Advances in the Mechanisms of the Effects of Pseudomonas spp. Proteases on Gelatinization of Dairy Products and Regulation Strategies

Authors

Shengjia LIU, Food Science College , Shenyang Agricultural University , Shenyang 110866 , China;Liaoning Food Fermentation Technology Engineering Research Center , Shenyang 110866 , China;Shenyang Key Laboratory of Microbial Fermentation Technology Innovation , Shenyang 110866 , China
Kairu HE, Food Science College , Shenyang Agricultural University , Shenyang 110866 , China;Liaoning Food Fermentation Technology Engineering Research Center , Shenyang 110866 , China;Shenyang Key Laboratory of Microbial Fermentation Technology Innovation , Shenyang 110866 , China
Hui LI, Inner Mongolia Mengniu Dairy (Group ) Joint-stock Limited Liability Company , Hohhot 010010 , China
Yongqiang LI, Mengniu Dairy (Ulanhot ) Limited Liability Company , Ulanhot 137400 , China
Xinyi LI, Mengniu Dairy (Qiqihar ) Limited Company , Qiqihar 161000 , China
Rina WU, Food Science College , Shenyang Agricultural University , Shenyang 110866 , China;Liaoning Food Fermentation Technology Engineering Research Center , Shenyang 110866 , China;Shenyang Key Laboratory of Microbial Fermentation Technology Innovation , Shenyang 110866 , China
Junrui WU, Food Science College , Shenyang Agricultural University , Shenyang 110866 , China;Liaoning Food Fermentation Technology Engineering Research Center , Shenyang 110866 , China;Shenyang Key Laboratory of Microbial Fermentation Technology Innovation , Shenyang 110866 , China
Sanjun ZHAO, Inner Mongolia Mengniu Dairy (Group ) Joint-stock Limited Liability Company , Hohhot 010010 , ChinaFollow

Corresponding Author(s)

赵三军（1983—），男，学士，工程师，主要从事质量安全追溯及乳制品加工研究。E-mail：zhaosanjun@mengniu.cn

Abstract

Gelatinization of dairy products has long been a common problem caused by heat-resistant protease contamination.Despite the high-temperature sterilization process,heat-resistant proteases secreted by Pseudomonas spp.can survive under extreme conditions.These enzymes can break down the peptide bonds of proteins in milk,resulting in the bitter taste and gelatinization of milk,which seriously affects the quality and shelf-life of dairy products.In view of the complex gelatinization mechanisms of dairy products and the lack of in-depth investigation,the author systematically outlined the characteristics,enzyme activity detection methods,and influencing factors of Pseudomonas spp.proteases,delved into the mechanisms underpinning the protease-induced gelatinization of dairy products,and summarized the control measures and novel regulatory strategies in the production process.The review is expected to provide a reference for the rapid identification of heat-resistant proteases and the inhibition of gelatinization of dairy products due to protease residues.

Publication Date

9-15-2025

First Page

12

Last Page

22

DOI

10.12441/spyswjs.20240626001

Recommended Citation

LIU, Shengjia; HE, Kairu; LI, Hui; LI, Yongqiang; LI, Xinyi; WU, Rina; WU, Junrui; and ZHAO, Sanjun (2025) "Advances in the Mechanisms of the Effects of Pseudomonas spp. Proteases on Gelatinization of Dairy Products and Regulation Strategies," Journal of Food Science and Biotechnology: Vol. 44: Iss. 9, Article 2.
DOI: 10.12441/spyswjs.20240626001
Available at: https://spsw.spyswjs.cnjournals.com/journal/vol44/iss9/2

