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Dynamic variation in biochemical properties and prebiotic activities of polysaccharides from longan pulp during fermentation process
International Journal of Biological Macromolecules 132 (2019) 915–921
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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Dynamic variation in biochemical properties and prebiotic activities of polysaccharides from longan pulp during fermentation process Fei Huang a, Ruiyue Hong a, Ruifen Zhang a, Lihong Dong a, Yajuan Bai a, Lei Liu a, Xuchao Jia a, Guangjin Wang b, Mingwei Zhang a,⁎ a Sericultural & Agri-Food Research Institute Guangdong Academy of Agricultural Sciences/Key Laboratory of Functional Foods, Ministry of Agriculture and Rural Affairs/Guangdong Key Laboratory of Agricultural Products Processing, Guangzhou 510610, PR China b College of Chemistry and Materials Science, Hubei Engineering University, Xiaogan 432000, PR China
a r t i c l e
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Article history: Received 13 January 2019 Received in revised form 24 March 2019 Accepted 4 April 2019 Available online 05 April 2019 Keywords: Longan polysaccharide Fermentation process Prebiotic activity
a b s t r a c t Lactic acid bacteria fermentation is an important processing technology for fruits and vegetables. Bioactive compounds such as polysaccharides are altered during the fermentation process. Polysaccharides from longan pulp (LPs) were extracted after different fermentation times and their physicochemical and prebiotic properties were investigated, such as longan polysaccharides named LP-0 and LP-12 means they were extracted from longan pulp fermented for 0 and 12 h, respectively. The yield, contents of neutral sugar and uronic acid, molecular weight (Mw), and monosaccharide composition of LPs were significantly changed with different fermentation times. Specially, the yield and uronic acid content of LPs were first increase and then decline. LP-12 contained the smallest Mw (108.71 ± 5.55 kDa) of the tested LPs (p b 0.05). When compared with unfermented LP-0, the glucose molar percentages of fermented LPs declined, while those of rhamnose and galactose increased, except for LP-6. Fermented LPs also exhibited a stronger stimulatory effect on Lactobacillus strain proliferation, with the proliferative effect of LP-12 being the strongest (p b 0.05). These results suggest that lactic acid bacteria fermentation can change the physicochemical properties and enhance the prebiotic activities of polysaccharides from longan pulp. © 2019 Published by Elsevier B.V.
1. Introduction Longan (Dimocarpus longan Lour.) is an edible fruit that is primarily found in subtropical areas. Longan pulp is rich in carbohydrates, protein, fiber, fat, vitamin C, amino acids, minerals, polyphenols, and volatile compounds [1]. Its pulp has a variety of uses, such as promoting healthy blood vessels and blood pressure, supporting a healthy metabolism, preventing memory loss, and relieving insomnia. Previous studies have shown that polysaccharide is the main functional ingredient and is likely tied to the health benefits of longan pulp [2–4]. An increasing number of studies have focused on longan polysaccharides (LP), including the isolation, purification, and characterization of LP [1]. For instance, the crude LP obtained from longan pulp with ultrasonic extraction had potent immune-modulatory effects in S180 tumor mice in vivo [5]. Four LP fractions have shown the immunomodulatory activities in vitro with up regulating lymphocyte proliferation and stimulating macrophage phagocytosis [4]. For the mechanism of immunomodulatory activities of LP fractions, an LP sub-fraction could ⁎ Corresponding author at: Sericultural and Agri-Food Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou 510610, PR China. E-mail address: [email protected] (M. Zhang).
https://doi.org/10.1016/j.ijbiomac.2019.04.032 0141-8130/© 2019 Published by Elsevier B.V.
stimulate macrophage activation via TLR4 and TLR2 receptor activation, followed by activation of p38 MAPK- and NF-κB-dependent signaling pathways [6]. As the biological activities of polysaccharides are closely related to their structure and physicochemical properties, some researchers have tried a variety of approaches to modify polysaccharides to improve their biological activities [7,8]. The modifications used for longan polysaccharides have centered on chemical ones, such as sulfation [9], acetylation, carboxymethylation [10] and phosphorylation [11]. Chemical modifications are able to change polysaccharide structures by introducing substituent groups, thereby affecting their respective bioactivities. However, it usually requires organic solvents, strong acids, or strong alkali reagents. These constraints have largely limited their application in actual production [7]. As such, other modification methods—such as biotransformation undertaken by microorganisms or enzymatic modification—have received increasing attention due to their high specificity, high efficiency, and few side effects [12]. Lactic acid bacteria fermentation is an important processing technology for fruits and vegetables, as it can keep and/or enhance the safety, nutrition, and shelf life properties of fruits and vegetables. The current research on the fermentation of fruits and vegetables by lactic acid bacteria has mainly focused on strain selection, optimization of strain
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culture conditions, and subsequent changes to the quality of fruits and vegetables [13]. Bioconversion of bioactive compounds caused by fermentation has also received some attention, such as phenolic compounds flavonoids, and γ-aminobutyric acid [14]. In our previous study, we found lactic acid bacteria fermentation could increase the amount of free and total phenolic contents, amino acids and reduce the contents of free amino acids with bitter taste (phenylalanine, tyrosine and leucine) for longan pulp [15]. However, there is little study on the effect of lactic acid bacteria fermentation on the polysaccharides from fruit pulp. Given this gap in our understanding, we used Lactobacillus fermentum to ferment longan pulp and compared unfermented and fermented longan pulp polysaccharides. It was that the fermentation treatment has profound effect on the immunomodulatory and prebiotic activities of longan pulp [16], but the change of molecular weight, neutral sugar, uronic acid and monosaccharide composition of longan pulp polysaccharide during the fermentation process have not yet been investigated. In addition, the dynamic variations of bioactivities of longan polysaccharides during fermentation process are unknown. Therefore, we continued using Lactobacillus fermentum to ferment longan pulp, and analyzed dynamic variation in physicochemical and prebiotic activities of polysaccharides from longan pulp with different fermentation degrees. 2. Materials and methods 2.1. Materials and chemicals 2.1.1. Chemicals and reagents Standard dextrans (including T-4: molecular mass, 4 × 103 Da, T-10: 1 × 104 Da, T-40: 4 × 104 Da, T-70: 7 × 104 Da, T-500: 5 × 105 Da, and T2000: 2 × 106 Da), rhamnose, arabinose, xylose, mannose, glucose, and galactose were all purchased from Sigma Chemical Co. (St. Louis, MO, USA). Man-Rogosa-Sharpe (MRS) was purchased from Guangdong Huankai Microbial Technology Co., Ltd. (Guangzhou, China). Fructooligosaccharides (DP 2-9) was purchased from Shanghai Ruiyong Biotechnology Co., Ltd. (Shanghai, China). All other chemicals used were of analytical grade and purchased from Guangzhou Qiyun Biological Co., Ltd. (Guangzhou, China). 2.1.2. Bacterial strains Bacterial strains included Lactobacillus fermentum GIM 1.191, Lactobacillus acidophilus GIM 1.731, Lactobacillus plantarum GIM 1.380, and Lactobacillus bulgaricus, GIM 1.189 were purchased from the Guangdong Provincial Microbial Culture Collection Center (Guangzhou, China). Strains were stored in MRS broth containing 10% glycerol at −80 °C until later use. Prior to all assays, bacteria were activated according to previously published methods [17]. 2.2. Logan pulp fermentation Fresh longan (cv. Chu-liang) fruits were provided by the Pomology Research Institute of the Guangdong Academy of Agricultural Sciences (Guangzhou, China). Fresh longan fruits were hot air-dried with an air flow velocity of 2.0 m/s at 70 °C for 72 h until the moisture content was 20 ± 2%. Dried longan was peeled and the pulp portion was homogenized with water (1:7 w/v) and the pH was adjusted to 5.8 ± 0.2. The resulting longan juice was then sterilized for 20 min at 121 °C. Fermentation experiments were conducted in aluminum foil sealed Erlenmeyer flasks, each containing 100 mL of pasteurized longan juice without any supplementary nutrients. Inoculum (1 mL) containing 7.0 log cfu/mL of activated Lactobacillus fermentum was added to each of the Erlenmeyer flasks. Fermentation was then allowed to proceed at 37 °C for 72 h. Samples were taken at 0, 6, 12, 24, 48 and 72 h for viable cell count, pH, and polysaccharides extraction as described below. Three duplicate samples were taken at each fermentation time point.
