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Gastrointestinal digestion fate of Tremella fuciformis polysaccharide and its effect on intestinal flora: an in vitro digestion and fecal fermentation study

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  • In this work, the gastrointestinal digestive outcome of Tremella fuciformis polysaccharide (TFP) was examined using in vitro simulated experiments, together with its effect on the intestinal microbiota. TFP did not significantly alter during the stage of oral digestion, according to an in vitro digestion investigation. Nevertheless, glycosidic connections of TFP were broken throughout the intestinal and stomach digesting phases, which resulted in the dissociation of macromolecular aggregates, a marked rise in decreasing sugar content (CR), as well as a drop in molecular weight (Mw). Additionally, microbial community analysis following fecal fermentation in vitro indicated that TFP might control the alpha and beta diversity of gut microbiota and change the genus- and phylum-level community composition. It increased the abundance of beneficial bacteria including Megasphaera, Phascolarctobacterium, and Bacteroides, and suppressed the growth of harmful bacteria like Escherichia-shigella and Fusobacterium, thus contributing to maintaining gut homeostasis. These results suggested that TFP could have a positive impact on health through enhancing the gut microbiota environment, giving a theoretical basis for its use as a prebiotic.
  • Passionfruit (Passiflora sp.) is primarily a tropical fruit, originating in South America with Passiflora edulis being the most important commercial species[1,2]. However, other species within Passiflora exist in subtropical and temperate areas. Some of these species produce fruit that may potentially be used in breeding for more cold hardy passionfruit vines. One of the most interesting of these species is P. incarnata, also called maypop. It is native to North America, especially the southeastern US[36]. It is most well-known for its pharmaceutical properties and is widely used as an anti-spasmodic and sedative[7, 8]. This diploid, herbaceous, perennial vine also can produce a sizable, edible fruit. In areas of the US where the vine is native, it is often considered a weed, as it will colonize disturbed areas within crop land or along fence rows[9]. Wild fruit of P. incarnata is highly variable in size and fruit quality and little effort has been made in selecting superior individuals for breeding. The distribution of the vine is broad, encompassing many different climates, from cold and wet (PA, NY, USA) to hot and dry (TX, USA)[6]. Evaluation of vines from across the spectrum of locations would enhance breeding efforts, but only limited work in this area has been performed.

    P. incarnata is also self-incompatible which further inhibits breeding progress[10]. Interspecific hybridization could potentially help with compatibility issues, although previous attempts have not proved successful. Knight[1113] tried for many years to cross P. edulis and P. incarnata for improved fruit quality, but a successful fruit-bearing cultivar was never released, only a colchicine-induced tetraploid ornamental ('Byron Beauty')[14].

    Passionfruit is gaining in popularity around the globe[15], but P. edulis cannot be grown everywhere successfully as it is tropical in nature and will not survive cold winter temperatures. Therefore, the purpose of this work is to develop baseline data on interspecific passionfruit hybrids involving P. incarnata that can survive subtropical and temperate winters while prospering in areas that have relatively long, hot summers.

    The study began in 2021 and fruit was collected in 2022 at the Mississippi State University South Mississippi Branch Experiment Station in Poplarville, Mississippi, USA (lat. 30°85'36" N, long. 89°49'94" W, elevation 97 m, USDA hardiness zone 8b). Passiflora incarnata parental vines were from Villa Park, Illinois (IL), Richton, Mississippi (MS), and Guthrie, Oklahoma (OK). P. incarnata were obtained from wild plants, either via seed or root cutting. Controlled crosses (P. edulis f. flavicarpa × P. incarnata, Fig. 1) that resulted in the seedlings tested in this study were performed under a high tunnel structure where P. edulis f. flavicarpa was planted in the ground. Controlled hand pollinations were done once a flower had opened, generally between 11:00 am and 1:00 pm. An anther was removed, and pollen was rubbed on the stigma. The pollinated flower was covered by a mesh bag. Evidence of positive fertilization was observed usually within 48 h, but final determination of success was not made until fruit fully developed. Successfully developed fruit were collected when ripe and seeds were extracted. Due to limitations imposed by the COVID-19 pandemic, seeds were stored in a cool, dry environment for up to 1 year before sowing. Collected seeds were submerged in hot water and allowed to sit for 24 h prior to sowing under intermittent mist in a greenhouse. Upon emergence, all seedlings remained in the greenhouse and/or fully enclosed screened high tunnel but were removed from intermittent mist once vining habit and tendril growth started. At that stage they were then re-potted into 11.4-L pots (3-gal) pots and hand watered as needed. The vines were eventually put onto a gravel-covered nursery pad in rows with pots spaced 1.83 m (6 ft) apart within the row and 2.44 m (8 ft) between rows after chance of frost had passed and were drip irrigated. All vines were fertilized with 16 g 13-13-13 (N-P-K, Magic Carpet, Agri-AFC LLC, Decatur, AL, USA) after installation on gravel pad. Vines were allowed to attach to wire fencing material up to 1.52 m (5 ft) tall. In total there were 35 vines. Once on the gravel pad, pollinations were allowed to be made naturally via insect visitation. Eastern Carpenter Bees (Xylocopa virginica) are common in Mississippi and around these vines. Therefore, no hand pollination was done. Fruits were collected regularly once they naturally abscised from the vine from 5 July to 7 Sept. 2022. All fruit were processed immediately where they were measured for length and width with a Mitutoyo Absolute Digimatic (Mitutoyo Corp., Kawasaki, Japan). Fruit were weighed both as entire fruit and once pulp was removed. Fruit density was determined by the formula: Fd = W/(H×D), where W = fruit weight (g), H = fruit height (mm), and D = fruit width (mm). Fruit shape was calculated as H/D as described in Md Nor et al.[16]. Fruits were rated based on feel on a scale of 1 to 5, with 1 = light, empty fruit to 5 = heavy, full fruit. A visual rating of pulp content was also done after the fruits were cut in half, also on the same 1 to 5 scale.

    Figure 1.  Representative flower types used in interspecific crosses to develop more cold hardy fruiting passionfruit vines. Passiflora incarnata flower (left), P. edulis f. flavicarpa flower (middle), and resulting hybrid flower (right).

