# Optimization, Probiotic Characteristics, and Rheological Properties of Exopolysaccharides from Lactiplantibacillus plantarum MC5

^{*}

## Abstract

**:**

_{700}) of EPS-MC5 were 73.33%, 87.74%, 46.07%, and 1.20, respectively. Furthermore, rheological results showed that the EPS-MC5 had a higher apparent viscosity (3.01 Pa) and shear stress (41.78 Pa), and the viscoelastic modulus (84.02 and 161.02 Pa at the shear frequency of 100 Hz). These results provide a new insight into the application of EPS in human health and functional foods, which could also improve theoretical guidance for the industrial application of EPS.

## 1. Introduction

## 2. Results and Discussion

#### 2.1. Identification of Strain MC5 and Culture Conditions Single Factor Test

#### 2.2. Optimization of Fermentation Conditions for EPS-MC5

^{2}− 68.19B

^{2}− 79.86C

^{2}− 100.95D

^{2}. The regression model was analyzed by variance analysis and reliability analysis, and the results were shown in Table 2.

^{2}and correction coefficient R

^{2}

_{Adj}were 0.9634 and 0.9268, respectively, indicating that the established regression equation had a good degree of fit and could successfully predict the response value. In addition, the coefficient of variation (C.V. = 10.00%) indicated that the experimental results had high precision and reliability. The model data showed that the primary term B, the interaction term BC, A

^{2}, B

^{2}, C

^{2}, and D

^{2}had a very significant impact on the EPS yield of the fermentation broth (p < 0.01), and the primary term C and D had a significant impact on the EPS yield (p < 0.05). To sum up, the order of the significant differences in the influence of the four factors was culture time > culture temperature > initial pH > inoculation size.

#### 2.3. Three-Dimensional Response Surfaces and Count Plots of Variables

#### 2.4. Verification Test of EPS-MC5 Yield

#### 2.5. Isolation and Purification of EPS-MC5

#### 2.6. In Vitro Resisting-Digestion Capacity of EPS-MC5 to α-Amylase and Simulated Gastrointestinal Juices

#### 2.7. In Vitro Antioxidant Activity of EPS-MC5

#### 2.8. Rheological Properties of EPS-MC5

#### 2.8.1. Apparent Viscosity of EPS-MC5

#### 2.8.2. Viscoelastic Properties of EPS-MC5

^{−5}–1.0 Pa [40], which was lower than that of EPS-MC5 in this study. These differences may be due to the different molecular weights, glycosidic bond type, monosaccharide composition, functional groups, and substituents [41].

## 3. Materials and Methods

#### 3.1. Materials

_{2}HPO

_{4}(2 g/L), sodium acetate (5 g/L), diammonium hydrogen citrate (2 g/L), MgSO

_{4}(0.2 g/L), MnSO

_{4}(0.08 g/L), and agar (15 g/L). It was sterilized at 121 °C for 20 min.

#### 3.2. Isolation and Determination of EPS-MC5

_{2}SO

_{4}, shaken for 10 min, and the absorbance was determined at 490 nm.

#### 3.3. Purification of EPS-MC5

#### 3.4. Single Factor Experiment of Culture Conditions for EPS Production from Lp. plantarum MC5

#### 3.4.1. Effects of Inoculation Size on EPS Production from Lp. plantarum MC5

#### 3.4.2. Effects of Culture Time on EPS Production from Lp. plantarum MC5

#### 3.4.3. Effects of Culture Temperature on EPS Production from Lp. plantarum MC5

#### 3.4.4. Effect of Initial pH Value on EPS Production from Lp. plantarum MC5

#### 3.5. Optimization of Lp. plantarum MC5 EPS Culture Conditions by Response Surface

#### 3.6. In Vitro Resisting-Digestion Capacity of EPS-MC5

#### 3.6.1. The Resisting-Digestion Capacity of EPS-MC5 to α-Amylase (RCA)

#### 3.6.2. The Resisting-Digestion Capacity of EPS-MC5 to Simulated Gastric Juice

#### 3.6.3. The Resisting-Digestion Capacity of EPS-MC5 to Simulated Intestinal Juice

#### 3.7. In Vitro Antioxidant Activity Analysis of EPS-MC5

#### 3.7.1. The Radical Scavenging Rate (RSR) of DPPH

_{j}), and ascorbic acid was used as a positive control. The RSR of DPPH free radical was calculated by the equation as follows:

_{j}: Absorbance of EPS solution (2 mL) + 95%-DPPH ethanol solution (2 mL);

_{i}: Absorbance of EPS solution (2 mL) + 95% ethanol solution (2 mL);

_{0}: Absorbance of 95%-DPPH ethanol solution (2 mL) + 95% ethanol solution (2 mL).

