Xin Wng, Yunyun Cui, Chowei Sng, Bin Wng, Yirong Yun,Lin Liu, Yhong Yun,*, Tinli Yue,c,*

a College of Food Science and Engineering, Northwest A&F University, Yangling 712100, China

b Laboratory of Quality & Safety Risk Assessment for Agro-products (YangLing), Ministry of Agriculture, Yangling 712100, China

c College of Food Science and Technology, Northwest University, Xi’an 710069, China

Keywords:

Fuzhuan brick tea

Fungi

Candidate probiotics

Probiotic properties

Bacteriostatic activity

Adhesion capacity

A B S T R A C T

Several of the fungal species associated with Fuzhuan brick tea (FBT) are considered as potential probiotics,but few studies have investigated the probiotic properties of these fungi.Here, we isolated 18 fungal strains from two types of FBT and identified these strains based on internal transcribed spacer (ITS) fragment sequence similarity to reference strains (sequence similarity > 98%).Of the 18 strains, 10 tolerated simulated human digestive conditions for sufficient periods in vitro: pH 2-3, 0.3%-0.5% (m/V) bile salts, and artificial gastrointestinal juices.We then measured the antimicrobial activity of the remaining 10 strains against 5 enteropathogenic bacteria and tested the bacteriostatic effects of the thalli and fermentation broth extracts.Of the 6 strains with strong bacteriostatic effects, we eliminated Eurotium cristatum S-9 due to its low hydrophobicity of (26.12 ± 0.35)%.Finally, 2 exhibited good adhesion abilities to human cells (> 100%).Notably, 2 strains can survive in vivo, because they can be isolated from C57BL/6 mice feces.Thus, 2 strains,Aspergillus cristatus H-1 and A.cristatus H-5, are herein identified as promising candidate probiotic strains.It may be put forward a novel research focus on evaluating potential probiotic fungi from FBT.

1.Introduction

Fuzhuan brick tea (FBT), a unique dark tea sold in compressed bricks, was commonly produced from old, coarse, rough leaves ofCamellia sinensisvar.sinensis[1].Unlike other types of teas, FBT contained a so-called ‘golden biota’, which was mainly generated by fungal fermentation [2].FBT has been shown to have inhibitory effects against several metabolic diseases, including hyperlipidemia [3],hyperglycemia [4], obesity [5], dysentery [6], bacterial infection [7],and oxidative damage [8]; FBT played a critical role in the maintenance of intestinal homeostasis due to orchestrated effects of gut microbiota, increasing the abundances of beneficial bacteria Clostridiaceae, Bacteroidales, and Lachnospiraceae,and decreasing the harmful Ruminococcaceae, Peptococcaceae,Peptostreptococcaceae, and Erysipelotrichaceae [9].Moreover,the health benefits of FBT attracted many tea consumers in China, South Korea, Japan, Russia, and other countries [10].The quercetin and kaempferol acylglycosides isolated from FBT can enhance hypoglycemic and hypolipidemic effects [3].Polysaccharides extracted from FBT reduced hyperlipidemia, arterial stiffening, and significantly ameliorated oxidative injuries [4].Water or ethanol FBT extracts reduced obesity in mice by modulating the gut microbiota [11].Notably, the health-promoting properties of FBT were inextricably linked to the microbial community [12].

Several studies have reported that the primary fungal genera associated with FBT fermentation wereEurotium,Aspergillus,Debaryomyces,Cyberlindnera,Candida, andPenicillium[13].Several of the dominant fungal genera in FBT are known to have beneficial health effects.For example,Eurotium cristatumwas a non-toxic potential probiotic fungus that can lose obesity and modulate gut microbiota in mice [14].What is more, Chan et al.[15]reported that okara (soy pulp) fermented byE.cristatumproduced anthraquinones, emodin and physcion to inhibit the enzyme of 11β-HSD1 and glucocorticoid selectively, thereby reducing blood glucose levels.Similarly,Angelica dahuricafermented withE.cristatumsubstantially improved its antioxidant properties [16];loose tea fermented withE.cristatumwas not only of better quality, but also enhanced hypolipidemic properties [10].Moreover,Hippophae rhamnoidesleaves fermented byE.amstelodami, greatly stimulated the release of more phenolic compounds and flavonoids compared to unfermented tea, which improved both the flavor and the beneficial efficacy of tea [17].Takenaka et al.[18]reportedAspergillus amstelodamimay produce extracellular hydrolytic enzymes including aspartic protease and lipases, participating in the decolorization of hemoproteins and hydrolysis of fats, respectively, which is good to reduce obesity.A.amstelodamialso can produce prenylated benzaldehyde derivatives, used as potent antimicrobial [19].A.cristatussecreted various enzymes metabolizing the pectin,cellulose, protein, and polyphenols within raw tea materials to generate the unique color and flavor of FBT [20].However, to our knowledge, few studies have yet examined the probiotic properties of the dominant fungal species in FBT.

