Nisin Production by a Mixed-Culture System Consisting of Lactococcus lactis and Kluyveromyces marxianus

To control the pH during antimicrobial peptide (nisin) production by a lactic acid bacterium, Lactococcus lactis subsp. lactis (ATCC11454), a novel method involving neither addition of alkali nor a separation system such as a ceramic membrane filter and electrodialyzer was developed. A mixed culture of L. lactis and Kluyveromyces marxianus, which was isolated from kefir grains, was utilized in the developed system. The interaction between lactate production by L. lactis and its assimilation by K. marxianus was used to control the pH. To utilize the interaction of these microorganisms to maintain high-level production of nisin, the kinetics of growth of, and production of lactate, acetate, and nisin by, L. lactis were investigated. The kinetics of growth of and lactic acid consumption by K. marxianus were also investigated. Because the pH of the medium could be controlled by the lactate consumption of K. marxianus and the specific lactate consumption rate of K. marxianus could be controlled by changing the dissolved oxygen (DO) concentration, a cascade pH controller coupled with DO control was developed. As a result, the pH was kept constant because the lactate level was kept low and nisin accumulated in the medium to a high level compared with that attained using other pH control strategies, such as with processes lacking pH control and those in which pH is controlled by addition of alkali.     

乳酸链球菌肽发酵动力学的研究

提出了在恒定不同pH的发酵条件下,乳酸链球菌SM526的菌体生长、底物消耗、乳酸及Nisin产生的动力学模型。菌体生长、乳酸及Nisin产生用逻辑方程描述,而底物消耗是菌体生长和乳酸产生速率的函数。模型表明,乳酸链球菌SM526菌体生长和乳酸产生的最佳pH为7．0,而Nisin产生的最佳pH却为6．5。     

Production of a nisin-like bacteriocin by Lactococcus lactis subsp. lactis A164 isolated from Kimchi

Lactococcus lactis subsp. lactis A164 was isolated from Kimchi (Korean traditional fermented vegetables). The bacteriocin produced by strain A164 was active against closely related lactic acid bacteria and some food-borne pathogens including Staphylococcus aureus, Listeria monocytogenes and Salmonella typhimurium. The antimicrobial spectrum was nearly identical to that of nisin. Bacteriocin activity was not destroyed by exposure to elevated temperatures at low pH values, but the activity was lost at high pH values. This bacteriocin was inactivated by pronase E and alpha, beta-chymotrypsin, but not by trypsin, pepsin, and alpha-amylase. Cultures of L. lactis subsp. lactis A164 maintained at a constant pH of 6.0 exhibited maximum production of the bacteriocin. It was purified to homogeneity by ammonium sulphate precipitation, sequential ion exchange chromatography, and ultrafiltration. Tricine-SDS-PAGE of purified bacteriocin gave the same molecular weight of 3.5 kDa as that of nisin. The gene encoding this bacteriocin was amplified by PCR with nisin gene-specific primers and sequenced. It showed identical sequences to the nisin gene. These results indicate that bacteriocin produced by Lactococcus lactis A164 is a nisin-like bacteriocin.     

Isolation, screening and characterization of bacteriocin-producing lactic acid bacteria isolated from traditional fermented food

100 lactic acid bacterial strains isolated from traditional fermented foods (yoghurt, milk cream, sour dough and milk) were screened for bacteriocin production. Twenty six strains producing a nisin-like bacteriocin were selected. Most of these isolates gave only a narrow inhibitory spectrum, although one showed a broad inhibitory spectrum against the indicator strains tested, this strain was determined as Lactococcus lactis. The influence of several parameters on the fermentative production of nisin by Lactococcus lactis was studied. Production of nisin was optimal at 30 degrees C and in the pH range 5.5-6.3. The effect of different sulphur and nitrogen sources on Lactococcus lactis growth and nisin production was studied. Magnesium sulfate and manganese sulfate were found to be the best sulphur sources while triammonium citrate was the best inorganic nitrogen source and meat extract, peptone and yeast extract were the best organic nitrogen source for nisin production.     

Influence of the carbon source on nisin production in Lactococcus lactis subsp. lactis batch fermentations.

