Fritsch Milling and Sizing

Milling affects volatile compound recovery in functional mushroom extracts

Dingding Xuan, Sajni Shah, Dr. Eric Janusson, Dr. Markus Roggen- Controlled Chemistry

Introduction

Fritsch milling equipment is an alternative to uncontrolled milling for sample preparation. Here we show a general method and assay for volatile constituents of functional mushroom extracts can be dramatically affected by milling conditions
Hand grinders, food processors, coffee grinders, and other milling methods with limited control settings are standard in laboratories. However, many biomolecules of interest are labile and could be consumed during these processes. Therefore, instruments that provide little control of milling parameters may yield inaccurate results for compound discovery and quantification. We investigated whether particle size, mill time, and blade speed influence analyte recovery and subsequent measurement of compound concentration in functional mushrooms. We used Lion’s Mane (Hericium erinaceus) as the test substrate and a Fritsch P11 bladed mill to precisely control the various milling factors. Using the most abundant components of these mushrooms (leucine, myrcene, alpha-hydroxyisobutyric acid, 2- acetylbutyrolactone, 2-diethylaminoethyl acetate, and mannitol), we monitored their concentration resulting from various milling procedures.

Experimental

Lion’s Mane mushroom extracts were prepared through a simple sample preparation and extraction analytical workflow. Experimental variables were minimized to emphasize differences in milling conditions. The extracts were surveyed by GCMS for compound discovery and quantification.

Sample Preparation

Vacuum-dried whole Lion’s Mane mushrooms were donated by Nammex (Gibsons, BC). Whole, dried, Lion’s Mane mushrooms were weighed, sectioned using a razor, milled using a Fritsch P11 bladed mill, and then run through a 7mm particle filter. Batches of material were milled at various rates programmed into the P11 mill (see milling parameters in Table 1). An aliquot of each batch of milled material was taken for particle size analysis and a separate aliquot was taken for liquid extraction.

Extraction

Milled Lion’s Mane (1.0g) was weighed into 20mL scintillation vials. The extraction was performed with 20.0 mL of a 1:1 mixture of HPLC grade methanol and deionized water containing 0.5% (v/v%) formic acid (Fisher Optima) at room temperature for 3 hours. The extracts were decanted, and an aliquot was filtered with 0.20 µm Nylon syringe filters into glass vials for analysis.
Table 1. Milling parameters for sample preparation experiments on Lion's Mane-min
Table 1. Milling parameters for sample preparation experiments on Lion's Mane

GCMS Analysis

The samples were analyzed by GCMS (Agilent Intuvo 9000 GC, 5975 MSD) using an oven ramp program designed to survey a broad range of analytes (Table 2). The injection volume was 1.0 µL and the GC column used was an HP-5ms ultra inert 30 m fused silica column. Suitable compounds were extracted from the GCMS raw data and identified using the Agile 2 peak identification algorithm. Compound fragmentation patterns, extracted ion areas, and retention times were extracted using MS-DIAL and assigned through comparison to our proprietary spectral database.

Results

Figure 1. Molecules recovered from extraction after milling at three different speeds programed into the P11 mill-min
Figure 1. Molecules recovered from extraction after milling at three different speeds programed into the P11 mill. Mill time fixed at 30 sec
Figure 2. Molecules recovered from extraction after milling for different time periods-min
Figure 2. Molecules recovered from extraction after milling for different time periods. Mill speed fixed at 2000 RPM
Figure 3. The top molecules identified at all mill times and speeds.-min
Figure 3. The top molecules identified at all mill times and speeds. Here we show their concentrations at three mill times at 2000 rpm.
Figure 4.The top molecules identified at all mill times and speeds.-min
Figure 4.The top molecules identified at all mill times and speeds. Here we show their concentrations at three mill speeds for 30 seconds.

Conclusions

The experiments above demonstrate that precise control of mill conditions is important in mushroom sample preparation. Lion’s mane mushroom samples were milled with a Fritsch P11 mill set to different parameters of mill speed and time, and then underwent liquid extraction. The analyte composition of the extracts was then identified as shown in figures 1 and 2. Our results show that mill speed and time correspond to changes in mushroom extract analyte composition. We could show that mill settings had a direct influence on analyte recovery from the mushroom (figures 3 and 4). 4-guanidinobutyrate is found in edible mushrooms and is a known fungal metabolite in many species.1,2 Hericium erinaceus is known to have 3.3% of its protein content made up of L-isoleucine. 3 Meanwhile, to the best of our knowledge, esculin and Ophiopogonoside A have not yet been described in these mushrooms. Among these compounds, lower mill speeds and shorter mill times corresponded to increased recovery. This indicates a Fritsch P11 mill would allow researchers to optimize their sample prep to ensure the highest yields.

References

  1. PubChem Compound Summary for CID 500, 4-Guanidinobutyric acid.
    National Center for Biotechnology Information
    https://pubchem.ncbi.nlm.nih.gov/compound/4-Guanidinobutyric-acid (2022).

