The Applications of Polysaccharides from Various Mushroom Wastes as Prebiotics in Different Systems

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The Applications of Polysaccharides fromVarious Mushroom Wastes as Prebiotics inDifferent SystemsWei-Ting Chou, I-Chuan Sheih, and Tony J. Fang

Abstract: The bases or stipes of mushrooms are normally discarded as low-economic value animal feed and compost.There are no known reports on deriving polysaccharides from these mushroom wastes for use as prebiotics. This studyshowed that the relatively low concentration (0.1% to 0.5%) of polysaccharides from Lentinula edodes stipe, Pleurotuseryngii base, and Flammulina velutipes base can enhance the survival rate of Lactobacillus acidophilus, Lactobacillus casei, andBifidobacterium longum subsp. longum during cold storage. The polysaccharides had synergistic effects with the peptidesand amino acids from a yogurt culture to maintain probiotics above 107 CFU/mL during cold storage, and they also hadsignificant protective effects on these probiotics in simulated gastric and bile juice conditions to achieve beneficial effectsin the host. These results showed that mushroom wastes, which are cheaper than other sources, could be an important,new, alternative source of prebiotics.

Keywords: mushroom, polysaccharides, prebiotic, wastes

Practical Application: Using the bases or stipes of mushrooms as prebiotics is less expensive than other food sources. Themushroom wastes can enhance the survival of probiotics during cold storage. The wastes also can improve the toleranceof probiotics in simulated gastric and bile juices.

IntroductionLactic acid bacteria are a group of organisms that promote

human health by maintaining intestinal microbial flora, immunemodulation, and promoting metabolism (Parvez and others 2006).Among these, Lactobacillus spp. and Bifidobacterium spp. are oftenused in several commercial products. To achieve beneficial probi-otic effects in the host, lactic acid bacteria must be alive and reachthe concentration of 106 to 108 CFU/mL in dairy-related prod-ucts (Lourens-Hattingh and Viljoen 2001). However, increasingacidity and oxidative pressure will decrease the viable number oflactic acid bacteria during the storage periods of dairy products.It has been reported that supplementation of prebiotics (Brunoand others 2002; Capela and others 2006; Guergoletto and others2010), utilization of microencapsulated technology in probiotics(Capela and others 2006), or directly screening gastric acid andbile-resistant lactic acid bacteria strains (Tuomola and others 2001)could enhance the survival of the microorganisms in fermenteddairy products.

Recently, bioactive polysaccharides from various foods, such ascereal (Michida and Pizzoferrato 2006), oats (Gokavi and others2005), mushrooms (Guo and others 2004; Aida and others 2009),herbs (Guo and others 2004), chicory root (Roberfroid 2000),

MS 20130054 Submitted 1/10/2013, Accepted 4/16/2013. Authors Chou andFang are with Dept. of Food Science and Biotechnology, Natl. Chung Hsing Univ.,250 Kuokuang Rd., Taichung city 40227, Taiwan, ROC. Author Sheih is withDept. of Food and Beverage Management, Ta Hwa Univ. of Science and Technology,No. 1 Dahua Rd., Qionglin Shiang, Hsinchu County 307, Taiwan, ROC. AuthorFang is also with Dept. of Nutrition, China Medical Univ., No. 91 Hsueh-ShihRd., Taichung city 40402, Taiwan, ROC. Direct inquiries to author Fang (E-mail:[email protected]).

citrus (Sendra and others 2008), soybeans (Crittendan and Playne1996), and potatoes (MacFarlane and others 2006), are gaining at-tention as new prebiotic alternatives. Mushrooms are edible foodsthat also are used as nutritional supplements, and their safety is wellestablished. Furthermore, many research studies also have indi-cated that polysaccharides from Pleurotus spp. (Synytsya and others2009), Lentinus edodes, Tremella fuciformis (Guo and others 2004),and Agaricus bisporus mushroom (Giannenas and others 2011)have prebiotic activity. β-Glucans, homo-glucans, and hetero-glucans with β(1→3), β(1→4), and β(1→6) glucosidic linkageswere thought to be responsible for their bioactivity (Manzi andPizzoferrato 2000). Among these, the carbohydrates of the ediblemushrooms, Pleurotus eryngii, L. edodes, and Flammulina velutipes,contain ribose, xylose, fructose, mannose, glucose, and trehalose.In addition, sucrose was observed only in L. edode, whereas bothP. eryngii and L. edode were constituted primarily of glucose. F.velutipes contains mainly xylose (Kim and others 2009).

