益生菌发酵乳低温酸性双重胁迫致益生菌损伤机制的研究进展
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摘要
益生菌是一类对宿主有益的活性微生物,是定植于人体肠道、生殖系统内,能产生确切健康功效从而改善宿主微生态平衡、发挥有益作用的活性有益微生物的总称。近年,有关益生菌发酵乳中低温酸性双胁迫致益生菌的损伤,发酵乳中益生菌活性的保护,以及组学技术推动下的菌株损伤相关代谢研究取得了一定进展。本文综述了益生菌低温酸性双胁迫下损伤应激和保护机制、菌株特性对发酵乳风味和质量的影响及益生菌发酵乳冷藏过程低温酸性双重胁迫下菌体活性的保护策略,为研究影响益生菌发酵乳中益生菌存活能力、菌体细胞膜特性及代谢酶活性的因素,明确益生菌发酵乳基质中益生菌低温酸性双胁迫损伤的分子机制,建立益生菌发酵乳加工和贮藏过程中益生菌的保护策略提供参考,也为未来改善益生菌发酵乳的功效和品质提供了科学依据。
Probiotics are a kind of active microorganisms beneficial to the host. They are the general term for active beneficial microorganisms that are located in the human intestines and reproductive system and can produce exact health effects to improve the balance of the micro-ecology of the host and play a beneficial role. In recent years, the prevention of probiotics caused by double stressing from low temperature and acidity in probiotic fermented milk, the protection of probiotic activity in fermented milk, and the research on the damage related to strain damage promoted by omics technology have made some progress. This paper reviewed the damage stress and protection mechanism of probiotics under low temperature and acidic stress, the effect of strain characteristics on the flavor and quality of fermented milk, and protection strategy of bacterial activity under low temperature and acidic double stress in probiotic fermented milk during cold storage, so as to provide references for studying factors affecting the viability of probiotics, cell membrane properties and metabolic enzyme activities in probiotic fermented milk, molecular mechanism of low temperature and acidic double stress damage of probiotics in fermented milk matrix of probiotics and establishing a protective strategy for probiotics during processing and storage of probiotic fermented milk. It also provided a scientific basis for improving the efficacy and quality of probiotic fermented milk in the future.
Probiotics are a kind of active microorganisms beneficial to the host. They are the general term for active beneficial microorganisms that are located in the human intestines and reproductive system and can produce exact health effects to improve the balance of the micro-ecology of the host and play a beneficial role. In recent years, the prevention of probiotics caused by double stressing from low temperature and acidity in probiotic fermented milk, the protection of probiotic activity in fermented milk, and the research on the damage related to strain damage promoted by omics technology have made some progress. This paper reviewed the damage stress and protection mechanism of probiotics under low temperature and acidic stress, the effect of strain characteristics on the flavor and quality of fermented milk, and protection strategy of bacterial activity under low temperature and acidic double stress in probiotic fermented milk during cold storage, so as to provide references for studying factors affecting the viability of probiotics, cell membrane properties and metabolic enzyme activities in probiotic fermented milk, molecular mechanism of low temperature and acidic double stress damage of probiotics in fermented milk matrix of probiotics and establishing a protective strategy for probiotics during processing and storage of probiotic fermented milk. It also provided a scientific basis for improving the efficacy and quality of probiotic fermented milk in the future.
引文
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[7]Akkasheh G,Kashani-Poor Z,Tajabadi-Ebrahimi M,et al.Clinical and metabolic response to probiotic administration in patients with major depressive disorder:A randomized,double-blind,placebo-controlled trial[J].Nutrition,2016,32(3):315-320.
[8]Passariello A,Agricole P,Malfertheiner P.A critical appraisal of probiotics(as drugs or food supplements)in gastrointestinal diseases[J].Curr Med Res Opin,2014,30(6):1055-1064.
[9]Lee CL,Liao HL,Lee WC,et al.Standards and labeling of milk fat and spread products in different countries[J].J Food Drug Anal,2018,26(2):469-474.
[10]Settachaimongkon S,Van-Valenberg HJ,Gazi I,et al.Influence of Lactobacillus plantarum WCFS1 on post-acidification,metabolite formation and survival of starter bacteria in set-yoghurt[J].Food Microbiol,2016,(59):14-22.
