Aspergillus metabolites

We were concerned about GI Aspergillus grown in our GI tracts. Can a good species ocntrol a bad one? WHhat about anti-fungal essential oil terpenes? What about species that produce terpenes? Cotty’s article 2021 paper: Degradation of Aflatoxins B1 by Atoxigenic Aspergillus flavus, Biocontrol Agents | Plant Disease [1]has inspired two ideas:

Maybe a healthy Aspergillus such as Aspergillus oryzae may be beneficial by either degrading the bad Aspergillus mycotoxins in one’s body or maybe competing with it (although less likely).
Microbiota transfer donors may already have “healthier” molds that either are less toxigenic (to compete), degrade mycotoxins, or have mycotoxins (that reduce virulence). (Definitely best to have single donor to decrease chance of any “bad” microbes).

This post was also an exploration of three terpenes from essential oils used in a pleasant mouth rinse to kill off bad fungi before introducing good fungi. Fungi also produce terpenes that might be fungicidal to other fungi.

Citronellol, geranial, and nerol have some anti-Aspergillis activity [2]
Menthol, carvacrol, and eugenol have Aspergillis biocidal activity. [3]
Limonene can be produced by Aspergillis fumigatus. In fact, many terpenes dected in these cultures have commonality with citronellol and menthol [4]

**Terpenes against Aspergillis species**

These are the terpenes that are being investigated for anti-aspergillis activity: citronellol, menthol, and limonene. PubMed was used as a searching reference.

Citronellol [2]

A Korean paper was retrieved examining individually terpenes of a common essential oil. Terprenes with no inhibition were removed from the table. A as being the best.
I think this would be so synergistically helpful with whatever approach is taken.

Compounds	Conc., ×10⁻³ mg/mL air	Inhibition rate (Mean±S.E., %)
Compounds	Conc., ×10⁻³ mg/mL air	*Aspergillus ochraceus*	*Aspergillus flavus*	*Aspergillus niger*
Isopulegol	56	34.6 ± 3.2^d	0^d	33.4 ± 2.2^cd
Citronellal	56	0f	0^d	16.2 ± 11.8^de
Citronellol	56	83.7 ± 3.3^b	90 ± 0.7^a	81.1 ± 9.0^b
	28	29.4 ± 3.0^de	37.7 ± 4.1^b	35.3 ± 7.0^c
Neral	56	100a	100a	100a
	28	100^a	26.9 ± 2.1^bc	0^e
	14	100^a	–	–
	7	34.1 ± 4.2^d	–	–
Geraniol	56	64.6 ± 7.5^c	36.7 ± 11.3^b	77.1 ± 9.8^b
	28	38.4 ± 4.4^d	–	0^e
Geranial	56	100^a	100a	100^a
	28	100^a	29.8±3.3^bc	0^e
	14	20.1 ± 3.8^e	22.3 ± 3.5^c	–

From Kim et al 2012. Different superscripts indicate different means at p<0.05 Note, there are aspects of this table that seem to be repeated information.

Menthol and more [3]

This study established the minimal inhibitory concentrations of four terpenes against 11 common food pathogens. This table summarizes the abstract only. All four of these compouns had anti fungal activity. These were the ones considered notable enough to make it into the abstract.

terpene	Aspergillis	Cladosporum
carvacrol	100 μg/mL
menthol	125 μg/mL	125 μg/mL
thymol		100 μg/mL
eguenol	350 μg/mL

Information from the abstract reference [3] was put in table form.

Aspergillus fumigatus fumes, go figure [4]

So far the search for peer reviewed evidence of fungicidal activity of terpenes in a kid friendly mouth rinse have been fruitful but not earth shattering. Somehow our focus had been on killing the fungi rather than simply controlling them. Fumes emitted by fungi have been used to detect invasive infections. A continued search for limonene yielded a PubMed report demonstrating that Aspergillus can produce limonene and other terpenes. This sorts of makes intuitive sense to anyone who has noted a fruity aroma to fungal growth even when the mold is on a piece of bread or something. Heddergott and coauthors used what seemed to be a minimal medium called Brian Medium which was supplemented with divalent cations and inhibitors of terpene synthesis. . -pinene,camphene, and limonene were consistently detected terpenes