Cell viable counts of the fermented longan juice were obtained as follows: A sample of the culture (1 mL) was transferred to sterile test tubes and serially diluted with sterile 1% (w/v) peptone solution. Then, 100 μL of each dilution was spread onto MRS agar-containing plates and then subjected to anaerobic incubation at 37 °C for 48 h. Bacteria were counted and expressed as log cfu/mL. The pH of the fermented longan juice was simultaneously measured during all cell counts using a standard pH meter (pH S-3C, Shanghai Precise Scientific Instrument Corp., China). 2.3. Longan polysaccharide extraction Fermented longan juice was boiled for 10 min at 100 °C to kill the live Lactobacillus. Sterilized products were then collected by adding distilled water to 1 L and holding the mixture at 80 °C for 4 h prior to filtering. This extraction was repeated, and the filtrates were collected, combined, and then vacuum concentrated at 65 °C. The extracted solution was then subjected to the Sevag method for 8 times to remove protein [18]. Four volumes of ethanol were added and precipitation was allowed to occur for 24 h at 4 °C. The resulting precipitate was collected using centrifugation (3000 ×g, 10 min), washed with ethanol, and lyophilized to obtain the final longan polysaccharides (LPs). The polysaccharides extracted from different time points during the longan pulp fermentation were named as follows: LP-0, LP-6, LP-12, LP24, LP-48 and LP-72, respectively. 2.4. Physicochemical properties of LPs 2.4.1. Chemical characteristics Neutral polysaccharide content was determined using the phenolsulfuric acid method according to previously published methods [19]. Glucose was used as the standard. Protein concentration was determined using the Bradford assay with a bovine serum albumin standard curve [20]. Uronic acid content was determined using a modified mhydroxydiphenyl method with galacturonic acid standards [21]. The monosaccharide composition of LPs was determined using gas chromatography–mass spectrometry (GC–MS) according to the methods of our previously published study [22]. Briefly, 10 mg of polysaccharide sample was dissolved in 4 moL/L trifluoroacetate (2 mL) and hydrolyzed at 110 °C for 6 h. The hydrolysate was dried under an N2 stream and then dissolved in 1 mL pyridine containing 10 mg hydroxylamine. The sample was then kept at 90 °C for 30 min, after which it was cooled to room temperature. Acetic anhydride (1 mL) was added and allowed to incubate at 90 °C for 30 min. The resulting acetylated hydrolysate was analyzed by GC–MS with an Agilent 6890 GC (Agilent Technologies Co., Ltd., Colorado Springs, CO, USA) equipped with a DB-1 column and an Agilent 5973 MS detector. The temperature program was set as follows: Initial column temperature was 190 °C, temperature was increased to 230 °C at 2 °C/min, held for 2 min, then increased to 240 °C at 5 °C/min, and held for another 2 min. The detector temperature was 290 °C and the vaporizing chamber temperature was set to 260 °C. GC/MSD ChemStation software was used for all analysis. Six monosaccharides (arabinose, mannose, rhamnose, galactose, xylose, and glucose) and inositol were used as external and internal standards respectively to identify polysaccharide composition. Each sample was analyzed in triplicate. 2.4.2. Analysis of molecular weights The average molecular weights of LPs were determined using an advanced polymer chromatography system (APC) equipped with a refractive index detector (RID). The analysis was completed using Acquity APC AQ 45, 125, and 450 columns (4.6 × 150 mm, Waters Corp., USA). The column oven temperature was set at 40 ± 0.2 °C and the column was eluted with H2O at a flow rate of 1.0 mL/min. Standard dextran including T-4 (molecular mass, 4 × 103 Da), T-10 (1 × 104 Da), T-40 (4
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× 104 Da), T-70 (7 × 104 Da), T-500 (5 × 105 Da), and T-2000 (2 × 106 Da) were used as molecular mass markers. 2.4.3. FT-IR spectroscopy According to our previous study [23], 2 mg polysaccharide samples were mixed with 100 mg potassium bromide (KBr) powder and pressed into 1 mm thick pellets for FT-IR measurements. FT-IR spectra were recorded on a Nexus 5DXC FT-IR (Thermo Nicolet, Austin, TX, USA) in the frequency range of 4000–400 cm−1 at 4 cm−1 resolution using 64 scans. 2.5. Prebiotic activity analysis Three Lactobacillus strains—L. acidophilus, L. plantarum, and L. bulgaricus—were used for investigating the prebiotic activity of the isolated LPs in vitro. Carbohydrate-free MRS broth supplemented with 0.05% (w/v) L-cysteine was used as the basal medium and as the blank control; fructooligosaccharide (FOS) was the positive control. All LP and FOS sugars were filter-sterilized and separately added to the basal medium to obtain a series of final concentrations (0.5, 1.0, 1.5, 2.0, and 3.0% [w/v]). The activated Lactobacillus strains were added to the medium (final concentration: 1 × 106 cfu/mL) and then incubated at 37 °C for 48 h under anaerobic conditions (85% N2, 10% CO2 and 5% H2). The numbers of Lactobacillus strains in the samples were then counted after incubating for 0 and 48 h. The pH values of all samples were simultaneously measured using a pH meter (pH S-3C, Shanghai Precise Scientific Instrument Corp., China). Bacterial counts were performed as previously described and bacteria were expressed as log cfu/mL. The increase in bacterial number between 0 and 48 h was calculated according to the following formula: Increase of bacterial number = log B − log A; Where A is the bacterial number at 0 h (cfu/ mL) and B is the bacterial number after 48 h incubation (cfu/mL). The fold-increase bacterial number which was relative to control group was presented as the result. 2.6. Statistical analyses All data are expressed as mean ± standard deviation (SD). Statistical significance was evaluated using a one-way ANOVA followed by the Student-Newman-Keuls test using SPSS 19.0 software. A p-value of 0.05 was chosen as the threshold for significance. 3. Results 3.1. Longan pulp fermentation The effect of fermentation time on bacterial number and pH is shown in Fig. 1. The bacterial number in the longan juice displayed an increasing trend during the fermentation process. The bacterial number started at 5.56 log cfu/mL, showed a slight increase until 6 h, and then rapidly increased to 9.12 log cfu/mL at 12 h. Bacterial number then maintained a slowly increasing trend until 48 h (9.68 log cfu/mL), after which it gradually decreased from 60 to 72 h (Fig. 1A). This increase in bacterial number indicated that Lactobacillus fermentum utilized the longan juice to grow. Correspondingly, the pH value of the longan juice showed a decreasing trend throughout the fermentation process. More specifically, pH started at 5.6, dropped slightly, and then quickly fell to 4.37 at 12 h. The pH then continued to slowly decline until 3.68 (72 h) (Fig. 1B). This change in pH also revealed that the carbohydrates contained in the longan juice were successfully metabolized to produce acids by Lactobacillus fermentum. 3.2. Preliminary characterization of LPs The yield as well as neutral sugar, uronic acid, and protein contents of the isolated LPs are summarized in Table 1. The polysaccharide yield increase first with 6 h fermentation and then decrease with the
Fig. 1. Effects of fermentation time on the bacteria number (A) and pH (B) of longan pulp at 0, 6, 12, 24, 48 and 72 h. The bacteria number of fermented longan pulp was measured with cell viable counts while its pH was measured using a standard pH meter.
fermentation time increase. LP-6 had the highest yield among all samples (p b 0.05). As for the neutral sugars, LP-72 had the highest content, while LP-24 had the lowest (p b 0.05). The trend of uronic acid content in LPs was similar with that of yields, which was increase first and then decrease. The polysaccharide protein content from non-fermented longan pulp was less than that from fermented samples (p b 0.05). The protein content of polysaccharides from fermented sample did not change with fermentation time. The molecular weights of all LPs were determined using APC and size exclusion columns. As shown in Table 2, LPs without fermentation had the largest average molecular weight (Mw), with LP-12 having the smallest Mw among the analyzed samples. The Mw of LP decreased
Table 1 The physicochemical properties of LPs. Sample
Extraction yield (%)
Neutral sugar (%)
Uronic acid (%)
Protein (%)
LP-0⁎ LP-6 LP-12 LP-24⁎ LP-48 LP-60 LP-72
2.40 ± 0.36c 2.65 ± 0.18cd 2.16 ± 0.09bc 1.49 ± 0.36ab 1.09 ± 0.15a 1.06 ± 0.02a 1.03 ± 0.13a
43.19 ± 1.43bc 43.01 ± 0.84bc 41.89 ± 0.70ab 41.50 ± 0.15a 43.40 ± 0.60c 43.74 ± 0.17c 46.81 ± 0.17d
35.23 ± 8.93cd 38.31 ± 1.18d 31.52 ± 1.36c 26.81 ± 0.25b 25.62 ± 1.25ab 23.52 ± 1.16a 22.63 ± 2.23a
0.88 ± 0.55a 1.56 ± 0.32b 1.50 ± 0.46b 1.37 ± 0.82b 1.52 ± 0.39b 1.51 ± 0.25b 1.57 ± 0.36b
Different lowercase letters represent significant differences among samples (p b 0.05). ⁎ Represents the data obtained from our previous work (Huang et al. [16]).
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Table 2 The molecular weights and distributions of LPs. Sample
LP-0 LP-6 LP-12 LP-24 LP-48 LP-60 LP-72
Mw (kDa)
Molecular weight distribution (%) 0–50 kDa
50–100 kDa
N100 kDa
61.34 65.54 84.09 80.69 70.11 62.48 55.76
10.09 5.49 3.13 2.18 13.26 18.31 24.45
28.57 28.97 12.78 17.13 16.63 19.21 19.79
221.63 ± 2.41d 152.98 ± 1.06c 108.71 ± 5.55a 109.62 ± 10.66a 136.83 ± 1.56b 139.42 ± 2.31b 152.31 ± 6.37c
Different lowercase letters represent significant differences among samples (p b 0.05).
from 0 h to 12 h and then increased as fermentation time increased from 12 h to 72 h. Of those with a Mw between 0 and 50 kDa, LPs were first increase and then decline and LP-12 had the highest percentage (84.09%); however, its percentage was the least of those with Mw N 100 kDa. For the fraction in the Mw range of 50–100 kDa, LPs were first decline and then increase. In the higher range of N100 kDa, LP-6 had the highest percentage (28.97%) and it had the largest Mw of all fermented LPs, but still smaller than that of LP-0 (p b 0.05). Our monosaccharide analysis revealed that all LPs were heteropolysaccharides and composed of different ratios of rhamnose, arabinose, xylose, mannose, glucose, and/or galactose (Table 3), which is consistent with our previous study [16]. The major monosaccharides in LP-0 were arabinose, galactose, and glucose. However, rhamnose became one of the main constituent monosaccharides when longan juice was fermented for longer than 60 h. When compared with nonfermented LP-0, the relative molar percentages of rhamnose increased in LPs that had been fermented for longer than 12 h. With the exception of LP-6, the relative molar percentage of glucose in the other fermented LPs was lower than that of the non-fermented LP-0. However, the percentage composition of galactose showed an opposite trend. The relative molar percentage of arabinose was the highest in all samples, with a minimum of 36.26% in LP-6 and a maximum of 48.55% in LP-24. The FT-IR images of LPs are shown in Fig. 2. All of LPs showed characteristic polysaccharide bands, including the hydroxyl group bands in the ranges of 3600–3200 cm−1 and 1075–1010 cm−1 and the alkyl group bands at approximately 2926, 2850 and 1458 cm−1 [16]. The absorption bands between 1100 and 1000 cm−1 were attributed to characteristic C\\O\\C glycosidic bond vibrations and ring vibrations overlapped with stretching vibrations of the side group C\\O\\H link bonds [24]. According to Fig. 2, the absorbance band at approximately 1550 cm−1 which was referred to the bending vibration of N\\H [25], appeared in LP-48, LP-60 and LP-72, but not in other samples. This indicated the protein existed in LP-48, LP-60 and LP-72, but did not exist or existed with very low content. This result is consistent with the protein results in Table 1 that LP-48, LP-60 and LP-72 had more protein than other samples.
Fig. 2. FT-IR spectra of LPs. The FTIR spectra of LPs were determined using a Fouriertransform infrared spectrophotometer over the frequency range of 4000–400 cm−1.