Probiotics are live microorganisms that confer a health benefit to the host when administered in adequate amounts [21].Probiotics generally tolerate extreme conditions after oral intake, including gastric juices in the stomach and bile salts in the small intestine [22].A few studies have evaluated the probiotic potential of given taxa based on resistance to the extreme conditions of the gastrointestinal tract (e.g., low pH), adhesion to the intestinal mucosa, prolonged and stable persistence in the intestinal tract, and competitive elimination of pathogenic microorganisms [23].In general, probiotics were first screened based on their ability to tolerate low pH levels and high concentrations of bile salts [24].This allows screening to beginin vitro, which is quicker and less expensive than screeningin vivo.

Thus, we herein aimed to assess the probiotic potential of the FBT fungal biota, including several different genera and species.First,we aimed to isolate and identify the dominant fungi in FBT.Second,these taxa were analyzedin vitroto evaluate a) their resistance to the extreme conditions of the gastrointestinal tract; b) their inhibitory effects on intestinal pathogenic bacteria; and c) their adhesion to human colon cells.These evaluations were performed as an initial step for screening candidate fungi with potential probiotic properties.

2.Material and methods

2.1 Isolation and identification

Hunan and Shaanxi have a long history of producing FBT in China.Thus, two types of commercial FBT (4-year-old tea)were obtained to study fungi probiotic properties produced from different fields: Hunan FBT was purchased from Yiyang Fu Cha Industry Development Co., Ltd.(Yiyang, Hunan Province, China);Shaanxi FBT was purchased from Xianyang Jingwei Fu Tea Industry Co., Ltd.(Xianyang, Shaanxi Province, China).Briefly,25 g of each FBT sample was homogenized in 225 mL sterile water for 30 min using a shaker (Shanghai Zhicheng Instrument Manufacturing Co., Ltd., Shanghai, China) at 120 r/min.After gradient dilution, 100 μL of above suspension was spread onto potato dextrose agar (PDA) and 20% Czapek Ddox agar (CDA)plate, respectively, which was then incubated at 28 °C [25].A single colony was subcultured on PDA medium (28 °C) to obtain pure culture and was used for subsequent identification [26].DNA extraction was carried out using a Biospin Fungus Genomic DNA Extraction Kit (Hangzhou Bioer Technology Co., Ltd., Hangzhou,China), following the manufacturer’s instructions.The primers were ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4(5’-TCCTCCGCTTATTGATATGC-3’) (Invitrogen).The PCR reaction system (50 μL) included 25 μL of PremixTaq(Takara Biotechnology Co., Ltd., Dalian, China), 2 μL of each primer(10 μmol/L), 2 μL of template DNA, and 19 μL of sterile distilled water.The amplification procedure as follows: pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min, 34 cycles, and final extension at 72 °C for 10 min.Then, 3 μL of PCR products was analyzed using 1.5% agarose electrophoresis to ensure that fragment (500-750 bp)had been amplified, among these, DL2000 DNA marker (Takara) and GelRed were used.PCR products were sequenced in Sangon Biotech Co., Ltd., Shanghai, China.

The Basic Local Alignment Search Tool (BLAST) was used to identify the sequences in the GenBank DNA database (http://www.ncbi.nlm.nih.gov/blast) most similar to the isolate sequences.

2.2 Acid tolerance

Fungal acid resistance was determined following the methods of Wang et al.[27], with minor modifications.In brief, fungal spores, scraping from the PDA, were harvested and resuspended in sterile water, adjusted to a concentration of 1 × 108spores/mL by hemocytometer [28].We then added 1 mL of above spore suspension to 9 mL aliquots of potato dextrose broth (PDB) previously adjusted to a pH of 1.0, 2.0, and 3.0 using HCl (0.1 moL/L) with PDB as control.After 2 h and 3 h of incubation (food usually stays in gastric juice for 2-3 h) respectively, 100 μL samples were removed from each mixture, evenly coated on separate PDA plates, cultured at 37 °C anaerobically for 3 days, and then the viable count was calculated by plate coating method [28].The acid resistance of fungal strain was expressed as percent survival as follow:

WhereN1was the total viable count on the experimental PDA plate (i.e., fungi isolates incubated in PDB with pH 1.0-3.0 for 2-3 h),andN0was the total viable count on the control PDA plate (i.e., fungi isolates incubated in PDB without pH adjustment).