#### 3.7.2. The Radical Scavenging Rate (RSR) of ABTS

_{j}), and ascorbic acid was used as a positive control. The RSA of ABTS was calculated using:

_{j}: Absorbance of EPS solution (600 uL) + ABTS solution (3 mL);

_{i}: Absorbance of EPS solution (600 uL) + deionized water (3 mL);

_{0}: Absorbance of deionized water (600 uL) + ABTS solution (3 mL).

#### 3.7.3. The Radical Scavenging Rate (RSR) of Hydroxyl

_{4}(2 mL), 1.8 mM salicylic acid (1.5 mL) and 0.3% H

_{2}O

_{2}(2 mL). After 30 min of standing at 37 °C and centrifugation (8000 rpm, 5 min), the absorbance of the supernatant was measured at 510 nm. Ascorbic acid was used as a positive control. The RSA of OH was calculated using:

_{j}: Absorbance of EPS solution + H

_{2}O

_{2};

_{i}: Absorbance of deionized water + H

_{2}O

_{2};

_{0}: Absorbance of salicylic acid was replaced by deionized water.

#### 3.7.4. The Ferric-Iron Reducing Power (IRP) of EPS-MC5

_{3}(0.1%, w/v). The absorbance was measured at 700 nm after 10 min. The IRP of EPS was calculated using:

#### 3.8. Analysis of Rheological Properties of EPS-MC5

#### 3.8.1. The Preparation of the EPS-MC5 Samples

#### 3.8.2. Apparent Viscosity and Flow Curves of EPS-MC5

#### 3.8.3. Amplitude and Frequency Sweep Tests of EPS-MC5

#### 3.9. Statistical Analysis

## 4. Conclusions

## Author Contributions

## Funding

## Institutional Review Board Statement

## Informed Consent Statement

## Data Availability Statement

## Acknowledgments

## Conflicts of Interest

## Abbreviations

EPS | Exopolysaccharide |

EPS-MC5 | Exopolysaccharide production from Lp. plantarum MC5 |

LAB | Lactic acid bacteria |

Lp. plantarum MC5 | Lactiplantibacillus plantarum MC5 |

RSA | Radical scavenging activity |

DPPH• | 1,1-Diphenyl-2-picrylhydrazyl, (free radical) |

ABTS• | 2,2’-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid ammonium salt) |