3.3. LP prebiotic activity The increased bacteria numbers of Lactobacillus strains after 48 h incubation with either LPs or FOS as their sole carbon source are shown in Fig. 3. For L. acidophilus, the bacterial populations were concentrationdependent in the range of 0.5–3.0%. And LPs stimulated the highest increased-fold bacterial populations at 3.0% of samples. Moreover, bacterial populations applied with fermented longan polysaccharides showed much higher than that of unfermented group. LP-12 had a significantly more beneficial effect on L. acidophilus proliferation than the other LPs at a similar concentration range (0.5–3.0%) (p b 0.05). As for L. bulgaricus, the bacterial number was greater when growing in any of the LPs or FOS when compared with that of the blank control. This indicated that all samples could stimulate the proliferation of that strain. Although there was no regularity in the relationship between bacterial population and polysaccharide concentration, FOS had the most beneficial effect on L. bulgaricus proliferation during the tested concentrations. Moreover, LP-12 had more prebiotic activity than any other LPs across the concentration range 0.5–1.5% (p b 0.05). The increased bacterial populations of L. plantarum stimulated with LPs and FOS was shown in Fig. 3C. Compared with blank control, all LPs and FOS stimulated more bacterial numbers. This indicated that all samples could stimulate the proliferation of that strain. When compared with non-fermented LP-0, fermented LPs showed a greater benefit on L. plantarum proliferation across the concentration range 1.0–3.0%; moreover, this was weaker than FOS. The increased bacterial
Table 3 The monosaccharide compositions of LPs. Monosaccharide composition (%)
Rhamnose
Arabinose
Xylose
Mannose
Glucose
Galactose
LP-0⁎ LP-6 LP-12 LP-24⁎ LP-48 LP-60 LP-72
8.790 ± 0.012b 7.193 ± 0.133a 7.187 ± 0.046a 9.294 ± 0.075c 9.769 ± 0.032d 11.915 ± 0.019f 11.100 ± 0.007e
43.628 ± 0.639c 36.263 ± 0.303a 43.472 ± 0.066c 48.549 ± 0.119f 47.460 ± 0.012e 45.235 ± 0.026d 41.308 ± 0.201b
1.529 ± 0.122ab 1.518 ± 0.316ab 2.303 ± 0.031d 1.831 ± 0.110bc 1.954 ± 0.050c 1.695 ± 0.087abc 1.462 ± 0.049a
2.347 ± 0.027a 2.824 ± 0.153c 3.590 ± 0.030f 3.280 ± 0.073e 3.118 ± 0.037de 2.917 ± 0.001cd 2.591 ± 0.167b
23.388 ± 0.442e 33.920 ± 0.002f 22.623 ± 0.029d 14.331 ± 0.072b 12.282 ± 0.131a 12.074 ± 0.061a 20.031 ± 0.010c
20.317 ± 0.334b 18.282 ± 0.030a 20.825 ± 0.007c 22.715 ± 0.066d 25.417 ± 0.000f 26.164 ± 0.154g 23.508 ± 0.086e
Different lowercase letters represent significant differences among samples (p b 0.05). ⁎ Represents the data obtained from our previous work (Huang et al. [16]).
F. Huang et al. / International Journal of Biological Macromolecules 132 (2019) 915–921
Fig. 3. Effects of LPs on the increased bacteria number after 48 h incubation at different concentrations (0, 0.5, 1.0, 1.5, 2.0 and 3.0%). (A) L. acidophilus, (B) L. bulgavicus, and (C) L. plantarum. The increased bacteria number was expressed as the fold of control group.
populations of L. plantarum cultured with LP-12 was greater than other LPs at the same concentration from 0.5% to 3% (p b 0.05), except 1%. Polysaccharide prebiotic activity depends on its hydrolysis catalyzed by glycosidase secreted from probiotics. Polysaccharides are hydrolyzed into monomers and further degraded into short-chain fatty acids and gases when they are the sole carbon source for bacteria. This ultimately results in a significant decrease in pH in the fermented broth. Given this, the effect of LPs on the acidifying activities of probiotics was further investigated. As shown in Fig. 4A, marked pH decreases were observed in L. acidophilus fermented broth after 48 h stimulation with LPs and FOS. Their pH values reached the minimum value observed at 3.0% concentration for all samples. Across the concentration range 0.5–2.0%, pH values of LP12 were the lowest of the LPs, but still larger than those of the
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Fig. 4. Effects of LPs on the pH of probiotics after 48 h incubation at different concentrations (0, 0.5, 1.0, 1.5, 2.0 and 3.0%). (A) L. acidophilus, (B) L. bulgavicus, and (C) L. plantarum. The pH of probiotic medium was measured using a standard pH meter.
positive control FOS. This was counter to the order of probiotic growth numbers when stimulated with polysaccharides (Fig. 3). LPs and FOS resulted in significant decreases of pH values after 48 h incubation with L. bulgaricus, with the exception of LP-0 across a concentration range of 0.5–1.5% (Fig. 4B). The minimum values we observed were obtained at 3.0% concentration and were in the following order: LP-60 N LP-48 ≈ LP-72 N LP-0 ≈ LP-12 N LP-6 ≈ LP-24 N FOS. The pH values of L. bulgaricus when incubated with LP-12 were lower than those of the other LPs across the 0.5%–2.0% concentration range (p b 0.05). This observation was consistent with the increased number of L. bulgaricus observed when incubated with LPs. As for L. plantarum, when compared with the blank control, the pH value of the media was significantly decreased after 48 h incubation with LPs and FOS (p b 0.05) (Fig. 4C). Moreover, the pH values of L. plantarum incubated with LP-12 and LP-24 were similar across the concentration range 1.0–3.0% and were both smaller than those of other LPs. However, they were still larger than those of the positive control FOS (p b 0.05).
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4. Discussion
4.2. Effect of fermentation on the prebiotic activity of LPs
4.1. Effect of fermentation on the physicochemical properties of LPs
Three Lactobacillus strains—L. acidophilus, L. plantarum, and L. bulgaricus—have found extensive use in the food industry and their probiotic functions in human health have been generally accepted [36]. As such, these three strains were used to evaluate the prebiotic activities of LPs in this study. Differences in stimulatory effects on the proliferation of Lactobacillus strains by LPs (Fig. 3) are related to both polysaccharide and probiotic properties. Different probiotics have different abilities to secrete enzymes, which causes differences in their ability to utilize available polysaccharides [37]. LPs were able to significantly stimulate L. acidophilus proliferation and exhibited a dose-dependent effect across the tested concentration range. The highest fold of increased L. acidophilus number was 5.18 while that of L. bulgavicus was 1.62 at the same concentration range (Fig. 3). The chemical and physical properties of polysaccharides can affect their utilization by probiotics and include monosaccharide composition, Mw, and glycosidic linkages. In present study, the polysaccharide degradation as indicated by decrease in Mw after fermentation treatment could result from the breaking of glycosidic bonds (Table 2). Thus, the chemical-structural properties of the polysaccharide might be altered. The fermented LPs were better utilized by bacteria than the non-fermented polysaccharides. Mw is another key factor influencing the prebiotic activity of polysaccharides. Generally, polysaccharides with a lower molecular weight have better access to probiotics. With this in mind, the proliferative stimulatory effect of LP-12 was the strongest, while LP-0 was the weakest (Fig. 3). This observation is presumably because their molecular weights were the smallest and the largest, respectively (Table 3). This conclusion coincided with the results of previous work examining the polysaccharides from lotus seed resistant starches [38]. Moreover, monosaccharide composition is also an important factor that influences the prebiotic activities of polysaccharides. Previous studies have shown that polysaccharides exhibit better prebiotic activities when they are composed of glucose, xylose, and galactose [39]. These include polysaccharides from bamboo shoots [40] and rapeseed [17]. LPs contained the aforementioned monosaccharides and promoted the growth of probiotics; moreover, the different molar ratios of the monosaccharides in LPs also likely contributed to their different prebiotic effects. However, it is likely that there are other mechanisms at work, which will need to be identified and studied in the future.
We first sought to explore the effect of lactic acid bacteria fermentation on longan pulp polysaccharides. To do this, we chose the commercial strain Lactobacillus fermentum, which is commonly used in fermented fruit juices, to ferment longan pulp for 72 h. The obtained polysaccharides were extracted from the longan pulp at different time points (0, 6, 12, 24, 48 and 72 h). The yield as well as neutral sugar and uronic acid content, Mw, and monosaccharide composition showed marked differences. Comparatively, protein content across these different fermentation time points showed no significant differences (Tables 1–3). Lactic acid bacteria or other microorganisms can affect polysaccharide composition by secreting different carbohydrate enzymes. These enzymes could actively assist the break down the cell walls of berry tissues, favoring the release of sugars. They produce hydrolytic enzymes to degrade the insoluble pecto-cellulosic cell wall of fruit into soluble polysaccharides, resulting in increased yield of water-soluble polysaccharides [26] as well as changes to Mw and monosaccharide composition of the extracted polysaccharides [27]. That is the reason for the increase in the yield, uronic acid and protein content of longan polysaccharide with 6 h fermentation. But for molecular weight change, we speculate it may be caused by two factors. For one is the production of pectin and AGPs with cell walls degradation [28], as their Mw is smaller than the polysaccharide obtained from longan pulp without treatment. The other is the cleavage of polysaccharide with enzymes, leading to decreases in observed Mw. So the average molecular weight of fermented longan pulp polysaccharides becomes smaller than that of LP-0. In addition, hydrolytic enzymes can also act on the glycosidic chains of watersoluble polysaccharides in the fruit pulp, hydrolyze them into oligosaccharides or monomers, which are then finally metabolized into shortchain fatty acids [29]. For example, the amount of pectin and AGPs in grape fermentation juice increased at the early stage of fermentation; however, as fermentation proceeds, these factions reduced [28]. This may be the reason the yield and uronic acid decrease as the fermentation time increase. LPs in the Mw fraction range of 0–50 kDa declined significantly from 12 h to 72 h may be related with bacteria utilization, as bacteria usually preferential use of polysaccharides with small molecular weight, such as wheat arabinoxylans with 66 kDa promoted Eubacteria growth better than those with 354 and 278 kDa [30]. The percentage of small Mw fraction decline and those of the large Mw fraction were relative increase. That may be the reason for the Mw of LP first decrease and then increase. Moreover, some lactic acid bacteria can produce extracellular polysaccharides when they utilize glucose and fructose in fruits [26,31]. Here, in order to more effectively study the effect of fermentation on the physicochemical and prebiotic activities of longan pulp polysaccharides, we chose Lactobacillus fermentum as the fermentation strain since it does not produce any extracellular polysaccharides. The glucose content of rice bran polysaccharides declines significantly with Grifola frondosa fermentation [32] and decreases in the relative percentages of arabinose and glucose in fermented litchi pericarp polysaccharides were also observed with Aspergillus awamori fermentation [33]. Pectinase treatment induces the cleavage of acidic polysaccharide from banana pulp and produces oligogalacturonic acids, leading to decreases in observed Mw [34]. Here, the change of glucose relative molar percentage, uronic acid content and Mw in LPs that had been fermented for longer than 12 h were consistent with previous observations [32–34]. Lactobacillus fermentum secretes rich extracellular enzymes during fermentation, including amylase, α-glucosidase, cellulose, lactate dehydrogenases, and peptidases [14,35]. It is likely that these enzymes are responsible for the changes in uronic acid content, monosaccharide composition, and Mw distribution.
