Journal of Functional Foods 115 (2024) 106120

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Journal of Functional Foods

journal homepage: www.elsevier.com/locate/jff

Effect of erinacine A-enriched Hericium erinaceus supplementation on

cognition: A randomized, double-blind, placebo-controlled pilot study
Maša Černelič Bizjak a, Zala Jenko Pražnikar a, Saša Kenig a, Matjaž Hladnik b, Dunja Bandelj b,
Andrej Gregori c, Katja Kranjc a, *
a
University of Primorska, Faculty of Health Sciences, Polje 42, SI-6310 Izola, Slovenia
b
University of Primorska, Faculty of Mathematics, Natural Sciences and Information Technologies, Glagoljaška 8, 6000 Koper, Slovenia
c
MycoMedica Ltd., Podkoren 72, 4280 Kranjska Gora, Slovenia

A R T I C L E I N F O A B S T R A C T

Keywords: Population aging has led to an increased interest in various dietary supplements in order to preserve cognitive
Erinacine A function. The aim of our study was to examine the effects of Hericium erinaceus supplementation (HE) on
Hericium erinaceus cognitive function and serum levels of Brain-Derived Neurotrophic Factor (BDNF) and Neuropeptide Y (NPY). An
Cognition
8-week double-blind comparative study involved 33 subjects, randomly assigned to a HE group and a placebo (P)
BDNF
group. Anthropometric measurements and analyses of selected biochemical parameters were performed before
Gut microbiota
and after the intervention. Simultaneously, faecal samples were collected to determine gut microbiota and
chitinase 3-like-1 (CHI3L1) levels. Cognitive function was assessed using two non-verbal speed tests. In the HE
group, a significant improvement in cognitive ability was observed when the corresponding cognitive scores at
baseline, CHI3L1 level, age, and gender were considered. Moreover, in the HE group there was a significant
increase in gut microbiota diversity that was positively correlated to NPY levels. The mechanism that could help
to explain the results on the relationship between the active ingredients of the H. erinaceus and the cognitive
parameters could point to the CHI3L1 activity that may enhance bioavailability of the bioactive ingredients.
Supplementing the diet with HE was recognised as a safe and well-tolerated intervention with a neurocognitive
benefit.

1. Introduction 2018). In this generalised slowing, the change of subcortical white

matter integrity in the whole brain may play a central role (Coelho et al.,
Cognitive functioning refers to the performance of mental processes, 2021; Madden et al., 2017).
including thinking, reasoning, perception, learning, memory, under­ Due to increased average life expectancy, we are confronted by a
standing, awareness, judgment, and intuition (Fisher et al., 2019). The higher number of older adults experiencing both normal age-related
speed of these mental processes dictates our responses to stimuli, called cognitive decline as well as a substantial number of individuals experi­
reaction time. Neural activity associated with processing speed plays a encing non-normal decline, such as mild cognitive impairment and de­
crucial role in cognitive performance and has been extensively investi­ mentia (Association, 2018). Cognitive (intellectual) ageing has become
gated as a cognitive marker in various neurocognitive disorders (Lu an important public health challenge attracting the attention of re­
et al., 2017). With aging, there is a natural decline in processing speed, searchers. As preserving cognitive function is an essential component of
leading to slower cognitive functioning. This normal (non-pathological, maintaining a healthy, active, and independent lifestyle, an expanding
normative) cognitive slowing or age-associated cognitive decline has body of research focuses on appropriate strategies to prevent the pro­
been shown to begin relatively early in middle age and progress more gressive cognitive decline (Dysken et al., 2014; Farina et al., 2017; Pinto
rapidly with advancing age (Meunier et al., 2014; Murman, 2015). The & Subramanyam, 2009) and slow age-related changes in cognitive
neuropathological cause of age-related cognitive slowing is related to functions. These conditions can be delayed or improved by lifestyle
the speed of neuronal transmissions and to myelination (Chopra et al., changes, such as healthier eating habits (Cohen et al., 2016; Ngandu

* Corresponding author.
E-mail addresses: [email protected] (M. Černelič Bizjak), [email protected] (Z. Jenko Pražnikar), [email protected] (S. Kenig), matjaz.
[email protected] (M. Hladnik), [email protected] (D. Bandelj), [email protected] (A. Gregori), [email protected] (K. Kranjc).

https://doi.org/10.1016/j.jff.2024.106120
Received 15 November 2023; Received in revised form 24 February 2024; Accepted 1 March 2024
Available online 7 March 2024
1756-4646/© 2024 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-
nc/4.0/).
M. Černelič Bizjak et al. Journal of Functional Foods 115 (2024) 106120