•OH | Hydroxyl (free radicals) |

IRP | Ferric-iron reducing power |

## References

- Faghfoori, Z.; Gargari, B.P.; Gharamaleki, A.S.; Bagherpour, H.; Khosroushahi, A.Y. Cellular and molecular mechanisms of probiotics effects on colorectal cancer. J. Funct. Foods
**2015**, 18, 463–472. [Google Scholar] [CrossRef] - Imran, M.Y.M.; Reehana, N.; Jayaraj, K.A.; Ahamed, A.A.P.; Dhanasekaran, D.; Thajuddin, N.; Alharbi, N.S.; Muralitharan, G. Statistical optimization of exopolysaccharide production by Lactobacillus plantarum NTMI05 and NTMI20. Int. J. Biol. Macromol.
**2016**, 93, 731–745. [Google Scholar] [CrossRef] - Kajala, I.; Shi, Q.; Nyyssölä, A.; Maina, N.H.; Hou, Y.; Katina, K.; Tenkanen, M.; Juvonen, R. Cloning and characterization of a Weissella confusa dextransucrase and its application in high fibre baking. PLoS ONE
**2015**, 10, e116418–e116437. [Google Scholar] [CrossRef] [Green Version] - Dilna, S.V.; Surya, H.; Aswathy, R.G.; Varsha, K.K.; Sakthikumar, D.N.; Pandey, A.; Nampoothiri, K.M. Characterization of an exopolysaccharide with potential health-benefit properties from a probiotic Lactobacillus plantarum RJF4. LWT-Food Sci. Technol.
**2015**, 64, 1179–1186. [Google Scholar] [CrossRef] - Sadishkumar, V.; Jeevaratnam, K. In vitro probiotic evaluation of potential antioxidant lactic acid bacteria isolated fromidli batter fermented with Piper betle leaves. Int. J. Food Sci. Technol.
**2017**, 52, 329–340. [Google Scholar] [CrossRef] - Mayer, M.J.; D’Amato, A.; Colquhoun, I.J.; Le Gall, G.; Narbad, A. Identification of genes required for glucan exopolysaccharide production in Lactobacillus johnsonii suggests a novel biosynthesis mechanism. Appl. Environ. Microbiol.
**2020**, 86, e2808–e2819. [Google Scholar] [CrossRef] [Green Version] - Loeffler, M.; Hilbig, J.; Velasco, L.; Weiss, J. Usage of in situ exopolysaccharide-forming lactic acid bacteria in food production: Meat products—A new field of application? Compr. Rev. Food Sci. Food Saf.
**2020**, 19, 2932–2954. [Google Scholar] [CrossRef] - Macedo, M.; Lacroix, C.; Gardner, N.; Champagne, C. Effect of medium supplementation on exopolysaccharide production by Lactobacillus rhamnosus RW-9595M in whey permeate. Int. Dairy J.
**2002**, 12, 419–426. [Google Scholar] [CrossRef] - Aslım, B.; Yüksekdag, Z.N.; Beyatli, Y.; Mercan, N.; Aslim, B. Exopolysaccharide production by Lactobacillus delbruckii subsp. bulgaricus and Streptococcus thermophilus strains under different growth conditions. World J. Microbiol. Biotechnol.
**2005**, 21, 673–677. [Google Scholar] [CrossRef] - Bryukhanov, A.L.; Klimko, A.I.; Netrusov, A.I. Antioxidant properties of lactic acid bacteria. Microbiology
**2022**, 91, 463–478. [Google Scholar] [CrossRef] - Seishima, R.; Wada, T.; Tsuchihashi, K.; Okazaki, S.; Yoshikawa, M.; Oshima, H.; Oshima, M.; Sato, T.; Hasegawa, H.; Kitagawa, Y.; et al. Ink4a/Arf-dependent loss of parietal cells induced by oxidative stress promotes CD44-dependent gastric tumorigenesis. Cancer Prev. Res.
**2015**, 8, 492–501. [Google Scholar] [CrossRef] [Green Version] - Matthew, R.B.; Andrew, B.; Rajesh, K. Reactive oxygen species-mediated diabetic heart disease: Mechanisms and therapies. Antioxid. Redox Signal.