5. Conclusions The present study sought to investigate the effect of lactic acid bacteria fermentation on the physicochemical and prebiotic activities of LP. The results showed that the yield as well as the content of neutral sugar and uronic acid, Mw, and monosaccharide composition of LPs changed with fermentation. Our prebiotic activity analysis revealed that the fermented polysaccharides had prebiotic activities when compared with unfermented polysaccharides. Moreover, that LP-12 exhibited the strongest prebiotic activity of the tested fermented polysaccharides. Overall, these results suggest that lactic acid bacteria fermentation is an effective method to modify longan polysaccharide and improve its prebiotic activity. Acknowledgements This work was supported by National Key Research Project of China (2017YFC1601002, 2018YFC1602105), National Natural Science Foundation of China (31801498), Guangdong Provincial Science and Technology Project (2017A030313163, 2016A030310321, 2017B090907022, 2018A050506050), and Scientific Research Program of Guangzhou (201804020064, 201704020039, 201803010079, 201803020014). This work was supported by China Scholarship Council (No. 201708440135).
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References [1] B. Yang, Y. Jiang, J. Shi, F. Chen, M. Ashraf, Extraction and pharmacological properties of bioactive compounds from longan (Dimocarpus longan Lour.) fruit — a review, Food Res. Int. 44 (7) (2011) 1837–1842. [2] Y. Bai, L. Liu, R. Zhang, F. Huang, Y. Deng, M. Zhang, Ultrahigh pressure-assisted enzymatic extraction maximizes the yield of longan pulp polysaccharides and their acetylcholinesterase inhibitory activity in vitro, Int. J. Biol. Macromol. 96 (2017) 214–222. [3] J. Zhang, G. Yang, Y. Wen, S. Liu, C. Li, R. Yang, W. Li, Intestinal microbiota are involved in the immunomodulatory activities of longan polysaccharide, Mol. Nutr. Food Res. 61 (11) (2017), 1700466. [4] Y. Yi, M.-W. Zhang, S.-T. Liao, R.-F. Zhang, Y.-Y. Deng, Z.-C. Wei, X.-J. Tang, Y. Zhang, Structural features and immunomodulatory activities of polysaccharides of longan pulp, Carbohydr. Polym. 87 (1) (2012) 636–643. [5] K. Zhong, Q. Wang, Y. He, X. He, Evaluation of radicals scavenging, immunitymodulatory and antitumor activities of longan polysaccharides with ultrasonic extraction on in S180 tumor mice models, Int. J. Biol. Macromol. 47 (3) (2010) 356–360. [6] Y. Yi, Hongxun Wang, Ruifen Zhang, Ting Min, Fei Huang, L. Liu, M. Zhang, Characterization of polysaccharide from longan pulp as the macrophage stimulator, RSC Adv. 5 (2015) 97163–97170. [7] S. Li, Q. Xiong, X. Lai, X. Li, M. Wan, J. Zhang, Y. Yan, M. Cao, L. Lu, J. Guan, Molecular modification of polysaccharides and resulting bioactivities, Compr. Rev. Food Sci. F. 15 (2) (2016) 237–250. [8] S.S. Ferreira, C.P. Passos, P. Madureira, M. Vilanova, M.A. Coimbra, Structure– function relationships of immunostimulatory polysaccharides: a review, Carbohydr. Polym. 132 (2015) 378–396. [9] J. Jiang, F.-Y. Meng, Z. He, Y.-L. Ning, X.-H. Li, H. Song, J. Wang, R. Zhou, Sulfated modification of longan polysaccharide and its immunomodulatory and antitumor activity in vitro, Int. J. Biol. Macromol. 67 (2014) 323–329. [10] J. Wang, Study on the Prepatation, Antioxidant and Immunomodulatory Activities of Acetylated and Carboxymethylated Polysaccahrides From Dimocarpus longan Pulp, Guangxi Medical University, Nanning, China, 2013. [11] Y. Ning, Study on the Prepatation, Immunomodulatory and Antioxidant Activities of Phosphorylated and Acid Hydrolyzed Polysaccahrides From Longan Pulp, Guangxi Medical University, Nanning, China, 2010. [12] P. Chen, Q. You, X. Li, Q. Chang, Y. Zhang, B. Zheng, X. Hu, H. Zeng, Polysaccharide fractions from Fortunella margarita affect proliferation of Bifidobacterium adolescentis ATCC 15703 and undergo structural changes following fermentation, Int. J. Biol. Macromol. 123 (2019) 1070–1078. [13] R. Di Cagno, R. Coda, M. De Angelis, M. Gobbetti, Exploitation of vegetables and fruits through lactic acid fermentation, Food Microbiol. 33 (1) (2013) 1–10. [14] S.J. Hur, S.Y. Lee, Y.-C. Kim, I. Choi, G.-B. Kim, Effect of fermentation on the antioxidant activity in plant-based foods, Food Chem. 160 (2014) 346–356. [15] S.A. Khan, L. Liu, T. Lai, R. Zhang, Z. Wei, J. Xiao, Y. Deng, M. Zhang, Phenolic profile, free amino acids composition and antioxidant potential of dried longan fermented by lactic acid bacteria, J. Food Sci. Technol. 55 (12) (2018) 4782–4791. [16] F. Huang, R. Hong, R. Zhang, Y. Yi, L. Dong, L. Liu, X. Jia, Y. Ma, M. Zhang, Physicochemical and biological properties of longan pulp polysaccharides modified by Lactobacillus fermentum fermentation, Int. J. Biol. Macromol. 125 (2019) 232–237. [17] X. Wang, M. Huang, F. Yang, H. Sun, X. Zhou, Y. Guo, X. Wang, M. Zhang, Rapeseed polysaccharides as prebiotics on growth and acidifying activity of probiotics in vitro, Carbohydr. Polym. 125 (1) (2015) 232–240. [18] M. Sevag, D.B. Lackman, J. Smolens, The isolation of the components of streptococcal nucleoproteins in serologically active form, J. Biol. Chem. 124 (2) (1938) 425–436. [19] M. Dubois, K.A. Gilles, J.K. Hamilton, P. Rebers, F. Smith, Colorimetric method for determination of sugars and related substances, Anal. Chem. 28 (3) (1956) 350–356. [20] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1) (1976) 248–254.
921
[21] N. Blumenkrantz, G. Asboe-Hansen, New method for quantitative determination of uronic acids, Anal. Biochem. 54 (2) (1973) 484–489. [22] F. Huang, Y. Liu, R. Zhang, L. Dong, Y. Yi, Y. Deng, Z. Wei, G. Wang, M. Zhang, Chemical and rheological properties of polysaccharides from litchi pulp, Int. J. Biol. Macromol. 112 (2018) 968–975. [23] Huang, Y. Guo, R. Zhang, Y. Yi, Y. Deng, D. Su, M. Zhang, Effects of drying methods on physicochemical and immunomodulatory properties of polysaccharide-protein complexes from litchi pulp, Molecules 19(8) (2014) 12760–12776. [24] W. Zhang, Biochemical Technology of Carbohydrate Complexes, Zhejiang University Press, Hangzhou, 1994. [25] S. Akbari-Alavijeh, S. Soleimanian-Zad, M. Sheikh-Zeinoddin, S. Hashmi, Pistachio hull water-soluble polysaccharides as a novel prebiotic agent, Int. J. Biol. Macromol. 107 (2018) 808–816. [26] A. Septembre-Malaterre, F. Remize, P. Poucheret, Fruits and vegetables, as a source of nutritional compounds and phytochemicals: changes in bioactive compounds during lactic fermentation, Food Res. Int. 104 (2018) 86–89. [27] S. Jégou, D.A. Hoang, T. Salmon, P. Williams, S. Oluwa, C. Vrigneau, T. Doco, R. Marchal, Effect of grape juice press fractioning on polysaccharide and oligosaccharide compositions of Pinot meunier and Chardonnay Champagne base wines, Food Chem. 232 (2017) 49–59. [28] Y. Gao, J.U. Fangel, W.G.T. Willats, M.A. Vivier, J.P. Moore, Dissecting the polysaccharide-rich grape cell wall changes during winemaking using combined high-throughput and fractionation methods, Carbohydr. Polym. 133 (2015) 567–577. [29] P. Filannino, R. Di Cagno, M. Gobbetti, Metabolic and functional paths of lactic acid bacteria in plant foods: get out of the labyrinth, Curr. Opin. Biotechnol. 49 (2018) 64–72. [30] S.A. Hughes, P.R. Shewry, L. Li, G.R. Gibson, M.L. Sanz, R.A. Rastall, In vitro fermentation by human fecal microflora of wheat arabinoxylans, J. Agric. Food Chem. 55 (11) (2007) 4589–4595. [31] A. Fessard, E. Bourdon, B. Payet, F. Remize, Identification, stress tolerance, and antioxidant activity of lactic acid bacteria isolated from tropically grown fruits and leaves, Can. J. Microbiol. 62 (7) (2016) 550–561. [32] Q. Liu, X. Cao, X. Zhuang, W. Han, W. Guo, J. Xiong, X. Zhang, Rice bran polysaccharides and oligosaccharides modified by Grifola frondosa fermentation: antioxidant activities and effects on the production of NO, Food Chem. 223 (2017) 49–53. [33] S. Lin, L. Wen, B. Yang, G. Jiang, J. Shi, F. Chen, Y. Jiang, Improved growth of lactobacillus bulgaricus and Streptococcus thermophilus as well as increased antioxidant activity by biotransforming litchi pericarp polysaccharide with Aspergillus awamori, Biomed. Res. Int. 2013 (2013) 1–7. [34] H. Liu, Y. Jiang, H. Yang, B. Yang, Structure characteristics of an acidic polysaccharide purified from banana (Musa nana Lour.) pulp and its enzymatic degradation, Int. J. Biol. Macromol. 101 (2017) 299–303. [35] M. Calderon Santoyo, G. Loiseau, R. Rodriguez Sanoja, J.P. Guyot, Study of starch fermentation at low pH by Lactobacillus fermentum Ogi E1 reveals uncoupling between growth and α-amylase production at pH 4.0, Int. J. Food Microbiol. 80 (1) (2003) 77–87. [36] P. Petrova, K. Petrov, Prebiotic–probiotic relationship: the genetic fundamentals of polysaccharides conversion by Bifidobacterium and Lactobacillus genera, Food Bioconversion 2017, pp. 237–278. [37] Y.J. Goh, T.R. Klaenhammer, Genetic mechanisms of prebiotic oligosaccharide metabolism in probiotic microbes, Annu. Rev. Food Sci. Technol. 6 (2015) 137–156. [38] H. Zeng, P. Chen, C. Chen, C. Huang, S. Lin, B. Zheng, Y. Zhang, Structural properties and prebiotic activities of fractionated lotus seed resistant starches, Food Chem. 251 (2018) 33–40. [39] G.T. Macfarlane, H. Steed, S. Macfarlane, Bacterial metabolism and health-related effects of galacto-oligosaccharides and other prebiotics, J. Appl. Microbiol. 104 (2) (2008) 305–344. [40] S. He, X. Wang, Y. Zhang, J. Wang, H. Sun, J. Wang, X. Cao, Y. Ye, Isolation and prebiotic activity of water-soluble polysaccharides fractions from the bamboo shoots (Phyllostachys praecox), Carbohydr. Polym. 151 (2016) 295–304.
