Expression of milk-derived angiotensin I-converting enzyme-inhibitory peptides in Lactococcus lactis

Angiotensin I-converting enzyme (ACE) plays a key role in the regulation of blood pressure. Currently, most single or tandem repeats of ACE-inhibitory (ACE-I) peptides have been expressed in Escherichia coli. However, in this study, a food-grade system was constructed using Lactococcus lactis (L. lactis) to simultaneously express four different milk-derived ACE-I peptides with antihypertensive activity. Mixed peptides (MPs) fused with green fluorescent protein (GFP) and eight histidines were synthesized. To ensure potent ACE inhibition by the MPs in human digestive juice, pepsin and trypsin cleavage sites were introduced among the four ACE-I peptides. The MP fusion gene was inserted into expression vector pSEC-E7 with nisin induction and expressed in L. lactis NZ9000, then purified by affinity chromatography. The transformants containing pSEC-MP:GFP were identified based on green fluorescence using Leica laser scanning confocal microscopy. The target proteins were detected by SDS-PAGE analysis and displayed obvious immunogenicity by western blot. After hydrolysis with digestive enzymes, the IC₅₀ of the MPs was 118.63 μM. These results suggested that multiple milk-derived ACE-I peptides with antihypertensive properties could be produced using a food-grade lactococcal expression system.     

乳酸菌两组分食品级选择标记复制子缺失质粒的构建

乳酸菌NICE系统的两组分食品级选择标记具有显性和互补选择标记的优点。含红霉素抗性选择标记的复制子缺失的伴生型质粒是依赖复制子完全且无选择标记的食品级克隆载体而复制的。构建的伴生型质粒的Emr和rep分别来源于pMG36e和pRAF800,经酶切、缺刻、补平和连接得到只能在红霉素选择压力下与θ型复制子的克隆载体共存的复制子缺失伴生型质粒。     

Enhancement of nisin production in milk by conjugal transfer of the protease‐lactose plasmid pLP712 to the wild strain Lactococcus lactis UQ2

Lactococcus lactis UQ2 is a wild nisin A producer isolated from a Mexican cheese that grows poorly in milk. Conjugal matings with L. lactis NCDO712 to transfer the Lac+ Prt+ plasmid pLP712 and selection with nisin and lactose yielded L. lactis NCDO712 NisA+. Naturally rifampicin resistant L. lactis UQ2Rif was isolated to provide an additional selective marker. The identity of a transconjugant L. lactis UQ2Rif Lac+ was confirmed by RAPD‐PCR fingerprinting, nisA PCR amplification, nisin production, presence of pLP712 and phospho‐β‐galactosidase activity. This strain performed well in milk and synthesised 200 IU/mL nisin, 40 times more than the original strain.     

Heterologous extracellular production of enterocin P in <Emphasis Type="Italic">Lactococcus lactis</Emphasis> by a food-grade expression system

In order to develop an entirely food-grade enterocin P expression system for the food industry, the enterocin P structural gene (entP) with or without the enterocin P immunity gene (entiP) was cloned in plasmid pLEB590 under control of the lactococcal constitutive promoter P₄₅. Introduction of the recombinant vectors in L. lactis MG1614 resulted in production of biologically active enterocin P in the supernatants of recombinant L. lactis MG1614. Moreover, coexpression of the entP and entiP genes could increase the production of enterocin P in all L. lactis MG1614 hosts. Recombinant enterocin P from L. lactis MG1614 (pLEB590-entP2) was purified by a three-step procedure involving ammonium sulfate precipitation, SP-Sepharose Fast Flow cation exchange, and hydrophobic adsorption chromatography. The purified bacteriocin protein concentration from recombinant L. lactis MG1614 (pLEB590-entP2) was 3.9-fold greater than that of E. faecium LM-2, and the final recovery of enterocin P activity from the supernatant of L. lactis MG1614 (40.2%) was dramatically improved compared with that of the native host strain (19.9%). Bacteriocin activity and Tricine-SDS–PAGE analysis revealed that purified recombinant enterocin P is biologically active and has a molecular mass corresponding to the native enterocin P from E. faecium LM-2, suggesting that the synthesis, process, and secretion of enterocin P progresses efficiently in recombinant L. lactis MG1614 hosts. The enterocin P was expressed successfully in this food-grade system.     