Nisin production by Lactococcus lactis subsp. lactis NIZO 22186 was studied in batch fermentation using a complex medium. Nisin production showed primary metabolite kinetics: nisin biosynthesis took place during the active growth phase and completely stopped when cells entered the stationary phase. A stringent correlation could be observed between the expression of the prenisin gene (nisA) and the synthesis of the post-translationally enzymically modified and processed mature nisin peptide. Moreover, it seemed likely that nisin had a growth control function. A physiological link is proposed between sucrose fermentation capacity and nisin production ability. Carbon source regulation appears to be a major control mechanism for nisin production.     

Characterization of Lactic Acid Bacteria Coexisting with a Nisin Z Producer in <Emphasis Type="Italic">Tsuda</Emphasis>-Turnip Pickles

Bacteriocin-producing lactic acid bacteria (LAB) are believed to be associated with many types of fermented food. The present study reports the identification of lactic acid bacterium MS27 producing a bacteriocin isolated from the Tsuda-turnip pickle, which is a Japanese fermented food, and characterization of LAB coexisting with the bacteriocin producers in the Tsuda-turnip pickle. The strain MS27 was identified as Lactococcus lactis subsp. lactis based on a partial 16S rRNA gene sequence and sugar fermentation pattern analyses. Mass spectroscopy and genetic analysis revealed that it produces nisin Z. Microbial population analysis revealed that the LAB community in the Tsuda-turnip pickle comprises nisin Z-sensitive and nisin Z-insensitive LAB (nonbacteriocin producers) and nisin Z producers at population rates of 52.5%, 37.5%, and 10.0%, respectively. This revealed that Leuconostoc spp. (nisin Z insensitive) is the dominant species among LAB microflora and that nisin Z insensitivity of a bacterial strain is proportional to its ability to dominate the population in Tsuda-turnip pickles. Competitive growth assay revealed that Leuconostoc spp. considerably suppressed the bacteriocin production of L. lactis MS27. These results suggested that Leuconostoc spp. contributes to the formation of the LAB community with a wide variety of microorganisms in Tsuda-turnip pickles.     

Identification and characterization of a mobilizing plasmid, pND300, in Lactococcus lactis M189 and its encoded nisin resistance determinant

A 60 kb conjugative plasmid, pND300, which encodes nisin resistance, was identified in Lactococcus lactis ssp. lactis (L. lactis ) M189. pND300 was found to mobilize the transfer of some other plasmids as indicated by the mobilization of plasmids encoding lactose utilization. The nisin resistance determinant from pND300 was initially subcloned on a 12 kb DNA fragment and subsequently reduced to 10.4 kb. Restriction analysis, PCR, Southern hybridization and sequencing illustrated that the nisin resistance of pND300 is very similar to that encoded by the transposon involved in nisin production. pND300 encodes nis R. as well as nis K and the recently reported nis F, nis E and nis G, but does not encode nis I. The DNA fragment encoding the nis genes is flanked by IS946 with a copy at each end in reverse orientation. The expression of these nis genes is probably controlled by a putative promoter upstream of nis R, which is composed of the TTGCAA hexanucleotide on the insertion sequence IS 946 and the TATAAT sequence 21 bp downstream.     

Growth of, and aflatoxin production by Aspergillus parasiticus when in the presence of either Lactococcus lactis or lactic acid and at different initial pH values

L uchese , R.H. & H arrigan , W.F. 1990. Growth of, and aflatoxin production by Aspergillus parasiticus when in the presence of either Lactococcus lactis or lactic acid and at different initial pH values. Journal of Applied Bacteriology 69 , 512–519.
Aspergillus parasiticus was grown in a modified Lab-Lemco tryptone broth both as a single culture and in association with Lactococcus lactis . Total aflatoxin (B1 + G1) production was higher in the mixed cultures. This stimulation persisted when different batches of media, inoculation procedures and makes of ingredients were used. Aflatoxin yields increased in media with an initial pH of 4.2 compared with a pH close to neutrality. Hydrochloric and/or lactic acid had little effect. The substitution of half the carbon content of the medium by lactate resulted in stimulation or reduction on aflatoxin production when the initial pH was 4.2 or 6.8, respectively.     