et al., 2015), following a nutritious diet (e.g., MIND diet based on the et al., 2021). As it is well known, gut microbiota is an important factor
dietary components of the Mediterranean or DASH diet) (Morris et al., influencing mental health since it can affect the gut-brain axis through
2015; Singh et al., 2014), caloric restriction (Bishop et al., 2010), regular regulation of different neuroactive modulators such as BDNF (Liaqat
exercise (Imaoka et al., 2019), and cognitive training practices (Pereira- et al., 2022). The consumption of H. erinaceus has already been shown to
Morales et al., 2018). promote the growth of beneficial gut bacteria with additional positive
Beneficial effects on cognitive functions of older individuals have effect on cognitive function in elderly mice model. Overall, authors
been confirmed by the intake of nutraceuticals and/or dietary supple­ suggest that the modulation of gut microbiota composition could trigger
ments (Iolascon et al., 2017). However, many of these interventions lack longevity-promoting effects, protecting from age-related cognitive
support from the published scientific literature regarding their efficacy decline (Priori et al., 2024).
or safety (Crawford et al., 2020). Favourable outcomes have also been In the present randomised clinical trial, we have thus investigated
observed from the consumption of edible mushrooms, which are the effects of daily consumption of erinacine A-enriched H. erinaceus
increasingly valued for their medicinal properties. These mushrooms are supplementation (HE) on cognitive functioning, serum biochemical
not used only as dietary food (functional foods), but also in the form of markers, faecal levels of chitinase, and gut microbiota composition.
dietary supplements, nutraceuticals, and mycotherapy products. The use
of mushrooms to promote and maintain health and to treat diseases has 2. Methods and materials
been known in traditional Chinese medicine since ancient times (Money,
2016). Owing to their health benefits, they are therefore widely used in 2.1. Preparation of erinacine A-enriched dietary supplement
complementary alternative medicine and complementary integrated
medicine (Venturella et al., 2021). During the R&D project entitled “First food supplement standardised
Numerous studies, both in vitro/vivo preclinical and clinical, have on erinacine A content”, also known as the ”ErinacinA project,“ more
demonstrated the effects and mechanisms of bioactive compounds than 50 strains of H. erinaceus were screened for erinacine A content. The
derived from Hericium erinaceus (Hetland et al., 2020; Venturella et al., most productive strains were later cultivated under optimised growing
2021). Both the fruiting body and mycelia of H. erinaceus contain a conditions with more than 2500 cultivation and extraction experiments
remarkable variety of structurally diverse bioactive components that conducted by MycoMedica Ltd, Kranjska Gora and PharmaHemp Ltd,
contribute to the prevention, alleviation, and treatment of various dis­ Ljubljana, Slovenia. As a result, a new food supplement “H. erinaceus
eases. Notably, antioxidative, anti-inflammatory, anticancer, immu­ heteropolysaccharides” was developed being sold under a brand name
nostimulant, antidiabetic, antimicrobial, hypolipidemic, and “GOBA® Hericium erinaceus”, containing standardised concentrations
antihyperglycemic properties of H. erinaceus have been reported (Khan of erinacine A. Since HE supplement used in this study is a commercial
et al., 2013). Through clinical trials, pharmacological activities and product for which a patent is currently pending only limited set of data
medical evidence of H. erinaceus have been demonstrated, showing its about cultivation is available. Briefly, H. erinaceus strain Her. Erin.
effectiveness in improving average cognitive impairment (Mori et al., (fungal culture collection of MycoMedica Ltd, Podkoren, Slovenia) was
2008), treating early Alzheimer’s disease (Li et al., 2020), alleviating cultivated in darkness at 24 ◦ C on Potato Dextrose Agar. When fully
symptoms of anxiety and depression, and improving sleep quality overgrown mycelium was dispersed in water (one 120 mm petri dish per
(Nagano et al., 2010; Okamura et al., 2015; Vigna et al., 2019). As its 1000 mL of sterilized water) using a Waring blender and further used as
applications are diverse, H. erinaceus is most commonly used for the liquid inoculum.
treatment of neurodegenerative diseases and cognitive impairment 5 kg of organic cellulose and supplements, containing a vegan,
(Chong et al., 2021; Kawagishi and Zhuang, 2008; Spelman et al., 2017). gluten- and allergen-free substrate with moisture content of 65 % (w/w),
Importantly, the bioactive metabolites of H. erinaceus, namely the eri­ were packed into polypropylene bags and sterilised at 121 ◦ C for 80 min.
nacines (a group of cyathin diterpenoids extracted from the mycelium) Following sterilisation, the substrate was cooled to room temperature
and hericenones (benzyl alcohol derivatives extracted from the fruiting and inoculated with 50 mL of liquid inoculum of H. erinaceus. The
body), can easily traverse the blood–brain barrier (Venturella et al., cultivation process took place at a temperature of 24 ◦ C for approxi­
2021). These compounds have been shown to enhance the synthesis of mately three months, after which the fungus was harvested. A portion of
trophic factors, such as nerve growth factor (NGF) and brain-derived the harvested fungus was extracted and combined with the initial batch,
neurotrophic factor (BDNF) (Chiu et al., 2018; Friedman, 2015; Kawa­ and then dried under vacuum for 24 h at temperatures below 60 ◦ C.
gishi et al., 1996; Mori et al., 2008), which exhibit neurotropic and During the drying process, a drying agent containing starch and cellu­
neuroprotective effects (Huang et al., 2021; Roda et al., 2021). The lose was used. Due to vigorous evaporation under vacuum of approxi­
evidences on H. erinaceus in stimulating NGF release, regulating in­ mately 60 mbar and elevated temperature (<60 ◦ C) the resulting
flammatory processes, reducing oxidative stress, and protecting nerve material achieved moisture content lower than 7 % in less than 24 h.
cells from apoptosis, shows its neuroprotective potential Subsequently, the material was finely powdered, with particle sizes
(Szućko-Kociuba et al., 2023). Furthermore, the presence of poly­ smaller than 250 μm, and further encapsulated into capsules containing
saccharides, such as chitin, is also important. Chitin is a β-(1,4)-linked 500 mg of material. The samples used in this study included erinacine A-
homopolymer of N-acetylglucosamine and is one of the most abundant enriched H. erinaceus supplement (HE), or corn starch in the case of
biopolymers found in fungi, serving as a primary component of their cell placebo (P) group. Both substances were filled into hydroxypropyl
walls (Shahidi & Abuzaytoun, 2005). Since chitin or chitooligo­ methylcellulose capsules and administered to the study participants.
saccharides are generally resistant to digestion by human enzymes, the
presence of chitinase-producing commensal bacterial in gut could 2.1.1. Analyses of erinacine A-enriched dietary supplement
facilitate the release and subsequent utilization of bioactive compounds For the analysis of erinacine A content in the material, extraction
from fungal cells. Moreover, the presence of chitinase-producing bac­ using 70 % ethanol was performed. Quantification was carried out using
teria is also necessary for the degradation of chitin and its utilisation as an Agilent 1260 Infinity II HPLC system with a Poroshell 120 SB-C18,
an energy source (Dohnálek et al., 2021). Conversely, certain bioactive 4.6 × 150 mm, 2.7 μm column and a DAD detector set at a wave­
compounds found in mushrooms, such as polysaccharides and phenolic length of 340 nm (Agilent, Santa Clara, CA, USA). The employed HPLC
compounds, have been shown to significantly affect gut microbiota. method was developed and validated in an ISO 17025 accredited labo­
These compounds can contribute to the enrichment of the gut micro­ ratory. Erinacine A standard was obtained from H. erinaceus by extract
biota, promote the growth of beneficial bacterial species, including fractionation using Flash/Prep in first dimension, followed by second
those that produce short-chain fatty acids (SCFAs), increase the Bacter­ dimension fractionation using HPLC-UV. The identity of the isolated
oidetes/Firmicutes ratio, and reduce the presence of harmful species (Li compound was proven by LC-MS/MS and the purity (against procedural

