Effect of the Intake of Oyster Mushrooms (Pleurotus ostreatus) on Cardiometabolic Parameters-A Systematic Review of Clinical Trials.

Cardiometabolic diseases are a leading global health challenge. Their incidence as well as progression is strongly affected by diet. Consumption of Pleurotus ostreatus (P. ostreatus), an edible oyster mushroom rich in functional ingredients (e.g., β-glucans), may improve glucose and lipid metabolism, blood pressure, body weight and appetite sensations. Hence, this systematic review aimed to provide an overview on the effects of P. ostreatus intake on cardiometabolic parameters from clinical trials, taking into account risk of bias (RoB). Relevant studies were investigated for details with consideration of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline. The Cochrane Collaboration’s tool was used to assess the RoB. In total, eight trials included observed beneficial effects of P. ostreatus intake on glucose metabolism (reduction in fasting and/or 2 h postprandial glucose) and lipids (decrease in total cholesterol, LDL-cholesterol and/or triglycerides), and some found a reduction in blood pressure. In contrast, body weight did not change. Appetite sensations were not assessed. In most studies, the RoB was high or unclear due to methodological weaknesses and/or inadequate reporting. Thus, P. ostreatus intake may improve cardiometabolic health, but evidence for this is low. Hence, further clinical trials with an adequate study design are warranted to validate these suggestions.


Introduction
Cardiometabolic diseases such as diabetes mellitus type 2 (DMT2), dyslipidemia, hypertension and obesity are associated with increased morbidity and mortality. They are known to be the major risk factors for cardiovascular diseases (CVDs), which are still the leading cause of death in modern societies worldwide. According to the World Health Organization (WHO), 17.9 million people died from CVDs in 2016, representing 31% of all deaths [1].
Incidence as well as the progression of these cardiometabolic diseases is strongly affected by behavioral factors such as tobacco use, physical activity and diet [1]. The latter explains current interest in functional food and food ingredients which may improve cardiometabolic health [2]. At the same time, the need for sustainable food production becomes more important due to growing populations and the effects of climatic change, which represents an additional global challenge [3].
In this respect, edible mushrooms which are part of the usual diet are interesting as an alternative source of nutrients, but also for ecological reasons [4,5]. Pleurotus ostreatus (P. ostreatus), also called oyster mushroom, is one of the most cultivated edible mushrooms worldwide after white button mushroom (Agaricus bisporus, A. bisporus) [5]. P. ostreatus is of particular interest as it is able to colonize and degrade which was performed by one reviewer (L.D.), was finished on 31 January 2020. Eligible articles which were published up to that date were considered for inclusion.
In addition, the reference lists of the included studies were perused to identify further studies of relevance that had not been identified in the PubMed database before.

Inclusion and Exclusion Criteria
This review focused on intervention studies which investigated the efficacy of P. ostreatus intake on cardiometabolic parameters in adults. Studies were included if they investigated (1) the effect of P. ostreatus as a whole mushroom (fresh, cooked or dried) as well as extracts; (2) parameters of glucose metabolism (e.g., glucose, insulin, HbA 1c ), lipid metabolism (e.g., triglycerides (TGs), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C)), cardiovascular health (e.g., systolic and diastolic BP) as well as BW and appetite sensations; (3) healthy subjects, subjects with overweight/obesity and/or cardiometabolic disorders (e.g., prediabetes, DMT2, hyperlipidemia, pre/hypertension, atherosclerosis). Studies were excluded for the following reasons: (1) treatment with other edible mushrooms than P. ostreatus; (2) treatment with isolated substances from P. ostreatus (e.g., pure β-glucans or mevinolin) or with mushroom powder or extract additionally enriched with vitamins or functional ingredients; (3) not being a clinical trial; (4) addressing other topics (e.g., anticarcinogenic effects); (5) investigating subjects suffering from severe diseases (e.g., concerning gut, liver, pancreas and kidneys, cancer), other types of diabetes than DMT2 or using medications which might favor cardiometabolic disorders (e.g., glucocorticoids, antiretroviral therapy).