The action mode of the polysaccharides might be relatedclosely to increased intestinal microbial activities and enhancedimmune function (Guo and others 2004). These noncellulosic β-glucans could be potent immunological stimulators. Among these,some now are used clinically in China and Japan (Chen and Se-viour 2007). However, there are no studies that have reportedthe prebiotic activity of polysaccharides derived from mushroomwastes.

The bases or stipes of mushrooms, because of their tough tex-ture, have been considered to be a waste product when mushroomsare harvested. These cut-off bases or stipes make up about 25%to 33% of the weight of fresh mushrooms, and they are normallyused to make low-economic value animal feed and compost. Theunderutilized wastes, which are good nitrogen sources, could be

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Mushroom polysaccharides as prebiotics . . .

bioconverted into value-added products, such as a novel alcoholicbeverage (Lin and others 2010). In addition, these mushroomwastes have been found to have a high content of polysaccharides,which exhibited prebiotic activity in our preliminary experiments.These preliminary results suggest that mushroom waste might be-come an important source of novel prebiotics.

Mushroom bases/stipes are comparatively cheap compared tothe sources of most prebiotics, which originate from costly foodsources, such as cereal, oats, intact mushrooms, herbs, chicoryroot, citrus, soybeans, and potatoes. In this study, the mushroomwastes from L. edodes stipe (LES), P. eryngii base (PEB), and F.velutipes base (FVB) were used to extract polysaccharides, andwe investigated their prebiotic effects on Lactobacillus acidophilus,Lactobacillus casei, Bifidobacterium longum, Streptococcus thermophilus,and Lactobacillus delbrueckii subsp. bulgaricus in various cultivationsystems during storage periods. In addition, we investigated theprotective effects of the polysaccharides derived from mushroomwastes by simulating the gastric acidity and bile that probioticstrains encounter.

Materials and Methods

Preparation of polysaccharides from mushroom wastesL. edodes, P. eryngii, and F. velutipes wastes were obtained from

a mushroom cultivator and processor in Taichung, Taiwan. Thesamples were dried, pulverized, and extracted twice with boilingwater at 1:15 (w/v) for 2 h (Guo and others 2003). The aqueoussolutions containing the extracts were mixed with an equal vol-ume of 0.8 M trichloroacetic acid at 4 ◦C for 3 h, after which thesolutions were centrifuged (5000 × g). The supernatant was mixedwith 95% ethanol at a ratio of 1:4 (v/v), and the obtained precip-itate was lyophilized. These precipitates were denoted as L. edodespolysaccharides (LEP), P. eryngii polysaccharides (PEP), and F. ve-lutipes polysaccharides (FVP). The percentage yield was defined as(weight of extract/initial weight of mushroom waste) ×100.

Microorganism and culture conditionsAll strains were obtained from the Bioresource Collection and

Research Center (BCRC) at the Food Industry Research andDevelopment Inst. (FIRDI) in Hsinchu, Taiwan. The lactic acidbacteria used in this study included L. acidophilus BCRC 10695,L. casei BCRC 14080, B. longum subsp. longum BCRC 14664,S. thermophilus BCRC 13680, and L. delbrueckii subsp. bulgar-icus BCRC 10696. Each strain was subcultured weekly in deMan Rogosa and Sharpe (MRS) broth supplemented with 0.05%L-cysteine.

Survival rate of probiotics in MRS systemsThree probiotic strains, that is, L. acidophilus, L. casei, and B.

longum subsp. longum, were inoculated in MRS broth with dif-ferent concentrations of mushroom polysaccharides (0.1%, 0.5%,1.0%, w/v) and incubated at 37 ◦C for 48 h. These samples werestored at 4 ◦C for 28 d, and the viability of the probiotic was de-termined during cold storage. The viabilities of L. acidophilus, L.casei, and B. longum were determined by MRS-sorbitol agar (Daveand Shah 1996), L. casei agar (Ravula and Shah 1998), and MRS-NNPL (MRS containing antibiotics nalidixic acid, neomycin sul-fate, paromomycin sulfate and lithium chloride) agar (Laroia andMartin 1991), respectively.