2.3 Bile tolerance

Intestinal bile concentration is approximately 0.3%-0.5% (m/V),and foods usually remain in the gastrointestinal tract approximately for 6–8 h [29].We tested fungal bile tolerance following Vinderola and Reinheimer [30], with minor modifications.In brief, fungi spore suspensions were prepared as described in section 2.2, and each was adjusted to a concentration of 1 × 108spores/mL by hemocytometer.We then added 1 mL of each spore suspension to 9 mL aliquots of PDB (pH 5.92) containing 0.3% or 0.5% (m/V) of bile salt (Sigma-Aldrich, St.Louis, MO, USA) with the PDB absence of bile salt as control.After 6 h and 8 h of incubation, the mixture was counted as shown in section 2.2.The bile resistance of fungal strains was expressed as percent survival as follow:

WhereN1was the total viable count on experimental PDA plate(i.e., fungi isolates exposed to 0.3%-0.5% (m/V) bile salt for 6–8 h),andN0was the total viable count on the control PDA plate (i.e., fungi isolates incubated in PDB absence of bile salt).

2.4 Gastrointestinal fluids tolerance

Artificial gastric juice (AGJ) was prepared by diluting 0.35 g pepsin (U, 1:3 000) in 100 mL of 0.2% physiological saline, adjusting the solution pH to 2.2 with HCl, and filtering the solution through a 0.45 μm membrane [31].Artificial intestinal juice (AIJ) was prepared by dissolving 0.1 g trypsin (U, 1:250) and 1.8 g bile salt in 100 mL of distilled water containing 1.1 g NaHCO3and 0.2 g NaCl, adjusting the solution pH to 8.0 with NaOH and filtering the solution through a 0.45 μm membrane [31].

To determine fungal viability in AGJ, 1 mL of spore suspension(1 × 108spores/mL) was inoculated into 9 mL AGJ, mixed for 10 s.After mixing evenly, 100 μL samples were removed, coated on PDA plates, and anaerobically incubated at 37 °C for 3 days.After the 3 h exposure to AGJ, 1 mL aliquots were removed from AGJ mixture,added to 9 mL AIJ (pH 8.0), and cultured at 37 °C for 4 h.After 4 h of incubation, the viable count of mixture was counted as shown in section 2.2.The total time exposed to AGJ and AIJ corresponded to total digestion time [29].Therefore, the survival percent of fungal isolates was expressed as follows (after 3 h AGJ exposure plus 4 h AIJ exposure):

WhereN1was the total viable count on the PDA plate at the end of digestion (i.e., the fungi isolate incubated after 3 h AGJ incubation and 4 h AIJ incubation), andN0was the total viable count on the control PDA plate at the beginning of digestion (i.e., the fungi isolate incubated in AGJ at 0 h).

2.5 Inhibition of enteropathogenic bacteria

2.5.1 In vitro antimicrobial effects of the FBT isolates thallus

We tested the bacteriostatic effects of the FBT isolates thallus according to the previously described bacterial block method [32].Five enteropathogenic bacteria (most common bacteria causing intestinal diseases), were selected as model pathogensStaphylococcus aureusATCC25923,Escherichia coliATCC25922,Shigellasp.ATCC12022,Enterococcus faecalisATCC10 andSalmonellasp.ATCC14028, purchased from the American Type Culture Collection(Virginia, USA).Enteropathogenic bacteria were cultured in a widely used Luria-Bertani (LB) medium at 37 °C for 24 h.Then, 100 μL of bacterial suspension was evenly coated on Luria-Bertani agar medium(LA) with a sterile scraper.Simultaneously, purified FBT isolate was incubated at 28 °C for 4 days, and then made into the round block with the diameter of 5 mm.Three blocks per isolate were gently placed on LA medium coated with enteropathogenic bacterium, such that the mycelium side of fungus block made direct contact with the medium.After incubation at 37 °C for 36 h, the inhibition zone width(mm) was measured by vernier calipers.

2.5.2 In vitro antimicrobial effects of the fermentation broth extract (FBE)

The antimicrobial effects of the isolate FBE were determined using the Oxford cup method, as described previously [33].Brie fly, 15 mL molten LA medium was poured into the plate and made it solidify.Sterilized Oxford cups were then placed on the medium, another 15 mL LA mixed with 600 μL (1 × 107CFU/mL) pathogenic bacteria was poured onto the solidified LA.The Oxford cups were carefully removed after the top layer of the medium solidified.Simultaneously,the isolate FBE was prepared as follows: fermentation broth was extracted with an equal volume of ethyl acetate three times,evaporated to dryness with a rotary evaporator, and dissolved in a small amount of methanol.Then, 200 μL isolate FBE was loaded into hole, and incubated at 37 °C for 36 h.Concurrently, 200 μL methanol(used as control) was treated in the same way.The experiment was carried out in triplicate.Inhibition zone widths (mm) were measured with the aid of a vernier calipers.