Complete genome sequence of Lactococcus lactis IO-1, a lactic acid bacterium that utilizes xylose and produces high levels of L-lactic acid

We report the complete genome sequence of Lactococcus lactis IO-1 (= JCM7638). It is a nondairy lactic acid bacterium, produces nisin Z, ferments xylose, and produces predominantly L-lactic acid at high xylose concentrations. From ortholog analysis with other five L. lactis strains, IO-1 was identified as L. lactis subsp. lactis.     

Production of L(+)-lactic acid from glucose and starch by immobilized cells of Rhizopus oryzae in a rotating fibrous bed bioreactor

A rotating fibrous-bed bioreactor (RFB) was developed for fermentation to produce L(+)-lactic acid from glucose and cornstarch by Rhizopus oryzae. Fungal mycelia were immobilized on cotton cloth in the RFB for a prolonged period to study the fermentation kinetics and process stability. The pH and dissolved oxygen concentration (DO) were found to have significant effects on lactic acid productivity and yield, with pH 6 and 90% DO being the optimal conditions. A high lactic acid yield of 90% (w/w) and productivity of 2.5 g/L.h (467 g/h.m(2)) was obtained from glucose in fed-batch fermentation. When cornstarch was used as the substrate, the lactic acid yield was close to 100% (w/w) and the productivity was 1.65 g/L.h (300 g/h.m(2)). The highest concentration of lactic acid achieved in these fed-batch fermentations was 127 g/L. The immobilized-cells fermentation in the RFB gave a virtually cell-free fermentation broth and provided many advantages over conventional fermentation processes, especially those with freely suspended fungal cells. Without immobilization with the cotton cloth, mycelia grew everywhere in the fermentor and caused serious problems in reactor control and operation and consequently the fermentation was poor in lactic acid production. Oxygen transfer in the RFB was also studied and the volumetric oxygen transfer coefficients under various aeration and agitation conditions were determined and then used to estimate the oxygen transfer rate and uptake rate during the fermentation. The results showed that the oxygen uptake rate increased with increasing DO, indicating that oxygen transfer was limited by the diffusion inside the mycelial layer.     

Controlled gene expression systems for lactic acid bacteria: transferable nisin-inducible expression cassettes for Lactococcus, Leuconostoc, and Lactobacillus spp.

A transferable dual-plasmid inducible gene expression system for use in lactic acid bacteria that is based on the autoregulatory properties of the antimicrobial peptide nisin produced by Lactococcus lactis was developed. Introduction of the two plasmids allowed nisin-inducible gene expression in Lactococcus lactis MG1363, Leuconostoc lactis NZ6091, and Lactobacillus helveticus CNRZ32. Typically, the beta-glucuronidase activity (used as a reporter in this study) remained below the detection limits under noninducing conditions and could be raised to high levels, by addition of subinhibitory amounts of nisin to the growth medium, while exhibiting a linear dose-response relationship. These results demonstrate that the nisin-inducible system can be functionally implemented in lactic acid bacteria other than Lactococcus lactis.     

Rapid differentiation of the closely related Kluyveromyces lactis var. lactis and K. marxianus strains isolated from dairy products using selective media and PCR/RFLP of the rDNA non transcribed spacer 2

PCR/RFLP of the NTS2 (IGS2) of rDNA was applied to differentiate two closely related yeast species, Kluyveromyces lactis var. lactis (referred to as K. lactis) and K. marxianus. Using specific primers, the NTS2 region was amplified from DNA of both K. lactis and K. marxianus type and collection strains. AluI restriction of amplified fragments generated patterns characteristic for each species. The NTS2 region from K. lactis var. drosophilarum and related species K. aestuarii, K. africanus, K. dobzhanskii, and K. wickerhamii could also be amplified with the same primers, but AluI patterns generated were clearly different. PCR/RFLP of the NTS2 appears thus to be a convenient method for rapid identification of K. lactis and K. marxianus, frequently found in dairy products. This test was validated therefore on K. lactis and K. marxianus from natural habitats. We showed that all yeast strains collected from whey samples and scoring blue on X-gal glucose plates were either K. lactis or K. marxianus. For application purposes, we propose here an approach for quickly screening for K. lactis/marxianus and Saccharomyces cerevisiae in dairy products using X-gal coloured and lysine growth media.     