Gastrointestinal toleranceThe gastrointestinal tolerance of the probiotic strains supple-

mented with mushroom polysaccharides was examined (Michidaand others 2006; Guergoletto and others 2010). The mushroomfiber was obtained from the mushroom samples by drying andpulverizing the samples, after which the pulverized material waspassed through a 20-mesh sieve. Probiotic strains were grown inthe MRS broth with 5% (w/v) mushroom fiber at 37 ◦C for24 h. The cell pellets were collected and washed twice with ster-ile PBS buffer. The mushroom fiber-entrapped cell pellets wereresuspended in 10 mL of sterile PBS buffer, and 1 mL of theabove cell pellets was added to 6.5 mL of the simulated gastricjuice (HCl solution at a pH of 2.0) or bile juice (3.0% (w/v)oxgall bile solution at a pH of 8.0), and the mixture was supple-mented with 0.5% (w/v) of mushroom polysaccharides. The pro-biotics were maintained at 37 ◦C for various time intervals, and theviability of the probiotics was determined. As a control treatment,we used probiotics that had no entrapped mushroom fiber and nopolysaccharides supplement.

Survival rate of probiotics in a fermented milk systemThe survival rates of the probiotic strains supplemented with

mushroom polysaccharides were examined according to Donkorand others (2007) and Sendra and others (2008) with minor mod-ifications. A volume of 150 mL of skim milk that contained 13%skim milk powder, 10% sucrose, and 0.67% mushroom polysac-charides was pasteurized at 90 ◦C for 30 min, after which it wasinoculated with 1% (v/v) S. thermophilus and L. delbrueckii subsp.bulgaricus. The samples were fermented at 42 ◦C until the pHreached 4.7, and they were used as starter cultures.

A volume of 50 mL of pasteurized skim milk was inoculatedwith 1% (v/v) of L .acidophilus, L. casei, and B. longum and fer-mented at 42 ◦C until the pH reached 4.7 for use as the probioticcultures. For B. longum, the skim milk medium was supplementedwith 0.05% L-cysteine (w/v) to enhance its growth.

Finally, 50 mL of the probiotic culture and 150 mL of the starterculture were mixed, and the viability of the probiotic cultures wasdetermined during cold storage for 28 d. The survival rate (%)was defined as probiotic viability after 28 d of cold storage/initialprobiotic viability) ×100.

Statistical analysisThe Statistical Analysis System (SAS) with Duncan’s multiple

range was used to determine the level of significance.

Results and Discussion

Polysaccharides concentrationThe viabilities of the probiotic cultures, that is, L. acidophilus

BCRC 10695, L. casei BCRC 14080, and B. longum BCRC 14664in different polysaccharide concentrations (0.1% to 1%, w/v) weretested, and the results are shown in Figure 1. The results showedthat the probiotic viabilities of all samples decreased with stor-age time, and the count of B. longum was the lowest among theprobiotics after 28 d of cold storage (Figure 1C). Probiotics are sus-ceptible to oxygen stress, pH, and decreased temperature (Bolducand others 2006; Rosburg and others 2010). Donkor and others(2007) also indicated that inulin sustained the metabolic activityof probiotics to maintain higher viable cell concentrations, but

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Figure 1–The viability of probiotics (A) L.acidophilus BCRC 10695, (B) L. casei BCRC14080, and (C) B. longum BCRC 14664 grownon MRS broth supplemented with differentconcentrations of L. edodes polysaccharide(LEP) during 28 d of cold storage. Each value isexpressed as mean ± SE (n = 3).

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Figure 2–The viability of probiotics grown onMRS broth supplemented with 0.5% mushroompolysaccharide for (A) L. acidophilusBCRC10695 and (B) L. casei BCRC 14080 and0.1% mushroom polysaccharide for (C) B.longum BCRC 14664 during 28 d of coldstorage. Each value is expressed as mean ± SE(n = 3). LEP, L. edodes polysaccharides; PEP, P.eryngii polysaccharide; FVP, F. velutipespolysaccharides.


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it also resulted in an increased production of primary metabo-lites, including lactic acid and acetic acid. However, the mediumsupplemented with 0.5% LEP significantly supported the growthof L. acidophilus and L. casei and 0.1% LEP supported the growthof B. longum compared to the groups without polysaccharides sup-plementation during 28 d storage at 4 ◦C. Previous reports haveindicated that citrus fiber, cereal fiber, modified corn starch, andβ-glucan had prebiotic effects at concentrations of 1% (Sendraand others 2008), 1.33% (Rosburg and others 2010), 5% (Michidaand others 2006), and 0.44% (Rosburg and others 2010), respec-tively. Our results showed that the polysaccharides from mushroomwastes supplemented at 0.1% to 0.5% showed protective effects forthe probiotics that we tested, indicating these polysaccharides werean effective prebiotic.