2.6 Cell surface hydrophobicity (CSH)

CSH was defined as the tendency of microorganisms to adhere to nonpolar sites.High CSH promoted bacteria to adhere to the cell surface and move from aqueous phase to organic phase [34].The hydrophobicity of the fungal cells was determined as described previously [35].Briefly, the isolates were grown in PDB at 28 °C for 5 days, and then fungal spores of isolates were harvested by centrifugation (12 000 ×g, 10 min at 4 °C), carefully washed twice in phosphate-buffered saline (PBS; pH 7.4, 50 mmol/L), re-suspended in PBS, and adjusted to 1 × 108spores/mL by hemocytometer.We added 1.5 mL of the fungal spore suspension to an equal volume ofn-hexadecane (Yuanye Bio-Technology Co., Ltd., Shanghai, China;98% purity).The solution was vortexed for 2 min and kept for 30 min at room temperature to allow phase separation.Control fungal spore suspensions were treated identically, but withoutn-hexadecane.The absorbance of the aqueous phase in solution was measured at 600 nm,and CSH was calculated as follows:

WhereA0was the absorbance of the spore suspension withoutn-hexadecane, andA1was the absorbance of the aqueous phase in the spore suspension containingn-hexadecane.

2.7 Cell adhesion assays

The adhesive abilities of the FBT isolates were measured using human colon cancer cell line HT-29 and Caco-2 following previous studies [36,37].In brief, the HT-29 and Caco-2 cell lines were respectively grown in RPMI 1640 and DMEM medium containing 15% fetal bovine serum (FBS; catalog no.900-108, lot A25F00H,Gemini) and 1% penicillin-streptomycin (100 ×).The culture medium was changed daily.The two cell lines were seeded onto 24-well tissue culture plates at a density of (8-9) × 105cells/mL by hemocytometer, and incubated at 37 °C in 5% CO2incubator (Thermo Fisher Scientific,Waltham, MA, USA).Simultaneously, spores were harvested from the FBT isolate cultures using centrifugation(12 000 ×gfor 10 min at 4 °C), resuspended in RPMI 1640 and DMEM medium respectively and diluted to (8-9) × 106spores/mL.Then, 1 mL above post-confluence spore suspension was added over the monolayer of HT-29 and Caco-2 cells in 24-well plates.The two cell lines and fungal spores were co-cultured at 37 °C in 5% CO2incubator for 60 min to adhere enough [38].Then, the plates were washed twice with sterile PBS (pH 7.4) to remove nonadhering fungal spores, and the numbers of fungal spores adhered to the HT-29 and Caco-2 cells were counted in 20 random microscope fields under an inverted microscope at 100 × magnification (Beijing ruihongcheng Technology Development Co., Ltd., Chongqing,China).Each adhesion assay was performed using cells from three successive passages in triplicate.Adhesive ability was expressed as %of adhesion (the number of fungal spores/HT-29 or Caco-2 cells) [36].

2.8 Animal experimental verification in vivo

Conventional C57BL/6 mice (7-week-old, male, SPF) were purchased from SLAC Jingda Laboratory Animal Company, Hunan,China.Every 5 mice were randomly assigned to one cage and placed in the animal facility under standard conditions (temperature(22 ± 2) °C, humidity at (50 ± 15)%, 12 h/12 h light-dark cycle).After 2 weeks of adaptive feeding, the animals were randomly divided into the following 3 groups (5 mice per group; normal control (NC),A.cristatusH-1 group,A.cristatusH-5 group).In treatment groups,400 μL, 103spores/mL live-spore suspension ofA.cristatusH-1 andA.cristatusH-5 was gavaged daily, and mice feces were collected after 31 days.Subsequently, 200 mg mouse feces were suspended in 1 mL sterile water, after mixing and centrifuging at 10 000 r/min,5 min, the supernatant was collected, spread onto M40Y medium, and cultured at 30 °C.

2.9 Statistical analysis

Graphical representations of the data were performed using Origin 8.0 and GraphPad Prism.Statistical analysis was performed using IBM SPSS 20.0.Results are represented as the mean ± SD (n= 3).Statistical differences were analyzed by independent samplettest.AP-value of less than 0.05 was considered statistically significant.Also,one way ANOVA with Duncan post hoc application was applied to analyze cell surface hydrophobicity and cell adhesion ability.