A food-grade cloning vector for lactic acid bacteria based on the nisin immunity gene nisI

A new food-grade cloning vector for lactic acid bacteria was constructed using the nisin immunity gene nisI as a selection marker. The food-grade plasmid, pLEB590, was constructed entirely of lactococcal DNA: the pSH71 replicon, the nisI gene, and the constitutive promoter P45 for nisI expression. Electroporation into Lactococcus lactis MG1614 with 60 international units (IU) nisin/ml selection yielded approximately 10⁵ transformants/μg DNA. MG1614 carrying pLEB590 was shown to be able to grow in medium containing a maximum of 250 IU nisin/ml. Plasmid pLEB590 was succesfully transformed into an industrial L. lactis cheese starter carrying multiple cryptic plasmids. Suitability for molecular cloning was confirmed by cloning and expressing the proline iminopeptidase gene pepI from Lactobacillus helveticus in L. lactis and Lb. plantarum. These results show that the food-grade expression system reported in this paper has potential for expression of foreign genes in lactic acid bacteria in order to construct improved starter bacteria for food applications. Electronic Publication     

A kinetic model describing Shewanella oneidensis MR-1 growth, substrate consumption, and product secretion

Aerobic growth of Shewanella oneidensis MR-1 in minimal lactate medium was studied in batch cultivation. Acetate production was observed in the middle of the exponential growth phase and was enhanced when the dissolved oxygen (DO) concentration was low. Once the lactate was nearly exhausted, S. oneidensis MR-1 used the acetate produced during growth on lactate with a similar biomass yield as lactate. A two-substrate Monod model, with competitive and uncompetitive substrate inhibition, was devised to describe the dependence of biomass growth on lactate, acetate, and oxygen and the acetate growth inhibition across a broad range of concentrations. The parameters estimated for this model indicate interesting growth kinetics: lactate is converted to acetate stoichiometrically regardless of the DO concentration; cells grow well even at low DO levels, presumably due to a very low K(m) for oxygen; cells metabolize acetate (maximum specific growth rate, micro(max,A) of 0.28 h(-1)) as a single carbon source slower than they metabolize lactate (micro(max,L) of 0.47 h(-1)); and growth on acetate is self-inhibiting at a concentration greater than 10 mM. After estimating model parameters to describe growth and metabolism under six different nutrient conditions, the model was able to successfully estimate growth, oxygen and lactate consumption, and acetate production and consumption under entirely different growth conditions.     

Growth of nisin-producing lactococci in cooked rice supplemented with soybean extract and its application to inhibition of Bacillus subtilis in rice miso

Lactic acid fermentation of cooked rice and rice koji by supplementation with soybean extract (SBE) and its application to rice miso fermentation were investigated. By supplementing the cooked rice with SBE, lactic acid bacteria (LAB) grew well without any unfavorable effects on the rice such as off-flavor or coloration. Lactococcus lactis subsp. lactis IFO12007 (Lc. lactis, a producer of the bacteriocin nisin) proliferated at 10(8 to approximately 9) cells/g after 24 h of incubation and produced high activity of nisin. The fermented rice with Lc. lactis strongly inhibited not only Bacillus subtilis ATCC19659 but also the other Bacillus strains. While some strains of LAB markedly inhibited the growth of Asp. oryzae, resulting in failure of koji fermentation, Lc. lactis did not affect the growth of these molds. When Lc. lactis was used for rice miso fermentation as a lactic acid starter culture, Lc. lactis rapidly proliferated and produced high nisin activity of 6,400 IU/g, in the steamed rice, resulting in complete growth inhibition of B. subtilis, which had been inoculated at the beginning of the koji fermentation. The rice miso after 12 weeks of aging had a suitable pH, and favorable taste and color. Furthermore, hyposalting of rice miso could be done without difficulty by lactic acid fermentation of both rice and soybeans.     