Prebiotic effects of different polysaccharidesThe mushroom wastes were prepared by extraction with boiling

water, trichloroacetic acid precipitation, and ethanol precipitation,and the yields were 3.37%, 6.36%, and 5.47% for LES, PEB, andFVB, respectively (data not shown).

Figure 2 shows that the probiotic viabilities of all samples de-creased rapidly from 14 to 28 d during cold storage, and thehardiness of the L. acidophilus and L. casei for low-temperaturestorage was better than that indicated by B. longum in this study.The groups with various polysaccharide supplements had signifi-cant protective effect, achieving beneficial probiotic effects in thehost, as described by Lourens-Hattingh and Viljoen (2001), andthe average survival rate above 107 CFU/mL was maintained for21 d. Among these, the FVP medium had higher prebiotic effectsfor L. acidophilus and L. casei than those observed for PEP andLEP media; the results were similar with citrus fibers (Sendra andothers 2008).

Among probiotics, the bifidobacteria are more susceptible tooxygen stress, pH, and decreased temperature than the other pro-biotics (Bolduc and others 2006; Rosburg and others 2010). Thepolysaccharides decreased the penetration of oxygen into the pro-biotics, which created a more protective environment for bifi-

dobacteria and increased their metabolic activity (Rosburg andothers 2010).

Gastrointestinal toleranceTo understand the stability of the tested probiotics against gastric

juice and bile juice, the mushroom fiber-entrapped probiotics andthe polysaccharides were incubated in simulated gastric juice orbile juice. Figure 3 shows that L. casei showed bile juice-resistantcharacteristics, and the PEP medium enhanced the viability of L.casei viability significantly in the simulated bile juice (P < 0.05).LEP supplementation increased the survival rate of B. longum andimproved its tolerance in the simulated bile juice (P < 0.05);but the groups supplemented with any quantity of mushroompolysaccharides showed almost no effect on the population of L.acidophilus (P > 0.05). However, in this study (data not shown),the probiotics all maintained concentrations of more than 109

CFU/mL during 240 min of exposure to bile juice, with/withoutpolysaccharide supplements, to achieve beneficial probiotic effectsin the host, as described by Lourens-Hattingh and Viljoen (2001).

Figure 4 shows that the probiotic viabilities of all samples de-creased rapidly during the 1st 30 min of incubation in gastricjuice. The control treatment, which had no entrapped mush-room fiber probiotics and supplemental polysaccharides, even-tually died out after 60 min of incubation. In this study, asignificant positive effect on gastric juice tolerance of probi-otics was found when the probiotics were immobilized in themushroom fiber and supplemented with mushroom polysaccha-rides, and the most protective effect was found in the LEPmedium, followed by the FVP medium and the PEP medium.The protective phenomenon also has been reported for the ad-dition of cereal extract and cereal fiber immobilized probiotics(Michida and others 2006).

Survival rate of starter culture and probiotic bacteria infermented milk

The effects of the mushroom polysaccharides on microbial pop-ulations of milk fermented with S. thermophilus and L. bulgaricus

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Figure 3–The survival rate of mushroom-fiberimmobilized (A) L. acidophilus BCRC 10695, (B)L. casei BCRC 14080, and (C) B. longum BCRC10695 in the bile juice supplemented with 0.5%mushroom polysaccharides for 240 min. Eachvalue is expressed as mean ± SE (n = 2). Theinoculated size of the respective trials isapproximately 108 CFU/mL. The survival ratewas defined as the ratio of bacteria counts ofthe respective trials in the bile juice for 240 minover the bacteria counts of the respective trialsin the bile juice for 0 min. LEP, L. edodespolysaccharides; PEP, P. eryngii polysaccharide;FVP, F. velutipes polysaccharides.


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Figure 4–Microbial counts of mushroom-fiberimmobilized (A) L. acidophilus BCRC 10695, (B) L.casei BCRC 14080 and (C) B. longum BCRC 10695in the gastric juice supplemented with 0.5%mushroom polysaccharides for 180 min. Eachvalue is expressed as mean ± SE (n = 3). LEP, L.edodes polysaccharides; PEP, P. eryngiipolysaccharide; FVP, F. velutipes polysaccharides.