3.Results and discussions

3.1 Isolation and identification of the dominant FBT isolates

In total, we isolated 18 fungal strains from the two FBT samples based on morphological observation (Fig.1) and theITSfragment(500-750 bp).It could be seen that the mycelium in the colony center was more developed than the marginal, that is developed mycelium secretes more pigment, leading to the colony color at the center deeper than edge [39].Significantly, a big difference in colony morphology was observed between PDA and 20% CDA, which attributed to different substrates produce a different impact on fungi [40].Thus, two widely applied medium were selected to enhance the number of potential isolates.Using BLAST, we identified the reference strains (NCBI) with the most similarITSfragment to that of each isolate (Table 1, Table S1, Fig.S1).All strains had high degrees of sequence similarity to the corresponding reference strains:16 with similarity ≥ 99%, and one strain with only 98.46% similarity toA.cristatus.Sequence similarity > 98% was considered to indicate conspecifics, while sequence similarity of 95%-98% was considered to indicate congenerics [41].Thus, all 18 FBT isolates were included in all subsequent experiments.

Fig.1 Morphological characteristics of 18 fungal isolates isolated from two types of FBTs.

Table 1Identifications of the 18 FBT isolates based on the percent sequence identity (% identity) between the ITS gene sequences of the reference strains and those of the isolates.

Across all 18 strains, 10 were identified asA.cristatus(56%),4 were identified asE.cristatum(22%), and 4 were identified asA.amstelodami(22%).Of these, 8 were isolated from Hunan FBT(4 strains ofA.cristatus, 2 strains ofE.cristatum, and 2 strains ofA.amstelodami), and 10 were isolated from Shaanxi FBT(6 strains ofA.cristatus, 2 strains ofE.cristatum, and 2 strains ofA.amstelodami).Xu et al.[13]used polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) to show thatEurotiumandAspergilluswere the dominant genera in FBT.Rui et al [42]also reported thatAspergillus(at genera level) was dominant fungi in FBT based on the Illumina MiSeq sequencing.These reports supported our results thatA.cristatusandE.cristatumwere identified as dominant fungi of FBT.Unlike previous work,both theA.cristatusandE.cristatumwere all identified at the species level, and we obtained the single colony, which can be used for further study.In addition, dominant fungi may play different functions due to strain-specific properties of probiotics [43,44], which need to be further investigated.

3.2 Tolerance of simulated gastrointestinal conditions

Probiotics were often screened based on their ability to tolerate low pH levels and high concentrations of bile salts [24], which generally resist the harsh conditions of the stomach and upper intestine to colonize the small intestine and affect host health [45].The acid resistance test was exhibited as follows.In terms of pH 1.0,the survival rates ofA.amstelodamiH-4,A.cristatusH-5,E.cristatumH-7 andA.cristatusS-10 were significantly improved more than (100 ± 10.65)% (P< 0.05) after 3 h of incubation, indicating that the 4 strains had a strong tolerance to extreme acid environment and good acid adaptability (Fig.2a).Similarly, at the condition of pH 2.0,the survival rates ofE.cristatumS-2 (92.85%) andA.cristatusH-5(50 ± 5.65)% increased to (213 ± 50.7)% and (127 ± 27.12)% after 3 h of incubation, respectively, exhibiting good acid adaptability.The survival rate ofE.cristatumH-8 andA.cristatusS-10 was greater than 100% both in 2 h and 3 h, showing better acid tolerance ability(Fig.2b).However, on the condition of pH 3.0, the survival rate ofA.cristatus H-2andA.amstelodami H-3was lower than other isolates, which significantly decreased after 3 h incubation (P< 0.01).Thus, the 2 strains had poor acid resistance (Fig.2c).On the whole,A.cristatusH-2 had poor tolerance to a simulated acid environment(4.69%) compared with all other isolates in acid tolerance assays.And the survival rate ofA.amstelodamiH-3 was significantly decreased after 3 h of incubation (P< 0.01), indicating bad acid tolerance.

Fig.2 Tolerance of simulated gastrointestinal conditions by fungal strains isolated from FBT, visualized as survival rates.(a) Acid resistance of pH 1.0; (b) Acid resistance of pH 2.0; (c) Acid resistance of pH 3.0; (d) 0.3% bile salt tolerance; (e) 0.5% bile salt tolerance; (f) Gastrointestinal tolerance.The survival rate was expressed as mean ± SD (n = 3), and statistical analysis was performed by ‘independent sample t-test’.*P < 0.05, #P < 0.01.