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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Dynamic variation in biochemical properties and prebiotic activities of polysaccharides from longan pulp during fermentation process Fei Huang a, Ruiyue Hong a, Ruifen Zhang a, Lihong Dong a, Yajuan Bai a, Lei Liu a, Xuchao Jia a, Guangjin Wang b, Mingwei Zhang a,⁎ a Sericultural & Agri-Food Research Institute Guangdong Academy of Agricultural Sciences/Key Laboratory of Functional Foods, Ministry of Agriculture and Rural Affairs/Guangdong Key Laboratory of Agricultural Products Processing, Guangzhou 510610, PR China b College of Chemistry and Materials Science, Hubei Engineering University, Xiaogan 432000, PR China
a r t i c l e
i n f o
Article history: Received 13 January 2019 Received in revised form 24 March 2019 Accepted 4 April 2019 Available online 05 April 2019 Keywords: Longan polysaccharide Fermentation process Prebiotic activity
a b s t r a c t Lactic acid bacteria fermentation is an important processing technology for fruits and vegetables. Bioactive compounds such as polysaccharides are altered during the fermentation process. Polysaccharides from longan pulp (LPs) were extracted after different fermentation times and their physicochemical and prebiotic properties were investigated, such as longan polysaccharides named LP-0 and LP-12 means they were extracted from longan pulp fermented for 0 and 12 h, respectively. The yield, contents of neutral sugar and uronic acid, molecular weight (Mw), and monosaccharide composition of LPs were significantly changed with different fermentation times. Specially, the yield and uronic acid content of LPs were first increase and then decline. LP-12 contained the smallest Mw (108.71 ± 5.55 kDa) of the tested LPs (p b 0.05). When compared with unfermented LP-0, the glucose molar percentages of fermented LPs declined, while those of rhamnose and galactose increased, except for LP-6. Fermented LPs also exhibited a stronger stimulatory effect on Lactobacillus strain proliferation, with the proliferative effect of LP-12 being the strongest (p b 0.05). These results suggest that lactic acid bacteria fermentation can change the physicochemical properties and enhance the prebiotic activities of polysaccharides from longan pulp. © 2019 Published by Elsevier B.V.
1. Introduction Longan (Dimocarpus longan Lour.) is an edible fruit that is primarily found in subtropical areas. Longan pulp is rich in carbohydrates, protein, fiber, fat, vitamin C, amino acids, minerals, polyphenols, and volatile compounds [1]. Its pulp has a variety of uses, such as promoting healthy blood vessels and blood pressure, supporting a healthy metabolism, preventing memory loss, and relieving insomnia. Previous studies have shown that polysaccharide is the main functional ingredient and is likely tied to the health benefits of longan pulp [2–4]. An increasing number of studies have focused on longan polysaccharides (LP), including the isolation, purification, and characterization of LP [1]. For instance, the crude LP obtained from longan pulp with ultrasonic extraction had potent immune-modulatory effects in S180 tumor mice in vivo [5]. Four LP fractions have shown the immunomodulatory activities in vitro with up regulating lymphocyte proliferation and stimulating macrophage phagocytosis [4]. For the mechanism of immunomodulatory activities of LP fractions, an LP sub-fraction could ⁎ Corresponding author at: Sericultural and Agri-Food Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou 510610, PR China. E-mail address: [email protected] (M. Zhang).
https://doi.org/10.1016/j.ijbiomac.2019.04.032 0141-8130/© 2019 Published by Elsevier B.V.
stimulate macrophage activation via TLR4 and TLR2 receptor activation, followed by activation of p38 MAPK- and NF-κB-dependent signaling pathways [6]. As the biological activities of polysaccharides are closely related to their structure and physicochemical properties, some researchers have tried a variety of approaches to modify polysaccharides to improve their biological activities [7,8]. The modifications used for longan polysaccharides have centered on chemical ones, such as sulfation [9], acetylation, carboxymethylation [10] and phosphorylation [11]. Chemical modifications are able to change polysaccharide structures by introducing substituent groups, thereby affecting their respective bioactivities. However, it usually requires organic solvents, strong acids, or strong alkali reagents. These constraints have largely limited their application in actual production [7]. As such, other modification methods—such as biotransformation undertaken by microorganisms or enzymatic modification—have received increasing attention due to their high specificity, high efficiency, and few side effects [12]. Lactic acid bacteria fermentation is an important processing technology for fruits and vegetables, as it can keep and/or enhance the safety, nutrition, and shelf life properties of fruits and vegetables. The current research on the fermentation of fruits and vegetables by lactic acid bacteria has mainly focused on strain selection, optimization of strain
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culture conditions, and subsequent changes to the quality of fruits and vegetables [13]. Bioconversion of bioactive compounds caused by fermentation has also received some attention, such as phenolic compounds flavonoids, and γ-aminobutyric acid [14]. In our previous study, we found lactic acid bacteria fermentation could increase the amount of free and total phenolic contents, amino acids and reduce the contents of free amino acids with bitter taste (phenylalanine, tyrosine and leucine) for longan pulp [15]. However, there is little study on the effect of lactic acid bacteria fermentation on the polysaccharides from fruit pulp. Given this gap in our understanding, we used Lactobacillus fermentum to ferment longan pulp and compared unfermented and fermented longan pulp polysaccharides. It was that the fermentation treatment has profound effect on the immunomodulatory and prebiotic activities of longan pulp [16], but the change of molecular weight, neutral sugar, uronic acid and monosaccharide composition of longan pulp polysaccharide during the fermentation process have not yet been investigated. In addition, the dynamic variations of bioactivities of longan polysaccharides during fermentation process are unknown. Therefore, we continued using Lactobacillus fermentum to ferment longan pulp, and analyzed dynamic variation in physicochemical and prebiotic activities of polysaccharides from longan pulp with different fermentation degrees. 2. Materials and methods 2.1. Materials and chemicals 2.1.1. Chemicals and reagents Standard dextrans (including T-4: molecular mass, 4 × 103 Da, T-10: 1 × 104 Da, T-40: 4 × 104 Da, T-70: 7 × 104 Da, T-500: 5 × 105 Da, and T2000: 2 × 106 Da), rhamnose, arabinose, xylose, mannose, glucose, and galactose were all purchased from Sigma Chemical Co. (St. Louis, MO, USA). Man-Rogosa-Sharpe (MRS) was purchased from Guangdong Huankai Microbial Technology Co., Ltd. (Guangzhou, China). Fructooligosaccharides (DP 2-9) was purchased from Shanghai Ruiyong Biotechnology Co., Ltd. (Shanghai, China). All other chemicals used were of analytical grade and purchased from Guangzhou Qiyun Biological Co., Ltd. (Guangzhou, China). 2.1.2. Bacterial strains Bacterial strains included Lactobacillus fermentum GIM 1.191, Lactobacillus acidophilus GIM 1.731, Lactobacillus plantarum GIM 1.380, and Lactobacillus bulgaricus, GIM 1.189 were purchased from the Guangdong Provincial Microbial Culture Collection Center (Guangzhou, China). Strains were stored in MRS broth containing 10% glycerol at −80 °C until later use. Prior to all assays, bacteria were activated according to previously published methods [17]. 2.2. Logan pulp fermentation Fresh longan (cv. Chu-liang) fruits were provided by the Pomology Research Institute of the Guangdong Academy of Agricultural Sciences (Guangzhou, China). Fresh longan fruits were hot air-dried with an air flow velocity of 2.0 m/s at 70 °C for 72 h until the moisture content was 20 ± 2%. Dried longan was peeled and the pulp portion was homogenized with water (1:7 w/v) and the pH was adjusted to 5.8 ± 0.2. The resulting longan juice was then sterilized for 20 min at 121 °C. Fermentation experiments were conducted in aluminum foil sealed Erlenmeyer flasks, each containing 100 mL of pasteurized longan juice without any supplementary nutrients. Inoculum (1 mL) containing 7.0 log cfu/mL of activated Lactobacillus fermentum was added to each of the Erlenmeyer flasks. Fermentation was then allowed to proceed at 37 °C for 72 h. Samples were taken at 0, 6, 12, 24, 48 and 72 h for viable cell count, pH, and polysaccharides extraction as described below. Three duplicate samples were taken at each fermentation time point.