[11]Settachaimongkon S,Van-Valenberg HJ,Winata V,et al.Effect of sublethal preculturing on the survival of probiotics and metabolite formation in set-yoghurt[J].Food Microbiol,2015,(49):104-115.
[12]Mohammadi R,Sohrabvandi S,Mohammad-Mortazavian A.The starter culture characteristics of probiotic microorganisms in fermented milks[J].Eng Lif Sci,2012,(12):399-409.
[13]于志会,张雪,李盛钰,等.植物乳杆菌C88在酸奶中的应用[J].中国乳品工业,2013,41(7):27-30.Yu ZH,Zhang X,Li SY,et al.Application of lactobacillus plantarum C88in yogurt[J].China Dairy Ind,2013,41(7):27-30.
[14]孙健,陈静,翁心刚.酸奶的储存温度对产品品质的影响[J].食品工业科技,2010,(12):140-141.Sun J,Chen J,Weng XG.Effect of storage temperature of yogurt on product quality[J].Food Ind Sci Technol,2010,(12):140-141.
[15]白凤翎.凝固型酸奶在贮藏过程中的微生物菌相演变研究[J].食品研究与开发,2005,(4):8-10.Bai FL.Microbiological evolution of solidified yoghurt during storage[J].Food Res Dev,2005,(4):8-10.
[16]高微娟,张富新,陈伟.贮藏温度对酸羊奶品质的影响[J].农产品加工(学刊),2011,(1):18-20.Gao WJ,Zhang FX,Chen W.Effect of storage temperature on quality of sour goat milk[J].Proc Agric Prod,2011,(1):18-20.
[17]Abdelazez A,Muhammad Z,Zhang QX,et al.Production of a functional frozen yogurt fortified with Bifidobacterium spp[J].Biol Med Res Int,2017,(6438528):1-10.
[18]Kim SH,Lim CH,Lee C.Optimization of growth and storage conditions for lactic acid bacteria in yogurt and frozen yogurt[J].J Korean Soc Appl Biol Chem,2009,52(1):76-79.
[19]Seo MH,Lee SY,Chang YH,et al.Physicochemical,microbial,and sensory properties of yogurt supplemented with nanopowdered chitosan during storage[J].J Dairy Sci,2009,92(12):5907-5916.
[20]Huang R,Pan M,Wan C.Physiological and transcriptional responses and cross protection of Lactobacillus plantarum ZDY2013 under acid stress[J].J Dairy Sci,2015,28(9):1-9.
[21]Broadbent JRRL,Larsen V,Deibel JL,et al.Physiological and transcriptional response of Lactobacillus casei ATCC 334 to acid stress[J].J Bacteriol,2010,(192):2445-2458.
[22]Zhao K,Xie Q,Xu D,et al.Antagonistics of Lactobacillus plantarum ZDY2013 against Helicobacter pylori SS1 and its infection in vitro in human gastric epithelial AGS cells[J].J Biosci Bioeng,2018,126(4):458-463.
[23]周方方,吴正钧,艾连中.蛋白组学技术在乳酸菌环境胁迫应激研究中的应用[J].综述与专题评论,2014,38(8):101-106.Zhou FF,Wu ZJ,Ai LZ.Application of proteomics technology in the study of environmental stress and stress of lactobacillus[J].Rev Spec Rev,2014,38(8):101-106.
[24]袁峥.嗜酸乳杆菌耐酸机制研究[D].新乡:河南科技学校,2013.Yuan Z.Study on acidophilus acidophilus resistance mechanism[D].Xinxiang:Henan University of Science and Technology,2013.
[25]Wu C,Zhang J,Chen W,et al.A combined physiological and proteomic approach to reveal lactic acid induced alterration in Lactobacillus casei Zhang and its mutant with enhanced lactic acid tolerance[J].Appl Microbiol Biotechnol,2012,93(2):707-722.
[26]Chen MJ,Tang HY,Chiang ML.Effects of heat,cold,acid and bile salt adaptations on the stress tolerance and protein expression of kefir-isolated probiotic Lactobacillus kefiranofaciens M1[J].Food Microbiol,2017,(66):20-27.
[27]Owaga EE,Chen MJ,Chen WY,et al.Oral toxicity evaluation of kefir-isolated Lactobacillus kefiranofaciens M1 in Sprague-Dawley rats[J].Food Chem Toxicol,2014,(70):157-162.