In addition, the variation of all isolates survival rate was basically consistent both in 0.3% and 0.5% bile salt (m/V).Obviously, the survival rate ofA.amstelodamiH-4 (4.62%) andA.cristatusS-8(4.51%) was lower than other isolates, exhibiting poor bile salt tolerance (Figs.2d, 2e).Thus, these 4 strains (A.cristatusH-2,A.amstelodamiH-3,A.amstelodamiH-4 andA.cristatusS-8) were not easy to survive in the condition of stomach acids and bile salts and were eliminated from follow-up trial.

The survival rates ofE.cristatumS-2 ((8 ± 1.41)%) andA.cristatusS-10 ((7.04 ± 0.71)%) were evidently lower than other isolates after 3 h exposure to AGJ and 4 h of AIJ (Fig.2f),suggesting that two strains were not suitable for surviving efficiently in the gastrointestinal tract.Besides, although the survival rates ofA.cristatusS-3 ((61 ± 2.12)%) andA.amstelodamiS-4 (33%) were high, the total viable counts in both strains were low (Table S2).Strains with poor viability should not be considered as probiotics, because probiotics provided health benefits to the host only when present and viable in the gastrointestinal tract in adequate numbers [21].Additionally, Kang et al.[5]had reported thatE.cristatumisolated from FBT could survive in the mouse intestine, which directly supported probiotic properties of the FBT dominant fungi.But due to the possible strain-specific of probiotics [44], the initial evaluation of probiotic properties is very necessaryinvitro.Therefore, these 4(E.cristatumS-2,A.cristatusS-3,A.cristatusS-4 andA.amstelodamiS-10) strains were also eliminated from consideration.

In general, based on their tolerance of acidic environments,bile salts, and gastrointestinal conditions, the following 10 strains were included in subsequent experiments:A.cristatusH-1,A.amstelodamiS-1,A.cristatusS-5,A.cristatusS-6,A.cristatusS-7,A.cristatusH-5,A.cristatusH-6,E.cristatumH-7,E.cristatumH-8, andE.cristatumS-9.

3.3 Inhibition of pathogenic intestinal bacteria

In order to screen out strains with broad-spectrum bacteriostatic effect [46], the following analysis were carried out.From the Table 2,it could be seen the thallus of 10 fungal isolates (A.cristatusH-1,A.amstelodamiS-1,A.cristatusS-5,A.cristatusS-6,A.cristatusS-7,A.cristatusH-5,A.cristatusH-6,E.cristatumH-7,E.cristatumH-8, andE.cristatumS-9) had no inhibitory effect onE.coliATCC25922 andSalmonellasp.ATCC14028, but could inhibitE.faecalisATCC10.Notably, there were diverse inhibitory effects onS.aureusATCC25923 andShigellasp.ATCC12022.Specifically speaking,A.cristatusH-1,A.cristatusS-6 andE.cristatumH-7 exhibited obvious inhibitory effect onS.aureusATCC25923, but no effect onShigellasp.ATCC12022.Four strains ofA.cristatusS-7,A.cristatusH-5,A.cristatusH-6 andE.cristatumS-9 showed inhibitory effects onS.aureusATCC25923 andShigellasp.ATCC12022, whileA.amstelodamiS-1 andE.cristatumH-8 had no inhibitory effect on them, suggesting a narrow bacteriostatic range on common pathogenic intestinal bacteria.Gaggìa et al.[47]reported that the benefic action of probiotics in gastrointestinal is the inhibition of pathogenic bacteria by competitive exclusion.Therefore, fungi isolate thallus probably gets more nutrition competitively to inhibit the growth ofS.aureusATCC25923,Shigellasp.ATCC12022 andE.faecalisATCC10.

Table 2Direct inhibition of intestinal pathogenic bacteria by FBT isolates thallus, based on bacteriostatic effectsa.