Optimization of Nisin Production by Lactococcus lactis UQ2 Using Supplemented Whey as Alternative Culture Medium

Abstract: Lactococcus lactis UQ2 is a nisin A-producing native strain. In the present study, the production of nisin by L. lactis UQ2 in a bioreactor using supplemented sweet whey (SW) was optimized by a statistical design of experiments and response surface methodology (RSM). In a 1st approach, a fractional factorial design (FFD) of the order 2^5-1 with 3 central points was used. The effect on nisin production of air flow, SW, soybean peptone (SP), MgSO₄/MnSO₄ mixture, and Tween 80 was evaluated. From FFD, the most significant factors affecting nisin production were SP (P = 0.011), and SW (P = 0.037). To find optimum conditions, a central composite design (CCD) with 2 central points was used. Three factors were considered, SW (7 to 10 g/L), SP (7 to10 g/L), and small amounts of added nisin as self-inducer (NI 34.4 to 74.4 IU/L). Nisin production was expressed as international units (IU). From RSM, an optimum nisin activity of 180 IU/mL was predicted at 74.4 IU/L NI, 13.8 g/L SP, and 14.9 or 5.11 g/L SW, while confirmatory experiments showed a maximum activity of 178 ± 5.2 IU/mL, verifying the validity of the model. The 2nd-order model showed a coefficient of determination (R²) of 0.828. Optimized conditions were used for constant pH fermentations, where a maximum activity of 575 ± 17 IU/mL was achieved at pH 6.5 after 12 h. The adsorption-desorption technique was used to partially purify nisin, followed by drying. The resulting powder showed an activity of 102150 IU/g. Practical Application : Nisin production was optimized using supplemented whey as alternative culture medium, using a native L. lactis UQ2 strain. Soybean peptone, SW, and subinhibitory amounts of nisin were successfully employed to optimize nisin production by L. lactis UQ2. Dried semipurified nisin showed an activity of 102150 IU/g.     

牛凝乳酶原基因在食品级乳酸乳球菌中的重组表达

将牛凝乳酶原基因连接pNZ8149载体,并转化乳酸乳球菌NZ3900,经乳酸链球菌素Nisin诱导,测得重组菌株胞内凝乳酶活力达到0.7 SU/mL,培养基中检测不到凝乳酶活力,实现了牛凝乳酶原基因在乳酸链球菌Nisin诱导基因表达系统（nisin controlled gene expression system,NICE）中活性表达。在此基础上,将分泌信号肽SPusp45连接于pNZ8149,构建了分泌型表达载体pNZ8149s,并实现牛凝乳酶原基因在NICE系统中分泌表达。当使用1 ng/mLNisin诱导5 h后,重组菌株胞内检测不到凝乳酶活力,培养基中凝乳酶活力为1.2 SU/mL,说明pNZ8149s能够促使凝乳酶原从乳酸乳球菌中分泌。该方法为重组牛凝乳酶在食品级菌株中重组表达提供了一种可行的方案。     

Suitability ofLactococcus lactissubsp.lactisATCC 11454 as a protective culture for lightly preserved fish products

This study is part of strategy to control the human pathogenListeria monocytogenesin lightly preserved fish products by using food-grade lactic acid bacteria. When the nisin- producingLactococcus lactissubsp.lactisATCC 11454 was cultured in the same vessel asL. monocytogenesScott A in brain–heart infusion broth (BHI) at 30°C, the pathogen declined from 5×10⁵to fewer than 5 cfu ml⁻¹within 31 h. The effect was not due to lactic acid inhibition. Growth and nisin production byL. lactisATCC 11454 were investigated under the conditions of temperature and salt used for light preservation. At 5°C in M17 broth, the organism grew well and produced nisin. In an infusion of cold-smoked salmon the organism did not grow at 5°C, although it did at 10°C. NaCl up to 4% allowed for efficient growth and nisin production, while 5% NaCl resulted in very slow growth and no detectable nisin. On slices of commercial cold-smoked salmon at 10°C, no net propagation ofL. lactisATCC 11454 could be detected within 21 days. However, when salmon slices were inoculated withL. monocytogenesat 10⁴cfu g⁻¹and a 300-fold excess of washed lactococcus cells, the pathogen's population declined a half log the first 1.5 days, then increased at a rate slightly lower than that of the control not inoculated with the lactococcus.     