Study Selection, Data Extraction and Risk of Bias Assessment
Two independent reviewers (L.D. and S.E.) identified relevant studies by analyzing the records using a self-made Excel template which considered the predefined eligibility criteria. First, all items were screened by title and/or abstract to exclude contributions that met exclusion criteria. Afterwards, all remaining studies were assessed for eligibility by reading the full-text article. Any discrepancies in the selection process were discussed until a consensus was reached. Finally, studies which were considered to be eligible were included in this systematic review.
Afterwards, both review authors extracted relevant data independently by using a self-made Excel template: study design, details of the intervention (amount, duration and application of P. ostreatus, including main ingredients based on food analysis) and of the control treatment, participants (sample size, dropout rate, demographics and baseline characteristics), outcomes (parameters on glucose and lipid metabolism, vascular function, BW, appetite sensations), times of measurement on outcome markers, and suggested mechanisms of action. Missing data were partly obtained from study authors. If available, the study registration was checked to complete data. Considering data extraction, discrepancies were also discussed by the authors.
Thereafter, both review authors independently assessed the quality of the included studies on the basis of the Cochrane risk of bias tool [23] to judge the RoB with regard to the following criteria: (1) adequate randomization method and generation of randomization list before the study (selection bias); (2) concealing treatment allocation from participants and investigators (allocation concealment); (3) blinding of participants and investigators (performance bias); (4) blinding of outcome assessment until completion of statistical evaluation (detection bias); (5) completeness of outcome data, reporting number and reasons of dropouts (attrition bias); (6) registration of study protocol, reporting full endpoints and outcomes according to registration (reporting bias). Furthermore, (7) controlled study design, details on intervention, investigation of outcome markers and on statistical evaluation, potential confounders, estimation of sample size, and comparison of baseline data were considered to assess other bias. Again, a self-made Excel template was used to check these criteria for each study. Discrepancies in the RoB assessment were also resolved through discussion.

Study Selection and Study Characteristics
After a systematic literature search, 94 records were retrieved from the PubMed database and another 11 publications were found through other sources. After the removal of duplicates, 102 records remained. Based on titles and/or abstracts, 94 records were excluded (reasons: intervention with other mushrooms than P. ostreatus, n = 65; intervention with isolated substances from P. ostreatus, n = 17; considering other topics not related to human health, e.g., cultivation conditions, n = 4; considering biomarkers related to other diseases than cardiometabolic disorders, e.g., cancer, n = 2; in vitro or animal studies, n = 3; no interventional study design, n = 1; investigating HIV-infected individuals under antiretroviral therapy, n = 1; full-text article not available, n = 1). After reading the full-text articles, a further publication was excluded which used another Pleurotus spp. than P. ostreatus for treatment. Finally, eight studies which were presented in seven original contributions were included in the present review. A flow diagram of the identification and selection of studies is shown in Figure 1.

Eligibility Identification
Additional records identified through other sources (n = 11) Records after duplicates removed (n = 102) Records screened (n = 102)

Full-text articles excluded (n = 1)
• other mushroom species (n = 1) 8 studies from 7 full-text articles included in qualitative synthesis Figure 1. Flow-diagram of study selection process according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement [21].

Glucose Metabolism
Khatun et al. [25] provided cooked P. ostreatus (150 g/d, trice daily 50 g) as part of a meal in exchange for vegetables for 7 days to hospitalized patients with DMT2 or impaired fasting glucose and dyslipidemia. The plasma glucose concentration in fasting state (fasting plasma glucose, FPG) and 2 h after breakfast (2hABF) was reduced on average by 22% and 23%, respectively, after the first treatment with oyster mushrooms. After 1 week without mushrooms, FPG as well as 2hABF increased on average by 13% and 20%, respectively. A second treatment with P. ostreatus for 7 days again induced a mean reduction in FPG by 23% and also in 2hABF by 16%. Sayeed et al. [24] also provided oyster mushrooms (200 g/d) in exchange for vegetables, but to women with drug-treated DMT2. All investigations were performed bimonthly during an intervention for 1 year. After treatment with P. ostreatus, FPG decreased approximately 12% after 2 months and up to 20% after 1 year compared to baseline but did not change in the control group. Comparable changes in postprandial glucose concentrations were detectable after mushroom treatment (mean reduction approximately 14% after 2 months and approximately 28% after 1 year), but not in the control group. In the uncontrolled study of Choudhury et al. [29], men with drug-treated DMT2 ingested capsules with sun-dried powdered P. ostreatus (3 g/d; thrice daily amounts of 1 g) for 3 months. After intervention, FPG as well as HbA 1c were reduced on average by 18% and 13%, respectively. Jayasuriya et al. [30] provided lyophilized powdered P. ostreatus (50 mg/kg BW) as suspension to different subgroups (healthy subjects and subjects with dietetically-treated DMT2). In healthy subjects, daily intake of the mushroom suspension for 14 days reduced FPG by 6%. A control treatment was lacking. In a further experiment with the same subjects, postprandial effects were examined by using an oral glucose tolerance test (OGTT) as glucose challenge. The first OGTT was provided 2 h after ingestion of water (control), following a daily intake of P. ostreatus for 14 days. Thereafter, a second OGTT was performed 2 h after bolus ingestion of the mushroom suspension. The glucose concentration in plasma 2 h after OGTT was 16% lower after the intake of the mushroom suspension compared to water, which reflects the combined effect of chronic and acute intake. A further substudy with diabetics investigated the effect of a single preload with mushroom suspension which was ingested 30 min before an OGTT compared to a preload of water (control). This diminished the 2 h glucose approximately 15% and increased the 2 h insulin approximately 22%. Both treatments were performed within a self-controlled trial and were separated by 1 week washout.

