Bacillus subtilis natto: a non-toxic source of poly-γ-glutamic acid that could be used as a cryoprotectant for probiotic bacteria

AMB Express - Tập 3 Số 1 - 2013
Aditya Bhat1, Victor U. Irorere1, T. Bartlett1, David J. Hill1, Gopal Kedia1, Mark R. Morris1, Dimitris Charalampopoulos2, Iza Radecka1
1University of Wolverhampton, Wolverhampton, UK
2University of Reading, Reading, UK

Tóm tắt

Abstract It is common practice to freeze dry probiotic bacteria to improve their shelf life. However, the freeze drying process itself can be detrimental to their viability. The viability of probiotics could be maintained if they are administered within a microbially produced biodegradable polymer - poly-γ-glutamic acid (γ-PGA) - matrix. Although the antifreeze activity of γ-PGA is well known, it has not been used for maintaining the viability of probiotic bacteria during freeze drying. The aim of this study was to test the effect of γ-PGA (produced by B. subtilis natto ATCC 15245) on the viability of probiotic bacteria during freeze drying and to test the toxigenic potential of B. subtilis natto. 10% γ-PGA was found to protect Lactobacillus paracasei significantly better than 10% sucrose, whereas it showed comparable cryoprotectant activity to sucrose when it was used to protect Bifidobacterium breve and Bifidobacterium longum. Although γ-PGA is known to be non-toxic, it is crucial to ascertain the toxigenic potential of its source, B. subtilis natto. Presence of six genes that are known to encode for toxins were investigated: three component hemolysin (hbl D/A), three component non-haemolytic enterotoxin (nheB), B. cereus enterotoxin T (bceT), enterotoxin FM (entFM), sphingomyelinase (sph) and phosphatidylcholine-specific phospholipase (piplc). From our investigations, none of these six genes were present in B. subtilis natto. Moreover, haemolytic and lecithinase activities were found to be absent. Our work contributes a biodegradable polymer from a non-toxic source for the cryoprotection of probiotic bacteria, thus improving their survival during the manufacturing process.

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Tài liệu tham khảo

Allaker RP, Douglas CWI: Novel anti-microbial therapies for dental plaque-related diseases. Int J Antimicrob Ag 2009, 33: 8–13. 10.1016/j.ijantimicag.2008.07.014

Anukam KC, Osazuwa EO, Osadolor HB, Bruce AW, Reid G: Yogurt containing probiotic Lactobacillus rhamnosus GR-1 and L. reuteri RC-14 helps resolve moderate diarrhea and increases CD4 count in HIV/AIDS patients. J Clin Gastroenterol 2008, 42: 239–243.

Bajaj I, Singhal R: Poly(glutamic acid) - An emerging biopolymer of commercial interest. Bioresour Technol 2011, 102: 5551–5561. 10.1016/j.biortech.2011.02.047

Beattie SH, Williams AG: Detection of toxigenic strains of Bacillus cereus and other Bacillus spp. with an improved cytotoxicity assay. Lett Appl Microbiol 1999, 28: 221–225. 10.1046/j.1365-2672.1999.00498.x

Benchimol E, Mack D: Probiotics in relapsing and chronic diarrhea. J Pediat Hematol Onc 2004, 26: 515–517. 10.1097/01.mph.0000133291.11443.a1

Benno Y, Mitsuoka T: Impact of Bifidobacterium longum on human fecal microflora. Microbiol Immunol 1992, 36: 683–694.

Buescher JM, Margaritis AM: Microbial biosynthesis of polyglutamic acid biopolymer and applications in the biopharmaceutical, biomedical and food industries. Crit Rev Biotechnol 2007, 27: 1–19. 10.1080/07388550601166458

Candela T, Fouet A: Poly-gamma-glutamate in bacteria. Mol Microbiol 2006, 60: 1091–1098. 10.1111/j.1365-2958.2006.05179.x

de LeBlanc ADM, Matar C, LeBlanc N, Perdigón G: Effects of milk fermented by Lactobacillus helveticus R389 on a murine breast cancer model. Breast Cancer Res 2005, 7: 477–486. 10.1186/bcr1032

de Moreno de LeBlanc A, Matar C, Perdigón G: The application of probiotics in cancer. Brit J Nutr 2007, 98: 105–110.