References

［1］ BRUZAROSKI S R，DE SOUZA CORREIA S，DA SILVA DEVARA L F，et al.Influence of the storage temperature of raw sheep milk on the spoilage potential of Pseudomon as spp.［J］.Small Ruminant Research，2023，224：106998.
［2］ GAGNON M，JEAN S，DE TORO-MARTÍN J，et al.Biofilm formation in dairy：a food safety concern：insights into the prevalence of Pseudomonadota and yeasts on milking system surface biofilms ［J］.Journal of Dairy Science，2025，108（8）：8141 -8156.
［3］ DAI L C，HU S Z，PANG X Y，et al.Community diversity of psychrophilic bacteria in dairy farm raw milk and its characteristic enzyme production at different temperature［J］.Food Bioscience，2023，54：102921.
［4］ 胡子毅.利用噬菌体控制原料乳中假单胞菌的研究［D］.哈尔滨：东北农业大学，2013.
［5］ 胡少震.原料乳中嗜冷菌污染源分析及快速检测方法的建立［D］.阿拉尔：塔里木大学，2023.
［6］ DU B Y，MENG L，LIU H M，et al.Diversity and proteolytic activity of Pseudomonas species isolated from raw cow milk samples across China ［J］.Science of the Total Environment，2022，838：156382.
［7］ MEJARES C T，HUPPERTZ T，CHANDRAPALA J.Thermal processing of buffalo milk：a review ［J］.International Dairy Journal，2022，129：105311.
［8］ STOECKEL M，LIDOLT M，ACHBERGER V，et al.Growth of Pseudomonas weihenstephanensis，Pseudomon as proteolytica and Pseudomonas sp.in raw milk：impact of residual heat-stable enzyme activity on stability of UHT milk during shelf-life ［J］.International Dairy Journal，2016，59：20-28.
［9］ 张熙，母智深.原料乳中产耐热蛋白酶细菌的鉴定及其酶学特性研究 ［J］.食品与生物技术学报，2013，32（3）：272-278.ZHANG X，MU Z S.Identification of a bacterium isolated from raw milk and study on its heat-stable protease ［J］.Journal of Food Science and Biotechnology，2013，32（3）：272-278.（in Chinese ）
［10］ ZHANG D，PALMER J，TEH K H，et al.16S rDNA high-throughput sequencing and MALDI-TOF MS are complementary when studying psychrotrophic bacterial diversity of raw cows ’ milk［J］.International Dairy Journal，2019，97：86-91.
［11］ 邓应旺，韩雪，涂远强，等.牛乳中嗜冷菌产耐热酶的研究进展［J］.食品工业，2021，42（6）：373-377.DENG Y W，HAN X，TU Y Q，et al.Research progress on thermostable enzymes produced by psychrophilic bacteria in milk ［J］.The Food Industry，2021，42（6）：373-377.（in Chinese ）
［12］ MAIER C，HUPTAS C，VON NEUBECK M，et al.Genetic organization of the aprX-lipA 2 operon affects the proteolytic potential of Pseudomonas species in milk ［J］.Frontiers in Microbiology，2020，11：1190.
［13］ AGUILERA-TORO M，KRAGH M L，THOMASEN A V，et al.Proteolytic activity and heat resistance of the protease AprX from Pseudomonas in relation to genotypic characteris tics［J］.International Journal of Food Microbiology，2023，391-393：110147.
［14］ MAIER C，HOFMANN K，HUPTAS C，et al.Simultaneous quantification of the most common and proteolytic Pseudomonas species in raw milk by multiplex qPCR［J］.Applied Microbiology and Biotechnology，2021，105（4）：1693 -1708.
［15］ MARTINS M L，PINTO U M，RIEDEL K，et al.Milk-deteriorating exoenzymes from Pseudomonas fluorescens 041 isolated from refrigerated raw milk ［J］.Brazilian Journal of Microbiology，2015，46（1）：207-217.
［16］ CALDERA L，FRANZETTI L，VAN COILLIE E，et al.Identification，enzymatic spoilage characterization and proteolytic activity quantification of Pseudomonas spp.isolated from different foods ［J］.Food Microbiology，2016，54：142-153.