Lipid Metabolism
Khatun et al. [25] also investigated the effects on serum lipids. The decrease in FPG after the first intervention with P. ostreatus was accompanied by a decrease in both TGs (28%) and TC (8%). After 1 week without mushrooms, TGs increased by 11% and TC by 6%. If treatment with mushrooms was repeated, TGs and TC were again reduced by 9% and 13%, respectively. In the study of Sayeed et al. [24], the decrease in FPG after consumption of oyster mushrooms was also accompanied by a decrease in TGs and TC. This effect was already significant after 2 months (mean reduction of TGs by 19% and of TC by 12%, respectively), with a similar reduction observed after 1 year (TGs 20%, TC 20%) compared to baseline. However, LDL-C remained unchanged. No changes in TGs, TC and LDL-C were observed in the control group. Throughout intervention, TGs, TC and LDL-C were significantly lower in the mushroom than in the control group. The RCT of Schneider et al. [27] investigated adults with moderate, untreated hyperlipidemia. Daily consumption of a soup providing 30 g of lyophilized P. ostreatus for 3 weeks decreased TGs on average by 23%, whereas TGs increased by 25% in the control group which received a mushroom-free tomato soup instead. The changes in TGs were significantly higher in the mushroom group by 0.8 mmol/Lcompared to the control group. Significant changes in TC and LDL-C were neither detectable within each group nor between the groups. P. ostreatus showed significant hypolipemic effects in an uncontrolled clinical trial of Kajaba et al. [26] in which participants with primarily combined types of dyslipidemia were treated with 10 g of lyophilized powdered P. ostreatus daily for 6 weeks. TGs, TC and the TC/HDL-C ratio decreased significantly compared to baseline by on average 36%, 22%, and 24%, respectively. A further uncontrolled trial of Choudhury et al. [28], who provided dried powdered oyster mushrooms (3 g/d for 90 days) to hypertensive men with overweight or obesity, found a mean reduction in TC and LDL-C by 12% and 19%, respectively. In contrast to Kajaba et al. [26], changes in TGs were not observed. HDL-C was determined in all trials which investigated lipid status, but changes were not detected in any study after intervention with P. ostreatus [24][25][26][27][28].

Blood Pressure
The trial of Choudhury et al. [29], already mentioned above, investigated BP in hypertensive subjects with DMT2. After a daily intake of capsules with powder from lyophilized P. ostreatus for 3 months, SBP and DBP decreased on average by 10% and 11%, respectively. Khatun et al. [25] also examined BP, but in normotensive subjects who received cooked oyster mushrooms daily for 7 days. Nevertheless, a significant reduction in SBP (4%) and DBP (2%) could also be observed. After consumption of vegetables instead of mushrooms for 7 days (control), DBP increased by 2%, whereas SBP remained unchanged. After retreatment with oyster mushrooms for one week, DBP decreased again by 1%, SBP remained unchanged. Sayeed et al. [24] provided fresh P. ostreatus to drug-treated women suffering from DMT2 (characteristics/data for BP were not provided) also in exchange for vegetables (control). BP was determined bimonthly during the one-year intervention period. Significant changes in SBP and DBP were not observed, neither within each group nor between the groups.

Pro-/Antioxidant Status
The uncontrolled study of Kajaba et al. [26] examined the pro-/antioxidant status in adults with primarily combined dyslipidemia in addition to lipids. The concentration of conjugated dienes (CD) in plasma decreased while the activity of glutathione peroxidase (GPX) and the concentration of glutathione (GSH) in erythrocytes increased after 6 weeks of P. ostreatus powder intake. In contrast, superoxide dismutase (SOD) and catalase (CAT) remained unchanged. Schneider et al. [27] observed a significant decrease in oxidized LDL (oxLDL) by approximately 11% in patients with moderate hyperlipidemia after 3 weeks of treatment with oyster mushrooms, but no changes in the control group. However, the changes in oxLDL between both groups were not significantly different.