De Simone C, Clardi A, Grassi A: Effect of Bifidobacterium bifidum and Lactobacillus acidophilus on gut mucosa and peripheral blood B lymphocytes. Immunotoxicol 1992, 14: 331–340.

Falagas ME, Betsi GE, Athanasiou S: Probiotics for the treatment of women with bacterial vaginosis. Clin Microbiol Infec 2007, 13: 657–664. 10.1111/j.1469-0691.2007.01688.x

From C, Pukall R, Schumann P, Hormazabal V, Granum PE: Toxin-producing ability among Bacillus spp. outside the Bacillus cereus group. Appl Environ Microbiol 2005, 71: 1178–1183. 10.1128/AEM.71.3.1178-1183.2005

Garrait G, Jarrige JF, Blanquet-Diot S, Alric M: Genetically engineered yeasts as a new delivery vehicle of active compounds to the digestive tract: In vivo validation of the concept in the rat. Metab Eng 2009, 11: 148–154. 10.1016/j.ymben.2009.01.001

Goto A, Kunioka M: Biosynthesis and hydrolysis of poly(γ-glutamic acid) from Bacillus subtilis IFO3335. Biosci Biotechnol Biochem 1992, 56: 1031–1035. 10.1271/bbb.56.1031

Heidebach T, Forst P, Kulozik U: Influence of casein-based microencapsulation on freeze-drying and storage of probiotic cells. J Food Eng 2010, 98: 309–316. 10.1016/j.jfoodeng.2010.01.003

Hlivak P, Odraska J, Ferencik M, Ebringer L, Jahnova E, Mikes Z: One-year application of probiotic strain Enterococcus faecium M-74 decreases serum cholesterol levels. Bratisl Lek Listy 2005, 106: 67–72.

Jagannath A, Raju PS, Bawa AS: Comparative evaluation of bacterial cellulose (nata) as a cryoprotectant and carrier support during the freeze drying process of probiotic lactic acid bacteria. LWT- Food Sci Technol 2010, 43: 1197–1203. 10.1016/j.lwt.2010.03.009

Kailasapathy K, Rybka S: L. acidophilus and Bifidobacterium spp. — their therapeutic potential and survival in yogurt. Aust J Dairy Technol 1997, 52: 28–33.

Kedia G, Hill D, Hill R, Radecka I: Production of poly-γ-glutamic acid by Bacillus subtilis and Bacillus licheniformis with different growth media. J Nanosci Nanotechnol 2010, 10: 5926–5934. 10.1166/jnn.2010.2614

Kligler B, Hanaway P, Cohrssen A: Probiotics in children. Pediatr Clin N Am 2007, 54: 949–967. 10.1016/j.pcl.2007.10.002

Lara-Villoslada F, Sierra S, Boza J, Xaus J, Olivares M: Beneficial effects of consumption of a dairy product containing two probiotic strains, Lactobacillus coryniformis CECT5711 and Lactobacillus gasseri CECT5714 in healthy children. Nutr Hosp 2007, 22: 496–502.

Lian W-C, Hsiao H-C, Chou C-C: Survival of bifidobacteria after spray-drying. Int J Food Microbiol 2002, 74: 79–86. 10.1016/S0168-1605(01)00733-4

Matarante A, Baruzzi F, Cocconcelli PS, Morea M: Genotyping and toxigenic potential of Bacillus subtilis and Bacillus pumilus strains occurring in industrial and artisanal cured sausages. Appl Environ Microbiol 2004, 70: 5168–5176. 10.1128/AEM.70.9.5168-5176.2004

Mitsuiki M, Mizuno A, Tanimoto H, Motoki M: Relationship between the antifreeze activities and the chemical structures of oligo- and poly(glutamic acid)s. J Agric Food Chem 1998, 46: 891–895. 10.1021/jf970797m