**2022**, 36, 608–630. [Google Scholar] - Zuo, L.; Rose, B.A.; Roberts, W.J.; He, F.; Banes-Berceli, A.K. Molecular characterization of reactive oxygen species in systemic and pulmonary hypertension. Am. J. Hypertens.
**2014**, 27, 643–650. [Google Scholar] [CrossRef] [Green Version] - Pessione, E.; Cirrincione, S. Bioactive molecules released in food by lactic acid bacteria: Encrypted peptides and biogenic amines. Front. Microbiol.
**2016**, 7, 1–19. [Google Scholar] [CrossRef] [PubMed] [Green Version] - Tallon, R.; Bressollier, P.; Urdaci, M.C. Isolation and characterization of two exopolysaccharides produced by Lactobacillus plantarum EP56. Res. Microbiol.
**2003**, 154, 705–712. [Google Scholar] [CrossRef] - Vereecke, D.; Fichtner, E.J.; Lambert, P.Q.; Cooke, P.; Kilcrease, J.; Stamler, R.A.; Zhang, Y.; Francis, I.M.; Randall, J.J. Colonization and survival capacities underlying the multifaceted life of Rhodococcus sp. PBTS1 and PBTS2. Plant Pathol.
**2021**, 70, 567–583. [Google Scholar] [CrossRef] - Degeest, B.; Vaningelgem, F.; De Vuyst, L. Microbial physiology, fermentation kinetics, and process engineering of heteropolysaccharide production by lactic acid bacteria. Int. Dairy J.
**2001**, 11, 747–757. [Google Scholar] [CrossRef] - Adesulu-Dahunsi, A.T.; Sanni, A.I.; Jeyaram, K. Production, characterization and in vitro antioxidant activities of exopolysaccharide from Weissella cibaria GA44. LWT-Food Sci. Technol.
**2018**, 87, 432–442. [Google Scholar] [CrossRef] [Green Version] - Zhao, X.F.; Liang, Q. EPS-Producing Lactobacillus plantarum MC5 as a compound starter improves rheology, texture, and antioxidant activity of yogurt during storage. Foods
**2022**, 11, 1660. [Google Scholar] [CrossRef] [PubMed] - Xia, M.; Zhang, S.; Shen, L.; Yu, R.; Liu, Y.; Li, J.; Wu, X.; Chen, M.; Qiu, G.; Zeng, W. Optimization and characterization of an antioxidant exopolysaccharide produced by cupriavidus pauculus 1490. J. Polym. Environ.
**2022**, 30, 2077–2086. [Google Scholar] [CrossRef] - Chaisuwan, W.; Jantanasakulwong, K.; Wangtueai, S. Microbial exopolysaccharides for immune enhancement fermentation, modifications and bioactivities. Food Biosci.
**2020**, 35, 100564–100601. [Google Scholar] [CrossRef] - Jiang, G.; He, J.; Gan, L.; Li, X.; Tian, Y. Optimization of exopolysaccharides production by lactiplantibacillus pentosus B8 isolated from sichuan PAOCAI and its functional properties. Appl. Biochem. Microbiol.
**2022**, 58, 195–205. [Google Scholar] [CrossRef] - Li, Y.; Liu, Y.; Cao, C.; Zhu, X.; Wang, C.; Wu, R.; Wu, J. Extraction and biological activity of exopolysaccharide produced by Leuconostoc Mesenteroides SN-8. Int. J. Biol. Macromol.
**2020**, 157, 36–44. [Google Scholar] [CrossRef] - Wang, X.; Shao, C.; Liu, L.; Guo, X.; Xu, Y.; Lü, X. Optimization, partial characterization and antioxidant activity of an exopolysaccharide from Lactobacillus plantarum KX041. Int. J. Biol. Macromol.
**2017**, 103, 1173–1184. [Google Scholar] [CrossRef] [PubMed] - Zhang, L.; Liu, C.; Li, D.; Zhao, Y.; Zhang, X.; Zeng, X.; Yang, Z.; Li, S. Antioxidant activity of an exopolysaccharide isolated from Lactobacillus plantarum C88. Int. J. Biol. Macromol.