    Data of fruit measurements were analyzed by JMP (version 12; SAS Institute, Cary, NC, USA) using a one-way analysis of variance and means were compared with standard error of the mean or Tukey's honestly significant difference (HSD) at the 0.05 level where appropriate. Pearson product-moment correlations were performed in the multivariate procedure.

    In total there were 35 seedling vines. Some of these had already been through one round of selection in the previous year (2021). Out of these 35, 11 were kept in 2022 as advanced selections (Table 1). The number of fruits collected per individual ranged from 4 (22R504) to 52 (20R104 and 21R308). The former had P. incarnata IL as a parent and the latter two had P. incarnata OK. Fruits were rated based on feel to gauge, non-destructively, the mass of the fruit. Most average ratings were between 3 and 4 but ranged from 1.59 (22R507) to 4.58 (20R314). After the feel rating, another the visual rating of the pulp was performed, also on a 1-5 scale. The purpose was to see how closely the ratings were aligned. The lowest ranked individuals were 22R502 and 22R504 (1.00) and the highest was 20R314 (4.50). These two ranking were significantly correlated (p < 0.0001) at 0.6586 (Table 2). In 25 out of 35 cases the feel rating was higher than the visual.

    Table 1.  Fruit related characteristics of interspecific hybrid Passiflora selections among both selections were kept for further evaluation and those that were eliminated from further analysis (not kept).
    SelectionParentageFruit #KeepFeel1Visual2Seed #Tot wt (g)Hull wt (g)HT (mm)Width (mm)Density (g/cm2)3Shape4Pulp wt (g)Pulp %5
    20R104P. edulis f. flavicarpa × P. incarnata OK52No2.381.985.6716.3712.4755.4041.430.691.342.2011.59
    20R114P. edulis f. flavicarpa × P. incarnata OK37Yes3.784.2721.9122.6112.6056.9739.100.991.469.5740.15
    20R305P. edulis f. flavicarpa × P. incarnata OK22Yes4.454.3625.5832.1719.1159.1644.591.181.3310.3633.21
    20R309P. edulis f. flavicarpa × P. incarnata OK14No3.933.149.7124.8915.1756.3842.300.981.333.1712.19
    20R314P. edulis f. flavicarpa × P. incarnata OK26Yes4.584.5022.7429.5417.0354.4942.391.211.299.5233.25
    20R426P. edulis f. flavicarpa × P. incarnata OK21No3.293.5511.2718.5614.2247.5735.711.011.355.0120.81
    21R203P. edulis f. flavicarpa × P. incarnata OK24Yes3.293.3314.5618.9411.9855.5240.650.821.375.2627.74
    21R206P. edulis f. flavicarpa × P. incarnata OK51Yes3.243.2210.3817.3811.1849.0233.080.991.495.3225.90
    21R207P. edulis f. flavicarpa × P. incarnata OK27No2.302.006.9612.039.1150.8334.390.661.492.6820.13
    21R215P. edulis f. flavicarpa × P. incarnata OK20No3.953.8513.4721.4714.4057.1438.950.941.475.4725.75
    21R217P. edulis f. flavicarpa × P. incarnata OK33No2.822.826.2015.7811.7453.0138.080.771.402.5014.36
    21R218P. edulis f. flavicarpa × P. incarnata OK33Yes3.063.3918.3318.149.6752.9137.650.871.416.4732.59
    21R221P. edulis f. flavicarpa × P. incarnata OK31No2.683.0015.5618.9713.1556.7340.180.821.415.5028.06
    21R301P. edulis f. flavicarpa × P. incarnata OK41No3.053.1715.5017.9312.1952.2837.830.871.397.3934.79
    21R303P. edulis f. flavicarpa × P. incarnata OK41Yes3.953.6617.1323.1214.6254.9040.660.991.356.8628.74
    21R304P. edulis f. flavicarpa × P. incarnata OK43Yes3.093.3014.6718.2111.7254.1037.500.861.456.0628.37
    21R308P. edulis f. flavicarpa × P. incarnata OK52No2.272.447.5915.0310.0653.5838.940.691.383.1419.50
    21R317P. edulis f. flavicarpa × P. incarnata OK6No2.331.332.0020.4820.0054.1744.910.831.200.852.35
    21R319P. edulis f. flavicarpa × P. incarnata OK41Yes3.414.0726.7122.2810.2756.5738.960.981.4510.9946.97
    22R306Frederick × P. incarnata IL30No2.432.379.0714.4010.2848.4339.870.741.223.9026.25
    22R307Frederick × P. incarnata IL25Yes3.363.8819.5718.6211.7852.8039.130.891.356.8736.87
    22R401P. edulis f. flavicarpa × P. incarnata IL16No3.132.196.9225.0315.3552.8046.630.941.143.3817.33
    22R402P. edulis f. flavicarpa × P. incarnata MS35No3.262.317.4220.6613.9053.1541.970.901.273.7219.77
    22R403P. edulis f. flavicarpa × P. incarnata MS5No3.001.400.0018.5113.0047.1936.521.041.310.000.00
    22R404P. edulis f. flavicarpa × P. incarnata MS12No3.753.5012.0021.3913.0950.3337.401.101.356.1426.96
    22R405P. edulis f. flavicarpa × P. incarnata MS20Yes3.853.3514.6425.7717.3155.3643.771.041.277.5529.30
    22R406P. edulis f. flavicarpa × P. incarnata MS20No3.452.4010.7521.5715.1249.3541.221.051.205.9324.84
    22R407P. edulis f. flavicarpa × P. incarnata MS16No3.753.2511.2023.0015.2248.5740.471.141.205.5827.77
    22R501P. edulis f. flavicarpa × P. incarnata IL23No1.611.131.0511.7311.1039.0434.340.861.130.492.39
    22R502P. edulis f. flavicarpa × P. incarnata IL9No2.111.002.0013.718.8045.4836.300.811.270.858.71
    22R503P. edulis f. flavicarpa × P. incarnata IL25No1.801.080.4111.8611.4339.2733.050.921.190.040.38
    22R504P. edulis f. flavicarpa × P. incarnata IL4No1.751.000.0013.117.7244.1032.530.811.360.000.00
    22R505P. edulis f. flavicarpa × P. incarnata IL12No2.921.675.2019.6014.3150.3241.750.961.212.4512.94
    22R506P. edulis f. flavicarpa × P. incarnata IL25No1.921.040.2115.3913.7448.2539.220.781.240.080.97
    22R507P. edulis f. flavicarpa × P. incarnata IL37No1.591.190.6310.7710.1241.9834.120.741.230.222.11
    1 Feel: (feel scale of 1−5, with 1 = light, empty fruit and 5 = heavy, full fruit). 2 Visual: (visual rating of pulp of 1−5, with 1 = light, empty fruit and 5 = heavy, full fruit). 3 Density: Fd = W/(H×D), where W = fruit weight (g), H = fruit height (cm), and D = fruit width (cm). 4 Shape: H/D, where H = fruit height (mm), and D = fruit width (mm). 5 Pulp percentage [(pulp weight/total weight) × 100].
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    Table 2.  Correlations of fruit characteristics among all interspecific Passiflora hybrids selections in 2022.
    Fruit feelVisualSeed #Hull wt (g)Pulp wt (g)Pulp %Density (g/cm2)Total wt (g)
    Fruit Feel11.000.65860.65240.51290.65920.65840.76770.7084
    Visual20.65861.000.68070.04420.72150.65860.51450.3972
    Seed #0.65240.68071.000.34410.96520.90690.56810.8095
    Hull wt (g)0.51290.04420.34411.000.30730.22600.61140.8024
    Pulp wt (g)0.65920.72150.96520.30731.000.89160.66040.7853
    Pulp %30.65840.65860.90690.22600.89161.000.63060.7314
    Density40.76770.51450.65810.61140.66040.63061.000.7818
    Total wt (g)0.70840.39720.80950.80240.78530.73140.78181.00
    1 Feel: (feel scale of 1−5, with 1 = light, empty fruit and 5 = heavy, full fruit). 2 Visual: (visual rating of pulp of 1−5, with 1 = light, empty fruit and 5 = heavy, full fruit). 3 Pulp percentage [(pulp weight/total weight) × 100]. 4 Density: Fd = W/(H×D), where W = fruit weight (g), H = fruit height (cm), and D = fruit width (cm).
     | Show Table
    DownLoad: CSV