Mizuno A, Mitsuiki M, Toba S, Motoki M: Antifreeze activities of various food components. J Agric Food Chem 1997, 45: 14–18. 10.1021/jf960422l

Nualkaekul S, Salmeron I, Charalampopoulos D: Investigation of the factors influencing the survival of Bifidobacterium longum in model acidic solutions and fruit juices. Food Chem 2011, 129: 1037–1044. 10.1016/j.foodchem.2011.05.071

Otero M, Espeche M, Nader-Macias M: Optimization of the freeze-drying media and survival throughout storage of freeze-dried Lactobacillus gasseri and Lactobacillus delbrueckii subsp. delbrueckii for veterinarian probiotic applications. Process Biochem 2007, 42: 1406–1411. 10.1016/j.procbio.2007.07.008

Park SK, Park DI, Choi JS, Kang MS, Park JH, Kim HJ, Cho YK, Sohn CI, Jeon WK, Kim BI: The effect of probiotics on Helicobacter pylori eradication. Hepato-gastroenterol 2007, 54: 2032–2036.

Pedersen PB, Bjornvad ME, Rasmussen MD, Petersen JN: Cytotoxic potential of industrial strains of Bacillus sp. Regul Toxicol Pharmacol 2002, 36: 155–161. 10.1006/rtph.2002.1574

Phelps RJ, McKillip JL: Enterotoxin production in natural isolates of Bacillaceae outside the Bacillus cereus group. Appl Environ Microbiol 2002, 68: 3147–3151. 10.1128/AEM.68.6.3147-3151.2002

Pochapin M: The effect of probiotics on Clostridium difficile diarrhea. Am J Gastroenterol 2000, 95: S11-S13. 10.1016/S0002-9270(99)00809-6

Saarela M, Virkajärvi I, Alakomi H, Mattila-Sandholm T, Vaari A, Suomalainen T, Mättö J: Influence of fermentation time, cryoprotectant and neutralization of cell concentrate on freeze-drying survival, storage stability, and acid and bile exposure of Bifidobacterium animalis ssp. lactis cells produced without milk-based ingredients. J Appl Microbiol 2005, 99: 1330–1339. 10.1111/j.1365-2672.2005.02742.x

SCAN: Opinion of the scientific committee on animal nutrition on the safety of use of Bacillus species in animal nutrition. European Commission, Health and Consumer Protection Directorate-General (SCAN) Scientific Committee on Animal Nutrition; 2000. Available at: http://ec.europa.eu/food/fs/sc/scan/out41_en.pdf

Schallmey M, Singh A, Ward OP: Developments in the use of Bacillus species for industrial production. Can J Microbiol 2004, 50: 1–17. 10.1139/w03-076

Shih I-L, Van Y-T: The production of poly-(γ-glutamic acid) from microorganisms and its various applications. Bioresour Technol 2001, 79: 207–225. 10.1016/S0960-8524(01)00074-8

Shih IL, Van YT, Sau YY: Antifreeze activities of poly(−glutamic acid) produced by Bacillus licheniformis . Biotechnol Lett 2003, 25: 1709–1712. 10.1023/A:1026042302102

Siaterlis A, Deepika G, Charalampopoulos D: Effect of culture medium and cryoprotectants on the growth and survival of probiotic lactobacilli during freeze drying. Lett Appl Microbiol 2009, 48: 295–301. 10.1111/j.1472-765X.2008.02529.x

Szymanski H, Pejcz J, Jawien M, Chmielarczyk A, Strus M, Heczko PB: Treatment of acute infectious diarrhoea in infants and children with a mixture of three Lactobacillus rhamnosus strains – a randomized, double-blind, placebo-controlled trial. Aliment Pharm Ther 2006, 23: 247–253. 10.1111/j.1365-2036.2006.02740.x

Wang Y-C, Yu R-C, Chou C-C: Viability of lactic acid bacteria and bifidobacteria in fermented soymilk aftery drying, subsequent rehydration and storage. Int J Food Microbiol 2004, 93: 209–217. 10.1016/j.ijfoodmicro.2003.12.001

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