VOC type and peak no.	Formula	Trivial name	CAS no.	Substance class	Probability
1	CO₂	Carbon dioxide	124-38-9		2.04
2	C₅H₈	Isoprene	78-79-5	Diene	3.74
3	C₆H₁₀	(Z,Z)-2,4-Hexadiene	6108-61-8	Diene	15.3
4	C₁₀H₁₆	α-Pinene	80-56-8	Monoterpene	15.7
5	C₁₀H₁₆	Camphene	79-92-5	Monoterpene	45.9
6	C₁₀H₁₆	Terpinolene	586-62-9	Monoterpene	28.1
7	C₁₀H₁₆	o-Cymene	527-84-4	Monoterpene	28.8
8	C₁₀H₁₆	d-(+)-Limonene	5989-27-5	Monoterpene	9.46
10a	C₁₀H₁₆	γ-Terpinen	99-85-4	Monoterpene	13.1
10b	C₉H₁₄N₂	2,3-Diethyl-5-methyl-pyrazine	18138-04-0	Pyrazine	24
11	C₁₅H₂₂	8,9-Dehydro-cycloisolongifolene		Sesquiterpene	24.9
12	C₁₅H₂₄	α-Santalene	512-61-8	Sesquiterpene	58.5
13	C₁₅H₂₄	α-Bergamotene	17699-05-7	Sesquiterpene	54.9
14	C₁₅H₂₄	(−)-β-Santalene	511-59-1	Sesquiterpene	67.2
16	C₁₅H₂₄	β-trans-Bergamotene	28973-97-9	Sesquiterpene	10.9
17	C₁₅H₂₄	β-Bisabolene	495-61-4	Sesquiterpene	41.2
18a	C₁₅H₂₃	4,5,9,10-Dehydro-isolongifolene	156747-45-4	Sesquiterpene	71.7
18b	C₁₅H₂₄	β-Vatirenene		Sesquiterpene	34.6

VOCs produced upon growth on Brian’s medium

Iron supplemented cultures

These are the volatiles produced in iron supplemented cultures. Unidentified peaks have been edited out of the table. What are the implications for fungal growth in the gingiva of humans? If the growth is bad and there is bleeding, there will also be iron.

3-Methyl-1-butanol, aka isoamyl alcohol, has potential anti fungal properties according to PubChem.“Isoamylol is an primary alcohol that is butan-1-ol in which a hydrogen at position 3 has been replaced by a methyl group. It has a role as a xenobiotic metabolite, a Saccharomyces cerevisiae metabolite and an antifungal agent. It is a primary alcohol, a volatile organic compound and an alkyl alcohol. It derives from a hydride of an isopentane.”

2,4-Diacetylphloroglucinol, according to PubChem, ” 2,4-diacetylphloroglucinol is a benzenetriol that is phloroglucinol in which two of the ring hydrogens are replaced by acetyl groups. It has a role as a bacterial metabolite and an antifungal agent. It is a diketone, an aromatic ketone, a methyl ketone and a benzenetriol. 2,4-Diacetylphloroglucinol is a natural product found in Pseudomonas, Pseudomonas fluorescens, and Pseudomonas protegens with data available.