Nisin and lacticin 481 coproduction by Lactococcus lactis strains isolated from raw ewes’ milk

Bacteriocin-producing lactococci were isolated from raw ewes’ milk samples obtained from 5 different Protected Designation of Origin Zamorano cheese manufacturers. Thirteen isolates with antimicrobial activity against Lactococcus lactis HP were selected. Eleven were identified by a PCR technique as L. lactis ssp. lactis and 2 were identified as L. lactis ssp. cremoris. They were grouped under 4 different pulsed-field gel electrophoresis patterns. The presence of structural genes of both nisin and lacticin 481 was detected in 10 L. lactis ssp. lactis isolates belonging to 2 different pulsed-field gel electrophoresis patterns. Coproduction of nisin and lacticin 481 was confirmed after semipurification by using selective indicators. The production of 2 bacteriocins by the same strain is an uncommon property, with relevance in food safety. Nisin and lacticin 481 L. lactis-producing strains might be used as adjunct cultures to the commercial starter in the manufacture of dairy products.     

Genetically modified Lactococcus lactis producing a green fluorescent protein–bovine lactoferrin fusion protein suppresses proinflammatory cytokine expression in lipopolysaccharide-stimulated RAW 264.7 cells

Lactoferrin (LF), an iron-binding glycoprotein distributed widely in the biological fluids of mammals, is believed to play an important role in host defenses against infection. Previous studies in animal models and humans demonstrated that combined administration of LF and probiotic lactic acid bacteria (LAB) can prevent sepsis. In this study, we genetically engineered a probiotic LAB strain, Lactococcus lactis, to produce recombinant bovine LF based on the green fluorescent protein (GFP)-fused expression system. Western blotting confirmed that the genetically modified L. lactis strain (designated NZ-GFP-bLF) produced a protein corresponding to a fusion of GFP and bLF in the presence of nisin, an inducer of target gene expression. The protein synthesized by NZ-GFP-bLF was fluorescent and thus we monitored the time-dependent change in the production level of the recombinant protein using fluorometric analysis. The utility of NZ-GFP-bLF in preventing sepsis was determined by investigating its anti-inflammatory property in lipopolysaccharide (LPS)-stimulated mouse macrophage RAW 264.7 cells. Pretreatment of RAW 264.7 cells with NZ-GFP-bLF significantly attenuated the LPS-induced mRNA expression and protein production of 3 proinflammatory cytokines (IL-1α, IL-6, and tumor necrosis factor-α) compared with pretreatment with a vector control strain of L. lactis. Our results suggest that NZ-GFP-bLF holds promise for the development of a new prophylaxis for sepsis.     

Novel conjugative plasmids from the natural isolate Lactococcus lactis subspecies cremoris DPC3758: a repository of genes for the potential improvement of dairy starters

A collection of 17 natural lactococcal isolates from raw milk cheeses were studied in terms of their plasmid distribution, content, and diversity. All strains in the collection harbored an abundance of plasmids, including Lactococcus lactis ssp. cremoris DPC3758, whose 8-plasmid complement was selected for sequencing. The complete sequences of pAF22 (22,388 kb), pAF14 (14,419 kb), pAF12 (12,067 kb), pAF07 (7,435 kb), and pAF04 (3,801 kb) were obtained, whereas gene functions of technological interest were mapped to pAF65 (65 kb) and pAF45 (45 kb) by PCR. The plasmids of L. lactis DPC3758 were found to encode many genes with the potential to improve the technological properties of dairy starters. These included 3 anti-phage restriction/modification (R/M) systems (1 of type I and 2 of type II) and genes for immunity/resistance to nisin, lacticin 481, cadmium, and copper. Regions encoding conjugative/mobilization functions were present in 6 of the 8 plasmids, including those containing the R/M systems, thus enabling the food-grade transfer of these mechanisms to industrial strains. Using cadmium selection, the sequential stacking of the R/M plasmids into a plasmid-free host provided the recipient with increased protection against 936- and c2-type phages. The association of food-grade selectable markers and mobilization functions on L. lactis DPC3758 plasmids will facilitate their exploitation to obtain industrial strains with enhanced phage protection and robustness. These natural plasmids also provide another example of the major role of plasmids in contributing to host fitness and preservation within its ecological niche.     