［17］ 张淑娟，李华军，乌鲁科晖，等.荧光假单胞菌 BJ10蛋白酶生成及乳蛋白降解研究 ［J］.食品加工与保鲜，2015，39（6）：2466 -2472.ZHANG S J，LI H J，WU L K H，et al.Studies on protease production and milk protein degradation by Pseudomonas fluorescens BJ10［J］.Food Processing and Preservation，2015，39（6）：2466 -2472.
［18］ WOODS R G，BURGER M，BEVEN C A，et al.The aprX-lipA operon of Pseudomonas fluorescens B52：a molecular analysis of metalloprotease and lipase production ［J］.Microbiology，2001，147（2）：345-354.
［19］ VON NEUBECK M，BAUR C，KREWINKEL M，et al.Biodiversity of refrigerated raw milk microbiota and their enzymatic spoilage potential ［J］.International Journal of Food Microbiology，2015，211：57-65.
［20］ 韦盘秋，黄丽，黄加祥，等.乳及乳制品中假单胞菌危害及控制研究进展 ［J］.食品科技，2021，46（9）：311-315.WEI P Q，HUANG L，HUANG J X，et al.Research progress on the harm and control of Pseudomonas in milk and dairy products ［J］.Food Science and Technology，2021，46（9）：311-315.（in Chinese ）
［21］ 王一恬.荧光假单胞菌耐热性蛋白酶特性及其对液态乳品质影响的研究 ［D］.哈尔滨：东北农业大学，2023.
［22］ 祁岩.液态乳中荧光假单胞菌生长及产酶情况分析［J］.食品科技，2023，48（7）：50-56.QI Y.Analysis of growth and protease production of Pseudomonas fluorescens in liquid milk ［J］.Food Science and Technology，2023，48（7）：50-56.（in Chinese ）
［23］ 任泽宇.假单胞菌对牛乳品质危害的种间差异分析及种水平检测技术开发 ［D］.无锡：江南大学，2022.
［24］ ZHANG S W，LI H J，ULUKO H，et al.Investigation of protease production by Pseudomonas fluorescens BJ- 10 and degradation on milk proteins ［J］.Journal of Food Processing and Preservation，2015，39（6）：2466 -2472.
［25］ BAGLINIÈRE F，MATÉOS A，TANGUY G，et al.Proteolysis of ultra high temperature-treated casein micelles by AprX enzyme from Pseudomonas fluorescens F induces their destabilisation ［J］.International Dairy Journal，2013，31（2）：55-61.
［26］ VOLK V，GLÜCK C，LEPTIHN S，et al.Two heat resistant endopeptidases from Pseudomonas species with destabilizing potential during milk storage ［J］.Journal of Agricultural and Food Chemistry，2019，67（3）：905-915.
［27］ MOHAMED AHMED I A，BABIKER E E，MORI N.pH stability and influence of salts on activity of a milk-clotting enzyme from Solanum dubium seeds and its enzymatic action on bovine caseins ［J］.LWT-Food Science and Technology，2010，43（5）：759-764.
［28］ 王学标，何云侠，刘强.福林酚试剂法与缩二脲试剂法测定饲用蛋白酶活力的比较分析 ［J］.畜牧与兽医，2019，51（3）：23-28.WANG X B，HE Y X，LIU Q.Comparison of the folinphenol reagent method and the biuret reagent method for determination of feed protease activity ［J］.Animal Husbandry & Veterinary Medicine，2019，51（3）：23-28.（in Chinese ）
［29］ QIN X，CHENG J Q，QI X H，et al.Effect of thermostable enzymes produced by psychrotrophic bacteria in raw milk on the quality of ultra-high temperature sterilized milk ［J］.Foods，2023，12（20）：3752.
［30］ YUAN L，SADIQ F A，LIU T J，et al.Spoilage potential of psychrotrophic bacteria isolated from raw milk and the thermo-stability of their enzymes ［J］.Journal of Zhejiang University：Science B （Biomedicine & Biotechnology ），2018，19（8）：630-642.
［31］ 杨旭，屈小玄，郭庆贺，等.牛奶中蛋白酶活力测定的方法比较［J］.中国乳品工业，2014，42（6）：48-51.YANG X，QU X X，GUO Q H，et al.Comparison of different determination methods of protease activity in the milk［J］.China Dairy Industry，2014，42（6）：48-51.（in Chinese）