[28]H?rmann S,Scheyhing C,Behr J,et al.Comparative proteome approach to characterize the high-pressure stress response of Lactobacillus sanfranciscensis DSM 20451[J].Proteomics,2006,6(6):1878-1885.
[29]Song S,Bae DW,Lim K,et al.Cold stress improves the ability of Lactobacillus plantarum L67 to survive freezing[J].Int J Food Microbiol,2014,(191):135-143.
[30]Tsakalidou.Stress responses of lactic acid bacteria[J].Food Microbiol Food Saf,2011,82(1-4):507-518.
[31]Fiocco D,Capozzi V,Goffin P,et al.Improved adaptation to heat,cold,and solvent tolerance in Lactobacillus plantarum[J].Appl Microbiol Biotechnol,2007,(77):909-915.
[32]刘闻.胆盐水解酶提高植物乳杆菌WCFS1肠道黏附的分子机理研究[D].南京:南京师范大学,2017.Liu W.Molecular mechanism of cholinolysis of brine to improve intestinal adhesion of lactobacillus plantagensis WCFS1[D].Nanjing:Nanjing Normal University,2017.
[33]Alcantara C,Zúniga M.Proteomic and transcriptomic analysis of the response to bile stress of Lactobacillus casei BL23[D].Microbiology,2012,(158):1206-1218.
[34]Wu C,He G,Zhang J.Physiological and proteomic analysis of Lactobacillus casei in response to acid adaptation[J].J Ind Microbiol Biotechnol,2014,(41):1533-1540.
[35]Aakko J,Sanchez B,Gueimonde M,et al.Assessment of stress tolerance acquisition in the heat-tolerant derivative strains of Bifidobacterium animalis subsp.lactis BB-12 and Lactobacillus rhamnosus GG[J].J Appl Microbiol,2014,(117):239-248.
[36]Belfiore C,Fadda S,Raya R,et al.Molecular basis of the adaption of the anchovy isolate Lactobacillus sakei CRL1756 to salted environments through a proteomic approach[J].Food Res Int,2013,(54):1334-1341.
[37]Zhai Z,Douillard FP,An H,et al.Proteomic characterization of the acid tolerance response in Lactobacillus delbrueckii subsp.bulgaricus CAUH1and functional identification of a novel acid stressrelated transcriptional regulator Ldb0677[J].Environ Microbiol,2014,(16):1524-1537.
[38]Hamon E,Horvatovich P,Marchioni E,et al.Investigation of potential markers of acid resistance in Lactobacillus plantarum by comparative proteomics[J].J Appl Microbiol,2013,(116):134-144.
[39]Wu R,Zhang W,Sun T,et al.Proteomic analysis of responses of a new probiotic bacterium Lactobacillus casei Zhang to low acid stress[J].Int JFood Microbiol,2011,147(3):181-187.
[40]Wei YX,Ye L,Liu DB,et al.Activation of the chromosomally encoded mazEF(Bif)locus of Bifidobacterium longum under acid stress[J].Int JFood Microbiol,2015,(207):16-22.
[41]Jin J,Zhang B,Guo H,et al.Mechanism analysis of acid tolerance response of Bifidobacterium longum subsp.longum BBMN68 by gene expression profile using RNA-sequencing[J].PLoS One,2012,(7):581-589.
[42]Jin J,Qin Q,Guo H,et al.Effect of pre-stressing on the acid-stress response in bifidobacterium revealed using proteomic and physiological approaches[J].PLoS One,2015,10(2):1-14.
[43]Chen MJ,Tang HY,Chiang ML.Effects of heat,cold,acid and bile salt adaptations on the stress tolerance and protein expression of kefir-isolated probiotic Lactobacillus kefiranofaciens M1[J].Food Microbiol,2017,(66):20-27.
[44]H?rmann S,Scheyhing C,Behr J,et al.Comparative proteome approach to characterize the high-pressure stress response of Lactobacillus sanfranciscensis DSM 20451[J].Proteomics,2006,6(6):1878-1885.
[45]Fiocco D,Capozzi V,Goffin P,et al.Improved adaptation to heat,cold,and solvent tolerance in Lactobacillus plantarum[J].Appl Microbiol Biotechnol,2007,(77):909-915.