食品级载体选择标记的研究进展

乳酸菌、芽孢杆菌等食品级微生物广泛应用于食品、医药、动物养殖等领域并发挥了重要作用。运用基因工程技术对这些微生物进行改良可使其具有更多的功能特性。这些微生物的克隆、表达载体往往含有1个或多个抗生素抗性筛选标记,其抗药性因子的可转移性将危害生物安全。用对人体安全的食品级选择标记替代载体上的抗生素抗性标记可解决这一问题。根据选择的方法,食品级选择标记可分为显性标记和互补标记2大类。文中主要对近十几年来有关食品级选择标记的研究进行综述。     

A food-grade expression/secretion vector for Lactococcus lactis that uses an alpha-galactosidase gene as a selection marker

A new food-grade expression/secretion vector for lactococci, pFMN30, was developed using an alpha-galactosidase gene (melA) of Lactobacillus plantarum as a selection marker. The 4.9-kb pFMN30 is a derivative of the lactococcal vector pMG36e containing a broad-host-range replicon of pWV01. In Lactococcus lactis, transformants carrying the vector were easily detectable by the appearance of a blue colony on a X-alpha-gal-containing medium and also by the growth on a medium containing melibiose as a sole carbon source. The expression/secretion vector was equipped with the controllable and strong nisA promoter. In addition, usp45 signal peptide was inserted for the efficient secretion of a foreign protein outside cells. The vector pFMN30 was used for the expression and secretion of alpha-amylase as a reporter gene, lacking a signal sequence derived from Bacillus licheniformis in L. lactis. These results show that the food-grade expression/secretion vector constructed in the present study could be used for the production of foreign proteins in L. lactis for the production food materials and also for the medicinal purposes.     

Effect of nisin‐producing Lactococcus lactis starter cultures on the inhibition of two pathogens in ripened cheeses

The effect of Lactococcus lactis nisin‐producing strains, isolated from Italian fermented foods, on the survival of two foodborne pathogens namely Listeria monocytogenes and Staphylococcus aureus was investigated in experimental cheese production. One of the three Lactobacillus lactis nisin innoculated as starters, Lactobacillus lactis 41FL1 lowered S. aureus count by 1.73 log colony‐forming units (cfu)/g within the first 3 days, reaching the highest reduction, 3.54 log cfu/g, by the end of ripening period of 60 days. There was no effect on L. monocytogenes. The application of L. lactis 41FL1 as bioprotective culture in controlling S. aureus shows considerable promise.     

Expression of bovine trypsin in Lactococcus lactis

Bovine trypsin is widely used in the production of bioactive peptides from milk proteins. The objective of this study was to express bovine trypsin in Lactococcus lactis to produce peptides by fermentation. The bovine trypsin with bias codon of L. lactis was synthesized, cloned into a secretory expressive vector pSEC-E7, and then introduced to L. lactis strain NZ9000 by electroporation. The expression of trypsin induced by nisin was identified by Western blot. Western blot analysis revealed a band in the protoplast fraction corresponding to SP_Usp45 (signal peptide of usp45)-trypsin. Biological activity of expressed trypsin was confirmed by the single substrate overlay assay. These results suggest that the expression of biologically active bovine trypsin has been detected in the protoplast fraction of L. lactis strain NZ9000 (pSEC:trypsin).     