［32］ BUTTON P D，ROGINSKI H，DEETH H C，et al.Improved shelf life estimation of UHT milk by prediction of proteolysis ［J］.Journal of Food Quality，2011，34（4）：229-235.
［33］ DACRES H，WEIHS F，WANG J，et al.Bioluminescence resonance energy transfer biosensor for measuring activity of a protease secreted by Pseudomonas fluorescens growing in milk ［J］.Analytica Chimica Acta，2023，1270：341401.
［34］ XIA N，LIU G，YI X Y.Surface plasmon resonance for protease detection by integration of homogeneous reaction［J］.Biosensors，2021，11（10）：362.
［35］ 王春燕，任彩霞，任向东，等.影响超高温灭菌乳货架期品质的相关蛋白酶类研究进展 ［J］.现代食品，2020，26（18）：40-47.WANG C Y，REN C X，REN X D，et al.Research progress of proteases related to shelf life quality of UHT milk［J］.Modern Food，2020，26（18）：40-47.（in Chinese）
［36］ STOECKEL M，LIDOLT M，STRESSLER T，et al.Heat stability of indigenous milk plasmin and proteases from Pseudomonas：a challenge in the production of ultra-high temperature milk products ［J］.International Dairy Journal，2016，61：250-261.
［37］ 王月，张荣先，张旦旦，等.碳氮源对 Bacillus pumilus K9产角蛋白酶的影响 ［J］.食品与生物技术学报，2014，33（10）：1077 -1083.WANG Y，ZHANG R X，ZHANG D D，et al.Effects of carbon and nitrogen sources on keratinase production from Bacillus pumilus K9［J］.Journal of Food Science and Biotechnology，2014，33（10）：1077 -1083.（in Chinese）［38］ 肖琳，吴安宁，许国超，等.恶臭假单胞菌的正丁醇耐受性驯化及比较基因组学分析 ［J］.食品与生物技术学报，2020，39（10）：65-72.XIAO L，WU A N，XU G C，et al.Adapting n-butanol tolerance of Pseudomonas putida and comparative genomics analysis ［J］.Journal of Food Science and Biotechnology，2020，39（10）：65-72.（in Chinese ）
［39］ 解万翠，杨亚东，杨锡洪，等.蜡状芽孢杆菌产中性蛋白酶发酵条件的优化 ［J］.食品与机械，2016，32（1）：26-30，129.XIE W C，YANG Y D，YANG X H，et al.Response surface methodology optimization of fermentation conditions for neutral protease production by Bacillus cereus［J］.Food & Machinery，2016，32（1）：26-30，129.（in Chinese ）
［40］ MACHADO S G，HEYNDRICKX M，DE BLOCK J，et al.Identification and characterization of a heat-resistant protease from Serratia liquefaciens isolated from Brazilian cold raw milk ［J］.International Journal of Food Microbiology，2016，222：65-71.
［41］ DU B Y，LU M，LIU H M，et al.Pseudomonas isolates from raw milk with high level proteolytic activity display reduced carbon substrate utilization and higher levels of antibiotic resistance ［J］.LWT-Food Science and Technology，2023，181：114766.
［42］ 乔传丽，金丹，蒋艾廷，等.传统发酵酸牛奶中产蛋白酶酵母菌的筛选及产酶条件优化 ［J］.食品与生物技术学报，2019，38（5）：73-78.QIAO C L，JIN D，JIANG A T，et al.Screening of yeast with high protease activities from traditional yogurt and optimization of enzyme conditions ［J］.Journal of Food Science and Biotechnology，2019，38（5）：73-78.（in Chinese）
［43］ ALVES M P，SALGADO R L，ELLER M R，et al.Temperature modulates the production and activity of a metalloprotease from Pseudomonas fluorescens 07A in milk［J］.Journal of Dairy Science，2018，101（2）：992-999.
［44］ CHANG G H，LI Q H，WANG T，et al.Characterization of Pseudomonas spp.contamination and in situ spoilage potential in pasteurized milk production process ［J］.Food Research International，2024，188：114463.
［45］ 张书文，刘鹭，李红娟，等.荧光假单胞菌胞外蛋白酶的纯化及特性研究 ［J］.食品科学，2012，33（9）：206-210.ZHANG S W，LIU L，LI H J，et al.Purification and properties of extracellular protease from Pseudomonads fluorescens ［J］.Food Science，2012，33（9）：206-210.（in Chinese）