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M. Černelič Bizjak et al. Journal of Functional Foods 115 (2024) 106120

blank) was determined by LC-CAD (Fig. S1). placebo (P) group. Randomisation with stratification was performed
Furthermore, HE was analysed for total polyphenols content and using a simple software, free open-source application MinimPy (Saghaei
antioxidant activity as described before (Atamanchuk and Bisko, 2023). & Saghaei, 2011), accessible through the website (https://sourceforge.
The content of total polyphenols in tested material was 3.17 ± 0.18 mg/ net/projects/minimpy). Stratified variables were gender and age. We
g of gallic acid equivalents and antioxidant activity of the material was used two randomly assigned separate sequences for males and females,
81.3 ± 0.43 % (free radical scavenging activity determined by 2,2- and three randomly assigned separate sequences for age groups (55–59
diphenyl-1-picrylhydrazyl (DPPH) assay). years; 60–65 years; 66–75 years).
The relative amounts of protein (AOAC Official method 954.01), Therefore, both groups (HE and P) were equal with regard to sex and
lipid (AOAC Official method 920.39), water (AOAC Official Method age. The study was double blind. Capsules were packed and labelled by a
934.01), ash (AOAC Official method 942.05) and fibre (AOAC Official research assistant that was blind to the aim of the study. Also, both
method 978.10) in HE were estimated by proximate analysis. The ana­ capsules were similar in colour, size and shape to ensure that they could
lyses were conducted at the Biotechnical Faculty, Chair of Nutrition, not be distinguished by the participants or researchers. The entire study
University of Ljubljana, Slovenia. Carbohydrate content in samples was consisted of 8 weeks of intervention period, during which subjects
calculated by difference (100 – water – lipids – proteins – ash). Since ingested two capsules after breakfast, lunch and dinner (6 capsules/day)
these results were not an object of our study, they are presented in with daily intake of 3.44 mg of erinacine A. Participants were advised to
Table S2. maintain their level of physical activity and diet, and to maintain their
daily life rhythm and habits. Anthropometric, cognitive, and biochem­
ical measurements were performed at baseline (measurement 1) and
2.2. Study design after 8 weeks of supplement intake (e.g. consumption of either HE or P)
(measurement 2). At this two time points, participants provided a stool
The study protocol was approved by the Slovenian National Medical sample, previously collected at home.
Ethics Committee (No. 0120–321/2017–4) and was registered at
ClinicalTrials.gov (Identifier: NCT04939961). All the participants
signed their informed consent prior to their inclusion in the study. The 2.3. Study participants
double-blind randomised, comparative trial was conducted at the Uni­
versity of Primorska, Faculty of Health Sciences Izola, Slovenia, between The Caucasian male and female volunteers were recruited through
June and September 2021. The study design is presented in Fig. 1. an advertisement posted on internet forums, sent via e-mail lists, or
Thirty-six participants were randomly assigned into 2 groups: to the published in local newspapers. Thirty-six asymptomatic adults were
erinacine A-enriched H. erinaceus supplementation (HE) group or to the recruited into the study. The inclusion criteria were age over 55 years,

Fig. 1. Participant flow diagram and design of the study. 36 participants were recruited to the study, out of which 33 met the inclusion criteria. All subjects
completed 8 weeks of intervention period, with daily intake of 3.44 mg erinacine A or placebo.

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M. Černelič Bizjak et al. Journal of Functional Foods 115 (2024) 106120