**2013**, 54, 270–275. [Google Scholar] [CrossRef] - Wang, J.; Zhao, X.; Tian, Z.; Yang, Y.; Yang, Z. Characterization of an exopolysaccharide produced by Lactobacillus plantarum YW11 isolated from Tibet Kefir. Carbohydr. Polym.
**2015**, 125, 16–25. [Google Scholar] [CrossRef] [PubMed] - Rajoka, M.S.R.; Jin, M.; Haobin, Z.; Li, Q.; Shao, D.; Jiang, C.; Huang, Q.; Yang, H.; Shi, J.; Hussain, N. Functional characterization and biotechnological potential of exopolysaccharide produced by Lactobacillus rhamnosus strains isolated from human breast milk. LWT-Food Sci. Technol.
**2018**, 89, 638–647. [Google Scholar] [CrossRef] - El-Dein, A.N.; El-Deen, A.M.N.; El-Shatoury, E.H.; Awad, G.A.; Ibrahim, M.K.; Awad, H.M.; Farid, M.A. Assessment of exopolysaccharides, bacteriocins and in vitro and in vivo hypocholesterolemic potential of some Egyptian Lactobacillus spp. Int. J. Biol. Macromol.
**2021**, 173, 66–78. [Google Scholar] [CrossRef] [PubMed] - Gan, D.; Ma, L.; Jiang, C.; Xu, R.; Zeng, X. Production, preliminary characterization and antitumor activity in vitro of polysaccharides from the mycelium of Pholiota dinghuensis Bi. Carbohydr. Polym.
**2011**, 84, 997–1003. [Google Scholar] [CrossRef] - Devi, P.B.; Kavitake, D.; Jayamanohar, J.; Shetty, P.H. Preferential growth stimulation of probiotic bacteria by galactan exopolysaccharide from Weissella confusa KR780676. Food Res. Int.
**2021**, 143, 110333–110340. [Google Scholar] [CrossRef] - Caggianiello, G.; Kleerebezem, M.; Spano, G. Exopolysaccharides produced by lactic acid bacteria: From health-promoting benefits to stress tolerance mechanisms. Appl. Microbiol. Biotechnol.
**2016**, 100, 3877–3886. [Google Scholar] [CrossRef] - Mao, Y.; Doyle, M.P.; Chen, J. Role of colanic acid exopolysaccharide in the survival of enterohaemorrhagic Escherichia coli O157:H7 in simulated gastrointestinal fluids. Lett. Appl. Microbiol.
**2006**, 42, 642–647. [Google Scholar] [CrossRef] - Khalil, M.A.; Sonbol, F.I.; Al-Madboly, L.A.; Aboshady, T.A.; Alqurashi, A.S.; Ali, S.S. Exploring the therapeutic potentials of exopolysaccharides derived from lactic acid bacteria and bifidobacteria: Antioxidant, antitumor, and periodontal regeneration. Front. Microbiol.
**2022**, 13, 803688–803699. [Google Scholar] [CrossRef] [PubMed] - Hao, L.; Sheng, Z.; Lu, J.; Tao, R.; Jia, S. Characterization and antioxidant activities of extracellular and intracellular polysaccharides from Fomitopsis pinicola. Carbohydr. Polym.
**2016**, 141, 54–59. [Google Scholar] [CrossRef] [PubMed] - Yamamoto, N.; Shoji, M.; Hoshigami, H.; Watanabe, K.; Takatsuzu, T.; Yasuda, S.; Igoshi, K.; Kinoshita, H. Antioxidant capacity of soymilk yogurt and exopolysaccharides produced by lactic acid bacteria. Biosci. Microbiota Food Health
**2019**, 38, 97–104. [Google Scholar] [CrossRef] [PubMed] [Green Version] - Miao, M.; Jia, X.; Jiang, B.; Wu, S.; Cui, S.W.; Li, X. Elucidating molecular structure and prebiotics properties of bioengineered α-D-glucan from Leuconostoc citreum SK24.002. Food Hydrocoll.
**2016**, 54, 227–233. [Google Scholar] [CrossRef] - Xu, Y.; Cui, Y.; Yue, F.; Liu, L.; Shan, Y.; Liu, B.; Zhou, Y.; Lü, X. Exopolysaccharides produced by lactic acid bacteria and bifidobacteria structures, physiofchemical functions and applications in the food industry. Food Hydrocoll.