[46]贺红军,李宜春,张雪婷,等.微胶囊法提高益生菌抗逆性的研究[J].食品工业,2017,38(2):188-193.He HJ,Li YC,Zhang XT,et al.Improvement of probiotics resistance by microencapsulation method[J].Food Ind,2017,38(2):188-193.
[47]Bosnea LA,Moschakis T,Nigam PS,et al.Growth adaptation of probiotics in biopolymer-based coacervate structures to enhance cell viability[J].LWT-Food Sci Technol,2017,(77):282-289.
[48]Carcoran BM,Stanton C,Fitsgerald G,et al.Life under stress:The probiotic stress response and how it may be manipulated[J].Curr Pharm Des,2008,(14):1382-1399.
[49]Muzammil HS,Javed I,Rasco B,et al.Viability of probiotics in frozen yogurt with different levels of overrun and glycerol[J].Int J Agric Biol,2015,17(3):648-652.
[50]EL-Sayed HS,Salama HH.Production of synbiotic ice cream[J].Int JChem Technol Res,2015,7(1):138-147.
[2]Rutella G,Tagliazucchi D,Solieri L.Survival and bioactivities of selected probiotic lactobacilli in yogurt fermentation and cold storage:New insights for developing a bifunctional dairy food[J].Food Microbiol,2016,(60):54-61.
[3]Lee A,Lee YJ,Yoo HJ,et al.Consumption of dairy yogurt containing Lactobacillus paracasei ssp.paracasei,Bifidobacterium animalis ssp.lactis and heat-treated Lactobacillus plantarum improves immune function including natural killer cell activity[J].Nutrients,2017,9(558):1-9.
[4]Pei R,Martin DA,DiMarco DM,et al.Evidence for the effects of yogurt on gut health and obesity[J].Critic Rev Food Sci Nutr,2017,57(8):1569-1583.
[5]Mirghafourvand M,Rad AH,Charandabi SM,et al.The effect of probiotic yogurt on constipation in pregnant women:A randomized controlled clinical trial[J].Iranian Red Crescent Med J,2016,18(11):1-12.
[6]Zamani B,Golkar HR,Farshbaf S,et al.Clinical and metabolicresponse to probiotic supplementation in patients with rheumatoid arthritis:Arandomized,double-blind,placebo-controlled trial[J].Int J Rheumat Dis,2016,19(9):869-879.
[7]Akkasheh G,Kashani-Poor Z,Tajabadi-Ebrahimi M,et al.Clinical and metabolic response to probiotic administration in patients with major depressive disorder:A randomized,double-blind,placebo-controlled trial[J].Nutrition,2016,32(3):315-320.
[8]Passariello A,Agricole P,Malfertheiner P.A critical appraisal of probiotics(as drugs or food supplements)in gastrointestinal diseases[J].Curr Med Res Opin,2014,30(6):1055-1064.
[9]Lee CL,Liao HL,Lee WC,et al.Standards and labeling of milk fat and spread products in different countries[J].J Food Drug Anal,2018,26(2):469-474.
[10]Settachaimongkon S,Van-Valenberg HJ,Gazi I,et al.Influence of Lactobacillus plantarum WCFS1 on post-acidification,metabolite formation and survival of starter bacteria in set-yoghurt[J].Food Microbiol,2016,(59):14-22.
[11]Settachaimongkon S,Van-Valenberg HJ,Winata V,et al.Effect of sublethal preculturing on the survival of probiotics and metabolite formation in set-yoghurt[J].Food Microbiol,2015,(49):104-115.
[12]Mohammadi R,Sohrabvandi S,Mohammad-Mortazavian A.The starter culture characteristics of probiotic microorganisms in fermented milks[J].Eng Lif Sci,2012,(12):399-409.
[13]于志会,张雪,李盛钰,等.植物乳杆菌C88在酸奶中的应用[J].中国乳品工业,2013,41(7):27-30.Yu ZH,Zhang X,Li SY,et al.Application of lactobacillus plantarum C88in yogurt[J].China Dairy Ind,2013,41(7):27-30.
[14]孙健,陈静,翁心刚.酸奶的储存温度对产品品质的影响[J].食品工业科技,2010,(12):140-141.Sun J,Chen J,Weng XG.Effect of storage temperature of yogurt on product quality[J].Food Ind Sci Technol,2010,(12):140-141.