Cell viable counts of the fermented longan juice were obtained as follows: A sample of the culture (1 mL) was transferred to sterile test tubes and serially diluted with sterile 1% (w/v) peptone solution. Then, 100 μL of each dilution was spread onto MRS agar-containing plates and then subjected to anaerobic incubation at 37 °C for 48 h. Bacteria were counted and expressed as log cfu/mL. The pH of the fermented longan juice was simultaneously measured during all cell counts using a standard pH meter (pH S-3C, Shanghai Precise Scientific Instrument Corp., China). 2.3. Longan polysaccharide extraction Fermented longan juice was boiled for 10 min at 100 °C to kill the live Lactobacillus. Sterilized products were then collected by adding distilled water to 1 L and holding the mixture at 80 °C for 4 h prior to filtering. This extraction was repeated, and the filtrates were collected, combined, and then vacuum concentrated at 65 °C. The extracted solution was then subjected to the Sevag method for 8 times to remove protein [18]. Four volumes of ethanol were added and precipitation was allowed to occur for 24 h at 4 °C. The resulting precipitate was collected using centrifugation (3000 ×g, 10 min), washed with ethanol, and lyophilized to obtain the final longan polysaccharides (LPs). The polysaccharides extracted from different time points during the longan pulp fermentation were named as follows: LP-0, LP-6, LP-12, LP24, LP-48 and LP-72, respectively. 2.4. Physicochemical properties of LPs 2.4.1. Chemical characteristics Neutral polysaccharide content was determined using the phenolsulfuric acid method according to previously published methods [19]. Glucose was used as the standard. Protein concentration was determined using the Bradford assay with a bovine serum albumin standard curve [20]. Uronic acid content was determined using a modified mhydroxydiphenyl method with galacturonic acid standards [21]. The monosaccharide composition of LPs was determined using gas chromatography–mass spectrometry (GC–MS) according to the methods of our previously published study [22]. Briefly, 10 mg of polysaccharide sample was dissolved in 4 moL/L trifluoroacetate (2 mL) and hydrolyzed at 110 °C for 6 h. The hydrolysate was dried under an N2 stream and then dissolved in 1 mL pyridine containing 10 mg hydroxylamine. The sample was then kept at 90 °C for 30 min, after which it was cooled to room temperature. Acetic anhydride (1 mL) was added and allowed to incubate at 90 °C for 30 min. The resulting acetylated hydrolysate was analyzed by GC–MS with an Agilent 6890 GC (Agilent Technologies Co., Ltd., Colorado Springs, CO, USA) equipped with a DB-1 column and an Agilent 5973 MS detector. The temperature program was set as follows: Initial column temperature was 190 °C, temperature was increased to 230 °C at 2 °C/min, held for 2 min, then increased to 240 °C at 5 °C/min, and held for another 2 min. The detector temperature was 290 °C and the vaporizing chamber temperature was set to 260 °C. GC/MSD ChemStation software was used for all analysis. Six monosaccharides (arabinose, mannose, rhamnose, galactose, xylose, and glucose) and inositol were used as external and internal standards respectively to identify polysaccharide composition. Each sample was analyzed in triplicate. 2.4.2. Analysis of molecular weights The average molecular weights of LPs were determined using an advanced polymer chromatography system (APC) equipped with a refractive index detector (RID). The analysis was completed using Acquity APC AQ 45, 125, and 450 columns (4.6 × 150 mm, Waters Corp., USA). The column oven temperature was set at 40 ± 0.2 °C and the column was eluted with H2O at a flow rate of 1.0 mL/min. Standard dextran including T-4 (molecular mass, 4 × 103 Da), T-10 (1 × 104 Da), T-40 (4
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× 104 Da), T-70 (7 × 104 Da), T-500 (5 × 105 Da), and T-2000 (2 × 106 Da) were used as molecular mass markers. 2.4.3. FT-IR spectroscopy According to our previous study [23], 2 mg polysaccharide samples were mixed with 100 mg potassium bromide (KBr) powder and pressed into 1 mm thick pellets for FT-IR measurements. FT-IR spectra were recorded on a Nexus 5DXC FT-IR (Thermo Nicolet, Austin, TX, USA) in the frequency range of 4000–400 cm−1 at 4 cm−1 resolution using 64 scans. 2.5. Prebiotic activity analysis Three Lactobacillus strains—L. acidophilus, L. plantarum, and L. bulgaricus—were used for investigating the prebiotic activity of the isolated LPs in vitro. Carbohydrate-free MRS broth supplemented with 0.05% (w/v) L-cysteine was used as the basal medium and as the blank control; fructooligosaccharide (FOS) was the positive control. All LP and FOS sugars were filter-sterilized and separately added to the basal medium to obtain a series of final concentrations (0.5, 1.0, 1.5, 2.0, and 3.0% [w/v]). The activated Lactobacillus strains were added to the medium (final concentration: 1 × 106 cfu/mL) and then incubated at 37 °C for 48 h under anaerobic conditions (85% N2, 10% CO2 and 5% H2). The numbers of Lactobacillus strains in the samples were then counted after incubating for 0 and 48 h. The pH values of all samples were simultaneously measured using a pH meter (pH S-3C, Shanghai Precise Scientific Instrument Corp., China). Bacterial counts were performed as previously described and bacteria were expressed as log cfu/mL. The increase in bacterial number between 0 and 48 h was calculated according to the following formula: Increase of bacterial number = log B − log A; Where A is the bacterial number at 0 h (cfu/ mL) and B is the bacterial number after 48 h incubation (cfu/mL). The fold-increase bacterial number which was relative to control group was presented as the result. 2.6. Statistical analyses All data are expressed as mean ± standard deviation (SD). Statistical significance was evaluated using a one-way ANOVA followed by the Student-Newman-Keuls test using SPSS 19.0 software. A p-value of 0.05 was chosen as the threshold for significance. 3. Results 3.1. Longan pulp fermentation The effect of fermentation time on bacterial number and pH is shown in Fig. 1. The bacterial number in the longan juice displayed an increasing trend during the fermentation process. The bacterial number started at 5.56 log cfu/mL, showed a slight increase until 6 h, and then rapidly increased to 9.12 log cfu/mL at 12 h. Bacterial number then maintained a slowly increasing trend until 48 h (9.68 log cfu/mL), after which it gradually decreased from 60 to 72 h (Fig. 1A). This increase in bacterial number indicated that Lactobacillus fermentum utilized the longan juice to grow. Correspondingly, the pH value of the longan juice showed a decreasing trend throughout the fermentation process. More specifically, pH started at 5.6, dropped slightly, and then quickly fell to 4.37 at 12 h. The pH then continued to slowly decline until 3.68 (72 h) (Fig. 1B). This change in pH also revealed that the carbohydrates contained in the longan juice were successfully metabolized to produce acids by Lactobacillus fermentum. 3.2. Preliminary characterization of LPs The yield as well as neutral sugar, uronic acid, and protein contents of the isolated LPs are summarized in Table 1. The polysaccharide yield increase first with 6 h fermentation and then decrease with the
Fig. 1. Effects of fermentation time on the bacteria number (A) and pH (B) of longan pulp at 0, 6, 12, 24, 48 and 72 h. The bacteria number of fermented longan pulp was measured with cell viable counts while its pH was measured using a standard pH meter.
fermentation time increase. LP-6 had the highest yield among all samples (p b 0.05). As for the neutral sugars, LP-72 had the highest content, while LP-24 had the lowest (p b 0.05). The trend of uronic acid content in LPs was similar with that of yields, which was increase first and then decrease. The polysaccharide protein content from non-fermented longan pulp was less than that from fermented samples (p b 0.05). The protein content of polysaccharides from fermented sample did not change with fermentation time. The molecular weights of all LPs were determined using APC and size exclusion columns. As shown in Table 2, LPs without fermentation had the largest average molecular weight (Mw), with LP-12 having the smallest Mw among the analyzed samples. The Mw of LP decreased
Table 1 The physicochemical properties of LPs. Sample
Extraction yield (%)
Neutral sugar (%)
Uronic acid (%)
Protein (%)
LP-0⁎ LP-6 LP-12 LP-24⁎ LP-48 LP-60 LP-72
2.40 ± 0.36c 2.65 ± 0.18cd 2.16 ± 0.09bc 1.49 ± 0.36ab 1.09 ± 0.15a 1.06 ± 0.02a 1.03 ± 0.13a
43.19 ± 1.43bc 43.01 ± 0.84bc 41.89 ± 0.70ab 41.50 ± 0.15a 43.40 ± 0.60c 43.74 ± 0.17c 46.81 ± 0.17d
35.23 ± 8.93cd 38.31 ± 1.18d 31.52 ± 1.36c 26.81 ± 0.25b 25.62 ± 1.25ab 23.52 ± 1.16a 22.63 ± 2.23a
0.88 ± 0.55a 1.56 ± 0.32b 1.50 ± 0.46b 1.37 ± 0.82b 1.52 ± 0.39b 1.51 ± 0.25b 1.57 ± 0.36b
Different lowercase letters represent significant differences among samples (p b 0.05). ⁎ Represents the data obtained from our previous work (Huang et al. [16]).
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Table 2 The molecular weights and distributions of LPs. Sample
LP-0 LP-6 LP-12 LP-24 LP-48 LP-60 LP-72
Mw (kDa)
Molecular weight distribution (%) 0–50 kDa
50–100 kDa
N100 kDa
61.34 65.54 84.09 80.69 70.11 62.48 55.76
10.09 5.49 3.13 2.18 13.26 18.31 24.45
28.57 28.97 12.78 17.13 16.63 19.21 19.79
221.63 ± 2.41d 152.98 ± 1.06c 108.71 ± 5.55a 109.62 ± 10.66a 136.83 ± 1.56b 139.42 ± 2.31b 152.31 ± 6.37c
Different lowercase letters represent significant differences among samples (p b 0.05).
from 0 h to 12 h and then increased as fermentation time increased from 12 h to 72 h. Of those with a Mw between 0 and 50 kDa, LPs were first increase and then decline and LP-12 had the highest percentage (84.09%); however, its percentage was the least of those with Mw N 100 kDa. For the fraction in the Mw range of 50–100 kDa, LPs were first decline and then increase. In the higher range of N100 kDa, LP-6 had the highest percentage (28.97%) and it had the largest Mw of all fermented LPs, but still smaller than that of LP-0 (p b 0.05). Our monosaccharide analysis revealed that all LPs were heteropolysaccharides and composed of different ratios of rhamnose, arabinose, xylose, mannose, glucose, and/or galactose (Table 3), which is consistent with our previous study [16]. The major monosaccharides in LP-0 were arabinose, galactose, and glucose. However, rhamnose became one of the main constituent monosaccharides when longan juice was fermented for longer than 60 h. When compared with nonfermented LP-0, the relative molar percentages of rhamnose increased in LPs that had been fermented for longer than 12 h. With the exception of LP-6, the relative molar percentage of glucose in the other fermented LPs was lower than that of the non-fermented LP-0. However, the percentage composition of galactose showed an opposite trend. The relative molar percentage of arabinose was the highest in all samples, with a minimum of 36.26% in LP-6 and a maximum of 48.55% in LP-24. The FT-IR images of LPs are shown in Fig. 2. All of LPs showed characteristic polysaccharide bands, including the hydroxyl group bands in the ranges of 3600–3200 cm−1 and 1075–1010 cm−1 and the alkyl group bands at approximately 2926, 2850 and 1458 cm−1 [16]. The absorption bands between 1100 and 1000 cm−1 were attributed to characteristic C\\O\\C glycosidic bond vibrations and ring vibrations overlapped with stretching vibrations of the side group C\\O\\H link bonds [24]. According to Fig. 2, the absorbance band at approximately 1550 cm−1 which was referred to the bending vibration of N\\H [25], appeared in LP-48, LP-60 and LP-72, but not in other samples. This indicated the protein existed in LP-48, LP-60 and LP-72, but did not exist or existed with very low content. This result is consistent with the protein results in Table 1 that LP-48, LP-60 and LP-72 had more protein than other samples.
Fig. 2. FT-IR spectra of LPs. The FTIR spectra of LPs were determined using a Fouriertransform infrared spectrophotometer over the frequency range of 4000–400 cm−1.