［46］ MU Z S，DU M，BAI Y.Purification and properties of a heat-stable enzyme of Pseudomonas fluorescens Rm 12 from raw milk ［J］.European Food Research and Technology，2009，228（5）：725-734.
［47］ 杨梦莹，庞坤容，潘江欣，等.一株可高效降解羽毛的铜绿假单胞菌的分离、鉴定、产酶条件优化及其酶活研究［J］.微生物学报，2022，62（3）：968-981.YANG M Y，PANG K R，PAN J X，et al.Isolation，identification，enzyme production optimization，and enzyme activity study of Pseudomonas aeruginosa strain capable of efficient feather degradation ［J］.Acta Microbiologica Sinica，2022，62（3）：968-981.（in Chinese）
［48］ ANEMA S G.Age gelation，sedimentation，and creaming in UHT milk：a review［J］.Comprehensive Reviews in Food Science and Food Safety，2019，18（1）：140-166.
［49］ ZHANG C Y，BIJL E，SVENSSON B，et al.The extracellular protease AprX from Pseudomonas and its spoilage potential for UHT milk：a review ［J］.Comprehensive Reviews in Food Science and Food Safety，2019，18（4）：834-852.
［50］ 汤恒，张娟，堵国成.高效降解牛奶过敏原蛋白酶菌种筛选及其重组植物过氧化氢酶的功能验证 ［J］.食品与生物技术学报，2021，40（3）：28-34.TANG H，ZHANG J，DU G C.Study on milk-allergen-degradation protease screening and its recombinant vegetative catalase function verification ［J］.Journal of Food Science and Biotechnology，2021，40（3）：28-34.（in Chinese）
［51］ WANG T，LI Y D，DE WITTE F，et al.Influence of calcium concentration on the re-assembly of sodium caseinate into casein micelles and on their renneting behavior［J］.Food Research International，2024，180：113991.
［52］ REITER M，REITMAIER M，HASLBECK A，et al.Acid gelation functionality of casein micelles in concentrated state：influence of calcium supplementation or chelation combined with enzymatic stabilization ［J］.Food Hydrocolloids，2023，143：108927.
［53］ 邵言蹊，刘大松，徐姝，等.低温诱导羊乳中 β-酪蛋白从胶束中解离的研究 ［J］.食品与生物技术学报，2021，40（8）：27-36.SHAO Y X，LIU D S，XU S，et al.Dissociation of β-casein from casein micelles in skimmed goat milk at low temperature ［J］.Journal of Food Science and Biotechnology，2021，40（8）：27-36.（in Chinese ）
［54］ 王辉，吕加平，迟玉杰.耐热蛋白酶对 UHT乳品质的影响及控制方法研究进展 ［J］.中国乳品工业，2007，35（9）：25-29.WANG H，LV J P，CHI Y J.Recent studies on effect of heat resistant enzymes on UHT milk quality and control measures［J］.China Dairy Industry，2007，35（9）：25-29.（in Chinese ）
［55］ 刘美凤，付瑞东，姚春艳，等.加压 CO 2对乳中荧光假单胞菌杀菌效果的研究 ［J］.中国乳品工业，2012，40（6）：39-42.LIU M F，FU R D，YAO C Y，et al.Sterilization effect of pressurized CO 2 on Pseudomonas fluorescens in milk［J］.China Dairy Industry，2012，40（6）：39-42.（in Chinese ）
［56］ ZHENG A R，WEI C K，LIU J，et al.Deciphering the mechanism by which carbon dioxide extends the shelf life of raw milk：a microbiomics- and metabolomics-based approach［J］.Molecules，2024，29（2）：329.
［57］ LO R，TURNER M S，WEEKS M，et al.Culture-independent bacterial community profiling of carbon dioxide treated raw milk ［J］.International Journal of Food Microbiology，2016，233：81-89.
［58］ SAHA S，MAJUMDER R，ROUT P，et al.Unveiling the significance of psychrotrophic bacteria in milk and milk product spoilage：a review［J］.The Microbe，2024，2：100034.
［59］ 张鹏博，李智星，魏勇，等.不同检测时间对原料乳中嗜冷菌检测结果的影响 ［J］.中国乳业，2023（1）：77-80.ZHANG P B，LI Z X，WEI Y，et al.Effect of different detection time on the detection results of psychrophilein raw milk［J］.China Dairy，2023（1）：77-80.（in Chinese ）