[15]白凤翎.凝固型酸奶在贮藏过程中的微生物菌相演变研究[J].食品研究与开发,2005,(4):8-10.Bai FL.Microbiological evolution of solidified yoghurt during storage[J].Food Res Dev,2005,(4):8-10.
[16]高微娟,张富新,陈伟.贮藏温度对酸羊奶品质的影响[J].农产品加工(学刊),2011,(1):18-20.Gao WJ,Zhang FX,Chen W.Effect of storage temperature on quality of sour goat milk[J].Proc Agric Prod,2011,(1):18-20.
[17]Abdelazez A,Muhammad Z,Zhang QX,et al.Production of a functional frozen yogurt fortified with Bifidobacterium spp[J].Biol Med Res Int,2017,(6438528):1-10.
[18]Kim SH,Lim CH,Lee C.Optimization of growth and storage conditions for lactic acid bacteria in yogurt and frozen yogurt[J].J Korean Soc Appl Biol Chem,2009,52(1):76-79.
[19]Seo MH,Lee SY,Chang YH,et al.Physicochemical,microbial,and sensory properties of yogurt supplemented with nanopowdered chitosan during storage[J].J Dairy Sci,2009,92(12):5907-5916.
[20]Huang R,Pan M,Wan C.Physiological and transcriptional responses and cross protection of Lactobacillus plantarum ZDY2013 under acid stress[J].J Dairy Sci,2015,28(9):1-9.
[21]Broadbent JRRL,Larsen V,Deibel JL,et al.Physiological and transcriptional response of Lactobacillus casei ATCC 334 to acid stress[J].J Bacteriol,2010,(192):2445-2458.
[22]Zhao K,Xie Q,Xu D,et al.Antagonistics of Lactobacillus plantarum ZDY2013 against Helicobacter pylori SS1 and its infection in vitro in human gastric epithelial AGS cells[J].J Biosci Bioeng,2018,126(4):458-463.
[23]周方方,吴正钧,艾连中.蛋白组学技术在乳酸菌环境胁迫应激研究中的应用[J].综述与专题评论,2014,38(8):101-106.Zhou FF,Wu ZJ,Ai LZ.Application of proteomics technology in the study of environmental stress and stress of lactobacillus[J].Rev Spec Rev,2014,38(8):101-106.
[24]袁峥.嗜酸乳杆菌耐酸机制研究[D].新乡:河南科技学校,2013.Yuan Z.Study on acidophilus acidophilus resistance mechanism[D].Xinxiang:Henan University of Science and Technology,2013.
[25]Wu C,Zhang J,Chen W,et al.A combined physiological and proteomic approach to reveal lactic acid induced alterration in Lactobacillus casei Zhang and its mutant with enhanced lactic acid tolerance[J].Appl Microbiol Biotechnol,2012,93(2):707-722.
[26]Chen MJ,Tang HY,Chiang ML.Effects of heat,cold,acid and bile salt adaptations on the stress tolerance and protein expression of kefir-isolated probiotic Lactobacillus kefiranofaciens M1[J].Food Microbiol,2017,(66):20-27.
[27]Owaga EE,Chen MJ,Chen WY,et al.Oral toxicity evaluation of kefir-isolated Lactobacillus kefiranofaciens M1 in Sprague-Dawley rats[J].Food Chem Toxicol,2014,(70):157-162.
[28]H?rmann S,Scheyhing C,Behr J,et al.Comparative proteome approach to characterize the high-pressure stress response of Lactobacillus sanfranciscensis DSM 20451[J].Proteomics,2006,6(6):1878-1885.
[29]Song S,Bae DW,Lim K,et al.Cold stress improves the ability of Lactobacillus plantarum L67 to survive freezing[J].Int J Food Microbiol,2014,(191):135-143.
[30]Tsakalidou.Stress responses of lactic acid bacteria[J].Food Microbiol Food Saf,2011,82(1-4):507-518.
[31]Fiocco D,Capozzi V,Goffin P,et al.Improved adaptation to heat,cold,and solvent tolerance in Lactobacillus plantarum[J].Appl Microbiol Biotechnol,2007,(77):909-915.
[32]刘闻.胆盐水解酶提高植物乳杆菌WCFS1肠道黏附的分子机理研究[D].南京:南京师范大学,2017.Liu W.Molecular mechanism of cholinolysis of brine to improve intestinal adhesion of lactobacillus plantagensis WCFS1[D].Nanjing:Nanjing Normal University,2017.