    Seed number was counted for each fruit and then averaged over all collected fruits for each individual vine. Some fruits produced no seeds (22R403 and 22R504) whereas others had more than 25 seeds per fruit (20R305 and 21R319) (Table 1). The presence of many seeds was an indicator of significant pulp presence. As would be expected with any segregating population only one generation from wild material, average total weight of the fruit varied greatly among the individual vines. Some were small, close to 10 g (22R507) and some were much larger, over 30 g (20R305). Of course, there are different components encompassing the total weight, namely hull weight and pulp weight (including seeds). Hulls of P. incarnata are thinner than those of P. edulis and that difference in thickness can affect postharvest shelf life. In general, hull weights ranged between 10 to 20 g, with a few less than 10 g and only one at 20 g (Table 1). Pulp weight ranged from 0 to 10 g, with two individuals above 10 g (20R305 and 21R319), both of which have P. incarnata OK as a parent. Pulp % [(pulp weight/total weight) × 100] was a strong indicator for advancing a selection. Many vines had poor pulp percentage (< 25%). A few were over 30% and two individuals were over 40% (20R114 and 21R319). Fruit density is a non-destructive measure of how full a fruit is of pulp while accounting for fruit size (g/cm2). Fruit densities were mostly < 1.00 but some were higher.

    In terms of harvested fruit, shapes were almost exclusively longer than wide. Most were greater than 40 mm and many over 50 mm and only two were less than 40 mm (22R501 and 22R503). The average width of fruit ranged between 32 and 46 mm. The overall shape (height/width) were similar among all fruits with all being over 1.00 (round) and they were categorized as oblong spheroid[16].

    All correlations of measured traits were significant at p < 0.05 except for hull weight and visual assessment (Table 2). Fruit density was positively correlated with feel and total fruit weight (r = 0.7677 and 0.7818, respectively). This would indicate that any of these three non-destructive measures could potentially be used equally. Total weight was highly correlated with seed number (r = 0.8095), pulp weight (r = 0.7853), and pulp percentage (r = 0.7314). Feel was also correlated with those same variables but lower (r = 0.6524, r = 0.6592, and r = 0.6584). Fruit density was similar to feel (r = 0.6581, r = 0.6604, and r = 0.6306). Therefore, total fruit weight, as a non-destructive measure, was the best indicator of seed number, pulp weight, and pulp percentage in this study (Table 2).

    When it came to influence of male parent on traits, IL was clearly behind both MS and OK (Table 3). Vines with IL as a parent produced fewer seeds, lower total fruit weight, shorter height, smaller width, lower fruit density, lighter pulp weight and pulp percentage. Those vines with OK as a parent made more seeds, longer height, and a more elongated shape than the other two pollen parents. MS-based vines had the greatest average total fruit weight, hull weight, fruit width, and fruit density. MS and OK were not different in pulp weight and pulp percentage.

    Table 3.  Fruit measurements by Passiflora incarnata male parent among all interspecific hybrid selections in 2022.
    MaleTotal wt (g)Hull wt (g)Height (mm)Width (mm)Density (g/cm2)2Shape3Pulp Wt (g)Pulp %4Seed #
    IL14.8 c11.5 b45.9 c37.6 c0.83 c1.23 c2.00 b11.9 b5.0 c
    MS22.1 a14.8 a51.6 b41.2 a1.02 a1.26 b5.24 a23.8 a10.3 b
    OK19.5 b12.3 b54.1 a38.8 b0.89 b1.40 a5.90 a27.2 a14.3 a
    P value1< 0.0001< 0..0001< 0.0001< 0.0001< 0.0001< 0.0001< 0.0001< 0.0001< 0.0001
    1 Means within a column followed by the same letter are similar according to Tukey's honest significant difference (HSD) at p ≤ 0.05. Means followed with different letters within a column are significantly different. 2 Density: Fd = W/(H×D), where W = fruit weight (g), H = fruit height (cm), and D = fruit width (cm). 3 Shape: H/D, where H = fruit height (mm), and D = fruit width (mm). 4 Pulp percentage: [(pulp weight/total weight) × 100].
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    DownLoad: CSV

    After selections were made, the individual selections kept were significantly higher/better in all traits categories except for fruit width (p = 0.3698) and hull weight (p = 0.2415) (Table 4). Eleven out of 35 vines were chosen as advanced selections. Those chosen to advance had much higher seed numbers and pulp weights, which are highly correlated (r = 0.9652). Fruit that was full of pulp had the highest priority in the selection process and that was borne out in the results (Table 4).