absence of chronic, cardiovascular, gastrointestinal, or liver diseases, (ALT), C-reactive Protein (CRP), and uric acid (UC), a Cobas c111 ana­
type 2 diabetes mellitus, or neurodegenerative diseases, and no taking of lyser (Roche, Basel, Switzerland) was used. Analyses were conducted
medications for lipid disorders, anti-inflammatory drugs, antidepres­ using fresh Cobas reagents and carried out following the manufacturer’s
sants, or anxiolytics, and absence of other brain diseases like brain instructions.
injury, brain atrophy, and stroke. Serum concentrations of Neuropeptide Y (NPY) and Brain-Derived
The exclusion criteria were mushroom allergies and use of antibiotics Neurotrophic Factor (BDNF) were determined in duplicate using
in the past three months. During the study, all participants refrained human ELISA kits (EMD Millipore Corporation, Missouri, USA). The
from taking antibiotics and other dietary supplements. assays were performed according to the manufacturer’s instructions.
The optical density of each well was measured using a microplate reader
2.4. Anthropometric measurements and dietary assessment (Tecan, Mannedorf, Switzerland). Assay sensitivity was 15 pg/ml for
BDNF and 2 pg/ml for NPY. Inter-assay and intra-assay CVs were typi­
Anthropometric measurements using a bioelectric impedance ana­ cally < 10 %.
lyser Tanita MC-980MA (Maeno-cho, Japan) were performed after an
overnight fasting between 7 a.m. and 9 a.m. in standardised conditions 2.7. Stool sample analysis
by the same examiner. Measured parameters included body weight,
body fat, fat free mass, muscle mass, visceral index, total body water, Participants were asked to collect stool samples at home and store
extracellular water, intracellular water, and phase angle. The body mass them at -20 ◦ C before taking them to the laboratory, where they were
index (BMI) was calculated as weight (kg) divided by height (m) kept at -80 ◦ C until analysis. Stool samples were weighed and divided for
squared. Participants recorded their food intake for three days before faecal chitinase 3-like-1 (CHI3L1) measurement and bacterial DNA
visit. Dietary data were analysed using the Open Platform for Clinical extraction. CHI3L1 faecal extraction, were conducted as described
Nutrition (OPEN), accessible through the website https://opkp.si/. before (Volkers et al., 2022) with some modifications. Briefly, Phosphate
Buffered Saline (PBS) was added to adjust weight-to-PBS ratios and
2.5. Cognitive tests vortexed into a homogeneous mixture. Samples were then centrifuged at
13,000g for 10 min and the resulting supernatants were used in the
A thorough cognitive assessment with two standardised tests was commercial ELISA kit (R&D Systems, Human Chitinase 3-like-1 DuoSet
performed at baseline and after an 8-week intervention. Both tests ELISA, Minneapolis, MN, USA), following manufacturer’s instructions.
measure cognitive performance: speed (rate of performance) and level For the assessment of the bacterial community composition, DNA
(accuracy of response). The primary outcome was a change in cognitive was extracted using the commercial QIAamp DNA Stool Mini kit (Qiagen
performance as measured by total test score (higher score indicates N.V., Venlo, The Netherlands) and analysed as described before (Petelin
better performance). et al., 2022; Šik Novak et al., 2022). Briefly, amplification of V4 16S
rRNA was performed with modified 515F and 806R. Primer 806R was
2.5.1. Test of perception speed “patterns” (THP) elongated with the sequence of P1 adapter and 515F primer was elon­
The THP is a 36-tasks non-verbal speed test to measure the percep­ gated with the sequence of linker, barcode and adapter at their 5′-end in
tion speed, mental speed, and in the less capable individuals also bio­ order to produce amplicons compatible for sequencing with Ion S5TM
logical intelligence (Pogačnik, 2012). In each task, there is one pattern System (Thermo Fisher Scientific, Waltham, MA, USA). Each sample was
on the left side and four more on the right side, only one of which exactly amplified with a unique barcode in triplicates to reduce PCR bias.
matches the pattern on the left. The participant must find and mark it as Negative control was performed with a unique barcode as well. PCR
quickly as possible. The test is limited to 4.5 min. The reliability products were checked on agarose gel and equal volume of pooled
calculated on various samples according to the KR-20 formula was 0.86 triplicates were joined in a final library. Final library was cleaned with
to 0.71 (Pogačnik, 2012). Agencourt AMPure XP magnetic beads (Beckman Coulter) using bead-
to-DNA ratio of 0.7:1. Quality of DNA library and DNA concentration
2.5.2. Test of series (TN-10-A) was determined on Agilent 2100 Bioanalyser using High Sensitivity DNA
The TN-10-A is a non-verbal speed test for measuring fluid intelli­ Assay kit (Agilent Technologies, Santa Clara, CA, USA). Ion 530™ chip
gence, which is relatively independent of education, experience and with DNA library and Ion S5™ calibration standard was prepared with
culture (Pogačnik, 2006), and reflects information processing capacity. Ion OneTouch™ 2 system using the kit Ion 520™ & Ion 530™ Kit-OT2
The result is primarily influenced by the ability to recognise relation­ and sequencing was performed on Ion S5™ System (Thermo Fisher
ships and legitimacy, working memory, the speed of information pro­ Scientific, Waltham, MA, USA). Reads were analysed with QIIME2
cessing, and mental and perceptual speed. The test thus measures a v.2021.8. DADA2 (qiime dada2 denois-pyro plugin) was used for
broad grouping of mental abilities. It consists of 45 series of tasks with denoising and determination of amplicon sequence variants (ASVs).
progression in difficulty. Each task contains a string of 15 characters on Taxonomy classification was made with feature classifier plugin based
the left side and five suggested answers on the right side, only one of on naive Bayes classifier trained on SILVA reference database, version
which continues the series correctly. The time for the test accomplish­ 138.1. Microbiota diversity was quantified using Shannon index, which
ment is 10 min, meaning that mental speed affects the scores. The reli­ reflects the richness of bacterial community (Kim et al., 2017).
ability reported for the TN-10 is 0.86 to 0.90 and by the test–retest
method (48 months) 0.90 (Pogačnik, 2006). 2.8. Statistics

2.6. Biochemical serum measurements Statistical analysis was performed using SPSS version 26.0 (IBM
Corp., Armonk, NY). The normality of the variables was tested using the
For venous blood withdrawal, the participants were instructed to Shapiro–Wilk test. Data are presented as the mean value with the
restrain from eating for 12 h prior to the blood withdrawal. The blood standard deviation, the median value with the minimum and maximum,
was collected into 5 mL vacuum blood serum collection tubes, and or a percentage. The effects of the interventions within each group were
serum samples were obtained as previously described (Tuck et al., analysed using Student’s paired samples t-test or the Wilcoxon signed-
2009). The serum samples were stored in polypropylene tubes at -80 ◦ C rank test, whereas the comparison of mean changes between the two
until further analysis. To determine the levels of serum glucose (GLU), groups was done using an independent t-test or Mann–Whitney U test.
Low-Density Lipoprotein (LDL), High-Density Lipoprotein (HDL), total Moreover, as age and sex have an impact on observed cognitive pa­
cholesterol (TC), triacylglycerols (TAG), Alanine Aminotransferase rameters, a univariate analysis of covariance (ANCOVA) with age and