3.3. LP prebiotic activity The increased bacteria numbers of Lactobacillus strains after 48 h incubation with either LPs or FOS as their sole carbon source are shown in Fig. 3. For L. acidophilus, the bacterial populations were concentrationdependent in the range of 0.5–3.0%. And LPs stimulated the highest increased-fold bacterial populations at 3.0% of samples. Moreover, bacterial populations applied with fermented longan polysaccharides showed much higher than that of unfermented group. LP-12 had a significantly more beneficial effect on L. acidophilus proliferation than the other LPs at a similar concentration range (0.5–3.0%) (p b 0.05). As for L. bulgaricus, the bacterial number was greater when growing in any of the LPs or FOS when compared with that of the blank control. This indicated that all samples could stimulate the proliferation of that strain. Although there was no regularity in the relationship between bacterial population and polysaccharide concentration, FOS had the most beneficial effect on L. bulgaricus proliferation during the tested concentrations. Moreover, LP-12 had more prebiotic activity than any other LPs across the concentration range 0.5–1.5% (p b 0.05). The increased bacterial populations of L. plantarum stimulated with LPs and FOS was shown in Fig. 3C. Compared with blank control, all LPs and FOS stimulated more bacterial numbers. This indicated that all samples could stimulate the proliferation of that strain. When compared with non-fermented LP-0, fermented LPs showed a greater benefit on L. plantarum proliferation across the concentration range 1.0–3.0%; moreover, this was weaker than FOS. The increased bacterial
Table 3 The monosaccharide compositions of LPs. Monosaccharide composition (%)
Rhamnose
Arabinose
Xylose
Mannose
Glucose
Galactose
LP-0⁎ LP-6 LP-12 LP-24⁎ LP-48 LP-60 LP-72
8.790 ± 0.012b 7.193 ± 0.133a 7.187 ± 0.046a 9.294 ± 0.075c 9.769 ± 0.032d 11.915 ± 0.019f 11.100 ± 0.007e
43.628 ± 0.639c 36.263 ± 0.303a 43.472 ± 0.066c 48.549 ± 0.119f 47.460 ± 0.012e 45.235 ± 0.026d 41.308 ± 0.201b
1.529 ± 0.122ab 1.518 ± 0.316ab 2.303 ± 0.031d 1.831 ± 0.110bc 1.954 ± 0.050c 1.695 ± 0.087abc 1.462 ± 0.049a
2.347 ± 0.027a 2.824 ± 0.153c 3.590 ± 0.030f 3.280 ± 0.073e 3.118 ± 0.037de 2.917 ± 0.001cd 2.591 ± 0.167b
23.388 ± 0.442e 33.920 ± 0.002f 22.623 ± 0.029d 14.331 ± 0.072b 12.282 ± 0.131a 12.074 ± 0.061a 20.031 ± 0.010c
20.317 ± 0.334b 18.282 ± 0.030a 20.825 ± 0.007c 22.715 ± 0.066d 25.417 ± 0.000f 26.164 ± 0.154g 23.508 ± 0.086e
Different lowercase letters represent significant differences among samples (p b 0.05). ⁎ Represents the data obtained from our previous work (Huang et al. [16]).
F. Huang et al. / International Journal of Biological Macromolecules 132 (2019) 915–921
Fig. 3. Effects of LPs on the increased bacteria number after 48 h incubation at different concentrations (0, 0.5, 1.0, 1.5, 2.0 and 3.0%). (A) L. acidophilus, (B) L. bulgavicus, and (C) L. plantarum. The increased bacteria number was expressed as the fold of control group.
populations of L. plantarum cultured with LP-12 was greater than other LPs at the same concentration from 0.5% to 3% (p b 0.05), except 1%. Polysaccharide prebiotic activity depends on its hydrolysis catalyzed by glycosidase secreted from probiotics. Polysaccharides are hydrolyzed into monomers and further degraded into short-chain fatty acids and gases when they are the sole carbon source for bacteria. This ultimately results in a significant decrease in pH in the fermented broth. Given this, the effect of LPs on the acidifying activities of probiotics was further investigated. As shown in Fig. 4A, marked pH decreases were observed in L. acidophilus fermented broth after 48 h stimulation with LPs and FOS. Their pH values reached the minimum value observed at 3.0% concentration for all samples. Across the concentration range 0.5–2.0%, pH values of LP12 were the lowest of the LPs, but still larger than those of the
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Fig. 4. Effects of LPs on the pH of probiotics after 48 h incubation at different concentrations (0, 0.5, 1.0, 1.5, 2.0 and 3.0%). (A) L. acidophilus, (B) L. bulgavicus, and (C) L. plantarum. The pH of probiotic medium was measured using a standard pH meter.
positive control FOS. This was counter to the order of probiotic growth numbers when stimulated with polysaccharides (Fig. 3). LPs and FOS resulted in significant decreases of pH values after 48 h incubation with L. bulgaricus, with the exception of LP-0 across a concentration range of 0.5–1.5% (Fig. 4B). The minimum values we observed were obtained at 3.0% concentration and were in the following order: LP-60 N LP-48 ≈ LP-72 N LP-0 ≈ LP-12 N LP-6 ≈ LP-24 N FOS. The pH values of L. bulgaricus when incubated with LP-12 were lower than those of the other LPs across the 0.5%–2.0% concentration range (p b 0.05). This observation was consistent with the increased number of L. bulgaricus observed when incubated with LPs. As for L. plantarum, when compared with the blank control, the pH value of the media was significantly decreased after 48 h incubation with LPs and FOS (p b 0.05) (Fig. 4C). Moreover, the pH values of L. plantarum incubated with LP-12 and LP-24 were similar across the concentration range 1.0–3.0% and were both smaller than those of other LPs. However, they were still larger than those of the positive control FOS (p b 0.05).
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4. Discussion
4.2. Effect of fermentation on the prebiotic activity of LPs
4.1. Effect of fermentation on the physicochemical properties of LPs
Three Lactobacillus strains—L. acidophilus, L. plantarum, and L. bulgaricus—have found extensive use in the food industry and their probiotic functions in human health have been generally accepted [36]. As such, these three strains were used to evaluate the prebiotic activities of LPs in this study. Differences in stimulatory effects on the proliferation of Lactobacillus strains by LPs (Fig. 3) are related to both polysaccharide and probiotic properties. Different probiotics have different abilities to secrete enzymes, which causes differences in their ability to utilize available polysaccharides [37]. LPs were able to significantly stimulate L. acidophilus proliferation and exhibited a dose-dependent effect across the tested concentration range. The highest fold of increased L. acidophilus number was 5.18 while that of L. bulgavicus was 1.62 at the same concentration range (Fig. 3). The chemical and physical properties of polysaccharides can affect their utilization by probiotics and include monosaccharide composition, Mw, and glycosidic linkages. In present study, the polysaccharide degradation as indicated by decrease in Mw after fermentation treatment could result from the breaking of glycosidic bonds (Table 2). Thus, the chemical-structural properties of the polysaccharide might be altered. The fermented LPs were better utilized by bacteria than the non-fermented polysaccharides. Mw is another key factor influencing the prebiotic activity of polysaccharides. Generally, polysaccharides with a lower molecular weight have better access to probiotics. With this in mind, the proliferative stimulatory effect of LP-12 was the strongest, while LP-0 was the weakest (Fig. 3). This observation is presumably because their molecular weights were the smallest and the largest, respectively (Table 3). This conclusion coincided with the results of previous work examining the polysaccharides from lotus seed resistant starches [38]. Moreover, monosaccharide composition is also an important factor that influences the prebiotic activities of polysaccharides. Previous studies have shown that polysaccharides exhibit better prebiotic activities when they are composed of glucose, xylose, and galactose [39]. These include polysaccharides from bamboo shoots [40] and rapeseed [17]. LPs contained the aforementioned monosaccharides and promoted the growth of probiotics; moreover, the different molar ratios of the monosaccharides in LPs also likely contributed to their different prebiotic effects. However, it is likely that there are other mechanisms at work, which will need to be identified and studied in the future.
We first sought to explore the effect of lactic acid bacteria fermentation on longan pulp polysaccharides. To do this, we chose the commercial strain Lactobacillus fermentum, which is commonly used in fermented fruit juices, to ferment longan pulp for 72 h. The obtained polysaccharides were extracted from the longan pulp at different time points (0, 6, 12, 24, 48 and 72 h). The yield as well as neutral sugar and uronic acid content, Mw, and monosaccharide composition showed marked differences. Comparatively, protein content across these different fermentation time points showed no significant differences (Tables 1–3). Lactic acid bacteria or other microorganisms can affect polysaccharide composition by secreting different carbohydrate enzymes. These enzymes could actively assist the break down the cell walls of berry tissues, favoring the release of sugars. They produce hydrolytic enzymes to degrade the insoluble pecto-cellulosic cell wall of fruit into soluble polysaccharides, resulting in increased yield of water-soluble polysaccharides [26] as well as changes to Mw and monosaccharide composition of the extracted polysaccharides [27]. That is the reason for the increase in the yield, uronic acid and protein content of longan polysaccharide with 6 h fermentation. But for molecular weight change, we speculate it may be caused by two factors. For one is the production of pectin and AGPs with cell walls degradation [28], as their Mw is smaller than the polysaccharide obtained from longan pulp without treatment. The other is the cleavage of polysaccharide with enzymes, leading to decreases in observed Mw. So the average molecular weight of fermented longan pulp polysaccharides becomes smaller than that of LP-0. In addition, hydrolytic enzymes can also act on the glycosidic chains of watersoluble polysaccharides in the fruit pulp, hydrolyze them into oligosaccharides or monomers, which are then finally metabolized into shortchain fatty acids [29]. For example, the amount of pectin and AGPs in grape fermentation juice increased at the early stage of fermentation; however, as fermentation proceeds, these factions reduced [28]. This may be the reason the yield and uronic acid decrease as the fermentation time increase. LPs in the Mw fraction range of 0–50 kDa declined significantly from 12 h to 72 h may be related with bacteria utilization, as bacteria usually preferential use of polysaccharides with small molecular weight, such as wheat arabinoxylans with 66 kDa promoted Eubacteria growth better than those with 354 and 278 kDa [30]. The percentage of small Mw fraction decline and those of the large Mw fraction were relative increase. That may be the reason for the Mw of LP first decrease and then increase. Moreover, some lactic acid bacteria can produce extracellular polysaccharides when they utilize glucose and fructose in fruits [26,31]. Here, in order to more effectively study the effect of fermentation on the physicochemical and prebiotic activities of longan pulp polysaccharides, we chose Lactobacillus fermentum as the fermentation strain since it does not produce any extracellular polysaccharides. The glucose content of rice bran polysaccharides declines significantly with Grifola frondosa fermentation [32] and decreases in the relative percentages of arabinose and glucose in fermented litchi pericarp polysaccharides were also observed with Aspergillus awamori fermentation [33]. Pectinase treatment induces the cleavage of acidic polysaccharide from banana pulp and produces oligogalacturonic acids, leading to decreases in observed Mw [34]. Here, the change of glucose relative molar percentage, uronic acid content and Mw in LPs that had been fermented for longer than 12 h were consistent with previous observations [32–34]. Lactobacillus fermentum secretes rich extracellular enzymes during fermentation, including amylase, α-glucosidase, cellulose, lactate dehydrogenases, and peptidases [14,35]. It is likely that these enzymes are responsible for the changes in uronic acid content, monosaccharide composition, and Mw distribution.
5. Conclusions The present study sought to investigate the effect of lactic acid bacteria fermentation on the physicochemical and prebiotic activities of LP. The results showed that the yield as well as the content of neutral sugar and uronic acid, Mw, and monosaccharide composition of LPs changed with fermentation. Our prebiotic activity analysis revealed that the fermented polysaccharides had prebiotic activities when compared with unfermented polysaccharides. Moreover, that LP-12 exhibited the strongest prebiotic activity of the tested fermented polysaccharides. Overall, these results suggest that lactic acid bacteria fermentation is an effective method to modify longan polysaccharide and improve its prebiotic activity. Acknowledgements This work was supported by National Key Research Project of China (2017YFC1601002, 2018YFC1602105), National Natural Science Foundation of China (31801498), Guangdong Provincial Science and Technology Project (2017A030313163, 2016A030310321, 2017B090907022, 2018A050506050), and Scientific Research Program of Guangzhou (201804020064, 201704020039, 201803010079, 201803020014). This work was supported by China Scholarship Council (No. 201708440135).