**2019**, 94, 475–499. [Google Scholar] [CrossRef] - Li, B.; Du, P.; Smith, E.E.; Wang, S.; Jiao, Y.; Guo, L.; Huo, G.; Liu, F. In vitro and in vivo evaluation of an exopolysaccharide produced by Lactobacillus helveticus KLDS1.8701 for the alleviative effect on oxidative stress. Food Funct.
**2019**, 10, 1707–1717. [Google Scholar] [CrossRef] - Zhao, D.; Liu, L.; Jiang, J.; Guo, S.; Ping, W.; Ge, J. The response surface optimization of exopolysaccharide produced by Weissella confusa XG-3 and its rheological property. Prep. Biochem. Biotechnol.
**2020**, 50, 1014–1022. [Google Scholar] [CrossRef] - Ayyash, M.; Abu-Jdayil, B.; Itsaranuwat, P.; Galiwango, E.; Tamiello-Rosa, C.; Abdullah, H.; Esposito, G.; Hunashal, Y.; Obaid, R.S.; Hamed, F. Characterization, bioactivities, and rheological properties of exopolysaccharide produced by novel probiotic Lactobacillus plantarum C70 isolated from camel milk. Int. J. Biol. Macromol.
**2020**, 144, 938–946. [Google Scholar] [CrossRef] - Zhou, Y.; Cui, Y.; Qu, X. Exopolysaccharides of lactic acid bacteria: Structure, bioactivity and associations: A review. Carbohydr. Polym.
**2019**, 207, 317–332. [Google Scholar] [CrossRef] - Fashogbon, R.O.; Adebayo-Tayo, B.; Sanusi, J. Optimization of extracellular polysaccharide substances from lactic acid bacteria isolated from fermented dairy products. Microbiol. J.
**2021**, 11, 1–11. [Google Scholar] [CrossRef] - Poespowati, T.; Mahmudi, A. Optimization of acid hydrolysis process on macroalga Ulva lactuca for reducing sugar production as feedstock of bioethanol. Int. J. Renew. Energy Res.
**2018**, 8, 466–475. [Google Scholar] - Al-Sheraji, S.H.; Ismail, A.; Manap, M.Y.; Mustafa, S.; Yusof, R.M.; Hassan, F.A. Fermentation and non-digestibility of Mangifera pajang fibrous pulp and its polysaccharides. J. Funct. Foods
**2012**, 4, 933–940. [Google Scholar] [CrossRef] - Vecchione, A.; Celandroni, F.; Mazzantini, D.; Senesi, S.; Lupetti, A.; Ghelardi, E. Compositional quality and potential gastrointestinal behavior of probiotic products commercialized in Italy. Front. Med.
**2018**, 5, 59–68. [Google Scholar] [CrossRef] [PubMed] [Green Version] - Li, E.; Yang, H.; Zou, Y.; Wang, H.; Hu, T.; Li, Q.; Liao, S. In-vitro digestion by simulated gastrointestinal juices of Lactobacillus rhamnosus cultured with mulberry oligosaccharides and subsequent fermentation with human fecal inocula. LWT-Food Sci. Technol.
**2019**, 101, 61–68. [Google Scholar] [CrossRef] - Wang, K.; Niu, M.; Song, D.; Song, X.; Zhao, J.; Wu, Y.; Lu, B.; Niu, G. Preparation, partial characterization and biological activity of exopolysaccharides produced from Lactobacillus fermentum S1. J. Biosci. Bioeng.
**2020**, 129, 206–214. [Google Scholar] [CrossRef] - Ayyash, M.; Abu-Jdayil, B.; Olaimat, A.; Esposito, G.; Itsaranuwat, P.; Osaili, T.; Obaid, R.S.; Kizhakkayil, J.; Liu, S.-Q. Physicochemical, bioactive and rheological properties of an exopolysaccharide produced by a probiotic Pediococcus pentosaceus M41. Carbohydr. Polym.
**2020**, 229, 115462–115471. [Google Scholar] [CrossRef]