Diversity Analysis,Batch Fermentation and Characterization of Nisin in Identified Strains of Lactococcus lactis spp. lactis

Thirty-one strains of Lactococcus lactis spp. lactis were identified out of 89 isolates of lactic acid bacteria (LAB) from dairy and nondairy sources. Of the 31 strains, 24 (46.1%) were obtained from dairy and seven (18.9%) from non-dairy sources. The cluster analysis of rep-PCR showed that (GTG)₅-PCR followed by ERIC-PCR exhibited more discriminating potential than BOX-PCR. The obtained banding patterns characterized the polymorphism among strains. The strain level polymorphism was also obtained by the combined cluster analysis of (GTG)_5, ERIC and BOX-PCR which exhibited a level of heterogeneity among strains but not with the sources of isolation. Among 31 strains, 17 strains were able to produce zones of inhibition against Lactobacillus acidophilus NCDC 015 and therefore considered as nisin producers. Nisin production by strains was further confirmed by PCR amplification of nisA/Z of 174 bp size. The nisin activity and cell growth observed to be higher in pH controlled batch fermentation than in uncontrolled fermentation. The nisin activity, cell concentration, and acidity were high in immobilized cell system than free cell batch fermentation. The hot acid and chloroform extraction method was found to be the efficient way for the partial purification of nisin from fermented broth.     

Rapid purification of nisin Z using specific monoclonal antibody-coated magnetic beads

A rapid method is described for the isolation and purification of nisin Z from Lactococcus lactis subsp. lactis biovar. diacetylactis UL719 culture supernatant using magnetic beads. Anti-nisin Z monoclonal antibodies were covalently bound to EDC-activated magnetic beads and the complex was incubated overnight at 4°C with the culture supernatant. Bound nisin Z was then eluted with urea 6 mformic acid 0.1 m mixture. Using this procedure, a high yield of 61.5% was obtained with a nisin Z specific activity of 2.4×10⁴ IU mg⁻¹. Magnetic beads were re-used for two other purification cycles during which a significant decrease of purification yield from 61.5 to 26.7% was observed. The immunomagnetic purification strategy developed in this work was shown to be efficient and offers an alternative rapid procedure for production of high-purity food-grade nisin Z.     

Potential of bacteriocinogenic Lactococcus lactis subsp. lactis inhabiting low pH vegetables to produce nisin variants

Antimicrobial behavior of lactic acid bacteria (LAB) has been explored since many years to assess their ability to produce bacteriocin, a natural preservative, to increase the shelf life of food. This study aims to characterize bacteriocin producing strains of lactic acid bacteria isolated from acidic to slightly acidic raw vegetables including tomato, bell pepper and green chili and to investigate their potential to inhibit food related bacteria. Among twenty nine LAB screened for antimicrobial activity, three exhibited antagonism against closely related bacterial isolates which was influenced by varying temperature and pH. They were identified up to strain level as Lactococcus lactis subsp. lactis TI-4, L. lactis subsp. lactis CE-2 and L. lactis subsp. lactis PI-2 based on 16S rRNA gene sequence. Their spectrum of inhibition was observed against food associated strains of Bacillus subtilis and Staphylococcus aureus. Moreover, L. lactis subsp. lactis PI-2 selected on the basis of higher antimicrobial activity was further evaluated for bacteriocin production which was detected as nisin A and nisin Z. These findings suggest the possible use of L. lactis strains of vegetable origin as protective cultures in slightly acidic as well as slightly alkaline food by the bio-preservative action of bacteriocins.     

Production of pediocin PA-1, and coproduction of nisin A and pediocin PA-1, by wild Lactococcus lactis strains of dairy origin

Heterologous production of pediocin PA-1 in nisin and non-nisin-producing Lactococcus lactis strains, which had been previously selected because of their technological properties for cheese making, was investigated. Plasmid pFI2160, which contains a hybrid gene (L-pedA) encoding the fusion between the lactococcin A leader and propediocin PA-1, and also the genes lcnC and lcnD, that encode the lactococcin A secretion apparatus, was introduced into L. lactis ESI 153 and L. lactis ESI 515 (Nis⁺). The pediocin production level of their respective transformants, L. lactis CL1 and L. lactis CL2 (Nis⁺), was approximately 600 and 400 ng mL⁻¹, respectively, which represents a 30% and a 20% of the quantity produced by the natural pediocin PA-1 producer Pediococcus acidilactici 347. Transformation of L. lactis ESI 515 with pFI2160 did not affect its ability to produce nisin. Pediocin bioassays showed the stability of pFI2160 in both heterologous hosts under selective and non-selective conditions.