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References [1] B. Yang, Y. Jiang, J. Shi, F. Chen, M. Ashraf, Extraction and pharmacological properties of bioactive compounds from longan (Dimocarpus longan Lour.) fruit — a review, Food Res. Int. 44 (7) (2011) 1837–1842. [2] Y. Bai, L. Liu, R. Zhang, F. Huang, Y. Deng, M. Zhang, Ultrahigh pressure-assisted enzymatic extraction maximizes the yield of longan pulp polysaccharides and their acetylcholinesterase inhibitory activity in vitro, Int. J. Biol. Macromol. 96 (2017) 214–222. [3] J. Zhang, G. Yang, Y. Wen, S. Liu, C. Li, R. Yang, W. Li, Intestinal microbiota are involved in the immunomodulatory activities of longan polysaccharide, Mol. Nutr. Food Res. 61 (11) (2017), 1700466. [4] Y. Yi, M.-W. Zhang, S.-T. Liao, R.-F. Zhang, Y.-Y. Deng, Z.-C. Wei, X.-J. Tang, Y. Zhang, Structural features and immunomodulatory activities of polysaccharides of longan pulp, Carbohydr. Polym. 87 (1) (2012) 636–643. [5] K. Zhong, Q. Wang, Y. He, X. He, Evaluation of radicals scavenging, immunitymodulatory and antitumor activities of longan polysaccharides with ultrasonic extraction on in S180 tumor mice models, Int. J. Biol. Macromol. 47 (3) (2010) 356–360. [6] Y. Yi, Hongxun Wang, Ruifen Zhang, Ting Min, Fei Huang, L. Liu, M. Zhang, Characterization of polysaccharide from longan pulp as the macrophage stimulator, RSC Adv. 5 (2015) 97163–97170. [7] S. Li, Q. Xiong, X. Lai, X. Li, M. Wan, J. Zhang, Y. Yan, M. Cao, L. Lu, J. Guan, Molecular modification of polysaccharides and resulting bioactivities, Compr. Rev. Food Sci. F. 15 (2) (2016) 237–250. [8] S.S. Ferreira, C.P. Passos, P. Madureira, M. Vilanova, M.A. Coimbra, Structure– function relationships of immunostimulatory polysaccharides: a review, Carbohydr. Polym. 132 (2015) 378–396. [9] J. Jiang, F.-Y. Meng, Z. He, Y.-L. Ning, X.-H. Li, H. Song, J. Wang, R. Zhou, Sulfated modification of longan polysaccharide and its immunomodulatory and antitumor activity in vitro, Int. J. Biol. Macromol. 67 (2014) 323–329. [10] J. Wang, Study on the Prepatation, Antioxidant and Immunomodulatory Activities of Acetylated and Carboxymethylated Polysaccahrides From Dimocarpus longan Pulp, Guangxi Medical University, Nanning, China, 2013. [11] Y. Ning, Study on the Prepatation, Immunomodulatory and Antioxidant Activities of Phosphorylated and Acid Hydrolyzed Polysaccahrides From Longan Pulp, Guangxi Medical University, Nanning, China, 2010. [12] P. Chen, Q. You, X. Li, Q. Chang, Y. Zhang, B. Zheng, X. Hu, H. Zeng, Polysaccharide fractions from Fortunella margarita affect proliferation of Bifidobacterium adolescentis ATCC 15703 and undergo structural changes following fermentation, Int. J. Biol. Macromol. 123 (2019) 1070–1078. [13] R. Di Cagno, R. Coda, M. De Angelis, M. Gobbetti, Exploitation of vegetables and fruits through lactic acid fermentation, Food Microbiol. 33 (1) (2013) 1–10. [14] S.J. Hur, S.Y. Lee, Y.-C. Kim, I. Choi, G.-B. Kim, Effect of fermentation on the antioxidant activity in plant-based foods, Food Chem. 160 (2014) 346–356. [15] S.A. Khan, L. Liu, T. Lai, R. Zhang, Z. Wei, J. Xiao, Y. Deng, M. Zhang, Phenolic profile, free amino acids composition and antioxidant potential of dried longan fermented by lactic acid bacteria, J. Food Sci. Technol. 55 (12) (2018) 4782–4791. [16] F. Huang, R. Hong, R. Zhang, Y. Yi, L. Dong, L. Liu, X. Jia, Y. Ma, M. Zhang, Physicochemical and biological properties of longan pulp polysaccharides modified by Lactobacillus fermentum fermentation, Int. J. Biol. Macromol. 125 (2019) 232–237. [17] X. Wang, M. Huang, F. Yang, H. Sun, X. Zhou, Y. Guo, X. Wang, M. Zhang, Rapeseed polysaccharides as prebiotics on growth and acidifying activity of probiotics in vitro, Carbohydr. Polym. 125 (1) (2015) 232–240. [18] M. Sevag, D.B. Lackman, J. Smolens, The isolation of the components of streptococcal nucleoproteins in serologically active form, J. Biol. Chem. 124 (2) (1938) 425–436. [19] M. Dubois, K.A. Gilles, J.K. Hamilton, P. Rebers, F. Smith, Colorimetric method for determination of sugars and related substances, Anal. Chem. 28 (3) (1956) 350–356. [20] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1) (1976) 248–254.
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[21] N. Blumenkrantz, G. Asboe-Hansen, New method for quantitative determination of uronic acids, Anal. Biochem. 54 (2) (1973) 484–489. [22] F. Huang, Y. Liu, R. Zhang, L. Dong, Y. Yi, Y. Deng, Z. Wei, G. Wang, M. Zhang, Chemical and rheological properties of polysaccharides from litchi pulp, Int. J. Biol. Macromol. 112 (2018) 968–975. [23] Huang, Y. Guo, R. Zhang, Y. Yi, Y. Deng, D. Su, M. Zhang, Effects of drying methods on physicochemical and immunomodulatory properties of polysaccharide-protein complexes from litchi pulp, Molecules 19(8) (2014) 12760–12776. [24] W. Zhang, Biochemical Technology of Carbohydrate Complexes, Zhejiang University Press, Hangzhou, 1994. [25] S. Akbari-Alavijeh, S. Soleimanian-Zad, M. Sheikh-Zeinoddin, S. Hashmi, Pistachio hull water-soluble polysaccharides as a novel prebiotic agent, Int. J. Biol. Macromol. 107 (2018) 808–816. [26] A. Septembre-Malaterre, F. Remize, P. Poucheret, Fruits and vegetables, as a source of nutritional compounds and phytochemicals: changes in bioactive compounds during lactic fermentation, Food Res. Int. 104 (2018) 86–89. [27] S. Jégou, D.A. Hoang, T. Salmon, P. Williams, S. Oluwa, C. Vrigneau, T. Doco, R. Marchal, Effect of grape juice press fractioning on polysaccharide and oligosaccharide compositions of Pinot meunier and Chardonnay Champagne base wines, Food Chem. 232 (2017) 49–59. [28] Y. Gao, J.U. Fangel, W.G.T. Willats, M.A. Vivier, J.P. Moore, Dissecting the polysaccharide-rich grape cell wall changes during winemaking using combined high-throughput and fractionation methods, Carbohydr. Polym. 133 (2015) 567–577. [29] P. Filannino, R. Di Cagno, M. Gobbetti, Metabolic and functional paths of lactic acid bacteria in plant foods: get out of the labyrinth, Curr. Opin. Biotechnol. 49 (2018) 64–72. [30] S.A. Hughes, P.R. Shewry, L. Li, G.R. Gibson, M.L. Sanz, R.A. Rastall, In vitro fermentation by human fecal microflora of wheat arabinoxylans, J. Agric. Food Chem. 55 (11) (2007) 4589–4595. [31] A. Fessard, E. Bourdon, B. Payet, F. Remize, Identification, stress tolerance, and antioxidant activity of lactic acid bacteria isolated from tropically grown fruits and leaves, Can. J. Microbiol. 62 (7) (2016) 550–561. [32] Q. Liu, X. Cao, X. Zhuang, W. Han, W. Guo, J. Xiong, X. Zhang, Rice bran polysaccharides and oligosaccharides modified by Grifola frondosa fermentation: antioxidant activities and effects on the production of NO, Food Chem. 223 (2017) 49–53. [33] S. Lin, L. Wen, B. Yang, G. Jiang, J. Shi, F. Chen, Y. Jiang, Improved growth of lactobacillus bulgaricus and Streptococcus thermophilus as well as increased antioxidant activity by biotransforming litchi pericarp polysaccharide with Aspergillus awamori, Biomed. Res. Int. 2013 (2013) 1–7. [34] H. Liu, Y. Jiang, H. Yang, B. Yang, Structure characteristics of an acidic polysaccharide purified from banana (Musa nana Lour.) pulp and its enzymatic degradation, Int. J. Biol. Macromol. 101 (2017) 299–303. [35] M. Calderon Santoyo, G. Loiseau, R. Rodriguez Sanoja, J.P. Guyot, Study of starch fermentation at low pH by Lactobacillus fermentum Ogi E1 reveals uncoupling between growth and α-amylase production at pH 4.0, Int. J. Food Microbiol. 80 (1) (2003) 77–87. [36] P. Petrova, K. Petrov, Prebiotic–probiotic relationship: the genetic fundamentals of polysaccharides conversion by Bifidobacterium and Lactobacillus genera, Food Bioconversion 2017, pp. 237–278. [37] Y.J. Goh, T.R. Klaenhammer, Genetic mechanisms of prebiotic oligosaccharide metabolism in probiotic microbes, Annu. Rev. Food Sci. Technol. 6 (2015) 137–156. [38] H. Zeng, P. Chen, C. Chen, C. Huang, S. Lin, B. Zheng, Y. Zhang, Structural properties and prebiotic activities of fractionated lotus seed resistant starches, Food Chem. 251 (2018) 33–40. [39] G.T. Macfarlane, H. Steed, S. Macfarlane, Bacterial metabolism and health-related effects of galacto-oligosaccharides and other prebiotics, J. Appl. Microbiol. 104 (2) (2008) 305–344. [40] S. He, X. Wang, Y. Zhang, J. Wang, H. Sun, J. Wang, X. Cao, Y. Ye, Isolation and prebiotic activity of water-soluble polysaccharides fractions from the bamboo shoots (Phyllostachys praecox), Carbohydr. Polym. 151 (2016) 295–304.