  2. Li, J., Wu, H., Wang, L., Huang, Y. & Wang, L. Key taste components in two wild edible Boletus mushrooms using widely targeted metabolomics.
    Biochemical Systematics and Ecology 96, 104268 (2021).

  3. Aparicio-Razo, M. & González-Pérez, M. ANALYSIS OF BIOMOLECULES OF THE FUNGUS HERICIUM ERINACEUS THROUGH THE THEORY OF ELECTRON
    TRANSFER OF QUANTUM CHEMISTRY AND ITS RELATIONSHIP WITH THE PRIMARY AMINO ACIDS. 9, 139–147 (2020).

Supporting

Table 2

Name Abundance in all experiments
4-guanidinobutanoate 5261052.235
L-leucine 1110933.091
3-amino-1,2-propanediol 425572.75
esculin 345937.8184
L-beta-homoleucine 326320.0547
tetrahydrofuran 272256.9375
dodecamethylcyclohexasiloxane 270464.666
cadaverine 244824.6094
ophiopogonoside A 243731.9395
hexazinone 240513.2041
citropen 223926.4727
4-oxo-3-phenyl-6-propyl-4h-chromen-7-yl acetate 195539.752
L-isoleucine 183783.2715
dehydroandrographolide 181819.543
N,N-dibutylnitrous amide 176344.2031
myrcene 134515.9443
4-hydroxybenzaldehyde 122221.9766
alpha-hydroxyisobutyric acid 120649.4336
N,N’-dicyclohexylthiourea 112791.6953
2-hydroxybutyric acid 94918.07813
2-((7-acetamido-1,2,3-trimethoxy-9-oxo-5,6,7,9- tetrahydrobenzo[a]heptalen-10-yl)amino)-n-(4-(5-(methoxymethyl)- 1h-1,2,4-triazol-3-yl)phenyl)acetamide 90886.3457
cyclododecanol 86997.33765
6-methoxy-4-methyl-2h-chromen-2-one 82209.84376
phenaceturic acid 66982.5
1-myristoyl-2-hydroxy-sn-glycero-3-phosphate (sodium salt) sodium salt 66748.45361
1-[(2e,4e)-6,7-dihydroxy-2,4-octadienoyl]prolyl-n-methylvalyl-n2- methylalaninamide  62692.64649
butanal 54538.58203
pyruvaldehyde 53863.91406
2-methylbutyrylglycine 53269.54297
cinchonine 52899.89649
sinapic acid 48724.09424
diffractaic acid 44918.83301
l-2,3-diaminopropionic acid 38335.02344
[(2r,3r,4s,5r,6s)-3,4,5-tris(acetyloxy)-6-[(7-tert-butyl-5,8-dihydroxy1,4-dioxo-3-{[(2s,3r,4s,5r,6r)-3,4,5-tris(acetyloxy)-6- [(acetyloxy)methyl]oxan-2-yl]sulfanyl}-1,4-dihydronaphthalen-2- yl)sulfanyl]oxan-2-yl]methyl acetate 36675.40137
pseudolaric acid a-o-beta-d-glucopyranoside 31419.36426
dl-coniine 28856.19677
ganoderol b 28060.43164
cyanate 25044.28906
allo-threonine 23335.97266
4-acetamidobutanoate 22061.03906
nerolidol 22051.06641
pseudopyronine b 21448.30469
dioctyl phthalate 21001.31372
daphnetin 20752.16382
mannitol 19570.46094
protocetraric acid 18046.0166
octylamine 16933.83765
n-acetyl-b-alanine 16850.11523
n-octyl-2-pyrrolidone 16671.71484
L-norleucine 14774.7041
flucarbazone 14044.42285
isoamylamine 12573.57324
bis(2-ethylhexyl) phthalate 12386.82568
2-amino-3-methylvaleric acid 11756.65332
formiminoaspartate 11709.78027
1-dodecyl-2-pyrrolidinone 11695.82129
sakuranetin 11362.86719
3-methyl-2-oxobutanoate 11235.17969
1-dodecanamine 11142.62036
isocucurbitacin b 10466.6709
guanidine 10229.51465
lutein 9612.479492
benzoic acid 9459.237305
lacosamide 8988.265625
dimethyl malate 8386.848145
1,2-dilauroyl-sn-glycero-3-phosphate monosodium salt 5801.692871
norfluoxetin 5688.64502
metformin 5674.167969
loureirin a 5657.355713
trimethylamine 5535.165039
mexiletine acetate 5474.612305
1-myristoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine 5455.387695
norfluoxetine 4984.3125
dl-norgestrel 3903.630127
aminoadipic acid 3554.445068
ethanolamine 2852.979736
diisopropyl phthalate 2370.876465
4-oxobutanoate 1833.481812
1,5-diaminopentane 268.306152