Additionally, the bacteriostatic effect of fungi FBEs was also studied (Table 3).It is obvious that 10 fungi FBEs had strong inhibitory effect againstS.aureusATCC25923 (inhibition zone width ≥ 2 mm),but had no inhibition onE.coliATCC25922 andShigellasp.ATCC12022 (no bacteriostatic circle), that is to say, fungi FBEs are more effective against gram-positive pathogenic bacteria.It is consistent with the literature that due to Gram-negative bacteria possess an impermeable outer membrane contributed to stronger tolerant [48].Unlike, other 8 fungal strains showed inhibitory effect onE.faecalisATCC10 except forA.cristatusS-5 andA.cristatusS-7.Moreover,A.cristatusH-5 displayed a weak antibacterial effect((0.58 ± 0.01) mm) onSalmonellasp.ATCC14028.As literature reported, the ethyl acetate extracts ofEurotiumsp.were tested for antibacterial activity against two Gram-positive (S.aureusandE.faecalis) [26].In particular, the anthraquinones emodin and physcion (two new prenylated indole carbaldehydes) detected in ethyl acetate extracts of strain, were found to significantly inhibit the production of bio film inS.aureusATCC 25923 [49].Moreover, 4 new indole alkaloids, namely cristatumins A–D (1–4), were also identified from the culture extract ofE.cristatum, showing antibacterial activity againstS.aureus[50].Accordingly, it is speculated that fungal isolate FBEs may contain amounts of antibacterial components against intestinal pathogens [51].However,A.cristatusS-5 andA.cristatusS-7 had no inhibitory effect on neitherE.faecalisATCC10 norSalmonellasp.ATCC14028, demonstrating a poor inhibition ability against common pathogenic intestinal bacteria.

Table 3Inhibition of intestinal pathogenic bacteria by the fermentation broth extracts of FBT isolates, based on bacteriostatic effectsa.

Interestingly, it can be found that 10 isolates of both thallus and FBEs were more likely to inhibit gram-positive bacteria (S.aureusATCC25923,E.faecalisATCC10) compared with Gram-negative bacteria (E.coliATCC25922,Shigellasp.ATCC12022,Salmonellasp.ATCC14028), which may be related to the cell wall composition(the content of protein and lipopolysaccharide) and structure (outer membrane) [52].Given the above, due to a narrow bacteriostatic range ofA.amstelodamiS-1,E.cristatumH-8,A.cristatusS-5 andA.cristatusS-7, the following isolates with relative broad-spectrum bacteriostatic effect were screened for further studies includingA.cristatusH-1,A.cristatusS-6,A.cristatusH-5,A.cristatusH-6,E.cristatumH-7 andE.cristatumS-9.

3.4 Isolate CSH

CSH was an important index to evaluate probiotic properties [53].Some previous studies reported that hydrophobicity of strain was a phenotype related to their adhesive capacity [54].High CSH benefits strain to adhere to intestinal cells such as Caco-2 and HT-29 to exert function.Thus, cell surface hydrophobicity was selected as a criterion to screen potential probiotics.

It can be seen thatA.cristatusS-6 showed the best hydrophobicity properties ((49.26 ± 0.16)%), followed byA.cristatusH-6,E.cristatusH-7,A.cristatusH-1, andA.cristatusH-5.And there was no significant difference among the 4 strains (Table 4).Nevertheless,the CSH ofE.cristatumS-9 ((26.12 ± 0.35)%) was significantly lower than that of other 5 strains (P< 0.05), indicating poor hydrophobicity properties.Microorganisms with high levels of CSH adhere to abiotic and biotic surfaces, and more easily penetrate host tissues to exert effect [55].As Meng et al.[56]reported that CSH reflects the affinity of components of the microbe’s cell surface.Obviously, the other 5 strains are more suitable for adhesion in gastrointestinal tract, except forE.cristatusS-9.Thus,E.cristatumS-9 was eliminated from subsequent experiments.

Table 4The cell surface hydrophobicity of the FBT fungi isolates.

3.5 Adhesion to human cells

Adhesion to intestinal epithelial cells or mucosa was a key selection criterion for effective probiotics because the longer probiotics persist in the gut, the more effective the function [57].In vitromodels using the human intestinal cell lines HT-29 and Caco-2 were common in preliminary investigations of the cell adhesion properties of various probiotics [56].These cell lines, exhibited characteristics of mature enterocytesin vitro, which are both derived from colon carcinomas correspond to the major cell phenotypes found in the human intestinal mucosa.

We found considerable variation in the ability of FBT isolate spores to adhere for HT-29 cells (Figs.3a, 4a):A.cristatusH-1 andA.cristatusH-5 spores strongly adhered to HT-29 cells with the adhesion rate of (125.74 ± 5.05)% and (111.53 ± 7.30)%, whileA.cristatusS-6,A.cristatusH-6, andE.cristatumH-7 adhered poorly with the adhesion rate of (28.88 ± 2.43)%, (30.05 ± 6.41)% and(56.58 ± 34.07)%, respectively.Notably, there were significant differences betweenA.cristatusH-1,A.cristatusH-5 and other 3 strains (P< 0.05), indicating thatA.cristatusH-1 andA.cristatusH-5 adhered more strongly to HT-29 cells.