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M. Černelič Bizjak et al. Journal of Functional Foods 115 (2024) 106120

sex as a covariate was performed to compare cognitive data among the cognitive outcome when age, sex, and baseline THP score have been
groups. The effects of the interventions were analysed using ANCOVA included in the model.
with the change at week 8 as the dependent variable, adjusted to the
corresponding values at baseline, and stratified for age and sex. P-values 3.4. Effects of HE on neurotrophic markers
< 0.05 were considered statistically significant.
In addition to cognitive function, we focused on the effect of HE on
3. Results serum levels of the neurotrophic factors BDNF and NPY. In the HE group,
we observed an increase in BDNF levels from baseline (7.47 ± 4.86) to 8
3.1. Baseline characteristics of study participants weeks (8.49 ± 3.28). This suggests that the intervention (HE) had a
positive effect on increasing serum BDNF levels. In contrast, the placebo
In this clinical study, 36 subjects started the experiments; one subject group demonstrated a notable decrease in BDNF serum levels over the
dropped out due to Covid-19, and two after the follow-up period. same period. The baseline BDNF level in the placebo group was 9.29 ±
Therefore, the final sample size was 33 subjects (67 % F; 33 % M), aged 3.65, which decreased to 7.01 ± 2.85 after 8 weeks. This decrease was
55–75. The baseline characteristic of the participants in the study are found to be statistically significant (p < 0.05) according to the results
presented in Table 1. At baseline, groups did not differ significantly with presented in Table 3. In summary, the study findings imply that the
respect to age, gender, BMI, and energy intake. intervention (HE) had a positive impact on increasing BDNF serum
levels, whereas the placebo group experienced a significant decrease in
BDNF levels over the 8-week period. Importantly, the difference in the
3.2. Effects of HE on anthropometric and biochemical parameters
intervention effect between the HE and placebo groups reached statis­
tical significance (p < 0.05). For NPY, there were no differences within
The consumption of HE or P had no major effect on anthropometric
or between the groups.
or basic biochemical parameters. No significant differences were found
between the groups, neither in the initial nor in the final values (with the
3.5. Effects of HE on faecal CHI3L1 levels and relation to cognitive and
respect to anthropometric and biochemical parameters, Table 2). BMI,
neurotrophic markers
visceral fat, glucose, total cholesterol, CRP, and liver enzyme ALT did
not change significantly in either group. Notably, the serum concen­
Due to the possibility of the chitinase effect on the bioavailability of
tration of TAG showed a significant increase in the P group (p ≤ 0.05). In
bioactive components of HE supplement, CHI3L1 levels in the stool
the HE group, there was an increase in serum LDL cholesterol and a
samples of each participant were measured. There were no statistically
decrease in HDL cholesterol and UA. Similar but not significant changes
significant differences in CHI3L1 levels between the groups and they
in these parameters were observed in the placebo group. When changes
were not observed either when the value after the intervention was
from baseline were compared, no significant differences between groups
compared with the initial value within the group (Table 3). Mean values
were detected.
from data before and after the intervention for each participant were
then calculated. We investigated the potential effect of CHI3L1 faecal
3.3. Effects of HE on cognitive function concentrations on cognitive abilities and neurotrophic markers.
After including the mean values of CHI3L1 as a covariate into our
The main focus of the present study was to test the effect of erinacine model, along with other relevant parameters (baseline values, age,
A-enriched HE supplement on the cognitive functioning of individuals. gender), a comprehensive analysis revealed a statistically significant
We were interested in whether the consumption of the supplement impact of the HE intervention on the parameters THP (F = 5.3, p =
during an 8-week intervention had an effect on improving certain 0.040), TN (F = 9.3, p = 0.010), and NPY (F = 5.3, p = 0.039). This
cognitive abilities. Initial cognitive test scores did not differ significantly underscores the significant influence of HE on these parameters, even
between the groups, nor did final scores or changes from baseline when accounting for the covariate CHI3L1. Conversely, in the P group,
(Table 3). Although a slight increase in THP and TN scores were CHI3L1 did not exhibit a statistically significant effect on any of the
observed in the HE group after the intervention, no significant differ­ parameters studied, emphasizing a differential impact compared to the
ences in participants’ cognitive performance were found using a simple HE group. Notably, similar results were replicated when incorporating
parametric tests. the post-intervention value of CHI3L1 instead of the average value in
Since gender differences in cognitive functioning had consistently stool into the model. This consistency strengthens the robustness of our
been reported in some cognitive tasks (Gurvich et al., 2020), it is findings, further supporting the notion that HE exerts a significant effect
important to consider age and sex differences when examining cognitive on THP, TN, and NPY parameters in contrast to the placebo group.
performance. Looking at the group difference between the HE group and
the placebo group, adjusted for baseline values and stratified by age and 3.6. Effect of HE on gut microbiota composition
gender, a statistically significant improvement in cognitive function in
the THP test/score (F = 4.6, p < 0.05) was observed in the HE group at The data of gut microbiota are presented for 32 subjects that is one
the end of the intervention (8 weeks). We thus noted a significant less than for other study results; one subject dropped out due to the
beneficial effect of the intervention with HE supplementation for the missing faecal sample in one of the time points. The relative abundance
of bacteria phyla before the intervention was the same across the groups
Table 1 and was the highest for Firmicutes (51.5 ± 17.4 vs. 49.6 ± 15.4 %).
Baseline characteristics of the subjects included in the clinical trial. There were also no statistically significant differences in genera distri­
HE group (N = 18) P group (N = 15) p value bution (with relative abundance ≥ 1 % that were found in more than 10
% of faecal samples) between the groups (Table S1).
Gender (% F, M) 67 % F, 33 % M 67 % F, 33 % M 1
Age (years) 63.1 ± 7.0 62.5 ± 7.6 0.841 The statistically significant differences in the HE group after the
BMI (kg/m2) 25.0 ± 3.8 25.7 ± 3.2 0.568 intervention were observed only for three genera with a relative abun­
Energy intake (kcal/day) 1967 ± 799 1833 ± 523 0.569 dance of less than 1 %. These genera were as follows: Intestinimonas (p =
Abbreviation: BMI, body mass index; F, female; HE, erinacine A-enriched 0.040), Gastranaerophilales (p = 0.038), and Tyzzerella (p = 0.028). A
H. erinaceus supplementation; M, male; P, placebo. higher abundance of Flavobacteriaceae family (p = 0.042) in the HE
Values are expressed as means ± SD. P-value denotes difference between groups group was also observed. When comparing the relative abundance of
using an independent samples t-test or Mann–Whitney U test. phyla and genera that were present at a relative abundance of more than