VOC type and peak no.	Formula	Trivial name	CAS no.	Substance class	Probability
19	C₂H₆O	Ethanol	64-17-5	Alcohol	90.5
20	C₅H₁₂O	3-Methyl-1-butanol	123-51-3	Alcohol	76.9
21	C₉H₁₄	3-Ethylidenecycloheptene			17.9
22	C₁₀H₁₆	α-Phellandrene	99-83-2	Monoterpene	61.5
23	C₈H₁₂N₂	2-Methyl-5-isopropylpyrazine	13925-05-8	Pyrazine	64.4
24	C₁₀H₁₆	α-Phellandrene	99-83-2	Monoterpene	33
25a	C₉H₁₈O	(E)-6-Nonen-1-ol	31502-19-9		28.5
25b	C₁₀H₁₆	Terpinolene	586-62-9	Monoterpene	23.6
28	C₁₁H₁₈N₂	2-(2-Methylpropyl)-3-(1-methylethyl)pyrazine		Pyrazine	81.2
30	C₁₅H₂₄	(Z,E)-α-Farnesene	26560-14-5	Sesquiterpene	20.2
33	C₁₅H₂₄	Cedr-8(15)-ene	11028-42-5	Sesquiterpene	11.6
34	C₁₅H₂₄	α-Curcumene	644-30-4	Sesquiterpene	53.2
36a	C₁₅H₂₄	Dihydrocurcumene	1461-02-5	Sesquiterpene	46.6
36b	C₁₅H₂₄	(+)-Epi-β-santalene	25532-78-9	Sesquiterpene	43.6
40	C₁₅H₂₄	α-Curcumene	644-30-4	Sesquiterpene	42.1
41	C₁₀H₁₀O₅	2,4-Diacetylphloroglucinol	2161-86-6	Polyketide	70.9
45	C₁₅H₂₄	α-Patchoulene	560-32-7	Sesquiterpene	20.9
46	C₁₅H₂₄	cis-α-Bisabolene	29837-07-8	Sesquiterpene	26.1
49	C₁₅H₂₄	β-Vatirenene		Sesquiterpene	19.1
51	C₁₅H₂₀	4,5,9,10-Dehydro-isolongifolene	156747-45-4	Sesquiterpene	74.8
54	C₁₅H₂₄O	Ledene oxide(II)		Sesquiterpene	20.3
56	C₁₅H₂₀	4,5,9,10-Dehydro-isolongifolene	156747-45-4	Sesquiterpene	79.9
57	C₁₅H₂₄O	Isoaromadendrene epoxide		Sesquiterpene	12.1
58	C₁₅H₂₄O	Santalol	11031-45-1	Sesquiterpene	17.6
59	C₂₀H₃₂	Biformene	5957-33-5	Diterpene	37.1
60	C₂₀H₃₁	Rimuene	1686-67-5	Diterpene	22.1
61	C₂₀H₃₂	(5α,9α,10β)-Kaur-15-ene	511-85-3	Diterpene	77.2
62	C₂₀H₃₂	13-Isopimaradiene	1686-56-2	Diterpene	55.3
63	C₂₀H₃₂	(8β,13β)-Kaur-16-ene	20070-61-5	Diterpene	42.8

This post has been rather wimpy in defining exactly what is meant by the probability. Finding reliable standards is not always easy.

Manganese and iron seem to promote terpene production while copper and zinc seem to inhibit production.

CuCl₂, Fe₂(SO₄)₃, FeSO₄, and MnCl₂ were added at a final concentration of 100 μM, and 1 mM ZnSO₄·7 H₂O was used.

fumagillin in spite of being produced by a fungi has anti-bacterial and protozoa activities as well as sellect anti-fungal properties
aspergillic acid also has antibacterial and anti-fungal properties
gibbereilic acid is a plant hormone

From Heddergott 2014. The structures of menthol and citronellol have been added. Limonene, in the lower right hand corner, has been starred. The red circles with the X on the side are metabolic deficient strains used in their study.

What does this mean?

The scientific community seems to be transfixed with terpenes from local foliage containing the latest and greatest anti microbial. It would seem that fungi produce plenty of metabolites to control each other. We know that those that do not produce aflatoxin can degrade the AF produced by other strains. [1] Is this true of terpenes as well? The change in metabolites in response to divalent cations has implications to our own oral microbiomes. Should we even consider a mouthrinse with EDTA?

References

Maxwell LA, Callicott KA, Bandyopadhyay R, Mehl HL, Orbach MJ, Cotty PJ. Degradation of Aflatoxins B₁ by Atoxigenic Aspergillus flavus Biocontrol Agents. Plant Dis. 2021 Sep;105(9):2343-2350. Free article
Kim E, Park IK. Fumigant antifungal activity of Myrtaceae essential oils and constituents from Leptospermum petersonii against three Aspergillus species. Molecules. 2012 Sep 3;17(9):10459-69. PMC free article
Abbaszadeh S, Sharifzadeh A, Shokri H, Khosravi AR, Abbaszadeh A. Antifungal efficacy of thymol, carvacrol, eugenol and menthol as alternative agents to control the growth of food-relevant fungi. J Mycol Med. 2014 Jun;24(2):e51-6. PubMed
Heddergott C, Calvo AM, Latgé JP. The volatome of Aspergillus fumigatus. Eukaryot Cell. 2014 Aug;13(8):1014-25 PMC free article

Aspergillus metabolites