**Figure 2.**Effect of culture conditions on the EPS production from Lp. plantarum MC5. (

**a**): Effect of inoculation size on the EPS production from Lp. plantarum MC5; (

**b**): Effect of culture time on the EPS production from Lp. plantarum MC5; (

**c**): Effect of culture temperature on the EPS production from Lp. plantarum MC5; (

**d**): Effect of initial pH on the EPS production from Lp. plantarum MC5. Error bars are represented the standard errors (se) of the mean value (n = 3). “a, b, c, d, e” indicate significant differences (p < 0.05).

**Figure 3.**3D response surface plot of the interaction of four factors. (

**a**) represents the 3D response surface plot of inoculation size and time; (

**b**) represents the 3D response surface plot of inoculation size and temperature; (

**c**) represents the 3D response surface plot of inoculation size and initial pH; (

**d**) represents the 3D response surface plot of time and temperature; (

**e**) represents the 3D response surface plot of time and initial pH; (

**f**) represents the 3D response surface plot of temperature and initial pH.

**Figure 4.**DEAE Sepharose Fast Flow elution curve of EPS-MC5 (

**a**) and UV full wavelength scan of EPS-MC5 eluent (

**b**). Data are represented as the mean values (n = 3).

**Figure 5.**(

**a**): The resisting-digestion capacity of EPS-MC5 in α-amylase solution; (

**b**): The resisting-digestion capacity of EPS-MC5 in simulative gastric juices (pH 2, 3, and 4); (

**c**): The resisting-digestion capacity of EPS-MC5 in intestinal juices (pH 6.8). Error bars represent the standard errors (se) of the mean value (n = 3). Different lowercase letters indicate the difference between different times of the same pH, different capital letters indicate the difference of different pH at the same time (p < 0.05).

**Figure 6.**Radical scavenging activity of DPPH (

**a**), ABTS (

**b**), OH (

**c**), and ferric-iron reducing power of EPS-MC5 (

**d**). Ascorbic acid (Vc) was used as a positive control. Error bars represent the standard errors (se) of the mean value (n = 3).

**Figure 7.**Apparent viscosity of EPS-MC5 at different shear rates and temperatures (

**a**,

**b**); shear stress and elastic modulus (G’) and viscous modulus (G”) of EPS-MC5 (

**c**,

**d**). Error bars represent the standard errors (se) of the mean value (n = 3).

**Table 1.**Design and results of response surface experiments. Data are represented as the mean (n = 3).

Run | Factor 1 (%) Inoculation Size | Factor 2 (h) Time | Factor 3 (°C) Temperature | Factor 4 Initial pH | EPS (mg/L) |
---|---|---|---|---|---|

1 | 4 | 24 | 37 | 6.40 | 356.92 |

2 | 5 | 24 | 37 | 6.00 | 187.33 |

3 | 4 | 24 | 34 | 6.80 | 154.49 |

4 | 4 | 24 | 37 | 6.40 | 356.92 |

5 | 5 | 18 | 37 | 6.40 | 194.38 |

6 | 4 | 24 | 37 | 6.40 | 356.92 |

7 | 5 | 24 | 34 | 6.40 | 165.39 |

8 | 4 | 24 | 34 | 6.00 | 194.61 |

9 | 3 | 24 | 40 | 6.40 | 136.76 |

10 | 3 | 30 | 37 | 6.40 | 177.21 |

11 | 3 | 24 | 34 | 6.40 | 188.13 |

12 | 4 | 24 | 37 | 6.40 | 356.92 |

13 | 4 | 18 | 34 | 6.40 | 171.54 |

14 | 4 | 30 | 40 | 6.40 | 158.81 |

15 | 3 | 24 | 37 | 6.80 | 139.14 |

16 | 4 | 30 | 37 | 6.80 | 205.19 |

17 | 4 | 18 | 37 | 6.80 | 151.87 |

18 | 5 | 24 | 37 | 6.80 | 148.58 |

19 | 4 | 24 | 37 | 6.40 | 356.92 |

20 | 4 | 24 | 40 | 6.00 | 210.74 |

21 | 4 | 24 | 40 | 6.80 | 169.37 |

22 | 3 | 24 | 37 | 6.00 | 176.22 |

23 | 5 | 30 | 37 | 6.40 | 183.02 |

24 | 4 | 18 | 40 | 6.40 | 208.02 |

25 | 4 | 30 | 37 | 6.00 | 224.72 |

26 | 3 | 18 | 37 | 6.40 | 143.59 |

27 | 4 | 18 | 37 | 6.00 | 175.74 |

28 | 4 | 30 | 34 | 6.40 | 345.98 |

29 | 5 | 24 | 40 | 6.40 | 182.90 |

Source | Sum of Squares | df | Mean Squares | F-Value | p-Value | Significance |
---|---|---|---|---|---|---|