Fig.3 Cell adhesion abilities of the fungal strains isolated from FBT.(a) The adhesion to HT-29 cells; (b) The adhesion to Caco-2 cells.

Consistent with the adhesion results for the HT-29 cells,A.cristatusH-1 andA.cristatusH-5 strongly adhered to Caco-2 cells with the adhesion rate of (146.78 ± 42.95)% and (143.84 ± 4.17)%,whileA.cristatusS-6,A.cristatusH-6, andE.cristatumH-7 adhered poorly with the adhesion rate of (16.09 ± 0.43)%, (37.27 ± 0.51)%and (57.21 ± 13.64)%, respectively (Figs.3b, 4a).The adhesion percentages ofA.cristatusH-1 andA.cristatusH-5 were significantly greater than those of other three isolates (P< 0.05), and there was no significant difference betweenA.cristatusH-1 andA.cristatusH-5(P> 0.05).Combined with the report of probiotics strain-specific,we speculated that different candidate probiotic fungi may also exist strain-specific, that is to say, microorganisms belonging to the same species may develop different mechanisms and show different characteristics [57].Other previous studies also reported that adhesion ability of probiotics such asLactobacillus fermentum,L.caseiandL.brevispossessed strain-specific [44,58].Hence, the evaluation of both wild and novel strains is essential, since isolates belonging to the same species may display different properties and probiotic mechanisms.Similarly, it is speculated that the different adhesion abilities ofA.cristatusH-1 andA.cristatusH-5 may be interpreted as strain-specific, rather than species-specific.

Due to their greater adhesion to both HT-29 and Caco-2 cells,we concluded thatA.cristatusH-1 andA.cristatusH-5 were fungal strains with better candidate probiotic properties, which provided a novel research focus in the field of screening potential probiotic fungi.These effects and edible safety ofA.cristatusH-1 andA.cristatusH-5 should be investigated furtherin vivo.

3.6 The presence of A.cristatus from mice feces

Probiotics are defined as live microorganisms that provide healthboosting effects to the host [24].Hence, to further confirm whether the two potential probiotics can survive in the gastrointestinal tract,the collected mice feces were used to isolateA.cristatus.Notably,both strains ofA.cristatusH-1 andA.cristatusH-5 were all isolated from feces (Fig.4b), indicating that they can tolerate extreme conditions and survive in gastrointestinal tract.Moreover, the body weight ofA.cristatusH-1 group showed an increasing trend and with no significant differences compared to the NC group, proving thatA.cristatusH-1 is non-toxic [5].Yet, from the 19thday, a significant difference was observed betweenA.cristatusH-5 group and NC,referring thatA.cristatusH-5 may play a critical role in obesity.But the specific mechanism needs further study.Concurrently, it puts forward a new research field on the health function ofA.cristatus.

Fig.4 The observation of adhesion assay and isolates from mice feces.(a) Enteropathogenic bacteria adhere to HT-29 and Caco-2 cell lines; (b) Isolation of A.cristatus from mice feces.

4.Conclusion

We identified 18 fungal strains, isolated from two types of FBT,based onITSfragment sequences.These strains were considered candidate probiotics.Ten of the 18 strains exhibited better tolerance of acidic environments, bile salts, and gastrointestinal conditions compared with other isolated strains.Of these, 6 strains had relatively broad-spectrum bacteriostatic effects against common intestinal pathogens either directly, via thallus, or FBEs (A.cristatusH-1,A.cristatusS-6,A.cristatusH-5,A.cristatusH-6,E.cristatumH-7,andE.cristatumS-9).The CSH ofE.cristatumS-9 significantly lower than other fungi isolates (P< 0.05), and thus eliminated from further evaluation.Of the remaining 5 strains, two (A.cristatusH-1 andA.cristatusH-5) adhered well to 2 different human colon cell lines Caco-2 and HT-29.Thus,A.cristatusH-1 andA.cristatusH-5 are promising candidate probiotic strains, which can be further studiedinvivofor working in the host.Interestingly, both two promising candidate probiotic fungi were from Hunan FBT, rather than Shaanxi FBT, that is, isolates from Hunan FBT are likely more suitable to survive in gastrointestinal tract.The animal experimental verification indicating thatA.cristatusH-1 andA.cristatusH-5 are easier to liveinvivoand may be produce a health effect, which put forward a novel research focus of potential probiotic fungi from FBT.

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgments

This research was supported by the Shaanxi Special Project of China (2018ZDXM-NY-084).

Appendix A.Supplementary data

Supplementary data associated with this article can be found in the online version, at http://doi.org/10.1016/j.fshw.2021.12.026.