［60］ ZHANG D，LI S Q，PALMER J，et al.The relationship between numbers of Pseudomonas bacteria in milk used to manufacture UHT milk and the effect on product quality［J］.International Dairy Journal，2020，105：104687.
［61］ 郭奇慧，白雪，胡新宇，等.UHT乳贮存期间蛋白变化的研究［J］.食品工业科技，2008，29（12）：143-144，147.GUO Q H，BAI X，HU X Y，et al.Study on protein changes of UHT milk during storage ［J］.Science and Technology of Food Industry，2008，29（12）：143-144，147.（in Chinese ）
［62］ 张书文.原料乳中耐热蛋白酶对 UHT乳品质的影响研究［D］.北京：中国农业科学院，2012.
［63］ 艾正文，徐致远，叶景锦.动态高压处理对牛乳中生物活性物质的影响 ［J］.食品与机械，2023，39（7）：210-214，222.AI Z W，XU Z Y，YE J J.Effects of dynamic high-pressure processing on bioactive components of milk ［J］.Food & Machinery，2023，39（7）：210-214，222.（in Chinese）
［64］ 艾正文.超高压均质对牛乳中微生物和酶的影响研究进展［J］.乳业科学与技术，2022，45（2）：42-46.AI Z W.Recent progress in understanding the effects of ultra-high pressure homogenization on microorganisms and enzymes in milk ［J］.Journal of Dairy Science and Technology，2022，45（2）：42-46.（in Chinese ）
［65］ DE OLIVEIRA M M，DE CASTRO LEITE B R，TRIBST A A L，et al.Use of high pressure homogenization to reduce milk proteolysis caused by Pseudomonas fluorescens protease［J］.LWT-Food Science and Technology，2018，92：272-275.
［66］ Khaled Sobhy Nassar Nassar.超高压处理改善羊乳及其酪蛋白胶束的理化和流变特性 ［D］.北京：中国农业科学院，2020.
［67］ 马蓉.超声处理对脱脂牛乳功能性质及蛋白结构的影响［D］.兰州：甘肃农业大学，2018.
［68］ WANG D，FRITSCH J，MORARU C I.Shelf life and quality of skim milk processed by cold microfiltration with a 1.4-μm pore size membrane，with or without heat treatment［J］.Journal of Dairy Science，2019，102（10）：8798 -8806.
［69］ LOTT T T，MARTIN N H，DUMPLER J，et al.Microbacterium represents an emerging microorganism of concern in microfiltered extended shelf-life milk products［J］.Journal of Dairy Science，2023，106（12）：8434 -8448.
［70］ VAN DER SCHAAF J M，GOULDING D A，FUERER C，et al.A novel approach to isolation of β-casein from micellar casein concentrate by cold microfiltration combined with chymosin treatment ［J］.International Dairy Journal，2024，148：105796.
［71］ BAULAND J，FAMELART M H，FAIVELEY M，et al.Rheological properties of enzymatic milk gel：effect of ion partitioning in casein micelles ［J］.Food Hydrocolloids，2022，130：107739.
［72］ RIVERA FLORES V K，DEMARSH T A，ALCAINE S D.Lactose oxidase：enzymatic control of Pseudomonas to delay age gelation in UHT milk ［J］.Journal of Dairy Science，2021，104（3）：2758 -2772.
［73］ WANG W，LI B Y，ZHANG M J，et al.Microfluidic emulsification techniques for controllable emulsion production and functional microparticle synthesis ［J］.Chemical Engineering Journal，2023，452：139277.
［74］ MUDGIL P，AL DHAHERI M K O，ALSUBOUSI M S M，et al.Molecular docking studies on α-amylase inhibitory peptides from milk of different farm animals［J］.Journal of Dairy Science，2024，107（5）：2633 -2652.
［75］ ASHOKBHAI J K，BASAIAWMOIT B，DAS S，et al.Antioxidative，antimicrobial and anti-inflammatory activiti es and release of ultra-filtered antioxidative and antimicrobi al peptides during fermentation of sheep milk：in-vitro，in-silico and molecular interaction studies ［J］.Food Bioscience，2022，47：101666.