Body Weight
BW did not change after consumption of P. ostreatus as well as after control treatment for 1 week [25] and 3 weeks [27].

Cardiometabolic Efficacy and Potential Mechanisms
To the best of our knowledge, this is the first systematic review on the effect of P. ostreatus on cardiometabolic parameters from clinical trials. This review considered eight trials. All studies which investigated markers of glucose metabolism found beneficial effects: these were observed after bolus

Cardiometabolic Efficacy and Potential Mechanisms
To the best of our knowledge, this is the first systematic review on the effect of P. ostreatus on cardiometabolic parameters from clinical trials. This review considered eight trials. All studies which investigated markers of glucose metabolism found beneficial effects: these were observed after bolus consumption with regard to glucose and insulin [30], but also after regular intake of P. ostreatus for 7-365 days with regard to FPG [24,25,29,30] and 2 h glucose after postprandial glycemic load [24,25,30], and in one trial with regard to HbA 1c [29]. These effects are plausible with respect to the mechanisms ascribed to β-glucans such as delaying gastric emptying as well as slowing down subsequent carbohydrate digestion and absorption due to the increased digesta viscosity [9]. However, the observed reduction in FPG and postprandial glycemic response after an intervention period of 365 days [24] does not reflect the long-term changes in glucose control by P. ostreatus treatment. Whether the decrease in FPG in the study of Sayeed et al. [24] was accompanied by a decrease in HbA 1c , as observed by Choudhury et al. [29], remains to be elucidated as HbA 1c was not determined. Moreover, it is not clear whether Khatun et al. [25] and Sayeed et al. [24] used a standardized breakfast as glucose challenge. Furthermore, the 2 h values in the study of Sayeed et al. [24] obtained before intervention were based on OGTT.
TGs were reduced in subjects with DMT2 in the controlled studies of Khatun et al. [25] and Sayeed et al. [24]. This may be explained by an improvement in glucose metabolism with regard to the reduction in FPG. In studies with non-diabetic subjects, TGs in fasting plasma were also diminished by treatment with P. ostreatus [26,27]. Only the study of Choudhury et al. [28], which was an uncontrolled study, did not observe significant changes in TGs. Of course, the reasons for the different results remain speculative, but the higher daily amount of P. ostreatus in studies which observed a reduction in TGs (150 g fresh mushrooms [25], 30 g powder (= 300 g fresh mushrooms) [27], 200 g fresh mushrooms [24], 10 g powder (= 100 g fresh mushrooms) [26]) compared to Choudhury et al. [28] who provided only 3 g P. ostreatus powder (= 30 g fresh mushrooms) may deliver an explanation. TC was reduced in fasting plasma in most studies [24][25][26]28] except that of Schneider et al. [27]. If LDL-C was also determined, a decrease in LDL-C was only observed in studies which found a reduction in TC [24,28], but not in the study of Schneider et al. [27]. This may be explained by the participants' state of general health: the subjects of Schneider et al. [27] had on average a higher HDL-C (1.7 mmol/L) and a lower age (26 y) and BMI (24.0 kg/m 2 ) compared to the subjects of other studies which found a reduction in TC and LDL-C (mean HDL-C: 0.9-1.2 mmol/L [25,26,28]; mean age: 43-46 y [25,26,28]; mean BMI: 27.6 kg/m 2 [26] and 28.2 kg/m 2 [28]; no data available for Khatun et al. [25] and Sayeed et al. [24]). The cholesterol-lowering effects by treatment with P. ostreatus may be mediated by β-glucans, which can increase the bile acid excretion and the activity of cholesterol 7-α-hydroxylase. In addition, short-chain fatty acids, particularly propionic acid, originating from microbial fermentation of β-glucans in the colon, can reduce de novo synthesis of cholesterol [9]. Mevinolin, a known HMG-CO A -reductase inhibitor, can be produced from P ostreatus [31], but it was not detectable in the oyster mushrooms provided by Schneider et al. [27]. Further studies did not determine mevinolin. The lack of changes in HDL-C [24][25][26][27][28] is not surprising as a meta-analysis on the impact of oat β-glucans on HDL-C could not detect any effects either [32].
All studies which investigated the effect of P. ostreatus treatment on BP investigated patients with DMT2. Khatun et al. [25] and Choudhury et al. [29] observed a reduction in SBP and DBP in contrast to Sayeed et al. [24]. The reasons for the different results remain unclear, as several methodological aspects (period of intervention, amount of P. ostreatus) which might be relevant for the results were not comparable. Moreover, the initial BP of the participants also differed (126/83 mm Hg [25]; 149/90 mm Hg [29]; Sayeed et al. [24] data not shown). β-glucans may induce antihypertensive effects by lowering insulin resistance [9], which, however, was not investigated in any study. However, an improvement in glucose metabolism was observed in all studies with regard to the results on FPG [24,25,29] and on HbA 1c [29].
The results on antioxidant status and lipid peroxidation are difficult to evaluate as these originate only from two studies which determined different biomarkers (oxLDL [27]; GPX, GSH, SOD, CAT, CD [26]). Furthermore, potential confounders such as smoking as well as dietary intake of antioxidants were not considered. The decrease in oxLDL [27] as well as in CD [26] after P. ostreatus treatment suggests a lower lipid peroxidation. However, there was no significance in the study of Schneider et al. [27] if the changes in the control group were also considered. Hence, the results of Kajaba et al. [26] have to be considered with caution due to the uncontrolled study design.
BW was only examined in two studies [25,27], but not as an outcome marker. With regard to the known effects of β-glucans, an impact of P. ostreatus on BW would be conceivable. Thus, investigation of BW would have been desirable in all long-term studies.