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Table 2
Anthropometric and biochemical parameters (means ± standard deviations) at baseline and after intervention (week 8).
HE group (N = 18) P group (N = 15)

Measure Baseline Week 8 Difference Baseline Week 8 Difference

BMI (kg/m2) 25.0 ± 3.8 25.0 ± 3.9 0.0 ± 0.4 25.7 ± 3.2 25.8 ± 3.0 0.1 ± 0.6
Visceral fat 8.7 ± 3.0 9.0 ± 3.2 0.3 ± 0.5 9.1 ± 2.1 8.9 ± 2.2 − 0.2 ± 0.8
GLU (mmol/L) 5.41 ± 0.77 5.49 ± 0.91 0.08 ± 0.41 5.34 ± 0.47 5.35 ± 0.57 0.01 ± 0.43
TC (mmol/L) 5.48 ± 0.99 5.92 ± 0.95 0.08 ± 0.49 5.30 ± 1.26 5.37 ± 1.22 0.07 ± 0.47
LDL (mmol/L) 4.35 ± 1.08 4.51 ± 1.00 a 0.17 ± 0.30 3.91 ± 1.28 3.98 ± 1.18 0.07 ± 0.41
HDL (mmol/L) 1.67 ± 0.32 1.60 ± 0.35 a − 0.07 ± 0.12 1.61 ± 0.31 1.54 ± 0.38 − 0.08 ± 0.19
a
TAG (mmol/L) 1.42 ± 0.71 1.44 ± 0.72 0.02 ± 0.30 1.22 ± 0.58 1.41 ± 0.57 0.19 ± 0.30
ALT (Ukat/L) 0.31 ± 0.11 0.32 ± 0.12 0.01 ± 0.10 0.42 ± 0.35 0.38 ± 0.37 − 0.04 ± 0.45
UA (µmol/L) 356 ± 65 332 ± 68 a − 24 ± 46 321 ± 72 310 ± 80 − 11 ± 35
CRP (mg/L) 1.65 ± 1.14 1.84 ± 1.45 0.19 ± 1.03 1.38 ± 0.88 1.28 ± 0.59 − 0.10 ± 0.92

Abbreviations: BMI, body mass index; GLU, glucose; TC, total cholesterol; LDL, low-density lipoprotein; HDL, high-density lipoprotein; TAG, triacylglycerols; ALT,
alanine aminotransferase; UA, uric acid; CRP, C-reactive protein; HE, erinacine.
A-enriched H. erinaceus supplementation; P, placebo.
a
Denotes a significant (p < 0.05) difference when compared with the initial value within the group using a Student’s paired samples t-test or Wilcoxon signed-rank test.

of study participants. Accordingly, there was a statistically significant

Table 3 higher abundance of Actinobacteria (p = 0.023) and Bacteroidetes (p =
Cognitive and neurotrophic markers, and CHI3L1 concentrations (means ±
0.016) phyla after the intervention in women.
standard deviations) at baseline and after intervention (8 week).
The Shannon diversity index per group at the genus level is shown in
HE group (N = 18) P group (N = 15) Table 4. The Shannon index significantly increased after the period of
Measure Baseline Week Difference Baseline Week Difference HE supplementation (t = 1.84, p = 0.042), whereas a significant
8 8 decrease in the P group (t = -2.16, p = 0.025) was observed. At baseline,
THP 20.3 ± 21.5 1.2 ± 4.1 19.8 ± 19.7 − 0.1 ± Shannon index did not differ between the groups (p = 0.098). However,
score 3.3 ± 5.0 †, #
3.5 ± 6.3 4.6 changes after the intervention period were significantly different (t =
TN score 15.4 ± 16.1 0.7 ± 4.0 17.9 ± 18.2 0.27 ± 4.0 2.84, p = 0.008). Furthermore, the increase in diversity was positively
#
4.1 ± 3.9 3.2 ± 3.4
BDNF 7.47 ± 8.49 1.02 ± 9.29 ± 7.01 − 2.28 ±
correlated to higher levels of NPY (r = 0.59, p = 0.012).
[pg/ 4.86 ± 3.28 3.38 3.65 ± 4.16b
mL] 2.85a 4. Discussion
NPY 9.67 ± 11.10 1.43 ± 13.17 13.35 0.19 ±
[pg/ 4.36 ± 6.84 5.52 # ± 7.76 ± 5.92 6.75
In this double-blind, placebo-controlled clinical trial we aimed to
mL]
CHI3L1 1.96 ± 4.36 2.40 ± 4.53 ± 3.66 − 0.87 ± investigate the effects of erinacine A-enriched HE supplement on
[ng/ 3.15 ± 6.7 3.56 6.93 ± 5.04 1.89 cognitive function, serum biochemical markers, faecal levels of CHI3L1,
mL] and gut microbiota composition in healthy adults (aged 62.9 ± 7.1). To
Abbreviations: THP, Test of Perception Speed Pattern; TN, Test of series; BDNF, emphasize, there were no significant differences between the groups in
Brain-Derived Neurotrophic Factor; NPY, Neuropeptide Y; CHI3L1, Chitinase 3 terms of age, anthropometric, and biochemical parameters (Table 1, 2).
like-1; HE, erinacine A-enriched H. erinaceus supplementation; P, placebo. In the light of available information, this is the first study revealing
a
Denotes a significant (p < 0.05) difference when compared with the initial diverse range of effects after the H. erinaceus supplementation. During
value within the group using a Student’s paired samples t-test or Wilcoxon the 8-week intervention, participants supplemented their normal diet
signed-rank test. with a daily intake of 3.44 mg of erinacine A.
b
Denotes a significant (p < 0.050) difference in changes between the H. erinaceus We hypothesised that the administration of HE exerts a positive ef­
and placebo groups. Changes were calculated by subtracting the initial values fect on the rapid and accurate processing of information in the brain.
from the final values.
(†) The speed of information processing serves as an indicator of central
Calculated from ANCOVA, adjusting for baseline values, and stratified for age
nervous system efficiency and represents a fundamental aspect of
and sex.
(#)
Calculated from ANCOVA, adjusting for baseline values and levels of CHI3L1, intellect and cognitive function, particularly in the terms of memory
and stratified for age and sex. efficiency. In this study, the applied cognitive test also includes
perceptual speed, i.e., the ability to perceive shapes in the field of view
1 %, there were no statistically significant differences detected between quickly and accurately, involving sensory and motor processes as well as
the HE and P groups. However, a difference in Flavobacteriaceae family various types of cognitive operations, such as comparison, substitution,
(p = 0.003) was observed between the groups after the intervention. As or transformation. The results revealed beneficial effects on cognitive
gut microbiota composition is influenced by a set of various factors, the performance observed as improved perceptual speed in the HE group,
effect of HE supplementation was valued also regarding age and gender when adjustments for age, gender, and baseline THP score were