    Table 4.  Resulting traits of interspecific Passiflora vine selections that were kept and those discarded during the 2022 season.
    Seed #Total wt (g)Height (mm)Width (mm)Density (g/cm2)2Shape3Pulp wt (g)Hull wt (g)
    Kept18.221.754.339.00.971.407.512.6
    Discarded7.016.950.438.70.841.313.012.2
    P value1< 0.0001< 0.0001< 0.00010.3698< 0.0001< 0.0001< 0.00010.2415
    1 Means within a column are different according to Tukey’s honest significant difference (HSD) at p ≤ 0.05. 2 Density: Fd = W/(H×D), where W = fruit weight (g), H = fruit height (cm), and D = fruit width (cm). 3 Shape: H/D, where H = fruit height (mm), and D = fruit width (mm).
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    Making selections in a new breeding program is challenging, especially when the crop is new or underutilized. The priority was to identify the most important traits to move a vine from general population to improved selection. In the case of interspecific hybrid passionfruit (i.e., Passiflora edulis x P. incarnata), there is insufficient literature to rely upon to establish fruit quality parameters. While cold hardiness is the primary goal, other traits are also important and easier to assess without being destructive and a level of increased cold tolerance is assumed at this stage. Based on prior observation of earlier populations of interspecific seedlings and knowledge regarding self-incompatibility in both Passiflora species, the most important trait to select for was fully filled fruit. Poor pollination can lead to fruit that has normal size but has few or no pulp or seeds[17]. Since the flowers were not hand-pollinated and pollination relied solely on insect pollinator activity, vines that produced well-filled fruit would be viewed as desirable in contrast to fruit that were only partially filled. The second consideration was fruit size. Average fruit size for P. incarnata can vary but may range between 30 and 40 g but may get to nearly 60 g[10]. Passiflora edulis also has a wide range depending on type. Purple fruit is usually far smaller than yellow or red fruit (Fig. 2), ranging from ~80g for purple to ~150 or more for yellow and red[1819]. Since these species were crossed, one would expect to see intermediate traits, including fruit weight; however, this was not the case. Fruit weights were smaller than either parent (Table 1), although fruit from some selections approached that P. incarnata. Arjona et al.[20] reported decreased fruit weight in container-grown P. incarnata vines in a greenhouse. Knight[11] found a wide range of fruit weights among Passiflora hybrids, some results similar to what was observed in the present study. However, in the study by Knight[11] the vines were tetraploid and thus were able to achieve larger size and heavier fruit weights. Conditions reported in other studies likely differed substantially from this one, yet the small fruit weight is a concern. It is important to have fruit that is not too small, such that consumers find it potentially a poor value. Because fruit fill and fruit size were the most important overriding factors in advancing selections in this study, other traits such as sugar and acid levels were not considered but will be in the next round of selection.

    Figure 2.  Fruit from P. edulis (purple and yellow) (left), P. incarnata (middle) and hybrid fruit (right). Hybrid fruit remained green similar to the P. incarnata parent.

    Fruit fill cannot be determined solely based on visual assessment of the whole fruit, nor by total fruit weight. Percent pulp can be measured via destructive measurement, but this is time consuming. A physical assessment of fruit fill can be done without measuring the amount of pulp, but it is subject to unmeasurable variables such as hull weight and thickness that make feel-based ratings a potentially inaccurate metric. Therefore, a non-destructive method to aid in the selection making process would be extremely useful. Fruit density proved to be similar to other methods (Table 2) for estimating fruit fill and could have potential as more data are collected to determine its appropriateness.

    Fruit shape was a less important consideration in the selection process as well. Most of the fruit was oblong, although not extremely so. Most P. edulis fruit are round or near-round and therefore that shape is the accepted standard for passionfruit[21]. With backcrossing, future selections may come closer to the round shape that is expected of passionfruit, but as the American consumer by-and-large has little knowledge of passionfruit it may be possible to introduce other fruit shapes into the marketplace.

    High seed number is desirable because an aril must have a seed to form (there are currently no seedless passionfruit although the possibility is being explored[22]), but seeds of P. incarnata are larger and harder than P. edulis. Hybrids produced intermediate sized seeds, but they were still considerably more noticeable than P. edulis seeds. Passiflora edulis seeds can be readily consumed, as they are small enough to not be noticed. Separating the seeds from the pulp is labor intensive as well. The future of passionfruit production in the US is in fresh fruit consumption and not the processing market due to the relatively inexpensive volume of juice and pulp that is imported from South America[22].

    Breeding with wild or native plant material can be a long-term endeavor. Numerous challenges exist within Passiflora that make progress difficult, especially in interspecific breeding efforts with P. incarnata. These obstacles include large seed size, poor fruit fill (poor pollination, self-incompatibility), low fruit quality, smaller fruit size, and short postharvest shelf life.

    One big advantage for using P. incarnata is the improvement in cold hardiness[11,13,19,23]. While there are problems with incompatibility and hybrid sterility[10,23] when using this species, successful crosses can be made. Since P. incarnata is an herbaceous vine, it will die to the ground starting in fall. Yet, early in the following spring it will emerge from the roots and within weeks it will produce flower buds. Conversely, the P. edulis vine is woody and can be severely damaged by cold temperatures. In south Mississippi, vines of P. edulis f. flavicarpa were completely killed by temperatures of 22 °F (−5.6 °C) even when grown under a high tunnel structure. Interspecific hybrid vines show intermediate growth habit, partially dying back in fall and winter. This could potentially leave them susceptible to damage at low temperatures aboveground. Observations of interspecific hybrids vines in south Mississippi have shown them to vary in their ability to handle cold temperatures, with some dying and some returning strongly from the roots. Appropriate selection for cold hardiness will be needed to progress breeding efforts.

