
• Full lab results and test certificates
• How to read a certificate of analysis (COA)
• β-glucan vs "total polysaccharides"
• Extraction ratio: what 1:3 says and what it does not
• Fresh or dried mushroom
• Fruiting body vs mycelium
• Research hub: reishi
• Functional mushrooms: the complete guide
• All extracts