Table 4
Gut microbiota diversity at baseline and after intervention (8 week).
HE group (N = 17) P group (N = 15)

Baseline Week 8 Difference Baseline Week 8 Difference

Shannon diversity index 2.75 ± 0.45 2.88 ± 0.34a 0.13 ± 0.28 2.99 ± 0.31 2.82 ± 0.44a − 0.16 ± 0.30b

Abbreviations: HE, erinacine A-enriched H. erinaceus supplementation; P, placebo.

a
Denotes a significant (p < 0.05) difference when compared to the initial value within the same group.
b
Denotes a significant (p < 0.050) difference in changes between the H. erinaceus and placebo groups. Changes were calculated by subtracting the initial values from
the final values.

6
M. Černelič Bizjak et al. Journal of Functional Foods 115 (2024) 106120

considered, that is the way information is organised, interpreted, and CHI3L1 has been recognised as one of the intestinal inflammatory
consciously experienced. Interestingly, H. erinaceus has been previously markers in relation to inflammatory bowel disease, with a cut-off value
reported to promote recovery of the sensory function after crush injury of 13.7 ng/g to differentiate healthy individuals (Aomatsu et al., 2011).
and improve peripheral nerve regeneration (Wong et al., 2015). In our study, there was no evidence of inflammation before or after the
The improvements in cognitive function observed in the HE group intervention, as indicated by mean faecal CHI3L1 of 4.03 ± 5.91 ng/mL
could be associated to altered serum levels of BDNF. In a recent study on and 3.16 ± 5.34 ng/mL, respectively. Although CHI3L1 lacks enzymatic
elderly hearing-impaired patients (Chan et al., 2022), BDNF levels were activity it possesses a binding ability to chitin and chito-oligosaccharides
elevated by the consumption of H. erinaceus that was however more (Fusetti et al., 2003). It has been shown that chitinase-producing bac­
effective for patients aged ≧ 65. It is known that BDNF is highly teria produce chitin-binding molecules such as CBP21, that can be
expressed in the hippocampus (Bathina & Das, 2015), a region critical involved in the bacterial adhesion to CECs (Vaaje-Kolstad et al., 2005).
for the maintenance of the cognitive function (Luo et al., 2017). One of Moreover, CHI3L1 can enhance the CBP21-mediated bacterial adhesion
the most empowering results about BDNF is its potent effects on syn­ to CECs in vitro (Kawada et al., 2008). We assume that a slight increase in
apses, making it an ideal and essential regulator of cellular processes CHI3L1 that was detected in the HE group after the intervention
that underlie cognition, playing a key role in modulating synaptic (Table 4) could promote the binding and accumulation of chitin-
transmission and plasticity (Bekinschtein et al., 2007). Thus, as BDNF is degrading commensal bacteria. The degradation of chitin in
one of the key molecules modulating brain plasticity and promoting H. erinaceus cells could increase the bioavailability of compounds and
axonal and dendritic growth, the altered BDNF levels in the HE group thus pronounce the effect of intervention on cognitive function. How­
could underlie the changes in structural connectivity detected in ever, further research is needed to better understand the exact role and
cognitive testing. Increased BDNF levels, which were observed in the HE mode of action of CHI3L1 and interaction to gut microbiota during the
group, but not in the P group in our study, could support the proposed intervention.
neurotropic and neuroprotective effects (Venturella et al., 2021) of the Furthermore, edible mushroom polysaccharides (EMPs) are consid­
bioactive metabolites of H. erinaceus. Researchers found that the her­ ered as prebiotics as they can be selectively fermented by colonised
icenones and erinacines present in H. erinaceus can easily cross the microbiota in the gastrointestinal tract (Ma et al., 2021). The potential
blood–brain barrier, and they have been reported to stimulate the pro­ regulation effects of EMPs of different origin on gut microbiota was
duction of trophic factors, such as BDNF (Chiu et al., 2018; Rupcic et al., shown, e.g., decreased Firmicutes/Bacteroidetes ratio and enriched faecal
2018). Erinacine A has been shown to increase the expression of sig­ microbiota diversity (Zhao et al., 2019). The effect of H. erinaeus on
nalling proteins, including NGF and BDNF, in the central nervous system microbiota was previously tested in vitro (Mitsou et al., 2020), using
in animal models (Shimbo et al., 2005). In a recent pilot study (Vigna animal models (Diling et al., 2017; Yang et al., 2021), and to our
et al., 2019) involving seventy-seven volunteers who suffered from knowledge in only one short-term pilot clinical study (Xie et al., 2021).
overweight or obesity and had one or more mood disorders, BDNF levels As shown by Xie et al. (2021), short-term supplementation of H. erinaeus
were tested before and after two months of daily supplementation with increased the alpha diversity and relative abundance of the SCFAs
H. erinaceus. An increase in circulating pro-BDNF levels was observed. producing bacteria, and downregulated some pathobionts. Despite our
As reviewed by Chong et al., 2021, based on neurotrophic and neuro­ expectations, only few changes in the gut microbiota composition were
genic pathophysiology of disease, H. erinaceus may be a potential observed after the intervention. This could be attributed to the study
alternative medicine also for the treatment of depression. Results of duration while the results of the short-term dietary interventions in
experiments on animal models have also revealed that H. erinaceus humans showed that alternations in gut microbiota are transient and do
supplementation increased neurogenesis in the dentate gyrus of the not persist (David et al., 2014; Leeming et al., 2019). Due to the variety
hippocampus (Brandalise et al., 2017) and improved hippocampus- of factors influencing the gut microbiota composition, participants in
dependent recognition memory with enhancement of neurotransmis­ our study were advised not to change eating, sleeping, or activity habits.
sion and neurogenesis in the hippocampus and cerebellum (Ratto et al., However, the effect of HE supplementation on gut microbiota compo­
2019). sition was shown to be influenced by age and gender of study partici­
The expression of neurotropic factors, including BDNF, has been pants. These results indicate the importance of considering the influence
shown to be regulated by NPY (Wirth et al., 2005) and conversely, BDNF of multiple factors that can significantly impact the composition of the
overexpression can significantly increase NPY mRNA and protein levels microbiota. After the intervention, the statistically significant differ­
(Croce et al., 2013). This suggests a cooperative relationship between ences in the HE group were observed only for three genera with a
BDNF and NPY in regulating the nutritional microenvironment and relative abundance of less than 1 %. Notably, after the intervention a
supporting neuronal development and survival. In the present 8-week higher abundance of Flavobacteriaceae family inside the HE group and
intervention with a daily intake of HE supplement, no significant ef­ also when comparing the HE group to the P group has been detected.
fect on serum levels of NPY was observed. However, subsequent ana­ Because members of Flavobacteriaceae family have been reported to
lyses revealed that the intervention had effect on cognitive performance digest insoluble chitin and many other polysaccharides (Kharade &
and NPY levels, accounting for corresponding values at baseline, age, McBride, 2014) the higher relative abundance of this family in the HE
gender, and faecal CHI3L1 levels. Therefore, we hypothesise that higher group could be connected to higher release of bioactive compounds and
levels of faecal CHI3L1 could lead to a more pronounced effect of sup­ possibly improving the effect on cognitive parameters. Moreover, it was
plementation due to the localisation of chitinase producing bacteria. To shown that the increase in relative abundance of the Flavobacteriaceae
our knowledge, this is the first study investigating the effect of HE family was associated to normal body weight and a healthy metabolic
intervention in relation to faecal CHI3L1 concentration. Until recently it profile, thus indicating a good metabolic health (Palmas et al., 2021).
was generally considered that due to the natural absence of chitin, Gut microbiota diversity had previously been linked to cognitive func­
chitinases are lacking in mammalian cells. However, the presence of tion in older adults (Canipe et al., 2021), but other benefits, such as
mammalian chitinases and chitinase-like proteins including CHI3L1 enhanced nutrient utilisation and a more effective immune system, were
(also known as YKL-40 or human cartilage glycoprotein 39 (HCgp-39)) shown. Following the intervention, a statistically significant difference
was observed (Hakala et al., 1993), whereas the exact mechanisms and (t = 2.84, p = 0.008) in bacterial diversity between the groups was
functions of the protein in the human gut are still being studied and are observed. Accordingly, the higher diversity was positively correlated to
not yet fully understood. CHI3L1 is a member of glycoside hydrolase higher levels of NPY in the HE group.
family 18 and is expressed by a multitude of cells, including colonic However, the relatively small sample size in the present study may be
epithelial cells (CECs), macrophages, and neutrophils (Krause et al., considered a limitation. We also note that our sample of participants
1996; Mizoguchi, 2006; Renkema et al., 1998; Volck et al., 1998). Faecal reflected the sex imbalances inherent in each group. The lack of greater