    There is reason to be cautiously optimistic about interspecific hybrid Passiflora involving P. incarnata. However, as seen in previous studies, the challenges are significant toward the production of a commercially viable fruit. In the case of this study, the fruit obtained was smaller than desired with lesser fruit quality than that of P. edulis. Therefore, creating more generations with backcrossing to P. edulis is the next logical step in the process with the expectation of incremental losses of cold hardiness. Additional proxy experiments of cold hardiness, such as differential thermal analysis and electrolyte leakage[24] will be performed as it becomes necessary to determine more precise estimates of tolerances to cold temperatures.

    The author thanks Jeremy Edwards, Robert Gabella, and Jennifer Sherrill, for providing Passiflora incarnata plant material and Jenny Ryals and Haley Williams for early reviews of the paper. The project was founded through a Specific Cooperative Agreement between Mississippi State University and USDA-ARS, supported by the Mississippi Agricultural, Forestry and Experiment Station and Mississippi State University Extension Service. This material is based upon work that is supported by the National Institute of Food and Agriculture, US Department of Agriculture, Hatch project under accession number MIS-211150.

  • The author declares that there is no conflict of interest.

  • Supplemental File 1 Changes in molecular weight of TFP during in vitro digestion. (a) TFP. (b) TFP-S. (c) TFP-G. (d) TFP-I.
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  • Cite this article

    Zhu X, Su J, Zhang L, Si F, Li D, et al. 2024. Gastrointestinal digestion fate of Tremella fuciformis polysaccharide and its effect on intestinal flora: an in vitro digestion and fecal fermentation study. Food Innovation and Advances 3(2): 202−211 doi: 10.48130/fia-0024-0018
    Zhu X, Su J, Zhang L, Si F, Li D, et al. 2024. Gastrointestinal digestion fate of Tremella fuciformis polysaccharide and its effect on intestinal flora: an in vitro digestion and fecal fermentation study. Food Innovation and Advances 3(2): 202−211 doi: 10.48130/fia-0024-0018

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ARTICLE   Open Access    

Gastrointestinal digestion fate of Tremella fuciformis polysaccharide and its effect on intestinal flora: an in vitro digestion and fecal fermentation study

Food Innovation and Advances  3 2024, 3(2): 202−211  |  Cite this article

Abstract: In this work, the gastrointestinal digestive outcome of Tremella fuciformis polysaccharide (TFP) was examined using in vitro simulated experiments, together with its effect on the intestinal microbiota. TFP did not significantly alter during the stage of oral digestion, according to an in vitro digestion investigation. Nevertheless, glycosidic connections of TFP were broken throughout the intestinal and stomach digesting phases, which resulted in the dissociation of macromolecular aggregates, a marked rise in decreasing sugar content (CR), as well as a drop in molecular weight (Mw). Additionally, microbial community analysis following fecal fermentation in vitro indicated that TFP might control the alpha and beta diversity of gut microbiota and change the genus- and phylum-level community composition. It increased the abundance of beneficial bacteria including Megasphaera, Phascolarctobacterium, and Bacteroides, and suppressed the growth of harmful bacteria like Escherichia-shigella and Fusobacterium, thus contributing to maintaining gut homeostasis. These results suggested that TFP could have a positive impact on health through enhancing the gut microbiota environment, giving a theoretical basis for its use as a prebiotic.

    • Prebiotic digestion and fermentation have drawn more attention in the past several years owing to the beneficial effects of prebiotics on host health[1]. Bioactive polysaccharides extracted from medicinal and edible plants and mushrooms exhibited prebiotic characteristics[2]. As a type of medicinal and edible fungus, Tremella fuciformis is rich in polysaccharides, proteins, dietary fiber, and other bioactive components[3]. In Tremella fuciformis, Tremella fuciformis polysaccharide (TFP) is the major bioactive substance[4] and exhibits various physiological activities such as antioxidant[5], anti-tumor[6], blood sugar control[7], anti-inflammatory[8], and immune-enhancing effects[9]. TFP has been confirmed by Xu et al. to prevent mice from colitis caused by dextran sulfate sodium (DSS), showing a reduction in colonic peroxidase and serum diamine oxidase activity, as well as alleviation of colonic tissue damage[10]. In addition, according to Yui et al., the major chain of TFP was identified as α-D-mannose, with β-D-xylobiose, β-D-gluconic acid, and β-D-xylose attached to the C-2 position of main chain[11]. Due to its excellent physiological activities and structure, the creation of medicinal goods and functional foods have made extensive use of TFP.

      It is common knowledge that the bioactivity of polysaccharides is largely associated with their digestion, absorption, and functional properties in the digestive system[12]. Absorption of polysaccharides is a crucial physiological step in the course of digestion and fermentation[13], involving the coordinated actions of various organs in the human digestive system, for instance the small intestine, stomach, and mouth. Eventually, the nutrients can be applied for subsequent fermentation via gut microbiota[14]. Due to technical difficulties and ethical limitations, conducting human experiments to determine the effects of polysaccharides are challenging[15]. Therefore, in vitro models that mimic the human gastrointestinal tract, including the stomach, intestines, and colon, are particularly important for assessing the fermentative and digestive properties of polysaccharides. A study by Wu et al. stated that the TFP was continuously degraded in the process of fermentation, and the total sugar, uronic acid content, molecular weight, and apparent viscosity of TFP decreases significantly with increasing fermentation time[16]. Studies on TFP features related to fermentation and digestion are few. Consequently, it is critical to examine the digestion mechanism of TFP and explore its actual effects in the human body, providing a basis for understanding the bioactivity mechanisms of TFP.

      Gut microbiota not only participate in physiological processes such as digestion, absorption, and metabolism of nutrients but also play important roles in immune regulation, biological defense, and maintaining intestinal homeostasis[17]. Intestinal inflammation and other diseases have been intimately linked to an imbalance in the gut microbiota, making the use of natural edible polysaccharides for intervention and regulation of gut diseases, obesity, and type II diabetes a current research focus. Since the human body lacks enzymes that are activated by carbohydrates, the majority of non-starch polysaccharides can only be fermented and utilized by the microbial community in the intestines to maintain microbial balance and diversity[18]. Edible fungal polysaccharides (primarily β-glucans) can reach the distal colon and be degraded by carbohydrate-active enzymes encoded by the gut microbiota, thus raising the number of beneficial bacteria (e.g., Phascolarctobacterium and Bacteroides) to modulate the composition of the intestinal microbiota[19]. As a result, knowledge of the interaction between the gut microbiota and TFP is essential for designing and manufacturing TFP-based functional health foods.