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cocultured with L. acidophilus, L. casei, or B. longum during 28-dcold storage are shown in Figure 5 and 6, respectively. Figure 5shows that the fermented milk supplemented with FVP apparentlyenhanced the survival of L. bulgaricus and L. acidophilus comparedwith the non-FVP supplemented group (P < 0.05); nevertheless,the addition of LEP and PEP had adverse effects on the probioticpopulation in the fermented milk. Figure 6 also shows that PEPhad the most positive effect among these polysaccharides on theviability of starter cultures (S. thermophilus and L. bulgaricus) in fer-mented milk and on the viability of the coculture of L. casei during28-d cold storage (P < 0.05), and, in this study, the protective ef-fect on probiotics can be described in the order of S. thermophilus >

L. bulgaricus > L. casei.

Figure 7 indicates that cold storage also caused considerablereduction of B. longum counts in fermented milk with or withoutpolysaccharides supplementation; but the combination of B.longum with yogurt starter cultures clearly enhanced its survivaland increased the viable counts above 107 CFU/mL, comparedwith that in MRS medium at 28 d of storage (Figure 2C).It was postulated that the higher levels of peptides and aminoacids in yogurt and the polysaccharides in the mushroomshad a synergistic effect to enhance the growth of B. longum(Donkor and others 2006). In addition, all of the probioticsmaintained concentrations of more than 107 CFU/mL during thecold storage period, with/without polysaccharide supplements,thereby achieving beneficial probiotic effects in the host; the

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Figure 5–The survival rate of starter cultures(S. thermophilus BCRC 13680 and L.delbrueckii subsp. bulgaricus BCRC 10696)and probiotic culture L. acidophilus BCRC10695 in fermented milk supplemented with0.5% mushroom polysaccharide stored at4 ◦C for 28 d. The inoculated size of therespective trials is approximately 108

CFU/mL. The survival rate was defined as theratio of bacteria counts of the respectivetrials at 4 ◦C for 28 d over the bacteria countsof the respective trials at 4 ◦C for 0 d. LEP, L.edodes polysaccharides; PEP,P. eryngii polysaccharide; FVP, F. velutipespolysaccharides.

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Figure 6–The survival rate of starter cultures(S. thermophilus BCRC 13680 and L. delbrueckiisubsp. bulgaricus BCRC 10696) and probioticculture L. casei BCRC 14080 in fermented milksupplemented with 0.5% mushroompolysaccharide stored at 4 ◦C for 28 d. Theinoculated size of the respective trials isapproximately 108 CFU/mL. The survival ratewas defined as the ratio of bacteria counts ofthe respective trials at 4 ◦C for 28 d over thebacteria counts of the respective trials at 4 ◦Cfor 0 d. LEP, L. edodes polysaccharides; PEP,P. eryngii polysaccharide; FVP, F. velutipespolysaccharides.


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Figure 7–The survival rate of starter cultures(S. thermophilus BCRC 13680 and L. delbrueckiisubsp. bulgaricus BCRC 10696) and probioticculture B. longum BCRC 14664 in fermentedmilk supplemented with 0.1% mushroompolysaccharide stored at 4 ◦C for 28 d. Theinoculated size of the respective trials isapproximately 108 CFU/mL. The survival ratewas defined as the ratio of bacteria counts ofthe respective trials at 4 ◦C for 28 d over thebacteria counts of the respective trials at 4 ◦Cfor 0 d. LEP, L. edodes polysaccharides; PEP, P.eryngii polysaccharide; FVP, F. velutipespolysaccharides.

LEP medium increased the viable counts of L. bulgaricus further(P < 0.05).

ConclusionsThe results acquired in this study indicated that a symbiotic in-

teraction occurred between the microbial bacteria and the polysac-charides from different mushroom wastes, clearly altering the bal-ance of the probiotics in the MRS medium and in the fermentedmilk; and these mushroom polysaccharides apparently had the ca-pability of retarding the death of the probiotics, allowing them tomaintain higher populations during cold storage. Furthermore, thetolerance and stabilities of the probiotics in simulated gastric juiceand bile acid also were improved significantly when they were sup-plemented with mushroom polysaccharides. The results showedthat polysaccharides extracted from inexpensive mushroom wasteshave significant potential for use as prebiotics.

AcknowledgmentsWe thank Agriculture and Food Agency, Council of Agricul-

ture, Executive Yuan, ROC (No. 100NK-3.1.3-LZ1 (5)) and Natl.Science Council, ROC NSC-99–2313-B-005–003-MY3 for fi-nancially supporting this research.

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The Applications of Polysaccharides from Various Mushroom Wastes as Prebiotics in Different Systems