7
M. Černelič Bizjak et al. Journal of Functional Foods 115 (2024) 106120

homogeneity within our sample could have a negative impact on the this study, students of the Faculty of Health Sciences for their help, and
validity of the results obtained from our sample. Due to the presence of MycoMedica Ltd. for providing the supplements.
wide range of bioactive compounds in mushrooms the results may also
be more relevant if placebo capsules contained the same material as Ethics statement
experimental group, without erinacine A. In addition, the daily intake of
supplements was self-administered, so the possibility of non-adherence This study was conducted in accordance to the guidelines laid down
and misreports of intake could not be excluded. Finally, it remains un­ in the Declaration of Helsinki and all procedures were approved by the
clear why serum BDNF levels decreased in the P group after the inter­ Slovenian National Medical Ethics Committee (No. 0120-321/2017-4).
vention. The significant decrease in BDNF levels in the P group may have The protocol for the present clinical study was registered at Clin­
several explanations, including stress, poor diet, genetics, environ­ icaltrials.gov (NCT 04939961).
mental factors and physical activity. One possible explanation is sea­
sonal variation, as the first measurements were taken in spring/summer Funding sources
and the second in autumn. There is evidence that BDNF levels fluctuate
with the seasons, increasing in spring/summer and decreasing in The authors would like to express the appreciation for the co-
autumn/winter. This could be due to the fact that sun exposure in­ financing received from the Republic of Slovenia and the European
fluences vitamin D production, which is known to be associated with Union through the European Regional Development Fund (ERDF;
BDNF. Sun exposure also influences circadian rhythms, which affects OP20.06532) to support a part of the research. This research was
neurotrophic factors. In addition, there are conflicting studies on the financially supported also by the Slovenian Research Agency (research
effects of physical activity on peripheral BDNF levels, although the program P1-0386 and infrastructure program I0-0035).
mechanisms are unclear. Further research is needed to fully understand
the relationship between intervention and BDNF levels. Appendix A. Supplementary data

5. Conclusion Supplementary data to this article can be found online at https://doi.

org/10.1016/j.jff.2024.106120.
As the elderly population increases, it is important to understand the
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