      In previous studies, in colitis-affected mice, TFP has been shown to affect the equilibrium of gut microbiota and protect the intestinal barrier. Nevertheless, TFP is a biopolymer that is difficult to absorb and digest, and its exact bioactivity mechanism is yet unknown. In this study, using an in vitro digestion model, the properties of TFP digestion during in vitro digestion were examined, followed by evaluating the interaction between poorly digestible TFP and gut microbiota using an in vitro fecal fermentation model. These findings provide a basis for clarifying the underlying digestive and fermentation mechanisms of TFP and give a theoretical basis for the mechanism of its bioactivity.

    • Tremella fuciformis and Inulin (> 98% purity) were provided by Gutian County, Fujian Province, China and Shanghai Yuanye Bio-Technology Co., Ltd (Shanghai, China), respectively. The other chemicals were all analytically graded.

    • Based on previous studies, the hot water extraction of polysaccharides from Tremella fuciformis were performed with slight modifications[20]. Details are supplied in the Supplementary File 1.

    • In accordance with previous methods, with slight changes, in vitro digestion of TFP was performed[2130]. As shown in Fig. 1a, TFP-I, TFP-G, and TFP-S are the names of TFP samples that were digested under various in vitro digesting circumstances, such as saliva-gastrointestinal, saliva-gastric, and saliva digestion, separately. Details were supplied in Supplementary File 1.

      Figure 1. 

      Flow diagram of (a) in vitro digestion and (b) fecal fermentation method of TFP.

    • The collection of fresh fecal samples, preparation of fermentation media, and methods for in vitro fermentation can be referenced from previous studies with slight modifications[31]. As shown in Fig. 1b, using TFP-I as the carbon source, it was added to the culture medium and subjected to in vitro fermentation using the collected gut microbiota from healthy individuals, named as TFP group. The group that had no carbon supply was designated as the blank control or BLANK group. To serve as a positive control, inulin (a recognized soluble polysaccharide serving as a prebiotic) was used as a substitute for TFP, named as FOS group. Details are supplied in the Supplementary File 1.

    • Measurement of CR, pH, carbohydrate content, and gas production during fermentation in vitro were conducted in accordance with previous studies[23]. Details are supplied in the Supplementary File 1.

    • The fermentation broth was subjected to low-temperature high-speed centrifugation to collect the bacterial pellet in cryovials, which were then stored at −80 °C after liquid nitrogen flash freezing. The gut microbiota investigation was carried out with the methodology outlined in our earlier research[32]. Details are supplied in the Supplementary File 1.

    • The data in the results were presented as 'mean ± SD'. Significant differences were indicated by letters (a−e) at p < 0.05.

    • Table 1 reveals that the total polysaccharide content in TFP were 89.62 ± 0.82%, indicating that polysaccharide is the main component of TFP and its content decreased significantly after gastrointestinal digestion[18]. Notably, the Mw of TFP decreased significantly in the digestion stage, and the content of CR increased significantly (Table 2), indicating that the glucoside bond was destroyed, leading to the decrease of Mw of TFP[33,34].

      Table 1.  Data summarization of TFP, TFP-S, TFP-G and TFP-I.

      TFP TFP-S TFP-G TFP-I
      Total polysaccharides (%) 89.62 ± 0.82a 88.90 ± 0.55ab 87.28 ± 0.78b 86.07 ± 1.03c
      Total uronic acids (%) 15.35 ± 0.80a 15.83 ± 0.36a 14.74 ± 0.11b 14.15 ± 0.33c
      Total proteins (%) 2.53 ± 0.05a 1.90 ± 0.01b 0.79 ± 0.01c 0.60 ± 0.02d
      Molecular weight
      Mw × 104 (Da) 2.0361 ± 0.0375a 1.9686 ± 0.0412a 1.7864 ± 0.0109b 1.6620 ± 0.0156c
      Mw/Mn 1.33172 1.2779 1.20094 1.36855
      Constituent monosaccharides and molar ratios
      Man 1.00 1.00 1.00 1.00
      GlcA 0.07 0.08 0.08 0.07
      Glc 0.86 0.73 0.75 0.58
      Xyl 0.42 0.44 0.40 0.41
      Fuc 0.19 0.19 0.20 0.20

      Table 2.  Variations in CR of TFP during in vitro digestion.

      Processes Time (h) CR (mg·mL−1)
      Origin 0.115 ± 0.001a
      Saliva digestion stage 0.25 0.113 ± 0.003a
      0.5 0.114 ± 0.002a
      1 0.116 ± 0.001a
      Gastric juice digestion stage 0.5 0.312 ± 0.032e
      1 0.364 ± 0.002d
      2 0.408 ± 0.010c
      4 0.583 ± 0.023b
      6 0.729 ± 0.016a
      Small intestinal juice digestion stage 0.5 0.809 ± 0.011c
      1 0.836 ± 0.024bc
      2 0.880 ± 0.039b
      4 0.931 ± 0.022ab
      6 0.950 ± 0.005a
    • Table 2 indicates that the content of CR did not vary considerably in the course of salivary digestion[35], but increased significantly after gastric digestion. This is due to the lower pH conditions in the stomach causing the glucoside bond to break, leading to an increase in the reducing end[36,37].

    • In Fig. 2a, the monosaccharide composition of TFP includes Fuc, Glc, Man, Gal, Xyl, GlcA, GalA, and Ara. Among them, the major monosaccharides in TFP are Glc and Man. Previous studies have shown that the main chain of TFP consists of (1→3)-α-D-mannopyranosyl residues and the side chains consist of Fucp, β-GlcAp, and β-Xylp residues[38]. The molar ratio of Glc declined after simulated digestion, which could be attributed to the lower pH causing degradation of the polysaccharide side chains[18,37], suggesting that in vitro digestion could affect the monosaccharide composition of polysaccharides.

      Figure 2. 

      Variations in structural characterizations of TFP during in vitro digestion. (a) Monosaccharide composition. (b) FT-IR. (c) Congo red staining. (d) Thermogravimetric curve. (e) Rheological properties. (f) Particle size and zeta potential.