Combined effect of bacteriocin-producing lactic acid bacteria and lactoperoxidase system activation on Listeria monocytogenes in refrigerated raw milk

The bactericidal activity of three bacteriocin-producing lactic acid bacteria alone and in combination with milk lactoperoxidase (LP) system activation against Listeria monocytogenes in refrigerated raw milk was studied. After 4 d at 4°C, the population of L. monocytogenes in milk inoculated with bacteriocin-producing Lactococcus lactis subsp. lactis ATCC 11454, L. lactis subsp. lactis ESI 515 or Enterococcus faecalis INIA 4 was reduced by 0·21–0·24 log units. Activation of the LP system did not enhance inhibition at this temperature. After 4 d at 8°C, L. monocytogenes levels in the non-activated LP system milk inoculated with L. lactis subsp. lactis ATCC 11454, L. lactis subsp. lactis ESI 515 or Ent. faecalis INIA 4 were reduced by 1·87, 1·54 and 1·11 log units compared to control milk, whereas in the activated LP system milk, this reduction was 1·99, 2·10 and 1·06, respectively. The higher nisin production by L. lactis subsp. lactis ESI 515 in milk with activated LP system than in non-activated LP system milk was responsible for the more pronounced decrease of L. monocytogenes counts in the former.     

Comparative effectiveness of nisin and bacteriocin J46 at different pH values

E. HUOT, C. BARRENA-GONZALEZ AND H. PETITDEMANGE. 1996. A Comparative study of the inhibitory activity of nisin, the well-known lantibiotic produced by certain strains of Lactococcus lactis subsp. lactis , and of the bacteriocin produced by L. lactis subsp. cremoris J46, a strain previously isolated from fermented milk, was conducted. For both bacteriocins, the activity against L. lactis subsp. cremoris decreased with increasing pH. In addition, the bacteriocin preparations were more stable at 4 than at 20°C. The influence of the storage temperature was more crucial for nisin. Essentially the same activity was observed for bacteriocin J46 stored for 3 h at 4 or 20°C. More interesting was the observed stability of bacteriocin J46 at pH values between 5.8 and 6.8. For example, about 23% of nisin activity was lost at pH 6.4 whereas no loss of bacteriocin J46 activity was observed.     

Inhibition of Listeria innocua in cheddar cheese by addition of nisin Z in liposomes or by in situ production in mixed culture

The effect of addition of purified nisin Z in liposomes to cheese milk and of in situ production of nisin Z by Lactococcus lactis subsp. lactis biovar diacetylactis UL719 in the mixed starter on the inhibition of Listeria innocua in cheddar cheese was evaluated during 6 months of ripening. A cheese mixed starter culture containing Lactococcus lactis subsp. lactis biovar diacetylactis UL719 was selected for high-level nisin Z and acid production. Experimental cheddar cheeses were produced on a pilot scale, using the selected starter culture, from milk with added L. innocua (10(5) to 10(6) CFU/ml). Liposomes with purified nisin Z were prepared from proliposome H and added to cheese milk prior to renneting to give a final concentration of 300 IU/g of cheese. The nisin Z-producing strain and nisin Z-containing liposomes did not significantly affect cheese production and gross chemical composition of the cheeses. Immediately after cheese production, 3- and 1.5-log-unit reductions in viable counts of L. innocua were obtained in cheeses with encapsulated nisin and the nisinogenic starter, respectively. After 6 months, cheeses made with encapsulated nisin contained less than 10 CFU of L. innocua per g and 90% of the initial nisin activity, compared with 10(4) CFU/g and only 12% of initial activity in cheeses made with the nisinogenic starter. This study showed that encapsulation of nisin Z in liposomes can provide a powerful tool to improve nisin stability and inhibitory action in the cheese matrix while protecting the cheese starter from the detrimental action of nisin during cheese production.     

Sequential gene integration for the engineering of Kluyveromyces marxianus

The attributes of the yeast Kluyveromyces marxianus (rapid growth rate at high temperature, utilization of a wide range of inexpensive carbon sources) make it a promising industrial host for the synthesis of protein and non-protein products. However, no stable multicopy plasmids are currently available for long-term culture of K. marxianus. To allow the stable genetic/metabolic engineering of K. marxianus, a method for integrating precise numbers of the same or different genes was developed for this yeast. A K. marxianus URA3 deletion mutant was constructed and the URA3 blaster (UB) reusable selection cassette from Saccharomyces cerevisiae was used to select sequential, untargeted chromosomal insertions of the Bacillus megaterium lactate dehydrogenase (LDH) gene. Following excision of the UB cassette from the chromosomes, the integrating vector was retransformed into the strain and a second copy of LDH was inserted, demonstrating the success of this method for sequential gene integrations in K. marxianus. LDH activity and lactic acid concentration increased with each gene insertion, further illustrating the success of this method.