Comparability of Mushroom Treatment
The nutritional composition of oyster mushrooms is affected by the P. ostreatus strain used [33], but also by cultivation conditions [4], storage, drying method [34] and processing [12]. These details except that of the drying method (if P. ostreatus was used in dried form [26][27][28][29][30]) were never provided. This limits comparability between studies. As the β-glucan content of P. ostreatus differs between caps and stipes [35], the amount of β-glucans ingested by mushrooms also depends on whether the whole fruiting body or only parts of it were used for intervention. Schneider et al. [27] used the whole fruiting body, but this remains unclear in further studies [24][25][26][28][29][30].

Study Quality
Most trials were not robust RCTs, which are considered as a gold standard for effectiveness research, and which imply blinding, concealment of allocation, intention-to-treat analysis and a sufficiently large sample size [36]. Moreover, there was no original contribution which completely fulfilled the Consolidated Standards of Reporting Trials (CONSORT) [37]. Consequently, several aspects remain to be elucidated, as many details were not reported. This explains why the RoB of the studies which were included in the present review was mostly rated as high or unclear (Figure 2).

Strengths and Limitations
This review provides a systematic overview on the effect of P. ostreatus on cardiometabolic parameters from human intervention studies. The consideration of methodological aspects in detail as well as the RoB allows a critical evaluation of the current findings. The inclusion of studies with different target groups and with uncontrolled study designs may be considered as weakness, but a stricter selection was not possible due to the limited number of studies on this topic of research. As DMT2, dyslipidemia and hypertension are usually related to obesity and mostly have a common pathophysiological basis, similarities between the target groups may be partly assumed considering the coexistence of at least two disorders in several studies [25,26,28,29] (not clear for Schneider et al. [27], Sayeed et al. [24] and Jayasuriya et al. [30], as further data were either not investigated or not reported). Furthermore, despite the systematic approach to the literature search, it is not clear whether all studies of relevance were completely identified, as the literature search was restricted to articles which had been published in English and had been listed in the PubMed database. However, the reference list of each suitable study found in PubMed was checked to identify additional trials of relevance.

Conclusions
All studies which were included in this systematic review observed beneficial effects of P. ostreatus intake on glucose and lipid metabolism, and partly on BP. This might be beneficial for cardiometabolic health. However, evidence for such effects is low, as the number of studies is small and the RoB is high due to methodological shortcomings and insufficient reporting. Therefore, current findings on the efficacy of P. ostreatus intake on cardiometabolic parameters can only be considered as suggestions of a beneficial effect.
Thus, further clinical trials with a well-controlled study design are necessary. These should be conducted as RCTs with consideration of potential confounders. Blinding is desirable but may be a challenge when using fresh mushrooms. Powder of dried P. ostreatus, either encapsulated or as part of a fortified food or meal, may ensure blinding and standardization of treatment (composition, dosing) and may improve long-term compliance with mushroom intake. If the suggestions on cardiometabolic benefits were confirmed in future studies, it would be important to reveal the functional ingredients of P. ostreatus and to identify mechanisms of action in humans, e.g., appetite sensations, gastrointestinal hormones or insulin resistance, which plays a key role in the pathogenesis of cardiometabolic disorders.