    • Figure 2b demonstrated that FT-IR spectra of TFP following the simulated digestion were comparable, suggesting that the RG-I backbone and other structural features of TFP were unaffected by the in vitro simulated digestion process[37]. In particular, the existence of carbohydrates was confirmed by the characteristic peak at 3,600−3,200 cm−1, which correlated with the O-H stretching vibration[39], and the range of 1,400−1,200 cm−1, which correlated with the C-H bending vibration[40,41]. The presence of uronic acids was indicated by the asymmetric stretching vibration of free carboxyl groups, which fell within the range of 1,590−1,644 cm−1[40]. Additionally, at 1,555 cm−1, there was no absorption peak, indicating a very low protein content in the polysaccharide samples. The peak at around 917 cm−1 presented the characteristic vibration of the non-symmetric ring stretching of pyranose[5].

    • TFP was described as a polysaccharide with a triple helix shape in Fig. 2c, and this structure held unchanged even after in vitro digestion was simulated. The TG curves in Fig. 2d did not reveal any discernible variations between various phases of TFP digestion. At temperatures between 25 and 600 °C, polysaccharides exhibited three stages of thermal degradation[42]. Notably, in the second stage (101−337 °C), there was a sharp decrease in weight, mainly because the anhydrous organic components gradually decompose under high-temperature heating. The rhamnogalacturonan chain was degraded, leading to carbonization and oxidation, causing the volatilization and loss of a large number of volatile small molecules. In the third stage (337−600 °C), aromatic carbon residues undergo combustion[43].

    • The apparent viscosities of TFP exhibited a typical Newtonian plateau at high shear rates, as presented in Fig. 2e[29]. Figure 2f exhibits that TFP-G had the lowest particle size, suggesting that TFP dissociates more readily in the acidic environment of the stomach. The highest charge was observed for TFP-G, indicating that the small intestine was able to absorb and utilize TFP-G-digested samples more easily.

    • Enzymes encoded by gut microbiota could break down carbohydrates into fermentable sugars, and the growth metabolism of gut microbiota could influence the content of total carbohydrates in the fermentation medium. As shown in Fig. 3a, the carbohydrate content of all substrates decreased most rapidly during the first 6 h, indicating that the fecal microbiota was in the logarithmic growth phase with the highest carbohydrate consumption[44]. With increasing fermentation time, the total sugar content in BLANK, TFP, and FOS groups all showed a decreasing trend, suggesting varying degrees of carbohydrate utilization and the presence of unfermentable components. Studies have found that the consumption of aloe polysaccharides after 48 h of fermentation was approximately 56%[45], and the total sugar consumption of loquat polysaccharides after fermentation was as high as 85%[46], In this experiment, after 48 h of fermentation, the FOS group consumed approximately 69.90% and the TFP group consumed approximately 66.08%. Therefore, TFP was a good fermentation substrate that could be effectively utilized by microorganisms.

      Figure 3. 

      Variations in CR, pH, residual carbohydrates and gas production during in vitro fermentation of TFP. (a) Total carbohydrates. (b) Reducing sugars. (c) pH value. (d) The amount of gas produced.

      As can be seen from Fig. 3b, throughout the entire process of fecal fermentation, the fermentation broth contained very few reducing sugars, ranging from 0.09 ± 0.03 mg/mL to 0.12 ± 0.04 mg/mL, suggesting that the gut microbiota can fully use the reducing sugars generated by TFP-I, with a dynamic balance between enzymatic hydrolysis rate and utilization rate[47].

      The pH level is a crucial signal throughout the fermentation process. Figure 3c exhibits that the pH values of the FOS and TFP groups were consistently lower than those of the BLANK group, owing to acidic substances like short-chain fatty acids (SCFAs) were produced throughout the fermentation process through the fermentation of polysaccharides. The development of pathogenic bacteria may be inhibited by the reduction in intestinal pH. Therefore, TFP and inulin could lower the colonic pH and maintain gut health.

      The gut microbiota tends to produce gases like CH4, H2 and CO2 while fermenting carbohydrates, which could cause adverse symptoms and were the main reason for the limitation of prebiotic application[48]. In Fig. 3d, the gas production of FOS, TFP, and BLANK groups gradually raised during fermentation. After fermentation for 48 h, the gas volume produced by TFP fermentation (0.53 mL) was significantly lower than that of inulin (1.08 mL), indicating that TFP was a more advantageous prebiotic biomass than inulin in terms of gas production.

    • Gut microbiota are crucial for the body's ability to absorb and store energy, perform a number of metabolic processes, and control the immune system, which are crucial for human health and disease. Previous studies have found that through altering gut microbiota, TFP reduced colitis caused by DSS in mice. Thus, it was essential to understand the connection between gut bacteria and TFP, as modulating gut microbiota could contribute to disease prevention and promote health.

      Figure 4ac indicated that most bacterial diversity in the samples was covered by the sequencing depth, indicating that the volume of sequencing data was appropriate. The findings in Fig. 4d & e revealed that there were remarkable differences in the gut microbiota composition between the BLANK, FOS, and TFP groups. Figure 4f demonstrated that between the three groups, there were more inter-group differences than intra-group differences, with intra-group differences being very minor. Figure 4g presented a certain distance between the samples in BLANK, FOS and TFP groups, indicating the specificity of bacterial distribution[49]. In summary, the inter-group differences of each experimental group were remarkably different from one another, and these differences outweighed the intragroup differences, suggesting that carbohydrates from different sources had different effects on the microbial community.

      Figure 4. 

      Correlation curve of species diversity and between-group similarity analysis of gut microflora in vitro fermentation for 48 h. (a) Rank-Abundance curve. (b), (c) Rarefaction curve. (d), (e) Hierarchical clustering tree based on OUT and Genus levels. (f) ANOSIM/Adonis analysis. (g) PLS-DA analysis.

      The samples in the TFP and FOS groups had much lower Sobs, Shannon, ACE, and Chao indices than the BLANK group, as presented in Figs. 5ad, indicating that supplying the gut microbiota with inulin and TFP as carbon sources can result in various degrees of decline in microbial abundance and diversity[43]. The FOS group samples had the lowest microbial diversity and richness, which was similar to the findings of Yu et al.[50]. These findings displayed that the microbial community composition might be changed by both FOS and TFP interventions. In addition, PCA analysis, PCoA analysis, and NMDS analysis (Fig. 5eg) demonstrated that the samples from the TFP, FOS, and BLANK groups exhibited a certain distance, suggesting that the gut microbiota compositions of the three groups varied. The results indicated that TFP, together with gut microbiota could change the microbial community composition, and the impact on gut microbiota varies when using carbohydrates from different sources for in vitro fermentation.