Model | 1.629 × 10^{5} | 14 | 11,638.92 | 26.34 | <0.0001 | Significant |

A-Inoculation size | 842.53 | 1 | 842.53 | 1.91 | 0.1890 | |

B-Time | 4400.29 | 1 | 4400.29 | 9.96 | 0.0070 | ** |

C-Temperature | 3799.94 | 1 | 3799.94 | 8.64 | 0.0109 | * |

D-Initial pH | 2152.58 | 1 | 2152.58 | 4.87 | 0.0445 | * |

AB | 505.80 | 1 | 505.80 | 1.14 | 0.3028 | |

AC | 1186.11 | 1 | 1186.11 | 2.68 | 0.1236 | |

AD | 0.70 | 1 | 0.70 | 1.578 × 10^{-3} | 0.9689 | |

BC | 12,504.83 | 1 | 12,504.83 | 28.30 | 0.0001 | ** |

BD | 148.11 | 1 | 148.11 | 0.34 | 0.5718 | |

CD | 87.89 | 1 | 87.89 | 0.20 | 0.6624 | |

A^{2} | 72,014.98 | 1 | 72,014.98 | 162.97 | <0.0001 | ** |

B^{2} | 30,161.36 | 1 | 30,161.36 | 68.26 | <0.0001 | ** |

C^{2} | 41,364.46 | 1 | 41,364.46 | 93.61 | <0.0001 | ** |

D^{2} | 66,101.51 | 1 | 66,101.51 | 149.59 | <0.0001 | ** |

Residual | 6186.39 | 14 | 441.88 | |||

Lack of fit | 6186.39 | 10 | 618.64 | |||

Pure error | 0.000 | 4 | 0.000 | |||

Cor total | 1.691 × 10^{5} | 28 | ||||

R^{2} | 0.9634 | |||||

Adj-R^{2} | 0.9268 | |||||

C.V. % | 10.00 |

Groups | OD_{600} | EPS (mg/L) |
---|---|---|

Verification value | 0.077 ^{a} | 345.98 ^{A} |

Initial value | 0.065 ^{b} | 140.34 ^{B} |

Strains | EPS (mg/L) | DPPH | OH | Isolation Source |
---|---|---|---|---|

L. plantarum C88 | 69.00 | 52.23% (4000 mg/L) | 85.21% (4000 mg/L) | Chinese traditional fermented dairy Tofu [25] |

L. plantarum YW11 | 90.00 | - | - | Kefir grains collected from Tibet [26] |

L. plantarum EP56 | 126.40 | - | - | Corn silage [15] |

S. thermophilus W22 | 127.00 | - | - | Village type yogurt [9] |

L. delbruckii subsp. Bulgaricus B3 | 263.00 | - | - | |

L. delbruckii subsp. Bulgaricus G12 | 238.00 | - | - | |

L. rhamnosus ATCC 9595 | 352.00 | - | - | Human breast milk [27] |

L. rhamnosus SHA114 | 461.00 | - | - | |

L. rhamnosus SHA113 | 549.60 | - | - | |

L. plantarum KX041 | 599.52 | 82.00% (6000 mg/L) | 82.64% (8000 mg/L) | Traditional Chinese pickle juice [24] |

L. plantarum NTMI20 | 827.00 | 91.86% (500 mg/L) | - | Milk sources [2] |

L. plantarum NTMI05 | 956.00 | 96.62% (500 mg/L) | - | |

L. plantarum KU985433 | 2030.00 | 88.00% (4000 mg/L) | - | Egyptian fermented food [28] |

L. rhamnosus RW-9595 M | 2767.00 | - | - | LAB research network culture collection [8] |

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## Share and Cite

**MDPI and ACS Style**

Zhao, X.; Liang, Q.
Optimization, Probiotic Characteristics, and Rheological Properties of Exopolysaccharides from *Lactiplantibacillus plantarum MC5*. *Molecules* **2023**, *28*, 2463.
https://doi.org/10.3390/molecules28062463

**AMA Style**

Zhao X, Liang Q.
Optimization, Probiotic Characteristics, and Rheological Properties of Exopolysaccharides from *Lactiplantibacillus plantarum MC5*. *Molecules*. 2023; 28(6):2463.
https://doi.org/10.3390/molecules28062463

**Chicago/Turabian Style**

Zhao, Xuefang, and Qi Liang.
2023. "Optimization, Probiotic Characteristics, and Rheological Properties of Exopolysaccharides from *Lactiplantibacillus plantarum MC5*" *Molecules* 28, no. 6: 2463.
https://doi.org/10.3390/molecules28062463