[33]Alcantara C,Zúniga M.Proteomic and transcriptomic analysis of the response to bile stress of Lactobacillus casei BL23[D].Microbiology,2012,(158):1206-1218.
[34]Wu C,He G,Zhang J.Physiological and proteomic analysis of Lactobacillus casei in response to acid adaptation[J].J Ind Microbiol Biotechnol,2014,(41):1533-1540.
[35]Aakko J,Sanchez B,Gueimonde M,et al.Assessment of stress tolerance acquisition in the heat-tolerant derivative strains of Bifidobacterium animalis subsp.lactis BB-12 and Lactobacillus rhamnosus GG[J].J Appl Microbiol,2014,(117):239-248.
[36]Belfiore C,Fadda S,Raya R,et al.Molecular basis of the adaption of the anchovy isolate Lactobacillus sakei CRL1756 to salted environments through a proteomic approach[J].Food Res Int,2013,(54):1334-1341.
[37]Zhai Z,Douillard FP,An H,et al.Proteomic characterization of the acid tolerance response in Lactobacillus delbrueckii subsp.bulgaricus CAUH1and functional identification of a novel acid stressrelated transcriptional regulator Ldb0677[J].Environ Microbiol,2014,(16):1524-1537.
[38]Hamon E,Horvatovich P,Marchioni E,et al.Investigation of potential markers of acid resistance in Lactobacillus plantarum by comparative proteomics[J].J Appl Microbiol,2013,(116):134-144.
[39]Wu R,Zhang W,Sun T,et al.Proteomic analysis of responses of a new probiotic bacterium Lactobacillus casei Zhang to low acid stress[J].Int JFood Microbiol,2011,147(3):181-187.
[40]Wei YX,Ye L,Liu DB,et al.Activation of the chromosomally encoded mazEF(Bif)locus of Bifidobacterium longum under acid stress[J].Int JFood Microbiol,2015,(207):16-22.
[41]Jin J,Zhang B,Guo H,et al.Mechanism analysis of acid tolerance response of Bifidobacterium longum subsp.longum BBMN68 by gene expression profile using RNA-sequencing[J].PLoS One,2012,(7):581-589.
[42]Jin J,Qin Q,Guo H,et al.Effect of pre-stressing on the acid-stress response in bifidobacterium revealed using proteomic and physiological approaches[J].PLoS One,2015,10(2):1-14.
[43]Chen MJ,Tang HY,Chiang ML.Effects of heat,cold,acid and bile salt adaptations on the stress tolerance and protein expression of kefir-isolated probiotic Lactobacillus kefiranofaciens M1[J].Food Microbiol,2017,(66):20-27.
[44]H?rmann S,Scheyhing C,Behr J,et al.Comparative proteome approach to characterize the high-pressure stress response of Lactobacillus sanfranciscensis DSM 20451[J].Proteomics,2006,6(6):1878-1885.
[45]Fiocco D,Capozzi V,Goffin P,et al.Improved adaptation to heat,cold,and solvent tolerance in Lactobacillus plantarum[J].Appl Microbiol Biotechnol,2007,(77):909-915.
[46]贺红军,李宜春,张雪婷,等.微胶囊法提高益生菌抗逆性的研究[J].食品工业,2017,38(2):188-193.He HJ,Li YC,Zhang XT,et al.Improvement of probiotics resistance by microencapsulation method[J].Food Ind,2017,38(2):188-193.
[47]Bosnea LA,Moschakis T,Nigam PS,et al.Growth adaptation of probiotics in biopolymer-based coacervate structures to enhance cell viability[J].LWT-Food Sci Technol,2017,(77):282-289.
[48]Carcoran BM,Stanton C,Fitsgerald G,et al.Life under stress:The probiotic stress response and how it may be manipulated[J].Curr Pharm Des,2008,(14):1382-1399.
[49]Muzammil HS,Javed I,Rasco B,et al.Viability of probiotics in frozen yogurt with different levels of overrun and glycerol[J].Int J Agric Biol,2015,17(3):648-652.
[50]EL-Sayed HS,Salama HH.Production of synbiotic ice cream[J].Int JChem Technol Res,2015,7(1):138-147.