      Figure 5. 

      Analysis of α and β diversity after 48 h of fermentation in vitro in the gut microbial community. (a)-(d) α diversity indices. (e) PCA analysis. (f) PCoA analysis. (g) NMDS analysis.

      Figure 6 displayed the changes in gut microbiota after 48 h of in vitro fermentation. As illustrated in Fig. 6a, c & e, the BLANK group at the genus level consisted mainly of Phascolarctobacterium, Bacteroides, Escherichia-Shigella, Klebsiella and Fusobacterium. In comparison with the BLANK group, Bacteroide proportion significantly increased in the TFP group. Bacteroides were one of the most significant genera of intestinal microbiota and could digest dietary fiber polysaccharides and host glycans. In addition, Bacteroides acted as a key player in the fight against obesity, immune disorders, and the alleviation of intestinal inflammation[51]. The proportion of Megasphaera and Phascolarctobacterium was elevated in the TFP group versus the BLANK group, which was in line with previous findings[37]. At the same time, Escherichia-Shigella and Fusobacterium were reduced in the TFP group, which suggested that TFP could facilitate the beneficial bacteria development and suppress the harmful bacteria development. In the FOS group, the relative abundance of Escherichia-Shigella significantly increased, as Escherichia-Shigella lacked carbohydrate-active enzymes and cannot utilize polysaccharides, whereas inulin, as a low-molecular-weight carbon source, facilitated its growth[52]. Furthermore, the relative abundance of Bifidobacterium, which could degrade and apply inulin to enhance the generation of fermentation end products might be greatly raised by inulin[37].

      Figure 6. 

      Analysis of gut microbial community composition during 48 h of in vitro fermentation. (a), (b) Relative abundance. (c), (d) Community heatmap analysis. (e), (f) Kruskal-Wallis H test bar plot.

      Figure 6b, d & f indicated that the major bacteria in the BLANK group at the phylum level were Proteobacteria, Firmicutes, Fusobacteriota, and Bacteroidetes. In comparison to the BLANK group, the TFP group had a much higher proportion of Bacteroidetes, but a markedly lower proportion of Firmicutes. Bacteroidetes was one of the main intestinal bacteria that were responsible for degrading polysaccharides[53]. When degrading substrates, it released polysaccharide hydrolases and glycoside hydrolases for the degradation of the Gal side chain structures along with the RG-I backbone of polysaccharides[37]. In addition, a rise in the ratio of Bacteroidetes to Firmicutes may reduce the risk of insulin resistance and obesity[54]. Therefore, TFP may play a role in anti-obesity and reducing insulin resistance by regulating the ratio of Bacteroidetes to Firmicutes. Fusobacteriota was generally considered to be associated with some opportunistic pathogens. The low proportion of Fusobacteriota in both TFP and FOS groups indicated that the addition of TFP and inulin could inhibit certain opportunistic pathogens. Proteobacteria and Actinobacteriota were the most varied bacterial phyla, which are often present in the fecal microbiota of healthy individuals[52]. The relative abundance of Actinobacteriota in the FOS group was substantially higher than the BLANK group. Actinobacteriota were Gram-positive bacteria that could convert carbohydrates into non-toxic acidic substances and were believed to promote intestinal health[55]. In summary, inulin and TFP have the potential to alter the gut microbiota composition, especially by encouraging the growth of beneficial bacteria. However, there were differences in their effects on gut microbiota. Compared to the FOS group, the addition of TFP had less interference with the normal community structure and was more conducive to maintaining gut homeostasis in the short term.

    • In conclusion, the present research demonstrated that TFP partially degraded under circumstances resembling salivary gastrointestinal digestion, leading to a notable rise in CR content and a fall in Mw. Furthermore, indigestible TFP-I may be extensively applied by the human gut microbiota during in vitro fecal fermentation. TFP demonstrated the ability to modulate both α and β diversity in the intestinal microbiota and induce changes in the community composition at the phylum and genus levels. This included a decrease in the growth of harmful bacteria for instance Escherichia-Shigella and Fusobacterium and a rise in the abundance of beneficial bacteria like Megasphaera, Phascolarctobacterium, and Bacteroides. These findings indicated that TFP had the potential to be a functional food that enhanced the intestinal microbiota environment, thereby promoting health and preventing disease, e.g., prebiotic.

    • The authors confirm contribution to the paper as follows: conceptualization, methodology, software, investigation, formal analysis, visualization: Zhu X; writing - original draft: Zhu X; writing - review & editing: Su J, Zhang L, Si F, Li D, Jiang Y, Zhang C; supervision: Jiang Y, Zhang C; resources: Zhang C. All authors reviewed the results and approved the final version of the manuscript.

    • This published article and associated supplementary information files contain all of the data generated or analyzed during this work.

      • This work was financially supported by the National Natural Science Foundation of China (31901644) and the University Innovation Team of Shandong Province (2022KJ243).

      • The authors declare that they have no conflict of interest.

      • Supplemental File 1 Changes in molecular weight of TFP during in vitro digestion. (a) TFP. (b) TFP-S. (c) TFP-G. (d) TFP-I.
      • Copyright: © 2024 by the author(s). Published by Maximum Academic Press on behalf of China Agricultural University, Zhejiang University and Shenyang Agricultural University. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (6)  Table (2) References (55)
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    Zhu X, Su J, Zhang L, Si F, Li D, et al. 2024. Gastrointestinal digestion fate of Tremella fuciformis polysaccharide and its effect on intestinal flora: an in vitro digestion and fecal fermentation study. Food Innovation and Advances 3(2): 202−211 doi: 10.48130/fia-0024-0018
    Zhu X, Su J, Zhang L, Si F, Li D, et al. 2024. Gastrointestinal digestion fate of Tremella fuciformis polysaccharide and its effect on intestinal flora: an in vitro digestion and fecal fermentation study. Food Innovation and Advances 3(2): 202−211 doi: 10.48130/fia-0024-0018

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